Abstract
Selected Oral Presentations
Eladocagene exuparvovec improves body weight and reduces respiratory infections in patients With aromatic l‐amino acid decarboxylase deficiency
1: National Taiwan University Hospital 2: PTC Therapeutics, Inc 3: PTC Therapeutics Swizterland, GmBH
Aromatic l‐amino acid decarboxylase (AADC) deficiency is caused by mutations in the dopa decarboxylase gene leading to reduced AADC enzyme activity. Patients with AADC deficiency often have feeding, swallowing, and gastrointestinal problems, which may contribute to low body weight. Upper respiratory tract infections (URTIs) and pneumonia are major causes of morbidity in these patients. Eladocagene exuparvovec is a recombinant adeno‐associated viral vector serotype 2 carrying the coding sequence for the human AADC gene.
Eladocagene exuparvovec was bilaterally infused into the putamena of 28 children with AADC deficiency in 3 clinical trials (AADC‐CU/1601 [8 patients, completed], AADC‐010 [10 patients, completed], and AADC‐011 [10 patients at 26 Feb 2020 cutoff date, ongoing]). Patients received 1.8 × 1011 vg (n = 21) or 2.4 × 1011 vg (n = 7; AADC‐011)]. Body weight was measured at baseline and at 12‐month follow‐up and compared with age‐ and gender‐matched values for children without AADC deficiency. Rate of URTI/pneumonia was measured annually for 5 years after therapy.
At baseline, most patients (83.3%, 20/24) had a body weight ≤3rd percentile. At 12 months, 95.9% maintained or gained weight relative to age‐ and gender‐matched children without AADC deficiency; 42% (10/24) shifted to a higher percentile, and 54% (13/24) maintained the same percentile as at baseline. The annual rate of URTI/pneumonia decreased from 2.41 at 1 year after treatment to 0.31 at 5 years after treatment.
These results demonstrate the efficacy of eladocagene exuparvovec in improving body weight and reducing respiratory infections in patients with AADC deficiency.
Inclusion of a degron reduces the levels of undesired inteins after AAV‐mediated protein
trans
‐splicing in the retina
1: Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli 80078, Italy 2: Medical Genetics, Department of Translational Medicine, Federico II University, Naples 80131, Italy 3: Eye Clinic, Multidisciplinary Department of Medical, Surgical and Dental Sciences, University of Campania L. Vanvitelli, Naples 80131, Italy 4: Medical Genetics, Department of Advanced Biomedicine, Federico II University, Naples 80131, Italy
Retinal AAV gene therapy is emerging as the leading approach to treat different forms of inherited blindness. However, the relatively small DNA packaging capacity of AAVs prevents their application to diseases due to mutations in genes with large coding sequences, such as ABCA4, the gene mutated in Stargardt disease (STGD1) which is the most common form of inherited macular degeneration in humans. Recently, intein‐mediated protein trans‐splicing (PTS) has been evaluated as a strategy to reconstitute large proteins via AAV vectors. However, non‐mammalian inteins persist as trans‐splicing by‐products, and this raises safety concerns for clinical application of AAV‐intein. In this study, we tested the ability of several degrons to selectively decrease levels of inteins after PTS in vitro and found that a version of E. coli Dihydrofolate reductase, shortened to better fit into AAV, is the most effective. AAV‐intein vectors including the degradation signal were co‐delivered to either mouse or pig retina and resulted in efficient full‐length ABCA4 reconstitution in the absence of significant intein persistence. Lastly, we show that subretinal administration of AAV intein armed with this short degron is both safe and effective in a mouse model of STGD1. This supports the use of optimized AAV‐intein for gene therapy of both STGD1 and other conditions which require transfer of large genes.
The Phase III REFLECT Trial: Efficacy and Safety of Bilateral Gene Therapy for Leber Hereditary Optic Neuropathy (LHON)
1: Cambridge Centre for Brain Repair and MRC Mitochondrial Biology Unit, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK. 2: Cambridge Eye Unit, Addenbrooke's Hospital, Cambridge University Hospitals, Cambridge, UK. 3: Moorfields Eye Hospital, London, UK. 4: UCL Institute of Ophthalmology, University College London, London, UK. 5: Sue Anschutz‐Rodgers University of Colorado Eye Center, University of Colorado School of Medicine, Aurora, CO, USA 6: Departments of Neurology and Ophthalmology, Wills Eye Hospital and Thomas Jefferson University, Philadelphia, PA, USA 7: Department of Ophthalmology, Taipei Veterans General Hospital, National Yang Ming Chiao Tung University, Taipei, Taiwan 8: Department of Ophthalmology, Ghent University Hospital, Ghent, Belgium 9: Department of Head & Skin, Ghent University, Ghent, Belgium 10: Division of Ophthalmology & Center for Cellular & Molecular Therapies, Children's Hospital of Philadelphia, Philadelphia, PA, USA 11: Department of Ophthalmology and Visual Sciences at Vanderbilt Eye Institute, Nashville, TN, USA 12: Monroe Carell Jr Children Hospital at Vanderbilt University, Nashville, TN, USA 13: Vanderbilt University Medical Center, Nashville, TN, USA 14: Departments of Ophthalmology, Neurology and Neurological Surgery, Emory University School of Medicine, Atlanta, GA, USA 15: IRCCS Istituto delle Scienze Neurologiche di Bologna, UOC Clinica Neurologica, Bologna, Italy 16: Unit of Neurology, Department of Biomedical and Neuromotor Sciences (DIBINEM), University of Bologna, Bologna, Italy 17: Department of Neuro Ophthalmology and Emergencies, Rothschild Foundation Hospital, Paris, France 18: Centre Hospitalier National d'Ophtalmologie des Quinze Vingts, Paris, France 19: Doheny Eye Institute / UCLA School of Medicine, Los Angeles, CA, USA 20: Ophthalmologyst at Ramon y Cajal Hospital, Madrid, Spain 21: Massachusetts Eye and Ear Infirmary, Boston, MA, USA 22: Department of Ophthalmology, Icahn School of Medicine at Mount Sinai, Nyew York, NY, USA 23: GenSight Biologics, Paris, France 24: Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France 25: Fondation Ophtalmologique A. de Rothschild, Paris, France 26: Department of Ophthalmology, The University of Pittsburgh School of Medicine, Pittsburgh, PA, USA 27: CHNO des Quinze‐Vingts, Institut Hospitalo‐Universitaire FOReSIGHT, INSERM‐DGOS CIC 1423, Paris, France
Leber Hereditary Optic Neuropathy (LHON) is an orphan blinding disease leading to bilateral vision loss. Lenadogene nolparvovec (rAAV2/2‐ND4) is a gene therapy developed for the treatment of LHON caused by a mutation in the ND4 gene.
REFLECT is a Phase III trial that enrolled 98 LHON subjects with the G11778A‐ND4 mutation and duration of vision loss ≤12 months. Each subject received an injection of lenadogene nolparvovec in their first‐affected eye, and either gene therapy or placebo in their second‐affected eye.
Forty‐eight subjects were treated bilaterally and 50 were treated unilaterally. At Week 78, the difference in BCVA improvement between second‐affected eyes was equivalent to +3 ETDRS letters in favor of lenadogene nolparvovec. The eyes treated with lenadogene nolparvovec showed a significant improvement from nadir of +19 and +16 ETDRS letters for the first and second affected eyes, respectively (p < 0.0001), compared to +13 ETDRS letters in eyes treated with placebo (p < 0.0001). There was a trend towards subjects treated bilaterally having a better final visual acuity than subjects treated unilaterally (+5 letters). The favorable safety profile of the gene therapy was confirmed in both unilaterally and bilaterally treated patients.
At 1.5 years post administration, a statistically significant improvement of BCVA was demonstrated from baseline for treated eyes and from nadir for all eyes. As in the RESCUE and REVERSE trials, a contralateral effect was reported in the placebo‐treated eyes. The results also point towards a dose effect with bilateral injection of lenadogene nolparvovec, supporting the benefit of bilateral treatment in LHON.
Frequent Aneuploidy in Primary Human T Cells Following CRISPR‐Cas9 cleavage
1: Tel Aviv University 2: Dotan Center for Advanced Therapies 3: Meir Medical Center
Multiple ongoing clinical trials use site specific nucleases to disrupt T cell receptor (TCR) genes in order to allow for allogeneic T cell therapy. In particular, the first U.S. clinical trial using CRISPR‐Cas9 entailed the targeted disruption of the TCR chains and programmed cell death protein 1 (PDCD1) in T cells of refractory cancer patients.
Here, we used the same guide RNA sequences and applied single cell RNA sequencing to primary human T cells, 4 days after CRISPR‐Cas9 transfection. Importantly, we found a high correlation between the chromosomal location of the targeted gene and the resulting aberration. In particular, cleaving the TCRα locus, near the chromosome 14 centromere, leads to functional aneuploidy, with up to 9% and 1.4% of the cell having a chromosome 14 loss or gain, respectively. Cleaving the TCRβ locus, in the middle of the chromosome 7 q‐arm, leads to the truncations in 9.9% of the cells, while cleaving the PDCD1 gene, near the chromosome 2 q‐arm telomere, expectedly has little effect on global gene expression. Loss of heterozygosity was further validated using fluorescent in situ hybridization and the temporal dynamics of cleavage and incomplete repair was monitored using digital droplet PCR.
We conclude that aneuploidy and chromosomal truncations are underappreciated, yet frequent, outcomes of CRISPR‐Cas9 cleavage in clinical protocols. Monitoring and minimizing these aberrant products is crucial for future applications of genome editing in T cell engineering and beyond.
Improved non‐viral CAR‐reprogramming of human T cells using CRISPR‐Cas and double‐stranded DNA
1: Charité University Medicine 2: Berlin Center for Advanced Therapies (BeCAT) 3: Berlin Institute of Health (BIH) Center for Regenerative Therapies 4: Integrated DNA Technologies, Inc.
In recent years, CAR T cells have become a potent treatment option for certain hematological malignancies. Therapeutic CAR transgenes are commonly delivered using viral vectors. Classical retroviral approaches have a random mode of transgene integration which imposes a risk for insertional mutagenesis. In order to achieve site‐specific integration of CAR‐transgenes, Eyquem et al combined adeno‐associated viruses with CRISPR‐Cas. Edited T cells which expressed the CAR from the TCR alpha chain constant locus (TRAC) showed improved functionality over their retrovirally transduced counterparts.
However, clinical‐grade viruses for gene transfer are associated with extensive costs. Following up on a study by Roth et al, we recently provided an improved virus‐free method for efficient CAR insertion into the TRAC locus of primary human T cells using CRISPR‐Cas and double‐stranded DNA. Previous studies (Roth et al, Schober et al) showed moderate editing efficiencies up to 15% when inserting tumor‐specific receptors. By pharmacological modulation of DNA sensing and repair pathways, we were able to dramatically increase editing outcomes with TCR‐to‐CAR replacement rates exceeding 50%. Single‐molecule real‐time sequencing showed homology‐independent integration of the DNA template at the on‐target locus in <1% of the edited cells.
TRAC‐replaced CAR T cells show antigen‐specific cytotoxicity and cytokine production in vitro. They show no alloreactivity and are less prone to exhaustion than lentivirally transduced control CAR T cells.
Thus, we provide a GMP‐compatible non‐viral platform technology that lays the foundation for clinical trials and fast‐track generation of novel CAR T cells applicable for autologous or allogeneic off‐the‐shelf use.
Expression of a pathogenic virulence factor enhances the efficacy of CAR‐T cell therapy against solid tumors
C Jin1 J Ma1 M Ramachandran1
1: Uppsala University
Chimeric antigen receptor engineered T cells (CAR‐Ts) targeting pan‐B cell marker CD19 are effective against hematologic malignancies. However, the heterogeneous expression of CAR target antigen and local immunosuppression impedes their clinical implementation in solid tumors. Here, we report expressing a pluripotent proinflammatory neutrophil‐activating protein (NAP) from Helicobacter pylori in CAR‐Ts drastically improved the CAR‐T therapy in solid tumors, and it is independent of tumor types and the targeted antigens. We first showed in several in vivo models with either endogenous or artificial‐expressed tumor‐associated antigens, CAR(NAP)‐Ts are more successful in reducing tumor growth and enhancing the survival of mice compared to conventional CAR‐Ts, even though they have similar killing capacity in vitro. Furthermore, in a mixed tumor model where CAR‐target antigen was expressed heterogeneously, CAR(NAP)‐Ts are also more effective compared to conventional CAR‐Ts. The therapeutic superiority of CAR(NAP)‐Ts are largely relying on CAR(NAP)‐T cell treatment can create an immunologically hot microenvironment, supporting dendritic cell maturation and induced bystander immunity via epitope spreading. This is further confirmed by a higher number of cytolytic proliferative OVA‐specific CD8+ T cells infiltration into tumor after CAR(NAP)‐T treatment targeting CD19, when evaluated in mice with an established solid tumor expressing both CD19 and Ova. Finally, we also confirmed that human T‐cell engineered with NAP also exert enhanced efficacy in various in vitro co‐culture assays. We believe the reported CAR(NAP)‐T platform warrants translation into the clinic and may facilitate efforts to explorer new non‐host derived immunomodulating factors to improve CAR‐T therapy.
CAR‐Tregs for the treatment of Systemic Lupus Erythematosus
1: Experimental Hematology Unit, Division of Immunology Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy 2: Innovative Immunotherapies, Division of Immunology Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy 3: Autoimmunity and Vascular Inflammation Unit, Division of Immunology Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy
Systemic Lupus Erythematosus (SLE) is characterized by an autoimmune response against nuclear antigens in which auto‐reactive B cells have a fundamental role. Regulatory T cells (Tregs) preserve the immune tolerance and are dysfunctional in SLE. Adoptive transfer of polyclonal Tregs proved unable to restore the immune tolerance in autoimmune diseases due to a low frequency of antigen‐specific cells. Chimeric Antigen Receptors (CARs) are capable of redirecting T cell specificity against target antigens. CAR‐Tregs proved effective in controlling the inflammation in mouse models of autoimmune diseases different from SLE.
We aim at employing the CAR technology to redirect Treg activity to restore immune tolerance in Systemic Lupus Erythematosus.
We sorted CD4+CD25+ cells from peripheral blood of Healthy Donors. We expanded Tregs for 14 days using anti‐CD3/CD28 magnetic beads, IL‐2 and rapamycin. To target B cells, we transduced CAR‐Tregs with a second‐generation anti‐CD19 CAR. GFP was employed as transduction marker. The functionality of engineered cells was evaluated with flow cytometry.
We efficiently generated CAR‐Tregs, which suppressed the proliferation of autologous T lymphocytes upon polyclonal stimulation. Noticeably, only engineered cells suppressed autologous B cell proliferation, demonstrating an antigen‐specific suppression capacity, without killing their targets. More strikingly, anti‐CD19 CAR‐Tregs suppressed conventional anti‐CD19 CAR‐T cell killing when co‐cultured together in the presence of a CD19+ tumor cell line. Finally, when tested in a humanized mouse model of SLE, only anti‐CD19 CAR‐Tregs delayed the occurrence of B cell leukopenia.
We proved the efficacy of anti‐CD19 CAR‐Tregs for B cell autoimmune diseases both in vitro and in vivo.
A phase 1/2 study of lentiviral‐mediated e
x‐vivo
gene therapy for pediatric patients with severe leukocyte adhesion deficiency‐I (LAD‐I): Interim results
1: Infection, Immunity, & Inflammation Department, UCL Great Ormond Street Hospital (GOSH) Institute of Child Health, London, United Kingdom 2: University of California, Los Angeles, Los Angeles, CA 3: Hematología y Hemoterapia, Fundación para la investigación Biomédica HIUNJ, Hospital Infantil Universitario Niño Jesús, Madrid, Spain and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER‐ISCIII), Madrid, Spain 4: Rocket Pharmaceuticals, Inc., Cranbury, NJ 5: Division of Hematopoietic Innovative Therapies, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER‐ISCIII), Madrid, Spain 6: Advanced Therapies Unit, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS‐FJD/UAM), Madrid, Spain
Leukocyte Adhesion Deficiency‐I (LAD‐I) is a rare disorder of neutrophil adhesion resulting from ITGB2 mutations encoding for the β2‐integrin component, CD18. Severe LAD‐I (CD18 < 2% of PMNs) is characterized by severe infections, impaired wound healing, and childhood mortality. Allogeneic hematopoietic stem cell transplant (alloHSCT) is potentially curative; efficacy is limited by donor availability and graft‐versus‐host disease (GVHD). A phase 1/2 open‐label trial is underway, employing autologous CD34+ cells transduced with a lentiviral vector (LV) carrying the ITGB2 gene (cDNA). Pediatric patients ≥3 months old with severe LAD‐I are eligible. Mobilized peripheral blood (PB) HSCs are collected via apheresis and transduced with the LV. Myeloablative therapeutic drug monitoring (TDM) busulfan conditioning precedes RP‐L201 infusion. Endpoints include safety, survival, and increase in PMN CD18 expression to ≥10%.
Seven patients (ages 5mos‐9yrs) have received RP‐L201; all with follow‐up ≥3 to 18 months. RP‐L201 cell doses ranged from 2.8x106 to 6.5x106 CD34+ cells/kg with VCN from 1.8‐3.8 copies/cell. No serious RP‐L201 related treatment‐emergent adverse events were reported. PB PMN CD18 expression in Patient 1 at 18‐months post‐treatment was 40% (vs. <1% at baseline), with PB VCN of 1.44. Baseline skin lesions resolved with no new lesions reported. CD18 expression in the subsequent 6 patients has been 25‐80% in neutrophils and stable for each patient from 3 to up to 9 months post‐treatment. No new infections have been reported in patients post‐infusion.
RP‐L201 leads to durable neutrophil CD18 expression and improved clinical course. Additional patient treatment is ongoing in 2021.
Investigational liver gene transfer of C1‐INH for the treatment of Hereditary Angioedema
1: Spark Therapeutics
Hereditary angioedema (HAE) is an autosomal dominant genetic disorder characterized by episodic attacks of acute angioedema that can have severe and sometimes fatal consequences. HAE is caused predominantly by deficiency in the first component of complement inhibitor (C1‐INH) that leads to aberrant production of bradykinin, driving painful and unpredictable episodes of subcutaneous or submucosal swelling. Treatment with C1‐INH‐replacement therapy increases survival, reduces attack frequency and severity, and is a current standard of care for HAE disease patients. However, current treatment modalities have several significant drawbacks such as the potential for breakthrough attacks, safety/tolerability, high patient burden, and potential for limited compliance, highlighting the need for new treatment options. Gene transfer for hereditary angioedema offers the potential for a one‐time therapeutic intervention with the potential to overcome some of these drawbacks. Here we show that a novel investigational liver‐directed adeno‐associated viral (AAV) gene therapy with an optimized C1‐INH encoding transgene results in sustained, dose‐dependent, circulating levels of C1‐INH in mice. Additionally, we show that AAV‐mediated C1‐INH expression in C1‐INH null mice both reduces bradykinin (the mediator of HAE attacks), stabilizes levels of circulating C4 (a diagnostic marker of HAE) and exhibits functional inhibition of serine proteases in the coagulation pathway. A single infusion of SPK‐10000 in non‐human primates at three ascending doses demonstrated dose‐dependent expression of functional hC1‐INH in plasma at therapeutically relevant levels. Taken together, our results support further evaluation of liver gene transfer with AAV‐hC1‐INH in the clinical setting.
CRISPR‐mediated genome editing to redirect T cells against Non‐Small Cell Lung Cancer
1: 1Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy 2: 2Department of Medical and Surgical Sciences for Children & Adults, University‐Hospital of Modena and Reggio Emilia, Modena, Italy
Adoptive therapy with engineered T cells to express tumour‐specific TCR to induce antitumor immunity, represents one the most promising strategy of precise cancer therapy for solid tumours. Clinical trials with TCR‐engineered T cells are ongoing to treat lung cancers, the leading cause of cancer‐related death worldwide. CRISPR‐mediated non‐viral TCR editing has been recently proposed to replace endogenous TCRs with tumour‐specific TCRs. In this study, we applied this strategy to engineer T cells derived from patients affected by non‐small‐cell lung cancers (NSCLC), to induce killing of cancer cells. A cohort of NSCLC patients was firstly HLA typed and analysed for the expression of tumour antigens frequent in NSCLC. Guide RNAs in complex to Alt‐R SpCas9 were delivered as ribonucleoproteins (RNPs) into primary T cells, to knock out endogenous TCR chains and simultaneously trigger the knock‐in of tumour‐specific TCR in the TRAC locus under the control of endogenous regulatory elements. Genomic and cytofluorimetric analyses showed highly efficient knock‐out of endogenous TCR, and precise HDR events leading to TCR replacement. Co‐culture experiments of TCR‐redirected T cells with tumour cell lines demonstrated that edited T cells were able to recognize and kill cells expressing the cognate tumour peptide. TCR‐engineered T cells derived from HLA‐A*0201‐typed NSCLC patients co‐cultured with NSCLC cells and analysed by flow cytometry for their killing activity, demonstrated that TCR editing enabled T cells to recognize and kill NSCLC cells. These data encourage the application of non‐viral TCR editing to generate tumour‐specific T cells able to kill lung cancer cells.
Consecutive genome editing in non‐human primate achieves durable production of human alpha‐1 antitrypsin at physiologic levels and reduction of the homologous native protein
C Boiselle1 P Sharma1 T Das1 K Zheng1 D Lizardo1 C Moroski‐Erkul1 T Yangdon1 E Kollarova1 V Doshi1 R Cole1 N Kolluri1 S Jaligama1 H Sampath1 D Kim1 S Soukamneuth1 S Parthasarathy1 A Whynot1 S Williams1 L Shaw1 A Amaral1 M Roy1 M Young1 D Guan1 N Gardner1 Y Xu1 L Sepp‐Lorenzino1 J Seitzer1
1: Intellia Therapeutics, Inc.
Using CRISPR/Cas9, we have developed a modular in vivo genome editing platform to knockout and/or insert genes within the liver. For alpha‐1 antitrypsin deficiency (AATD), both knockout and insertion may be beneficial. Mutations in the SERPINA1 gene cause pulmonary pathology due to deficiency of the alpha‐1 antitrypsin (AAT) protein. Additionally, in some AATD patients, liver pathology can ensue due to aggregation of mutant AAT protein. As proof‐of‐concept for a treatment addressing both disease manifestations, we performed consecutive genome editing in a non‐human primate (NHP) model. To knockout endogenous SERPINA1 expression, we used lipid nanoparticles (LNPs) to deliver Cas9 and a guide RNA targeting the gene. After 4 weeks, we saw significant reduction in NHP AAT protein. To express functional human AAT, we inserted a construct encoding SERPINA1 into the albumin locus, using LNPs to deliver Cas9 and a guide RNA targeting intron 1 of the albumin gene, along with adeno‐associated virus to deliver a promotorless transgene template. Precise insertion downstream of the albumin promoter led to production of AAT protein within the normal physiologic range, stable for over 38 weeks in an ongoing study, without impacting albumin expression. These results demonstrate that our in vivo CRISPR/Cas9 platform can address both manifestations of AATD through gene knockout or insertion, either individually or in combination. Our results further highlight the power of our modular gene insertion approach to produce durable, high‐level expression of therapeutic proteins, beyond what has been achieved using traditional gene therapy.
MMEJ‐mediated IDLV knock‐in via CRISPR/Cas9 in human hematopoietic stem/progenitor cells
1: Genethon, UMR_S951, Inserm, Univ Evry, Université Paris Saclay, EPHE
Ex‐vivo gene correction of hematopoietic stem and progenitor cells (HSPCs) represents as a promising therapeutic approach for inherited human blood disorders. Nuclease mediated DNA targeted integration (TI) is currently achieved via homologous recombination of AAV delivered donor DNA (HDR), a repair pathway that is inefficient in HSPCs.
Here, we describe a novel micro‐homology mediated joining (MMEJ) based platform for CRISPR‐Cas9 mediated TI of integrase defective lentiviral vector (IDLV). This platform can work in HSPCs, as MMEJ is active in all cell cycle phases, and allow integration of a bigger DNA cargo size (∼9kb of IDLV compared to ∼4.7kb of AAV). As proof of concept, a reporter gene was targeted to clinically relevant genomic loci (HBA, IL2RG) and safe harbor site (AAVS1) using IDLV and gRNA/Cas9 ribonucleoprotein complexes in K562 and T‐cell lines. After investigating the TI mechanism and optimizing different parameters (such as timing of IDLV transduction and DNA homology length), we demonstrated efficient TI and transgene expression by PCR and FACS analysis. Compared to NHEJ mediated IDLV TI, the presence of micro‐homology favored on‐target integration, correct cassette orientation and seamless TI. Efficient reporter TI and expression was observed also in human HSPCs for both HBA and AAVS1 loci. Finally, we demonstrated the potential therapeutic application of this strategy by integrating IL2RG transgene to correct SCID‐X patient's derived T‐cells.
Overall, our study demonstrates that MMEJ‐mediated TI represents an effective alternative to HDR‐based TI in hematopoietic cells and primary HSPCs.
Preclinical development of TALEN®‐based genome editing in T cells for the treatment of Hyper‐IgE‐Syndrome
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: Cellectis Inc, New York, NY 10016, USA 4: Institute of Medical Bioinformatics and Systems Medicine, Medical Center – University of Freiburg, Freiburg, 79110, Germany 5: German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Partner Site Freiburg, Freiburg, 79106, Germany 6: Institute for Immunodeficiency, Medical Center – University of Freiburg, Freiburg, 79106, Germany 7: Cellectis S.A., Paris, 75013, France
Hyper‐IgE‐syndrome (HIES) is a rare immunodeficiency characterized by recurrent skin and pulmonary abscesses, elevated IgE serum levels, and a severe impact on the quality of life in general. Disease‐causing mutations in HIES patients are mainly found in exons 10‐24 of the tightly regulated STAT3 locus, which is expressed in two isoforms, a STAT3a oncogene and a STAT3b tumor suppressor. These mutations affect dimerization or DNA binding of STAT3 and result, inter alia, in the failure of naïve T cells to differentiate to Th17 cells. This lack of IL‐17 and IL‐22 secreting cells renders HIES patients highly susceptible to infections. Due to the risk of graft‐versus‐host disease allogeneic hematopoietic stem cell transplantation is not indicated. In order to provide HIES patients with a therapy, we focused on genetically correcting long‐lived patient‐derived T cells. To restore expression of both STAT3 isoforms under control of the endogenous promoter, we used TALEN® technology to target integration of a smart STAT3 donor cassette comprising exons 10‐24 into intron 9. Analyses of edited healthy donor or patient‐derived T cells confirmed high activity of the employed TALENs® (>85% of edited alleles) and high targeted integration frequency (at ∼50% of alleles). STAT3 transgene expression was observed in ∼50% of long‐lived T cells, and alternative splicing from the smart transgene cassette was maintained at the appropriate STAT3a to STAT3b ratio. Furthermore, upon activation of the T cells, we observed the expression of STAT3 downstream targets, such as SOCS3 and IL‐17, thus highlighting the therapeutic potential of our strategy.
Tim‐3, LAG‐3 and 2B4 disruptions differentially regulate anti‐tumor response of TCR gene edited memory stem T cells
1: Experimental Hematology Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, 20132, Italy 2: Innovative Immunotherapies, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, 20132, Italy 3: Gene transfer technologies and new gene therapy strategies Unit, San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, 20132, Italy 4: Bone‐Marrow Transplantation Unit, San Raffaele Scientific Institute, Milan, 20132, Italy 5: San Raffaele Telethon Institute for Gene Therapy (HSR‐TIGET)
Exhausted T cells infiltrating tumors express multiple inhibitory receptors (IRs) that are exploited by cancer cells to evade immune attacks. Monoclonal antibodies blocking IR pathways proved successful in selected tumors but cause unwanted autoimmune manifestations because they unleash T cell responses on the entire T cell repertoire, including autoreactive clones.
We aim at simultaneously redirecting T cell specificity by TCR gene editing and permanently disrupting IRs by CRISPR/Cas9 system in long‐living memory stem T cells (TSCM) for adoptive cell therapy.
With CRISPR/Cas9, we simultaneously inactivated the endogenous TCR (>98% of NHEJ) and LAG‐3, Tim‐3 or 2B4 genes with an efficiency >80%. TCR disrupted‐IR‐KO cells were efficiently transduced (> 50%) with a lentiviral vector encoding for a NY‐ESO1157‐165‐specific TCR and retained high expansion capacity and an early differentiated TSCM/TCM phenotype.
Upon chronic stimulation with multiple myeloma cells, we observed that the disruption of Tim‐3 or 2B4 genes sustain a higher degranulation capacity in TCR‐edited T cells, while LAG‐3 disruption prevents the upregulation of additional inhibitory receptors.
In immunodeficient murine models of aggressive multiple myeloma, TCR‐edited‐Tim3‐KO and TCR‐edited‐2B4‐KO T cells caused higher immunological pressure and were enriched in a higher proportion of highly activated T cells than TCR‐edited‐IRCOMP‐cells, while TCR‐edited‐LAG‐3‐KO, but not TCR‐edited‐IRCOMPcells efficiently eliminated cancer cells upon tumor rechallenge.
Overall, we generated innovative tumor‐specific cellular products resistant to inhibitory signals by means of CRISPR/Cas9 reagents and we found that multiple IRs differentially enhance anti‐tumor responses.
Clinical feasibility and treatment outcomes with unselected autologous tumour‐infiltrating lymphocyte (TIL) therapy in patients with advanced cutaneous melanoma
1: Instil Bio, Inc. 2: The Christie, NHS Foundation Trust 3: Division of Cancer Sciences, University of Manchester
The intrinsic antitumour activity and broad neoantigen‐specific T‐cell receptor repertoire of unselected autologous TILs may provide advantages over other treatments in advanced melanoma. In this retrospective analysis of a single‐centre experience, patients with advanced cutaneous melanoma and no other treatment options received lymphodepleting chemotherapy (cyclophosphamide × 2 days, fludarabine × 5 days [Cy/Flu]), followed by TIL infusion and post‐TIL high‐dose IL‐2 on a compassionate use basis. Efficacy was reported by investigator assessment of CT/MRI per RECIST v1.1; additional patients were followed using non‐RECIST v1.1 imaging (PET) and clinical monitoring. Clinically significant adverse events (AEs) were reported. Between October 2011 and August 2019, 21 patients were treated. With a median follow‐up of 52.2 months, the overall response rate (ORR) in imaging‐evaluable patients (n = 15) was 53% (13% complete response rate). Durable responses were also observed in patients followed by PET and clinical monitoring (n = 6). Responses were generally consistent across subgroups, including brain metastases and prior PD‐1, BRAF, and MEK inhibitor. Median survival was 21.3 months for all treated patients. AEs were generally self‐limited and consistent with Cy/Flu and high‐dose IL‐2. Common any‐grade AEs were thrombocytopenia (62%), pyrexia (57%), and rigors (43%); no treatment‐related deaths were observed. The high response rate observed in this series highlights the successful bench‐to‐bedside application of unselected autologous TILs to address unmet medical need in advanced melanoma. Use of tumour digests as starting material for manufacturing of TILs demonstrates the feasibility of this approach. A multicentre phase 2 trial of this therapy in advanced melanoma is planned for 2021.
Physiological (TCR‐like) regulated lentiviral vectors for the generation of improved CAR‐T cells
1: Pfizer‐University of Granada‐Junta de Andalucía Centre for Genomics and Oncological Research (GENYO) 2: LentiStem Biotech 3: Maimonides Institute of Biomedical Research in Córdoba (IMIBIC), Cellular Therapy Unit, Reina Sofia University Hospital, University of Córdoba, Córdoba, Spain 4: Department of Hematology, ICMHO, Hospital Clínic de Barcelona, Spain. 5: Biopathology and Regenerative Medicine Institute (IBIMER), Centre for Biomedical Research (CIBM), University of Granada, Spain
CAR T cells against CD19 have achieved impressive outcomes for the treatment of relapsed/refractory B lineage neoplasms. However, important limitations remained due to severe side effects and lack of efficiency in 40‐50% of the patients. Most CAR‐T products are generated using retroviral vectors with strong promoters that lead to high CAR expression levels, tonic signaling, premature exhaustion and over‐stimulation, reducing therapy efficacy and increasing side effects. TCR‐like expression of the CAR through genome editing resulted in enhanced anti‐tumor potency, reducing tonic signaling and improving CAR‐T phenotype. Here, we searched for LVs that mimic the TCR expression pattern as a closer‐to‐clinic alternative for the generation of improved CAR‐T cells. Our data showed that LVs expressing the transgene through a WAS gene chimeric promoter (AW) mimic closely the TCR/CD3 expression pattern kinetic upon antigenic stimulation. We then compared second generation αCD19‐4‐1BB‐CAR generated with standard EF1α‐driven LVs (ARI‐0001), versus those generated with AW‐LVs (AWARI). AWARI‐CAR‐T cells exhibited a higher proportion of naïve/stem cell memory T cells and less exhaustion after efficient in vitro and in vivo killing of CD19+ cells. AWARI‐CAR‐T cells also showed lower tonic signaling and reduced secretion of pro‐inflammatory cytokines. In adition, AWARI‐CART cells showed enhanced tumor lysis against CD19+ pancreatic cancer cells. We finally demonstrated the feasibility of large‐scale manufacturing of AWARI‐CAR‐T cells in GMP‐like conditions. In summary, we propose the use TCR‐like LVs based on the WAS‐gene promoter as an alternative to strong‐promoter for the generation of CAR‐T products with lower tonic signaling, improved phenotype and safer profile.
A novel strategy for off‐the‐shelf T cell therapies evading host T cell and NK cell rejection
1: Intellia Therapeutics, Inc
Despite the clinical success of autologous chimeric antigen receptor (CAR) T cells, barriers to more widespread use of this potentially curative therapy include manufacturing challenges and the high cost of individualized production, leading to a strong desire for “off‐the‐shelf” engineered cell therapies. Here, we report on an allogeneic T cell strategy leveraging Intellia's CRISPR/Cas9 platform and innovative sequential gene‐editing process. By knocking out select HLA class I and class II genes, the engineered cells were not recognized by host CD4+ and CD8+ T cells from different donors. Edited T cells were also protected from host NK cells, both in vitro and in an in vivo model engrafted with functional human NK cells. T cell receptor (TCR) edited donor T cells did not induce GvHD in an immune‐compromised mouse model over the 90‐day evaluation period. Using an improved T cell engineering process, we successfully generated allogeneic T cells with multiple sequential KOs and insertion of a tumor‐specific TCR or CAR with high yield. Importantly, these allogeneic T cells had comparable functional activity to their autologous T cell counterparts in in vitro assays. In summary, we have successfully developed a differentiated “off‐the‐shelf” approach for CAR‐T and TCR‐T cell therapies, which is a key step towards a safe, cost‐effective, and durable therapy that avoids long‐term or aggressive lymphodepletion. This promising strategy is being applied to our ex vivo pipeline candidates.
Pre‐clinical proof of concept of an AAV5‐GLA gene therapy for Fabry disease resulting in cross‐correction in GLA‐KO mice and non‐human primates in target organs
1: uniQure biopharma B.V. 2: Leiden University 3: University of Amsterdam
Fabry disease is an X‐linked hereditary metabolic disorder due to mutations in the alpha‐galactosidase A (GLA) gene. Deficiency of GLA‐enzyme leads to a constant accumulation of globotriaosylceramide Gb3 and its deacylated derivative globotriaosylsphingosine (lysoGb3) in cells and plasma, resulting in cell abnormalities and organ dysfunction affecting heart, kidney and brain. Current enzyme‐replacement therapy has limited therapeutic effects, mainly because of poor cross‐correction, revealing an unmet medical need.
We explore the use of AAV gene therapy expressing GLA under control of a proprietary strong liver‐specific promoter and a low immunogenic AAV serotype 5. Injection of AAV5‐GLA vector at different dosages in GLA‐knockout mice showed significant dose dependent increase in GLA‐activity and lowering of (lyso)Gb3 in plasma and target organs, such as liver, kidney, heart and brain. Tissue staining confirmed the presence of GLA‐protein and reduction of lipid accumulation. Moreover, image quantification showed a dose dependent increase of GLA‐protein in kidney glomeruli. Additionally, already 10 weeks after AAV5‐GLA injection GLA‐knockout mice significantly improved phenotypically on nociception.
In non‐human primates (NHP), administration of AAV5‐GLA was well‐tolerated and did not show formation of anti‐GLA antibodies. One‐time AAV5‐GLA treatment in NHP resulted in 30‐times elevated GLA‐activity levels in plasma. Also, the target organs had increased GLA‐activity above basal levels, indicating cross correction capability of the AAV5‐GLA vector. Thus, the phenotypic improvement in GLA‐knockout mice, sustained GLA expression with cross‐correction in mice and NHP and favorable profile of AAV5 gene therapy expressing GLA, warrants further investigation as an attractive approach for Fabry disease.
AAV8 gene therapy as a potential treatment for adults with late‐onset ornithine transcarbamylase (OTC) deficiency: updated results from a phase 1/2 clinical trial
1: University of Birmingham, Birmingham, UK 2: University of Santiago de Compostela, Santiago de Compostela, Spain 3: Cruces University Hospital, Biocruces Bizkaia Health Research Institute, Barakaldo, Spain 4: Oregon Health & Science University, Portland, OR, USA 5: University of Calgary, Calgary, Canada 6: Icahn School of Medicine at Mount Sinai, New York, NY, USA 7: Boston Children's Hospital, Harvard Medical School, Boston, MA, 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: Hôpital Femme Mère Enfant, Lyon, France 11: Ultragenyx Gene Therapy, Cambridge, MA, USA
OTC deficiency is an X‐linked urea cycle disorder resulting in episodic hyperammonemia that can be life threatening and cause cumulative neurocognitive damage. DTX301, an AAV8 vector containing the OTC transgene, is being investigated for OTC deficiency.
CAPtivate (NCT02991144) is an ongoing global, open‐label, phase 1/2 trial evaluating DTX301 safety and efficacy in adults with late‐onset OTC deficiency. The primary endpoint is incidence of adverse events (AEs). Patients received one IV infusion of DTX301. Cohort 1 received 3.4x1012 Genome Copies (GC)/kg; Cohort 2, 1.0x1013 GC/kg; Cohort 3, 1.7x1013 GC/kg. Data cutoff was 15 Dec 2020. Complete responders discontinued all ammonia‐scavenging drugs and protein‐restricted diet. Responders had ≥50% reduction in baseline disease management.
In Cohorts 1‐3, no treatment‐related serious AEs or dose‐limiting toxicities were reported; all AEs during study were mild or moderate (grade 1 or 2). Seven patients experienced treatment‐emergent AEs (TEAEs) related to DTX301, five of whom experienced asymptomatic ALT increases that were managed with oral corticosteroids. Overall, 6 of 9 patients responded to DTX301: 3 complete responders, and 3 responders. All 9 treated patients maintained or improved ammonia control. Cohort 1 had one complete responder; Cohort 2, 1 complete and 1 responder; Cohort 3, 1 complete and 2 responders. The longest‐treated responders are showing durable responses 3 years post‐treatment.
In Cohort 4 (1.7x1013 GC/kg with prophylactic oral steroid taper), dosing is complete, and data are being collected.
DTX301 has shown promising results to date. Continued work is planned to study DTX301 as a potential new treatment for patients with OTC deficiency.
In vivo
Base‐Editing Corrects Metabolic Defects in Glycogen Storage Disease Type‐Ia
1: Beam Therapeutics, Inc. 2: National Institutes of Health
Glycogen storage disease type Ia (GSD‐Ia) is an autosomal recessive disorder caused by mutations in the G6PC gene that inactivate glucose‐6‐phosphatase‐α (G6Pase‐α or G6PC), a key enzyme in maintaining interprandial euglycemia. GSD‐Ia patients manifest a phenotype of impaired glucose homeostasis and life‐threatening fasting hypoglycemia. The most prevalent pathogenic mutation identified in Caucasian GSD‐Ia patients is G6PC‐p.R83C containing a single G > A transition mutation in the G6PC gene. Adenine base editors (ABEs) enable the programmable conversion of A•T to G•C in genomic DNA. Here, we evaluate the efficacy of a newly engineered ABE to correct the G6PC‐p.R83C mutation in a mouse model of GSD‐Ia. We first generated a homozygous huR83C mouse strain carrying the entire human G6PC‐p.R83C coding sequence and confirmed that the mutant mice lacked hepatic G6Pase‐α activity. The huR83C mice manifested growth retardation, hypoglycemia, hyperlipidemia, hyperuricemia, and hepatomegaly mimicking the abnormal metabolic phenotype of human GSD‐Ia. We then treated newborn huR83C mice with lipid nanoparticles (LNP) containing guide RNA and mRNA encoding ABE and showed that the treated mice grew normally to 3 weeks of age without hypoglycemia‐induced seizures. The treated huR83C mice displayed editing efficiencies up to ∼60% in total liver extracts, significant levels of hepatic G6Pase‐α activity, and normalized serum metabolites. In addition, LNP treatment of adult heterozygous huR83C mice restored hepatic G6Pase‐α activity to that of wild‐type levels. Taken together, our data demonstrate the potential of base‐editing to correct the G6PC‐p.R83C mutation and the metabolic defects associated with GSD‐Ia.
AAV‐mediated gene therapy for Wilson disease using split‐intein technology
1: Telethon Institute of Genetics and Medicine
Wilson disease (WD, OMIM#277900) is a rare genetic disorder of copper homeostasis, caused by mutations in the copper transporter ATP7B. Gene therapy with recombinant adeno‐associated vectors (AAV) holds promises for WD treatment, although AAV limited cargo capacity has hampered its use for ATP7B gene replacement. To overcome this limitation, we designed a dual AAV vector approach using split‐intein technology.
Split‐inteins catalyze seamless ligation of two separate polypeptides in a highly specific manner. We selected intein of DnaE from Nostoc punctiforme (Npu) recognizing a specific tripeptide in human ATP7B coding sequence. We generated two AAVs expressing the 5’‐half of a codon optimized human ATP7B cDNA followed by N‐teminal Npu DnaE intein and C‐terminal Npu DnaE intein followed by the 3’‐half of ATP7B cDNA, respectively, under the control of a liver specific promoter. Intra‐venous co‐injection of these vectors in wild‐type and Atp7b ‐/‐ mice resulted in efficient reconstitution of full‐length ATP7B in the liver. Moreover, Atp7b ‐/‐ mice treated with 1x1013gc/Kg of each AAV‐intein‐ATP7B vector showed absence of liver fibrosis and necroinflammation by histological analysis and no increase in serum transaminases at twelve weeks post‐injection, while showing a significant amelioration of liver injury and reduction of serum transaminases at the dose of 2.5x1012gc/Kg compared to Atp7b ‐/‐ mice treated with a control vector.
Taken together these data demonstrate the ability 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 WD.
Exchange of alveolar macrophages with
ex vivo
generated macrophages restores pulmonary immunity by niche specific adaption
1: Hannover Medical School 2: TWINCORE, Centre for Experimental and Clinical Infection Research 3: Telethon Institute of Gene Therapy (HSR‐TIGET) 4: Boston Children's Hospital
Macrophages (MΦ) have become an increasingly attractive cell type for cell therapy. Given the importance of MΦ in various disease entities, we aimed to develop a MΦ transfer strategy to replace dysfunctional MΦ populations by swapping the pulmonary MΦ pool with ex vivo generated MΦ. We employed clodronate liposomes to deplete endogenous alveolar MΦ (AMs) in mice and transferred ex vivo‐generated bone marrow (BM)‐derived MΦ into the lungs 7 days post‐depletion, leading to a robust engraftment of 10‐15% among total CD45+. 14 days after transfer, BM‐MΦ had adapted a surface marker profile similar to AMs (CD45+CD11c+SiglecF+CD11b‐). Single‐cell RNA‐sequencing revealed that the transplanted MΦ clustered away from the BM‐MΦ and showed stepwise adaptation to an AM‐associated transcriptome. Interestingly, in both primary AM and BM‐MΦ 14 days post‐transfer, we found an exclusive subset of cells that expressed Ki67, indicating first adaption, followed by local proliferation. As an application of the adoptive MΦ transfer, we used Ifngr1‐/‐ mice, a model of Mendelian Susceptibility to Mycobacterial Disease, and swapped their dysfunctional AMs with healthy MΦ yielding engraftment levels of ∼10%. Replacement of dysfunctional MΦ protected these mice from subsequent intra‐pulmonary infection with Bacillus Calmette‐Guérin, leading to 3‐fold lower number of colony forming units in the spleen. In summary, we developed an adoptive pulmonary transfer scheme for ex vivo generated MΦ that results in engraftment and adaption of the cells to the lung. Transferring this scheme to a disease model led to clinical benefit and could be transferred to other diseases and organs.
Ex vivo transduced macrophages produce therapeutic levels of secreted protein when transplanted to the lung
1: Imperial College London
We are assessing the feasibility of cell therapy‐based treatments for respiratory diseases. We optimised transduction of murine bone marrow‐derived macrophages (BMDM) with our proprietary F and HN pseudotyped lentivirus (rSIV.F/HN) and following pulmonary transplantation, demonstrated that these gene modified BMDM can function as factories for production of secreted proteins.
Proof‐of‐concept studies for this platform technology in a disease model were performed in a mouse model (GM‐CSF knockouts) of auto‐immune pulmonary alveolar proteinosis (aPAP). aPAP is caused by the development of anti‐GM‐CSF antibodies, which leads to defective surfactant clearance and deposition in alveoli, resulting in breathing difficulties and increased likelihood of infection. BMDM of donor mice were transduced with rSIV.F/HN carrying GM‐CSF or Gaussia luciferase (Gluc) cDNA (MOI 20) and transplanted into lungs of PAP mice through oropharyngeal delivery (6.3e6 cells/mouse, n = 6/group). Four weeks post transplantation turbidity of broncho‐alveolar lavage fluid (BALF) was significantly (p < 0.05) reduced (optical density: GM‐CSF: 2.24 ± 0.74, Gluc: 4.12 ± 0.89) indicating a clearance of the surfactants. We also detected a trend in reduction of surfactant protein‐D in BALF (GM‐CSF: 2504 ± 3084 ng/ml, Glux: 4987 ± 1852 ng/ml). In animals treated with BMDM expressing Gluc, we also showed that protein expression was stable for at least 4 weeks (week 2: 16951 ± 5455 RLU, week 4: 13783 ± 1810 in 10ul BALF). In summary, we have demonstrated that lentivirus transduced BMDMs secrete therapeutically relevant levels of recombinant proteins following pulmonary transplantation.
Prevention of Premature Lethality and Reversal of Cardiac Hypertrophy with an Optimized MYBPC3 Gene Therapy
1: Tenaya Therapeutics
Cardiomyopathy is the number‐one cause of sudden cardiac arrest in children under 18. Hypertrophic cardiomyopathy (HCM) affects 0.5 million Americans, potentially resulting in heart failure or sudden death. Loss‐of‐function mutations in Myosin Binding Protein C3, MYBPC3, are the most common genetic cause of HCM. The majority of MYBPC3 mutations causative for HCM result in truncations, via nonsense, frameshift or splice‐site mutations. The sarcomeric pathophysiology of the majority of HCM patients with MYBPC3 mutations appears to be due to haploinsufficiency, as the total amount of MYBPC3 protein incorporated into sarcomeres falls significantly below normal.
The clearest path to the treatment of haploinsufficiency is the restoration of the insufficient gene product; in this case wild‐type MYBPC3. Thus, we have successfully engineered an AAV vector (TN‐201) with superior properties for selective restoration of MYBPC3 to cardiomyocytes upon systemic delivery. Critically, we have demonstrated for the first time with AAV the ability of both a mouse surrogate and TN‐201, which encodes the human gene, to reverse cardiac dysfunction and hypertrophy and improve survival in a symptomatic murine model of disease. Dose‐ranging efficacy studies exhibited restoration of wild‐type MYBPC3 protein levels and saturation of cardiac improvement at the clinically relevant dose of 3E13 vg/kg. Further, pilot safety studies in adult and infant mice injected with >10X an efficacious dose exhibited no clinical observations and no alterations in cardiac function. Finally, we have established stable cardiac benefit for greater than one year post‐injection, as well as reversal of cardiac dysfunction even in late‐stage homozygote disease.
Artificial miRNA‐mediated glycogen synthase silencing as effective substrate reduction therapy in glycogen storage diseases mouse models
1: Genethon, UMR_S951, Inserm, Univ Evry, Université Paris Saclay, EPHE 2: UPMC ‐ Paris 6
Diseases characterized by multi‐organ glycogen accumulation are collectively named Glycogen storage diseases (GSDs). Among them Pompe (GSDII) and Cori (GSDIII) diseases are characterized by abnormal glycogen accumulation in cardiac and skeletal muscle tissues resulting in muscle weakness, cardiomyopathy, and respiratory failure. Enzyme replacement therapy is a life‐saving treatment in GSDII, however its efficacy is being questioned due to immunogenicity, as well as its poor biodistribution in particular in adults and the high costs associated to the high doses administered. No curative treatment exists for GSDIII. Substrate reduction therapy (SRT), aimed at the reduction of the glycogen synthesis, represents an alternative strategy to address GSDs. To this purpose, we downregulated muscle glycogen synthase, the enzyme responsible for glycogen synthesis, in the two mouse models of muscular GSDs, namely GSDII and GSDIII. Artificial miRNA targeting specifically muscular transcripts of glycogen synthase (miR‐Gys1) were developed and validated in murine myoblasts. Then, miR‐Gys1 were packaged in an AAV vector with high tropism for muscle, and delivered by intravenous injection to adult and newborn Pompe and Cori mice. This resulted in efficient and specific down regulation of glycogen synthase expression in cardiac and skeletal muscle. Importantly, in mice treated as neonates, the reduction of Gys1 led to decreased glycogen accumulation in all the muscle tissues analyzed. This work provides proof‐of‐concept of the efficacy of an AAV‐mediated artificial miRNA therapy in the prevention of glycogen build up in muscle of newborn mice and confirms SRT as a promising therapeutic strategy for infantile‐onset GSDs.
Intracisternal delivery of an AAV gene therapy candidate for the treatment of GM1 Gangliosidosis
1: Lysogene
GM1 gangliosidosis is a rare, inherited neurodegenerative disorder caused by mutations in the GLB1 gene which encodes the lysosomal hydrolase β‐galactosidase (Beta‐gal). Beta‐gal deficiency leads to toxic accumulation of GM1 ganglioside, predominantly in the central nervous system (CNS), resulting in progressive neurodegeneration. No treatment has been approved so far for this fatal disease. LYS‐GM101 is an AAVrh.10 based gene therapy vector carrying the GLB1 cDNA, administered via a one‐time injection into the cisterna magna. The efficacy of intra‐CSF injection of LYS‐GM101 analogs was demonstrated in GM1 mouse and cat models. Biodistribution and safety of LYS‐GM101 was assessed in a GLP study in nonhuman primates. An interventional, multicenter, single‐arm, 2‐stage adaptive design study of LYS‐GM101 (ClinTrials.gov, NCT04273269) was initiated in June 2021. This clinical trial includes a safety phase and a confirmatory efficacy phase. The trial will enroll 16 patients with a diagnosis of early or late infantile GM1 gangliosidosis at sites in the US and Europe. This talk will focus on non‐clinical pharmacology and toxicology studies and elaborate on the current status of the ongoing clinical trial.
Phase 1/2 Trial of AXO‐AAV‐GM1 Gene Therapy for the Treatment of Infantile‐ and Juvenile‐onset GM1 Gangliosidosis
1: National Institutes of Health 2: National Human Genome Research Institute 3: National Institute of Mental Health 4: Sio Gene Therapies Inc.
Data is presented from an ongoing open‐label, single‐arm, dose‐ranging Phase 1/2 trial (NCT03952637) of an investigational gene therapy AXO‐AAV‐GM1 (AAV9‐GLB1) for treatment of GM1 gangliosidosis which is a rare, fatal, neurodegenerative lysosomal storage disorder. Subjects are administered AXO‐AAV‐GM1 intravenously following immune modulation with rituximab, sirolimus and glucocorticoids. Seven subjects have been enrolled in 2 cohorts: low‐dose (1.5x1013 vg/kg), n = 4 late‐infantile‐onset and n = 1 juvenile‐onset, follow‐up 12‐months; high‐dose (4.5x1013 vg/kg), n = 2 juvenile‐onset, follow‐up 6‐months. AXO‐AAV‐GM1 was generally safe and well‐tolerated. There were two serious adverse events unrelated to AXO‐AAV‐GM1. Six subjects had adverse events of AST or ALT elevations (≤5X above baseline) that did not require clinical intervention or have associated clinical sequelae and no adverse events of elevations in GGT or bilirubin were reported. At 6‐months, CSF GM1 ganglioside was reduced to published normal levels and serum β‐gal enzyme activity increased above the lower limit of normal reference levels in the high‐dose subjects. At 12‐months, the CSF GM1 ganglioside remained below baseline levels in all low‐dose subjects and serum β‐gal activity remained above baseline levels in two of five subjects. Volumetric MRI data in low‐dose subjects showed maintenance of brain volume in four of five subjects at 12‐months. There was no clinical evidence of disease progression in four of five low‐dose subjects at 12‐months or in the high‐dose cohort at 6‐months post‐treatment as assessed by Vineland‐3, Upright/Floor Mobility Score, Clinical Global Impression, and neurological examination. Further evaluation of these subjects is ongoing.
Therapeutic advantage of combined gene/cell therapy strategies in the murine model of Sandhoff disease
1: San Raffaele Telethon Institute for Gene Therapy (SR‐Tiget), IRCCS San Raffaele Scientific Institute 2: Department of Chemistry, Biology and Biotechnology, University of Perugia 3: Department of Medical Biotechnology and Translational Medicine, University of Milano 4: Department of Neuroscience, IRCCS San Raffaele Scientific Institute
Sandhoff disease (SD) is a rare neurodegenerative Lysosomal Storage Disease (LSD) caused by genetic deficiency of the β‐N‐acetylhexosaminidase (Hex) enzyme. The consequent neuronal accumulation of GM2 ganglioside has devastating effects on the central nervous system (CNS). The infantile forms of SD are the most aggressive and currently lack therapeutic options. Thus, there is an urgent need to develop novel therapies able to metabolically correct the CNS with appropriate timing to counteract the rapid disease progression. The combination of intracerebral lentiviral vector (LV)‐mediated gene therapy (IC GT) and bone marrow transplant (BMT) significantly benefit the murine model of globoid cell leukodystrophy, a severe neurodegenerative LSD (Ricca et al., 2015). Here, we show the therapeutic advantage of coupling neonatal injection of LVs coding for the functional Hex enzyme to BMT in the SD murine model. Treated mice show a significant increase of lifespan (up to 8 months) and normalization of the pathological phenotype. This benefit correlates with increased Hex activity and remarkable reduction of GM2 storage in neural tissues. Our results suggest that the early enzymatic supply provided by IC GT is crucial for the proficient activity of HSPC progeny, which reduces neuroinflammation and provides a reservoir of functional enzyme, overall delaying the onset and slowing the progression of the disease. The proof of concept of efficacy and tolerability of combining LV‐mediated IC GT and BMT in SD mice support the rationale of refining CNS‐targeted gene/cell‐based approaches to treat GM2 gangliosidosis and possibly other LSDs.
Bicistronic AAV gene therapy for Tay‐Sachs and Sandhoff diseases in a sheep model
1: University of Massachusetts Medical School 2: Auburn University 3: Tufts Cummings School of Veterinary Medicine
Tay‐Sachs and Sandhoff diseases (TSD, SD) are fatal neurodegenerative disorders, caused by mutations in alpha or beta subunit of enzyme Hexosaminidase (Hex), respectively, and result in GM2 ganglioside storage and neuronal death. Here we describe therapeutic efficacy of a bicistronic AAV9 vector construct in naturally occurring sheep model of TSD. TSD sheep were injected with 1E14 vg total intravenously (IV, n = 5) or via cerebrospinal fluid (CSF, n = 9). Sheep treated IV survived to 18 ± 5 months and CSF treated sheep are ongoing with the oldest now more than 3 years old. Untreated TSD sheep reach humane endpoint at ∼9 months. GM2 levels in CSF of sheep treated by CSF administration were normalized starting at 1 month after treatment and remained in normal range up to 2 years, however, GM2 levels in IV cohort remained above normal. CSF treatment resulted in HexA activity at above normal levels in central nervous system (CNS), however, for IV cohort only lumbar spinal cord reached normal level. HexA expression in peripheral nervous system (PNS) was similar for both cohorts. GM2 levels in CNS and PNS were in line with HexA levels. Vector genome content in CNS was consistent with HexA levels, however, IV treatment except for optic nerve resulted in higher number of virus particles in PNS. Magnetic resonance spectroscopy of treated sheep thalamus showed normalization of neuronal health markers, myelination and metabolism in CSF cohort and slight improvements in IV cohort. Both cohorts exhibited marked attenuation of neurologic disease and normal cognition in neurological examination.
CRISPR‐Cas9 globin editing can induce megabase‐scale partial uniparental disomy with imprinting defects in hematopoietic cells
J Boutin1 J Rosier1 D Cappellen1 F Moreau‐Gaudry1
1: INSERM U1035 2: Université de Bordeaux
The ON‐target genotoxicity of CRISPR‐Cas9 nuclease due to DNA double‐strand breaks has received little attention and is probably underestimated. We previously reported that CRISPR‐Cas9 nuclease can induce megabase‐scale truncations targeting UROS in chromosome 10 in HEK cell lines. Here, we looked at whether these unexpected large rearrangements can occur targeting globin genes in chromosome 11. We observed that genome editing targeting globin genes induces megabase‐scale losses of heterozygosity (LOH) from the globin CRISPR‐Cas9 cut site to the telomere (5.2Mb), suggesting that CRISPR‐mediated large rearrangements are not locus‐dependent. In established lines, CRISPR‐Cas9 nuclease induced frequent (up to 20%) terminal chromosome 11p truncations and rare copy‐neutral LOH. In primary hematopoietic progenitor/stem cells, we detected 1.1% of clones (7/648) with acquired megabase LOH induced by CRISPR‐Cas9. In‐depth analysis by SNP‐array revealed the presence of copy‐neutral LOH from the cut site to the telomere, but not deletion. So, cell‐type seems to be crucial to determine rearrangement‐type after CRISPR‐Cas9 DSB. In hematopoietic stem cells, copy‐neutral LOH led to 11p15.5 partial uniparental disomy, comprising two Chr11p15.5 imprinting centers (H19/IGF2:IG‐DMR/IC1 and KCNQ1OT1:TSS‐DMR/IC2) and impacting H19 and IGF2 expression. This genotoxicity could be a safety concern for CRISPR clinical trials and have to be confirmed in other loci and in vivo. Anyway, these new data highlight the necessity to develop preclinical tests to evaluate carefully not only the well‐known OFF‐target but also ON‐target genotoxicity risk.
BRILLIANCE: A Phase 1/2 Single Ascending Dose Study of EDIT‐101, an
in vivo
CRISPR Gene Editing Therapy, in
CEP290
‐Related Retinal Degeneration
1: Ocular Genomics Institute, Massachusetts Eye and Ear, Boston, MA 02114, USA 2: Casey Eye Institute, Oregon Health & Science University, Portland, OR 97239, USA 3: Bascom Palmer Eye Institute, Miami, FL 33136, USA 4: University of Michigan Kellogg Eye Center, Ann Arbor, MI 48105, USA 5: Editas Medicine Inc., Cambridge, MA 02141, USA 6: Flaum Eye Institute, University of Rochester Medical Center, Rochester, NY 14642, USA
Centrosomal protein 290 (CEP290)‐related retinal degeneration is a rare autosomal recessive inherited disease that causes severe visual impairment from infancy. It is most commonly associated with a mutation in intron 26 (IVS26) of CEP290, which results in photoreceptor dysfunction. A single subretinal administration of EDIT‐101 aims to restore photoreceptor function by removing the IVS26 mutation using in vivo CRISPR gene editing technology. Based on preclinical dose response studies in transgenic mice, where we observed an average productive excision of approximately 54% at a dose of 3.0E+12 vg/ml, we would anticipate clinically meaningful and robust productive editing at the comparable dose ranges in a clinical setting.
To evaluate the clinical utility of CRISPR‐mediated excision of the IVS26 mutation, BRILLIANCE (NCT03872479) is a Phase 1/2 single ascending dose, first‐in‐human trial, which is currently assessing three EDIT‐101 doses, 6.0E+11 vg/ml (low dose), 1.1E+12 vg/ml (middle dose), and 3.0E+12 vg/ml (high dose), in up to five cohorts; and is enrolling adults (≥18 years) and children (3–17 years) with CEP290‐related retinal degradation due to a c.2991 + 1655A>G mutation in IVS26. The primary objective is to evaluate the safety and tolerability of EDIT‐101. Determining a maximum tolerated dose and evaluating patients for improvement in visual function are secondary objectives.
Six patients in Cohorts 1 (adult low dose, n = 2) and 2 (adult middle dose, n = 4) have been enrolled as planned and treated with EDIT‐101 so far. No dose‐limiting toxicities or serious adverse events have been reported in these patients to date. Efficacy and safety evaluations are ongoing.
A versatile platform for ADAR‐mediated RNA editing
in vivo
in preclinical models
1: Wave Life Sciences
The ADAR (adenosine deaminases acting on RNA) family of enzymes catalyze adenine (A) to inosine (I) changes in RNA, which are read as guanine (G) by the translational machinery. Recruiting endogenous ADAR enzymes using chemically modified oligonucleotides to direct specific editing holds great promise for treating human disease. PRISMTM, Wave Life Sciences' proprietary discovery and drug development platform, enables us to develop stereopure oligonucleotides—those in which the chiral configuration of backbone linkages (Rp or Sp) are precisely controlled at each position—that direct sequence‐specific RNA editing using endogenous ADAR enzymes. We will present preclinical in vivo data in mouse and non‐human primates (NHPs) showing that stereopure oligonucleotides are effective when delivered as naked oligonucleotides or GalNAc‐conjugates in the absence of any delivery vehicle via systemic subcutaneous, intracerebroventricular or intrathecal administration. We show editing in myriad tissues, including liver, multiple regions of the CNS, kidney, and lung. The most promising oligonucleotides support up to 50% editing in vivo. Finally, we will illustrate our application of this technology to AATD (a1 antitrypsin deficiency), a genetic disorder with lung and liver manifestations that has potential to be corrected with an ADAR editing therapeutic. Stereopure oligonucleotides support dose‐dependent increases in SERPINA1 RNA editing in vitro and in vivo, resulting in a 3‐fold increase in serum AAT levels, and restoration of AAT protein function. These preclinical investigations lay the foundation for development of RNA‐editing therapeutics with potential to treat human genetic disease.
Preclinical development of a TALEN®‐based genome editing therapy for RAG1 deficiency
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: Cellectis Inc, New York, NY 10016, USA 4: Institute of Medical Bioinformatics and Systems Medicine, Medical Center – University of Freiburg, Freiburg, 79110, Germany 5: German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Partner Site Freiburg, Freiburg, 79106, Germany 6: Cellectis S.A., Paris, 75013, France
Severe combined immunodeficiency (SCID) is a diverse group of genetic defects, which lead to impaired T cell development or function with varying effects on the B‐/NK‐cell compartments. Newborn screening revealed that mutations within the RAG1 gene accounted for about 16% of reported SCID cases in California between 2010‐2017. To date, hematopoietic stem cell (HSC) transplantation remains the only curative option. Attempts to treat RAG1 deficiency by conventional gene therapy have produced controversial results. In early studies, RAG1‐/‐
mice transplanted with HSCs, which were transduced with a RAG1 transgene, presented with Omenn‐like symptoms. This highlights the need of a tight spatio‐temporal control of RAG1 expression as key for functional restoration. Here, we present a genome editing strategy based on TALEN
Gene therapy for Fanconi anemia [group a]: Interim results of RP‐L102 clinical trials
1: Hematología y Hemoterapia, Fundación para la Investigación Biomédica HIUNJ, Hospital Infantil Universitario Niño Jesús, Madrid, Spain 2: Center for Definitive and Curative Medicine, Stanford University, Stanford, CA 3: Div. of Pediatric Hematology/Oncology/Stem Cell Transplant and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 4: Stanford Children's Hospital, Palo Alto, CA 5: UCL Great Ormond Street Institute of Child Health, London, UK 6: Instituto de Innovación Biomédica, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spain 7: Centro de Investigación Biomédica en Red de Enfermedades Raras, Madrid, Spain 8: Unidad Mixta de Terapias Avanzadas, Instituto de Investigación Sanitaria Fundación Jiménez Díaz, Madrid, Spain 9: Rocket Pharmaceuticals, Inc., Cranbury, NJ 10: Blood and Marrow Transplant Program, Dept. of Pediatrics, University of Minnesota, Minneapolis, MN
Fanconi anemia (FA) is a disorder of defective deoxyribonucleic acid (DNA) repair resulting in bone marrow failure (BMF). Allogeneic hematopoietic stem cell transplant (alloHSCT) is potentially curative for BMF; however efficacy is limited by human leukocyte antigen (HLA)‐matched sibling donor availability and toxicities. Lentiviral mediated gene therapy utilizing autologous FA‐A hematopoietic stem cells (HSCs) confers a proliferative advantage to gene‐corrected HSCs as demonstrated in preclinical studies and the FANCOLEN‐I clinical trial. We report results from ongoing RP‐L102 studies. Patients (pts) with a FANCA gene 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+ HSCs are transduced with a lentiviral vector and infused fresh without antecedent conditioning. Pts are followed for safety and efficacy 3 years post‐infusion. As of March 2021, 9 pts have received RP‐L102. Evidence of engraftment has been identified in 6 pts with ≥6 months of follow up as indicated by PB VCN. 2 of 3 pts with follow up of ≥12 months have shown increased BM CFU MMC resistance. 1 pt developed BMF requiring alloHSCT after influenza B infection. 1 pt had a serious Grade 2 transient RP‐L102 infusion‐related reaction. Updated data for pts with ≥12 months of follow up will be presented.
RP‐L102's safety profile remains favorable. Increasing evidence of engraftment has been confirmed in 6 subjects as demonstrated by PB VCN; without conditioning, 12+ months of follow up is likely required to observe the proliferative advantage of transduced HSCs.
Pre‐clinical evaluation, including genomic off‐target analysis, of VOR33: a clinic‐ready CRISPR/Cas9 engineered hematopoietic stem cell transplant for the treatment of acute myeloid leukemia
1: Vor Biopharma, Cambridge, MA 2: Irving Cancer Research Center, Columbia University Medical Center, Columbia University, New York, NY 3: Myelodysplastic Syndromes Center, Columbia University Medical Center, Columbia University, New York, NY
Treatment of acute myeloid leukemia (AML) with targeted therapies has been limited because the lack of tumor specific antigens results in “on‐target, off‐tumor” toxicity. To unlock the full potential of targeted treatments, we create treatment‐resistant hematopoietic stem progenitor cells (HSPCs) by genetically ablating CD33 from healthy, human leukocyte antigen‐matched donor HSPCs for hematopoietic stem cell transplant. The reconstituted hematopoietic compartment of patients receiving CD33‐null HSPCs (VOR33) is expected to be resistant to cytotoxicity induced by the anti‐CD33 antibody‐drug conjugate Mylotarg™. Here, we describe the preclinical data and process scale‐up of VOR33 for a first‐in‐human clinical trial.
The manufacturing process yielded clinically relevant doses of VOR33 under GMP‐like conditions. Using CRISPR/Cas9, we routinely achieved CD33 gene knockout efficiency >70% (90% biallelic). Cell viability, differentiation, and function were unaffected by the loss of CD33. Pharmacology studies using NOD/SCID‐gamma mice showed long‐term engraftment, multilineage differentiation, and persistence of gene editing. Importantly, CD33 protein loss conferred selective protection to VOR33‐derived myeloid cells from Mylotarg™ in vitro (>65‐fold) and in vivo (>60‐fold). A GLP toxicology study of >40 tissues revealed no tumorigenicity or notable changes in toxicology parameters. Off‐target editing assessment using unbiased cell‐based GUIDE‐seq and sequence homology‐based hybrid capture NGS analysis revealed no significant or reproducible off‐target editing events. These studies set the stage for a first‐in‐human clinical trial of VOR33 to evaluate its safety and efficacy in patients with AML.
AAV‐intein mediated Factor VIII trans‐splicing for gene therapy of Haemophilia A
1: Fondazione Telethon ‐ TIGEM 2: Universita del Piemonte Orientale 3: Universita' di Napoli Federico II
Haemophilia A (HemA) is the most common X‐linked bleeding disorder (1:5000 males), caused by deficiency of human clotting factor VIII (FVIII). The current management of HemA involves administration of either recombinant FVIII, or of a recently approved bispecific antibody, Emicizumab. However, lifelong intravenous infusions of either of the two are still required.
Liver gene therapy with adeno‐associated viral (AAV) vectors holds great promise to provide long‐term FVIII expression after a single administration. However, HemA poses a great challenge to AAV‐delivered gene therapy due to the size of the FVIII coding sequence (∼7 kb) that exceeds the canonical AAV cargo capacity (∼ 4.7 kb). Some of the B‐domain deleted (B‐dd) FVIII variants tested in gene therapy clinical trials also are at the limit of AAV packaging capacity.
To overcome this limitation, we developed a protein trans‐splicing (PTS) strategy using two AAVs, each encoding one half of the large and highly active B‐domain deleted (B‐DD) N6‐FVIII variant (∼5 kb) flanked by split inteins. We show that AAV‐intein and a codon optimized version of these constructs, successfully reconstitute the full‐length N6‐FVIII both in vitro and in mouse liver and achieve therapeutic activity levels of FVIII (∼150 IU/dl) in the plasma of hemophilic treated mice in the absence of anti‐FVIII antibodies.
Therefore, systemic administration of AAV‐intein N6‐FVIII is a potential therapeutic option for HemA.
Promoterless gene targeting approach combined with CRISPR/Cas9 efficiently corrects hemophilia B phenotype in neonatal mice
1: ICGEB 2: Center of Research in Myology, Sorbonne University, Paris, France 3: Genethon, Evry, Paris 4: IRCCS San Rafaele Hospital, Milan, Italy 5: Tel Aviv University 6: Stanford University 7: Spark Therapeutics
Many inborn errors of metabolism require life‐long treatments and, in severe conditions, organ transplantation remains the only curative treatment. Non‐integrative AAV‐mediated gene therapy has shown to be efficient in adult patients of hemophilia A and B. However, treatment in pediatric or juvenile settings may result in the rapid loss of episomal viral DNA and, consequently, therapeutic efficacy. This limitation is a major concern since a re‐administration is not possible due to the presence of anti‐AAV neutralizing antibodies.
We used a gene‐targeting approach to target the human coagulation factor IX (FIX) cDNA into the genome of a mouse model of hemophilia B. Two AAV vectors were used: a promoterless AAV vector with arms of homology for the albumin locus, and an AAV carrying the CRISPR/SaCas9 with the sgRNA to increase recombination rate. A single injection of the AAV cocktail into neonatal FIX KO mice leads to long‐term and stable expression of above‐normal levels of hFIX. Mice were subjected to tail clip analysis in which the bleeding time was comparable to wild‐type animals demonstrating the complete normalization of the disease phenotype. However, in adult FIX KO mice the targeting rate was less efficient and it did not lead to a correction of the coagulation activity.
With this study, we demonstrate an alternative gene targeting strategy exploiting the use of the CRISPR/Cas9 platform that can be potentially applied for the treatment of pediatric patients suffering from hemophilia, also supporting its application to other monogenic diseases of the liver.
Cellular senescence and inflammatory programs are unintended consequences of CRISPR‐Cas9 gene editing in hematopoietic stem and progenitors cells
1: San Raffale Telethon Institute for Gene Therapy (SR‐TIGET), Milan, Italy 2: Vita‐Salute San Raffaele University, Milan, Italy 3: CUSSB‐University Center for Statistics in the Biomedical Sciences, Vita‐Salute San Raffaele University, Milan, Italy 4: National Research Council, Institute for Biomedical Technologies, Segrate, Italy
Gene editing (GE) by artificial nucleases holds promise for Hematopoietic Stem and Progenitor Cells (HSPC)‐based gene therapy applications. Despite advances in increasing the yield of gene corrected HSPC, their repopulating potential upon transplantation remains limited. We previously showed that GE procedure with DNA repair template for Homology Directed Repair (HDR) by AAV6 cause the activation of the p53‐mediated DNA Damage Response (DDR) pathway, constraining HSPC proliferation and yield (Conti*, Schiroli* et al. CSC 2019), suggesting that DDR‐related cellular programs may inadvertently contribute to HSPC dysfunctions upon GE. By integrating transcriptional analysis with innovative imaging‐based cellular assays we reported induction of cellular senescence markers (p16 and SA‐β‐Gal) and pro‐inflammatory programs, including activation of the IL1 axis (upstream mediator of DDR‐dependent inflammation) and NF‐kB pathway across edited HSPC subtypes and in vivo in the human graft. Transcriptional activation of inflammatory cytokines in edited HSPC was dependent on the apical DDR kinase ATM and partly mitigated by p53 inhibition. Temporary inhibition of IL1 and NF‐kB pathways at the time of GE resulted in an increase in edited HSPC clonogenicity in vitro and ameliorated the long‐term hematopoietic reconstitution in xenotranspanted mice, with a concomitant decrease in senescence markers. In vivo clonal tracking of HDR‐edited HSPC by a barcoding‐based strategy revealed that IL1 inhibition improved polyclonal reconstitution and better preserved self‐renewal and multi‐potency of individual edited HSPC. Our findings pave the way for the development of novel strategies based on senescence suppressors and anti‐inflammatory molecules to overcome adverse cellular responses for efficient HSPC‐based clinical applications.
Nuclease‐free, promoterless recombinant AAV‐mediated genome editing restores function of hepatocytes leading to selective advantage and repopulation in mouse models with liver disease
1: LogicBio Therapeutics
Many genetic defects cause liver diseases, leading to liver tissue regeneration. This biological process poses great challenges to liver‐targeted, non‐integrative gene therapies that get diluted as the liver grows or regenerates. We have developed GeneRide™, a nuclease‐free, promoterless recombinant AAV‐based genome editing technology to treat diseases with durable transgene expression. A liver‐targeted vector mLB‐001 was designed to specifically insert a copy of mouse Mmut open reading frame sequence into the mouse albumin locus via homologous recombination. The chimeric mRNA produces a functional methylmalonyl‐CoA mutase (MMUT) protein and a tagged albumin protein, albumin‐2A, which serves as a circulating pharmacodynamic biomarker for site‐specific genomic integration. To mimic the dietary restrictions used in methylmalonic acidemia (MMA) patients, we developed a novel protein dietary regimen for our preclinical studies when treating a Mmut‐deficient mouse model of MMA. After administering mLB‐001 to the MMA mice, we observed a time‐dependent increase in circulating albumin‐2A, suggesting expansion of the corrected hepatocytes. Immunohistochemistry showed MMUT‐expressing hepatocytes increased from an initial ∼0.5% to 20‐25% at 6 months post dosing. The expansion of the healthy hepatocytes was associated with a statistically significant improvement in animal health and survival even when challenged with a high protein diet. Importantly, we have now observed selective expansion of GeneRide‐edited hepatocytes in several other murine models of human diseases. The repopulation rate correlates with the degree of liver damage in the varied disease models studied to date. These results support the development of GeneRide vectors to durably treat genetic diseases with liver dysfunction.
In vivo
deletion of glycolate oxidase (GO) using double‐nicking CRISPR/Cas9 for an efficient and safer treatment of Primary Hyperoxaluria type I (PH1)
1: Gene Therapy and Regulation of Gene Expression Program, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain 2: Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona, Spain 3: Bioinformatics core, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain 4: Cell Therapy Program, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain 5: Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana‐Champaign, Urbana, IL, USA 6: Biomedical Research Networking Center on Rare Diseases (CIBERER), Institute of Health Carlos III, Madrid, Spain 7: Hospital Universitario de Canarias, San Cristóbal de La Laguna, Tenerife, Spain 8: Grousbeck Gene Therapy Center, Schepens Eye Research Institute, Mass Eye and Ear, Boston, MA, USA; Ocular Genomics Institute, Mass Eye and Ear, Boston, MA, USA; Department of Ophthalmology, Harvard Medical School, Boston, MA, USA
Liver‐kidney transplantation is the only curative therapy for PH1, an inherited disorder associated with mutations in the AGXT gene that cause deficiency in hepatic alanine‐glyoxylate aminotransferase (AGT), leading to insoluble oxalate accumulation in kidneys causing renal failure and systemic oxalosis.
An attractive therapeutic strategy for diseases associated with the accumulation of a toxic metabolite is substrate reduction therapy (SRT). The inhibition of GO, the enzyme that synthesizes glyoxylate (oxalate precursor), using RNA interference has proven to be an efficacious SRT for PH1. Recently, we demonstrated the therapeutic efficacy of an AAV‐CRISPR/Cas9 system to disrupt the Hao1 gene, which encodes GO in a PH1 mouse model.
To reduce the likelihood of off‐targets, we investigated the administration in PH1 mice of two AAV‐CRISPR/Cas9(D10A)‐nickases, each with a guide RNA, targeting nearby regions on the opposite strands of Hao1. Simultaneous on‐target nicking resulted in undetectable GO levels (WB and IHC). Individual nicks were faithfully repaired, as detected by next generation sequencing and normal GO expression, evidencing the inability of a single nickase to generate indels, and therefore reducing the probability of off‐target events. Next, we combined the dual nickase‐gRNA system into a single vector (all‐in‐one) allowing us to reduce the effective minimal dose by three‐fold in comparison with the administration of the two independent vectors. Furthermore, a single administration in PH1 mice of the all‐in‐one vector showed a significant therapeutic effect, preventing oxalate accumulation.
In conclusion, we have designed a safer CRISPR/Cas9‐mediated GO disruption by reducing the potential off‐target effects, maintaining the editing efficacy of WT Cas9.
Correction of a Factor VIII genomic inversion with designer‐recombinases
1: Dresden University 2: Cincinnati Children's Hospital Medical Center 3: German Center for Neurodegenerative Diseases 4: Max‐Planck‐Institut für molekulare Biomedizin, Münster 5: University Hospital Dresden
Haemophilia A is the most common blood clotting disorder affecting 1:5000 new‐born males. Currently there is no cure for Haemophilia A and severely affected individuals receive injections of recombinant Factor VIII several times a month. Treatment is not only expensive, but has a major impact on the patient's quality of life. Moreover, one‐third of the treated individuals develop antibodies against recombinant Factor VIII making their treatment ineffectual.
A genetic correction resulting in only 5% of F8 gene expression would be a curative approach for severely affected individuals. Some promising results were recently obtained by gene replacement therapy where a modified version of the F8 gene is expressed ectopically with the aid of AAVs. Though, there are still some unanswered safety concerns using AAVs.
The ideal treatment would correct the endogenous F8 gene. However, it remains challenging because 50% of the severe cases are caused by large gene inversions that are difficult to rectify using nucleases such as CRISPR/Cas9.
An alternative tool to actively invert genomic DNA are site‐specific recombinases. Here we present evolved Cre‐like recombinases that recognize a specific sequence left and right of the inverted F8 gene causing severe Haemophilia A. Expression of these recombinases result in the precise correction of the inversion in human cells with an efficiency of up to 30%. More importantly, corrected patient derived endothelial cells express up to 6% of the F8 gene after treatment with recombianses. Our system provides a possible blueprint for a curative correction of large gene inversions causing human diseases.
A novel effective vaccine against SARS‐CoV‐2 based on recombinant adeno‐associated virus serotype 5 (AAV5)
1: BIOCAD
Coronavirus disease 2019 (COVID‐19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) currently is a major public health issue worldwide. In order to prevent further spread of the disease the COVID‐19 prophylaxis vaccines are in urgent need. Currently there are more than 180 vaccines based on different platform technologies under development all over the world. Replication‐incompetent recombinant viral vectors expressing SARS‐CoV‐2 spike (S) protein or its receptor‐binding domain (RBD) represent promising system for the vaccine development. Particularly, recombinant adeno‐associated viruses (AAV) seem to be promising vector for SARS‐CoV‐2 vaccine development. In this study, we describe a novel vaccine candidate against SARS‐CoV‐2 based on AAV5 vector which expresses RBD of SARS‐CoV‐2 S‐protein as an antigen molecule. We showed that AAV5 viral vector with unique codon optimized RBD gene sequence (AAV5‐RBD) allows to transduce the cells with high efficiency leading to the high level of RBD protein expression. Moreover, our results indicated that immunization with AAV5‐RBD induces high titer of antibodies specific to RBD antigen in mice, rabbit and non‐human primates (NHP). It was shown that produced antibodies may neutralize the native SARS‐CoV‐2 virus with high efficacy which was complemented by functional memory T‐cells response. Overall findings allow us to consider developed AAV5‐RBD vaccine candidate as an effective prophylaxis against COVID‐19.
MAVS‐/‐ mesenchymal stem cells as oncolytic adenovirus ICOVIR‐5 carriers for cancer treatment
1: Cellular Biotechnology Unit, Instituto de Salud Carlos III 2: Unidad de Terapias Avanzadas, Hospital Infantil Universitario Niño Jesús
Celyvir is an immunotherapy that uses mesenchymal stem cells (MSCs) as carriers for the oncolytic adenovirus ICOVIR‐5. Early clinical trials in patients with relapsed or refractory solid tumors showed positive results and excellent safety profiles. In these trials, we observed that autologous MSCs from responder patients presented lower expression of the mitochondrial antiviral signaling protein (MAVS).
In this work, we hypothesize that this lower expression of MAVS in the MSC may be partially responsible for the improved effect of Celyvir. We performed studies with mouse MSCs knockout for mavs infected with ICOVIR‐5 (Celyvir MAVS‐/‐), characterising their intracellular signaling and secretion of proinflammatory cytokines. We also performed several in vivo assays to determine the antitumor efficacy and tumor immune infiltration induced by Celyvir MAVS‐/‐. The results showed that the use of MAVS ‐/‐ mMSCs in vitro did not differ from mMSCs wild‐type after infection with ICOVIR‐5. However, in in vivo models, Celyvir MAVS‐/‐ significantly reduced tumor growth kinetics, improving the efficacy of Celyvir wildtype. Celyvir MAVS‐/‐ also increased the tumor immune infiltrate of T cells –especially CD8+ T cells– and B cells.
In conclusion, the manufacture of Celyvir with MSC with low expression of MAVS induces a better antitumor effect than using wild‐type MSC. Therefore, we present an approach to obtain better antitumor efficacy in future clinical trials by targeting those human allogeneic MSCs less activated for the MAVS pathway.
A universal platform to generate chimeric antigen receptor macrophages from human stem cell sources
D Paasch1 2
1: Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany 2: REBIRTH Research Center for Translational and Regenerative Medicine, 30625 Hannover, Germany 3: Clinic for Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, 30625 Hannover, Germany 4: Department of Gynecology and Prenatal Medicine, Hannover Medical School, 30625 Hannover, Germany 5: Center for Molecular Medicine Cologne, University of Cologne, 50931 Cologne, Germany 6: Regensburg Center for Interventional Immunology (RCI), Dev. Genetic Immunotherapy and University Hospital Regensburg, 93053 Regensburg, Germany 7: Division of Hematology/Oncology, Children's Hospital Boston, Harvard Medical School, Boston, MA, USA
While immunotherapy using chimeric antigen receptor (CAR) technology has shown great efficacy to enhance T cell cytotoxicity in hematologic malignancies, the treatment of solid tumors remains challenging. In this context, CAR‐macrophages (CAR‐Mac) recently have been introduced as an additional tool. We here demonstrate the feasibility to use human hematopoietic stem and progenitor cells (HSPCs) as a source to generate functional CAR‐Mac, which target the carcinoembryonic antigen (CEA) expressed on pancreatic, colorectal and other tumors (CEA‐Mac). Additionally, we employ induced pluripotent stem cells (iPSCs) to generate CAR‐Macs targeting the CD19 antigen (CD19‐iMac), an approach allowing for the sustained production of CAR‐Mac effector cells in scalable quantities. CEA‐Mac and CD19‐iMac both showed effective CAR expression and exhibited morphology, phenotype (CD45+, CD11b+, CD14+, CD163+) and basic functionality similar to unmodified control macrophages. Two CEA‐Mac constructs differing in their CAR signal transduction domain (DAP12/ CD3ζ) were investigated. Both showed antigen specificity and displayed pro‐inflammatory cytokine secretion only upon contact to CEA‐positive target cells. Furthermore, CD3ζ expressing CAR‐Macs showed increased phagocytosis of CEA‐positive HT1080 cells compared to CEA‐negative control cells. With regard to CD19‐iMac, we were able to generate these cells from our iPSC‐based continuous differentiation platforms without loss of CAR‐transgene expression or activity. Of note, co‐culture systems showed that CD19‐iMacs were capable of targeting CD19‐ positive Raji cells by increased phagocytosis. In summary, we here provide a universal tool for the generation of highly functional HSPC‐ or iPSC‐derived CAR‐Macs, thus paving the way for future CAR‐Mac‐based immunotherapy strategies.
Haematopoietic reconstitution dynamics of mobilized peripheral blood‐ and bone marrow‐derived haematopoietic stem/progenitor cells after gene therapy
1: San Raffaele Telethon Institute for Gene Therapy (SR‐TIGET), IRCCS San Raffaele Scientific Institute, Milan, 20132 Italy. 2: Pediatric Immunohematology and Stem Cell Programme, IRCCS San Raffaele Scientific Institute, Milan, 20132 Italy 3: Università Vita‐Salute San Raffaele, Milan, 20132 Italy 4: Center of Biostatistics for Clinical Epidemiology, University of Milano—Bicocca, Monza, 20900, Italy
Haematopoietic Stem/Progenitor Cells (HSPC)‐gene therapy (GT) is based on infusion of genetically‐modified autologous HSPC obtained from bone marrow (BM) or mobilized peripheral blood (MPB). In haematopoietic stem cell transplantation (HSCT), MPB is usually preferred due to higher HSPC yields and faster haematopoietic reconstitution in the recipient.
Despite their wide clinical use, there is limited information on the kinetics of HSPC reconstitution occurring after GT using these two sources.
Here, we evaluated haematopoietic reconstitution dynamics, engraftment and clonality in 13 pediatric Wiskott‐Aldrich syndrome (WAS) patients treated with autologous lentiviral‐vector transduced HSPC derived from MPB (n = 7), BM (n = 5) or BM+MPB (n = 1). MPB and BM gene‐corrected HSPC displayed similar vector copy number on liquid culture and percentage of transduced colonies. MPB‐GT patients showed faster neutrophil and platelet recovery, higher number of engrafted clones and increased gene correction in the myeloid lineage, which correlated with higher amount of primitive and myeloid progenitors contained in MPB HSPC. All patients recovered in a timely manner and similar reconstitution was observed starting from 1 year post‐GT. In vitro differentiation and transplantation studies in mice showed that primitive HSPC from both sources have comparable engraftment and multilineage differentiation potential. Overall, the results of our work provide a comprehensive picture of the haematopoietic reconstitution after GT using BM or MPB as HSPC cell sources, generating new references for future interpretation of clinical data and transplantation outcome.
CRISPR/Cas9‐mediated rescue of osteoclast function in a stem cell model of osteopetrosis
1: University Medical Center Göttingen 2: Charité ‐ Universitätsmedizin Berlin
Autosomal recessive osteopetrosis (ARO) is a severe hereditary bone disease characterised by non‐functional osteoclasts resulting in fractures, immunological and neurological problems, and in bone marrow failure if untreated. Osteoclasts are multinucleated bone‐resorbing cells generated by fusion of hematopoietic progenitors. One of the major causes of ARO are mutations in the CLCN7 gene encoding the Cl‐/H+‐exchanger ClC‐7, which is essential for extracellular acidification by the osteoclast. Studies in mouse models demonstrated normal bone homeostasis at 30% remaining Clcn7 expression. To date, the only curative treatment for ARO is allogenic stem cell transplantation, which is limited by the availability of a suitable donor, the risk of engraftment failure, and requires lifelong treatment.
Therefore, we developed a gene therapeutic strategy for ex vivo correction of stem cells with the CRISPR/Cas9 system. We used a patient‐derived induced pluripotent stem cell (iPSC) line carrying compound heterozygous mutations in exon 10 and exon 14 of the CLCN7 gene. CRISPR/Cas9 combined with single stranded DNA oligonucleotides was used to correct the mutations in ARO iPSCs. Deep sequencing of the transfected bulk cultures revealed 30% and 38% targeted correction of the pathogenic mutations in CLCN7 exon 10 and 14, respectively. Analysis of the bone resorption capacity of osteoclasts differentiated from edited iPSCs showed a partial rescue of the osteoclast function of 30% compared to a healthy control.
Our study provides a proof of concept for the rescue of osteoclast function by genetic manipulation with CRISPR/Cas9, indicating the possibility of a gene therapeutic strategy for the treatment of ARO.
Transcriptional and epigenetic identity of hiPSC‐derived neural stem/progenitor cells: implications for cell therapy approaches
1: San Raffaele Telethon Institute for Gene Therapy (SR‐Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy 2: Computational and Chemical Biology, Fondazione Istituto Italiano di Tecnologia, Genoa, Italy 3: Institute for Biomedical Technologies (ITB), National Research Council (CNR), Segrate, Italy 4: Institute of Genetic and Biomedical Research, UoS of Milan, National Research Council (CNR), Rozzano, Italy 5: Genomic Unit, IRCCS Humanitas Clinical and Research Center, Rozzano, Italy 6: Laboratory of chromatin and gene regulation during development, INSERM UMR1163, Imagine Institute, Paris, France
Human iPSC‐derived neural stem/progenitor cells (hiPSC‐NPCs) are a promising source for cell/gene therapy approaches to treat neurodegenerative disorders with unmet clinical needs. A comprehensive characterization of hiPSC‐NPCs identity is key to improve efficacy and safety criteria for prospective clinical application. To this end, we coupled the global evaluation of the transcriptional and epigenetic signatures marking the hiPSC to hiPS‐NPC commitment with functional studies aimed to assess the behavior of hiPSC‐NPC upon transplantation in the host central nervous system. RNA‐seq and ChIP‐seq data revealed a strong downregulation of pluripotency, cell cycle, and cancer‐related pathways and concomitant acquisition of a distinct “neural signature” in hiPSC‐NPCs. We highlighted a dramatic change in the usage of cell‐specific enhancers and super‐enhancers during neural commitment, suggesting their major role in the generation and maintenance of hiPSC‐NPCs. Transcriptional and epigenetic differences between hiPSC‐NPCs and human fetal NSCs (hfNSC, used as reference) were not associated with abnormal differentiation or pluripotent “memory” reactivation. Single cell RNA‐seq analyses confirmed the heterogeneity of hiPSC‐NPC lines, which are mainly composed of radial glia‐like cells and committed progenitors. Upon intraventricular transplantation in neonatal mice, hiPSC‐NPCs showed robust engraftment, widespread migration in the brain parenchyma, and predominant differentiation into oligodendrocytes and astrocytes, with no evidence of abnormal proliferation up to 12 months after transplant. Results of this study support the use of fully characterized hiPSC‐NPC populations for cell‐based approaches to treat neurodegenerative and demyelinating disorders.
Extracellular vesicles produced by mesenchymal stromal cells inhibit rather the progression than initiation of fibrosis.
1: Institute for regenerative medicine, Medical Research and Education Center, Lomonosov Moscow State University, Moscow, Russia 2: Faculty of Medicine, Lomonosov Moscow State University, Moscow, Russia
The regenerative effects of the mesenchymal stromal cells (MSC) secretome on tissue repair after damage and development of fibrosis were demonstrated in multiple studies. However, which stages of fibrosis were mostly targeted by different MSC secretome components, remained unclear.
To clarify this issue, we evaluated the effects of the extracellular vesicle (EV‐MSC) fraction and non‐vesicular fraction of MSC secretome using bleomycin‐induced pulmonary fibrosis model in mice. After the simultaneous administration of the investigated components and bleomycin, there were no significant therapeutic effects. Moreover, in the group with EV‐MSC administration, the severity of fibrosis was increased. However, after administration of MSC secretome components 14 days after bleomycin exposure, EV‐MSC, but not the non‐vesicular fraction, significantly decreased signs of fibrosis. Only in the group with EV‐MSC administration the area of collagen deposition and the severity of fibrosis according to the Ashcroft scale decreased significantly, and the area of functional lung tissue increased by 40‐45%. Also, using these models, we showed that the effect of EV‐MSC on fibrosis progression was based on their ability to regulate transdifferentiation of stromal cells into activated fibroblasts and myofibroblasts ‐ the main drivers of fibrosis.
Our results indicate that EV‐MSC can inhibit and reverse the developed fibrosis, but not prevent its initiation and progression in the early stages of injury. Apparently, the therapeutic effect of MSC secretome or its components on fibrosis could be closely related to the time‐point of their administration. The study was funded by RFBR grants #20‐04‐60487 and 20‐315‐90120.
Process‐related impurities in the ChAdOx1 nCov‐19 vaccine
1: Ulm University
To fight the SARS‐CoV2 pandemic, the ChAdOx1 nCov‐19 vaccine has been developed and approved. ChAdOx1 nCov‐19 is based on the chimpanzee adenovirus Y25 with modifications of the E1, E3 and E4 region and encodes for the SARS‐CoV2 spike protein. Following vaccination, rare cases of vaccine‐induced immune thrombotic thrombocytopenia (VITT) have been reported. The initial trigger for these serious adverse events has not been determined. Initially intended to exclude any product‐related issues, we analysed the ChAdOx1 nCov‐19 vaccine by biochemical and proteomic methods. Unexpectedly, we found that the vaccine, in addition to the adenovirus vector, contained substantial amounts of both human and non‐structural viral proteins. Among the human proteins, heat‐shock proteins and cytoskeletal proteins were particularly abundant. We propose that the often‐observed strong clinical reaction one or two days after vaccination might be associated with the detected protein contaminants. A linkage to later immune related adverse events is also conceivable. The here reported identification of specific classes of protein impurities should guide and accelerate efforts to improve the purity of adenoviral vector‐based vaccines and quality assessment methods to increase safety and efficacy.
Multicentre, prospective research protocol for development of a clonal neoantigen‐reactive T cell therapy pipeline across multiple tumour types
1: Achilles Therapeutics UK
Adoptive cell therapies derived from tumour‐ infiltrating lymphocytes (TIL) can generate durable responses in solid tumours, with most clinical data in melanoma. We are developing precision TIL therapies targeting clonal neoantigens, using proprietary platforms to identify (PELEUSTM) these targets and expand (VELOSTM) clonal neoantigen‐reactive T‐cells (cNeT). To explore the potential of cNeT across indications, it is necessary to characterise baseline tumour, TIL intermediates and final cNeT products in different malignancies, and to evaluate factors that can affect these. The Material Acquisition Platform (MAP) study (NCT03517917) is a prospective research protocol collecting tumour tissue and blood in a range of cancers (Lung, Melanoma, Head and Neck, Renal, Bladder and Breast) across 7 sites in the UK and US, with 13 tumour procurement channels. Ninety patients are currently enrolled; median age is 67 years; 54 (60.0%) are male. 59 (65.6%) had tissue procurement at first diagnosis, 31 at relapse. 19 (21.1%) had received prior systemic anti‐cancer therapy. Mutational burden reflected previously published experience across tumour types. From samples containing TIL, the median number of TIL was 45.9x106 (range: 0.14x106‐161x107). There was no significant difference in TIL numbers between newly diagnosed and recurrent (p = 0.307), or treatment‐naïve and pre‐treated (p = 0.149) patients. Expansion of cNeT was feasible across all tumour types evaluated and further detail on protocols and product characteristics (phenotype, reactivity and cytokine production) will be shared in the presentation. MAP facilitates continued process development and initial analyses demonstrate TIL extraction from multiple tumour types is not significantly impacted by previous therapies.
Development of a novel lipid nanoparticle with widespread photoreceptor delivery of ceDNA & mRNA cargos
1: Generation Bio
Viral‐based gene therapy in the retina has been limited to a subset of inherited retinal diseases (IRD) due to the limited AAV packaging capacity of ∼4.7Kb. Non‐viral delivery of large nucleic acid cargos can overcome this capacity barrier for gene replacement or correction, yet poor tolerability and expression profiles for existing polymer and lipid nanoparticle (LNP)‐based delivery systems has historically limited their use for ocular indications. We have developed a new class of cell targeting LNPs (ctLNPs) that avoid local immune cell uptake to improve tolerability and achieve efficient, uniform transduction of photoreceptors and RPE. While other classes of LNPs lead to severe outer nuclear layer (ONL) loss with chronic inflammation with either mRNA or DNA cargos, both mouse and rat showed no chronic degeneration or inflammation at Day 21 following subretinal ctLNP delivery. Long‐term maintenance of GFP transgene expression and tolerability with ctLNP‐DNA was demonstrated out to Day 63. Expression levels were consistent with AAV5.GFP in the retina. ctLNP/nucleic acid delivery led to widespread photoreceptor transduction as detected by GFP immunohistochemistry, unlike AAV.GFP that showed uneven ONL expression. Retina transduction was enhanced using ligand‐receptor pairs that facilitate LNP uptake and was confirmed using receptor knockdown and competition assays in ARPE19 cells. Initial findings suggest ctLNPs are well tolerated in the retina, avoid immune cell uptake, and efficiently deliver nucleic acid cargos across species with a uniform transduction pattern in photoreceptors and RPE. Based on this favorable profile future work will explore ctLNP‐based therapeutic applications for Stargardt disease.
Modelling cardiogenesis using intravital hydrogel bioprinting and tissue engineering identifies novel roles for hyaluronan and ECM mechanics during human trabeculogenesis
1: University College London 2: Università degli Studi di Padova 3: Shanghai University
Advances in bioengineering now enable complex tissue assembly and simulations of morphogenic events in vitro, creating robust modelling platforms of human development and congenital disorder manifestation. The embryonic myocardium of the primary heart tube undergoes rapid and asymmetrical architectural transformation initiated by polarising cues from the adjacent endocardium and the cardiac‐specific extracellular matrix (ECM), termed cardiac jelly. The signals that deliver a polarising instruction in a three‐dimensional system can now be emulated in a controllable laboratory format using established technologies of pluripotent stem cell‐derived cardiovascular tissue engineering and emerging ones of intravital bioprinting.
Using human induced pluripotent stem cell (hiPSC)‐derived cardiac progenitor cells (CVPs) seeded onto multiphoton‐bioprinted cardiac jelly‐mimicking scaffolds we have engineered the embryonic heart tube to explore how the myocardium becomes polarised in utero. Scaffolds are designed with geometries, biochemical composition and biophysical properties of embryonic heart ECM, upon which hiPSC‐CVP on‐scaffold differentiation to cardiomyocytes closely mimics heart tube formation; namely transmural matrix contact and exogenous simulations of endocardial‐to‐myocardial signalling in a highly biomimetic model. Motile CVPs seeded onto bioprinted scaffolds differentiate functionalised contractile tubular tissues, maintaining lumens that occlude with contractions and displaying organised sarcomeric alignment. Engineered Cardiac Tubes host spontaneous hyaluronan (HA) secretions reminiscent of cardiogenesis, and exhibit a morphogenic priming in the form of proliferation and trabecular fate bias in response to the endocardial signalling mitogen Neuregulin‐1β (NRG1β). We anticipate reverse engineering the human embryonic heart will emerge as a versatile method for uncovering human data in a field populated almost exclusively by animal studies.
Integration site analysis in patients with cerebral adrenoleukodystrophy (CALD) treated with elivaldogene autotemcel (Lenti‐D; eli‐cel) gene therapy
1: Division of Pediatric Blood and Marrow Transplantation & Cellular Therapy, Department of Pediatrics, University of Minnesota Masonic Children's Hospital, Minneapolis 2: Division of Hematology/Oncology, Department of Pediatrics, Boston Children's Hospital, Harvard Medical School 3: Children's Cancer and Blood Disorders, Boston Children's Hospital and Dana‐Farber Cancer Institute 4: Department of Neurology, Massachusetts General Hospital and Harvard Medical School 5: Clinical Research Development, bluebird bio, Inc. 6: Severe Genetic Diseases Research, bluebird bio, Inc.
As of October/November 2020, 51 patients with CALD treated with elivaldogene autotemcel gene therapy (eli‐cel; Lenti‐D lentiviral vector [LVV]‐transduced autologous CD34+ cells) were followed for median 29.1 (range 0.1‒82.7) months in studies ALD‐102, ALD‐104, and long‐term follow‐up in LTF‐304. Of evaluable patients, 27/30 (90%) were alive and major functional disabilities (MFDs)‐free at Month‐24 and without MFDs in LTF‐304.
The highest number of unique integration sites (IS) in blood/patient at any visit was median 5277 (range 552‒15683) (n = 42). As of 9 August 2021, 3 predominant clones, defined as >50% IS contribution to blood or any lineage, were identified in 3 patients: one at Month‐65 (ALD‐102/LTF‐304), two at Month‐6 (ALD‐104). These clones, with insertions into and dysregulated expression of MECOM, also had insertions in additional genes.
Multiple bone marrow (BM) evaluations of the ALD‐102/LTF‐304 patient showed benign clonal expansion. Both ALD‐104 patients experienced platelet engraftment >Day‐100 and persistent thrombocytopenia. BM evaluations at months‐12/14 (ALD‐104 Patient‐1) and months‐22/24 (ALD‐104 Patient‐2) showed normo‐ or hypo‐cellularity with dysplastic megakaryocytes without other cytogenetic or molecular abnormalities characteristic of leukemic transformation. However, based on BM analysis and persistent thrombocytopenia, at Month‐14, ALD‐104 Patient‐1 was diagnosed with myelodysplastic syndrome (MDS) with unilineage dysplasia, likely mediated by Lenti‐D LVV insertion.
These findings highlight a potential role for MECOM dysregulation in clonal expansion post‐Lenti‐D LVV gene therapy, which can evolve to MDS. As most patients have clinically benefitted from eli‐cel, such events need to be considered in the context of the known morbidity/mortality of the currently available treatment option of allogeneic transplantation.
Monitoring of vector integration sites in in vivo gene therapy approaches by Liquid‐Biopsy‐Integration‐Site‐Sequencing
1: San Raffaele Telethon Insitute for Gene Therapy (HSR‐TIGET) 2: Laboratory of Human Lympho‐hematopoiesis, INSERM, France 3: Institut de Génétique Moléculaire de Montpellier, CNRS, France 4: Dr. von Hauner Children's Hospital, LMU, Germany 5: Department of Microbiology, UPeNN, USA
Clonal tracking techniques by monitoring the fate of transduced cells enable the assessment of safety and efficacy of GT procedures. However, these studies are limited in in vivo GT context since required invasive tissue biopsies.
Recently, we developed Liquid Biopsy Integration Site sequencing (LiBIS‐seq), a PCR technique optimized to quantitatively retrieve vector integration sites (IS) from cell‐free DNA (cfDNA) which is released into the bloodstream by dying cells residing in diverse tissues. This approach enabled the early detection of a T‐cell lymphoma triggered by insertional activation of LMO2 in a patient enrolled in a γ‐retroviral‐based clinical trial for X‐SCID, while conventional IS analyses on circulating cells failed to detect such aberrant expansion. Furthermore, in 75% of newborn mice systemically injected with genotoxic LVs, LiBIS‐seq was able to detect the presence of hepatocellular carcinoma and histiocytic sarcoma induced by insertional mutagenesis (N = 14). Finally, by using a specific multiplexed PCR protocol, LiBIS‐seq was able to identify AAV IS in cfDNA of ZAP70 knock‐out mice that were intra‐thymically injected by an AAV8 expressing the therapeutic transgene (N = 8). More than 800 AAV IS were retrieved from lymph‐node DNA and cfDNA revealing an enrichment of IS in TCR genes, thus reflecting the high level of RAG‐mediated recombination events occurring in these loci during T cell differentiation.
In conclusion, LiBIS‐Seq allows to study the clonal repertoire of genetically modified cells residing in solid organs in LV‐ and AAV‐based in‐vivo GT applications without the need of invasive tissue biopsies.
Sequencing of genotoxic recombinant adeno‐associated viral integrations events found in murine hepatocellular carcinomas reveals conservation of specific transgene elements
1: National Institutes of Health
Hepatocellular carcinoma (HCC) in mice after recombinant adeno‐associated virus (rAAV) treatment was first reported some two decades ago, but until recently these findings were largely considered a species specific phenomenon. Recent reports of an association between human HCC and wildtype AAV infections, clonal rAAV integrations in hemophilic dogs, and the occurrence of HCC patient following rAAV treatment have again raised concerns about the possibility of rAAV integrations causing insertional mutagenesis in humans and highlight the importance of understanding the mechanisms by which rAAV causes HCC in mice. Previously, we reported that rAAV integrations in the Rian locus resulted in HCC in mice. Here, we report 5 complete and 6 partial HCC‐rAAV integrations sequences from 11 different mice. The rAAV integration events completely sequenced range in size from 428 to 1,216 base pairs. Interestingly, none of the 11 rAAV integrations contained sequences from the coding region of the transgene. All the rAAV integrations possessed a portion of the 5’ ITR, a wildtype AAV enhancer element and the enhancer element from the transgene. The presence of the AAV and transgene enhancers in all 11 AAV integrations suggest that these elements are likely the primary drivers of the upregulation of proximal genes and tumorigenesis. Studying insertional mutagenesis in animals as we have done here could help determine if HCC is the result of the underlying disease being treated or related to rAAV toxicity, and would help inform risks after human gene therapy with AAV vectors.
High‐resolution cellular and molecular follow‐up of lysosomal disorder patients treated with hematopoietic stem cell lentiviral gene therapy
1: AVROBIO Inc, Cambridge, MA 2: Division of Genetics, Department of Pediatrics, University of California, San Diego, La Jolla, CA 3: John Hopkins Medicine, Baltimore, MD, USA 4: Advanced Centre for Biochemical Engineering, University College London, London, UK 5: Great Ormond Street Institute of Child Health Faculty of Population Health Sciences, London, UK
Lysosomal disorders (LD) are inborn errors of metabolism causing buildup of toxic material in patients' cells. We will present interim analysis of exploratory studies on LD patients treated with ex vivo lentiviral (LV) gene therapy (GT) utilizing AVROBIO's plato® platform where plerixafor and G‐CSF mobilized peripheral blood (PB) CD34+ cells are genetically modified with LV vectors to insert the therapeutic genes in a closed, automated system and infused after personalized busulfan conditioning deploying precision dosing.
We designed and conducted a high‐resolution cellular and molecular follow‐up of bone marrow (BM) and PB samples after GT. Thirteen BM subpopulations at multiple differentiation stages and seven PB subtypes were sorted (average purity 99%) and analyzed by ddPCR‐vector copy number and integration site (IS).
We used clonal tracking data, combining three separate methods for abundance estimation and non‐linear regression models, to explore the dynamics of each blood cell population after GT. We designed network analyses to estimate progenitor cell clonal output by means of IS sharing based on the most recent concepts on hematopoietic hierarchies, and for the first time used BM and PB baso/eosinophils as surrogate markers for the megakaryoid/erythroid‐lineage output. Lastly, we implemented high‐throughput scRNA‐Seq analyses of BM CD34+ cells isolated before and after GT (to date 60,718 single‐cell transcriptomes), achieving a uniquely granular view of hematopoietic progenitor states after transplantation and in different LD backgrounds.
These are providing high‐resolution insight on the fate of genetically engineered hematopoietic stem and progenitor cells and their progeny during reconstitution after GT.
Durable efficacy of AAV‐based gene therapy for PFIC3 (VTX‐803) in mice at different disease stages
1: Vivet Therapeutics S.L., Pamplona, 31008, Spain 2: Division of Gene Therapy and Regulation of Gene Expression, Cima Universidad de Navarra, Pamplona, 31008, Spain 3: Vivet Therapeutics S.A.S., Paris, 75008, France 4: Instituto de Investigación Sanitaria de Navarra (IdISNA), Pamplona, 31008, Spain
Gene addition based on liver‐directed AAV delivery is emerging as a successful option for treating rare disorders caused by inborn genetic errors. Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare metabolic disease caused by inherited mutations in ABCB4, which encodes for MDR3, a membrane‐bound phosphatidylcholine (PC) floppase involved in the regulation of bile composition. Without fully functional MDR3, insufficient PC is transported into the bile, which results in toxicity to hepatocytes and cholangiocytes, leading to cholestasis, cirrhosis and end‐stage liver disease requiring transplantation. We have developed an AAV vector delivering a codon optimized MDR3 transgene under the control of a liver specific promoter (VTX‐803), and tested it with an AAV8 serotype in preclinical studies. Therapeutic efficacy was observed with intravenous administration to two‐week‐old (pre‐fibrotic liver) or five‐week‐old (fibrotic liver) Abcb4−/− mice as seen in the normalization of serum biomarkers, hepatosplenomegaly and liver fibrosis. The same therapeutic effect was obtained in older animals (16 weeks) presenting severely fibrotic/cirrhotic livers, without an increase in dose. Long‐term studies have shown the vector to be safe and the effect to be durable to 12 months. The efficacy was comparable between 5‐week‐old and 16‐week‐old mice associated with equivalent levels of MDR3 mRNA‐positive hepatocytes. Finally, it was found that the diseased state of the PFIC3 mouse liver affects transduction efficiency since wildtype mice of the same background were more efficiently transduced with the vector at equivalent doses. These results confirm the strong potential of VTX‐803 as a candidate therapy for this devastating childhood disease.
Administration during liver growth improves the efficiency of lentiviral vector based gene therapy in mice
1: San Raffaele Telethon Institute for Gene Therapy (HSR‐TIGET) 2: Universita Vita‐Salute San Raffaele 3: San Raffaele Scientific Institute
The liver is a relevant target organ for in vivo gene therapy, given the opportunity to treat metabolic and haematological diseases. We previously developed a lentiviral vector (LV) platform for liver‐directed gene therapy in the context of coagulation disorders, obtaining efficient gene transfer in adult immune‐competent mice, dogs and non‐human primates, with a dose‐dependent reconstitution of clotting factors IX (FIX) or VIII. We now aim to investigate the stability of liver‐directed LV gene therapy in mice during post‐natal growth and homeostasis. We treated mice by intravenous delivery of LV.FIX and observed the highest transgene output in mice treated at 2 weeks of age, intermediate levels in newborn‐treated mice and the lowest in adult‐treated mice. The low transgene output in adult compared to young treated mice is explained by a reduced transduction of hepatocytes, paralleled by an increased uptake by non‐parenchymal cells. We also observed a switch from peri‐central to peri‐portal hepatocytes transduction bias at the age of weaning, which seems to be independent from LV phagocytosis by tissue‐resident macrophages. Furthermore, we exploited AlbCreERT2/Rosa26‐Confetti mice to assess hepatocyte clonal proliferation during post‐natal liver growth. We observed that 25% of hepatocytes proliferates and contributes to form most of the adult liver and experiments are ongoing to characterize this subpopulation. We also show that transduced and untransduced cells have comparable proliferation rate. These studies indicate that timing of administration impacts on efficiency of LV gene transfer, may also be relevant for genome editing strategies and inform future developments for application to paediatric patients.
Hepatocyte‐directed AAV gene transfer drives efficient long‐term rescue of the metabolic impairment in a mouse model of glycogen storage disease type Ia
1: Université Lyon 1 2: GENETHON 3: Université Paris‐Saclay
Glycogen storage disease type Ia (GSDIa) is a rare disease due to mutations in the glucose‐6‐phosphatase catalytic subunit 1 (G6PC1) gene, leading to a decreased activity of glucose‐6‐phosphatase (G6Pase). G6Pase hydrolyzes glucose‐6‐phosphate into glucose. In GSDIa patients, reduced hepatic G6Pase activity results in severe hypoglycemia, hepatomegaly, and hepatocellular adenomas development. Preclinical and recent clinical studies (NCT03517085) have shown the potential of AAV for the treatment of GSDIa. However, although glycaemia correction may require a low G6pase activity, the normalization of the metabolic impairment and the prevention of tumor development may require a larger percentage of hepatocytes transduction.
In this study, we evaluated long‐term efficacy of AAV9 vectors expressing G6PC1 under the control of a liver specific promoter, or the human G6PC1 promoter (hGPE), or its murine version (mGPE), in a liver‐restricted model of GSDIa, the L.G6pc‐/‐ mice. Mice were injected with 8x1012 vg/kg and were fed a standard diet, or a high fat/high sucrose (HF/HS) diet known to promote tumor formation. In standard diet conditions, we observed a complete correction of glycaemia and a normalization of the metabolic status 6 months post‐injection. Under the HF/HS diet, the vector expressing G6Pase under the control of the liver specific promoter achieved the highest G6Pase activity and the best metabolic rescue associated with reduced tumor formation.
This study suggests that enhanced and specific hepatocyte targeting can rescue the metabolic impairment in a relevant GSDIa preclinical model and supports the use of a liver specific promoter for the development of a gene therapy approach for GSDIa.
AVR‐RD‐01, an investigational lentiviral gene therapy for Fabry disease: interim results from Phase 1 and Phase 2 studies
1: Royal Perth Hospital, WA 6000, Australia 2: Royal Melbourne Hospital, Parkville, VIC 3050, Australia 3: AVROBIO, Inc., Cambridge, MA 02139, USA 4: University of Florida, Gainesville, FL 32611, USA 5: Advanced Centre for Biochemical Engineering, University College London, London WC1E 6BT, UK 6: John Hopkins Medicine, Baltimore, MD 21224, USA
Fabry disease (FD) is a monogenic lysosomal disorder associated with impaired alpha‐galactosidase A (AGA) enzyme activity and pathological accumulation of substrates and metabolites, including globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso‐Gb3), which can lead to chronic kidney and cardiac diseases.
Treatment with AVR‐RD‐01, an investigational gene therapy, involves infusion of autologous stem cells genetically modified ex vivo with a lentiviral vector containing a ribonucleic acid (RNA) transcript that, after reverse transcription, results in codon‐optimized complementary deoxyribonucleic acid (cDNA) that, upon its integration into the human genome, encodes for functional human AGA.
In the ongoing Phase 1 investigator‐sponsored study of AVR‐RD‐01, five adult male FD patients, who were receiving enzyme replacement therapy (ERT), were treated with a single infusion of AVR‐RD‐01. To date, three out of five patients have elected to no longer receive ERT after treatment with AVR‐RD‐01.
As of June 30, 2021, eight adult treatment‐naïve classic FD male patients have been treated with AVR‐RD‐01 in the ongoing AVROBIO‐sponsored Phase 2 study. The two Phase 2 participants with evaluable kidney biopsies at 48‐weeks post infusion demonstrated an 87% and 100% reduction in renal peritubular capillary Gb3 inclusions compared to baseline.
This presentation will include updated safety data for all patients, including data on those patients in the Phase 2 trial conditioned with a precision dose of busulfan 90 mg.h/L target concentration intervention (TCI).
AVR‐RD‐01 has generally been well tolerated. Adverse events have been assessed as not related to the drug product but related to the conditioning regimen, underlying disease or pre‐existing conditions.
In vivo
evaluation of novel synthetic promoters for CNS gene therapy
1: Gene Transfer Technology Group, EGA Institute for Women's Health, University College London, UK 2: AskBio (Europe), Roslin Innovation Centre, Edinburgh, UK 3: Wits/SAMRC Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
Neurological diseases are diverse with different brain regions and cell types affected. Gene therapy for previously untreatable neurological diseases is now a clinical reality, however the use of ubiquitous promoters may not be suitable for all indications. The development of customised design vectors is required before gene therapy can address the diversity of neurological diseases. Using AskBio's Synpromics bioinformatics‐based promoter design platform, novel synthetic central nervous system promoter candidates were generated and evaluated in vivo.
Thirteen novel promoters driving expression of GFP reporter gene were evaluated in rAAV9 vectors. P0 CD1 mice received titre matched vector by intracerebroventricular or intravenous delivery and were euthanised at 5 weeks to assess GFP expression. CNS distribution and cell type GFP expression were characterised in detail by free‐floating whole brain immunohistochemistry and immunofluorescence. Systemic organ biodistribution and brain regional vector copy numbers were evaluated by qPCR. Human Synapsin 1 promoter was used as a control. All novel constructs were active in the CNS with rostrocaudal gradient. Within these novel promoters, we identified a candidate with higher activity in midbrain dopaminergic neurons. We generated a gene therapy construct to validate this novel promoter in the dopamine transporter knockout (DAT‐KO) mouse as a model of dopamine transporter deficiency syndrome: infantile parkinsonism dystonia. Heterozygous mice were time‐mated to generate DAT‐KO pups that received intravenous gene therapy at P1. Untreated littermates were used as controls (n = 10/group). Here, we present biodistribution of 13 novel CNS promoters, and therapeutic validation data of a novel promoter active in midbrain dopaminergic neurons.
Developing a gene‐replacement therapy for CDKL5‐deficiency disorder
1: University of Pennsylvania 2: Children's Hospital of Philadelphia
CDKL5‐deficiency disorder (CDD) is a rare neurodevelopmental disorder that affects children and is caused by loss‐of‐function mutations in the X‐linked cyclin‐dependent kinase‐like 5 (CDKL5) gene. Symptoms include early‐onset seizures, intellectual disability, stereotypies, and motor impairment. There is currently no cure for CDD. Gene therapy is a promising curative approach in which administration of an adeno‐associated virus (AAV) encoding human CDKL5 could restore CDKL5 protein expression.
We show here that gene therapy with an AAV vector comprising an AAVrh91 capsid and a codon‐optimized human CDKL5 transgene restored CDKL5 expression and kinase activity in a CDD mouse model. Neonatal intracerebroventricular injection of AAV‐CDKL5 vector resulted in up to 80% of cortical neurons expressing CDKL5 for at least five months. Treated Cdkl5‐knock‐out mice performed significantly better than the untreated cohort in a battery of neurobehavioral assays. In a separate cohort, we investigated resting and evoked electroencephalogram responses.
To test our AAV‐CDKL5 gene‐therapy vector in a larger animal, we conducted a two‐month study on non‐human primates. Optimized infusion into the cerebrospinal fluid via the cisterna magna achieved vector distribution throughout the brain at 1‐5 vector copies per cell. Including a dorsal‐root ganglia (DRG) de‐targeting cassette in the AAV‐CDKL5 vector prevented DRG‐mediated toxicity. All three non‐human primates tolerated AAV‐CDKL5 gene therapy well.
Here we present pre‐clinical evidence that CDKL5 gene therapy provides a lasting curative benefit to model mice and is well tolerated in non‐human primates. Further optimizations may offer an option for clinical intervention in children affected by CDD.
Therapeutic AAV‐mediated homology‐independent targeted integration in the retina
1: Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli 80078, Italy 2: Eye Clinic, Multidisciplinary Department of Medical, Surgical and Dental Sciences, University of Campania L. Vanvitelli, Naples 80131, Italy 3: Next Generation Diagnostic srl, Pozzuoli 80078, Italy 4: Medical Genetics, Department of Translational Medicine, Federico II University, Naples 80131 Italy
Retinitis pigmentosa (RP) is a heterogeneous group of inherited ocular diseases with a prevalence of approximately 1 in 4000 individuals. Thirty‐40% of cases of RP have an autosomal dominant (Ad) pattern of inheritance. Mutations in the rhodopsin (RHO) gene are responsible for ∼20% of AdRP cases worldwide.
Homology‐independent targeted integration (HITI), based on CRISPR/Cas9 cleavage followed by non‐homologous end‐joining repair, allows precise integration of a donor DNA at a desired genomic locus. We have exploited HITI mediated by AAV vectors as an allele‐independent approach to target RHO gain‐of‐function mutations.
We subretinally co‐injected in either mice or pigs, a clinically‐relevant large animal model, one AAV encoding SpCas9 and one carrying both a donor DNA with the fluorescent DsRed reporter and an expression cassette for a Rho‐specific gRNA. In both mouse and pig photoreceptors we observed ∼5% efficiency of integration at the Rho locus which was precise. After exchanging the coding sequence of DsRed with that of RHO, we found that HITI provides therapeutic benefit in a mouse model of AdRP resulting in significant improvement of both retinal function and morphology. This study provides a proof‐of‐concept of the therapeutic potential of HITI for RP as well as for other inherited retinal diseases.
AAV‐mediated gene therapy to treat Niemann‐Pick Type C2
1: CBATEG ‐ UAB 2: Department of Biochemistry and Molecular Biology (UAB) 3: Department of Animal Health and Anatomy (UAB) 4: CIBERDEM 5: Department of Nutritional Sciences, Rutgers University 6: Rutgers Center for Lipid Research
Niemann‐Pick type C2 (NPC2) disease is a rare autosomal recessive lysosomal storage disorder caused by deficiency of NPC2 gene, involved in the egress of unesterified cholesterol from the endo‐lysosomal compartment. NPC2 deficiency leads to pathologic accumulation of cholesterol in lysosomes. Patients develop severe, progressive neurological disorders together with relatively mild peripheral pathology. Death usually occurs during the first or second decade of life. Treatment of NPC2 disease represents an unmet medical need. In vivo gene therapy with adeno‐associated vectors (AAV) may offer the possibility of lifetime treatment following a single intra‐CSF administration. Here, we first developed a new mouse model of NPC2 disease, generated by targeted disruption of Npc2 gene, that recapitulated the main hallmarks of the disease. Efficacy of intra‐CSF delivery of AAV9‐Npc2 vectors was assessed in Npc2‐/‐ mice. AAV9‐Npc2 gene transfer resulted in significant decrease of unesterified cholesterol storage and lysosomal pathology in CNS, leading to increased myelination and reduced neuroinflammation and neurodegeneration (Purkinje cells). Moreover, AAV9‐Npc2 vectors also transduced the liver, providing a source of therapeutic protein that corrected peripheral cholesterol storage and lysosomal pathology. Finally, AAV9‐Npc2 treatment also normalised locomotor deficits, improved body weight and prolonged survival. Altogether, our results demonstrated that intra‐CSF administration of AAV9‐Npc2 vectors led to widespread correction of both CNS and peripheral pathology in a NPC2 mouse model. This study supports clinical translation of AAV9‐Npc2‐based gene therapy to treat human patients.
Lentiviral mediated gene therapy for pyruvate kinase deficiency: Interim results of a global phase 1 study for adult and pediatric patients
1: Hospital Universitario Fundación Jiménez Díaz, Madrid, Spain 2: Unidad Mixta de Terapias Avanzadas, Instituto de Investigación Sanitaria Fundación Jiménez Díaz, Madrid, Spain 3: Center for Definitive and Curative Medicine, Stanford University, Stanford, CA 4: Div. of Pediatric Hematology/Oncology/Stem Cell Transplant and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 5: Stanford Children's Hospital, Palo Alto, CA 6: Centro de Investigación Biomédica en Red de Enfermedades Raras, Madrid, Spain 7: Instituto de Innovación Biomédica, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spain 8: Hematología y Hemoterapia, Fundación para la investigación Biomédica HIUNJ, Hospital Infantil Universitario Niño Jesús, Madrid, Spain 9: Rocket Pharmaceuticals, Inc., Cranbury, NJ
Pyruvate kinase deficiency (PKD) is an inherited hemolytic anemia caused by mutations in the PKLR gene resulting in decreased red cell pyruvate kinase activity and impaired erythrocyte metabolism. Manifestations include anemia, reticulocytosis, splenomegaly and iron overload. Current treatments are limited to blood transfusions, chelation therapy, and splenectomy which are associated with significant side effects. Preclinical studies in a murine model have demonstrated that infusion of gene‐modified Lin− bone marrow (BM) cells may ameliorate PKD phenotype. Based on compelling preclinical data, a global Phase 1 clinical trial RP‐L301‐0119 (NCT04105166) is underway to evaluate the safety of lentiviral mediated gene therapy in subjects with severe PKD.
Six adult and pediatric patients with severe PKD (defined as severe and/or transfusion‐dependent anemia despite prior splenectomy) will be enrolled. Peripheral blood (PB) hematopoietic stem cells (HSCs) are collected via apheresis, transduced with PGK‐coRPK‐WPRE lentiviral vector, and cryopreserved. Following myeloablative busulfan conditioning, RP‐L301 is then infused. Patients are followed for safety and efficacy assessments for 2 years.
As of May 2021, 2 adult patients with severe anemia have received RP‐L301 and have normal‐range hemoglobin (Hb), improved hemolysis markers, and have required no red blood cell transfusions post‐engraftment at 9‐ and 6‐ months follow‐up. Both report improved quality of life. No serious adverse events have been attributed to RP‐L301. Updated safety and efficacy data will be presented.
HSC mobilization is feasible and effective in adult PKD patients. Normalized Hb is associated with engraftment as measured by PB and marrow (BM) vector copy number (VCN).
Homology Independent Gene Editing Strategies to Correct Hematopoietic Stem Cells from Fanconi Anemia A Patients
1: Division of Hematopoietic Innovative Therapies, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIEMAT/CIBERER), Madrid 28040, Spain; Advanced Therapies Unit, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS‐FJD, UAM), Madrid 28040, Spain. 2: Department of Biology, ETH Zurich, Zurich, Switzerland
Gene editing has become a promising strategy for acutely correct a wide range of disease related mutations. Although Homologous Directed Repair (HDR) is the conventional mechanism used to correct specific mutations, in the case of disorders associated with marked defects in this DNA repair mechanism, such as Fanconi anemia (FA), the application of base and prime editing, that are independent of HDR and do not generate double strand breaks in the cell could be a great opportunity. Our results show that base editing constitutes a promising and very efficient therapeutic strategy to correct specific base substitutions in hematopoietic stem and progenitor cells (HSPCs), in which we have observed up to 85 % specific editing efficiencies in healthy donor CD34+ cells analyzed by next generation sequencing (NGS). Using this strategy, we have also demonstrated the feasibility to efficiently generate therapeutic base conversions in FA derived HSPCs, reaching clinically applicable efficiencies and demonstrating the phenotypic correction of a very frequent mutation described in FA patients from Spain (FANCA c.295C>T). Additionally, for those mutations that cannot be corrected by base editing, such as the most frequent mutation in FA patients worldwide (FANCA c.3788_37909delTCT), prime editing approach was selected. NGS analysis revealed 5% initial editing efficiency that, showed a marked proliferation advantage with 97% wild type sequence and the complete reversion of the FA phenotype 30 days after in vitro culture.
Altogether our results open new perspectives for the application of precise gene editing approaches to correct FA mutations in HSPCs.
A novel DNA oligo‐based repair strategy for the functional correction of Shwachman‐Diamond Syndrome
1: Dana‐Farber Cancer Institute 2: Boston Children's Hospital 3: Massachusetts General Hospital 4: University of Massachusetts Medical School 5: Harvard Medical School
The autosomal‐recessive bone marrow failure syndrome Shwachman‐Diamond syndrome (SDS) is associated with cytopenia and an increased risk of developing leukemia. The vast majority of patients harbor the 258 + 2T>C splice donor mutation in intron 2 of the Shwachman‐Bodian‐Diamond syndrome (SBDS) gene on chromosome 7.
Here, we explored a short dsDNA oligo‐trapping strategy to re‐establish correct splicing by providing a functional splice donor. When co‐delivering the repair oligo with Cas9, the oligo integrates into the cut‐site via non‐homologous end‐joining (NHEJ). To determine the best repair oligo sequence composition for efficient splicing, a partially randomized oligo was introduced into SDS patient‐derived fibroblasts. The selective advantage mediated by optimal splicing of SBDS led to the enrichment of oligo sequences which are similar to the human splice donor consensus sequence. Based on these findings we designed 30bp oligos which achieved insertion rates of 51 ± 0.83% in SDS patient‐derived cells. Using Cas9‐mSA (monomeric streptavidin) tethering a 30bp biotinylated oligo, the correction rate was further increased to 66 ± 2.67%, which recovered the correct mRNA splicing and SBDS protein expression to approximately 90% of the wildtype. In primary CD34 HSCs, insertion rates of 30 ± 1.54% were achieved.
Our findings show that the most common 258 + 2T>C mutation in SBDS can be efficiently repaired by NHEJ‐mediated insertion of short DNA oligos. This repair mechanism is highly active in quiescent HSC, making this approach potentially more efficient than HDR‐based therapies for indications where target cells are postmitotic or in a quiescent state.
Assessing stealth and sensed base editing in human hematopoietic stem/progenitor cells
1: San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, 20132 Milan (MI), Italy. 2: Vita‐Salute San Raffaele University, 20132 Milan (MI), Italy. 3: National Research Council, Institute for Biomedical Technologies, 20090 Segrate (MI), Italy. 4: Equally contributing Authors.
Engineered nucleases enable targeted genetic manipulation in human hematopoietic stem/progenitor cells (HSPCs) for therapeutic purposes. Still, nuclease‐induced DNA double‐strand breaks (DSBs) trigger p53‐dependent DNA damage response affecting HSPC properties and may lead to chromosomal rearrangements. Base editing (BE) allows for precise editing while bypassing DSBs. However, a comprehensive characterization of efficiency, tolerability and genotoxicity of cytidine (C‐) and adenine (A‐) BE in human HSPCs is lacking and is required to instruct the rationale towards clinical translation. Here, we developed an optimized mRNA‐based protocol for BE in HSPCs and compared CBE4max, ABE8.20‐m and Cas9 nuclease by targeting B2M locus using the same sgRNA. Common outcome is disruption of targeted gene expression measured by FACS and NGS. ABE8.20‐m showed higher efficiency than CBE4max and Cas9 (up to 90, 40 and 50%), which was consistent across HSPC subpopulations and cell sources. Transcriptional analyses uncovered that CBE4max, but not ABE8.20‐m, triggered p53 pathway activation, albeit at lower extent as compared to Cas9 and presumably consequent to a fraction of nicks turning into DSB upon replication. Additionally, BE, and particularly CBE4max, upregulated the expression of interferon‐stimulated genes, which was not ascribed to mRNA delivery. Remarkably, despite edited HSPCs showed long‐term multilineage capacity in xenotransplanted mice, CBE4max edited cells decrease over time pointing to some detrimental response of long‐term HSC subset. Overall, our results prompt further investigation on BE sensing. On‐going studies are aimed to investigate clonal dynamics and genome integrity of base‐edited HSPCs with the final goal of building confidence for their perspective clinical translation.
Administering high‐dose AAV9 vector in wild‐type mice leads to complement activation, liver deposition, and concomitant liver‐transaminase elevations
1: University of Pennsylvania
For many single‐gene disorders, adeno‐associated virus (AAV) gene‐therapy treatments require transduction and expression of the therapeutic protein across a large tissue area. To achieve this, preclinical studies and ongoing clinical trials deliver high AAV doses via systemic injection. However, case reports from these trials describe unanticipated toxicities like thrombocytopenia, liver damage, and complement activation. These complications highlight the need for better preclinical modeling of high‐dose AAV gene therapies. Here, we systemically administered AAV9 at doses in excess of those delivered in the clinic. We injected wild‐type C57BL/6J mice with 3E14 genome copies/kilogram and sacrificed them over several timepoints up to 10 days post‐vector administration. Beginning at 24 hours, and peaking on day 3, mice injected with AAV9 showed elevated C3b levels, indicative of complement activation, compared to vehicle‐administered mice. Plasma C3b elevations correlated with the deposition of complement membrane attack complex C5b‐9 in the liver. Peak liver deposition occurred on day 3, which was associated with single‐cell necrosis as revealed through histopathological analysis. Liver serum transaminases were elevated after high‐dose AAV9 and reached maximum levels by day 3. In summary, we observed some of the clinical hallmarks of systemic high‐dose AAV toxicities in wild‐type mice. This study design can serve as a model for future therapeutic manipulations to mitigate such toxicities.
Prevalence study of cellular capsid‐specific immune response to AAV9 reveals an unconventional T cell immunity
R Xicluna1 2 3 M Devaux1 2 3 C Vandamme1 2 3 C Couzinié1 2 3 N Jaulin1 2 3 A Charrier1 2 3 M Guilbaud1 2 3
1: Université de Nantes 2: Inserm U1089 3: CHU de Nantes
With the recent approvals for Luxturna® and Zolgensma®, recombinant adeno‐associated virus (rAAV) vectors appear, more than ever, to be efficient viral vectors for in vivo gene transfer. Nevertheless, pre‐existing immunity to AAV capsid in humans remains one of the major limits for a successful clinical translation. Whereas pre‐existing humoral response to AAV capsid is well documented, the prevalence of pre‐existing capsid‐specific T cell responses still needs to be studied and characterized. Because of the scarcity of circulating AAV‐specific T cells, optimization of different tools to increase their detection and allow their characterization is necessary. In a previous study, the use of tetramer‐associated magnetic enrichment allowed us to evidence that healthy donors carry a terminally‐differentiated effector memory cell (TEMRA) fraction pool of AAV8‐specific T cells with a CD45+CCR7‐ phenotype. This cell population may play a role in AAV‐mediated cytotoxicity in patients. In the present study, we confirmed the presence of these cells by another assay based on IFNγ‐secreting CD3+ T cells phenotyping after their enrichment. We also extended our study to AAV9 serotype and surprisingly, we detected a capsid‐specific population of naive T cells in addition to memory T cells suggesting an unconventional T cell immunity for this particular serotype compared to AAV2 or AAV8. The characterization of these cells is currently ongoing. Altogether, these results suggest that pre‐existing immunity to AAV may vary depending on the serotype and support the necessity of using multiparametric monitoring methods to better characterize anti‐capsid cellular immunity and predict its impact in rAAV‐mediated clinical trials.
Intramuscular delivery of rAAV in non‐human primates results in a chronic local inflammation in situ without the loss of transgene expression
1: Inserm U1089
Recombinant adeno‐associated virus‐derived vectors (rAAV) provide a clinically relevant platform for efficient and sustained gene therapy. However, studies in large animal models have reported a potential immune‐induced loss of the transgene expression, in particular following intramuscular (IM) vector delivery.
Following IM delivery of a rAAV expressing an immunogenic transgene in the macaque model, we have recently reported that transgene expression can be observed up to five years following gene transfer after an unexpected initial reversible transitory loss. Functional viral genomes were detected at the site of rAAV injection at 5 years post‐injection despite a peripheral humoral and cellular immune response against the transgene product and the presence of CD8 T cell infiltrates in the injected muscle.
Here, we report that this long‐term persistence of rAAV functional genomes is associated to a chronic inflammation. After a RNAseq‐based transcriptomic analysis of micro‐dissected muscle cell infiltrates around the site of IM vector delivery, we have indeed observed a significant upregulation of a number of inflammation genes. Interestingly, this in situ chronic inflammation was associated to the presence of FoxP3‐expressing CD4 T regulatory cells.
Our results suggest that rAAV‐mediated gene transfer can lead to a non deleterious immune response in situ allowing the persistence of viral genomes in situ despite an inflammatory environment.
Adeno‐associated virus vector mediated gene therapy for Crigler Najjar syndrome: results of safety and efficacy from the dose escalation phase of the CareCN clinical trial
1: Hospital Papa Giovanni XXIII Bergamo, Italy 2: Amsterdam University Medical Centers, The Netherlands 3: ICGEB Trieste, Italy 4: TIGEM, University Federico II, Naples, Italy 5: Hannover Medical School, Germany 6: Hopital Antoine‐Béclère Paris, France 7: Genethon 8: Inserm, Integrare research unit UMR_S951, Univ Evry, Université Paris Saclay, EPHE
Patients with Crigler‐Najjar syndrome (CN) lack hepatic uridine diphosphoglucuronate glucuronosyltransferase 1A1 (UGT1A1) leading to severe unconjugated hyperbilirubinemia that can cause irreversible neurological injury and death. Prolonged, daily phototherapy (PT) partially and temporarily controls jaundice, while the only definitive cure is liver transplantation. We report the outcomes of the dose escalation part of a phase 1/2, international, multicentre, open‐label study evaluating safety and efficacy of a single intravenous infusion of GNT0003, an adeno‐associated virus vector serotype 8 (AAV8) encoding UGT1A1 under the control of a hepatocyte‐specific promoter, in patients with severe CN aged ≥18 years who require PT.
Five patients were treated with increasing doses. Two received 2x1012 vector genomes (vg)/kg (Cohort 1) and three received 5x1012 vg/kg (Cohort 2). Primary endpoints included safety, tolerability, and efficacy defined by serum bilirubin levels <300 μmol/l measured at week 17 after one week of PT discontinuation.
No GNT0003‐related serious adverse events were reported. Four patients experienced vector‐related adverse events (grade 1 transaminase elevation and grade 2 Gamma‐GT elevation) treated with a steroid course. In Cohort 1 efficacy was lost by week 16. In Cohort 2, patients 3 and 4 had a persistent reduction of bilirubin levels >80% from baseline, that allowed to stop PT successfully. Patient 5, currently at week 8 post‐treatment, also experienced clearance of jaundice with >90% bilirubin reduction.
These results suggest that, in patients with Crigler Najjar syndrome, GNT0003 at the dose of 5x1012 vg/kg is safe and restores UGT1A1 expression to levels allowing safe phototherapy withdrawal. (ClinicalTrials.gov, NCT03466463).
An AAV‐based, room‐temperature stable, single dose COVID‐19 vaccine
1: Harvard Medical School 2: Mass Eye and Ear/Harvard Medical School 3: IMVA‐HB/IDMIT, Université Paris‐Saclay 4: University of Pennsylvania 5: Novartis Gene Therapies 6: Massachusetts General Hospital/Harvard Medical School 7: NEIDL/Boston University 8: Ragon Institute/Harvard Medical School 9: Institut Pasteur 10: Albamunity
AAVs are extensively used as gene therapy vectors but their potential as vaccine platform has barely been studied. AAVrh32.33 is a chimera of two natural isolates from rhesus macaques that can induce functional anti‐viral immunity upon IM injection in mouse and NHPs in a vaccine context. We have developed a COVID‐19 vaccine candidate, AAVCOVID19‐1 (AC1), that consists of AAVrh32.33 carrying the stabilized full‐length SARS‐CoV‐2 Spike.
Potent and durable (up to one year) humoral immune responses to Spike were achieved after a single dose IM administration of AC1 in two mouse strains and in rhesus macaques. Robust T cell responses were also detected by IFN‐γ ELISpot, while no IL‐4 response was detected. Cynomolgus macaques immunized with 1012gc were challenged on week 9.5 with 105pfu of SARS‐CoV‐2 virus. Viral and subgenomic RNA analyses revealed near sterilizing immunity in the upper and lower respiratory tract of vaccinated animals, except some low but detectable transient breakthroughs. Additionally, PET/CT scans revealed no major lesion or lymph node activation in the lungs of the vaccinated animals. Neutralizing antibody responses to the capsid revealed slow kinetics and overall low titers. Importantly, no cross‐reactivity was detected against AAV serotypes 1,2,5,8 and 9. AC1 demonstrated stability for 1 month at room temperature and for at least 3 months at 4 degrees in liquid formulation.
In conclusion, AAVCOVID presented the desired features to potentially become a competitive vaccine candidate. Ongoing optimizations to the expression cassette show a 10‐fold increase in potency and adaptability to variants of concern.
Long‐term, sustained efficacy and safety from a phase 1/2 clinical trial of an AAV8‐mediated liver‐directed gene therapy in adults with glycogen storage disease type Ia
1: Hospital Clínico Universitario de Santiago de Compostela, Santiago de Compostela, Spain 2: Montreal Children's Hospital, Montreal, Quebec, Canada 3: University of Michigan, Ann Arbor, Michigan, USA 4: University of Groningen, Groningen, The Netherlands 5: University of Connecticut, Farmington, Connecticut, USA 6: University of Texas McGovern Medical School, Houston, Texas, USA 7: Ultragenyx Pharmaceutical Inc., Cambridge, Massachusetts, USA
Glycogen storage disease type Ia (GSDIa) results from a deficiency of glucose 6‐phosphatase (G6Pase). DTX401 is an adeno‐associated virus serotype 8 vector expressing the human G6PC gene. This global, open‐label, phase 1/2, dose escalation gene therapy trial (NCT03517085) is evaluating the safety and efficacy of a single DTX401 intravenous infusion in adults with GSDIa.
Three patients in Cohort 1 received DTX401 2.0x1012 gene copies (GC)/kg, and three each in Cohorts 2, 3, and 4 received 6.0x1012 GC/kg. Cohort 4 includes a prophylactic steroid regimen to prevent transaminase elevation.
In the nine patients enrolled in Cohorts 1‐3, mean (SD) total daily cornstarch intake reduction from baseline to Week 52 was 65.5% (22.3) and to last visit (range: 60‐131 weeks) was 78.8% (20.1), both p < 0.0001. In Cohort 3, continuous glucose monitoring was implemented; an average 61% cornstarch intake reduction from baseline to Weeks 49‐52 was associated with a mean 8.4% increase in time spent in euglycemia (blood glucose 60‐120 mg/dL). At the time of the data cutoff for this analysis, one Cohort 4 patient completed prophylactic steroids; two continue to taper steroids. No DTX401 infusion‐related or treatment‐related serious adverse events (SAEs) were reported. All unrelated SAEs were classified as serious due to hospitalizations; all resolved.
DTX401 showed a positive and sustained long‐term efficacy and safety profile in all treated patients, in favor of the 6.0x1012 GC/kg dose (dose by ddPCR 1.0x1013 GC/kg) as the optimal biological dose for a pivotal phase 3 trial expected to start in the second half of 2021.
Enhanced level and durability of AAV transgene expression and mitigation of anti‐capsid neutralizing antibodies by ImmTOR tolerogenic nanoparticles in nonhuman primates
T K Kishimoto1 S Elkins1 T Capela1 G Rizzo1 P O Ilyinskii1
1: Selecta Biosciences
Immune responses to the capsid or transgene product can lead to the loss of transgene product and the formation of neutralizing anti‐AAV8 antibodies (NAb), which prevent the ability to re‐dose patients. ImmTOR nanoparticles encapsulating rapamycin have been shown to selectively mitigate AAV immunogenicity and enable vector redosing. Here we explored the impact of different dosing regimens of AAV8 encoding human secreted embryonic alkaline phosphatase (AAV8‐SEAP) and ImmTOR nanoparticles on NAb formation and SEAP activity in nonhuman primates. As expected, the control group had an early anti‐AAV8 IgM response that transitioned to an anti‐AAV IgG response and strong NAb titers by day 84. SEAP activity peaked at day 28 and rapidly declined by day 84, suggestive of an anti‐SEAP antibody response. In contrast, the addition of a single dose of ImmTOR delayed anti‐AAV8 IgG antibody formation until at least day 56 and reduced NAbs on day 84 in some animals. ImmTOR treatment led to increased and sustained SEAP activity in comparison to the control group. The impact of ImmTOR was most striking in groups investigating 3 monthly doses of ImmTOR, in which anti‐AAV8 IgM, IgG and neutralizing antibodies were mitigated. Five of 6 animals had NAb titers <1:5 and the sixth animal had a weak titer of 1:11. Combined with the enhanced and sustained expression of SEAP in these animals, these results indicate that 3 monthly doses of ImmTOR may enhance the level and durability of transgene expression, while inhibiting the formation of NAbs and enabling the possibility of vector re‐administration.
Optimizing IdeS treatment regimen for enhanced AAV transduction in individuals with pre‐existing anti‐AAV neutralizing antibodies
1: Vivet Therapeutics S.L. 2: Centro de Investigacion Medica Aplicada (CIMA) 3: Institute for Sanitary Research (IdiSNA) 4: Vivet Therapeutics S.A.S.
Pre‐existing neutralizing antibodies (NAbs) to adeno‐associated viruses (AAVs) remain an impediment for successful systemically administered AAV‐mediated gene therapy treatment in many patients and various strategies have been under investigation to overcome this limitation. Here, IgG degrading (Ide) enzymes derived from bacteria of the genus Streptococcus were tested for their ability to cleave human IgG. The selected candidate, IdeS, was found to be highly efficient to cleave human IgG, although 5‐10‐fold less efficient on non‐human primate (NHP) IgG and inefficient on mouse IgG. Employing passively immunized mice or NHP with naturally occurring anti‐AAV NAbs, we showed that the best transduction levels were achieved when the AAV vector is administered once IgG NAb digestion products are cleared from the circulation and before natural restoration of circulating NAb. Thus, preconditioning with IdeS represents a unique treatment opportunity for gene therapy clinical trial patients primarily excluded from participation due to elevated circulating anti‐AAV NAbs levels. However, careful determination of the optimal IdeS dose and timing of AAV administration are essential for optimal AAV‐mediated transduction.
Novel AAV capsid variant for muscle‐directed gene therapy
1: Université Paris‐Saclay, Univ Evry, Inserm, Généthon, Integrare research unit UMR_S951, 91000, Evry‐Courcouronnes, France 2: Institute of Experimental Hematology, Hannover Medical School
Muscular dystrophies are inherited genetic disorders due to mutations in genes responsible for the structure and function of skeletal muscle fibers. A promising treatment for these diseases is gene therapy using recombinant Adeno‐Associated Vectors (rAAVs). Capsid engineering to improve muscle targeting is a powerful tool to decrease costs and possible adverse effects of rAAV‐based gene transfer. Here, we performed a high throughput peptide display AAV2 library screening by intravenous injection in Dmdmdx mice (a model for Duchenne muscular dystrophy) and in healthy controls. Three peptides (O1, O2 and O3) were selected according to their occurrence in the target tissue (Tibialis Anterior) and based on target‐to‐noise ratio (Tibialis Anterior/liver). The peptides were then cloned into AAV2 and additional capsids such as AAV8, AAV9. The vectors were intravenously injected in healthy and Dmdmdx mice. O2 fusion in rAAV2 increased muscle‐specific transduction by 1 log and transgene expression by 3 log, while liver transduction and transgene expression were decreased by 1 log. O3‐rAAV2 also showed a slight increase in muscle‐specific targeting. Surprisingly, O2 and O3 peptides in rAAV8 and rAAV9 did not increase the efficiency of transduction and transgene expression in muscle, but O2 decreased by 1 log the liver‐specific transduction. In rAAV9rh74, O2 peptide reduced liver transduction by 3 log and transgene expression by 2 log. These results showed that virus entry does not always correlates with transgene expression, and the efficiency of capsid engineering still depends on the AAV serotype used, giving important insights for future design strategies.
Engineered AAV‐based vaccines against SARS‐CoV‐2
1: Department of Ophthalmology, University Hospital, LMU Munich, Munich, Germany 2: Department of Veterinary Sciences, Ludwig‐Maximilians‐Universität München, Munich, Germany
Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) – the cause of Covid‐19 – shook the world and led to a still ongoing pandemic with high morbidity and mortality. The high medical need sparked global research activities and led to the development of mRNA‐ and vector‐based vaccines against SARS‐CoV‐2 at unprecedented speed.
Here, we explored engineered adeno‐associated virus (AAV) capsids as a novel platform technology for vaccines. We have chosen SARS‐CoV‐2 for feasibility testing and to exemplify the capabilities of this technology. In particular, we inserted approximately 200 amino acid long sequences covering the receptor‐binding domain (RBD) of the S‐spike protein of SARS‐CoV‐2 into the surface‐exposed variable loop region IV of the AAV2 or AAV9 capsid. The corresponding engineered AAVs termed HtW could be produced and purified by standard methods as full or empty particles. HtWs expressing eGFP showed higher transduction levels on ACE2‐overexpressing cell lines confirming that the modification endowed the AAVs with this SARS‐CoV‐2‐like property. Moreover, HtW‐immunized rabbits showed a strong immune response already after the first booster injection. Sera from immunized rabbits strongly cross‐reacted with HtWs, but showed only weak affinity for wildtype AAVs. In addition to the induction of high levels of anti‐SARS‐CoV‐2 RBD antibodies in rabbits, HtWs also induced specific human cellular immune responses in vitro with PBMC of individuals vaccinated with an mRNA‐based SARS‐CoV2 vaccine. These results suggest that capsid‐modified AAVs carrying large sequence insertions could be potent immunogens for the development of novel vaccines.
rAAV sensing in microglial cells
1: Inserm U1089
Adeno associated virus (AAV)‐mediated transgene expression in microglial cells shows limited efficiency, possibly due to enhanced viral sensing in these cells. It has been shown that while recombinant AAVs can transduce the cell, the virion particles are disrupted and fail to reach the nucleus. There is a risk that AAVs used for retinal and CNS gene therapies may have a bystander effect in the microglia. In this case, even though vector cargo may not be expressed in the microglia, the status of the cell may change upon sensing the viral genome. To examine this, we tested a range of AAV serotypes in primary and immortalized HMC3 microglial cells. We have assessed the expression levels of five viral DNA sensors as well as the activation status of the cells in response to the different vectors. First, we find differential transduction efficiencies for the HMC3 immortalised line compared to primary microglia with transgene expression supported for rAAVs with AAV2, AAV5, AAV6 and AAV8 capsids in HMC3 cell line, while only rAAVs with AAV6 capsid achieved expression in primary microglial cells. Furthermore, we observe a divergent sensing and activation response of the cells to the different AAV serotypes. This may have implications for the optimal selection of safe vectors for retinal and CNS gene therapies.
The Endopeptidase IdeS Degrades Anti‐AAV IgG and Enables Liver‐Directed AAV Gene Therapy in Animal Models of Humoral Immunity
1: Spark Therapeutics
Despite its promise to treat a myriad of genetic diseases, systemic AAV gene therapies continue to be challenged by limited patient access caused by pre‐existing neutralizing antibodies (NAbs) in target patient populations. Numerous approaches, such as plasmapheresis, capsid engineering, and immunomodulation, have been evaluated to circumvent the barrier of pre‐existing humoral immunity, but all have either limited efficacy or potential safety concerns. IdeS is an endopeptidase that cleaves and inactivates IgG with high efficiency and substrate specificity. Here, we evaluate the potential of IdeS to degrade anti‐AAV NAbs and to enable liver‐directed AAV gene therapy. Our data show that IdeS reduces levels of anti‐AAV IgG in purified pooled human IgG and in human plasma. In a passively immunized mouse model, IdeS reduces anti‐AAV antibodies and rescues liver‐directed transgene expression. Moreover, IdeS is well tolerated and enables both functional AAV transduction of actively‐immunized rabbits and non‐human primate models. Taken together, these results suggest treating NAb‐positive patients with IdeS may facilitate AAV gene therapy and warrants clinical development.
Characterization of a reporter gene cell line for the easy assessment of therapeutic AAV vectors – a case study
1: Svar Life Science
Due to recent breakthrough innovations within the gene therapy field, this is the light at the end of the tunnel for many previously untreatable conditions. With the use of gene therapy, defective genes can be replaced by functional genes, with the aim to restore normal function. Advanced therapeutic platforms like gene and cell therapies offer tremendous potential for treating these unmet medical needs. However, they are extremely challenging to develop successfully.
We have developed a technology, based upon the reporter gene assay format, that allows to assess and quantify the presence, activity, and function of therapeutic AAV vectors. This technology can be adapted for many applications throughout the drug development phases including drug manufacturing processes such as the quantification of vector potency and functionality or the detection of neutralizing anti‐drug antibodies (NAb).
Here we showcase, using a proprietary developed reporter gene cell line – the iLite cGMP/GC, examples from the development, validation and production of this custom genetically modified cell line and subsequent assay using it in an assay ready format.
Efficient suppression of IgG antibody responses to high doses of AAV8 capsids by single and multiple administrations of ImmTOR nanoparticles
1: Selecta Biosciences 2: AskBio 3: UNC Gene Therapy Center
Achieving durable systemic AAV gene therapy may require repeat AAV dosing. Currently, re‐dosing is prevented by the formation of neutralizing antibodies. We have previously demonstrated that tolerogenic ImmTOR nanoparticles encapsulating rapamycin mitigate AAV immunogenicity and enable vector redosing in mice and nonhuman primates at moderate vector doses of ∼2e12 vg/kg. Here we evaluated the ability of ImmTOR to block IgG formation using higher doses of AAV8 empty capsids (AAV8‐EC). A single dose of 100 μg ImmTOR completely abrogated IgG responses to 2e13 vector particles (vp)/kg AAV8‐EC through day 62 in Balb/C mice and in the majority (10/12) of C57BL/6 mice at 2‐6e12 vp/kg AAV8‐EC. However, ImmTOR was less efficient at 2e13 vp/kg in C57BL/6 mice, with delayed breakthrough of antibodies observed in most animals at 200 μg. Higher doses of ImmTOR (300 μg) inhibited IgG formation in the majority of mice (9/12) at 2E13 vp/kg. We next evaluated administration of two additional monthly doses of ImmTOR. Mice treated with 2E13 vp/kg capsid and three 200‐300 μg monthly ImmTOR doses developed little or no IgG through Day 84. Thus, repeated administration of ImmTOR provides more durable suppression of antibodies against higher viral capsid doses. Monthly dosing of ImmTOR has been shown to be well tolerated and effective in mitigating immunogenicity of a fungal‐derived uricase therapy in gout patients and is currently in Phase III clinical trials.
ImmTOR combined with B cell‐targeted therapies provides synergistic activity in mitigating anti‐AAV capsid antibody responses and enables repeated vector dosing
1: Selecta Biosciences
ImmTOR tolerogenic nanoparticles encapsulating rapamycin have been demonstrated to mitigate immunogenicity of AAV vector and enable vector redosing (Meliani et al., Nature Commun 2018; Ilyinskii et al., Science Adv 2021). While ImmTOR has been shown to directly inhibit germinal center plasmablasts, the primary mechanism of action is thought to be the induction of tolerogenic antigen‐presenting cells that induce antigen‐specific regulatory T cells. However, ImmTOR only partially inhibits the initial T cell‐independent B cell IgM antibody response and inhibits subsequent class‐switching to IgG. The residual anti‐capsid IgM response can have neutralizing activity and affect the efficiency of vector re‐administration. Here we evaluated the combination of ImmTOR with currently available B cell targeting agents to mitigate the IgM response and increase the efficiency of re‐dosing. ImmTOR combined with a monoclonal antibody (mAB) directed against B cell activation factor (BAFF), a B cell survival factor, synergistically reduced anti‐AAV IgM antibodies, provided more durable suppression of anti‐AAV IgG antibodies, and enabled multiple re‐administrations of an AAV8 vector. Similar effects were observed when combined with ibrutinib, a Bruton's tyrosine kinase inhibitor. While ImmTOR alone had little or no effect on total splenic B cells or immature pre‐B cells, anti‐BAFF mAB reduced total B cells by ∼50% and increased pre‐B cells by ∼2‐3 fold. The combination of ImmTOR and anti‐BAFF mAb showed a synergistic effect in increasing splenic pre‐B cells and reducing B cell plasmablasts. These results suggest that ImmTOR could be combined with belimumab, an anti‐BAFF mAb, or ibrutinib to further mitigate anti‐AAV antibody responses.
Evaluation of prophylactic immune suppression and application to repeat dose administration of AAV‐mediated gene therapy in mice
1: BioMarin Pharmaceutical
Systemic administration of AAV mediated gene therapy results in the stimulation of high titer, durable capsid‐specific antibody responses which substantially diminish the efficacy of repeat gene therapy dose administrations with the same or related capsids. The application of prophylactic immune suppression was evaluated to determine the effect on AAV5 capsid specific humoral and cellular immune responses following gene therapy dose administration in C57BL/6 mice. The aim was to mitigate adaptive immunity to allow for successful repeat dose administration. This study evaluated three distinct immunosuppression regimens for effectiveness in preventing or diminishing the AAV5 capsid‐specific antibody response (AAV5 TAb) following gene therapy dose administration: Abatacept, a CTLA‐4‐Ig fusion protein, Abatacept in combination with anti‐CD20 antibody, and anti‐CD20 in combination with Rapamycin administered 1 week prior to gene therapy dose administration. A single 6E13vg/kg dose of AAV5‐bCG was administered at Day 0 to all mice as an intravenous tail vein injection followed by a challenge dose of 6E13 vg/kg AAV5‐FIX on Day 45. Prophylactic immune suppression resulted in reduction of AAV5 TAb titers up to 94% relative to untreated controls. In addition, mice treated with Abatacept had a reduced AAV5‐specific cellular immune response in an IFN‐gamma ELISpot assay of spleen mononuclear cells. Despite the diminished immune response to AAV5, repeat gene therapy dose administration was generally unsuccessful with plasma human FIX levels remaining below or near the limit of detection. Further studies are needed to evaluate the effect of modifying the dosing regimen and concomitant use of additional immune suppression treatment modalities.
Novel MSD‐based screening assays to quantify pre‐existing antibodies against various AAV serotypes
1: Research Beyond Borders, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany 2: Drug Discovery Sciences, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany 3: Boehringer Ingelheim Venture Fund GmbH, Ingelheim am Rhein, Germany
Adeno‐associated virus (AAV)‐based gene transfer has been successfully progressed into the clinic during the last two decades. However, AAV vector applications are often limited by capsid‐directed humoral immune responses, mainly through neutralizing antibodies (NAbs) which are present throughout the human population due to natural AAV infections. Therefore, pre‐screening, and close monitoring of biologically relevant NAb‐titers is critical for product development. Equally, assessment of neutralizing antibodies is needed for animal experiments in preclinical settings and for capsid engineering approaches. Currently, NAb levels are often quantified via ELISA‐based protocols or by cellular NAb assays, less frequently by in vivo NAb assays in passively immunized mice. These methods need optimization for each serotype tested and are often not applicable to AAV variants with poor in vitro transduction. To tackle these limitations, we have established Meso Scale Discovery® (MSD)‐based assays for the quantification of binding antibodies (BAb) and neutralizing antibodies (NAb) against the three most commonly used AAV serotypes 2, 8 and 9. Both assays detect anti‐AAV‐IgG1‐3 with high sensitivity and robustness and show strong correlation. After validation of both assays with the serum of 40 healthy donors, a cohort of 42 NHPs was successfully screened for seronegative animals. Moreover, the MSD‐based BAb assay protocol was extended to a panel of 14 different AAV serotypes but is further suitable for characterization of any natural or engineered AAV variants irrespective of transduction efficiency. In summary, our platform allows rapid and quantitative assessment of the immunological properties of any given AAV capsid and enables high‐throughput screens.
Neutralizing antibody prevalence toward a hematopoietic stem cell‐derived AAV and immunoassays for clinical trial enrollment
1: Charles River 2: Homology Medicines, Inc.
Screening patients for pre‐existing neutralizing antibodies (NAbs) against AAV capsids prior to enrollment in gene therapy clinical trials is standard practice to mitigate potential efficacy and safety risks. The typical screening approach identifies neutralizing seropositive samples as those exhibiting greater than 50% transduction inhibition (TI) relative to a seronegative control. The TI threshold is often derived from the analytical system used, and not statistically from the biological variation of the population under study, as recommended by regulatory guidance. In a study population using the TI method, there was relatively low prevalence of NAbs to AAVHSC15, one of Homology Medicines' adeno‐associated viruses derived from human hematopoietic stem cells. Here, we show correlation between the TI method and a validated three‐tier NAb assay for identifying anti‐AAVHSC15 NAb positive serum samples. The prevalence of confirmed positive samples in commercial sera was 16% in the three‐tier assay versus 24% using the TI method. All of the NAb‐positive samples identified in the three‐tier assay had previously been identified as positive using the TI approach. Of interest, two samples were positive for exhibiting neutralization in the absence of detectable total antibodies. Determining an anti‐capsid NAb screening strategy for patient exclusion is method and vector dependent; however, there was close agreement in reported results between the two approaches used in this instance. A standardized validation of anti‐capsid NAb assays, per regulatory guidance and statistical considerations for seropositivity thresholds, is recommended for enrollment in gene therapy clinical trials.
Increasing Gene Therapy Vector production using Viral Sensitizer Molecules
1: Virica Biotech
Inefficient virus manufacturing processes can have several important implications during gene therapy product development. The most obvious is cost of goods, driven upwards through consumption of raw materials, longer times to produce target amounts of virus in GMP, more substantial needs in personnel, physical space, and investments in infrastructure. Inefficient manufacturing can also limit therapeutic efficacy by imposing a “maximum feasible dose”, a significant concern for systemically delivered viral products.
Virica's Viral Sensitizers (VSETMs) are a proprietary collection of small molecules that enhance the growth of viruses by transiently and efficiently overcoming cellular antiviral defenses. VSEs can be used in a range of applications such as improving virus manufacturing yield, improving tumor infection by oncolytic viruses, or transduction of cells by common gene therapy vectors like AAV, adenovirus, and lentivirus. Owing to different molecular mechanisms through which they operate, VSEs can be combined, adapted, and formulated for specific uses.
As a demonstration of the technology's application in the context of gene therapy vectors, we employed high throughput screening and DOE methodology to develop a VSE formulation that significantly improved transfection‐based production of 3rd generation lentivirus in adherent HEK293T cells. Enhancements in lentivirus production were observed over 72 hours, without a requirement to resupply compounds. Different transfection reagents, and production scales as well as commonly used lentivirus production enhancers were tested and found to be compatible. Improvements in yield observed using the formulation were condition‐dependent but in pre‐optimized lentivirus production conditions led to >5X increase in TU/ml.
Change in capsid‐HSPG interaction through peptide insertion in variable loop VIII in liver‐targeting AAV vectors
1: Institute for Drug Discovery, University Leipzig Medical School, Leipzig 04109, Germany 2: Institute for Experimental Hematology, Hannover Medical School, Hannover, 30625, Germany 3: authors contributed equally 4: authors contributed equally
Adeno‐associated virus (AAV) vectors have shown promise in human clinical gene therapy trials, and three AAV vector‐based in vivo gene therapy products have obtained market approval. The transduction process of AAV is complex and engineering new AAV vectors to target specific cells remains a major goal for AAV‐based gene therapy. Here, we combine capsid engineering with structural modeling to analyze how molecular interactions shape tropism of two newly described AAV vectors that show preferential targeting of liver cells and efficient transduction of hepatocytes. Inserting a peptide at position 587 of the AAV2 capsid separates two key residues of the heparan sulfate proteoglycan (HSPG) binding motif and thus is expected to impact AAV2's ability to bind to its primary attachment receptor. When analyzing the vectors in comparison to AAV2, no or moderate binding to a heparin affinity column was observed. However, in vitro transduction by the variants, although less sensitive than AAV2, is inhibited by the addition of soluble heparin. We modeled the structure of the engineered capsids and examined their ability to bind HSPG through docking studies. We find that the conformation of our candidate peptides, when inserted in the loop, does not affect the native structure of the HSPG binding motif. Nonetheless, the conformation of the peptide containing VIII loop sterically hinders HSPG binding, therefore lowering affinity and binding capacity compared to AAV2. In conclusion, structural analysis can complement capsid engineering efforts, revealing the molecular basis of newly observed features and enabling rational design choices to improve AAV vectors.
Interferon‐α gene delivery by tumor‐associated macrophages improves function and prevents exhaustion of B7‐H3‐redirected CAR T cells in glioblastoma
1: San Raffaele Telethon Insitute for Gene Therapy (HSR‐TIGET) 2: Universita Vita‐Salute San Raffaele
Despite the identification of relevant target antigens, the tumor microenvironment (TME) limits trafficking and killing capability of chimeric antigen receptor (CAR) T cells in solid tumors, particularly glioblastoma multiforme (GBM). As previously shown, TIE2‐expressing macrophages can be exploited to deliver interferon (IFN)‐α in the TME through a hematopoietic stem cell (HSC)‐based gene therapy to specifically reprogram it toward immunoactivation. Therefore, a combination approach may unleash CAR T‐cell function against GBM.
We evaluated the expression of candidate targets at transcriptional and protein levels in a new murine GBM model (mGB2), closely recapitulating the human disease. mGB2 cells expressed the immune‐checkpoint B7‐H3, which shares high human‐mouse homology. We generated B7‐H3‐redirected (CARB7‐H3) and control human_CD19‐redirected (CARhCD19) mouse CAR T cells to be systemically administered in syngeneic C57BL/6 mice, previously transplanted with engineered HSCs (Tie2‐Ifna or control) and orthotopically injected with mGB2 cells.
While circulating levels of CARB7‐H3 dropped more than CARhCD19 ones, higher levels of CARB7‐H3 were retrieved from the tumor infiltrate of both control and Tie2‐Ifna mice, suggesting a target‐driven homing or local expansion. CARB7‐H3 T cells from Tie2‐Ifna mice displayed improved activation, evaluated as CD69 and Granzyme‐B positivity, and reduced exhaustion, assessed by co‐expression of PD‐1, LAG‐3 and CTLA‐4, as compared to control mice. These changes accounted for the lowest tumor burden achieved in the combination therapy group, while no effect was detected with CARB7‐H3 T cells only by magnetic resonance imaging.
In conclusion, IFN‐α‐driven TME reprogramming can overcome some key limitations of systemic CAR T cells against GBM, restoring their anti‐tumor functions.
CRISPR/Cas9‐mediated CD39 disruption can be combined with TCR editing in T cells to improve the adoptive T cell therapy of colorectal cancer
1: San Raffaele Scientific Institute 2: University of Milano‐Bicocca
Colorectal cancer (CRC) is the 2nd cause of cancer‐related death. Despite standard therapies, more than 50% of patients experience relapse, eventually with metastatic disease. The CRC microenvironment is densely infiltrated by T‐cells, which presence correlates with improved overall survival, thus sustaining the rational for immunotherapy. By high‐dimensional flow cytometry coupled with an advanced pipeline of data handling by dimensionality reduction and clustering algorithms we described the phenotype and the exhaustion status of T‐cells retrieved from the healthy, peritumoral and neoplastic tissue of treatment‐naïve primary CRCs and from the peritumoral and tumoral tissue of CRC patients undergoing surgery for liver metastasis. Populations of TILs defined by a peculiar inhibitory receptors signature were enriched both in primary and metastatic CRCs. Of note, the signatures retrieved from primary and metastatic CRC overlapped for the upregulation of CD39, thus underlying this molecule as a relevant target for T‐cells engineering. By CRISPR/Cas9 we disrupted CD39 in T cells with >80% efficiency. We combined CD39 knock‐out with the genetic disruption of both the alpha and beta chain genes of the endogenous TCR, observing >90% efficiency, thus generating triple‐knockout T‐cells that could be then engineered by LV to express tumor‐reactive TCRs. By repetitively stimulating healthy donors' peripheral blood mononuclear cells with autologous antigen‐presenting cells loaded with a pool of peptides selected to be immunogenic and expressed by CRC, we obtained a library of anti‐tumor TCRs to redirect T cell specificity. Preliminary experiments showed a functional advantage for CD39‐disrupted vs CD39‐competent TCR‐redirected T‐cells in recognizing and killing CRC target cells.
Administration of Ad5/chitosan/PEG‐aptamer vector delivering PDGF‐D shRNA decreases tumor growth in rat breast cancer model
1: Pharmaceutical Biotechnology Department, Marmara University, Istanbul, 34854, Turkey 2: Pharmaceutical Technology Department, Medipol University, Istanbul, 34810, Turkey
Breast cancer, with different subtypes and signaling pathways included, is a good candidate for gene therapy strategies. Platelet derived growth factor‐D (PDGF‐D) is known plays roles in angiogenesis, metastasis and epithelial‐mesenchymal transition in different cancer types including breast cancer. While Ad5 is one of the most commonly used viral vector in gene delivery, evolving hybrid vectors with non‐viral systems is a promising strategy. In this study, we prepared Ad5/chitosan/PEG hybrid vector retargeted with epidermal growth factor receptor (EGFR) aptamer to deliver shPDGF‐D and evaluated in rat breast cancer model. We showed that intratumoral administration of AdshPDGF‐D/chitosan/PEG‐aptamer reduced tumor volume approximately 85% compared to control and 45% compared to AdshPDGF‐D after 35 days. ELISA and RT‐PCR were performed to evaluate therapeutic efficiency and results showed that AdshPDGF‐D/chitosan/PEG‐aptamer group decreased PDGF‐D expression significantly compared to control both in protein (65.80%) and mRNA (50.18%) level. On the other hand, IL‐6 and IFN‐α levels was measured to investigate immune response and it was observed that coating Ad5 vector with chitosan/PEG reduced IL‐6 and IFN‐α levels in serum. While IL‐6 protein levels reached 173.41 pg/ml and IFN‐α levels reached 7.59 ng/L after AdshPDGF‐D administration, IL‐6 concentration was 50.68 pg/ml and IFN‐α concentration was 2.72 ng/L in AdshPDGF‐D/chitosan/PEG‐aptamer group. Together, results showed that shielding Ad vectors with polymers like chitosan/PEG and retargeting with aptamer increase therapeutic efficiency and also enables to evade immune response.
Towards a single‐shot prime‐boost AAV‐based vaccine for cancer immunotherapy
1: Hannover Medical School 2: Université de Rouen Normandie 3: University Hospital Leipzig 4: equally contributed first author 5: equally contributed senior author
Cancer immunotherapies, like CAR‐cell‐based strategies, bi‐specific T‐cell engagers, or immune checkpoint inhibitors, have demonstrated profound efficacy in the treatment of distinct cancer entities. Novel vaccine‐based approaches are promising as an alternative, independent strategy to increase immunogenicity and to broaden the applicability of immunotherapy approaches already in clinical use.
Here, we report on the development of a novel anti‐tumor vaccine based on our single‐shot prime‐boost AAV vector platform. Specifically, we combined tumor antigen display on the capsid surface and vector‐mediated tumor antigen overexpression, inducing thereby a profound and long‐lasting T‐ and B‐cell immune response directed against tumor antigens. The tumor inhibitory potential of the vaccine was investigated using the syngeneic B16/F10‐Ova and EG‐7 (Ova+) tumor model, which differ in immunogenicity. Mice that received the prime‐boost vaccine construct exhibited some level of immune protection against tumor growth in both models, even when tumor cells were injected 80 days post‐vaccination. Specifically, we observed either an impaired tumor growth or a lack of tumor engraftment.
In summary, we successfully established a novel AAV‐based anti‐tumor vaccine strategy, which can be used to efficiently target therapeutically relevant tumor antigens.
In‐vivo gene‐based immune reprogramming of liver metastases enables protective T cell responses
1: San Raffaele Telethon Institute for Gene Therapy (HSR‐TIGET) 2: San Raffaele Scientific Institute 3: Cancer Research Institute Kanazawa University
The liver hosts an immunosuppressive microenvironment, which favours metastatic seeding of cancer cells. Therefore, developing strategies to reprogram the liver tumor microenvironment towards an immune activating state is of crucial importance.
We have developed a lentiviral vector (LV)‐based platform that enables selective genetic engineering of resident and tumor‐associated macrophages (TAMs) for the delivery of therapeutic molecules to LMS. Transgene expression is driven by leveraging a macrophage specific promoter and fine‐tuned by microRNA target sequences.
Upon systemic delivery of the LV, we observed selective transgene expression in liver resident macrophages and in TAMs surrounding LMS. To address therapeutic efficiency we equipped the LV with an IFNα‐encoding sequence, a cytokine with pleiotropic immune effects. Long term analysis of mice treated systemically with IFNα LV showed a rapidly established, vector dose‐dependent and sustained IFNα expression, with no signs of adverse events. In two relevant mouse models of LMS, treatment with the IFNα LV led to a significantly delayed tumor growth up to complete response. Upon treatment, we observed upregulation of IFNα responsive genes and altered activation/polarization profile in tumor infiltrating cells indicating robust immune reprogramming of the LMS microenvironment.
In summary, we have developed an innovative gene‐based platform that upon a single well‐tolerated intravenous LV infusion rapidly establishes a protective response against LMS through promotion of macrophage reprograming and adaptive immune activation.
CRISPR‐mediated targeting of fusion oncogenes in combination with chemotherapy enhances the efficacy of cancer cells elimination and tumor growth reduction in xenograft models.
1: Centro Nacional de Investigaciones Oncológicas 2: CIEMAT/CIBERER 3: Josep Carreras Leukemia Research Institute 4: ICREA y Universitat Autonoma Barcelona 5: Institut de Recerca Sant Joan de Deu
Fusion oncogenes (FOs) are common in many cancer types and are powerful drivers for several human cancers, including leukemia, lymphoma, and sarcoma. Because their expression is restricted to cancer cells, FOs are perfect therapeutic targets. We have developed a simple, efficient, and clinically relevant genome editing strategy for targeting fusion oncogenes using CRISPR/Cas9 technology. Our strategy is based on targeting two different introns of the genes involved in the rearrangement; this simple but efficient strategy, allows us to specifically disrupt FO in cancer cells without altering exonic sequences or protein expression of non‐rearranged alleles. We have shown that the combination of CRISPR/Cas9 targeting FO with standard‐of‐care chemotherapy present additive effects in vitro and in vivo enhancing tumor regression when comparted with monotherapy in ewing sarcoma xenograft.
Membrane coating for nonreplicating adenoviral vectors aids gene delivery in a murine model of melanoma
1: University of São Paulo School of Medicine
Melanoma is the most aggressive and lethal type of skin cancer. Our group has shown that the combined gene transfer of p19Arf and IFNβ delivery by nonreplicating adenovirus is an efficient means to induce both cell death and an immune response in melanoma model. To overcome some limitations, we are evaluating the use of membranes derived from the B16 mouse melanoma cell as a coating for our adenoviral vector in order to improve transduction efficiency, avoiding the generation of neutralizing antibodies. AdRGD‐PG‐Luciferase vector was extruded in the presence of membranes isolated from B16 cells. The extraction of cell membrane and its integrity was evaluated by Nanotracking analysis and western blot. B16 cells were injected in C57BL/6 and treated intratumorally with 1x109 PFU/each dose of naked or encapsulated‐adenovirus. The virus trafficking was evaluated by in vivo bioluminescence imaging. For the neutralizing antibody assay, AdRGD‐PG‐eGFP virus was incubated for 1h at 37°C in the presence of serum recovered from these animals and then used to transduce cells and, 48h later, the transduction rate was measured by flow cytometry. The presence of viral DNA in the tumors was assessed by qPCR. Preliminary results from ongoing experiments indicate that the cell membrane coating confers efficient on‐target transduction in both in vivo and in vitro models and a reduction in neutralizing antibody production. These results show advantages for intratumoral delivery, but also encourage the exploration of systemic delivery. Currently, we are evaluating membrane‐coated adenoviral vectors as a means to potentiate our melanoma gene therapy strategy.
Prostratin as a small molecule inducing agent for improving lentiviral vector titres.
1: Oxford BioMedica
Improving lentiviral (LV) vector titres by overcoming bottlenecks in manufacturing can determine whether a new gene therapy product is commercially viable or not. Hence, a considerable amount of research aims to establish new technologies that can improve the productivity or recovery of upstream or downstream platform processes, respectively.
Induction is a key stage in LV production that typically involves introducing the histone deacetylase inhibitor, sodium butyrate, to production cells to enhance the expression of LV plasmids by decondensing transiently delivered DNA. By performing small molecule screening to explore alternative inducing agents, we identified prostratin as a candidate drug compound that can be added in combination with sodium butyrate to enhance LV titres by 2‐ to 5‐fold, in a product‐dependent manner.
Prostratin is a non‐tumour promoting agonist of protein kinase C that has previously demonstrated promising in vitro properties for reactivating latent reservoirs of HIV‐1 via the NF‐κB pathway. Here, we describe how prostratin is an effective supplementary inducing agent that can be used in both transient and packaging cell‐lines, and can be combined synergistically with a new technology developed in‐house based on a modified U1 snRNA to increase titres of LV‐CAR >10‐fold at bioreactor scale. Furthermore, these benefits are achieved without detriment to the viability of production cells or quality attributes of the product, and residual prostratin is removed from the vector product following downstream processing. Having established the benefits of introducing prostratin to our platform process, we now intend to transfer this technology to GMP manufacturing for commercial products.
Development of a bio‐inspired nanovector for targeted cancer gene therapy
1: Université de Nantes 2: Inserm 3: CRCINA
Over the past few decades, the field of nanovectorization has emerged to address the main limitations of current anti‐cancer drugs: (i) systemic toxicity due to low tumor selectivity; (ii) low plasma stability; (iii) limited tumor uptake resulting in a poor therapeutic efficacy, and eventually (iv) poor solubility. Synthetic nanoparticles are currently the most represented nanovectors in clinical trials but interest in new protein‐based carriers such as virus‐like particles (VLPs) is also growing due to their better biocompatibility. VLPs can be derived from animal, plant or bacteria viruses and consist in a non‐replicative viral capsid free of viral genome that can be further engineered to target tumor cells. Thanks to their viral origin, VLPs are particularly suited for nucleic acids delivery. In this context, we derived the filamentous bacteriophage M13 to obtain non‐replicative VLPs actively targeted to mesothelin, a tumor antigen widely overexpressed in several cancers. These VLPs contain a eukaryotic ORF for transgene expression in human cells. At this point, we showed that these VLPs are selectively internalized by MSLNpos tumor cells, but not healthy cells, by flow cytometry and in co‐culture experiments by confocal microscopy. We also demonstrated that those VLPs can be used to express a reporter transgene in human tumor cells. Moreover, we showed that these VLPs can be easily conjugated to a fluorochrome for an easy tracking in vitro. Following those encouraging results, we will assess VLP biodistribution in tumor models in vivo and evaluate a therapeutic transgene for cancer‐specific gene therapy.
Assessment of Natural Killer (NK) cell activity for immunotherapy using a novel flow cytometry‐based killing assay
1: Bio‐Techne
Human NK cells have shown promise as therapeutics in clinical trials of patients with therapy‐resistant or advanced‐stage blood cancers. Currently, over 100 NK cell and induced NK cell‐based clinical trials are ongoing for blood cancers and refractory solid tumors.
We sought to develop novel, clinically relevant, and robust methods for human NK cell expansion. In addition, to simplify the processing of cells for characterisation, we developed a flow cytometry based killing assay to monitor NK activity. Therefore, within the same experiment, we can analyze NK cell phenotype profiles with specific antibody panels while also assessing NK cell killing activity.
Using optimized serum‐ and xeno‐free conditions with minimal cell manipulations, we have expanded highly purified NK cells up to 500‐fold in 14 days from human PBMCs using Cloudz™ Human NK Cell Expansion (NK Cloudz™) microspheres or anti‐NKp46 antibody. To characterise PBMCs after expansion with optimized cytokines and NK Cloudz™, we used a unique antibody panel to characterize the NK cells by flow cytometry. Within the same experiment, NK killing assays against K562 cells can be performed using a flow‐based killing assay, which has the advantages of using one instrument for all analyses, shortened processing times, and reproducibility. Using this workflow, we can identify different culture conditions that adversely affect NK activity.
Together, this data presents a comprehensive and clinically relevant workflow for expanding highly purified NK cells and assessing their killing potential. Finally, this workflow can be used for screening the activity of CAR expressing immune cells against specific cancer antigens.
Actively targeted chitosan‐based vector for efficient CRISPR‐Cas9 mediated
Tenascin‐C
gene editing in triple negative breast cancer
1: Marmara University, Istanbul 2: Cancer Research Centre (IBMCC/CSIC/USAL/IBSAL), Salamanca Spain 3: Eastern Mediterranean University, Famagusta 4: Inonu University
Despite the advances in diagnostic and therapeutic methods, the prognosis of the triple negative breast cancer (TNBC) hasn't improved substantially. Thus, innovative therapeutic approaches, such as gene therapy should be urgently developed against TNBC. Tenascin‐C (TN‐C) silencing is a promising TNBC oncotherapy strategy because of its restricted expression and its role in tumor survival, metastasis, and immune escape. In this study, we investigated the effect of TN‐C downregulation in the human TNBC cell line, MDA‐MB 231 using the potent genome editing tool CRISPR‐Cas9, and subsequently developed a chitosan‐based targeted gene delivery vector for safe and efficient in vivo application. Herein, the cells were transfected by CRISPR‐Cas 9 containing plasmids that target three regions on the TN‐C gene by a commercial lipid‐based reagent. Within 72 hours, the multiplex sgRNA therapy lowered TN‐C mRNA expression by 40%. This transfection, when compared to the transfection with scrambled guide RNA, increased the percentage of apoptotic cells by three folds and reduced the percentage of cells in G2/M phase accompanied with an increase in G1/G0. Afterwards, a nanoplex was prepared by grafting polyethylene glycol to cationic chitosan polymer, and by adding oxytocin to the vector as an active breast targeting molecule. FT‐IR, electron microscopy, and dynamic light scattering were used to characterize the nanoplex harboring the CRISPR/Cas9 plasmid. The nanoplex lowered the expression of TN‐C mRNA by 30‐40%. Considering the results herein, we consider the developed targeted non‐viral vector for TN‐C editing by CRISPR‐Cas9 as a viable approach to lower TNBC proliferation and survival.
The research was funded by Inonu University, Department of Scientific Research Projects (TOA‐2019‐1543).
Evaluation of transduced cultures of MCF‐7 cells with a first‐generation adenoviral vector expressing the caspase‐3 gene as a model for breast cáncer.
1: Pharmaceutical Biotechnology Laboratory. Faculty of Pharmacy. Autonomous University of Morelos, Morelos, México. 2: Direction of Chronic Infections and Cancer, Research Center in Infection Diseases, National Institute of Public Health, Morelos, México.
Breast cancer continues to be a public health challenge, with significant incidence and mortality rate in women worldwide generating the need of novel and focused therapies. Caspase‐3 is a relevant protein because of its role to induce programmed cell death by both extrinsic and intrinsic pathways in cells. Thus, in the present study, the molecular construction of a first‐generation adenovirus with the ability to express the casp‐3 gene (Ad5‐Casp3) to activate apoptosis in MCF‐7 breast cancer cells was performed. Cultures of MCF‐7 cells were transduced with the Ad5‐Casp3 vector. The effect of viability was assessed by trypan blue and MTS techniques. Caspase‐3 expression by RT‐PCR and Western blot assays. Apoptosis was analyzed by AO/IP staining, DNA fragmentation, cell cycle analysis and annexin V flow cytometry. Transduction of MCF‐7 cells with Ad5‐Casp3 showed expression of the gene and caspase‐3 protein, induction of DNA fragmentation and typical apoptotic cell morphology. These observations indicate that the Ad5‐Casp3 vector has the ability to activate both extrinsic and intrinsic pathways of apoptosis in MCF‐7 cells becoming a potential gene for breast cáncer treatment.
Transcriptional and functional assessment of CAR‐T cells targeting CD33 from AML patients and healthy donors.
1: Area de Terapia Celular, Clinica Universidad de Navarra. IdiSNA. Pamplona, 31008, Spain. 2: Servicio de Hematología, Complejo Hospitalario de Navarra. IdiSNA. Pamplona, 31008, Spain. 3: Departamento de inmunología e inmunoterapia, Clinica Universidad de Navarra. Pamplona, 31008, Spain. 4: Programa de Hemato‐Oncología, CIMA Universidad de Navarra. IdiSNA. Pamplona, 31008, Spain. 5: Departamento de Hematología y hemoterapia, Clinica Universidad de Navarra. IdiSNA. Pamplona, 31008, Spain. 6: Programa de inmunología e inmunoterapia, CIMA Universidad de Navarra. IdiSNA. Pamplona, 31008, Spain. 7: Centro de Investigación Biomédica en Red de Cáncer (CIBERONC)
Patients with R/R AML, the most common form of acute leukemia in adults, present a very poor prognosis, thus the development of new therapies is required. Notwithstanding great successes of CAR‐T therapies against hematological malignances, there are still considerable challenges in AML. Here we identified factors contributing to optimal CAR‐T response in AML patients.
Using a reporter system, we selected a CAR targeting CD33 with high specific activation without tonic signaling. In contrast to the enriched memory phenotype observed in CAR‐T from adult healthy donors (<40yr), that correlates with improved efficacies in clinical trials, CAR‐T from AML patients displayed a predominantly effector phenotype, with increased PD1 and LAG3 expression. However, all CAR‐T showed strong proliferation, cytokine production and cytotoxicity, regardless of the origin. After continuous stimulation, CAR‐T from AML patients increased their differences in PD1 and LAG3. Transcriptional studies provided molecular insights into the differences between CAR‐T cells form AML patients compared not only with adult healthy donors but also with age‐matches donors.
All CAR‐T cells demonstrated in vivo antitumoral efficacy in xenograft AML models, increasing the survival of treated mice. Moreover, comparing 41BB and CD28 CAR‐T cells, no significant differences were observed regarding their basal phenotype and expression profile, but interestingly, reduced antitumoral efficacy was observed in CD28 CAR‐T, being more significant in CAR‐T from AML patients.
In conclusion, we have identified several factors influencing the functionality of CAR‐T cells from AML patients, and the comprehension of the mechanistic insights offer an important information for the development of improved therapies.
Optimization of the CD123 CAR T‐cell scFv for the treatment of patients with Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN)
1: UMR1098‐RIGHT/EFS BFC/UFC, Besançon, 25000, France 2: CHRU Jean Minjoz, Besançon, 25000, France 3: Invectys, Paris, 75013, France
Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) is an aggressive malignant hemopathy characterized by constant overexpression of the CD123 antigen on 100% of blasts identifying CD123 as an antigenic target for the development of adoptive cell therapy. However, some healthy cells (hematopoietic stem cells, endothelial cells, monocytes) weakly express CD123, making them potential target cells. We have developed four 3rd generation CD123 CAR‐T (CD28, 4‐1BB, CD3z) composed of a single chain fragment variable (scFv) with a distinct affinity for CD123 in order to better discriminate BPDCN cells from healthy cells and reduce on‐target/off‐tumor toxicity. We showed strong CAR expression by T cells for three of four CD123 CAR‐T as well as strong cytotoxicity of CD123 CAR‐T against BPDCN (CAL‐1, Gen2.2, PDX) but not on CD123 negative cell line (Daudi) and very low cytotoxicity on healthy cells (HMEC‐1 and monocytes). CD123 CAR‐T did not affect significantly hematopoetic stem cell survival and progenitor's clonogenicity. Thanks to two preclinical models, we showed that two of four CD123 CAR‐T increased mouse overall survival (untransduced T‐cell: 32days; CAR01: 57days and CAR03: 56days in the CAL‐1 Luc+ model; untransduced T‐cell: 67days; CAR01: 157days; CAR03: 162days in the Patient Derived Xenograft model). To conclude, we emphasized that scFv selection is an essential step for the development of a CD123 CAR‐T with good efficacy/safety profile to bring it to patients.
Development of
in vivo
anti‐CD19 CAR T‐cell therapy based on chemically encapsulated lentiviral vectors
F Mourlane1 P Tesson1 C Magliocco1 C Duchene1 M Girardon1 M Lhuaire1 L Leclere1 E Maunichy1 R Pacherie1 R Vaillant1
1: Ixaka
With Yescarta®, Kymriah®, Tecartus® and Breyanzi® marketed therapies for B cell hematological malignancies and more than 300 ongoing clinical trials, genetic modification of T cells with chimeric antigen receptors (CARs) recognizing surface antigens on tumor cells has emerged as a revolutionary therapeutic strategy in immuno‐oncology. Despite impressive clinical benefits, the complex logistics required to manufacture ex vivo CAR‐T cells from individual patients and the associated costs represent major hurdles to the widespread use of these therapies.
Ixaka is a preclinical‐stage biotechnology company developing in vivo CARs based on hybrid nanoparticles consisting of lentiviral vectors (LV) encoding anti‐CD19 CARs encapsulated in oligopeptide‐modified poly(beta‐amino ester)s (OM‐PBAEs) biodegradable polymers.
Here we report on a microfluidics‐based method that consistently manufactures hybrid nanoparticles (NPs) able to reprogram peripheral blood mononuclear cells to express a functional CAR.
The biodistribution of NPs has been investigated in immunocompetent mice after multiple intravenous administrations or infusions and showed a different profile compared to pseudotyped LVs. Repeat administration was safe and well tolerated as no obvious sign of distress, body weight loss, change in blood cell count or cytokine levels were reported. The pronounced tropism for blood cells observed in vivo with Hybrid NPs provides an obvious advantage for CAR T‐cell therapy of blood malignancies. This technology shows great potential for further preclinical and clinical development of in vivo universal CAR T‐cell therapy.
CAR density influences CAR‐T antitumoral efficacy and correlates with clinical outcome
1: Hemato‐Oncology Program. CIMA Universidad de Navarra. IdiSNA. Pamplona, 31008. Spain. 2: Computational Biology Program. CIMA Universidad de Navarra. IdiSNA. Pamplona, 31008. Spain. 3: Hematology and Hemotherapy Department. Clínica Universidad de Navarra. Pamplona, 31008. Spain. 4: Hematology Service, Complejo Hospitalario de Navarra. IdiSNA. Pamplona, 31008, Spain. 5: Cell Therapy Area. Clínica Universidad de Navarra. IdiSNA. Pamplona, 31008. Spain. 6: Immunology and Immunotherapy Department, Clínica Universidad de Navarra. Pamplona, 31008. Spain. 7: Department of Hematology, Hospital Clinic de Barcelona, IDIBAPS, Barcelona, 08036, Spain 8: Department of Immunology, Hospital Clinic de Barcelona, IDIBAPS, Barcelona, 08036, Spain 9: Immunology and Immunotherapy Program. CIMA Universidad de Navarra. IdiSNA. Pamplona, 31008. Spain. 10: Centro de Investigación Biomédica en Red de Cáncer (CIBERONC). Madrid, 28029. Spain.
Recent studies have shown that control of CAR expression influences CAR‐T efficacy. Therefore, we hypothesized that CAR density directly affects CAR‐T cell function. In this study we characterized the in vitro and in vivo antitumoral efficacy of FACS‐isolated subpopulations of CAR‐T cells with different CAR densities targeting BCMA.
CARHigh‐T cells presented increased basal activation together with more differentiated phenotypes, increased cytotoxicity and cytokine production against MM cell lines. This effect was also observed in the infusion products of a clinical trial, where products enriched in CARHigh‐T cells presented increased cytotoxicity. After antigen‐driven activation, higher presence of terminally differentiated effector cells was observed in CARHigh‐T cells, along with increased exhaustion. In vivo, CARLow‐T cells presented increased persistence, suggesting that higher CAR levels could reduce long‐term efficacy.
Transcriptomic analysis revealed completely different expression profiles, with increased tonic signal in CARHigh‐T cells. We generated a gene signature associated to CAR density to annotate CARHigh‐T cells in scRNA‐seq analysis to further infer regulatory dissimilarities driven by different CAR densities, using novel computational methods.
Finally, to evaluate the impact of CAR density in the clinical outcome of CAR‐T therapies, we applied the inferred gene signature to score infusion products from a clinical trial, observing absence of complete responders in those products enriched on CARHighsignature.
Our data demonstrate that CAR density plays important roles in CAR‐T activity with impact on clinical outcome. Comprehension of regulatory mechanisms driven by CAR densities at the single cell level offer an important tool for the development of improved therapies.
Development of T‐CAR and NK‐CAR using a nonviral, nonintegrating, episomal DNA vector
G M Aguiar3 K RS Gomes3 G E Mulia1 M L Figueiredo1 A Athanassiadou2 D T Covas3
1: Department of Basic Medical Sciences, Purdue Center for Cancer Research, and Purdue Institute for Drug Discovery, Purdue University College of Veterinary Medicine, West Lafayette, IN 47907, USA. 2: Department of General Biology, Medical School, University of Patras, 26504 Rion, Patras, Greece. 3: Center for Cell‐based Therapy (CTC), Regional Blood Center of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil.
CAR‐T cell therapy has proven its importance in cancer treatment and currently there are five approved products on the market, all based on viral vectors, that bear the drawbacks of insertional mutagenesis, immune response and, importantly, high production cost. The development and use of extrachromosomal vectors (episomes) ‐ suitable for the efficient and stable transfection of NK or T cells ‐ is a valid alternative objective for the field of immunotherapy with CAR‐modified. We used an episomal vector carrying i) the scaffold/matrix attachment region (S/MAR)that mediates the episomal maintenance, ii) the replication‐Initiation Region (IR) from the β‐globin locus, as a replication enhancer, (pEP‐IR) and iii) a CAR anti CD19 (pEP‐IR‐CAR). We electroporated non‐activated T cells and observed that higher plasmid concentration increased the proportion of transfected cells, but it decreases cell viability. Transfections of T cells with 20 ug of control pEP‐IR‐GFP plasmid reached 47 ± 21.83% of GFP+ cells and viability around 89% depending of the culture condition used after electroporation (FBS% and IL2). We accessed the efficacy of pEP‐IR‐CAR, CAR expression was confirmed in 3.2% of transfected cells and 97.5% of viability. Cells expressing CAR on their surface were selected with anti‐CAR antibody Biotin‐SP‐conjugated and sorted by anti‐Biotin microbeads, achieving an enrichment of 54.9% CAR+ cells. Thus, we established an optimized protocol that maintains cell viability resulting in an acceptable transfection efficiency. The positive cells can be isolated and used in vitro cytotoxicity and in vivo assays in the next steps.
Lentiviral vector/GM‐CSF Gene Therapy for Autoimmune Pulmonary Alveolar Proteinosis
1: Imperial College London 2: University of Oxford
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. Alternatively, recombinant GM‐CSF protein has been shown to outcompete the auto‐GM‐CSF antibodies and stimulates clearance of pulmonary surfactant by alveolar macrophages, but a recent Phase 3 trial showed only a modest benefit when administered daily. We are therefore assessing whether gene therapy may overcome the limitations of the current therapies.
We have developed a lentiviral vector pseudotyped with the F and HN proteins from Sendai virus (rSIV.F/HN). Here, we assessed efficacy of rSIV.F/HN‐mediated gene transfer in a mouse model of pulmonary alveolar proteinosis (PAP). Lungs of GM‐CSF knockout mice were transduced with a single dose of rSIV.F/HN expressing murine (m)GM‐CSF (1e5‐92e7 TU/mouse). mGM‐CSF expression in murine lungs was dose‐related and persisted for the duration of the experiment (∼9 months). Biomarkers of PAP disease were rapidly and persistently ameliorated. Expression of high levels of GM‐CSF caused histopathological changes in various organs at the highest doses, but correction of some PAP biomarkers was also achieved with very low doses of vector (1e5 TU/mouse), which did not induce histopathological changes over a 11 months study period. This study also provides a pathfinder for other lung diseases that may benefit from prolonged and stable expression of secreted proteins.
Development of in vitro transcribed mRNA therapeutics for cystic fibrosis
1: University College London 2: University of Pennsylvania 3: St George's University of London
Cystic fibrosis (CF) is a relatively common recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Absent/non‐functional CFTR leads to thick, sticky mucus in the lung which results in chronic bacterial infection and inflammation. In vitro transcribed (IVT) mRNA has emerged in the last few years as a new approach for CFTR protein replacement therapy but problems of delivery must be overcome.
We have developed novel, receptor‐targeted nanocomplex (RTN) formulations consisting of liposomes and receptor‐targeting peptide for delivery of CFTR IVT mRNA. The RTNs were first optimised in transfections of primary cystic fibrosis bronchial epithelial cells (CFBE) using reporter IVT mRNAs. We then assessed delivery efficiency of CFTR mRNA in CFBE cells at submerged culture and ALI culture and assessed uptake by qRT‐PCR and demonstrated protein expression by Western blot.
Interestingly, we found that co‐delivery of the CFTR corrector, VX‐809, with CFTR IVT mRNA improved expression of the transfected CFTR protein in CFBE cells in Western blots by approximately 2 to 2.5‐fold. CFTR protein expression was also shown in transfections of ALI culture of CFBE cells by functional analysis of ion transport in Ussing chamber. The ΔIs was 3‐fold larger in cells transfected with CFTR mRNA than the untransfected cells before/after Forskolin added.
In conclusion, RTN mediated delivery CFTR IVT mRNA is a promising therapeutic approach for cystic fibrosis. In addition, the lipid compartment of the RTN allows co‐delivery of VX809 with CFTR mRNA, which significantly improved CFTR expression.
Functional characterisation of an engineered next generation lentivirus vector for the treatment of cystic fibrosis
1: Boehringer‐Ingelheim Pharma GmbH & Co. KG 2: UK Respiratory Gene Therapy Consortium 3: National Heart and Lung Institute, Imperial College London 4: Centre for Genomic and Experimental Medicine, IGMM, University of Edinburgh 5: Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford 6: The Roslin Institute & R(D)SVS, Easter Bush Campus, University of Edinburgh
Despite the success of CFTR‐modulator therapies there is still a need for therapies that address all CFTR mutations. In collaboration with Boehringer Ingelheim, the UK Gene Therapy Consortium is developing a novel gene therapy approach using a lentiviral vector pseudotyped with the F/HN proteins from Sendai virus to introduce a wildtype copy of the CFTR gene into lung epithelial cells of CF patients.
Here, we demonstrate the ability of the lentiviral vector to restore CFTR current to physiological levels in primary human airway epithelial cells with F508del mutation grown in air liquid interface (ALI). The restoration effect was dose dependent based on multiplicity of infection (MOI) of the vector: MOI 3 resulted in 64% of CFTR current restoration, while MOI 10 resulted in 141% of restoration. Current modulator therapies were also tested and gave 36‐122% of restoration. We next assessed the effect of the lentiviral vector in Class 1 null ALI cultures. Transduction at MOI 3 and 10 was able to restore the CFTR current to 101% and 133% respectively. In contrast, modulator therapies alone did not alter the CFTR current. From a safety perspective, the genomic integration pattern of the lentiviral vector showed no hotspots of integration into the host genome.
Overall, the generated results underscore the potential of lentiviral vector mediated CFTR expression as a promising treatment option for all CF patients independent of the underlying genotype.
Single cell tools for measuring mRNA and protein expression for gene therapy
1: Imperial College London 2: University of Oxford
As more gene therapies reach clinical trials, better tools for testing the efficacy of these therapeutics are needed; methods that can accurately and sensitively measure RNA and protein from patient samples. As part of our lung gene therapy programme using an rSIV pseudotyped with F and HN proteins from Sendai virus, we developed three assays to quantify the number of transduced cells, and vector derived expression in single lung epithelial cells: an in‐situ hybridisation assay, a single cell reverse transcriptase droplet digital PCR (RT‐ddPCR) assay, and a single cell digital proximity ligation assay (dPLA). In situ hybridization, using RNAscope™, detected lentiviral‐derived RNA in transduced A549 cells, mouse nasal epithelium, and ex vivo transduced human nasal brushings on a single cell basis. Quantification with an automated ImageJ macro found that a median of 33.2% (range 15.3‐53.5%) of mouse nasal epithelial cells contained vector‐derived transcripts 7 days after transduction. Similarly, in ex vivo human nasal brushings, a median of 34.7% (range 7.6‐54.8%) of cells were transduced. Using ddPCR and dPLA, lentiviral‐derived RNA and EGFP were simultaneously quantified in single HEK293T cells, air liquid interface cultures, and mouse airway epithelial cells. In mouse nasal epithelial cells, a median of 951 (range 624‐1620) RNA molecules and 9980 (range 5336‐10299) EGFP molecules were detected in single cells. In conclusion, we have demonstrated that single cell tools can be used to determine transduction efficiency in lung epithelium on a single cell basis.
Modifying signal peptides for respiratory gene therapy with secreted proteins
1: Imperial College London
Using a recombinant SIV lentiviral vector pseudotyped with Sendai virus F and HN proteins allows for robust and sustained transduction of murine lungs. However, transduction of the human lung will require large amounts of virus. Technologies that improve protein secretion could decrease the necessary dose, and even modest increases (2x) could reduce manufacturing costs by half. Signal peptides are heterogenous sequences at the N‐terminus of secreted proteins that can be modified to improve protein secretion. Here we explore if synthetic and endogenous signal peptides are a viable strategy for boosting the secretion of therapeutically relevant proteins such as alpha‐1 antitrypsin (AAT) and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). Bioinformatic analysis of proteins secreted from the lungs produced five candidate signal peptides: tetranectin (CLEC3B), cartilage acidic protein 1 (CRTAC1), alpha‐2‐macroglobulin (A2M), uteroglobin (SCGB1A1) and pulmonary surfactant‐associated protein A2 (SFTPA2). Additionally, a strong synthetic signal peptide, secrecon, was selected from the literature. In HEK293T cells secrecon reduced secretion of AAT from 41.07 to 16.75 ng/mL (0.4x) and GM‐CSF from 213.70 to 33.74 pg/mL (0.2x) compared to the endogenous signal peptides. However, CRTAC1 significantly increased GM‐CSF secretion from 213.70 to 383.7 pg/mL (1.8x, p = 0.024 Kruskal‐Wallis test with Dunn's multiple test correction). In conclusion, signal peptides are a viable approach for increasing protein secretion. Further evaluation of lung signal peptide candidates, in models that better represent the lungs such as air‐liquid interface cultures and mice, could identify an optimal signal peptide for respiratory gene therapy.
Regulation of lentivirus‐mediated expression in a human airway model
1: Imperial College London
The UK Respiratory Gene Therapy Consortium, has developed a lentiviral vector pseudotyped with Sendai virus envelope proteins F and HN (rSIV.F/HN) producing high level pulmonary gene transfer. 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 gene expression. To enhance the efficacy/toxicity window we aimed to generate a lentiviral vector carrying a regulatable expression cassette, which will initiate gene expression following administration of an inducer molecule.
To establish proof‐of‐concept that lentivirus‐mediated expression can be regulated, a commercial mifepristone‐inducible system (GeneSwitch, Invitrogen) was cloned into a lentiviral backbone and VSV‐G vectors generated. The commercial expression cassette was kept in the original configuration, encoded by two vectors (2V), and additionally cloned into one vector (1V). First HEK293T cells were transduced with 1V or 2V, carrying gaussia luciferase (GLux) as the transgene, and expression induced by administration of the drug mifepristone 24 hours post‐transduction (10−8M mifepristone, MOI 10, n = 6 wells/condition). Cells transduced with 1V showed a 12.8‐fold induction in GLux expression, compared to a 519.6‐fold induction observed by 2V. Similarly, human air‐liquid interface cultures were transduced with inducible vectors encoding the therapeutic transgene GMCSF, and expression induced with mifepristone (10−8M mifepristone, MOI 50, n = 3 ALIs/condition). GMCSF expression increased by 27.8‐fold in 1V cultures compared to 575.9‐fold with 2V cultures. In summary, we have demonstrated proof‐of‐concept that lentivirus‐mediated gene expression can be regulated in a human airway model, with superior induction observed by the 2V system.
Preclinical pharmacology of KB408, an HSV‐1‐based gene therapy vector, for the treatment of alpha‐1 antitrypsin deficiency
1: Krystal Biotech, Inc.
Alpha‐1 antitrypsin deficiency (AATD) is a rare autosomal co‐dominant inherited genetic disorder resulting from mutations in the gene encoding alpha‐1 antitrypsin (AAT). AAT is a secreted α1‐glycoprotein and the most abundant serine protease inhibitor in human plasma, whose principal substrate is neutrophil elastase in the lungs. Despite causing both severe lung and liver disease, for the large majority of AATD patients, lung disease is of the greatest clinical importance as it often results in life threatening, progressive pulmonary impairment and severe respiratory insufficiency. Augmentation therapy, consisting of weekly intravenous infusions of recombinant or plasma‐derived AAT, remains the only FDA approved therapy for AATD. Its clinical efficacy in preventing progressive lung dysfunction has not been established, hence novel effective treatments targeting pulmonary disease are being explored. To this end, we developed KB408, an engineered replication‐defective herpes simplex virus type 1 (HSV‐1)‐based gene therapy vector encoding full‐length human AAT for the treatment of AATD lung disease. Preliminary data indicated that KB408 efficiently transduced human cells, including clinically relevant primary human small airway epithelia cells (SAECs), resulting in production and secretion of full‐length human AAT. Moreover, KB408 effectively targeted the respiratory tract of immunocompetent mice after inhalation, promoting expression and secretion of detectable levels of human AAT in both the serum and lung lining fluid while maintaining a robust safety profile, thus suggesting proper trafficking through the interstitial space. Taken together, these observations support the application of KB408 as a novel gene therapy for the treatment of AATD lung disease.
Proteomic changes induced by intrapericardial administration of secretomes from menstrual blood stromal cells on the infarcted areas of porcine myocardium
M Pulido1
1: Jesús Usón Minimally Invasive Surgery Centre, Cáceres, Spain 2: CIBER de Enfermedades Cardiovasculares, Madrid, Spain 3: Immunology Unit, University of Extremadura, Cáceres, Spain.
Acute myocardial infarction (AMI) is a multifactorial disease that causes ischemia in myocardial tissue. Myocardial reperfusion injury activates a wide range of cellular and molecular processes including inflammation, neutrophil infiltration, oxidative stress, intracellular calcium overload, increased mitochondrial permeability, and, consequently, apoptosis. The administration of the secretome of mesenchymal stem cells has been suggested to be a promising therapeutic approach. The biological composition of these secretomes seems to be an effective tool to modulate altered molecular mechanisms in AMI. In this work, we aimed to evaluate the effect of secretome from menstrual blood stromal cells (S‐MenSCs) on the infarcted areas of porcine myocardium. Myocardial infarction models were generated using a closed chest myocardial occlusion‐reperfusion procedure in 8 pigs. After 72 hours, animals were intrapericardially administered with S‐MenSCs (AMI/S‐MenSCs, n = 4) and with vehicle (AMI/Placebo, n = 4). Seven days post‐therapy, hearts were harvested and infarcted tissues were collected for proteomic analyses. Proteomic analyses identified 113 differentially regulated proteins when infarcted tissue of AMI/S‐MenSCs was compared with the AMI/Placebo biogroup. Functional enrichment and protein‐protein interaction analysis was performed using OmicsBean. Our results showed that the intrapericardial administration of S‐MenSCs may improve the cardiac function targeting proteins involved in pathways, as energy derivation by oxidation of organic compounds, electron transport chain, muscle organ development, muscle contraction, and heart morphogenesis. In conclusion, this study suggests that S‐MenSCs administration may have a beneficial effect in restorating cardiac function after acute myocardial infarction. However, more preclinical studies should be performed to confirm S‐MenSCs potential as a successful therapeutic tool.
Hypoxic extracellular vesicles protect human cardiomyocytes from oxidative damage
1: Jagiellonian University 2: Freiburg University
Cardiomyocytes (CMs) damage associates with various heart diseases, including myocardial infarction and in a consequence may lead to heart failure. Notably, disorders of the cardiovascular system are the leading cause of mortality and morbidity in the developed countries. Despite available treatment options, there is still no cure for cardiac diseases, which constitutes a serious socio‐economic burden. Thus, there is strong need to develop novel and effective therapies to solve this problem. Here, we show a strong cytoprotective effect of extracellular vesicles (EVs) isolated from human induced pluripotent stem cells (hiPSCs) cultured in low oxygen condition (5% O2) on human CMs undergoing hypoxia‐reoxygenation (HR) in vitro. Hypoxic EVs triggered pro‐survival pathways in stressed CMs, including activation of Akt and MAPK kinases and reduced apoptosis, as confirmed by fluorescence microscopy, Western blot and qPCR methods. A proteome analysis revealed that hypoxic EVs contained anti‐oxidant proteins, such as PRDX6 and GSTP1 on a higher level, in comparison to EVs derived from normoxic (21% O2) conditions. In conclusion, we provide evidence that hypoxic hiPS‐EVs exert protective function on CMs in a hypoxia‐reoxygenation model. Such data may constitute a first step in the development of a future therapy of the cardiovascular system. This work was supported by the grant UMO‐2016/23/D/NZ3/01310 from the National Science Centre of Poland.
Live‐tracking of biomaterial distribution using CT‐visible microspheres after non‐invasive delivery into the heart
1: University College London
The therapeutic goal in heart failure patients is to restore cardiac function. Cell therapy is a potential treatment for cardiac regeneration. However, two major problems of cell therapies are low cell retention during cell delivery, and the inability to visualise the delivery and distribution of the therapy.
To address these problems, a trackable and injectable biomaterial was designed as a substrate for cellular attachment and growth. The biomaterial was delivered using ultra‐sound guided intramyocardial injections for targeted delivery.
Highly porous microspheres were fabricated from 2% (w/v) 75:25 DL‐lactide/glycolide co‐polymer and 20% (w/v) barium sulphate (BaSO4) using Thermally Inducted Phase Separation (TIPS).
In vitro, L929 fibroblasts were exposed to BaSO4 microspheres and showed minimum toxicity for up to 7 days (12% vs 5% vehicle control). In vivo, using a murine model (n = 5), BaSO4 microspheres were administered via ultrasound‐guided intramyocardial injections. Computed tomography scans were acquired to demonstrate persistent visualizations of the BaSO4 microspheres at the injection site with retained contrast signal measured immediately after injection, 2, 4 and 6 days later. A minority of injected BaSO4 microspheres were also visualised in off‐target organs and the findings were confirmed by post‐mortem histology.
The findings suggest that the microspheres can be used as a novel tool to improve the training, delivery and tracking of therapeutic biomaterials for targeted delivery.
iPSC‐derived macrophages from Cystic Fibrosis patients for infection models and drug screening platforms.
1: Clinic for 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), Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany 4: German Center for Lung Research (DZL), Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Hannover, Germany
Cystic fibrosis (CF) is an autosomal recessive disease driven by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, leading to increased lung infection susceptibility. The increasing number of pulmonary infections caused by multidrug resistant Pseudomonas aeruginosa is of particular concern for CF patients. Alveolar macrophages (AMs) form the first line of defence against airborne bacteria. However in CF, AMs are hyperinflammatory and suboptimal phagocytes. To dissect the role of macrophages in CF and to evaluate novel treatment scenarios, an in vitro macrophages infection platform would be highly valuable. We present the development of an infection model using induced pluripotent stem cells (iPSCs) from CF patients and thereof derived primitive macrophages (iMac). IMac can be generated on demand, in a reproducible and scalable manner. Human iPSCs from a ΔF508 (most common CFTR mutation) CF patient and healthy donor (WT) were differentiated into iMac in a similar quantity of approx. 0.5x106 cells/week/well. Both CF and WT‐iMac showed typical macrophage morphology and expressed macrophage markers such as CD11b, CD14 and CD163. Notably, CF‐iMac showed impaired phagocytic capacity in comparison to WT‐iMac when exposed to P. aeruginosa for 6h at MOI 1 and 10. Similarly, CF‐iMac showed a delay of approximately 1 hour before effectively initiate bacterial clearance, whereas WT‐iMac degraded more than 50% of initial infection dose of P. aeruginosa within the first hour of infection. In summary, we are able to provide a new platform to generate primitive macrophages from CF patients, which demonstrate impairment in bacterial phagocytosis and clearance.
FGF7 treatment improves survival and proliferation of injured primary lung epithelial cells
1: Imperial College London
Fibroblast Growth Factor 7 (FGF7) is secreted by mesenchymal cells during normal lung development and repair, and binds specifically to epithelial cells to promote survival, proliferation, and migration. Therefore, there is interest in supplementing this growth factor during lung injury. Systemic delivery of recombinant human FGF7 (rhFGF7) has demonstrated therapeutic benefit in a human model of ALI but failed to improve acute respiratory distress syndrome. Recombinant proteins are limited by short half‐life, poor bioavailability, and systemic toxicity. To overcome some of these issues, lung delivery of exogenous messenger RNA (mRNA) has demonstrated local, transient, and dose‐responsive translation of encoded protein. We hypothesised that FGF7 mRNA transfection will facilitate survival, proliferation, and healing of lung epithelial cells under physiological stress. To elucidate this effect, primary bronchial epithelial cells were seeded at low densities and treated with PBS or rhFGF7. We observed a 2‐fold increase in confluency of rhFGF7 treated cells at 40 hours post‐treatment (P < 0.0001), an improvement in cell survival, and a 74.6% reduction in cytotoxicity compared to PBS treated cells (P < 0.01). Next, we established an mRNA transfection system and found that primary lung epithelial cells and primary lung fibroblasts successfully translate exogenous FGF7 mRNA for up to 72 hours without reduction in cell viability. This system is being applied to injured lung cells to elucidate the therapeutic potential of FGF7 mRNA for lung cell survival and wound healing.
Alleviated inflammatory response in infarcted tissue after intrapericardial administration of secretome of prime menstrual blood stromal cells
E López1
1: Jesús Usón Minimally Invasive Surgery Centre, Cáceres, Spain 2: CIBER de Enfermedades Cardiovasculares, Madrid, Spain 3: Immunology Unit, University of Extremadura, Cáceres, Spain.
Myocardial ischaemia‐reperfusion injury triggers molecular changes in cardiac tissue, activating inflammatory response. This response involves activating signals such as cytokine, chemokines and growth factors and the recruitment of inflammatory cells such as neutrophils, platelets, lymphocytes and macrophages. In this scenario, stem cell therapy becomes a promising therapeutic option. Moreover, priming cell strategies improve the immunomodulatory capacity of their secretome. Therefore, the aim of this work was to evaluate proteomic changes in infarcted tissue after intrapericardial administration of secretome of primed menstrual blood stromal cells with IFNγ and TNFα (S‐MenSCs*). Myocardial infarctions were induced by 90‐min balloon occlusion of the mid‐LAD in 8 pigs. Via a mini‐thoracotomy performed 72 hours after, animals receive S‐MenSCs* (AMI/S‐MenSCs*, n = 4) and vehicle (AMI/Placebo, n = 4). Seven days post‐therapy, hearts were harvested and infarcted tissues were collected for proteomic analyses. Proteomic results showed 107 differentially expressed proteins after S‐MenSCs* treatment in infarcted tissue. Pathway enrichment using Reactome database revealed that these differentially expressed proteins were related with pathways such as Platelet activation, signaling and aggregation (R‐HSA‐76002) or Neutrophil degranulation (R‐HSA‐6798695). Most of the genes classified in these pathways were down regulated in AMI/S‐MenSCs*. These results suggest that secretome content of MenSCs* can reduce the activation of infiltrated platelets and neutrophils, and therefore, cause a reduction of pro‐inflammatory signaling. In conclusion, the immunomodulatory capacity of S‐MenSCs* may improve the prognosis of myocardial infarction, alleviating the exacerbated immune response in infarcted tissue after myocardial infarction.
miRNA‐4732‐3p as a possible therapeutic tool for the treatment of myocardial ischemia
1: Instituto de Investigacion sanitaria La Fe
Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSC) have emerged as an alternative to parental cells in cell free‐based cardiac therapies for the treatment of a variety of diseases including ischemic diseases. In this sense, the identification of EVs functional cargoes is an important key to move into the clinical area. Thus, we analyzed dental pulp MSC‐EVs miRNA cargo and identified the miR‐4732‐3p as a novel molecule regulated by hypoxia. The effect of a miR‐4732‐3p mimic was tested compared to MSC‐EVs into in vitro models using cardiomyocytes (CM), fibroblasts (Fb) and endothelial cells under oxygen glucose deprivation (OGD) conditions during 7h. After OGD, cardiomyocytes treated with either MSC‐EVs or miR‐4732‐3p mimic increased the survival rate by reduction of cleavage caspase 3 expression, lower levels of ROS and LDH activity and increasing the beating frequency. The expression of the myofibroblast markers α‐SMA and Col1A was also downregulated in Fb stimulated with TFGβ. In addition, miR‐4732‐3p mimic induced angiogenesis in an in vivo model of matrigel plug angiogenesis assay in NOD/SCID mice. It should be noted that intramyocardially injection of miR‐4732‐3p mimic also reduced the scar tissue formation and preserved cardiac function in infarcted nude rats. Our results showed that the miRNA‐4732‐3p can reduce myofibroblast differentiation and exert cardioprotective features under ischemic conditions. In addition, it was able to induce angiogenesis and preserve cardiac function in different animal models, which could have potential applications in cell‐free based therapeutic strategies.
Krüppel‐Like Factor 4 as a novel gene therapy target contributes to Hypoxic Pulmonary Hypertension
1: DKFZ 2: Christian‐Albrechts‐Universität zu Kiel 3: Klinik für Innere Medizin III‐ UKSH 4: Labor Molekulare Kardiologie ‐ UKSH
Hypoxic Pulmonary hypertension (HPH) is caused by persistent low oxygen tension (hypoxia), resulting in pulmonary vascular inflammation and remodeling and blood flow impairment. Thus, understanding and modulating factors involved in pulmonary endothelial inflammation could contribute to potential therapeutic approaches. We investigated the therapeutic potential of overexpression of the transcription factor KLF4 to prevent pulmonary hypertension. In vitro experiments were performed using AAV‐mediated gene transfer of KLF4 into human umbilical vein endothelial cells (HUVECs). Inflammation was significantly reduced in the AAV‐KLF4 treatment group compared with the AAV‐EGFP group. Hypoxia‐induced endothelial dysfunction was repaired by overexpression of KLF4. Besides, hypoxic reduced endothelial cell‐cell adhesion was improved in AAV‐KLF4. Firstly, in vivo experiments aimed to demonstrate the efficiency and safety of overexpressing KLF4 in a mouse model. Specifically, as KLF4 was shown to act as an oncogene regulator, the tumor‐regulating properties of KLF4 are associated with p53 and p21. The results demonstrated that overexpression of Klf4 did not activate oncogene (p53, p21, p300) expression. Overall, the AAV‐mediated KLF4 overexpression enables significant protection of endothelial cells from hypoxia‐induced stress in vitro and does not result in side effects when expressed in murine pulmonary artery endothelial cells in vivo. This study's results enable further investigations on AAV‐mediated KLF4 overexpression in pulmonary artery endothelial cells in a murine model of chronic hypoxia‐induced pulmonary hypertension.
Cardiac AAV:PKP2 gene transfer prevents development of arrhythmogenic cardiomyopathy in a
PKP2
‐deficient mouse model
1: Tenaya Therapeutics
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiac disease associated with ventricular arrhythmias and sudden cardiac death. Mutations in the desmosome gene PKP2, Plakophillin‐2, are the most common genetic cause of ARVC. Currently there is no known effective treatment available for ARVC patients. Here we report an AAV‐based gene transfer approach preventing ARVC development in a cardiac specific knock‐out mouse model of PKP2. Early proof‐of‐concept studies using PKP2‐deficient human iPSC‐derived cardiomyocytes show cellular recovery of desmosome components and rescue of contractility upon expression of exogenous PKP2. Cardiac AAV:PKP2 gene delivery significantly improves life span of PKP2‐cKO ARVC mice by 1) preventing adverse cardiac remodeling; 2) maintaining ventricular functions; 3) reducing arrhythmia event frequency and severity. Our results indicate that AAV:PKP2 is a viable gene transfer approach to address the major genetic cause of ARVC.
Gene therapy with eladocagene exuparvovec improves cognition and language in patients with aromatic l‐amino acid decarboxylase deficiency
P W‐L Hwu1 Y H Chien1 N C Lee1 S H Tseng1 A Wang2 J Wang2 E T Leonardi2
1: National Taiwan University Hospital 2: PTC Therapeutics, Inc 3: PTC Therapeutics Switzerland GmBH
Aromatic l‐amino acid decarboxylase (AADC) deficiency is caused by mutations in the dopa decarboxylase gene leading to reduced AADC enzyme activity. Patients with AADC deficiency may experience delayed cognitive and speech development. Eladocagene exuparvovec is a recombinant adeno‐associated viral vector serotype 2 carrying the coding sequence for the human AADC gene.
Eladocagene exuparvovec was administered via bilateral infusions to the putamen of 28 children with AADC deficiency in 3 clinical trials (AADC‐CU/1601 [8 patients, completed], AADC‐010 [10 patients, completed], and AADC‐011 [10 patients on 26 February 2020 cut‐off date, ongoing]). Patients received 1.8 × 1011 vg (n = 21) or 2.4 × 1011 vg (n = 7; AADC‐011)]. Cognition and language changes were assessed using Comprehensive Developmental Inventory for Infants and Toddlers (CDIIT; N = 8) and Bayley Scales of Infant Development, 3rd edition (Bayley‐III; N = 20). Both tools measure paediatric development and include cognitive and language subscales.
CDIIT showed improvements in cognitive and language skills as early as 6 months, which were maintained up to 60 months. Bayley‐III showed gradual, sustained improvement up to 60 months. Mean change from baseline, total language score was 46.5% after 12 months (n = 17), 62.7% after 24 months (n = 15), 80.5% after 36 months(n = 10), 108.3% after 48 months (n = 8), and 110.7% after 60 months (n = 4). Significant improvements in subscale scores were observed 24 months post‐treatment.
Results demonstrate the efficacy of eladocagene exuparvovec in improving cognition and communication in patients with AADC deficiency, indicating that gene therapy may successfully target neurotransmitters affected by AADC deficiency and may improve quality of life.
Eladocagene exuparvovec gene therapy improves motor development in patients with aromatic l‐amino acid decarboxylase deficiency
P W‐L Hwu1 Y H Chien1 N C Lee1 S H Tseng1 A Wang2 J Wang2 E T Leonardi2
1: National Taiwan University 2: PTC Therapeutics, Inc 3: PTC Therapeutics Switzerland, GmBH
Aromatic l‐amino acid decarboxylase (AADC) deficiency is caused by mutations in the dopa decarboxylase gene leading to reduced AADC enzyme activity; it is characterized by motor impairments and inability to attain developmental milestones. Eladocagene exuparvovec is a recombinant adeno‐associated viral vector serotype 2 carrying the coding sequence for human AADC.
Eladocagene exuparvovec was infused bilaterally in the putamina of 28 children with AADC deficiency in 3 clinical trials (AADC‐CU/1601 [8 patients, completed], AADC‐010 [10 patients, completed], and AADC‐011 [10 patients on 26 February 2020 cut‐off, ongoing]). Patients received a total of 1.8 × 1011 vg (n = 21) or 2.4 × 1011 vg (n = 7; AADC‐011)] and were assessed for the motor milestone attainment using the Peabody Developmental Motor Scale, 2nd edition (PDMS‐2) and Alberta Infant Motor Scale (AIMS). PDMS‐2 contains subscales for interrelated motor abilities and AIMS contains subscales for elements of movement in different positions.
All patients treated with eladocagene exuparvovec had clinically meaningful increases in total PDMS‐2 and total AIMS scores, which were maintained or improved over time, up to 60 months (LS mean change from baseline [SE] 15.0 [8.54] and 27.5 [2.62] respectively, at 60 months, the last measured timepoint). Similar increases were noted for PDMS‐2 and AIMS sub scores. Clinically meaningful increases from baseline in PDMS‐2 total scores were seen as early as 3 months post‐treatment and extended to at least 60 months.
The data indicate that eladocagene exuparvovec can provide a durable, positive impact on motor development in patients with AADC deficiency.
Efficacy of C9orf72 ALS gene therapy using miQURE® and widespread distribution in cortical and spinal regions in non‐human primates
1: uniQure biopharma B.V.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of the upper and lower motor neurons, leading to muscle atrophy and paralysis. There is currently no curative therapy for ALS. The most common genetic cause of familial ALS is an expanded hexanucleotide repeat in the first intron of chromosome 9 open reading frame 72 (C9orf72) gene. Cellular toxicity due to RNA foci and dipeptide repeat proteins underlie the pathogenesis of the disease. Therapeutic microRNAs (miQUREs) were developed to silence C9orf72 mRNA. miQURE candidates silencing efficacy was tested on luciferase reporters that endogenously expressed C9orf72 mRNA. Selected candidates were incorporated into adeno‐associated virus serotype 5 (AAV5) and their target engagement was investigated in human‐derived iPSC neurons and in transgenic C9orf72 mice. AAV delivered miQUREs expression in iPSC derived neurons resulted in ∼40% lowering of sense intronic C9orf72. Intrastriatal injection of AAV‐miQUREs in transgenic mice resulted in high expression of mature miRNAs and significant lowering of sense C9orf72 intronic transcripts in the cortex and striatum. As a consequence of C9orf72 lowering, significant reduction of toxic RNA foci in the cortex was observed. To support translation to humans, AAV‐miQURE was administered directly into the cerebrospinal fluid of non‐human primates. Vector DNA distribution and expression of mature miRNAs were achieved in relevant brain area (cortex) and spinal cord. Taken together, these data provide pre‐clinical proof‐of‐concept for silencing of C9orf72 using AAV‐miQUREs and warrants further investigation as an attractive approach to target ALS.
Modulation of brain cholesterol metabolism through CYP46A1 overexpression for Rett syndrome
E Audouard1 F Nobilleau1 O Bouazizi1 S Stanga2 A Lamaziere3 B Meha1 N Cartier1
1: NEUROGENCELL, Institut du Cerveau et de la Moelle épinière, INSERM U1127, Hôpital Pitié‐Salpêtrière, 47 bd de l'Hôpital, 75013, Paris, France 2: Neuroscience Institute Cavalieri Ottolenghi, University of Turin, 10043 Orbassano (TO), Italy 3: LBM, Sorbonne Université, Faculté de Médecine, AP‐HP, Hôpital Saint Antoine, 75012, Paris, France
Rett syndrome is a rare neurodevelopmental disorder caused by mutation in the MECP2, a ubiquitously expressed transcriptional regulator. Rett results in intellectual disabilities. It is characterized by normal early growth and development followed by severe problems with language and communication, learning, coordination, and other brain functions. Currently, there is no curative treatment for Rett. Thus, it is crucial to develop new therapeutic approaches for Rett patients. Studies suggest that Rett is link with defects in cholesterol homeostasis. Moreover, our laboratory has demonstrated therapeutic efficacy of CYP46A1 overexpression, enzyme involved in cholesterol pathway, in several neurodegenerative diseases. Based on the studies reported with abnormal cholesterol metabolism in Rett models and statins studies but also phenotype associated with Rett syndrome, we strongly believe that CYP46A1 could be a relevant therapeutic target for Rett disorder. Our goal was to investigate the restoration of neuronal cholesterol metabolism through the overexpression of CYP46A1 as a therapeutic option for Rett patients. Herein, we evaluated the effects of intravenous AAVPHP.eB‐hCYP46A1 delivery in KO‐MECP2 mice. The AAVPHP.eB‐hCYP46A1 transduced essentially neurons of central nervous system (CNS). No toxicity of vector was observed in peripheral organs and CNS. Preliminary data suggest that CYP46A1 overexpression alleviates behavioral alterations. No inflammation was observed in CNS of treated mice. In addition to evaluate the therapeutic potential of CYP46A1 gene therapy in mouse model, mechanisms by which CYP46A1 could act to have a therapeutic effect will are be presented. Altogether our results strongly suggest that CYP46A1 could be a relevant target in Rett syndrome.
Development of chimeric GALC enzymes with improved bioavailability to refine gene therapy strategies for Globoid cell Leukodystrophy.
1: San Raffaele Telethon Insitute for Gene Therapy (HSR‐TIGET) 2: Vita‐Salute San Raffaele University 3: Department of Chemistry, Biology and Biotechnology, University of Perugia
Globoid Cell Leukodystrophy (GLD) is an untreatable lysosomal storage disorder (LSD) caused by the deficiency of β galactosylceramidase (GALC). The infantile forms display a rapid and severe central and peripheral nervous system (CNS, PNS) dysfunction. The difficulty of achieving and maintaining therapeutic levels of GALC in CNS and PNS tissues hampers the development of effective gene therapy (GT) strategies for GLD. We hypothesize that the availability of GALC enzymes with increased tissue biodistribution may overcome this issue. We generated lentiviral vectors expressing murine (m)GALC enzymes engineered with the signal peptide of the iduronate‐2‐sulfatase (IDSsp) to promote secretion and including the low density lipoprotein receptor (LDLr)‐binding domain to favor transport across the blood‐brain barrier (BBB). We showed safe supraphysiological expression and enzymatic activity of chimeric mGALC enzymes in GLD murine neural and hematopoietic stem/progenitor cells and progeny (relevant cell types in the context of GT platforms), with an advantage given by IDSsp. The chimeric enzymes were secreted, recaptured, and delivered to the lysosomes of GALC‐deficient neural cells, which were metabolically cross‐corrected. The expression of LDLr in GLD neuron/glial cells reinforces the use of chimeric GALC enzymes to enhance the GALC supply in CNS cells. These results support the rationale of testing the safety and efficacy of chimeric GALC enzymes in ex vivo and in vivo GT approaches in GLD animal models, with the final goal of developing novel and more effective GT strategies for this untreatable disease.
Local delivery of AAV9 vector in peripheral nerves of mouse, rat and macaque allows a safe, well‐tolerated, high and specific delivery to myelin forming cells
1: Inserm 2: Nervosave Therapeutics
The large majority of peripheral nerve diseases such as Charcot‐Marie‐Tooth diseases or diabetic peripheral neuropathy affects the myelin sheath that surrounds motor and sensory axons disseminated in our body. The roles of the myelin sheath are to accelerate the propagation of action potentials and to protect and nurture axons along their way. Myelin defects result in abnormal myelin sheath deposition, myelin sheath loss, myelin repair failure and finally axon loss. Schwann cells are the myelin forming cells of the nerves and targeting these cells remains a challenge in the design of treatments for these diseases. While all nerves are affected by diseases, symptoms mainly result from the degeneration of the longest nerves that are located in arms and legs. We therefore investigated the possibility to treat these nerves through a local injection of AAV9 and rh10 in young and adult mouse and rat and in adult macaque. We found that AAV9 is the most efficient to transduce myelinated Schwann cells through a local injection. Up to 90% of the target cells were transduced with a specificity above 87%. Vector diffusion along nerves was significative after a single injection in rodents. Several regularly spaced injections allowed a significant coverage of several nerves of the macaque. These injections, even multiple in the same nerve, were shown to be safe and well‐tolerated both in rodents and macaques. This opens a novel avenue for the treatment of myelin‐related peripheral nerve diseases using gene therapy approach.
Innovative and Regulated Lentiviral Promoter for the Gene Therapy of Neurodegenerative Diseases
Y Ciervo1 L Bucciarelli1
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
Neuroinflammation is characterized by microglial cell‐activation that plays a major role in the pathogenesis and progression of several neurodegenerative Lysosomal Storage Disorders (LSDs). The beneficial effect of ex vivo gene therapy (GT), based on the use of genetically modified hematopoietic stem cells (HSCs), relies on functionally microglia‐equivalent cells derived from the transplanted HSCs into the recipient's Central Nervous System (CNS) where they exert homeostatic and scavenging functions, normalize CNS homeostasis and reduce neuroinflammation. An ideal lentiviral vector (LV) for neurodegenerative LSDs should drive a regulated expression of the therapeutic transgene in the HSC‐derived microglia‐like cells: upregulated in the presence of neuroinflammation and downregulated under homeostatic CNS conditions. To this aim, we designed a novel LV promoter, based on the human HLA‐DRA promoter region, able to induce basal levels of gene expression in microglial cells, at mRNA and protein level, increasing up to 4‐5 folds upon cell‐activation. The LV promoter faithfully reproduces the transcriptional pattern of the endogenous promoter in vitro and induces a high transgene expression in vivo, in myeloid/microglia cells derived from transplanted HSPCs, suggesting its capability to maintain the correct regulatory profile upon pluripotent‐cell differentiation to microglial cells, not being subjected to epigenetic silencing or other forms of undesired regulation. These results strongly support the usage of this new hHLA promoter in GT approaches for pathological conditions of the CNS characterized by neuroinflammation.
Minimizing toxicity of AAV‐based gene therapy for translation in a pediatric patient population with congenital hearing loss
1: Decibel Therapeutics, Inc 2: Regeneron Pharmaceuticals, Inc
AAV continues to elicit wide interest as a therapeutic modality, but reports of unwanted off‐target effects are becoming more common, with prominent examples of toxicity in the dorsal root ganglia and retinal pigmented epithelium. These effects can be mitigated using two major strategies. First, AAV delivery may be restricted to enclosed compartments, and second, expression of the therapeutic transcript may be restricted to target cell types via promoter or other vector engineering. For inner ear therapeutics, both strategies are likely to be important, as most genetic hearing disorders manifest only in the enclosed inner ear and require correction only in a small subset of the 40+ cell types in the organ. In the non‐human primate, we have observed minimal systemic biodistribution after direct inner ear AAV delivery. We and others have observed local toxicity when expressing candidate inner ear therapeutic transgenes (GJB2, OTOF, TMC1) under control of a ubiquitous promoter, manifesting as loss of sensory cells over several weeks post‐transgene infusion in mice. Local toxicity may be driven by transgene‐directed immunity due to expression in antigen‐presenting cells, at least in the case of the OTOF transgene. In developing a therapeutic for congenital deafness due to OTOF deficiency (DB‐OTO) we synthesized a hair cell‐specific promoter to minimize off‐target toxicity. We observed that local delivery of DB‐OTO in the disease model durably instated hearing in congenitally deaf mice and was well‐tolerated in exploratory toxicology studies. We intend to initiate a Ph1/2 clinical trial in 2022.
Anc80L65 results in more widespread gene transfer in the CNS of non‐human primates compared to AAV9
1: Affinia Therapeutics 2: Grousbeck Gene Therapy Center, Mass Eye and Ear 3: Harvard Medical School
Naturally‐occurring AAV capsids typically have poor distribution in the central nervous system (CNS) and do not efficiently transduce the target cell populations critical for the treatment of CNS diseases. To overcome limitations of current AAV capsids in the development of CNS gene therapies, we used ancestral sequence reconstruction to rationally design the novel capsid Anc80L65. We compared brain and spinal cord distribution and transduction of Anc80L65 to AAV9 expressing eGFP, in adult male cynomolgus macaques. Each animal received a 2ml injection of 2E13 vg/ml, administered either via lumbar puncture (LP; L3‐L4) or intracisterna magna (ICM); 3 animals per capsid per route of administration received vector and 1 animal per route of administration received vehicle control. Fourteen days post‐administration we found greater GFP expression by IHC and RT‐ddPCR throughout the cortex, spinal cord and deep brain nuclei in animals administered Anc80L65 than in those administered AAV9. In most CNS regions, Anc80L65 administered via LP showed greater expression than AAV9 administered via ICM. We also found transduction in both neurons and astrocytes throughout the cortex with Anc80L65, as well as transduction of a small number of oligodendrocytes in cortical regions. AAV9 transduced both neurons and astrocytes but did not show any evidence of oligodendrocyte staining in the brain sections examined. We found little to no evidence of microglial transduction with either Anc80L65 or AAV9 vectors. Together these data show superior CNS transduction and distribution of Anc80L65 over AAV9 and support continued development of Anc80L65 AAV gene therapy for CNS diseases.
Complete rescue of BBS1 neurometabolic syndrome, brain ventriculomegaly and obesity with a unilateral intracerebroventricular delivery of an AAV9 expressing a codon‐optimised
BBS1
sequence
J Jeyabalan Srikaran3 A Abarrategui3 6 M M Chawda3 SCP De Castro3 A L Gomes7 D Ma8 R Ruiz3 6 D Reichert2 9 10 S Bernard7 C Lane5 P A Hamblin4 5 P L Beales1 3
1: Axovia Therapeutics Inc. 2: Brunel University London 3: Institute of Child Health Great Ormond Street ‐ UCL 4: GlaxoSmithKline 5: Apollo Therapeutics 6: CICbiomaGUNE 7: The Francis Crick Institute 8: Simon Fraser University 9: National Institutes of Health 10: Johannes Gutenberg University
Bardet‐Biedl syndrome (BBS) is a rare autosomal recessively inherited monogenic ciliopathy, in which cilia function is impaired. The most clinically significant feature of BBS for patients is childhood obesity, which may be associated with other serious medical comorbidities, driven by central nervous system (CNS) hypothalamic dysfunction and other brain abnormalities also associated with cognitive and memory deficits and ventriculomegaly. Obesity can often require lifetime care and carries long‐term metabolic risk, increasing risk factors for type 2 diabetes and cardiovascular disease. There is currently no cure for BBS, and symptomatic treatments will only manage downstream complications of the disease. The most commonly mutated BBS gene is BBS1 and the most prevalent mutation worldwide is the missense M390R mutation, a change of a methionine for an arginine. BBS1 is a highly evolutionary conserved protein and the Bbs1 mouse model carrying the M390R mutation (Bbs1 M390R/M390R) recapitulates the same neurometabolic phenotype found in BBS patients including weight gain and CNS ventriculomegaly. Here we describe the use of a novel gene therapy used to treat and rescue neurometabolic manifestations of BBS. Intracerebroventricular (ICV) delivery of an Adeno‐associated virus 9 (AAV9) expressing human BBS1 (hBBS1) is able to ameliorate the neurometabolic phenotype in the Bbs1 M390R/M390R mice. Following perinatal ICV dosing of Bbs1 M390R/M390R mice, we observed halting of weight gain and obesity, restoration of brain ventricular volume and normalisation of circulating leptin hormone levels. We found no response to transgene expression in wild‐type controls with a sustained treatment effect in all treated Bbs1 M390R/M390R cohorts.
A human iPSC‐based model of Globoid Cell Leukodystrophy uncovers early neurodevelopmental defects.
1: HSR TIGET, San Raffaele Telethon Institute for Gene Therapy 2: Department of Chemistry, Biology and Biotechnology, University of Perugia, Italy 3: Pathology Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy 4: IFOM‐FIRC Institute of Molecular Oncology, Milan, Italy 5: Humanitas Clinical and Research Center‐IRCCS, Rozzano, Milan, Italy 6: National Research Council (CNR)‐IRGB/UOS of Milan, Milan, Italy
Globoid Cell Leukodystrophy (GLD) is a lysosomal storage disorder caused by mutations in the b‐galactocerebrosidase (GALC) gene. GALC deficiency causes the accumulation of toxic galactosphingolipids (e.g. psychosine), which promote a rapid and severe CNS and PNS demyelination/neurodegeneration. The mechanisms of disease onset/progression and disease correction upon treatments are still poorly elucidated in human neural cells. We studied the impact of GALC deficiency and lentiviral (LV) vector‐mediated GALC rescue/overexpression in a panel of GLD hiPSC‐derived neural stem/progenitor cells (hiPSC‐NPCs) and their neuronal/glial progeny.
GLD iPSCs showed undetectable GALC activity. Psychosine accumulation increased during NPC differentiation and normalized dupon LV‐mediated delivery of a functional hGALC. Still, LV‐mediated rescue of GALC activity only partially rescued the defective oligodendroglial and neuronal cell commitment/differentiation of GLD NPCs, whose severity was cell type‐ and patient‐dependent. These results suggested that multiple mechanisms besides psychosine storage are involved in GLD pathogenesis. Indeed, global lipidomic profiling showed a profound and peculiar unbalance in the relative levels of structural and bioactive lipids in GLD cells. The altered lipidome was associated with a senescence phenotype displayed by GLD neurons/glial cells and possibly contributed to the impaired neural differentiation program. Our data also revealed that GALC overexpression negatively impacts the neuronal/glial differentiation of healthy donor iPSC‐NPCs, suggesting that regulated GALC expression is crucial for proper human neural cell commitment and maturation. This work uncovers unforeseen mutation‐ and cell‐type‐specific early pathogenic events that may contribute to GLD neuropathology, paving the way to tailored gene/cell therapy strategies to enhance disease correction in GLD CNS cells.
Development of an AAV‐based model of tauopathy in the mouse visual pathway to study the role of microglia in Tau protein propagation
C Duwat1 P Leal1 A Vautheny1 G Auregan1 C Josephine1 M C Gaillard1 C Jan1 A S Herard1 E Brouillet1 P Hantraye1 G Bonvento1 K Cambon1
1: MIRCen, CEA
During Alzheimer's diseases (AD) tauopathy follows a precise spatio‐temporal progression, which is highly correlated with symptoms severity. This progression is due to the spread of pathological forms of Tau between connected neurons and in which microglia and neuroinflammation could play a role. Our main objective is therefore to develop relevant in vivo models to study the molecular and cellular determinants of pathological Tau spreading. We developed AAV vectors overexpressing human Tau (hTau) in order to trigger a focal tauopathy in transduced neurons after in vivo administration. We selected the visual system, a neural circuit with strictly separated afferent and efferent neurons. To do so, we transduced retinal ganglion cells (RGCs) following intravitreal injection of serotype 2 AAV. We first characterized tauopathy, neuroinflammation and neurodegeneration in wild‐type mice. We then performed similar analyses in Trem2‐/‐ mice (an innate immune receptor expressed by microglia and whose polymorphisms are associated to AD) to challenge the role of microglial activation in our experimental paradigm. One month after injection, we found hTau in RGCs, optic nerve and superior colliculi but not in efferent neurons projecting to the primary visual cortex. Morphological analysis highlighted a moderate but significant toxicity induced by hTau in RGCs from wild‐type mice. Preliminary results indicated that TREM2 deficiency reduced microglial activation and protected RGCs from hTau‐induced toxicity. Results obtained in Trem2‐/‐ animals suggest that tauopathy‐triggered neuronal cell death does not rely solely on cell‐intrinsic mechanisms. In this context, reducing TREM2 activity appears as a relevant therapeutic target in AD.
Preclinical testing of subretinal gene supplementation therapy for
PDE6A
‐linked retinitis pigmentosa
1: Department of Ophthalmology, University Hospital, LMU Munich, Munich, Germany 2: Veterinary Medical Center, Michigan State University, Michigan, USA 3: Department of Pharmacy ‐ Center for Drug Research, Ludwig‐Maximilians‐Universität München, Munich, Germany 4: Department of Chemistry, Ludwig‐Maximilians‐Universität München, Munich, Germany
PDE6A‐linked retinitis pigmentosa (RP; also referred as RP43) is an autosomal‐recessively inherited retinal disorder caused by loss‐of‐function mutations in the rod photoreceptor phosphodiesterase 6 alpha subunit. Rods lacking PDE6A activity are non‐functional and degenerate over time. Secondary to rods, cone photoreceptors also degenerate causing a classical RP phenotype. Gene supplementation therapy using recombinant adeno‐associated virus (rAAV) vectors represents a promising and potentially curative therapeutic option.
Here, we will present an overview on the preclinical testing of rAAV8.hPDE6A a translatable vector optimized for specific and efficient expression of full‐length human PDE6A in rods under the control of a human rhodopsin.
Therapeutic effects were assessed after subretinal delivery of rAAV8.hPDE6A in the Pde6a‐mutant dog model at different doses and at different timepoints. The outcome was monitored in‐life using spectral‐domain optical coherence tomography (SD‐OCT) and electroretinography (ERG) for assessing effects on morphology and function, respectively. Effects on vision were tested using a four‐choice vision test. After necropsy, the treatment effect was analysed via immunohistochemistry and mass spectrometry.
Treatment with rAAV8.hPDE6A achieved a long‐term, dose‐dependent and cell type‐specific transgene expression and resulted in a gain of rod function and improvement of rod‐mediated vision under dim‐light conditions. In addition, the treatment resulted in a structural preservation halting the primary degeneration of rod photoreceptors and preventing the secondary degeneration of cone photoreceptors.
In conclusion, preclinical studies showed that subretinal treatment with rAAV8.hPDE6A vector leads to a long‐term and dose‐dependent morphological and functional benefit. These results led to the initiation of a Phase I/II clinical trial in RP43 patients.
From embryo to sheep: Generation of a large animal model of sialidosis
1: University of Massachusetts Medical School 2: St. Jude Children's Research Hospital 3: National Institutes of Health 4: Tufts Cummings School of Veterinary Medicine
Sialidosis is a rare lysosomal storage disease caused by mutations in the NEU1 gene that encodes the lysosomal sialidase neuraminidase 1 (NEU1), essential for the catabolism of sialylated glycoproteins. Deficiency in NEU1 function causes toxic accumulation of sialylated glycopeptides in tissues throughout the body. There are two types of sialidosis, type I is the less severe form with onset in early adulthood and symptoms such as, myoclonus, seizures, muscle twitches and cognitive dysfunctions. Type II is the most severe form and is categorized by congenital or infantile onset that includes facial and bone deformities, hepatosplenomegaly, gingival hyperplasia, hearing loss and severe developmental delay. Both types are fatal and there is no effective treatment for sialidosis. Therefore, we aim to develop an adeno‐associated viral (AAV) gene therapy to treat sialidosis. Large animal models are crucial in evaluating therapies for human disease, however no large animal model of sialidosis exists to test our AAV gene therapy. We have utilized CRISPR‐Cas9 gene editing in single cell sheep embryos, to induce a mutation in the NEU1 gene to generate a sheep model of sialidosis. Our experiments resulted in ∼80% editing in sheep embryos that were implanted in donor recipient ewes. Results of these experiments produced affected lambs containing mutations in NEU1 that exhibited similar phenotypes to children with sialidosis. Characterization of affected lambs, establishing a breeding colony and implanting edited embryos are ongoing. These experiments along with a natural history study in sialidosis patients will inform future clinical trials for this devastating disease.
Reductions in oculogyric crisis duration and frequency in children with aromatic l‐amino acid decarboxylase deficiency treated with eladocagene exuparvovec gene therapy: results from 3 clinical trials
P W‐L Hwu1 Y H Chien1 N C Lee1 S H Tseng1 A Wang2 J Wang2 E T Leonardi2
1: National Taiwan University Hospital 2: PTC Therapeutics, Inc 3: PTC Therapetuics Switzerland, GmBH
Aromatic l‐Amino Acid Decarboxylase (AADC) deficiency is a rare autosomal recessive disorder resulting in marked dopamine loss, impeding normal motor development. Oculogyric crises (OGC) are involuntary eye movements characteristic of AADC deficiency, and are linked to decreased dopamine levels. OGCs may be accompanied by limb stiffness, torso rigidity, and autonomic signs.
Eladocagene exuparvovec was administered as a bilateral infusion in the putamen of 28 children with AADC deficiency in 3 clinical trials (AADC‐CU/1601 [8 patients, completed], AADC‐010 [10 patients, completed], and AADC‐011 [10 patients to date; ongoing]). Patients received a total of 1.8 × 1011 vg (n = 21) or 2.4 × 1011 vg (n = 7; AADC‐011)]. Duration (h/wk) and frequency (episodes/wk) of OGC episodes were calculated at baseline and 3 to 12 months after gene therapy.
At baseline, the mean duration of OGC episodes was 12.58 h/wk (n = 22). OGC s were reduced from baseline by a mean of 2.08 h/wk at 3 months (n = 20), 2.24 h/wk at 6 months (n = 12), 3.2h/wk at 9 months (n = 12), and 3.64 h/wk at 12 months (n = 8). At baseline, mean frequency of OGC was 2.63 episodes/wk (n = 22). By month 3, the mean frequency decreased to 1.93 episodes/wk (n = 20), by month 6, it was 1.9 episodes/wk (n = 12), and remained at ∼2 episodes/wk from months 9 to 12.
These results indicate a pattern of steady and sustained decrease over time in OGC episodes after eladocagene exuparvovec gene therapy.
Sustained, long‐term, correction of neuropathology in a mouse model of Hunter Disease following stem cell gene therapy with an LV.IDS.ApoEII vector.
1: University of Manchester 2: University College London 3: Erasmus University Medical Center
Hunter disease is a lysosomal disorder caused by mutations in the Iduronate‐2‐sulfatase (IDS) gene leading to the loss of IDS enzyme and accumulation of heparan sulfate and dermatan sulfate in lysosomes, leading to a multi‐system disease with severe neurodegeneration in two thirds of patients. Current therapeutic options fail to correct the neurological manifestations. Intravenously delivered enzyme (enzyme replacement therapy or ERT) can relieve non‐neurological disease but is unable to cross the blood‐brain barrier (BBB). Haematopoietic stem cell transplant (HSCT) also fails, presumably due to insufficient enzyme production from cells engrafting in the brain. Previously, we have demonstrated that haematopoietic stem cell gene therapy (HSCGT), using a lentiviral vector containing IDS fused to the BBB‐targeting peptide sequence ApoEII (LV.IDS.ApoEII), can correct neuropathology in IDS‐/‐ mice 6 months post‐infusion. Here we present our findings at 10‐12 months post‐infusion. Normalisation of neuropathology was only seen in the LV.IDS.ApoEII group, providing significantly enhanced correction compared to untagged LV.IDS, despite lower levels in the brain. Staining of retinal sections demonstrated better preservation of retinal structure with LV.IDS and LV.IDS.ApoEII compared to controls. Histopathological analysis of multiple organs was performed independently and revealed no safety concerns as a result of LV.IDS.ApoEII HSCGT. Transduction of human CD34+ stem cells was effective with both LV.IDS and LV.IDS.ApoEII showing VCN and IDS expression above normal. In conclusion, HSCGT with an LV.IDS.ApoEII vector provides benefit in IDS‐/‐ mice up to 12 months post‐infusion and is well tolerated in human CD34+ stem cells, demonstrating proof of principle for clinical trial development.
Complete correction of brain and spinal cord pathology in Metachromatic Leukodystrophy mice
E Audouard1 V Oger1 B Meha1 N Cartier1 C Sevin1 2
1: NeuroGenCell, Institut du Cerveau et de la Moelle épinière, ICM, Inserm U 1127, CNRS UMR 7225, Sorbonne Université, F‐75013, Paris, France 2: Bicêtre Hospital, Neuropediatrics Unit, Le Kremlin Bicêtre, 94275, Paris, France
Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder characterized by accumulation of sulfatides in both glial cells and neurons. MLD results from an inherited deficiency of arylsulfatase A (ARSA) and myelin degeneration in the central and peripheral nervous systems. Currently, no effective treatment is available for the most frequent late infantile (LI) form of MLD after symptom onset. The LI form results in rapid neurological degradation and early death. ARSA enzyme must be rapidly and efficiently delivered to brain and spinal cord oligodendrocytes of patients with LI MLD in order to potentially stop the progression of the disease. We previously showed that brain gene therapy with adeno‐associated virus serotype rh10 (AAVrh10) driving the expression of human ARSA cDNA alleviated most long‐term disease manifestations in MLD mice but was not sufficient in MLD patient to improve disease progression. Herein, we evaluated the short‐term effects of intravenous AAVPHP.eB delivery driving the expression of human ARSA cDNA under the control of the cytomegalovirus/b‐actin hybrid (CAG) promoter in 6‐month‐old MLD mice that already show marked sulfatide accumulation and brain pathology. Within 3 months, a single intravenous injection of AAVPHP.eB‐hARSA‐HA resulted in correction of brain and spinal cord sulfatide storage, and improvement of astrogliosis and microgliosis in brain and spinal cord of treated animals. These results strongly support to consider the use of AAVPHP.eB‐hARSA vector for intravenous gene therapy in symptomatic rapidly progressing forms of MLD. Data of dose response evaluation and delivery in non‐human primate will be presented.
Transplantation of gene‐modified haematopoietic stem cells and their application in murine models of neurodegenerative disease
1: UCL 2: University Medical Center Utrecht 3: AVROBIO
Transplantation of lentiviral vector gene‐modified haematopoietic stem cells provides clinical benefit for rare neurological diseases including metachromatic leukodystrophy and cerebral adrenoleukodystrophy because gene‐modified cells engraft in both the periphery and the brain to provide a functional gene on both sides of the blood brain barrier. This suggests that haematopoietic stem cell transplantation gene therapy (HSCT‐GTx) could also be applied to more common diseases with neurological and peripheral symptoms, like Parkinson's disease and dementia. Here, we assess the biodistribution, histological, and single‐cell transcriptional characteristics of a murine model of HSCT‐GTx using multiple routes of administration. We observed that intravenous (IV) administration of gene‐modified HSCs led to robust engraftment throughout the periphery, but this was not seen when cells were administered directly to the brain via intracerebroventricular (ICV) injection. Both IV and ICV administration resulted in long‐term engraftment of gene modified microglia‐like cells that bear many hallmarks of endogenous microglia (Iba1), but can be distinguished by a number of key markers (Apoe, Tmem119). We further show in murine models of GBA‐Parkinson's disease and Progranulin‐associated frontotemporal dementia (GRN‐FTD) that HSCT‐GTx can provide therapeutically relevant levels of transgene to both the periphery and the brain as measured by vector copy number, transgene expression, associated protein levels, and relevant lysosomal activity assays. Moreover, we've demonstrated in a model of GRN‐FTD that treatment can reverse downstream molecular changes associated with disease, including lysosomal dysfunction. Taken together, this work suggests the potential utility of lentiviral HSCT‐GTx in more common neurological diseases.
Gene therapy results in long‐term rescue of photoreceptor function in a mouse model for Bardet‐Biedl Syndrome 1
1: Axovia Therapeutics Inc. 2: Brunel University London 3: Institute of Child Health Great Ormond Street ‐ UCL 4: Institute of Ophthalmology ‐ UCL 5: Kings College London
Bardet‐Biedl syndrome (BBS) is a rare autosomal recessively inherited disorder associated with primary cilia dysfunction. The most common mutation of this ciliopathy, accounting for a third of all BBS cases, is caused by a homozygous single missense (M390R) mutation in the BBS1 gene. BBS1 patients' lives may be drastically affected by the disease's characteristic phenotype where patients typically develop obesity, renal dysfunction, cognitive impairment and retinal dystrophy, one of the most debilitating aspects of the disease. The present study aims to explore the therapeutic potential of an ocular gene therapy to rescue and further prevent progression of retinal degeneration associated with BBS1 deficiency. We found that although Bbs1 M390R/M390R mice presented primarily with photoreceptor degeneration as seen in patients, supplementation of BBS1 in photoreceptors alone was not always effective or indeed sufficient to prevent retinal degeneration, as assessed by electroretinography and histology. The low levels of expression of BBS1 in photoreceptors may explain the inability to successfully rescue the retinal phenotype. We therefore explored the therapeutic effect of delivering BBS1 to the Retinal Pigmented Epithelium (RPE) cells, which are highly ciliated and crucial for photoreceptor survival. The targeting of both photoreceptors and RPE was able to halt the degeneration of the photoreceptors. This study suggests that although supplementing BBS1 in photoreceptors and RPE leads to the rescue of the retinal degeneration observed, further understanding of the function of BBS1 in photoreceptors and RPE is essential to develop a successful and efficient ocular gene therapy for BBS patients.
Non‐invasive AAV delivery to the brain using focused ultrasound and microbubbles
1: Biological Sciences, Sunnybrook Research Institute, Toronto, Canada 2: Department of Laboratory Medicine and Pathobiology, Temerty Faculty of Medicine, University of Toronto, Toronto, Canada 3: Sanofi, Framingham, MA 01701‐9322, USA 4: Physical Sciences, Sunnybrook Research Institute, Toronto, Canada 5: Department of Medical Biophysics, Temerty Faculty of Medicine, University of Toronto, Toronto, Canada
Gene delivery using adeno‐associated virus (AAV) for neurological disorders is gaining momentum with the recent European Medicine Agency approval of an AAV‐based gene therapy treatment for spinal muscular atrophy. Effective AAV delivery to the central nervous system is, however, challenged by the blood‐brain barrier (BBB) and requires invasive intracranial injections or high intravenous doses.
We use non‐invasive, transcranial focused ultrasound combined with intravenous microbubbles, gas‐filled lipid vesicles, to transiently increase the permeability of the BBB for 4‐6 hours. Ultrasound induces microbubble oscillation, which causes mechanical stress on the BBB endothelium resulting in a decrease in tight junction protein levels and an increase in transcytosis in targeted areas.
We conducted a comprehensive study using ultrasound and microbubbles to deliver intravenously injected naturally occurring AAV serotypes to targeted brain regions. We achieved gene delivery to neuronal and glial cells with high spatial resolution, including the ability to target multiple or single brain regions. In addition, we combined ultrasound and microbubble‐mediated delivery with novel engineered AAV serotypes. AAVrg, a retrograde AAV2 variant, allowed for gene delivery to multiple connected brain areas by targeting only a single region with ultrasound. AAV2‐HBKO, which shows greater parenchymal diffusion than AAV2, provided therapeutically relevant gene delivery efficacies in large brain areas by only applying ultrasound to 30% of the transduced area.
Recent demonstrations of safe BBB opening in humans using ultrasound and microbubbles are paving the way for non‐invasive AAV delivery. Our results establish critical parameters to consider for successful AAV delivery with this promising drug delivery platform.
Gene therapy for Dopamine transporter deficiency syndrome: Infantile Parkinsonism‐dystonia
1: University College London 2: Medical University Vienna 3: University of Oxford 4: King's College London 5: National Hospital for Neurology and Neurosurgery 6: Great Ormond Street Hospital
Most inherited neurodegenerative disorders are incurable, and often only palliative treatment is available. Precision medicine has great potential to address this unmet clinical need. We explored this paradigm in Dopamine Transporter Deficiency Syndrome (DTDS), caused by biallelic mutations in SLC6A3, which encodes the dopamine transporter (DAT). Patients present with early infantile hyperkinesia, severe progressive childhood parkinsonism and raised cerebrospinal fluid dopamine metabolites. The absence of effective treatments and relentless disease course frequently leads to death in childhood. Using patient‐derived induced pluripotent stem cells (iPSCs), we generated a midbrain dopaminergic (mDA) neuron model of DTDS. Lentiviral gene transfer restored DAT activity and prevented neurodegeneration in all patient‐derived mDA lines. To progress towards clinical translation, we utilised the knockout mouse model of DTDS. It recapitulates human disease, exhibiting parkinsonism features, including tremor and bradykinesia and premature death. Neonatal intracerebroventricular injection of adeno‐associated virus (AAV) vector provided neuronal expression of human DAT which ameliorated motor phenotype, lifespan and neuronal survival in the substantia nigra (SN) and striatum, though off‐target neurotoxic effects were seen at higher dosage. These were negated with stereotactic delivery of AAV2.hDAT gene therapy targeted to the midbrain of adult knockout mice, which rescued both survival and motor phenotype to 1 year in‐life study, clearly demonstrating the feasibility of future clinical trials of targeted AAV gene therapy for DTDS patients.
VY‐NPC101:
in vitro
and
in vivo
validation of a novel AAV gene replacement therapy to treat Niemann‐Pick disease Type C1
E Knoll1 B Ezell1 E Liu1 A Sayed‐Zahid1 M Li1 H Patzke1 J Tocci1 J Brown1 K Bales1
1: Voyager Therapeutics
Niemann‐Pick Disease Type C (NPC) is a rare, progressive, autosomal recessive, neuro‐visceral disease which often results in death between 10 and 25 years of age. In 95% of cases, there is a loss of function mutation in the NPC1 gene. The subsequent loss‐of‐function in the NPC1 protein causes abnormal cholesterol and sphingolipid metabolism in the periphery and CNS. Replacement of the mutated NPC1 protein using gene therapy has the potential of being the first treatment to change the disease progression for patients battling this disease. We designed a potent NPC1 transgene following a comprehensive investigation into multiple transgene enhancement features that could be delivered via AAV. We tested these features using multiple disease relevant human cell lines. We uncovered specific enhancements that resulted in robust transgene expression that also improved cholesterol metabolism in patient derived cell lines. We then tested several of these optimized transgenes in wildtype mice using AAV transduction. Our top hits showed robust NPC1 expression within the CNS and the periphery compared to a reference vector that lacked these features. We next tested our top hits in a rodent model that carried a LOF mutation in NPC1 and measured peak body weight and survival. VY‐NPC101 was identified after providing robust improvement in these efficacy measures. In summary, the rational design of VY‐NPC101 and validation in a NPC1 disease‐relevant mouse model supports its continued development and translation into the clinic.
In vivo potency testing of subretinal rAAV5.hCNGB1 gene therapy in Cngb1 mouse and dog models of retinitis pigmentosa
1: Veterinary Medical Center, Michigan State University 2: Gene Therapy, Rare Diseases, Sanofi, Framingham 3: Department of Ophthalmology, University Hospital, LMU Munich 4: Department of Pharmacy ‐ Center for Drug Research, Ludwig‐Maximilians‐Universita¨t Mu¨nchen
Loss‐of‐function mutations in the cyclic nucleotide‐gated channel beta 1 subunit (CNGB1) gene are known to cause the autosomal‐recessive CNGB1‐linked Retinitis pigmentosa (RP), a severe inherited blinding disease characterized by a progressive retinal degeneration affecting rod photoreceptors and subsequently cone photoreceptors. Gene supplementation therapy using recombinant adeno‐associated virus (rAAV) vectors represents a promising and potentially curative therapeutic option.
In this study, we developed the novel translatable rAAV5.hCNGB1 vector optimized for specific and efficient expression of full‐length human CNGB1 in rods under the control of a short human rhodopsin promoter and we evaluated its transspecies efficacy in the Cngb1‐/‐ mouse model and the Cngb1‐deficient dog model.
We assessed therapeutic effects after subretinal delivery of rAAV5.hCNGB1 at different doses and different timepoints for analysis. We monitored retinal morphology in vivo using spectral‐domain optical coherence tomography (SD‐OCT) while we analyzed retinal function by electroretinography (ERG). Transgene expression was investigated via immunohistochemistry. Effects on vision were tested using a visual Water Maze in mice and a four‐choice‐vision‐test in dogs.
Treatment with rAAV5.hCNGB1 achieved dose‐dependent and cell type‐specific transgene expression and formation of functional chimeric CNG channels in rod outer segments. This reconstitution led to gain of rod function and improvement of rod‐mediated vision under dim‐light conditions while causing deceleration of primary retinal degeneration and prevention of secondary cone photoreceptor degeneration.
In conclusion, subretinal treatment with the novel rAAV5.CNGB1 vector resulted in successful gene supplementation and led to a dose‐related functional benefit and long‐term rescue of photoreceptors in the mouse and dog models of CNGB1‐linked RP.
Dose dependent lowering of alpha‐synuclein and rescue of motor phenotype by miRNA‐based AAV gene therapy
1: uniQure biopharma B.V. 2: University Medical Center Groningen
Parkinson's disease (PD) is a progressively debilitating neurodegenerative disease with an increasing prevalence with age. Only symptomatic therapies are available which do not tackle the underlying disease mechanism. One of the underlying causes of PD is aggregation of alpha‐synuclein protein (α‐syn), encoded by the SNCA (Synuclein Alpha) gene. Aggregated α‐syn is one of the main components of Lewy Bodies (LBs), a key neuropathological hallmark in PD. Typically, LB pathology originates in brainstem and extends to midbrain and cortical regions with disease progression, in parallel with neurodegeneration of nigro‐striatal dopaminergic circuits and other neurotransmitter systems. Our hypothesis for a disease modifying therapy is that lowering α‐syn protein levels in relevant brain regions may reduce aggregation and degeneration, ultimately halting disease progression.
We are developing an adeno‐associated virus (AAV) gene therapy to deliver SNCA‐targeting miRNAs (miSNCAs) engineered to decrease α‐syn mRNA and protein levels. Several miSNCA candidates were designed, targeting different SNCA splicing variants. After initial screening with luciferase‐based assays, selected AAV‐miSNCA candidates were assessed by small RNA sequencing. The lead miSNCAs were all correctly processed and expressed within endogenous miRNA levels. We then tested efficacy in both in vitro and in vivo model systems, achieving a dose‐dependent lowering of α‐syn mRNA and protein. Finally, the potential phenotypic improvement by miSNCA was evaluated in a C. elegans model of PD. Lead miSNCA candidates decreased α‐syn mRNA levels and rescued the motor phenotype in this model. These results support the potential therapeutic value of α‐syn RNAi‐based gene therapy for disease modification in PD.
CL002, An AAV9 vector expressing engineered miRNA targeting knockdown of GluK2‐containing kainate receptors as a novel gene therapy approach for treating intractable temporal lobe epilepsy
C Boileau1 S Deforges2 A Giles3 A Peret1 D Scavarda4 F Bartholomei5 N Partouche6 J Gautron6 J Viotti2 G Penchet7 C Marchal8 A Trebuchon5 N Villeneuve5 J Rumi2 T Marissal1 R Khazipov1 I Khalilov1 A Lepine5 M Milh5 D Figarella‐Branger9 E Dougy9 S Tong9 R Appay9 A Mercer3 J Smith3 O Danos3
1: INMED, INSERM UMR1249, Aix‐Marseille University, Marseille, France 2: Univ. Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, F‐33000 Bordeaux, France 3: REGENXBIO, Rockville, MD 20850, USA 4: APHM, INSERM, Aix Marseille Univ, INS, Timone Hospital, Pediatric Neurosurgery, Marseille, France 5: APHM, INSERM, Aix Marseille Univ, INS, Timone Hospital, Epileptology Department, Marseille, France 6: Corlieve Therapeutics SAS, 75008 Paris, France 7: AP‐HM, CRB, Timone Hospital, Marseille, France 8: PELLEGRIN ‐ CHU, Bordeaux, France 9: APHM, CRB, Timone Hospital, Marseille, France
No targeted treatments for temporal lobe epilepsy (TLE) exist, and the majority of patients are resistant to approved drug therapies. Recurrent mossy fiber sprouting from dentate granule cells (DGCs) is an anatomical hallmark of TLE, which leads to an aberrant excitatory network between DGCs that operates via ectopic kainate receptors (KARs) composed of GluK2/GluK5 heterodimers.
Our prior demonstration that GluK2 knockout mice are protected from pilocarpine‐induced seizures led us to explore the tractability of an anti‐Gluk2 approach to address TLE therapeutically. We have designed AAV9 vectors expressing engineered miRNA designed to knockdown the grik2 mRNA, with the goal of reducing GluK2 subunit expression in the hippocampus and thus ameliorating the epileptic phenotype.
Using this approach, we observed a reduction in GluK2 protein levels following transduction into primary neuronal cultures from both mice and rats. Furthermore, direct hippocampal delivery in pilocarpine‐induced epileptic mice significantly reduced the number of epileptic seizures in these mice, and normalized the pathological hyperlocomotion phenotype associated with this model. Finally, transduction of organotypic hippocampal tissue surgically resected from TLE patients showed an almost complete suppression of recorded epileptiform discharges.
These data support the pivotal role of GluK2/GluK5 in triggering the epileptic seizures human TLE, and that a reduction in their expression may be expected to reduce the epileptic phenotype. We believe that AAV‐mediated RNAi targeting the expression of GluK2/GluK5‐KARs in DGCs is a viable approach to reduce seizures associated with intractable TLE and to address unmet needs in this patient population.
Blood‐brain‐barrier crossing leads to long term efficacy in the CNS of HMI‐203: Gene therapy development candidate for Mucopolysaccharidosis Type II (MPS II), or Hunter Syndrome
1: Homology Medicines Inc
Hunter syndrome is a rare X‐linked lysosomal storage disorder caused by mutations in the iduronate‐2‐sulfatase (IDS) gene, resulting in loss of I2S enzyme activity. This loss leads to subsequent systemic (including the central nervous system, or CNS) lysosomal accumulation of glycosaminoglycans (GAGs). Currently approved intravenously (IV) administered enzyme replacement therapies cannot efficiently cross the blood‐brain barrier (BBB) and therefore do not improve neurocognitive deficits in MPS II patients. Herein, we summarize our CNS preclinical data for HMI‐203, a gene therapy development candidate utilizing AAVHSC to deliver IDS in the MPS II murine model. Following a single IV dose of HMI‐203, successful crossing of the BBB was observed, accompanied by widespread distribution and a dose‐response in vector genomes, transcripts, I2S activity, and GAG reduction in brain tissue. At highest doses examined, I2S activity levels were comparable to wild‐type (WT) mice and normal adult human brain tissue. Two highly correlated disease‐relevant biomarkers, GAG‐heparan sulfate and lysosomal‐associated membrane protein, were reduced in a dose‐ and time‐dependent fashion throughout the CNS. Lastly, HMI‐203 prevented loss of cerebellar Purkinje neurons and progression of vacuolization in the brain. Long term and robust IDS expression observed in peripheral organs reduced tissue and urine GAGs down to WT levels with functionally active I2S protein sustained in the serum out to 52 weeks (end of study). These preclinical studies demonstrated BBB crossing with a single IV injection and support HMI‐203 as an investigational gene therapy for the treatment of MPS II with potential to address CNS‐related manifestations.
AAV‐RNAi gene therapy for GNAO1 c.607 G > A encephalopathy in iPSC‐derived neurons
E A Luchkina1 2 E A Lunev1 2 3 E A Volovikov4 5 D M Spirin5 I M Savchenko2 3 A A Shmidt1 2 3 T V Egorova1 2 M A Lagarkova4 5 6
1: Institute of Gene Biology Russian Academy of Sciences, Moscow, Russia 2: Marlin Biotech LLC 3: Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology Russian Academy of Sciences, Moscow, Russia 4: Federal Research and Clinical Center of Physical‐Chemical Medicine, Federal Medical Biological Agency, Moscow, Russia 5: Lomonosov Moscow State University, Russia 6: Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Federal Research and Clinical Center of Physical‐Chemical Medicine, Federal Medical Biological Agency, Moscow, Russia
Heterozygous de novo mutations in GNAO1 are associated with an ultra‐rare neurodevelopmental disease (GNAO1 encephalopathy) featuring infantile epilepsy and/or movement disorder. Missense variant c.607 G > A represents a mutation hotspot and increasing evidence suggests dominant negative activity of the protein product. Previously, we proposed allele‐selective RNA interference (RNAi) for downregulating c.607 G > A transcripts as a potential therapeutic approach. Small hairpin RNAs (shGNAO1) targeting mutant transcript were screened based on silencing efficacy and selectivity. Here, the two best RNAi candidates for targeting c.607 G > A were incorporated in recombinant adeno‐associated virus vectors (rAAVs). AAV‐mediated reduction of mutant, but not wild‐type, mRNA and protein was confirmed for both shGNAO1 candidates in stable cell lines overexpressing GNAO1 isoforms. To further assess the efficacy of the AAV‐RNAi approach, we established a neuronal model of GNAO1 c.607 G > A encephalopathy derived from patient‐specific induced pluripotent stem cells (iPSCs). iPSCs were differentiated into GABAergic neurons and cerebral organoids with endogenous levels of mutant and wildtype GNAO1 expression at an approximate ratio 1:1. Following transduction with AAV‐shGNAO1, the accumulation of mutant GNAO1 transcripts was reduced leading to 1.5‐2 fold increase in the relative abundance of wild‐type allele expression. Our findings demonstrate the feasibility of AAV‐RNAi for selective silencing of GNAO1 transcripts with a single nucleotide substitution c.607 G > A in patient‐specific neurons. For preclinical development AAV‐shGNAO1 vector configuration needs to be further modified and therapeutic benefit has to be evaluated by functional assays.
Development of an
ex vivo
Gene 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 a severe and fatal adult neurodegenerative condition devoid of any cure or specific approved treatment. About 20% of familial FTD cases are caused by pathogenic loss‐of‐function mutations in the progranulin gene (GRN). 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. Autologous Hematopoietic Stem/Progenitor Cell (HSPC) Gene Therapy (GT) based on the use of lentiviral vectors (LVs) for gene transfer offers the potential benefit of long‐lasting delivery of therapeutics to the brain. The potential therapeutic efficacy of HSPC‐GT in FTD is based on the ability of the genetically engineered, transplanted HSPCs to repopulate the myeloid Central Nervous System compartment, including microglia, with a transplant‐derived, mature progeny able to express robust GRN levels and exert neuro‐immunomodulatory and neuroprotective functions. We developed and validated therapeutic LVs able to safely deliver multiple copies of the human GRN gene in GRN knock‐out cell models, inducing overexpression of the therapeutic protein which is then correctly secreted and taken up by target GRN knock‐out cells. Preliminary in vivo data showed that GRN‐transduced murine HSPCs, transplanted via the intracerebroventricular route after myeloablative Busulfan treatment, are able to engraft and repopulate the brain myeloid compartment of FTD mice, and to locally deliver GRN as measured by ELISA. We are currently proceeding with the in vivo proof‐of‐concept study to evaluate the safety, feasibility, and efficacy of the therapeutic strategy.
Preclinical development of Hematopoietic Stem and Progenitor Cell Gene Therapy for the treatment of Multiple Sclerosis
R Milazzo1 2
1: Division of Pediatric Hematology, Oncology and Stem Cell Transplantation, Woman's and Child Health Department, University of Padova, Padova, Italy 2: Altheia Science srl, Milan, Italy 3: Department of Biology&Biotechnology L. Spallanzani, University of Pavia, Pavia, Italy 4: Gene Therapy Program, Boston Children's Dana Farber Cancer and Blood Disorder Center, Boston, USA 5: International Center for T1D, Pediatric Clinical Research Center Romeo ed Enrica Invernizzi, DIBIC, Università di Milano, Milan, Italy 6: Division of Endocrinology, ASST Fatebenefratelli‐Sacco, Milan, Italy 7: Nephrology Division, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA
Multiple sclerosis (MS) is a debilitating neurodegenerative disease characterized by an abnormal immune response mediated by auto‐reactive activated T cells inducing chronic inflammatory demyelination in the central nervous system (CNS). Current therapeutic strategies are mainly aimed at controlling symptoms, and do not provide a stable remission of the disease. The transplantation of autologous hematopoietic stem/progenitor cells (HSPCs) in conjunction with the administration of an immune and myeloablative regime has been explored in the context of randomized multicenter clinical trials as a treatment option for autoimmune diseases, including MS, to eliminate self‐reacting clones and to induce self‐tolerance upon renewal of the immune system. We designed a strategy aimed at enhancing the immune‐regulatory property of HSPCs by upregulating the immune‐regulatory factor programmed death‐ligand 1 (PD‐L1), by lentiviral vector (LV) transduction and reprogramming an auto‐aggressive immune system towards a de novo self‐tolerant immune repertoire. To properly design such an approach for clinical translation, we investigated in the animal model of MS, generated by inducing experimental autoimmune encephalitis (EAE), the effects on disease phenotype of PD‐L1 over‐expressing HSPCs transplantation. We explored different routes of cell administration and the actual need of a pre‐transplant conditioning. Interestingly, our preliminary data indicated that transplantation of LV‐transduced, PD‐L1 over‐expressing HPSCs can significantly reduce the severity of disease manifestations, decreasing the extent of neuroinflammation, particularly when cells are delivered directly in the CNS and even in the absence of pre‐transplant conditioning, thus paving the way for the further development of the approach towards clinical testing.
Development of CNS‐directed
GBA1
gene replacement therapy for IV delivery via Blood Brain Barrier Penetrant AAV Capsid
1: Voyager Therapeutics
Adeno‐associated viral vector‐based gene‐replacement therapies can be used to achieve sustained correction of lysosomal storage disorders affecting the central nervous system. GBA1 gene encodes the lysosomal enzyme b‐glucocerebrosidase [GCase] and GBA1 loss of function (LOF) results in GCase deficit and cellular build‐up of glycosphingolipid‐substrate. Homozygous GBA1 LOF mutations manifest as Gaucher disease, the most common lysosomal storage disorder, whereas heterozygous GBA1 LOF mutations and reduced GCase activity are strong genetic risk factors for Parkinson disease and Lewy Body dementia. Enzyme replacement therapy can have clinical impact in the periphery but fails to adequately cross the blood‐brain barrier. Here, we report development of an AAV‐based GBA1‐gene replacement therapy that delivers GCase to a widespread CNS‐footprint and periphery following intravenous (IV) dosing.
Several classes of enhanced GBA1 transgenes with favorable attributes, including promoter and gene optimization; cell and lysosomal targeting; tissue‐detargeting were designed. Following expression validation, GBA1 transgenes were packaged into AAV for testing on patient‐derived cells for in vitro target engagement. Multiple AAV‐GBA1 constructs demonstrated dose‐dependent GCase activity increases, GBA protein normalization, and correction of glycosphingolipid levels to match healthy human comparator cells.
Single IV‐dosing of our optimized AAV‐GBA1 vector in WT mice resulted in widespread vector genome CNS distribution and GCase activity increases in CSF and multiple relevant brain regions [up to ∼5x over baseline]. We are currently evaluating the optimized AAV‐GBA1 vectors in a relevant rodent model of GBA1 LOF.
In summary, we present our strategy to address gaps and accelerate development of an IV‐delivered, CNS‐directed AAV‐GBA1 gene replacement therapy.
Efficient and precise processing of the optimized pri‐amiRNA in a Huntingtin‐lowering AAV gene therapy: from cultured cells to nonhuman primates
W Wang1 P Zhou1 X Wang1 F Chen1 E Christensen1 J Thompson1 X Ren1 A Kells1 L Stanek2
1: Voyager Therapeutics 2: Sanofi
Huntington's Disease is a neurodegenerative disorder caused by an inherited mutation in the huntingtin gene (HTT) comprising an expanded trinucleotide repeat sequence that results in a pathogenic huntingtin protein. AAV gene therapy containing a primary artificial microRNA (pri‐amiRNA) specifically targeting HTT mRNA has the potential to provide long‐lasting therapeutic benefit via durable reduction of mutant HTT expression after a single administration. The efficiency and precision of processing of the pri‐amiRNA precursor to the mature guide strand by transduced cells is critical for specific and potent HTT lowering. The selection of our clinical candidate VY‐HTT01 comprised a comprehensive series of studies that revealed the predictivity of certain cell culture systems and rodent models for nonhuman primates with respect to some, but not all, key features of pri‐amiRNA processing. Our results show that processing to the mature guide strand can differ greatly across variant scaffolds and sequences, while providing indistinguishable levels of target lowering. Importantly, our data demonstrate a combinatorial effect on pri‐amiRNA processing arising from guide and passenger strand selection in the context of a specific scaffold, where different guide and passenger strand sequences within the same scaffold dramatically alter processing. Taken together, our results highlight the importance of optimizing not only target lowering, but also efficiency and precision of pri‐amiRNA processing across cultured cells, rodent models and nonhuman primate systems. VY‐HTT01 was selected using this approach, resulting in a highly potent and selective AAV gene therapy that harnesses the endogenous miRNA biogenesis pathway for HTT lowering.
Real‐Time MR tracking of AAV gene therapy with β‐gal activated MR probe
1: University of Massachusetts Medical School 2: Northwestern University 3: Worcester Polytechnic Institute 4: Auburn University
Transformative results of AAV gene therapy in patients have demonstrated unequivocally the potential of in vivo gene transfer to treat monogenic diseases. However, the field lacks a non‐invasive way to assess gene expression and its duration. Here we measure β‐gal enzyme activity after AAV gene therapy encoding lysosomal acid β‐galactosidase (β‐gal) using a self‐immolative β‐gal responsive probe. This gadolinium‐based agent has an exposed arm that is enzymatic target of therapeutic, which, upon cleavage, results in MR signal enhancement. GM1 mice (β‐gal‐/‐) treated with AAV encoding β‐gal, show global distribution, with highest β‐gal levels in CNS after intracranial delivery and in liver after intravenous (IV) administration. Intrathecal (IT) administration of β‐gal‐responsive probe, 1 month after intracranial AAV treatment (1010 vg, thalamus‐unilateral, n = 4), resulted in strong MR signal enhancement in CSF and parenchyma. CSF enhancement occurred rapidly after intrathecal administration with an increasing ratio of parenchymal/CSF signal. Distribution of probe within brain was greatest on ventral aspect with a gradient projecting dorsally. Intraperitoneal administration of contrast agent, 1 month after AAV9 treatment (IV, 3X1011 vg, n = 4), resulted in sharp rise of liver signal over first 40‐50 minutes after which it plateaued for ∼2 hours. The detection range was linear and extended to at least 3‐logs with strong correlation between enhancement and enzyme activity within brain (R2 = 0.81) and liver (R2 = 0.94) for AAV treated, wild type and untreated. This class of contrast agents can be modified to interact with active sites of other enzymes independent of treatment approach.
Comparison between CRISPR‐mediated HITI and MITI for Opto‐mGluR6 expression.
1: University of Bern
Retinitis Pigmentosa (RP), an inherited form of retinal degeneration, is characterized by a progressive loss of rods and subsequent degeneration also of cones leading to blindness. However, the remaining neural part of the retina (bipolar and ganglion cells) remains anatomically and functionally intact for an extended time period. A possible treatment consists in rendering these remaining cells photosensitive using optogenetic tools like, for example, Opto‐mGluR6, a chimeric protein of human origin developed in our lab. In previous studies, we have demonstrated that the AAV vector mediated expression of Opto‐mGluR6 in ON bipolar cells (ON‐BPC) can restore visual function in otherwise blind RP mouse models.
Classical gene addition still suffers of drawbacks such as specificity of expression, control of expression levels and promoter silencing. To overcome these issues, we are using two approaches based on the use of the CRISPR/Cas technology namely HITI (Homologous‐Independent Target Integration, based on spCas9 or saCas9) and MITI (MIcro‐homology Targeted Integration, based on Cpf1) to knock‐in (KI) Opto‐mGluR6 downstream to an ON‐BPC specific promoter. This should lead to a specific and physiological level of expression of our construct.
After having selected the best performing sgRNA target sequences for each system, we confirmed, in vitro, the possibility of an efficient KI through both HITI and MITI. Next, we prepared dual AAV vectors for testing both HITI and MITI in vivo with the aim to express Opto‐mGluR6 selectively in ON‐BPC of C57Bl/6 mice.
Ablation of a prodegenerative gene confers histological and functional protection in a mouse model of retinal degeneration
1: Trinity College Dublin 2: Royal Victoria Eye and Ear Hospital, Dublin
Axons of the retinal ganglion cells (RGCs) form the optic nerve, delivering visual signals to the brain. Degeneration of RGCs and the optic nerve is seen in conditions including Leber hereditary optic neuropathy and glaucoma, disrupting connectivity between the eye and brain and impairing visual function. The heterogeneity of these conditions represents a hurdle for the development of gene‐specific therapies. Fortunately, mechanistic commonalities between conditions reveal key pathways that may, in principle, be targeted for the development of gene‐independent therapies that may be applicable to a wider cohort of patients. One such pathway involves the degeneration of axons in response to injury. Here we evaluate the modulation of this pathway as a means to preserve the injured optic nerve and associated RGCs. We demonstrate histological and functional protection of ablation of a pro‐degenerative gene in a mouse model of retinal degeneration. Wild type mice receiving rotenone, a complex I inhibitor, exhibit RGC death and loss of axon density in the optic nerve, while knockouts exhibit RGC death to a lesser extent than their wild type counterparts and show preservation of axon density in the optic nerve. Accordingly, optokinetic response measurements are significantly higher in knockouts receiving rotenone, with this protection of spatial vision preserved over time and across age groups and sexes.
Development of editing technologies for allele‐specific silencing or precise correction of mutation hotspots affecting Alexander disease's patients
1: Telethon Institute of Gene Therapy (HSR‐TIGET)
Alexander disease (AxD) is an autosomal dominant neurodegenerative disorder caused by missense mutations in the glial fibrillary acidic protein (GFAP) gene. Mutations are mainly clustered in two hotspots affecting arginines at positions 79 and 239 of the structural helical coiled‐coil rod domains. Accumulation of mutated GFAP compromises astrocyte functions and alters the homeostasis of the central nervous system (CNS). Currently, this orphan disease lacks a cure. Our long‐term goal is to develop novel, single‐dose therapeutic treatments exploiting allele‐specific gene editing strategies to target GFAP mutation hotspots and recover pathological phenotypes. Here, we report the optimization of in vitro models and reagents that are key for the project. We engineered cell lines to express mutated and wild‐type (WT) murine Gfap coding sequences fused to reporter genes for a fast screening of single guide RNAs recognized by different Cas9 nucleases. We identified CRISPR/Cas9 systems efficiently inducing the allele‐specific knock‐out of Gfap sequences harboring the R76H and R236H mutations, homologues of the human mutation hotspots. Also, we identified adenine base editors to correct R‐to‐H hotspot mutations avoiding the potential activation of DSB‐induced DNA damage response. These editing platforms will be then tested in in vitro disease models. Results of these in vitro data pave the way to translational studies aimed at delivering safely and effectively the selected editing systems in the CNS of AxD murine models by means of AAV vectors or nanoparticles. Novel editing platforms for in vivo targeting of CNS astrocytes could benefit AxD and other diseases characterized by astrocyte degeneration/dysfunction.
NG2‐producing cells in the ventral horns with increasing distance from the spinal cord injury site
D H Sabirov1 A V Timofeeva1 A A Rizvanov1
1: Kazan Federal University
The area of spinal cord injury (SCI) contains numerous NG2‐producing glial cells and is externally bounded by a compact layer of reactive astrocytes that form a glial scar. The formation of glial scar depends not only proliferating NG2 glial cells, but also pericytes. Dividing NG2 glia outnumber dividing NG2 pericytes up to 30‐fold, but are restricted to the glial scar and spared tissue, whereas dividing NG2 pericytes enter lesions concomitant with angiogenesis. Taking into account the special role of NG2‐producing cells, it seems important to assess the reaction of these cells, in an area remote from the injury site to provide extended axon growth in it.
In rat contusion model of severe spinal cord injury at the Th8 the number of NG2 cells, as well as the expression of NG2 proteoglycan was studied in ventral horns at different distances in caudal direction from the epicenter of injury. At a distance of 3‐5 mm the number of NG2+/Olig2+ cells increased by 7 days post injury (dpi) and remained elevated at 30 dpi. The spatiotemporal dynamics of NG2–/Olig2+ cells are similar to that of NG2+/Olig2+ cells, but with smaller deviations. A decrease in NG2 proteoglycan mean intensity correlates with a decrease in the number of NG2+ cells at a distance of 6‐8 and 10‐12 mm. The results indicate significant cell reactions not only in the area of the gray matter damage, but also in adjacent and remote areas, which is important for assessing the possibility of long distance axonal growth.
Codon‐optimisation for Bardet‐Biedl Syndrome 1 (BBS1) and Bardet‐Biedl Syndrome 10 (BBS10) genes for AAV constructs
SCP De Castro3 J Jeyabalan Srikaran3 MM Chawda3 P A Hamblin4 5 P L Beales1 3
1: Axovia Therapeutics Inc. 2: Brunel University London 3: Institute of Child Health Great Ormond Street ‐ UCL 4: GlaxoSmithKline 5: Apollo Therapeutics
The use of Adeno‐Associated Virus (AAV) as vectors for genes therapies has increased dramatically in preclinical and clinical studies. To achieve effective transgene expression levels, higher AAV doses have been tested through a variety of delivery routes. Concerns that use of high titres might lead to undesired immunological and toxicological adverse effects is an open debate. Codon‐optimisation of the gene of interest sequence and cassette may offer a possible solution to improve transgene expression. To support the investigation of AAV‐delivered gene therapy for the multi‐ syndromic ciliopathy Bardet‐Biedl Syndrome (BBS), a series of DNA vectors were designed and constructed based on different promoters and codon‐optimised human expression cassettes. We developed diverse codon‐optimised sequences for the most prevalent BBS genes, BBS1 and BBS10. The expression levels of BBS1 and BBS10 from the panel of vectors was assessed following transient transfection in HEK293 cells followed by quantitative PCR for RNA expression analysis and Western Blot for protein levels. Based on the results of the transient transfections and on a direct comparison between the wild‐type human cDNA, we found in all cases that all different codon‐optimised BBS1 and BBS10 sequences consistently yielded the highest mRNA expression levels. We also tested several strong promoter combinations driving constitutive expression, with the Cytomegalovirus (CMV) and the synthetic CAG promoters yielding the highest expression levels. Our results show that a precise analysis of different constructs and codon‐optimised sequences, is a requisite first step to test new constructs for AAV vectors before assessing for actual pharmacological efficacy.
Intrathecal delivery of an AAV encoding human ABCD1 shows dose‐responsive expression and activity in a mouse model of adrenomyeloneuropathy
H Park1 T del Rio1 V Vasireddy1 T Lutz1
1: SwanBio Therapeutics 2: Massachusetts General Hospital 3: Amsterdam UMC
X‐linked adrenoleukodystrophy (ALD) is an inherited neurodegenerative disease caused by mutations in the ABCD1 gene. Adrenomyeloneuropathy (AMN) is the most frequent clinical manifestation of ALD affecting virtually all males and >80% of females. AMN is characterized by a slowly progressive muscle weakness leading to loss of mobility, incontinence, and debilitating pain. The ABCD1 gene encodes a peroxisomal membrane protein that transports very long‐chain fatty acids (VLCFA) into the peroxisome. In the absence of functional ABCD1, affected cells have increased VLCFA levels. Similar to human AMN, the Abcd1 knockout mouse model of AMN exhibits increased VLCFA levels and changes in mitochondrial DNA levels as a result of oxidative stress. We examined the ability of SBT101, a novel AAV9‐hABCD1 vector, to impact markers of disease in the Abcd1‐knockout mouse. In mixed glial cultures derived from Abcd1 knockout mice, SBT101 transduction resulted in a dose‐dependent lowering of VLCFA and achieved approximately wild‐type levels. Following an intrathecal bolus delivery of SBT101 in adult Abcd1 knockout mice, expression levels of the hABCD1 transgene in the spinal cord were dose‐dependent, persisted for 6 months and did not result in observable spinal cord pathology. Furthermore, delivery of SBT101 restored mitochondrial DNA levels and significantly improved VLCFA levels in the spinal cord in a dose dependent manner. Therefore, successful targeting of the spinal cord with SBT101 was demonstrated together with dose‐dependent improvement of disease markers in a mouse model of AMN. These data support further preclinical investigation of AAV9‐hABCD1 as a potential therapeutic for the treatment of AMN.
Three‐Month Preclinical Safety Data of AAV9‐hABCD1 following intrathecal delivery in Non‐human Primates
1: SwanBio Therapeutics
The inherited neurodegenerative disorder adrenomyeloneuropathy (AMN) is a form of X‐linked Adrenoleukodystrophy (ALD), characterized by a slowly progressive muscle weakness leading to loss of mobility, incontinence, and debilitating pain. While mutations in the ABCD1 gene encoding an ATP Binding Cassette subfamily D member‐1 protein were identified to be responsible for AMN, there currently remains no treatment for AMN. SBT101 is an AAV9‐based gene therapy encoding a functional human ABCD1 transgene, which has demonstrated activity in mouse models of AMN. To evaluate the safety of SBT101, male cynomolgus monkeys (n = 3/dose) received one‐time intrathecal infusions of SBT101 at 1.5E13, 3.5E13 or 7.5E13 vector genomes/animal and were observed for 3 months.
SBT101 was well tolerated with no mortality or SBT101‐related clinical observations. Mild to moderate transient elevations in alanine aminotransferase activity were observed through day 28 with no histopathological correlate, while both cardiac markers and electrocardiogram remained within normal limits. There were no macroscopic pathological findings, and presence of AAV9 vector genomes was found to be dose dependent in the spinal cord and dorsal root ganglia (DRG). Microscopic histopathological evaluations revealed slight changes in the spinal cord, DRGs and peripheral tissue, consistent with or less than previously described for AAV9 alone, or in all animals irrespective of treatment following intrathecal delivery.
These results suggest SBT101 is tolerable and without toxicity at a range of doses in nonhuman primates, supporting potential translation to humans and further investigation of SBT101 as a potential treatment for AMN.
CRISPECTOR: an accurate tool for measuring CRISPR‐Cas‐induced translocation and adverse off‐target activity
1: The Institute for Advanced Materials and Nanotechnology, The Mina and Everard Goodman Faculty of Life Sciences, Bar‐Ilan University, Ramat‐Gan 5290002, Israel 2: Arazi School of Computer Science, Interdisciplinary Center, Herzliya 4610101, Israel. 3: Department of Computer Science, Technion ‐ Israel Institute of Technology, Haifa 32000, Israel. 4: Integrated DNA Technologies Inc., Coralville, IA 52241, USA.
One of the central challenges that the CRISPR technology must overcome to become widely applicable in medical practice is limiting off‐target editing activity. This potential lack of specificity can be particularly problematic when the CRISPR system is applied to human cells as gene therapy. Simultaneous editing at numerous, unintended, genomic sites can lead to the accumulation of mutations, such as deletions and insertions or other adverse chromosomal aberrations such as translocations. Existing algorithms for analyzing off‐target activity do not provide statistical quantification, are not sufficiently sensitive in separating signal from noise in experiments with low editing rates, and do not address the detection of translocations. We developed a software tool called CRISPECTOR that represents progress over the current algorithms in four main aspects. First, our tool provides increased accuracy based on treatment vs control experiments enabled by the use of a statistical model comparison approach. This technique produces better false‐negative rates in sites with weak yet significant off‐target activity. Second, our tool supports detection of alternative cut‐sites in off‐target loci. Third, CRISPECTOR is capable of analyzing multiplex‐PCR and NGS data to detect adverse structural variations and translocation events occurring in an editing experiment. Finally, we report statistical confidence intervals for inferred off‐target activity rates. We demonstrated CRISPECTOR's superior performance in multiple different loci and numerous cell types. Utilizing CRISPECTOR as part of a gRNA selection process has the potential to significantly enhance and improve the accurate use of genome‐editing in biotechnology and therapeutic applications.
Mesenchymal stromal cells improve the transplantation outcome of CRISPR‐Cas9 gene‐edited HSPCs
1: San Raffaele Telethon Insitute for Gene Therapy (HSR‐TIGET) 2: KU Leuven 3: Dana‐Farber Cancer Institute 4: Cagliari University‐Hospital 5: National Research Council, Dept. Biomedical Technologies 6: Vita‐Salute San Raffaele University 7: San Raffaele Scientific Institute
Hematopoietic Stem and Progenitor Cells (HSPCs) reside in a specialized microenvironment in the bone marrow (BM), named the hematopoietic niche. Mesenchymal Stromal Cells (MSCs) are key elements of the BM niche, regulating HSPC function by direct contact and through the release of paracrine factors. Due to these properties, MSCs have been employed in vitro to support HSPC expansion, and in vivo to promote HSPC engraftment and hematopoietic reconstitution in preclinical models and clinical trials of HSPC transplantation (HSCT). Based on these studies, we developed an MSC‐based co‐culture system to improve the outcome of transplantation with CRISPR‐Cas9 gene‐edited (GE)‐HSPCs, whose efficiency in stem cells requires further advancement to fully exploit its therapeutic potential. Indeed, GE‐HSPCs activate a DNA damage cascade in response to nuclease‐induced double‐strand break and delivery of DNA repair template, which constrains HSPC proliferation and long‐term repopulating capacity. Here, we show that MSCs produced several hematopoietic supportive and anti‐inflammatory factors, capable to reduce the proliferation arrest and mitigate the apoptotic and inflammatory programs activated in GE‐HSPCs, ultimately improving GE‐HSPC expansion and clonogenic potential in‐vitro. This resulted in superior human engraftment and an increased number of GE‐HSPC clones contributing to early reconstitution phases in transplanted mice. Moreover, we demonstrated that MSCs favored the engraftment of limited doses of GE‐HSPCs when co‐transplanted in vivo. In conclusion, our work poses the basis for the clinical use of MSCs as a novel strategy to promote the engraftment of CRISPR‐Cas9 gene‐corrected HSPCs and improve the transplantation outcome.
Development of a double shmiR lentivirus effectively targeting both BCL11A and ZNF410 for enhanced induction of fetal hemoglobin to treat β‐hemoglobinopathies
1: Boston Children's Hospital 2: Dana‐Farber Cancer Institute
A promising treatment for β‐hemoglobinopathies is the de‐repression γ‐globin expression leading to increased fetal hemoglobin (HbF). Decreasing the expression of γ‐globin repressor BCL11A results in a coordinated increase in γ‐globin expression and reduction in β‐globin expression. Here, we aim to improve a lentivirus vector (LV) containing a single BCL11A shmiR (SS) to further increase γ‐globin induction. We engineered a novel LV to express two shmiRs simultaneously targeting BCL11A and the γ‐globin repressor, ZNF410. Erythroid cells derived from human HSCs transduced with the double shmiR (DS) showed up to 80% reduction of both BCL11A and ZNF410 proteins and there was a consistent and significant (p < 0.05) additional about 10% increase in induction of HbF compared to targeting BCL11A alone. Erythrocytes differentiated from SCD HSCs transduced with the DS demonstrated significantly reduced in vitro sickling phenotype (p < 0.001) compared to the SS. Erythrocytes differentiated from transduced HSCs from β‐thal major patients demonstrated restored globin chain balance by increased γ‐globin to more than 60% with reduced microcytosis. Finally, reconstitution of DS‐transduced cells from Berkeley SCD mice was associated with a statistically larger reduction in peripheral blood hemolysis markers compared with the SS vector: erythroid precursor cells 28.9%, 7.2% and 5.1%; reticulocytes 31.2%, 10.4% and 7.7%; sickled cells 48% 11.9% and 6.6% for untransduced, SS‐ and DS‐transduced, respectively. Overall, these results indicate that the DS LV targeting BCL11A and ZNF410 can enhance HbF induction in treating β‐hemoglobinopathies and could be used as a model to simultaneously and efficiently target multiple gene products.
Novel dual mRNA trans‐splicing rAAV vectors for efficient reconstitution of split dCas9‐VPR in neurons
1: Center for Drug Research, Ludwig‐Maximilians‐Universität München, Munich, Germany
Recombinant adeno‐associated viral (rAAV) vectors are currently the gold standard for gene therapy. However, their limited genome packaging capacity (4.7 kb) impedes their application for large gene expression. Many CRISPR‐associated (Cas) fusion proteins with high translational potential, such as various modules designed for gene activation (CRISPRa), exceed the rAAV packaging capacity. One of the most efficient CRISPRa modules is Cas9‐VPR. By using split‐intein‐based dual rAAV vectors to reconstitute dCas9‐VPR in a mouse model of retinal degeneration, we recently provided a proof‐of‐principle for activation of functionally equivalent genes as a novel gene therapy strategy. Although currently most efficient for large gene reconstitution, split‐intein approaches bear the risk of substantial immunogenicity due to the generation of an alien protein (intein) as a by‐product of the reconstitution process. To overcome this limitation, we recently introduced a novel intein‐free dual rAAV vector system based on reconstitution at the mRNA level. Here, we used these dual rAAV vectors to functionally reconstitute split dCas9‐VPR and activate genes in murine retina, primary neurons, and brain. In particular, we show that our dual rAAV vector approach can be used to activate Myo7b as a functional equivalent of Myo7a. Considering that mutations in the MYO7A gene cause Usher syndrome, the most common form of deafblindness worldwide, such an approach would represent a novel strategy to treat this devastating disease. Finally, we show that our dual rAAV vector approach can be used for simultaneous gene activation and gene knockdown or for reconstitution of prime editors, highlighting its great translational potential.
Counteracting culture stress for efficient genetic engineering in hematopoietic stem and progenitor cells (HSPCs)
1: San Raffaele Telethon Institute for Gene Therapy (SR‐TIGET), IRCCS San Raffaele Scientific Institute, Milan, Italy 2: CUSSB—University Center for Statistics in the Biomedical Sciences, Vita‐Salute San Raffaele University, Milan, Italy. 3: National Research Council, Institute for Biomedical Technologies, Segrate, Italy. 4: Vita‐Salute San Raffaele University, Milan, Italy.
Prolonged time in culture is still a prerequisite to reach sufficient levels of gene correction by homology‐driven repair (HDR) for HSPC‐based clinical applications. We recently discovered that gene‐editing by co‐delivery of nuclease‐induced DNA double strand break (DSB) and corrective DNA templates via AAV6 in cultured HSPCs triggers a strong p53‐mediated DNA damage response (DDR) affecting their functionality post‐transplant. Instead, DDR was transient when HSPCs were edited upon short time in culture, suggesting that prolonged time in culture makes edited HSPCs to exceed a threshold of tolerable DDR with adverse cellular outcomes. We found that activated HSPCs accumulated physical DNA damage, ROS and signs of DNA replication stress by accumulation of yH2AX and the single strand DNA binding protein pRPA nuclear markers. We identified as an upstream mediator of the observed culture stress the activation of the p38‐MAPK and found that its chemical inhibition prior to gene editing mitigate culture stress and enhanced the clonogenic potential of edited HSPCs, especially of the most primitive subset. Edited HSPCs pre‐treated with p38i displayed higher engraftment and repopulation capacity upon transplantation in immunocompromised mice. Moreover, in vivo clonal tracking revealed that p38 inhibition increased the number of HDR‐corrected clones in the human graft. Altogether, our findings identified a novel molecular pathway in activated human HSPCs linking physical DNA damage, p38 and premature exhaustion of HSPCs upon genetic engineering. Pharmacological manipulation of such a pathway ameliorated HSPC functionality and may be exploited to further advance HSPC‐based gene and cell therapies.
Multiplex base editing of NK cell to enhance cancer immunotherapy
1: Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota, 55455, USA 2: Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, 55455, USA 3: Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, 55455, USA
Natural killer (NK) cells have many unique features that have gained attention in cancer immunotherapy. NK cells can kill in antigen independent and dependent fashion, can be used as an allogeneic product, and perform antibody‐dependent cell‐mediated cytotoxicity (ADCC). However, NK cell function is regulated by many activating and inhibitory receptors, which cancer cells take advantage of to avoid being killed by NK cells. Previously, we reported a high efficiency method for multiplex engineering of T cells using base editor (BE) technology and thus reasoned that applying similar concepts in NK cells may offer a chance to alter many genes simultaneously through multiplex base editing. We thus selected a panel of genes bearing critical rolesin NK cell function for immunotherapy, including AHR, CISH, KLRG1, TIGIT, KLRC1, PDCD1, and CD16A. CD16A encodes an Fc receptor responsible for ADCC and cleaved upon NK activation. Non‐cleavable CD16A improves ADCC and can be achieved through single‐base substitution with BE. Using ABE8e, we achieved up to 95% base editing in NK cells both at single and multiple gene editing levels. Further functional evaluation also confirmed the functional changes induced by base editing in NK cells. Our end goal is to achieve high efficiency multiplex editing in CAR‐expressing NK cells to improve NK cell activity and toxicity for cancer immunotherapy.
HMI‐103: An investigational gene editing vector for Phenylketonuria (PKU)
1: Homology Medicines, Inc
PKU is a rare autosomal recessive monogenic disorder. Greater than 98% of cases are due to mutations in the phenylalanine hydroxylase (PAH) gene, resulting in deficient activity of PAH, a hepatic enzyme that catalyzes the formation of tyrosine, a precursor to neurotransmitters, from phenylalanine (Phe). Untreated PKU results in progressive, irreversible neurological impairment during infancy and early childhood. Restricting protein and Phe intake is standard of care for most PKU patients.
HMI‐103 is an investigational gene editing vector packaged in AAVHSC15 designed to deliver functional copies of human PAH to hepatocytes with the potential to restore PAH activity and normalize Phe metabolism. HMI‐103 contains locus‐ and human‐specific homology arms (HA) flanking the PAH sequence (cDNA), which are designed to guide the cDNA to the PAH locus and integrate through non‐nuclease‐based, AAV‐mediated homologous recombination.
IND‐enabling studies of HMI‐103 showed integration into the PAH locus without introducing de novo single nucleotide variants, insertions or deletions in hepatocytes in humanized‐liver xenograft mice. Fidelity of integration has been verified by long‐read sequencing. No off‐target integration was detected via molecular analysis.
A mouse surrogate gene editing vector was used in GLP toxicity studies in the Pah enu2 PKU murine model and germline transmission studies in C57BL/6J mice. Blood Phe was normalized in Pahenu2 mice at all doses tested and there were no test‐article related findings. There was no evidence of germline transmission.
These data demonstrated integration, fidelity, and HA‐specificity for the PAH locus and preclinical safety of HMI‐103 and are supportive of clinical trial initiation.
Targeted Genome Editing of Haematopoietic Stem Cells for Treating Recombination Activating Gene 1 (RAG1) Immunodeficiency
1: San Raffaele‐Telethon Institute for Gene Therapy (SR‐Tiget), IRCSS San Raffaele Scientific Institute, Milan, Italy. 2: Milan Unit, Istituto di Ricerca Genetica e Biomedica (IRGB), Consiglio Nazionale delle Ricerche (CNR), Milan, Italy. 3: Translational and Molecular Medicine (DIMET), University of Milano‐Bicocca, Monza, Italy 4: Laboratory of Clinical Immunology and Microbiology, NIAID, NIH, Bethesda, MD, USA 5: Gene Therapy Program, Dana‐Farber/Boston Children's Cancer and Blood Disorders Center, Harvard Medical School, Boston, MA, USA 6: Vita‐Salute San Raffaele University, Milan, Italy 7: # Contributed equally
Recombination Activating Genes (RAG) are tightly regulated during lymphoid differentiation and their mutations result in a broad spectrum of severe immunological disorders. The only curative treatment is haematopoietic stem/progenitor cell (HSPC) transplantation, but donor availability is limited, and unsatisfactory outcomes have been described with partially HLA‐matched donors. By performing competitive transplant experiments into Rag1‐/‐ mice we found that low proportion of wild‐type HSPC corrects immune defects. Thus, we developed a “one‐size‐fits‐all” gene editing (GE) strategy, based on high‐fidelity CRISPR/Cas9 and AAV6, to integrate a corrective codon‐usage optimized RAG1 sequence into its endogenous locus. Functionality of the edited RAG1 gene was confirmed in a RAG1‐KO cell line, which showed restored RAG1 recombination activity in vitro. We then applied the GE procedure on human mobilized peripheral blood HSPC from healthy donors and two patients carrying different hypomorphic RAG1 mutations and we achieved high homology directed repair (HDR) efficiency and comparable between healthy donor and patient‐derived HSPC, with no impact on cell phenotype in vitro. Gene editing, coupled with transient p53‐inhibition (GSE56), did not impact the engraftment and multilineage differentiation capability of edited HSPC in NSG mice, compared to unedited HSPC. Of note, we observed a higher percentage of circulating edited cells in NSG mice transplanted with treated patient‐derived HSPCs as compared to that measured in the bone marrow, possibly indicating selective advantage of RAG1‐corrected cells. Overall, our findings confirmed the development of an efficient and promising GE platform for the correction of RAG1 deficiency.
Development of a polyfunctional editing strategy for adoptive T‐cell immunotherapy of cancer
1: San Raffaele Telethon Institute for Gene Therapy (HSR‐TIGET) 2: University Vita‐Salute San Raffaele
Adoptive transfer of genetically engineered T lymphocytes has provided remarkable clinical benefits for the treatment of cancer. In this context, autologous or allogenic T cells are redirected against tumor‐related antigens through de novo expression of a transgenic TCR or a CAR. Although endowed with powerful new properties, transplanted cells often fail to exert their biological function, because limited tumor trafficking, premature exhaustion and/or deactivation in the tumor microenvironment. Molecular dissection of these roadblocks is providing a wealth of novel actionable targets that can be exploited to devise more effective and safer engineered T cell products. These advances, however, pose the challenge of implementing innovative tools able to perform simultaneous orthogonal editing (i.e., knock‐in/out and transcriptional modulation) at multiple genomic sites without causing genotoxicity. To this end, we have extended the scope of permanent epigenetic silencing with engineered transcriptional repressors to include genome editing. These novel polyfunctional editors based on the CRISPR/Cas9 system proved to be effective in mediating epigenetic silencing and genome editing at multiple loci, including B2M and TRAC, without inducing reciprocal chromosomal translocations, both in cell lines and in human primary T lymphocytes. To achieve this goal, we established cell manipulation procedures and tailored platform composition to reach high rates (> 60%) of long‐term stable epigenetic silencing in primary T cells. We are currently evaluating the functional properties of the edited T lymphocytes in vitro and in vivo. Overall, this polyfunctional editing platform will pave the way to safe multiplexing of T cells and beyond.
Baboon envelope pseudotyped “Nanoblades” loaded with Cas9/gRNAs complexes allow efficient genome editing in human T, B cells, HSCs and donor DNA knock‐in provided by AAV‐6 in HSCs
1: CIRI; Inserm U1111 2: CONICET, Buenos Aires, Argentina 3: Université de Nice 4: GENyO‐ Centro de Genomica e Investigacion Oncologica: Pfizer / Universidad de Granada / Junta de Andalucia 5: LBMC, Inserm, U1210 6: KU Leuven 7: Inserm UMR 1089
Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into human blood cells can be challenging. Here, we have utilized “nanoblades”, a new technology based on a modified Murine Leukaemia Virus (MLV) or HIV‐derived virus like particle (VLP), in which the viral structural protein Gag is fused to Cas9. These VLPs are thus loaded with Cas9 protein complexed with the guide RNAs (gRNAs). 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. A brief incubation of human T and B cells with nanoblades incorporating two gRNAs resulted in 40% and 15% edited deletion in the Wiskott Aldrich Syndrome (WAS) gene, respectively. HSCs treated with the same nanoblades allowed 30‐40% edited deletion in the WAS gene and 70% INDELS when a single gRNA is used. Importantly, upon nanoblades incubation neither toxicity nor phenotypic changes were detected. Moreover, off‐target site INDELS were not detected by ICE and TIDE analysis. When HSCs were treated with nanoblades together with a donor template encoding rAAV‐6 vector, up to 40% of stable expression cassette knock‐in into the WAS gene was observed. To still improve CD34+ entry efficiency of donor DNA, AAV2 and AAV6 were chemically bio‐conjugated with a HSC‐targeting ligand, resulting in higher gene knock‐in and cell survival of CD34+ cells. Summarizing, the nanoblades are simple to implement and highly flexible and efficient in hematopoietic cells such as HSCs.
Ex vivo editing of human hematopoietic stem cells for LAL‐D therapy.
1: Integrare research unit UMR_S951, Genethon, Inserm, Univ Evry, Université Paris‐Saclay, 91000, Evry, France
Lysosomal acid lipase deficiency (LAL‐D) is an autosomal recessive disorder caused by mutation in the LIPA gene and characterized by fats accumulation in organs and tissues leading to multi‐organ failure and premature death. The only treatment consists in weekly systemic injection of recombinant LAL that is uptaken by affected cells (cross‐correction), but it is not curative and is associated with risk of developing neutralizing antibodies.
Here, we developed an alternative platform for erythroid‐specific expression of proteins via ex vivo targeted integration of therapeutic transgenes in hematopoietic stem/progenitor cells (HSPCs).
Using CRISPR‐Cas9, we integrated different therapeutic transgenes under the transcriptional control of the endogenous α‐globin promoter to achieve robust expression of enzymes (FIX, LAL, IDUA, etc.) by the erythroid lineage. In particular, erythroblasts derived from targeted HSCs secreted LAL enzyme, which retained enzymatic activity and cross‐corrected patients' fibroblasts. To further improve the expression and the secretion of the enzyme, we engineered LAL cDNA sequence by changing its signal peptide sequence and optimizing the codon usage and obtained an 8‐fold increase in enzymatic activity in the medium of erythroid differentiated hHSPCs. To test if this HSPC platform works in vivo, we have modified HSPC from a mouse model humanized for the α‐globin locus (∼120kb) and transplanted them in a wild type mouse to quantify the level of LAL expression in the blood and in a LAL‐D mouse model to assess its therapeutic potential. These results and the ongoing in vivo analyses will be presented.
Novel CRISPR‐Cas13d system to target highly conserved sequences of SARS‐CoV‐2 and other human coronaviruses
1: Academic Medical Center
The current SARS‐CoV‐2 pandemic is a major global health burden. Although protective vaccines are available, concerns remain as new viral variants continue to appear worldwide. Gene editing‐based antiviral approaches, such as CRISPR‐Cas, may offer an attractive alternative as it can be rapidly adjusted to new viral sequences. This study aimed at using CRISPR‐Cas13 to target highly conserved viral RNA sequences, not only among different SARS‐CoV‐2 variants, but also among the other human coronaviruses, thus preparing for future zoonotic outbreaks of novel coronaviruses.
Complete genome sequences of SARS‐CoV‐2 variants were aligned in order to identify conserved sequences. Moreover, SARS‐CoV‐2 sequences were aligned with those of other human coronaviruses to identify broadly conserved sequences. Based on the selected target sequences, crRNAs were designed. The knock‐down efficiency of the crRNAs was tested in vitro in transfected cells with a designed multi‐target luciferase reporter construct. The antiviral activity was subsequently validated by scoring the crRNA knock‐down efficiency in cells transfected with the SARS‐CoV‐GFP and SARS‐CoV‐2‐mNeonGreen replicons.
We identified a panel of crRNAs targeting highly conserved SARS‐CoV‐2 sequences with a high predicted activity. The majority of these crRNAs induced a strong reduction in luciferase signal. Selected crRNAs demonstrated efficient targeting of the SARS‐CoV and SARS‐CoV‐2 replicons, confirming that the designed crRNAs exhibit broader anti‐coronavirus activity in a biologically relevant setting.
Overall, this study demonstrates potent targeting of highly conserved SARS‐CoV‐2 sequences by the novel CRISPR‐Cas13 system and emphasizes the importance of rationally designing gene‐editing based antiviral strategies to prepare for future outbreaks of novel coronaviruses.
Lentivector carrying Cas9 protein, guide RNA and transgene for simultaneous “hit‐and‐run” gene disruption and transgene delivery
1: University of Veterinary Medicine, Vienna
CRISPR/Cas9‐based genome editing offers the potential to treat genetic diseases and to generate more advanced immunotherapy products such as allogeneic CAR‐T cells or CAR‐T cells with enhanced antitumor function. To achieve this goal, highly efficient and, at the same time, safe methods for the delivery of the CRISPR/Cas9 complex together with gene(s)‐of‐interest to cells has to be developed. Lentiviral vectors, due to their high transduction efficiency, are commonly used to deliver exogenous genes to cells. However, the limited payload capacity does not allow the generation of “all‐in‐one” lentivectors for simultaneous transduction of the bulky cas9 gene together with a transgene. In addition, the designer nuclease should be delivered in a “hit‐and‐run” fashion that narrows the window of activity of the protein to minimize the risk of genotoxicity. Here, we developed an “all‐in‐one” lentivector carrying the Cas9 protein and templates for the expression of sgRNA and transgene. Direct packaging of the Cas9 protein (via fusion with Vpr) freed a large space (∼4.2 kb) in the transfer vector that can be filled with exogenous genes. For the “proof‐of‐concept” study, we transduced HEK293GFP cells with the three‐component lentivector containing Cas9, sgRNA directed against gfp gene, and a red fluorescent protein gene (DsRed). Single transduction with the unconcentrated lentivectors resulted in robust gfp disruption (∼90%) with a concurrent gain of the DsRed expression in ∼99% of cells. The results demonstrate that lentivectors can be engineered into a versatile and highly efficient multicomponent delivery tool for concurrent genome editing and exogenous DNA delivery.
Innate immunity signaling inhibitors for stimulation of homology‐directed repair at
CCR5
locus during CCR5‐Uco‐TALEN‐mediated genome editing in primary human hematopoietic stem cells.
1: RM Gorbacheva Research Institute of Pediatric Oncology, Hematology and Transplantation, Pavlov University 2: Research Centre for Medical Genetics 3: Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, UMC Hamburg‐Eppendorf
Targeted insertion of protein‐coding sequences into the genome of hematopoietic stem cells (HSCs) mediated by homologous recombination (HR) after targeted introduction of double‐strand breaks is a promising platform for gene therapy of monogenic diseases. Increasing the HR efficiency may be achieved through the modulation of DNA damage‐induced innate immune response mechanisms.
The aim of this work is to evaluate the effects of small‐molecule inhibitors of TLR9/AIM2/cGAS, STING and caspase antagonists A151, H151, and Z‐VAD‐FMK on the HR rate at the CCR5 locus after CCR5‐Uco‐TALEN mRNA transfection of primary human HSCs.
After magnetic selection and initial activation, HSCs were transfected with 25 μg/ml CCR5‐Uco‐TALEN mRNA by electroporation in the presences of small‐molecule inhibitors A151 (4 μg/ml), H151 (0.5 μg/ml), or FMK (25 μg/ml) added 3 hours previously. The proportion of non‐homologous end joining (NHEJ) events was estimated by digital‐droplet PCR (ddPCR) as previously described (Mock et al., 2015) five days after transfection. The frequency of HR events was estimated as the difference between reference (EPOR) and CCR5 gene copy numbers by ddPCR.
The obtained total CCR5 gene knockout efficiency ranged from 9.0 to 53.5%. FMK addition affected both NHEJ (27.2%) and HR (26.3%) rates at CCR5 compared to control (15.7% and 13.7% respectively). In the presence of A151, the CCR5 knockout efficiency was significantly reduced (9%), and 95.6% of k.o. alleles were repaired by NHEJ. FMK seems to be the most promising agent in terms of HR stimulation.
Hematopoietic stem cell gene editing for the treatment of IL7R‐SCID
1: UCL Institute of Child Health
Interleukin 7 Receptor ‐ Severe combined immunodeficiency (IL7R‐SCID) is a life‐threatening syndrome characterized by a complete block in T cell development that has a fatal outcome in children by the second year of life. The disease, which represents the third most common form of SCID, is caused by a mutation in the IL7R gene. Although gene therapy has proved to be a powerful tool to treat rare genetic diseases affecting the hematopoietic system, pre‐clinical studies using lentiviral vectors to introduce a correct copy of IL7R in haematopoietic stem and progenitor cells (HSPCs) showed constitutive and unregulated gene expression predisposing to leukaemia. CRISPR/Cas9 based genome editing has the potential to correct the mutated IL7R locus by site‐specific insertion of a correct IL7R cDNA, thus restoring a physiological gene expression. To test this strategy, we electroporated healthy HSPCs with an optimized gRNA‐Cas9 complex targeting the IL7R locus followed by transduction with an AAV6 donor template containing a GFP cDNA flanked by IL7R homology arms, reaching an average of 40% of targeted integration of the reporter cassette. Moreover, by delivering a therapeutic AAV6 donor vector encoding a codon‐optimized IL7R cDNA in place of the GFP reporter cassette, we successfully restored IL7R expression in IL7R‐deficient cells. The final goal of this study is to correct patient‐derived IL7R‐SCID HSPCs with our CRISPR/AAV6 platform and restore a physiological T‐cell development. For this purpose, we are now gene editing IL7R‐deficient HSPCs and assessing correct lymphopoiesis through an in vitro T‐cell differentiation platform and in vivo xenotransplantation assays.
Modelling Congenital Dyserythropoietic Anemia Type II in Human Hematopoietic Stem and Progenitor Cells using Gene Editing
1: CIEMAT/CIBERER 2: Instituto de Investigación Sanitaria Fundación Jiménez Díaz 3: Universitat Internacional de Catalunya 4: Hospital de Sant Pau 5: Bloodgenetics
Congenital Dyserythropoietic Anemia type II (CDAII) is a rare inherited disorder that affects red blood cell development. CDAII is caused by mutations in the SEC23B gene. CDA patients show anemia of variable degrees and 20% of them are transfusion dependent. Current treatments for CDAII may involve blood transfusions, iron chelation and splenectomy. However, the only described definitive therapy is allogeneic bone marrow transplantation, which implies additional side effects to the patients.
Availability of a CDAII cellular model will boost the development of new therapeutic approaches. We have developed an efficient CRISPR/Cas9 system to alter SEC23B open reading frame in healthy human hematopoietic stem and progenitor cells (CD34+) that has been tested both in vitro and in vivo in immunodeficient NBSGW mice. SEC23B expression in gene edited CD34+ cells was diminished, which led to an impaired cell growth and an increase of binucleated erythroblasts after in vitro erythroid differentiation. Similarly, the in vivo erythroid differentiation of CD34+ cells in NBSGW mice showed an impairment of terminal erythroid differentiation with an increment in the percentage of binucleated erythroblasts. Surprisingly, frequency of frameshift indels in SEC23B was lessened throughout the erythroid differentiation, which may point out an advantage of SEC23B wild type alleles over the mutant ones.
In summary, CRISPR/Cas9 system has been used to model CDAII in human hematopoietic progenitors through the knock‐out of SEC23B gene. Our CDAII model will be an effective tool for the development of new gene therapy curative strategies in this disease.
CRISPR/Cas9‐mediated gene editing for treatment of the Wiskott‐Aldrich Syndrome
1: University of Gent 2: UTHealth 3: Rice University
The Wiskott‐Aldrich Syndrome (WAS) is a severe X‐linked primary immunodeficiency caused by a variety of mutations in the WAS gene. This leads to altered or absent WAS protein (WASp) expression and function, resulting in thrombocytopenia, eczema, recurrent infections, auto‐immunity and an increased risk of leukaemia. Gene therapy approaches using lentiviral‐mediated delivery of WAS cDNA to CD34+ hematopoietic stem/progenitor cells (HSPCs) show promising results. However, a delay in reconstitution of certain hematopoietic lineages, most notably platelets, is observed. In addition, there is a potential risk of genotoxicity. Thus, gene editing instead of gene addition approaches would offer the potential advantage of a reduced number of transgene integrations and physiological regulation of transgene expression. We have developed a nuclease‐based gene‐editing strategy for site‐specific integration of a corrective WAS gene into the endogenous WAS chromosomal locus. We were able to integrate a WAS2‐12 ‐construct into intron 1 of the endogenous WAS gene of primary CD34+ hematopoietic stem/progenitor cells (HSPCs), as well as WASp‐deficient B cell lines and WASp‐deficient primary T cells. Not only did we efficiently restore WASp expression in treated cells, this targeted integration also restored WASp‐dependent functions, such as proliferation, actin filament formation, and cytokine production (for T cells). Furthermore, HSPCs with targeted WAS2‐12 integration retained the ability to differentiate in vitro into various hematopoietic cell types including erythroid, megakaryocyte, NK, B, monocyte and granulocyte lineages. Taken together, these results are promising for a gene‐editing based, autologous cell therapy, potentially applicable to the vast majority of WAS patients.
Simultaneous high‐efficiency base editing and reprogramming of patient fibroblasts
1: University of Helsinki 2: Department of Pediatrics; Helsinki University Hospital 3: Department of Clinical Genetics; Helsinki University Hospital
Human induced pluripotent stem cells (hiPSC) allow in vitro study of disease mechanisms and hold potential for personalised stem cell therapy. By modifying precisely targeted loci without altering the genetic background, gene editing serves to study genetic diseases, introducing or correcting DNA mutations into patient‐derived hiPSC. However, the process of hiPSC generation and subsequent gene editing can take many months and require a significant investment in time and reagents. Here, we describe a highly efficient method for simultaneous gene editing and reprogramming of fibroblasts employing a CRISPR‐Cas9 adenine base editor. As a proof‐of‐concept, we apply this approach to generate gene‐edited hiPSC from skin biopsies of four patients carrying a Finnish‐founder pathogenic point mutation either in NOTCH3 or LDLR genes. Starting from primary patient‐derived fibroblasts, this method yields tens of on‐target edited isogenic hiPSC lines in less than six weeks, not compromising their genetic integrity nor their pluripotency quality. Without any selection or sorting, the on‐target editing efficiency consistently remained above 96%, while considerably diminishing the cell culture time and thus the reagent spences and the risk for in vitro alterations.
This approach can significantly simplify and enhance the work of researchers and biobanks, providing a cleaner and more efficient way to convert A•T to G•C base pairs, which could potentially correct a large portion of human pathogenic SNPs.
Assessment of functional activity of AAV9 encoding ARSA in the minipig model
in vivo
1: Kazan Federal University 2: Kazan State Academy of Veterinary Medicine named after N.E. Bauman
Metachromatic leukodystrophy is a severe demyelinating lysosomal storage disease arising due to ARSA enzyme deficiency. The aim of this work is to create a gene drug based on a recombinant adeno‐associated virus serotype 9 (AAV9).
In this study AAV9‐ARSA has been created, which contains a unique codon‐optimized ARSA sequence. The resulting AAV9‐ARSA was intravenous and intrathecal administered into mini pigs. Each group contained 3 pigs, the results were compared to the group of intact animals. ARSA dynamic activity was analyzed in cerebrospinal fluid (CSF) and plasma samples. Central nervous system (CNS) parts were used to analyze ARSA expression using qPCR and IHC. The changes in Alt, Ast, Bilirubin, Creatine‐J in serum were analyzed. The enzymatic activity of ARSA in the cell lysates, plasma, SCF, in the homogenate of CNS parts of animals was analyzed using pNCS. WB and IHC were performed using monoclonal antibodies to ARSA.
An increase in ARSA activity in plasma was found after drug administration. In the CSF an increase in ARSA activity was found only after intrathecal administration (by 13‐74%). In the homogenates of the CNS organs an increase in ARSA activity was also found in the experimental groups. The presence of ARSA mRNA and protein in the CNS was confirmed using qPCR and IHC. Biochemical analysis showed a decrease in the levels of AST after intravenous administration.
Thus, we have shown that after intravenous and intrathecal administration, AAV9‐ARSA reaches the CNS and leads to the ARSA overexpression.
In depth assessment of off‐target editing by CRISPR/Cas9 in VOR33, an engineered hematopoietic stem cell transplant for the treatment of acute myeloid leukemia
1: Vor Biopharma
VOR33 is an engineered allogeneic hematopoietic stem cell transplant for treatment of acute myeloid leukemia (AML) in which the CD33 surface antigen is removed by CRISPR/Cas9 editing, thus enabling post‐transplant immunotherapeutic targeting of CD33‐expressing leukemic cells while sparing the CD33 gene‐edited graft. To ensure safety of gene‐edited CD34+ hematopoietic stem and progenitor cells (HSPCs), off‐target editing must be minimized. Here, we present a rigorous appraisal of potential CRISPR/Cas9 off‐target editing across multiple VOR33 batches via multiple independent in silico and experimental methods. Analysis of on‐target structural variation by long‐read sequencing revealed low frequencies of large deletions and insertions across VOR33 batches with no perceivable impact on safety or efficacy. To identify potential off‐target indels, both homology‐dependent in silico prediction as well as GUIDE‐seq were performed. Potential off‐target indel frequencies were quantified by hybrid capture NGS across multiple VOR33 batches manufactured at both research and clinical‐scales and revealed no significant and reproducible editing. Lastly, karyotyping revealed no detectable gross chromosomal abnormalities across multiple batches, indicating that VOR33 maintains genomic stability. Importantly, our findings show the VOR33 engineering process is robust and reproducible, with no discernable differences in off‐target frequencies or patterns in multiple independent VOR33 batches. This assessment of off‐target editing establishes a clinically translatable safety framework to evaluate genotoxicity in CD34+ HSPC‐based cell therapies for AML treatment.
*equal contribution
CAST‐Seq discloses low frequency of chromosomal rearrangements in double‐nickase based genome editing in primary human cells
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: EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, 5020, Austria 4: Institute of Medical Bioinformatics and Systems Medicine, Medical Center – University of Freiburg, Freiburg, 79110, Germany 5: German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Partner Site Freiburg, Freiburg, 79106, Germany
Unwanted off‐target effects remain a major challenge in therapeutic genome editing. The use of a catalytically modified version of the CRISPR‐Cas9 system, Cas9 nickase (Cas9n), was postulated to reduce the risk of off‐target mutagenesis significantly. We previously established that a Cas9n based double‐nicking strategy mediates high gene correction frequencies in primary keratinocytes at COL7A1 and COL17A1 loci, two genes linked to Epidermolysis bullosa. Whereas Cas9 generated DNA double‐strand breaks are known to incite chromosomal translocations, it has not been characterized whether Cas9n introduced single‐strand nicks trigger genomic aberrations in primary human cells. To address this question we applied CAST‐Seq, an unbiased method to identify and quantify chromosomal rearrangements in gene‐edited cells with high sensitivity. In primary keratinocytes we compared the genotoxic profiles of Cas9 nucleases to dual and single Cas9n strategies targeting the three loci COL7A1, COL17A1, and LAMA3. CAST‐Seq identified 11 off‐target mediated translocation (OMT) events upon COL7A1 editing with either of two Cas9 nucleases. While concomitant application of two Cas9 nickases caused large rearrangements at the on‐target site, no OMT events could be detected. Likewise, no aberrations were identified upon transfer of a single Cas9n. A similar trend was observed for Cas9 and Cas9n‐mediated editing at COL17A1 and LAMA3. Taken together, we demonstrate that Cas9n‐induced nicks induce chromosomal rearrangements at the on‐target site in clinically relevant human cells. At the same time, CAST‐Seq revealed that double‐nickase strategies combine efficient genome editing with strongly reduced genotoxic side effects.
Development of a gene editing therapeutic approach for the treatment of
RPL5
‐deficient Diamond‐Blackfan anemia patients
1: CIEMAT/CIBERER 2: IIS‐Fundación Jimenez Diaz 3: Hôpital Robert‐Debré 4: Hospital Sant Joan de Déu 5: Hospital Gregorio Marañón
Diamond‐Blackfan Anemia (DBA) is an inherited bone marrow failure (IBMF) syndrome mainly characterized by red cell aplasia, congenital abnormalities and increased risk of cancer. Mutations in more than 20 genes have been associated with DBA, being RPS19 is the most frequently (25%), followed by RPL5 (11%). A lentiviral gene therapy approach for the treatment of RPS19‐deficient is under development in our laboratory. However, this strategy might not be the most convenient one to correct RPL5 mutations, since this protein directly interacts with MDM2, an inhibitor of p53. Thus, a directed GT approach using a homology‐directed repair (HDR) has been used for the knock‐in of RPL5, maintaining the tight endogenous regulation of this gene.
In this context, we have developed a potentially therapeutic approach based on CRISPR/Cas9 system and adeno‐associated virus (AAV) viral vector harbouring a codon optimized version of RPL5 gene. Designed sgRNAs allowed us to achieve high percentage of indels (>90%) with different single‐guide RNA (sgRNA) in CD34+ cells from healthy donor cord‐blood (CB) and also from bone marrow from RPL5‐deficient patients. Transduction with CoRPL5‐AAV was then optimized to achieve clinically relevant HDR percentages in CB‐HD CD34+ cells.
AAV‐mediated liver‐directed homology‐independent targeted integration provides stable therapeutic levels of transgene expression in newborn mice with a lysosomal storage disease.
1: Telethon Institute of Genetics and Medicine 2: Federico II University, Deparment of Translational Medicine 3: Telethon Institute of Genetics and Medicine, Armenise/Harvard Laboratory of Integrative Genomics 4: Next Generation Diagnostic srl 5: Federico II University, Department of Advanced Biomedicine 6: University of Campania L. Vanvitelli, Department of Precision Medicine
One of the major limitations of liver gene therapy with adeno‐associated viral (AAV) vectors is the episomal nature of their genome, which undergoes dilution during hepatocyte proliferation, thus representing a drawback for treatment of pediatric subjects. Similarly, liver injury followed by regeneration might cause loss of transgene expression in adult patients.
To overcome these issues, we applied SpCas9‐mediated homology‐independent targeted integration (HITI) by using two AAV vectors: one encoding SpCas9 under the control of a liver‐specific promoter; the other encoding for: i) a gRNA targeting the murine Albumin (mAlb) locus and ii) a donor DNA with the transgene to be integrated.
Upon neonatal administration in mice, HITI efficiency ranged between 2‐4% in hepatocytes. Also, HITI was mostly precise without detectable gRNA or HITI off‐targets.
Next, we delivered systemically AAV‐HITI to newborn mice with mucopolysaccharidosis type VI (MPS VI), a rare lysosomal storage disorder. We observed stable serum levels of the therapeutic enzyme arylsulfatase B and phenotypic improvement, similar to that obtained with traditional gene replacement therapy which is currently under clinical investigation in a gene therapy clinical trial (Clinicaltrials.gov identifier: NCT03173521) from which MPS VI subjects younger than 4 years‐old are excluded because of potential hepatocyte proliferation associated with liver growth.
These results lay the groundwork for neonatal treatment of MPS VI and other metabolic diseases that demand early intervention, and for targeting adult damaged liver with hepatocyte proliferation.
COL17A1 editing in junctional epidermolysis bullosa keratinocytes via CRISPR/Cas9 induced homology‐directed repair
1: EB 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: Cell Therapy Institute, SCI‐TReCS, Paracelsus Medical University, 5020 Salzburg, Austria 3: Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University Salzburg, 5020 Salzburg, Austria
Junctional epidermolysis bullosa (JEB) is an autosomal recessive skin disorder associated with mutations in the genes LAMA3, LAMB3, LAMC2, ITGA3, ITGA3, ITGB4 and COL17A1, which encode dermal‐epidermal junction proteins within the skin. Type XVII collagen, encoded by COL17A1, is a transmembrane protein that connects basal keratinocytes with the lamina lucida of the basal membrane zone (BMZ) between the epidermis and dermis of the skin. In this study, we employed homology‐directed repair CRISPR/Cas9‐based strategies to precisely correct a mutation within exon 52 of COL17A1 in JEB keratinocytes. We compared the efficacy of double‐strand breaks or paired nicks induced by the wild‐type Cas9 or Cas9n, followed by homology‐directed repair with symmetric or asymmetric single‐stranded oligonucleotides that served as repair templates. Wild‐type Cas9 ribonucleoproteins delivered together with HDR oligonucleotides resulted in a high homologous recombination efficiency of 59%, while via double nicking an editing efficiency of 23% was achieved as revealed by next generation sequencing. Highly efficient type XVll collagen restoration was also confirmed at RNA, protein and cellular level. Furthermore, the correct localization of type XVll collagen was detectable within the basal membrane zone of generated skin equivalents. The proposed approach could serve as a good strategy for precise COL17A1 editing and a potential clinical application for JEB.
Artificial Thymic Organoids as an
in vitro
platform to evaluate the T‐cell differentiation potential of gene edited hematopoietic stem/progenitor cells
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 20900, Italy. 3: Milan Unit, Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Milan, Italy. 4: Laboratory of Clinical Immunology and Microbiology, NIAID, NIH, Bethesda, MD, USA. 5: Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, Univ. of California, Los Angeles (UCLA), Los Angeles, CA, USA. 6: Gene Therapy Program, Dana‐Farber/Boston Children's Cancer and Blood Disorders Center, Harvard Medical School, Boston, MA, USA. 7: Vita‐Salute San Raffaele University, Milan, Italy. 8: Contributed equally.
Artificial Thymic Organoid (ATO) is an in vitro three‐dimensional platform that mimics thymic environment sustaining T‐cell development from hematopoietic stem/progenitor cells (HSPC). Recently, it has been demonstrated that ATO system is a suitable tool to study early T‐cell differentiation defects. Since this system requires minimal amounts of HSPC, it represents a unique model to assess T‐cell differentiation potential of gene engineered HSPC and complement in vivo xenotransplantation. However, when healthy donor (HD)‐derived HSPC, edited with CRISPR/Cas9 ribonucleoprotein and adeno‐associated vector carrying PGK‐GFP cassette to monitor edited cells, were co‐cultured with MS5‐hDLL4 cells, we found that the editing procedure strongly reduced cell viability and T‐cell differentiation. Thus, we edited HSPC in combination with a transient p53‐inhibitor (GSE56) and/or the Ad5‐E4orf6/7 protein, reported to dampen the impact of AAV6 transduction and enhance editing efficiency by forcing cell‐cycle progression, respectively. The addition of GSE56 alone partially restored the viability of ATO cells, but the T‐cell output remained poor. Remarkably, when adding Ad5‐E4orf6/7 we observed a dramatic improvement not only in viability and editing efficiency but also in T‐cell differentiation, up to TCRαβ+ CD3+ stage. We applied the optimized ATO platform to the GE procedure developed for correction of RAG1 deficiencies confirming the beneficial effect of GE enhancers (Ad5‐E4orf6/7 and GSE56) on ATO generation with edited T‐cells. Overall, by coupling HSPC editing with the ATO system we established a robust and efficient platform that can be broadly applied to disease modelling and validation of therapeutic efficacy.
CRISPR/Cas9 allele‐specific design to inactivate a dominant‐negative mutation in COL6A1 causing Ullrich muscular dystrophy
1: Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy 2: University Hospital S. Anna of Ferrara, Ferrara, Italy 3: Unit of Neuromuscular and Neurodegenerative Disorders, Department of Neurosciences, Bambino Gesù Children's Research Hospital IRCCS, Rome, Italy 4: Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy 5: Molecular Genetic Institute, Bologna‐CNR, Bologna, Italy
Ullrich muscular dystrophy (UCMD) is a congenital disorder caused by defects in collagen VI genes (COL6A1, COL6A2 and COL6A3). Collagen VI is a multimeric essential component of the extracellular matrix and dominant negative mutations are frequently implicated in UCMD pathogenesis (50‐75% of UCMD patients). CRISPR/Cas9 approach aimed at selectively knocking out mutated COL6A alleles could represent a therapeutic intervention. The genome editing strategy was assessed in primary fibroblasts from a UCMD patient carrying a heterozygous dominant mutation in the COL6A1 gene. This mutation is responsible for a marked reduction of collagen VI protein, thereby even a mild genomic correction should increase protein production and secretion in CRISPR‐treated fibroblasts, ameliorating the disease phenotype. To specifically knock out the mutated allele, we electroporated patient's fibroblasts with ribonucleoprotein (RNP) carrying the high‐fidelity SpCas9 and the mutation‐specific gRNA. The indels frequency was scored by TIDE analysis and Sanger sequencing of the targeted genomic region. We also predicted possible genome wide off‐targets by COSMID webtool. The top ranked off‐target sites showed undetectable frequency of indels by TIDE analysis. Patient's fibroblasts were analyzed for functional collagen VI expression. RT‐PCR demonstrated the decrease of mutated transcript respect to the untreated cells. Immunofluorescence analysis showed recovery of collagen VI secretion and, finally, ultrastructural analysis by Rotary‐shadowing electron microscopy demonstrated the restoration of collagen VI microfibrillar network in the gene edited fibroblasts. In conclusion, these results support the application of CRISPR/Cas9‐mediated genome‐editing approach to knock out COL6A mutated alleles and rescue UCMD phenotype in patient‐derived primary fibroblasts.
CRISPR‐Cas9 mediated correction and generation of iPSC clones derived from a Cornelia de Lange Syndrome (CdLS) patient with a c.5483 G > A substitution in NIPBL
1: Department CIBIO, University of Trento, via Sommarive 9, 38123 Trento, Italy 2: Laboratory of Molecular Biology, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, via Mario Negri 2, 20156 Milano, Italy
Cornelia de Lange syndrome (CdLS) is an autosomal dominant disease caused by alteration of the cohesin pathway. Most of the mutations are located in the NIPBL gene, including the missense c.5483 G > A substitution found in a CdLS patient. Here, we developed an efficient and precise CRISPR‐Cas based approach to correct the NIPBL mutation through homology directed repair (HDR). The efficacy of the gene correction strategy that we have developed was validated in patient derived human induced pluripotent stem cells (hiPSCs). The isogenic iPS clones carrying the corrected NIPBL showed a regular karyotype and preserved pluripotency as demonstrated by the expression of specific markers and by proper differentiation into the three‐germ layers. With this study we provided the first genome editing proof‐of‐concept for the correction of a mutation in NIPBL causing CdLS. Through our genome editing strategy we produced isogenic primary cellular models representing valuable disease models to study CdLS molecular mechanism and develop new therapeutic strategies.
Development of a Gene Editing Strategy for Cystic Fibrosis
We aim to develop a genome editing treatment for cystic fibrosis using homology independent targeted integration (HITI), a strategy that relies on non‐homologous end joining (NHEJ) after targeted double strand breaks by CRISPR. This will enable editing of non‐dividing lung epithelial cells, where homology‐directed repair (HDR) is not an option. HITI potentially enables targeted insertion of large pieces of cDNA, such as CFTR, into the genome, rescuing function regardless of genotype. We are targeting the safe harbour AAVS1 site, with the potential of more robust expression from the PPP1R12C gene promoter than possible in the CFTR locus with its the tightly‐regulated, relatively weak promoter. We first developed a primary cell model by transduction of basal epithelial cells carrying a CFTR nonsense mutation (R709X) and shown lack of CFTR activity by Ussing chamber analysis. For the HITI strategy, linearised donor plasmids containing a promoterless GFP along with a guide RNA for AAVS1 were transfected into HEK293T cells, and GFP expression detected as a readout for successful genomic integration. Potential off‐target editing was investigated by sequencing of PCR products at sites identified by in silico screening for gRNA homology. Of the off‐target sites successfully sequenced, none have shown any evidence of undesired editing. In addition, transfection of Cas9 RNP using a lipid‐peptide, receptor‐targeted nanoparticle was evaluated and found to effectively cut the genome at the desired site by ICE indel analysis.
Engineered human neonatal mesenchymal stromal cells expressing FGFb: assessment of immunophenotype, proliferation, differentiation and immunosuppressive effects
in vitro
1: Curexsys GmbH, Göttingen, 37079, Germany 2: Instituto Distrital de Ciencia Biotecnología e Innovación en Salud, Bogotá, 111221, Colombia 3: PhD Programme in Biomedical and Biological Sciences, School of Medicine and Health Sciences, Universidad del Rosario, Bogotá, 111221, Colombia
Clinical efficacy of Mesenchymal Stromal Cells (MSC) ‐based therapy remains a critical aspect for improvement. MSC engineering platforms represent an opportunity to provide more effective cell therapy strategies, by ex‐vivo delivering of therapeutic genes into cells. Basic fibroblast growth factor (bFGF) is widely considered as critical MSC growth and differentiation factor. However, the role of bFGF gene transfer into human Wharton's Jelly MSC (hWJ‐MSCs) has not been evaluated. In this study we evaluated a gene engineering approach based on lentiviral (LV) vector expression of bFGF and assessed its impact on innate characteristics and functions of WJ‐MSC‐based cell products. To this end, LV encoding bFGF‐Flag and GFP were generated and used to transduced hWJ‐MSC. Immunophenotypic expression of surface markers, proliferation, osteogenic and immunosuppressive potency of engineered MSC were assessed. Efficiency of LV transduction ranged between 9.7 – 30.3% when using a MOI of 5. LV transduction did not affect immunophenotype and osteogenic differentiation of WJ‐MSC. bFGF expression was significantly higher in WJ‐MSCbFGF (5‐15‐fold) as compared to WJ‐MSCGFP controls. Despite that additional constitutive expression of bFGF did not improve cell proliferation and immunosuppressive effect on T cell proliferation as compared to GFP‐MSC controls, bFGF improved osteogenic differentiation in WJ‐MSCbFGF. Together these data confirmed the feasibility of LV‐engineering of WJ‐MSC. bFGF transfer did not alter the phenotype, proliferation and immune suppression function of WJ‐MSC but could significantly improve osteogenic differentiation in vitro. Gene transfer technology applied to WJ‐MSC products, might offer a potential delivery system to improve clinical efficacy of MSC‐based cell therapies.
Preclinical optimization of gene editing for adoptive T cell therapy with high fidelity CRISPR/Cas9 ribonucleoproteins
1: MaxCyte 2: Aldevron
Adoptive cell therapy with T lymphocytes harnesses the immune system for cancer treatment. T lymphocytes engineered with transgenic T cell receptors (TCR) specific for tumor antigens are in clinical development for various cancers. Knockout of endogenous TCR gene may improve expression of TCR transgene, and knockout of PD‐1 may reduce T cell exhaustion induced by tumor expressed PD ligands. Gene editing with CRISPR/Cas9 via electroporation offers a rapid, clinically scalable approach for multiplexed knockout. High editing efficiency may reduce the timeline and cost of cell therapy manufacturing, and increase the proportion of edited cells. Additionally, editing with Cas9 ribonucleoprotein (RNP) may reduce off‐target edits relative to Cas9 nucleic acid substrates, particularly with high fidelity Cas9. We transfected high fidelity Cas9 RNP (SpyFi™ Cas9 Nuclease, Aldevron) into activated human T cells to knockout TCR using MaxCyte's scalable, GMP‐compliant electroporation technology. High editing efficiency was observed by flow cytometry using RNPs complexed with SpyFi or wild type enzyme (wt SpCas9, Aldevron). Editing efficiency was dependent on RNP concentration and electroporation energy. Following electroporation, viability improvement and resumption of proliferation were observed within 5 days. Co‐electroporation of RNPs directed to TCR alpha chain, TCR beta chain, and PD‐1 resulted in dual knockout of TCR and PD‐1, with comparable editing efficiency for wt or SpyFi Cas9 RNPs. The frequency of off target editing was compared at multiple genomic loci by next generation sequencing. Taken together, the results support editing of T lymphocytes lacking endogenous TCR and PD‐1 with high fidelity Cas9 RNPs.
Surveying sequence specificity of tyrosine site‐specific recombinases
1: Technical University Dresden
Tyrosine‐type site‐specific recombinase (SSR) systems, are successfully used as genome engineering tools in various model organisms. Through protein engineering and directed evolution, it has become possible to redirect the target site specificity of SSRs to recombine naturally occurring sites in genomes. To facilitate faster development of new designer‐recombinases as safe, genome editing agents, it is not only important to understand their general specificity determinants but also to be able to rapidly test their activity on large sets of potential off‐targets. Current methods are either limited to measuring recombination activity on single substrates of interest, or use in vitro assays of recombination between partially randomized DNA libraries and a single on‐target sequence.
We describe a high‐throughput, in vivo assay to profile the target site specificity of naturally occurring and designer‐SSRs on DNA libraries of non‐randomized target sequences. The functional screen requires minimal experimentation and is carried out in a bacterial system, eliminating possible artifacts from in vitro assays. Target‐site profiles are determined using deep sequencing, yielding accurate and highly reproducible results.
We validate our pipeline using the Cre/loxP system by designing a loxP based library to profile the target site specificity of this recombinase. Our results remained consistent across multiple experiments and matched previously published data. Using the obtained data, we generated a sequence specificity model of Cre to predict potential human genomic off‐target sites. Our results also facilitate nomination of new target sites for Cre‐based designer‐SSRs.
Computationally defined human genomic safe harbour loci validated in vitro for stable transgene expression
1: Genome Institute of Singapore 2: National University of Singapore 3: Institute of Molecular and Cell Biology
Controlled expression of transgenes is essential in both therapeutic and research applications. Targeting using viral vectors leads to random integration of introduced transgenes with the potential risks for insertional mutagenesis as well as transgene silencing. Technologies such as CRISPR/Cas9 allow directed transgene integration into the genome. Ideal integration sites are genomic safe harbour (GSH) sites, which allow for controlled expression without perturbing endogenous gene expression. To date, only a limited number of targeted integration sites have been reported in human. However, none of these meet stringent requirements for a GSH locus.
To discover putative human GSH, we conducted a computational analysis of publicly available data. The criteria used filtered for sites where integration will not lead to interference of the native transcriptome and are stable and accessible in most cell and tissue types. Our analysis yielded 25 putative human GHS that pass all the set criteria. Using CRISPR/Cas9 in H1 human embryonic stem cells (hESC), we have targeted and validated transgene expression in three shortlisted GSH. Transgenes in the targeted GSH are expressed stably and at a high level in hESC and cells differentiated to all three germ lineages. Furthermore, their integration shows no significant change in native gene expression levels. The uncovered GSH may enable targeted and controllable transgene expression in a cell therapy setting, without the risk of interference to the native transcriptome.
Evaluation of the efficiency of targeted transgene integration into the CCR5 gene locus using HDR and HITI.
A S Tikhonov1 R R Mintaev1 2 D V Glazkova1
1: Centre for Strategic Planning of FMBA of Russia 2: I.Mechnikov Research Institute of Vaccines and Sera
Recently, a novel method of transgene knockin, known as homology‐independent targeted integration (HITI), has been proposed as an alternative to the common site‐specific integration utilizing the homology‐directed repair (HDR). Suzuki and colleagues demonstrated that HITI was more efficient for DNA integration in dividing HEK293T cells and non‐dividing mouse primary neurons than method based on HDR. We compared targeted integration of the GFP reporter gene into the CCR5 gene region of HT1080 cells using HDR and HITI. A double‐stranded break in the CCR5 locus was introduced by spCas9 +gRNA (corresponded to 99‐118 bp of CDS) delivered to the cells by plasmid transfection. On the next day donor DNA was delivered using AAV vector which contained EGFP expression cassette either flanked by CCR5 locus homology arms for HDR or by gRNA target sequences for HITI. The percent of EGFP positive cells was measured on the 21‐st day after AAV transduction, when DNA that failed to integrate was diluted out during cell division. In our experiment the efficiency of targeted integration mediated by HDR was higher than the one mediated by HITI – 33.6% and 12.7%, respectively. Additionally, the HITI method was tested using the Minicircle plasmid for the delivery of donor DNA, as was described in Suzuki at all. However, the frequency of knockin was still lower than that mediated by HDR with AAV delivery (23.19% vs 33.6%). In conclusion it has been found that in HT1080 cells HDR provides a higher frequency of transgene integration into the CCR5 locus than HITI.
Efficient
In utero
RNA lipid nanoparticle‐mediated gene editing for correction of type 1 tyrosinemia without the AAV‐associated genotoxicity
1: Children's Hospital of Philadelphia 2: University of Pennsylvania
Adeno‐associated viral vectors (AAV) and lipid nanoparticles (LNP) have emerged as a great potential for the treatment of genetic diseases using gene editing approaches, such as CRISPR‐Cas9 technology. Safe and efficient in utero delivery of gene editing molecules is critical for the success of these treatment strategies. In this study, we compare LNP and AAV mediated in utero delivery of CRISPR‐Cas9 molecules and then, evaluate AAV‐related genotoxic events. Inhibition and knockout of HPD enzyme have been reported as therapeutically beneficial for treating type 1 tyrosinemia. We separately administered, via the vitelline vein, AAVs (AAV.Hpd.Cas9) or LNPs (LNP.Hpd.Cas9) harboring Cas9 mRNA and Hpd‐targeting crRNA‐tracrRNA, to Fah‐/‐ E16 fetuses. The livers of injected and untreated control mice were harvested two weeks after birth and were evaluated for gene editing and AAV integration events.
The on‐target editing at 2 weeks of age, without any survival advantage due to NTBC removal, by AAV.Hpd.Cas9 and LNP.Hpd.Cas9 was 37.76 ± 6.67% and 24.53 ± 3.98% respectively. Moreover, we found several AAV‐associated integration events in AAV.Hpd.Cas9 injected samples, especially near the target site of Hpd targeting sgRNA. However, there was no evidence of such integration events in LNP.HPD.Cas9 treated livers.
This study demonstrates that LNP‐mediated in utero gene editing can be a safe platform for in utero gene editing, without any AAV‐associated genotoxic events. Moreover, this opens up implications for the development of safer delivery strategies of gene editing molecules for the treatment of genetic diseases other than liver disease, specifically lung, brain, and heart diseases.
Generation of Glutaric Aciduria type‐I cellular models and phenotype rescue by GCDH targeted gene edition
1: IDIBAPS 2: CIBERER 3: Hospital Clinic Barcelona
Glutaric Aciduria type I (GA‐I) is a rare autosomal recessive disorder caused by the deficiency of the enzyme glutaryl‐CoA dehydrogenase (GCDH). Currently there is neither an efficient therapy that reverses the neurological damage caused by the clinical debut, nor cellular models of the disease. The aim of this work has been to develop cellular models as valuable tools to study the complexity of the disease and to evaluate the potential of gene targeting therapeutic strategies. We generated GCDH‐deficient neuronal cells using CRISPR/Cas9 technology, via NHEJ (SH‐SY5Y‐GCDH‐KO), enabling to explore the neurological features of the disease. In parallel, we generated a prevalent conformational GCDH mutation using HAP1 haploid cells using CRISPR/Cas9, via HDR (HAP1‐GCDH‐Val400Met). Functional studies corroborated that both cellular models recapitulate the main biochemical characteristics of GA‐I patients, showing a loss in the GCDH enzymatic activity and, especially in the KO cell line, an accumulation of toxic metabolites from lysine catabolism. Furthermore, we developed a gene‐targeting strategy to rescue the GCDH activity through targeted insertion of the GCDH gene in the AAVS1 "safe harbor" genomic locus. GCDH targeted gene edition rescued GCDH enzymatic activity, while reduced the accumulation of toxic metabolites in SH‐SY5Y‐GCDH‐KO cells. Our results provide two cellular models of GA‐I that can be important in the study of the molecular pathogenesis of the disease and show their interest as platforms to evaluate the potential benefit of gene‐targeting correction and other therapeutic strategies.
Exon skipping by Polypurine Reverse Hoogsteen hairpins
1: Universidad de Barcelona
Exon skipping strategies are aimed to mediate the elimination of mutated exons containing premature stop codons and to restore the reading frame of the affected protein. We explored the capability of polypurine reverse Hoogsteen hairpins (PPRHs) to cause exon skipping at the genomic level in a cell line carrying a dihydrofolate reductase (dhfr) minigene pD22 with a duplicated exon 2 that causes a frameshift abolishing DHFR activity.
Three putative PPRH target regions were identified in the pD22 minigene, localized in the promoter, exon 3 and exon 6, respectively. Each PPRH core was extended at its 5’ end by a sequence tail homologous to 20 nt upstream and/or 20 nt downstream of a PstI restriction site, which corresponded to the insertion site of the additional exon 2. The different Editing‐PPRHs were transfected in NB6 cells followed by incubation in DHFR selective medium lacking hypoxanthine and thymidine. PPRHs carrying the whole homologous tail including both the 20 nt upstream the Pst I site and the 20 nt downstream of this same site originated colonies in selective medium. DNA sequencing results proved that the dhfr sequence corresponded to the wild type with just one copy of exon 2. The skipping of the additional exon was confirmed at the mRNA level, DHFR protein was restored, and it showed high levels of DHFR activity.
We conclude that editing‐PPRHs are able to cause exon skipping at the DNA level and could be applied as a possible therapeutic tool for genetic diseases such as Duchenne muscular dystrophy.
Towards clinical translation of hematopoietic cell gene editing for treating Hyper‐IgM Type 1
1: San Raffaele Telethon Institute for Gene Therapy (HSR‐TIGET), Milan, Italy 2: Vita‐Salute San Raffaele University, Milan, Italy 3: CNR, Milan, Italy 4: Fondazione Unimi, Milan, Italy
In HIGM1, mutations in CD40L impair B‐cell activation and Ig class‐switching induced by activated CD4 T‐cells. We developed an editing strategy to insert a corrective CD40L cDNA in the first intron of the endogenous gene, restoring physiologic expression, conditional to targeted insertion, avoiding constitutive CD40L expression, which leads to lymphoproliferation/lymphoma. By applying this strategy to T‐cells, we obtained ∼40% correction and restored a regulated, although partial, CD40L expression, sufficient to provide contact‐dependent activation of B‐cells. To increase the purity, we coupled the corrective cDNA with a selector. Enriched T‐cells engrafted in NSG mice; furthermore the new cassette fully rescued the level of CD40L expression. In human HSCs we reached 15‐30% editing. We then modelled T‐cell and HSC therapy by infusing WT murine cells in HIGM1 mice. Administration of WT T‐cells alone or admixed with HIGM1 T‐cells into HIGM1 mice resulted in partial rescue of antigen‐specific IgG response; mice transplanted with functional cells alone had the highest response, indicating competition between WT and HIGM1 cells. Infusion of T‐cells from mice pre‐exposed to P. murina, mimicking pneumocystis pneumonia, protected recipient mice from the infection. Intriguingly, transplanting 10‐25% WT HSC along with HIGM1 ones – mirroring the editing efficiencies achieved in human HSC ‐ rescued antigen‐specific IgG response and protected from pathogen comparably to T‐cell therapy. T‐cell therapy does not require myeloablation and provides significant clinical advantages; therefore, we started assessing GMP‐compliant reagents and protocols for T‐cell editing and developing a scalable manufacturing process.
CRISPR/Cas9 correction of Finnish gyrate atrophy mutation in patient‐derived iPSCs
1: University of Helsinki 2: Helsinki University Hospital
Hyperornithinaemia with gyrate atrophy of the choroid and retina (HOGA) is a severe disease of autosomal recessive inheritance. It causes muscular degeneration and retinochoroidal atrophy, which progresses to blindness in adulthood. The current treatment strongly restricts dietary protein and includes creatine supplementation to manage the symptoms. This delays the progression of the disease but hardly results curative. HOGA arises from loss‐of‐function mutations in the ornithine aminotransferase (OAT) gene, leading to highly increased levels of circulating ornithine and subsequent imbalance of related metabolites. The most common mutation, present in homozygosity in nearly one‐third of all the patients worldwide, is the Finnish founder mutation c.1205T>C (p.Leu402Pro). Here, we have corrected this loss‐of‐function OAT mutation in patient‐derived induced pluripotent stem cells (iPSCs) using CRISPR/Cas9, establishing a novel model for the Finnish disease. The correction restored OAT expression in stem cells. Consequently, we observed a decrease in the disease's characteristic elevated ornithine levels and normalisation of related pathways. These results show an efficient recovery of OAT function in iPSC, encouraging further mechanical research and exploration of novel long‐term therapies for the disease.
Modeling rare monogenic disorders as a translational tool: CRISPR/CAS9 editing of Pompe Mutations
I Ramos‐Hernández1 A Aguilar‐González11 E Barriocanal1 P Muñoz1 F Martín1
1: GENyO‐ Centro de Genomica e Investigacion Oncologica: Pfizer / Universidad de Granada / Junta de Andalucia 2: FIBAO‐Fundación para la investigación biomédica de Andalucía oriental – Alonso Otero
Despite extensive efforts on developing efficient therapies for rare disorders, most of these patients can only rely upon non‐curative palliative treatments. Pompe Disease (PD) patients suffer a rare monogenic metabolic disorder caused by insufficient GAA activity, resulting in progressive dystonia. A wide variety of mutations in the GAA gene are linked to a vast diversity of phenotypes from severe Infantile cases (IOPD) to milder late onset adult patients (LOPD). Current treatments are utterly unsatisfactory, yet new‐drug discovery is hampered by scarce sample availability and limited disease models.
Our objective is to apply gold‐standard gene editing technologies to reproduce PD‐causing mutations described in human patients. Hence, we engineered several CRISPR/CAS9 guides with a discrete DNA repair pattern to accurately reproduce relevant IOPD mutations. Our system allows for rapid gene editing on clinically relevant primary tissues (demonstrated on HSPCs) while preserving viability and engraftment features.
We demonstrated that CRISPR/CAS9 efficiently reproduce specific PD mutations with high accuracy. Moreover, we evaluated these PD‐edited cells as disease model for testing standard and experimental therapies for the rescue of GAA activity. Noteworthy, our editing resulted in efficient PD modelling and could be exploited for the generation of humanized mouse PD models, allowing for a more informative translational platform for forthcoming PD drugs.
These results set the foundation of a rapid and upscalable system to accelerate drug discovery and translation in PD, but also, our design can be easily carbon‐copied to address other rare monogenic and metabolic disorders bypassing limiting access to rare diseases patients' samples.
Genome editing with TALEN, CRISPR Cas9 and Cas12a and AAV6 homology donor restores T cell function for XLP
1: UCL Institute of Child Health 2: University Medical Center Freiburg 3: Garvan Institute of Medical Research
X‐linked lymphoproliferative disease (XLP) is a rare inherited immune disorder that arises due to mutations or deletions in the SH2D1A gene, which encodes an intracellular adaptor protein SAP. In the absence of SAP, the immune system is dysregulated, leading to a spectrum of clinical manifestations associated with significant mortality and morbidity. A haematopoietic stem cell transplant (HSCT) can be curative, but outcomes are limited in the mismatched donor setting. This unmet clinical need could be fulfilled by an autologous gene‐corrected HSC therapy, however, SAP has a tightly controlled expression profile ‐ limited to NK, NKT, and T cells ‐ that is challenging to replicate using lentiviral vector technology. Genome editing offers an alternative approach that maintains more of the endogenous regulatory elements that govern SAP expression, potentially providing a safer therapy. We have developed a gene editing platform in T cells, comparing the ability of TALEN, CRISPR/Cas9 and Cas12a nucleases coupled with a homology donor in AAV6 format to drive targeted insertion of a SAP cDNA at the SH2D1a locus, achieving over 45% gene insertion across multiple donors, with minimal off‐target nuclease activity. Corrected T cells from XLP patients demonstrated SAP protein expression and SAP‐dependent immune functions restored to the level of healthy controls, including T follicular helper cell signalling, sensitivity to re‐stimulation induced cell death (RICD), and cytotoxicity to EBV + B cell lines. To provide a potentially life‐long cure and correction of SAP‐dependent function in all affected immune lineages, we are now developing an HSC gene editing strategy.
Optimizing genome editing approaches for safe transgene expression in monogenic disorders
1: GENyO‐ Centro de Genomica e Investigacion Oncologica: Pfizer / Universidad de Granada / Junta de Andalucia
Most patients suffering from motor and neurodegenerative monogenic disorders as Pompe Disease (PD), have to hold for non‐curative treatments, slowing down disease progression at best. The CX3CR1 locus is known to be active in Haematopoietically Stem Cells (HSCs) and high expressed on their myeloid progeny. It is, therefore, an attractive locus for hosting therapeutic genes to be expressed in a systemic way. Our objective is to implement advanced gene editing technologies to achieve an endogenous rescue of the GAA activity. Hence, we engineered several donors based on CRISPR‐Cas9 system targeting CX3CR1 locus to characterize which DNA repair mechanism contributes to a safer and more efficient addition of therapeutic GAA cassettes on HSCs. We targeted CX3CR1 and CCR5, as control safe‐harbour locus, and evaluate both loci regarding toxicity, efficiency and DNA repair fidelity. High targeting (achieving more than 90% of edition) was obtained on HSCs without toxicity. Host loci expression was not compromised and donor reporter expression was maintained for both safe‐harbours. Besides, we investigated several donors including sequences promoting Homologous Direct Recombination (HDR), Non‐Homologous End Joining (NHEJ) or a combination of both to increase knock‐in efficacy in HSCs. Our aim is to use this specific cassette insertion into a safe Stem/Myeloid locus applying gene therapy strategies for cross‐correction of PD. These results open the possibility to apply this blueprint to other metabolic monogenic diseases in which differentiated HSCs could reach damaged target tissues to deliver locally therapeutic genes
Folate‐targeted Cas9/sgRNA Ribonucleoprotein Delivery for Dual Immune Checkpoints Disruption in Colorectal Carcinoma
1: Department of Pharmacy and Center for NanoScience (CeNS), Ludwig‐Maximilians‐Universität, Munich, Germany. 2: Faculty of Life Sciences, University of Vienna, Vienna, Austria.
The CRISPR‐Cas9 system, as an RNA‐guided genome editing tool, is triggering a revolutionary change in cancer immunotherapy with its versatility and ease of use. Immune checkpoint blockade (ICB) as a form of cancer immunotherapy can release inherent limits on the activation of T cell effectors and revert tumor‐induced immune suppression. However, only a portion of patients can respond to this treatment. To overcome this limitation, a folate‐targeted delivery vector was developed for the effective intracellular transport of Cas9/sgRNA ribonucleoproteins (RNP) anddual immune checkpoint gene knockout. The lipopeptide‐based carriers are decorated with folic acid (FolA) and polyethylene glycol (PEG) to specifically deliver RNP in folate receptor α overexpressing cancer cells. In vitro studies showed that FolA‐modified formulations significantly enhance the cellular uptake and editing efficiency of RNP in HeLa and colon carcinoma CT26 cells compared to PEG‐ and unmodified formulations. Due to the improvement by FolA‐modification, the genome editing system can induce effective PD‐L1 (CD274) and PVR (CD155) knockout in CT26 cells simultaneously. Notably, PVR knockout in CT26 cells was not only intended to revert tumor immunosuppression, but also mediated strong direct tumor cell killing. We expect that the established receptor‐targeted Cas9/sgRNA RNP delivery system can provide a facile strategy to increase the efficacy of ICB therapy by combined immune checkpoints disruption.
Base and prime editing as potential gene correction therapy for cystic fibrosis
1: KU Leuven 2: UZ Leuven
Cystic Fibrosis (CF) is Europe's most common monogenic and lethal disease affecting >85.000 people worldwide. CF is caused by recessive mutations in the CFTR (Cystic Fibrosis transmembrane conductance regulator) gene, which codes for the CFTR protein, a chloride and bicarbonate channel in the apical membrane of secretory epithelia. The defective CFTR channel causes severe dysfunction in multiple organs, although most of morbidity and mortality can be attributed to progressive lung disease. Following the promising developments in the gene editing field, we applied base and prime editing to two drug‐refractory CF‐causing CFTR mutations, G85E and L227R. For L227R, we were able to design and optimize a prime editing (PE) strategy capable of correcting an integrated CFTR cDNA with ∼20% efficiency in HEK293T cells. This genomic correction correlated with an equal number of corrected cells expressing plasma membrane localized CFTR (immunocytochemistry, flow cytometry), as well as with a rescue in ion channel function (assessed by halide‐sensitive YFP quenching). We also applied PE, as well as adenine base editing (ABE) to G85E. We were however not able to achieve any significant gene correction by either ABE or PE, indicating target specific determinants of correctional activity for both enzymes. To validate our approach in a more relevant and translational model, we delivered the PE enzyme, together with its required guides to patient‐derived intestinal organoids via lentiviral vectors. This resulted in a correction of the endogenous CFTR gene and a corresponding functional recovery of CFTR activity as measured by forskolin‐induced organoid swelling.
Development of translational activation tool using designer PPR (Pentatricopeptide Repeat) proteins
1: University of Kyushu
Gene editing technology opens new insight of basic and applied biology. Recent advances enable to target RNA in addition to DNA. Pentatricopeptide Repeat (PPR) protein is an attractive platform for RNA targeting technology, due to the programmable RNA binding selectivity. In this study, the designer PPR protein was utilized to develop translational activation tool, by fusing the translation initiation factor eIF4G. c‐myc and p53 mRNAs were chosen as target molecules for investigation of the utility of PPR‐based translational activation tool. Because, the translation activation can be evaluated by activation and inhibition of cell proliferation by targeting c‐myc and p53, respectively. Sixty‐four and seventeen PPR proteins of 18 bases recognition were designed to cover the 5'UTR of c‐myc and p53 mRNAs, respectively. The plasmid encoding PPR‐eIF4G was transiently transfected into mammalian cultured cells, and then amount of the proteins and RNA, and the cell growth were analyzed. As the results, the cells expressing PPR‐eIF4G that targets the middle region of 5'UTR of c‐myc or p53 increased expression of c‐myc or p53 proteins, without changing their RNA amounts. The increased expression of c‐myc induced activation of cell growth, and the increased expression of p53 lead to inhibition of cell growth, as expected. Moreover, the effects of PPR‐eIF4G were dependent on both the target position and the presence of eIF4G. This study demonstrated that PPR‐eIF4G is able to activate translation of endogenous mRNAs and control the following cell fate, strengthened the future therapeutic application.
Polymer Nanoparticle Delivery of Firefly Luciferase mRNA Shows Distribution of Bioluminescence in the Brain
1: GenEdit, Inc.
Developing targeted gene therapies for clinical CNS diseases which are specific, safe, and effective have been an arduous area of research. While they can be effective for certain tissue types, clinically available adeno‐associated viruses (AAV's) have limitations of acquired immunity, off‐target transfection, and genome integration. Our proprietary polymer nanoparticles offer the potential for a non‐viral delivery system which can be engineered for specificity to target specific structures within the CNS. Our initial effort has demonstrated efficacy of intrathecal administration of our PNPs into the cerebrospinal fluid to deliver mRNA cargo to the brain with limited penetration into the parenchyma. A recent screening of a new library of nanoparticles administered via IT injection have shown a broader distribution of bioluminescence across the brain, suggesting the potential for engineering PNP's which can further penetrate into the parenchyma to treat diseases effecting deeper brain structures. Further research is required to determine which properties are most critical to achieve a more targeted distribution to those deeper structures within the brain.
Preclinical studies for a hematopoietic stem cell gene editing platform to treat Wiskott‐Aldrich Syndrome.
1: UCL Institute of Child Health 2: NIHR Great Ormond Street Hospital Biomedical Research Centre, London, U.K
Wiskott‐Aldrich syndrome (WAS) is an X‐linked recessive disease caused by mutations in the WAS gene resulting in the development of microthrombocytopenia, eczema and increased susceptibility to infections, tumors and autoimmune disorders. Allogeneic stem cell transplantation is the treatment of choice for WAS patients, however, it offers a selective benefit as a certain percentage of patients cannot find HLA matched donors. Besides HSCT, lentiviral gene therapy has proved effective, although full immune and platelet reconstitution and physiological WAS protein expression is not always achieved in treated patients
Interaction of polyelectrolytes with DNA for gene delivery application
1: Kazan Federal University
The concept of gene therapy developed for the past two decades and conclude that for the treatment of various diseases it is necessary to correct the genome of the patient. Growing popularity among the global medicinal community, will allow gene therapy has become an attractive market for companies and investors. The target of gene therapy may be hereditary, viral, autoimmune or oncological diseases. The main barrier on the way of DNA to the cell nucleus is the double nuclear envelope. Only small molecules can pass through the nuclear pore complex by passive diffusion. It has been found that about 50% of the injected DNA degrades in the cytosol as early as 1‐2 hours after administration. Obviously, finding a synthetic polycationic carrier that meets all these requirements is an extremely difficult task, and therefore a search for factors that make it possible to increase the efficiency of transfection of the polymer carrier is of great interest. Here we report the study of in vitro cytotoxicity of a variety water‐soluble polycations considered as drug delivery systems and non‐viral vectors for gene transfer in 2D and 3D cell cultures. In this study, we determined using atomic force microscopy technique the structures of polyelectrolyte‐DNA complexes, which used to form due to electrostatic interactions. This work was supported by Russian Foundation of Basic Reserarch (grant 20‐015‐00353 A).
In vitro therapeutic evaluation of the lentiviral gene therapy vector encoding vasoactive intestinal peptide for retinal dystrophies
1: The Department of Gene and Cell Therapy, Akdeniz University Faculty of Medicine, Antalya, 07058, Turkey 2: GenomCare Ltd. Co., Akdeniz University Technopark, Antalya, 07058, Turkey 3: The Department of Ophthalmology, Akdeniz University Faculty of Medicine, Antalya, 07058, Turkey 4: Intergen Center for Genetic Diseases, Ankara, 06510, Turkey
Retinal dystrophies result from the degeneration of photoreceptor cells leading to blindness. Hypoxic conditions and oxidative stress due to the excessive and long‐term microglial activation lead to cytokine production and inflammatory responses in the retina. By this token, gene therapy approaches hold promise to stop or prevent photoreceptor degeneration. Accordingly, we aimed to study neuroprotective and anti‐inflammatory effects of Vasoactive Intestinal Peptide (VIP) using gene delivery. To accomplish this, we first constructed a transfer plasmid carrying VIP encoding gene (pLentiVIP) using Gateway cloning technology. Transient transfection of the transfer and packaging plasmids into 293T cells via CaPO4 transfection resulted in the production of an HIV based third generation lentiviral vector (LentiVIP). The transduction efficiencies of the LentiVIP in Human Microglial (HMC3) and Human Retinal Pigment Epithelial (ARPE‐19) cell lines were determined by ELISA assay. According to MTT assay results, VIP expression decreased cell death induced by a hypoxia mimicking agent CoCl2. Based on immunofluorescence staining, CoCl2‐induced HIF‐1α expression levels were reduced by LentiVIP transduction. Lastly, exogenous VIP expression suppressed CoCl2‐induced microglial activation and proinflammatory cytokine release (TNFα and IL‐6). TUBİTAK‐218S543
Combining distinct phenotypes of AAV capsid to simultaneously target and detarget specific tissues in mouse models
1: Affinia Therapeutics 2: Grousbeck Gene Therapy Center, Mass Eye and Ear 3: Harvard Medical School
AAVs that simultaneously target specific tissues while detargeting others could have enormous potential. To this end, we sought to understand the effect of combining specific AAV variations that target muscle and simultaneously detarget liver. Library screens using ancestral sequence reconstruction identified a single amino acid change in the Anc80 library that resulted in distinct groups of liver tropic or liver detargeted AAVs. AAVmod1, which applied this change to AAV9, showed the same liver detargeting phenotype. We developed AAVmod1.5 by inserting a peptide into AAV9 to enhance muscle targeting and AAVmod2 by combining the liver detargeting and muscle targeting modifications. We evaluated AAV9, AAVmod1, AAVmod1.5, and AAVmod2 carrying CAG.GFP administered intravenously in C57BL/6 mice. Twenty‐eight days post administration we found that, compared to AAV9, the AAVmod1 vector showed decreased liver expression and no increase in muscle expression; AAVmod1.5 showed similar expression to AAV9 in liver with increased muscle expression; and AAVmod2 showed decreased liver expression and increased muscle expression. A comparison of AAVmod1 and AAVmod2 in BALB/c mice showed that the AAVmod2 vector had no increase in liver tropism, with higher expression than AAVmod1 in quadriceps; AAVmod2 showed similar enhancement in heart, triceps surae, and diaphragm. There were no differences between AAVmod1 and AAVmod2 in spleen or spinal cord. These data show that we can modify AAVs to both detarget liver and target muscle in mouse models, supporting the idea that designed AAVs can simultaneously target and detarget specific tissues. It will be important to confirm these findings in non‐human primates.
A first‐in‐human, phase 1, open‐label trial of FX201, an intra‐articular, helper‐dependent adenoviral gene therapy for osteoarthritis – preliminary evaluation of clinical activity in 8 patients from the mid‐dose cohort of a single ascending dose study design
1: Altoona Center for Clinical Research 2: Flexion Therapeutics, Inc.
FX201 is a helper‐dependent adenovirus serotype 5 expressing the anti‐inflammatory protein interleukin‐1 receptor antagonist (IL‐1Ra) in clinical development as intra‐articular (IA) gene therapy for osteoarthritis (OA); it is designed to produce IL‐1Ra in the presence of inflammation via a nuclear factor‐kappa B‐responsive promoter. Following positive preclinical experience with FX201, we initiated a single ascending dose trial (NCT04119687) of FX201 in patients with knee OA. Here we report on the mid‐dose cohort through 24 weeks. Eight adult patients with moderate‐to‐severe knee OA (Western Ontario and McMaster Universities Osteoarthritis Index [WOMAC] pain score ≥5.0 and ≤9.0 [0‐10 numeric rating scale], Kellgren‐Lawrence Grade 2‐4 radiographic severity), and prior failure of ≥2 other OA treatments were enrolled. FX201 at a dose of 1.4E11 genome copies was administered to the index knee via ultrasound‐ guided IA injection. Safety and tolerability were assessed. Clinical activity of FX201 was assessed by measuring change in pain (WOMAC‐A) and function (Knee Injury and Osteoarthritis Outcome Score‐Activities in daily living [KOOS‐ADL]) and substantial improvement was evaluated using the Initiative on Methods Measurement, and Pain Assessment in Clinical Trials (IMMPACT) criteria. FX201 was generally well tolerated in the 8 patients. Three Grade 2 adverse index knee events managed conservatively and one Grade 3 adverse event treated successfully with an intra‐articular steroid injection were observed. 7 of 8 patients showed reduction in pain (WOMAC‐A) and functional improvement (KOOS‐ADL) at 24 weeks following administration of mid‐dose FX201, 4 of whom showed substantial improvement by IMMPACT criteria for Weeks 8‐24.
Combinatorial AAV Capsid Library Enables Multidimensional Study of Vector Biology
1: Grousbeck Gene Therapy Center, Schepens Eye Research Institute, Massachusetts Eye and Ear, Boston, MA, USA 2: Ocular Genomics Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA 3: The Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA 4: Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, USA 5: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, 6: Paul F. Glenn Center for the Biology of Aging, Harvard Medical School, Boston, MA 02115, USA 7: Joslin Diabetes Center, Boston, MA, 02215
Gene therapy is emerging as a modality in 21st century medicine. Adeno‐associated viral (AAV) gene transfer is a leading technology to achieve efficient and durable expression of a therapeutic transgene. However, the structural complexity of the capsid has constrained efforts to engineer the particle towards improved clinical safety and efficacy. In this work, we generated a curated library of 2048 barcoded AAVs with mutations across a variety of functionally relevant motifs. We then screened this library in vitro and in vivo in mice and nonhuman primates, enabling a broad, multi‐parametric assessment of every vector within the library. Among the results, we note a single residue which, when mutated, profoundly reduces transduction in both murine and non‐human‐primate liver. We validate this finding by traditional clonal approaches, confirming that the mutation is associated with the liver‐detargeting phenotype first observed in the library setting (reducing expression by over 500‐fold in mice) while only modestly reducing expression in the mouse quadricep (2.93‐fold). We demonstrate that this liver‐detargeting mutation can be grafted into AAV9 to reduce transgene expression in the mouse liver (also over 500‐fold) while only modestly reducing expression in skeletal muscle and brain. Furthermore, we graft the liver‐targeting mutation into AAV3B, increasing the observed expression in mouse liver by more than 20‐fold. We then conducted a structural meta‐analysis and present a hypothesis that AAV‐AAVR interactions may underly this (and other published) liver‐detargeting vector phenotypes.
Splicing modulation therapy for a variety of ABCA4 mutations underlying Stargardt disease
1: Department of Pediatrics, Radboud University Medical Center 2: Department of Human Genetics, Radboud University Medical Center 3: Center for Medical Genetics, Ghent University and Ghent University Hospital 4: UCL Institute of Ophthalmology 5: Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center 6: Radboud Institute for Molecular Life Sciences, Radboud University Medical Center
Our proof‐of‐concept work on the correction of a recurrent splicing defect underlying CEP290‐associated Leber congenital amaurosis (LCA) has led to the initiation of a clinical trial, with recently published positive results, demonstrating safety and efficacy upon intraocular antisense oligonucleotide (AON) delivery in LCA patients. Here, we expand the use of AONs for the treatment of another subtype of inherited retinal disease caused by mutations in the ABCA4 gene: Stargardt disease (STGD1).
We established a panel of ABCA4 midigenes encompassing the complete ABCA4 gene, with which the effect of any ABCA4 variant on pre‐mRNA splicing can readily be assessed in vitro. For variants resulting in aberrant ABCA4 splicing, AONs were designed and co‐transfected with the midigenes to assess their correction efficacy. For several variants, patient‐derived fibroblasts and/or induced pluripotent stem cell (iPSC)‐derived photoreceptor progenitor cells (PPCs) were treated with the AONs, and rescue efficacy was determined by RT‐PCR analysis.
We identified more than twenty different ABCA4 variants that affect pre‐mRNA splicing. The majority of variants reside in introns, leading to a pseudoexon insertion, in some cases in a retina‐specific manner. Others cause exon exclusion or exon elongation. In all the cases, the splicing defect disrupts the reading frame, creating a premature stop codon. Using AON technology, most of these splicing defects were corrected and the reading frame restored.
Altogether, AONs appear to be an effective and versatile tool to correct different types of splicing defects that are caused by ABCA4 mutations but potentially also for other retinal diseases.
Non‐integrative and integrative gene‐based strategies for the treatment of Citrullinemia type I mouse model
1: ICGEB
Citrullinemia type I (CTLN1) is a severe liver monogenic disorder caused by the deficient activity of the ASS1 gene, coding for the third enzyme of the urea cycle, leading to toxic accumulation of blood ammonia and citrulline. The neonatal form of the disease shows the first signs of hyperammonemia right after birth and, if not treated, can lead to encephalopathy and death.
In our work, we developed a gene targeting strategy by introducing the hASS1 cDNA into the albumin locus of the CTLN1 mouse model (ASS1fold/fold) using the Crisp/Cas9 platform to enhance the recombination rate. After a single injection at day 2, mice had a decrease in citrulline and ammonia levels lasting up to 5 months of age (time of sacrifice), although not enough to reach healthy wild‐type values, completely rescuing mortality in the ASS1fold/fold animals. We then treated juvenile (P30) mice with an episomal hASS1 AAV8 vector or with the gene targeting strategy applied previously to neonates. Mice treated with the non‐integrative gene therapy vector completely rescued the diseased phenotype, reducing plasma ammonia and citrulline to wild‐type values up to 3 months post‐administration. Juvenile mice treated with gene targeting strategies had an improvement in their lifespan.
Altogether, our data demonstrate that non‐integrative gene therapy may be successfully applied to CTLN1 juvenile mice, but not to neonate animals, ensuring a stable therapeutic effect. The treatment of neonatal mice with integrative approaches is effective. However, further improvements in the targeting rate to completely correct the diseased phenotype are required.
Gene therapy for IL‐1‐mediated systemic autoinflammatory diseases
1: San Raffaele Telethon Institute for Gene Therapy (SR TIGET), IRCCS San Raffaele Scientific Institute, Milan, Italy 2: Vita‐Salute San Raffaele University, Milan, Italy 3: Pediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy
Systemic autoinflammatory diseases (SAIDs) are rare but severe conditions caused by a dysregulation of the innate immune system. The leading group of SAIDs comprises hereditary periodic fever syndromes characterized by interleukin‐1β (IL‐1β)‐mediated systemic inflammation. Although IL‐1 blocking biologics have shown a clinical benefit in patients, they are not curative and can lose their efficacy in a consistent cohort of patients due to poor tissue biodistribution and low stability. We, therefore, envisaged a lentiviral vector (LV)‐mediated gene therapy approach based on transplantation of hematopoietic stem‐progenitor cells (HSPCs) expressing the human IL‐1 receptor antagonist (IL‐1RA). Toxicity studies demonstrated that LV‐IL‐1RA transduction did not alter the clonogenic potential of mouse lineage‐negative HSPCs in vitro and in vivo. IL‐1RA‐expressing cells proliferated and differentiated similarly to HSPCs transduced with a control GFP vector. Bone marrow chimeras reconstituted with IL‐1RA‐ and GFP‐transduced HSPCs showed a similar immune reconstitution of lineage‐committed cells over time. IL‐1RA chimeric mice released high and stable amounts of human IL‐1RA in the plasma. Notably, LV‐derived IL‐1RA could effectively suppress IL‐1‐mediated inflammation by diminishing neutrophil accumulation following monosodium urate crystals treatment in vivo. Our findings demonstrate that ex vivo IL‐1RA gene therapy may be a feasible and durable treatment to reduce IL‐1‐mediated events in patients with SAIDs refractory to conventional therapy. On‐going studies investigate the efficacy of this approach in suppressing the exacerbated IL‐1 activity in patient's cells and a mouse model recapitulating SAIDs phenotype.
Targeting Therapeutic Proteins to Alpha Granules for Platelet‐mediated Transport and Delivery
V MA Woods2
1: Paul‐Ehrlich‐Institute 2: Hannover Medical School
Platelets are anucleate, discoid shaped blood cells released from megakaryocytes (MK). They regulate hemostasis, support wound healing and are involved in innate immune responses. They fulfill their functions by local secretion of bioactive substances from their storage granules after the activation via specific surface receptors. Alpha granules (αG) are the most abundant granules and therefore of particular interest for a targeted cell therapy approach to store, hide and specifically deliver transgenic proteins. We propose an adaptable platform for targeting transgenic proteins to platelet αG based on third generation self‐inactivating lentiviral vectors. Expression from the murine platelet factor 4 promoter ensured MK‐lineage restricted transgene expression. Fusing the transgene to the short sorting signal peptide (10 aa) of the human cytokine RANTES, promoted specific transport to αG. We prove effective targeting of GFP to αG in murine and human in vitro‐differentiated MKs with ∼70‐80% co‐localization with αG marker proteins (P‐selectin and von Willebrand factor). In murine platelets, GFP was retained in the resting state and secreted after activation with high concentrations (40 ng/ml GFP from ∼5x107 platelets). Next, we generated lentiviral vectors that target IFNα to αG. Targeted IFNα‐expression in vivo did not impair hematopoietic recovery after transplantation. Furthermore, IFNα secretion from activated platelets effectively inhibited measles virus replication in vitro in a dose‐dependent manner up to 92.5 ± 1.2% which is comparable to 10U recombinant IFNα. Our vectors create a platform for numerous applications for cell therapy by utilizing platelets as vehicle to deliver therapeutic proteins.
Towards a clinical trial of gene therapy to target retinitis pigmentosa associated with Usher syndrome type IB
1: Telethon Institute of Genetics and Medicine 2: Federico II University, Department of Translational Medicine 3: ReiThera srl 4: Federico II University, Department of Advanced Biomedicine
Usher syndrome type IB (USHIB), due to biallelic mutations in the MYO7A gene, is a severe form of USH, the most common inherited combination of retinitis pigmentosa and deafness.
Due to AAV packaging limitations, to transfer the large MYO7A gene to the retina, we have developed a dual adeno‐associated viral vector (dual AAV.MYO7A) which reconstitute full‐length MYO7A to therapeutic levels in the USHIB mouse model. In view of clinical translation, we performed a GLP non‐clinical study to assess the safety of dual AAV.MYO7A.
A 13‐weeks study was conducted in non‐human primates (NHPs) using a GMP‐like dual AAV.MYO7A lot. NHPs received a single sub‐retinal injection of either formulation buffer as control or dual AAV.MYO7A at one of two doses corresponding to 1.6X and 4.3X the highest dose proposed for the clinical trial.
Sub‐retinal injection of formulation buffer was well‐tolerated. Dual AAV.MYO7A‐treated eyes showed retinal alterations that were dose‐dependent in terms of severity and improved at the end of the study compared to the interim evaluation. Consistently, electroretinography showed a dose‐dependent reduction of retinal function, which improved over time with some eyes in the low dose‐group falling in the range of normality at the end of the study.
In conclusion, our data showed that sub‐retinal administration of dual AAV.MYO7A at the low dose results in a mild toxicity which improved over time. Therefore, we believe that this safety study, once completed by biodistribution, expression and immunology data, will pave the way for a gene therapy clinical trial for USHIB retinitis pigmentosa.
Gene therapy‐mAb platform targets Complement Protein 5 using AAVHSCs
1: Homology Medicines, Inc.
Many diseases necessitate chronic dosing of therapeutic monoclonal antibodies (mAbs). Patients present with compliance‐fatigue, discomfort and repeat‐dosing related complications such as infusion‐related reactions, and in minor instances, incomplete disease control during mAb troughs. A single dose of AAV‐mediated gene therapy delivering vectorized mAb may mitigate these complications.
Establishing Homology's gene therapy‐mAb (GTx‐mAb) platform, we designed an AAV‐mediated anti‐C5 antibody for the treatment of the complement‐related disorder paroxysmal nocturnal hemoglobinuria. Vector designs expressing C5mAb using liver‐specific promoters were evaluated in vivo and in vitro and delivered by AAVHSCs (AAV capsids isolated from human hematopoietic stem cells).
We previously demonstrated that a single dose of GTx‐mAb expressing anti‐C5 antibody resulted in robust, sustained, functional antibody levels in NOD‐SCID (C5‐deficient) and in FRG® liver‐humanized mice (reconstituted with human hepatocytes and expressing humanC5) for 26 and 12 weeks, respectively. Dose response studies with our best design achieved IgG levels >20 mg/mL at the highest dose examined (Sharma et al, ASGCT‐2021).
Here, we have focused on design optimization around coding and non‐coding elements. Fully assembled mAbs were highly expressed in vitro in plasmid‐transfected hepatoma cells and AAVHSC‐transduced primary hepatocytes, and in vivo, in NOD‐SCID mice. We achieved a further 3‐fold increase in IgG expression at steady state with our lead construct, reflected in higher serum IgG/liver vector genome ratios.
In summary, we demonstrated that one‐time treatment with an AAVHSC construct expressing a full anti‐C5 antibody results in sustained therapeutic levels of serum mAbs that may alleviate the inconvenience and potential side‐effects of repeat dosing.
In vivo translation of adeno‐associated vectors for the treatment of inner ear disorders
1: Sensorion
Gene therapy emerges as a novel viable strategy for congenital non‐syndromic deafness cases with genetic causes, that represent half of hearing disabilities. Adeno‐associated viral vector (AAV) demonstrates efficacy in preclinical models of inner ear disorders. Nevertheless, the broad tropism of the vector and limited volume injectable in the inner ear are existing barriers to the use of AAV. Indeed, the inner ear is constituted by hyperspecialized cells sensitive to both intracellular and extracellular changes. Thus, it is important to restrain protein expression in targeted inner ear cells and identify cell‐specific gene therapy components (AAV capsid, regulatory sequences) efficient at a low dose. Moreover, the surgical procedure allowing good vector distribution across the organ with minimal side effects is challenging but mandatory for clinical translation. Here we describe Sensorion's approaches to develop and translate innovative AAV therapeutic products from bench to bedside. We have established a high throughput AAV candidate screening and selection process, using an ex vivo rodent cochlear explants culture platform combined with fully automatized imaging acquisition of immunostainings (includes 3D and live imaging). In vivo validation in mice and non‐human primates is performed using the round window injection approach and completed by an audiological evaluation on Sensorion's audiology platforms. In vivo safety, tolerability and efficacy data are supported by morphological and immunohistochemical examination, in mice and primates. Our approach allowed us to compare several technics in both models and, select the safest and most efficient surgical procedure and most suitable AAV vector for our human inner ear gene therapies programs.
Enhancing cell specificity of AAV gene therapy vectors by using membrane‐protein specific nanobodies
1: University Medical Center Hamburg‐Eppendorf 2: Heidelberg University Hospital 3: University of Rouen Normandy
The use of adeno‐associated virus (AAV) vectors in gene therapy is limited by high production costs and lack of cell specificity. We aim to enhance the cell specificity of AAV and to reduce the amount of AAV needed to transduce cells of interest using membrane‐protein specific nanobodies. To this end, we pursue two strategies that harness the small size and high solubility of nanobodies which enables simple reformatting into fusion protein: 1) we directly inserted a membrane protein‐specific nanobody into a surface loop of the AAV capsid and 2) we generated bispecific adaptors to enhance binding of AAV to target‐expressing cells by genetically fusing a membrane protein‐specific nanobody to an AAV‐specific nanobody. Using nanobodies against structurally distinct membrane proteins ‐ P2X7, a multispan ion channel; CD38, a single‐span transmembrane protein; and ARTC2.2, a GPI‐anchored ecto‐enzyme ‐ both strategies achieved specific transduction of cells expressing the respective membrane protein with GFP‐encoding AAVs. We show that both strategies can be adapted to a broad range of AAV serotypes. Nanobody‐enhanced targeting of AAV provides a highly efficient approach that may open new avenues for cell and gene therapy.
Supported by DFG No310/13 and the BMBF (COMMUTE).
Single cell RNA‐seq analysis of multipotent mesenchymal stromal cells revealed polarly expressed genes during myofibroblast and adipogenic differentiation
1: Institute for Regenerative Medicine, Lomonosov Moscow State University, Moscow, Russia 2: Faculty of Medicine, Lomonosov Moscow State University, Moscow, Russia
Fibrotic replacement of damaged tissue is an unresolved medical challenge as it provokes multiple severe diseases. The main cellular mechanism of fibrosis pathogenesis is an activation of myofibroblast differentiation and reinforced synthesis of extracellular matrix (ECM). This process may acquire self‐induction manner due to profibrotic microenvironment. Accumulating data suggests that myofibroblast accumulation can be interrupted by induced adipogenic differentiation. We investigated the pattern of genes activated in multipotent mesenchymal stromal cells (MSCs) after the induction of adipogenic or myofibroblast differentiation using single cell RNA high throughput sequencing (scRNA‐seq). Human adipose tissue derived MSCs were cultivated for 4 days with insulin, dexamethasone and IBMX for adipogenic differentiation (MSC_adipo) or in the presence of a key profibrotic factor TGFβ on decellularized ECM produced by fibroblasts for myofibroblast differentiation (MSC_fibr). scRNA‐seq analysis (10xGenomics) was performed to identify the differentially expressed genes in MSCs_fibr vs. control and MSCs_adipo vs. control. Further, we revealed 120 polarly expressed genes induced by myofibroblast‐ (65 genes) and adipogenic (55 genes) differentiation that could be involved in the regulation of MSC response to profibrotic stimuli. These results could be further used to develop approaches to the resolution of fibrosis. The study was supported by RFBR (#21‐315‐70002).
Dissecting bone remodelling mechanisms and hematopoietic stem cell gene therapy impact in Mucopolysaccharidosis type I Hurler bone defects
1: San Raffaele Telethon Insitute for Gene Therapy (HSR‐TIGET) 2: DIMET, University of Milano‐Bicocca, School of Medicine and Surgery, Monza 3: Pediatric Immunohematology and Bone Marrow Transplantation Unit, San Raffaele Scientific Institute 4: Department of Molecular Medicine Sapienza University, Rome 5: Tissue Engineering Department of Biomedicine, University Hospital Basel 6: Cell and Tissue Engineering Laboratory, IRCCS Galeazzi Orthopaedic Institute, Milan 7: Vita‐Salute San Raffaele University Milan 8: Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Milan
Mucopolysaccharidosis type I Hurler (MPSIH) is characterized by deficit of α‐L‐iduronidase enzyme, involved in the degradation of glycosaminoglycans (GAGs), leading to their accumulation. It results in organ dysfunction and skeletal abnormalities. Although hematopoietic stem cell transplantation represents the standard of care, skeletal manifestations progress over time.
Results of the phase I/II gene therapy (GT) clinical trial show peripheral blood supraphysiologic enzyme levels, near normalization of urinary GAGs, and reduced joint stiffness.
As the pathogenesis of bone defects is still not understood and there is still limited knowledge of bone correction after GT, we investigated the role and properties of mesenchymal stromal cells (MSCs), osteoblasts (OBs), chondrocytes and osteoclasts (OCs) of healthy donors and MPSIH patients before and after GT.
OC differentiation and bone resorption were not impaired by IDUA absence. Characterization of MSCs before GT was also not altered, although a trend of reduced CD146+ cell frequency was assessed. MPSIH‐MSCs normally undergo osteogenic and adipogenic differentiation. Although patient‐derived MSCs did not show GAG accumulation, when differentiated into OBs and adipocytes, they showed an increase in intracellular GAGs.
After GT, patient‐derived OCs and their supernatant show supraphysiologic enzyme activity. MPSIH‐MSCs and OBs were able to uptake the missing enzyme when exposed to the supernatant of gene‐corrected OCs, rescuing GAG engulfment found in MPSIH‐OBs. We will investigate the interplay between OBs and OCs with a 3D bone remodelling model and chondrocyte differentiation of MPSIH‐MSCs by exploiting a 3D model of hypertrophic cartilage. In addition, bone biopsies of MPSIH patients will be analysed.
Transient and lentiviral delivery of CRISPR/Cas13 system to knockdown oncogenes
1: Centro Nacional de Investigaciones Oncológicas 2: CIEMAT
Contrary to traditional CRISPR/Cas9, the novel RNA‐targeting CRISPR/Cas13 system does not modify the genes themselves, making the changes reversible and temporally, and spatially more controllable. Therefore, the newly described RNA‐targeting CRISPR system opens up the possibility of a selective knockdown of oncogenes of interest. It has several advantages, it has high knockdown efficiencies (up to 96%) and is not associated with detectable off‐target effects, in contrast to shRNAs. Moreover, CRISPR/Cas13 system can be implemented into adeno‐associated viruses because Cas13 enzymes are among the smallest class 2 CRISPR effectors. Because of all of the above, this CRISPR/Cas13 system can be more robust than current RNA knockdown methods and safer than DNA‐targeting CRISPR systems for future therapeutic applications. Although the potential use of CRISPR/Cas13 system in therapeutic settings would be facilitated through a viral delivery, our first attempts in the use of this CRISPR RNA‐targeting system through lentiviral vectors have shown that efficient knockdown of transcripts is achieved only with very high MOIs. Because of that, we aim to develop a viral delivery of CRISPR/Cas13 system for a high‐throughput RNA knockdown in cancer cell lines, without deleterious effects due to very high MOIs. In this work, we show our preliminary results in the optimization process of the CRISPR/Cas13 for this purpose.
Characterisation of novel naturally occurring site‐specific recombinase systems
1: Medical Systems Biology, Medical Faculty, Technical University Dresden
Precise genome engineering is instrumental for biomedical research and holds great promise for future therapeutic applications. Site‐specific recombinases (SSRs) such as the Cre/loxP system, are valuable tools for genome engineering due to their exceptional ability to mediate excision, integration and inversion of genomic DNA at single nucleotide precision. With recent efforts to change Cre's specificity towards other therapeutically relevant targets by means of directed evolution, it was shown that utilisation of SSRs can be expanded towards medical application. This ever‐increasing need for sophisticated genome engineering tools, is driving efforts to identify novel SSR systems with different properties. In this work, we discover and characterise new naturally occurring Cre‐like SSRs in order to acquire better insight in DNA‐specificity determinants and increase the potential of more focused and faster design of novel therapeutically relevant enzymes. We characterised 8 new SSR systems and tested their activity in bacterial and human cells. Furthermore, we examined novel and previously known SSRs for their ability to cross‐recombine each other's target sites, which enabled us to profile target‐site selectivity among all so far described Cre‐like recombinases. We propose that these novel recombinases are a substantial addition to the genetic engineering toolbox, and by combining them or using them as different starting points for directed‐evolution, we should be able to significantly shorten future development efforts and increase number of potential therapeutically relevant targets in the human genome that can be addressed.
Design, Generation, and Investigation of Novel Dumbbell‐Shaped DNA Minimal Vectors for Suicide Gene Therapy
1: Department of Medicine, Addenbrooke's Hospital, University of Cambridge 2: Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore
Spliceosome‐mediated RNA trans‐splicing allows endogenous labelling of target pre‐mRNAs with an artificial trans‐splicing RNA triggering the formation of chimeric RNAs and proteins. Hence, the technology enables genetic repair on the pre‐mRNA level by exon replacement, labelling of nascent transcripts with fluorescent proteins for diagnostic imaging, and specific reprogramming of cellular functions. We developed a programmable RNA trans‐splicing‐based suicide gene therapy approach that can destroy any cell type based on based on the expression of distinct pre‐mRNA biomarkers. As a medical target, we selected hepatocellular carcinoma (HCC). We explored suicide RNAs that can target multiple HCC biomarkers thereby increasing the overall target concentration as well as the trans‐splicing and suicide activities. To deliver the suicide RNAs, non‐viral redosable dumbbell‐shaped DNA vectors were designed, manufactured, and tested in human tissue culture cells and in three‐dimensional spheroids formed by patient‐derived xenograft cells. Our latest multi‐targeting constructs targeting up to five different HCC pre‐mRNA biomarkers exhibited the highest cell death activity even at 330‐fold reduced GCV concentrations. We found that RNA secondary structure design improves on‐target activity by the selection of multiple, unstructured binding domains which harbour target mismatches. The inclusion of an internal safety domain suppresses off‐target trans‐splicing. In summary, we achieved effective and selective destruction of liver cancer cells without evidence for off‐target cell killing. The conjugation of dumbbell vectors with (GalNAc)3 residues enabled targeted delivery into HCC‐derived human tissue culture cells. This project is highly translational, offering promising perspectives towards suicide gene therapy of cancer or incurable infections with integrating viruses.
Gene silencing of
c‐MYC
and
K‐RAS
oncogenes by targeting G‐quadruplexes using Polypurine Reverse Hoogsteen Hairpins
1: University of Barcelona
G‐quadruplex (G4s) are involved in the transcriptional and translational regulation of cellular genes. We observed that targeting the complementary strand of G4 forming sequences (G4FS) in the 5’‐UTR of the Thymidylate synthase (TYMS) was very effective in reducing the viability, mRNA and protein levels of this target. Here we used PolyPurine Reverse Hoogsteen hairpins (PPRHs) against polypyrimidine tracts in the genes coding for either c‐MYC or K‐RAS complementary to G4FS to promote G4 folding thus increasing gene silencing. PPRHs hairpins are polypurine strands linked by a thymidine loop running in antiparallel orientations. These molecules that are bound by intramolecular Hoogsteen bonds which bind to their polypyrimidine target region of DNA or RNA by Watson‐Crick bonds provoking strand displacement. We searched putative targets of PPRHs in C‐MYC and K‐RAS genes using the Triplex‐forming Oligonucleotide Target Sequence Search software. We selected those forming G4 structures in silico using the QGRS mapper. Subsequently, we designed the corresponding PPRHs against the target genes. These PPRHs reduce the cell viability of neuroblastoma, prostate, breast, and cervix cancer cell lines. For K‐RAS, HpKRAS‐PR‐C was the most effective PPRH in all the cell lines tested and for C‐MYC, HpMYC‐I1‐WT‐T showed a decrease higher than 95% of viability in prostate cancer cells. Our results show that PPRHs targeting G4 regions in oncogenes induce cell death on cancer cells. Therefore, PPRHs can be considered as a new type of molecules to selectively target G4 structures associated with the regulation in different oncogenes that could be used for cancer treatment.
Peptide‐based carriers with anionic coating as a delivery system for gene therapy of uterine leiomyoma.
1: D.O.Ott Research Institute of Obstetrics, Gynecology and Reproductology, Saint‐ Petersburg, Russian Federation 2: Saint‐Petersburg State University, Department of Genetics and Biotechnology, Saint‐Petersburg, Russian Federation
Uterine leiomyoma (UL) is the most common benign tumor of the female reproductive tract. Precise ultrasound localization makes the disease a perfect target for suicide gene therapy in situ. Tumor microenvironment is a formidable barrier for gene delivery due to complicated structure and extracellular matrix (ECM) enzymes accumulation. Coating with polyanions can provide colloidal stability of the polyplexes and resistance to ECM. We developed αvβ3‐integrin‐targeted peptide‐based carriers and anionic peptide coating for DNA delivery into primary UL cells.
Arginine‐rich peptide carriers conjugated with cycloRGD‐ligand were synthesized. Physicochemical properties of DNA complexes with anionic coating were tested. Transfection efficacy of polyplexes with an anionic coating at different charge ratios was studied in the experiments on αvβ3‐integrin‐expressing PANC‐1 cells. Suicidal gene therapy with pPTK‐1 and subsequent ganciclovir treatment was held for primary UL cells at early passages. Injections of polyplexes were carried out into leiomyoma nodes obtained after myomectomy.
Negatively charged coating increased polyplexes stability to polyanions and allowed successful transfection in the presence of serum. Proliferative AlamarBlue and TrypanBlue exclusion tests showed a decline of proliferative activity among primary UL cells transfected with pPTK‐1 carrying complexes in comparison with pCMV‐lacZ transfected cells. A green glow on sections of leiomyoma nodes after injections of polyplexes with the GFP gene encoding plasmid and anionic coating indicates successful transfection.
The study shows that the utilization of peptide carriers modified with cycloRGD‐ligand and negatively charged peptides is a promising approach for the development of DNA delivery systems for UL gene therapy.
The study was supported by RSF grant 21‐15‐00111.
The development of Hutchinson‐Gilford progeria syndrome gene therapy using RNA interference
1: University of Wroclaw
Hutchinson‐Gilford progeria syndrome (HGPS) is a severe genetic disorder leading to premature aging. The most frequent HGPS‐causing mutation c.1824 C > T in LMNA gene encoding lamin A/C leads to splicing alteration, resulting in synthesis of toxic protein lacking 50 amino acids, called progerin. The aim of our study is to develop the gene therapy treatment for progeria. The ultimate goal, the most beneficial for patients, would be the removal of progerin without disturbance of non‐mutated allele encoding for correct lamins A/C as they have an essential role in mammalian cells.
To design a therapy, we decided to use a RNA interference approach. To ensure effective and quantitative drug screening, we created novel cell lines, derived from Hela line, that contain only a single copy of gene coding for fluorescent fusion proteins: GFP‐lamin A and GFP‐progerin. This approach allowed us to obtain an expression level similar to observed in patients' cells. We designed a set of siRNA sequences aimed at mutation site and we selected the molecules specifically and efficiently down‐regulating progerin only, but not lamins A/C. Then we prepared the expression vectors based on microRNA‐9 known to silence lamins A in neurons by interacting with 3’UTR of its transcript. Using RNA modeling in silico, we designed a set of microRNA with a progerin‐specific siRNA in an active site. We have evaluated our sequences with flow cytometry, fluorescence microscopy and western blotting, focusing on their efficiency and specificity to select the most suitable for preclinical models and further delivery to patient's cells.
Novel riboswitches regulate AAV delivered transgene expression in mammals via small molecule inducers
1: Gene Regulation, MeiraGTx, New York, NY 10016, USA 2: Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599‐3290, USA 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
Regulation of proteins or RNA expression is critical to the control of gene therapy, and riboswitches represent a superior class of RNA‐based molecular switches. Riboswitches are widely used in bacteria, but have not been found in mammalian cells. Here, we report that by linking synthetic aptamers or aptamers from natural bacterial riboswitches to an alternative splicing gene expression platform, we have created a potent synthetic riboswitch cassette that regulates gene expression in response to small molecule inducers. The splicing‐based expression platform creates an ‘on’ switch in the presence of the small molecule, by sequestering the 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. 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 a platform for using unique aptamer ligand pairs to regulate genes in mammals. This platform enables both temporal and spatial control of gene expression, as mediated by gene therapy.
Extinction of all infectious HIV in cell culture by the CRISPR‐Cas12b system
1: Amsterdam UMC
The CRISPR‐Cas9 system has been used successfully for genome editing of various organisms. We previously reported inhibition of the human immunodeficiency virus (HIV) in cell culture infections and subsequent viral escape when a single guide RNA (gRNA) was used, but complete inactivation of all infectious HIV with certain combinations of two gRNAs. The recently described CRISPR‐Cas12b system may provide an even more promising tool for genome engineering with increased activity and specificity. Full HIV inactivation in cell culture was achieved with only a single gRNA (called crRNA). We disclose that DNA cleavage by the Cas12b endonuclease and subsequent DNA repair causes mutations with a sequence profile that is distinct from that of Cas9. Both CRISPR systems can induce the typical small deletions around the site of DNA cleavage and subsequent repair, but Cas12b does not induce the pure DNA insertions that are routinely observed for Cas9. We propose that the different architecture of the Cas9 versus Cas12b endonuclease explains this effect. Then we tested the antiviral activity of dual crRNA combinations and analyzed the HIV proviral genomes at the target sites. We demonstrated that dual crRNA combinations can exhibit more robust antiviral activity than a single crRNA attack and, more importantly, that the dual‐crRNA therapy can prevent virus escape in long‐term cultures. We did not detect excision of HIV sequences located between the two Cas12b cleavage sites in dual strategy, but efficient HIV inactivation by ”hypermutation” at both sites as a result of DNA cleavage and subsequent error‐prone DNA repair.
Using CRISPR/Cas9 technology for the specific elimination of dominant mutations associated with rare diseases
D Gómez‐Domínguez1 S Amarilla1 C Epifano2 A Martín1 B Vilaplana‐Marti2 I Hernández1 M Pallares1
1: Instituto de Salud Carlos III 2: Fundación Andrés Marcio, niños contra la laminopatía
CRISPR/Cas9 technology is successfully used for the generation of KI and KO cell lines and organisms. This gene editing methodology relays on a two component machinery that consists on a endonuclease guided for a small sequence of RNA (sgRNA) to its target. Specificity of the CRISPR/Cas9 activity depends on different factors including a 20 nucleotide sequence complementary to the sgRNA and a three nucleotide sequence named PAM (protospacer adjacent motif). Since single changes in the sgRNA sequence could avoid the activity of Cas9, we hypothesized that the use of sgRNAs containing point dominant mutations related with genetic diseases might be a way to specifically destroy those mutations while wild type alleles are let unedited. We tested our hypothesis in two different rare diseases: the LMNA‐related congenital muscular dystrophy and the FOXL2‐associated granulosa cell tumors. Our results, obtained in human and mouse cell lines as well as mouse embryos, confirm our working hypothesis and open the use of mutation‐specific sgRNAs for the potential, gene therapy based, treatment of these rare diseases.
The GuardOne clinical trial: a first in‐human, open‐label, multinational phase 1/2 study of AVR‐RD‐02
ex vivo
lentiviral vector, autologous gene therapy for Gaucher disease
E Ridha1
1: AVROBIO, Inc. 2: University of Calgary Cumming School of Medicine 3: University College London, London, UK
Gaucher disease results from the abnormal function of the GBA gene leading to multi‐organ pathology with lower life expectancy when untreated. The impact of reduced endogenous activity of the enzyme glucocerebrosidase (GCase) is the basis for standard treatment with ERT and SRT which require regular and life‐long use of these medications with disease regression as soon as they are discontinued. For many patients, standard therapies are not completely effective and are also not a realistic option in many parts of the world leaving an unmet need for a more innovative solution.
GuardOne is a Phase 1/2, open label, single arm study in adult patients with Gaucher Type‐1. The study is evaluating a third‐generation lentiviral vector to transduce CD34+ stem cells for autologous transplant as an investigational product (AVR‐RD‐02) to correct GCase levels in progenitor cells with multi‐lineage capacity. This treatment aims to provide a correction of reduced GCase activity with a single dose of product. Objectives of the study include assessment of the safety and tolerability of treatment with AVR‐RD‐02, assessment of engraftment of gene‐modified cells, and evaluation of early signals of efficacy as reflected by organ volumes, hemoglobin level, platelet count, lyso‐Gb1 and chitotriosidase levels. Patients will be followed for 2 years and then enrolled in a 15 year long‐term follow‐up study.
The first patient has been dosed in GuardOne and the trial is active in the US, Canada and Australia, with a site in Israel to open in 2021. Early clinical data will be provided.
RNA‐PolyPurine Reverse Hoogsteen Hairpins as tool for gene silencing
1: University of Barcelona 2: Institute of Nanoscience and Nanotechnology 3: ICREA y Universitat Autonoma Barcelona 4: Institute of Neurosciences at UAB, and Vall d'Hebron Institute of Research (VHIR)
PolyPurine Reverse Hoogsteen Hairpins (PPRHs) are gene silencing DNA‐oligonucleotides developed in our laboratory. They are formed by two antiparallel polypurine mirror repeat domains bound intramolecularly by Hoogsteen bonds, which can hybridize to polypyrimidine targets in the genomic DNA. Among all the technologies developed for nucleic acids delivery, Adenoviruses (AdVs) vectors present high transduction efficiencies on a variety of cells without integrating with the host genome. Since in viral vectors the PPRH sequence would be transcribed into RNA, we evaluated the effect of a PPRH targeting survivin as an RNA species (synthetic RNA‐PPRH). We demonstrated the ability of the RNA‐PPRH to bind to its ssDNA or dsDNA target sequence. We showed that the RNA‐PPRH induced a decrease on cell viability in a dose‐dependent manner and an increase of apoptosis in PC‐3 and HeLa cells. We also determined that both the synthetic RNA‐PPRH and an RNA‐PPRH intracellularly generated upon transfection of a plasmid vector were able to reduce survivin mRNA and protein levels of PC‐3 cells. Once validated that RNA‐PPRHs induced survivin silencing, we confirmed that an AdV5 encoding the PPRH against survivin was able to decrease survivin mRNA and protein levels, leading to a reduction in HeLa cell viability. In contrast, no effect was observed in cells infected with the negative control AdV‐GFP. In this work we demonstrated that PPRHs can also work as RNA species, using RNA chemically synthesized, plasmid and viral expression vector. Therefore, we proved that adenoviral vectors can be considered as a delivery system of PPRHs.
Lipid based vectors containing mRNA for corneal inflammation treatment
1: Pharmacokinetic, Nanotechnology and Gene Therapy Group (PharmaNanoGene), Faculty of Pharmacy, Centro de investigación Lascaray ikergunea, University of the Basque Country UPV/EHU, Paseo de la Universidad 7, 01006 Vitoria‐Gasteiz, Spain; Bioaraba, PharmaNanoGene, Vitoria‐Gasteiz, Spain 2: Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, via Pietro Giuria 9, 10125 Torino, Italy
Interleukin‐10 (IL‐10) is an anti‐inflammatory cytokine considered as an efficient treatment for corneal inflammation, although its low ocular bioavailability and short half‐life limit its therapeutic use. Messenger RNA (mRNA) delivery in ocular tissues by non‐viral systems could be an efficient and safe alternative to induce IL‐10 production in the cornea. The aim of this work was the development of solid lipid nanoparticles (SLNs) to deliver IL‐10 mRNA to treat corneal inflammation. Systems were evaluated in vitro in human corneal epithelial (HCE‐2) cells and in vivo after administration as eye drop instillation to mice. Three different SLNs were compared: solvent evaporation/emulsification (SLNEE), hot‐melt emulsification (SLNHM) and coacervation (SLNC). mRNA encoding green fluorescent protein (GFP) or human IL‐10 was complexed with protamine, a polysaccharide (dextran (DX) or hyaluronic acid (HA)) and SLNs. Transfection efficacy, intracellular disposition and production of IL‐10 were evaluated in HCE‐2 cells. Additionally, in vivo mRNA expression of vectors containing 1% polyvinyl alcohol (PVA) was evaluated in wild type mice. SLNEE‐ and SLNHM‐based vectors transfected a higher percentage of cells (over 11%) than SLNC‐vectors (2%). SLNEE‐vectors, specifically DX‐SLNEE vector, induced higher extracellular levels of IL‐10 in HCE‐2 cells. All formulations produced GFP in the corneal epithelium of mice, although SLNEE‐ and SLNHM‐based vectors showed higher intensity and surface transfection. All formulations also produced IL‐10 in the corneal epithelium. Moreover, DX‐SLNEE vector exhibited IL‐10 in deeper layers of the epithelium. This work shows the potential utility of mRNA‐SLN‐based vectors as delivery systems to treat the corneal inflammation by gene therapy.
Development of isogenic myoblast‐based models to study cystinosis myopathy
1: KU Leuven
Cystinosis is an autosomal recessive lysosomal storage disease caused by defective cystinosin (CTNS), leading to cystine accumulation. Renal transplantation, cysteamine treatment and improved medical care resulted to a better quality of life and life‐expectancy for cystinosis patients. However, the latter also led to the emergence of additional systemic phenotypes like myopathy. Muscle weakness forms a major concern leading to life‐threatening events like swallowing difficulties and respiratory insufficiency. These complications affect over 80% of the untreated patients by the age of 40, but also affect cysteamine‐treated patients although the incidence of this complication is decreased by cysteamine. Moreover, the etiology of cystinosis myopathy remains to be elucidated. We aim to study the role of CTNS in cystinosis myopathy by employing human muscle cell models. Patient‐derived typical development immortalized myoblast cells together with CRISPR‐nanoblades and viral vector technology was used to generate polyclonal CTNS depleted (KO) isogenic muscle models. Genomic DNA analysis revealed a frame‐shift, and metabolomic analysis further demonstrated elevated cystine levels, the robust hallmark of cystinosis. In a first assessment, these myoblasts were subjected to differentiation into multi‐nucleated myotubes. CRISPRed CTNS KO myoblast showed a reduced fusion index compared to control myoblasts. As a next step, the potential of CTNS cDNA addition using viral vectors will be assessed.
Targeting cost‐effective large‐scale manufacturing for r‐AAV vectors by transient transfection using Pall's Allegro™ STR bioreactor
M L Collignon1 A Pedregal1 D Mainwaring1
1: Pall Life Sciences 2: Yposkesi
To date there is enormous pressure on biomanufacturing facilities due to the high demand for the manufacturing of gene therapy products such as recombinant adeno‐associated viral vectors (r‐AAV) under current good manufacturing practice conditions.
Yposkesi is a leading Contract Development and Manufacturing Organization in Europe for r‐AAV and lentivirus production, and is investing significantly to improve the productivity of viral vector manufacturing processes.
Yposkesi has evaluated the Allegro STR50 bioreactor for its usability and performance by conducting a proof of concept study of the triple transient transfection r‐AAV process, using a proprietary transfection reagent, a high‐productivity HEK293T cell line, and a custom medium. The process tech transfer was performed by keeping the power input per unit volume and the superficial gas velocity constant.
These process conditions resulted in foam production and required the addition of some antifoam. Slower cell growth and a lower r‐AAV yield was obtained, compared to the original stirred tank reactor (STR) platform. The process was then redeveloped by keeping a constant low sparger air flow rate and oxygen in the dissolved oxygen cascade, to avoid foam generation. The resulting yields were significantly higher than the original STR platform.
The user‐friendliness of the system is a strong point, for both biocontainer installation and software operations, and the close collaboration between Yposkesi and Pall scientific supporting teams, resulted in a favourable increase in production yields. Moving forward, the process is planned to be scaled up to 1000 L, in order to further increase cost‐effectiveness of large‐scale manufacturing for r‐AAV vectors.
Preclinical efficacy and safety of lentiviral (LV)‐based gene therapy in IMO support clinical trial initiation
1: Department of Molecular Medicine and Gene Therapy, Lund Strategic Center for Stem Cell Biology, Lund, Sweden 2: Rocket Pharmaceuticals, Inc., Cranbury, NJ 3: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany 4: University of California, Los Angeles, Los Angeles, CA
Infantile malignant osteopetrosis (IMO) is a rare and severe form of osteopetrosis characterized by impaired osteoclast bone resorption. Mutations in the T cell immune regulator 1 (TCIRG1) gene are the most frequent cause. IMO is largely fatal in the first decade of life unless successfully treated with allogeneic hematopoietic stem cell transplantation (HSCT), with overall survival following HSCT lower than in other non‐malignant hematologic disorders. Preclinical studies were performed to assess the potential safety and efficacy of TCIRG1‐based gene therapy (GT) using autologous hematopoietic stem cells (HSCs) as a potential definitive treatment for this disorder. Function, efficacy, feasibility, and safety of lentiviral vector (EFS.hTCIRG1‐LV)‐ mediated GT for IMO were evaluated. Pharmacology studies were performed using HSCs from both oc/oc osteopetrotic mice and human IMO patients to assess phenotypic correction. Murine toxicology studies evaluated genotoxicity, clinicopathology, and histopathology.Oc/oc mice receiving ex vivo GT with EFS.hTCIRG1‐LV‐transduced HSPCs showed correction of the disease phenotype, with increased long‐term survival, tooth eruption, weight gain, and normalized bone resorption. EFS.hTCIRG1‐LV‐transduced HSCs from IMO patients transplanted into immunodeficient mice exhibited normal engraftment and enabled correction of osteoclast function ex vivo. Patient‐derived PB CD34+ cells were also efficiently transduced. Toxicity studies indicated that the drug product (transduced HSCs) is likely to be safe and present minimal clinical risk relative to potential benefit. Results from these nonclinical studies unequivocally supported the initiation of the first‐in‐human clinical GT trial for IMO. The clinical trial is currently underway and the first patient received investigational infusion in May 2021.
Efficacy assessment of lentiviral vector gene therapy in the immune dysregulated hypomorphic
Rag1
mice
1: San Raffaele‐Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, 20132, Italy 2: Milan Unit, Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Milan, 20090, Italy 3: Humanitas Clinical and Research Centre IRCCS, Rozzano (MI), 20089, Italy 4: IRCCS Istituto G. Gaslini, Genoa, 16147, Italy 5: Laboratory of Clinical Immunology and Microbiology, NIAID, NIH, Bethesda, MD, 20852, USA 6: Leiden University Medical Centre, Leiden, 2333ZA Netherlands
Recombinase activating genes (RAG) 1 and 2 defects are the most frequent form of severe combined immunodeficiency (SCID). Patients with residual RAG activity have a spectrum of clinical manifestations ranging from Omenn Syndrome to combined immunodeficiency associated with granulomas and/or autoimmunity (CID‐G/AI). Lentiviral vector (LV) gene therapy (GT) has been proposed as an alternative to the hematopoietic stem cell transplant and a clinical trial for RAG1 SCID patients recently started (NCT04797260). However, GT in Omenn syndrome or CID‐G/AI patients poses additional risks, because of RAG1 endogenous expression. Therefore, we performed GT in 2 hypomorphic Rag1 murine models (Rag1F971L/F971L and Rag1R972Q/R972Q), exploiting a LV encoding RAG1 under the control of MND promoter.
Starting 6 weeks after transplant, GT mice showed the decrease of myeloid cells and the concomitant increase of B, T and white blood cells. However, counts remained lower than mice transplanted with WT Lin‐ cells. Analysis of TNP‐KLH‐specific IgM and IgG serum levels showed an amelioration of antibody response after in vivo challenge. At termination (six months post GT), we observed: the immune subset redistribution in the spleen; the appearance of immature and mature recirculating B cells in bone marrow; the overcome of double negative block with a modest improvement in the medulla/cortex ratio in the thymus. Moreover, B‐cell activating factor decreased to normal levels, while autoantibody profile did not show gross changes. Overall, GT mice showed improved immune functions without overt signs of immune dysregulation.
Project funded from the European Union's Horizon 2020 programme (RECOMB, grant agreement 755170).
Kinetics and composition of haematopoietic stem/progenitors mobilized cells upon G‐CSF and Plerixafor administration in transplant donor or patients undergoing autologous gene therapy
1: San Raffaele Telethon Institute for Gene Therapy (SR‐TIGET), San Raffaele Scientific Institute, Milan, Italy 2: Pediatric Immunohematology and Bone Marrow Transplantation Unit, San Raffaele Scientific Institute, Milan, Italy 3: Università Vita‐Salute San Raffaele, Milan, Italy 4: Division of Immunology, Transplantation, and Infectious Diseases, Immunohematology and transfusion medicine, San Raffaele Scientific Institute, Milan, Italy 5: Division of Experimental oncology, Onco‐hematology, San Raffaele Scientific Institute, Milan, Italy
Mobilised Haematopoietic Stem/Progenitor Cells (mHSPC) are becoming a major cell source for autologous HSPC‐gene therapy (GT) due to accessibility and number of collected cells. However, there is limited information on the composition and kinetics of responses of distinct HSPC subsets to the mobilizing agents Granulocyte Colony‐Stimulating Factor (G‐CSF, G) and C‐X‐C Chemokine receptor type 4 (CXCR4) antagonist Plerixafor (P). We studied HSPC mobilisation kinetics in 25 patients with different inherited disorders whose autologous mHSPC were collected for the purpose of gene therapy or backup, as well as in 12 adult healthy subjects who donated for allogeneic transplantation. We quantified HSPC subsets in the BM and peripheral blood (PB) at steady state and in the PB after administration of G and G+P. Primitive and myeloid progenitors showed the highest mobilisation capability after G, while all HSPC mobilized after G+P. We also found differential CXCR4 expression among the distinct HSPC subpopulations in BM before mobilisation, that inversely correlates with the subset‐specific mobilisation propensity. Conversely, higher CXCR4 expression correlated with higher migration capability in response to CXCR4 ligand in vitro. By reproducing patients' mobilisation protocol in immunodeficient mice transplanted with human HSPC, we confirmed that the response to mobilising agents of each HSPC subset depends on its CXCR4 expression. We are currently comparing mHSPC numbers and phenotype after mobilisation with the haematopoietic reconstitution in patients receiving GT. This information will be important to define the best strategy for mHSPC collection for transplantation and gene therapy and help to identify characteristics influencing haematopoietic reconstitution.
Improving the efficacy of liver directed lentiviral gene therapy for hemophilia
1: San Raffaele Telethon Insitute for Gene Therapy (HSR‐TIGET) 2: Vita‐Salute San Raffaele University 3: San Raffaele Scientific Institute
Lentiviral vectors (LV) are attractive vehicles for liver‐directed gene therapy, by virtue of their ability to stably integrate into the target cell chromatin, being maintained as cells divide. We have developed LV that achieve stable expression of coagulation factor IX (FIX) or VIII (FVIII) in the liver after intravenous administration to small and large animal models. However, LV‐mediated transfer of FVIII remains more challenging due to the large transgene size and lower vector infectivity. Moreover, increasing the efficiency of LV gene transfer is crucial to achieve the same therapeutic efficacy at lower doses, thus alleviating concerns related to possible LV‐dose dependent toxicities and reducing the costs and burden of manufacturing. To achieve this goal, we exploited inhibitors of known anti‐viral pathways, such as the proteasome and type‐I interferon (IFN) pathways. We found that a single administration of the clinically approved proteasome inhibitor Bortezomib or of an IFN receptor blocking antibody prior to LV administration increased hepatocyte transduction and transgene output up to 3‐fold, in wild‐type mice and mouse models of hemophilia A and B. In addition, we show a stable 2‐3 fold increase in both FIX and FVIII output when mice were fasted for 24 hours prior to LV administration. Since LV particles are about 120 nm in diameter, their accessibility to hepatocytes may be dependent on submicroscopic features of liver sinusoids, which may change upon fasting, potentially explaining the observed outcome. Overall, here we present several clinically viable strategies that increase the potency of LV‐mediated liver gene therapy.
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 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 neurodegeneration and severity of 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 a therapeutic Lentiviral Vector (LV) able to safely and efficiently deliver multiple copies of a codon‐optimized GLB1 gene in patient‐derived cells, express dose‐dependent supra‐physiological levels of therapeutic enzyme, which is correctly secreted and uptaken by target cells. LV transduction determines a stable genetic and metabolic correction of patient fibroblasts, proven by the rescue of storage material as early as two weeks post‐transduction. We are currently proceeding with the in vivo validation of the GT strategy, in a feasibility and efficacy study performed in the animal model of the disease.
Lentiviral‐mediated gene therapy for the treatment of adenosine deaminase 2 deficiency
M Zoccolillo1 C Mesa Nuñez1 A Brix2 I Brigida1 F Barzaghi3 S Scala1 R Jofra Hernandez1 L Basso‐Ricci1 M Colantuoni1 4 S Cesaro5 F Conti6 A Pession6 F Benedetti7 M Gattorno8 L Naldini1 4 M P Cicalese3 A Pistocchi2 A Aiuti1 3 4
1: San Raffaele Telethon Institute for Gene Therapy (SR‐Tiget), IRCCS San Raffaele Scientific Institute 2: Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano 3: Pediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute 4: Vita‐Salute San Raffaele University 5: Pediatric Hematology‐Oncology, Ospedale della Donna e del Bambino 6: Pediatric Unit, IRCCS Azienda Ospedaliero‐Universitaria di Bologna 7: CTMO Ematologia Azienda Ospedaliera Universitaria integrate 8: Centro Malattie Autoinfiammatorie e Immunodeficienze, IRCCS Istituto Giannina Gaslini
Adenosine deaminase 2 deficiency (DADA2) is a recently defined inborn error of immunity caused by loss‐of‐function mutations in the ADA2 gene. Clinical manifestations include vasculitis, ischemic strokes, intracranial haemorrhages, immunological and haematological abnormalities. The therapeutic benefit of the current treatments is unsatisfactory. Lentiviral vector (LV)‐mediated gene therapy based on transplantation of ADA2‐corrected hematopoietic stem‐progenitor cells (HSPCs) may represent a valid therapeutic option for treating patients with DADA2. We initially demonstrated that severe neutropenia and HSPC defects are a direct cause of DADA2. Indeed, ADA2 knock‐down in zebrafish – as rodents do not harbour an ADA2 orthologue gene – caused a significant decrease in neutrophil and HSPC numbers, reminiscent of patients' phenotype, which was effectively corrected by administering human recombinant ADA2. We used a third‐generation LV to restore constitutive ADA2 expression in HSPCs. Transduction of healthy donors' HSPCs allowed efficient delivery of the functional ADA2 enzyme with no toxicity. Supranormal ADA2 expression in healthy donors' and patients' HSPCs was well‐tolerated and did not impact HSPC multilineage differentiation potential in vitro and in vivo. We also assessed whether LV‐derived ADA2 could correct the hyperinflammatory M1 macrophage phenotype characteristic of DADA2. ADA2 reconstitution in patients' macrophages led to the normalization of IL‐6 and TNF release. Similar results were obtained using M1 macrophages differentiated from ADA2‐transduced HSPCs. Altogether, our findings indicate that HSPC gene therapy is a promising approach to re‐establish stable ADA2 activity and correct the haematological and inflammatory manifestations in patients with DADA2.
Preclinical studies for lentiviral‐mediated gene therapy of
RPS19
‐deficient Diamond‐Blackfan anemia
1: CIEMAT/CIBERER 2: IIS‐Fundación Jimenez Diaz 3: ULB‐Cancer Research Center 4: Hospital del Niño Jesús 5: Hôpital Robert‐Debré 6: Hospital Sant Joan de Déu 7: Hospital Gregorio Marañón
Allogenic hematopoietic stem cell transplantation (HSCT) currently represents the only curative treatment for the bone marrow failure (BMF) of patients with Diamond Blackfan Anemia (DBA). Mutations in RPS19 are the most frequent, 25% of all DBA patients. To develop a gene therapy program for RPS19‐deficient DBA patients, we have generated a clinically applicable lentiviral vector (LV) which encode a codon‐optimized version of RPS19, driven by the PGK promoter. First, gene therapy studies were conducted in K562 cells in which RPS19 was down‐regulated with sh‐RPS19 LVs. In these studies, we observed that transduction with the therapeutic LV restored the expression of RPS19 and corrected the ribosomal biogenesis defects characteristic of sh‐RPS19 transduced cells. The therapeutic efficacy of this LV was confirmed in primary CD34+ cells from RPS19‐deficient patients. Transduction of these CD34+ cells with the therapeutic LV significantly increased the number of BFU‐E colonies. Moreover, the therapeutic LV reverted the red blood cell differentiation defect characteristic of DBA CD34+ cells, increasing the output of CD71+/CD235+ mature erythroid cells. Remarkably, CD34+ DBA cells transduced with the therapeutic LV were also capable of repopulating the hematopoiesis of NSG mice. In addition, no evident changes in the repopulating function of HD CD34+ cells were observed after transduction with the therapeutic vector, revealing the safety associated with the ectopic expression of RPS19. Taken together, the preclinical studies conducted in this work support that the lentiviral‐mediated gene therapy of RPS19‐deficient DBA patients should constitute an efficient and safe approach for the treatment of DBA patients.
Mesenchymal stromal cells stably expressing CXCR4 and IL10 as therapy for acute graft versus host disease.
1: CIEMAT‐CIBERER/IIS‐FJD
While previous experimental and pre‐clinical data have shown the efficacy of mesenchymal stromal cells (MSCs) to revert graft versus host disease (GvHD) after allogeneic transplantation, the therapeutic efficacy of these cells have not been reproducibly demonstrated in clinical trials. With the purpose of increasing the anti‐GvHD effect of MSCs, we constructed a bicistronic lentiviral vector carrying the genes encoding for CXCR4 and IL10, two molecules involved in cell migration to inflamed sites and with potent anti‐inflammatory properties, respectively. In vitro experiments showed that the stable expression of these molecules in adipose tissue derived human MSCs (CXCR4‐IL10‐MSCs) efficiently enhanced the migration of MSCs towards SDF1 when compared to unmodified MSCs. CXCR4‐IL10‐MSCs also displayed enhanced capacity to inhibit the proliferation of activated T cells, concomitant with their polarization from a pro‐inflammatory to an anti‐inflammatory T cell profile. Using a humanized GvHD mouse model generated by the transplantation of human peripheral blood mononuclear cells into immunodeficient NSG mice, a decreased GvHD score was observed in mice treated with a single dose of CXCR4‐IL10‐MSCs as compared to unmodified MSCs. CXCR4‐IL10‐MSCs also induced a significant reduction in the proliferation of Th1 and Th17 cells, compared to unmodified MSCs. Moreover, a significant increase in the number of regulatory T cells (Tregs) was observed in mice infused with CXCR4‐IL10‐MSCs. Taken together, our preclinical studies strongly suggest that the lentiviral‐medicated expression of CXCR4 and IL10 should improve the anti‐GvHD properties of MSCs in the clinic.
Targeting CD34+ Hematopoietic Stem cells: a potential promise for Hemophilia A
1: Research Department, Sidra Medicine, PO Box 26999, Doha, Qatar 2: College of Health and Life Sciences, Hamad Bin Khalifa University, Doha, Qatar 3: Department of Health Sciences, Università del Piemonte Orientale ‘‘A. Avogadro’’, 28100, Novara, Italy 4: Icahn School of Medicine at Mount Sinai, Division of Hematology and Medical Oncology, Hess Center, New York, NY 10029
Hemophilia A (HA) is a monogenic bleeding disorder, due to deletions/mutations in Factor‐VIII (FVIII) gene causing defective FVIII production. Clinical studies of Hemophilia Gene Therapy (GT) directed to the liver proved to be efficient, yet with limited patient eligibility. Here, we aim to correct HA through FVIII‐transduced CD34+ Hematopoietic stem cells (HSCs). Recently, we developed a flow‐cytometry protocol to assess FVIII expression in CD34+ progenitors before and after FVIII‐gene transfer. Fresh CD34+ cells produced a negligible FVIII rate of 0.37% ± 0.05 (Mean% ± SEM, n = 2). LV mediated transduction of CD34+ with FVIII transgene increased FVIII protein level to 19.64% ± 5.34 at 100 MOI (n = 2), with a clinical targeted transduction protocol (TPO, Flt3L, IL3 & SCF) and UM171 (38 nM) which is a novel HSCs self‐renewal agonist. Further, using a transparent Zebrafish (ZF) transgenic reporter line, Tg(fli1:EGFP) expressing a vasculature with green fluorescent protein, for xeno‐transplantation, we showed the successful engraftment of CD34+ cells transduced with a LV‐GFP/LV‐Tomato‐9 traceable marker with the same clinical protocol. UM171‐treated CD34+ had 2‐fold higher engraftment and higher self‐proliferation as compared to cells treated with cytokines only. This correlates with previously published data in mice but is first shown here in ZF. Interestingly, we could follow through time‐lapse video analyses the dynamics of engraftment and spatial relationships of CD34+ with endothelial cells, and quantify their in‐vivo expansion. In conclusion, human CD34+ treated with UM171 can successfully be transduced with an LV‐carrying‐FVIII transgene and show abundant in‐vivo engraftment, potentially serving as GT target in HA.
Hematopoietic reconstitution and lineage commitment in HSC GT patients are influenced by the disease background
1: San Raffaele Telethon Insitute for Gene Therapy (HSR‐TIGET) 2: Bernoulli Institute for Mathematics, University of Groningen 3: University Vita‐Salute San Raffaele 4: Università della Svizzera Italiana, Institute of Computational Science
Lentiviral vector (LV) ‐based hematopoietic stem cell (HSC) gene therapy (GT) applications have shown a favorable efficacy and safety profile for the treatment for a variety of genetic diseases. To understand the impact of patient‐specific factors and disease background on the hematopoiesis after transplantation, we studied the clonal reconstitution and multilineage potential over time in 48 HSC‐GT patients affected by 3 different diseases: 29 with metachromatic leukodystrophy (MLD), 10 with Wiskott‐Aldrich syndrome (WAS) and 9 with β‐thalassemia (β‐Thal).
We analyzed clonal dynamics by tracking LV genomic Integration Sites (IS) over time (>3 million unique IS observed). All trials showed highly polyclonal reconstitution, multilineage marking and no signs of genotoxicity in all patients.
Regardless of disease background, all patients showed a similar pattern of hematopoietic reconstitution over time. Differences across diseases were found when we analyzed the multilineage potential of individual clones and their commitment towards a specific lineage over time. In MLD patients multilineage clones reached a proportion of 75% on the total engineered clones, decreased over time to 50%, remaining stable thereafter. Myeloid‐committed clones increased over time and stabilized at 40%. The decrease in multilineage clones over time was also observed in WAS and β‐Thal patients. However, in WAS patients we did not observe a concomitant increase in myeloid committed cells rather an increase of the commitment in the T‐cell lineage, as expected by the selective advantage in T cells, whereas in β‐Thal patients several clones showed erythroid commitment.
Investigating Mechanisms of Variability of AAV5‐hFVIII‐SQ Expression in Mice
1: BioMarin Pharmaceutical
Valoctocogene roxaparvovec (AAV5‐hFVIII‐SQ) is an investigational gene therapy for the treatment of severe hemophilia A. Similar levels of inter‐subject variability have been observed across species (mouse, NHP and human) treated with AAV5‐hFVIII‐SQ. We hypothesize that variations in host factors involved in many steps, from AAV5 transduction to transgene expression, contribute to inter‐subject variability. Samples from male mice administered AAV5‐hFVIII‐SQ were utilized to determine levels of plasma FVIII protein, liver FVIII DNA, RNA, protein and various host factors. First, we determined if expression of AAV5 receptors correlated with AAV5 vector transduction. There was a significant correlation between levels of FVIII DNA and PDGFRa expression (r = 0.5036; p = 0.0017) but not AAVR (r = 0.1796; p = 0.7726). Artemis, a DNA repair enzyme that facilitates vector genome processing and episome formation, significantly correlated with vector DNA levels (r = 0.6822; p = 0.0052). Next, we identified factors that may contribute to vector transcriptional variability. There was a significant correlation between FVIII transcripts and mRNA levels of RNF121 (r = 0.7188; p 0.003), Phf5A (r = 0.5970; p = 0.0154) and HNF1α (r = 0.7158; p = 0.0054). Overall, we demonstrated that inter‐subject variability may be driven by multiple contributing mechanisms. Additional studies that further investigate the mechanistic drivers of AAV5 gene therapy variability are ongoing and may help identify predictive biomarkers of transgene expression.
Fanconi anemia mesenchymal stromal cells have reduced differentiation capacity and impaired HSC maintenance
1: CIEMAT/CIBERER/IIS‐FJD 2: Fundación Biomédica Hospital Niño Jesús
The human bone marrow niche is composed by a variety of non‐hematopoietic cells. Among them, mesenchymal stromal cells (MSCs) have an important role supporting and regulating hematopoietic stem cells (HSCs). In addition to these functions, MSCs are characterized by important immunomodulatory properties and differentiation capacity into different mesodermal lineages. Fanconi Anemia (FA) is a complex genetic disorder with a high incidence of bone marrow failure and cancer predisposition, being allogeneic bone marrow transplant the only curative treatment. FA‐HSCs are well characterized, however, FA BM stroma has not been deeply characterized. With the aim of analyzing the functionality of the FA stroma, in this study we have characterized BM‐MSCs from Fanconi Anemia patients. Similar to FA‐HSCs, we have observed that FA‐MSCs show high sensitivity to the intercross‐linking agent mitomycine‐C, increased ROS production and high percentage of apoptotic cells compared with healthy donors (HD) MSCs. We have also observed a decreased bone and adipose differentiation capacity due to a decreased expression of genes involved in this process. Additionally, FA‐MSCs showed a reduced migration capacity to SDF‐1 gradient and impaired immunomodulation properties. Significantly, MSCs from FA patients revealed an impaired support of HSCs, as revealed by the reduced percentage of CD34+/CD38‐/CD90+/CD45RA‐cells and the lower number of CFU‐GMs observed in hematopoietic samples co‐cultured with FA‐MSCs, compared to co‐cultures with HD‐MSCs. Taken together, our results suggest an impairment in FA BM niche, at least due to defective MSC compartment, which may contribute to the bone marrow failure characteristic of FA patients.
A novel γ‐globin lentiviral vector pseudotyped with the HF glycoproteins restores the thalassemic maturation bottleneck and displays a safe integration pattern
1: Laboratory of Cell and Gene Therapy, Centre for Basic Research, Biomedical Research Foundation of the Academy of Athens (BRFAA), Athens, Greece 2: Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus and Cyprus School of Molecular Medicine, Nicosia, Cyprus 3: GeneWerk GmbH, Heidelberg, Germany 4: Centre of Basic Research, Biomedical Research Foundation of the Academy of Athens (BRFAA), Athens, Greece 5: Greek Genome Center, Biomedical Research Foundation of the Academy of Athens (BRFAA), Athens, Greece 6: Centre International de Recherche en Infectiologie, Université de Lyon, Université Claude Bernard Lyon 1, INSERM, U‐1111, Centre National de la Recherche Scientifique (CNRS), UMR 5308, Ecole Nonmale Supérieure de Lyon, Lyon, France 7: Université Côte d'Azur, INSERM U‐1065, Centre Méditerranéen de Médecine Moléculaire, Nice, France
The β‐thalassemia syndromes are characterized by ineffective erythropoiesis, primarily due to early erythroid maturation bottleneck. To further ameliorate the β‐thalassemia phenotype in vitro, we generated a novel γ‐globin lentiviral vector GGHI‐mB‐3D/HF, pseudotyped with the HF envelope glycoproteins, previously shown to display increased tropism towards hCD34+ cells. The new vector was assessed in vitro in CD34+ cells from normal donors and thalassemia patients of either β+ or βo genotype, at MOI 25. We demonstrate that the vector transduction leads to increased F‐cell percentage, both in normal (14.2% ± 7.3, p = 0.04, n = 4) and thalassemia (7.2% ± 5.0, p = 0.04, n = 5) cells, followed by a 30.7% ± 81.5, (p < 0.0001, n = 4) increase in the number of orthochromatic erythroblasts with a VCN/cell 0.9 ± 0.9 (n = 4), suggesting an amelioration of the erythroid maturation bottleneck. To further investigate the molecular basis of this effect, we analyzed the transcriptome of normal (n = 4), β+‐thalassemia (n = 4) and βo‐thalassemia (n = 2) erythroid progenitors, before and after transduction, employing RNASeq analysis. Principle Component Analysis showed that following transduction, thalassemia transcriptomes converge towards the respective normal ones, while modules like TNFα‐mediated apoptosis and heme metabolism are alleviated. Remarkably, although β+ and βo‐thalassemia cells share a similar transcriptome pattern before treatment, they display robust differences following transduction, enabling to identify specific genes responsible for this differential response, which may present putative targets for a rational and effective treatment of β‐thalassemia. Furthermore, integration site analysis of the vector, revealed no significant integration hotspots neither near cancer genes nor regulatory active elements, documenting the safety and effectiveness of the novel vector for β‐thalassemia gene therapy.
Role of peripheral blood circulating haematopoietic stem/progenitor cells during physiological haematopoietic maturation and after gene therapy
1: San Raffaele Telethon Institute for Gene Therapy (HSR‐TIGET), Milan, Italy 2: Vita‐Salute San Raffaele University, Milan, Italy 3: Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy 4: University of Milan, Department of Clinical Sciences and Community Health, Milan, Italy 5: Pediatric Immunohematology and Bone Marrow Transplantation Unit, San Raffaele Scientific Institute, Milan, Italy 6: Pediatric Department, San Raffaele Scientific Institute, Milan, Italy 7: Orthopaedic Unit, San Raffaele Scientific Institute, Milan, Italy
Although most Haematopoietic Stem/Progenitor Cells (HSPC) reside in the bone marrow (BM), few circulating HSPC (cHSPC) traffic in peripheral blood (PB) of un‐mobilized individuals and their role is poorly characterized. Combining multi‐parametric phenotyping and single‐cell RNAseq with in vitro and in vivo functional assays, we studied cHSPC properties in physiological conditions and after gene therapy (GT). We detected changes in cHSPC subset counts during aging in healthy donors (HD) and different composition in PB vs. BM HSPC, suggesting diverse recirculation capacities of distinct HSPC subsets. Applying matched phenotyping and transcriptional analyses, we found consistency between phenotype and differentiation‐associated signatures among the distinct cHSPC subsets. PB vs. BM HSPC displayed lower cell cycle activity and enriched expression of gene sets related to lympho/erythroid differentiation and cell‐cell adhesion. HD PB and BM HSPC had comparable in vitro differentiation rate and BM homing potential after transplantation in immunodeficient mice. Of note, we detected higher erythroid output of PB vs. BM HSPC in single‐cell multi‐lineage differentiation assay and after transplantation, consistently with their enriched erythroid transcriptional signature. Following hematopoietic recovery in GT‐patients, we observed an increased cHSPC count in the first 2 months post‐GT, with high recirculation of primitive subsets, suggesting an active role in early reconstitution. We are currently analyzing integration sites retrieved from BM and PB HSPC overtime after GT to investigate their relationship and differentiation potential in vivo in humans. These studies will help to unveil the functional role, the recirculation mechanisms and the hematopoietic output of cHSPC in humans.
Patient‐derived, exhausted tumor‐specific T cells can be manipulated to establish a T‐cell receptors library for adoptive T cell therapy
1: San Raffaele Scientific Institute 2: IRCSS San Raffaele 3: Vita‐Salute San Raffaele University
Despite adoptive T cell therapy with genetically engineered T cells (ACT) is gaining ground as a cancer treatment option, its applicability is limited by the low number of known tumor‐reactive T‐Cell Receptors (TCRs).
To broaden the array of exploitable TCRs, n = 13 T cell clones reactive against MAGE‐A2, WT1, Survivin and hTERT immunodominant peptides were purified from allo‐transplanted blood cancer patients. Despite documented in vitro enrichment and expansion, T‐cell cultures failed to lyse target cell lines pulsed with the appropriate tumor‐specific peptide, suggesting that T cells were hypo‐functional. Accordingly, an ex vivo phenotypic analysis underlined that tumor‐specific T cells express multiple inhibitory receptors. To circumvent hypo‐functionality, healthy donors T cells were engineered with those newly isolated tumor‐specific TCRs after endogenous TCR KO via the CRISPR/Cas9 system. Noteworthy, engineered T cells proficiently eliminated peptide‐pulsed cell lines in vitro.
We next exploited the tumor‐specific exhaustion signature to identify novel tumor‐reactive TCRs. The T cell fraction positive for multiple inhibitory receptors (IR+) was isolated from three Acute Myeloid Leukemia patients and then stimulated with autologous Leukemic‐Antigen Presenting Cells. In all patients, in the IR+ but not in the control IR‐ group we documented the expansion of a dominant TCR clonotype. Donor T cells engineered with such dominant TCRs efficiently lysed autologous leukemia in vitro while sparing healthy bone marrow cells.
In summary, our data suggest that the exhausted, tumor‐specific T cell pool circulating in patients after transplant can be exploited to isolate novel tumor‐specific TCRs to be employed in ACT.
Development of physiological and inducible 4th generation CAR (iTRUCK)‐T cells using the Lent‐On‐Plus platform
1: LentiStem Biotech 2: GENyO‐ Centro de Genomica e Investigacion Oncologica: Pfizer / Universidad de Granada / Junta de Andalucia
Chimeric Antigen Receptor (CAR)‐T cells have become a new tool for treating refractory blood cancers. However, its effectiveness in solid tumours is less than 5%, due to inability of CAR‐T cells to infiltrate into tumour, overcome tumour microenvironment (TME) and survive for long enough. One approach to overcome this is developing 4th generation CARs (TRUCKs) co‐expressing chemokine receptors, immuno‐stimulatory molecules or genes involved in survival/stemness. However, continuous expression of these molecules may cause unacceptable side effects in patients. Other way to increase potency/survival of CAR‐T cells is expressing CAR physiologically, following the TCR‐kinetics. We used Lent‐On‐Plus (LOP)‐technology to regulate highly‐active molecules and our AW‐platform (TCR‐like expression) to express CARs on T cells. LOP is a transactivator‐free system able to generate inducible primary T cells that are highly‐responsive to very low doses (0.1ng) of doxycycline (dox). AW‐platform showed previously ability to improve phenotype and killing efficacy of CAR‐T cells by expressing CAR physiologically. Here we combine expression of an antiCD19‐CAR through AW‐promoter and IL‐18 though inducible LOP‐system to generate a first‐in‐class AW‐driven (and EF1α‐driven) iTRUCK. Both iTRUCKs express IL‐18 in presence of dox, with low leaking in absence. This increment in IL‐18 secretion correlates with an increase in killing activity against a pancreatic ductal adenocarcinoma model (KRAS/p53/p16/SMAD4‐mutated) artificially expressing CD19. Importantly, AW‐iTRUCK improved killing activity compared to EF1α‐iTRUCK in a serial challenging, showing better phenotype with higher proportion of stem cell memory/central memory T cells. These data indicate that AW/LOP‐platform is an interesting platform for developing inducible‐TRUCKs for treating solid tumours.
Improvement of “off‐the‐shelf” allogeneic CAR‐T cells
1: GENyO‐ Centro de Genomica e Investigacion Oncologica: Pfizer / Universidad de Granada / Junta de Andalucia 2: IMIBIC Instituto Maimónides de Investigación Biomédica de Córdoba 3: Hospital Universitario Reina Sofía de Córdoba
Therapy based on T cells expressing chimeric antigen receptors (CAR) is a novel treatment for lymphoid neoplasms, which has already proven promising results. Currently antitumor T cells grafts are obtained from autologous T cells, which increases the cost and reduces the efficacy of the treatment. Additionally, several clinical trials involving CAR T cells have been impacted by T cells intrinsic factors, mainly associated with their phenotype. To avoid these major drawbacks, the use of allogeneic products in combination with the selection of defined CAR T cells subsets with greater in vivo persistence is being considered and tested. In this project, we expect to generate off‐the‐shelf allogeneic CAR‐CD19 T cells with a defined phenotype.
We first generated universal HLA‐I and T‐cell receptor (TCR) double‐knockout (dKO) T cells, by simultaneous disruption of B2M and TRAC genes through Cas9/sgRNA ribonucleoporation. When transduced with a CD19 CAR‐T, the dKO‐CAR T retained similar antitumoral activity in comparison with unedited CAR T cells. Ongoing work is focused in testing whether our dKO‐CAR T cells reduce both rejection and graft versus host disease, and in the improvement of the persistence and antitumoral activity of these cells, by selection of specific T cells subsets.
To sum up, the combination of gene editing of CAR‐T lymphocytes by TCR and B2M loci disruption, with the isolation of specific T subsets is an in vitro procedure developed with the translational objective of improving the clinical results of CAR T cells infusions in patients with refractory or relapsing B neoplasms.
Efficient AAV/CRISPR‐based genetic engineering strategy for the production of allogeneic NKG2D CAR T cells in a single step process
1: Institute of Biology and Molecular Genetics (IBGM), University of Valladolid (UVa)‐CSIC, Valladolid, Spain. 2: Fundación para la Investigación Biomédica H12O, H12O‐CNIO Hematological Malignancies Clinical Research Group, CIBERONC, Madrid, Spain. 3: Department of Pathology and Laboratory Medicine. University of Pennsylvania, Philadelphia, USA
Chimeric antigen receptor (CAR)‐T cells have shown impressive clinical efficacy in patients with hematological malignancies expressing CD19. However, only autologous CAR‐T therapies are commercially available until now. Our work focuses on the generation of allogeneic CAR‐T cells, which allows reduction of manufacturing costs and avoids delays in patient treatment. We use the atypical NKG2D‐CAR, which recognizes eight ligands expressed mainly on cancer cells. To generate allogeneic NKG2D‐CAR‐T cells, we used CRISPR/Cas9 technology in combination with AAV6 vectors carrying the NKG2D‐CAR flanked by TRAC homology arms. Twelve days after transfection 90% of the cells were NKG2D‐CAR positive and TCRneg. Additional multiplexed knockout of MHC‐I, MHC‐II and/or PD‐1 were included in successive experiments obtaining the following efficacies determined by flow cytometry: 90% of NKG2Dpos/TCRneg/MHC‐Ineg; 7% of NKG2Dpos/TCRneg/MHC‐Ineg/MHC‐IIneg; 10% NKG2Dpos/TCRneg/MHC‐Ineg/PD‐1neg and 3% of NKG2D+/TCRneg/MHC‐Ineg/MHC‐IIneg /PD‐1neg. We are currently analysing the cytotoxic potential of these edited cell populations. So far, we have tested the NKG2Dpos/TCRneg cells through MTT cytotoxic assay using four different human tumour cell lines (HeLa, HT29, HCT116 and U2OS) and found approximately 80% of cytotoxicity at 48 hours and 90% at 72 hours after co‐culturing with our NKG2D‐CAR‐T cells. Overall, the results demonstrate a high in vitro antitumor efficacy of our NKG2D‐CAR‐T cells against a variety of cancer cells. CAR‐T cells expressing the NKG2D‐CAR are broadly useful against many different cancer types and are less prone to resistance, since it targets eight different ligands. Our results represent a further step towards developing allogeneic NKG2D‐CAR‐T cells for clinical application.
Engineering and functional characterization of anti‐PSMAxCD3 bispecific aptamers
1: Ixaka, Villejuif, France
Bispecific therapies redirecting cytotoxic effector T cells to malignant cells by simultaneous binding to CD3, the signaling component of the T‐cell receptor, and tumor‐associated antigens have demonstrated striking activity in patients across different cancers (blinatumomab and catumaxomab). Currently over 80 bispecific antibody fragments/derivatives are under active clinical developments in oncology, half of which involves CD3 recognition.
Ixaka is developing CD3‐specific aptamers as new targeting agents to specifically deliver in vivo anti‐cancer therapeutics to T lymphocytes. These G‐quadruplex‐based binders with nanomolar affinities recognize epitopes on the CD3 receptor that are different from OKT3 and other T‐cell activating antibodies. They exhibit remarkable cross specificity with human, mouse and cynomolgus isoforms and extended serum stability.
In order to further explore the therapeutic potential of these leads, we engineered by click chemistry PSMAxCD3 bispecific aptamers to develop new anticancer agents able to recruit cytotoxic T cells to induce killing of prostate‐specific membrane antigen (PSMA)‐positive tumor cells. By combining biophysical and on‐cell binding methods, we demonstrated that dimerization did not alter the specificity and affinity of PSMA and CD3 monomers. The serum stability of the PSMAxCD3 heterodimers was not any different from that of individual monomers. In vitro cytotoxicity assays revealed that engineered PSMAxCD3 aptamers were able to recruit effector T lymphocytes to PSMA‐positive cells and trigger cytolysis when control monomer lacking the CD3 binding moiety were inactive. Based on these promising properties, PSMAxCD3 aptamers have been selected for further preclinical and clinical development as prostate cancer therapeutics.
Non‐Viral Engineering of Human iPS Cells to Manufacture Rejuvenated TCR T Cells for Cancer Immunotherapy
M Osawa1 M Akizuki1
1: Thyas Co., Ltd. 2: Kyoto University 3: National Cancer Center 4: University of Toyama 5: MaxCyte, Inc.
Engineered, human induced pluripotent stem cells (iPSCs) offer a replenishable source to differentiate large numbers of T cells for cancer immunotherapy applications. However, the transduction efficiency of iPSCs by retroviral and lentiviral vectors is suboptimal, and there are safety concerns owing to the presence of viral gene products. Therefore, we explored using a non‐viral, cGMP compliant, electroporation platform to transfect TCR‐expressing piggyBac® transposons into human iPSCs. Three promoters were evaluated for optimal expression of a surface marker, and iPSCs were analyzed 14 days after electroporation by flow cytometry to determine the transposition efficiency. Remarkably, up to ∼47% of cells expressed the surface marker protein without selection. Subsequently, iPSCs were electroporated to compare several TCR molecules. TCR‐expressing iPSCs were differentiated into T cells and then evaluated for cytotoxic activity when co‐cultured with peptide‐pulsed target cells. Potent antigen‐specific cytotoxicity was observed. Finally, iPSC‐derived T cells expressing several TCR candidates were injected multiple times into mice engrafted with a luciferase‐expressing liver cancer cell line. Significant tumor reduction was observed by Day 23 after the initial tumor cell introduction. These results demonstrate efficient TCR DNA transposition into iPSCs, followed by differentiation into T cells with functional activity both in vitro and in vivo.
Human syncytins can pseudotype lentiviral vectors, enabling gene transfer into B cells
M Ferrand1 2 Y Coquin1 2 A Seye1 2 S Frin1 2 E Renaud1 2 Y Danger3 4 M Cogné3 4
1: Genethon 2: Université Paris‐Saclay, Univ Evry, Inserm, Genethon, Integrare research unit UMR_S951 3: EFS‐Rennes 4: Inserm U 1236, University Hospital Rennes, Faculty of Medicine, Rennes
The viral glycoproteins that pseudotype recombinant lentiviral vectors (LV) are critical for cell entry and for the immunogenicity of the particles. In an attempt to endogenize LV, we used human syncytin‐1 or syncytin‐2. These fusogenic cellular proteins are found at high level in placenta and originate from endogenous retrovirus envelope sequences. Each of the syncytins were able to pseudotype LV, generating particles that were infectious in the presence of a transduction additive, vectofusin‐1 (VF1). Syncytin‐pseudotyped LVs selectively transduced some human cells lines, such as 293T cells, but not others, for instance HCT116 cells. The vectors transduced human lymphocytes and dendritic cells to various levels. Syncytin‐pseudotyped LV showed a remarkable ability to transduce peripheral blood B cells including naive B cells, activated B cells, plasmablasts as well as several B cell lines. The receptor for human syncytin‐1, ASCT2, and the receptor for syncytin‐2, MFSD2a, were detected at high levels in B cells, also in dendritic cells and at a lower level in T cells, correlating with the differential transduction levels in these cell types. In vivo, the intravenous injection of LV pseudotyped with syncytin‐1 to PBMC‐reconstituted NSG mice was able to transduce a small percentage of B cells (3‐10%). Syncytin‐pseudotyped LV provide the first evidence that endogenized particles can be obtained. In addition, this is the first time that interactions between syncytins and B cells are described leading to novel perspectives in immunology and new tools for gene delivery to B cells.
A novel immunopeptidomic‐based pipeline for the generation of personalized oncolytic cancer vaccines
1: University of Helsinki 2: Valo Therapeutics 3: Karolinska Institutet
Beside the isolation and identification of MHC‐I restricted peptides from the surface of cancer cells, one of the challenges is eliciting an effective anti‐tumor CD8+ T cell mediated response as part of therapeutic cancer vaccine. Therefore, the establishment of a solid pipeline for the downstream selection of clinically relevant peptides and the subsequent creation of therapeutic cancer vaccines are of utmost importance. Indeed, the use of peptides for eliciting specific anti‐tumor adaptive immunity is hindered by two main limitations: the efficient selection of the most optimal candidate peptides and the use of a highly immunogenic platform to combine with the peptides to induce effective tumor‐specific adaptive immune responses. Here, we describe for the first time a streamlined pipeline for the generation of personalized cancer vaccines starting from the isolation and selection of the most immunogenic peptide candidates expressed on the tumor cells and ending in the generation of efficient therapeutic oncolytic cancer vaccines. This immunopeptidomics‐based pipeline was carefully validated in a murine colon tumor model CT26. Specifically, we used state‐of‐the‐art immunoprecipitation and mass spectrometric methodologies to isolate >8000 peptide targets from the CT26 tumor cell line. The selection of the target candidates was then based on two separate approaches: RNAseq analysis and the HEX software. The candidates peptides were then tested in our previously described oncolytic cancer vaccine platform PeptiCRAd, a vaccine platform that combines an immunogenic oncolytic adenovirus (OAd) coated with tumor antigen peptides. The results demonstrated that our pipeline can be exploited for personalised cancer therapeutic vaccine.
Efficient and safe delivery of multiple mRNA using non‐integrative bacteriophage‐chimeric retrovirus‐like particles for cell therapy and vaccination perspectives.
1: Flash Therapeutics
Gene therapy approaches show that there is no universal delivery tool for all therapeutic strategies. Compared to DNA delivered‐therapies mediated by integrative lentiviral vectors and AAV, RNA therapies are expected to be more versatile, cover a broad range of applications with minimal regulatory concerns and thus address a large variety of diseases. The technology targets applications in which a transient expression is expected. Once engineered, patient's cells become the effective medicine.
RNA delivery mediated by a lentiviral particle is an attractive approach as it combines most of the inherited properties of lentiviral vectors (cell entry and tropism) without the potential adverse effects from long‐lasting expression or genomic integration. From a therapeutic perspective, a great advantage of such system is its ability to carry different RNA species.
Here, we show the potential of bacteriophage‐chimeric retrovirus‐like particles called LentiFlash®
Characterization of CAR‐mediated T cell dysfunction in mice with solid tumors
1: IDIBAPS 2: Hospital Clinic of Barcelona 3: Centre for Genomic Regulation (CNAG‐CRG)
Despite the unprecedented results of CAR‐T cells in hematologic malignancies, efficacy of CAR‐T cells in solid tumors remains poor. A key question is how T cell fitness impacts on CAR‐T cell‐mediated tumor elimination. Here we hypothesized that continuous antigen exposure in solid tumors leads to a CAR‐T cell intrinsic program of dysfunction that prevents tumor control.
To test this hypothesis, we generated a xenograft mouse model of Her2+ solid tumors in which anti‐Her2 CD28‐based CAR‐T cells go through two different phases: i) an initial phase of T cell activation that leads to tumor regression (named “effective CAR‐TILs”) and ii) a second phase of progressive loss of T‐cell function and tumor progression (named “dysfunctional CAR‐TILs”). The phenotype, function and transcriptional signature of tumor infiltrating T cells was assessed after T cell isolation from tumors at effective and dysfunctional phases using cell sorting. PD‐1 was found to be expressed in both effective and dysfunctional TILs, whereas Ki67 was only expressed in effective CAR‐TILs. Effective but not dysfunctional CAR‐TILs displayed cytotoxic activity and released IFNγ when co‐cultured with tumor cells ex vivo. Contrary to previous reports, this loss of effector functions in dysfunctional CAR‐TILs was not reversible after overnight resting.
Transcriptome analysis by RNA sequencing revealed significant differences between effective and dysfunctional CAR‐TILs, with more than 2.000 differentially expressed genes. Enriched gene signatures will be discussed. Altogether, our results suggest that CAR‐T cell dysfunction needs to be addressed to unleash the full potential of CAR‐T cell therapy in solid tumors.
In vivo
CAR T cell generation in humanized NSG‐SGM3 mice
1: Paul‐Ehrlich‐Institut 2: Frankfurt Cancer Institute, Goethe University
Humanized pre‐clinical mouse models are a powerful tool to evaluate novel immunotherapies. For example, CD34+ humanized NSG‐SGM3 (huSGM3) mice develop human cells not only of the lymphoid but also of the myeloid lineage due to transgenic expression of human SCF, GM‐CSF and IL‐3. Therefore, this model better reflects the human immune system than the widely used humanized NSG model (huNSG). In here, we assessed the huSGM3 model for in vivo generation of CD19‐CAR T cells by single intravenous injection of T cell specific lentiviral vectors (LVs) CD4‐LV and CD8‐LV. 6 of 12 CD4‐LV and 8 of 14 CD8‐LV injected mice developed CAR T cells detectable in blood directly and/or by depletion of CD19+ B cells 1‐3 weeks after LV injection. Overall, in vivo CAR T cell generation was about two times less efficient in huSGM3 than in huNSG mice. Interestingly, the CD4‐LV group showed the least robust CAR T cell development, which correlated with cytokine patterns, such as increased IL‐15 and decreased GM‐CSF, that are typical for monocyte and macrophage‐associated activities. Further in vitro assays identified macrophages as potential interceptor for in vivo gene transfer. Refining CD4‐LV and CD8‐LV with a less immunogenic surface by using β2M‐/‐/CD47 overexpressing vector production cells, indeed improved transduction efficiencies of lymphocytes cultivated in presence of macrophages. Finally, the optimized CD4‐LV mediated in vivo CAR T cell generation, solely in CD4+ lymphocytes, in all injected huSGM3 mice. The data emphasize the relevance of innate immune responses for the in vivo generation of CAR T cells.
TEM‐GBM: an open‐label, Phase I/IIa dose‐escalation study evaluating the safety and efficacy of genetically modified Tie‐2 expressing monocytes to deliver IFN‐a within glioblastoma tumor microenvironment
1: San Raffaele Telethon Institute for Gene Therapy (HSR‐TIGET) 2: Hematology and Bone Marrow Transplant Unit ‐ San Raffaele Hospital – Milano 3: Genenta Science 4: Neuro‐Oncology Unit ‐ Istituto Neurologico Carlo Besta – Milano 5: Neurosurgery Unit ‐ Policlinico Gemelli – Roma 6: Neuroradiology Unit ‐ Istituto Neurologico Carlo Besta – Milano 7: Neurosurgery Unit ‐ Istituto Neurologico Carlo Besta – Milano 8: Neuropathology Unit ‐ Istituto Neurologico Carlo Besta – Milano 9: Neuro‐Oncology Unity ‐ San Raffaele Hospital – Milano
Temferon, is a macrophage‐based treatment relying on ex‐vivo transduction of autologous HSPCs to express immune‐payloads within the TME. Temferon, targets IFN‐a to a subset of tumor‐infiltrating macrophages the Tie‐2 expressing macrophages (TEMs).
As of 31st May 2021, 15 patients received Temferon (D + 0) with follow‐up of 3 – 693 days. After conditioning neutrophil and platelet engraftment occurred at D + 13 and D + 13.5. Temferon‐derived differentiated cells were found within 14 days post treatment and persisted albeit at lower levels up to 18‐months. Very low concentrations of IFNa in the plasma (8.7 pg/ml‐D+30) and in the CSF (1.6 pg/ml‐D+30) were detected. 5deaths occurred at D + 322, +340, +402, +478 and +646 due to PD, and 1 at D + 60 due to complications following the conditioning regimen. 8patients had PD (D‐11 to +239) as expected for this tumor type. SAEs include GGT elevation (possibly related to Temferon) and infections, venous thromboembolism, brain abscess, hemiparesis, seizures, anemia and general physical condition deterioration, compatible with ASCT, concomitant medications and PD. 4patients underwent 2ndsurgery. Recurrent tumors had gene‐marked cells and increased expression of ISGs compared to first surgery. In one patient, a stable lesion had a higher proportion of T cells and TEMs within the myeloid infiltrate and an increased ISGs than in the progressing lesion, detected in the same patient. Tumor‐associated clones expanded in the periphery. TME characterization by scRNA and TCR‐sequencing is ongoing. To date, Temferon is well tolerated, with no DLTs identified. The results provide initial evidence of Temferon potential to activate the immune system of GBM patients.
Real‐world experience with axicabtagene ciloleucel in aggressive B‐cell lymphoma confirms correlation of
in‐vivo
expansion with treatment outcome
1: UMC Hamburg‐Eppendorf 2: Stem Cell Transplantation 3: Institute for Transfusion Medicine 4: Department of Intensive Care Medicine 5: Department of Hematology and Oncology 6: Department of Neurology
A direct association between axicabtagene ciloleucel (axi‐cel) in‐vivo expansion and patient outcome was suggested from clinical‐trial data, but real‐world data has largely been missing. We recently described dedicated digital‐PCR assays facilitating precise monitoring of CAR‐T‐cell numbers in vivo. Using the axi‐cel assay we prospectively quantified CAR‐T cells in 21 consecutive patients treated for aggressive B‐cell lymphoma at our clinic. CAR‐T cell numbers per μL were correlated with clinical outcome as assessed per institutional practice. Statistical analyses were performed using SPSS software. We applied the calculated median peak CAR‐T‐cell count (16.14 per μL) to divide the 21 patients into two groups ‐ “strong expanders” peaked at ≥16.14/mL, whereas “weak expanders” peaked below the median. Strong expansion was associated with significantly more day‐30 objective responses (91% vs. 40%, P = 0.02). Besides peak CAR‐T cells ≥16.14 (P < 0.001), normal platelet counts at start of lymphodepletion (P < .001), no prior stem cell transplantation (P = 0.04), and peak CAR‐T cells as continuous variable (P = 0.03) were associated with better progression‐free survival (PFS) in univariate analysis. Notably, weak CAR‐T expansion remained associated with shorter PFS (RR 0.15, 95% CI 0.04‐0.59, P = 0.007) after adjustment for platelet counts and prior SCT, as also did low platelet counts. In conclusion, our data confirms the immediate correlation of axi‐cel levels and clinical outcome in a real‐world setting. The observed cut‐off allowing segregation of weak and strong expanders with respective outcomes will need confirmation and fine tuning in larger patient cohorts.
IL23R‐CAR‐Tregs ‐ a novel approach to treat Crohn's disease patients
1: Sangamo Therapeutics
Crohn's disease (CD) is a painful and debilitating autoimmune disease that may lead to life‐threatening complications. It is caused by inflammation of the digestive tract, leading to abdominal pain, severe diarrhea, weight loss, and malnutrition, severely impacting patients' lives. Clinical and preclinical data suggest a critical role of regulatory T cells (Tregs) and deregulation of the IL‐23/TH17 axis in disease pathogenesis. Accordingly, the IL‐23/TH17/Treg axis is targeted by anti IL‐23 monoclonal antibodies to treat inflammatory bowel disease (IBD), including CD patients. We investigated expression of the IL23‐receptor (IL‐23R) in the gut of CD samples from biobank versus control samples (disease‐free gut). We observed that IL23R is overexpressed in the gut of CD patients, making this receptor an attractive target for a chimeric antigen receptor (CAR). Therefore, we are developing an allogenic IL23R‐CAR‐Treg to treat CD patients. The IL23R‐CAR lead candidate is composed of an anti‐IL23R‐scFv identified through a large library screening which obtained more than 100 potential hits. This CAR showed high specificity to IL23R with an undetectable tonic signaling, and a specific CAR‐dependent suppressive activity in vitro. A dextran sodium sulfate (DSS)‐IBD‐mouse model confirmed these findings in vivo. In summary, our data suggest that an IL23R‐CAR‐Treg product candidate could represent a promising therapeutic approach for the treatment of CD. Its mode of action could also pave the way for treating other IBD and chronic autoimmune pathologies involving IL23R.
Engineering Regulatory T cells with constitutive expression of FOXP3 as a phenotype‐lock enhances efficacy and safety of Treg cell therapies
1: Quell Therapeutics 2: University College London
Regulatory T cells (Tregs) can modulate immune responses through multiple mechanisms; therefore, their therapeutic use has been proposed to induce immune tolerance in transplantation, autoimmune diseases, and chronic inflammatory conditions. FOXP3 is considered the master transcription factor of Tregs, driving expression of CD25, CTLA‐4 and repression of pro‐inflammatory cytokines such as IL‐2. It has been shown that some cells that bear all the hallmarks of Tregs can, under certain conditions, lose expression of FOXP3 and acquire pathogenic features, such as expression of effector cytokines. This outcome would be particularly problematic in the context of antigen‐specific Treg therapies – posing a significant safety issue. Here we show that transduction of bona fide Tregs with the FOXP3 gene increases and stabilises the expression of FOXP3 without impacting cell survival and proliferative capacity. Importantly, we show that under in vitro and in vivo conditions of cell stress that can drive loss of FOXP3 expression, constitutive expression of FOXP3 maintains Treg phenotype and function. In addition, the antigen‐specific suppressive function of engineered CAR‐Tregs is improved by the enhanced expression of FOXP3. Overall, we show that engineering bona fide Tregs to constitutively express FOXP3 offers a safety mechanism for Treg cell therapy with no disadvantage in cell growth or function, providing a significant benefit for the clinical application of antigen‐specific Treg products.
CAR T cell derived exosomes: structural and functional characterization.
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 3: Haematology Unit, Reina Sofia Hospital, Cordoba 4: Biopathology and Regenerative Medicine Institute (IBIMER), Centre for Biomedical Research (CIBM), University of Granada 5: Department of Human Anatomy and Embryology, Faculty of Medicine, University of Granada 6: Biomedical Research Institute (IBS. Granada)
Adult patients diagnosed with mature B cells neoplasms usually present modest outcomes after standard therapies. In these cases, the use of immunotherapy using chimeric antigen receptors (CARs) led to a complete remission of advanced B lineage malignancies or partial remission in patients with chronic lymphocytic leukaemia (CLL). Even though the impressive results, a high tumour mass at the time of treatment with CAR T cells is normally associated with worse results mainly associated, among other factors, with the high concentration of exosomes (EXOs) from tumour cells in the peripheral blood of patients. While the role of EXOs as diagnostic markers is more than evident, their therapeutic use is much more limited, due to the lack of specificity towards cancer cells, the existence of HLA molecules in EXOs that may induce rejection, and the low efficiency of EXOs isolation and purification. We suggest an innovative application in the field of nanotechnology and immunotherapy producing CAR+ EXOs from anti CD19‐CAR T cells. We intend to solve the limitation associated with the production of EXO‐CAR Ts by setting up a proof‐of‐concept protocol, using CAR T cells, and modulating the expression of genes involved in exosome biosynthesis. Preliminary data will be presented showing that the deletion a specific gene involved in EXOs biogenesis induces an increase in the amount of cytotoxic exosomes production according to AFM, SEM and NanoSight analysis.
Exploiting genome editing and WT1‐specific T cell receptors to redirect T lymphocytes against acute myeloid leukemia
1: Experimental Hematology Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy 2: Intellia Therapeutics, Cambridge, MA, USA 3: Immunogenetics, Leukemia Genomics and Immunobiology Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy 4: Immunohematology and Transfusion Medicine, San Raffaele Scientific Institute, Milan, Italy 5: Hematology and Bone Marrow Transplantation Unit, San Raffaele Scientific Institute, Milan, Italy 6: Pathology Unit, San Raffaele Scientific Institute, Milan, Italy 7: Vita Salute San Raffaele University, Milan, Italy
Dressing up T lymphocytes with anti‐tumor T‐cell receptors (TCR) is a promising approach for targeting every surface and intracellular tumor target. However, the selection of the ideal tumor‐specific TCR and the need of clinically compliant manufacturing protocols, remain major hurdles in the TCR immunotherapy field. Focusing on Wilms Tumor 1 (WT1), a tumor antigen expressed on a multitude of blood and solid malignancies, and by longitudinal monitoring T cell functionality and dynamics in 14 healthy donors, we isolated 19 WT1‐specific TCRs recognizing peptides restricted by different HLA alleles and displaying a wide range of functional avidities, including some in the low nM range. Of interest, 7 receptors recognized HLA‐A*02:01‐restricted peptides naturally processed by primary leukemic blasts (2 TCRs specific for a less explored, though immunogenic, portion of the WT1 protein). By applying a funnel selection strategy, we selected 2 high avidity TCRs for further investigation. TCR targeted integration into the TRAC locus combined with TRBC knock‐out, was successfully employed to generate a T cell product with an improved biosafety profile while preserving T cell fitness. A nearly complete redirection of T cell specificity was achieved also when circulating T cells harvested from AML patients were used as starting material. Of note, one TCR showed antigen‐specific responses in CD4 and CD8 T‐cells, and efficiently eliminated patients' blasts in vitro and in vivo in the absence of off‐tumor toxicity. WT1‐TCR engineered T‐ are currently being advanced into clinical development for AML immunotherapy and potentially other WT1‐expressing tumors.
Lipid nanoparticles (LNPs) as a superior CRISPR/Cas9 delivery modality for highly efficient multiplex gene editing of T cells for adoptive cell therapy
1: Intellia Therapeutics 2: San Raffaele Scientific Institute
Despite clinical success of adoptive cell therapy in hematological cancers, technical and biological barriers prevent this approach from achieving full potential. Genome editing of T‐cells using CRISPR/Cas9 has the potential to address many of these limitations, enabling efficient gene knockout (KO) or targeted insertion (TI) of transgenes. Traditionally, CRISPR/Cas9‐editing of T‐cells has involved electroporation of Cas9/sgRNA‐RNPs, however electroporation impacts T cell viability, expansion, and alters gene expression. Additionally, performing multiple gene edits in a single electroporation step can lead to chromosomal translocations across target sites. Herein we report the use of lipid nanoparticles (LNPs) to deliver CRISPR/Cas9 to T‐cells, obviating the need for electroporation. Relative to electroporation, T‐cells engineered using LNPs possessed similar editing rates with a 3‐fold increase in viable cell expansion, a favorable memory phenotype, and increased functional activity in vitro and in vivo. Lower toxicity associated with LNP delivery permitted sequential editing of target genes, reducing target‐to‐target translocation rates to near background levels. Using this technology, we developed a modular platform for sequential multiplex genome engineering, encompassing 4 or 5 KOs and up to 2 TIs with high efficiency and yield, enabling manufacture of autologous or allogeneic T‐cells, or T‐cells with customizable edits for solid tumor indications. These processes consistently achieve >80% frequency of T‐cells harboring all intended edits, including >95% KO of TRAC, TRBC, HLA Class‐I and Class‐II genes, and >80% TI of TCR or CAR sequences, while maintaining high rates of expansion with a favorable early memory phenotype and potent functional activity.
Pre‐clinical development of CAR‐T Cells directed against the tumour antigen 5T4 for the treatment of Acute Myeloid Leukaemia
1: Oxford BioMedica 2: University of Birmingham
Trophoblast glycoprotein (5T4) is a transmembrane oncofoetal antigen that is expressed on a wide range of solid tumours, but has limited expression on normal tissue. This, together with the fact that 5T4 is expressed on cancer stem cells, suggests that 5T4 could make an excellent target for cancer immunotherapy. In spite of data showing expression of 5T4 on most solid tumours, very little is known about the expression of 5T4 on haematological malignancies. To investigate this, we screened samples collected from patients with Acute Myeloid Leukaemia (AML) for cell‐surface expression of 5T4 by flow cytometry. Our data demonstrate that 5T4 is expressed on AML blasts as well as on the putative leukaemic stem cell (LSC) population. As a result, Oxford Biomedica has developed OXB‐302 a CAR‐T therapy specifically targeting cells expressing 5T4. To demonstrate that OXB‐302 cells are effective against AML cells, we performed a co‐culture assay against 5T4‐positive AML cell lines Kasumi‐1 and THP‐1. We show that OXB‐302 cells produce interferon‐gamma and granzyme B, and induce cell killing against both 5T4‐expressing cell lines. In addition, we identified a LSC population within Kasumi‐1 cells and were able to demonstrate the killing of these LSCs following short‐term co‐culture with OXB‐302 cells and subsequent assessment in colony‐forming assays. We conclude that OXB‐302 cells could be a promising treatment for patients with AML. OXB‐302 is now in development with efficacy and safety studies ongoing.
Cytolytic activity of CAR T cells and maintenance of their CD4+ fraction is critical for optimal antitumor activity in preclincial solid tumor models
M Csaplar1 J Szollosi1 S Gottschalk2 G Vereb1
1: University of Debrecen 2: St. Jude Children's Research Hospital
Correlative studies of clinical studies for hematological malignancies have implicated that less differentiated, CD8+ dominant CAR T cell products have greater antitumor activity. Here, we have investigated if the differentiation status of CAR T cell products affects their antitumor activity in preclinical models of solid tumors. We explored if different activation/expansion protocols, as well as different co‐stimulatory domains in the CAR construct influence the short and long term efficacy of CAR T cells against HER2‐positive tumors. We generated cell products that range from the most differentiated (CD28.z; OKT3‐antiCD28 expansion) to the least differentiated (41BB.z; OKT3‐RetroNectin/ LymphoONE expansion) as judged by cell surface expression of the differentiation markers CCR7 and CD45RA. While the effect of differentiation status was variable with regard to antigen‐specific cytokine production, the most differentiated CD28.z CAR T cell products, which were enriched in effector memory T cells, had the greatest target‐specific cytolytic activity in vitro. These products also had a greater proliferative capacity and maintained CD4+ T cells upon repeated stimulation in vitro. In vivo, differentiated CD28.z CAR T cell also had the greatest antitumor activity, resulting in complete response. Our results highlight that it is critical to optimize CAR T cell production and that optimal product characteristics might depend on the targeted antigen and/or cancer.
Allogeneic NKG2D‐CAR T cells as universal anticancer treatment: new manufacturing strategy based on CRISPR/Cas9
1: Institute of Biology and Molecular Genetics (IBGM), University of Valladolid (UVa)‐CSIC, Valladolid, Spain. 2: Fundación para la Investigación Biomédica H12O, H12O‐CNIO Hematological Malignancies Clinical Research Group, CIBERONC, Madrid, Spain. 3: Department of Pathology and Laboratory Medicine. University of Pennsylvania, Philadelphia, USA.
One of the most promising advances in cancer treatment is chimeric antigen receptor (CAR)‐T cell therapy, in which genetically‐modified T cells are redirected against the tumour antigen of interest. Autologous anti‐CD19 CAR‐T cells are successfully used against haematological malignancies; however, high costs, manufacturing delay and CD19‐negative relapses are major limitations of these therapies.
To address these challenges, we developed a novel allogeneic CAR‐T cell therapy based on NKG2D receptor, which targets eight different ligands upregulated in both solid and haematological tumours, thus being less prone to resistance. Moreover, the allogeneic approach will result in reduced manufacturing costs and immediate availability. Additionally, culturing was optimized to maintain memory T cell phenotype, as it improves in vivo CAR‐T survival, expansion and long‐term persistence.
To that end, T cells were isolated from peripheral blood mononuclear cells of healthy donors, activated, and cultured with different interleukins. Then, CRISPR/Cas9 technology was used to eliminate TCR and HLA‐I to avoid the risk of graft‐versus‐host disease and immune rejection, respectively. Subsequently, TCR‐deficient T cells were purified and lentiviral vector was used to integrate the NKG2D‐CAR. The CAR T cell product was characterized by flow cytometry.
Our strategy resulted in approximately 40% TCR‐/HLAI‐ T cells, from which 60% express the NKG2D‐CAR. Of these allogeneic NKG2D‐CAR‐T cells, 60‐80% maintain the central memory T cell phenotype. Further research is ongoing to assess the potential utility of this novel CAR‐T therapy as universal anticancer treatment.
Novel personalized cancer vaccine platform based on Bacillus Calmette‐Guèrin
E Ylosmaki1 2 3 4
1: University of Helsinki 2: Laboratory of Immunovirotherapy, Drug Research Program, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland 3: TRIMM, Translational Immunology Research Program, University of Helsinki, Finland 4: iCAN Digital Precision Cancer Medicine Flagship, University of Helsinki 5: Department of Molecular Medicine and Medical Biotechnology and CEINGE, Naples University 24 Federico II, 80131, Naples, Italy 6: Serum Institute of India Pvt Ltd, Calcutta, India 7: ValoTherapeutics Oy, Helsinki, Finland 8: Pharmaceutical Biophysics Research Group, Drug Research Program, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland
Intratumoural bacillus Calmette‐Guérin (BCG) therapy, one of the earliest immunotherapies, can lead to infiltration of immune cells into a treated tumour. Here, we have developed a novel cancer vaccine platform based on BCG that can direct BCG‐induced immune responses against tumour antigens. By physically attaching tumour‐specific peptides onto the mycobacterial outer membrane, we were able to induce strong systemic and intratumoural T cell‐specific immune responses towards the attached tumour antigens. These therapeutic peptides can be attached to the mycobacterial outer membrane using a cell‐penetrating peptide sequence derived from human immunodeficiency virus Tat, N‐terminally fused to the tumour‐specific peptides. Alternatively, therapeutic peptides can be conjugated with a poly‐lysine sequence N‐terminally fused to the tumour‐specific peptides. Using two mouse models of melanoma and a mouse model of colorectal cancer, we observed that the anti‐tumour responses of BCG can be significantly improved by coating the BCG with tumour‐specific peptides. In addition, by combining this novel cancer vaccine platform with anti‐PD‐1 immune checkpoint inhibitor therapy, the number of responders to anti‐PD‐1 immunotherapy can be significantly increased.
Functional characterization of CD3‐specific DNA aptamers
1: Ixaka
A number of therapeutic strategies that modulate T cell immunity by targeting CD3 protein, the signaling component of the T‐cell receptor, has been used in clinics to develop immunosuppressive agents in transplantation (OKT3), autoimmune type I diabetes and psoriasis (teplizumab and otelixizumab). More recently, bispecific therapies retargeting the cytotoxic activity of effector T cells by binding to CD3 to tumors expressing tumor‐associated antigen have demonstrated striking activity in patients across different cancers (blinatumomab and catumaxomab).
Ixaka is developing CD3‐specific aptamers as new targeting agents to specifically deliver in vivo anti‐cancer therapeutics to T lymphocytes. We have screened and selected G‐quadruplex‐based DNA aptamers that bind to CD3 ɛ/γ or CD3 ɛ/δ protein complexes expressed on human T lymphocytes. By combining biophysical and on‐cell binding methods, CD3‐specific interactions with nanomolar range affinities were reported with the best candidates. Sequence‐optimized derivatives have been engineered with abasic sites, base substitutions or N‐terminal functionalization that showed improved affinity, specificity and serum stability. They exhibited remarkable cross‐specificity with human, mouse and cynomolgus isoforms and recognized an epitope on CD3 different from the epitopes covered by OKT3 and other T‐cell activating antibodies. Accordingly, these aptamers did not induce any activation of primary human T lymphocytes nor the internalization of CD3 receptors upon binding, which is of particular interest for the development of inert targeting agents without any immunomodulating properties. All together these lead CD3 aptamers exhibit unique properties and qualify for further preclinical and clinical development of CD3‐targeting therapeutics.
Single‐cell RNA‐seq informs efficient reprogramming of human somatic cells to cross‐presenting dendritic cells
F F Rosa1 2 3
1: Wallenberg Centre for Molecular Medicine, Lund University, Lund, 221 84, Sweden. 2: Molecular Medicine and Gene Therapy, Lund Stem Cell Centre, Lund University, Lund, 221 84 Sweden. 3: Centre for Neuroscience and Cell Biology, University of Coimbra, Coimbra, 3004‐517, Portugal. 4: Skolkovo Institute of Science and Technology, Moscow, 143026, Russia. 5: Department of Pathology and Immunology, University of Geneva Medical School, Geneva, CH‐1211, Switzerland. 6: Division of Molecular Hematology, Department of Laboratory Medicine, Lund University, Lund, 221 84, Sweden. 7: Department of Hematology, Skåne University Hospital, Lund, 221 84, Sweden.
Type‐1 dendritic cells (cDC1s) are critical for anti‐tumor immunity, as response to checkpoint blockade and adoptive T‐cell transfer rely on their ability to prime CD8+ T‐cells. Vaccination strategies to harness this unique DC subset for immunotherapy have been limited by their rarity in peripheral blood and lack of homogeneous cell sources. We have previously identified PU.1, IRF8 and BATF3 transcription factors as sufficient to induce cDC1 fate in mouse fibroblasts but reprogramming efficiency in human cells was low. Here, we investigated single‐cell transcriptional dynamics during the reprogramming of human embryonic fibroblasts to cDC1. Human induced cDC1s (hiDC1s) acquired global cDC1 transcriptional profile but not signatures characteristic of other subsets. Inflammatory signaling and the DC1‐inducing transcription factor network were identified as key drivers of the successful reprogramming process. Combining IFN‐γ, IFN‐β and TNF‐α with constitutive expression of the three factors improved reprogramming efficiency by 190‐fold and allowed efficient hiDC1 generation from human adult cells with xeno‐free protocols. HiDC1s uptake dead cells, respond to stimuli, secrete inflammatory cytokines and perform antigen cross‐presentation. Mechanistically, PU.1 shows dominant and independent chromatin targeting at promoter and enhancer regions in open chromatin and recruits IRF8 and BATF3 to shared binding sites, silencing fibroblast genes and activating cDC1 genes. These findings provide insights into human cDC1 specification and reprogramming and represent a platform for generating patient‐specific cDC1s, a long‐sought DC subset for cancer immunotherapy.
Chimeric Antigen Receptor (CAR)‐T cells targeting EpCAM for the treatment of pediatric Germ Cell Tumors (pGCTs)
1: Institute of Experimental Hematology, Hannover Medical School, Hannover, 30625, Germany 2: REBIRTH Center for Translational Regenerative Medicine, Hannover Medical School, Hannover, 30625, Germany 3: Research Group‐Reprogramming and Gene Therapy, Institute of Experimental Hematology, REBIRTH, Hannover Medical School, Hannover, 30625, Germany 4: Division of Immune Receptors and T Cell Activation, Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, 1090, Austria 5: Department of Pediatric Hematology and Oncology, University Hospital Bonn, Bonn, 53105, Germany 6: Regensburg Center for Interventional Immunology (RCI), Dept. Genetic Immunotherapy, University Hospital Regensburg, Regensburg, 93053, Germany 7: Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA
Pediatric Germ Cell Tumors (pGCTs) represent ∼3% of all malignancies found in children and arise in ovaries, testes and other locations, including the abdomen and chest. Most pGCTs overexpress the Epithelial Cell Adhesion Molecule (EpCAM) which is associated with poor prognosis. To develop a CAR‐T cell therapy approach for pGCTs we analyzed EpCAM expression in a panel of GCT cell lines (2102EP, JAR, GCT27 and NCCIT). Moreover, we cloned a 2nd generation CAR construct containing an anti‐EpCAM single chain variable fragment (scFv) into a SIN lentiviral vector. CAR expression was confirmed in Jurkat and human primary T cells by flow cytometry and Western blot analyses. A triple parameter reporter T cell line (TPR) expressing specific fluorescent proteins in response to NF‐kB, NFAT and AP‐1 activation was used to demonstrate the ability of the EpCAM‐CAR to induce signaling upon antigen recognition. In addition, IL2 and IFNγ secretion as well as the expression of T cell‐specific activation markers were detected upon stimulation of primary CAR‐T cells with EpCAM‐positive tumor cell lines. Bioplex analysis of 48 human cytokines revealed significant secretion for almost all cytokines after CAR‐T cell stimulation. Moreover, cytotoxicity against tumor cells was shown by measuring the Lactate Dehydrogenase release and correlated to the level of EpCAM expression on the surface of target cells. Cytotoxicity was confirmed by the xCELLigence assay as well. Currently, the functionality of newly generated EpCAM‐specific TRUCK constructs is tested and compared to the 2nd generation CAR construct to further improve CAR/TRUCK T cell‐based immunotherapy for pGCTs.
Targeted Nano‐particles (TNP) technology as a universal and versatile platform for gene therapy.
1: IXAKA
Intracellular delivery of nucleic acids to mammalian cells using non‐viral gene delivery remains a challenge both in vitro and in vivo, with transfections often suffering from variable efficacy and limited stability. Ixaka's TNP platform is a unique technology that combines the versatility of negative and positive oligopeptide‐modified poly(beta‐amino ester)s (OM‐PBAEs) as biodegradable and safe transfection material together with the gene transfer efficiency of lentiviral vectors (LV). Here we report on the microfluidics‐based formulation of LV particles lacking the VSV‐G protein (“bald”) encapsulated with OM‐PBAE polymers. Biophysical methods (NTA, DLS, Videodrop) have been implemented and are used in routine to monitor nano‐particles size, concentration and aggregates. The nano‐particles (NP) showed a remarkable stability at room temperature as confirmed by transduction efficiency and biophysical methods. In vitro, targeted NP efficiently transduced a wide range of mammalian cells including human and mouse cell lines and primary immune cells, without pronounced cell toxicity. In addition, the use of a cell‐specific promoter drives the expression of the transgene in the cells of interest. Importantly, in vitro and in vivo transduction efficiency was achieved without the need of CD3/CD28 activation and cytokines addition normally required with VSV‐G pseudotyped LV for immune cells transduction. Cell reprogramming was achieved when intracellularly expressed reporter system (GFP) and surface receptor (Chimeric Antigen Receptor) were used as transgenes. Thus, Ixaka's versatile platform offers the potential to target and reprogram a broad range of therapeutic cells not limited to the immuno‐oncology field but also various genetic disorders, neurological and ocular diseases.
A personalized anti‐cancer vaccine for melanoma based on an approved vaccine against measles, mumps, and rubella
E Ylosmaki1 2 3 4 7
1: University of Helsinki 2: Drug Research Program (DRP) ImmunoViroTherapy Lab (IVT), Division of Pharmaceutical Biosciences, Faculty of Pharmacy, Viikinkaari 5E, University of Helsinki, 00790 Helsinki, Finland 3: Helsinki Institute of Life Science (HiLIFE), Fabianinkatu 33, University of Helsinki, 00710 Helsinki, Finland 4: Translational Immunology Program (TRIMM), Faculty of Medicine Helsinki University, Haartmaninkatu 8, University of Helsinki, 00290 Helsinki, Finland 5: Pharmaceutical Biophysics Research Group, Drug Research Program, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland 6: Department of Molecular Medicine and Medical Biotechnology, Naples University “Federico II”, S. Pansini 5, Italy 7: iCAN Digital Precision Cancer Medicine Flagship, University of Helsinki
Common vaccines for infectious diseases have been repurposed as cancer immunotherapies. Intratumoural administration of these repurposed vaccines can induce immune cell infiltration into the treated tumour. Here, we have used an approved trivalent live attenuated measles, mumps, and rubella (MMR) vaccine in our previously developed PeptiENV anti‐cancer vaccine platform. Intratumoural administration of this novel MMR‐containing PeptiENV anti‐cancer vaccine significantly increased both intratumoural as well as systemic tumour‐specific T cell responses. In addition, PeptiENV therapy, in combination with immune checkpoint inhibitor therapy, significantly improved tumour growth control and survival as well as increased the number of mice responsive to immune checkpoint inhibitor therapy.
Feasibility of generating universal and physiological CAR T cells for the treatment of type B malignances.
1: Fundacion Publica Andaluza Progreso y Salud/Genyo 2: Hospital Clínic in Barcelona 3: Unidad de Terapia Celular IMIBIC‐HURS, Hospital Reina Sofía, Córdoba
Autologous T cells expressing the Chimeric Antigen Receptor (CAR) have been approved as medicaments against type Bmalignances. In spite of the impressive benefit, there are still important limitations. A main drawback comes from its autologous nature that impedes that all the selected patients can be treated and that also precludes the standardization as a medicament. Off‐the‐shelf CAR‐T cells can overcome the histocompatibility problem, however they present reduced persistence. Another downside of actual CAR‐T cells is the strong and unregulated expression of the CAR that can lead to T cell activation in the absence of target (tonic signalling), cytokine release syndrome (CRS) and early CAR‐T cell exhaustion. All together reduces efficacy and increase the risk of side effects. Here we investigated the feasibility of generating TCRKO CAR‐T cells expressing the CAR physiologically in order to reduce toxicity and increase the survival of TCRKO allogeneic CAR‐T cells. We first analysed the efficacy and potential safety issues that arise during disruption of the TCR gene using CRISPR/Cas9. We found that efficient TCRKO leads to on‐target large deletions by microhomology‐mediated repair mechanism (alt‐EJ) indicating a potential safety risk of this procedure that need monitoring. Importantly, TCRKO‐T cells maintain similar phenotype to WT T cells. We next generated physiological TCRKO‐CAR‐T cells using lentiviral vectors (LVs) expressing the CAR through the WAS gene promoter and showed efficient anti‐tumor activity and a reduced tonic signalling compared to TCRKO‐CAR‐T expressing the CAR through the EF1alfa promoter.
Manufacturing of tightly controllable Adapter CAR T cells using the CliniMACS Prodigy® platform
1: Miltenyi Biotec
Chimeric antigen receptor T cells (CAR T cells) represent an effective therapeutic modality for B cell malignancies. However, its wide application in other indications, including patients with solid tumours, is hampered by the unavailability of single tumor‐specific antigens, antigen escape, T cell exhaustion, and overall lack of efficacy and control of CAR T cell responses.
Adapter CAR T cells (AdCAR T cells) offer a possibility to address some of these challenges by using an adapter molecule to redirect AdCAR T cells to specific tumour antigens, enable multitargeting, switch cytotoxic responses on and off, as well as finetune the activity of T cells by adjusting the adapter concentration. The AdCAR system used in this study, harbors an scFv which is specific to a neo‐epitope like structure derived from biotin, which can be easily conjugated to a variety of therapeutic proteins, like monoclonal antibodies or fragments.
In this study manufacturing of AdCAR T cells using the fully closed and automated CliniMACS Prodigy® platform was investigated. AdCAR T cells generated on this platform showed efficient, strictly adapter‐dependent tumour cell lysis in in vitro assays as well as in pre‐clinical mouse models. Overall, our results suggest that highly functional AdCAR T cells can be manufactured using the CliniMACS Prodigy® platform.
Selecting AAV capsids for optimised homology‐dependent repair in T cells.
1: Children's Medical Research Institute, University of Sydney 2: UCL Institute of Child Health 3: Kid's Research, Sydney Children's Hospital Network 4: Military Institute of Medicine, Poland
T‐cells are promising targets for the development of gene therapies for immune‐deficiencies and cellular immune‐therapies for malignant tumours. The latter has been successfully applied in the context of chimeric antigen receptor (CAR) T‐cell therapies. Conventionally, lentiviral vectors have been used to semi‐randomly insert the CAR coding sequence into the T‐cell genome. To improve safety and minimise exhaustion of CAR T‐cells, novel approaches utilising targeted gene editing with the help of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 were developed. This approach, pioneered by Eyquem et al., 2017, uses CRISPR/Cas9‐driven homology‐dependent repair (HDR)‐mediated targeted insertion of a CAR cassette delivered by an adeno‐associated viral vector (AAV). However, we hypothesised that because AAV6, the natural serotype frequently used to target human primary T‐cells, has not evolved for those biomedical applications and might not be the best vector to mediate an optimal level of HDR.
Therefore, we developed an AAV bioengineering platform to allow for AAV selection based on their ability to support on‐target CRISPR/Cas9‐mediated HDR into the TRAC locus in human T‐cells. We performed a screen of three highly diverse AAV capsid libraries and identified several variants that show improved levels of on‐target HDR. In addition, the new AAV variants led to decreased level of toxicity and improved T‐cell expansion, when compared to AAV6 and other AAV variants recently developed for T‐cell targeting. We are currently performing studies that aim to evaluate the ability of our new capsid variants support generation of highly functional CAR T‐cells and their activity against various tumour types.
Combination of nanomedicine and immunotherapy for the treatment of Neuroblastoma
1: Unidad de Terapias Avanzadas, Oncología, Hospital Infantil Universitario Niño Jesús, Madrid, 28009, Spain 2: Fundación de Investigación Biomédica, Hospital Infantil Universitario Niño Jesús, Madrid, 28009, Spain 3: Instituto de Investigación Sanitaria La Princesa, Madrid, Spain 4: Dpto. Materiales y Producción Aeroespacial, ETSI Aeronáutica y del Espacio, Universidad Politécnica de Madrid, Madrid, 28040, Spain
Treating solid tumours with chimeric antigen receptors‐modified T cells (CAR‐T cells) has shown limited efficacy due to the lack of cancer‐type specific antigens. To solve this, our team has developed a neuroblastoma (NB)‐specific targeting strategy based on a family of analogs of Metadodobenzylguanidine (MIBG). Two versions of these analogs, differing in the substitution of the aromatic ring that forms the targeting element (1) have been conjugated with FITC to be recognized by second generation CAR‐T cells (2). The CAR sequence carries a human‐derived version of scFv against FITC. Transduced T lymphocytes were characterized attending to CD4 and CD8 composition, effector/memory phenotypic and activation markers and VCN. NB cells incubated with MIBG analogs were co‐culture with anti‐FITC‐CAR‐T cells at different ratios. The analog with a substitution in para showed the best CAR‐T activation capacity, resulting in higher NB killing (43,48% versus 24,8%) in a 24h culture assay. In vivo experiments are currently in progress, testing the best MIBG analog candidate to estimate the efficacy and toxicity of the therapy. Our initial results suggest that this specific therapy for NB tumour nanotargeting combined with CAR‐T cells may become an immunotherapy strategy against solid tumours.
Induction of immunogenic cell death in canine melanoma model in response to combined p14ARF and interferon‐beta gene transfer
1: University of São Paulo 2: Instituto do Câncer do Estado de São Paulo
Advanced melanoma is an aggressive cancer type often difficult to treat. Activation of wild‐type p53, frequently retained in melanoma, represents an attractive therapeutic opportunity. To this end, we have studied the use of p14ARF transduction to activate endogenous p53 and induce cell death. However, this approach would be improved by including a means to activate anti‐tumour immune responses. In order to overcome this obstacle, our group has successfully used an improved adenoviral vector for the delivery of p14ARF combined with IFNβ, resulting in the induction of immunogenic cell death (ICD) in in vivo mouse and ex vivo human models. Encouraged by these finding, we next 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 encoding 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 further evaluating the ICD potential of our vectors in vitro and soon we will perform in situ gene therapy studies in xenograft models using CMCLs. With the success of the current study, we will propose the treatment of canine melanoma in the veterinary setting. Funding: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).
Decitabine induces IDO1 expression on human CD4+ T cells
1: University of Patras Medical School 2: The Center for the Study of Haematological Malignancies, Nicosia, Cyprus
As previously demonstrated by our group, decitabine treatment induces de novo expression of HLA‐G on T cells through demethylation of the HLA‐G proximal promoter. Decitabine‐treated HLA‐G+CD4+ T cells acquire potent in vitro immunosuppressive function largely dependent, but not exclusive, on HLA‐G, suggesting that a complementary suppressor function is mediated by other genes.
Here, following the isolation of 5 paired samples of decitabine‐treated HLA‐G+CD4+ and HLA‐G‐CD4+T cells by FACSsorting, RNA was isolated using the miRNeasy micro kit (Qiagen). Next Generation Sequencing was performed using the HiSeq 2000 sequencing platform (Illumina) and reads were aligned against the whole human genome (Homo sapiens: hg19 GRCh38). Flow cytometry validation was then performed in three independent experiments.
The principal component analysis (PCA) along with the gene expression profile depict a clear separation between both populations. Furthermore, a statistically significant increase of the IDO1 gene transcripts was revealed in the HLA‐G+CD4+ population (log2 fold change = 2.55, padj = 0.028). Subsequent flow cytometry experiments validated the upregulation of IDO‐1 in the HLA‐G+CD4+ compartment via the increase of the IDO1 Median Fluorescence Intensity‐MdFI (HLA‐G+CD4+IDO1‐MdFI range = 4680‐7441, median = 6114 and HLA‐G‐CD4+IDO1‐MdFI range = 4070‐6561, median 5096, paired t‐test p = 0.02).
Taken together, we herein present the upregulation of IDO1 expression on CD4+ T cells upon hypomethylation treatment. IDO1 is known for its potent immunomodulatory properties and could thus complement the function of HLA‐G+CD4+ T cells. Ongoing experiments by our group aim to elucidate the mechanism of IDO1‐mediated immunosuppression of IDO1highHLA‐G+CD4+ T cell through molecular, immunological and functional assays.
Antitumor responses of anti‐FITC CAR‐T cells targeting B7‐H3+ sarcoma tumor cells
1: Cellular Biotechnology Unit, Instituto de Salud Carlos III
Immunotherapy with CAR‐T cells has changed the treatment of hematological malignances but they are still a challenge for solid tumors, including pediatric sarcomas. We have tested the expression of different targets for CAR‐T cells in several osteosarcoma and primary condroblastoma cell lines. All the tumor cell lines analyzed express B7‐H3, an immune checkpoint inhibitor overexpressed in different tumor types.
We have evaluated the activity of CAR‐T cells against several cell lines expressing B7‐H3 using universal anti‐FITC CAR‐T cells. For this system, a FITC adaptor molecule is necessary. Here, we use a monoclonal antibody against B7‐H3 conjugated to FITC. In vitro, specific lysis and cytokine release has been quantified after coculture at different CAR‐T : Tumor cell ratios. Anti‐FITC CAR‐T cells show strong antitumor activity against B7‐H3‐FITC positive tumor cells, even at low ratios. Moreover, osteosarcoma tumor cell lines attract in vitro anti‐FITC CAR‐T cells.
In vivo, FITC conjugated B7‐H3 monoclonal antibody penetrates specifically in the tumor and binds 143B osteosarcoma tumor cells. However, anti‐FITC CAR‐T cells barely infiltrate the tumor. Despite the ineffective homing, anti‐FITC CAR‐T cell therapy shows antitumor effect in an osteosarcoma mouse model. Since the trafficking of CAR‐T cells to the tumor is critical, we are working on strategies to improve the homing to the tumor site and the antitumor efficacy of CAR‐T cells.
Creation of next generation CAR‐T by Repurposing endogenous immune pathways
1: GENyO‐ Centro de Genomica e Investigacion Oncologica: Pfizer / Universidad de Granada / Junta de Andalucia 2: IMIBIC 3: Hospital Reina Sofia, Córdoba 4: Lentistem, Granada
For refractory patients from leukaemia or lymphomas wich not respond to polychemotherapy, immunotherapy based on Adoptive Cell Transfer is an alternative. Among these, genetically engineered T cells expressing Chimeric Antigen Receptors (CARs), has emerged as a successful therapy for type B malignancies. CARs are fusion proteins formed by the combination of an extracellular region for specific binding to the tumour, a spacer, a transmembrane domain and a cytoplasmatic domain responsible for signalling upon antigen recognition. 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 in CRISPR/Cas9 to repurpose TCR and PD1, two major players of the T cell activation pathway. Our final aim is to engineer the TRAC locus to express a CD19‐CAR (generating universal and physiological CAR‐T cells) and the PD1 locus to express Il15 (generating T cells that respond to PD‐L1 increasing their activity). Our first results showed the appearance of GFP+CD3‐ and GFP+PD1‐ T cells when using the appropriate donor indicating specific insertion in the target locus. This strategy allows generating CAR‐T cells with exhaustion/activation dependent IL15 secretion, which should lead to improved therapeutic efficacy. We observed a high proportion of death cells as consequence of GE, probably due to the use of PCR product as donors. The same donors will be delivered as AAV in next experiments as an alternative to reduce cytotoxicity of the approach.
Exploiting epigenetic therapy to improve viral‐based platforms for cancer immunotherapy.
1: University of Helsinki 2: Drug Research Program (DRP) ImmunoViroTherapy Lab (IVT), Division of Pharmaceutical Biosciences, Faculty of Pharmacy, Viikinkaari 5E, University of Helsinki, 00790 Helsinki, Finland. 3: Helsinki Institute of Life Science (HiLIFE), Fabianinkatu 33, University of Helsinki, 00710 Helsinki, Finland. 4: Translational Immunology Program (TRIMM), Faculty of Medicine Helsinki University, postal code Haartmaninkatu 8, University of Helsinki, 00290 Helsinki, Finland. 5: Department of Molecular Medicine and Medical Biotechnology, Naples University “Federico II”, S. Pansini 5, Italy.
During the last decade, oncolytic viruses (OVs) have been recognized as a promising tool for cancer therapy since their tumour‐selectively replication and lysis as well as immunosuppression disruption. However, using OVs as a single agent unmet clinical expectations for poorly immunogenic cancers due to low MHC‐I tumoral MHC and lack of suitable antigens to trigger anti‐tumour immunity. In this context, one interesting solution to reprogram tumour´s immunogenicity is represented by epigenetic therapy. Epigenetic drugs such as Decitabine and Azacytidine, are approved for treating haematological malignancies and represent an exciting solution to reprogram the immunogenicity of those cancers that are poorly responsive to conventional immunotherapies. Indeed, epi‐drugs can induce de‐repression of human endogenous retroviruses (HERVs) and cancer‐testis antigens (CTAs), a source of HLA‐I ligands that may increase patient's eligibility for therapeutic cancer vaccines and adoptive cell therapies. Taken together, this evidence makes us claim a potential synergism between epigenetic therapy and oncolytic virotherapy either for antigen discovery and improved vaccine development or as a combinatorial treatment to overcome viral‐based immunotherapy's limitations.
Expansion of natural killer (NK) cells using an FBS‐free and feeder cell‐free culture protocol
1: Bio‐Techne, 3F Gill Street Woburn, MA 01801, USA
Generating clinical doses of NK cells remains a significant hurdle for commercializing of NK and chimeric antigen receptor (CAR)‐NK therapies. Many manufacturing protocols use fetal bovine serum (FBS) and irradiated feeder cells to improve NK cell expansion. However, these methods suffer from regulatory concerns, clinical translation challenges, and licensing constraints. Defined reagents in serum‐free and feeder‐cell‐free workflows are advantageous because they minimize variability in clinical manufacturing environments. We previously showed that the Cloudz™ Human NK Cell Expansion Kit expanded NK cells 250 ± 90.8‐fold to 92.4 ± 3.4% purity from a PBMC population after 10 days in culture containing 10% FBS. The expansion, purity, and cytotoxicity achieved in NK cells expanded with the Cloudz™ Human NK Cell Expansion Kit were similar to NK cells expanded with K562_41BBL_mbIL21 feeder cells. In the current work, we studied the effect of media and FBS alternatives: Human AB serum, Platelet Lysate, and commercial immune serum replacements on NK expansion, purity, and phenotype using the Cloudz™ Human NK Cell Expansion Kit. The result is a feeder‐cell‐free and FBS‐free protocol for the expansion of NK cells.
Analysis if CD markers of immune cells after interaction with melanoma cells membrane vesicles
1: Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia
Cytochalasin B induced membrane vesicles (CIMVs) of tumor cells, due to their ability to fuse with recipient cells through endocytosis and release their contents into the cytoplasm of recipient cells, are considered as a promising vector for targeted delivery of various antitumor agents. Genetic modification of human melanoma M‐14 cells was carried out with recombinant lentiviruses LV‐IL‐2 to obtain stable cell lines with overexpression of IL‐2. Peripheral blood monocytes (PBMCs) were isolated by Ficoll gradient centrifugation (1,077 g/cm3). Differentiation of DCs from PBMCs was reached by cultivation of PBMCs with a cocktail of cytokines. Co‐cultivation of DCs and CIMVs was carried out for 2 days, and then PBMCs were added. Afterward PBMCs were stained with the antibodies containing a fluorescent label. The results were analyzed by flow cytometry. We analyzed the interaction of activated mature DCs with PBMCs. It was shown that the number of activated HLA‐DR CD38+ T‐lymphocytes increased by 2.5 times after culturing PBMCs with DCs loaded with CIMVs from M‐14‐IL‐2 (experimental group) compared to PBMCs that were cultured with native mature DCs (control group). It was also shown a 1.5‐fold decrease in the number of T‐helper 1 in the experimental group compared to the control group. Thus, due to the ability of CIMVs to present tumor antigens to DCs and activate the antitumor immune response, CIMVs of tumor cells are a promising object for the development of therapeutic antitumor vaccines. However, further studies are needed in this area to study possible ways of modulating the immune response.
AAVCOVID, a single dose AAV‐based vaccine, elicits potent and broad SARS‐CoV‐2 neutralizing response
1: Grousbeck Gene Therapy Center, Schepens Eye Research Institute, Mass Eye and Ear, Boston, MA 2: IMVA‐HB/IDMIT, Université Paris‐Saclay, Inserm, CEA, Fontenay‐aux‐Roses, France 3: Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 4: Novartis Gene Therapies, USA 5: Department of Microbiology and NEIDL, Boston University School of Medicine, Boston, MA 6: Albamunity, Boston, MA 7: Center for Computational & Integrative Biology, Massachusetts General Hospital, Boston, MA 8: Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA 9: Department of Medicine, Harvard Medical School, Boston, MA
AAVCOVID1(AC1) is a novel room temperature stable single dose AAV based vaccine against SARS‐CoV‐2. AC1 comprises of pre‐fusion stabilized Wuhan spike packaged in AAV serotype Rh32.33. As the pandemic progressed, there was emergence of several neutralization escape SARS‐CoV‐2 variants. To assess levels and breadth of cross‐reactive antibodies in AC1 vaccinated animals in different contexts, serum was tested with a pseudotyped lentivirus bearing variant spike. We measured cross‐reactivity against alpha, beta, gamma, and delta variants of concern (VOC). In mice vaccinated with 1011gc AC1, humoral response was durable through week 48 and cross‐reactive response increased slightly with time. In mice, a heterologous boost with 1011 gc AAV1‐AC1 increased the cross‐reactive humoral response suggesting that re‐encounter with the antigen increased breadth of antibodies for VOC. Cynomolgus monkeys immunized with 1012 gc dose of AC1 were protected from SARS‐CoV‐2 challenge and neutralized predominant VOCs. In rhesus macaques, 1012 gc dose of AC1 elicited neutralizing antibodies through 1 year. We also observed increase in neutralizing antibodies breadth against VOC over time. To benchmark titers from our vaccine candidates, we obtained convalescent plasma with varying severity of disease. The levels of cross‐neutralizing antibodies were dependent on severity of infection. In summary, AAVCOVID in mouse and non‐human primates demonstrated single‐dose protection from SARS‐CoV‐2 challenge and durable neutralizing titers against currently predominant VOCs. We are currently assessing if better expression cassette and different antigen types can induce a better cross‐reactive response against VOCs.
Primary B cell restricted eCD4‐Ig expression through lentiviral vector delivery supports HIV‐1 neutralisation
1: University of Washington 2: Seattle Children's Research Institute 3: Scripps Research Institute, CA 4: Scripps Research Institute, FL 5: UCSD
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. A different strategy is to neutralise HIV‐1; the neutralising therapy would dampen viral spread by targeting emerging virus. A recent advancement has been the development of the broadly neutralising eCD4‐Ig immunoadhesin (HIV IC50<0.05μg/mL). AAV delivery of eCD4‐Ig transgene to the muscle of non‐human primates was previously shown to control SIV infection. To provide a potentially longer lasting and less immunogenic eCD4‐Ig treatment, we developed an eCD4‐Ig transgene restricted to B cell expression for lentiviral vector (LV) delivery to human B cells.
VSV‐G pseudotyped LVs can transduce B cells only up to 15%, limiting their potential in B cell gene therapy applications. To improve B cell transduction, a measles‐pseudotyped LV packaging methodology was optimised. This allowed improvement of LV B cell transduction levels up to 75%. LV eCD4‐Ig transduced primary B cells were found to secrete eCD4‐Ig that can neutralise distinct HIV‐1 isolates comparably efficient as the broadly HIV‐1‐neutralising VRC01 antibody. Modification of eCD4‐Ig in the CH3‐Ig domain reduced B cell IgG heterodimer formation and increased eCD4‐Ig mediated HIV‐1 neutralisation 3‐fold. Astonishingly, these transduced primary B cells were found to secrete eCD4‐Ig at ∼40pg/cell/day, indicating expression levels comparable to endogenous‐IgG.
Overall, our findings support the potential for B cell lineage‐restricted expression of eCD4‐Ig as a therapeutic approach for HIV‐1 infection and suggest transgene delivery to B cells may be a useful approach for therapeutic protein production.
Novel vector systems towards a cure for HIV/AIDS
1: Laboratory of Experimental Virology, Department of Medical Microbiology, Amsterdam UMC, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
Many potent antiviral drugs are currently available to treat HIV infection and combination therapy has saved many lives. However, antiviral therapy has to be continued life‐long because HIV rebounds from an established reservoir when therapy is interrupted. HIV DNA in the reservoir can be targeted by the CRISPR‐Cas genome‐editing tool. Our laboratory demonstrated potent and durable HIV inhibition in a combinatorial attack with two gRNAs. Although remarkable, the large size of CRISPR‐spCas9 transgene cassettes impedes their implementation in gene therapy applications with vectors that have a limited packaging capacity, including lentiviral vectors. There is a serious need for more simple/smaller CRISPR‐Cas vector designs.
We propose to minimize the size of the lentiviral vector by a) adopting the small H1‐Pol‐III promoter that we recently found to exhibit both Pol‐III and Pol‐II promoter activity for the production of both the gRNA and Cas9‐encoding mRNA and b) incorporating a smaller saCas9 or cjCas9 nuclease. These measures will reduce the vector size and likely increase the vector titer.
We have compared different CRISPR‐Cas systems for their efficiency in terms of antiviral activity and viral titer. Superior antiviral activity is reported for saCas9 compared to cjCas9, which can achieve full HIV inactivation in cell culture with only a single gRNA. We demonstrated that reduction of the vector size (smaller Cas9 nuclease and dual‐polymerase active H1‐promoter) increases the vector titer. This greatly facilitates the use of viral vectors with a limited packaging capacity. These results are important in the path towards the formulation of a cure strategy.
Construction of a lentiviral vector‐carrying SARS‐CoV‐2 spike gene for COVID‐19
1: The Department of Gene and Cell Therapy, Akdeniz University Faculty of Medicine, Antalya, 07058, Turkey 2: Department of Medical Genetics, Cukurova University Faculty of Medicine, Adana, Turkey 3: The Department of Immunology, Bursa Uludag University, Faculty of Medicine, Bursa, Turkey
A new pandemic disease named COVID‐19 has emerged in December 2019 in China spreading rapidly to other countries. This highly contagious disease is transmitted through respiratory droplets mostly affecting the lungs of the infected individuals. Consequently, the development of an effective vaccine is urgently needed to prevent the spread of SARS‐CoV‐2 virus. As suggested previously, genetic immunization is one of the most effective tools for vaccination. The ex vivo transfer of viral antigens to dendritic cells was the first method used in dendritic cell‐based vaccines against infectious diseases. With the discovery of viral vectors, antigen encoding genes are delivered to dendritic cells by recombinant viruses. Among the viral vectors tested lentiviral vectors with improved biosafety profile appeared superior in the transduction of dendritic cells. We chose spike protein of SARS‐CoV‐2 as the antigen due to its role in infectivity and its potential to induce an effective immune response as shown in previously studies concerning SARS‐CoV and MERS‐CoV. A multisite gateway reaction was set up between entry and destination vectors to generate an expression clone carrying Spike gene (pLentiSpike). DNA sequencing and restriction enzyme analysis were utilized to confirm the orientation and the sequence of the transgene. Then, immunocytochemical staining of transfected cells with an Anti‐SARS‐CoV‐2 spike glycoprotein antibody demonstrated Spike protein expression from the expression plasmid. Transient transfection of the transfer and packaging plasmids resulted in the generation of a lentivirus carrying spike protein encoding gene.
Combained antibacterial effect of polyethyleneimine and halloysite nanotubes
1: Kazan Federal Universiry
The search for agents inhibiting the growth and development of pathogenic microorganisms is promising. It is known that polycation polyethyleneimine and its derivatives inhibit the growth of biofilms, in particular, the growth of Candida albicans. In addition, it has been shown to inhibit the growth of Bacillus subtilis biofilms on a substrate made of halloysite mineral and surface‐active compounds. However, there are no studies evaluating the combined effect of polyethyleneimine and halloysite nanotubes on the growth and development of E. coli. The aim of this work was to study the effect of the combined effect of halloysite nanotubes at a concentration of 600 μg / ml, as well as polyethyleneimine (1%) on the growth of the planktonic form of E. coli for 96 hours. The object of the study was the gram‐negative bacterium Escherichia coli. There are certain strains of E. coli that can cause infections by colonizing medical devices such as urethral and intravascular catheters, joint prostheses and shunts, and prostheses. Therefore, it is important to study the effect of various therapeutic agents to inhibit the growth of E. coli. Initially, using laser Doppler velocimetry, data on the zeta potential and hydrodynamic parameters of the studied compounds were obtained, which amounted to 423.9 ± 8.351 nm and ‐36 ± 0.819 mV for halloysite nanotubes and 55.63 ± 1.241 nm and +11.7 ± 0.289 mV for polyethyleneimine. The reported study was funded by Russian Foundation of Basic Research (grant 18‐29‐25057).
Adenoviral vector design and construction for SARS‐CoV‐2 vaccine development
1: The Department of Gene and Cell Therapy, Akdeniz University, Faculty of Medicine, Antalya, 07058, Turkey 2: GenomCare Ltd. Co., Akdeniz University Technopark, Antalya, 07058, Turkey
COVID‐19 is a contagious and a fatal respiratory illness caused by SARS‐CoV‐2. The outbreak was declared a public health emergency of international concern on January 30 2020, and a pandemic on March 11 2020 by The World Health Organization. Since, vaccination is the most effective way for protection against such infectious diseases, outstanding efforts from various laboratories have focused on the development of effective and safe vaccines. The production of a vaccine requires the understanding of the molecular structure of the virus, as the selection of a proper antigen and the vector type is essential to induce an optimum immune response following vaccination. Compared to other structural proteins, the spike protein of SARS‐CoV‐2 appeared to be the most promising antigen candidate in vaccine development due to its essential role in viral transduction through interaction with the Angiotensin‐Converting Enzyme 2 (ACE2) receptors. Viral vector‐based vaccines induce both an effective cellular immune response and a humoral immune response owing to their natural adjuvant properties via transduction of immune cells. Accordingly, adenoviral vectors with well‐known structure, serve as effective gene carriers to induce immune response via gene transfer. To generate a viral vector based vaccine candidate, we first constructed an adenoviral expression vector (pAd5Spike) encoding spike protein via Gateway cloning technology. 293T cells were transfected with pAd5Spike to confirm spike protein expression by immunostaning. The pAd5Spike plasmid was then used to produce the first generation adenovirus vector encoding spike protein (Ad5Spike).
Investigation of the antibacterial activity of a composite based on halloysite nanotubes and silver nanoparticles
Y S Cherednichenko1
1: Kazan Federal University
Due to the ability of silver nanoparticles to damage the cytoplasmic membrane, respiratory enzymes of microorganisms and prevent the growth of gram‐positive and gram‐negative bacteria, fungi and viruses, they are widely used in pharmacy and medicine as an antibacterial component. Using ultrasound, the authors obtained a composite based on silver nanoparticles and halloysite nanotubes. The obtained nanocomposite was visualized using various types of microscopy (dark‐field, transmission electron, and atomic‐force) and its elemental composition was investigated, which contains oxygen, silver, silicon, and aluminum. Studies of the antibacterial activity of the nanocomposite have shown its effectiveness against bacteria Serratia marcescens (S. marcescens) at a concentration of 1.0 mg/ml and 1.5 mg/ml. Based on the data of the growth curve, it was found that the concentration of bacteria was reduced by more than two‐fold compared to the control at a nanocomposite concentration of 1.0 mg/ml. The antibacterial effect of the obtained nanocomposite was observed from the first hours and did not decrease for 48 hours. At a nanocomposite concentration of 1.5 mg/ml, the death of bacteria was observed 2 hours after the start of the study.
The obtained nanocomposite has the ability to suppress the quorum sensing of S. marcescens bacteria, which is promising for medical applications.
The research was funded by the Russian Science Foundation grant No. 21‐74‐10034.
Hematopoietic stem cell gene therapy corrects lysosomal storage in CNS in murine model of GM1‐gangliosidosis
1: Department of Pediatrics, Jikei University School of Medicine 2: Division of Gene Therapy, Research Center for Medical Science, The Jikei University School of Medicine 3: Dementia Research Unit, Osaka Psychiatric Research Center, Osaka Psychiatric Medical Center 4: Institute of Clinical Medicine and Research, Research Center for Medical Science, The Jikei University School of Medicine 5: Division of Neuroscience, Research Center for Medical Science, The Jikei University School of Medicine 6: Department of Human Health Science and Therapeutics, The Jikei University School of Nursing
GM1 gangliosidosis (GM1) is progressive neurodegenerative glycoshingolipidosis due to a mutation in GLB1 gene, causing the deficiency of lysosomal enzyme β‐galactosidase (β‐gal), which leads to the abnormal accumulation of GM1 ganglioside primarily in the central nervous system (CNS). In this study, we evaluated the therapeutic efficacy of ex vivo lentiviral gene therapy on the GM1 mice. We constructed the recombinant lentivirus vector (LV) with the normal GLB1 cDNA or enhanced green fluorescent protein (eGFP). Bone marrow cells from GM1 mice were harvested, and transduced with LV and administered through the tail vein of 8 weeks‐old mice(LV‐GLB1 or LV‐eGFP). Non‐treated model mice (KO) and wild type mice (WT) were used as control. Enzyme activity, substrate accumulation, histological and behavioral analysis was evaluated. LV‐GLB1 group displayed a superphysiological level of β‐gal activity in visceral organs. In CNS, LV‐GLB1 group showed a significant increase in β‐gal activity (p < 0.05) and a decrease in accumulation of GM1 ganglioside in cerebrum and cerebellum (p < 0.05) compared to KO group. In behavioral analysis, a rota‐rod test at 32 weeks old detected an improvement in motor function for LV‐GLB1 group compared with LV‐eGFP group (p < 0.05). In histological analysis, reduction in GM1 ganglioside content and improvement in neuroinflammatory response was observed, especially in the cerebrum. These results suggest that ex vivo gene therapy with LV can ameliorate biochemical abnormalities in impaired organs, inhibit motor function decline and neurodegeneration in GM1 gangliosidosis mice. We have shown that ex vivo gene therapy could be a feasible therapeutic strategy in GM1 gangliosidosis.
Lentiviral vector mediated
in vivo
gene transfer into liver organoid forming cells in mice and non‐human primates
1: San Raffaele Telethon Institute for Gene Therapy (SR‐TIGET) 2: University Vita‐Salute San Raffaele 3: The Gurdon Institute 4: MRC LMCB, University College of London 5: Max Planck Institute of Molecular Cell Biology and Genetics
Liver gene therapy with adeno‐associated viral (AAV) vectors delivering clotting factor transgenes into hepatocytes has shown multi‐year therapeutic benefit in adults with haemophilia. However, the mostly episomal nature of AAV challenges their application to paediatric patients. Moreover, their ability to sustain efficacy in the context of damage‐driven proliferation remains to be addressed. We developed a complementary strategy based on integrating lentiviral vectors (LV) and showed safety and efficacy in mice, dogs and non‐human primates (NHP). We now set out to investigate LV gene transfer to liver cell types that have been shown to regenerate the liver following damage. Among them, cholangiocytes have been described to be able to generate self‐renewing liver organoids in vitro and to trans‐differentiate into hepatocytes. We systemically administered LV.GFP to newborn mice and showed efficient transduction of intrahepatic cholangiocytes that retain their ability to form liver organoids in vitro. We also derived organoids from the liver of LV‐treated NHP and showed LV marking in around 2% of the obtained organoids. These organoids can be cultured on different substrates to skew differentiation towards hepatocytes or mature cholangiocytes and produce the transgene encoded by the LV that was systemically administered to the NHP. Finally, we showed similar engraftment of organoids derived from the liver of either treated or untreated NHP in immunodeficient mice. Our work suggests that LV‐transduced cholangiocytes may sustain stable gene transfer even in the context of disease or tissue damage and may also shed light on the role of different cell populations in liver regeneration.
Sustained correction of Glycogen Storage Disease Type Ib mice by gene therapy
1: Glycogen Storage Disease Program Department of Pediatrics University of Connecticut School of Medicine
Glycogen storage disease type Ib (GSD‐Ib) is an autosomal recessive disorder caused by deficiencies in a glucose‐6‐phosphate (G6P) transporter (G6PT). GSD‐Ib patients manifest fasting hypoglycemia, growth retardation, hepatomegaly, nephromegaly, elevated levels of blood metabolites and immune cell related diseases including neutropenia. Dietary therapies support patients to maintain normoglycemia, however, the patients still exposed to the risk of life‐threatening hypoglycemia and long‐term complications such as hepatocellular adenoma (HCA) and kidney disease. In this study, to treat GSD‐Ib, we developed a recombinant adeno‐associated virus (rAAV) vector carrying codon‐optimized human G6PT directed by native human G6PT promoter (GTP‐co‐opt‐hG6PT) and investigated the strategies of gene delivery with different serotype combinations of AAV vectors. The GSD‐Ib mice received AAV8 vector neonatally and the metabolic profiles at age 4 and 12 weeks were evaluated. And then the mice received either AAV9 or AAV2‐QuadYF vectors at age 12 weeks as 2nd dose. In the results, neonatal treatment with rAAV8‐GTP‐co‐opt‐hG6PT vector enabled the GSD‐Ib mice survived through adult age with normal blood glucose level and metabolic profiles despite the expected vector dilution effect with age‐related liver growth. In addition, the 2nd dose achieved the fasting blood glucose levels of GSD‐Ib mice similar to wild type control and showed its capability to treat neutropenia. Our results suggest that the rAAV‐GTP‐co‐opt‐hG6PT vectors are efficacious in correcting metabolic abnormalities as well as neutropenia in murine model of GSD‐Ib.
Preclinical development of an
in vivo
lentiviral 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: Programa de Medicina Regenerativa, CIMA Universidad de Navarra. Instituto de Investigación Sanitaria de Navarra, IdiSNA, Pamplona, Spain. 4: San Raffaele Telethon Institute for Gene Therapy (SR‐Tiget), Italy. “Vita‐Salute San Raffaele” University, 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 metabolic disorder caused by mutations in the AGXT1 gene, which codes for alanine‐glyoxylate aminotransferase, expressed in hepatic peroxisomes. PH1 patients suffer from oxalate overproduction that can result in end‐stage renal disease and life‐threatening oxalosis. Thirty percent show infantile onset and the only curative treatment is liver and kidney double‐transplant. Consequently, new therapeutic approaches are needed. In vivo gene therapy using lentiviral vectors (LV) is a promising approach for liver monogenic diseases. Its integrative capacity together with the low pre‐existing immunity in the human population makes it a good tool for the treatment of paediatric patients.
A preclinical study of in vivo LV gene therapy in a PH1 mouse model has been conducted. Adult Agxt1 KO mice were intravenously injected with different doses of a LV expressing the AGXT gene in hepatocytes. Three weeks later mice were subjected to an ethylene glycol challenge to induce overload in oxalate production and PH1 phenotype was analysed. According to the PH1 endpoints, treated mice showed a significant reduction in urine oxalate overproduction, prevention of weight loss and no signs of nephrocalcinosis, achieving a partial pathological phenotype reversion. Due to the cell‐autonomous character of PH1, transduction efficiency is a key parameter to define. Mice treated in parallel with a reporter LV revealed a transduction percentage up to 10% at equivalent LV doses.
The observed phenotype reversion with such a limited number of corrected cells supports the feasibility of this approach for the treatment of PH1.
A novel therapeutic strategy for skeletal disorders: proof‐of‐concept of gene therapy for X‐linked hypophosphatemia
1: GENETHON 2: Université Paris‐Saclay 3: Université Paris Descartes 4: Hôpital Bicetre 5: Université Lyon 1 6: Hôpital Bretonneau 7: Hôpital Cochin
X‐linked hypophosphatemia (XLH) is a rare genetic form of rickets due to mutations in the PHEX gene. Although the mechanism leading to increased fibroblast growth factor 23 (FGF23) secretion from bones is unknown, it is considered the main physiopathological feature of the disease. Increased circulating FGF23 leads to phosphate wasting and reduced active vitamin D synthesis in kidneys, associated with severe skeletal deformities and short stature. Conventional treatment relies on phosphate and active vitamin D analogs supplementation, and may results in severe long‐term side effects. Burosumab (Crysvita®, Ultragenyx and Kyowa Kirin), an anti‐FGF23 monoclonal antibody, represents an alternative treatment.
Based on the central role of overactive FGF23 pathway in the pathophysiology of XLH, we devised a liver‐targeting AAV gene therapy strategy to inhibit FGF23. Our strategy is based on the competition of the C‐terminal FGF23 (cFGF23) for the binding of FGF23 to its renal receptor. Engineering of the cFGF23 by its fusion with a stabilizing moiety through a cleavable linker allowed for efficient secretion and stable circulating levels of this molecule reportedly unstable in vivo. We then evaluated the efficacy of the AAV8‐cFGF23 in a murine model of XLH, the Hyp‐Duk mice. Secretion of the cFGF23 by the liver significantly reduced phosphate wasting in kidney, normalized osteomalacia, and led to restoration of the skeletal structure, and bone and joint alterations in Hyp‐Duk mice.
Taken together, our data provide proof‐of‐concept to the use of AAV liver gene therapy for the treatment of XLH and support the translation of this approach to the clinic.
Dose finding study of AAV‐LSP‐MMUT in a mouse model of MMA and efficient suppression of anti‐capsid antibody responses by single and multiple administrations of ImmTOR nanoparticles
1: Selecta Biosciences 2: National Human Genome Research Institute, NIH
A wide dose range of MMA‐101, an AAV vector encapsidating the methylmalonyl‐CoA mutase (MMUT) gene under control of a liver‐specific promoter, was tested in juvenile (14 and 28‐day old) Mmut‐/‐;TgINS‐MCK‐Mmut (MCK‐MMUT) mice, a model of methylmalonic acidemia. A rapid and dose‐dependent decrease of plasma methylmalonic acid (pMMA), a key disease‐associated metabolite, was seen at doses ranging from 2E11 to 5E12 vg/kg in 28‐day old mice maintained on a low protein diet. Similar efficacy was observed in 14‐day old mice at MMA‐101 doses of 1E12 vg/kg to 1E14 vg/kg, although pMMA partially rebounded at 1E12 vg/kg. ImmTOR, nanoparticle‐encapsulated rapamycin, combined with 1E13 vg/kg of MMA‐101 reduced anti‐capsid IgG antibody responses. Additional monthly doses of ImmTOR doses led to further and more durable IgG suppression at high vector dose. No adverse effects were seen after AAV‐MMUT +/‐ ImmTOR administration. Histological analysis of key target organs showed improvement in MMA‐related vacuolization in the liver with MMA‐101 +/‐ ImmTOR treatment, and, in addition, 1‐C‐13 propionate oxidation was improved in the treated MMA mice. Thus, MMA‐101 +/‐ ImmTOR corrected the metabolic defect in MCK‐MMUT mice and showed no adverse effects in MCK‐MMUT model. Repeated administration of ImmTOR provided more durable suppression of antibodies against AAV capsid. Monthly dosing of ImmTOR has been shown to be well tolerated and effective in mitigating immunogenicity of a fungal‐derived uricase therapy in Phase 2 clinical trials in gout patients and is currently in Phase 3 clinical testing.
In vivo
lentiviral gene therapy for argininosuccinic aciduria
1: Genetics and Genomics Medicine Group, Great Ormond Street Institute of Child Health, University College London, London, UK 2: Gene Therapy for Metabolic Diseases, NIHR Great Ormond Street Hospital Biomedical Research Centre, London, UK 3: Gene Transfer Technology Group, 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
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 with an incidence of 1/70.000. Patients present neonatal‐ or late‐onset hyperammonaemia, which causes coma and death if untreated, and a high risk of neurological sequelae. Standard of care relies on protein‐restricted diet, arginine supplementation and ammonia scavengers. Curative liver transplantation can be performed in severe cases but is complicated by procedure‐related mortality/morbidity and requires lifetime immunosuppression. Preclinical studies have shown efficacy of in vivo lentiviral gene therapy, targeting the liver, for haemophilia B. We aimed to test in vivo lentiviral gene therapy in ASL‐deficient (AslNeo/Neo ) mice. We initially performed a single neonatal intravenous injection in wild‐type mice of a lentiviral vector containing GFP and liver‐specific promoter (CCL.GFP); liver expression persisted up to at least 12 months. A lentiviral vector containing a codon‐optimised human ASL gene (CCL.cohASL) was injected intravenously into neonatal AslNeo/Neo pups (n = 8) with 4x10e7 transducing units per mouse compared to wild‐type (n = 11) and CCL.GFP‐injected AslNeo/Neo littermates (n = 5). Although CCL.GFP‐injected animals died within 4 weeks post‐injection, CCL.cohASL‐injected animals survived (log‐rank test p < 0.001) and normalised their abnormal fur coat pattern. Growth and plasma ammonia levels were not significantly different in CCL.cohASL‐injected mice compared to wild‐type levels at 8 weeks post‐injection. Plasma argininosuccinate (p < 0.001) and citrulline (p < 0.01) were significantly normalised in CCL.cohASL‐versus CCL.GFP‐injected AslNeo/Neo mice. This study demonstrates proof of concept of in vivo lentiviral gene therapy for ASA.
Re‐Administration of AAV expressing MDR3 (VTX‐803) treats progressive familial intrahepatic cholestasis type 3 (PFIC3) in juvenile
Abcb4−/−
mice when co‐administered with ImmTOR
1: Vivet Therapeutics, S.L., Pamplona, 31008, Spain 2: Division of Gene Therapy and Regulation of Gene Expression, Cima Universidad de Navarra, Pamplona, 31008, Spain 3: Selecta Biosciences, Watertown, MA, 02472, United States 4: Vivet Therapeutics, S.A.S., Paris, 75008, France 5: Instituto de Investigación Sanitaria de Navarra (IdISNA), Pamplona, 31008, Spain
AAV gene therapy targeting the liver is effective at treating genetic disorders, such as hemophilia, in adult patients. However, gene therapy for many inborn errors of metabolism, such as progressive familial intrahepatic cholestasis type 3 (PFIC3), would be most effective if administered in infancy or early childhood to prevent irreversible damage. A key challenge for AAV gene therapy in pediatric PFIC3 patients is the potential for therapeutic benefit to wane as the non‐replicating AAV vector is diluted over time, and the formation of persistent neutralizing antibodies precludes re‐administration. Here, we tested the effectiveness of tolerogenic ImmTOR nanoparticles carrying the immunomodulating drug rapamycin to enable repeated intravenous administration of an hepatotropic AAV vector carrying human ABCB4 cDNA (VTX‐803) in 2‐week‐old juvenile Abcb4 −/− mice when ImmTOR is co‐administered with the first AAV treatment. VTX‐803 co‐injected with ImmTOR allowed for a successful re‐administration of VTX‐803 alone two weeks later and resulted in robust and stable correction of the disease phenotype lasting 8 months, while repeat dosing of the vector alone (at a sub‐therapeutic dose) or together with empty nanoparticle did not exhibit a therapeutic effect. In males, a temporary therapeutic effect was observed following only a single treatment of VTX‐803 with ImmTOR, suggesting that ImmTOR increases vector transduction and/or transgene expression at the first dose. Vector treatment without ImmTOR resulted in the production of AAV‐specific neutralizing antibodies (NAbs), while vector coadministration with ImmTOR prevented NAb production. Thus, this provides further evidence that redosing AAV gene therapy can be achieved through coadministration with ImmTOR.
GCDH gene therapy shows efficacy in the glutaric aciduria type I mouse model.
1: IDIBAPS 2: CIBERER
Glutaric aciduria type I (GA‐I) is a rare metabolic inherited disorder in the catabolic pathways of lysine, hydroxylysine and tryptophan. It is caused by the deficiency of glutaryl‐CoA dehydrogenase (GCDH). The enzymatic defect results in the accumulation of glutarate, hydroxyglutarate and glutarylcarnitine in tissues and body fluids. Clinically, GA‐I patients display macrocephaly, progressive dystonia and dyskinesia. Dietary lysine restriction, carnitine supplementation and intensified emergency treatment during catabolism are effective for some individuals. Unfortunately, one‐third of affected children do not respond to therapy and experience irreversible brain damage.
We have explored the feasibility of in vivo gene therapy in the preclinical model of the disease, the Gcdh ‐/‐ mice, following an intravascular delivery of AAV‐GCDH in adult and newborn mice. We observed that GCDH expression was restored in the liver of adult mice and was maintained for six months after treatment. Consequently, the accumulation of glutarylcarnitine and glutaric acid metabolites were significantly reduced in liver and serum of treated mice. AAV‐GCDH gene therapy in newborn mice also showed GCDH expression in brain. Interestingly, when these mice were exposed to a lysine overload to enhance the severity of the disease, we identified a reduction in the content of glutarylcarnitine in brain. Moreover, whereas a 40% of Gcdh ‐/‐ mice succumbed from lysine overload, newborn AAV‐GCDH gene therapy prevented the aggressive phenotype and all mice survived to lysine intake. In summary, these data suggest that AAV‐mediated gene therapy can prevent from the devastating consequences of GA‐I.
A minimal BSEP promoter allows bile acid‐driven physiological regulation of transgene expression from an AAV vector
1: CIMA Universidad de Navarra 2: Instituto de Investigación Sanitaria de Navarra 3: Vivet Therapeutics S.L.
Bile acid (BA) homeostasis is mainly regulated by the bile salt excretory pump (BSEP), a hepatocyte transporter of BA to the bile. BSEP expression is tightly regulated by BA levels via activation of farnesoid X receptor transcription factor, which binds to IR‐1 element in BSEP promoter. Although the natural physiological regulation of transgene expression can improve gene therapy, the large sizes of some endogenous promoters limit this approach. We have evaluated the use of minimal versions of human and mouse BSEP promoters (ihPr and imPr, respectively) containing IR‐1 element, to regulate expression of luciferase using an adeno‐associated viral vector with liver tropism (AAV8). imPr showed significantly higher levels of BA‐mediated expression and inducibility in human and mouse hepatic cells than ihPr or the full‐length BSEP promoters. In addition, AAV8 containing luciferase downstream of imPr was efficiently regulated in mice by providing a BA‐enriched diet. Next, this vector was able to express significantly higher luciferase levels in Abcb4‐/‐ mice, which possess high natural levels of BAs, compared to wild‐type mice or mice receiving AAV8 expressing luciferase downstream of a constitutive alpha‐1 antitrypsin promoter. In both mouse models, the AAV vector with ihPr showed very low expression levels in the absence or presence of BA. Finally, we have improved imPr by adding three extra IR‐1 repeats to its 5’ end, which significantly increased transgene expression in vitro and in vivo. Altogether, our data show that imPr represents a useful tool for gene therapy approaches in which physiological regulation by BA is desired.
rAAV‐mediated gene therapy in combination with short‐term nitrogen‐scavenger treatment corrects biochemical and behavioral abnormalities and increases lifespan in infant Citrullinemia Type 1 (CTLN‐1) mice
1: Division of Gene Therapy and Regulation of Gene Expression, Cima Universidad de Navarra, Pamplona, Spain. 2: Vivet Therapeutics, S.L., Pamplona, Spain. 3: Division of Metabolism and Children's Research Centre (CRC), University Children's Hospital Zurich, Switzerland. 4: Vivet Therapeutics, S.A.S., Paris, France.
Citrullinemia type I (CTLN‐1) is a rare autosomal recessive genetic disorder caused by mutations in the Argininosuccinate Synthase 1 (ASS1) gene that catalyzes the third reaction of the urea cycle. CTLN‐1 patients suffer from toxic accumulation of circulating ammonia and urea cycle byproducts that causes metabolic encephalopathy and death at a very young age. Standard of care (SOC) management of CTLN‐1 consists of daily nitrogen‐scavenger administration and lifelong low‐protein, high‐calorie diet. Early restoration of hepatic ASS1 expression based on recombinant adeno‐associated virus (rAAV) gene therapy represents an attractive prospect for treatment.
Vivet Therapeutics is developing VTX‐804, a rAAV vector expressing the human ASS1 enzyme under the control of a liver‐specific promoter. The long‐term therapeutic efficacy of VTX‐804 (6 months post‐vector administration) was evaluated in 3‐week‐old CTLN‐1 mice (Ass1fold/fold ) treated or untreated from birth to weaning with nitrogen‐scavenger agents as per SOC in patients. Combination of VTX‐804 with SOC restores CTLN‐1 mice survival, normalizes weight gain and ammonia levels, while mice receiving VTX‐804 alone present 90% survival, did not normalize weight gain and ammonia levels are normalized just transiently.
Similar to CTLN‐1 patients; CTLN‐1 mice showed several behavioral abnormalities such as anxiety and reduced welfare and innate behaviors that were notably improved when they were treated with VTX‐804, and even more with the SOC in combination that improved rAAV‐transduction and hASS1 transgene expression at 3 and 6 months post‐VTX‐804 injection. Therefore, combination of the SOC prior to gene therapy administration represents a very promising therapeutic strategy for very young CTLN‐1 patients.
Gene supplementation of
CYP27A1
in the liver restores bile acid metabolism in a mouse model of Cerebrotendinous Xanthomatosis
1: CIMA 2: Universidad de Navarra 3: Hospital Universitario Miguel Servet 4: Vivet Therapeutics 5: CIBERehd 6: IdiSNa 7: University of Salamanca
Background & Aims. Cerebrotendinous Xanthomatosis (CTX) is an autosomal recessive disease caused by mutations in the CYP27A1 gene, encoding the sterol 27‐hydroxylase. The enzymatic defect causes disruption of the hepatic bile acid biosynthesis pathway and the accumulation of toxic metabolite precursors such as cholestanol and 7α‐hydroxy‐4‐cholesten‐3‐one (7αC4). Apart from chronic diarrhea and bilateral cataracts, deposition of cholestanol and cholesterol in different tissues (xanthomas) leads to progressive motor and neurophsychiatric alterations. Approach & Results. We performed gene transfer of the CYP27A1 coding sequence in the liver of Cyp27a1 knock‐out mice using Adeno‐Associated Viral vectors (AAV). We found that a vector equipped with a strong liver‐specific promoter is well tolerated and shows therapeutic effect at relatively low doses, when less than 20% of hepatocytes over‐express the transgene. The vector was able to restore bile acid metabolism and to normalize bile acid composition in plasma. Conclusions. Over‐expression of CYP27A1 in a fraction of hepatocytes using AAV vectors is well tolerated and provides full metabolic restoration in a CTX mouse model.
Lentiviral gene therapy for arthrogryposis‐renal dysfunction‐cholestasis (ARC) syndrome
1: Gene Therapy for Metabolic Diseases, NIHR Great Ormond Street Hospital Biomedical Research Centre, University College London, London, WC1N 1EH, United Kingdom
Arthrogryposis‐renal dysfunction‐cholestasis (ARC) syndrome is a rare autosomal recessive disorder. There is a variable degree of severity but most patients die before 12 months of age. ARC patients present at birth with hallmark symptoms including arthrogryposis, renal tubular acidosis and neonatal cholestatic jaundice. They can also display ichthyosis, platelet abnormalities and recurrent sepsis. About 75% of the patients display mutations in the Vacuolar protein sorting 33 homolog B (VPS33B) gene. In patients' hepatocytes, these mutations lead to mislocalisation of several transmembrane proteins involved in bile formation, explaining the cholestatic phenotype. No curative treatment is available for these patients and liver transplantation is not offered due to the multi‐organ nature of the disease. Lentiviral gene therapy has proven efficacy in ex vivo treatments of several inherited monogenic disorders and recent studies in animal models suggest that lentiviral gene therapy may also be safely used in vivo. This project aims to develop in vivo lentiviral gene therapy for the liver phenotype of ARC syndrome. Thus, a lentiviral vector containing the VPS33B gene has been created and validated in vitro in an ARC syndrome HepG2 VPS33B ‐/‐ cell model created by CRISPR/Cas9 technology. VPS33B expression was checked by RT‐qPCR and western blotting, while its function was verified by co‐expression of CEA and MRP2 at pseudo‐bile canaliculi formed between HepG2 cells. Our results have shown that VPS33B lentiviral gene therapy restores VPS33B expression and function. In future studies, the vector will be tested in vivo in a murine model for the ARC syndrome liver phenotype.
Baculoviral transduction of 3T3‐L1 adipocytes with CRISPR/dCas9 system activating UCP‐1 expression increases glucose uptake
1: Lomonosov Moscow State University 2: National Medical Research Centre for Cardiology 3: National Tsing Hua University
Nowadays beige‐like phenotype of adipocytes is considered to be a perspective therapeutic target to treat metabolic disorders, such as type 2 diabetes. Main function of these types of adipose tissue is to dissipate the proton gradient on mitochondria membrane via uncoupling protein 1 (UCP1) which leads to heat production. Activation of this protein expression in adipocytes could help obese people to dissipate extra energy and lower blood glucose level. Therefore, the purpose of this work was to investigate glucose uptake in cells that were transduced by baculovirus containing CRISPR/dCas9 system activating UCP‐1 expression. 3T3‐L1 preadipocytes were used as a beige adipocytes model. We carried out baculovirus transduction on 3T3‐L1 beige adipocytes according to a standard protocol. After this transduction we extracted RNA and performed reverse transcription reaction and real‐time PCR. After performing these steps we measured glucose uptake with [3H]‐deoxyglucose. We observed a significant increase in UCP‐1 gene expression in cells transduced with activating UCP‐1 system compaired to control group of non‐transduced cells and cells transduced with baculovirus without activating system. We showed that transduction with baculovirus decreased glucose uptake of 3T3‐L1 adipocytes compared to control, while glucose uptake of 3T3‐L1 adipocytes transduced with baculovirus containing CRISPR/dCas9 system activating UCP‐1 increased to the level of control group. Therefore, we can assume that activation of UCP‐1 increases glucose uptake due to the increase of cell energy needs and can be a possible therapeutic gene target to treat obesity.
This work was supported by Russian Science Foundation grant #20‐45‐08003.
Activation of UCP‐1 expression in 3T3‐L1 cells via CRISPR/dCas9 system leads to decrease of cytochrome c oxidase and superoxide dismutase genes expression
1: Lomonosov Moscow State University 2: National Medical Research Center for Cardiology 3: National Tsing Hua University
Today obesity is one of the most widespread diseases among population. It is associated with white adipose tissue overgrowth. Brown‐like adipocytes called beige are differentiated from the same progenitor cells as white, but also are capable of producing heat. By switching white genotype to a beige, we could reprogram adipocytes to thermogenesis. Uncoupling protein 1 (UCP1) is the major player in this process; it decreases the proton gradient and produces heat. The aim of our work was to enhance UCP‐1 expression and investigate electron transport chain complexes' genes expression in 3T3‐L1 beige adipocytes after baculovirus transduction by CRISPR/dCas9 system for UCP‐1 gene activation. We differentiated 3T3‐L1 preadipocytes into beige adipocytes and performed baculovirus transduction via CRISPRa activating UCP‐1 according to standard protocols. Genes' expression was measured via Real‐time PCR. We have showed that indeed this activating system enhances UCP‐1 expression. Next thing we observed is a decrease of cytochrome c oxidase (Cox7) gene that can be explained by lowered activity of electron transport chain due to enhanced UCP‐1 expression. We also showed decrease of superoxide dismutase (SOD2) gene expression. We assume it can be explained by the fact that superoxide is a side product of cytochrome c oxidase activity, therefore when cytochrome c oxidase expression lowers the production of superoxide decreases. Thus in this work we have showed that enhanced UCP‐1 gene expression leads to decrease of cytochrome c oxidase and superoxide dismutase genes expression.
This work was supported by Russian Science Foundation grant #20‐45‐08003.
A novel endonuclease‐free genome editing technology to edit hepatocytes in vivo led to a full molecular liver transplant and cured mice in preclinical models of Tyrosinemia Type 1
C K Ko1 S Amarwani1 J Vora1 L Freland1 N Ramesh1 Y Chen1 N Walsh1 A Bastille1 J Liao1 S Zhang1 G Preston1 L Drouin1 R C Thompson1 M Hebben1 D Gruskin1 M Franken1 M Nacht1
1: LogicBio Therapeutics
GeneRideTM is a novel AAV‐based, nuclease‐free, genome editing technology that is designed to precisely insert a therapeutic transgene into the genome via homologous recombination. GeneRide has the potential to provide durable transgene expression regardless of cell proliferation and tissue growth, and GeneRide‐corrected hepatocytes show selective expansion in the presence of intrinsic liver damage due to genetic defects. Hereditary Tyrosinemia Type 1 (HT1) is a neonatal‐onset metabolic disorder caused by loss‐of‐function mutations in FAH gene, which, if untreated, results in acute liver failure and death. Mouse models of HT1 also show acute liver damage and we used these to demonstrate that GeneRide‐corrected hepatocytes can repopulate the entire liver within four weeks post dose (figure below), achieving a complete “molecular liver transplant”. GeneRide‐treated HT1 mice were non‐reliant on NTBC or diet restrictions (standard of care therapies), restored normal body growth and liver function, and had undetectable succinylacetone level (LLoQ = 10 nM). Compared to NTBC treatment (∼2 mg/kg/day in drinking water), GeneRide treatment showed superior succinylacetone reduction and lower alfa‐fetoprotein, a clinically validated biomarker for hepatocellular carcinoma and another risk‐factor for untreated HT1. These data demonstrate strong preclinical proof‐of‐concept for GeneRide.
Phase 1/2 Open‐label Dose Evaluation Study of AXO‐Lenti‐PD Gene Therapy for Parkinson's Disease: Efficacy, Safety, and Tolerability Data up to 24 Months
1: Sio Gene Therapies 2: UCL Institute of Neurology 3: Addenbrooke's Hospital 4: Groupe Hospitalier Henri‐Mondor
AXO‐Lenti‐PD uses a lentiviral vector that includes the three genes required for endogenous dopamine synthesis (TH, CH1 and AADC) and is delivered via one‐time direct bilateral infusions into each putamen. Six subjects have been enrolled in 2 cohorts in the Phase 1/2 SUNRISE‐PD study (NCT03720418): low‐dose (4.2E+6 TU), n = 2, follow‐up 24‐months; mid‐dose (1.4E+7 TU), n = 4, follow‐up 12‐months. AXO‐Lenti‐PD was generally well‐tolerated with no hypersensitivity, immune response or endotoxicity related adverse events. Two subjects experienced 5 serious adverse events, all unrelated to AXO‐Lenti‐PD. Subjects continued to show improvements from baseline in UPDRS Part III (Motor) OFF: 14‐27 points in low‐dose subjects at 24‐months and 5‐23 points in two UPDRS‐evaluable mid‐dose subjects at 12‐months. Changes from baseline in UPDRS Part II (Activities of Daily Living) OFF of 8‐19 points in low‐dose subjects at 24‐months and 0‐8 points in two UPDRS‐evaluable mid‐dose subjects at 12‐months were reported. One low‐dose subject completed the Hauser diary at 24‐months and had an increase from baseline in good ON time (ON without dyskinesia plus ON with non‐troublesome dyskinesia) of 2.0 hours and a mean decrease from baseline in OFF time of 0.5 hours. Mid‐dose subjects at 12‐months reported a mean increase from baseline in good ON time of 1.8 hours and a mean decrease from baseline in OFF time of 2.1 hours. Further evaluation of AXO‐Lenti‐PD is planned using a higher dose/volume open label cohort followed by a sham‐controlled study.
Evaluation of a CSF biomarker for gene therapy that discriminates neuronopathic and non‐neuronopathic forms of MPS II
1: REGENXBIO 2: University of Adelaide 3: Université Toulouse III and Laboratoire de Biochimie 4: University of Naples Federico II 5: Hospital de Clinicas de Porto Alegre
Mucopolysaccharidoses (MPSs) are a heterogenous group of inherited lysosomal storage disorders characterized by genetic variants inactivating lysosomal enzymes and subsequent accumulation of partially degraded complex carbohydrates, glycosaminoglycans, leading to progressive widespread cell and tissue damage. Neuronopathic forms of MPS manifest central nervous system abnormalities and neurocognitive impairment. Currently, there are no predictive or prognostic biomarkers for MPS phenotype capable of differentiating neuronopathic from non‐neuronopathic forms of MPS.
A bioanalytical mass spectrometry method was developed and validated to quantify the total amount of the glycosaminoglycan heparan sulfate (t‐HS) as the sum of four disaccharides (D2S6, D0A0, D0S0, D0A6) in cerebrospinal fluid (CSF) following enzymatic digestion. CSF samples from healthy controls (n = 31), MPSI (Neuronopathic n = 8, Non‐neuronopathic n = 2) and MPSII (Neuronopathic n = 11, Non‐neuronopathic n = 5) patients were used to determine t‐HS and HS composition in CSF. Significant increases in t‐HS were observed in all MPSI and MPSII patients compared to healthy controls. When comparing the composition of the disaccharides measured, concentration of D2S6 in CSF separated neuronopathic from non‐neuronopathic MPSII. D2S6 is a highly sulfated disaccharide generated exclusively from the N‐sulfated domains of HS known to mediate interactions with protein ligands in critical physiological processes. Increased HS sulfation has been shown to be pathogenic in MPS brain tissue; therefore, CSF D2S6 may provide a diagnostic biomarker to differentiate neuronopathic and non‐neuronopathic MPSII in presymptomatic individuals. Additionally, D2S6 is a direct substrate of iduronate‐2‐sulfatase, the deficient lysosomal enzyme in MPSII, and may provide a direct estimate of enzyme activity with potential utility in therapy monitoring.
RNAScope™ analysis of non‐human primate liver after AAVS3 delivery shows widespread liver transduction and periportal transgene expression
1: Freeline, Stevenage Bioscience Catalyst, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2FX UK 2: Katharine Dormandy Haemophilia Centre & Thrombosis Unit, Royal Free Hospital, Rowland Hill Street, London, NW3 2PF, UK
The use of adeno‐associated virus (AAV)‐based vectors for liver‐directed gene therapy has been shown to provide clinical benefit. Cellular characterization of vector uptake and transcriptional activity will increase insight into the therapeutic potential of AAV‐based products. In situ hybridisation technologies, such as RNAScope™, represent powerful methods to track AAV uptake and transgene expression on a cellular level across complex tissues. Here we describe results from RNAScope analysis of archival non‐human primate (NHP) liver tissue after AAVS3‐mediated delivery of human factor IX (hFIX) and human alpha‐galactosidase A (hα‐Gal A) transgenes. Liver tissue from NHPs dosed at either ∼3e13 vg/kg (hFIX and hα‐Gal‐A) or 6e12 vg/kg (hα‐Gal‐A) were stained with probes detecting vector DNA and mRNA. Using a combination of semi‐quantitative histological scoring and quantitative image analysis, we observed on average 50% and 30% of cells displayed a positive signal for transgene mRNA and vector DNA, respectively, in the highest dose cohort. The discordance between mRNA and DNA signal is consistent with an increased abundance or accumulation of mRNA per cell compared to vector DNA, and different limits of detection for the two signals from a single focal plane during microscopy. Transgene expression was heterogeneous across the liver, with a trend for higher transduction in the periportal region. The ability to estimate both the percentage and distribution of hepatocytes functionally transduced allows for a deeper understanding of gene therapy products and may help improve the accuracy of preclinical dose predictions.
Reducing anti‐vector humoral immunity through modulation of surface glycoprotein density
1: National Heart and Lung Institute, Imperial College London 2: Retroviral Immunology Lab, Francis Crick Institute
Immunity to delivery vectors for in vivo gene transfer or expression applications limits therapy efficacy upon repeat vector administration. Lentiviral vectors have a low immune profile which is particularly apparent when delivered to the lung. A lentiviral vector pseudotyped with the Fusion (F) and Haemagglutinin‐Neuraminidase (HN) proteins of Sendai virus (SeV) was capable of transducing the lungs of mice after 3 doses of vector with comparable efficacy to a single dose of vector. We studied anti‐vector immune responses to this vector further and compared these responses to those induced by matching doses of the standard lentiviral pseudotype, Vesicular Stomatitis Virus glycoprotein (VSVg). Anti‐glycoprotein neutralising antibodies (NAbs) against SeV F/HN‐lentivirus were induced later and at lower titres than for VSVg‐lentivirus after repeat dosing of mice with lentiviral vectors. Binding antibody and NAb titres correlated, indicating a lack of immune stimulation rather than a poor‐quality humoral response. We identified glycoprotein density as a key determinant of vector immunogenicity through western blot and mass spectrometry analysis of virions. SeV F/HN‐pseudotyped virions had a considerably lower glycoprotein density than VSVg‐pseudotyped virions, likely due to a modification of the cytoplasmic tail of the HN protein required for pseudotyping. Modulation of VSVg density reduced the immunogenicity of the vector but had a detrimental effect on transduction potential. This work highlights the importance of controlling the density of pseudotyping glycoproteins on the surface of virions during vector production to minimise anti‐vector immune responses, whilst permitting efficient transduction for therapeutic benefit.
Development of a scalable adeno‐associated virus production process by transient transfection in suspension cells
1: Cytiva
Use of viral vectors to transfer genes for specific tissue or cell type modifications has grown. Of the viruses that have been investigated for their use in cell and gene therapy, adeno‐associated virus (AAV) has emerged as the main vector for gene therapies. In this study, an efficient and scalable cell culture process for AAV production was developed by evaluating and optimizing each process step. HEK 293T cells were successfully adapted to suspension culture in a serum‐free medium without any animal‐derived components. Triple plasmid transfection using polyethylenimine (PEI) was optimized for different parameters and conditions using a design of experiments (DoE) strategy in shake flasks. The optimal concentrations and ratio of plasmids, PEI, temperature, transfection volume, and incubation times were evaluated for transfection efficiency and virus productivity. The conditions were further developed for production in single‐use bioreactor systems. A qPCR assay, Biacore technology and ELISA was used to quantify total virus capsid titer. The percentage of full capsids was calculated as the ratio between viral genomes and viral capsids. An optimized transfection protocol in shake flasks was generated based on the DoE studies for AAV2, with titers above 1014 viral capsids/L. Thus, a scalable, robust, and reproducible AAV production process was developed from small‐scale shake flask production up to 20 L in single‐use bioreactor systems.
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GMP‐compatible, automated production of
CCR5
‐edited CD4+‐T cells for the treatment of HIV‐positive patients
1: Dept. of Stem Cell Transplantation, Research Dept. Cell and Gene Therapy, University Medical Center Hamburg‐Eppendorf 2: Miltenyi Biotec 3: German Centre for Infection Research (DZIF)
The progress in ex‐vivo gene therapies pave the way for new strategies in immunotherapies. One of those approaches is directed at the protection of CD4+‐T cells from HIV infection in HIV‐positive individuals by knockout of the HIV co‐receptor C‐C motif chemokine receptor 5 (CCR5). We recently optimised a CCR5‐targeting TALE nuclease, CCR5‐Uco‐hetTALEN, which facilitates high CCR5 knockout rates with reduced off‐target activity in primary human T cells. Clinical translation of this approach requires up‐scaling of the manufacturing process in accordance with good manufacturing practice (GMP). Here we describe a GMP‐compatible mRNA electroporation protocol for the automated production of CCR5‐edited CD4+‐T cells in the closed CliniMACS Prodigy system. The automated T Cell Engineering (TCE) process reliably produced high numbers of viable CCR5‐edited CD4+‐T cells (1.65 ‐ 5.00 × 109 cells with >60% CCR5 editing) within 12 days. Notable, about 40% of total large‐scale produced cells showed a biallelic CCR5 editing, and between 25 ‐ 42% of produced cells had a central memory T‐cell phenotype. Titration of TALEN mRNA used in the TCE process resulted in high CCR5 editing with low (<2%) CCR2 off‐target activity reducing the risk of chromosomal aberrations. Furthermore, CCR5‐edited CD4+‐T cells showed no impairment of cytokine release or proliferation capacity. In addition, biallelic CCR5‐knockout resulted in a reduced susceptibility of TCE produced cells towards transduction with HIV‐enveloped pseudotyped lentiviral vectors. In conclusion, transfection of primary T cells with CCR5‐Uco‐hetTALEN mRNA is readily scalable for GMP‐compatible production and suitable for application in HIV gene therapy.
A second‐generation two‐plasmid packaging system for manufacturing of AAV vectors further improves quality and yield
1: Freeline, Stevenage Bioscience Catalyst, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2FX UK
We previously reported the development of a proprietary first‐generation, two‐plasmid packaging system that combines the adeno‐associated virus (AAV) rep gene and helper virus genes on one plasmid, and the AAV cap gene and a transgene expression cassette flanked by inverted terminal repeats on a second plasmid. This system is being used in early clinical programs and offers cost of goods and quality benefits such as high vector yields with low product‐related impurities, minimised generation of replication competent AAV (rcAAV), and an ability to modulate the ratio of vector genome‐containing particles to total particles.
By optimization of the AAV rep gene function on the ‘helper’ plasmid, we developed a proprietary second‐generation packaging system. Here, we provide data demonstrating that this system further enhances the performance over its predecessor in several respects. These include increases in cell‐specific yields and improvements in critical quality parameters such as the ratio of vector genome‐containing particles to total particles and the level of DNA mispackaging. Advantages regarding quality and yield are particularly pronounced for larger AAV vectors such as FLT210, used in a haemophilia A gene therapy program with a vector genome size of 4.7 kb. Applicability of the second‐generation plasmid system was demonstrated in small‐ and large‐scale formats (from dish to iCELLis® bioreactor).
In summary, the second‐generation plasmid system enables the production of AAV vectors with a further improved quality and yield compared with the first‐generation.
Functional Roles of the Novel Membrane‐Associated AAV Protein MAAP
1: Kuopio Center for Gene and Cell Therapy 2: FinVector Vision Therapies Ltd 3: University of Jyväskylä 4: Kuopio University Hospital 5: A.I.Virtanen Institute
The adeno‐associated virus (AAV) has evolved over‐lapping genes to maximize its genome use, as with the cap gene, which encodes the membrane‐associated accessory protein (MAAP) located in the same genomic region as the VP1/2 unique domain. To study the role of MAAP in wild‐type AAV2, we made point mutations along the MAAP ORF while keeping overlapping capsid proteins ORFs intact. In cells co‐transfected with plasmids encoding AAV2 genome and adenovirus helper genes, MAAP localized in the plasma membrane, as well as intracellular membranes. Both inactivation and truncation of MAAP translation affected the emergence and intracellular distribution of the AAV capsid proteins. Some MAAP‐modifications resulted in substantially improved virus yields and capsid integrity. The same MAAP modified cap gene improved recombinant AAV production yields and affected cellular egress. Altogether, our results reveal some MAAP functions having important implications for better‐quality and quantity production of AAV vectors for therapeutic purposes.
Efficient large‐scale production of AAV using a high cell density and low volume transfection process
1: Cell and Gene Therapy Catapult 2: MaxCyte
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.
Comparison of Lentiviral Vector Production from two Stable Packaging Cell Lines
1: CSL Behring Innovation GmbH
Production of lentiviral vectors for clinical gene transfer trials is challenging due to the cost of acquiring GMP‐grade plasmids and the labour‐intensive nature of making vectors via transient transfection. To facilitate clinical grade lentiviral vector production, we developed two packaging cell lines, GPRG and GPRTG, which were used to make a fully stable producer cell line for expression of lentiviruses. GPRG expresses codon‐optimized HIV‐1 gag‐pol, rev and VSV‐G and GPRTG expresses additionally Tat. Expression of Rev, Tat and VSV‐G are regulated by a Tet‐Off system. The performance of the GPRTG cell line was improved relative to GPRG by including the expression of Tat while decreasing the expression of Rev.
We have compared GPRG versus GPRTG packaging cell lines using stable transfection of concatamer encoding a selectable marker and WASp as the gene of interest. We have generated six stable pools per packaging cell line and have compared titer, lentivirus quality as well as transduction efficiency and dose response.
Producer pools originating from both packaging cell lines are producing high infectious titer to a different extent. Lentivirus titer based on virus‐associated p24 ELISA was similar between pools originating from both packaging cell lines, while differences were observed in the RNA content via ddPCR. A comparative assessment of the transduction efficiency and dose response in hCD34+ cells of lentiviruses originating from each of the GPRG and GPRTG cell lines was also conducted as well as assessment of quality parameters of the lentivirus.
Genetic engineering of a virus producer cell line to generate high‐titer and high‐quality BaEVRless‐pseudotyped alpha‐retroviral particles
1: Gene Therapy Program, Dana Farber/Boston Children's Cancer and Blood Disorders Center, Harvard Medical School 2: Institute of Experimental Hematology, Hannover Medical School 3: Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School 4: Centre International de Recherche en Infectiologie (CIRI), Université Lyon, Université Claude Bernard Lyon 1, INSERM, U1111, CNRS, UMR 5308, Ecole Normale Supérieure de Lyon 5: Université Côte d'Azur, INSERM U1065, Centre Méditerranéen de Médecine Moléculaire
The baboon endogenous retrovirus (BaEV) glycoprotein binds to the neutral amino acid transporters ASCT1 and ASCT2 for cell entry and is superior to VSV‐G regarding transduction of resting hematopoietic stem cells and serum resistance. The derivative BaEVRless (without R domain) produces higher viral titers compared to BaEV but its vector production is impaired by syncytia formation and cell death. Here, we used CRISPR‐Cas9 to knockout the entry receptors ASCT2 or ASCT1/2 in HEK293T cells to prevent superinfection and syncytia formation. The loss of ASCT2 is sufficient to abrogate the infectivity of BaEV‐pseudotyped viral particles in HEK293T cells. The elimination of syncytia formation is associated with reduced cell death and lower accumulation of toxic debris in concentrated viral supernatants as shown by a 3‐fold reduction of apoptotic cells after transduction of K562 cells. Improved cell fitness and reduced sequestration of viral particles led to a 2.2‐fold increase in viral titers using ASCT2 KO producer cells. The alpha‐Gag/Pol expression plasmid was stably integrated into the ASCT2 KO cells to generate a stable packaging cell line. Titers of BaEVRless‐pseudotyped alpha‐retroviral particles were comparable between the packaging cell line and the parental ASCT2 KO cells. For potential in vivo application, CD47 was overexpressed on the ASCT2 KO cell line, which reduced non‐specific macrophage‐mediated uptake of viral particles by 2‐fold compared to WT cells. In summary, we engineered a stable alpha‐retroviral packaging cell line to produce high‐titer and high‐quality BaEVRless‐pseudotyped alpha‐retroviral particles with the potential of in vivo application.
Application of Aber's FUTURA♦ Biomass Probes to Inform Transfection and Cell Lysis in iCELLis® Bioreactor‐based AAV Manufacturing
R Alfano2 S A Pezoa2 A Pennybaker2
1: Pall Life Sciences 2: InVitria
Utilization of classical adherent formats in large scale viral vector manufacturing can have significant setbacks due to the lack of scalability of production vessels typically used at small scale. The iCELLis fixed bed bioreactor has emerged as an enabling technology to efficiently scale adherent‐based processes in a controlled and highly integrated environment. This technology has been developed for the clinical manufacture of lentiviral and adeno‐associated vectors with commercially viable yields. Aber's FUTURA Biomass probes, which induce polarization of cells and measures the resulting capacitance of the medium in pF/cm, can be employed with the iCELLis bioreactor to provide direct online information on cell biomass during a viral vector production run. Routine utilization of these probes can provide invaluable online information regarding cell growth and health, control of feed/perfusion rate, and the identification of optimal time for transfection or harvest. Here, we utilized Aber's FUTURA Biomass probes in iCELLis bioreactor runs to produce an AAV‐2 GFP vector in OptiPEAK
Development of an Anion Exchange Chromatography Method to Assess Percent Full Capsids for Chimeric Capsid AAV‐LK03
1: LogicBio Therapeutics
Novel chimeric capsids have been discovered that have the potential to greatly increase transduction efficiency or target specific organs that would otherwise be inaccessible with adeno‐associated virus (AAV) gene therapy. In particular, AAV‐LK03 has been shown to have significantly higher transduction efficiency in human hepatocytes compared to wild‐type capsids and is now being used in clinical trials to treat diseases such as Methylmalonic Acidemia and Hemophilia A. However, the use of these chimeric capsids often requires significant optimization to develop a robust manufacturing process and accompanying analytical methods. Recent advancements in anion exchange (AEX) columns have allowed for the reliable separation of empty and full capsids for both large‐scale purification as well as for analytical characterization. When coupled with AEX, high pressure liquid chromatography (HPLC) offers a high throughput analytical method to measure the percentage of filled capsids (%Full) in a given sample. Of particular interest is the use of an AEX column with a tetramethylammonium chloride salt elution to improve the separation compared to a typical sodium chloride gradient. In this study, linearity is determined by injecting samples of varying %Full and the suitability of the method for stability and forced degradation studies is assessed. Various in‐process samples ranging from crude lysate to bulk drug substance are tested to show the utility of the method. Finally, a comparison is conducted between orthogonal %Full methods including analytical ultracentrifugation and vector genome/capsid particle titer (ddPCR/ELISA) to assess the practicality of the assay for routine sample analysis.
Development of a scalable adeno associated virus purification process and an SPR titer assay
1: Cytiva
Adeno Associated Virus (AAV) is the main vector for gene therapy and there is need for scalable, cost‐efficient and robust filtration and chromatography‐based purification processes. Key for a successful process are high overall yields with empty capsid reduction and efficient impurity removal. In this study, AAV2 and AAV5 serotypes were produced by triple plasmid transfection of HEK 293T cells in suspension. Each of the different steps from harvest, clarification, concentration and buffer exchange, affinity capture and polishing were evaluated and optimized. At the time of harvest the cells were lysed by using detergent and the DNA was fragmented by DNA nuclease and finally treated with low pH to induce a precipitate that could be removed by sedimentation before clarification by normal flow filtration. Next step was concentration and buffer exchange using ultrafiltration/diafiltration comparing 100 and 300 MWCO hollow fiber. The 300 MWCO hollow fiber filters gave similar high yields but better impurity reduction compared to 100 MWCO filters. The retentate was applied to affinity chromatography using Capto AVB and virus could be eluted with high recovery and excellent impurity reduction. Polishing to reduce empty capsid was evaluated with different ion exchange and multimodal resins. Viral genome titer was determined by qPCR and the viral capsid titer was assessed by ELISA and SPR assay using Biacore. The results were used to determine the % full capsids. In conclusion, a scalable and efficient AAV purification process was developed using filtration, chromatography and new analytics.
AAV production using a closed, semi‐automated hollow fibre bioreactor
1: Cell Therapy Catapult
As in vivo gene therapy applications continue to demonstrate efficacious clinical data, smaller scale manufacturing (<1e15 vg) generally still requires large footprint facilities and relies upon plastic based, heavily manual processes. Here, after an initial cost of goods (CoGs) and risk analysis (RA) in the context of a small fixed footprint GMP cleanroom (60 m2), we aimed to develop an automated, closed, end‐to‐end process for adherent AAV production. This process was benchmarked against standard plastic‐based manufacturing, using the same CoGs and RA approaches to evaluate different productivity ranges against batch size requirements. The quantum hollow fibre bioreactor (QHFBR) from Terumo BCT was identified to provide closed and semi‐automated expansion of cells, thawed and inoculated directly into the system without the need for a prior cell build. AAV2 vectors were transiently produced within HEK293T cells expanded within the QHFBR, lysed in situ and harvested into a low final volume (1.2 L) containing approximatively five billion lysed cells and ∼1e14 vg. To demonstrate the processability of the high biomass harvest, this complex crude lysate was then processed through a series of depth filters and ultrafiltration/diafiltration using tangential flow filtration, achieving 90% viral particles recovery and reducing process related impurities; clearance of 97% for turbidity, 89% for DNA and 30% for host cell proteins (HCP). Subsequent purification demonstrated the workflow capable of delivering purified (99% clearance of HCP and DNA; DNA <7.8ng/mL) and infective product (5046:1 vg:TU), suitable for drug substance specification.
Optimized Lentiviral Transduction Process for
ex vivo
CD34+ Hematopoietic Stem Cell Gene Therapy Drug Product Manufacture
1: Orchard Therapeutics 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 commercially available 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 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.
Combining highest rAAV manufacturing performance with highest quality standards to support CGT industry
1: Polyplus Transfection
The number of ATMP therapeutic‐based medicines for inherited genetic disorders is in constant growth, with a global 32% increase in new clinical trials in the last 4 years. ATMPs have demonstrated their success with already more than ten approved for commercialization. The success of AAV as the most promising viral vector for gene therapy is due to low immunogenicity, broad tropism and non‐integrating properties. One major challenge for translation of promising research to clinical development is the manufacture of sufficient quantities of AAV. Transient transfection of suspension cells is the most commonly used production platform, as it offers significant flexibility for cell and gene therapy development. However, this method presents some limitations in large scale bioreactors: inadequate transfection protocol, reduced transfection efficiency and lower productivity. To address this concern, we present data on a novel transfection reagent showing: i) increased AAV titers, ii) Cost‐effectiveness with reduced cost per dose for therapy affordability, iii) improved transfection protocol for large scale bioreactors and iv) reproducibility of viral titers at different production scale. With our continuous commitment in supporting CGT manufacturers, FectoVIR‐AAV is available at GMP grade, the highest quality grade for ancillary materials aligned with quality standards recommended by health authorities to ensure patient safety.
Separation of full and empty AAV capsids using membrane chromatography
A Hejmowski1 K Boenning1 A Kavara1 J Huato1
1: Pall Life Sciences
Adeno‐associated virus (AAV) is a widely used vector for gene therapies and there is an ever‐growing need within the industry to meet impurity thresholds and standardize AAV sample formulation. One major limiting factor in developing a consistent AAV therapeutic is the presence of “empty” AAV capsids which lack the gene of interest and may be considered a process impurity.
Here we present the progress in implementing our novel conductivity step elution method using anion exchange chromatography (AEX) with Mustang® Q XT membrane for full AAV capsid enrichment. We previously developed this technique using AAV5 and here describe purification of the additional serotypes AAV8 and AAV9. By comparing Absorbance measurements (260nm vs 280nm) the elution conditions for obtaining optimal amounts of full capsids from an affinity purified pool were determined. PCR and intact capsid ELISA, showed the majority presence of full capsids in these elution fractions. This approach for enrichment of full AAV capsids can be used as a rapid process development tool. Using information from the multistep elution method enables the implementation of a two step elution for production and manufacturing settings. This work demonstrates the promise of membrane chromatography as an AAV polishing step: It can supply full capsid enrichment, remove host cell protein and DNA contaminants and can be operated at high flow rates, up to 10 MV/min. The ability to operate a polishing step at high flow rates faciltates process intensification as typically a large dilution is required after AAV affinity chromatography prior to this step.
A DoE Approach to optimize AAV production and purification using FectoVir®‐AAV.
1: Andelyn Biosciences
Numerous strategies are currently being implemented to improve production and purification of Adeno‐Associated Virus (AAV) vectors for gene therapy. A novel transfection reagent for HEK293 suspension cultures, FectoVIR®‐AAV, has recently been released by Polyplus‐transfection® with claims of improved AAV yields compared to Polyethyleneimine (PEI), the current transfection reagent of choice for many platforms. This study describes the Design of Experiments approach used to optimize AAV yield in a suspension cell platform using FectoVIR®‐AAV. Using AAV with eGFP transgene, full factorial and mixture designs were developed to screen and optimize multiple factors including cell density, total amount of plasmid DNA, the ratio between the FectoVIR®‐AAV and DNA, and the ratios between the plasmids. These conditions were tested using a multitude of different serotypes in shake flasks (25mL and 62.5 mL cultures). AAV yields were 2 – 10 times higher with FectoVIR®‐AAV than that produced using PEI, depending on serotype. Flow cytometric analyses revealed >85% transfection efficiency for all serotypes compared to 50‐60% with PEI. The size of the transfection complex, monitored using dynamic light scattering, ranged between 500 – 900 nm. The optimized conditions were used to assess scalability and purification from larger cultures in flasks (1L, 2L) and PALL STR50 bioreactor (50L). The final product purity at all scales was >99%. At the bioreactor scale, AAV9 yields were 2‐ fold higher than historical averages for 50L productions using PEI. These studies confirm the utility of FectoVIR® as an effective transfection reagent for the suspension platform.
Isolation and characterization of cGMP monoclonal HEK293T master cell bank to manufacture lentiviral vector in adherence‐based bioreactors under serum‐free conditions
A Arrasate1 R López de Maturana1 J C Ramírez1
1: VIVEbiotech
VIVEbiotech is a CDMO specialized in LVV manufacturing to worldwide based clients ongoing phase I/II clinical trials and soon reaching commercial phase.
Growing interest in the use of lentiviral vectors (LVV) for gene and cell therapy applications has resulted in demand for production processes combining state of the art production rates and high regulatory standard compliancy.
The framework of cGMP manufacturing limits the features of manufacturing processes and among others, promt to the use of monoclonal master cell bank for lentiviral productions to increase predictability and robustness reducing batch to batch variability.
Starting from a cGMP batch of HEK293T we have isolated monoclonal cultures that have been screened by LVV production criteria. Strong variability from 7 to 15‐fold higher yields was observed between the isolated clones. In addition we have evaluated the behaviour of the more productive clones in both standard condition with media supplemented with fetal calf serum (FCS) and with the specific formulation (SerLESS, VIVE biotech named) that allows to VIVEbiotech to produce LVV in the absence of serum. Importantly we have demonstrated that production in the latter outperforms the standard production in FCS‐containing media with the polyclonal MCB either at low scale or in bioreactor.
We will present data on the flowchart followed to isolate and characterize the clones and the major steps in transferring from standard to SerLESS media conditions focusing on the relative yields production of reporter gene‐containing transfer plasmids and therapeutic ones as well.
Nanofiltration of AAV Cell Culture Media
N Marchand1
1: Pall Life Sciences
In bioreactor applications, there is risk of adventitious viruses in cell culture media, which could cause contamination. These are low‐occurrence but high‐impact events as they result in loss of a batch and costly decontamination, and if gone undetected, can affect patient safety. That risk can be mitigated by running the media through virus retention filters. While previous work has indicated there is no impact of Pall Pegasus Prime virus filtration on cell culture media properties affecting growth of e.g. Vero cells, we now address the open question on whether there is an impact on gene therapy media and cultures. In this work we tested Pall's Pegasus™ Prime virus removal filter in series with a Supor® EKV sterilizing‐grade filter against the sterile filter alone by running gene therapy cell culture media through each filter train separately. Using this sterile filtered media, we then cultured and transfected cells with AAV5 in the iCELLis® Nano bioreactor with a run lasting a total of ten days. The results showed that the Pegasus Prime filter had no impact on the growth, metabolites, or AAV5 viral production of the cells as compared to a Supor EKV filter.
Modified plasmid and transfection optimization in suspension HEK293 cells lead to scalable high‐yield process for AAV manufacturing
1: LogicBio Therapeutics
Transient transfection of suspension HEK293 cells is a robust and well‐established process for AAV manufacturing. However, it is usually considered to be poorly cost effective due to its low yield and because of the price of GMP grade plasmids. Based on the high demand of vector for clinical dosage, the process yield requires a quantum leap to make AAV gene therapies economically viable for patients.
To increase AAV titers, initial efforts focused on the plasmid design and the transfection parameters. Our optimized plasmids led to a 3 to 5‐fold increase of titers compared to the original 3‐plasmid system developed by Xiao et al. Additionally, the use of a novel transfection reagent allowed for a further 3 to 5‐fold increase in titer compared to the gold standard PEI. Using a design of experiment (DOE) approach to determine optimal transfection conditions, the combined technologies resulted in a titer increase of 10 to 25‐fold using LK03 capsid.
Finally, the scalability of our new process conditions was assessed using bench scale bioreactors (250 mL and 2 L) and 50 L single use reactors. Very consistent growth kinetics across all scales were demonstrated using automated cell counting and metabolite analysis. Comparable AAV titers were observed at any scale, in the range of 5E11 vg/mL, measured by droplet digital PCR in the crude harvests.
An extensive analytical characterization has been performed to demonstrate the comparability of the vector attributes between the historical process and the optimized conditions.
Pushing the right buttons: genetic silencing in producing cell lines to boost lentiviral vector production led to metabolic disfunction counteracting vector requirements
1: VIVEbiotech
Lentiviral‐derived vectors (LVV) are becoming one of the cornerstones of gene therapy, although its scaled‐up manufacturing is currently the main bottleneck.
Lentiviruses produced worldwide are mainly pseudotyped with the VSVg protein that drives the entry into the target cell by interaction with the low‐density lipoprotein receptor (LDLR) and the LDLR‐related protein family (LRP) that are expressed by producer HEK293 cells. We have demonstrated that more than 50% of the virus present in the harvest is lost by re‐entry into the cell during continuous LVV production. To overcome these unwanted phenomena, we have generated using a gene‐editing strategy an LDLR‐/‐ HEK293T‐derived KO cell line and by targeting the chaperone RAP involved in proper vesicular trafficking of most LRPs, the double KO cell line LDLR‐/‐_RAP‐/‐.
Using this cell line (LDLR‐/‐_RAP‐/‐) we have demonstrated a 2‐fold decrease in LVV transduction, corroborating the major role of LDLR members in VSV pseudotyped‐LVV during entry. However, and unexpectedly the cell line LDLR‐/‐_RAP‐/‐ albeit being capable of reducing reentry of LVV in the producer cells, did not increase net LVV production.
The key role of the LDLR members in the cholesterol homeostasis prompted us to measure its impact during vector production. We have shown that limited cholesterol supply in both KO cells compromises net LVV yield, demonstrating the importance of cell metabolism, and vector‐cell interactions during vector production. Hence, it is fundamental to understand the metabolic stress that may undergo upon genetic modification of the producer cells and the particular impact that in boosting vector yields.
Meeting Market Needs with Scalable AAV Manufacturing
1: AGCBiologics
AGC Biologics is a leading global CDMO, providing world‐class development and manufacture of therapeutic proteins, plasmid DNA, viral vectors and genetically engineered cells. Our technical know‐how allows us to bring small‐scale processes to scalable industrial manufacturing, ensuring process robustness and commercial viability. Because the application of AAV products is so diverse across indications and virus volumes needed, there is a market need for a platform offering that allows for manufacture at different scales. The availability of an efficient and scalable AAV vector production process is often a critical manufacturing challenge, for both the clinical and commercial phases. To address this, AGC Biologics is developing a platform for various scales (50L‐500L) of AAV production with different serotypes (AAV‐6; AAV‐8; AAV‐2; AAV‐9), based on transient transfection in HEK293 and HEK293T cells. This will be achieved on an adherent cell line using Pall iCELLis® fixed‐bed disposable bioreactors. The upstream process has been optimized with different producer cell line and identifying the best transfection setting, harvest and lysis strategy and timing for each step. The downstream process consists of a capture step with affinity chromatography, followed by concentration and formulation steps based on TFF. A polishing step increases the full to empty particle ratio.
AGC Biologics presents process development data, including potency and vector purity at each production step for the different serotypes to ensure quality standards. This platform is flexible in terms of AAV serotypes and Gene of Interest (GOI) and it is available at scales ranging from 50L‐500L.
Superior precision and reliability of multidimensional ddPCR for accurate analysis of rAAV vectors
1: Charité ‐ Universitätsmedizin Berlin, Germany
As the most widely used vector in gene therapy, recombinant Adeno‐Associated Virus (rAAV) is rapidly progressing to the clinic. For regulatory approval many critical quality attributes (CQAs) have to be defined and reproducibly quantified, such as AAV stability, purity, aggregates, empty/full particles ratio and rAAV genome titre. Droplet digital PCR (ddPCR) is becoming the tool of choice for quantification of rAAV.
We have identified several critical parameters that could impair the accuracy of AAV titration with ddPCR: dilution factors are critical and primers/probe location within the rAAV genome could directly affect results. In addition, physico‐chemical properties of the sample, such as the presence of AAV aggregates or residual DNA, could also impact characterisation.
Exploiting the unique ddPCR option of multidimensional single particle analysis, we developed analytical tools for in depth rAAV characterisation. We validated the tools on the acknowledged AAV8 reference standard material (AAVRSM8), as well on our own AAV1 batches. Dependent on the location of primers/probe, we found up to 30% variation in titre. Multidimensional ddPCR further allowed for characterisation of AAV genome integrity on the single particle per droplet level. To our surprise only 50% of packaged genomes were full length in a single AAV, both in the standard reference material as well as in newly produced rAAV. Collectively, our results show that ddPCR allows for precise and extensive characterisation of rAAV vectors on a single particle level.
Ixaka versatile platform for the bioproduction and characterisation of lentiviral vectors: from R&D quality up to pilot‐grade batches
1: IXAKA
Recent market approval of CAR‐T cell therapies and the continuously growing number of gene‐based ex vivo and in vivo therapies evaluated in clinical trials has created a huge constraint on the supply of high quality grade lentiviral vectors (LV). This results in higher manufacturing costs and longer timelines, not sustainable for early‐stage development programs.
Ixaka has built up a versatile manufacturing platform for high‐quality grade LV material designed to cover customers' needs from R&D in vitro and in vivo evaluation. Our facility based in Paris offers manufacturing capacities in mammalian cell suspensions from 150mL to 3L of bulk produced with synthetic medium. After two purification and concentration steps, the vector batches are sterile‐filtered and stored at ‐80°C. Here we report improvements on process optimization and control on our routine process that allows the bioproduction of 60 mL drug product within 5 days with increasing physical titers and process handling.
We succeed transferring the cell culture steps from Shake Flask to Single use, fully automated Bioreactors and to implement in line parameter monitoring.
Adapted quality controls have been developed, analytically validated and implemented to ensure the characterization of the products. In addition, we have implemented biophysical methods compatible with in‐process and in line controls. All of this will allow a tight management of operations and bioproduction costs through increase process automation reliability and batch to batch comparability. Ixaka is thus able to provide an integrated solution to support biotech companies and academic laboratories throughout the development of their gene therapy programs.
Stability and proliferative potential of hematopoietic stem cells is maintained over time after thawing in albumin solution
1: Sidra Medicine, Doha, Qatar
Effective cryopreservation and thawing protocols are essential for the efficacy and safety of cellular products as hematopoietic stem cells (HSCs). While the HSC cryopreservation has been already standardized, the thawing procedures have been poorly studied. The aims of our study were to validate the thawing and washing protocol of cord blood (CB)‐derived HSC and determine CD34+ cells' stability over time.
Three cryopreserved CB products were thawed and washed in 3 different solutions (10% Dextran40 in NaCl equally prepared with 5% human albumin solution (HAS); 5% HAS in PBS/EDTA; and normal saline) and stored at 4°C. The mononuclear cell (MNC) count, CD45+/CD34+ cell enumeration, and cell viability tested at 0, 2, 4, 6, 8, 12, 24, 36, and 48 hours showed that the 5% HAS was the most suitable thawing solution. The protocol with the selected solution was further validated on additional 10 CB samples. Above paremeters and colony‐forming unit (CFU) assay were analyzed at time points 0, 2, 4, 6, and 8 hours.
MNCs were stable up to 4 hours (p = 0.009), viable CD45+ cells were unstable even at 2 hours (p = 0.013), and viable CD34+ cells were stable until 6 hours (p = 0.019). The CFU assay proved the proliferative potential up to 8 hours, although significantly decreased after 4 hours (p = 0.013) and correlated with the viable CD34+ cell counts.
We demonstrated that the expiration time of post‐thawed and washed HSCs using 5% HAS was validated up to 4 hours.
Lentiviral CAR‐T backbone promotor configuration determine vector production yield and vector functionality by modulating viral genome production and packaging yields
M Fertin1 G Gavilanes1 M Ugalde1
1: VIVEbiotech
VIVEbiotech is a CDMO specialized in lentiviral vectors (LVV) manufacturing to worldwide based clients ongoing phase I/II clinical trials.
One of the most powerful optimization strategies employed in VIVEbiotech for titer improvement relies on the optimization of the features of the therapeutic/transfer plasmid. Backbones of transfer plasmid carrying the transgene, contain at least two promoters. While external promoter has been previously described as a key factor for LVV titer, the internal promoter driving expression of the transgene is usually selected under a transcription‐on targeting criterion. However, the crosstalk between both promoters in LVV production yields is poorly understood and data in the literature scarce. We have studied such interaction in a collection of CAR‐expressing transfer plasmids in an overall identical LVV backbone focused on the impact of the different combinations on (i) the biological titre (VCN/TU), (ii) viral particles (p24), and (iii) genome packaging and analyzing the corresponding performances.
Our data indicate that these two elements have a major impact on LVV yield allowing to increase up to 50‐fold vector production by changing the internal promoter. In addition, we demonstrated that at least unbalance of genomic and transgene mRNA synthesis during production is involved in such variation. We will present conclusive data supporting the idea that certain combinations i.e., EF‐1alpha and CMV must be avoided to gain a significant increase in LVV production and quality. Then a correct design of the transfer plasmid in early studies is key to guarantee success in onwards scaled manufacturing at late stages.
High added value of DoE and Ambr®15 automated bioreactor for determining critical parameters of AAV vector production in mammalian and insect suspension cells
1: INSERM UMR 1089, University of Nantes, CHU of Nantes 2: Sartorius Stedim Biotech
Adeno‐associated viruses (AAVs) are commonly used vehicles to deliver therapeutic DNA, supporting more than 250 clinical trials to date. Manufacturers face challenges to respond to the growing need of high quality AAV vectors. Scalable processes based on mammalian and insect suspension cells have already been implemented. However, to tackle the current limitations and increase productivity, process optimization is essential. Here, we have used Sartorius Ambr® 15 microbioreactor system and a design of experiment (DoE) strategy to determine the critical parameters for AAV8 vector production in both HEK293 and Sf9 suspension cells. Ambr® 15 is an automated system allowing to run up to 48 parallel bioreactors of 15 mL. Taking advantage of this high throughput system, several parameters related to the growth conditions and plasmid transfection have been particularly scrutinized in two HEK293 cell lines. The DoE plan was designed with the Sartorius MODDE® software to test different combinations of process variables, e.g transfection reagents, media, pH, plasmids concentration. Amongst those parameters, the choice of the transfection reagent has a greater impact on the rAAV titer. Demonstrating the versatility of the Ambr® 15 system, optimization of the dual‐baculovirus/Sf9 platform has also been realized. In particular, models have been drawn for cell density and baculovirus MOI variables. The optimal conditions in Sf9 cells allowed to reach a yield of 1.2 x 1012 vector genomes per mL at harvest. This work highlights the advantages of using Ambr® 15 for process development activities in the context of rAAV manufacturing in suspension cultures.
Development of a HEK293‐based fully stable, helper virus‐free ELEVECTA production system for rAAV
1: Cevec Pharmaceuticals GmbH, Cologne
We have recently developed a stable helper‐virus free rAAV production platform named ELEVECTA®. This AAV production platform consists of mammalian suspension cells which have stably integrated all components necessary to produce AAV. Initially, this platform was based on immortalized human amniocytes (CAP cells). In order to expand our portfolio, HEK293 cells were chosen for the generation of a second ELELVECTA® AAV production platform.
Adherent HEK293 cells were adapted to serum‐free suspension growth in animal component‐free and chemically defined culture medium. Subsequent single cell cloning of these newly generated suspension 293 cells resulted in a clonal cell line, which grows in single cell suspension mode to high density. Benchmarking experiments against competitor 293 cell lines revealed favorable rAAV production characteristics of this cell line in a fully transient production setting.
For the generation of the suspension HEK293 ELEVECTA® cell line, cells were modified to express AAV Rep as well as adenoviral helper functions under control of a Tet‐inducible promotor system. Subsequently, single cell clones were generated, which displayed high titer rAAV production upon activation of the inducible promotor system and transient transfection of packaging as well as transgene elements. In a final step, a proof of concept 293 producer cell line was generated by genetically integrating capsid and transgene elements into the cellular genome.
A same lentiviral vector manufacturing process for both research and clinical application
1: Flash Therapeutics
Needs of Advanced Therapy Medicinal Products have exploded, in particular for CAR‐T and CAR‐NK treatments. Even though lentiviral vectors (LV) are used as starting material for ex vivo clinical trials, providing high‐class quality in terms of purification level is a success factor for the development of cell and gene therapy products. Here we describe a robust process of LV manufacturing for both research and clinical applications, using Hyperstack® technology, leading to highly consistent process yield & quality attributes on transduced target cells. This case study was performed on a CAR‐expressing LV using either R&D or GMP qualified raw materials used at pilot and GMP manufacturing scales. A detailed analytical characterization of product titer, process impurities & quality attributes was established, showing the removal of 99% of proteins and DNA impurities and led to equivalent product specifications through different process steps, either considering integrated genome (IG/mL) or transgene expression using immortalized cell line (TU/mL). Physical particles over transducing units were low, ensuring a high infectivity. We also showed that productivity was maintain between the 2nd and 3rd generation of LVs, and benzonase treatment had no impact on infectious titer. T‐lymphocytes transduction efficiency and transgene expression level were also assessed with both products originating from different process steps and purity grades, showing there was no difference between raw materials grades, and increased through process purifications steps. These robust and consistent results strengthen the claim of an easy and prompt transition from R&D activities to phases 1/2 clinical studies.
A synthetic biology approach to large scale AAV production
1: Fondazione Telethon ‐ TIGEM 2: Università degli Studi di Napoli Federico II
Vectors based on the AAV (Adeno Associated Virus) are the safest and most effective for in vivo gene therapy. However, large‐scale AAV vector production is inefficient and this represents a bottleneck for AAV clinical development. The most common method for AAV production relies on mammalian cell transfection of three different plasmids, which is expensive and difficult to scale. Efforts to generate cells with stable expression of the AAV production machinery have failed because of viral genes toxicity. Here, we describe a synthetic biology approach to create a stable AAV producer cell line. First, using bicistronic mRNA elements, we developed a microHelper (μHelper) plasmid encoding only for two helper genes, thus resulting in a construct that is half the length of the original Helper plasmid commonly used to produce AAVs. Second, we designed a synthetic circuit to block the expression of μHelper and Rep/Cap genes unless an inducer molecule is added to the medium. Unlike previous efforts based on inducible promoters, whose leakiness has prevented the establishment of robust producer cell lines, our approach combines post‐transcriptional and post‐translational feedback regulation by means of RNA‐binding proteins to achieve this goal. We are currently generating cells stably expressing our synthetic construct to compare our producer clones with the triple transfection approach. This new production technology will allow for an easy and economic scale‐up, avoiding the requirement for expensive plasmids supply under good manufacturing practices. This could be useful in both academic and industrial contexts where large clinical lots of AAV may be required.
Enhancing transduction efficiency of Adeno‐Associated Virus 9 by cell line gene engineering for gene therapy potency assay
1: Toolgen Inc.
AAV‐mediated gene therapy holds significant promises to treat or potentially cure various human diseases. Although AAV holds promise for their significant therapeutic potential, batch‐to‐batch differences can exist from manufacturing and therefore, a potency assay is required for clinical development of AAV. Among different serotypes, due to its ability to cross blood‐brain‐barrier and wide‐spread transduction capability in vivo upon systemic administration, AAV9 has been widely utilised for the development of treatment of neurological disorders. However, as AAV9 is known to show poor transduction in vitro, establishing a robust in vitro potency assay have been difficult. To this end, we engineered HEK293T and Schwann‐like cell lines to overexpress 2 different previously identified common AAV receptors (AAVR, LamininR) or 2 different endogenous host factors (GPR108, TM9SF2) that can increase infectivity of AAVs in an attempt to increase transduction capability of AAV9. Out of these, we found that GPR108 engineered HEK293T showed increase in AAV9 transduction the most whereas AAVR engineered Schwann‐like cell line showed increase in AAV9 transduction the most. These results indicate that different cell lines may require different gene engineering to increase AAV9 infectivity. Using these engineered cell lines, we established and validated of AAV9‐based potency and neutralizing antibody assay with at least 10‐fold lower amount of AAV9 required for each assays, indicating a versatility of this engineered cell lines for AAV9‐based gene therapy in vitro assays.
Form follows function – or vice versa?
The effect of plasmid quality and transfection reagent on transfection, cytotoxicity and AAV production in HEK cells
1: Xell AG 2: Bielefeld University 3: PlasmidFactory GmbH & Co. KG
With the rapid developments in the field of gene therapy, the use of viral vectors to treat various monogenic, inherited diseases in human patients is gaining momentum. Nonetheless, there are still open questions to be addressed in order to fully understand and optimize the production of viral vectors in mammalian cells. Factors to be considered include the effect of plasmid quality and transfection reagent on cell growth, cytotoxicity and yield of vectors such as adeno‐associated viruses (AAV).
We therefore evaluated different types and qualities of expression vector and helper & packaging plasmid in combination with several transfection reagents for production of AAV‐5 in commercial HEK cell lines. We compared Research Grade (RG) DNA and further purified ccc (covalently closed circular) DNA, which contains fewer to no defective, e.g., open circular (oc) plasmid DNA and significantly reduced chromosomal DNA. Additionally, minicircle DNA, which almost exclusively contains the gene of interest, was included in the experiments. Cell growth and cytotoxicity were analyzed via automated cell counting and lactate dehydrogenase assay, while transfection efficiency was assessed through flow cytometry. AAV‐5 titer was determined by droplet digital PCR and ELISA.
Our results indicate that the effect of DNA quality (RG vs. ccc) on cell growth and AAV titer is neglectable in comparison to the effect caused by different transfection reagents. These led to variations in transfection complex size, measured by dynamic light scattering, which correlated with transfection efficiency and total AAV‐5 titer. Overall, the observed interactions are more complex than “form follows function” implies at first.
Efficient Lentiviral Vector Production in a Chemically Defined, Blood‐Free and Serum‐Free Medium, Scalable to the iCELLis® Technology
S A Pezoa2 R Alfano2 A Pennybaker2
1: Pall Life Sciences 2: InVitria
Retroviral vectors are a promising candidate for the treatment of rare, monogenic diseases. Lentivirus — a type of retrovirus based on HIV — is currently being clinically evaluated in stage 3 trials for the treatment of rare blood disorders in addition to the genetic modification of human T cells in oncology applications. While the efficacy looks promising in the clinic, numerous questions surrounding the feasibility of large‐scale manufacturing of lentivirus remain. Traditionally, production of these retroviral vectors has been performed using adherent platforms that rely on the use of fetal bovine serum for the adherence and growth of HEK cells used to produce lentivirus vectors. At scale, fetal bovine serum presents numerous problems including but not limited to lot‐to‐lot variation, constraints on the global supply chain, and increasing cost due to global demand. To overcome these limitations, we have developed OptiPEAK♦ HEK serum‐free, chemically defined cell culture medium that is free from any blood‐derived proteins and supports adherent HEK cells in 2D and 3D formats. With OptiPEAK HEK cell culture medium, we are able to achieve equivalent growth kinetics and viral titer compared to medium supplemented with serum. We also demonstrate that OptiPEAK HEK medium can be readily scaled up to an iCELLis Nano bioreactor, achieving high viral titers without the addition of serum. The presented data here demonstrate that high titer, retroviral vectors can be manufactured without the constrictions brought on by the inclusion of serum in cell culture medium.
♦OptiPEAK is a trademark of InVitria
Robotics & Miniaturization enable fast serotype adaptation of AAV DSP‐Platform
1: Baxalta Innovations GmbH, a part of Takeda
Adeno‐Associated Virus (AAV) hold an immense promise for in vivo cell therapy as well as in vitro gene delivery in cell therapy applications. However, different AAV serotypes and engineered capsids pose a challenge for process development. To simplify and accelerate process development, Takeda has implemented a platform approach. Despite wide serotype compatibility, adjustments and optimization of specific process parameters, such as chromatography binding capacity, may be needed.
In this work we optimized the binding capacity of polishing chromatography using a microscale robotic system with minimal sample volume. High throughput experimentation was combined with fast analytical methods, such as UV absorption together with Static/Dynamic Light Scattering, to enable fast AAV‐particle breakthrough and thus maximal resin utilization. The feasibility of the robotic approach was further verified by comparing the results obtained against a breakthrough experiment performed in a chromatography workstation (industry standard).
Our approach enabled a 7‐fold increase in chromatography capacity and was performed within 24 h, which contributes for platform flexibility to deal with different serotypes and engineered capsids. The combination of robotics, automation and fast analytics provides a path for process development and optimization under strict timelines and increasing cost limitations.
Integrated & continuous processing: a proven solution to tackle gene therapy manufacturing challenges
1: Univercells Technologies
Over the last decade, the monoclonal antibody (mAb) industry has seen several evolutions. Working toward a continuous approach, mAb production processes were redefined to reduce manufacturing footprint and increase overall productivity. This effort initially started with increasing upstream productivity before moving to overcome subsequent midstream and downstream processing challenges. These unit operations would eventually be combined into continuous and automated manufacturing platforms.
With so much progress made in mAb production, the industry is now seeking answers for newer modalities like gene therapies (GTs). Though demand for GTs is skyrocketing, these products are dependent on the availability of viral vectors for commercialisation. The technology legacy acquired from mAbs results in low GT titers and limited performance, constraining developers to eventually scale out their process rather than scale up. Running parallel operations increases the overall volume and footprint needed to achieve commercial production scales.
Recent technology innovation offers a paradigm shift in integrated continuous manufacture of viral vectors with next‐generation equipment purposefully designed to overcome such challenges. These technologies apply the principles of process intensification and chaining to enable integrated continuous processing and redefine scalability in GT manufacture.
This presentation will show how leveraging lessons learned from the mAbs industry enable to reach capacity increase and manufacture acceleration for GTs. Inspired by the continuous processing approach applied to mAbs, a novel technology tackling GT challenges was designed. Case studies demonstrating its superior performance will be shared at the event.
Scale Up of a Lentiviral Production Process from the iCELLis® Nano Bioreactor to the iCELLis 500 + Bioreactor
I Pelletier2 V Pasupuleti2 P Agnihotri2 Y Do2 Z Sandalon2
1: Pall Life Sciences 2: Advanced Bioscience Laboratories
Lentivirus is a type of retrovirus that has a unique ability to infect non‐dividing cells, giving it the potential to be used in a wide range of applications. Traditional adherent methods of growing cells and producing lentivirus are cumbersome at the large scales required to obtain enough doses for patients. In addition, traditional flatware methods often have open handling steps that increase the likelihood of contaminations. The iCELLis bioreactor technology addresses the need for a scalable adherent system that has process controls and is closed, reducing the risk contamination risk from open handling. Further, by keeping factors such as linear speed, perfusion rates, fixed bed height, and fixed bed compaction constant, processes can be scaled from the bench‐scale iCELLis Nano bioreactor to the large‐scale iCELLis 500 + bioreactor. The data presented here shows that a process producing lentivirus was effectively scaled from a 0.53 m2 iCELLis Nano bioreactor to the 66 m2 iCELLis 500 + bioreactor. Utilizing perfusion, the iCELLis Nano achieved a titer of 1.01x108 copies/cm2 and the iCELLis 500 + achieved a titer of 3.47x108 copies/cm2, showing that an efficient large‐scale process can be adapted quickly using the iCELLis fixed bed bioreactor technology.
Development of manufacturing system for GMP‐grade iPSC lines
E Burns1 R Gilbert1 S Glen1
1: RoslinCT, Nine Edinburgh BioQuarter, 9 Little France Road, Edinburgh
Pluripotent stem cells have the ability to differentiate into almost any cell type found in the human body. With the identification by Yamanka of key transcriptional factors for reversing specialised somatic cells into the pluripotent state, a new avenue in the field of cell therapies has opened and with it the opportunity to advance iPSC‐based therapies to clinical application. To address this challenge, expertise in iPSC technology, process development, manufacturing, and the regulatory requirements of Good Manufacturing Practice (GMP) were brought together to establish a robust manufacturing system for the generation of clinical‐grade iPSC lines.
The GMP‐compliant manufacturing system comprises a fully qualified process for iPSC line derivation, expansion and banking which has been adapted to requirements of the clean‐room environment, and a customised Quality Control (QC) strategy with appropriately qualified QC assays which are accompanied by the full suite of documentation required for GMP‐compliance.
The manufacturing system has been developed around a process utilising the Sendai reprogramming method to generate iPSCs, with consideration for ethical, legal, and technical requirements for GMP manufacture as well as with a strong emphasis on commercial feasibility.
Improving targeting and yield of AAV by capsid and cell engineering
1: Royal Institute of Technology 2: AstraZeneca 3: Vironova
Targeting genetic disorders by gene delivery into patients has become a reality during the past five years with the approval of for instance adeno‐associated virus(AAV)‐based drugs such as Luxturna and Zolgensma. Regardless the promising outlook for AAV‐related therapies, major limitations related to low production efficiencies and high dosage requirements must be overcome to make treatments more accessible. With the goal to tackle such issues by cell line and AAV vector engineering, we have mapped the production and structural differences between seven AAV serotypes in both adherent and suspension HEK293 cell lines. Based on these data AAV hybrid variants were designed generating variants with either improved production capacities or improved secretion properties from producing cells. Moreover, transcriptome analysis of adherent and suspension AAV‐producing HEK293 cells identified host genes for cell line engineering purposes of an AAV producer cell line.
AAV VP1, VP2 and VP3 protein standards – Value of reliable standards for AAV vector manufacturing
H Besir1 R Wetzel1
1: PROGEN
A growing number of academic and industrial labs are using AAV vectors for the development of gene therapy products. This leads to an increase in the demand for effective and reliable analytical AAV tools for R&D and manufacturing. A crucial but often neglected factor in AAV vector manufacturing is the comprehensive monitoring of the complete manufacturing process. The establishment of a comprehensive monitoring can reduce time and money by the identification of the ideal production and purification conditions to ensure high yield and high quality of the final gene therapy product. However, the lack of reliable standards for AAV has been a major challenge for the gene therapy community.
For this reason, PROGEN developed suitable AAV2 capsid protein standards, i.e. recombinant VP1, VP2 and VP3 proteins for the determination of AAV capsid protein identity and optimization of particle yield during the initial production process. Depending on the method of choice, there are critical factors that need to be corrected in order to generate reliable data, e.g. differences in transfer efficiency from gel to membrane in Western Blot or labeling efficiencies with fluorescent dyes in capillary electrophoresis. PROGEN´s recombinant AAV capsid proteins enable proper comparison of VP1:VP2:VP3 ratios in an AAV preparation including the correction of artifacts with different methods.
This poster presents different areas of application and emphasizes the value of reliable AAV capsid protein standards for AAV vector manufacturing.
Major splice donor‐mutated HIV‐1 lentiviral vectors (LVs) and enhancement of their production by co‐expression of LV RNA‐targeted U1 snRNA.
1: Oxford BioMedica
To advance the utility of LVs it will be necessary to further improve the quantity and quality of vectors produced. The HIV‐1 genome is structurally complex, with cis‐acting elements including the major splice donor (MSD), packaging signal (Psi), and rev‐response element (RRE) governing production of unspliced/spliced RNA. Standard LVs retain minimal HIV‐1 sequences including Psi and RRE, in such a configuration that theoretically MSD/splice acceptor (SA) activity is suppressed by rev, leading to unspliced cytoplasmic vRNA for packaging. However, we've observed 30‐95% aberrant splicing from the MSD to strong or cryptic SAs within transgene expression cassettes, even in the presence of rev during LV production. Whilst only the unspliced vRNA is packaged into vector particles, the aberrantly spliced mRNAs can encode transgene protein, explaining why transgenes can be highly expressed during LV production, even when using weak/tissue specific transgene promoters. In addition, the TRiP systemTM (Maunder et al 2017) was less able to repress transgene expression from these aberrantly spliced mRNAs. One way of addressing this is to inactivate the MSD; however this leads to titre reduction, activation of cryptic SDs in Psi, and confers tat‐dependent production of LVs, which is undesirable for clinical use.
We will report on new MSD‐mutated LV genomes that do not aberrantly splice, and enhance TRiP repression. Tat‐independent production of MSD‐mutated LVs can be realised by co‐expression of a modified U1 snRNA molecule targeted to Psi. These new genomes are being assessed for stable producer cell development, where unspliced vRNA is often the limiting component.
In depth analysis of AAV‐containing fractions extracted from CsCl ultracentrifugation gradient
1: Department of Biotechnology and Systems Biology, National institute of Biology, Vecna pot 111, Ljubljana, 1000, Slovenia 2: Niba Labs Ltd., Litostrojska 52, Ljubljana, 1000, Slovenia
In the production of recombinant AAVs, the harvest always contains a mixture of viral particles and contaminants. Downstream purification (e.g., CsCl gradient ultracentrifugation) then focuses on removing impurities, which may not be sufficient and may reduce the expected therapeutic effect. In our work, we performed an in‐depth comparison of four AAV‐containing fractions from the 2nd CsCl ultracentrifugation gradient (called heavy, empty, intermediate and empty), which surprisingly showed similar transduction efficiencies. We implemented a holistic approach and analysed the distribution of intact bioactive AAV particles and contaminants such as unwanted DNA molecules, host cell proteins, unwanted AAV viral particles, and aggregates. Many analytical methods (TEM, transduction efficiency assay, ddPCR, qPCR, ELISA, MADLS and HTS) were tested side by side on two different rAAV9 (single‐stranded AAV and self‐complementary AAV) produced at International Centre for Genetic Engineering and Biotechnology in Triste, Italy. The results showed that the fractions were relatively similar in terms of relative vector and impurities content, except for the heavy fraction, in which we observed a high proportion of single‐stranded host cell DNA and many aggregates. As expected, the optimal fraction containing the highest amount of full AAVs and the lowest amount of impurities was the full fraction. Interestingly, the intermediate fraction, which is normally discarded, also contained a high proportion of full virus particles. Although the empty fraction contained the highest proportion of empty and damaged viral particles, the viral genome titer was relatively high. In addition, we evaluated the integrity of the vector genome using ddPCR.
Development and scale‐up of a transient AAV production process using a suspension‐adapted HEK293 clone
1: Merck Millipore
Adeno‐associated virus (AAV) has become an increasingly popular viral vector for gene therapy. Currently, there are two approved AAV‐based gene therapies and the number of clinical trials is steadily increasing. As with most viral vector therapies, their production is labor intensive and expensive due to the use of adherent cell culture production processes. For process intensification, the industry has begun to utilize bioreactors. Here, we highlight our development of a suspension based AAV upstream production process to provide a more efficient and cost‐effective bioprocessing solution for large scale production.
LVV Production At‐Scale: From Cell Factory™ Systems to iCELLis® 500 Bioreactor
F Bellinatani1 M Neri1 F Rossetti1 L Crippa1 S Corbetta1 F Lorenzetti1 M Cota1 M Brunati1 L Allievi1 M Rausa1
1: AGC Biologics
AGC Biologics is a leading global CDMO, providing world‐class development and manufacture of, plasmid DNA, viral vectors and genetically engineered cells. We developed a robust, scalable, high quality and quantity process for the industrial scale production of LV vectors: upstream in iCELLis® bioreactor, downstream purification and concentration by chromatography and TFF steps and sterile filtration and filling in different formats. Upstream process was developed in iCELLis® Nano system where key process parameters were screened to increase productivity, producing a bulk vector with consistently low contaminant profile. Data obtained on full‐scale iCELLis® 500 system confirmed full scalability and equivalent performance of the process. The downstream process was designed to remove main process related contaminants maintaining vector infectivity. The process consists of an DEAE step enabling 80% recovery while removing HCPs, DNA and BSA. Eluted vector is then concentrated and diafiltered with TFF using hollow fibers. With this step, the vector is further concentrated with 80% recovery, and additional contaminant removal is achieved. The vector is then 0.2μ sterile filtrated and filled. Total process recovery is approximately 30%. Final purified vector data shows a similar impurity profile as the established 48L CF's process. The system has already been successfully implemented for the manufacture for different vectors with different Gene of Interest. With the ability to produce a volume of 4x without a loss in quality, the iCELLis® 500 platform offers a scalable and cost‐effective solution with regards to the number of patients treated versus the cost of production and qc.
Make Gene Therapy Benefit All Mankind
1: University of Iowa
OBiO Technology (Shanghai) Corp., Ltd. is a gene therapy‐focused biotechnology company founded in 2013. OBiO offers holistic gene therapy CRO/CDMO services across vector construction, gene expression and function study, drug efficacy study, process development, and testing, chemistry manufacturing and controls for Investigational New Drug Application (IND‐CMC), GMP manufacturing for Phase I‐III clinical trials, and GMP manufacturing for commercialized products. Providing high‐quality service as our responsibility, OBiO is committed to accelerating the exploratory research and drug development of recombinant viral vectors, oncolytic viruses, and CAR‐T cell therapies." Gene medicine, made in China, by OBiO", OBiO is dedicated to promoting the gene therapy industry for the benefit of populations around the world.
By doing the following three things, we believe OBiO is your first option for CDMO.
Together, we can speed up your drugs' development and benefit more patients.
Details, please contact
AAV9 gene replacement therapy in two mutant mouse models of CMT1X demyelinating neuropathy
1: Neuroscience Department, The Cyprus Institute of Neurology and Genetics and Cyprus School of Molecular Medicine 2: Department of Biochemistry & Molecular Biology, Institute of Neurosciences, Universitat Autònoma de Barcelona 3: Center for Neuromuscular Disorders, The Cyprus Institute of Neurology and Genetics and Cyprus School of Molecular Medicine
X‐linked Charcot‐Marie‐Tooth disease (CMT1X) is a common form of inherited demyelinating peripheral neuropathy resulting from mutations affecting the gap junction protein connexin 32 (Cx32). Patients with CMT1X express mutant forms of Cx32 in Schwann cells, that could potentially interact with virally delivered wild type (WT) Cx32 through dominant‐negative effects. Previous lentiviral delivery of the human Cx32 gene rescued the demyelinating neuropathy in ER but not in Golgi retained mutants. In order to study the efficacy of AAV9 to rescue the demyelinating neuropathy in CMT1X models with Golgi‐retained Cx32 mutants, we delivered by intrathecal injection an AAV9 carrying the GJB1 gene under the myelin protein zero promoter (Mpz) in 2‐month‐old mutant mice expressing the R75W or N175D mutations on a Gjb1‐null background. Expression analysis of Cx32 was performed using immunofluorescence staining. Assessment of therapeutic effect was performed 6 months after treatment by behavioral, electrophysiological and morphological studies. Virally delivered WT Cx32 correctly localized in the non‐compact myelin areas while mutants were localized in the perinuclear compartment of myelinating Schwann cells. AAV9‐Mpz.GJB1 treated R75W/Gjb1‐null and N175D/Gjb1‐null mice showed improved motor performance, along with lower ratios of abnormally myelinated fibers and reduced numbers of inflammatory cells in all tissues examined compared to mock‐treated animals. Motor nerve conduction velocities were also improved in both lines following intrathecal delivery of the viral vector. This study provides additional proof of principle for a clinically translatable gene therapy to treat CMT1X even in the presence of endogenously expressed Golgi‐retained Cx32 mutants, using a clinically translatable vector, AAV9.
CRISPR/Cas9‐mediated glycogen synthase silencing as effective substrate reduction therapy in glycogen storage diseases mouse models
1: Genethon, UMR_S951, Inserm, Univ Evry, Université Paris Saclay, EPHE 2: UPMC ‐ Paris 6 3: Universidad de Navarra
Glycogen storage diseases (GSDs) are a group of rare inborn metabolic disorders, characterized by mutations in enzymes having a role in glycogen metabolism. Among them, Pompe (GSD type II) and Cori (GSD type III) are diseases with a prominent neuromuscular phenotype due to abnormal glycogen accumulation in cardiac and skeletal muscles, muscle weakness, cardiomyopathy and respiratory failure. Enzyme replacement therapy, the standard of care for Pompe disease has a limited efficacy due to immunogenicity and poor biodistribution of the enzyme to skeletal muscle. No treatment is available for Cori disease. Several therapeutic approaches are being proposed for GSDs. Among them, substrate reduction therapy (SRT) represents a promising modality. To this aim, we developed a genome editing strategy to downregulate the expression of muscle‐specific glycogen synthase (Gys1), a key enzyme for glycogen synthesis in these tissues. Staphylococcus aureus Cas9 and a guide RNA targeting Gys1, were packaged in an single AAV vector, and delivered by intravenous injection to adult and newborn Pompe and Cori mice. This resulted in specific editing of Gys1 alleles, leading to a significant reduction of glycogen synthase expression in cardiac and skeletal muscle tissues. Interestingly, efficient Gys1 genome editing in mice treated as neonates, resulted in significant reduction of glycogen accumulation in the heart of both Pompe and Cori mice. This work provides proof‐of‐concept of AAV‐mediated CRISPR/Cas9 therapy in neuromuscular GSDs and further support the substrate reduction therapy potential in this family of diseases.
A Novel AAV8‐Based Gene Therapy for Duchenne Muscular Dystrophy: Preclinical Studies in the Mdx Mouse
S Kim1 N Buss1 H Patel1 L Yang1 K Kim1 G Chan1 C Qiao1 Y Liu1 M Fiscella1
1: REGENXBIO Inc
Duchenne muscular dystrophy (DMD) is an X‐linked developmental disorder caused by mutations in the dystrophin gene leading to progressive muscular weakness and premature death due to respiratory and/or cardiac failure. RGX‐202 is a recombinant adeno‐associated virus of serotype 8 (AAV8) with an optimized human microdystrophin transgene with a promoter designed to increase expression in muscle (Spc5‐12). The RGX‐202 microdystrophin transgene is designed to encode key elements of full‐length dystrophin including extended coding region of the c‐terminal (CT) domain. The CT domain is known to recruit α‐dystrobrevin and α‐syntrophin to the dystrophin‐associated protein complex (DAPC) and contributes to maintaining membrane integrity and cellular signaling during muscle contraction and relaxation. RGX‐202 was administered intravenously to young male mdx mice and evaluated over 26 weeks. RGX‐202‐administered mdx mice showed a dose‐proportional increase of RGX‐202 vector DNA and microdystrophin protein in the skeletal and cardiac muscles. RGX‐202‐administered mdx mice exhibited significant improvements in muscle strength and gait, respectively, measured by in vitro force and fine motor kinematic analysis. Additionally, a dose‐dependent reduction of hyperintense lesions associated with muscle edema was observed using T2 magnetic resonance imaging. Consistent with these results, the dystrophic pathology (i.e., inflammation, degeneration, regeneration, fibrosis) was improved in the RGX‐202‐administered mdx mice. Immunostaining also showed restoration of the DAPC. Our data demonstrates that treatment with RGX‐202 resulted in the expression of an active microdystrophin, which improves muscle function and reduces dystrophic pathology in mdx mice. We are currently developing RGX‐202 as a candidate for microdystrophin gene therapy for DMD.
Large animal safety studies demonstrate no drug‐related toxicity following high doses of AT132
1: Astellas Gene Therapies 2: Tox and Text Solutions, LLC 3: PCMeunier Consulting 4: GENETHON 5: INTEGRARE, Genethon, Inserm, Univ Evry, Universite Paris‐Saclay 6: Department of Pathology and Laboratory Medicine and Neuroscience Research Center, Medical College of Wisconsin 7: Department of Rehabilitation Medicine, University of Washington
X‐linked myotubular myopathy (XLMTM) is a congenital myopathy caused by null‐mutations in the MTM1 gene that presents with muscle weakness, hypotonia, feeding and breathing difficulties, and in some instances, death. AT132 is an investigational AAV8‐based gene therapy that contains the coding sequence of the human MTM1 gene under the control of a desmin promoter (rAAV8‐Des‐hMTM1), intended for the treatment of XLMTM. Three of seventeen XLMTM patients dosed with 3e14 vg/kg AT132 died after developing severe hyperbilirubinemia and decompensated liver disease. Herein, we present the large animal safety data that supported clinical testing of AT132. Juvenile dogs with an MTM1 missense mutation received a single intravenous injection of ≤5e14 vg/kg of a canine version of AT132 (rAAV8‐Des‐cMTM1). In addition, infant cynomolgus monkeys received a single intravenous injection of 8e14 vg/kg rAAV8‐Des‐hMTM1. In both studies, rAAV8‐Des‐MTM1 was well tolerated with no drug‐related safety findings as assessed by clinical observations, clinical pathology, histopathology, and immunogenicity endpoints. In dogs, rAAV8‐Des‐cMTM1 vector genomes were highest in liver (ranging 3‐7 vg/dg) followed by cardiac muscle (1‐5 vg/dg), while corresponding myotubularin protein expressed highest in cardiac muscle (1‐20‐fold compared to WT), followed by skeletal muscle (1‐2‐fold). In monkeys, vector genome concentrations were highest in liver (214‐368 vg/dg) followed by cardiac muscle (13‐23 vg/dg), while myotubularin protein was highest in skeletal muscle (9‐51‐fold compared to WT) followed by cardiac muscle (6‐24‐fold). Importantly, there was no evidence of hyperbilirubinemia or other liver‐associated toxicities following administration of relatively high doses of rAAV8‐Des‐MTM1 to XLMTM dogs or cynomolgus monkeys.
Improvement of skeletal muscle function in the Dba2.mdx mouse following administration of AAV‐microdystrophin
1: Royal Holloway, University of London 2: UCB Celltech
Duchenne muscular dystrophy (DMD) is a severe, progressive muscle degenerative disease caused by mutations in the DMD gene, leading to the loss of its protein product, dystrophin, in skeletal and cardiac muscle. Recently, the new mdx/DBA/2J mouse has been characterised and described in terms of the disease progression and fibrosis. It appears to be a more severe and therefore a more reflective model of DMD than the more widely used mdx/C57BL/10 mouse. To further validate the model, we have conducted here a short‐term in vivo study in the Dba2.mdx mouse using a single intravenous administration of an AAV‐microdystrophin. We show a significant improvement in muscle function with the AAV‐microdystrophin treatment through treadmill exhaustion tests (time to fatigue is ∼50% higher than that seen in the non‐treated group) and in situ muscle force measurement of tibialis anterior muscles (significantly higher specific force seen at 80, 100, 120 (each p = <0.05),150 and 180 Hz (both p = <0.005) in treated vs non‐treated mice). Furthermore, we show a normalisation of muscle mass back to wild type levels, most importantly in that of the diaphragm, the most severely affected muscle in this model. Studies are continuing to assess the changes in the cellular and molecular phenotype of muscles with treatment, particularly in relation to fibrosis. Our findings further confirm the reliability of this mouse as a disease model and provide additional evidence of the efficacy of AAV‐microdystrophin gene addition for DMD.
Characterization of a new Duchenne Muscular Dystrophy rat model with an out‐of‐frame deletion of exon 45
1: Université Paris‐Saclay, Univ Evry, Inserm, Généthon, Integrare research unit UMR_S951, 91000, Evry‐Courcouronnes, France
Duchenne Muscular Dystrophy (DMD) is a recessive X‐linked genetic neuromuscular disorder characterized by progressive muscle degeneration leading to ambulatory, respiratory and cardiac functions impairment. It can also be associated with cognitive disabilities. DMD is caused by mutations in the gene coding for the dystrophin protein. In over 95% of cases, when the mutation causes a shift in the gene's reading frame, dystrophin is not expressed, leading to a severe DMD phenotype. In some cases, a background level of alternative splicing can restore the frame, leading to a variable but low dystrophin expression.
Here we present a DMD rat model generated by CRISPR/Cas9 excision of Dmd exon 45, the most common single exon deletion in DMD patients. A characterization of this model demonstrated the presence of muscle wasting and inflammation, respiratory deficiency, and cardiac damage. Additionally, our model shows signs of cognitive impairment and high levels of anxiety when compared to its WT peers.
While the model presents an out‐of‐frame mutation, a significant age‐dependent increase of dystrophin expression is observed within its muscles. We determined that the unexpected level of dystrophin expression is due to an inner exon‐skipping mechanism that restores the gene's reading frame.
This rat model could offer new insights into the exon splicing machinery of the DMD gene and new possible therapeutic targets. In addition to being a muscular dystrophy model, our DMD model can be useful for a better understanding of the involvement of dystrophin in brain function.
Effects of Smad7 gene transfer in a DMD mouse model
1: Université Paris‐Saclay, Univ Evry, Inserm, Généthon, Integrare research unit UMR_S951, 91000, Evry‐Courcouronnes, France
While the majority of therapeutic approaches for Duchenne muscular dystrophy (DMD) are only targeting the genetic defect, combining such an approach to the targeting of the secondary pathological cascade could improve the efficiency of this therapy. The TGFβ/Smad2/3/4 pathway plays a central role in the DMD pathological cascade, particularly in the generation and maintenance of fibrosis following the binding of TGFβ1, and in regulation of muscle mass following the binding of myostatin. Therefore, by negatively regulating the TGFβ/Smad2/3/4 pathway, we could combine the decrease of fibrosis and the increase of muscle mass and strength. In this study, we assessed the potential benefits of this kind of approach, combined or not with an AAV9 vector coding for microdystrophin, using an AAV9 vector coding for Smad7, a negative regulator of the TGFβ/Smad2/3/4 pathway, in the DBA‐mdx mouse model.
We first evaluated the effects of Smad7 gene transfer on fibrosis following intramuscular injection of the vector. Histological and molecular analyses did not show any improvement of fibrosis following treatment. However, overexpression of Smad7 led to hypertrophy of injected muscles as demonstrated by the increase of fibre diameter. We then assessed Smad7 AAV transfer following systemic injection to investigate its effects at the functional level. Results showed an enhanced integration and expression of the microdystrophin transgene when co‐injected with Smad7. However, no amelioration of the functional parameters, i.e. global strength, endurance and activity, were observed following Smad7 transfer, alone or combined with microdystrophin. We conclude that this approach is not of interest for DMD.
Dose escalation preclinical study with FKRP gene therapy and benefits on dysregulated pathways
1: Genethon, UMR_S951, Inserm, Univ Evry, Université Paris Saclay, EPHE
The Fukutin‐related protein (FKRP) is a ribitol 5’phosphatase of the Golgi apparatus, which contributes to the α‐dystroglycan (αDG) glycosylation. This latter protein is a hyper‐glycosylated membrane component participating to the linkage between extracellular matrix and the cytoskeleton. In striated muscle, this linkage is crucial for stability of the fibers and protection against the mechanical stress induced by muscle contraction. Mutations in the FKRP gene lead to αDG hypoglycosylation and disruption of these functions. Depending on the nature of the mutation, FKRP deficiency results in a whole range of pathologies such as Limb Girdle Muscular Dystrophy R9 (LGMDR9), Congenital muscular dystrophy type 1C, Muscle‐Eye‐Brain disease and Walker Warburg Syndrome. To date, there is no cure for these diseases except palliative care. We previously described the in vivo potential of rAAV mediated FKRP gene therapy in LGMDR9 mouse model. To gain insight and knowledge about the potential of the FKRP gene therapy developped, we performed a dose escalation preclinical study comparing wild‐type, FKRP deficient mice and rAAV‐FKRP treated animals. Using RNA‐seq technologies in combination with bioinformatic analyses, we first identified dysregulated pathways in FKRP deficient mice model. We next studied benefits and rescue on these modified pathways in FKRP gene therapy treated mice. Modifications of the inflammatory, metabolic and mitochondrial pathways were noted. This study may contribute to identify key pathways and regulators and demonstrate benefits of the FKRP‐based gene therapy.
Generation of Pompe disease cellular models to analyze gene therapy aproaches using optimized GAA
1: GENyO‐ Centro de Genomica e Investigacion Oncologica: Pfizer / Universidad de Granada / Junta de Andalucia 2: Department of Medicinal and Organic Chemistry, Faculty of Pharmacy, University of Granada
Pompe disease is a rare disease caused by mutations in the gene coding for the acid alpha‐glucosidase enzyme (GAA) that in the most severe cases, cause premature death in early infancy. Gene therapy (GT) can theoretically cure Pompe patients by introducing the correct version of the GAA gene in affected tissues. Most GT strategies also rely on the cross‐correction of unmodified cells through the capture of GAA secreted by corrected cells. In this work, we have designed three different chimeric GAA proteins (named 2G, IFG and IFLG) with the aim to increase their expression, secretion and uptake. To test the improvements of the optimized GAAs, we generated several GAA‐KO murine muscle cell lines mimicking the infantile form of Pompe disease and also used different myeloid and muscle cell lines from mouse and human origin. As expected, all GAA chimeras showed improved behaviour, although the IFG chimera containing the leader peptide of the IFNβ1 gene presented the best characteristics in terms of expression and secretion in most cells lines. The uptake of IFG proteins by GAA‐KO muscle cells was also improved compared with the other GAA chimeras, pointing to this protein as the best option for gene therapy approaches for Pompe disease. Interestingly, our results also showed that murine cells are not always an appropriate model to determine improvement in secretion since the analysis using myeloid human cells indicated the best chimera was the 2G, which incorporate the leader peptide and the first 88 aminoacids from the IGF2 protein.
Development of a utrophin modulation CRISPR‐Cas9 strategy for Duchenne Muscular Dystrophy
1: Genethon, UMR_S951, Inserm, Univ Evry, Université Paris Saclay, EPHE 2: Department of Cell and Developmental Biology and Centre for Stem Cells and Regenerative Medicine, University College London, London, UK
Duchenne muscular dystrophy (DMD) is a lethal X‐linked neuromuscular disorder caused by mutations in the dystrophin gene. In the absence of the dystrophin protein, the link between the cytoskeleton and extracellular matrix is impaired resulting in loss of muscle strength, flexibility and stability. DMD patients are restricted to wheelchair by the age of 12 years and usually succumb to cardiac or respiratory complications in their late twenties. Despite exhaustive clinical management, there is currently no effective treatment for DMD although various promising approaches are progressing through human clinical trials. A promising therapeutic approach, applicable to all DMD patients irrespective to their genetic defect, consists in upregulating utrophin, a structural and functional paralogue of dystrophin. In the present study, we developed a CRISPR/Cas9 approach to transcriptionally and/or post‐transcriptionally upregulate the expression of the endogenous full‐length utrophin gene. Using a reporter system, we studied and compared deletions and point mutations to modulate utrophin expression at the DNA level. We next screened guides RNA targeting several repressor domains on utrophin gene in immortalized DMD myoblasts and myotubes. Our data demonstrate that CRISPR‐Cas9 system can modulate the endogenous full‐length utrophin expression up to 7‐fold in these cells. Based on molecular, cellular and functional analyses, 3D dimensional studies using artificial skeletal muscle tissue derived from DMD patient cells and treatment in mdx animals will allow us to assess the in vivo therapeutic potential of this approach. This study presents an innovative CRISPR strategy for all DMD patients irrespective of their dystrophin mutation.
Pol II‐driven Dicer‐independent shRNAs provide efficacy and improved safety for RNAi‐based gene therapy
1: Aarhus University, Department of Biomedicine 2: University of Southern Denmark, Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences 3: Aarhus University Hospital, Department of Ophthalmology
RNAi‐based gene therapy has the potential to regulate a selected target gene potently and specifically. However, concerns regarding the therapeutic use of RNAi include the risk of off‐target effects, disruption of the endogenous miRNA processing, or eliciting an immune response by the expression of exogenous small RNAs. We have designed Pol II‐driven pri‐miRNA embedded Dicer‐independent shRNAs (miR‐agshRNAs) which are processed by Ago2 into a single functional strand and importantly, appear to offer advantages over canonical short hairpin RNAs (shRNAs) in terms of specificity and safety.
As our target we have selected vascular endothelial growth factor (VEGF), the expression of which is pathologically elevated in exudative age‐related macular degeneration. With the Pol II‐driven miR‐agshRNAs we have achieved on‐target knockdown efficacies of ∼90 % while we observe no passenger strand off‐target effect, which is often observed from canonical shRNAs. We have also confirmed that the knockdown efficacy of the miR‐agshRNAs is not reduced in the absence of Dicer. Furthermore, we have discovered a notable trend indicating that the agshRNAs cause less disruption of the processing of endogenous miRNAs compared to corresponding canonical shRNAs. Altogether, our data indicate that the miR‐agshRNAs provide a superior safety profile. Lastly, we have shown that the miR‐agshRNAs can be delivered using a lentiviral vector system, and that we can achieve tissue specific expression of the miR‐agshRNAs in the retinal pigment epithelium cells in vivo, after subretinal injections in mice.
These combined features make the Pol II‐driven miR‐agshRNAs a valuable tool for gene therapy purposes.
AAV‐Sponge‐mediated modulation of miR‐181a/b as a gene‐independent therapeutic approach for inherited retinal diseases (IRDs)
1: Telethon Institute of Genetics and Medicine (TIGEM) 2: Medical Genetics, Department of Precision Medicine, University of Campania "L. Vanvitelli", Naples, Italy. 3: Medical Genetics, Department of Translational Medical Science, University of Naples "Federico II", Naples, Italy. 4: Institute for Genetic and Biomedical Research (IRGB), National Research Council (CNR), 20090, Milan, Italy
IRDs are a significant cause of visual impairment in the Western population and are mainly characterized by progressive photoreceptor (PRs) cell death. Their broad genetic heterogeneity constitutes an important limitation to the development of gene‐specific therapies. Therefore, the implementation of gene‐independent strategies targeting common dysregulated pathways, which can be applied to a significant fraction of IRD patients, is highly desirable. Since mitochondrial dysfunction seems to be an early and common pathogenic event in IRDs, therapies targeting basic mitochondrial functions hold great promise. We previously discovered that miR‐181a/b downregulation enhances mitochondrial turnover in the retina and protects from cell death in models of a mitochondrial disease affecting the inner retina, the Leber's hereditary optic neuropathy (LHON). Here, we demonstrate that miR‐181a/b modulation exerts a protective effect also in IRDs independently of the underlying genetic cause. We found that miR‐181a/b downregulation in the transgenic RHO‐P347S, a model for an autosomal dominant form of IRD, results in improvement of visual function, preserves PRs from death, ameliorates PR outer segment structures and PR marker expression, and rescues mitochondrial fragmentation, mitochondrial mass, and morphology. The therapeutic potential of the subretinal delivery of an adeno‐associated viral vector (AAV) carrying a miR‐181a/b inhibitor sequence (AAV2/8‐GFP‐Sponge‐miR‐181a/b) was validated in RHO‐P347S and in other IRD animal models, supporting the gene‐independent protection exerted by miR‐181a/b inhibition on PR degeneration. Overall, our data highlight the potential use of AAV2/8‐GFP‐Sponge‐miR‐181a/b vectors as innovative therapeutic tools for these conditions, both in slowing down disease progression and in supporting gene‐specific therapeutic procedures.
Trapping miR‐223 leads to overexpression of NLRP3 in colorectal cancer
1: Kazan Federal University 2: Bursa Uludag University 3: University of Nevada
NOD‐like receptor pyrin domain‐3 (NLRP3) inflammasome is a multiprotein complex that can trigger inflammation through IL‐1β secretion. Studies demonstrated that low expression of NLRP3 is a better predictor of colorectal cancer (CC) worse outcome. In contrast, high expression of this inflammasome could indicate resistance to fluoropyrimidine‐based chemotherapy. Therefore, NLRP3 could be a potential therapeutic target for the treatment of CC. MicroRNA (miRNA) post‐transcriptionally regulates protein expression. Among these miRNAs, miR‐223 was shown to bind the UTR sites of NLRP3. However, the effect of miR‐223 on NLRP3 expression in CC remains largely unknown. In this study, the regulatory effect of miR‐223 on NLRP3 expression in CC was investigated. A CC cell line, HCT‐116 cells was transfected with AB.pCCL.sin.cPPT.U6.miR‐223‐Decoy.hPGK.GFP.WPRE to inhibit miR‐223. To activate NLRP3, miR‐223‐Decoy expressing and non‐transfected HCT‐116 cells were treated with 1 μg/mL of LPS for 3 hours as an initial activator of NLRP3 and 5mM ATP for 25 min as the second stimulus. Inflammasome activation was assessed by analyzing NLPR3 protein expression and IL‐1β secretion using western blot and ELISA. Statistical analyses were done using one‐way ANOVA and Tukey's analyses in SPSS 20 software. Our results demonstrated that NLRP3 expression and IL‐1β secretion were significantly higher in miR‐223‐Decoy expressed HCT‐116 cells than non‐transfected HCT‐116 cells (p < 0.05). Our findings suggest that inhibiting miR‐223 results in the loss of control over the NLRP3 translation and enhances secretion of pro‐inflammatory cytokine IL‐1β. These findings provide a piece of evidence of miR‐223 direct involvement in the regulation of NLRP3 expression in CC.
AAV‐mediated inhibition of miR‐181a/b as gene‐independent therapeutic tool for mitochondrial diseases
1: TIGEM 2: University of Campania L. Vanvitelli 3: University of Naples Federico II 4: CNR / IRGB
Mitochondrial Diseases (MDs) are a group of inherited disorders caused by defective oxidative phosphorylation, resulting in a wide range of clinical phenotypes. They show extreme genetic heterogeneity that makes the development of efficient treatments particularly difficult. Indeed, despite progress made lately, no effective therapies are still available. Gene/mutation‐independent approaches acting downstream of the genetic defect are thus highly desirable.
microRNAs (miRNAs) represent a promising therapeutic tool due to their capability to simultaneously regulate different pathways. We recently demonstrated that the inactivation of miR‐181a and miR‐181b (miR‐181a/b) exerts a protective action on different MDs models associated with neuronal degeneration through the enhancement of mitochondrial turnover. Therefore, we decided to test if miR‐181a/b downregulation may be exploited as a therapeutic strategy in MDs. To achieve long‐term loss‐of‐function of miR‐181a/b in vivo, we generated an Adeno Associated Viral (AAV) vector expressing a miRNA sponge able to specifically bind miR‐181a/b and inhibit their activity. We are currently studying the effect of miR‐181a/b sponge in the Ndufs4 KO mouse, which is a widely used murine model of both Leber Hereditary Optic Neuropathy (LHON) and Leigh syndrome, two of the most frequent MDs affecting the eye and the brain, respectively. To achieve an efficient transduction of the CNS we used the AAV.PHP.B serotype that successfully overcome the blood‐brain barrier. Interestingly our data show a significant amelioration of the visual function and an improvement of the locomotor activity in mice treated with the miR‐181a/b sponge. These findings highlight the therapeutic potential of the sponge therapy for MDs.
Characterization of a novel OX40 ligand and CD40 ligand‐expressing oncolytic adenovirus used in the PeptiCRAd cancer vaccine platform
E Ylosmaki1 2 3 7 L Ylosmaki4
1: University of Helsinki 2: Laboratory of Immunovirotherapy, Drug Research Program, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland. 3: TRIMM, Translational Immunology Research Program, University of Helsinki, Finland 4: Valo Therapeutics Oy, Helsinki, Finland. 5: Department of Oncology and Hematology, Krankenhaus Nordwest, Frankfurt am Main, Germany 6: Pharmaceutical Biophysics Research Group, Drug Research Program, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland 7: iCAN Digital Precision Cancer Medicine Flagship, University of Helsinki, Helsinki, Finland 8: Department of Molecular Medicine and Medical Biotechnology and CEINGE, Naples University, 24 Federico II, 80131 Naples, Italy.
Oncolytic viruses (OVs) have been shown to induce anti‐cancer immunity and enhance cancer immunotherapies, such as immune checkpoint inhibitor therapies. OV therapies can be further improved by arming OVs with immunostimulatory molecules, including various cytokines or chemokines. Here, we have developed a novel adenovirus encoding two immunostimulatory molecules: cluster of differentiation 40 ligand (CD40L) and tumor necrosis factor receptor superfamily member 4 ligand (OX40L). This novel virus, designated VALO‐D102, is designed to activate both innate and adaptive immune responses against tumors. CD40L affects the innate side by licensing antigen‐presenting cells to drive CD8+ T cell responses, and OX40L increases clonal expansion and survival of CD8+ T cells and formation of a larger pool of memory T cells. VALO‐D102 and its murine surrogate VALO‐mD901, expressing murine OX40L and CD40L, were used in our previously developed PeptiCRAd cancer vaccine platform. Intratumoral administration of PeptiCRAd significantly increased tumor‐specific T cell responses, reduced tumor growth, and induced systemic anti‐cancer immunity in two mouse models of melanoma. In addition, PeptiCRAd therapy, in combination with anti‐PD‐1 immune checkpoint inhibitor therapy, significantly improved tumor growth control as compared to either monotherapy alone.
Expression of galectin‐3 inhibitors from a self‐replicating RNA vector can inhibit the growth of pediatric osteosarcoma
1: CIMA Universidad de Navarra 2: Clinica Universidad de Navarra 3: Health Research Institute of Navarra (IDISNA)
Osteosarcoma is an aggressive bone tumor, primarily arising in the pediatric age, characterized by its highly immunosuppressive environment. Inhibition of galectin‐3 (Gal3), a protein involved in immunosuppression, adhesion of tumor cells and metastases, has been shown to reduce tumor progression in different tumor models, including osteosarcoma. On the other hand, virotherapy based on recombinant Semliki Forest Virus (SFV), a self‐replicating RNA virus, has shown therapeutic effect in orthotopic osteosarcoma mouse models. We hypothesized that using this type of vector to express inhibitors of Gal3 might have a potent antitumor effect against osteosarcoma. For that purpose, we generated SFV vectors expressing truncated forms of Gal3, including its carboxy‐terminal domain (SFV‐Gal3‐C) and its amino‐terminal domain alone (SFV‐Gal3‐N) or fused to the Gal3 inhibitor peptide C12 (SFV‐Gal3‐N‐C12). An additional construct expressed the C12 peptide (SFV‐C12). Orthotopic osteosarcoma tumors, induced by intratibial injection of K7M2 murine cells, were treated with SFV vectors expressing Gal3 inhibitors or luciferase or with PBS (control). Treatment with SFV‐Gal3‐N‐C12 showed the highest therapeutic efficacy, reducing tumor growth significantly, leading to 47% of complete regressions, and decreasing spontaneous lung metastasis. Furthermore, pro‐inflammatory macrophages were significantly reduced in tumors of treated mice. Nevertheless, no protection against tumor rechallenge was observed in cured mice, indicating the lack of a memory antitumor immune response. These data suggest treatment that with SFV‐Gal3‐N‐C12 might benefit from combination with other immunostimulatory approaches. In summary, our data underscore the potential therapeutic value of SFV‐Gal3‐N‐C12 vector as treatment for patients affected by osteosarcoma.
Hexon modification of HAdV‐5 vectors enables efficient transduction of human multipotent mesenchymal stromal cells
1: Ulm University
Viral vectors based on human adenovirus type 5 (HAdV‐5) show promise for oncolytic tumour therapy. However, clinical success so far has been limited. One likely reason is the inefficient vector delivery to tumor tissue upon systemic administration, resulting from several cellular and non‐cellular vector sequestration mechanisms. The use of human multipotent mesenchymal stromal cells (hMSCs) as carrier cells is being developed as strategy to improve tumor‐directed delivery of oncolytic adenoviruses. It has been shown that MSCs naturally have the ability to migrate to tumour tissue ‐ the primary prerequisite to function as a carrier cell for oncolytic viruses. However, as hMSCs lack Coxsackie and Adenovirus Receptor (CAR) expression, they are hardly transduced with non‐modified HAdV‐5 vectors. We recently showed that transduction enhancers with positive charge can be used to enhance HAdV‐5 mediated transduction of hMSCs. Based on this observation, we genetically introduced amino acids with positive charge into hypervariable and solvent‐exposed regions of HAdV‐5. One of these vectors, referred to as HAdV‐5‐HexPos3, enabled strongly improved transduction of hMSCs. Additionally, we found that HAdV‐5‐HexPos3 enables CAR‐independent transduction of various other cell types (including tumour cell lines), highlighting the potential of this vector for clinical applications. An in‐depth analysis of the transduction mechanism revealed an involvement of heparan sulfate proteoglycans (HSPGs) in the vector uptake. Finally, we investigated the migration ability of efficiently transduced hMSCs towards tumour cells in vitro and in vivo.
Development of oncolytic and gene therapy vectors based on Adenovirus serotype 4 as an alternative to Adenovirus serotype 5
1: Witten/Herdecke University
Adenoviral vectors are among the most frequently used vectors for gene therapy and cancer treatment. Most adenoviral vectors are derived from human Adenovirus serotype 5 (AdV5) despite limited applicability caused by preexisting immunity and unfavorable liver tropism, while the other 103 known serotypes remain largely unused.
Screening a library of reporter‐gene‐expressing Adenovirus representative of the natural Adenovirus diversity, 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.
Therefore, we aim to assess the potential of AdV4 for improved muscle transduction or oncolysis of HPV‐induced tumors. To produce first generation AdV4 gene therapy vectors, we deleted its E1 and E3 genes. The E1‐E3‐deleted vector was replication‐competent in HEK293 cells, whereas other cancer cell lines including Hela and Siha were not permissive. Since only HEK293 cells stably express AdV5‐E1, this suggests that AdV5‐E1 can complement for AdV4‐E1, enabling the rescue of E1‐deleted AdV4. Transient AdV5‐E1 expression in non‐permissive cells enabled E1‐deleted AdV4 replication, further supporting our findings of trans‐species E1 complementation in Adenoviruses.
In order to increase AdV4 uptake into Papillomavirus induced cancer cells we replaced the AdV4 fiber knob with the AdV5 fiber knob. However, pseudotyping did not alter vector infectivity nor replication.
We hope that our work will lead the way for the development of improved AdV vectors based on non‐canonical serotypes for a broad range of clinical applications.
Directed evolution generates novel oncolytic H‐1 parvoviruses with improved therapeutic efficacy in virus‐resistant pancreatic cancer cells
P Garcin1 2 M Kazamimanesh1 2 H Lulka1 2 N Dusetti1 3 G Labrousse1 2 E Benuzzi1 2 L Buscail2 4
1: Inserm 2: Cancer research center of Toulouse 3: Cancer research center of Marseilles 4: CHU de Toulouse
Despite considerable promise and emerging clinical success, several challenges impede the broader implementation of oncolytic virus(OV)‐based gene therapy, including for patients with pancreatic cancer (PDAC). One of such challenge is inter‐patient variability that may impact on OV selectivity and killing efficacy for tumor cells. For this study, we selected the rat parvovirus H‐1 (H‐1PV) that is nonpathogenic in humans and has a natural oncolytic activity in several cancer models. The safety and tolerability of H‐1PV was recently demonstrated in early clinical trials for glioma and PDAC. However, we report here that H‐1PV infection, oncolytic and pro‐apoptotic activity are limited in PDAC cells, including patient‐derived primary cells. To address this concern, we applied a directed evolution strategy to generate H‐1PV variants with specific activity towards PDAC cells. Following selection using patient primary cells, we managed to isolate clonal, PDAC‐adapted H‐1PVs that induce PDAC cells lysis as compared to parental H‐1PV while infection of normal pancreatic cells remained negligeable. Genome sequencing of the tumor adapted virus reveals mutations in promoting and viral capsid sequences. In vivo, the tumor‐adapted H1PV demonstrates greater anti‐tumor effect than parental H‐1PV, following intravenous administration in an experimental model of orthotopic pancreatic tumors engrafted in immunodeficient mice. To our knowledge, we report here for the first time the production of highly selective and potent OV using directed evolution to override PDAC resistance to virotherapy. While the molecular mechanisms involved are still under investigation, this project is a first step towards precision medicine strategies based on OV
The combinatory therapy of oncolytic adenovirus armed with co‐stimulatory molecules and anti‐PD1 as a promising malignant mesothelioma treatment strategy
M Garofalo3 M Staniszewska2 M Wieczorek1 K W Pancer1 M Prygiel1 A Zasada1 S Salmaso3 V Cerullo5 B Rinner4
1: National Institute of Public Health NIH – National Research Institute 2: Warsaw University of Technology 3: University of Padova 4: Medical University of Graz 5: University of Helsinki
Malignant mesothelioma is a treatment‐refractory malignancy to current therapies with no efficacious curative options for advanced cancer patients. 2‐year overall survival rates is 41% in the nivolumab plus ipilimumab group and 27% in the chemotherapy group. The low incidence of mesothelioma has for a long time limited the discovery of new agents. Therefore, new treatment modalities are in high need. Single therapy is rarely successful in treating cancer, especially in metastatic or advanced cancer, and survival rates are generally poor. The combination of multiple therapies to treat cancer has already shown profound results. This strategy utilizes the combination of both conventional and novel therapies. Oncolytic viruses exhibit unique anti‐cancer mechanism compared to conventional therapies, allowing the possibility for additive or synergistic anti‐cancer effect. In this project it has been hypothesized that by combining novel oncolytic adenovirus expressing CD40L and ICOS ligands with check point inhibitors we could improve anti‐cancer efficacy through synergistic anti‐cancer effect. Therefore, the virus may efficiently prime anti‐tumour immunity through its enhanced immunogenicity combined with oncolysis. Blockade of PD‐1 with anti‐PD‐1 antibody releases the brakes of anti‐tumour immunity and enhances the oncolysis primed responses against tumour antigens. We demonstrated that the combinatory therapy of novel oncolytic adenovirus with anti PD‐1 can induce immunogenic cell death of mesothelioma cell lines, infiltration of various immune cell subsets resulting in improved anti‐tumor activity in both humanized and immunodeficient H226 malignant pleural mesothelioma xenografts models. Therefore, if successful, will pave the way towards further clinical application.
A novel oncolytic virus (ISC301) effectively inhibits tumor growth based on the intratumoral activation of the immune system
1: Cellular Biotechnology Unit, Instituto de Salud Carlos III
Oncolytic virotherapy uses viruses designed to selectively replicate and kill cancer cells. Besides the specific oncolytic action, the effect of the therapy in patients seems to be mediated by the activation of the immune response, and therefore the capacity of the treatment to induce high tumor infiltration. Here we have developed and validated a new human oncolytic adenovirus, named ISC301, based on the oncolytic adenovirus ICOVIR‐5. Our new virus incorporates different modifications in the RGD motifs that increase its antitumor efficacy due to a higher intratumoral activation of the immune response.
Only two days after systemic administration in an immunocompetent mouse model of lung adenocarcinoma, the antitumor efficacy of ISC301 was significantly increased compared to ICOVIR‐5 and control group. We then studied the activation of the NF‐κB pathway in the tumor microenvironment using a luciferase reporter system. Interestingly, in vitro and in vivo results showed an increased early activation of the pro‐inflammatory pathways in tumors treated with ISC301 compared to those treated with ICOVIR‐5 or PBS. As a result, tumors treated with ISC301 also presented an increased number of tumor‐infiltrating lymphocytes (TIL) and higher percentages of CD8+ T cells at end point than those treated with PBS.
In conclusion, the new oncolytic adenovirus ISC301 presents significant antitumor efficacy, which seems to be based on a high intratumoral activation of the immune system. These results highlight the importance of the immune response in oncolytic virotherapies, which represents an area that has been poorly studied to date.
Turning melanoma cold tumors into hot by enhancing T‐cell infiltration
L Kuryk1 M Staniszewska2 M Wieczorek1 K W Pancer1 S Salmaso3
1: National Institute of Public Health NIH – National Research Institute 2: Warsaw University of Technology 3: University of Padova
Oncolytic virotherapy is a promising anticancer strategy able to induce anti‐tumor immunity. Priming with oncolytic vectors can transform a “cold” tumor microenvironment into a “hot” one by enhancing infiltration of various innate and adaptive immune cells into a tumor lesion. Therefore, the combinatory therapy with checkpoint inhibitors is most effective in a tumor microenvironment with a presence of lymphocytic infiltrates. Such critical concepts have the potential to play a promising future role as enablers of immunotherapies involving oncolytic vectors. Previously we have shown that combination approaches exhibit enhanced anti‐cancer ability and immunogenic cell death in in vitro melanoma models. Furthermore, oncolytic vector combined with anti PD‐1 revealed a fall in tumor volume and 100% survival in in vivo syngeneic melanoma mouse model, thus suggesting improved efficacy and survival. Importantly, our results showed enhanced infiltration of various immune cells subsets in treated tumors such as cytotoxic CD8 T cells able to counterplay with immunosuppression. Therefore, the presence of tumor‐infiltrating lymphocytes in melanoma tumor microenvironment resulted in a better prognosis and durable tumor regression observed in response to the combinatory therapy. Collectively, obtained results suggest a promising future role for oncolytic vectors as immune activators able to synergize with checkpoint inhibitors resulting in improved clinical efficacy.
Chorioallantoic Membrane tumour model for evaluating oncolytic viruses
1: Ulm University
Oncolytic viruses are promising anticancer agents and a plethora of promising new virus vector variants are designed each year. All these vectors require conscientiously and encompassing preclinical investigation, though. Especially in vivo evaluation of oncolytic viruses using murine syngeneic or xenograft tumour models is laborious, cost‐intensive and time‐consuming.
To address this drawback, we evaluated the chicken chorioallantoic membrane (CAM) assay as a method for the analysis of oncolytic viruses. The immune‐compromised CAM model is characterized by easily accessible and fast growing tumours and it supports many cancer characteristics such as angiogenesis, invasion, and remodelling of the microenvironment. In addition, eggs are of low cost in acquisition and maintenance and ethical approval of experiments is not required.
Here human adenovirus type 5 was used as a showcase to analyse known interactions of viral particles with cellular and non‐cellular blood components in vivo, investigate biodistribution patterns and compare the murine and the chick model. We thoroughly describe the tumour establishment and careful characterization and optimization of the CAM model. Moreover, we show that it allows both intratumoral and intravenous virus administration and facilitates the assessment of tumour targeting efficiencies.
In summary, we consider the CAM tumour model as an applicable, inexpensive and quick high‐throughput tumour model system that enables a feasible and conclusive in vivo investigation of relevant virus‐host interactions, biodistribution patterns, and tumour‐targeting profile of modified viral vectors.
The application of rapid molecular methods to overcome challenges in biosafety testing of oncolytic virus products
1: Merck KGaA (BioReliance Ltd)
Oncolytic virotherapy is a rapidly growing modality for the treatment of a variety of cancers. However adventitious agent testing of these virus products is challenging due to their ability to replicate in both in vitro cell culture and in vivo assay systems. This can lead to false positives or invalid results in critical biosafety assays for the detection of contaminating virus and mycoplasma species. Traditionally neutralising anti‐serum has been used to overcome this issue, but this is time consuming, costly and may have variable success depending on the product virus being investigated.
Here we present rapid and sensitive molecular based approaches including Next Generation Sequencing and Real time PCR detection of specific pathogens, including Mycoplasma. These methods can be employed to minimise the requirement for neutralising anti‐serum and at the same time significantly decrease the overall duration of adventitious agent testing. Aspects including method overview, assay performance, validation, comparability data and regulatory expectations will be discussed. The need for rapid testing during development of COVID‐19 vaccines has accelerated the regulatory acceptance of NGS based approaches as a replacement for in vivo assays, which also addresses ethical concerns in biosafety testing.
In addition, we have evaluated the viral clearance potential of manufacturing processes for non‐enveloped viral vectors. Data will be shown that demonstrate steps from the purification process of many non‐enveloped viral vectors that can provide effective clearance of enveloped viruses. This is another crucial aspect which contributes to the overall risk assessment of oncolytic virotherapies.
CG0070 for the treatment of non‐muscle invasive bladder cancer (NMIBC) unresponsive to Bacillus Calmette‐Guerin (BCG)
R Li1 E Uchio2 G D Steinberg3 N Shore4 P D Anderson5 T Ben6 A Kamat7 M Keel8 V Packiam9 J McAdory8 J H Kim8 M Chisamore10 P Grandi8
1: H. Lee Moffitt Cancer Center, Tampa, FL, USA 2: UCI University, Orange CA, USA 3: NYU Langone Health, NYC, NY, USA 4: Carolina Urologic Research Center, Myrtle Beach, SC, USA 5: Royal Melbourne Hospital, Melbourne, Australia 6: Peter MacCallum Cancer Centre, Merlburne, Australia 7: University of Texas MD Anderson Cancer Center, Houston, TX, USA 8: CG Oncology, Irvine, CA, USA 9: University of Iowa Health Care , Iowa City, IA, USA 10: Merck & Co., Inc., Kenilworth, NJ, USA
CG0070, an oncolytic vaccine available as an intravesical (IVE) therapy, is a serotype 5 adenovirus engineered to express GM‐CSF and replicate in tumor cells with mutated or deficient RB. Initial response rates (RR) of approximately 45% have been observed in patients with recurrent NMIBC following BCG failure.
A single arm phase 3 global study (BOND‐3 NCT04452591) was launched to confirm the clinical activity of CG0070 monotherapy. The trial is enrolling 110 patients with BCG‐unresponsive CIS with or without concurrent Ta or T1 disease. Patients will be treated with intravesical CG0070 at a dose of 1x1012 vp.
In a second study (CORE‐1 NCT04387461), 35 patients with BCG‐unresponsive CIS with or without concurrent Ta or T1 disease will be treated with IVE CG0070 (1x1012 vp) in combination with pembrolizumab. Intravenous pembrolizumab was recently FDA approved for patients with BCG‐unresponsive CIS unable or unwilling to undergo radical cystectomy with a complete RR of 41% at 3 months and 12 months duration of response rate of 46%. This phase 2 study will assess the potential synergy between the two agents in the treatment of BCG‐unresponsive CIS containing NMIBC.
The primary endpoint for the BOND3 trial is CR at any time on study. Secondary endpoints include CR rate at 12 months. For CORE 1, the primary endpoint of the study is CR rate at 12 months. Secondary endpoints will include CR at any time.
Both trials are currently enrolling. An update on enrollment and preliminary data will be provided at the time of presentation
Anelloviruses are highly diverse, ubiquitous commensal denizens of the human virome, and show promising properties as novel gene delivery vectors
D M Nawanar1 C A Arze1 J Timpona1 N Boisvert1 M Trivedi1 F Diaz1 K Lebo1 C Prince1 C Scano1 S Springer1 G Bounoutas1 R Fusco1 G Dudas2 S Patel1 A Bhattacharyya1 H Swaminathan1 S Liou1 K Swanson1 T Ong1 A Kahvejian1 3 Y Echelard1 3 E G Weinstein1 3 R J Hajjar1 3 K G Andersen4 S Delagrave1
1: Ring Therapeutics 2: Gothenburg Biodiversity Centre 3: Flagship Pioneering 4: Scripps Research Translational Institute
The field of gene therapy is striving to develop gene delivery vectors offering new properties such as tropism to novel cell types, the ability to re‐dose, or evasion of pre‐existing immunity. Anelloviruses, a viral family comprising negative‐sense single stranded circular DNA genomes, are known to be diverse, ubiquitous, immunologically stealthy, and abundant in the viromes of healthy humans and other animal species. These properties, and the commensal nature of anelloviruses in humans, suggest their untapped potential as a gene therapy platform if anelloviruses can be vectorized. To better understand their genetic diversity, molecular virology, and properties as vectors, a multidisciplinary study addressing these questions was carried out. First, we employed in‐depth sequencing of blood transfusion donor‐recipient pairs coupled with public genomic resources for a large‐scale assembly of anellovirus genomes to characterize global and personal anellovirus diversity through time. This work illuminates parenteral transmission dynamics, reveals much greater diversity than expected, and identifies recombination as the principal mechanism for diversification. We also used recombinant methods to rescue human and non‐human anelloviruses and characterized the resulting particles biochemically and by electron microscopy. This and other work enabled the creation of anellovirus‐derived vectors, or Anellovectors (ANV), capable of transducing human and non‐human cell lines. Pre‐existing immunity in humans was low using neutralization assays against IVIG and a serum panel. In vivo, anelloviruses or Anellovectors were well tolerated, targeted multiple tissues, and elicited no neutralizing response when delivered intramuscularly. These results suggest that Anellovectors have the potential to open new avenues in gene therapy.
Effect of prophylactic corticosteroid treatment on adeno‐associated virus (AAV)‐mediated gene expression
1: BioMarin Pharmaceutical
Adeno‐associated virus (AAV)‐based gene therapy may elicit immune responses that could limit AAV‐mediated expression. Corticosteroid treatment may modulate these immune responses and improve gene therapy outcomes. In a previous murine study, prednisolone treatment initiated one week after valoctocogene roxaparvovec (AAV5‐hFVIII‐SQ) administration did not affect FVIII expression in mice. Here, we investigated whether corticosteroid treatment initiated before AAV dosing could promote transgene expression in mice.
Mice were treated daily with prednisolone (2 mg/kg) or water for 4 weeks starting 1 day or 2 hours before administration of 6x1013 vg/kg of an AAV5 vector delivering human alpha‐1 antitrypsin transgene (AAV5‐HLP‐hA1AT) as a reporter. Starting 6 weeks post‐AAV dosing, prednisolone‐treated mice showed significantly higher serum hA1AT protein levels (1.5‐2.2‐fold) compared to the non–prednisolone‐treated groups and a trend toward lower interindividual variability. These serum hA1AT protein levels significantly correlated with liver full‐length vector genome DNA (r = 0.68; P < 0.0001) and transgene RNA. In addition, the number of hepatocytes transduced with vector DNA was higher in prednisolone versus non–prednisolone‐treated groups.
Overall, corticosteroid use before AAV administration increased liver‐directed AAV5 expression in mice and may represent a potential strategy to reduce interindividual variability in AAV‐mediated gene therapy.
Antitumor effect of a self‐amplyfing RNA expressing interleukin‐12 in combination with in vivo electroporation
1: Universidad de Navarra
Alphavirus vectors based on self‐amplifying RNA (saRNA) generate high and transient levels of transgene expression and induce innate immune responses, constituting an interesting tool for antitumor therapy. These vectors are usually delivered as viral particles, but it is also possible to administer them as RNA. We have evaluated this possibility by in vivo electroporation, using Semliki Forest Virus (SFV) saRNA for local treatment of murine colorectal MC38 subcutaneous tumors. First, we optimized SFV saRNA in vivo tumor electroporation, using a vector coding for luciferase. This study showed that optimal in vivo transfection was achieved by intratumoral injection of saRNA followed by eight 0.1 ms long pulses of 1200 V/cm. Then, we evaluated the therapeutic potential of this approach using a saRNA coding for interleukin‐12 (SFV‐IL‐12), a proinflammatory cytokine that has a potent antitumor effect. The delivery of SFV‐IL‐12 saRNA by electroporation led to a decrease in tumor growth and to significant higher survival compared to the control group that received saline. In addition, this treatment promoted tumor‐specific CD8 T‐cells infiltration, as well as immunological memory, demonstrated in tumor rechallenge experiments. saRNA administration or electroporation as single therapies did not show significant antitumor effects compared to the control group. Finally, the antitumor effect of SFV‐IL‐12 RNA was increased by combination with an anti‐PD1 antibody. Our results suggest that delivery of saRNA by electroporation could represent an attractive strategy for local cancer immunotherapy. This approach could have an easy translation to the clinical practice, especially for tumors that can be accessed percutaneously.
Intravenous administration of F/HN pseudotyped lentiviral vector
1: Imperial College London
The UK Respiratory Gene Therapy Consortium developed a lentivirus pseudotyped with Sendai virus envelope proteins F and HN (rSIV.F/HN) for efficient pulmonary gene transfer. Contrary to AAV and adenoviral vectors, rSIV.F/HN generates stable gene expression after a single dose (approximately 2 years in mice) and additionally retains efficiency after repeated topical administration. Topical administration of rSIV.F/HN to the lungs leads to release of proteins into the circulation. Here, we characterised vector biodistribution after intravenous (IV) delivery and compared efficacy of topical and IV delivery for production of secreted proteins.
Vector encoding gaussia luciferase (rSIV.F/HN‐gLux, n = 6/group) was administered by IV or topical administration to mice. IV delivery of rSIV.F/HN led to persistent (12 months) transgene expression in serum, approximately one‐log higher than topical. To characterise biodistribution, vector encoding firefly luciferase (rSIV.F/HN‐Lux) was administered and bioluminescent imaging performed 28‐days post‐dosing. Significant transgene expression was observed in the spleen, lungs, liver and heart following IV (1927 ± 1585 area under curve (AUC), mean signal±SD, n = 8), in contrast to highly localised lung expression following topical administration (16725 ± 6047 AUC, n = 6). Finally, transduction efficiency was compared between mice receiving rSIV.F/HN‐Lux, and mice delivered 2X control vector followed by rSIV.F/HN‐Lux at monthly intervals (n = 6/group). Mean transgene expression was 77% lower in the repeat IV group, suggesting differential anti‐vector immunogenicity. In summary, IV delivery of rSIV.F/HN achieves persistent levels (12 months) of secreted protein, with multiple organs contributing expression. Reduced efficacy following repeated vector administration may impact IV delivery for chronic diseases but potentially is suited to those requiring single administration.
Next generation split intein‐based protein splicing to reconstitute large genes for gene therapy.
1: SpliceBio, C/Baldiri Reixach 10, Barcelona 08028, Spain. 2: Program of Gene Therapy and Regulation of Gene Expression, Foundation for Applied Medical Research, University of Navarra, Pamplona, Spain. 3: IdiSNA, Navarra Institute for Health Research, Pamplona, Spain.
AAVs have been successfully used as delivery vectors in numerous small and large animal studies and have yielded the first successful gene therapeutic products on the market. A major hurdle for AAV‐mediated gene therapy is the limited cargo capacity (about 5.0 kb). To effectively deliver the full gene, large constructs need to be split into two‐AAVs and re‐joined in the cells.
Split intein mediated protein trans‐splicing (PTS) has demonstrated it is possible to reconstitute functional proteins in vivo and shows superiority to alternative dual AAV approaches. However, natural split‐inteins used in these studies were suboptimal: low expression levels and strict sequence requirements limit their therapeutic potential.
After 20‐plus years of pioneering intein research, SpliceBio co‐founders designed and engineered the next‐generation of split‐inteins (SB inteins) to overcome the limitations of natural split‐inteins. SpliceBio's engineered inteins result in a 3‐fold higher splicing rate, 10‐30‐fold higher protein expression yields, and 30‐fold higher sequence flexibility.
In this study, we tested the ability of AAV‐SB‐intein‐ABCA4 constructs to reconstitute ABCA4 in cells and in vivo in Abca4‐/‐ mice. We demonstrate that AAV vectors containing SB‐inteins can achieve unprecedented ABCA4 expression in Abca4‐/‐ mice, comparable to wild‐type ABCA4 levels.
In addition, we demonstrate that SB‐inteins‐mediated PTS can also reconstitute a split reporter gene in mice in the central nervous system.
For the first time, gene therapy becomes a viable therapeutic strategy for a wide range of monogenic diseases caused by large genes that could not be treated with AAVs, such as is the case of Stargardt disease.
Delivery of disease‐responsive mini‐intronic plasmids with cationic liposome vectors for rheumatoid arthritis
1: UCL Institute of Child Health 2: Queen Mary University
Rheumatoid arthritis (RA) is an autoimmune disorder affecting the joints. Current treatments risk serious infection while some patients respond poorly. Hence, we aim to develop a targeted gene therapy that modulates RA disease without life‐threatening immunosuppression. Our approach uses novel cationic, receptor‐targeted nanoparticles (RTN) to deliver therapeutic DNA to the inflamed synovium, where the promoter regulating transgene expression is activated by joint inflammation.
Mini‐intronic plasmids (MIPs) were investigated for their reportedly high and sustained gene expression. Transfection efficiency was compared to conventional expression plasmids derived from pCI containing the same promoters and luciferase gene as the MIPs. Transfected luciferase expression at 24‐72 hours was comparable for pCI and MIPs at equimolar ratios in rabbit synoviocytes (HIG‐82) and HEK293Ts. At equal weight ratios, MIPs outperformed pCIs, due, presumably, to their higher copy number. Similarly, naked MIP or plasmid DNA delivered intra‐muscularly by electroporation in Balb/c mice demonstrated similar levels of luciferase expression, by in vivo imaging that was sustained for 28‐days.
Next, an NF‐κB‐responsive promoter was subcloned into MIPs and its response to inflammatory cues assessed in transfections of HeLa cells. Luciferase expression was increased by stimulation with inflammatory markers, TNFα, IL‐1β or PMA. IL‐6 expression confirmed stimulation. However, IL‐6 expression was induced by transfection itself in primary human synoviocytes so, promoter responsiveness could not be evaluated, although expression from the NF‐κB‐responsive MIP was greater than from a conventional plasmid with the same promoter.
Thus, MIP‐encoded therapeutic genes delivered by RTNs provides the basis for a disease‐responsive RA gene therapy with selective gene expression.
Comparison of the exon skipping efficacy of different chemically modified antisense oligonucleotides within the mouse retina
1: Department of Human Genetics, Radboud university medical center, Nijmegen (The Netherlands) 2: Donders Institute for Brain, Cognition and Behavior, Radboud university medical center, Nijmegen (The Netherlands) 3: Department of Pediatrics, Department of Human Genetics, Amalia Children's hospital and Radboud Institute of Molecular Life Sciences (RIMLS), Radboud university medical center, Nijmegen (The Netherlands)
Antisense oligonucleotides (AONs) are short RNA molecules that can modulate pre‐mRNA splicing, and have shown promising results for the treatment of selected subtypes of retinal diseases. Despite their therapeutic potential, little is known about the effect of the chemical modifications that AONs present concerning uptake, biodistribution and toxicity in the retina.
We selected different genes that are exclusively expressed in specific retinal cells that are often affected in retinal diseases (rod, cone, bipolar and retinal pigment epithelium cells) to compare the splicing modulation capacity of AONs harbouring three different chemical modifications [2’‐O‐methyl‐phosphorothioate (2‐OMe), 2’‐O‐methoxyethyl‐phosphoriate (2‐MOE), and phosphorodiamidite morpholino oligomers (PMO)] in vitro and in vivo.
In vitro results revealed that 2’OMe and 2’MOE modified AONs were able to redirect splicing in a similar way. However, 2’MOE AONs showed a higher splicing modulation efficacy for almost all the genes. PMO modified AONs, in general, showed much less efficacy. Currently, we are testing these molecules in vivo by comparing their effects in wild‐type mouse retina. Our preliminary data show interesting differences amongst the different chemically modified AONs in vivo. At this moment, we are conducting in depth analyses at RNA (RT‐PCR) and morphological (IHC) levels to determine the efficacy and toxicity of these molecules.
Overall, our results showed different behaviour of AONs with respect to chemical modifications inside the retina. Thus, this work constitutes the first steps towards a more comprehensive optimization of AON‐based approaches to treat inherited retinal diseases.
Clinical and regulatory strategies to support the marketing authorisation of regenerative medicine products in Japan
1: Universitat Autònoma de Barcelona
Several regenerative medicine products (RPs) have been approved in Japan. This study analyses the most relevant features of the regulatory strategies and clinical development that led to their approval in this market. A systematic review until May 31st, 2021 was carried out. A total of 10 RPs were approved for 12 indications, 6 of the products being cell therapies and 4 of them being gene therapies. Six of the therapies obtained an orphan designation and 2 obtained a Sakigake designation. The mean (SD) time required from submitting the marketing authorisation (MA) application to approval was 14.72 (10.63) months, and 10.5 (2.51) months for those products with a Priority Review designation. Three out of 11 (27.27%) analysed indications received a conditional and time‐limited approval. Thirteen main clinical trials (CT) were conducted to support the MA for those products specifically developed in Japan (n = 7). Of these studies, all except one were small, uncontrolled and single arm. Eleven (84.62%) used intermediate variables to evaluate the primary efficacy. A total of 4 (30.79%) were Phase III or Phase II/III, while 9 (69.23%) were Phase I/II or II. Finally, the median (IQR 25‐75) number of patients enrolled was 10 (5‐16). RP regulations in Japan allow adaptive licensing and constitute shortcut through the clinical development to the approval. Evidence shows that RPs have been mainly approved so far based on inconclusive efficacy and limited safety, prioritising the unmet medical needs of the target diseases, and therefore, the early access for patients.
The small heat shock protein
Al
IbpA from mycoplasma
Acholeplasma laidlawii
prevents the formation of amyloid structures
1: Kazan Federal University
Heat shock proteins (HSPs) inhibit the aggregation of a wide range of target proteins. Interaction of HSPs with aggregating proteins that precipitate in amorphous or fibrillar form, is associated with various neurodegenerative diseases, the transfer of prions, drug resistance and biofilm formation.
In this study, we investigated the ability of the small heat shock protein AlIbpA from phytopathogenic mycoplasma Acholeplasma laidlawii to influence the process of biofilm formation and formation of amyloid structures.
For these purposes, we used Escherichia coli BL21 HSP deletion strains (ΔEcIbpA, ΔEcIbpB, ΔEcDnaK and ΔEcClpB) and overexpression of AlIbpA with deletions of putative functional terminal motifs (AlIbpAΔN12, AlIbpAΔC14, AlIbpAΔN12C14). Biofilm staining with crystal violet showed that the deletion of any HSP leads E. coli cells to form dense biofilms. To assess the formation of amyloids, E. coli strains were grown on a medium with Thioflavin S and Congo red dyes. Removal of one of the physiological HSPs in E. coli leads to enhanced formation of amyloid structures, which was also confirmed by cross‐sectional microscopy of the colonies. An increased level of amyloids was observed during overexpression of AlIbpAΔN12 in ΔEcIbpA and AlIbpAΔC14 in ΔEcIbpB cells, respectively. Amyloids were also detected during overexpression of AlIbpAΔN12C14 in any HSP mutant strain.
Thus, we hypothesize that HSPs control amyloidogenic processes; overexpression of AlIbpA can compensate for the lack of EcIbpA, significantly reducing the amounts of amyloids in the matrix, and for this requires its full N‐terminus.
The work was supported by RFBR grant 20‐34‐90066.
Curcumin‐capped Poly‐L‐Lysine modified gold nanoparticles for delivery of mRNA to cervical cancer cells
1: University of KwaZulu‐Natal
Cervical cancer is a leading cause of female death in developing communities, with a mortality rate of 265 653 annually. The non‐communicable disease shows immense complexity at the epigenetic, genetic and cellular levels, limiting conventional treatment. Immunotherapeutic nanoparticle‐based platforms enable researchers to co‐deliver immunomodulatory agents, target tumours, and improve pharmacokinetics while minimising collateral toxicity to healthy cells. In this preliminary study, gold nanoparticles (AuNPs) were biologically synthesised and capped with curcumin, an aqueous extract of C. longa, that possesses an array of additional anti‐carcinogenic properties. The green synthesis of nanoparticles has been shown to reduce cytotoxicity and enhance economic and environmental benefits. The nanoparticles were further functionalised using the cationic polymer, poly‐L‐lysine (PLL) and stabilised with polyethylene glycol (PEG). Nanocomplexes were characterised using UV‐vis, and Fourier transform infra‐red (FTIR) spectroscopy, transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA). Fluc‐mRNA binding, compaction and nuclease protection was assessed using the band shift, dye displacement and nuclease digestion assays, respectively. The degree of cytotoxicity of the nanocomplexes in the human embryonic kidney (HEK293) and cervical carcinoma (HeLa) cells was evaluated using the MTT assay, and transgene expression monitored using the luciferase reporter gene assay. Results highlighted the favourable properties of the PLL‐PEG‐AuNP nano‐system, such as small size, colloidal stability, efficient binding and protection against nucleases, low cytotoxicity, and significant transgene expression. This proof of principle study has shown potential for safe and efficient delivery of mRNA, necessitating further studies using a therapeutic mRNA molecule for the immunotherapeutic intervention in cervical cancer.
Influence of monthmorillonite adsorbents on the efficacy of removing of pharmaceuticals from water
1: Kazan Federal University
Hydrophobic montmorillonite modified with trimethyl stearyl ammonium and untreated montmorillonite were used to adsorb carbamazepine, ibuprofen and paracetamol. The efficiency of adsorption was investigated under static conditions depending on the pH of the solution, temperature, contact time, the initial concentration of pharmaceuticals and the mass ratio of adsorbents. In the course of the experiments, the optimal conditions for the use of adsorbents were selected. Of the adsorbents tested, untreated montmorillonite is less effective than hydrophobic montmorillonite, which has a higher adsorption capacity to pharmaceuticals in the following order: carbamazepine → ibuprofen → paracetamol. In the course of the experiments, the optimal conditions for the use of adsorbents were selected. Within the concentration range of 10‐50 μg/ml, the most optimal mass ratio of adsorbents is a ratio of 1:67, in the pH range of 6, at a temperature of 25°C, the contact time is not less than 24 hours. Hydrophobic montmorillonite is the most promising adsorbent for further research on the removal of organic pollutants from wastewater. The research was funded by the Russian Science Foundation grant No. 21‐74‐10034.
Disease Correction by delivery of FBX‐101, an Intravenous AAV Gene Replacement Therapy, Administered with Unrelated Umbilical Cord Blood Transplantation for the Treatment of Infantile Krabbe Disease
1: University of Pittsburgh 2: Forge Biologics 3: Thomas Jefferson University 4: Nationwide Children's Hospital
Infantile Krabbe disease is a devastating neurodegenerative disease caused by a lack of functional galactosylceramidase (GALC) enzyme leading to cytotoxic buildup of metabolites such as psychosine. Myelin‐producing cells are particularly sensitive to these toxic metabolites, resulting in rapid loss of white matter in the central nervous system and progressive demyelination in peripheral nerves (PNS). The current standard of care for presymptomatic patients with infantile Krabbe is unrelated umbilical cord blood transplantation (UCBT) which can significantly improve long term outcomes but is not curative. Peripheral nerve disease initially improves after transplantation but will deteriorate over time. We have developed an AAV gene therapy, FBX‐101, that will be systemically delivered by intravenous administration to augment correction of the PNS in patients receiving UCBT. This combined approach has been evaluated in murine and canine models of the disease, demonstrating superior correction of both CNS and PNS disease at the molecular and functional level, and significantly extending lifespan of treated animals. Safety of the treatment approach has been demonstrated in a long term rodent GLP toxicology study. In conclusion, the administration of FBX‐101 with UCBT is a safe and highly effective approach to address the CNS and PNS symptoms of Krabbe disease. FBX‐101 is entering the clinical phase in 2021.
pH‐responsive multicomponent system for charge masking of cationic nanoparticles for gene delivery
1: Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena 2: Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena 3: Leibniz Institute for Age Research, Fritz Lipmann Institute Jena
Cationic non‐viral gene vectors have been extensively studied and show great potential in overcoming the physiological barriers of the gene delivery process. However, the prevention of cytotoxic effects and non‐specific interactions following systemic application remains a challenge. By the introduction of “stealth” polymers such as polyethylene glycol (PEG), biocompatibility and circulation time can be enhanced. However, this often impairs transfection efficiency by impeding cellular uptake and intracellular trafficking. Within this study, a multicomponent polymer‐based gene delivery system was developed comprising highly transfecting cationic hydrophobic particles (P(nBMA‐co‐MMA‐co‐DMAEMA), (PBMD)) and the pH‐responsive block copolymer poly((4‐acryloylmorpholine)‐b‐(2‐(carboxy)ethyl acrylamide)) (P(NAM74‐b‐CEAm74), PNC). While the anionic PCEAm block interacts with the PBMD polymer, PNAM serves as “stealth” moiety. PNC‐masked particles stably encapsulated pDNA at diameters below 200 nm and showed reduced interaction with human erythrocytes at extracellular conditions (pH 7.4, blood) whereas acidic conditions (pH 6, endosome) resulted in membrane leakage. Both, adherent (HEK293T) and difficult‐to‐transfect suspension cells (K‐562) expressed reporter genes comparably or even better if treated with pDNA‐loaded PNC‐masked particles compared to linear polyethylenimine (LPEI). Cytotoxicity was significantly reduced by PNC‐masking in K‐562 cells and erythrocytes. Preliminary in vivo studies further showed transfection of bone marrow blood cells in mice without signs of toxicity. Taken together, the pH‐responsive design provides biocompatibility and stability under extracellular conditions (pH 7.4) while maintaining endosomal escape and transfection efficiency at acidic pH values. Therefore, these results demonstrate the high potential of multicomponent pH‐responsive "stealth" systems, especially for the transfection of difficult‐to‐transfect suspension cells in culture and in vivo.
Optimizing polymeric micelles for non‐viral pDNA delivery
1: Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Germany 2: Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Germany
In recent decades, the development of non‐viral nanocarriers for the (targeted) delivery of genetic material has made significant progress, most recently due to the urgent need for effective vaccines. Polymeric micelles represent a promising class of nanomaterials, offering a broad spectrum of functionalities due to their variable composition of different polymer blocks. While the hydrophobic polymer block induces the assembly in water and forms the micellar core, the hydrophilic shell provides the cationic charges for the binding of the genetic material. However, these charges are not only beneficial, as they bear the risk of increased cytotoxicity and interaction with serum proteins. To circumvent this dilemma, cationic micelles containing tertiary amines were modified with complementary anionic polymers featuring additional stealth blocks. These modifications were introduced either by layering with different anionic and/or hydrophilic (co)polymers or by incorporation of anionic monomers. As another functionality, the impact of redox‐sensitive monomers was analysed by incorporation of lipoic acid into the core of other cationic micelles.
All assemblies were investigated regarding pDNA‐delivery and interaction with the biological environment. Especially, a triblock anionic‐cationic micelle proved to be superior showing high viability of the cells and no significant loss in efficiency. Full recovery of viability and acceptable efficiency were achieved by coating cationic micelles with anionic stealth copolymers. By contrast, the incorporation of lipoic acid into micelles stood out with superior transfection efficiency in difficult‐to‐transfect leukemia cells outperforming the commercial poly(ethyleneimine) (PEI). These results clearly demonstrate the high potential of micellar variability for gene delivery.
Using bone marrow permeabilisation instead of chemotherapy as a conditioning regimen for HSC gene therapy of RAG2‐SCID
1: Hacettepe University
RAG2 severe combined immunodeficiency (SCID) is a rare, inherited primary immunodeficiency (PID), characterized by a T‐/B‐/NK+ phenotype and is treated with haematopoietic stem cell (HSC) transplantation. In cases where no suitable donor is available, these patients have no other treatment options. Therefore, it is important to develop new treatment strategies, such as gene therapy (GT), where the patient's own autologous cells can be used. For SCID patients, usually reduced conditioning regimens (RIC) are used to support homing and long‐term engraftment of the HSCs in the bone marrow (BM). However, since even RIC protocols may cause significant toxicity and unwanted side effects, there is a need to develop alternative conditioning regimen protocols that support adequate engraftment but give minimal toxicity. Here we assessed the effects of granulocyte‐colony stimulating factor (G‐CSF) and very late antigen 4 inhibitor (VLA4I) on BM permeabilisation and HSC engraftment after transduction with LV‐SF‐RAG2co and LV‐UCOE RAG2co and transplantation into RAG2 deficient mice and compared the results with Busulfan (BU) conditioning. The methylation‐resistant UCOE promoter displayed better results than the stronger SF promoter, and both CD3 and CD19 cells could be detected in peripheral blood and spleen tissue 6 months after GT in transplanted RAG2 mice. Although the engraftment kinetics after HSC transplantation and HSC GT of mice treated with G‐CSF or VLA4I were different from the mice treated with BU, use of both G‐CSF and VLA4I was shown to be at least as effective as BU in terms of immune recovery at 6 months after transplantation.
Ex vivo proof‐of‐concept study evaluating STAT1 gain‐of‐function gene therapeutic approaches
1: Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven 2: Department of Microbiology, Immunology and Transplantation, Allergy and Clinical Immunology Research Group, KU Leuven 3: Department of Microbiology, Immunology and Transplantation, Immunogenetics Research Group, KU Leuven 4: Leuven Viral Vector Core, KU Leuven
Autosomal dominant (AD) signal transducer and activator of transcription 1 (STAT1) gain‐of‐ function (GOF) is a primary immunodeficiency caused by over 100 mutations and presenting a broad, mostly unexplained phenotype. Its mutational heterogeneity and GOF nature make it challenging to tackle via regular gene therapy strategies. Here, we propose a mutation‐independent knock‐out/knock‐in gene therapy approach to correct this genetic defect for most, if not all, mutations. Proof‐of‐concept studies were performed in HeLa cells. First, we employed lentiviral vector (LV) technology to stably deliver the CRISPR components into the target cells to knock‐out both endogenous alleles of STAT1. Knock‐out and functional suppression of the IFN‐γ signaling pathway were assessed via western blot, genomic DNA analysis and relevant interferon stimulated genes (ISGs) expression (qPCR) upon IFN‐γ stimulation. Homozygosity of the knock‐out was studied by characterization of single clones. Subsequently, a functional codon optimized copy of STAT1 wild type (WT), driven by an EF1α promoter was delivered into the STAT1 knock‐out cells via LVs. Rescue of the phenotype was achieved. In addition, we evaluated the effect of STAT1 WT overexpression in wild‐type cells on ISG. Upon IFN‐γ stimulation most studied ISGs were not significantly upregulated compared to WT (p < 0.001) or the overexpression of a STAT1 GOF (N574I). These proof‐of‐concept studies in cell lines underscore the potential of a knock‐out/knock‐in approach for STAT1 GOF that could be applied for autologous stem‐cell gene therapy.
Correction of RAB27A gene from Griscelli Syndrome Type II derived Mesenchymal Stem Cells
1: Hacettepe University 2: UMC Utrecht
Griscelli Syndrome type II (GS‐2) is a rare autosomal recessive disease caused by a mutation in the RAB27A gene. GS‐2 patients show typical clinical signs, including silver grey hair, hypopigmentation, recurrent fever and infections. Currently, the only curative treatment for GS‐2 patients is haematopoietic stem cell (HSC) transplantation, in the absence of a suitable donor patients may die at an early age. Here, we aimed to develop lentiviral gene therapy for GS‐2. GS‐2 mesenchymal stem cells (MSCs) were transduced with lentiviral vectors (LVs) carrying RAB27Aco under the control of two different promoters (PGK and UCOE). Transduction was confirmed using flow cytometry, qRT‐PCR, western blot and immunofluorescence staining. RAB27A protein expression was higher with the PGK promoter than with UCOE. Similarly, RAB27A gene expression was found to be increased when using the PGK promoter in comparison to UCOE. Although transduction efficiency increased with higher MOI, transduction efficiency of UCOE never reached levels of PGK. In conclusion, both PGK and UCOE promoters supported overexpression of RAB27A in GS‐2 MSCs, although effects of PGK were more distinct. Using these LV vectors and CRISPR/Cas9 we now aim to increase RAB27A expression in GS‐2 derived induced pluripotent stem cells (iPSCs).
Bridging the gap: use of rapid quality control test methods for cell and gene therapies as alternatives to traditional methods
1: Merck KGaA
Many advanced therapy medicinal products have a short shelf life from the point of manufacture to administration to the patient. Furthermore, they may be produced in relatively low volumes for small scale clinical trials or small patient populations. Within this limited period and with this low product volume, it is necessary to complete regulatory required quality control (QC) testing, be that for early or late phase clinical trials, or for licensed drug product.
Two of the biggest challenges with QC testing of cell and gene therapies using traditional test methods is time to results, due to short shelf‐life, and availability of sufficient sample, due to low production volumes.
So how can these challenges be overcome while meeting regulatory expectations?
Here we discuss and review suitable methods for rapid testing of short‐life cell and gene therapies that may also help conserve limited amounts of product. In particular we look at the regulatory expectations for using rapid test methods in place of classical or compendial test methods and how to bridge the gap between the two. We provide examples and data from our own experience, as a contract testing organisation, with a range of methods including rapid tests for sterility, mycoplasma and replication competent virus.
Application of InGetox to assess the genotoxicity of LV and AAV vectors
1: 3. Brunel University London, Department of Life Sciences, Uxbridge, United Kingdom 2: 1. Genewerk GmbH, Heidelberg, Germany 3: 2. University Heidelberg, Medical Faculty, Heidelberg, Germany 4: 5. King's College London, Department of Infectious Diseases, London, United Kingdom 5: 6. University College London, Infection & Immunity, London, United Kingdom 6: 7. The Natural and Medical Sciences Institute, Target Expression Systems, Kusterdingen, Germany
RV and LV have been shown associated with genotoxic events in vitro, in vivo and in the clinic. AAV have also been identified previously associated with liver cancers in mice and clonal development in dog studies for the treatment of haemophilia. Current models to assess the safety of LV include immortal cell lines and small and large animal models. Models for AAV risk assessment are rudimentary, mainly because these vectors are considered safe. However, it is now known that AAV integration occurs in the host genome albeit less frequently than LV. Hence, the development of model that can identify potential LV and AAV side effects would be desirable.
Currently a number of models for RV and LV exist, however, they are either oversensitive with genotoxic bias or have long lead in times and may limit vector entry to the clinic. Furthermore, they are not of human origin and none have been accepted by FDA, EMA or MHRA regulators.
Using a human induced pluripotent stem cell/liver derivative platform, we have generated hepatocyte like cells in 2D and 3D organo‐culture formats as a personalised approach to identify the currently accepted virus‐associated contributory factors to oncogenesis for LV and AAV vectors. We present this human‐based model using positive and negative control vectors and our findings specifically directed to vector integration sites and their associated gene expression changes, vector splicing and readthrough generating novel vector/host oncogene truncations, epigenetic changes and a clonal tracking assay.
Dissecting the non‐cell‐autonomous effects of oncogene activation on hematopoiesis
1: Telethon Institute of Gene Therapy (HSR‐TIGET)
Oncogene activation in hematopoietic stem and progenitor cells (HSPCs) increases the risk of leukemia in patients and thus is a major concern in the gene therapy field. Moreover, we recently showed for the first time that transplantation of oncogene (BRAFV600E) expressing human HSPCs in immune‐deficient (NSG) mice causes histiocytosis and lethal bone marrow failure. Oncogene‐induced senescence was the source of these detrimental effects, rousing a senescence‐associated secretory phenotype that also affected bystander cells.
We sought to understand whether this aggressive phenotype was also due to inefficient clearance of senescent cells in NSG recipient mice. For this reason, we transplanted mouse wild‐type (WT) HSPCs expressing human BRAFV600E or a mouse form of activated Braf (mBraf‐trunc) into WT or NSG mice and monitored them over time. Strikingly, neither NSG nor WT mice showed any lethality. However, in WT recipients transplanted with human BRAFV600E‐expressing mouse HSPCs, we observed myeloid skewing and counter‐selection of oncogene‐expressing lymphoid cells at 4 weeks post‐transplant, while in NSG recipients counter‐selection was observed only after 24 weeks. On the other hand, transplantation of mouse HSPCs expressing mBraf‐trunc in NSG mice induced an expansion of oncogene‐expressing B cells, yet without major impairment on hematopoiesis.
Our results highlight that the immunological condition of recipient mice impacts the tolerance toward BRAFV600E‐expressing cells, while also providing a strong indication of differences between mouse and human biology. Ultimately, these studies might uncover mechanisms of selection and impairment of oncogene‐expressing cells and perhaps suggest ways to eliminate them.
Finding integrations of a clinical gene therapy vector in NGS data with isling
1: Commonwealth Scientific and Industrial Research Organisation 2: Children's Medical Research Institute
Viral vectors are a critical technology underlying gene therapy, and verifying the the safety of every vector is an important precursor to clinical application of gene therapy. Indeed, recent evidence links integrations of a liver‐specific enhancer‐promoter element in the AAV2 3’ untranslated region 3 near oncogenes within the human liver to hepatocellular carcinoma 1,2 . There is therefore a need to characterise the integration behaviour of AAV and other gene therapy vectors. To investigate viral integration in gene therapy, as well as integrations of wild‐type viruses, we developed isling, a flexible, scalable tool to identify host/virus junctions in sequencing data. Isling out‐performed other viral integration tools on both simulated and previously published benchmarking datasets 4 –6 . We then applied isling to sequencing data resulting from a study of a pre‐clinical vector for Ornithine Transcarbamylase Deficiency. This vector was found to be integrated in macaque hepatocytes and xenografted human hepatocytes in the FRG mouse liver, and isling identified millions of integrations in each case. Analysis of these integration sites will identify any safety concerns for this clinical vector, and inform our knowledge about the mechanisms of AAV vector integration.
LATE – a novel sensitive cell‐based assay for the study of CRISPR‐Cas9 related Long‐term Adverse Treatment Effects
1: Research Dept. Cell and Gene Therapy; Dept. of Stem Cell Transplantation, University Medical Centre Hamburg‐Eppendorf, Hamburg (Germany)
Programmable DNA‐modifying enzymes, based on the CRISPR/Cas9 system, provide precise and facile molecular mechanisms for editing genomes of single cells, tissues, organs, and whole organisms. However, fast translation into clinical testing has raised some concerns. Indeed, before broad clinical use, deeper insights into the toxicity profile of large‐scale application of designer nucleases in general and CRISPR/Cas9, in particular, will be required. Whereas frequencies and locations of off‐target cutting have been broadly addressed, little is known about their potential biological consequences. We have developed a Long‐term Adverse Treatment Effect (LATE) in‐vitro assay that addresses the potential toxicity of designer nucleases by assessing cell‐transformation events. Our assay is based on genetically marked indicator cells and flow‐cytometry as well as digital‐PCR‐based read‐outs to detect growth advantage. In small‐scale proof‐of‐principle experiments, we reproducibly detected low‐frequency (<0.5%) growth‐promoting events in primary and immortalized cells resulting in off‐target cleavage in the TP53 gene as well as in other tumor suppressor genes. Importantly, some of those off‐target effects would not have been predicted by commonly used online tools. Whereas the initial assay was relatively time‐consuming (up to 8 weeks), we have meanwhile introduced modifications to significantly shorten the readout. We suppose that our method might become useful for preclinical toxicity assessment in the expanding field of clinical genome editing.
Development of a new all‐in‐one
in vitro
safety assay for gene therapy to detect lymphoid and myeloid insertional mutants
1: Institute of Experimental Hematology, Hannover Medical School 2: REBIRTH ‐ Research Center for Translational Regenerative Medicine, Hannover Medical School 3: Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School
Safety prediction in retroviral vector‐mediated gene therapy remains challenging. Insertional mutagenesis led to lymphoid and myeloid leukemia in early clinical trials for severe combined immunodeficiency, chronic granulomatous disease, and Wiskott‐Aldrich syndrome. The In Vitro Immortalization Assay (IVIM) and Surrogate Assay for Genotoxicity Assessment (SAGA) are currently used for risk assessment of integrating vectors. Murine hematopoietic stem and progenitor cells (mHSPC) transduced with mutagenic vectors acquire a proliferation advantage under limiting dilution (IVIM) and activate stem cell‐ and cancer‐related gene expression pathways (SAGA). However, both assays present an inherent myeloid bias due to culture conditions. To detect lymphoid 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 an early progenitor stage (>30%), while untransduced mock cultures and samples transduced with safer vector designs fully differentiated. Arrested samples upregulated the leukemogenic transcription factors Lmo2 and Mef2c and contained vector integrations in their vicinity. We then combined the lymphoid and myeloid readouts by transferring samples that started in lymphoid conditions to conventional myeloid culture medium. RSF91‐transduced samples from the combinatorial assay still showed immortalization in standard IVIM and SAGA analysis and contained high‐risk integrations close to Mecom and Prdm16. In sum, our all‐in‐one approach enables the detection of retroviral vector genotoxicity in the myeloid and lymphoid lineages and reproduced the risk pattern observed in early clinical trials. Further fine‐tuning of this assay could lead to a better understanding and risk assessment of insertional mutagenesis.
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: Tettamanti Research Center 4: University of Milano‐Bicocca 5: Institut de Genetique Moleculaire de Montpellier 6: Dr. von Hauner Children's Hospital, LMU 7: Gene Center, LMU 8: Universita Vita‐Salute San Raffaele 9: Pediatric Immunohematology and BMT, San Raffaele Hospital,
Sonication Linker mediated‐ PCR (SLiM‐PCR) is a method for quantitative Integration Site (IS) retrieval in which the vector/genome junction is specifically amplified after the genomic DNA is sonicated and ligated to a linker cassette. Vector marked clones can be quantified by counting the number of different fragments containing the same IS.
We validated this method on samples composed by DNA extracted from two purified cell clones carrying one and six lentiviral vector (LV) integrations in known genomic positions and DNA extracted from a polyclonal LV‐marked cell population, mixed at different ratios.
The validation showed a high correlation of the abundance estimates of the 2 clones with the theoretical estimations (±5%), a high correlation between the number of retrieved and expected IS (R2 ≃0.9), and a ∼10‐fold higher efficiency in IS retrieval when compared to other methods.
SLiM‐PCR has been successfully applied on clinical and preclinical studies using different vector platforms. It allowed the detection and quantification of (pre)malignant clones in 2 patients of a γRV‐based clinical trial for Wiskott‐Aldrich syndrome. It was highly efficient in retrieving AAV IS from lymphoid tissues collected from Zap70‐/‐ immune‐deficient mice intra‐thymically injected with AAV. It also allowed to study the response to tumor overtime of CD19 CAR T‐cells engineered by the sleeping beauty transposon in a clinical trial for B cell acute lymphoblastic leukemia.
Our results show that SLiM‐PCR is a powerful method for IS retrieval and accurate clonal abundance estimation, and that can be applied to a broad spectrum of gene therapy platforms.
Qualification of an NGS‐based assay for the identification and quantification of encapsidated sequences in rAAV batches
1: Genewerk GmbH Heidelberg
Massive parallel sequencing allows to identify and quantify process‐ and host‐related impurities encapsidated in recombinant adeno‐associated viral vector (rAAV) batches lacking the primer binding bias of PCR methods. Performance of NGS‐based approaches is still undercharacterized and we aimed at providing a detailed assay qualification for the detection and quantification of encapsidated DNA species.
Preliminary work was performed on a research‐grade AAV8 reference batch (n = 3) to determine the precision of the species quantification. Packaged sequences were extracted and, after second strand synthesis, DNA fragments were sheared and library prepped for sequencing on the Illumina platform. Sequencing data were analyzed through an in‐house pipeline to identify and quantify the species. The majority of reads mapped to the rAAV genome (95.13‐95.36%), followed by the plasmid backbone (2.74‐2.77%) and the human genome (1.90‐2.11%). Precision was measured as the percentage coefficient of variation and, considering both total sequence counts and relative frequencies, CV ranged from 0.12% to 5.39% thus showing the low inter‐replicate variability.
Accuracy was determined in a control generated by spiking 250 copies of human genome into 2E+11 copies of rAAV plasmid (“rAAV” + “backbone”). Considering the total nucleotides expected from the copies spiked, accuracy was ‐23.67%, ‐14.82% and 22.70% for each of the three references (rAAV, backbone and human genome, respectively).
These data show that the assay is able to provide a precise and accurate quantification of eventual DNA impurities within vector batches. Additional work is currently being perform on additional reference material and by systematic comparison to PCR approaches.
Standardization of lentiviral vector copy quantification and vector insertion site detection using cellular clones.
1: Genethon 2: Université Paris‐Saclay, Univ Evry, Inserm, Genethon, Integrare research unit UMR_S951
The evaluation of potency and safety of lentiviral gene therapy vectors during their preclinical development, their manufacture and their use in humans, relies on a precise determination of lentiviral vector copy number (VCN) in transduced cells as well as quantifying clonal contribution by genomic insertion site analysis. Despite recent development, these methods lacks standardization and reference materials to determine their sensitivity. We here describe the generation of reference cellular material that can be used for the development, the calibration and the qualification of analytical methods. Using this reference material, we determined the sensitivity of our vector copy number quantification methods based on digital droplet PCR and improved the method specificity for integrated provirus. We also benchmarked the sensitivity of the vector insertion site analysis protocol based on LM‐PCR. The standards also serve for quality assurance through longitudinal recording of test values. Such cellular standards can contribute to an international standardization effort in gene therapy.
Lentivirus tethering to the genome is associated with transcription factor binding sites that favour virus survival
1: Department of Life Sciences, College of Health, Medicine & Life Sciences, Brunel University London, Uxbridge, UK 2: Department of Computer Science, College of Engineering Design and Physical Sciences, Brunel University London, Uxbridge, UK 3: Genewerk GmbH, Heidelberg, Germany 4: University Heidelberg, Medical Faculty, Heidelberg, Germany 5: Division of Infection and Immunity, University College London, London, UK 6: Division of Advanced Therapies, National Institute for Biological Standards and Control, Potters Bar, United Kingdom 7: Gene Transfer Technology, EGA Institute for Women's Health, University College London, London, UK 8: NCT and DKFZ, Department of Translational Oncology, Heidelberg, Germany 9: Division of Ecology and Evolution, Department of Life Sciences, Imperial College London, London, UK
Lentiviral vectors offer effective and permanent therapeutic gene delivery into the host genome for gene therapy. However, integration can result in insertional mutagenesis and the mechanisms by which LV integrate should be understood further. Insertion site tethering involves host cellular proteins such as PSIP1/LEDGF/p75, which binds to the virus pre‐integrase complex and aids targeting of the virus genome to host chromatin. Transcription factors that bind to specific sequences in the vector LTR and host genome are also thought to influence this process. To investigate the association between LV of different LTR configuration and host genome site selection, we used LV carrying native or self‐inactivated LTRs to infect induced pluripotent stem cells and their hepatocyte‐like derivatives. We identified predicted transcription factor binding sites common to each LV LTR and the IS chosen by each vector. We identified significant enrichment for sites recognised by TF important to HIV‐1 life cycle and virus survival which were also present in the viral LTRs. These sites were also found to align to the IS chosen by HIV‐1 in infected patient and murine genomes after infection by native and SIN configuration LV respectively. As these pTFBS also appear close to genes known to be associated with LV genotoxic events, further modifications to the LTRs of LV should be considered to avoid integration in undesirable sites.
Standardization and Validation of a Probe‐Based qPCR Assay for Lentiviral Vector Copy Number Determination
1: GeneWerk GmbH Heidelberg 2: Medical Faculty, University of Heidelberg
As integrating platforms, lentiviral vectors (LV) require copy number monitoring to enable proper risk assessment and there is an unmet need for standardized copy number quantification procedures. We developed and validated a highly accurate and sensitive probe‐based qPCR assay for the quantification of LV vector copy numbers within human samples.
To facilitate standardization, the assay was established and validated using a synthetic DNA fragment bearing the target LV gag sequence, as well as a human genomic control region (RNAseP) for normalization purposes. Assay validation comprised a set of calibration standards together with quality control (QC) samples and the lower limit of quantification (LLOQ), all generated by spiking the synthetic DNA into a blank matrix of untransduced genomic DNA.
The assay exhibited a dynamic range from 1E+7 to 10 copies with an LLOQ of 30 copies into 100ng of genomic DNA and an LOD of 10 copies with a 100% detection rate. Calibration standards revealed an average PCR efficiency of 91.76% with all standards quantified within the ±15% accuracy threshold of the nominal value. QC samples and LLOQ ranged between 30 and 800,000 copies and all cases met the ≤5% (LLOQ: ≤6.67%) precision threshold and the ±15% accuracy, for both within‐run and between‐run parameters. In addition to the work on synthetic controls, performance is being cross‐validated on the reference panel available from the WHO.
Therefore, the assay enables the accurate and highly sensitive determination of LV copy numbers to monitor vector persistence.
Safety Profile of Conditioning Regimens of AVROBIO's Investigational ex vivo Lentiviral Gene Therapy for Fabry Disease
1: Royal Perth Hospital, WA 6000, Australia 2: Royal Melbourne Hospital, Parkville, VIC 3050, Australia 3: AVROBIO, Inc., Cambridge, MA 02139, USA
AVR‐RD‐01 is investigational ex vivo lentiviral gene therapy for Fabry disease, which involves three steps: mobilization with apheresis, myeloablative conditioning, and infusion of patient's own genetically modified hematopoietic stem cells carrying the therapeutic gene.
Conditioning enables stem cell engraftment and potentially facilitates durable distribution of functional protein. In AVR‐RD‐01's clinical development program, two currently available conditioning agents, low‐dose melphalan or busulfan (Bu90‐TCI), have each been used as single agents. Safety is evaluated with adverse events (AEs), vital signs, physical exams, ECGs, laboratory assessments and clonal expansion monitoring by integration site analysis (ISA). Here we present new safety data for 12 patients (out of 14 dosed in Phase 1 and 2) who had ≥30 days of follow‐up post gene therapy infusion.
Duration of observation was from Day 1 of conditioning through Week 48 post gene therapy. The most common AEs in these patients related to the conditioning agents were neutropenia, nausea, alopecia, leukopenia, thrombocytopenia, vomiting, diarrhea, and fatigue. Of the AEs assessed as related to conditioning and occurring in ≥50% of patients, all AEs had onset between Day 1 of conditioning and 30 days post gene therapy with 97% (66/68) of these AEs being Non‐Serious (2 Serious AEs were nausea and vomiting), 60% (41/68) Grade 1 or 2 in Severity, and 74% (50/68) resolving within the 30 days of onset without sequelae. Preliminary data suggest that use of both conditioning agents for gene therapy has predictable and well‐tolerated acute and sub‐acute safety profile. Additional data, including ISA, will be presented.
Clinical Trial Update:
Ex‐vivo
autologous haematopoietic stem cell gene therapy in MPSIIIA
S A Jones4 J L Kinsella2 J Potter2 A J Thrasher3 C Booth3 K F Buckland3 N Izotova3 S Rust2 D Weisberg2 H J Church4 K L Tylee4 K L Brammeier4 C Jones4 H Lee5 L Ford5 A Sadegholnejat5 R J Holley1 S Ellison1
1: The University of Manchester 2: Royal Manchester Children's Hospital 3: Great Ormond Street Institute of Child Health 4: Saint Mary's Hospital, Manchester 5: Manchester Royal Infirmary
Mucopolysaccharidosis type IIIA (MPSIIIA) is a devastating lysosomal storage disorder. Mutations in SGSH lead to substrate accumulation with affected children presenting in toddlerhood with developmental delay and progressive loss of previously acquired skills, attributed to brain inflammation and neurodegeneration. There are currently no approved treatments. Here we report the status of children with rapid progressive MPSIIIA treated at 6‐24 months of age with ex‐vivo autologous stem cell gene therapy as part of a phase I/II clinical trial (NCT04201405/EudraCT#2019‐002051‐42).
As of June 2021, we have treated four patients and enrolled a fifth with no serious adverse events linked to the investigational medicinal product (IMP). Mobilised patient‐specific haematopoietic stem cells were transduced ex‐vivo with a lentiviral vector containing the SGSH gene driven by the CD11b promoter and cryopreserved (the IMP). All four IMPs had vector copy numbers between 3.54‐8.91 copies/cell, with cell doses of 7.67‐22.7x106 CD34+/kg. Haematological engraftment following busulfan‐conditioning was rapidly achieved, with demonstrable platelet/neutrophil/haemoglobin counts.
Supraphysiological SGSH enzyme in leukocytes was measured at 38‐90‐fold above normal at one‐month post‐transplant. In patients with available follow‐up, plateauing from three‐months post‐transplant. Supraphysiological SGSH is also detectable in CD15 lineages, plasma and bone marrow, with CSF SGSH within the normal range by six‐months post‐transplant. Marked reductions in heparan sulphate levels in urine (>90% reduction) and plasma (>75% reduction) at three‐months post‐transplant were recorded. Patients will be followed‐up for three years post‐transplant and neurocognitive outcomes assessed.
Thus, ex‐vivo autologous stem cell gene therapy was generally well tolerated, providing an exciting potential treatment for severe MPSIIIA, warranting further investigation.
Artificial membrane vesicles isolated from mesenchymal stem cells with IL2 overexpression activate CD8+ T‐killers to kill triple negative breast cancer cells
1: Kazan Federal University
Almost all human cells release extracellular vesicles (EVs) participating in intercellular communication. EVs are rounded structures surrounded by the cytoplasmic membrane, which embody cytoplasmic contents of the parent cells. Therefore, it is EVs that are of interest as a therapeutic tool for cell‐free therapy that has targeted delivery.
In this study, human MSCs were isolated from adipose tissue (hADSCs) and genetically modified to overexpress interleukin‐2 (IL2) (hADSCs‐IL2). Extracellular vesicle release from native and genetically modified hADSCs was induced by cytochalasin B treatment. To evaluate immunomodulating properties of membrane vesicles from native (native CIMVs) or hADSCs‐IL2 (CIMVs‐IL2), peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood and co‐cultured with CIMVs (50 μg/ml) for 72 hours. Activation of PBMC populations was determined by flow cytometry using staining with antibodies specific for surface markers of various human immune cell populations.
The number of activated T‐killers (CD3+CD8+CD4‐CD38+HLA‐DR+) was significantly increased after incubation with CIMVs‐IL2 (205.6 ± 4.5%) compare to native PBMCs (100.0 ± 9%) and PBMCs incubated with native CIMVs(116.7 ± 4.5%). The number Th1 cells was decreased in the sample of PBMCs incubated with CIMVs‐IL2 (92.3 ± 1.6%) compared with native PBMCs (100.0 ± 1%), while the number of Th2 cells (109.0 ± 0.1%) was increased compared to native PBMCs (100.0 ± 1%), number of Tregs remained unchanged.
The use of CIMVs‐IL2 can be effective in the treatment of triple negative breast cancer, since CIMVs‐IL2 are able to activate and stimulate the proliferation of T‐killers. However, further studies of CIMVs efficiency in animal tumor models are required.
High throughput characterization of CD34+ cell states and dynamics upon genetic engineering
1: AVROBIO Inc., Cambridge MA 2: University College London, London, UK 3: Great Ormond Street Institute of Child Health Faculty of Population Health Sciences, London, UK
Human CD34+ cells are key components of ex vivo lentiviral gene therapy (GT) drug products (DP), therefore understanding the changes occurring in hematopoietic stem and progenitor cells (HSPC) upon genetic engineering is critical to determining the potency of GT. However, key surface molecules normally used to assess CD34+ cells become unreliable during ex vivo manipulation, and a full characterization of the DP remains virtually inaccessible. Here, we combined state‐of‐the‐art multiparametric flow cytometry and next generation single‐cell (sc) technologies for a systematic characterization of the main sources of human HSPC and their fates upon genetic engineering. Analysis of 60,900 sc transcriptomes unveiled source‐specific transcriptional landscapes, cell cycle states, and gene expression patterns such as the ones differentiating single‐ vs double‐mobilized peripheral blood (MPB) cells. Focusing on G‐CSF+Plerixafor MPB as the most clinically relevant source, we FACS‐sorted and individually cultured 7 HSPCs subsets, capturing at high resolution (100,578 single cells) the progenitor‐specific transcriptional response to common cytokine cocktails. Using flow cytometry RNA detection (PRIMEFLOW), we are validating candidate markers of HSPC differentiation in culture. We then investigated the cell states over 7 days of expansion in 191,980 single cells from bulk HSPCs and FACS‐sorted hematopoietic stem cells (HSC), unveiling potential early effects of the HSC‐agonist UM171 on CD34+ cells priming, differentiation, and vector insertion site (IS) distribution (94,362 unique IS). To our knowledge, our analytical platform represents the most comprehensive transcriptional characterization of CD34+ cells upon genetic engineering and may provide a long needed step‐change in the characterization of GT DP.
KCNV2 retinal organoid disease model for KCNV2 AAV gene therapy development
1: MeiraGTx 2: NIHR Biomedical Research Centre at Moorfields Eye Hospital
Microvesicles transduced with NGF ameliorate pathological outcomes of EAE mouse model
1: Kazan Federal University
Multiple sclerosis (MS), is a chronic neuroinflammatory demyelinating disease that leads to physical or cognitive incapacitation. One of the promising therapies of MS is mesenchymal stromal cells (MSCs). Extracellular microvesicles (MV) have been identified as a therapeutic alternative to MSCs and delivery vectors of therapeutic factors. Nerve growth factor (NGF) effect was evaluated upon it's delivery in a MSCs ‐induced MV to the EAE model as a strategy for subcellular therapy of MS. In a mice MOG35‐55 EAE model, the effectiveness of adipose tissue MSCs (ADMSCs), ADMSCs cytochalazine B‐ induced MV both native transduced with NGF, was studied. A morphometric analysis and the level of blood cytokines was evaluated. There was a significant decrease in the area of demyelination and the number of reactive astrocytes after the introduction of both types of MV compared with groups without treatment, and MV with NGF compared to native MV. The levels of TNF‐a, IL‐6, MCP1 and IL‐1b decreased after the introduction of native MV and MV with NGF. The level of anti‐inflammatory cytokines IL‐4 and IL‐10 was increased after the introduction of MV with NGF. Thus, the treatment of EAE using native MV or MV with NGF is not inferior to therapy with ADMSCs. The use of MV with NGF showed better results, which gives grounds for the development of a subcellular therapeutic strategy.
EVs derived from HIF‐overexpressing MSC primed with a pro‐inflammatory cocktail promote M2 macrophage polarization and reduce fibrosis and endothelium activation
in vitro
and in a mouse ulcerous colitis model.
1: Grupo de Regeneración y Trasplante Cardiaco, Instituto de Investigacion sanitaria La Fe, Valencia, Spain 2: Sección departamental de Biología Celular, Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain 3: Instituto de Investigación Sanitaria, Hospital Clínico San Carlos, Madrid, Spain 4: Departamento de Regeneración y Terapía Celular. Centro Andaluz de Biología Molecular y Medicina Regenerativa‐CABIMER, Junta de Andalucía‐Universidad Pablo de Olavide‐ Universidad de Sevilla‐CSIC, Sevilla, Spain.
Extracellular vesicles therapy (EVs) derived from Mesenchymal Stem Cell (MSC) is a potential advance treatment to healing of multiple immune disorders like ulcerous colitis due to their regenerative and immunosuppressive properties. Currently, EVs therapies have shown improvements compare to MSCs transplantation in terms of biosafety and production in clinical grade conditions. However, for its clinical translation it is important to boost EVs effectiveness. To increase therapeutic potential of EVs source we have developed an immortalized MSCs line that overexpress HIF‐1α (MSC‐HIF). Prior to EVs isolation, these MSCs were cytokine preconditioned (EVMSC‐T‐HIF c) to simulate an inflammatory environment. The aim of this study was to prove the immunoregulatory properties of EVs derived from the MSC‐HIF on macrophages differentiation, fibroblasts and endothelial cells. We have observed that EVMSC‐T‐HIF c induce the repolarization of monocytes from M1 toward M2‐like macrophages, with a reduction in inflammatory cytokine release. In addition, EVMSC‐T‐HIF c were also able to reduce adhesion of PBMCs on activated endothelium and they decrease fibrosis caused by TGFβ. Finally, we observed that the EVMSC‐T‐HIF c treatment promote healing into a mouse TNBS‐induced colitis model. Also, the administration of EVMSC‐T‐HIF c increase colon length, partially restores the physiological tissue structure of large intestine, reduce M1/M2 ratio infiltration and fibrillar collagen proportion. In this work, we have demonstrated that boosted MSC‐EVs have effective anti‐inflammatory properties, making them potential therapeutic agents safer than MSCs for the treatment in immune and inflammatory diseases.
Funding: PI16/00107, PI19/00245, RD16/0011/0002, RD16/0011/0004 and ACIF/2018/254, cofunded by FEDER “una manera de hacer Europa”.
Clonal analysis of ex vivo expanded mobilized peripheral blood hematopoietic stem and progenitor cells in xenografts proves symmetric HSC division in culture
1: San Raffaele Telethon Insititute for Gene Therapy (SR‐TIGET) 2: National Research Council, Institute for Biomedical Technologies 3: Hematology & Bone Marrow Transplant Unit, IRCCS San Raffaele Hospital
Ex vivo expansion of HSPC is an unmet medical need in gene therapy and regenerative medicine. We have optimized a small molecule‐based expansion protocol for genetically‐engineered mPB HSPC (abstract by Galasso I et al.). We here report clonal analysis of an expanded (EX) versus a minimally‐manipulated (MM) HSPC graft. A lentiviral vector library, whereby each particle contains a unique barcode sequence (BAR‐LV), was generated, and validated for NGS detectability & complexity. MPB CD34+ HSPC were transduced with BAR‐LV yielding on average two copies per cell. Part of the culture was xenotransplanted within 14 hours from transduction (MM), while the other part was expanded for 7 days before transplantation (EX). Human CD45+ engraftment at 16 weeks was similar between MM and EX when the same starting cell dose equivalent was transplanted. Graft clonality was assessed by 2 independent techniques, namely LV integration site analysis and barcode sequencing. Both approaches yielded strikingly similar results. Engraftment was polyclonal, without evidence for clonal dominance. The number of unique clones was higher in the EX group compared to the MM group at the same starting dose equivalent, and similar when 1/3 of the dose was transplanted in the EX group. We next assessed the degree of sharing of unique barcodes/integration sites between different mice. Differently from the MM group, a noticeable fraction of individual barcodes/integration sites was found in more than one mouse after ex vivo expansion, consistent with symmetric HSC division in culture. These novel readouts will facilitate the development of improved expansion protocols.
Subtype‐specific differentiation of atrial and ventricular cardiomyocytes from human iPSCs
1: Instituto de Investigacion sanitaria La Fe 2: Universidad Politécnica de Valencia
Cardiovascular disease is the leading cause of death worldwide and advanced insights into disease mechanisms and therapeutic strategies require deeper understanding of the healthy heart's molecular processes. Human pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) offer a human‐based model for disease modelling and drug discovery. Nevertheless, most of the differentiation protocols available are focused on ventricular cardiomyocytes. Generation of homogeneous populations of subtype‐specific cardiomyocytes (CMs) differentiated from iPSCs and their comprehensive phenotyping is crucial for a better understanding of the subtype‐related disease mechanisms and as tools for the development of chamber‐specific drugs. The goal of this study is to provide new insights into the development of the human atrial and ventricular lineages from hiPSC that enable the generation of highly enriched, functional cardiomyocyte populations. We applied a simplified protocol for differentiation of human iPSCs into defined populations of atrial or ventricular CMs subtypes in monolayer and feeder‐free culture conditions with the addition of retinoic acid (RA) for the hiPSC‐aCMs population. Our study demonstrated that the RA treatment during a specific time window of the differentiation, lead to an overexpression of the atrial biomarkers CACN1D, MYL7, KCNJ3 and NR2F2 and a downregulations of the ventricle genes GJA1, MYL2, IRX4 and KCNJ2 with a consequently effect on the action potential frequency. Finally, the hiPSC‐aCMs showed identical morphology and troponin expression respect to the hiPSC‐vCMs, underling the fact that the treatment with RA does not alter the differentiation efficiency and providing a unique opportunity to study atrial physiology in a human‐relevant in vitro model.
Development of cell‐mediated gene therapy method for metachromatic leukodystrophy using modified mesenchymal stem cells
1: Kazan (Volga Region) Federal University 2: Kazan State Academy of Veterinary Medicine named after N.E. Bauman
Metachromatic leukodystrophy is an autosomal recessive lysosomal storage disease arising due to arylsulfatase A enzyme (ARSA) deficiency, that results in a buildup of sulfatides leading to the destruction of myelin sheaths and progressive demyelination of central and peripheral nervous system. The aim of this work is to develop a treatment method for Metachromatic leukodystrophy using modified mesenchymal stem cells (MSCs).
Recombinant adeno‐associated viruses encoding ARSA gene were used to transduce MSCs, which were then intravenously injected into mini pigs. Results were compared to a group of intact animals. Enzymatic activity was analyzed in cerebrospinal fluid (CSF) and plasma samples. Homogenates of CNS organs were used to analyze ARSA expression using qPCR and Immunohistochemistry analysis (IHC). Changes in biochemical parameters were also studied in serum samples of pigs before and after MSC‐ARSA administration.
High ARSA activity was observed in all plasma and CSF samples, followed by a later gradual decrease. Enzymatic activity showed also an increase of values in CNS lysates. Analysis of biochemical parameters showed an increase in ALT and AST levels on the 7th day after administration. However, all parameters decreased later to normal values.
Obtained results suggest that MSC‐ARSA expresses a functionally active enzyme ARSA and can be considered as a candidate for treatment of metachromatic leukodystrophy.
Lentiviral fluorescent genetic barcoding allows real‐time fate tracking of different hematopoietic stem cell populations
in vivo
1: Hannover Medical School 2: Boston Children's Hospital
Hematopoietic stem cells (HSC) represent a rare cell population of particular interest for biomedical research. When we performed phenotyping of murine HSC with two established marker panels (LSK‐SLAM [Lin‐Sca‐1+cKit+CD48‐CD150+] & ESLAM [CD45+EPCR+CD48‐CD150+]), we noticed a subdivision of LSK‐SLAM into ESLAM and LSK‐SLAM‐EPCR‐ (LSK‐SLAM‐) HSCs with unknown implications. We thus aimed to characterize the ESLAM and LSK‐SLAM‐ populations in a reduced number of mice, and therefore developed a lentiviral multiplexing fluorescent genetic barcoding (FGB) system enabling the parallel characterization of color‐coded cells. Previous barcoding experiments showed limited in vivo persistence of transduced cells, presumably due to vector design or transduction conditions. Thus, we established a set of 6 new lentiviral FGB vectors utilizing Azami Green, mCherry, and YFP as single and bicistronic constructs. Optimized gene transfer rates were achieved by overnight transduction of pre‐stimulated HSC. We next performed competitive in vivo assays by co‐transplanting 3 uniquely color‐coded ESLAM and LSK‐SLAM‐ samples each per irradiated recipient yielding 30 data points per condition in two independent experiments (each n = 5). The first experiment was hampered by data fluctuations, but increasing cell numbers and exchanging the spleen focus‐forming virus‐derived U3 promoter by the elongation factor one alpha (short)‐derived promoter led to 74.4±15.0% chimerism, with 87.1±13.3% of color‐coded cells originating from ESLAM HSCs in the second experiment. Importantly, ESLAM HSC produced 88.1% of myeloid cells indicative of long‐term repopulation potential. Our work underlines the importance of the EPCR marker for long‐term engraftment and the applicability of our FGB system for the effective characterization of HSCs.
PKNOX2 can be a new player in NF‐κB/p65 signaling in bone marrow mesenchymal stem cell senescence
1: Hacettepe University
Mesenchymal stem cells undergo replicative senescence by cultivating in vitro and as the passage extends lose their stem cell properties. Senescence in the culture conditions can be stimulated by exogenous factors such as hydrogen peroxide(H2O2), an agent that induces oxidative stress. Aims of this study were 1)to investigate the changes in PKNOX2 and p65/RelA protein levels of BM‐MSCs aged by serial passaging in culture and oxidative stress 2) to confirm the interaction of PKNOX2 and NF‐κB/p65(RelA), the interaction which was obtained in PKNOX2 proteomic analysis in our previous study, and 3) to examine interaction changes during the aging process if there is any.
Replicative senescence and oxidative stress were induced by serial passaging and H2O2 respectively. Replicative senescence was assesed by SA‐β‐Gal staining and p16, p21 protein levels obtained by western blot analysis. Genomic stress was assessed by γ‐H2AX levels obtained by immunofluorescence imaging. Changes in PKNOX2, NF‐κB/p65 were demonstrated by western blot analysis. PKNOX2‐NF‐κB/p65 interactions was obtained in vitro using co‐IP followed by western blot and in situ using proximity ligation assay (PLA) technique.
Analysis of western blotting showed that PKNOX2 protein levels tended to decrease with replicative senescence and NF‐κB/p65 protein levels overall increased in BM‐MSCs exposed to oxidative stress induced by 200 μM H2O2. PKNOX2 and NF‐κB/p65 interactions were confirmed by both co‐IP and PLA techniques. PLA analyzes showed that interactions of NF‐κB and PKNOX2 were present both in the cytoplasm and nucleus of BM‐MSCs, and the levels of interaction increased at passage‐9 and decreased at passage‐12.
The influence of cytochalasin B‐induced membrane vesicles derived from mesenchymal stem cells overexpressing TRAIL on the expression of apoptotic genes in triple‐negative MDA‐MB‐231 breast cancer cells
T V Pukhalskaia1
1: Kazan Federal University
TNF‐related apoptosis‐inducing ligand (TRAIL) selectively induces apoptosis in tumor cells by activating TRAIL‐mediated apoptosis inducing pathway. Transport of TRAIL in extracellular vesicles is believed to be highly efficient method for TRAIL delivery to the tumor microenvironment and apoptosis induction.
Membrane vesicles were isolated from native mesenchymal stem cells (MSCs) and MSCs overexpressing TRAIL using Cytochalasin B (CIMVs), a drug that disrupts the cell cytoskeleton. In order to assess the effect of isolated CIMVs on the induction of apoptosis in triple negative MDA‐MB‐231 breast cancer cells, CIMVs (50 μg) were added to 5 × 104MDA‐MB‐231 and cells were cultured for 24 h and then the expression of apoptotic genes CAS3, Bcl2, BAX was assessed using qPCR.
It was shown that after 24 h of cultivation, the expression of CAS3 gene mRNA in the CIMVs‐TRAIL sample was increased by 2.1 times compared to native MDA‐MB‐231. The expression of the Bcl2 gene, which is responsible for suppressing apoptosis, was 2.7‐fold increased in the CIMVs‐TRAIL sample compared to the native MDA‐MB‐231. The mRNA expression of the pro‐apoptotic BAX gene was also increased by 3.1 times, which indicates the activation of the apoptotic pathway and cell death stimulation.
The results obtained indicate the activation of the pathway responsible for the induction of apoptosis in MDA‐MB‐231 cells when co‐cultured with CIMVs‐TRAIL. Thus, the use of CIMVs‐TRAIL can be effective in the therapy of breast cancer.
Fully defined iPSC‐macrophages to assess pyrogens in parenteral drugs and medical products
1: Clinic for Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, 30625Hannover, Germany 2: REBIRTH Research Center for Translational and Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany 3: Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany 4: German Centre for Lung Research (DZL), Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), 30625 Hannover, Germany 5: RESIST, Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany 6: Microbiological Safety, Paul‐Ehrlich‐Institute, Federal Institute for Vaccines and Biomedicines, Langen, Germany
The field of “Advanced Therapy Medicinal Products (ATMPs)”, including cell and gene therapy products is constantly growing. While the isolation of cells is challenging, proper measures to verify product safety and the absence of pyrogens have to be developed prior infusion of cell‐products into the patient. According to the European Pharmacopoeia, the Monocyte‐Activation‐Test (MAT) is one option to evaluate medical products for a potential contamination with pyrogens. Given the current limitations of the MAT with respect to the use of primary peripheral blood mono‐nuclear cells (PBMCs) or the use of monocytic cell lines, we here demonstrate the generation of monocyte/macrophages from human induced pluripotent stem cells (iMac), which are fully defined and can be produced in reproducible quality and quantity from the same donor. Using a scalable differentiation platform, iMac showed typical macrophage like morphology and stained positive for CD‐14, CD‐163, CD‐45, CD‐11b, and for several toll like receptor (TLRs) such as TLR‐2, TLR‐5, TLR‐4. Furthermore, our results indicate that iMacs were sensitive to both endotoxin and non‐endotoxin‐based pyrogens at a high dynamic range, when using these cells in an MAT format. In addition, produced iMacs were also more reactive to pyrogens. Stimulation with e.g. MALP‐2(1μg/ml) resulted in secretion of >1000 pg/ml IL6 in iMacs, which was 5‐10 fold less secreted by macrophages from PBMCs or the MonoMac6 cell line, respectively. Of note, iMac showed increased sensitivity to a broad range of pyrogens as well, highlighting the suitability of iMac within the MAT to evaluate parenteral drugs and ATMPs in the near future.
IFNg‐mediated immunomodulation on Mesenchymal Stromal Cells is driven by genetic and epigenetic mechanisms
D Kizhakayil1 M Elanbari1 S M Herrera1 G Gentilcore1 C Lachica1 E E Elgaali1 A Makki2 I Ahmed1 J C Grivel1
1: Sidra Medicine, Doha, Qatar 2: Plastic Surgicenter, Doha, Qatar
Mesenchymal Stromal Cells (MSCs) immunosuppressive properties can be enhanced by priming with interferon (IFN)g.
In this study we analyzed the effect of IFNg priming on 3 clinical grade adipose tissue‐derived MSCs. We report on the priming effect on gene expression, surfaceome and miRNA expression (Nanostring panel for 800 human miRNAs).
The analysis of the expression of 361 surface proteins revealed that IFNg significantly induced or upregulated the expression of 18 proteins. The strongest markers were implicated in immune modulation/activation (CD38, CD40, CD317), intercellular adhesion and migration (ICAM‐1, integrina3) and immunological checkpoints (CD274, HVEM).
At the gene expression level, immunomodulatory markers were significantly upregulated, with IDO being the most increased, followed by CXCL9, CXCL10, RANTES, TLR3, TLR4 and HGF.
Out of 138 expressed miRNAs, 12 showed more than a 2‐fold change between primed and non‐primed MSCs: 11 were upregulated (miR‐199b‐5p; miR‐221‐3p; miR‐136‐5p; miR‐218‐5p, miR‐93‐5p; miR‐337‐3p; miR‐148a‐3p; miR‐145‐5p; miR‐337‐5p; miR‐450b‐5p; miR‐16‐p) and 1 downregulated (miR‐221‐5p).
Both IPA and miRNA enrichment (MIENTURNET) analyses were conducted, to build enrichment and miRNA‐target interaction networks, which identified for miR‐148a‐3p and miR‐145‐5p two nodes converging on SKP1 (ubiquitination of proteins involved in cell cycle progression, signal transduction and transcription) and AGFG1 (vesicle docking or fusion, RNA trafficking or localization).
Among these miRNAs, some have been previously demonstrated to target both the expression of the surface markers and immunomodulatory genes respectively identified by Biolegend assay and gene expression analysis.
Knowing mechanisms and pathways underlying the development of “enhanced” MSCs might support the manufacturing of more homogenous and potent products for clinical applications.
Stem cell‐derived microvesicles transfer surface receptors to epithelial cells
1: Kazan Federal University
Microvesicles (MVs) are mediators of intercellular communication. It was demonstrated that stem cell‐derived MVs deliver genetic information and biologically active factors leading to reprogramming of target cells. The role of stem cell‐derived MVs in reprogramming of epithelial cells and induction of epithelial‐mesenchymal transition is not fully understood. The aim of our study was to reveal the role of MVs in transfer of mesenchymal stem cells (MSCs) surface receptors to epithelial cells HEK293FT cells.
MSCs were treated with cytochalasin B and subjected to vortexing to induce MVs pinching‐off. Target cells HEK293FT were pre‐stained with membrane dye DiO and incubated 24 hours with DiD labeled MVs (10μг/ml). Expression of CD90 is specific for MSCs and absent on the surface of HEK293FT cells. Transfer of CD90 surface receptor from donor MSCs to HEK293FT was detected using monoclonal antibody, flow cytometry and confocal microscopy.
We found fusion of MSC‐derived MVs with HEK293FT cells and transfer CD90 surface receptor to HEK293FT cells. Regions of membrane with double staining DiD and CD90 were found on the surface of target HEK293FT cells treated with MSC‐derived MVs. HEK293FT cells treated with MVs acquired a CD90‐positive phenotype due to internalization of MVs membrane into the cytoplasmic membrane of recipient cells. We observed that 99.14% of the target HEK293FT cells acquired the CD90‐positive phenotype.
Acquisition of the stem cell‐like immunophenotype by differentiated target cells might lead to their reprogramming.
M2‐macrophage‐induced chronic inflammation promotes mesenchymal stromal cell senescence.
1: Faculty of Medicine, Lomonosov Moscow State University, Moscow, Russia 2: Institute for Regenerative Medicine, Lomonosov Moscow State University, Moscow, Russia
Mesenchymal stromal cells (MSCs) regulate tissue homeostasis and repair after damage stimulating regeneration and preventing fibrosis, mostly due to the production of specific secreted factors and noncoding RNAs within extracellular vesicles. However, MSCs exposed to the cytokines released during age‐associated chronic inflammation, particularly, by macrophages, may become senescent and how it affects their regenerative properties remains poorly understood. We studied the role of alternatively activated macrophages (M2) in human adipose tissue derived MSC senescence. Conditioned medium from M2 (M2‐CM), derived from human peripheral blood monocytes, increased the number of p21+ MSCs and stimulated the secretion of key components of senescence associated secretory phenotype (SASP), IL‐6 and MCP‐1, stronger, than reference prosenescent agent hydrogen peroxide. Extracellular vesicles produced by MSCs exposed to M2‐CM were unable to promote the antifibrotic effects in cell models of myofibroblasts differentiation compared to the control. Thus, M2‐induced MSC senescence can affect their regulatory functions. The study was supported by RFBR (#19‐29‐04172).
Osteoconductive properties of nanomodified scaffolds
1: Kazan Federal University
To maintain high titers of stem cells in the area of bone tissue defect, it is promising to use three‐dimensional biopolymer matrices as carriers. Composition and mechanical properties of scaffolds can be controlled using various types of polymers, dopants, including nanomaterials, as well as methods of matrix formation. Currently, various types of scaffolds suitable for transplantation have been developed using artificial and natural polymers. Addition of nanomaterials to the polymer composition represents a promising approach to matrix formation. Nanocomposites while retaining all the advantages of polymeric materialsand increase their biocompatibility, functionality and, thus, to expand their field of application. We used biopolymer scaffold doped with mineral nanotubes loaded with forscolin as osteoconductive material. Using von Kossa staining we have demonstrated the possibility of MSCs grown on 3D polymeric scaffolds to transform into osteoblasts without specific media. In this system nanotubes were used as nanocontainers for the slow release of forskolin, which is a known as stimulator of bone morphogenic protein‐2 formation by activating cAMP synthesis. The introduction of halloysite nanotubes containing forskolin into a biopolymer hydrogel significantly affects the process of osteodifferentiation of MSCs. Cells cultured on scaffolds with forskolin nanotubes exhibited slight enchancement in stiffness. Higher rigidity of transformed cells observed due to nodules formed by calcium salts in the intercellular space, which were detected using von Kossa staining and confirms the process of osteodifferentiation under the influence of nanomodification of a biopolymer hydrogel with forskolin‐loaded nanotubes. This work was funded by Russian Foundation of Basic Research (grant 20‐015‐00353A).
Targeting the blood‐brain barrier to increase haematopoietic stem cell engraftment for neurological lysosomal storage disease gene and cell therapy
1: UCL Institute of Child Health, London, UK 2: Orchard Therapeutics Ltd., London, UK 3: NIHR Great Ormond Street Hospital Biomedical Research Centre, London, UK
Lysosomal storage disorders (LSDs) are monogenic, multisystem diseases caused by deficient lysosomal enzyme function which leads to accumulation of toxic macromolecules. Many LSDs have significant central nervous system (CNS) involvement which begins at an early age and rapidly progresses. Transplantation of haematopoietic stem and progenitor cells (HSCs) partially attenuates neurological symptoms in LSD patients. The key effector mechanism is believed to be migration of a HSC subpopulation into the CNS and engraftment as microglia, which can supply surrounding defective cells with functional lysosomal enzyme and thereby partially restore lysosomal function. Delivering a greater number of HSCs across the blood‐brain barrier (BBB), which controls substance exchange between the systemic circulation and the CNS, may improve therapeutic effect in the brain. By modulating the BBB, the subpopulation of cells with migratory capacity and indeed their contribution to resident cell populations can be investigated, with a view to identifying method(s) to increase CNS engraftment and perhaps therapeutic benefit for LSD patients. In vitro transwell migration assays with a monolayer of brain endothelial cell line bEnd.3 have provided insight into the migratory behaviour of murine lineage negative (lin‐) HSCs and monocytes. An in vivo murine model of HSC transplantation which utilises the chemotherapeutic pre‐conditioning agent busulfan prior to lin‐ HSC injection demonstrated high levels of contribution to tissue‐resident macrophage populations in a range of somatic tissues, and much lower contribution in the CNS. These findings provide deeper insight into current approaches, and support further investigation of BBB disruption for CNS‐targeted LSD treatment options.
Characterization of hiPSC‐CMs derived from pediatric cancer patients treated with anthracyclines that suffered cardiotoxicity
1: Instituto de Investigacion sanitaria La Fe 2: IDIBELL‐ICO
The improvement of antineoplastic treatments and early diagnosis have significantly increased the lifespan of cancer patients. However, this has led to an increment of cancer survivors facing the adverse effects caused by cancer therapies. Doxorubicin is an anthracycline effective against several types of cancer, but its use is limited due to the cardiotoxicity that patients develop after their treatment. This is especially relevant in the case of pediatric cancer patients treated with anthracyclines. Actually, it is not possible to predict which patients will suffer doxorubicin‐induced cardiotoxicity. To study the susceptibility of developing cardiotoxicity, we have obtained human induced pluripotent stem cells (hiPSCs) derived from three pediatric cancer patients that underwent cardiotoxicity due to their doxorubicin treatment. In this work, we have set up a differentiation protocol to obtain human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) from each cell line. We have characterized the hiPSC‐CMs against doxorubicin‐induced damage, comparing them with hiPSC‐CMs derived from children that do not developed doxorubicin‐induced cardiotoxicity (control). Our results indicate that hiPSC‐CMs from patients with cardiotoxicity were more sensitive to doxorubicin treatment comparing with control cells in terms of cell viability and apoptosis.
Immunomodulatory capacity of equine mesenchymal stem cells (MSCs) is influenced by inflammation, differentiation and compatibility for the major histocompatibility complex (MHC)
A Cequier1 F J Vázquez1 2 S Fuente1 2 A Vitoria1 2 A Romero1 2 C Rodellar1
1: Laboratorio de Genética Bioquímica LAGENBIO (Universidad de Zaragoza), Instituto Agroalimentario de Aragón– IA2 ‐ (Universidad de Zaragoza‐CITA); Instituto de Investigación Sanitaria de Aragón (IIS), Zaragoza, 50013, Spain 2: Servicio de Cirugía y Medicina Equina, Hospital Veterinario, Universidad de Zaragoza, Zaragoza, 50013, Spain
Immunomodulatory properties of mesenchymal stem cells (MSCs) influence their therapeutic efficacy and safety. The horse is a valuable model to developing and translating treatments to humans, but equine MSC immune properties need further investigation. This study aimed at analysing the changes elicited by equine MSCs on subpopulations' distribution and proliferation of lymphocytes under different conditions: inflammation, differentiation, and compatibility for the major histocompatibility complex (MHC). Equine MSCs in basal conditions (MSC‐B), pro‐inflammatory primed (MSC‐P) or chondrogeneically differentiated (MSC‐D) were co‐cultured with either autologous or allogeneic MHC‐matched/mismatched lymphocytes activated with phytohemagglutinin and stained with CFSE. After 3 days, lymphocytes were collected and stained with antibodies for flow cytometry analysis. Overall, autologous and allogeneic MHC‐matched MSCs further reduced the proliferation of different lymphocyte subsets. The total percentage of CD3+ T cells did not change but CD8+ cytotoxic T cells were increased and CD4+ helper T cells were reduced. CD3+ proliferation was suppressed in all co‐cultures, especially by MSC‐P. However, MSC‐B did not decrease CD8+ proliferation and showed less suppressive potential for CD4+ proliferation. CD4+/CD25high Treg percentage was increased by MSC‐B and MSC‐P, but all co‐cultures reduced Treg proliferation. Percentage of B cells (CD3‐/Pan‐Ig+/CD21+) tended to increase upon MSC‐B and MSC‐P exposure, but their proliferation was suppressed in all co‐cultures and particularly by MSC‐P. In conclusion, MSC regulatory abilities are maintained after differentiation and are activated by inflammation, whereas MHC‐mismatching can reduce this suppressive capacity. This study shows that equine MSC immunomodulation is influenced by different conditions and differentially act on lymphocyte subpopulations.
Effects of chlorpyrifos on stem cell biology: cell death and phenotypic specification
1: Instituto de Salud Carlos III 2: Universidad de Alcalá
Chlorpyrifos (CPF) is one of the most widely used organophosphate pesticide in agriculture. Inhibition of acetylcholinesterase is the best described mechanism for CPF neurotoxicity.
Chemical exposure during developmental stages can interfere with the proper development of the nervous system resulting in functional alterations or diseases during the lifespan of the individual and thus, resulting in developmental neurotoxicity (DNT). Despite the large body of results on animals, these studies are costly, time consuming. It remains clear that there is a growing necessity for developing alternative methodologies that can better identify and assess chemical substances with the potential to induce neurotoxicity during brain development and maturation.
Human stem cells are currently being a model that promises to be very useful in evaluating this type of toxicity and may be a valuable tool for DNT. In this study, the cell line hNS1 was used to evaluate the effects of CPF. hNS1 cells were exposed to different concentrations of the pesticide and cell death, proliferation and cell fate specification were analyzed under differentiation conditions. The results showed that this compound induces apoptotic cell death at the highest doses tested and promoted the differentiation of hNS1 cells into glial cells by increasing the pool of proliferating glia progenitors.
In addition, we used brain organoids derived from human induced pluripotent stem cells (hiPSCs) to see if CPF has the same effects as in hNS1. We found that CPF induced cell death at the high dose tested and a decrease in the number of neurons and glial cells.
Evaluation of induced membrane vesicles effect on tumor spheroids
1: Kazan Federal University
Extracellular vesicles (EVs) play an important role in the tumor microenvironment and communication between cells. Therefore, the study of the vesicle effect on tumor cells is relevant. The aim of this study is to establish the effect of cytochalasin B‐induced membrane vesicles (CIMVs) on tumor spheroids in vitro.
Colon cancer cell line (HCT‐15) was used to create tumor spheroids. Fourth passage of adipose derived MSCs were used to isolate CIMVs (MSC CIMVs). MSC CIMVs were isolated using 10 μg/ml of cytochalasine B, series of sequential centrifugations and filtration. MSC CIMVs to tumor spheroids were added at concentrations of 1 μg, 2 μg and 5 μg. Transmission electron microscopy was used to confirm the MSC CIMV production. The effect of MSC CIMVs was analyzed using annexin V assay and real‐time PCR.
MSC CIMV fraction consisting of rounded cellular components surrounded by a membrane was obtained. The addition of 5 μg MSC CIMVs decreased cell viability on the fourth day of spheroid culture. The level of mRNA expression of Sox2 and Nanog was significantly increased after the addition of 1 μg and 2 μg of MSC CIMVs to tumor spheroids. Oct4 expression was decreased compared to control. After the addition of 5 μg of MSC CIMVs the expression of Sox2, Nanog, Oct4 did not change or was reduced relative to the untreated spheroids. Thus, these data may indicate the effect of vesicles on the stemness‐related genes and require further study.
Development of cell‐mediated gene therapy for Tay‐Sachs disease
1: Kazan Federal University
The Tay‐Sachs disease is a lysosomal storage disease which is caused by various mutations in the HEXA gene, which leads to the HEXA enzyme deficiency. HEXA impairment leads to the accumulation of GM2 in neurons, which leads to severe neurodegeneration. To date, there is no treatment for the disease.
AAV‐HEXA and AAV‐HEXB, consisted of unique codon‐optimized HEXA and HEXB gene sequences, respectively, were used for co‐transduction of mesenchymal stem cells (MSCs). Wistar rats were intravenously administered with 4 х 106 MSC‐HEXA‐HEXB (n = 5), the control groups were administered intravenously native MSCs (n = 5) or PBS (n = 5). Before the injection and every 7 days, blood was collected to analyze the enzymatic activity of HexA.
In the conditioned medium from MSCs‐HEXA‐HEXB, HEXA activity was 5 times higher compared to control samples. WB showed the presence of bands corresponding to the expected molecular weights of the α‐ and β‐subunit of the HexA enzyme in the MSCs‐HEXA‐HEXB. It was shown that 21 days after the injection of MSCs‐HEXA‐HEXB, the activity of HexA was 1.5‐fold significantly increased in the rat plasma. The injection of MSCs‐HEXA‐HEXB did not affect the percentage of alive cells in immune organs, a ratio of various types of leukocytes and the level of cytokines as compared with control groups.
Thus, we have shown that the MSCs‐HEXA‐HEXB express the functionally active HexA enzyme, detectable in vitro and in vivo, its intravenous injection does not cause an immune response in animals. These data allow assuming that this gene‐cell grug is functional and safe.
The cellular immune response to allogeneic equine mesenchymal stem cells (MSCs) induces changes in their immunomodulatory and immunogenic profiles
1: Laboratorio de Genética Bioquímica LAGENBIO (Universidad de Zaragoza), Instituto Agroalimentario de Aragón– IA2 ‐ (Universidad de Zaragoza‐CITA); Instituto de Investigación Sanitaria de Aragón (IIS), Zaragoza, 50013, Spain 2: Servicio de Cirugía y Medicina Equina, Hospital Veterinario, Universidad de Zaragoza, Zaragoza, 50013, Spain
Therapeutic potential of mesenchymal stem cells (MSCs) is mainly based on their immunomodulatory properties and the horse is one of the most suitable animal model for its development. Allogeneic application presents several advantages, but recipient's immune response can be influenced by donor's major histocompatibility complex (MHC)‐matching/mismatching and MHC‐expression level, which can be modified by inflammation. The aim of this study was to analyse the gene expression of molecules related to MSC immunoregulatory and immunogenic profiles in equine MSC‐naïve and pro‐inflammatory primed (MSC‐primed), after their co‐culture in vitro with lymphocytes from either autologous or allogeneic MHC‐matched/mismatched animals in a one‐way modified mixed lymphocyte reaction (MLR) during 5 days. MSC gene expression was assessed by RT‐qPCR for immunomodulatory (Vascular Cell Adhesion Molecule‐1, VCAM‐1; Cyclooxygenase‐2, COX‐2; indoleamine‐2‐3‐dioxygenase, IDO; inducible nitric oxide synthase, iNOS; interleukin‐6, IL‐6) and immunogenicity (MHC‐I, MHC‐II, CD40, CD80) genes. Gene expression of both immunomodulatory and immunogenicity‐related genes was higher in MSC‐primed over MSC‐naïve alone. In MSC‐primed, all the co‐culture conditions increased their expression of immunomodulatory genes and downregulated the immunogenic ones. Specifically, IL‐6, VCAM, MHC‐I and MHC‐II were significantly upregulated in MSC‐primed exposed to both MHC‐matched and mismatched allogeneic lymphocytes, compared to the same MLRs with MSC‐naïve. These results suggest that inflammation increases both the immunogenic and immunomodulatory MSC profiles, which are maintained after allogeneic exposure to lymphocytes regardless of MHC‐matching or mismatching. Further studies are needed to clarify how these changes may influence the in vivo immune recognition with clinical implications.
Bilayer fibrin‐coated surgical meshes seeded with menstrual‐blood derived stromal cells show immunomodulatory properties
in vitro
1: Centro de Cirugía de Mínima Invasión Jesús Usón 2: Universidad de Extremadura 3: CIBER‐CV
Hernioplasty is the gold standard treatment for abdominal and groin hernia. Cell therapy is currently the focus of attention to counteract the adverse effects associated to non‐absorbable surgical mesh implantation. Here we propose a bilayer coating for polypropylene (PP) meshes to allow viability and adhesion of human menstrual blood‐derived mesenchymal stromal cells (MenSCs). Moreover, we evaluated the immunomodulatory properties of these fibrin‐coated meshes. Fibrinogen and thrombin from a commercial fibrin sealant were diluted in PBS to obtain 5 mg/ml and 5.5 IU/ml solutions, respectively. High‐density monofilament PP meshes were coated twice with fibrinogen and thrombin solutions and uniaxial tensile tests were performed. MenSCs (n = 3) were seeded on fibrin‐coated meshes and cultured for 24 h for CCK‐8 viability tests and fluorescence microscopy. For immunomodulatory assays, human peripheral blood lymphocytes (PBLs, n = 3) were stained with CFSE, stimulated with a T cell activation/expansion kit, and co‐cultured with fibrin‐coated meshes seeded with MenSCs for 4 days, then CD4+ and CD8+ T cells were analysed by flow cytometry. Mesh mechanical parameters were not changed by the bilayer fibrin coating, which also allowed MenSC adhesion on the meshes, as confirmed by Hoechst 33342 staining. Cell viability on fibrin‐coated meshes was significantly increased, compared to uncoated meshes (p < .001). Finally, CD4+ and CD8+ T cell proliferation was significantly decreased by MenSCs seeded on fibrin‐coated meshes, compared to in vitro stimulated PBLs (p < .0001). Hence, these bilayer fibrin‐coated meshes with MenSCs should be considered to reduce mesh‐related inflammation after hernia surgery.
Therapy free of cells vs human mesenchymal stem cells from umbilical cord strome. Effect of miR21 to treat the inflammation in Osteoarthritis
M Morente‐López1 R Mato‐Basalo1 P Miranda1 J Fafián‐Labora1 C Gil2 M Carrera3
1: Universidade da Coruña 2: Complutense University 3: Institute of Marine Research (IIM)
Mesenchymal stem cells and the extracellular vesicles produced by them have been shown to be a powerful alternative for the treatment of inflammatory processes in different diseases associated with aging. Mesenchymal stem cells from human umbilical cord strome and their derived extracellular vesicles were modified by lentiviral expression to inhibir their miR‐21‐5p. Shotgun proteomic analysis was performed in the extracelular vesicles miR21‐/‐ vs extracelular vesicles control to check which proteins were modified because of inhibition of miR21. A total of 1.861 proteins were identified at least with a unique peptide and we have able to establish the proteomic profile of miR21‐/‐ extracelular vesicles vs no modified extracelular vesicles. 112 proteins were modulated by miR21‐/‐ related to inflammation. On the other way, these modified mesenchymal stem cell and their extracellular vesicles were intraperitoneally injected in an Osteoarthritis animal model by twice. One month after treatment, the animals were eutanized to check SASP and cytokine markers and which therapy was the most affective to reduce inflammation and SASP comparing with animals untreated. Both treatments presented the same efective to reduce SASP and cytokine levels. So, miR21 is modulating SASP, inflammation and extracelular vesicles production which make it a good candidate to generate a new anti‐inflammatory therapy.
Three‐dimensional hepatocellular cultures for modeling diseases and assessing the efficacy of drugs and their hepatotoxicity
1: Kazan Federal University
Despite the high regenerative capabilities of the liver in vivo, the expansion of liver cells and their growth in a two‐dimensional culture are difficult. In this regard, the importance of studies is sharply increasing, where a three‐dimensional organoid, which has genetic stability and a high potential for expansion, can be used as a model of the disease. Organoids are self‐organizing multicellular structures, which replicate the structure and function of organs, and can be used to model the development, maintenance and repair of organs ex vivo. Thus, an organoid can be defined as a three‐dimensional organ‐like structure formed from organ‐specific progenitor cells. However, cells cultured under two‐dimensional conditions can often exhibit different behavior and morphology compared to cells under three‐dimensional conditions. In this work, three‐dimensional models of hepatic organelles and tumoroids of various types have been obtained and will be used to model diseases and study efficacy of nanopreparations for the treatment of diseases. Multicellular spheroids from various types of cells of the native liver and mesenchymal stem cells were obtained. Tumor‐like structures ‐ tumoroids ‐ were obtained on the basis of liver cancer cell lines and other types of cells (fibroblasts and endothelial cells). In addition, directed differentiation of iPSCs will be carried out in the direction of creating normal liver tissue and the formation of organelles together with endothelial cells and mesenchymal stem cells to form the so‐called embryonic niche using matrix and matrix‐free technology. This work was funded by Russian Foundation of Basic Research (grant 20‐015‐00353 A).
Developing of glucose‐consuming adipocytes by interleukin 4 gene delivery
1: NMRC of Cardiology, Moscow 2: Lomonosov Moscow State University
Energy metabolism of adipose tissue is essential for whole body glucose homeostasis. Adipocytes' insulin sensitivity and storage function is strongly regulated by inflammatory status of the tissue and can be altered during latent inflammation, developing in obesity and type 2 diabetes. Using lentiviral gene delivery we produced adipocytes 3T3‐L1 expressing anti‐inflammatory cytokine interleukin 4 (IL‐4), and closely examined their adipogenic differentiation and insulin sensitivity.
3T3‐L1 cells were transduced before or after differentiation to determine the effect of IL‐4 on preadipocytes and mature cells. White adipogenic differentiation was performed with insulin, dexamethasone, isobutylmethylxanthine and rosiglitazone; during beige adipogenic differentiation isoproterenol and triiodothyronine were added. Insulin sensitivity was analyzed by 3H‐2deoxyglucose uptake and immunoblotting. Adipogenic differentiation was determined by OilRedO staining and real time PCR.
Adipocytes expressing IL‐4 exhibited higher basal and insulin‐stimulated glucose consumption, elevated phosphorylation of insulin signaling proteins. IL‐4 production during white and brown adipogenic differentiation increased expression of respiratory chain proteins (NDUFA1, Cox7) and mitochondrial biogenesis factor PGC1a, but had no effect on lipid metabolism (hormone sensitive lipase, perilipin, fatty acid synthase) and lipid accumulation.
This suggests that IL‐4 activates oxidative metabolism through mitochondria potentiation. Furthermore, it increases glucose uptake by adipocytes without an increase of lipid droplets content. These results emphasize that IL‐4 is a mediator of oxidative metabolism in adipocytes and a potential activator of glucose‐consuming adipocytes, beneficial for type 2 diabetes prevention.
This work was supported by RFBR grant #20‐015‐00100.
Successful generation of induced pluripotent stem cells from HEK‐293T cells via sodium butyrate and codon‐optimized mini intronic plasmids encoding reprogramming factors
1: The Department of Gene and Cell Therapy, Akdeniz University, Faculty of Medicine, Antalya, 07058, Turkey
Induced pluripotent stem cell (iPSC) technology has been widely recognized for its potential to contribute greatly to the development of novel gene and cell therapies. iPSCs are developmentally immature cells with self‐renewal capacity and the ability to transform into any type of cell in the body. Various vectors are utilized for iPSC generation, including codon‐optimized mini‐intronic plasmids (CoMiPs) that are advantageous for their efficiency, cost‐effectiveness and safety. iPSC generation protocols are also usually potentiated via small molecules and various modifications in the process for optimum efficiency. We have generated iPSCs from HEK‐293T cells by CoMiPs encoding OSKM factors (Oct4, Sox2, Klf4, c‐Myc) along with a short hairpin RNA against p53, applied together with CoMiPs encoding Lin28 and Nanog. Sodium butyrate was used to support the process. Cells were transfected efficiently via electroporation at 110V carried out as a single pulse or via Lipofectamine 3000. DMEM, E7 and E8 mediums were used throughout the process, with sodium Butyrate (10mM) added at every medium change. Cells were transferred to Vitronectin‐XF coated 6‐well plates at day 7, after which iPSC colonies started to emerge. Pluripotency of the newly formed colonies was tested by Alkaline Phosphatase, Tra‐1‐60, Tra‐1‐81, Oct4, Klf4, c‐Myc, and Nanog stainings and embryoid body formation. With this protocol, we generated iPSCs from HEK‐293T cells via an effective and relatively short‐duration process. This protocol is yet to be tested and we believe is likely to be effective in other cell types, particularly in various primary cells (TUBITAK Grant No: 218S617).
Optimising culture of iPSC‐derived cardiomyocytes for delivery into the heart
1: University College London
Cell therapy is a potential novel treatment for cardiac regeneration and numerous studies have attempted to transplant cells to regenerate the myocardium lost during myocardial infarction. To date, only minimal improvements to cardiac function have been reported in clinical trials. This is likely to occur from low cell retention following delivery and high cell death after transplantation.
The current study aims to improve the delivery and engraftment of viable cells by using an injectable biomaterial which could provide an implantable, biodegradable substrate for attachment and growth of cardiomyocytes derived from induced pluripotent stem cells (iPSC).
Highly porous microspheres were fabricated from 2% (w/v) 75:25 DL‐lactide/glycolide co‐polymer using Thermally Inducted Phase Separation (TIPS). A range of parameters were investigated for optimising conditions for iPSC attachment to TIPS microspheres. This included pre‐conditioning with a protein solution containing vitronectin (VTN‐N) ranging from 0‐20 μg/ml and ‘wetting’ with solutions of HBSS or F10 medium. The microspheres pre‐conditioned with 0.5 μg/ml VTN‐N and HBSS enhanced iPSC attachment and supported proliferation, with cell number doubling within 24 hours. Attachment of iPSC to the microspheres retained target iPSC pluripotent phenotype, as demonstrated by positive expression of pluripotent markers, SOX2, OCT4, TRA‐1‐60 and SSEA4. Pluripotency was further demonstrated by the differentiation of iPSC into a mixed cardiomyocyte‐like population which showed a beating phenotype for up to 40 days in culture.
This work demonstrates that TIPS microcarriers offer a supporting matrix for culturing and differentiating iPSC and may provide an injectable biomaterial for cardiac regeneration.
In vivo and in vitro optimization of protocols for the expansion of genetically‐engineered human hematopoietic stem and progenitor cells
1: Telethon Institute of Gene Therapy (HSR‐TIGET) 2: Universita Vita‐Salute San Raffaele 3: Hematology and Bone Marrow Transplantation Unit, IRCSS Ospedale San Raffaele
Hematopoietic stem cell (HSC) expansion remains an unmet goal for ex vivo gene therapy/editing. However, transferring expansion protocols optimized for cord blood to genetically‐engineered mobilized peripheral blood (mPB) cells is not trivial. We first evaluated 2 HSC agonists, UM171 and SR1, and found that combination of both maintained the highest engraftment capacity of mPB CD34+ cells in NSG mice, with a particular benefit on short‐term (ST) engraftment as compared to UM171 alone. Next, we investigated the inclusion of IL3 and IL6 in the SCF, TPO, and FLT3L‐based cytokine cocktail. IL3 increased total cell number (33.6 vs. 8.2 fold expansion), but decreased the proportion of cells with a primitive immunophenotype, while IL6 increased the proportion of such cells but supported only limited growth. The combination of IL3 and IL6 resulted in a net gain in CD34 + 90 + 45RA‐201+ITGA3+ cells, which translated into higher engraftment, particularly for ST‐HSC. Notably, lentiviral transduction was not entirely neutral, even when using purified vectors, counteracting the expansion of primitive cells. Transduction enhancers (TEs) had variable effects, with some molecules exhibiting strong antagonism on HSC expansion. Optimizing the choice of TEs and the timing of transduction allowed us to neutralize the negative impact of the genetic engineering step. Preliminary results suggest the possibility to implement new base editor technology in our optimized expansion protocol. In conclusion, we present a revisited expansion protocol based on clinically validated compounds and tailored to genetically‐engineered mPB HSC, towards harnessing the full potential of ex vivo HSC expansion in the gene therapy context.
Role of Amyloid Precursor Protein (APP) in the Cell Fate Specification of Human Neural Stem Cells
1: Instituto de Salud Carlos III 2: Universidad de Alcalá
The pathological implication of Amyloid Precursor Protein (APP) in Alzheimer's disease has been widely documented due to its involvement in the generation of amyloid‐β (Aβ) peptide. However, the physiological functions of APP are still poorly understood.
APP is a transmembrane glycoprotein widely expressed in the central nervous system and is encoded by a single gene located on chromosome 21. Due to its role in a wide variety of processes, APP is considered by various authors as a multimodal protein. Specifically, APP seems to be implicated in neural development of CNS, playing a key role in the proliferation, differentiation, cell fate specification and maturation of neural stem cells (NSCs).
We have examined the endogenous APP expression in hNS1 cells, a model cell line of human NSCs, both under proliferation and throughout the differentiation period. Our results show elevated APP‐immunoreactivity in hNS1 cells and, to investigate the potential function that APP plays in biology (proliferation, differentiation, cell fate specification and cell death) of human NSCs, we performed a loss‐of‐function study. To achieve the down‐expression of APP, we used a commercial siRNA against human APP and we transfected it into hNS1 cells. Our data indicate that low levels of APP induce hNS1 cell fate towards a neuronal phenotype, while decreasing glial differentiation. Moreover, according our results, these effects could be, in part, mediated by β‐catenin protein.
The knowledge of physiological functions of APP, as well as the possible signaling pathways that could be implicated, are essential to advance the understanding of the pathogenesis of AD.
GUCY2D retinal organoid disease model for AAV gene therapy development
1: Meira GTx UK II, 34‐38 Provost Street, London N1 7NG 2: NIHR Biomedical Research Centre at Moorfields Eye Hospital, London
To evaluate potential AAV gene replacement therapies for GUCY2D mutations, CRISPR/Cas9 gene editing was used to generate human GUCY2D Knockout (GUCY2D KO) and isogenic human control retinal organoids. These human retinal organoids were assessed for the expression of GUCY2D by immunostaining, Western blotting, and qPCR. Total cGMP levels following light stimulation and recovery in the dark were assessed using a FRET‐based assay. In addition to a loss of GUCY2D protein in outer segments, GUCY2D KO organoids showed reduced cGMP levels and decreased expression of the phototransduction protein PDE6β.
Four AAV vectors were designed to enable restoration of GUCY2D‐mediated signalling in photoreceptor outer segments via gene replacement. Both CMV and Rhodopsin kinase (RK) promoters were tested, and the presence or absence of Woodchuck Hepatitis virus post translational regulatory element (WPRE) was assessed. All vectors were packaged into 7m8 capsids.
Reverse of mesenchymal stromal cell senescence by senolytics in vitro
1: Faculty of Medicine, Lomonosov Moscow State University, Moscow, Russia 2: Medical Research and Education Center, Lomonosov Moscow State University, Moscow, Russia 3: Faculty of Biology, Lomonosov Moscow State University, Moscow, Russia
Multipotent mesenchymal stromal cells (MSCs) are considered as critical regulators of tissue homeostasis and regeneration after damage. However, MSCs are liable to aging and regenerative properties of senescent MSCs could be significantly declined. Targeted elimination of senescent cells may be achieved using senolytics (e.g. quercetin, fisetin, curcumin, etc.), but if they affect senescent MSCs remain unclear. We investigated in vitro effects of senolytics on adipose tissue‐derived MSCs from young (age <40, nonSEN_control) and aged (age >65, SEN_control) patients by the addition of quercetin (SEN_Q), taxifolin (SEN_Tax) or experimental water‐soluble dihydroquercetin (SEN_QH) in adjusted concentrations. Senolytics stimulated apoptosis of senescent MSCs measured by ethidium homodimer real‐time staining using BioSpa Live Cell Analysis System, but didn't affect viability or proliferation as well as b‐galactosidase activity in cells from all groups. However, only in SEN_Tax and SEN_QH groups number of p21‐positive MSCs significantly decreased compared to SEN_control with moderate advantage in SEN_QH. Thus, senolytics could affect senescent cells within MSCs, and water‐soluble dihydroquercetin targets senescent MSCs more effectively. Further research is required to confirm these effects in vivo. The study was performed under the State Assignment of Lomonosov Moscow State University.
The proliferative activity and cytokine secretion changes in mesenchymal stem cell and neuroblastoma cells during co‐culture under hypoxic conditions
1: Kazan Federal University
The hypoxic condition one of the key components that affect the cellular interactions inside tumor tissue and associate with aggressive cancer development. We investigated the effect of hypoxia (3% of oxygen) on mesenchymal stem cell (MSCs) derived from human bone marrow and human neuroblastoma cells (SH‐SY5Y) during co‐culture, the effect of cisplatin (CDDP) on cell proliferation in hypoxia and normoxia, and the changes in cytokine profile in co‐culture. To create the co‐culture, cells were mixed at 1:1 ratio, after 48 hours of co‐cultivation, the CDDP was added. Hypoxic conditions were created using hypoxia (anaerobic) station Bactrox. The proliferative activity of co‐cultures was significantly lower after CDDP‐treatment (10 μg/mL), in particular, cells cultured under normoxic conditions reduced proliferation by 80%, and cells cultured under hypoxic conditions by 68%. Despite a significant decrease in proliferation in both groups, the decrease in cell proliferation in normoxia was more pronounced (80% vs 68%). Also, after 72 hours of cultivation the conditioned media was collected and analysed using multiplex cytokine, the concentration of CXCL5 showed 2‐fold increase after CDDP treatment during hypoxic conditions (133,9 ng/mL vs 276 ng/mL), the same results were observed in secretion of IL6 (362,5 ng/mL vs 630,8 ng/mL), and 3‐fold increase was observed in concetration of IL8 (86,9 ng/mL vs 286,74 ng/mL) in comparison with co‐cultures after CDDP treatment in normoxia. Was shown that hypoxic conditions reduce the sensitivity of cells to anticancer drug and contribute to increase in the secretion of pro‐inflammatory cytokines in co‐culture containing tumor and stem cells.
Effect of short‐term Hypoxic Culture Conditions on Menstrual Blood Stromal cells
1: Jesús Usón Minimally Invasive Surgery Centre, Cáceres, 10071, Spain 2: CIBER de Enfermedades Cardiovasculares, Madrid, 28029, Spain 3: Immunology Unit, University of Extremadura, Cáceres, 10003, Spain
Menstrual blood‐derived stromal cells (MenSCs) are a promising candidate in the field of cell‐based therapies due to their biological activities. Here we hypothesize that hypoxic preconditioning may modify the functional characteristics of MenSCs cultured under standard conditions (20.9% O2). Hence, the aim of this study was to evaluate the characteristics of MenSCs cultured under two different hypoxia ranges: physioxia (1 – 2% O2) and acute hypoxia (0.1 – 1% O2) at 24, 48, and 72 h (short‐term culture). For this purpose, cell proliferation and cell viability were measured by CCK8 and live/dead assays, respectively; and the release of two immunomodulatory molecules (IDO‐1 and TGF‐β) was determined in the supernatants by ELISA. Moreover, a phenotypic characterization was performed by flow cytometry in 30 surface markers at 72h. Our results demonstrated that an increase in proliferation rate was observed under physioxia exposure, while MenSCs cultured under acute hypoxia proliferated slower than cells in normoxia. No changes in viability were detected. The highest release of IDO1 and TGF‐b was observed at 48h under acute hypoxia condition. In addition, cell phenotypic characterization revealed a significant increase of CD117 and CD279 in physioxia, while CD29, CD44, CD49a, CD49e, and CD105 were significantly decreased in acute hypoxia. Our results demonstrate that acute hypoxia during 48h seems to be an optimal condition to evaluate the therapeutic characteristics of preconditioned MenSCs. In conclusion, oxygen concentration and timing of culture modulate the phenotypic profile of MenSCs.
Prediction of a surface marker for cardiac progenitor cells derived from human pluripotent stem cells Prediction of a surface marker for cardiac progenitor cells derived from human pluripotent stem cells
1: 1‐ Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, 1665659911, Iran.
itritriCardiac progenitor cells (CPCs) have a great potential for cardiovascular regenerative medicine. While they retain proliferative capacity, CPCs can be differentiated into all three cardiovascular lineages i.e. cardiomyocytes, endothelial cells and smooth muscle cells. Despite great potential, their clinical application was hindered by lack of a specific surface marker. Furthermore, in vitro studies were influenced by the absence of an approved highly specific marker/s for CPCs. The aim of the current study was to identify a surface marker for CPCs' enrichment using in situ analysis. Microarray data of in vitro differentiated human MESP1+ cells were analyzed using GEO2R tool and differentially expressed genes (DEGs) were identified in comparison to Flk1+/Pdgfra+ cells. Key genes were extracted based on p‐value <0.05 and log2‐fold change ≥1. Finally, gene ontology analysis of DEGs spotted ADGRL2 (Adhesion G Protein‐Coupled Receptor L2) as a surface marker of CPCs. To verify the in situ predicted marker, we checked the expression pattern of ADGRL2 gene during in vitro generation of CPCs derived from human pluripotent stem cells. ADGRL2 expression was doubled at day two of differentiation and substantially increased at day 3, reaching the 2.5‐fold expression at day four of differentiation. Cells at day 4 showed the molecular signature of CPCs when checked for expression of other CPCs markers. In summary, ADGRL2 can be used as a surface marker to obtain a homogenous CPCs population for research and clinical application.
Keyboard: CPCs, Cell surface marker, ADGRL2, Bioinformatics analyses
Differentially expressed miRNAs in human Wharton's Jelly mesenchymal stromal cells‐derived extracellular vesicles: revealing potential mechanisms of action in immune modulation
1: Curexsys GmbH, Göttingen, 37079, Germany 2: Advanced Therapies Unit, Instituto Distrital de Ciencia Biotecnología e Innovación en Salud, Bogotá, 111221, Colombia 3: Faculty of Medicine, University EAN, Medellín, Colombia 4: PhD Programme in Biomedical and Biological Sciences, School of Medicine and Health Sciences, Universidad del Rosario, Bogotá, 111221, Colombia
Extracellular vesicles (EV) have been identified as mediators of mesenchymal stromal cell (MSC) immune suppressive and pro‐regenerative activities in several disease models. To shed light on the potential of EV, further research focused on its content remains to be clarified. The scope of this study was to characterize exosomes (Exo) and microvesicles (MV) secreted by Wharton's Jelly (WJ) MSC in terms of membrane markers, immune suppression and miRNA profile. For EV production, supernatants (SN) were collected from cultures and characterized by flow cytometry and Nanoparticle Tracking Analysis (NTA). Both nanoparticles displayed the EV tetraspanins CD81, CD9 and CD63 and exhibited concentrations of 2.4 ± 0.4x106 (MV) and 29.1 ± 5.9x106 (Exo) particles/mL. Furthermore, in vitro immune assays confirmed the suppression of CD3 proliferation (29 ± 2% and 25 ± 2%). Finally, expression profile of small RNA was evaluated in isolated vesicular RNA by RNAseq. EV‐contained miRNA profile revealed 167 and 65 IsomiR differentially enriched in vesicular as compared to cellular miRNA. Importantly, there were no striking differences between miRNA content in both nanoparticles. We identified five IsomiR mostly enriched in MSC‐EV: miR‐146a‐5p, miR‐409‐3p, miR‐423‐3p, miR‐423‐5p and miR‐92b‐3p. A dual‐fluorescence reporter system confirmed the biological activity of these IsomiRs. Accordingly, enriched MSC‐EV IsomiRs were found to be related to immunological processes. In summary, WJ‐MSC‐derived Exo and MV were successfully isolated and characterized, pointing to a potential immune suppressive effect in vitro. Remarkably, we identified a set of 5 differentially expressed microRNAs and confirmed their biological activity. Subsequent functional studies will reveal mechanisms of action in immunomodulation and inflammation processes.
Isolation of extracellular vesicles derived from mesenchymal stem cells by ultracentrifugation and tangential flow filtration followed by size‐exclusion chromatography: a functional comparison
1: Grupo de Regeneración y Trasplante Cardíaco, Instituto de Investigación Sanitaria La Fe, Valencia, 46026, España
Mesenchymal stem cell (MSC)‐derived extracellular vesicles (EVs) have been shown to recapitulate the therapeutic effects of the MSCs in different in vitro and in vivo studies. One of the limitations of EV based therapies is the number of vesicles needed to perform the treatments. This problem is further exacerbated when investigating their effect in relevant preclinical models or in humans. Currently, one of the most widely used methods to obtain EVs is serial ultracentrifugation, however, the amount of EVs obtained with this technique is limited. In order to optimize the EVs extraction process, we have developed an isolation method based on tangential flow filtration followed by size‐exclusion chromatography (TFF‐SEC). EVs obtained by this method were characterized by nanoparticle tracking analysis (NTA), Western Blot and electronic microscopy. When this method was compared with ultracentrifugation, we observed that EVs obtained by both techniques were effective in macrophage polarization assays. In both cases, the EVs were able to promote macrophage polarization towards a pro‐resolving or M2 phenotype. In addition, they were also able to increase efferocytosis. In order to estimate the efficiency of the different isolation methods, the useful dose related to the total amount of isolated EVs was calculated. We found that the yield was at least 10‐fold higher when the TFF‐SEC method was used. In conclusion, the TFF‐SEC method is more efficient in terms of purification yield while maintaining therapeutic properties of the EVs extract. These results will make it possible to obtain a sufficient number of particles for clinical purposes.