Abstract
Oral Presentations
Safety and clinical benefit of lentiviral haematopoietic stem and progenitor cell gene therapy in 23 patients with Wiskott‐Aldrich Syndrome with up to 10.5 years of follow‐up
1: Pediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy 2: San Raffaele Telethon Institute for Gene Therapy (SR‐Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy 3: Vita‐Salute San Raffaele University, Milan, Italy 4: Bicocca Bioinformatics Biostatistics and Bioimaging B4 Center, School of Medicine and Surgery, University of Milano ‐ Bicocca, Monza, Italy 5: Temerty School of Medicine, University of Toronto, Toronto, Ontario, Canada 6: Unity Health Toronto (St. Michael's Hospital) and the Hospital for Sick Children, Toronto, Ontario, Canada 7: Centre hospitalier universitaire Felix Guyon, service d'immunologie clinique, 97405, Saint Denis Cedex Ile de la Reunion, France 8: Department of Oncohematology, Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy 9: Department of Pediatric Hematology and Oncology, ARNAS Civico Di Cristina and Benfratelli Hospital, Palermo, Italy 10: Division of Molecular Pathogenesis, Department of Human Genetics, National Center for Child Health and Development, Tokyo, Japan 11: Department of Pediatrics, Division of Immunology and Allergy, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada 12: Department of Pediatric Respiratory Medicine, Immunology and Critical Care Medicine, Charité ‐ Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt‐Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany 13: BIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health (BIH), Berlin, Germany 14: Labor Berlin GmbH, Department of Immunology, Berlin, Germany 15: Berlin Institute of Health at Charité – Universitätsmedizin Berlin, Berlin, Germany 16: Department of Human Genetics, National Center for Child Health and Development, Tokyo, Japan 17: Orchard Therapeutics (Europe) Limited, London, UK 18: Department of Hematology and Stem Cell Transplantation, IRCCS San Raffaele Scientific Institute, Milan, Italy
Allogeneic haematopoietic stem cell transplantation (HSCT) can be curative for Wiskott‐Aldrich Syndrome (WAS). However, despite outcome improvements, HSCT is still limited by donor availability and higher risk of complications in older subjects. Gene therapy (GT) is being studied as an alternative treatment. Between 2010 and 2020, 23 WAS patients were treated with OTL‐103, as part of phase I/II (n = 8) or III (n = 6) clinical trials or Expanded Access Program (EAP) (n = 9), at a median age of 3.1 years (range: 1.0‐35.1). OTL‐103 is an investigational autologous haematopoietic stem and progenitor cell (HSPC) GT composed of CD34+ HSPCs transduced ex vivo with a self‐inactivating lentiviral vector encoding human WAS cDNA under endogenous promoter control. Seventeen subjects received fresh OTL‐103, while six received its cryopreserved formulation. Before OTL‐103, subjects received rituximab and reduced‐intensity conditioning. At data cut, median follow‐up was 3.6 years (range: 0.4‐10.5). All were alive, except one EAP subject who died early post‐GT due to pre‐existing neurological condition deterioration. To date, no signs of insertional mutagenesis or GT‐related adverse events have been observed. Efficacy analysis performed in trials' subjects confirmed sustained engraftment of gene‐corrected cells, leading to substantial increase of WASP expression in lymphocytes and platelets. This resulted in ameliorated T‐cell functionality and platelet count, with marked reduction of severe infections and moderate/severe bleedings. Antimicrobial and bleeding prophylaxis was stopped. Eczema improved and clinical autoimmunity resolved. Our data suggest that lentiviral GT is well tolerated and leads to sustained clinical benefit, underlying its potential as an alternative therapeutic option for WAS.
Experience of genome editing patient haematopoietic stem cells to treat X‐linked Agammaglobulinemia
1: Molecular and Cellular Immunology section, UCL Great Ormond Street Institute of Child Health, University College London 2: NIHR Great Ormond Street Hospital Biomedical Research Centre
CRISPR/Cas technology has improved in recent years and human trials of this technology have been reported in cell‐based cancer therapies and monogenic disorders. For patients with primary immunodeficiencies, gene editing has the potential to rescue genetic defects via the correction and transplantation of autologous haematopoietic stem cell (HSCs).
We report our experience trialling a gene‐editing technique on HSCs from patients with X‐Linked Agammaglobulinemia (XLA). This disease is caused by mutations in Bruton's Tyrosine Kinase (BTK) and results in a cessation of B‐Cell development and an absolute antibody deficiency.
Our universal strategy involves inserting codon optimised BTK using an AAV vector in a Cas9 mediated break at the endogenous gene location. This would correct almost all XLA causing gene mutations with the transgene remaining under the control of local regulatory factors.
Cell line optimisation experiments revealed successful transgene integration and expression demonstrated in a BTK knockout version of a B cell line (DG75). Gene editing on HSCs donated from 3 XLA patients showed restored ability to differentiate into immature CD19+IgM+ B cells in vitro. Editing efficiencies of up to 60% were achieved (via droplet digital PCR).
Assessment of edited cells in a NSG mouse model showed persistence of cells 12 weeks post transplantation with restored proportions of human B cells compared to mice transplanted with non‐edited patient HSCs.
Our work demonstrates the potential of this strategy as a future curative therapy for XLA.
Enhanced transduction and immunophenotyping demonstrates preclinical safety and efficacy of haematopoietic stem cell gene therapy for Mucopolysaccharidosis II (MPSII) using an IDS.ApoEII brain targeted therapy
1: University of Manchester
MPSII (Hunter Syndrome) is a debilitating paediatric lysosomal disorder caused by mutations in the iduronate‐2‐sulphatase (IDS) gene. The majority of patients manifest with cognitive symptoms that cannot be treated by conventional means such as enzyme replacement therapy, since native IDS does not readily cross the blood–brain barrier. In a proof‐of‐concept murine study we demonstrated that a brain‐targeted hematopoietic stem cell gene therapy (HSCGT) approach using lentiviral IDS fused to ApoEII (IDS.ApoEII) was able to normalise brain pathology and behaviour of MPSII mice, providing significantly enhanced correction compared to mice receiving HSCGT with a lentiviral vector expressing normal IDS or treatment with a conventional allogeneic bone marrow transplant. Here we describe additional preclinical studies conducted in order to accelerate translation of brain‐targeted MPSII HSCGT to the clinic and provide additional safety and efficacy data. We performed Luminex immunophenotyping on a range of organs including the brain, liver, serum, spleen, brain, heart, lung, spinal cord and skeletal muscle using a 23‐plex mouse cytokine panel and observed correction of a number of elevated disease markers following HSCGT treatment in support of previous findings. In addition, we developed an optimised clinical stem cell manufacturing protocol, with the inclusion of the transduction enhancers protamine sulphate and LentiBOOSTTM, resulting in a 5‐fold reduction in the amount of IDS.ApoEII lentiviral vector required per treatment achieving a vector copy number (VCN) in the 3‐6 copy range. This work paves the way for a phase I/II HSCGT clinical trial in MPSII patients anticipated to start late 2022.
Preclinical development of an ex vivo gene therapy for Mucopolysaccharidosis type II
S Das1 F Rruga2 L Rigon2 J Patel1 R Milazzo2
1: Dana‐Farber/Boston Children's Cancer and Blood Disorders Center, Harvard Medical School, Boston, USA 2: Department of Pediatric Hematology and Oncology Center, Woman's and Child Health, University of Padova, Padua, Italy
Mucopolysaccharidosis type II (MPSII) is a X‐linked inherited lysosomal storage disorder caused by absence or reduced activity of the lysosomal enzyme iduronate‐2‐sulfatase (IDS). MPSII causes a progressive glycosaminoglycan (GAG) accumulation in nearly all cell types, with the most severe clinical manifestations in the central nervous system (CNS).
We designed a gene therapy (GT) approach based on the autologous transplantation of hematopoietic stem/progenitor cells (HSPCs) modified by lentiviral vector (LV) gene transfer to restore IDS expression. The GT approach tested in pre‐symptomatic and symptomatic MPS II mice rescued behavioral, skeletal, biochemical and pathological disease manifestations and significantly increased animal survival, with all GT‐treated mice out‐surviving the control mock‐transplanted mice. GT leads to IDS activity restoration up to 50% of the normal level and reduction of GAG accumulation in the brain of treated animals. Based on these efficacy results in MPSII mice, we are currently proceeding with the preclinical development of our GT strategy, performing a toxicology/biodistribution study in humanized immunodeficient mice (NSG). Human HSPCs, transduced with two different protocols intended for clinical use, were transplanted in NSG mice to assess human‐cell engraftment, biodistribution, hematopoietic reconstitution and vector integration profile. The results of the NSG study will be instrumental to a rapid progression toward the clinical development of our GT strategy for MPSII.
Skeletal damage and cross‐correction in MPSIH HSPC‐gene therapy
1: San Raffaele Telethon Institute for Gene Therapy (HSR‐TIGET) 2: Pediatric Immunohematology and Bone Marrow Transplantation Unit, San Raffaele Scientific Institute, Milan, Italy 3: Department of Molecular Medicine Sapienza University, Rome Italy 4: M. Tettamanti Research Center, Monza, Italy 5: Tissue Engineering Department of Biomedicine, University Hospital Basel, Switzerland 6: Cell and Tissue Engineering Laboratory, IRCCS Galeazzi Orthopaedic Institute, Milan, Italy 7: Milan Unit, Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Milan, Italy.
Mucopolysaccharidosis type I Hurler (MPSIH) patients, missing the α‐L‐iduronidase (IDUA) enzyme, show widespread accumulation of glycosaminoglycans (GAGs), leading to skeletal abnormalities that remain uncorrected by allogeneic hematopoietic stem cell transplantation. Interim results from a Phase I/II ex vivo gene therapy (GT) clinical trial in 8 patients have shown potential metabolic and skeletal correction. Osteomedullary biopsies obtained from MPSIH patients displayed a reduced expression of type I collagen and a morphological disorganization of chondrocytes (CH). To further investigate CH differentiation and the endochondral ossification process, we are exploiting an in vitro 3D model of hypertrophic cartilage to evaluate whether patient‐derived mesenchymal stromal cells (MSCs) are capable of generating de novo bone tissue. MPSIH‐MSCs isolated before and after GT normally underwent intramembraneous ossification in vitro, although osteoblasts (OBs) showed intracellular accumulation of GAGs. Cross‐correction of patient‐derived OBs was feasible due to their ability to uptake the IDUA enzyme when exposed to the supernatant of patient gene‐corrected osteoclasts. Moreover, osteomedullary biopsies of MPSIH patients before GT showed enthesopathy, with enlarged and vacuolated cells, that was completely reversed in the post GT samples. To better characterize this aspect, the Achilles tendons of untreated MPSI mice at different ages were harvested, showing a progressive vacuolization, matrix mineralization and calcification. In addition, MPSI mice showed a reduction of OBs number but an increased bone formation rate compared to healthy mice. These muscoloskeletal features will be further investigated in GT‐treated patients and mice to provide a broader undestanding of disease‐specific pathogenetic mechanisms and skeletal correction after GT.
Using a systems biology approach to unravel the Immunogenicity of AAV8 empty capsids in healthy volunteers
1: AskBio HQ 2: AskBio Europe 3: Selecta Biosciences 4: The University of North Carolina at Chapel Hill
High vector doses of AAV are associated with an increased risk of immunogenicity and toxicity. Empty AAV capsids, devoid of transgene sequences, are a byproduct of AAV manufacturing and can comprise a significant fraction of the total capsid mass even though they are not included in the calculated vector dose. The contribution of empty capsids to immunogenicity, and potential toxicity, in humans is unknown. Here we report data from a healthy volunteer study in which eight participants were administered 2e12 viral particles/kg of AAV8 empty capsids. Using a systems biology approach, we employed whole blood transcriptomic, serum proteomic, and peripheral blood mononuclear cell flow cytometric analyses to comprehensively characterize the developing immune response from pre‐treatment to 90 days post‐dosing. Traditional immunologic assessments measuring neutralizing and total anti‐AAV IgG/M antibodies, and IFNγ ELISpot were also deployed. Empty capsids were well‐tolerated, and no severe adverse events were observed. Prophylactic medications and empty capsids induced robust changes in peripheral blood mRNA expression and serum protein levels within hours post‐dose, with upregulation of markers associated with innate immune responses, complement activity, and cell proliferation. Anti‐AAV8 neutralizing antibodies were induced in all participants, while capsid‐specific T‐cell IFNγ responses were induced in 7 of 8 participants. These responses peaked 7‐14 days post‐dose and were correlated significantly with proteomic and flow cytometric measures. We believe our data highlight that empty capsids are strongly immunogenic even in the absence of transgene‐encoded CpG/immunostimulatory motifs. These results suggest that empty capsids could contribute to AAV immunogenicity and toxicity.
ImmTOR tolerogenic nanoparticles combined with Treg‐selective IL‐2 mutein induces massive expansion of antigen‐specific regulatory T cells and synergistically inhibit formation of anti‐AAV antibodies to high vector doses
1: Selecta Biosciences
ImmTOR nanoparticles encapsulating rapamycin, an inhibitor of the mTOR pathway, have been shown to mitigate immunogenicity of AAV vectors and enable re‐dosing. However, delayed immune responses can result in breakthrough of anti‐AAV antibodies in some animals, particularly at higher vector doses. Here we demonstrate that a single dose of ImmTOR and antigen administered the same day as a regulatory T cell (Treg)‐selective interleukin‐2 (IL‐2 mutein) results in a profound synergistic expansion of antigen‐specific Treg in an OTII T cell transfer model, with ∼45% of OTII cells expressing Foxp3. We next tested the ability of ImmTOR and IL‐2 mutein to enable more durable inhibition of antibody responses to co‐administered AAV gene therapy vectors. Mice were administered two doses of AAV8 vector with or without ImmTOR +/‐ IL‐2 mutein on Days 0 and 56. As expected, mice treated with a therapeutic dose of ImmTOR (200 μg) inhibited the formation of anti‐AAV antibodies; however, by Day 91, 19 days after the second dose of AAV, some mice showed delayed development of anti‐AAV antibodies. Treatment with IL‐2 mutein alone resulted in only a modest reduction in antibodies. In contrast, the combination of ImmTOR + IL‐2 mutein completely inhibited antibody formation through Day 117, even at sub‐optimal doses of ImmTOR. These results show that the combination of ImmTOR and IL‐2 mutein has the potential to provide more durable antigen‐specific immune tolerance to mitigate immunogenicity of AAV gene therapy vectors even at high vector doses up to 5E13 vg/kg.
Sustained factor IX activity levels and bleeding protection following etranacogene dezaparvovec administration in people with haemophilia B without and with adeno‐associated virus serotype 5 neutralizing antibodies
1: University Hospital Frankfurt, Frankfurt, Germany 2: Erasmus MC, University Medical Center, Rotterdam, Netherlands 3: Oregon Health & Science University, Portland, OR, United States 4: University of North Carolina, Chapel Hill, NC, United States 5: Center for Bleeding Disorders and Coagulation, Careggi University Hospital, Florence, Italy 6: uniQure BV, Amsterdam, Netherlands/uniQure Inc. Lexington, MA, USA 7: CSL Behring, King of Prussia, PA, United States 8: University of Michigan, Ann Arbor, MI, United States
Pre‐existing adeno‐associated virus (AAV) neutralizing antibodies (NAbs) have limited the use of AAV‐based gene therapy in clinical applications, including in haemophilia. Uniquely, in the Phase 3 HOPE‐B trial (NCT03569891), 54 adult male participants with ≤2% factor IX (FIX) activity levels received a single intravenous infusion of 2 × 1013 gc/kg etranacogene dezaparvovec, a liver‐directed AAV5 vector expressing FIX Padua, regardless of their AAV5 NAb status at baseline (day of dosing). At baseline, 33/54 (61.2%) had undetectable AAV5 NAbs (NAb‐); 21/54 (38.8%) had measurable NAbs (NAb+) with a median (Q1–Q3) titer of 56.9 (23.3–198.9), among which 1/21 (5%) had a high titer of 1:3212 and 20/21 (95%) had titers <1:700. Two participants did not express FIX and pursued FIX prophylaxis; 1 with the highest NAb titer and 1 (titer: 1:198.9) who received only ∼10% of the etranacogene dezaparvovec dose. At 18 months post‐dose, no clinically meaningful correlation between the individual's AAV5 NAb titer and FIX activity was identified. NAb+ <1:700 and NAb‐ participants presented sustained median (min–max) FIX activity levels of 32.0% (10.3–57.9) and 35.0% (4.5–122.9), respectively. NAb+ <1:700 and NAb‐ subgroups demonstrated low annualized bleeding rates (ABR) of 1.30 and 0.93, respectively; this was significantly improved compared with the respective ABRs of 4.29 (p = 0.0005) and 3.8 (p < 0.0001) during the ≥6‐month lead‐in period where participants received standard‐of‐care FIX prophylaxis. The safety profile of etranacogene dezaparvovec was acceptable and similar between NAb groups. Etranacogene dezaparvovec demonstrated efficacy in participants without and with AAV5 NAbs <1:700 titer.
Investigating the role of innate immune signaling in cell toxicity upon AAV‐mediated gene transfer in hiPSC‐derived CNS models
1: San Raffaele Telethon Institute for Gene Therapy (SR‐TIGET), Milan, Italy 2: Spark Therapeutics, Philadelphia, PA, USA
Gene therapy with adeno‐associated viral (AAV) vectors is gaining ground as a promising treatment for genetic neurodegenerative diseases for which there is currently no cure. Nonetheless, high AAV vector doses are still required for clinical efficacy. Recent lethal adverse events and neurotoxicity associated with high AAV doses in clinical trials and non‐human primate studies have raised great concern regarding dose‐dependent vector toxicity. Links between innate immunity, cell toxicity and transduction efficacy are emerging, but the mechanisms of innate immune sensing and potential antiviral restriction of AAV remain largely unknown. Given the species‐specificity of vector tropism and innate signaling, and the lack of studies in a human context, we have exploited human iPSC‐derived models to assess innate sensing of AAV in cells of the CNS, in complex environments including mixed neural‐glial cultures and 3D organoids. Functional studies and genome‐wide analysis of transcriptional changes triggered by different AAV serotypes revealed early activation of DNA damage, unfolded protein responses and induction of apoptosis, followed by upregulation of inflammatory and antiviral signaling, in particular in glial cell subsets. Innate responses and cellular toxicity were further enhanced in mixed cultures, analyzed by single‐cell RNAseq, as compared to the transduction of the various cell types individually, suggesting an active interplay between the different CNS cell subsets in the establishment of a detrimental inflammatory environment. Together, our studies shed light on the mechanisms leading to AAV sensing and neurotoxicity, informing the development of more stealth gene therapy and gene correction strategies.
CRISPR/Cas9 based disease modelling and functional correction of Interleukin 7 Receptor Severe Combined Immunodeficiency
1: University College London
Interleukin 7 Receptor Severe Combined Immunodeficiency (IL7R‐SCID) is a life‐threatening disorder caused by a homozygous mutation in the IL7R gene. Defective IL7R expression prohibits survival, proliferation and differentiation of human precursor T cells during lymphopoiesis. This results in complete absence of functional mature T cells in new‐borns, who succumbs to severe infection and early death. Previous attempts to tackle IL7R‐SCID by gene therapy demonstrated constitutive and unregulated IL7R expression predisposing leukaemia. This disparity in clinical safety has evoked the effort to apply targeted genome editing to insert Wild‐type IL7R copy within the endogenous locus to mediate physiological protein expression. Here, we have devised a highly efficient CRISPR/Cas9 editing platform targeting IL7R locus to correct the molecular, phenotypic and functional defect of IL7R knock‐out T cells and Haematopoietic Stem Cells (HSCs). Gene editing with AAV6 vector containing IL7R cDNA linked with either GFP and/or mCHERRY reporter cassette yielded an average of 20% and 10% mono‐allelic as well as 10% and 5% bi‐allelic correction in knock‐out T cells and HSCs, respectively. Bi‐allelic integration of AAV6 repair template restored IL7R expression and rescued functional signalling pathways in T cells as demonstrated by phosphorylation of STAT5 protein. Differentiation of IL7R knock‐out HSCs in Artificial Thymic Organoid recapitulated the defective lymphopoiesis block observed in IL7R‐SCID patients while physiological T cell development was rescued in bi‐allelic gene edited HSCs. In vivo xenotransplantation of IL7R corrected HSCs into immunodeficient mice are ongoing that will confirm the safety, efficacy and feasibility of our CRISPR/Cas9 strategy to treat IL7R‐SCID.
CoCas9: a compact nuclease from the human microbiome for efficient and precise editing
1: University of Trento, Department CIBIO, Laboratory of Molecular Virology, Trento, Italy 2: University of Trento, Department CIBIO, Laboratory of Computational Metagenomics, Trento, Italy 3: Alia Therapeutics s.r.l, Trento, Italy 4: Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy
*authors contributed equally
The efficacy of CRISPR‐Cas genome editing strongly depends on cellular delivery. In vivo genome modifications in clinical applications are mostly achieved through viral vectors, especially adeno‐associated vectors (AAVs). Nonetheless, AAVs can only accommodate transgenes smaller than 4 kbp, thus hardly compatible with the most used Cas9 from Streptococcus pyogenes (SpCas9). In this study we interrogated a massively expanded microbiome database (Pasolli et al., Cell 2019) to identify novel Cas9 proteins with reduced molecular size. We discovered a highly active nuclease from an unknown species belonging to the Collinsella genus, CoCas9 (1004 amino acids). CoCas9 showed robust editing levels in human cells, with up to 50% of cleavage efficiency. A direct comparison with SpCas9 using overlapping sgRNAs showed a similar editing profile and increased precision, measured by GUIDE‐seq. Moreover, the adenine base editor using CoCas9 resulted in high levels of base editing (up to 60% of A‐to‐G conversion), resembling those detected with a base editor derived from SpCas9. Finally, we evaluated the compatibility of CoCas9 with an all‐in‐one AAV approach and compared its activity with compact Cas9s (SaCas9 and NmeCas9) suitable for AAV delivery. We found that CoCas9 is efficiently delivered together with its sgRNA through AAV, inducing up to 20% of InDels in the RHO gene. Here we generated a metagenomic‐experimental pipeline to enrich the CRISPR‐Cas9 toolbox. We identified CoCas9, a compact and efficient Cas9, which is compatible with all‐in‐one AAV delivery. CoCas9 will contribute to the advancement of genome editing by improving deliverability and precision.
Inhibition of p38‐MAPK counteracts DNA damage induced by ex vivo expansion of hematopoietic stem and progenitor cells for efficient genetic engineering
1: San Raffaele Telethon Insitute for Gene Therapy (HSR‐TIGET) 2: Universita Vita‐Salute San Raffaele 3: National research Council, Institute for Biomedical technologies, Milan, Italy 4: University Center for Statistics in the Biomedical Sciences, Milan, Italy 5: Department of Chemistry, Materials and Chemical Engineering “G. Natta”, Politecnico di Milano, Milano, Italy 6: New York Stem Cell Foundation Robertson Investigator
For HSPC‐based clinical applications, prolonged time in culture together with nuclease‐induced DNA double strand break (DSB) and exposure to high vector doses is required to reach sufficient levels of gene editing (GE) by homology‐driven repair (HDR). Although current culture conditions have been tailored to ensure high efficiencies while preserving the long‐term repopulating capacity of HSPCs, we recently discovered that activated stem cells accumulate physical DNA damage, replication stress and ROS, resulting in stem cell dysfunctions. Interestingly, we identified as a key mediator of the observed culture stress the activation of the p38‐MAPK and found that its chemical inhibition diminished mitogenic ROS, thus counteracting the uncontrolled proliferation of HSPCs that ultimately leads to DNA damage. Consequently, p38 inhibition prior to GE increased HSPC clonogenic potential and their multi‐lineage output at single‐cell resolution. Single‐cell RNA‐seq of p38i treated GE‐cells showed an expansion of the most primitive compartment and downregulation of proliferation, oxidative response, and differentiation‐related genes. In vivo clonal tracking of transplanted p38i‐treated GE‐cells revealed a higher number of HSPC clones that underwent NHEJ and HDR gene correction, pointing to the preservation of HSC subsets responsible for long‐term hematopoietic reconstitution. Importantly, we showed that GE‐HSPC functionality was enhanced when cells were cultured on advanced tri‐dimensional substrates before ex vivo genetic manipulation, thus suggesting that uncontrolled proliferation and DNA damage accumulation are mitigated by mimicking geometric constraints of the bone marrow niche. Our findings indicate that overcoming unintended cellular responses linked to HSPC ex‐vivo activation improves yield and quality of genetically engineered cells.
An in vivo CAST‐Seq workflow identifies and quantifies off‐target activity as well as chromosomal translocations in organs edited in vivo with CRISPR‐Cas nucleases or nickases
1: Institute for Transfusion Medicine and Gene Therapy, Medical Center – University of Freiburg, Freiburg, 79106, Germany 2: Center for Chronic Immunodeficiency (CCI), Medical Center University of Freiburg, Freiburg, 79106, Germany 3: Gene Therapy, Clinical Pharmacology & Safety Sciences, R&D, AstraZeneca, Cambridge, UK 4: Gene Therapy and Regulation of Gene Expression Program, Center for Applied Medical Research (CIMA), University of Navarra, IdiSNA, Pamplona, 31008, Spain 5: Institute of Medical Bioinformatics and Systems Medicine, Medical Center – University of Freiburg, Freiburg, 79110, Germany 6: German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Partner Site Freiburg, Freiburg, 79106, Germany 7: Gene Therapy, Clinical Pharmacology & Safety Sciences, R&D, AstraZeneca, Gothenburg, Sweden
Genome editing‐associated genotoxicity, such as off‐target (OT) activity and chromosomal translocations, must be carefully assessed before clinical application of gene‐edited products. Three methods have been employed to evaluate OT activity in vivo: VIVO uses in vitro identification of candidate OTs that must be validated in vivo, DISCOVER‐Seq relies on ChIP to nominate OTs based on the recruitment of MRE11 to double‐strand breaks (DSBs), and GUIDE‐tag requires the co‐delivery of a DNA donor to ‘tag’ DSBs for subsequent capture. We recently described CAST‐Seq, a method to nominate OT activity by detecting CRISPR‐Cas‐induced chromosomal aberrations. Here, we show that in vivo CAST‐Seq can identify OT sites and chromosomal translocations in in vivo edited mouse livers with unprecedented sensitivity. CAST‐Seq was performed on genomic DNA isolated from livers upon adenoviral or AAV‐based delivery of different CRISPR‐Cas nucleases or nickases, respectively. For instance, OT analyses of Pcsk9‐edited livers confirmed that CAST‐Seq outperformed the other three methods in terms of numbers of confirmed OTs: VIVO 19, DISCOVER‐Seq 26, GUIDE‐tag 40 vs. CAST‐Seq 75. Subsequent analysis by rhAmp‐Seq verified mutagenesis at these OTs, allowed us to establish the frequencies of chromosomal translocations between the on‐target site and the various OTs, and demonstrated that each OT translocates with at least one other OT site. In conclusion, CAST‐Seq followed by rhAmp‐Seq provides a workflow to identify and quantify genome editing‐associated OT activity as well as gross chromosomal rearrangements in vivo, with a process that is applicable to any tissue, including human biopsies during therapeutic in vivo genome editing.
Targeting the hepatitis B cccDNA with a sequence‐specific ARCUS nuclease to eliminate Hepatitis B virus in vivo
1: Precision BioSciences, Inc. 2: Gilead Sciences, Inc. 3: Acuitas Therapeutics
Persistence of chronic Hepatitis B (CHB) is attributed to maintenance of the intrahepatic pool of the viral covalently closed circular DNA (cccDNA), which serves as the transcriptional template for all viral gene products required for replication. Current nucleos(t)ide therapies for CHB prevent virus production and spread but have no direct impact on cccDNA or expression of viral genes. We describe a potential curative approach using a highly specific engineered ARCUS nuclease (ARCUS‐POL) targeting the Hepatitis B virus (HBV) genome. Through iterative rounds of nuclease engineering, ARCUS‐POL nucleases were optimized to exhibit high levels of on‐target editing with minimal off‐target activity. Transient ARCUS‐POL expression in HBV‐infected primary human hepatocytes produced >75% reductions in both cccDNA and Hepatitis B surface antigen (HBsAg). After transient delivery of ARCUS‐POL into cells containing integrated HBV DNA, >80% on‐target editing was achieved with subsequent HBsAg reductions. To evaluate ARCUS‐POL in vivo, novel episomal adeno‐associated virus (AAV) mouse and non‐human primate (NHP) models were developed containing a portion of the HBV genome serving as a surrogate for cccDNA. Clinically relevant delivery was achieved through systemic administration of lipid nanoparticles ARCUS‐POL mRNA. In both mouse and NHP models, a significant decrease in total AAV copy number and high on‐target indel frequency was observed. In the case of the mouse model, which supports HBsAg expression, circulating surface antigen was durably reduced by 96%. Together, these data support a gene editing approach for elimination of cccDNA toward an HBV cure.
AAV‐mediated induction of immunogenic cell death to promote anti‐tumor immune responses
1: Normandie Univ, UNIROUEN, INSERM, U1234, Pathophysiology, Autoimmunity and Immunotherapy, Normandie University, Rouen, France 2: Department of Oncology, Gastroenterology, Hepatology, Pulmonology, and Infectious Diseases, University Cancer Center Leipzig (UCCL), University of Leipzig Medical Center, Leipzig, Germany 3: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany 4: REBIRTH Research Center for Translational Regenerative Medicine, Hannover Medical School, Hannover, Germany 5: Department of Immunology and Biotherapy, Rouen University Hospital, Rouen, France
Recombinant adeno‐associated virus (AAV) vectors represent a safe and efficient tool for in‐vivo gene transfer in cancer cells as well as in the cells of the tumor microenvironment. The aim of this study was to evaluate the ability of immunogenic cell death (ICD) executioners to promote anti‐tumor immune responses. For that, we developed expression plasmids and AAV2 vectors coding for engineered active forms of GSDMD, GSDME, or MLKL, corresponding to recently identified executioners of pyroptosis or necroptosis. We first demonstrated that plasmids coding for active forms of ICD‐executioners induced, as expected, i) rapid and efficient death of transfected B16F10 melanoma cells within the first 24 hours and, ii) the exposition of prototypical proinflammatory molecules associated with immunogenic cell death (as calreticulin, ATP and HMGB1). Next, we demonstrated that B16F10 cells transfected with our constructs not only does not form a primary tumor when injected subcutaneously in vivo, but also protected mice against a secondary tumor challenge. This suggested efficient priming of the anti‐tumor immune responses by the in vivo release of tumor antigens and proinflammatory signals from the dying tumor cells. Finally, we showed that intratumoral injection of AAV2 vectors coding for the active forms of GSDME or GSDMD, combined with anti‐PD1 blocking antibody, significantly inhibited tumor growth. Overall, our strategy offers the considerable potential, when combined with established immunotherapeutic approaches, to increase tumor immunogenicity even in immunologically “cold” tumors that still represent a clinical challenge.
Ex Vivo Immunotherapeutic NK Cell Production From Cord Blood Or Mobilized Peripheral Blood CD34+ Cells Using Notch Ligand Delta‐Like 4 Culture System
1: Université de Paris, Imagine Institute, Human Lymphohematopoiesis Lab, INSERM UMR 1163, Paris, France 2: Smart Immune, Paris, France 3: Department of Biotherapy Clinical Investigation Center, AP‐HP, Hôpital Necker‐Enfants Malades, Paris, France.
NK cells are being investigated as an attractive alternate candidate to T‐cells for cancer immunotherapies as they neither cause graft‐versus‐host disease nor induce cytokine release syndrome. However, the clinical use of NK cell immunotherapy is restricted by the limited availability of ex vivo clinical‐grade methods for genetic modification and expansion of NK cells. Here, we developed an efficient, feeder cell‐free and clinically compatible method for producing large numbers of NK cells from CD34+ cells based on our recently published TNFα‐supplemented immobilized DL‐4 culture system. Upon DL‐4/TNFα culture, followed by a feeder cell‐ and DL‐4‐free NK cell differentiation culture, we could obtain a large number (up to 10000/CD34+ cell) of highly pure (up to >90%) NK cells from CB or mPB CD34+ cells with an efficient transduction within a short period of 14 to 21 days. The NK cells expressed activation receptors, cytotoxic molecules (perforin and granzyme B) and NK cell transcription factors. They underwent degranulation and expressed IFNγ and TNFα upon stimulation with myelogenous leukaemia cell line K562 cells. Subsequently, we showed that CD19‐CAR NK cells, that are able to kill NALM‐6 cells efficiently can be generated from CB CD34+ cells. Therefore, the unique combination of our DL‐4 culture system and a feeder cell‐ and DL‐4‐free NK cell differentiation/expansion culture may provide an efficient and clinically applicable method to generate large numbers of immunotherapeutic pure NK and CAR NK cell population. These results lay a groundwork towards an effective NK cell immunotherapy platform for cancers and viral infections.
Nanoblades allow high‐level genome editing in organoids
V Tirolle2 A Krug2 E Bokobza2 M Bulcaen3 M M Ensinck3 A Gutierrez‐Guerrero1 L Medaer3 R Gijsbers3 M S Carlon3 F Bost2
1: CIRI; Inserm U1111, Lyon, France 2: C3M, Inserm U1065 Nice , France 3: KU Leuven, Leuven, Belgium
Genome engineering has become in the last few years more accessible thanks to the RNA programmable endonucleases such as the CRISPR‐Cas9 system. However, using this editing technology in synthetic organs called ‘organoids' is still very inefficient. This is due to the delivery methods used for the CRISPR‐Cas9 machinery, which are mainly performed by electroporation of CRISPR/Cas9 DNA, mRNA or ribonucleoproteins (RNPs) containing the CAS9‐gRNA complex, procedures toxic to some extent for the organoids. Here we describe the use of the ‘Nanoblade’ technology to accomplish genome editing in murine and human tissue derived organoids. Nanoblades outperformed by far knock‐out (KO) levels achieved with other techniques used to date for delivery of the gene editing machinery. We reached up to 80% of gene knockout in organoids after treatment with nanoblades. We achieved high‐level nanoblade‐mediated KO for the androgen receptor (AR) encoding gene and the cystic fibrosis transmembrane conductance regulator (CFTR) gene with single gRNA or dual gRNA containing nanoblades in murine prostate and colon organoids. Likewise, nanoblades achieved high levels of gene editing in human organoids ranging between 20 and 50 %. Most importantly, in contrast to other gene editing methods, this was obtained without toxicity for the organoids. Moreover, it requires only four weeks to obtain stable lines of a gene KO in organoids and no obvious unwanted INDELS in off‐target sites in the genome were detected.
In conclusion, nanoblades simplify and allow rapid genome editing in organoids for cancer and gene therapy preclinical studies with little to no side‐effects.
Identification of marker genes to monitor residual iPSCs in iPSC‐derived products
1: Novartis Pharma AG 2: University of Basel
Engineered tissues and cell therapies based on human induced pluripotent stem cells (iPSCs) represent a promising approach for novel medicines. However, iPSC‐derived cells and tissues may contain residual undifferentiated iPSCs that could lead to teratoma formation after implantation into patients. As consequence, highly sensitive and specific methods to detect residual undifferentiated iPSCs are indispensable for safety evaluation of iPSC‐based therapies. Here, we used RNA‐seq data to identify potential marker genes for iPSC contaminations in iPSC‐derived cells. Identifying such iPSC marker genes for each cell type individually provided a larger and more specific set of potential marker genes than considering all cell types in the analysis. Thus, we focused on one cell type for validation experiments. By spiking different amount of iPSCs into iPSCs‐derived cardiomyocytes (iCMs), we evaluated the sensitivity of the selected candidate genes by RT‐qPCR, and compared their performance to the previously suggested marker LIN28A. ESRG, LINC00678, CAMKV, IDO1, CNMD, L1TD1, LIN28A, LCK, VRTN and ZSCAN10 detected contaminant iPSCs amongst iCMs with a limit of detection ranging from 0.001 to 0.1%, depending on the gene and the iCM batch used. In conclusion, using the example of iCMs, we provide an approach to identify a set of highly specific and sensitive markers that can be used for quality assessment of iPSC‐derived products.
Utilising CRISPR‐Cas13 systems to target frontotemporal dementia and amyotrophic lateral sclerosis‐causing C9orf72 repeat expansion‐containing RNA
1: UK Dementia Research Institute, UCL 2: Institute for Stem Cell Biology and Regenerative Medicine, India 3: Francis Crick Institute, UK 4: Sheffield Institute for Translational Neuroscience, University of Sheffield, UK 5: Department of Cell and Developmental Biology, UCL, UK 6: VIB‐KU Leuven Center for Brain & Disease Research, Belgium 7: National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA 8: Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA.
The most common genetic cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is a G4C2 repeat expansion in intron 1 of the C9orf72 gene. The hexanucleotide repeat expansion undergoes bidirectional transcription and repeat‐associated non‐ATG translation producing sense and antisense RNA foci and five dipeptide repeat proteins (DPRs), with poly‐GR and poly‐PR shown to be the most toxic. Recent discoveries of RNA‐targeting CRISPR‐Cas13 systems open promising avenues for novel therapeutics. Here, we demonstrate that CRISPR‐Cas13 systems can successfully target both the sense and antisense transcripts of the C9orf72 hexanucleotide expansion repeat resulting in a reduction of sense and antisense RNA foci, and poly‐GR and poly‐PR to background levels in a transient human cell culture model. Using lentiviruses expressing our CRISPR‐Cas13 system, we effectively target the endogenous repeat‐containing C9orf72 transcripts in three patient‐derived iPSC neuron lines reducing repeat‐containing transcripts and DPRs by ∼60% in 5 days, whilst leaving non‐repeat ‐containing C9orf72 transcripts intact. We next developed a viral strategy to treat C9orf72 mouse models with a single adeno‐associated virus (AAV) therapy that contains the full CRISPR‐Cas13 system and guides targeting both sense and antisense repeat‐containing transcripts. This AAV strategy effectively reduces repeat‐containing transcripts in vivo. We demonstrate a reduction in the pathological hallmarks of C9orf72 FTD/ALS in patient‐derived iPSC neurons, and in vivo in a C9orf72 mouse model, suggesting CRISPR‐Cas13 systems as a viable therapeutic approach for C9orf72 FTD/ALS.
Final safety and efficacy of a phase 1/2 trial of DTX301 in adults with late‐onset ornithine transcarbamylase deficiency (OTCD)
1: University of Birmingham, Birmingham, UK 2: Oregon Health & Science University, Portland, OR, USA 3: University of Santiago de Compostela, Santiago de Compostela, Spain 4: Boston Children's Hospital, Harvard Medical School, Boston, MA, USA 5: University of Calgary, Calgary, Canada 6: Cruces University Hospital, Biocruces Bizkaia Health Research Institute, Barakaldo, Spain; 7: Icahn School of Medicine at Mount Sinai, New York, NY, USA 8: University Hospitals of Cleveland Medical Center, Cleveland, OH, USA 9: University of CO School of Medicine and the Children's Hospital Colorado, Aurora, CO, USA 10: Reference Centre in Inherited Metabolic Diseases, HCL, Lyon, France 11: Ultragenyx Gene Therapy, Cambridge, MA, USA
OTCD is an X‐linked urea cycle disorder resulting in life‐threatening episodic hyperammonemia that can cause cumulative neurocognitive damage. DTX301, an AAV8 vector containing the OTC transgene, is being investigated for potential treatment of OTCD.
CAPtivate (NCT02991144) was a global, open‐label phase 1/2 trial evaluating DTX301 safety and efficacy in 11 adults with late‐onset OTCD. The primary endpoint was incidence of adverse events (AEs). Patients received one DTX301 IV infusion: Cohort 1 received 3.4x1012 Genome Copies (GC)/kg; Cohort 2, 1.0x1013 GC/kg; Cohort 3, 1.7x1013 GC/kg, and Cohort 4, 1.7x1013 GC/kg with prophylactic steroids. Complete responders discontinued all ammonia‐scavenging drugs and protein‐restricted diet. Responders had ≥50% reduction in baseline disease management. Final data from the main study are presented with a 21Mar2022 cutoff.
No treatment‐related serious AEs, infusion‐related reactions, or dose‐limiting toxicities were reported. All AEs during the main study were mild or moderate (grade 1 or 2), except one patient with grade 3 hyperammonemic crises, assessed as related to OTCD and unrelated to DTX301. Eight patients experienced treatment‐related AEs. All eight experienced asymptomatic ALT increases managed with oral corticosteroids. Other treatment‐related AEs were photophobia, headache, hypertension, and hypophosphatemia.
Seven patients responded to DTX301. Cohort 1 had one complete responder; Cohort 2; one complete responder and one responder; Cohort 3, one complete responder and two responders; and Cohort 4, one complete responder. The longest‐treated responder has a durable response 4.5 years post‐treatment.
Safety and efficacy will continue to be studied during the 6‐year extension study and in the Phase 3 trial (NCT05345171).
Preliminary results of STAAR, a Phase I/II study of isaralgagene civaparvovec (ST‐920) gene therapy in adults with Fabry disease and long‐term follow‐up
1: Addenbrooke's Hospital, Cambridge, UK 2: The Icahn School of Medicine at Mount Sinai, New York, NY, USA 3: Lysosomal and Rare Disorders Research and Treatment Center, Fairfax, VA, USA 4: Cincinnati Children's Hospital Medical Center , Cincinnati, OH, USA 5: University of Cincinnati College of Medicine, Cincinnati, OH, USA 6: University of Iowa, Iowa City, IA, USA 7: Emory University School of Medicine, Atlanta, GA, USA 8: Sangamo BioSciences, Inc.
Fabry disease (FD) is caused by mutations in the GLA gene leading to deficiency of alpha‐galactosidase A (α‐Gal A) and accumulation of globotriaosylsphingosine (lyso‐Gb3). ST‐920 is an investigational gene therapy using a recombinant AAV2/6 vector containing the human GLA cDNA. STAAR (NCT04046224) is an ongoing Phase 1/2 multicenter, open‐label, dose‐ranging trial evaluating the safety and tolerability of a single ST‐920 administration. After STAAR, subjects transition into a 4‐year long‐term follow‐up study (LTFU, NCT05039866). As of May 19, 2022, nine male classic FD subjects were dosed at 0.5e13 vg/kg (C1S1‐ERT, C1S2‐pseudo‐naïve), 1e13 vg/kg (C2S1‐pseudo‐naïve, C2S2‐ERT), 3e13 vg/kg (C3S1‐ERT, C3S2‐ERT, C3S3‐ERT), and 5e13 vg/kg (C4S1‐naïve, C4S2‐naïve) in STAAR and the first four subjects entered LTFU. The longest follow‐up was 18 months in C1. No serious treatment‐related AEs were reported. Most treatment‐related AEs were mild with one moderate pyrexia. No subject experienced ALT elevations requiring steroid treatment. Six subjects produced supraphysiologic α‐Gal A activity from 4‐ to 21‐fold of normal; pending data for the latest dosed subjects C3S3, C4S1, and C4S2. Three subjects (C1S1, C2S2, C3S1) withdrew from ERT; C2S2 had 28 weeks of follow‐up of ERT withdrawal and elevated α‐Gal A activity was maintained with no major increases in lyso‐Gb3. C2S1 showed reduction of plasma lyso‐Gb3 from 83.7 ng/mL at baseline to 48.4 ng/mL (average, weeks 10‐52 after dosing). ST‐920 was well tolerated while achieving sustained supraphysiologic α‐Gal A activity in plasma.
In vivo lentiviral gene therapy restores ureagenesis in the urea cycle defect argininosuccinic aciduria
1: Great Ormond Street Institute of Child Health, University College London, London, UK 2: NIHR Great Ormond Street Hospital Biomedical Research Centre, London, UK; 3: EGA Institute for Women's Health, University College London, London, UK 4: Metabolic Medicine, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK 5: School of Biomedical Engineering and Imaging Sciences, King's College London, UK
The liver‐based urea cycle enables nitrogen waste and clearance of neurotoxic ammonia. Argininosuccinic aciduria (ASA) caused by argininosuccinate lyase (ASL) deficiency is the second most common inherited urea cycle defect. Patients present either with neonatal‐ or late‐onset hyperammonaemia, which causes coma and death if untreated, and a high risk of severe cognitive impairment and epilepsy. Curative liver transplantation can be performed in severe cases but requires lifetime immunosuppression. We aimed to test in vivo lentiviral gene therapy in ASL‐deficient (AslNeo/Neo ) mice. We tested whether injection of lentiviral vector delivering a codon‐optimised human ASL (LV.coASL) could provide correction of lethal AslNeo/Neo mice for 3 months. Control littermates received the same vector with GFP instead of coASL. LV.coASL‐injected animals survived the whole experiment whilst control mice died within 4 weeks (p < 0.001). Growth (p < 0.01), fur coat pattern, ammonia (p < 0.001), plasma arginosuccinate (p < 0.001), citrulline (p < 0.01), and orotate (p < 0.05) were normalised to those in wild‐type mice. Significantly increased ASL expression (+300%; p < 0.01) and activity (+30%; p < 0.05) were observed in treated AslNeo/Neo livers compared to controls. Oxidative stress is a key feature of ASA disease. We assessed the activity of membrane transporter xCT, an indirect oxidative stress marker, with [18F]FSPG positron emission tomography (PET) in tissues of AslNeo/Neo and wild‐type mice. [18F]FSPG had a significantly higher retention (p < 0.0006) in AslNeo/Neo versus wild‐type livers, correlating with overexpressed xCT. Similarly, [18F]FSPG retention was significantly reduced (p < 0.002) in LV.coASL‐injected AslNeo/Neo mice compared to controls. This study demonstrates proof of concept of in vivo lentiviral gene therapy for ASA.
Gene Therapy to Treat Methylmalonic and Propionic Acidemia Using the Novel Adeno‐associated Viral Capsid 44.9
1: National Institutes of Health 2: National Human Genome Research Institute 3: National Institute of Dental and Craniofacial Research
Methylmalonic acidemia (MMA) and propionic acidemia (PA) are severe and rare autosomal recessive metabolic disorders caused by defects in the mitochondrial catabolism of propionyl‐CoA to succinyl‐CoA. In the case of MMA, mutations in the methylmalonyl‐CoA mutase (MMUT) gene are the most common, and in PA, mutations in propionyl‐CoA carboxylase alpha (PCCA) or subunit beta (PCCB) genes are causative. MMA and PA can both be fatal despite dietary and medical management, which has motivated the development of new therapies. Here, we assayed systemic gene delivery using a novel AAV44.9 capsid in two murine models of MMA and one of PA. AAV44.9 treatment rescued neonatal lethality and reduced disease related metabolites in both the MMA and PA models. Tissue biodistribution and transgene expression studies showed high levels of transduction and transgene expression in the liver and heart after gene therapy. In addition, a neutralizing antibody (NAb) survey was conducted and demonstrated a relatively low seroreactivity to the AAV44.9 capsid in both adult blood donors and patients with MMA. In aggregate, our studies establish that AAV44.9 serotyped vectors are highly potent in mouse models of the common organic acidemias, MMA and PA, and display the pronounced hepato‐cardiac trophism following systemic delivery. The use of pseudoserotyped AAV44.9 vectors might be especially suited for the treatment of disorders where hepatic and cardiac targeting is needed, such as propionic acidemia, glycogen storage disorders and fatty acid oxidation defects, and perhaps more widely given the low seroprevalence of AAV44.9 NAbs in humans.
Fusion of designer‐recombinases for efficient and specific correction of a Factor VIII genomic inversion
L Mukhametzyanova1
1: Dresden University
The precise mechanism of inversion, integration, and excision of DNA makes tyrosine site‐specific recombinases (SSRs), such as the Cre/loxP system, attractive candidates for therapeutic applications. Substrate‐linked directed molecular evolution (SLiDE) allows to change the specificity of Cre to a defined genomic target, enabling the use of evolved SSRs as novel gene editing tools. Moreover, two different SSRs can be evolved to cooperatively recombine an asymmetric target site, thereby greatly expanding the targetability of genomic sites. However, using two SSRs instead of one increases the risk for potential off‐target activity. Theoretically, each individual monomer can recombine symmetric off‐targets or both monomers can recombine asymmetric off‐targets cooperatively. We hypothesized that the generation of a system that enforces heterodimer formation and therefore abolishes homodimer formation would reduce off‐target activity. To test this hypothesis, we used a recombinase heterodimer evolved in our laboratory to correct the large inversion of the F8 gene, causing hemophilia A. We fused the heterodimer monomers with a linker library and used the SLiDE platform to screen for variants with no activity on the symmetric targets. We were able to identify linked heterodimers with greatly improved specificity without losing on‐target activity. Strikingly, transient treatment with the fused heterodimer showed up to 30% inversion of the F8 target sequence in HEK293T cells. Furthermore, we were able to revert the F8 inversion in hemophilia patient‐derived endothelial cells. Overall, our results show that fusion of heterospecific recombinases is a simple and efficient method to create SSR‐systems with enhanced specificity for therapeutic genome editing.
AAV‐mediated homology‐independent targeted integration leads to sustained secretion of therapeutic proteins from new‐born liver
1: TIGEM 2: Department of Advanced Biomedical Sciences, Federico II University, Naples
Mucopolysaccharidosis type VI (MPS VI) and Haemophilia A (HemA) are two inherited, life‐threatening disorders due to the deficiency of secreted proteins, arylsulfatase B (ARSB) and clotting factor VIII (hFVIII), respectively. The current treatment of both conditions is protein replacement therapy which requires frequent intravenous infusions for the entire lifespan of patients.Liver gene therapy with adeno‐associated viral (AAV) vectors holds great promise to provide long‐term expression and secretion of therapeutic transgenes after a single administration. However, AAV vectors are predominantly non‐integrating, leading to loss of transgene expression over time due to liver proliferation or liver damage.We took advantage of AAV‐mediated homology‐independent targeted integration (HITI) by CRISPR/Cas9 to integrate transgenes of interest at the 3’ end of mouse Albumin (mAlb) which is highly expressed in hepatocytes. Proof‐of‐concept in new‐born mice using the reporter gene Discosoma red (DsRed) shows stable DsRed expression in about 20% of hepatocytes. Exchange of DsRed with either hARSB and a B domain‐deleted hFVIII, resulted in circulating supraphysiological levels of hARSB up to 1 year after neonatal delivery, or therapeutic levels of FVIII which however declined after one month due to the development of anti‐hFVIII antibodies, as observed with F8 protein replacement therapy. MPS VI mice treated with HITI had a significantly improved phenotype.Overall, AAV‐HITI shows promising results for the integration of small and large coding sequences with stable, high expression levels of therapeutic transgenes from liver with hepatocyte proliferation.
Assessment of FVIII in the hematopoietic system: UM171‐expanded CD31+ monocytes show the highest GT potential to correct HA
M Elnaggar1 5 A Al‐Mohannadi1 W Hasan1 D Abdulrahman1 M J Al‐Kubaisi1 I Pavlovski1 G Gentilcore1 A Sathappan1 D Kizhakayil1 A I Ali1 S Mohan2 D Olagunju2 C Cugno1 J C Grivel1 C Borsotti3 A Follenzi3 S Da'as1 4
1: Sidra Medicine, Research Department, 26999, Doha, Qatar 2: Sidra Medicine, Department of Obstetrics and Gynecology, 26999, Doha, Qatar 3: Università del Piemonte Orientale ‘‘A. Avogadro’’, Department of Health Sciences, 28100, Novara, Italy 4: Hamad Bin Khalifa University, College of Health and Life Sciences, Doha, Qatar 5: Icahn School of Medicine at Mount Sinai, Division of Hematology and Medical Oncology, Hess Center, New York, NY 10029
HA Gene Therapy trials are investigating the possibilities of expressing FVIII by correcting Hematopoietic Stem Cells (HSCs). Although platelets, monocytes/macrophages, and mesenchymal stromal cells express transgenic‐FVIII, a systematic assessment of FVIII extrahepatic production in blood lineages is presently lacking. Previously, we were able to optimize a robust flow‐cytometry screening approach for detecting intracellular FVIII (Elnaggar, 2019). Now, we used it to map native and transgenic FVIII production capacity of HSCs‐derived blood cells with the aim to identify the best candidates for HA gene correction among HSC lineages. Total CD34+ cells, common‐myeloid and common‐lymphoid progenitors express 0.4%, 0.3%, and 0.6% FVIII (n = 4). However, culturing CD34+ cells in serum‐free‐medium for 7‐10 days enhanced FVIII basal production to 12,2% (P = 0.04, n = 4). Among mature blood subsets, monocytes produce the highest FVIII amount, and show better ability to maintain transgene overexpression of 7.9% compared to native 3.1% in long‐term cultures (P = 0.02, n = 3). The addition of UM171 stem‐cell renewal compound to the transduction expanded an intriguing subset of CD14+/CD31+ cells, expressing transgenic‐FVIII up to 31.2%, after 7‐10 days of culture (n = 5). Additionally, we modeled through an antisense RNA a transient MO‐f8 HA zebrafish to functionally evaluate the corrective potential of FVIII‐transduced HSCs, specifically CD14+/CD31+ cells. Injection of FVIII‐transduced CD14+/CD31+ cells into HA zebrafish larvae, corrected bleeding after laser‐induced caudal vein puncture, significantly reducing bleeding‐time to 11 seconds, compared with 14 seconds in control injections of untransduced CD14+/CD31+ cells (P = 0.03). In conclusion, FVIII‐transduced CD31+ monocytes represent a good target for HA phenotype correction in HA zebrafish.
IFNalpha by in vivo‐engineered macrophages abates liver metastases and triggers counter regulatory responses limiting treatment efficacy
1: San Raffaele Telethon Institute for Gene Therapy (HSR‐TIGET) 2: San Raffaele Scientific Institute 3: Kanazawa University
The liver hosts an immune suppressive environment favouring metastatic seeding and proliferation of cancer cells. Pharmacological treatments, including immunotherapies, fail in presence of liver metastases (LMS). Therefore, identifying new interventional tools and key targetable players involved in the immunosuppressive environment is of pivotal importance.
Here, we describe a novel LV‐based platform, termed KC‐LV, to selectively engineer liver macrophages, termed Kupffer cells (KCs), in vivo with the goal of delivering bio‐therapeutics specifically to LMS. Indeed, upon systemic delivery of the KC‐LV, we observed selective transgene expression in KCs which was enhanced in macrophages located in areas surrounding LMS.
We equipped the KC‐LV with an IFNα‐coding sequence, a cytokine with pleiotropic immune effects. Long‐term analysis in mice showed LV dose‐dependent, sustained and well‐tolerated IFNα expression. Remarkably, therapeutic IFNα‐LV treatment significantly delayed LMS growth reaching a complete response in up to 50 % in a colorectal cancer (CRC) organoid‐based syngeneic mouse model of LMS containing molecular and histopathological hallmarks of the human disease.
Single cell omics and spatial transcriptomics of LMS from IFNα‐LV‐treated mice showed upregulation of IFNα‐responsive genes, macrophage skewing towards an immune‐activating polarization state, and expansion as well as reduced exhaustion of LMS‐associated antigen specific CD8 T‐cells especially in treatment responsive mice. Treatment resistant mice instead were characterized by accumulation of immunosuppressive T‐regulatory‐type‐1 (TR1)‐like cells in the tumor microenvironment. Interestingly, we also found a positive correlation between IFNα and TR1‐like cell signatures in human LMS, thus suggesting that Tr1‐like cells might favour LMS immune evasion in the presence of IFNα signaling.
Development of a tumour selective precision immunovirotherapy expressing immune checkpoint inhibitors targeting LAG3
1: Cardiff University
Oncolytic viruses (OV) have significant potential to induce immunogenic cell death (ICD), turning immunologically “cold” tumours “hot”. This potential can be enhanced when combined with immunotherapies, such as immune checkpoint inhibitors (ICI), although systemic administration of ICIs can induce toxicities as has been observed for example with anti‐CTLA‐4 or anti‐PD‐1. More recently, LAG3 has been highlighted as a promising target for cancer therapy. Combining OV and ICI into single agents capable of targeting tumour cells following intravenous delivery and mediating high level, tumour selective overexpression of ICI locally within the tumour microenvironment may enhance “on‐target” and minimise “off‐target” activity of immunotherapies.
We previously described a refined, tumour selective, intravenously targetable OV, Ad5NULL‐A20, which transduces cells expressing αvβ6 integrin. We designed an antibody fragment targeted against LAG3 and engineered this into Ad5NULL‐A20 controlled by a CMV promoter and a CD33 secretion signal (Ad5NULL‐A20.LAG3). αvβ6 integrin expressing cancer cells infected with Ad5NULL‐A20.LAG3 secreted high quantities of the scFv‐Fc into the supernatant. We purified anti‐LAG3 scFv‐Fc and characterised its functionality. The scFv‐fc was biologically active, blocking the interaction between MHC‐II/LAG3 in engineered cells. Additionally, the scFv‐Fc bound LAG3 with equal or better affinity as an anti‐LAG3 control antibody demonstrated by both SPR and AlphaScreen. Specificity was confirmed by flow cytometry using engineered JRT‐3 cells expressing LAG3, hence, validating our approach as new precision virotherapy.
Taken together, these data highlight the potential for precision immunovirotherapies expressing ICIs to “heat up” the tumour microenvironment, enhancing anti‐tumour immunity and warranting further preclinical investigation prior to clinical translation.
AAV9‐mediated gene therapy in a knock‐in mouse model of infantile neuroaxonal dystrophy
S Whaler1
1: University College London 2: University of the Witwatersrand 3: Great Ormond Street Hospital
Infantile neuroaxonal dystrophy (INAD) is a rare lethal pediatric neurodegenerative disease. It is caused by mutations in PLA2G6, which codes for the enzyme calcium‐independent phospholipase A2. Patients present with neurological symptoms between six months and three years of age, with mortality typically occurring by 10 years old. No disease modifying treatments are available and there is an urgent need to develop new therapies. We conducted an in‐depth characterization of the pla2g6‐inad knock‐in mouse model. Following characterization, we investigated the therapeutic potential of an AAV9.hPLA2G6 vector administered by various routes of administration to neonatal pla2g6‐inad mice. The average lifespan of the model is reduced to 97 days, with weight loss and behavioral decline from 9 weeks old. Neuropathology studies showed neuronal loss and neuroinflammation in the brain and spinal cord, along with autophagic dysfunction and lysosomal accumulation. Neonatally administered AAV9.hPLA2G6 gene therapy resulted in a significant improvement in all parameters measured including survival, weight, locomotor function, and neuronal counts in both the brain and spinal cord. Furthermore, the autophagic function was restored and lysosomal accumulation was significantly reduced. Interestingly, an intravenous administration of the vector had no effect on survival. However, when combined with the intracerebroventricular administration it was able to enhance survival beyond an intracerebroventricular administration alone. This study provides novel insights into INAD disease pathology and cellular dysfunction in the CNS and suggests an effective therapy with potential for clinical development.
Gene therapy in APOE4 homozygote Alzheimer's disease – interim data
1: Department of Neurological Surgery, Weill Cornell Medical College, New York, NY USA 2: Department of Genetic Medicine, Weill Cornell Medical College, New York, NY USA 3: Department of Neurology, Weill Cornell Medical College, New York, NY USA 4: Department of Radiology, Weill Cornell Medical College, New York, NY USA 5: Departments of Neurology and Psychiatry, Icahn School of Medicine at Mt. Sinai, New York, NY USA 6: LEXEO Therapeutics, Inc., New York, NY USA
Alzheimer's disease (AD) is associated with a strong genetic risk from polymorphisms of the apolipoprotein E (APOE) gene. APOE4 homozygotes have a 15‐fold greater risk of developing AD compared with APOE3 homozygotes, while APOE2 have reduced risk.
LX1001 is an adeno‐associated viral vector (AAV) investigational gene therapy (AAVrh.10hAPOE2) designed to deliver the protective human apolipoprotein E2 (hAPOE2) gene into the central nervous system of APOE4 homozygous AD subjects to halt or slow disease progression, mediated by the APOE4 allele.
This first in human trial is designed to evaluate the safety and feasibility of converting the cerebral spinal fluid (CSF) from the APOE4 homozygous profile to an APOE4/E2 profile as a biomarker of successful gene delivery. Additional secondary endpoints include analysis of other CSF AD biomarkers, including amyloid‐beta (Aß42), total tau (T‐tau), and phosphorylated tau (P‐tau).
This is a Phase 1, open label, 52‐week dose‐finding study evaluating the safety and tolerability of LX1001 in three ascending single‐dose cohorts (5.0E10, 1.6E11 and 5.0E11 gc/ml CSF) in APOE4 homozygous AD subjects.
In low dose cohort 1 (5.0E10 gc/ml CSF), LX1001 was well‐tolerated with no serious adverse events. APOE2 was expressed in CSF in all 4 subjects with follow‐up data ≥3 months. Reductions in CSF t‐tau and p‐tau were observed in subjects with 12‐month data (n = 2); Mid‐dose cohort is ongoing.
LX1001 is the first investigational gene therapy to directly address APOE4, a well‐recognized genetic risk factor of AD. Initial data support further exploration of APOE2 gene therapy as a potential therapeutic approach.
Reelin Gene Therapy for Fragile X Syndrome
N K Morrill1 A Joly‐Amado1 Q Li1 S Prabhudeva1
1: University of South Florida
Fragile X Syndrome (FXS) is the most prevalent form of inherited intellectual disability. FXS patients have a reduction in Fragile X Mental Retardation Protein (FMRP) expression. FMRP is critical for synaptic plasticity, spatial learning, and memory. We have previously demonstrated a reduction in the extracellular matrix protein Reelin in Fmr1 knock‐out (KO) mice, but more importantly, we demonstrated that protein supplementation with the central fragment of Reelin could improve Fmr1 KO cognitive deficits. Here we explore the potential of a novel construct of Reelin repeats 3 and 6, termed R36, which is small enough to be used in a recombinant adeno‐associated virus (rAAV) vector. We show that Reelin signaling enhancement via a single intracerebroventricular injection of R36 protein can profoundly rescue cognitive deficits in hidden platform water maze and fear conditioning, as well as hyperactivity during the open field maze for Fmr1 KO mice. Additionally, we demonstrate the same recovery of cognitive deficits in behavioral assays following 2 months of expression of R36 when Fmr1 KO mice were given a single intracerebroventricular injection of a rAAV expressing R36. Our data suggest that a gene therapy approach directed at increasing Reelin signaling via the R36 construct, could offer a novel therapeutic approach for improving cognition in FXS.
Genetically retargeting E3 ligases to enhance CAR T cell therapy
1: Massachusetts General Hospital Center for Cancer Research, Boston, MA, USA 2: Massachusetts General Hospital Department of Pathology, Boston, MA, USA 3: Harvard Medical School, Boston, MA, USA 4: Broad Institute of Harvard and MIT, Cambridge, MA, USA 5: Department of Medical Oncology, Dana‐Farber Cancer Institute, Boston, USA 6: Howard Hughes Medical Institute, Dana‐Farber Cancer Institute, Boston, MA, USA
Chimeric antigen receptor (CAR) T cell therapies are medical breakthroughs in cancer treatment. However, CAR T cell dysfunction and poor proliferation often cause treatment failure. Novel approaches are needed to program CAR T cells to overcome multiple inhibitory signals that limit anti‐tumour potency. To this end, we developed bifunctional fusion “degrader” proteins containing an E3 ligase‐binding domain and a target‐binding domain that recruit the target(s) to the E3 for subsequent ubiquitination and degradation. For precision applications, we developed degrader proteins that are constitutively active, drug‐inducible, and cell state‐regulated. We then sought to use this generalisable platform to degrade endogenous proteins that limit CAR T cell potency. TGFβ signalling is a major cause of CAR T cell dysfunction that is difficult to perturb due to the complexity of the pathway. Multiple TGFβ‐family ligands and receptors transduce signals that converge on a small number of T cell‐suppressive SMAD transcription factors. We engineered a degrader to block SMAD‐dependent TGFβ signalling using a domain that binds both SMAD2 and SMAD3. The SMAD degrader efficiently depleted endogenous SMAD2 and SMAD3, and suppressed SMAD‐dependent transcriptional activity. We then integrated our SMAD‐based degrader into a conventional CD19 CAR. In multi‐day co‐culture experiments with tumour cells, the SMAD degrader‐CAR T cells demonstrated increased cytotoxicity and dramatically enhanced proliferation in the presence of TGFβ. Here, we demonstrate a clinically suitable synthetic biology platform to reprogram E3 ligases for conditional, multi‐specific endogenous protein degradation. We further show that SMAD2/SMAD3 degradation enhances CAR T cell function in suppressive conditions.
Immunotherapy treatment with FAP‐specific CAR‐T cells can reduce skeletal muscle fibrosis in a murine model of Duchenne muscular dystrophy
1: Genethon, UMR_S951, Inserm, Univ Evry, Université Paris Saclay, EPHE 2: Université Franche Conté, Besançon, France
Fibrosis is a prominent pathological feature of Duchenne muscular dystrophy (DMD), a progressively degenerating muscle disorder leading to skeletal and cardiac muscle impairment. A murine model of DMD (DBA2mdx mice) shows a rapid occurrence of severe skeletal muscle fibrosis with over‐expression of the fibroblast activation protein alpha (FAP) and collagen type III (Col3) genes in the limb muscles at 2, 3 and 4 months of age compared to their aged‐matched DBA2 littermates. Recently, others reported the use of autologous FAP‐specific CAR‐T cells to reduce induced cardiac fibrosis in mice. This prompted us to test if FAP‐specific CAR‐T cells could also reduce the spontaneously‐occurring skeletal muscle fibrosis in DBA2mdx mice. We generated a CAR bearing a single‐chain variable fragment (ScFv)FAP antibody coupled to a CD28‐41BB‐CD3zeta domain. The CAR‐specificity was validated on FAP‐overexpressing 3T3 cells. The FAP‐CAR vector was used to transduce T cells from DBA2 spleens and to generate FAP‐specific cytotoxic CD8 T cells. Two consecutive administrations of high or low doses of FAP‐CAR T cells were given to DBA2mdx mice to measure the effects on fibrosis, 2 weeks post‐injection, by histological and molecular analyses. Only high doses of FAP‐CAR T cells significantly reduced FAP and Col3 RNA levels in limb muscles and heart but not in the diaphragm, a finding supported by reduced histological staining of collagen fibers. These results provide a novel therapeutic option to treat fibrosis in muscular dystrophies and future experiments aim to evaluate the effects of combined cell and gene therapy in this model.
Generation of first‐in‐class doxycycline‐inducible IL‐18 releasing CAR‐T cells targeting solid tumors
1: GENyO‐ Centro de Genomica e Investigacion Oncologica: Pfizer / Universidad de Granada / Junta de Andalucia 2: LentiStem Biotech S.L 3: Universidad de Granada 4: Hospital Clínic
Solid tumors treatment with Chimeric Antigen Receptor (CAR)‐redirected T cells remains challenging. Clear limitations include inhibitory tumor microenvironment (TME), inability to infiltrate into the tumor and poor persistence of CAR‐T cells. Arming CAR‐T cells with immunomodulatory molecules (T cells redirected for antigen‐unrestricted cytokine‐initiated killing or TRUCKs) seems crucial to achieve therapeutic efficacy. Previous publications showed that IL‐18 releasing CAR‐T cells enhanced antitumor effect but also lighted up potential toxicity problems. We have previously developed the first all‐in‐one doxycycline (dox)‐inducible, transactivator‐free lentiviral vector (LV) (Lent‐On‐Plus). Here, we describe the first‐in‐class anti‐CD19‐CAR‐T cells engineered to release IL‐18 only in presence of dox (αCD19‐iTRUCK‐IL‐18). These iTRUCKs tightly controlled IL‐18 expression upon dox addition and exhibited enhanced antitumor potency against a metastatic pancreatic ductal adenocarcinoma model expressing different levels of CD19. Interestingly, dox‐induced IL‐18 increased T stem cell memory and T central memory phenotype and imprinted a reduced exhaustion state on T cells. Additionally, αCD19‐iTRUCK‐IL‐18 were able to polarize M2 (pro‐tumor) to M1 (anti‐tumor) macrophages in a dox‐dependent manner. Finally, dox‐induced αCD19‐iTRUCK‐IL‐18 induced stronger tumoricidal responses in vivo in an orthotopic CD19+ pancreatic tumor model compared with those without dox. These results showed that Lent‐On‐Plus LVs can generate iTRUCKs controlling functional IL‐18 release, constituting a promising tool for future iTRUCKs development against solid tumors.
CRISPR‐coupled CAR engineering of universal donor T cells for paediatric B‐ALL
1: University College London 2: Great Ormond Street Hospital NHS Trust
Genome editing of allogeneic T cells can help overcome HLA barriers and provide “off‐the‐shelf” alternatives to autologous chimeric antigen receptor (CAR) T cell therapies. Disruption of T cell receptor alpha chain (TRAC) to prevent graft versus host disease (GVHD), and removal of CD52 provides edited CD52‐ CAR19 T cells a survival advantage in the presence of alemtuzumab. Here we deployed next generation CRISPR/Cas9 editing and linked CAR expression to multiplexed DNA editing of TRAC and CD52 through incorporation of self‐duplicating CRISPR guide RNA expression cassettes within the 3’Long terminal repeat (LTR) of a CAR19 lentiviral vector. Lentiviral transduction was followed by electroporation of Cas9 mRNA and stringent bead mediated depletion of residual TCRαβ cells through a semi‐automated and compliant manufacturing process. Three cell banks of TT52CAR19 T cells were generated and cryopreserved, with mean CAR19 expression of 92.8% and residual TCRαβ expression of 0.7%. Next generation sequencing was used to quantify editing signatures and screen top‐scoring sites of predicted sgRNA off‐target activity. In vitro functional assessments confirmed CD19 specific cytotoxicity and cytokine responses and in vivo potency was measured by inhibition of CD19+ human leukaemia engraftment in immunodeficient mice. A Phase 1 clinical trial is being conducted in children with relapsed/refractory CD19‐positive B‐cell acute lymphoblastic leukaemia (B‐ALL) (NCT04557436). Four of the first six children infused exhibited TT52CAR19 T cell expansion and achieved flow cytometric remission within 28 days, and then proceeded to allogeneic stem cell transplantation. The study provides evidence of feasibility, safety and therapeutic potential of CRISPR engineered immunotherapy.
CAR‐Tregs for Systemic Lupus Erythematosus
1: San Raffaele Scientific Institute 2: Universita Vita‐Salute San Raffaele 3: San Raffaele Hospital
Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by an abnormal inflammatory response against nuclear antigens with consequent tissue damage. Autoreactive B cells and auto‐antibodies have a fundamental role in SLE pathogenesis. Regulatory T cells (Tregs) physiologically maintain the immune tolerance and are impaired in SLE. Polyclonal Treg trnasfer obtained unsatisfactory results due to the low number of disease‐relevant antigen‐specific cells. Chimeric Antigen Receptors (CARs) are molecules capable of redirecting T cell specificity. CAR‐Tregs proved effective in pre‐clinical mouse models of autoimmunity.
We aimed at developing a CAR‐Treg based product to be employed in SLE.
We isolated Tregs from Healthy Donors Peripheral Blood Mononuclear Cells (PBMCs) and expanded them with IL‐2 and rapamycin. We transduced Tregs with a Lentiviral Vector encoding for a second‐generation anti‐CD19 CAR, considering the relevant role of autoreactive B cells and autoantibodies in SLE. Engineered cells retained their immune suppressive capabilities upon polyclonal stimulation. Noticeably, they acquired new antigen‐specific suppressive capacities, being able to block autologous B cell proliferation. We set up a humanized mouse model of SLE. In vivo, CAR‐Tregs delayed the occurrence of B cell lymphopenia, producing immunomodulatory cytokines and without showing toxicity or reprogramming towards Th17 pro‐inflammatory cells. In inflamed organs, CAR‐Tregs restored the normal composition of the immune system.
In conclusion, we efficiently generated anti‐CD19 CAR‐Tregs and proved their efficacy both in vitro and in an in vivo humanized mouse model of lupus.
Development of novel gene therapeutic approaches for protection from drug‐induced ototoxicity
1: Hannover Medical School 2: University of Kansas School of Medicine 3: Boston Children's Hospital
More than 200 drugs have ototoxic side effects and may cause sensorineural hearing loss (SNHL) in patients, including aminoglycosides (e.g., Neomycin and Gentamicin) and chemotherapeutic agents (e.g., Cisplatin). Previous studies have revealed that ototoxicity is closely related to the accumulation of reactive oxygen species (ROS) and apoptosis of important cochlear cell types, such as hair cells (HC) and spiral ganglion neurons. Despite various strategies to combat ototoxicity, an FDA‐approved prevention or treatment regimen is still lacking. Therefore, we have developed a gene therapy concept to protect cochlear cells from drug‐induced toxicity. For this, self‐inactivating lentiviral (LV) vectors were used to ectopically express proteins that are naturally involved in ROS degradation or should prevent apoptosis in affected cell types. First, it was demonstrated that the LV vectors efficiently transduced HC‐like cell lines and in‐vitro differentiated auditory neurons. The vectors were next screened for their ability to decrease cytotoxicity, and importantly, overexpression of the selected genes achieved an up to 3‐fold reduction in drug‐induced cytotoxicity. In direct comparison, genes interfering with apoptosis showed stronger effects than those promoting ROS degradation. The most promising candidates were further evaluated in rat cochlea explants and demonstrated protection from Cisplatin‐mediated tissue destruction. Strikingly, in‐vivo application of anti‐ototoxic LV vectors improved hearing and increased survival of HC in Cisplatin‐treated mice. In summary, we have established a gene therapeutic approach to protect patients from ototoxicity, which will next be fine‐tuned for clinical application to reduce disease severity and the number of patients undergoing drug‐induced SNHL in the future.
Clinical benefit of PD‐L1 hematopoietic stem and progenitor cell gene therapy in an animal model of multiple sclerosis
1: Division of Pediatric Hematology, Oncology and Stem Cell Transplantation, Woman's and Child Health Department, University of Padova 2: Altheia Science srl, Milan 3: Gene Therapy Program, Boston Children's Dana Farber Cancer and Blood Disorder Center, Boston 4: Department of Biology&Biotechnology L. Spallanzani, University of Pavia 5: International Center for T1D, Pediatric Clinical Research Center Romeo ed Enrica Invernizzi, DIBIC, University of Milan
Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease of the central nervous system (CNS) due to an aberrant attack of the immune system to the myelin leading to demyelination and neurodegeneration. Current strategies are mainly aimed at controlling symptoms not providing a stable remission. Transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) coupled with immunosuppressive/myeloablative conditioning provides some benefits in selected patients but its use is controversial.
Here, we designed an innovative lentiviral‐based gene therapy approach aimed at enhancing HSPC immune‐regulatory properties by their genetic engineering for human factor programmed death‐ligand 1 (hPD‐L1).
Several evidence suggest that the PD‐1/PD‐L1 axis plays a key role in the restrain of self‐reactive T cells, inhibiting T cell activation and favoring their exhaustion. Thus, in the experimental autoimmune encephalomyelitis (EAE) MS animal model, we investigated the ability of hPD‐L1 HSPCs to mitigate the clinical disease course upon transplantation by means of standard intravenous or innovative intra‐CNS cell administration routes. Interestingly, transplantation of hPD‐L1 HSPCs resulted in a significant benefit, mitigating the disease severity and inducing faster recovery of treated mice as compared to mock or untreated controls. Interestingly, intra‐CNS cell transplantation resulted more effective in limiting EAE disease severity than standard intravenous injection. Consistently, PD‐L1 HSPC transplanted mice showed overall lower CNS inflammation along with lower demyelination. Overall, these data provide the first evidence that PD‐L1 HSPC gene therapy may represent a valuable therapeutic option for MS, thus paving the way for the development of the approach towards clinical testing.
AAV‐delivered NDI1 improves mitochondrial function and provides benefit in AMD models
1: Trinity College Dublin 2: 3: Queen's University Belfast 4: Sorbonne Université, INSERM, CNRS, Institut de la Vision 5: The Research Foundation, Royal Victoria Eye and Ear Hospital, Dublin
Exudative (wet) and non‐exudative (dry) age‐related macular degeneration (AMD), is a debilitating multifactorial condition, affecting ∼10% of people over 65 years, typically leading to progressive loss of the retinal pigment epithelium (RPE) and subsequent loss of photoreceptors and vision. There are currently no effective treatments for dry AMD, accounting for ∼90% of cases. Features of dry AMD that have emerged recently include mitochondrial dysfunction, oxidative stress, chronic inflammation and increased reactive oxygen species (ROS) levels, which are believed to cause oxidative damage to the RPE. We investigated the beneficial effects of a yeast mitochondrial complex I equivalent, NADH‐quinone oxidoreductase (NDI1), a gene therapy directly modulating mitochondrial function, in disease models including Cfh ‐/‐ and NaIO3‐induced murine models of AMD and in ARPE19 and primary porcine RPE cell models. AAV2/2, AAV2/5 and AAV2/8 recombinant serotypes with NDI1 gene therapies were investigated. Critically, benefit has been demonstrated in all disease models. Functional benefit assessed via electroretinography and optokinetic analysis together with histological benefit were obtained in vivo. The study represents the first demonstration globally of a gene therapy for dry AMD directly targeting mitochondrial dysfunction.
A combinatorial gene therapy for temporal lobe epilepsy based on NPY and one of its receptors
1: Universita Vita‐Salute San Raffaele 2: San Raffaele Scientific Institute 3: Institute of Neuroscience, National Research Council (CNR), 20854 Vedano al Lambro, Italy 4: Department of Neuroscience and Rehabilitation, Section of Pharmacology, University of Ferrara, 44121 Ferrara, Italy.
Epilepsy is a common neurological disease characterized by a persistent predisposition to generate seizures. About 30% of patients are drug‐resistant, that is, refractory to currently available anti‐epileptic drugs. There is therefore a major need to identify new therapeutic strategies. Gene therapy may represent a doable option in this respect.
Neuropeptide Y (NPY) can act as an endogenous anticonvulsant and, therefore, NPY‐based gene therapy may represent an effective approach for the treatment of epilepsies. NPY expression is increased both in the rodent and human epileptic hippocampus, despite a strong loss of hilar GABAergic interneurons that physiologically express it. To potentiate the protective effect of NPY, we developed a combinatorial gene therapy based on the expression of NPY and its receptor Y2. Since Y2 receptors act mainly pre‐synaptically to reduce glutamate release, we drove transgenes expression using the minimal CamKII promoter, biasing their expression in excitatory neurons.
We characterized the ability of our lentiviral vectors to express NPY and its Y2 receptor by using biochemical and immunocytochemical techniques in hippocampal neurons (immunofluorescence, Ca2+ measurements, western blotting) and mouse brain (immunofluorescence, western blotting). Telemetry video‐EEG monitoring was then used to assess the effect of the therapeutic genes on the epileptic phenotype of a genetic mouse model (synapsin triple KO).
We found that the combined expression of NPY and Y2 is sufficient to reduce both frequency and duration of seizures. These data suggest that the combined delivery of NPY and Y2 may be successful for the treatment of drug‐resistant epilepsy.
A Mutation‐Independent CRISPR/Cas9‐based ‘Knockout and Replace’ Strategy to Treat Rhodopsin‐Associated Autosomal Dominant Retinitis Pigmentosa
C H Liu1 P Wolf1 R Dong1 Y Huang1 D Tabbaa1 E Marco1 B Duke1 A Pinilla1 A Pant1 R D'Souza1 J Newmark1 G Giannoukos1 K Zhang1 A Timmers1 M S Shearman1
1: Editas Medicine
Rhodopsin‐associated autosomal dominant Retinitis Pigmentosa (RHO‐adRP) is an inherited retinal disease causing photoreceptor degeneration. Over 150 mutations in the RHO gene are known to cause RHO‐adRP. Using a dual AAV system, Editas Medicine is exploring CRISPR/Cas9‐based (KO&R) therapy to knockout endogenous RHO and replace it with an exogenous RHO. The specificity of a human and non‐human primate (NHP) cross‐reactive Cas9 guide was tested using human retina explants. Pharmacokinetics and pharmacodynamics of the KO&R were assessed in humanized mRhohRHO /+ mice. Efficacy was measured in NHPs by comparing knockout (KO) only and KO&R versions of the dual AAV system. A RHO promoter drives Cas9 and RHO expression restricting editing and RHO replacement expression to rods. No off‐target editing was observed in human retina explants after transduction with the KO&R. The KO&R achieved maximal levels approximately 6 weeks post‐dose and remained stable for at least 13 weeks post‐dose in mRhohRHO /+ mice. The KO&R levels displayed a dose‐response plateauing at a dose of 3E12 vg/ml. Studies in NHP demonstrated nearly 100% knockout of endogenous RHO, and replacement RHO produced over 30% of normal RHO protein. The KO&R‐injected eyes showed restoration of RHO expression and retention of normal photoreceptor structure and function compared to the KO‐injected eye. In conclusion, Editas demonstrated high efficacy of a CRISPR/Cas9‐based KO&R therapeutic strategy for RHO‐adRP. The experimental therapy is mutation‐agnostic and a potential one and done treatment for RHO‐adRP. The efficacy of our KO&R gene editing therapy in NHP supports continued advancement of this approach toward treating patients.
In vivo adenine base editing corrects the HFE C282Y mutation and improves iron metabolism in hemochromatosis mice
1: Hannover Medical School 2: BioQuant; University of Heidelberg 3: ETH Zürich
Hereditary Hemochromatosis (HH) is one of the most common genetic diseases in the white population, with a prevalence of 1:200/400. Among the four different types, the most common form is Type 1, a homozygous p.C282Y mutation in the HFE gene, in which a guanosine is replaced by an adenosine (c.845 G>A). This mutation results in the misfolding of the HFE protein, which can no longer reach the cell membrane of the hepatocytes, thereby losing its ability to work as a sensor for the iron content in the bloodstream. This ultimately causes the accumulation of iron in various organs, mostly in the liver, heart and pancreas, thus leading to the development of chronic diseases. Here, we developed a strategy to correct the G > A point mutation in the HFE gene in a permanent way, by using the adenine base editor ABE7.10. The generation of an in vitrosystem tailored to the HFE sequence allowed us to screen various sgRNAs. The best performing guide, together with the base editor, was then applied in 129‐Hfe tm.1.1Nca mice using the AAV8 split‐vector technology. Our treatment led to a base conversion rate in the HFE gene of >10% and it improved iron metabolism in the liver. This proof‐of‐concept study demonstrates the therapeutic potential of adenine base editor for HH.
Non‐viral DNA delivery associated to TALEN® gene editing leads to highly efficient correction of sickle cell mutation in long‐term repopulating hematopoietic stem cells
1: Cellectis Therapeutics 2: Imagine Institut
Sickle cell disease stems from a single point mutation in the HBB gene which results in sickle hemoglobin. For patients who are not eligible for an allogeneic stem cell transplantation, nuclease‐based gene therapy approaches provide a relevant therapeutic alternative to restore functional hemoglobin production.
Here, we leveraged TALEN® technology to develop a gene editing process leading to highly efficient HBB gene correction via homology directed repair, while mitigating potential risks associated to HBB gene knock‐out. Furthermore, we compared viral (TALEN‐V) and non‐viral (TALEN‐NV) DNA template delivery strategies in mobilized healthy donor (HD) or non‐mobilized homozygous sickle patient (HbSS) hematopoietic stem and progenitor cells (HSPCs).
Both strategies led to high and comparable efficiencies of HBB gene correction in vitro in HD and HbSS, without affecting viability, purity or clonogenic potential of corrected HSPCs. Moreover, they both elicited high and similar expression of normal adult hemoglobin in red blood cells differentiated from edited HbSS HSPCs.
Interestingly, when evaluated in vivo using an immunodeficient mouse model, transplanted TALEN‐NV edited HSPCs showed higher levels of engraftment and gene correction compared to TALEN‐V edited HSPCs. Further characterization of edited HSPCs by single‐cell RNAseq enabled us to identify transcriptomic signatures that could explain discrepancies between the two DNA template delivery methods tested.
Overall, these results show that non‐viral DNA delivery associated to TALEN gene editing reduces the toxicity usually observed with viral DNA delivery and allows high levels of HBB gene correction in long‐term repopulating hematopoietic stem cells.
EMD‐101, an autologous, allele‐specific gene‐edited hematopoietic stem cell product, for treating ELANE‐mediated severe congenital neutropenia
1: EmendoBio, Inc. 2: Department of Medicine, University of Washington
Severe congenital neutropenia (SCN) is a life‐threatening disorder usually caused by dominant heterozygous mutations in ELANE. Current treatments consist of lifelong daily injections of G‐CSF or allogeneic transplantation, involving increased risk of hematopoietic malignancies and graft failure or acute graft‐versus‐host disease, respectively. Potential gene therapy platforms have been proposed, but these cannot provide the accurate, allele‐specific editing required in targeting ELANE. Using a proprietary high‐fidelity CRISPR‐associated nuclease, optimized as an allele‐ and target‐specific editing tool, we have developed a novel strategy that precisely targets the heterozygous sites of single nucleotide polymorphisms (SNPs) associated with most of ELANE mutated alleles. Hematopoietic stem cells (HSCs) in which a single ELANE allele was selectively knocked‐out form an autologous cell therapy products termed EMD‐101. In‐vitro, EMD‐101 HSCs present alleviated cellular abnormalities associated with the disease resulting in 50‐70% increase in normally functioning neutrophils. Genomic analysis confirmed about 80‐90% knock‐out of ELANE mutated allele without any detectable off‐targets or translocations. Transplanting EMD‐101 into immunocompromised mouse model resulted in efficient engraftment of the edited cells that gave rise to multiple lineages in the mouse bone marrow and spleen. These results support the development of EMD‐101 as a autologous product for more than 75% of ELANE‐related SCN patients and present a highly target‐specific, allele‐selective editing strategy that could be applicable to other autosomal dominant disorders.
Sharpening the adenine and cytosine base editing outcome in hematopoietic stem cells
1: Imagine Institute, INSERM UMR1163, Paris, France 2: Necker Hospital, Paris, France
β‐hemoglobinopathies are caused by mutations affecting the adult hemoglobin (Hb) β‐chain. The co‐inheritance of mutations causing fetal γ‐globin expression in the adult life (HPFH: Hereditary Persistence of Fetal Hb) ameliorates the disease phenotype. We previously reported adenine and cytosine base editing (BE) mediated introduction of HPFH mutations with minimal off‐target activity, that led to therapeutic HbF levels in red cells differentiated from SCD or β‐thalassemia hematopoietic stem/progenitor cells (HSPCs).
Serial xenotransplantation experiments showed BE in long‐term HSCs; however, the efficiency was reduced compared to input HSPCs. RNA‐seq analysis showed no alteration for ABE‐treated HSPCs, but activation of the p53 pathway and dysregulation of genes involved in HSC biology in CBE‐treated samples. We also performed single‐cell RNA‐seq analysis to unravel the BE impact on bona fide HSCs. Furthermore, FACS‐sorting of HSPC subpopulations showed that base editing efficiency was similar in HSC‐enriched versus progenitor subpopulations, but did not exceed ∼50%, highlighting the need to increase the overall BE efficiency.
To increase the fitness of edited HSCs and editing efficiency, we optimized the RNA delivery of the BE machinery and used histone deacetylases, RNases and apoptosis inhibitors. Moreover, we developed a strategy to enrich edited cells. By way of example, two rounds of mRNA electroporation led to increased BE efficiency with minimal toxicity. Furthermore, highly processive base editors outperformed the original enzymes even upon only one round of electroporation. In conclusion, we identified key elements that improved the BE efficiency in HSCPs to enable the clinical development of BE strategies for the treatment of β‐hemoglobinopathies.
Correction of Congenital Dyserythropoietic Anemia Type II using Lentiviral Gene Therapy
M Dessy‐Rodriguez1 2 3 S Fañanas‐Baquero1 2 3 V Venturi4 R Sanchez‐Dominguez1 2 3 O Alberquilla1 2 3 Z Garcia de Castro1 2 3 S Payan5 C Tornador6 G Hernández4 P Bianchi7 M Sanchez4 6 J C Ramirez8 J C Segovia1 2 3
1: CIEMAT 2: IIS‐FJD 3: CIBERER 4: Universitat Internacional de Catalunya 5: Hospital Universitario Virgen del Rocío 6: Bloodgenetics 7: Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico 8: VIVEbiotech
Congenital dyserythropoietic anemia type II (CDAII) is a rare inherited disorder that affects red blood cell development. CDAII patients show hypercellular bone marrow, erythroid hyperplasia and the presence of binucleated erythroid cells. CDAII is caused by mutations in SEC23B gene. SEC23B is involved in protein processing and Golgi‐reticulum trafficking. CDAII management is generally limited to blood transfusion and iron chelation, being allogeneic hematopoietic stem cell transplant (HSCT) the only curative option. Consequently, autologous HSCT of genetically corrected cells can offer a definitive treatment for CDAII.
To develop a gene therapy for CDAII, we have designed two lentiviral vectors carrying the wild type or the codon optimized SEC23B cDNA (wtSEC23B LV and coSEC23B LV). We have generated SEC23B knock‐out human hematopoietic progenitors (SEC23BKO CD34+ cells) using CRISPR/Cas9 system to assess the efficacy of the lentiviral vectors. SEC23BKO CD34+ cells reproduced the erythroid defects observed in CDAII patients. Moreover, LV transduced SEC23BKO CD34+ cells increased SEC23B protein expression restoring their normal capacity for in vitro erythroid differentiation. Similarly, in vivo erythroid differentiation in NBSGW mice showed a selection of the transduced cells, accompanied by a reduction in binucleated cells with a better terminal erythroid differentiation. Furthermore, SEC23B LV transduction of peripheral blood CD34+ cells from CDAII patients improved their erythroid differentiation potential with a reduction in the percentage of binucleated erythroid cells.
In summary, SEC23B LVs compensate for SEC23B deficiency in SEC23BKO CD34+ cells and in patients' hematopoietic progenitor cells, which opens the possibility of future treatment of CDAII by gene therapy.
Reconstitution of Humoral and Cellular Immunity in Thalassemic Patients Treated with HSC LV‐mediated Gene Therapy Following Myeloablation
1: San Raffaele Telethon Insitute for Gene Therapy (SR‐TIGET) 2: IRCCS San Raffaele Scientific Institute 3: Università Milano Bicocca 4: San Gerardo Hospital Monza 5: Fondazione IRCCS Ca' Granda, Milan 6: University of Cagliari 7: Università degli studi della Campania “Luigi Vanvitelli”, Napoli 8: Universita Vita‐Salute San Raffaele
Transfusion‐dependent beta‐thalassemia is due to mutations in the ß‐globin chain gene of haemoglobin A, leading to severe anemia and lifelong transfusion dependence. In TIGET BTHAL trial 9 patients (3 adult and 6 pediatric), treated with autologous CD34+ cells engineered by SIN lentiviral vector expressing human ß‐globin, have completed 2yrs follow‐up and entered in the LTFU study. Overall, no adverse events related to investigational product and highly polyclonal engraftment with no clonal dominance have been reported at the latest update. Persistent engraftment was observed in 6 out of 9 patients, reaching up to 80.0% marking in BM CD34+ progenitors. Clinical outcome correlated with the efficiency of transduced CD34+ cells in vivo and the VCN distribution measured in single CFU, suggesting a threshold of >40% marking may be required for transfusion independence. Similar VCN were retrieved in myeloid and B cells while lower values were observed in CD3+ cells. In particular CD4+ T cells remained below the normal range for a prolonged period in all patients, resulting in an inverted CD4/CD8 ratio, similar to results from reported allogeneic bone marrow transplant. Phenotypic, TREC and KREC evaluation showed a progressive recovery of innate and adaptive immunocompetent cells with slower kinetics in adult patients vs pediatric. All patients showed a good response to mitogens and pathogens with adequate antibody titer in response to vaccinations Overall, these results showed that immunological reconstitution is achieved at normal levels following gene therapy with G‐CSF and Plx mobilized autologous CD34+ cells in adult and pediatric patients.
Characterization and GMP manufacturing of epigenetically reprogrammed HLA‐G expressing, T regulatory cells (iG‐Tregs)
1: Institute of Cell Therapy, University of Patras, Rio, Greece 2: Gene and Cell Therapy Center, George Papanikolaou Hospital, Thessaloniki, Greece 3: Department of Genetics, School of Biology, Aristotle University of Thessaloniki, 4: Center for the Study of Hematological and other Malignancies, Nicosia, Cyprus 5: University of Washington, Seattle
Immunotherapy with T‐regulatory cells (Tregs) stands as an alternative treatment for graft‐versus‐host disease (GvHD), a major complication of allogeneic hematopoietic cell transplantation. We have shown that HLA‐G expression, known to allow the maternal‐fetal tolerance, can be induced by pharmacological hypomethylation (Decitabine‐Dec) of T‐cells, generating T‐cells with immunoregulatory properties (iG‐Tregs). Herein, we elucidated the molecular characterization of iG‐Tregs, assessed their safety and efficacy in vitro and in vivo and validated their GMP manufacturing. iG‐Tregs were generated after a 3‐day exposure of anti‐CD3/CD28‐activated T cells to Dec. RNA‐seq revealed a distinct expression profile of FACS‐sorted HLAG+CD4+cells over their HLAG‐CD4+ counterpart, in terms of regulatory genes (CCL17/CCL22/CXCL9) and the myeloid suppressor gene IDO‐1. iG‐Tregs demonstrated a favorable safety profile over untreated‐control cells in vitro, by producing decreased levels of GvHD‐associated cytokines upon PMA/ionomycin stimulation (IL‐2/IFNγ/IL‐17a) and exerting diminished alloreactivity against allogeneic PHA‐blasts (p < 0.0001). Safety was also confirmed in vivo; all control mice succumbed by d35 from histologically confirmed GvHD whereas 67% of iG‐Treg‐treated mice survived until sacrifice (d84) (p = 0.0019). As regards efficacy, co‐administration of iG‐Tregs with donor lymphocyte infusions (DLIs) delayed or prevented the onset of GvHD versus DLI‐treated‐mice. Two large‐scale iG‐Treg products were generated and validated under GMP conditions; an average of 8.6x107 iG‐Tregs were produced from 2 healthy volunteers, enriched for HLA‐G+cells (26 ± 0.04%) and lacking alloreactivity. Overall, we demonstrate the feasibility of GMP generating clinically relevant doses of well‐characterized iG‐Tregs. A close to initiation, phase I/II clinical trial (EUDRACT:2021‐006367‐26) will evaluate iG‐Tregs for the prevention and treatment of steroid‐refractory GvHD.
Sonication Linker Mediated‐PCR (SLiM‐PCR), an efficient method for quantitative retrieval of vector integration sites
1: San Raffaele Telethon Institute for Gene Therapy (HSR‐TIGET) 2: GlaxoSmithKline 3: University Vita‐Salute San Raffaele 4: Pediatric Immunohematology and BMT, San Raffaele Hospital
Specialized PCR‐based techniques combined with next‐generation sequencing (NGS) and bioinformatics analyses allow the identification of vector integration sites (IS) in the genome of transduced cells. IS characterization in gene therapy (GT) applications allows to track transduced cells and their progeny in different tissues or cell lineages over time, evaluate the clonal composition of the engrafted population, identify the targeted genes and quantify the relative abundance of IS to detect or exclude clonal expansions.
Here, we present our method, Sonication Linker mediated‐ PCR (SLiM‐PCR), in which IS are PCR amplified from sheared genomic DNA, sequenced, and mapped on the target genome and quantified by custom bioinformatics software.
SLiM‐PCR was validated in an experimental framework in which the genomic DNA from a monoclonal cell line harboring one lentiviral vector (LV) IS in a known genomic position was mixed at different ratios (70‐0%) with that of a bulk‐transduced cell line with LV IS randomly distributed in the genome. SLiM‐PCR showed a high correlation between the number of retrieved and expected IS (R2 ≃0.9) and a detection limit of 0.16% genomes over the total.
SLiM‐PCR was successfully applied on thousands of samples coming from 7 GT clinical trials (97 patients) and more than 30 research and preclinical studies using different vector types like gamma‐retroviruses, LV, transposons and adeno‐associated vectors.
Our results showed that SLiM‐PCR is a reproducible, accurate and versatile method that combined with a state‐of‐the‐art infrastructure, automated laboratory procedures and dedicated bioinformatics tools allows to perform fast and high‐throughput IS analyses.
Application of novel AAV purification and analysis column with Adeno‐Associated Virus Receptor (AAVR)
1: Tosoh Corp.
Adeno‐associated virus (AAV) vectors are widely used as gene therapy drugs. There are a variety of serotypes and most infect cells via binding to the AAV receptor (AAVR; KIAA0319L). Utilizing AAVR, we have succeeded in development of an affinity chromatography column (AVR column) with AAVR as a ligand. After analyzing various AAVs, the purified AAV showed a single peak and the wide range of serotypes (AAV1, 2, 5, 8, 9, rh10, etc.) that infect via AAVR were analyzable. On the other hand, AAV4 which infects without AAVR didn't adsorbed on the column. In addition, the analysis of AAV mutants revealed that a correlation was observed between elution time and infectivity of the various mutants, suggesting that the infectivity via AAVR can be evaluated. Next, AAV in culture supernatants and cell lysates was successfully detected, enabling detection of AAV production in culture without purification. The tryptophan fluorescence (excitation 280 nm/emission 350 nm) can analyze even AAV at 2 x 10^9 cp and quantify to 10^14 cp. Furthermore, this column can elute AAV under neutral conditions was confirmed by buffer solutions containing chelating agents or high concentrations of salts. The purified AAV had higher infectivity than acid‐eluted AAV. Based on these results, the AVR column is expected to be used not only for isolation of AAV, but also for evaluation of infectivity of AAV without reporter gene, screening of mutant AAV and productivity monitoring.
Vector genome loss and epigenetic modifications impact long‐term transgene expression of AAV5 vectors produced in mammalian HEK293 and insect Sf cells
1: BioMarin Pharmaceutical
Recombinant adeno‐associated virus (rAAV) vectors can be produced in HEK293 cells or Spodoptera frugiperda (Sf)‐based cell lines. We produced a rAAV5‐human alpha‐1 anti‐trypsin vector in HEK293 and Sf cells and investigated factors affecting long‐term expression. C57BL/6 mice were administered with 6x1013 vg/kg of vector and collected blood, and liver samples through week 57. Peak expression for vector produced in both systems was at week 12‐24, and their expression declined by ∼50% at final time point. For HEK293‐produced vector, liver vector genomes decreased continuously from week 1 through 57 and significantly correlated with circulating A1AT protein levels. Further, RNA‐Seq analysis of liver tissues revealed that HEK293 vector‐treated mice showed significantly higher inflammatory response at 12 weeks post‐dose compared to Sf vector‐treated mice. Together, this data suggest that heightened innate immunity and subsequent continual vector genome loss might mediate the decline of transgene expression in HEK293‐vector‐treated mice. In contrast, decrease in vector genomes from peak expression to final time point was not observed in Sf‐vector‐treated mice; instead, vector genome accessibility significantly decreased from peak to final time point and correlated with transgene RNA production. In parallel, vector DNA interactions with active histone marks (H3K4me3/H3K27Ac) were significantly reduced, consistent with decline of transgene expression in Sf‐vector‐treated mice. Our data shows that AAV5 vectors produced in HEK293, and Sf cells showed similar long‐term durability of expression; despite distinct mechanisms contributing to decline in transgene expression over time. Additional analysis of epigenetic regulations on rAAV vectors would deepen our understanding of AAV‐mediated transgene expression.
Nanobodies as versatile ligands to retarget AAV
1: Institute of Immunology, University Medical Center, Hamburg, Germany 2: Vector Core facility, University Medical Center, Hamburg, Germany 3: Sirion GmbH, Martinsried, Germany 4: Normandie Univ, UNIROUEN, INSERM, U1234, Pathophysiology, Autoimmunity, and immunotherapies (PANTHER), 76000 Rouen, France
Low target cell specificity and high costs of production remain limitations of AAV vectors. We have developed a technology that may overcome this limitation by enhancing the cellular specificity and reducing the amount of AAV needed to transduce cells of interest. For this, we exploit the small size and high solubility of nanobodies, single immunoglobulin variable domains derived from camelid heavy‐chain‐only antibodies. Using genetic engineering we inserted a membrane protein‐specific nanobody into the GH2/3 or GH12/13 loop of the VP1 capsid protein of AAV9. Display of a nanobody on the AAV capsid dramatically enhanced the transduction of cells expressing the cognate membrane protein compared to both, the parental AAV without a nanobody and to cells that do not express the respective membrane protein. We have verified this technology for several structurally distinct membrane proteins, including PD‐L1 and CD38, a single‐pass type I and type II transmembrane protein, respectively, P2X7, a multi‐pass ion channel, and CD73, a GPI‐anchored ecto‐enzyme. Furthermore, we show that it is possible to simultaneously insert two different nanobodies (αCD38 and αPD‐L1) into the GH2/3 and GH12/13 loops of the same VP1 capsid protein. The resulting bispecific nanobody‐displaying AAV enhanced transduction of cells expressing either CD38, PD‐L1 or both. Our results demonstrate the versatility of nanobodies as targeting moieties to improve AAV gene therapy.
Capsid‐engineered adeno‐associated virus (AAV) vector for neurotrophin gene therapy in inner ear disease
1: Hannover Medical School 2: University of Kansas 3: Harvard Medical School
Hearing loss affects more than 460 million people worldwide and often leads to adverse educational and social consequences that significantly affect quality of life. Currently, cochlear implants (CI) are the standard of care for restoring severe to profound hearing loss. Although contemporary CIs are generally highly successful, outcomes vary greatly among individuals due to differences in auditory nerve survival. Loss of neurotrophic factor (NF) production is thought to be the main reason for the degeneration of spiral ganglion neurons (SGN). Supplementation of NFs, including brain‐derived neurotrophic factor (BDNF), have been proposed for improving SGN survival and enhancing cochlear implant outcomes. However, current delivery systems, including AAV vectors, are inefficient in transducing cell types of the inner ear, such as SGNs.
To overcome this challenge, we developed novel capsid‐engineered AAV vectors displaying heptamer peptide sequences previously derived from a phage display library screen. Capsid variants were analyzed in vivo and in vitro regarding transduction efficiency and specificity of gene transfer into distinct cell types of the inner ear. For a neurotrophic‐directed gene therapy approach, we focused on variants with tropism for SGNs. Our best‐performing variant is currently assayed in a therapeutic setting using overexpression of BDNF in SGN after loss of hair cells in mice as therapeutic strategy. In conclusion, our novel AAV vector demonstrated superior properties relevant for more efficient SGN transduction, which might significantly improve neural survival and improve outcomes in CI recipients upon cochlear gene therapy.
Endogenous human SMN1 promoter‐driven gene replacement improves the efficacy and safety of AAV9‐mediated gene therapy for spinal muscular atrophy (SMA) in mice
Q Xie1 H Ma1 X Chen1 Y Zhu2 Y Ma2 L Jalinous2 Q Su1 P Tai1
1: Horae Gene Therapy Center, UMass Chan Medical School, Worcester, MA USA 2: CANbridge Pharmaceuticals, Cambridge, MA USA
Physiologically regulated transgene expression is a major challenge in gene therapy. Zolgensma®, an FDA‐approved gene therapy for SMA, represents a significant breakthrough for treating neuromuscular diseases. This biotherapy is an scAAV9‐based vector that delivers a ubiquitously expressed human SMN1 transgene under the control of the cytomegalovirus enhancer/chicken β‐actin promoter (CMVen/CB‐hSMN1). However, high doses of the vector are required to achieve therapeutic benefit, leading to liver damage in >90% patients and hematologic complications in some patients. We first attempted to create a more potent rAAV9 ubiquitously expressing codon‐optimized hSMN1 transgene (rAAV9‐CMVen/CB‐co‐hSMN1) for efficacious but non‐toxic gene therapy at reduced doses. Surprisingly, when we treated 1‐day old SMNdelta7 mice with this potent vector at 3.3E+14 VG/kg by facial vein administration, all treated animals (n = 6) died before untreated SMA mice. We found that the early lethality was associated with super‐physiological levels of overexpressed hSMN1 in the liver. We hypothesize that restoration of physiological levels of hSMN1 expression in the appropriate tissue/cell types may be essential for a safer and more efficacious SMA gene therapy. To this end, we developed a second generation (2nd gen) scAAV9 vector expressing co‐hSMN1 from an endogenous hSMN1 promoter (scAAV9‐SMN1p‐co‐hSMN1). Head‐to‐head comparison studies between our 2nd gen vector and benchmark vector whose design is identical to Zolgensma® (scAAV9‐CMVen/CB‐hSMN1) demonstrated that the 2nd gen physiologically regulated vector is indeed more efficacious and safer than the benchmark vector as indicated by prolonged life span, expanded therapeutic window, elimination of liver toxicity and lethality, and improved restoration of motor functions.
Mutagenesis of capsid surface residues for improvement of AAV vectors with retinal tropism
1: Department of Ophthalmology, University Hospital, LMU Munich, 80336 Munich, Germany 2: Laboratory for Infection Biology and Gene Transfer, Institute of Experimental Haematology, Hannover Medical School, Hannover, Germany
Recombinant adeno‐associated virus (AAV) vectors are the gold standard in gene therapy for the treatment of retinal disorders. Despite the success, there are still some obstacles such as the requirement for challenging injection routes and the risk of vector‐induced immune responses. To overcome such hurdles, AAVs with optimised properties are needed.
Here, we sought to further improve the tropism of AAV capsids previously identified in a directed evolution screen by rational design. In particular, starting from the next generation AAV capsids, AAV2.GL and AAV.NN, we aimed to optimise cell‐specific targeting and/or minimise immunogenicity. We modified AAV2.GL and AAV2.NN at five previously described antibody‐recognition epitopes on the capsid surface to prevent antibody binding and neutralisation. The resulting variants led to high transduction efficiencies in vitro (e.g. 66.19% and 72.08% at a MOI of 250). In an ex vivo human retinal explant model these engineered vectors led to high levels of eGFP expression, validating their transducing potential for human retinal cell types. Escape from neutralisation was assessed using an in vitro neutralisation assay. Despite the improved transduction, the novel capsids were neutralised to a similar extent as the parent variants. Their promising transduction potential was further validated in vivo by intravitreal injection in wild type mice followed by confocal scanning laser ophthalmoscopy and immunohistochemistry.
These results suggest that modification of known antibody‐binding epitopes of the AAV capsid surface can further enhance tropism but may not be sufficient to prevent neutralisation.
Novel conditioning enables high and stable repopulation of macrophage/microglia niches by wild type and genome‐edited hematopoietic cells
1: Stanford University
Autologous transplantation of gene‐modified hematopoietic stem and progenitor cells (HSPCs) is showing great promise in clinical studies for neurometabolic diseases. The efficacy of the approach relies on the life‐long repopulation of hematopoietic and non‐hematopoietic organs with corrected/engineered HSPC‐derived cells. However, the high dose chemotherapy required to condition the central nervous system (CNS) and achieve stable bone marrow (BM) engraftment limits the wide‐spread applicability of the approach. Moreover, the slow CNS repopulation kinetics limits its benefit in rapidly progressive neurological diseases.
We developed a conditioning regimen that enables fast, efficient, and stable engraftment of HSPC‐derived microglia‐like cells in the CNS with unperturbed hematopoietic reconstitution and neurobehavior in mice. The regimen combines Busulfan and PLX3397, a CSF1R inhibitor, which specifically depletes microglia/macrophages. In transplants of C57BL/6 mice with BM cells from CAG:GFP/GFP mice, the combination regimen resulted in a 15‐ to 4‐fold increase in the fraction of BM‐derived GFP+ microglia‐like cells in the brain compared to Busulfan (89 ± 5% vs. 6 ± 2.4% at 3 months, 86 ± 4% vs. 23 ± 17% at 7 months). Non‐myeloablative Busulfan doses plus PLX3397 also led to significant BM‐derived cell engraftment in the brain. Single‐cell RNA‐seq analyses of brain‐engrafted BM‐derived and host CD45+CD11b+ cells showed tissue‐specific cell signatures and a predominant homeostatic microglia‐like phenotype. In immunocompromised NSG‐SGM3 mice, conditioning with Busulfan plus PLX3397 resulted in increased human cell chimerism in the brain without detrimental effects on hematopoietic reconstitution. Our findings provide proof‐of‐concept of efficiency and safety of a novel conditioning regimen to improve the efficacy of autologous HSPC transplantation for neurometabolic diseases.
Targeting CX3CR1 Gene To Improve Microglia Reconstitution And Transgene Delivery Into The CNS Upon Hematopoietic Stem And Progenitor Cell Transplant
1: Gene Therapy Program, Dana‐Farber/Boston Children's Cancer and Blood Disorders Center, Harvard Medical School, Boston, MA, USA 2: Division of Pediatric Hematology, Oncology and Stem Cell Transplantation, University of Padua, Padua, Italy
Transplantation of engineered hematopoietic stem/progenitor cells (HSPCs) has shown curative potential for multiple pathologic conditions upon repopulation of hematopoietic compartments, including microglia. To foster the engraftment and differentiation of transplanted engineered HSPCs into microglia‐like cells (MLCs), we focused on CX3CR1, a microglia chemokine receptor that regulates microglial recruitment to sites of neuroinflammation and microglia ontogeny.
Firstly, we showed that, in competitive transplantation settings, CX3CR1 haploinsufficient HSPCs resulted in a more robust engraftment (60% vs 40% in hematopoietic organs; 80% vs 20% in brain) and generation of mature MLCs as compared to wild type (WT) HSPCs. Moreover, CX3CR1 haploinsufficient brain‐engrafted MLCs displayed a greater extent of ramifications and complexity compared to WT cells, suggesting more rapid acquisition of microglia‐like morphology and phenotype.
Next, to develop a therapeutic strategy combining the improved features of CX3CR1 haploinsufficient HSPC transplantation with microglia specific transgene expression, we developed a CRISPR/Cas9 gene editing/addition strategy and a promoter‐less cassette allowing efficient editing (up to 45% of targeted alleles) and targeted gene addition at the CX3CR1 locus of human HSPCs. Edited hHSPC successfully engrafted primary and secondary myeloablated immunodeficient mice, showing higher transgene expression in human MLCs compared to hematopoietic organs, as per physiologic CX3CR1 expression. Interestingly, expression of the transgene driven by CX3CR1 promoter in engrafted MLCs was stronger than the expression driven by a conventional PGK promoter.
Overall, we identified and validated CX3CR1 as key target to enhance the ability of HSPCs to generate a microglia‐like progeny upon transplantation, allowing a specific and robust transgene expression in the CNS.
A negative regulatory region in WAS intron 1 controls megakaryocytic differentiation
1: Universidad de Granada 2: Lentistem 3: Fundacion Publica Andaluza Progreso y Salud/Genyo 4: Fundación para la Investigación Biosanitaria de Andalucia Oriental‐ Alejandro Otero 5: GENyO‐ Centro de Genomica e Investigacion Oncologica: Pfizer / Universidad de Granada / Junta de Andalucia
The Wiskott‐Aldrich syndrome (WAS) gene encodes for a hematopoietic specific protein that is a key regulator of haematopoietic homeostasis and function. WAS gene expression is regulated through two promoters and a 3’ enhancer, although other regions have also been reported. Here, we describe a new WAS gene regulatory region located in the first intron containing several transcription factor binding sites (TFBS) involved in hematopoietic regulation such as Ets1, Stat1 and Hmg1. Interestingly, two of the Ets1 binding sites are palindromic, a configuration previously shown to be associated with transcriptional silencing in tissue‐specific genes. Disruption of these TFBS by CRISPR/Cas9 increases WAS gene expression on K562 cells (megakaryocytic origin) but not in other hematopoietic cell types, suggesting a cell‐type‐specific negative regulation. The negative‐regulation activity of the intron 1 of the WAS gene was also observed when inserted downstream of an unrelated promoter on K562 cells. We therefore investigated the role of this regulatory region during MK differentiation and showed that targeting WAS gene intron 1 with CRISPR/Cas9 on K562 and HSCs accelerated the appearance of CD41 and CD42 MK markers and increased their granularity and size. Finally, we demonstrated that disruption of this regulatory region altered the WAS gene expression pattern during the MK differentiation, suggesting that the phenotypic alterations observed are a consequence of the inappropriate control of WAS expression. In summary, our results showed that the intron1 of the WAS gene acts as a transcriptional repressor and this repression is important to control megakaryocytic differentiation.
Therapeutic changes of cis and trans regulatory elements
R Maritato1 E D'Andretta1
1: Department of Translational Medical Sciences (DiSMeT) University of Naples “Federico II”, Italy Via Pansini, 5 ‐ 80131 ‐ Napoli
Protein trans‐acting factors (TAFs) interacting with DNA Cis regulatory sequences (CREs) within promoters and enhancers encode timing, location, and magnitude of gene expression. Along with Transcription factors (TF), which mediate direct interaction with CREs (TF‐DNA), TF‐TF interactions and multiple epigenomic remodeling ensure maintenance of physiological gene expression. Currently, each of these mechanisms can be controlled to lower or enhance transcriptional output. DNA binding modules (DBM) based on ZFs, TALEs, and CRISPRs, can be designed to target endogenous CREs loci. Once docked to selected CRE sequence the effector domain fused to DBMs dictate one of three mechanisms controlling transcriptional outputs. i) DBMs fused to nucleases change CRE sequences composition, ii) DBM coupled to activator or iii) repressor domains up or down regulate transcription, respectively. We conceived an additional mode of transcriptional repression iv), relaying on the exclusive use of DBMs (ZF), which operate without the aid of repressor domains. Using in vivo DBMs‐mediated Rhodopsin (RHO) repression via AAV vector photoreceptors delivery as a paradigm, we demonstrated that the sole binding of these DBMs to certain CREs block RHO expression, with both limited off targeting and engagement of cellular systems. Furthermore, in vivo functional studies on both RHO CREs and DBMs show that the kay determinants of RHO repression output are i) the selection of the CREs targeted by DBMs, ii) the sequence composition and orientation of CREs, and iii) the binding properties of DBMs. Thus, selecting, and rewriting CREs along with the design of their cognate DBMs impact therapeutic transcriptional modulations.
Base editing in hematopoietic cells corrects the disease underlying intronic mutation in a mouse model of familial hemophagocytic lymphohistiocytosis
1: Institute for Transfusions Medicine and Gene Therapy, Medical Center – University of Freiburg, Freiburg, Germany, 2: Center for Chronic Immunodeficiency, Medical Center – University of Freiburg, Freiburg, Germany, 3: Institute for Immunodeficiency, Medical Center – University of Freiburg, Freiburg, Germany, 4: Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA, 5: Department of Pathology, Harvard Medical School, Boston, MA, USA.
Familial hemophagocytic lymphohistiocytosis (FHL) is an immunohematologic disorder caused by hyperactivated T cells and macrophages that lead to severe inflammation and multiorgan damage. The uncontrolled immune activation is a result of impaired lysis of antigen‐presenting cells due to defective cytolytic activity of T cells and NK cells. The disease‐causing mutations in FHL type 3 (FHL3), accounting for one third of FHL cases, are found in the UNC13D locus. Given that current protocols, including allogeneic hematopoietic stem cell (HSC) transplantation, still show high mortality, novel treatment options are needed. Here, we developed a base editing strategy to amend the disease‐underlying mutation in Jinx mice – a preclinical model for FHL3. We generated several adenine (ABE) and cytosine base editors (CBE) to target the cryptic splice donor site in Unc13d intron 26. Screening in specifically generated reporter cells resulted in functional correction of 65–96% of cells. The best‐performing base editors were applied to primary hematopoietic cells. To this end, ABE/CBE‐encoding mRNA and corresponding guide RNAs were transferred to Jinx CD8+ T cells and HSCs by electroporation, resulting in 71% and 61% of edited Unc13d alleles for the CBE, respectively. Functional assays confirmed reestablished cytolytic activity of edited T cells. The Unc13d‐edited HSCs have been transplanted in conditioned Jinx mice to demonstrate functionally relevant restoration of lymphocyte cytotoxicity in the murine FHL3 model. The results of these in vivo experiments as well as the evaluation of off‐target effects, which revealed unexpectedly both CBE‐mediated off‐target mutagenesis and chromosomal translocations, will be presented.
Assessing Stealth and Sensed Base and Prime Editing in Human Hematopoietic Stem/Progenitor Cells
1: San Raffaele Telethon Institute for Gene Therapy (HSR‐TIGET) 2: Vita‐Salute San Raffaele University 3: Institute for Biomedical Technologies, Italian National Research Council 4: Equally contributing author
Base (BEs) and Prime Editors (PEs) enable small‐range edits in human hematopoietic stem/progenitor cells (HSPCs) bypassing the need for DNA double‐strand breaks (DSBs). However, comprehensive characterization of efficiency, tolerability and genotoxicity of these platforms is lacking and required for clinical application. Here, we performed a multiparameter comparison by electroporating late‐generation cytidine (CBE4max) or adenine (ABE8.20‐m) BEs, or Cas9‐nuclease, as mRNAs, and targeting different loci using gRNAs compatible with all of them. ABE8.20‐m outperformed CBE4max and Cas9, reaching ∼90, 30 and 50% editing, respectively. Cas9, but not BEs, reduced clonogenicity compared to mock‐electroporated cells, indicating milder impact of BEs. Accordingly, Cas9‐edited HSPCs displayed the lowest engraftment capacity, despite all treatments maintained multilineage reconstitution and no output skewing, as assessed by clonal tracking. Cas9 editing, however, showed moderate shrinkage of clonal complexity in xenotransplanted mice. Editing in xenografts was <15% for CBE4max‐edited cells, pointing to constrained efficiency in engrafting HSPCs and detrimental response to treatment. Transcriptomic analyses uncovered p53‐activation by CBE4max, albeit to lesser extent than Cas9. Additionally, both BEs, but not Cas9, upregulated interferon‐stimulated genes, suggesting sensing of exogenous long‐mRNAs. BE mRNA engineering enabled reaching >80% editing for all platforms in xenografts, decreased the effective dose and abolished interferon response without aggravating p53‐activation. Remarkably, when applying such mRNA engineering to PE, we reached >50% editing, maintained in xenografts across lineages. However, PE shrank graft size due to upregulation of p53‐related genes, interferon signalling and unfolded protein response. On‐going studies are investigating genome integrity upon BE/PE to build confidence for clinical translation.
Chimeric phagemid/AAV for targeted gene delivery and cancer immunotherapy
1: Imperial College London 2: Chiang Mai University 3: Mahidol University
A fundamental challenge in the broad application of immunotherapies for cancer is uncontrolled modulation of the immune system. Therefore, the ability to target delivery of immunotherapies is central to the development of safe and effective cancer treatment. In this study, we designed and characterised a systemic gene delivery system through transmorphic encapsidation of human recombinant adeno‐associated virus DNA using coat proteins from a tumour‐targeted phagemid, so called transmorphic phagemid/AAV (TPA). We show that our transmorphic particles provide superior delivery of transgenes over the previous advanced phage‐based vector through better diffusion across the extracellular space and improved intracellular trafficking. We used our transmorphic system to target the delivery of cytokine‐encoding transgenes for interleukin‐12 (IL12), newly designed isoforms of interleukin‐15 (IL15) or tumour necrosis factor alpha (TNF‐alpha) via systemic administration in different tumour models. This resulted in particles capable of selective and highly efficient immunotherapy against different types of solid tumours, without harming healthy tissues. Our findings provide a promising modality to deliver safe and effective cancer immunotherapies.
Gamma‐retroviral hotspots integration in human primary keratinocytes: potential implication for clinical application
1: University of Modena e Reggio Emilia 2: University of Padua 3: Holostem Terapie Avanzate s.r.l. 4: IGA Technology Services s.r.l.
Gene Therapy (GT) achieved remarkable results in treating monogenic diseases deemed incurable by conventional medicine. Although successful clinical trials have demonstrated the feasibility of GT for hematological and skin genetic disorders, genotoxicity concerns on the use of gamma‐retroviral vectors (γRV) still exist. In‐depth characterization of γRV integration pattern unveiled insertional mutagenesis events, particularly in hematopoietic stem cells (HSC), highlighting hotspots of integration in proto‐oncogene enhancer regions. Today, ten patients affected by Epidermolysis Bullosa (EB), a congenital skin disease, have been grafted with γRV‐transgenic epidermal sheets, with no signs of insertional mutagenesis. Although integration preferences for open‐chromatin regions have been reported in γRV‐transduced keratinocytes, no hotspots of integration have been yet described. In this work, we integrated two different Integration Site (IS) analysis, obtained through Ligation‐Mediated Polymerase Chain Reaction (LM‐PCR) and Amplification‐free Integration Site sequencing (AFIS‐Seq), to better characterize integration profile across the genome of eight γRV‐transduced human primary keratinocytes, either from normal or EB‐diseased donors. Thus, a highly conserved integration pattern has been identified. Combination of epigenomic and IS data suggests that keratinocytes chromatin landscape is instrumental in directing γRV integration. Furthermore, the use of epigenetic signatures allowed us to label each IS to assess the γRV safeness in keratinocytes. Compared to HSC, keratinocytes present a completely different γRV IS profile, in terms of hotspots and targeted genomic regions, which might explain the absence of genotoxic events in skin‐GT patients. This knowledge, combined with the use of SIN‐γRV vectors, could lead to definitively support γRV in skin‐GT.
Study of the potential of gene therapy approaches for the treatment of lmna‐related congenital muscular dystrophy
C Epifano1 2 D Gómez‐Domínguez1 B Vilaplana‐Marti1 I Hernández1 M Sena‐Esteves3
1: Instituto de Salud Carlos III 2: Fundación Andrés Marcio, niños contra la laminopatía 3: University of Massachusetts
LMNA‐related congenital muscular dystrophy (L‐CMD) is a genetic disease without cure causally associated with point mutations in the LMNA gene. Our main goal is to advance in the knowledge of this disease and to obtain effective therapies to fight against it. Here, we show the results obtained while exploring the potential of two CRISPR‐based strategies. One consists on the use of sgRNAs specific for the most common L‐CMD associate mutation, LMNA c.745C>T. The other implies the homology independent targeted insertion (HITI) of a wild type, exon3‐12‐STOP cassette that could overpass the expression of any LMNA mutation located downstream of exon 3. We have successfully tested these two strategies in mouse embryos and fibroblasts, as well as human myoblasts carrying the LMNA c.745C>T mutation. In addition, for both approaches, AAV‐mediated gene therapy studies have showed significant survival improvement of the early lethality associated with the LmnaR249/R249W mice. These results confirm the therapeutic potential of these two CRISPR‐based strategies for the treatment of L‐CMD.
Identification of barriers to AAV transduction in the heart: focus on receptor availability and intracellular vector processing
1: Jagiellonian University
Efficient transgene delivery is one of the major obstacles in translating AAV‐based therapies from animal models to patients. As demonstrated in clinical trials, this issue seems especially challenging when targeting human cardiac tissue. In this study, our objective was to identify and overcome the main cellular barriers to successful transduction in the heart, using iPSC‐derived cardiomyocytes (iPSC‐CMs), cardiac fibroblasts (iPSC‐CFs), and primary endothelial cells to model vector‐host interactions. We found that inhibition of CK2 protein kinase activity is an efficient and versatile strategy to enhance transduction rate in all analysed cell types. Transient inhibition of CK2 improved the trafficking of AAVs through the cytoplasm and impaired DNA‐damage response through destabilisation of Mre11, sensor of dsDNA breaks, that directly binds the ITRs of the vector genome. Since downregulation of CK2 activity was comparably effective for single‐stranded and self‐complementary vectors, we also examined the stability of AAV genomes after transduction. Despite very low transgene expression in iPSC‐CFs, we found long‐term retention of AAV genomes in these cells in a silent but functional form that allows transgene expression after inhibition of CK2, even 1 week after initial exposure to AAV. Additionally, using spheroid co‐cultures of iPSC‐CMs and iPSC‐CFs, we established that cell‐cell interactions in 3D environment may alter receptor availability for cardiotropic AAV serotypes in iPSC‐CMs. In summary, our data provide a deeper insight into the biology of the vector‐host interaction in cardiac cells and offer a promising strategy to improve the outcome of AAV‐based therapy in the future.
Poster Presentations
Chemically‐engineered recombinant Adeno‐Associated Virus: how chemistry improve rAAV liver gene delivery
1: Nantes Université, CHU de Nantes, TaRGeT ‐ Translational Research in Gene Therapy, INSERM UMR 1089, F‐44200 Nantes, France 2: Nantes Université, CNRS, CEISAM UMR 6230, F‐44000 Nantes, France. 3: INRAE, Oniris, PAnTher, APEX, Nantes, France.
Results from gene therapy clinical trials for hemophilia demonstrate the potential of rAAV to target the liver. However, to achieve therapeutic transgene expression, high vector doses are often required which raises safety concerns.
To address this issue and increase the therapeutic index of rAAV, we developed a strategy of covalent grafting of chemical molecules on the surface of rAAV capsids. In order to target asialoglycoprotein receptors (ASPGR) expressed on the surface of hepatocytes, a family of GalNAc ligands associated with a phenylisothiocyanate coupling function was synthesized and conjugated to AAV2 vectors to generate chemically‐engineered AAV vectors (ChemLiver‐AAV).
After validation of the chemical coupling using a panel of analytical assays, we evaluated the in vivo efficiency of ChemLiver‐AAV for liver cell transduction. Mice were injected with either unmodified AAV2 or ChemLiver‐AAV2 vectors both carrying an eGFP reporter gene expression cassette. One month post injection, ratios between eGFP mRNA levels (RQ) and vector copy numbers (vg/dg, vector genomes per diploid genome) analyzed in liver resulted in a value of 0.37 vs 30 respectively with non‐modified AAV2 and ChemLiver‐AAV. Said differently, one copy of ChemLiver‐AAV vector expressed up to 80 times more eGFP mRNA than one copy of AAV2. Furthermore, ChemLiver‐AAV vectors showed a moderate reduced interactions with pre‐existing capsid neutralizing factors when compared to AAV2 vectors, which, if confirmed, would be of interest in individuals with preexisting humoral anti‐AAV immunity.
Taken together, our findings reveal the great potential of creating a liver specific engineered AAV platform via chemical coupling to improve AAV‐mediated liver gene therapy.
Characterisation of AAVs in NHPs and rodents using the AskBio Capsid screening (ABCs) platform
1: Asklepios BioPharmaceutical (AskBio), Heidelberg, Germany 2: Asklepios BioPharmaceutical (AskBio), Edinburgh, United Kingdom 3: Asklepios BioPharmaceutical (AskBio), Durham, NC 4: Asklepios BioPharmaceutical (AskBio), San Sebastian, Spain 5: Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC
We have implemented a multiplexed AAV capsid variant screening pipeline, in which unique DNA barcodes are linked to specific capsids for detection and quantification of physical and functional transduction in a high throughput strategy. Capsid pooling enables us to accurately assay many different capsids in a single experiment more rapidly than traditional directed evolution approaches and generates a quantitative biodistribution for AAV capsid variants based on association with a given barcode. Such pooling is cost and time efficient, reducing the number of animals required to generate reliable data. We have used the AskBio Capsid screening (ABCs) platform to screen a pool of nearly 50 capsids consisting of ∼two dozen peptide‐display and chimeric AAV candidates, as well as several previously published benchmarks and natural serotypes.
Using the ABCs platform, we have generated data from: i) C57BL/6J; ii) C57BL/6 B6N albino mouse strains, and iii.) one male and one female non‐human primate (Macaca fascicularis). RNA and DNA were collected from select target organs which were analyzed via Illumina and Oxford Nanopore sequencing. Amplification of sequences that contained the unique barcodes allowed us to confirm the predicted and expected biodistribution pattern for the benchmark AAVs. We believe identification of a conserved capsid expression pattern across different species and strains increases the likelihood that capsid tropism will translate to human studies. Additional data related to these experiments will be presented along with complementary data including reporter detection in tissue slices showing AAV expression on a cellular level.
Sustained efficacy and safety up to 3.5 years in adults with glycogen storage disease type Ia (GSDIa): longer‐term results from a phase 1/2 clinical trial of DTX401, an AAV8‐mediated, liver‐directed gene therapy
1: Montreal Children's Hospital, Montreal, Quebec 2: University of Groningen, Groningen, The Netherlands 3: University of Connecticut, Farmington, Connecticut, United States 4: University of Texas McGovern Medical School, Houston, Texas, United States 5: University of Michigan, Ann Arbor, Michigan, United States 6: Hospital Clínico Universitario de Santiago de Compostela, Santiago de Compostela, Spain 7: Ultragenyx Pharmaceutical Inc., Cambridge, Massachusetts, United States
Glycogen storage disease type Ia (GSDIa) results from a deficiency of glucose 6‐phosphatase which is essential for glycogenolysis and gluconeogenesis. DTX401 is an investigational adeno‐associated virus serotype 8 vector expressing the human G6PC gene under transcriptional control of the native promoter.
This global, open‐label, phase 1/2, dose escalation gene therapy trial is evaluating the safety and efficacy of a single DTX401 intravenous infusion in adults with GSDIa. Three patients in Cohort 1 received DTX401 2.0 x 1012 genome copies (GC)/kg, and three patients each in Cohorts 2, 3, and 4 received 6.0 x 1012 GC/kg.
In the 12 patients enrolled in Cohorts 1 through 4, mean (SD [range]) total daily cornstarch intake reduction from baseline to last available timepoint (∼3.5 years for three patients) was 73.8% (23.5 [29‐100%]), p < 0.0001. From Cohort 3 onward, continuous glucose monitoring was implemented. In Cohort 3, the average percentage of time in euglycemia (60‐120 mg/dL) from Baseline to Weeks 77 to 80 increased 14%, despite a reduction in average cornstarch intake of 65% and remained stable (+0.5%) in Cohort 4 from Baseline to Weeks 49 to 52, despite a reduction in average cornstarch intake of 51%. At Week 52 exit interviews, patients reported improved mental clarity, improved glycemic control independent from cornstarch, improved sleep quality, and general quality of life improvements from cornstarch intake reduction. All serious adverse events (Grade 1 or Grade 2: n = 17; Grade 3: n = 2) were determined to be unrelated to study drug by both the investigator and study sponsor; all resolved.
A novel dual AAV approach for intravitreal gene therapy of Stargardt disease
1: ViGeneron GmbH 2: University of Zurich 3: University of Munich (LMU)
Recombinant adeno‐associated virus (AAV) vectors are the leading platform for gene therapy to date. However, large genes require dual AAV vectors for efficient in vivo delivery, reconstitution, and full‐length protein expression. The reconstitution efficiency of dual AAV vectors in the target tissue can be influenced by various factors including the reconstitution mechanism and capsid choice. Here, we present a novel dual AAV vector technology based on reconstitution via mRNA trans‐splicing (REVeRT) in combination with engineered vgAAV capsids, which enable efficient expression of large genes in retinal photoreceptors after intravitreal (IVT) injection.
To test the efficacy of our IVT‐REVeRT approach, we chose to reconstitute the ATP‐binding cassette of subfamily A member 4 (ABCA4) in a mouse model of Stargardt disease. For this, the split AAV genomes, each encoding the 5’ and 3’ parts of human ABCA4, were packaged with vgAAV capsids and co‐injected intravitreally into two‐week‐old Abca4 ‐/‐/Rh8 ‐/‐ mice. Ten weeks post injection, mice were analysed by electroretinography and optical coherence tomography. The observed functional effects match the ABCA4 protein expression detected by Western blotting and immunohistochemistry. Moreover, qRT‐PCR analysis revealed trans‐spliced ABCA4 transcript levels equal to or exceeding the endogenous Abca4 transcript level in wildtype mice.
In summary, this study demonstrates functional replacement of a large gene that exceeds AAV packaging capacity following IVT administration of dual AAV vectors. Successful implementation of this approach in clinical trials is expected to benefit many patients with inherited retinal dystrophies.
Selection of clinical doses for SBT101, an AAV9‐hABCD1 vector for the treatment of adrenomyeloneuropathy
1: SwanBio Therapeutics, Inc., PA 19004, USA 2: The Massachusetts General Hospital; Harvard Medical School, MA 02115, USA 3: IDIBELL‐Hospital Duran I Reynolds, 08908 Barcelona, Spain 4: Institute of Neurosciences, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain 5: Laboratory Genetic Metabolic Diseases, Amsterdam UMC, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands
Adrenomyeloneuropathy (AMN) is an inherited neurodegenerative disease caused by pathogenic variants in the ABCD1 gene which encodes a peroxisomal transporter for very long‐chain fatty acids (VLCFA). Currently, there is no treatment for AMN. SBT101 is an adeno‐associated virus serotype 9 (AAV9)‐based gene therapy candidate intended to deliver a functional copy of the human ABCD1 gene in development as a treatment for patients with AMN. Here we present safety, efficacy, and biodistribution data in rodents and non‐human primates (NHPs) to inform SBT101 dose selection for a planned first‐in‐human Phase 1/2 clinical study. In Abcd1‐/y mice, intrathecal SBT101 dose‐dependently increased human ABCD1 protein levels in spinal cord, and significantly improved mitochondrial DNA levels at a dose of 2.0E10 vector genomes/animal (vg/an) versus untreated controls. In Abcd1‐/y/Abcd2‐/‐ mice, increased grip strength (equivalent to wild‐type) and dose‐dependently reduced VLCFA levels (versus control mice; AAV9‐Null) 7–8 months after receiving SBT101 (3.3E10 and 3.3E11 vg/an). The minimum effective dose in mice was 2.0E10 vg/an; which translates to 7.5E13 vg/person in humans. In NHPs, intrathecal rAAV9‐CBA‐GFP‐WPRE (encoding a reporter gene) administered at 1.1E13 and 3.4E13 vg/an led to reporter gene expression in 25–100% of spinal cord and dorsal root ganglia neurons. One‐time intrathecal SBT101 at doses ≤7.6E13 vg/an in NHPs (≤ 8.8E14 vg/person in humans) was well tolerated, with a high‐dose safety margin of approximately 2.75‐fold. Based on these findings, a first‐in‐human proposed dose range of SBT101 was calculated to be 1.0E14–3.0E14 vg/person.
This study was sponsored by SwanBio Therapeutics.
Gene Replacement Therapy for Spastic Paraplegia 47
1: University of Sheffield
Mutations in any one of the four subunits (ɛ, β4, μ4 and σ4) comprising the AP‐4 complex results in a form of Hereditary Spastic Paraplegia (HSP), often termed AP‐4 deficiency syndrome. This deficit of AP4 function disrupts neuronal intracellular trafficking, resulting in severe intellectual disability and progressive spasticity of the lower limbs of patients. Through a gene therapy approach we aim to replace the mutated subunit and restore some degree of function for these patients, in particular we look at Ap4b1 subunit deficiency, known as SPG47. Our in vitro studies on patient's fibroblast and Ap4b1 deficient neurons show restoration of AP4 complex and ATG9A (a known cargo of the AP‐4 complex, that is upregulated and mislocalised in the trans‐golgi network through AP4 disfunction). We have established a CRISPR‐mediated Ap4b1‐knockout mouse model (Ap4b1‐/‐) presenting the neuropathological hallmarks of AP‐4 deficiency syndrome, this includes corpus callosum thinning, lateral ventricle enlargement, striking mislocalisation of ATG9A, motor co‐ordination deficits, hyperactivity, a hindlimb clasping phenotype associated with neurodegeneration, and an abnormal gait. Cisterna magna delivery of AAV9‐mediated expression of Ap4b1 in SPG47 mice has shown strong evidence for the overexpression of the Ap4b1 in relevant tissues. While behavioural and biochemical assessments for this gene replacement therapy revealed promising results including reducing ventricle enlargement and corpus callosum thinning in SPG47 mice. This study has provided us with a platform for progressing this approach towards clinical development where preclinical safety studies have already shown no adverse effects of the treatment.
Optimization of intrathecal delivery of an infused AAV9 vector for delivery of a gene therapy candidate for adrenomyeloneuropathy in non‐human primates
V Vasireddy1 S W Clark1
1: SwanBio Therapeutics, Inc., PA 19004, USA
Adrenomyeloneuropathy (AMN) is an inherited neurodegenerative disease caused by pathogenic variants in the adenosine triphosphate binding cassette sub‐family D Member 1 (ABCD1) gene, and is characterized by a dying‐back axonopathy affecting spinal cord (SC) tracts, ultimately leading to loss of mobility. We are developing SBT101, an adeno‐associated virus serotype 9 (AAV9)‐based gene therapy encoding a functional human ABCD1 (hABCD1), as a candidate treatment for AMN. Prior research suggests that a 24‐hour intrathecal administration of AAV9 achieved biodistribution to the entire SC/dorsal root ganglia (DRG). We have therefore assessed multiple infusion parameters in non‐human primates using an AAV9‐GFP reporter vector to further understand intrathecal delivery infusion parameters and their effect on biodistribution within the SC/DRG. Animals received either a bolus (20 min) or extended (6‐ or 24‐hour) infusion of AAV9‐GFP at either cervical or lumbar sites at 1.06E13 to 3.38E13 vector genomes per animal (total delivered volume, 2.5–10 mL) followed by a 14‐day survival and immunohistochemical analysis of GFP throughout multiple tissues. The 24‐hour lumbar infusion delivered widespread biodistribution to the entire SC/DRG compared with cervical or bolus delivery. Furthermore, biodistribution within SC/DRG was equivalent with either a 6‐ or 24‐hour lumbar infusion and total volume delivered did not significantly influence biodistribution. These results provide evidence that a 6‐hour intrathecal lumbar infusion of an AAV9 vector can deliver widespread biodistribution to the SC/DRG that is comparable to or potentially greater than a 24‐hour or bolus infusion respectively, at doses predicted to be clinically relevant in patients.
Hardwiring tissue‐specific AAV transduction in mice through engineered receptor expression
1: Department of Microbiology and Immunology, Stanford University School of Medicine 2: Baxter Laboratory for Stem Cell Biology, Stanford University School of Medicine 3: Department of Radiology, Stanford School of Medicine 4: Department of Comparative Medicine, Stanford University School of Medicine
The development of transgenic mouse models that express genes of interest in specific cell types has transformed our understanding of basic biology and disease. However, generating these models is time‐ and resource‐intensive. Here, we describe a new model system, SELective Expression and Controlled Transduction In Vivo (SELECTIV), which enables efficient and specific expression of transgenes by coupling adeno‐associated virus (AAV) vectors with Cre‐inducible overexpression of the multi‐serotype AAV receptor, AAVR. We demonstrate that transgenic AAVR overexpression greatly increases the efficiency of transduction of many diverse cell types including muscle stem cells, which are normally refractory to AAV transduction. We saw a >4‐fold increase in local transduction of MuSCs with local transduction of AAV2, while systemic delivery of AAV9 resulted in approximately 30‐fold increases in transduction of MuSCs. Superior specificity is achieved by combining Cre‐mediated AAVR overexpression with whole‐body knockout of endogenous Aavr (SELECTIV‐KO). Using the SELECTIV‐KO system we were able to demonstrate selective transduction in heart cardiomyocytes and liver hepatocytes with systemic delivery of AAV9 with near complete detargeting of other tissues. We were also able to specifically target cholinergic neurons after systemic delivery of AAV‐PHP.eB with no transduction seen in other neuronal cells or the liver. The enhanced efficacy and exquisite specificity of SELECTIV has broad utility in biomedical research as well as in testing AAV vectors for human gene therapy.
Characterization of engineered AAV capsids in the retina of mice and in two novel human retina ex vivo models
1: Boehringer‐Ingelheim Pharma GmbH & Co. KG 2: University of Iowa 3: Eberhard Karls University Tübingen
The development of Adeno‐associated virus (AAV) capsids enabling efficient gene delivery after intravitreal injection remains a significant challenge for retinal gene therapies. Characterization of novel capsid variants includes the use of human derived models like cell lines, retinal organoids, and human explants. Here, we applied next‐generation‐sequencing (NGS) guided screenings of random AAV peptide libraries in mice. Three rounds of iterative screening recovery of viral genomes from the retina and retinal pigment epithelium were performed. Enriched virus candidates were evaluated as single variants or in a pooled barcoded approach for their transduction efficiency. We identified one candidate (R195‐003) that outperformed the current benchmarks for intravitreal applications AAV2, 7m8 and R100 (4D) with regards to retinal transduction efficacy. Remarkably, R100 did not result in any expression after intravitreal injection to mice at all. In addition, we could show that NGS based quantification of RNA expression from 10 different capsids in the barcode library are directly correlating to data generated by single capsid variant expression levels. Subsequently, the transduction efficacy of R195‐003 was characterized in two newly established human ex vivo systems: I) the stem cell‐based retina on chip model incorporating retinal organoid and retinal pigment epithelium and II) a modified explant model, simulating clinical intravitreal administrations by ex vivo application into the vitreous of intact eyes. Taken together, we identified R195‐003 ‐ a capsid for transduction of the mouse retina after intravitreal injection and for human retina ex vivo systems.
Barcode screening identifies AAV‐KP1 as an AAV capsid that efficiently transduces pancreatic islets with minimal off‐target consequences following retrograde pancreatic duct injection in non‐human primates
K Adachi1 M Horikawa1 C Dorrel1 H R Baggett1 G Dissen2 T Hobbs2 P Kievit2 M Brissova3 A C Powers3 C T Roberts2 M Grompe1
1: Oregon Health & Science University 2: Oregon National Primate Research Center 3: Vanderbilt University
Pancreatic islet cells are an attractive target for AAV vector‐mediated in vivo gene therapy for diabetes and mouse studies have already shown proof of concept. However, for successful clinical translation, a method has yet to be established that can transduce islets safely and effectively in large animal models such as non‐human primates. Here we show that, when administered by retrograde pancreatic duct (PD) injection in rhesus macaques (RMs), AAV‐KP1 can transduce islet cells effectively with minimal vector dissemination to off‐target organs while AAV9 vector transduces acinar cells more preferably and causes substantial vector spillover from the pancreas. In the study, we first established a real‐time image‐guided retrograde PD injection method using RMs. We then performed a next‐generation sequencing‐based screening of 45 different AAV capsids in RMs using an barcoded AAV library, and identified AAV‐KP1 as an AAV capsid that outperforms other AAV capsids and transduces islet cells in RMs when delivered via PD. A subsequent single AAV capsid validation study using CAG‐tdTomato as a payload confirmed that AAV‐KP1 vector transduces the pancreas substantially better than AAV9 with preference toward islets. Importantly, although all AAV capsids could leak into the bloodstream following PD injection, AAV‐KP1 vector concentrations in the blood after PD injection were substantially lower than those of AAV9, leading to a very limited vector dissemination to non‐target organs including the liver. These observations indicate that retrograde PD injection of AAV‐KP1 vector is a promising approach for AAV vector‐mediated in vivo gene delivery to pancreatic islets with minimal off‐target effects.
DART‐AAVs as new gene delivery platform in tumor immunotherapy
1: Paul‐Ehrlich‐Institut 2: Ernst Strüngmann Institute for Neuroscience 3: Frankfurt Cancer Institute, Goethe University
Adeno‐associated virus (AAV) vectors are the leading platform for in vivo gene delivery. As many other vector systems, off‐target activity leads to transduction of therapy‐irrelevant tissues (especially liver) which can cause toxicity and restricts applicability.
To overcome this problem, we present here an advanced platform for DARPin‐targeted AAVs (DART‐AAVs). DART‐AAVs display high‐affinity designed ankyrin repeat proteins (DARPins) on their surface, which mediate precise attachment to the cell type of interest. Compared to the first generation of targeted AAVs, DART‐AAVs exhibit a substantial increase in gene delivery without being compromised in selectivity. Mouse and human T lymphocytes were targeted with CD4‐ and CD8‐specific DARPins. CD4‐AAV and CD8‐AAV transduced specifically CD4‐ and CD8‐positive human and mouse lymphocytes, respectively. Notably, they were not only highly selective but up to 3‐fold increased in their transduction capacity over unmodified AAV2.
Towards tumor cell targeting, we have generated HER2‐targeted AAV2 (HER2‐AAV2) which transduced murine GL261 cells only when they expressed human HER2/neu. In nude mice subcutaneously engrafted with HER2‐positive SKOV‐3 cells, HER2‐AAV2 delivered luciferase solely into tumor tissue completely sparing liver. Its gene delivery activity was at least 3‐fold higher than that of the first generation HER2‐AAV2.
When equipped with the coding sequence for the checkpoint‐inhibitor Nivolumab, transduced tumor cells released functional Nivolumab at high amounts. Experiments are ongoing to show the potential of HER2‐AAV2 as novel therapeutic tool for tumor‐specific delivery of immune checkpoint inhibitors and cytokines into the tumor microenvironment.
Evaluation of mRNA trans‐splicing dual AAV‐vectors for treatment of Usher syndrome 1B
1: Department of Pharmacy – Center for Drug Research, LMU Munich, Munich, 81377 Germany 2: Department of Ophthalmology, Leiden University Medical Center (LUMC), 2333 ZA, Leiden, Netherlands 3: Department of Ophthalmology, University Hospital Zurich, University of Zurich, Zurich, CH‐8952 Schlieren, Switzerland
Usher syndrome (USH) is the most common form of inherited deafblindness. Patients with Usher syndrome 1B (USH1B) carry mutations in the MYO7A gene and display the most severe symptoms. Currently, there is no therapy to halt or slow down retinal degeneration in USH patients. Since the MYO7A coding sequence (6.7 kb) exceeds the packaging capacity of recombinant adeno‐associated‐viral (rAAV) vectors, alternative strategies including dual AAV approaches are required for gene supplementation. Here, we use a novel dual AAV vector strategy based on reconstitution of the split fragments at the mRNA level (REVeRT) to reconstitute MYO7A. Reconstitution was successfully evaluated in vitro in 661W and HEK293 cells co‐transfected with the dual plasmid vectors using qRT‐PCR and sequencing. Dual AAV8 (Y733F) capsids were used for transduction of mouse photoreceptors and human retinal organoids. C57BL/6J mice were subretinally injected at postnatal day 30. Tissues were isolated 4 weeks post injection for subsequent Western blotting and immunohistochemistry experiments. Human retinal organoids were transduced at differentiation day 224 and examined by immunohistochemistry. We achieved 56 % MYO7A expression relative to the endogenous Myo7a in vivo, suggesting a high reconstitution efficiency of our approach. We could also confirm a correct localisation of reconstituted MYO7A in murine photoreceptors and in human retinal organoids. Further experiments on Myo7a‐deficient animals are currently under investigation. Taken together our study provides an important basis for future MYO7A gene supplementation studies in USH1B patients.
Dose‐response evaluation of 9P801, an engineered AAV capsid with high BBB penetration and CNS transduction in non‐human primates
1: Voyager Therapeutics, Cambridge, MA
Engineering of gene therapy vectors has emerged as an exciting strategy to enable clinically relevant expression levels of a therapeutic gene at vector doses low enough to avoid undesirable events. Using TRACER™ RNA‐driven directed evolution of Adeno‐Associated Virus (AAV) capsids, we have recently generated 9P801, an AAV capsid variant with an unprecedented capacity for CNS transduction in adult non‐human primates (NHPs) following intravenous delivery. Here we attempted to determine the minimal dose of 9P801 vector sufficient for near‐physiological expression of a therapeutic payload in the CNS of adult macaques via systemic delivery. A 9P801 vector containing a hemagglutinin‐tagged NHP protein under a ubiquitous promoter was injected to male NHPs at various doses spanning a 30‐fold range for a duration of 28 days. Widespread transgene protein expression was detected in the spinal cord and the brain of high‐ and medium‐dose animals, especially in the putamen, thalamus, globus pallidus and brainstem. Viral DNA and mRNA were readily detectable in all animals and showed a consistent dose response. Strikingly, the lowest dose of 9P801 allowed higher mRNA and protein expression than a 30‐fold higher dose of AAV9. Comparison of transgene mRNA with the matching endogenous transcript indicated that a dose of 2e12VG/kg was sufficient to achieve supraphysiological levels in the CNS, while showing low transduction in the liver and the dorsal root ganglia (DRG). Together, our data suggest that engineered AAV vectors have the potential to achieve a large improvement of their therapeutic index by retaining strong efficacy at low dose.
Intrathecal administration of AAV9‐hABCD1 in a rodent model of adrenomyeloneuropathy delivers biochemical and functional improvement
A Pujol1 A Bosch2 I ME Dijkstra3 S Kemp3 S Fourcade1 C Guilera1 M Leal‐Julia2 A Onieva2 V Vasireddy4 S W Clark4
1: Neurometabolic Diseases Laboratory, IDIBELL‐Hospital Duran I Reynals, 08908 Barcelona, Spain 2: Department of Biochemistry and Molecular Biology and Institute of Neurosciences, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain 3: Department of Clinical Chemistry, Amsterdam UMC location University of Amsterdam, 1105 AZ Amsterdam, The Netherlands 4: SwanBio Therapeutics, Inc., Philadelphia, PA 19104, USA
Adrenomyeloneuropathy (AMN) is an X‐linked inherited neurodegenerative disease caused by pathogenic variants in the ABCD1 gene encoding a peroxisomal membrane protein that transports very long‐chain fatty acids (VLCFAs) into the peroxisome. In the absence of functional ABCD1, affected cells accumulate increased VLCFA levels which can result in oxidative stress at the cellular level and inflammatory demyelination in the spinal cord. There are no therapies available for AMN, leaving patients with progressive neurodegeneration and lifelong disability. We are developing SBT101, an adeno‐associated viral serotype 9 (AAV9)‐based gene therapy delivering a functional copy of the human ABCD1 (hABCD1) gene, as a candidate treatment for patients with AMN. Utilizing the Abcd1/Abcd2 double knock‐out (Abcd1‐/y /Abcd2 ‐/‐; DKO) mouse model of AMN which exhibits key behavioral features of the human disease and increased VLCFA levels, we evaluated the effect of SBT101, as compared to AAV9‐Null vector, following intrathecal delivery at doses up to 3.3E11 vector genomes per animal. Administration of SBT101 in DKO mice demonstrated robust biodistribution, neuronal expression of ABCD1, dose‐dependent improvement in 4‐paw grip strength, and reduction in VLCFA levels and concomitant modulation of inflammatory markers in the lumber spinal cord and dorsal root ganglia, as compared to AAV9‐Null‐treated DKO mice. These data support further preclinical and clinical development of AAV9‐hABCD1 as a potential treatment for patients with AMN.
From AAV virus to AAV vector: characterization of a collection of AAV capsid variants isolated from human liver
1: Genethon, Evry, France 2: Paris‐Saclay University, Evry University, Inserm, Integrare research unit UMR_S951, Evry, France 3: Sorbonne University, INSERM, Institut de Myologie, Centre de Recherche en Myologie, Paris, France 4: Centre de Recherche des Cordeliers, Sorbonne Université, INSERM, University of Paris, Paris, France 5: Functional Genomics of Solid Tumors laboratory, équipe labellisée Ligue Nationale contre le Cancer, Labex OncoImmunology, Paris, France 6: Avicenne Hospital, Paris‐Seine‐Saint‐Denis university hospital, APHP, Bobigny, France 7: European hospital Georges Pompidou, AP‐HP, Paris, France
Adeno associated viral (AAV) vectors represent the leading platform for gene therapy. The high versatility of this tool is mostly due to the large number of available capsid variants, each varying for transduction properties. In 2020, we screened a cohort of 1319 human liver tissues and isolated 59 new capsid variants equally distributed in two distinct subtypes: AAV2 and AAV2‐13 hybrids. AAV vectors derived from these new capsids were characterized in terms of manufacturability, in vitro and in vivo transduction efficiency. The 70% of the capsids passed the first selection criteria based on manufacturing yields and was infectious in cells. AAV capsid production was correlated to mutations in structural and non‐structural proteins highlighting the presence of some indels within the VP sequences, which might destabilize the VPs structure, and mutations in AAP and X gene with a potential impact on vector production yields. In vivo biodistribution in C57BL/6 mice revealed a marked muscles tropism for the majority of the capsids opening the way for a possible muscle‐directed gene therapy application. Interestingly, some capsids were able to reach the central nervous system (CNS) suggesting the capacity to bypass the blood‐brain barrier, a remarkable feature for CNS transduction via systemic administration. The CNS targeting was confirmed by using a PGK‐GFP vector highlighting an efficient transduction of motoneurons and glial cells. This study provides important insights in the structure/function of human wild‐type AAV capsids and support the therapeutic potential of natural AAV capsids for central nervous system and muscle targeting.
A Tale of Two Capsids: TREM2 gene therapy reduces pathological amyloid‐beta in 5xFAD mouse model
1: Eli Lilly 2: Prevail Therapeutics
TREM2 (Triggering receptor expressed on myeloid cells‐2) is a cell surface transmembrane glycoprotein expressed primarily in cells of myeloid lineage, including microglia. TREM2 directly binds to amyloid‐beta, facilitating microglial congregation around plaques, limiting plaque accumulation and promoting phagocytosis. TREM2 mutations associated with increased risk of AD decrease microglial response to amyloid plaques. In mouse models of AD, increasing TREM2 levels enhances microglia activation, promoting their clustering around plaques, amyloid‐beta uptake and plaque compaction. AAV‐hTREM2 is a gene therapy drug candidate designed to reduce deposition and toxicity of amyloid‐beta in AD by increasing TREM2 levels. The hTREM2 vector was packaged in two different capsids, AAV9 and AAV‐PRV, a novel capsid which shows broader CNS biodistribution and increased microglial transduction compared to AAV9. In the 5xFAD mouse model of AD, we evaluated the effects of AAV9‐hTREM2 and AAV‐PRV‐hTREM2, administered by intracerebroventricular (ICV) injection. Post‐treatment, animals were assessed for biodistribution, TREM2 protein levels, and amyloid‐beta levels. hTREM2 dosing in this model resulted in broad biodistribution and dose‐dependent increases in TREM2 protein levels for both capsids, but significant reduction of amyloid‐beta isoforms and aggregates only in mice treated with the AAV‐PRV capsid. This study demonstrates the potential of a TREM2‐based therapy for AD and highlights the critical importance of the optimal capsid for targeting disease‐associated cell types.
Chemical engineering of AAV capsids improves their biodistribution and transduction properties in neural tissues
1: Coave Therapeutics
Adeno‐associated virus (AAV) vector‐mediated gene therapy represents one of the most promising therapeutic avenues to address rare inherited disorders and acquired diseases affecting nervous tissues (brain, eye). Yet, delivering genetic materials to the tissue areas most relevant to the disease remains a challenge. Local routes of administration to the brain or the eye, despite their invasiveness, have proven to be more efficient than systemic approaches. A limitation of these delivery routes, however, is linked to the limited ability of most AAV serotypes to spread within tissues from the point of delivery.
To tackle this issue, we rationally designed and engineered new vectors from existing AAVs of various serotypes, by adding monosaccharides of choice to their capsids through chemical conjugation. The resulting ScoAAVs were screened and selected upon their biodistribution and transduction properties after being locally delivered to rodent and non‐human primate neural tissues. Top‐performing ScoAAV2s are presented herein, that showed superior tissue coverage and transgene expression in neural tissues compared to unconjugated AAVs. In particular, microdosed S01coAAV2 variants delivered to the brain parenchyma managed to provide sustained transgene expression to 50% to 80% of basal ganglia structures, pointing to their enhanced therapeutic potential to treat diseases affecting motor control.
Together, these data indicate that chemical conjugation can effectively transform existing AAVs into more potent vectors, in particular for local use in neural tissues. As they are simple to produce, coAAVs thus represent great and unique additions to the toolbox for gene therapy applications.
Engineering novel AAV capsids for cardiac gene delivery
1: Tenaya Therapeutics
Gene therapy is an emerging treatment option for both acquired and inherited cardiac disorders. While certain known adeno‐associated virus (AAV) serotypes can achieve moderate transduction of the heart, the requirement of high doses and the substantial viral load to the liver or off‐target cell types raise the critical need of novel AAV capsids with improved properties. We have established an in‐house AAV capsid engineering platform and successfully screened over 30 diverse, proprietary AAV libraries (rational design, peptide insertion, variable region, chimeric, scanning, etc.) representing more than one billion of unique capsids in multiple in vitro, in vivo, and in silico models to discover novel AAV capsids that can target the different types of cells in the heart through different routes of administration. Targeting cardiomyocytes following systemic delivery is critical to enable gene therapy treatments for many cardiac conditions and AAV9 has become the workhorse capsid for this type of gene therapy. To identify novel capsids that outperform AAV9, we performed two rounds of directed evolution studies in NHPs with AAV libraries built on multiple parental capsids focusing on selecting novel capsids that efficiently transduce the heart while de‐target from the liver. We further validated 102 candidates in NHPs, mice, and human induced pluripotent stem cell‐derived cardiomyocytes and identified multiple novel AAV capsids with superior properties including improved (up to 7‐fold compared to AAV9) heart‐to‐liver ratio, improved cardiomyocyte transduction, and excellent consistency between different species. Together, these novel AAV capsids may enable more efficacious and safer gene therapies for cardiac disorders.
A Novel Engineered AAV2 Capsid Variant CereAAVTM for Efficient In Vivo Gene Transduction into Mouse and Marmoset Brain
1: Takara Bio Inc.
Recombinant adeno‐associated virus vectors (AAVs) have been widely used as vehicles to deliver gene of interest for research tool as well as for therapeutic purpose. Recently, AAV9 have been commonly used for gene delivery into brain including central nervous system (CNS) and several AAV9 derivatives such as AAV‐PHP.B, have been reported to transduce CNS efficiently. However, there is still no AAV2 variant is reported to transduce CNS across the BBB efficiently. To overcome shortcoming of AAV2 property, we addressed to develop a novel brain‐targeting AAV vectors based on AAV2 serotype. For this purpose, we performed directed evolution using capsid library inserted random 7‐mer peptide and identified a brain targeted AAV2 variant named as CereAAVTM in rodent model. To confirm the transduction capability, we injected the CereAAVTM into mouse and marmoset intravenously and then determined the transgene expression by immunohistochemistry. CereAAVTM showed stronger transgene expression in the whole brain than AAV2 and AAV9 in both mouse and marmoset brain. In addition, CereAAVTM efficiently transduced neurons and microvascular endothelial cells in the mouse brain. On the other hand, in marmoset brain, astrocytes and some neurons were transduced by CereAAVTM systemic administration. Now, we are preparing to confirm the gene transduction capability of CereAAVTM in non‐human primate model, such as cynomolgus macaque and trying to reveal the mechanism of efficient gene transfer in brain. CereAAVTM may have a benefit to use as a transduction tool for research use, but also clinical studies against brain diseases.
Novel AAV capsid variant for efficient and effective inner ear gene therapy
A Rossi1
1: Hannover Medical School 2: Harvard Medical School 3: University of Kansas
Sensorineural hearing loss (SNHL) is a common disorder affecting more than 360 million individuals worldwide. Currently, therapies for hearing loss range from sound amplification using hearing aids to cochlear implantation in severe cases. Advances in genetic diagnostics have demonstrated that over 150 genes are causing non‐syndromic SNHL. The monogenic nature of many forms of SNHL makes it an attractive target for gene therapy treatment. However, the inefficiency of current vector systems is still a challenge in gene therapy. Current research has focused on adeno‐associated virus (AAV) vectors as a promising delivery system, which, however, requires engineering to use its full potential for gene therapy of inner ear disease. The Vandenberghe team, who reported on AAV‐Anc80, already provided proof‐of‐concept for the potency of capsid‐engineered AAV vectors for the inner ear. We report on the development of a potent capsid‐engineered AAV vector that efficiently and consistently transduces a wide variety of cell types within the inner ear in vivo, in all turns of the cochlea, and in vitro in long‐term with great efficiency, clearly outperforming AAV‐Anc80. Based on these findings, we are currently investigating our novel capsid‐engineered AAV in a therapeutic setting in our recently developed TMPRSS3 mutant mouse model, which faithfully reproduces human progressive hearing loss.
Comparative analysis of tropism of a large pool of AAV capsids in cell culture, mice, and non‐human primates
1: ReGenX Biosciences
Hundreds of naturally occurring and engineered adeno‐associated virus (AAV) capsids with a range of characteristics have been described. Novel capsids are evaluated in model systems (e.g. in vitro cell lines, rodents, and non‐human primates) before they can be translated to the clinic; yet, there is limited information on the consistency of model systems in terms of capsid distribution and tropism. Here we present data on a pool of 118 natural and engineered AAVs. We individually produced each vector with a DNA barcode in the packaged genome, pooled all vectors, and then tested this pool in various model systems. Extensive tissue sampling was done in rodents and NHPs, while cell lines of different origins were treated with the vector pool. Extraction of DNA and RNA followed by next generation sequencing of the samples allowed for assessment of the relative activity of each capsid. For well described serotypes we were able to replicate previously reported results. For example, AAV2 robustly transduced cultured cells, AAV4 was heavily enriched in rodent lung, and AAV8 successfully transduced NHP liver. Some engineered capsids, such as our previously described AAV9 variant BC029, showed improved properties in both rodent and NHP. However, we found a lack of correspondence between rodent and NHP biodistribution for many of the capsids in the pool. Furthermore, in vitro data was similar across the cell lines tested regardless of their origins. The data presented here support screening AAV capsids in multiple systems to evaluate translatability and provide rationale for development towards the clinic.
Identification of a cell surface receptor utilized by an engineered BBB‐penetrant capsid family with enhanced brain tropism in non‐human primates and mice
1: Voyager Therapeutics, Cambridge, MA
The blood‐brain barrier (BBB) represents a significant limitation to AAV‐mediated gene therapies for CNS indications. Natural AAVs typically cross the BBB with low efficiency, requiring high systemic doses or invasive direct injection into the CNS for therapeutic efficacy. Thus, considerable efforts have been undertaken to engineer capsids with enhanced BBB‐crossing properties. These efforts have recently yielded several AAV capsid variants with enhanced CNS tropism in non‐human primates (NHP). The mechanisms utilized by these engineered capsids to cross the BBB are still unknown. We have previously reported the generation of an engineered capsid class exhibiting 50‐fold increased BBB penetrance in both macaques and mouse. Here we report the identification of a surface receptor specifically bound by this engineered capsid class. Ectopic overexpression of the human isoform of this receptor in cultured cells led to a significant increase in capsid binding and transduction while no difference was observed with the parental capsid, AAV9. Direct capsid‐receptor interaction was confirmed by additional biochemical analyses, which strongly supports a role as a primary attachment receptor. Immunostaining and single‐cell RNA‐seq experiments performed in mouse brain suggest that the receptor expression pattern and capsid tropism are correlated, further supporting a role in the transport of these cross‐species capsids across the BBB. Our data provide mechanistic insights into the enhanced brain transduction by these novel BBB‐penetrant capsids and raise exciting possibilities for the prediction of capsid behavior in humans based on receptor cross‐species conservation and expression pattern.
Tyrosine chemically modified AAV capsids to achieve ever more efficient retinal transduction
1: Université de Nantes 2: CHU de Nantes
Inherited retinal dystrophies (IRDs) affect more than 2 million people worldwide. Gene therapy (GT) for IRDs using recombinant Adeno‐Associated Virus (rAAV), is to date the preponderant approach. However, the safety and efficiency of GT for IRDs requires improvement, as doses high enough to provoke inflammation are often necessary to achieve efficient transgene expression. Modifications of the rAAV capsid might overcome this issue by improving its efficacy in transducing retinal cells. Here, we describe chemically‐modified rAAV2 or rAAV8 capsids. The bioconjugation onto the rAAV capsid is achieved using synthetic molecules composed of mannose (Man), and a reactive function specific to tyrosine residues (Y‐Man.rAAV). After validation through analytical assays, these vectors expressing EGFP were tested in mice. Funduscopy and flatmounts of retinas from mice subretinally injected with Y‐Man.rAAV2‐EGFP show stronger fluorescence compared to un‐modified rAAV2 in all retinal layers, demonstrating the high efficacy and penetrance of this chemically modified capsid. A second group of mice, this time injected intravitreally using Y‐Man.rAAV2 and Y‐Man.rAAV8 capsid vectors showed EGFP expression comparable to unmodified vectors. However, modified rAAV2 capsids was overall more effective intravitreally compared to modified rAAV8 vectors. The data demonstrates the potential of chemical modifications to keep improving rAAV vectors for better and safer gene therapies.
Can machine learning predict which AAV capsids are viable after 7mer insertion?
1: Institut de la Vision 2: Sorbonne Université 3: Kyoto University 4: Inserm 5: CNRS
Directed evolution (DE) has been broadly used to improve adeno‐associated viral vectors (AAV) for gene therapy. Using this technique, AAVs have been bioengineered to efficiently target specific tissues. DE for AAVs starts from a highly‐diverse library with millions of different mutants which is screened by applying a selection pressure over multiple rounds, eventually converging towards a few ‘evolved’ variants. Clearly, if the initial viral library is not diverse enough AAV variants with advantageous properties might be missed. One of the most utilized protocols for producing a high diversity capsid library consists in inserting a random 7mer at amino acid position 588 of the AAV2 capsid. Although this approach generates many different variants with interesting properties, some of the modified genomes do not code for viable capsids, resulting in a decrease of the initial viral library diversity, and therefore of the DE throughput. As a consequence, understanding whether a certain insertion is viable or detrimental can improve the overall efficiency of future DE screens. Starting from massive datasets previously collected, we trained different machine learning models capable of predicting which sequences are viable and which are not. We focused on both supervised and unsupervised methods and benchmarked them against each other and against previously proposed approaches. Our results show promise to improve DE of AAV by providing initial conditions which are more likely to lead to capsids with desiderable properties.
Engineering CNS‐targeted AAV capsids from massively diverse libraries using machine learning
1: Shape Therapeutics
Targeted biodistribution of AAV remains a critical challenge for human gene therapies. The AAVidTM platform solves multiple limitations of other capsid engineering approaches with a) massive diversity libraries that enable unprecedented coverage of the AAV sequence landscape; b) direct‐to‐non‐human‐primate (NHP) screening to accelerate clinical translation; and c) profiling of library biodistribution in >50 NHP tissues to power computational extraction of sequence features that determine target tissue tropism.
We successfully applied the AAVid platform to engineer novel, tissue‐tropic AAV5 variants. A combinatorial variant library disrupting the AAV5 natural ligand binding pocket and containing >1 billion unique variants was administered to 4 NHPs by intravenous injection. After 4 weeks of in vivo biological selection, NGS recovered >30 million unique AAVid capsid sequences from >50 distinct tissues. Most individual variants were found in only one tissue; however, the deep cross‐tissue datasets enable computational clustering and machine‐learning analyses that discriminate rare tropism patterns. To validate the ML‐guided discovery approach, we generated a validation library of AAVid variants predicted to have increased CNS, muscle or liver tropism. This validation library was designed to measure DNA and RNA and included wild type AAV controls for direct quantitative comparison of functional transduction. Our results show that ML model predictions significantly correlated with observed AAV tropism for CNS, muscle and liver. Notably, top CNS candidates showed up to 1000‐fold CNS enrichment compared to wild type capsids while de‐targeting the liver. These results highlight the power of the AAVid platform to identify tissue‐tropic AAVs for many clinically‐relevant tissue types.
Whole genome siRNA and microRNA high throughput screenings to shed light on cellular determinants of the successful AAV vector transduction
1: ICGEB 2: King's College London
The efficacy of adeno‐associated vectors (AAVs) in transducing many different targets in vivo and in vitro is widely recognized, still incomplete information is available on the molecular determinants of AAV tissue permissivity.
To systematically identify the host cell factors involved in virus fate after infection, we performed a high‐throughput screening using a genome‐wide siRNA library (Mano, 2015) identifying components of the double‐stranded DNA break repair as regulators of rAAV genome processing.
More recently, we performed a high‐throughput screening using a genome‐wide library of human microRNA mimics (988 mature sequences, miRBase 13 ‐ Dharmacon) in AAV2‐Luciferase transduced HeLa cells. Using this approach, 51 microRNA mimics were shown to increase AAV transduction by more than 4‐fold (up to 23‐fold change) while 26 microRNA mimics significantly decreased AAV transduction. Hsa‐miR‐329 and hsa‐miR‐362‐3p, which share the same seed‐sequence, were identified as the most effective microRNAs at increasing AAV transduction in HeLa cells. The extent of this effect is, however, dependent on the cell line tested, underlining the complexity of the vector‐cell interaction.
Transcriptomic analysis on total RNA extracted from HeLa cells transfected with 3 top hit microRNAs identified several hundred transcripts significantly downregulated. The direct comparison of these results with those obtained from the screening of siRNAs for AAV2‐mediated transduction, highlighted the involvement of proteins that may interfere with virus endocytosis or nuclear import. Work in progress, aimed at determining the biological relevance of these findings, will open new perspectives for the exploitation of RNAi/miRNA strategies to improve AAV vectors and in vivo transduction.
Should I be satisfied by my directed evolution experiment? A machine learning approach
1: Kyoto University 2: Institut de la Vision
Directed evolution (DE) is a versatile protein‐engineering strategy to engineer and optimise proteins like enzymes, antibodies, or viral vectors for gene therapy. Starting from a library containing billions of random variants, DE screens them against a chosen task over multiple rounds until they converge to a few evolved variants. Today, DE benefits from the tremendous progress in DNA sequencing techniques (deep sequencing) allowing us to read millions of distinct DNA sequences at each round of the DE screening iterations. Nevertheless, deep sequencing data are noisy and to understand if the selection process has yet converged into reliable results is often difficult. As a consequence, experimentalists are compelled to increase the number of costly and time‐consuming DE rounds or even to replicate the experiments. In this work we propose a method to increase the amount of information inferred from deep sequencing data of DE experiments. In particular we provide a method to: (i) reliably estimate the selectivity of individual sequences and its statistical error using the data from all available rounds; (ii) identify which variants among the best ones are worth keeping for further in‐detail analyses; (iii) quantify if enough DE rounds are performed to understand when to end the experiment. To showcase our method, we re‐analyse public datasets ranging from DE of viral vectors to phage‐display experiments. Lastly, we benchmark our method against previously proposed approaches in the literature.
Evaluation of tissue specific promoters for liver or muscle gene transfer
1: ReGenX Biosciences
Development of potent tissue specific promoters is essential for the advancement of systemic AAV gene therapy. Here, we utilized next generation sequencing (NGS) to comprehensively compare a selection of promoters in liver or muscle.
Fourteen liver promoters were assessed for transcriptional activity in primary human hepatocytes, mice, minipigs, and non‐human primates (NHP) via a barcoded AAV library. An engineered liver promoter, ELP1, was consistently a top performer in all animal models tested, indicating its potential for driving liver expression regardless of species. Other liver promoters were variable in ranking, such as a variant of the G6PC promoter, which ranked first in NHPs, but nearly last in mouse. Universally, the promoters examined had significantly less activity in NHP versus mouse liver, suggesting transcription from AAV genomes in NHP liver is impaired for all tested promoters. Rank order of promoters in primary human hepatocytes differed substantially from animal models but showed a similar magnitude of suppressed activity as NHP liver.
Expanding the toolbox of available muscle promoters with unique properties is desirable for muscle‐directed gene therapy. Through bioinformatics guided design and NGS, we discovered a novel skeletal muscle‐specific promoter, Mus022, that only drives expression in mouse skeletal muscle but not heart. Another muscle promoter, EMP1, displayed 5 to 10‐fold more activity in mouse skeletal muscle, heart, and diaphragm when compared to CK7, despite being around half its length.
In conclusion, we have identified several novel liver or muscle specific promoters that are potentially useful for improving the efficacy of systemic AAV gene therapies.
Establishing a directed evolution approach to generate novel tissue‐specific AAV variants
1: Bayer AG, Research and Development, Gene Therapy 2: Asklepios Biopharmaceutial, Inc
Adeno‐associated viruses (AAVs) have become a leading tool in the field of gene therapy in the past few years. Their low immunogenicity, broad tropism, and ability to infect both dividing as well as quiescent cells makes them ideal delivery vehicles. Given the general need for suitable AAV variants for the in vivo manipulation of various tissues to treat cell‐type specific disorders, this project aims to establish a directed evolution approach to discover novel tissue‐specific variants. We generated AAV peptide display and shuffled barcoded libraries which we screened in vivo in mice. The libraries were generated to contain features that allow us to identify functional variants by expressing in each variant a barcoded mRNA, which linked the transgene to the capsid sequence. Following the first in‐vivo library injection and RNA‐seq analysis our results show enrichment of certain AAV variants in different tissue types. Our results demonstrate that our chosen workflow holds the potential to engineer novel tissue‐specific AAV variants following further rounds of selection and validation in vivo.
A two‐step bioconjugation methodology based on click chemistry for the modification of the adeno‐associated virus capsid
1: Nantes Université, CHU de Nantes, TaRGeT ‐ Translational Research in Gene Therapy, INSERM UMR 1089, F‐44200 Nantes, France 2: Nantes Université, CNRS, CEISAM UMR 6230, F‐44000 Nantes, France
Adeno‐associated viruses (AAV) are currently extensively investigated as vectors for gene therapy. Two limitations still remain concerning this type of vectors which are: an important tropism resulting in difficult targeting of the cells of interest, and loss of efficacy considering pre‐existing neutralizing antibodies. In the purpose of improving the AAV's efficacy, our group has developed expertise in direct chemical bioconjugation of their capsid. Modifications of lysine or tyrosine with isothiocyanate or diazonium salt anchors respectively were developed to allow covalent linkage of ligands with targeting properties. In particular, AAV functionalisation with carbohydrates such as N‐acetyl galactosamine and mannose resulted in considerably improved gene delivery to the targeted cells in both in vitro and in vivo transduction studies.
Despite promising results, it is now of particular relevance to be able to introduce easily and efficiently a broad range of functions (small molecules, peptides, biomolecules …) to exploit AAV‐conjugates as vectors in a broad spectrum of pathologies. In this matter, this study consists in the evaluation and characterization of a two‐step bioconjugation methodology of AAV capsid, based on strain‐promoted azide‐alkyne cycloaddition (SPAAC). Tyrosine and lysine targeting molecules bearing anchors such as azides and strained octynes were synthesized and their bioconjugation on AAV capsid is being evaluated, as well as the second step ligation of synthetic biorthogonal ligands (fluorescein, biotin, carbohydrate…). With this methodology, ready‐to‐use AAVs were developed that can be easily and quickly modified.
In vivo evaluation of novel synthetic promoters for CNS gene therapy
1: UCL 2: AskBio (Europe) 3: University of the Witwatersrand
Gene therapy for previously untreatable neurological diseases is a clinical reality but key challenges in control of level and location of gene expression, and improving delivery and distribution remain. There is need to develop customised vectors with next generation promoters to address the diversity in neurological diseases. Using Synpromics‐AskBio's bioinformatics‐based promoter design platform, novel synthetic CNS promoter candidates were generated and evaluated in vivo.
Ten novel promoters and three controls driving GFP expression were evaluated in rAAV9 vectors. Neonatal CD1 mice received titre‐matched vector by intracerebroventricular or intravenous delivery and were euthanised at 5 weeks. CNS distribution and cell‐type GFP expression were characterised by immunohistochemistry and immunofluorescence. Systemic organ expression was evaluated by qPCR. Human synapsin 1 (hSyn) promoter was used as a neuronal selective control. All novel constructs were active in the CNS with rostrocaudal gradient. We identified an intriguing candidate promoter with superior activity in midbrain dopaminergic neurons following intravenous delivery. We evaluated therapeutic efficacy of the novel candidate by treating the dopamine transporter knockout (DAT‐KO) mouse model of Dopamine transporter deficiency syndrome infantile parkinsonism‐dystonia. DAT‐KO neonates received intravenous gene therapy with GFP‐injected littermates as controls (n = 10/group). Gene therapy showed significant differences in anxiety and motor coordination tests. Immunofluorescence showed low human DAT expression in both dopaminergic neurons and non‐dopaminergic expression that may account for partial amelioration of DAT‐KO phenotype. This gene therapy study presents data from the initial development of novel CNS promoters that transduce the midbrain non‐invasively.
In silico immunogenicity assessment of FLT201: an investigational gene therapy for Gaucher disease Type 1 encoding an engineered variant of the GCase enzyme
1: Freeline Therapeutics
In Gaucher disease Type 1 (GD1), mutations in the GBA1 gene attenuate or abrogate the activity of the lysosomal enzyme glucocerebrosidase (GCase). FLT201, a clinical‐stage investigational gene therapy for GD1, consists of an engineered AAV capsid (AAVS3) containing an expression cassette encoding a novel engineered GCase variant (GCase‐var85) protein. GCase‐var85 contains two novel amino acid substitutions, which result in a 6‐fold increase in half‐life in human serum and a 20‐fold increase in GCase half‐life at lysosomal pH conditions, but similar catalytic parameters (KM) to those of wild‐type human GCase (GCase‐WT). In vitro and in vivo studies have shown that GBA1‐var85 produces robust expression of human GCase in the liver, leading to metabolic cross‐correction of target organs and substrate clearance in tissues including liver, spleen, bone marrow and lung. Here we report the results of an in silico evaluation to predict the immunogenicity of the GCase‐var85 relative to GCase‐WT. In silico binding affinity calculations were performed for the peptide sequences derived from both proteins and human major histocompatibility complex (MHC) class I and II allotypes. The calculations were performed using the computational tools available in the Immune Epitope Database and Analysis Resource. Affinity cut‐off values were then used to categorize the peptides as “strong” or “intermediate” binders and the number of binders was then compared between the two proteins. This immunogenicity assessment showed that GCase‐var85 generally had fewer binders than GCase‐WT. In conclusion, based on the in silico assessment, GCase‐var85 and GCase‐WT have comparable immunogenicity profiles.
Analytical comparison of AAV manufactured by triple‐plasmid transfection/HEK293 and double‐baculovirus infection/SF9 processes
M Ng1 S Nelson1 S Kakkar1 A Arnaout1 T Fenn1 G Danniels1 J Zhou1 P Raut1 C Daniels1 L Higgins1 S Kamalakaran1 Y Dai1 M L Hayashi1
1: Prevail Therapeutics
Triple‐plasmid transfection/HEK293 adherent and double‐baculovirus infection/Sf9 suspension are two widely used GMP AAV manufacturing processes. However, direct comparison between the two platforms, using clinical manufacturing process with the same vector sequence, has not been well studied. We recently completed a comparability exercise in which these two manufacturing processes were evaluated based on product quality attributes, including release testing, characterization testing, and product stability. Both processes yield viral capsid proteins with identical mass and presented similar post‐translational modifications. Furthermore, final products from both processes showed comparable analytics regarding safety, identity, achievable strength, biological activity, stress stability and AAV purity. In addition, differences in the impurity profiles, which are inherent to the processes, were observed. Overall, the baculovirus/Sf9 process presented significantly fewer AAV partial viral particles and contained less total DNA residual impurities compared to triple‐plasmid transfection/HEK293 process. The main contributor to the residual DNA in the triple‐plasmid transfection/HEK293 process was plasmid DNA. In contrast, no detectable infectious baculovirus or particle associated Sf9‐Rhabdovirus RNA were present in the Sf9 produced drug substance. In conclusion, the double‐baculovirus infection/Sf9 process was able to upscale AAV production and deliver a comparable AAV product to the AAV material manufactured from the triple‐plasmid transfection/HEK293 adherent process.
The Ambr® 15 bioreactor system is a suitable down‐scale model for rAAV suspension cell manufacturing platform optimisation
R Derler1 S Werner1 K Breunig1 S Lange1 S Reichl1 M Ohme1 A Youssef1 M Hörer1 F Sonntag1
1: Freeline Therapeutics
Recombinant adeno‐associated virus (rAAV) is among the most widely used viral vectors for gene therapy. While different large‐scale adherent production systems, such as the iCELLis® 500, are extensively used, there are several reasons to adapt rAAV production processes to suspension. Suspension processes allow flexibility regarding scale per batch (scale‐up instead of scale‐out) resulting in lower operational costs. Furthermore, suspension processes eliminate the need for serum and the associated negative effects. Scalability of the process at comparable quality and potency must be demonstrated throughout development.
Previously, we presented the development of a 96‐well, plate‐based, high‐throughput system for the optimisation of our suspension cell‐based manufacturing platform. The possibility of parallel processing of many setups enables the use of this system for process screening, plasmid development, and program candidate selection. However, this system cannot mimic the features and process control provided by larger scale bioreactor systems. Therefore, we established an Ambr® 15 automated bioreactor system for parallel cultivations at a microbioreactor scale as a down‐scale model for rAAV production. Using design of experiment (DoE) methodology, we identified the process parameters that allow down‐scaling of rAAV manufacturing with comparable yield and quality relative to larger scale bioreactor platforms such as Ambr® 250 and 50L single‐use bioreactors.
This research demonstrates that the Ambr® 15 bioreactor system is applicable as a down‐scale model for larger bioreactor systems, enabling process optimisation and platform evolution (e.g. plasmid design) to close the gap from miniature to production scale bioreactors for suspension cell‐based rAAV manufacturing.
SVec: A superior platform for next generation gene therapies targeting genetic disorders and autoimmune diseases
1: Amarna Therapeutics
To date, adeno‐associated virus (AAV) vectors are the most popular for use in in vivo gene therapy. However, clinical studies using AAV vectors revealed that administration of vector particles elicits innate and adaptive immune responses against the viral and transgene proteins in the vast majority of treated patients, leading to decreasing expression levels of the therapeutic transgenes over time and elimination of the transduced cells from the body, compromising re‐administration of patients with AAV vectors. For these reasons, an alternative non‐immunogenic vector is highly desired.
Replication‐defective simian virus 40 (SV40) vectors could be an attractive alternative to AAV vectors. The non‐immunogenicity in humans and capacity to induce immune tolerance to transgene proteins render SV40 vectors highly attractive for use in gene replacement and immunotherapies.
Amarna's SV40‐based SVec vectors are safe by design and non‐immunogenic in humans, inducing long‐term transgene expression. The lack of immunogenicity and capacity to induce immune tolerance opens the possibility of repeated administration with SVec vectors delivering the same or different transgenes. Amarna's SVec platform, therefore, fulfils the requirements of a superior platform for the development of safe and effective gene replacement therapies for genetic disorders such as hemophilia A & B and primary hyperoxaluria type 1. Besides genetic disorders, its non‐immunogenicity and capacity to induce immune tolerance to the transgene proteins in humans render SVec vectors ideally suited for the development of effective tolerization therapies to treat autoimmune diseases such as neurodegenerative and psychiatric diseases, atherosclerotic cardiovascular disease, diabetes mellitus, arthritis, and chronic obstructive pulmonary disease.
Selectivity Analysis for Identifying the Best Performing Variants in Directed Evolution Screens
1: Institut de la Vision
Directed evolution of adeno‐associated viral vectors (AAV) has been part of the gene therapy toolkit for almost two decades. By putting selective pressure on diverse libraries of AAV variants it is possible to select variants capable of performing specific desirable tasks, such as infecting all retinal layers through intravitreal injections or transducing endothelial cells in the brain's vasculature after intravenous injections. Variants such as AAV2.7m8 and AAV‐BR1 have been selected as some of the most abundant sequences after several rounds of selection as part of in vivo directed evolution. However, recent data suggests that the most abundant sequences might not be the most performant ones. With next generation sequencing (NGS) it is possible to identify and assess the behaviors of less frequent variants. We developed a selectivity analysis method whereby the increase in a variant's presence in successive rounds of selection can pinpoint its ability to perform a given task, even if its overall frequency is low in comparison to other variants. After six rounds of selection in human retinal explants and subsequent NGS sequencing, 11 variants of interest were identified through selectivity analysis. The infectivity of these variants as well as their transduction capacity was compared in vitro. Here we show that selectivity analysis can pinpoint less frequent variants with superior transduction efficiency compared to natural serotypes and engineered variants previously identified through directed evolution including the most frequent variant (variant 1) identified in our human retina screen.
Development of novel AAV variants for improved transduction of retinal microglia
1: Department of Ophthalmology, University Hospital, LMU Munich, 80336 Munich, Germany
Recombinant adeno‐associated virus (AAV) vectors have been widely used in gene therapy applications for central nervous system (CNS) diseases with favourable preclinical and clinical results. Indeed, these vectors can successfully transduce post‐mitotic cells, e.g, neurons and astrocytes. Given the emerging central role of microglia, the major resident immune cells in the CNS, in the pathobiology of neurodegenerative disorders, these cells are increasingly becoming the focus of the development of new treatment approaches. However, microglia are generally refractory to transduction by viral and non‐viral vectors. Here, we introduce a novel microglia culture protocol from mouse retina, which allows for good reproducibility, high cell numbers and long in vitro viability. We used this model as a screening platform to evaluate CMV‐mediated GFP transgene expression of different modified capsids based on AAV6, the AAV serotype with the highest reported efficiency in microglia transduction in vivo. In addition, we screened the vectors on retinal macroglia cells. The engineered variants carry single‐point mutations of surface‐exposed tyrosine, lysine, threonine, serine residues, and/or arginine residues, or the peptide insertions of AAV2.GL and AAV2.NN, two previously described AAV2‐based peptide insertions variants with enhanced transduction of retinal cells. Our results indicate that the two novel variants AAV6.GL and AAV6.NN show an increased transduction efficiency of primary mouse retinal microglia (2 to 4‐fold) compared to AAV6 wild type or the recently published triple mutant AAV6 TM6. Overall, our study establishes a new in vitro platform for assessing microglial transduction and identifying novel AAV capsids with improved microglial transduction properties.
Production of AAV vectors using synthetic, enzymatically produced linear DNA
C Winckler1 W Thompson1 Á Picher1 H Lanckriet1
1: 4BaseBio
Adeno‐associated virus (AAV) remains a popular vector for gene therapy. However, AAV manufacture faces several challenges to keep pace with the demand for large scale, high quality batches. The standard plasmid triple‐transfection presents several issues including availability and cost of GMP grade plasmid, sequence fidelity of the inverted terminal repeats (ITR), and safety concerns over non‐specific encapsidations. 4basebio has developed a proprietary, scalable synthesis process for the production of linear closed DNA constructs via its Trueprime™ amplification technology. The hpDNA™ produced is devoid of any bacterial backbone and circumvents cumbersome fermentation processes required for plasmid DNA. The process is size and sequence independent, allowing for large scale production of linear DNA with high yield and purity in less than a week. Here, we compared the production of AAV vectors using hpDNA™ encoding for the typical Adenovirus helper functions, rep and cap genes, and an expression cassette consisting of AAV2 ITRs and a reporter gene driven by an ubiquitous promoter. Conventional plasmid triple‐transfection was used as a control. We achieved equivalency in viral genome titres, full:empty ratios and infectivity between the two production methods. We have demonstrated that functional AAV vectors can be produced using hpDNA™, which could greatly accelerate therapeutic development of gene therapy programmes. The technology could overcome the difficulties associated with complex ITR structures required for AAV production, which are inherently difficult to synthesise via bacterial propagation systems. Moreover, the lack of plasmid backbone sequences such as antibiotic resistance genes enhances the safety profile of the AAV product.
Analytical and Biophysical Characterization of a rAAV9 vector for in‐vivo gene therapy to treat Dilated Cardiomyopathy
1: Renovacor Inc. 2: BioAnalysis LLC
We are developing a rAAV gene therapy for the treatment of BAG3‐associated dilated cardiomyopathy (DCM) caused by mutations in BCL2‐associated athanogene 3 (BAG3) gene, which lead to reduced levels of cardiac BAG3 protein and the onset of cardiac dysfunction in patients. As part of CMC development, in addition to the usual release methods for strength, purity, identity, safety and quality, we conducted additional in‐depth characterizations of the rAAV9 vector using LC‐MS, AUC, SEC‐MALS and DLS technologies.
LC/MS was used to determine intact mass as well as post‐translational modification and potential degradation via peptide mapping. Post translational modifications (PTMs) such as phosphorylation, lysine acetylation, methylation and N‐terminal acetylation of VP proteins were characterized. Additional PTMs such as asparagine and glutamine deamidation and methionine oxidation were also characterized.
The percent full capsid is widely recognized as a critical quality attribute and was determined by sedimentation velocity analytical ultracentrifugation (SV‐AUC). These data were also compared to the percent full capsid determined by the ratio of vector titer to capsid titer to understand trends. The hydrodynamic radius of the vector was determined by Dynamic Light Scattering (DLS) to understand monodispersity of the vector. Additional characterization with SEC‐MALS was conducted to further characterize the vector properties.
While rAAV gene therapy for genetic diseases continues to be a rapidly growing precision medicine approach, fundamental analytical and biophysical characterization is becoming increasingly common in early CMC development to gain improved product and process understanding and for analytical comparability studies to support process changes in the future.
AAV9 biodistribution and expression in the PNS of Non‐Human Primates
1: Neuroscience Department, The Cyprus Institute of Neurology and Genetics, Nicosia, 2371, Cyprus 2: Neuroscience Department and Center for Neuromuscular Disorders, The Cyprus Institute of Neurology and Genetics, Nicosia, 2371, Cyprus 3: Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas Texas, 75390, USA 4: School of Medicine, National Primate Research Center, Tulane Univeristy, Covington LA, 70112, USA
While biodistribution and expression of AAV vectors has been extensively studied in the CNS of NHPs, data regarding AAV biodistribution in the PNS and especially to myelinating Schwann cells, remain limited. Our aim is to examine the efficacy of AAV9 and its variant to transduce Schwann cells of PNS tissues of NHPs following intrathecal injection.
In order to study the efficacy to target Schwann cells in the PNS of NHPs, we delivered by lumbar intrathecal injection the AAV9 vector and the AAV9 variant vector C204. All vectors carried the plasmid AAV‐CBh‐EGFP. PNS tissues were collected 8 weeks post‐injection for DNA extraction and vector biodistribution analysis, as well as for evaluation of EGFP expression. For PNS expression, we studied the anterior lumbar roots, proximal and distal parts of sciatic nerve, femoral and ulnar nerves. VGCNs were determined in these tissues. A non‐injected control was used in all evaluations.
Biodistribution analysis showed relatively low efficacy of the vectors to transduce PNS tissues. EGFP was detectable mostly in PNS axons, but was also present in myelinating Schwann cells in all groups examined. Although both vectors resulted in Schwann cell expression, EGFP was detected at higher percentages in NHPs injected with the AAV9 compared its variant.
This study provides evidence of successful biodistribution into the PNS of NHPs including the distal nerves and reporter gene expression in myelinating Schwann cells of intrathecally injected AAV9 and one of its variants. Further studies using optimal vector doses and Schwann cell specific expression plasmids are warranted.
Comparative study of AAV vector serotypes 1, 2, 5, 6, 8 and 9 after intracardiac gene delivery
1: University of Eastern Finland, Kuopio 2: Kuopio University Hospital
Cardiovascular diseases (CVD) are the leading cause of death and remain a global health problem with a huge economic burden worldwide. Although treatment options have improved over the last years, up to 30 percent of the patients are not suitable for current treatments due to the diffuse chronic disease or comorbidities. Therefore, a clear need for improved or novel therapies to treat CVD is existing. Cardiac gene therapy provides a promising tool and Adeno‐associated viruses (AAV) are widely used vectors since they do not induce any human disease and lead to long‐term transgene expression. So far, several AAV serotypes have been identified out of which AAV‐1, 6, 8 and 9 were shown to be cardiotropic in preclinical studies. However, the data on transduction efficiency after cardiac gene transfer are inconsistent.
In this study, the transduction efficiency after ultrasound guided intramyocardial administration of 6 different AAV‐serotypes (AAV‐1,2,5,6,8 and 9) was compared 30 days after gene delivery in C57Bl/6J mice. In addition, cardiac performance was assessed by echocardiography before myocardial gene transfer and 30 days afterwards.
We observed that all AAV serotypes after intramyocardial gene transfer led to transgene expression in cardiac tissue with varying transduction efficiencies. In addition, we detected variable effects on cardiac performance 30 days after gene transfer with different AAV serotypes. Together these data can be used to determine the best suitable AAV serotype for cardiac gene delivery.
AAV‐specific nanobodies as tools to enhance cell specificity
1: University Medical Center Hamburg‐Eppendorf
Nanobodies, single variable immunoglobulin domains derived from camelid antibodies, are small, highly soluble, and easy to reformat into fusion proteins. We have shown that a membrane protein‐specific nanobody displayed on the capsid of AAV vectors can dramatically enhance their transduction of specific target cells. Here we show that AAV‐transduction can also be enhanced using soluble, bispecific nanobody‐dimers. We selected AAV‐specific nanobodies from immunized llama IgH‐transgenic mice using classic hybridoma and direct cloning technologies. The nanobody‐coding region was cloned into a eukaryotic expression vector upstream of hinge, CH2 and CH3 domain of rabbit IgG. Specific binding of the resulting recombinant rabbit IgG heavy chain antibodies to AAV1, 2, 6, 8 and 9 was verified by immunofluorescence microscopy. To generate bispecific adaptor proteins, we genetically fused a membrane protein‐specific nanobody via a glycine‐serine linker to an AAV‐specific nanobody and produced the dimers as secretory proteins in HEK cells. These recombinant adaptors specifically enhance AAV transduction of target cells expressing the respective membrane protein on the cell surface. Such nanobody‐based adaptors are valuable tools for improving AAV transduction in the lab and, if validated in vivo, possibly also in the clinic.
IsoTag™AAV – An Innovative, Scalable, and Non‐Chromatographic Method for Streamlined AAV Manufacturing
1: Isolere Bio, Inc.
Demand for gene therapies capable of treating previously inaccessible targets has risen precipitously in the past decade. Adeno‐associated viruses (AAVs) are a preferred vector for delivery because of their more favorable safety profile and tissue tropism but they have significant manufacturing challenges with end‐to‐end yields sometimes as low as 10%. IsoTag™AAV was developed to combat these low yields and improve the scalability of the affinity capture step. IsoTag™AAV combines affinity and size separation in one unit operation by leveraging liquid‐liquid phase separation combined with a standard tangential flow filtration (TFF) process which replaces the standard of TFF concentration followed by affinity chromatography that exists today. IsoTag™AAV improves upon traditional affinity yields and product quality with minimal optimization. A head‐to‐head comparison of IsoTag™AAV purification and traditional affinity purification was performed at the 1L scale. This comparison showed yield improvements up to 20% unoptimized and comparable purity in half the time. Process cost modeling showed that IsoTag™AAV can reduce cost of goods for a commercial AAV program by as much as 50%.
Large therapeutic gene delivery with dual‐AAV vectors: enhancing intein protein trans‐splicing systems
1: IBET‐Instituto de Biologia Experimental e Tecnológica 2: ITQB NOVA‐ Instituto de Tecnologia Química e Biológica António Xavier
Adeno‐associated viral (AAV) vectors represent one of the leading platforms for gene delivery. Nevertheless, their small packaging capacity presents a major limitation restricting its use for diseases requiring large‐gene delivery. To overcome this, dual‐AAV vector systems relying on protein trans‐splicing were explored, being the split‐intein Npu DnaE (Nostoc Punctiforme) system the most used. However, it presents low reconstitution efficiencies requiring the administration of higher vector doses. This work seeks to overcome inefficient reconstitution rates by applying novel split‐inteins with unmatched trans‐splicing rates.
Studies were conducted between Npu DnaE and split‐inteins: engineered‐consensus (Cfa) and cyanophage‐like Gp41‐1. AAV expression cassettes were developed encoding a frGFP terminal half fused to a split‐intein. Full‐length frGFP constructs comprising the remaining intein a.a. (Scar‐frGFP) after split‐intein reaction were developed. Protein reconstitution was evaluated by transient transfection and dual‐AAV co‐infection.
Results demonstrated a 2‐fold decrease in fluorescence intensities with Scar‐frGFP constructs revealing that intein residues impact protein function. Cfa and GP41‐1 inteins presented superior (2‐fold) reconstitution rates than Npu DnaE and almost 100% frGFP reconstitution. Initial dual‐AAV2 co‐infections were performed to determine optimal transduction conditions by testing different vector doses (1x104‐5x104 V.G./cell). Results showed 40% decrease in fluorescence intensity compared to single infections, suggesting vector competition. Cfa and GP41‐1 split‐inteins dual‐AAV co‐infections resulted in 10% more frGFP reconstituted positive cells than Npu DnaE.
This work showed improved protein reconstitution efficiencies, revealing Cfa and Gp41‐1 as superior split‐inteins for dual‐AAV delivery. Further work optimizing trans‐splicing and transduction is ongoing aiming to reduce vector dose use in therapeutic administrations.
rAAV capsid enrichment: Chromatography and iodixanol gradient ultracentrifugation improve product quality across multiple serotypes
1: OXGENE
Quality of adeno‐associated viral vectors (AAV), especially proportion of full capsids, has been increasingly more important in development of gene therapies. Downstream processing of AAV plays a critical role in determining quality of the final product and can become a major bottleneck in gene therapy development. We contrast downstream processing of several AAV serotypes using conventional iodixanol density gradient ultracentrifugation (IDGUC) and chromatographic methods being developed for the primary capture and AAV capsid enrichment.
We developed IDGUC through the optimisation of buffering and osmolality conditions to increase recovery. To counter‐act IDGUC limited scalability and to improve overall processing capacity, we explored a pre‐treatment using a PEG‐salt precipitation step directly from crude material. An alternative method is the primary capture using AAV affinity chromatography and then capsid enrichment using ion‐exchange chromatography.
We applied rational approach to optimisation of several AAV serotypes to account for different interactions of each serotype with the chromatography media. This has been achieved using variant salt and buffer conditions as well as both gradient and stepped elution, to maximise recovery and peak separation and to produce a scalable purification platform. We established capsid enrichment process based on anion exchange chromatography for AAV2 resulting in significant peak separation, over 70% step recovery and 3‐fold increase in proportion of full capsid.
Enhancing efficacy and specificity of rAAV5 via small, intermediate and large peptide insertions
1: uniQure biopharma B.V.
Rational capsid design is a potential method for enhancing the targeting profile of AAVs. rAAV5 has several advantages relative to other AAV serotypes including its relative safety and reduced immunogenicity which allow it to be dosed even in the presence of neutralizing antibodies. One limitation of rAAV5 is its limited tropism which restricts it to delivering cargoes to the liver when administered IV or to the brain with intraparenchymal administration. Here we present several effective approaches for enhancing the transduction of target tissues and for re‐directing rAAV5's tropism towards cells that are usually not transduced. We demonstrate that rAAV5 tolerates the insertion of peptides of varying sizes into surface‐exposed loops in a way that affects neither capsid assembly nor genome packaging. Engineered capsids remain infectious across different cells lines and display altered transduction profiles compared to wild‐type AAV5. Remarkably, insertion of high‐density lipoprotein (HDL) binding peptides (6‐30 amino acids) into surface‐exposed loops influenced the binding of the AAV5 capsid to liver cells by exploiting the HDL particle as a carrier to improve liver biodistribution. Similarly, introducing larger peptides (∼160 amino acids) with epitope binding properties (such as single‐chain antibodies) into the capsid surface enabled targeting of cell types that express the target epitope. When presented with mixed populations of cells, rAAV5 displaying the epitope binding peptide specifically targeted the cells expressing the cognate epitopes. Taken together, these approaches allow for the creation of rAAV5 with custom targeting profiles that significantly extend the range of applications of rAAV5 in gene therapy.
Development of a human‐based platform to assess adeno‐associated viral vector genotoxicity
1: Brunel University London 2: Testavec 3: Genewerk 4: University Heidelberg 5: The University of Edinburgh 6: University College London 7: National Institute for Biological Standards and Control 8: University of the Witswatersrand 9: NCT and DKFZ 10: Imperial College London 11: Sydney Children's Hospitals Network and Children's Medical Research Institute
Gene therapy is an effective treatment for genetic disorders by delivery of therapeutic genes to tissues, such as the liver. Gene transfer can use viral vectors such as adeno‐associated virus, where, once delivered to the nucleus can remain episomally or integrated into the host DNA. Recently, adeno‐associated viruses have shown to been found associated with hepatocellular carcinoma in mice, which is likely due to vector effects on gene(s) local to the site of integration. Models to determine potential gene therapy vector genotoxicity have been developed, however, these often use animals, cells of murine origin or immortal cells that may be oversensitive in predicting human genotoxic risk. To circumvent this, we developed a human based model using induced pluripotent stem cells and their hepatocyte‐like cell derivatives. Cells were profiled for markers of pluripotency and terminal differentiation, respectively, then transcriptomically demonstrating hepatocyte‐like cell expression aligning with primary human hepatocytes. Cells were infected with adeno‐associated vectors using the strong Chicken β‐actin (CB7) or weak Apo‐lipoprotein (ApoE) promoters to drive GFP expression and investigated for integration by EPTS/LM‐PCR that showed twice as many insertions in cancer genes by the ApoE vector than the CB7 vector. RNASeq profiling of infected cells also identified cancer gene fusion transcripts between adeno‐associated virus and host cells and analysis of methylation changes in the infected cells showed differences in global methylation profiles by each vector. We present this model as useful to identify and measure the genotoxic potential of these vectors on the host.
Improving the efficiency of AAV gene delivery vectors via capsid design and bioprocess engineering
1: UCL 2: Pall Corporation
Various AAV capsid serotypes have been identified which differ in their tropism, which makes AAV a highly useful system for transduction of a wide range of tissue and cell types. Despite the ability of the current available capsid serotypes to transduce a variety of target tissues, there are several limitations, including a lack of tissue‐specificities that are not within the coverage of currently available serotypes, low transduction efficiencies and inherent difficulty in manufacturing some serotypes at scale with sufficiently high titres. Improving AAV vector efficiency, tropism and titre is critical to meeting the growing clinical and commercial requirements of AAV‐mediated gene therapies for more challenging disease indications. However, it is necessary to adopt an interdisciplinary approach, combining biological expertise in vector capsid design and bioprocess engineering to deliver functional, efficient, and scalable vectors. The focus of this project is to design and manufacture a clinically relevant AAV vector with a novel capsid design resulting in enhanced tropism and improved gene transfer efficiency at commercially relevant scales. The project adopts a dual, complementary approach: (1) Design and manufacture a clinically relevant AAV vector with a novel capsid design via the capsid DNA family shuffling directed evolution approach and (2) Design and development of improved bioprocess control and medium feeding strategies supporting production of the selected novel vector.
Novel Adeno‐Associated Virus (AAV) Capsid Variants for Improved Liver‐Directed Gene Therapy
1: Hannover Medical School 2: Center for Molecular Medicine Cologne 3: University of Sydney 4: University of Leipzig Medical School 5: Center for Scalable Data Analytics and Artificial Intelligence ScaDS.AI 6: Institut national de la santé et la recherche médicale 7: Twincore Centre for Experimental and Clinical Infection Research 8: German Red Cross Blood Donor Service 9: University Hospital of Cologne 10: University Hospital Frankfurt 11: University Hospital Bonn 12: Goethe University Frankfurt 13: Ghent University 14: Laboratory of Molecular Oncology and Innovative Therapies
Adeno‐associated virus (AAV)‐based liver‐directed gene therapies still require novel next‐generation vectors with improved efficacy. In this regard, capsid engineering is one of the most promising strategies for developing AAV vectors that meet efficiency thresholds for therapeutic impact. Here, we report on two novel capsid variants, MLIV.K and MLIV.A, both derived from a high‐throughput in vivo AAV2 peptide display selection screen in mice. The variants prove to transduce primary murine and human hepatocytes at comparable efficiencies. Concurrently, MLIV.K and MLIV.A show significantly improved liver transduction efficacy outperforming parental AAV2 in curing haemophilia B mice (up to 400% normal FIX) and in targeting human hepatocytes in humanized FRG mice (6 to 7‐fold more cDNA reads compared to AAV2). Furthermore, the MLIV variants are signifcantly superior to AAV8 in human hepatocyte transduction. Remarkably, these capsid variants with reduced HSPG‐binding affinity tailor more efficient expression of their delivered genomic payload as cDNA to DNA ratios are significantly improved in human and mouse hepatocytes (9‐fold > AAV2). In conclusion, MLIV.K and MLIV.A are the first AAV peptide display variants with improved across‐species hepatocyte tropism and transduction efficiency. Therefore, the translatability of liver‐directed gene therapy approaches mediated by our MLIV variants bears strong potential.
High‐throughput AAV screening platform on retinal organoids to target cell types for gene therapy
1: Institute of Molecular and Clinical Ophthalmology Basel
Recombinant adeno‐associated viral vectors (rAAVs) are the most widely used tool for therapeutic gene delivery to treat retinal diseases. The ability to drive high transgene expression in the disease critical cell types is the most important feature when selecting AAV promoter variants for clinical applications. The current individual testing of AAV promoter variants on post‐mortem human retina explants provides accurate results, but the limited availability and significant labour restrict the number of variants that can be compared. Human retinal organoids closely resemble most of the cell types of the human retina and can be generated in large amounts. Based on this more available model system, we developed a semi‐automated workflow to pre‐screen AAV promoter variants for the most therapeutically relevant cell types in a high throughput system. This proof‐of‐concept study shows the generation of 96 AAV plasmids harbouring different promoter variants in parallel and their subsequent AAV production in small scale to infect retinal organoids in quadruplicates. Finally, AAV promoter variants were evaluated by fluorescent imaging and ranked for further evaluation on human retina explants by the mean density of GFP expressing cells per organoid. The present study confirmed promoters already reported for cell type specific expression in human retina and demonstrates the feasibility of retinal organoids as a high‐throughput screening platform for AAV variants.
Isolation of novel porcine AAV serotypes for retinal transduction
1: Department of Translational Medicine, University of Naples Federico II, Naples, Italy 2: Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli (NA), Italy 3: Institute for Veterinary Medical Research, Hungária krt. 21, H‐1143 Budapest, Hungary 4: Department of Advanced Biomedicine, Federico II University, 80131 Naples, Italy
Due to their excellent efficacy and safety profile, vectors derived from the adeno‐associated virus (AAV) are the leading platform for retinal gene therapy. PCR‐based isolation of AAVs from biological samples represents an important source of new AAV variants with unique biological features. In our study, we aim at isolating from pig tissues novel AAV variants with potential improved retinal transduction capabilities compared to existing ones. To this aim, we screened by PCR porcine blood (n = 244) and liver (n = 274) samples for the presence of AAVs, using primers complementary to conserved and flanking hypervariable regions of the CAP gene. We found that about 23% of blood and 28% of liver samples contained AAV sequences. Aminoacidic sequence alignment revealed five novel AAV variants from blood which have 85‐97% homology with the closest porcine AAV5 (AAVpo5) capsid sequence. One of them, AAVpoaa1, transduced pigs retinal pigmented epithelium and photoreceptors to levels comparable to the gold‐standard AAV8 upon subretinal injection. The isolation, cloning and characterization of additional variants of pig origins is ongoing.
Development of refined helper plasmid: one step closer to the next generation rAAV vectors
1: Polyplus Transfection
Harnessing rAAVs as viral vectors
Here we present our state‐of‐the art approach to designing
The use of these tailor designed DNA plasmids with an optimized scalable transfection protocol allows to
Targeting glioblastoma with nanobody‐displaying AAV9
1: University Medical Center Hamburg Eppendorf 2: Department of Experimental Medicine, University of Genova, Italy 3: Sirion GmbH, Martinsried, Germany
Nanobodies, single variable immunoglobulin domains derived from camelid heavy‐chain antibodies, are small, highly soluble, and easy to reformat into fusion proteins. We developed a technology to insert a membrane protein‐specific nanobody into the VP1 capsid protein of AAV vectors that enhances their transduction of target cells expressing the respective membrane protein. We now report application of this technology to specifically transduce glioblastoma cells that express the GPI‐anchored ecto‐enzyme CD73. Gliobastoma is the most common and deadliest type of primary brain tumor and highly resistant to therapy. CD73 is often overexpressed on mouse and human glioblastoma cells and is negatively correlated with patient survival. We equipped AAV9 as vector of choice for the CNS with a CD73‐specific nanobody by genetic fusion of the nanobody into the GH2/GH3 loop of VP1. AAV9 displaying the CD73‐specific nanobody strongly enhanced the transduction of mouse and human glioblastoma cells in vitro.
Managing Process‐related Residual Testing Requirements in Complex Viral Gene Therapeutics
1: Merck
Impurities can have critical undesirable impact on the safety of viral gene therapies and effective removal of process‐related residuals is an important step in the development of such sophisticated and complex therapeutics. It is key to gain knowledge of any process‐related residuals early in your development activities to ensure a clear understanding of your process, all while maintaining an acceptable product yield, to avoid future pitfalls. Examples of impurities critical to consider may include transfection reagents, contaminants from the host cell DNA or proteins, other process‐derived impurities (such as BSA, DNAse, detergents), among others. Further complexity arises due to the diversity of chemical structures and behaviours of these molecules. and given their presence is typically at trace levels in potentially challenging matrices making detection and quantitation challenging.
This poster will present data utilising a range of methods, utilising an expansive toolbox of methodologies to evaluate a number of these process‐related residuals.
Aura+: High Throughput, Low volume Product Stability and Purity Analysis for Gene and Cell Therapies
A Ross1
1: Halo Labs Ltd
Aura+ is the latest instrument designed specifically to detect, count, and characterize subvisible aggregates and extrinsic materials for product quality measurements in both gene and cell therapy applications. Aura+ outputs images, count, size, particle identification and morphological information with 100% sampling efficiency requiring as little as five microliters of sample volume through Backgrounded Membrane Imaging and Fluorescence Membrane Microscopy (FMM). FMM identifies cellular, protein, viral aggregates or extrinsic in a high throughput format for rapid analysis of your sample. Specifically, Aura+ has the capability to detect SYBR® Gold stain, which is used to detect the presence of DNA in AAV aggregation to understand the role of leaky capsids in subvisible particle formation often leading to reduced AAV transduction efficiency and adverse patient responses.
miRNA coexpression during vector production to increase vector yield and transduction efficiency
1: AskBio
We performed a literature search to identify published improvements to AAV production systems which could further increase vector yield and transduction efficiency. Based on this search we decided to test coexpression of miR‐431 and miR‐636 during vector production which was previously reported to increase yields during vector production by transient transfection (PubMed ID 32108448). We will present data describing our observations of the effects of these construct designs on vector yield and transduction efficiency.
Evaluation of an Early, Late, Very Late Expressed Rep in a Recombinant Baculovirus to Produce a More Potent AAV‐based Gene Therapeutic in Insect Cells
1: Voyager Therapeutics
The baculovirus expression vector (BEV) insect cell platform is an economical way to produce AAV‐based human therapeutics at scale for large patient populations. The common method to produce AAV is to co‐infect insect cells with a Rep/Cap and ITR‐transgene BEVs. In this regime Rep and Cap genes are synchronously expressed from very late baculovirus polh and p10 promoters respectively. We have investigated asynchronously expressing Rep earlier than Cap to initiate ITR‐transgene replication prior to the production of capsid proteins. This was achieved by expressing Rep from a synthetic early, late, very late (ELVL) baculovirus promoter based combining the OpMNPV gp64 promoter with the AcMNPV polh promoter. When this ELVL‐Rep design was compared it to the common polh‐Rep design in BEV infected insect cells we observed comparable AAV titers and productivity. However, the AAV capsids generated by ELVL‐Rep were more potent than the AAV capsids generated by the common polh‐Rep design.
Development and production of recombinant Adeno Associated Virus (rAAV) at 1 to 1,000L scale using a Platform process
F Agbogbo1 S Wesel1 G Krishnamoorthy1 A Craddick1 A Davis1
1: Forge Biologics
Recombinant Adeno Associated Virus (rAAV) is a leading platform for the treatment of genetic diseases. The demand for rAAV for clinical trials requires the development of a robust and scalable process that yields high purity vectors at high titers. Transient transfection of HEK293 in suspension culture is a standard approach used at Forge Biologics to produce rAAV vectors. In the HEK293/transient transfection manufacturing process, the final production culture is taken through cell lysis which results in cellular debris, including cell‐derived nucleic acids, proteins, and lipids. These process contaminants are removed during downstream purification unit operations to yield a highly pure concentration of rAAV vector (the active ingredient) in a defined final formulation buffer. Forge Biologics has developed a platform process to produce rAAV and this has helped in decreasing time and effort required in process development and allows projects to move into cGMP production more rapidly. In this talk, data will be presented on work that has been executed at small‐scale (1L), mid‐scale (50L), and large‐scale (500L and 1,000L) for a variety of rAAV serotypes. The overview of the process; seed train, cell growth and transfection in the bioreactor, lysis, clarification, chromatography, and final formulation will be shared. In addition, the analytical characterization data for rAAV vectors produced including GOI titer by ddPCR, residual plasmid DNA by ddPCR, residual host cell DNA, residual host cell protein, purity by SDS‐PAGE, genome integrity by alkaline gel electrophoresis, aggregation by DLS, empty:full particle ratio, potency, and endotoxin will be presented.
Advancing AAV: Novel Sequencing Solutions for Quality Control in Gene Therapy
1: Azenta Life Sciences
Interest in cell and gene therapy‐based disease prevention and treatment has grown rapidly over the last decade, however there are still hurdles to overcome in the field. Recombinant Adeno‐Associated Viruses (rAAV) have become the vector of choice for virus‐mediated gene therapy due to their non‐replicating nature, high‐titers, low immunogenicity, and genotoxicity. Extensive quality control (QC) throughout the entire development and manufacturing process is essential. A robust QC process expedites safe and effective commercialization of the final product.
Next‐generation sequencing (NGS) offers an effective high‐throughput approach for monitoring AAV quality, from construct assembly to analysis of the encapsulated product. Both Illumina® short‐read and PacBio® long‐read technologies offer distinct advantages including sequencing of the entire AVV genome and inverted terminal repeat (ITR) regions, with detection of potential mutations, truncations, and contaminants. Both platforms require conversion of the single‐stranded genome to double‐stranded DNA prior to library preparation; however, high‐fidelity protocols for this step are lacking. Additionally, efficient bioinformatics pipelines are needed to facilitate interpretation from massive NGS data amounts.
Here we describe a novel proprietary combined NGS solutions that alleviates high throughput AAV sequencing constraints and enhances the QC process. The latter combined with our GLP Sanger sequencing method enable sequence confirmation of both transfer plasmid and final packaged product for improved AAV manufacturing in advance of regulatory filings.
Characterization of extracellular vesicles that incorporate adeno‐associated viruses
1: Oxford University
Adeno‐associated viruses (AAVs) are one of the leading gene therapy vectors for clinical applications. However AAV gene therapy suffers from several limitations: targeting and immune response. AAV serotypes are limited by their inherent tropisms to specific tissues, and presence of neutralizing antibodies (NAbs) in a patient's bloodstream can prevent efficient biodistribution of AAVs and make repeated treatment challenging. A small proportion of the AAVs incorporate into extracellular vesicles (EVs) to evade immune detection. These EV‐AAVs have several advantages including protection from NAbs, and ability to co‐package additional molecules for targeting or immunomodulation. However EV‐AAV candidacy for clinical therapy is limited by low AAV incorporation into vesicles. EVs are lipid nanoparticles produced by cells that can be subdivided into three groups based on origin: exosomes, microvesicles and apoptotic bodies. It is the goal of this study to characterize which EVs naturally incorporate AAVs, to inform future engineering strategies in increasing EV‐AAV yield. Due to marked differences in secretion to the extracellular media, serotypes AAV2, 8 and 9 were evaluated, and incorporation of AAVs into vesicles was found to be serotype independent. Based on titration and differential ultracentrifugation larger vesicles (20k pellet) were found to associate/incorporate higher amount of AAVs when compared to smaller vesicles (100k pellet). Proteomic analysis of vesicles collected from AAV producer cells compared to non‐producer cells revealed upregulation of CHMP1A and B, MBLAC2, and SUMO‐2, among other proteins. Future investigation of upregulated targets will reveal possible mechanisms that can be exploited for active loading strategies, improving EV‐AAV yield.
Optimising the genetic modification of macrophages using a lentiviral vector for application in cellular therapies
1: Imperial College London
We are developing a platform technology utilising ex vivo modified macrophages as protein secretion factories for respiratory disorders. We have already demonstrated in vivo proof‐of‐concept using murine bone marrow derived macrophages (BMDMs) and are now optimising this platform. In vitro, our proprietary F and HN (rSIV.F/HN) pseudotyped lentiviral vector has comparable transduction efficiencies to a VSV‐G pseudotype (F/HN: 10.7 ± 1.9%, VSV‐G: 12.9 ± 5.6%, MOI ∼50). We found addition of the cationic polymer Polybrene (4 μg/ml) enhanced transduction efficiency (median % GFP positive cells with Polybrene: 46.3% [range 42.8‐49.1%]; virus only: 34.5% [range 31.1‐38.3%]). However, “spinfection” (transduction using centrifugation) did not increase transduction efficiencies when spun for 30 or 120 minutes at 1,000 rcf. The strength of ubiquitous promoter CMV‐ and EF1α‐driven transgene expression was compared to our proprietary chimeric hCEF promoter consisting of a CMV enhancer and EF1α promoter. In vitro, following an initial high burst of expression from CMV (day 3), CMV‐ and hCEF‐driven expression of Gaussia luciferase was comparable on day 7 (median: 58800 [range 53229‐79702] RLU and 37538 [range 29189‐52383] RLU, respectively) whereas EF1α was significantly lower (median: 5442 [range 3920‐7043] RLU). Finally, we found transducing bone marrow precursor monocytes versus mature BMDMs resulted in similar transduction efficiencies across a range of MOIs (1‐50) without affecting differentiation, but reduced vector requirements by a third owing to proliferation during differentiation. Macrophages are often refractory to gene modification and here we have identified promising ways to improve macrophage transduction and transgene expression for application in our system and general macrophage studies.
LentiVEX: Development of lentiviral vector producer cell lines
1: Oxford Genetics Ltd 2: WuXi Advanced Therapies
Lentiviral vectors (LVV) are of increasing interest in the field of cell and gene therapy, but high costs and variable vector productions continue to limit patient access. The industry standard LVV production procedure requires four‐plasmid transfection, which contributes to high cost and titre variability. We sought to address this by developing LVV producer cell lines that yield LVV titres as high as the four‐plasmid method, but at reduced cost and process variability as they do not require plasmid transfection during production. We developed producer cell lines by transposon‐facilitated integration of lentiviral genome into our suspension packaging cell lines. These encode TetR‐regulated, codon optimised lentiviral packaging components (VSV‐G, GagPol and Rev) with regions of DNA homology deleted to decrease risk of inter‐chromosomal recombination. Transposon‐based integration was chosen to increase lentiviral genome copy number and transcriptional activity compared to our previous developments, for which random integration was used. Initially, we developed cell lines that produce GFP‐encoding LVV. Later, we trialled production of LVV encoding several different therapeutically relevant genes‐of‐interest. Further research work is ongoing to achieve regulated expression of GOI in our producer cell lines to maximise titre. GFP‐encoding LVV was produced at 1‐2E+08 TU/mL (infectious titre measured by flow cytometry). This titre was consistently achieved during producer cell stability tests over 30 passages. In summary, our producer cell lines produce LVV at titres comparable to the industry standard four‐plasmid method. This is likely to reduce costs and titre variability, and thus improve patient access to LVV‐based cell and gene therapies.
Comparison of CRISPR‐assisted knock‐in and prime editing to integrate landing pad sequences of the Bxb1 integrase for precise delivery of large genetic cargo
1: BIH‐Center for Regenerative Therapies (BCRT), Berlin Institute of Health at Charité – Universitätsmedizin Berlin, Germany 2: Max Planck Institute for Molecular Genetics, Berlin, Germany 3: Berlin Center for Advanced Therapies (BeCAT), Charité – Universitätsmedizin Berlin, Germany 4: Institute of Transfusion Medicine Charité
Integration of large DNA sequences at a specific location is currently a challenge in virus‐free genetic engineering. Programmable nucleases such as CRISPR‐Cas can be designed to precisely insert a desired sequence via homology‐directed repair (HDR), but they have limited efficacy when integrating sequences larger than 4 kb. Integrases and transposases overcome this cargo limitation and facilitate transfer of large transgenes greater than 20 kb, but they lack the programmability of CRISPR nucleases. To overcome this current limitation of gene transfer technologies, we seek to combine site‐specific gene editing of the CRISPR‐Cas system and take advantage of the large cargo capacity of serine integrases. In a first step, we compared the efficacy of different CRISPR‐based knock‐in strategies to install the landing pad (attB site) of the Bxb1 integrase at the ACTB and TRAC loci in HEK293 cells. The efficacy of CRISPR‐ assisted HDR and microhomology‐based end joining (MMEJ) was compared to prime editing approach in the desired loci. Knock‐in efficacies of all approaches were analyzed using digital droplet PCR (ddPCR). The presence of the landing pad was also examined using conventional PCR and gel electrophoresis. Our first findings indicate the presence of a BXB1 landing pad in TRAC locus. Methods involving HDR and MMEJ show a better knock‐in rate as compare to prime editing. Additionally, a single band can be observed in conventional PCR, demonstrating a site‐specific integration of our desired sequence.
linQURETM platform: Development of novel scaffolds for multi‐transcript targeting towards the treatment of complex genetic disorders
1: uniQure biopharma B.V.
Given the vast aetiological array of genetic disorders that could be addressed with AAV‐based gene therapy, sustained innovation of its two main constituents – the capsid and the encapsulated cargo – is of utmost importance. Through adaptation of the delivered cassette – or cargo – it is possible to regulate the expression profile of genes involved in the pathogenesis of various genetic diseases. By targeting the toxic transcripts of defective genes using our miRNA‐based miQURE® technology, progression and severity of those genetic diseases can be reduced. Importantly, we further improved our gene silencing strategy by concatenating several miQURE® molecules in a single construct, resulting in our novel linQURE™ platform. This approach could both allow for the augmented targeting of a unique transcript and also pave the way for a multi‐targeting approach, enabling our gene therapy products to adapt to more complex multigenic indications. Here we show the range of targeting strategies with our new linQURE™ technology which includes single‐transcript, multi‐transcript and sense‐antisense transcript targeting. We deliver the proof‐of‐mechanism that our optimised linQURE™ technology enables the expression of multiple synthetic miRNAs, leading to efficient downregulation of their disease‐causing targets.
Efficient and targeted transduction of hepatocytes using Oxford Biomedica's lentiviral vector system
1: Oxford BioMedica
A principal goal of liver‐directed gene therapy is the specific expression of the transgene in the target cell. Transgene expression in non‐target cells has been proposed to reduce the therapeutic efficacy. Lentiviral vectors (LV) are well suited for gene replacement strategies because these can carry therapeutic cargoes of significant size and integration can result in life‐long expression of the therapeutic transgene.
We have demonstrated that Oxford Biomedica's (OXB) HIV‐based LentiVector® can consistently transduce the liver in different mouse models. Here we demonstrate that our LentiVector® platform efficiently transduces both human hepatic cell lines and primary hepatocytes in vitro. Restricted transgene expression in hepatocytes is achieved by using a liver‐specific promoter. Pseudotyping OXB's LV with gp64 reduced transduction of non‐hepatocytes. Incorporating the target sequence for the hematopoietic miR 142.3p (miR 142.3pT) reduced expression of transgenes in haematopoietic transduced cells. Incorporating OXB's technologies reduced the presence of the recombinant transgene in the final vector further reducing the possibilities of raising an undesired immune response to the transgene product. Lastly, we demonstrate species‐specific differences in LV transduction of primary hepatocytes. The combination of these technologies will generate a hepatocyte‐targeted LV for gene therapy and together with the ability to produce high quality, large quantities of clinical grade vector provides a platform from which to target genetic liver disorders.
These studies underpin the successful use of LV in vivo for the life‐long correction of disorders from a single administration in which hepatocytes are the targeted cell type.
INTRONPLUS‐ PKR knock‐out of viral producer cell line enables stable lentiviral vector integrity
M L Meijueiro1 I Bravo1 A Arrasate1 C Lopez‐Robles1 S Baños‐Mateos1 M J Fertin1 C Albo1
1: VIVEbiotech S.L.
Lentiviral vectors (LVs) are generally used as gene transfer tools for gene and cell therapy (GCT). LV production is a challenging process, which includes complex steps encompassing from minacious transgene design to efficient purification and aliquoting of the final product. The former is occasionally an arduous task because the standard LV backbone contains HIV sequences that include multiple splicing donor (SD) and acceptor sites (AS). Since the generation of viral RNA follows the producer cell transcription pathway, intronic sequences present a high risk of being processed, compromising viral genome integrity. On the other hand, internal promoters are usually selected upon target cell expression criterion, and strong promoters that include intronic sequences are often selected. To overcome specifically the latter hurdle, several research groups have opted to switch the transgene direction to the reverse form. Although this strategy successfully avoids systematic processing of intronic sequences, the transcription of two complementary and opposite RNAs belonging to the external and internal promoter tends to anneal, creating double‐stranded RNAs (dsRNAs). The formation of dsRNAs triggers a cellular innate alert and activates PKR gene expression, inhibiting whole cellular translation, and subsequently, reducing viral titer. In this study, VIVEbiotech proposes to knock‐out PKR gene utilizing CRISPR/Cas9 to increase, not only the lentiviral titer, but also the integrity of the lentiviral particles. This strategy will enable those clients that are willing or currently performing their preclinical experiments with promoters‐containing intronic sequences to produce safer lentiviral particles with better virus integrity.
Non‐viral CAAR T cell generation utilizing the hyperactive Sleeping Beauty platform
1: MDC 2: Charité University Medicine
Ex vivo autologous immunotherapies have seen tremendous development and success. To date, viral vectors are most commonly used for gene therapies in clinical trials. However, they have several drawbacks such as safety concerns regarding immunogenicity and insertional mutagenesis. Moreover, the generation of autologous transgenic immune cells is time‐, cost‐ and labour‐intensive. As an alternative, non‐viral vectors have shown promising results and are being developed for a plethora of new immuno‐ and gene therapies. Here we show the usage of the non‐viral, hyperactive transposase Sleeping Beauty 100x (SB100x/pT4) system to generate chimeric autoantigen receptor (CAAR)‐harbouring T cells for the treatment of autoimmune encephalitis. The SB100x technology is able to integrate large genetic cargos into the genome with a close‐to‐random integration pattern. We used miniaturized DNA that carries the gene of interest but is devoid of unnecessary bacterial genes. Using the SB100x technology, we achieved similar gene transfer efficacies in human T cells compared with viral gene transduction. The advantageous close‐to‐random integration pattern of SB100x and the miniaturized DNA limits unwanted side effects, like insertional mutagenesis or immunogenicity, to a minimum compared with current state‐of‐the‐art viral gene transfers. Furthermore, treatment costs and production time of required components can be dramatically reduced because SB100x is delivered as in‐vitro‐transcribed mRNA. Therefore, the SB100x transposon system is a promising technology to enable cost‐efficient and safe gene therapies in the future.
Phage hybrid vector mediating TRAIL gene therapy in osteosarcoma
1: Thailand Excellence Center for Tissue Engineering and Stem Cells, Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand 2: Cancer Phage Therapy Group, Department of Brain Sciences, Faculty of Medicine, Imperial College London, London, United Kingdom 3: Musculoskeletal Science and Translational Research Center, Department of Orthopedics, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand 4: Center of Multidisciplinary Technology for Advanced Medicine (CMUTEAM), Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
Neoadjuvant chemotherapy, a standard treatment for osteosarcoma (OS) which is an aggressive bone tumour with highly metastatic incidence, increased five‐year survival rate only 10‐30% in metastatic patients. One of hopeful strategies for OS treatment is gene therapy, but a major developed Transmorphic phage/AAV (TPA) to target OS. The vector composes of an AAV transgene cassette integrated inside the intergenomic region of M13 bacteriophage genome, also displaying a tumour cell targeting ligand, RGD4C peptide, on pIII capsid. Flow cytometric analysis showed that αvβ3 and αvβ5 integrins, RGD4C targets, were expressed on 143B and U2OS human OS cell lines. Impressively we found that TPA can deliver luciferase gene into OS cells. We then chose tumour‐necrosis factor related apoptosis‐inducing ligand (TRAIL), which selectively trigger apoptotic tumour cell death through binding death receptors, as a therapeutic gene for OS treatment. We engineered TPA carrying two different forms of TRAIL, transmembrane (tmTRAIL) or soluble (sTRAIL), and compared their OS killing efficiency using cell viability assays. The results showed a significant cell dead in both forms of TRAIL particularly in TPA‐TRAILs treated 143B cells. Next, we evaluated biodistribution of transgene in different organs after systemic administration of TPA carrying human sTRAIL gene in immunodeficient mice (BALB/c nu/nu) bearing 143B‐derived xenografts. We found that mRNA level of sTRAIL transgene significantly increased only in tumours of the targeted RGD4C/TPA‐sTRAIL treated group. All these findings indicate advantages of TPA on cancer targeted gene therapy.
Investigating infectivity of HIV‐1 Env lentiviral vectors
1: Kings College London 2: GlaxoSmithKline
Exploitation of HIV‐1's lifecycle holds the potential to optimise current methods of gene delivery. Target cell isolation and subsequent alteration outside of their natural environment is a lengthy, laborious and expensive step in some gene therapies. Therefore, exploring envelope pseudotyping in lentiviral vectors may supersede ex vivo gene therapy. Here we aim to utilise the natural tropism of HIV‐1 to CD4+ T lymphocytes, ultimately broadening the potential of lentiviral vectors for in vivo gene delivery.
Expression and production of envelope proteins post‐transfection in the three‐plasmid system is driven by internal promoters and regulatory controls designed to boost overall production and therefore infectivity. We tested a variety of expression plasmids each with their own unique advantages to investigate expression of the CCR5 envelope JR‐FL, and the CXCR4 envelope NL4.3, in both their wild type and codon optimised sequence. Here we show that envelope expression plasmids can affect lentiviral infectivity in target cells. We hypothesise that lentiviral vector infectivity can be enhanced by increasing expression and stability of HIV‐1 envelopes, as well as incorporation into mature viral particles. We demonstrate that envelope expression plasmid transfected into producer cells correlates with increased infection of target cells. We are now further dissecting this phenotype.
These data provide solid groundwork into the promise of utilising HIV‐1 Env vectors for further analysis in peripheral blood mononuclear cells and their potential use in in vivo gene therapy. Optimisation of transduction would lead to the production costs and efforts being significantly reduced.
Next generation sequencing: a powerful tool for validation of viral genome integrity provided by alternative viral production platforms
1: OXGENE
ABSTRACT WITHDRAWN
1: Center for Neuroscience and Cell Biology 2: Center for Innovative Biomedicine and Biotechnology 3: ViraVector–Viral Vector for Gene Transfer Core Facility, University of Coimbra 4: Faculty of Pharmacy, University of Coimbra 5: Institute for Interdisciplinary Research (III), University of Coimbra 6: Molecular Neurogenetics Unit, Massachusetts General Hospital, Harvard Medical School
Exosome‐enveloped adeno‐associated viral vectors (exo‐AAVs) emerged as a new opportunity for non‐invasive gene therapy for the central nervous system (CNS). Since EVs are naturally formed cell vesicles that can transport different biomolecules and travel through the circulation without being eliminated by the immune system, the combination of EVs with AAVs seems to dramatically improve AAVs transduction in different organs such as the brain. Despite the remarkable results obtained in previous reports, only AAV serotypes with already known ability to cross the blood‐brain barrier (BBB) have been used.
In this work, we aimed at developing a size exclusion chromatography (SEC) protocol for the isolation of natural and biologically active exo‐AAVs, from any AAV serotype, which could be used as non‐invasive gene delivery vectors to the CNS. Moreover, we also aimed at performing a direct comparation between exo‐AAVs isolated by different centrifugation (UC) and SEC.
Exo‐AAVs were produced following the standard protocol for AAVs production and subsequently isolated by UC and SEC. Characterization was performed by WB, NTA, qPCR and TEM and infectivity was evaluated both in vitro and in vivo.
SEC allowed a higher recovery of exo‐AAVs, free of cell contaminating proteins and with less solo AAVs than those isolated by UC. Exo‐AAVs were also more infectious in vitro than solo AAVs. When intravenously injected in mice, exo‐AAVs were able to cross the BBB and the blood‐cerebrospinal fluid barrier without AAV's contribution and transduce mouse brains.
In conclusion, SEC‐purified exo‐AAVs are a promising gene delivery vector for therapy of brain disorders.
An Integrated AAV cis‐Acting Replication Element Assembles a Highly Processive Rep‐dependent Replication Complex
1: OXGENE a WuXi Advanced Therapies company 2:
The Adeno‐Associated virus (AAV) cis‐acting replication element (CARE) mediates ITR‐independent site‐specific integration and amplification of integrated AAV sequences. CARE spans the AAV region encompassing the p5 promoter and the 5’ region of Rep. The HeLaRC32 cell line, which is widely used for rAAV vector titration, contains a chromosomally integrated AAV2 sub‐genomic sequence that lacks the left and right ITRs, and thereby is dependent on CARE for amplification of Rep and Cap following infection by adenovirus helper. Here, we examine the temporal kinetics and extent of Rep‐dependent CARE‐mediated amplification in HeLaRC32 cells.
Employing Targeted Locus Amplification (TLA) analysis, we found that the AAV RepCap sequences have integrated into chromosome 19 (Chr 19) close to the AAVS1 site, a known hotspot for site‐specific integration of wild type AAV. Interestingly, HeLaRC32 cells were observed to have a 12kb duplication of Chr19 upstream of the AAVS1 locus. At least twenty copies of RepCap are estimated to be present within the integration site. Amplification of integrated AAV sequences occurs within 48 hours post adenoviral infection, with RepCap copy numbers continuing to increase 96 hours post infection. Importantly, this process is not limited to AAV sequences but extends asymmetrically into adjacent host chromatin.
Our results indicate that CARE‐dependent amplification is not constrained to AAV sequences, but instead extends across the integration site and into neighbouring host chromatin. The replication complex assembled on CARE is highly processive and may extend for tens of kilobases from the site of initiation.
In vitro screening of niosome formulations for the effective gene transfer of human mesenchymal stem cells
1: Centro de Investigacións Científicas Avanzadas (CICA), Universidade da Coruña
Genetic modification of human mesenchymal stem cells (hMSCs) constitutes a promising therapeutic tool for regenerative medicine. Niosomes are self‐assembled vesicular nanocarriers consisted of cationic lipids, non‐ionic surfactants and helper lipids that have recently emerged as new candidates for gene delivery applications.
The objective of this study was to elucidate the most suited niosome formulations to genetically modify immortalised MSCs. Niosomes were synthesised by thin‐film hydration method varying both compositions and ratios from cationic lipid (DOTMA), non‐ionic surfactants (polysorbate 80, P80; polysorbate 60, P60; polysorbate 20, P20; and poloxamer 407, PX) and helper lipids (cholesterol, CH; chloroquine, CQ; and squalene, SQ). Transfection assays were performed in hMSCs monolayers using the commercial reagent Lipofectamine (LPF) as positive control.
Formulation of niosomes with P20 as non‐ionic surfactant with CQ as helper lipid (P20CQ) showed a superior transfection efficiency of MSCs when compared with those based on CH or SQ. However, niosomes containing P60 or P80 reached the highest levels of transgene expression when combining with CH (P60CH and P80CH). Thus, the highest performance of niosome formulations as nanocarriers were reached at cationic lipid: non‐ionic surfactant: helper lipid ratio of 1:4:4 for P20CQ and P80CH formulations and 1:10:4 for P60CH niosomes. Incorporation of PX to the P20CQ niosomes reached similar levels of transfection to those achieved with LPF. Viability of cells transfected with nioplexes was always higher than that reached with LPF, at all mass ratios studied. In conclusion, niosomes constitute promising carriers with high biosafety and tunable properties for the effective gene transfer of hMSCs.
HIV‐ 1 lentivirus tethering to the genome is associated with transcription factor binding sites found in genes that favour virus survival
1: Brunel University London 2: Testavec 3: Protagene 4: University Heidelberg 5: University of Edinburugh 6: University College London 7: National Institute of Biological Standards and Controls 8: University of Witswatersrand 9: NCT & DKFZ 10: Imperial College London
Lentiviral vectors are attractive for permanent and effective gene delivery as they can integrate into the host genome. However, this can cause insertional mutagenesis highlighting the importance to further understand the integration process. Insertion site tethering is believed to involve cellular proteins such as PSIP1/LEDGF/p75, which binds to virus pre‐integration complexes to target the virus genome to that of the host. Transcription factors that bind both the vector LTR and host genome are also suspected influential to this. To determine the role of TF in the tethering process, we mapped predicted transcription factor binding sites near to IS chosen by HIV‐1 LV in infected human induced pluripotent stem cells and their hepatocyte‐like cell derivatives. These sites were also found in both the native and self‐inactivated LTR configurations and significant enriched in transcription factors essential to HIV‐1 life cycle and virus survival. These sites also reside in HIV‐1 patient IS and in mice infected with SIN LTR configuration HIV‐1 based recombinant LV. Our in silco data analysis suggests pTFBS present in the virus LTR and IS sites selected by HIV‐1 LV are important to virus survival and propagation.
A novel muscle‐specific promoter to improve muscle targeting while reducing liver overload
1: Genethon, UMR_S951, Inserm, Univ Evry, Université Paris Saclay, EPHE
Muscle‐directed transgene expression with reduced off‐targets is one of the biggest challenges for AAV‐based gene therapy. After intravenous infusion, a large portion of AAV vectors is retained by the liver. Muscle targeting is also complicated by the fact that this tissue represents around 30‐40% of the body weight and gene transfer occurs only at high doses that may be associated with toxicities. The existing limitations for an efficient and specific expression in muscles led us to develop novel muscle promoters.
Tandem liver‐muscle promoter (LiMP) beside expression in both tissues unexpectedly showed increased transgene expression in muscle (Colella et al. 2018). Due to its large size (1.1Kb), different combinations of enhancers and promoters were tested to identify smaller promoters with similar characteristics. Through the combination of liver‐specific enhancers and muscle promoters, we identified a promoter with the size ranging 600‐800bp and up to 15‐fold increase in transgene expression in skeletal muscle compared to the parental muscle promoter. Following these results, more complex combinations of promoters were tested. A second generation of promoters was derived, named double muscle promoter (DMP). When compared to the ubiquitous and strong CAG promoter, DMP showed a superior efficacy in transgene expression in muscles, with a 10‐fold reduction in liver expression.
To conclude, tandem promoters allows identifying promoters with unexpected properties representing a new method for promoter generation starting from promoter with a known safety and efficacy profile. Experiments are ongoing to validate promoter expression in human cells and efficacy and immunogenicity in models of neuromuscular diseases.
Characterization of AAV9 infectivity when it presents CY5.5 linked to the capsid at positions 470 and 471
1: CIMA 2: Universidad de Navarra
Lately there is a great effort focused on the generation of new adeno‐associated virus (AAV) variants to obtain vectors with specific tropism, more efficient infectivity and to avoid recognition by neutralizing antibodies present in treated patient. AAV capsid‐directed evolution strategies have started to generate new AAV variants with improved tropisms and infectivity opening the possibility to obtain enhanced gene therapy vectors. Another approach to modify recombinant AAV properties is to attach moieties to their surface, however this strategy has been neglected and there are few cases exploring this approximation. We have examined the generation of new versions of AAV9 replacing residues N470 and M471 by the unnatural amino acid azido‐lysine (AzK). Interestingly we have obtained mutant AAV9 viral productions with similar titers to the AAV9 WT. Incorporation of AzK to AAV9 capsid enables to perform cupper free click chemistry reactions linking molecules coupled with a DBCO group to the AAV9 capsid. To determine the conditions and effects of coupling moieties to AAV9 capsid we explored covalent attachment of Cy5.5‐DBCO molecules to the virus surface. We have observed a reduction of GFP transgene expression in modified AAV9 mutants being this effect stronger when there were more Cy5.5 molecules coupled to the capsid. In addition, some modified AAVs presented an accumulation of viral genomes in infected cells that was not translated in a stronger transgene expression. Therefore, the substitution of amino acids N470 and M471 by an AzK in the AAV9 capsid interferes with transported transgene expression.
Versatile gene delivery targeting via Ultrasound and gas Microbubbles
1: Bracco Suisse SA
The COVID‐19 crisis and the rapid development of highly effective mRNA vaccines opened a new era for gene therapy. While viral vectors were for a long time the only tool for efficient delivery, new non‐viral vectors have recently emerged, spawning new opportunities (indications, tissues, etc.). A new one is set to take off thanks to its safety profile, its specificity toward tissues, and its versatility toward both genetic materials and indications. Gas‐filled microbubbles (MB), clinically used as ultrasound (US) contrast agents, have proven their benefits in various animal models and clinical applications for targeted delivery of drugs/genes. Herein, we disclose the development of new MB formulations allowing the delivery of various genetic materials at a specific location under the control of an ultrasound probe. We set forth a study to elicit the expression of a foreign enzyme in a liver mouse model. To this aim, MB were systemically co‐injected with a Luciferase pDNA (6 to 65 μg) in the tail vein, then Ultrasound were delivered at MB arrival in the liver. The effective pDNA transfection was observed by bioluminescence 24 hours after treatment. Mice were divided into three groups: pDNA alone; pDNA with US; pDNA with US and MBs (n ≥ 5). The use of our MB allowed increasing the signal up to 5 folds in comparison to the US alone. These results highlight the potential of MB plus US to efficiently deliver locally genetic material without any safety concerns.
Evaluation of a novel dopaminergic neuronal promoter design candidate in neonatal mice
1: UCL EGA Institute for Women's Health 2: University of the Witwatersrand 3: University of Toronto
Viral gene therapy is a compelling field of therapeutic development for neurological disorders. Recombinant adeno‐associated virus (rAAV) has been widely employed in clinical and preclinical trials, that often utilise ubiquitous promoters to target the CNS. However, as different brain regions and cell types can be the therapeutic targets, there is clinical need to develop tissue‐specific or cell‐type selective viral vectors. In this study we focus on promoter candidates for dopaminergic neuronal selectivity.
To express transgenes in specific cells, promoters for endogenous genes are commonly created by truncating the sequence upstream of the transcriptional start site. We have previously evaluated different versions of the rat tyrosine hydroxylase (rTH) promoter in rAAV9, expressing GFP reporter gene for dopaminergic neuronal selectivity. Two versions of rTH promoter (298bp and 2.5kb) were injected into neonatal mice by intracerebroventricular delivery, and brain transduction evaluated by immunohistochemistry. We observed that the significant promoter truncation resulted in loss of selectivity and expression efficiency, but the larger version precludes use due to restricted rAAV packaging capacity.
Newer methods of promoter candidate design have been developed, in order to reduce promoter size and retain cell‐type selectivity by truncating regions with high palindromic density. This approach has shown promoter candidates can retain expression levels and selectivity in vitro. This study evaluates this new promoter design approach in vivo. A new rTH promoter design was evaluated in rAAV9, delivered by intracerebroventricular injection to wild‐type neonatal mice, and collected at P35 for brain immunohistochemistry and double labelled immunohistofluorescence for TH and GFP positive cells.
Development of a novel modular encapsulation system for targeted gene delivery
1: The University of Queensland
Targeted nanoparticles for gene therapy provide the potential for more effective treatments. We have developed a modular targeting system using “caveospheres”; 50nm vesicles produced in E. coli by expression of chimeric proteins based on mammalian caveolins. In this study we incorporated the Z‐domain from protein A to provide a specific binding region on the caveosphere surface for monoclonal antibodies (for targeting) and fluorescent secondary antibodies (for labelling). Using this platform, we investigated the specificity of targeting to cancer cell lines in vitro and to induced cancers in vivo in mouse models. Here, we show that functionalized caveospheres presenting anti‐EGFR antibodies on the surface membrane are specifically internalized in A431 cells in culture and show greater accumulation in breast cancer tumours in a mouse xenograft model compared to non‐functionalised controls. In addition, we have developed sonication methods that enable the loading of cDNA into functionalized caveospheres for tumour targeting in order to enhance accuracy and decrease immunogenicity. We demonstrate induced expression of GFP from DNA loaded caveospheres in A431 cells. Furthermore, caveospheres functionalized with anti‐EGFR antibodies gave a greater percentage of cells expressing the DNA cargo than those transfected with commercial lipofectamine, albeit at a lower expression level. The ability to produce caveospheres in large quantities in bacteria from a single cDNA represents a rapid, simple, and tuneable method for precise delivery of genes to specific cell types. This technology may be adapted for novel cancer treatments, gene therapy and vaccine development.
Cross‐packaging a vector genome containing densovirus ITR into AAV capsid serotype 2 or 8: possible or not?
1: Inserm UMR 1089
Recombinant Adeno‐associated vectors (rAAV) are composed of an icosahedral capsid defining its serotype, inside which an expression cassette replaces the initial viral genome. Two Inverted Terminal Repeats (ITR), at genome extremities, are essential for rAAV production and have to be kept. Two main ways of rAAV manufacturing exist: transient plasmids transfection of mammalian HEK293 cells or baculovirus infection of insect Sf9 cells. In both ways, the expression cassette and ITR, and the Rep/Cap genes, are brought separately, either by two plasmids or by two baculoviruses.
Production of rAAV of all serotypes is technically possible, only by changing the Cap sequence. However, ITR and Rep operate in a complex binding partnership, essential for genome replication and encapsidation. Rep from serotype 2 (Rep2) allows the production of vector genomes containing ITR from all serotypes, except ITR5 which requires its Rep5, into capsid of many AAV serotypes and even into other Parvovirus serotypes : so called cross‐packaging.
We studied the cross‐packaging possibility of vector genomes containing very different Parvovirus ITRs (ITRX) into AAV2 and AAV8. We tested the production of such vectors in the two platforms, in presence of Rep2 or RepX. Rep expression was confirmed by RTqPCR. Vector genome copy numbers were assessed by qPCR. However, in HEK293 nor in Sf9, no replication or encapsidation occurred. This questions the existence of additional proteins or co‐factors essential for production. Moreover, this work gives insight into the replication and encapsidation mechanisms of rAAV and limits the range of production possibilities.
Properties of adenovirus vectors with increased affinity to DSG2 and the potential benefits for oncolytic approaches and gene therapy
1: University Witten/Herdecke 2: University of Washington
Carcinomas are characterized by a widespread upregulation of intercellular junctions, which create a barrier to immune response and drug therapy. Desmoglein 2 (DSG2) represents such a particular junction protein and serves as one of the adenovirus receptors. Importantly, the interaction between the human adenovirus type 3 (Ad3) and DSG2 leads to shedding of the binding domain followed by the decrease of the junction protein expression and transient tight junction opening. Junction opener 4 (JO‐4), a small recombinant protein derived from the Ad3 fibre knob, was previously developed with higher affinity to DSG2. JO‐4 has proven to enhance the effect of antibody‐ and chemotherapy and is now considered for clinical trials. However, the effect of the JO4‐mutation in the context of a virus remains insufficiently studied. Therefore, we introduced the JO4‐mutation to various adenoviral vectors to explore their infection properties. From current experimental settings and investigated cell lines, the JO4‐containing vectors showed no enhanced transduction compared to the parental vectors in DSG2‐high cell lines. Moreover, in DSG2‐low cell lines, the JO4 vectors presented rather weakened effect. Interestingly, DSG2‐negative cell line MIA PaCa‐2 even showed resistance to JO4 vectors' infection, possibly due to the negative effect of JO4 mutation on the usage of another Ad3 receptor‐CD46. Together, our observations suggest that the JO4 vectors may have an advantage to prevent CD46‐mediated sequestration, thereby achieve DSG2‐specific transduction.
Use of Evader‐AAV8 for repeat dosing in mice and non‐human primates
A Rosario1 K Paolilli1 M Levinson1 C Ceballos Diaz1 A Mishra1 K Shenoy1 M Miller1 C A Maguire2 3 P A Fitzpatrick1
1: Chameleon Biosciences 2: Department of Neurology, The Massachusetts General Hospital 3: Harvard Medical School
We have developed the EVADER platform to allow repeat dosing with the use of extracellular vesicle (EV) bound immune system modulators (ISMs). We explore the use of EVs with ISMs (PD‐L1 and CTLA‐4) associated with AAV8 encoding human coagulation factor IX (Evader‐AAV8‐hFIX) in both mice and non‐human primates (NHPs) to mediate efficient liver transduction. Dual sequential fixed intravenous dosing studies in mice resulted in EVADER‐AAV8‐hFIX (STD) successfully expressing hFIX at twice the initial injected dose (p = 0.01) compared to an insignificant increase (p = 0.28) for STD. EVADER results in lower CD4+ T cells in murine liver than with STD by 60%. EVADER immune suppression also reduces anti‐AAV8 IgG capsid antibodies by 3‐fold. In NHPs injected with EVADER or STD at 5e10 vg/kg there was an average 3.4‐fold increase in vector genomes in the liver in the 2x dosed cohort with EVADER compared to the 1x dosed cohort with EVADER. For STD, there was no increase in vector genomes in the liver for the 2X dosed cohort compared to the 1X dosed cohort. This suggests that redosing with EVADER is feasible in NHPs and mediates higher levels of transgene expression than STD. In addition, at 5e11 vg/kg dosing, FIX levels at study termination were 0.7 to 8.0 fold higher for the EVADER group compared to the STD group. At a lower dose of 5e10 vg/kg, FIX expression was detected in 2/3 of injected NHPs, while STD levels of FIX were below background.
AI‐driven promoter optimization at MeiraGTx
1: MeiraGTx
Promoters are crucial elements for regulating potency and specificity of transgene expression. Whilst many promoter sequences have been characterized/optimized through conventional low‐throughput analyses (rational design) or newer high‐throughput methodologies (e.g., MPRA), these approaches still require an expensive and time‐consuming in‐vitro optimization. With better data and more advanced artificial intelligence models, we have repurposed and optimized a convolutional neural network (CNN) to predict promoter potency. We computationally constructed a library of synthetic promoters by cloning all known enhancer elements reported in the ENCODE database upstream of a potent, compact constitutive promoter and used the said CNN model to predict their potency. Subsequently, we optimized the correct spacing between the top enhancer elements and the promoter sequence. In parallel, we also performed an in‐silico saturated mutability, whereby we introduced all possible point mutations within the promoter sequence and selected those with the best impact on promoter's potency. Top performers were printed and tested in vitro. While most of the elements exhibited better or equal performance than the original promoter, we selected and combined the best enhancer elements and point mutation in a second optimization round. After synthesizing and testing this new set in vitro, we obtained an enhanced promoter that exhibits increased potency. This approach shows the power of AI methods in narrowing down the search space in the promoter optimization process while at the same time reducing cost and time.
Promoter discovery: identification of novel cell type‐specific and highly active synthetic promoters for gene therapy vectors
1: OXGENE
A primary objective of successful viral vector‐mediated gene therapy is long‐lasting expression of the therapeutic gene at physiologically relevant levels that is restricted to the tissue of interest. Promoters commonly used in viral vectors often fail to meet these requirements and are typically constitutive and ubiquitously active, are frequently silenced in vivo, and due to promoter‐driven overexpression frequently result in toxicity or unwanted immune responses against the transgene. By contrast, most native cellular promoters have sizes that exceed the packaging capacity of viral vectors and are frequently sensitive to genomic context.
To overcome such obstacles, we have developed a strategy for synthetic promoter design, which is based on in‐house‐generated transcriptomic data, epigenetic datasets and bioinformatic sequence analyses. Using either known or de novo identified native cis‐regulatory elements functional in target tissues, we have created novel combinatorial libraries ranging from a few hundred variants up to >1 million promoter variants. Evaluation of promoter libraries, in LV and AAV vectors, was conducted in some of the major targets for gene and cell therapies using high‐throughput fluorescence‐activated cell sorting (FACS) and luciferase assays. Top promoter candidates were subsequently validated for strength and specificity of reporter gene expression against target and a broad range of non‐target cell types. Champion promoters demonstrated robust cell‐ or tissue‐specific patterns of expression ‐ 10‐100 times stronger in target cells. Our data indicate that rational promoter design coupled with screening campaigns can be valuable tools for improving the performance of viral vectors and the on‐target safety of gene therapies.
Novel adenovirus vectors for efficient transduction of human hematopoietic stem and peripheral blood cell subsets
1: Witten/Herdecke University 2: Ensoma
Over the recent years, a gene therapy approach for direct in vivo hematopoietic stem cell (HSC) transduction was established. This approach combines mobilization of HSC from the bone marrow into the peripheral blood followed immediately by intravenous viral vector administration for efficient stem cell transduction. For direct in vivo transduction of HSCs, a chimeric adenovirus (Ad) type 5 vector carrying an optimized Ad35 fiber knob from species B (Ad5F35++) for increased CD46 binding and HSC transduction was established. However, to improve HSC transduction, we explored 9 alternative species B derived Ads (Ad3, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad35, Ad50), 3 species D Ads (Ad26, Ad37, Ad48), and Ad52 from species G. Ad5 from species C and Ad5F35++ were included as controls. In vitro transduction was assessed in primary CD34+ HSCs from three healthy donors by flow cytometry and qPCR. We found that Ad7, Ad11, Ad16, Ad34 and Ad35 show robust transduction efficiencies. Next, we converted these wild‐type candidate viruses into E1‐deleted first generation vectors and confirmed these transduction results in HSCs. To explore transduction of additional hematopoietic cells that can be relevant for in vivo gene therapy, we transduced peripheral blood mononuclear cells from healthy donors with these vectors and found that in contrast to Ad5, vectors based on Ad11, Ad16, Ad34, and Ad35 show high transduction efficiencies in monocytes, NK‐ and T‐cells. This study identified novel viral vectors, which can be explored for in vivo HSC gene therapy as well as direct transduction of other blood lineage subsets.
Lentiviral vector platform development for improved Chimeric Antigen Receptor (CAR) T cell applications
1: University College London 2: National Institute for Biological Standards and Control
Development of technologies improving the production and function of lentiviral vectors (LV) are essential to meet the increasing demand for high‐quality vectors used in gene‐modified cell therapies. Chimeric Antigen Receptor (CAR) T cell therapies reported highly effective clinical results against blood cancers, with response rates of up to 90%. Most commercially available CAR T cell therapies rely on LV or retroviral vector transduction for CAR transfer into the target T cells.
CAR T cell therapy requires a highly complex bioprocess, resulting in high manufacturing costs, limiting access to these lifesaving therapies. An essential bioprocessing step is the immune activation of T cells to allow efficient LV vector transduction as well as cell expansion. This is typically achieved through mitogen receptor targeting using microbeads conjugated to anti‐CD3 and anti‐CD28 antibodies. This technique is effective but can lead to T cell overactivation and cellular exhaustion.
We have investigated the use of lentiviral particles called LentiSTIM (patent number PCT/GB2016/050537), capable of targeting mitogen receptors to simultaneously activate and transduce the target T cells. The mitogens are expressed in engineered HEK293T producer cell lines as membrane bound proteins. We tested several combinations of mitogens to improve T cell phenotype and limit their overactivation in three healthy donors. Compared to conventional activation and transduction, the LentiSTIM approach resulted in higher transduction levels from 24% up to 34% and in a lower activation and exhaustion profile, when transferring a clinically relevant anti‐CD19 CAR construct.
Efficient and flexible mRNA trans‐splicing dual AAV vectors for large gene delivery
1: Department of Pharmacy ‐ Centre for Drug Research, LMU Munich, Munich, 81377, 2: Lab for Retinal Gene Therapy, Dept. Ophthalmology, University Hospital Zurich, University of Zurich, Zurich, 8952, Switzerland 3: ViGeneron GmbH, Planegg, 82152, Germany 4: Department of Ophthalmology, Leiden University Medical Centre (LUMC), Leiden, 2333 ZA, Netherlands 5: Netherlands Institute for Neuroscience, Institute of the Royal Netherlands Academy of Arts and Sciences (KNAW), Amsterdam, 1105 BA, Netherlands 6: Department of Ophthalmology, University Hospital, LMU Munich, Munich, 80336, Germany 7: German Centre for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, Munich, 81377, Germany
Recombinant adeno‐associated viral (rAAV) vectors are currently the gold standard for efficient in vivo gene delivery and long‐term gene expression. However, many disease‐associated large genes or CRISPR‐Cas modules exceed the packaging capacity of rAAVs. To overcome this limitation, different dual rAAVs strategies have been developed. Although they all work in principle, they have some decisive disadvantages, including low reconstitution efficiency, production of alien proteins, or low flexibility in split site selection. To circumvent these constraints, we developed a novel dual rAAV vector technology based on
Development of oncolytic and gene therapy vectors based on Adenovirus serotype 4 as an alternative to Adenovirus serotype 5
1: Witten/Herdecke University 2:
Adenoviral vectors are explored for gene therapy and cancer treatment. Despite limited applicability caused by preexisting immunity and unfavorable liver tropism, most adenoviral vectors are derived from human Adenovirus 5 (AdV5). We therefore screened an Adenovirus library representing the natural Adenovirus diversity to identify potential vector candidates.
To compare different AdVs infectivity and AdV mediated and reporter‐gene expression we assessed GFP or luciferase activity in variety of cell lines and further tested oncolytic activity using viability assays. Candidate oncolytic vectors were tested on Siha and Hela mini‐organoids.
We found that AdV4 transduced muscle and heart cells more efficiently than AdV5. Moreover, despite low transduction efficiency, AdV4 showed efficient oncolytic activity, gene expression, genome replication and progeny virus production in papillomavirus (HPV) induced tumor organoids.
We constructed a first generation AdV4 vector platform by deleting E1 and E3 genes. The E1 and E1‐E3‐deleted vectors were replication‐competent in HEK293 cells, whereas other cancer cell lines including Hela and Siha were not permissive, suggesting that AdV5‐E1 can complement for AdV4‐E1, enabling the rescue of E1‐deleted AdV4. Purified E1, E3 deleted vectors showed transduction efficiencies as the parental vector.
To increase AdV4 uptake into Papillomavirus induced cancer cells we replaced the AdV4 fiber knob with the AdV5 fiber knob, but pseudotyping did not improve vector infectivity nor replication.
Our new AdV4 vector will be further explored for oncolytic or muscle gene therapy leading the way for the development of further alternative AdV vectors for a broad range of clinical applications.
An AAV gene therapy for the treatment of geographic atrophy by targeting the alternative pathway of the complement system
1: Gyroscope Therapeutics
Age‐related macular degeneration (AMD) is the most common cause of blindness among the elderly in the industrialised world. Approximately 5 million people globally have geographic atrophy (GA) in at least one eye. Evidence implicates activation of the complement system as one of the factors affecting AMD pathogenesis. Variants in complement genes, including human complement factor I (hFI) have been shown to be associated with an increased risk of AMD.
GT005 is a recombinant adeno‐associated viral (rAAV) serotype 2 vector expressing hFI under the control of a cytomegalovirus (CMV) enhancer and chicken beta‐actin (CAG) promoter. High expression of hFI was detected in HEK293 cells transduced with GT005 and was shown to be functionally active in a C3b cleavage assay. Subretinal delivery of GT005 in mice led to detectable levels of hFI in ocular fluids and ex vivo demonstration of increased C3b degradation. Immunostaining of the mouse retina demonstrated predominant localisation of hFI in the retinal pigment epithelium (RPE) and inner/outer segment of photoreceptors. GT005 showed a statistically significant reduction of CNV leakage area in a dose‐dependent manner at day 7 in the mouse laser‐induced CNV model.
The pre‐clinical data presented here demonstrate the potential of GT005 in slowing disease progression of GA by limiting complement‐mediated inflammation in the posterior segment of the eye in a laser induced CNV mouse model compared with a control group. These studies paved the way for the current evaluation of GT005 in Phase I and II clinical studies for GA secondary to AMD.
Design considerations for circular RNA overexpression vectors
1: University of Eastern Finland, Kuopio
Circular RNAs are a novel non‐coding RNA species with a long half‐life and largely uncharacterised functions. They have been speculated to be important pieces of regulatory networks controlling cellular function as such are interesting novel therapeutic candidates. One strategy for utilising potentially beneficial circular RNAs as therapeutic agents is overexpression gene therapy from a viral vector. For this purpose, understanding what factors can be used to promote circular RNA formation from a vector construct is crucial. Adeno‐associated virus (AAV) vectors have low immunogenicity and low risk of genotoxicity, while promoting long term sustained expression, making them an excellent tool for in vivo gene delivery. Here, we have studied how the efficiency of circular RNA formation from AAV vectors and AAV vector plasmids is affected by intron and exon length, intron complementarity, and splice site strength. We found that longer introns with longer complementary regions promoted more efficient circularisation, while no effect was associated with exon length. Additionally, the exonic elements known to affect splicing efficiency of linear mRNA were also associated with more efficient circularisation. These findings offer new insights into solving the mechanistical challenges of circular RNA mediated gene therapy.
Directed evolution of human Adenoviruses to improve vectored vaccines properties
1: Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, Via Pansini 5, 80131 Naples 2: Nouscom srl, Via di Castel Romano 100, 00128 Rome, Italy 3: CEINGE Biotecnologie Avanzate S.C.aR.L., Via G. Salvatore 486, 80145 Naples, Italy
Replication incompetent human Adeno (hAd)‐based vectors are able to induce potent adaptive immune responses in animal models and humans, being of interest for both infectious diseases and cancer therapy or prevention. Different Adenovirus‐based vaccines have been approved for human use, including those developed to contrast Covid‐19 pandemic. Immunological potency of hAd‐vectors based on different serotypes can be influenced by the extent of pre‐existing host immunity, different cell tropism and intracellular trafficking, also impacting the extent of innate immunity induction. Vaccines based on species C hAd are among the most potent, yet significant seroprevalence in humans limits their application into the clinic, with pre‐existing neutralizing antibodies negatively affecting vectored vaccine immunogenicity and efficacy. Error prone PCR represents an efficient method to introduce random mutations by reducing the DNA polymerase fidelity, allowing directed evolution and improvement of target proteins. Since the majority of hAd neutralization determinants are located in the hypervariable regions (HVR) of the hexon, the major capsid protein, we generated seven different hexon‐variant libraries with mutations in single or multiple HVRs from Group C hAd, through error prone PCR. Such libraries have been used to produce complex repertoires of hAd vectors potentially including variants with novel properties such as lower recognition by anti‐hAd antibodies present in humans or reduced unwanted side effects in the vaccinees. These libraries will be surveyed with different screening strategies to identify novel variants with improved properties for both prophylactic and therapeutic vaccine applications.
MaxPax: a next generation Lentiviral Vector with increased capacity, rationalised backbone and rev‐independence
J Wright1 B M Alberts1 D D Chipchase1 T M Evans1 C Nogueira1 J A Holley1 F Zambon1 E Guzman1 S Ferluga1 M Martin‐Urdiroz1 K A Mitrophanous1 N G Clarkson1
1: Oxford BioMedica
Retroviral and lentiviral vectors (LVs) remain the standalone vector system of choice when considering approaches to stably deliver larger, intact genetic payloads to target cells. Nevertheless, the need for multiple/longer transgenes is increasing. Unlike other viral vectors, the ∼9kb size limit of LVs is a ‘soft’ one, with a linear decline in titre as vector genome/vRNA length increases; this is strongly correlated to the steady‐state pool of packageable vRNA. Since LV output titre is a major contributor to commercial viability, the ability to reduce total vRNA size without compromising on functionality may lower this viability threshold. We previously reported on a new class of LVs wherein the major splice donor (MSD) is mutated, which ablates aberrant splicing into transgene sequences, that would otherwise lead to transgene‐encoding mRNA, limiting full length vRNA production. MSD mutation leads to a significant attenuation in vRNA production/stability but this is rescued by co‐expression of a re‐directed U1 snRNA ‘chaperone’ that binds to the vRNA packaging region. Here we report a second member of this new class of MSD‐mutated LVs – called MaxPax ‐ harbouring a ‘Vector‐Intron’ (VI) in the backbone. Splicing‐out of the VI stabilises the vRNA during production without impacting genome length. This novel configuration allows full deletion of the rev‐response element (RRE) and the majority of the gag region retained in the packaging signal, liberating an additional 1kb for transgene cargo. Since MSD‐mutation enables ‘clean’ VI splicing, the MSD‐null/VI paradigm provides a unique ‘symbiotic’ molecular relationship, enabling independence from both rev/RRE‐ and ‘U1‐chaperone’.
Homology Mediated End Joining Enables Efficient Non‐viral Targeted Integration of Large DNA Templates in Primary Human T Cells
1: University of Minnesota 2: Intima Bioscience
Adoptive cellular therapy using genetically engineered immune cells holds tremendous promise for the treatment of advanced cancers. While the number of available receptors targeting tumor specific antigens continues to grow, the current reliance on viral vectors for clinical production of engineered immune cells remains a significant bottleneck limiting translation of promising new therapies.
Here, we describe methods for efficient CRISPR‐based, non‐viral engineering of primary human T cells that overcome limitations of previous approaches. By synergizing temporal optimization of reagent delivery, reagent composition, and integration mechanism, we have achieved targeted knock‐in of cargo up to 3Kb at rates up to 70% with post‐editing cell viability of over 80%; efficiencies nearing those of viral vector platforms. Notably, integration of DNA donor molecules by homology mediated end joining (HMEJ) with short homology arms (48bp) consistently outperformed the use of 1Kb homology arms and traditional HR.
As proof of concept, we engineered CAR‐T cells and transgenic TCR T cells using a splice acceptor gene construct and gRNA specific to the TRAC locus. Using this approach, we consistently achieved integration rates of over 35% for CAR‐T cells, over 30% for TCR transgenic T cells, and over 25% for super‐sized genetic cargo (>6.3Kb).
CAR‐T cells generated using our approach are highly functional and elicit potent anti‐tumor cytotoxicity in vitro and in vivo. Our method is readily adaptable to cGMP compliant manufacturing and clinical scale‐up, offering a near‐term alternative to the use of viral vectors for production of genetically engineered T cells for cancer immunotherapy.
Use of tumour‐targeting transmorphic phage AAV particles to deliver IL‐15 cytokine as a therapy for solid tumours
1: Imperial College London
IL‐15 cytokine has gained popularity as a cancer therapeutic as it shows a similar lymphocyte stimulation and survival profile to the clinically approved IL‐2 but lacks the ability to stimulate regulatory T cells, making it an even mo interesting candidate. Despite these attractive characteristics, systemic administration of cytokines is linked to toxicity limitations and targeted delivery is required. Here we present a targeted vector delivery system for IL‐15 transgenes: the Transmorphic Phage AAV particles (TPAs). These particles use a modified bacteriophage M13 capsid to deliver a phagemid‐based genome encoding the therapeutic transgene to the tumour site thanks to the display of the RGD motif in their pIII surface proteins. In this work, we have used these TPA particles to deliver a secretion‐optimized version of the mouse IL‐15. The particle's targeting specificity and transduction efficiency were first tested in vitro with an ELISA assay, and the biological activity of the modified cytokine was tested with a CTLL2 proliferation assay. In vivo biodistribution of the therapeutic particles confirmed their specific localization to the tumour following systemic administration and their therapeutic capacity was proven by tumor size and viability reduction as well as animal survival. In view of these successful results, we sought to investigate if the use of an IL‐15 superagonist based on the fusion between the IL‐15Ra sushi domain and the IL‐15 would further improve therapy. In vitro studies show the improved efficiency of this cytokine at stimulating lymphocyte proliferation and their in vivo potential is under study.
Characterisation of the safety and efficacy of integrase‐endonuclease fusion protein lentivirus vectors
1: University of Eastern Finland
Lentivirus vectors (LVs) enable long‐term transgene expression by integrating their cargo into the genome of the host cell, which makes LVs a valuable tool for gene therapy applications. However, uncontrolled transgene integration leaves gene therapy prone to genotoxic side‐effects. To combat this, we have developed targeted vectors containing the I‐PpoI endonuclease fused to the viral integrase (IN). The IN‐I‐PpoI fusion protein directs transgene integration to the I‐PpoI recognition site in the 28S ribosomal RNA gene. Furthermore, the targeting molecule and the transgene can be packaged into the same vector particle. To enhance the targeting efficiency and to enable homologous recombination (HR) to the target site, we designed transgene constructs with 500 bp homologous arms (HA) flanking the transgene. We developed a droplet digital PCR ‐based method to measure the efficiency of integration targeting, as this approach allows for direct quantitation of all correctly integrated transgene copies, including HR events. We also characterised the durability of transgene expression, and the effects of the vectors on chromosomal integrity, to assess the functionality and safety of the targeted LVs. We found that the HA transgene construct enhances integration targeting to 28S rRNA, and improves the persistence of transgene expression, but only when combined with the correct IN‐I‐PpoI targeting construct. Our study shows that IN‐I‐PpoI‐LVs can be used to induce safe, long‐term transgene expression, and that detailed analysis methods are needed to confirm the chromosomal integrity of treated cells after double‐strand break repair ‐mediated transgene integration.
Increased in vitro transgene expression driven by rAAV expression cassette modifications is not predictive of the in vivo outcome
1: Universidad de Navarra 2: Vivet Therapeutics
The levels of rAAV vector‐mediated transgene expression, and consequently the vector potency, can be improved by introducing variations in the expression cassettes, such as codon optimization, transcription enhancers or transcript‐stabilizing sequences like introns and polyadenylation signals. Comparative analyses of the different constructs generated are more easily performed in vitro than in vivo. However, attention should be paid to any discrepancies in transgene expression between in vitro and in vivo analyses as poor translatability of vector potency can hamper the development process. We designed several constructs by incorporating codon optimization, an enhancer, or an intron and tested them in vitro in a comparative expression assay. Transgene expression levels in HepG2 cells infected with rAAV vectors containing a CpG‐reduced codon optimization of the transgene, an enhancer element, or an intron were higher compared to the original construct. Next, rAAV vectors harboring the modified cassettes were tested in Abcb4 knockout (KO) and wild type (WT) mice in parallel. Therapeutic efficacy was substantially reduced in all modified constructs except the CpG‐reduced codon optimized construct. Interestingly, in KO mice, despite presenting similar transduction levels, transgene expression of vectors containing the enhancer or the intron decreased after day 7 post‐treatment, while the original and CpG‐reduced constructs without the enhancer or intron continued to increase at least up to day 14. These two constructs also induced the highest transgene expression in WT mice. This in vitro / in vivo discordance points to the importance of testing in multiple systems prior to selecting final rAAV candidates.
Regulated expression of secreted transgenes by pulmonary transplanted macrophages in mice
1: Imperial College London
We are assessing the feasibility of cell therapy‐based treatments for respiratory diseases. Pulmonary transplantation of murine bone marrow‐derived macrophages (BMDM) demonstrated that gene‐modified BMDM can lead to in vivo production of secreted proteins. However, not all target diseases require constitutive expression of the therapeutic gene and diseases such as pulmonary alveolar proteinosis (PAP) may only require short, intermittent bursts of expression. To investigate regulated gene expression in BMDM, a commercial mifepristone‐inducible system (GeneSwitch, Invitrogen) was packaged into dual VSV‐G vectors, consisting of a regulated promoter upstream of reporter gaussia luciferase (GLux), and a mifepristone‐inducible activator complex. BMDM transduced in vitro with regulated vectors demonstrated a >100 fold‐induction in GLux expression upon mifepristone addition. Importantly expression returned to baseline upon mifepristone withdrawal and comparable induction levels were achieved after repeat stimulation. Next, donor mice BMDM transduced with regulated vectors (MOI 20:40 transgene:activator) were transplanted into lungs of wildtype mice (1e6 cells/mouse, n = 6/group). Gene expression was induced by intraperitoneal mifepristone injection (four consecutive days of 0.5 mg/kg/dose, one week post transplantation) and GLux levels were subsequently analysed in the lungs. Mice stimulated with mifepristone achieved a median induction in GLux expression of 7‐fold (range 5‐37 fold‐induction, bronchoalveolar lavage fluid) and 10‐fold (range 5‐13 fold‐induction, lung homogenate) compared to minimal leakiness observed in unstimulated mice. Data shown here demonstrates that regulation of gene expression from pulmonary transplanted BMDM can be achieved through mifepristone stimulation. This promising initial data has progressed to ongoing studies applying inducible macrophages to a PAP mouse model.
Optimized human regulatory sequences achieve targeted expression in brain and decreased off‐target liver and dorsal root ganglion expression
B Zhao1 S Tan1 S Liu1 M Lopez1 P Dhanota1 T Solanki1 S Levine1 J Griffin1 M Chen1 J McLaughlin1 G Lucey1 H Lejmi1 A Kim1 M C Vila1 A Tanenhaus1
1: Encoded Therapeutics
Adeno‐associated virus (AAV)‐mediated gene therapy (GT) has demonstrated transformative potential in treating genetic disorders, but off‐target transgene expression remains an important safety issue. Human genomic regulatory elements (REs) produce exquisite control of gene expression, but efficient identification of sequences that confer cell‐selective expression is challenging. We developed a next generation sequencing (NGS)‐based platform that combines functional screening and computational modeling to discover REs to regulate transgene expression. Here, we have applied our method in vivo to uncover 3’UTR REs that decrease transgene expression in liver and/or dorsal root ganglion (DRG), while maintaining brain expression. In two first‐generation (GEN1) screens for liver and DRG, over 20,000 putative de‐targeting variants comprising human‐derived 3’ UTR REs were tested in multiplexed AAV libraries in mice. Individual validation of the top candidates demonstrated robust expression in brain with substantially reduced expression in liver/DRG. To further optimize these REs, we developed a second‐generation (GEN2) AAV library containing combinations and sequence variants of the top REs for each tissue, resulting in improved de‐targeting. Simultaneous de‐targeting was achieved in a separate library combining liver and DRG de‐targeting REs. Finally, we show that data from our libraries support predictive modeling to identify the core sequence features contributing to targeted expression profiles to improve the safety and efficacy of GT.
Lentiviral vectors pseudotyped with Canine Distemper Virus envelope proteins: A novel tool for targeted transduction
1: Miltenyi Biotec
Selective gene delivery to the cell type of interest utilizing targeted lentiviral vectors (LV) provides an efficient and safe strategy for immunotherapeutic applications such as the chimeric‐antigen‐receptor (CAR) T cell therapy. Previously, we described the development of an indirect system based on universally targeted LV referred to as Adapter‐LV. Here the viral attachment protein (H) of the LV is fused to a single chain variable fragment (scFv) specific for an artificial tag present on adapter molecules, which in turn mediate binding to the target cell. This results in selective transduction with broad flexibility and control of the genetic transfer. However, targeted LVs that are based on measles virus envelope proteins exhibit lower productivity and are efficiently neutralized due to pre‐existing immunity.
In order to overcome these limitations we evaluated the glycoproteins of the canine distemper virus (CDV) for pseudotyping of LVs. Therefore, a truncated H protein of CDV was fused to the scFv specific for an artificial tag accordingly to the established Adapter‐LV technology. We demonstrate that CDV‐Adapter‐LVs confer improved productivity while maintaining equal selectivity and flexibility compared to the MV‐Adapter‐LVs. Functional CAR T cells with varying CD4/CD8 ratio were successfully generated as confirmed by in vitro assays. Additionally, reduced neutralization in presence of human serum using CDV‐Adapter‐LVs was observed.
In summary, we established a novel and flexible pseudotype for LVs. The improved productivity and reduced neutralization will enable the use of adapter‐mediated transduction in a variety of applications in the field of cell and gene therapy.
Identification and validation of target receptors for AAV9
1: University of Sheffield
Adeno‐associated virus (AAV) vectors are the major delivery vector utilised in gene therapy. Unfortunately, treatment of neurological disorders with the use of AAV gene therapy is hampered by the limited neurotropism of many serotypes. Among the AAV capsid serotypes identified, AAV serotype 9 (AAV9) shows strong neurotropism and blood brain barrier penetrability and is therefore the leading serotype for neuronal gene therapy. However, the exact mechanism by which AAV9 interacts with and transduces neuronal cells is not well known. To identify protein targets necessary for AAV9 transduction, we performed microarray analysis of porcine endothelial brain cells (PEBCs) infected with AAV9. Dysregulated targets were further validated by qPCR. Our preliminary data revealed AAV9 infection resulted in the upregulation of Syndecan‐4 (SDC4) and Solute Carrier Family 7 Member 2 (SLC7A2). We therefore hypothesise that these targets may facilitate AAV9's wide neurotropism. To investigate whether SDC4 and SLC7A2 are involved in AAV9 transduction, we established a transduction efficiency assay in AAV9 permissive cells using scAAV9‐EGFP and modulated target protein expression via targeted siRNA. Using AAV receptor (AAVR) knockdown as a positive control for reduced transduction efficiency, we found that targeted siRNA knockdown of SDC4 significantly reduced AAV9 transduction efficiency in permissive cells, while targeted knockdown of SLC7A2 did not. These data identify SDC4 as a potential protein target required for AAV9 transduction and help to further our understanding of AAV9's neurotropism which could assist future improvements to the capsid and the development of enhanced variants.
Re‐creation of stable Lentivirus Packaging Cell Lines resulting in improved viral vector production and cell growth parameters
1: Oxford Genetics Ltd 2: WuXi Advanced Therapies
Current lentiviral vector manufacturing approaches primarily use fully transient and adherent processes which, although well‐established, present challenges of robustness, scalability, and high cost‐of‐goods. To address these, we are developing suspension HEK293 cell lines for both transient and stable manufacture of lentivirus.
After adaptation of HEK293 to suspension culture and high‐throughput screening of clones, we have isolated a cell line with improved growth characteristics and transfectability, able to consistently produce lentiviral vectors at the titres of 2x108 TU/mL with our 4‐plasmids system (eGFP as Gene‐Of‐Interest). This new platform v2.0 is the optimal starting material for generating more productive packaging cell lines.
Lentivirus packaging components VSV‐G, GagPol and Rev were simultaneously integrated into the genome of platform v2.0 under a Tetracycline‐regulated promoter. Using a fully traceable, animal component‐free and optimised cell line development process, the LentiVEX Packaging cell line v2.0 was identified. It only requires transfection of the LV genome plasmid and doxycycline induction to achieve yields similar to the four‐plasmid system (2x108 TU/mL), which is also a four‐fold improvement on the previous generation, LentiVEX Packaging Cell Line v1.0. Long term stability in growth and lentiviral production of the new cell line as well as scaling up to stirred tank and testing of various therapeutically relevant GOIs are currently being assessed.
With a reduced plasmid cost and a simplified transfection process, the LentiVEX Packaging Cell Line v2.0 is a flexible platform to support both early‐phase discovery studies with easy screening of multiple GOIs and large‐scale lentiviral vector manufacture.
Engineering adenoviral vectors for a targeted delivery of therapeutic payloads
1: University of Zurich
Adenoviral vectors are the most commonly used delivery vectors for clinical gene therapy due to their favorable characteristics: (i) they do not integrate into the host cell genome and are therefore considered safe, (ii) they efficiently transduce dividing and non‐dividing cells, and (iii) they have a large packaging capacity of up to 37 kilobase pairs. However, targeted gene delivery in vivo is still compromised because of the endogenous adenoviral tropism and interactions with the host immune system.
To overcome this limitation, we developed a generic adenoviral de‐/retargeting platform consisting of a designed ankyrin repeat protein (DARPin)‐based adapter mediating cell‐specific transduction, and a single chain variable fragment (scFv)‐based shield to reduce off‐targeting as well as immune‐mediated vector clearance. In an autocrine delivery approach, we previously demonstrated efficient cancer drug delivery to tumor cells in vivo by applying tumor cell‐specific DARPin adapters to our shielded adenoviral vector. Building on this approach, we are aiming to expand our platform and enable alternative targeting strategies to achieve efficient adenoviral gene delivery for various therapeutic areas.
Here, we present the selection and development of novel DARPin‐based adapters suitable for the retargeting of human adenovirus 5‐based vectors in a variety of disease areas, including cancer and inflammatory disorders. We demonstrate efficient vector retargeting to target cells in vitro and in vivo, and show successful adenoviral delivery of therapeutic payloads to target tissues in in vivo models.
New non‐viral biocompatible gene delivery nanometric platform for controlled expression of foreign genes
1: Bar Ilan University, Dept. of Chemistry
We have developed a panel of synthetic nanohydrogels (NHGs) that complex nucleic acids with no cell toxicity. The final size of the complexes is pre‐determined by the size of the nanohydrogel. Thus we can make 50, 200 and 400 nm sized NHGs/DNA complexes.
Interestingly, the complexes can be incubated with cells for undetermined period of time with no detectable cell toxicity.
We acknowledge that gene expression becomes significant 1 week after incubation. Expression of GFP and m‐cherry plasmids last for at least 28 days in vitro and in vivo.
Unlike conventional lipo/polyplexes, our system can be incubated with cells for long periods without toxicity in vitro.
Our in vivo results suggest a continues release of plasmids from the complexes with sustained protein expression along long period of time.
The possibility of using our system for gene delivery of immunogenic proteins (vaccination) or for secreted proteins is now rationally supported by our in vivo results obtained after sub‐cutaneous administration of the complexes and their sustained expression for at least 28 days.
ADEVO: Proof‐of‐Concept of Adenovirus Directed EVOlution by Random Peptide Display on the Fiber Knob
1: Witten/Herdecke University 2: Children's Medical Research Institute 3: University College London 4: Pulawy Military Institute of Hygiene and Epidemiology
Directed evolution consists in generating a random library of vector variants and selecting the improved variants according to pre‐defined selection criteria. It already led to breakthroughs in Adeno‐associated virus vectors, but yielded limited success in Adenovirus vector (AdV) development, mainly due to technical difficulties in constructing large random libraries.
Meanwhile, AdVs applications as gene therapy or oncolytic vectors are hampered by the predetermined tropism of natural serotypes. Vector retargeting can be achieved through peptide insertions in the fiber knob domain.
Here we designed novel directed evolution protocols for AdV retargeting. We selected two protocols, both based on high‐throughput insertion of random oligonucleotides in the fiber gene, using an intermediary shuttle plasmid in protocol 1 or directly in the AdV genome in protocol 2. We constructed libraries based on adenovirus serotype 5 (Ad5) and tested if we could select variants able to infect A549‐ΔCAR cells, that lack Ad5's primary receptor and are non‐permissive for wild‐type vectors.
Protocol 1 library contained around 1.2e9 virions and 4.5e4 unique variants, against 3.8e8 virions and 3.9e5 unique variants for protocol 2 library. Variants able to infect efficiently A549‐ΔCAR cells were enriched during the selection phase by serial passaging on A549‐ΔCAR cells and could be purified and characterized.
ADEVO, our directed evolution workflow for Adenoviruses, facilitates the construction of highly diverse variant libraries and the selection of improved vectors with user‐friendly protocols. It may contribute to the development of clinically relevant adenoviral vectors with more specific tropism and higher transduction ability in target cells.
Development of new non‐viral systems for genetic modification of senescent cells
1: Centro de Investigacións Científicas Avanzadas (CICA), Universidade da Coruña 2: Instituto de Investigación Biomédica de A Coruña (INIBIC)
Niosomes are self‐assembled non‐viral nanocarriers with important advantages over conventional gene transfer vectors, due to their elevated cytocompatibility, versatility and cost‐efficiency. In this work, we explored for the first time the use of niosomes as potential carriers for genetic modification of senescent umbilical cord‐derived mesenchymal stem cells (UC‐MSCs).
Two different formulations of niosomes, containing DOTMA (cationic lipid), polysorbate 20 and poloxamer 407 (non‐ionic surfactants, 1:1 ratio), andsqualene (SQ) or cholesterol (CH) as helper lipids, were proposed. Increasing volumes of these niosome formulations were complexed with pEGFP reporter plasmid, originating assembled structures called nioplexes. The resulting niosomes and their corresponding nioplexes were characterized in terms of size and zeta potential.
Transfection assays were performed in primary cultures of non‐senescent or senescent UC‐MSCs generated using a TIS model. The transfection efficiency and cytotoxicity profiles of nioplexes were evaluated at 24 and 72 hours post‐transfection by FACS, using the commercial reagent Lipofectamine as positive control of transfection.
Niosome formulations based on SQ showed a superior transfection efficiency of non‐senescent cells when compared with those based on CH. In contrast, the highest levels of transgene expression in senescent cells were achieved with CH nioplexes. All formulations led to significantly higher levels of cell viability than those achieved by the commercial reagent Lipofectamine, regardless the cell phenotype transfected (non‐senescent vs senescent UC‐MSCs).
In conclusion, niosomes constitute effective vectors for genetic modification of senescent cells preserving their viability and providing new tools for the prevention and/or treatment of age‐related diseases.
Promoter Engineering Platform at MeiraGTx
J Sullivan1 D Lee1 C Jin1 J Schmerler1 N Fabela1 E Mossotto1 E Hernandez1 K Kytidou1 S Van der Sanden1 M Zamatkesh1 D Lokhorst1 M During1 A Forbes1
1: MeiraGTx
Promoters are essential components of a gene therapy that control the expression level and durability of a therapeutic gene. Promoters can drive cell‐specific expression of transgenes independent of capsid choice. Incorporation of stronger promoters can increase potency and efficacy at lower viral vector doses, thereby potentially decreasing safety risks, immune responses, and reducing cost of vector production. Moreover, reducing promoter size while maintaining strength and specificity allows efficient packaging of larger transgenes or expression cassettes. We have built libraries of synthetic promoters to improve the safety, efficacy, and durability of our gene therapies. Here we highlight our efforts in promoter engineering in the muscle for both constitutive and tissue‐specific promoter classes. We have developed enhanced CAG‐based promoter variants that are both stronger and smaller than the original CAG sequence. Iterative evolution of a small constitutive promoter resulted in a compact promoter that has significantly stronger muscle expression than in the liver. High‐throughput screening approaches also allow us to generate a multitude of synthetic regulatory elements that we incorporate into our promoters to boost their expression in the muscle. Our robust and diverse promoter engineering platform has yielded numerous synthetic promoters that can be applied to therapeutic transgenes. We have also successfully applied this platform to various other tissues and cell types of interest, including the central nervous system and liver.
Performance assessment of a next generation sequencing‐based assay for the quantification of DNA sequences packaged in AAV vectors
1: ProtaGene CGT GmbH, Heidelberg, 69120, Germany
In the last decade, next generation sequencing based approaches provided a better understanding of nucleic acid sequences packaged within viral vectors, allowing for the identification and monitoring of potentially oncogenic sequences. Therefore, there is a need for a deeper characterization of the performance of these methods to allow for their standardization. Here, we present a study investigating the performance of an NGS‐based workflow for the quantification of DNA species packaged within rAAV vectors.
Control samples were generated by mixing pre‐defined quantities of two research grade rAAV batches (AAV9 and AAV‐DJ) with the AAV‐DJ batch acting as a matrix (N = 3). In a first study, the precision and accuracy of the quantification were assessed, while the second focused at establishing the assay's limit of detection. The workflow started with the extraction of the nucleic acid fragments, double‐strand conversion, sonication and, finally, library preparation for sequencing on the Illumina MiSeq platform. The assay exhibited a high precision (CV ranging 0.012%–0.991% for AAV9 and 0.002%–0.468% for AAV‐DJ) and accuracy (ranging ‐22.126%–1.660% and ‐0.553%–1.677%, respectively) in the quantification of species present at frequencies ranging from 6.25% to 100.00%. The LOD was set at 5E+03 vg (species present at a frequency of 1E‐08%), with an average of 3.667 reads.
The method was shown to reliably quantify packaged sequences in vector, even in presence of high quantities of matrix interference. These data will contribute to the standardization of these approaches providing background information useful to compare assay performance throughout protocols and testing laboratories.
Interim Results from an Ongoing Global Phase 1 Study of Lentiviral Mediated Gene Therapy for Pyruvate Kinase Deficiency
1: Hospital Universitario Fundación Jiménez Díaz, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS‐FJD) 2: Unidad Mixta de Terapias Avanzadas, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS‐FJD) 3: Center for Definitive and Curative Medicine, Stanford University 4: Stanford University School of Medicine 5: Lucile Packard Children's Hospital 6: Hospital Infantil Universitario Niño Jesús (HIUNJ) 7: Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) 8: Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) 9: Rocket Pharmaceuticals, Inc.
Pyruvate kinase deficiency (PKD) is an inherited hemolytic anemia caused by mutations in the PKLR gene. Manifestations include anemia, splenomegaly and iron overload, which may be life‐threatening. Current treatments are limited to a recently‐approved enzyme activator, and also blood transfusions, chelation therapy and splenectomy which are associated with significant side effects. Based on compelling preclinical data, a global Phase 1 clinical trial RP‐L301‐0119 (NCT04105166) is underway to evaluate lentiviral mediated hematopoietic stem and progenitor cell (HSPC)‐targeted gene therapy for adults and children with severe PKD. Splenectomized patients with severe PKD (severe and/or transfusion‐dependent anemia) are eligible. Following apheresis, HSPCs are transduced with PGK‐coRPK‐WPRE lentiviral vector, and cryopreserved. Myeloablative therapeutic drug monitoring‐guided busulfan is administered over 4 days and the gene therapy product (RP‐L301) is thawed and infused. Patients are followed for safety and efficacy for 2 years post‐infusion. As of April 2022, 2 adult patients (age 31 and 47 years at enrollment) with severe anemia have received RP‐L301. At 18 months post infusion, both patients have sustained transgene expression, normalized hemoglobin, improved hemolysis and no red blood cell transfusion requirements post‐engraftment. Other parameters of hemolysis and anemia (LDH, bilirubin, erythropoietin) are improved. Both patients have anecdotally reported improved quality of life, also demonstrated by increases in both the FACT‐An and SF‐36 scores, with particularly marked improvement in SF‐36 energy/fatigue, physical functioning, and general health components. No serious adverse events (SAEs) have been attributed to RP‐L301. Updated safety and efficacy and initial pediatric experience will be presented.
The paradigm shift of gene therapy for haemophilia: impact on the patient journey
1: Het Med Occupational Health Consultancy, Amsterdam, The Netherlands 2: Department of Vascular Medicine, Amsterdam Cardiovascular Sciences, Amsterdam, The Netherlands 3: CSL Behring Europe, Hattersheim am Main, Germany
Haemophilia is an inherited bleeding disorder for which current standard‐of‐care relies on lifelong regular IV infusions of coagulation factor products (prophylaxis) from early childhood. A single IV infusion of gene therapy (GT) has shown efficacy in preventing bleeds for years in patients included in clinical trials, inducing a paradigm shift for the care of patients with haemophilia. As some GT products are likely to receive market authorization within months, a patient journey for haemophilia including GT was designed. A mixed system‐approach including ethnographical interviews of patients, family members and health care professionals (HCPs, ie. physicians, nurses or else) with experience in haemophilia care with or without experience in GT clinical trials, market research, literature screening, and peer‐to‐peer discussions, were used to identify the critical steps and the persons involved. Based on the insights from 399 patients and 270 HCPs in 7 different European countries, 12 steps were identified from diagnosis of haemophilia to long‐term follow‐up post‐administration of GT, including GT‐specific steps such as eligibility assessment, shared‐decision making process, informed consent obtention as well as patient's commitment to short‐ and long‐term monitoring. The minimum number of persons involved were 2‐7 per step, in a multidisciplinary setting. Continuous education of both patients and HCPs, and patient experience sharing were identified as conditions to help considering GT in a well‐informed and safely manner. Multidisciplinary perspective, including patient experience for designing the patient journey for haemophilia, is key to improve quality of care, especially in case of treatment paradigm shift such as GT.
Engineering of Factor IX‐secreting B cells using ZF‐Nuclease/AAV6 editing technology in a GMP‐compatible medium
M David1 D Monteferrario1 G Saviane1 C Jeanneau1 I Marchetti1 C Dupont1 C Dumont1 J Fontenot1 M De La Rosa1 2
1: Sangamo Therapeutics France, Les Cardoulines HT1, Allée de la Nertière, Valbonne Sophia‐Antipolis, 06560, France. 2: Co‐Senior authors
B cells have the capacity to differentiate into plasmablast/plasma cells (PCs), cellular factories capable of producing high amounts of antibodies for years. Highly specific ZF‐nuclease editing opens the possibility of engineering plasmablast/PCs capable of secreting sustained and high levels of therapeutic proteins. We established an advanced in vitro culture system using a serum free GMP‐compatible medium, optimizing cell viability, expansion rate and differentiation of B cells into plasmablast/PCs. Using this culture system, highly specific editing of B cells was obtained by combining electroporation of ZF‐nuclease mRNA with AAV6 donor DNA, to achieve site‐specific insertion of the human Factor IX Padua (FIX‐B cells) into the silent TRAC locus. In vitro analysis of FIX‐B cells revealed high levels of secreted human immunoglobulins (huIg) and up to 30 ng/mL of FIX‐Padua suggesting fully functional engineered FIX‐B cells. To assess the therapeutic potential of FIX‐B cells in vivo, FIX‐B cells were administered intravenously to NSG mice pre‐engrafted with human memory T cells. After four weeks, human PCs were detected in spleen and bone marrow, indicating successful and potential long‐term engraftment in mice. Moreover, a stable and functional engraftment of FIX‐B cells is suggested since we observed high levels of huIg (up to 2500 μg/mL IgM) and therapeutic FIX‐Padua levels in mouse plasma (up to 90 ng/mL). The plasma FIX levels correlate with about 15% of normal human FIX activity. This study highlights the potential of engineered B cells as a new cell therapy platform to treat a variety of protein deficiencies.
In vivo delivery of CRISPR‐Cas9 using lipid nanoparticles enables antithrombin gene editing for sustainable hemophilia A and B therapy
1: Seoul National University 2: Ewha Woman's University 3: Toolgen Inc.
Hemophilia is a hereditary disease that remains incurable. Although innovative treatments such as gene therapy or bispecific antibody therapy have been introduced, substantial unmet needs still exist with respect to achieving long‐lasting therapeutic effects and treatment options for inhibitor patients. Antithrombin (AT), an endogenous negative regulator of thrombin generation, is a potent genome editing target for sustainable treatment of patients with hemophilia A and B. In this study, we developed and optimized lipid nanoparticles (LNPs) to deliver Cas9 mRNA along with single guide RNA that targeted AT in the mouse liver. The LNP‐mediated CRISPR‐Cas9 delivery resulted in the inhibition of AT that led to improvement in thrombin generation. Bleeding‐associated phenotypes were recovered in both hemophilia A and B mice. No active off‐targets, liver‐induced toxicity, and substantial anti‐Cas9 immune responses were detected, indicating that the LNP‐mediated CRISPR‐Cas9 delivery was a safe and efficient approach for hemophilia therapy.
Correction of haematopoietic stem cells from Bernard‐Soulier Syndrome patients and disease models by overexpression of GP9 with lentiviral vectors
1: GENYO. Centre for Genomics and Oncological Research: Pfizer / University of Granada / Andalusian Regional Government 2: University of Granada 3: Department of Haematology and Medical Oncology, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB‐Arrixaca, CIBERER‐U765, 30008 Murcia, Spain.
Platelets recognize injured vascular vessels by the interaction between GPIB‐V‐IX platelet receptor and von Willebrand factor (VWF), contained in the exposed subendothelial matrix of the damaged area. The absence of this receptor impedes proper plug formation because platelets cannot recognize damaged vessels. This is the origin of an extremely rare disease denominated Bernard‐Soulier Syndrome (BSS). Mutations of GPIB‐V‐IX receptor genes avoid its migration to the membrane and consequently clot formation.
Last decades, several gene therapy approaches for hematologic diseases have been successfully carried out. Our lab has developed a novel treatment for BSS subtype C based on the use of Lentiviral Vectors (LVs) to revert the disease originated by GP9 mutations.
Firstly, we have reverted the disease phenotype on a GP9‐Knock‐out megakaryoblastic cell line (DAMI), which regularly expresses the GPIB‐V‐IX complex on its surface. Secondly, we have rescued a BSS model based on iPSCs from BSS patients. This model allows us to analyze our LVs behaviour along a differentiation process. Finally, we have isolated Hematopoietic Stem Cells from BSS peripheral blood samples and treated them with our LVs. The reappearance of the complex after a differentiation towards megakaryocytic lineage indicates the reversal of BSS in vitro. All together our results demonstrated that we can revert GPIX expression on human BSS models.
Next, we will complete rescue experiments in a GP9‐knock‐out BSS animal model. We consider that all these results will allow us to propose an ex vivo curative LV‐based gene therapy on Hematopoietic Stem Cells from BSS patients.
Young mice administered adult doses of AAV5‐hFVIII‐SQ achieve therapeutic factor VIII expression into adulthood
1: BioMarin Pharmaceutical
Hemophilia A (HA) is an X‐linked genetic bleeding disorder caused by a deficiency in the activity of coagulation factor VIII (FVIII). Valoctocogene roxaparvovec (AAV5‐hFVIII‐SQ) gene transfer provided reduced bleeding for adult clinical trial participants with severe hemophilia A. However, pediatric clinical feasibility and outcomes are unknown. Using a mouse model of hemophilia A, we investigated the effect of vector dose on transgene production and persistence in neonatal vs adult mice. We dosed AAV5‐hFVIII‐SQ to neonatal and adult mice based on body weight or at a fixed dose and assessed human factor VIII SQ‐variant (hFVIII‐SQ) expression through 16 weeks. AAV5‐hFVIII‐SQ dosed per body weight in neonatal mice did not result in meaningful plasma hFVIII‐SQ protein in adulthood. When treated with the same total vector genomes per mouse as adult mice, neonates expressed therapeutic levels of hFVIII‐SQ protein into adulthood. Quantitative polymerase chain reaction (qPCR) showed neonatal mouse livers were capable of taking up the same amount of vector genomes as adults when given the same absolute dose. No features of hepatotoxicity or endoplasmic reticulum stress were observed. These data suggest that young mice require the same total vector genomes as adult mice to achieve and sustain meaningful hFVIII‐SQ plasma levels.
Genome editing‐mediated knock‐in of therapeutic gene ameliorate disease phenotype in hemophilic model
1: Graduate School of International Agricultural Technology and Green and Institute of Green BioScience and Technology, Seoul National University; Pyeongchang, Gangwon, 25354, Korea 2: Toolgen Inc.; Geumcheon‐gu, Seoul, 08501, Korea 3: WCU Biomodulation Major, Department of Agricultural Biotechnology, Seoul National University; Gwanank‐gu, Seoul, 08826, Korea
Recently, clinical trials of adeno‐associated virus‐mediated replacement therapy have suggested long‐term therapeutic effects for several genetic diseases of the liver, including hemophilia. However, there are still concerns regarding decreased therapeutic effects when the liver is regenerated or when physiological proliferation occurs. Although genome editing using the clustered regularly interspaced short palindromic repeats/Cas9 system provides an opportunity to solve this problem, low knock‐in efficiency may limit its application for therapeutically relevant expression. Here, we identified a novel gene, APOC3, in which a strong promoter facilitated the expression of knocked‐in genes in hepatocytes. We also investigated the effects of APOC3 editing using a small Cas9 protein derived from Campylobacter jejuni (CjCas9) in a hemophilic model. We demonstrated that adeno‐associated virus‐mediated delivery of CjCas9 and donor led to moderate levels of human factor 9 expression in APOC3‐humanized mice. Moreover, knock‐in‐driven expression induced substantial recovery of clotting function in mice with hemophilia B. There was no evidence of off‐target editing in vitro or in vivo. Collectively, our findings demonstrated therapeutically relevant expression using a precise and efficient APOC3‐editing platform, providing insights into the development of further long‐term therapeutics for diverse monogenic liver diseases.
AAV Amplification and DNA Sequencing of Factor VIII and Factor IX Concentrates
1: Pfizer Ltd 2: Pfizer Inc 3: *currently at Moderna 4: Siemens Healthineers
Neutralizing antibodies (NAbs) to adeno‐associated virus (AAV) may exclude individuals from AAV‐based gene therapy. The aim of this work was to analyze a range of commercially available plasma and recombinant Factor VIII (FVIII) and Factor IX (FIX) products to determine if AAV sequences could be detected.
Plasma‐derived FVIII and FIX lots and recombinant FVIII and FIX lots were sourced. A nested and tailed PCR primer approach was utilized for amplification ahead of short‐read DNA sequencing. Oligonucleotides were designed in locations conserved across multiple AAV serotype reference sequences. The assay was selected through in‐silico analysis and functional performance evaluations. Spike‐in controls based on an AAV4 reference sequence and synthetic sequence tags were developed for QC and detection of cross‐contamination
One plasma‐derived concentrate lot produced 5 positive results from 7 reactions to amplify AAV. DNA sequencing reads were high‐quality and consistent across all positive replicates. While the AAV sequence was distinct from our control material, the amplified region was too short to unambiguously match to a single serotype.
This exploratory work was conducted to determine whether there could be a mechanistic basis to a previously reported clinical observation which reported increased seroprevalence of NAbs to AAV in some patients who had received plasma‐derived blood products. A highly sensitive assay was developed, and preliminary results indicate a potential for the presence of AAV and other viral serotypes in some of the plasma‐derived blood products. Further analysis to determine the consequences of these findings would be required to confirm this hypothesis.
RBD‐CURE: A new gene therapy platform for Rare Bleeding Disorders
1: Sanquin Research 2: SanaGen, B.V. 3: Netherland Institute for Neuroscience
Deficiencies of coagulation factors other than factor VIII or IX are the cause of rare bleeding disorders (RBDs) with prevalence in the general population of 1:500,000. The only treatment option, when available, is a recurrent and lifelong factor concentrate infusion, which is both a burden for both patients and healthcare system. Our aim is to generate an AAV‐mediated, liver‐directed gene therapy treatment, which has the potential to be a one‐time, durable and more efficient cure for multiple type of RDBs.
Our optimization approach is based on: 1) adapting the AAV capsid to be more liver specific; 2) improving efficiency and specificity of the therapeutic expression cassette; and 3) adapting the therapeutic efficacy of the recombinant therapeutic factor.
Preliminary results suggested that our rational capsid engineering of surface‐exposed loops successfully generated at least two stable AAV candidates with specific in vivo hepatic localization. Furthermore, in vitro selection of novel rationally designed liver‐specific promoters also showed multiple candidates with high liver‐specificity. Moreover, differential in vitro expression suggested different codon‐optimized strategies may have an impact on gene expression levels. Additionally, proof‐of‐concept in vivo studies showed that AAV‐directed gene therapy can successfully express and release biologically active hFVII protein into the circulation. hFVII antigen levels were such that they could potentially rescue the bleeding phenotype.
Our results support further (pre)clinical development of the project. Development of the platform technology to other liver‐specific disorders is currently ongoing.
Lentiviral‐mediated Gene Therapy for Patients with Fanconi Anemia [Group A]: Results from Global RP‐L102 Clinical Trials
1: Hospital Infantil Universitario Niño Jesús (HIUNJ) 2: Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) 3: Center for Definitive and Curative Medicine, Stanford University 4: Stanford University School of Medicine 5: Lucile Packard Children's Hospital 6: UCL Great Ormond Street Institute of Child Health 7: Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) 8: Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS‐FJD) 9: Rocket Pharmaceuticals, Inc. 10: University of Minnesota
Fanconi anemia (FA) is an inherited DNA repair disorder that results in childhood bone marrow failure (BMF) in 80%. Allogeneic hematopoietic stem cell transplant (alloHSCT) is potentially curative for FA‐related BMF. While survival approaches 90% in children in experienced transplant centers, late effects, including increased cancer risk, limit overall success. The current gene therapy studies utilize autologous FA‐A CD34+ enriched hematopoietic stem and progenitor cells (HSPCs), and rely upon the proliferative advantage of gene‐corrected FA HSPCs, enabling engraftment without antecedent conditioning, as demonstrated in pre‐clinical studies and the FANCOLEN‐I clinical trial. We report results from global RP‐L102 studies. Patients (pts) with a FANCA mutation, age ≥1 year with no HLA‐matched sibling donor and at least 30 CD34+ cells/μL in bone marrow (BM) are eligible. Following leukapheresis, CD34+ cells are transduced with a lentiviral vector carrying the FANCA gene, and infused fresh without conditioning. Pts are followed for 3 years post‐infusion for safety and efficacy evaluations. As of April 2022, 12 pts age 2 to 6 years received RP‐L102. Sustained engraftment has been demonstrated in 6 of 9 evaluable pts with ≥12 months of follow up as indicated by PB VCN. Five of these 6 have increasing BM CFC MMC resistance to levels ≥20%, accompanied by hematologic stability across all lineages. One pt had progressive BMF and underwent successful alloHSCT. One pt had a transient serious Grade 2 RP‐L102 infusion‐related reaction which resolved without sequelae. Updated safety and efficacy data for pts with ≥12 months of follow up will be presented.
Therapeutic genome editing based bypassing strategy for Hemophilia A and B
1: Toolgen Inc.
Hemophilia is an inherited bleeding disorder where affected patients lack coagulation factor for normal blood clotting. Recombinant coagulation factor treatment options are available for these patients, however, repeated intravenous administration urged gene replacement therapy development for Hemophilia. Although, AAV‐based FVIII or FIX gene replacement therapy for Hemophilia A or B respectively showed promise in clinics for longer term therapeutic efficacy over protein drugs, these therapeutic candidates may lose their transgene expression over time and cannot be utilised for subset of patients with FVIII or FIX. To overcome this, we targeted antithrombin, an endogenous negative regulator of downstream thrombin generation, antithrombin, an endogenous negative regulator of downstream thrombin generation, in an attempt to re‐balance coagulation by bypassing upstream FVIII or FIX pathway to generate thrombin. We developed a CRISPR/Cas9‐based antithrombin editing strategy and delivered to mouse liver by lipid nanoparticle (LNP). Efficient and specific editing of antithrombin was achieved and no aberrant toxicity was found. Most importantly, when delivered to both hemophilia A and B mice, our therapeutic LNP showed significant thrombin generation followed by improvements in bleeding‐associated phenotypes. Our study holds promise to treat both Hemophilia A and B and may also be applicable to patients with FVIII or FIX inhibitors.
Hematopoietic Gene Editing of the Telomerase RNA Component: An approach for the treatment of autosomal dominant dyskeratosis congenita
1: Division of Hematopoietic Innovative Therapies, Biomedical Innovation Unit, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas (CIEMAT), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) and Advanced Therapies Unit ‐ Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS‐FJD/UAM). Madrid, 28040. Spain. 2: Instituto de Investigaciones Biomédicas “Alberto Sols” (CSIC/UAM) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER). Madrid, 28029. Spain. 3: Division of Epithelial Biomedicine, Biomedical Innovation Unit, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas (CIEMAT), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER). Madrid, 28040. Spain.
Dyskeratosis congenita (DC) is a rare inherited bone marrow failure (BMF) syndrome caused by mutations in genes involved in telomere maintenance, thus leading to premature telomere shortening. One of the most prevalent mutated gene in DC is TERC, which encodes the telomerase non‐coding RNA component (TERC) and exhibits autosomal dominant inheritance (AD‐DC). The allogeneic transplant of hematopoietic stem cells (HSCs) is the only curative treatment of the BMF of AD‐DC patients. However, several limitations hamper the success of these transplantations in a high percentage of DC patients. In order to develop an alternative curative treatment for BMF characteristic of AD‐DC we propose a gene editing approach based on the ex vivo correction of autologous HSCs using CRISPR/Cas9 nucleases and adeno‐associated vectors (AAVs). CRISPR/Cas9 system produced a double strand break (DSB) that would be repaired by homologous recombination (HR) using TERC homology arms delivered by the AAVs. Under optimized conditions, the HDR efficiency in HD cord blood CD34+ cells ranged between 60‐90%, as deduced from analyses of the 3’ region of the TERC donor in total cells and also in colony forming units. Analysis of the RNA expression and maturation pattern of edited HD CD34+ cells showed that none of these processes were affected as consequence of editing approach. More recently, edited CD34+ cells have also been transplanted into immunocompromised mice to evaluate the repopulation capacity and the editing efficiency in edited repopulating cells. Next steps will aim to demonstrate phenotypic correction in TERC‐mutated deficient models and HSC from TERC‐deficient patients.
Improved efficacy of Mesenchymal Stromal Cells stably expressing CXCR4 and IL10 in a preclinical Graft versus Host Disease Humanized Mouse Model
R Hervás‐Salcedo1
1: Division of Hematopoietic Innovative Therapies, Biomedical Innovation Unit, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas (CIEMAT), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) and Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS‐FJD, UAM), Madrid 28040. Spain.
Mesenchymal stromal cells (MSCs) currently constitute one of the cell types more frequently used in advanced therapies. While previous pre‐clinical data have shown that MSCs could reduce graft versus host disease (GvHD) after allogeneic hematopoietic stem cells transplantation, only moderate therapeutic effects have been observed in clinical trials. With the aim of increasing the anti‐GvHD effect of these cells, adipose tissue derived human MSCs (Ad‐MSCs) were transduced with a bicistronic lentiviral vector carrying the cDNAs of CXCR4, a molecule involved in cell migration to inflamed sites, and IL10, a cytokine with potent anti‐inflammatory properties. In vitro experiments showed that the stable expression of these molecules in Ad‐MSCs (CXCR4‐IL10‐MSCs) efficiently enhanced their migration towards SDF1 when compared to unmodified‐Ad‐MSCs. Moreover, CXCR4‐IL10‐MSCs displayed enhanced capacity to inhibit activated T cells proliferation. Using a humanized GvHD mouse model generated by human peripheral blood mononuclear cells infusion into immunodeficient NSG mice, we demonstrated a significant reduction in the clinical and histological signs severity of the disease in mice treated with a dose of CXCR4‐IL10‐MSCs compared to unmodified‐Ad‐MSCs. Additionally, CXCR4‐IL10‐MSCs reduced pro‐inflammatory Th1 and Th17 cells proliferation, and induced a polarization of T cells towards an anti‐inflammatory profile (CD3+‐IL10+ cells). Importantly, a significant increase in the number of regulatory B and T cells was observed after CXCR4‐IL10‐MSCs infusion compared to unmodified‐Ad‐MSCs. Taken together, our preclinical studies strongly suggest that the lentiviral‐mediated expression of CXCR4 and IL10 should improve the clinical efficacy of MSC‐based cell therapies to treat GvHD in patients transplanted with allogeneic hematopoietic grafts
Therapeutic Base and Prime Editing in Fanconi Anemia Hematopoietic Stem and Progenitor Cells
L Ugalde M E Karasu B Olalla S Siegner L Garcia A Clemens L Alvarez I Peña J A Bueren J E Corn
Current clinical studies show the therapeutic potential of lentiviral‐mediated gene therapy in patients with Fanconi anemia (FA). In an attempt to develop alternative gene therapy approaches in FA, we have previously shown that NHEJ‐CRISPR/Cas9 mediated gene editing can remove/compensate specific mutations in different FA genes.
To improve the safety and the efficacy of gene editing in FA HSPCs, we have now focused on the use of Base (BE) and Prime editing (PE) strategies to edit FA genes, since they do not generate double strand breaks (DSBs) in the cell genome and do not require a donor template. As a proof of concept we focused on a frequent mutation present in FA patients from Spain, which results in a stop codon in exon 4 of FANCA (c.295 C>T). Using BE technology we generated a therapeutic base conversion in >50% BM HSPCs from FA patients that harbor this mutation. The efficacy of the genetic conversion was confirmed by the correction of the characteristic phenotype of FA cells. Using an optimized Prime editing approach, combining the use of a PEmax architecture in synergy with the PE3 system, the FA mutated sequence was precisely corrected to the WT sequence in up to 40% of cells from a FA‐A lymphoblastic cell line. As expected, corrected cells showed a marked proliferative advantage together with an evident phenotypic correction. Altogether, our results suggest that BE and PE may constitute alternative therapeutic approaches for the treatment of bone marrow failure in FA patients carrying specific mutations.
Chemically surface modified AAV6 vectors combined with nanoblades are a promising alternative for gene knock‐in in HSCs
1: CIRI; Inserm U1111 2: Nantes Université, CNRS, CEISAM UMR 6230 3: CHU de Nantes, TaRGeT, INSERM UMR 1089 4: Université de Nice, INSERM C3M
Nanoblades, a new technology based on a modified murine leukaemia virus, where the viral structure protein gag is fused to Cas9 and the viral particles are loaded with the Cas9 protein complexed with the gRNA and devoid of any viral genome. We showed that nanoblades were remarkably efficient for entry into human T, B and HSCs thanks to their surface co‐pseudotyping with baboon retroviral and VSVG envelope glycoproteins. Incubation of rAAV‐6 vector containing 2 homologous arms to the Wiskott‐Aldrich syndrome (WAS) locus flanking a GFP expression cassette with nanoblades, resulted in up to 40 % of stable expression cassette knock‐in into the WAS gene locus in HSC. However, higher rAAV‐6 doses increased HSC cell death. Comparing rAAV6 with rAAV2 encoding the donor DNA, we demonstrated that at high doses, AAV2 was much less toxic and gave higher transduction levels in HSCs. To improve donor template delivery at low vector doses, rAAV2 and rAAV6 were chemically bio‐conjugated with a ligand, via the amino‐acid residues exposed at the capsid surface, in different positions and quantities. Initial results showed high level transduction of HSCs with ligand coupled rAAV6 vectors accompanied by a remarkable lower toxicity compared to the WT‐rAAV6. Bio‐conjugated rAAV6 combined with nanoblades increased gene knock‐in and survival of HSCs from 40% to 80% as compared to the WT‐rAAV6.
To sum up, the introduction of a specific ligand on rAAV6 increased donor mediated gene knock‐in and significantly increased HSC survival, an important feature for clinical translation of HSC gene editing strategies
Transient overexpression of engineered engraftment enhancers to increase hematopoietic stem cell homing and retention in a chemotherapy‐free conditioning regimen
1: San Raffaele Telethon Institute for Gene Therapy (HSR‐TIGET) 2: University Vita‐Salute San Raffaele 3: Equally contributing authors
In current HSPC gene therapy, ex vivo genetically corrected HSPCs are infused back to the patient after conditioning regimens aiming to deplete the resident cells in the bone marrow (BM), creating space for the corrected cells. Recently, we showed that HSPC mobilization by G‐CSF, AMD3100 and BIO5192 creates a window of opportunity for engraftment of ex vivo‐cultured cells, which effectively outcompete those mobilized in the circulation to repopulate the depleted BM niche. Transient overexpression of the CXCR4 homing receptor endowed transplanted cells with further competitive advantage over the mobilized recipient ones, resulting in higher exchange. We also demonstrated how this strategy could be implemented in a standard gene editing protocol without requirement of additional cell manipulation, resulting in increased engraftment efficiency.
To further improve the advantage of donor cells, here we investigated the use of CXCR4 variants resistant to its antagonist ligand AMD3100. Transient over‐expression of these variants by an optimized mRNA platform led to increased CXCR4 surface expression, endowing human HSPCs with partial resistance to CXCR4 antagonists in an in vitro migration assay. Preliminary results support the advantage of these variants in enhancing in vivo engraftment in immunodeficient mice. Ongoing studies also investigate the synergistic effect of transient overexpression of more than one engraftment enhancer, as c‐KIT and ITGA4, or anti‐phagocytosis signals, as CD47, which already proved to increase engraftment efficiency when used alone.
Coupling HSPC mobilization with transient overexpression of engineered engraftment enhancers resistant to the mobilizers might ultimately broaden mobilization‐based HSCT efficiency and applicability beyond autologous HSCT.
Analyzing gene editing enhancers and hematopoietic stem and Progenitor cell clonality using DNA barcoding for the treatment of Pyruvate Kinase Deficiency
I Ojeda‐Perez1 2 A Bustos3 S Fañanas‐Baquero1 2 M Dessy‐Rodriguez1 2 V Lang4 O Alberquilla1 2 A Garcia‐Torralba1 2 R Torres‐Ruiz2 5 6 S Rodriguez‐Perales5 R Sanchez‐Dominguez1 2 C Trigueros4 R Mayo‐Garcia3 O Quintana‐Bustamante1 2
1: Cell Technology Division. CIEMAT‐CIBERER. Madrid. Spain 2: Unidad Mixta Terapias Avanzadas. IIS‐FJD. Madrid. Spain 3: Scientific IT. CIEMAT. Madrid. Spain 4: Viralgen. San Sebastian. Spain 5: Molecular Cytogenetics and Genome Editin Unit. CNIO. Madrid. Spain 6: Division Terapias Avanzadas en Sistema Hematopoyetico. CIEMAT‐CIBERER. Madrid. Spain
Pyruvate kinase deficiency (PKD) is an autosomal recessive disorder caused by mutations in PKLR gene. PKD‐erythrocytes suffer from energy imbalanced caused by reduction of erythroid pyruvate kinase (RPK) activity. PKD is associated with reticulocytosis, splenomegaly and iron overload, and may be life‐threatening. We developed a knock‐in gene editing strategy by combining ribonucleoprotein electroporation and adeno‐associated viral vector (rAAV6) donor delivery, getting 40% editing in hematopoietic stem and progenitor cells (HSPCs). To further increase gene editing efficacy, we divided the procedure in different parts (AAV transduction, homology repair pathway, or maintenance of the stem cell properties), and assessed how different modifications could enhance HDR efficiency. Since HDR gene editing is a homogeneous modification of the genome, clonality studies of edited cells cannot be ruled out. DNA barcoding allows individual tracking of cell progeny. We developed a donor DNA barcode AAV6 library and used it to further study the different conditions. Improved conditions, including reduction in cell concentration and addition of SR1 reagent, reached a high in vitro targeting efficiency, up to 70% targeted HSPCs. Gene editing efficiencies in cell engrafting NSG mice were slightly reduced. Analysis of clonal output in vivo showed up to 300 individually marked HSPCs, with 9 to 12 unique HSPCs clones per mouse responsible for 90% of the human hematopoietic engraftment. No evidence of progressive clonal dominance in any of the conditions tested was observed.
Our data define optimized conditions to get high HDR efficacy and clonality in HSPCs moving PKD gene editing towards its clinical applicability.
Towards lentiviral‐mediated gene therapy for RPS19‐Diamond Blackfan anemia patients
Y Giménez1 2 M Palacios1 2 R Sanchez1 2 C Zorbas3 J Peral1 2 A Puzik4 L Ugalde1 2 O Alberquilla2 P Río1 2 E Galvez5 L Da Costa6 M Strullu6 A Catalá7 A Ruiz7 J C Segovia1 2 J Sevilla5 B Strahm4 C Niemeyer4 C Beléndez8 T Leblanc6 D L.J Lafontaines3 J A Bueren1 2
1: CIEMAT/CIBERER 2: Instituto de Investigación Sanitaria‐Fundación Jiménez Díaz 3: ULB‐Cancer Research Center (U‐CRC), Université Libre de Bruxelles 4: University Medical Center Freiburg 5: Hospital del Niño Jesús 6: Hôpital Robert‐Debre 7: Hospital San Joan D´Deu 8: Hospital Gregorio Marañón
Diamond Blackfan anemia (DBA) is a rare bone marrow failure syndrome (BMFS) (prevalence 7/1,000,000) with the clinical hallmark of anemia due to erytroblastopenia. Allogenic hematopoietic stem cell transplantation (HSCT) represents the only curative treatment for DBA hematological signs. Mutations in ribosomal protein gene RPS19 are present in 25% of DBA patients. Therefore, we have developed a clinically applicable lentiviral mediated ex vivo hematopoietic gene therapy approach for the treatment of RPS19‐haploinsuficient DBA patients. First we demonstrated the therapeutic efficacy of two different therapeutic LVs (PGK.CoRPS19.Wpre* and EF1α(s).CoRPS19.Wpre*‐LVs) in primary CD34+ cells from RPS19‐deficient patients. This was deduced from the observation that transduction with either therapeutic vector significantly increased the number erythroid progenitors and also reverted the red blood cell differentiation defect characteristic of DBA CD34+ cells, as inferred from the increased output of CD71‐/CD235+ mature erythroid cells. Remarkably, CD34+ DBA cells transduced with the PGK.CoRPS19.Wpre* LV had a preserved hematopoietic repopulating potential in immunodeficient mice, compared to healthy donor (HD) CD34+ cells, and also improved the erythroid differentiation compared to samples transduced with a control EGFP‐LV.. The health status of transplanted immunodeficient recipients and the polyclonal integrational pattern of the therapeutic provirus in transplanted hematopoiesis evidenced the safety of this therapeutic approach. Taken together, these preclinical studies support the notion that the lentiviral‐mediated gene therapy should constitute an efficient and safe approach for the treatment of RPS19‐deficient DBA patients.
Efficient knockout of both CD33 and CLL‐1 by multiplex genome editing of human hematopoietic stem cells enhances the potential of next‐generation transplants for acute myeloid leukemia (AML) treatment
J X Ferrucio1 G Angelini1 S Krishnamurthy1 N Patel1 M Pettiglio1 T Collingsworth1 A Halfond1 C Cummins1 J Etchin1 Y Keschner1 G Mundelboim1 H Qiu1 R Wang1 G Ge1 J Lydeard1
1: Vor Biopharma
AML is the most common form of adult acute leukemia characterized by abnormal clonal expansion, with median 5‐year survival rate <30%. The standard of care for AML treatment is allogeneic stem cell transplant, yet ∼40% of patients relapse post‐transplant, highlighting the need for new therapeutic approaches. CD33 and CLL‐1 are expressed in >85% of AML cells, thus targeting both can address disease heterogeneity and potential for antigen escape. Both antigens are also expressed on normal hematopoietic cells, thereby impeding immunotherapy due to myelotoxicity. We propose deleting both CD33 and CLL‐1 from allogeneic stem cell grafts and consequently, confining these antigens exclusively to AML cells to enable subsequent immunotherapies. CD34+ human hematopoietic stem and progenitor cells (hHSPCs) were sequentially gene edited in order to delete CD33 and CLL‐1, resulting in biallelic deletion of both genes in >80% cells without impacting viability (>90%), myeloid differentiation, phagocytic function or cytokine secretion. Importantly, multiplex‐edited hHSPCs showed successful long‐term engraftment with unaltered multilineage reconstitution in xenotransplantation models. CD33 and CLL‐1 were simultaneously ablated from >95% of myeloid cells, confirming long‐term persistence of biallelic editing at both genes with no impact on myeloid differentiation in vivo. Next‐generation sequencing revealed high bone marrow editing frequencies at 16 weeks post‐engraftment. These data indicate that dual‐engineered hHSPCs can persist long‐term without being counter‐selected. We also demonstrated that CD33 and CLL‐1 dual‐edited cells were resistant to CD33 and/or CLL‐1 CAR‐T cytotoxicity, validating the core concept of killing tumor cells with immunotherapy while protecting the edited graft.
Lentiviral vector mediated in vivo gene transfer into hematopoietic stem and progenitor cells
1: San Raffaele Telethon Institute for Gene Therapy (SR‐TIGET) 2: Vita‐Salute San Raffaele University
Lentiviral vector (LV) mediated ex vivo gene therapy in hematopoietic stem and progenitor cells (HSPC) has shown clinical benefit in patients with different genetic diseases. However, ex vivo manipulation and patient conditioning still pose challenges to broad access to HSPC gene therapy. To investigate the feasibility of in vivo LV‐mediated gene transfer into HSPC, we systemically administered GFP‐expressing LV to adult mice or newborns and found only in the latter stable GFP expression in 1% of all blood lineages up to 1‐year. We observed comparable GFP marking in hematopoietic organs and in HSPC harvested from the bone marrow (BM), which were able to engraft long‐term in busulfan‐conditioned mice. LV integration site analysis confirmed common origin of different hematopoietic lineages from multiple clones. These findings indicate successful in vivo gene transfer into bona fide HSC favoured by unique features of newborn hematopoiesis, such as access to hepatic hematopoietic niches persisting after birth, and HSC‐trafficking to the BM. To increase gene transfer efficiency we exploited phagocytosis‐shielded CD47‐high LV and showed 3‐fold higher gene marking in the blood and BM of treated mice. Biodistribution studies showed transgene‐positive cells in liver, spleen, lung, kidney, and brain, comprising hematopoietic, endothelial, and liver parenchymal cells. As a systemic disease model, we administered LV expressing adenosine deaminase (ADA) enzyme to ADA‐SCID newborn mice and showed rescue from lethal phenotype and reconstitution of lymphocyte counts with selective advantage of LV‐transduced cells. Further studies are ongoing to explore in vivo LV gene transfer into HSPC in other disease models.
Phenotypic correction of hematopoietic stem and progenitor cells from Fanconi anemia group A knockout mice using non‐viral gene therapy with Sleeping Beauty transposon vectors
1: Paul Ehrlich Institute 2: CIEMAT/CIBERER 3: Francisco de Vitoria University
Lentiviral‐mediated gene therapy of human CD34+ hematopoietic stem cells (HSCs) derived from Fanconi anemia subtype A (FA‐A) patients confers engraftment and proliferation advantage of phenotypically corrected HSCs in immunodeficient mice and non‐conditioned FA‐A patients. We have demonstrated the applicability of the non‐viral Sleeping Beauty (SB) transposon system for efficient and stable gene delivery in human CD34+ cells. Here we address the feasibility of using this system for preclinical gene therapy in a Fanca‐/‐ mouse model. Nucleofection of wild‐type Lineage negative (Lin‐) cells with a minicircle (MC) SB transposon vector expressing the Venus fluorescent protein together with mRNA encoding the hyperactive SB100X transposase resulted in >70% Venus+ cells 14 days post‐electroporation. Venus+ cells maintained their ability to proliferate and differentiate in vitro and efficiently engrafted primary and secondary recipient mice. Following MC‐based delivery of a SB transposon vector expressing FANCA and GFP transgenes in murine Fanca‐/‐ Lin‐ cells, approximately 20% of the cells maintained GFP expression for more than 26 days in liquid culture and corrected mytomicin C (MMC) hypersensitivity. Preliminary in vivo data indicate that SB‐mediated gene therapy of Fanca‐/‐ HSCs results in a progressive expansion of genetically corrected cells in lethally irradiated Fanca‐/‐ mice. MMC treatment of mice with poor initial engraftment levels leads to an increase in frequency of GFP+ cells in peripheral blood. The SB transposon system is a very promising non‐viral alternative for gene transfer in HSCs that could be employed in future clinical gene therapy protocols for patients with inherited hematopoietic diseases such as FA.
Adenosine Deaminase 2 Deficiency: from the Understanding of the Bone Marrow Pathogenesis to the Development of Hematopoietic Stem Cell Gene Therapy
1: Telethon Institute of Gene Therapy (HSR‐TIGET) 2: University Vita‐Salute San Raffaele 3: Pediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute
Deficiency of adenosine deaminase 2 (DADA2) is an inborn error of immunity caused by loss‐of‐function mutations in the ADA2 gene. Manifestations include vasculopathy and immunological and hematological abnormalities. The current clinical management is a life‐long anti‐TNF therapy, and hematopoietic stem and progenitor cell (HSPC) transplantation is recommended only in the most severe patients. There are no data on how lack of ADA2 causes bone marrow (BM) failure. Therefore, understanding these mechanisms is essential for developing new precise therapies. Phenotypic analysis of the BM from DADA2 patients revealed that the HSPC compartment was significantly reduced compared with age‐matched healthy donors (HDs). Although patients' HSPCs were reduced in number, their clonogenic or multilineage differentiation capacities remained unaltered. To investigate the BM defects in DADA2, we focused on the mesenchymal stromal cells (MSCs). DADA2 MSCs expressed low levels of a primitive marker while senescence markers increased compared with HD's MSCs. To assess whether HSPC gene therapy could constitute a valuable treatment to ameliorate disease manifestations, we developed a third‐generation lentiviral vector (LV). Transduction of HSPCs allowed efficient delivery of functional ADA2 in patients' CD34+ cells. Colony‐forming cell assay exhibited high transduction efficiency with a concomitant absence of toxicity. Transduced HD's HSPCs infused into immunodeficient mice supported a multilineage reconstitution with a polyclonal integration pattern, and integration site analysis confirmed the absence of genotoxicity. Our results indicate that LV‐mediated ADA2 reconstitution is efficient in re‐establishing ADA2 activity and correcting the hematological manifestations in DADA2.
Hematopoietic stem cell behavior in gene therapy treated beta‐thalassemia patients
1: San Raffaele‐Telethon Institute for Gene Therapy (SR‐TIGET), San Raffaele Scientific Institute Milan, Italy 2: Wellcome ‐ MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK 3: Institute for Biomedical Technologies, National Research Council, Segrate, Italy 4: Haematology and BMT Unit, San Raffaele Scientific Institute, Milan, Italy 5: Pediatric Immunohematology, San Raffaele Scientific Institute, Milan, Italy, 6: University Vita‐Salute San Raffaele, Milan, Italy
Hematopoiesis is a continuous differentiation process and is maintained by distinct stem/progenitor cell populations in native and stress states, indicating that the hematopoietic hierarchy roadmap is flexible to adapt to different conditions. Recently, it has been described that stress and inflammatory signals influence hematopoietic stem cell (HSC) fate and blood output. The stressed bone marrow microenvironment, present in Beta‐thalassemia (Bthal), could have an impact on the lineage commitment of HSCs leading to a unique model of hematopoiesis. Our aim is to understand how the priming and kinetics of HSCs undergoing lineage specification are modulated by a stress environment. Our transcriptome analysis reveals a downregulation of several pathways involved in stem cell quiescence in Bthal HSCs compared with healthy donor (HD) ones, along with an upregulation of some erythroid transcription factors, highlighting a marked erythroid signature. Immunophenotype and functional studies show an increased proportion of multipotent progenitors (MPPs) in Bthal patients compared to healthy donors with an enhanced erythroid potential in the multipotent (Ery/My/Ly) Subset component in response to specific signals.Preliminary scRNAseq analysis reveals that Bthal HSCs harbored different primitive subsets compared with healthy donors. To evaluate the effect of gene therapy (GT) on the ability of primitive cells to reconstruct differentiation trajectories, we also profile single HSPCs isolated from Bthal patients after GT. Differences in the outcome of hematopoietic reconstitution mirror the composition of the primitive compartment pre‐GT. The definition of the landscape shape will offer crucial insights to efficiently target the cell population relevant for genetic correction.
High Frequency Of AAV Integration At Double Strand Breaks Induced In Preclinical Model Of Gene Therapy As Well As In Edited Long‐Term Engrafted HSPCs
A Calabria1 C Cipriani1 G Spinozzi1 S Esposito1 F Benedicenti1 A Albertini1 S Ferrari1 A Jacob1 M C Castiello1 2 M Pouzolles3 M Luoni4 S Giannelli4 V Broccoli4 N Taylor3 V Zimmermann3 A Villa1 L Naldini1 E Montini1
1: San Raffaele Telethon Institute for Gene Therapy (Sr‐TIGET), IRCCS OSR, Milan, Italy 2: Istituto di Ricerca Genetica e Biomedica, CNR, Milan, Italy 3: Institut de Génétique Moléculaire de Montpellier, CNRS, France 4: Stem Cell and Neurogenesis Unit, Division of Neuroscience, IRCCS OSR, Milan, Italy
Recombinant Adeno Associated Viral (rAAV) vectors have been exploited in gene therapy (GT) applications to treat several genetic disorders. Despite rAAVs have being considered episomal vectors, AAV DNA can integrate within the genome of host cells leading to hepatocellular carcinoma and clonal expansion events in some preclinical gene therapy (GT) models. Hence, a deeper assessment of rAAV integration events is required. Here, we analyzed the genomic distribution of rAAV integrations in T‐cells (N = 443) and liver tissues (N = 197) of immune‐deficient ZAP70 knock‐out mice that were intra‐thymically injected with a rAAV expressing the therapeutic transgene. Moreover, we investigated the distribution of AAV IS in two clinically relevant gene editing (GE) contexts based on human hematopoietic stem cells (HSPCs) edited at two different genomic loci and engrafted long‐term in immune‐deficient mice (N = 182). By this analysis, we identified that >80% of the IS retrieved from T‐cells of ZAP70‐treated mice clustered at double‐strand breaks produced by RAG enzymes within T cell receptor genes (TCR) during T cell maturation. In edited‐HSPCs, AAV integration preferentially occurred at the editing sites and at some predicted off‐target loci. Overall, Inverted Terminal Repeats were mainly involved in those integration events, revealing their intrinsic and strong tendency to be captured at DNA damage sites by non‐homologous end‐joining mechanisms. Hence, these studies provided insights into the genomic integrity of transduced cells in AAV‐based GT and GE applications revealing that AAV integrations can confer a long‐term therapeutic benefit or be an inadvertent consequence of homology‐directed repair protocols in GE applications.
Development of an in vitro assay to predict retroviral vector‐induced genotoxicity in lymphoid cells
1: Institute of Experimental Hematology, Hannover Medical School 2: REBIRTH, Hannover Medical School 3: Division of Hematology / Oncology, Boston Children's Hospital, Harvard Medical School
Insertional mutagenesis is still a significant bottleneck in retroviral vector‐mediated gene therapy. The dysregulation of proto‐oncogenes neighboring the vector´s insertion sites triggered lymphoid and myeloid leukemia in some gene therapy clinical trials. The In Vitro Immortalization Assay (IVIM) and Surrogate Assay for Genotoxicity Assessment (SAGA) quantify the genotoxic risk of integrating vectors on murine hematopoietic stem and progenitor cells (mHSPCs). Mutagenic vectors induce a proliferation advantage under limiting dilution conditions (IVIM) and the activation of cancer‐related and stem cell‐like gene expression programs (SAGA). However, both assays are limited by an intrinsic myeloid bias due to culture conditions. To detect lymphoid insertional mutants, we differentiated mHSPC to mature T cells using the OP9‐DL1 co‐culture system. Transduction with the LTR‐driven RSF91 mutagenic vector induced a block in T cell differentiation at the early double‐negative 2 (DN2) progenitor stage, in contrast to fully differentiated untransduced mock cultures. Arrested samples upregulated the leukemogenic transcription factor Lmo2 and harbored vector insertions in its vicinity, as frequently observed in the patients from the clinical trials with severe adverse events. Using the SAGA pipeline, we identified a distinct transcriptional profile in the DN2‐blocked T cell progenitors. The lymphoid SAGA classifier (“SAGA‐XL”) showed a promising model performance when predicting retroviral vector‐induced genotoxicity in bulk cultures based on this immortalization signature (AUC: 0.96; sensitivity: 0.92; specificity: 1.00). The new lymphoid branch of IVIM/SAGA complements the already accepted myeloid approach and contributes to a safer clinical translation of gene therapy strategies.
A83‐01 enhances murine HSPC expansion for gene therapy and pre‐clinical biosafety assays
1: Institute of Experimental Hematology, Hannover Medical School, Germany 2: REBIRTH ‐ Research Center for Regenerative Translational Medicine, Hannover Medical School, Germany 3: Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Massachusetts, USA.
Ex vivo gene modification with retroviral vectors requires the cultivation of hematopoietic stem and progenitor cells (HSPCs) outside their niche environment. Undesired differentiation processes during culture before retroviral transduction can lead to uncontrollable modification of late progenitors instead of the target cell type – hematopoietic stem cells. Cytokines such as SCF and FLT3‐L maintain stemness features in culture, while myeloid cytokines (IL‐3, IL‐11) enhance proliferation. In combination, these cytokines (S3F11) massively expanded murine HSPCs 20,000‐fold after seeding highly purified LSK SLAM (lin‐Sca1+ckit+CD48‐CD150+) cells in low‐cell density. However, the expanded population differentiated first to myeloid progenitors expressing CD11b/GR1+ and subsequently to mast cells expressing FcɛR1α. We examined the effect of the small compounds A83‐01, pomalidomide and UM171 (APU) in S3F11‐containing medium and revealed the attenuation of mast cell differentiation by A83‐01. While preserving a more stem cell‐like phenotype, S3F11+APU also significantly increased the expansion of HSPCs compared to S3F11. Remarkably, S3F11+APU‐expanded cells were transduced as efficiently as fresh bone marrow‐derived lineage‐negative (lin‐) cells with a LTR‐driven gammaretroviral vector and even with increased transduction efficiency using self‐inactivating lentiviral vectors. After transduction, expanded cells were further tested in pre‐clinical biosafety assays. Here, we detected in vitro immortalization events due to typical integrations near Mecom. The surrogate assay for genotoxicity assessment (SAGA) revealed that these RSF91‐transduced expanded samples dysregulated a typical oncogenic gene expression signature. Ultimately, replacing fresh lin‐ cells with S3F11+APU‐expanded material could reduce the number of experimental animals needed for biosafety studies on viral vectors for gene therapy.
Design of novel non‐viral vectors for the ex vivo gene therapy for β‐hemoglobinopathies
L Buccairelli1 C Fabris1 T Diem2 A Biffi1 F Mavilio3 Z Ivics2
1: Department of Pediatric Hematology and Oncology Center, Woman's and Child Health, University of Padova, Padua, Italy 2: Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany; 3: Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy 4: Pediatric Research Institute “City of Hope”; Padua, Italy 5: Dana‐Farber/Boston Children's Cancer and Blood Disorders Center, Harvard Medical School, Boston, USA
β‐hemoglobinopathies are the most common monogenic diseases worldwide, caused by defects of β‐globin expression or structure. They lead to transfusion‐dependent anemia, multi‐organ damage and early death. ZYNTEGLO™ by Bluebird Bio, i.e. autologous CD34+ cells transduced with the LentiGlobin, is the first GT medicinal product approved in Europe for transfusion‐dependent β‐thalassemic non‐β0/β0 patients. It provided remarkable benefit in non‐β0/β0 β‐thalassemic patients, but failed to control the disease in complete β‐globin absence. This is mainly due to a suboptimal lentiviral β‐globin production, from a limited number of erythrocytes, and to inner LV restraints. In addition, the inability to reach a consensus around a fair therapy pricing with the European authorities highlighted the need for more cost‐effective therapeutic strategies for β‐hemoglobinopathies, which still remain a crucial unmet clinical need.
To overcome these limitations, we designed a GT strategy for β‐hemoglobinopathies based on a gene‐cassette optimization for enhanced production of a potent anti‐sickling (AS) β‐globin, embedded in a hyper‐functional transposon‐vector (TV) to be delivered by nucleofection as minicircle (MC) DNA, for a cost‐effective and scalable vector‐production. Preliminary data of TV and transposase nucleofection in human CD34+ cells showed vector integration and detectable levels of AS β‐globin mRNA in CD34‐derived erythrocytes by qPCR. We are currently developing alternative cassettes combining small, epigenetically‐identified core enhancer elements derived from the 30‐kb long β‐globin LCR, optimized in size and sequence to be delivered as TVs, and investigating the chromatin state corresponding to the integrated TV in the human hematopoietic stem cell genome.
Correcting causal mutations in Fanconi Anaemia group A patient‐derived cells by prime editing
1: Childhood Leukemia Investigation Prague, Department of Hematology and Oncology, Second Faculty of Medicine, Charles University and University Hospital Motol, V Uvalu 84, Prague 15006, Czech Republic 2: Department of Hematology and Oncology, University Hospital Motol, V Uvalu 84, Prague 15006, Czech Republic
Fanconi anaemia (FA) is a rare genetic disorder caused by mutations in genes involved in DNA repair. Most FA patients undergo bone marrow failure (BMF) early in childhood. The current treatment for BMF in FA is allogeneic hematopoietic stem cell transplantation, which can be associated with severe side effects. Genome‐editing technologies hold great potential to change the treatment paradigm for FA, whereby the underlying genetic cause is corrected ex vivo in the patient's haematopoietic stem cells which are then returned to the patient body upon BMF. Prime editing (PE) is an advanced genome‐editing technology that, unlike the traditional CRISPR/Cas9 system, does not require the activity of homology‐directed repair pathway (HDR). We designed pegRNAs for the correction of two compound heterozygous FANCA mutations identified in a patient with FA (c.1A>G and c.4010 + 1‐c.4010 + 18del) and tested their efficiency using different forms of pegRNA and prime editor molecules (plasmid/RNA delivery, engineered molecules) in the patient‐derived fibroblasts. For the c.1A>G, we obtained up to 15% of precisely edited DNA (imprecise editing ≈0.5%) and detected full‐length FANCA protein in edited cells. The level of corrected DNA increased over time in culture (up to fourfold over two weeks) in accordance with the hypothesized restoration of FA pathway. The PE tools designed for correction of the c.4010 + 1‐c.4010 + 18del generated up to 1% of precisely corrected DNA (imprecise editing ≈0.4%). Our results show that prime editing can be successfully used for the correction of FANCA mutations in FA patient‐derived cells.
Supported by KOG‐202108‐00904 and Next Generation EU, LX22NPO5102.
Non‐human primate (NHP) models for intrauterine therapy
1: KK Women's and Children's Hospital 2: National University of Singapore 3: National University Health System 4: Duke NUS Graduate Medical School 5: Institute of Cell and Molecular Biology 6: University College London 7: The Children's Hospital of Philadelphia 8: Université Lyon 1
Intrauterine hematopoietic stem cell transplantation (IUHSCT) has the potential as a non‐myeloablative approach for prenatal treatment of a variety of congenital hematological and immunological disorders. Here we applied IUHSCT in the context of a fetal Macaque model.
Maternal HSC were harvested from bone marrow aspirates, magnetically enriched for CD34+ cells, and labeled with eGFP using a simian immnodeficiency virus (20‐70% GFP+). Transduced CD34+ cells were CD26 inhibited prior to intravenous injections at ∼1x 108 cells/ estimated fetal weight (kg) with a final 1‐3% CD3+ in the inoculum into 0.4‐0.5 gestation fetal macaques. Following transplant, fetal subjects (n = 6) were delivered via cesarean section and hand‐reared.
Fetal tissue harvested at 12 weeks post‐IUHSCT showed chimerism levels ranged from 0.05% to 3.31%. Whereas cord blood collected at birth was evaluated for presence of GFP cells by both qPCR and FACs, demonstrating levels to be very low if not absent (<0.05%). Mixed lymphocyte reaction between treated offspring and same‐donor (maternal) CD45 cells showed evidence of tolerance of towards maternal cells. Postnatal same donor boosting offspring is underway with peripheral chimerism showing some evidence of improvement. Further results will be discussed.
A prenatal maternal donor IUHSCT with postnatal boosting has been shown to be efficacious in murine and canine models. A preclinical fetal macaque model is essential in establishing its value prior to clinical translation.
The novel lentiviral vector IFNβ/HF displays therapeutic efficacy as a gene therapy‐based approach for multiple myeloma
1: Laboratory of Cell and Gene Therapy, Biomedical Research Foundation of the Academy of Athens, Athens, Greece 2: Plasma Cell Dyscrasia Unit, Department of Clinical Therapeutics, University of Athens School of Medicine, Alexandra Hospital, Athens, Greece 3: Department of Hematology and Bone Marrow Transplantation Unit, University of Athens School of Medicine, Athens, Greece 4: International Centre for Infectiology Research, INSERM, U‐1111, Université de Lyon, Lyon, France 5: INSERM, U‐1065, Université Côte d'Azur, Nice, France 6: Laboratory of Biology, University of Athens School of Medicine, Athens, Greece
In the context of developing novel therapeutic approaches for Multiple Myeloma (MM), we investigated the efficacy of an IFNβ‐expressing lentiviral vector, pseudotyped with the measles virus H and F glycoproteins (IFNβ/HF) in myeloma cell lines and in primary cells of MM patients.
Cytotoxicity was evaluated using CCK‐8 assay. Apoptosis and cell cycle were estimated by Annexin V/7‐AAD and PI staining, respectively. Expression of apoptotic genes was determined by qPCR and a Human Apoptosis Antibody Array. IFNβ secretion and its paracrine action were determined by ELISA and Transwell® co‐culture. Autophagy gene expression was assessed employing qPCR and confocal microscopy, while serum neutralizing antibody activity was determined by flow cytometry on JJN3 cells transduced with GFP/HF.
IFNβ/HF‐transduction (ΜΟΙ = 1), led to a dramatic reduction of cell survival followed by a marked increase of apoptosis 90.3% (p ≤ 0.001) in H929, 74.1% (p ≤ 0.001) in JJN3, 91.82% (p = 0.004) in U266, and 92.59% (p = 0.002) in RPMI‐8226 cells. Notably, the increase of Caspases 3 and 9 highlights the crucial role of the intrinsic apoptotic pathway in the IFNβ‐induced apoptosis, whereas a decrease of autophagy gene expression (e.g. Beclin‐1, Atg4b, Atg5) was detected. After Transwell® co‐culture, IFNβ secreted by the transduced cells, was capable of significantly decreasing the viability of untransduced cells. Furthermore, IFNβ/HF efficiently transduced primary cells, decreasing cell survival, with a 51.8% increase of apoptosis (p = 0.01) and induction of cell cycle arrest.
Overall, our findings suggest that IFNβ/HF is a promising therapeutic candidate for the treatment of MM.
Development of a universal prime editing strategy for the treatment of β‐hemoglobinopathies
1: Imagine Institute, INSERM UMR1163, Paris, France
β‐hemoglobinopathies are caused by mutations affecting adult β‐globin production. Disease severity is alleviated by the co‐inheritance of mutations in the HBG promoters that generate activator binding sites (aBSs), leading to elevated fetal γ‐globin in adulthood. Base editors (BEs) and prime editors (PEs) are double‐strand‐break‐free editing technologies. While BEs introduce selected point mutations, PEs can “rewrite” genomic sequences.
Here, we intend to develop a universal strategy to modify hematopoietic stem cells (HSCs) and reactivate γ‐globin. Insertion of one aBS in HBG resulted in potent γ‐globin reactivation. Interestingly, BE‐mediated sequential introduction of KLF1 and TAL1 aBSs in HUDEP‐2 erythroid cells led to a higher γ‐globin expression than the insertion of individual aBSs. However, BEs cannot allow the simultaneous introduction of multiple mutations, an approach that is desirable in primary HSCs.
Thus, we attempted to use PEs to insert aBSs in HBG. Experiments in K562 cells allowed us to select pegRNAs and ngRNAs that efficiently bind the HBG promoters. The insertion of the KLF1 aBS was significantly improved by the use of ngRNAs and optimized PEs. However, the simultaneous introduction of KLF1 and TAL1 aBSs was poorly efficient. We are currently testing PE enhancers, PAM‐less PE strategies (to shorten pegRNAs' length known to affect PE efficiency) and highly processive PEs to improve PE efficiency. Finally, we optimized PE efficiency in patient HSCs by testing RNA or protein delivery and by inhibiting factors impairing PE activity.
Our long‐term goal is to provide sufficient proof of efficacy for the application of PEs for β‐hemoglobinopathies.
Common Dendritic Progenitors are responsible for Tolerance Induction following Allogeneic In Utero Hematopoietic Cell Transplantation
1: University College London, UK 2: Children's Hospital of Philadelphia, USA 3: Federal University of Parana, Brazil 4: Wellcome Sanger Institute, Cambridge, UK
In utero hematopoietic cell transplantation (IUHCT) achieves durable mixed chimerism and donor‐specific tolerance. Our previous work demonstrates that purified HSC are unable to achieve this: cells are not present in the fetal thymus following transplant and are consequently rejected. This work aimed to explore this further in an allogeneic setting (B6‐GFP to BALB/c).
Iterative FACS sorting of donor BM‐MNC and subsequent transplantation of isolated populations of cells in their respective proportions demonstrates that cKit‐/Sca1‐ cells within the Lin‐ fraction of BM‐MNC are present in the fetal thymus in large numbers 48 hours following transplantation. tSNE analysis reveals that a common dendritic progenitor (CD115+CD135+) lies within this population. FACS phenotyping of the cells that are present in the fetal thymus at this time point suggests that the majority of these cells are CD11c+ DCs; scRNAseq suggests that these donor‐derived DCs have a distinct transcriptomic phenotype to recipient fetal thymic DCs or donor BM‐derived DCs.
Transplantation of a purified population of CDP was sufficient to induce tolerance to postnatal transplantation with BM‐MNC; and a mixed population of CDP and HSC produces long‐term microchimerism. In both settings, tolerance can be demonstrated with donor‐derived skin grafts. Finally, FACS analysis suggests that MHC peptides may be exchanged and cross presented by both recipient and donor dendritic cells (Class I >Class II).
Identification of a single tolerogenic cell may allow the avoiding of transplanting of cells that may be harmful to the fetus or pregnancy. Specific MHC‐focused non‐cellular therapies may also provide a novel route to donor‐specific tolerance induction.
Characterization of the risks of oncogene‐induced‐senescence in HSPCs gene therapy
1: San Raffaele Telethon Insitute for Gene Therapy (HSR‐TIGET)
In hematopoietic stem and progenitor cell (HSPC) gene therapy (GT), insertional mutagenesis may result in oncogene activation, increasing the risk of leukaemia in patients. Moreover, BRAFV600E‐expressing human HSPCs transplanted into immune‐deficient (NSG) mice, even in small numbers, induce lethal bone marrow (BM) failure. This aggressive phenotype is due to the engagement of senescence, characterized by cell cycle blockade and a senescence‐associated secretory phenotype, affecting also non‐mutated bystander cells.
Thus, it is fundamental to characterize oncogene activation in HSPCs, since it poses risks well before malignant transformation. To investigate the fate of senescent cells in the presence or absence of an active immune system, we transplanted NSG or immune‐competent (WT) mice with mouse (m) HSPCs transduced with lentiviral vectors expressing mBrafV600E, an N‐truncated version (mBraf‐trunc), or GFP as control.
In NSG recipients, both mBraf‐trunc and mBrafV600E expression caused dose‐dependent lethality and reduced cellularity, the latter resulting in a more aggressive phenotype. Differently from the humanized model, only mBrafV600E‐lymphoid cells were impaired but not bystander cells. Moreover, transcriptional profiling showed upregulated TNFα signaling and downregulated ribosome biogenesis, hallmarks similar and different from the humanized model, respectively. Instead, in WT recipients, we observed reduced lethality (only 60%) and complete clearance of oncogene‐expressing cells in the surviving mBrafV600E mice, suggesting that the immunological competence of recipients promotes a more efficient clearance of senescent cells.
This study aspires to identify species‐specific or universal biomarkers for pre‐clinical and clinical GT safety studies and to recognize factors determining the resilience/clearance of senescent cells, suggesting strategies for their elimination.
HBBIVSI‐110(G>A)‐specific gene editing as advanced therapy for β‐thalassemia
1: Cyprus Institute of Neurology and Genetics 2: Thalassemia Clinics, SHSO, Nicosia
β‐Thalassemia is brought about by defective β‐globin (HBB) formation and can lead to patients dependent on regular blood transfusions and iron chelating agents for survival. A safer alternative to double‐strand break (DSB) DNA‐editors, base editors (BEs) catalyse base transitions at a targeted base and are of interest for clinical translation. Cytosine BEs enable C > T transitions, whereas adenine BEs enable A > G transitions. Four recently published BEs with relaxed protospacer adjacent motif (PAM) requirements are being evaluated for their ability to correct the common Cypriot HBB[IVSI‐110(G>A)] splice mutation. ABEs and CBEs were obtained from Addgene, and the T7 promoter inserted, to allow in vitro mRNA transcription. Editors were delivered into primary hematopoietic cells by nucleofection. Clonal cell models were plate‐sorted on a BD FACS Aria III after DSB‐based editing. Editing efficiency was assessed at the DNA level and, after erythroid differentiation, at the protein level. DecodeR and EditR were used to assess DSB‐based and base editing, respectively. The strategies considered aim for precise correction of the mutated A (i) or G (ii) of the aberrant AG splice motif, and alteration of upstream sequence elements critical for aberrant splicing (iii). Efficiency of the ABEs for HBB[IVSI‐110(G>A)] target sites has been confirmed by correction of β‐globin expression. Furthermore, upon removal of the GFP reporter, on‐target efficiency for the SPRY editor doubled. DSB‐based precision editing was applied to create a HBB[IVSI‐110(G>A)]‐homozygous cell model, displaying decreased β‐globin expression, as confirmed by HPLC analysis, and same‐site deletion models, based on HUDEP‐2 cells, as tools for further analyses.
Editing of human hematopoietic stem cells with CRISPR/Cas: A gene correction approach for sickle cell disease
1: Cell and Gene Therapy for Hematological Disorders Unit, Department of Oncology‐Hematology, IRCCS Bambino Gesù Children's Hospital, Rome, Italy 2: Boston Children's Hospital, Harvard Medical School, Boston, MA, United States
β‐hemoglobinopathies are highly burdening severe monogenic diseases and Sickle Cell Disease (SCD) is the most common. The established potentially curative option is HLA‐matched Hematopoietic Stem Cell Transplantation, feasible for a minority of patients because of the lack of immune‐genetic compatible donor availability. For these reasons, several gene therapy approaches have emerged and some of them are currently under clinical investigation. Among them, the gene‐editing approach based on the BCL11A gene knock‐down with CRISPR‐Cas9 technology allows a restoration of fetal hemoglobin, with great clinical benefits, but does not allow a correction of the genetic defect. Since in SCD there is a specific section of the gene interested by the pathogenic mutation (first exon), we designed a novel gene‐editing strategy addressing this fragment. This project proposes a virus‐free platform that aims to correct the first exon of the β‐globin gene using a CRISPR‐Cas9/12 nuclease system and single‐stranded donor templates (ssODN) as delivery system. As to avoid an unwanted disruption of the β‐globin gene, the chosen regions for the target integration are non‐coding ones. Firstly, we have demonstrated the feasibility of this approach in K562 cell line, selecting the best performing gRNAs to move on the next experimental stages. As for the integrating potential, we achieved an Homology Directed Repair efficiency of 24‐36% both with Cas9 and Cas12.This approach might lead to the correction of the genetic defect with a significant reduction of the production costs and the possibility to overcome the safety limits of a virus‐based approach.
Development of gene therapy vectors for Ataxia Telangiectasia (A‐T)
1: Gene Modification in Stem Cells, Division of Veterinary Medicine; Paul‐Ehrlich‐Institut, Langen, Germany 2: Institute of Medical Microbiology and Virology, University Hospital and Medical Faculty, Technische Universität Dresden, Germany 3: Department for Children and Adolescents, Goethe University Hospital, Frankfurt am Main, Germany
Ataxia Telangiectasia (A‐T) is a rare multi‐organ, monogenetic, autosomal recessive disorder caused by mutations in the ataxia telangiectasia mutated (ATM) gene. The encoded ATM protein kinase plays a major role in DNA damage response and coordinates DNA repair, cell cycle arrest and apoptosis. Progressive cerebellar degeneration, telangiectasia, immunodeficiency (impaired B‐ and T‐cell development), recurrent sinopulmonary infections, radiation sensitivity, premature aging, and a predisposition to cancer are the most prominent clinical signs. As curative treatment, gene therapy with genetically modified autologous hematopoietic stem and progenitor cells is a promising strategy.
Because of the large coding sequence of Atm (∼9.2 kb), the packaging capacity of most retroviral vectors is reached. To find the best performing vector platform, we generated lentiviral, gammaretroviral (both VSV‐G pseudotyped) and foamy viral (pseudotyped with simian foamy viral envelope) vectors for gene transfer of Atm. The vector genome lengths vary between 12.5 – 13.5 kB. Lentiviral vectors were superior to the other platforms, with viral titres of 9x105 IP/ml after concentration (gammaretroviral: 2x105 IP/ml, foamy viral: 3x105 IP/ml). Next, we transduced murine Atm‐deficient fibroblasts with lentiviral Atm vectors and could demonstrate the expression of Atm protein in these cells. Moreover, to show the function of the introduced Atm protein, the phosphorylation potential of downstream substrates and the correction of cell cycle abnormalities seen in Atm‐deficient cells were analysed. Transduction with the Atm expressing lentiviral vectors restored the phosphorylation of H2AX, a direct Atm target.
Safety and clinical findings 12‐months following bilateral putaminal convection enhanced delivery of AAV2‐GDNF in early and moderate stages of Parkinson's Disease
1: Asklepios BioPharmaceutical Inc. 2: University of California, San Francisco 3: Ohio State University 4: University of California, Irvine 5: Ohio State University Wexner Medical Center 6: University of Arizona‐ Banner University Medical Center
Glial cell line‐derived neurotrophic factor (GDNF) gene therapy's neurorestorative potential for Parkinson's Disease (PD) is evaluated in this Phase 1b study, the first to investigate gene therapy in an early PD population. An optimized neurosurgical technique utilizing MR‐imaging and convection enhanced delivery was used to administer high volumes of AAV2‐GDNF to the brain. In this ongoing open‐label study, 6 early (<5 years; motor rating score on MDS‐UPDRS III <32 while OFF PD‐medications) and 5 moderate (>4 years; MDS‐UPDRS III 33‐60) stage PD participants received up to 1.8mL of AAV2‐GDNF (3.3E12 vg/mL)/gadoteridol (2mM) co‐infusions per putamen. Average putaminal coverage was 62.6% ± 1.8% and comparable between cohorts. All 11 participants tolerated the neurosurgical procedure well and 10 participants are >12 months post‐treatment. Asymptomatic, unilateral neuroimaging findings (3 of 22 infusions) were identified on scheduled 6‐month MRIs.
Intraocular gene therapy improves retinal morphology in a mouse model of gyrate atrophy of the choroid and retina
1: TIGEM 2: Department of Translational Medicine, “Federico II” University 3: Department of Advanced Biomedicine, “Federico II” University
Gyrate atrophy of the choroid and retina (GACR) is due to ornithine aminotransferase (OAT) deficiency and hyperornithinemia leading to retinal pigment epithelium (RPE) toxicity and impaired vision. An arginine‐restricted diet has been shown to decrease hyperornithinemia but not vision loss. Here we set to test if restoration of retinal OAT expression preserves vision in a mouse model of GACR. We developed an AAV 8 vector expressing human OAT (AAV8.hOAT) from a ubiquitous promoter in both RPE and photoreceptors. A single subretinal administration of AAV8‐hOAT in GACR mice improved RPE abnormalities and thickness of the outer nuclear layer up to 12 months of age. However, retinal electrical activity remained reduced, suggesting that hyperornithinemia, which is not reduced following intraocular AAV delivery, impairs retinal function. Our data suggest that the combination of intraocular gene therapy with an arginine‐restricted diet has the potential to treat vision loss in GACR patients.
Update on AAVance, a Phase 2/3 Clinical Trial of LYS‐SAF302 Gene Therapy in Children with MPS IIIA
1: Lysogene 2: Great Ormond Street Hospital for Children 3: Weill Cornell Medical College 4: Baylor College of Medicine 5: Hôpital Trousseau 6: University Medical Center Hamburg‐Eppendorf 7: Children's Hospital of Orange County 8: University of Minnesota 9: Amsterdam UMC
Mucopolysaccharidosis type IIIA (MPS IIIA) or Sanfilippo syndrome type A (OMIM 252900) leads to early‐onset neurodegeneration and premature death. AAVance is a Phase 2‐3, single‐arm, international study of AAVrh.10 carrying the human SGSH cDNA (LYS‐SAF302, olenasufligene relduparvovec), delivered directly to the brain via intraparenchymal infusion, for the treatment of MPS IIIA (NCT03612869). Eight clinical sites in the US and Europe are participating in this trial. The trial is fully enrolled, with 19 subjects dosed from February 2019 to March 2020. The primary objective of AAVance is to assess the drug efficacy in improving or stabilizing the neurodevelopmental status of patients compared to the expected evolution based on natural history data. Safety, tolerability, biomarkers, effect on behavior, sleep and quality of life are secondary endpoints. An update on safety and efficacy of LYS‐SAF302 will be presented.
Phase 1/2 Clinical Trial of Combined Bilateral Intrathalamic/Intracisternal/Intrathecal Delivery of a rAAVrh8 vector in Infantile and Juvenile Tay‐Sachs and Sandhoff Disease: Report of Ongoing Studies
1: UMass Chan Medical School 2: Massachusetts General Hospital 3: Washington University St. Louis
Tay‐Sachs Disease (TSD, HEXA mutation) and Sandhoff disease (SD, HEXB mutation) result in lysosomal storage of GM2 gangliosides with severe neurodegenerative consequences. Infantile TSD and SD are fatal within the first several years. Juvenile‐onset patients regress after age 2 and also have poor outcomes related to progressive movement disorders. Previously, a two‐vector rAAVrh8 gene therapy (rAAVrh8‐HexA/HexB) showed bioactivity and partial phenotypic correction in animal models and in 2 SD infants. In this trial, 6 patients (3 infantile and 3 juvenile) were treated with bilateral intrathalamic delivery (BiTh) to exploit axonal transport, along with intracisternal/intrathecal (CSF) injection. Patients were treated with rituximab, corticosteroids and sirolimus, the latter two for 3‐ and 6 months post. Three dose levels were tested: starting BiTh dose (STD) of
Gene therapy in a novel large animal model of Stargardt disease
1: Telethon Institute of Genetics and Medicine 2: University of Campania Luigi Vanvitelli 3: University of Bologna 4: Avantea 5: Federico II University
Stargardt disease type 1 (STGD1), due to bi‐allelic mutations in ABCA4, is the most common form of inherited macular degeneration, for which no therapeutic options are currently available. We have recently developed a gene therapy approach for STGD1 which relies on the use of two adeno‐associated viral vectors (AAV) each encoding for one of the two halves of the ABCA4 protein flanked by split‐inteins, which catalyse trans‐splicing of the two ABCA4 half proteins following AAV administration. We have shown that subretinal administration of the AAV intein vectors results in precise reconstitution of the full‐length ABCA4 protein and in therapeutic efficacy in Abca4‐/‐ mice. However, the mouse retina largely differs from that of humans both in terms of size and retinal architecture. Given the similarity between the swine and human retina, we have recently generated pigs through somatic cell nuclear transfer from primary fibroblasts in which ABCA4 has been knock‐out (KO) using CRISPR/Cas9. We have found that ABCA4 KO pigs have increased lipofuscin accumulation in the retinal pigmented epithelium, as it also occurs in STGD1 patients, up to 24 months, the longest time‐point evaluated. Subretinal delivery of AAV‐ABCA4 intein vectors in ABCA4 KO pigs results in efficient reconstitution of the ABCA4 protein at 2 months post‐injection. Further analysis of the impact of subretinal delivery of AAV intein on lipofuscin accumulation will provide important insights towards the clinical translation of this platform for gene therapy of STGD1.
Neuroprotective effects of adipose‐derived stromal vascular fraction on acute spinal cord injuries in rats
1: Institut des Sciences du Mouvement : Étienne‐Jules MAREY, UMR 7287 ‐ Aix‐Marseille University & CNRS, France 2: VetAgro Sup, Veterinary Campus of Marcy l'étoile ‐ Clermont‐Auvergne University and Lyon‐1, France 3: Department of Neurosurgery, La Timone Hospital, Assistance Publique ‐ Hôpitaux de Marseille, France 4: Cell Therapy Unit, La Conception Hospital, Assistance Publique ‐ Hôpitaux de Marseille, France
Spinal cord injuries (SCI) lead to functional alteration with important consequences such as motor and sensory disorders. The repair strategies developed to date remain ineffective. The adipose tissue‐derived stromal vascular fraction (SVF) is composed of a cocktail of mesenchymal and hematopoietic stem cells with trophic, pro‐angiogenic and immunomodulatory effects. Numerous therapeutic benefits were shown for tissue reconstitution, peripheral neuropathy and for the improvement of neurodegenerative diseases.
Our strategy is based on an autologous injection of the SVF within 4 hours after SCI. To check our hypothesis, we conducted a pre‐clinical study in adult male rats. Contusions performed at thoracic level T10 using an impactor, all the animals were paraplegic. The epididymal fat removed in a second time, then the autologous SVF is purified (>90% of viability), before 1 million of cells are reinjected into the peri‐medullary space in front of the lesion.
Autologous SVF implantation promotes 1) locomotor recovery (BBB test, Ladder rung walking test), 2) H‐reflex normalization, 3) and ventilatory frequency adjustment to an isometric exercise. 4) In vivo 7T MRI, shows a significant regeneration and vascularization in the periphery of the spinal contusion. 5)These results were also confirmed by immunohistology analysis (neurofilament labeling, signs of neuronotrophic and neuritogenic), that also revealed axonal regeneration within the SCI at 3 months. However, our results are not complete, some biochemical analyses are still being processed, these encouraging results demonstrate significative recovery at 3 months post‐autologous SVF implantation that could be improved by treadmill exercise.
AAV‐miQURE®‐mediated targeting of hexanucleotide repeat expansion‐containing transcripts in ALS C9orf72 mouse models
1: uniQure biopharma B.V.
Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, neurodegenerative disorder that affects the upper and lower motor neurons in the brain and corticospinal tract leading to eventual muscle atrophy and paralysis. The most common genetic cause of familial ALS is a hexanucleotide repeat expansion in the first intron of the chromosome 9 open reading frame 72 (C9ORF72) gene. The bi‐directionally transcribed, expanded, repeat‐containing C9orf72 mRNA causes cellular toxicity due to RNA foci and dipeptide repeat protein production, both of which are the pathogenetic hallmarks of the disease.
A new generation of therapeutic microRNAs (miRNAs) was developed to selectively silence mutant C9orf72 transcripts using our miQURE® and linQURE™ platforms, which enable the expression of one or multiple therapeutic miRNAs from a single cassette, respectively. The most potent miRNA candidates, targeting the sense and/or antisense mutant C9orf72 mRNAs were tested, resulting in significant human C9orf72 mRNA lowering in vitro.
The AAV‐miQURE® candidates were then tested in vivo for their efficacy in lowering mutant intronic C9orf72 mRNAs in ALS mouse models. Intrastriatal delivery of AAV‐miQURE® resulted in a dose‐dependent decrease of intronic C9orf72 mRNA expression.
In conclusion, we have selected potent AAV‐miQURE® candidates and provided in vivo proof‐of‐mechanism of selectively targeting mutant C9orf72 transcripts.
Overexpressing a protective variant while simultaneously lowering toxic APOE as potential treatment for Alzheimer Disease
L Hentschel1
1: uniQure
Alzheimer Disease (AD) is the most common form of dementia in people over 65 years (∼50 million), manifesting as progressive memory loss and cognitive function. The strongest genetic risk factor for late onset AD (LOAD) is the Apolipoprotein E4 (APOE4) variant. This lipoprotein is expressed in the liver and brain, facilitating lipid transport and homeostasis. APOE4 is present in 45‐60% of all AD cases and is associated with increased risk and decreased age of onset, while APOE2 is deemed neuroprotective. Depletion of APOE4 in previous preclinical studies in humanized (h) APOE‐AD animal models prevented neurodegeneration at various stages of pathology.
uniQure is developing an AAV gene therapy approach to treat patients with LOAD by silencing toxic APOE4 and overexpressing a protective APOE variant. We recently demonstrated that miRNAs against APOE using our silencing miQURE® platform effectively silence APOE in vitro.
These APOE‐targeting miQURE® molecules were encapsidated in AAV vectors and tested in vivo via intrastriatal delivery. In transgenic mice carrying the hAPOE4 gene, expression of miQURE® molecules resulted in potent reduction of both hAPOE4 mRNA and protein in the brain.
Additionally, we screened various protective APOE‐overexpression variants in human‐derived cells and detected transgene expression and secretability. Furthermore, we created a combinatorial approach in which miQURE® molecules and APOE‐overexpression protective variants are incorporated.
In conclusion, these potent silencing miQURE® molecules could be implemented in a gene therapy approach for AD silencing toxic APOE and overexpressing a protective APOE variant individually or in combination.
Assessment of safety and biodistribution of AAVrh.10hCLN2 following intracisternal administration in nonhuman primates for the treatment of CLN2 Batten disease
1: Weill Cornell Medicine, New York, NY USA 2: Memorial Sloan Kettering Cancer Center, New York, NY USA 3: LEXEO Therapeutics, New York, NY USA
CLN2 (Batten) disease is a fatal, childhood autosomal recessive neurodegenerative disorder caused by mutations in CLN2 gene, encoding lysosomal enzyme, tripeptidyl peptidase 1 (TPP‐1). Loss of TPP‐1 activity leads to accumulation of storage material in lysosomes and resultant neuronal cell death with progressive neurodegeneration. Genotype/phenotype comparisons suggest that the severe phenotype should be ameliorated with increase of TPP‐1 levels to 5‐10% of normal with wide CNS distribution. Our previous clinical studies showed that intraparenchymal (IPC) CNS administration of AAVrh.10hCLN2, an AAV serotype rh.10 encoding human CLN2, significantly slowed, although it did not stop the progression of the disease, suggesting that IPC route of administration (ROA) may not be sufficient to distribute the therapy throughout the CNS. Subsequently, a safety study in nonhuman primates (NHPs; African Greens) was conducted with intracisternal ROA to the CSF with AAVrh.10hCLN2 [5 x1013 genome copies (gc), n = 1M/1F] or PBS (n = 2M). In the 2 treated NHPs, 42% and 66% of brain had >1000 vector genomes, compared to 0% in the 2 PBS NHP. TPP‐1 activity was >2X SD above controls in 47% and 51% of treated brains. In 1 out of 2 animals treated with AAVrh.10hCLN2, dorsal root ganglia showed mild‐to‐moderate mononuclear cell infiltrates and neuronal degeneration. No abnormal behavior was noted in any of the NHPs; histopathology and clinical chemistry data were unremarkable. Collectively, these data indicate that AAVrh.10hCLN2 delivered by intracisternal ROA is safe and widely distributes TPP‐1 in brain and CSF at levels that are potentially therapeutic.
Towards a cure for Creatine Transporter Deficiency: promises and challenges of gene therapy
1: Department of Developmental Neuroscience, IRCCS Stella Maris Foundation, 56128, Pisa, Italy 2: Institute of Neuroscience, National Research Council (CNR), I‐56124, Pisa, Italy 3: Department of Neuroscience, Psychology, Drug Research and Child Health NEUROFARBA, University of Florence, 50135, Florence, Italy 4: Functional Neuroimaging Laboratory, Center for Neuroscience and Cognitive Systems @UniTn, Istituto Italiano di Tecnologia, Corso Bettini 31, 38068 Rovereto (TN), Italy 5: Department of Biology, University of Pisa, I‐56126, Pisa, Italy 6: BIO@SNS Lab, Scuola Normale Superiore, 56126 Pisa, Italy
Creatine Transporter Deficiency (CTD) is an X‐linked neurodevelopmental disorder caused by mutations in the Creatine Transporter (CrT) gene. It typically presents with brain creatine (Cr) depletion, intellectual disability, epilepsy and behavioural problems, and no treatment is currently available. To evaluate gene replacement therapy as a possible solution to reverse CTD pathology we developed an adeno‐associated viral vector carrying a functional copy of the CrT gene and administered it to newborn CrT knockout mice through intracerebroventricular injection. After six weeks we found that treatment induced transgenic CrT expression throughout the whole brain, increased brain Cr levels and improved cognitive function. However, we also observed dose‐dependent toxicity, neuroinflammation and neurodegeneration likely caused by Cr overload. This suggests that therapeutic Cr levels are a matter of a fine balance, and we are now optimising the vector design to yield lower CrT expression. Our results provide proof‐of‐concept evidence that gene therapy has potential applications for treating CTD and suggest that further steps of vector engineering to finely tune CrT expression may be pivotal for maximising safety and efficacy.
Towards first‐in‐human of subretinal administration of dual AAV vectors for Usher syndrome type 1B
1: TIGEM 2: Innovavector S.R.L. 3: Reithera S.R.L. 4: AAVantgarde Bio 5: Department of Advanced Biomedicine, “Federico II” University
Gene therapy of Usher Syndrome type 1B (USH1B) due to mutations in the large Myosin7A (MYO7A) gene is limited by the packaging capacity of vectors based on the adeno‐associated virus (AAV). We developed dual AAV serotype 8 vectors encoding human MYO7A (dual AAV8.hMYO7A) and showed that their subretinal delivery improves the retinal defects of the shaker1 (sh1) mouse model of USH1B. To translate this to subjects with USH1B, a good manufacturing practice (GMP)‐like lot of dual AAV8.hMYO7A was used subretinally in sh1 mice to define the therapeutic range of dual AAV8.hMYO7A. Dose‐dependent increase in both hMYO7A levels and improvement of the retinal phenotype was seen in this model. Biodistribution and shedding studies in non‐human primates (NHPs) revealed the presence of vector DNA mainly in ocular tissues and human MYO7A mRNA predominantly in the retina. Based on these results and GLP‐Toxicology data in NHPs, three doses have been selected for evaluation in a first‐in‐man clinical study.
Gene therapy for ALS by specifically overexpressing a pleiotropic chronokine, secreted α‐Klotho, in skeletal muscles
1: Institut de Neurociències (INc), Universitat Autònoma de Barcelona (UAB) 2: Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona (UAB) 3: Departament de Biologia Cel·lular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona (UAB) 4: Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas (CiberNed), Instituto de Salud Carlos III 5: Unitat Mixta UAB‐VHIR, Vall d'Hebron Institut de Recerca (VHIR) 6: Institut Català de Recerca i Estudis Avançats (ICREA)
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the loss of cortical and spinal motoneurons (MNs). Denervation of endplates and axonal retraction is thought to lead, in a “dying‐back” pattern, to the death of MNs and subsequent muscle atrophy. ALS neuropathology is associated with oxidative stress, inflammation, excitotoxicity, and mitochondrial dysfunction while chronokines like α‐Klotho (α‐KL) may counteract some of these pathways.
In the SOD1G93A mouse model we have found decreased mRNA levels of α‐KL in skeletal muscles, motor cortex and lumbar spinal cord. Furthermore, in rat spinal cord organotypic cultures, the overexpression of α‐KL protects spinal MNs from glutamate‐induced excitotoxicity.
Given the pleiotropic beneficial properties of α‐KL, we hypothesized that boosting the expression in skeletal muscles through a gene therapy treatment would protect muscles from atrophy and prevent neuronal loss in SOD1G93A mice. Our results show that the overexpression of secreted α‐ KL in muscles enhances motor function and delays disease onset as evidenced by rotarod and grip strength tests. Improvement of the functional outcome is corroborated by increased compound muscle action potential amplitudes of the tibialis anterior and the plantar interossei muscles compared to non‐treated controls. Increased amplitude of motor evoked potentials also reflects the preservation of central connectivity between upper and lower MNs. All this correlates with a protection of motoneurons, a higher number of innervated neuromuscular junctions and a heavier gastrocnemius muscle. These findings indicate that increasing the secretion of α‐Klotho by muscles is a promising approach for promoting functional improvement in ALS.
A Novel AAV Gene Therapy for Rett Syndrome through Reactivation of the Silent X Chromosome
S Powers1 S Lou2 S Nardini1 S Likhite1 A Sierra Delgado1 M Baird1 F Roussel1 A Kaleem1 M Schwartz1 S Sinha Ray1 S D'Costa3 S Bhatnagar2
1: Abigail Wexner Research Institute Nationwide Children's Hospital 2: UC Davis 3: Alcyone Therapeutics 4: The Ohio State University
Rett syndrome (RTT) is an X‐linked dominant neurodevelopmental disorder caused by loss of function of the transcription factor Methyl‐CpG‐binding protein 2 (MeCP2). RTT patients are mainly female and experience loss of developmental milestones and cognitive function. X chromosome inactivation (XCI) plays an important role in RTT disease progression. XCI is a developmentally regulated process, wherein one X chromosome per cell is inactivated through accumulation of XIST RNA. In Rett patients, this process leads to a mosaic of cells expressing either the mutant or healthy MeCP2 gene depending on which chromosome is active. Importantly, all cells that express the non‐functional copy of MeCP2 contain the healthy copy on the silenced chromosome. Thus, therapies that reactivate the silent X chromosome in defective cells are a promising therapeutic strategy. microRNA miR106a is a potent regulator of X chromosome inactivation and that knockdown leads to re‐activation of the silent X chromosome. We have developed an AAV9 carrying a miR106a sponge (miR106aSP) construct as a treatment for X‐linked dominant disorders including but not limited to Rett Syndrome. In a multicenter study, we demonstrated that AAV9.miR106aSP significantly ameliorates RTT‐like phenotypes in two mouse models. The treatment was safe and well tolerated in wt mice and two non‐human primates. Moreover, we developed a RTT in‐vitro model using direct conversion that can be used to test therapies in the context of patient mutations. AAV9.miR106aSP treatment successfully ameliorated classical phenotypes such as neurite length. These results may help to determine ideal patient populations for future clinical trials.
A baculovirus‐produced gene therapy vector LY3884961 (formerly PR001A) demonstrates efficacy in mouse models of Gaucher Disease and tolerability in NHPs
1: Prevail Therapeutics
Gaucher disease (GD) is caused by biallelic mutations in the glucocerebrosidase (GBA1) gene. Severe alleles lead to neurological manifestations (neuronopathic Gaucher disease) whereas moderate alleles cause Gaucher disease type 1, a primarily peripheral disease. GBA1‐encoded glucocerebrosidase (GCase) is a lysosomal enzyme required for the metabolism of glycolipids. GCase deficiency causes the accumulation of glycolipids, resulting in lysosomal dysfunction that leads to inflammation and other pathological changes. We are developing LY3884961 (formerly PR001A), a one‐time gene therapy, which is designed to increase GCase activity in GD patients to ameliorate lysosomal dysfunction and treat disease manifestations. Our original LY3884961 product, produced on a HEK‐based platform, is currently in use in the Phase 1/2 PROPEL and PROCEED clinical trials. To leverage advances in AAV manufacture, we have produced an Sf9‐baculovirus LY3884961 product. To demonstrate the comparability of LY3884961 produced by the two platforms, we performed preclinical studies assessing efficacy and safety in the CBE and 4L/PS‐NA mouse models of GD, where insufficient GCase enzyme activity results in glycolipid accumulation, motor behaviour deficits, microgliosis and astrogliosis. When administered by intracerebroventricular administration, both LY3884961 products reduced key GD‐related phenotypes. Safety endpoints (body weight, multiple organ histopathology) revealed no adverse LY3884961‐related histopathological findings. A 6‐month GLP toxicology study in nonhuman primates (NHPs) revealed no adverse in‐life or histopathological findings. Overall, baculovirus‐produced LY3884961 was well‐tolerated in both mouse and NHP and demonstrated safety and efficacy comparable to HEK‐produced LY3884961.
A transposon‐based gene therapy approach to treat avascular Age‐Related Macular Degeneration (AMD) using mRNA transposase: results of the in vivo local and systemic toxicity study
1: University of Geneva 2: University Hospitals of Geneva
Gene therapy studies try to prolong the one‐month‐lasting therapeutic effect of anti‐VEGF drugs in neovascular AMD. However, 90% of patients suffer from avascular AMD (aAMD) for which no treatment exists. We developed an approach to halt neuroretinal degeneration in aAMD by subretinally transplanted iris (IPE) or retinal (RPE) pigment epithelial cells transfected using the Sleeping Beauty (SB100x) transposon system with the pigment epithelium‐derived factor (PEDF) and granulocyte macrophage‐colony stimulating factor (GM‐CSF) genes. Safety was confirmed in healthy rabbits. Rabbit IPE/RPE cells were transfected by the SB100x mRNA and transposons carrying the PEDF/GM‐CSF genes. 20’000 cells resuspended in 30μL BSS‐Plus were transplanted subretinally (n = 4/group); rabbits were monitored for 1h‐6 months evaluating repeatedly welfare, blood, intra‐ocular pressure (IOP), fundus and retina (ocular coherence tomography, OCT). Retinas were analysed (immuno‐)histologically (hemalum/eosin, Iba‐1, GFAP, Caspase‐3, CD68, rhodopsin, RPE65, CaB‐5, MAP‐1/2, Ki‐67, Pancytokeratin, Venus, PEDF, TUNEL). All 68 rabbits showed excellent welfare (score: 9.9 ± 0.1 from 10; weight: Δ0.3 ± 0.3kg [0d‐6 months]) and normal IOP; right: 11.7 ± 2.3 (0d) and 11.8 ± 1.2mmHg (6 months); left: 11.8 ± 2.5 (0d) and 12.5 ± 1.7mmHg (6 months). Fundoscopy and OCT visualized successful transplantation by the retinal bleb that reattached within 7d; no pathological alterations were detected. The 37 blood values measured indicated no toxicity or inflammation (e.g., GLDH: 2.8 ± 3.65U/l [0d], 2.2 ± 3.5U/l [6 months]; lymphocytes: 55.2 ± 12.6% [0d], 58.310.5% [6 months]. Tissue analysis excluded inflammation, apoptosis, tumorigenicity and confirmed normal morphology. Data proved excellent local and systemic tolerability of PEDF‐GM‐CSF‐transfected IPE/RPE cells using the mRNA transposase supporting the hypothesis their transplantation would be safe to aAMD patients.
The stress granules protein G3BP1 mitigates pathological deficits in two polyglutamine diseases
1: Algarve Biomedical Center Research Institute 2: Programa Doutoral em Ciências Biomédicas, Faculdade de Medicina e Ciências Biomédicas, Universidade do Algarve, Faro, Portugal 3: Faculdade de Medicina e Ciências Biomédicas, Universidade do Algarve, Faro 4: Center for Neuroscience and Cell Biology, Universidade de Coimbra, Coimbra, Portugal 5: Champalimaud Research Program, Champalimaud Center for the Unknown, Lisbon, Portugal 6: Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal
Polyglutamine diseases are a group of nine different neurodegenerative disorders characterized by abnormal expansions of the CAG trinucleotide, withing the coding region of each different disease associated gene. The mutant genes are translated into toxic proteins bearing abnormally elongated polyglutamine tracts that are responsible for neuronal death and consequently a myriad of highly debilitating motor symptoms. In this work, we showed that expression of the stress granules protein G3BP1 mitigates the phenotype of two different polyglutamine diseases, spinocerebellar ataxia type 2 (SCA2) and type 3 (SCA3), which are caused by abnormal expansions in the ataxin‐2 and ataxin‐3 proteins, respectively.
In cellular models, we found that the G3BP1 expression reduces the levels of both mutant ataxin‐2 and mutant ataxin‐3. Moreover, it also decreased the formation of pathological aggregates of both proteins.
Next, we used a lentiviral mouse model of SCA2 and SCA3 that display aggregation of mutant ataxin‐2 and mutant ataxin‐3, respectively, with loss of neuronal markers. Our results demonstrated that lentiviral‐mediated expression of G3BP1 reduced neuronal markers loss in both models. Accordingly, we observed a decrease of mutant ataxin‐2 and mutant ataxin‐3 aggregates upon G3BP1 expression. To further support this therapeutic impact of G3BP1 expression, we used a SCA3 transgenic mouse model, characterized by marked neurodegeneration and highly severe motor deficits. The lentiviral‐mediated expression of G3BP1 led to Purkinje cell preservation and reduced mutant protein aggregates. Importantly, mice treated with G3BP1 significantly improved motor performance. Altogether, our results show that G3BP1 could be a novel therapeutic target for SCA2 and SCA3 diseases.
Intravenous gene therapy using AAVPHP.eB for metachromatic leukodystrophy
E Audouard1 N Khefif1 C Mansat1 O Nelcha1 A Lamaziere2 C Sevin1 3
1: Neurogencell, ICM, PARIS, France 2: Saint Antoine Hospital, PARIS, France 3: APHP Hopital Kremlin Bicetre
Metachromatic leukodystrophy (MLD) is a rare, autosomal recessive disease caused by deficient activity of the lysosomal enzyme arylsulfatase A (ARSA), resulting in sulfatide accumulation and subsequent demyelination and neuronal loss within the central peripheral nervous system. Three clinical forms of MLD have been described, based on the age of symptom onset. Among them, early‐onset forms comprise a continuum between late‐ infantile MLD most frequent accounting for 50‐60% of MLD patients) and early juvenile MLD. In these early‐onset forms, first symptoms typically develop between 1 and 4 years of age and progress rapidly towards severe motor and cognitive regression and premature death.
we proposed a gene therapy approach based on intravenous delivery of an AAVPHP.eB encoding the human ARSA gene with an HA‐tag. We demonstrated a broad transduction of brain and spinal cord leading to a complete correction of sulfatide storage both in brain and spinal cord in symptomatic animals (ARSA KO animals) as well as a significant improvement of neuroinflammation in mouse model of the disease. A dose response study has been done in ARSA KO mouse model and will be presented. More importantly in term of translationnal study to establish a phase I/II clinical trial, we will present the impressive data of expression in the Non human primate that we recently obtained and demonstrated a broad expression of the transgene in the CNS.
Innovative and regulated lentiviral promoter for the Gene Therapy of Neurodegenerative Diseases
1: Division of pediatric Hematology, Oncology and Stem Cell Transplantation, Woman's and Child Health Department, University of Padova, Padova, Italy 2: Altheia Science, Milan, Italy 3: Gene Therapy Program, Boston Children's Dana Farber Cancer and Blood Disorder Center, Boston, USA
Neuroinflammation is characterized by microglia cell‐activation, playing a major role in the pathogenesis and progression of neurodegenerative disorders. The clinical benefit of ex vivo gene therapy (GT), i.e. autologous transplantation of genetically modified hematopoietic stem cells (HSCs), for these diseases derives from functional transplant‐derived microglial cells engrafted into the recipient's Central Nervous System (CNS). Once established, they may exert homeostatic and scavenging functions, normalize CNS homeostasis, reduce neuroinflammation and deliver therapeutic proteins, such as lysosomal enzymes in the case of lysosomal storage diseases. An ideal lentiviral vector to be employed in this setting should drive a regulated expression of the therapeutic transgene in the HSC‐derived microglia‐like progeny: upregulated in the presence of neuroinflammation and downregulated under homeostatic CNS conditions.
We designed a novel synthetic promoter (sHLA), based on the human leukocyte antigen ‐ DR alpha (HLA‐DRA) promoter region, able to drive a basal level of transgene expression in resting microglia cells, responsive to cell activation. Once embedded in a lentiviral vector, the sHLA promoter faithfully reproduces the transcriptional pattern of the endogenous promoter in vitro, and induces a moderate transgene expression in vivo in resting myeloid/microglia cells derived from HSPCs transplanted in wild type mice. Whereas, in mice with induced experimental autoimmune encephalomyelitis, a strong transcriptional upregulation of sHLA promoter (6‐fold increase) was observed in multiple immune cell types upon cell‐activation. These results strongly support investigating this new promoter in HSC GT approaches for pathological conditions of the CNS characterized by neuroinflammation.
A novel Adeno‐associated viral gene therapy treatment protocol in sheep for use in MPSIIIC and other neurological disorders
C O'Leary1 G Forte1 N L Mitchell3 4 A S Youshani1 A Dyer2 M Wellby4 K N Russell4 S J Murray4 N Jolinon2 K Stacey1 D Davis1 D N Palmer4 E Henckaerts2 5 I Kamaly‐Asl6
1: University of Manchester 2: Kings College London 3: University of Otago 4: Lincoln University 5: KU Leuven 6: Royal Manchester Children's Hospital
Current delivery routes for Adeno‐Associated Virus (AAV) vectors are inadequate at targeting the entire human brain, which is essential in diseases characterised by global pathology such as Mucopolysaccharisdosis (MPS) IIIC. AAV based gene therapies that are effective in mouse brains can fail in human trials due to differences in morphology and size. We have previously shown full neurological and behavioural correction of the mouse model of MPSIIIC from two intrastriatal injections of AAV expressing the missing HGSNAT enzyme using either AAV9 or AAV truetype capsids. Here we describe a novel approach of AAV delivery using Brainlab targeted catheter placement for convection enhanced delivery (CED) in sheep. Using an AAV vector containing a green fluorescent protein (GFP), AAV9‐GFP, we found that Brainlab cranial navigation is optimal for gene therapy using targeted catheter placement and that intraparenchymal CED gives better distribution compared to intracerebroventricular delivery (with same vector volumes). We can effectively deliver functional heparan acetyl‐CoA:alpha‐glucosaminide N‐acetyltransferase (HGSNAT) in up to 36.5% of a 140g gyrencephalic sheep brain using AAV9‐HGSNAT. AAV serotype may also be important, as AAVTT‐GFP displayed moderately better transduction compared to AAV9‐GFP. Overall, we found the primary attribute for efficient brain delivery is catheter design. These data highlight key aspects for consideration using direct delivery to the human brain in clinical settings.
Clinical effect of intrathecal infussion of allogeneic mesenchymal stem cells derived from umbilical cord Wharton's jelly (WJ‐MSC) in adults with spinal cord injury (SCI) treated at BioXcellerator, Medellín, Colombia
F A Barrios‐Arroyave1 J Polo1 J España1
1: Research group, BioXcellerator, Medellin, 050021, Colombia
Stem cell therapy could improve SCI by modulating the inflammatory response. The aim was to describe the clinical effect of intrathecal WJ‐MSC therapy. A retrospective cohort was followed by using ASIA scale at 3, 6, 9 months post‐treatment. WJ‐MSC primary cultures were obtained using the explant method and expanded in culture medium supplemented with 10% human Platelet Lysate (hPL) until passage 7. Cell‐markers expression, in vitro differentiation to mesodermal lineage and microbiological tests were conducted. WJ‐MSC were cultured in cerebrospinal fluid (CSF). 48‐hours cell markers expressions of Tubulin β‐III, NeuN, Neurofilament light (NfL) protein and MAP2 were evaluated. WJ‐MSC positive markers expression was over 90% and hematopoietic markers were express in less than 2.5%. Differences in neuronal markers expression in cells growth with CSF were found related to the control cells growth in regular medium. Treatment protocol included two intrathecal applications of 40*106 WJ‐MSC and 20*106 WJ‐MSC, repeated two to four times every three months. A total of 23 patients were included (October/2019‐January/2022), 78% men, 74% aged ≤40 years. 56.5% had cervical, 34.8% thoracic and 8.6% lumbar injuries. 82% had ASIA grade A. In fourth therapy sequence, the median pin‐prick and light‐touch scores increased between 10 to 20 (p = 0.05) and 14 to 16 points (p = 0.08). The lower and upper limb strength medians of scores increased 2 and 22 points (p = 0.06, p = 0.09), respectively. No serious adverse events were reported. This study was approved by an ethics committee. WJ‐MSC are safe and seem to have valuable clinical effects in SCI patients.
Severity of histopathological findings in spinal cord of non‐human primates following intra‐CSF AAVrh10 vector administration are associated with neurofilament light chain concentrations in both cerebrospinal fluid and serum
1: Lysogene
LYS‐GM101 is an AAVrh.10 based gene therapy vector carrying the GLB1 cDNA, which encodes the lysosomal hydrolase acid β‐galactosidase (βgal), administered via a one‐time injection into the cisterna magna. Biodistribution and safety of LYS‐GM101 were assessed in a Good‐Laboratory‐Practice (GLP) study in Non‐Human Primates (NHP) to support the initiation of a clinical study with LYS‐GM101 for the treatment of GM1 gangliosidosis. Asymptomatic vector‐induced microscopic changes in sensory pathways of the spinal cord and dorsal root ganglia were observed at both tested dose levels (1.0E+12 and 4.0E+12 vg/mL of CSF) with similar incidence and severity after 3 or 6 months. Severity of changes in the spinal cord, consisting of minimal to moderate vacuolation and axonal necrosis in the white matter (dorsal funiculi) at all anatomical levels, correlate with the tissue viral genome levels. Increase of neurofilament light chain (NF‐L) concentrations in cerebrospinal fluid and serum was observed at 3 months, followed by a decrease to levels close to baseline after 6 months. CSF NF‐L concentrations were strongly associated with the severity of vacuolation and axonal degeneration in the spinal cord at 3 months. In summary, these data support the transient nature of spinal cord and DRG toxicity observed after intracisternal AAV administration in NHP and the usefulness of NFL assay in serum and CSF as a promising biomarker to follow its evolution.
Exploring the identity and safety of hiPSC‐derived neural stem/progenitor cells: implications for cell therapy approaches
M Luciani1 V Meneghini1
1: San Raffaele Telethon Institute for Gene Therapy (SR‐Tiget), Ospedale San Raffaele, Milan, 20132, Italy 2: Computational and Chemical Biology, Fondazione Istituto Italiano di Tecnologia, Genoa 3: Institute for Biomedical Technologies (ITB), National Research Council (CNR), Segrate 4: Institute of Genetic and Biomedical Research, UoS of Milan, National Research Council (CNR), Rozzano 5: Genomic Unit, IRCCS Humanitas Clinical and Research Center, Rozzano 6: Laboratory of chromatin and gene regulation during development, INSERM UMR1163, Imagine Institute, Paris
Cell therapies using human induced pluripotent stem cell‐derived neural stem/progenitor cells (hiPSC‐NPCs) have the potential to achieve long‐lasting benefit for neurodegenerative diseases with an unmet clinical need. Still, the clinical translation of these approaches requires a rigorous evaluation of the cell source in terms of composition, functionality, and safety. Our previous RNA‐seq and ChIP‐seq analyses revealed a downregulation of pluripotency, cell cycle, and cancer‐related pathways and the acquisition of a distinct “neural signature” in hiPSC‐NPCs. Here, we integrated these transcriptomic and epigenetic profiles with molecular (at the single‐cell level), phenotypic, and functional studies to i) investigate mechanisms underlying the hiPSC to neural differentiation and ii) define a comprehensive hiPSC‐NPC signature and safety profile. A deep analysis of transcriptomic data identified several transcription factors (TFs) with a potential key role in neural commitment/maintenance, based on their peculiar kinetic of expression along the hiPSC to neural differentiation. Loss‐of‐function studies are currently ongoing to test this hypothesis. Single‐cell RNA‐seq highlighted the heterogeneity of the hiPSC‐NPC populations, which are mainly composed by radial glia‐like cells and more committed neuronal/glial progenitors without residual pluripotent cells. Indeed, upon intracerebral transplantation in neonatal immunodeficient mice, hiPSC‐NPCs engrafted robustly and dispersed widely in the brain parenchyma, differentiating predominantly into glial cells with no evidence of abnormal proliferation or presence of cells expressing pluripotency markers at 12 months after transplant. Results of this study provide insights into the safety and identity profile of hiPSC‐NPC populations and support their use for cell‐based approaches in neurodegenerative and demyelinating disorders.
Biodistribution and safety of a novel AAV9 gene therapy for treatment of temporal lobe epilepsy shown in non‐human primates
1: Corlieve Therapeutics SAS a fully owned subsidiary of uniQure NV 2: ReGenX Biosciences 3: Integrated Biologix
Temporal lobe epilepsy (TLE) is a chronic disorder of the nervous system characterized by recurrent spontaneous seizures. Many TLE patients are drug‐resistant to available anti‐seizure medications resulting in a high unmet medical need for new therapeutic options. In a pilocarpine mouse model, we observed anti‐seizure activity with a novel AAV9 vector containing an engineered micro‐RNA against the GluK2 subunit of kainate receptors.
Here we report on the biodistribution of an AAV9‐hsyn1‐eGFP vector after local injection into cynomolgus monkeys using MRI guided convection‐enhanced delivery (CED). Monkeys were dosed with a range of vector concentrations into the tissue. Vector biodistribution was assessed 4 weeks post‐dosing in brain, spinal cord and peripheral organs. Immunohistochemical studies with eGFP revealed strong, dose‐dependent transgene expression throughout the injected site in all animals. Further, the GFP expression under the control of the hSyn1 promoter was largely restricted to neurons. We then designed an AAV9 vector containing a double expression cassette of engineered miRNAs targeting the GRIK2 gene. Following the same protocol as previously, after 4 weeks, the new vector was safe and well tolerated in monkeys after local administration using CED. Brain tissue biodistribution of the miRNA vector was similar to the GFP vector. RNAScope visualization of brain slices demonstrated robust reduction of the GRIK2 mRNA in neurons associated with high miRNA.
These non‐human primate data show that local administration is a viable strategy for delivering AAV9 vectors to the brain and represent a promising novel approach for treatment of refractory TLE.
3D brain imaging to predict gene therapy efficacy for neurological disorders
1: Lausanne University Hospital 2: Université de Lausanne
The development of tools targeting specific neuronal circuits combined with refined quantitative analytic methods will facilitate the establishment of gene therapies for neurological disorders. We developed a 3D‐based imaging analytic workflow to predict treatment efficacy of a novel therapeutic strategy for Huntington's disease (HD). The strategy consists in the delivery of an AAV‐based KamiCas9 to inactivate the disease‐causing gene (mutant huntingtin; mHTT) in the cortico‐striatal neuronal network affected in HD by co‐injecting AAV2/10 and AAV2retro in the striatum. Given the AAV‐KamiCas9 neuronal tropism, we theoretically anticipated a maximal transduction efficiency of 65% in the striatum and 25‐27% in the cortical striatal‐connected areas based on data from the cortico‐striatal connectome and Blue Brain Cell Atlas. While AAV2/10‐mCherry extensively transduced the striatum, AAV2retro‐GFP predominantly transduced cortical neurons with striatal projections. To estimate the transduction efficiency and predict therapeutic efficacy, we then co‐registered 3D‐imaged brains to the Allen Brain Atlas using the MIRACL algorithm. Our analysis predicted a HTT editing efficiency of 60% in the striatum and 12% in cortical transduced areas. As predicted, the AAV‐KamiCas9 injection resulted in the inactivation of 57% and 11% of the HTT alleles on punches from striatal and cortical transduced areas, respectively. Considering the maximal neuronal transduction in these regions, we inferred that HTT gene was inactivated in 87% of striatal neurons and in 42% of cortical projecting neurons. These results not only validate our therapeutic strategy but also demonstrate the power of this 3D‐based quantitative workflow to predict outcomes of gene therapy strategies for neurological disorders.
Antisense oligonucleotide‐induced exon‐skipping as treatment for retinitis pigmentosa caused by mutations in USH2A
1: Radboud University Medical Center 2: Donders Institute for Brain, Cognition and Behaviour 3: Inserm U1298 4: University of Montpellier
Loss‐of‐Function mutations in the USH2A gene are among the most common causes of syndromic and non‐syndromic retinitis pigmentosa (RP). We previously presented skipping of USH2A exon 13 as a promising treatment paradigm for USH2A‐associated RP. However, RP‐associated mutations are often private, and evenly distributed along the USH2A gene. We therefore expanded our approach to another exon of USH2A in which unique loss‐of function mutations have been reported*. Skipping of this exon from the USH2A mRNA is predicted to result in a slightly shortened usherin protein with residual function. Using CRISPR/Cas9, we first generated stable zebrafish mutants carrying either a loss‐of‐function mutation in the orthologous ush2a target exon, or a genomic deletion of the entire target exon to force exon‐skipping. Excision of the target exon restores expression of an usherinΔexon protein, which displays proper subcellular localization in photoreceptors, and rescues hallmark RP phenotypes including visual dysfunction and photopigment mislocalisation. To translate these findings into a future treatment in man, we employed in‐vitro assays to identify an antisense oligonucleotide (AON) with a high, sequence‐specific exon‐skipping potential. We furthermore showed that this AON is able to induce skipping of the exon of interest from USH2A mRNA, and the subsequent restoration of usherin protein expression in matching patient‐derived 3D retinal organoids. In conclusion, the obtained in vitro and in vivo data demonstrates AON‐induced exon skipping to be a highly promising treatment option for RP caused by mutations in USH2A.
*Target exon will be disclosed at ESGCT 2022 (after patent filing)
Neuronal transduction of AAV‐Progranulin rescues pro‐inflammatory microglial morphology in a mouse model of progranulin deficiency
1: University of Alabama at Birmingham
Of the more than 70 GRN mutations associated with FTD, nearly all cause progranulin haploinsufficiency. Therefore, progranulin replacement is a straightforward therapeutic approach for the FTD‐GRN patient population. There are two AAV‐Progranulin gene therapeutics currently in clinical trials. PR006, manufactured by Prevail Therapeutics, is a human progranulin transgene product packaged in an AAV9 capsid which transduces both neurons and astroglia. PBFT02, manufactured by Passage Bio, is human progranulin packaged in an AAV1 capsid, which selectively transduces neurons. Our lab has demonstrated that mouse progranulin packaged in AAV1 rescues microglial lysosomal dysfunction in progranulin homozygous knockout (Grn –/–) mice as measured by CD68 immunostaining, and reduces microglial soma size, as measured by IBA1‐positive particle analysis of DAB micrographs. Here, we further characterized microglial morphological phenotypes of Grn –/–mice and determined if neuronal restoration of progranulin influences microglial morphology. A blinded experimenter used 3D Slicer to manually segment and perform 3D reconstruction of microglia from 40X Z‐stacks of IBA1‐stained sections of Grn –/–and Grn +/+ brains. We also used a MATLAB‐based script which segments and skeletonizes microglia based on a threshold set by the user. We repeated these analyses with Grn –/– mice that received AAV1mGrn or AAV1GFP. Grn –/– mice had age‐dependent increases in microglial cell volume, territorial volume, and average branch length and AAV1mGrn treatment rescued these morphologic phenotypes. Exogenous progranulin was detected only in neurons, not microglia. Thereby, we conclude that neuronal transduction of AAV‐Progranulin rescues pro‐inflammatory microglial morphology in Grn –/– mice.
Development of an ex vivo ene Therapy for Frontotemporal Dementia (FTD)
1: Division of pediatric Hematology, Oncology and Stem Cell Transplantation, Woman's and Child Health Department, University of Padova, Padova, Italy 2: Gene Therapy Program, Boston Children's Dana Farber Cancer and Blood Disorder Center, Boston, USA
Frontotemporal Dementia (FTD, OMIM:607485) is the second most common dementia in people under the age of 65 after Alzheimer Disease and is devoid of any cure or specific approved treatment. The incidence of pathogenic loss‐of‐function mutations in the progranulin gene (GRN) accounts for approximately 5% of all familial FTD cases. GRN is a secreted lysosomal protein that functions as neurotrophic factor and regulator of neuroinflammation. Raising GRN levels in the brain of FTD patients, and in particular in microglia, may result in therapeutic benefit. Hematopoietic Stem/Progenitor Cell (HSPC) Gene Therapy (GT) based on the use of lentiviral vectors (LVs) for gene transfer could thus represent a valuable treatment option for GRN‐FTD patients as it could generate a long‐lasting source of GRN in their brains through its myeloid, microglia‐like CNS progeny. We developed and extensively tested therapeutic LVs with novel myeloid/microglia‐specific promoters able to safely deliver multiple copies of the human GRN cDNA in GRN knock‐out (ko) cell models, inducing over‐expression of the therapeutic protein which is then correctly secreted and taken up by target GRN knock‐out cells. The most promising among these constructs have been tested in vivo in optimized transplant conditions enabling CNS‐specific and selective engraftment of the transplanted cells and of their progeny. Thus far, we showed that this approach efficiently delivers human GRN to the brain of transplanted GRN‐ko mice as measured by ELISA. We are currently monitoring cohorts of transplanted GRN‐ko mice to accumulate evidence supporting this potential therapeutic approach.
Identification and evaluation of microRNAs involved in photoreceptor degeneration
1: TIGEM 2: University of Campania “Luigi Vanvitelli” 3: University of Naples Federico II
Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous group of disorders, characterized by progressive photoreceptor degeneration and incurable loss of vision. MicroRNAs (miRNAs), a class of non‐coding RNAs with post‐transcriptional regulatory properties, are known to play a major role in retinal function, both in physiological and pathological conditions. Since miRNAs are capable of simultaneously modulating multiple molecular pathways, they represent promising tools to therapeutically tackle disorders with high genetic heterogeneity, such as IRDs. In the present work, a high‐throughput screening approach was employed to study, in a systematic manner, the impact of miRNAs on a photoreceptor‐like cell line undergoing light‐induced degeneration. For this approach, more than 1200 miRNAs were transfected and assayed for their putative protective action in light‐stressed 661W cone photoreceptor cells. Top‐performing miRNAs were tested with secondary in vitro methods and one of them displayed a significant protective role. Additionally, in vivo overexpression of this miRNA in the retina of the Rho‐P23H mouse, a model for an autosomal dominant form of IRD, showed preservation of retinal function as well as protection of cone photoreceptors from degeneration. Our results confirm the effectiveness of the designed in vitro screening method both to shed further light on the contribution of miRNAs to photoreceptor degeneration and, possibly, to the development of novel therapeutic approaches for IRDs.
Adeno Associated Vector‐Based Gene Therapy for the Autosomal Recessive Non‐Syndromic Deafness 9 (DFNB9)
G Olivier1
1: Sensorion, Montpellier, France 2: Genetics and Physiology of Hearing Laboratory, Institut de l'Audition/Pasteur, Paris, France
The autosomal recessive deafness 9 (DFNB9) is due to pathogenic mutations in the gene encoding Otoferlin (OTOF) that is expressed in the inner hair cells (IHC) where it is critical for synaptic vesicles fusion with the presynaptic membrane. OTOF gene defect leads to the failure of synaptic transmission between IHC and the auditory nerve, resulting in profound deafness. Today, cochlear implantation is the only option proposed to young patients. Although the medical device improves the quality of life and language acquisition, hearing quality is limited, and a treatment for DFNB9 is necessary to address this unmet medical need.
Adeno‐associated virus (AAV) is the vector of choice for in vivo gene therapy. Nevertheless, the size of the OTOF coding sequence largely exceeds AAV packaging capacity. To overcome this limitation, we adopted a dual AAV vector strategy, with OTOF coding sequence being split into two AAV vectors. The therapeutic candidate was validated through qualitative and quantitative evaluation of otoferlin expression and integrity upon reconstitution of the full‐length sequence. Then, it was injected into the cochlea of congenitally deaf DFNB9 mice. Long‐term auditory‐evoked brainstem response (ABR) restoration, up to 1‐year post‐injection, was demonstrated. Good preliminary tolerability, restricted diffusion outside the injected ear, and IHC‐restricted Otoferlin expression were shown both in mice and non‐human primates (NHP). In NHP, the selected AAV vector components allowed to efficiently target IHC at levels compatible with therapeutic intervention in humans, which constitutes a major step toward future clinical trials in DFNB9 patients.
CRX gene therapy for treating mouse models of dominant CRX‐associated retinopathies and beyond
1: CNRS 2: Universite Paris Saclay 3: Retina France
Efficacy and safety of gene therapies for retinal diseases (RDs) have been proven with preclinical success translated into clinical effectiveness. Based on our previous published work, we developed a mutation‐independent AAV vector that could circumvent the clinical and genetic heterogeneity of CRX mutations in the transcription factor CRX. We also tested the neuroprotective potential for CRX‐independent retinopathies.
AAV‐CRX is an AAV2/5 vector allowing the expression of human CRX specifically in photoreceptors. AAV‐CRX injection led to specific expression of CRX in photoreceptors with no toxicity. Three months after subretinal injection in CrxRip/+ mice, a mouse model of Leber Congenital Amaurosis with congenital blindness, we observed: i) a rescue of rod and cone opsin expression, ii) a rescue of outer segment formation, iii) some degree of ERG response whereas it remained flat in controls iv) a fully restored behavioral response to light stress. Tg(CRXR41W) transgenic line characterization, carrying CRXR41W mutation causing cone dystrophy, revealed a dose‐dependent deleterious effect of CRXR41W supporting the relevance of increasing the amount of CRXWT to counteract the dominant‐negative effect of mutant CRX. The beneficial effects of AAV‐CRX were observed in Tg(CRXR41W) mutant mice with a cone only retina (Nrl‐/‐ background). Finally, AAV‐CRX has also a beneficial effect on CRX‐independent RD by preserving rod photoreceptors in rd10 mice, a mouse carrying a Pde6b mutation.
Overall, our gene therapy shows promising results for treating CRX‐associated RDs, as well as CRX‐independent retinopathies. It also highlights the potential interest of gene therapy to treat patients with RD carrying dominant‐negative mutations.
Ablation of NADase SARM1 is protective in a mouse model of retinal ganglion cell degeneration induced by mitochondrial dysfunction
1: Trinity College Dublin 2: The Research Foundation, Royal Victoria Eye and Ear Hospital
NAD+ has traditionally been viewed as a cofactor and electron acceptor in redox reactions. More recently, its role in axonal maintenance has been established. SARM1 is an NADase with a key role in axon degeneration. The axons of the retinal ganglion cells (RGCs) form the optic nerve, delivering visual signal to the brain. Degeneration of these cells and the optic nerve is seen in inherited conditions such as Leber hereditary optic neuropathy and complex conditions including glaucoma. Mitochondrial dysfunction is a common feature of such degenerations. As such, therapies protecting against this dysfunction may be applicable to numerous conditions. Here, SARM1 ablation was explored as a therapeutic strategy in the treatment of mitochondrial dysfunction in RGCs. Rotenone, a complex I inhibitor, was delivered intravitreally to Sarm1‐/‐ and wild type mice to induce mitochondrial dysfunction primarily in the RGCs and associated optic nerve. Sarm1‐/‐ mice insulted with rotenone had significantly higher optokinetic responses than wild types. This protection of spatial vision was observed in both sexes and preserved over time. Wild type mice insulted with rotenone exhibited greater RGC death than knockout counterparts. Similarly, axonal density in the optic nerves of Sarm1‐/‐ mice was preserved following rotenone insult whereas axonal density was significantly reduced in optic nerves of insulted wild type mice. In vitro, Sarm1‐/‐ primary fibroblasts demonstrated increased mitochondrial bioenergetics compared to wild type cells. These data highlight SARM1 suppression as a potential therapeutic target for the treatment of retinal degenerations where mitochondrial dysfunction is a feature.
Assessment of Safety of miniMECP2 AAV9 vector (TSHA‐102) for Gene‐replacement Therapy of Rett Syndrome in Rats
1: Department of Research & Development, Taysha Gene Therapies, Dallas, Texas, USA 2: Department of Pediatrics; University of Texas Southwestern Medical Center, Dallas, Texas, USA
Rett syndrome is mainly caused by loss‐of‐function mutations of the methyl‐CpG‐binding‐protein 2 (MECP2) gene. A miniMECP2 gene packaged in a self‐complementary AAV9 vector (TSHA‐102) and utilizing the miRARE platform, designed to regulate MECP2, is in development as an investigational gene‐replacement therapy for Rett syndrome. Forty Sprague Dawley rats (20 male and 20 females, approximately 3‐6 weeks old) were assigned to one of four groups to receive either vehicle or a total dose of TSHA‐102 (4.81x1011, 9.62 × 1011, or 3.85 × 1012 vg/animal corresponding to 2.5x1014, 5x1014, 2x1015 vg/participant human equivalent dose [HED], respectively) via a single lumbar intrathecal infusion. Scheduled assessments (20 rats/sex/group) were performed at Weeks 1, 4, 13 and 26. There were no adverse effects on neurobehavioral and macroscopic observations, clinical pathology findings or organ weight changes associated with TSHA‐102 treatment through Week 26. Sensory nerve conduction studies of the caudal and sural nerves demonstrated no significant reduction in nerve conduction function following treatment with TSHA‐102. In addition, the amplitude and duration of the sensory nerve action potential as well as motor nerve conduction in the tibial nerve remained comparable to age‐matched controls. Microscopic evaluations revealed a low treatment‐related incidence of minimal to mild axonal degeneration in the sciatic, tibial, and sural nerves that was deemed not to be adverse. There was no obvious TSHA‐102 dose relationship in the incidence or severity of the microscopic changes in the nerves. Single intrathecal infusion of TSHA‐102, at equivalent planned clinical doses, was well tolerated in Sprague Dawley rats.
Co‐transduction of GDNF and RET protects substantia nigra pats compacta neurons from alpha‐synuclein toxicity in vivo
1: Lund University
Glial cell‐line derived neurotrophic factor (GDNF) has not been able to protect substantia nigra pars compacta neurons (SNpc) from alpha synuclein (αsyn) toxicity in rat vector‐based synucleopathy models of Parkinson's Disease (PD) so far. Literature suggests that there may be a GDNF signalling deficit in these models. Therefore, we postulated that expressing the RET receptor together with GDNF could restore GDNF signalling and neuroprotective activity in rat vector‐based synucleopathy models of PD.
After thorough in vivo titration, AAV expressing GFP, αsyn or αsyn+hRET were delivered to SNpc, while lentiviral vectors expressing GFP or GDNF were delivered to the striatum of adult Sprague‐Dawley rats in a total of 5 experimental groups. Nine weeks after viral vector delivery, the animals were euthanized and their brains processed for histology. Assessment of GDNF signalling pathways using phosphorylated ribosomal protein S6 (pRPS6) indicated that GDNF was active in animals expressing αsyn in SNpc and GDNF in striatum and in animals expressing αsyn+RET in SNpc and GDNF in striatum. Histological quantification of SNpc neurons using dopaminergic markers such as TH and vesicular monoamino transporter 2 (VMAT2) indicated that in animals expressing αsyn in SNpc and GFP in the striatum had approximately 75% surviving neurons. This level of neuronal loss was similar in the remaining groups, except in animals expressing αsyn+RET in SNpc and GDNF in the striatum. Further histological analysis is currently ongoing to fully characterize the observed neuroprotective effect.
A novel CRISPR nuclease targeting an Usher syndrome deep‐intronic mutation restores USH2A splicing
1: Alia Therapeutics 2: University of Trento
Usher syndrome is an autosomal recessive disease representing the most common cause of inherited deaf‐blindness and is classified into three types based on clinical symptoms, among which the most prevalent is type 2 (USH2). The most commonly mutated gene in USH2 is USH2A, encoding for the Usherin protein.
We focused on the USH2A c.7595‐2144A>G mutation, a deep intronic mutation generating a high‐quality donor splice site causing the incorporation of a 152bp‐pseudoexon (PE40) into the mature transcript, leading to premature termination of translation. Our strategy exploits AIK, a newly identified CRISPR nuclease, and two sgRNAs to introduce a deletion spanning the pseudoexon to correct the USH2A splicing and restore the expression of wild‐type Usherin, which is expected to revert the phenotype in USH2 patients. We identified sgRNAs for AIK showing high levels of editing and very limited off‐targets, as measured by genome‐wide GUIDE‐seq analysis. Our best candidates were tested in combination in cells expressing minigene models recapitulating USH2A wild‐type and aberrant splicing, showing >50% splicing restoration in the mutated allele with no effects on its wild‐type counterpart. We further validated the efficacy of our strategy in USH2 patient‐derived fibroblasts carrying the c.7595‐2144A>G mutation.
Given the possibility to fit AIK and its two sgRNAs into a single AAV vector, the high efficacy and the safety profile of our approach, we propose our strategy for further development of an AAV‐based therapeutic to restore photoreceptor function in the retina of USH2 patients harboring the targeted mutation.
A gene therapy approach utilising a matrix metalloproteinase secreted from the corneal endothelium to treat glaucoma
1: Trinity College Dublin 2: Virscio 3: Exhaura 4: A.K. Consulting
The primary risk factor for glaucoma is ocular hypertension, which is caused by increased resistance to the outflow of aqueous humour (AH) from the eye. Gene therapies targeting the outflow tissue hold promise for decreasing intraocular pressure (IOP); these therapies offer the additional advantage of built‐in compliance, which is a problem with eyedrops. Here we demonstrate that intracameral delivery of AAV9 expressing matrix metalloproteinase‐3 (MMP3) – a therapeutic modulator of ECM – is safe and efficacious in mouse models, and well tolerated in the non‐human primate (NHP).
Efficacy studies were performed using two murine models of glaucoma, where mice were intracamerally injected with AAV9 encoding murine MMP3 in one eye, while the contralateral eye was injected with vehicle. These studies resulted in decreased (IOP) by 2mmHg, with a concomitant 50% increase in outflow facility. Ultrastructural images of the outflow tissue revealed optically empty spaces that indicate reduced ECM at key sites.
A preliminary safety study was performed in NHP, where AAV9 encoding hMMP3 (1.2x1012vg in 50μL) was administered as above. At 2‐week intervals, ophthalmic examinations were performed alongside routine general wellbeing assessment. Inflammatory and Corneal safety markers were similar for AAV9 and vehicle injected eyes at all time points. Immunohistological imaging showed efficient transduction of the corneal endothelium in AAV9‐treated eyes. Outflow facility was measured in vivo at 8 months and was significantly elevated in treated eyes by 53%.
This pre‐clinical data demonstrates the potential of genetic intervention in the anterior chamber for the treatment of anterior segment conditions such as glaucoma.
A self‐regulating regulatory gene therapy ameliorates multiple phenotypic domains in mice modelling Rett syndrome
1: University of Edinburgh 2: Neurogene Inc.
Rett syndrome (RTT) is a neurological disorder caused by mutations in the MECP2 gene. The disorder is characterised by a regression of previously acquired developmental milestones including loss of speech, gross and fine motor skills and purposeful hand function. Other features include breathing abnormalities and seizures. Many of these core phenotypes are recapitulated in mice modelling RTT where we tested NGN‐401, an AAV9 vector delivering a unique self‐regulating MECP2 expression circuit termed EXACT. Mecp2‐/y mice dosed neonatally by intracerebroventricular injection with NGN‐401 (1e11 to 3e11 vg/mouse) revealed a dramatic extension of survival (median survival = 9 weeks in vehicle treated versus 37 weeks in NGN‐401 treated high dose cohort) and a significant amelioration of cardinal RTT‐like phenotypes. Clinical scoring showed significant improvement across domains including mobility, gait, abnormal breathing and limb clasping. Having shown efficacy, we then conducted a tolerability study in female mice that were heterozygous and thus mosaic for functional MeCP2 expression and representative of the predominant RTT patient population. In this study, NGN‐401, at doses equivalent to the therapeutic study, were well tolerated with no adverse findings. In contrast, mice dosed with a similar gene therapy cassette but missing the EXACT control circuit showed severe toxicity and were euthanized. Overall, the results demonstrate that NGN‐401 regulated gene therapy product can achieve significant therapeutic benefit while avoiding overexpression toxicity.
Morc2a p.S87L mutation develops peripheral and central neuropathies accompanying neuronal DNA damage and apoptosis
1: Graduate School of International Agricultural Technology and Institute of Green Bio Science and Technology, Seoul National University, Korea 2: Department of Neurology, SAIHST, Sungkyunkwan University School of Medicine, Korea 3: Department of Health Science and Technology, SAIHST, Sungkyunkwan University School of Medicine, Korea 4: Department of Laboratory Animal Medicine, College of Veterinary Medicine, Konkuk University, Korea 5: Stem Cell and regenerative medicine institute, Samsung Medical Center, Korea 6: WCU Biomodulation Major, Department of Agricultural Biotechnology, Seoul National University, Korea
Microrchidia (MORC)‐family CW‐type zinc finger 2 (MORC2) gene is related to DNA repair, adipogenesis, lipid homeostasis, and epigenetic silencing via the human silencing hub complex. MORC2 missense mutation was known to cause Charcot‐Marie‐Tooth type2 Z (CMT2Z) of peripheral axonal neuropathy. However, there were reports of central neuropathy in patients with a missense mutation in the ATPase module of MORC2. It could be co‐categorized by developmental delay, impaired growth, dysmorphic facies, and axonal neuropathy (DIGFAN) syndrome. The etiology of MORC2 mutation‐mediated neuropathy remains uncertain. The mouse orthologue of human MORC2 is Microrchidia 2A (Morc2a). This study established Morc2a p.S87L mutant mice using CRISPR mediated single‐stranded template repair and analyzed neuropathy progress and its etiology. B6.Morc2a S87L/+ animals displayed the clinical symptoms expected in human CMT2Z patients, like delayed nerve‐conducting velocity, axonal neuropathy with unmyelinated axons, and locomotive dysfunction with skeletal muscle weakness. Notably, we observed severe central nervous system neuropathy with cerebella ataxia and motor neuron degeneration in the anterior horn of the spinal cord, and this seemed to be evidence of DIGFAN syndrome. B6.Morc2a S87L/+ exhibited an accumulation of DNA damage in neuronal cells, followed by p53/Cyto c/Caspase 9/Caspase 3‐mediated apoptosis. Here, we present a new mouse model of CMT2Z and DIGFAN with a Morc2a p.S87L mutation. Morc2a missense mutation causes DNA damage accumulation, especially in neuronal cells, and that this is related to neuronal degeneration via apoptosis. We suggest that neuronal apoptosis is a possible target for the therapeutic approach in MORC2 missense mutation.
Biodistribution, efficacy, and safety of a tissue‐restricted AAV‐hFXN vector
M S Goodwin1 M Hamm1 N Rutherford1 J McCoy1 P Chaudhuri1 T McParland1 D Falk1
1: Lacerta Therapeutics, Inc
Friedreich's ataxia (FA) is an autosomal recessive disease that results from the deficient expression of the frataxin protein (FXN gene). We are developing a tissue‐restricted adeno‐associated virus (AAV) gene therapy to deliver the hFXN gene to tissues affected in FA, with a primary focus on cardiac tissue, cerebellar dentate nuclei, and spinal dorsal root ganglia. An expression cassette comprised of a synthetic promoter and regulatory elements of the FXN gene was packaged into an AAV vector and its pharmacotoxicology profile evaluated in rodents and primates. In mouse models that recapitulate the cardiac dysfunction [MCK‐Cre/FA mice] or neuropathophysiology [PV‐Cre/FA mice] of FA, delivery of the test article [AAV‐hFXN] fully restored cardiac function and blocked progression of CNS dysfunction as shown by in‐life and endpoint measures. These observations were reproduced using multiple doses and following delivery of AAV‐hFXN at different ages. No phenotypic or histopathological abnormalities were detected in long‐term studies, indicating that a tissue restrictive approach promotes the safety of AAV‐hFXN. The biodistribution and toxicology of AAV‐hFXN were further evaluated in Chlorocebus aethiops (African green monkey). AAV‐hFXN was well tolerated for up to 180‐days with minimal histopathological findings reported at the two doses tested. A battery of biochemical and histological assessments showed a broad distribution of the AAV‐hFXN vector and tissue‐restricted expression of human frataxin in relevant tissues at or above the expected therapeutic threshold. The AAV‐hFXN gene therapy program described here is now entering IND‐enabling studies to support its application as a CNS/Cardiac targeted drug for FA patients.
Characterization of KT‐A261 an AAV vector encoded to express adalimumab intended for the treatment of patients with non‐infectious uveitis
A Pappas1 D Fellner1 P Prentiss1 A Bassiri1 R Roeloffs1
1: Kriya Therapeutics
Non‐infectious Uveitis (NIU) accounts for approximately 10% of cases of preventable blindness in Europe. First‐line treatment for NIU involves topical and systemic corticosteroids; however, prolonged usage results in significant systemic and ocular side effects including cataract formation. Systemic administration of adalimumab (Humira®), a recombinant antibody targeting human tumor necrosis factor‐alpha (TNFα), is the only biologic agent approved for the treatment of NIU. KT‐A261 is an adeno‐associated virus (AAV) vector that encodes for adalimumab and is intended to be administered via a one‐time intravitreal (IVT) injection. As this route of administration bypasses the blood‐retina barrier at the injection site, IVT administration of KT‐A261 is expected to produce increased ocular concentrations of adalimumab compared to subcutaneous dosing.
Characterization of the expressed adalimumab from KT‐A261 was performed prior to initiating in vivo studies. In vitro transduction assays demonstrated quantifiable KT‐A261 derived adalimumab following transduction of HEK293 cells using an enzyme‐linked immunosorbent assay (ELISA) kit developed for Humira. Subsequent neutralizing antibody experiments utilizing HEK‐Blue™ TNFα cells indicated that adalimumab expressed from KT‐A261 neutralized TNFα to the same extent as recombinant adalimumab.
Initial in vivo studies conducted in mice that received a single (0.5 μL) bilateral IVT injection of 1E9 or 1E10 vg of KT‐A261 resulted in durable and dose dependent intraocular adalimumab at concentrations that exceeded physiological levels of TNFα. Collectively, these studies indicate that KT‐A261 expressed adalimumab possesses similar binding characteristics, neutralizing properties, and ocular exposure levels as compared to systemic Humira. These findings support further evaluation of KT‐A261 in non‐human primates.
Silencing of mutant ATXN3 through antisense oligonucleotides rescues molecular and neuropathological features in Machado‐Joseph disease mice
1: CNC ‐ Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal 2: CIBB ‐ Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal 3: Institute for Interdisciplinary Research, University of Coimbra, Coimbra, Portugal 4: ViraVector, Viral Vector for Gene Transfer Core Facility, University of Coimbra, Coimbra, Portugal 5: Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal 6: Wave Life Sciences Ltd., MA, USA 7: Equal contribution
Machado‐Joseph disease (MJD) is a neurodegenerative disorder characterized by an abnormal CAG trinucleotide expansion in the ATXN3 gene. This results in a toxic gain‐of‐function for the encoded ATXN3 protein and eventually in neurodegeneration. Currently, no disease‐modifying therapies are available. Importantly, silencing of mutant ATXN3 has shown promising results in several disease models. Moreover, antisense oligonucleotides (ASOs) have been under investigation as powerful gene silencers. In the present study, we aimed at investigating the therapeutical potential of ASOs targeted to mutant ATXN3 in cellular and mouse models of MJD. First, silencing capacity and selectivity of mutant ATXN3‐targeting ASOs was assessed in mouse neuronal cell lines stably expressing human mutant ATXN3 or wild‐type ATXN3. Our results showed that lead ASO‐2.1 displayed potent and selective silencing capacities as demonstrated by a dose‐dependent reduction of mutant ATXN3, while maintaining wild‐type ATXN3 levels. Next, a single intracerebroventricular bolus injection of ASO‐2.1 in a lentiviral‐based mouse model of MJD resulted in a dose‐dependent reduction of the levels of human mutant ATXN3 soluble form, confirming target engagement in vivo. Lastly, we investigated whether an improved chemistry to the previous ASO (ASO‐2.2) would rescue molecular and neuropathological features in a severely impaired transgenic mouse model of MJD. An important improvement of disease features in the cerebellum was observed following ASO‐2.2 administration, including reduction in the levels of ATXN3 soluble and aggregated forms and number of ATXN3 inclusions, as well as increase in a Purkinje cell marker (calbindin). Altogether, these data highlight the therapeutic potential of ASOs for MJD.
Supplementation of NDI1 via AAV in in vitro models of Stargardt disease and age‐related macular degeneration
1: Trinity College Dublin
Stargardt disease (STGD1) and age‐related macular degeneration (AMD) are retinal degenerations causing serious vision loss. STGD1 is an inherited disease with a prevalence of 1 in 10000 caused by loss‐of‐function mutations in the ABCA4 gene, which leads to accumulation of bisretinoids and lipofuscin, causing toxicity, which ultimately leads to degeneration of retinal pigment epithelium (RPE) cells. AMD is a multifactorial disorder affecting 190 million people worldwide. Neither of these disorders currently have approved treatments available. The yeast homolog of human mitochondrial complex 1, NDI1, increases ATP production in cells. In this study we investigated whether supplementation with Ndi1 via AAV could rescue metabolic parameters in cell models of STGD1 and AMD. A construct carrying the NDI1 gene driven by a CAG promoter (AAV.CAG.Ndi1) was created and packaged in recombinant AAV vectors. ARPE19 cells and primary porcine RPE cells were treated with NaIO3, an oxidizing agent that induces cellular damage, to model aspects of STGD1 and AMD. Cells were transduced with AAV for 48 hours and then insulted with 5mM NaIO3. In both disease models, mitochondrial respiration and ATP production were significantly reduced by NaIO3 insult and significantly improved in AAV.CAG.NDI1‐transduced cells. Immunocytochemistry showed that mitochondrial fragmentation and oxidative stress markers were reduced if NaIO3‐treated cells were transduced with AAV.CAG.NDI1. Our data demonstrate that AAV‐mediated delivery of the yeast NDI1 gene to ARPE19 and primary porcine RPE cells improved their bioenergetic profiles after NaIO3 insult.
Funding: EU Horizon 2020 (813490), SFI (16/IA/4452), Fighting Blindness Ireland (FB20DOC), HRCI‐HRB (MRCG‐2016‐14; HRCI‐HRB‐2020‐007).
Gene transfer rescues audiogenic seizures in a mouse model of Fragile X syndrome
1: Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, EH8 9XD, UK 2: Neurogene Inc., New York, NY 1001, USA
Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by silencing of the FMR1 gene. FXS is considered a good candidate for gene therapy as it is a monogenic disorder with no disease modifying treatment options. The aim of the study was to establish efficacy and safety of a novel AAV9/FMR1 gene therapy vector in a mouse model of FXS. The novel cassette contained a dominant human FMR1 isoform and native elements of the FMR1 promoter and 3’UTR sequence. Neonatal WT and Fmr1‐/y mice were injected with vehicle or the therapeutic vector via intracerebroventricular injections (5x109‐3x1011 vg/mouse) and assessed at 2 months post injection. Fmr1‐/y mice showed a profound genotype effect when assessed for audiogenic seizures with high a penetrance of seizures (>80% of subjects) in vehicle treated Fmr1‐/y mice relative to WT littermates (<10%). Cohorts treated with the vector showed a strong and dose dependent reduction in the occurrence and severity of induced seizures with the higher doses rescuing the induced seizure burden to WT levels. Behavioral analysis revealed improvements in marble burying for Fmr1‐/y mice treated with the two highest doses but no clear treatment effect in the open field or elevated plus maze tests. The present study supports the continued development of an AAV9/FMR1 gene therapy vector utilizing native regulatory elements in ameliorating disease‐related phenotypes in FXS.
An EXACT single gene circuit improves the safety of vector‐derived MeCP2 expression in vivo
1: Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, EH8 9XD, UK 2: Histology Department, Faculty of Medicine, Tanta University, Egypt 3: Neurogene Inc., New York, NY 10001, USA
A major limitation of conventional AAV gene therapy is its highly variable biodistribution, with excessive uptake in high affinity tissues such as the DRG and liver. This attribute limits gene therapy for dosage sensitive indications, which require a narrow range of gene expression to avoid overexpression toxicity. We have developed a second‐generation gene therapy technology (EXACT), designed to auto‐regulate transgene expression to desired levels and limit expression in highly transduced cells. The exemplar gene used to develop EXACT is MECP2, which is highly dosage sensitive, whereby both deficiency and duplication of the gene are associated with severe neurological disease. This study compared vector‐derived MeCP2 biodistribution and expression levels as well as tolerability using EXACT and conventional gene therapy in wild type mice. Neonatal mice were dosed 1‐4E11 vg/mouse via ICV and adult mice 1E12 vg/mouse via IV administration with AAV9 packaged EXACT and conventional cassettes. The vector genome distribution to various tissues were comparable between EXACT and conventional constructs. There was a marked reduction in transgene expression in high AAV9 affinity tissues in the EXACT treated cohorts compared to conventional treated cohorts at comparable doses and administration (brain and DRG for ICV cohorts, and liver for IV cohort). Moreover, significant toxicity was observed in the high dose ICV conventional gene therapy cohort, while EXACT treated animals showed no tolerability concerns. These data provide strong proof of principle for EXACT to expand gene therapy as a treatment modality for dosage sensitive indications currently not addressable by conventional gene therapy.
AAV delivery of G protein gated K+ channel increases cone‐mediated vision in the rd10 mouse model of Retinitis Pigmentosa
1: SparingVision, 5/7 avenue Percier, 75008 Paris, France 2: Department of Zoology and Neurobiology, Ruhr‐University Bochum, Bochum, Germany 3: EyeCRO, Oklahoma City, OK, USA 4: Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, 75012 Paris, France
Retinitis Pigmentosa (RP) is a debilitating disease causing irreversible loss of rod photoreceptors (PRs), leading to night blindness. A secondary degeneration of cone PRs occurs, starting with the progressive degradation of their outer segments, responsible for light capture. We previously described a cone‐reactivation strategy to restore function in degenerating cones. This gene therapy approach restores light responses by providing a target G protein‐gated Inward Rectifying K+ (GIRK) channel which relays the light response transduced by the cone opsins. Light‐induced currents were elicited with ratGIRK2 upon opsin activation by light stimulation, both in vitro and in vivo.
To move this approach towards human application, we selected a previously described human GIRK1 variant with a point mutation (GIRK1‐F137S). We demonstrated its ability to generate light‐induced currents when GIRK1‐F137S and an opsin are co‐expressed in vitro. We then tested whether cone function could be improved in vivo using this human GIRK channel in the rd10 mouse model of RP. GIRK1‐F137S was expressed using an AAV8 under the control of a PR1.7 cone promoter after subretinal injection. Animals were dosed at eye opening and significant visual improvements were observed in GIRK1‐F137S‐treated mice at three weeks post injection compared to control mice. Visual improvements were transient likely due to the low number of cones remaining at the later timepoints in this fast‐progressing model.
These encouraging in vivo data support exploring the potential of AAV‐hGIRK1 F137S in other models with the aim of developing a vision restoration gene therapy product for late‐stage RP patients.
Systemic delivery of the brain‐targeting AAV‐PHPeB encoding the cholesterol hydroxylase CYP46A1 into a mouse model of Machado‐Joseph disease: a promising therapeutic strategy
B Serra1 S P Duarte1 2 N Cartier3 S Alves3 L P de Almeida1 4
1: Center for Neuroscience and Cell Biology, 3004‐504 Coimbra, Portugal 2: Institute of Interdisciplinary Research, Coimbra 3030‐789, Portugal 3: AskBio, Paris Brain Institute (ICM), 47 boulevard de l´Hôpital, 75013 Paris, France 4: Faculty of Pharmacy, University of Coimbra, Coimbra 3000‐548, Portugal
Machado‐Joseph disease (MJD) is the most prevalent autosomal dominant spinocerebellar ataxia (SCA) leading to premature death. Deregulation of brain cholesterol homeostasis has been linked to neurodegenerative disorders, including MJD. We previously showed that intracerebellar injection of AAVs encoding CYP46A1 (the key enzyme involved in brain cholesterol turnover), into MJD mouse models, was neuroprotective, reducing mutant ataxin‐3 accumulation and alleviating motor impairments associated with the disease.
We aimed at investigating whether a non‐invasive delivery of CYP46A1 was effective at ameliorating the disease phenotype and neuropathology in a transgenic MJD mouse model, with established pathology.
Post‐symptomatic MJD mice were injected with 5 × 1011vg of PHPeB‐AAV‐CYP46A1 through retro‐orbital injection. Motor assessment was performed every 3 weeks until 9 weeks post‐administration. At 10 weeks the animals were sacrificed and expression of CYP46A1 in the cerebellum, reduction of mutant ataxin‐3 aggregates and amelioration of the neuropathology of treated mice was investigated.
Results showed a tendency for motor performance improvement in MJD mice, 9 weeks post‐AAV‐PHPeB‐CYP46A1 treatment and revealed that CYP46A1 promoted the clearance of mutant ataxin‐3 aggregates. Furthermore, we observed increased levels of cerebellar CYP46A1 in treated mice, indicating that the transgene effectively reached the brain. Data obtained by immunofluorescence also suggested a reduction in ubiquitin‐positive aggregates in the cerebellum of CYP46A1 treated animals, compared to controls.
These data corroborate the beneficial role for CYP46A1 and brain cholesterol metabolism in MJD. Thus, non‐invasive CYP46A1 delivery holds great promise as a relevant therapeutic approach not only for MJD but also for other SCAs.
Regulated Chondroitinase gene therapy combined with reaching and grasping rehabilitation for restoration of upper limb function following spinal cord injury
1: King's College London 2: University of Alberta 3: Netherlands Institute for Neuroscience
Chondroitinase ABC (ChABC) is a potent neuroplasticity‐promoting agent that breaks down inhibitory extracellular matrix components formed after spinal cord injury (SCI). Combining this treatment with rehabilitation may enhance neuroplasticity induced recovery. In this study, intraspinal delivery of a second generation, immune evasive, doxycycline‐inducible lentiviral vector encoding a mammalian‐compatible ChABC transgene (dox‐i‐ChABC) was investigated in a rodent model of spinal cord injury. In Phase 1, presented here, dox‐i‐ChABC combined with reaching and grasping rehabilitation was assessed up until 26 weeks post injury (wpi) in the following experimental groups: GENE‐OFF (contusion + dox‐i‐ChABC without dox), GENE‐ON‐OFF (contusion + dox‐i‐ChABC with dox administered from time of injury until 19wpi and withdrawn from 19‐26wpi); No treatment (contusion, no dox‐i‐ChABC, no rehabilitation). Phase 2 (26‐41 wpi) is presented in linked poster (Spejo et al). Reaching and grasping rehabilitation was initiated four weeks post‐injury (10 min per day/5 days a week) and functional recovery assessed over 26 weeks post‐injury (skilled pellet retrieval, ladder stepping, grip strength). Early improvements in the pellet retrieval task in the GENE‐ON‐OFF group were not maintained after initiation of reaching and grasping rehabilitation, suggesting a potential negative interaction between treatments. The GENE‐ON‐OFF group exhibited improved ladder stepping at 10 and 14 wpi, which was reversed upon turning off ChABC expression for 8 weeks. Grip strength remained unaltered in either treatment group. At Phase 2 end point (41 wpi), cervical spinal cord, muscle and brain were collected and are currently being analysed for markers of neuroplasticity gene expression. Study funded by WFL.
AAV‐mediated gene therapy treatment of newly generated hereditary spastic paraplegia type 52 (SPG52) mouse model
1: Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona 2: Institut de Neurociències (INc), Universitat Autònoma de Barcelona 3: Unitat Histologia Mèdica, Universitat Autònoma de Barcelona 4: Vall d'Hebron Research Institute (VHIR) 5: Unitat producció de Vectors (UPV), Universitat Autònoma de Barcelona 6: Institució Catalana de Recerca i Estudis Avançats (ICREA)
Hereditary spastic paraplegia type 52 (SPG52) is an ultra‐rare inherited neurological disorder characterized by lower limb spasticity, weakness, global developmental delay, intellectual disability, and seizures. SPG52 is caused by a biallelic mutation in AP4s1 gene, which encodes a subunit of the adaptor protein complex 4 (AP‐4). Mutations in any of its 4 subunits result in complex destabilization and degradation of all subunits, leading to a shared pathophysiology and symptomatology. We have characterized patient‐derived cells and successfully treated them with viral vectors carrying a correct copy of the AP4s1 gene. This treatment enables restoration of the expression of all complex subunits. Therefore, to further assess gene therapy as a successful treatment for SPG52 we have developed the first rodent animal model. We have generated a knock‐out (KO) mouse using CRISPR‐Cas technology targeted to abolish AP4s1 expression. In the interest of both characterizing the animal model and testing the efficacy of the treatment, cognitive and physical skills batteries have been performed. The animal's motor deficiency is evaluated through grip strength, RotaRod and clasping tests, whereas the elucidation of cognitive impairment is studied with Open Field, nesting test and Morris Water Maze. Neuroanatomical changes in KO mice are being assessed by magnetic resonance imaging (MRI) and histological techniques. Currently, we are treating KO animals systemically with neurotropic AAV vectors carrying a correct copy of the AP4s1 gene. The establishment of a mouse model and its treatment with a gene therapy approach are key milestones on the road to a successful treatment for SPG52.
Long term correction of Dopamine transporter deficiency syndrome following midbrain gene therapy
1: UCL EGA Institute for Women's Health 2: UCL School of Pharmacy 3: Zayed Centre for Rare disease Research, UCL GOSH Institute of Child Health 4: Great Ormond Street Hospital for Children
Dopamine transporter deficiency syndrome (DTDS) is an inherited neurotransmitter disease due to biallelic mutations in the SLC6A3 gene. The children present with a progressive infantile parkinsonism‐dystonia and do not respond to currently available medical or neurosurgical treatments for movement disorders. The SLC6A3 gene encodes for the dopamine transporter (DAT) that is integral to dopamine homeostasis and neurotransmission. In this study, we assessed the long‐term efficacy and safety of rAAV mediated gene therapy to treat DTDS.
We transduced the midbrain of Dopamine transporter (DAT) knockout mice with rAAV2.hSLC6A3 gene therapy under the control of human synapsin promoter in a 2 log dose ranging study. Wildtype and knockout control animals received stereotactic midbrain injections of rAAV2.GFP vector (n = 8 per group). Animals were monitored for regularly for general well‐being, body weight and assessed serially for locomotor behaviours associated with DAT knockout mouse phenotype. Animals were sacrificed at 12months to evaluate long‐term transgene expression and neuropathology.
We observed a dose‐dependent therapeutic effect of rAAV2.hSLC6A3 manifested by normalisation of locomotor behaviours in open field, vertical pole and foot fault tests. The locomotor response to amphetamine, exclusively dependent on functional DAT, was restored in treated knockout animals. Brain tissue analysis showed sustain DAT expression in the midbrain of treated animals with absence of neuropathological features at 12months.
Our data demonstrate that stereotactic midbrain delivered rAAV2.SLC6A3 gene therapy provides long‐term correction of DAT function and safety in a clinically relevant animal model of DTDS, providing support for clinical trial application.
Gene‐independent strategies for cone preservation in inherited rod‐cone dystrophies
1: SparingVision, Paris, France 2: SparingVision, Philadelphia, PA, USA 3: Centre Hospitalier National d'Ophtalmologie des Quinze‐Vingts, Paris, France 4: Institut de la Vision, Paris, France 5: University of Pittsburgh, School of Medicine, Department of Ophthalmology, Pittsburgh, PA, USA
Most retinal gene therapies target the genetic variant responsible for retinal degeneration, however clinical development of genotype‐dependent therapies will not be possible for all causative genes, so gene‐independent therapeutic approaches could represent a broader treatment option.
A first approach is the co‐expression of rod‐derived cone viability factor (RdCVF) and RdCVF‐Long (RdCVFL). RdCVFL is a thioredoxin that mitigates the damage caused by accumulated reactive oxygen species in photoreceptors. Splice variant RdCVF is a neurotrophic factor secreted by rods, that binds cones to increase glucose entry and stimulate aerobic glycolysis, thereby promoting cone function. When rod cells degenerate in rod‐cone dystrophies, the lack of RdCVF and increased oxidative stress —caused by elevated oxygen levels, as rods no longer consume it— trigger cone dysfunction and degeneration. Supplementing the retina with RdCVF and RdCVFL could therefore help slow down vision loss.
A second approach is the expression of the G protein‐gated inwardly rectifying K+ (GIRK) channel in dormant cone cells that lost their outer segment and ability for phototransduction. Light stimulation of cone opsins generates a short phototransduction cascade that activates GIRK, inducing membrane hyperpolarization and generation of electrical current within the dormant cone cell body.
RdCVF/RdCVFL and GIRK gene therapies have respectively demonstrated preliminary efficacy in slowing down cone degeneration or restoring their function in animal models of retinitis pigmentosa. As these approaches are independent from the underlying genotype, they have the potential to treat a wide range of rod‐cone dystrophies, thereby addressing the high unmet need in treating this disease.
Modulation of migration and neuronal differentiation of iPSC‐derived Neuroepithelial Stem Cells to improve cell therapy in Machado‐Joseph Disease
1: CNC ‐ Center for Neuroscience and Cell Biology, University of Coimbra, Portugal 2: CIBB ‐ Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Portugal 3: Faculty of Pharmacy, University of Coimbra, Portugal 4: Institute of Interdisciplinary Research, University of Coimbra, Portugal 5: Algarve Biomedical Center Research Institute (ABC‐RI), University of Algarve, Faro, Portugal
Machado‐Joseph disease is a neurodegenerative disease caused by a mutation in the ATXN3 gene that presents several neuropathological features, including extensive cerebellar neuronal loss. Therefore, cell replacement strategies constitute a promising approach to tackle this disease. In our lab, we obtained promising results with the cerebellar transplantation of Neuroepithelial Stem cells (NESC) derived from iPSC. Nevertheless, we also observed limited cell migration and differentiation into cerebellar neurons upon transplantation. To overcome such limitations, we studied cellular pathways related to NESC migration and neuronal differentiation and identified drugs potentially able to modulate these pathways. The identified drugs were tested in NESC for cytotoxicity using Resazurin Reduction and Fluorescein Diacetate/Propidium Iodide assays. The impact on mRNA levels of genes related to migration (NOTCH1 and MSI1) and neuronal differentiation (MAP2 and TUBB3) were evaluated by RT‐qPCR. Moreover, differentiation into neurons was evaluated by immunocytochemistry for neuronal markers and cell migration was assessed through the Matrigel migration assay. Our data indicate no major cytotoxicity for the drug doses used. One of the drugs selected to induce neuronal differentiation promoted a 2‐fold increase in MAP2 and TUBB3 mRNA levels, and a robust increase in β3 Tubulin‐ and MAP2‐positive cells compared to control. Regarding cell migration, the most promising drug triggered a 50% increase in Msi1 mRNA levels and a 2‐fold increase in NOTCH1 mRNA levels, although preliminary results did not show significant cell migration. Our data indicate that the studied drugs are able to induce NESC neuronal differentiation, while cell migration is still under investigation.
Nonclinical safety and pharmacokinetic assessment of SPVN06, an AAV‐based gene therapy for the treatment of rod‐cone dystrophies
M Marie1
1: SparingVision, Paris, France 2: Institut de la Vision, Paris, France 3: University of Pittsburgh, School of Medicine, Department of Ophthalmology, Pittsburgh, PA, USA
Rod‐Cone dystrophies (RCD) are inherited retinal diseases characterized by progressive loss of the rod photoreceptors, followed by cone photoreceptor degeneration, eventually leading to total blindness. 71 genes are identified in RCD, mostly affecting the rods. SPVN06 aims at slowing down the degeneration of cones by restoring RdCVF trophic support normally provided by functioning rods, and by promoting RdCVFL antioxidant activity. RdCVF and RdCVFL are encoded in the same AAV‐based vector. SPVN06 subretinal administration is expected to slow down cone degeneration in RCD patients independently of the causative mutation. SPVN06 nonclinical safety was evaluated in cynomolgus monkeys at dose levels ranging from 6E9 to 3E11 vg/eye. A standard toxicological assessment was conducted including an exhaustive battery of ocular testing. Immunogenicity assessments included total antibodies against AAV and RdCVF/L as well as T‐cell mediated toxicity. Biodistribution analysis demonstrated that SPVN06 vector genome and transgenes persisted up to 3 months following SPVN06 administration, the longest timepoint evaluated. There were no drug‐related systemic effects. SPVN06‐related adverse findings were dose‐related, noted at ≥1E11 vg/eye, and limited to the photoreceptors and RPE cells. These findings were characterized by reduced ffERG amplitude with microscopic correlates. Uveitis was transient and the presence of immune infiltrate minimum. In absence of an immune response to the transgenes and consistently with their mechanism of action, this toxicity was attributed to cell exhaustion and/or supraphysiological levels of transgenes in a healthy retina. The NOAEL was determined at 6E10 vg/eye.
Developing gene therapy for FAM161A‐associated retinitis pigmentosa in a murine model
1: Jules‐Gonin Eye Hospital, UNIL 2: Hadassah‐Hebrew University Medical Center 3: Institute of Molecular and Clinical Ophthalmology Basel
Defects in the FAM161A gene encoding for two isoforms of a ciliary protein have been proven to cause autosomal recessive retinitis pigmentosa (RP28). Currently, no effective cure is available. In order to develop a gene augmentation therapy, we have tested different constructions of adeno‐associated virus (AAV) vectors to drive expression of two different isoforms of wildtype FAM161A cDNA. Plasmids were first validated in vitro to localize FAM161A expression in transfected cells. Following vector administration by subretinal injection, we monitored gene expression and retinal functionality in a Fam161a‐/‐ murine model. Depending on the promoter used, human FAM161A proteins could be expressed in mouse retina, preferentially localized to the connecting cilium of photoreceptors or with high expression in both the photoreceptor connecting cilium and cell body. Three months post‐injection, correct expression is accompanied by a maintenance of retina activity monitored by ERG and by a marked rescue of photoreceptor cells. However high and ectopic expression of FAM161A was associated with a good morphological rescue, but with transient improvement of retina activity. Further work is under process to define the best conditions for morphological and functional rescue. Our preliminary results inferred a therapeutic effect of human FAM161A expressed in murine photoreceptors.
MicroRNAs as cornerstones in Machado‐Joseph disease pathogenesis: Development of a microRNA‐based gene therapy
D Santos1 2 M I Santos1 2 R Baganha1 2 3 R J Nobre1 2 3 M Figiel4 L Pereira de Almeida1 5
1: CNC ‐ Center for Neuroscience and Cell Biology, University of Coimbra, Rua Larga, 3004‐504 Coimbra, Portugal 2: IIIUC ‐ Instituto de Investigação Interdisciplinar, University of Coimbra, 3030‐789 Coimbra, Portugal 3: ViraVector, Viral Vector for Gene Transfer Core Facility, University of Coimbra, Rua Larga, 3004‐504 Coimbra, Portugal 4: Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61‐704 Poznan´, Poland 5: Faculty of Pharmacy, University of Coimbra, 3000‐548 Coimbra, Portugal
Machado‐Joseph disease or Spinocerebellar ataxia type‐3 (MJD/SCA3) is a genetic neurodegenerative disorder caused by a CAG over repetition in the ATXN3 gene. MJD patients have premature death and no treatment is yet available. MicroRNAs (miRNAs) dysregulation is documented in MJD, however knowledge on this subject is quiet scarce. This work aimed at identifying dysregulated miRNAs that contribute decisively to MJD pathology and to develop a miRNA‐based therapeutic strategy. MiRNA expression profiling was evaluated by Small RNAseq in the cerebella of transgenic (Tg‐Q69) and Ki91 knock‐in (Ki91) MJD mouse models. Tg‐Q69 mice were submitted to bilateral stereotaxic injection in cerebellum with adeno‐associated viral vectors encoding for miRNAs. MiRNA levels were validated by qRT‐PCR. Balance and motor coordination were assessed over time, brains processed for PCR, WB, and IHC analyses and miRNA‐targetome has been explored using bioinformatic tools. Interestingly, a higher number of differentially expressed miRNAs was identified in Tg‐Q69 mice than in Ki91 mice with a large number of upregulated miRNAs being detected. Out of a top 60 dysregulated miRNAs, 30 were highly conserved in mice/humans and over 10 appeared significantly altered over time in Tg‐Q69. Reversion of two abnormally downregulated miRNAs in Tg‐Q69 mice resulted in significant upregulation and improvement in motor performance was observed for one of those miRNAs at 9 weeks post‐injection. In addition, miRNA‐targetome was unraveled to further elucidate miRNA‐mediating neuroprotective effect. Overall, novel dysregulated miRNAs implicated in MJD pathogenesis were identified and restoration of altered miRNA levels showed promise as a therapeutic strategy for MJD.
CYP46A1 as a relavant target to treat ALS pathology independant from its origin
G Wurtz1 H Yousef Zadeh1 C Herrmann1 E Audouard1 A Lamaziere2 N Cartier1
1: Neurogencell, Paris Brain Institute, PARIS, France 2: Saint Antoine Hospital, PARIS, France
Amyotrophic Lateral Sclerosis (ALS) is the most common motor neuron disease and is characterized by the progressive loss of upper and lower motor neurons, leading to paralysis and death. Accumulation of cholesterol in the central nervous system (CNS) has been reported to actively contribute to the disease progression in Alzheimer, Huntington, Spinocerebellar ataxia and more recently ALS. Cholesterol is essential for myelin compartment, but also for its functional and structural role in plasmatic membrane. However, in the CNS, cholesterol is synthetized in situ and is not able to freely cross the blood brain barrier (BBB). Cholesterol‐24‐hydroxylase (CYP46A1) allows the conversion of cholesterol to 24‐ hydroxycholesterol, able to cross the BBB, thus regulating cholesterol homeostasis. Furthermore, this enzyme is a key neuronal stress response such as oxidative stress or protein aggregation. Therefore, we hypothesized that CYP46A1 could be relevant for a therapy in ALS to target both familial and sporadic forms of ALS independently from their genetic origin. In the severe SOD1G93A model, we demonstrated the proof of concept of our approach. However, our therapy could be applied to any ALS form independant from its origin, we thus performed an evaluation in the C9ORF72‐ 500 repeats and obatined significant imporvements that will be presented.
Reversing MPS IIIA neuropathology through an iPSC‐based therapeutic approach
1: University of Manchester
The mucopolysaccharidoses (MPSs) are a rare heterogenous family of lysosomal storage disorders (4.5:100,000 live births) which result from deficiency in the enzymes involved in the catabolism of glycosaminoglycans. These long‐chain sugars progressively accumulate within the lysosome, compromising cellular function and leading to severe somatic and brain degeneration. MPS type IIIA, one of the most frequent types of MPSs, is mostly marked by extensive neurological decline and behavioural abnormalities. The rapid cognitive degeneration observed in these patients, together with frequent diagnostic delay, makes MPSIIIA particularly challenging, to the point that no effective disease‐modifying therapy is available. Therefore, we hypothesise that a direct repopulation of the CNS with cells expressing the SGSH enzyme could be the best option to rescue such advanced neuropathology. In this regard, we generated iPSCs from MPS IIIA compound heterozygous (R245H/c1284del11) patient dermal fibroblasts and a healthy control (WT) by a single transfection with oriP/EBNA‐based episomal vectors. Subsequently, we differentiated both cell lines into neural progenitor cells (NPCs), which were genetically modified using lentiviral vectors (LVs) overexpressing SGSH and luciferase. Finally, the genetically modified NPCs were injected into the striatum of MPS IIIA/NSG mice, a highly immunodeficient mouse model of disease, in an attempt to achieve both neurological and behavioural correction. When comparing the treatments, injection of WT LV.NPCs led to complete correction of the spatial working memory deficit observed in MPS IIIA/NSG mice; conversely, injection of MPS IIIA LV.NPCs did not rescue the neurological deficit, yet it led to aggravation of the hyperactive phenotype.
Generation of three distinct non‐human primate inducible‐models of retinal degeneration
1: Institut de la Vision 2: ESIEE‐Paris, Université Paris‐Est 3: CHNO des Quinze‐Vingts
The differences between the retinas of humans and most animal models pose a challenge for testing novel therapies. Non‐human primate (NHP) retina is physiologically and anatomically closest to the human retina. However, there is a lack of relevant NHP models for retinal degeneration that can be used for preclinical studies of vision restoration strategies. To address this unmet need we aimed to generate inducible NHP models of retinal degeneration. We generated cynomolgus macaque models – 1. by AAV‐mediated optogenetic ablation of rods; 2 by using a Crispr‐Cas9 system to disrupt the rhodopsin gene and 3. by creating a physical separation between the photoreceptors and Retinal Pigment Epithelium (RPE) using a polymer patch. In‐vivo degeneration was evaluated by fundus imaging, optical coherence tomography (OCT) and electroretinogram (ERG). The retinal tissues were fixed and evaluated by immunolabelling after sacrifice. Proof‐of‐concept experiments were first conducted in rodents to optimise doses, light levels and polymer materials. In the ‘light‐induced’ and ‘Crispr model’ we observed punctuate areas of degeneration in the injected area marked by disorganization of outer segments, loss of rod photoreceptors and thinning of the outer nuclear layer (ONL). In the ‘physical model’ the degeneration was faster and more severe but restricted to the area of the patch. In all three models, there was a general trend in the reduction of ERG wave. The three distinct NHP models for retinal degeneration that we have generated can be used for testing different gene and cell‐based therapies.
Development of a stable autologous regulatory T cell‐based cell therapy for multiple sclerosis
1: Hasselt University 2: Maastricht University Medical Center
In autoimmune diseases, FOXP3+ regulatory T cells (Tregs) skew towards a pro‐inflammatory, non‐suppressive phenotype and are unable to control the exaggerated autoimmune responses. This may largely impact the success of autologous Treg therapy which is currently under investigation for treatment of autoimmune diseases, including multiple sclerosis (MS). Our recent findings provide for the first time evidence that the inflamed blood‐brain barrier (BBB) affects human Treg stability. Using functional in vitro assays, we found that the suppressive capacity of migrated Tregs was affected. Transcriptome analysis indicated that migrated human Tregs of healthy donors and MS patients are less suppressive, have a pro‐inflammatory Th1/17 signature and upregulate the mTOR signaling pathway. In vitro treatment of migrated Tregs with the clinically‐approved mTOR inhibitor rapamycin restored the loss of suppressive function. In MS‐derived Tregs specifically, Th17‐related pathways were increased while amphiregulin was downregulated. This suggests a pre‐existing susceptibility towards inflammation and lost regenerative capacity in MS‐derived migrated Tregs. These insights and specific target identification can help in significantly improving the efficacy of autologous Treg therapy of MS. The aim is to target this BBB‐induced Treg instability in patient‐derived Tregs by genetic manipulation. By this, we restore and stabilize their immunosuppressive and regenerative function and use these designer Tregs as a powerful, autologous cell therapy for MS.
Gene therapy against amyotrophic lateral sclerosis: a bicistronic AAV vector for RNAi against mutated SOD1
P Valdés2 P Colin2 J P Gaudry2 A Aebi2
1: Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland 2: Bertarelli Platform for Gene Therapy, EPFL, 1202 Geneva, Switzerland
Amyotrophic Lateral Sclerosis (ALS) is a fatal disease caused by motoneuron degeneration. In some inherited and sporadic forms of ALS, the misfolding and aggregation of Superoxide Dismutase 1 (SOD1) lead to the gain of toxic protein activities. Our approach is based on RNA interference to reduce the level of SOD1 mRNA expression and limit the toxic effects of misfolded SOD1. When considering gene therapy against ALS, the co‐targeting of neurons and astrocytes has been shown to be important to maximize treatment efficiency. Therefore, we designed a gene therapy platform to target these two cell types using a single AAV9 vector specifically expressing a miRNA directed against the human SOD1 mRNA. This bicistronic AAV vector contains two cassettes controlled by CNS‐specific promoters driving expression in astrocytes (gfaABC1D promoter) and neurons (hSynapsin1 promoter). Intrathecal injection of AAV9‐bicistronic‐miR SOD1 in 5 months‐old high‐copy SOD1 G93A ALS mice improved motor function, rescued neuromuscular junction occupancy and significantly enhanced the survival of motor neurons until disease end stage. Furthermore, intracerebroventricular injection of AAV9‐bicistronic‐miR SOD1 in neonatal ALS mice prolonged median survival from 156 days to more than 300 days, depending on the injected vector dose. Preliminary data in intrathecally injected non‐human primates show expression of the miRNA against SOD1 throughout the spinal cord with minimal expression per AAV genome copy in the liver, consistent with the CNS‐restricted activity of the promoters. Overall, these results demonstrate the advantage of targeting RNA interference to key CNS cell types using a bicistronic vector combining neuron‐ and astrocyte‐specific promoters.
Assessment of Safety and Biodistribution of a miniMECP2 AAV9 Vector for Gene‐replacement Therapy of Rett Syndrome in Non‐human Primates (NHPs)
1: Department of Research & Development, Taysha Gene Therapies R&D, Dallas, Texas, USA 2: Department of Pediatrics, The University of Texas Southwestern Medical Center, Dallas, Texas, USA
Rett syndrome is mainly caused by loss‐of‐function mutations of the methyl‐CpG‐binding‐protein 2 (MECP2) gene. A miniMECP2 gene packaged in a self‐complementary AAV9 vector (TSHA‐102), associated with the miRARE platform, is in development as an investigational gene‐replacement therapy for Rett syndrome. Twenty‐four female non‐human primates (NHPs), aged approximately 2 years, received either a single intrathecal administration of vehicle or a total dose of TSHA‐102 (2.88x1013, 5.77x1013, or 2.31x1014 vg/animal, corresponding to 2.5x1014, 5x1014, 2x1015 vg/participant human equivalent dose [HED], respectively). Clinical pathology, anatomic pathology, behavior, clinical signs, body weight, and food consumption were assessed throughout the in‐life portion of the study. At the scheduled assessment time points of 90‐ and 180‐days there were no TSHA‐102‐related abnormal clinical observations, or changes in clinical pathology, body weight or food consumption, or neurobehavioral signs noted. Animals receiving TSHA‐102 demonstrated minimal to moderate hepatic infiltration of lymphocytes and macrophages without functional deficiency. By histopathology, dose‐related minimal to moderate nerve degeneration in dorsal tracts of the cervical, thoracic, and lumbar spinal cord was observed. Minimal nerve fiber degeneration was present in the sciatic, tibial, and radial nerves in mid‐ and high‐dose groups, with minimal mononuclear cell infiltrate in the cervical and lumbar dorsal root ganglion, all of which were considered non‐adverse. Two animals, each in Day 90 and 180 cohorts, developed anti‐miniMECP2 antibodies, but none showed a T cell response. Single IT administration of TSHA‐102 doses of up to 2.31 x 1014 vg/animal (HED 2x1015 vg/participant) were well tolerated in NHPs, with no adverse treatment‐related findings.
Assessing gene transfer in a mouse model of SYNGAP1 haploinsufficiency associated disorder
1: Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, The University of Edinburgh, Edinburgh, EH8 9XD, UK 2: Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA
SYNGAP1 haploinsufficiency causes severe neurodevelopmental disorder including intellectual disability, epilepsy and autism traits (MIM #603384). SYNGAP1 protein is a component of postsynaptic density and affects synaptic plasticity through the regulation of AMPA receptor trafficking.
The aim of this study was to assess the potential for gene transfer to correct disease‐related phenotypes in mice modelling SYNGAP1 haploinsufficiency. A gene therapy cassette incorporating a human SYNGAP1 isoform driven by a neuronal‐specific promoter, was packaged into AAV9 and delivered to postnatal Syngap1+/‐ and wild‐type mice by intracerebroventricular injection. Mice treated with vector (doses: 5E10 and 1E11vg/mouse) or excipient were subsequently exposed to a battery of neurobehavioral assessments at 7 and 15 weeks post‐treatment.
Results in vector treated Syngap1+/‐ mice revealed a dose‐dependent amelioration in the frequency of departures in a platform departure test compared to untreated controls. In contrast, no treatment effect was observed in other tests including open field, elevated plus maze and MoSeq. Post hoc immunoblot analysis of whole cortex and hippocampus showed presence of vector derived protein. An increase in total SYNGAP1 was detected in cortex in the range of 0.4‐1x wild‐type levels. Analysis of synaptosomal preparation from hippocampal tissue revealed correct translocation to the synapse of vector derived SYNGAP1.
In conclusion, we demonstrate the ability of gene transfer to produce a subtle amelioration of a risk‐taking phenotype but no clear treatment effect on hyperactivity phenotypes in MoSeq analysis.
Early distribution of 18F‐labeled AAV9 vectors in the cerebrospinal fluid after intracisternal infusion in non‐human primates
1: Jichi Medical University 2: Hamamatsu Photonics KK 3: The University of Tokyo
The delivery of adeno‐associated virus (AAV) vectors via cerebrospinal fluid (CSF) has emerged as a useful method for widespread transduction in the central nervous system. While infusion into the cerebral ventricles is a common protocol in preclinical studies of small animals, cisterna magna has been recognized as an alternative target for clinical studies that can be reached in a less invasive manner using an intrathecal catheter via the subarachnoid space from a lumbar puncture. Unlike the classical circulation theory that the CSF moves as a unidirectional bulk flow from the choroidal plexus to subarachnoid villi, recent in vivo magnetic resonance imaging revealed the dynamic motion of the CSF influenced by various factors including cardiac pulsations and pulmonary respiration. We herein evaluated the early distribution of fluorine‐18‐labeled AAV9 vectors ([18F]FB‐AAV) infused into the cisterna magna of non‐human primates by positron emission tomography (PET). Positron emitter‐labeled tyrosine‐mutant AAV9 expressing green fluorescent protein (GFP) was prepared by amino group‐modification of capsid protein using N‐succinimidyl 4‐[fluorine‐18] fluorobenzoate. The [18F]FB‐AAV were infused via the cisterna magna in two macaques. Accumulation of [18F]FB‐AAV was analyzed at 30‐min intervals over 4 hours by PET. [18F]FB‐AAV vectors were distributed into subarachnoid space around the spinal cord as well as basal cisterns within the initial 30 min. On histological examination of GFP expression, neurons in the cervical spinal cord were efficiently transduced. For gene therapy that primarily targets the spinal cord and brainstem such as amyotrophic lateral sclerosis, intracisternal magna infusion is expected to result in effective vector distribution.
Amelioration of retinal mitochondrial dysfunction with AAV‐ophNdi1.
1: Trinity College Dublin 2: Royal Victoria Eye and Ear Hospital
Many ocular disorders have a mitochondrial involvement from primary mitochondrial disorders such as Leber hereditary optic neuropathy (LHON), predominantly due to mutations in genes encoding subunits of complex 1, to Mendelian and multifactorial ocular conditions such as dominant optic atrophy, glaucoma and age‐related macular degeneration. In this study, we have optimised the nuclear yeast gene, NADH‐quinone oxidoreductase (NDI1), which encodes a single subunit complex I equivalent, creating a candidate gene therapy to improve mitochondrial function, agnostic to the genetic mutation driving disease. Optimisation of NDI1 (ophNdi1) should allow for a significant reduction in the therapeutic dose required to achieve benefit, reducing the potential of an immune response when delivered virally. ophNdi1 substantially increased expression in vivo, protected RGCs and increased visual function, as assessed by optokinetic and photonegative response, in a rotenone‐induced murine model of mitochondrial dysfunction. In addition, ophNdi1 increased cellular oxidative phosphorylation and ATP production, counteracting two of the hallmarks of mitochondrial dysfunction, and protected cells from rotenone insult to a significantly greater extent than wild type NDI1. Notably, AAV‐ophNdi1 also rescued the reduced ATP and oxygen consumption rates observed in LHON patient‐derived fibroblasts, demonstrating the potential of ophNdi1 as a candidate therapy for ocular disorders where mitochondrial deficits comprise an important feature.
Gene‐based therapeutics for C9ALS/FTD
1: Sheffield Institute for Translational Neuroscience (SITraN), Department of Neuroscience, University of Sheffield, UK 2: The Institute of Neuroscience, University of Sheffield, Sheffield, UK
A hexanucleotide repeat expansion of GGGGCC (G4C2) in the first intron of C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), termed C9ALS/FTD. Repeat associated non‐AUG (RAN) translation of the repeat expansion leads to the aberrant formation of five toxic dipeptide repeat (DPR) proteins. Furthermore, the repeat expansion and associated DPR proteins also lead to increased DNA double strand breaks. Thus, genome instability is a potential contributing factor in C9ALS/FTD pathogenesis.
RuvBL1 and RuvBL2 are members of the AAA+ (
UPF1 delivered by novel expression‐enhanced promoters protects cultured neurons in a genetic ALS model
1: MeiraGTx 2: University of Michigan
Regulator of nonsense transcripts 1 (UPF1) is an RNA helicase important for nonsense mediated decay (NMD) of mRNA transcripts that contain premature stop codons. Studies have demonstrated that UPF1 overexpression in animals protects against neurodegeneration in models of ALS involving TDP‐43 and FUS toxicity. We aimed to enhance the potency of our UPF1 expression vectors while decreasing their size, since greater potency will allow lower MOIs of virus to be used clinically, while the smaller construct size improves AAV packaging and manufacturing efficiency. First, we enhanced the expression of UPF1 by rationally optimizing cis‐regulatory elements such as the promoter. In three different mammalian cell lines, the top promoter construct, UPF1‐11, drove greater UPF1 expression than the controls, including the myc‐UPF1 construct used in the original mouse neuroprotection experiments. Next, we tested the function of the best candidates to rescue toxicity in a TDP‐43 cellular model of iPSC‐induced neurons (iNeurons). The AAV vector containing UPF1‐11 protected iNeurons at lower MOIs than the myc‐UPF1 vector containing the original promoter, indicating a functional potency enhancement. At 4.9 kb in size, UPF1‐11 is 1.5 kb smaller than the original construct, and thus offers AAV packaging and manufacturing benefits as well. UPF1 delivered by MeiraGTx vectors shows promise in treating ALS with TDP‐43‐related proteinopathy, and animal studies are ongoing.
Generation and characterization of cerebellar neuronal progenitors for Machado‐Joseph Disease treatment
1: CNC ‐ Center for Neuroscience and Cell Biology, University of Coimbra, Portugal. 2: CIBB ‐ Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Portugal. 3: Institute of Interdisciplinary Research, University of Coimbra, Portugal. 4: Faculty of Pharmacy, University of Coimbra, Portugal. 5: University of Copenhagen, Copenhagen, Denmark.
Machado‐Joseph disease (MJD) is the most common dominantly inherited ataxia worldwide characterized by widespread neuronal death, mainly in the cerebellum. As by the time of diagnosis, MJD patients usually present extensive neuronal death, we anticipate that cell replacement will be required to promote robust recovery. For MJD treatment, we have obtained very promising results upon cerebellar transplantation of induced pluripotent stem cell‐derived neuroepithelial stem cells (iPSC‐derived NESC). However, limited cell migration and differentiation into cerebellar neurons were observed. The transplantation of the right type of cells is critical for the success of cell replacement strategies and the transplantation of cerebellar neuronal progenitors (CNPs) into the cerebellum can potentially improve the engraftment success. Here, we describe the generation and characterization of iPSC‐derived CNPs for cerebellar transplantation. Markers of progenitors of granule cells (ZIC1), Purkinje cells (FOXP2), Gabaergic (PTF1A) and glutamatergic (ATOH1) neurons were selected for CPNs markers. The mRNA levels of these cell markers were evaluated in the iPSC‐derived CNPs generated with a protocol adapted from a previous study (Nolbrant et al. Nat. Protoc. 2017) at different time points (t = 0, 9, 11, and 16 days). ZIC1, FOXP2, PTF1A, and ATOH1 mRNA levels of the CNPs were found significantly increased. Additionally, increased levels of ZIC1 and ATOH1 at t = 16 were confirmed through immunocytochemistry. Furthermore, iPSC‐derived CNPs presented 5 times higher PTF1A and 3.8 times increased FOXP2 mRNA levels compared with iPSC‐derived NESC. Data suggests a successful generation of CNPs, which will be further tested for cell replacement strategies in MJD mouse models.
AAV2 expressing truncated form of soluble VEGF receptor‐2 reduces the growth of choroidal neovascularization in mice
1: A.I.Virtanen Institute, University of Eastern Finland 2: School of Pharmacy, University of Eastern Finland 3: Kuopio Center for Gene and Cell Therapy 4: Drug Research Program, Division of Pharmaceutical Biosciences, University of Helsinki 5: Gene Therapy Unit, Kuopio University Hospital
Pathological angiogenesis in the eye can lead to the vision‐threatening diseases such as neovascular age‐related macular degeneration. Because vascular endothelial growth factors (VEGFs) and their receptors regulate angiogenesis, antiangiogenic agents such as aflibercept have become a mainstay in treating neovascular ocular diseases. However, for these conditions, most patients require frequent injections into the eye, burdening the health care system. Therefore, new therapies with long duration of treatment response are necessary. In this study, we investigated the effect of a novel gene therapy of adeno—associate virus 2 expressing soluble vascular endothelial growth factor receptor 2 (AAV2‐sVEGFR‐2‐Fc) on neovascularization in the laser—Induced choroidal neovascularization (CNV) mouse model. Optical coherence tomography and fluorescein angiography were used to assess the formation of neovascularization in the eye. The immune response of the injection was determined by immunostaining of macrophages. As a result, a single intravitreal injection of AAV2‐sVEGFR‐2‐Fc significantly suppressed fluorescein leakage at 7, 14, 21 and 28 days post‐lasering compared to the control group and caused no changes in the retinal morphology. After the injection of AAV2‐sVEGFR‐2‐Fc and phosphate saline buffer (PBS) macrophage infiltration was observed in the eye. In conclusion, AAV2 mediated gene delivery of sVEGFR‐2‐Fc efficiently reduces the formation of CNV and could be developed to a therapeutic compound for such diseases.
Safety study for a non‐viral gene therapy approach to treat dry AMD: In vitro exclusion of tumorigenicity of IPE cells transfected with the genes coding for PEDF and GM‐CSF
1: University of Geneva 2: University Hospitals of Geneva
Multiple gene therapy approaches are under investigation as treatment for ocular disorders like age‐related macular degeneration (AMD). Though viral gene therapy is accompanied by several challenges, most approaches still use adenoviral‐associated virus vector systems. We suggest to transfect retinal‐(RPE) or iris pigment epithelial (IPE) cells with the genes coding for pigment epithelium‐derived factor (PEDF) and granulocyte macrophage‐colony stimulating factor (GM‐CSF) using the non‐viral Sleeping Beauty (SB100X) transposon system. Here, we excluded tumorigenicity of the final cell product in a soft‐agar assay for 14d. Colony formation of transfected porcine IPE cells was compared to human Y79 retinoblastoma and immortalized (transfected with the pSV3‐neo plasmid) porcine RPE cells as positive controls (n = 6/group); non‐transfected human RPE cells were chosen as negative control. Cells were transfected by 0.03μg of the pFAR4‐CMV SB100X SV40 transposase and 0.47μg of the pFAR4‐ITRs‐CMV‐PEDF‐BGH and pFAR4‐ITRs‐CMV‐GM‐CSFS‐BGH transposons using electroporation (1’350V, 20ms, 2 pulses); transfection efficiency was confirmed by ELISA determining PEDF (678.44 ± 91.81 ng/h/104cells) and GM‐CSF (8.96 ± 2.44ng/h/104cells) concentrations in the medium of transfected IPE cells. Cell growth was examined microscopically at days 0,6,9,12. At 14d, colonies were stained with crystal violet, microscopically documented, and counted using the ImageJ software. Y79 cells showed high proliferation with 10.25 ± 2.88 CFU/image. Immortalized RPE cells showed lower but detectable CFU‐formation capabilities (2.3 ± 1.02 CFU/image). In contrast, transfected IPE cells could not grow in soft‐agar (0 ± 0 CFU/images) similar to not‐transfected RPE cells (0 ± 0 CFU/image). The data demonstrates that PEDF‐GM‐CSF double‐transfected IPE cells do not grow in soft agar and do not show any tumorigenic potential.
Establishing a robust sgRNA expression screening assay for CRISPR activation in vitro
1: Lund University
Gene therapy represents an attractive option for tackling the complex pathology of neurodegenerative disorders (NDs), as well as directly targeting the underlying cause of a condition. Indeed, there is great therapeutic potential in increasing endogenous expression of neuroprotective genes through CRISPR activation (CRISPRa). Transcription Factor EB (TFEB) is a master regulator with a central role in autophagic response and lysosomal biogenesis and has been highlighted as a potential therapeutic target through improved clearance of neuronal plaques and aggregates commonly found in NDs. However, reliable, and flexible models are required to assess the expression activity of sgRNAs for potential therapeutic target sites. In this project, we present a robust screening assay for determining the gene expression activity of sgRNAs using CRISPRa in vitro. Additionally, we provide a comprehensive cloning workflow for seamless tandem assembly of up to eight sgRNA candidates in a single vector using the GoldenGate cloning technique. This strategy allows for efficient activity assessment of single‐ or multiple targeted sgRNA candidates simultaneously through standard cell‐line transfection and fluorescence‐based quantification. We determined numerous promising sgRNA‐candidates, demonstrating a 3.1 to 6.9‐fold increase of TFEB expression across multiple cell lines. This data illustrates the reliability and multiplexing potential of our workflow and presents a robust method for easily assessing sgRNA activity against genes of interest in vitro. Additionally, it opens for further assay development that may be used to also assess the functional effect of selected CRISPRa sgRNAs through fibril‐based in vitro models of NDs.
AAV‐ARSA mediated gene replacement for the treatment of Metachromatic Leukodystrophy
1: Sanofi
Metachromatic leukodystrophy (MLD) is an autosomal recessive neurodegenerative disorder caused by mutations in the arylsulfatase A (ARSA) gene, resulting in lower sulfatase activity and the toxic accumulation of sulfatide in the central and peripheral nervous system. In MLD patients, this leads to progressive demyelination, cerebral atrophy, peripheral neuropathy, and shortened life expectancy. We present an AAV‐mediated gene replacement strategy to treat MLD and report therapeutic benefit in a MLD mouse model treated at different neuropathologic stages, and biodistribution in non‐human primates (NHPs). We demonstrate that AAV‐hARSA mediated gene replacement via bilateral intracerebroventricular (ICV) dosing in MLD mice (Arsa ‐/‐) at different neuropathologic stages resulted in the reversal of MLD‐associated pathology. AAV‐hARSA treated mice demonstrated increased sulfatase activity in the brain and spinal cord and a concomitant reduction in sulfatide levels (LC‐MS) in the brain, spinal cord, CSF and plasma. Treated MLD mice exhibited prominent hARSA expression, secretion, and uptake by non‐transduced cells (cross‐correction) leading to improved myelination in the corpus callosum, recovery in brain oligodendrocyte cell numbers (snRNAseq), and normalization of plasma Nf‐L levels. We also report a NHP study evaluating hARSA expression and biodistribution by intra‐CSF administration of AAV‐hARSA. Broad and dose‐dependent supra‐physiological sulfatase activity was noted in NHP brain. Furthermore, hARSA in situ hybridization and immunohistochemistry confirmed broad biodistribution and robust transgene expression in NHP brain and spinal cord. Our results indicate that AAV‐hARSA mediated gene replacement is a viable approach to achieve broad and therapeutic levels of ARSA in the CNS and PNS, with potentially meaningful therapeutic impact.
Optimization and scale‐up of AAV2‐AQP1 Production using a novel transient transfection agent
1: MeiraGTx
Transient transfection in adeno‐associated viral vector production is a complex and multimodal operation requiring multi‐stage optimization to yield robust and predictable outcomes at manufacturing scale. A manufacturing process for the production of AAV2‐AQP1 was developed using HEK293 suspension cells and triple plasmid transfection in single use stirred tank reactors. The novel transfection reagent showed improved productivity and full capsids ratio compared to the current reagent in an initial screening and was selected for further optimization. Optimization of the transfection unit operation using the novel transfection reagent was performed using a 5‐factorial DOE at 250 mL scale and aiming at maximizing productivity and full capsids ratio whilst minimizing cost‐of‐goods and enabling a robust transfection operation at the planned commercial scale of 200L. The transfection setpoints selected following 250 mL scale optimization were tested at 10L scale and further scaled up during two 50L production bioreactor runs. Cell growth and viability performance targets were maintained during scale up and reproducible viral genome titers above 3x1014 VG/L were obtained across the entire range of bioreactor scales.
Bioprocess optimization and nutrient supplementation for improved production of lentiviral vectors in serum‐free suspension cultures
1: IBET ‐ Instituto de Biologia Experimental e Tecnológica 2: ITQB NOVA ‐ Instituto de Tecnologia Quimica e Biologica Antonio Xavier
The use of lentiviral vectors for gene therapy applications has been growing in recent years. These vectors are currently a first choice for applications requiring long‐term transgene expression. The most common method for lentiviral vector production relies on transient transfection of mammalian cells with plasmid DNA. This method presents high batch‐to‐batch variability and is often performed in adherent cell culture systems in the presence of animal‐derived serum, limiting scalability and increasing downstream processing burden. This work describes the establishment and optimization of a new protocol for scalable production of lentiviral vectors in serum‐free suspension cultures. Key bioreaction parameters were evaluated and a comprehensive study of nutrient supplementation was performed to further improve productivity and robustness.
293T cells were sequentially adapted to different serum‐free medium formulations and characterized for cell growth and viability. Transfection parameters were optimized to maximize lentiviral vector yields. Increasing cell density at transfection increased titers by up to 12‐fold. A set of nutrient supplementation strategies was evaluated to further improve lentiviral vector production. Lipid supplementation was the most relevant, further increasing productivity by up to 3‐fold. To understand the main factors leading to high variability in transient lentiviral vector production, different experimental parameters were monitored. Cell concentration steps and, specifically, the time that cells spend pelleted before transfection was identified as a major source of variability, impacting transfection efficiency and titers.
With this work, a new scalable production platform was established and key metabolic insights and bioreaction parameters were identified, enabling to debottleneck lentiviral vector manufacture.
Achieving AAV vector productivity >1E15vg/L and scaling suspension transfection to 2,000L through upstream process optimization
1: Oxford Biomedica Solutions
Transient transfection of plasmids into suspension HEK293 cells for producing AAV provides great flexibility and speed to move drug candidates into clinical trials. Having a platform model allows for efficient implementation of continuous innovation. Traditionally, a batch process transfected with three plasmids at 2E6 viable cells/mL is used. To improve bioreactor vector genome (vg) productivity and percentage intact capsids, OXB Solutions have developed a high cell density process with a novel transfection method and cutting‐edge dual plasmid transfection. This new process demonstrates substantial increases in bioreactor vg titer by about 10 folds and increases the percentage of intact capsids in the affinity product by about 2 folds. The new process is highly reproducible and allows for plug‐and‐play use, now tested in over 9 different capsid serotypes (clades A, B, E & F), in most cases resulting in 2L bioreactor vg titer close to or above 1E15vg/L and intact capsids in the affinity product close to or above 50%. Furthermore, we successfully scaled up the new platform process from 2L to 50L and 2,000L bioreactor with consistent vg productivity and Drug Substance product quality, confirming 90% fully intact vector. To push the bar even higher, we have been developing our next‐ generation high cell density transfection process and are reporting a bioreactor vg titer of over 2.5E15vg/L from our preliminary 2L studies.
Stable and inducible AAV producer cell lines to support large‐scale AAV manufacturing
S Ross1 T K Liu1 A K Parker1 A M Green1 J R Studer1 R T Oglesby1 J Carrillo1 N Kruer‐Zerhusen1 E Johnson1 J Wright1 V Oden1 I Zolotukhin1 A Darling1 E Butterworth1 I Finger‐Baker1 J McCoy1 P Chaudhuri1 E Rodriguez‐Lebron1 K H Warrington1
1: Lacerta Therapeutics
Innovative strategies are required to develop large‐scale manufacturing solutions and provide commercially viable AAV products to large clinical populations. We have developed the first stable and inducible AAV producer cell line system using the OneBac IC‐BEVs platform. OneBac producer cell lines are engineered with inducible promoters, enhancer motifs, and proprietary kozak sequences to enhance capsid stoichiometry, overcoming limitations of reduced potency of AAV vectors derived from the IC‐BEVs system. Stable integration of these elements into the Sf9 genome facilitates induction of AAV production at high volumetric titers by active infection using a recombinant baculovirus devoid of AAV elements that mitigates BEV genome stability issues during scale‐up. The system is adaptable to multiple AAV capsid serotypes and effectively packages self‐complimentary and single‐stranded AAV genomes. Image verified clonal producer cell populations can be rapidly screened to identify rhabdovirus free candidates capable of exceeding E5 AAV gc/cell or E14 AAV gc/L. The performance of OneBac producer cell lines is reproducible and consistent over at least 20 passages post‐thaw, supporting the linear scalability of the system. As reported by others, different patterns of post‐translational modifications are observed in the capsid proteins of IC‐BEV derived AAVs compared to those of mammalian origin. Importantly, OneBac‐derived AAVs are as potent and effective as their mammalian‐derived counterparts in vivo. In conclusion, this novel producer cell line system is a highly promising solution for low‐cost, robust, and highly reproducible large‐scale AAV manufacturing. Lacerta is now partnering with Contract Drug Manufacturing Organizations to provide access to this next‐generation technology.
AAV titer and integrity quantification by multiplex digital PCR
1: Gene Therapy Analytics, Baxalta Innovations GmbH, a part of Takeda.
Vector genome titer quantification using digital PCR (dPCR) is a state‐of‐the‐art analytical method in gene therapy for determining the viral concentration. A single target approach can provide a consistent concentration of the product however this approach lacks important information of the DNA insert including the data of the full‐length of the transgene. Multiplex digital PCR allows to quantify multiple targets of the vector genome, delivering a broad versatile and meaningful read out. This analytical method delivers the vector genome titer, allows the monitoring of the depletion of truncated inserts during the production process and in addition the assessment of the presence of full‐length or only nearly full‐length inserts in the final product. Our investigation, including nine different process step samples, convinced us that the multiplex dPCR method is an important method to monitor the production of gene therapy products, especially in process development but not exclusively. In addition, it delivers a good understanding on the best target for the vector genome quantification for product release testing to deliver reliable titers for dosing. On top it might be a suitable method to further investigate the packaging model for AAV, that still leaves open questions.
CARE‐dependent DNA amplification of integrated AAV rep and cap genes in stable packaging cells is dependent on Adenovirus late proteins
1: Oxford Genetics Ltd 2: 3: University of Oxford
Adeno‐associated virus (rAAV) is the vector of choice for in vivo gene therapy, however, scalable production of rAAV to meet demands remains a challenge. rAAV‐packaging cell lines containing rep‐cap have been explored extensively and remain an efficient approach for rAAV production. rAAV production is induced by infection with wild‐type adenoviruses to promote amplification of integrated rep‐cap genes (100 to 1000‐fold), essential to supply sufficient Rep‐Cap proteins for rAAV production. This amplification effect is dependent on a cis‐acting replication element (CARE). Adenovirus proteins that play a role in mobilizing CARE remain to be fully defined.
We recently described a new helper adenovirus system entitled ‘TESSA’ wherein the Major Late Promoter (MLP) was modified in situ to enable promoter self‐repression for contaminant‐free manufacture of rAAV. TESSA was capable of providing helper functions to enable rAAV replication when the rep‐cap genes were provided in trans but unable to support rAAV production from stable packaging cells.
Using rAAV‐packaging cell line HeLaRC32, we show that expression from the adenovirus MLP is required for rep‐cap amplification and efficient rAAV production. We show that expression of the adenovirus L4 22/33K unit is essential and can be titrated away from the CARE amplification process by replicating adenoviral genomes. Importantly, siRNA‐knockdown of the adenovirus DNA‐polymerase or use of a temperature‐sensitive DNA‐polymerase mutant in TESSA (TESSA‐tsDNA) significantly decreased its genome replication, and recovered rep‐cap amplification for rAAV production. We propose TESSA‐tsDNA as an efficient helper virus for contaminant‐free manufacture of rAAV from stable packaging cell lines that warrant further exploration.
rAAV doses: impact of titer determination methods
1: Charité ‐ Universitätsmedizin Berlin, Germany
With an increasing number of on‐going clinical trials in gene therapy for recombinant Adeno‐Associated Virus (rAAV) thorough investigation and full characterisation of Critical Quality Attributes are mandatory. These include precise assays to identify known biases for rAAV vector genome titration. While AAV titration was initially determined using qPCR, in recent years the shift has moved to digital droplet PCR (ddPCR) due to its higher accuracy and lower variability. Using ddPCR we have identified previously undetected biases in the quantification of intact rAAV vector genomes either with single‐stranded or self‐complementary AAV vectors. The percentage of partial AAV genomes double be considerable, goes undetected by qPCR and impacts the determination of real, transducing vector genomes. We propose a newly developed bias‐free method to quantify truncated and full‐length rAAV genomes by multidimensional ddPCR. Based on the position of the primers/probe within the genome. we detect a 30% variation in genome titer for ssAAV and more than 50% for scAAV based. The identification of inter‐ and intra‐assay variations calls for updated standards in AAV genome titer and dose reporting for consistent dose determination of clinical trial preparations.
Towards the generation of pre‐clinical grade macrophages from human iPSC for upcoming cell‐based immunotherapies
A Rafiei Hashtchin1 2
1: Department of Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Hannover, Germany 2: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany 3: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), REBIRTH, Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany 4: Novo Nordisk A/S, Måløv, Denmark 5: Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Hannover, Member of the German Center for Lung Research (DZL)
Macrophages are crucial cells with a key role in tissue homeostasis of different organs. Thus, macrophages are considered a valuable therapeutic cell type and a plethora of clinical and pre‐clinical attempts are underway to prove macrophage‐based immunotherapies. To fulfill the growing demand and to pave the clinical translation of macrophages as an investigational advanced therapy medicinal product, human induced pluripotent stem cell (hiPSC) and thereof derived macrophages in fully defined, GMP quality represent a promising technology. We here show the first steps towards the generation of macrophages from human iPSC in preclinical‐grade, which is suitable for the stepwise scale‐up of macrophage production using industrial‐compatible bioreactors.
As the first step, iPSC expansion was followed by combined aggregation and mesoderm priming in small‐scale suspension culture, using GMP‐grade components and media. Primed aggregates were exposed to direct differentiation, which resulted in successful production of macrophages (iMacGMP), along with the definition of defined quality control criteria. As the next step, the established procedure was advanced to larger process volume, which allowed for the efficient and continuous production of iMacGMP from day 4 of differentiation for more than 12 weeks. Generated iMacGMP were comparable across all productions and represented classical macrophage morphology, with more than 95% viability and purity defined by CD45+CD11b+CD14+ expression. The Initial potency of iMacGMP was validated by high phagocytic capacity of bacterial bioparticles. As of now, we are further advancing the procedure toward the bioreactor system, which can provide sufficient quantity of macrophages for cell therapies targeting different diseases.
Development of a Robust Process and Analytics for scAAV9 Manufacture
D Jesus1 M Gosse1 C Troche1 M Breedlove1 K Meyer1 2 3
1: Alcyone Therapeutics, Lowell, Massachusetts 2: Abigail Wexner Research Institute Nationwide Children's Hospital, Columbus, Ohio 3: The Ohio State University, Columbus, Ohio
Viral vector gene therapy is a fairly novel therapeutic approach for several disease indications, most of which are rare diseases involving an accelerated pathway to the clinic. One of the major challenges of this approach is building a robust and scalable manufacturing process early in the product's lifecycle with limited time and investment. In an attempt to improve productivity, scalability and manufacturability, our team is focused on taking a quality by design (QbD) approach. Utilizing a suspension process, design of experiment (DoE) and state‐of‐the‐art reagents, we have shown improvements in productivity. These improvements in productivity are scalable from small scale studies to bioreactor. We have also focused our attention on two critical assays – dosing (vg titer by ddPCR) and potency, in parallel to process development. Early analytical development is imperative to not only help progress process development, but also to ensure assays will be ready for qualification and timely release of product. The biology of our transgene (significant secondary structure, non‐coding short RNA) has made the development of a canonical vg titer assay and functional potency assay challenging. We will share results that address and solve for these issues, including ddPCR troubleshooting and a phase appropriate approach for potency assay development
Designing and screening formulations to improve manufacturability and distribution of AAV gene therapies
1: MeiraGTx UK II
AAV gene therapies in a liquid formulation are rarely considered stable under refrigeration and are typically stabilized through freezing at ‐80°C. However, the process of freezing and thawing may compromise product quality, while transportation and storage of frozen materials is often complicated and expensive. Furthermore, the use of ‐80°C reduces the number of available drug delivery device options, e.g., pre‐filled syringes, which can hinder accessibility of the product. In this work, we designed and screened a number of formulations that would maintain AAV2 product quality at various storage and freeze / thaw conditions over time. We demonstrate that an appropriate selection of excipients, and the balance of ionic strength and osmolality can lead to reproducible and stable product quality at room temperature, at 4°C, and the ability to withstand 10 freeze / thaw events. The results show that the reliance on the ‐80°C cold chain can be reduced for improved manufacturability and distribution, and alternative product containment options can be implemented for improved product accessibility.
Contributing factors to loss of lentiviral vectors during scale‐up of membrane processes for cell and gene therapy manufacturing
1: Department of Biochemical Engineering, University College London 2: Division of Infection and Immunity, University College London 3: Biotherapeutics and Advanced Therapies, Scientific Research and Innovation, Medicines and Healthcare products Regulatory Agency (MHRA)
Multiple Lentiviral vectors (LV) based Cell and Gene Therapies (CGT) are now advancing past phase I/II. The need for scalable, high‐yield and robust manufacturing processes is thus becoming necessary. Membrane processes are employed in the clarification, concentration and formulation of LV. However, unoptimised purification steps expose the fragile enveloped LV to potentially harsh or unsuitable processing conditions with membrane fouling and increasing shear forces. These parameters have been hypothesised to cause LV functional titre loss.
This presentation will focus on a novel examination of the impact of shear stress on LVs during membrane processing. It will discuss this interaction during pre‐chromatography concentration (ultrafiltration) and buffer exchange (diafiltration) using constant flow and pressure‐controlled tangential flow filtration (TFF). This was achieved using an ultra‐scale down TFF model for high throughput small‐scale studies and scaled up using commercial KrosFlo®KR2i TFF. The LV feed was produced using transient transfection with three envelope proteins (VSV‐G, RDpro and Cocal‐G) and stable WinPAC_RDpro packaging cells.
This study demonstrated that shear alone does not lead to significant loss irrespective of the envelope protein (p = 0.8324). For VSV‐G, 90% of functional titre is recovered at high shear. However, introducing a membrane surface reduces LV recovery to 40% for VSV‐G and 20% for Cocal‐G post ultrafiltration. This suggests that the interaction between the LV and the membrane surface may lead to detrimental interfacial shear stress. Utilising this we have studied the recovery and productivity further by considering other factors such as the sequence of TFF unit operations.
Tetracycline Enabled Self‐Silencing Adenovirus (TESSA) enables efficient rAAV manufacture from CARE‐complement HEK293 stable producer cells
1: Oxford Genetics Ltd
Recombinant adeno‐associated virus (rAAV) is the vector of choice for in vivo gene therapy, but it remains challenging to scale up rAAV manufacture in a cost‐effective manner to meet clinical demands. We recently described a new self‐silencing helper adenovirus system entitled ‘Tetracycline‐Enabled Self‐Silencing Adenovirus' (TESSA) for the delivery of AAV rep and cap genes, and the ITR genome for efficient rAAV manufacture without adenovirus contamination.
Here we developed HEK293‐based stable producer cell lines to further simplify the rAAV manufacturing process using TESSA. We explored the use of the AAV cis‐acting replication element (CARE), nucleotide sequences mapping to the p5 promoter of the rep gene, which acts as an origin of replication for AAV and is well‐known for promoting rep/cap gene amplification from stable rAAV packaging cells upon adenovirus infection. We modified an rAAV genome encoding EGFP with the CARE sequences. Using the phiC31 system, we integrate the AAV genome into a cGMP‐compliant suspension serum‐free HEK293 cell line. High‐producer single‐cell clones were effectively screened by simple infection with a single TESSA expressing Rep and Cap, banked, and expanded for rAAV manufacture using TESSA. Importantly, replication of the stably‐integrated rAAV transfer genome exceeded >1x105‐fold upon infection with TESSA‐RepCap and yielded ∼1.5 x1011 and ∼4.0 x1010 vector genomes per mL of rAAV8 and rAAV1. While replication of the CARE DNA mirrored amplification of the rAAV genome, packaging of CARE sequences into AAV capsids was observed at low levels of 0.6%. We propose this as a simple and high‐yielding approach for scalable rAAV manufacture.
What's in your AAV ‐ Oxford Nanopore Sequencing and SEC MALS reveal serotype‐specific differences
1: Sartorius Xell GmbH
In the past years, recombinant adeno‐associated viruses (rAAV) have gained an outstanding reputation in gene therapy. Evidence of a dose‐dependent immune response with systematic use renders a comprehensive understanding of AAV quality characteristics essential. AAV serotypes 2, 5 and 8 were produced in bioreactor scale in a commercially available HEK293 suspension cell line. Quality attributes from crude AAV samples after chemical lysis followed by alluvial filtration, including AAV aggregation, as well as vector and capsid titers were assessed by size exclusion chromatography (SEC) combined with multiangle light scattering (MALS). The data obtained were also aligned by ddPCR and capsid ELISA, showing a high proportion of empty capsids with the lowest full:empty ratio for AAV2. To determine the proportion of AAVs that do not contain the desired vector transgene, AAVs were purified by affinity chromatography followed by Proteinase K and DNase I digestion. AAV vector genomes were isolated from purified samples by capsid disruption and a silica‐based DNA purification method. Sequencing was performed using the Oxford Nanopore MinION device along with the Rapid Sequencing Kit. Among the more than 3% of mispackaged sequences, resistance markers for the amplification of plasmids in E. coli as well as host cell DNA were identified. Among the packaged vector sequences, truncated vector genomes, as well as fusion products were observed. The applied methods give insights into AAV vector preparations, which will be helpful for future developments of safe gene therapy products.
Use of anion exchange chromatography in weak partitioning mode to provide high empty AAV capsid removal and product yields
1: MeiraGTx UK II
Removal of product‐related impurities is an important part of adeno‐associated viral vector purification. Although separation of the empty capsids from the full capsids is achievable when using ultracentrifugation, scalable chromatography methods have proven more challenging. As an alternative, anion exchange chromatography in bind and elute mode has commonly been investigated in an attempt to reproduce the high full capsid ratios achieved by ultracentrifugation. However, the similar properties of the full and empty capsids lead to a sharp trade‐off between product yield and full capsid ratio which makes selecting optimal conditions difficult. This is also hindered by complex interactions between the AAV capsids and chromatography matrices which often lead to further product losses.
In this study different surfactant types and concentrations were screened to maximise the product yield of the AAV2. Exploring the design space led to weak partitioning mode being identified as an alternative to bind‐and‐elute mode, and conditions that achieved the removal of >50% of the empty capsids were identified. The results show that a full capsid ratio of >80% as confirmed by AUC, and a product yield >50% can be robustly achieved at the conditions identified in this study. Understanding the design space and identifying the process failure points meant that the process was reproducible at manufacturing scale.
Production and characterisation of mesenchymal stromal cell‐derived extracellular vesicles in a 3D bioreactor system
1: Discipline of Surgery, Lambe Institute for Translational Research, National University of Ireland Galway, Galway H91 YR71, Ireland. 2: Regenerative Medicine Institute, National University of Ireland Galway, Galway H91 YR71, Ireland 3: CÚRAM, SFI Research Centre for Medical Devices, National University of Ireland Galway, Galway H91 YR71, Ireland
Mesenchymal stromal cell‐derived extracellular vesicles(MSC‐EVs) have garnered increased interest as cancer therapeutics due to a potential tumour homing capacity and immune privilege. However, In Vitro production is challenging due to yield and scalability issues. This study aimed to produce and characterise large quantities of MSC‐EVs through 3D culture in a serum‐free hollow fiber bioreactor(FiberCell Systems Inc). Following pre‐treatment with fibronectin, 1x 109 MSCs were inoculated into a 20 kDa Fibercell bioreactor for 3D culture. Glucose consumption was monitored daily to guide media replacement. Once culture stabilised, chemically defined serum‐free media was introduced. Cell conditioned media(CCM) was harvested every two days for seven weeks with cell samples withdrawn. CCM EVs were isolated by size exclusion chromatography(qEV10 Izon) and characterised by Nanoparticle Tracking Analysis, Transmission Electron Microscopy and Western Blot(WB). MSCs were successfully cultured in the bioreactor and sampling at a variety of timepoints revealed viable cells with normal morphology and proliferation when transferred to culture flasks. Glucose readings revealed limited MSC proliferation in 3D culture. Isolated EVs were in the size range of 139‐197nm with a characteristic lipid bilayer. EV concentration per harvest ranged from 5.47x109 to 1.39x1010 particles/ml. Expression of EV‐associated CD63, CD82 and TSG‐101, and absence of Calnexin was confirmed by WB at a range of timepoints. MSCs were successfully cultured in a 3D serum free bioreactor system for 7 weeks, providing considerably higher yields of EVs relative to conventional culture. This approach may circumvent some challenges and support scale up for therapeutic application.
Influence of downstream processing parameters on post‐translational modifications of the AAV capsid
1: Group of Cell Culture Technology, Faculty of Technology, Bielefeld University, Bielefeld, 33615 Germany 2: Biofidus AG, Bielefeld, 33615 Germany
Various purification strategies for adeno‐associated virus (AAV) gene therapy vectors co‐exist with vastly different physicochemical parameters to which AAV capsids are subjected during purification, like pH and processing time. While it is well known from other biologicals, like monoclonal antibodies, that these parameters influence the abundance of spontaneously occurring post‐translational modifications (PTMs), like deamidation of asparagine, the subject has not been extensively discussed in the published literature on AAV. The matter is pressing because AAV dosage in clinical settings must be reduced as much as possible to avoid inflammatory responses to the vector in some cases, and PTMs can adversely affect protein stability and function, thereby reducing efficacy. In addition, currently published protocols for PTM analysis of AAV by LC‐MS/MS rely on protease digestion for prolonged time periods and may overestimate deamidation and oxidation events. Up to 100% deamidation of multiple sites has been reported elsewhere. Here, using an optimized quick‐digest protocol, and in contrast to previous reports, we show that deamidation is limited to one site of the AAV2 capsid after affinity purification with elution at pH 2.2, and comprises at most 6% of peptides. Deamidation and oxidation increased slightly with an extension of the elution time, showing that process parameters are critical to avoiding PTMs. Further purification strategies, like ultracentrifugation and ion‐exchange chromatography, will be discussed. In conclusion, avoidance of spontaneous PTM formation during AAV downstream processing is presented as one puzzle piece towards reducing dosage and increasing the safety and efficacy of AAV therapeutics.
Gain and Loss‐of‐function screens identify targets with >10‐fold improvement in AAV production
C A Reid1 F Grafton1 L Leveque‐Eichhorn1 H Griffiths1
1: Ascend GCTx
Recombinant AAV is one of the most widely used vectors for in vivo gene delivery. There are currently over 900 ongoing pre‐clinical and clinical programs using AAV which all require significant manufacturing capacity. Inefficiencies in current manufacturing techniques has ultimately resulted in extremely high cost of gene therapy drug products. This severely limits the widespread use of AAV‐mediated gene therapies for common and rare indications. There are two dominant AAV production platforms: transient transfection of mammalian cells or baculovirus infection of Sf9 insect cells. Both academic and industry‐led studies have shown Sf9 cells produce higher titers of rAAV, but it comes at a cost of vector quality and potency. Few studies have reported on systematically engineering cell systems to enhance their viral production capacity and potency. To address this issue, we performed a series of targeted and unbiased genetic and small molecule screens in suspension HEK293 cells and identified targets that showed greater than a 10‐fold increase in rAAV production. Examples of targets include transmembrane and DNA repair proteins, cell‐cycle, and epigenetic modulators. Current efforts are underway to validate these hits in a scale‐up model and identify targets that will have a synergistic effect on rAAV production and potency.
Bioprocess Modelling of Upstream Viral Vector Production Enhancement
A Vervoort1
1: Virica Biotech
Viral‐based cell and gene therapies are a highly promising advancement for the treatment of complex and rare diseases. However, high manufacturing costs and limited manufacturing capacity of viral vectors is insufficient to support the global demand for clinical grade products. This can lead to delayed product development of ground‐breaking treatments and subsequently, therapies that are unaffordable and inaccessible to the public. To mitigate these challenges, many manufacturers use process additives to optimize product yield, the cost of which needs to be assessed. Using bioprocess modelling, the impact of process additives on manufacturing costs and yield can be evaluated.
Virica's Viral Sensitizers (VSEsTM) are upstream process additives that offer a solution to high manufacturing costs and low viral vector yields. VSEs TM are proprietary small molecules that enhance the growth of viruses by transiently attenuating cellular antiviral defenses. VSEsTM have been shown to improve viral vector manufacturing yield in a wide variety of contexts, including both lentivirus and AAV production where fold enhancements in yield of up to 7x were seen.
Using BioSolve Process 8.3 Software (Biopharm Services Ltd.), Virica assessed yearly upstream cost of goods sold (COGS) savings associated with VSETM‐mediated yield enhancement. Manufacturing process modifications enabled by a 2‐fold increase in yield including reductions in the number and size of bioreactors required to produce a target yield were assessed. Findings of the modelling demonstrated that annual COGS savings of 33% could be achieved, in addition to a reduction in water and plastics usage of 6% and 24%, respectively.
Overcoming Barriers in Viral Vector Manufacturing: Small Molecule Targeting of Antiviral Defences
1: Virica Biotech
Manufacturing of viral‐based cell and gene therapies is complex, often resulting in insufficient yields and delayed product development. While the last decade has seen major advances in viral platform and bioreactor design, as well as cell line and media formulation optimization, the industry still faces challenges with production consistency and yield. An often overlooked and underappreciated aspect of cell culture‐based manufacturing of viral vectors are the cellular innate antiviral immune pathways which remain partially, if not fully, intact in producer cells. These pathways are often triggered via the detection of foreign nucleic acid, and as such, blunt viral replication/assembly in both infection and transfection‐based manufacturing strategies. Due to the breadth of cellular innate antiviral defenses, even manufacturing cell lines genetically designed to attenuate the cellular antiviral defenses cannot overcome all major antiviral signaling hubs.
Virica's Viral Sensitizers (VSEsTM) encompass a proprietary collection of small molecules that enhance the production of viruses by transiently overcoming cellular antiviral defenses. Leveraging high‐throughput methods, Virica has assembled a library of over 130 small molecules which enhance viral production by transiently antagonizing a broad range of cellular innate antiviral pathways.
Using Virica's unique high‐throughput methodology, a custom VSETM formulation was developed to produce 3rd generation Lentiviruses in adherent HEK293T cells. Treatment of HEK293T cells with an optimized VSETM formulation resulted in over 7‐fold enhancement compared to untreated cells. VSETM enhanced viral vector production was also demonstrated using suspension HEK293 cells to produce AAV. Multiple VSEsTM tested resulted in significant improvements in AAV functional titre.
Production of in vitro transcribed mRNA using synthetic, enzymatically produced linear DNA
Z Whiffen1 A Dhir1 C Lal1 A Picher1
1: 4basebio
The manufacture of high‐quality, GMP grade DNA is a major bottleneck in the production of mRNA for use in gene therapy and vaccines. 4basebio has developed a proprietary, scalable synthesis process for the production of linear closed DNA constructs via our Trueprime™ amplification technology. The opDNA™ produced is devoid of any bacterial backbone and the manufacturing process circumvents cumbersome fermentation processes required for plasmid DNA. The process is size and sequence independent, enabling the incorporation of polyA tails >120 bp, and allows for large scale production of linear DNA with high yield and purity in less than a week.
Here, we compared the synthesis of in vitro transcribed (IVT) mRNA using opDNA™ versus linearised plasmid DNA, and achieved significantly higher mRNA yields irrespective of construct size, ranging from 1.8kb reporter genes to 9.5kb samRNA constructs. Proinflammatory cytokine/chemokine levels in primary human PBMCs were comparable to mRNA produced from linearised plasmid, as was 3‘ heterogeniety of the transcipts and levels of dsRNA impurities. Finally, reporter gene expression across a range of cell lines (HEK293, C2C12, primary human chondrocytes and primary human hepatocytes) was equivalent to mRNA produced from linearised plasmid.
We have demonstrated that opDNA™ templates can be used for the production of IVT mRNA, which could greatly accelerate the therapeutic development of gene therapy and vaccine programmes. Moreover, the technology could overcome the difficulties associated with complex polyA tails for mRNA constructs, which are inherently difficult to synthesise via bacterial propagation systems.
Optimized lentiviral transduction process for ex vivo CD34+ hematopoietic stem cell gene therapy drug product manufacture
M del Mar Masdeu1 S Elavazhagan1 G Crawford1 L Du1 F Enjalbert1 A Georgiou1 S Janakan1 V Pennucci1 S Wantuch1 C Whiting1 C Recchi1 D A Carbonaro‐Sarracino1 A J Thrasher2 J Wolfe1 H B Gaspar1 F Mavilio1
1: Orchard Therapeutics Europe Ltd 2: UCL Institute of Child Health
Reducing vector requirements and cost of goods presents a key challenge in commercializing ex vivo CD34+ hematopoietic stem cells (HSC) gene therapies. Several enhancer compounds are already routinely applied in clinical gene and cell therapy manufacture to improve viral transduction. To develop an optimized protocol for lentiviral (LV) transduction of HSCs, we screened over 20 commercial and novel candidate compounds for enhancement activity. Our comprehensive survey of improved transduction efficiency (TE) and vector copy number (VCN) achievable by these enhancers was conducted with both scale‐down high‐throughput and clinical‐scale transduction processes for mock HSC gene therapy drug product manufacture.
Here we describe J‐Boost, a novel class of transduction enhancers (diblock copolymers, PCT/US20/56123) which facilitates viral entry. When used in concert with Protamine Sulphate (PS) and high‐density cultures, J‐Boost results in up to ∼9 fold increases in VCN and ∼4 fold increases in TE, enabling 50‐70% reduction in LV vector for HSC transduction to achieve desired target drug product profiles. Our results demonstrate that J‐Boost/ PS enhancers are largely inert, inducing minimal alteration of HSC gene expression, HSC phenotype and multilineage progenitor function, while retaining comparable qualitative and quantitative HSC engraftment potential in NOD‐scid IL2Rgnull mice. Importantly, we show compatibility of this optimized transduction protocol with therapeutic LV vectors and manufacture methods currently in use for drug products under development. In vitro and in vivo demonstration of the safety, efficacy and comparability of this optimized transduction protocol validates its potential for clinical application and the reduction in vector usage and manufacturing costs.
Employing a bioreactor system for efficient production of engineered extracellular vesicles (EVs) to support tracking transfer in the breast cancer setting
1: Discipline of Surgery, Lambe Institute for Translational Research, National University of Ireland Galway, H91 V4AY Galway, Ireland 2: CÚRAM, the SFI Research Centre for Medical Devices, National University of Ireland Galway, H91 W2TY Galway, Ireland 3: Anatomy & Regenerative Medicine Institute, School of Medicine, College of Medicine Nursing and Health Sciences, National University of Ireland Galway, H91 TK33 Galway, Ireland;
Extracellular vesicles(EVs) have emerged as promising versatile vehicles for delivery of therapeutic agents, however further understanding of EV migration and transfer between cell populations is required. The aim of this study was to establish 3D serum‐free culture of engineered breast cancer cells in a hollow fiber bioreactor(HFBR) system to support isolation of high yields of fluorescent labelled EVs.MDA‐MB‐231 cells were transduced with a lentiviral vector containing GFP fused to the tetraspanin CD63(pCT‐CD63‐GFP) supporting packaging into EVs. Following puromycin selection, 1x109 CD63‐GFP MDA‐MB‐231 cells were seeded into a 20kD HFBR(FiberCell Systems Inc.). Once culture was established, chemically defined media was introduced. Multiple harvests of GFP‐EVs were collected over the course of 5 weeks, isolated using size exclusion chromatography(Izon‐qEV 10) and characterized using Nanoparticle Tracking Analysis(NTA), western blot and Transmission Electron Microscopy(TEM). GFP expression was demonstrated in transduced cells by fluorescence microscopy and western blot, with longitudinal expression confirmed in cell harvests taken from the fibercell at different timepoints. NTA and TEM revealed that small EVs in the size range of 30‐200nm with an intact lipid bilayer were successfully isolated from repeat harvests of conditioned media. Initial harvests contained subpopulations of EVs in a higher size range which disappeared within a few days of serum withdrawal, highlighting initial serum contamination. Western blot confirmed the expression of EV markers CD63, TSG101, CD81 and GFP in sequential samples.The fibercell bioreactor system supported efficient and reproducible scale up of GFP labelled EV production which will facilitate tracking EV transfer in the cancer setting.
Lentiviral Vector large scale manufacturing using suspension platform
M Neri1
1: AGC Biologics
AGC biologics is a leading global Contract Development and Manufacturing Organization (CDMO), providing world‐class development and manufacture of mammalian and microbial‐based therapeutic proteins, plasmid DNA (pDNA), viral vectors and genetically engineered cells.
Lentiviral vectors (LVV), produced with transient quadri‐transfection in 293T cell line are extensively used for genetic modification in gene therapy. In the context of advanced clinical studies or commercial phase, an important manufacturing challenge is the optimization of vector production in large‐scale platforms. To address this issue and to obtain a scalable and robust process, we are developing a system with suspension cells in a culture system without animal derived reagents in stirred tank bioreactor.
Process final scale is 200L with the possibility to further increase the bulk volume.
A proprietary suspension 293T cell line has been developed and banked in AGC. Process development has been performed in small scale (3L) STR bioreactors working on several parameters included seeding cell density, DNA concentration, timing and volumes of harvest, and also the clarification phase has been optimized. Final vectors were characterized for infectious viral titre (TU/mL), particle content (ng p24), and process related impurities (HCP and total DNA).
Downstream process was developed in order to accommodate larger volumes of vectors from bioreactor. Single use Tangential flow Chromatography and DEAE chromatography step were successfully implemented, with good removal of process related contaminants and no detrimental impact on vector infectivity and stability.
Scale up of the process to 50L bioreactor has been already performed and results will be presented.
A robust downstream strategy for high quality AAV using an all‐column purification method
1: Andelyn Biosciences, 5185 Blazer Parkway, Dublin, OH, USA
Andelyn Biosciences has recently developed a robust scalable suspension platform for production of high yields of Adeno Associated Virus (AAV) for preclinical and all phases of clinical production. This platform is based on a quality by design approach that ensures scalability and quality at different stages of development. The standard purification strategy involves use of ultracentrifugation for enrichment of full capsids. An alternative all‐column chromatography technique will be presented which replaces ultracentrifugation for downstream purification of AAV. The process flow includes concentration of the clarified harvest followed by two ion exchange (IEX) chromatography steps – one for capture and another for enrichment of full capsids. Data will be presented showing reproducibility and robustness of the strategy, with consistency in recovery and purity at each stage. Compared to the standard ultracentrifugation approach for enriching full capsids, the all‐column method results in 75% reduction in processing time. The IEX procedure results in 50% recovery which consists of 88‐98% full capsids with final purity of 99‐100% as evaluated using HPLC, AUC and capillary electrophoresis.
Development of an entire downstream process to efficiently purify AAV
1: Sartorius Stedim Biotech GmbH, Göttingen, 37079, Germany 2: Sartorius Stedim Switzerland, Tagelswangen, 8317, Switzerland 3: BIA Separations, Ajdovscina, 5270, Slovenia 4: iBET – Instituto de Biologia Experimental e Tecnológica, Oeiras, 2780‐157, Portugal
With the potential diversification of adeno‐associated virus (AAV) capsid serotypes for gene therapies applications, a downstream processing platform, that can purify a variety of serotypes with similar recovery and purity is of particular interest to reduce process development timelines and manufacturing complexity.
When developing an AAV downstream process, firstly, AAV particles must be efficiently separated from the producing cells which can be challenging due to an obligatory lysis of the cells. In our study we are showing the potential of a double‐layer cellulose‐based depth filter to efficiently clarify AAV2 from lysed cells.
Next, we have been optimizing the tangential‐flow‐filtration (TFF) step. The early selection of high performing filter as a base for the development of a tangential flow filtration can represent a particular challenge when feed material is scarce and the development timeline short. The comparison of multiple flat sheet cassettes and hollow‐fibers performances as well as the optimization of process parameters for an AAV concentration and diafiltration TFF process will be presented.
Lastly, to efficiently purify a variety of AAV serotypes and capsid configurations, an alternative to an affinity‐based capture chromatography was developed. Strong cation exchange chromatography has the possibility to become a universal capture platform where binding, wash and elution conditions can be quickly optimized for each new AAV serotype instead of relying on the development of a new affinity ligand. We will show the development journey of a capture chromatography method based on the CIMmultus SO3 monolith where multiple parameters have been optimized by DOE.
rAAV production revisited ‐ from plasmid design to nanopore sequencing and proteomics
M T Radukic1 D T Le1 S Golm1 C Rothschild‐Gronau1 R Hoffrogge1
1: Bielefeld University
Recombinant adeno‐associated virus (rAAV) therapy celebrates the 10th anniversary of the first marketing authorization and additional applications are added each year. However, the success is limited by production challenges and product heterogeneities. We set out to illuminate ‘known unknowns' of rAAV genesis with synthetic and modern analytical approaches to tackle production issues with a focus on the three‐plasmid system in HEK‐293 cells and in‐vitro assembly. We re‐evaluated classical design spots such as ITRs deletions, P5 promoter variations and a minimal set of pHelper components. In addition, we analyzed several variants of the Rep‐cassette from mutations via larger C‐terminal deletions to knock outs. The main read out was DNase resistant viral genomes and, depending on the project, cell growth, transduction, expression by western blot, empty capsid fraction and capsid composition. To pinpoint more effects, we established long‐read nanopore sequencing of rAAVs for the unbiased detection of genetic impurities and recombinations. These studies were complemented by proteome analyses of the nuclear fraction during production and the deployment of in‐vitro assembly of AAV capsids from purified proteins. Collectively, out data enabled us to semi‐quantitatively judge the effect of variations with respect to critical production parameters. The combination of beneficial traits improved production several fold compared to that of established settings.
The Need for Speed: Maximising Adeno‐Associated Virus (AAV) vector productivity with high throughput (HTP) platforms
1: Pharmaron
Pharmaron has established a multi‐faceted purification toolbox approach using small scale upstream processing (USP) and downstream processing (DSP) HTP systems, to deliver rapid process optimisation and manufacture of a wide range of Adeno‐Associated Virus (AAV) serotypes and products. With utilisation of Ambr15 and Ambr250 bioreactor systems, Pharmaron can provide an initial small‐scale assessment of a product's performance in their platform process at a fraction of the cost, space, and resource of a full lab scale study. Equally the use of an automated liquid handling robot in DSP, allows Pharmaron to conduct an early assessment of purification for a gene therapy product. They can then concentrate on a targeted HTP screening approach utilising DoE to assess, for example, varying seeding densities and transfection conditions in USP, to varying chromatography buffers and resins in DSP. This targeted approach drastically reduces chemistry, manufacturing, and controls (CMC) development timelines, maximises productivity and drives results. Despite this, the use of HTP and automated processes can cause bottlenecks in an end‐to‐end platform which delivers gold standard analytics as an end point; therefore, Pharmaron has developed a number of HTP analytical methods to match this output. Through integration of size exclusion (SEC) and anion exchange (AEX) ultra‐performance liquid chromatography (UPLC) systems for fast titre generation and Empty:Eull capsid quantification, Pharmaron has developed a testing strategy for rapid screening of hundreds of samples in a 24‐hour period. Here they show that high throughput platforms provide advanced technical solutions which drive reproducibility, productivity, and reduced process times.
Design of experiment, a powerful tool to simplify viral production process development
1: Exothera S.A. 2: SD Analytics
Viral vector production is a complex process, with many steps requiring process optimization. Process development activities can be time‐consuming as each single step and process parameters need to be optimized. A complete development of a transfection step should include as many as 16 parameters in flask and lead to, with a one‐factor‐at‐a‐time approach, a lengthy development still not meeting optimal experimental conditions. Design of experiment (DoE) is a powerful statistical tool that allows efficient planning, execution, analysis, and data interpretation to model the factors that control the value of parameter(s). We have studied the manufacturing of adeno‐associated viruses focusing on transfection steps, endonuclease treatment and chromatography using the DoE methodologies. Experiments were performed in scale‐down models in anticipation of a manufacturing transfer. The virus titre and process impurities were evaluated as main process output for the DoE. Experiments were designed with the Custom Design platform of SAS JMP 15 allowing to estimate the main effects or 2‐way interactions, or alternatively, the continuous effects: linear or non‐linear effects. Based on statistical analyses, best conditions were performed at larger scale (bioreactor) to mimic industrial production. We analysed the protocol sensitivity for different steps to the reagent used and the operating conditions. Our results have shown a 2.5 logs variation of viral titre during transfection or between 1 to 3 logs difference in host cell DNA reduction during endonuclease treatment, the selection of the enzyme as critical parameter. DoE has shown its effectiveness giving additional assurance on process operating ranges and robustness.
Automated, closed, GMP‐compliant manufacture of TCR‐engineered T cells for therapy
I Elias Yonezawa Ogusuku1 S Lennartz1 C Brandes1 V Herbel1 K Teppert1 T Schaser1 G Koutsoumpli2 G Griffioen2 I Johnston1
1: Miltenyi Biotec 2: Leiden University Medical Center
Dissemination of T cell receptor (TCR)‐engineered T cell therapies to large patient numbers is limited by a complex and laborious manufacturing procedure. Therefore, this study established a novel protocol for the manufacture of genetically modified T cells expressing a transgenic TCR using the CliniMACS Prodigy. The protocol enables an automated, closed and Good Manufacturing Practice (GMP)‐compliant process. Contrary to the well‐known production of chimeric antigen receptor (CAR) T cells on this device starting from CD4/CD8 co‐enriched T cells, our TCR process focused on CD8 T cells since most TCRs are dependent on the CD8 co‐receptor for making a high affinity interaction with the peptide‐HLA complex. For manufacturing, we used the dNPM1 TCR, which recognizes an HLA‐A*02:01 neoantigen on acute myeloid leukemia. By adjusting culture conditions, we reduced the manufacturing time for a clinical relevant yield of engineered T cells from twelve to eight days. During the process, CD8 T cells were enriched from a frozen leukapheresis, subsequently activated, transduced, expanded and finally formulated. Our protocol resulted in a highly viable T cell product at clinical scale with a preferred early memory phenotype. Furthermore, we confirmed the cytolytic potential of dNPM1 TCR‐engineered T cells in vitro as well as in vivo. In summary, we demonstrated that our CD8 process is robust and reproducibly yielded clinical relevant numbers of potent dNPM1 TCR‐engineered T cells to treat patients with acute myeloid leukemia.
Process C: A novel, perfusion‐based lentiviral vector manufacturing process demonstrating increased productivity and enhanced purity
1: Oxford Biomedica
For over 25 years, Oxford Biomedica (OXB) has been a pioneer in the development of products and innovative technologies based on lentiviral vectors. To meet the forecast on vector demand for gene and cell therapies, OXB has recently introduced a next‐generation transient lentiviral manufacturing process incorporating innovative process modifications which simultaneously enhance lentiviral process yields and improve vector quality attributes. Designated “Process C”, this new process takes advantage of advances in perfusion technology to support vector production at higher cell densities than have been associated with traditional batch and fed batch production approaches for lentiviral vectors, allowing for significant process intensification without the need for increased bioreactor volume. Harvesting of lentiviral particles secreted into the bioreactor milieu via continuous perfusion also allows stabilisation of generated vector at lower temperatures, leading to improved preservation of functional lentivirus in the final product.
Importantly, Process C adopts a plug and play approach facilitating incorporation of small molecule enhancers, some of which have already been identified by OXB. For example, incorporation of U1 (RNA based enhancer) during vector production using Process C led to increased cell‐specific productivity. Overall, process yield improvements ranging from 2 to 10 fold have been demonstrated for a range of therapeutic lentiviral vectors with process scalability demonstrated in stirred tank bioreactors up to 200 L in GMP.
The new manufacturing platform offers significant benefits for clinical production of lentiviral vectors and will further enable OXB to support the continuing global demand for high quality gene and cell therapy products.
A rhabdovirus‐negative Sf9 cell line (Sf‐RVN®) platform for improved safety profile
1: Merck Millipore
Spodoptera frugiperda (Sf) cell lines are widely used as hosts for baculovirus expression vector systems (BEVS) to produce vaccines and gene therapy vectors. However, the majority of Sf9 and Sf21 cell lines contain a Sf‐rhabdovirus which is considered a contaminant and must be removed during downstream processing. To improve the safety profile of the BEVS production method, a robust Sf‐rhabdovirus‐negative platform, composed of a Sf9 rhabdovirus‐free cell line (Sf‐RVN® Insect Cell Line developed by GlycoBac LLC) with an optimized chemically defined medium, was created. Most insect media formulations contain hydrolysates which often have lot‐to‐lot variability of trace metals and amino acid content. This variability can cause fluctuations in cell growth and productivity. To increase the performance and reliability of the platform, a chemically defined insect medium was developed.
The new chemically defined formulation supports the growth of Sf9 cell lines equivalently to hydrolysate‐containing media, with Sf21, S2, Tni and C6/36 cell lines also performing well. For the Sf‐RVN® Insect Cell Line particularly, we found that the companion chemically defined medium enabled improved cell growth and higher productivity compared to other commercially available media. The Sf‐RVN® Platform provides increased productivity which is free of Sf‐rhabdovirus and thus improves the safety profile of bioprocesses.
Automated systems leading solution to increase LVV titration capacity
F Rossetti1
1: AGC Biologics 2: Hamilton Company
AGC biologics is a leading CDMO, providing world‐class development and manufacture of therapeutic proteins, plasmid DNA, viral vectors and genetically engineered cells.
Lentiviral vectors (LVV) are efficient vehicles for gene delivery that play an important role for ATMPs. The efficiency of the production process and the performance of the final LVV is monitored through potency methods. In AGC Biologics, the determination of the infectious viral titre is based on the detection of proviral DNA copy number integrated into the cell genome. The selected reference cell line, deeply characterized and full representative of target primary cells, is transduced with serial dilutions of vectors and tested with a qPCR assay. The number of samples to be analysed represents a significant bottleneck for process characterization. To increase the throughput, automated system based on Hamilton liquid handlings full integrated with Sealer and Real Time PCR has been designed and optimized to perform different steps of the method as LVV dilutions, pellet preparation and qPCRs.
Hamilton automated solutions brought to 5‐fold increase in number of samples daily tested, greatly reducing the timing of analysis. Results generated with automatic stations on different purification steps or final purified LVV are comparable to the manual ones (p = 0.61), with a repeatability ≤15% and a reproducibility with manual execution system ≤19%.
The automated stations, compliance to CFR21 and qualified according to ICH Q2 (R1), allow to have a complete automatic and traceable flow, from sample vial to result, that makes the difference in laboratory capacity.
Development of a stable, inducible (N‐1) cell line for the efficient and high titer production of lentiviral vectors
1: Merck Millipore
Lentiviral vectors are one of several viral based gene delivery systems used for gene therapy and gene‐modified cell therapy applications. Currently, lentiviral vectors have been approved for several ex‐vivo therapy applications (CAR‐T therapy), with many clinical trials in progress to expand use for in vivo gene replacement therapy as well.
The current processes used for the manufacturing of lentiviral vectors are transient transfection and expression processes where multiple plasmids are used to transfect HEK293T cells that are grown in adherent or suspension growth formats.
We have developed an “N‐1” cell line where all the necessary lentiviral packaging genes have been stably integrated within the genome of a suspension adapted HEK293T cell line grown in a chemically defined and animal component free medium. The viral packaging genes Gag/Pol and VSV‐G have been placed under TET‐ON inducible promoters to reduce viral protein toxicity during the growth of the cells allowing viral production to be initiated by the addition of the inducer agent doxycycline. Only the lentiviral transfer plasmid containing the therapeutic gene of interest has to be transfected into the cells to initiate functional viral production, which significantly reduces the cost of the plasmids and transfection reagents required. The inducible, stable lentiviral producer cell line we have developed has achieved functional viral titers exceeding 1x10e7 transducing units/mL from selected pools when using GFP as the gene of interest. Single cell cloning and screening for clones with higher titers from these pools are in progress.
An optimised protocol to generate high titre lentiviral vectors by extended HEK293T culture following transient transfection and suspension culture
1: Brunel University London 2: Imperial College London 3: TestAVec 4: University of Manchester
HIV‐1 based lentiviral viruses are considered powerful and versatile gene therapy vectors to deliver therapeutic genes to patients with hereditary or acquired diseases. These vectors can efficiently transduce a variety of cell types when dividing or non‐dividing to provide permanent delivery and long‐term gene expression. Demand for scalable manufacturing protocols able to generate enough high titre vector for widespread use of this technology is increasing. Current methods for LV production either use transient transfection of producer cell lines or production from isolated clonal producer cells engineered to generate vector permanently. Cells can be grown at scale either in 2D relying on culturing producer cells in multi‐tray flasks or in roller bottles or cells can be adapted to grow in 3D suspensions in large batch fermenters. Transient transfection is, however, ideal to rapidly generate vector to test for efficacy. In this study, we compared cell survival and LV titres using three different transfection reagents: polyethyleneimine, Fugene® 6 and Genejuice®. LV was produced routinely at titres of 109 TU/ml by traditional monolayer conditions for up to 10 days instead of 72 hours using Genejuice® with little drop in titre due to low cellular toxicity. Genejuice® also enabled rapid conversion of HEK293T from 2D to 3D suspension cultures generating titres of 108 TU/ml in glass bottles. We propose, these simple changes in vector production enables the generation of larger volumes of high titre vector in a cost‐effective manner.
Optimized media, transfection, and bioreactor parameters for HEK293 production of AAV‐2
1: Merck Millipore
Adeno‐associated virus (AAV) has become a prominent vector for gene delivery to treat life threatening genetic diseases. One of the major limitations for product development and treatment availability is the need for high levels infectious AAV particles, therefore, media and process optimization is vital to achieve high AAV titers.
Specific raw materials found in many cell culture media formulations, such as iron citrate, are known to interfere with PEI transfections and must be removed/reduced to enable efficient transfection. Therefore, media development work for HEK293 production of AAV via PEI transfections should target maximizing viable cell density (VCD) and AAV titer, while also enabling PEI transfection. To maximize AAV titers across multiple HEK293 cell lines, media development for AAV production was performed with three HEK293 cell lines with different growth rates. Following media development, transfections were optimized with Design of Experiment (DoE) studies using multiple commercially available transfections reagents. Finally, bioreactor parameters for growth, PEI transfection, and AAV production were evaluated separately in an Ambr® 15. With this approach, the highest VCD and AAV titers can be achieved without the bottleneck of a non‐optimized step in the AAV production process. Optimized transfection and Ambr® 15 parameters were then evaluated with HEK293 cultures expanded in a perfusion process vs conventional passaging scale‐up.
Generation and Characterization of Stable Recombinant Baculoviruses for large‐scale production of rAAV
1: VectorY
Industrial‐scale production of clinical‐grade, high‐quality, recombinant adeno‐associated virus (rAAV) remains a challenge in the field of Gene Therapy. Production of rAAV using the baculovirus/insect cell system is compatible with large‐scale production (up to 20,000 L), but mutant viruses such as defective interfering particles or deletion mutants can emerge during each baculovirus propagation step of the scale‐up process. These mutants can rapidly outgrow the transgene‐containing baculoviruses, resulting in a baculovirus seed which is incapable of producing high yields of fully packaged AAV. Hence, the scalability of this system for AAV manufacturing is highly dependent on the genetic stability of the recombinant baculoviruses. The baculovirus genome used for homologous recombination to insert transgenes can be genetically engineered to improve the genetic stability. Here we present a head‐to‐head comparison of the genetic stability of different baculovirus genomes (so‐called bacmids) during serial passage. Different AAV transgenes (Cap, Rep and Gene of Interest) were inserted into various bacmids by homologous recombination and the resulting plaque‐purified baculoviruses were passaged up to Passage 6. The baculovirus harvests were characterized by infectious titer determination (TCID50), end‐point PCR to determine transgene presence and size, and quantitative PCR to determine viral genome titer, as well as their ability to produce rAAV. We show that the choice of bacmid markedly influences baculovirus stability and rAAV production yields. This work will enable the setup of an efficient and scalable rAAV production process with stable recombinant baculoviruses.
Evaluation of Lentiviral Vector Aggregation During the Downstream Manufacturing Process
1: Merck Millipore
Understanding the impact of process parameters on vector quality (e.g., aggregation) is an important consideration during downstream process development. During the manufacturing process, lentiviral vectors (LVV) are exposed to conditions that drive product aggregation and lead to decreased vector viability and reduction in process yield. Conditioning of vector feed by salt addition during chromatography, buffer exchange, vector concentration during tangential flow filtration (TFF), and creation of areas of highly concentrated vector during AEX elution and sterile filtration can all result in the formation of aggregates. Dynamic light scattering (DLS) was used to determine levels of product aggregation across a typical LVV downstream manufacturing process consisting of AEX chromatography, diafiltration using TFF and sterile filtration. Process conditions that led to changes in vector aggregation state were identified and the effects of buffer composition were evaluated. This study demonstrates the value of technologies like DLS for charting vector aggregation and highlights the need to understand and address the specific impact of the manufacturing process on vector quality and yield.
π‐AlphaTM High‐yield AAV Production Platform with improved scalability and biosafety
B Liu1 L Sheng1 H Hu1
1: Guangzhou PackGene Biotech. Inc., Guangzhou, 510000, China.
Adeno‐associated virus (AAV) has been extensively utilised as a gene therapy vector. While in large‐scale commercial manufacturing procedures, the yield, scalability, and biosafety of AAV remain obstacles. To address these problems, we optimised the plasmid design, production conditions and the scale‐up strategies of the AAV production in triple plasmid transfection of HEK293 cells. By adding the non‐coding regulatory elements to rep/cap plasmid, the genome titre of packaged AAV increased by 3‐8 times among different serotypes. In the bioprocessing, design of experiments (DoEs) on central process parameters significantly increased the yield of AAV by about 10 times, achieving 7.3E16vg, with the total recovery rate of 30%. With these optimisations, the genomic yield of AAV produced in suspension HEK293 system can be stably scaled up to 200L suspension production. At the same time, the quality of AAV was also largely improved. The residue host cell DNA was as low as 30‐90 ng/1E13vg in different serotypes, while the residue of encapsulated plasmid DNA can be reduced by 90%. As to the empty capsid ratio of AAV, the optimised downstream process using scalable ultracentrifuge rotors largely decreased the empty capsid ratio to < 5%, while the scalable anion exchange chromatography process increased the full capsid ratio up to 75‐94% in AAV5, AAV8 and AAV9. In conclusion, we managed to optimise the π‐AlphaTM High‐yield AAV Production Platform with enhanced yield and biosafety and improved application at various scales of production.
Small molecule chemical additives for enhanced recombinant AAV production in HEK293 cells
1: University of Sheffield 2: REGENXBIO Inc.
Recombinant adeno‐associated virus (rAAV) has established itself as a highly efficacious gene delivery vector with a well characterised safety profile and an extensive toolbox of both naturally occurring and engineered serotypes, allowing broad clinical application. Recent successes in rAAV‐mediated gene therapy clinical trials and subsequent regulatory approval of rAAV products will continue to drive demand for high titre, high volume vector preparations but have also highlighted the need for improved rAAV production processes to reduce costs. Nocodazole (an anti‐mitotic agent) and M344 (a selective histone deacetylase inhibitor) were identified as positive regulators of rAAV8 genomic titre in a multi‐well plate screening assay. Addition of nocodazole to triple‐transfected HEK293 suspension cells producing rAAV arrested cells in G2/M phase, increased average cell volume, and reduced viable cell density relative to untreated rAAV producing cells at harvest (72 hours post‐transfection (HPT)). Final crude genomic vector titre from nocodazole treated cultures was >2‐fold higher compared to non‐treated cultures, as determined by digital droplet PCR (ddPCR). Further investigation showed nocodazole addition to cultures to be time critical, with optimal addition at 4 HPT. Genomic titre improvement was found to be scalable (from multi‐well plate to E125 shake flasks) and serotype independent across two distinct rAAV serotypes, rAAV8 and rAAV9. Furthermore, a combination of M344 and nocodazole produced a positive additive effect on rAAV8 genomic titre, resulting in a 3‐fold increase in genomic titre compared to untreated cells.
In silico tools to accelerate process development of cell and gene therapies
1: Takeda
Cell and gene therapy are enabling us to move towards a new golden age of medicine. The excellent clinical results coupled with the first medicines approved by the regulatory authorities have sparked fever of these new modalities and many leading pharmaceutical companies are investing efforts to deliver the best‐in‐class cell and gene therapy products. However, developing a platform to produce virus‐like particles (VLP) or engineering appropriate cells requires mastery of different disciplines, not only molecular and cell biology, biochemistry or bioengineering but also computational biology, bioinformatics or even mechanical engineering.
In accordance with the new Industry 5.0 European recommendation, Takeda has implemented smart in silico predictive tools to accelerate process development at different platform development stages while increasing efficiency and reducing development‐derived costs. In this presentation and by means of real case studies, different in silico tools and approaches to further develop VLP and cell therapy platforms will be discussed.
Efficient large‐scale production of AAV using a high cell density and low volume transfection process
1: Cell Therapy Catapult
The generation of reliable and cost‐effective AAV for clinical application within the ATMP industry is of strategic importance. Current commercial‐scale production relies upon costly chemical transfection reagents within sub‐optimal transfection workflows, highlighting the need for more efficient alternatives. We propose a strategy for incorporating an integrated fluidised‐bed centrifugation technology for cell concentration and washing (kSep® Sartorius), coupled with a high‐throughput flow electroporation system to transfect large numbers of cells in a closed automated process (VLX® MaxCyte), to deliver affordable high‐quality product in a repeatable and robust manner. The MaxCyte technology is enabled by concentration of cells to high‐density (1x108 cells/mL). Following an iterative process, we characterised the operating capacity of the kSep® 400, and consistently achieve high‐density cell concentration, whilst maintaining high cell viability (>95%). We also investigated previously identified critical process parameters (CPPs) to optimise the electroporation energy, DNA quantity and ratio, and media for vector production at small scale, using the OC‐400 Processing Assembly (PA) with the MaxCyte STX® transfection system (4x107 cells electroporated). Once identified, optimal CPPs were scaled up to test the performance of the CL‐1.1 PA on both STX® and VLX® platforms (3.5 x108 cells electroporated) integrated with the kSep® 400 concentration step; then further scaled to incorporate the CL‐2 PA on the STX® instrument (2x109 cells electroporated). Final demonstration indicative of manufacturing scale (10 L BIOSTAT® RM) was demonstrated utilising the VLXD PA on the VLX® platform (1x1010 cells electroporated), highlighting the promise this workflow holds for future continuous clinical scale production platforms.
Advanced characterization of enzymatically amplified doggybone DNA* for rAAV manufacturing
C Sallares1 A Asenjo1
1: TAAV Biomanufacturing Solutions, S.L.U.
*dbDNA technology has been licensed from Touchlight Genetics Ltd. and dbDNA™ and doggybone™ are trademarks of Touchlight Genetics Ltd.
Doggybone™ DNA (dbDNA™) is a novel enzymatic DNA technology aimed to efficiently produce rAAV. dbDNA™ is amplified by an in vitro dual enzyme process: (i) Phi29 polymerase for rolling cycle amplification of the precursor plasmid template, and (ii) the protelomerase TelN for covalent closure of both ends. The process also includes exonucleases and chromatographic and tangential flow filtration steps to clear enzymes and DNA residuals. Analytical assays to release of dbDNA™ final product includes purity of the product determined by agarose gel electrophoresis, process related impurities measured by ELISA for TelN, and construct identity by Sanger sequencing, among others.
In this work, we provided dbDNA™ final product characterization with higher resolution techniques. Ultra‐High Pressure Liquid Chromatography was utilized to determine purity of final product and showed high purity. We developed two analytical methods for the detection of residual activity of process enzymes and demonstrated that Phi29 and the exonucleases are removed in the downstream process. High‐throughput sequencing methods were used to confirm construct identity and to interrogate Phi29 error rate during amplification. Furthermore, we barely detected residual plasmid backbone in the final product.
In summary, we showed dbDNA™ final product characterization, demonstrating that dbDNA™ may be a good raw material for rAAV production due to elimination of residual bacterial sequences and efficient characterization techniques, thus providing a high purity product.
Derivation of a clonal HEK293 suspension cell line for high yield AAV production
1: Andelyn Biosciences, 5185 Blazer Parkway, Dublin, OH, USA
Cells are critical components of biotherapeutic productions and implementation of clonal cell lines is highly recommended in manufacturing processes. Cell line clonality mitigates the genetic heterogeneity inherent to most biological cell lines. Andelyn Biosciences utilizes a robust suspension platform for AAV production using a clonal HEK293 suspension cell line developed from an adherent HEK293 parental line. We subjected an existing adherent line of HEK293 to a selection series with decreasing serum concentrations before switching it to growth in serum‐free media. These processes resulted in a family of suspension‐adapted, serum‐free HEK293 cell lines that were the parental lines for subsequent isolation of single‐cell clones. Clonal selection was performed with the Solentim Verified In‐Situ Plate Seeding system which delivers Day 0 assurance of clonality. Clonal cell lines were initially assessed for AAV production in 125 mL shake flasks. Promising candidates were further assessed with the Ambr® 250 modular and then subjected to 2 L yield assessments. The final candidate was selected from a panel of six comparable clones. It was scaled to a 50 L production with AAV Serotype A using an Allegro STR50 bioreactor and purified to final fill. At all stages of production, AAV yields exceeded up to four‐fold those produced from the commercially available control cell line. We will discuss further optimization of this newly developed cell line, including viral titers of AAV serotypes with our production platform.
Scalable Downstream Purification of Recombinant Adeno‐associated Viral Vectors
M Schofield1 N Marchand1 A Kavara1 A Hejmowski1 S Tansey1 R Channawar1 M Olson1 A MacIntyre1 J Huato1 K Boenning1 M Collins1
1: Pall Life Sciences
Scalable manufacturing technologies are essential for ensuring modern medicines can be produced to meet the increasing needs of clinical trials, process development, and commercial manufacture. Recent advances in in vivo gene therapies have resulted in regulatory approval of rAAV vector‐based therapies for gene transfer in humans.
In this work, we evaluate the use of existing single‐use filtration and separation technologies for downstream purification of an rAAV5 viral vector. rAAV5 vector was produced by transient transfection of HEK293 cells in the Pall iCELLis® Nano bioreactor. Bioreactor harvest lysis material was clarified using direct flow filtration with both depth and sterilizing grade filters. The product was concentrated 10 fold using 100 kD OmegaTM flat‐sheet TFF before primary purification using affinity chromatography. The rAAV5 vector was then polished using Mustang® Q membrane chromatography to enrich for full capsids. A second TFF step was performed to concentrate and buffer exchange with flat sheet TFF with the same 100 kD Omega membrane. Final sterile filtration was performed using Supor® EKV 0.2 μm sterilizing grade filters.
All downstream unit operations resulted in acceptable performance. Feasibility of a complete downstream process was established with a theoretical whole process yield of ∼25%. This process results in a very low contaminant profile as host cell protein (HCP) and host cell DNA were reduced to near and below the assays' limits of quantitation during purification. Of particular interest, Mustang Q membrane polishing resulted in enriching the ratio of full capsids to empty capsids by 4.5 fold.
Implementation of 3D Biomimetic Scaffolds for Expansion of γδ T Cells In Vitro
1: UCL
Gamma delta (γδ) T cells possess both innate and adaptive immune characteristics. They recognise and eliminate foreign cells in a major histocompatibility complex independent way. These unique features make γδ T cells an appealing candidate for more robust allogenic off‐the‐shelf T cell therapy products. However, low γδ T cell yield and inadequate cancer killing capacity caused failures during some recent early phase clinical trials. Although T cells are considered as optimal under static condition, conventional 2D flat‐plate suspension system is impractical to use to satisfy the abounding clinical cell yield requirement. Few publications since late 1990s have reported remarkable benefits in αβ T cells' proliferation and cytotoxicity via co‐stimulation of cytokines and biomimetic materials. Better consideration of 3D physical culturing environment may benefit the γδ T cell scaled‐up expansion development by producing sufficient γδ T cell yield with satisfactory functionality for future allogenic therapy applications. Compared to biological activation and stimulation solely by zoledronate and IL‐2, natural material scaffolds doubled the activated γδ T cell quantity from human PBMCs after 13 days. Subset phenotypes, functional phenotypes and cancer killing capability of expanded γδ T cells were not negatively influenced by the scaffold implementation via examinations utilising flow cytometry and killing assay. The scaffolds induced a significantly greater γδ T cell expansion than suspension cultures under identical biological condition, similar trend was also observed in scaled‐up dynamic setting. Our data thus suggest biomimetic scaffolds are able to intensify the γδ T expansion capacity whilst retaining comparable functional capability.
Enhancement of exo‐AAV secretion pathways by metabolic gene engineering
1: Universidad Autónoma de Barcelona (UAB) 2: Novo Nordisk Foundation Center for Biosustainability
Recombinant adeno‐associated virus (rAAVs) have emerged as one of the vectors of choice for gene therapy applications due to its desirable characteristics. However, production at large‐quantities remains a key challenge for the field, resulting in high production costs. The standard method requires rAAV to be harvested by cell lysis. A fraction of rAAV vectors can be collected from the supernatant as they are secreted associated with exosomes (exo‐AAV). Exo‐AAVs are interesting both as a strategy for high‐efficient deliver and to improve manufacturing.
In this study, a multiplexed quantitative proteomic approach was conducted to study exo‐AAV secretion pathway. Three different conditions were compared: a HEK293 cell growth condition and two rAAV production conditions (triple transfection and sustained expression).
Based on the increase of exo‐AAV generation upon sustained production, biological processes that significantly changed in this condition were analyzed. Vesicle transport processes were upregulated, and proteins involved in exosome generation were selected to enhance exo‐AAV generation by metabolic gene engineering. Combinations of these genes were overexpressed during transient transfection process and rAAV titer at supernatant was quantified showing improvements in comparison to control condition. In order to clarify exo‐AAV secretion pathway, shRNA targeting proteins involved in the secretion pathway were added during rAAV production by triple transfection and extracellular titers were assessed.
These findings will contribute to the optimization of rAAV production method by increasing the prodction of exo‐AAV through modulation of rAAV secretion pathway.
IsoTag™ LV ‐ A faster and more effective purification solution for lentiviral applications
1: Isolere Bio
The rapidly growing cell and gene therapy field has the potential to target a wide range of previously incurable diseases and cancers, due in no small part to the use of lentiviral vectors (LVs). LVs can target a variety of dividing and non‐dividing cell types and are capable of carrying large genetic loads predominantly used for ex vivo transgene delivery. However, the fragility of LV envelopes, a critical component to their high potency, makes manufacturing difficult at each step of the production workflow. This leads to expanding upstream production volume, exponentially increasing the demand for expensive raw materials. These costs are pushed off onto clients, and individuals intending to use LVs in research are left to purchase prohibitively expensive viral vectors or create an in‐house small scale production system. We developed the IsoTag™LV to meet the purification needs of researchers and to streamline gene delivery to ex vivo cellular targets. Compared to industry leading affinity and PEG concentrating products, IsoTag™LV was able to concentrate crude LV 10x between two and 15 times quicker. The IsoTag™LV system also purified out 50% more contaminants than the PEG‐based product and required no additional materials or equipment. The process can be performed anywhere, requiring only a basic centrifuge for operation. Furthermore, IsoTag™LV improved vector stability during freeze‐thaw and improved transduction efficiencies in T cells 5x. IsoTag™LV is a single step, single reagent process capable of binding and purifying LV and stabilizing LV during all manipulations, hence maximizing the functional product from each batch twenty‐fold.
Novel AAV drug product formulation achieving: long‐term liquid state stability (2‐8°C) and high titer preparations (>E15vg/mL)
1: Oxford Biomedica Solutions
Oxford Biomedica Solutions has a proprietary plug‐and‐play Process and Manufacturing Platform to develop and manufacture AAV gene therapies. As part of our proven ability to create commercial‐ready programs, we have developed a strong emphasis on Drug Product development. This includes a comprehensive formulation development capability to enable long term storage and shipping at liquid storage temperatures (2‐8°C), robustness to multiple freeze‐thaw cycles, ability to stabilize very high titer presentations, all leading to improved ease of clinical use.
AAV products have a long‐held reputation as both difficult to produce and store, which presents a significant challenge for distribution and administration. To address this, we have developed a novel drug product development system and proven platform formulation that can successfully deliver long term liquid phase stability, which has now been established out to 12 months.
With our proprietary platform formulation, stability was achieved at concentrations greater than 1E14 vg/mL for at least 1 year at 2‐8°C or ‐80°C and 3 months at 25°C. Furthermore, our proprietary platform formulation has been demonstrated to be stable at extremely high concentrations in excess of 1E15vg/mL, also in the liquid state at 2‐8°C.
This data highlights the value that an early and thorough focus on Drug Product sciences can bring to an AAV product by providing long expiries, reduced administration volumes, 2‐8°C supply chains, and clinical and commercial ready products. These Drug Product innovations will ultimately allow for a much simpler commercial supply chain to support high volume and globalization of Gene Therapy products.
Safety assessment of gene edited CD4+ T‐cells for the treatment of Hyper‐IgM 1
1: Vita‐Salute San Raffaele University 2: San Raffaele Telethon Institute for Gene Therapy (HSR‐TIGET) 3: Pediatric Immunohematology Unit and BMT Program, IRCCS San Raffaele Hospital 4: IRCCS Humanitas Research Hospital 5: UOS Milan Unit, Istituto di Ricerca Genetica e Biomedica (IRGB)
We are developing a gene correction strategy to treat Hyper‐IgM1, a combined immunodeficiency caused by mutations of the CD40LG gene. CD4+ T‐cells are edited with Cas9 nuclease targeting the CD40LG locus and an integrase‐defective lentivirus bearing the corrective template for homology‐driven repair (HDR) to restore physiological expression of CD40LG. Here we present the safety assessments we designed anticipating clinical translation.
We quantified CD40LG targeting, HDR and on‐site deletions with droplet digital PCR copy number variation assays tiling LNC00892 and ARHGEF6, i.e. the closest annotated genes flanking CD40LG. The integrity of the Xq chromosome arm was estimated with MECP2, a telomeric gene. We detected a small percentage of LNC00892 deletions early after editing, especially in female T‐cells, that were spontaneously purged in culture. Notably, we validated the rationale and portability of this approach in the AAVS1 locus of hematopoietic stem cells.
Our previous investigations did not detect off‐target activity of the selected guideRNA using a high‐fidelity Cas9. Nevertheless, we decided to unbiasedly address genome integrity by 100‐metaphase karyotyping and high‐coverage optical mapping to detect unanticipated large‐scale genomic events. To assess the sensitivity and specificity of optical mapping we generated a cell line harboring multiple genomic rearrangements and admixed it to different proportions with the unmanipulated parental cell line. For functional assessment, we confirmed the physiological expression kinetic of CD40LG after editing, its ability to bind CD40, and inducing intracellular signaling. No cytokine‐independent growth was observed.
In summary, we present a comprehensive assessment of safety and genome integrity for gene‐edited drug products.
Optimized set up for purification of extracellular vesicles from human induced pluripotent stem cells
1: Pall Life Sciences 2: Laboratory of Regenerative Medicine, Department of Transfusion Medicine and Hematology, Fondazione IRCCS
Human induced pluripotent stem cells (hiPSC) possess tri‐lineage differentiation potential, thus represent the most promising cell source for tissue‐replacement clinical research. Furthermore, hiPSCs secrete extracellular vesicles (EVs) for long‐distance intercellular communication and biomolecule horizontal transfer. Therefore, hiPSC‐EVs may act as carriers of nucleic acids or other molecules for gene therapy or regenerative medicine. Yet, the study of EV functionality needs an improved isolation strategy, because commonly used ultracentrifugation‐based methods are time‐ and labor‐intensive. In this study, the optimization of a scalable and standardizable hiPSC‐EV purification platform was achieved, testing different filters for clarification, tangential flow filtration (TFF) and sterile filtration.
EVs were initially purified using glass fiber prefilter PreFlowTM UUA to remove cell debris and large contaminant protein. EVs were then diafiltered to remove soluble contaminant proteins and concentrated with OmegaTM Membrane 30kD under gentle operation conditions. The final EV‐solution was sterile‐filtered with 0.2 μm Supor® EKV polyethersulfone membranes.
The process was monitored at each step for yield and size distribution by Nanoparticle Tracking Analysis and for purity calculating the particle to protein ratio. Initial clarification step reached 85% yield, while TFF retained 80% of hiPSC‐EVs and final sterile filtration reached 78% yield.
Large scale production of therapeutic lentiviral vectors from producer cell lines: Process D
1: Oxford BioMedica
Market approval of Lentiviral Vector (LV) gene therapies together with the continuous emerging number of LV based products has created demand for large quantities of LV. Production of LV using producer cell lines (PCLs) is desirable due to the reduced costs achieved by the ability to scale to large volumes, improved batch consistency and increased streamlined production process compared to standard transient transfection processes. Oxford Biomedica (OXB) has successfully developed a cell line platform that has been used to generate suspension PCLs capable of producing high titre LV for various cell and gene therapies. These new PCLs have been combined with a new manufacturing process which uses innovative technologies and process modifications to enable large scale LV production from PCLs. This new process, Process D, takes advantage of advances in perfusion technology to support LV production at higher cell densities than those associated with traditional batch and fed batch production approaches. This allows for significant process intensification without the need for increased bioreactor volume. It also incorporates an improved downstream processing approach to specifically target unwanted protein:DNA complexes implicated in LV aggregation. In summary, the integration of OXB technologies has led to the identification of suspension PCLs that are capable of producing high titre LV at large‐scale. It is anticipated that this PCL LV production platform will support increased yields at reduced cost enabling more patients to be treated with these life changing cell and gene therapies.
Applying process intensification and integration throughout the value chain for manufacturing of gene therapies
1: Univercells Technologies
The gene therapy industry is facing a number of biomanufacturing challenges which represent significant obstacles to the commercialization of these life‐changing therapies, such as scalability and reproducibility hurdles, high production costs, and capacity bottlenecks. These stem from the use of inherited production technologies, such as flatware and stirred‐tank bioreactors, limited in scalability and capacity, which constrains developers to scale out rather than scale up. The principles of process intensification and continuous manufacturing, long implemented in the biologics industry, may ease the impact that these challenges have on the gene therapy industry.
Univercells Technologies has been pioneering the application of process intensification and integrated manufacturing to tackle the above issues, through the development of innovative bioprocessing solutions. In this presentation, the benefits of structured fixed‐bed bioreactor technology will be explored, with applications throughout the manufacturing value chain, from ultra‐small to large scale.
More specifically, the speaker will: Provide a summary of the advantages of structured fixed‐bed bioreactors, enabling scalable and intensified viral manufacturing with increased productivities in various applications. Demonstrate the benefits of using fixed‐bed bioreactors for intensified and automated seed train generation (a key bottleneck of large‐scale adherent‐based production), as well as their suitability for suspension‐based production. Address how integration of upstream and midstream unit operations within a single manufacturing platform can provide significant flexibility while decreasing costs at commercial manufacturing scale.
AAV Manufacturing Challenges and Platform Development for Full Capsid Enrichment
1: PTC Therapeutics, Inc.
Recent advances in adeno‐associated virus (AAV) vectors have resulted in the growth of AAV manufacturing in the gene therapy field. However, scaling up AAV production continues to be challenging, especially for full capsid enrichment at a high percentage level (≥50%), since typical triple transfection produces low percentage full (≤10%) and traditional ultracentrifugation can only be scaled out. To address the challenges, a scalable full capsid enrichment process is evaluated by using various platform technologies such as polishing chromatography, monolith separation, and continuous ultracentrifugation. Process optimization and characterization are performed to develop a robust and manufacturing friendly procedure. The scalability of the process is accessed through pilot and manufacturing scales (up to 1000 L). This work can shed light on the solutions of purification challenges for scalable full capsid enrichment as well as provide strategies on process improvement in the manufacturing through quality by design (QbD).
Designing for scalability to accelerate bioprocess development of gene therapies
1: Univercells Technologies
Bringing gene therapies to the market is an expensive and high‐risk endeavor, and developers must rapidly prove early efficacy and safety to secure further development funds. Many candidate therapies are competing in the race to market, in which scalability can be critical for succeeding and achieving return on investment. In light of this, bioprocessing choices made early in the development process have a profound impact on both commercialization speed and final process economics. Univercells Technologies uses scalability by design and engineering characterization to develop its scale‐XTM and NevoLineTM product range, ensuring that bioprocesses can be successfully translated from pre‐clinical, small‐scale studies to commercial scale in record times.
In this presentation, the speaker will: Provide a summary of the key scaling parameters for a structured fixed‐bed bioreactor and how they contrast with those for stirred‐tank bioreactors. Describe how characterization of the environment within the scale‐X fixed‐bed bioreactors (mixing, gas exchange) leads to reproducible performance during scale‐up. Illustrate scalability through case studies that demonstrate reproducibility across scales for a range of cell types and viral applications.
AAV large scale manufacturing
M Neri1 F Rossetti1
1: AGC Biologics
AGC Biologics is a leading global CDMO, providing world‐class development and manufacture of mammalian and microbial‐based therapeutic proteins, plasmid DNA, viral vectors and genetically engineered cells.
Adeno Associate Vectors (AAV) produced with transient transfection in HEK293 cells are used for gene therapy in ex‐vivo and in‐vivo treatments and an important manufacturing challenge is the availability of an efficient and scalable off‐the‐shelf AAV vector production process.
To address this goal AGC Biologics is developing two different systems for AAV production at large scale, in adhesion and suspension, flexible to be adapted to different serotypes.
The adherent system is based on adherent cell line, using Pall iCELLis® fixed‐bed disposable bioreactors. The process has been optimized and a productivity of 2.6E+09 vg/cm2 has been obtained with AAV6. DSP steps, have been studied at small/medium scale leading to a 30% total yield with an optional polishing step to increase full/empty particles ratio.
These data were confirmed in the scale up to iCellis500 333m2 .
The suspension system exploits a proprietary suspension cell line in stirred tank bioreactor.
AAV8 production at 2L‐scale has been optimized obtaining 1E+11 vg/ml with 63% ratio full/total and a 50% DSP total yield. These data were confirmed in the scale up in 50L STR bioreactor and the platform is ready to be scaled up to 2000L.
AAVs have been characterized with an orthogonal strategy including Viral Genome/Particles quantification, infectious viral titer, core protein characterization and process related impurities demonstrating high potency and quality suitable for ex‐vivo and in‐vivo applications.
Removal of contaminating DNA in downstream processing of lentiviral vectors
1: University College London 2: National Institute for Biological Standards and Controls
Recent advances in cell and gene therapy have sharply increased demand of high‐quality gene transfer technologies, including lentiviral vectors (LV). A common form of LV production is achieved through transient transfection of plasmid DNA into a producer cell line. The LV are subsequently produced by budding from transfected cells into the supernatant along with impurities such as free nucleic acids. These impurities pose a challenge to downstream purification and are required to be removed to achieve clinical grade LV.
The removal of free nucleic acid is achieved by enzymatic digestion prior to downstream processing (DSP). We have tested the requirement and efficiency of nuclease enzymes against undigested LV harvest. These underwent clarification using 0.45 μm dead‐end filtration followed by tangential flow filtration (TFF). We tracked the removal of double stranded DNA (dsDNA) by picoGreen assay and determined the functional titre of the LV. Nanoparticle size distribution was measured by Nanoparticle Tracking Analysis (NTA).
We demonstrated the importance of dsDNA removal for dead‐end filtration and its impact on recovery. Without enzymatic digestion, dead‐end filtration achieved 65% of dsDNA removal as compared to treated samples which had 89% of dsDNA contents degraded by enzymatic digestion and 3% removed by the filtration step. Furthermore, clarification via dead‐end filtration of LV harvests without nuclease treatment resulted in a significant loss of functional titre (p = 0.0179) and an insignificant loss in enzyme‐treated samples (p = 0.0647). This was further reflected on nanoparticle size distribution as digested samples showed a less polydispersed profile post‐clarification.
Improvement of adeno‐associated viral vector Production using “ HEK+ ” cells, deleted of SV40 large T antigen encoding sequences
1: Yposkesi 2: Genethon 3: Genethon, INTEGRARE, UMR_S951
The majority of in vivo gene therapy clinical trials require the production of recombinant AAV vectors with high purity and potency. We have previously isolated a high producing clone derived from the human embryonic kidney (HEK) 293T cell line which grows in suspension in serum‐free media, but the presence of the SV40 large T antigen may pose safety concerns. We have recently generated a new clonal cell line (called HEK+) derived from the HEK293Tclone by removing the SV40 T antigen‐encoding sequences via CRISPR‐Cas9 genome editing. In this study, we have evaluated the performance of this cell line. Firstly, regardless of AAV serotypes (2, 6, 8 or 9), we have observed a two‐fold increase in AAV productivity in comparison to the parental HEK293T clone in Shake Flasks (100mL) and also in 10L bioreactors, with titers higher than 1E11 VG/mL in the crude lysate. Secondly, we have evaluated the stability of this cell line for rAAV vector productivity up to 39 cell passages by measuring E1A and E1B mRNA expression. Finally, we have produced rAAV2/8 vector with a transgene of interest at the 50L bioreactor scale and confirm the ability to obtain high titers of purified rAAV vectors without SV40 T antigen expression.
Adeno‐Associated Virus (AAV) Production: A collaborative approach to accelerate process optimisation
1: Pharmaron Biologics UK Ltd
Pharmaron believes that forming effective partnerships are critical to advance the chemistry, manufacturing and controls (CMC) development of viral vectors to bring these life changing medicines to patients. By combining expertise and knowledge with collaborative working with industrial and academic partners, Pharmaron has developed a scalable and robust multi‐serotype AAV manufacturing platform. Effective partnering with upstream experts has enhanced their transfection system for large scale manufacture of AAV viral vectors in suspension cell culture and driven a significant increase in viral titre and infectious particles. Further, through collaborations on purification science, Pharmaron has developed a toolbox approach to purify different AAV serotypes and products. Through this development they have carefully balanced the purity of AAV vectors, the separation of genome containing (full) from genome‐free (empty) AAV capsids and improved process recovery using Pharmaron's state‐of‐the‐art AAV platform analytics. In this presentation they will show how working collaboratively with strategic industrial partners is key to continuously improving Pharmaron's processes and advancing gene therapy innovation into commercial solutions.
A New Application Utilizing Atomic Force Microscopy in Analysis of Adeno‐associated Virus Full and Empty Capsids
1: Korea Advanced Institute of Science and Technology (KAIST) 2: Yonsei university
One challenge in standard adeno‐associated virus (AAV) vector production is an unavoidable mixture of full and empty capsids, where the ratio of empty capsids ranges from 20% to 80%. Herein, we suggest a new application of atomic force microscopy (AFM) in distinction between full and empty AAV capsids. A working hypothesis in this study is that a relatively large portion of empty AAV capsids can be found in upper band of 40% iodixanol gradient in ultracentrifugation. In contrast, a relatively large portion of full AAV capsids can be exist in the lower band. Interestingly, height information of AAV particles measured by AFM reveals that upper portion is mostly consist of 17 nm sized particles compared to those in lower portion mainly showing 21 nm sized particles. The result was confirmed by quantitative PCR, in which the titration showed 4.6 × 107 vg/μl and 6.9 × 108 vg/μl for upper and lower band respectively. Finally, Transmission Electron microscopy (TEM) showed that the upper portion of AAV capsids is indeed empty capsid dominant. In contrast, the lower portion of AAV capsids is full capsid dominant. Our study shows that the utility of AFM can be expanded for rapid analysis/monitoring of the ratio between AAV full and empty capsid rather than just nano‐indentation to study mechanical properties of AAV particles.
Multivariate analysis for increased understanding of MeiraGTx upstream process
1: MeiraGTx UK II
The production of adeno‐associated viral vector is a complex multi‐stage cell culture process that generates a combination of multivariate time‐course data and end point measurements. Data obtained in cell culture processes can be challenging to analyze due to multicollinearity, multidimensionality and measurement uncertainty. A partial least squares (PLS) regression was used to analyze data generated from 66 bioreactor runs. Data included 63 runs performed at 250 mL scale and 3 runs performed at 10L scale using a manufacturing process for the production of one AAV product in HEK293 suspension cells, triple plasmid transfection and in single use stirred tank reactors. A 3 factor PLS model was generated to explain VG titer and F:E ratio in the lysate using biomass, metabolites, pH, DO and transfection parameters as X variables. Results obtained from the PLS model allowed for the understanding of the edges of failure of the process and for the identification of transfection parameters contained in a robust area of the explored design space.
Investigating the extracellular microenvironment as a potential target to monitor and control large‐scale haemopoietic differentiation
1: Cell Therapy Catapult
Allogeneic stem cell‐derived therapies are being investigated for several indications. In‐vitro haematopoietic differentiation of pluripotent stem cells (PSCs) holds great promise for the development of novel therapies, however in many cases requires large cell numbers per dose. To support reproducible and rapid scale‐up of cell cultures to meet clinical needs, manufacturing must abandon planar flask technologies and adopt automated bioreactor systems. Despite the drive towards automation, cell therapies are inherently more prone to variability, with increased risk of batch failure and costs if processes are not sufficiently controlled. In‐process analytics can help maintain control and reduce risks of failure. However, it is difficult to apply analytics that are used for monitoring expansion and differentiation in planar flasks to bioreactor systems.
The extracellular microenvironment is composed of myriad molecules consumed and produced by cells during differentiation, potentially alluding to cell state and cell fate. Here, we have characterised the cell secretome with respect to metabolites, miRNAs secreted proteins, and performed multi‐parametric analysis in conjunction with single‐cell transcriptomics to determine potentially novel biomarkers for monitoring future large‐scale differentiation processes.
The ELEVECTA® HEK293 platform – An innovative, fully stable and helper virus‐free AAV production system
1: Cevec Pharmaceuticals GmbH, Cologne
To address the growing demand for industrial scale AAV vector production, we have recently launched an innovative stable, helper‐virus free AAV production platform called ELEVECTA®. The ELEVECTA® stable producer cell lines harbor all relevant components for AAV production stably integrated into the cellular genome, which upon addition of an inducing agent enable high‐titer AAV production. Initially, our platform was based on immortalized human amniocytes (CAP cells). Here, we describe the successful transfer of the ELEVECTA® system to the HEK293 cell substrate, a well‐established cell line for the industrial scale production of AAV‐based gene therapy products by transient transfection.
Adherent HEK293 cells were adapted to suspension growth mode under serum‐free and chemically defined culture conditions. A clonal cell line was isolated and served as base cell substrate for ELEVECTA® cell line development. Stable integration of Rep as well as adenoviral helper genes under control of a Tet‐inducible promotor system resulted in polyclonal ELEVECTA® HEK293 alpha cells. Subsequently, these cells were used for the generation of a clonal alpha cell line.
The resulting clonal alpha cell line showed high‐performance growth characteristics and produced AAV to high titers after transient transfection of packaging as well as transgene elements with excellent stability over 100 cell doublings. Finally, we were able to successfully generate highly productive monoclonal HEK293 producer cells by genetically integrating capsid and transgene elements, proving the feasibility of our ELEVECTA® HEK293‐based production system.
UHPLC and CE for in‐process controls in the manufacturing of linear covalently closed doggybone DNA* for rAAV production
1: TAAV Biomanufacturing Solutions, S.L.U.
*dbDNA technology has been licensed from Touchlight Genetics Ltd. and dbDNA™ and doggybone™ are trademarks of Touchlight Genetics Ltd.
Doggybone™ DNA (dbDNA™) is a linear covalently closed DNA molecule industrially produced through an enzymatic process that can be used for rAAV manufacturing. dbDNA™ has many advantages over plasmid DNA, which include rapid production, scalability and absence of Kanamycin resistant gene copies. In industry, analytical techniques for large DNA molecules lag those used for proteins. Production of dbDNA™ is controlled by analyses of in‐process controls (IPCs) using agarose gel electrophoresis or UV absorbance. The current work was done to characterize each step of the manufacturing process (UPS and DPS) and dbDNA™ final product of the manufacturing process with Ultra‐High Pressure Liquid Chromatography (UHPLC) and Capillary Electrophoresis Laser Induced Fluorescence (CE‐LIF). The results obtained with reverse phase UHPLC showed that the technique can be used for successful separation of DNA and monitorization of the steps during manufacturing. IPC´s analyzed by CE‐LIF with a modified dsDNA 1000 Kit showed higher signals than for UHPLC, however, peaks overlapped, making their interpretation more complex.
In conclusion, the UHPLC was capable of identifying dbDNA™ from backbone fragments and other elements in the reaction with high sensitivity. Using UHPLC instead of agarose gel electrophoresis, it was possible to control the purification steps during dbDNA™ manufacturing and consequently the purity of final product.
Upscaling AAV production for research purposes: development of HEK293 suspension cell‐based AAV production protocol
1: Netherlands Institute for Neuroscience 2: Vrije Universiteit Amsterdam
Adeno‐associated viral vectors (AAVs) are an amazing tool to deliver therapeutic genes to the central nervous system (CNS). Novel capsids, including AAV.PHPeB developed by the Gradinaru laboratory can achieve widespread transduction of the CNS by intravenous injection. A higher titre (1x1011 GC/mouse) is needed compared to direct injection in the CNS parenchyme (1x109 GC/μl injection site) to achieve similar transduction. Currently, we produce AAVs, including AAV.PHP.eB, using adherent HEK293T cells and the triple transfection method. Achieving high titres required for intravenous delivery with adherent cells is labour and material intensive. To improve viral vector yields a protocol for suspension‐based cell (A35827, Thermo Fisher) culture in TubeSpins bioreactors was developed. TubeSpins can culture up to 300 ml, are compact saving both space and plastics. Several important parameters, including culture medium, plasmid quantities, and harvest conditions were tested in addition to transfection reagents Polyethylenimine “Max” (PEI‐MAX) (Polysciences) and TransIT‐VirusGEN (Mirus). AAVs generated in adherent cells (12 x 15 cm2 plates) were compared to the suspension production method (300 ml). With the adherent method, we achieved on average 2.6x 1012 GC total, using the suspension method PEI‐MAX 7.7x 1012 GC total, and TransIT‐VirusGEN 2.4x1013 GC total. Preliminary data indicates there is no difference in in‐vivo transduction efficiency between vectors produced with adherent compared to a suspension cell system. By culturing in suspension we reduced the amount of time and space needed for vector production whilst achieving almost 10‐fold higher yields using components which are available for research purposes from commercial vendors.
Final Formulation and Filtration of Lentivirus
N Marchand1 J Arena1 L McCarney1 A Lambropoulos1 M Lindberg1
1: Pall Life Sciences
In recent years, development in the gene therapy industry has grown rapidly, with lentivirus making up one of the largest classes of viral vectors. A critical step in the manufacture of lentivirus is the final formulation and filtration, which processes the vector into its final concentration and buffer matrix and ensures drug substance sterility. In recombinant protein manufacturing, final formulation and filtration has been platformed to use flat sheet tangential flow filtration (TFF) cassettes and 0.2 μm sterilizing‐grade filters. However, lentiviral vectors present unique processing challenges at these steps due to their relative instability and large size (∼0.12 μm).
In this work we demonstrate the flux benefits of flat sheet TFF devices and share data on how molecular weight cutoff and operation conditions can impact processing time, yield, and impurity removal. At the final filtration step several filters were evaluated varying in chemistry and symmetry. We also share insights on how filter filtration performance can be impacted by process parameters (flux, scale), lentivirus concentration, and impurity concentration (aggregates, HCP, DNA). The findings presented here should provide some guidance for which parameters to prioritize when developing these key process steps.
In‐process controls for plasmid isoform characterization
1: PlasmidFactory GmbH & Co. KG
During plasmid production, the purity of the plasmid DNA increases with progressing downstream processing stage. Apart from the major contaminants such as chromosomal DNA and RNA, plasmid isoforms are also present and need to be removed. The covalently closed circular conformation of plasmid is highly efficient in traversing the cell and nuclear membrane resulting in better transfection rates. Even where the plasmid DNA acts as a starting material, for example for the in vitro production of mRNA, the ccc conformation ensures that no single strand break is present in critical points of the plasmid that could otherwise lead to premature length mRNA. Thus it is very important to analyze plasmid quality during production through in‐process controls so that the percentage of supercoiled plasmid can be continuously monitored and to ensure that impurities are contained within the limits defined for the corresponding downstream stage. Capillary Gel Electrophoresis is the established gold standard method for this analysis which gives a clear result in percentage of ccc and open circular (oc) form of plasmid. As an auxiliary method, High Performance Liquid Chromatography has been tested in this work to explore its suitability as a method for routine analysis. The results show that with the current conditions, it is possible to demonstrate a good resolution between the ccc and oc forms of the final plasmid product. Initial quantitative measurements of the respective isoforms showed correlation with the CGE data. Adapting this method as an in‐process control will involve testing samples of varying purity levels.
Novel HPLC applications for rapid in‐process characterization during AAV platform development
1: Andelyn Biosciences, 5185 Blazer Parkway, Dublin, OH, USA
Andelyn Biosciences has developed a robust scalable suspension platform for Adeno Associated Virus (AAV) production suitable for all phases. It relies on utilizing a quality‐by‐design (QbD) approach to ensure both scalability and quality of AAV. Development of in‐process analytics for tracking of AAV purity and quality at different stages of production is an integral part of the platform. HPLC can allow for rapid and accurate analysis which are critical to enable fast, robust development cycles. Various HPLC methods will be presented supporting the characterization of in‐process AAV development samples. One of the described HPLC methods uses SEC/UV that provides capsid quantitation information in addition to protein impurity profiling. This method can analyze crude up through final samples. In a second method, anion exchange (AEX) chromatography is used to resolve and quantitate empty and full capsids. AEX data at final stages of the suspension process showing the separation of empty and full capsids will be presented. Additionally, process characterization AEX data will be presented that correlate with outsourced AUC data but requiring only a day for data turnaround. Thirdly, multiple in‐process impurities are monitored using a single reversed‐phase HPLC method. Data that tracks these impurities throughout the downstream purification process will be presented.
Challenges in non‐GMP pilot scale production of rAAV to support gene therapy development
1: Baxalta Innovations GmbH, a part of Takeda
Recombinant adeno‐associated viruses (rAAV) have become the vehicle of choice for in vivo gene therapy applications. However, manufacturing processes for high‐quality rAAV vectors are generally low yielding compared to processes for other biologics. Therefore, providing sufficient material to support pre‐clinical studies as well as other activities such as analytical method development and qualification, comparability, and stability studies is challenging, especially for high dose applications. In this poster, we will discuss challenges during scale up and the establishment of a robust and high yielding rAAV production process as well as strategies to meet the increasing material demands.
Optimization of lentiviral vector mediated transduction of T cells for clinical use
1: AGC Biologics
Genetic modification of T cells represents a straightforward technology to generate cell‐therapy‐based drugs, which showed promising clinical efficacy in cancer patients.
Indeed, we demonstrated the feasibility of retroviral and lentiviral vector‐genetically modified T cells production for clinical use.
Lentiviral vectors (LVV) are a platform to deliver and permanently express therapeutic genes and have displayed great potentiality in clinical trials.
Here we present proof of concept results concerning the setting up of optimal T‐cell transduction conditions with a highly‐purified VSV‐G pseudotyped LVV encoding for a CD44v6‐CAR construct in order to obtain higher transduction efficiency and process yields. Briefly, donor‐derived peripheral blood cells were activated with anti‐CD3‐anti‐CD28 nanomatrix and cultured in chemically defined T‐cell medium supplemented with interleukin‐7 and interleukin‐15. Different CD3/CD28‐based activators and T cells specific serum free media were tested to define the best protocol for T cells activation and expansion. Transduction was performed after 24 hours or 48 hours at MOI from 2 to 6 and cells were maintained in culture up to nine days. Transduction efficiency was optimal when infection was performed at shorter time after activation and with higher cellular concentration. Retronectin didn't significantly improve the transduction rate.
In addition, the small scale protocol was scaled‐up to establish a GMP‐compliant process for LVV‐transduced T cells manufacturing. In particular, to this end, it was investigated the introduction of G‐Rex platform for cell expansion. The optimal performance in G‐Rex 100M was achieved at seeding density of 1x105 cells/cm2 in presence of a specific feeding regimen.
A novel, chemically defined, serum‐free cell culture medium successfully expands transfected T cells
M Zander1 C Stoia1 A Toell1 S Harvey2
1: Lonza Cologne 2: Lonza Walkersville
The manufacture of T cell therapy products requires ex vivo culture and expansion of cells, to reach target doses. A key element for cell proliferation in media is the presence of human AB serum (HAB) or fetal bovine serum (FBS). These components risk to expose the final product to pathogens, experience significant lot variability and supply chain shortage.
Without the addition of serum, T cells, especially when patient derived, fail to grow optimally and exhibit reduced efficiencies of gene transfer, irrespective of the transfection method applied. In the transfection arena, the Nucleofector® Technology offers a powerful non‐viral delivery method for T cell engineering.
Lonza will soon launch TheraPEAK® T‐VIVO® Medium, a chemically defined, xeno‐free cell culture medium which robustly expands T cells in the absence of serum and works well in combination with the Nucleofector® Technology. We present preliminary data generated for the upstream expansion and proliferation after Nucleofection® of human T cells cultured in TheraPEAK® T‐VIVO® Medium. Results were generated with activated T cells obtained by stimulating CD3 cells or PBMC. The growth kinetics observed were comparable to cells cultured in media with serum. The expansion profiles after Nucleofection® show that cells recover from the transfection procedure and proliferate.
TheraPEAK® T‐VIVO® Medium can be used alone or with the addition of chemically defined supplements which help cell recovery post Nucleofection®, to achieve greater expansion rates.
These initial results suggest that TheraPEAK® T‐VIVO® Medium is a good candidate to support GMP manufacturing of clinical grade T cell therapies.
Characterisation of mRNA expression patterns of different HEK293 cell lines used for virus‐like particles production
M Pistek1 C I Kahlig1 B Kraus1
1: Takeda
HEK293 cell lines used for virus‐like particles (VLPs) are characterised by a high variability and diversity in respect of both physiological properties such as transfectability or growth and production relevant properties relating to productivity and product quality. The heterogeneity is derived to a large extent from uncontrolled integration of endogenous genes into the genome. Evaluation of molecular characteristics that determine the differential behaviour of these cell lines is often hampered by the fact of the aforementioned heterogeneity results in a variety of genes being differentially regulated, thus obscuring the determining genes that bring about a certain change in physiology.
In this study, we evaluated the transcriptomics profiles from different HEK293 cell lines with different growth and rAAV productivity patterns along production batch processes. Our data showed significant differences at transcriptional level only 4 hours after a 3‐plasmid‐based transient transfection protocol between rAAV high and low producer HEK293 cells. The evaluation of these transcriptomics profiles combined with collected in‐process control parameters and titers shed some light on potential cell engineering targets to maximise transient production of rAAV in HEK293 cells.
Scaling the manufacture procedure of human monocyte derived macrophages to deliver a novel drug product for the resolution of inflammatory liver damage
1: Resolution Therapeutics
Inflammatory liver damage is a growing global concern with the only course of treatment for end stage disease being transplantation. Recent studies have investigated the potential of autologous cell therapies, harnessing the pro‐restorative function of human monocyte derived macrophages (hMDM) with the MATCH clinical trial showing safety in phase I.
To produce an autologous macrophage cell therapy, monocytes are extracted from the blood and differentiated into macrophages. By nature, both cell types are non‐proliferative posing a challenge in developing a drug product with each handling step leading to cell loss from a finite starting number of cells. The manufacturing procedure must therefore pay close attention to every intervention to ensure recovery and vitality of the product, whilst also showing scalability to reach as many patients as possible.
Building upon the work in MATCH trial, Resolution Therapeutics has developed a manufacturing procedure to improve cell yield, maintain vitality, and phenotype. We have demonstrated the feasibility of this approach showing effective translation from small scale to large scale engineering whilst maintaining macrophage function and vitality. We have further ensured our cell therapy product shows promise to reach a wider patient population by developing a cryopreservation method compatible with GMP‐manufacture.
Our manufacturing procedure enables a fully scaled, GMP compatible, and closed system for manufacturing of this novel macrophage product to treat chronic liver disease.
Addressing logistical challenges – Improving storage and stability of cells
1: Albumedix
During ATMP manufacture, cryopreservation of the therapeutic cell product is currently a necessity. However, even with this there are limitations on how quickly the therapy can be used and extremely complex logistical solutions are needed to ensure therapy and patient are ready at the same time. The effect of cryopreservation on the post thaw viability of the cell product can affect the efficacy of the therapy. In addition, cryopreservation adds the requirement of a cryoprotective agent such as DMSO, which can cause negative side effects if administered, or requires an extra washing step to remove it from the cell product prior to administration. Recombumin® Elite is a GMP grade, defined, consistent, animal free, safe recombinant human albumin manufactured for use in the ATMP space. In this work Recombumin Elite was utilised in the cryopreservation and post thaw hold of T cells. Cells were expanded and cryopreserved in different media with and without the use of Recombumin Elite and the viability and growth post thawing were investigated. We will demonstrate that the addition of Recombumin Elite to the cyropreservation media increases the stability of the cells upon thawing increasing the cell viability window and therefore allows greater flexibility in administration of the therapy to the patient. In addition the inclusion of Recombumin Elite can aid in the reduction of DMSO to 5% thus enabling better patient care and increasing the opportunity for a successful therapy.
Tackling scalability challenges of tetracycline‐enabled self silencing adenovirus (TESSA) vector manufacture
1: Oxford Genetics Ltd
Tetracycline‐Enabled Self Silencing Adenovirus (TESSA) vectors allow scalable and efficient production of recombinant adeno‐associated virus (rAAV). At lab scale, TESSA vectors were produced from adherent HEK293 cells in HYPERflask®s, lysed by freeze‐thaw cycles and purified using caesium‐chloride (CsCl) gradient centrifugation. Almost all these steps present challenges in scaling‐up manufacture. To improve scalability of TESSA vector production, our Process Development team developed a production process using suspension HEK293 cells with chemical lysis, followed by chromatography‐based purification. We compared genome copy (GC) number and infectious (I) titres of TESSA‐AAV‐EGFP vectors produced by original and scalable processes, with either CsCl gradient centrifugation or chromatography‐based purification. We observed comparable titres from upstream productions in the two processes, but the chromatography‐based purification yielded vectors with 200‐fold lower I/GC ratio compared to CsCl gradient centrifugation. This indicates that we've developed a successful scalable upstream process for TESSA vector manufacture, but still need to further optimise the scalable downstream process. The analytical bottleneck is TESSA titration via TCID50 assay; a lengthy and technically intensive step, taking 14 days per batch. By automating serial dilutions, up to 16 batches of TESSA can be accurately titrated. Reproducibility remains comparable to manual operation, while reducing FTE requirement by 60%. Plaque scoring is another intensive step, requiring an operator to monitor samples over 4‐5 days. We applied qPCR to facilitate plaque scoring, which reduced assay length by 2 days, requiring half of an operator's time and producing results within 15% variance from visual results.
Analytical assay development for TGT‐001, AAV‐CRISPR‐based therapeutic candidate for Charcot‐Marie‐Tooth type 1A (CMT1A) : AAV titration using ddPCR and a quantitative in vitro potency assay for AAV vectors encoding CRISPR
1: ToolGen
AAV‐mediated gene therapy holds significant promises to treat or potentially cure various human diseases. Two important aspect of AAV QC assay is titration and potency assay. AAV genome titre is traditionally determined by qPCR; however, assay precision is not optimal despite extensive efforts. More recently, droplet digital PCR(ddPCR) emerged as a powerful alternative that offers excellent accuracy and precision. ddPCR has become emerging technique for the sequence‐specific detection and quantification of nucleic acids for various applications. Although, ITR targeting primers can be utilised as an universal primers for AAV titration, transgene‐specific primers should be utilised to take empty capsid into consideration. Upon verifying genome titre of AAV, in vitro potency assay should be performed to test batch‐to‐batch differences. TGT‐001 utilises AAV‐CRISPR to precisely edit PMP22 TATA‐box through non‐homologous end joining. In part of establishment of manufacturing of TGT‐001, AAV‐CRISPR‐based therapeutic candidates for CMT1A, we developed an AAV titration assay to target the transgene (SpCas9, gRNA) using ddPCR. To validate this assay, we performed linearity assessment; 8 concentration values, each values done in 4 replicates and performed by 3 analysts. Furthermore, we developed a cell‐based, quantitative potency assay that detects capsid entry, transgene expression and gene editing activity of AAV‐Cas9 and AAV‐gRNA vector. To increase AAV entrance in vitro, we engineered cell line by stably overexpressing AAV Receptor (AAVR). Based on this cell line, we analysed different batches of TGT‐001 manufactured. Together our results facilitates analytical assay for TGT‐001.
Utilization of the Ambr® 15 microbioreactor system and a DOE approach for adeno‐associated virus upstream process development
A Ushijima1
1: Merck Millipore
Recombinant adeno‐associated virus (rAAV) has become a leading modality for in vivo gene therapy. Currently, there are two approved AAV‐based therapeutics and the number of rAAV clinical trials is steadily increasing. As bioprocessing of AAV therapies industrializes, tools commonly utilized in traditional biotherapeutics process development, such as the Ambr® 15 microbioreactor system and a DOE based approach to experimentation, can be adopted for efficiency. Specifically, these tools can be applied to transfection parameter optimization where the screening of multiple parameters happens simultaneously, and their potential interactions can be gleaned. The work summarized here demonstrates this high throughput method for transfection optimization and the scale‐up of the optimized process to bench scale bioreactors.
HEK293 Transcriptome evaluation for efficient AAV production
1: Takeda
Since the early development of gene therapeutic applications, the once with Ad5 virus genome modified HEK293 cell line developed into a reliable efficient production cell line for several applications. Numerous derivates and sub‐clones are generated from the initial parental origin and every descendant has its own beneficials. Nevertheless, in comparison screenings some HEK293 cell lines demonstrated higher potential for maximum titer AAV production than other HEK293 cell lines. Up to now there is still a lack of knowledge how different cellular pathways are influencing the transfection efficiency and the virus reassembling process. Modern high throughput next generation sequencing methods and standardized genome mapping protocols are useful instruments to enable an overview about active cellular pathways. The comparison of efficient and less efficient AAV producer cell lines at different timepoints before and after transfection and in non‐transfected condition show the cellular metabolism development in virus production batch mode. Specific bioinformatic evaluations of mRNA distribution at different time points during transfection allows to assign activated hallmark pathways and GO terms in high producer cells. Comparing the transcriptome of a transfected cell against a non‐transfected cell supports the characterization of normal cellular batch activity compared to the stressful virus production condition. Creating an insight into the transient transfected cells transcriptomics supports the process of disentangling ongoing cellular processes and is an essential tool to scan for cell line improvement starting points.
Stability of lentiviral vectors in in vitro biopotency
P Andorfer1 B Mußnig1
1: Baxalta Innovations GmbH, a part of Takeda
Lentiviral vectors (LV) are a suitable tool to stably deliver therapeutic genes into a host's genome. The weakness of LV is their envelope which makes them less robust than viral vectors derived from parvovirus and others. LV manufacturing for gene therapy includes time consuming process steps. Therefore, we investigated the short‐term stability of LV and challenged the LV by freeze‐thaw cycles, different buffer conditions and temperature. Third generation LVs were manufactured by transient transfection of HEK293T cells with pLV‐EGFP reporter vector, pVSV.G, pRev and pGag/Pol and purified by ion exchange followed by ultra‐centrifugation and dialyses. Biopotency is most impactful to assess LV stability. Supernatants and purified material were determined by HEK293 cell infection followed by flow cytometry measurements. Additionally, LV capsid titer were determined by anti‐p24 ELISA. Two freeze thaw‐cycles reduced the LV potency by half when formulated in PBS. Temperature conditions as 4°C, RT and 37°C decreased the activity to 50% after more than two weeks, three days and 10 hours, respectively. Inactivation of LV was achieved by 95°C treatment which reduces the S2 safety classification and alleviates pursuing analyses. In general, the p24 capsid detected by ELISA indicates a more moderate harm of the LV particles than the biopotency. Taken together, it is worthwhile to control temperature, buffer conditions and freeze‐ thaw steps during production to provide for a maximal potent LV. All authors are all employees of Baxalta Innovations GmbH, a part of Takeda companies, which are involved in the development of gene therapy products. The presented work was funded by Baxalta Innovations GmbH, a Takeda company.
Good Manufacturing Practice (GMP) Adeno‐Associated Virus (AAV) Manufacturing Process Using Transient Transfection in a 2000 L Bioreactor (Theoretical approach)
1: Pall Life Sciences 2: Polyplus Transfection
The approval of gene therapies delivered by Adeno‐Associated Viruses (AAVs) has resulted in an increased commitment to adopt AAV as the vector of choice to address a broad range of diseases. To meet the demand in production, the industry has made investments to build dedicated facilities in parallel to working to scale‐up process development. Currently, transient transfection is used as the gold standard method because it brings with it key advantages of the fastest process development and highest productivity. Designing a Current Good Manufacturing Practice (cGMP) compliant AAV manufacturing process using large‐scale transfection is not an easy task, thus critical supplier partnerships and input are early indicators of success.
In this poster, we detail the method for achieving end‐to‐end AAV manufacturing process at the 2000 L bioreactor scale. From raw material supply, through upstream processing (USP)/downstream processing (DSP) manufacturing, right down to analytics, we review the different process stages and provide guidance for the steps that require particular focus.
Transcriptomics of suspension 293 cells during AAV vector production
R Palacios1
1: AskBio
Improving the yield and infectivity of AAV vector obtained from current manufacturing processes could increase the number of treatments available to patients and enable a larger number of cells to be transduced with the same vg/kg dose. The goal of this project is to identify aspects of AAV, helper virus or 293 host cell transcription occurring during vector production that could be manipulated to increase vector yield and/or infectivity. To characterise host cell transcriptomes during vector production we generated AAV vector by transient transfection in two different suspension 293 cell lines and harvested cell samples at various timepoints for RNAseq analysis. We will present data describing changes in producer cell transcriptome observed during these production campaigns.
Impact of bioreactor control parameters on AAV production
1: uniQure
Optimal cell culture environment is critical for improving recombinant AAV production; arguably the most critical factor to control in the bioreactor is oxygen supply. The effect of bioreactor parameters over baculovirus infected insect cells (ExpresSF+) was assessed in DoE‐format in bench scale single‐use stirred tank reactors. Parameters such as cascade, stirring speed, gassing and dissolved oxygen setpoint were tested and as outcome, the best conditions were identified to achieve the optimal AAV production process.
Development and qualification of a relative potency assay for SPVN06, a recombinant AAV vector for the treatment of inherited retinal disorders
A Rogue1 G Massonnet1 E Dubreucq‐Laine1 L Bauduin1
1: Charles River Laboratories Evreux, Route de Pacy, 27930 Miserey, France 2: SparingVision, 5/7 avenue Percier, CS 40230 – 75008, Paris
SPVN06 is an AAV‐based, mutation‐agnostic, ocular gene therapy being developed for the treatment of inherited retinal degeneration. The drug product (DP) contains cDNAs encoding for a trophic factor, Rod‐derived Cone Viability Factor (RdCVF) and a thioredoxin enzyme, Rod‐derived Cone Viability Factor Long (RdCVFL). Here we describe the development and qualification of a phase‐appropriate, relative potency assay for SPVN06, measuring the levels of mRNA transcripts for RdCVF and RdCVFL compared to a standardized reference control vector. The assay includes three steps: (1) Transduction of Weri‐Rb1 cells with varying MOIs of SPVN06 DP and the reference control vector. (2) Cell harvest, lysis and RNA extraction. (3) Determination of transcript levels using two distinct RT‐qPCR assays, one for each form of RdCVF. For each analysis, log MOI (vg/cell) is plotted against log quantity of the transcript (copies/μg of RNA) and fitted using a linear model. Linear concentration–response curves are then compared between the reference standard and the DP using an analysis of variance (ANOVA). Potency assay performance parameters such as precision, parallelism, linearity, specificity, and robustness are evaluated. Intermediate precision for RdCVF and RdCVFL is determined to be 11.2% and 20.4%, respectively. The assay shows reproducible linearity within a 50‐200% range as compared to the reference DP, with MOIs ranging from 5 x 103 to 3 x 105 vg/cell. Based on the results and the performance parameters, the applicability of the assay for release and comparability of several SPVN06 batches, as well as stability studies, is discussed.
The FNIH Accelerating Medicines Partnership Bespoke Gene Therapy Consortium
1: Pfizer Ltd
Approximately 25–30 million people in the United States live with the devastating effects of rare diseases. There are over 5,000 rare diseases that are caused by genetic defects. These patients frequently lack access to effective treatment, as knowledge about many rare diseases, as well as funding for research, often lag behind more common diseases. For the approximately 80 percent of rare genetic diseases caused by a single defective gene, adeno‐associated virus (AAV) gene therapy is one very promising treatment modality. Gene therapies can be tailor‐made, or “bespoke,” for a very small population or even a single individual, but the development process is complex, expensive, and hampered by a lack of common biologic, manufacturing, and regulatory standards. The Bespoke Gene Therapy Consortium (BGTC) aims to make gene therapy more accessible by creating a platform approach to deliver novel therapies for many different genetic disorders. The BGTC is one of the latest initiatives to emerge from the Accelerating Medicines Partnership® (AMP®) Program, a public private collaboration among the NIH, the FDA, the pharmaceutical industry, and patient organizations—coordinated by the FNIH—to speed drug development across different diseases. At a cost of $80.5 million over six years, the BGTC is the largest effort of its kind to streamline the development of therapies for rare diseases, bringing together the resources of 34 partner organizations spanning the public, private, and nonprofit sectors.
Human stem‐cell based models to study innate immunity and neuroinflammation in the central nervous system
1: IBET‐Instituto de Biologia Experimental e Tecnológica 2: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa
The development of gene therapies for central nervous system (CNS) disorders has been hindered by the lack of human‐relevant models in which the neuro‐immune axis is depicted. Upon infection, glial cells undergo activation, triggering a neuroinflammatory process that leads to neuronal death. Still, the mechanisms that initiate and sustain human glial activation remain unknown. Here we propose a human innate immunocompetent CNS model that recapitulates neuroinflammatory hallmarks and is a suitable platform for preclinical development. Human induced pluripotent stem cell (hiPSC)‐derived neural progenitors were cultured in perfusion stirred‐tank bioreactors and differentiated into 3D neurospheroids composed of neurons, astrocytes, and oligodendrocytes. A co‐culture between hiPSC‐derived microglia (iMGL) and neurospheroids was established to mimic the neuro‐immune axis. iMGL efficiently infiltrated the neurospheroid structure, adopting a ramified phenotype and maintaining the expression of typical markers (e.g., TMEM119). Prototypical neuroinflammatory factors were used to challenge the neurospheroids. Transcriptomic and proteomic profiling revealed both modulation of key inflammatory pathways, namely NF‐κB pathway, and secretion of several proinflammatory molecules (e.g., CXCL8), key players in neuroinflammation. Adeno‐associated virus (AAV) serotype 9, a candidate for gene therapy able to cross the blood‐brain barrier, was used to assess viral vector immunogenicity. Neurospheroids were transduced with AAV9‐eGFP at different MOI and the transgene expression, and cell toxicity was evaluated. Ongoing work focuses on characterizing glial cell innate immune response to the viral vectors. Hence, we propose the human neurospheroid model as a useful preclinical model to accelerate the development of advanced therapies. Acknowledgements: FCT/MCTES(PT):LA/P/0087/2020; UIDB/04462/2020&UIDP/04462/2020; PTDC/BTM‐ORG/29580/2017; UI/BD/151253/2021. EU/EFPIA/IMI JU:No.945473.
Continuous generation of fully functional feeder‐free human iPSC‐derived natural killer cells for clinical applications
1: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany 2: Department of Pediatric Pneumology, Allergology, and Neonatology, Hannover Medical School, Hannover, Germany 3: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany 4: Institute of Transplant Immunology, Hannover Medical School, Hannover, Germany 5: Department of Rheumatology and Clinical Immunology, Hannover Medical School, Hannover, Germany 6: Institute of Transfusion Medicine and Transplant Engineering, Hannover Medical School, Hannover, Germany
The use of primary human Natural Killer cells (NKs) has recently gained interest due to their capabilities to target a variety of haematological malignancies and solid tumours. Most ongoing clinical trials use primary cells isolated from either peripheral blood or umbilical cord blood. However, these sources greatly depend on donor variability and comprise a high heterogeneity in the NKs sub‐populations. In contrast, human induced pluripotent stem cells (hiPSC) may offer an interesting alternative source of NKs for immunotherapy. hiPSC‐derived NKs entail a more homogeneous cell population that can be genetically engineered and scaled‐up at clinical grade. These characteristics make iPSCs‐derived NKs a desirable “off‐the‐shelf” product for immunotherapy. Here we studied the feasibility to continuously generate hiPSC‐derived NKs. For this purpose, a >38 day differentiation protocol was established. Using feeder‐free hiPSCs, we generated NK cell‐producing aggregates using Essential‐8 media supplemented with BMP4, VEGF, SCF and IL‐3 in small‐scale suspension cultures for mesoderm induction. Furthermore, mesoderm primed aggregates were cultured in X‐VIVO15 media supplemented with hematopoietic differentiation growth factors (IL‐3, IL‐7, IL‐15, SCF and Flt3) for NKs production. Starting on day 28 of hematopoietic differentiation, regular harvests of NKs were performed achieving a purity of >85%. Additionally, the generated hiPSC‐NKs depicted classical morphology, homogenous surface marker profile of CD45+CD3‐CD56+CD16‐, with intact degranulation and killing capacity against K562 cells. In summary, we introduce a new method for the continuous generation of mature, homogenous, fully functional human iPSC‐derived NKs that might serve as a robust alternative to blood‐isolated NK as a cell‐based immunotherapy.
Establishment of primary paediatric low‐grade glioma (lgg) cultures: in need of a novel 3D platform for advanced preclinical studies of innovative and immunotherapeutic approaches
1: Hematology, Oncology and Cell and Gene Therapy Department, IRCCS Bambino Gesù Children's Hospital, Rome, Italy 2: Pediatrics Residency Program, University of Rome Tor Vergata, Rome, Italy 3: Catholic University of the Sacred Heart, Department of Life Sciences and Public Health, Rome, Italy
Reproducing the structural complexity of solid tumors is crucial to obtain a more reliable in vitro model for studying these tumors. Paediatric low‐grade glioma (pLGG), in particular, is extremely challenging to maintain in culture, owing to the activation of senescence pathways. We evaluated the ability of an extremely versatile, fibrin‐based hydrogel to sustain 3D‐cultures of pLGG by culturing in parallel, in conventional 2D platforms and in 3D, 24 samples obtained by tumor biopsies at OPBG. Cell lines identity was confirmed by short tandem repeats (STRs) and immunohistochemical characterization (H&E, Ki67, tumor and differentiation markers) in 3D cultures revealed phenotype, cellular organization and proliferative rates closer to those observed in the onset samples, as compared to the 2D. Whereas in the 3D‐setting we observed a regular growth between passages, in 2D cells showed an irregular growth between passages. The analysis of cell senescence using ß‐galactosidase assay revealed a lower senescence in the 3D cultures (3D: 12,28% +4,3% of the cells vs 2D: 50,69% +18,46%; p = 0,008). Lastly, we tested the responses to radiotherapy, chemotherapy and to an innovative immunotherapeutic approach represented by oncolytic adenovirus (OA). By MTS assay, we observed that 2D‐cultured cells are significantly more sensitive to these treatments than 3D, suggesting an overestimation of the efficacy of these treatments by the 2D‐setting, therefore reducing the predictive power. In conclusion, the 3D‐culture offers a more representative model of solid tumors, offering an innovative platform for biological and therapeutic studies.
Human brain organoids as a tool to study the effects of mutations potentially associated with ASD
1: Genomic Medicine, Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), University of Santiago de Compostela, Santiago de Compostela, 15706, Spain 2: ZebraBioRes, Zoology, Genetics and Anthropology Department, University of Santiago de Compostela, Lugo, 27002, Spain 3: C005, Instituto de Investigación Sanitaria de Santiago (IDIS), Santiago de Compostela, 15706, Spain 4: U711, Center for Biomedical Network Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Madrid, 28029, Spain 5: Glial and Neuronal Biology, Lund Stem Cell Center, Lund University, Lund, 221 84, Sweden 6: Fundación Pública Galega de Medicina Xenómica (FPGMX), Hospital Clínico Universitario, Santiago de Compostela, 15706, Spain.
*contributed equally
#corresponding author
Autism Spectrum Disorders (ASD) are characterized by behavioural and communicational alterations. Although some environmental and genetic factors have been associated with ASD, their aetiology has not been fully described. Next‐Generation Sequencing techniques have been used to identify new de novo genomic variants in ASD cases, which are potentially associated with the disorder. However, the functional role and disease implication of most of them is unknown.
In this communication, we will explore how to assess the functional role of de novo mutations selected from ASD‐affected individuals, with the final goal of improving our understanding of the molecular bases of the disorder and the genetic counselling offered to the families, as well as proposing new therapeutical targets.
Human induced pluripotent stem cells (hiPSCs) were selected as an in vitro model based on their differentiation capability into neuronal cell lines. After selecting the genes to study, CRISPR/Cas9 was applied to knock them out, and the obtained mutant lines were characterized. Then, the edited lines and their isogenic control were differentiated into human brain organoids using published protocols. The generated organoids were analyzed at several time points during development, using techniques such as immunostaining, qPCR and calcium imaging, to identify how the mutations of interest alter neurodevelopment and neural function. Close attention was paid to the presence of alterations in the maturation times of the cell lineages present in the organoid, and to the presence of an imbalance between GABAergic and glutamatergic neurons as both have been previously associated with ASD development.
Modelling Machado‐Joseph Disease in patient‐derived isogenic lines by CRISPR/Cas9‐mediated correction of ATXN3‐CAG expansion
1: CNC ‐ Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal 2: CIBB ‐ Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal 3: Institute for Interdisciplinary Research, University of Coimbra, Coimbra, Portugal 4: ViraVector, Viral Vector for Gene Transfer Core Facility, University of Coimbra, Coimbra, Portugal 5: Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal
Machado‐Joseph disease (MJD) integrates the group of polyglutamine (polyQ) disorders, regarded as the most dominant form of spinocerebellar ataxia. Caused by an abnormal expansion of the CAG (cytosine‐adenine‐guanine) tract within the ATXN3 gene, MJD is characterized by the formation of toxic polyQ‐expanded Ataxin‐3 species that interfere with numerous physiological mechanisms and culminate in neuronal loss.
Patient‐derived induced pluripotent stem cells (iPSC) are valuable models to recapitulate MJD disease mechanisms in a more physiologically relevant context and investigate novel therapeutics, yet these are limited by the high phenotypic variability inherent to inter‐individual comparison. To address this issue, we established an efficient and precise genome editing approach to correct the ATXN3‐CAG expansion in MJD iPSC. Our approach relies on CRISPR/Cas9‐mediated homology directed repair to integrate a non‐expanded CAG tract within the endogenous ATXN3 locus and thus generate isogenic iPSC lines that share an identical genetic background. Opposed to previous efforts to edit polyQ‐associated genes, this approach can attain unprecedent levels of targeted recombination without requiring selection‐based markers. MJD isogenic lines were obtained with biallelic and seamless ATXN3‐CAG correction, confirmed by the absence of mutant Ataxin‐3 expression and undetectable off‐target genomic modifications. Homogenous isogenic neuronal cultures were generated using a rapid differentiation protocol based on pro‐neural Neurogenin overexpression to characterize MJD disease mechanisms and validate phenotypic rescue. In summary, our work provides novel human in vitro models of MJD models to accurately investigate therapeutic interventions and establishes a precise method to correct CAG expansion potentially translatable for autologous cell therapy of polyQ disorders.
Human iPSC‐derived hepatic organoids – a model for blood coagulation research and therapy
1: Oslo University Hospital 2: University of Oslo 3: Universidad de Murcia
Inherited blood clotting disorders are monogenic diseases that cause severe bleeding or thrombosis in affected individuals. To date, investigations into the pathophysiology of these mutations have relied upon overexpression studies in cell lines, however, a relevant physiological model is lacking.
Human induced pluripotent stem cells were differentiated into hepatic organoids (iHO) using a small molecule approach. mRNA expression was measured with RT‐qPCR, while protein was detected by ELISA, multianalyte bead profiling, Western blotting, confocal immunofluorescence microscopy and mass spectrometry. Thrombin generation was measured with the Calibrated Automated Thrombogram assay. Glycosylation was studied using N‐glycosylase F (PNGase F) and neuraminidase digestion. Human primary hepatocytes (PH) or human plasma pool were used as controls.
mRNA expression of factor (F) 2 (prothrombin), F7, F8, F9, F10, protein C, antithrombin and fibrinogen was detected in the iHO, at levels ranging from 0.5‐500% of the PH control. The organoids expressed mRNA levels of the hepatic markers alpha‐1‐antitrypsin (A1AT) and hepatic nuclear factor‐4 (HNF‐4) comparable to the control cells. Protein expression of FII (prothrombin), FVII, FIX, FX, protein C, protein S, and antithrombin were detected, at levels ranging from 50‐300% of control cells. FVII produced by iHO showed similar glycosylation pattern as plasma FVII, and its activity was comparable to that of primary hepatocytes. The organoid media induced thrombin generation in FVII deficient plasma with similar lag time and time to peak as normal plasma.
Human iHO constitutes a relevant model to investigate the physiology of hepatic coagulation proteins in both normal and diseased states.
Induced Pluripotent Stem Cells from patients with congenital defects of hematopoiesis for modelling hematologic disorders
1: Medical University of Lodz, Poland 2: Institute of Medical Biology of PAS, Poland
Induced pluripotent stem cells are obtained from somatic cells that have been engineered to reprogram their epigenome and convert them into a state of pluripotency. Such cells can differentiate into any cell of the human body and can therefore be used in regenerative medicine, disease modelling, and for a better understanding of physiological mechanisms.
The study aimed to obtain iPS cells under xenogeneic factor‐free culture conditions and then differentiate them into hematopoietic cells, myeloid precursors and ultimately granulocytes. Urinary tract epithelial cells and mesenchymal stromal cells were derived from two patients with mutations in the CLBP gene, a patient with Shwachman‐Diamond syndrome and healthy control. These cells were reprogrammed with oriP/EBNA‐1 episomal vectors. The induction of the lateral plate mesoderm cells was performed in the presence of the factors FGF2 and BMP4; for the specification of hematopoietic progenitor cells VEGF, SCF and IL‐3 were used, and the process of maturation of precursor cells was carried out in the presence of IL‐3 and G‐CSF.
Urinary tract epithelial cells showed ∼150 × higher reprogramming efficiency than MSC cells and increased iPSC colony formation kinetics by 7‐10 days. The induced cells showed morphology characteristic of ESCs, expression of pluripotency‐associated transcription factors and surface markers. They also demonstrated the potential to differentiate into cells of the three germ layers. The directed differentiation of iPS cells into granulocytes demonstrated the presence of CD34/CD45‐positive hematopoietic progenitor cells, CD13‐positive cells in the myeloid precursor population, and segmented nuclei in terminally differentiated cells.
Tracing mouse early retinal progenitor cells in optic‐cup organoids
1: 2: 3: University of Lausanne
Retinal organoids (RO) become a powerful tool to delve deeper into retinal diseases causes and development. However, one limitation is the heterogeneity of opticup organoid development synchrony, likely caused by interference with non‐retinal tissues. To address this shortcoming, we hypothesize that derivation of RO from population of early retinal progenitor cells (RPC), instead of pluripotent stem cells, will reduce RO heterogeneity. To trace and isolate RPC, we used CRISPR nickase gene editing system to create a reporter mouse embryonic stem cell line, in which expression of RPC‐specific gene Rax (aka Rx) drives the expression of the mCherry gene. For this modification, we used Crx‐GFP mouse embryonic stem cell line to trace photoreceptors. We expect that the mCherry‐positive cell population contains RPC as well as cells forming the niche for the eye field induction. After ES cell editing, and single‐cell clone selection, we analyzed 5 lines with PCR and sequencing. One line had desired insert in one allele. Immunohistochemistry analyzes of RO slices revealed appearance of mCherry‐positive retinal progenitor cells starting from forth day after aggregation. We will further characterize the progenitor cells to confirm their identity. This new cell line can serve in the future for retinal studies aiming at tracing retinal progenitors and mature rods to study their biology in different conditions.
Developing next‐generation neuromuscular therapeutics using quasi vivo 3D human skeletal muscles
1: The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK 2: Universite´ Paris‐Saclay, Univ Evry, Inserm, Genethon, Integrare research unit UMR_S951, 91000 Evry, France. 3: Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of Child Health and Great Ormond Street Hospital for Children, London, UK 4: University College London, Department of Cell and Developmental Biology, London, WC1E 6DE, UK
Skeletal muscle is the most abundant human tissue. It is composed of large, post‐mitotic, multinucleated fibres innervated by motoneurons and supported by several other cell types. Skeletal muscle function is impaired in muscular dystrophies: genetic disorders characterised by muscle wasting, impaired mobility and premature death, which remain incurable. Although preclinical and clinical evidence position genetic therapies amongst key emerging treatments for several genetic conditions, no gene therapy or editing strategy has been approved for any muscular dystrophies yet. The lack of robust, human(ised) models enabling precise development of such therapies is a barrier towards clinical translation of muscle gene therapies. Here we generate advanced in vitro models of muscular dystrophy to test and develop gene therapy vectors and genome editing strategies with high efficacy, low toxicity and selective tropism. We first explored the suitability of our 3D muscle platform to be permissive for transduction with adeno‐associated viral vectors (AAVs; leading neuromuscular gene therapy clinical products). Dose response was measured by live imaging of fluorescent proteins expressed by AAVs. Different AAV serotypes were then tested on 3D human muscles containing both myofibres and motoneurons, enabling assessment of AAV cell specificity for human tissues in vitro. The selected serotype was then utilised to deliver therapeutic transgenes to 3D muscles from patients with Duchenne muscular dystrophy (DMD). Finally, we validated our platform beyond conventional gene therapy and provide evidence of its translational potential in a novel strategy to upregulate the expression of an endogenous gene known to compensate for dystrophin deficiency in DMD.
Integrating preclinical data using causal networks to build clinical dose predictors
1: Prevail Therapeutics a wholly owned subsidiary of Eli Lilly and Company
Clinical dose translation from pre‐clinical data is an important but challenging step in gene therapy development. Closed‐form equations for dose translation fail to use all available pre‐clinical data, forcing heuristic dose translation based on selected subsets of preclinical studies. We have developed a causal network framework for synthesis and integration of pre‐clinical data into mechanistic models of gene delivery on downstream biochemical pathways, which could potentially serve as clinical dosing predictors. We deploy these causal networks in the context of PR001, a single‐dose intravenous AAV9 gene therapy for Type 1 Gaucher Disease patients harboring GBA1 mutations. First, we constructed a knowledge‐derived causal network that directs causal connections between dose, biodistribution, glucocerebrosidase (GCase) enzyme activity, and lipid accumulation (i.e., the Sequential‐GCase Model [SGM]). Second, we demonstrated that the SGM can be recovered from our mouse model pre‐clinical data. Third, we expanded SGM to model time effects and enable cross‐species data integration. Fourth, we built a SGM simulator, programming it to find doses that lead to pre‐specified average reductions in lipids along with the causally linked changes in biodistribution and GCase activity. Importantly, for each dose, the SGM simulator outputs distributions which affords characterization of distributional moments (e.g., the mean and variance) of each causal link. Thus, the SGM simulator is a dosing simulator that provides expectations of how individuals may respond to a given dose at each node in the network. We demonstrate the utility of knowledge‐driven causal networks as clinical dosing predictors capable of providing mechanistic insights into therapeutic efficacy.
Development of AAV‐UPF1 gene therapy to rescue ALS pathophysiology using microfluidic platforms
1: MeiraGTx
Development of a liver fibrosis disease model in a triple immunodeficient mouse strain
1: Resolution Therapeutics Ltd.
Deaths from liver cirrhosis continue to rise in contrast to other chronic health conditions. Orthotopic liver transplantation remains the only therapeutic option for patients with end‐stage disease with alternative therapies urgently sought. Macrophages are professional phagocytes that have potent tissue remodeling properties and have been shown to be a promising cell‐based therapy in pre‐clinical models of liver fibrosis. Autologous human macrophage therapy is now under evaluation in Phase 2 clinical trials. Modelling human cell‐based therapies in pre‐clinical species represents a significant translational challenge. Immunocompromised strains must be utilised to prevent acute rejection of human cell products in vivo. However, immunocompromised strains can be highly sensitive to toxins and require careful dosing and monitoring. Here, we tested if several commercially available immunocompromised mouse strains could support the carbon tetrachloride (CCl4)‐induced liver fibrosis model. Each strain was administered CCl4 (i.p.) twice weekly for up to 12 weeks. Liver fibrosis was assessed at different times during the 12‐week protocol and quantified using picrosirius red (PSR) staining and image quantitation. Finally, engineered human macrophages were examined in a pharmacokinetic study using a fibrotic triple immunodeficient mouse strain. Human macrophages were detected in liver tissue for several days following intravenous administration. In summary, immunocompromised strains are compatible with liver fibrosis protocols and can be used to understand the biodistribution and persistence of human cell‐based therapies in vivo.
Apoptosis and autophagy in the acute phase of a traumatic disease of the spinal cord
M E Baltin2
1: Ernst Strüngmann Institute for Neuroscience in Cooperation with Max Planck Society 2: Mechanobiology laboratory, Institute of fundamental medicine and biology, Kazan Federal University
Here we investigated the pathogenesis of autophagy and apoptosis following spinal cord injury (SCI). The study was performed on 28 adult Sprague‐Dawley rats of both sexes, weighing 200‐240 g. All experiments were performed in compliance with bioethical standards. Contusion SCI was applied using the Allen method. Autophagy and apoptosis markers were determined in spinal cord tissue and peripheral blood leukocytes using Western blotting. The results showed that the level of LC3‐II in the spinal cord on day 1 after SCI increased 5‐fold compared to the control. On day 3, a decrease in the content of the LC3‐II protein was observed, as well as in the peripheral blood. Increased expression of LC3B‐II on day 1 after SCI correlated with increased cell death. An increase in the expression of the apoptotic marker Caspase‐3 in the spinal cord tissue was observed on day 3 after SCI compared with the intact group. Positive p62 inclusions formed up to 21 days after injury. On day 21 after SCI, an increase in the level of LC3B and beclin‐1 proteins was observed, which indicates the induction of autophagy. We can conclude that the process of autophagy and apoptosis can proceed at the same time, depending on each other after SCI. In literature, this is referred to as “molecular cross‐talk” between autophagy and apoptosis, however, it is difficult to determine which process, apoptosis or autophagy, contributes more to the neurodestructive effects associated with SCI.
Genetic engineering of human NK‐cells for CAR‐Enhanced immunotherapy of haematological malignancies
1: Department of Pediatrics III, University Children's Hospital Essen, Germany 2: Department of Otorhinolaryngology, Head & Neck Surgery, University Hospital Düsseldorf, Germany 3: Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich‐Heine University Düsseldorf, Germany
With their highly effective and antigen‐selective cytotoxic responses, chimeric antigen receptor (CAR) T‐cells finally made their way into approved therapy for haematological malignancies. As a promising alternative with shorter in vivo persistence and the potential of off‐the‐shelf manufacturability, NK‐cells are currently tested in numerous clinical trials. However, standard techniques for the development of suitable CAR NK‐cell products still need improvement. Therefore, we systematically improved lentiviral transduction of primary human NK‐cells by testing different internal viral or cellular promoters for robust transgene expression and comparing lentiviral pseudotypes as well as viral entry enhancers to promote NK‐cell transduction. Additionally, we modified CAR constructs recognising common ALL and AML target antigens to harbour our previously described CD34‐derived hinge that allows CD34 microbead‐assisted selection of transduced NK‐cells with over 95% purity and also facilitates efficient recognition of CAR NK‐cells in vitro and in vivo. All in vitro cultures were performed in a commercial GMP‐compatible chemically defined medium. Since most leukemic blasts are a priori immunogenic for activated primary human NK‐cells, we blocked six activating NK‐cell receptors and thereby were able to verify target‐specific and CAR‐driven killing of ALL and AML cells by our CAR NK‐cells. Finally, in an ALL xenograft model in NSG mice, we demonstrated that CD19 CAR NK‐cells critically rely on NK‐cell‐produced soluble or membrane‐bound IL‐15 for persistence and ultimately leukaemia control in vivo. In summary, this study provides important improvements for the generation of pure and highly active allogeneic CAR NK‐cells, thereby bringing CAR NK‐cell therapy closer to the clinic.
EDIT‐202, an AsCas12a and SLEEK™ gene‐edited iPSC‐derived NK cell therapy maintains prolonged persistence, high cytotoxicity and enhanced in vivo control of solid tumors
1: Editas Medicine
EDIT‐202 is an engineered induced pluripotent stem cell (iPSC)‐derived natural killer (iNK) cell product under development to address the unmet need for treating solid tumors. EDIT‐202 is engineered with AsCas12a and SLEEKTM gene editing tool to knock in CD16 and membrane bound (mb) IL‐15, and knock out TGFβR2 and CISH, to prolong persistence, enhance antibody‐dependent cellular cytotoxicity (ADCC), and resist TGF‐β‐mediated immunosuppression in the tumor microenvironment.
In vitro, EDIT‐202 persisted significantly longer than unedited iNKs in the absence of IL‐2 or IL‐15. EDIT‐202 demonstrated significantly enhanced ADCC against multiple human solid tumor cell lines and showed significantly better serial killing that was maintained in the presence of TGF‐b. In addition, activated EDIT‐202 secreted 15‐fold higher IFN‐g compared with unedited iNKs.
EDIT‐202 induced significantly enhanced anti‐tumor efficacy in vivo when evaluated using an SKOV3 HER2+ metastatic ovarian cancer model. Tumor bearing mice were treated intravenously (IV) with an isotype control, trastuzumab (TRA) alone, EDIT‐202 alone, or EDIT‐202 iNKs in combination with TRA. Treatment with 20x106 EDIT‐202 plus ADCC‐mediating TRA induced significant reduction in tumor burden compared with TRA alone resulting in complete tumor clearance in 100% of mice by day 5. Combination treatment significantly increased survival to 100% at day 82 compared with 0% with TRA alone. Monotherapy with 15x106 EDIT‐202 reduced tumor burden, however combination treatment with ADCC‐mediating TRA resulted in significantly more tumor reduction with as few as 1.7x106 EDIT‐202 cells. These data support the development of EDIT‐202 as a potential allogeneic cell‐based medicine for treating solid tumors.
Validation of 5T4 as an immunological target antigen for the treatment of acute myeloid leukaemia
1: Oxford Biomedica 2: University of Birmingham
Acute Myeloid Leukaemia (AML) is a rare blood cancer, representing 1% of all new cancer cases; it affects mainly adult patients (the median age at diagnosis being 68); relapse occurs in 50% of patients with AML with a five‐year survival rate of 30%. Identification and validation of antigens expressed specifically on the surface of tumour cells is a critical step in the development of new and highly specific treatments for cancer. 5T4 is a tumour‐associated antigen expressed on the cell surface of most solid cancers, however very little is known about its expression in haematological malignancies. In this study we present our data from the target validation of 5T4 in leukaemias using multi‐parameter flow cytometry and mass cytometry (Cytometry by Time‐of‐Flight, CyTOF), all the way to in vitro assessments of safety and efficacy of 5T4 CAR‐T cells against HSCs and several leukaemia cell lines. Our data showed that 5T4 expression was seen in about 50% of AML cases. Moreover, work undergoing in the lab is evaluating the expression of 5T4 in different molecular subsets of AML and co‐expression with other known AML‐associated antigens. Leukaemic stem cells (LSC) were also positive for 5T4 expression. 5T4 CAR‐T cells efficiently and specifically killed AML tumour cell lines including the LSC population. Co‐culture of 5T4 CAR‐T cells with HSC from healthy donors showed no impact on subsequent colony formation. These results demonstrate that 5T4 is a promising target for immune intervention in AML.
Characterization of DSG3‐CAART cells prior to & following adoptive transfer in mucosal Pemphigus Vulgaris
1: Cabaletta Bio 2: University of Pennsylvania 3: Stanford University 4: University of California, Davis
Mucosal‐dominant pemphigus vulgaris (mPV) is a painful autoimmune blistering disease mediated by anti‐desmoglein 3 autoantibodies (anti‐DSG3 Ab). The current standard of care for mPV includes broadly immunosuppressive therapies that have risks of serious or life‐threatening infection. We are evaluating the safety and activity of a novel cellular therapy consisting of gene‐modified autologous T cells (DSG3‐CAART) engineered to eliminate DSG3 reactive B cells in mPV. We previously reported on the translational and clinical data from mPV patients receiving lower doses of DSG3‐CAART. Here, we expand on those preliminary assessments by including both patients that received higher doses of DSG3‐CAART and through more robust translational studies. Briefly, pre‐infusion, DSG3‐CAART eliminated target cell lines and were comprised of a heterogenous mixture of effector and memory cells. Following infusion in the absence of lymphodepletion, gene‐modified T cells were detected in the blood of all patients within the first 29 days post‐infusion with a dose dependent increase in peak vector concentration & AUC also within the first 29 days post‐infusion. Persisting cells were predominantly of the central memory or stem cell memory phenotype. In patients receiving higher doses of DSG3‐CAART cells, transient elevations of T cell cytokines (ex: IFNγ) were detected in patient sera. No clear pattern in clinical disease scores or anti‐DSG3 Ab levels from pre‐infusion to post‐infusion were observed (at doses ≤2.5 x 109 DSG3‐CAART cells). These data suggest that DSG3‐CAART is not eliminated by pre‐existing anti‐DSG3 immunity present in mPV & support further exploration of DSG3‐CAART in mPV for safety & efficacy.
MTOR as a selectable genomic harbor for CRISPR‐engineered CAR‐T cell therapy
1: Centre Hospitalier Universitaire de Québec Research Center – Université Laval 2: Trev and Joyce Deeley Research Centre, BC Cancer 3: Department of Biochemistry and Microbiology, University of Victoria
Chimeric antigen receptors (CARs) reprogram T cells to recognize and target cancer cells. Despite remarkable responses and remission rates observed with CAR‐T cell therapy in patients with hematological malignancies, relapses are common, and personalized strategies are needed to improve clinical outcomes. Here, we devised a CRISPR‐based strategy to simultaneously introduce a rapamycin resistance mutation and a CAR gene cassette to the MTOR locus via intron nesting to generate rapamycin‐resistant CAR‐T cells. Considering that the PI3K‐AKT‐mTOR pathway is hyperactive in many cancers, we hypothesized that combinatorial therapy with rapamycin, a clinically used mTORC1 inhibitor, could improve CAR‐T cell therapy outcomes.
We first demonstrated that therapeutic transgenes could be targeted to the MTOR locus in K562 cells, allowing the marker‐free selection of up to 99% of rapamycin‐resistant cells expressing the transgenes of interest. Using a fluorescent mTORC1 signaling reporter, we confirmed that mTORC1 signaling remains functional in the presence of rapamycin after targeted transgene integration to the MTOR locus. We then targeted a CD19‐CAR to the MTOR locus in primary T cells from different healthy donors, allowing the enrichment of up to 85% of rapamycin‐resistant CAR‐T cells. Using luciferase‐based and FACS‐based cytotoxicity assays, we confirmed that our CAR‐T cells could efficiently target CD19+ leukemia cells in the presence of rapamycin. We are currently assessing the impact of combinatorial CAR‐T cell therapy with rapamycin in a humanized mouse model of acute lymphoblastic leukemia. We foresee that our strategy could both facilitate the enrichment of CRISPR‐engineered CAR‐T cells and improve tumor eradication.
Novel chimeric antigen receptor against JAG1 with Ag‐specific cytotoxicity
1: IBET‐Instituto de Biologia Experimental e Tecnológica 2: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa 3: Bayer Portugal, Carnaxide, Portugal
The Notch‐signaling ligand JAG1 is a key oncogene in subsets of various aggressive solid tumors and correlates with poor clinical prognosis. Overexpression of JAG1 in these tumors contributes to tumor growth, angiogenesis, metastasis, anti‐tumor immunity, recurrence, and drug resistance. Hence, therapies targeting oncogenic JAG1 hold great potential for the treatment of aggressive tumors. Here we developed anti‐JAG1 specific antibodies (Abs) and anti‐JAG1 chimeric antigen receptors (CARs) and generated JAG1 CAR‐T cells exerting cytotoxicity against JAG1‐positive cells. Anti‐JAG1 single‐chain variable fragments (scFv) were selected from phage display libraries, reformatted into full‐length Abs and produced in mammalian cells. Characterization of the purified Abs identified two candidates with specific binding to JAG1 with nanomolar affinities. Cloning of the respective scFv sequences in our second and third‐generation CAR backbones, containing CD28 or/and 4‐1BB costimulatory domains, resulted in a panel of six anti‐JAG1 CAR lentiviral constructs in various configurations. Anti‐CD19 CARs with matching configurations were made as controls. Expression of these CARs in Jurkat cells confirmed correct scFv display and revelated four anti‐JAG1 CARs resulted in induction of activation marker CD69 in co‐culture assays with two JAG1‐positive cell lines. Studies in primary T cells demonstrated that one anti‐JAG1 CAR induced effective activation in the presence of JAG1‐positive but not JAG1 knockout tumor cells, as well as specific killing of JAG1‐positive tumor cells. Therefore, this new anti‐JAG1 CAR presents as a promising candidate against JAG1‐positive tumors, namely, for combinatorial therapeutic strategies including additional tumor antigens to increase specificity and overcome tumor heterogeneity issues.
Universal allogeneic CAR‐T cells engineered with Sleeping Beauty transposons and CRISPR/Cas9 for cancer immunotherapy
1: Vrije Universiteit Brussel (VUB) 2: Universitair Ziekenhuis (UZ) Brussel 3: Center for Molecular & Vascular Biology, Department of Cardiovascular Sciences, University of Leuven
Allogeneic CD19‐specific chimeric antigen receptor (CAR)‐T cells with inactivated donor T cell receptor (TCR) expression can be used as an ‘off‐the‐shelf’ therapeutic modality for lymphoid malignancies, thus offering an attractive alternative to autologous, patient‐derived T cells. Current approaches for T cell engineering mainly rely on the use of viral vectors. Here, we optimized and validated a non‐viral genetic modification platform based on Sleeping Beauty (SB) transposons delivered with minicircles to express CD19‐28z.CAR and CRISPR/Cas9 ribonucleoparticles to inactivate allogeneic TCRs. Compared to other conventional transposon systems using plasmid/mRNA‐based site‐specific nucleases for genetic ablation, our current platform enabled relatively more efficient TCR gene disruption with minimal cytotoxicity and with attainment of robust and stable CD19‐28z.CAR expression (∼88% CAR+/TCRneg T cells following 2‐week expansion/early CAR enrichment). We demonstrated that CRISPR/Cas9 did not affect the SB transposon integration profile and was not associated with any selective clonal amplification of transposon‐engineered T cells. The CAR‐T cells were responsive to CD19+ tumor cells with antitumor activities that induced complete tumor remission in NALM6 tumor‐bearing mice while significantly reducing TCR alloreactivity and Graft‐versus‐Host‐Disease (GvHD) development. Single CAR signaling induced similar T cell signaling signatures in TCR‐disrupted CAR‐T cells and control CAR‐T cells. In contrast, TCR disruption inhibited T cell signaling/protein phosphorylation compared to the control CAR‐T cells during dual CAR/TCR signaling. This non‐viral SB transposon‐CRISPR/Cas9 combination strategy serves as an alternative with relatively robust efficiency for generating next‐generation CD19‐specific CAR‐T while reducing GvHD risk and easing potential manufacturing constraints intrinsic to viral vectors.
Therapeutic potential of CAR Treg cellular therapy for neurodegenerative diseases
1: Quell Therapeutics Ltd
Neuroinflammation is a common pathological hallmark of many neurodegenerative diseases such as Huntington's Disease, Amyotrophic Lateral Sclerosis and Multiple Sclerosis. In the absence of a cure, there is significant need for the development of therapies to slow disease progression, improve patient quality of life and extend survival. Regulatory T cells (Treg) have the ability to suppress inflammation, modulate local immune micro‐environments and play a key role in neuroinflammation. Changes in Treg phenotype and disease site infiltration are associated with the progression of many neurodegenerative diseases during which Tregs gradually lose their regulatory function, contributing to disease exacerbation. The prospect of stopping or even reversing this deterioration of immune tolerance sparked an interest in the development of Treg based therapies. Indeed, cell therapy using polyclonal Tregs yielded promising results in the treatment of inflammatory and neurodegenerative diseases. To increase Treg cell therapy efficacy, we genetically engineered human and murine Tregs to express a Chimeric Antigen Receptor (CAR) to induce their activation and expansion at the site of inflammation while avoiding unwanted side effects such as systemic immunosuppression. Here we present the development and validation of novel CARs against a unique target protein highly expressed in the CNS during neuroinflammation. We demonstrate that human and murine CAR Tregs respond in an antigen‐specific manner in vitro and further show efficacy in a model of neuroinflammation. Our expanding data illustrates the great potential to use our novel CAR Treg for the treatment of neurodegenerative diseases to improve immune regulation and control disease.
Base edited “universal” CAR T cells against childhood AML
1: UCL Institute of Child Health
Major hurdles in the development of efficacious and safe chimeric antigen receptor (CAR) T‐cell therapies for childhood acute myeloid leukaemia (AML) include disease heterogeneity and a lack of disease‐specific target antigens that discriminate healthy cells. The receptor CLEC12A/CLL‐1 exhibits upregulated expression on myeloid blasts and chemo‐resistant leukaemic stem cells (LSCs) but is usually absent on healthy haematopoietic stem cells (HSCs). Several CLL‐1 targeting CAR T cell therapies are under clinical development for childhood AML, although these are limited to the autologous setting where difficulties in obtaining high‐quality T‐cell starting material due to lymphopenia, and the risk of transducing and masking blast populations, remain. “Universal” allogeneic formulations were investigated alongside CAR T‐cells targeting the widely expressed AML antigen, CD33, in anticipation of combinatorial treatment approaches. The cytidine deaminase base‐editor BE3 was employed to disrupt genes encoding the ab T‐cell receptor to prevent graft‐versus‐host disease (GVHD), and a shared T‐cell antigen to prevent fratricide. Healthy‐donor T‐cells were transduced with lentiviral vector expressing CAR and a sgRNA cassette against TRBC1, operational in combination with transiently expressed BE3 mRNA. High levels of CAR expression (CLL‐1CAR 82.9%, CD33CAR 79.3%, n = 5) were achieved and end‐of‐process products were further enriched for CAR+TCRab ‐ cells (CLL‐1CAR 96.0%, CD33CAR 96.5%, n = 5) with minimal TCRab + cells remaining (CLL‐1CAR 0.5%, CD33CAR 0.5%, n = 5). In vitro, CLL‐1 and CD33 CAR T‐cells exhibited cytokine release and target antigen specific cell lysis in Cr51 release assays. Anti‐leukaemic comparisons in humanised xenograft murine models of AML are underway ahead of translational studies.
Multiplex base editing protects allogeneic solid tumor targeting CAR‐T cells from inhibition by extracellular adenosine in the tumor microenvironment
1: Northeastern University 2: Beam Therapeutics
Allogeneic, off‐the‐shelf Chimeric Antigen Receptor T (CAR‐T) cells promise to deliver effective cell therapies for the treatment of cancer and could overcome barriers of autologous CAR‐Ts limiting patient uptake and accessibility. However, efficacy of CAR‐T therapies for solid tumors has been hindered by the complexity of the tumor microenvironment (TME). Failure of current‐generation CAR‐T cells to reject solid tumors is due in part to the hostile, tumor‐supportive, immunosuppressive TME, characterized by hypoxia‐driven accumulation of extracellular adenosine. Signaling by adenosine through A2A adenosine receptors (A2AR) on T cells inhibits cytokine secretion and anti‐tumor cytotoxicity. To address this biochemical barrier, we leveraged base editing to generate solid tumor targeting
Investigation of chimeric FcγRIIIa receptor (cFcR) engineered ‘Universal’ T cells in combination with anti‐CD20 monoclonal antibody
1: UCL Institute of Child Health
‘Universal’ antibody‐mediated cell therapy (uACT) comprises T cells edited with CRISPR/Cas9 to enable use in an allogeneic setting via genomic disruption of TCR and HLA genes, and incorporates lentiviral transfer of a chimeric FcγRIIIa receptor (cFcR). The cFcR contains second generation chimeric antigen receptor (CAR) activation domains derived from CD3ζ and 41BB. In combination with existing therapeutic monoclonal antibodies (mAbs), uACT was investigated as a strategy for enhanced antibody‐directed cytotoxicity. Such an approach could have broad applicability with advantages over conventional CAR therapies, including reduced production costs if combined with existing mAbs and reduced risk of toxicity through mAb withdrawal. Healthy donor cells were transduced with a self‐inactivating lentiviral vector, designed to couple cFcR expression with simultaneous disruption of TCR and β2‐microglobulin (MHC‐I) expression, characterised by flow cytometry and molecular sequencing. Subsequent magnetic bead‐based enrichment provided a highly homogenous cFcR+ (>94%) TCR‐ (>99%) MHC‐I‐ (>99%) cell product. In functional assays, uACT specifically killed CD20+ Daudi lymphoma cells in combination with anti‐CD20 mAb but had minimal activity when combined with human immunoglobulin. The approach was further investigated in a humanised immunodeficient murine model, comparing the uACT/anti‐CD20 mAb combination with conventional CAR T cells expressing a CAR20 with identical scFv region, and sharing similar hinge and activation domains. In both cases, effectors displayed comparable levels of tumour inhibition by serial bioluminescence over a 5‐week period. Disease burden was examined at necroscopy with similar findings in both groups. uACT offers an off‐the‐shelf approach to improve and exploit established mAb therapies.
CD247 (CD3ζ) gene editing as a novel platform technology for off‐the‐shelf Chimeric Antigen Receptor reprogrammed immune cells
1: Berlin Institute of Health (BIH) at Charité ‐ Universitätsmedizin Berlin, BIH Center for Regenerative Therapies, Charitéplatz 1, 10117 Berlin, Germany 2: Berlin Center for Advanced Therapies (BeCAT), Charité ‐ Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt‐Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany) 3: Institute of Medical Immunology, Campus Virchow‐Klinikum, Charité ‐ Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt‐Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany) 4: Institute of Transfusion Medicine, Charité ‐ Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt‐Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany 5: Division of Medical Oncology, Department of Medicine, and Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA. 6: USC/CHLA Cell Therapy Program, University of Southern California, and Children's Hospital Los Angeles, Los Angeles, CA 90033, USA.
The adoptive transfer of chimeric antigen receptor (CAR)‐reprogrammed immune cells has become an important modality in anti‐cancer therapy and holds great promise for many other disease entities. However, personalized cell manufacturing and costly viruses for gene transfer are major roadblocks to accessible cell therapies. Recently developed virus‐free gene editing represents an alternative tool to engineer immune cells. It allows for site‐specific transgene integration, thereby reducing the risk of insertional mutagenesis. Here, we present virus‐free CAR‐reprogramming via integration of truncated CAR‐transgenes into the human CD247 (CD3ζ) gene as a novel strategy to abolish T cell mediated allo‐reactivity through functional replacement of the T cell receptor (TCR). In our approach, incomplete CAR‐transgenes with no main signaling domain of their own are fused in frame with the endogenous CD247 (CD3ζ) gene, leading to the assembly of complete, signaling‐competent CAR molecules via endogenous splicing while at the same time preventing TCR assembly. This strategy reduces CAR‐transgene size by 30% which decreases knock‐in toxicity and permits the parallel transfer of a second therapeutic transgene using bicistronic constructs. CD247‐CD19‐CAR redirected T cells display comparable in vivo leukemia control to virus‐transduced and TRAC‐replaced CD19‐CAR T cells in xenograft mouse models. In contrast to TRAC gene editing, CD247‐modification not only allows for CAR‐reprogramming of all T cell types, but also enables reprogramming of NK cells to enhance their cytotoxicity in a CAR‐dependent fashion without affecting missing‐self recognition. Our approach can serve as a universal virus‐free platform technology to redirect lymphocytes for allogeneic off‐the‐shelf applications.
Disruption of CD40L and GM‐CSF encoding loci in CAR T cells mitigates cytokine release syndrome without affecting their cytolytic activity
1: Institute for Transfusion Medicine and Gene Therapy & Center for Chronic Immunodeficiency, Medical Center – University of Freiburg, 79106 Freiburg, Germany
Chimeric antigen receptor (CAR) T cell therapy has shown promising results for hematological malignancies but was associated with undesirable side effects, including Cytokine Release Syndrome (CRS). CRS is triggered by CAR T cell‐based activation of monocytes, leading to massive release of proinflammatory cytokines. Monocyte activation is mediated inter alia by direct interaction with T cells via the CD40L–CD40R axis, or via uptake of GM‐CSF released from activated CAR T cells. While mouse models were able to predict CRS‐associated toxicity to some extent, we established a human cell‐based in vitro assay in accordance with the 3R principle to replace and reduce animal experiments. It involves the co‐culturing of leukemic B cells with CD19‐targeting CAR T cells and monocytes from the same donor. We show that upon exposure to leukemic B cells, the CD19‐CAR T cells upregulated CD40L and GM‐CSF expression, which in turn activated the monocytes to upregulate proinflammatory cytokines. We further demonstrate that a CRISPR‐Cas9 based ‘double hit’ strategy led to efficient disruption (∼80%) of the CD40L and/or GM‐CSF encoding loci in CAR T cells. Off‐target effects, including translocations between the two target sites, were determined by CAST‐Seq. Notably, edited CAR T cells maintained their antitumor activity towards leukemic B cells but led to significantly reduced cytokine secretion from the co‐cultured monocytes. We conclude that our CRS in vitro model allowed us to evaluate genome editing based mitigation strategies, including the assessment of the antitumor activity of gene‐edited CAR T cells as well as their propensity to trigger CRS.
HER2/neu‐specific CAR expression level in human T cells dictates antigen‐independent differentiation during in vitro expansion
1: Wagner Lab, Charité – Universitätsmedizin Berlin, BIH‐Center for Regenerative Therapies (BCRT), Berlin Institute of Health at Charité – Universitätsmedizin Berlin, Berlin, 13353, Germany 2: R&D, Berlin Center for Advanced Therapies (BeCAT), Charité – Universitätsmedizin Berlin, Berlin, 13353, Germany 3: LIT Leibniz Institute for Immunotherapy, Div. Genetic Immunotherapy, and University Regensburg, Regensburg, 93053, Germany
Chimeric antigen receptor (CAR) T cell therapy is successful in fighting haematological diseases, but it is less effective in solid tumours. The human epidermal growth factor receptor 2 (HER2/neu) has emerged as a suitable target antigen for CAR T cell therapy against several solid tumours such as breast cancer. Yet, one challenge is the limited persistence of the CAR T cells in vivo, potentially due to terminal differentiation and exhaustion. Using non‐viral CRISPR‐Cas9 gene editing, we generated multiple HER2/neu‐specific CAR T cells by exploiting different integration sites with endogenous or transgenic promoters. Expression of the second‐generation CAR from the endogenous TRAC promoter resulted in a surprisingly high CAR expression in primary human T cells. It exceeded the expression level driven by a strong EF1α‐LTR promoter from the AAVS1 locus. In contrast, transgene insertion into the CD247 locus resulted in a lower CAR expression. Those CD247.HER2/neu‐CAR T cells showed a more naïve‐like phenotype profile, expressed less canonical markers of exhaustion and were less prone to activation‐induced cell death upon CAR engagement compared to TRAC and AAVS1.HER2/neu‐CAR T cells. This supports the notion that the CAR expression level is a key driver for antigen‐independent differentiation in vitro. Analysis of the cytotoxicity of the different HER2/neu‐CAR T cells are currently ongoing. In particular, future in vivo experiments could inform on the persistence of the CD247.HER2/neu‐CAR T cells. Maintaining a naïve‐like phenotype during the manufacturing process of the CAR T cells may be superior for prolonged anti‐tumour activity.
Development of transposon‐based CAR‐T cells for the treatment of patients with lymphoma
1: Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas/Centro de Investigación Biomédica en Red de Enfermedades Raras (CIEMAT/CIBERER), Unidad Mixta de Terapias Avanzadas, IIS Fundación Jiménez Díaz, Madrid, Spain 2: Hematology and Cell Therapy Department, Clínica Universidad de Navarra, IdiSNA, Pamplona, Spain 3: Hemato‐Oncology Program, Cima Universidad de Navarra, IdiSNA, CIBERONC, Pamplona, Spain 4: Servicio de Hematología, Hospital Universitario de Navarra, IdiSNA, Pamplona, Spain 5: Hematology Department, University Hospital of Salamanca (HUS/IBSAL), CIBERONC and Center for Cancer Research‐IBMCC (USALCSIC), Salamanca, Spain 6: Division of Hematology and Hemotherapy, IIS Fundación Jiménez Díaz, Madrid, Spain 7: Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Department of Immunology and Cell Immunotherapy Platform HSJD‐HCB. UB, Hospital Clínic de Barcelona, Barcelona, Spain 8: Deparment of Hematology, Stem Cell Transplant and Cell Therapy Unit, IMIB‐Arrixaca, Virgen de la Arrixaca University Hospital, University of Murcia, Murcia, Spain 9: Transposition and Genome Engineering, Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany 10: Immunology and Inmunotherapy Department, Clínica Universidad de Navarra, Pamplona, Spain
TranspoCART is a project conceived to develop an innovative CAR‐T cell therapy for lymphoma patients based on a viral vector‐free gene‐transfer strategy. A Sleeping Beauty transposon minicircle (SBmC) carrying a CAR consisting of the 4‐1BB CAR endodomain, an anti‐CD19 ScFv and a truncated EGFR ‐as a safety switch‐ was designed for this clinical trial. Our optimized manufacturing protocol showed transposition efficacies ranging between 30 and 60% in both healthy donors and patients' T cells, with efficient cell expansion rates (between 29.6x and 31.8x), compatible with clinical applications. Extensive in vitro characterization of TranspoCART19 cells showed an enriched stem‐cell memory/central memory phenotype, no signs of cell exhaustion and high level of specific cytotoxicity against tumour cell lines and primary tumour cells expressing CD19. No detectable levels of both the SBmC and the transposase protein were found at the end of the expansion period. NALM6 xenograft models using immunodeficient NSG mice were used to evaluate the in vivo antitumoral efficacy of TranspoCART19 cells. Treated animals showed an improved survival compared to untreated mice, similar to the one observed in mice treated with lentivirally‐produced CAR‐T cells. Biodistribution and toxicity studies revealed that TranspoCART19 cells administration is safe. Additionally, cetuximab administration in CAR‐T cell treated NALM6 xenograft mice depleted TranspoCART19 cells, proving its efficacy. Finally, two independent GMP facilities carried out TranspoCART19 cell manufacturing GMP validations, and showed that the manufacturing procedure is highly efficient and reproducible. These encouraging results support the initiation of a clinical trial with the TransproCART19 cell product.
Adapter CAR™ T cells produced on the CliniMACS Prodigy™ platform show potent and controllable anti‐tumour activity in different human leukaemia xenograft mouse models
1: Miltenyi Biotec B.V.
Chimeric antigen receptor (CAR) T cell therapy for haematopoietic cancers has achieved great results in the clinic. However, FDA‐approved therapies are still limited to B‐cell malignancies. Targeting other types of haematopoietic cancers such as acute myeloid leukaemia (AML) has shown to be more difficult requiring a more versatile toolbox to overcome potential issues with safety and efficacy. With the Adapter CAR™ (AdCAR) system we have developed a versatile, flexible, redirectable, tuneable, and switchable platform using biotin‐labelled adapter molecules to crosslink the AdCAR T cells to target cells. Here we study the functionality of AdCAR T cells manufactured using our fully closed and automated CliniMACS Prodigy™ system. AdCAR T cells generated on this platform showed efficient tumour cell lysis in vitro as well as effectivity against different types of haematopoietic cancers including AML and Non‐Hodgkin lymphoma in xenograft NSG mouse models. Importantly, AdCAR‐mediated anti‐tumour responses were strictly adapter‐dependent and could be modulated by the adapter dose, adapter injection interval, and variations in adapter design, offering multiple ways to fine‐tune AdCAR T cell activity. In conclusion, our data demonstrates that highly functional AdCAR T cells can be manufactured using the CliniMACS Prodigy platform, offering the potential for an improved control and safety of CAR T cells in vivo.
CAR‐NK cell approaches targeting HER1 and CD44v6 in HNSCC
1: Hannover Medical School 2:
Head and neck squamous cell carcinoma (HNSCC) arises from the epithelium of the upper aerodigestive tract and is the sixth most common type of cancer worldwide. The global incidence of HNSCC is approximately 600,000 new cases annually, which will increase further, according to the Global Cancer Observatory. Patients suffering from HNSCC have a 40% 5‐year survival rate, highlighting the need for new therapeutic strategies. To exploit the inherent immunosurveillance and killing activity of natural killer (NK) cells paired with their potential as off‐the‐shelf products, we used chimeric antigen receptors (CAR) to redirect NK cell cytotoxicity by targeting tumor‐associated antigen HER1. Screening of third‐generation anti‐HER1 CARs led to the selection of two candidates based on stable CAR expression and lack of tonic signaling. Furthermore, anti‐HER1 CAR‐NK‐92 cells effectively killed HNSCC cell lines SCC‐4 and SCC‐25 at effector‐target (E:T) ratios (5:1, 10:1). Next, we characterized primary HNSCC (pHNSCC) samples and found high percentages of CD45+ immune cells, including T cells, B cells, NK cells and macrophages. To prevent contamination with fibroblasts and immune cells, an ex vivo expansion protocol that includes sorting for epithelial marker EpCAM+ cells was established. Upon coculture of pHNSCC with our CAR‐NK cells, strong killing was evident at 0.5:1 E:T ratio in our flow cytometry‐based cytotoxicity assay. Analysis of the remaining pHNSCC after CAR‐NK coculture showed loss of HER1+ and HER1+CD44v6+ populations with an expansion of CD44v6+ populations, indicating the need for dual‐targeted CAR‐NK therapies that can be combined with chemo‐ and radiotherapy.
Potent, CRISPR‐engineered off‐the‐shelf CAR‐T and TCR‐T cells evading host T and NK cell rejection
Y Zhang1 A Prodeus1 U Yetley1 Y Tan1 U Ranade1 I Balwani1 D Liu1 I Miller1 B Zhang1 I Dutta1 P Sharma1 S Goel1 V Malik1 P Venkatesan1 M Keenan1 M Lam1 S Arredouani1
1: Intellia Therapeutics, Inc
Despite the clinical success of autologous CAR‐T cells, manufacturing challenges and the high cost of individualized production make “off‐the‐shelf” therapies a desirable alternative approach. Graft‐versus‐host disease (GvHD) with allogeneic T cells has largely been solved by efficient knockout (KO) of the endogenous T cell receptor (TCR). However, both immune rejection via mismatched HLA alleles and NK cell‐mediated rejection due to loss of the ‘missing self’ signal (HLA‐I KO) remain a challenge. We developed a differentiated allogeneic strategy, leveraging our proprietary sequential, LNP‐based CRISPR/Cas9 editing platform to abrogate expression of HLA‐A (cIass I) and all HLA class II alleles (CIITA KO), insert a CAR or TCR into the TRAC locus, and match HLA‐B and HLA‐C alleles using HLA‐B/C homozygous donors (2/2 match). T cells with >99% TCR KO did not induce GvHD in vivo over a 90‐day nonclinical study. HLA‐A/CIITA KO T cells from HLA‐B and HLA‐C matched allogeneic donors were not recognized by host CD4+ or CD8+ T cells and were protected from host NK cells, both in vitro and an in vivo animal model engrafted with human NK cells. Allogeneic T cells retained their viability, cell expansion, and in vitro and in vivo functional characteristics. This approach was tested in the context of a CD30 CAR‐T as well as a WT1‐specific TCR‐T. It is projected that a range of 3‐20 product types with different HLA‐B/C combinations will cover 40‐90% of the population.
Generation of CD19‐specific CAR NK cells using virus‐free CRISPR‐Cas9 gene editing
1: Wagner Lab, BIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health at Charité – Universitätsmedizin Berlin, 13353 Berlin, Germany 2: Research and Development unit, Berlin Center for Advanced Therapies (BeCAT), Charité – Universitätsmedizin Berlin, 13353 Berlin, Germany 3: Berlin‐Brandenburg School for Regenerative Therapies (BSRT), Charité – Universitätsmedizin Berlin, 13353 Berlin, Germany 4: Berliner Hochschule für Technik (BHT), 13353 Berlin, Germany 5: Institute of Biotechnology, Technische Universität Berlin, 10623 Berlin, Germany
Natural killer (NK) cells are innate lymphoid cells, which are important in the control of viruses and elimination of damaged or malignant cells. Previous clinical studies have demonstrated that the anti‐tumor efficacy of NK cells can be enhanced by transduction with tumor‐specific chimeric antigen receptors (CAR). To date, most clinical studies with CAR NK cells use retroviral transduction for CAR gene transfer, however, retroviruses are associated with costs and the risk of insertional mutagenesis. To overcome these challenges, we established a method to equip NK92 cells with CAR via non‐viral CRISPR‐Cas9 gene editing. We integrated an anti‐CD19.CD28.zeta CAR into the AAVS1 and the CD247 gene in the NK92 cells. Both gene‐edited CAR NK92 cells lysed CD19+ Nalm‐6 cells significantly more efficiently than unmodified NK92 cells and the CD247.CAR NK92 cells showed also a significant higher CAR‐mediated cytotoxicity against the Nalm6 cells than the AAVS1.CAR NK92 cells in all tested effector cell to target cell ratios (p = 0,001, two‐way ANOVA and Tukey test, n = 12). Furthermore, CD247‐edited CAR NK92 cells demonstrated higher cytotoxicity towards CD19‐negative K562 cells than AAVS1.CAR NK92 cells, indicating intact missing self‐induced activation. These results highlight that the endogenous CD247 gene may be an integration site to create highly functional CAR‐NK cells with gene editing. Currently, we are optimizing the virus‐free gene editing in healthy donor‐derived NK cells to validate the findings from NK92 cell line in primary cells. In preliminary experiments, we achieved up to 10% CAR knock‐in in ex vivo expanded peripheral blood derived NK cells.
Nanobody‐based CD38‐specific CARs and BiKEs sent into race against multiple myeloma
1: University Medical Center Hamburg Eppendorf
While chimeric antigen receptors (CARs) and bispecific T cell engagers (BiTEs) are already well established in cancer therapy, CAR‐expressing NK cells and bispecific killer cell engagers (BiKEs) are also quickly gaining importance. CD38 is a highly effective target for immunotherapy of multiple myeloma. Llama‐derived CD38‐specific nanobodies allow easy reformatting into mono‐, bi‐, and multispecific proteins. We have generated nanobody‐based CD38‐specific CAR NK cells (nano‐CAR NK cells) and half‐life extended BiKEs (HLE‐nano‐BiKEs). To construct nano‐CAR NK cells, we fused a CD38‐specific nanobody to a classical CAR backbone and expressed the nano‐CAR on NK92 cells via retroviral transduction. We generated nano‐BiKEs by fusing a CD38‐specific nanobody to a CD16‐specific nanobody to mediate engagement of NK cells (via the Fc‐receptor CD16 on NK cells). We fused this nanobody dimer to a third, albumin‐specific, nanobody to extend the half‐life of this construct in vivo. Both the CARs and BiKEs mediated specific dose‐ and time‐dependent cytotoxicity against CD38‐expressing cell lines and CD38‐expressing myeloma cells in primary human bone marrow samples. Even at the lowest effector‐to‐target ratio tested (1:1), both CD38‐specific nano‐CARs, as well as HLE‐nano‐BiKE‐engaged NK cells, mediated more than 50% lysis of CD38+ myeloma cells in vitro. In conclusion, nano‐CAR NK cells and HLE‐nano‐BiKEs may be a promising new technology for multiple myeloma therapy and should be investigated in further clinical trials.
Supported by DFG grants No310/16 and Ba5893/7
Virus‐free production of CD20‐targeted CAR‐T cells via CRISPR‐Cas9‐mediated transgene insertion
1: UCL Institute of Child Health
Integrating gamma‐retroviral and lentiviral vectors have been widely applied in the manufacture of CAR‐T cell therapies, but supply has been a major bottleneck and expense for these therapies. Furthermore, viral vectors generally employ constitutive promoters and integration variegation effects can lead to clonal dominance. Alternatively, CRISPR‐Cas9 mediated knock‐in of a CAR transgene into the TRAC locus of TCRab T‐cells has been shown to simultaneously enable CAR expression and disrupt endogenous TCRab expression. Transcriptional activity mediated through the endogenous TRAC promoter machinery may favour better regulated CAR‐T cell function. In the autologous setting, reduced TCR‐mediated competition may improve CAR T cell longevity. In the context of allogeneic T cells, disruption of TCRab mitigates against GVHD.
A virus‐free manufacturing protocol for anti‐CD20 directed CAR‐T cells (TRAC‐CAR20 cells) was developed using SpyCas9 ribonucleoproteins (RNPs) with an sgRNA targeting the TRAC locus and a dsDNA donor template encoding a self‐cleaving CAR20 transgene cassette and TRAC homology arms. Following activation and electroporation, primary T‐cells yielded a mean of 41% CAR20 expression (range 19.4‐60.4%, n = 5) and TCR expression was disrupted by 89.9‐98.7% (n = 5). Removal of residual TCRab T cells was achievable by magnetic bead column depletion. Molecular analysis confirmed appropriate site‐specific integration and disruption of TCR expression. In vitro TRAC‐CAR20 cells exhibited 51Cr release cytotoxicity against CD20+ Daudi B cells comparable to lentiviral CAR20‐T cells.
Overall, CRISPR‐Cas9 mediated CAR20 integration using dsDNA donor homology‐flanked templates offers a route to virus‐free cell therapy, including in a universal ‘off‐the‐shelf’ formulation.
A new synthetic circuit for B cell reprogramming to cure cancer
1: CIRI; Inserm U1111 2: Ecole Normale Supérieure de Lyon 3: CNRS, UMR5308 4: Université Claude Bernard Lyon 1
The expansion of techniques for genetic engineering has brought a new dimension for synthetic immunology. Immune cells are perfect candidates for such approaches because of their ability to patrol, to interact with many cell types, to proliferate upon activation and differentiate in memory cells. In particular, for cancer cure, they can be modified to eradicate tumor cells while counterbalancing the immunosuppressive tumor microenvironment.
The goal of our study is to implement a new synthetic circuit in B cells by lentiviral vector transduction, allowing the expression of therapeutic molecules in a temporally and spatially restricted manner controlled by the presence of tumor antigens. These molecules will increase inflammation locally and promote the differentiation of effector immune cells, thereby mediating tumor clearance.
We developed a synthetic circuit encoding a “sensor” (a membrane‐anchored B‐cell receptor, BCR, targeting a tumor antigen), a “transducer” (a promoter that can be induced by activated BCR) and “effector” molecules (Interleukine 18). We isolated a 734 bp‐long fragment of the NR4A1 promoter, which is specifically activated by the BCR signaling cascade in a fully reversible manner. By co‐delivery of lentiviral vectors encoding respectively the transducer/effector and the sensor targeting ovalbumine, we achieved antigen‐specific induction of the circuit. Indeed, the recognition of ovalbumine on the ectopic sensor specifically induced the activation of the NR4A1 promoter and the expression of the effector, demonstrating the circuit functionality in vitro. We are currently evaluating the ability of reprogrammed B cells to mediate tumor clearance in immunodeficient mice bearing human melanomas.
Dasatinib potently enhances transduction by CD3‐targeted lentiviral vectors and counteracts CAR T cell exhaustion
1: German Cancer Consortium (DKTK) 2: Paul‐Ehrlich‐Institut
CD3‐targeted lentiviral vectors (LVs) mediate selective targeting of human T lymphocytes in vitro as well as in vivo while simultaneously activating the targeted cells. Cross‐linking of CD3:TCR induces CD3 phosphorylation by the Src kinase LCK, leading to T cell activation and downregulation of CD3:TCR. Previously, we have demonstrated that CD3‐LV, due to its agonistic nature, leads to downmodulation of the CD3:TCR complex. We therefore hypothesized that inhibition of CD3 phosphorylation by Src/Abl tyrosine kinase inhibitors such as dasatinib results in enhancement of gene delivery by T cell‐targeted LVs.
Remarkably, dasatinib treatment of T cells prior to incubation with CD3‐LV increased reporter gene delivery by 3‐ to 10‐fold, when applied at the optimal concentration of 50 nM. Moreover, the presence of dasatinib enhanced selective transduction into non‐activated target cells present in whole blood. When combined with the delivery of the CD19‐CAR gene, dasatinib increased CAR T cell numbers by 4‐ to 9‐fold. Importantly, dasatinib‐mediated inhibition of T cell activation during CD3‐LV transduction did not interfere with the killing activity of the resulting CAR T cells but instead led to less exhausted CAR T cells with a more naïve phenotype even upon killing.
Our data suggest that dasatinib prevents CD3‐LV‐induced phosphorylation and CD3:TCR intake, increasing the amount of CD3‐LV bound to the cell surface. This allows efficient CD3‐LV cell entry, resulting in substantially increased transduction efficiencies and refined CAR T cells. This is the first description of dasatinib as transduction enhancer, an activity particularly relevant for CAR T cell generation with CD3‐LV.
Identification of efficacious CARs by in vitro screening using a reporter cell line and primary T cells
L Kochsiek1 N Werchau1 M Assenmacher1 T Schaser1
1: Miltenyi Biotec B.V.
Redirection of T cells by chimeric antigen receptor (CAR) expression to target cell surface‐expressed tumour antigens has developed into promising options to treat patients suffering from cancer. Strategies to select effective and safe CAR molecules towards alternative or novel tumour cell‐surface antigens require lead CAR candidate identification from numerous CAR constructs in in vitro head‐to‐head comparisons. However, isolation and expansion of primary T cells for CAR assessment is labour‐ and time‐intense.
Here, we devise an attempt to identify functional CAR molecules over non‐functional constructs in vitro by employing co‐cultures with target cells expressing the relevant target epitope. CAR‐transduced J76‐based reporter cells were analysed for GFP expression in dependency of CAR functionality by NFAT/AP‐1‐mediated GFP expression after 1‐day co‐culture. Additionally, CAR‐expressing primary T cells were assessed in repeated co‐cultures using the same target cell line.
T cells expressing effective CARs were identified by serial target‐cell killing in all three rounds and could be distinguished from CAR T cells that showed cytotoxicity in one or two rounds, or complete absence of functionality. The ability to kill was associated with cytokine production and T‐cell expansion in primary T cells. Most functional CARs could be identified by induced GFP expression in the reporter cell line.
Our results emphasize the relevance of thorough in vitro screening of CAR molecules to identify lead candidates, and reporter cell lines may help reduce the number of CAR candidates funnelled into assessments in primary T cells.
Titratable and reversible control of CAR‐T cell receptor and activity by riboswitch via oral small molecule
1: 1. Gene Regulation, MeiraGTx, New York, NY 10016, USA
Chimeric antigen receptor (CAR)‐T cell therapy is a promising therapy against cancer. However, uncontrolled CAR expression, CAR‐T expansion and activity causes severe CAR‐T cell‐associated toxicity and CAR‐T cell exhaustion, limiting the success of this living drug. Here, we present Ribo CAR, a mammalian synthetic riboswitch‐based gene regulation system for regulating CAR expression via small molecule inducer. Unlike previously reported regulatable CAR platforms that utilize viral protease or chemical induced protein dimerization, Ribo CAR contains an ON aptamer riboswitch in the coding sequence of a CAR transgene, in which, the aptamer functions as a sensor for small molecule. Upon binding with a small molecule ligand, the aptamer RNA adopts a conformation change that triggers splicing, resulting in the expression of intact functional CAR molecule. The CAR gene with the riboswitch only expresses CAR protein in the presence of the small molecule inducer. Further, CAR expression is titratable by titrating the dose of the small molecule inducer. With an optimal dose of small molecule, the induced level of CAR can reach the level of constitutively expressed CAR. Consistent with small molecule induced expression of the CAR, CAR triggered‐activation of CAR‐T cells and the production of IL‐2 and INFγ are also controlled by small molecule inducer in a dose dependent manner. With a bioavailable small molecule inducer, CAR‐T activity can be precisely tuned and “remotely” controlled in vivo, both temporally and spatially, thus providing a safety mechanism for CAR‐T cell therapy as well as the ability to finely modulate CAR‐T activity over time.
CRISPR‐mediated targeting of IL15 to PD1 locus improves CD19‐CAR‐T Cell phenotype
1: GENyO‐ Centro de Genomica e Investigacion Oncologica: Pfizer / Universidad de Granada / Junta de Andalucia 2: GC 14 Cell Therapy, IMIBIC. University of Cordoba, Reina Sofia University Hospital, Córdoba, 14004, Spain. 3: Hematology Unit, Reina Sofia Hospital, Cordoba, Cordoba, 14004, Spain. 4: Lentistem Granada, 14016, Spain. 5: Hospital Clinic, Barcelona, 08036, Spain
Adoptive Cell therapy (ACT) is an emerging field that shows promise for refractory leukemia or lymphoma patients. Among these, genetically engineered T cells expressing Chimeric Antigen Receptors (CARs), have highlighted as a successful therapy for type B malignancies. Although CAR‐T cell therapies hold great promise, still face multiple challenges, including toxicity, inactivation by the tumour microenvironment and low persistence in patients. In the present study, we explore the application of genome editing (GE) approach based on CRISPR/Cas9 to repurpose TCR and PD‐1 loci, two major players of the T cell activation pathway.
Our final aim is to engineer the TRAC locus to avoid Graft versus host disease (GVHD) (generating universal CAR‐T cells) and the PD‐1 locus expressing IL‐15 (generating CAR‐T resistant to PD‐L1‐mediated immunosuppression) under PD‐1 endogenous promoter. For proof‐of‐principle, we first designed a donor DNA to insert IL‐15 into PD‐1 locus. First experiments of Knock‐In were carried out with PCR product us donor delivery of IL‐15. Finally, we used AAV6 as donor delivery tool and RNP nucleofection with CRISPR/Cas9 to generate CD19‐CART DPD‐1_IL‐15 final product. Our preliminary results point to an attenuated expression of exhaustion markers and increase in the proliferative capacity of edited cells. In addition, edited cells exhibited a reduction in Pro‐Apoptotic proteins like BIM, and an increase of Anti‐Apoptotic proteins like Bcl‐2 and Bcl‐xL.
Our strategy shows that the expression of IL15, under the PD1 transcriptional machinery improve the CD19‐CART general fitness which should lead to improved therapeutic efficacy.
Adapter CAR™ T cells specific to a mutated human peptide tag demonstrate efficacy in preclinical models
1: Miltenyi Biotec B.V. & Co. KG
During the past years, chimeric antigen receptor (CAR) T cell therapy has become a powerful therapeutic strategy to treat cancer patients suffering from hematologic tumours. But despite its success, CAR T cell therapy still has major limitations limiting its therapeutic efficacy like tumour antigen escape, and can be associated with life‐threatening toxicities such as cytokine release syndrome. To overcome these hurdles, we developed an Adapter CAR (AdCAR) system consisting of CAR T cells recognizing a short peptide tag extracted from a human protein frequently mutated in cancer patients. AdCARs were selected to bind specifically to the mutated peptide to prevent activity against the human wild‐type protein. The short peptide tag can be readily fused to a variety of biological molecules that bind tumour antigens, which makes the system easily applicable to various indications. Here we present preclinical data demonstrating the anti‐tumour efficacy of AdCAR T cells in CD20+ Non‐Hodgkin lymphoma and CD33+ acute myeloid leukaemia models in vitro as well as in mouse models. Importantly, the AdCAR T cells showed high specificity to the tag and no cross‐reactivity to the human wild‐type protein. By titrating the adapter, the anti‐tumour response of the AdCAR T cells could be fine‐tuned or switched off completely, which could become important to prevent severe toxicities. This work demonstrates the utility of an AdCAR system using short peptide tags derived from mutated human proteins, and its potential to improve safety and controllability of CAR T cell therapy.
Comparison of cytidine deaminase base editors for multiplexed editing of T cells
1: UCL Institute of Child Health
Genome‐editing has been employed to remove T cell receptors, HLA and other molecules required for generation of ‘universal’ CAR T cells, or to confer fratricide resistance when targeting shared T lineage antigens. Studies have described multiplexed edited CAR T cells generated using CRISPR/Cas9 knockout or cytidine deaminase base‐editors (CBE) where conversion of C•G>T•A nucleotides introduces premature stop codons or disrupts splice sites. Here we undertake comparisons of humanised CBE versions with original rat APOBEC1 iterations.
Transient delivery of guide RNA and CBE mRNA by electroporation was compared for rat APOBEC1 (BE3), human activation‐induced cytidine deaminase (hAID‐BE) and human APOBEC3A (hA3A‐BE). Serial Western blot detected transient expression of each editor at 16‐96 hours after electroporation. A window of deaminase activity was anticipated at cytidine (C) positions within an editor‐specific window set within a defined range from the target PAM sequence.
Editing of both TRBC and CD7 loci in primary T cells exhibited efficient disruption; TCRαβ negative cells: (BE3 90‐94%, hAID‐BE 88‐90%, and hA3A‐BE 95‐97%) vs unedited control (7‐8%), and CD7 negative cells: (BE3 66‐78%, hAID‐BE 36‐62%, and hA3A‐BE 86‐94%) vs unedited control (4‐4.3%) by flow cytometry (n = 4). Sanger sequencing confirmed C > T conversions at ‘on‐target’ sites for all editors, with hA3A‐BE yielding the highest levels of editing at both TRBC (C5:65%, C6:75%) and CD7 (C8:98%). However, this editor was also associated with high levels of wider C > T conversion outside the predicted editing window (TRBC: 71%C3, and CD7: 74%C3), highlighting both efficiency and fidelity of editing as considerations for downstream applications.
HSC‐CAR Treg gene therapy as a treatment for severe chronic autoimmune disorders through delivery of stable antigen‐specific suppression of autoimmunity
1: Orchard Therapeutics Europe Ltd
Haematopoietic stem cell (HSC) gene therapy offers the potential to provide stable and targeted immunotherapy, through the ability of HSCs to differentiate into T regulatory cells (Treg) engineered to express chimeric antigen‐specific receptors (CAR). This approach combines the proven durability of HSC GT with the specific suppressive activity of CAR Tregs, providing a potential alternative to current treatments which fail to effectively control chronic autoimmune diseases such as Multiple Sclerosis (MS). MS is challenging to treat due to its complex immune pathology, requiring regulation of multiple immune effector subsets with evolving autoreactivity, and suppression of neuroinflammation. Autologous HSC transplantation has shown efficacy for some clinical forms of MS, by providing an ‘Immune Reset’ function, but does not address the autoimmune degenerative component of progressive MS in these patients. Several studies have successfully applied adoptive transfer of CAR‐ or TCR‐ antigen specific Tregs to prevent MS in murine disease models, however it is unknown whether these approaches translate to sustained suppression of chronic autoimmunity.
Here we describe our HSC‐CAR Treg GT approach, using lentiviral vector constructs designed to transduce HSC and allow expression of a CAR cassette under the control of Treg specific regulatory elements. Using both in vitro and in vivo methods, we demonstrate the generation of Tregs armed with CAR and evaluate their antigen‐specific suppressive activity in primary mouse and human cells. Realisation of the potential of HSC‐CAR Treg GT for immunomodulatory therapies presents an opportunity to provide lifelong treatments for a broad spectrum of severe chronic autoimmune disorders.
Generation of universal CAR T cells with a memory phenotype: safety and efficacy studies
1: GENyO‐ Centro de Genomica e Investigacion Oncologica: Pfizer / Universidad de Granada / Junta de Andalucia 2: Maimonides Biomedical Research Institute of Cordoba (IMIBIC) 3: Hospital Reina Sofía (Cordoba) 4: Center for non‐coding RNA in Technology and Health, Department of Veterinary and Animal Sciences, University of Copenhagen, Thorvaldsensvej 57, 1871 Frederiksberg, Denmark.
Engineered T cells therapies such as chimeric antigen receptor (CAR) expressing T cells (CAR T cells) have showed a great potential to treat many human diseases and display promising results specially against B cell malignancies. However, these treatments are based on autologous T cells, which increases the cost and reduces the efficacy of the therapy. It is also known that efficacy of these therapies is directly correlated with the phenotype of the infused cells.
In order to reduce these effects, we have generated functional universal antiCD19 CAR T cells with a defined memory phenotype. We have eliminated the B2M and TRAC genes in CAR T cells to avoid both graft versus host and host versus graft reactions and selected the less differentiated T cells (CD45RA+/CD45RO‐) to obtain more stable and persistent universal CAR T cells. We evaluated the safety of our strategy, by analyzing different genotoxicities that genome editing could cause.
Our data show efficient generation of universal off‐the‐shelf CAR T cells by the elimination of TCR and HLA‐I from the surface of T cells (THKO‐CAR‐T cells). This procedure reduces the bidirectional allogeneic response between cells from different donors. Edition of THKO‐CAR‐T cells persists over time and does not affect neither phenotype of cells nor CAR expression. Universal CAR T cells with a memory phenotype proved to be equally lytic as regular CAR carrying cells, and besides presented a fitter phenotype over time in co‐culture with tumoral cells.
Pin‐pointTM: A versatile editing platform driving cell therapies
1: Horizon, a PerkinElmer Company
Horizon's modular Pin‐point base editing system efficiently and precisely converts target nucleotides in the genome by recruiting DNA modifying deaminases via an aptamer encoded in the guide RNA (gRNA) of the sequence‐targeting Cas component. We optimized design and delivery conditions of synthetic gRNAs and mRNA to apply base editing to therapeutically relevant cell types including T cells, induced pluripotent stem cells (iPSC) and hematopoietic stem cells (HSC). In T cells, we applied multiple base editing to the development of engineered CAR‐T cells. We targeted a set of therapeutically relevant genes (B2M, CD52, TRAC and PDCD1) using APOBEC1 combined with Cas9 nickase and achieved greater than 70% knockout efficiency and high purity at all sites simultaneously. Engineered CAR‐T cells retain their proliferative and cytotoxic activity in vitro, and, compared to Cas9, multiplex gene knockout with Pin‐point is associated with less gRNA‐dependent off‐target editing and negligible occurrence of chromosomal translocations. We additionally demonstrate that Pin‐point technology enables robust simultaneous targeted knock‐in and multiplex knockout in individual T cells. We also applied the Pin‐point system to gene knockout in iPSCs demonstrating that edited cells maintain pluripotency and normal differentiation potential. The Pin‐point platform also allows for simultaneous knockout of multiple gene targets in iPSCs with significantly enhanced cell viability and efficiency compared to Cas9. The ability to perform complex genome editing in multiple cell types safely, efficiently, and precisely opens the door to the application of the Pin‐point system in a range of advanced cell therapies.
Identification of molecular mechanisms governing CAR‐T cell response in MM patients using single cell transcriptomics
1: Hemato‐Oncology Program. Cima Universidad de Navarra. IdiSNA. Pamplona, Spain. 2: Computational Biology Program. Cima Universidad de Navarra. IdiSNA. Pamplona, Spain. 3: Flow Cytometry Core. Cima Universidad de Navarra. IdiSNA. Pamplona, Spain. 4: Immunology and Immunotherapy Program. Cima Universidad de Navarra. IdiSNA. Pamplona, Spain. 5: Department of Hematology, IMIB‐Virgen de la Arrixaca University Hospital. University of Murcia. Murcia, Spain. 6: Hematology Department, IBSAL‐University Hospital of Salamanca. University of Salamanca. Salamanca, Spain. 7: Department of Hematology. Hospital Clinic de Barcelona. IDIBAPS. Universidad de Barcelona. Barcelona, Spain. 8: Department of Immunology. Hospital Clinic de Barcelona. IDIBAPS. Universidad de Barcelona. Barcelona, Spain. 9: Instituto de Investigación Sanitaria‐Fundacion Jimenez Diaz. Madrid, Spain. 10: Department of Hematology, University Hospital Virgen del Rocio‐IBIS. Universidad de Sevilla. Sevilla, Spain. 11: Hematology and Cell Therapy Department. Clinica Universidad de Navarra, IdiSNA. Pamplona, Spain. 12: Immunology and Immunotherapy Department. Clinica Universidad de Navarra. Pamplona, Spain. 13: Centro de Investigacion Biomedica en Red de Cancer (CIBERONC). Madrid, Spain. 14: Data Science and Artificial Intelligence Institute (DATAI). Universidad de Navarra. Pamplona, Spain. 15: Red RICORS TERAV. Madrid, Spain
Therapeutic strategies based on CAR‐T cells represent a promising advanced therapy for relapsed/refractory (R/R) multiple myeloma (MM) patients. Nevertheless, despite the high remission rates observed with BCMA CAR‐T cells, not every patient achieves a complete response, and a significant number of patients still relapse. The application of single‐cell technologies to study CAR‐T cells has helped to shed light on important aspects of their expansion and evolution after administration. In this work, we used single‐cell RNA sequencing (scRNA‐seq) coupled with SimiC, a machine learning algorithm that infers gene regulatory networks (GRNs) from scRNA‐seq data, to interrogate CAR‐T cells isolated from bone marrow (BM) and peripheral blood (PB) of MM patients at different times after treatment. We found that most of the CAR‐T cells remaining after infusion were CD8+ cells with effector/effector memory phenotype. Interestingly, CAR‐T cells infiltrating BM presented increased cytotoxic and exhausted features, compared to their PB counterparts.Moreover, combination of SimiC and trajectory analysis allowed the identification of key TFs regulating these differences and their dynamics. Further analysis of TCR repertoire using single‐cell TCR sequencing (scTCR‐seq), integrated with scRNA‐seq, allowed the identification of a hyperexpanded CAR‐T cell clone in the BM of a relapsed patient. Deeper characterization showed that this clone presented increased IL10 expression and a specific transcriptional profile with immunosuppressive features. In summary, our results show that multiomic analysis are useful tools not only to characterize CAR‐T cells and their dynamics after infusion, but also to identify possible mechanisms of relapse that are currently being validated.
Selective transduction of T cell subsets using adapter based lentiviral vectors
1: Miltenyi Biotec 2: Institute for Transfusion Medicine and Gene Therapy, Medical Center ‐ University of Freiburg 3: Faculty of Medicine, University of Freiburg
The success of chimeric antigen receptor (CAR) expressing T cells to treat B cell malignancies is limited by a considerable number of relapses. CAR T cells from defined T cell subsets may provide superior potency overcoming this limitation. To study synergistic effects of T cell subsets, e.g. CD4+/CD8+ T cells, state‐of‐the‐art methods require enrichment, separate genetic modification and subsequent mixing of the cell products. In contrast, targeted lentiviral vectors (LV) have been developed to generate CAR T cells with varying CD4/CD8‐ratios directly from PBMC by mixing of LVs.
We previously established a universally targetable LV system (Adapter‐LV) that binds an artificial tag present on adapter molecules that in turn are specific for the desired antigen. Adapter‐LVs were only functional in presence of tagged adapter molecules. By applying adapter mixtures simultaneous transduction of CD4+ and CD8+ T cells was achieved using one LV only.
Here, we used the Adapter‐LV to generate CD20‐CAR T cells with varying CD4/CD8‐ratios in a single transduction step. Using a co‐culture assay, these CAR T cells were analyzed for differences in cytolytic activity, cytokine secretion and exhaustion/activation markers. Comparable cytolytic activities were observed independently of the CD4/CD8‐ratio of the CAR T product. In contrast, cytokine secretion and exhaustion/activation marker expression varied between CD4+ and CD8+ CAR T cells but also between different CAR T products.
In summary, the Adapter‐LV technology is easily used to adjust the composition cell products enabling the evaluation of synergistic effects of cellular subsets for various applications without additional isolation steps.
Developing a high order multiplex ddPCR assay for single cell vector copy number assessment of transduced T‐cells
1: Cell Therapy Catapult
Lentiviral vectors (LVs) are widely used for the generation of gene‐modified cell therapies with their main applications in immuno‐oncology and rare diseases. Yet, the variability of the transduction process may affect the consistency of the manufacturing process causing product heterogeneity. Therefore, controlling the integration of LVs into the host genome is critical to mitigate a risk for insertional mutagenesis. We have previously developed a single‐cell vector copy number (VCN) assay which allowed for an in‐depth measurement of the heterogeneity of vector transduction, enhancing on a population VCN analysis. However, the workflow currently used is limited by a labour‐intensive droplet digital PCR (ddPCR) step, relying on several 3‐plex ddPCR reactions to quantify 5 different targets in transduced single cells. In this study, we demonstrate how the ddPCR analytical burden can be streamlined with a high‐order multiplexing strategy, whereby 5 targets are measured simultaneously in one single ddPCR reaction using ratio‐based mixing of channel1 and channel2 fluorochrome‐conjugated probes. This method improvement allows for faster and cheaper single cell VCN analysis, unlocking the potential application of this assay to a larger number of samples or higher number of single cells, enabling greater monitoring of the integration of LVs in cell therapy products. Simplification of analytical assays for the characterisation of complex cell therapy products will facilitate the adoption of novel, enhanced assays in the field, which in turn will help the design and manufacturing of safer, higher quality products.
Development of an in vitro testing platform for functionality of macrophage cell therapies for liver fibrosis
1: Resolution Therapeutics
The estimated number of people living with chronic liver disease (CLD) is 1.5 billion worldwide. Currently, the only treatment for patients with severe CLD is a liver transplant. This has major limitations, namely, the lack of donor organs available to treat patients. Therefore, it is fundamental that alternative treatment options are explored.
Macrophages are key immune cells involved in tissue inflammation and repair. Peripheral injection of macrophages into mice with modelled liver fibrosis, shows that macrophages home to the liver and stimulate liver regeneration via host immune cell recruitment and scar degradation. In humans, a phase I clinical trial for autologous macrophage therapy for liver fibrosis demonstrated safety and feasibility.
At Resolution Therapeutics (RTx), we are building a pro‐restorative macrophage cell therapy to promote organ repair. To ensure we develop the optimal therapy for patients, we have built a screening‐platform to assess macrophage phenotype and function. Herein, we will show you preliminary data on the establishment of the platform. This platform allows us to develop and manufacture the best pro‐restorative macrophages to treat patients with CLD.
Loss of BCL11B induces the development of highly cytotoxic innate T cells out of IL‐15 stimulated peripheral blood αβ CD8+ T cells
1: Internal Medicine C, University Medicine Greifswald 2: Pediatric Oncology, University Medicine Greifswald 3: Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald
BCL11B, an essential transcription factor for thymopoiesis, regulates also vital processes in post‐thymic lymphocytes. Increased expression of BCL11B was recently correlated with the maturation of NK cells, whereas reduced BCL11B levels were observed in native and induced T cell subsets displaying NK cell features. We show that BCL11B‐depleted CD8+ T cells stimulated with IL‐15 acquired remarkable innate characteristics. These induced innate CD8+ (iiT8) cells expressed multiple innate receptors like NKp30, CD161, and CD16 as well as factors regulating migration and tissue homing while maintaining their T cell phenotype. The iiT8 cells effectively killed leukemic cells spontaneously and neuroblastoma spheroids in the presence of a tumor‐specific monoclonal antibody mediated by CD16 receptor activation. These iiT8 cells integrate the innate natural killer cell activity with adaptive T cell longevity, promising an interesting therapeutic potential. Our study demonstrates that innate T cells, albeit of limited clinical applicability given their low frequency, can be efficiently generated from peripheral blood and applied for adoptive transfer, CAR therapy, or combined with therapeutic antibodies.
A scalable method for the production of exosomes derived from CAR‐T cells
1: GENyO‐ Centro de Genomica e Investigacion Oncologica: Pfizer / Universidad de Granada / Junta de Andalucia 2: GC14 Cell Therapy, IMIBIC. University of Cordoba, Reina Sofia University Hospital, Cordoba 14004, Spain. 3: Department of Hematology, Reina Sofía University Hospital, Córdoba, Spain.Biomedical Research Institute (IBS. Granada), Granada, 18012, Spain. 4: Biopathology and Regenerative Medicine Institute (IBIMER), Centre for Biomedical Research (CIBM), University of Granada, Granada, 18016, Spain. 5: Department of Human Anatomy and Embryology, Faculty of Medicine, University of Granada, Granada, 18016, Spain. 6: Biosanitary Research Institute of Granada (ibs.GRANADA), University Hospitals of Granada ‐ University of Granada, Granada, 18016, Spain
Adoptive cell therapies (ACTs) based on modified T cells with chimeric antigen receptors (CAR) have shown remissions in hematopoietic neoplasms but not in solid tumors. This is mainly due to the complexity of the tumor microenvironment in this kind of tumors. Unlike disseminated liquid tumors, solid tumors present an impenetrable physical barrier, which prevents the arrival of therapeutic cells to their targets, reducing the effectiveness of CAR‐T cells in such type of tumors. Recently, the peculiar properties of nanomaterials have made possible to improve current cell therapies against cancer, particularly exosomes have emerged as a potential therapeutic agent in the field of immunotherapy. One innovative application is the production of exosomes from CAR‐T cells (EXO‐CAR‐T), presenting CAR on their surface and allowing a specific lysis of the tumor, which could be used as complementary therapy alongside the CAR‐T cells.
However, there are some limitations in the use of exosomes in clinics since these therapies require a high amount of these particles so to develop a therapeutic effect. On the other hand, if exosomes are to be used in an allogeneic setting, the presence of HLA molecules in their surface could led to graft rejection by the immune system of the patient. We aim to increase the exosome production and to eliminate HLA from the final therapeutic product. Our preliminary data showed that exosomes derived from modified CAR‐T cells are functional and express exosomes markers and, by using B2M knock‐out cells as exosomes source we demonstrated a reduction of the ex‐vivo alloreactivity.
Delivery of mRNA and CRISPR‐Cas9 RNP to primary Natural Killer cells and T cells using the Flowfect®, a new transfection technology
1: Kytopen 2: Massachusetts Institute of Technology
Cell and gene therapy manufacturing relies on robust and consistent delivery of payloads, such as mRNA, DNA, and CRISPR‐Cas RNPs to primary cells. Static electroporation delivers these payloads to cells by the electrical disruption of cellular membranes, which can also disrupt normal cell function, resulting in poor process efficiency and cell recovery. Kytopen's Flowfect® is a transfection technology platform that combines continuous fluid flow with electric fields to deliver payloads to cells of interest. The technology provides added benefits over existing payload delivery techniques by seamlessly scaling high throughput screening used in discovery to large scale manufacturing of cell and gene therapy products.
We previously demonstrated high efficiency and recovery when delivering mRNA payloads to primary T cells, however optimal conditions for mRNA delivery are not universal for all payload and additional cell types. To further expand the possible applications of Flowfect®, we explored multiple parameters to modulate Flowfect® configurations for delivery of CRISPR‐Cas9 RNP payloads to primary T cells and CD34+ cells, and used a similar approach to identify optimal configurations for the delivery of CRISPR‐Cas9 RNP and mRNA to Natural Killer (NK) cells. Our work demonstrated increased efficiency and cell recovery relative to previous configurations and to static electroporation technology. These data provide compelling evidence for the use of Flowfect® to enable mRNA and RNP delivery to cells for immune‐based therapies at both small‐scale discovery and at‐scale manufacturing stages of cell therapy development and production.
Preclinical evaluation of CAR20(NAP)‐T cells for B cell lymphoma
T Saren1 J Ma1
1: Uppsala University
CD19‐targeted CAR T cell therapy has shown striking results against B cell malignancies, which has led to approval of four CD19CAR T cell products. However, in long‐term follow up evaluations it has become evident that many patients relapse after CD19 CAR‐T cell treatment and then in many cases present with CD19‐negative tumors. Our previous study showed that CAR‐T cells armed with immunomodulatory neutrophil‐activating protein (NAP) from Helicobacter pylori (termed CAR(NAP)‐T) can trigger the endogenous T‐cell mediated immune response and further eliminate “CAR‐target‐antigen‐negative” tumor cells. Here, we report the development of CD20‐targeting (targeting moiety from Rituximab) CAR20‐T cells, as well as the NAP‐armed CAR20(NAP)‐T cells and their pre‐clinical evaluations in a murine lymphoma model. CAR20‐T cells were as efficient in killing as conventional CAR19‐T cells against multiple lymphoma cell lines in vitro. Importantly, primary mantle cell lymphoma cells isolated from a patient who relapsed after Rituximab treatment, can also be recognized and killed by CAR20‐T cells. In addition, mice with mouse lymphoma cells engineered to express human CD20 (A20‐hCD20) treated with CAR20(NAP)‐T cells showed delayed tumor growth and prolonged survival. Furthermore, NAP did not induce any toxicity in a human whole blood loop assay, indicating a safe profile as a therapeutic transgene. CAR20(NAP)‐T cells are worth further investigation especially in patients relapsing with CD19‐negative tumors after CD19 CAR T cell therapy.
Genome editing in hematopoietic stem cells restores lymphocyte‐mediated cytotoxicity and prevents hemophagocytic lymphohistiocytosis in a murine model of Munc13‐4 deficiency
1: Institute for Transfusions Medicine and Gene Therapy 2: Center for Chronic Immunodeficiency 3: Institute for Immunodeficiency, Medical Center – University of Freiburg, Freiburg, Germany
Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disorder characterized by life‐threatening cytokine storm and immunopathology. Familial HLH type 3 (FHL3) accounts for 30% of all inborn HLH cases. It is caused by mutations in the Munc13‐4 encoding UNC13D locus and results in compromised T and NK cell‐mediated killing. Since current treatment, including allogeneic hematopoietic stem cell (HSC) transplantation, still shows 30‐40% mortality, we sought to evaluate a curative genome editing strategy in Jinx mice, a mouse model for FHL3 that harbors a cryptic splice donor site (cSD) in Unc13d intron 26. To restore T and NK cell function in vivo, Jinx HSCs were edited by a CRISPR‐Cas9 ‘double hit’ strategy, simultaneously targeting the cSD upstream and downstream to excise it. Subsequently, the edited HSCs were transplanted into busulfan‐conditioned Jinx to establish a graft‐derived immune system. NGS‐based genotyping of the Unc13d target site and CAST‐Seq to characterize ‘double hit’‐associated off‐target effects was performed on DNA isolated from peripheral blood cells at different time points. This genetic analysis corroborated efficient and stable gene editing (>95% of Unc13d alleles) and the absence of detectable off‐target activity. Finally, reconstituted mice were challenged with lymphocytic choriomeningitis virus, a potent trigger of lethal hyperinflammation in Jinx mice. We observed rapid virus clearance and protection from HLH in Jinx mice transplanted with Unc13d‐edited HSCs while Jinx mice that received mock‐edited cells developed an acute and fatal HLH. In conclusion, our efficient CRISPR‐Cas9‐based ‘double hit’ protocol allowed us to fully restore defective cytotoxicity in a familial HLH disease model.
Specific elimination of m.3243A>G mutant mitochondrial DNA using mitoARCUS in cultured cells and a novel xenograft mouse model
1: Precision BioSciences, Inc. 2: University of Miami
Pathogenic mutations in the multi‐copy mitochondrial DNA (mtDNA) are commonly heteroplasmic, with both wild‐type and mutant genomes co‐existing in the same cell. Clinical symptoms manifest once the proportion of mutant mtDNA exceeds a particular disease threshold, resulting in multi‐systemic, complex disorders. Taking advantage of the lack of double‐strand break repair within mitochondria, we propose a therapeutic approach to correct these mutations by utilizing a mitochondrial‐trafficked ARCUS nuclease (mitoARCUS) to selectively cleave and eliminate mutant mtDNA. This specific targeting would leave the wild‐type mtDNA to repopulate the cell, resulting in a shift in heteroplasmy. We engineered mitoARCUS to target one of the most common pathogenic mtDNA mutations, m.3243A>G, which is responsible for >80% of cases of MELAS. It was observed that mitoARCUS efficiently localized to mitochondria, resulting in undetectable levels of nuclear off‐target editing. As the mutation only differs from the wild‐type sequence by one nucleotide, the nuclease was optimized to prevent cutting of wild‐type mtDNA while maintaining activity against mutant mtDNA. This enhanced specificity allowed for robust shifts in heteroplasmy in m.3243A>G cybrid cells and concomitant improvements in cellular oxygen consumption rates and mitochondrial ATP production. Due to the lack of animal models, a novel xenograft mouse model was generated to evaluate efficacy of mitoARCUS delivered systemically by AAV. Dose‐dependent shifts in heteroplasmy were observed, despite low tumor transduction compared to disease‐relevant tissues. Together, these data support the development of mitoARCUS as an in vivo gene editing therapeutic for the treatment of disease‐causing heteroplasmic mtDNA mutations.
FaDe‐Cas9: a fast degrading SpCas9 with reduced cellular immunity
1: AstraZeneca BioPharmaceutical R&D
There is growing interest in the therapeutic use of CRISPR‐Cas9 to correct monogenic human disorders, with many medicines progressing rapidly into clinical trials. However, Cas9 isolated from Streptococcus Pyogenes (SpCas9), considered the “golden standard” endonuclease, is known to trigger host immunogenicity. The recent reports describing the negative impact of SpCas9‐induced immunity in healthy human cells and animal studies underline a potential barrier to CRISPR/Cas9, especially in the context of multiple dosing. Evading the cellular immunosurveillance could therefore represent a major advancement to broaden the clinical applications of CRISPR. We rationally engineered SpCas9 with a single amino acid substitution in the Rec2 domain to introduce chaperone‐mediated autophagy (CMA) binding sequence. The CMA‐moiety enables the recognition of Cas9 by the HSC70 chaperone that directs it to lysosomal degradation. Lysosomal degradation makes SpCas9 less presented to the MHC‐I, thus reducing the CD8+ T‐cell activation. This mutation led to a substantial decrease in Cas9 persistence in primary and immortalized cells; hence we named it “Fast Degrading Cas9, or FaDe Cas9” (fdCas9). In vivo, fdCas9 presented the same high rapid turnover observed in vitro and comparable editing efficiency with the wtCas9.
Moreover, we found a markedly reduced activation of the cellular immunity measured by cytotoxic CD8+ T‐cell in livers of mice exposed to FaDe‐Cas9. Our data confirmed a novel version of SpCas9 able to circumvent activation of host immunity, making it an attractive enzyme for human therapies.
Application of ADAR‐mediated RNA editing to modulate protein‐protein interactions
1: 2: Wave Life Sciences
At Wave Life Sciences, we develop stereopure AIMers—chemically modified oligonucleotides in which the chiral configurations of backbone linkages are precisely controlled at each position—that direct sequence‐specific adenine (A) to inosine (I)/guanine (G) RNA editing using endogenous ADAR enzymes. In addition to correcting disease‐causing mutations, we have applied AIMers to modulate protein‐protein interactions. Successful application of AIMers to modulate biological pathways at the protein‐protein interaction level may expand the scope of human diseases we can address with this technology. We will present AIMers designed to modulate the NRF2‐KEAP1 system, a conserved intracellular defense pathway to counter cellular oxidative stress. NRF2 is a transcriptional regulator of antioxidant and metabolic genes that is negatively regulated through direct interaction with KEAP1. Through this interaction, KEAP1 sequesters NRF2, blocking its translocation into the nucleus and promoting its degradation by the proteasome. By disrupting this interaction, AIMers can activate expression of NRF2‐dependent gene expression. We have designed AIMers to modify amino acids at the interface between KEAP1 or NRF2. We show AIMers direct up to 80% editing of NRF2 and KEAP1 transcripts in multiple human and mouse cell types in vitro (primary astrocytes, primary hepatocytes) and show that editing leads to dose‐dependent activation of NRF2‐dependent gene expression. We have applied some of these AIMers in vivo in mice expressing human ADAR1 and observe ≥40% transcript editing and activation of Nrf2‐dependent gene expression in the liver. Together, these data provide preclinical proof of concept that we can apply AIMers to modulate protein‐protein interactions.
Contemporary modulation of two major immune checkpoints expression in CAR T cells via hit‐and‐run epigenome editing
1: Institute for Transfusion Medicine and Gene Therapy 2: University of Freiburg
CAR (Chimeric Antigen Receptor) T cells represent a promising immunotherapy approach to treat cancer. However, cancer cells have developed strategies to evade the immune system by overexpressing ligands that, upon binding to the corresponding immune checkpoint (IC) receptors expressed by CAR T cells, promote their exhaustion ultimately impairing their killing capacity. Immune checkpoint blockers have shown promise inhibiting this interaction, thus preventing exhaustion. Moreover, IC combinatorial targeting elicits synergistic effects. However, the occurrence of severe immune‐related side effects due to their systemic activity, and the adaptation of the tumor microenvironment that can eventually lead to resistance, are still unresolved. We have devised a strategy to render CAR T cells devoid of two major immune checkpoints via hit‐and‐run epigenome editing. Designer Epigenome Modifiers (DEMs) targeting PDCD1 or LAG3 led to the deposition of de novo cytosine methylation in their respective promoters, sustainably and simultaneously inducing silencing of the two genes both in primary T cells and in CAR T cells directed against the Prostate‐Specific Membrane Antigen (PSMA). The epigenetically induced silencing was persistent upon repetitive CAR T cell activation through direct contact with PSMA‐positive target cells. Furthermore, the epigenetically edited CAR T cells were fully functional and indistinguishable from their parental unmodified cells in an array of in vitro assays, demonstrating the safety of epigenome editing in CAR T cells. Our results propose this technology as a novel approach to modulate diverse T cells inhibitory pathways in a simultaneous and targeted manner needless of DNA sequence modification.
Epigenome‐editing strategies to enhance oligodendroglial differentiation from human induced pluripotent stem cells
1: San Raffaele Telethon Institute for Gene Therapy (HSR‐TIGET) 2: Vita‐Salute San Raffaele University, Faculty of Medicine, 20132 Milan (MI), Italy
The generation of human induced pluripotent stem cell (hiPSC)‐derived oligodendroglial cells (OL) at yield, purity, and homogeneity required for translational applications in demyelinating disease is still a challenge. Current protocols to generate hiPSC‐derived OL are time‐consuming, lead to heterogeneous OL populations and, if based on transient and/or stable overexpression of OL‐related Transcription Factors (TFs), could lead to gene misregulation and/or genotoxicity.
Here, we exploited novel epigenome‐editing technologies to timely and physiologically modulate the expression of OL‐related genes in human iPSC‐derived neural stem/progenitor cells (hiPSC‐NPCs) through Artificial Transcriptional Activators (ATAs; based on dCas9 fused to transcriptional activator domains). After selection of target regulatory regions, we confirmed the capability of ATAs to robustly and transiently up‐regulate key OL genes in hiPSC‐NPCs, which were then differentiated using an established OL differentiation protocol. Gene expression, cytofluorimetric, and immunofluorescence analysis showed increased numbers and enhanced branched morphology of OPC and OL derived from edited cells as compared to untreated (UT) controls. Upon intracerebral transplantation in immunodeficient neonatal mice, edited OPC showed robust engraftment, widespread migration, and acquisition of a mature/myelinating OL phenotype. The potential functional advantage of edited OPC over UT counterparts is under evaluation.
The results of this study suggest that transient modulation of endogenous OL regulatory network in hiPSC‐NPCs favors their oligodendroglial commitment/differentiation. Enrichment for edited cells and regulated timing of OL gene overexpression during differentiation could be applied to further improve the yield, homogeneity, and purity of edited OL populations to be used for basic and pre‐clinical research.
Complex gene editing of T cells without translocations
1: Berlin Center for Advanced Therapies, Charité University Medicine 2: BIH Center for Regenerative Therapies, Charité University Medicine 3: Institute of Transfusion Medicine, Charité University Medicine 4: Institute of Medical Immunology, Charité University Medicine
CRISPR‐Cas mediated gene editing is a promising tool to engineer universal immune cell therapies. It can be used to replace the endogenous T cell receptor (TCR) with a chimeric antigen receptor (CAR) by insertion into the T cell receptor alpha constant (TRAC) locus. TRAC‐replaced CAR T cells have a reduced risk to induce graft‐versus‐host‐disease, due to the lack of the endogenous TCRs. Recent clinical trials demonstrated that allogeneic CAR T cells fail to persist in patients due to HLA‐barriers by allo‐immunity. Therefore, knock‐out of B2M and CIITA during CAR knock‐in may enhance the performance of CAR‐T cell therapy in an allogeneic setting by elimination of HLA‐class 1 and 2, respectively. We demonstrate that simultaneous knock‐in and knock‐out with conventional CRISPR‐Cas9 editing can be achieved efficiently, however, it led to high rate translocations between different DNA double strand breaks (>2,8% detected by ddPCR). To overcome this challenge, we employed a Cas9‐derived adenine base editor (ABE8.20‐m) that enables gene silencing without DNA cleavage. We show that co‐electroporation ABE8.20‐m mRNA with Cas12a Ultra nuclease and dsDNA repair templates for CAR knock‐in avoided guide RNA exchange between the CRISPR‐editors and reduced translocations by 20‐fold, without significant difference to the background noise of the assays (edited: 0.14%, mock: 0.05%; ns, paired t test, p = 0.0553, n = 4). The resulting triple‐gene edited CAR T cells were resistant to allo‐reactive T cell attack. Thus, we demonstrated a solution for safer complex gene‐edited cell products as a step towards off‐the‐shelf CAR T cell therapies.
Base editing strategy to repair the CFTR 2789 + 5G>A splicing mutation
1: University of Trento, Department CIBIO, Laboratory of Molecular Virology, Trento, Italy 2: KU Leuven, Department of Pharmaceutical and Pharmacological Sciences, Laboratory for Molecular Virology and Gene Therapy, Leuven, Belgium 3: Telethon Institute of Genetics and Medicine, Pozzuoli, Italy 4: KU Leuven, Department of Development and Regeneration, CF Reference Center, Leuven, Belgium 5: UZ Leuven, Department of Pediatrics, Leuven, Belgium 6: KU Leuven, Department of Chronic Diseases and Metabolism, BREATHE laboratory
The 2789 + 5G>A mutation in the CFTR gene is among the most frequent defects causing cystic fibrosis (CF). This mutation determines an aberrant splicing which results in the production of a non‐functional CFTR protein. In this study we applied CRISPR adenine base editors (ABE) to precisely correct this point mutation. To set the most effective base editing strategy we generated a minigene model mimicking the splicing defect in HEK293 cells. We obtained up to 40% correction of the genetic defect by inducing an A‐to‐G conversion in the mutated minigene model through lentiviral delivery of the ABEmax base editor. The efficacy of this genome editing strategy was tested in intestinal organoids derived from a CF patient (2789 + 5G>A/G542X). In this CF model we obtained up to 10% A‐to‐G conversion which correlated with the restoration of correct splicing and functional recovery of CFTR measured by forskolin‐induced swelling assay. We finally validate the strategy in primary human bronchial epithelial (HBE) cells isolated from a compound heterozygous patient for the mutation (2789 + 5G>A/F508del). In HBE cells we obtained 10% base correction by delivering the base editor components as RNA to limit unwanted A‐to‐G conversions near the target site (bystander activity). Short‐circuit current experiments confirmed the restoration of CFTR channel activity after gene correction through CRISPR base editing.
These results demonstrate that CRISPR‐mediated base editing is an efficient method to restore the 2789 + 5G>A splicing defect and is potentially extendable to other CFTR mutations.
Development and characterization of GNTI‐122, an autologous engineered human regulatory T cell therapy for type 1 diabetes
J Y Yam1 P S Saikumar‐Lakshmi1 A E Landuyt1 G I Uenishi1 T Guo1 T F Chen1
1: GentiBio, Inc.
GNTI‐122 is a novel autologous Treg therapy in non‐clinical development for type 1 diabetes. It is engineered from bulk CD4 cells with CRISPR‐Cas9 to knock‐in an MND promoter to stabilize FOXP3 expression, and an islet antigen‐specific TCR into the TRAC locus (knocking‐out endogenous TCRalpha), and introducing a rapamycin‐activated, chemically induced IL‐2 signaling complex (CISC). GNTI‐122 addresses key limitations of sorted Treg therapies through stabilized FOXP3 expression, tissue‐specificity, and built‐in IL‐2 signaling. Following gene editing of CD4+ T cells, GNTI‐122 is highly enriched to >90% purity through selective IL‐2 signaling via CISC. GNTI‐122 cells demonstrate a robust Treg suppressor phenotype (CD25, CTLA4, EOS, CD39, CD27, TNFRII, and low CD127 and CD70). Upon stimulation, GNTI‐122 maintains low expression of IL‐2, TNF‐α, and IFN‐γ, and increased LAP and GARP. GNTI‐122 displays potent immunosuppressive activity in islet antigen‐specific and polyclonal T cell suppression assays over controls, supportive of MoA. In vitro, CISC activation by low dose rapamycin promotes GNTI‐122 survival without exogenous IL‐2. Concurrent CISC and TCR signaling are required for GNTI‐122 proliferation, indicating that CISC will selectively promote GNTI‐122 expansion only in the context of auto‐antigen presentation. A dose dependent increase in GNTI‐122 engraftment was shown in NSG mice receiving subtherapeutic rapamycin dose. Non‐clinical proof of concept has been successfully demonstrated in vitro and in vivo through indirect suppression assays and T1D mouse models, respectively. Thus, GNTI‐122 demonstrates the potential to overcome key limitations of sorted Treg therapies and provides a novel therapeutic modality to restore immune tolerance in T1D patients.
Multiplex deletion of myeloid antigens by base editing in human hematopoietic stem and progenitor cells (HSPCs) enables potential for next generation transplant for acute myeloid leukemia (AML) treatment
1: Vor Bio
Targeted immunotherapy of AML has been limited due to lack of true tumor‐specific antigens. This “on‐target, off‐tumor” effect leads to severe cytopenia. To unlock the full potential of targeted treatments, we create treatment‐resistant HSPCs by genetically ablating target antigens from healthy, human leukocyte antigen‐matched, donor‐derived HSPCs for hematopoietic stem cell transplant. This allows compatible immunotherapy to specifically target only the target‐antigen bearing leukemic cells while protecting the target antigen‐null allogeneic graft. Targeting multiple antigens simultaneously to help avoid potential antigen escape, address the issue of antigen heterogeneity of tumor cells, and subsequently, expand therapy to more patients may be the future of AML treatment. In such scenarios, we need to ablate multiple target antigens from the HSPC graft by multiplex genome editing to protect the transplanted healthy graft from multi‐pronged immunotherapies. Cytosine base editors, to introduce premature stop codons or adenosine base editors, to inactivate splice sites, were used to disrupt multiple genes, achieving >80% editing at all loci while 80% of differentiated myeloid cells were multi‐knockouts. No on‐target translocations were detected using rhAmpSeq. Pharmacology studies using NOD/SCID‐gamma mice showed unaltered long‐term engraftment and multilineage differentiation in the multiplex‐edited cohorts. NGS analysis revealed no change in total editing between dual knockout input and bone marrow cells post‐engraftment, indicating the edits in dual‐engineered cells persisted long‐term and loss of multiple antigens was well‐tolerated. We demonstrated that base editing provides an efficient and safe strategy for multi‐gene disruptions in HSPCs and can enable next‐generation AML treatments.
Enrichment of CRISPR/Cas‐mediated transgene integrations by transient CRISPR activation of a silent reporter gene
1: Department of Biomedicine, Aarhus University, Aarhus, Denmark 2: Aarhus Institute of Advanced Studies, Aarhus University, Aarhus, Denmark
Site‐specific integration of transgenes by homology‐directed repair (HDR) using CRISPR/Cas has a wide range of applications within gene‐ and cellular therapies. However, primary cells often suffer from low editing efficiencies potentially impeding such therapies. Various approaches have been developed to enrich for cells with transgene integrations, but challenges for each strategy remain. One enrichment strategy relies on the inclusion of a surface reporter gene that can be used for immunomagnetic selection. However, stable expression of a reporter gene may perturb cell homeostasis and lead to unwanted effects. Here we develop a broadly applicable and versatile strategy for enriching cells carrying a transgene by harnessing the capability of CRISPR activation (CRISPRa) to transiently induce expression of a therapeutically relevant reporter gene that can be used for immunomagnetic enrichment. We demonstrate that this strategy is readily adaptable to both cell lines and primary cells, including primary human T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). We achieved enrichment up to 3.7‐fold and 4.2‐fold in primary human T cells and CD34+ HSPCs, respectively. Furthermore, we demonstrate that T cells expressing a chimeric antigen receptor (CAR) can be enriched 2.5‐fold to >80% CAR + T cells, which consequently improves cytotoxicity. This novel enrichment strategy expands the possibility to enrich for transgene integrations in therapeutically relevant settings and may improve gene and cellular therapies.
Harnessing mobile genetic elements to write DNA sequences with RNA
1: Tessera Therapeutic
The ability to introduce new DNA sequences into the genome with high specificity and efficiency would provide a critical complement to existing gene editing approaches. Naturally occurring mobile genetic elements, such as transposases, retrotransposases, and recombinases, afford a rich reservoir of activities that can be exploited to introduce genetic information. These highly abundant, and functionally diverse elements vary in their nucleic acid sequence, biochemical mechanism of substrate integration, and the efficiency and specificity with which they integrate their substrate. We have identified, prioritized, and tested thousands of previously uncharacterized mobile genetic elements for their ability to mediate the introduction of genes into the human genome. We conducted a systematic in silico analysis of tens of thousands of naturally occurring mobile genetic elements from across the tree of life and developed screening strategies to evaluate the activity of these newly characterized enzymes in human cells. We further engineered these elements to generate synthetic molecules that enhance and expand their natural activities.
This screening paradigm and further high thought put engineering resulted in the development of several enzymes that are capable of catalyzing a broad spectrum of editing reactions from introduction of large DNA sequence to small single nucleotide changes. These edits can be achieved with single RNA delivery in primary cell and in vivo. Here we will present our screening and engineering platform as well as the application of these tools for the correction of a Phenylketonuria (PKU) mouse model to restore therapeutically relevant levels of phenylalanine hydroxylase (PAH).
Novel riboswitches regulate AAV‐delivered transgene expression in mammals via oral small molecule inducers
1: 2. Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599‐3290, USA 2: 1. Gene Regulation, MeiraGTx, New York, NY 10016, USA 3: 3. Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599‐7363, USA
Controlled expression of delivered transgene is critical for gene therapy. Here, we report that by linking a synthetic aptamer to an alternative splicing gene expression platform, we have created a robust, synthetic mammalian riboswitch cassette that regulates gene expression tightly and dynamically in response to small molecule inducers. The splicing‐based expression platform creates an “on” switch in the presence of the small molecule by sequestering a splice site of an alternative exon. This switch has a high dynamic range which has allowed us to screen, identify and modify novel aptamers that bind and respond to novel small molecules. With these riboswitches, we were able to tightly regulate expression in vivo, using oral small molecules, of hormones such as human growth hormone, growth factors such as erythropoietin (Epo), therapeutic antibodies such as anti‐PD1 and anti‐HER2 antibodies, chimeric antigen receptors (CARs), and nucleases such as CasRx protein. Riboswitches that respond to these novel small molecule inducers regulate transgene expression with high dynamic range in a dose‐dependent manner. When delivered through an AAV vector to the liver or the muscle in mice, the engineered riboswitches reversibly regulate transgene expression via an orally delivered small molecule inducer, providing precise control of transgene expression. Thus, our potent gene regulation system provides the first synthetic aptamer riboswitch that is capable of controlling therapeutic gene expression with precise dose control through orally available small molecule inducers. This platform enables both temporal and spatial control of gene expression for gene and cell therapies.
Generation of AGXT gene‐corrected induced‐hepatocytes with restored glyoxylate metabolic capacity as potential treatment for Primary Hyperoxaluria type 1
V Nieto‐Romero1 A García‐Torralba1 A Molinos‐Vicente1 R García‐Escudero2 E Salido3 J C Segovia1
1: Biomedical Innovation Unit, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) and Instituto de Investigación Sanitaria Fundación Jiménez. (IIS‐FJD, UAM), 28040 Madrid, Spain. 2: Molecular Oncology Unit‐CIEMAT and Centro de Investigación Biomédica en Red de Cancer (CIBERONC), 28040 Madrid, Spain. 3: Pathology Department, Hospital Universitario de Canarias, Universidad La Laguna. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 38320 Tenerife, Spain.
Primary Hyperoxaluria Type 1 (PH1) is a rare genetic disorder characterized by oxalate overproduction in the liver, which could result in renal damage. It is caused by mutations in the AGXT gene, codifying for alanine:glyoxylate aminotransferase (AGT), a hepatic enzyme involved in glyoxylate metabolism.
Liver transplantation is currently the only curative treatment for many inherited liver disorders and, in the case of PH1 patients, both liver and kidney transplant is required. We propose the generation of autologous phenotypically healthy induced hepatocytes (iHeps) as an alternative to whole organ or allogenic cell transplantation.
We combined site‐specific gene correction and hepatic direct cell reprogramming to generate iHeps from PH1 patient‐derived fibroblasts. First, we obtained specific AGXT gene corrected cells by applying two different CRISPR/Cas9 based strategies in patient's fibroblasts: 1) Accurate point mutation (c.853T‐C) correction by homology‐directed repair with a ssODN harboring the wild‐type sequence; 2) Knock‐in of an enhanced version of AGXT cDNA near the start codon of the endogenous gene, an almost universal correction strategy for PH1 mutations. Then, we generated iHeps by lentiviral overexpression of hepatic transcription factors, directly from AGXT‐corrected fibroblasts, avoiding the undifferentiated step of iPSCs. iHeps showed a hepatic gene expression profile and hepatocyte functionality. Most important, AGXT‐corrected iHeps exhibited in vitro reversion of oxalate accumulation compared to non‐edited PH1‐derived iHeps.
This innovative therapy set up a potential alternative cellular source to replace deficient hepatocytes with autologous functional corrected cells for genetic liver disorders. Moreover, patient‐derived iHeps constitute a useful personalized in vitro disease modeling.
Development of a miniaturized CRISPR activation for a single AAV delivery method for muscular dystrophy
1: Div. of Genetic and Genomic Medicine, Dept. of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA USA 2: Dept. of Human Genetics, School of Public Health, University of Pittsburgh, Pittsburgh, PA USA 3: Dept. of Neuroscience, Kenneth P. Dietrich School of Arts and Sciences, University of Pittsburgh, PA USA 4: Dept. of Neuroscience, Macalester College, Saint Paul, MN USA 5: Div. of Pulmonology, Dept. of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA USA
LAMA2‐related dystrophies (LAMA2‐RD) are a subtype of congenital onset muscular dystrophy caused by mutations in the LAMA2 gene encoding laminin‐α2, an extracellular protein essential for skeletal muscle and Schwann cell functions. Individualized correction is hampered by the mutation heterogeneity in patients. In contrast, upregulation of compensatory gene LAMA1 can serve as a mutation‐independent approach. We previously showed that upregulation of Lama1 via AAV‐mediated CRISPR‐activation (CRISPRa) rescues disease phenotypes in mice. However, it required the use of dual‐AAVs, which directly contributes to the high dose, toxicity, and cost.
In this project, we aim to generate a mini‐CRISPRa system suitable for a single AAV9 packaging and improve the clinical relevance of this technology.
First, we swapped a commonly‐used CMV promoter with a novel, 7.5x‐shorter synthetic promoter 4NRF1 to drive SadCas9 expression. Subsequently, we substituted previously used 2xVP64 transcriptional activators with miniaturized tripartite VP64, P65, and RTA activators (miniVPR), and coupled it with a single gRNA targeting the mouse Lama1 promoter.
We observed that the mini‐CRISPRa system induces Lama1 upregulation in LAMA2‐RD myoblasts at a 1.88‐fold higher level than the original CRISPRa. Subsequently, we treated a LAMA2‐RD mouse with an intramuscular injection of 1.5E+12 AAV9 viral genomes carrying the mini‐CRISPRa and observed successful Lama1 upregulation in tibialis anterior muscles.
Current experiments involve systemic upregulation of Lama1 in vivo, in which the AAV biodistribution, neuromuscular functions, and immune responses are being examined. Successful completion will lead to new therapeutic avenues based on the upregulation of compensatory genes applicable for other neuromuscular disorders and haploinsufficiency‐related diseases.
CRISPR‐induced on‐target large deletions can be controlled by modulating PolQ and RPA
1: King Abdullah University of Science and Technology
CRISPR‐Cas9, an efficient genome editing tool, has been widely used in research and holds great promise in the clinic. However, the large unintended rearrangements of the genome after CRISPR‐Cas9 editing occur frequently and their potential risk cannot be ignored. In this study, we detected large deletions (LDs) induced by Cas9 in human embryonic stem cells (hESCs) and found micro‐homology end joining (MMEJ) repair pathway plays a predominant role in LD. We genetically targeted PARP1, RPA, PolQ and Lig3, which plays critical roles in MMEJ, during CRISPR‐Cas9 editing. We found that knocking down PARP1 and Lig3 does not alter LD frequency, while knocking down or inhibiting PolQ dramatically reduces Cas9‐induced LD frequency. Knocking down RPA increases LD frequency, and consistently, overexpression of RPAs reduces the frequency of LD. In conclusion, RPAs and PolQ play opposite roles in Cas9‐induced LD and may be promising targets for reducing large rearrangement frequency during genome editing.
Writing new DNA sequences into the genome via engineered mobile genetic elements
1: Tessera Therapeutics, Inc.
Mobile genetic element enzymes, such as transposases, retrotransposases, and recombinases, provide a rich reservoir of biochemical activities that in principle can be exploited to introduce new genetic information into the genome without the introduction of DNA breaks or use of DNA repair pathways. To leverage this large class of elements, we conducted a systematic in silico analysis of tens of thousands of mobile genetic elements from across the tree of life and then established a high throughput screening strategy to evaluate their activity in human cells.
This screening paradigm resulted in the identification of enzymes that catalyze the introduction of new DNA sequences into the genome of human primary cells via a process that we call “writing”. Using an engineered retrotransposon, a class of elements that write new DNA into the genome using an RNA template, we observed stable integration of a GFP marker cassette in up to 40% of human primary T cells co‐transfected with RNA encoding the engineered enzyme and GFP template. We also showed T cells could be re‐targeted to actively kill tumor cells by introducing an RNA template to stably integrate a chimeric antigen receptor. This process was well‐tolerated with little‐to‐no impact on cell viability and proliferation and without activation of P53 or interferon responses. Here we will present further on the engineering and application of the Gene Writing platforms to expand the genome engineering toolbox for creating new genetic medicines by enabling gene addition using all‐RNA compositions.
ARCUS gene editing of Apolipoprotein C3 results in substantial reduction in serum triglycerides in vivo
1: Precision BioSciences, Inc.
Familial Chylomicronemia Syndrome (FCS) is a rare genetic disease that results in dangerously high levels of plasma triglycerides (TGs). Patients suffering from FCS are unable to correctly metabolize lipids, resulting in TG accumulation in the bloodstream and increased risk of pancreatitis. Apolipoprotein C3 (ApoC3), a secreted glycoprotein synthesized primarily in the liver, is known to regulate plasma TG levels by interfering with hepatic clearance of TG‐rich lipoproteins. ApoC3 is associated with elevated plasma TG levels and cardiovascular disease (CVD) risk, and patients with a naturally occurring null mutation in ApoC3 exhibit cardioprotective effects. We propose a gene editing approach to treat FCS by using an engineered ARCUS nuclease to disrupt expression of ApoC3, ultimately resulting in a decrease in circulating TG levels. High levels of on‐target editing (>50%) within the ApoC3 gene were observed in HEK 293 cells treated with ARCUS. ARCUS mRNA was formulated into a lipid nanoparticle (LNP) and delivered systemically to ApoC3 transgenic mice. At three weeks post‐LNP administration, 11.84% ± 3.43% indels were detected at the target site in the liver. ApoC3 mRNA, ApoC3 protein, and serum TG levels were decreased by 67.25% ± 12.55%, 69.46% ± 16.58%, and 70.00% ± 16.42%, respectively, compared to PBS‐treated mice at three weeks post‐LNP administration. Through iterative protein engineering, an optimized ARCUS enzyme was generated that maintained high on‐target activity with reduced off‐target editing. Together, these data support a gene editing approach for inactivation of ApoC3 to treat FCS, or as a preventative measure against the development of CVD.
CRISPR/Cas9‐based gene editing for Rett syndrome therapy
1: Medical Genetics, University of Siena, Siena, Italy 2: Med Biotech Hub and Competence Center, Department of Medical Biotechnologies, University of Siena, Siena, Italy 3: Central Institute of Mental Health (ZI), Department for Translational Brain Research, Mannheim, Germany 4: Vall d'Hebron Research Institute (VHIR), Barcelona, Spain 5: Institut Clinique de la Souris, ICS/MCI, PHENOMIN, GIE CERBM, IGBMC, CNRS, INSERM, 1 Rue Laurent Fries, 67404 Illkirch‐Graffenstaden Cedex, France 6: Genetica Medica, Azienda Ospedaliero‐Universitaria Senese, Siena, Italy 7: Division of Child and Adolescent Neuropsychiatry, University Hospital of Siena, Siena, Italy 8: Pediatric Surgery, Department of Women and Children, S. Maria alle Scotte Hospital, University of Siena, Siena, Italy 9: Core Research Laboratory, ISPRO Institute for Cancer Research, Prevention and Clinical Network, 50139 Firenze, Italy 10: Institute of Clinical Physiology, National Research Council, 56124 Pisa, Italy 11: Catalan Institution for Research and Advanced Studies (ICREA), Spain 12: Institute of Neurosciences (INc) Biochemistry and Molecular Biology Department Autonomous University of Barcelona, Spain
Rett syndrome is one of the most common causes of intellectual disability in girls, with severe cognitive and physical disabilities. The classic form is caused by mutations in the transcriptional regulator MECP2. There are currently no effective therapies and CRISPR/Cas9‐based gene editing appears an appealing option for the development of new therapeutics. We engineered a gene editing toolkit and demonstrated its ability to efficiently correct the MECP2 mutation hotspot c.473C>T (p.(T158M)), in patient cells. Based on these results, we have designed and we are currently validating in patient fibroblasts constructs for other hotspots, namely c.502C>T (p.(R168*)), c.763C>T (p.(R255*)), c.916C>T (p.(R306C)). We are reprogramming iPSCs for 2 patients for each variant to generate brain organoids for further testing. Preliminary analyses on organoids for one variant demonstrated interesting differences in mutated organoids compared to isogenic WT ones. As delivery system, we selected AAVs. We tested available chimeric serotypes, AAV7m8 for human cells and AAV9P31 with increased selectivity for mouse brain, demonstrating their functionality. However, even with these serotypes, a significant portion of virus spreads to peripheral organs reducing efficiency and increasing the risk of toxicity. We are thus working to develop new serotypes with higher brain specificity and selectivity. Colonies of KI mice harboring the 4 variants are also being established to further test the approach in vivo. Altogether, these experiments will allow us to demonstrate the full potential of gene editing as a therapeutic option for RTT and possibly other neurodevelopmental disorders currently lacking an effective treatment.
Differential accessibility of chromatinized rAAV vector is a major regulator of variable expression in mouse and NHP liver
1: BioMarin Pharmaceutical
Understanding the mechanism(s) of variability of transgene expression within and between species is important for improved prediction and translation of rAAV gene therapy to the clinic. In mouse and non‐human primate (NHP) models, variability in vector transcription (DNA to RNA) is greater than for vector translation (RNA to protein), and transcriptional efficiency (expressed as RNA/DNA) is lower in NHP than in mouse. Remarkably, transcriptional efficiency varies by a factor of more than 500 between these species. We investigated chromatin accessibility as a possible mechanism for variability of RNA production and transcriptional efficiency.
Vector DNA and RNA from multiple rAAV vector constructs were quantified by droplet‐digital PCR (ddPCR) to accurately measure transcriptional efficiency in liver tissue of recipient animals. Assay for Transposase‐Accessible Chromatin (ATAC) sequencing was performed to evaluate the relationship between accessibility of chromatinized vector and transgene expression. Differential accessibility to promoter‐enhancer regions of chromatinized vector DNA correlated with vector RNA production across multiple constructs, and accounted for variability within each species. The fraction of accessible vector DNA (accessible/total vector DNA) correlated with transcriptional efficiency (RNA/DNA) and accounted for most of the variability between mouse and NHP. Longitudinal analysis demonstrated that differential accessibility of rAAV vector DNA was established in the first weeks after dosing. Chromatin Immunoprecipitation sequencing (ChIP‐seq) identified active and repressive histone marks that corresponded to chromatin accessibility.
We conclude that >70% of the variability in rAAV transgene transcriptional efficiency can be attributed to regulation of promoter‐enhancer accessibility, both within and between pre‐clinical species.
Tracking chromosomal aberrations in designer nuclease edited primary cells using ddPCR
1: UCL Institute of Child Health
Understanding the extent of unintended aberrations following designer nuclease gene editing is fundamental in moving therapeutic applications forward. Various sequencing strategies have been used to assess the frequency, location, and type of such chromosomal aberrations to predict their genotoxic potential. While these techniques provide a high degree of sensitivity, they are relatively expensive, time‐consuming, and infer a narrow, and sometimes biased, set of information. Here, we describe Multipurpose Editing and Genotoxicity Assessment (MEGA), a novel diagnostic method based on digital PCR to detect and characterize a wide category of mutations at the on‐ and off‐target sites. To demonstrate the applicability of MEGA, we have used multiple designer nuclease platforms, alongside AAV transduction, to edit various therapeutic and model targets in human‐derived haematopoietic stem and progenitor cells (HSPCs) and T cells. We find that MEGA effectively quantifies indels, large deletions, chromosomal loss, and homologous recombination following editing. Time course analyses also reveal the kinetics of DNA cleavage and repair across the different mutations. In addition, targeted GFP knock‐in using AAVs shows a much lower percentage of indel formation relative to nuclease‐only editing, and that episomal AAV material is retained in cells longer than the GFP can be detected with flow cytometry. In summary, MEGA fulfills a niche within the growing toolbox of post‐editing analysis platforms by providing a fast and specific overview of the integrity of genomes following modern gene‐editing techniques.
A truncated reverse transcriptase enhances prime editing by split AAV vectors
1: Aarhus University
Prime editing is a new CRISPR‐based genome editing technology that enables versatile and precise genome editing independent of double‐strand DNA breaks and exogenous donor template DNA. Prime editing consists of three components: the prime editor (PE) ‐ a fusion protein of Cas9‐nickase and M‐MLV reverse transcriptase (RT), a prime editing guide RNA, and a nicking gRNA. Despite of its versatility, the large PE gene (∼6.4 kb) challenges prime editing for efficient in vivo delivery. Here, we make advancements to the RT moiety of PE to mitigate issues with delivery by AAV vectors. These attempts include RT variant screening, codon optimization, and PE truncation by removal of the RNase H domain and further trimming. This led to a codon‐optimized and size‐minimized PE that has an expression advantage (1.4 × fold) and size advantage (621 bp shorter). In addition, we optimize the split intein PE system and identify Rma‐based Cas9 split sites (573/4 and 673/4) that combined with the truncated PE delivered by dual AAVs result in superior AAV titer and prime editing efficiency. We also show that this minimized PE gives rise to superior lentiviral vector titers (46 × fold) over the regular PE in an all‐in‐one PE lentiviral vector. We finally deliver the minimized PE to mouse liver by dual AAV8 and show up to 6% precise editing of the PCSK9 gene, demonstrating the value of this truncated split PE system for in vivo applications.
Optimisation of mutation‐specific TALEN‐ and CRISPR/Cas9‐based gene correction therapy for β‐thalassaemia
1: Cyprus Institute of Neurology and Genetics 2: Thalassemia Centre, Cyprus Ministry of Health, Cyprus 3: Kings College London 4: University Medical Center Freiburg
Functional correction of abnormal splicing for the common HBBIVSI‐110(G>A) β‐thalassemia mutation may be achieved by efficient non‐homologous end joining (NHEJ)‐mediated disruption of the aberrant regulatory elements (DARE) associated with the mutation. With bulk editing efficiencies in hematopoietic stem/progenitor cells (HSPCs) surpassing those for homology‐directed repair, with functional correction potentially superior to that achieved by universally applied NHEJ‐based approaches and with remaining uncertainties over alternative editing technologies, the approach has potential for clinical translation. DARE correction was evaluated in patient‐derived HSPCs by nucleofection of in vitro synthesized TALEN mRNAs and of CRISPR/Cas RNA‐guided nuclease (RGN) ribonucleoprotein complexes. Correction was assessed for RNA splicing (RT‐qPCR), protein (HPLC) and morphology (differential counts), and off‐target activity by Sanger and targeted deep sequencing. RGNs and TALENs both reached high bulk disruption efficiencies and high levels of normal β‐globin. Significant correction across primary samples of β‐globin synthesis and late‐stage erythroid differentiation as key parameters of β‐thalassaemia pathology was accompanied by detectable TALEN off‐targeting for HBD and high‐level RGN off‐targeting of the unrelated, non‐coding RNF219_AS1 gene. Evaluation in patient‐derived HSPCs of alternative TALEN and RGN designs achieved variable reduction of off‐target behavior. Moreover, comparison of different RGNs showed that the same level of β‐globin chains was achieved in patient‐derived HSPCs, irrespective of whether 90% or 50% of mutant alleles were disrupted. Our study shows for the correction of HBBIVSI‐110(G>A) thalassemia that efficiency and biosafety data warrant advanced analyses, such as of genome integrity and long‐term repopulation potential of treated cells, towards clinical translation.
Engineering of allogeneic regulatory T cells expressing a CAR (Allo‐CAR‐Tregs) using ZFN‐mediated editing and AAV6 targeted integration
1: Sangamo Therapeutics France, Valbonne Sophia‐Antipolis, Les Cardoulines HT1, Allée de la Nertière, 06560, France 2: Co‐first author 3: Co‐senior author
Cell‐based therapies using genetically modified regulatory T cells armed with a chimeric antigen receptor (CAR‐Tregs) is an attractive potential treatment option for autoimmune diseases. Here, we describe the generation of allogeneic CAR‐Tregs using Zinc Finger Nuclease (ZFN) editing technology to knockout the cell‐surface expression of major histocompatibility complex class I (MHC‐I). MHC‐I is the crucial mediator of the immune mediated allograft rejection in patients. A clinical grade ZFN targeting the Beta 2‐Microglobulin (B2M) gene was designed and was combined with adeno‐associated virus type 6 (AAV6)‐mediated delivery of donor DNA to insert the CAR expression cassette into the B2M locus through homologous recombination. An AAV6‐based toolbox, comprising several CAR expression cassettes harboring different promoters (PGK, EF1a, PGK100, EFS, FXP3.1 and FXP3.2) were evaluated to efficiently mediate targeted integration (TI) into the B2M locus and to allow robust expression of our CAR candidates. Our results showed potent MHC‐I knockout ranging between 70‐80% with no impact on viability and phenotype of edited CAR‐Tregs. Furthermore, all the tested AAV6‐CAR cassettes showed a comparable TI (40‐50%), resulting in the generation of 41‐80% of CAR+/MHCI‐ Tregs with variable CAR expression levels depending on the chosen promoter. Importantly, edited CAR‐Tregs showed a robust increase of CD69 activation marker following the CAR engagement with specific ligands, regardless of the CAR expression cassette used, indicating that our genome editing strategy does not compromise CAR functionality. In conclusion, our molecular toolbox can be applied to engineer allogeneic CAR‐Tregs for clinical applications.
Functional characterization of a dCas9‐MutH endonuclease
1: Justus‐Liebig‐University Giessen
Genome editing applications employ endonucleases to introduce double strand breaks (DSBs). When introducing two nicks in close proximity, a staggered DSB is generated and subsequently repaired. The aim of this study was to characterize a paired nicking endonuclease approach based on a fusion protein between the dead(d) Cas9 protein and the sequence specific cutting domain of MutH. The design of the paired nicking approach was that the MutH specific sequence GATC would be cut on both strands simultaneously through co‐transfection with specific gRNAs. The distance between binding site of dCas9 and cutting site of MutH was varied within the target sequence. Expression of proteins was verified by western blot analysis and activity of the endonuclease was assessed by use of the Bioluminescence resonance energy transfer (BRET) based DNA repair sensor in HEK293 cells. DCas9‐MutH was expressed in HEK293 cells and was found to be active as single as well as paired fusion protein only in a distance of 9 bp between binding and cleavage site. The activity was comparable to a control paired nicking endonuclease generated with (n)Cas9 variants targeting the same sequence. The sequence specific MutH‐dCas9 endonuclease generates DSB repair events when utilized as a paired nicking approach. Since both steps, DNA binding and cleavage, are sequence specific, this endonuclease represents an alternative with potentially lower off target activity for future applications in therapeutic genome editing.
Base editing of HLA for the generation of ‘universal’ human keratinocytes
1: UCL Institute of Child Health
Failure to repair wounds can result in chronic non‐healing ulcers with long‐term consequences in diabetes, vascular disorders, burn injuries, and certain genetic skin diseases. There is a notable social and economic burden on healthcare providers managing such complications. Skin autografts can be applied to refractory wounds, but applications are limited, and donor wound sites may be problematic. Allogenic keratinocyte sheets and sprays of keratinocytes with fibroblasts have been shown to reduce the healing duration of skin ulcers associated with chronic venous insufficiency.
‘Universal’ epidermal sheets from healthy donor keratinocytes devoid of HLA molecules may provide improved therapeutic strategies. Keratinocytes constitutively express HLA class I and upregulate HLA class II in response to cytokines like IFN‐γ. A third‐generation cytosine deaminase base editor was used to simultaneously disrupt B2M and RFXANK with relevant base changes of above 95% and 80%, respectively, resulting in significantly reduced HLA class I and II expression by flow cytometry to less than 5% and approximately 20%, respectively. Molecular studies characterised editing signatures and quantified possible translocations between sites of multiplexed editing. Immunological stealth was investigated in vitro and revealed blunted T cell activation, proliferation, and cytotoxic responses against HLA‐knockout (KO) compared to unedited keratinocytes. Despite HLA class I absence, natural killer cell responses against HLA‐KO keratinocytes were negligible in degranulation and cytotoxicity assays. Universal keratinocytes retained architectural potential in vitro in 3D skin equivalent models, with normal morphology and appropriately formed stratified epithelia. Additional functional and safety profiling investigations are underway ahead of preclinical modelling of therapeutic applications.
Characterization of CRISPR/Cas9‐induced genomic alterations and AAV targeted integration in human HSPC
1: Genethon, UMR_S951, Inserm, Univ Evry, Université Paris Saclay, EPHE
CRISPR/Cas9 technology has revolutionized our ability of genome editing and is a promising tool for gene therapy. However, recent studies show that it can result into extensive on‐target DNA damage. Additionally, little is yet known about the CRISPR mediated targeted integration outcomes in cell lines or clinically relevant cells. Here, we present a detailed analysis of the genomic events occurring upon CRISPR/Cas9 or Cas9 D10A mediated gene editing, alone or in combination with targeted integration of a therapeutic donor DNA. We profile in HUDEP‐2 and the clinically relevant Hematopoietic Stem/Progenitor Cells (HSPCs) the different outcomes of genome editing on our target site and the therapeutic knock‐in (AAV6 encoding for β‐globin directed to the editing site by homology arms) repair/integration outcomes. We perform long read sequencing of the targeted genomic locus using Oxford Nanopore Technology and the CRISPR/Cas9‐based enrichment procedure, avoiding PCR amplification and its potential biases. Cas9‐enrichment of a 14kb region of interest is performed and sequenced with an average 100X enrichment. We establish a bioinformatical pipeline to process the generated data and explore the structural variations (large deletions, insertions, etc) to evaluate the precision of our genome engineering. We demonstrate that large deletions occur at similar frequencies, ∼15%, in HUDEP and HSPC and we reveal the existence of complex rearrangements of integrated AAV genomes, ∼25% of the integrations. These data were confirmed by independent approaches as ddPCR and FACS analyses. This knowledge will be important in future clinical applications of the CRISPR/Cas9 gene‐editing tool.
Multiplex HDR for Modeling and Correction of RAG2 Severe Combined Immunodeficiency by CRISPR‐Genome Editing in Human CD34+ HSPCs
1: The Institute for Advanced Materials and Nanotechnology, The Mina and Everard Goodman Faculty of Life Sciences, Bar‐Ilan University, Israel 2: Pediatric Department A and the Immunology Service, Jeffrey Modell Foundation Center, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel 3: Sackler Faculty of Medicine, Tel Aviv University, Israel 4: The Division of Hematology and Bone Marrow Transplantation, Chaim Sheba Medical Center, Tel‐Hashomer, Israel
Severe combined immunodeficiencies (SCIDs) are a set of life threatening genetic diseases in which patients are born with mutations in one of more than twenty known genes and are unable to develop functional immune systems. While allogeneic bone marrow transplantation can be curative for these diseases, there remain significant limitations to this approach. In contrast to using viral vectors to deliver transgenes in an uncontrolled fashion, we are working towards developing CRISPR genome editing to correct the RAG2 disease‐causing mutations by precisely modifying the genome. Here we first developed a RAG2‐SCID disease model using CRISPR‐Cas9 and rAAV6 gene‐editing through biallelic knock‐out in healthy donor (HD)‐derived CD34+ HSPCs. Additionally, we established a knock‐in/knock‐out (KI‐KO) strategy to develop a proof‐of‐concept gene correction in HD CD34+ HSPCs. Lastly, we show successful gene correction of RAG2‐SCID patient‐derived CD34+ HSPCs which developed into CD3+ T cells with diverse TCR repertoires. Our system outlines an approach for the study of human lymphopoiesis and will allow researchers to determine the optimal configuration for CRISPR‐Cas9 gene correction for SCIDs and additional recessive primary immunodeficiency and blood disorders.
Enhancing SaCas9 target specificity by rational directed mutagenesis
1: OXGENE, Medawar Centre, Oxford Science Park Oxford, OX4 4HG, United Kingdom
Continued development of the CRISPR/Cas9 system is making therapeutic gene editing a viable biomedical tool. However, delivery of Streptococcus pyogenes (SpCas9) by adeno‐associated viral vectors (AAV) is challenging due to the small packaging capacity of AAV (∼4.7 kb). This can be overcome by using the minimal SpCas9 ortholog Staphylococcus aureus Cas9 (SaCas9). SaCas9 is compact enough to be packaged into AAV and therefore commonly used for in vivo genome editing. Nevertheless, the lack of fidelity remains a major barrier to clinical translation. While many high‐fidelity SpCas9 variants have been reported, far fewer SaCas9 variants have eliminated off‐target editing, leaving off‐target cleavage of unintended genomic sites a critical issue to be resolved for this ortholog.
Here, we developed a traffic light reporter plasmid that examine on‐ and off‐target editing efficiency and used it to screen rationally engineered SaCas9 variants. Using this system, we identified SaCas9 variants (variants harbouring mutations at position Y256, R314, Q414 and R654) with enhanced‐accuracy and single base mismatch discrimination whilst maintaining robust on‐target activity comparable to WT‐SaCas9. Using sanger and next generation sequencing we confirmed 50 to 95% reduced off‐target effects of our variants compared to WT‐SaCas9 at the three genomic loci. Finally, when delivered by an AAV vector our variants showed no detectable off‐target activity when targeting SHLD1 locus in HeLa cells compared with WT‐SaCas9. Thus, our variants could be used for genome editing applications where high precision is required.
FiCAT (find and cut‐and‐transfer), a programmable transpose for mammalian genome editing
1: Universitat Pompeu Fabra
Different technologies have been developed in the last few years for small allele genome editing opening a new path on cell and tissue engineereing and gene therapy. Nevertheless, targeted integration of large DNA fragments in mammalian genomes are still missing, while already in development in bacteria. We developed a gene delivery tool (FiCAT) combining the precision of a CRISPR‐Cas9 (find module), and the payload transfer efficiency of an engineered piggyBac transposase (cut‐and‐transfer module). FiCAT combines the functionality of Cas9 DNA scanning and targeting DNA, with piggyBac donor DNA processing and transfer capacity. We engineered PiggyBac functional domains to provide increased on‐target integration and reduced off‐target events.
FiCAT technology is applied in vivo in mouse models to target muscle and liver and deliver large payloads as a proof of concept, such as Lama2 gene in a Lama2 defficient mouse model by using lipid nanoparticles as carriers of the complete gene and the gene editing machinery.
Allele‐Specific gene editing approach for a dominant form of Epidermolysis Bullosa Simplex
1: University of Modena e Reggio Emilia 2: Holostem Terapie Avanzate s.r.l.
Epidermolysis Bullosa Simplex (EBS) is a rare skin dominant inherited disease. Patients display a skin fragility that leads to non‐scaring blisters and erosions caused by minor mechanical trauma. 75% of all EBS cases display mutations in keratin 5 (KRT5) and keratin 14 (KRT14) genes, that perturb the intermediate filament network formation in EBS basal epithelial cells. Besides palliative treatments, there is still no cure for this orphan disease and effective treatment may be achieved only through stem cell tailored gene editing approach. Here we propose a personalized gene editing strategy to correct human primary keratinocytes derived from a seven‐years‐old patient (EBS01), carrying a de novo monoallelic mutation in KRT14 gene. Via the CRISPR/Cas9 system we obtained a specific cleavage only on the mutant allele, restoring a corrected cellular phenotype and intermediate filament network in EBS01 primary cells. Gene editing also rescued primary keratinocytes' mechanical stress resilience, thereby leading to a complete restoration of mechanical properties. Due to a remarkable editing efficiency and in the absence of Off‐Target events that would determine genotoxicity in primary keratinocytes, we demonstrated a fully correction of epidermal stem cells, which play a crucial role in the regeneration of a permanently corrected epithelium. Furthermore scRNA‐sequencing data also outlined the restoration of inferred pathway after allele‐specific gene editing, delineating a wider impact of this molecular approach. These overall data suggest a potential safety use of gene editing approach in adult somatic stem cells.
Optimization of a shuttle peptide‐cargo conjugate for in vivo delivery to airway epithelial cells
1: Feldan Therapeutics 2: Université Laval / Département de Génie chimique
The delivery of biologic cargoes to airway cells offers great therapeutic opportunities but is challenged by the complex barrier properties of lung epithelia. We previously reported that non‐covalently combining amphiphilic shuttle peptides with proteins or CRISPR‐associated nuclease ribonucleoproteins enables their rapid and efficient delivery into human and mouse airway epithelial cells. In this work, we further improved in vivo airway delivery by chemically attaching the shuttle peptide to a cargo through a cleavable link allowing the cargo to be released once internalized. Here we present the synthesis and purification processes of shuttle‐cargo conjugates, using a nuclear localizing Cy5 labeled peptide as a cargo, and their delivery efficiency quantification in vitro using HeLa cells and in vivo in mouse lungs. Using a shuttle peptide‐cargo conjugate, fluorescence microscopy analysis showed an improved biodistribution up to the alveolar region of mouse lungs and an increase in the percentage of epithelial cells delivered from 30% to 60% when compared to the non‐covalently linked shuttle peptide and cargo. Enabling delivery of membrane‐impermeable cargoes to airway epithelial cells will pave the way to the development of unexplored therapeutic avenues to treat lung diseases.
In vivo dissection and correction of the tyrosine catabolic pathway using CRISPR‐Cas9
1: Centre Hospitalier Universitaire de Québec Research Center and Faculty of Medicine 2: Service de Génétique médicale, Département de Pédiatrie, Centre Hospitalier Universitaire de Sherbrooke 3: Sainte‐Justine University Hospital Center
In vivo somatic genome editing in animal models of human diseases is a powerful approach to provide insights into biological pathways and fuel the development of novel therapeutics. Programmable nucleases can be harnessed to elucidate and treat defects associated with rare metabolic disorders. In this study, we show that liver‐directed genome editing along the tyrosine catabolic pathway can modulate and correct the disease phenotype in a murine model of tyrosinemia type 1. Neonatal gene delivery using rAAV8 vectors expressing Staphylococcus aureus Cas9 (SaCas9) under the control of a liver‐specific promoter led to efficient gene disruption and metabolic rewiring at many enzymatic steps along the pathway with systemic effects. An intron targeting approach using Streptococcus thermophilus Cas9 (St1Cas9) that could correct most mutations known to cause hereditary tyrosinemia type I in humans also led to disease correction. We finally show that targeting the whole human FAH therapeutic gene to the 5′ untranslated region of the albumin locus also leads to disease correction in the treated animals. Our findings show that in vivo CRISPR‐Cas9 editing can profoundly rewire and restore a defective molecular pathway, fueling drug discovery and enabling novel therapeutic avenues for the treatment of inborn metabolic diseases.
Expanding the CRISPR‐Cas toolbox through interrogation of a massive metagenomic dataset coupled with protein evolution platform
M Demozzi1 E Pedrazzoli1
1: University of Trento, Department CIBIO, Laboratory of Molecular Virology, Trento, Italy 2: University of Trento, Department CIBIO, Laboratory of Computational Metagenomics, Trento, Italy 3: Alia Therapeutics s.r.l, Trento, Italy
Despite the enormous advancement in genome editing with the development of CRISPR‐Cas technology, several hurdles still limit its use for clinical purposes. Rational design strategies were proven valid tools to optimize Cas proteins by enhancing precision and activity. Nevertheless, data on Cas engineering show that improving specific Cas traits deteriorates others and molecular size cannot be modified. To circumvent these limitations our study aims at discovering and developing a novel CRISPR‐Cas repertoire responding to genome editing requirements through the interrogation of a massively expanded dataset of metagenome and virome assemblies by Pasolli et al. (Cell 176‐2019). By identifying a novel variety of Cas orthologs we may identify Cas with diverse PAM recognition sequences and reduced molecular size facilitating their delivery. From the interrogation of this database we currently selected 12 unreported CRISPR‐Cas nucleases (< 1100 amino acids), which showed variable editing efficacy in mammalian cells (both EGFP and endogenous loci). The most active Cas orthologs with molecular weight compatible with all‐in‐one AAV delivery were further tested for genome editing. Variants with desirable editing properties but reduced activity were introduced into an eukaryotic yeast platform to evolve into highly active variants. With this work we are setting a CRISPR‐Cas search pipeline to identify and optimize through directed evolution new tools for genome editing responding to the complexity of gene therapy approaches.
Methylation of rAAV vector DNA is not a mechanism for differential transgene RNA expression from rAAV gene therapy in mouse and NHP liver
1: BioMarin Pharmaceutical
In vivo delivery of recombinant AAV (rAAV) gene therapy has resulted in successful expression of encoded gene products, but variability between individual animals who received the same product and dose has reached 10‐fold. A substantial portion of the observed variability may be attributed to the number of RNA molecules produced per DNA molecule (the RNA/DNA ratio). We investigated vector DNA methylation as a candidate mechanism for variability of RNA production and transcriptional efficiency in mouse and non‐human primate (NHP) models. We hypothesized that CpG methylation in vector regulatory regions would inversely correlate with RNA expression.
DNA methylation was analyzed by bisulfite sequencing, and results were expressed as the ratio of methylated to non‐methylated CpG on the vector as a whole and at individual base resolution. Average CpG DNA methylation in host liver harvested up to one‐year post‐dose was 10‐ to 20‐fold lower on rAAV vector DNA than on genomic DNA. On the vector, CpG methylation was highest at the Inverted Terminal Repeats (ITRs). The CpG methylation was lowest in the enhancer‐promoter regions, the same regions with highest chromatin accessibility. Methylation at intermediate but still quite low levels was observed in downstream segments of the transgene. CpG methylation levels in downstream vector regions increased over time, reaching up to 40% at one‐year post‐dose, while methylation levels in the enhancer‐promoter region remained under 3%. Regression analysis of rAAV vector methylation and RNA expression do not support a substantial role for the methylation of CpG sequences in regulating transcription within or between species.
Targeted, non‐viral delivery of gene editing technologies for the treatment of DMD
A Bouchareb2 E Dyankova2 T Mahadeva2 A V Hangu2
1: 4BaseBio 2: 4Basebio
Duchenne muscular dystrophy (DMD) is a severe monogenic disease caused by mutations in the gene encoding dystrophin. While several trials are on‐going, including AAV‐mediated delivery of a truncated micro‐dystrophin, no clinically effective therapy is available to date. The emergence of CRISPR‐Cas and associated editing technologies provide an attractive therapeutic option for the treatment of DMD, however delivery to muscle remains a challenge.
4basebio has developed a proprietary, non‐viral nanoparticle that targets muscle cells with high specificity and low immunogenicity, allowing for repeat dosing. The Hermes™ particle is payload agnostic and has been used to deliver DNA, mRNA and proteins to muscle cells both in vitro and in vivo.
In this study, we utilised the muscle targeting particle to deliver Cas9 protein with an sgRNA targeting the DMD gene in C2C12 myoblasts and myotubes. Particles had highly favourable biophysical characteristics, stability, encapsulation of payload, and achieved high KO efficiency in relevant cell models.
To investigate a gene‐integration approach using our plasmid‐free synthetic DNA technology, we designed an oeDNA™ template encoding the cDNA sequence of the Dystrophin gene with flanking homology arms. The co‐delivery of template DNA along with the RNP complex encapsulated in our Hermes™ nanoparticle resulted in a successful integration of the oeDNA template, and enhanced protein expression in vitro. Mouse studies confirming these findings are ongoing.
The present study provides proof‐of‐concept for the use of Hermes™ particles to deliver RNP + donor templates to reframe or fully replace a dysfunctional gene, offering a promising approach for DMD therapy.
Base editing as a potential therapeutic strategy for motor neuron diseases
1: 2: Imagine Institut
Amyotrophic Lateral Sclerosis (ALS) is caused by a number of mutations, with C9orf72 repeat expansions as the most common genetic cause. Due to the limitations of current pharmacological therapies, there is a strong need to develop innovative approaches. The goal of this study is the development of genome editing approaches aimed at correcting the disease‐causing mutations. The genomic engineering strategy adopted is a CRISPR‐Cas9‐based genome editing technology that allows the introduction of point mutations in the DNA without generating DSBs, using recently developed, highly precise enzymes: cytidine base editors (CBEs) and adenine base editors (ABEs) allowing C > T and A > G conversions, respectively. Several single‐guide RNAs (sgRNA) were designed to target C9orf72 within the region containing the repeat expansions with the aim of boosting C9orf72 expression. This strategy was validated in the K562 cell line. In particular, at least three optimal sgRNA candidates resulted in up to 45% ± 5 base editing efficiency, that consequently resulted in 2‐fold change increase of the C9orf72 transcripts levels. This strategy is currently being validated in induced pluripotent stem cell line (iPSCs) derived from ALS patients and differentiated to motor neurons in order to determine whether increasing the expression of C9orf72 will lead to amelioration of several cellular phenotypes in motor neuron cultures. We will also carry out proteomics and metabolomics analyses to define molecular pathways and targets in vitro that are altered upon increased expression of C9orf72 to further establish the potential of genome editing as a therapeutic strategy for ALS patients.
Characterization and deployment in vivo of FiCAT genome writer for therapy
J Martínez de Lagos1 2 N Robles1 J Jaraba‐Wallace1 M Güell1 2
1: Integra Therapeutics 2: Universitat Pompeu Fabra
Find Cut‐and‐Transfer (FiCAT) is a robust gene writing platform, combining the precision of CRISPR‐Cas9 systems, with an engineered piggyBac transposase with donor DNA processing and high transfer capacity. Here, with the aim to demonstrate FiCAT activity in vivo, we performed precise gene delivery to mouse models liver cells using viral‐free delivery vectors via systemic administration. We were able to efficiently deliver FiCAT to mice liver targeting genomic safe harbors, together with a reporter transposon in DNA form. Stable transduction was confirmed by qPCR analysis of the inserted transgene and at 6 weeks after transduction. Lipid nanoparticles (LNPs) have been extensively validated both pre‐clinically and clinically for the delivery of different nucleic acid cargos. Once optimized FiCAT mRNA‐based LNP formulation, specific cut capacity in mice liver was analyzed by targeted NGS. In this work, we were able to successfully deliver mRNA cargos via LNPs in vivo, and optimize DNA cargos delivery for codelivery with FiCAT machinery; for enabling gene writing in vivo. Our work validates the feasibility of doing viral free stable gene transfer in vivo; which is one of the challenges in our desire to deliver safely and efficiently therapeutic genes into the target cells and achieve precise and efficient in vivo targeted insertion of DNA fragments in mammalian genomes.
Evaluation of gene expression regulation and function after CRISPR/Cas9‐based genome editing strategies for the treatment of RAG1 defects
1: San Raffaele‐Telethon Institute for Gene Therapy, IRCSS San Raffaele Scientific Institute, Milan, Italy. 2: Translational and Molecular Medicine (DIMET), University of Milano‐Bicocca, Monza, Italy. 3: Milan Unit, Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Milan, Italy. 4: Vita‐Salute San Raffaele University, Milan, Italy 5: IRCCS Humanitas Research Hospital, Rozzano, Italy. 6: National Research Council, Institute for Biomedical Technologies, Segrate, Italy. 7: Gene Therapy Program, Dana‐Farber/Boston Children's Cancer and Blood Disorders Center, Department of Pediatric Oncology, Harvard Medical School, Boston, MA, USA. 8: Laboratory of Clinical Immunology and Microbiology, NIAID, NIH, Bethesda, MD, USA.
Recombination‐activating gene 1 (RAG1) is a key player of the V(D)J recombination process, which occurs during B and T cell differentiation. RAG1 mutations are responsible for a variety of clinical phenotypes, caused by partial/complete absence of recombination activity. With the aim of correcting the RAG1 gene on patient‐derived haematopoietic stem/progenitor cells, we exploited a NALM6 RAG1 knock out (KO) cell line to develop and optimise two alternative CRISPR/Cas9‐based editing strategies: one targeting the intron, the other targeting the coding exon. NALM6 RAG1 KO cells edited with a specific ribonucleoprotein and an adeno‐associated vector carrying a corrective donor template for each strategy were subcloned by single cell sorting and screened for mono‐ or biallelic editing. Levels of correction were characterised by performing gene expression analysis and recombination assays. With the intronic strategy, we obtained a high variability between edited clones: some missed restoration of expression and function, while others showed a good level of correction. On the contrary, all edited clones with the exonic strategy displayed a significant expression and function of RAG1. To further investigate the variability among our intronic edited clones, we extended our studies, including karyotype analysis to exclude the presence of chromosomal abnormalities caused by the editing procedure. Since the majority of reported and successful CRISPR/Cas9 editing strategies target exonic regions of different loci, we are going to assess the transcriptional and translational stability of both intronic and exonic edited clones with epigenetic studies to deeply define chromatin conformational changes and accessibility after genome editing.
Gene editing for the New Zealand clinic
C Malhi1 J Hunt1 A du Rand1 S Meidinger1 D Verdon1 R Dunbar1 K Lenhert1 Y Michaels2 D Knapp3 D Purvis4 V Feisst1
1: The University of Auckland 2: The University of British Columbia 3: The University of Montreal 4: Auckland District Health Board
Conditions that affect either skin or blood cells are attractive targets for gene editing applications due to their ease of access.
Skin ‐ we are targeting epidermolysis bullosa (EB), a severe, monogenetic fragile skin condition. Using our patented skin‐growing techniques we aim to engineer gene edited, patient‐specific skin in vitro, which can be used to permanently cover chronic EB wounds. Our optimised CRISPR/Cas9 editing methods yield >95% non‐homologous end joining (NHEJ)‐mediated repair efficiencies in patient‐derived primary keratinocytes and fibroblasts. These rates allow for a successful exon‐skipping approach to remove defective exons and thus repair collagen VII in cells derived from NZ patients. Other EB‐causing mutations require a homology directed repair (HDR) approach to make specific edits in the gene of interest. We can achieve targeted edits in >30% of a patient‐derived keratinocyte or fibroblast cell population. Off target analysis (using nanopore sequencing to rapidly deconvolute edits in compound heterozygote mutations) and functional assays are in progress.
Blood ‐ we are dissecting immune checkpoint inhibitor biology by editing primary human T cells and melanoma cell lines to fine‐tune key molecules involved in these pathways (specifically PD‐1 / PD‐L1). We can achieve high knockout rates in both cell types (>95%) and targeted knockin rates (HDR) in >65% of a T cell and >98% of a melanoma cell population. The functional consequences of modulating these pathways are currently being analysed. Results from this research may be used to improve checkpoint inhibitor therapies and to engineer optimal cancer‐killing T cells for use in adoptive immunotherapies.
Development of a clinically relevant gene‐editing strategy to correct the Finnish‐founder pathogenic mutations causing Familial Hypercholesterolemia and ASL
1: University of Helsinki 2: Department of Pediatrics, Helsinki University Hospital 3: Department of Clinical Genetics, Helsinki University Hospital
This project aims to develop the first gene‐editing platform to correct the Finnish‐founder mutations causing Familial Hypercholesterolemia (FH‐Pogosta) and Argininosuccinate lyase deficiency (ASL). These two independent diseases present an exceptionally high prevalence in Finland and represent a life threat to hundreds of patients. In these diseases, a C•G base‐pair substitutes the original T•A. Therefore, the CRISPR adenine base editor (ABE), capable of correcting the C•G>T•A mutations, is a strong candidate to tackle the genetic cause of these illnesses.
Our first objective was to collect skin biopsies from four patients, correct the genetic mutations in vitro and generate patient‐derived induced pluripotent stem cells (hiPSCs). To this end, we have developed a novel approach for simultaneous hiPSC generation and single base correction with unprecedented speed and efficiency (Jalil et al., 2021). Our current goal is to design and compare clinically relevant delivery systems that allow an in vivo therapeutic application of the ABE. For this purpose, we test and optimise different viral vectors and RNA Lipid Nanoparticles (LNPs) capable of efficiently targeting patient hiPSC‐derived hepatocytes.
As an essential metabolic centre, the liver plays a central role in FH‐Pogosta and ASL. By correcting the pathogenic mutations in patient hiPSC‐derived hepatocytes and assessing their phenotypic restoration, we seek to understand the therapeutic potential of the ABE to treat FH‐Pogosta and ASL.
CX3CR1 intron 4 targeting provides a novel safe harbour for Trojan Horse strategies to treat monogenic disorders
1: Universidad de Granada 2: GENyO‐ Centro de Genomica e Investigacion Oncologica: Pfizer / Universidad de Granada / Junta de Andalucia 3: Fundación Pública Andaluza para la Investigación Biosanitaria en Andalucía Oriental Alejandro Otero 4: Fundacion Publica Andaluza Progreso y Salud
Success of ongoing ex‐vivo gene editing clinical trials exploiting Haematopoietic Stem Progenitor Cells (HSPCs) is undeniable. However, there are still many concerns regarding editing safety and repopulation efficacy that need further refinement. We have previously identified CX3CR1 as locus with primed myeloid expression, with potential to be use for the insertion of therapeutic cassettes on ex‐vivo approaches. Herein, we targeted efficiently the 4th intron of this locus with CRISPR/Cas9 system on HSPCs (>90% indels). Next, we ranked several AAV6 donor templates based on different insertional DNA repair mechanisms to set an optimized targeting protocol on HSPCs and successful gene editing was achieved (>50% stable GFP expression). However, despite using a strong viral promoter GFP expression presented a myeloid skew pattern. Furthermore, we observed increased levels of CX3CR1 protein and mRNA in HSPCs, but after macrophage differentiation this modulation of CX3CR1 is lost. Curiously, no colonies from CFU's assays, T cells nor THP1 cells showed any CX3CR1 increment after targeted addition with same AAV6. We propose that this CX3CR1 increment restricted to stem states of HSPCs, could be beneficial for the migration of these cells and their derivates towards inflamed tissues, including CNS. Thus, CX3CR1 targeting could improve current Trojan Horse strategies for monogenic diseases. Our preliminary results encourage the use of CX3CR1 as a safe harbour for CRISPR/Cas9 targeted insertion. Hence, we are testing the safety and biodistribution of our modified HSPCs in‐vivo to help a future translation of this strategy for the expression of therapeutic transgenes in monogenic disorders.
Enabling stable expression of cytotoxic envelope glycoproteins through virus cell receptor knock‐out for continuous lentiviral vector production
1: IBET ‐ Instituto de Biologia Experimental e Tecnológica 2: ITQB NOVA ‐ Instituto de Tecnologia Química e Biológica António Xavier
Lentiviral vectors (LV) recently became the most used viral vector in gene therapy clinical trials with several market approved products. Nonetheless, their manufacture still faces several challenges, namely the low yields and poor scalability of current production systems, hindering the transition from clinical to large‐scale production settings. Therefore, establishing efficient production processes based on high‐titer constitutive producer cells is paramount to sustain the increasing demand of LV‐based gene therapies.
Herein, we describe a new approach for establishing constitutive LV producer cells stably expressing GaLVΔR, an engineered, high‐titer envelope glycoprotein. GaLVΔR promotes a 5‐fold increase in LV titers relative to the original glycoprotein. However, due its fusogenic properties, GaLVΔR leads to syncytia formation. To overcome GaLVΔR cytotoxicity, we used CRISPR‐Cas9 gene editing to establish a producer cell host knocked‐out for the cell receptor of GaLV (inorganic phosphate transporter‐1, PiT‐1). PiT‐1 knock‐out (KO) cells supported GaLVΔR stable expression without syncytia formation, while maintaining transient LV titers. These cells were further explored for the development of constitutive LV producer cells. To that end, PiT‐1 KO clones were isolated by limiting dilution and selected by a functional screening based on resistance to GaLVΔR transduction. From 37 clones, 6 supported stable transfection with GaLVΔR, which are currently being used to develop new constitutive LV producer cells.
This work demonstrates the feasibility of stably expressing fusogenic envelopes by abolishing the expression of the corresponding receptors in the producer cell host. It also provides new cell substrates currently being explored to enable constitutive LV production.
CRISPR‐Cas9 mediated knockdown of PIK3CA to restore the therapeutic response via apoptotic pathway in breast cancer
1: Cukurova University AGENTEM (Adana Genetic Diseases Diagnosis and Treatment Center), Adana, Turkey 2: Cukurova University, Institute of Natural and Applied Sciences, Dept. of Biotechnology, Adana, Turkey 3: Cukurova Technopolis InfoGenom, Adana, Turkey 4: Cukurova University, Faculty of Medicine, Dept. of Medical Genetics, Adana, Turkey
CRISPR‐Cas9 is pioneering the cancer studies including carcinogenesis mechanisms, cancer therapeutics and biomarker discovery among all genome modification methods. Thus, the implementation of this technology makes it hotspot for novel therapeutic strategies in cancer. In this study, PIK3CA gene knockdown via CRISPR‐Cas9 have been tested to enlighten the role on apoptosis and cell proliferation via PI3K/Akt1/mTOR signalling pathway as the most promising target in hormone‐positive and HER2‐negative breast‐cancer due to the recent developments of pan‐PI3K/isoform specific‐inhibitors or combine inhibitor therapies. Exon‐9 of PIK3CA gene was modified on MCF‐7 breast‐cancer cell line using CRISPR‐Cas9 through our previously optimized electroporation protocol. Cell death was tracked using Annexin‐V/PI flow cytometry assay for 3‐days in different timelines of transfection(24‐48‐72h), and cells were examined under inverted microscope for adhesion and morphology changes. Then, the expression profile was examined for PIK3CA, AKT1, MTOR and NFKB1 genes via RT‐PCR(RT2 Profiler PCR array, Qiagen). As the result of the study, modified cells have undergone apoptosis after 24‐hours, and early apoptotic cell population became its highest at hour‐48 with 40% of total cell population. We also detected significantly reduced PIK3CA(fold‐change = 0.50), AKT1(fold‐change = 0.15) and MTOR(fold‐change = 0.44) expressions at hour‐30 which may lead to cell death while NFKB1 gene had 1.57 fold‐change when compared to the non‐modified MCF‐7 control group. In conclusion, the possible therapeutic outcomes of an implemented CRISPR‐Cas9 strategy to knockdown PIK3CA for inducing apoptosis. The stand‐alone PIK3CA inhibitor treatment for wider‐range of breast‐cancers together with CRISPR‐Cas9 strategy as a novel combinatorial therapy should be considered according to our findings.
Next‐generation ex‐vivo gene editing of human hematopoietic stem cells by optimized delivery platforms and selection of cells bearing the intended edits
1: San Raffaele Telethon Institute for Gene Therapy (HSR‐TIGET) 2: Vita‐Salute San Raffaele University 3: Pediatric Immunohaematology Unit and BMT Program, San Raffaele Scientific Institute 4: Inserm UMR1089 5: Institute for Genetic and Biomedical Research, Italian National Research Council 6: Institute for Biomedical Technology, Italian National Research Council 7: Equally contributing author
Gene editing by homology‐directed repair (HDR) in human hematopoietic stem/progenitor cells (HSPCs) is a promising therapeutic strategy. However, p53‐mediated DNA damage response, low HDR efficiency and poor characterization of unintended genotoxic outcomes challenge its clinical translation. Here, we show that the most common HDR template vehicle, Adeno‐Associated Vector (AAV), causes prolonged Mre11‐Rad50‐Nbs1‐dependent p53 activation upon accumulation of AAV genomes, which frequently integrate as transcription‐competent ITR fragments at Cas9 nuclease on‐/off‐target sites. Optimized Integrase‐Defective Lentiviral Vector (IDLV)‐based gene editing outperforms the AAV‐based one by improving editing efficiency and reducing the intracellular load of viral DNA copies, which shortens p53 induction and lowers the frequency of DNA fragments integration. Furthermore, bypassing electroporation using lipid nanoparticle (LNP)‐based delivery of the nuclease strongly alleviated the toxicity of the procedure, improving HSPC clonogenic potential and preserving similar editing efficiencies. To overcome the limitations to future therapeutic applications imposed by the large fraction of HSPCs that do not undergo HDR upon editing treatment and to simultaneously purge out those carrying on‐target long‐range genomic deletions (>10%), we developed a versatile platform for the positive selection of HSPCs bearing the intended HDR edit based on transient Selector expression by Means of Artificial Transcriptional activators (SMArT). SMArT enriched HSPCs edited at ∼30% to nearly 100% and significantly reduced the frequency of long‐range deletions. The selected cells yielded a fully edited human graft in immunodeficient mice with the selector no longer detectable. Overall, we envisage that these strategies will advance HDR gene editing towards safer and more effective clinical translation.
Targeted Locus Amplification: an unbiased NGS technology for genetic characterization and QC of genetically modified cells
J Bergboer1 I Sergeeva1 E Splinter1 M Yilmaz1 J Swennenhuis1 M Cangiano1 G Georgiou1
1: cergentis 2:
Targeted Locus Amplification (TLA) is a NGS technology based on proximity ligation that allows for extensive, precise genetic characterization of genetically engineered cells. By targeting the vector and generating broad sequencing coverage into the host genome, it can detect integration hypothesis‐free and with high sensitivity. At the same time, the method will detect any small or structural variants in the vector sequence or in the host genome around integration sites.
TLA is frequently used for the genetic validation of stable vector producing cell lines for cell and gene therapy, to detect integrations by random transposon‐ or viral vector‐based gene transfer as well as targeted genome editing technologies such as CRISPR/Cas9 and TALEN, whether 1 or 10’s, whether partial or intact copies, whether on‐target or homology‐independent. In a study of different genetic engineering methods in CAR‐T cells the integration site distribution determined by TLA was shown to correlate well with TCR expression and functionality.
TLA has also been used for genetic risk assessment and release testing of cell therapy medicinal products. TLA can proof a heterogeneous genome‐wide integration pattern without dominant integration sites. Simultaneously, the method will reveal any dominant SNVs in the integrated sequence. Results can be further assessed with regard to e.g. insertional oncogenesis.
Altogether, TLA has proven to be a powerful technology which can address several genetic questions at the same time. This is especially helpful for characterization of CGT products, which usually have limited quantities, while extensive characterization is required.
Beyond conventional CRISPR screening approaches – pooled screens using Cas12a
1: Oxgene
In the past, most CRISPR screening methods within drug target discovery campaigns were based on Cas9. However, while Cas9 is highly efficient in introducing single mutations, its multiplex application is still limited by its guide RNA (gRNA) processing. Cas12a can outperform Cas9 in multigene screens as it harbors an intrinsic RNase activity that enables multigene editing using a single RNA transcript.
At OXGENE, a WuXi Advanced Therapies Company, we have established a highly efficient Cas12a system that is suitable for combinatorial large‐scale loss‐of‐function screens. The editing activity of multiple Acidaminococcus Cas12a (AsCas12a) expression cassettes was tested in HEK293 cells by targeting the essential gene, CDK1, and non‐essential gene, STAG2. We observed high mutation rates within the STAG2 locus and could associate a lethal phenotype response with AsCas12a editing within the CDK1 locus. A pilot library, consisting of 100 guide arrays with four guides each, was successfully validated in a CRISPR pooled screen using an optimised next‐generation sequencing strategy. In addition, we have optimised a previously published genome‐wide knock‐out Cas12a guide library, allowing us to target up to 17,500 human genes.
The use of our here‐established AsCas12a multiplex screening system minimises the quantity of constructs needed, thereby reducing the number of cells required for CRISPR screens. Moreover, it has been reported that AsCas12a has higher fidelity compared to Cas9, which reduces the risks of off‐target effects. Ultimately, our AsCas12a system will boost therapeutic explorations by drawing connections between genomics, disease phenotypes and therapeutic targets.
Precise Gene editing as a promising therapeutic strategy for hematopoietic diseases
1: Institute for Transfusion Medicine and Gene Therapy & Center for Chronic Immunodeficiency, Medical Center – University of Freiburg, 79106 Freiburg, Germany
Genome editing has emerged as a promising strategy to treat various disorders affecting the hematopoietic system. In principle, gene editing of the underlying defect by homology‐directed repair (HDR) can be attempted to revert the specific disease‐causing mutation. However, therapeutic applications of DNA nucleases suffers from the intrinsically low frequency of HDR compared to NHEJ at target site. We have demonstrated that by complementing the necessary effectors with Cas9 we can inhibit NHEJ and increase HDR events at targeted site. Using our HDR‐CRISPR we were able to achieve 5 fold increase in precise genome editing as compared to the normal Cas9. To improve this platform we developed a novel linked‐template HDR CRISPR system (LT‐HDR‐CRISPR) that is capable of enriching the repair template at cleavage site further promoting HDR‐mediated repair. Combining the LT system with HDR‐CRISPR, we achieved more than 35% HDR events in reporter cell line, 7‐fold higher than a normal Cas9 and 1.7‐fold higher than the previously established HDR‐CRISPR. Ongoing experiments explore the use of LT‐HDR‐CRISPR to correct Fanconi Anemia (FA) specific mutations in‐patient‐derived hematopoietic stem cells. We believe LT‐HDR system offers the opportunity to pursue precise genome editing when DNA repair mechanisms are impaired as a consequence of the underlying genetic defect. Further development of the LT‐HDR‐system will certainly promote the future application of precise genome editing to develop novel therapies for hematopoietic defects.
Establishment of erythroid cellular models recapitulating β‐thalassemic mutations by base‐editing techniques
1: Genethon, UMR_S951, Inserm, Univ Evry, Université Paris Saclay, EPHE 2: Cell and Gene Therapy for Hematological Disorders Unit, Department of Oncology‐Hematology, IRCCS Ospedale Pediatrico Bambino Gesù
β‐thalassemia is one of the most common monogenic disorders worldwide caused by several mutations in the β‐globin gene (HBB). Mutations in HBB result in either reduced or absent synthesis of the β‐globin chain, which is normally coupled with two α‐globin chains to form the hemoglobin tetramer. Excess of free α‐globin chains causes severe red blood cell dysfunctions including ineffective erythropoiesis, anemia and impaired oxygen delivery.
Genome and base editing (BE) paved the way in reverting disease‐causing mutations for many genetic disorders. The development of base editors enables the efficient single‐nucleotide conversion at the desired target site even in non‐dividing cells. However, the use of BE for β‐thalassemia is limited by the paucity of cellular models harboring the exact mutation responsible for the disease.
Here, we used BE technologies to introduce a spectrum of β‐thalassemia‐causing mutations in erythroid‐specific cellular models. We reproduced mutations in the promoter, initiation codon, intronic and exon regions of the HBB gene known to affect different stages of β‐globin production. Delivery of BE systems resulted in an efficient target of the desired loci, with no toxic effects, which led to a either reduced or abolished β‐globin expression. Moreover, preliminary results on erythroid differentiation revealed a reduced erythropoiesis ability of mutation‐harboring cells compared to untreated ones.
With this study, we established a cohort of mutation‐specific cellular models with the dual purpose of (i) finding new insights about cellular mechanism of b‐thalassemia; and (ii) testing therapeutic BE protocols for the reversion of β‐thalassemia‐causing mutations.
A high throughput platform for CRISPR/Cas12a mediated manufacture of engineered induced pluripotent stem cell lines
1: Oxford Genetics Ltd
Human induced pluripotent stem cell (hiPSC) technology has propelled the stem cell biology field; the ability of iPSCs to give rise to almost any cell type in the body offers opportunity for modelling diseases, drug discovery and cell therapy. Gene editing hiPSCs can be challenging due to their sensitivity to spontaneous differentiation and their reported low editing efficiency. Here we present an automated, streamlined pipeline for generation of simple and complex gene modifications in iPSCs. We first optimise transfection conditions, recovery from single cell sorting and determine copy numbers of target genes. In‐house software, GRNADE and PRIMAPE, enables optimal sgRNA and primer design. We use ssODNs, the preferred template for HDR applications, for precise genome edits, and ascertain on‐target cleavage and HDR efficiencies using in‐house CRITIC software. We single cell sort transfected cells and use CellMetric® imaging software for automated monitoring to assure single cell clonality. Hamilton‐robotics facilitate automated clone transfer for effective expansion. Clone screening is performed by CRITIC, followed by NGS analysis for validation. We assess quality of generated clones through expression analysis of the pluripotency markers, and through karyotype assessment. Using this pipeline, we have generated knockouts and knock‐ins in multiple different stem cell culture systems, for multiple applications from basic biology studies to complex disease modelling. We routinely achieve >95% editing efficiencies and up to 30% HDR efficiency using our optimised conditions. Coupling CRISPR/Cas technology with high‐throughput robotics facilitates scaling of simple complex genetic modifications, enabling large scale exploration of disease models.
Targeting two common pathogenic CRB1 variants with RNA‐editing
1: Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, OX9 3DU, United Kingdom 2: Oxford Eye Hospital, Oxford University Hospitals, NHS Trust, Oxford, OX3 9DU, United Kingdom
Mutations in the CRB1 gene are associated with early‐onset retinal degeneration for which no therapy currently exists. RNA‐editing with CRISPR‐associated protein 13b (Cas13b) coupled to a deaminase domain (ADARDD) has been shown to correct single nucleotide variants (SNVs) in vivo and in vitro. The functional consequence of the most common pathogenic CRB1 variant, c.2843G>A, located in the first nucleotide of exon 9, as well as the pathogenic splice variant c.2842 + 5G>A, located in the splice donor site of intron 8, was evaluated. The feasibility of RNA‐editing to correct these SNVs was then assessed with a dual luciferase assay containing an out‐of‐frame CRB1 exon 8‐intron 8‐exon 9 DNA sequence, correct splicing of which would lead to an in‐frame sequence and firefly luminesce. The splice variant c.2842 + 5G>A resulted in a read‐through of the splice donor site, generating no firefly luminescence. RNA‐editing attempts targeting this SNV did not result in a significant restoration of firefly luminescence, while firefly luminescence was restored by up to 30% when targeting a nonsense variant upstream of the splice site. The pathogenic variant c.2843G>A resulted in a significant reduction of firefly luminescence due to a read‐through of the splice acceptor site in 60% of the splice products. RNA‐editing targeting the pre‐mRNA did not restore firefly luminescence, while RNA‐editing targeting the mRNA achieved editing rates up to 80%. These results indicate the most common CRB1 missense variant may also affect splicing. Results suggest low efficiency of RNA‐editing when targeting splice variants and good efficiency for correcting SNVs in exonic sequences.
Significant transgene repression using the dCas9‐KRAB‐MeCP2 repressor system
1: Imperial College London
Lentiviral vectors are RNA‐based gene transfer agents widely used in the field of gene and cell therapy. Packaging of lentiviral vectors encoding certain transgenes can negatively impact virus titres which represents a significant hurdle faced in vector manufacturing. Repression of such transgenes during vector production has been shown to rescue titres. However, most repressor systems incorporate exogenous DNA sequences within the transgene expression cassette, increasing the risk of immune recognition when introduced into the host system. To investigate transgene repression, we used a clustered‐regularly‐interspaced‐short‐palindromic‐repeats (CRISPR) deactivated Cas9 (dCas9) repressor system that consists of the Krüppel‐associated‐box (KRAB) and methyl‐CpG‐binding‐protein‐2 (MeCP2) transcriptional repressors fused to dCas9; a fusion complex that was shown previously to efficiently repress genes in mammalian cells. A guide RNA (gRNA) directing the dCas9‐KRAB‐MeCP2 complex to a ubiquitous promoter driving transgene expression were cloned together with the repressor complex onto the Rev packaging plasmid required for lentiviral production. Co‐transfection of dCas9‐KRAB‐MeCP2 with a Firefly luciferase reporter plasmid in HEK293T cells resulted in a median repression of 94.1% (range 85.6‐95.8%) and 92.8% (range 90.1‐94.2%) compared to a control plasmid omitting the gRNA (n = 6 wells per condition, two independent repeats). Assessment of the functionality of dCas9‐KRAB‐MeCP2 repressor system in the context of lentiviral vector production needs to be investigated in future studies. Preliminary data shows the CRISPR‐dCas9 repressor platform could be optimised and scaled‐up generating higher titres and lowering costs.
Response Surface Methodology for rapid optimisation of gene editing
1: Molecular and Cellular Immunology, UCL Great Ormond Street Institute of Child Health, University College London, London, UK 2: NIHR Great Ormond Street Hospital Biomedical Research Centre, London, UK
Development of new gene therapy approaches for primary immunodeficiencies generally requires extensive pre‐clinical optimisation of the correction parameters in order to balance the insertion of the desired sequence with deleterious effects such as cell death, growth arrest, and genotoxicity. Generally, one factor is varied at a time. This optimisation process can be open‐ended, time‐consuming, and recursive due to interactions between factors, and the criteria for choosing the best‐performing conditions are not always strictly defined. We have developed a mathematically defined optimisation strategy based on Response Surface Methodology, which allows complete and definitive multi‐dimensional optimisation of multiple continuous variables in a single experiment and reveals the landscape of the correction/fitness trade‐off.
A dual‐central composite design allows a multidimensional response surface curve to be fitted to continuous variables (MOI, Cas9, and gRNA:Cas9 ratio), with the % integration, loss of cells compared to unedited and loss of colony‐forming units (CFU) compared to unedited evaluated as responses. We found that there was a continuous decline in cell number and CFU when increasing MOI and nuclease activity, but that construct integration rapidly saturated. We optimised % integration against CFU and cell number losses to maximise the potential of a single apheresis. The optimum conditions were consistent for two unrelated constructs and across unrelated donors despite variable donor correction levels and sensitivity. When optimum conditions were applied to CD34+ cells for treatment of p67‐deficient Chronic Granulomatous Disease or Wiskott‐Aldrich Syndrome, we obtained high levels of correction with a minimal loss of cells and colony‐forming units.
Kilobase‐scale directed knock‐in with all‐in‐one lentivector carrying Cas9 protein, gRNA, and donor DNA
D Lichtscheidl1 I Hoger1
1: University of Veterinary Medicine Vienna
Site‐specific introduction of kilobase‐sized foreign DNA into endogenous loci is a long‐standing goal in the field of genome editing. However, current tools are limited by low efficiency and the need for multiple vectors. To address these drawbacks, we developed a lentivector carrying all the components needed for site‐directed knock‐ins including the Cas9 protein, a template for both the guide RNA and donor DNA carrying multiple genes and the guide RNA target site. Transduction of the “all‐in‐one” lentivector into target cells resulted in simultaneous cleavage of the genomic and donor DNA, followed by the donor insertion into the double‐strand chromosomal breaks via the non‐homologous end‐joining pathway. Further modification ‐ the introduction of a single homology arm into the donor DNA ‐ allowed site‐directed insertions via the non‐canonical homology‐directed repair (ncHDR) pathway. Although less efficient than the NHEJ‐mediated insertions, the ncHDR pathway‐repaired junction sites contained virtually no indels, which were frequently detected in the NHEJ‐corrected junctions. This work establishes that lentivectors can be engineered into a versatile modular tool for kilobase‐scale directed knock‐ins.
Development of CRISPR‐Cas‐based strategies for spinocerebellar ataxia type 2
1: Algarve Biomedical Center Research Institute (ABC‐RI), Portugal 2: Faculdade de Medicina e Ciências Biomédicas, Universidade do Algarve, Portugal 3: PhD program in Biomedical Sciences, Faculdade de Medicina e Ciências Biomédicas, Universidade do Algarve, Portugal 4: Faculdade de Farmácia, Universidade de Coimbra, Portugal 5: Center for Neuroscience and Cell Biology
CRISPR‐Cas‐mediated genome engineering allows one to precisely alter any genomic sequence specified by specific stretch of guide RNA. This is particularly advantageous for neurodegenerative diseases caused by genetic mutations. Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant disorder caused by an abnormal expansion of the trinucleotide CAG in coding region of the ATXN2 gene. This is translated into an abnormally long tract of glutamines in the ataxin‐2 protein, which leads to a gain of toxic function. Besides, the pathogenic mechanisms triggered by ataxin‐2 dysfunction, previous studies showed that abnormally expanded CAG repeats display toxicity already at the RNA level, contributing for disease pathogenesis. Considering these data, it is important to design therapeutic approaches that act at the most upstream point of the pathogenic cascade. Therefore, the present work focuses on the development of CRISPR‐Cas‐based strategies to silence the expression of ATXN2. For that, we designed a CRISPRi strategy whereby inactive, but still targetable Cas9 is fused to a transcriptional repressor, KRAB, and is directed to four sites of the ATXN2 promoter, hindering the activity of the transcription machinery. Additionally, we designed a second strategy based on the Cas9 activity directed to two early sites of ATXN2 gene, to excise the intervening region and compromise the integrity of the reading frame. Although SCA2 still remains an incurable disorder, silencing the pathogenic expression of ATXN2 seems to be a promising therapeutic approach for treatment or even cure of SCA2 patients.
Development of splice switching antisense oligonucleotides targeting midkine
J M Cale1 M T Aung‐Htut1 S D Wilton1
1: Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Western Australia 2: Lyramid Ltd, Sydney Australia
Midkine is a multi‐functional growth factor/cytokine that is highly expressed in many organs during embryonic development followed by down‐regulation at birth. However, in most disease settings midkine expression increases in local tissues with systemic levels elevated 10 to 100‐fold. Midkine mediates diverse cell‐cell interactions and signaling pathways, and thereby contributes to metastasis, resistance to immune checkpoint inhibitors and angiogenesis in tumours.
Biologicals including siRNA, aptamers and antibodies can inhibit midkine in animal models of cancer, autoimmunity and chronic inflammatory diseases in various organs. However, splice switching antisense oligonucleotides (SSOs) have not been evaluated for their ability to not only reduce the levels of full length midkine protein, but also generate non‐functional protein lacking regions encoded by critical exons. Guided by SpliceAid to identify splice enhancer binding motifs and consideration of RNA sequence bias constraints, SSOs were designed to predicted splice motifs in exons 3 and 4 of the midkine mRNA and synthesized using 2’OMe‐PS chemistry. Exon skipping was initially assessed by RT‐PCR with primers flanking exons 2 and 5. Transfection into midkine‐expressing human Huh7 liver and SHSY5Y neuroblastoma cancer cells elicited up to 30% of mRNA missing the targeted exons. Optimisation of lead SSOs through microwalking, cocktails of SSOs and PMO chemistry resulted in >90% exon skipped midkine mRNA, with a corresponding increase in truncated midkine protein. Studies are underway to examine the functional outcomes of midkine SSOs on cancer cell behaviour.
The lead midkine SSOs will then be assessed for their ability to alter in vivo tumour growth and metastasis as a prelude for further pre‐clinical development.
In vitro characterization of Decitabine‐induced CD4+HLAG+ suppressor cells
1: The Center for the Study of Haematological and other Disorders 2: Institute of Cell Therapy, University of Patras 3: The Karaiskakio Foundation, Nicosia, Cyprus
We have previously shown that HLA‐G+ T‐cells from Decitabine‐treated T‐cell cultures display potent suppression functions. As DNA hypomethylation is associated with increased gene expression, we hypothesize that other regulatory function genes may also be induced. RNA sequencing was performed on Dec‐induced sorted CD4+HLA‐G+ and CD4+HLA‐G‐ cells for the identification of upregulated gene transcripts.
Enriched T‐cells were bead‐activated and further incubated with IL‐2 and Decitabine or PBS. Sorting of CD4+HLA‐G+ and CD4+HLA‐G‐ cells was performed, followed by RNA extraction and cDNA synthesis. Real‐Time PCR was performed for expression analysis of genes identified to be upregulated in Dec‐induced sorted CD4+HLA‐G+ through RNA sequencing. In another instance, sorted CD4+HLA‐G+ and CD4+HLA‐G‐ cells were cultured with or without TCR stimulation and the supernatants were used in multiplex immunoassays for the investigation of cytokine/chemokine secretion profiles.
TaqMan® Gene Expression Assays, confirmed HLA‐G expression, as well as the upregulation of IDO‐1 (p = 0.0476), CCL22 (p = 0.0328), CCL17 (p = 0.0490) and CXCL9 (p = 0.0389). Supernatants were used in multiplex immunoassays, enabling the interrogation of cytokines/chemokine cell signalling molecules in complex extracellular events and signalling pathways. At a resting state IL‐4 (p = 0.002), IL‐6 (p = 0.011), IFN‐γ (p = 0.045) and CCL17 (p = 0.027) and at a stimulated state IL‐4 (p = 0.019), IL‐13 (p = 0.011), IL‐6 (p = 0.003), CCL17 (p = 0.0019) and CCL22 (p = 0.0021) were significantly increased in CD4+HLA‐G+ cells, compared to controls. IDO‐1 protein expression was also investigated and was found to be increased in CD4+HLA‐G+ cells compared to both CD4+HLAG‐ (p = 0.001) and PBS‐treated controls (p = 0.014).
The possible contribution of other regulatory genes will be assessed through functional studies.
Resistance is futile: evaluating and modifying cellular pathways during AAV vector expression in HEK293 cells to increase viral titers
1: Sartorius Xell GmbH, Bielefeld 33689, Germany 2: Sartorius Xell GmbH, Schloss Holte‐Stukenbrock 33758, Germany 3: Cell Culture Technology, Faculty of Technology, Bielefeld University, Bielefeld 33615, Germany
The demand for Adeno‐Associated Virus (AAV) as a vector for gene therapy grows consistently and will do so for the coming years. Despite the recent discoveries regarding AAV biology and its production in cell culture, there is still a lot to unravel. Numerous works focus on the identification of host cell characteristics that are beneficial for product yield in viral vector production processes. There are different intertwined mechanisms and pathways triggered during AAV production, consisting of receptors, signaling molecules and effectors. Here, we present a rational approach to understanding and potentially altering different cellular pathways, based on the analysis and interpretation of RNA‐Seq data we collected from two different commercially available HEK293 cell lines. Samples were taken 18 h after transfection with a two‐plasmid system, harboring the necessary genes for AAV‐2 production. With the use of bioinformatic tools, we identified genes that were significantly up and down regulated in transfected cells, as well as enriched pathways that play a role during viral vector production.
Development of a gene editing approach for the treatment of RPL5‐deficient Diamond‐Blackfan anemia patients
1: Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER 2: Advanced Therapies Unit, IIS‐Fundación Jiménez Díaz (IIS‐FJD, UAM) 3: CIMA, Universidad de Navarra 4: Hôpital Rober‐Debré 5: Hospital Clinico Universitario Virgen de la Arrixaca. University of Murcia. Spain 6: Hospital Gregorio Marañón
Diamond‐Blackfan Anemia (DBA) is a ribosomopathy included in the group of inherited bone marrow failure (IBMF) syndromes. Among clinical manifestations are red cell hypoplasia, congenital abnormalities and increased incidence of cancer. The two most commonly mutated genes associated with DBA are RPS19 (25%) and RPL5 (11%). A lentiviral gene therapy approach for the treatment of RPS19‐deficient patients is under development in our lab. RPL5 might require a tight endogenous regulation due to its direct interaction with MDM2, P53 master regulator. Therefore, we have developed a homologous recombination (HR) gene editing strategy for the treatment of RPL5‐deficient patients based on the use of CRISPR/Cas9 system and single‐stranded adeno‐associated viral vector 6 (ssAAV6) harboring a codon optimized sequence of the RPL5 cDNA. To optimize the efficacy of HDR‐mediated gene editing, healthy donor CD34+ cells from bone marrow (BM) and cord blood (CB) were transduced with CoRPL5‐ssAAV6 at different multiplicities of infection (MOIs). Up to 31% and 14% of homologous recombination has been achieved in CB‐CD34+ cells transduced at MOIs of 104 and 3x103 genome copies per cell, respectively. Experiments to test in vivo gene editing efficacy in NBSGW mice using these conditions are currently ongoing. The same strategy for BM‐CD34+ showed a higher toxicity and lower efficacy. Therefore, in order to reduce toxicity and increase gene editing efficacy in BM‐CD34+ cells, a self‐complementary version of the adeno‐associated viral vector 6 (scAAV6) has been developed and its performance is under evaluation.
Development of a DNA‐Editing Based Strategy to Treat Cystic Fibrosis
1: University College London 2: St. George's University of London
We aim to develop a genome editing strategy for cystic fibrosis (CF) using homology independent targeted integration (HITI) after targeted double‐strand breaks by CRISPR‐Cas9. HITI relies on non‐homologous end joining (NHEJ), enabling editing of non‐dividing cells. HITI also enables genomic integration of large pieces of cDNA, such as a CFTR‐encoding superexon. We are targeting the safe harbor AAVS1 site for two reasons: firstly, we anticipate more robust expression from the endogenous PP1R12C gene promoter than at the CFTR locus with its tightly regulated promoter; secondly, editing at a site distant from the CFTR locus reduces the risk of damaging any residual activity in the case of indel formation.
We first characterized basal epithelial cells carrying a CFTR nonsense mutation (R709X). Cells were expanded and differentiated into a pseudostratified epithelium via air‐liquid interface (ALI) culture. Using this model, we showed lack of CFTR activity by Ussing chamber analysis, and mucosal phenotypes that recapitulate in vivo symptoms of cystic fibrosis via scanning ion conductance microscopy. For HITI, a donor construct was created including two gRNA cut sites flanking a promoterless GFP donor. Donor was introduced alongside AAVS1‐targeting gRNA and Cas9. GFP expression was detected via fluorescence activated cell sorting (FACS) as a readout for genomic integration. The HITI donor was found to integrate with approximately 40% efficiency. Genotypic integration was verified via PCR analysis, confirming integration. GFP expression was monitored for one month; the AAVS1 endogenous promoter showed inconsistent levels of activity, as measured by fluctuations in the percentage of GFP‐expressing cells.
Traceless gene editing in skin diseases
1: 1EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University Salzburg, 5020 Salzburg, Austria 2: Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University Salzburg, 5020 Salzburg, Austria
Prime editing is a novel, highly promising tool for traceless gene editing. This technique requires a prime editing guide RNA (pegRNA) as well as a Cas9 nickase (Cas9n)‐reverse transcriptase (RT) fusion protein. An RT (reverse transcriptase) template sequence within the pegRNA enables the insertion of new genetic information at the target site.
The monogenetic skin disease epidermolysis bullosa (EB) is characterized by the formation of blisters and lesions upon minimal mechanical stress induction. Here we use prime editing for the restoration of gene function in recessive dystrophic EB (RDEB). The underlying mutation in the patient cells is an A > G transition in exon 3 (425A>G) of COL7A1.
RDEB patient keratinocytes were nucleofected with plasmids encoding Cas9n‐RT and patient‐specific pegRNA(s). Upon blasticidin selection cells were analysed via immunofluorescence (IF) staining, Western blot and next‐generation sequencing (NGS) analysis. IF stainings of type VII collagen (C7) revealed the correction of ∼10 % of treated keratinocytes. Western blot analysis revealed the presence of full‐length C7. Furthermore, NGS analysis confirmed the correction of >10 % of alleles without any insertions or deletions (indels).
Classical reframing and homology‐directed repair (HDR) approaches with CRISPR/Cas9 are often accompanied by inaccuracies. In contrast, prime editing is a highly precise gene editing tool that is easily adaptable to most of the mutations present in DEB patient cells. Our initial experiments confirmed the complete absence of indels at the target site. Thus, we assume prime editing to be highly suitable gene editing tool for epidermolysis bullosa and for genodermatoses in general.
CRISPR/Cas9 corrected iPSC model for gyrate atrophy
1: University of Helsinki 2: Helsinki University Hospital
Hyperornithinemia with gyrate atrophy of the choroid and retina (HOGA) is a severe recessive inherited disease, that causes type‐II‐ muscle fibre degeneration and progressive chorio‐retinal atrophy to blindness. HOGA arises from loss‐of‐function mutations in the ornithine aminotransferase (OAT) gene. Although it classifies as a rare disease, more than 30% of the known patients in the world present the Finnish founder mutation OATc.1205 T > C p.(Leu402Pro) in homozygosity. Using CRISPR/Cas9, we have corrected this OAT mutation in patient‐derived induced pluripotent stem cells (iPSCs). The genetic correction restored OAT production in stem cells, which in turn normalised the elevated ornithine levels in cell media and cell lysates. These results show an efficient recovery of protein function in iPSC, encouraging the development of advanced therapies for gyrate atrophy.
CRISPR approach for allele‐specific targeting of SNPs associated with Age‐related Macular Degeneration in ARPE19 cells
1: University of Oxford 2: Oxford Eye Hospital
Age‐related Macular Degeneration (AMD) is a major cause of sight loss in the developed world with known predisposing single nucleotide polymorphisms (SNPs). CRISPR‐mediated genome editing enables the possibility of targeting negatively associated SNPs in an allele‐specific manner, investigation into which requires a relevant cell model. AMD is characterised by the formation of deposits (drusen) that accumulate between Bruch membrane and the retinal pigment epithelium (RPE), which drives inflammation by stimulating the immune system. The ARPE19 cell line is a spontaneously immortalised model that grows in a stable monolayer, expresses RPE‐specific markers and exhibits a typical RPE morphology, including the formation of tight junctions. The complement factor system and SNPs in genes associated with this pathway are believed to play a pivotal role in AMD pathology. A number of AMD‐related SNPs were identified as being present in the ARPE19 cell line, which were then selected as targets to assess the allele‐specific targeting ability of CRISPR constructs. Guide RNA sequences cloned into a CMV‐driven SpCas9 vector via BsmBI cloning sites and packaged into lentivirus. Targeting efficiency of the AMD‐related SNPs was evaluated with TIDE analysis after lentivirus transduction of ARPE19 cells. The levels of different complement factor proteins were then evaluated by ELISA. For instance, CFH rs_340199 SNP revealed up to 7.8% targeting efficiency with elevated levels of the CFH proteins compared to the control. These results show a capability to manipulate the complement factor system in an AMD model with a therapeutic potential to target different disease predisposing SNPs.
Base editing to induce a ‐113A>G HPFH mutation, results in efficient HbF reactivation in CD34+ cells of patients with hemoglobinopathies
1: Aristotle University of Thessaloniki, School of Biology, Thessaloniki, Greece 2: Gene and Cell Therapy Center, Hematology Department, George Papanicolaou Hospital, Thessaloniki, Greece 3: University of Washington, Department of Medicine, Division of Medical Genetics, Seattle, WA 4: Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA 5: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA 6: Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA 7: University of Thessaly, Department of Computer Science and Biomedical Informatics, Lamia, Greece 8: Hematological Laboratory, Second Department of Internal Medicine, Faculty of Health Sciences, School of Medicine, Aristotle University of Thessaloniki, Hippokration General Hospital, Thessaloniki, Greece 9: Stem and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA 10: University of Washington, Department of Pathology, Seattle, WA 11: Hematology Department, George Papanicolaou Hospital, Thessaloniki, Greece
Genome editing approaches have led to new therapeutic opportunities for gene therapy of beta‐hemoglobinopathies. Most of these strategies involve nucleases inducing double‐strand breaks (DSBs), which may cause critical side‐effects. In contrast, base editors efficiently install precise nucleotide substitutions, without creating DSBs. Given that the hereditary persistence of fetal hemoglobin (HPFH) greatly ameliorates the clinical phenotype of β‐thalassemia and sickle cell disease (SCD), we used a non‐integrating HDAd5/35++ vector expressing a highly efficient adenine base editor (ABE8e) to install a ‐113A>G HPFH mutation in CD34+cells from beta‐thalassemia and SCD patients. To expand the transduced cells, our vector contained an mgmtP140K gene allowing for enrichment of edited cells after O6BG/BCNU treatment. Following 48‐hour transduction, cells were treated or not, with 50μΜ Ο6BG & 35μΜ BCNU and seeded in erythroid differentiation (ED) medium. The editing rate was evaluated by next generation sequencing and the γ‐globin expression by FCM and HPLC. After selection, the ‐113A>G conversion rates reached ∼60%. At the end of ED, the percentage of HbF+ cells was significantly higher in HDAd‐EF1a.ABE8e‐transduced over the untransduced cells while selection further increased their frequency in enucleated red cells (% HbF+/NucRed‐ cells; Thal:60%, SCD: 85%) and the levels of g‐globin chains relative to b‐ and a‐globin chains (up to 34%). HbF reactivation significantly reduced the ROS levels in GlyA+ cells and phenotypically corrected the patients' erythroid cells in cytospins. Overall, we present an alternative method of precise genome editing for HbF reactivation that potentially overcomes critical obstacles to clinical translation of gene editing for hemoglobinopathies.
Tissue‐specific gene‐editing via tissue‐specific guide RNAs as double lock safety for in vivo CRISPR Cas based gene editing
1: Helex 2: University of Hyderabad
Safety remains a critical concern in CRISPR Cas mediated in vivo gene editing with respect to unintended edits in the genome, and in non‐target tissues. The reliance on hundred percent targeted vector delivery continues to be a challenge. Helex's Hele‐GUIDE enables double lock safety system where targeted vector delivery (first lock) is supported by tissue‐specific gRNAs (second lock) to prevent editing in non‐target tissues despite unwanted vector biodistribution. The Hele‐GUIDE platform identifies unique cell and tissue‐specific biomarkers that regulate given target genes, determine their amenability to editing by an epigenetic analysis including chromatin accessibility, histone marks etc. and design gRNAs around the identified unique sequences to edit the target genes in a tissue specific manner. We have experimentally validated these gRNAs for multiple genes targeting editing in liver cells while considering lung, colon, skin and RPE cells as off‐target cells where editing should not occur. Our results have shown between 30‐50% editing in liver cells, with minimal to no editing in off‐target cells when all cells were transfected with the same RNP (gRNA and Cas9) complex via lipofectamine. These results hold promise for enhancing safety via site specific editing and enabling greater control on editing for translation into therapeutics.
Discovering cell‐line and tissue specific enhancers and promoters
1: Oxford Genetics Ltd
Once a gene therapy is designed and delivered, it must be expressed where desired. It is imperative that it is not mistargeted, so does not interfere with otherwise healthy tissues and functions. R&D practitioners are constantly seeking new enhancers and promoters to modulate gene expression in cell‐lines. Even when successful, downstream applications pose additional constraints. Achieving physiological or constitutive expression of gene products in a target tissue in vivo requires consideration of a much more complex regulatory landscape than in the model cell‐lines.
The genome sequence does not vary across different tissues; therefore, the epigenome, which distinguishes different cell‐lines and tissues, must be interrogated. The quantity of data available to address this challenge required the use of computational methods. We mined and processed epigenetic data to identify regulatory regions in different samples. Exclusion‐based filtering methods were used to find regions exclusive to the individual cell‐lines and tissues. This ensures that genes paired with identified regulatory elements would exert their influence in the desired targeted tissue and not others.
The resulting pipeline identified an average of approximately nine thousand enhancers in each of fifteen samples of interest. These were narrowed down using sample‐specific expression data for validation in the laboratory. The richness and abundance of the data, additionally, presents an opportunity to gain insight from machine learning models. Sequence data paired with epigenetic information can be used to train models to identify cell‐line and tissue ‐specific regulatory elements and better understand the complex regulatory landscape that governs tissue‐specific gene expression.
Genetic alteration and etiology of HCC in Egypt
1: Academic Medical Center
Hepatocellular carcinoma (HCC) is one of the most prevalent cancer, and it is highly aggressive with high fatality. Viral hepatitis is a major etiological factor causing HCC, with factors such as advanced age, diabetes, hypertension, may contribute to progression of HCC. The ATM gene us essential in the repair of DNA double strand breaks (DSB), which is caused by a variety of factors including HCAim of work: To identify types of mutations in ATM genes in Egyptian HCC patients using NGS based cell free DNA and to evaluate viral hepatitis. Materials and Methods; Clinicopathological data and 5ml of blood were obtained from each 21 primary HCC patients. After extraction of cell free DNA the library construction was prepared and sequenced using NGS
Results: Among 21 HCC patients the male was predominant in which representing 18 (85%) patients while only (14%) 3 patients were females. Four patients (19%) had a positive family history of cancer. The hepatitis C were reported in 18 patient (85%) and one of them had coinfection (HCVand HBV). Medical history of bilharziasis in 13 (61%), diabetes in (7%) and hypertension (14%) After sequencing of
ATM by NGS, a54 somatic mutstions were detected to be non synonymous mutations, most of the frequently identified were novel mutations, which represent 35.1 of all mutations, followed by synonymous mutations, intron mutations, copy number mutations Conclusion: Our study was in agreement with the results of previous HCC studies including incidence of HCC in Egypt.
Keywords: ATM, risk factors, HCC, NGS
Single‐cell Individual Full‐length mtDNA Sequencing Enables Quantitative Haplotype‐resolved Variant Analysis in Native and Genome‐Edited Mitochondria
C Bi1 L Wang1 Y Fan3 Y Yu4 J Izpisua Belmonte2
1: King Abdullah University of Science and Technology 2: Salk Institute for Biological Studies 3: The Third Affiliated Hospital of Guangzhou Medical University 4: Peking University Third Hospital
The ontogeny and dynamics of mtDNA heteroplasmy remain unclear due to limitations of current mtDNA sequencing methods. We developed
The Impact of Manufacturing Process Changes on the Quality of AAV Gene Editing Products
1: LogicBio Therapeutics
During the course of drug development, changes are occasionally made to the manufacturing process to yield a more productive and robust process. Along these lines, the manufacturing process for LB‐001, a gene editing vector designed to treat MMA patients, was optimized to simplify and streamline the purification process and increase final product yield. An analytical comparability assessment was performed to evaluate the AAV gene editing material pre‐ and post‐manufacturing process change. Here we show that the process changes induced an improvement in product quality and provide supporting 2D ddPCR data, Nanopore sequencing, and in vitro potency analysis. The root cause for this change was investigated and linked to modifications in the upstream manufacturing process, demonstrating a correlation with transgene integrity and subsequent implications on product potency.
A base editor screening platform to characterize performance of the modular components of Horizon's Pin‐pointTM base editing system
P Russell1 J Harbottle1 A Hale1 L Nantie1 H Machado1 K Marshall1 J Stombaugh1
1: Horizon Discovery Ltd, 8100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
Horizon's Pin‐pointTM base editing technology utilizes an RNA aptamer embedded within the sgRNA to enable the recruitment of an effector module, e.g., cytidine deaminase. The resultant modularity enables high degree of flexibility within the system which can be exploited to alter critical aspects of editing behavior including efficiency, editing window and nucleotide context preference.
We report on the development of a base editing arrayed screening platform to assess and characterize the impact of different deaminases on the editing profile of the Pin‐point base editor system. The high‐throughput nature of our screening platform allows for the characterization of the base editor against a more comprehensive set of target loci. Here we demonstrate the utility of the screening platform in multiple cell lines, using five different cytidine deaminases and three structurally distinct tracrRNAs, and assess each configuration of the Pin‐point system at seventy target sites.
The generated data clearly show the utility of the base editor screening platform to characterize the resultant genomic editing profile obtained with each of the editor architectures within the Pin‐point system. In utilizing the arrayed screening platform, we demonstrate significant impact upon editing efficiency, editing window size, and context preference with different deaminases and sgRNA/aptamer architectures. In summary we present the Pin‐point platform as a tuneable, modular system whose functionality can be readily adapted to address diverse editing requirements. Further, the ability to comprehensively screen multiple iterations of Pin‐point in a high‐throughput manner promises to enable customization to meet unique researcher base editing requirements.
HIV‐resistant anti‐HIV CAR T cells as a functional cure
1: Aarhus University 2: Aarhus University Hospital
HIV is a chronic infection that, despite effective antiretroviral therapy, cannot be cured due to latently infected cells. Current efforts to cure HIV has not yet led to profound reductions in the size of the viral reservoir. There is therefore an urgent need for novel approaches to eliminate latently infected cells. Chimeric Antigen Receptor (CAR) T cells have revolutionized treatment of hematological malignancies by enhancing the patient's own immune system to kill diseased cells. The CAR consists of an extracellular antigen‐binding domain fused to intracellular signaling domains. This enables the CAR T cell to MHC‐independently kill target cells. Because of the autologous origin, the T cells can engraft and persist in the patient lifelong. This project utilizes single‐chain variable fragments from clinically potent broadly neutralizing antibodies against HIV envelope to effectively target the CAR T cells towards latently infected cells. Furthermore, we have used the CRISPR/Cas9 system to integrate the expression cassette into the CCR5 locus, a co‐receptor necessary for viral entry into cells. This leads to concurrent knock‐out of CCR5 making the engineered CAR T cells resistant to infection. These anti‐HIV CAR T cells effectively kill HIV‐infected CD4+ primary human T cells ex vivo and are currently being evaluated in vivo using a humanized mouse model of HIV infection. Ultimately, the goal is to develop HIV‐resistant anti‐HIV CAR T cells that can effectively target HIV‐infected cells and persist in patients lifelong. This will remove the need for antiretroviral therapy leading to a functional cure.
Investigating Mechanisms of Variability of AAV5‐hFVIII‐SQ Expression in vitro
1: BioMarin Pharmaceutical
Valoctocogene roxaparvovec (AAV5‐hFVIII‐SQ) is an investigational gene therapy for treatment of hemophilia A. Significant inter‐subject variability has been observed in AAV5‐hFVIII‐SQ gene expression across species and trials, however, the mechanistic drivers of variability remain unknown. We hypothesize that variation in host factors involved in AAV5 transduction, vector genome metabolism, FVIII RNA expression and FVIII protein expression/secretion contribute to inter‐subject variability. Previously, we systematically investigated and identified 15 host factors whose expression correlated with AAV5‐hFVIII‐SQ transgene expression in mice. Here, we performed additional studies using a HepG2 in vitro hepatocyte model to identify drivers of AAV5 gene therapy variability. To test the effect of each of these candidates on AAV5‐hFVIII‐SQ‐mediated expression, we employed lentiviral delivery of shRNAs targeting each gene followed by AAV5‐hFVIII‐SQ transduction, and subsequently quantified hFVIII‐SQ expression levels. Knockdown of genes involved in second‐strand synthesis, transcription and protein folding/secretion resulted in significant reductions in transgene RNA and protein. Specifically, FKBP4, a negative regulator of viral DNA second‐strand synthesis; Phf5a, a protein involved in transcriptional elongation and pre‐mRNA splicing; and GRP78, an ER chaperone involved in protein folding, all resulted in significant reductions of transgene RNA and protein after shRNA knockdown. The effects seen when modulating the expression of these three genes highlight the complexity of successful AAV transduction and transgene expression. Further work to elucidate mechanistic drivers and mechanisms underlying inter‐subject variability may help identify predictive biomarkers of response and approaches to optimize outcomes of AAV gene therapy.
siRNA silencing of cellular entry proteins as a gene therapy strategy for Covid‐19
1: UCL Institute of Child Health 2: UCL
SARS‐CoV‐2 remains a significant public health threat, causing severe respiratory illness in susceptible individuals. Several effective Covid‐19 vaccines have been developed but novel SARS‐CoV‐2 variants continuously emerge that are more transmissible and have potential to evade vaccine immune responses. We are developing a novel therapy that does not depend on an immune response, based on siRNA‐mediated silencing of Angiotensin‐converting enzyme 2 (ACE2) receptor and Transmembrane Serine Protease 2 (TMPRSS2). SARS‐CoV‐2 requires these host proteins to enter respiratory epithelial cells at the cell surface, through binding and priming of its Spike protein. As a cell model for SARS‐CoV‐2 infection, we have utilised primary nasal epithelial cells (NHNE), as well as HEK293T cells overexpressing ACE2 and TMPRSS2. siRNA transfection in NHNE cells led to a 78%‐88% knockdown of ACE2 and TMPRSS2, as determined by qRT‐PCR and western blot data. TMPRSS2 knockdown in the overexpressing HEK293T cells resulted in an 87% reduction in infectivity from SARS‐CoV‐2 Spike‐pseudotyped lentiviruses expressing a luciferase transgene, indicative of a significant reduction in virus entry (p < 0.0001 by one‐way ANOVA). We are now working to confirm these results with live SARS‐CoV‐2 and to test lipid nanoparticle delivery of the siRNAs to air‐liquid interface grown NHNEs to more accurately model the respiratory airway. This siRNA approach could provide a novel therapy for immunocompromised individuals who do not gain sufficient protection from SARS‐CoV‐2 vaccines. Additionally, by targeting host proteins rather than virus components, our therapy is likely to remain effective in spite of emerging SARS‐CoV‐2 variants that circumvent pre‐existing immune responses.
A non‐viral CRISPR/Cas9 gene‐editing approach to express anti‐HIV broadly‐neutralizing antibodies in human B cells for HIV immunotherapy
1: ART‐TG, Inserm, Corbeil‐Essonnes, France 2: Vaccine Research Institute, IMRB Inserm U955, UPEC, Créteil, France 3: Division of Immunology and Allergy, CHUV, Lausanne, Switzerland
A definitive cure for HIV infection has never been achieved. Antiretroviral therapy has considerably improved the management of the disease but does eradicate the HIV reservoir. Immunotherapies may represent novel treatment options that could prevent viral re‐emergence. Passive administration of broadly neutralizing antibodies (bnAbs) or expression of bnAbs by AAV gene transfer has already been tested in patients, showing promising results but with limitations. Recently, genome editing strategies have been developed to genetically engineer the antibody repertoire of human B cells, providing a possibility to durably express high affinity HIV bnAbs in patients. We have established a simple non‐viral approach for ex vivo CRISPR/spCas9 gene‐editing human B cells. A homology‐directed repair template coding for bnAbs variable regions is inserted in the human IgH gene and can be spliced into IgH constant regions. The production of the single strand DNA template is obtained by an optimized PCR with increased yield and purity. Gene editing efficacy and specificity was validated in cell lines and in human primary B cells. Typically, 2‐5% of B cells can express membrane bnAbs 3BNC117 and VRC01 which target the HIV‐1 CD4 binding site. In vitro edited human blood B cells respond by antigen‐induced proliferation, suggesting the possibility to combine this strategy with vaccination. Novel bnAbs targeting different sites on the HIV‐1 envelope were identified and cloned by our team, providing novel paratopes for broad HIV neutralization and protection. Thus, gene editing bnAbs in B cells may represent a useful gene therapy approach for HIV immunotherapy.
Development of a lentiviral vector mediated B cell gene therapy platform for the delivery of therapeutic anti‐HIV‐1 eCD4‐Ig immunoadhesin
1: Scripps Research Institute 2: Seattle Childrens' Research Institute 3: University of Washington
A barrier to clinical anti‐HIV‐1 gene therapy has been the large number of target cells that must be modified to be resistant to HIV‐1 to successfully reduce infection. An alternative strategy would be to neutralise HIV‐1; to investigate this approach, we developed LVs that express the potent anti‐HIV‐1 eCD4Ig immunoadhesin and chose B cells as target cells due to their longevity and specialised antibody expressing machinery.
To overcome the low transduction efficiency associated with VSV‐G‐LV eGFP‐eCD4Ig primary B cell delivery, a measles‐pseudotyped‐LV (MV‐LV) packaging methodology was optimized, achieving B cell transduction efficiency of up to 75%. Sustained and B‐lineage restricted eCD4Ig expression was achieved using the B cell‐specific EμB29 enhancer/promoter. We improved the eCD4‐Ig protein for B cell expression by mutating the CH3 Fc IgG domain of eCD4Ig (KiHR), which reduced eCD4Ig/endogenous IgG heterodimerisation and resulted in increased eCD4Ig‐KiHR neutralising potency of 1.5‐4‐fold depending on the HIV‐1 isolate. EμB29 regulated eCD4Ig‐KiHR expression was stable and found to increase with B cell differentiation to plasma cells, eventually outperforming the strong constitutive viral MND promoter in plasma cells. Finally, unlike previously reported AAV‐eCD4‐Ig delivery to non‐human myocytes, eCD4‐Ig produced in B cells did not require co‐delivery of TPST2, a tyrosine sulfation enzyme required for eCD4Ig function, suggesting that the B cell environment is optimal for eCD4Ig expression and allowing for a simpler and safer vector design.
Our strategy and improvements resulted in the development of a robust B cell gene delivery platform with the potential for therapeutic protein production against a plethora of diseases.
Combined cellular and gene therapy to treat Primary Ciliary Dyskinesia
1: Institute for Regenerative Medicine and Biotherapy, Inserm, Montpellier 2: Department of respiratory diseases, CHU Montpellier
Primary Ciliary Dyskinesia (PCD) is a genetic disease caused by mutations that alter cilia beating, including in the respiratory airways, resulting in impaired mucus clearance and severe morbidity as well as increased mortality.
We hypothesized that human induced pluripotent stem cells (iPSC) could help restoring bronchial cilia beating. Genetically corrected iPSC could be differentiated into air‐liquid interface bronchial epithelium model derived from iPSC (iALI), providing an unlimited source of cells for clinical intervention.
The main candidate cell types for lung regeneration are p63+/KRT5+ basal cells due to their self‐renewal ability and their capacity to differentiate in the different cell types of the bronchial epithelium. The identification of the most specific extracellular marker, allowing cell sorting of this cell type, is a priority concern for the project. Transcriptomic data allowed us to identify specific basal cell surface marker candidates which can be validated with co‐staining with p63 for flow cytometry analysis.
A second issue is the best mean to erode the bronchi prior to cell therapy. A first preliminary experiment compared mechanical, chemical and enzymatic erosion. Results suggest a better efficiency with enzymatic erosion, showing a homogeneous detachment of the cells and a better engraftment of cells from a GFP cell line.
In conclusion, promising extracellular markers should allow to sort basal cells from iALI in order to engraft eroded model. We have to asses now the ability of the basal cells from a healthy or corrected cell line to functionally repair a PCD model.
Measurement of rAAV infectivity using TESSA‐Rep Enabled AAV titration (TREAT)
C Fustinoni1 L Holland1 A Dooner1 M Burridge1 S Wynde1 J Lampreia1 R Leydon1 C Greenwood1 H Wickens1 N Lopez‐Anton1 S Pollack1 Q Liu1 R Cawood1
1: Oxford Genetics Ltd
Determining the infectious titre of rAAV is a key attribute of preclinical development for AAV‐mediated gene therapies. However, it has been challenging to determine AAV infectious titres for a range of serotypes due to their low transduction efficiency in vitro. Specifically engineered cell lines that contain AAV rep‐cap (such as HeLaRC32) are commonly used with wild‐type adenoviruses to supply the ‘helper’ components to determine the infectious titre of rAAVs. Requirements for wild‐type adenovirus can cause safety concerns and limitations of a small number of modified cell lines bring into question how representative the titres are compared to target cell types or tissues.
We recently described a new self‐silencing helper adenovirus entitled ‘TESSA’ that enables the delivery of AAV rep‐cap for efficient and contaminant‐free manufacture of rAAV. Here we developed a novel rAAV infectious assay ‘TESSA‐Rep Enabled AAV titration’ (TREAT) based on TESSA. TREAT employs a self‐repressing TESSA virus that expresses all adenoviral helper genes alongside Rep to enable DNA amplification of the rAAV within infected cells for quantification via the Tissue Culture Infectious Dose (TCID50) assay. We show robust genome amplification and detection of the rAAV genome from single‐infection events using TREAT.
TREAT enables titration of various serotypes of rAAV in a wide range of target cell types and provides a more informative infectious titre for research and clinical applications. To streamline the process and reduce operational variability, we also deployed a Hamilton liquid handler for automation of the TREAT workflow, increasing the accuracy and reproducibility for infectious titration of rAAV.
CRISPR/Cas9‐mediated inhibition of human adenovirus replication
Z Didara1 F Reithofer1
1: University of Applied Sciences Krems
Patients with an impaired immune system or solid organ and particularly hematopoietic stem cell transplant recipients are at high risk of undergoing life‐threatening infections with adenoviruses. The efficacy of drugs to treat adenovirus infections is limited and alternative drugs are still under investigation. Hence, given the fact that numbers of solid organ and hematopoietic stem cell transplant recipients are constantly rising, investigations into possible, alternative, treatment options are needed. We investigated if CRISPR/Cas9 has the capability to target the DNA of replicating, human, adenoviruses. We provide proof‐of‐principle that during a lytic infection the adenoviral DNA is in a state that allows to be recognized by CRISPR/Cas9. Our data indicated that targeting of the viral E1A gene with two different gRNAs is enough to significantly decrease the output of infectious virus from infected cells. However, combining four gRNAs further increased the targeting efficiency and led to a decrease of numbers of infectious virus particles by about two orders of magnitude. Molecular analyses showed that the introduction of deletions into E1A mediated by targeting with two more gRNAs was by far more effective than the introduction of indels by a single gRNA. Taken together our data suggested that adenoviral DNA can be a substrate for CRISPR/Cas9 even during active replication, that the time window before the viral DNA is again packaged into capsids is large enough to allow efficient recognition by CRISPR/Cas9, and that CRISPR/Cas9 is potent enough to cope with the sheer amount of viral DNA generated during lytic adenovirus infection.
The mammalian lectin galectin‐3 is upregulated and promotes viral RNA synthesis during influenza virus infection: therapeutic implication for influenza
1: Ditmanson Medical Foundation Chia‐Yi Christian Hospital, Taiwan
Influenza is one of the most prevalent and significant viral infections worldwide. Due to the high mutation rate and emergence of drug resistance of influenza virus, antiviral strategies targeting viral RNA‐dependent RNA polymerase (RdRp), which is highly conserved among all subtypes and strains of influenza virus, are attractive. Here, we explored the role of galectin‐3 in viral life cycle and pathogenesis of influenza A virus (IAV). We used galectin‐3 knockout mice and wild‐type C57BL/6 mice, as well as knockdown and overexpression approaches to investigate the effects of intracellular galectin‐3 on different stages of viral life cycle. The interactions between galectin‐3 and viral proteins were examined via co‐immunoprecipitation and immunofluorescence. Our results show that the levels of galectin‐3 were increased in the bronchoalveolar lavage fluid and lungs of mice infected with IAV. There was a positive correlation between galectin‐3 levels and viral loads. Mice deficient in galectin‐3 were resistant to lethal IAV infection. Knockdown of galectin‐3 significantly reduced the production of viral proteins and virions. Furthermore, galectin‐1 did not affect viral entry; however, it enhanced nuclear import of the viral ribonucleoprotein (vRNP) and the RdRp activity required for viral transcription and replication. Notably, galectin‐3 interacts with the PA subunit of the RdRp, thus promoting viral RNA synthesis. In conclusion, our results demonstrate that galectin‐3 can enhance IAV production through increases in the nuclear import of the vRNP, RdRp activity, and viral replication and transcription. Our findings also identify galectin‐3 as a potential target for the development of anti‐influenza drugs.
High‐dose systemic adeno‐associated virus causes acute hepatocellular and liver sinusoidal endothelial cell injury in macaques
1: Gene Therapy Program, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
Most high‐dose adeno‐associated virus (AAV) clinical trials have shown liver enzyme elevations and platelet reductions, with occasional evolution to life‐threatening thrombotic microangiopathy. We previously reported the development of acute thrombocytopenia and transaminitis in nonhuman primates (NHPs) within days of receiving high intravenous (IV) AAV doses, with occasional progression to a fatal adverse event.
We administered AAV9 or an engineered capsid (AAV‐PHP.eB) encoding green fluorescent protein (GFP) at 1 or 2 x 1014 genome copies/kg IV to ten rhesus macaques and monitored acute toxicity for two weeks post‐administration via bloodwork, complement activation, and histopathology. Toxicity was characterized by transaminitis, thrombocytopenia, and alternative complement activation that peaked three days post‐AAV dosing. Although most animals recovered, some developed ascites, hyberbilirubinemia, or coagulopathy, leading to unscheduled necropsies.
To investigate the mechanism of toxicity, we took advantage of our cohort in which most animals presented biological anomalies and a minority developed fatal events. We interrogated the factors characterizing the most severe events using hypothesis‐driven immunohistopathological investigations and unbiased single‐nucleus RNA sequencing.
All liver cell types expressed high levels of GFP transcripts by day three, followed by a subsequent decrease. Thrombocytopenia coincided with sinusoidal platelet microthrombi and endothelial injury. Liver sinusoidal cells presented an acute upregulation of pro‐thrombotic and pro‐fibrotic genes. These findings of sinusoidal injury, liver platelet sequestration, and transaminitis observed in NHPs are similar to some types of drug‐induced endothelial injury syndromes. These findings highlight a possible toxic effect of high‐dose AAV on the liver microvasculature and warrant further preclinical investigations in NHPs.
IL‐1RA gene therapy prevents systemic and tissue inflammation in immune‐mediated diseases
1: Telethon Institute of Gene Therapy (HSR‐TIGET) 2: Vita‐Salute San Raffaele University 3: Pathology Unit, Department of Experimental Oncology, IRCCS San Raffaele Scientific Institute, Milan, Italy 4: GLP Test Facility, San Raffaele Telethon Institute for Gene Therapy (SR‐Tiget) 5: Pediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy. 6: Neuroimmunology Unit, INSpe, Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy.
Interleukin‐1 (IL‐1) is a proinflammatory cytokine with multiple functions in the immune response. When its production is dysregulated due to genetic or environmental factors, it causes a series of severe inflammatory and immune‐related disorders. Anti‐IL‐1 blockade represents the first‐line therapy for patients affected by these disorders. However, these therapeutics are life‐long treatments, and their tissue penetration is poor. To limit IL‐1‐mediated inflammation at the tissue level, we envisaged a lentiviral vector (LV)‐mediated approach targeting IL‐1 receptor antagonist (IL‐1RA) expression in hematopoietic stem‐progenitor cells (HSPCs). When transplanted in mice, IL‐1RA‐expressing HSPCs engrafted and generated all immune cell types similarly to control mice in amplitude and kinetic. HSPC‐derived expression of IL‐1RA was well tolerated in vivo. An in‐depth analysis of the bone marrow and peripheral blood revealed a normal distribution of primitive and progenitor cells and immune cells. The efficacy of the IL‐1RA gene therapy approach was tested in three mouse models of inflammatory diseases. LV‐derived IL‐1RA successfully suppressed acute neutrophil recruitment in chimeric mice treated with an inflammatory trigger. IL‐1RA gene therapy significantly reduced the clinical signs associated with Cryopyrin‐associated periodic syndrome (weight loss, leucocytosis, serum IL‐6 production, and multi‐tissue inflammation). IL‐1RA production prevented disease‐associated mortality and reduced the severity of symptoms in the experimental autoimmune encephalomyelitis model of multiple sclerosis. In conclusion, our data demonstrated that LV‐directed IL‐1RA gene therapy successfully reduces disease susceptibility and warrants further development as a new therapeutic opportunity for IL‐1‐mediated immune disorders.
Prophylactic corticosteroid treatment in mice improved AAV5 transgene expression through multiple mechanisms
1: BioMarin Pharmaceutical
Adeno‐associated virus (AAV)‐based gene therapy can activate immune responses that interfere with AAV‐mediated expression. Corticosteroid treatment may improve gene therapy outcomes by reducing these immune responses. Here, we investigated the effects of prophylactic corticosteroids on AAV‐mediated expression. Mice received daily prednisolone or water (non‐prednisolone control) for 4 weeks starting before I.V. administration of an AAV5 vector delivering human alpha‐1‐antitrypsin (hA1AT) under a hepatocyte‐specific promoter. Starting 6 weeks through study end at 12 weeks post‐AAV dosing, circulating hA1AT levels were significantly higher in prednisolone versus non‐prednisolone‐treated mice. To analyze potential mechanisms leading to improved expression, we performed RNA‐Seq analysis and showed that AAV5 treatment activated acute innate immune responses which were dampened by prophylactic corticosteroids. Surprisingly, a significant increase in PDGFRα expression was detected in livers of prednisolone‐treated mice. Immunohistochemistry analysis demonstrated upregulation of PDGFRα correlated with increased AAV5 uptake in hepatocytes. Lastly, prednisolone treatment increased PDGFRα expression and PDGFRα knockdown decreased AAV5 transduction in human hepatocyte culture.
Overall, prophylactic corticosteroid treatment improved liver‐directed AAV5 transgene expression by increasing hepatocyte transduction through multiple mechanisms including suppression of innate immune responses and upregulation of the AAV5 co‐receptor PDGFRα.
Seroprevalence of anti‐anti‐adeno‐associated virus 3B neutralizing antibodies in an international cohort of healthy donors
1: Vivet Therapeutics SAS 2: Vivet Therapeutics SL
Anti‐adeno‐associated virus (AAV) neutralizing antibodies (NAbs) pose a major challenge for AAV‐based gene therapies by limiting vector transduction efficiency and hence treatment success. An assessment of anti‐AAV immunity is therefore necessary for determining the primary eligibility of patients for inclusion in clinical trials. As part of the development of VTX‐801, an AAV3B gene therapy product for the treatment of Wilson's Disease, we studied the seroprevalence of pre‐existing immunity against the capsid in a cohort of 200 healthy donors by using a cell‐based transduction inhibition assay, validated in accordance with ICH guidelines. AAV3B NAb titers were determined as the reciprocal dilution of serum at the validated assay's cut point. Samples were considered negative whenever transduction efficiency was superior to the cut point at a dilution of 1:5. Results showed an AAV3B seroprevalence of 35.3%, with only 18% of donors exhibiting high NAb titers (≥1:40). Seroprevalence showed to increase with age, from 20.6% in the 18–30‐year‐old group to 52.2% for donors over 50. No gender differences were observed. Among the different races/ethnicities, the White subgroup presented the largest percentage of seronegative donors (82.4%, n = 56), followed by Asian (52.94%, n = 17), Black (52.79%, n = 36) and Hispanic (60.94%, n = 91) subgroups. Altogether, these results support the use of AAV3B as a gene therapy vector.
A strategy to mitigate toxicities associated with AAV doses of 1E14 vg/kg or higher by enabling repeated vector administrations at 5E13 vg/kg
1: Selecta Biosciences
High vector dose toxicities have been observed in humans, typically at a threshold dose of 1E14 vg/kg or higher. One potential strategy is to enable repeat dosing of lower vector doses to achieve similar levels of activity. ImmTOR nanoparticles encapsulating rapamycin have been shown to mitigate immunogenicity of AAV vector, elevate transgene expression and enable vector redosing at moderate vector doses (∼2E12 vg/kg). However, breakthrough antibodies have been observed in some animals at higher vector doses. Here we evaluated the combination of ImmTOR with B cell targeting agents to mitigate antibody responses and enable re‐dosing at vector doses up to 5E13 vg/kg. ImmTOR combined with a monoclonal antibody against B cell activation factor (aBAFF) showed a synergistic effect in reducing splenic mature B cells, plasmablasts and plasma cells more than 5‐10‐fold while elevating the fraction of pre‐/pro‐B cells. The combination of ImmTOR and aBAFF synergistically reduced anti‐AAV IgM and IgG antibodies and enabled four successive AAV administrations at doses up to 5E12 vg/kg and at least two AAV administrations at 5E13 vg/kg. Importantly, the level of transgene activity after two doses of 5E13vg/kg with ImmTOR and aBAFF was similar to that observed with a single dose of 1E14 vg/kg in control animals. Similar, but weaker effects were observed when ImmTOR was combined with ibrutinib, a Bruton's tyrosine kinase inhibitor. These results suggest that ImmTOR could be combined with belimumab, a humanized aBAFF, to enable repeated vector administrations with the aim to mitigate toxicities associated with vector doses above 1E14 vg/kg.
Immunotherapy with an anti‐PD‐1 antibody expressed by a viral vector improves with agents reprogramming tumor associated myeloid cells
1: Oncoimmunology Research Unit, Navarrabiomed‐Fundación Miguel Servet, Universidad Pública de Navarra (UPNA), Complejo Hospitalario de Navarra (CHN), Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona, Spain. 2: Division of Gene Therapy and Regulation of Gene Expression, Cima Universidad de Navarra and Instituto de Investigación Sanitaria de Navarra (IdISNA), Pamplona, Spain 3: Medical Oncology Unit, Complejo Hospitalario de Navarra (CHN), Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona, Spain. 4: Proteomic Research Unit, Navarrabiomed‐Fundación Miguel Servet, Universidad Pública de Navarra (UPNA), Complejo Hospitalario de Navarra (CHN), Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona, Spain.
Therapies based on PD1/PD‐L1 blockade have shown remarkable clinical outcomes and durable responses, but are not efficient in all patients. One approach to increase the potency of immunotherapy is to modulate the tumor microenvironment (TME) by reprogramming myeloid cells to stimulate anti‐cancer activity, since these cells constitute the major TME component. For this purpose, it is important to identify molecular signatures associated to cancer‐promoting myeloid cells. In this work, we have defined the phenotype and proteome of tumor associated myeloid cells (TAMCs) generated ex vivo using a lung cancer model (3LL). We identified differences in proteomic signatures between monocytic myeloid‐derived suppressor cells (MDSC), granulocytic‐MDSC, and tumor‐associated macrophages (TAMs) related to lineage and cancer‐driven polarization. The proteomic atlas of tumor‐associated cells revealed important routes that could be altered to reprogram cancer‐associated myeloid cells. Based on these data we evaluated the effects of several compounds at the differentiation, maturation, and immunosuppressive levels in MDSCs and TAMs. Furthermore, we confirmed the effects of selected compounds in myeloid cells from non‐small cell lung cancer patients in vitro. Finally, to assess this therapy in vivo, we evaluated the combination of selected compounds, administered intraperitoneally, with immunotherapy based on a Semliki Forest virus (SFV) vector expressing an anti‐PD‐1 antibody locally in 3LL tumors in mice. This combination strategy showed higher antitumor effects than each individual agent. Moreover, we observed similar effects when combining the most effective compound with systemic delivery of anti‐PD‐1 mAb in lung and colon adenocarcinoma tumor models.
Adeno‐associated virus induced microglia activation differs for males and females in a dose dependent manner
1: UCL Institute of Ophthalmology 2: Academic Unit of Ophthalmology, University of Bristol 3: Wyss Unit for Biologically Inspired Engineering, Harvard University 4: NIHR Biomedical Research Centre of Ophthalmology, Moorfields Eye Hospital
Adeno‐associated virus (AAV) gene therapy is growing in the treatment of ophthalmic disorders. The potential for treating monogenic and multifactorial disorders has made this an alluring therapeutic method, with clinical trials ongoing for hereditary disorders and age‐related macular degeneration. However, despite advances there are still important considerations for translation of gene therapy. Whilst AAV is considered less immunogenic, dose‐dependent intraocular inflammation is reported across trials, particularly with intravitreal delivery. In these adverse effects of treatment there is no reporting of sex effect, despite the knowledge of differences in immune response and, the eyes resident immune cells, microglia, between males and females. Here, we have used male and female Cx3cr1CreER:R26‐tdtomato (C57BL/6) heterozygous mice with flow cytometry to investigate the impact of intravitreally injected AAV2.null or a secreted cytokine mouse IL‐33 transgene, on microglia activation. At four weeks post‐injection of high dose (2E9 gc/eye), male and female eyes had clinical signs of posterior inflammation and flow cytometry analysis revealed activation of microglia by AAV treatment, with no interaction between treatment and gender. Injection of mid (2E9 gc/eye) or low (2E8 gc/eye) doses reduced the clinical severity of inflammation, however AAV treatment affected male and female microglia activation differently. AAV‐mediated inflammation was similar, regardless of null or IL‐33 transgene expression. These data confirm dose‐dependent ocular inflammation caused by AAV vectors, but more importantly reveal sex differences in the resident microglia response to different AAV doses. Such differences will need to be investigated further to aid safe gene therapy treatment in males and females.
Global neutralising antibody seroprevalence against adeno‐associated virus (AAV) serotypes of relevance to gene therapy: the ARGUS study design
J Rasko5 6 G Bashirians2 C J Petropoulos4 T Wrin4 Y Paliwal2 P V Henstock1 S Somanathan2 M Casey2 C da Fonseca Pereira3 I Winburn1
1: Pfizer Ltd 2: Pfizer Inc. 3: Pfizer Australia 4: Labcorp 5: University of Sydney 6: Royal Prince Alfred Hospital
Recombinant adeno‐associated virus (rAAV) vectors are used to deliver potentially curative gene therapies to patients. However, preexisting immunity can limit patient eligibility, viral gene transfer efficiency, and sustained transgene expression. There is currently limited published information on the geographic prevalence of neutralising antibodies (NAbs) spanning multiple AAV capsids. We describe here the design of an observational, retrospective, cross‐sectional, epidemiologic study to estimate the prevalence of NAbs against 6 AAV serotypes in 10 countries (Australia, Canada, France, Germany, Italy, Japan, Korea, Spain, UK, and USA) among paediatric and adult individuals who participated in clinical studies between 2015 and 2019 with informed consent for further exploratory studies. The primary objective was to estimate the titer of NAbs against AAV1, AAV5, AAV6, AAV8, AAV9, and AAV‐Spark100. Secondary objectives were to estimate the AAV NAb seroprevalence in subgroups of adults stratified by geographic region (North America, Europe, Asia‐Pacific), biological sex (male, female), and race (white, Asian, African, other). Exploratory objectives included the seroprevalence of NAbs in clinical trial participants stratified by location in 4 US regions (Northeast, Midwest, South, West), indication of original trial, immunomodulation (exposed, non‐exposed), cross‐reactivity, and thresholds of seropositivity in the study population. Serum samples were analysed by a central laboratory using an anti‐AAV neutralisation assay. Prevalence of NAbs at thresholds of 1:1, 1:2, 1:4, 1:8, 1:16, 1:80, 1:400, 1:2000, 1:10,000, and 1:50,000 were estimated. The results from this study are being analysed and prepared for future presentations.
Neutralising antibodies towards AAV based gene therapy can be depleted by imlifidase treatment
1: Hansa Biopharma AB
Pre‐existing neutralising antibodies towards adeno associated virus (AAV) based gene therapy is a major problem, as they are present in 30‐60% of patients, making them ineligible for successful gene therapy treatment. However, these neutralising antibodies can be effectively depleted by imlifidase, an IgG cleaving cysteine protease. Imlifidase, with the tradename Idefirix®, has been granted a conditional marketing authorisation in Europe* for desensitisation of highly sensitised adult kidney transplant patients. In addition, there are several ongoing clinical trials in a variety of indications, with IgG antibodies and Fc‐mediated effector responses as their common denominator. The clinically approved dose for desensitisation; 0.25 imlifidase mg /kg body weight, effectively depletes >95% of all IgG, within a few hours after dosing. Newly synthesised IgG starts to show 5‐7 days after dosing of imlifidase, thus creating an antibody‐free window of approximately one week. IgG levels are normally restored to their initial levels after 2‐6 months. This pattern has been confirmed in clinical samples and in vitro with different antibodies, including donor specific antibodies (DSA), antibodies towards human leukocyte antigen (HLA) and antibodies towards different vaccines. All in all, antibody depletion with imlifidase could offer a simple and elegant way to enable gene therapy in seropositive patients. The promising effects of imlifidase established in various indications are now further evaluated in collaboration with gene therapy companies utilising AAVs, including Sarepta Therapeutics and AskBio.
*EU/EEA, UK and Switzerland
Gene‐therapy based modulation of antigen‐presenting cells within hyperproliferative epithelium to restore immunity
1: The University of Queensland
Persistent human papillomaviruses (HPV) infecting basal keratinocytes (KCs) can cause DNA mutations and lead to malignant transformation of epithelium. Local immune suppression is often associated with HPV infection and epithelial transformation. Previously, we observed that antigen‐presenting cells (APC) of epithelium, are impaired in antigen‐presentation and induction of adaptive immunity in the transgenic K14E7 mouse model of HPV‐induced dysplastic epithelium. APC impairment is correlated with reduced production of a key APC‐instructing cytokine by hyperproliferative KCs, and low expression is also associated with poor prognosis in epithelial cancers. The aims of this study were to decipher the transcriptional regulation of this key cytokine in hyperproliferative epithelial cells, and to develop tools to restore expression in hyperplastic epithelium. Using Assay for Transposase‐Accessible Chromatin sequencing, we identified significant changes in the chromatin accessibility landscape of KCs and APCs in K14E7 epithelium. We identified ∼30.000 differential enriched sequences (DES) between K14E7 and wildtype KCs, and between K14E7 and wildtype APCs. These included DES within regulatory gene loci of the APC‐instructing cytokine and its corresponding receptor loci. Furthermore, we generated lentiviruses expressing the APC‐instructing cytokine with the aim to restore the function of APCs in hyperproliferative epithelium. The lentiviruses will allow further investigations into the potential of APC‐instructing cytokines as gene‐therapies for epithelial cancers.
Pyro‐drive jet injection of synthetic stem‐loop RNA (sl‐RNA) fragment derived from Sendai virus genome for inducing antitumor immunities
1: Osaka University 2: Daicel Corpolation
In this study, we developed synthetic stem‐loop (sl‐) RNA fragment which induces strong antitumor immunities. The sl‐RNA sequence is derived from Sendai virus DI (defective interfering) particle genome. Sin‐sl‐RNA (114nt) and sl‐RNA‐57 (57nt) have been developed as a third generation of synthetic RNA fragment which originated in Sendai virus DI particle genome. Sin ‐sl‐RNA is a single fragment which has complemental base sequence (25nt) at both ends of fragment. The complemental base sequence helps to forms double strand stem and single strand loop structure. In other words, sin‐sl‐RNA can form stem‐loop structure by a single RNA fragment. On the other hand, sl‐RNA‐57 (57nt) is a half size of the sin‐sl‐RNA. Two sl‐RNA‐57 fragments are needed to be annealed to form the stem‐loop structure. The sin‐sl‐RNA and sl‐RNA‐57 were injected to B16F10 (mouse melanoma) tumor by pyro‐drive jet injector (PJI) three times. The PJI is a novel injector system capable of injection depth adjustment. The tumor growth was strongly suppressed in sin‐sl‐RNA and sl‐RNA‐57 injected group. Chemokine and cytokine array revealed that MCP‐2, IP‐10, RANTES and MIP‐2 secretions were increased in the B16F10 tumor tissues after sl‐RNA‐57 injections. Additionally, macrophage infiltrations and macrophage polarization to M1 (anti‐tumorigenic) were observed in the sl‐RNA‐57 injected B16F10 tumor sections by F4/80 and NOS‐2 immunostaining. Further investigations revealed that the core of antitumor immunity induction part might be the sl‐RNA fragment derived from Sendai virus genome. These findings provide a novel nucleic acid medicine for the cancer treatment.
Anti‐tumour effect of double‐stranded RNA transfection is mediated by RIG‐I and TLR‐3 dual immune pathway
1: Osaka University, Graduate School of Medicine, Department of Device Application for Molecular Therapeutics
Cancer immunotherapy is dependent on immunostimulation to reorganise the tumour microenvironment (TME) and increase anti‐tumour effect of immune cells. There are currently many immunostimulants applicable to cancer therapy, such as antibodies, molecular drugs and PAMP activators. Not only is the immunostimulant choice important, method of delivery of treatment is equally as important as different modes may stimulate different immune pathways. Polyinosinic:polycytidylic acid (poly I:C) is known to interact with toll‐like receptor 3 (TLR3) when taken up by endocytosis, but when directly transfected into cells, poly I:C stimulates retinoic acid‐inducible gene I (RIG‐I) instead. Here, we treated 4T1 mammary tumour‐bearing mice with poly I:C by electroporation (PE) or syringe injection (SI) and monitored for tumour growth. Our results showed that PE induced stronger anti‐tumour effect than SI. This PE‐treated anti‐tumour effect could be suppressed by RIG‐I and TLR3 pathway inhibitors, but not in SI. In TME analysis, we found higher neutrophil infiltration and increase in neutrophil anti‐tumour N1 marker Fas expression in PE‐treated tumours. Neutrophil depletion decreased PE‐induced anti‐tumour effect, but not in SI, confirming that PE anti‐tumour effect is mediated by neutrophils. And even though 4T1 is a PD‐1‐resistant tumour model, PE combined with anti‐PD‐1 antibody treatment induced stronger anti‐tumour effect compared to either treatment alone. This suggests that PE can work in synergy with anti‐PD‐1 antibody treatment in 4T1 tumour model. We have demonstrated that poly I:C could induce different anti‐tumour effects dependent on treatment delivery method.
Sendai virus Fusion gene therapy induced anti‐tumour effects mediated by T cell chemokine
1: Osaka University
Inactivated Sendai virus particle (or hemagglutinating virus of Japan envelope: HVJ‐E) has previously been reported to possess tumour suppressive effects that activate anti‐tumour immunity. Of the two glycoproteins on the viral surface, only the fusion (F) glycoprotein, and not hemagglutinin‐neuraminidase (HN), when reconstituted onto liposomes showed anti‐tumour activity by inducing IL‐6 secretion in dendritic cells, which suggests that F protein alone can elicit anti‐tumour effects. In this study, we examined the application of F gene therapy on the B16F10 mouse melanoma model and the potential anti‐tumour effects. Plasmid encoding F gene (pCY4B‐F) was delivered into B16F10 tumours on backs of C57BL6 mice by electroporation and we demonstrated that F gene therapy suppressed tumour growth, increased CD4+ and CD8+ T cell infiltration into tumours and induced tumour‐specific IFN‐gamma T cell responses. However, F plasmid‐mediated anti‐tumour effect was not abrogated with neutralisation of IL‐6R signaling. Instead, we found that F plasmid treatment resulted in significant increase in the secretion of the chemokine RANTES (regulated upon activation, normal T cell expressed and secreted) by tumour‐infiltrating T cells. Neutralising antibody against RANTES was sufficient to abolish the anti‐tumour effect of F plasmid treatment. Thus, F gene therapy can elicit anti‐tumour effects by upregulating the secretion of RANTES which promotes the infiltration of T cells into B16F10 tumors, and may show promise as a novel therapeutic for single or combined cancer immunotherapy.
Anti‐adenovirus serotype 6 neutralizing and binding antibodies among a cohort of donors within the United States
A Khatri1 C Oh1 M Bazile1 K Patel1 D Rybin2
1: Rare Disease Research Unit, Pfizer, Cambridge, MA, 02139, USA 2: Early Clinical Development, Pfizer, Cambridge, MA, 02139, USA
The promise of treating all patients with AAV gene therapy is hindered by the presence of preexisting antibodies in a subset of subjects that inhibits vector transduction. These antibodies, reminiscent of a previous infection with a wild‐type virus, abrogate vector transduction and reduce clinical efficacy. Hence, patients with preexisting anti‐AAV antibodies above a threshold are often excluded from clinical trials. Here we investigated AAV6 seroprevalence in healthy subjects within the United States using two commonly used assays to evaluate seroprevalence; a cell‐based transduction inhibition assay (TI) assay, that quantified neutralizing antibodies (NAb), and an ELISA‐based assay (BAb), that quantified binding antibodies. First, AAV6 NAb seroprevalence was evaluated in a cohort of donors (n = 100) representing the three major races (White, Black, and Hispanic). NAb seroprevalence was 33% and skewed towards minority races. Cross‐reactivity was 100% against AAV1, AAV3B, and AAV9 capsids. A second cohort of donors (n = 100) were screened using both the NAb and BAb assays. Lack of sufficient Caucasian donors prevented us from accurately evaluating overall seroprevalence. However, increased NAb seroprevalence was noted among minority Hispanic donors. In addition, BAb values were good predictor of NAb positivity among all donors (C‐statistic 0.73). Interestingly, 20% of NAb positive donors had non‐immunoglobin neutralizing factors that prevented AAV6 transduction. Our data here suggests the presence of novel anti‐AAV6 non‐Ig neutralizing factor/s that requires further investigation. In addition, the observation of a significant correlation between the NAb and BAb AAV6 assays suggests either assay is suitable to identify patients with preexisting neutralizing factors.
Safety of Adeno‐Associated Viral Gene Therapies in Neurology: a Systematic Review
1: The University of Oxford 2: University College London 3: National Institutes of Health 4: University of Iowa
Adeno‐Associated Virus (AAV) gene therapies are generating much excitement in the rare disease field, particularly for previously untreatable neurological conditions. Efficacy has been claimed for several gene therapy products and the number of trials is rapidly increasing. There is also an increasing awareness of associated serious adverse events, some of which have been fatal. These adverse events remain poorly characterised and information on prevention, monitoring and treatment is urgently needed. We therefore undertook to systematically review publicly available data on AAV gene therapies for neurological disorders in order to collate existing information on both safety and efficacy. Results presented will be updated immediately prior to the congress, in view of the rapidly evolving field. To date, 61 trials for 52 different gene therapy products have been identified, many of which show promising initial efficacy information. There have, however, been concerning reports of adverse events, including 7 deaths. Serious adverse events include thrombotic microangiopathy (which is now recognised), myopathy, cardiomyopathy, acute liver failure and meningioradiculiits. In this review, we discuss where the serious adverse events have been seen as well as strategies that have been used for mitigation. As more trials emerge and participants are dosed, there is a real risk of further, possibly fatal, harm. Without tackling these safety issues carefully, the potential benefit of these novel therapies will also remain unrealised. There is therefore an urgent need to collaborate across sponsors in order to better understand, treat and prevent significant adverse events.
Development of an improved assay for NAb against AAV capsids
1: Jichi Medical University
Gene therapy using adeno‐associated virus (AAV) vector is currently applied in clinical stages. However, a presence of neutralizing antibody (NAb) against AAV capsids is known to suppress its efficacy significantly. Therefore, it is important to establish a highly sensitive assay to detect NAb. Current in vitro cell‐based NAb assays may not be sufficiently sensitive to detect low‐titer NAb due to the lack of sensitive cells for transduction, particularly for AAV8 or AAV9. In this study, we designed to develop a sensitive assay for NAb against a variety of AAV serotypes, which utilizes commonly available cell lines such as HEK293 and HuH‐7. To enhance the transduction efficiency of AAV, we tested to add a wide variety of compounds to the cell at various timing and found that the saccharides induced transgene expression efficiently in a variety of cells regardless of AAV serotype. The choice of reporter gene is also important; use of nanoluc, a highly sensitive luminescence reporter, further increased the sensitivity. As a result, we developed a serotype‐universal in vitro cell‐based NAb assay with greatly reduced amount of AAV vector to determine NAb titer precisely
Curcumin‐capped gold nanoparticles for Interleukin‐12 (IL‐12) gene delivery to a cervical cancer cell model
1: University of KwaZulu‐Natal
Cervical cancer (CC) is a leading cause of death in developing communities, with an annual mortality rate of 265 653. This non‐communicable disease shows immense complexity limiting conventional treatment. Immunotherapeutic approaches using Interleukin‐12 (IL‐12), a pleiotropic cytokine, induce the proliferation of IFN‐γ, which destroys malignant cells. However, the systematic application of IL‐12 was thwarted by adverse side effects. Localized delivery using nanotechnology enhances the spatiotemporal distribution of IL‐12 within the tumor microenvironment, promoting antitumor activity. This study aimed to elucidate the synergistic anticancer properties of IL‐12‐conjugated curcumin‐capped gold nanoparticles (AuNPs) in CC. AuNPs were biologically synthesized and capped using curcumin, functionalized with poly‐L‐lysine to facilitate IL‐12‐DNA binding, and stabilized by PEGylation. Nanoparticle characterization using UV‐vis and Fourier‐Transform Infrared (FTIR) spectrophotometry confirmed the synthesis and functionalization of the AuNPs. Nanoparticle Tracking Analysis (NTA) and Transmission Electron Microscopy (TEM) conferred that the spherical AuNPs had good colloidal stability and were the ideal size for gene therapy. Effective compaction and nuclease protection of the IL‐12 gene by the nanoparticles was confirmed by binding and the nuclease protection assays. The 3‐[4,5‐dimethylthiazol‐2‐yl]‐2.5‐diphenyltetrazolium bromide (MTT) cytotoxicity assay revealed high cell viability in vitro. Proof of principle studies preceding the delivery of IL‐12 also elucidated great promise for the use of this nanodelivery system in gene therapy. The IL‐12 gene was successfully transfected into the HeLa cells with significant expression determined by qPCR and ELISA. Overall, these novel biosynthesized nanoparticles worked synergistically with the IL‐12 gene, enhancing its therapeutic effect in a CC cell model.
Imlifidase treatment facilitates AAV8 liver transduction in SCID mice injected with high dose IVIg
E Erlandsson1 C Freiburghaus1 M V Jain1 H Jernmark Nilsson1 M Lindvall1
1: Hansa Biopharma AB
Imlifidase is an enzyme that cleaves IgG at the hinge region, creating one Fc‐fragment and one F(ab’)2 fragment. In the clinical setting, pre‐treatment with imlifidase creates a one‐week antibody free window, thus potentially offering an attractive solution to pre‐existing antibodies inhibiting the effective administration of gene therapies. Moreover, due to the rapid effect of this enzyme, the Fc‐mediated effector response is also inhibited. However, the neutralising effect of the IgG cleaving products are not yet established. In vitro data show that F(ab’)2 fragments have an inhibitory effect on AAV8 transduction in HEK293 cells, but to a much lesser extent than both single‐cleaved and intact IgG. In this study, we aim to clarify the impact of F(ab’)2 on AAV transduction in vivo, by analysing plasma and target organs from CB17 SCID mice treated with AAV8‐CMV‐LUC and IVIg in the presence or absence of imlifidase. In the liver lysates from mice treated with imlifidase, the transduction is significantly higher compared to the non‐imlifidase mice, where the transduction is very close to or below measurable levels, confirming that the inhibitory role of the imlifidase cleaving products is less prominent than their intact counterparts. Comparing transduction data for individual mice with their plasma F(ab’)2 levels, there is a clear negative correlation. Experiments are ongoing to further understand and elucidate this.
Therapy free of cells vs human mesenchymal stem cells from umbilical cord stroma to treat the inflammation in OA
M Morente‐López1 2 3 R Mato‐Basalo1 2 3 S Lucio‐Gallego1 2 3 L Silva1 4 A García‐Sánchez1 2 4 F J De Toro1 4 J A Fafián‐Labora1 2 3
1: Universidade da Coruña 2: INIBIC 3: CICA 4: CHUAC
In the last decade, extracellular vesicles in combination with the use of miRNAs to modify post‐transcriptional expressions of multiple genes have shown their utility in new therapies to treat inflammatory diseases. This work delves into the anti‐inflammatory effect of extracellular vesicles derived from mesenchymal stem cells previously modified so that the expression of miR‐21 is inhibited. We compare the efficacy of two treatments, mesenchymal stem cell with their miR‐21 inhibited through lentiviral transfection and their derived extracellular vesicles, against inflammation in a new OA animal model. The modified mesenchymal stem cell and their extracellular vesicles were intraperitoneally injected in an OA animal model by twice. One month after treatment, the animals were eutanized to check which therapy was the most affective to reduce inflammation compares with animals untreated. Treated OA model sera were analyzed for cytokines and chemokines. Subsequently, different organs were analyzed to validate the results obtained. Extracellular vesicles were the most efective treatment to reduce SASP as well as chemocines and cytokines in serum of OA animals compared with mesenchymal stem cells alone in a statistically significant way. Besides, miR‐21 is acting through ERK1/2 pathway which is envolved into inflammation. Syndecan 1 seems to be involved into this communication between SASP and inflammation as well as extracelular vesicles production, which make it a good candidate to generate a new anti‐inflammatory therapy.
Anti‐monocyte chemoattractant protein‐1 gene therapy in collagen antibody‐induced arthritis
1: Institute of Molecular Biomedicine, Comenius Unive
It has been shown that the monocyte chemoattractant protein‐1 (MCP‐1) is crucial in the pathogenesis of experimental models of arthritis as well as in clinical rheumatoid arthritis. An N‐terminal deletion mutant MCP‐1 protein (7ND) has been shown to act as a dominant‐negative inhibitor of cell migration into the inflammation site. The aim of our study was to analyse the effects of intra‐articular 7ND gene delivery on the course of collagen antibody‐induced arthritis (CAIA) in mice. CAIA was induced in adult DBA/1J male mice untreated or treated with either sterile PBS or 8μg of 7ND plasmid DNA using intra‐articular delivery enhanced with in vivo electroporation (four unipolar 100V square wave pulses). Experimental model CAIA lasted 8 days in total, sterile PBS or 7ND plasmid DNA treatment and electroporation were performed on day 3. Joint inflammation was assessed using the arthritis score, plethysmography and thermal imaging every second day. CAIA was induced successfully. Arthritis was confirmed with all used methods. No significant differences in the arthritis score, paw volume and chemiluminescent signal emission of paws were found between CAIA and 7ND treated CAIA groups as well as between PBS and 7ND treated joints. Further experiments should test a higher dose of the plasmid. Additional in vitro experiments are needed to prove successful transgene expression and its effects on the chemotaxis of macrophages and neutrophils relevant for the pathogenesis of CAIA.
Induction of immunogenic cell death in a canine melanoma model in response to p14ARF and interferon‐beta co‐transduction by adenoviral vectors
1: Universidade de São Paulo 2: Instituto do Câncer do Estado de São Paulo
Melanoma is an aggressive cancer type whose advanced cases are frequently challenging to treat. Wild‐type p53 protein reactivation, usually retained in melanoma, represents an attractive therapeutic opportunity. Seeking this end, we have studied the use of p14ARF gene transfer to activate endogenous p53 and induce cell death. Still, this approach may be improved by including a way to trigger anti‐tumour immune responses. In order to overcome this obstacle, our group has successfully used an improved adenoviral vector for the delivery of p14ARF in combination with IFNβ, resulting in the induction of immunogenic cell death (ICD) in in vivo murine and ex vivo human models. Encouraged by these findings, we seek evidence supporting the efficacy of our approach in clinically relevant models, including those that develop spontaneous tumours and metastases. The treatment of melanoma in dogs would achieve this goal. Here we show preliminary evidence suggesting that our adenoviral vector platform delivers canine p14ARF and IFNβ cDNAs induce ICD in canine melanoma cell lines (CMCLs). Upon treatment of the CMCLs, accumulation of hypodiploid cells revealed the induction of cell death while impaired clonogenicity suggests reduced cellular proliferation. Additionally, ATP release, a critical ICD marker, was confirmed. Currently, we are evaluating extra ICD markers in vitro and we performed some preliminary in situ gene therapy studies in xenograft models. With the success of the current study, we will propose the future treatment of canine melanoma in a veterinary environment.
A novel in vivo model to simultaneously assess efficacy and toxicity of Chimeric Antigen Receptor T‐Cell immunotherapy
J Yang1 W Lee1 J Jiao1 D Cai1
1: The Jackson Laboratory, Sacramento, CA 2: Seattle Children's Research Institute, Seattle, WA
Chimeric antigen receptor T (CAR T)‐cell therapy has emerged as a novel treatment option for certain hematologic malignancies, with several producing highly efficacious responses from patients. However, wider adoption of CAR T‐cell therapy is challenged by the potential development of life‐threatening toxicities including cytokine release syndrome (CRS). CRS symptoms can range from mild fever to life‐threatening events, including death. Preclinical animal models to assess the efficacy and toxicity of CAR T‐cell therapies have been lacking. Here, we developed a novel mouse model to assess these outcomes simultaneously, in two different PBMC humanized NSG™ variants. NSG‐MHC Class I/II double knock‐out strain is known to have a delayed onset of GvHD, and NSG‐SGM3xIL15 strain shows a higher engraftment level of human NK and myeloid cells, in addition to human T cells. Mice were humanized using PBMCs and treated with CAR T cells, and efficacy and toxicity were assessed. Compared to control, CD19 CAR T‐cells caused a decrease in the human CD19+ cell population in blood/spleen, and induced both T‐cell and myeloid cytokine release (IFNγ, IL‐10, RANTES, and MIP‐1α). The platform can also be used to determine individual PBMC/CAR T donor differences and show the extent of efficacy and toxicity of each PBMC donor treated with autologous and allogeneic CAR T‐cell therapy. We further confirmed anticancer efficacy using luciferase‐tagged human B‐cell lymphoma Raji tumor cells, which highly express CD19. In summary, we have developed a novel in vivo model to test the efficacy and toxicity of autologous and allogeneic CAR T‐cell therapy simultaneously.
Validity and safety preclinical in vivo study of gene therapy for hypophosphatasia using ARU‐2801 (AAV8 vector expressing TNALP‐D10)
1: Aruvant Sciences 2: Roivant Sciences 3: Nippon Medical School
Hypophosphatasia (HPP) is an inborn error of metabolism resulting from loss of function mutations in the tissue‐nonspecific alkaline phosphatase (TNAP) gene. Asfotase alfa is an approved enzyme replacement therapy for HPP, while effective, it requires chronic multiple injections up to 6 times per week. We have developed a one‐time gene therapy drug (ARU‐2801: an adeno‐associated viral vector expressing TNAP‐D10) for HPP and examined the safety and efficacy of ARU‐2801 in Alpl‐/‐ HPP mice. To develop the ARU‐2801 for clinical use, we evaluate the efficacy and safety of ARU‐2801 in non‐human primates. Juvenile WT macaques were injected intramuscularly with either saline or 5.0E12, 1.0E13, 2.0E13, and 4.0E13 vector genome/kg dose levels of ARU‐2801. Following treatment with a single dose of ARU‐2801, durable high plasma ALP levels (100‐10,000 U/L) were achievable with normal physical activity and a healthy appearance. The clinical chemistry parameters of these animals did not show signs of liver toxicity, which is consistent with histopathology examination. ARU‐2801 DNA was detected in only injected side muscle but not other organs by quantitative PCR analysis. Toxicities of chronic exposure to TNAP‐D10 examined by Von Kossa staining and CT scan of the animal did not show any ectopic calcification. Durable transgenic plasma ALP levels without any toxicities are achievable with ARU‐2801 in NHPs, at levels that potentially could be efficacious in the clinic. ARU‐2801, which can be administered as a single dose, has the potential to improve the quality of life for HPP patients by eliminating chronic administration.
Targeting the blood brain barrier for haematopoietic stem cell gene therapy in neurological lysosomal storage disorders
1: Great Ormond Street Institute of Child Health, University College London, London, UK 2: Orchard Therapeutics Ltd, London, UK 3: NIHR Great Ormond Street Hospital Biomedical Research Centre, London, UK
Lysosomal storage diseases (LSDs) are multisystem inherited metabolic disorders caused by dysfunctional lysosomal enzymes, resulting in the accumulation of undegraded macromolecules in a variety of organs/tissues, including the central nervous system (CNS). Current treatments include enzyme replacement therapy and haematopoietic stem/progenitor cell gene therapy (HSC‐GT). In the last decade, HSC‐GT has been effective in attenuating LSD patients' neurological symptoms; a subpopulation of HSCs can cross the BBB, engraft the damaged CNS as microglia, and secrete enzymes that are taken up by enzyme‐deficient cells, hence preventing further neurodegeneration. One poorly understood but essential component of this process is the passage of HSCs across the BBB. Manipulation of BBB permeability, specifically by targeting endothelial tight junctions (TJ), may increase HSC transmigration thereby improving HSC‐GT therapeutic effect. To investigate the role of TJs, using CRISPR‐Cas9 we knocked out Junction Adhesion Molecule A (JAM‐A), a key protein in TJ formation and function, in murine brain endothelial cells and performed transendothelial migration assays with lineage negative (lin‐) HSCs and monocytes. Monocyte, but not lin‐, migration was significantly increased when a JAM‐A KO brain endothelium was used, implicating JAM‐A in BBB cellular migration. We also investigated the effect on the BBB of pre‐conditioning agent busulfan, currently used as a myeloablative regime prior to lin‐ HSC transplantation in neurological LSDs, and present a detailed analysis of CNS vasculature and interactions with other BBB components. These findings provide deeper insight into HSCs' transmigration and conditioning regime in HSC‐GT for neurological LSDs, and support further investigation of the BBB.
Identification of novel peptide sequences to fuse to Iduronate‐2‐Sulfatase for the Treatment of Mucopolysaccharidosis Type II
1: University of Manchester
Mucopolysaccharidosis II (MPSII) is a lysosomal storage disease caused by a mutation in the IDS gene, resulting in heparan sulfate (HS) and dermatan sulfate (DS) accumulation. This leads to skeletal and cardiorespiratory disease with severe neurodegeneration in two thirds of suffers. Enzyme replacement therapy is ineffective at treating neurological disease, as intravenous IDS is unable to cross the blood‐brain‐barrier (BBB). Haematopoietic stem cell transplant is also unsuccessful, presumably due to insufficient enzyme production from transplanted cells engrafting in the brain. We have previously demonstrated that a haematopoietic stem cell gene therapy (HSCGT) strategy, using a lentivirus containing IDS fused to ApoEII (LV.IDS.ApoEII), can correct neuropathology in MPSII mice. Here we present data using two peptides, RVG (rabies virus glycoprotein) and gh625 (Herpes virus glycoprotein derived peptide), fused to IDS and delivered via HSCGT in MPSII mice. HSCGT with LV.IDS.RVG and LV.IDS.gh625 was compared to LV.IDS.ApoEII, LV.IDS, wild‐type and un‐treated MPSII littermates. Reductions in pathology were encouraging, with both RVG and gh625 peptides, but some toxicity is seen with these products. Furthermore, enzyme levels in the brain were lower than ApoEII (<20% of ApoEII for both). We have therefore also screened several other peptide constructs (IDS.HS2 and IDS.HS5) for their ability to increase uptake into the brain using an in vitro BBB uptake assay. When compared to our gold standard ApoEII peptide, HS5 was able to improve BBB uptake over ApoEII (65% increase) and also improved uptake into multiple cell types including cardiomyocytes (37%), retinal pigment epithelium (105%) and osteoblasts (116%).
Targeted integration of a promoterless mini‐ATP7B, enhanced by copper‐mediated DNA damage, confers proliferative advantage to ATP7B‐deficient hepatocytes and corrects Wilson disease
1: Telethon Institute of Genetics and Medicine, Pozzuoli (NA), Italy 2: Department of Chemical Sciences, Federico II University of Naples , Naples, Italy 3: Pathology Unit, A. Cardarelli Hospital, Naples, Italy
Wilson disease (WD) is a life‐threatening autosomal disorder, due to mutations in ATP7B gene, a P‐type copper transporting ATPase exerting a key role in copper metabolism. Loss of ATP7B function results in severe and progressive liver and brain diseases due to copper accumulation. Gene therapy with recombinant adeno‐associated vectors (AAV) holds promises for WD treatment. Nevertheless, several hurdles remain toward the development of effective and safe gene therapy for WD, especially for the treatment of paediatric patients.
We applied a liver‐directed nuclease‐free genome editing approach, based on the AAV‐mediated targeted integration of a promoterless human mini‐ATP7B cDNA into the albumin locus of Atp7b ‐/‐ mouse. Once integrated by homology directed repair (HDR), mini‐ATP7B is expressed by robust liver‐specific albumin promoter and is inherited by daughter cells if proliferation occurs, thus avoiding AAV dilution effect. Administration of AAV‐Alb‐mini‐ATP7B alone, or in combination with copper‐chelating drug, rescued Atp7b ‐/‐ mouse survival. Genome‐edited hepatocytes repopulated up to 90% of the liver and this was associated to amelioration of liver injury and copper metabolism, and rescue of serum ceruloplasmin activity. The extensive liver repopulation by genome‐edited hepatocytes observed in Atp7b‐/‐ livers may be also driven by a higher integration efficiency of donor DNA. Consistently, transcriptomic analysis revealed upregulation of genes involved in HDR in ATP7B‐deleted cells and livers compared to controls, that was associated to copper‐induced DNA double strand breaks.
In summary, promoterless nuclease‐free genome editing provide a significant and sustained therapeutic benefit in WD and may represent a safer alternative to classic gene replacement strategies.
Full‐length ATP7B reconstituted through protein trans‐splicing corrects Wilson disease in mice
1: Telethon Institute of Genetics and Medicine, Pozzuoli (NA), Italy 2: Division of Cardiovascular Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom 3: Centre for Advanced Discovery and Experimental Therapeutics (CADET), Manchester Academic Health Sciences Centre, Manchester, United Kingdom 4: Pathology Unit, A. Cardarelli Hospital, Naples, Italy 5: School of Biological Sciences, Faculty of Science, University of Auckland, Auckland, New Zealand
Wilson disease (WD) is a genetic disorder of copper homeostasis, caused by deficiency of the copper transporter ATP7B. Gene therapy with recombinant adeno‐associated vectors (AAV) holds promises for WD treatment. However, the full‐length human ATP7B gene exceeds the limited AAV cargo capacity, hampering the applicability of AAV in this disease context. To overcome this limitation, we designed a dual AAV vector approach using split‐intein technology. Split‐inteins catalyse seamless ligation of two separate polypeptides in a highly specific manner. We selected a DnaE intein from Nostoc punctiforme (Npu) that recognises a specific tripeptide in the human ATP7B coding sequence. We generated two AAVs under the control of a liver‐specific promoter that express either the 5’‐half of a codon optimized human ATP7B cDNA followed by the N‐teminal Npu DnaE intein or the C‐terminal Npu DnaE intein followed by the 3’‐half of ATP7B cDNA. Intravenous co‐injection of the two vectors in wild‐type and Atp7b ‐/‐ mice resulted in efficient reconstitution of full‐length ATP7B protein in the liver. Moreover, Atp7b ‐/‐ mice treated with intein‐ATP7B vectors were protected from liver damage and showed improvements in copper homeostasis.Taken together, these data demonstrate the efficacy of split‐intein technology to drive the reconstitution of full‐length human ATP7B and to rescue copper‐mediated liver damage in Atp7b ‐/‐ mice, paving the way to the development of a new gene therapy approach for Wilson disease.
AAV‐mediated hepatic delivery of miR‐34b/c reduces fibrogenesis, promotes fibrolysis and improves liver fibrosis
1: Telethon Institute of Genetics and Medicine, Pozzuoli (NA), Italy 2: Pathology Unit, A. Cardarelli Hospital, Neaples, Italy 3: Department of Translational Medicine, Federico II University of Naples, Naples, Italy
Liver fibrosis is a major health problem worldwide and currently available treatments are only supportive. The microRNA‐34 (miR‐34) family is upregulated in response to chronic liver injuries, and miR‐34b/c downregulates the platelet derived growth factor signaling pathway. Here, we found that mice deleted of miR‐34b/c are susceptible to liver fibrosis and miR‐34b/c reduces TGF‐β‐mediated activation of human hepatic stellate cells and expression of type I collagen and several other genes involved in collagen biosynthesis. Adeno‐associated viral vector‐mediated hepatic delivery of miR‐34b/c ameliorated established and advanced liver fibrosis/cirrhosis in mice. Mechanistically, the underlying mode of action of the miR‐34b/c included inhibition of hepatic stellate cells and type I collagen expression, and enhancement of fibrotic scar degradation by direct downregulation of plasminogen activator inhibitor type 1, a major player in tissue fibrosis. In summary, the anti‐fibrotic activity of miR‐34b/c occurs at multiple levels as it reduces fibrogenesis and promotes fibrolysis. In conclusion, this study may pave the way toward the development of novel microRNA‐based therapies against hepatic fibrosis.
A novel AAV9 gene therapy for producing GCase enzyme with high mannose 6‐phosphate content to treat Gaucher disease
1: M6P Therapeutics
AAV gene therapy for soluble lysosomal enzymes to treat lysosomal storage disorders largely relies on enzyme cross‐correction of non‐transduced cells to achieve high efficacy during the treatment. Different approaches are employed to improve the targeting of the overexpressed enzymes (such as: enzyme fusion with IGF2 peptide, or an antibody fragment) are under investigation in the field. Data from our previous publications show that a truncated GlcNAc‐1‐phosphotransferase (designated as S1S3 PTase), greatly increases the levels of mannose 6‐phosphate (M6P) on lysosomal enzymes for efficient cellular uptake via the cation‐independent mannose 6‐phosphate receptor (CI‐MPR) that is the broadly expressed on most cells including neurons and other cell types in the CNS. Mutations in the GBA gene lead to loss of β‐glucocerebrosidase (GCase) enzyme activity and build‐up of glucosylceramide and related substrates in cells resulting in Gaucher disease. Here, we report a novel AAV gene therapy approach using a dual promoter construct design for co‐expression of GBA gene with the truncated S1S3 PTase to produce highly phosphorylated GCase as a potential treatment for Gaucher disease. The produced GCase with the S1S3 PTase co‐expression is shown to have high M6P content and enhanced binding to CI‐MPR as compared to the enzyme produced without S1S3 PTase. GCase uptake and distribution in the CNS was also evaluated by immunohistochemical staining. More details will be presented in the 2022 ESGCT meeting.
Low‐density lipoprotein (LDL) receptor liver gene transfer with lentiviral vectors achieves full normalization of LDL cholesterol and protection from atherosclerosis in a mouse model of familial hypercholesterolemia
1: San Raffaele Telethon Insitute for Gene Therapy (HSR‐TIGET) 2: Vita‐Salute San Raffaele University 3: San Raffael Scientific Institute
Familial hypercholesterolemia (FH) is characterized by high LDL‐cholesterol, due to impairment in its clearance by LDL receptor (LDL‐R). Liver gene therapy may represent a definitive cure, particularly if delivered during childhood. Lentiviral vectors (LV) integrate in the genome of target cells and may allow stable liver gene transfer even in pediatric patients following a single intravenous (i.v.) administration. We produced vesicular stomatitis virus glycoprotein (VSV.G)‐pseudotyped LV encoding LDL‐R and observed 10‐fold lower infectious titer, compared to other transgene‐encoding LV. Since LDL‐R is the main entry route of VSV.G LV, their interaction during LV production and re‐infection of producer cells were likely causing this drop. Titer was rescued abrogating transgene expression in producer cells. We then assessed VSV.G LV liver gene transfer in Ldlr‐/‐ mice. Surprisingly, hepatocyte gene transfer was >2 fold higher than in wild‐type (WT) mice. We thus treated juvenile Ldlr‐/‐ mice with LDL‐R LV at 3.5‐4e10 transducing units/kg and achieved complete normalization of LDL cholesterol for more than 6 months. LDL‐R RNA and protein analysis of treated mice suggests that liver expression should be higher than WT to normalize cholesterol. Mice were then challenged with high‐fat/high‐carbohydrate “Western” diet (WD) for 3 months and while LDL‐cholesterol increased in Ldlr‐/‐, it remained normal to near‐normal in LV‐treated mice. Accordingly, histopathology analysis showed aortic atherosclerosis only in Ldlr‐/‐ mice. Liver steatosis was observed in WT and LV‐treated mice after WD challenge and it was lower in mice with the highest LDL‐R positive area. Our results encourage further investigation towards clinical translation.
Liver‐directed gene targeting as a potential therapy for Fabry disease
1: International Centre for Genetic Engineering and Biotechnology, Trieste, Italy 2: Institute of Clinical Pathology, Department of Laboratory Medicine, University Hospital of Udine 3: Regional Coordinator Centre for Rare Diseases, University Hospital of Udine, Udine, Italy
Fabry disease (FD) is an X‐linked inherited, lysosomal storage disorder caused by mutations in the Alpha Galactosidase‐A (GLA) gene encoding for GLA enzyme responsible for the catabolism of glycosphingolipids like Globotriaosylceramide (Gb3). Accumulation of Gb3 in lysosomes results in systemic clinical manifestations and reduced lifespan. Enzyme Replacement Therapy (ERT) and chaperone therapy are the available treatments for FD however, noncurative and with limitations.
We developed a potential therapeutic approach based on the permanent genetic modification of hepatocytes to express the hGLA enzyme by targeting the albumin locus in vivo. Juvenile P30 Fabry KO mice were treated with 3.0E13 vg/kg of an AAV8 donor vector containing mAlb homology arms and the hGLA cDNA and were sacrificed 4 months post‐treatment. This integrative approach was coupled with CRISPR/SaCas9. Efficacy was assessed in plasma, liver, kidney, and heart. Lyso‐Gb3 accumulation was reduced by 70%‐80% compared to untreated mutant mice. To increase safety, we performed a nuclease‐free approach in the presence of fludarabine, an HR‐enhancer drug. Plasma GLA activity was 4‐5‐fold higher than donor‐only‐treated mice but did not reach donor+Cas9‐treated mice values. Immunofluorescence analysis showed the absence of lyso Gb3 accumulation in kidneys. A comparative analysis with commercial ERT and AAV‐mediated liver gene therapy is ongoing. To test translatability, AAV‐LK03 vectors containing hALB homology arms will be tested in tissue culture human liver cell lines, in primary cultures of human hepatocytes, and in humanized mice. This data is inclined toward a promising one‐shot therapy using a safer gene‐targeting approach to ameliorate FD phenotype.
Liver fibrosis impairs hepatocyte transduction by AAV vector
1: Telethon Institute of Genetics and Medicine, Pozzuoli (NA), Italy
Adeno‐associated viral vectors (AAVs) are the most promising tools for liver‐directed gene therapy. However, integrity of hepatic architecture has been considered pre‐requisite for efficient gene delivery and clinical studies have been addressed toward patients with no or negligible hepatic damage and fibrosis. Preliminary evidence suggests that AAV‐mediated gene transfer to hepatocyte may be hampered by liver fibrosis, but knowledge about AAV vector interactions with fibrotic livers are still very limited.
We investigated transduction and biodistribution of AAV8‐based vectors, that are commonly used in liver‐directed gene therapy clinical trials, in mouse models of induced and genetic liver fibrosis. Wild‐type mice were treated with thioacetamide (TAA) to induce hepatic fibrosis and then intra‐venously injected with an AAV8 vector expressing GFP under the control of an hepatocyte‐specific promoter. TAA‐treated fibrotic mice showed delayed clearance of blood‐borne vector and reduced hepatic GFP expression, associated with reduced vector genome content, compared to non‐fibrotic controls. We next evaluated AAV‐mediated transduction in a mouse model of Wilson disease (WD), an inherited disorder of copper metabolism characterized by liver fibrosis. WD mice showed reduced hepatic transgene expression and vector genome copies at two weeks post‐AAV administration compared to controls. However, analysis of livers at two days post‐injection revealed similar AAV genome content in WD and control mice, suggesting increased vector scavenging by non‐parenchymal cells in fibrotic livers.
These findings demonstrated that liver fibrosis impairs AAV‐mediated gene transfer to hepatocytes and highlight the relevance of the limitations posed by liver fibrosis to efficient and safe gene transfer.
Pre‐clinical development of an ex‐vivo lentiviral‐based Hematopoietic Stem/Progenitor Cells (HSPC)‐gene therapy (GT) approach for Mucopolysaccharidosis type IVA (MPSIVA)
1: San Raffaele Telethon Insitute for Gene Therapy (HSR‐TIGET) 2: Vita‐Salute San Raffaele University 3: GLP‐ San Raffaele Telethon Institute for Gene Therapy (HSR‐TIGET) 4: Telethon Foundation 5: Pediatric Neurology Unit, Meyer Children's Hospital 6: Rare Metabolic Disease Unit, San Gerardo Hospital 7: Pediatric Immunohematology and Bone Marrow Transplantation Unit, San Raffaele Scientific Institute
Mucopolysaccharidosis type IVA (MPSIVA) is a lysosomal storage disease (LSD) caused by the deficiency of the lysosomal enzyme N‐acetylgalactosamine‐6‐sulfatase (GALNS), characterized by severe skeletal dysplasia, which the currently available therapies fail to cure (1‐2). Our study aims at developing hematopoietic stem progenitor cells (HSPC)‐gene therapy (GT) for MPSIVA, as part of an innovative GT platform approach for LSDs with skeletal involvement, based on pre‐clinical and clinical data proving the safety and efficacy of HSPC‐GT for the treatment of other LSDs (3‐6). To this aim, we generated a 3rd‐generation lentiviral vector encoding for the GALNS enzyme (LV‐GALNS) to transduce human mobilized peripheral blood (mPB) CD34+ cells. Transduced cells showed proper clonogenic and proliferative capability in vitro and significantly overexpressed and released the GALNS enzyme. Using the conditioned medium from LV‐GALNS transduced myeloid culture, we restored GALNS expression and activity in patient‐derived fibroblasts, mesenchymal stromal cells (MSCs), and MSC‐derived osteoblasts suggesting the cross‐correction of non‐hematopoietic cells in the presence of superphysiological enzyme levels. Importantly, LV‐GALNS human mPB CD34+ cell derived‐osteoclasts secreted GALNS, possibly functioning as a resident source of enzyme for skeletal cross‐correction. When we transplanted LV‐GALNS mPB CD34+ cells in sub‐lethally irradiated NSG mice, we observed human multilineage reconstitution in mice PB over time and in the hematopoietic organs at termination. Importantly, we detected a significantly higher level of GALNS enzyme in the bone marrow of mice treated with HSPC‐GT. Altogether, our preliminary preclinical data support the development of HSPC‐GT approach for MPSIVA treatment.
Liver directed lentiviral gene therapy provides long term phenotypic correction of methylmalonic acidemia in a mouse model
1: San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy 2: IRCCS San Raffaele Scientific Institute, Milan, Italy 3: Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence, Italy 4: Vita‐Salute San Raffaele University, Milan, Italy
Methylmalonic acidemia (MMA) is a severe inborn error of metabolism caused by deficiency of methylmalonil‐coA mutase (MUT), leading to accumulation of the toxic methylmalonic acid. The significant MMA‐related morbidity and mortality in infancy led to exploit liver transplantation as elective treatment, and to explore gene therapy in MMA mouse models as an alternative therapeutic option. We exploited lentiviral vector (LV) mediated liver gene therapy, which would potentially allow for a stable gene transfer even in paediatric patients at the first disease stages by virtue of LV genomic integration. We generated and validated LV encoding a human MUT transgene under the control of a hepatocyte‐specific expression cassette (LV.MUT). We assessed LV‐mediated liver gene therapy in the Mut‐/‐ mice, expressing a compensatory MUT transgene in muscles (MCK‐Mut‐/‐). We administered 2.5x1010 transducing units (TU)/kg of LV.MUT intravenously to 2‐week‐old MCK‐Mut‐/‐ mice (n = 8) and monitored them over 6 months. The treatment resulted in a complete rescue of the survival rate and growth of MCK‐Mut‐/‐ animals, comparable to that of wild‐type controls, with a rapid and sustained decrease of circulating methylmalonic acid (cMMA; mean of 175 μM and 1000 μM in treated vs. untreated animals at 6 months post‐LV). End‐point immunohistochemistry and electron microscopy analyses on liver samples of MCK‐Mut‐/‐ treated animals revealed ∼15‐20% of transduction, resulting in the correction of the mitochondriopathy (median mitochondrial area and shape comparable to that of wild‐type mice). Our data provide evidence for therapeutic benefit in MMA by LV gene therapy and encourage further pre‐clinical research towards clinical translation.
Neonatal gene therapy in a Mucopolysaccharidosis type I (MPSI) mouse model
1: San Raffaele Telethon Institute for Gene Therapy (HSR‐TIGET) 2: M. Tettamanti Research Center, Department of Pediatrics, University of Milano‐Bicocca 3: Experimental Neuropathology Unit, INSPE, San Raffaele Scientific Institute 4: Department of Molecular Medicine, Sapienza University 5: Department of Biomedical Research, Alfred I. duPont Hospital for Children 6: Department of Pediatrics, University of Milano‐Bicocca 7: Pediatric Immunohematology and Bone Marrow Transplantation Unit, San Raffaele Scientific Institute 8: Vita‐Salute San Raffaele University
Mucopolysaccharidosis type I (MPSI) is rare infantile disease caused by mutations in the IDUA gene encoding for the lysosomal enzyme a‐L‐iduronidase, involved in glycosaminoglycans (GAGs) metabolism. Most newborns with the most severe form of MPSI (Hurler Syndrome, MPSIH) are asymptomatic at birth, but severe disease manifestations appear precociously in life. Since a superior clinical outcome could be obtained with early intervention and lentiviral vector gene therapy (LV‐GT) has been performed in adult MPSI mice and MPSIH patients, we tested a neonatal GT approach in MPSI newborn mice. One‐2 days‐old MPSI pups were transplanted with MPSI BM Sca1+ cells, transduced with LV‐IDUA. We monitored IDUA activity and vector copy number (VCN) over time in mice's peripheral blood (PB), resulting linearly correlated. At sacrifice, we evaluated disease correction over MPSI untreated mice and normalisation over WT mice. For what concerns IDUA activity, GT‐treated MPSI mice displayed supraphysiological values in the PB and median levels above the WT's range in almost all compartments analysed, with normalisation of the metabolic alteration. Substrate accumulation was significantly reduced and reached values found in plasma, liver, heart, kidneys and lungs of healthy mice. Furthermore, the skeleton, resistant to standard therapies, displayed a significant improvement both at radiographic and histological level. Normalisation of metaphyseal trabecular and cortical bone morphometry parameters analysed through mCT studies was achieved after GT. Moreover, ameliorations were observed in vacuolisation of brain cortex. Further analyses on the GT effects are ongoing and will provide a more precise knowledge of neonatal GT for MPSI mice.
Characterization of KT‐A112, a dual AAV vector therapy expressing human insulin and glucokinase in an STZ mouse model of Type 1 Diabetes Mellitus
1: UAB 2: Kriya
Type 1 diabetes (T1D) is a chronic condition in which the pancreas produces little or no insulin. T1D has no cure and limited therapies to manage blood glucose without complications. Several studies have shown that co‐administration of two AAV1 vectors utilizing a CMV promoter and an insulin (INS) and glucokinase (GCK) gene injected into skeletal muscle of diabetic mice can achieve significant levels of glycemic control without inducing hypoglycemia. We have designed a novel gene therapy comprised of CpG‐optimized human insulin (AAV1‐hINS) and human glucokinase (AAV1‐hGCK) which has shown dose‐dependent, safe glycemic control in a model of T1D. In this study, we interrogated the relationship between AAV dose, functional endpoints, and gene expression to guide dose selection in future studies. Mice were administered streptozotocin (STZ, 40 mg/kg) or buffer and allowed to develop frank diabetes over 4 weeks. At that time, intramuscular injections of three doses, a low‐, mid‐, or high‐dose of AAV1‐hINSIns + AAV1‐hGCK (0.45, 0.6 and 1.2 E + 11 vg/kg ) were made bilaterally in the quadriceps, gastrocnemius and tibialis anterior muscles and the mice followed for 8‐wks for glycemic control endpoints. Muscles were collected at the end of the study and assayed to determine transduction efficiency (vgs/μg DNA), message (mRNA/μg RNA), ELISA‐reactive protein and glucokinase catalytic activity. Results confirmed glycemic control, transduction, mRNA expression, exogenous protein and enzyme activity in injected muscles of treated animals. Patterns of gene expression depended on injected muscle and varied with dose. No background signal was observed in untreated controls.
Bloodspot sulphatide concentration in suspected lysosomal storage diseases including metachromatic leukodystrophy
H Brown1 H Church1
1: Manchester University NHS Foundation Trust 2: Orchard Therapeutics 3: University of Washington
Metachromatic leukodystrophy (MLD) is a progressive lysosomal storage disorder (LSD) caused by pathogenic mutations in the ARSA gene, resulting in low activity of arylsuphatase A and accumulation of sulphatides. Recently, ex vivo autologous gene therapy, Libmeldy, has been approved by the European Medicines Agency for the treatment of MLD. Early diagnosis and intervention may only be possible and most effective with newborn screening (NBS). To prepare for an MLD NBS pre‐pilot study, dried blood spots (DBS) from patients referred for LSD investigations were analysed, using a validated UPLC‐MS/MS assay. Diagnostic samples received between 2020‐2022 were stored as DBS at ‐20°C. Sulphatides were extracted from one 3mm diameter DBS punch in D5‐hexadecanoylsulphatide internal standard (D5‐ISTD) and analysed using a Waters ACQUITY UPLC, Xevo TQ‐S mass spectrometer. Quantification of hexadecanoylsulphatide (C16:0‐S) was validated according to ISO 15189‐compliant procedures. C16:0‐S was quantified using qNMR‐verified D5‐ISTD. Twelve‐hundred and seven diagnostic, and 177 anonymised NBS samples were analysed. The assay, linear to ≤1000nM, showed inter/intra‐assay imprecision of <10%CV. Signal‐to‐noise ratios in chromatograms from low concentration NBS samples was >30. The average multiple‐of‐median (defined for year of life in an unaffected control group) in five cases of confirmed MLD was 2.6 (1.9‐3.5). The median C16:0‐S concentration in the neonatal samples was 81.9nM (53.5‐195.3). The validated UPLC‐MS/MS is suitable for analysis of sulphatide in the investigation of MLD. Initial results from anonymised NBS samples suggests the method is fit for purpose for our NBS pilot study.
Development of optimized ATP7B gene therapy vectors for the treatment of Wilson's Disease with increased potency
J Sullivan1
1: MeiraGTx
Wilson's Disease (WD) is caused by autosomal recessive, loss‐of‐function mutations in the ATP7B gene which lead to pathological accumulation of copper in the liver, brain, and other tissues. Symptoms of WD include Parkinson's Disease like neurological defects and hepatologic defects associated with cirrhosis. ATP7B is a transmembrane copper pump that is too large to effectively package into adeno‐associated virus (AAV). As such, efforts have been made to engineer ATP7B minigenes that can be used as gene therapies for WD. By applying our vector optimization platform to ATP7B, we created a construct that far exceeds the expression of published gene therapy approaches. Here we show that by altering various components within the construct, we can achieve higher expression in various in vitro models and over 100‐fold higher expression in the liver of ATP7B‐null animals. This may allow us to use a lower viral dose which could improve safety outcomes in patients and lower manufacturing hurdles including cost. Moreover, AAV8‐mediated expression of our miniATP7B in ATP7B‐null mice reduced ALT activity and spleen size, suggesting improved liver function, and decreased immune response. Together, these results indicate a potent and effective new gene therapy for the treatment of WD.
A CNS‐targeted gene therapy for the treatment of obesity
1: MeiraGTx
BDNF is a key molecular regulator of metabolic function and body weight. Monogenic mutations in brain‐derived neurotrophic factor, BDNF, or its receptor, NTRK2, cause obesity in humans. BDNF signaling is also downstream of the leptin‐melanocortin pathway, which controls appetite and body weight. Here we present an optimized BDNF gene therapy for the treatment of obesity. We show that our optimized BDNF gene therapy, designed by altering various cis‐regulatory components, achieves significantly higher expression compared to a previously published construct, in head‐to‐head comparisons in primary mouse cortical neurons as well as in various immortalized neuronal cell lines. Furthermore, AAV‐mediated delivery of our BDNF gene therapy to the hypothalamus of mice is more effective than the published CAG‐BDNF construct at preventing weight gain induced by high‐fat diet. By designing a highly expressing BDNF gene therapy, we can drive efficacy at lower viral vector doses and potentially lower immune responses and decrease safety risks. Taken together, these results indicate a potent and effective gene therapy for the treatment of rare obesity populations as well as more prevalent forms of obesity that arise from polygenic or environmental factors.
Systemically administered HIV‐based lentiviral vectors are capable of transducing liver cells and human hepatocytes in disease‐relevant mouse models
C Khoo1 A Barnard1 S Iqball1 G Devarajan1 R Powles1 R Jordan1 S Lauchande1 E Guzman1
1: Oxford BioMedica
The liver is an important target for gene therapy vectors. There are several genetic disorders where restoration of the expression of a defective protein in the liver would be therapeutically effective. In paediatric disorders, where early intervention is essential, a vector which integrates into the genome giving rise to long term expression, from a single administration, would have an advantage.
We have evaluated our HIV LentiVector® platform in both wild‐type mice and a mouse model of a metabolic disease. Administration of our HIV vector has shown consistent liver transduction and increasing vector copy number (VCN) with increasing vector dose. Using luciferase to assess in life transgene expression demonstrated strong expression in the liver with higher expression observed in males over females which was consistent with VCN analysis. To test human hepatocyte transduction efficiency we delivered Lentiviral GFP vector IV in a mouse model which has a liver repopulated with 70% human hepatocytes. Flow cytometry analysis demonstrated at least 10% of the human hepatocyte population was GFP positive.
Lentiviral vectors, with their ability to stably integrate and provide long term gene expression from a single administration have an advantage over episomal AAV and have a key role to play in liver‐targeted gene therapy. We have demonstrated that our HIV LentiVector® platform can consistently transduce the liver in different mouse models, together with the ability to produce high quality, large quantities of clinical grade vector provides a platform from which to target genetic liver disorders.
TALEN®‐mediated engineering of HSPC enables systemic delivery of IDUA
1: Cellectis Therapeutics
Mucopolysaccharidosis type I (MPS‐I) is caused by deficiencies in the alpha‐L‐iduronidase (IDUA) gene. Current treatments are limited to enzyme replacement therapy usually preceded by allogenic bone marrow transplantation. Gene editing of hematopoietic stem and progenitor cells (HSPCs) followed by transplantation offers unique therapeutic advantages including local delivery into the brain and could be a therapeutic strategy for MPS‐I and other lysosomal storage diseases.
We established a TALEN®‐based ex vivo gene editing protocol to insert an IDUA‐expression cassette into a specific locus of HSPC. In vitro, edited HSPC were 90% viable and maintain differentiation potential. Level of gene insertion was up to 60% allelically in bulk edited population, and up to 80% of clones from colony forming unit assay. Edited cells displayed 60‐ and 80‐fold higher IDUA secretion than unmodified cells at the HSPC and myeloid level, respectively.
Edited HSPCs were functionally evaluated in 2 immunodeficient mouse models: NSG and NSG‐SGM3. Edited HSPC maintained differentiation potential toward myeloid and lymphoid lineages in vivo, with no differences with unmodified cells. Editing rates in vivo were 6‐9% sixteen weeks after injection, depending on the tissue analyzed (blood, spleen, bone marrow). Lastly, 8.3% of human cells were edited in the brain compartment.
In conclusion we established a safe TALEN®‐based gene editing protocol procuring IDUA‐edited HSPCs able to engraft, differentiate into multiple lineages and reach multiple tissues, including the brain. These results pave the way towards targeted gene therapy‐mediated treatment of MPS‐I.
Systemic AAV9 gene therapy results in a durable clinical and biochemical improvement in a severe adult murine model propionic acidemia with a novel human missense mutation
1: National Human Genome Institute 2: National Institutes of Health
Propionic acidemia (PA) is a severe autosomal recessive metabolic disorder caused by mutations in either PCCA or PCCB genes, and patients experience high mortality and disease related morbidity despite conventional dietary and medical management. However, testing new therapies and the recapitulation of PA related clinical phenotypes in the existing murine models of PCCA deficiency of PA has been limited by immediate lethality in the Pcca knock‐out mice, or alternatively, the lack of clinically significant disease‐related phenotypes in the hypomorphic PCCAA138T transgenic model. We therefore selected a human PCCA missense mutation (MS) that, when present in the homozygous state, was characterized as less severe to introduce the into murine Pcca gene. Pcca MS/MS mice manifest pronounced growth retardation and fragility, yet typically survive past weaning, and display elevated plasma 2‐methylcitrate (∼10uM compared to ∼0.5uM in wildtype control mice), growth failure, diminished Pcca RNA levels (∼ 50% of wildtype), decreased PCCA protein (∼ 20% of wildtype), impaired 1‐C‐13 propionate oxidation. In addition, Pcca MS/MS mice show phenotypic improvement with dietary therapy. Treatment of adult Pcca MS/MS mice with ∼7x1013 gc/kg of AAV9‐CAG‐PCCA resulted in decreased plasma 2‐methylcitrate and increased growth in comparison to untreated Pcca MS/MS mice. This PA model will be useful to study the spectrum of clinically important postnatal features of PA in the juvenile period and beyond, and afford facile testing of new treatments, including small molecules, mRNA therapy, genome editing, cell therapy, gene therapy and as demonstrated here, systemic AAV gene therapy.
Brain‐targeted lentiviral haematopoietic stem cell gene therapy for severe Mucopolysaccharidosis Type I: peptide tag optimisation for more efficient delivery across the blood‐brain‐barrier.
1: University of Manchester
Mucopolysaccharidosis Type I (MPSI‐Hurler) is a rare inherited lysosomal storage disease which arises due to defective α‐L‐iduronidase (IDUA) enzyme, leading to accumulation of undegraded glucosaminoglycans in lysosomes throughout the body and multi‐organ disease. The current treatment options (enzyme replacement therapy and allogeneic haematopoietic stem cell therapy) only partially correct the neurological manifestations in MPSI‐Hurler. Haematopoietic stem cell gene therapy (HSCGT) is a promising therapeutic strategy to treat MPSI‐Hurler, as it circumvents the blood‐brain‐barrier (BBB). The efficacy of HSCGT may be further improved by the use of BBB‐crossing peptides to enhance uptake across the BBB. In the related condition MPSII, we fused IDS (IDS in the enzyme missing in MPSII) to the brain‐targeting peptide ‐ApoEII and we showed that IDS:ApoEII mediated complete normalization of brain pathology and behaviour, providing significantly enhanced correction compared to the untagged IDS enzyme. Here, we employ a similar strategy for IDUA. We designed IDUA:peptide fusions on the same basis as for IDS:ApoEII and tested them in vitro for their ability to cross the BBB. We identified two IDUA:peptide candidates (IDUA:ApoEII and IDUA:H5) for their ability to be uptaken into brain endothelial cells and cross the BBB. However, these two candidates had very poor secretory profiles (76.4% decrease in secretion). We analysed the protein structure of these IDUA:peptide candidates and compared them to the structure of IDS:ApoEII. We were then able to optimize the design of the IDUA:peptides based on differences between the two enzymes. Our data with the re‐designed IDUA:peptides show a normal secretory profile.
Validation of therapeutic strategies in a novel compound heterozygote model of Methylmalonic Acidemia
1: Duke University School of Medicine 2: Duke Molecular Physiology Institue 3: Duke University Trinity College of Arts and Sciences 4: Duke University Pratt School of Engineering 5: Duke Cancer Institute 6: National Human Genome Research Institute, National Institutes of Health 7: Duke Global Health Institute
Methylmalonic acidemia (MMA) is a common organic acidemia primarily caused by mutations in the methylmalonyl‐CoA mutase (MMUT) gene. Newborns with MMA typically present in “crisis”, with metabolic acidosis and hyperammonemia. Despite aggressive dietary management and vigilant care, the patients experience substantial disease related morbidities and increased mortality. Hence, there is an urgent need for better therapies to treat this disorder. We generated a novel compound heterozygote murine model using orthologous mut 0 (MMUT p.R108C) and mut ‐ (MMUT p.M700K) mutations. MmutR106C / M698K mice survive to adulthood with a mild failure to thrive phenotype but, when challenged with a precursor enriched diet, develop a severe clinical syndrome characterized by weight loss, lethargy, and death. The inducible metabolic crisis displayed by MmutR106C / M698K mice make them ideal for testing new therapies for MMA, including small molecule inhibitors of branched‐chain amino acid catabolism and CRISPR gene therapy approaches. We will further detail the characterization of this new model of MMA and preliminary results of our therapeutic approaches.
Systematic literature review on biomarkers associated with metabolic decompensation events in propionic acidemia
1: Semmelweis University 2: Syreon Research Institute 3: Moderna Therapeutics Inc.
Propionic acidemia (PA) is an inborn metabolic disorder caused by the deficiency of propionyl‐CoA carboxylase, that can manifest in severe metabolic decompensation events (MDEs). In this study we investigated the biomarker changes related to MDEs in PA patients. Two separate systematic literature reviews were performed. The first focused on the association of biomarker changes and clinical outcomes in studies presenting data on clinical outcomes in ≥5 PA patients. The second collected the biomarker changes and clinical outcomes in PA patients undergoing liver transplantation, knowing that in this group MDEs are eliminated in most of the cases. 2,455 publications were screened of which 26 articles reported both on biomarkers and clinical outcomes in ≥5 PA patients, and 27 studies were found that published biomarker data on 61 PA cases with transplantation. Articles that presented 3‐hydroxypropionate (3‐HP) levels during MDEs indicated elevated levels of 3‐HP, while all the identified studies reporting on transplanted patients presented substantial decrease in 3‐HP levels after transplantation. Articles presenting data on plasma ammonia indicated a significant increase in ammonia level during MDEs and a decrease post‐transplantation in most cases. Change in fibroblast growth factor 21, methycitrate and acetylcarnitine levels were inconclusive in relation to MDEs and no clear tendency was reported for transplanted patients. In conclusion, the identified data point to the direction that 3‐HP and plasma ammonia may have a reasonably associated role as a surrogate biomarker in relation to MDEs in PA patients.
Riboswitch‐controlled delivery of therapeutic hormones for gene therapy
1: Gene Regulation, MeiraGTx, New York, NY 10016, USA
AAV‐mediated gene transfer is a promising therapy for many diseases. However, excessive amounts of transgene—especially hormones or growth factors—from unregulated vectors may not be desirable and may limit the success of gene therapy. A genetic switch or gene control mechanism would provide a gene therapy approach that can be safely controlled and is applicable to a broader range of disease areas. Here, we present the development of regulated hormone expression, e.g., erythropoietin (Epo) and parathyroid hormone (PTH), whose expression is controlled by riboswitch via small molecule inducer. Different from previously reported gene regulation systems that use exogenous protein components, our riboswitch platform utilizes RNA element that contains aptamer as a sensor for small molecule ligand/inducer. In our aptamer riboswitch system, aptamer/ligand binding alters transgene splicing, turning gene on or off in a dose dependent fashion. In the absence of the small molecule inducer, Epo or PTH gene with the riboswitch does not express protein. In the presence of the small molecule inducer, Epo or PTH is robustly produced in an inducer dose‐dependent manner. When Epo gene with riboswitch was delivered in AAV to mice with kidney disease‐associated anemia, orally dosed small molecule‐induced Epo increased hematocrits to normal levels, indicating the induced transgene expression reaches efficacious therapeutic levels. Similarly, when PTH gene with riboswitch was delivered in AAV to mice, oral small molecule treatment induced‐PTH increased blood calcium levels. Our data provide evidence that our riboswitch platform is effective for small molecule‐controlled gene therapy for treating hormone deficiency.
An innovative platform approach for the development of ex‐vivo gene therapies for the treatment of lysosomal storage diseases with skeletal involvement
1: Telethon Foundation, Rome, Italy 2: San Raffaele Telethon Institute for Gene Therapy (SR‐TIGET), IRCCS San Raffaele Scientific Institute, Milan, Italy 3: Pediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy 4: Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy 5: Vita‐Salute San Raffaele University, Milan, Italy 6: GLP‐ San Raffaele Telethon Institute for Gene Therapy (SR‐TIGET), Milan, Italy 7: Bicocca Bioinformatics, Biostatistics, and Bioimaging Center, School of Medicine and Surgery, University of Milano‐Bicocca, Monza, Italy 8: Regional Coordinating Center for Rare Diseases, European Reference Network for Hereditary Metabolic Diseases (MetabERN), University Hospital Udine, Udine, Italy 9: Rare Metabolic Disease Unit, San Gerardo Hospital, Monza, Italy
Lysosomal storage diseases (LSDs) are characterized by accumulation of undigested macromolecules causing severe organ damage, including neurological and skeletal involvement, which are not targeted by the currently approved treatments. Moreover, ultra‐rare LSDs are disregarded by major industry investments resulting in few/no therapies available for these indications. Previous data obtained by our Institute demonstrated that Hematopoietic Stem and Progenitor Cell‐Gene Therapy (HSPC‐GT) is capable of cross‐correcting non‐hematopoietic cells more effectively than enzyme replacement therapy and HSC transplantation, since the hematopoietic progeny of gene corrected HSPCs function as hyper‐functional enzyme factories for storage clearance. So far, HSPC‐GT approaches for the treatment of LSDs have provided clinical evidence of metabolic correction in Metachromatic Leukodystrophy and Mucopolysaccharidosis type I, Hurler as part of SR‐TIGET research programs subsequently out‐licensed. Building on this experience and on the common pathological mechanism of LSDs, we are proposing HSPC‐GT for the treatment of a group of rare/ultra‐rare LSDs with progressive skeletal involvement (Mucopolysaccharidosis IVA, Mucopolysaccharidosis IVB, Alpha‐Mannosidosis) by using a standardized process in the framework of an innovative platform approach. CMC (Chemistry, Manufacturing and Controls), Non‐clinical and Clinical development plans have been optimized to generate a platform dataset complemented by specific disease data with the goal to generate a single combined Clinical Trial Application for the platform clinical testing. This innovative platform approach has the ambition to move from the “1‐to‐1 sequential” drug development to the “simultaneous and parallel” development approach, to help deliver on the promise of gene therapy to patients with rare diseases of limited commercial interest.
Efficacy improvement of an in vivo lentiviral vector gene therapy for the treatment of Primary Hyperoxaluria Type 1
1: Biomedical Innovation Unit, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain 2: Unidad Mixta de Terapias Avanzadas. Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS‐FJD, UAM), Madrid, Spain. 3: San Raffaele Telethon Institute for Gene Therapy (HSR‐TIGET), Milan, Italy 4: Vita‐Salute San Raffaele University, Milan, Italy 5: Pathology Department, Hospital Universitario de Canarias, Universidad La Laguna. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Tenerife, Spain
Primary Hyperoxaluria Type 1 (PH1) is a rare genetic disorder caused by mutations in the AGXT gene which encodes for the liver enzyme alanine‐glyoxylate aminotransferase. PH1 patients suffer from oxalate overproduction that can result in end‐stage renal disease and life‐threatening oxalosis. Thirty percent patients show infantile onset. The only curative treatment is liver and kidney double‐transplant. In vivo lentiviral vector (LV) gene therapy has emerged as a promising therapy for liver monogenic diseases, especially in pediatric patients, taking advantage of its integrative capacity. We have previously reported a partial pathological phenotype reversion in in vivo LV treated PH1 mice. However, this strategy needs to be improved to achieve therapeutic levels.
LVs could induce an IFNalpha‐mediated immune response when injected in vivo. Therefore, we explored the sporadic use of immunosuppressors as transduction enhancers. First, C57BL/6 adult mice were treated with a reporter LV expressing eGFP in combination with cyclosporin H, an inhibitor of a protein involved in the LV entry. These results showed no improvement in terms of vector integration or transduction percentage. Next, we combined the treatment of C57BL/6 adult mice with the reporter LV with dexamethasone, a well‐known immunosuppressor, obtaining a reduction in the vector integration in the whole liver. Interestingly, transduction efficacy in the hepatocytes was tripled, reaching up to 20% transduction.
Overall, we identified dexamethasone as a potential transduction enhancer in the context of in vivo LV gene therapy, which supports the feasibility of this strategy in the context of PH1 patients.
Intravascular delivery of AAV9 vectors into adult and neonatal mice display transgene expression differences in transduced organs
1: IDIBAPS 2: CIBERER 3: Hospital Clinic Barcelona 4: Universitat de Barcelona
Inherited metabolic diseases, represents a collection of around 700 genetic disorders. For some of these conditions adeno‐associated virus (AAV) mediated gene therapy has shown promising results in the clinic. AAV serotype 9 (AAV9) has gained interest in gene therapy applications due to its high tropism in peripheral tissues, such as liver, spleen and heart, following intravascular delivery and its capacity to cross the blood brain barrier and transduce neuronal and glial cells. In the current study, we compared the transduction efficiency and the persistence of transgene expression following systemic administration of AAV9 vectors in adult and neonatal mice. Intravenous delivery was performed by tail vein injection in adult mice and through temporal vein at postnatal day1 (P1) in neonatal mice. Viral genome quantification and transgene expression were analysed in several organs and brain regions at 1 and 6 months post‐viral administration. Injection into adult mice confirmed a widespread distribution of the virus and good expression levels specially in liver that were maintained at six months. Expression in brain structures were detected at low levels and slightly decreased with time. Interestingly, neonatal administration resulted in high expression in peripheral tissues and brain regions. Over time, transgene expression was reduced in liver, but remained at high levels in the different brain regions. These results suggest that AAV9 intravascular treatment in adults can be more appropriate for diseases affecting peripheral organs, whereas rare conditions with brain pathology might benefit from temporal vein administration in neonatal mice.
Quantification of hexadecanoylsulphatide in dried blood spots using liquid chromatography tandem mass spectrometry
H Brown1 M Gelb2
1: Manchester University NHS Foundation Trust 2: University of Washington 3: Orchard Therapeutics
Interest is growing in the characterisation of sulphatides for newborn screening (NBS) for metachromatic leukodytrophy. Quantitative concordance will enable meaningful method comparisons between studies when exchange of ultra‐rare positive control samples is a challenge. Method validation, and the evaluation of harmonisation practices are presented. Sulphatides were extracted from a dried blood spot (DBS) punch in methanol containing D5‐hexadecanoylsulphatide internal standard (D5‐ISTD) and analysed in <2 minutes by negative electrospray ionisation by LC‐MS/MS using a novel, low surface‐binding UPLC column. Quantification of hexadecanoylsulphatide (C16:0‐S) using qNMR‐verified D5‐ISTD was validated according to ISO 15189‐compliant procedures; other species response ratios were monitored but not validated. Assay control was verified by healthy adult donor C16:0‐S QC, and DBS enriched with 13C8‐C16:0‐S in the neonatal range. An external C16:0‐S calibrator and a qNMR‐verified Response Factor Solution (RFS) of equimolar C16:0‐S, D5‐ISTD and 13C8‐C16:0‐S was analysed for normalisation and harmonisation, and to support the use of 13C8‐C16:0‐S as a surrogate QC. Anonymised, presumed negative control NBS samples (n = 125) were analysed to assess analytical sensitivity. Assay performance met laboratory validation specifications. Median C16:0‐S concentration in NBS samples was 100nM (66‐232) and 84nM (55‐194) with RFS adjustment. The median normalised C16:0‐S was 0.40 (0.26‐0.93). Initial results from anonymised samples suggest the method is fit for purpose for our NBS pilot study. Performance of the novel column was satisfactory and peak shape resulted in improved signal to noise ratios. Control sample C16:0‐S and normalised results were comparable with published pilot studies. Wider participation in the harmonisation evaluation is encouraged.
Development of an ex vivo Gene Therapy for Infantile GM1‐Gangliosidosis
1: Pediatric Hematology and Oncology Center, Woman's and Child Health Department, University of Padova, Padova, Italy 2: Altheia Science, Milan, Italy 3: Harvard Medical School, Harvard University, Boston, USA
GM1‐gangliosidosis (GM1, OMIM #230500) is a rare, recessive Lysosomal Storage Disorder (LSD) caused by mutations in the GLB1 gene encoding the β‐galactosidase enzyme. The enzymatic deficiency causes lysosomal accumulation of undegraded metabolites and secondary mechanisms of cell damage. The rapid and severe neurodegeneration in GM1, requires a therapy providing a prompt and robust enzyme delivery to the central nervous system, possibly associated to reduction of local inflammation. Ex vivo Gene therapy (GT), i.e. the autologous transplantation of Hematopoietic Stem/Progenitor Cell (HSPCs) genetically‐corrected by lentiviral gene transfer, may represent a valuable therapeutic option for GM1, as proven for other LSDs in multiple preclinical and pivotal clinical studies. The rationale is based on the replacement of patient's microglia with a metabolically‐competent myeloid progeny of the transplanted HSPCs, which restore the metabolic defect and a normal scavenging function, rescuing the storage and mitigating tissue damage.
To this aim, we developed therapeutic lentiviral vectors (LVs) expressing a codon‐optimized sequence of the human GLB1 gene alone or in combination with an immunomodulatory molecule i.e.metallothionein 1G (MT1G). Both LVs can be produced at high titer and infectivity, and efficiently deliver multiple gene copies in human and murine HSPCs. LV‐transduced cells express dose‐dependent levels of the therapeutic enzyme, which is correctly secreted and uptaken in cell linens and primary patient‐derived fibroblasts, whose transduction with the therapeutic LVs resulted in the rescue of the storage disease phenotype. We are currently evaluating the efficacy and feasibility of our GT strategy in the disease animal model.
RNA editing based on CasRx system is an alternative substrate reduction therapy for Primary Hyperoxaluria type I
1: Center for Applied Medical Research 2: Instituto de Investigación Sanitaria de Navarra 3: Grousbeck Gene Therapy Center 4: Mass Eye and Ear 5: Harvard Medical School
Primary Hyperoxaluria type I (PH1) is a rare monogenic disease associated with a deficiency of the hepatic alanine‐glyoxylate aminotransferase (AGXT) enzyme that leads to oxalate overproduction and its accumulation in the kidneys, causing renal failure and systemic oxalosis. Inhibition of glycolate oxidase (GO) by targeting the Hao1 gene with CRISPR/Cas9 has been demonstrated to be an efficacious substrate reduction therapy in a PH1 mouse model, preventing oxalate accumulation and renal damage. However, potential undesirable side effects associated with permanent modifications of the genome remain a safety concern. Here we have explored the use of CRISPR/CasRx, a programmable RNA‐guided, RNA‐targeting CRISPR system that allows targeted gene knockdown without altering the genome. First, guide‐RNAs (gRNAs) targeting Hao1 mRNA were tested in vitro in transfected mammalian cells. All gRNAs showed similar GO protein reduction efficacy. Moreover, CasRx‐bystander cleavage of exogenous non‐targeted mRNAs was observed as shown by a significant reduction of reporter protein (GFP) expression, while the expression of endogenous proteins remained unaltered. Next, the gRNAs associated with the lower bystander effect were combined in an array and tested in vitro showing a significant reduction of GO protein expression. These promising results supported the in vivo evaluation of CasRx combined with single or multiple gRNAs. In summary, CasRx represents an attractive system to reduce protein expression without altering the cellular genome. In vivo studies are ongoing to test the therapeutic efficacy of the AAV8‐CasRx system in PH1 mice.
Development of a new therapeutic strategy based on RNA interference for lysine catabolism diseases
1: Institut d'Investigacions Biomediques August Pi i Sunyer (IDIBAPS) 2: CIBERER 3: Hospital Clínic Barcelona
Glutaric aciduria (AG1) and pyridoxine‐dependent epilepsy (PDE) are two inherited disorders caused by enzymatic defects in the lysine catabolism pathway. Both are characterized by the accumulation of toxic metabolites in the central nervous system. Currently, the available treatment consists mainly on dietary restriction of lysine and, in the case of PDE, pyridoxine is administered. Despite a good response to treatment, it still remains residual neurological damage. There is a need to develop more effective treatments that can reverse the neurological damage in both diseases. In this study we propose to explore the silencing of the first enzyme of the lysine degradation pathway, alpha‐aminoadipic semialdehyde synthase (AASS), as a common therapeutic strategy for AG1 and PDE. The blockade of this pathway would allow the reduction of the accumulation of neurotoxic metabolites, although it would cause an AASS deficiency and consequently hyperlysinemia, which is a benign entity in humans. We are developing a therapy based on interfering RNA (iRNA) using adeno‐associated viruses (AAV). We have explored different approaches, which consist of activating the iRNA mechanism. We have screened candidate sequences studying the in vitro silencing efficiency and have identified sequences capable of inducing the silencing of AASS transcripts in human and mouse cells. As a consequence of the silencing, inhibition of the AASS enzymatic activity is observed, slowing down the catabolism of lysine. Our results suggest that a therapy based on the use of iRNA against AASS is attractive to evaluate in vivo in AG1 and PDE models.
Development of new AAV hybrid capsids with enhanced liver gene transfer properties
1: Genethon, UMR_S951, Inserm, Univ Evry, Université Paris Saclay, EPHE
Adeno‐associated vectors (AAV) are currently the platform of choice in gene therapy for many monogenetic diseases. One of the key advantages is their ability to naturally infect human cells and persist at long term in non‐dividing cells. Moreover, the AAV capsid plays a key role in the tropism by interacting with the host cell receptors thus determining the tissue specificity of the targeting. With the increased number of gene transfer clinical trials targeting the liver, it is crucial to develop new AAV capsids more hepato‐specific in order to achieve an efficient gene transfer with a low dose of vector.
In this context, domain swapping was used to design new AAV hybrid capsids in order to improve liver targeting. In vivo biodistribution, assessed after intravenous injection in C57BL/6 mice, highlighted some regions of interest in the capsid sequence that may improve the liver targeting. To confirm this hypothesis, the engineered capsids were combined with a liver specific promoter showing an increase of transgene expression in the liver compared to AAV8. Interestingly, no variation in the vector genome copy number per diploid cells were detected, suggesting a direct role of the capsid in enhancing the transgene expression.
This work provided new understanding on capsid modification and liver targeting paving the way for a new generation of AAV capsids with improved properties.
Generation and characterisation of a novel mouse model for Mucopolysaccharidosis type IVA
1: Universita Vita‐Salute San Raffaele 2: Telethon Institute of Gene Therapy (HSR‐TIGET) 3: Good Laboratory Practice (GLP) test facility, San Raffaele Telethon Institute for Gene Therapy (SR‐Tiget) 4: Preclinical imaging Facility, San Raffaele Scientific Institute, Milan, Italy. 5: Osteoporosis and bone and mineral metabolism Unit, San Raffaele Scientific Institute, Milan, Italy. 6: Mouse Behavior Facility, San Raffaele Scientific Institute, Milan, Italy. 7: Pediatric Immunohematology and Bone Marrow Transplantation Unit, San Raffaele Scientific Institute, Milan, Italy 8: University Vita‐Salute San Raffaele, Faculty of Medicine, Milan, Italy. 9: Pathology Unit, Department of Experimental Oncology, IRCCS San Raffaele Scientific Institute, Milan, Italy
Mucopolysaccharidosis type IVA (MPSIVA) is a lysosomal storage disease caused by mutations in the GALNS gene with consequent deficiency of N‐acetylgalactosamine‐6 sulphate sulphatase (GALNS) enzyme. This leads to an accumulation of Keratan Sulphate (KS) and Chondroitin‐6‐Sulphate (C6S) in tissues, causing a severe skeletal phenotype. Vimizin, an enzyme‐replacement therapy, is the only registered treatment, although not curative. An ex‐vivo gene therapy (GT) approach could represent a therapeutic option for MPSIVA patients; however, available models are not representative of the human disease, making therapy development challenging. Therefore, we propose to establish a new mouse model for MPSIVA. In C57Bl/6J mice, we generated a mutation by CRISPR‐Cas9, obtaining five different variants. Galns‐/‐ (knockouts, KOs) for each mutation are being examined at different time‐points under a biochemical (enzymatic activity, KS accumulation, complete blood count), morphological (weight‐gain, CT‐scan), behavioural (rotarod, grip and open‐field tests) and histopathological point of view to select the most representative model. Biochemical analyses have confirmed all KOs to have no GALNS activity. In parallel, KS accumulation was observed in the peripheral blood of KO mice, with differences among the mutants. We also observed KOs to be smaller than wild‐type mice at 6 weeks of age and to show a decreasing trend in the long bones' dimensions, suggesting a skeletal involvement. Overall, these results will allow us to select the most representative model of MPSIVA for further characterisation and testing of novel therapeutic approaches, such as ex‐vivo GT. Moreover, the underlying molecular mechanisms involved in MPSIVA skeletal progression will be investigated.
Extracellular vesicle‐based delivery of silencing sequences for the treatment of Machado‐Joseph disease (MJD)/ Spinocerebellar Ataxia type‐3 (SCA3)
1: CNC ‐ Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; 2: CIBB – Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal 3: Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal 4: Institute for Interdisciplinary Research, University of Coimbra, Portugal
Effective chondrogenic differentiation of human mesenchymal stem cells via hyaluronic acid‐based gene‐activated cryogels
1: Centro de Investigacións Científicas Avanzadas (CICA), Universidade da Coruña.
Gene‐activated matrixes (GAMs) constitute promising tools for cartilage tissue engineering due to their capacity to host a target cell population, while promoting in situ transfection. The main objective of this study was to design a GAM to promote the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) by combining a shape‐memory hyaluronic acid‐based cryogel and a new niosome formulation complexed with a plasmid encoding for the therapeutic transcription factor SOX9 (psox9 nioplexes). SOX9‐activated cryogels were cultured in chondrogenic medium for 21 days and chondrogenesis efficiency was evaluated by biochemical, histological and immunohistochemical analyses. SOX9‐activated cryogels generated with the commercial reagent Lipofectamine (psox9 lipoplexes) and cryogels containing only hMSCs, were used as positive and negative controls, respectively. Cryogels were prepared by radical polymerization at low temperatures, showing a porosity percentage up to 90% and shape memory capacity over 99%. SOX9 overexpression in cryogels led to increased proteoglycans contents compared with control constructs. Moreover, both protein and DNA contents were higher in cryogels with psox9 nioplexes compared with those comprising psox9 lipoplexes.
Effective chondrogenesis via SOX9‐activated cryogels was further confirmed by histological/immunohistochemical analyses showing an enhanced expression of hyaline‐like cartilage markers (SOX9, type‐II collagen, proteoglycans), with reduced expression of hypertrophy and osteogenic differentiation markers (type‐X and type‐I collagen). These results confirm the potential of hyaluronic acid cryogels activated with psox9 nioplexes to promote the effective chondrogenesis of hMSCs, while preserving their viability. Further studies will determine the potential of these GAMs for cartilage repair in an ex vivo model of cartilage defect.
Magnetic targeting of iron oxide nanoparticles to the kidneys
C J Saande2
1: Centre of Surface Engineering and Advanced Materials, Asociación de la Industria Navarra 2: Department of Gene Therapy and Regulation of Gene Expression, Center for Applied Medical Research, CIMA, University of Navarra
Many kidney‐targeted therapies present poor pharmacokinetic profiles, such as low delivery and retention. Magnetic nanoparticles (MNP) have been successfully applied in several fields to localize therapies to a target organ; thus, the present study aimed to characterize the renal retention of iron oxide MNP in the presence of a magnetic field gradient localized to the kidneys. MNP were administered to C57BL/6 mice intravenously (IV; 6.5 μg MNP/g body weight) via the retro‐orbital vein or locally to the left kidney (1.3 μg MNP/g body weight) via the renal vein or the ureter. Mice were randomly assigned to non‐magnetic control conditions, or to be placed for 1 hour with an external Neodymium N42 magnet localized to both kidneys or to the injected kidney, respectively, following IV or local administration. All mice were sacrificed 1 hour‐post administration. The brain, lungs, heart, liver, kidneys, spleen and bladder were excised and fluorescence was measured using the Photonimager Optima system (Biospace Lab). MNP accumulation in the kidneys increased significantly 1 hour‐post IV administration through application of an external magnetic field. MNP accumulation in the injected kidney was significantly higher relative to the other organs analysed after ureteral administration, and after renal vein administration compared to all organs except the lungs; however, no differences were observed between magnetic and non‐magnetic conditions. These data demonstrate that application of an external magnetic field significantly increased renal MNP accumulation after IV administration. These findings suggest that MNP may be an effective means to deliver therapies to the kidneys.
Extracellular vesicles‐mediated delivery of CRISPR/Cas9 systems to treat Machado‐Joseph Disease
1: Center for Neuroscience and Cell Biology 2: Center for Innovative Biomedicine and Biotechnology (CIBB) 3: Institute for Interdisciplinary Research 4: Faculty of Pharmacy, University of Coimbra
Machado‐Joseph Disease (MJD) is a neurodegenerative dominantly‐inherited disorder caused by abnormal over‐repetition of a CAG tract within the ataxin‐3 gene. To correct this and other genetic defects, CRISPR/Cas9 systems allow precise genome editing. However, a major challenge of CRISPR/Cas9 therapeutics is their long‐term expression in target tissues. To counteract this, extracellular vesicles (EVs) may be promising vehicles to transiently deliver CRISPR/Cas9 tools.
Therefore, in this work, the goal was to generate an EV‐based delivery platform for CRISPR/Cas9 systems to treat MJD.
An EV‐packaging signal (EVPack) was engineered with Cas9 and its nuclease activity evaluated. Enrichment of EVPack‐Cas9 protein and sgRNA in EVs and their transient EV‐mediated delivery to recipient cells was evaluated by western blot and qPCR. Modifications on EVs surface were performed to improve cargo delivery in vitro. Then, engineered EVs packaging an enriched Cas9/sgRNA cargo were incubated in recipient cells. Ataxin‐3 knock‐out was evaluated at DNA and protein level, as well as by a luminescent reporter system.
Western blot and immunocytochemistry results showed that EVPack signal association did not impair Cas9 activity. Moreover, EVPack‐modified cells significantly enriched Cas9 protein and sgRNA in EVs. Furthermore, modification of EVs surface improved delivery of CRISPR/Cas9 tools to recipient cells and led to significant knock‐out of ataxin‐3 expression in recipient cells.
These results provide evidence that our EV‐based platform efficiently packaged and transiently delivered CRISPR/Cas9 tools, significantly knocking‐out ATXN3 expression. This system allows alleviation of MJD and potentially other disorders, while mitigating the deleterious effects of CRISPR/Cas9 long‐term expression in eukaryotic systems.
Characterising the Immunotherapeutic Capabilities of Bacterial Outer‐Membrane Vesicles
1: UCL 2: UCL Institute of Child Health 3: Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences 4: University of Hong Kong
Increasing evidence suggests that outer‐membrane vesicles (OMVs) shed by Gram‐negative bacteria are highly immunogenic nanoparticles that can facilitate the recognition and destruction of malignant cells. At present however, the mechanistic pathways underlying the anti‐tumour response remains poorly understood. Herein, we sought to investigate OMV‐mediated immune interactions, elucidating key cells able to be leveraged in the context of potential cell therapy.
OMVs were isolated from a hypervesiculating E.coli K‐12 MG1655 strain expressing a penta‐acylated LPS, achieved via pal and lpxM deletions. Co‐cultures were performed using PBMCs from healthy donors, with cytokine and cell marker expression determined using ELISA and flow cytometry. Negatively‐isolated OMV‐activated lymphocytes were co‐cultured with various cancer cell lines and cytotoxicity investigated using the MTS assay and flow cytometry.
We observed an increase in activation markers on NK and αβ T cells, whilst γδ T cells were the predominant cell type to proliferate, expanding from 3% to 40% of the total lymphocyte population. Noticeably the majority of γδ T cells were of the Vγ9Vδ2 type, which possess the ability to respond to both bacterial metabolites as well as stress markers present on malignant cells. We observed robust cytolytic activity of Vγ9Vδ2 T cells against both breast cancer (SkBr3) and leukaemia (Nalm6) cell lines. Our findings support the hypothesis that Vγ9Vδ2 T cells are a crucial component of the OMV‐anti‐tumour immune response, cells that may be used to improve future immunotherapies.
Gold nanoclusters mediate effective knockdown of the TNF‐α gene in a triple negative breast cancer cell model
1: University of KwaZulu‐Natal
The tumour necrosis factor α (TNF‐α) is a cytokine that plays an integral role in mediating the inflammation process. Its dysregulation has been implicated in promoting the growth of several cancers, include triple negative breast cancer, and thus presents a novel target for knockdown. Gold nanoclusters (AuNC) are ultrasmall nanoparticles that have recently gained attention as potential gene and drug carriers. Their unique optical properties and favourable biocompatibility facilitates their use as dual imaging and delivery vectors. This study aimed to develop AuNC vectors, and evaluate their efficacy as anti‐TNF‐α siRNA delivery agents. AuNC were synthesised and functionalised with chitosan and polyethylene glycol in weight ratios of 1 and 2%. The functionalised AuNC (FAuNC) showed favourable physical characteristics and interactions with siRNA, being capable of efficiently condensing the siRNA into nanocomplexes <200 nm, and protecting it from RNase digestion. In vitro toxicity studies were conducted in the HEK293, MCF‐7, SKBR‐3, and MDA‐MB‐231 cell lines. FAuNC were found to be well‐tolerated in all cell lines tested, indicating their safety as delivery vehicles. Gene expression studies, conducted in the triple negative MDA‐MB‐231 cell line, showed FAuNC vectors to mediate a significant reduction in TNF‐α mRNA and protein levels. The effects of knockdown in these cells was further investigated using flow cytometry. TNF‐α knockdown, particularly by PEGylated FAuNC, was observed to lead to slightly increased apoptosis and oxidation levels, and a shift to the G0/G1 phase. Overall, the FAuNC showed their potential as delivery vectors, and require further investigation in vivo.
Production of extracellular vesicles(EVs) enriched with the sodium iodide symporter(NIS) in a serum free continuous culture bioreactor
1: Discipline of Surgery, Lambe Institute for Translational Research, National University of Ireland Galway, H91 V4AY Galway, Ireland 2: CÚRAM, SFI Research Centre for Medical Devices, National University of Ireland Galway, Galway H91 YR71, Ireland
The sodium iodide symporter(NIS) facilitates uptake of iodide by thyroid follicular cells. This is exploited for imaging and treatment of thyroid disease using radiolabelled iodide, and NIS transfer to tumours has been investigated as a means of imaging and ablating cancer. This study aimed to engineer breast cancer cells to express NIS and study transfer to extracellular vesicles(EVs) released by the cells in a 3D fibercell bioreactor system. MDA‐MB‐231 breast cancer cells were transduced with lentivirus containing hNIS(MDA‐hNIS) and expression confirmed via PCR, immunohistochemistry and western blot. MDA‐hNIS were seeded into a FiberCell Bioreactor® and once target glucose consumption was achieved, serum‐free media was employed. Sequential harvests of cell conditioned media(CCM) and cells were performed. EVs were isolated from CCM using size exclusion chromatography(Izon qEV10) and analysed via nanoparticle tracking analysis(NTA) and western blot targeting EV‐associated proteins and hNIS. MDA‐hNIS cells were successfully maintained in 3D serum‐free continuous culture for 6 weeks. PCR and western blot revealed that cells maintained robust hNIS expression throughout, with membrane targeting demonstrated through immunohistochemistry. Sixteen sequential harvests of CCM and the EVs within were performed producing a consistently high yield of EVs over time. Isolated EVs were found to be 50‐200nm in size and expressed EV associated proteins CD63, CD81 and TSG101. hNIS protein was also detected in EVs and was efficiently transferred to wild type recipient cells. This study demonstrates successful and efficient scale up and longitudinal production of engineered EVs, and is an important first step in investigating EV‐NIS transfer.
Redox‐sensitive mesoporous silica nanoparticles exhibit enhanced tumour‐targeting ability for the co‐delivery of c‐myc siRNA and 5‐fluorouracil
1: The University of KwaZulu‐Natal
The asperity of conventional cancer treatment options calls for the development of improved and tailored cancer strategies that can improve current clinical diagnoses and be commercially relevant for developing nations. Polymeric mesoporous silica nanoparticles (MSNs) possess a rigid framework and large surface area that can incorporate supramolecular constructs and varying metal species that allow for stimuli‐responsive controlled release functions that may actively target the tumour microenvironment by use of an endogenous trigger (redox, pH) or an externally applied exogenous trigger (light, magnetic stimuli). In this study, MSNs of distinct structural integrity, size (36‐90 nm), morphology and relevant redox‐sensitive polymeric surface modifications was produced for the explicit co‐delivery of the popular anti‐cancer drug, 5‐fluorouracil (5‐FU) and small interfering RNA (siRNA) targeting the overexpressed tumour proto‐oncogene c‐myc. Favourable drug loading (0.3119 mg5‐FU/mgfmsn), and release profiles (>40 % ; >7 mM GSH) of these 5‐FU‐MSN nanoconjugates were obtained over 72 hours in vitro. Cytotoxicity evaluations alluded to a cell specific cytotoxicity profile, with the cancer cells (Caco‐2, MCF‐7 and HeLa) showing reduced cell viability (≥50 %) over the non‐cancer (HEK293) cells. Enhanced cellular uptake of the siRNA nanocomplexes was observed which corresponded to successful silencing of the c‐ MYC gene (> 65% decrease in c‐myc gene expression and >60% decrease in c‐myc protein expression). Hence, these redox‐sensitive MSN formulations show promising potential for the safe and enhanced synergistic delivery of conventional anti‐cancer drugs and siRNA in vitro.
Dual treatment of gliomas through IDH1 gene knockout via CRISPR/Cas9 and surface modified selenium nanocarriers
1: University of KwaZulu‐Natal
The metabolic alterations to cancer cells are immense but play a crucial role in understanding the disease. Oxidative stress has been a fundamental phenotype of cancers due to the high metabolic activities of these cells. NAD(P) + /NAD(P)H play a role in the redox relief of oxidative stress within a cell. The IDH1 gene belongs to a family of genes that control NAD(P) + /NAD(P)H within cells through the Krebs cycle, with IDH1 gene being most active within the cytoplasm. Mutations to the gene have been shown to impair cell growth. This study aimed to alter/silence the functional protein site at exon 4 through CRISPR/Cas9 mediated knockout in gliomas. Intern using a SeNP vector to deliver the CRISPR/Cas9 molecule in the form of a plasmid. SeNPs are known to alleviate oxidative stress in cells and increase ROS activity in cancer cells compared to normal cells. To achieve this SeNPs were surface modified with chitosan, polyethylene glycol and transferrin, and fully characterised by transmission electron microscopy, nanoparticle tracking analysis UV‐visible and Fourier transform infra‐red spectroscopy. Guide RNA was selected through in vitro digestion of a PCR fragment. SeNP plasmid binding and protection was assessed through intercalation, band shift and nuclease protection assays. Cytotoxic effects of SeNP treatment were assessed through MTT, oxidation, cell cycle arrest, apoptosis, and DNA damage assays. Knockout of the IDH1 gene was confirmed through the T7E1, qPCR and ELISA analysis. U87 cells showed a significant reduction in growth indicating a novel strategy for treating gliomas.
The Potential of Titanium Dioxide Nanoparticles for Cancer Gene Therapy: Suitable Gene Delivery Vehicles?
1: The University of KwaZulu‐Natal
Genetic aberrations are the leading cause of cancer formation and progression worldwide. With great advancements in nanomedicine over the last decade, it has the potential, together with gene‐based therapies, to be a major pillar in cancer therapy. However, there is the need for non‐toxic, biocompatible nanoparticles (NPs) for the delivery of therapeutic genes in cancer therapy. Titanium dioxide (TiO2) NPs, commonly used in cosmetics, pharmaceutics, and food products, have gained much interest for biological applications due to their high surface area, low toxicity, and stability, however, their use in cancer gene therapy has not been yet explored. In this study, curcumin reduced TiO2 nanoparticles were synthesized and functionalized with the cationic polymer, chitosan (CS). Physicochemical characterization with UV‐vis spectroscopy, DLS and TEM have shown that CS functionalization influenced the size, zeta potential, and PDI of the TiO2 NPs. The band shift, and nuclease protection studies showed the ability of the NPs to bind and protect the DNA. Minimal toxicity of the NPs and their nanocomplexes with DNA was exhibited on the HEK293, HeLa, MCF‐7 and HepG2 cell lines, following the MTT assay, with cell survival over 80%. The transfection ability of these TiO2nanocomplexes with pCMV‐Luc pDNA was confirmed using the luciferase reporter gene assay. This study provides preliminary evidence of the ideal physicochemical and biocompatible nature of CS functionalized TiO2 NPs with enhanced transfection efficiency which makes them promising and attractive vehicles for cancer gene therapy.
Novel biocompatible nanohydrogels for gene delivery
1: Department of Chemistry, Laboratory of Nano‐Biotechnology, Bar Ilan University, 52900, Ramat Gan, Israel
Here in, we have developed novel biocompatible nanohydrogels (NHG's) for nucleic acid packaging and delivery towards gene expression and regulation. The NHG's are obtained from mixtures of N‐isopropylacrylamide, acrylonitrile, di‐block, and tri‐block jeffamine macro‐monomers in the presence of a cross‐linker and radical initiator. The mechanism of NHG formation includes the formation of a self‐assembly obtained by heating the thermo‐responsive monomer's mixture. The final size of the final NHG is dictated by the size of the intermediary self‐assembly. Sizes are tuned by combining different ratios of the starting monomeric mixtures which upon heating form self‐assemblies of varied sizes. Initiator is then added at high temperature and the polymerization gives place to the formation of NHG's. The obtained NHG's are then chemically reduced generating NHG's with positive zeta potential. The obtained reduced NHG's of 50, 200 and 400 nm were extensively characterized including DLS, zeta potential, TEM, AFM and NMR. The NHG's are highly biocompatible and can complex pDNA to form polyplexes devoid of cell toxicity as assessed by XTT assays. The in‐vitro results reveal that the NHG's can deliver DNA to cells and mediate long‐term gene expression. Additionally, results obtained after in‐vivo administration of m‐Cherry/NHGs polyplexes indicate a delayed but prolonged expression of the marker gene.
Subsequent transfection with BMP‐2 and BMP‐7 chemically modified mRNAs induces higher protein production in hMSCs
1: cBITE Department, MERLN Institute for Technology‐Inspired Regenerative Medicine, Maastricht University 2: Ethris GmbH 3: Mayo Clinic 4: Department of Orthopaedic, Trauma, and Reconstructive Surgery, RWTH Aachen University Hospital
Gene transfer applications are not limited to treating genetic diseases. In the context of regenerative medicine, gene therapy could also deliver gene products that stimulate regeneration of damaged tissues. Non‐healing fractures are among the biggest challenges associated with bone diseases due to their high frequency, expensive treatments and reduced patient's quality‐of‐life. Considerable progress has been made for the delivery of pDNA to stimulate bone regeneration. Nevertheless, traditional gene therapy has yet to generate a clinical product. This technology is associated with limitations including safety issues and affordability. Transcript therapy uses mRNA instead of pDNA to deliver genetic material. This new type of therapy promises to combine safety with affordability, and is thus attractive in the gene therapy field. We studied chemically modified mRNA (cmRNA) mediated gene delivery of two potent osteoinductive growth factors, namely bone morphogenetic proteins‐2 and ‐7 (BMP‐2 and BMP‐7). The cmRNA lipoplexes obtained featured sizes of 90‐100 nm and negative Z potentials of ‐10 to ‐15 mV. Transfection of human mesenchymal stromal cells with BMP‐2 and BMP‐7 cmRNA alone or in combination was performed with Lipofectamine™ MessengerMAX™. Interestingly, the highest peak of BMP‐7 protein production was observed when cells were transfected with BMP‐2 cmRNA and subsequently, after 5 days, with BMP‐7 cmRNA. This successive transfection also yielded double alkaline phosphatase activity at day 14 compared to the cmRNAs used individually. This work highlights the relevance of a dual cmRNA regime that may overcome the shortcomings of protein delivery while circumventing issues of traditional pDNA‐based gene therapy.
Development of in vitro transcribed mRNA therapeutics for cystic fibrosis
1: UCL Institute of Child Health
Cystic fibrosis (CF) is an autosomal recessive disorder caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The dysfunctional CFTR protein leads to production of a thick sticky mucus, chronic bacterial infection, and loss of lung function. Modulator drugs provide effective therapies for some CFTR genotypes but approximately 20% of patients remain untreatable. For this group we propose to develop in vitro transcribed CFTR messenger RNA (mRNA) as a novel therapy. We have developed an epithelial cell targeting nanoparticle to improve mRNA delivery comprising targeting peptides and lipids. The optimal design for transfection of 16HBE epithelial cells comprised the cationic lipid, ditetradecyl trimethyl ammonium propane (DTDTMA), the neutral helper lipid, 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine (DOPE), and peptide Y (K16GACYGLPHKFCG) with luciferase mRNA which led to high transfection efficiency. The second part of this project is to optimize the in vitro transcription process and mRNA structural elements to produce synthetic mRNA with high translation efficiency, stability, and low inflammatory potential. The in vitro transcription protocol was optimized with the RNA cap structure analog, 3’‐O‐Me‐m7G(5’)ppp(5’)G, added during in vitro transcription to allow co‐transcriptional capping. Unmodified uridine‐5’‐triphosphates (UTPs) were replaced by modified UTPs, N1‐methylpseudouridine‐5’‐triphosphate. Double strand RNA contaminants were removed by cellulose‐base purification. According to this optimised protocol, the luciferase mRNA produced transfected 16HBE cells with similar efficiency to commercially available luciferase mRNA. Taken together, this work provides the basis for the development of targeted gene therapy for cystic fibrosis that precisely delivers mRNA to epithelial cells in the lung.
Electroporation of plasmid DNA into plasma‐derived extracellular vesicles
1: Comenius University
Non‐viral vectors for gene therapy have limited applications especially due to low efficiency. Extracellular vesicles (EVs) could improve the gene transfer of plasmids by protecting the naked DNA against degradation. The aim of our study was to optimize the protocol for electroporation‐mediated plasmid DNA delivery into EVs and analyze its nuclease resistance.EVs from human plasma were isolated using ultracentrifugation, characterized and mixed with isolated plasmid DNA. Cuvette‐based Gene Pulser system was used for the electroporation. Voltage settings have been modified in a published electroporation protocol, while pulse timing and number of pulses remained stable. Sytox Green was used for the quantification of DNA outside of the vesicles. DNase I was added to prove the resistance of plasmid DNA against cleavage. DNA fluorescence did not reveal conclusive results indicating that Sytox Green is not suitable for the evaluation of insertion efficiency of plasmid DNA into EVs in this experiment. However, DNase removed most of the DNA regardless of the voltage settings used suggesting that most of the plasmid DNA was not protected by the EVs. Further optimization should focus on the preparation of plasmid DNA, isolation of a specific subtype of vesicles such as exosomes and also on the ratio of plasmid DNA to EVs. In addition to electroporation, sonication, saponification or alternating freeze‐thaw cycles should be tested. The final goal is the evaluation of plasma‐derived EVs as gene therapy vehicles in in vivo experiments.
A Universal Formulation for Several Adeno‐Associated Virus Serotypes Protects Against Thermal Degradation
1: Asklepios Biopharmaceutical, Research Triangle, 27709, USA 2: The University of North Carolina at Chapel Hill
The stability of gene therapy vectors is vital to the successful distribution, transportation, and administration of these therapeutics. Adeno‐associated virus (AAV) is an excellent candidate for gene therapy applications because of its average melting temperature (Tm) of 75°C. However, the Tm fluctuates by ±20°C, depending on the serotype and formulation buffer utilised. This fluctuation creates significant complications when formulating drug products, since stability of all AAV vectors must be confirmed prior to patient administration. Our aim was to develop a single formulation that could stabilise and protect four different serotypes of AAV from thermal degradation. Formulation optimisation was done by comparing the impact of two different buffer systems with varying concentrations of either a multivalent salt, sugar, and/or surfactant on the presence of aggregates (DLS), vector genome titre (qPCR), and viral particle titre (SEC). Top performing formulations were further evaluated for accelerated stability by monitoring the presence of free ssDNA, using a fluorescent‐based free DNA assay. Additionally, physical titre, capsid titre, and particle size were monitored to evaluate the trend between the presence of free ssDNA and the stability of AAVs in different formulations. In conclusion, we have identified a universal formulation and the critical role that multivalent salt concentration plays in stabilisation of AAVs.
Development of a novel genetically modified HAdV‐5 vector for improved infection of human mesenchymal stromal cells
1: Ulm University
Human multipotent mesenchymal stromal cells (hMSCs) are of significant therapeutic interest for different clinical applications. Due to their inherent ability to migrate to tumor tissue, they became relevant carrier cells for oncolytic adenoviral vectors. Thereby, extensive off‐target sequestration of adenoviral particles by cellular and non‐cellular barriers can be avoided enabling efficient systemic administration. However, viral vectors based on human adenovirus type 5 (HAdV‐5) show inefficient transduction of hMSCs. To circumvent this bottleneck, we aimed to generate a novel genetically‐modified HAdV‐5 vector. While most modifications of adenoviral capsids focus on the protruding Fiber protein, we altered distinct surface‐exposed amino acids in Hexon, the most abundant protein in the adenoviral capsid. Strikingly, one of the generated vectors (HAdV‐5‐HexPos3) transduced hMSCs more efficiently than any other adenovirus‐based strategy published so far and enhanced the transduction of various tumor cell lines. An in‐depth analysis of the uptake mechanism revealed heparan sulfate proteoglycans (HSPGs) to be crucial for the HAdV‐5‐HexPos3‐mediated gene transfer.
Moreover, several conditionally replicating HAdV‐5 vectors containing the HexPos3 mutation were generated. Although designed to prevent HAdV‐5 replication in healthy cells, both E1A‐delta24bp and E1B‐delta19K life cycle modified virus showed efficient replication in hMSCs. Interestingly, viruses carrying the E1B‐delta19K mutation showed significantly faster release of infectious viral progeny, which might negatively impact clinical outcomes due to a shortened migration time. Taken together, with the HAdV‐5‐HexPos3 mutant we present a novel vector that impressively enhances infection of hMSCs and might pave the way for more efficient hMSC‐based oncolytic virus therapy.
A novel cancer vaccine for melanoma based on an approved vaccine against measles, mumps, and rubella
1: University of Helsinki 2:
Common vaccines for infectious diseases have been repurposed as cancer immunotherapies. The intratumoral administration of these repurposed vaccines can induce immune cell infiltration into the treated tumor. Here, we have used an approved trivalent live attenuated measles, mumps, and rubella (MMR) vaccine in our previously developed PeptiENV cancer vaccine platform. The intratumoral administration of this novel MMR‐containing PeptiENV cancer vaccine significantly increased both intratumoral as well as systemic tumor‐specific T cell responses. In addition, PeptiENV therapy, in combination with immune checkpoint inhibitor therapy, improved tumor growth control and survival as well as increased the number of mice responsive to immune checkpoint inhibitor therapy. Importantly, mice pre‐vaccinated with the MMR vaccine responded equally well, if not better, to the PeptiENV therapy, indicating that pre‐existing immunity against the MMR vaccine viruses does not compromise the use of this novel cancer vaccine platform.
Development of an αvβ6 integrin selective oncolytic virotherapy based on the low seroprevalence human adenovirus, HAdV‐D10.
1: Division of Cancer and Genetics, School of Medicine, Cardiff University, Cardiff, CF14 4XN, UK. 2: Department of Genetics and Microbiology, Faculty of Science, Charles University, Vinicná 5, 128 44 Prague 2, Czech Republic. 3: Division of Molecular Biology, Ruder Boškovic Institute, Bijenicka cesta 54, 10000 Zagreb, Croatia. 4: European Cancer Stem Cell Research Institute, Cardiff University, Cardiff, CF24 4HQ, UK. 5: Division of Infection and Immunity, School of Medicine, Cardiff University, Cardiff, CF14 4XN, UK. 6: Velindre Cancer Centre, Whitchurch, Cardiff, CF14 2TL, UK.
Human adenoviruses (HAdV) have a broad range of clinical applications. Species C, HAdV‐C5 serotype is best characterized, but efficacy may be hampered by high prevalence of pre‐existing immunity and “off‐target” interactions resulting in dose limiting toxicities. Vectors derived from adenovirus species with lower rates of pre‐existing immunity may be advantageous for clinical applications. We generated a low seroprevalence HAdV‐D10 serotype vector, containing deletions in E1 and E3. We produced a 2.5Å structure of the HAdV‐D10 fiber knob domain and modelled potential receptor interactions. Surface plasmon resonance studies confirmed low affinity binding with known adenoviral receptors; CAR, CD46 and DSG2. Biological assays suggest HAdV‐D10 engages CAR and sialic acid but not CD46. In addition, HAdV‐D10 does not bind blood coagulation Factor X, potentially eliminating hepatic sequestration in vivo. HAdV‐D10 has limited tropism and retargeting is required for use as an effective virotherapy. A20, a peptide which binds αvβ6 integrin with high affinity and selectivity was incorporated into the DG loop of HAdV‐D10 fiber knob. Assays in multiple αvβ6+ cancer cell lines, demonstrated significantly increased transduction, confirmed microscopically. HAdV‐D10.A20 resisted neutralisation in the presence of HAdV‐C5 neutralising sera. Systemic delivery of HAdV‐D10.A20 demonstrated a significant increase in GFP expression in BT20 tumours. Replication competent HAdV‐D10.A20 demonstrated significant targeted cell killing with high specificity to αvβ6+ tumours both in vitro and in vivo. In summary, HAdV‐D10 possesses characteristics of a promising adenoviral vector including lower seroprevalence, weaker affinity receptor interactions, reduced off‐target effects and can be effectively retargeted into a tumour selective virotherapy.
KPC pancreatic cancer cells are a novel immune‐competent murine model supporting human adenovirus replication
1: IDIBAPS 2: Hospital Clínic Barcelona 3: Universitat de Barcelona 4: CIBERER 5: CIBEREHD
Pancreatic cancer remains one of the most lethal cancers, being oncolytic adenoviral therapy a promising approach for the treatment of this neoplasia. However, the lack of a suitable immune‐competent mouse model to study oncolytic adenovirus (OA) antitumor activity precludes the full evaluation of the virotherapy. Human OA are generally very limited to produce infectious viral progeny in murine cancer cells what hinders the study of the immunological activity linked to OA replication.
In the current work we established the murine KPC cell line from pancreatic tumors developed in LSL‐KRasG12D ; LSL‐Tp53R172H ; Pdx‐Cre mice. KPC cells were susceptible to adenoviral infection as shown by EGFP expression in KPC cells infected with the non‐replicative adenovirus (AdTL). The infectivity levels were superior to other murine lines tested, although lower than those reached in human pancreatic cancer cells (PANC‐1). KPC infected cells with a replicative‐competent adenovirus expressing the EGFP (Adwt_EGFP_late) displayed a noticeable cytopathic effect and generated a progeny of infective virions. Viral replication, analysed by E3 gene quantification revealed a viral release 5‐fold lower than that detected in human PANC‐1 cells but 103‐fold higher than that of CT26 murine cells. Moreover, Adwt and the oncolytic adenovirus AdNuPARmE1A triggered cytotoxic effects in KPC cells in a dose‐dependent manner. Interestingly, AdNuPARmE1A replication was also observed in KPC subcutaneous xenografts developed in immunodeficient and immunocompetent mice.
In summary, our data shows that KPC cells are semi‐permissive to human OA replication rendering KPC syngeneic xenografts an interesting model to evaluate the multifaceted antitumor activities of OA for pancreatic cancer.
A surface‐charge modified oncolytic adenovirus combines substantially reduced toxicity with improved tumor targeting and anti‐tumor efficacy after a single intravenous injection
1: University of Ulm 2: University Hospital of Ulm
Oncolytic viruses (OVs) based on human adenovirus type 5 (HAdV‐5) are under investigation as anti‐cancer therapeutics. A systemic route of OV administration would be beneficial as it might allow to also reach metastases. However, rapid blood clearance of HAdV‐5‐based vectors after intravenous (i.v.) injection limits the therapeutic potential of OVs and particle uptake by the liver, additionally, may result in hepatotoxicity. By genetic modification of the major capsid protein Hexon, we generated an HAdV‐5 oncolytic virus (HAdV‐5‐HexPos3) with a reduced negative surface charge. In vitro, HAdV‐5‐HexPos3 vectors exhibited significantly enhanced transduction efficiencies in cancer cells, even if binding to the primary receptor coxsackie‐ and adenovirus receptor (CAR) was ablated. In vivo biodistribution studies in tumor‐bearing mice revealed significantly reduced off‐target transduction in all analyzed tissues including in the liver. I.v. injection of HAdV‐5‐HexPos3 further led to substantially increased intratumoral vector loads leading to a 31‐fold elevated tumor‐to‐liver ratio compared to an unmodified vector control. Following single i.v. injection of HAdV‐5‐HexPos3 in a conditionally replicating virus format, we observed prolonged survival of tumor‐bearing mice, intratumoral viral loads detectable for up to 56 days after treatment and substantially reduced hepatotoxicity. Thus, HAdV‐5‐HexPos3 represents a promising candidate for future anti‐cancer therapy combining significantly reduced vector toxicity with improved anti‐tumor efficacy, the main limiting factors of systemically administered HAdV‐5‐based OVs.
Development of αvβ6 integrin specific “Precision Immunovirotherapies” expressing bispecific immune cell activators
1: Cardiff University
We previously described the construction of a highly engineered, tumour‐selective virotherapy, Ad5NULL‐A20, devoid of all native tropisms, but able to infect via αvβ6 integrin expressed in multiple aggressively transformed epithelial cancers. To enhance the activation of tumor infiltrating T‐cells and NK cells at tumor sites we engineered the Ad5NULL‐A20 platform to express bi‐specific molecules targeting either CD3, CD16, NKG2D via single chain variable fragments or NKG2D using its ligand MICA and the tumour‐antigen Epidermal growth factor receptor (EGFRscfv) to encourage immune cell activation and redirect an effective immune response to tumour sites. Incorporation of bispecific transgenes did not alter virus particle formation, infectivity nor oncolytic activity of the viruses. Secretion of bispecific molecules into the supernatant of virally infected cells was demonstrated by Western blot. CHO and CHO‐EGFR cells co‐cultured with NK cells in the presence of virally infected supernatants containing the CD16/NKG2D/MICA‐EGFR targeting bispecifics were analysed for CD107a degranulation. CD107a, indicative of NK cell‐mediated lysis was significantly upregulated (50‐60%) on the surface of NK cells co‐cultured with CHO‐EGFR cells. Similarly, CHO‐EGFR cells co‐cultured with a NF‐κB/Jurkat/GFP™ Transcriptional Reporter Cell Line in the presence of CD3‐EGFR supernatants saw a 35‐45% increase in GFP expression representing the upregulation of NF‐κB signalling. The bispecific constructs tested demonstrated both their specificity to the target antigen and proficiency to induce activation pathways in vitro. We demonstrate the arming of a targeted tumour agent, Ad5NULL‐A20 to express bispecific immune cell engagers designed to immunologically “heat up” the Tumour Microenvironment. This approach has significant translational potential.
Characterisation of a panel of wild type species D adenoviruses for suitability as oncolytic vectors
1: Cardiff University Division of Cancer and Genetics
In the field of oncolytic viruses (OV), Adenovirus (Ad) 5, a species C Ad, has proven popular due to its high lytic activity and the relative ease with which it can be manipulated. While promising results have been achieved, pre‐existing immunity against Ad5 and the rapid development of anti‐vector immunity limit the current potential of Ad5 based OVs. Species D Ads present lower levels of pre‐existing immunity in the population, representing a relatively untapped repository: Phylogenetically, species D Ads encompass the largest and most diverse species of Ad and might therefore prove to be a good alternative oncolytic platform.
We investigated a panel of species D Adenoviruses for their suitability as oncolytic vectors.
We studied wild type species D Ads in regard to cytotoxicity, innate immune activation, tropism and infective patterns compared to wild type Ad5. Both cytotoxicity and immune activation were found to be significantly lower in species D Ads, and differences in infective patterns were observed, indicating that their use as OV may require further engineering and optimisation.
Whilst cytotoxicity, which may induce immunogenic cell death, is a potentially advantageous trait for an OV, higher levels of cell killing may be achieved through the expression of inserted transgenes. As rapid cytotoxicity may interfere with therapeutic transgene expression, species D Ads may be well suited to this approach. The immune evasion (both innate and adaptive) observed in species D Ads make them interesting platforms as OV, suitable for onward engineering and optimisation.
Development of novel immunotherapy:virotherapy combinations for head and neck cancer
1: Division of Cancer and Genetics, Cardiff University, Cardiff, CF14 4XN 2: Immunocore Ltd, Milton Park, Abingdon, OX14 4RY 3: University Hospital of Wales, Heath Park Cardiff, CF14 4XW 4: Velindre Cancer Centre, Velindre Road, Cardiff, CF14 2TL
Head and neck cancer (HNC) is the 6th most common cancer globally. ImmTACs are bi‐specific molecules consisting of a monoclonal T‐cell receptor (mTCR) binding peptide:HLA on cancer cells, and anti‐CD3 scFv that binds and activates T‐cells. The mTCR binds cancer peptide antigens such as NY‐ESO‐1, a HNC biomarker. ImmTACs are restricted to HLA.A*02:01 patients, meaning >50% of the population are refractive to this therapy. Furthermore, the benefit of such immunotherapies is limited by immunosuppressive tumour microenvironments. Oncolytic virotherapies (OVs) infect and lyse tumour cells, stimulating Immunogenic cell death. Adenoviruses (Ads) are attractive OV platforms due to their ease of manipulation. Our precision OV Ad5NULL‐A20 is tropism‐modified to exclusively infect tumour cells expressing αvβ6 integrin.
Using flow cytometry and immunohistochemistry, we demonstrate that HNC expresses Ad receptors and αvβ6 integrin. Having confirmed the selectivity of Ad5NULL‐A20 to αvβ6 integrin in transduction assays, we engineered Ad5 and Ad5NULL‐A20 expressing anti‐NY‐ESO‐1 ImmTACs for local delivery in HLA.A*02:01 patients. We confirmed the expression and secretion of ImmTACs by western blot. We also engineered Ad5 and Ad5NULL‐A20 vectors to produce HLA.A*02:01 presenting NY‐ESO‐1 peptide on the cell surface, and confirmed production by western blot. Finally, we established HNC patient‐derived organoids and demonstrate these organoids express common Ad receptors and αvβ6 integrin.
We demonstrate the potential of using virotherapies to express either ImmTACs or peptide:HLA complexes to sensitise patients of alternative HLA types to ImmTAC therapy. Both strategies present a powerful new approaches to harness patient immunity against HNC, presenting an opportunity for clinical translation.
Development of a self‐amplifying RNA vector expressing a high affinity PD‐1 domain for cancer therapy
A Gónzalez de Zárate1 A Covo‐Vergara1 A Igea‐Sucunza1 N Silva‐Pilipich1 S Cuenca1 E Blanco2 G Herrador‐Cañete1 J Martínez‐García1
1: Division of Gene Therapy and Regulation of Gene Expression, Cima Universidad de Navarra and Instituto de Investigación Sanitaria de Navarra (IdISNA), Pamplona, Spain 2: Oncoimmunology Research Unit, Navarrabiomed‐Fundación Miguel Servet, Universidad Pública de Navarra (UPNA), Complejo Hospitalario de Navarra (CHN), Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona, Spain
Cancer research has been revolutionized by immune checkpoints inhibitors based on monoclonal antibodies (mAbs). However, mAbs, which are given systemically, have poor tissue/tumor penetrance, can have Fc‐effector detrimental functions that lead to depletion of immune cells, and could be immunogenic. We reasoned that a soluble fragment of the PD‐1 ectodomain (sPD‐1) could be used as a competitive antagonist of PD‐L1. Having 14 kDa, sPD‐1 would be approximately ten‐fold smaller than a mAb and lack an Fc moiety. The aim of our study was to use a non‐antibody based strategy based on local expression in tumors of sPD‐1 using a self‐amplifiying RNA vector derived from Semliki Forest virus (SFV). We have previously shown that SFV vectors can be used to express locally cytokines and immunomodulatory mAbs, resulting in potent antitumor responses. We have now developed SFV vectors expressing three different variants of sPD‐1, including the human, a human high‐affinity version (HAC‐sPD‐1) containing ten mutations, and the murine sequences. BHK cells infected with SFV viral particles or electroporated with SFV RNA showed good expression and secretion levels of recombinant ligands, being murine sPD‐1 the one expressed at higher levels. HAC‐sPD‐1 produced by SFV was able to efficiently bind human and murine PD‐L1 in a binding ELISA assay, being more efficient in the first case. Interestingly, HAC‐sPD‐1 was very effective to block human PD‐1/PD‐L1 interactions, analysed by a competitive ELISA assay, even at very low concentrations. We believe that the SFV vector expressing HAC‐sPD‐1 could have potential therapeutic activity against human cancer.
Generation of precision αVβ6 tumour‐targeted species D adenovirus fiber‐knob proteins
1: Division of Cancer and Genetics, Cardiff University School of Medicine, Cardiff, CF14 4XN 2: Division of Infection and Immunity, Cardiff University School of Medicine, Cardiff, CF14 4XN
The use of adenoviruses as oncolytic virotherapy vectors requires their precision targeting to tumour cells. Achieving this is predicated upon an intimate understanding of the native interactions mediated by adenoviral capsid proteins, and the development of engineering strategies to both ablate these and confer novel tumour selective binding properties. The distal C‐terminal knob domain of the trimeric adenovirus fiber mediates the primary virus interactions with cell surface receptors. Well‐established methods to disrupt native fiber‐knob binding and incorporate tumour targeting moieties exist for the frequently used human adenovirus type‐5 (Ad5). However, the high seroprevalence of Ad5 and resulting presence of pre‐existing neutralising antibodies can significantly diminish the efficacy of these therapeutics. We have employed homology modelling using fiber‐knob crystal structures from less seroprevalent species D adenoviruses to identify novel sites suitable for incorporation of retargeting elements in adenovirus types‐15, 24, 25 and 30. We demonstrate that the DG loop is a surface exposed, flexible loop capable of tolerating such insertions, including of the peptide A20, which retargets binding to αvβ6 integrins overexpressed on aggressive epithelial carcinomas. Initial characterization of recombinant species D Ad.DG.A20 knob proteins confirmed their selective binding to αvβ6 expressing cells in vitro. Further studies will elucidate their interactions with native adenoviral receptors, and reveal mechanisms to ablate these. Such fiber‐knobs could subsequently be employed to achieve tumour‐specific targeting of novel oncolytic adenovirotherapy vectors, or be used in isolation as companion diagnostic imaging tools and drug‐conjugates to facilitate tumour‐selective delivery of chemotherapy agents.
AAV‐mediated expression of mouse IL‐12 shows potent anti‐tumor activity through innate immunological activation in syngeneic murine cancer models
D Fellner1 P Prentiss1 J Liu1 M Stone1 M Stone1 R Roeloffs1
1: Kriya Therapeutics
Interleukin‐12 (IL‐12) is a pleiotropic potent pro‐inflammatory type 1 cytokine that has demonstrated substantial promise as an anti‐tumor agent; however, therapeutic success has been hampered by severe systemic toxicities. IL‐12 elicits antitumor activity through the activation of both natural killer and CD8+ T cells along with differentiation of CD4+ T cells to the TH1, pro‐inflammatory subtype. KT‐A281 is an adeno‐associated virus vector (AAV) designed to convert an injected tumor into a biofactory for continuous IL‐12‐driven immunostimulation of the tumor microenvironment. Direct intratumoral (IT) injection is expected to limit IL‐12‐driven toxicity seen with systemic administration of recombinant IL‐12. Adaptive immunological memory induced by local IL‐12 activity via KT‐A281 was demonstrated in multiple syngeneic mouse models (MC38, H22, and CloudmanS91). Additionally, IT delivery of KT‐A281 alone or in combination with anti‐PD‐1 was well tolerated.
Durability of effect and long‐term immunity were demonstrated in tumor rechallenge experiments using mice that demonstrated complete response as assessed by palpation following treatment with a murine version of IL‐12 in KT‐A281 alone or in combination with anti‐PD‐1. Mice were rechallenged with the same tumor line with which they were originally implanted. In all models, newly implanted tumors failed to grow or had delayed growth in previously treated tumor‐free mice; however vigorous growth of the same tumor in naïve control animals was observed. These results are consistent with published literature of IL‐12 therapy and suggestive of long‐term antitumor T cell memory response that can potentially translate to a durability of response clinically.
Investigating sialic acid usage in species D human adenoviruses
1: Division of Cancer and Genetics, Cardiff University School of Medicine, Cardiff, CF14 4XN, UK 2: Division of Infection and Immunity, Cardiff University School of Medicine, Cardiff, CF14 4XN, UK
Human adenoviruses are phylogenetically divided across seven species, A‐G, causing transient mild illnesses, except in immunocompromised individuals. Their double stranded DNA genome is amenable to genetic manipulations, enabling development of highly engineered virotherapies. Species D adenoviruses have naturally low seroprevalence rates, an important trait in avoiding neutralising anti‐vector immunity.
We previously demonstrated that HAdV‐D26, the platform of the Janssen SARS‐CoV2 vaccine, uses sialic acid as a primary cell entry receptor. Here, we structurally and biologically investigated sialic acid usage across species D.
We solved multiple structures of species D adenovirus fiber knob proteins alone and in complex with sialic acid, identifying a conserved binding pocket common with known sialic acid binders HAdV‐D26 and 37. Using fiber‐knob pseudotyped viruses, we demonstrate significantly reduced transduction in cells treated with neuraminidase to remove sialic acid residues in HAdV‐D26 and 53, with HAdV‐D15, 24 and 29 also demonstrating non‐significant reductions. IC50 data also showed highlighted binding to CAR, although at a significantly lower affinity compared to the CAR‐binding HAdV‐C5.
Improved understanding of the usage of sialic acid as a receptor will enable better exploitation of the species D adenoviruses as therapeutic vectors. Our findings raise the possibility of a conserved sialic acid binding pocket within species D adenoviruses resulting in varying affinity levels. Further evaluation of specific glycan binding patterns used by these viruses, as observed between HAdV‐D37 and GD1a glycan, will better inform the design of appropriate antivirals to contain adenovirus outbreaks as well as the engineering of targeted vectors for translational applications.
Exploiting epigenetic therapy to improve viral‐based vaccines for cancer immunotherapy
1: University of Helsinki
Viral‐based vaccines are an attractive tool for cancer immunotherapy due to their tumour‐selective replication and stimulation of both innate and adaptive immune responses. However, viral‐based vaccines as single agents fail when poorly immunogenic cancers express low MHC levels, impairing antigen presentation and therefore T‐cell recognition, which is crucial for successful immunotherapy‐based treatments. On the other hand, epigenetic drugs are approved for treating haematological malignancies and represent an exciting solution to reprogram cancer cells. Indeed, besides acting like chemotherapy agents, epi‐drugs are known to increase cancer immunogenicity by enhancing the MHC machinery for antigen presentation. In these settings, FDA‐approved de‐methylating agents like Azacytidine (AZA) and Decitabine (DAC) may increase patients' eligibility for therapeutic cancer vaccines and adoptive cell therapies. Therefore, we claim a potential synergism between epigenetic therapy and immuno‐virotherapy for improved anti‐cancer vaccine development and to overcome viral‐based immunotherapy's limitations.
The new European regulation on Health Technology Assessment: what changes for Advanced Therapy Medicinal Products?
1: Aix‐Marseille University 2: Université Paul Sabatier 3: CNRS 4: Université de Bourgogne Franche‐Comté 5: INSERM UMR 1295 6: UMR 7318 (DICE) CERIC 7: Université de Toulon
The new European regulation (EU) 2021/2282 on Health Technology Assessment (HTA), “a multidisciplinary process that summarises information about the medical, patient and social aspects and the economic and ethical issues related to the use of a health technology in a systematic, transparent, unbiased and robust manner”, has been adopted on 15 December 2021. It ensures an efficient use of resources and strengthens the quality of common HTA across the European Union, because until now, the assessment has been realized independently by each Member State. It will thus contribute to the improvement of the availability of health innovative technologies for EU patients that has been widely highlighted by the Council of the European Union and the European Parliament. It mainly establishes a Member State Coordination Group on HTA, a stakeholder network, whom the joint works and exchanges will be facilitated thanks to an IT platform. It also provides rules for joint clinical assessments, joint scientific consultations, horizon scanning for emerging technologies, further voluntary cooperation notably on non‐clinical assessments on health technologies, i.e. “a medicinal product, a medical device, or medical and surgical procedures as well as measures for disease prevention, diagnosis or treatment used in healthcare”. The regulation shall apply progressively from 2025 to different types of health technologies, Advanced Therapy Medicinal Products (ATMPs) being the first targeted with cancer medicines. This poster will discuss the expected improvements for patients' access to ATMPs regarding the forthcoming implementation of this new regulation.
This work has been supported by ANR‐funded I‐BioLex project (ANR‐20‐CE26‐0007‐01)
Targeted exon skipping using conjugated tricyclo‐DNA antisense oligonucleotides: A therapeutic strategy for recessive dystrophic epidermolysis bullosa
1: Université Paris Cité 2: Imagine Institute 3: SQY Therapeutics 4: Université Versailles Saint Quentin en Yvelines 5: Curie Institute
Dystrophic Epidermolysis Bullosa (DEB) is a group of rare inherited genodermatoses causing skin and mucosal blistering and a wide range of local and systemic severe complications. DEB can be recessively (RDEB) or dominantly (DDEB) inherited and is caused by mutations in COL7A1 encoding type VII collagen (C7) the component of anchoring fibrils (AFs) which are essential for dermal‐epidermal adhesion. COL7A1 consists of 118 small exons of which the 82 encoding the central collagenous domain are in frame, making it an attractive target for antisense oligonucleotide (AON)‐mediated exon skipping. Among them, exon 73 carries the largest number of recessive and dominant mutations. We have designed a conjugated tricyclo‐DNA (tc‐DNA) AON targeting COL7A1 exon 73. Tc‐DNA AONs present higher affinity to pre‐mRNA and reduced toxicity compared to 2’OMePS AONs, allowing for more efficient systemic delivery. We showed that this AON induces efficient skipping of exon 73 (up to 72%) and ex vivo re‐expression of C7 in transduced primary homozygous and heterozygous RDEB keratinocytes and fibroblasts. We provide evidence that two subcutaneous injections of 1mg of this tc‐DNA induce efficient in vivo exon skipping in a transgenic murine model (mCol7a1‐/‐ ; TghCOL7A1) carrying the human COL7A1 genomic locus. Further investigations using local and systemic administrations of this AON in mice model grafted with human RDEB skin equivalents to demonstrate in vivo C7 expression restoration and AFs formation are currently ongoing. Our data demonstrate the potential relevance of tc‐DNA AON mediated‐exon 73 skipping of COL7A1 mRNA as a therapeutic strategy for RDEB and DDEB.
Using IVIM/SAGA as screening tools during lentiviral vector lead selection for detection of clinically translatable insertional transformational risk
R Pfeifer3
1: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany 2: Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA 3: AVROBIO, Cambridge, MA USA
Since the early 2000s, self‐inactivating (SIN) lentiviral vectors (LVVs) have been used to treat thousands of patients with LVV‐modified CD34+ cells or T cells, informing potentially favorable benefit/risk profiles. Recently, questions have surfaced around the potential of SIN‐LVVs with internal MND promoters to cause insertional oncogenesis, after reports from two clinical trials where dominant clones followed by onset of myelodysplastic syndrome (MDS) were observed in three participants.
Here, we will share in vitro immortalization (IVIM) assay and Surrogate Assay of Genotoxicity Analysis (SAGA) data from six LVVs to inform vector selection. Using IVIM assays for five SIN‐LVVs with internal EFS promoter, no C1 wells were detectable. In the MTT‐assay, these five LVVs had six of 40 plates above the Q1‐quantification threshold, incidence statistically different from assay‐day positive control (RSF91) values and metadata and indistinguishable from non‐transduced cells. Mean replating frequencies were lower compared to metadata of RSF91 reaching statistical significance. SAGA showed that two of 36 (0.06%) transduced vector samples had NES >1. In all cases, mean NES was statistically different from the assay‐day positive control and metadata (RSF91) and was not statistically different from metadata of SIN LVVs with internal EFS promoter. In contrast, for SIN‐LVV with internal MND promoter, replating frequency and incidence was not statistically different from the assay‐day positive control (RSF91). Using SAGA, four of nine (44%) transduced samples showed gene enrichment associated with insertional oncogenesis.
In summary, IVIM/SAGA evaluation performed early in preclinical development may inform LVV selection to inform potential risk profile for clinical programs.
Lentiviral MYO7A gene therapy rescues hearing and balance function in the Shaker‐1 mouse model of Usher syndrome type‐1B
1: Hannover Medical School 2: University of Kansas 3: Harvard Medical School
Hearing loss is the most common sensory disorder in humans, with a high and currently unmet social and clinical need for therapeutic options. Sensorineural hearing loss (SNHL) may be accompanied by balance loss, as the underlying defect often affects both the auditory and the vestibular sensory cells. While hearing aids and cochlear implants can attenuate the hearing loss phenotype in some patients, there is currently no treatment to address balance dysfunction. As the majority of SNHL cases and syndromes are due to genetic reasons, gene therapy appears as a promising novel treatment option.
Using a third‐generation self‐inactivating lentiviral vector platform, we demonstrate efficient transduction of the vestibular organ and the cochlea, including the major treatment‐relevant cell types, i.e. hair cells and spiral ganglion neurons. In the Shaker‐1 mouse model of Usher syndrome type‐1B, a combined blindness / deafness / balance loss syndrome caused by mutation of the myosin‐7a (Myo7a) gene, we achieved rescue of function through lentiviral gene therapy. The large MYO7A transgene (∼6.7 kb) was successfully packaged, transferred and expressed, which is not possible with most alternative vector systems due to their lower coding capacity. The early‐onset hearing‐loss / impaired‐balance phenotype manifesting in homozygous Shaker‐1 knockout animals was partially rescued upon lentiviral Myo7a vector administration through canalostomy. In heterozygotes, the observed late‐onset hearing loss was completely rescued to wild‐type hearing levels.
Altogether, we demonstrate the first therapeutic option for genetic forms of balance loss and the first gene therapy option addressing hearing loss using high‐capacity lentiviral vector technology.
Imposing a pro‐inflammatory state to solid tumor microenvironment through an autologous macrophage‐based immunotherapy: results from the TEM‐GBM study
1: San Raffale Telethon Institute for Gene Therapy (SR‐TIGET) 2: Hematology and Bone Marrow Transplant Unit ‐ San Raffaele Hospital – Milano 3: Neuro‐Oncology Unit ‐ Istituto Neurologico Carlo Besta – Milano 4: Neurosurgery Unit ‐ Policlinico Gemelli – Roma 5: Neuro‐Oncology Unity ‐ San Raffaele Hospital – Milano 6: Genenta Science‐Milano 7: Genenta Science‐New York
Temferon is an autologous HSC‐based platform that target IFNa into the tumor microenvironment (TME) via Tie‐2 expressing monocytes, by a transcriptional & post‐transcriptional control mediated by miRNA target sequences. As of June 2022, 4 doses of Temferon (0.5‐3.0x106/kg) have been Infused in 16 unmethylated MGMT GBM patients. To date, no dose limiting toxicity has occurred. Seven recurrent tumors were collected and where enough fresh material was available gene‐marked cells were identified in the CD45+ tumor infiltrate in 2/2 specimens (3%‐5%). scRNA analysis of the myeloid TME compartment (n = 4 Temferon patients) detected a broad induction of an IFN, TNF/NFkB and hypoxia response relative to n = 6 standard‐of‐care treated patients and unveiled an overrepresentation of pro‐inflammatory macrophage clusters. Strikingly, this closely resembled the microenvironmental changes observed in a murine GBM model treated with IFN gene therapy (Birocchi, Sci Transl Med, 2022), where a shift in the M2 to M1 macrophage balance was associated with tumor responses. Notably, the highest proportion of pro‐inflammatory macrophages was detected in a stable lesion biopsied from a patient that had a contemporaneous progressing lesion, which instead contained the lowest pro‐inflammatory macrophage quantity. Analysis of the Tcell compartment (scRNAseq+TCRseq) of the stable lesion highlights the presence of specific CD8+ T cells clones characterized by an effector phenotype with an inflammatory profile (IFNa/g response,TNFa signaling). These data support the hypothesis that Temferon, by acting on the M1‐M2 balance, favors a pro‐inflammatory state that, as predicted by preclinical studies, induces an immune system reset that may favor containment of GBM growth.
Non‐viral Gene Therapy for Autoimmune Pulmonary Alveolar Proteinosis
1: National Heart and Lung Institute, Imperial College London 2: Royal Brompton Hospital 3: University of Oxford 4: UK Respiratory Gene Therapy Consortium
Autoimmune pulmonary alveolar proteinosis (aPAP) is a rare lung disease characterised by the accumulation of surfactant in the alveoli due to anti‐granulocyte macrophage colony‐stimulating factor (GM‐CSF) antibodies. The standard of care is whole lung lavage (WLL), an invasive procedure that only treats symptoms. Recombinant GM‐CSF protein can outcompete the auto‐GM‐CSF antibodies and stimulates clearance of pulmonary surfactant, but a recent Phase 3 trial showed only a modest benefit when administered daily. We previously assessed whether lentiviral vector‐mediated GM‐CSF expression could correct aPAP biomarkers in a mouse model of the disease (GM‐CSF‐knockout mice). We showed that biomarkers were rapidly and persistently ameliorated. However, persistent high expression of GM‐CSF (over 11 months after single dose) caused histopathological changes in various organs, suggesting that transient expression (shorter bursts) is likely to improve the efficacy/toxicity window.
Here we show proof‐of‐concept studies using non‐viral (transient) gene therapy. Lungs of PAP mice were transfected with phCEFI‐mGM‐CSF complexed to the cationic lipid GL67A (80 μg pDNA/mouse) (n = 3‐5/group). Low levels of mGM‐CSF were detectable for at least 6 months but were undetectable 10 months after transduction. PAP biomarkers (bronchoalveolar lavage fluid turbidity, surfactant protein D levels and surfactant deposition) were significantly (p < 0.05 reduced) 3 months after transfection, efficacy was lost at later time points, with only BALF turbidity remaining significantly lower at 10 months post dosing. No histopathological changes were observed in treated animals 10 months after treatment. These data suggests that non‐viral based GM‐CSF gene therapy may provide a wider toxicity:efficacy window suitable for clinical translation.
Investigation of the Tumour Suppressive Potential of the mir‐379 Locus in Breast Cancer
1: Discipline of Surgery, Lambe Institute for Translational Research, National University of Ireland Galway 2: Cúram, SFI Research Centre for Medical Devices, National University of Ireland Galway
Members of the microRNA‐379 family located on the C14 gene megacluster are believed to have tumour suppressor potential in cancer. This study focuses on the potential anti‐cancer synergism of miRNAs from this family in breast cancer.
Breast cancer cells (MDA‐MB‐231, HCC‐1954) were transduced with a lentivirus construct containing miR‐379‐5p. Wildtype and transduced cells were subsequently transfected with a miR‐758‐3p mimic or scramble sequence using Lipofectamine® RNAiMAX. The impact of miRNA expression individually or in combination on cell viability was determined. Changes in cell cytoskeleton or migration were determined using phalloidin staining of F‐actin, and corning transwell inserts respectively. Secretion of pro‐angiogeneic factors was detected via angiogenesis array and ELISA, with tubule formation in response to secreted factors also assessed.
Significant elevation of miR‐379‐5p and miR‐758‐3p was confirmed in cells with no change in cell viability observed in the presence of one or both miRNAs. Phalloidin staining revealed a change in intracellular distribution of F‐actin. While control cells maintained elongated morphology with well‐polarized actin bundles, miR‐758 transfected cells displayed increased volume and loss of polarity. A decrease in migration was observed in MDA‐758 and MDA‐379 cells with the greatest decrease observed in cells engineered to express both miRNAs simultaneously. The miRNAs also resulted in decreased secretion of proangiogenic factors TIMP1/2 and VEGF, with a consequent decrease in tubule formation observed.
The data showcases a combined effect of miR‐379‐5p and miR‐758‐3p in breast cancer, targeting pathways involved in cancer metastasis and angiogenesis. The synergistic effects observed may have important implications for development of miRNA therapy.
Advanced characterization of human hepatocytes xenotransplanted mice as predictive pharmacological tool system for human liver‐targeted gene therapy
1: Evotec GT, part of Evotec International GmbH 2: Evotec International GmbH
Primary human hepatocytes xenotransplanted into mice represent an increasingly interesting system to investigate the translational potential of liver‐targeted gene therapy solutions and can in many cases provide an alternative to the use of non‐human primates during early efficacy evaluation.
Here we present a standardized workflow for the screening of AAV‐derived viral vectors in xenotransplanted mice, with a focus on improved human liver tropism and species‐specific transduction of the payload. We describe the application of histological and molecular techniques to study biodistribution and pharmacodynamics with resolution not limited to the tissue‐level but refined to the individual human hepatocyte.
In particular, we have established techniques to monitor in vivo as well as ex vivo expression of a reporter payload in mice, and precisely within the xenografted (human) vs. resident (murine) populations of liver cells. The combination of immunofluorescent labelling of the payload and detection of cell‐identifying markers further allows for the analysis of spatial biodistribution within the organ, such as cell‐type targeting and zonation effects.
For a deep molecular characterization, single‐nuclei RNA sequencing is also presented as an innovative technology allowing molecular identification of transduced cell populations and associated disturbances to the gene expression profile, with the potential to highlight altered molecular pathways that can inform either pharmacodynamic or toxicological outcomes.
In summary, we disclose the application of xenotransplanted mice and associated analytics vastly improving the translational value of the model system in the development of gene therapies.
Integrating and Non‐Integrating Lentiviral Vector‐mediated Induced Pluripotent Stem Cell Generation from Human Fibroblast Cells and Differentiation into Pancreatic Beta Cell‐Like Insulin‐Producing Cells
1: Akdeniz University Center for Gene and Cell Therapy
Induced pluripotent stem cells (iPSCs) hold great potential for regenerative medicine applications due to their ability to self‐renew and differentiate into different cells including beta‐like cells for potential use in diabetes treatment. Lentiviral transfer of OSK (Oct4, Sox2, Klf4) reprogramming factors is an effective way for generation of iPSCs from a variety of cell types. Studies aiming establishment of new beta cell sources from pluripotent stem cells have allowed for direct methodologies for differentiation of iPSCs into beta cell‐like insulin‐producing cells (IPCs) via use of small molecules to activate key signaling pathways in beta‐cell development. Yet these protocols still need optimization in terms of efficiency and safety. We generated and characterized iPSCs via transduction of HFF‐1 and BJ fibroblast cells with integrating LV‐OSK and non‐integrating, integrase‐deficient IDLV‐OSK vectors. We present an improved approach for generation of IPCs from IPSCs via use of key small molecules plus soluble TNF‐Related Apoptosis‐Inducing Ligand (sTRAIL) molecule. The process of IPC generation was confirmed by expressions of Sox17 and FoxA2 (definite endoderm); PDX‐1 and insulin (pancreatic differentiation); and PDX‐1, insulin, NeuroD1, Pax6 and Islet‐1 (differentiation into IPCs). Use of sTRAIL in the process displayed increased insulin and c‐peptide secretion in differentiated IPCs. We believe that our combined protocol can be useful as an improved approach and may contribute further to revealing of the potential of TRAIL as a possible therapeutic molecule in diabetes. (TUBITAK Grant No: 218S617).
ProTcellTM exhibits thymic regenerative properties
1: Smart Immune SAS 2: The University Children's Hospital of Basel 3: Imagine Institute
Thymic hypoplasia secondary to congenital immune deficiency, cyto‐ablative chemo‐radio therapy or senescence causes a restricted T‐cell repertoire and consequently impaired immunity. Enhancing intrathymic T cell development and selection are therefore critical to replenishment of the peripheral T‐cell compartment. Our team developed a clinical grade, feeder‐free process to manufacture ex vivo human early T cell precursors, designated ProTcellTM. These progenitors (CD34‐CD7+) seeded the thymus of NSG mice where they gave rise to a polyclonal T cell repertoire in a shorter time and a more efficient fashion when compared to human CD34+ hematopoietic stem and progenitor cells. Furthermore, the infusion of allogeneic ProTcellTM did not cause Graft‐versus‐Host disease as the recipient's thymic stromal microenvironment imparted normal negative thymocyte selection and thus tolerance to an individual's self antigens. A detailed analysis of ProTcellTM demonstrated the expression of markers known to be involved in thymic homing and regeneration such as CXCR4, CCR9 and RANKL. Following engraftment, ProTcellTM gave rise within a month to double positive (CD4+CD8+) thymocytes and CD3+TCRab+ CD4+ or CD8+ (i.e. single positive) T cells. NSG mice reconstituted with human ProTcellTM had an enlarged thymus and displayed medullary islets (keratin 14+), morphological features characteristic for effective thymopoiesis and typically absent in uninjected NSG mice. Taken together, these preclinical results demonstrate the capacity of ProTcellTM to reconstitute the thymus of immune deficient mice where they give rise to mature T cells. ProTcellTM progenitors are currently tested in clinical trials to accelerate immune reconstitution after an allogeneic stem cell transplant.
Macrophage inhibitor clodronate enhances liver transduction of lentiviral but not AAV vectors in vivo
1: Great Ormond Street Institute of Child Health, University College London, London, UK 2: NIHR Great Ormond Street Hospital Biomedical Research Centre, London, UK 3: EGA Institute for Women's Health, University College London, London, UK 4: Metabolic Medicine, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK
Over the last 2 decades, liver‐directed gene therapy has shown clinical success in treating genetic diseases. Adeno‐associated viral (AAV) vectors are currently considered as the vector of choice for liver targeting, delivering mainly an episomal transgene, which can be lost during cell division. Anti‐AAV immune response currently prevent AAV re‐injections in humans. Alternatively integrative lentiviral vectors are being developed to enable long‐term transgene expression, especially during liver growth in paediatrics. Preclinical studies have reported reduced efficacy of lentiviral gene therapy in vivo due to high uptake by liver and splenic macrophages. Clodronate, a bisphosphonate prescribed to inhibit bone resorption in cancer, achieves 80‐90% transient macrophage depletion in liver and spleen after systemic administration. We assessed liver transduction after systemic administration of clodronate prior to lentiviral and AAV injections, in neonatal and young adult wild‐type mice. Both neonatal and adult clodronate‐treated C57Bl/6J mice showed significant increase of LV‐transduced hepatocytes, assessed by immunostaining (+150%, p < 0.01). Adult clodronate‐treated C57Bl/6J mice showed significantly increased lentiviral vector genome copies (VCN) in the liver (+130%, p < 0.05), and significantly reduced lentiviral VCN in the spleen (‐350%, p < 0.001). These results were replicated in neonatal and adult CD1 mice. AAV‐treated neonatal and adult CD1 mice did not show significant increase of liver VCN or hepatocyte transduction following clodronate injection. This study demonstrates significant improvement of liver transduction following systemic clodronate administration prior to injection of liver‐targeting lentiviral but not AAV vectors. These findings will have translational application for liver‐targeting gene therapy programmes.
Riboswitch‐controlled delivery of therapeutic antibodies for gene therapy
1: 1. Gene Regulation, MeiraGTx, New York, NY 10016, USA
AAV‐mediated gene transfer is a promising therapy for many diseases. However, excessive amounts of transgene from unregulated vector may not be desirable, and may limit the success of gene therapy. A genetic switch or gene control mechanism would provide a gene therapy approach that can be safely controlled and is applicable to a broader range of disease areas. Here, we present the development of regulated antibody genes, whose expression is controlled by riboswitch via small molecule inducer. Different from previously reported gene regulation systems that involve the use of exogenous protein components, our gene expression platform utilizes a riboswitch which is an RNA element that contains an aptamer as sensor for small molecule ligand/inducer. In our aptamer riboswitch system, aptamer/ligand binding alters transgene splicing, turning gene expression on or off in a dose dependent fashion. In the absence of the small molecule inducer in vitro, antibody gene with riboswitch cassette does not express antibody protein, whereas in the presence of small molecule inducer, antibody is robustly produced . When antibody gene with riboswitch was delivered in AAV to mice, orally dosed small molecule induced antibody expression, which subsequently diminished and returned to baseline level following withdrawal of the small molecule inducer. Our data indicate that our synthetic mammalian riboswitch works efficiently in vivo and can provide precise control of therapeutic antibody expression by controlling the dose of orally administered small molecule.
CRISPR/Cas13‐based detection and silencing of cancer fusion oncogenes
1: Centro Nacional de Investigaciones Oncológicas 2: CIEMAT 3: Instituto de Biomedicina de Sevilla‐IBiS 4: Universidad de Sevilla
Studies conducted over the last few decades have shown that approximately 20% of human cancers have specific fusion oncogenes (FOs). Due to their tumor‐specific expression, FOs offer unique advantages as diagnostic and therapeutic targets.
In this regard, the discovery of type II Cas13 nuclease, an RNA‐targeting CRISPR enzyme, has made it possible to selectively cleave FO transcripts, opening the door to the development of new targeted therapeutics and extremely accurate diagnostic solutions. Contrary to the CRISPR/Cas9 method, cell‐based RNA targeting with Cas13 results in reversible and temporally controllable alterations without modifying the DNA. Furthermore, compared to shRNAs, the CRISPR/Cas13 method is associated with high knockdown efficiency and no off‐target effects, offering unique advantages when used for therapeutic purposes. Diagnostic methods based on CRISPR/Cas13 provide rapid RNA detection with attomolar sensitivity and single‐base mismatch specificity. We show our results in i) the set‐up of a Cas13 system for selective degradation of FO RNA inducing efficient elimination of cancer cells, and ii) the development of a platform for highly‐sensitive, specific, affordable, and instrument‐free diagnostic test for FO detection by Cas13.
Cell type‐specific genetic therapy of cancer, virus infection and mitochondrial disease
1: Department of Microbiology and Immunology, National University of Singapore, Singapore 2: Department of Medicine, University of Cambridge, United Kingdom
Cell type specificity remains one of the biggest challenges for gene delivery. To mitigate potential off‐target effects, we developed a programmable cell type‐specific RNA trans‐splicing‐based approach for gene replacement therapy or suicide gene therapy that can selectively express therapeutic proteins or suicide signals based on the cell's endogenous pre‐mRNA expression profile. Using the liver as a medical target organ, we developed a hepatocellular carcinoma (HCC)‐specific trans‐splicing‐based Herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) suicide gene therapy approach. We designed RNAs that can target multiple HCC‐related pre‐mRNA biomarkers thereby increasing the overall target concentration. Our latest multi‐targeting constructs exhibited the highest cell death activity even at a GCV concentration of only 0.3 uM. For cell‐type specific delivery, the conjugation of dumbbell vectors with GalNAc3 residues enabled targeted delivery into hepatoblastoma‐derived human tissue culture cells. To deliver nucleic acids into the human mitochondria, we studied the long non‐coding β2.7 RNA of the human cytomegalovirus which was previously reported to colocalise with mitochondrial complex I. Using thermodynamic profiling, we identified four thermodynamically conserved structural subdomains that are responsible for mitochondrial‐targeting activity. A vector comprising eight mitochondrial‐targeting domains exhibited a three times higher mitochondrial‐targeting activity as compared with the parental β2.7 RNA. The amalgamation of mitochondrial‐targeting RNA and trans‐splicing allows cell type‐specific transcription of mRNA which can be delivered into mitochondria for mitochondrial gene therapy. Trans‐splicing‐based gene therapy is highly translational, offering promising perspectives towards suicide gene therapy of cancer or incurable infections with integrating viruses and may enable cell type‐specific mitochondrial gene therapy.
Improving gene therapy approaches for globoid cell leukodystrophy: pre‐clinical testing of chimeric GALC enzymes with enhanced bioavailability in human neural and hematopoietic cells
1: San Raffaele Telethon Institute for Gene Therapy (HSR‐TIGET) 2: Vita‐Salute San Raffaele University 3: University of Perugia
Globoid cell leukodystrophy (GLD) is a fatal and still untreatable lysosomal storage disease caused by the genetic deficiency of β‐galactosylceramidase (GALC) and characterized by severe central and peripheral nervous system (CNS and PNS) dysfunction. The difficulty in achieving supranormal levels of GALC activity in affected tissues limits the clinical exploitation of gene therapy (GT) approaches. Chimeric GALC enzyme with enhanced secretion and increased capability to cross the blood‐brain barrier may potentiate the bioavailability of GALC in CNS and PNS tissues and overcome the limitation of current experimental GT approaches. Here, we tested the safety and efficacy of optimized transduction of lentiviral (LV) constructs encoding for engineered chimeric GALC enzymes in CD34+ hematopoietic stem and progenitor cells (HSPCs) and in neural cells generated from GLD patient‐derived induced pluripotent stem cells, the closest human in vitro models of target and effector cells, respectively, in GT approaches. LV‐transduced human cells safely expressed high levels of transgenic mRNA and supranormal GALC activity. Importantly, the chimeric GALC enzymes produced by HSPC‐derived progeny were efficiently secreted, recaptured, and delivered to the lysosomes of GLD patient‐derived neurons/glial cells, which were metabolically cross‐corrected. These data support the rationale of testing the safety and potential functional advantage of chimeric GALC enzymes in ex vivo GT approaches in GLD mice. Improving GALC bioavailability may reduce the need for GALC overexpression in effector cells, simplifying the development of clinical protocols and refining their safety profile
Comparison of the effects of lentiviral vectors and of rAAV6 on CD34+ cells at the transcriptional and DNA methylation levels
1: Integrare research unit UMR_S951, Genethon, Inserm, Univ Evry, Université Paris‐Saclay, 91000, Evry, France
Gene transfer viral vectors such as lentiviral vectors (LV) or adeno‐associated viruses (rAAV) are efficient gene‐modifying tools but it has become clear that they may induce unwanted biological effects on target cells. We have reported that some lentiviral vector (LV) batches, especially integration‐defective LV (IDLV), can rapidly and reproducibly induce genome‐wide DNA methylation changes in vitro in adult human CD34+ hematopoietic stem cells from apheresis. These findings were confirmed in CD34+ cells from umbilical cord‐blood. To evaluate if these epigenetic changes perturb gene expression, we exposed CD34+ HSC to methylation‐inducing preparations of LV in vitro and measured CpG DNA methylation and gene expression levels by Illumina sequencing. We identified around 200 genes involved in developmental and stem cells pathways for which the CpG in their promoter regions were differentially methylated. In contrast, RNA‐seq showed no significant differential gene expression across the different LV batches tested. In parallel, exposure of CD34+ cells to rAAV6 showed no DNA methylation changes but revealed dramatic inductions of P53 related genes, thereby confirming observations made by others. To further characterize the novel LV‐induced CpG methylation effects, we selected a subset of differentially methylated genes and developed a targeted sequencing approach to examine the triggering mechanisms. Preliminary results suggest that high levels of capsid in the LV preparation induce hyper‐methylation. These results emphasize the need to control vector quality to reduce the sensing of viral components by cells during gene modification. Functional consequences of these effects remain to be determined.
Improved survival of injected mesenchymal stromal Cells in immunodeficient mice
1: Imagine Institut 2: Military Blood Transfusion Center, Clamart
Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin disease caused by loss‐of‐function mutations in COL7A1 encoding type VII collagen (C7). Patients with RDEB suffer since birth from skin and mucosal blistering and develop severe local and systemic complications resulting in poor prognosis. Mesenchymal stromal cells (MSC) demonstrated potential to improve wound healing and skin inflammation in RDEB patients due to their capacity to express C7 and their anti‐inflammatory properties. The aim of this work is to optimize in vitro conditioning of human bone marrow‐derived MSC (hBM‐MSC) prior to their injection in murine models to improve their survival. We first transduced healthy hBM‐MSC with a fluorescent and bioluminescent reporter lentiviral vector without modifying their MSC phenotype. Transduced hBM‐MSC were cultured under hypoxic or normoxic conditions and were supplemented with either platelet lysate or foetal calf serum prior to their subcutaneous (SC) or intravenous (IV) injections in immunodeficient mice. Survival and distribution of BM‐MSC in mice were assessed using the bioluminescent reporter by in vivo imaging. After SC injection, the bioluminescence remained localized to the site of injection for at least 8 months. Following IV injection, the bioluminescence was mainly detected in the lungs and was lost after 10 days. Optimization of in vitro conditioning and routes of administration of BM‐MSC to improve their biodistribution and survival in murine models should contribute to develop efficient and persistent treatment of wounded skin and mucosae in the perspective of clinical translation for RDEB.
Improving AAV in vitro transducibility for cell‐based potency assay development
1: MeiraGTx
Development, optimisation and GMP implementation of a fully automated platform for determining lentiviral vector titre
1: Oxford Biomedica (UK) Ltd., Windrush Court, Transport Way, Oxford, OX4 6LT, United Kingdom
Oxford Biomedica's LentiVector® platform is at the forefront of lentiviral gene therapies and forms the basis of several innovative treatments, including CAR‐T products.
In the production of lentiviral vectors at Oxford Biomedica, each batch of LentiVector® undergoes multiple tests to determine identity, RNA and protein composition, vector titre, biological potency, and replication competency (biological safety), in addition to standard QC tests such as sterility and purity.
The commercial scale manufacture of lentiviral vectors (LV) presents unique challenges in QC release testing with many of the methods requiring days or weeks to complete, with multistep cell culture combined with quantitative analytical readouts. Lengthy protocols, operator‐to‐operator variability, and a heavy dependence on manual setup are common shortfalls. Automated liquid handlers integrated with other robotic lab equipment are an obvious choice to alleviate many of these burdens and future‐proof increasing demand.
We will describe how we have successfully implemented and validated a fully GMP‐compliant automated workflow of a key QC release assay for lentiviral vectors: the determination of vector titre. This assay consists of cell culture and vector transduction, nucleic acid extraction, qPCR setup and data analysis. Automation has significantly reduced manual operator time, increased throughput while meeting a high demand with improvements in precision and robustness. We will detail some of the challenges faced and the solutions implemented, encompassing the approach taken for both instrument and method qualification.
Austrian healthcare professionals are positive about gene therapy, but expressed a high educational need
1: Pfizer Corporation Austria, Vienna, 1210, Austria
Gene therapy development is fast‐paced with a steadily increasing number of clinical trials and expected further approvals. To better understand the knowledge among health care professionals in Austria about gene therapy, we conducted a survey between April and June of 2021. A semi structured questionnaire containing 25 open and closed question was completed by 65 hospital physicians, including 13 experts in areas for which gene therapies have already been approved or launches are expected in the near future, 23 pediatricians and 29 internal medicine specialists. The need for gene therapy education was rated as high or very high by 88% of the participants. Moreover, 68% indicated they would be unable to explain gene therapy to another colleague, and more than 50% expressed having not enough knowledge to adequately answer gene therapy related questions from patients. However, 72% were open to suggest this therapeutic option to eligible patients, provided that there are positive study results, no alternative therapy options, a positive risk‐benefit ratio as well as a good safety profile. In general, more than half of the participants had a positive attitude towards gene therapy but expressed a need for more detailed information on the technology itself and disease‐specific gene therapies. In conclusion, this survey of Austrian healthcare professionals revealed a gap in their gene therapy knowledge but desire for further education.
The current revision of the orphan medicinal products regulation in the European Union: what is at stake for gene and cell therapy?
1: UMR 7318 DICE CERIC, CNRS, Aix‐Marseille University, Toulon University
In the European Union, gene and cell therapy medicinal products are mainly regulated by the specific legal framework applicable to Advanced Therapy Medicinal Products (ATMPs): regulation (EC) n°1394/2007. Yet most ATMPs that have been authorised for marketing in the European Union are also classified as orphan medicinal products. The latter are regulated by a different but also specific legal instrument : Regulation (EC) n°141/2000, which aims to promote the development of innovative medicines for orphan diseases thanks to a set of incentives for investigators. However, after twenty years of implementation, results are still unsatisfactory for several reasons. One of these reasons is that the market of orphan medicinal products had “started to look more similar to ‘standard’ medicines” than innovative medicines like ATMPs. Moreover, it has also been observed that even when marketed, orphan medicinal products fail in being equally accessible to patients in the different Member States. The European Commission has started a long and thorough revision process of the pharmaceutical legislation in order to give a new impulse to the development of, and access to, truly innovative orphan medicinal products, which may have a positive impact for patients as well as for investigators working on gene and cell therapies. In this poster, we will discuss what aspects of the revision of the regulation on orphan medicinal products may have an impact on the development of gene and cell therapy in the European Union.
This work has been supported by ANR‐funded I‐BioLex project (ANR‐20‐CE26‐0007‐01, coord. A. Mahalatchimy).
Bringing gene and cell therapies from lab to patients: the EuroGCT research pathways
1: Aix‐Marseille University 2: The University of Edinburgh 3: CNRS
Bringing gene and cell therapies from lab to patients raises many challenges from the development of these therapies to patient access. Therapy development has to comply with many regulatory requirements, some being binding, other being guidance only, in order to primarily ensure patients' safety. These requirements have been established throughout the life cycle of these therapies from fundamental research to post‐administration vigilance, through manufacturing, clinical trials, marketing authorisation, patentability, or pricing & reimbursement. While the majority are governed by European Union law, several aspects remain regulated at national levels. The legal classification of these therapies also leads to different regulatory pathways. Therefore, their development is not only complex scientifically, it is also complex regarding their regulatory landscape, which is made up of a combination of general and specific rules and guidance depending on competent institutions (e.g. EU and Member States regulatory agencies or health technology assessment bodies), types of products, and steps of their developments. To address this complexity, the European Consortium for Communicating Gene and Cell Therapy Information (EuroGCT), is building an online tool to provide research pathways' information, that will be presented in this poster, to facilitate better decision‐making at key points in development of new therapies and thus enable improved product development, by providing the research community and regulatory and healthcare authorities with an information source on the practical steps needed for cell and gene therapy development.
This work has been funded by the European Union's Horizon 2020 research and innovation programme under grant agreement No 965241.
Risk management considerations and advanced therapy medicinal products
1: Paul‐Ehrlich‐Institut
Advanced therapeutic medicinal products (ATMP) offer promising new strategies to treat haematologic or rare genetic disorders but are associated with the risk of serious adverse reactions. An unmet medical need for the specific indications results in accelerated approval processes based on limited data only. Features intrinsic to the respective ATMP and limitations of clinical trials pose challenges for safety considerations in the clinical translation process and for risk management in the real‐life setting. A key question is how regulatory activities can support the safe use of ATMP with the aim to make new treatment options available to patients early while ensuring proper risk management and continued data collection. For example, cancer therapy by chimeric antigen receptor (CAR) T cells has been associated with serious short‐term toxicities in a considerable proportion of patients and incomplete long‐term data at time of approval. Proper risk management is considered key for a positive benefit‐risk balance especially in the early post‐approval phase of CAR T cell therapy products. Measures should involve all stakeholders and include educational materials, a controlled distribution programme and post‐authorisation safety studies. First experiences of treatment with CAR‐T cells are presented from a German regulatory perspective.
Investigating the influence of mRNA encoded transcription factor delivery on human bronchial epithelial cell differentiation
1: Imperial College London
Synthetic messenger RNA (mRNA) represents a promising tool for transient, dose‐dependent expression of any protein of interest, when efficiently delivered to target cells. Culture of primary human bronchial epithelial cells (HBECs) at air‐liquid interface (ALI) facilitates differentiation towards a polarised, pseudostratified epithelium containing basal, ciliated and goblet cells. This model provides a powerful tool for studying the function of HBECs and for manipulating genes to modulate differentiation. mRNA‐based therapeutics are being evaluated in clinical trials, however, the impact of delivery and translation of synthetic mRNA on epithelial cell differentiation and barrier integrity remains unclear. To determine how non‐viral mRNA delivery impacts the ability of primary HBECs to form a differentiated epithelium at ALI, histological analysis, immunostaining and qPCR was conducted on transfected and non‐transfected cells, together with an assessment of barrier integrity using paracellular permeability and tight junction protein expression. Reporter protein expression was observed in HBECs 12 to 48 hours post transfection, with epithelial cell differentiation and barrier integrity unaffected. Cilia dysfunction is observed in several lung diseases, including cystic fibrosis and primary ciliary dyskinesia. Therefore, we aimed to manipulate mucociliary differentiation and subsequent ciliogenesis by transfecting HBECs with an mRNA encoding a key transcription factor involved in ciliogenesis, Forkhead box J1 (FOXJ1). Preliminary data shows expression of FOXJ1 protein following transfection, and upregulation of several downstream genes including CETN2 and DNAH11. Ongoing studies will utilise HBEC ALI as a model to understand how the exogenous delivery of mRNA encoded transcription factors can control differentiation and function of HBECs.
Improving access to innovative medicines: proposals from national authorities to revise the European legislation
1: UMR 7318 DICE CERIC, CNRS, Aix‐Marseille University, Toulon University 2: CERPOP (Center for Epidemiology and Research in POPulation health), UMR 1295 Inserm, Université Paul Sabatier‐ Toulouse 3 3: CREDIMI, Université de Bourgogne
Gene and cell therapies are currently considered as the main drivers of innovation in the treatment of genetic diseases and cancer therapies. They offer new hopes to rare disease patients as 80% of rare diseases are of genetic origin in the European Union. Hence, many gene and cell therapies have obtained the orphan medicine status under European regulation (EC) n° 141/2000, which provides incentives to promote the development of orphan medicines. The European Union tries to find a balance between the interests of industry, which should be encouraged to invest, patients' access to medicines, and the sustainability of health systems. Nevertheless, the current legal regime needs adaptation. Thus, the European Commission launched two public consultations in 2021: a first one specifically on the revision of the orphan medicines legislation, and then, another one on the revision of the general pharmaceutical legislation. Out of the 303 and 478 answers received, 12 and 30 respectively came from national authorities. On the basis of an analysis of their answers, this poster will discuss the criteria for orphan designation (e.g., the significant benefit which needs clarification and modification), as well as the incentives implemented, and patient access. It will highlight several proposals emphasized by national authorities that might be retained in the future legislations: to reward mainly truly innovative medicines, a transparency obligation on R&D costs before granting incentives to sponsors, or an obligation to launch the medicine in most Member States.
This work has been supported by ANR‐funded I‐BioLex project (ANR‐20‐CE26‐0007‐01).
A lentiviral vector intranasal dosing strategy to control local and systemic expression of intracellular and secreted transgenic proteins in vivo
1: National Heart and Lung Institute, Imperial College London 2: UK Respiratory Gene Therapy Consortium 3: University of Oxford
Targeting of the nasal epithelium to allow sustained expression of a secreted therapeutic protein represents a potential non‐invasive lentiviral vector application strategy. Using a combination of reporter‐imaging, molecular, and radiopharmaceutical tracing methods in mice, we have developed an intranasal dosing strategy for near exclusive nasal transduction with our proprietary Sendai‐virus envelope glycoprotein pseudotyped lentiviral vector (rSIV.F/HN). Using multiple (up to 10) small volume (5 μl) intranasal pipette bolus applications we have shown in technetium radiotracer experiments that ∼90% of the liquid is retained in mouse heads, resulting in minimal reporter gene expression in the lung (0.28 ± 0.21% of total radioactivity at the maximum dose). Nasal‐tissue expression levels were dose‐related up to 2.4e8TU vector titre (mRNA to protein Spearman correlation, r = 0.9783, p < 0.0001). There was negligible acute toxicity associated with nose delivery, as judged by histopathology. Next, we compared levels of Gaussia luciferase (Glux) in serum of mice 6‐months following administration of equal titres (1.8e8 TU/mouse) of rSIV.F/HN‐Glux administered to the nose or given intravenously. Systemic protein levels were detected following nose‐targeted delivery of the vector and were statistically similar to levels achieved after intravenous administration (Nose: 812 ± 345, IV: 1701 ± 566‐SEM arbitrary‐units, Kruskal‐Wallis p > 0.05, n = 7 + 5/group). We are currently assessing whether transduction of nasal epithelium with rSIV.F/HN expressing GM‐CSF, leads to sufficient circulating levels of GM‐CSF to correct biomarkers in a mouse model of pulmonary alveolar proteinosis. We conclude that non‐invasive administration of our proprietary rSIV.F/HN vector to the nasal epithelium may provide a safe and convenient route for the production of secreted transgenic proteins.
Age of administration impacts the efficiency of lentiviral vector‐mediated hepatocyte transduction in vivo and its distribution in the liver lobule
1: San Raffaele Telethon Institute for Gene Therapy (HSR‐TIGET) 2: Universita Vita‐Salute San Raffaele 3: San Raffaele Scientific Institute
The liver is an attractive target organ for in vivo gene therapy since it offers the possibility to treat metabolic and coagulation disorders. We have previously shown efficient gene transfer to hepatocytes of adult mice, dogs and non‐human primates by intravenous (i.v.) delivery of lentiviral vectors (LV), achieving stable clotting factor expression in animal models of hemophilia. Here we investigated LV‐mediated liver gene therapy in newborn and juvenile mice, towards potential application to pediatric patients. We observed that i.v. administration of LV in mice at post‐natal day 1 (D1) or week 2 (W2) led to reduced uptake of vector particles by non‐parenchymal cells, increasing transduction of hepatocytes compared to adult‐treated mice. Moreover, LV distribution in the liver lobule was affected by the age at treatment, with preferential transduction of central area in young‐treated mice and of portal area in adult‐treated mice. By exploiting factor IX as a secreted reporter, we observed life‐long maintenance of transgene output and that W2‐treated mice have a 2‐3‐fold higher transgene output compared to D1‐treated mice, despite similar efficiency of hepatocyte transduction. However, by changing promoter, we achieved higher transgene output in D1‐treated mice compared to W2‐treated mice, suggesting differences in promoter activity of transduced hepatocytes' subsets. Overall, our results indicate that LV‐based liver gene therapy is more efficient in young mice, and that age of treatment impacts on LV transduction efficiency, spatial distribution of transduced hepatocytes and transgene output. These studies will inform further development of liver‐directed LV gene therapy towards application to pediatric patients.
Clinical effect of intradiscal application of allogeneic mesenchymal stem cells derived from umbilical cord Wharton's jelly (WJ‐MSC) in adults with degenerative disc disease (DDD) treated at BioXcellerator, Medellín, Colombia
1: Research group, BioXcellerator, Medellin, 050021, Colombia
Mesenchymal stem cell therapy could improve DDD symptoms by promoting intervertebral disc matrix natural repair processes. The aim was to analyze clinical effects of intradiscal WJ‐MSC cell therapy. A retrospective cohort was followed. Clinical outcomes measures included: Short Form‐12 questionnaire, Pain Visual Analog Scale (VAS), Oswestry disability Index (ODI), Neck Disability Index (NDI). Follow‐ups were at pretherapy time and 3, 6, 12 months after intradiscal application of a single dose of 5x106/mL cells per disc as a treatment protocol. WJ‐MSCs were expanded in culture medium supplemented with 10% human platelet lysate (hPL) up to passage 7. Cell marker expression, in vitro differentiation to mesodermal lineage, and microbiological tests were performed. 53 patients were included (August/2019‐June/2022), 62% men. Median age = 48 (IQR = 28‐64). 53.8% had cervical, 21.02% thoracic, 41.7% lumbar disc injuries. 91.4% had multiple disc injuries. MODIC changes = 29.3%. The most frequent Pfirrmann grade was II (26.3%). The SF‐12 scores were increased (0‐month = 47, 3‐month = 67, 6‐month = 84, 12‐month = 78, Wilcoxon p‐value = 0.04). VAS, ODI and NDI scores were reduced (0 = 8,60,72, 3 = 7,48,65, 6 = 5,40,60, 12 = 4,28,45, Wilcoxon p‐values = 0.10, 0.03 and 0.09 respectively). The fit predictors at Multivariate GEE model mainly associated with the final result of the SF‐12 at twelve months were: age (b = ‐0.278,p = 0.03), Pfirrmann grade (b = ‐0.019,p = 0.04) and WJ‐MSC treatment (b = 0.157, p = 0.02). In relation to ODI at one year: sex (male) (b = ‐0.098,p = 0.02), MODIC changes (present) (b = 0.61,p = 0.01) and WJ‐MSC‐treatment (b = ‐0.099,p = 0.04). Approval was obtained by an ethics committee. No serious adverse events were reported. We conclude that intradiscal application with WJ‐MSC is safe and has shown relevant clinical effects on DDD.
Preclinical optimisation of lentiviral gene therapy delivery for surfactant deficiencies in piglets
C McDougall1 C McLaughlin1 E Thornton1 K M Miah2 E Castells2 R J Dean2 M A Viegas2 S C Hyde2 D R Gill2
1: The Roslin Institute, University of Edinburgh 2: University of Oxford
Surfactant protein deficiencies account for 10% of childhood interstitial lung disease and the prognosis depends on the protein that is deficient. Infants with surfactant protein B (SP‐B) deficiency or double null mutations in ABCA3, present shortly after birth with respiratory distress and failure. Despite ventilation and treatment with surfactant replacements, they rarely live beyond the first few months of life. We have delivered vector expressing EGFP by bronchoscopic instillation via an endotracheal tube to the lungs of ventilated neonatal piglets to model delivery of vector to the lungs of ventilated children. We compared multisite instillation with small volumes of concentrated vector at two doses with lavage‐like delivery of the same highest dose of vector in a larger volume. Multisite instillation with small volumes resulted in dose‐dependent expression of GFP with significant heterogeneity (47% and 39% captured images with no GFP at low and high dose) and efficiency ranging from 0% to 98% in individual images. Delivery of the high dose in larger volumes resulted in only 17% images with no GFP signal and a range from 0% to 17%. The overall average efficiency in the latter study was 4%. Further studies in piglets will help to optimise dose and delivery protocols and will aid calculations of the required dose projected to reach the therapeutic level of expression in the target cells in clinical trials.
Advanced humanized immune system mouse models: An efficient pre‐clinical mouse model to evaluate immune targeted therapies in cancer research
K Y Ho Wang Yin1
1: TransCure bioServices
Engraftment of CD34+ haematopoietic stem cells in NCG mouse allows the reconstitution of a complete human immune system, with mature T, NK cells and macrophages. The human immune system could be further enhanced by the expression of human cytokines that will improve the immune system development. This mouse model provides a relevant in vivo context, to assess the therapeutic efficacies of immune targets in the oncology field such as checkpoint inhibitors, bi‐specific antibodies, oncolytic viruses or cell‐engineered based therapies. At TransCure bioServices, we have developed a comprehensive database of CDX engrafted in advanced humanized immune system mouse models. The tumor infiltrating leukocytes were analysed by flow cytometry, revealing high differences of infiltration between the different CDX models and the importance of the tumor micro‐environment to drive the immune infiltration. Thanks to this pre‐clinical mouse model, we were able to demonstrate efficacy of drugs targeting macrophages polarization or T cell activation.
Production of ATMPs: What are the specificities for quality aspects?
1: UMR 7318 DICE CERIC, CNRS, Aix‐Marseille Univ., Toulon Univ. 2: University of Oxford 3: Marseille Public Hospital (AP‐HM) 4: Research Unit Microbes Evolution Phylogeny and Infection (MEPHI) Aix‐Marseille Université 5: C2VN INSERM INRAe 1263 AIX Marseille University
Advanced Therapy Medicinal Products (ATMPs) is a European classification of medicinal products based on genes, cells and tissues that have been specifically regulated in the European Union (EU) from 2007. Their manufacturing (i.e. their production) raises specific challenges for ensuring quality and complying with regulatory requirements in order to obtain manufacturing and marketing authorizations. For this reason, detailed guidelines on Good Manufacturing Practices specific to ATMPs have been adopted by the European Commission, and have been enforceable since 2017. They are distinct from other guidelines on GMP applicable to other kinds of biological medicinal products. Separate GMP guidelines, covering manufacture of biological active substances and medicinal products for human use including biological active substances were revised in June 2018 with the objective of preventing overlaps in scope with the ATMP GMP guidelines. Our hypothesis is that the biological nature that commonly characterizes ATMPs and biologicals may give rise to significant similarities in the manufacturing aspects as addressed by the respective guidelines. Through a comparative textual analysis of the GMP guidelines for biological medicinal products and ATMPs, this poster will highlight the key areas of similarities and differences. This analysis reveals why we have two different texts and whether they are based on substantial differences regarding production between ATMPs and other types of biological medicinal products.
This work has been supported by ANR‐funded I‐BioLex project (ANR‐20‐CE26‐0007‐01, coord. A. Mahalatchimy).
Specific guidelines requirements for clinical trials with Advanced Therapy Medicinal Products in the European Union
1: UMR 7318 DICE CERIC, CNRS, Aix‐Marseille University, Toulon University 2: Centre for Health Law and Emerging Technologies (HeLEX), University of Oxford 3: Faculty of Pharmacy, Aix‐Marseille University, Research Unit Microbes Evolution Phylogeny and Infection (MEPHI) Aix‐Marseille Université, IRD 4: Marseille Public Hospital (AP‐HM), Biotherapies Clinical Investigation Center 5: C2VN INSERM INRAe 1263 Aix‐Marseille University, Cell therapy research centre at Marseille Public Hospital (AP‐HM)
Advanced Therapy Medicinal Products (ATMPs), a European legal classification of medicinal products based on genes, cells and tissues, raise specific issues in the context of clinical trials. In comparison to more traditional medicinal products, ATMPs have been subject to specific regulatory provisions in the European Union (EU) since Regulation (EC) n°1394/2007. Yet for the clinical trials with ATMPs, the general regime laid out in Regulation 536/2014 on clinical trials (which came into effect on 31 January 2022) applies, together with the ICH E6 Guidelines on Good Clinical Practice (GCP). For clinical trials conducted in the EU, compliance with GCP is mandatory. The European Commission has also adopted and published 2019 Guidelines on GCP specific to ATMPs, as required by Article 4 of Regulation (EC) n°1394/2007 on ATMPs. These guidelines both adapt the ICH guidelines to ATMPs' characteristics and provide additional measures that have been considered necessary. However, they are not exhaustive as they explain only some specificities of ATMPs and they remain complementary to the general rules. After having analysed these documents, we will highlight the specificity of requirements for investigational ATMPs in order to reveal the specific challenges they are addressing and why these challenges warrant separate regulation in order to obtain clinical trials' authorisation for investigational ATMPs.
This work has been supported by ANR‐funded I‐BioLex project (ANR‐20‐CE26‐0007‐01, coord. A. Mahalatchimy).
Pre‐clinical safety evaluation for mRNA‐vaccine development in mouse model
1: Seoul National Unversity College of Medicine 2: Seoul National University Hospital 3: The Catholic University of Korea 4: Brain Science Institute Korea Institute of Science and Technology 5: Seoul National University College of Veterinary Medicine
Messenger RNA (mRNA) vaccine has emerged as an attractive agent for prevention of infectious disease and anti‐cancer therapy. However, there is a fatal risk that the safety evaluation for mRNA vaccine have not been fully studied yet. In this study, we evaluated the safety of four type of COVID‐19 S‐protein targeting mRNA vaccines with different compositions (C2/LNP90, C2LNP128, C3LNP90 and C3LNP128). Theses vaccines were intramuscularly injected to 6‐wk old male and female ICR mice with twice at an interval of 2 wks. The necropsy was carried out on 2 days or 14 days after secondary injection. The results showed that the body weight was decreased for 2days after the first injection in C2/LNP128 and C3/LNP128‐injected mice, but it was almost recovered at 7day post injection (dpi). At 2 dpi after secondary injection, the endpoint blood analysis of demonstrated that C2/LNP128 and C3/LNP128 decreased the number of lymphocytes, monocytes and reticulocytes carrying the abnormal level of liver function indicator such as albumin, AST, ALT and total protein. Additionally, C2/LNP128 decreased the number of platelet and C3LNP128 decreased the number of red blood cells, respectably. Spleen and inguinal lymph node were enlarged in all experimental group. Notably, C2/LNP128 and C3/LNP128 induced severe edema in injection site, femoris muscle. At 14 dpi after secondary injection, the toxicity that was observed at 2 dpi after secondary injection was recovered. These results suggest that the potential side effects of mRNA vaccines must be systematically evaluated with multiple aspect of toxicology.
Optimisation of lentiviral vectors for gene therapy of pulmonary fibrosis
1: CGEM, University of Edinburgh, EH4 2XU, UK 2: Gene Medicine Research Group, NDCLS, University of Oxford, OX3 9DU, UK 3: Roslin Institute & R(D)SVS, University of Edinburgh, Roslin, EH25 9RG , UK 4: UK Respiratory Gene Therapy Consortium
Pulmonary fibrosis is a progressive and usually idiopathic disease characterised by exertional dyspnea and lung scarring. Prognosis is poor, with a post‐diagnosis median survival of 2‐5 yrs. We are developing a therapeutic lentiviral gene augmentation approach for the delivery of antifibrotic genes, in the first instance decorin (TGF‐β inhibition) and TRIM72 (cell membrane repair). qRT‐PCR was used to measure transgene expression from plasmids containing wildtype and codon‐optimised ORFs of each gene under control of several promoters in transfected human airway epithelial cell lines (hAECs) A549 and H441. Lentiviral derivatives of the top expressers, containing wt ORFs driven by PCMV and hCEF, were made bearing the lung‐optimised rSIV.F/HN pseudotype. Using qRT‐PCR, Western blotting and immunohistochemistry, expression was examined after transduction in vitro using hAECs in adherent and air‐liquid interface cultures, and ex vivo in sheep precision‐cut lung slices. Transgene expression was confirmed in all model systems, with decorin expression consistently exceeding that of TRIM72. We are now preparing to assess the efficacy of constructs in appropriate models of pulmonary fibrosis.
Tissue nuclease activity affects the degradation of naked plasmid DNA
1: Institute of Molecular Biomedicine, Comenius University
Our preliminary data showed that there is high variability of nuclease activity between different organs. This might limit the applicability of plasmid‐mediated gene therapy since naked DNA is sensitive to degradation. The aim of our study was to compare the degradation of naked plasmid DNA in various organs in mice. Organs (liver, spleen, kidneys and parts of the gastrointestinal tract) were harvested from adult C57BL/6J mice. Isolated plasmid DNA (pcDNA3‐GFP) was added to the homogenates and the degradation kinetics was assessed using Sytox‐mediated fluorescence and gel electrophoresis. The results confirm that high nuclease activity observed in the kidneys and small intestine leads to rapid plasmid DNA degradation ex vivo. On the contrary, plasmid DNA seems to be relatively stable in liver homogenates. Analysis of different púarts of the gastrointestinal tract suggests that the proximity to the duodenum and, thus, the pancreatic DNase determines the nuclease activity of the intestines. Tissue nuclease activity varies considerably and affects plasmid DNA degradation. This variability should be taken into account in non‐viral gene therapy. Targeting the kidneys might require additional protection with deoxyribonuclease inhibitors, vesicles or at least a higher dose. The outcomes require verification in in vivo experiments.
MenSCs‐derived secretomes as a new therapeutic approach for inflammatory‐based diseases thought modulating M1/M2 polarization
1: Stem Cell Therapy Unit, Jesús Usón Minimally Invasive Surgery Centre, Cáceres, 10004, Spain 2: RICORS‐TERAV Network, ISCIII, Madrid, 28029, Spain 3: Institute for Tumor Immunology, Center for Tumor Biology and Immunology (ZTI), Philipps University, Marburg, 35043, Germany 4: Immunology Unit, University of Extremadura, Cáceres, 10003, Spain 5: Institute of Molecular Pathology Biomarkers, University of Extremadura, Cáceres, 10003, Spain
Activated macrophages are frequently classified into M1 (involved in proinflammatory responses) and M2 (anti‐inflammatory responses) according to their surface marker expression and their cytokine secretion. Macrophages can be influenced by a range of factors that can shift their phenotype, thereby affecting their function. Thus, ameliorating the inflammatory environment may be an effective method for disease treatment via macrophage immunomodulation. In this context, the secretome derived from Menstrual blood‐derived stromal cells (MenSCs) has been suggested as a promising therapeutic approach. Therefore, the aim of this study was to evaluate the effect of MenSCs‐derived secretomes (S‐MenSCs) on macrophage polarization in vitro. S‐MenSCs were collected after pro‐inflammatory priming of MenSCs (IFNγ‐ and TNFα‐treatment) or basal conditions. In this work, human monocytes were isolated from the peripheral blood healthy donors (n = 3) and macrophages were generated in vitro undergoing classical, M1 (GM‐CSF+LPS+IFN‐γ) or alternative, M2 (M‐CSF+IL‐10) activation. Both macrophage subtypes were treated with primed or basal S‐MenSCs at a concentration of 100 μg/mL. Phenotype switch was determined by the analysis of representative surface markers by flow cytometry: CD80, CD86, and HLA‐II for M1 and CD163, CD206, and CD16 for M2. Our results have shown a macrophage phenotype switch after treatment with both S‐MenSCs. In M1‐polarized macrophages, M1 markers decreased and M2 markers increased, pointing out an attenuation of the proinflammatory profile. In contrast, M2‐polarized macrophages showed opposite results. In conclusion, S‐MenSCs delivery could improve the inflammatory environment by modulating M1/M2 polarization and hence be considered as an alternative and effective treatment for inflammatory‐based diseases.
Not the same for all: Distinction of environmental risks of different types of gene therapy products and recombinant vaccines and how this difference effects clinical trial and market authorisation applications
1: Jenal & Partners Biosafety Consulting
The environmental risk assessment (ERA) forms a significant part of clinical trial and market authorisation applications for in vivo or ex vivo gene therapies, oncolytic therapies or immune therapies as well as vaccination with genetically modified organisms (GMO). A multitude of different GMOs are used in an ever increasing number of applications. This number will likely increase when considering possibilities for genome editing using CRISPR/Cas technology. Likewise, the experience with ERAs by both applicants and competent authorities will increase, co‐evolving in question and answer processes.
Using a number of examples of investigational products, this presentation addresses the most frequent and tricky issues related to an ERA and highlights the significant questions for different types of products to be solved, taking into account the range of questions and concerns raised by different competent authorities.
While definitely not all types of products, applications, and application locations require the same ERA, the goal of this presentation is to define a recurrent theme for them all.
AAV Lateral Flow Assays for Rapid Titer Determination
C Querfurth1 K I Pfrepper1 R Wetzel1 L Mutz1 C Odenwald1
1: PROGEN
Adeno‐associated viruses (AAV) represent the most commonly used viral vector for the delivery of therapeutic transgenes in academia and industry. However, the gene therapy community is facing major challenges like the high demand for effective and reliable analytical AAV tools for gene therapy manufacturing and development. The use of multiple orthogonal methods is recommended to ensure a comprehensive characterization of AAV‐based gene therapeutics. However, rapid testing of individual and crude AAV samples during the development and manufacturing process is desirable but often difficult. Most precise analytical AAV tools are not convenient for single sample measurements and the faster biophysical methods often require highly purified samples. Therefore, we introduce our new AAV lateral flow assays (Dip'n'Check AAV) that are available for different AAV serotypes to (i) estimate AAV titers in crude and purified AAV preparations, (ii) compare AAV titers of different AAV preparations, (iii) proof the binding of common AAV antibodies to an AAV capsid variant and (iv) monitor the stability of AAV preparations. We demonstrate the good correlation of estimated AAV capsid titers by the lateral flow assays with data obtained by the more accurate AAV ELISA as well as qPCR, and the clear visual discrimination of a 2‐fold concentration difference within the dynamic range of 1.0E+09 to 1.0E+11 p/ml in diluted samples. In conclusion, the new Dip'n'Check AAV lateral flow assays provide excellent analytical tools to rapidly estimate AAV titers and a fast evaluation of the general antibody binding capacity to AAV capsid variants in any process step.
Considerations for biosafety testing of cell and gene therapies
1: SGS
Regulatory authorities such as the US Food & Drug Administration (FDA) and the European Medicines Evaluation Agency (EMEA) impose stringent limits on the amount of microbial contaminants and impurities present during the manufacturing of biological medicines and vaccines, and present in cell and gene therapy products. These regulations ensure sterile products and thus patient safety. To establish that the testing procedures are accurate, regulatory authorities require proof of testing before clinical trials can be approved. Consequently, all components of the manufacturing process must undergo extensive safety testing to demonstrate identity, stability, and purity. This talk will review general approaches to biosafety testing, with specific focus related to cell (for example CAR‐T cell therapies) and gene therapies.
Cell bank & Virus Seed Biosafety & Characterisation, including a brief overview of Identity Testing Genetic Stability Purity (freedom from bacteria, fungi and mycoplasma) Virus Safety will be the focus of the talk, considering Broad specificity approaches in vitro, in vivo, NGS non‐targeted Retroviruses – infectivity /EM/RTase/PCR
Species specific ‐ PCR / targeted NGS / 9CFR / MAP / HAP
How are viruses detected? Considerations for viral vectors
Validation of an efficient electroporation protocol for the genetic modification of MCF‐7 cells
O Sonmezler1 2 C Mujde1 3
1: Cukurova University AGENTEM (Adana Genetic Diseases Diagnosis and Treatment Center), Adana, Turkey 2: Cukurova University, Institute of Natural and Applied Sciences, Dept. of Biotechnology, Adana, Turkey 3: Cukurova Technopolis InfoGenom, Adana, Turkey 4: Cukurova University, Faculty of Medicine, Dept. of Medical Genetics, Adana, Turkey
Electroporation is one of the safest and easiest non‐viral transfection technique for transportation of CRISPR‐Cas9 elements into cells. Variable electroporators with different specifications are accessible for in vitro genome modification studies that are offering different impacts on cell viability and transfection efficiency. Even though electroporation protocols are provided by the manufacturers for any desired cell line, all the parameters should be subjected to preliminary optimization. In the present work, we share our results for MCF‐7 cell‐line electropermeabilization validation study in comparison with manufacturers' recommended protocol for Lonza 4D‐Nucleofector System (Lonza, Switzerland) prior to CRISPR‐Cas9 mediated gene modification assay. We performed a triplicate experiment consisting of Lonza's cell line optimization assay template (together with their suggested protocol for MCF‐7, EN‐130) via manufacturer's suggested electroporation buffer and supplement solution, in comparison of the same template via Opti‐MEM (Thermo Fisher Scientific, USA) as electroporation medium without supplement solution. Cell viability and efficiency of pmaxGFP plasmid (Lonza, Switzerland) transfection were examined under florescence microscopy and flow cytometry. Among these electropermeabilization protocols, three of them (DS‐109, EL‐110, DC‐100) succeeded with more cell viability while one of them (DS‐109) have showed the most accurate results overall. The optimization procedure of an electroporation protocol even for the closed systems involving the electroporation medium, pulse type, capacitance, voltage, pulse number and durations becomes inevitable to achieve an efficient solution. As a result of this study, the optimized protocol reported provides as the suitable, cost‐effective and are accessible way that facilitating the widespread adaption/use of this technology.
Improvement of Polyethylene Glycol‐cell fusion efficiency by transient pressure using Pyro‐drive Jet Injector
1: Osaka University , Graduate School of Medicine, Department of Device Application for Molecular Therapeutics
Cell fusion is not only a physiological process but also an important cell engineering technology applicable to various fields, such as regenerative medicine, antibody engineering, genetic engineering and cancer therapy. There are many methods of cell fusion, one of which is polyethylene glycol‐mediated cell fusion (PEG). PEG‐mediated cell fusion method is an economical and convenient approach for cell fusion which does not require expensive instrumentation. Although PEG cell fusion is simple and cost effective, there are still some shortcomings, such as high cytotoxicity and low fusion efficiency. To improve these defects of the PEG fusion method, here, we used Pyro‐drive Jet Injector (PJI) in combination with PEG fusion method. In this PJI‐PEG fusion method, instead of gentle agitation to increase the chance of cell‐to‐cell contact and shorten the distance between cells in the process of cell fusion, PJI exerts transient acute pressure on the cells to achieve similar outcomes. Our results showed that PJI‐PEG fusion method not only decreased cell cytotoxicity during the fusion process, but also increased fusion efficiency in comparison to the traditional PEG method. Using this method, we successfully fused cells for dendritic cell‐tumour cell vaccines and hybridoma production which were functionally indistinguishable from those fused using the traditional PEG method. The PJI‐PEG fusion method is a fast and efficient method to induce cell fusion and has potential to improve both basic research and clinical protocols that require cell‐cell fusion.
Comparative proteomic analysis in remote myocardium of infarcted pigs after administration of secretome from Menstrual blood‐derived stromal cells (S‐MenSCs) in different priming conditions
1: Jesús Usón Minimally Invasive Surgery Centre, Cáceres, 10004, Spain 2: RICORS‐TERAV Network, ISCIII, Madrid, 28029, Spain 3: Ciber de enfermedades cardiovasculares, 28029, Madrid, Spain 4: Immunology Unit, University of Extremadura, Cáceres, 10003, Spain 5: Institute of Molecular Pathology Biomarkers, University of Extremadura, Cáceres, 10003, Spain
Environmental factors can modulate the biology of cell in culture, in particular, an inflammatory environment can enhance their therapeutic potential. In stem cell based‐therapies, this strategy is widely accepted to increase the beneficial effects of the treatments. Diseases with high mortality rates, such as myocardial infarction, require the investigation of new approaches to mitigate the progress of disease. Thus, the aim of this study was to compare the biological effects of secretome from Menstrual blood‐derived stromal cells (S‐MenSCs) under different priming conditions in remote myocardium after myocardial infarction. For this purpose, secretomes were concentrated from Menstrual blood‐derived stromal cells after pro‐inflammatory priming (IFNγ‐ treatment) (S‐MenSCs*) or basal conditions (S‐MenSCs) and administered intrapericardially in a closed‐chest swine model of myocardial infarction. Seven days post‐therapy, hearts were harvested and remote tissue was collected for proteomic analyses. Comparative results showed 62 genes differentially expressed (FDR < .05) between S‐MenSCs and S‐MenSCs* treatment in remote myocardium. Protein‐protein interaction analysis, using STRING as a biological database, revealed that the most significant Gene Ontology Cell Component terms in level 6 were: contractile fiber (GO:0043292), mitochondrion (GO:0005739). Most genes associated with these terms are up‐regulated in tissue of animals administered with S‐MenSCs*. These results suggest that secretome cargo of MenSCs* has more potential to regulate the gene expression of protein related to contractile function of cardiac remote tissue contributing to alleviate cardiac dysfunction. In conclusion, priming strategies could be considered new approaches to increase the therapeutic potential of stem cell based‐therapies.
rAAV‐based gene therapy formulations ‐ current challenges and future solutions
1: Hoffmann‐La Roche Ldt
The marketed recombinant adeno‐associated virus (rAAV) based gene therapy products have proved the tremendous potential of this approach, thanks to their transformative therapeutic outcomes. However, to be able to fulfil their potential, there is an urgent need to improve the stability of rAAV formulations to increase their resistance to stresses related to handling and transportation. In this work, we assess how formulation excipients, such as salts, sugars and surfactants affect the behaviour of rAAVs in terms of aggregation, titre recovery, full‐to‐empty ratio, potency, and DNA release. Further, we present how a panel of different analytical techniques, with a distinct pattern of strengths and limitations, can be put together to provide a comprehensive evaluation of rAAV formulations, and therefore support the formulation development process. Overall, this work contributes to a growing body of knowledge regarding the optimisation and analytical assessment of rAAV formulations, which is essential for the development of next generation gene therapies, able to withstand handling and transport‐related stressors.
The secretome of cardiosphere‐derived cells primed with IFNg and TNFa enhances the anti‐inflammatory and pro‐angiogenic effect of porcine M2 macrophages in vitro
M Pulido1
1: Stem Cell Therapy Unit, Jesús Usón Minimally Invasive Surgery Centre, Cáceres, 10004, Spain 2: RICORS‐TERAV Network, ISCIII, Madrid, 28029, Spain 3: Immunology Unit, University of Extremadura, Cáceres, 10003 Spain 4: Institute of Molecular Pathology Biomarkers, University of Extremadura, Cáceres, 10003 Spain
Activated macrophages are usually divided into M1 (pro‐inflammatory profile) and M2 (anti‐inflammatory profile), based on surface marker expression and cytokine secretion. Both are ‐involved in inflammatory diseases such as myocardial infarction (MI), being key in both the initial inflammatory response and subsequent wound healing. Therefore, ameliorating the inflammatory scenario by modulating M1/M2 polarization may be an effective approach for inflammatory‐based diseases.
Cardiosphere‐derived cells (CDCs) have been suggested as a promising therapeutic approach, as well as their secretomes. Our aim was to enhance the therapeutic potential of porcine CDCs by priming them with IFNg and TNFa and to evaluate the effect of their secretome (S‐CDCs*) on macrophage polarization in vitro.
Pigs are widely used as animal models in the field of cardiovascular diseases due to their similarity to the human heart, so porcine macrophages were isolated from the peripheral blood of healthy pigs and polarized them to M1 or M2 by adding hGM‐CSF + LPS + IFNg or hM‐CSF + IL‐4 to the medium, respectively. In addition, M2 were treated with 100 mg/ml of S‐CDCs* for 24 h.
The phenotype switch was determined by surface receptor expression and cytokine expression analysis by flow cytometry and qPCR.
After S‐CDC* treatment, M2 macrophages showed a decrease in pro‐inflammatory markers, and an increase in the expression of anti‐inflammatory markers and the proangiogenic gene VEFGA.
In conclusion, administration of S‐CDCs* may have a beneficial effect on the inflammatory and reparative phases of MI.
Contracts for better access to Advanced Therapy Medicinal Products (ATMPs)
1: Université de Bourgogne Franche‐Comté
The greater part of ATMPs is manufactured in a customized way with high technicity to offer promising health benefits with mostly a one‐shot treatment for incurable, often orphan diseases. The scope of use of these therapies is quite narrow and they also raise safety issues for patients. These are some of the reasons why ATMPs are extremely expensive and why they do not fit in the current regulatory and economic framework. If we want as many patients as possible to benefit from these medicines, we need to set a fair price for health insurance systems. On the basis of the Managed Entry Agreements (MEAs), the parties can negotiate on the performance of the product instead of signing a classic contract. The OECD distinguishes between two main types of agreements: financial and performance‐based MEAs. Focusing on the latter, this paper will highlight its potential to facilitate access to ATMPs. The “coverage with evidence development (CED) contracts” allow treatment to be temporarily covered by the payer while a study evaluates its performance. Based on the results, coverage can be continued, withdrawn or extended, or prices are adjusted. The “pay‐for‐results contract” considers the uncertainty of results and organizes a “satisfied or reimbursed” policy. Here, the risk of innovation seems to lie with the laboratory and not with the community. Unfortunately, confidentiality is a barrier to evaluating objectively the strengths and weaknesses of these agreements. Greater transparency and an extended collaboration between States could help them negotiate better with drug laboratories.
ATMP ‐ Oligonucleotides and AAV analytics
1: Solvias
Analysis of nucleic acids used for cell and gene therapeutics is a complex and challenging task. Starting from simple formulation and small size of oligonucleotides (OGN), the complexity is increased with added modifications, higher size of e.g. mRNA or the need for an additional delivery system as LNP or a viral vector. The high variability of products leads to the necessity to choose suitable methods for the product‐specific analysis.
Therapeutic OGN are sized from 15‐50 nucleotides either present in single or double stranded form. They commonly include several modifications to the phosphate backbone, the ribose moiety, or the nucleobases. Identity confirmation including site‐specific confirmation of modifications is performed at Solvias either applying conventional ion‐pairing RP‐MS conditions or using a novel ion pairing free HILIC‐MS based approach. This analysis is also applied to larger OGNs as for example sgRNA analysis for CRISPR/Cas therapy.
At increasing size of RNA or DNA (1000‐10.000 nt), distinct analytics for larger molecules e.g. general DNA/RNA integrity by capillary electrophoresis or mass spectrometry is applied. Furthermore, the use of delivery systems such as lipid nanoparticles or viral vectors add additional target attributes to be analyzed.
Combining orthogonal physicochemical, CE, LC and MS‐based analytical approaches OGN or AAV can be characterized.
Elimination of cancer cells by targeting the complement cascade
1: Uppsala University
Complement is an inseparable part of the immune system crosslinked with both innate and adaptive immunity. The complement system is effective in clearing offensive pathogens and damaged host cells but complement activation in the tumour microenvironment has been shown to usually support tumour growth and metastasis. No therapeutic strategy has been proposed aiming at inducing potent complement activation in cancer till now. We aim at provoking the complement cascade by expressing Properdin, the only known positive regulator in the complement cascade, and a sugar modification, which can be rejected by the pre‐existing antibodies in human, on cancer cells. Properdin was expressed in either a secreted form or a membrane‐bound form with a stable transmembrane anchored to its C‐terminal. On the surface of cancer cells with engineered sugar modification, membrane‐bound properdin induced a significantly higher C3b deposition which also led to stronger complement‐dependent cytotoxicity in vitro. In the mouse pancreatic cancer model, additional properdin expression in cancer cells significantly slowed down the tumour growth and prolonged mice survival while showing a higher flux of tumour‐associated neutrophils and a decreased expression of iNOS in tumour‐infiltrating myeloid cells. Contrary to the widely‐accepted pro‐tumoural role of complement activation, our results indicated that the complement cascade can be manipulated to actively eliminate cancer cells.
Intravenous FBX‐101 (AAVrh10.hGALC) following Hematopoietic Stem Cell Transplantation increases GALC activity, supports brain development and improves motor function in Patients with Infantile Krabbe Disease: RESKUE Phase 1/2 Clinical Trial
1: University of Pittsburgh 2: Forge Biologics
RESKUE is a first‐in‐human, open‐label Phase 1/2 dose‐escalating trial to evaluate safety and efficacy of FBX‐101 after hematopoietic stem cell transplantation (HSCT) for the treatment of patients with Infantile Krabbe disease (IKD). IKD is a severe neurodegenerative disorder caused by galactocerebrosidase (GALC) deficiency which leads to death by 2 years. HSCT improves outcomes in asymptomatic infants, but motor function declines due to peripheral neuropathy. FBX‐101 is an intravenously administered AAVrh10‐hGALC vector designed to rescue peripheral nerve disease. Nonclinical studies demonstrated that FBX‐101 significantly increased survival and corrected peripheral neuropathy in Krabbe mice and dogs when given after HSCT. FBX‐101 safety and biodistribution were established in a GLP rat toxicology study.
The first 2 subjects in the RESKUE low dose cohort received FBX‐101 at a dose of 3.0x1013 vg/kg 25 and 29 days after HSCT. FBX‐101 was well tolerated, with no treatment‐related serious adverse events observed up to Day 180 and Day 90, respectively. No antibodies to the capsid have developed so far after FBX‐101 administration. Both subjects have engrafted with full chimerism. We report a significant increase in plasma and CSF GALC enzyme activity following gene transfer, improvement in motor skills measured by PDMS‐II, and normal brain white matter growth measured by MRI‐DTI. Administration of FBX‐101 after HSCT is enhanced by a safer immune environment that allows efficient AAV transduction, ensuring that GALC enzyme is available to support both brain and peripheral nerve development in patients and represents a novel gene therapy strategy for metabolic diseases amenable to treatment by HSCT.
ADCC‐ and BCR‐inspired receptors for antigen‐specific NK cell activation
1: Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland
Antibody‐dependent cellular cytotoxicity (ADCC) in Natural Killer (NK) cells boosts clinical efficacy of immunoglobulin gamma (IgG) therapeutic antibodies. In ADCC, soluble IgG antibodies bind to an antigen on the surface of a target cell and engage, via their Fc‐domain, a CD16 receptor on the NK cell surface, leading to target cell death. The three‐component nature of this process, however, limits its efficiency. We hypothesised that the immobilisation of an antibody on an NK cell would result in antigen‐specific NK cell therapeutics. To test this approach, we tethered an HER2 antibody to the membrane of CD16POS NK‐92 cells via a transmembrane domain of a B cell receptor (BCR). We found that the modified NK cells engage in a strong ADCC‐like cytotoxic response against HER2‐positive cancer cells, surpassing canonical ADCC. Next, we eliminated the need for CD16 by coexpressing the membrane‐bound antibody with BCR‐associated CD79A/B chains fused to CD3z signalling domains. NK cells augmented with these receptors induce strong antigen‐specific cytotoxicity against target cells without the need for CD16. Through further incorporation of FceRIg signaling domains to the cytosolic tails of the membrane‐tethered antibody we engineered a multi‐chain receptor architecture comprising four transmembrane chains, each capable of accommodating an independent signaling domain. This modification drastically increases NK cell cytotoxicity against HER2‐positive cancer cells. Our approach to ADCC‐ and BCR‐inspired receptor architectures offers a highly modular and programmable platform to engineer NK cells with the desired antitumor specificity and potency, and opens the way to a novel class of cell therapeutics.
Preclinical development of an efficient and safe gene editing platform for the treatment of Wiskott Aldrich Syndrome
1: UCL Institute of Child Health 2: NIHR Great Ormond Street Hospital Biomedical Research Centre, London, UK
Wiskott‐Aldrich syndrome (WAS) is a rare, X‐linked primary immunodeficiency disease caused by mutations in the WAS gene which encodes the WAS protein (WASp). Affected individuals suffer inflammatory complications, increased incidence of autoimmunity and are at risk of developing lymphoproliferative disorders. We have recently developed a CRISPR‐Cas9 and AAV6‐based gene editing platform to knock‐in a correct WAS cDNA in the WAS locus in hematopoietic stem and progenitor cells (HSPCs), providing specificity, toxicity and efficacy data supportive of continued development of the platform to treat WAS. We have now progressed this gene editing‐based therapy to preclinical studies to assess its clinical‐scale feasibility. By manufacturing HSPCs at clinical scale with GMP‐compliant protocols we achieved reproducible high rates of targeted integration, while preserving HSPCs viability and colony forming efficiency. Cells were capable of long‐term engraftment in NSG mice with reconstitution of haematopoiesis and retention of the correct WAS gene in all hematopoietic organs. Histopathological analysis revealed no evidence of tumorigenicity or abnormal haematopoiesis in treated mice. A comprehensive assessment of off target cutting and of chromosomal aberrations imputable to gene editing was carried out at different time points both in vitro and in vivo and confirmed the safety of the platform. Furthermore, evaluation of potential AAV integration revealed the presence of rare events of donor vector integration although not in proximity of tumour‐associated genes. Altogether, our preclinical safety and efficacy data provide evidence of the feasibility of the platform and support its translation into early phase clinical studies.
A first‐in‐human phase 1 clinical gene therapy trial for the treatment of non‐ischemic heart failure using a novel rationally designed cardiotropic adeno‐associated vector targeting calcium cycling
1: AskBio 2: Christ Hospital Cincinnati 3: Minneapolis Heart Institute 4: University of Texas, Dallas 5: University of Cincinnati 6: Flagship Pioneering
Congestive heart failure (CHF) is a disease in need of innovative therapies. A key aspect in CHF and other cardiomyopathies is abnormal intracellular Ca2+ handling and increased protein phosphatase 1 activity (PP1). Inhibition of PP1 by a constitutively active inhibitor‐1 (I‐1c) has been shown to enhance cardiac function in large animal models of heart failure. We developed a novel cardiotropic adeno‐associated vector that delivers I‐1c (AAV2i8.I‐1c) and used it in a first‐in‐human dose‐escalation gene‐therapy trial to evaluate safety and feasibility of a single coronary artery infusion of AAV2i8.I‐1c in patients with nonischemic cardiomyopathy. Eight patients received either 3E13 (Cohort 1; n = 3) or 1E14 (Cohort 2; n = 5) viral genomes of AAV2i8.I‐1c. In Cohort 1, there were no investigational product‐related adverse events after 12 months. AAV2i8‐I‐1c demonstrated high myocardial transduction efficiency in a human heart biopsy sample taken 13 months post‐intracoronary gene transfer (n = 1). In the human left ventricular sample, there were 1‐3 viral copies per diploid genome and the phospholamban (PLN) phosphorylation at Ser 16, which is depressed in CHF, was comparable to normal heart left ventricular control samples. Compared with baseline, all 3 patients in Cohort 1 showed clinically meaningful improvements in left ventricular ejection fraction and New York Heart Association score at month 12. Twelve‐month follow‐up of Cohort 2 is ongoing. Results suggest that AAV2i8‐I‐1c may provide encouraging safety and efficacy results in patients with non‐ischemic CHF. Additionally, other cardiomyopathies caused by mutations to PLN (eg R14del‐PLN), leading to cardiomyopathies in carriers, may also benefit from treatment with AAV2i8.I‐1c,
Analysis of Vector Performance in Patient Heart Biopsy for Congestive Heart Failure Provides Support for Rationally Designed Capsids
1: AskBio, Research Triangle Park, NC 2: Gene Therapy Center and Department of Pharmacology, University of North Carolina, Chapel Hill, NC 3: The Christ Hospital, Cincinnati, Ohio 4: University of Cincinnati, Cincinnati, Ohio
Congestive heart failure (CHF), a widespread progressive disease with a critical unmet need is characterized by the heart's inability to pump adequately. Advances in understanding the molecular basis of myocardial dysfunction culminated in AAV gene therapy (GT) clinical trials for heart failure. Cardiac tissue analysis from earlier trials that failed to show clinical efficacy revealed low vector transduction in heart at <0.012 vg/dg(vector genomes/diploid genome). To overcome poor vector transduction, a novel cardiotropic capsid, AAV2i8 detargeting liver was developed through rational design. Our clinical trial for non‐ischemic cardiomyopathy uses the AAV2i8 capsid to deliver a constitutively active protein phosphatase 1 inhibitor 1 (I‐1c). Evaluation of left ventricular tissue from a subject enrolled in our trial showed 1.19 vg/dg, 30‐fold higher than in the pre‐clinical porcine model. Abnormal calcium handling via the sarcoplasmic reticulum plays a central role in the pathophysiology of CHF. Decreased SERCA2a Ca2+ transport is in part due to dephosphorylation of its inhibitor, phospholamban (PLN), because of increased protein phosphatase 1 (PP1) activity. I‐1c is a constitutively active inhibitor of PP1 and augments PLN phosphorylation and cardiac contractility in pre‐clinical models of heart failure. Analysis of biopsy sample showed that Ser16 phosphorylation of PLN is restored to levels within normal range. Planned analyses of additional biopsy specimens will provide further insights into vector performance in humans. These data underscore the importance of obtaining quantitation of vector performance for novel capsids in human tissues, and also support the validity of our GT approach towards the treatment of heart failure.
Intracoronary therapy with cardiosphere derived cell secretome after myocardial infarction. Experimental validation of the effects on cardiac function and ventricular tachycardia inducibility
C Baez‐Díaz1 2 3 V Blanco1 2 3 FM Sánchez‐Margallo1 2 3 A Torrescusa1
1: Jesús Usón Minimally Invasive Surgery Centre, Cáceres, Spain 2: RICORS‐TERAV Network, ISCIII, Madrid, Spain 3: CIBER de Enfermedades Cardiovasculares, Madrid, Spain
Patients after myocardial infarction (MI) are at high risk for malignant arrhythmias and sudden cardiac death due to the development of ventricular tachycardia (VT) which is caused by viable myocytes located within the infarct scar tissue. Our aim was to evaluate the effects of an intracoronary (IC) administration of cardiosphere derived cell secretome (S‐CDCs) on cardiac function and VT inducibility in a porcine MI model. Four weeks after experimental MI, pigs received an IC injection of saline (G1; 5mL; n = 7) or S‐CDCs (G2; 9.16 mg in 5mL of saline; n = 7). VT inducibility and magnetic resonance (MR) studies were performed pre‐ and 16 weeks post‐injection calculating ejection fraction (EF), infarct size (IS) and indexed end diastolic and systolic volumes (EDVi, ESVi). No differences were seen between groups in any MR‐derived parameter and VT was inducible in all cases before injection. IC infusion was successful in all animals. At 16 weeks EF was higher in G2 (35 ± 10% versus 29 ± 10%) although differences between groups were not statistically significant. Conversely, IS was significantly lower in G2 (12 ± 3% versus 16 ± 2 (p = 0.03)). A trend towards lower ventricular volumes (EDVi: 83 ± 18mL/m2 versus 88 ± 29mL/m2 and ESVi: 56 ± 20mL/m2 versus 64 ± 27mL/m2) and lower inducibility rates (57% versus 100%; p = 0.05) were detected in G2 compared to G1. In conclusion, an IC injection of S‐CDCs seems to reduce infarct size in this porcine MI model. Moreover, S‐CDCs administration suggests a beneficial effect of this therapy on cardiac function as well as a potential reduction on post‐MI VT development.
Comparative evaluation of genome editing and RNA interference for substrate reduction therapy in two animal models of glycogenosis
1: Genethon, UMR_S951, Inserm, Univ Evry, Université Paris Saclay, EPHE 2: Centro de Investigacion Medica Aplicada (CIMA)
Pompe disease (PD) is an inherited metabolic disorder, characterized by a pathological build‐up of glycogen in lysosomes. PD affects tissues throughout the body and particularly cardiac and skeletal muscles, resulting in severe progressive myopathy and respiratory failure. Enzyme replacement therapy that represents the standard of care, has limited efficacy. Substrate reduction therapy (SRT), aimed at the reduction of glycogen synthesis, represents a potential therapeutic approach to address this disease. Here we show the results of a comparative study evaluating the efficacy of the downregulation of muscle glycogen synthase (GYS1), the enzyme responsible for glycogen branch elongation, in a PD mouse model. We first developed a genome editing strategy using CRISPR/Cas9 technology. The S.aureus Cas9 and a guide RNA targeting the Gys1 gene were packaged in an AAV vector and delivered intravenously in newborn mice. This approach demonstrated efficacy in the heart of treated mice, where we observed, 6 months after vector injection, a significant reduction of GYS1 expression, and almost no glycogen accumulation. A second SRT approach was developed, based on a RNA interference mechanism mediated by an AAV encoding for a Gys1‐specific artificial microRNA. After systemic delivery in newborn Pompe mice, this approach prevented glycogen accumulation in skeletal muscles. To validate this results in a different disease model, we demonstrated that the same SRT reduced glycogen accumulation in muscles of mice affected by Cori disease, another glycogen storage disorder (GSD). These results highlight the therapeutic potential of SRT for all muscular GSDs.
Adeno‐associated virus vector combination gene therapy with C‐type natriuretic peptide and GALNS enzyme in Mucopolysaccharidosis IVA mouse model
1: Department of Molecular Biology, Faculty of Biology, University of Gdansk, Gdansk 80‐308, Poland 2: Skeletal Dysplasia Research Lab, Nemours Children's Health, Wilmington 19803 DE, USA 3: Department of Biological Sciences, University of Delaware, Newark, 19716 DE, USA 4: Department of Pediatrics, Thomas Jefferson University, Philadelphia 19107 PA, USA
Mucopolysaccharidosis type IV A (MPS IVA) is caused by a deficiency of GALNS enzyme responsible for degradation of glycosaminoglycans (GAGs). The progressive accumulation of GAGs leads to various skeletal abnormalities (short stature, knock knees, hypoplasia, tracheal obstruction) as well as several symptoms in the peripheral tissues (respiratory problems, valvular heart disease, hearing impairment). To date, no treatment is effective for patients with bone abnormalities. Enzyme replacement therapy was found to improve quality of life without bone lesion improvement. More recent studies indicated that modified C‐type natriuretic peptide (CNP) is an effective treatment for short stature in Achondroplasia patients. To improve bone pathology, we propose a novel combination treatment with gene therapy and a CNP peptide as a growth‐promoting agent for MPS IVA. MPS IVA mice were treated for 12 weeks with AAV expressing GALNS combined with AAV expressing CNP. After combination therapy, bone growth in mice was induced together with increased enzyme activity in both tissues (bone, liver, heart, lung) and plasma. Moreover, there were significant changes in bone morphology in CNP‐treated mice with increased CNP activity in plasma. Our therapy confirmed that the enzyme might not easily penetrate growth plate region in the bone, but CNP can change bone morphology. Combination therapy with CNP and GALNS gene therapy in MPS IVA more effectively induceed bone growth in mice than GALNS gene therapy alone.
Gene Transfer of SCN10A‐short Improves Cardiac Conduction in WT and Scn5aHet Mice
1: Amsterdam UMC
Cardiac voltage‐gated sodium channel NaV1.5 is an important therapeutic target to treat conduction block and ventricular tachycardia. However, the large size of SCN5A encoding NaV1.5 imposes a substantial challenge to gene therapeutic interventions. We previously reported that a naturally occurring short transcript encoding a C‐terminal portion of neuronal SCN10A (SCN10A‐short) increases sodium current in an SCN5A‐dependent manner. In this study we evaluated the potential of SCN10A‐short as a therapeutic gene for cardiac conduction disorders. First, we studied the effect of overexpressing SCN10A‐short in cardiomyocytes using single cell patch‐clamp in human iPS‐derived cardiomyocytes. Cells transduced with SCN10A‐short showed significantly higher sodium channel‐dependent action potential upstroke velocities compared to the GFP control. A similar effect was observed in cardiomyocytes isolated from mouse hearts treated with adeno‐associated virus (AAV)‐mediated SCN10A‐short gene therapy. We next studied if SCN10A‐short gene transfer could improve cardiac conduction in vivo. Wildtype (WT) and heterozygous Scn5a mutant (Scn5aHet ) mice were systemically injected with AAV vectors containing SCN10A‐short or GFP. Two weeks post injection, hearts were isolated and perfused on a Langendorff setup, where optical‐mapping was performed to measure epicardial conduction patterns. In both mouse models, gene transfer of SCN10A‐short led to a ∼30% increase in conduction velocity in the left ventricle, when compared with the GFP controls. Notably, AAV‐mediated SCN10A‐short overexpression in Scn5aHet mice brought the conduction velocity back to WT animal's level. In sum, our results highlight the potential of SCN10A‐short gene delivery as an effective therapeutic intervention to normalize cardiac conduction in inherited and acquired conduction disorders.
Feasibility and safety of repeated intracoronary administration of Cardiosphere derived cells or their secretome in a porcine model of myocardial infarction
V Crisostomo1 2 3
1: Jesús Usón Minimally Invasive Surgery Centre, Cáceres 10004, Spain 2: RICORS‐TERAV Network, ISCIII, Madrid 28029, Spain 3: CIBER de Enfermedades Cardiovasculares (CIBERCV), 28029 Madrid, Spain
Regenerative therapies with cell‐derived products, such as cardiosphere‐derived cells (CDCs), and their secretome (S‐CDCs) may be beneficial in patients suffering from myocardial infarction (MI). However, single‐dose trials have yielded moderate results at best. We aimed to evaluate feasibility and safety of 3 intracoronary doses of CDCs or S‐CDCs performed 1, 2, and 3 weeks after MI in swine. Safety was determined during repeated administration and in the immediate postoperative periods. Cardiac MR studies were performed before first dose infusion and at 10 weeks, and processed blindly to obtain left ventricular function parameters (Ejection fraction (EF), end diastolic (EDVi) and systolic volumes (ESVi)).
Infarcted animals were randomly allocated to receive a volume of 20 ml containing 30x106 CDCs (n = 6), 9.16mg S‐CDCs (n = 6) or saline (n = 6) slowly injected into the left anterior descending coronary artery. No changes to the ECG or postoperative fibrillation or deaths were observed in any animal, irrespective of the group. There were no differences in troponin or C‐reactive protein when comparing before or after injection.
Treatment effects were consistently, if not significantly, better in S‐CDCs animals, while CDCs‐treated animals showed a trend towards worsening cardiac function. Over time, EF changed 3 ± 7% vs 6 ± 5% vs 4 ± 7%, EDVi 9 ± 18mL/m2 vs 1 ± 11mL/m2 vs 9 ± 20mL/m2, ESVi 4 ± 14mL/m2 vs ‐4 ± 12mL/m2 vs 4 ± 17mL/m2 and infarct size ‐6 ± 3% vs ‐8 ± 5% vs ‐5 ± 5% in control compared to S‐CDCs or CDCs, respectively. Therefore, although the repeated administration of regenerative therapies appears to be safe in this experimental model, only S‐CDCs may be beneficial for improving cardiac function.
Novel designed VEGF‐B186R127S gene therapy induces angiogenesis in mouse heart without significant side effects
1: University of Eastern Finland, Kuopio 2: Kuopio Center for Gene and Cell Therapy, Kuopio, Finland
Myocardial ischemia is the most common clinical consequence of cardiovascular disease, due to reduced blood supply to the affected region(s) of the heart. Despite the reperfusion treatments (conventional bypass surgery or catheter mediated interventions), subendocardial blood supply is commonly insufficient. Therefore, novel therapeutic strategies for promoting capillary and collateral vessel growth are in high demand. Previous studies have indicated that vascular endothelial growth factor B186 (VEGF‐B186) supports coronary vascular growth in normal and ischaemic myocardium. However, previous studies also indicate that induction of ventricular arrhythmias is a severe side effect preventing the use of VEGF‐B186 in cardiac gene therapy, possibly mediated by binding to neuropilin 1 (NRP1). We have designed a novel VEGF‐B186 variant, VEGF‐B186R127S, which is resistant to proteolytic processing and unable to bind to NRP1. Here, we studied its effects on mouse heart to explore the mechanism of VEGF‐B186‐induced vascular growth along with its effects on cardiac performance. The proteolytically resistant VEGF‐B186R127S significantly induced vascular growth in mouse heart without causing any side effects, such as arrhythmias.
ABSTRACT WITHDRAWN
1: Institute of Virology and Microbiology, Center for Biomedical Education and Research (ZBAF), Department of Human Medicine, Faculty of Health, Witten/Herdecke University, 58453 Witten, Germany 2: Department of Neurology, University Hospital Bergmannsheil, Heimer Institute for Muscle Research, 44789 Bochum, Germany 3: Sorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, F‐75013 Paris, France.
The heterogeneity of muscular tissues complicates the development of targeted gene therapy. Depending on the therapy approach and disease background restriction to one, or expression in all muscle types may be warranted. Target tissue specificity can be achieved using tissue specific promotors. A promoter for muscle gene therapy should exhibit limited activity in non‐targeted tissue, while conferring specific, long‐term sustained and physiological expression in the desired muscle types. Here we present a direct comparison of muscle specific Desmin and MCK‐based chimeric MHCK7 promoter, that are highly active in murine and human skeletal myocytes as well as in cardiomyocytes. In contrast, the microRNA206 and Calpain3 promoter provide gene expression mainly restricted to skeletal myocytes. Since both, the activity of human promoters and the complexity of protein related physiological processes may differ in the mouse model compared to the human organism, we present an in vitro model based on electrical pulse stimulation (EPS) to provoke sarcomere formation in 2D cell culture for quantification of promoter activity in far differentiated mouse and human myotubes. We found that Desmin and MHCK7 promoters showed stronger reporter gene expression levels in proliferating and differentiated myogenic cell lines than miR206 and CAPN3 promoter. However, Desmin and MHCK7 promoters promoted gene expression also in cardiac cells whereas miR206 and CAPN3 promoter expression was restricted to skeletal muscle. Our study provides insights helping to choose an optimal muscle specific promoter for an intended treatment approach with regard to tissue specificity, expression strength and duration.
Pre‐clinical development of an ex‐vivo Hematopoietic Stem/progenitor Cells‐gene therapy for α‐Mannosidosis
P Quaranta1 A A Rilievo1 L Basso‐Ricci1 R J Hernandez1 C Forni4 I Visigalli5 P Albertini5 A Paoli6 A Morrone6 R Parini7 B Gentner1 S Crippa1 M E Bernardo1 2
1: San Raffaele Telethon Institute for Gene Therapy (SR‐TIGET), IRCCS San Raffaele Scientific Institute, Milan, Italy 2: Pediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan,Italy 3: Università Vita‐Salute San Raffaele, Milan, Italy 4: Telethon Foundation, Rome, Italy 5: GLP‐ San Raffaele Telethon Institute for Gene Therapy (SR‐TIGET), Milan, Italy 6: Pediatric Neurology Unit, Meyer Children's Hospital, Florence, Italy 7: Rare Metabolic Disease Unit, San Gerardo Hospital, Monza, Italy
α‐Mannosidosis is a rare autosomal recessive lysosomal storage disease (LSD) caused by deficiency of lysosomal α‐mannosidase (MAN2B1), which results in accumulation of mannose‐enriched oligosaccharides and progressive skeletal and organ damage. Ex‐vivo lentiviral (LV)‐based Hematopoietic Stem/progenitor Cells (HSPC)‐gene therapy (GT) proved its efficacy in correcting the clinical phenotype of other LSDs, including Metachromatic leukodystrophy and Mucopolysaccharidosis type I, restoring the deficient enzyme also in non‐hematopoietic cells in the diseased tissues (e.g. CNS and bones) through cross‐correction. Based on these results, we developed LV encoding for both the wild‐type (WT) and codon optimized (OPT) forms of MAN2B1 cDNA for the treatment of α‐Mannosidosis, as part of a GT platform program for LSDs with skeletal involvement. Our preliminary data show that MAN2B1 gene‐transfer in human healthy donor(HD)‐derived CD34+ cells do not alter their clonogenic and proliferation capability in vitro. LV‐MAN2B1 transduced CD34+ cells show increased production of α‐mannosidase and extracellular release of both precursor and lysosomal forms of the enzyme resulting in 2.5‐fold higher MAN2B1 intracellular and extracellular enzymatic activity as compared to untransduced control. Moreover, the secreted MAN2B1 precursor can be internalized and processed by α‐Mannosidosis patients‐derived fibroblasts restoring the protein content at level comparable to HD controls. Altogether, our preliminary data show that both WT and OPT MAN2B1 gene‐transfer is well tolerated by human CD34+ cells, increasing the availability of extracellular MAN2B1 for cross‐correcting non‐hematopoietic cells. In vivo studies will complement these data paving the way for the clinical development of HSPC‐GT for α‐Mannosidosis.
Modelling multi‐tissue involvement in Duchenne muscular dystrophy using advanced in vitro models of human striated muscle
1: The Francis Crick Institute 2: Department of Cell and Developmental Biology, University College London 3: Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of Child Health & Great Ormond Street Hospital for Children
Muscular dystrophies are a heterogenous group of inherited disorders characterised by skeletal muscle wasting and weakness. Importantly, several muscular dystrophies are also characterised by cardiac muscle involvement, such as Duchenne muscular dystrophy (DMD), which is the most common paediatric muscular dystrophy. However, this cardiac phenotype is poorly recapitulated in most DMD animal models. Although there are multiple in vitro DMD modelling platforms, none of them has been designed to look at phenotypic readouts of both skeletal and cardiac muscle involvement in parallel. Furthermore, very few DMD experimental therapies can target both striated muscles simultaneously. To address these limitations, we are developing isogenic DMD skeletal and cardiac 3D muscle platforms as well as analysing maturation properties of the engineered muscles. We have differentiated human induced pluripotent stem cells (hiPSCs) into cardiomyogenic cells using a small molecule‐based, transgene‐free protocol and generated 3D engineered heart tissue. Molecular and electrophysiological analyses indicate that 3D cardiac tissue shows enhanced tissue maturation than conventional monolayer cultures of cardiomyogenic cells. In parallel, to develop a similar approach also for skeletal muscle, we have tested various transgene‐free hiPSCs differentiation protocols to produce skeletal myogenic cells. Future plans include validation of both DMD striated muscle platforms for disease and therapy modelling, focusing on testing small molecules and advanced molecular therapies such as antisense oligonucleotides.
The effect of utrophin deficiency on the electrophysiological properties of DMD hiPSC‐derived cardiomyocytes
1: Jagiellonian University
Duchenne muscular dystrophy (DMD) is an X‐linked genetic disease caused by mutations in the gene encoding dystrophin. Loss of this protein leads to progressive muscle weakness followed by respiratory failure and cardiomyopathy, which is a major cause of death in DMD patients. However, the molecular background of the disease‐associated heart failure has not been fully described. Utrophin, a protein that shares a high structural and functional homology with dystrophin, was proposed in the previous studies as the potential therapeutic target for DMD through its compensatory mechanism. Although many intensive studies have been carried out on the role of utrophin in skeletal muscles, its role in the heart has never been fully understood. In the present study, our objective was to verify how the utrophin deficiency affects the dystrophin‐deficient human induced pluripotent stem cells (hiPSC) – derived cardiomyocytes (hiPSC‐CM). For this approach, the double dystrophin/utrophin‐deficient hiPSC were generated using CRISPR/Cas9 and differentiated to hiPSC‐CM. The electrophysiological properties of hiPSC‐CM were evaluated using calcium oscillations measurements, which revealed severely impaired oscillations pattern and decreased oscillations frequency in dystrophin‐deficient hiPSC‐CM, and this effect was exacerbated in dystrophin/utrophin‐deficient hiPSC‐CM. However, the characterization of electrophysiological properties of single cardiomyocytes, performed by patch‐clamp, did not demonstrate any significant abnormalities. This discrepancy may be explained by the localization of utrophin in cardiomyocytes, which, in addition to the sarcolemma, was also found in intercalated discs, and its lack may worsen the proper signal transduction between the cells, but it does not affect the electrophysiological properties of individual cells.
Intracoronary administration of CDCs or their secretome is not enough in a porcine model of severely depressed cardiac function post myocardial infarction
V Crisostomo1 2 3
1: Jesús Usón Minimally Invasive Surgery Centre, Cáceres 10004, Spain 2: RICORS‐TERAV Network, ISCIII, Madrid 28029, Spain 3: CIBER de Enfermedades Cardiovasculares (CIBERCV), 28029 Madrid, Spain
Nearly one third of patients with Ejection Fraction (EF) below 30% after myocardial infarction (MI) die within two years in absence of cardioverter implantation. Regenerative therapies with cardiac‐derived products, such as cardiosphere‐derived cells (CDCs), and their secretome (S‐CDCs) may be advantageous in these patients. Our main objective was to evaluate whether the intracoronary administration of CDCs or S‐CDCs could be beneficial in an experimental model of severely impaired heart function secondary to MI in swine.
Large‐White pigs with EF <30% after a 90‐min balloon occlusion of the mid‐left anterior descending coronary artery, were randomly allocated to blindly receive 30x106 CDCs (n = 5), 9.16mg S‐CDCs (n = 5) or the same volume of vehicle (n = 5) via the infarct related artery one week after MI. Cardiac MR studies were performed immediately before and 10 weeks after therapy administration, and processed blindly to obtain left ventricular function parameters (EF, end diastolic (EDVi) and systolic volumes (ESVi)).
While control animals performed slightly better and CDCs‐treated animals showed a trend towards worse cardiac function over time, no significant differences between groups were found at the end of the study. Specifically, EF was 31 ± 3% vs 23 ± 2% vs 25 ± 3%, EDVi was 88 ± 9mL/m2 vs 110 ± 13mL/m2 vs 98 ± 3mL/m2, ESVi was 62 ± 8mL/m2 vs 89 ± 14mL/m2 vs 73 ± 4mL/m2 and infarct size 9 ± 1% vs 11 ± 2% vs 11 ± 2% in control compared to CDCs‐ or S‐CDCs‐treated animals, respectively.
In this experimental model of severely impaired cardiac function, the administration of heart‐derived regenerative products as performed herein does not appear to have any beneficial effect on cardiac function.
Developing advanced human myo‐fibrogenic 3D models for disease modelling and therapy development
1: UCL 2: The Francis Crick Institute 3: NIHR Great Ormond Street Hospital Biomedical Research Centre 4: UCL Institute of Child Health
Skeletal muscle extracellular matrix (ECM) is a three‐dimensional (3D) and highly organised scaffold which provides structural stability and supports contractile force transmission, whilst also regulating myogenesis, homeostasis and tissue regeneration. Mutations in crucial muscle ECM constituents such as collagen type VI (COL6) cause a spectrum of muscle disorders of which Ullrich congenital muscular dystrophy (UCMD) is the most severe. UCMD remains incurable and disease mechanisms are still poorly understood. Robust human models capable of recapitulating key UCMD hallmarks will facilitate studying disease pathogenesis whilst also providing platforms to develop therapies. To this aim, we are harnessing our expertise in bioengineering human skeletal muscles to develop advanced models of ECM‐muscle cell interactions. We hypothesise that by equipping these models with myofibres and ECM secreted from fibrogenic cells, we can recapitulate key aspects of human skeletal muscle which are dysfunctional in COL6‐related disorders, including development of typical contractures associated with UCMD. This novel myo‐fibrogenic 3D model can then be used to reduce the use of animal models in UCMD drug discovery while also facilitating development of precision medicine strategies for the whole family of COL6‐related disorders.
Editing the Nuclear Envelope: Using Human iPS Cells and CRISPR‐Cas Technology to Develop Novel Therapies for Skeletal Muscle Laminopathies
1: UCL 2: The Francis Crick Institute 3: Kings College London 4: Institut de Myologie 5: Great Ormond Street Institute of Child Health
Laminopathies are a heterogenous group of diseases caused by mutations in the LMNA gene, encoding nuclear lamin A/C proteins. Together with lamins B1 and B2, these intermediate filaments form a meshwork structure residing beneath the inner nuclear membrane called the nuclear lamina that plays important roles in maintenance of nuclear shape and integrity, chromatin organisation and gene expression regulation. Patients with LMNA mutations show a wide range of clinical phenotypes, with a large proportion affected by severe and incurable muscular dystrophies. Current efforts to research disease mechanisms and therapeutic strategies are hindered by lack of robust, high‐fidelity human disease models. Previous work in our laboratory demonstrated the potential of using patient‐derived induced pluripotent stem (iPS) cells to model hallmark laminopathy phenotypes such as abnormal nuclear shape in vitro. Here, we use this strategy to model laminopathy‐associated skeletal muscle phenotypes in vitro in both standard 2D cultures and 3D bioengineered muscles using a transgene‐free, small molecule‐based protocol for differentiation. Results show that myogenic capacity is unaffected in LMNA‐mutant lines and nuclear shape abnormalities are present in a mutation‐specific manner. Further, we utilise this platform to develop mutation‐specific, CRISPR‐based therapeutic strategies. Pathogenic LMNA mutations were targeted for correction or removal using homology directed repair, permanent exon‐skipping or prime editing to restore expression of healthy lamin A/C. Edited pluripotent cells will then be differentiated and amelioration of the aforementioned nuclear morphometric readouts will be used as an initial screen of efficacy of this approach for this severe and incurable group of muscle disorders.
CRISPR gene editing in muscle cells to study RyR1‐related myopathies
1: University Grenoble Alpes, INSERM, U1216, CHU Grenoble Alpes, Grenoble Institut 9 Neurosciences, Grenoble, France
Type 1 ryanodine receptor (RyR1) is the intracellular calcium channel responsible for skeletal muscle contraction. Mutations in RYR1 gene can lead to RyR1‐related myopathies (RyR1‐RM), a group of genetic disorders. They are characterised by abnormal calcium homeostasis and muscle weakness of variable severity. Currently, there is no treatment for these pathologies.
We have developed a protocol to perform RYR1 gene editing using Clustered Regularly Interspaced Short Palindromic Repeats associated protein 9 (CRISPR/Cas9) in immortalised skeletal muscle cells. This protocol can be used to develop tools to further study pathophysiological mechanisms and for therapeutic development.
Screening siRNA efficacy using high throughput fluorescence system in vitro
1: William Harvey Research Institute, Queen Mary University of London
Osteoarthritis (OA) is characterized by loss of articular cartilage, bone remodelling and pain. It is a leading cause of disability in older people. Currently, there are no disease modifying drugs to stop progression of OA. One bottleneck in clinical translation is that the cartilage extracellular matrix is avascular and impenetrable for large macromolecules. siRNA induces highly specific gene silencing and can be used for targets that are “undruggable”. However, efficacy of “naked” siRNA is limited as it is instantly targeted for degradation by nucleases. To improve utility of siRNA as a therapeutic modality, chemical modifications can be introduced to the siRNA backbone, and it can be coupled with carrier molecules. An efficient high throughput fluorescence system that can be used universally has been optimised to screen siRNA in vitro. A plate reader and inverted microscope are used to determine the efficacy of GFP siRNA in GFP transduced Cos7 cells. The fluorescence system uses a 96‐well plate and allows for repeat measurements over time, without the need to harvest samples at each timepoint. This system was used to screen the effects of modifications to the GFP siRNA backbone. The GFP siRNA modification that yielded highest efficacy was selected and Intra‐articular injections of the stabilised GFP siRNA were given to GFP transgenic mice to determine the efficacy and deposition of siRNA. Overall, a system to measure the pharmacokinetics of siRNA in vitro for a long time has been developed to aid drug discovery and can be applied beyond the musculoskeletal field.
Enhancement of AAV Transduction in Muscle Cells In Vitro
1: Solid Biosciences
Adeno‐associated virus 9 (AAV9) serves as an efficient vector for gene delivery to muscle in vivo. However, in vitro transduction of terminally differentiated skeletal muscle cells (myotubes) is inefficient and necessitates high titers to observe gene expression. Achieving adequate transduction in vitro is important for the development of potency assays to support gene therapy clinical studies. Therefore, we sought to identify agents that enhance myotube transduction by AAV9.
Several agents are reported to increase AAV2 transduction in HeLa or HEK293 cells, but few have been evaluated for AAV9, or in myotubes. To identify agents that increase transduction of AAV9 in myotubes, a small panel of agents was screened in a dystrophin deficient C2C12 cell line. Differentiated C2C12 myotubes were pre‐treated with test agents having various mechanisms of action and were then transduced with AAV9 expressing luciferase using a CK8 promoter. Four days post transduction cells were harvested, and luminescence was measured.
Most agents evaluated had little impact on AAV9 transduction; however, etoposide (topoisomerase II inhibitor) and neuraminidase (sialic acid cleaving enzyme) significantly enhanced transduction, with a 2‐4 or 2‐5‐fold increase in luciferase expression over control, respectively. Dual treatment resulted in increased transduction greater than either single agent alone, demonstrating a combination effect of these agents, each with a different mechanism of action.
These studies demonstrate the potential to enhance AAV9 transduction in difficult to transduce myotubes, furthering study of AAVs effects in these cells, and supporting development of in vitro potency assays in muscle cells for clinical AAV9‐based gene therapies.
Skeletal muscle atrophy under the conditions of hindlimb unloading
A O Fedianin2
1: Ernst Strüngmann Institute for Neuroscience in Cooperation with Max Planck Society,Germany 2: Mechanobiology laboratory, Institute of fundamental medicine and biology, Kazan Federal University
Here we evaluate the atrophy of rat skeletal muscle under conditions of hindlimb unloading (HU, up to 35 days, Morey‐Holton Model). The study was performed on 21 adult Sprague‐Dawley rats of both sexes, weighing 160‐200 g. All experiments were performed in compliance with bioethical standards. Cryostat transverse serial sections of the soleus, gastrocnemius, and tibialis anterior muscles were stained with primary polyclonal antibodies to dystrophin and nNOS. The results showed that dystrophin immunofluorescence in control rats was present in all muscles. Dystrophin immunofluorescence was present evenly in all muscles in rats on days 7 and 14 of HU, however, on day 35 of HU, a decrease in the amount of dystrophin and the appearance of muscle fibers with a violation of the dystrophin layer were observed, which was manifested by a decrease in color intensity by 15% compared to the control in all muscles, which is also confirmed by nNOS immunofluorescence. During the readaptation period (up to 35 days after HU), the level of dystrophin increased slightly, the color intensity was higher on average by 6% compared with the groups during the HU period but did not recover to the control level. Thus, we have shown that the atrophy of muscle fibers in skeletal muscles develops by the 35th day of HU. Readaptation within 35 days is not enough to restore the integrity of muscle fibers. Probably, to achieve full structural adaptation of animal muscles after hypogravity conditions, longer rehabilitation is required.
Title: The impact of TLR‐2 stimulation on human mesenchymal stem cells antimicrobial capacity
1: School of Cellular and Molecular Medicine, Faculty of Life Sciences, University of Bristol, UK 2: 3:
Implications of novel Adenovirus‐Host interactions in Thrombosis with Thrombocytopenia Syndrome
1: Mayo Clinic 2: Cardiff University 3: Arizona State University 4: Michigan State University 5: University of Illinois 6: Glasgow University 7: AstraZeneca 8: University of Maryland
In this study we investigated a link between adenovirus‐based vaccines, deployed to fight the SARS‐CoV‐2 pandemic, and life‐threatening thromboembolisms after vaccination. Post‐marketing surveillance showed that, following vaccination, Vaxzevria (ChAdOx1 based, AstraZeneca) and Jcovden (Adenovirus type 26 based, Johnson & Johnson) are associated with reduced platelet counts (thrombocytopenia) and blood clots (thrombosis) in some individuals. This extremely rare condition, with a rate between 1:50,000 – 1:350,000 cases per vaccinated individual, is above background rates of thrombosis in the population and can lead to fatal ischemic events including cerebral venous thrombosis, intracranial haemorrhage, and pulmonary embolism. It has been termed vaccine induced thrombotic thrombocytopenia (VITT) or thrombosis with thrombocytopenia syndrome (TTS).
Heparin induced thrombocytopenia (HIT) is another condition with a similar clinical presentation to TTS. In HIT, immuno‐aggregates are formed due to the presence of strong anti‐self‐antibodies directed against Platelet Factor 4 (PF4). When similar anti‐PF4 antibodies were detected in TTS patients, we investigated whether there could be a link between the adenovirus vectors used in the vaccines and PF4.
This study demonstrates a direct interaction between adenovirus capsids and PF4 using surface plasmon resonance. We then utilized an integrative structural biology workflow including cryo‐electron microscopy and molecular dynamics to characterize and demonstrate the mechanism of this interaction.
These results demonstrate a previously unknown adenovirus‐host interaction and provide critical clues as to the underlying mechanism which causes TTS, including how these pathogenic anti‐PF4 antibodies may be induced. We are therefore able to present a hypothesis as to the route of pathogenesis in TTS.
In vivo genetically engineered liver macrophages expressing tumor antigens and immunomodulatory molecules trigger adaptive immunity against tumors
1: San Raffaele Telethon Insitute for Gene Therapy (HSR‐TIGET)
In an effective adaptive immune response against cancer, tumor antigens (TAs), which are mostly released by apoptotic or dying cancer cells, are internalized, processed and presented by antigen presenting cells (APC) to T cells in lymph nodes. Upon priming and activation, T cells reach tumors and exert cytotoxic activity leading to cancer cell killing. However, in the presence of tumors, APC and T cell functions are impaired, thus limiting the efficacy of anti‐tumoral adaptive immune responses. Building on this observation, we developed a lentiviral vector‐based platform to activate TA‐specific T cells by enforcing TA and immunomodulatory molecule expression selectively in liver resident macrophages, termed Kupffer cells (KCs). We used ovalbumin (OVA) as a surrogate tumor antigen and found that gene‐based delivery of OVA to KCs by systemic LV administration promoted the generation of a high number of OVA‐reactive CD8 T cells. Upon delivery of high doses of LV, T cells showed an exhausted‐like phenotype, and were not found in circulation or in tumors. However, delivery of lower doses of LV, or co‐delivery of OVA with immunomodulatory molecules, partially reinvigorated CD8 T cells, leading to higher number of memory‐like cells, which reached the tumor and cleared cancer cells. Further studies are necessary to dissect the genetic profile of KC‐activated T cells and the mechanism(s) whereby these cells overcome the immunosuppressive tumor microenvironment, kill cancer cells and, due to their liver putative priming, may prevent the onset of liver metastasis.
A novel platform for engineered AAV‐based vaccines
1: Department of Ophthalmology, University Hospital, LMU Munich, 80336 Munich, Germany 2: Department of Veterinary Sciences, Ludwig‐Maximilians‐Universität München, Munich, Germany
The still ongoing pandemic has highlighted the unmet need for more innovative and rapidly adaptable vaccine platforms. Here, we introduce a novel vaccine platform which uses adeno‐associated virus (AAV) capsids as scaffolds for large immunogenic epitopes to induce strong and specific immune responses.
The structural viral proteins (VP) of AAVs are known to allow small peptide insertions in specific surface exposed regions. In order to allow for a better immune response, we introduced larger insertions of approximately 200 amino acids in the variable loop IV of the AAV2 or the AAV9 capsid. We produced and characterised AAV particles with or without genome. Empty, virus‐like particles (VLP) were administered intramuscularly in adult rabbits and tested for their ability to induce an immune response delivered against the immunogenic protein sequence presented on the capsid surface. The immunised rabbits showed elevated levels of binding and neutralising IgG antibodies against the administered antigen. Moreover, VLPs presenting a large SARS‐CoV‐2 antigenic protein on the capsid surface were efficiently neutralised by serum from Comirnaty mRNA‐vaccinated individuals. These VLPs also strongly activated T‐ and B‐cell responses in PBMCs of individuals vaccinated with mRNA‐based vectors as evident by the induction of CD4, CD8 and CD19 markers.
This next‐generation vaccine platform based on AAV capsids with large insertions of immunogenic sequences enables strong and specific immune responses without the need for genomically encoded immunogens, thus reducing the risk of potentially pathogenic intracellular processes associated with viral vector genomes and prolonged transgene expression.
Circumventing anti‐vector immunity towards adenoviral vectored vaccines
1: Cardiff University 2: InBio
Replication deficient (RD) adenoviruses (Ad) are the most widely administered viral vectors, with licensed SARS‐CoV‐2 vaccines using vectors derived from human Ad type 5 (Ad5) and 26 (Ad26), and chimpanzee Ad “ChAdOx1”. Ad vectored vaccines generate robust cellular and humoral immunity, against both the transgene‐encoded protein and the Ad vector itself. It's unclear how many times a single Ad vector can be re‐administered before this anti‐vector immunity impairs generation of the desired transgene‐specific adaptive responses. Anti‐vector immunity also arises from naturally acquired Ad infections. In the absence of anti‐Ad5 immunity, Ad5 is a gold‐standard vector with robust vaccine immunogenicity, however widespread Ad5 seroprevalence hampers its use as a vector for the global population.
We developed novel pseudotyped Ads as RD vectored vaccines encoding SARS‐CoV‐2 spike protein. These vectors exhibit fiber knob swaps from low seroprevalence Ads grafted onto an Ad5 backbone. We characterised innate immune responses following administration of these vectors in mice, and spike‐specific adaptive responses three weeks later. Furthermore, we quantified the effects of anti‐vector humoral immunity against these vectors in an in vitro transduction assay using human plasma. The pseudotyped vectors exhibit many desirable vaccine characteristics as the equivalent Ad5 vector, including CD4+ and CD8+ T cell responses against multiple spike epitopes. Importantly, fiber knob pseudotyping can substantially circumvent the direct, humoral, anti‐vector immunity induced through Ad exposure in humans. These data indicate the fiber knob plays an important role in anti‐vector immunity, and can be manipulated for evasion of such responses without hampering vaccine immunogenicity.
Virus‐cell interactions of the ChAdOx1 viral vector give insights into vaccine‐induced immune thrombotic thrombocytopenia
1: Division of Cancer & Genetics, Cardiff University School of Medicine 2: Medicines Discovery Institute, Cardiff University School of Biosciences 3: School of Pharmacy and Pharmaceutical Science, Cardiff University College of Biomedical and Life Sciences 4: Division of Infection and Immunity, Cardiff University School of Medicine
The ChAdOx1 nCoV‐19 vaccine (AZD1222/Vaxzervia) adapted from the chimpanzee adenovirus Y25 (ChAd‐Y25) has been critical in combatting the severe acute respiratory coronavirus 2 (SARS‐CoV‐2) pandemic. However, as part of the largest vaccination campaign in history, a potentially life‐threatening clotting disorder, thrombosis with thrombocytopenia, resembling heparin‐induced thrombocytopenia (HIT), has been observed in a minority of AZD1222 patients following the first but not the second dose. Vaccine‐induced immune thrombotic thrombocytopenia (VITT) is characterised by development of thromboses at uncommon sites such as the cerebral venous sinuses and the splanchnic veins, with concomitant thrombocytopaenia. Therefore, to determine how ChAdOx1 may contribute to this novel disorder, it is critical to investigate the vector‐host interactions of ChAdOx1. Structural and in vitro analysis of the fiber knob responsible for the primary virus‐cell interaction suggests that coxsackie and adenovirus receptor (CAR) is the primary ChAdOx1 receptor. However, ChAdOx1 infection of CAR(‐) human vascular endothelial cells has been demonstrated in vitro, suggesting ChAdOx1 may be using additional receptors. Dual tropism has been demonstrated in other human adenoviruses, with HAdV‐D26 and HAdV‐D37 both using sialic acid and CAR for transduction. Furthermore, coagulation factor X (FX), a factor demonstrated to bind to the hexon and facilitate human adenovirus type 5 (HAdV‐C5) transduction via a CAR‐independent pathway does not increase ChAdOx1 infection, with amino acid alignment between the hexon proteins suggesting ChAdOx1 is unable to bind FX. Taken together, these findings suggest ChAdOx1 uses additional as yet unknown mechanisms for transduction, which may further contribute to the pathogenesis of VITT.
Characterization of adenovirus interactions with PF4 using a novel ELISA‐qPCR technology
1: Witten/Herdecke University
The COVID vaccines Janssen and AstraZeneca, based respectively on adenovirus (AdV) serotypes AdV26 and ChAdOx1, have been associated with rare cases of vaccine‐induced thrombotic thrombocytopenia (VITT). It was recently demonstrated that the AdVs of the vaccines can bind to the blood protein platelet factor 4 (PF4), an interaction very likely to be involved in VITT.
Since there are hundreds of known AdV serotypes, we hypothesized that certain serotypes have a lower affinity for PF4. We therefore aimed to screen a library comprising dozens of serotypes from different AdV species. For this purpose, we established the ELISA‐qPCR technology. Like in standard ELISA, AdV viral particles are allowed to specifically interact with PF4 proteins coated on a plate. However, the revelation is not performed by antibody staining, but by qPCR after the genomes of bound AdVs are released through alkaline heat lysis. This technology enables fast, accurate and unbiased assessment of virus molecular interactions.
Unlike most tested serotypes, the species D AdV37, AdV69 and AdV70 did not bind to PF4. Even though the ELISA‐qPCR technique is not sensitive enough to detect potential low‐affinity interactions, these serotypes may avoid or decrease the risk of VITT and represent safer candidates for vaccine or gene therapy vector development.
In order to gain deeper insights into the mechanism of virion binding to PF4, we tested how AdV5 affinity for PF4 was affected by genetic removal or PEGylation of different hyper‐variable regions (HVR) of the hexon protein of the capsid.
Peptide Tools for the Development of Immunotherapies
1: JPT Peptide Technologies GmbH 2: 3B Pharmaceuticals GmbH
Cell‐ and immunotherapies are gaining tremendous attention as promising approach to fight cancer as well as infections. One of the main challenges with these novel treatments is the selection of optimal DNA / mRNA / protein / peptide sequence(s) to produce antigen‐specific immunotherapeutic agents. Among others, this task is largely hampered by sequence diversity in the target organisms as a result of mutations, isoforms, splice variants, polymorphisms, and post‐translational modifications.
In the present communication we describe a workflow that combines bio‐informatic algorithms to exploit known sequence information, an ultra‐high throughput peptide synthesis concept and synergistic high‐content assay formats. The process is applied to the establishment of efficient procedures for deep epitope discovery, immune monitoring and immunotherapy approaches.
Examples are presented for both major immune response mechanisms:
1. Humoral immune response: High‐density peptide microarrays provide an efficient parallel assay format and accommodate vast numbers of sequence variations for the characterization of antibody repertoires. Based on a novel sequence selection workflow, an HIV‐library with comprehensive proteome coverage was developed. Incubations of plasma samples with the respective microarrays allowed the detailed characterization of humoral immune responses for guidance of vaccine development and dose finding.
2. Cellular immune response: Peptides find widespread application to stimulate antigen specific T‐cell responses for immune monitoring and therapeutic applications. To increase stimulating efficiency complex antigen‐spanning overlapping peptide pools (PepMixTM and PepMixTM Ultra) were developed. Examples include the utilization of peptide pools for the generation of T cells against HIV and broad‐spectrum antiviral (AdV, EBV, CMV, BKV, HHV6) treatment.
Synthetic, enzymatically produced DNA for use in DNA vaccines
1: 4basebio 2: Neomatrix Biotech
4basebio osDNA™ is devoid of any plasmid backbone, and its manufacturing process circumvents cumbersome fermentation processes, allowing for the rapid production of GMP‐grade, linear DNA in less than a week. Moreover, the DNA can be modified to enhance its immunostimulatory properties, making it ideally suited for use in DNA vaccines.
In a collaboration with Takis Biotech and Neomatrix, we have demonstrated that osDNA™ constructs can elicit a meaningful immune response and slow tumour growth in an immunocompetent mouse model.
We synthesised osDNA™ constructs encoding the Neomatrix’ proprietary neoantigen cancer vaccine. Adult C57Bl/6 mice were vaccinated intramuscularly at day 0 and day 14 by DNA‐Electroporation (EP). On day 21, isolated PBMCs were stimulated overnight against a pool of neoantigen peptides and neoantigen‐specific immune responses were measured by flow cytometry. Mice vaccinated with osDNA™ constructs showed an increase in CD8+IFNγ+, CD8+TNFα+, and CD8+IFNγ+TNFα+.
One month post‐immunization, mice were challenged with 3x105 MC38 tumour cells. On day 9, osDNA™ vaccinated mice showed a significant reduction in tumour growth (*p < 0,05) compared to the control group. The percentage of tumour‐free mice vaccinated with osDNA™ was statistically significant compared to mice vaccinated with supercoiled pDNA at an equimolar ratio.
We have demonstrated that osDNA™ can facilitate dose reductions compared to plasmid DNA, whilst eliciting an enhanced immune response, correlating with a significant reduction in tumour growth and an increase in tumour‐free mice. The use of osDNA™ could greatly decrease the time required to generate personalised cancer vaccines encoding tumour specific neoantigens.