Abstract
Elevator Pitch & Invited Abstracts
Gene transfer technologies
: VIVEbioTECH
VIVEbioTECH is a company specialising in gene transfer technologies which is focused exclusively in the design and manufacture of lentiviral vectors for pre-clinical and clinical studies. The team has long experience in the development and manufacturing of lentiviruses for in vitro and in vivo research studies. The manufacturing process of lentiviral vectors has been optimized and the final yield increased more than 5 times compared with regular protocols. Facilities, equipment and manufacturing protocols comply with the current GMP standards. VIVEbioTECH is client-oriented and highly flexible to their needs, being a one-stop company from lentiviral design to manufacturing and aseptic filling and finishing, highly competitive in prices and delivery times.
: PSNResearch
Regulation (EC) No 1394/2007 has provided the starting on common European regulatory framework for the marketing of advanced therapy medicinal products (ATMP). The Agency's Committee for Advanced Therapies (CAT) plays a central role in the scientific assessment of ATMP, providing the expertise needed to evaluate and assessing the quality, safety and efficacy of ATMPs, and to follow scientific developments in the field. It also assists scientifically in the elaboration of any documents related to the fulfillment of the objectives of mentioned regulation. Companies and researchers developing ATMPs must ensure that the standards of good practices (GCP, GDP, GMP) are applied when carrying out clinical trial studies. If an ATMP contains genetically modified organisms, companies must follow relevant legislation on the deliberate release of genetically modified organisms into the environment. Hanna et al. 2016 and Maciulaitis et al. 2012 reported that more than 50% of the trials in Europe belong to Phase I-I/II and were sponsored by non-commercial sponsors. It is clear that ATMPs development is further hindered by the fact that researchers or small companies usually lack appropriate regulatory expertise to successfully navigate through the regulatory framework. PSNResearch, a global clinical research services company aware of this need, has a team of professionals with experience in ATMPs development. Therefore, our company is capable of support ATMPs developers within the European regulatory framework such as preliminary authorization for GMO voluntary release, preparation of clinical regulatory documentation, specialized service on Good Distribution Practice, risk based monitoring, and consulting services as needed.
: Orchard Therapeutics, London
Orchard Therapeutics is a clinical-stage Biotech company dedicated to the development and commercialization of transformative gene therapies for patients with serious and life-threatening disorders.
Our pipeline includes ex-vivo autologous lentiviral gene therapies in primary immune deficiencies and inherited metabolic disorders
: Progen
Adeno-associated viral (AAV) vectors are powerful tools in gene therapy research and development. Several recombinant AAV vectors (rAAV) corresponding to the different viral serotypes have successfully been tested in recent years as universal gene shuttle in human cells. The increasing interest in rAAV in clinical applications demands dependable instruments to study the biology of AAV infection and reproducible quantification of accurate rAAV titers to ensure a safe and reliable gene transfer.
In the past 20 years, PROGEN has established a unique portfolio of AAV tools for basic and clinical research as well as for pharmaceutical applications. Among them are specific antibodies to study AAV assembly or the course of an infection. A line of reliable AAV quantification ELISAs for different serotypes utilizing PROGEN's portfolio of capsid-specific AAV antibodies completes PROGEN's AAV based portfolio.
PROGEN offers its broad expertise in antibody & ELISA technologies, protein interaction and purification and welcomes collaborations with academic and industrial institutions to advance basic AAV research. In addition, the company aims to establish partnerships with academic, pharmaceutical or medical institutions for the development of AAV-based tools and therapies, e.g. for standardization and validation steps in clinical trials.
: University College London
This presentation summarizes the clinical considerations for early phase clinical trials with advanced therapies focusing specifically on the European Medicines Agency (EMA) guidance documents and reflection papers referenced below 1,2,3. Following the tragic incidents in the Phase I first-in-human clinical trial in Rennes, France, in January 2016, the EMA initiated a review of the guidelines describing first-in-human clinical trials and the data needed to enable their appropriate design and allow initiation 4. EMA's review focuses on best practices and guidance and takes into account the findings from two in-depth investigations into what went wrong during the trial in Rennes, which resulted in the death of one participant and hospitalized five others. This presentation highlights the key findings from the concept paper which (at the time of writing) is anticipated in July 2016, and which concludes EMA's review and summarizes its proposals to further minimise the risk of similar accidents happening. 1. Guideline on strategies to identify and mitigate risks for first –in-human clinical trials with investigational medicinal products (EMEA/CHMP/SWP/28367/07). 2. Guideline on human cell-based medicinal products (EMEA/CHMP/410869/2006). 3. Reflection paper on stem cell-based medicinal products (EMA/CAT/571134/2009). 4.
: Novasep
Novasep, as a custom development & manufacturing organization (CMO) active in the field of biopharmaceuticals, will present its perspectives on the key steps to ensure starting new projects in the most efficient and successful way. Novasep's experience related to process risk assessment, analytical transfer and process transfer will be discussed.
: Cellular Dynamics International
Over the past 12 years, Cellular Dynamics International (CDI) has established itself as a world leader in providing Pluripotent Stem Cell (PSC)-derived cells for research applications including drug discovery, disease modeling and toxicity testing. CDI has partnered with pharmaceutical companies and innovators to develop 13 cryopreserved cellular products that are produced to high quality specifications for these applications. Many of these were developed through Contract Development and Manufacturing Organization (CDMO) relationships between CDI and global pharmaceutical companies. In addition to producing and testing standard cell types including cardiomyocytes, neurons, hepatocytes, myoblasts, endothelial cells, and hematopoietic progenitors, CDI has produced PSCs from donors with healthy and disease backgrounds providing valuable in vitro models for studying a range of diseases. CDI has leveraged this wealth of experience in developing scalable manufacturing processes and Quality Control test methods for PSC-derived cells to move into Regenerative Medicine applications where we are advancing several cell therapy candidates toward human clinical trials. My presentation will cover examples of several of these projects including CDI's cell development process and lessons learned as we transitioned from research applications into producing and testing cells in compliance with current Good Manufacturing Practice (cGMP) regulations for pre-clinical animal testing and human clinical trials. Projects that will be discussed include a collaboration with the U.S. National Eye Institute to develop an autologous RPE cell therapy for AMD as well as a program to develop Dopaminergic Neurons for treating Parkinson's Disease. CDI plans to continue to expand capabilities as both a supplier of cells for research and therapeutic applications as well as a partner in developing cell therapies by providing comprehensive CDMO services.
: Centre for Immunodeficiency UCL Institute of Child Health/Great Ormond Street NHS Trust
Primary immunodeficiencies have played a major role in the development of gene therapy for monogenic diseases of the bone marrow. The last decade has seen convincing evidence of long term disease correction as a result of ex vivo viral vector mediated gene transfer into autologous haematopoietic stem cells. The success of these early studies has been balanced by the development of vector related insertional mutagenic events. More recently the use of alternative vector designs with self inactivating (SIN) designs which have an improved safety profile has led to the initiation of a wave of new studies which are showing early signs of efficacy. These studies in SCID-X1, ADA-SCID, Wiskott-Aldrich syndrome and Chronic Granulomatous disease are all multi-centre studies using lentiviral vectors and have the potential to recruit patients rapidly and to show efficacy and safety. The ongoing development of safer vector platforms and gene editing technologies together with improvements in cell transduction techniques and optimised conditioning regimes is likely to make gene therapy amenable for a greater number of PIDs. If long term efficacy and safety are shown, gene therapy will become a standard treatment option for specific forms of PID. These technologies may also be important for other monogenic disorders of the haematopoietic system.
: NICE
Over the last two years cell and gene therapies became significantly more noticeable on the NICE's (National Institute for Health and Care Excellence) radar. As more products are reaching maturity in their development phases, market access issues are starting to be increasingly important not just to the industry but also to decision- and policy-makers. Where high unmet medical need exists, patients and the public want effective medicines to become available as quickly as possible without having to compromise on safety. Cash strapped health economies see many financial and clinical risks associated with cell and gene therapies. To enable adoption of these technologies, multiple stakeholders need to come together to develop innovative solutions for evidence generation and evaluation as well as design practical payment methods. The objective is to achieve fair risk sharing mechanisms where the lack of robust evidence results in high levels of uncertainty for decision-makers. NICE is making a number of steps to face and address these challenging problems. This talk will cover NICE's experience to date in the evaluation of cell and gene therapy products, scientific advice given to ATMP (advanced therapy medicinal products) developers from the HTA (health technology assessment) perspective and methods work commissioned by NICE to explore key issues associated with evidence requirements and HTA decision-making for cell and gene therapies.
: IRDIRC
Advanced Therapies are a new type of drugs that could change the way patients are treated. The European and US rules norm them as pahrmaceutical products, but they are in fact a brand new cathegory, being either living cells or parts of them, that require deep understanding and specific approaches for their successful development. Quality and manufacturing standards are unevenly applied across countries and market access is still to be ascertained for these drugs, making them a faulty investment for the time being. Holoclar, the first stem-cell based product ever approved in Europe, has undergone all the hurdles to obtain the Regulatory approval in Europe, but still has to access the European markets. We will review its story and some lessons learned for developing tissue-engineered stem cell-based products.
: GlaxoSmithKline
: University of Oxford
Taking forward new therapies from the laboratory to a clinical trial is often a daunting process that can limit the rate of progress in any medical field. For gene and cell therapy, the problem is acutely felt with the particularly high cost of performing first in human studies.
Commercial investment can, in principle, bring the level of capital necessary to initiate a clinical development programme but just like sizeable grant funding it can be difficult to attract and secure.
Typically, the demonstration of encouraging pre-clinical data alone is not sufficient to attract commercial investment, because the perception of risks associated with the clinical development process, rather than the science itself, can be too high.
A new biotech company should therefore concentrate on eliminating or managing risks such as product manufacture or patient recruitment rates for example in order to expose investors only to the question of clinical efficacy. With these risks covered, investors can keen to support new technology and help move through the clinical trial process in a timely and efficient manner.
In this talk I will describe from my own experience of taking a new technology forward from grant funded research to investor backed trials.
: Istituto Superiore di Sanità
Advanced Therapies represent a significant emerging field in which new treatment opportunities are offered to patients. Due to their complexity, they are regulated under the umbrella of medicine provisions.
Recent success in gene and cell therapy field has resulted into a number of Advanced Therapy Medicinal Products already available on the EU market, generated by a great deal of research at preclinical and clinical level.
Regulatory expectations for safe and efficacious Advanced Therapy Medicinal Products can be met by developers by means of a deep understanding of their product and of careful planning of its development, in continuous and close collaboration with regulatory bodies, taking also advantage of research infrastructures that European Commission has put in place to facilitate an efficient translation of research discoveries into effective ATMP.
This presentation will discuss the scientific and regulatory challenges found when developing Advanced Therapy Medicinal Products and how to overcome them to accelerate translation of a bright research idea into a clinically safe and efficacious Advanced Therapy Medicinal Product.
: Fondazione Telethon - TIGEM
Telethon is an Italian charity funding excellent research on genetic diseases with the goal of advancing biomedical research until therapies for genetic disorders are identified, developed and made accessible to all patients. On May 27th 2016 EMA approved the Marketing Authorization for Strimvelis, the first ex-vivo stem cell gene therapy to treat patients with a very rare disease called ADA-SCID (Severe Combined Immunodeficiency due to Adenosine Deaminase deficiency), developed up to clinical proof of concept (PoC) within the San Raffaele-Telethon Institute for gene therapy (SR-Tiget), an Italian research institute created through a partnership between Telethon and Ospedale San Raffaele. Clinical PoC was not sufficient to make the therapy accessible to patients, hence Telethon together with Ospedale San Raffaele signed in 2010 a strategic partnership with GlaxoSmithKline that allowed the deployment of the economic resources, the technical expertise and the infrastructures required so to complete research development and achieve pharmaceutical production at the industrial level. The key elements of the success have been obtained through a combination of basic research, platform technology and disease pathogenesis study, preclinical and clinical research spanning from natural history trials throughout interventional clinical studies. Strimvelis confirms the translational potential of nonprofit research and should encourage academic researchers to produce data in a format and with such soundness as to be easily acquired by pharma and biotech companies, to ensure patients accessibility to innovative therapies.
: CHDI Foundation inc.
Huntington's disease is a monogenic inherited disorder characterized by widespread degeneration of cortical regions and basal ganglia nuclei. Molecular therapies targeting Huntingtin are in late-stage clinical development or are being tested in patients for safety evaluation. My presentation will briefly describe the main therapeutic strategies being developed to lower the causative protein, and will describe challenges that need to be addressed in terms of biomarker development and in the context of increasing exposure of therapeutics through nucleic acid and viral engineering.
: Institute of Experimental Hematology, Hannover Medical School, Hannover
: Department of Gene Therapy & Regenerative Medicine, Free University of Brussels
The development of next-generation gene therapies requires efficient expression of the therapeutic genes to minimize the doses of the gene therapy vector and limit the risk of inadvertent immune responses. This can be accomplished by using gain-of-function transgene (e.g. coagulation factor IX Padua) that have an increased functional activity over conventional wild-type transgenes, designing efficient tissue-specific promoters or a combination of both strategies. To boost the transcriptional activity of tissues-specific promoter we have developed and validated new genome-wide computational approaches that resulted in the identification of transcriptional cis-regulatory modules (CRM). When used in the context of a gene therapy vector (e.g. AAV, transposon, mini-circle), these tissue-specific CRMs caused a significant increase in gene expression in the desired target tissue (e.g. liver, heart, skeletal muscle). These tissue-specific CRMs offer an unprecedented opportunity to obtain relatively efficient in vivo gene editing (i.e. in hepatocytes) using the CRISPR/Cas9 (S. pyogenes) system without any significant off-target effects.
Funding: FWO, EU, VUB IOF GEAR & GENEFIX (Grower), AFM
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: Paul-Ehrlich-Institut
In gene therapy we distinguish between ex vivo and in vivo gene delivery. The latter is particularly challenging for vector technology, especially when regarding systemic vector administrations and the delivery of crucial genes that should only be expressed in the therapy-relevant cell type. Apart from engineering regulatory sequences on the vector genome, modifying gene vector cell entry is of special interest since this may avoid loss of particles to irrelevant cells. Particle surface engineering resulting in the use of a predefined cell surface marker of choice as receptor for cell entry is regarded as one key issue for vector improvement to reach this ambitious goal. Based on recent progress, different vector types such as lentiviral vectors, AAV vectors and oncolytic viruses can be manipulated in a flexible and rationally based approach to enter into cells expressing the targeted transmembrane protein at their cell surface. Binding to the surface marker is mediated by targeting ligands displayed on the vector particle surface, which can be polypeptides, single-chain antibodies or designed ankyrin repeat proteins. Improvements and challenges in vector engineering as well as potential applications of receptor-targeted vectors will be presented.
: University of Naples Federico II
p63, a p53 family member, is a master regulator of epidermal gene expression program, directly regulating genes involved in cell proliferation, cell adhesion, cellular architecture and terminal differentiation. Heterozygous p63 mutations in Ankyloblepharon- Ectodermal defects Cleft lip/palate (AEC) syndrome cause a severe skin phenotype with extensive long-lasting skin erosions, bleeding and infection that are currently treated with palliative care. Using constitutive and conditional knock-in mice expressing an AEC mutation in the epidermis to model the human disorder, we have dissected signaling pathways downstream of p63. During embryonic development p63 positively controls expression of the Fibroblast Growth Factor Receptor 2 (FGFR2), thereby causing an expansion of progenitor cells that influence the number of stem cells in postnatal life. Reduced FGFR2 signaling in AEC syndrome is the likely cause of slow repair of skin erosions. The balance among different p63 protein isoforms determines the number of stem cells and/or their proliferative potential. In addition, we find that AEC mutations cause focal skin erosions due to reduced resilience to mechanical stress caused by a selective decrease in a subset of basal keratins and reduced desmosome formation. Skin erosions lead to progressive skin and systemic inflammation accompanied by massive induction of thymic stromal lymphopoietin (Tslp), an interleukin-7 like cytokine. Genetic ablation of Tslp in the epidermis significantly reduces, improving general health. Mechanistically, massive Tslp induction is accompanied by NFAT accumulation and by downregulation of Notch signaling. Therefore p63 controls an increasing number of crucial signaling pathways in the epidermis.
: Hannover Medical School
Direct reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is a promising approach for the derivation of disease-specific cells for molecular analyses and the evaluation of new therapeutic strategies. Therefore, we aimed to identify microRNAs, whose modulation improve the efficacy of iPS cell generation. Moreover, we evaluated a cytokine and small molecule based protocol for direct differentiation of human pluripotent stem cells into hepatic cells that could be visualized and selected by a lentiviral albumin-GFP/Neo construct. Functional characterization of these cells allowed the recapitulation of the disease phenotype for further studies of underlying molecular mechanisms of the respective disease. With respect to stem cell-based therapies, we investigated a xenograft-accepting mouse strain, which serves as competitive liver repopulation model. Because our results suggested that the transplant maturity has a major effect on repopulation efficiencies, we screened a set of microRNAs supporting the maturation of stem cell-derived hepatic cells and discovered that inhibition of microRNA199a-5p during the differentiation process of pluripotent stem cells eventually yielded in transplantable cells exhibiting long-term repopulation capabilities in xenograft-accepting animals. In a cell transplantation setting for hereditary disorders, patient-specific iPSCs must undergo repair prior to autologous cell transplantation and long-term functional capabilities of the gene-corrected cells needs to be ensured. Aiming at a sustained knock-down or repair of a disease causing gene in iPSCs and their differentiated derivatives, we investigated novel approaches for the expression of a therapeutic shRNA in α1-antitrypsin deficiency (A1AT)-specific iPSC as well as CRISPR/Cas9-mediated precise genome editing tools.
: University of Trento
The human brain cortex is composed by many distinct neuronal subtypes spatially and functionally organized that originate during development by an elaborate sequence of events that, if altered, can lead to neuropsychiatric diseases. Efficient derivation of large-scale cortical neurons from human pluripotent stem cells (hPSCs) is thus pivotal to the understanding of human cortex development, modelling of neuropsychiatric disorders in vitro and potential development of new therapies. Here, I'll describe a culture system to rapidly and homogenously convert hPSCs into neural progenitors and to induce their rapid and highly efficient differentiation into mature cortical excitatory neurons and GABAergic interneuron subtypes by tightly modulating neural patterning temporally at a previously undefined primitive neural progenitor stage.
: San Raffael Scientific Institute
T cells genetically engineered with chimeric antigen receptors (CARs) are attaining spectacular results in haematological malignancies. For CAR-T cells to become viable therapeutic products, however, a better understanding of their mode of anti-tumor action (pharmacodynamics, PD) and of bio-distribution and persistence (pharmacokinetics, PK) is urgently needed. In this educational presentation, I will review current knowledge on PK/PD relationships of CAR-T cells and propose innovative tools for their clinical pharmacology modelling.
: Research National Council, Institute of Neuroscience, Via Vanvitelli 32, 20129, Milan, Italy
Direct cell reprogramming methods offer a straightforward and accelerated system to convert fibroblasts into neuronal and glial cells. In particular, multiple sets of transcription factors (TFs) have been recently identified that enable the generation of a wide range of different neuronal subtypes. Induced neurons can mature in vitro and acquire functional properties very convenient for disease modeling. I will overview the numerous molecular and metabolic pathways recently identified that can facilitate neuronal cell reprogramming to increase overall efficiency and generate more mature and functional cells. In addition, glial cells can be generated as well with selected combinations of lineage-specific developmental TFs. I will report on a new procedure to convert fibroblasts into Schwann cells competent to generate myelin sheaths in vitro and in vivo. Neuronal conversion has been recently obtained using exclusively cocktails of small-molecules. I will discuss these advancements in respect to their application for the conversion of human cells and the generation of more mature and reliable induced neurons.
: University of Minnesota
The discovery of genes has revolutionized our understanding of the biological world. Now, the ability to edit genes has re-defined the way we think about medicine. Gene editing is based on the concepts of nucleic acids transmitting information via a digital code, and the natural mutating variance of nucleic acids as a substrate for evolutionary selection. Human genetic disorders are clinical representations of these variations. Gene editing offers—for the first time—a technology whereby the disease-causing mutations can be eliminated by re-coding the affected gene. Synthetic molecules with the dual functions of identifying and cutting the DNA at a specific genomic location proximal to the targeted mutation are one of the most elegant examples of current biology's emerging ability to engineer clinically relevant interventions at the site of the problem, i.e., at a molecular level. Gene editing offers the additional advantages that: [1] the gene restored to function remains under the physiological control of the native promoter, [2] these gene-editing molecules can be multiplexed to modify several molecular targets at once, and [3] when paired with activation or repression, they can be used to regulate gene expression. Gene editing offers an improved future for patients with untreatable or incurable genetic disorders—freedom from the limitations of their disease. As one of the most critical tools to be combined with stem cell biology, regenerative medicine, and transplantation biology—gene editing will govern our practice of medicine and experience of life in the 21st century.
: Massachusetts General Hospital
CRISPR-Cas nucleases are readily programmable genome editing reagents that have become widely adopted for research and are being developed as novel therapeutics. Here I will provide a general introduction to this platform and how it can be deployed for various research and therapeutic applications. I will also discuss recent advances in defining and minimizing off-target effects of these nucleases in human cells as well as efforts to expand the targeting range of these reagents.
Lgr5 stem cell-based organoids in human disease
: Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences & University Medical Centre Utrecht and Princess Maxima Center for pediatric oncology, Utrecht, the Netherlands
The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We originally defined Lgr5 as a Wnt target gene, transcribed in colon cancer cells. Two knock-in alleles revealed exclusive expression of Lgr5 in cycling, columnar cells at the crypt base. Using lineage tracing experiments in adult mice, we found that these Lgr5+ve crypt base columnar cells (CBC) generated all epithelial lineages throughout life, implying that they represent the stem cell of the small intestine and colon. Lgr5 was subsequently found to represent an exquisitely specific and almost ‘generic’ marker for stem cells, including in hair follicles, kidney, liver, mammary gland, inner ear tongue and stomach epithelium.
Single sorted Lgr5+ve stem cells can initiate ever-expanding crypt-villus organoids, or so called ‘mini-guts’ in 3D culture. The technology is based on the observation that Lgr5 is the receptor for a potent stem cell growth factor, R-spondin. Similar 3D cultures systems have been developed for the Lgr5+ve stem cells of human stomach, liver, pancreas, prostate and kidney. Using CRISPR/Cas9 technology, genes can be efficiently modified in organoids of various origins.
: The Salk Institute, La Jolla, California
Cellular programming and reprogramming technology (CPART) has provided a new way to investigate human development and disease. This technology is particularly useful for diseases in which the affected tissue is not available for cell purification and where aspects of cell development are crucial for the pathology. The central nervous system (CNS) is a good example of tissue that falls into this category. Modeling human brain diseases using induced pluripotent stem cells (iPSC) or induced neural cells (iN) has remarkable potential to generate insights into understanding disease mechanisms and opening new avenues for clinical intervention. Researchers now have the opportunity to study human disease in living, developing neural cells that carry the disease-specific genetic variants that are present in the patient. In addition, CPART represents a fresh approach for developing original diagnostic tools and obtaining novel drug candidates for CNS therapy. In order for CPART to be successful/useful, our underlying assumption is that, cellular deficiencies can be measured in vitro, recapitulating the phenotype that is relevant to human brain disease. Success requires that in vitro modeling is robust enough to detect a reliable and statistically meaningful difference between phenotypically normal and abnormal cells derived using CPART. This is particularly important in our view because there could be diseases where the variability of phenotypes will be too large to achieve reliable statistical significance. I will present several potential uses for modeling neurological and psychiatric diseases, as well as highlighting areas of caution and opportunities for improvement.
: RIKEN, CDB
The first in man application of iPS-derived cells started in September 2014 targeted the retinal disease called age-related macular degeneration (AMD). The grafted iPS-derived retinal pigment epithelial (RPE) cell sheet is survived well and good in color, that means no immune rejection occurred without immune suppression. Her visual acuity is stable, compare to the past history of deterioration even with multiple anti-VEGF injections. Primary endpoint, the safety was achieved at one year point. We evaluated plasmid remnant & gene alteration using WGS, epigenetic characteristics and purity using single cell RT-PCR other than our original quality control (QC). From these experiences, we think we should distinguish between basic research and regulatory science in order to promote regenerative medicine promptly. Since autologous transplantation is time consuming and the cost is high, it is necessary for making standard treatment to prepare allogeneic transplantation using HLA three loci homozygous iPS cell lines (iPS cell stocks) as well as autologous transplantation. It is known that RPE cells suppress the activation of T-cells, so that RPE cells appeared most suitable for such kind of allogeneic transplantation. We confirmed in vitro and in vivo that human iPS-derived RPE cells also have such function. It is possible that the rejection is considerably small by using the iPS-RPE cell with matched three loci of HLA.
: Lund University
Considerable progress has been made in generating fully functional and transplantable dopamine (DA) neurons from human embryonic stem cells (hESCs). We have performed a comprehensive preclinical assessment of hESC-derived midbrain DA neurons in a rat model of PD. We show long-term survival and functionality using clinically relevant imaging techniques, and demonstrate efficacy in restoration of motor function with a potency comparable to that seen with human fetal DA neurons. Furthermore, we show that hESC-derived DA neurons can project sufficiently long distances for use in humans, integrate into the host brain circuitry, fully regenerate midbrain-to-forebrain projections, and innervate correct target structures. This provides strong preclinical support for translation of hESC-derived DA neurons. A major challenge when developing such therapies is that transplantation is performed with immature progenitors that undergo phenotypic and functional maturation after transplantation, and there is a lack of markers reliably predicting yield and functional maturation of the cells in vivo. To tackle this, we took an unbiased approach to identifying markers expressed in DA progenitors that predict successful graft outcome in an animal model of PD through gene expression analysis of >30 batches of grafted hESC-derived progenitors. We found that many commonly used markers did not accurately predict in vivo subtype specific maturation, and identified a novel set of markers associated with the caudal midbrain, which correlated with high dopaminergic yield after transplantation in vivo. Using these markers, we developed a GMP differentiation protocol for highly efficient and reproducible production of transplantable DA progenitors from hESCs.
: Case Western Reserve University
Oligodendrocyte progenitor cells (OPCs) are endogenous stem cells in the central nervous system that serve as the predominant source of myelinating oligodendrocytes. Oligodendrocyte loss or dysfunction can lead to significant motor and cognitive disability in patients due to myelination deficits. We have developed technologies that enable the rapid and robust generation of OPCs from pluripotent stem cells and via direct cell reprogramming technologies. These in vitro generated OPCs serve as a powerful platform to understand oligodendrocyte development and to discover therapeutic compounds for enhancing myelination. I will discuss our recent efforts to use high-throughput phenotypic screening of pluripotent stem cell-derived OPCs to uncover new aspects of oligodendrocyte biology.
: Princess Margaret Cancer Centre, University Health Network, University of Toronto
For decades, hematopoiesis has been described as a cellular hierarchy maintained by self-renewing hematopoietic stem cells (HSCs) that proceed through a series of multipotent, oligopotent and then unipotent progenitors to make blood. The presence of oligopotent intermediates is crucial to the model since they define the path from multipotent cells to unipotent cells. Although the standard model is still used extensively as an operational paradigm, further cell purification and functional clonal assays have led to key revisions of the model. Previously, we identified human multilymphoid progenitors (MLP) as the earliest lymphoid differentiation precursor with concomitant lymphoid (T, B, NK) and myelomonocytic potential, rather than common lymphoid progenitors (CLP). Through novel cell-sorting and more sensitive single cells assays to assess multilineage My, Er, and Mk fate potential contemporaneously we now provide a new framework to understand this arm of human hematopoiesis (Notta et al Science 2016). These studies indicate that oligopotent progenitors are a negligible component of the hierarchy of adult BM suggesting that multipotent cells differentiate into unipotent cells directly. These data support the concept of a ‘two-tier’ human blood hierarchy: a top-tier containing multipotent cells (HSCs and MPPs) and a bottom-tier composed of committed unipotent progenitors. Also the blood hierarchy changes between prenatal and adult stages. To gain a deeper understanding of the molecular basis for the two tier hierarchy, transcriptional, proteomic and ATAC-seq data will be presented providing mechanistic insight into how human hematopoiesis is regulated across development.
: Boston Children's Hospital
Blood stem cells proliferate or differentiate and have been used as a source for transplantation. We have used the zebrafish as a model system to study this process. Stem cells arise in the developing aorta of the zebrafish and sequentially colonize other sites of hematopoiesis similar to processes that happen in mammals. We have investigated interactions between stem cells and their niches. Such studies have led to the discovery that prostaglandin E2 can stimulate stem cell production in zebrafish. PGE2 stimulates better engraftment in marrow transplantation in mice. PGE2 was tested in a clinical trial in patients with leukemia who were treated by cord blood transplantation. Using a competitive transplantation experiment akin to the mouse competitive repopulation assay, a clinical trial was designed (Cutler et al., 2013) in which patients with leukemia receive two cord blood units: one unit was treated with dmPGE2, the other was untreated. DNA polymorphisms in white blood cells revealed preferential engraftment and earlier return of neutrophils and platelets in 10 out of 12 patients with treated cord bloods. This work has progressed to a Phase II clinical trial with 48 patients. Other eicosanoid pathways also regulate stem cell engraftment. EETs, another lipid derived from arachidonic acid, stimulates hematopoiesis. We demonstrate that eicosinoids induce a long-term memory in stem cells. Our work has implications for new therapies for blood diseases and cancer.
: SR-Tiget, San Raffaele Telethon Institute for Gene Therapy
Long-term follow-up of patients treated by lentiviral HSC gene therapy shows stable and extensive genetic engineering of hematopoiesis with polyclonal reconstitution by gene modified HSC, no evidence of genotoxicity and substantial therapeutic benefit. These results open the way to design new gene therapy strategies applicable to more common diseases such as cancer. We have devised a gene therapy strategy for the targeted delivery of interferon alpha to tumors by gene-modified tumor infiltrating macrophages. The local release of interferon at the tumor site reprograms the tumor microenvironment towards a permissive state for the generation and deployment of immune effector responses against tumor associated antigens, leading to tumor clearance and resistance to re-challenge. More precise genetic engineering can be achieved by correcting disease-causing mutations in situ, thus restoring both the function of the gene and its physiological expression control. Targeted gene editing, however, is constrained in HSC by quiescence and low expression of the DNA repair machinery. We could overcome these barriers by culture conditions that induce proliferation while preserving long-term engraftment capacity and provide evidence of correction of SCID-X1 causing mutations in the IL2RG gene. We have validated this approach in an ad hoc humanized SCID-X1 mouse model to support the scientific rationale and safety of the proposed treatment, and identify the conditioning regimen and degree of chimerism with edited cells required to correct the disease.
: Hôpital Necker- Paris Descartes
: Yale University
Tissue repair is fundamental to our survival as tissues are challenged by recurrent damage. During mammalian skin repair, cells respond by migrating and proliferating to close the wound. However, the coordination of cellular repair behaviors and their effects on homeostatic functions in a live mammal remains unclear. Here we capture the spatiotemporal dynamics of individual epithelial behaviors by imaging wound re-epithelialization in live mice. Differentiated cells migrate while the rate of differentiation changes depending on local rate of migration and tissue architecture. Cells depart from a highly proliferative zone by directionally dividing towards the wound while collectively migrating. This regional co-existence of proliferation and migration leads to local expansion and elongation of the repairing epithelium. Finally, proliferation functions to pattern and restrict the recruitment of undamaged cells. This study elucidates the interplay of cellular repair behaviors and consequent changes in homeostatic behaviors that support tissue-scale organization of wound re-epithelialization.
: ETH Zürich
Stem cell systems are highly complex and dynamic, and consist of large numbers of different cells expressing many molecules. Despite intensive research, many long-standing questions in stem cell research remain disputed. One major reason is the fact that we usually only analyze populations of cells — rather than individual cells — at very few time points of an experiment. Tracking of individual cells would be an extremely powerful approach to improve our understanding of molecular cell fate control. We are therefore developing imaging systems to follow the fate of individual cells over many generations. We program new software to help recording and displaying the divisional history, position, properties, interaction, etc. of all individual cells over many generations. In addition, novel microfluidics devices are designed and produced to allow improved observation and manipulation of individual cells. Our approaches also allow continuous long-term quantification of protein expression or activity in individual living cells. The resulting novel kind of continuous quantitative single cell data is used for the generation of improved models describing the molecular control of stem cell fates. I will discuss how we try to find answers for long standing questions in stem cell research.
: Center for Regenerative Medicine-University of Modena
Breakthroughs in regenerative medicine have generated enthusiasm and many efforts to explore new therapeutic potentials of both somatic and pluripotent stem cells. About 30 years passed since a discovery of a method of producing a great number of human epidermal keratinocytes by cultivation from a small skin biopsy, many possibilities are now envisaged for therapeutic application of different cultured cell types. The importance of stem cell content was proven for many tissues or organs in different pathologies. Ocular burns cause depletion of limbal stem cells, which lead to corneal opacification and visual loss. Most of available treatments are palliative and focused on the relief of the devastating clinical picture. The human corneal epithelium was reconstructed in vitro and transplanted on hundreds of patients. All findings provided improvement and standardization of the cure for this disabling disease. A long-term follow-up (6.5 years) of a phase I/II clinical trial envisaging the use of autologous genetically modified cultured epidermal stem cells for gene therapy of junctional epidermolysis bullosa, reported the safety and long-term persistence of genetically modified epidermis. These data pave the way for the safe use of Advanced therapy medicinal products, including stem cells or in combined cell and gene therapy for genetic skin diseases.
: Inserm
Retinal degenerative diseases are a leading cause of irreversible blindness. Retinal cell death is the main cause of vision loss in genetic disorders such as retinitis pigmentosa, Stargardt's disease and Leber's congenital amaurosis, as well as in complex age-related diseases such as age-related macular degeneration (AMD). For these blinding conditions, gene and cell therapy approaches offer therapeutic intervention at different stages of the disease. Classical gene replacement therapy has been effective in rare diseases where the causal mutation is known and the cells bearing the mutation are still present. To go beyond these diseases, the development of new gene and cell therapies is necessary. The focus of this talk will be the developments in gene and cell therapy technologies to prevent vision loss and restore vision in advanced stage retinal degenerations. In late stages of retinal degeneration gene therapy can be used to promote the survival of retinal neurons or to reanimate the retina through the expression of optogenetic modulators of membrane potential. Such protective and restorative gene therapies can be combined to extend therapeutic benefit. In the most advanced stages of disease, once the photoreceptor cells have completely degenerated, cell therapy can provide therapeutic benefit provided that functional photoreceptors are transplanted. A new methodology to generate functional cones in vitro from human induced pluripotent stem cells will be discussed in view of its utility in advanced stages of retinal disease.
: Gene Therapy Program Dana-Farber/Boston Children's Cancer and Blood Disorders Center
: Division of Neurology, Department of Medicine Jichi Medical University
Adeno-associated virus (AAV) vectors transduce neurons efficiently and express therapeutic genes for a prolong period without significant toxicity. Recently, robust transductions of neurons in adult animals were achieved by the systemic delivery of tyrosine-mutant AAV9/3 vectors containing neuron-specific promoters. We developed gene therapy for neurodegenerative diseases using AAV vectors. In advanced Parkinson disease, the most efficient treatment approach is to restore the local production of dopamine in the striatum by delivering dopamine-synthesizing enzymes. Concomitant with persistent expression of transgene, motor symptoms of the patients were ameliorated in open-label clinical studies after the gene transfer of aromatic L-amino acid decarboxylase (AADC) into the putamen. The beneficial effects of AADC gene transfer have also been shown in children with AADC deficiency. It would be advantageous to deliver genes for L-dopa synthesis, such as tyrosine hydroxylase and guanosine triphosphate cyclohydrolase I, in addition to the AADC gene in order to reduce motor fluctuation associated with intermittent L-dopa intake. In sporadic amyotrophic lateral sclerosis patients, the downregulation of the RNA-editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) is a death-causing molecular abnormality that occurs in motor neurons. Gene delivery of ADAR2 using tyrosine-mutant AAV9/3 vectors in conditional ADAR2 knockout mice effectively prevented progressive motor dysfunction. Further proof-of-principle studies establishing that viral delivery of disease-specific miRNA can rescue disease phenotypes have been performed in mice models of spinal and bulbar muscular atrophy and spinocerebellar ataxia type 6. Therefore, intra-thecal infusion of tyrosine-mutant AAV9/3 vectors is a feasible approach in clinical trials for most neurodegenerative diseases, as global transduction of the central nervous system is necessary.
: Leiden University Medical Center
The first beating cardiomyocytes were derived from human embryonic stem cells in 2002 and the differentiation protocols proved directly transferable to induced pluripotent stem cells (hiPSC). Understanding the underlying developmental mechanisms that control differentiation of pluripotent cells to their derivatives and mimicking these in defined culture conditions in vitro has proven essential for moving the field forward. This has been aided by a valuable series of transgenic lineage reporter hPSC lines. From initial methods that yielded only ventricular like cells, we can now specify atrial and pacemaker fates and derive epicardial-like cells that normally cover the outer surface of the heart. Heart cells from hPSC are an area of growing interest as a way of modelling cardiac and vascular diseases with view to understanding underlying mechanism for drug target identification. Challenges to applications of these cells in disease modelling, drug discovery and safety pharmacology however remain their limited expansion in culture and immature phenotype after differentiation. We discuss approaches to addressing this through culture modification, complex cell combinations and biophysical strain and show examples of the impact on determining the phenotype of hiPSC-derivatives in genetic diseases of the cardiovascular system.
: International Centre for Genetic Engineering and Biotechnology (ICGEB) Trieste, Italy
There is an impelling need to develop innovative therapies that promote cardiac repair in patients with myocardial infarction and heart failure. In contrast to other species that are able to regenerate the heart throughout their entire life, post-natal damage to the myocardium in mammals is repaired through formation of a scar. Copious evidence nonetheless indicates that the capacity for myocardial renewal, albeit limited, also exists in adult individuals. During the last years, my laboratory has become deeply interested in developing methods to search for factors able to foster this cardiac regenerative capacity. We follow two parallel strategies, both based on unbiased, functional screenings. In one strategy, we have generated two libraries in AAV, one corresponding to the secretome (all secreted factors encoded by the genome, approximately 1200 cDNAs) and the second one to the miRNAome (800 pri-miRNA genes). We developed a procedure (named FunSel) for the functional selection of therapeutic factors after in vivo administration of pools from this AAV library to the heart after myocardial infarction. FunSel has so far generated a number of novel and, in some cases, unexpected molecules able to protect the heart and, in some instances, to induce its regeneration.
With the other strategy, we searched for small RNAs inducing cardiac regeneration by the ex vivo, high throughput screening of miRNAs and miRNA-inhibitors able to promote primary cardiomyocyte proliferation using whole genome libraries. We identified at least 8 microRNAs that increase cardiomyocyte proliferation in mice, rats and pigs, as well as in human cardiomyocytes from fetal hearts, or derived from hES cells. Delivery of two of these miRNAs in vivo, either using AAV9 vectors or as synthetic mimics after myocardial infarction markedly reduced infarct areas and improved cardiac function in both mice and pigs. These microRNA function by directly activating the proliferative potential of differentiated cardiomyocytes thus bypassing the requirement of stem cell expansion and differentiation.
Taken together, these results indicate that finding novel biotherapeutics for myocardial infarction and heart failure is an attainable goal, including proteins or small RNAs capable to induce cardiac regeneration.
: University Health Network, Toronto
Human embryonic stem cells (hESCs) have a number of potential advantages for use in myocardial infarct repair, including a tremendous capacity for expansion in the undifferentiated state and the ability to differentiate into phenotypically unambiguous cardiomyocytes. Our group has contributed to the development of efficient, reliable protocols to generate large quantities of hESC-derived cardiomyocytes (hESC-CMs), and we have shown that the transplantation of hESC-CMs can partially “remuscularize” the infarct scar with new, electrically-integrated myocardium in small and large animal models of myocardial infarction. This is a sine qua non for true heart regeneration; however, a number of important challenges remain to the successful development of hESC-based solutions for heart failure. In particular, we need to 1) optimize the large-scale, clinical-grade production of mature, ventricular hESC-CMs, 2) develop methods to avoid graft cell death and immune rejection, and 3) address important safety concerns about the risks of tumor formation and graft-related arrhythmias. In this presentation, I will provide an update on efforts to overcome each of these barriers, including our recent work to understand and improve the electrophysiological function of hESC-CM grafts and outcomes from transplantation studies in a highly relevant porcine infarct model.
: Université Versailles Saint-Quentin
The proof of concept for splice switching therapies has now been provided for several neuromuscular disorders, such as Duchenne muscular dystrophy (using exon skipping to rescue DMD-mRNA ORF) as well as Spinal muscular atrophy (SMN synthesis following inclusion of exon-7 of the SMN2 gene) with miscellaneous AON chemistries tested in murine models and several clinical trials being performed. Major issues concern either efficient widespread delivery of AON to target tissues after systemic delivery or toxicity, which eventually hampers its use at high dose that would enable higher splice switching capacity. To overcome these limitations, we investigated the therapeutic potential of a new class of conformationally constrained DNA analogues, the tricyclo-DNAs (tcDNA), for dystrophin rescue following exon skipping in the mdx mouse model of Duchenne muscular dystrophy (DMD). We demonstrated extensive restoration of dystrophin in the mdx mouse following systemic injections of tcDNA annealing the donor splice site of the dystrophin exon 23. Remarkably, this treatment also leads to restoration of dystrophin in the cardiac muscle and detection of exon skipping in the brain, improving their muscle function and alleviating the dystrophic condition. These findings suggest great potential for these compounds in the systemic treatment of many neuromuscular disorders where it is critical to target the whole skeletal musculature, the heart and incidentally the CNS such as in DMD.
: Stanford University
I will describe our work on developing methods for generating functional 3D forebrain cultures starting from human induced pluripotent stem cells cells. Specifically, I will describe the derivation of neural spherical structures resembling the human cerebral cortex, called human cortical spheroids. These floating spheroids grow up to 5 mm in diameter, and include deep and superficial layer pyramidal neurons of the cortex, as well as astrocytes. After developing in culture for ∼10 weeks, cortical spheroids display transcriptional characteristics of late mid-fetal human cortex. Neurons display spontaneous electrical activity and form functional synapses, and the emerging neural network activity can be probed in preparations similar to slice recordings of the animal brain. I will discuss the transcriptional and functional maturation of astrocytes, as well as viral infection in 3D cultures. Neural spheroids allow a detailed interrogation of human cortical development, function and disease, and represent a versatile platform for generating other neuronal and glial subtypes in vitro.
: Cold Spring Harbor Laboratory
Pancreatic Cancer remains the most deadly of the common malignancies, due the lack of a viable early detection method and the limited activity of most drugs. To increase our basic understanding of this disease and advance medical approaches, we have recently co-developed with Hans Clevers mouse and human pancreatic cancer organoid model systems (1). Organoid cultures can be robustly prepared from either normal pancreatic ducts or any stage of neoplasia, including preinvasive carcinomas. Additionally, orthotopically transplanted neoplastic organoids remarkably demonstrate cellular plasticity and model the progression of pancreatic cancer from a low-grade preinvasive carcinoma to an invasive and metastatic carcinoma over several months. Thus, the in vitro and in vivo organoid models provide a platform to explore new diagnostic and therapeutic strategies, facilitate the systematic analysis of neoplastic biochemical cascades, and also enable the dissection of tumor microenvironment interactions. The organoid series have allowed us to query the function of important mediators of pancreatic cancer oncogenesis. We have found that Nrf2, a principal regulator of redox metabolism, maintains proliferation in KRAS mutant pancreatic ductal cells by lowering the levels of reactive oxygen species to promote protein translation, providing new therapeutic approaches for pancreatic cancer (Chio et al, in press). Finally, the co-culture of organoids with pancreatic stellate cells promotes the activation of each cell type and results in the production of extracellular matrix. Co-cultures reveal distinct subtypes of stellate cells and provide an opportunity to identify and target novel mediators of the tumor microenvironment (Olund et al, personal communication).
: Department of Oncology and Hemato-Oncology University of Milan
The in vitro recapitulation of human development, largely propelled by cell reprogramming, is transforming medicine by making genetic variation and disease predisposition experimentally tractable. This is especially true for disorders of the nervous system, for which cell reprogramming is particularly relevant due to the inaccessibility of relevant tissues, and whose main disease-associated genes are strongly enriched for transcription factors and chromatin regulators. Transcriptional and chromatin dysregulation is thus a priviledged entry point into the mechanistic underpinnings of neurodevelopemntal conditions, yielding relatively rapid molecular insight for bridging genetic or environmental lesions to in vivo phenotypes. Here I present novel insights from an integrated platform of 2D and 3D stem cell-based models of neural development through which we investigate convergent chromatin dysfunction brought about by genetic and environmental causes of intellectual disability. Specifically, we first define new empirically validated benchmarks for induced pluripotent stem cell (iPSC)-based experimental designs by combining: i) a uniquely informative cohort of 40 iPSC lines and neural derivatives, that we established from patients with neurodevelopmental and autism spectrum disorders caused by the most frequently mutated regulators belonging to inter-related chromatin pathways; ii) a comprehensive meta-analysis of iPSC transcriptomes; iii) novel approaches to measure the contribution of genetic, epigenetic, and technical variations to transcriptional variance. We then use this framework to assess the neurotoxic effect of mixtures of endrocine disrupting compounds, a key concern for environmental and health policy, harnessing the deconvolution of chemicals-to-phenotypes pathways from the two largest epidemiological studies on the environmental impact on fetal neural development.
: Center for Regenerative Medicine in Barcelona (CMRB), Barcelona, Spain
The reprogramming of somatic cells to induced pluripotent stem (iPS) cells using delivery of defined combinations of transcription factors is a groundbreaking discovery that opens great opportunities for modeling human diseases, including Parkinson's disease (PD). iPS cells can be generated from patients and differentiated into disease-relevant cell types, which would capture the patients’ genetic complexity. Furthermore, human iPS-derived neuronal models offer unprecedented access to early stages of the disease, allowing the investigation of the events that initiate the pathologic process in PD. Recently, human iPS-derived neurons from patients with familial and sporadic PD have been generated and importantly they recapitulate some PD-related cell phenotypes, including abnormal α-synuclein accumulation in vitro, and alterations in the autophagy machinery. I will introduce our efforts to generate PD iPS-based models and discus the potential future research directions of this field.
: A.I.Virtanen Institute
Therapeutic vascular growth is a potentially useful strategy for ischemic heart disease and peripheral arterial occlusive disease. It involves generation of new capillaries, collateral vessels and lymphatic vessels in ischaemic muscles using either recombinant growth factors or their genes. Arteriogenesis is a process caused by increased sheer stress at the arteriolar level resulting in the formation of large conduit vessels from preexisting small vessels whereas angiogenesis and lymphangiogenesis refers to generation of new vascular structures in vivo. Most commonly used growth factors for therapeutic angiogenesis are members of the vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) families. Some other cytokines and growth factors can also have angiogenic effects. Improved perfusion and functional parameters can be achieved by angiogenesis and arteriogenesis in large animal chronic ischemia models and in man. Safety of the clinical gene therapy of cardiovascular diseases has been excellent with long-term follow-up to 10 yrs after the therapy. Small non-coding RNAs can also be used for angiogenic gene therapy. Most promising results have so far been obtained with direct catheter-based intramyocardial injections of VEGF-D genes with adenovirus and AAV vectors. References: Yla-Herttuala S, Bridges C, Katz MG, Korpisalo P. Angiogenic Gene Therapy in Cardiovascular Diseases: Dream or Vision? Eur Heart J 2016, in press.
: The University of Edinburgh
Our research targets pathological vascular remodelling, for example associated with failure of coronary artery bypass grafts, in in-stent restenosis and in the development of pulmonary hypertension. We have developed a gene therapy approach to limit vessel remodelling, through overexpression of TIMP-3 using adenovirus vectors. We have evaluated the expression, regulation and function of non-coding RNA in these settings and have been able to identify key non-coding RNA regulators of vascular cell function in vitro and in vivo. For example, we have shown that loss of miR-21 in vein graft tissue leads to a substantial reduction in neointima formation. In the setting of pulmonary hypertension, we have shown that both miR-143 and miR-145 impact on smooth muscle function and that cell:cell communication is important following pathological stimulation. Recently, we have begun to understand the regulation of long non-coding RNA, and have identified SMILR (Smooth Muscle cell Induced LncRna) that regulates the proliferation of vascular smooth muscle cells. SMILR is located upstream of HAS2 and controls its expression, as evidenced by siRNA studies. SMILR expression was also associated with human vascular pathology and is secreted and found in the plasma of patients with cardiovascular disease. Collectively, these studies suggest that non-coding RNA are fundamental regulators of cell function in vascular pathology and are therefore important targets for therapeutic intervention.
: Sorbonne Université, UPMC Univ paris 06, INSERM umrs959
The immune system fails to spontaneously eradicate tumors while its activation by checkpoint inhibitors unleashes therapeutic immune responses in some patients. Better understanding of the tumor tolerant environment should help design more efficient immunotherapies of cancer. We developed a discovery strategy based on a systems biology approach and the concept that maternal-fetal and tumor tolerance share similarities (1). We found that tumor-expressed TGF-beta and VEGF cooperatively control the Treg-mediated tumor tolerant environment. Inactivation of these genes in tumor cells led to major reduction of tumor growth, with even some tumor rejection, and greatly improved check-point inhibitors efficiency as well as immuno-cell therapies (2). We also discovered molecular signatures of the immune tolerant environment of tumors, which could predict cancer patient's survival from tumor biopsies sampled at the time of diagnosis (3). This indicates that the immune environment of tumors is a major determinant of cancer outcome. 1) Nehar et al., Journal of Immunology 2015 2) Courau et al., Journal of Clinical Investigation insight 2016 3) Nehar et al., submitted
: University College London
T-cells can be engineered using lentiviral vectors to express chimeric antigen receptors against CD19 (CAR19), an important antigen in B cell leukemia. While efficacy data accumulates, there are challenging imitations for manufacturing autologous therapies, including difficulties in generating sufficient cell yields from heavily treated patients. The incorporation of gene editing steps offers the prospect of producing ‘off-the-shelf’ universal cell therapies from healthy HLA mismatched donors. In 2015, we manufactured a bank of universal healthy donor CAR19 T-cells by lentiviral transduction and simultaneous gene-editing of T cell receptor (TRAC) and CD52 gene loci. This was achieved by electroporation mediated delivery of mRNA for two pairs of Transcription Activator-Like Effector Nuclease (TALEN). Subsequently, two infants with CD19+ B-cell acute lymphoblastic leukemia who had relapsed after first allogeneic stem cell transplantation were treated with these cells under compassionate use arrangements. Both received lymphodepleting chemotherapy and anti-CD52 serotherapy before cell infusion, and achieved molecular remissions within 28 days. Evidence of CAR-T cell persistence based on flow cytometry, chimerism and vector copy number was documented until successful second allogeneic stem cell transplant procedures. Phase 1 trials are now underway to further assess this approach as a bridge to transplantation in patients with refractory B cell leukaemia.
: Department of Microbiology and Immunology, UNC Chapel Hill
The adoptive transfer of CD19-specific CAR-T cells has shown remarkable anti-tumor activity in patients with B cell derived acute lymphoblastic leukemia and lymphomas. Alternative targets are being explored in phase I studies in hematological malignancies. Specifically, to reduce the B cell aplasia associated with effective and long-term persisting CD19-specific CAR-T cells, at Baylor College of Medicine, Houston, TX, we have implemented a phase I clinical trial with CAR-T cells targeting the κ-light chain of human immunoglobulin, to selectively eliminate κ+ tumors whilst sparing normal λ+ B lymphocytes. To target Hodgkin lymphoma and other CD30+ lymphomas, we have also implemented a phase I study with CAR-T cells targeting the CD30 antigen. The outcome of these studies, challenges and future directions will be presented at the meeting.
: Program in Immunology, Fred Hutchinson Cancer Research Center
The development of adoptive immunotherapy with engineered T cells that are genetically modified to express tumor-targeting chimeric antigen receptors (CARs) has resulted in complete remissions in some patients with advanced B cell malignancies and represents the first successful application of the principles of synthetic biology for cancer therapy. At this nascent stage, the field has largely focused on the contributions of signaling domains in the receptor to enhance in anti-tumor function. Our studies have analyzed the contribution of distinct T cell subsets and conducted the first studies of CAR-T cells targeting CD19 in B cell malignancies in which the CD4 and CD8 T cell composition of the CAR-T cell product is uniform in all patients. This has resulted in excellent antitumor activity with high rates of complete remission in ALL, NHL, and CLL and provided insight into CAR-T cell dose/response and dose/toxicity relationships. These insights are being applied to target molecules expressed on common epithelial cancers with CAR-T cells. Our preclinical work has also extended the principles of receptor design to include sequences that provide multiple functions that facilitate optimal signaling, in vivo tracking and elimination and clinical manufacturing. We show that the incorporation of a small polypeptide tag (Strep-tag II) into various locations of the extracellular domain of the CAR provides a universal intrinsic marker for identification of CAR expression on cells in vitro and in vivo, allows flexibility in tailoring spacer length to optimize CAR function, and enables both the rapid selection of highly purified CAR-T cell products for immediate in vivo administration or their large scale in vitro expansion. This approach, which can be universally applied for CARs with different scFv specificity and costimulatory domains, also provides a target for antibody mediate elimination of transferred CAR-T cells and can reverse toxicity in in vivo models. Because StrepTactin reagents are being used in clinical cell processing, this novel CAR design uniquely endows CAR-T cells with properties that facilitate performing cGMP manufacturing of cell products with markedly shorter culture times.
: Center for Cellular Immunotherapies and Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, 19104; Children's Hospital of Philadelphia, and Novartis Institute for Biomedical Research, Cambridge, MA
The immune system evolved to distinguish non-self from self to protect the organism. As cancer is derived from our own cells, immune responses to dysregulated cell growth present a unique challenge. This is compounded by mechanisms of immune evasion and immunosuppression that develop in the tumour microenvironment. The modern genetic toolbox enables the adoptive transfer of engineered T cells to create enhanced anticancer immune functions where natural cancer-specific immune responses have failed. Genetically engineered T cells, so-called ‘living drugs’, represent a new paradigm in anticancer therapy. Recent clinical trials using T cells engineered to express chimeric antigen receptors (CARs) or engineered T cell receptors (TCRs) have produced stunning results in patients with relapsed or refractory hematological malignancies. Most CAR single chain variable fragment (scFv) domains, including that of CTL019, are of murine origin; therefore, anti-mouse reactivity is one potential cause of immune-mediated rejection that may be overcome by fully human or humanized CAR designs. We developed a humanized anti-CD19 scFv domain and have conducted a phase I trial with humanized CD19-directed CAR T cells (CTL119) in patients with relapsed/refractory acute lymphoblastic leukemia. In patients with no prior exposure to a CD19 CAR T cell product, MRD-negative CR was achieved in 17/17 patients (100%), and in 64% of patients previously treated with CD19-directed murine CAR T cells. Further investigation into CAR T cell persistence and anti-CAR responses will be vital to improve durable remission rates in this highly refractory population.
: Università Vita-Salute San Raffaele and Ospedale San Raffaele Scientific Institute
T-cell based cellular therapy exploits the ability of T lymphocytes to recognize and destroy specific targets, on microbes or tumors, leading to efficient killing and long-term protection against diseases. Genetic engineering can be implemented to tame toxicity of T cells, through the use of suicide genes, or to modify T cell specificity. In particular, TCR genetic engineering allows the transfer in patients’ T cells of genes encoding for rare tumor-specific TCR. TCRs recognize antigen-derived peptides processed and presented on HLA molecules, thus allowing targeting both extracellular and intracellular molecules expressed by cancer cells. However, the simple transfer of tumor specific TCR genes into T cells is affected by some limitations: genetically modified T cells shall express four different TCR chains, that might mispair, leading to unpredictable toxicity and to an overall dilution of the tumor specific TCR on lymphocyte surface, thus limiting the efficacy of therapeutic cellular product.
To overcome these issues, we developed a TCR gene editing procedure, based on the knockout of the endogenous TCR genes by transient exposure to alfa and beta chain specific Zinc Finger Nucleases (ZFNs), followed by the introduction of tumor-specific TCR genes by lentiviral vectors. The TCR gene editing technology, proved safer and more effective than conventional TCR gene transfer in vitro and in animal studies. Early differentiated T cells, such as memory stem T cells and central memory T cells, cells endowed with long term persistence capacity, can be genetically engineered by TCR gene transfer and TCR gene editing, thus allowing to produce long-lasting living drugs, with the aim of eliminating cancer cells and patrol the organism for tumor recurrence
: University of California San Francisco
Hematopoietic stem cell (HSC) activation is accompanied by mitochondria activation and a shift in metabolic activity from glycolysis to OXPHOS, which provides energy and increases the production of ROS and other mitochondrial metabolites that act as substrates/co-activators for epigenetic enzymes. Metabolically activated HSCs are poised to undergo lineage priming and produce different types of lineage-biased multipotent progenitors (MPP). However, activated HSCs must also return to quiescence to maintain the stem cell pool and autophagy plays an essential role by clearing activated mitochondria to allow OXPHOS-driven HSCs to efficiently revert to a mostly glycolysis-based quiescence metabolism. At steady state, blood production reflects the differential generation by HSCs of a small number of myeloid-biased MPP2/3 and a large number of lymphoid-biased MPP4, both giving rise to granulocyte macrophage progenitors (GMP) and contributing to myeloid output. In contrast, during blood regeneration, HSCs are induced to overproduce MPP2/3, and MPP4 are reprogrammed towards almost exclusive myeloid output in large part due to cytokine stimulations. An important consequence of the activation of this myeloid regeneration axis is the formation of defined GMP clusters in the bone marrow (BM) cavity, which drive the local overproduction of granulocytes. This process is tightly regulated by the timed release of important BM niche signals and transient activation of a progenitor self-renewal network. Altogether, the metabolic activation of HSCs, remodeling of the MPP compartment and induction of GMP clusters represent emergency myelopoiesis pathways that are transiently activated during regeneration, and are continuously triggered in myeloid disease conditions.
: HI-STEM
Ageing of the hematopoietic system in humans is characterized by peripheral blood cytopenias and bone marrow aplasia (termed anemia of ageing) as well as increased incidence of hematologic malignancies. These phenomena are likely caused by gradual attrition of the hematopoietic stem cell (HSC) compartment. Unfortunately, the standard experimental mouse model of hematopoiesis does not recapitulate these important clinical features of ageing. We have recently shown that prolonged exposure to inflammatory stress leads to premature hematologic ageing in wild type mice, caused by depletion of the HSC pool (Walter et al. Nature, 2015). We have now extended these studies in order to characterize whether HSCs recover following inflammatory stress and to what degree this impacts upon blood cell production. Surprisingly, we find no evidence for HSC regeneration following challenge and that exposure to chronic inflammation in early to mid-life provokes the onset of peripheral blood cytopenias and bone marrow aplasia in old age. Our data contradict the canonical view that HSCs demonstrate extensive self-regenerative capacity following injury, but instead are progressively depleted by hematologic stress in a manner consistent with an age-associated decline in function. We would propose that the absence of profound hematologic ageing phenotypes in laboratory mice could be explained by their lack of exposure to environmental stress agonists, including infection.
: Tigenix NV
One of the hallmarks of Mesenchymal Stem or Stromal cells is their capability to interface broadly with the immune system. In case of overt inflammation, it has been shown that MSC exert generally an anti-inflammatory response. We have explored the therapeutic potential of expanded Adipose Stem Cells in several settings of inflammatory disease. First, the anti-inflammatory activity of ASCs was demonstrated in vitro, showing a clear reduction of the pro-inflammatory status of activated T cells. Then, the translation of this anti-inflammatory control in the context of in vivo inflammation was demonstrated in several models of inflammatory disease like rheumatoid arthritis or inflammatory bowel disease. Finally, these supportive preclinical observations were confirmed in two human inflammatory diseases, complex perianal fistulas in Crohn's patients and refractory rheumatoid arthritis, demonstrating the therapeutic benefit of ASC treatment in human inflammatory disease.
: Leiden University Medical Center
Mesenchymal stromal cells (MSCs) comprise a heterogeneous population of multipotent cells that can be isolated from various human tissues. Due to the low frequency of MSCs in human tissues, MSC are extensively expanded ex-vivo before being used clinically. Expanded MSCs are mainly characterized by immune phenotype by a combination of positive (CD73, CD90, CD105, CD271) and negative (CD14, CD31, CD34, CD45) markers. MSCs display unique immune modulatory properties that have been first demonstrated in-vitro and subsequently also in-vivo in both animal models and in humans. MSCs suppress T lymphocyte proliferation in-vitro, induced by allo-antigens and mitogens. MSC polarize T cells towards a regulatory phenotype, which serves as an important mechanism through which MSC may suppress inflammatory responses. Co-infusion of MSCs and Umbilical Cord Blood-derived CD34+ cells was associated with enhanced engraftment of human HSCs in the BM, in particular when relatively low doses of HSCs were infused. Infusion of MSC has also been shown to enhance HSC mobilization induced by G-CSF. These effects may be mediated by the production of soluble facors that modulate the niche. The immunomodulatory properties of MSCs have been employed clinically in disorders associated with allogeneic or autologous immune responses, including severe aGVHD developing after allogeneic stem cell transplantation or donor lymphocyte infusion, solid organ, in particular renal transplantation and Crohn's disease or type I diabetes mellitus. The collected data indicate that infusion of MSCs is safe and that treatment is potentially effective, although pivotal clinical studies have yet to be completed.
: Genethon, Evry
Gene transfer approaches based on the adeno-associated virus (AAV) vector platform have shown great promise both in preclinical animal models of several diseases. In the clinic, sustained therapeutic levels of transgene expression have been observed in severe hemophilia gene transfer trials following systemic vector administration to target the liver. However, immunogenicity associated with the transgene product or the vector poses a limitation to the safety and efficacy of in vivo gene transfer with AAV vectors. For example, pre-existing neutralizing antibodies (NAb) to AAV can drive vector clearance and results lack of efficacy. Additionally, anti-AAV NAbs develop at high titers following vector administration and persist for several years, making vector re-administration hard if not impossible.
This presentation will provide an overview of the current issues related to immune responses in AAV gene transfer and of possible strategies to overcome AAV vector- and transgene-related immunogenicity.
: Spark Therapeutics
This presentation will review findings from several clinical trials, with the goal of defining characteristics of an optimized vector for hemophilia gene therapy. The effects of vector dose on human immune response will be discussed, as well as the effect of manufacturing methods on vector doses required. The hemophilia B trial sponsored by Spark Therapeutics was designed to test the hypothesis that a high specific activity vector can drive therapeutic levels of FIX activity at doses low enough to avoid triggering the cell-mediated immune response seen in earlier studies. Findings from this study will be reviewed. Pre-clinical data in support of the hemophilia A trial will also be reviewed.
: Stanford University
Most common cancers arise in organs that contain a small population of stem cells that constantly replenish the mature cells of the tissue. Stem cells are defined by the ability to divide and give rise to a new stem cell (self-renewal), as well as the ability to give rise to the differentiated cells of an organ, and thus are the only long-lived cell population in many tissues. Recent evidence suggests that colon and breast cancers arise from normal stem or early progenitor cells. Our laboratory was the first to isolate breast and colon cancer stem cells, demonstrating that these cells are critical for the growth and metastasis. These findings have implications for the treatment of these cancers. We reasoned that the frequency of cancer stem cells in early tumors could be used to identify high-risk Stage-II colon carcinomas patients who need adjuvant treatment after surgery. We used a novel bioinformatics approach to search for robust, individual biomarkers of colon epithelial differentiation. We found that the transcription factor CDX2 could be used to quantify the cancer stem cell content of tumors and asked whether it could be used to predict chemotherapy benefit in Stage-II colon cancer patients. Lack of CDX2 expression appears to identify a subset of high-risk Stage-II colon cancer patients who appear to benefit from adjuvant chemotherapy. To our knowledge, this is the first time that the investigation of cancer stem cells has led to improvement of patient outcomes and has informed therapeutic decisions in the clinic.
: The Francis Crick Institute
Acute myeloid leukemia (AML) is a hematologic malignancy, arising within the bone marrow, which is characterized by the uncontrolled proliferation of leukemic blasts, often in association with a disruption of normal hematopoiesis. Like their normal counterparts, AML cells depend upon both cell-intrinsic and -extrinsic regulatory signals generated by their surrounding microenvironment, for their survival and proliferation. AML has long been considered a hematopoietic-cell autonomous disorder in which disease initiation and progression is driven by hematopoietic cell intrinsic genetic events. Recent experimental findings in diverse model systems have challenged this view, implicating different stromal cells of the bone marrow in disease pathogenesis. Thus it is now accepted that leukemic hematopoiesis can turn the BM niche into a “leukemic niche” which promotes leukemic stem cell (LSC) function and impairs the maintenance of normal HSC. However, much remains to be understood about how different leukemic cells impacts the BM microenvironment and, in turn, how changes in the activity of specific BM niche cells contribute to AML pathogenesis. This talk will highlight some of the current understanding of the alterations of BM niche components and how the dialogue between leukemic and stromal cells participated in leukemogenesis.
: Laboratory of Genetics, The Salk Institute, La Jolla, California, USA
Glioblastomas are the most common and lethal form of intracranial tumors. They account for approximately 70% of the 22,500 new cases of malignant primary brain tumors diagnosed each year in adults in the United States. Although relatively uncommon, malignant gliomas are associated with disproportionately high morbidity and mortality. These cancers exhibit a relentless malignant progression characterized by widespread invasion throughout the brain, resistance to traditional and newer targeted therapeutic approaches, destruction of normal brain tissue, and certain death. The median age of patients at the time of diagnosis is 64 years. Despite optimal treatment and improving standard of care, the median life expectancy is only 12 to 15 months for patients with glioblastomas.
Recently, we have shown that glioblastomas can originate from differentiated cells in the central nervous system (CNS), including cortical neurons. Transduction by oncogenic Cre-inducible lentiviruses in the hippocampus of GFAP-Cre or the cortex of SynapsinI-Cre transgenic mice induced the formation of gliomas. Interestingly, these tumors mostly expressed markers of the three major lineages of the CNS (glia, neuron and oligodendrocytes), progenitor/neural stem cells, Nestin and Sox2. In a study aimed at following the kinetic expression of some of these markers during tumor development, we observed that at early stages differentiation, markers are progressively diminished while Nestin, a marker of NSC, that was hardly detectable a few days after transduction, increased significantly as tumors developed. We proposed that the transformed mature/differentiated cell, acquires the capacity to dedifferentiate to a cell that has all the attributes of a stem-like cell, which can then not only maintain its pluripotency but also give rise to the heterogeneous cell populations observed in malignant gliomas. We further argued that the genetically altered differentiated cell acquires the capacity to reprogram to a stem cell state, and that tumor progression probably requires a permissive microenvironment composed of cell types and molecular signals that can sustain both differentiation of tumor cells and the maintenance of CSCs. These data support the view that dysregulation of specific genetic pathways, rather than cell-of-origin, dictates the emergence and phenotype of high-grade gliomas.
We were very surprised to detect endothelial cells in mouse gliomas which originated from tumors. The transdifferentiated endothelial cells (TDECs) are present mainly in hypoxic areas of tumors and do not express VEGF receptors and are therefore insensitive to anti-VEGF therapy. In fact, treatment with VEGF inhibitors led to an increase in TDECs. These data provide a rationale to investigate molecular mechanisms of tumor plasticity and transdifferentiation to identify novel targets for therapy.
: Human Gene and Cell Therapy Program, University of California, Los Angeles
10 ADA-SCID patients were enrolled and treated in a Phase II study (NCT00794508) between 2009–2012 transplanting autologous CD34+ cells modified with the MND-ADA gammaretroviral vector (γRV) after conditioning with low dose busulfan and ERT cessation. Patients were treated between 3 months and 15 years of age (median 11.5 months) and follow-up ranges from 57–86 months. With the exception of the oldest patient (15 y/o at gene therapy), all others remain off ERT with normalized PBMC ADA activity. All nine remaining off ERT show normal proliferative responses to mitogens and three of nine were able to discontinue IVIg. MND-ADA is detected in PBMC (0.1-2.6 VCN) and in granulocytes (0.01-0.3 VCN) at most recent visit. Integration site analysis shows limited clonal expansion with clustered integration sites, as has been described for other trials for ADA-SCID with γRV. Clonal diversity correlated with busulfan dose intensity (AUC), based on both integration site analysis and TCR deep sequencing.
A Phase I/II trial opened in May 2013 (NCT01852071) using a self-inactivating lentiviral vector (EFS-ADA LV) developed with Thrasher/Gaspar to correct autologous CD34+ cells from ADA-SCID patients, using a similar clinical approach with low dose busulfan as in the prior γRV trial. Preclinical comparisons of this LV to ADA γRVs used previously demonstrated better gene transfer to CD34+ cells with a shorter ex vivo transduction period, higher expression of ADA enzyme activity, and no in vitro clonal expansion of murine HSC (IVIM) by EFS-ADA. Twenty subjects have been enrolled and treated. The subjects with the longest follow-up have normal or above PBMC and RBC ADA activity and higher levels of gene marking in myeloid cells with more effective immune reconstitution compared to MND-ADA γRV trial subjects. Follow-up is ongoing.
A Phase I/II clinical trial opened in September 2015 for X-linked Chronic Granulomatous Diseases (XCGD) (NCT02234934), using an LV (G1XCGD) and protocol (NCT02757911) developed by investigators Thrasher, Grez, at Genethon and others. The G1XCGD lentiviral vector expresses a codon-optimized gp91phox cDNA under transcriptional control of a novel chimeric myeloid-restricted enhancer/promoter, designed to have reduced risks of enhancer activation in HSC. The US trial has enrolled two subjects to date (Boston Children's Hospital and NIH Clinical Center, NIAID). They received pharmacokinetic-adjusted myeloablative conditioning with busulfan and LV-transduced PBSC CD34+ cells and tolerated the treatment well with rapid engraftment. Both show circulating granulocytes with restored neutrophil oxidase function at frequencies expected to eliminate risks of severe and recurrent infections.
These studies show the continued success and safety with applications of gene therapy with HSC for primary immune deficiencies.
: National Heart, Lung, and Blood Institute
Natural killer (NK) cells recognize and eliminate infected and malignant cells. Their life histories are poorly understood, particularly in humans, due to lack of informative models. Here we apply transplantation of barcoded rhesus macaque hematopoietic cells to interrogate the landscape of NK cell production and expansion at a clonal level. We identify rhesus NK cells with signaling protein expression and epigenetic profile analogous to human CMV-associated adaptive NK. Via barcoding, we demonstrate that these adaptive NK are oligoclonal, with expansion and contraction of sets of clones over time, in a pattern uncoupled from other hematopoietic lineages, including canonical NK. These distinct patterns persist longterm, including after profound in vivo NK depletion and regeneration. Clonal mapping suggests these adaptive NK cells are not produced in the bone marrow or lymph nodes. Our findings offer the first direct evidence for NK cell clonal expansions, providing a mechanism for adaptive NK immunological memory.
: University of Pennsylvania
Gene delivery remains a major hurdle for successful genome editing in vivo. Vectors based on AAV have demonstrated success in more traditional gene replacement strategies. We have explored the use of AAV in delivering CRISPR/Cas9 to edit genes in vivo. Our initial work focused on gene editing of hepatocytes in mouse models. We can successfully achieve gene knockdown in newborn and adult mice through non-homologous end joining (NHEJ) although efficiency of homology directed repair (HDR) is higher in newborns than in adults. Attempts to gene edit in vivo in large animal models are underway. We are also exploring the potential of AAV to gene edit in other organs such as muscle and CNS.
: San Raffaele Telethon Institute for Gene Therapy (SR-Tiget) and Vita-Salute San Raffaele University, Milan, Italy
Gene silencing represents a powerful strategy to interrogate gene function and holds great promise for therapeutic applications. We repurpose the Endogenous RetroViruses (ERVs) silencing machinery of embryonic stem cells to stably silence endogenous genes in somatic cells. This approach was achieved by transiently expressing combinations of Engineered Transcriptional Repressors (ETRs), chimeric proteins containing a custom-made DNA binding domain fused to the effector domain of chromatin-modifying enzymes involved in the ERVs silencing. The ETRs bind to and synergize at the target locus to instruct repressive histone marks and de novo DNA methylation, thus ensuring long-term memory of the repressive epigenetic state. Silencing was highly specific, as shown by genome-wide analyses, sharply confined to the targeted locus without spreading to nearby genes, resistant to activation induced by cytokine stimulation and artificial transcriptional activators, and relieved only by targeted DNA demethylation. We demonstrate the portability of this technology by multiplex gene silencing, adopting different DNA binding platforms and interrogating thousands of genomic loci in different cell types, including primary T lymphocytes. We conclude that targeted epigenome editing might have broad application in research and medicine.
: The Broad Institute
Advances in genome sequencing technology have accelerated the rate at which we can identify genetic variants associated with phenotypes related to human health and disease, but functionally interrogating these variants remains time intensive. Being able to quickly find the causative variants in a sea of a natural variation is essential to the goal of personalized medicine. To this end, new genome editing tools adapted from the microbial CRISPR-Cas system can be employed to rapidly screen through variants for functional effects as well as to model diseases based on patient-specific mutations. I discuss here how the CRISPR-Cas system can be deployed as a powerful discovery platform, highlighting recent findings from CRISPR screens, and describe our efforts to improve and refine these tools. Finally, I present recent work exploring the next generation of genome editing technologies beyond Cas9, and how these new tools will further expand our ability to connect genotype to phenotype and, ultimately, treat human disease.
: Murdoch Childrens Research Institute
Recent studies investigating the directed differentiation of human pluripotent stem cells have observed the spontaneous 3D patterning of tissue organoids, including brain, optic cup, stomach and intestine. Such protocols are based on a deep understanding of the normal processes of embryonic patterning of the tissue to be generated and upon an inherent capacity for self-organisation of cells within developing tissues. Development of the mammalian kidney involves reciprocal signalling between critical progenitor populations to drive the formation of a branching ureteric tree and a nephron-producing surrounding mesenchyme. Based on detailed temporospatial transcriptional and morphological analyses of kidney development, we have developed a protocol for the differentiation of human pluripotent stem cells into kidney organoids containing collecting duct epithelium, patterned and segmenting nephrons, surrounding interstitium and vasculature (1). This involves the stepwise induction of posterior primitive streak, anterior and posterior intermediate mesoderm and ultimately progenitors of all epithelial and non-epithelial components of the final organ. The development of this protocol opens the door on disease modelling and drug screening using organoids as a model of the kidney. In the longer term, such an approach may provide the requisite cell types for new therapies for kidney disease, including cellular therapy and bioengineered renal tissue. 1. Takasato et al, Nature, 2015
The cerebral cortex consists of several hundreds of different types of neurons, organized into specific cortical layers and areas, that display specific profiles of gene expression, morphology, excitability and connectivity.
Embryonic stem (ES) and induced (IPS) pluripotent stem cells constitute a promising tool for the modelling and treatment of human neural diseases. Here we will describe how corticogenesis from pluripotent stem cells can be used to identify novel mechanisms underlying human neurodevelopmental diseases and how the transplantation of ES/iPS cell-derived cortical neurons can lead to functional integration into developing and damaged cortical circuits.
Wherever you go – there you are: tracking DNA
: National Center for Tumor Diseases (NCT) Heidelberg, Heidelberg
Therapeutic use of gene transfer requires the detection, quantification and validation of the transferred nucleic acid in target cells and tissues. Effectiveness, efficiency, toxicity and longevity of this process need to be determined, and mechanistically understood. This gene transfer can inadvertently or intentionally change the biological properties of its target cells, and thereby the distribution of the modified cells in the target tissues and target organisms, which for clinical validation must be documented and understood. Such analyses comprise the most important portion of the pharmacokinetics and pharmacodynamics of gene therapy. In addition to the measurement and visualization of transgene expression, vector copy number and vector insertion sites can be determined by a variety of PCR-based, and, more recently, direct sequencing based methodologies. Gene editing technologies by CRISPR Cas9 or other systems pose additional challenges because their clinical use requires the detection of on and off target activities that has been followed by homology repair or non homologous end joining, and is usually devoid of any vector sequences. The technological aspects of tracking DNA modifications in target cells, tissues and organisms will be discussed.
: San Raffaele Telethon Institute for Gene Therapy (SR-Tiget)
Retroviral/lentiviral vectors have been used for efficiently and stably delivering therapeutic genes to mature T cells and hematopoietic stem/progenitor cells (HSPC) upon ex vivo gene therapy (GT). As viral integrations occur in a semi-random fashion, upon gene correction each transduced cells becomes univocally tagged by an individual insertion site (IS) that can be monitored for safety through LAM-PCR + high throughput sequencing. Our group has been focusing on using IS as molecular barcodes to address basic biology questions on the fate of hematopoietic clones in vivo in humans. We comprehensively studied human T-cells dynamics analyzing samples from adenosine deaminase deficient-SCID patients treated with HSPC or T cell GT. By tracing >1700 individual clones we discovered that engineered T memory stem cells can functionally last for decades in human beings in absence of supply by hematopoietic progenitors. We more recently tracked in vivo in humans the dynamics of hematopoietic reconstitution after HSPC GT in Wiskott Aldrich syndrome patients. Analyzing >89.000 clones belonging to 15 hematopoietic populations we described repopulating waves, populations’ dynamics and hierarchical relationships among lineages. We are further expanding this study collecting >10.000 IS from 7 different HSPC subtypes isolated from the bone marrow of GT patients, to address survival and activity of primitive and committed hematopoietic progenitors directly in vivo in humans. Concomitantly, we are characterizing vector-host interaction in vitro on the HSPC and T cell products uncovering specific insertional features upon in vitro manipulation. These studies are expanding our understanding of the biology of engineered human hematopoiesis.
: University Medical Center Freiburg
Targeted genome editing with designer nucleases has heralded a new era in gene therapy. Genetic disorders, which have not been amenable to conventional “gene-addition-type-gene-therapy” approaches, such as disorders with dominant inheritance or diseases caused by mutations in tightly regulated genes, can now be treated by precise gene surgery. Moreover, designer nuclease technology allows for novel genetic interventions to fight infectious diseases, including chronic viral infections. We have developed highly specific designer nucleases based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system. In preclinical models we demonstrate that TALENs and CRISPR-Cas nucleases can be employed to generate cells resistant to infection with human immunodeficiency virus type 1 (HIV-1) or to correct primary immunodeficiencies. Strategies to translate therapeutic gene editing into the clinic will be discussed.
: Duke University
The advent of genome editing technologies, including the RNA-guided CRISPR/Cas9 system, has enabled the precise editing of endogenous human genes. We have applied these tools to the correction of mutations that cause genetic disease. For example, we engineered CRISPR/Cas9-based nucleases to correct the human dystrophin gene that is mutated in Duchenne muscular dystrophy patients. When we delivered these nucleases to cells from patients with this disease, the correct gene reading frame and expression of the functional dystrophin protein were restored in vitro and following cell transplantation into mouse models in vivo (Ousterout et al., Nature Communications 2015). When delivered directly to a mouse model of this disease, gene editing by the CRISPR/Cas9 system led to gene restoration and improvement of biochemical and mechanical muscle function (Nelson et al., Science 2016). More recently, we have developed novel animal models of this disease for the preclinical development of therapies that will correct human disease-causing mutations. New constructs have been developed and validated to have significant activity levels in this model. Collectively, these studies demonstrate the potential of modern genome editing technologies to correct the underlying genetic cause of devastating hereditary diseases.
: Institut Catala d'Oncologia
Oncolytic viruses propagate selectively in tumor cells. Oncolytic viruses derived from Herpes Simplex Virus, Vaccinia Virus, Reovirus, and Adenovirus have advanced to late stage clinical trials. Limited clinical efficacy indicate that these viruses need to be improved at the levels of tumor targeting, intratumoral spread, and their ability to spark anti-tumor immune responses. Adenoviruses have specific properties in their life cycle and epithelial cell infectivity suitable for oncolysis of solid tumors. This talk will present strategies to improve oncolytic adenoviruses using cell vehicles, genetic modifications of the capsid, expression of transgenes that digest the tumor stroma, fusion of tumor epitopes to virus proteins, and expression of transgenes that retarget Tcells to tumor cells.
: Okayama University
Prostate is an ideal target organ for the gene therapy as a translational research (TR). It has advantages as follows: prostate is not a life keeping organ, can be approached easily by ultrasound as a routine clinical technique and PSA is a potent tumor marked for the evaluation of clinical response. Many clinical gene therapy trials for prostate cancer with Herpes Simplex Virus Thymidine Kinase (HSV-tk) gene therapy have been conducted in many institutes showing clinical safety and efficacy including Japanese trial (Molecular Therapy 15:834, 2007). Intraprostatic therapeutic gene transduction (in situ gene therapy) is one of the potent therapeutic option for prostate cancer gene therapy aiming at antimetastatic benefits through the generation of immune cell-mediated cytotoxic activities that affect not only the primary tumor but also metastatic lesion. As a preclinical study, new therapeutic gene REIC/Dkk3 has been proved to be potential therapeutic gene for prostate cancer as it has induces local apoptosis and systemic immune activation. (Cancer Research 65:9617, 2005, Cancer Gene Therapy 14:765,2007, J Bio Chem 284:14236,2009, Int J Mol Med 24:789, 2009). With positive experimental results, a phase I/II study of adenovirus-mediated REIC/Dkk-3 gene therapy for prostate cancer was initiated from January 2011 in Okayama university and completed with positive safety profile and clinical response. In this session development of in situ gene therapy for prostate cancer in Japan will be reported. I would like to discuss about the present problem and future direction for the development of prostate cancer gene therapy.
: Weill Cornell Medical College
The immune system plays a critical role in host defense, but can also be detrimental, either when deficient in an important component, or when activated in an abnormal or excessive fashion. We have developed gene therapy strategies for both categories. Hereditary angioedema is an autosomal, dominant, hereditary disorder secondary to mutations in C1-esterase inhibitor (C1EI), resulting in upregulation of bradykinin, causing life-threatening edema via leaky vessels. In the first strategy, we administered to the liver an AAV vector coding for C1EI to compensate for the deficiency. We developed C1EI-deficient mice in which the heterozygotes have low C1EI levels, elevated bradykinin levels, and leaky blood vessels, all of which reverse on a persistent basis with a single administration of therapy with AAVrh.10h C1EI. In the second strategy, severe food allergy results from abnormal levels of food antigen-specific IgE; when the food is consumed, anaphylaxis results. To treat this condition, we created a humanized, peanut-allergic mouse by reconstituting immunodeficient mice with blood mononuclear cells from peanut-allergic individuals and sensitizing the mice with peanut extract. Untreated, these mice exhibited high levels of peanut-specific IgE and evidence of anaphylaxis when exposed to peanuts. However, when treated with AAVrh.10h anti-IgE, a vector expressing an anti-IgE monoclonal, the therapy suppressed free IgE levels, blocked IgE-mediated histamine release, and protected the mice from death secondary to anaphylaxis. In both models, the AAV-mediated persistence of relevant therapy reversed the abnormalities in the immune system, providing long-term protection against potentially fatal responses to immune system dysfunction.
: Division of Stem Cell Transplantation and Regenerative Medicine, Department of Pediatrics, and ISCBRM, Stanford School of Medicine, Stanford, California, USA
T regulatory (Treg) cells play an essential role in maintaining tolerance. Due to their suppressive properties, Treg cells have been used as a cellular therapy for preventing excessive immune responses or abrogating autoimmune diseases. A number of clinical trials have been conducted with in vitro expanded Treg cells. However, a major limitation in this Treg cell therapy approach is the instability of the Treg cells under inflammatory conditions and the inability to track them in vivo. We investigated the use of lentiviral vector (LV) gene transfer to convert T effector (Teff) into Treg cells and to generate a stable and functional pool of Treg cells for clinical use. We showed that LV-mediated gene transfer of FOXP3 in human CD4+ T cells, converts Teff cells into Treg-like (CD4FOXP3) cells with potent in vitro and in vivo suppressive activity. Ag-specific CD4FOXP3 T cells stably expressing FOXP3 can also be generated. Furthermore, LV-mediated gene transfer of wild-type FOXP3 in CD4+ T cells from IPEX patients carrying different mutations in the FOXP3 gene, resulted in the generation of functional Treg cells, which displayed potent suppressive activity in vitro and in vivo.
Similarly, enforced IL-10 expression converts human CD4+ T cells into Tr1-like (CD4IL-10) cells that express the Tr1 markers, suppress Teff cells and selectively kill myeloid cell lines and blasts in a HLA-class I- and Granzyme B-dependent manner in vitro. CD4IL-10 cells mediate anti-leukemia effects and prevent xenogeneic GvHD in humanized models in vivo.
These findings provide a strong rationale for adoptive immunotherapy with genetically engineered Treg cells for the treatment of T-cell mediated disorders of genetic or unknown origin.
: San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), Milan, Italy
Severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID) is an ultra- rare disorder reported in only 0.23–0.68 per 100,000 live births. 1–4 If not diagnosed and treated promptly, ADA-SCID often proves fatal within the child's first years of life.
In this GSK-sponsored symposium, the expert faculty will provide an overview of recent advances in treatment of patients with ADA-SCID for whom no suitable hematopoietic stem cell transplant donor is available.
1. Ryser O, et al. J Clin Immunol 1988; 8: 479-85.
2. Verbsky JW, et al. J Clin Immunol 2012; 32: 82-8.
3. Vogel BH, et al. J Clin Immunol 2014; 34: 289-303.
4. Yee A, et al. Pediatr Allergy Immunol 2008; 19: 298–302.
: TIGEM
Mucopolysaccharidosis type VI (MPS VI) or Maroteaux-Lamy syndrome is a rare lysosomal storage disorder caused by arylsulfatase B (ARSB) deficiency. This results in widespread accumulation of dermatan sulfate (DS) in various tissues and organs without primary central nervous system involvement. Enzyme replacement therapy (ERT) is the standard-of-care for MPSVI however ERT requires weekly infusions of costly enzyme and has limited efficacy. We have recently shown that a single systemic injection of an adeno-associated viral (AAV) vector 8 expressing ARSB from a liver-specific promoter results in significant biochemical, pathological and functional improvement in MPS VI cats. ARSB expression and DS clearance is sustained for at least 6 years after vector administration in cats. In addition, we have shown in a mouse model of MPS VI that a single high-dose vector administration of AAV8 results in therapeutic efficacy similar to weekly infusions of recombinant enzyme. Interestingly, similar results are achieved combining low doses of AAV8 with monthly ERT. Based on our pre-clinical data we have recently received EU funding to develop a clinical trial to test the safety and efficacy of a single administration of AAV8 in MPS VI patients.
: Departments of Pediatrics and Genetics Stanford University Stanford, CA USA
Recombinant AAV vectors (rAAV) are beginning to show promise in early clinical trials. However, when moving from animals to humans a number of unanticipated responses have occurred that resulted in lower than expected efficacy. Based on our published (Lisowski et al., Nature 2014) and more recent unpublished studies, we propose that murine-human organ xenograft models represent a robust system to more closely predict clinical trial outcomes. Moreover, such models used in combination with multi-species DNAse-shuffled AAV capsid libraries have allowed us to select for AAV vectors that show 10–20 times enhanced transduction of both human liver and muscle. Using new libraries, we have identified new “humanized” liver capsids with high (human selective) transduction properties. The sequence of these new variants are quite different than our original isolates. We have also used newer capsid libraries to isolate chimeric capsids that show enhanced transduction of human and rhesus skeletal fibers. New high-throughput sequencing and bioinformatic algorithms have allowed us to optimize selection screens and potentially identify a larger number of capsids that have clinically important properties. The episomal nature of the vector genomes in transduced cells restrict classical rAAV-mediated gene transfer to quiescent tissues. Moreover, even with the low rate of AAV integration, high rates of hepatocellular carcinoma resulting from promoter activation of oncogenic loci in young mice, and loss of episomal AAV genomes (and hence transgene expression) with normal growth and development has raised concerns about treating infants. To circumvent these concerns and provide a viable approach to treating infants, we have developed an AAV promoterless gene targeting approach without the use of nucleases (Barzel et al. Nature 2015) and use this tactic to successfully treat mice with hemophilia B. We are now expanding this approach for treating a variety of diseases as well as developing novel methods for increasing gene targeted-mediated transgene expression. The discovery and characterization of novel AAV vectors as well as their use in both classical gene transfer and genome-editing approaches broadens their application in both biological discovery and therapeutic applications.
: Technische Universität München
Adoptive transfer of antigen-specific T cells has demonstrated astonishing clinical results in the treatment of infections and some malignancies. The definition of optimal targets and antigen receptors as well as the differentiation status of transferred T cells are emerging as crucial parameters for generating cell products with predictable efficacy and safety profiles. Our laboratory has demonstrated that defined subsets within the memory CD8+ T cell compartment fulfill all key characteristics of adult tissue stem cells and are essential for robust and long-term maintained responses upon adoptive transfer. Enrichment of these memory stem T cells for adoptive T cell therapy, using primary (unmodified) or genetically engineered T cells, can be highly effective, even when low numbers of T cells are transferred. Since such potent T cell products also bear some risk of toxicity, safeguards that allow selective depletion of transferred cells in the case of un-tolerable side effects may be needed. In this context, we explored the capacity of a truncated version of Epidermal Growth Factor Receptor (EGFRt) co-expressed with adoptively transferred T cells. EGFRt is functionally inert, as it cannot bind EGFR-ligands and lacks signaling components, and is non-immunogenic. However, it still binds to Cetuximab, an EGFR-specific antibody already used for clinical applications. We can show in a pre-clinical animal models that EGFRt-expressing engineered T cells can be effectively depleted via Cetuximab treatment in vivo. For example, B cell aplasia, which is a common long-lasting side effect of CD19 CAR-T cell treatment, can be reverted by antibody-mediated in vivo depletion.
: San Raffaele Telethon Insitute for Gene Therapy (SR-Tiget)
Immuno-oncology is an emerging discipline aimed towards exploiting the body's immune system to fight cancer overcoming the profound state of immunosuppression inherent to the tumor and its microenvironment. Adoptive T cell therapy and checkpoint blockade have recently demonstrated the power of immunotherapy in curing advanced malignancies. We are developing novel therapies based on monocytes/macrophages, important mediators of tumor angiogenesis, invasion and metastasis on one hand, and central components of the adaptive immune system on the other. We have developed a platform to genetically engineer hematopoietic stem and progenitor cells (HSPC) with a transcriptionally and post-transcriptionally regulated cassette driving the expression of anti-tumor molecules preferentially into tumor-infiltrating myeloid cells (TIMs) differentiating from stably engrafted HSPC. Type 1-Interferon (IFN) delivered via TIMs inhibited tumor growth in multiple experimental models including breast-, colorectal cancer, myeloma and leukemia. Importantly, locally-delivered IFN augments T cell function leading to cure in a fraction of mice when appropriate immunogenic tumor models are used. We are now preparing a clinical IFN gene therapy trial for patients affected by multiple myeloma, for which we have conducted preclinical toxicity and biodistribution studies. Additional strategies using alternative payloads will be discussed.
: Sangamo BioSciences
Zinc finger nucleases (ZFNs) provide an attractive platform for therapeutic gene editing, as they can be targeted to cleave virtually any chosen sequence and may be optimized to very high levels of activity and specificity. This talk will describe our strategies for generating ZFNs for therapeutic applications, including development of new, highly active ZFN architectures in which the FokI-ZFP domain order has been reversed (i.e. the FokI domain is linked to the amino terminus of the designed ZFP). The development of these architectures has further increased the range of genome sites that can be targeted with highly active and specific ZFNs. Examples will be provided from current programs including development of the albumin locus as a safe harbor for transgene insertion and the knockout of T-cell receptor expression for enhancing allogeneic adoptive T-cell therapies.
In addition, this talk will describe development of a genome editing approach for the treatment of monogenic diseases, including the lysosomal storage disease Mucopolysaccharidosis Type I (MPS I), which is caused by deficiency of the enzyme α-L-iduronidase (IDUA). The current standard of care for MPS I is enzyme replacement therapy (ERT), in which regular infusions of the missing enzyme are required for the lifetime of the patient. ERT is a costly and time consuming treatment for MPS I patients.
Our strategy is to use an in vivo gene editing approach as a one-time treatment, using ZFN-mediated integration of a corrective copy of the human IDUA (hIDUA) gene into the albumin ‘safe harbor locus’ in hepatocytes. After delivery by AAV, ZFN-mediated integration of the hIDUA transgene at the albumin locus allows stable expression of therapeutic protein from the liver, secretion into the plasma, and uptake into secondary tissues for cross-correction of disease phenotypes. Proof of concept data for this “In Vivo Protein Replacement Platform” approach in an MPS I mouse model will be presented.
: Harvard Medical School
MicroRNAs are small non-coding RNAs that regulate gene expression to control important aspects of development and metabolism such as cell differentiation, apoptosis and lifespan. miR-21, miR-155, let-7 and miR-34 are microRNAs implicated in human cancer. Specifically, human let-7 and miR-34 are poorly expressed or deleted in lung cancer, and over-expression of let-7 or miR-34 in lung cancer cells inhibits their growth, demonstrating a role for these miRNAs as tumor suppressors in lung tissue. let-7 and miR-34 regulate the expression of important oncogenes implicated in lung cancer, suggesting a mechanism for their involvement in cancer. We are focused on the role of these genes in regulating proto-oncogene expression during development and cancer, and on using miRNAs to suppress tumorigenesis. In contrast, miR-21 and miR-155 are oncomiRs and up-regulated in many cancer types. We are also developing effective strategies to target these miRNAs as a novel anti-cancer approach. Lastly we are examining the non-coding portions of the genome for mutations and variants that are likely to impact the cancer phenotype. We have successfully resequenced the 3’UTRome and microRNAome from cancer patients with a family history of cancer.
: MolMed S.p.A.
Gene therapy is accumulating evidence for the long-sought promise of being able to dramatically modify the natural history of very severe clinical conditions. Herein, the most recent example is Zalmoxis (MolMed S.p.A.), for which the European Commission granted a Conditional Marketing Authorisation. Zalmoxis is the first immunogene therapy, as patient-specific adjunctive treatment in haploidentical HSCT for adult patients with high-risk haematological malignancies. Zalmoxis is based on genetically engineering donor immune system T cells to carry an inducible “suicide gene”. These cells foster an anti-leukemia effect by eliminating post-transplant immunosuppression prophylaxis and inducing a rapid immune reconstitution, while the suicide gene allows to readily control GvHD, the most significant and serious adverse event in haplo-identical transplantation. Zalmoxis significantly increases long-term survival, regardless of disease status at transplant, thus making HSCT from haplo-donors safer and more effective. In this context, even the most striking scientific breakthrough needs translation to become clinically useful. This is a complex multi-challenge task integrating competences and resources from different players. These include patients&patient organisations to focus on unmet needs, academia and hospitals for cutting-edge discovery and clinical development, biotech and industry to develop processes, technologies and products at large scale, and Regulatory Authorities to frame an efficient set of regulations to guarantee the safe access of innovative therapies to patients. Cell and gene therapy holds promise also thank to this integrated effort; several challenges associated with process development, manufacturing and control still need resolution to make gene therapy available and viable to significant numbers of patients.
: Miltenyi Biotec
Cellular therapies have never been more in vogue than in the last few years. A number of scientific breakthroughs and successful clinical pilot studies have been followed by enormous interest from the industrial sector. Whereas during the development of numerous novel cell and gene therapies, academic institutions have been more than capable of handling investigator-sponsored trials, driving these therapies towards commercialization requires a very different infrastructure. While academic trials are mainly funded by public organisations, where the costs of therapy equal the costs of manufacture, the manufacturing process and concept have to be completely reworked to enable a cell or cellular gene therapy product to be commercially viable. Increasing the scale of production while significantly reducing the cost of goods are prerequisites for commercial success. Automation enables a single employee to manufacture a number of autologous cell products simultaneously. Using closed system manufacturing reduces the risks of contamination, can reduce the requirements for certain in process controls and may in the future reduce clean room requirements. Using the examples of CAR T cell-based immunotherapies and autologous stem cell therapies, it will be demonstrated how production of cell products can be fully automated in a single-use closed system, the CliniMACS Prodigy®. Use of closed systems and automation will allow an easy establishment of robust processes at academic institutes and an effective scaling out of these procedures for commercialization.
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: INSERM U1169, MIRCEN CEA and Université Paris-Sud, Université Paris Saclay, 91400 Orsay, France
The role of cholesterol metabolism in neurodegenerative diseases, particularly Alzheimer's disease and Huntington's disease is increasingly recognized. Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by abnormal polyglutamine expansion in huntingtin (Exp-HTT) leading to degeneration of striatal neurons. Recent evidences indicate that cholesterol homeostasis is altered in both HD patients and HD animal models. Although sterol biosynthesis is reduced in HD patients brains, increased cholesterol content in the caudate has been reported. The major exportable form of brain cholesterol is 24-hydroxycholesterol (24-OHC) generated by the neuronal cholesterol 24-hydroxylase enzyme (CYP46A1). Reduction of cholesterol conversion to 24-OHC is among the mechanisms that could account for this apparent paradox. Indeed, plasma 24-OHC levels are reduced in HD patients who exhibit motor symptoms. A likely explanation is that attenuated production of 24-OHC in HD-affected brains may be a result of reduced synthesis. To address this issue, we investigated (1) the levels of CYP46A1 in post-mortem biopsies from HD patients, (2) the effects of CYP46A1 knock-down in normal mice and (3) the beneficial effects of CYP46A1 overexpression in the striatum of HD mice models using striatal injections of AAV-CYP46A1. We conclude that striatal restoration of CYP46A1 using AAV-CYP46A1 brain delivery holds promise as a therapy to counteract HD.
: Center for Gene Therapy Nationwide Children's Hospital
AVXS-101 is a gene therapy designed to deliver a functional copy of the SMN gene by utilizing the adenovirus associated virus serotype 9 (AAV9) viral vector. In this ongoing phase 1 trial, 15 patients with SMA Type 1 confirmed by genetic testing (with 2×SMN2 copies) were enrolled. Patients received an intravenous dose of AVXS-101 at 6.7 × 10^13 vg/kg (Cohort 1, low dose, n = 3) or 2.0 × 10^14 vg/kg (Cohort 2, proposed therapeutic dose, n = 12). The primary study objective is safety and tolerability, and secondary outcome is time to death or need for ≥16 h of ventilator assistance per day continuously for ≥2 weeks. CHOP-INTEND scores and motor milestone development are also determined as exploratory objectives. As of April 1, 2016, AVXS-101 has shown a favorable safety profile and was generally well tolerated. AVXS-101 appears to demonstrate a positive impact on the disease course of SMA Type 1. All patients in both dosing cohorts remain free of ventilator dependence as defined by the secondary objective, with the median age of all 15 patients at 14.9 months. All patients have seen increases in their CHOP-INTEND scores from baseline: Cohort 1 and Cohort 2 had average increases of 8.7 points and 19.2 points, respectively. Several patients in Cohort 2 have achieved developmental milestones essentially never seen in SMA Type 1 patients. Moreover, AVXS-101 appears to have reduced the expected need for nutritional and pulmonary support. A clinical update will be given at the time of presentation.
: Boston Children's Hospital
Pluripotent stem cells can be cultured from mammalian blastocysts as embryonic stem cells (ESCs) or derived via reprogramming of somatic cells (iPSC). Through their differentiation in vitro, ESC/iPSC represent tractable resources for the study of embryonic hematopoietic development and hold promise for modeling genetic diseases of the blood like immune deficiency, bone marrow failure, and hemoglobinopathy. While current protocols for directing hematopoietic differentiation faithfully recapitulate myeloid lineages, and there have been encouraging reports of NK, B and T cell development, recapitulating the various stages of hematopoietic ontogeny and producing bona fide hematopoietic stem cells (HSC) has proven elusive. Novel strategies to achieve the production of specific hematopoietic lineages, and to achieve the ultimate goal of HSC derivation will be discussed, alongside illustrations of the utility of ESC/iPSC in disease modeling.
: UCL London
In 2011, our group showed that it was possible to achieve therapeutic expression of coagulation factor IX (FIX) at between 1–5% in 10 severe haemophilia B patients following a single administration of a self-complementary, serotype 8 pseudotyped, adeno-associated viral (AAV) vector. The only toxicity observed was transient subclinical transaminitis at the high dose level, which resolved following corticosteroid treatment. FIX expression has remained stable in the majority of patients for >5years permitting these patients to discontinuation of FIX prophylaxis without increasing the risk of spontaneous haemorrhage.
Further advance has been made recently with the use of AAV vectors containing the naturally occurring gain-of-function Padua mutation in the FIX gene. Two on-going Phase I-II studies report a ∼8-fold enhancement of FIX catalytic activity to 25–40% without antibody formation to FIX. The next major breakthrough is likely to emerge from the use of engineered capsids designed to improve the efficiency of AAV gene transfer to the human liver, thus enabling therapeutic gene transfer in humans using substantially lower vector doses. This should further improve safety, whilst easing pressure on vector production, which remains a big challenge.
Progress has also been made with haemophilia A, a more challenging target for gene therapy. Using our codon optimised AAV-FVIII expression cassette a BioMarin sponsored study recently showed Factor VIII expression of between 12–271% in 7 severe haemophilia A patients recruited to the high dose cohort.
Therefore, after decades of failure, rapid progress is being made in the field of haemophilia gene therapy, which should in time spur success with other monogenetic disorders.
Selected Oral Presentation
: Institute of Biomedicine of the University of Barcelona (IBUB), University of Barcelona
Parkinson's disease (PD) is an incurable, progressive disorder leading to premature invalidity and death. Cellular and molecular mechanisms responsible for PD pathogenesis have been proposed based on genetic studies or brain pathology. However, a major impediment to testing PD hypotheses has been the lack of human cell models. Recently, we developed a humanized Parkinson's disease in vitro model using iPSC from patients suffering sporadic or familial PD, and age-matched controls. We found that patients’ DA neurons show PD-relevant phenotypes such as abnormal alpha-synuclein accumulation, alterations in the autophagy machinery, and increased susceptibility to undergo neurodegeneration upon long-term culture. Here we investigated the emergence of early pathogenic events resulting in functional alterations occurring in DAn differentiated from PD patient-specific iPSC. Using calcium fluorescence imaging assays, that allows for the interrogation of how human-derived neurons behave complexively in circuits, we recorded their activity at early stage of dopaminergic differentiation, as well as at long span culture. Interestingly, we found that after 35 days, the total number of spikes and the total amount of active cells in CTRL and PD neuronal culture, were similar. On the contrary, as the cultures matured, while the number of spikes increased in CTRL DA neurons, the numbers of spikes in PD DA neurons did not, leading to a significant difference between PD and control cultures. After 80 days, we also detected synchronized bursts from CTRL neurons that was never found from PD neurons, suggesting an impairment of network formation in PD-derived neuronal cultures.
: University of Manchester
Utilising nanomatrials for an effective and non-invasive in vivo nucleic acid delivery holds great promise for the development of modern therapeutics. Recent advances in nanomaterial-mediated effective siRNA transport leading to therapeutic gene silencing at localised brain foci following stereotactic administration are very promising. Here, we report nanoconstructs comprising amino-functionalised multi-walled carbon nanotubes (f-CNTs) or positively charged liposomes complexed with Bcl-2 targeting siRNA (siBcl-2) stereotactically injected into the striatum of C57BL/6J mice. The aim was to assess its potential as a targeted neuronal therapy to replace surgical resection in focal neurodegenerative diseases. Nanomaterial transport can increase cellular internalisation and residence time of siRNA into a diverse group of neuronal cells when compared to siRNA alone. Following deep-brain gene silencing of the pro-survival Bcl-2 was correlated with localised cellular apoptosis at the site of injection by TUNEL staining. To comparatively assess the spatial distribution of the induced apoptotic effect by both the nanotube- and liposome-based vectors, 3D imaging and reconstruction of the TUNEL-positive apoptotic region was developed. We are proposing the design and application of nanoscale, non-viral vectors as efficacious and targeted vectors for siRNA-mediated gene silencing deeply and focally in the CNS. Compared to surgical resection, this technology and approach could provide safer and less aggressive methods to achieve ablation of neuro-pathological structures.
: UCL Institute of Ophthalmology
Retinal degenerations caused by loss of photoreceptors leads to permanent visual impairment. Attempts to replace rod photoreceptors by transplantation have been studied extensively, but few investigations have focused on cone photoreceptors. Due to their involvement in visual acuity, daylight and color vision, it is of importance to develop stem cell-based therapies for the treatment of blindness caused by loss of cone photoreceptors. In this study we sought to unequivocally establish if human pluripotent stem cell (hPSC)-derived cone photoreceptors can integrate into mouse models of retinal degeneration following transplantation. In order to generate and isolate cone photoreceptors for transplantation, we have adapted two recently reported differentiation protocols that generate neuroretina from hPSCs (Reichman et al., 2014, Zhong et al., 2014). We performed a comprehensive characterisation of hPSC-derived photoreceptor development in vitro, showing that it resembled in vivo human photoreceptor genesis. The hPSC-derived neuroretinas gave rise to ONL-like structures containing photoreceptors that matured in vitro and developed inner segments, connecting cilia and outer segments, as well as ribbon synapses. Importantly, this differentiation system generated cone-rich neuroepithelia that allowed for the first time the isolation of a pure population of human cones for transplantation purposes. Here, we show that a population of M/Lopsin. GFP+ human cone precursors can survive and integrate within degenerate retinas of adult mice. This study is the first to demonstrate the restorative potential of hPS-derived cone precursors for retinal cell therapy.
: GenSight Biologics, Paris, France
LHON is caused by point mutations in mtDNA, the most common of which is the G11778 ND4 mutation. LHON manifests with severe bilateral central vision loss. GS010 is a recombinant AAV2/2 encoding the human wt-ND4 protein. GS010 supports allotopic expression by utilizing the cis-acting elements of the human COX10 gene. A first-in-man, open-label, dose-escalation, Phase I/IIa safety study of GS010 was performed (NCT02064569). Each patient received a single intravitreal injection of GS010 in their worst-seeing eye. Fifteen patients were included and 1-year follow-up is now completed. Eighty-one treatment emergent AEs were documented, 34/81 were considered related to the study drug and 18/81 were considered related to study procedures. One SAE occurred and was not considered related to GS010/procedures. No CTCAE grade 3 or 4 AE occurred. Most (75/81) AEs were of mild intensity. No AE lead to study discontinuation. The most common side effects were transient IOP elevations and ocular inflammations. These side effects were well tolerated, without sequelae and treatment responsive with resolution. No treatment-related loss of vision occurred. Patients with relatively shorter disease duration and better baseline visual function showed trends of treatment effect. For 5 patients with vision loss duration below 2 years and with baseline LogMAR acuity ≤2.79, the difference in change from baseline to week 48 was −0.338 LogMAR, indicating a mean treatment effect of 17 ETDRS Letters improvement of the treated, relative to the untreated eye. Phase III RESCUE and REVERSE trials of GS010 are recruiting LHON patients with vision loss for ≤1 year.
: Weill Cornell Medical College
As neurorestorative approaches based upon stem cell technologies are currently starting or are in planning for clinical trials for Parkinson's disease (PD), it is critical to understand long term outcomes of prior attempts at cell transplant. We performed neuroimaging studies and detailed clinical batteries in 5 surviving patients who underwent embryonic tissue transplant derived from the ventral mesencephalon, as a source of dopaminergic neuroblasts, between 1997–1998 for advanced PD (Freed et al 2001). 11C-PE2i PET imaging (n = 3) was used to detect dopamine active transporter as a marker of dopaminergic neurons, and demonstrated robust irregularly distributed signal in the bilateral putamen, corresponding to the site of transplant. PK11195 uptake measured by PET (n = 3), reflecting microglial activation, was marked in 2 subjects. Motor function was assessed by standardized rating scales in the “off’ state, and by Parkinson's KinetigraphTM continuous monitoring (an accelerometer based wearable device), demonstrating an unexpectedly high level of motor function in a subset of subjects (n = 2) at 28 years and 35 years PD duration. Video-recorded motor examinations will be presented. A subset of subjects also manifested graft-induced dyskinesia (n = 2) and graft-induced dystonia (n = 1). We conclude that in a subset of surviving patients 17–18 years post-transplant, there is evidence of graft survival both radiologically and clinically. There is scarce long term follow up in the published literature of such patients, and our findings both provide support for going ahead with neurorestorative approaches in PD and reinforce the need for sustained surveillance for long term and latent effects of therapy.
: Imperial College London
Most current therapies for Alzheimer's disease (AD) are only symptomatic and fail to improve memory decline and reverse neuronal loss. PPARγ-coactivator-1α (PGC-1α) is a transcriptional regulator of metabolic genes, oxidative phosphorylation and mitochondrial biogenesis. We previously reported that PGC-1α is decreased in AD patients. We also showed that overexpressing PGC-1α in a neuroblastoma cell line increased the neurotrophic sAPPα and reduced Aβ generation by regulating transcription of BACE1. In the present study, we explored the potential therapeutic potential of PGC-1α by generating an HIV-1 based lentiviral transfer vector bearing the human PGC-1α cDNA downstream of the human cytomegalovirus immediate early gene enhancer promoter (CMV). High titer lentiviral vectors expressing either eGFP or PGC-1α pseudotyped with the rabies virus glycoprotein (RVG) were produced. These were stereotactically administered bilaterally in hippocampus and cortex of APP23 transgenic mice at 8-months of age, before plaque formation and behavioral deficits appear. Four months post injection, APP23 mice treated with the PGC-1α vector showed improved spatial and recognition memory and significant reduction in Aβ deposition and microglial activation. These effects were accompanied by marked preservation of CA3 pyramidal neurons and increased expression of neurotrophic factors (BDNF, NGF) in hippocampus and cortex. Wild-type mice that received the PGC-1α vector also demonstrated increased expression of BDNF and NGF, indicating that PGC-1α can regulate these growth factors. Our data suggest that induction of PGC-1α gene expression holds promise as a potential therapy in AD-related neurodegeneration.
: Department of Cell and Developmental Biology, University College London, London WC1E 6DE, UK
The generation of artificial 3D human skeletal muscles could provide an instrumental tool for tissue replacement, disease modelling and development of new therapies. This is indeed becoming a reality in other tissues, where organoid technology is revolutionizing drug development and regenerative medicine. Nonetheless, skeletal muscle tissue engineering is very challenging, due to the complexity of the tissue and its multiple functions. Some laboratories have reported the production of artificial muscles, but all have used tissue-derived cells from invasive biopsies. Although these models could be used for simple studies, they hold the drawback of reduced availability of myogenic cells for large-scale tissue replacement or high-throughput screening of small molecules and gene therapy vectors. Here we present the generation of 3D human skeletal muscles from ES and iPS cells derived from healthy donors and from patients with Duchenne and Limb-Girdle muscular dystrophies. We differentiated human pluripotent stem cells to the skeletal myogenic lineage within a 3D matrix under tension to induce alignment. These human pluripotent cell-derived, disease-specific, artificial muscles recapitulate features of adult muscle tissue and, crucially, efficiently engraft into immunodeficient mice subjected to volumetric muscle loss. Finally, we show that additional cell lineages present in skeletal muscle (e.g. endothelial cells) can be derived from the same pluripotent stem cell source and can be combined within the same patient-specific artificial muscle. This platform lays the foundation for a multi-functional, human skeletal muscle “organoid” for regenerative medicine and therapy screening.
: Laboratory for Viral Vector Technology and Gene Therapy, Division of Molecular Medicine, KU Leuven, Leuven, Belgium
To date, the majority of cystic fibrosis patients receive symptomatic treatment. Gene therapy holds promise for a curative mutation-independent treatment applicable to all patients. First, we studied the longevity of gene expression of rAAV2/5-Fluc to mouse airways. This resulted in detectable gene expression up to 15 months, a substantial proportion of the lifespan of a mouse, with only a 4-fold decrease in bioluminescent signal in lungs. Readministration of rAAV2/5 to the airways was feasible and increased gene expression 14 months after primary vector administration, despite the presence of neutralizing antibodies. We opted for a truncated CFTR which allows insertion of an additional promoter to enhance gene expression levels. First, we evaluated its functionality in highly translational intestinal organoids, which are stem-cell based and self-organize into closed gut-like structures. Upon CFTR activation, the organoids swell allowing quantification which correlates with the amount of CFTR in the luminal plasma membrane. Our data demonstrate that the therapeutic vector rAAV-CFTRΔR is functional and that the ion transport-induced organoid swelling is CFTR-specific. Finally, we administered rAAV2/ 5-CFTRΔR to CF mice by nasal instillation. 4 weeks later, we demonstrated a response to low-chloride and forskolin perfusion in 6 of 8 mice by nasal potential differences, indicating restoration of CFTR activity. In conclusion, we obtained sustained reporter gene expression in murine airways and demonstrated restoration of the CF phenotype in human organoids and CF mice. Our results underscore the therapeutic potential of rAAV-CFTRΔR for CF gene therapy opening new avenues towards a definitive cure for all CF patients.
: UCL Institute of Child Health
Inherited retinal dystrophies resulting in the death of the light-sensitive rod and cone photoreceptor cells are a major cause of incurable blindness. Cone loss has the greatest impact on vision as although less abundant than rods, cones are indispensable for colour vision and high visual acuity. Human pluripotent stem cell differentiation cultures provide a powerful platform to model retinal diseases and a potential renewable source of cone photoreceptors for cell replacement therapy. However, little is currently known about the developmental expression profile of human cone photoreceptors. This project aimed to isolate and compare cone photoreceptors from the human foetal retina with those from human induced pluripotent stem cell (hiPSC)-derived retinal cultures, in addition to identifying novel cell surface markers of cones to develop a cell isolation strategy. A human iPSC-derived retinal organoid culture system was established that showed upregulation of cone gene transcripts from 5 weeks to 28 weeks of culture. An adeno-associated viral vector carrying an L/M-opsin.GFP reporter was used to specifically label human foetal L/M-opsin cones and hiPSC-derived retinal cells expressing photoreceptor markers, Recoverin and Crx. Single cell and total RNA seq was performed on FAC-sorted L/M-opsin.GFP labelled human retinal cells and differential biomarker expression was explored. Cell surface markers that label L/M-opsin.GFP expressing cells were identified using high-throughput antibody screening panels. Combinations of cell surface markers applied in FAC-sorting experiments provided enrichment for L/M-opsin cones compared to unsorted cells. These data provide the first comparative transcriptomic analysis of in vivo and in vitro generated human cone photoreceptors.
: Department of Oncology and Hemato-oncology, University of Milan, c/o Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, Milan 20139, Italy
Induced pluripotent stem cells (iPSCs), coupled with recent advances in differentiation protocols, are becoming a transforming tool for drug discovery, allowing the creation of complex and phenotypically accurate cellular models of disease for preclinical and translational research. In this context, High Throughput Screenings (HTS) of small molecules in human iPSC-derived cells promise to shape a new landscape for the identification of therapeutic agents, but they still face substantial challenges in order to adapt and validate the complexities of sophisticated differentiation paradigms for a high-throughput scale. Here, we addressed these challenges for two neurodevelopmental disorders caused by symmetrical copy number variations at 7q11.23: Williams-Beuren (WBS) and 7q-microduplication syndrome, characterized by, respectively, the hemyzygous deletion or duplication of a genetic interval encompassing 26-28 genes. Specifically, we present the integration of the following key innovations: i) a panel of stable monoclonal Neurogenin-2 engineered patient derived-iPSC lines along with a modified differentiation protocol for conversion into mature cortical glutamatergic neurons without the aid of astrocytes; ii) miniaturization and automation of the whole process into 96-well microplates, where both iPSCs and iPSCs-derived NGN-2 neurons retain the same relevant differences in gene expression. Our results establish a foundational resource for a small molecule HTS campaign in patient-derived NGN-2 neurons, with the aim of identifying compounds capable of modulating the expression of WBS interval genes, as an innovative platform for the discovery of new leads or the repositioning of available drugs for a critical area of unmet medical need such as intellectual disability and autism.
: San Raffaele Scientific Institute
Schwann cells (SCs) generate the myelin wrapping the peripheral nerve axons. Transplantation of SCs is an interesting therapeutic opportunity for the treatment of injuries and demyelinating diseases of the peripheral nervous system (PNS). However, to date a renewal source of SCs is lacking. In this study, we established a genetic system based on the minimal combination of two transcription factors, supporting the direct conversion of skin fibroblasts into induced Schwann cells (iSCs). iSCs strongly resembled primary SCs in global gene expression profiling and PNS identity. Remarkably, iSCs wrapped axons generating compacted myelin sheaths with regular nodal structures. Conversely, iSCs from Twitcher mice showed a severe loss in the myelinogenic potential indicating iSCs as an attractive system for in vitro modeling of PNS diseases. In vivo studies on a rat model of peripheral nerve injury demonstrated that grafted iSCs sustained nerve regeneration in an autologous transplantation strategy. Generating iSCs through direct conversion of somatic cells disclose significant opportunities for in vitro disease modeling as well as cell regenerative therapies.
: Dept of Biomedical and Biotechnological Sciences (BIOMETEC), University of Catania Medical School, Catania 95125, Italy
Stem cells residing within the adult brain represent a promising tool for treating Parkinson's disease (PD), a neurodegenerative disorder characterized by the progressive loss of dopamine producing midbrain (mDA) neurons. Within their specialized germinal niches, populations of local astrocytes instruct neural stem/progenitor cells (NSCs) via complex cell-cell interactions and signaling cascades, which include the activation of the Wnt/β-catenin pathway. We have recently uncovered that the midbrain aqueduct (Aq)-periventricular regions (PVRs) SVZ act as a natural niche for mDA progenitors. Accordingly, mDA neuron death induced by the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) promotes an early activation of these Aq-PVR-DA progenitors, but a lack of appropriate niche environmental signals restrict their neurogenic potential and compromise neuronal survival/rescue. Given that transplanted NSCs possess intrinsic capacity to ameliorate the injured microenvironment and to rescue dysfunctional neurons, here we used adult green fluorescent protein (GFP)+ NSCs as a graft source for unilateral transplantation above the subtantia nigra (SN) of MPTP mice. Remarkably, grafted GFP-NSC survived and proliferated within the SN, in situ. Spatio-temporal analyses showed a significant protection/restoration of SN-TH+ cell bodies. Additionally, GFP+-NSCs were seen to accumulate at the Aq-SVZ niche where they induced a profound remodelling of host GFAP+ astrocytes and β-catenin over-expression thus suggesting activation of astrocyte-dependent Wnt signaling. Increased β-catenin expression was also observed in SN-repairing neurons together with a robust striatal reinnervation, thereby uncovering a critical role of NPC crosstalk with the host niche and DA neurons via astrocytes for DA neuroprotection and neurorestoration, with implications for cell-based therapies for PD.
: Case Western Reserve University
Pelizaeus-Merzbacher disease (PMD) is an X-linked orphan disease caused by genetic aberrations in the proteolipid protein 1 gene, resulting in loss of myelinating oligodendrocytes. Patients exhibit a range of neurological symptoms, and the disease is invariably fatal. Unfortunately, no treatment options exist, owing to our lack of understanding for the drivers of disease in PMD. To probe these mechanisms, we generated induced pluripotent stem cells (iPSCs) from the jimpy mouse, a spontaneous model of the severe connatal form of PMD, and identified previously unrecognized disease pathophysiology. In vitro, time-lapse imaging analysis of differentiating iPSCs showed a severe loss of jimpy oligodendrocytes resulting from death of oligodendrocyte progenitor cells as they commit to an oligodendrocyte fate. Strikingly, the rare jimpy oligodendrocytes that escape this critical period appear morphologically normal, suggesting that the enhancement of jimpy oligodendrocyte progenitor cell differentiation may provide a new therapeutic modality for PMD. To identify chemical therapeutics for PMD, we performed an in vitro, high content imaging-based phenotypic screen on over 3,000 bioactive compounds. We validated six compound hits that significantly enhanced successful differentiation and survival of jimpy oligodendrocytes in vitro, restoring oligodendrocyte number to wild-type levels. Administration of the top hit to postnatal jimpy pups resulted in a significant enhancement of oligodendrocyte number and myelination. Our work provides the rationale for clinical advancement of candidate small molecule therapies for PMD patients and more broadly, demonstrates the power of pluripotent stem cell technologies to reveal previously unknown disease mechanisms and provide a platform for genotype-specific precision medicine.
: Institute for Neuroscience, Medical University Innsbruck
Friedreich's ataxia (FRDA) is a severe autosomal recessive neurodegenerative disease caused by an intronic mutation in the nuclear-encoded frataxin gene, which results in reduced levels of the frataxin protein in mitochondria. Most affected cell types comprise sensory neurons and cardiomyocytes. We previously reported a FRDA cellular model based on human sensory neurons generated via the iPSC technology. We showed increased levels of frataxin during the differentiation of the control but not of the FRDA sensory neurons. Here we address bioenergetic and mitochondrial functions during in vitro differentiation and maturation of the sensory neurons. High resolution respirometric measurements were complemented with mitochondrial membrane potential and cell death assessments. ATP-linked respiration, proton leak, maximal respiration, spare respiratory capacity and non-mitochondrial respiration were determined by measuring the oxygen consumption rate together with modulators of cellular respiration in both intact and permeabilized cells. We found that sensory neurons were especially shifted to mitochondrial respiration. Lower respiratory and mitochondrial membrane potential values were found in FRDA cells compared with control cells, with the highest difference observed in sensory neurons. Uncoupling the proton gradient established by the electron transfer chain of the inner mitochondrial membrane with the protonophore FCCP produced a massive cell death in FRDA sensory neurons, but not in control neurons or in non-sensory populations. These results confirm a mitochondrial disease phenotype in FRDA sensory neurons. Our cell-specific and functional FRDA model is yet prone to screening and optimising drugs and gene therapy (Project P26886-B19 FWF Austria).
: Celixir Limited
In heart failure, non-reversible or Fixed defect (FD) myocardial scar tissue formed after a myocardial infarction is identified by Late Gadolinium Enhanced MRI (LGE-MRI) or rest-redistribution SPECT imaging as metabolically inactive tissue. Rest-redistibution SPECT distinguishes between hibernating myocardium that can be improved with current treatments (CABG Bypass) and true fixed defect (FD) myocardial scarring that is impervious to current therapy. The Heartcel Phase II clinical study investigated the effect of intramyocardial injection of iMP cells (immune-Modulatory Progenitor cells, Celixir Ltd UK, at the time of CABG in advanced heart failure patients. Pre- and post-operatively rest-redistribution SPECT imaging assessed FD scar and segmental disease severity. Post-hoc randomized and blinded analysis assessed 140 patient segments, identifying 108 segments contained FD scar pre-operatively. After iMP treatment (post-op) 83 of the 108 FD segments showed improvement. 28 segments were clear of FD scar and 53 had reduced FD scarring. The reported SPECT error margin is 4%-9%. The observed improvement of (77%, P < 0.01) is surprising and very encouraging for the Heart failure.
: iBET/ITQB
The production of human pluripotent stem cell derived cardiomyocytes (hPSC-CM) holds great promise for cardiotoxicity drug testing, disease modeling and cardiac regeneration. However, the complex nets of signaling pathways involved in cardiomyogenesis compromises the effectiveness of the existing differentiation protocols to reproducibly produce high-quality hPSC-CM. Produced hPSC-CM are immature compared with adult CMs, express typical fetal cardiac genes, use glucose as major energy source and have immature structural and electrophysiological properties. In this study we aim to overcome these hurdles by devising an integrated strategy for production and maturation of functional hPSC-CM. hPSC (hiPSC and hESC lines) were differentiated into CMs, using a novel directed differentiation protocol. CM differentiation was monitored by flow cytometry, qRT-PCR and proteomic analyses. Different medium compositions were tested aiming to improve hPSC-CM maturation. Metabolome, fluxome and transcriptome analyses were used to evaluate the impact of feeding strategies on hiPSC-CM fate. Structure, ultrastructural and functionality of the hPSC-CMs were also analyzed. We showed that our differentiation protocol originates pure 2D monolayers and 3D aggregates of hiPSC-CMs that present typical CM transcriptome and proteome profiles. Also, we demonstrated that hiPSC-CMs cultivated in glucose depleted medium supplemented with fatty acids display more aspects of maturation than hiPSC-CMs cultured in glucose rich medium namely: increased oxidative capacity, up-regulation of cardiac specific genes; more elongated morphologies; organized sarcomeric structures; higher myofibril density and alignment; faster calcium-transient kinetics; higher contractile force; enhanced AP durations and upstroke velocities; all signs of a more mature CM phenotype.
: Department of Health Sciences, Università del Piemonte Orientale, Novara, 28100, Italy
Hemophilia A (HA) is an X-linked bleeding disorder caused by factor VIII (FVIII) gene mutations. We generated iPSCs from healthy and hemophilic donors peripheral blood CD34+ cells, by reprogramming with a Cre-Lox lentivector (LV) carrying OCT4-SOX2-KLF4 and miRNA302/367. Reprogrammed CD34+ cells originated ESC-like-iPSCs colonies positive for stem cell markers, increased telomeres length and normal karyotype. EBs expressed germ layers specific markers and differentiated in osteogenic, chondrogenic and adipose tissues. iPSCs were differentiated into endothelial cells (EC) with an optimized protocol, acquired endothelial-like morphology, expressed ECs markers and were able to form tubules when cultured in matrigel. IPSC-derived ECs were transduced with a LV carrying GFP driven by an endothelial-specific promoter (VEC). Cells transplanted intraportally in NOD/SCID-γNull (NSG) mice, engrafted and proliferated in the livers up to 12 weeks and confirmed by FACS analysis to be GFP and CD31+ representing the 30% of liver non-parenchymal cells. Moreover, transplanted cells formed vessels-like structure in the host liver. These results demonstrated that healthy iPSCs-derived ECs were able to engraft and proliferate in mouse liver after transplantation. Finally, we transplanted HA-IPSC-derived ECs corrected by LV-VEC-FVIII in NSG-HA mice to evaluate FVIII secretion and phenotype correction. Transplanted mice showed a reduced bleeding time and a stable 5% FVIII activity measured by aPTT until 12 weeks. Taken together, these results demonstrated that hemophilic phenotype could be rescued by transplantation of ECs derived from HA-iPSCs and corrected by LV carrying FVIII under the control of an endothelial-specific promoter confirming the suitability of this approach for HA gene-cell-therapy.
: Inserm
Right ventricular (RV) failure is a major concern in grown-up congenital heart diseases. Cell-based myocardial repair is an innovative approach. The aim is to regenerate the failing RV myocardium in a repaired Tetralogy of Fallot pig model. After 4 months of a combined overloaded RV, cell therapy was surgically administrated in 6 pigs using human NKX2.5+ cardiac progenitor cell-seeded collagen patches (3 patches,107 cells within each patch) applied on the epicardium. Myocardial function was assessed by standard and strain echocardiography before cell therapy, 1 month (n = 4) and 2 months (n = 2, ongoing) after cell graft. In the last two pigs, both a programmed ventricular stimulation and an intramyocardial electrophysiological study were performed. The fate of progenitors was studied using antibodies directed against an anti-human mitochondrial marker, -troponin T (TnT), -Nkx2.5, -CD31, Ki67, actinin. After cell therapy, RV dilatation slowed down in all pigs. Median values of tele-diastolic area slightly decreased of 8.8 %. Global RV function improved: median values of indexed TAPSE, FAC and S’ increased of 34%,16%, and 21%, respectively. The RV free wall strain improved in all pigs (26% increase of the median value). No arrhythmia was induced, nome of electrophysiological parameters was modified. Differentiating human cardiac progenitors were found in the patch and migrated within the myocardium close to the endocardium. Although still immature, some cells featured TnT+ sarcomeric units. Human cardiac progenitors migrated toward endocardium and differentiated within interstitial area, contributing to maintain RV adaptation to overload and to improve systolic function without any rhythmic adverse effects
: San Raffaele University, Milan
: UCL Cancer Institute
T-cell lymphomas are aggressive, treatment-resistant and associated with poor prognosis. Translation of immunotherapeutic approaches has been limited by a lack of target antigens discriminating malignant from healthy T-cells. Unlike B-cell depletion, pan T-cell aplasia would be prohibitively toxic. One potential target, the alpha/beta T-cell receptor (TCR), is expressed on >90% of tumours but also on all normal T-cells. However, following TCR recombination, each T-cell assembles a unique beta-chain. Cells of a cancer all express the same clone-specific chain. While generating a bespoke therapeutic for each cancer is impractical, a compromise is possible. The TCR β-constant region comprises two functionally identical genes: TRBC1 and TRBC2. Each T-cell clone expresses only one. We identified an antibody with unique TRBC1 specificity and used this to demonstrate that while polyclonal (Figure 1a) and viral-specific normal T-cells contain both TRBC1 (1/3) and TRBC2 (2/3) populations, cell lines and primary T-cell tumours (Figure 1b) are entirely restricted to one compartment. We developed anti-TRBC1 chimeric antigen receptor (CAR) T-cells. CARs have shown dramatic recent results in the treatment of advanced B-cell cancers. Anti-TRBC1 CART-cells specifically recognised and killed normal and malignant TRBC1 and not TRBC2 cells in vitro and in a murine model of disseminated leukaemia. Given the relatively similar presentation and nature of B- and T-cell cancers, we hypothesise that anti-TRBC1 CAR could prove also efficacious in T-cell disorders. Unlike non-selective approaches targeting the entire T-cell population, our strategy could eradicate a T-cell malignancy while preserving sufficient normal T-cells to maintain cellular immunity.
: Massachusetts General Hospital
Glioblastoma, the most common primary brain tumor in adults, is invariably fatal. Glioblastoma contains cancer stem cells, likely important in tumor progression, heterogeneity, recurrence and resistance. We used a new mouse syngeneic glioblastoma stem cell (GSC) tumor model with 005 GSCs, isolated from activated H-Ras and Akt induced, Tp53+/- gliomas in C57Bl/6 mice. 005 GSCs are highly tumorigenic, relatively non-immunogenic, and recapitulate the hallmarks of human glioblastoma. Immunovirotherapy arises from the natural inflammatory responses to virus infection and oncolytic virus-induced cancer cell death to drive antitumor immunity. Oncolytic herpes simplex viruses (oHSVs) are genetically engineered to selectively replicate in cancer cells and not normal cells. We have combined G47Δ-mIL12, an oHSV expressing murine IL-12, a critical cytokine involved in adaptive and innate immune responses as well as anti-angiogenesis, with blocking antibodies to immune co-inhibitory molecules like CTLA-4 and PD-1. Mice with established 005 GSC-derived brain tumors were treated with a single intratumoral injection of G47Δ-mIL12 followed by systemic administration of anti-mPD-1, anti-mPD-L1, anti-mCTLA-4, or isotype control antibodies. Monotherapy modestly improved survival compared to mock treatment. The combination of G47Δ-mIL12 with a single checkpoint inhibitor further extended survival. However, the triple combination of G47Δ-mIL12 with two checkpoint inhibitors cured most of the mice and protected them from tumor rechallenge. We have examined the immune cells infiltrating the tumor that are associated with this response. In this representative GSC model, the combination of intratumoral oHSV expressing IL12 and two systemic checkpoint inhibitors was necessary to eliminate established tumors.
: San Raffaele Scientific Institute
In patients with acute myeloid leukemia (AML), relapse is the major cause of death after allogeneic HSCT. To investigate if T-cell dysfunction is associated to post-transplant relapse, we longitudinally analyzed bone marrow (BM) and peripheral blood (PB) samples of 30 AML patients receiving HSCT from HLA-identical (HLAid, n = 18) or HLA-haploidentical (haplo, n = 12) donors. The expression of inhibitory receptors (IRs) on T-cell subsets was investigated by multi-parametric flow cytometry. BM and PB were collected at 60 days and at relapse (median 219d; n = 16) or at 1 year in case of complete remission (CR; n = 14). After haplo-HSCT, PD-1, CTLA-4, 2B4 and Tim-3 were upregulated in BM- and PB-T cells at all time-points, compared to healthy donors (HD) and independently from the clinical outcome. Conversely, after HLAid-HSCT, relapsing patients displayed higher frequencies of BM-infiltrating T cells expressing PD-1, CTLA-4 and 2B4 than CR patients and HD (p < 0.05). In the BM of HD, IR expression was confined to effector memory and effectors. While a similar IR distribution was observed in CR, at relapse, PD-1, 2B4 and Tim-3 were also significantly upregulated in BM-infiltrating central memory and memory stem T cells. Interestingly, at relapse, leukemias expressed PD-L1 (10/10 cases) and Galectin-9 (7/10). Results were confirmed also by using the bh-SNE algorithm, an unbiased computational method for the analysis of FACS data. In conclusion, after HLAid-HSCT, exhausted T-cell signature in relapsing patients includes PD-1, CTLA-4, 2B4 and Tim-3. IR expression on early-differentiated T cells at relapse suggests a wide immunological dysfunction mediated by AML relapsing blasts.
: San Raffaele Telethon Insitute for Gene Therapy (HSR-TIGET)
Although hematopoietic stem/progenitor cells (HSPC) are widely exploited in clinical gene therapy (GT) the contribution of each progenitor to engineered hematopoiesis after transplant remains unknown. Combining phenotypic characterization and integration sites (IS) tracking we studied for the first time in humans at the clonal level the dynamics after transplant of 7 HSPC subtypes including hematopoietic stem cells (HSC), multi- potent progenitors (MPP) and lymphoid- or myeloid-committed precursors through CD34, CD38, CD90, CD45RA, CD7, CD10 and CD135 surface markers. We isolated bone marrow (BM) progenitors from 7 Wiskott-Aldrich syndrome (WAS) patients treated with autologous lentiviral transduced BM and/or mobilized peripheral blood (MPB) HSPC (1 BM+MPB, 5 BM and 1 MPB) and detected 10.394 unique IS at early (1–3 months) and steady-state (2–5 years) reconstitution phases post-GT. Phenotypic characterization revealed early enrichment of committed progenitors followed by stabilization of HSPC composition starting from 1–2 years post-GT. Measurement of HSPC clonal output through IS suggests a segregation of tasks of early vs. steady-state progenitors after transplant. Interestingly, IS shared among HSPC subtypes were also highly represented in mature lineages, indicating our capability to track in real-time active progenitors in vivo. When comparing MPB vs. BM HSPC cells dynamics, we unveiled a higher number of neutrophils clones in MPB-GT at early time-points, in line with faster myeloid reconstitution. We are investigating the relationships between HSPC subtypes isolated from two distal BM sites within the same patient and time-point post-GT. Overall, this study would provide key information on HSPC behavior after transplant in vivo in humans.
: CIRI; Inserm U1111, Lyon, France
A challenge in the field of gene therapy is the transduction of HSCs with high efficiency without compromising their stemness. Here we show that after a single application, Measles virus envelope pseudotypes LVs (MV-LVs) transduced 100% of hCD34+- cells prestimulated with a mild cytokine cocktail and up to 70% of quiescent hCD34+-cells, while VSV-G-LVs reached only 5 % of transduction. Importantly, reconstitution of primary and secondary recipient NSG mice with these MV-LV transduced hCD34+- cells demonstrated that this high transduction levels were maintained in all hematopoietic lineages. Notably, we found increased transduction levels (up to 100%) in all human cells in these secondary recipients for the resting MV-LV transduced hCD34+ cells, emphasizing that these vectors preferentially gene-modified HSCs. Fanconi anemia (FA) is a rare monogenic inherited disease characterized by bone marrow failure, congenital abnormalities and cancer predisposition. HSC-based gene therapy has been proposed for FA treatment but the reduced reservoir of HSCs in FA patients together with the significant loss of HSCs associated with CD34+ cell isolation and cytokine stimulation results in reduced numbers of HSCs for re-infusion in the patients. MV-LVs showed a marked increase in the transduction efficiency and FA correction of CD34+ cells from unfractionated BM from FA patients in comparison with classical VSVG-LVs. This reinforces the relevance of conducting gene therapy approaches using unfractionated BM combined with minimal stimulation to avoid the loss of hematopoietic progenitors in many diseases but in particularly in bone marrow failure syndromes, such as FA.
: University Health Network, Toronto, Ontario, Canada
Better understanding of normal blood differentiation programs during ontogeny was achieved recently (F.Notta et al, Science, 2016) by applying single-cell differentiation assay of highly purified HSPC populations. Our knowledge of the key genes action involved in HSPC development or disease progression requires their introduction to the particular cell populations through gene transfer. We apply single-cell erythro-myeloid stroma-based differentiation assay to interrogate differentiation and proliferative potentials of human HSPC following introduction of neutral marker gene. The major differentiation characteristics of HSC (CD34+CD38-CD45RA-CD90+CD49f+) and multipotent progenitors (CD34+CD38-CD45RA-CD90-CD49f-), i.e. their ability to produce myeloid, erythroid, erythro-myeloid and erythro-myelo-megakaryocitic colonies were not significantly changed by the gene transfer procedures. The investigation of proliferative potential of HSC revealed their huge heterogeneity in cell production after 15–17 days of cell culture. Thus, the size of the colonies produced by single cells varied from 850 to 313000 cells pointing out that HSC and its progeny might proliferate with the rate of more than 1 division per day. Moreover, heterogeneity of HSC proliferative potential was not dramatically changed by gene transfer procedure raising the question about HSC functional homogeneity. The interrogation of differentiation and proliferation potentials of HSPC following the introduction of two genes involved in cell sphingolipids metabolism revealed that their overexpression led to significant lost of HSC proliferative potential mainly due to the substantial decrease of the erythroid cells produced by single cell. In progenitor populations the effect of overexpression of this two genes was less evident demonstrating their importance for HSC function
: Institute of Medical Science, University of Tokyo
Cord blood (CB) has proven its value as a donor source in hematopoietic cell transplantation. Despite its utility, the use of CB transplantation (CBT) is often limited due to low graft cell numbers, which would lead to delayed engraftment. To overcome this cell dose barrier, we sought to ask if multiple units in combination could be used to improve CBT. To this end, we first modeled an “insufficient dose” CBT setting in lethally irradiated mice. In this model, a mixture of hematopoietic stem/progenitor cells (HSPCs) derived from 4 mouse allogeneic strains rescued recipients and significantly accelerated hematopoietic recovery, irrespective of marked MHC disparities. Enhanced early hematopoiesis was demonstrated in leukocytes, erythrocytes, and platelets, with the evidence of donor cell contribution by allogeneic cells. Of note, inclusion of T cells from one strain was shown to achieve stable single-donor chimerism through graft versus graft reactions, without affecting early hematopoietic recovery supported by a mixed allogeneic HSPCs. Furthermore, we succeeded in setting up clinically relevant procedures that enabled isolation of a mixture of CD34+ cells from 9 frozen CB units at one time regardless of HLA-type disparities. We demonstrated that a mixture of CD34+ cells obtained from multiple units could enhance early human hematopoiesis when supplied to an “insufficient dose” xenotransplantation model (NOG mice). We here provide proof of concept that combined multiple units of allogeneic HSPCs are capable of early hematopoietic reconstitution while allowing single-donor hematopoiesis by a principal graft. Published online J Exp Med. 2016 Aug 8. doi: 10.1084/jem.20151493
: Bambino Gesù Children Hospital, Department of Onco-Haematology and Trasfusional Medicine
: apceth GmbH & Co. KG, D-81377, Munich, Germany
Chronic obstructive pulmonary disease (COPD) is a devastating progressive lung disease characterized by airflow limitation, chronic bronchitis, emphysema, and small airway remodeling. It is currently the third leading cause of death worldwide. There is no cure for COPD as yet, thus innovative alternative treatments are desperately needed. We developed a novel cell product composed of MSCs genetically modified to express the protease inhibitor AAT, combining the potent immunomodulatory and cytoprotective properties of MSCs and of AAT. After generation of an optimized AAT expression cassette and confirmation of in vitro functionality by a neutrophil elastase inhibition assay, we established a process for large scale transduction and expansion of gene-modified MSCs. To evaluate the therapeutic benefits of AAT-MSCs in vivo, a mouse model of elastase-induced emphysema was performed. Treatment of the mice with AAT-MSCs resulted in significant improvement of pulmonary function parameters, while a slight functional amelioration was observed after treatment with non-modified MSCs. Histopathologic examination of AAT-MSC-treated mice revealed a significant decrease of airspace enlargement, indicating regeneration of pulmonary tissue. Again, the effect was smaller after treatment with non-modified MSCs, demonstrating a synergistic mechanism of AAT and MSCs. In conclusion, the present study provides the first in vivo proof of concept for the treatment of emphysematous COPD with AAT-MSCs.
: Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, 28040 Madrid, Spain
The efficacy of Mesenchymal Stromal Cells (MSCs) to improve the HSC engraftment has been previously reported in xenogeneic and specific allogeneic transplants. Also, we have recently demonstrated that the co-infusion of MSCs with low numbers of purified HSCs significantly improve the short- and long-term hematopoietic reconstitution in an autologous HSCT experimental model with sublethal conditioning (5Gy). With the aim of approaching to a more clinically relevant model, we have studied the effect of MSCs co-infusion in a mouse model of hematopoietic gene therapy of Fanconi anemia (Fanca-/- mice), with risk of engraftment failure. In these experiments, the infusion of low numbers of WT LSK cells (1,500 LSK) in Fanca-/- mice resulted in 30% graft failure, which was prevented when 6.105 Ad-MSCs were co-infused. When 1,500-3,000 Fanca-/- LSK cells transduced with a therapeutic lentiviral vector (PGK-FANCA-wPRE*) were transplanted, the infusion of similar cell doses resulted in more than 50% of engraftment failure, which decreased to 30% only when more than 10,000 gene-corrected LSK were infused. Once again, Ad-MSCs co-infusion prevented engraftment failures in the short and long-term after the infusion with the same number of gene-corrected LSK cells. Taken together, our results demonstrate the potential of Ad-MSCs to avoid graft failure in a clinically relevant model of hematopoietic gene therapy with risks of engraftment failure.
: University of bristol
Lack of growing and remodelling potential of available grafts is the bottleneck of current congenital heart defect treatment in paediatric surgery. We tested the possibility of solving this problem by engineering a decellularised graft with human umbilical cord blood stem cells (HUCB-SCs) or newborn swine peripheral blood SCs (SPB-SCs), and the feasibility in a large animal model. HUCB-SCs and SPB-SCs were isolated, expanded and characterised in vitro. Cells were then seeded onto the decellularised porcine small intestinal sub-mucosa and incubated in a bioreactor for 10 days. Non cell seeded or swine cell-seeded scaffolds were shaped into a conduit and implanted into the left pulmonary artery of 12-15 kg piglets. Six months after surgery, grafts were harvested and analysed. Both HUCB-SCs and SPB-SCs displayed mesenchymal stem cell-like phenotype and were capable of differentiating into adipocytes, osteocytes, chondrocyte and smooth muscle cells (SMCs). Graft-implanted piglets recovered well and grew at normal rate. All grafts appeared patent at echocardiography 6 months after implantation. However, while the acellular-graft implanted animals showed light stenosis and higher blood flow velocity through the pulmonary artery, no abnormal feature was observed in the celullarised-graft group. At histology, the explanted cellularised-grafts revealed lumen endothelialisation and a multi-layer of smooth muscle-like cells within the vessel wall. The acellular graft exhibited a relative patchy luminal cell layer and a thinner SMC layer. Results indicate that umbilical cord blood is a valuable stem cell source for tissue engineering of grafts to correct cardiac defects, especially in the paediatric surgery.
: University of Massachusetts Medical School
Alpha-1 antitrypsin (AAT) deficiency is a monogenic disorder resulting in emphysema due principally to the unopposed effects of neutrophil elastase. We previously reported achieving plasma wild-type (M) AAT concentrations at 2.5–3.8% of the purported therapeutic level at 1 year after a single intramuscular (IM) administration of recombinant adeno-associated virus serotype 1 (rAAV1)-AAT vector in AAT-deficient patients. Here, we report sustained expression at 2.0–2.5% of the target level from years 1–5 in these same patients without any additional rAAV1-AAT administration. In addition, we observed partial correction of disease-associated neutrophil defects, including neutrophil elastase inhibition, markers of degranulation and membrane-bound anti-neutrophil antibodies. There was also evidence of an active Treg response (similar to the 1 year data) and an exhausted cytotoxic T-cell response to AAV1 capsid. These findings suggest that muscle-based AAT gene replacement is tolerogenic and that stable levels of M-AAT may exert beneficial neutrophil effects at lower concentrations than previously anticipated.
: San Raffaele Telethon Institute for Gene Therapy (SR-TIGET)
Lentiviral vectors (LV) are attractive vehicles for liver-directed gene therapy by virtue of their ability to stably integrate in the genome of target cells and the absence of pre-existing anti-vector immunity in most humans. Over the past years, we have developed LV that can achieve stable transgene expression in the liver, induce transgene-specific immune tolerance and establish correction of hemophilia in animal models upon systemic administration. This LV is designed to stringently target transgene expression to hepatocytes through transcriptional and microRNA-mediated regulation. We have recently evaluated our gene therapy approach in a canine model of hemophilia B. We treated 4 dogs and observed stable reconstitution of canine factor IX (FIX) activity up to 9% of normal with >10 years of cumulative follow up. To reduce the immunogenicity of LV for in vivo administration, we disrupted the beta-2 microglobulin gene, thus obtaining cells devoid of surface-exposed MHC-I that produced MHC-free LV. These LV showed significantly reduced immunogenicity on human primary T cells co-cultured with autologous monocytes exposed to LV, from several healthy donors. Moreover we have generated LV with increased levels of a phagocytosis inhibitor on the vector surface, that show decreased transduction of human macrophages in vitro and spleen macrophages in mice and may have several advantages for in vivo delivery, in reducing innate and adaptive immune response to the viral particles. Overall, our studies position LV for further pre-clinical development and clinical translation to complement other available vectors for liver-directed gene therapy of hemophilia and conceivably other diseases.
: School of Biological Sciences, Royal Holloway, University of London, Egham, UK
Among triplet expansion diseases, oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant, late-onset muscle disorder characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. OPMD is caused by a short trinucleotide repeat expansion in the polyadenylate-binding protein nuclear 1 (PABPN1) gene that results in an N-terminal expanded polyalanine tract. PABPN1 controls several biological processes such as the length of mRNA poly(A) tails, the mRNA export from the nucleus and the alternative poly(A) site usage. OPMD is characterized by nuclear aggregates of expanded PABPN1, fibrosis and muscle atrophy. Our phase I/IIa clinical study (NCT00773227) using autologous myoblast transplantation in adult OPMD patients showed safety, tolerability and efficacy. However the protocol involved the autologous transplantation of unmodified cells from spared muscles still carrying the genetic mutation. Here we demonstrate using an OPMD mouse model that a gene therapy strategy based on DNA-directed RNA interference to silence the endogenous expPABPN1, combined with the re-expression of a healthy sequence-optimized human PABPN1 gene, significantly reduced the amount of nuclear aggregates in affected muscles, decreased the intramuscular fibrosis, reverted the muscle strength to the level of healthy wild-type muscles and completely normalized the muscle transcriptome. Importantly the efficacy of the combined treatment was also verified in cells derived from OPMD patients. These results obtained in a relevant mammalian animal model of OPMD and in patient cells pave the way for the clinical application of gene and cell therapies as a treatment for OPMD patients.
: UCL Institute of Child Health
Treatments aimed to target skeletal muscles are a big challenge nowadays. As an alternative to conventional medications, which predominately slow down disease progression and relief symptoms in patients, gene therapy opens new promising horizons of a personalised medicine and therapeutic treatments1. Pompe's disease is a metabolic disorder caused by mutations in the acid-alpha-glucosidase (GAA) gene2. The deficiency of GAA dramatically impairs skeletal muscle, cardiac and pulmonary function which untimely leads to death before patients turn 1-year old2. The aim of this study is to develop a haematopoietic-stem-cell (HSC) gene therapy to obtain a long-lasting systemic expression of GAA thereby restoring GAA functions in the entire muscular system. To achieve this, we generated a third-generation-lentiviral-vector encoding the GAA gene under the transcriptional control of EFS-promoter and β-globin-LCR-enhancer which allows overexpression in the erythroid component3. In-vitro studies show that the vector increases the expression and activity of GAA in murine-erythroleukemia, K562 and CD34+-cells differentiated into erythrocyte-like cells. As proof of concept, HSCs from males of a GAA-/- murine colony were treated ex-vivo with the construct and then transplanted in female recipients. Mice receiving gene-corrected-HSCs show high enzymatic activity in plasma and reduced disease biomarker in urine. Treated and untreated animals were then tested for muscular and cardiac function at 6-months showing a recovering of function in treated mice. These preliminary results suggest that red blood cells overexpressing GAA may be a strategic carrier to achieve a systemic and capillary distribution of the defective gene in the wide-spread skeletal muscles throughout the body.
: Vita-Salute San Raffaele University
MicroRNA-126 reinforces HSC quiescence by dampening PI3K-AKT signaling. We recently reported key functions of miR-126 in acute leukemia: in AML, it was required to maintain leukemic stem cell quiescence (Lechman&Co, Cancer Cell 2016), while its ectopic expression in mouse HSC induced B-ALL that fully regressed when switching a tetracycline-repressible miR-126 cassette (Tet.126) off (Nucera&Co, Cancer Cell 2016). RNAseq suggested that miR-126 prevented (1) p53-dependent senescence, (2) B-cell differentiation and (3) sustained oncogenic/prosurvival pathways typically associated with stem cells (Kit, Wnt, Thy1, Jak/Stat, Bcl2). We functionally interrogated these pathways by expressing dominant negative p53 (GSE56), Ikaros (IK6) or BCRABL in Tet.126-ALL. Doxy-treatment cured mice transplanted with Tet.126-ALL. However, GSE56 delayed early blast clearance, which was further slowed when combined with IK6 or BCRABL, confirming that they played a relevant but not exclusive role in our model. We are currently performing Crispr/Cas9-mediated, combinatorial knock-out of candidate master regulators responsible for miR-126-dependence in B-ALL. We are also consolidating a role for miR-126 in primary human B-ALL. We measured biological miR-126 activity at single cell level with the help of a miR-126 reporter-lentivirus. In 7 (BCRABL+)ALL, we detected substantial miR-126 activity that was stable and disease-specific over serial transplantation in NSG mice. Interestingly, the miR-reporter identified ALL subpopulations that differed in miR-126 activity within single patients. RNAseq analysis of sorted miR-126 (high) and miR-126 (low) subpopulations pointed to common, miR-126-dependent pathways in the mouse model and human disease. Our data reveal an unexpected function of a stem cell-microRNA in B-ALL, uncovering hierarchically-linked or subclonally-related subpopulations that can be prospectively isolated.
: Memorial Sloan Kettering Cancer Center
: National Institute of Allergy and Infectious Diseases
We report outcome to date of a clinical trial (NCT01306019) of SIN-lentivector gene transduced autologous CD34+ hematopoietic stem cell (HSC) therapy with non-myeloablative busulfan (6mg/kg) conditioning to treat older children and young adults with SCID-X1 who had received a T-cell depleted haploidentical HSC transplant without conditioning as infants, but had waning immunity, chronic medical problems and supplemental IgG dependence. We previously reported long term outcome efficacy from 2 patients (P1 and P2) (P2 died at 28 mo post-treatment of a pulmonary bleed from long standing end stage bronchiectasis) and early (3-6 mo) vector marking data from 3 additional patients (P3, P4, and P5) (De Ravin SS, et al. Sci Transl Med. 2016, 8:335ra57; all authors on this paper also contributed to this abstract). We have treated a 6th patient (P6) and herein we report the continued increase in immune reconstitution and improved clinical status of the 5 living patients. P1, P3-P6: age at treatment (23,7,16,10,23 yrs); months from treatment to last assessment (42,12,12,9,3 mos); % marking last assessment (myeloid: 8,6,27,38,30%) (B cell: 38,22,83,70,35%) (T cell: 22,5,40,>90,1); note- patients retain haplo-donor T cells). P1 and P2 achieved independence of IgG supplement and achieved protective titers to immunization, and P4 and P5 are having IgG held and are being immunized. P1, P2, P4 and P5 all achieved cure of multiyear chronic norovirus infection.
: Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER
We have initiated a hematopoietic gene therapy trial in Fanconi anemia A (FA-A) patients using plerixafor and G-CSF mobilized CD34+ cells exposed to a short transduction with a PGK-FANCA.Wpre* lentiviral vector. Five patients (3–5 years-old) out of seven mobilized patients showed more than 5 CD34+ cells/microliter PB. A median number of 2.0×106 (range: 0.85–5.1×106) CD34+ cells/kg was collected from these patients by apheresis, then purified, and in most cases cryopreserved. Small aliquots of mPB CD34+ cells were transduced with the therapeutic vector, showing transduction efficacies between 17–45%. The in vivo repopulating ability of these samples was evaluated in NSG mice conditioned with 1.5 Gy. Most of the transplanted animals showed human hematopoietic engraftment in BM(1–10% of recipient BM cells were hCD45+/mCD45-). Moreover, an evident in vivo selection advantage of corrected CD34+ FA-A cells was demonstrated in transplanted mice. Two not conditioned FA patients have been recently infused with transduced mPB CD34+ cells. The first patient was infused with 1.2x106 fresh CD34+ cells/Kg (estimated transduction efficiency, 17%), while the second patient was infused with 0.8×106 cryopreserved CD34+ cells/Kg (estimated transduction efficiency: 45%). Our preliminary results show that plerixafor/G-CSF efficiently mobilize CD34+ cells in FA patients in early stages of the disease, with no severe adverse events for up to 2.5 years of follow-up. An update of the recently opened FA gene therapy trial will be presented.
: Lund University, Stem Cell Center
Infantile malignant osteopetrosis (IMO) is an autosomal recessive disorder characterized by nonfunctional osteoclasts. About 50% of the patients have mutations in the TCIRG1 gene, encoding for a subunit of the osteoclast proton pump. Gene therapy could be an alternative treatment to allogeneic stem cell transplantation. We tested two mammalian promoters: Elongation factor 1α short (EFS) and a chimeric myeloid promoter (ChimP), using the pRRL backbone. The EFS promoter gave rise to higher rescue levels compared to ChimP, therefore we proceeded with the LV-EFS/TCIRG1 vector. CD34+ cells from peripheral blood of 3 IMO patients were transduced, expanded and differentiated to mature osteoclasts on bone slices. LV-EFS/TCIRG1 corrected IMO osteoclasts restored Ca2+ release to 92% and levels of the bone degradation product CTX-1 to 95% in the media compared to cord blood derived osteoclasts. IMO CD34+ cells transduced with LV-EFS/TCIRG1 were transplanted into sublethally irradiated NSG mice (n = 8), 12 weeks after transplantation bone marrow was harvested, vector copy number per human cell ranged between 0.2-2. Human CD34+ cells were selected, expanded and seeded on bone slices. Vector corrected IMO osteoclasts showed completely restored Ca2+ release and levels of the bone degradation product CTX-1 in the media were 33% compared to cord blood derived osteoclasts, whereas non-corrected IMO osteoclasts failed to resorb bone. Here we provide evidence for rescue of IMO osteoclasts generated from NSG-engrafting hematopoietic cells transduced with a TCIRG1 expressing vector containing a mammalian promoter and without a marker gene. This supports the continued clinical development of gene therapy for IMO.
: San Raffaele-Telethon Institute for Gene Therapy (SR-TIGET)
In genetic diseases affecting the differentiated progeny of hematopoietic stem cells (HSCs), such as beta-thalassemia, the bone marrow (BM) microenvironment is altered by the expansion of immature precursors and stress signals. In particular, thalassemic BM is characterized by the compensatory expansion of erythroid progenitors, secondary to ineffective erythropoiesis. Whether other hematopoietic subpopulations and/or HSCs might be affected by such a stressed environment is still ignored. Our hypothesis is that peculiar disease-related aspects may influence the maintenance of thalassemic HSCs. We analyzed BM hematopoietic primitive and committed progenitors in th3/+ mutant mice, a murine model of severe beta-thalassemia intermedia, and we observed a lower frequency of LSK CD48-CD150+ HSCs in comparison to age-matched wt animals. Competitive transplantation experiments demonstrated a disadvantage in the engraftment capacity of thalassemic HSCs. These data were supported by cell cycle analyses: th3/+ cells are more cycling and displayed impaired progression through S-phase, resembling the behavior of stressed or aged HSCs. Serial BM transplantations proved an accelerated exhaustion of cells engrafted in thalassemic niche, showing an active role of recipient BM microenvironment. Analyses of BM stromal cells revealed an altered organization of Nestin+ mesenchymal cells in th3/+ niche. Our results uncover a previously ignored defect of HSCs, highlighting the impact of disease-related alterations of microenvironment on HSC function. These data for the first time change the conception of an inherited dyserythropoietic disorder to a disease affecting the whole hematopoiesis. Further investigation will unveil the role of cellular and soluble niche components potentially affecting in trans HSC functions.
: TIGEM
Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism due to mutations of AGXT gene encoding the peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT) which catalyzes the conversion of glyoxylate to glycine. In PH1 patients, glyoxylate cannot be converted into glycine and is oxidized to oxalate, resulting in systemic oxalosis with deposition of insoluble calcium oxalate in kidneys and in other tissues, leading to kidney failure and systemic tissue damage. Combined liver/kidney transplantation is the only therapy available to prevent disease progression. We investigated whether helper-dependent adenoviral (HDAd) vector-mediated hepatic overexpression of glyoxylate reductase/hydroxypyruvate reductase (GRHPR), that converts glyoxylate into glycolate, will steer glyoxylate towards alternative pathways to diminish oxalate production in Agxt−/− mice. Intravenous injection of HDAd-GRHPR resulted indeed in significant reduction of hyperoxaluria and concomitant increase of serum glycolate that was not associated with signs of toxicity. Glutamate-pyruvate transaminase (GPT) in the cytosol catalyzes the transamination of glyoxylate using glutamate and alanine as amino-group donors. The intravenous injection of HDAd vector expressing murine GPT (HDAd-GPT) steered glyoxylate towards transamination and resulted in sustained reduction of hyperoxaluria in Agxt−/− mice. Interestingly, co-administration of HDAd-GRHPR and HDAd-GPT resulted in further reduction and normalization of hyperoxaluria. In summary, we show that metabolic diversion towards non-toxic metabolites has potential for treatment of PH1 and potentially other forms of hyperoxalurias, both primary and secondary. In addition, this study shows that HDAd vectors can be used to functionally validate therapeutic enzyme targets in inherited metabolic diseases.
: Généthon
Adeno-associated viruses (AAV) are one of the most efficient vectors for liver gene therapy. Results obtained in the first hemophilia B clinical trials demonstrated the long-term efficacy of this approach in humans, showing efficient targeting of hepatocytes with both self-complementary (sc) and single-stranded (ss) vectors. However, to support clinical development of AAV-based gene therapies, efficient and scalable production processes are needed. In an effort to translate to the clinic an approach of AAV-mediated liver gene transfer to treat Crigler-Najjar (CN) syndrome, we developed a ssAAV vector carrying the UDP-glucuronosyltransferase-1-isotype-A1 (UGT1A1) transgene under the control of a liver-specific promoter. We compared our construct with similar scAAV vectors expressing UGT1A1, showing comparable potency in vitro and in vivo. Conversely, ssAAV-UGT1A1 vectors showed superior yields and product homogeneity compared with their sc counterpart. We then focused our efforts in the scale-up of a manufacturing process of the clinical product ssAAV8-UGT1A1 based on the triple-transfection of HEK293 cells grown in suspension. Large scale production of this vector yielded to high-quality vector preparations with characteristics identical to those of small scale vector produced in adherent cells. This process will be used to produce clinical-grade vector for a proposed clinical trial for CN syndrome. Preclinical studies in animals models of the disease and a GLP toxicology/biodistribution study were also conducted using vectors produced with this scalable platform. These studies demonstrated the safety and efficacy profile of gene transfer with ssAAV8-UGT1A1, thus supporting the clinical translation of this gene therapy approach for the treatment of CN syndrome.
: Centro de Investigacion Medica Aplicada (CIMA)
Wilson's disease (WD) is an autosomal recessive inherited disorder due to mutations in the ATP7B gene that causes hepatic and neurologic symptoms. Current treatments are based on lifelong copper chelating drugs, which may cause side effects and do not restore normal copper metabolism. We have recently demonstrated that the administration of an AAV vector expressing ATP7B under the control of a liver specific promoter into Wilson disease male mice induce full restoration of copper homeostasis. However, the size of the vector genome surpass the optimal size for AAV genome packaging preventing the use of bigger promoters or additional regulatory sequences such as introns or bigger poly A sequences. In the present work we have designed two truncated versions of the ATP7B protein and analyzed their therapeutic efficacy. The administration of recombinant AAV vectors carrying the truncated proteins under the control of a liver specific promoter to Wilson disease mice showed that both proteins are able to reduce liver damage, copper in the urine and liver, however while the T2 was as efficient as the whole ATP7B protein, T1 has only a partial effect. Furthermore T2 was more efficient than WT in restoring ceruloplasmin oxidase activity in serum. In conclusion, we have identified a fully functional truncated version of the ATP7B protein that will allow to reduce the size of the therapeutic AAV vector genome and the introduction of additional regulatory elements.
: Genosafe
Recombinant AAV (adeno-associated virus) vectors have proven their efficacy as gene therapy vectors in different settings and are now coming close to marketing application. Anti-AAV antibodies are however a great concern to such therapeutic programs with seroprevalence to some serotypes reaching up to 80% of the normal population. These antibodies can be binding or neutralizing, the latter being a major concern as they can partially or completely prevent the biotherapeutic to exert its biological effects. Assessment of the immune response is then crucial at different levels: either to determine whether a patient may be part of the clinical assay (screening phase) or to follow-up the patient's response to the treatment. Most of the time, analysis of the immune response to the treatment is required by regulatory agencies as part of the IND (investigational new drug) submission file. One of the challenges is that only validated assays can be used to analyze patient's samples. A full validation means that the assay is designed to suit the clinical protocol requirements, that every critical parameter of the assay is challenged, and that assay performance can be monitored and guaranteed over time. During this presentation, we will focus on the validation of immunoassays such as ELISA or cell-based assays used to detect anti-AAV antibodies.
: NCT DKFZ Heidelberg
Although recombinant adeno-associated viral vectors (rAAV) are cleared out from serum within 4–8 weeks, a sustained vector persistence was found in peripheral blood mononuclear cells (PBMC) from patients receiving a rAAV-2/5 aimed to treat acute intermittent porphyria. To unravel the nature of rAAV persistence in PBMC, quantitative and linear amplification-mediated (LAM)-PCR were performed on PBMC collected 6 and 12 months following intravenous rAAV-1 or rAAV-5 administration to non-human primates (NHP). Similarly, bone marrow mononuclear cells (BMNC) collected 12 months after vector injection were analyzed to investigate whether PBMC vector persistence could arise from bone marrow transduction. rAAV genomes persisted within PBMCs and, although 187 integration sites (IS) were retrieved, they mainly remained episomally. At the later timepoint, we found a decreased vector copy number (VCN) that correlated with a loss of episomal forms (88.6 and 75.1% of episomes at 6 and 12 months, respectively). In BMNC, we found a ∼2-fold higher VCN when compared to PBMC. This also correlated with a higher IS retrieval (342 IS), although the vector persisted again mainly episomally. Importantly, isolation of BMNC CD34+ and CD34- cells revealed rAAV presence in both fractions with higher VCN within the hematopoietic progenitor compartment. Notably, we found no integration hotspots nor targeting of cancer-related genes. Here we show rAAV-1 or rAAV-5 long-term persistence within NHP hematopoietic progenitors with neither clonal outgrowth nor signs for malignant transformation. This indicates the safe integration profile of these vectors within this compartment and places rAAV as potential candidates for bone marrow gene therapy.
: Adverum Biotechnologies, Inc.
: Hannover Medical School
The number of gene therapy vectors for a multitude of different diseases has increased tremendously over the years. A bottleneck of transition from basic research to clinical application is the test for safety. Instead of laborious in vivo models with limited predictive value, scientists try to employ in vitro assays to screen for insertional mutagenesis. A decade ago, our lab developed the in vitro immortalization (IVIM) assay to quantify the genotoxic potential of vectors. Many academic and industrial partners have used IVIM to complete their preclinical safety documentation. Despite general acceptance in the field of haematopoietic gene therapy, bias for insertional mutants of the myeloid lineage and a low sensitivity are clear limitations. We now developed the molecular surrogate assay for genotoxicity assessment (SAGA). The new test is more robust, sensitive and biologically informative. Instead of replating cells for later analysis of proliferating mutants, we isolated total mRNA of the bulk cultures and performed microarrays (n = 86). With the help of machine learning algorithms, we can identify mutagenic vectors by the deregulation of an oncogenic gene expression signature. This core set was used to quantify the mutagenic potential of several benchmark vectors by gene set enrichment analysis. Gammaretroviral vectors MFG.yc and CMMP.WAS, which elicited leukaemia in past clinical trials, showed a significant mutagenic enrichment score. We also tested currently used self-inactivating lentiviral vectors (EFS.IL2RG and EFS.ADA) which performed much safer compared to constructs with strong viral promoters (SFFV.eGFP). We present the results for these vectors side-by-side either using IVIM or SAGA.
: San Raffale Telethon Institute for Gene Therapy (HSR-TIGET)
We studied the in vivo safety profile of four validated CCCTC-binding-Factor (CTCF)-based Chromatin Insulators (CI) by cloning them within the Self-Inactivating (SIN) Long-Terminal-Repeats of a Lentiviral-Vector (LV) harboring a strong enhancer-promoter (CI.SIN.LVs) and tested them in the Cdkn2a-/- and Cdkn2a+/- in vivo genotoxicity assays. These models allow measuring vector-induced genotoxicity as accelerated tumor onset vs. Mock and Integration Sites (IS) retrieval from tumors followed by Common Integration Sites (CIS) analysis enable identifying the mechanisms of insertional mutagenesis. In Cdkn2a-/- mice all CI.SIN.LVs displayed slight, but not statistically-significant, improvement in the median survival-time vs. the uninsulated-SIN.LV counterpart, while in Cdkn2a+/- mice the median survival-time for two CI.SIN.LVs-treatment groups was significantly different from the uninsulated-SIN.LV (p-values: 0.0135 and 0.0063) and not different from mock-controls. We analyzed >14000 IS and identified different CIS. In Cdkn2a-/- mice, uninsulated-SIN.LV-induced tumors harbored predominantly activating integrations targeting Map3k8-oncogene, while tumors from mice treated with two CI.SIN.LVs significantly reduced the frequency of tumors with Map3k8-activating insertions. The reduced Map3k8-targeting was accompanied in one case by a skewing towards inactivating-integrations targeting Pten or other tumor-suppressors, as genotoxicity escape mechanism. In Cdkn2a-/+ mice we identified different predominant CIS by the four CI.SIN.LVs, suggesting that this model allows discriminating between more subtle shades of genotoxicity. Interestingly, merging the results from both in vivo assays we observed that one CI displayed superior safety profile in terms of significant improvement in the median survival-time and of reduced oncogenic CIS identified. In summary, we validated novel insulator elements able to block SIN.LV-genotoxicity in vivo.
: University of California Los Angeles (UCLA)
X-linked hyper-IgM syndrome (XHIM) is a primary immunodeficiency due to mutations in CD40 ligand (CD40L). Previous gene therapy-based studies have investigated the use of retroviral vectors for delivery of CD40L cDNA. Due to uncontrolled expression of the gene, this resulted in abnormal lymphoproliferation in mouse models, emphasizing the need for alternative strategies. Site-specific genome modification provides the promise for correction of the gene under the regulation of its endogenous enhancer and promoter. To this end, we have designed TALENs and CRISPRs targeting the 5’ UTR of the CD40L gene and demonstrated allelic disruption levels ranging from 30–50% in K562 cells, patient primary T cells, and CD34+ hematopoietic stem and progenitor cells. XHIM T cells electroporated with either TALEN mRNA or CRISPR guide RNA/Cas9 mRNA and subsequently transduced with an AAV vector to deliver a codon-optimized CD40L cDNA repair template resulted in CD40L expression ranging from ∼3% at rest and increasing up to 17% with immune stimulation by flow analysis. Rates of gene insertion as measured by ddPCR paralleled that of CD40L expression by flow cytometry. Given this, similar experiments were been carried out in normal cord blood and peripheral blood stem cells. Targeted gene integration rates by ddPCR quantification range from 8–40% depending on the site-specific endonuclease. Treated HSPC have been transplanted into neonatal NSG mice with analysis pending. This work provides the foundation for moving forward with methods for site-specific modification at CD40L as a potential therapy for immune reconstitution in XHIM.
: University of Trento
CRISPR nucleases are efficient tools to edit cellular genomes in a variety of organisms. However, the in vivo application of this technology is still severely limited by unwanted genomic cleavages leading to unpredictable results. Recent efforts towards improved specificity include the development by rational design of SpCas9 variants with increased fidelity [1,2]. To explore a wider mutational space for the identification of novel SpCas9 variants we developed a yeast-based assay to simultaneously measure the nuclease on- versus off-target activity towards two synthetic genomic targets. By screening a library of random mutants in our reporter yeast strain we identified several aminoacid substitutions that preserved efficient nuclease activity and significantly reduced off-target cleavages. We obtained hits with increased on/off ratios and further combined their mutations within a single variant to isolate the best performing nuclease. The selection was further refined by structure-assisted prediction of protein-DNA and -RNA contacts. Genome-wide and targeted deep-sequencing analyses revealed a significant reduction of off-targets cleavages towards standard and repetitive target loci. Side by side analyses with previously reported rationally designed variants [1,2] demonstrated a significant improvement in fidelity of our SpCas9 mutant. Compared to structure-guided directed mutagenesis, our in vivo screening increases the likelihood to identify the best combination of aminoacid substitutions to obtain an error-free SpCas9. Altogether, this study proves that our approach represents a valid strategy to enhance the specificity of SpCas9 as well as other RNA-guided nucleases.
1. Slaymaker et al., Science, 2015.
2. Kleinstiver et al., Nature, 2016.
: Cleveland Clinic
Toca 511 (vocimagene amiretrorepvec) is a retroviral replicating vector, encoding a modified yeast cytosine deaminase (CD) that converts the well-tolerated anti-fungal drug, 5-fluorocytosine (5-FC) into the antineoplastic drug, 5-fluorouracil. The vector appears highly selective for tumor cells in animal experiments and in human resected tumor tissue. We have treated more than 120 recurrent high grade glioma patients in three Phase 1 studies by intratumoral injection (NCT01156584), injection into the tumor-bed post-resection (NCT01470794), or by IV administration (NCT01985256) followed by extended release 5-FC (Toca FC); in all studies there is good evidence of selective tumor infection by PCR, RT-PCR and IHC against the CD protein. Clinical data in the first two trials (with 39 and 43 evaluable subjects respectively) show a favorable safety profile and extended overall survival (OS). In the resection study, median OS was 13.6 months compared to 7–8 months in matched historical controls, and the OS at 24 months in the higher dose cohorts was 40% compared to around 10% historically. A tumor RNA expression signature, not normally predictive of survival, predicts long-term survival. In animals the treatment leads to strong, tumor-clearing, antitumor immunity with local elimination of immunosuppressive myeloid cells. Trials data are consistent with the immune response playing a significant role. Toca 511 and extended-release 5-FC (Toca FC) apparently lead to selective tumor destruction. Based on results from these trials, a phase 2/3 trial (Toca 5) is underway and recruiting patients (NCT02414165). The combination of preclinical and clinical data supporting this decision will be reviewed.
: Targovax Oy, Helsinki, 00180, Finland
Malignant mesothelioma is a rare cancer type with no effective treatment therapies so far. Therefore, new and more efficient treatment strategies against mesothelioma are in high demand. Ad5/3-d24-GM-CSF (ONCOS-102) is a double targeted, chimeric oncolytic adenovirus, coding for human GM-CSF. The use of ONCOS-102 as cancer vaccine aims to i) oncolyse tumor cells and ii) trigger a tumor-specific immune response that targets the patient's unique tumor antigen repertoire. We have evaluated the anticancer activity of ONCOS-102 in i) combined therapy: first-line chemotherapy (Pemetrexed, Cisplatin, Carboplatin) with the virus in a human malignant mesothelioma xenograft model in BALB/c mice, and ii) in a Phase I study, where malignant pleural mesothelioma patients, were treated with ONCOS-102. ONCOS-102 eradicated all human mesothelioma cell lines in vitro and exhibited anti-tumor activity in tested refractory H226 mesothelioma xenograft model. Furthermore, current standard of care chemotherapy regimens for malignant mesothelioma did not show anti-tumor efficacy. Importantly, a synergistic anti-tumor effect was shown, when ONCOS-102 was combined with chemotherapy regimens. In clinical settings, the treatment with ONCOS-102 resulted in massive infiltration of CD8+ T cells to tumor, induction of systemic anti-tumor CD8+ T cells and Th1 type polarization. These results indicate that ONCOS-102 shows ability to be used in mesothelioma treatment and to sensitizes tumors to other immunotherapies by modulating a T cell positive phenotype to an initially T-cell negative tumor.
: San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy
Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency characterized by thrombocytopenia, eczema, high susceptibility to autoimmune manifestations and malignancies. Although thrombocytopenia is one of the main causes of death, the pathogenesis of platelet (PLT) defect is poorly understood. Here, we evaluated the role of WAS protein (WASp) in PLTs in a new conditional mouse model (CoWas) lacking WASp only in the megakaryocytic (MK) lineage. We observed an increased number of MKs and their progenitors both in CoWas mice and complete Was-/- mice (WKO), as well as normal in vitro PLT production by Was-/- MKs. Upon in vivo depletion of PLTs, WKO and CoWas mice were able to restore PLT count with kinetics comparable to wild-type (WT) mice, suggesting no defect in thrombopoiesis. Of note, WASp-deficient PLTs both in WKO and CoWas mice had a shorter half-life and a hyper-activated status before and after ADP stimulation. We also found that CoWas mice develop anti-PLT autoantibodies against Was-/- PLTs. In line with this result, we found that WASp-deficient PLTs show a different proteomic profile compared to WT PLTs and express higher amounts of CD40L and sCD40L, a key regulator of B cell autoreactivity. Moreover Was-/- PLTs release a higher amount of the sCD40L and we found that supernatants of Was-/- PLTs were able to induce B-cells activation in in vitro. In parallel we are evaluating PLT reconstitution in WAS patients treated with lentiviral vector-mediated gene therapy, to assess whether this treatment is able to restore PLT function.
: Université Laval, Chemical Engineering Department, Quebec, Canada
The Zmapp cocktail contains chimeric neutralizing antibodies (c13C6, c2G4 and c4G7) against Ebola virus. It is among the most promising experimental approaches for treating Ebola infections. However, because of high doses required of purified antibodies, its use on large populations poses a manufacturing challenge and is of economic concern in developing countries. To address these potential issues, recombinant vectors derived from adeno-associated virus (rAAVs) are very attractive. They 1) can be produced in large quantity, 2) permit long-term expression thus, reducing the number of treatments, 3) are highly stable to storage conditions and 4) are efficiently administered intranasally. Our main goals are to develop a treatment based on rAAVs to deliver genes coding for Zmapp antibodies and to scale-up the manufacturing at reasonable costs. In this study, three rAAVs (serotypes 9 and DJ) expressing one of the three antibodies were produced in shake flasks (200 mL) and in WAVE bioreactors (10 L). rAAVs were produced by transfection using our patented cGMP compatible HEK293 cell line in suspension culture without serum. Light and heavy chains were expressed under the same expression cassette by using a 2a peptide and furin cleavage sequences. rAAVs productions were either directly purified by an iodixanol step-gradient or concentrated first by tangential flow filtration. Titers were obtained by qPCR. Antibodies produced in transduced HEK293 and CHO cells were characterized by western blot, LC-MS/MS and a functional assay. The efficacy of rAAV-c2G4 for preventing Ebola infections is currently under evaluation in a mouse model challenged with the virus.
: Bluebird Bio
B cell maturation antigen (BCMA) is expressed on most multiple myeloma (MM) cells, yet normal tissue expression is limited to plasma and some B cells. We developed lentiviral vectors expressing chimeric antigen receptors (CARs) derived from multiple anti-BCMA single chain variable fragments (scFv) using 4-1BB and CD3zeta T cell signaling domains. To evaluate CARs for eventual clinical application we exploited a rapid screening method based on cell surface expression, antigen-specific recognition, lack of antigen-independent signaling, and activity in xenograft models. A number of CAR constructs with high effector cytokine production and strong cytolytic activity also displayed antigen-independent tonic signaling. Tonic signaling was associated with increased activation marker expression (HLA-DR and CD25) and decreased expression of memory-cell associated marker CD62L. In MM xenograft models, tonic signaling CARs were less effective in treating established tumors than CARs without antigen-independent reactivity. One anti-BCMA CAR (bb2121) out of >30 unique constructs was selected for clinical development. A multi-center phase I clinical study of centrally-manufactured bb2121 for the treatment of MM was initiated in February 2016 (ClinicalTrials.gov, NCT02658929). In experiments designed to improve anti-tumor activity, we determined that CAR T cells cultured with a PI3K inhibitor demonstrated memory-like properties (including increased memory-associated markers CD62L, CD127, and CD197) and showed enhanced efficacy in two stringent anti-tumor models involving treatment of advanced disease and tumor rechallenge. These data demonstrate that a potent, memory-like anti-BCMA CAR T cell can be produced with an industrial-scale manufacturing process, which represents an attractive option for next-generation CAR T cell products.
: Centro Ricerca Tettamanti, Clinica Pediatrica, Universita' Milano Bicocca, Osp. San Gerardo/Fondazione MBBM, Monza (MB), Italy
Chimeric Antigen Receptors (CARs)-redirected T lymphocytes are a promising immunotherapeutic approach, nowadays object of accurate preclinical evaluation also for Acute Myeloid Leukemia (AML) targeting. We recently developed a CAR against the CD123 antigen, found to be over-expressed on AML blasts and leukemic stem cells (LSCs). However, the potential recognition of low CD123-positive healthy tissues, through the so called “on-target-off-organ” effect, limits the safe clinical employment of CAR-T cells. According to CAR design variables, CAR-T cell functional profiles can be modulated by tuning the binding affinity to the target antigen. Therefore, we developed a novel integrated model for the functional screening of in silico-selected CAR affinity mutants, starting from predicted antibody binding properties, that have been then in vitro validated based on CAR-redirected T cell biological requirements. By exploiting a panel of anti-CD123 CAR affinity mutants, we defined both “lytic” and “activation” antigen thresholds showing that, while the early cytotoxic activity is not affected neither by CAR expression nor by CAR affinity tuning, the CAR expression represents the main variable impairing later effector functions. All these variables are essential for a further clinical translation of this approach, and the lowest affinity mutant could represent the one with an affinity threshold granting a proper balance between safety and efficacy profiles, below which the antileukemic efficacy could be impaired.
: TIGEM
Transcription factors (TFs) function by the combined activity of their DNA-binding domains (DBDs) and effector domains (EDs). Here we show that in vivo delivery of an engineered DNA-binding protein uncoupled from the repressor domain entails complete and gene-specific transcriptional silencing. To silence RHODOPSIN (RHO) gain-of-function mutations, we engineered a synthetic DNA-binding protein lacking canonical repressor domains and targeted to the regulatory region of the RHO gene. AAV-mediate retinal delivery at a low dose (AAV2/8-CMV-ZF6-DBD, 1×10e10 vector genomes, vg) in the porcine retina resulted in selective transcriptional silencing of RHO expression. The rod photoreceptors (the RHO expressing cells) transduced cells when isolated by FACS-sorting showed the remarkable 90% RHO transcriptional repression. To evaluate genome-wide transcriptional specificity, we analysed the porcine retina transcriptome by RNA sequencing (RNA-Seq). The differentially expressed genes (DEGs) analysis showed that only 19 genes were perturbed. In this study, we describe a system based on a synthetic DNA binding protein enabling targeted transcriptional silencing of the RHO gene by in vivo gene transfer. The high rate of transcriptional silencing occurring in transduced cells supports applications of this regulatory genomic interference with a synthetic trans-acting factor for diseases requiring gene silencing in a large number of affected cells, including for instance a number of neurodegeneration disorders. The result support a novel mode of gene targeted silencing with a DNA-binding protein lacking intrinsic activity.
: IGBMC
Centronuclear myopathies (CNM) are severe non-dystrophic muscle diseases characterized by muscle weakness and hypotrophic fibers with centralized nuclei. The most severe and neonatal X-linked form is known as myotubular myopathy and caused by loss-of-function mutations in Myotubularin (MTM1), while the main autosomal-dominant form is due to mutations in Dynamin 2 (DNM2). However, no specific therapy is available for patients. We previously showed that genetic reduction of Dnm2 in the myopathic Mtm1 Knockout (KO) mice restores a normal lifespan with improved muscle structure and function. This genetic proof-of-concept highlights epistasis between Mtm1 and Dnm2, and validates the concept of cross-therapy where downregulation of a CNM gene rescues the loss of another CNM gene. However, translation for therapeutic application requires a deliverable compound. Here we develop a translated approach targeting Dnm2 pre-mRNA and demonstrate that systemic delivery of antisense oligonucleotides (ASO) into Mtm1KO at early phase of the disease reduces efficiently Dnm2 protein level and prevents the myopathy progression while rescuing both lifespan and body weight, and correcting muscular mass, histology and force. More importantly, systemic injections of ASO into strongly affected mice reverted different myopathy signs and prolonged lifespan. Thus, ASO-mediated knockdown towards a target that is not the mutated gene can efficiently correct physiological and cellular muscles defects by controlling DNM2 level, and provides an attractive therapeutic strategy for non-dystrophic muscle diseases.
: Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, 50931 Germany
Adeno-associated viral (AAV) vectors are among the most widely used gene transfer systems and have been applied in more than 160 clinical studies. Yet, manufacturing of pure AAV vector preparations remains a difficult task. Specifically, prokaryotic backbone sequences including antibiotic resistance genes which stem from producer plasmids are packaged into AAV capsids. These encapsidated DNA impurities cannot be removed by state-of-the-art purification strategies. We investigated whether in a dual plasmid system, both AAV vector and helper & packaging plasmids could be replaced by minicircles (MC). MC are produced from a parental plasmid through an intramolecular recombination step and contain no functional prokaryotic sequences. Both single-stranded (ssAAV) and self-complementary (scAAV) vectors could be produced using MC constructs without negatively impairing capsid titer, genomic titer, and transducing titer of vector preparations, in comparison to vectors produced by standard plasmid transfection. Indeed, MC-derived scAAV vectors even outperformed plasmid-derived scAAV up to 30-fold regarding transduction efficiencies. Importantly, replacing both, helper & packaging and vector plasmids by MC reduced the content of prokaryotic backbone sequences to below quantification limit. Thus, MC technology offers an easy to implement modification of standard AAV packaging protocols, which significantly improves the quality of AAV vector preparations. This is of particular interest in scAAV, where plasmid backbone-derived sequences amount up to 26.1% of total vector genomes (compared to up to 2.9% in ssAAV).
: Bluebirdbio Inc
Successful ex vivo lentiviral gene therapy relies in part on the generation of a sufficient proportion of gene-modified cells. The protein kinase inhibitor staurosporine was found to enhance the lentiviral vector (LVV) transduction of mobilized peripheral blood (mPB) CD34+ cells both and in vivo. CD34+ cells treated with staurosporine rapidly decrease cofilin phosphorylation and F-actin staining, suggestive of actin treadmilling, which has been suggested to be important for LVV entry. Staurosporine treatment increased LVV entry about 1.5-fold by BlaM assay, supporting alleviation of an LVV entry barrier, and increased vector copy number (VCN) about 2-fold in vitro. Staurosporine also enhanced VCN in long-term repopulating cells in vivo in a NSG transplant setting, with no observed toxicities. In addition, the combination of staurosporine and PGE2 further enhanced VCN. PGE2 does not affect LVV entry and therefore, PGE2 and staurosporine likely increase LVV transduction by alleviating two different LVV restriction steps. Based on these observations, the use of staurosporine to enhance LVV transduction of human CD34+ cells, either alone or in combination with PGE2, is a promising method to improve the potency of gene therapy.
: The Research Institute at Nationwide Children's Hospital
Spinal muscular atrophy (SMA) Type 1 is the most common genetic cause of infant death with mutations or deletions of survival motor neuron gene (SMN1) on chromo 5q13 affecting 1 in 10,000 live births. SMN1 is necessary for SMN protein production and motor neuron survival. In the most severe form of the disease, SMA Type 1, 92% of infants die or rely on permanent ventilation support by 20 months of age. Gene therapy has been a potential therapeutic strategy for genetic neurological diseases, such as SMA, but multiple barriers such as effective targeting of cells throughout the nervous system have limited potential. In the case of SMA, motor neurons reside at all levels of the spinal cord, including the brainstem. Adeno-associated virus (AAV) has emerged as an ideal viral vector given its safety profile and long-term persistence in pre-clinical and clinical studies to date. The discovery of AAV serotype 9 crossing the blood brain barrier or cerebrospinal fluid barrier has opened the potential for less invasive ways to target the brain and spinal cord efficiently. This talk will specifically focus on two routes of administration, systemic and intrathecal, describing the pre-clinical dose ranging and safety studies in multiple species that have advanced to first-in-human studies for SMA patients.
: Jichi Medical University
: SR-Tiget, San Raffaele Telethon Institute for Gene Therapy
Genome editing by artificial nucleases has brought the goal of site-specific gene inactivation or in situ gene correction within the reach of gene therapy. With the first strategy we established the proof-of-principle of TCR editing as a novel means of T-cell therapy, in which a new biological function is instructed to an immune effector cell by genetically re-writing its endogenous antigen specificity. These studies allow for the first time to effectively re-direct the specificity of CD8 T cell against a tumor-associated antigen without the risks associated with TCR mispairing. The power of precise genetic engineering can also be exploit to directly correct disease-causing mutations, thus restoring both the function and the physiologic expression control of the targeted gene. Gene editing, however, is specifically constrained in the primitive HSPC subset by quiescence and low expression of the DNA repair machinery. By tailoring delivery platforms and explore culture conditions that induce proliferation while preserving engraftment capacity, we partially overcame these barriers and provide evidence of targeted integration in human HSCs by long-term multilineage repopulation of xeno-transplanted mice. We demonstrate the therapeutic potential of our strategy by targeting a corrective cDNA into the IL2RG gene of HSCs from healthy donors and subjects with SCID-X1. We have validated this approach in an ad hoc humanized SCID-X1 mouse model to support the scientific rationale and safety of the proposed treatment, and identify conditioning regimen and degree of chimerism required to correct the disease phenotype. These results open new avenues for treating SCID-X1 and other diseases.
Poster Presentations
: The University of Texas Health Science Center at Houston
Emerging evidences suggest that long noncoding RNAs (lncRNA; >200bp) play important roles in cell fate determination. However, it is challenging to identify lncRNA comprehensively, since lncRNAs are often expressed at lower levels and are more cell type-specific than protein-coding genes. In this study, we performed ab initio transcriptome reconstruction using nine purified cell populations from mouse cortex and detected more than 5,000 lncRNAs. Predicting lncRNAs' function using cell type specific data revealed their potential functional roles in central nervous system development. ENCODE DNase I digital footprint data and Mouse ENCODE promoters were utilized to infer transcription factor (TF) occupancy. By integrating TF binding and cell-type specific transcriptomic data, we constructed a novel framework useful for systematically identifying lncRNAs that are potentially essential in brain cell fate determination. Based on this integrative analysis, we identified lncRNAs that are regulated during Oligodendrocyte Precursor Cell (OPC) differentiation from Neural Stem Cells (NSCs) and likely to be involved in oligodendrogenesis. The top candidate lnc-OPC shows highly specific expression in OPCs and remarkable sequence conservation among placental mammals. OLIG2-binding sites in the upstream regulatory region of lnc-OPC were identified by ChIP (chromatin immunoprecipitation)-Sequencing and validated by luciferase assays. Furthermore, loss-of-function experiments confirmed that lnc-OPC plays a functional role in OPC genesis. Overall, our results substantiated the role of lncRNA in OPC fate determination and provided an unprecedented data source that will advance the knowledge of this major class of non-coding genes and their potential roles in neurological development and diseases (
: CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Rua Larga, 3004-504 Coimbra, Portugal
Machado-Joseph disease or spinocerebellar ataxia type 3 (MJD/SCA3) is a genetic neurodegenerative disorder associated with expansion of the number of CAGs within the coding region of the MJD1 gene, which translates into an expanded polyglutamine tract within ataxin-3 protein. MJD patients have severe clinical manifestations and premature death and there is no treatment available to modify disease progression. We and others provided evidence that autophagy impairments contribute to MJD pathogenesis with autophagosome accumulation, reduction of autophagy-associated protein levels, accumulation of mutant ataxin-3 and neurodegeneration. Recently, we also brought evidence that let-7 microRNA is a key regulator of autophagy with particular relevance in polyglutamine disorders. In this work we aimed at investigating let-7 de-regulation in MJD, dissecting let-7 role in the regulation of the autophagy pathway in MJD, and evaluating its potential as a new therapeutic approach to MJD. Preliminary results suggest that let-7 levels are reduced in a lentiviral MJD mouse model. Injection of lentiviral vectors encoding for let-7 resulted in a 20% increase of let-7 levels in mice striatum. LC3 immunoblot analysis revealed increased levels of LC3-II relative to actin upon let-7 treatment. A significant let-7-mediated reduction of ubiquitin-positive inclusions and neuronal dysfunction was observed at 4 weeks. Hence, let-7 activates autophagy in the mammalian brain, promotes increased turnover of mutant ataxin-3 protein in mouse CNS and reduces neuronal dysfunction. Therefore, autophagy activation mediated by let-7 may represent a new efficient therapeutic approach for MJD.
: Medical Genomics and Molecular Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Betrhesda Maryland, U.S.A.
Niemann-Pick disease, type C (NPC) is a heritable lysosomal storage disease characterized by a progressive neurological degeneration that causes disability and premature death. To test the potential efficacy of gene therapy for NPC, we constructed an adeno-associated virus serotype 9 (AAV9) to deliver the human NPC1 gene under the transcriptional control of the neuronal-specific (CamKII) or a ubiquitous (EF1a) promoter and treated a murine model of NPC (Npc1-/-) with these AAV9 vectors. Similar to the severe phenotype of NPC observed in humans, Npc1-/- mice display a rapidly progressing form of NPC disease, characterized by weight loss, ataxia, increased cholesterol storage, loss of cerebellar Purkinje neurons and early lethality (mean survival of 67 days). The Npc1-/- mice received a retro-orbital injection with either a dose of 2.6×1011GC of AAV9-CamKII-NPC1 in the neonatal period, 1×1012GC of AAV9-CamKII-NPC1 at 3 weeks or 1×1012GC of AAV9-EF1a-NPC1 at 3 weeks. All groups of treated Npc1-/- mice exhibited an increased life span, and a delayed weight and motor decline. In addition, cholestrol storage and purkinje neuron loss were reduced in a subset of AAV9 treated Npc1-/- mice at 9 weeks. While there was no significant difference in efficacy for gene delivery of AAV9-CamKII-NPC1 at different time points, AAV9-EF1a-NPC1 when compared to AAV9-CamKII-NPC1 treatment resulted in a significant increase in survival (P < 0.05; mean survival >147 and 105 days, respectively). Our results demonstrate for the first time the potential efficacy of AAV gene therapy as a therapeutic option in NPC patients.
: MIRCen, CEA
Parkinson's disease (PD) is treated with oral dopaminergic treatments that are initially highly efficacious but over time lead to debilitating long-term side effects that seriously impact on their effectiveness. OXB-102 is a lentiviral-based vector that delivers the genes for three key enzymes in the dopamine biosynthetic pathway; tyrosine hydroxylase, aromatic L-amino acid decarboxylase (AADC), and GTP-cyclohydrolase, to non-dopaminergic striatal neurons of the sensorimotor putamen. These cells are then able to synthesise and release their own dopamine. The effectiveness of this strategy has been demonstrated in rodents, non-human primates (NHPs) and PD patients (Palfi et. al, Lancet 2014) with ProSavin®, a precursor vector. OXB-102 is an improved vector with increased dopamine production per genetically modified cell. The efficacy of full-strength and 1/5th dose of OXB-102 has been compared to full-strength ProSavin® in the ‘gold standard’ MPTP NHP model of PD. Clinical rating scores and locomotor activity were recorded before and after vector administration. Both doses of OXB-102 were as efficacious as ProSavin®; indicating improved dopamine production from OXB-102. Positron emission tomography using 18F-FMT, a presynaptic biomarker that acts as a substrate of AADC, showed a significant increase in FMT signal in OXB-102 treatment groups compared to baseline that was greater than the ProSavin® group. A 6-month NHP toxicology and biodistribution study indicates OXB-102 is safe and well tolerated following stereotactic administration and vector does not significantly spread beyond the site of administration. GMP manufacture of OXB-102 is complete and a clinical trial in PD patients will initiate in 2016.
: INSERM U1169, Le Kremlin Bicêtre, MIRCen/CEA, Fontenay aux Roses, France
Growing evidences suggest the role of cholesterol in Alzheimer's disease (AD) progression. Since the blood-brain-barrier (BBB) prevents peripheral cholesterol to enter the brain parenchyma, excess of cholesterol cannot be directly eliminated. To be degraded, the cholesterol in excess must be exported to the peripheral circulation. The conversion to 24-hydroxycholesterol, (24-0HC) by the key neuronal enzyme cholesterol 24-hydroxylase (CYP46A1), represents the main mechanism by which cholesterol in excess is eliminated from the brain. Here, we first demonstrate that CYP46A1 is decreased in hippocampal biopsies from AD patients. In a therapeutic perspective we overexpressed CYP46A1 by mean of an AAV9 vector, which was demonstrated to efficiently transduce hippocampal neurons in mice. We then treated APP23 mice before the onset of plaque load and showed that AAV9-CYP46A1 injection increased hippocampal 24-OHC content, strongly prevented amyloid plaques deposition, and decreased Aß peptides and astrogliosis. Notably, this tissue remodeling was associated with recovery of memory deficits, as demonstrated in the Morris water maze. Strikingly, APP/PS1 knock-in mice having severe and established early AD pathology treated with AAV9-CYP46A1, not only showed reduced amyloid plaques load and reduced astrogliosis, but also complete rescue of impaired electrophysiological and spine defects. In addition, we demonstrated the efficacy, neuronal tropism and tolerability of the AAV9 vector in the non-human primate hippocampus. Overall, our data strongly suggest AAV-CYP46A1 brain delivery as a relevant therapy for AD, therefore opening new avenues for the treatment of early severely affected Alzheimer patients.
: Center for Neuroscience and Cell Biology, University of Coimbra, Portugal
Machado-Joseph disease (MJD) is the most common dominantly-inherited ataxia. Although there is no cure, our group and others showed that RNA interference holds great promise for its treatment. However, these experiments involved craniotomy and in situ injection in the brain. There is therefore a need for less invasive procedures. The aim of the present study was to develop an AAV-based system that enables: delivery of RNA interference-based treatments to the brain, the specific silencing of mutant ataxin-3 (mutATAX3) and alleviation of the disease by intravenous injection. For that, we generated AAV9 vectors encoding an artificial microRNA that targets the mutant form of ataxin-3 mRNA (AAV9-mirATAX3). Its efficacy and specificity were firstly confirmed in neuronal cell models and the therapeutic potential was then tested in a severely impaired transgenic mouse model. Mice were intravenously injected at postnatal day one (PN1); were submitted to behavioral tests at 3 different ages and were sacrificed at PN95. AAV9-mirATAX3 vectors efficiently spread throughout the brain, transducing regions affected in MJD, such as the striatum, cerebellum, brainstem and spinal cord. AAV9-mirATAX3’s treatment also reduced the number of mutATAX3 aggregates and cerebellar neuropathology, and treated animals showed a better performance in all behavioral tests. Overall, this study provides compelling evidence that a single iv injection of AAV9-mirATAX3 at PN1 is able to transpose the BBB, silence mutATAX3 and alleviate MJD motor phenotype. To our knowledge, this is the first time that a non-invasive iv administration of rAAV9 had significant impact on motor deficits of a polyglutamine disorder.
: Lausanne University Hospital
Neurons are evolving in an environment composed of multiple cell populations with close relationships to ensure an adapted brain functioning. In particular, neuron-glia interactions are crucial for numerous processes ranging from brain homeostasis to synapse maintenance and their role in the neuronal vulnerability observed in neurodegenerative diseases is now recognized. Precise characterization of the contributions of these subpopulations in the central nervous system is essential to improve our comprehension of brain complexity in normal and pathological contexts. Currently, cell-type specific characterization is facing a major challenge in the isolation of cellular populations from adult animals with techniques compatible with high-throughput OMICS. Here we describe a highly efficient and flexible method for dissection and isolation of thousand of cells, compatible with high-throughput OMICS analysis. We used this tool to generate a transcriptome-wide profile of coding (mRNA) and non-coding RNA (miRNA) of striatal projecting neurons (Drd1 and Drd2 neurons), astrocytes and microglia. We identified specific molecular signatures revealing the implication of each cell-type in specialized functions but also in multi-cellular pathways. Co-analysis of mRNA and miRNA profiles highlighted differential regulation of gene expression across cell populations. Furthermore, comparison of our database with a previous transcriptomic study in Huntington's disease patients show the potential of this method for discovering new cell-type specific transcriptional dysregulations in a neurodegenerative state. This resource provides a powerful database unravelling specific roles of neurons and glial cells in the striatum with a great potential to improve our comprehension of the central nervous system.
: Lausanne University Hospital
Huntington‘s disease (HD) is a rare and dominant inherited disorder caused by the expansion of CAG repeats within the first exon of the huntingtin gene (HTT). Effective treatment for this devastating disease remains unavailable but HTT lowering strategies targeting the HTT mRNA have been developed and a clinical trial is underway. The recent development of the CRISPR/Cas9 system with its ease of design and high efficiency is broadening our ability to perform gene disruption or gene correction strategies. In this first application of gene editing in the central nervous system, the gene responsible for HD was targeted in adult mouse neuronal and glial cells. We designed an sgRNA (sgHTT1) targeting a region close to the translation start site of the human HTT gene, with the aim of permanently blocking HTT expression. Indels were highly frequent around the target site with more than 75% of the indels detected resulting in frameshifts. Furthermore, hHTT-82Q editing decreased by approximately 65% the aggregation of the mutant HTT protein while preserving neuronal functions. Precision and safety are prerequisites for the use of genome editing tools in the study of brain disorders, and particularly for potential therapeutic applications. Blocking Cas9 expression after HTT editing might significantly improve the final outcome of the approach. We therefore developed of a self-inactivating CRISPR/Cas9 system, kamiCas9, which trigger a transient expression of the Cas9 protein without altering its efficiency. These results demonstrate the considerable potential of the CRISPR/Cas9 system for applications in the context of genetic disorders of the brain.
: Charité University Medicine
With a worldwide prevalence of 1–2% epilepsy represents the most common chronic CNS disease. Roughly 70% of epilepsies are focal in origin and the high incidence of drug-resistant focal epilepsies poses an unmet medical challenge. Only selected patients benefit from surgical removal of the epileptogenic focus, a potentially risky and invasive procedure without guarantee of success. The majority of patients with focal epilepsy cannot permanently be freed from seizures and on the long run develop devastating cognitive comorbidities. The importance of endogenous peptides in seizure control is widely acknowledged, however the releasing neurons often degenerate during disease progression. We have developed a novel gene therapy as alternative treatment for refractive focal epilepsy. “Proof of concept” was provided with a neuropeptide-expressing AAV vector stereotactically applied to the epileptic focus in the established kainic acid mouse model for temporal lobe epilepsy. This led to complete and lasting suppression of seizures over several months, whereas control vectors did not alter disease progression. Behavioral tests for learning, memory, anxiety and depression-like behavior showed, that the typical long-term sequelae of repeated seizures did not develop in treated mice, whereas untreated epileptic mice deteriorated, as expected. Work is ongoing to develop the model towards clinical application as gene therapy for patients suffering from refractory mesial temporal lobe epilepsy and other types of intractable, focal epilepsy.
: Instituto de Biomedicina de Sevilla-IBiS, Hosp. Univ. VR/CSIC/Universidad de Sevilla, Sevilla, 41013, Spain
Intrastriatal carotid body (CB) grafts produce trophic protection and restoration of the dopaminergic nigrostriatal pathway in rodent and primate models of Parkinson's disease (PD), which is mediated by high levels of glial cell line-derived neurotrophic factor (GDNF) produced by CB implants. Phase I/II open trials showed that CB autotrasplantation improve motor symptoms in PD patients. However, the efficiency of CB cell therapy observed in clinical trials is lower than in experimental models, being patient age one of the factors influencing the clinical outcome. To explore limiting factors that affect the efficacy of human CB transplants, we have studied how aging and chronic hypoxia present in intracerebral grafts can modify CB GDNF expression. Chronic hypoxia induced an up-regulation of CB GDNF expression in young mice, while the same treatment in aged mice decreased CB GDNF expression. This age-related differential regulation of GDNF is also present in the intrastriatal graft and affects the efficacy of mice antiparkinsonian CB cell therapy. Moreover, human CB xenografts from young (≤40 years) donors induced an important protection of the nigrostriatal dopaminergic neurons of parkinsonian mice, while CB implants from aged (≥60 years) donors failed to produce a significant effect. Finally, we performed a study of the methylation status of human and murine GDNF promoter from young and aged CBs, identifying hypoxia-related regions that could explain the differential regulation of GDNF expression. These findings provide a molecular explanation of the outcome of previous clinical trials and offer insights for the design of new antiparkinsonian cell therapy treatments.
: Imagine Institut
Human fetal-derived neural stem/progenitor cells (hfNSCs) are under clinical evaluation for several neurodegenerative diseases. These cells display a favorable safety profile but require immunosuppression upon allogeneic transplantation in patients. In this scenario, obtaining bona-fide NSC populations from human induced pluripotent stem cells (hiPSCs) may be relevant to develop autologous ex-vivo gene therapy approaches to treat neurological disorders. We have recently generated a collection of hiPS-NSCs sharing molecular, phenotypic and functional identity with hfNSCs, which we used as a “gold standard” in a side-by-side comparison to validate the hiPS-NSC phenotype in vitro and in vivo (Meneghini et al., submitted). We are currently performing a genome-wide mapping of regulatory elements, integrating RNA-seq and ChIP-seq data, providing: (i) the comprehensive profile of genes differentially expressed among hiPSCs, hiPS-NSCs and hfNSCs; (ii) the broad description of promoter and enhancer usage characterizing the transition from pluripotent to neural restricted stem cells; (iii) the comparison of regulatory elements activated in hiPS-NSCs and hfNSCs. The identification of markers of critical steps in neural lineage commitment will aid strategies for increasing efficiency and consistency of hiPSC to neural differentiation. Furthermore, the definition of hiPS-NSC epigenetic and transcriptional signatures in comparison to hfNSCs is an essential step to better define their cell identity and safety profile.
: Ospedale San Raffaele
Epilepsy is a severe neurological disorder associated with hyperexcitability of neuronal brain networks. Since 1/3 of the patients is not responsive to common pharmacological treatments the development of alternative therapeutic strategies represents an urgent medical need. Transmembrane ionic channels are the major determinants of neuronal excitability, therefore modulating their expression in specific neuronal subtypes could represent a valid approach to restore the physiological excitation/inhibition balance in altered neural circuits. Our project took advantage of the innovative CRISPRa technology to induce the transactivation of the voltage-gated ion channels Kv1.1 and Nav1.1 in different neuronal populations in order to reduce the activity of excitatory glutamatergic neurons or to enhance the inhibitory control mediated by GABAergic interneurons, respectively. To this aim we first designed a set of sgRNAs directed to the promoter region of the two genes, and we screened their efficacy in inducing transcriptional activation of the target genes in P19 cell line and in mouse primary neurons. For each gene we identified an efficient sgRNA able to induce robust overexpression both at the transcript and at the protein level, after lentiviral transduction of the activatory dCas9-VP160 and the specific sgRNA. On the contrary we didn't detect any aspecific effect on predicted off-targets. Furthermore preliminary data show that electrophysiological activity of treated neurons changes in response to channel overexpression, suggesting that CRISPRa could be a valuable tool to modulate neuronal activity in vitro and encouraging future in vivo studies on epileptic mouse models to define the therapeutic potential of such an approach.
: Humanitas Clinical and Research Center
Current therapies in Parkinson's disease (PD) are exclusively symptomatic, treating the motor dysfunctions without altering the course and progression of the disease processes. Therefore, new therapeutic strategies that could directly counteract the pathological mechanisms are needed. Recent studies have shown that PD progression is strictly associated with the accumulation and propagation of alpha-Synuclein (α-Syn) toxic aggregates throughout the brain. In fact, over time α-Syn accumulates in neurons leading to their severe intoxication resulting in neuronal dysfunctions and eventual degeneration. This process is slow and can take years in humans, leaving enough time for applying new treatments aiming to block this deadly α-Syn accumulation and spreading. One of the potential targets for a gene-therapy approach for PD is the lysosomal enzyme Glucocerebroside (GCase). GCase viral transduction in brain, in fact, has proven to strongly enhance α-Syn degradation, but the mechanism is far from being fully understood. In this work we conducted an extensive study on the mechanism of action of GCase, and its role in modulating aggregation and diffusion of α-Syn aggregates in neurons. We also gained new knowledge on GCase intra- and extra-cellular trafficking, which could increase its therapeutic potential. These findings will be tested in vivo, in A53T-α-Syn transgenic mice, to validate the efficacy of the GCase gene-therapy approach in α-Syn toxic species degradation and rescuing of motor defects.
: Auburn University College of Veterinary Medicine, Scott-Ritchey Research Center
GM1-gangliosidosis is an autosomal recessive disease caused by mutations in the GLB1 gene encoding for the lysosomal acid beta-galactosidase (βgal) enzyme. The resulting enzymatic deficiency leads to accumulation of GM1-ganglioside in neurons and eventual massive neurodegeneration. Previously we have shown that intracranial injection of AAV-βgal vectors in GM1 mice and cats reduces lysosomal storage throughout the CNS. Additionally AAV gene therapy extends survival dramatically with >50% of AAV-βgal treated GM1 cats alive at 6 years of age compared to untreated cats that succumb to disease by 7–8 months of age. In our pre-clinical development program we are conducting two dose-ranging studies in 6–8 week-old GM1 mice injected with AAVrh10-βgal vector in thalamus (1E9, 3E9, 1E10, 3E10 vg) or intracerebroventricular (1E10, 3E10, 1E11 vg). These studies will determine the minimum effective dose to reduce GM1-ganglioside in cerebrum, cerebellum and spinal cord by >90% compared to age matched untreated controls and identify a maximum tolerated dose (MTD). Quantitative outcome measures are enzymatic activity and GM1-ganglioside content. The spatial distribution of βgal and lysosomal storage will be assessed in tissue sections. Previously we showed that overexpression of βgal in can lead to de novo lysosomal storage in injected structures in the brain and cause neurodegeneration in normal mice. A dose escalation study is being conducted in normal mice injected with AAVrh10-βgal to determine MTD. These studies will determine the dose ranges for long-term efficacy and safety studies in support of an IND application for an AAVrh10-βgal based clinical trial in GM1 gangliosidosis patients.
: IBET-Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal
Viral vectors derived from canine adenovirus serotype 2 (CAV-2) are considered promising tools for neurodegenerative disorders gene therapy. These vectors can bypass the clinical disadvantages of human adenoviruses, having demonstrated in rodent models attractive features, such as a preferential neuronal tropism and high level of axonal retrograde transport. However, accurate pre-clinical evaluation of efficacy and safety is critical for clinical translation. Human stem cells have great potential as complementary tools, bridging the gap between animal models and clinical trials. Herein we describe the assessment of helper-dependent CAV-2 (hd-CAV-2) efficacy and safety for gene delivery in a human stem cell-derived 3D neural in vitro model. Human neural progenitor/stem cells were cultured as neurospheres in dynamic culture systems. Differentiated neurospheres enriched in neurons, astrocytes and oligodendrocytes, presented increased expression of mature neuronal markers, along with synaptic functionality, neurotransmitter synthesis/release and were able to elicit voltage- and ligand-activated currents. Assessment of hd-CAV-2 transduction was performed at different MOIs, by evaluation of transgene expression and toxicity by cell viability and impact on neuronal gene expression. Under optimized conditions, hd-CAV-2 transduction led to stable long-term (up to 30 days) transgene expression with low toxicity. The evaluation of vector specific tropism showed that hd-CAV-2 has an increased neuronal tropism in contrast to hAd5 that preferentially transduced the glial cell population. This work demonstrates, in a physiologically relevant 3D model, that hd-CAV-2 vectors are efficient vehicle for gene delivery to human neurons, with stable long-term transgene expression and minimal cytotoxicity.
: Auburn University College of Veterinary Medicine
A deficiency of lysosomal β-galactosidase (βgal) causes the rapidly progressive and fatal neurologic disease, GM1 gangliosidosis, for which no treatment exists. Adeno-associated viral (AAV) gene therapy is effective in GM1 cats, with a greater than 6 fold increase in lifespan after bilateral injection of the thalamus and deep cerebellar nuclei. Delivery routes and AAV purification methods were tested to maximize safety and efficacy for upcoming clinical trials. To avoid injection of the cerebellum and improve cortical distribution, 3 cerebrospinal fluid (CSF)-based routes were tested: cisterna magna (CM), bilateral intracerebroventricular (ICV), or lumbar cistern (LC). GM1 cats were treated with 1e12 vector genomes/kg body weight with AAVrh10 expressing a feline βgal cDNA. After LC injection, enzyme was limited to the spinal cord, where it reached a maximum of 0.7-fold normal in the lumbar region. CM or ICV delivery restored βgal activity to 0.5–1.4 fold normal in the spinal cord and up to 0.7-fold normal in the cerebellum and caudal cerebrum, with no statistical difference between CM and ICV routes. A long-standing question regarding the influence of AAV production methods was tested by injecting the thalamus and lateral ventricle of GM1 cats with vector purified by cesium chloride or iodixanol centrifugation. All cats showed salutary effects of treatment, and clinical disease progression between groups will be presented. Based on the results of this study, injection of the thalamus and CSF (CM or ICV) should prove beneficial in clinical trials. Combining CM and ICV injection could further enhance therapeutic effect.
: Paik Institute for Clinical Research, Inje University College of Medicine, Busan 614-735, Republic of Korea
Neural crest stem cells (NCSCs) are widely distributed in a set of neural crest-derived adult tissues, but not in the adult peripheral nerve (PN). We represent here the existence of multipotent adult NCSCs in PN and their neurotrophic and neuroprotective roles in a spinal cord injury model of rat. NCSCs isolated by niche-preserving organ culture of PN showed robust clonogenic potential, uniformly expressed neural crest-specific markers, and had the ability to differentiate into multiple mesodermal and neuroglial lineage cells in vitro. NCSCs were able to secrete neurotrophic, neuroprotective, and anti-inflammatory factors, all of which led to promoting cell growth, neurite outgrowth, and synaptogenesis, but inhibiting ROS-induced cell death and inflammatory response in vitro. Through implantation of multilayered cell sheet, NCSCs promoted functional and structural recovery of injured spinal cord in a cell-number dependent manner. These results suggest that adult NCSCs residing in PN opens new opportunities for spinal cord repair.
: Imperial College London
The progressive phase of multiple sclerosis (MS) is characterised by accumulating grey matter (GM) pathology. The presence of immune cell infiltrates in the meninges is associated with lymphoid tissue development, greater cortical demyelination, shorter disease duration and significant neuronal loss. Analysis of isolated meninges of MS cases has shown increased gene expression for the pro-inflammatory cytokines: tumour necrosis factor (TNF), lymphotoxin- (LT) and interferon-(IFN). We tested the hypothesis that chronic production of pro-inflammatory cytokines in the meningeal compartment and diffusion into underlying GM can drive MS GM pathology in a rat. For this we cloned the human cytokine genes downstream of the full CMV promoter and produced high titer HIV-1 based VSVG pseudotyped lentiviral vectors. These were stereotactically injected into the sagittal sulcus of DA rats so as to deliver continuous transgene expression in the meninges. Efficient transduction of meningeal cells resulted in cytokine expression for up to 3 months. Injection of vectors for TNF or LT, in combination with IFN, induced the formation of large immune cell aggregates in the meninges by 28 days, which remained at 3 months. Subpial demyelination was observed accompanied by widespread microglial activation underlying these cellular aggregates. A decrease in neurofilament expression in regions with subpial demyelination was observed. RT-PCR on cortical RNA from TNF and IFN vector injected animals showed an increase in expression of TNFR1 and downstream necroptotic genes, compared to control animals. Our results indicate that this somatic rat model is useful for delineating mechanisms relevant to the pathology of MS.
: University of Manchester
Non-viral vector design for the CNS using nanomaterials has demonstrated effective therapeutic siRNA-mediated gene silencing at localized brain foci. In this study, nanoscale constructs comprising amino-functionalised multi-walled CNTs (f-CNTs) complexed with siRNA against the pro-survival Bcl-2 mRNA (siBcl-2), stereotactically targeted the striatum and subthalamic nucleus of parkinsonian rodents. Following injection of the f-CNT:siBcl-2 vectors, expression was successfully silenced and this was in agreement with cellular apoptosis on TUNEL staining. To evaluate functional rehabilitation, f-CNT:siBcl-2 vectors were stereotactically administered into the STN of hemiparkinsonian rats induced by 6-OHDA injection at the medial forebrain bundle (MFB). This was aimed to suppress pathological neuronal over-activation by induction of localised apoptosis. Efficacious in vivo Bcl-2 gene silencing was afforded by f-CNT:siBcl-2 at the mRNA and protein levels. Bcl-2 gene knockdown led to significant functional recovery of hemiparkinsonian symptoms compared to controls without adverse events or neuroinflammatory responses. This proof-of-concept study offers promising evidence for the application of f-CNT:siBcl-2 as safe neurosurgical tools that can achieve therapeutic gene silencing at localised brain structures for management of focal neurological pathologies.
: Qingdao University Affiliated Hospital
The intracranial transplantation of the dopaminergic neurons to treat Parkinson depends on the number of viable donor cells. The hypoxia limits the donor cells seeding in the recipient brain. Hypoxia-inducible factor 1 alpha (HIF-1α) have been found to associate with cell survival related genes. Based on our previous studies, our hypothesis was that the lower level of HIF-1α in the differentiated neural cells could be explained by vulnerability to hypoxia conditions. Immature and mature neural cells, and HIF-1α antisense oligo-deoxynucleotides (AODs) were engaged in this investigation. The results of these experiments were observed: (1) after O2 was changed from 95% to 50% for 2 hours, the HIF-1α mRNA expression increased about 3 x, 4 x and 1.5 x in precursor, glial and neuronal cultures respectively; (2) meanwhile, the mRNA levels of caspase 3 and 9 were elevated significantly in the neuronal cultures; (3) with AODs, HIF-1α decreased, the levels of both caspase 3 and 9 were increased in all cell cultures; (4) cells with high levels of HIF-1α are able to overcome the hypoxia pressure such as astrocytes. The data suggests that the expression characters of HIF-1α depend upon the type of neural cells. It is likely the hypoxia pressure is more lethal to the cells with low HIF-1α expression ability. Taken together, the potential of HIF-1α expression is one of the necessary native protection signals for neuronal cell survival. Limitation of hypoxia pressure and the promotion of the donor cells to express HIF-1α deserve further studies.
: Lund University
Parkinson's disease is a neurodegenerative disorder that affects approximately 1% of the population aged 60 and above [1]. The treatment of the disease primarily consists of Levodopamine (L-DOPA). Long-term treatment with L-DOPA does however sooner or later lead to dyskinesia. L-DOPA induced dyskinesia is a disorder in which striatal serotonin terminals are thought to inappropriately release dopamine, which activates incorrectly long-term potentiated Dopamine receptor 1 (DRD1) medium spiny neurons (MSN) [2]. We therefore sought to find a way to efficiently and stringently target DRD1 MSN instead of Dopamine receptor 2 (DRD2) MSN. We intended to do this using microRNA target sites (miRT). In order to be able to select the correct miRTs we first had to sequence the microRNA (miRNA) population of the DRD1 and DRD2 MSN. To do this, we decided to use microRNA immunoprecipitation (miRIP) [3] and the fact that the DRD1 and DRD2 MSN project to different nuclei in the brain. We wanted to inject a vector with a high degree of retrograde transport into either the globus pallidus or substantia nigra, expressing a GFP-Ago2 fusion protein. The striatum would then be collected in order to perform miRIP on the transduced neurons. We therefore tested a few novel AAV capsids developed by Tomas Björklund's group to test their transduction and retrograde transport efficiency from the substantia nigra to the striatum.
1. Wirdefeldt, K., et al., Eur J Epidemiol, 2011.
2. Carta, M., et al., Prog Brain Res, 2008.
3. He, M., et al., 2012.
: San Raffaele-Telethon Institute for Gene Therapy (SR-Tiget)
Over the past decades outcomes of clinical hematopoietic stem cell transplants have established a relationship between the sources of infused hematopoietic stem cells (HSCs) and their differential homing and engraftment properties. We investigated the molecular and functional features of human primitive HSCs from different sources to unravel their reconstitution potential. We show that HSCs from different sources, i.e. BM and peripheral blood following mobilization by growth factor granulocyte-colony stimulating factor (G-CSF) and/or Plerixafor, are endowed with specific properties. Plerixafor mobilized cells possess the higher ability to home to hematopoietic niches and engraft in immunodeficient NOD/ShiLtSz-scid/IL2Rγnull mice. The higher content of CXCR4+ and CD49f+ cells correlates with this feature. Furthermore, global gene expression profiling highlights the superior in vivo reconstitution activity of Plerixafor mobilized cells. This “stemness” signature is attenuated by the combined use with G-CSF, which is added for higher cell yield in the apheretic product. These data indicate that a qualitative advantage accounts for the superior performance of Plerixafor mobilized cells. These findings provide the rationale for using a suboptimal dose of more primitive HSCs when target cell number for transplantation is limited, or when harvesting by G-CSF mobilization is too risky, like in patients affected by sickle cell disease. Moreover, CD34+ cells mobilized by Plerixafor alone or with the combination of G-CSF are efficiently transduced by a lentiviral vector encoding for human ß-globin gene (GLOBE LV) and are able to engraft and differentiate in vivo, supporting their use for gene therapy applications.
: Josep Carreras Leukemia Research Institute
Human hematopoiesis is finely regulated by gene regulatory networks controlled by few master transcription factors (TFs). GATA2 is considered one of the master regulators of hematopoiesis, highlighted by the fact that genetic deletion of GATA2 in mice leads to severe anemia and embryonic lethality. New findings implicate GATA2 haploinsufficiency with some familial cases of myelodysplastic syndrome (MDS), bone marrow failure, aplastic anemia and MonoMac syndrome; however, the specific role of GATA2 during human early hematopoietic development remains unknown. To investigate the role of GATA2 on human hemogenic progenitors (HEPs) specification and their further differentiation into hematopoietic progenitors (HPCs) we generated hiPSC lines in which GATA2 signaling could be induced by doxycycline. The ectopic GATA2 activation at the point of origin of mesoderm and hemogenic endothelium (from day 2 to day 7 of embryoid body differentiation) significantly enhanced the number of HEPs (CD31+CD34+CD43-CD45-; ∼5 fold) and promoted their further commitment into HPCs (CD34+CD43+CD45+; ∼5 fold), without altering proliferation and survival of cells. Functionally, GATA2 enhanced the clonogenic potential of hematopoietic-derived cells (∼4 fold); however GATA2 on its own was not sufficient to confer in vivo long-term engraftment potential to hiPSC-hematopoietic derivatives. RNA-seq analysis showed that GATA2 establishes the transcriptional programs that are critical for specifying HEPs and HPCs during hiPSCs differentiation. Finally, we generated GATA2 knockout iPSC lines by CRISPR/Cas-mediated genome editing and the analysis of their blood differentiation potential is now ongoing. Our findings indicate that GATA2 promotes hiPSC-hematopoietic development, suggesting this regulator is critical for human HSCs emergence.
: Paul Ehrlich Institute
Thrombopoietin (Thpo)/Mpl-signaling regulates hematopoietic stem cell (HSC) function, megakaryopoiesis and platelet production. Mpl knockout(-/-) mice develop thrombocytopenia, have reduced HSC numbers and show repopulation defects after transplantation, reflecting the human phenotype in MPL deficient patients. Previously, we identified several Thpo target genes in models of HSC regeneration and aplasia using transplantations in the Mpl-/- mouse. The endothelial protein C receptor (Epcr; CD201) was found to be downregulated in Mpl-/- or Thpo-/- LSK cells (Kohlscheen S et al., PlosOne, 2015) and its surface expression to be upregulated after Thpo stimulation in vitro. Different candidates were overexpressed by lentiviral in vitro transduction of Mpl-/- HSC and subsequent transplantation into Mpl-/- recipient mice. Epcr overexpression (Mpl-/-/Epcr(td)) improved peripheral blood and LSK cell chimerism 18 weeks post transplantation (mean 82%, n = 3) compared to the negative control (Mpl-/-/GFP(td), mean 41%; n = 6, p < 0.05). Notably, Epcr expanded phenotypic LT-HSCs (LSKCD150+CD48-) 28- to 31-fold compared to Mpl-/-/GFP(td) and similar to the positive control (Mpl+/+/GFP(td), 43-fold). Megakaryopoiesis was also increased, however, MK did not efficiently produce platelets in the primary mice. Secondary transplanted BM of Mpl-/-/Epcr(td) mice engrafted more efficiently with still 8-fold increased LT-HSC numbers compared to Mpl-/-/GFP(td) transplanted HSC. Quantitative real-time PCR of sorted Mpl-/-/Epcr(td) LSK cells revealed increased expression of the anti-apoptotic gene Bcl-xL and the cell cycle inhibitor Cdkn1a. Recently, the aPC/Epcr/Par-1-pathway was shown to regulate HSC niche retention (Cohen SG et al., Nat. Medicine, 2015), which could add up to a possible mechanism of Mpl-/- HSC expansion.
: Hacettepe University Center for Stem Cell Research and Development
Endocannabinoids are endogenous morphine ligands and present widespread receptor-mediated effects at physiological and pathological levels on many systems (1). These effects are partially realized through mechanisms affecting cell growth, differentiation, apoptosis and migration at the molecular level (2). Endocannabinoid ligands (AEA, 2-AG) are released from mesenchymal stem cells (MSCs) and their receptors (CB1, CB2) are located on human bone marrow hematopoietic stem cells (HSCs). Endocannabinoids play a role in cytokine-mediated hematopoietic stem cell migration, the effects of these mediators on human bone marrow derived HSC mobilization have not been compared and their relationship with the sympathetic nervous system is still remains unknown. During mobilization, endocannabinoids and the sympathetic nervous system may be working together. In order the test this hypothesis, the migration of HSCs towards (i) SDF-1, (ii) AMD3100, (iii) norepinephrine (iv) adrenergic receptor antagonist (SR59230), (v) endocannabinoid ligands, (vi) CB1 receptor antagonist (AM281) can CB2 receptor antagonist (AM630) have been investigated. In addition, migration of HSCs towards MSCs was studied in the presence of various antagonists. As a source of HSCs, apheresis product collected from healthy donors were used. Hematopoietic cell mobilization towards SDF-1 and epinephrine was inhibited by AMD3100 and SR59230, respectively. Mobilization in presence of the endocannabinoid ligands AEA and 2-AG was increased compared to SDF-1 alone and inhibited when using the endocannabinoid receptor antagonists AM281 and AM630. HSCs migration towards MSCs is inhibited by AMD3100, SR59230, AM281 and AM630. Thus, endocannabinoids may be candidate for clinical use of to enhance or facilitate/accelerate G-CSF mediated HSC mobilization.
: Fundacion Publica Andaluza Progreso y Salud/Genyo
Human embryonic stem cells (hESCs) are a unique model to study early human development. Runx1 is a master hematopoietic transcription factor essential for definitive HSC emergence. Runx1-deficient mice die during early embryogenesis due to the inability to establish definitive hematopoiesis. Here we analyzed the role of RUNX1 in human embryonic hematopoiesis. RUNX1a, b and c were expressed in CD45+ hematopoietic cells; however, RUNX1c was the only one expressed in hemato-endothelial progenitors (HEPs). Constitutive expression of RUNX1c in hESCs enhanced the appearance of HEPs and promoted subsequent differentiation into blood cells. Conversely, specific deletion of RUNX1c virtually abrogated the hematopoietic potential of HEPs, indicating that RUNX1c is a master regulator of human hematopoietic development. Gene expression profiling of HEPs revealed a RUNX1c-induced pro-inflammatory molecular signature, supporting previous studies demonstrating pro-inflammatory signaling as a regulator of hematopoietic stem cell emergence. Collectively, RUNX1c orchestrates hematopoietic specification of hESCs, likely in cooperation with pro-inflammatory signaling.
: Stem Cell Neuroplasticity Research Group, Kyungpook National University, Daegu, Korea
Hematopoietic stem/progenitor cell (HSPC) mobilization is an essential homeostatic process regulated by the interaction of cellular and molecular components in bone marrow niches. It has been shown by others that neurotransmitters released from the sympathetic nervous system regulate HSPC egress from bone marrow to peripheral blood. In this study we investigate the functional role of neuropeptide Y (NPY) on this process. NPY deficient mice had significantly impaired HSPC mobilization due to increased expression of HSPC maintenance factors by reduction of matrix metalloproteinase-9 (MMP-9) activity in bone marrow. Pharmacological or endogenous elevation of NPY led to decrease of HSPC maintenance factors expression by activating MMP-9 in osteoblasts, resulting in HSPC mobilization. Mice in which the Y1 receptor was deleted in osteoblasts did not exhibit HSPC mobilization by NPY. Furthermore, NPY treatment in ovariectomized mice caused reduction of bone loss due to HSPC mobilization. These results suggest a new role of NPY on HSPC mobilization, as well as the potential therapeutic application of this neuropeptide for stem cell-based therapy.
: San Raffaele Telethon Insitute for Gene Therapy (SR-Tiget)
Human hematopoiesis is a hierarchically organized system where distinct cell types with diverse properties are continuously generated from primitive Hematopoietic Stem/Progenitor Cells (HSPC). Several hematological disorders cause unbalances of immature and mature cell composition. We designed a novel protocol capable of analyzing in a single test-tube both progenitors and all major mature cell lineages on either bone-marrow (BM) or peripheral blood (PB) samples by multi-parametric flow-cytometry, called “Whole Blood Dissection” (WBD) (under patent filing). Starting from 100ul and 500ul of BM and PB whole blood respectively, WBD allows unambiguously identifying up to 23 different blood cell types including HSPC subtypes and all the major myeloid and lymphoid lineage compartments at different stages of maturation, through a combination of 18 surface markers and 1 viability cell marker. With our multi-parametric staining we are able to cover, on average, 99,7% of each sample in a reproducible, standardized, cost- and time-efficient manner. We validated our technology performing morphological evaluation and analyzing BM and PB samples from adult (n = 9) and pediatric (n = 10) healthy donors highlighting age-related shifts in blood cells composition. We tested the efficacy of our protocol on clinical samples from patients with aberrant hematopoietic cell composition unveiling known and novel unbalances on 6 immune-deficient patients and on 4 patients with lymphoid or myeloid acute leukemia, Overall, our WBD protocol provides a one-shot comprehensive evaluation of human hematopoietic system for the characterization of hematopoietic disorders and for the monitoring of hematopoietic reconstitution and HSPC maintenance in patients after transplant and gene therapy.
: Hacettepe University
Hematopoietic stem cells (HSCs) collected from umbilical cord blood (UCB) are limited in number and ex vivo expansion is desirable to shorten reconstitution time after transplantation. We have developed an approximately 15-fold expansion method of repopulating stem cells by stimulating CD34+ cells with SCF/TPO/aFGF/IGF-2/Angptl3 (STIFA3). In the present study, research grade StemMACS™ HSC Expansion Medium has been compared to a pre-lot clinical grade HeMAtiCS GMP Medium ((Miltenyi Biotec). CD34+ UCB cells were cultured for 7 days, either stimulated with STIFA3 or the StemMACS HSC Expansion Cocktail containing SCF/TPO/Flt3L (STF), supplemented with IGF-2 and Angptl3. Progenitor cell frequency (MethoCult™, STEMCELL Technologies Inc.) and CD34/CD38/CXCR4 expression by FACS were analysed before and after culture. STF/IGF-2/Angptl3 outperformed STIFA3 with respect to total cell expansion, on average 20.1 ± 4.4 and 25.4 ± 8.5 fold expansion in, respectively, the research grade and the clinical grade medium after stimulation with STF/IGF-2/Angptl3. The CD34+/CD38- population, which contains the repopulating stem cells, expanded in the STIFA3 stimulated cultures respectively 4.0 ± 1.2 and 5.1 ± 17 fold. Significant differences were not found in the frequencies of the progenitor cells BFU-E, CFU-GM or CFU-GEMM within the four groups, indicating a similar potency of cells. CXCR4 expression, a determinant of stem cell homing, was relatively higher in cultures treated with STF/IGF-2/Angptl3 irrespective of the medium used. In conclusion, the clinical grade pre-lot HeMAtiCS medium performs as well as the research grade medium, opening the way to clinical application of stem cell expansion using the developed technology.
: Hacettepe University
Biogenic amines (dopamine and serotonin) protect cells against hypothermia-induced damage (1). The newly developed compound ROKEPIE®-S01 (Sulfateq BV) has been successfully demonstrated for hypothermic storage and transportation of cell lines (2), but not of primary hematopoietic stem cells (HSC). Hypothermic preservation of HSC could be preferred over short-term cryopreservation, to prevent cell loss during freezing/thawing and could be particularly useful for short-term storage, e.g., during conditioning of transplant patients or transport of HSC transplants. Here, we assessed optimal doses of this compound and culture conditions to preserve CD34+ cord blood HSCs. HSCs were supplemented with TPO, TPO+SCF, TPO+SCF+Flt3L or TPO+SCF+aFGF+IGF2+ Angptl3 (STIFA3) for 2 days (analogous to our gene transfer method) or 7 days (analogous to our HSC expansion method). The compound was then added directly to the medium, the plates closed airtight and stored up to 7 days at 4°C. After rewarming, the frequency of apoptotic cells and cell cycle status were assessed. Two day cultures with TPO resulted in the lowest frequency of apoptotic cells after hypothermic storage in the presence of 1:10 or 1:20 diluted compound. After 7 days of culture/hypothermic storage, cell viability was 10–30% higher in cultures containing the compound, whereas numbers of cycling cells were 10–20% lower, in particular when stimulated with STIFA3. Thus, ROKEPIE®-S01 effectively promotes cell viability of primary HSCs during short-term hypothermic storage, and is currently assessed for its effects on long-term repopulating HSC. (1) Talaei F et al., PLoS One. 2011;6(7):e22568 (2) Talaei F et al. Patent application WO2011128458 A1
: Fundacion Publica Andaluza Progreso y Salud/Genyo
Platelet transplantations are essential to treat patients with thrombocytopenias who show a decrease in the number of circulating platelets in the bloodstream. Nowadays, peripheral blood from healthy donors is the only source of platelets in clinic. Human platelet production from several hematopoietic stem cells sources has been succesfully reported. Human Pluripotent Stem Cells (hPSCs) represent a potential donor-independent source of platelets which could constantly provide hospitals with these blood components, due to their unlimited growth capability in vitro. Although there have been some advances in this field, the production of platelets from hPSCs is still inefficient and more effective protocols are required. A posibility may be trying to reproduce physiological conditions found in the bone marrow niche where megakaryopoiesis occurs. We analyse the role of hypoxia in megakaryopoiesis from hPSCs in vitro. Our results demonstrate that hypoxia reduces cell viability of hPSCs selecting progenitors with a higher megakaryopoietic potential. Importantly, an increase in the number of both mature megakaryocytes and platelets derived from hPSCs was observed. Recently, our group showed that SCL-overexpression potentiated megakaryopoiesis from hPSCs. However, hypoxia reduces megakaryocyte production in SCL-overexpressing hPSCs. A more detailed molecular analysis has allowed us to demonstrate that hypoxic conditions accelerate SCL degradation through MAPK mediated-phosphorylation and proteasome degradation. These results have allowed us not only to increase our understanding of the molecular mechanisms controlling human megakaryopoiesis from hPSCs, but also to improve our current megakaryocytic differentiation protocols
: Department of Hematology and Blood Banking, Faculty of medical Sciences, Tarbiat Modares University, Tehran, IRAN
Hepatocyte Growth Factor (HGF), a pleiotropic growth factor, plays pivotal role in hematopoiesis, motility, growth and mobilization of hematopoietic stem/ progenitor cells (HSPCs). It has been reported that HGF is constitutively produced by bone marrow mesenchymal stem cells (MSCs). Previous studies demonstrated that MSCs expressed erythropoietin (EPO) receptor. In current study we aimed to assess the effect of EPO on HGF secretion in Bone marrow-derived MSCs (BM-MSCs). Following Characterization cells were treated with EPO (4 IU/ml) for 6, 24 and 48 hours. After treatment at defined time point, HGF expression and protein levels assessed using quantitative real time PCR (qRT-PCR) and Enzyme-linked immunosorbant Assay (ELISA). In order to show the effect of secreted HGF on migration of HSPCs, CD34+ hematopoietic stem cells (HSCs) were isolated from umbilical cord blood and evaluated using Transwell migration assay system. We observed a significant increase in level of HGF in cell supernatant after 48h compared to control group (P < 0.05). Also, qRT-PCR results demonstrated a significant elevation in HGF expression level after 24 and 48 hours treatment with EPO compared to control group (P < 0.05). Finally, migration assay results show a significant increase in migration of HSCs in the group exposed to EPO and MSCs after 48 hours. Our Data indicate that EPO could play important role in inducing migration of HSCs by altering critical genes in the process of stem cell mobilization. Keywords: Hematopoietic stem cells, Bone marrow, Mesenchymal stem cells, Erythropoietin, Hepatocyte Growth Factor
: StemCell2MAX, Biocant Park Núcleo 04 Lote 02, 3060-197 Cantanhede, Portugal
Haematopoiesis is a developmental process that generates all blood cell lineages in health and disease. This relies on quiescent haematopoietic stem cells (HSCs) that are able to differentiate, self renew and expand upon physiological demand. HSCs have great interest to regenerative medicine, including haematological malignancies, immunodeficiencies and metabolic disorders. However, the limited yield from existing HSC sources drives the global need for reliable techniques to expand harvested HSCs at high quality and sufficient quantities. With the extensive use of cord blood progenitors for clinical applications there is a demand for a safe and efficient expansion protocol that is able to overcome the limitations of the cord blood as a source of HSC. StemCell2MAX developed a technology that enhances the survival, proliferation and transplantation efficiency of HSC, leading the way to a more widespread use of HSC for research and clinical purposes. In recent studies, StemCell2MAX MIX was shown to efficiently support the selective expansion of CD34+ hematopoietic stem and progenitor cells (both from enriched and non-enriched cord blood cell populations), while maintaining the multipotent differentiative potential. The technology developed by StemCell2MAX opens new horizons for the usage of expanded hematopoietic progenitors for both research purposes and clinical applications.
: Gene Regulation, Stem Cells and Development Lab. GENyO: Pfizer-Universidad de Granada-Junta de Andalucia Centre for Genomics and Oncological Research. Granada
Bernard Soulier syndrome (BBS) is a rare platelet disorder characterized by thrombocytopenia and frequent bleeding. It is caused by mutations in one of the three genes coding for the glycoprotein complex in platelets GPIb-IX-V. These mutations inhibit expression of this complex on the membrane and prevent its binding to the Von Willebrand factor, resulting in defects in platelet adhesion and aggregation. When severe bleeding occurs, platelet transfusion is the only available treatment. However, recurrent transfusions can induce platelet refractoriness. Restoration of the mutated gene in hematopoietic stem cells might represent a powerful treatment for BBS patients. Induced pluripotent stem cells (iPSCs) are an excellent tool for human disease modeling. We generated iPSCs derived from peripheral blood cells of three BSS patients containing three different mutations in the GPIX gene (iPSC-BSS). iPSC-BSS generated were free from reprogramming vectors, preserved a normal karyotype and maintained stemness and plurypotency. We also designed gene therapy tools to correct iPSC-BSS mutation following two approaches: 1) using (CRISPR)-Cas9 system to repair the endogenous gene by homologous recombination; 2) expressing wt GPIX driven by a constitutive promoter in lentiviral vectors. By inducing in vitro megakaryocytic differentiation in iPSC-BSS lines and corrected ones, we will analyze whether megakaryocytes and platelets keep the same functional alterations that occur in patients. Moreover, we will evaluate whether gene correction restores both the number and the functionality of the platelets in corrected iPSC-BSS in vitro. Our results may open the possibility of applying gene therapy for curative treatment in BBS patients.
: Experimental and Clinical Cell Therapy Institute, Paracelsus Medical University, Salzburg, Austria
Leukotrienes are important response mediators. Their receptors are expressed on various cell types including immune cells, hematopoietic stem/progenitor cells (HSPCs), mesenchymal stem/progenitor cells (MSPCs) and endothelial progenitors. Leukotriene receptor antagonists are used for asthma treatment but were also shown to modulate HSPC migration and promote neurogenesis. Aiming to better understand injury-induced and drug-induced stem/progenitor cell mobilization, we asked whether cysteinyl-leukotriene receptor antagonism influences circulating stem cells. Blood samples from five healthy adults taking an LTD4 antagonist daily for one week were analyzed at four time-points, defining baseline, early (4–6h after 1st medication), intermediate (day 3–4) and late (day 7) mobilization by complete blood count and high-resolution 10-color flow cytometry focusing on HSPCs, MSPCs and endothelial colony-forming progenitor cells (ECFCs). Human bone marrow aspirate and umbilical cord blood served as technical positive controls. We found a significant mean 1.9-fold mobilization of lineageNEG/CD34+/CD38-/CD45RA-/CD90+ hematopoietic stem cells (HSCs; baseline = 79 ± 18; day seven = 147 ± 36/mL; mean± SEM) after seven days of drug intake. There were no significant differences in total CD34+ and various HPCs analyzed (multipotent progenitors, common myeloid progenitors, megakaryocyte/erythrocyte progenitors, granulocyte/macrophage progenitors, common lymphoid progenitors and lymphoid-primed multipotent progenitors) or in leukocyte count, subsets (neutrophils, monocytes, lymphocytes) or lymphocyte subsets (T cells, B cells, NK cells) before day seven. No detectable levels of MSPCs or ECFCs were mobilized by leukotriene inhibition (detection threshold 10-6). While inhibition of leukotrienes induced no significant changes in the composition of mature blood cells and their progenitors, it may have a previously unobserved mobilizing effect on the most immature pluripotent HSCs.
: Universidad Nacional Autónoma de México
Induced pluripotent stem cells (iPSC) have potential to become useful research tools to model diseases, cell therapy and regenerative medicine. Dilucidating differentiation of human induced pluripotent stem (iPS) cells is important for understanding both normal and pathological hematopoiesis. Primitive hematopoiesis is thought to derive from a multipotent progenitor called the hemangioblast identified by the phenotype CD34+CD309+CD235-. iPSC were derived from a primary culture of dental pulp mesenchymal cells of a healthy donor in a serum/feeder free system and then transduced with lentiviral vectors containing the transcription factors Oct4, Sox2, Klf4 and C-Myc. Pluripotency was assessed by expression of Oct4, Sox2, Nanog and Tra1-60/80 and teratoma formation in nude mice. iPS and ES Differentiation was achieved in a monolayer serum free system with media containing BMP-4. Once mesodermal markers T and Sox17 were present, media was changed into differentiation media containing hematopoietic cytokines. Day 6 marks the multipotent stage, as shown by the LT-HSC phenotype, CFC cultures and NK/lymphoid lineages, however our evidence shows that the presence of CD34+ occurs only in a reduced window of time since further differentiation into mature lineages (with exception of erythroid cells) it's not possible from day 8 on with/without EPO due to the absence of mature populations. This findings show that is possible to obtain hematopoietic stem cells from a KDR+ CD34+ population via mesodermal induction in a monolayer free serum system without embryoid bodies allowing access to enriched populations for future studies in iPSC hematopoietic differentiation and generation of hematopoietic progenitors in vitro.
: Yousei University
Differentiation of human induced pluripotent stem cells (hiPSCs) by reprogramming endometrium cell in to the erythroid lineage of cells offers a novel opportunity to study hematopoietic and erythroblast development in vitro. We describe an approach for the efficient generation of erythroblast from hiPSC using an OP9 coculture system to induce hematopoietic differentiation followed by selective expansion of erythroid cells in serum-free media with erythropoiesis-supporting cytokines. We showed that endometrium-derived transgenic hiPSCs generated using retrovirus-based vectors can be differentiated into hematopoietic and erythroblast. To induce hematopoietic differentiation by day 9 of hiPSC/OP9 coculture, we have 12% ∼ 13% of CD43+, 3% of CD34+ hematopoietic marker presents on all coculture cells (no isolation). Analysis of the erythroid cultures by morphologic analysis Wright-Giemsa stained cytospins revealed that after 7–17 days of expansion. All hiPSCs showed terminal maturation into polychromatic and orthochromatic erythroblasts. Overall, these results demonstrate the feasibility production of erythroid cells from endometrium-derived hiPSCs.
: Pirogov Russian National Research Medical University
MRI combined with the labeling of cells with superparamagnetic iron oxide (SPIO) particles is one of the methods of visualization of transplanted cells in live animals, currently under development. The goal of this study was to quantitatively re-evaluate the sensitivity of different MRI techniques in SPIO labeled cells tracing studies. Cultured mesenchymal stromal cells from human placenta loaded with fluorescent SPIO microspheres were injected into collagen phantoms or stereotaxically into the rat striatum. Labeled cells were visualized in the live brain or phantom using T2 weighted imaging (T2WI), T2* weighted imaging (T2*WI), and susceptibility-weighted imaging (SWI). In vivo MRI data were verified by histological methods. SWI ensured detection of 101 cells in vitro and 102 cells in vivo, superior to T2WI and T2*WI techniques and better than claimed in any published paper. Actually, SWI allowed detection of MR signal even from 101 cells injected into the brain tissue, but it could not be statistically differentiated from MR signal produced by traumatic microhemorrhages at the injection site. Conclusion: SWI, due to its high sensitivity and resolution, is the most sensitive pulse sequence for visualization of SPIO labeled cells. Despite some restrictions, such as the blooming effect at high SPIO concentrations and inability to differentiate low concentrations of the SPIO label from intra-tissue iron deposits, it is the method of choice for cell tracking studies.
: University College London
Using novel imaging algorithms and optical oxygen sensors integrated in a microfluidic device, we demonstrate (1) automated culture of embryonic stem cells in volumes similar to volumes of 96-well plates, (2) on-line establishment of cell growth curves via rapid segmentation of phase contrast microscopy images, and (3) real-time determination of specific oxygen uptake rates. The data is obtained label-free and without disrupting the culture. The non-invasive and real-time determination of the growth and oxygen kinetics of adherent stem cell cultulres will inform future studies in stem cell biology and stem cell-based therapies.
: Karolinska Institute
: Humboldt University
Engineered T cells carrying transgenic T cell receptor (TCR) genes are effective in cancer immunotherapy. However, their modification requires ex vivo gene transfer, which most often is performed using retrovirus vectors (RV). This approach is laborious, expensive and not suitable for personalized immunotherapy targeting mutated, tumor-specific antigens. We modified the Sleeping Beauty (SB) transposon system to achieve efficient gene transfer into human T cells. Since the electroporation of large DNA amounts results in T cell damage, we replaced the transposase encoding DNA plasmid vector by in vitro-transcribed RNA and the TCR-encoding transposon DNA plasmid vector by a minicircle vector. Due to these modifications, T cell viability increased from 5 to 50% and stable gene transfer of about 50%, resulting in functional T cells, was achieved. Thus, SB-mediated gene transfer can be used to produce TCR-engineered T cells in a short time for personalized immunotherapy. We also developed targeted RV capable of transducing subsets of T cells. We modified the measles virus (MV) envelope glycoprotein hemagglutinin (H) by adding sequences encoding single-chain antibody fragments, derived from CD8α or CD4 hybridoma. The generated CD8α (MVm8) and CD4 (MVm4) targeted RV exclusively transduced CD8-positive and CD4-positive murine T cells, respectively. By i.v. injection of the CD8-targeted RV, encoding an ovalbumin- (OVA) specific TCR, MVm8 RV mediated specific in vivo transduction of CD8-positive T cells. Upon in vivo CD8 T cell engineering by MVm8, mice were challenged in an infection model with OVA-expressing bacteria and developed protective immunity.
: Paul-Ehrlich-Institute
Cancer immunotherapy has raised a lot of attention due to substantial clinical benefit obtained in end stage cancer patients. Antibodies directed against checkpoints such as PD-1 (programmed death-1) and PD-L1 (programmed death-1 ligand 1) are under clinical development or have reached the market, especially for the therapy of malignant melanoma. While the clinical response is at least in some cancer patients impressive, therapy is often associated with a number of immune-related adverse events. Therefore, our aim is the specific delivery of immune checkpoint inhibitors precisely to sites of tumor growth. This way, we aim at reducing the side effects and enhancing the therapeutic benefit of checkpoint inhibition. As vehicle for the delivery of checkpoint inhibitors tumor-targeted AAV vectors are used. Her2-AAV selectively transduces Her2/neu-positive tumor cells. Her2-AAV is completely redirected from liver to tumor lesions when injected i.v. and enabled the safe systemic administration of cytotoxic genes. A similar tumor-restricted biodistribution in tumor-bearing BALB/c mice was obtained for the recently generated CD105-AAV, which binds to the tumor marker endoglin for cell entry. Both vectors are currently assessed for the delivery of checkpoint inhibitors. For this purpose, we have packaged scFv-Fc fusion proteins of α-PD-1 and α-PD-L1 into AAV vectors. Upon transduction of tumor cells the antibodies were readily detectable in the cell culture supernatant and showed specific binding to their target antigen, respectively. In vivo, expression of the AAV-encoded checkpoint inhibitors was detected in subcutaneously growing RENCA tumor cells. Biodistribution analyses of systemically injected AAV-encoded checkpoint inhibitors are ongoing.
: Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, 97078, Germany
Adoptive therapy with CD19-specific chimeric antigen receptor (CAR) T cells that were gene-modified with gamma-retroviral and lentiviral (LV) vectors has shown efficacy in pilot clinical trials. However, there are biosafety concerns related to viral vectors, and their production cost and regulatory demands are a roadblock for broad clinical implementation of CAR T-cell therapy. In this study, we engineered CAR T cells through non-viral Sleeping beauty (SB)-transposition from minimalistic DNA vectors called minicircles (MCs) to encode CAR transposon and SB100X transposase. The stable gene transfer rate and cell viability was 4-fold and 1.5-fold higher respectively when MCs rather than conventional plasmids were used (n = 7; p < 0.01). In aggregate, this translated into a 6-fold higher yield of CAR + T cells and therapeutic doses could be obtained without the need for feeder cell expansion within 14 days of culture. Importantly, CD19-CAR T cells modified by MC-based SB-transposition were equally effective as LV transduced CD19-CAR T cells in vitro and mediated lymphoma eradication in a xenograft model. To address biosafety, we performed the most comprehensive genomic insertion site analysis performed with SB in T cells to date. The data show a close-to-random integration profile of MC-derived CD19-CAR transposons, without preference for highly expressed genes or oncogenes. Intriguingly, a higher proportion of SB compared to LV integrations had occurred in genomic safe harbors (7-fold; p < 0.01). The high gene-transfer rate of MC-based SB-transposition and superior safety profile compared to viral vectors position our novel approach to become a preferred gene-transfer strategy in advanced cellular and gene-therapy
: Miltenyi Biotec GmbH
Recent breakthroughs in the treatment of lymphoid malignancies using chimeric antigen receptor (CAR)-redirected T cells have generated intense interest to transfer this technology to the solid tumor setting. However, therapeutic success has been hampered largely due to the lack of truly tumor-specific antigens. In our approach to develop a CAR-based cancer treatment modality for solid tumors that is both effective and curative, we have identified the sialyl glycolipid stage-specific embryonic antigen (SSEA-4) as a promising target. Since its expression strongly correlates with chemoresistence and metastasis in various types of solid tumors but shows restricted distribution in normal tissues, a combinatorial therapeutic approach using chemotherapy followed by CAR T cell therapy holds great promise to improve the treatment outcome of cancer patients. For this study, we constructed several second generation anti-SSEA-4 CARs and compared their potency in T cell activation by analyzing the degranulation capacity, cytokine production, and killing ability of the effector cells upon antigen engagement. Focus of the analysis was the impact of the single chain variable fragment (scFv) orientation as well as the spacer type and length on CAR functionality. Having assessed the performance of different anti-SSEA4 CAR constructs, we next generated a panel of CARs containing humanized scFvs and assessed their performance in primary T cells. CAR T cells with humanized scFv can overcome potential anti-mouse immune-mediated rejection and prolong in vivo persistence, thereby improving the efficacy and safety of CAR T cells. Current studies are now focusing on evaluating the in vivo functionality using mouse models.
: Institut Necker Enfants Malades, Inserm U1151
Recombinant adeno-associated viral (rAAV) vectors exhibit interesting properties as vaccine carriers for their ability to induce long-lasting antibody responses. However, rAAV-based vaccines have been suggested to trigger functionally impaired long-term memory CD8+ T cell responses, in part due to poor dendritic cell (DCs) transduction. Such results, albeit limited to intramuscular immunization, undermined the use of rAAV as vaccine vehicles against intracellular pathogens. We report here that intradermal immunization with a model rAAV2/1-based vaccine drives the development of bona fide long-term memory CD8+ T cell responses. The intradermal route of immunization and the presence of potent MHC Class II responses showed synergistic effects on the overall quantity and quality of both tissue-resident and systemic long-term effector-memory transgene-specific CD8+ T cells being generated. Of key interest, we further found that the phenotype and protective potential of intradermally-induced memory CTL responses were fully preserved in the absence of transgene expression in DCs, as cross-presentation appeared highly enhanced for skin-expressed transgenes. Overall our results provide a rationale for the use of a novel intradermal administration route in the future design of rAAV-based vaccines aimed at inducing potent memory CD8+ T cell responses.
: Università Politecnica delle Marche
Tumor and inflammation are strictly related. Neoplastic cells can take advantage of the paracrine effect in an inflamed microenvironment, using trophic factors to interfere with the anti-tumor response. Among the others, mesenchymal stem cells (MSCs) participate to the microenvironment creation sustaining inflammation, which in turn can affect MSCs immunobiology. In this study, influence of inflammation on immunobiology of MSCs and paracrine effect of MSCs in tumor survival and progression were evaluated. MSCs were isolated from periprosthetic capsule caused by breast implant, affected by inflammation (I-MSCs). Contralateral tissue of the same patient, not inflamed, was used as control (C-MSCs). A panel of secreted cytokines was analyzed by Real-Time PCR and ELISA. I- and C-MSCs show a different profile of cytokines expression, revealing that inflammation affects MSCs immunobiology. Then, I-MSCs and C-MSCs were co-cultured with MCF-7 or KI-JK derived from breast adenocarcinoma and anaplastic large cell lymphoma respectively, tumors perhaps associated with breast implants use. Analyses on proliferation rate and cytokines expression were performed. Cytokines analyses revealed I- and C-MSCs show the same variation in cytokines expression after co-culture highlighting that inflammation does not affect the behaviour of MSCs against cancer. However, MCF-7 and KI-JK have a different behavior in co-culture, indicating that they use different strategy to evade the host immune response. In conclusion, our study absolves completely the breast implants from the insult to enforce the risk of breast related tumor through inflammation.
: Selecta Biosciences
Gene transfer approaches based on the adeno-associated virus (AAV) vector platform have shown great promise both in preclinical animal models and in the clinic. However, there may be circumstances where re-treatment may become necessary, particularly for systemic applications in pediatric patients. In addition, it would be desirable to be able to boost expression in patients who have inadequate expression levels. Currently vector re-administration is limited by the formation of neutralizing antibodies to AAV, which mediate vector clearance and inhibit efficacy. Additionally, CD8 immunity against the AAV capsid or the transgene can reduce or eliminate expression from transduced cells. We have recently developed a novel strategy to induce immune tolerance to protein biotherapeutic drugs based on the co-administration of biodegradable nanoparticles containing rapamycin (SVP-Rapamycin; Kishimoto et al., Nature Nanotechnology, 2016 in press). SVP-Rapamycin has been shown to mitigate the formation of anti-drug antibodies against a pegylated uricase enzyme in a Phase 1b trial. Here we demonstrate that SVP-Rapamycin added on to AAV8-base gene therapy inhibits the formation of anti-AAV8 antibodies and enables successful vector re-dosing in both mice and nonhuman primates. The effect of SVP-Rapamycin was antigen-specific, as treated mice showed normal immune responses to subsequent challenge with unrelated antigens. In addition, co-administration of AAV with SVP-Rapamycin inhibited CD8+ T cell infiltrates in the liver and inhibited the ex vivo recall responses of CD8+ T cells. In conclusion, co-administration of SVP-Rapamycin with AAV vector mitigates immunogenicity, enabling AAV vector re-administration.
: Institute of Stem Cell and Translational Cancer Research, CGMH
Glycosphingolipids (GSLs) are membrane lipids consisting of a polar oligosaccharide chain attached to a hydrophobic sphingosine-containing ceramide moiety. Antibodies against several GSLs designated as SSEAs have been widely used to characterize ES cells. Since the cross-reactivities of these antibodies with multiple glycans can yield misleading interpretations, a few laboratories including ours have employed advanced mass spectrographic technologies to define the dynamic changes of GSLs. We had delineated the changes in GSLs systematically during differentiation of ES cells into various derivatives. In addition to the well-known markers SSEA-3 and SSEA-4, several previously undisclosed globo/lacto-series GSLs (Gb4Cer, Lc4Cer, fucosyl Lc4Cer, Globo H, and disialyl Gb5Cer) were identified in the undifferentiated ES cells. A switching of GSL core structures from globo/lacto- to ganglio-series during ES differentiation was revealed. Lineage-specific differentiation was also marked by alterations of specific profiling of GSLs. These dynamic changes in GSL profiles were associated with altered expression of key glycosyltransferases in GSL biosynthetic pathways, and were clearly linked to lineage specificity and differentiation of ES cells. Finally, with better understanding of the relevance of GSL signatures in ES cells to cancer, new anti-cancer immunotherapy targeting tumor associated glycans which are expressed in ES cells/progenitors is gaining international attention. Until recently, all approved cancer immunotherapies target proteins but not glycans. While GD2 targeted immunotherapy of neuroblastoma marks the first new agent targeting a GSL molecule to receive FDA approval for standard medical care, Globo H targeted immunotherapy of breast cancer has generated promising results in the on-going multinational clinical trials.
: San Raffaele Telethon Insitute for Gene Therapy (SR-Tiget)
The tumor microenvironment dampens the efficacy of anti-cancer treatments. To reprogram this microenvironment, we generated a stable source of Interferon-alpha (IFNa)-expressing tumor-infiltrating macrophages by reconstituting mice with genetically-engineered hematopoietic stem/progenitor cells and showed efficacy against breast cancer (Escobar et al, Sci .Transl.Med 2014). We now employ a primary B-lymphoblastic leukemia (B-ALL) model (Nucera et al, Cancer Cell 2016) to study the immuno-modulatory effects of IFNa gene therapy. We generated an immunogenic B-ALL variant expressing the ovalbumin model antigen (OVA-B-ALL). We show effective OVA-B-ALL growth inhibition in IFNa mice accompanied by induction of OVA-specific T-cells, which exhibited increased cytotoxic activity. Consistently, depletion of CD8+ T-cells abrogated efficacy, confirming their prominent contribution to the anti-tumor response. Of note, we observed long-term responses in a fraction of IFN-treated mice, which remain disease-free even upon successive tumor challenges with both OVA-expressing and OVA-negative B-ALL, suggesting that IFNa-treatment promoted antigenic spreading in surviving mice. Conversely, control mice that induced OVA-specific T-cells experienced high rates of immune-escape due to early loss of OVA expression. When transgenic OVA-specific T-cells (OT-I) were adoptively transferred into B-ALL-bearing mice, we show effective OT-I expansion and B-ALL containment in IFN mice only. Analysis of the tumor microenvironment revealed increased CXCL10, IL12, IL2 and IFNg cytokines and a shift toward pro-inflammatory monocytes, consistent with an immune-functional signature in IFN-treated mice. In addition, we show a direct anti-leukemic effect of IFNa gene therapy in a xenograft model of primary human Ph+B-ALL, which was additive to TKI treatment, building a strong rationale for clinical development.
: Department of Academic Haematology, University College London Cancer Institute, 72 Huntley Street, London, WC1E 6DD, U.K.
Blinatumomab, the most advanced bispecific T-cell engager (BiTE) antibody against CD19/CD3, has shown impressive results in patients with relapsed/refractory B cell acute lymphoid leukaemia. However, as CD19 is exclusively expressed on B-lymphocytes it is not possible to extend this success to other common cancers with significant unmet treatment need. Receptor tyrosine kinase like Orphan Receptor 1 (ROR1) is an onco-foetal antigen expressed on a range of haematological and solid malignancies, including pancreatic, melanoma, glioblastoma, ovarian, prostate, breast and hepatic cancers, but virtually absent on normal adult tissue, making it an attractive therapeutic target. We have generated a novel anti-ROR1 BiTE (ROR1xCD3) able to target ROR1 positive cell lines and induce T cell mediated cytotoxicity. Focusing on pancreatic cancer we demonstrated that our ROR1xCD3 resulted in potent T cell mediated, antigen-specific cytotoxicity against a panel of pancreatic cancer cell lines in vitro at exceedingly low concentrations (0.1ng/mL; p < 0.001). We next assessed the wider applicability of our ROR1xCD3 BiTE against cell lines representative of melanoma, glioblastoma, ovarian, prostate, breast and hepatic cancers but not a ROR1 negative breast cancer cell line. Finally transitioning our in vitro studies to animal models we showed ROR1xCD3 significantly retarded growth of established pancreatic (p < 0.05) xenografts. Overall our ROR1 BiTE has great potential for immunotherapy in an array of malignancies, especially poor prognosis solid tumours for which novel therapies are urgently needed.
: Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University, Salzburg, Austria
The production of cell therapeutics requires human alternatives for animal serum, such as pooled human platelet lysate (pHPL). To prevent pHPL-supplemented media from fibrinogen-induced clotting, the addition of porcine heparin is common. Even though heparin is safely used in the clinic, its impact on mesenchymal stromal/progenitor cells (MSPCs) remains elusive. Therefore we investigated the effect of heparin on the global gene expression of MSPCs derived from umbilical cord (UC) and white adipose tissue (WAT). UC and WAT-MSPCs (each n = 3) were cultured using different alpha-MEM medium compositions (+/- heparin). For WAT-MSPCs, there were no significant differences of cell proliferation and clonogenicity in all different pHPL media. Proliferative and clonogenic capacity of UC-derived MSPCs however, was increased in the absence of fibrinogen and presence of heparin in early passages (p1-p2). Flow cytometric profiles and differentiation assays did not depend on different medium compositions. Whole genome expression profiling revealed a total of 299 upregulated and 114 downregulated genes by the presence of heparin (p < 0.05). Confirmative qRT-PCR was done. Classification into signaling pathways identified upregulation of several pathways including integrin signaling, pathways concerning cytoskeletal regulation by Rho-GTPase, inflammation (chemokine/cytokine), FGF, PDGF and the Notch signaling pathway by heparin. These results corroborate the known interactions between heparin and FGF/PDGF signaling and provide a link between the Notch signaling pathway and the glycosaminoglycan heparin for the first time. Notch and heparin are known stemness factors of progenitor cells. However, further investigation is needed to characterize the biologic relevance of this proposed interaction.
: Department of Biomedicine, Aarhus University, Aarhus, Denmark
Previously, we have demonstrated that functional mesenchymal stem cells (MSC) can be simply and efficiently derived from human induced pluripotent stem cells (iPSC) by culturing iPSCs in MSC medium. However, the molecular mechanism and lineage commitments during iPSCs differentiation into MSCs are unclear. In this study, we reported an optimized method for simple deriving iPSC-MSCs based on feeder-free cultivation system. Meanwhile, using RNA sequencing, we characterized the gene expression profiles, gene signatures, molecular pathways, and lineage commitments during iPSCs differentiation into MSCs. Two human iPSCs and H9-ESC were cultured in VTN-coated plate with E8 medium. For feeder-free MSC derivation, iPSCs at 80% confluence were treated with low-glucose MSC medium for 2 weeks, with medium switched every other day, followed by serial passaging by trypsinization. MSCs were characterized by flow cytometry analysis of MSC markers, and tri-lineage differentiation. Total RNAs from cells at 0, 7, 14, and 21 days of differentiation, and iPSC-MSCs at passage 5 were conducted by RNA sequencing using the complete genomics platform. The iPS-MSCs were morphologically similar to BM-MSCs, positive for all MSC markers, and proliferated up to 20 passages without losing typical MSC characteristics. These iPSC-MSCs retain robust osteogenic and chondrogenic capacity as BM-MSCs. Whole-genome transcriptome analysis revealed iPS-MSCs possessed similar transcriptomic profiles as BM-MSC. The Hedgehog, WNT, TGF-beta, Calcium, MAPK and VEGF signaling pathways play crucial roles during iPSCs differentiation into iPSC-MSCs. Notably, our transcriptome analysis revealed that differentiation of iPSCs into MSCs is following the lineage commitment of the epithelial-mesenchymal transition (EMT) process.
: Tsinghua University Graduate School at Shenzhen
Three dimensional (3D) culture has been shown to improve pluripotent gene expression in mesenchymal stem cells (MSCs), but the underlining mechanisms are poorly understood. Here we found that the relaxation of cytoskeleton tension of MSCs in 3D culture was critically associated with the up-regulation of Nanog. Cultured in spheroids, MSCs showed decreased integrins-based cell-matrix adhesion but increased cadherins-based cell-cell interaction. Different from that in 2D culture, where MSCs exhibited branched and multiple-directed F-actin stress bundles at the cell edge and strengthened stress fibers transversing the cell body, MSCs cultured in spheroids showed compact cell body, relaxed cytoskeleton tension with very thin cortical actin filament outlining the cell, and increased expression of Nanog along with reduced levels of Suv39h1 (H3K9 methyltransferase) and H3K9me3. Notably, pharmaceutical inhibition of actin polymerization with cytochalasin D or silencing Suv39h1 expression with siRNA in 2D cultured MSCs elevated the expression of Nanog via H3K9 demethylation. Thus our data suggest that 3D culture increases the expression of Nanog through the relaxation of actin cytoskeleton which mediates reduced Suv39h1 and H3K9me3 levels.
: Cell Factory, Unit of Cell Therapy and Cryobiology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano
Mesenchymal stem cells (MSC) can be isolated from several tissues and recently also cord blood (CB) has made its appearance in the regenerative medicine field as a source of MSC. In a recent work, our group showed the existence of at least two distinct MSC subsets within the cord blood: one mainly short-living and less proliferative (SL-CBMSC), the other long-living, with higher growth rate, and, very importantly, with significantly longer telomere (LL-CBMSC). Since in an allogenic cell therapy setting, there is a strong need of healthy, proliferative, stable, highly characterized cells able to sustain a cell culture scaling-up for therapeutic applications, we examined whether LL-CBMSC possessed peculiar paracrine properties able to affect angiogenesis or inflammatory processes. Isolated CBMSC possess all the features of the most commonly used adult MSC with striking and specific features such as their proliferative and pro-angiogenic capacities by releasing high levels of soluble cytokines and mRNA precursors embedded in membrane vesicles. The combination of this primary (proteic factors interacting with surface receptors) and delayed (mRNA transferred via vesicle fusion and cargo release) interaction in endothelial target cells resulted in strong blood vessel induction with the development of capillary-like structures. In addition, in different in vitro models LL-CBMSC could dynamically modulate their release of extracellular vesicles and rescue tissue damage. These data led us to exploit CBMSC as paracrine effectors of tissue regeneration for clinical applications.
: International Centre for Genetic Engineering and Biotechnology (ICGEB)
Adeno-Associated Viruses (AAVs) are popular in the gene therapy arena due to their safety and efficiency. Yet, the molecular correlates of transduction with these vectors remains poorly understood. We wanted to identify the cellular genes regulating AAV efficiency by high throughput screening using a whole genome siRNA library (18,120 siRNAs). We discovered that, among the most effective siRNAs downregulating AAV transduction, was the one against the 3’-exoribonuclease ERI-1, which degrades endogenous miRNAs and histone mRNAs. ERI-1 knockdown markedly decreased single-stranded (ss) but not self-complementary (sc) AAV transduction, whereas its overexpression increased ssAAV gene transfer up to 7 times. Efficiency of AAV transduction negatively correlated with histone mRNA levels. Chromatin immunoprecipitation studies showed that ERI-1 overexpression reduced the association of ssAAV with H3 and H4, without altering chromatin formation at scAAV DNA. Accordingly, the downregulation of CAF-1 (a replication-dependent histone chaperone) induced an over 20-fold increase in ssAAV transduction. ERI-1 overexpression also significantly decreases association, with the viral genomes, of proteins of the cellular DNA damage response), which we previously described as inhibitory of AAV transduction. Hydroxyurea, a drug inducing DNA damage and increasing AAV transduction, also determined histone mRNA degradation through ERI-1. Supporting a suppressive role of chromatin on AAV DNA, we also found that the knock down of different histone deacetylases increased AAV transduction up to 15 folds. This work underlines the importance of histone dosage and chromatin conformation of AAV episomal DNA and identifies the exoribonuclease ERI-1 as a major determinant of AAV transduction.
: UC3M
Congenital generalized lipodystrophies (CGL) are rare disorders characterized by severe loss of adipose tissue. CGL patients show insulin resistance, hypertriglyceridemia, esteatohepatosis and adipokines deficiency. Current treatment of CGL is based on the control of triglycerides and cholesterol to avoid metabolic complications and surgery for the physical defects. Also, leptin replacement has beneficial pharmacological effects, but not curative. Although the genetic causes of different CGL types have been established, less is known on the downstream events leading to impaired adipogenesis. Therefore, new models allowing the study of CGL pathogenesis mechanisms and therapy are needed. We thus sought to develop an adipogenesis-deficient MSC cell line carrying a deletion in the AGPAT2 gene by a CRISPR/Cas9 gene editing strategy. We have confirmed the efficiency of the strategy in: a) 293T cells, being able to produce a deletion in the AGPAT2 locus, similar to a mutation found in type I CGL patients and b) in a pool of MSC suitable to isolate homozygous clones for the AGPAT2 deletion. In order to evaluate a potential therapeutic approach we tested the ability of PPARγ1-overexpressing dermal fibroblasts from normal donors and CGL patients to sustain adipogenesis in vivo. We have developed a bioengineered-adipose tissue containing PPARγ1-transduced cells and different combinations of adipogenic and angiogenic helper cells. Upon transplantation to immunodeficient mice we were able to regenerate adipose tissue expressing the molecular biomarkers of functional adipose tissue. The new models and approaches presented here will be beneficial for the understanding and potential cure of this severe metabolic disorder.
: BREONICS, Inc.
: Institute of Biophysics Carlos Chagas Filho - Federal University of Rio de Janeiro
Mesenchymal Stem Cells (MSC) are adult stem cells that have unique immunomodulatory properties. These cells can be isolated from different niches, as bone marrow, adipose tissue and Whartonńs Jelly of the umbilical cord. Whartonńs Jelly derived MSC (WJ-MSC) have the advantages of been collected in a non-invasive, painless way and devoid side effects, as accumulation of cellular damage or loss of regenerative functions, associated with older donor tissue. Moreover, an increasing number of clinical trials demonstrates the safety of WJ-MSC therapy. For those reasons, interest in WJ-MSC research and clinical use has been rising. However, questions regarding potential benefit of different MSC sources or efficiency of stress preconditioning have yet not been answered. In the present study, we search for molecular signatures that could predict a better regenerative output in WJ-MSC therapy. To that end, WJ-MSC have been incubated in control (presence of 10% SFB), deprivation stress (without SFB) or neuronal damage stress (without SFB and in the presence of an injured retina) conditions. Total RNA, miRNA and protein have been isolated from cells and secreted vesicles in each condition. And transcriptome, miRNAome and proteome have been analyzed by microarrays, next-generation sequencing and mass spectrometry technique. Combinatorial and comparative analysis reveals significant difference in the molecular expression profile of these cells and secreted vesicles, suggesting that MSC therapy of different diseases or damages could require particular preconditioning.
: Institute of Biomedical Chemistry (IBMC)
We aimed to develop a tissue engineering approach for bone regeneration, combining polymer-mineral composite scaffolds fabricated by surface selective laser sintering (SSLS) with mesenchymal cells derived from the pulp of human exfoliated deciduous teeth. Evidence for the presence of mesenchymal stem cells (MSCs) in the culture was obtained from the analysis of the surface marker expression profile. Osteogenic differentiation was confirmed by a significant increase in both alkaline phosphatase activity and matrix mineral deposition shown by alizarin red staining. Adipogenesis was confirmed by oil red staining of intracellular lipids. Chondrogenic differentiation in micromass cultures was demonstrated by positive alcian blue staining indicating the accumulation of glycosaminoglycans. We used a SLS-100 printer, developed by us, to fabricate biodegradable porous mineral-polymer composite scaffolds consisting of polylactoglycolide (80%) and TCPC (20%). The cells successfully populated the scaffolds. Cell viability was evaluated by the CytoTox LDH assay. SEM imaging was performed using a novel supravital lanthanoid staining kit (BioRee) which gives excellent preservation of cell morphology and, hence, allows the elucidation of cell-scaffold interactions. In vitro experiments followed by in vivo implantation showed that the scaffolds have very low cytotoxicity and good biocompatibility as well as possess osteogenic properties. The data obtained suggest that the pulp of human deciduous teeth can be regarded as a very efficient and easily accessible source of MSCs and the polymer-mineral scaffolds under development are promising cell carriers for the use in bone tissue engineering.
: Blood Group Serology & Transfusion Medicine, Paracelsus Medical University, Salzburg, Austria
Efficient propagation of mesenchymal stem/progenitor cells (MSPC) could be established using pooled human platelet lysate (pHPL) alternatively to fetal bovine serum (FBS). In this study the influence of fibrinogen-depleted modifications of pHPL on MSPC biology was analyzed, enabling avoidance of porcine heparin for xeno-free culture. According to our recent protocol (J Transl Med. 2015) we prepared medium-clotted pHPL (mcpHPL) by adding pHPL to culture medium without heparin, and pHPL serum (pHPLS) by adding CaCl2 to pHPL. Preparations were tested for fibrinogen. White adipose tissue (WAT; n = 3) and umbilical cord (UC; n = 4) derived MSPC were cultured in pHPL-, mcpHPL-, pHPLS- or FBS-medium (4 passages). Growth factors were analyzed in different media (d0, d5). Proliferation, clonogenicity, trilineage differentiation capacity and surface markers were analyzed. In all different pHPL-media cumulative population doublings were significantly higher than in FBS-medium. Until passage 4 in all pHPL media clonogenicity declined continuously, but remained constant in FBS-medium. MSPC revealed characteristic markers and differentiation potential. Fibrinogen decreased from mean 776 μg/ml in pHPL to <1 μg/ml in mcpHPL and pHPLS. PDGF-BB (285; 217-317 pg/ml), EGF (127; 118-154 pg/ml) and BDNF (828; 640-985 pg/ml) were similar in all pHPL media on d0 and were exhausted until d5. In pHPL media VEGFa increased until d5 to 18,448 pg/ml compared to 12,741 pg/ml in FBS media. In conclusion, MSPC culture was feasible in a completely animal-component free system without fibrinogen and heparin. Our results may contribute to optimization of clinical-grade MSPC expansion enabling applications in somatic cell therapy and tissue engineering.
: Center of Excellence in Stem Cell Research, Faculty of Medicine, Thammasat University
Cancer has been considered a serious global health problem and a leading cause of morbidity and mortality worldwide. Despite recent advances in cancer therapy, treatments of advance stage cancers are still mostly ineffective resulting in poor survival rate of the patients. Several recent evidence suggest that the multipotent human mesenchymal stem cells (hMSCs) play important roles in growth and metastasis of several cancers by enhancing their engraftment and inducing tumor neovascularization. However, the effect of hMSCs on cancer cells is still controversial because there are also evidence demonstrating that hMSCs inhibited growth and metastasis of some cancer cell types. In the present study, we investigated the effects of cytokines derived from bone marrow and gestational tissue-derived hMSCs on the proliferation of various human cancer cell lines, including C3A, HT29, A549, Saos-2 and U251. We herein report for the first time that the effect of hMSCs on cancer cell proliferation depend on hMSC sources as well as cancer cell types. We also found that the cancer-derived cytokines did not affect the cancer suppressive capacity of hMSCs. Moreover, different types of hMSCs use distinct mechanisms to suppress the proliferation of different cancer cell types. Using protein fractionation and mass spectrometry analysis, we have identified several novel hMSC derived factors that might be able to suppress the proliferation of cancer cells. We believe that the strategy developed in our study could be used to systematically identify therapeutically useful molecules in the secretomes of various hMSC sources for the future clinical applications.
: Paracelsus Medical University Salzburg
Human mesenchymal stem/progenitor cells (MSPCs) are currently tested in multiple clinical trials evaluating their immunomodulatory and regenerative capacity. Restricted availability and limited life cycle of MSPCs confine both clinical applicability and mechanistic studies that are urgently needed to better understand their mode of action. In this study induced pluripotent stem cell (iPSC) lines were generated from healthy parental bone marrow (BM)-MSPCs and umbilical cord blood (UCB)-MSPCs to optimize GMP-compliant iPSC differentiation and study mechanisms of iPSC-derived mesenchymal progenitor cells (i-MPCs) function. MSPCs were established under animal serum-free conditions. After primary large scale culture, purity and identity of the cells were characterized by clonogenicity (CFU-F), immune phenotype and three-lineage differentiation capacity, before reprogramming into iPSCs (Sendai virus-OKSM). Differentiation of iPSCs along mesenchymal lineage was initiated by mesoderm induction, followed by i-MPCs differentiation in pooled platelet lysate (pHPL)-supplemented media. Small molecules targeting signaling pathways involved in stem cell self-renewal, oxygen sensing, immune modulation and cell adherence were added to promote differentiation into functional i-MPCs. Immune phenotype, clonogenicity, differentiation capacity and immune modulatory potential of i-MPCs were compared to their parental MSPCs. Under feeder-free conditions, iPSCs differentiated into expandable and cryo-preservable CD73+/CD105+/Tra-1-81- MSPC-like cells lacking immunosuppressive potential. Successive passaging was required for reaching full immunosuppressive competence comparable to parental MSPCs. This demonstrates that mature immunophenotype and function of i-MPCs can be established in a stepwise fashion under animal serum-free conditions. This strategy builds the basis for clinical grade GMP-compliant iPSC-derived functional MSPC propagation.
: Paracelsus Medical University Salzburg
There is an urgent need for the selection of potent pre-tested donor cells for allogeneic immunotherapy. To generate a reliable potency assay for evaluating the immune modulatory function of MSPCs we hypothesized that pooling responder leukocytes as well as MSPCs would enable compensation of individual donor variation. PBMCs from 10 different donors were pooled and pre-labeled with CFSE before cryopreservation. To determine donor and organ variation human MSPCs from 5 independent bone marrow (BM), white adipose tissue (WAT) and umbilical cord (UC) donors were tested either freshly thawed or cultured for 3 days with pooled human platelet lysate (pHPL) replacing fetal bovine serum. The potential of these cells to inhibit phytohaemagglutinin (PHA)-mediated proliferation of CD3+ cells (day 4) or to prevent the allo-response of the pooled PBMCs (day 7) was examined by flow cytometry. By pooling PBMCs a ‘general responder’ could be created showing more stable stimulation of CD3+ cells than PBMCs derived from single donors. All analyzed MSPCs displayed immune modulatory potential at day 4 in particular at a 1:3 modulator:responder ratio with a profound degree of variability. Significant differences between the 5 donors were found in each organ-derived MSPC group which could be compensated by pooling. Impaired immune modulation after thawing was donor-variable and could be corrected by ‘rescue culture’. Inhibition of the allo-response was generally less potent compared to inhibition of mitogenesis.
: Experimental and Clinical Cell Therapy Institute, Spinal Cord & Tissue Regeneration Center, Paracelsus Medical University, Salzburg, Austria
Currently there are app. 600 clinical trials registered (
: Laboratory of Molecular Oncology, Cancer Research Institute, Biomedical Research Center of Slovak Academy of Sciences, Bratislava, Slovakia
Gene-directed enzyme/prodrug therapy (GDEPT) using therapeutic mesenchymal stromal cells (MSC) attracts a special attention because it allows selective conversion of non-toxic compounds (prodrugs) into cytotoxic drugs in the vicinity of the tumor. Despite significant success at the preclinical level, there is still much work ahead to find out to what extent genetically modified MSC are able to target aggressive chemoresistant subpopulations of tumor cells. Yet, no study evaluating the efficacy of GDEPT approaches on a model of chemoresistant colorectal carcinoma has been conducted. Given the clinical significance of chemoresistance, we chose to prepare the 5-Fluorouracil-resistant cell line from a chemosensitive parental HT-29 cells, and to investigate their molecular and phenotypic alterations, as well as to compare the efficiency of GDEPT therapeutic regimen. Based on our results, we demonstrated that resistance to 5-Fluorouracil significantly alters cells` features, assignes them with naturally metastatic capability, and negatively affects the GDEPT outcome. The resistance to therapeutic approach increases with the number of passages in vitro and has been correlated with increased activity of aldehyde dehydrogenase. We propose that resistance to GDEPT can also be hidden in enzymes related to pyrimidine metabolism thus it became of our interest to examine the role of inhibition of these enzymes in overall response. Financial support Slovak Research and Development Agency under the contracts No. APVV-0052-12 and APVV-0230-11; VEGA grants No. 2/0171/13, 2/0130/13 and 2/0087/15. This work was partially supported by the WAC and RFL programs funded by the Slovak Cancer Research Foundation.
: University of Florence
Bone-marrow-derived mesenchymal stromal cells (BM-MSCs) have the potential to significantly contribute to skeletal muscle healing through the secretion of paracrine factors that support proliferation and enhance participation of the endogenous muscle stem cells in the process of repair/regeneration. However, MSC-derived trophic molecules have been poorly characterized. We have found that sphingosine 1-phosphate (S1P) is secreted by BM-MSCs and represents an important factor by which these cells exert their stimulatory effects on myoblasts and satellite cell proliferation. In addition, S1P is able to exert an autocrine action on BM-MSCs. In fact, S1P through the activation of S1P1 receptor exerts a trophic action by controlling extracellular matrix remodelling, stress fibre formation and cell proliferation. Our data may have important implications in the optimization of cell-based strategies to promote tissue regeneration.
: University of Genova
SSeveral works suggest that stem cells might exert therapeutic beneficial effects due to their paracrine potential, rather than by trans-differentiation when transplanted in vivo. Growing interest has been driven to the characterisation of the stem cells secretome and of the stem cell-derived extracellular vesicles (EV)as mediators of such modulatory effect. Human amniotic fluid stem cells (hAFS) have been recently described as immature mesenchymal progenitors with a distinct secretory profile and significant regenerative paracrine potential. In this work, we aim at characterising the regenerative potential of the hAFSEV (hAFS-EV) for future paracrine therapy. hAFS were isolated by the expression of c-kit from left over samples for prenatal diagnosis and cultured in normoxia (20% O2) versus hypoxia (1% O2) for 24h in serum-free medium. hAFS cells showed to secrete vesicles ranging in size from of 100 to 1000 nm. Western Blot on hAFS-EV confirmed expression of exosomal canonical markers like TSG101, Alix, CD81 while lacking GRP94. In vitro analysis using HDF and C2C12 cells suggested the role of hAFS-EV as biologically mediators of paracrine effects stimulating cell proliferation and sustaining survival following oxidative stress. Preliminary results on Smn(F7/F7) mice suggested a modulatory and protective role of the hAFS-EV in decreasing skeletal muscle inflammation. The analysis of small RNAs within the hAFS-EV isolated under hypoxic conditions showed an up-regulation of specific miRNAs involved in regenerative and repair processes. In conclusion these encouraging findings suggest a novel translational approach based on exploiting the regenerative potential of hAFS-EV for future regenerative medicine.
: University of A Coruña
Extracellular Vesicles (EVs) have been found to mediate interaction between cells, mediate non-classical protein secretion, facilitating processes such as antigen presentation, in trans signalling to neighbouring cells and transfer of RNAs and proteins. The detection of low copy numbers of mRNA and small RNAs, including micro-RNAs (miRNA), in EVs from mouse and human has added much research interest impetus to the field. To advance in development EVs-based therapies to reduce the risks and drawback associated with cell-based therapies we decided study whether miR-21 was affecting the Toll-like receptors (TLRs) which are an important component of innate and adaptive immune responses expressed in MSCs and their derived Evs and their damage-associated molecular patterns (DAMPs) (S-100A4, S100A6 and HMGB1) which are molecules that have a physiological role inside but acquire additional functions when they are exposed to the extracellular environment and they can be secreted or exposed by living cells undergoing a life-threatening stress. MSCs from pre-pubertal group was transitory transfected with miRVana miR-21-5p. Western blot and RT-PCR analysis of DAMPS associated with TLR4 indicated that miR-21 inhibition produced a statistically significant decreased of S100A4, S100A6 and HMGB1 with respect control. The inhibition of miR-21 was affecting TLR4 through AKT/mTOR pathway because mTOR and pAKT were down-regulated. We concluded these data might point that miR-21 could be a negative regulator of TLR4 signalling.
: GENyO- Centro de Genomica e Investigacion Oncologica: Pfizer / Universidad de Granada / Junta de Andalucia
Multipotent mesenchymal stromal cells (MSCs) are a promising therapeutic strategy in regenerative medicine, transplantation and autoimmune diseases. Basic studies on their proliferative, immunomodulatory and differentiation capacities are needed for their future success in therapy. We recently found that human MSCs express glycoprotein A repetitions predominant (GARP) on their membrane. The highest GARP levels were found on human adipose tissue-derived MSCs (ASCs) and its expression was rapidly induced upon in vitro culturing. Silencing GARP expression in ASCs, using lentiviral vectors (LVs) encoding for GARP-specific shRNAs, reduced the proliferative capacity and increased their apoptosis compared to non-transduced or control shRNA-transduced ASCs. A qPCR-based quantification of the expression of GARP-specific and control siRNAs showed equal levels in the transduced cells. Overexpression of GARP in ASCs, using a LV encoding a codon-optimized GARP cDNA did not affect their proliferation. However, overexpressing GARP in GARP-silenced ASCs partly restored their proliferative capacity. As a continuation, we performed a microarray comparing the gene expression profile of GARP-silenced ASCs with control and non-transduced ASCs. Several genes promoting proliferation (Runx1, E2F2, Cyclin B2) were downregulated in GARP-silenced ASCs whereas genes involved in the inhibition of the cell cycle or promotion of apoptosis were increased (p27Kip1, E2F6, DAXX,). In summary, we have found that GARP is an essential molecule for MSC biology, regulating their proliferative activity.
: Department of Physiology, School of Medicine, University Road, National University of Ireland Galway, Ireland
The sub-dermal panniculus carnosus (PC) skeletal muscle layer found in most mammals with remnants in humans is unique in its highly regenerative capacity and unclear physiological function. Here, we morphologically and functionally characterise the PC muscle in muscular dystrophic mdx mice compared to wild-type mice. PC muscle was examined in 6 weeks, 12 weeks and 1-year-old mice. We show that PC has 8% central nucleated muscle fibres (CNF), as compared to ∼1% CNFs in limb muscles in 6 week old normal mice. CNFs are elevated to 30% in 6-week-old mdx PC. The accumulation of CNFs further increases in PC with age in both wild-type (12%) and mdx (53%) quantified at 12 weeks. Muscle fibre size distribution (Ferrets diameter) was heterogeneous in wild-type PC and further accentuated in aged mdx mice. PC vascular bed was examined in different aged mice by deriving capillary density and capillary to fibre ratio. In order to examine the regenerative activity of dermal PC, we derived muscle precursor cells from the back skin of wild-type and mdx mice. Dermis-derived PC cells were capable of forming striated contractile myotubes from both wild-type and mdx mice. Myogenic activity as indicated by qPCR detection of myogenic factors Pax7, Myogenin and embryonic / adult myosin were not significantly different between genotypes. We conclude that PC muscle is uniquely highly regenerative which is further perturbed in diseased muscle and makes for an ideal accessible tissue model to study aspects of the vascular-myogenic niche relevant to DMD therapy.
: UMR 7365 IMoPA CNRS/Université de Lorraine
: Department of Clinical Biochemistry, Faculty of Medicine, Hormozgan University of Medical Sciences, Bandar Abbas, Iran
: Instituto de Investigacion sanitaria La Fe
The ability of human Mesenchymal Stem Cells (hMSC) to repair damaged tissues is known. Our group demonstrated that infusion of hMSC overexpressing HIF-1α (HIF-MSC) promotes myocardial healing in an experimental model of myocardial infarction in rats. HIF-1 is the main mechanism of eukaryotic cells to provide an appropriate O2 homeostasis. We studied the crosstalk of HIF-1α and NOTCH signalling pathway. NOTCH modulates transcriptional activity in an environment and time dependent manner. In this work we demonstrate the steps underlying HIF-NOTCH crosstalk in hMSC and this interaction results in: a) a major auto-activation of NOTCH pathway, b) an increase of NOTCH ligands expression, c) increase in NICD nuclear translocation and d) increase on migration, invasiveness and cell proliferation. The ubiquitin family member SUMO has important functions in many cellular processes including DNA repair, transcription and cell division and increased SUMO1 protein levels have been reported following hypoxic stimulation. To investigate if SUMOylation process was involved on HIF/NOTCH crosstalk, we cultured HIF-MSC/MSC in presence or absence of Anacardic Acid, a SUMOylation inhibitor. We observed that both, proliferation and migration were abolished. This effect was stronger in HIF-MSC. Immunoprecipitation studies showed the SUMOylation of N1ICD, what contributed to the activation of NOTCH pathway favoring the nuclear translocation of N1ICD. In summary we have described for the first time that N1ICD is SUMOylated and this modification is potentiated by HIF pathway. These phenomena could be of relevance to deep into the therapeutic mechanisms elicited by MSC in hypoxia or in conditions of HIF-1α stabilization.
: Hospital La Fe/Centro de Investigación Príncipe Felipe
Acute Myocardial Infarction (AMI) still an unsolved issue nowadays and the good results obtained over the last years in the treatment of AMI with mesenquimal stem cells (MSC) make them an alternative therapy to be considered in the future. Our group showed that overexpression of the Hypoxia-inducible factor-1α (HIF-1α), a master regulator of the adaptive response to hypoxia, improves the therapeutic potential of MSC. Since the paracrine hypothesis is the most extended to explain the therapeutic effect of the MSC, in this work we study the implication of exosomes from MSC and MSC-HIF in the angiogenic process focusing in the Notch pathway. We observed that MSC-HIF derived exosomes are loaded with greater amounts of the Notch ligand Jagged 1 than MSC wild type. Indeed, when MSC-HIF derived exosomes were added to endothelial cells (HUVEC; ECs), they induced changes in the transcriptional activity of notch pathway and in the angiogenic process, and both effects were mediated by jagged-1 ligand expressed in MSC-HIF and MSC exosomes. Furthermore we pointed out that exosomes from MSC and MSC-HIF are loaded with a broad repertoire of miRNA in a HIF-1α expression dependent manner with related enhancement of the angiogenesis. In summary, we propose that the increased angiogenic potential of MSC-HIF is consistent with the paracrine hypothesis and could be mediated, at least in part, by an exosomes Notch crosstalk mechanism, between MSCs and ECs. These types of approaches aiming to characterize functionality of MSC-HIF derived exosomes are crucial to understand their therapeutic effect.
: Hacettepe University
In vitro assays for T/B/NK lymphopoietic differentiation could be helpful to assess the differentiation potential of hematopoietic stem cells (HSCs) from patients with immune deficiencies before and after genetic correction. Current protocols involve the use of fetal thymus organ cultures or the use of the murine Op9/Op9-DL1 cell lines. Use of either protocol is suboptimal for differentiation of human HSCs. NOTCH1 signaling occurs through its ligands Delta-Like 1 (DL1) and is important for T cell differentiation. Here, we aimed to develop an immortalized human cell line using bone marrow-derived mesenchymal stem cells and 3rd generation self-inactivating lentiviral vectors carrying a codon-optimized gene for human telomerase reverse transcriptase (hTERTco) and eGFP. Subclones were transduced with codon-optimized human hDL1co-Tomato. The hTERTco cell line (52% GFP expression) was used for B-cell differentiation; the hTERTco/hDL1co cell line (37,4% Tomato expresion) was used for T-differentiation. We previously found that B and T-cell differentiation on human MSCs was hampered by the excretion of M-CSF by these cells, which support the differentiation of CD3neg/CD4dim myelomonocytic lineages rather than lymphocytic lineages. Therefore, we added small molecule inhibitors of the M-CSF receptor, c-fms inhibitor III (1 uM) and c-fms inhibitor IV (80 nM), to suppress signaling. CD34+ umbilical cord blood cells were co-cultured with the hTERTco cell line. After 4 weeks of co-culture 28% of hematopoietic cells expressed CD10 and 2.6% was CD10+/CD19+. In conclusion, use of immortalized MSCs with c-fms inhibitors supports in vitro B-cell differentiation. Co-cultures on hTERTco/DL1 cell lines for T-cell differentiation are still ongoing.
: FSBI “CCH with Outpatient Health Center”, Moscow, Russia
Multipotent mesenchymal stromal cells (MMSC) derived from various intraoral sources attracts attention of an increasing number of researchers due to availability and some features making them different from MMSC of bone marrow or adipose tissue. It is well known that MMSC derived from buccal mucosa, periodontal ligament, dental pulp, apical papilla and jaw periosteum can differentiate in myogenic direction. Such feature was not described for gingival mucosa derived MMSC (gMMSC) before now. The study conducted by us has demonstrated the possibility of isolation of gMMSC possessing the high proliferative potential and the ability to differentiate efficiently in canonical (adipo-, chondro-, osteogenic) directions. Moreover, we observed for the first time gMMSC induced differentiation in myogenic direction both at early and late passages. Further experiments showed that anatomical peculiarities play important role. Only gMMSC derived from alveolar but not attached part of gingiva are able to differentiate in myogenic direction. Similar features were observed for rabbit gMMSC, but not rodent gMMSC. Identified subpopulation of cells was fully characterized by us using immunocytochemical staining, ELISA, microarray. Comparative analysis of gMMSC and MMSC obtained from another sources testified the advantages of using these cells. Our results indicate that subpopulation of gMMSC derived from alveolar part of gingiva are perspective candidates for clinical usage in patients with skeletal muscle disorders. Preclinical experiments on a model of local muscle injury are ongoing. The study was funded by Russian Science Foundation (project No 14-25-00166)
: Hacettepe University
Mesenchymal stromal cells (MSCs) are heterogeneous stem cell populations with varying differentiation potentials that are controlled by different transcriptional proteins. HOX transcription factors are one of these proteins that affect osteogenic and adipogenic differentiation. We hypothesized that HOX gene expression profile changes between bone marrow-derived MSCs (BM-MSCs) with or without osteogenic differentiation potential. Herein, BM-MSCs (n = 11) were characterized by immunophenotyping and differentiation capacity using histochemical staining and their HOX gene (n = 39) expression profile was measured using qPCR. BM-MSCs had similar adipogenic lineage specifications while they varied in their osteogenic lineage potential. Expression of 12 HOX genes increased and of 9 HOX genes decreased in the BM-MSCs, cultured in osteogenic differentiation medium. Osteogenic differentiation was assessed by Alizarin Red S (ARS) staining of the cells on day 21 and the level of staining was quantified by ARS extraction to sub-group the cells according to their differentiation potential. 6 of those had osteogenic differentiation and five was deficient for osteogenesis. Cells lacking osteogenic differentiation potential had significantly lower HOXA5 and HOXB5 expression levels compared to the ones with good osteogenic differentiation capacity (P < 0.05). In conclusion, low HOXA5 and HOXB5 expression in osteogenic induced BM-MSCs may have an effect on the failure of osteogenesis. This study was suported by TUBITAK 110S021 and EU COST BM0805.
: Laboratory of Dermato-Immunology, Catholic Research Institute of Medical Science, College of Medicine, The Catholic University of Korea, Seoul, 06591, Republic of Korea
Mesenchymal stem cell (MSCs) therapy have been proposed in several autoimmune diseases and successfully tested in animal models, but their contribution to atopic dermatitis is poorly understood. In addition, a powerful antioxidant extracellular superoxide dismutase (SOD3) that catalyses dismutation of reactive oxygen species and is known to have anti-angiogenic, anti-chemotactic, and anti-tumor activities, have been recently demonstrated for its therapeutic efficacy to treat or prevent autoimmune arthritis, ovalbumin-induced allergic airway inflammation and UVB-induced skin inflammation. However, effects of SOD3 as potential biomedicine for treatment of atopic dermatitis is unknown. Here, we explore anti-allergic effect of SOD3 overexpressed in MSCs on OVA-induced atopic dermatitis-like skin inflammation in mice, define the role in regulating keratinocyte and mast cell functions during pathogenesis of atopic dermatitis. Moreover, we co-cultured SOD3-transduced MSCs or MSCs with human CD4+ T cells isolated from atopic dermatitis patients and explored the use of extracellular superoxide dismutase (SOD3)-transduced MSCs as a novel therapeutic approach in atopic dermatitis and investigated poorly understood underlying mechanism. Our in-vitro and in-vivo results from co-culturing of SOD3-MSCs or MSCs with human mast cells, human epidermal keratinocytes, CD4+ T cells and ovalbumin (OVA)-induced mouse model of atopic dermatitis, respectively, showed that SOD3-transduced MSCs can have stronger immunosuppressive and immunomodulatory effect that could possibly be a potential and novel approach to treat atopic dermatitis likely through a suppression of proliferation and infiltration of various effector cells into skin with a concomitant modulated cytokines and chemokines expression, and inhibition of underlying signaling pathways.
: UMR 7365 IMoPA CNRS/Université de Lorraine
: Bambino Gesù Children's Hospital, Department of Onco-Haematology and Trasfusional Medicine, Rome
: Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Linz/Vienna 1200, Austria
Human adipose tissue is an attractive and abundantly available source of adult stem cells used in regenerative medicine and tissue engineering. The adipose derived stromal/stem cells (ASC) isolated from adipose tissue show a high regenerative potential due to their capacity to secrete molecular factors which are involved in wound healing. But nothing is known about the stromal vascular fraction (SVF). On the other hand, extracorporeal shockwave therapy (ESWT) demonstrated beneficial effects on wound healing especially due to neovascularization and an early upregulation of angiogenesis-related growth factors. Our analysis of the SVF supernatant showed a positive secretion of epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF) and vascular endothelial growth factor (VEGF) -family members. Significantly enhanced protein secretion after ESWT compared to untreated cells was detected for IGF-1 and placental growth factor (PLGF). Moreover, SVF treated with ESWT showed significantly enhanced osteogenesis and increased ATP release. The detected secretion of different proteins as well as the significantly increased PLGF secretion after ESWT supports the possible therapeutic effect of SVF cells. PLGF is, like the other members of the VEGF family, involved in angiogenesis. Therefore, SVF combined with ESWT could be a promising tool to treat disorders, which require neovascularization such as wound healing and soft tissue repair.
: Bambino Gesù Children Hospital, Department of Onco-Haematology and Trasfusional Medicine, Rome
: Swansea University
: Chulalongkorn University
Basic fibroblast growth factor (bFGF) regulates pluripotent marker expression and cellular differentiation in various cell types. However, the mechanism by which bFGF regulates REX1 expression in stem cells isolated from human exfoliated deciduous teeth (SHEDs) remains unclear. The aim of present study was to investigate the regulation of REX1 expression by bFGF in SHEDs. SHEDs were isolated and characterized. The mRNA and protein expression levels were determined using real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. In some experiments, chemical inhibitors were added in the culture condition to impede target signalings. Cells isolated from dental pulp tissues of human exfoliated deciduous teeth expressed mesenchymal stem cell surface markers (CD44, CD73, CD90, and CD105). These cells were able to differentiate into osteogenic and adipogenic lineages upon induction. Addition of bFGF induced REX1 mRNA expression and this effect was attenuated by pretreatment with FGFR or Akt inihibitor. Cycloheximide pretreatment inhibited bFGF-induced REX1 expression, implying the involvement of intermediate molecule(s). Further, it was demonstrated that the addition of neutralized antibody against IL-6 attenuated bFGF-induced REX1 expression by SHEDs. In conclusion, bFGF enhanced REX1 expression by SHEDs via FGFR and Akt signaling pathway. Moreover, IL-6 also participates in bFGF-induced REX1 expression in SHEDs.
: The Catholic University of Korea
Lin28 protein affects mRNA translation and specifically represses the processing of let-7 miRNAs into mature forms that mediate ESC differentiation, thereby modulating self-renewal and pluripotency. However, it has not been reported that Lin28 can be automatically translocated into cell. Its basic amino acids and zinc-finger structure have a major contribution for cell-penetration. We here introduce that Lin28-derived peptide has much more excellent cell-penetrating ability for intracellular delivery compared with Tat-like CPP in the presence of serum. Correspondingly, Lin28-derived peptide of its own show growth stimulation, wound repair and deviated neuronal cell differentiation when treating with the exogenous peptide. These findings shed a new light on the development for regenerative drugs.
: Cancer Research Institute, Biomedical Research Center of SAS
The efficacy of gene-directed/enzyme prodrug therapy (GDEPT) mediated by cellular vehicles was proved in many preclinical studies, and first clinical studies were initiated. Our previous studies demonstrated that adipose tissue-derived mesenchymal stromal cells (AT-MSC) retrovirally transduced with cytosine deaminase::uracil phosphoribosyltransferase (CD::UPRT-MSC) exhibit significant therapeutic effect in the presence of prodrug 5-Fluorocytosine (5-FC) in vivo. On the other hand it was demonstrated that mesenchymal stromal cells can contribute to tumour stroma, and they have pleiotropic effect on tumour cells including supporting effect on chemoresistance and tumourigenicity. Despite promising in vitro data, CD::UPRT-MSC were not able to mediate therapeutic effect on intravenously administered colorectal cancer-derived cell line LS 180 Moreover, AT-MSC exhibited supportive effect on proliferation of tumour cells in vivo. Subsequent analysis revealed that cultivation of LS 180 cells in media containing AT-MSC-derived soluble factors induced expression of stem cells` markers, and markers of epithelial-to-mesenchymal transition. Our findings indicate the importance to comprehend the impact of MSC on tumour cells in order to validate the safety and efficacy of cell based therapies. Financial support: Slovak Research and Development Agency under the contracts No. APVV-0052-12 and APVV-0230-11; VEGA grants No. 2/0171/13, 2/0130/13 and 2/0087/15. This work was partially supported by the WAC and RFL programs funded by the Slovak Cancer Research Foundation.
: Hacettepe University, Dept of Obstetrics and Gynecology
CD271 is expressed by a small fraction of bone marrow Mesenchymal Stem Cells (MSCs), but can be used effectively for prospective isolation of MSCs. We previously showed that CD271+ bone marrow cells display a significantly higher clonogenic potential than plastic adherent MSCs and that these cells effectively support expansion of CD34+ umbilical cord blood cells in vitro. However, selected cell populations using anti-CD271 microbeads, contain high numbers of CD271dim/CD45+ cells, which we believed are hematopoietic cells, that do not contribute to the clonogenic capacity of the total CD271+ BM cells. Here, we assessed the effects of CD45 depletion followed by CD271 selection on clonogenic capacity and phenotype of CD271+ cells and tested whether expanded CD271+ cells support in vitro lymphopoiesis. CD34+ cells were co-cultured with CD271+ or CD271bright/CD45- passage 3 cells for 7 days in serum-free medium with cytokines, followed by differentiation on OP9 and OP9/DL1 cell lines for B/T-cell differentiation. CD271+ cells and CD271bright/CD45- cells showed clonogenic capacity, whereas only CD45- or CD45+ cells did not. CD271+ cells and CD271bright/CD45- cells showed a similar morphology, FACS profile and clonogenic potential and most likely represent the same stem cell group. In comparison to BM-MSCs, both populations display superior support of ex vivo expansion of CD34+ cells. B-cell differentiation stabilized around day 30 with up to 13% CD10+/CD19+ cells. Maximum T-cell differentiation was observed at day 60 with 2,5% of cells being CD3+/CD4+/CD8+. In conclusion, the CD271 antigen can be used effectively to prospectively isolate BM-MSCs, which support lymphohematopoiesis in vitro.
: Laboratory of Bioengineering and Regenerative Medicine (BioReM), Kazan (Volga region) Federal University, Kazan, 420008, Russia
A combination of biomaterial and autologous cells represents the best replacement option for tissue engineering tubular tissues like the bladder, trachea and blood vessel. We here evaluate the in vitro cytotoxicity of different electrospun biomaterials in a blinded trial. Six 2D electrospun scaffolds made of polyamide-6, polyethylene terephthalate and polyurethane, with fiber size ranging from 1.68 to 4.4 μm and density range, 7.1 to 36.6%, were sterilized with γ radiation (air dose 15 kGy). No significant biomechanical differences were observed. In vitro cytotoxicity was evaluated in a blinded trial using bone-marrow derived rat mesenchymal stromal cells (BM-MSCs). Primary cell isolation and in vitro culture were performed. Cytotoxicity was assessed by evaluating cell metabolic activity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromid, MTT) and viability (live/dead viability/cytotoxicity test) after 48 and 72 h of static 2D scaffolds cultivated with 20,000 BM-MSCs. None of the 2D scaffolds significantly interfered with the MTT reagents. MTT assay and live/dead staining showed that polyamide-6 scaffolds displayed the best cyto-compatibility after 48 and 72 hours. In particular, polyamide-6 with fiber diameter range of 1.6–3.6 μm; density, 7.1%, thickness, 0.77mm displayed the best in vitro cyto-compatibility when evaluated blindly against the other biomaterials tested here. Further in vivo studies will determine its biocompatibility for possible clinical translation. Work was funded by the Russian Science Foundation (Grant No. 14-45-00018)
: Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Biotechnology, 34660, Istanbul, TURKEY
The combinations of scaffold structures and cells are a promising strategy for tissue engineering and cellular therapy. Scaffold structures have been widely used differentiation, proliferation and absorption of cells. An ideal scaffold should be not immunogenic and cytotoxic. Furthermore, it must be biodegradable, biocompatible and sterile. In this study, we aimed to investigate effects to cell proliferation and differentiation and to characterization and prepare of different scaffold structures using binary and ternary combinations of chitosan, alginate and protamine. In our study, biodegradable natural polymers included chitosan, alginate, and protamine were used and their binary and / or ternary combinations were prepared for making a scaffold. Prepared scaffolds were characterized (SEM, FT-IR, mechanic stability test, swelling and biodegradability profile studies) and investigated the effect on HEK-293 stable cell line GFP expressing (MTT assay, fluorescence microscopy). The characteristic peaks showing interaction of alginate, chitosan and protamine were observed by FT-IR analysis. In the swelling data, swelling ratios of binary scaffold structures were higher than ternary scaffolds. In the biodegradability profile study demonstrated that the increase of chitosan concentration was enhanced mechanical strength of scaffold. The binary and ternary scaffolds were showed increase to cell proliferation by MTT study. The increase cell adsorption and proliferation of scaffold structures was observed by fluorescence microscopy in HEK-293 stable cell line GFP expressing. In conclusion, we suggested to be use of chitosan/alginate/protamine ternary scaffolds in tissue engineering.
: 4MEDi-CBTD
Transplantation of adipose-derived stem cells (ASC) is an emerging therapeutic option for addressing intractable diseases such as critical limb ischemia (CLI). Evidence suggests that therapeutic effects of ASC are primarily mediated through paracrine mechanisms rather than transdifferentiation. These secreted factors can be captured in conditioned medium (CM) and concentrated to prepare a therapeutic factor concentrate (TFC) comprised of a cocktail of beneficial growth factors and cytokines. The ability of TFC to promote reperfusion in a rabbit model of CLI was evaluated. A total of 27 adult female rabbits underwent surgery to induce ischemia in the left hindlimb. An additional five rabbits served as sham controls. One week after surgery, the ischemic limbs received intramuscular injections of either: (1) placebo (control medium), (2) low dose TFC or (3) high dose TFC. Limb perfusion was serially assessed with a Doppler probe. Blood samples were analyzed for growth factors and cytokines. Tissue was harvested postmortem at day 35 and assessed for capillary density by immunohistochemistry. At one month after treatment, tissue perfusion in ischemic limbs treated with high dose TFC was almost double (P < 0.05) that of the placebo group (58.8 + 23 relative perfusion units [RPU] versus 30.7 + 13.6 RPU; mean + sd). This effect correlated with greater capillary density in the affected tissues and with transiently higher serum levels of the angiogenic and pro-survival factors VEGF and HGF. Results of this study demonstrate that TFC represents a potent therapeutic combination for patients with CLI.
: Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Linz/Vienna 1200, Austria
One of the mainstays of facial rejuvenation strategies is volume restoration which can be achieved by autologous fat grafting. In our novel approach, we treated the adipose tissue harvest site with extracorporeal shock wave therapy (ESWT) in order to improve the quality of the regenerative cells in situ. The latter was demonstrated by characterizing the cells of the stromal vascular fraction (SVF) in the harvested liposuction material regarding cell yield, ATP content, proliferative capacity, surface marker profile and differentiation potential. While SVF cell yield was only slightly enhanced, viability and ATP concentration of freshly isolated cells as well as proliferation doublings after 3 weeks in culture were significantly increased in the ESWT compared to the untreated group. Likewise, cells expressing mesenchymal and endothelial/pericytic markers were significantly elevated concomitant with an improved differentiation capacity towards the adipogenic lineage. Hence, in situ ESWT might be applied in the future to promote cell fitness and adipogenesis within the fat graft for successful facial rejuvenation strategies with potential long-term graft survival.
: National Tsing Hua University
Delayed union/non-union resulting from bone fractures or serious trauma remains a challenging problem for orthopaedic surgeons. Conversely, cartilage defect or degeneration due to trauma or joint diseases can lead to debilitating pain and physical impairment. These problems have inspired the development of tissue engineering, which combines cells, biomaterials and biological signals, to stimulate cartilage/bone regeneration. Over the past decade, gene therapy has converged with bone engineering, by which an increasing number of therapeutic genes are explored to stimulate cartilage and bone repair. These genes can be administered to cells via in vivo or ex vivo approaches using either viral or nonviral vectors. This presentation will focus on the use of baculovirus, a promising and effective viral vector, for genetic engineering of mesenchymal stem cells for bone regeneration.
: Institut de Recherche Biomedicale des Armées
The cutaneous radiation syndrome observed after a local high dose irradiation, is characterized by necrosis and poor revascularization of the skin, extensive inflammation, muscle inflammation and fibrosis. Due to their paracrine secretion properties, adipose tissue derived stem/stromal cells sub-cutaneous injections have shown favorable effects on skin wound healing in a minipig model of gamma acute local irradiation (50Gy). However a persistent muscle fibrosis remained, due to radio-induced muscles inflammation. Based on the pro-myogenic and immunomodulation potential of stromal/stem cells, a new protocol combining subcutaneous and intra-muscular injections of adipose tissue derived stem/stromal cells have being evaluated on tissue recovery. Six minipigs were locally irradiated at the dose of gamma 50 Gy and randomly divided into 2 groups. Three controls animals received the vehicle (phosphate-buffer-saline solution) and three animals received 3 injections of 75 × 106 adipose tissue derived stromal/stem cells. The muscle regeneration pathway, the tissue remodeling marker and the polarization of the inflammatory response of irradiated muscle were analysed by western-blot and immunohistochemistry 76 days after irradiation. All minipigs exhibited a homogeneous skin evolution. Irradiated muscles observation showed prominent fibrosis and necrosis areas in PBS group o highlighted a macrophage (M2) polarization of the inflammatory response, muscle regeneration pathway activation and a recruitment of myofibroblasts after intramuscular injections. Globally, intramuscular injections of adipose tissue derived stromal/stem cells favour biological processes implied in muscle regeneration in the context of cutaneous radiation syndrome (minipig model). Further work is ongoing to evaluate this therapeutic strategy on a larger cohort with a longer clinical follow-up.
: Academic Medical Center
Barrett's esophagus(BE) is a metaplastic abnormality in which the normal stratified squamous epithelium of the oesophagus is replaced by columnar epithelium. BE predisposes for esophageal adenocarcinoma. A novel therapeutic option involves targeting the Bone Morphogenetic Proteins (BMPs), a family of growth factors that orchestrate tissue architecture, intestinal homeostasis and stemcell differentiation. Using a novel in vivo organoid model of human BE, we have started to investigate the potential to reverse the metaplastic process using novel anti BMP Dwarfbodies® (Calpe et al., Mabs 2016). Endoscopic biopsies of BE were implanted into immunocompromised mice using an intramuscular transplantation technique (Read et al., 2016), in combination with either BMP inhibitor or control. Implants were then cultured for a period of three months in order to form in vivo organoid structures. These structures were then assessed both histologically and immunohistochemically using panels of both squamous and intestinal markers. Barrett's organoids were lined by a functional epithelial layer containing Goblet cells and recapitulated the crypt and villous regions seen within Barrett's glands. Immunohistochemical validation confirmed that the xenograft structures were of human origin and expressed the appropriate markers of intestinal differentiation (CK8, CDX2 and villin). Following treatment with BMP inhibition, organoids demonstrated a tendency to form a multi-layered epithelium that expressed the squamous marker p63. Preliminary results demonstrate a trend towards the generation of organoids with a squamous-like phenotype following treatment with BMP inhibitors. These pre-clinical results may be translated to the clinical setting in order to prevent the development of oesophageal adenocarcinoma.
: Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, P.O. Box: 19395-4644, Tehran, Iran
Cardiovascular diseases are the leading global cause of death, accounting for 17.3 million death annually. hPSC-derived cardiomyocytes are nominated as new tool for studying mechanisms underlying cardiovascular diseases and drug development. The lack of maturity of hPSC-derived cardiomyocytes may reduce the suitability of drug discovery, applications in disease modeling and engraftment into the heart. Recently, structures resembling whole organs, termed organoids, have been generated from stem cells through the development of 3D culture systems. Organoids are derived from hPSCs, isolated organ progenitors or multicell-types that differentiate to form an organlike tissue. In this study we used 3D culture systems for organoid formation. We cocultured hPSC-derived cardiac progenitor cells (hPSC-CPC) with human umbilical vein endothelial cells (HUVECs) and hPSC-derived mesenchymal stem cells (hPSC-MSCs) on agarose coated U-bottomed 96-well plates. Organoids were characterized by RT-PCR for lineage cardiac genes, immunostainig for cardiac and vascular markers and Multielectrode array technique for electrophysiological properties at day 7 culture. Beating in organoid was observed 72 hour after seeding. Organoid size increased by time, reached to 800 μm at day7. RT-PCR analysis revealed substantially higher expression of early cardiac marker genes such as NKX2.5, MEF2c, TBX5 in cardiac organoid. Immunohistochemistry analysis showed that cardiac organoid contains cTNT+ and CD31+ cells. Electrophysiological recordings showed the organoid's beating properties. In summary, we developed a method to generate beating cardiac organoid by co-culture of three cell populations. Cardiac organoid may result in fully mature cells once transplanted providing an advanced platform for disease modeling, drug testing or transplant strategies.
: Institut de Biomedicina Universitat de Barcelona
Parkinson's disease (PD) is an incurable chronically progressive neurodegenerative disease that affects between 8–10 million people worldwide. Despite decades of intense research, the pathogenic mechanisms behind PD are for the most part still unknown, owing to the lack of experimental models that recapitulate the main features of the disease. Induced pluripotent stem cell (iPSC) technology allows for in vitro recapitulative modeling of complex diseases. By taking advantage of a new genuinely human PD model, here we investigated whether ventral midbrain dopaminergic neuron (vmDAn) degeneration in PD is truly a cell-autonomous phenomenon, or whether it is influenced by an altered crosstalk between vmDAn and glial cells. Four patient-specific iPSC-derived astrocytic lines (two from healthy controls with no history of neurological disease and two from PD-patients carrying the familial LRRK2 mutation) were generated. PD related phenotypes, including dysfunctional autophagy and altered mitochondrial dynamics, as well as a progressive accumulation of α-synuclein were evident in our LRRK2-PD astrocytes. LRRK2-PD astrocytes were found to compromise neuronal survival when co-cultured with LRRK2-PD iPSC-derived vmDAn, suggesting an important role for astrocytes during PD pathogenesis. By accurately recapitulating PD related phenotypes in a progressive manner through the use of iPSC technology, several unknown mechanisms will be unveiled to aid in the future development of specific PD-targeted therapies.
: Stem Cell and Neurogenesis Unit, Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy
New emerging evidences link neurodegenerative disorders, such as Parkinson Disease (PD) with mitochondrial dysfunction. The OPA1 gene encodes for a dynamin-related protein of the inner mitochondrial membrane that regulates mitochondrial fusion and cristae remodelling. OPA1 mutations are associated with dominant optic atrophy. New OPA1 missense mutations were reported in patients with age-related PD and cognitive impairment. These mutations affects highly conserved amino acids in the GTPase domain of the protein. We reprogrammed skin fibroblasts into transgene-free iPSCs from three independent patients and unrelated healthy donors. hIPSCs were then differentiated into stable neural progenitor cell (NPC) lines and dopaminergic neurons. Interestingly, OPA1 mutant NPCs showed in basal conditions a reduced content of mitochondria with a more fragmented morphology compared to control cells. Moreover, OPA1 mutant NPCs exhibited a decrease in cellular respiration, heightened oxidative stress and overall reduction in mitochondrial DNA content. Finally, in glucose-deprived medium, OPA1 mutant NPCs had reduced proliferation and vitality. Importantly, reintroduction of OPA1 functional gene was sufficient to rescue mitochondria dysfunctions in patients’ cells. NPCs have been differentiated into dopaminergic neurons and tested for mitochondrial homeostasis and function as well as for vitality in basal and stress conditions. These experiments will unveil the OPA1-dependent molecular mechanisms of neurodegeneration and possibly the bases for the accentuated vulnerability of dopaminergic neurons. This study establishes a new human in vitro model for neurodegenerative disorders ideal for revealing the relation between mitochondrial dysfunctions and neuronal death as well as particularly attractive for developing new approaches of neuroprotection and translational therapies.
: University Roma Tre
Alteration of adult neurogenesis has been reported in several Alzheimer's disease (AD) animal models, nevertheless a causal link between this phenomenon and oligomeric A-beta (Aβ), the most neurotoxic species in AD, has not been demonstrated yet. We aimed to address this issue in Tg2576 transgenic mice, which represent a well characterized animal model for AD and Aβ accumulation. We analyzed in vitro the biology of adult neural stem cells (aNSCs) derived from the SVZ of 1–2 months old Tg2576 mice, that is considered a pre-symptomatic age in terms of Aβ accumulation. We showed that Tg2576 aNSCs express high level of Aβ oligomers and proliferate significantly less, with respect to their control counterparts. Tg2576 neurospheres are enriched in DCX+ neuroblasts, which failed to terminally differentiate, and give rise to less GFAP+ astrocytes with an aberrant morphology. These defects have been confirmed also in vivo, where a defective olfactory bulbs neurogenesis is observed. Reduced proliferation and the differentiation impairment of Tg2576 progenitors are likely to be due to Aβ accumulation, as both defects can be rescued in vitro by the expression of scFvA13 intrabody, that interferes with intracellular Aβ oligomers (Meli et al, 2014). Strikingly, we have preliminary results that in vivo expression of scFvA13 antibody in Tg2576 SVZ rescues SVZ-derived aNSCs proliferation both in vivo and in vitro. Altogether, our results demonstrate a causal link between Aβ accumulation and failed neurogenesis occurring prior to neurodegeneration. This information might provide useful tool to develop new cell therapy strategies for AD treatment.
: Stanford University
Mutations in the Neuroligin 4 gene (NLGN4) are associated to Autism Spectrum Disorder (ASD) and have almost complete penetrance. The effect of these mutations has been problematic to study because of the poor conservation and very low expression levels of the NLGN4 gene in rodents. Here we report the generation of engineered isogenic human neurons derived from embryonic stem cells (ESC) carrying mutations in the NLGN4 gene. Mutations were introduced by adeno associated virus AAV – mediated homologous recombination with efficiency up to 40%. Synaptically competent excitatory and inhibitory neurons were generated by using specific transcription factor cocktails developed in our laboratory. We found that the NLGN4 mature protein is expressed by inhibitory and only a subset of excitatory neurons. We found that the both NLGN4 knock-out and NLGN4-R704C neurons have exaggerated excitatory post-synaptic currents (EPSC) but normal inhibitory (IPSC). Interestingly, this is in sharp contrast with previous studies in mouse neurons. The mutant neurons have normal cell morphology but abnormal synapses number suggesting a role of NLGN4 also during synaptogenesis. Moreover, Co-Immunoprecipitation (Co-IP) shows the interaction of NLGN4 with a subunit of the AMPA receptor. This is the first report of the study of NLGN4 in the context of human neurons. Our data suggest that NLGN4 mutations predispose to autism, at least partially, by inducing an abnormal synaptic activity by modulating the AMPA receptor that may be amenable to pharmacological intervention.
: Institut de Génomique Fonctionnelle
In vitro corticogenesis from embryonic stem cells (ESCs) is a model of corticogenesis and a promising tool for cortical therapy. It is unknown to which extent epigenetic mechanisms crucial for cortex development and function, such as parental genomic imprinting, are recapitulated by in vitro corticogenesis. We have recently performed genome-wide transcriptomic and methylation analyses on hybrid mouse tissues and cells and observed a high concordance of imprinting status between in vivo and ESCs-derived cortices. Notably, in vitro corticogenesis strictly reproduced the in vivo parent-of-origin dependent expression of 41 imprinted genes. Parent-of-origin dependent DNA methylation was also largely conserved. Combined, our data provide evidence for broad conservation of the epigenetic mechanisms at imprinted loci in ESC-derived cortical tissue. We are using uniparental ESCs to tackle the role of imprinting during corticogenesis. Uniparental ESCs with two paternal genomes (androgenetic, AG-ESCs) or two maternal genomes (parthenogenetic, PG-ESCs) are tools to measure the impact of defective imprinting. Additionally they have been proposed for stem cell-based therapy. Whether AG-ESCs and PG-ESCs have a corticogenesis potential compatible with cortical development and therapy is unknown. To address this we are testing the corticogenesis potential of mouse AG- and PG-ESC lines relative to control ESC lines, in vitro, and in vivo using chimeras. Our preliminary findings indicate that PG-ESCs have a relatively normal imprinting and potential of corticogenesis. By contrast the defective imprinting of AG-ESC correlates with less corticogenesis potential. Thus, PG-ESC but not AG-ESC might be a tool for cortical therapy.
: University of Copenhagen
Early-onset Alzheimer's disease (AD) is a form of progressive dementia often found in people younger than age 65 and it is associated with two neuropathological hallmarks: extracellular plaque deposits of Aβ and neurofibrillary tangles of hyperphosphorylated tau. Most early-onset cases are caused by an inherited change in one of three genes including presenilin 1 (PSEN1). PSEN1 encodes the catalytic subunit of the γ-secretase complex which generates Aβ. To date, over 100 mutations in PSEN1 are identified and marked phenotypic heterogeneity among different mutations is reported. Elucidation of the molecular mechanisms underlying the PSEN1 mutations and the generation of physiologically relevant systems for cellular analysis and disease modelling will propel the development of effective AD therapies. Here, we use patient-derived induced pluripotent stem cells (iPSCs) to better understand the function of PSEN1 and its role in AD pathogenesis. We generated iPSCs from six AD patients carrying distinct PSEN1 mutations and non-demented individuals as controls. CRISPR/Cas9-mediated gene editing is further used to generate isogenic mutation-corrected lines. The iPSCs are subsequently differentiated into AD-affected cell types (e.g., the forebrain cholinergic neurons) that are characterized by expression profile and functionality. Comparative studies of patient-derived neurons and controls are underway with systematic assessments of disease-related neuronal atrophy and processes include Aβ production, Notch processing, and tau phosphorylation. Responses of neurons to pharmacological agents (e.g., γ-secretase modulator) will also be tested. This study underscores the value of iPSC-derived neurons as a platform for investigating essential features in the molecular pathogenesis of AD and preclinical drug screening.
: Hertie Institute for Clinical Brain Research, Tübingen, Germany
Hereditary spastic paraplegia (HSP) is a rare monogenetic neurodegenerative disorder characterized by progressive lower limb spasticity and weakness due to axonal degeneration of the corticospinal tract. Spastic paraplegia gene type 5 (SPG5) is an autosomal recessive subtype of HSP caused by mutations in CYP7B1, a gene encoding for the cytochrome P-450 oxysterol 7-α-hydroxylase, essential for the liver-specific alternative pathway in bile acid synthesis. Mutations within CYP7B1 lead to a decreased enzyme activity and consecutively to an accumulation of oxysterol substrates (e.g. 27-hydroxycholesterol) in plasma and cerebrospinal fluid (CSF) of patients. Research into molecular pathogenesis of HSP is limited by the restricted access to primary cell material from patients. Derivation of disease-specific induced pluripotent stem cells (iPSCs) provide an unlimited cell population which can give rise to any somatic cell type. Therefore, we reprogrammed primary fibroblasts of SPG5-patients using non-integrative episomal plasmids. Differentiation into iPSC-derived cortical neurons and hepatocyte-like cells could be established, leading to a disease-specific cell model. In cultures of cortical neurons a toxic effect as well as an impact on neurite outgrowth of 27-hydroxycholesterol could be demonstrated and supports the hypothesis that the accumulation of oxysterols leads to progressive axonal degeneration. We aim to use this cell model to study further molecular mechanisms of SPG5 via lipidomic analysis of supernatant and cell pellets of differentiated hepatocytes and neurons. These studies will improve our insight in pathogenesis of SPG5 which is an essential step towards a safe and effective treatment strategy for HSP.
: ICOB, Academia Sinica
The early neural population that differentiates from human pluripotent stem cells (hPSCs) consists of various embryonic neural stem cells and progenitors (ENSCs/ENPs) with broad neural developmental propensity, and these cells are thus an excellent source for the development of novel clinical interventions. Here, we sought to directly convert human somatic cells into cells with ENP-like phenotypes through the use of hESC-derived ENP-enriched neural transcription factors (hESC-ENP-TFs). By combining hESC-ENP-TF overexpression, small molecule stimulation, and NPC reporter selection, we demonstrated that induced ENP (iENP) populations could be efficiently converted from human fibroblasts using two nTF combinations. The iENPs exhibit the hallmark characteristics of hESC-derived NPs, including morphological traits, self-renewal, and ENP marker expression. Importantly, the iENPs are able to give rise to astrocytes, oligodendrocytes, and functional neuronal subtypes of CNS and PNS in vitro and in vivo. Nevertheless, our analysis further revealed that these two types of iENP differ in terms of their proliferation ability, differentiation propensity, and regional preference. Finally, we demonstrated that the iENPs can be readily converted from HD and AD patients’ fibroblasts, and the diseased iENP-derived neurons exhibit the hallmark pathological features of AD and HD, respectively. Collectively, our study provides a novel strategy to generate functional iENPs for the purpose of disease modeling and cellular therapy of neurodegenerative diseases.
: European institue of oncology
Kabuki syndrome (KS) is mainly caused by heterozygous mutations on KMT2D, a major -monomethylase with a key role in enhancer regulation. To understand the role of KMT2D in KS pathogenesis and to identify dysregulated molecular networks caused by KMT2D heterozygous mutations, we model the disease by induced pluripotent stem cells (iPSC) generated with non-integrative methods, starting from skin biopsies of patients and matched un-affected relatives. In primary fibroblasts, KMT2D heterozygous mutations lead to transcriptional dysregulation of relevant pathways correlated with the clinical characteristics. Moreover, primary fibroblasts H3K4me1 ChIP-seq shows a clear difference between relatives and affected in distal intergenic regions. Therefore, we show that KMT2D heterozygous mutations lead to transcriptional and epigenetic alteration in primary cell types. The correlation between transcriptome and H3K4me1 in primary fibroblasts suggests an indirect effect of the mutation. The dysregulation could have mainly impaired the maturation of more clinically relevant cell types. According to this hypothesis, iPSCs transcriptomes show high homogeneity across samples. In addition, enhancer profiling (H3K4me1 and H3K27Ac) shows that the impact of heterozygous KMT2D mutations at the pluripotent stage is very selective. To address the transcriptional and epigenetic impact of KMT2D mutations in relevant cell types, with a focus on enhancer markers and KMT2D occupation, neural crest stem cells and cortical neurons have been obtained from iPSCs. Therefore, we are able to study the disease in non-accessible tissues mostly affected in KS, thus improving the knowledge of the molecular pathogenesis and paving the way for pharmacological studies.
: University of Queensland
We previously reported a thermo-responsive polymer to maintain hESC, enzyme-free, as 3D suspension cultures. Data demonstrated increased proliferation compared to 2D cultures while maintaining pluripotency, differentiation ability and normal karyotype. Here we investigated hESC aggregates induced towards the neuronal lineage. Current culture methods do not allow for bulk cultures required for therapeutic applications or pharmacological investigations. Previously reported methods for aggregate formation and differentiation (dual inhibition, EGF/bFGF induction) were used for initial culture. Aggregates were then passaged on a weekly basis with polymer components replaced at each passage and cultures maintained in neural basal media. Analysis was performed on differentiated aggregates for population doublings (PD), live-dead cell analysis and staining. Data demonstrated that 3D aggregate width increased from 300 to 700um during the period of induction. Additionally, cumulative PD demonstrated >6 fold increase in proliferation and live/dead imaging showed >80% viability. Aggregate width, measured after passage, showed a reduction to 390um, this increased to 670um over the next 7 days. However, terminal aggregate width decreased to 470 and 330 um over the following 2 passages. At d10, staining demonstrated that all cells were PAX6+/OCT4- demonstrating that cells had differentiated towards the neural lineage. Cells maintained these markers for 3 passages. No significant differences in PD, viability, or aggregate size was found between polymer and non-polymer conditions. However, over time it became necessary to increase shear force applied during passaging to aggregates without polymer to facilitate disaggregation. This suggests the polymer may play a role aiding disaggregation of cell-cell interactions during passaging.
: Ospedale San Raffaele
The CRISPR/Cas9 system is a rapid and customizable tool for gene editing in mammalian cells. In particular, this approach has widely opened new opportunities for genetic studies in neurological disease. Human neurons can be differentiated in vitro from hPSC (human Pluripotent Stem Cells), hNPCs (human Neural Precursor Cells) or even directly reprogrammed from fibroblasts. Here, we described a new platform which enables the simultaneous rapid and efficient CRISPR/Cas9-mediated genome targeting in a single-step procedure during these three paradigms of in vitro generation of neurons. This system was employed to inactivate two genes associated with neurological disorder (TSC2 and KCNQ2) and achieved up to 85% efficiency of gene targeting in the differentiated cells. In particular, we devised a protocol that, combining the expression of the CRISPR components with neurogenic factors, generated functional human neurons highly enriched for the desired genome modification in only 5 weeks. This new approach is easy, fast and that does not require the generation of stable isogenic clones, practice that is time consuming and for some genes not feasible.
: Laboratory of Neurogenetics and Neuroscience, Institute Giannini Gaslini, Genova, Italy
Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including benign familial infantile seizures, paroxysmal kinesigenic dyskinesia/choreoathetosis and migraine. A large number of PRRT2 nonsense, frameshift, and missense mutations have been associated with diseases with a variable phenotypic spectrum, ranging from mild forms that improve with age to severe phenotypes. We established iPSC lines from fibroblasts of patients carrying mutations in PRRT2 gene using RNA-Sendai viruses expressing the reprogramming genes Oct4, Sox2, c-Myc and Klf4. Neural differentiation was achieved by inducing the generation of rosette-like structures, that were GFP-infected and co-cultured with rat embryonic cortical neurons. Morphological and immunofluorescence analyses showed that, at about 4 weeks of differentiation, GFP+ hiPS-derived neurons reached a mature state with neuronal-like morphologies and expression of neuronal markers such as microtubule-associated protein 2 and alpha1 subunits of voltage-gated Na+channels. The electrophysiological analysis of hiPS-derived neurons showed that these cells were able to generate action potentials in response to depolarizing current and revealed at the same time the presence of both K+ and Na+ voltage-gated currents. The large and rapidly activating inward currents evoked by voltage steps protocol were completely blocked by tetrodotoxin. The analysis of hiPS PRRT2-patient derived neurons showed an increased of intrinsic neuronal excitability and an enhancement of Na+ current density. These features may contribute to the epileptic phenotype observed in PRRT2 mutated patients.
: Institut de Genetique Moleculaire de Montpellier
Lysosomal storage disorders (LSD) are caused by dysfunction of the greater lysosomal system and accumulation of undigested macromolecules within the cell. LSD mucopolysaccharidosis type VII (MPSVII) is a monogenic disorder caused by deficiency of β-glucuronidase (β-gluc) activity leading to glycosaminoglycans (GAGs) accumulation. No effective treatment is available. Although mutations in the GUSB gene encoding β-gluc are known, the molecular pathways leading from GAGs accumulation to cognitive impairment are not yet understood. Induced pluripotent stem cell (iPSC) technology offers the opportunity to model human disease by generating iPSC from patient somatic cells and continuing differentiation into disease-relevant cell types. In this study, we set out to test whether patient-specific iPSC-derived neurons could be used to investigate the existence of functional hallmarks of the disease. For this purpose, MPSVII patient-specific iPSC derived neurons were generated. MPSVII-iPSCs were fully reprogrammed to pluripotency as judged by colony morphology, long-term passaging, karyotype stability, alkaline phosphatase staining, expression of pluripotency associated TFs and surface markers, silencing of retroviral transgenes, as well as displaying in vitro and in vivo pluripotency differentiation ability. MPSVII-iPSCs were successfully differentiated into mature neurons expressing neuronal maturity markers, synapse formation (vGLUT and PSD95 colocalisation) and functionally by measuring calcium-dependent activity. Importantly, MPSVII-iPSC-derived neurons showed defects associated to MPSVII disease, such as GAGs accumulation, high content in Lamp1+vesicles and expanded endocytic compartment. These results confirm the validity of iPSC-based technology to model human diseases, and widening the spectrum of potential applications of somatic cell reprogramming for biomedical research.
: San Raffaele Telethon Institute for Gene Therapy (SR-Tiget)
Globoid Cell Leukodystrophy (GLD) is a lysosomal storage disorder caused by genetic defects in the activity of β-galactocerebrosidase (GALC), a key enzyme for the catabolism of myelin-enriched sphingolipids. GLD is characterized by progressive demyelination and neurodegeneration of the CNS and PNS. Although the genetic defect and the primary hallmark of disease (lysosomal storage) are known, the pathological cascade leading from primary substrates accumulation to cell damage and death is poorly elucidated, due to the lack of relevant human-derived experimental models. To fill this gap, we took advantage of a novel integration-free reprogramming approach (Luni et al. 2016) to generate bona-fide induced pluripotent stem cells (iPSC) lines from GLD-affected patients (n = 5; different mutations) and unrelated normal donors (ND; n = 3). Biochemical characterization of GLD hiPSCs revealed undetectable GALC activity and marked accumulation of psychosine, a cytotoxic GALC substrate. Nevertheless, we did not observe lysosomal expansion or accumulation of Galactosylceramide (primary substrate). Lentiviral vector (LV)-mediated transfer of the functional human GALC gene fully reconstituted enzymatic activity in GLD hiPSCs, and completely cleared psychosine buildup. We have differentiated ND, GLD and GLDLV.hGALC hiPSCs into phenotypically and functionally characterized neural stem/progenitor cells (hiPS-NSC) that have been ultimately differentiated into neuronal- and oligodendroglial-enriched cultures. Thus, we have established a reliable hiPSC-based experimental platform that we are currently exploiting to investigate: 1) the molecular mechanisms at the basis of GLD onset and progression in relevant neural cell types; and 2) the efficacy of gene therapy and/or pharmacological strategies to ameliorate/revert the disease-associated features.
: Department of Neurology and Epileptology, Hertie-Institute for Clinical Brain Research, University of Tübingen
We have analyzed electrophysiological properties of single neurons obtained using two differentiation protocols, viral-driven and embryoid body-based, and compared them to the electrophysiology data coming from human biopsied adult cortical tissue[1] as well as the tissue obtained postmortem from the human fetal cerebral cortex during the second trimester of gestation [2]. Our results reveal that particularly the input resistance (Ri) can reflect temporal maturation of neurons. The 22nd week fetal tissue had Ri of 2240+-310 MOhm and the adult human neurons 70+-6 MOhm. Both iPSC differentiation protocols yielded cells with Ri values in the mentioned range. Neurons from the virus-driven differentiation reached Ri of 1382+-117 MOhm and the embryoid bodies-derived neurons had Ri 889+-75 Mohm. Whereas the former, patched between days 16 and 21 of differentiation, showed stable values of Ri, the latter differentiation revealed continual decline in input resistance from 1400 MOhm to 346 MOhm between 3 and 6 month after the start of the differentiation. Using least square fit, we could interpolate the “biological age’ of differentiated cells.
[1] Testa-Silva G, Verhoog MB, Linaro D, de Kock CP, Baayen JC, Meredith RM, De Zeeuw CI, Giugliano M, Mansvelder HD. High bandwidth synaptic communication and frequency tracking in human neocortex. PLoS Biol. 2014 Nov 25;12(11)
[2] Moore AR, Filipovic R, Mo Z, Rasband MN, Zecevic N, Antic SD. Electrical excitability of early neurons in the human cerebral cortex during the second trimester of gestation. Cereb Cortex. 2009 Aug;19(8)
: KAIST
Joubert syndrome (JBTS) is a recessive genetic disorder caused by dysfunction of primary cilia. The patients show phenotypic diversity, including hypotonia, ataxia, developmental delays and intellectual disability. Although primary cilia are involved in diverse signaling pathways such as Wnt, Shh, and PDGF which are critical for neurodevelopment in eukaryotic cells, how primary cilia are directly associated with abnormal neurodevelopment in the patient remains still elusive. By infection of Yamanaka factors (OCT4, SOX2, c-MYC and KLF4) using retroviruses, in this study, induced pluripotent stem cells (iPSCs) were generated from human dermal fibroblasts derived from three patients who have different mutation sites on TMEM138 locus, respectively. The JBTS- iPSCs normally expressed pluripotency-associated genes at the transcriptional and protein levels, like wild type (WT)-iPSCs. To explore whether JBTS phenotype appears in the neurodevelopmental process, JBTS-iPSCs were differentiated into the neuronal lineage. JBTS-iPSCs-derived neuronal cells showed neuron-like morphology with neurite extension and normally expressed neural precursor markers such as MAP2 and TUJ1. Whether the JBTS-iPSCs-derived neurons may recapitulate defective cilliogenesis during neurodevelopment is in process.
: Masaryk University, Czech Republic
The invention of human embryonic stem cell (hESC) and induced pluripotent stem cell (hiPSC) technologies have shifted the research approach from the adaptation of conventional animal models to the human pluripotent stem cell (hPSC)-based modeling, which offers potential for studying human conditions using human systems. However, despite hPSCs holding great potential, it is imperative to validate how faithful hPSC-based neural developmental modeling is in recapitulating the developmental process in vivo. This study undertook to substantiate the validity of hPSC-based neural developmental modeling by comparing it with many facets of neural developmental events occurring in mice. We found that the hPSC-based system mimicked the process from the neural plate to the neural tube, then to the neocortex, during which the system captured characteristics of brain development. We also showed that the system reserved the similar neural stem cell (NSC) niche residing in the ventricular region of the cortex. Moreover, FGF2 signaling exhibited a different repertoire of regulatory activities depending upon neural stage progression.
: Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Belvaux, L-4367, Luxembourg
Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. PD is a complex multifactorial disease and can be divided in familial and idiopathic PD. Apart from being a neurodegenerative disease it has been proposed that PD is having an important neurodevelopmental component. Evidence supporting this hypothesis was provided from several labs, including ours, through studies in transgenic mice and neural stem cell in vitro experiments. Since previous studies in this field focused on mice, the aim of this study is to investigate if neuronal development is impaired in human patient specific cells. Here, we are focussing on the Leucine-rich-repeat-kinase 2 (LRRK2) carrying the G2019S mutation. In an in vitro approach using 13 iPSC derived patient specific human neuroepithelial stem cell (NESC) lines we are elucidating the deregulation of NESC characteristics at an embryonic stage. In age and gender matched NESC compared to isogenic controls we found reduced viability coming along with mitochondrial and autophagosomal phenotypes. Additionally, phenotypes concerning the differentiation into neurons (“in vitro neurogenesis”) were detected. Specifically surprising are the different marker dynamics between healthy and G2019S-PD in the differentiation process. Further, we are systematically dissecting LRRK2-G2019S from genetic-background dependent phenotypes to fully exploit the potential of the isogenic controls.
: The Chinese University of Hong Kong
: Molecular and Cellular Immunology, Institute of Child Health, University College London (UK)
Clinical studies of adoptive cell immunotherapy with chimeric antigen receptor (CAR) therapy against CD19 show considerable promise in treating relapsed/refractory B-cell leukaemia. However, CD19 CAR therapy is associated with substantial immunotoxicity. Further, engraftment failure by CAR T-cells is associated with relapse. While most efforts at improving outcome are focused on CAR signalling domains and CAR T-cell production, we have explored the contribution of the CD19 targeting domain on function. We constructed a new CD19-CAR based on a novel anti-human CD19 scFv (CAT) in the 41BB/Z format described by Campana. We selected the CAT scFv since we believed its fast on rate and fast off rate determined by Biacore might favour serial killing and improved CAR T-cell engraftment. CAT-CAR was compared to CARs based on either the FMC63 or 4G7 anti-CD19 scFvs (both currently in clinical studies). CAT-CAR transduced T-cells showed enhanced cytotoxic responses to the CD19+ cell line SUPT1-CD19 by 51Cr release. Moreover, CAT+ T-cells demonstrate an increased proliferative capacity following CD19 stimulation and increased capacity to produce IL-2 and TNFα. Flow based cytotoxicity assays against a cell line engineered to express CD19 at very low levels demonstrated increased cytotoxicity by CAT+ T-cells. Similarly, CAT+ T-cells showed greater killing of NALM 6 cells at very low effector:target ratios suggesting improved serial killing. Imaging studies showed increased CAT-CAR T-cell motility. In conclusion – we have developed a CD19 CAR with improved in-vitro characteristics. Our work suggests that very particular scFv binding kinetics influence CAR T-cell function.
: Division of Gene Therapy, CIMA, University of Navarra, Pamplona, 31008 Spain
Alphaviruses are enveloped viruses containing a positive-strand RNA genome packaged into an icosahedral nucleocapsid. Viral budding is driven by specific interactions between pre-assembled nucleocapsids and viral envelope proteins. However, we have recently reported that Semliki Forest virus (SFV) and Sindbis virus lacking capsid sequences propagate in mammalian cells. Capsid-less propagation is mediated by infectious microvesicles (iMVs) coated by viral envelope proteins and containing viral RNA. iMVs are non-pathogenic in mice when injected intravenously, but infect organs like lungs and heart, suggesting they could be used as vectors. However, as generation of iMVs is not efficient (10e5 infection units/ml) we packaged SFV genomes devoid of capsid into viral particles (VPs) by providing the capsid protein in trans. Optimization of this methodology led to production of propagative viral particles (pVPs) at high titers (>10e9 pVP/ml). To evaluate the potential use of pVPs as expression vectors, GFP and single-chain interleukin-12 (IL-12) genes were cloned into the SFV genome devoid of capsid downstream a second subgenomic promoter. In cell culture, SFV-GFP pVPs were able to mediate efficient spreading of GFP expression, while SFV-IL-12 pVPs showed higher IL-12 expression than non-propagating SFV-IL-12 VPs. Interestingly, SFV-IL-12 pVPs mediated a higher antitumor effect than SFV-IL-12 VPs in a subcutaneous model of murine colon adenocarcinoma when given intratumorally at a dose of 10e6 VP. When the dose was increased 10-fold the antitumor effect was more potent although both types of vectors showed similar efficacy. These results highlight the potential use of these new vectors in cancer gene therapy.
: San Raffaele Scientific Institute
: San Raffaele Telethon Institute for Gene Therapy (SR-Tiget)
Metachromatic Leukodystrophy (MLD) is a fatal neurodegenerative lysosomal storage disorder due to genetic deficiency of arylsufatase A (ARSA), the enzyme responsible for the catabolism of cerebroside 3-sulphate (sulfatide). While primary genetic and biochemical defects are described in MLD, the paucity of available human-derived CNS models has limited so far the assessment of downstream events in disease-relevant cell types. In this work, we established induced pluripotent stem cells (iPSC) from fibroblasts of two MLD patients and from two normal donors (ND) via lentiviral-mediated reprogramming. Biochemical and immunohistochemical analysis revealed lysosomal expansion and impaired lysosomal trafficking in MLD iPSCs as compared to ND counterparts. These primary dysfunctions resulted in generalized cellular stress, as indicated by alteration of Golgi activity and reactive oxygen species (ROS) production. Importantly, these pathological hallmarks were ameliorated in lentiviral vector-transduced ARSA-overexpressing MLD iPSCs. By applying specific culture conditions we differentiated ND, MLD, and ARSA-overexpressing MLD iPSCs into neural stem/progenitor cells (hiPSC-NSC) and their neuronal and glial progeny, with a particular focus on oligodendrocytes (the most affected cell type in MLD). We are currently assessing the presence (and potential exacerbation) of disease hallmarks in iPSC-derived neural populations, also testing the efficacy of gene transfer in reverting the complex disease phenotype. Preliminary data revealed storage of myelin-specific sulfatide species in MLD iPSC-derived oligodendrocytes as compared to ND counterparts, which are cleared in ARSA-overexpressing cells. These results indicate iPSC-derived neural cells as a reliable tool to comprehensively model the degenerative mechanisms underlying MLD pathology.
: Department of Pathology, Haukeland university hospital, Bergen, Norway
Previously we have shown that lentiviral vector mediated herpes simplex virus thymidine kinase (HSV-Tk) / ganciclovir (GCV) therapy is a very promising therapeutic option for glioblastoma (GBM) treatment by using a patient derived xenograft model. However, tumor recurrence was observed in treated animals and interestingly a fraction of recurrent glioma cells were found to express tk-GFP, indicating that a subpopulation of the transduced cells had survived the 3-week prodrug administration period. We hypothesize that short-term prodrug delivery fails to eliminate a fraction of gliomas cells, which are slow-proliferating; and thus, a longer period of prodrug administration would provide better survival benefit. As long-term prodrug we used valganciclovir (valGCV), which is similar to GCV, but tailored for oral administration. After orthotopic implantation of patient derived glioma spheroids and visible growth on MRI, we intratumorally injected lentiviral vectors expressing HSV-Tk.007-GFP, a highly active mutant of HSV-tk. Animals were treated with either short-term GCV (3 weeks) or long-term valGCV (3 months). Although both treatment groups showed remission and subsequent tumor recurrence, animals in the valGCV treatment group survived significantly longer and and had a longer recurrence-free time period. Hereby we report that valGCV is a compatible prodrug in the modus operandi of HSV-Tk mediated suicide gene therapy that can effectively cross the blood brain barrier and upon long-term administration results in better therapeutic benefit compared with short-term GCV treatment. We are currently characterizing the recurrent tumors to identify new targets that potentially could lead to further development of the suicide therapy for GBM.
: University College London
Receptor Tyrosine Kinase Like Orphan Receptor (ROR1) is expressed on a variety of haematological malignancies including Chronic Lymphocytic Leukaemia (CLL) and Acute Lymphoblastic Leukaemia (ALL) as well as an array of solid malignancies including pancreatic, ovarian and breast cancer. Its expression on normal tissue is limited therefore making it an attractive target for immunotherapy with chimeric antigen receptor (CAR) modified T-cells. We isolated 13 novel anti-ROR1 antibodies targeting the membrane proximal Immungolobulin and intermediate Frizzled domain following a rat immunisation programme. These were converted into a single chain variable fragment (ScFv) format which resulted in 10 novel ScFv binders all of which demonstrated specific cytotoxicity against ROR1 positive cell lines. Iterative optimisation has led to two leading candidates that showed significant cytotoxicity against SKW6.4 and Jeko1 suspension cell lines and primary CLL cells, which all express ROR1 at low levels as well as a multitude of ROR1 positive solid cell lines. Efficacy was further demonstrated in a murine model of mantle cell lymphoma. Overall we report a novel ROR1 CAR therapeutic with a potential to treat an array of malignancies. Ongoing work focuses on improving its efficacy through affinity refinement, humanisation and detailed in vivo assessment to generate a best in class ROR1 CAR.
: Department of Genetics, Hyogo College of Medicine
Retroviral replicating vectors (RRVs) have been shown to achieve efficient tumor transduction and enhanced therapeutic benefit in a wide variety of cancer models. Here we evaluated two different RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), which utilize different cellular receptors for viral entry, in human osteosarcoma and malignant mesothelioma cells. We analyzed 1) expression levels of the cellular receptors for GALV (PiT-1) and AMLV (PiT-2) by quantitative RT-PCR, 2) Viral replication, and 3) RRV-mediated suicide gene therapy in human osteosarcoma and malignant mesothelioma cell lines, as well as normal cells. Both receptors were expressed and RRVs expressing the green fluorescent protein gene efficiently infected and replicated in most tumor cell lines tested, but not in normal cells. Among the osteosarcoma cell lines tested, low PiT-1 but high PiT-2 expression was observed in HOS and MG-63 cells, and AMLV but not GALV spread efficiently in these cells. Among the mesothelioma cell lines tested, high PiT-1 but low PiT-2 expression was observed in ACC-MESO-1 cells, and GALV but not AMLV spread efficiently in these cells in culture and in subcutaneous ACC-MESO-1 tumors xenografted in nude mice. Furthermore, RRVs expressing the cytosine deaminase suicide gene showed differential cytotoxicity that correlated with the results of viral spread. The development of multiple RRVs that utilize different cellular receptors for entry may be highly useful for RRV-mediated suicide gene therapy against different types of solid tumors, by enabling customization of virotherapy on the basis of cellular receptor expression profiles.
: Institut d'Investigacions Biomediques August Pi i Sunyer (IDIBAPS)
Pancreatic ductal adenocarcinoma (PDAC) is characterized by a high mortality rate due to late detection, metastatic capacity and resistance to conventional therapies. Patients with this neoplasia could benefit from oncolytic adenoviral (OA) therapy. Despite the progress in the generation of tumor-selective adenoviruses, more potent OA are needed. Cell/virus interactions are key determinants to impact the virus outcome. Cancer cells have acquired a hallmark of traits that may interfere with viral propagation. Since microRNAs (miR) influence many biological processes, and are profoundly deregulated in PDAC, we hypothesized that restoring specific miR activity in PDAC may enhance oncolytic activity. In this study, we generated a library in an Adwt5 EGFP genome expressing up to 243 human microRNAs. The miR-adenoviral library was bioselected through different infection passages in PANC-1 cells and different microRNAs that sensitized cells to adenoviral oncolysis were identified. Three microRNAs, miR-A miR-B and miR-C, were selected for their capacity to increase adenoviral fitness in several PDAC cell lines. Significant increase in viral release was obtained when compared to the parental virus. Interestingly, this effect was tumor-specific since it was not observed in the non-tumoral pancreatic cells HPDE. In line with increased viral production, enhanced cytotoxicity was also observed. In vivo bioselection in patient-derived xenografts confirmed that Adwt5 EGFP miR-A replicated better than the parental virus. Bioinformatic analysis of miR-A, miR-B and miR-C targets identified candidate genes susceptible to modulate adenoviral fitness. Currently, we are studying the molecular mechanisms implicated in the miR-candidates adenoviral sensitization.
: Department of Integrated Life Sciences, Research Institute of Advanced Omics, Dankook University, Yongin 16890, Korea
Chimeric antigen receptors (CARs) that bind to tumor specific antigen have been developed to re-direct T-cells to kill cancer cells. However, negative regulators of T-cell immune function, such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD-1), can restrain anti-cancer activities of engineered CAR-expressing T-cells. To overcome these constraints, here we describe a novel genetic approach based upon trans-splicing ribozyme that can selectively induce CAR targeting tumor-associated glycoprotein 72 (Tag 72) which is a glycoprotein found on the surface of many cancer cells through replacement of CTLA-4 RNA and can remove the CTLA-4 RNA by cleavage activity at the same time. Using in vitro and intracellular mapping strategy, we identified the most accessible site of the CTLA-4 RNA for group I intron-based ribozymes. The ribozyme was designed to trans-splice the 3′ tagging sequence that is CAR targeting Tag 72 to the most accessible target site of the CTLA-4 RNA. To develop this ribozyme as clinically relevant anti-cancer agent, we employed retroviral system to deliver the CTLA-4 targeting ribozyme into T-cells. Retroviral delivery of CTLA-4 targeting ribozyme to T-cells efficiently reduced CTLA-4 RNA level and simultaneously induced CAR expression at surface of T cells. Moreover, anti-cancer efficacy of CTLA-4 targeting ribozyme-transduced T cells was observed in LS174T cells, which express Tag 72, at the level comparable to that of CAR-transduced T cells. CAR expression by the CTLA-4 RNA targeting trans-splicing ribozyme could be a more potent anti-cancer approach through repressing negative regulators of T-cell immune function.
: Retroviruses and molecular evolution, IBMC, CNRS, Université De Strasbourg
We have recently developed an original approach for carrying out molecular evolution of genes in human cells, exploiting the error prone nature of the replication system of HIV-1 (Rossolillo et al 2012). Using this approach, we have focused on the generation of variants of the human deoxycytidine kinase (hdCK) that sensitises cells to treatment with low doses of nucleoside analogues used in anticancer therapy. The goal was to generate a protein to use as security protein to introduce in cells genetically engineered and transplanted in patients. So far, the protein used for this purpose is the thymidine kinase of herpes virus (hvTK). The hvTK presents clinical drawbacks, though, due to its viral origin. We have generated and characterized two mutants of the hdCK: G12 and M36 (Rossolillo et al 2012 and Coulibaly et al 2015). The most efficient mutant (M36) decreases the IC50 for cancer cells in culture to doses of gemcitabine and cytarabine (AraC) that, for certain cell types, is as lower as a factor 180 for gemcitabine and 10, 000 for AraC (Coulibaly et al., 2015). G12 is characterized by three mutations and M36 by four, all located in regions of the protein distant from the active site and totally unpredictable on a rational basis. Sensitisation of cells in culture is achieved through a change in substrate specificity, apparently due to a change in the position of the substrate in the active site caused by a long-range change in the structure of the protein.
: University Hospital of Munich
The sodium iodide symporter (NIS) as reporter and therapy gene allows non-invasive imaging of functional NIS expression by 123I-scintigraphy and 124I-PET as well as therapeutic treatment using 131I. Based on their excellent tumor-homing capacity, genetically engineered mesenchymal stem cells (MSCs) show promising potential as tumor-selective delivery vehicles for NIS, as shown in our previous studies. As a next step towards clinical application, tumor specificity of MSCs was investigated in an advanced genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC). Syngeneic mouse MSCs were stably transfected with NIS driven by the CMV-promoter (CMV-NIS-MSC). CMV-NIS-MSCs were characterized in vitro by analysis of functional NIS expression using iodide uptake assay, qPCR and Western blot. CMV NIS MSCs were injected systemically in PDAC mice and tumoral iodide uptake was monitored by 123I-scintigraphy and 124I-PET. Resected tumors were analyzed by NIS-immunohistochemistry. Iodide uptake studies revealed a 20-fold increase in NIS-mediated perchlorate-sensitive iodide uptake in CMV NIS MSCs compared to wild-type MSCs based on functional NIS expression. In vivo experiments showed significant tumor-specific accumulation of radioiodide after i.v. application of CMV-NIS-MSCs. Tumor-selective NIS protein expression was confirmed ex vivo in resected tumors by NIS specific immunoreactivity. We have successfully established MSCs highly expressing NIS for the application as gene delivery vehicles in an advanced endogenous mouse model of PDAC. In vivo studies demonstrate high stromal targeting of NIS by selective recruitment of NIS-expressing MSCs after systemic application, which sets the stage for therapeutic application of MSC-mediated NIS radionuclide therapy in this clinically highly relevant model.
: INSERM UMR-S1147-Paris Descartes University
Gene-directed enzyme pro-drug therapy (GDEPT) consists of expressing, in tumor cells, a suicide gene which converts a pro-drug into cytotoxic metabolites, in situ. In a previous work, we demonstrated that the combination of the suicide gene CYP2B6TM-RED (a fusion of a triple mutant of CYP2B6 with NADPH cytochrome P450 reductase) and cyclophosphamide (CPA) constituted a powerful treatment for solid tumors mainly due to i) an optimized suicide gene able to metabolize efficiently CPA, ii) an efficient bystander effect, and iii) the development of an anti-tumor immune response. In this work, we investigated the use of mesenchymal stem cells (MSCs) as cellular vehicles for the delivery of our suicide gene. MSCs were genetically engineered ex-vivo to stably express CYP2B6TM-RED. Ex vivo and in vivo investigations showed that MSCs expressing CYP2B6TM-RED were able 1) to bioactivate CPA and produce local cytotoxic metabolites in tumor sites and 2) to destroy neighboring tumor cells through a by-stander effect. Intratumoral injections of CYP2B6TM-RED-MSCs and CPA completely eradicated tumors in 33% of mice without recurrence after 6 months. Rechallenge experiments demonstrated an efficient immune response. These data suggest that MSCs expressing CYP2B6TM-RED with CPA hold promise as a highly effective treatment for solid tumors in humans.
: Tettamanti Research Center, Department of Pediatrics, University of Milano-Bicocca, San Gerardo Hospital/Fondazione MBBM, Monza, Italy
Adoptive transfer of lymphocytes modified with anti-CD19 chimeric antigen receptor (CAR) by viral vectors has been proposed as advanced treatment for B cell-malignancies. To overcome high manufacturing costs, regulatory hurdles and scale-up complexities, we previously developed an improved Sleeping Beauty (SB) transposon platform to engineer Cytokine-Induced Killer cells (CIKs) by using a single stimulation with irradiated PBMCs. In this study, we evaluated the preclinical efficacy and safety of SB modified CARCIK-CD19 cells. The feasibility of large scale manufacturing process was verified starting from 30-60X10^6 PBMC, reaching efficient expansion with an average of 69.3 ± 15.2-fold increase and stable expression of CD19CAR (average 65%). Immunophenotypic analysis showed the typical enrichment of the CD3+/CD56+ cell subset, and maintenance of naïve and central memory CD8+ and CD4+ T cells. Modified cells displayed specific and effective cytotoxicity and proliferation towards CD19+ tumor targets. Furthermore, CD19.CAR CIK cells showed a dose-dependent antitumor response in patient-derived xenograft models, bearing the high-risk features of MLL–ENL and Ph-like (PAX5/AUTS2) gene rearrangements. Frozen/thawed CD19.CAR CIK cells remained active in vitro and in vivo. Finally, with a follow-up of 3 months, persistence of CARCIK-CD19 cells in the hematopoietic and post-injection perfused organs were observed in NOD-SCID-γchain-/- (NSG) mice treated with CAR+ cells to evaluate toxicity and biodistribution. Our findings describe a novel donor-derived non-viral CAR approach characterized by efficient cell transfection and expansion that may widen the range of applications of adoptive immunotherapy. We are currently designing a phase I/II study for relapsing and refractory ALL post Hematopoietic Stem Cell Transplantation.
: Institute of Child Health
There is accumulating evidence that umbilical cord blood (UCB) T cells can mediate superior anti-leukaemic effects compared to conventional peripheral blood mononuclear (PMBC) T cell sources. Following allogeneic transplantation, naïve UCB T cells undergo rapid peripheral expansion with memory-effector differentiation with favourable graft versus leukaemia/ graft versus host effects. Here we investigate the engineering of UCB T-cells with CD19 specific chimeric antigen receptors (CAR) as a targeted treatment for B-cells malignancies. Lymphocytes isolated from fresh UCB donations by ficoll gradient centrifugation and were activated with anti-CD3/CD28 microbeads and IL-2. Cells were transduced with a third generation self-inactivating lentiviral vector expressing a second generation CD19 CAR and this resulted in gene transfer efficiencies of 40–60% in the CD3+ populations. T cells expanded around 50-fold (n = 3) over a 12 day period. Both transduced and non-transduced UCB cells retained naive and memory markers based on CCR7, CD62L and CD45RA expression and exhibited low expression of the exhaustion marker PD-1. Functionally CD19 CAR+ UCB T-lymphocytes demonstrated specific lysis of target CD19+ malignant B-cells in vitro at levels comparable levels to CD19 CAR+ PBMC T-cells. In vivo anti-leukaemic effects were assessed by serial bioluminescent imaging of human: murine chimeric model using luciferase expressing Daudi B cell tumors in immunodeficienct NOD/SCID/Gc- mice. Our data supports the notion that UCB T-cells can be engineered and expanded to produce an effective anti-leukaemic cell therapy and this is now being assessed in scaled up experiments.
: UCL Cancer Institute
CD160 is a glycosylphosphatidylinositol (GPI) anchored protein expressed on circulating NK cells, TCRgamma-delta lymphocytes, intestinal intraepithelial T lymphocytes and neo-vasculature. CD160 is expressed on B-cell chronic lymphocytic leukaemia (B-CLL) and cancer neo-vasculature, making it a potential immunotherapeutic target. We set out to develop a set of novel binders against CD160 for the purpose of generating Chimeric Antigen Receptors (CARs) as well as other therapeutic approaches. To this aim, we developed a hybrid DNA vaccination / phage display platform which allows rapid identification of high-quality binding domains. Three rats were vaccinated by plasmids coding for human CD160 impregnated onto gold-nanoparticles administered into the abdominal musculature by gene-gun. An immune rat Single Chain Variable Fragment (scFv) library was generated and expressed on the pIII protein of m13 phage. The library was subsequently panned twice against Strep-Tactin beads coated with recombinant strep-tag II tagged CD160. A total of 17 unique scFvs were isolated. Heavy chain variable usage was either IGHV2 or IGHV5. Five of these were selected for further analysis. Biacore showed affinities ranging from 0.206 to 233 KD(nM). These scFvs were cloned into CAR format. Normal donor peripheral blood mononuclear cells (PBMCs) were transduced with CD160 CARs and showed a high degree of killing of CD160 positive cells with some differences in function between CAR T-cells. In conclusion, we developed a set of novel scFvs targeting CD160 leading to functional CARs using a rapid scFv discovery platform.
: Oxford Genetics Ltd
Lentiviral vectors are central to the application of many exciting gene therapy strategies, including gene-supplementation (e.g SCID) and immunotherapies such as CAR T cells in the treatment of cancer. Factors currently limiting wider development of these approaches is lentivirus yield and their relatively low infectivity in T cells. We set out to systematically improve these limitations. Lentivirus Cell Line Development: Stable packaging cell lines for lentiviruses have been difficult to develop because lentivirus protein cytotoxicity, meaning they are usually provided in trans, or using inducible promoters. We explored a new approach by co-expressing cytotoxic proteins with inhibitors of cell death pathways. We screened >50 fifty different death pathway inhibitor proteins and identified particularly powerful combinations, allowing the stable expression of VSV G in adherent and suspension HEK-293 cells. Using this approach, we have engineered a range of packaging lines expressing different lentivirus components that enable versatile systems for lentivirus production. Transduction of T and B cells: Lentiviruses are typically pseudotyped with the VSVG glycoprotein, which can infect a range of cell types. However, we reasoned that alternative glycoproteins may be superior for infection of primary T and B cells. We screened >60 different virus glycoproteins known to infect PBMC subsets pseudotyped onto lentivirus vectors. Several of these proteins gave higher levels of infection of primary T and B cells, with some doubling infectivity compared to VSVG. We believe that the systematic evaluation of previously untested glycoproteins will enable significant improvements in lentivirus infectivity in a range of PBMC subsets.
: SISSA, Trieste, 34136, Italy
Glioblastoma is a highly heterogenous and devastating CNS tumour for which no cure is presently available. The brain patterning gene Emx2 normally inhibits proliferation of astrocyte progenitors. Moreover, it is poorly expressed by human glioblastomas compared to surrounding healthy tissue. Therefore, we wondered if its overexpression may be employed for therapy of these tumors. We found that Emx2 overexpression induced the collapse of seven out of seven in vitro tested glioblastoma cell lines, by antagonizing proliferation and stimulating apoptosis. Next, it suppressed four out of four of these lines in vivo, upon their transplantation into neocortical parenchyma of immunocompetent mouse neonates. Finally, it increased the survival rate of juvenile immunodefective mice, orthotopically transplanted by U87MG glioblastoma cells. Interestingly, the antioncogenic activity displayed by Emx2 was overtly correlated with its expression level. As proven by dedicated rescue assays, Emx2 activity originated from Emx2 impact on a variety of metabolic nodes, including the stem-state-linked genes Sox2 and Hes1, the early-G1 check point machinery, EGF, PDGF, FGF and BMP signalling machineries, and the translation factor eIF4E. This may account for the robustness of such activity despite glioblastoma heterogeneity. Last but not least, in two out of two tested lines, the tumor culture collapse was also achieved when Emx2 was driven by a neural stem cell-specific promoter, likely active within tumor-initiating cells. All that points to Emx2 as a novel, promising tool for therapy of glioblastoma and prevention of its recurrencies.
: UMC Utrecht
: Graduate School of Pharmaceutical Sciences, Osaka Univ.
Mammalian reovirus type 3 Dearing (hereafter reovirus), which is a non-enveloped, double-stranded RNA virus, has received much attention as an oncolytic virus. More than 10 protocols of clinical trials using reovirus for various types of cancers are internationally going. In cancer therapy, not only cancer cells but also cancer-associated fibroblasts (CAF), which differentiate from various types of normal cells, including fibroblasts and endothelial cells, via stimulation with several cytokines in the cancer, are important target cells in cancer therapy because CAF are highly involved in the growth, survival, and metastasis of cancer cells. In addition, large amounts of extracellular matrices, including collagens, are produced by CAF. The extracellular matrices prevent the distribution of anticancer drugs in cancer. However, it remained to be clarified whether reovirus efficiently killed not only cancer cells but also CAF. In this study, we examined oncolytic efficiencies of reovirus in mouse primary CAF. The primary CAF expressed higher levels of cathepsins B and L, which are lysosomal cysteine proteases crucial for endo/lysosomal escape of reovirus, and CAF markers, including alpha-smooth muscle actin (a-SMA), than normal fibroblasts. Inoculation with reovirus at multiplicity of infection (MOI) 10 decreased the viabilities of CAF to about 60%. Oncolytic efficiencies of reovirus in the CAF significantly declined in the presence of inhibitors of cathepsin B or L. Reovirus also efficiently killed CAF in the tumor following intratumoral injection. These results indicate that reovirus mediated efficient oncolysis of not only cancer cells but also CAF, leading to superior anticancer effects.
: Dankook University
Carcinoembryonic antigen (CEA) is a glycoprotein and overexpressed by many types of cancer cells including colorectal carcinoma. CEA overexpression affects the resistance to anoikis and hepatic metastasis of colon cancer cells. 5-Fluoruracil (5-FU) is a widely used chemotherapeutic drug and leucovorin is a chemotherapeutic adjuvant against colon cancer patients. However, major hurdle of the chemotherapeutic drugs is the occurrence of chemoresistance. Previously, we identified a CEA-specific RNA aptamer which can inhibit hepatic metastasis of colon cancer cells in vivo. In this study, we explore the diverse applicability of the aptamer as anticancer tool such as; chemosensitivity enhancer and drug delivery vehicle. (I) Compared with 5-FU or leucovorin alone, triple treatment using the aptamer combined with 5-FU and leucovorin can efficiently enhance cancer cell death. Moreover, the aptamer efficiently reduces half maximal inhibitory concentration (IC50) of 5-FU. Furthermore, triple treatment efficiently enhanced chemosensitivity, and hence increased cell death rate in 5-FU resistant colon cancer cells. (II) RNAi-based therapeutic applications have been vastly attempted. Nevertheless, a critical technical hurdle for RNAi-based therapeutics is the delivery of siRNAs across the plasma membrane of cells in vivo. We generated a chimeric RNA which consists of CEA aptamer and siRNA. Chimeric RNA specifically delivered siRNA into CEA-overexpressed colon cancer cell lines and inhibited target gene expressions. Chimeric RNA-mediated cell death was detected and siRNA-mediated target gene specific cleavage was confirmed. Multiple functions of the CEA-specific RNA aptamer would be applicable to therapeutic tools for colon cancer patients.
: Paul-Ehrlich-Institute
Hodgkin lymphoma (HL) is a hematopoietic malignancy with a characteristic cellular composition. The tumor mass is made up mainly of infiltrated lymphocytes and other cells of hematologic origin but only very few neoplastic cells (<2%) that are identified by the diagnostic marker CD30. While most patients can be cured by standard therapy, approximately 20% relapse, suffer from progressive disease and develop secondary cancers. Here, we suggest a novel therapeutic concept relying on oncolytic viruses that selectively destroy the CD30-positive HL tumor cells. Relying on a recently described CD30-specific scFv, which we displayed on the measles virus (MV) hemagglutinin for lentiviral vector targeting, we have generated CD30-targeted MV (MV-CD30) and vesicular stomatitis virus (VSV-CD30), the latter being the first example of a fully retargeted VSV. For VSV-CD30 the VSV glycoprotein G reading frame was replaced with the CD30-targeted MV glycoproteins. Both viruses were found to be highly selective for CD30-positive cells as demonstrated by infection of co-cultures of target and non-target cells as well as through blocking infection by soluble CD30. Notably, VSV-CD30 replicated significantly faster to much higher titers than MV-CD30 and resulted in a more rapid and efficient killing of cultivated HL cells. Initial data from in vivo mouse tumor models show that intratumorally injected VSV-CD30 replicates in subcutaneously transplanted HL cells and completely eliminates the tumor burden (N = 2). Moreover, infectious VSV-CD30 was detected in HL tumors also after systemic administration. Side-by-side comparisons of the therapeutic activities of MV-CD30 and VSV-CD30 are ongoing.
: Cancer Gene Therapy Group, Department of Pathology, University of Helsinki, Helsinki, Finland
Dendritic cell (DC) therapy is currently considered as a promising therapeutic option for treatment of cancers. However, tumor induced immunosuppression impairs the function of dendritic cells. Therefore, human clinical trials with DC therapy have often been disappointing. Interestingly, oncolytic adenoviruses have been shown to activate immune response by enhancing the release of tumor-specific antigens and trigger danger signals at the tumor site. To achieve optimal activation of the transferred dendritic cells, we armed adenoviruses with CD40L, a surface receptor known for its capacity to trigger multifaceted signals in dendritic cells, leading to cytotoxic T-cells activation. Therefore, we constructed a novel virus Ad3-hTERT-CMV-hCD40L features Ad3 for enhanced tumor transduction, human telomerase reverse transcriptase (hTERT) promoter for enhancing tumor selective replication and CD40L, a potent stimulator of dendritic cells and to enhance antitumor efficacy. Moreover, human and animal data have shown the ability of Ad3 to reach tumors through the intravenous route. To deeply analyze the ability of CD40L-encoding adenovirus to modulate the tumor microenvironment, we generated a murine version of the virus (Ad5/3-CMV-mCD40L). In syngeneic studies in immunocompetent model, DC therapy in combination with Ad5/3-CMV-mCD40L showed potent antitumor activity and triggered significant antitumor immune response. The improved therapeutic effect by the adenovirus expressing CD40L and DCs combination treatment correlated with increased numbers of tumor infiltrating lymphocytes and the reduction of immunosuppression in the tumor stroma. These findings support the development of clinical trials where dendritic cell therapy is enhanced with oncolytic adenovirus.
: University of Helsinki
A large part of the population has been pre-exposed to adenoviruses, hence pre-existing immunity (PEI) represents a recurrent concern. Although considered to counteract the efficacy of oncolytic adenoviruses, the role of PEI in the context of cancer immunotherapy has not been clearly elucidated. Challenging this assumption, we investigated the role of PEI on the combination of PD-L1 blockade with a novel oncolytic vaccine platform (PeptiCRAd). In B16 melanoma bearing mice the combination therapy significantly increased the median survival (43 days for Combo therapy versus 35 and 27.5 days for the anti-PDL1 and PeptiCRAd monotherapies respectively). When re-challenged with a second tumor, the Combo therapy led to a significantly slower tumor growth (p = 0.0248), highlighting that immunological memory was induced. Next, all surviving mice were subjected to a boost-treatment with a PeptiCRAd targeting TRP2 and gp100 tumor antigens. Interestingly, mice with an anti-adenovirus PEI (Combo therapy) showed a reduction in tumor growth compared to mice näive to Adenovirus (anti-PDL1 monotherapy). This finding suggested that pre-existing immunity to adenoviral vector might play a positive role in anti-cancer immunotherapy. Interestingly, we found a positive correlation between anti-adenoviral and anti-tumor T-cell responses, measured by an ELISPOT assay. Finally, we performed an additional experiment where we induced anti-adenovirus-PEI by subcutaneous injections of OAds. Then, 14 days after the first immunization, we implanted B16 melanomas and once tumors developed, we treated these mice with the combination of PeptiCRAd and anti-PDL1. The results of the comparison between pre-immunized and non-pre-immunized mice will be presented in the ESGCT meeting.
: ICGEB
Functional screening of expression libraries in vivo offers the possibility of identifying novel biotherapeutics without a priori knowledge of their biochemical function. Here we used a procedure called FunSel, based on the in vivo delivery of arrayed cDNA libraries corresponding to the mouse secretome using Adeno-Associated Vectors (AAV9), for the functional selection of factors inhibiting cancer cell invasion. We have injected 24 pools of AAV encoding for secreted factors into the mouse skeletal muscle, followed by implantation of invasive Lewis Lung Cancer (LLC) cells to exert a selective pressure. The genes present in the fibers that survived from tumor invasion were amplified by PCR and sequenced by NGS. The results indicate enriched genes that protected resisting fibers from being invaded by cancer cells. Preliminary results confirmed the feasibility and reliability of this FunSel procedure and identified some enriched genes previously associated with anti-cancer activity. Among the identified genes is glypican 6 (Gpc-6), which overexpression has been correlated to increased survival of patients with ovarian cancer. Moreover, another enriched gene was N-acetylgalactosaminyltransferase 2 (Galnt-2), known for suppressing malignant phenotypes in gastric adenocarcinoma and neuroblastoma. Of interest, several genes enriched in our screening have never been characterized and could represent novel, potent soluble factors capable to inhibit cancer invasion.
: Chong Hin Loon Memorial Cancer and Biotherapy Research Center National Yang-Ming University, Taipei, Taiwan
Treatment of unresectable pancreatic ductal adenocarcinoma (PDCA) remains a great challenge nowadays. Updated related knowledge revealed adenovirotherapy may be a promising novel and safe virogenetic therapeutics for PDCA. TP53 mutation occurs in 75% of human PDAC following an initiating activating mutation in the KRAS gene (100%). In 2010, we have constructed a novel mutant KRAS-regulated promoter, Δp53REP2, derived from human double minute 2 (hdm2) P2 promoter with a deletion of p53 response elements. The promoter activation is stringently modulated where the cells owning an activating mutation of KRAS but not p53 mutation. So the property of this specific promotor has highly potential application for the adenovirotherapy of PDAC. In addition, E1B-55kD-deleted adenoviruses have been widely used as conditionally replicative adenoviruses (CRAds) for therapeutic purposes in tumors with loss-of-function p53 mutation. Here, we combine the advantage of mutant KRAS-specific promoter with E1B-55kD-deleted adenoviruses to create a CRAd specifically against the PDAC with double KRAS and p53 mutations. We also make some point mutations in the fiber of adenovirus genome to attenuate the binding of Coxsackievirus and adenovirus receptor (CAR). Additionally, the plectin-1 targeting ligand, which could specifically target PDAC, is also incorporated into the H-I loop of Ad fiber knob to increase the virus tropism. The oncolytic activity, cancer tropism and underlying mechanisms of CRAd are examined to evaluate its therapeutic potential in PDAC.
: IBET-Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal
Basal-like breast cancer (BBC) is mainly comprised by triple-negative breast cancers which display an aggressive and metastatic phenotype. These tumors rapidly acquire resistance to chemotherapy and have no targeted therapies. Herein, we aimed to develop a novel gene therapy-based treatment for BBC based on viral vector-mediated delivery of shRNA targeting previously identified BBC dependency genes, such as PSMA2. pAAV2-shRNA plasmids were generated by cloning shRNA sequences, together with GFP reporter, between AAV2 inverted terminal repeats in an AAV plasmid backbone. AAV2-shRNA vectors were produced by 2-plasmid co-transfection of HEK293T cells, with high titers and high purity. Transduction of two BBC cell lines (MDA-MB-468 and HCC1954) with AAV2-PSMA2sh decreased the expression of its target gene by 80%, as compared to control cells transduced with AAV2 vector encoding a scramble shRNA. In MDA-MB-468 cells, knockdown of PSMA2 gene was associated with significant decrease in cell viability and 2-fold increase in the percentage of apoptotic cells. In vivo studies to assess the anti-tumorigenic effect of AAV2-PSMA2sh in mouse orthotopic BBC cell xenografts showed that intratumoral injections caused a significant decrease in tumor growth when compared to negative scramble shRNA control vector or PBS injected mice. No major macroscopic alterations were observed in the liver of mice treated with AAV-shRNA vectors as compared to PBS control. Assays are currently in progress to further characterize the tumors, e.g. proliferation and apoptotic status. Our results indicate that AAV2-PSMA2sh vector decreases BBC tumorigenesis and suggest that PSMA2 gene may represent a potential therapeutic target for BBC therapy.
: Pall Life Sciences
Today, gene therapy offers a wide range of perspectives for incurable genetic disorders and industrialization of viral vector production becomes a key challenge for the biotechnology industry. Genethon's mission is to design and produce gene therapy products for rare disease in order to provide innovative treatments to patients. In this context, Genethon and Pall combined their respective expertise to develop an efficient viral vector production process in the single use iCELLis™ fixed bed bioreactor system which is scalable up to 500m2. Starting from bench scale process, rAAV-8 production process by triple PEI-mediated transfection of HEK-293 cells was successfully developed, optimized and scaled up in iCELLis™ Nano bioreactor. Results showed that medium exchange post-transfection with DMEM leads to a 7 fold increase of productivity and that the DNA amounts used for transfection can be reduced by 50% while maintaining productivity. Fixed-bed height and harvest method scaling-up also gave promising results. Today, cutting-edge and cost-efficient process of rAAV production has been developed at iCELLis™ Nano scale leading to promising perspectives to industrialize within iCELLis™ 500 bioreactors (up to 500m2).
: Chiba University
A majority of malignant mesothelioma, about 80% of the clinical specimens, had a homologous deletion in the INK4A/ARF region which contains the p14ARF and the p16INK4A genes, while the p53 genotype was wild-type. Loss of p14 protein augmented a p53 degradation process through increased MDM2 activities, and lack of p16 activity hyper-phosphorylated pRb through uninhibited CDK4/6 actions. The genetic alterations thereby down-regulated functions of the major human tumor suppressor genes, but restoration of p53 expression by itself can also down-regulate pRb phosphorylation through p21 induction. We showed that adenoviruses (Ad) expressing the p53 gene achieved cytotoxicity on INK4A/ARF-defective, p53 wild-type mesothelioma. Moreover, MDM2 inhibitors, nutlin-3a and RITA, which stabilized the endogenous p53 protein by suppressing MDM2-mediated ubiquitination, likewise induced growth suppression and cell death in the mesothelioma. These data indicated that augmented p53 levels produced cytotoxicity in the mesothelioma. We therefore examined cytotoxicity of replication-competent Ad which were devoid of the p53-binding E1B-55kDa molecules (Ad-delE1B), and demonstrated that Ad-delE1B activated the endogenous p53 and produced anti-tumor effects on the mesothelioma by inducing apoptotic cell death. We next examined effects of the Mdm2 inhibitors on the cytotoxicity by Ad-delE1B since up-regulated p53 induced premature cell death and consequently blocked Ad replication. We found that the p53-augmenting agents produced synergistic cytotoxicity with Ad-delE1B in mesothelioma bearing the wild-type p53 despite reduced production of viral progenies. These data indicated that elevation of endogenous p53 expression plays an important role in oncolytic virotherapy.
: National Cheng Kung University Medical College
Environmental stress conditions may induce nuclear accumulation of YB-1, which occurs in multidrug-resistant and adenovirus-infected cancer cells. Overexpression and nuclear localization of YB-1 are associated with poor prognosis and tumor recurrence in various cancers. Nuclear YB-1 transactivates the multidrug resistance 1 (MDR1) genes through the Y-box. Here, we developed a novel E1B55K-deleted adenovirus driven by the MDR1 promoter, designed Ad5GS3, and tested the feasibility of exploiting YB-1 to transcriptionally upregulate Ad5GS3 replication in cancer cells. In cancer cells infected with Ad5WS1, an E1B55K-deleted adenovirus driven by the E1 promoter, E1A enhanced YB-1 expression, and then further phosphorylated Akt, which, in turn, triggered nuclear translocation of YB-1. Ad5GS3 in combination with etoposide facilitated nuclear localization of YB-1, upregulated the MDR1 promoter activity, enhanced Ad5GS3 replication in cancer cells, as well as synergistically suppressed tumor growth and prolonged survival of NOD/SCID mice bearing human lung tumor xenografts. Thus, E1A, YB-1, and the MDR1 promoter form a positive feedback loop to enhance Ad5GS3 replication in cancer cells, and this regulation can be further augmented when chemotherapeutic agents are added. In clinical lung cancer cells, advanced tumors expressed high levels of MDR1 and nuclear YB-1 were susceptible to Ad5GS3-induced cytolysis. In conclusion, our results indicate that Ad5GS3 may have therapeutic potential for cancer treatment. More importantly, advanced tumors may be good target for Ad5GS3 treatment, especially in combination with chemotherapy. Because YB-1 is expressed in a broad spectrum of cancers, this oncolytic adenovirus may be broadly applicable.
: University of KwaZulu-Natal
In the last decade Selenium has found a new role in life sciences as a potential gene delivery vehicle. Selenium, an essential requirement in the function of certain enzymes is known for its cancer chemoprevention and chemotherapeutic properties. In this study Chitosan, a positively charged natural polysaccharide was used to functionalize selenium nanoparticles (SeNPs). Further conjugation to folate was accomplished, for selective tumour targeting in vitro. Physico-chemical studies were conducted on all nanoparticle systems using TEM, UV spectroscopy, FTIR and NTA. This study clearly showed that chitosan functionalization and addition of a targeting moiety to the SeNPs, influences nanoparticle size, distribution and charge. Band shift and nuclease protection studies confirmed their ability to bind, condense and protect the DNA cargo from degradation. Evaluation of the effects of all SeNPs on the cell viability and related transgene expression, in the HT-29 and Caco-2 (human colon cancer), and HEK293 (human embryonic kidney) cell lines was studied using the MTT and luciferase reporter gene assays respectively. All NPs and their respective nanocomplexes with pDNA demonstrated low cytotoxicity in both cell lines with percentage cell survival over 70 %. Folate receptor targeted SeNPs (Fol-SeNPs) exhibited higher transgene expression at the optimum binding ratio (w/w) in the Caco-2 cell line, compared to the other cell lines and the non-targeted SeNPs. These results suggest that the low cytotoxicity and significant gene expression, coupled with small particle sizes, make these NPs potentially suitable non-viral gene delivery vectors. Further modifications could enable their extension into in vivo delivery.
: Barts Cancer Institute, Queen Mary University of London
Late stage metastatic prostate cancer (PCa) remains incurable due to the rapid development of resistance to all available therapies. Cytotoxic drugs currently used in the clinic to treat PCa, frequently activates cellular autophagy that may contribute to the development of resistance by promoting cell survival. Our recently developed replication-selective adenoviral mutant AdΔΔ, deleted in E1ACR2 and the anti-apoptotic E1B19K, specifically sensitises cancer cells to apoptosis-inducing cytotoxic agents. In preclinical PCa models, AdΔΔ synergises with the clinical anti-PCa chemotherapeutics, mitoxantrone and docetaxel, by increasing drug-induced apoptosis and efficiently kills drug-insensitive PCa cells. Here, we demonstrate that the enhanced apoptosis correlates with AdΔΔ-mediated attenuation of drug-induced autophagy. We found that mitoxantrone induces autophagy in the human PCa cell lines PC-3, PC-3M and 22Rv-1, determined as increased LC3II/I ratios and increased number of acidic vesicles. Simultaneous infection with AdΔΔ increased mitoxantrone-dependent mitochondrial depolarisation (apoptosis) 2-fold, and restored LC3II/I ratios to basal levels. Addition of the autophagy-inducer rapamycin prevented AdΔΔ-mediated sensitization in PC-3 cells and increased mitoxantrone EC50-values 3-fold. In contrast, the late-stage autophagy-inhibitor chloroquine further sensitized the cells to the combination-treatment, decreased mitoxantrone EC50-values in 40% and increased apoptotic cell death 2-fold. Silencing of anti-apoptotic and anti-autophagic Bcl2 increased basal autophagy and apoptotic death while AdΔΔ-mediated sensitization to mitoxantrone-dependent cell killing was prevented. These data suggest, that AdΔΔ attenuates drug-induced cell survival/rescue mechanisms such as autophagy, promoting the elimination of cancer cells through apoptosis and viral lysis. Validation of our findings in 3-dimensional co-culture models of PCa will be presented.
: Osaka University
Reovirus, which has genome consisting of 10-segmented double-stranded RNA (dsRNA), has received much attention as an oncolytic virus. Clinical trials of cancer therapies using reovirus, including phase III clinical trials, are ongoing internationally. Reovirus efficiently replicates and induces tumor cell lysis via several mechanisms. In addition, reovirus has been demonstrated to induce down-regulation of HIF-1α which contributes to antitumor effects of reovirus. However, the mechanism remains to be elucidated. In this study, we examined the mechanism of reovirus-mediated down-regulation of HIF-1α. Reovirus mediated down-regulation of HIF-1α in not only reovirus-permissive tumor cells but also reovirus-resistant tumor cells. Reovirus-mediated down-regulation of HIF-1α resulted in the decline of expression of HIF-1α target genes, including glucose transporter 1 (Glut1). Furthermore, down-regulation of HIF-1α in the tumor was also found following intravenous injection of reovirus into nude mice transplanted with human tumor cells containing a HIF-responsive luciferase expression cassette. These results indicate that reovirus induces down-regulation of HIF-1α in not only in vitro cultured cells but also mouse subcutaneous tumors. Down-regulation of HIF-1α in tumor cells was found by treatment with not only reovirus but also reovirus dsRNA genome. A synthetic dsRNA polyI:C also efficiently induced down-regulation of HIF-1α indicating that dsRNA plays a crucial role in the HIF-1α down-regulation. dsRNA-mediated HIF-1α down-regulation was efficiently induced when retinoic acid-inducible gene-I (RIG-I), which is a pattern recognition receptor recognizing dsRNA, was knocked down. These results indicate that RIG-I-mediated innate immunity is not involved in reovirus-mediated down-regulation of HIF-1α.
: Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
Mesenchymal Stem Cells(MSCs) are widely use in cell based therapies. Differentiation, Low risk of allogeneic rejection, immune suppression and induction of T-regulatory function made MSCs as a major source in regenerative medicine. Whereas, the HLA molecules control immunologic reactions in recipients, here we compared the HLA-ABC, D PQR and G molecules expression in Placenta, cord, amniotic membrane and bone marrow derived MSCs, to find the low immune reactive source for cell therapy. Five samples of each sources were collected from consent healthy donors. MSCs were isolated and expanded under similar culture conditions. Cells from passage 4 were characterized immunophenotypically, also osteogenic and adipogenic differentiation. The surface expression of HLA-ABC, D PQR and cytoplasmic expression of HLA-G were investigated by flowcytometry via Mean Fluorescent Intensity(MFI) detection of Fluorescein Iso ThioCyanate conjugated monoclonal antibodies. The Photo Multiplier Tube voltages were fixed during all analysis. The results were analyzed with t-test. HLA-ABC in Amiotic membrane, Cord and BM derived MSCs were significantly lower than Placenta derived MSCs. Amniotic membrane derived MSCs showed the highest expression of HLA-G, that was meaningfully higher than placenta and BM derived cells (P = 0.02, P = 0.04). HLA-D PQR had lower expression in amniotic membrane derived MSCs. However it did not showed any meaningful differences within all sources. According to the important role of HLA-ABC in immunologic reactions, also, HLA-G in immunomodulatory effect of MSCs, it is suggested that Amniotic membrane derived MSCs are the privilege source for different applications in cell therapy.
: Dankook University
Japanese encephalitis virus (JEV) and dengue virus (DENV) are the most common etiological agents of worldwide epidemic viral disease. They are a member of the flavivirus in the family flaviviridae and harbor an enveloped, single positive-stranded RNA genome of approximately 11 kb. They encode a single methyltransferase (MTase) domain located at the N-terminal region of the RNA replicase. MTase catalyzes methylation of the viral RNA cap, producing m7GpppAm type I cap structure by using S-adenosyl-L-methionine as a methyl donor. MTase methylates only viral RNA by recognizing its 5’ UTR RNA sequence. Viral RNA cap methylation occurs in the cytoplasm in contrast to that of human mRNA methylation that occurs in the nucleus, suggesting that MTase is a useful target for antiviral therapy. Aptamers are oligonucleotides that specifically and avidly interact with diverse target molecules including proteins. In this study, we identified specific and RNase-resistant RNA aptamers against the viral MTase. RNA aptamers were isolated with modified 2’-O-methyl and 2’-fluro pyrimidines that bind JEV and DENV type II MTase with high affinity and specificity, respectively. These flavivirus MTase aptamers efficiently inhibited the cap methylation activity in vitro. Moreover, DENV type II MTase aptamer could not only bind but also inhibit methylation activity of DENV type III MTase. Inhibition of JEV replication as well as that of the methylation activity were observed by JEV RNA aptamers in cell culture system. Activity of the DENV type II aptamer to inhibit DENV production in cells will be discussed.
: Francisco de Vitoria University
The Na/ I symporter gene (hNIS) is expressed in the thyroid and allows the accumulation of iodine from the diet, to form T3 and T4 hormones. Moreover, it is used (i) as a reporter gene for molecular imaging (when the positron emitter isotope is I124 for PET or Tc99 for SPECT) or (ii) as a therapeutic gene for cancer therapy, mediated by the accumulation of I131. An unresolved challenge is how to direct this gene specifically to the tumoral area. Previously, our group demonstrated the migratory capacity of placental mesenchymal stem cells (MSCs) towards tumors, carrying adenovirus expressing hNIS. However as hNIS is expressed at the placental tissue (because vvit transfers iodine to the foetus from the maternal blood) we decide to study whether those placental MSCs might express hNIS endogenously. Therefore here, we studied whether those MSCs (1) expressed hNIS endogenously (2) reached the tumoral areas when they were intratumorally injected. Here we present the theragnostic capacity of those of placental cells for cancer therapy and imaging at the same time in tumour bearing mice and how the cell passages might influence those effects.
: Division of Pharmaceutical Biosciences and Centre for Drug Research, University of Helsinki
Efficient anticancer agent and its targeted delivery into the tumor mass is a key prerequisite for the successful cancer therapy. Oncolytic virotherapy is emerging as a potential approach to treat cancer, using viruses, which are specifically engineered to selectively infect, replicate in and kill cancer cells without causing damage to normal cells. However this approach has also disadvantages like high virus toxicity, production of neutralization antibodies against virus. Additionally oncolytic viruses are administered intratumorally, thus many solid tumors cannot be treated using this approach. Its's known that extra vesicles (EVs), which are naturally occurring cargo delivery agents have a potential to be used as vehicles for drug delivery. Therefore EVs can be used for targeted delivery of the therapeutic agents into the tumor cells and to finally decrease drug toxicity. We have found that oncolytic adenoviruses encapsulated into EVs loaded with therapeutic agents enhance specific tumor targeting thus improving efficacy of the cancer treatment.
: INSERM U1147 - University Paris Descartes
: Hanyang University
Receptor for advanced glycation endproduct (RAGE) and its ligands, including S100 protein and high mobility group box 1 (HMGB1), are highly expressed in most tumor cells especially in glioblastoma (GBM). Interaction of RAGE and its ligands has an importance role in tumor cells such as invasion, angiogenesis. Blockage of the interaction can inhibit RAGE signaling, resulting in anti-tumor effect such as angiogenesis. In this study, RAGE targeting carrier was developed using polyethylenimine (PEI) conjugated with RAGE binding peptide (RBP) that was originated from HMGB1. Gel retardation assays showed that PEI-RBP formed a complex with a plasmid DNA at or above 1:2 weight ratio (pDNA:carrier). Luciferase assays showed that PEI-RBP had the highest transfection efficiency at a weight ratio of 1:5, which was higher than that of PEI in C6 rat glial cell. In a RBP competition assay, the transfection efficiency of PEI-RBP was decreased by addition of free RBP. In addition, expression of VEGF was reduced in RBP and PEI-RBP transfected C6 cell. These observations suggest that interaction of RAGE and RBP may lead to endocytosis of pDNA/carrier complex and inhibit RAGE signaling associated with angiogenesis. Accordingly, PEI-RBP may be useful for the treatment of glioblastoma as a RAGE targeting gene delivery carrier. *This work was supported by the grant of BK21plus program from the Ministry of Education in Korea.
: MolMed S.p.A
Retroviral vector (RVV) are used in cell and gene therapy studies. For early clinical phases, RVV can be manufactured at medium scale by different open and manual systems. Moving to advanced clinical and commercial phases, one of the major bottlenecks is the difficulty to find an adequate large-scale platform for vector production from adherent cells. In order to address this issue, a development study was performed in iCELLis® fixed-bed disposable bioreactor in the context of the MM-TK therapy, constituted of T lymphocytes genetically modified ex vivo with the γ retroviral vector SFCMM-3 Mut2 #48. The role of culture regulation parameters on cell growth were investigated by evaluating key metabolites during cell expansion. The RVV vectors have been characterized for infectious viral titre and viral particle content. In two different runs in iCELLis® nano the results in terms of viral titre and infectivity were comparable to the ones obtained with the no-disposable NLF-32 bioreactor. The values calculated for the NLF-32 bioreactor versus the iCELLis® nano bioreactor were respectively: infectious viral titre 5.5x10^5 TU/cm^2 vs 7.5x10^5 TU/cm^2; infectivity 6.1 x10^3 TU/ng p30 vs 4.4 x10^3 TU/ng p30. Considering this, the scale up of the process in the iCELLis 500 could permit to obtain about 1.0x10^12 total TU for T cells transduction for about 150 patients. A purification step was also introduced consisting in 10-20 fold vector concentration by hollow fiber tangential flow filtration followed by sterilizing filtration step. The scale-up of the developed process in iCELLis 500 is currently on-going.
: MolMed S.p.A.
The MM-TK is a medicinal product constituted of T lymphocytes genetically modified ex vivo with the γ retroviral vector SFCMM-3 Mut2#48 to express HSV-TK Mut2 and ΔLNGFR genes, developed as adjunctive therapy in leukaemia patients undergoing stem cell transplantation. The manufacturing process consists in stimulation of PBMCs (about 3 × 109 cells) isolated from the haploidentical donor, with anti-CD3 mAb and IL-2, transduction with retroviral vector in RetroNectin® coated bags and immunoselection of TK cells using the customized CD271-LS Reagent (Miltenyi Biotec). TK cells are then further expanded for 7 days before freezing. The process lasts 13 days and allows the generation of MM-TK doses in a closed large-scale, semi-automated culture system using the CliniMACS Prodigy® with a specific software. A validation study has been performed to demonstrate that i) the process was robust and reproducible, ii) suitable levels of TK+ cells could be manufactured, iii) the final product was free of bacterial and mycoplasma contamination and RCR. The validation requirements in terms of T cell expansion, transduction efficiency, biological activity, testing and release criteria were met for all three validation batches: cell recovery and viability were >80% at each step; total process yield was 100%; purity of CD3+ and LNGFR+ cells was >94%. Moreover, MM-TK products have been evaluated for product- and process-related impurities, potency and identity. The results have supported the discussion with the Regulatory Authorities before the introduction of the new optimized closed manufacturing process in the late phase of the phase III clinical trial and for the market.
: University of KwaZulu-Natal
: Bacteriology and Virology Department, Shiraz University of medical sciences, Shiraz, Iran
: Laboratorio di Terapia Cellulare e Genica Stefano Verri, ASST-Monza, Ospedale San Gerardo, Monza (MB)
Acute lymphoblastic leukemia (ALL) is a malignant disorder with a long-term remission of less than 50% of adult patients and of nearly 80% of children. Adoptive transfer of T cells engineered to express a chimeric antigen receptor (CAR) has shown striking responses in highly refractory populations. Unmodified Cytokine-Induced Killer (CIK) cells have demonstrated a high profile of safety in ALL patients. Aim of the present report was to demonstrate the feasibility and reproducibility of a GMP-compliant protocol to obtain CD19.CAR-expressing CIK cells using the Sleeping Beauty (SB) transposon. Peripheral blood mononuclear cells (PBMCs) were isolated and modified with simultaneous electro-transfer of the SB GMP-grade DNA plasmids CD19.CAR/pTMNDU3 and pCMV-SB11 using 4D-Nucleofector (Lonza). The manufacturing process was performed in a GMP academic cell factory. We manufactured three lots by seeding 46.0x106, 56.8x106 and 140.4x106 PBMCs respectively. After 21–22 days of culture, we harvested 15.9x109, 1.4x109, and 3.8x109 total nucleated cells respectively, with a mean viability of 97.23%. CD3+/CAR+ cell fold increase was 725, 93, 40. At the end of expansion, more than 94% of total nucleated cells were CD3+. The median expression of CD3+CD19.CAR + cells was 46.90% (range 31.27% – 65.45%), and the median expression of CD56+ was 49.80% (range 48.29% – 63.10%). In all three lots, CARCIK cells demonstrated specific and effective cytotoxicity towards the CD19+ REH cell line. These results represent solid bases for the application of this platform in a future Phase I clinical trial for patients with relapsed/refractory B-cell precursor ALL post HSCT.
: Hasselt University
: Yonsei University, Seoul, Korea
Adeno-associated viruses (AAVs) have gradually become a promising vectors for safe gene delivery, but the efficacy of AAV delivery tends to be reduced by the neutralization of pre-existing antibodies. We found that the genetically modified AAV vectors have better ability of evading from antibodies and show the higher transduction efficiency to the cancer cells. To strengthen the both of properties, we applied biocompatible polymers to the genetically modified vectors and confirmed that engineered AAVs were protected from antibodies’ attack and transduction efficiency was also enhanced. Finally, these vectors were utilized as the effective therapeutic gene carriers for the cancer gene therapy.
: Laboratory of Morphology, Biomedical Research Institute (BIOMED), Hasselt University, Diepenbeek, Belgium
The survival rate of patients with ‘head and neck squamous cell cancers (HNSCC)’ has remained incredibly poor and unchanged during the past 30 years. Our goal is to validate whether human dental pulp stem cells (DPSCs) could be used as vehicle to deliver gene therapy to HNSCC. The effect of DPSCs on tumor growth in a HNSCC xenograft mouse model was investigated. 1x106 squamous carcinoma cells were injected into the dorsal flank of nude mice, 2 tumors per mice. 10 days later, one of the tumors received injection of 1x106 DPSCs. No difference in tumor volume between DPSC-inoculated or control tumors was observed during 21 days (n = 14). qPCR analysis revealed no development of epithelial mesenchymal transformation (EMT). In addition, both qPCR of the endothelial cell marker CD31 as well as immunohistochemistry demonstrated no difference in blood vessel count between control and DPSC-treated tumors. In the transwell system, DPSCs were able to migrate towards tumor cells and to SDF-1 but not towards VEGF, FGF-2, IL-8 and IL-6 (n = 6). High patient variability was observed. Our data suggest that DPSCs can be applied for gene delivery in cancer as they do not promote tumor growth nor angiogenesis in the applied model and time frame and migrate towards tumor cells in vitro.
: IBGM - University of Valladolid
The novel CRISPR-Cas9 system is widely used to engineer the genomes of various cells and organisms. This technique is highly efficient but generates many off-target effects. An improved CRISPR –Cas9 nuclease has been developed to create a catalytic mutant Cas9 (D10A) nickase (CRISPR-Cas9n), which minimize off-target effects. Here we show the application of CRISPR-Cas9n to eliminate FASTK gene. We have demonstrated, in collaboration with the laboratory of Dr. Martinou (University of Geneva), that the lack of FASTK causes the virtual absence of mature mRNA ND6 and this causes a 60% decrease in mitochondrial complex I activity (Cell Reports 2015 vol 10: 1110-1121). The use of transmitochondrial cybrids, several in vitro studies as well as in vivo studies using mouse xenograft models of tumor cell lines, have revealed the importance of mitochondrial complex I in metastatic and migratory ability of both cervical and breast cancer cells. Here we decided to explore the effect of the deletion of FASTK gene in the aggressiveness of cervical cancer cells (HeLa). By transfecting HeLa cells with Cas9n, a guide-RNA (gRNA) and donor DNA carrying the blasticidin gene which replaces the FASTK gene, we induced homologous recombination (HR) of this gene and efficiently obtained three different FASTK knock-out cells. We checked the absence of FASTK by PCR, RT-qPCR and western blot. This new FASTK knock-out cell line will help us to identify the link between FASTK and cancer progression by means of in vitro and in vivo migration and invasion assays.
: Non-viral Gene Delivery Laboratory, Discipline of Biochemistry, University of KwaZulu-Natal, Private Bag X54001, Durban 4000, South Africa
The rapidly developing field of nanotechnology has provided a strong support for the development of nanoparticle mediated gene delivery systems. Amongst these, gold nanoparticles (AuNPs) have attracted much attention as gene and drug delivery vehicles due to their low toxicity, ease of synthesis, small size and the ability to be functionalized with suitable polymers or targeting molecules. In this study, AuNPs were functionalized with chitosan which has a high positive charge density. These AuNPs were further modified by the addition of the steric stabilizing polymer, polyethylene glycol (PEG). Physicochemical characterization using TEM, UV spectroscopy and NTA revealed that surface modification of the AuNPs with chitosan and PEG influenced the size, colloidal stability, polydispersity and zeta potential. The ablility of these nanoparticles to bind and afford protection to siRNA was determined using the band shift, dye displacement and RNase A protection assays. The cytotoxicity of the modified AuNPs and their respective nanocomplexes with siRNA, evaluated using the MTT and Alamar Blue assays in the HEK 293, Caco-2 and MCF-7 cell lines, showed minimal toxicity with cell survival over 75%. Cellular uptake studies utilizing fluorescently labelled siRNA, confirmed that there was a good correlation between pegylation/non-pegylation, and internalization and eventual transfection ability of these nanocomplexes. Ideal physicochemical properties, low cytotoxicity and the relative ease of cellular uptake for transfection, make these pegylated-chitosan functionalized AuNPs promising delivery vehicles for siRNA and other therapeutic molecules. Further research into their optimization and modification may be required before they can be extended to in vivo studies.
*
: Universidad Francisco de Vitoria
On a previous work, our group demonstrated the migratory capacity and the differences of several mesenchymal stem cells (MSCs) on their mgration towards tumors. On this work, we intensively studied the different characteristics of several types of MSCs by (1) surface markers (2) migration on wound healing assays (3) pluripotency markers (4) adenoviral infection (5) cell growth(6) microarray and qPCR assays. As placental MSCs were the cells of our choice due to the easy culture conditions, migratory potential and lack of tumour growth, we decided to study on deep those cells. The studies performed included the same as previously but considering passages, oxigen conditions and confluency. Here we confirm the extensive potential of placental MSCs for cell therapies and the importance of the cell culture conditions
: Pall Life Sciences
Over the past 15 years, cell therapies have begun to reach the market. Some companies have faced challenges to achieving scalable, cost-effective, and robust cell therapy manufacturing — leading to notable failures caused by manufacturing concerns, such as high CoG. Cell-based therapies are however gearing up to have extensive impact on the healthcare field in coming years. In order to capitalize on the potential of these therapies, there must be efficient methods for growing the adherent stem cells at larger scales. This has set the stage for high productivity manufacturing technology with an emphasis on safety, reproducibility and compliance to GMP standards while maintaining the comparability of the products released and keeping the cost of goods under tight control. In this presentation, we will discuss the review the factors influencing your bioprocess scale, design optimized manufacturing strategies for allogeneic cell-based therapies, evaluate and select the most cost-effective technology according to lot size to align the process development and product roadmaps.
: Department of Biomedical and Clinical Science, University of Florence, 50134 Italy
The cell-based therapies and the nanomedicine have advanced significantly over the past decade and are poised to become a major pillar of modern medicine. Tumor progression of many types of cancer, including melanoma, is characterized by increased expression of the membrane-associated plasminogen activation system (urokinase-type plasminogen activator, uPA; uPA receptor, uPAR; uPA inhibitor type-1, PAI-1). We engineered EPCs with a lentivirus encoding the uPAR-degrading enzyme MMP12 to induce a lentivirus-dependent “gain of function” of MMP12 activity in EPCs. We demonstrated that intravenous injected 111In-oxine labelled EPCs are able to home into tumor mass by exploiting the CXCR4/SDF1 axis. We demonstrated that the i.v. injected MMP12- engineered “commandos EPCs” were able to control melanoma progression, angiogenesis and metastasis and to cleave uPAR on tumor cells and endothelial cells of the tumor microenvironment in human melanomas transplanted in nude mice. We showed that administration of autologous 111In-oxine labelled MMP12- lentiviral modified EPCs provide an effective melanoma “personalized therapy” without rejection. Gold nanoparticles (AuNPs) showed great promise for the treatment of cancer for their non-cytotoxic nature, ease of synthesis and functionalization and their tunable plasmonic properties. This allows AuNPs to cause local heating when exposed to near-infrared (NIR) light. We loaded EPCs with gold nanoparticles, assessed cellular uptake and retention using inductively coupled plasma mass spectrometry (ICP-MS) and TEM microscopy and finally tested the effect on cytotoxicity. Gold nanoparticle does not alter EPC biological activity. Therefore, upon laser irradiation, AuNP-loaded EPCs could thermally ablate melanoma cells providing a promising theranostic approach for melanoma.
: Cell Info, Inst. Adv. Med. Res., Keio Univ. Sch. Med.
Tumor-initiating cells thought to drive brain cancer are embedded in a complex heterogeneous histology. In this study, we isolated primary cells from 21 human brain tumor specimens to establish cell lines with high tumorigenic potential and to identify the molecules enabling this capability. The morphology, sphere-forming ability upon expansion, and differentiation potential of all cell lines were indistinguishable in vitro However, testing for tumorigenicity revealed two distinct cell types, brain tumor-initiating cells (BTIC) and non-BTIC. We found that macrophage migration inhibitory factor (MIF) was highly expressed in BTIC compared with non-BTIC, indicating that this molecule may play a role in tumor initiation. MIF gene knockdown decreased the cellular proliferation of BITC in vitro. Transcriptome analysis using RNA sequencing showed the down regulation of glioma-related genes (PDGFA, PDGFB, NLGN3) in MIF gene knockdown BTIC. In addition, we observed the higher expression of some glycans in BTIC compared to human neural stem cells using lectin array. Intriguingly, MIF knockdown increased the transcription of p53 target genes (p21, BAX) in p53 wild-type BITC. In contrast, in p53 mutant-type BITC, MIF knockdown increased the accumulation of p53 into mitochondria, inducing apoptosis in a transcription independent fashion. Moreover, MIF knockdown inhibited tumor formation by BTIC in a xenograft mouse model, and led to increased overall survival of the mice. Our findings suggest that MIF plays a role in the regulation of cell proliferation in BTIC.
: Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
Bone marrow niche is a spatial structure which composed of mesenchymal stem cells (MSCs), their progeny and extra cellular matrix that regulate hematopoietic stem cells (HSCs) development. Capacity of MSCs in supporting HSCs becomes attenuated in leukemia and provides a decent bed for leukemic progression. Different behavior of stem cells shows the pivotal role of BM niche studies to find the mechanisms that lay behind it. In this study two model of co-culture conditions; 2D and 3D microfluidic devices were tested in order to analyze the effect of different culture conditions on leukemic cells properties. K562 cell line was cultured on BM derived MSCs as a feeder layer, in 2D and 3D microfluidic devices. To demonstrate the cell morphology, scanning electron microscopy was used and carboxyfluorescein succinimidyl ester test was performed to proliferation evaluation. In order to compare the cell viability and apoptosis rate in 2D and 3D culture conditions, Annexin V and Propidium Iodide reaction was carried out and bcl2 expression level was assayed by qRT-PCR. Our results show that proliferation rate of k562 cells were significantly higher in 3D in comparison to 2D microfluidic devices (P value <0.05). The viability of cells had a meaningful reduction in 2D in contrast with 3D culture condition and bcl2 level verified this result (P value <0.05). On balance, 3D microfluidic devices would provide an opportunity to follow up the molecular and cellular behavior of normal and malignant stem cells in their niche and this condition can be closer to their real microenvironment.
: Department of Molecular Genetics, Tehran Medical Branch, Islamic Azad University, Tehran, Iran
It becomes evident that long non coding RNAs (lncRNA) play important roles in cell physiology and pathology. Although all the facts about lncRNAs are much remained to be elucidated, but they regulate cells behavior via epigenetic, transcriptional and post-transcriptional modifications. Studies show that in diverse malignancies from leukemia to breast cancer, lncRNAs have been dysregulated and have a crucial role in tumorigenesis. Plasmacytoma variant translocation 1 (PVT1) oncogene, located in chromosome 8 encodes an lncRNA, which have an interaction with important oncogene c-myc, overexpressed in different malignancies such as gastric, breast and ovarian cancers. Therefore, we determined to assess the role of pvt1 in proliferation and apoptosis in acute lymphoblastic leukemia cell line. In this study, Jurkat cells (Acute T cell leukemia cell line) were treated with Anti PVT1 siRNA in order to minimize the level of PVT1. The expression of PVT1 was evaluated by qRT-PCR before and after siRNA treatment. The proliferation rate using carboxy fluorescein succinimidyl ester, cell cycle with Propidium Iodide and apoptosis assay by utilizing Annexin-PI were carried out. The Level of PVT1 in Jurkat cells was much higher than normal T cells and PVT1 knockdown, significantly inhibited proliferation and increase apoptosis in Jurkat cell line (P value <0.05). Meanwhile cell cycle analysis revealed reduction of S-phase population in comparison to the control group (P value <0.05). On balance, PVT1 is a major contributing factor in leukemic progression and by considering this fact, we can utilize it as a decent target for ALL treatment studies.
: Université Bordeaux Segalen
Iatrogenic teratogenesis potential is a critical hurdle to the application of induced pluripotent stem cells (hiPSCs) use in hematology. To reduce this risk our goal was to evaluate the best system to purge residual hiPSCs before graft, without compromising hematopoietic repopulation capability. Teratoma risk after systemic injection of hiPSC expressing the reporter gene luciferase was assessed for the first time. Teratoma formation in NSG mice was tracked by in vivo bioimaging. We observed that systemic injection hiPSCs produced multisite teratoma as little as 5 weeks post-injection. To eliminate hiPSCs before graft, we tested the embryonic-specific expression of suicide genes under the control of the pmiR-302/367 promoter. This promoter was highly active in hiPSCs, but not in differentiated cells. The gene/prodrug iCaspase-9/AP20187 was more efficient and rapid than thymidine kinase/ganciclovir, fully specific, and without bystander effect. We observed that inducible Caspase-9 (icaspase-9) expressing hiPSCs died in a dose-dependent manner with AP20187, without reaching full eradication in vitro. Unexpectedly non-specific toxicity of AP20187 on iCaspase-9 negative hiPSCs and on CD34+ cells was evidenced in vitro. This toxic effect strongly impaired CD34+-derived human hematopoiesis in adoptive transfers. Survivin inhibition is alternative to suicide gene approach because hiPSCs fully rely on survivin for survival. Survivin inhibitor YM155, an analog of quercetin, was more efficient to kill hiPSCs than AP20187/iCaspase-9, without toxicity on CD34+ cells, in vitro and in adoptive transfers. hiPSCs purge by survivin inhibitor fully eradicated teratoma formation in NSG mice. This will be useful to improve the safety-management for hiPSCs-based medicine.
: Unit of Bone and Mineral Diseases, Department of Surgery and Translational Medicine, University of Florence, Florence, 50134, Italy
Ewing Sarcoma (EWS), a member of the Ewing family of tumors, is a malignant mesenchymal-derived tumor, which usually arises in bone and rarely in soft tissues. Despite the intensive combined therapy, the EWS is the second cause of cancer related death in childhood and in youth. Nowadays, the hope for patients affected by EWS is the development of novel and more effective therapies based on the molecular characterization of this cancer. An innovative prospect in this battle has emerged from research on cancer stem cells (CSCs). Their presence offers a possible explanation as to why many treatments seem to be initially effective, but the patient later relapses because the subset of CSCs was not completely eradicated. In this study we have set up three primary finite cell lines of human EWS, from which we have respectively isolated the CSCs by the sarcosphere assay. The three EWS-CSCs isolated have been marked as, ES1-, ES2- and ES3-CSCs. Their CSC phenotype has been confirmed by the study of their mesenchymal stem phenotype, by studying their capacity to differentiate into osteoblasts and into adipocytes and their profile of mesenchymal stem markers. At the same time, we have studied and confirmed their embryonic stem phenotype. In conclusion, our results show the presence of CSCs in the EWS, establishing a possible in vitro model to study the biology of EWS tumors and to find new molecular targets for the discovery of new therapies.
: Unit of Bone and Mineral Diseases, Department of Surgery and Translational Medicine, University of Florence, Florence, 50134, Italy
Osteosarcoma, the most common primary bone tumor, presents several aggressive subtypes between we can find the small cell osteosarcoma (SCO). In this study, the presence of the cancer stem cells (CSCs) in a human SCO is showed. SCO sample was collected at the “S.O.D Ortopedia Oncologica e Ricostruttiva”, AOU Careggi, Florence, with informed consent approved by the local Ethical Committee. From the primary finite cell line of human SCO we have isolated the CSC. Consequently, we have evaluated in vitro their mesenchymal and embryonic stem phenotype and we have also evaluated their neoplastic phenotype. We have set up a primary cell line of SCO, from which we have isolated and established a SCO-CSCs line, called OSA3-CSCs. The CSC phenotype of OSA3-CSC line was confirmed by observing their capacity to differentiate into osteoblasts and into adipocytes, by showing the presence of the mesenchymal stem cells markers and by studying their clonogenic and tumorigenic capacities. We have also confirmed, their embryonic stem phenotype by verifying the presence of the expression of the embryonic stem cells marker genes together with CD133 gene and other genes involved in the pluripotency of CSCs (i.e. MYC) and in the metastatic process (i.e AXL, EZR). This research provides, for the first time, the existence of CSCs in human SCO and highlights the establishment/characterization of a SCO-CSCs line at cellular and molecular level, setting up a first in vitro model to study/find new molecular targets to develop a molecular therapy against this rare aggressive osteosarcoma.
: CIRI; Inserm U1111, Lyon, France
The malignant transformation of normal T-cell progenitor cells into self-renewing leukaemia-initiating cells causes the development of T-cell Acute Lymphoblastic Leukaemia (T-ALL), an aggressive hematologic cancer presenting unfavourable clinical features. Leukaemia-initiating cells are linked to chemotherapy resistance and relapse but the lack of tools to manipulate them prevents identification of their immunophenotypic and molecular features, thus precluding the development of novel targeted therapies. Our study reveals that lentiviral vectors (LVs) pseudotyped with a baboon retrovirus envelope (BaEV-LVs) are excellent tools to genetically manipulate T-ALL leukaemia initiating cells. These LVs enabled high-level transduction at low multiplicity of infection (MOI = 10) since they use ASCT1 and ASCT2, two aminoacid transporters which are highly expressed on the T-ALL cells. The transduced blasts engrafted cohorts of primary and secondary NOD/SCID gamma mice, with typical T-ALL features, thus demonstrating that BaEV-LVs target leukaemia-initiating cells. Competitive xenografts models demonstrated that the BaEV-LV do not alter leukemic self-renewal, as the transduced blasts did not acquire a growth advantage, nor were they outcompeted by untransduced cells. Furthermore, our BaEV-pseudotyped lentiviral vectors will facilitate chimeric antigen receptor expression and the development of genomic editing strategies for primary T-ALL cells. BaEV-LVs are thus excellent tools to genetically manipulate LICs to aid revealing novel therapeutic avenues.
: Miltenyi Biotec
Human pluripotent stem cells (hPSCs) hold great promise for clinical use and cell therapy applications. To ensure highest quality and consistency of the resulting cellular products, suitable hPSCs lines have to be maintained under standardized cultivation conditions and procedures. In this context, we have developed the xenofree iPS-Brew GMP Medium following the recommendations of USP 1043 on ancillary materials, thus enabling expansion of hPSC for clinical research use. For qualification of the iPS-Brew GMP Medium, hiPSC were expanded for 20 passages on recombinant Laminin-521 using a pilot lot of this medium. Resulting cells displayed a normal karyotype and were highly pluripotent as assessed by multi-color flow cytometry and in vitro differentiation assays. To increase the level of process standardization and product consistency we developed a procedure for cultivation of hPSCs using the integrated cell-processing platform CliniMACS Prodigy®. PSCs could be expanded by a factor of 25 to 60 in a single passage using automated coating, feeding and harvesting procedures in a closed, single use tubing set (TS730) under adherent culture conditions. Subsequently, these cells could be replated and differentiated into neuroectodermal progenitor cells within the closed system, illustrating the feasibility of an automated cell production for future clinical cell manufacture.
: ICGEB
Innovative therapies for patients with myocardial infarction (MI) are urgently needed. Screening of expression libraries in vivo offers the possibility of identifying novel biotherapeutics without prior knowledge of their function. Here we describe a procedure for the functional selection (FunSel) of protective factors based on the in vivo delivery of cDNA libraries from the mouse secretome using AAV vectors. We generated 1200 AAV-plasmids, each one coding for a different factor, and organized them into 24 AAV9-pools, composed by 50 factors each. All the clones contain a specific, 10-bp barcode sequence allowing direct identification by Next Generation sequencing (NGS). Encouraged by preliminary findings obtained on a subset of clones (Ruozi et al., Nature Comm-2015) we applied FunSel to the whole secretome library to identify cardioprotective factors after MI. The pools were injected in the myocardium and selective pressure was induced by coronary artery ligation. After three weeks, the viral genomes were recovered from the infarcted hearts and the barcode frequency of each vector was determined by NGS. Approximately 200 factors resulted enriched over statistical significance, of which 25 factors 3 folds or more over neutral effect. Supporting the efficacy of our approach, among these hits we found both known cardioprotective factors and unknown factors related to tissue regeneration (including VEGFB, midkine and relaxin1). Most notably, among the selected factors, there are new interesting molecules, which had never been previously investigated. Efficacy of the top hits from the screening is currently investigated both after AAV9 delivery and as infusion of recombinant proteins.
: Kyushu University
Critical limb ischemia (CLI) has a poor prognosis and adversely affects patients’ QOL. The aim of this study is to investigate the effects of the first-in-man Phase I/IIa clinical trial of gene therapy using a non-transmissible recombinant Sendai-virus vector carrying the fibroblast growth factor-2 (SeV/dF-hFGF2: product name of DVC1-0101) on improving QOL in patients with CLI. QOL was assessed using the Short Form 36 health survey (SF-36) for 12 patients, who participated in the gene therapy, on pre-administration, and 28 days, 3, 6, and 12 months after administration. We examined differences between pre and post administration QOL scores and correlations between QOL scores and vascular parameters. Post-treatment scores showed significant improvements in physical functioning at 3 and 6 months, in role-physical at 3, 6 and 12 months, in bodily pain at 1, 3, 6 and 12 months, in vitality at 1, 6 and 12 months, and in physical component summary (PCS) at 6 and 12 months (p < .05). There were no positive correlations between improvements in changes of SF-36 summary scores and changes of absolute claudication distance, ankle–brachial index, and pain scales after DVC1-0101 administration, however there were positive correlation between PCS and toe-brachial index at 12 months and thermography at 6 months (r = .88 and r = .86, respectively, p = .001). This result was updated for details from previous report. Our findings indicate that DVC1-0101 gene therapy may improve or preserve the physical component for CLI patients in a long-term period.
: University of Melbourne Australia
Pulmonary arterial hypertension is a debilitating disease that results in obstruction of pulmonary vessels resulting in right heart failure. Patients diagnosed with Group I heritable pulmonary arterial hypertension (HPAH) have a mean survival rate of 3 years and young women in particular are predisposed to this fatal disease. 70–80% of HPAH patients have been found to harbour a mutation in the bone morphogenetic receptor 2 (BMPR2) gene, however it is only 20% penetrant. To account for the lack of penetrance several hypotheses have been proposed, such as haploinsufficiency, loss of function, gain of function, dominant negative and environmental insults leading to the progression of disease. Currently, these hypotheses have been investigated in animal models that do not fully recapitulate PAH in humans. We derived an induced pluripotent stem cell from patient fibroblasts with a rare BMPR2 mutation (2504delC), resulting in truncation of protein missing the cytosolic tail domain. Using this cell line, we corrected the mutation utilizing the clustered regularly interspaced short palindromic repeats (CRISPR) associated Cas system. Isogenic controls permit elucidation of the mechanisms behind PAH by studying the differentiation and function of cardiovascular cell types. Furthermore, we generated 2504delC in another human embryonic cell line (HES3) that has eGFP reporter tag in one allele of NKX2-5. We also generated several knockouts of BMPR2 in NKX2-5eGFP/w cell line, a complete null, kinase region knockout and a tail domain knockout via CRISPR. These cell lines facilitate modelling of Pulmanary Aterial Hypertension in a disease.
: Human Stem Cells Institute, Moscow, Russia
The objective of this study is evaluation of safety and efficacy of pl-VEGF165 transfer in patients with neuroischemic type of diabetic foot syndrome (NCT02538705). The pilot study included 27 diabetic patients with neuroischemic foot ulcers (Wagner stage 1–2) who were not candidates for revascularization procedures. The mean age was 63.9 ± 7.6 years, the duration of diabetes mellitus – 12.1 ± 2.4 years, and mean duration of foot ulcers – 3.2 months. The primary efficacy endpoint was the surface area of the ulcers (sq.cm), the secondary endpoints were Tcp02, ABI, neuropathy disability score (NDS) and neuropathy symptoms score (NSS). Adverse events were monitored throughout the study. At 6 months the mean surface area of the ulcers decreased from 3.2sq.cm to 0.45sq.cm. Complete healing of ulcers was achieved in 18 patients. 2 patients developed healthy granulation tissues. No changes were found in 3 patients. 4 patients underwent major amputations due to the progression of ischemia and infectious complications. Positive changes in reparative regeneration of the foot tissues were associated with improved perfusion: within 6 months tcp02 value increased by 38% as compared to baseline. The mean value of ABI increased by 24% and reached 1.12. NDS improved within 6 months as compared to baseline. Both NSS and NDS improved, but did not reach statistical significance. No adverse events, oncology cases or visual impairment were observed during the monitoring. The study show that the use of pl-VEGF165 gene transfer in combination therapy allows for complete healing of neuroischemic diabetic foot ulcers in the majority of patients.
: CEDOC, NOVA Medical School, Lisboa, Portugal
Perturbations on the Left-Right axis establishment lead to laterality defects, with frequently associated Congenital Heart Diseases (CHDs). Indeed, in the last decade, it has been reported that the etiology of isolated cases of CHDs or cases of laterality defects with associated CHDs is linked with variants of genes involved in the Nodal signaling pathway. With this in mind, we analyzed a cohort of 38 patients with CHD that can arise from initial perturbations in the formation of the Left-Right axis. Genomic DNA was extracted from buccal epithelial cells, and variants’ screening was performed by PCR and direct sequencing. We identified two CHD patients with a DAND5 heterozygous non-synonymous variant (c.455G>A) in the functional domain of the DAND5 protein (p.R152H), a master regulator of Nodal signaling. A functional Nodal-dependent luciferase assay showed a significant decrease in the activity of p.R152H variant when compared to its wild-type counterpart. Epithelial cells from those patients were used to generate iPS cell lines to study the role of DAND5 in cardiomyocyte proliferation and function. Altogether, our results refined the roles of Cerl2/DAND5 in the regulation of the signaling mechanism(s) underlying cardiomyocyte proliferation in vitro and in vivo. At the end, we will better understand the disease mechanism of patients with a mutation in DAND5 and similar pathologies aiming to find possible therapies.
: Cardiology Division & Experimental Cardiology Lab, Fondazione IRCCS Policlinico San Matteo, Pavia
In view of a potential therapeutic use of autologous bone marrow-derived mesenchymal stromal cells (BM-MSC), we investigated whether patients’ left ventricular dysfunction may affect cell growth, phenotype and functionality. Marrow aspirates were collected during cardiothoracic surgery from subjects with left ventricular ejection fraction (LVEF) <40% (Chronic Heart Failure = CHF) and LVEF >50% (NON-CHF) or during orthopedic surgery (controls = CTR). The corresponding BM-MSC were cultured in alpha MEM containing 10% FBS and characterized at passage 4; growth curves were then evaluated up to passage 8. BM-MSC cultures could be generated by all three groups of subjects: CHF (n = 10), non-CHF (n = 14) and CTR (n = 10). All the cultures displayed the typical MSC morphology and surface phenotype (CD90/CD73/CD105 positive, CD14/CD20/CD34/CD45/HLA-DR negative). Of notice, cultures from CHF patients showed reduced growth rate in respect to non-CHF patients and CTR (doubling time: CHF 206 ± 106 hours, NON-CHF 171 ± 115, CTR 122 ± 48; T-test CHF vs CTR p < 0.05); similar data were obtained after cell freezing and thawing. Conditioned media (CM) from CHF cultures showed reduced cytoprotective activity on neonatal rat cardiomyocytes in vitro, compared to CM from NON-CHF (assays: cell viability, p < 0.01; apoptosis, p < 0.05; cytotoxicity p < 0.01); comparison with CTR cultures is ongoing. In conclusion, BM-MSC from CHF patients are similar to those from NON-CHF and CTR in terms of morphology and immunophenotype, but show slower growth. Functional in vitro results suggest that ventricular dysfunction may adversely affect the cytoprotective properties of BM-MSC.
: Mayo Clinic
Hypertrophic cardiomyopathy (HCM) is the most common familial heart disease, with the prevalence between 0.2 to 0.5 %, characterized by an increase in left ventricular (LV) mass with thickening of the interventricular septum (IVS), interstitial fibrosis, and sudden death. The cardiac hormone, B-type natriuretic peptide (BNP) is a guanylyl cyclase A (GC-A) agonist. BNP shows direct cardioprotective properties including anti-hypertrophic and anti-fibrotic effects. We have recently found that genetic BNP ablation results in progressive cardiac hypertrophy with substantial upregulations in HCM-associated genes in rats. To test our hypothesis that sustained BNP treatment may preserve cardiac function and structure in HCM, here, we assessed the influence of long-term BNP therapy in a mouse model of HCM, expressing a mutated α-myosin heavy chain transgene. Adeno-associated virus serotype 9 (AAV9) vectors were utilized to over-express BNP. At 6, 9, 12, 15 and 25 weeks after AAV vector administration, left ventricular mass by echo was significantly higher in the untreated HCM group than the AAV-BNP-treated HCM and wildtype C57bl/6 mice. Significant increases in IVS and posterior wall thickness were also observed in control HCM group. At 11, 19, and 25 weeks after AAV vector administration, exercise capacity, monitored by treadmill tolerance, was significantly lower in the control HCM group than the AAV-BNP-treated HCM mice. HCM AAV9-BNP treated mice showed a trend towards a lower blood pressure than both C57bl/6 and HCM mice. Our data demonstrate that AAV9-mediated BNP over-expression blocks progressive LV enlargement and reduces exercise capacity in a mouse model of HCM.
: International Centre for Genetic Engineering and Biotechnology ICGEB)
Loss of functional cardiomyocytes is a major determinant of heart failure after myocardial infarction. Through a genome-wide microRNA (miRNA) screening, we identified a few human miRNAs capable of promoting neonatal and adult cardiomyocyte proliferation and cardiac regeneration in small and large animal models. Analysis of the transcriptional profile of miRNA-treated cardiomyocytes revealed an involvement of the Hippo-Yap pathway, which is known to regulate cell proliferation during development. Experimentally, we found that 9 out of the 10 most effective miRNAs in driving cardiomyocytes proliferation also activated Yap-mediated transcription, as concluded from the analysis of Tead gene reporter analysis, increased transcription of Yap responsive genes and nuclear import of active Yap. In particular, miR-199a-3p, one of the most effective miRNAs, directly downregulated at least three mRNA targets impinging on the Hippo pathway, namely the upstream inhibitory kinase Taok1, the E3 ubiquitin ligase destroying Yap, bTrCP, and the regulator of actin cytoskeleton dynamics, Cofilin2. We also discovered that cardiomyocyte treatment with either miR-199a-3p or a siRNA targeting Cofilin2 induced actin polymerization concomitant with cell proliferation. Specific inhibition of F-actin formation strongly decreased Yap activation. Taken together, these results indicate that activation of the Yap transcriptional coactivator in the Hippo pathway and modulation of the acting cytoskeleton are major components of the pro-proliferative effect of miR-199a-3p and other miRNAs inducing cardiomyocyte re-entry into the cell cycle.
: Heart Institute (InCor), University of São Paulo Medical School, São Paulo, Brazil
Urine Stem Cells or Progenitor Cells (UPCs) display high proliferation and multi-differentiation capacities, such as MSCs. However, since UPCs have been recently described, their characteristics remain to be studied. Additionally, despite encouraging studies using fetal renal cells for kidney repair, the potential of adult UPCs in this context remains unknown. We firstly performed large in vitro human UPC characterization using urine samples from around 60 control individuals and patients. Using our simplified UPC isolation protocol, we found that UPC isolation success rate varies among urine samples. By qPCR, immunostaining and flow cytometry, we quantified inter-individual (control and patients) and intra-individually (using clonal growth system) heterogeneity. Results showed that polyclonal or monoclonal UPC lines are highly variable in terms of proliferation capacity, spontaneous differentiation state and expression of renal tubular markers. Additionally, we observed various UPC stemness profiles (CD24, CD133, SSEA4 and TRA1-81 expression) that we are linking with susceptibility to differentiation and/or reprogramming into hiPSCs. Furthermore, we injected control UPCs seeded on 3D-nanofibers scaffolds (biocompatible and biodegradable) in rats, and found that UPCs were still present at least 2 weeks after renal subcapsular transplantation. We are currently testing UPCs potential benefits in a non-invasive rat model of chronic kidney disease (high sodium-oxalate diet) characterized by glomerular filtration rate reduction (60%), increased blood urea nitrogen and tubular atrophy. Altogether, these results highlight that “UPCs”, besides being a non-invasive and unlimited cell source, is a heterogenous cell type, and this may have to be considered to design cell therapy strategies.
: Celixir Limited
Immuno-modulatory progenitor cells (iMP cells), are a novel and distinct mesodermal progenitor cell that do not meet the International Society for Cellular Therapy mesenchymal stromal cell (MSC) definition, but do exhibit an immune-modulatory (MIC A/B, CD178, CD289, CD99 and EGF-R) and cardiac specific phenotype (CD181, CD126, CD304, CD363 and CD182). We previously reported the completion in 2014 of a phase II (Heartcel) clinical trial of advanced heart failure patients incompletely re-vascularized (ICR) by Coronary Artery Bypass Graft (CABG). Concomitant with CABG, iMP cells were injected intra-myocardially into the areas of hypo-kinetic myocardium bypass which would not re-vascularise. SPECT imaging was used pre-operatively to identify these iMP injection sites and post-operatively to monitor/measure change in viability and contractility. The study met all endpoints: 1-year MACE-Free survival in all patients (18–24 months) and clinically and statistically significant mean improvement in LVEF (30%), LV scar size (40%) and quality of life (50%). We are now pleased to report that all patients remain alive to date (100% MACE-Free survival 34–44 months). These results suggest the potential for in situ myocardial regeneration to mitigate the effect of incomplete revascularization in heart failure and to improve a patient's quality of life in the long term.
: ICGEB
The reasons why a hypoxic tumor forms its own vasculature, mainly through the secretion of the factor Vascular Endothelial Growth Factor (VEGF), whereas an ischemic heart cannot, still remain obscure. The injection of an Adeno-Associated Vector (AAV) encoding for the VEGF165 isoform into either the adult skeletal muscle or the embryonic/neonatal heart resulted a massive formation of capillaries and arterioles, as detected by anti-CD31 and anti-a-SMA immunostaining. However, the adult heart was not responsive to VEGF and only a modest increased in the number of arterioles was observed, with no endothelial cells proliferation. To understand whether the different angiogenic potential of the three organs depending on intrinsic properties of endothelial cells or rather on any inhibitory factor present in the adult heart, we purified endothelial cells from EGFP+ hearts using CD31 magnetic beads. Cardiac endothelial cells contained a sub-population of VEGFR++, CD105++ cells (possibly tip cells) before but not after birth. Neonatal endothelial cells injected in vivo into the skeletal muscle of syngeneic mice were proliferated and formed capillaries after 20 days. However, the same cells stop proliferating and were not present in the adult heart at 1 week, suggesting that the adult heart exhibits anti-angiogenic activity. The expression of a soluble form of VEGFR-1 (sFLT-1), a potent VEGF antagonist, progressively increased in the post-natal heart, at both mRNA and protein level, similar to what happens in the avascular cornea. Collectively, our data support a relevant role of sFLT-1 in inhibiting tip cell formation and angiogenesis in the adult heart.
: University Hospital Heidelberg
Systems to regulate gene expression from Adeno-associated viral (AAV) vectors are widely used. In most cases, the transgene expression has to be switched on by applying a drug. In terms of safety of gene therapy, a shut-off system for AAV vectors would be beneficial to silence gene expression in case of side-effects, ideally by destruction of the vector. Therefore, we aimed to develop a system for elimination of gene expression from an AAV vector after systemic injection using an inducible Cre/loxP system that leads to excision of DNA fragments flanked by loxP sites in the presence of tamoxifen. We generated AAV9 vectors consisting of the inducible CreERT2 recombinase, a luciferase reporter gene, and loxP sites inserted at different positions within the vector genome. Four weeks after intravenous vector injection of 10(12) vg per mouse into adult mice, tamoxifen was administered intraperitoneally (1 mg daily, for 5 days) resulting in an up to 10-fold decrease in luciferase activity in heart samples. We further found out that a second tamoxifen administration for another 5 days led to an up to 26-fold reduction in luciferase levels. However, overall expression levels were reduced by insertion of loxP sites (between 2.4 and 5.4-fold). Taken together, delivery of an inducible CreERT2 allows efficient inactivation of AAV-mediated gene expression on the expense of reduced overall expression levels due to insertion of loxP sites. Our results contribute to the generation of a novel shut-off system for AAV-mediated gene transfer applicable for the use of various promoters and serotypes.
: Human Stem Cells Institute, Moscow, Russia
A plasmid VEGF65 (pl-VEGF165) gene therapy drug was approved in Russia for the treatment of atherosclerotic peripheral arterial disease (PAD) after an randomized, controlled, multicenter clinical study in 2011 that enrolled 100 patients with limb ischemia stage II-III according to the Fontaine-Pokrovsky classification. 40 patients gave their consent to participate in the long-term 5-year follow-up study. By the end of the 5th year the value of PWD increased in pl-VEGF165 patients (n = 28) by 297.5m (278%) as compared to baseline (from 107.5 ± 14.2m to 405 ± 47.9m, respectively; p < 0.01). The largest increase in the PWD value by 200.3 m (186%) was observed at the end of the first year. The opposite results were obtained in the control group (n = 12): PWD decreased by 25.0 m (22%) during the follow-up period as compared to baseline. ABI in the test group increased by 6% (p > 0.05) and decreased in the control group by 17% (p < 0.01). TcPo2 value in the pl-VEGF165 group increased by 18 mmHg (24%, p < 0.01) within the 1st year and slightly decreased during the next 4 years by 9%. No AE, SAE, or significant laboratory abnormalities were observed in either study group during follow-up period. Limb salvage rates at 5 years were 91.7% in the test group and 67% in the control group. Thus, pl-VEGF165 intramuscular gene transfer is an effective and safe method of treatment of moderate to severe claudication due to chronic lower limb ischemia. The therapeutic effect of pl-VEGF165 retained during 5 years and allow significantly improve functional status of patients.
: Chung Ang University College of Medicine
: Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, P.O. Box: 19395-4644, Tehran, Iran
Systemic scleroderma (SSc) is a complex autoimmune disease with multi-organ targets resulting in high mortality and morbidity. The common manifestations of this disease include vascular disturbance, immune response and extensive fibrosis of the skin and internal organs. However, pulmonary and cardiac diseases have been recently reported as the primary cause of death in scleroderma. Primary cardiac phenotype may involve myocardial damage, fibrosis of the conduction system or pericardial/valvular diseases. It has been suggested that primary myocardial involvement might result from vasculopathy-induced ischemia. However, the underlying mechanism is not fully understood. In the current study we aimed to use cardiomyocytes differentiated from SSc-specific induced pluripotent stem cells as an in vitro model to study molecular, cellular and functional SSc cardiac phenotype. Skin biopsies from patients and healthy individuals were collected and induced pluripotent stem cells (iPSC) were generated using Yamanaka factors. The iPS cells exhibited pluripotency markers in immunocytochemistry, flow cytometry and RT-PCR experiments. The functional properties of multicellular cardiomyocyte colonies was evaluated using multi-electrode array. Beating frequency was measured as 65 ± 10 And 72 ± 10 In SSc and control cardiomyocyte colonies, respectively. Action potential duration was significantly prolonged in SSc as reflected in longer APD50 measured in whole cell patch-clamp experiments. Moreover, the incidence of EAD and DAD was higher in SSc compared to control. Relative expression of triadin, a component of Ca2+ handling machinery, was significantly enhanced in SSc cardiomyocytes compared to control. These analyses suggests that SSc cardiomyocytes might innately possess some electrophysiological properties that infer to whole heart phenotype.
: CABD, CSIC, Universidad Pablo de Olavide
Mouse foetal liver (FL) hemato/vascular cells from day 12 of gestation (E12), expressing the Stem Cell Leukaemia (SCL) gene enhancer transgene (SCL-PLAP+ cells), had robust endothelial engraftment potential when transferred to the blood stream of newborns or adult conditioned recipients, compared to the scarce vascular contribution of adult bone marrow (BM) cells. We present data showing that long-term reconstituting endothelial cell (LTR-EC) activity is restricted to the SCL-PLAP+VE-cadherin+CD45- (P+V+C-) cell population, devoid of hematopoietic reconstitution activity and largely composed by Lyve1+ endothelial-committed cells. P+V+C- cells contributed to the liver sinusoidal endothelium and also to the heart, kidney and lung microvasculature. LTR-EC activity was detected at different stages of FL development, yet marginal activity was identified in the adult liver, revealing unknown functional differences between foetal and adult liver endothelial/endothelial progenitors. Importantly, the observations that expanding donor-derived vascular grafts preferentially co-localize with proliferating hepatocyte-like cells and participate in the general circulation, support their functional integration in young livers. These findings offer new insights into the engraftment, phonotypical, and developmental characterization of a novel endothelial/endothelial progenitor cell subtype with multi-organ LTR-EC activity, potentially instrumental for the treatment/genetic correction of vascular diseases.
: Russian Cardiology Research and Production Complex
Technology of cell sheet (CS) engineering resulted in significant advance in regenerative medicine in experimental and clinical development. Cell sheets comprise of viable cells along with extracellular matrix and can be used as a minimal construct for transplantation of cells and tissue repair. Furthermore, cells within CS can be modified by viral transduction, which is a feasible and effective way to “tune up” their therapeutic potential. Our group has extensively developed CS from adult mammalian stem cells focusing on mesenchymal stromal (MSC) and cardiac stem cells (CSC) using CS assembly without thermoresponsive dishes and viral expression of growth factors. Our findings in hindlimb ischemia model supported the concept of CS being superior to dispersed cells injection due to higher survival of cells. Furthermore, we established a method for viral expression of VEGF165 in CS and found them to exceed control CS or injected cells in angiogenesis and tissue protection. Another developed application was CS delivery of MSC or CSC to infarcted myocardium in animal models where we found them to significantly induce angiogenesis, positively influence ventricular morphology and reduce fibrosis giving them a prospect for cardiac plastics during surgical procedures. Furthermore, we addressed the “fate” of transplanted CS and found them to interact with the host undergoing vascularization and certain extent of proliferation in skeletal muscle and myocardium. Finally, we attempted to generate decellularized CS to study the role of matrix components in regenerative functions and discuss probable application of CS object for study of cellular interactions and tissue formation.
: Stem Cell and Molecular Biology Lab, Bhupat and jyoti Mehta School of Biosciences, Department of Biotechnology, Indian Institute of Technology Madras, Chennai-36. Tamil Nadu, India
Epigenetics is postulated to be a central regulatory network which organizes stem cell linage commitment and cellular differentiation. The ability of stem cells to self-renew and differentiate into downstream lineages is dependent on specialized chromatin signatures that keep stem cell-specific genes active and key differentiation factors repressed but poised for activation. Recent studies suggested that epigenetic regulation plays a vital role in modulating stem cell differentiation and maintaining the expression of specific genes for specific cell types. However, the knowledge about the epigenetic regulatory mechanism with respect to DNA methylation profiles in differentiation of stem cells toward cardiac lineage is not fully studied. Hence, the study was aimed to characterize the DNA methylation profiles of cardiac specific genes like GATA4, NKX-2.5, MEF2C, TBX5, HAND2, SERCA, Connexin 43, MYL6, and MYL7 in native and induced state. Further, methylation data has been validated with qRT-PCR study and Western analysis. Nevertheless, more studies are needed to understand the molecular events that control the mechanism of differentiation via epigenetically and probing the mechanism culminates in the identification of suitable cell types which has the potential to become functional cardiomyocytes, and that can be utilized for transplantation, and various pre-cardio toxic pharmacological studies.
: Kazan (Volga region) Federal University. Kazan, 420008, Russia
Prevalence of peripheral arterial disease has been increasing over decades, that stimulates interest to indirect revascularization methods. Cell and gene-based therapeutic angiogenesis showed controversial results, while combination of both approaches may enhance therapeutic effect. The aim of our project was to study influence of transduction with Ad5-VEGF165, Ad5-FGF2 and Ad5-EGFP on survival and differentiation of adipose-derived mesenchymal stem cells (ADMSC) in rat hindlimb ischemia model. ADMSC were isolated from adipose tissue of Wistar rats by collagenase digestion. Cells of the 3rd passage were transduced with combination of Ad5-EGFP, Ad5-VEGF165 and Ad5-FGF2. Expression of vegf165 and fgf2 was analyzed by qRT-PCR compared to native ADMSC on the 7th day after transduction. Hindlimb ischemia model was performed on Wistar rats by hindlimb vessels ligation. On the 7th day after the second stage of the operation gastrocnemius muscle was injected by 2*106 of ADMSC transduced with combination of Ad5-EGFP, Ad5-VEGF165 and Ad5-FGF2 or Ad5-EGFP only, diluted in 200 μl of saline. The calf muscles of the operated and intact limbs were taken on the 3, 7, 14, 21, 28th days after injection. Paraffin sections were stained with H&E, immunohistochemically with antibodies to EGFP and CD31. RT-PCR analysis showed 2.58*103-fold vegf165 and 2.51*106-fold fgf2 increased expression in genetically modified ADMSC. EGFP immunostaining demonstrated better survival of VEGF165-FGF2-EGFP-ADMSC compared to EGFP-ADMSC (21 days vs 7 days respectively) and increased capillary-to-muscle ratio in ischemic gastrocnemius muscle 28 days after intramuscular injection (3.67 ± 1.25 vs 2.15 ± 0.80 respectively). This work was funded by a Russian Science Foundation grant (14-15-00916).
: University of Heidelberg
An overexpression of immunomodulatory therapeutic genes in Duchenne muscular dystrophy (DMD) seems to be a promising therapeutic option. Interleukin 10 (IL-10) appears to be a promising candidate for a gene therapy approach to treat dystrophin-associated cardiomyopathy since IL-10 modulates macrophage function which plays a central role in inflammation processes in muscle tissue of Duchenne mouse models (mdx). Aim of this study was to test whether an adeno-associated virus (AAV)-mediated cardiac overexpression of IL-10 may delay or even prevent the development of a cardiomyopathy in a mdx mouse model. Therefore 10(12) particles of an AAV9 vector containing IL-10 transgene under transcriptional control of a CMVenh-MLC0.26 promoter (AAV9/IL-10) were injected through the tail vein into 2 months old male mdx mice and wild-type mice (C57Bl/6NRj). Two weeks after vector application treated mice started to lose weight compared to the untreated control group. Five weeks after application, mice were euthanized for animal welfare reasons. In both mouse models a systemic inflammation response was found, but the affected organs differed in dystrophic and wild-type mice. While heart and skeletal muscle tissue of mdx mice showed severe inflammation in histological staining, wild-type mice revealed comparably minor inflammation in muscle, but rather in pancreas and liver. Systemic expression pattern of inflammatory cytokines was different in both models. In summary it can be said that an IL-10 gene transfer in this mouse models is not as beneficial as shown in other treatment approaches and thus care should be taken when considering immune modulating strategies in DMD patients.
: Kazan (Volga region) Federal University, Kazan, Russia
The therapeutic effect of gene therapy of the peripheral arterial disease with vegf165 gene shown in experimental models remains for a short time and demands reinjection of a plasmid. We assume that the combination of vegf165 and sdf1a will facilitate therapeutic effect of gene therapy by induction of necessary cellular resources recruitment in the neoangiogenesis area. The model of hindlimb ischemia was performed on Wistar rats by two stages of hindlimb vessels ligation. Gastrocnemius muscle was injected by the 200 μg pBUDK-coVEGF165-coSDF1 plasmid diluted in 200 μl of saline or by 200 μl of saline only. The calf muscles of the operated and intact limbs were taken on the 3, 7, 14, 21, 28 days after plasmid injection. Analysis of the vegf165 and sdf1a expression was performed using the qRT-PCR. Paraffin sections were stained with H&E, Mallory trichrome, immunohistochemically with antibodies to CD31, fast/slow myosin heavy chains, Ki-67. Expression of vegf165 and sdf1a was 50–100-fold and 10000–12000-fold increased on the 7th and 14th day after injection in ischemic limb compared with that in the intact limb. The effect of plasmid demonstrated increase in capillary density (2.11 ± 0.55-fold on the 28th day after injection), the earlier regeneration and return of the original biochemical profile of damaged muscle (14th vs 28th day after injection in control), higher rate of endothelium proliferation. Combination of vegf165 and sdf1a genes allows also to decrease the speed of fibrogenesis (fibrosis area by day 28 9.15 ± 7.78% vs 11.89 ± 5.53% in control). This work was funded by a Russian Science Foundation grant (14-15-00916).
: GlaxoSmithKline - Cell and Gene Therapy
Development of packaging and producer cell lines for production of lentiviral vectors has always been a labour-intensive and lengthy process. Improved transcription of self-inactivating vectors leading to high titers has been attempted in different ways with the intent to find a high stable producer clone. In this project, we studied the use of lentiviral vectors as a tool to target and identify high-transcribing loci in the genome of our host cells for lentivitral packaging cell line development. Third generation lentiviral vectors carrying eGFP under the control of an endogenous clinically-tested promoter (short EF1α) were produced, containing a variable DNA sequence tag (barcode) in their long terminal repeat (LTR). The aim of the barcode is to uniquely tag, identify and track a particular clone within the heterologous expressing population. Human embryonic kidney cell lines (HEK-293) were transduced with a barcoded lentiviral library at a low multiplicity of infection. We demonstrated that integration site analysis and next-generation sequencing of lentiviral barcoded vector junctions by ligation-mediated PCR (LM-PCR) coupled with RNA-Seq allows for quantification of the relative abundance of each barcode variant in each specific genomic position. Expression cassettes containing lentiviral vector components were then site-specifically integrated into these genomes sites using the CRISPR-Cas9 technology. The barcoded lentiviral system allows for rapid and high resolution and throughput screening of gene expression in a large number of genomic positions naturally targeted for optimal vector expression but also of lower expressing sites in order to meet lentiviral cytotoxicity and stoichiometric constraints.
: Hanyang University
Adiponectin (APN) is a secreted protein with many biological functions from the adipose tissue. It has been reported that APN has an anti-inflammatory and cytoprotective effects. Thus, APN may have a positive effect in the acute lung injury (ALI) animal model. In this study, dexamethasone conjugated PEI2k (PEI2k-Dexa) and dexamethasone conjugated PAMAM (PAM-Dexa) were evaluated as a carrier of the APN gene in an animal model of lipopolysaccharide (LPS) ALI. Gel retardation assays showed that PEI2k-Dexa and PAM-Dexa formed stable complexes with plasmid DNAs (pDNAs). In vitro transfection to the L2 lung epithelial cells, PAM-Dexa showed higher transfection efficiency and lower cytotoxicity than PEI2k-Dexa. In the in vivo delivery of the luciferase plasmid into the lungs by intratracheal injection, PAM-Dexa had higher DNA delivery efficiency than PEI2k-Dexa in the lung. According to the results, PAM-Dexa was used as a carrier of the APN gene in the ALI animal model. The PAM-Dexa/the APN plasmid (pAPN) complexes were administered to mice via intratracheal injection. The results showed that the PAM-Dexa/pAPN complex decreased TNF-α, IL-6 and IL-1β in the lung tissue and bronchoalveolar lavage (BAL) fluid. These results suggest that delivery of the APN gene into the ALI animal model reduced the inflammation in the lungs. Therefore, delivery of pAPN using PAM-Dexa may be useful for the treatment of ALI.
: UCL
Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disease caused by mutations in the dystrophin gene. Several experimental therapies are under investigation. Among these, gene and cell therapy is promising but complex (e.g. large size of dystrophin gene). The combination of both Human Artificial Chromosome-based gene correction and iPS cell-derived transplantable myogenic cells has been pioneered in our lab and can overcome some major hurdles. However safer strategies to translate these technologies into clinical trials still need to be developed. This projects aims at generating safe transplantable DMD iPS cell-derived myogenic cells genetically corrected with episomal HACs containing the entire human dystrophin locus. First, DMD iPS cells have been generated using genomic integration-free strategies (e.g. Sendai virus and mRNAs). They have been subsequently differentiated into expandable cells similar to skeletal muscle pericyte-derived mesoangioblasts, which have the advantage over myoblasts to be systemically deliverable. Finally DMD iPS cell-derived mesoangioblast-like cells have been induced to terminal myogenic differentiation using integration-free myogenesis regulators (e.g. MyoD mRNA). Additionally a novel HAC capable to provide genetic correction and MyoD-mediated differentiation without genomic integration was also developed. These data provide the foundation for a new, safer iPS cell and HAC-based approach for ex vivo gene therapy of DMD.
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: GENyO- Centro de Genomica e Investigacion Oncologica: Pfizer/Universidad de Granada/Junta de Andalucia
Integrative deficient lentiviral vectors (IDLV) have been described as an effective platform for transgene expression in quiescent cells and also in actively dividing cells. IDLV show several advantages compared with other delivery methods, including their capacity to transduce virtually all mammalian cells types and their low cytotoxicity. In counterpart, both titer and expression levels of IDLV are significantly reduced compared to their integrative counterpart. This observation is, in part, attributed to epigenetic transcripitional silencing via histone-deacetylation (HDAC). In this regard we have previously reported that the inclusion of chimeric chromatin insulator (IS2) based on the chicken β-globin locus control region hypersensitive site 4 (HS4) and synthetic scaffold/matrix attachment regions (SAR2) in the LV backbone enhances expression level in cell lines including pluripotent stem cells. In the presents study we measured vector titers and expression levels of IDLV harboring IS2, HS4 and WPRE on K562 cells, Neural Progenitor Cells (NPCs) and induced Pluripotent Stem Cells (iPS). Our results indicate that the inclusion of our insulators enhances the relative titer and expression levels in all cell types. Additionally, our results suggest that the insulators enhance the transgene expression level via HDAC-independent mechanism. Finally, we investigated if the inclusion of the different insulators improves the properties of the IDLV as a delivery vehicle for CRISPR-CAS9 nucleases. Our preliminary data showed that the IDLV-IS2-Cas9-gRNA achieved higher expression level of Cas9 proteins and higher levels of gene disruption. Therefore, we propose the IDLV–IS2 as suitable platform for transient expression of these genome editing technologies.
: Nature Technology Corporation
The bacterial backbone spacer region (SR) flanking the transgene expression cassette within a canonical plasmid DNA silences transgene expression following transfection into quiescent tissues such as the liver. DNA vectors such as minicircles that are devoid of the SR, or have SRs less than 1000 bp, are not silenced and can provide 10 times higher sustained levels of transgene expression compared to a canonical plasmid containing the same expression cassette. We report development of novel plasmid vectors with less than 500 bp SRs. These new generation vectors utilize antibiotic-free (AF) selection using the RNA-OUT antisense RNA and have two general designs, either: 1) mini-intronic plasmid (MIP) vectors in which the components required for plasmid propagation in bacteria (bacterial replication origin and RNA-OUT selectable marker) are positioned within an intron of the expression cassette; or 2) NanoplasmidsTM in which the traditional large 1000 bp pUC replication origin is replaced with novel temperature inducible miniorigins. Unlike minicircle vectors, MIP and NanoplasmidTM vectors are compatible with high yield plasmid fermentation processes and standard plasmid production methodologies. Like minicircles, MIP and NanoplasmidTM vectors avoid transgene silencing observed with plasmids after hydrodynamic delivery to the liver. Significantly these vectors have 2–10 times higher levels of transgene expression compared to minicircle vectors. These results suggest improved expression level compared to canonical plasmids is a general property of short SR plasmids. These novel MIP and NanoplasmidTM AF vectors have great potential for enhancing transgene expression level and duration to improve nonviral gene therapy and DNA vaccine performance.
: Vectorologie et Thérapies Anticancéreuses, UMR 8203, CNRS, Univ. Paris-Sud, Gustave Roussy, Université Paris-Saclay, Villejuif, France
In vitro and in vivo gene transfer into cells and tissues using electric pulse application is more and more attractive due to its safety, versatility and efficiency of improved protocols. However, uncertainties remain about which mechanism(s) are actually involved in the DNA penetration during electrotransfer. To learn more about the process(es) implicated for another large molecular object, we have applied electric pulses with plate electrodes during infection of cells in suspension with a recombinant adenovirus (rAd) bearing GFP gene. We observed an important increase of recombinant adenovirus-mediated GFP gene transfer (AMGT) when low voltage (LV) millisecond pulses were applied on cells otherwise poorly infectable with rAd. This effect is proportional to the number and amplitude of the LV pulses applied. It can only be explained by the role played by the high quantities of metallic particles generated from the electrode during this type of pulse. Indeed, if the virus suspension is pulsed separately, and later on added to unpulsed cells, the same increase in AMGT is observed. Moreover, after centrifugation of the pulsed infection solution, the virus was found mainly associated with the metallic particles in the pellet. Preliminary results show that association of rAd capsids with the metallic particle network formed during the pulse application is the key element for the AMGT increase. We are currently investigating further the endocytosis mechanism(s) involved in the enhancement of adenovirus entry into cells under these conditions.
: Ludwig Boltzmann Institute for Experimental and Clinical Traumatology
Efficient delivery, appropriate gene expression and optimal potency of regulatory elements are crucial for gene therapeutic approaches in tissue regeneration. Especially in the field of non-viral gene therapy, several challenges arise when aiming for improvement of plasmid systems in order to compensate for low expression levels or low efficiency of gene transfer. In this work, we present newly developed hybrid vector constructs, suited for overexpressing bone morphogenic protein 2 (BMP-2), while simultaneously down regulating several inhibitory genes of the osteogenic differentiation cascade. This was achieved by designing a double-active plasmid, harboring, aside from a BMP-2 gene cassette, an additional regulatory element with specific pro-osteogenic microRNAs (miRNAs) under a separate promoter, suitable for high expression of bioactive miRNAs. This arrangement was executed in order to enhance the osteoinductive, and thereby potential therapeutic effect in osteogenic regeneration. Several constructs incorporating various miRNAs, showed multiple-fold higher potency to induce osteogenic differentiation of target cell lines and mesenchymal stem cells compared to conventional BMP-2 vectors. By combining the most promising candidates, even higher differentiation rates could be achieved, compensating for low transfection efficiencies, as common in non-viral approaches. Finally, osteoinductive effect of the hybrid vectors was verified in an in vivo ectopic bone mouse model using magnetofection, followed by microtomography quantification. Altogether, we present a hybrid system resulting in highly efficient vectors and demonstrate their impact on osteogenic differentiation, which can be used in viral as well as non-viral gene therapeutic applications, and moreover can easily be transferred to other fields of gene therapy.
: Telethon Institute of Gene Therapy (SR-Tiget)
Hematopoietic stem cell (HSC) gene therapy (GT) applications exploits retroviral vectors, such as HIV-derived lentiviral vectors (LVs), to safely deliver the therapeutic transgene. We recently developed a new bioinformatics method that can handle ISs landing in repetitive elements (∼30% of the overall ISs in GT patients), previously discarded by other tools. Repetitive DNA sequences cover ∼50% of the human genome and are associated with species evolution. The characterization of ISs landing in repeats may add useful results to shed light on viral vector integration mechanisms. In our ongoing clinical trial for metachromatic leukodystrophy with a self-inactivating lentiviral vector we retrieved more than 5 million of ISs from 3 gene therapy patients with a follow up of 18 months after treatment. The analysis of ISs landing in repetitive elements compared with the whole genome distribution in GT patients revealed an enrichment in Alu-SINE repeats (∼23% of total ISs, p < 0.0001). On the other hand, we observed an under-representation of ISs landed in L1-L2 LINE repeats (5% of the ISs versus 22%, p < 0.0001) and in LTR repeats (observed 2.5%, expected 9%, p < 0.0001). We also observed an INT-motif (∼5% of Alu) TG-(N)5-7-CA in the genomic sequences flanking the IS. The distribution of ISs within the prototype Alu sequence showed the highest pick (14.8%) surrounding the A-Box of the RNA-polymerase-III promoter corresponding to the INT-motif. These results may suggest new transcription binding factor sites, beyond the transcriptional co-activator LEDGF/p75 of HIV-integrase, linked to LV integration machinery.
: Department of Ophthalmology, University Hospital RWTH Aachen, 52074 Aachen, Germany
The current treatment of neovascular age-related macular degeneration is based on pharmaceutical applications of vascular endothelial growth factor antibodies. The TargetAMD consortium has developed an alternative non-viral gene therapeutic approach to inhibit choroidal neovascularisation. Autologous pigment epithelial cells will be genetically modified by stably transferring the anti-angiogenic pigment epithelium-derived factor (PEDF) gene. Pre-clinical animal studies verified the safety profile of the vectors used, consisting of the Sleeping Beauty (SB100X) transposon system cloned into miniplasmids free of antibiotic resistance markers (pFAR4). Soft agar colony formation assay and integration site analysis were performed to analyze a possible tumorigenicity of the transfected cells in vitro. Freshly isolated and pre-cultivated iris (IPE) and retinal (RPE) pigment epithelial cells of C57/BL6 mice were transfected using 30 ng of pFAR4-SB100X transposase and 470 ng of pFAR4-PEDF transposon plasmid and seeded on a 2-layer soft agar assay. The capability of anchorage-independent growth was observed for 21 days referring to HeLa cells as positive tumorigenic control. Integration site analysis was performed and compared to a computationally generated random set of genomic integrations. No colony formation was detected for transfected mouse IPE and RPE cells, whereas HeLa cells developed 78.69 ± 10.49 colonies at day 21. Integration profile studies showed a close to random distribution of transposon insertions in the mouse RPE cell genome. Both methods exclude any evidence of tumorigenic potential for Sleeping Beauty transposon-modified pigment epithelial cells. These findings indicate that pFAR4/SB100X-mediated PEDF gene delivery fulfils the safety criteria necessary for the transfer to a clinical application.
: Synpromics Ltd
Synthetic Biology is a relatively new discipline and is firmly established as a means to introduce engineering principles to improve and exploit bio-based processes. The vision is that genetic parts can be standardised and taken “off-the-shelf” to build complex biological systems to improve upon various industrial processes. The key to this vision is being able to identify parts that perform in a predictable fashion in any given biological system, and this remains its greatest challenge. At Synpromics we have developed a systematic way in which to construct synthetic promoters that mediate unprecedented control of gene expression. By adopting complex bioinformatics analysis of functional genomics datasets we are able to create promoters that are active under pre-defined transcription profiles. Here we present data showing the applicability of this technology to cell and gene therapy. We have constructed panels of synthetic promoters driving cell-type specific gene expression in a number of different tissues. We present data from in vitro and in vivo models illustrating improved efficiency and specificity of gene expression. Our results demonstrate the power of employing a synthetic biology approach to better facilitate construct design and enhance the potency of therapeutic expression cassettes.
: Institute for Women's Health, UCL
Germ-line light producing transgenic mice, where luciferase expression is controlled by a surrogate promoter or transcription factor binding elements, are used to provide in vivo readouts of disease processes. However, germ-line transgenics are expensive to generate and maintain. Using a standard Gateway® cloning system we previously established a library of more than 25 lentivirus biosensors which we validated in the context of luciferase-expressing lentiviral vectors. Administration of these vectors to neonatal mice permitted monitoring of signalling pathways by whole body bioluminescence imaging. To achieve even wider transduction we ported this system to the adeno-associated viral (AAV) backbone. In this study, we aimed to obtain preliminary validation of this versatile system by delivering NFκB driving a luciferase reporter construct to the nervous system of neonatal mice to generate somatic-transgenic mice using AAV vectors. AAV8 serotyped vector was injected intracranially to outbred CD1 neonatal (P1) mice and luciferase expression was monitored continually by whole body bioluminescence imaging of conscious mice. Intracranial injection showed a widespread luciferase expression throughout the brain and along the length of the spinal cord. Interestingly, even when we administered AAV8 NFκB biosensor intravenously at P1, luciferase expression was still strongest in the brain and spine. This is surprising since previous studies have shown that AAV8 serotype transduces most organs after neonatal injection. Our observations demonstrate the feasibility of using AAV vectors to generate somatic transgenic light producing transgenic mice. Moreover, they also reveal a surprisingly high degree of NFκB signalling in the nervous system compared with other tissues.
: Mayo Clinic
Through screening of an siRNA library, we recently identified U2 snRNP as a post-entry restriction factor for AAV vector transduction, which occurs after the second-strand synthesis, independently of the cellular splicing machinery. Genetic and pharmacological inhibition of U2 snRNP and associated proteins potently enhanced AAV vector transduction (up to 400-fold). Here, we further studied the underlying mechanism(s). To assess the possible influence of U2 snRNP inhibition on AAV vector transcripts, we determined the abundance and appearance of alternatively spliced transcripts by RNA-seq in AAV vector-infected cells, treated by a U2 snRNP inhibitor Pladienolide B (PladB) or SF3B1-siRNA. Both PladB and SF3B1 siRNA treatments significantly enhanced the abundance of AAV vector transcripts by 170- and 20-fold, respectively. Although some alternatively spliced AAV vector transcripts emerged upon PladB or SF3B1 siRNA treatments, the majority of transcripts remained the same as those found in controls. Thus, U2 snRNP inhibition enhances AAV vector transduction through increased abundance of AAV vector transcripts. We next assessed the epigenetic modifications of the AAV2 genome in the presence or absence of PladB. Through chromosome immunoprecipitation (ChIP) assays we mapped histone recruitment patterns at different regions of the AAV vector epigenome. PladB treatment reproducibly enhanced recruitments of histone H2b and H3 proteins to the AAV CMV promoter, luciferase transgene, and polyA regions up to 3-fold. U2 snRNP inhibition also increased the levels of H3K36me3 on the AAV vector genome. Our results therefore suggest that U2 snRNP proteins inhibit AAV vector transduction through chromatin regulation of AAV vector epigenome.
: University Hospitals of Geneva
The production of a gene therapy medical product under GMP-conditions requires compliant products and devices. The Target AMD project aim is the performance of a phase Ib/IIa clinical trial to treat neovascular age-related macular degeneration (nAMD) by transplanting iris-pigment epithelial cells transfected with the PEDF gene ex vivo using electroporation and the Sleeping Beauty Transposon (SB100X). For the transfection of small cell numbers (5,000–10,000 cells), an electroporation device (Cliniporator®), approved for in vivo gene delivery in humans, has been modified and micro-cuvettes have been developed for high transfection efficiency and cell viability. An electroporation buffer has been developed for plasmid manufacturing and cell transfection under GMP-conditions. Human primary iris (IPE) and retinal (RPE) pigment epithelial cells were transfected with a Venus-encoding plasmid to quantify transfection efficiency. Electroporation has been performed using the newly developed Cliniporator®, buffer and a mixture of two pFAR4-miniplasmids, encoding the transposase SB100X and the gene of interest. Transfection efficiency was increased significantly using the newly developed buffer (IPE 26.9%, RPE 21.9%) compared to commercial buffers (IPE 19.5%, RPE 14.9%). and efficiency was increased to 35.2% using microcuvette developed for the TargetAMD project, selecting appropriate electroporation parameters, volume and plasmid concentrations. The modified electroporation device allows for efficient ex vivo gene transfer to small cell numbers using a newly developed buffer and pFAR4-miniplasmids. The transfection protocol presented here will meet the requirements for GMP-production of the gene therapy medical product necessary to perform the phase 1b/2a clinical trial, which is planned for 2017.
: CNRS
Nucleic acids electrotransfer has been shown to be a safe and efficient non-viral technique in a wide variety of cells (including primary cells) even with large nucleic acids. Using a small 3.5 kbp GFP reporter plasmid we previously obtained electrotransfer efficiency of up to 90%, with around 70% cell viability, in Mesenchymal Stem Cells (MSCs). However, with larger plasmids (about 15 kbp), we observed highly decreased cell viability and electrotransfer efficacy in MSCs. Our results suggest that this could be the consequence of the increased time necessary for larger plasmids to cross the plasma membrane, resulting in a longer permeabilization and a stronger sensibility to the external medium composition (especially calcium). Our results shed light on some of the mechanisms involved in gene electrotransfer as well as provide means to enhance electrotransfer efficacy and cell viability for small and large plasmids.
: Telethon Institute of Gene Therapy (SR-Tiget)
The analysis of the genomic distribution of viral vector genomic integration sites (IS) has a key role in gene therapy applications, allowing to assess both the safety and the efficacy of the treatment and to study the basic aspects of hematopoiesis and stem cell biology. Vector integration sites are identified by high-throughput sequencing of PCR products containing the DNA junction between cellular and integrated proviral genome. Mapping the cellular genomic portion to the reference genome allows to accurately locate IS on the genome. We developed VISPA2 (Vector Integration Site Parallel Analysis, version 2), an improved version of VISPA (Calabria et al., Genome Medicine 2014), optimized in time-space consuming and in IS identification. The bioinformatics pipeline consists of several sequential steps, from raw sequencing reads pre-processing to the creation of the final list of IS with genomic context annotation. The major improvements regard (1) the use of the paired-end read aligner BWA-MEM, (2) a more robust and accurate filter of low quality reads, (3) repetitive elements analysis, and (4) a new intuitive web user interface. Moreover, VISPA2 performances showed increases of ∼2X in terms of memory, ∼6X in HD space and ∼7X time, in test datasets. We measured VISPA2 precision and recall both on simulated and cell line experimental data and compared its results with other available tools (MAVRIC, SeqMap, QuickMap, VISPA) showing best performances. Finally, VISPA2 was successfully used for the IS identification in our gene therapy clinical trials and preclinical studies.
: Fraunhofer IBMT
In the majority of cases, primary and stem cells are targeted in gene and cell therapy. These cells are notorious for being hard-to-transfect and solely efficiently modified by viral vectors. Unfortunately, viral vectors can carry safety risks for patients by promoting severe immune response or activating oncogenes due to the integration into the host genome. A safe but challenging alternative are non-viral nanoparticle-based gene delivery systems. Therefore, a biocompatible, biodegradable nanoparticular delivery system based of human serum albumin (HSA-NP) was developed, optimized and evaluated. In general, these HSA-NP can be loaded with various drugs or nucleic acids. For the establishment of a gene delivery system, plasmid DNA coding for a green fluorescent reporter gene (GFP) was incorporated. To promote cellular uptake of the HSA-NP, they were ligand-functionalized with the peptide TAT. The transfection efficiency as well as cellular uptake, intracellular distribution and gene expression of the differently modified HSA-NP was evaluated in vitro. Flow cytometry analysis and confocal laser scanning microscopy (CLSM) studies revealed efficient binding and uptake of unmodified and TAT-modified HSA-nanoparticles by human mesenchymal stem cells and BJ-fibroblasts. Unmodified HSA-NP led to hardly any gene expression of the GFP. In comparison, TAT-modification remarkably increased GFP expression in HEK293T cells and BJ-fibroblasts. This qualifies ligand-functionalized HSA-NP for further studies with functional genes. In summary, ligand-functionalized HSA nanoparticles represent a promising tool for efficient and safe gene therapy and could open up broad future possibilities and markets.
: Ludwig Boltzmann Institute of Experimental and Clinical Traumatology
Delivery of minicircle DNA allows increased trangene expression and offers great advantages over conventional plasmid DNA. This is due to the lack of bacterial sequences, thereby reduced size, providing safe usage and improved performance for tissue regeneration. To date, the strong osteoinductive potential of bone morphogenetic protein 2 (BMP-2) was hindered through weak gene delivery and brief expression peaks restricting the therapeutic effect. Minicircle DNA is a powerful strategy in terms of enabling elevated and persistent gene expression. We report the application of an innovative technology regarding efficient minicircle DNA biosynthesis with an improved BMP-2 gene for fast manufactory in high purity and amounts appropiate for clinical use. C2C12 cells transfected with BMP-2 minicircle in vitro result in significant elevated expression of osteocalcin, increased alkaline phosphatase (ALP) activity and higher BMP-2 protein amount. In addition, when introduced into human bone marrow stem cells, higher levels of ALP and comparable extend of mineralization can be achieved. The BMP-2 minicircle was also delivered into chinese hamser ovary (CHO) cells showing elevated levels of robust BMP-2 protein production up to at least 3 weeks. Finally, we show that additional proccessing of minicircles improves performance in vitro underlining the importance of maximal purity. Herein, we present a highly bioactive BMP-2 minicircle with the potential to fulfill requirements for clinical translation in the future.
: Genetics & Regenerative Medicine Research Centre, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, 43400 UPM, Malaysia
Transient transgene expression following lentiviral (LV) gene delivery in mammalian cells is a major setback for a successful somatic cells gene therapy. The use of pluripotent cells, which lack of DNA methylation profile, may circumvent this issue. Here, we assessed the duration of Green Fluorescence Protein (GFP) expression from lentivirus driven by either human elongation factor 1α (EF1α) or cytomegalovirus (CMV) promoter in mouse induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). The resultant GFP expressing cells were sorted by FACSAria III, which was then followed by GFP expression time point study for 30 days. Percentage of GFP positive cells and mean fluorescent intensity (MFI) were determined by FACSCanto. Both LV/CMV and LV/EF1α transduced iPSCs exhibited significant GFP expression (80% expressing cells) with persistent level of MFI for up to 30 days and beyond. Surprisingly, ES cells presented lower percentage of GFP expression, with LV/EF1α showing significantly higher MFI when compared to LV/CMV. Functional pluripotency analysis indicated that the cells were able to form good quality Embryoid bodies (EBs) and expressed markers for self-renewal indicating that the cells retained their pluripotency potential through out the study period. The findings of this study could provide an insight for the application of pluripotent stem cells, especially iPSCs, for persistent correction of genetic disorders using gene therapy technology.
: CIEMAT/CIBERER
Hematopoietic stem cells (HSCs) are well-characterized primitive precursor cells that acquire and lose different surface markers during their differentiation. Among the markers that characterize the HSC population there is a consensus indicating that these cells are CD34+ CD38- CD90+ CD45RA-. In this study we have investigated the expression of the β2 integrin, CD18, in HSCs from cord blood samples. Our data show that most of the CD34+ CD38- CD90+ CD45RA- cells were CD18low/-. In addition, colony forming cell assays showed the presence of a high proportion of CFU-GEMM progenitors in the CD18low/- fraction, as compared to the CD18+ population. Additionally, an increased population of cells in the G0 phase of the cell cycle was found in the CD18low/- fraction. Finally, in vivo transplantation studies in NSG mice showed the highest repopulation ability, both in primary and secondary recipients in the CD34+ CD18- population as compared to the CD34+ CD18+ one. All together, these results demonstrate for the first time that primitive hematopoietic repopulating cells have negative or low expression of CD18. This observation will constitute a new approach for the enrichment of HSCs in gene therapy applications.
: San Raffaele Telethon Institute for Gene Therapy (SR-Tiget)
Omenn syndrome (OS), caused by hypomorphic mutations in RAG1 or RAG2 genes, is a severe combined immunodeficiency associated with autoimmunity. Gene therapy (GT) may represent a valid alternative to classical bone marrow (BM) transplantation. We tested the efficacy of lentiviral vector (LV) GT in the murine model of OS (Rag2R229Q/R229Q). OS Lin- cells were transduced with a LV encoding for human RAG2 gene, driven by the ubiquitous chromatin opening element (UCOE), and then injected into irradiated OS recipients. Control mice were transplanted with wild-type (BMT-WT) or untransduced (BMT-OS) Lin- BM cells. We analyzed immune reconstitution in peripheral blood overtime from 6 weeks post GT, observing partial redistribution of immune subsets: appearance of B cells, increase of T lymphocytes and decrease of myeloid cells. In serum, the elevated IgE levels declined to concentration comparable to BMT-WT controls, while IgM, IgA and IgG increased to normal levels, indicating functional B cells. Moreover, B-cell activating factor (BAFF), overexpressed in autoimmunity, decreased to normal values. At sacrifice, B cell progenitors in BM and marginal zone and follicular B cells in spleen of GT mice were present. We observed the reconstitution of intestinal mucosal immunity, with the presence of IgA- and IgG-coated bacteria. Importantly, partial restoration of thymic architecture was visible. T cells developed normally in thymus and spleen, showing polyclonal TCR repertoire. Notably, GT mice positively responded to in vivo challenges with T-dependent or T-independent antigens, demonstrating efficient restoration of immunological functions. Concluding, our results demonstrated the feasibility and efficacy of GT for OS.
: RG Reprogramming and Gene Therapy, Cluster of Excellence REBIRTH
Hereditary pulmonary alveolar proteinosis (herPAP) is a rare lung disease caused by mutations within the α- (CSF2RA) or β-chain (CSF2RB) of the GM-CSF receptor gene, resulting in the inability of alveolar macrophages (Mφ) to clear the alveolar spaces from surfactant material. Given the limited treatment options in herPAP, we evaluated the suitability of murine iPSC-derived Mφ (iPSC-Mφ) as a source for an innovative cell replacement therapy. First, we established an efficient and robust protocol to obtain mature and functional Mφ from healthy murine iPSCs. These Mφ closely resemble their counterparts generated in vitro from bone marrow cells with regard to morphology, surface phenotype and function. We next evaluated the feasibility, safety and clinical benefit of intra-tracheal application of healthy iPSC-Mφ. Following single pulmonary Mφ transplantation (PMT) of 4x106 iPSC-Mφ, specific engraftment was observed in the alveolar spaces for up to 8 weeks in Csf2rb-/- mice. Moreover, cells displayed donor-specific CD45.1 expression and typical Mφ morphology in vivo and upon re-isolation. No teratoma formation or tissue toxicity was detected in the organs of transplanted mice. Most importantly, following PMT a significant improvement of disease parameters such as reduced protein, M-CSF, GM-CSF and surfactant protein-D (SP-D) concentration was shown in the bronchoalveolar lavage fluid. We also observed a decrease in PAS-positive material in lung sections and a reduction in lung opacity in computer tomography scans. Thus, we here introduce iPSC-derived Mφ as an innovative, highly promising source for cell therapy and demonstrate PMT of iPSC-Mφ as an innovative cell therapy for herPAP.
: GENETHON
X-linked severe combined immunodeficiency (SCID-X1, OMIM #300400) is caused by mutations in the interleukin-2 receptor γ-chain gene (IL2RG) resulting in the absence of T- and NK-cell development, and impairment of B-cell function. Previous SCID-X1 clinical trials based on MLV-based retroviral vectors expressing the IL2RG gene successfully restored immunity in most patients, demonstrating the efficacy of the gene therapy despite the occurrence on insertional T leukemias in some patients. To overcome these side effects, we developed a SIN lentiviral-vector carrying a codon-optimized human IL2RG cDNA under the control of the human EF1α-S promoter, and we tested it in a preclinical ex-vivo gene therapy study for SCID-X1 on IL2RG-deficient mice. In the treated mice, a normal level of T, B and NK cells and lymphoid organs (thymus and spleen) were restored, without the occurrence of hematopoietic malignancies. Then, the potential genotoxicity of LV-IL2RG was evaluated in vivo by analyzing viral integration sites in transduced hematopoietic stem/progenitor cells before and after transplantation in treated mice, and in vitro by an IVIM assay. Here we present the results of the extensive insertion site analysis performed on bone marrow, thymus and peripheral blood of individual animals or groups of mice, in order to detect any clonal abnormality or skewing, and any deviation from a normal lentiviral integration pattern. These studies will enable a multicenter phase-I/II/registration clinical trial aimed at establishing the safety and clinical efficacy of LV-mediated gene therapy for SCID-X1.
: UMR_S951, Evry
Intracellular pathways that regulate the efficacy of HIV-1-based lentiviral vectors (LVs) gene transfer into human cells are not entirely defined. Compounds capable of overcoming the restriction factors for hCD34+ hematopoietic stem/progenitor cells (HSPCs) transduction with LVs would ameliorate gene therapy efficacy, reduce toxicity and costs of vectors for clinical trials. Recently, Tat-Beclin1 (TB1), a new autophagy and autosis cell death-inducing peptide was reported to inhibit HIV-1 replication, prompting us to investigate if autophagy or related pathways regulate LV transduction. Contrary to expectations, TB1 at low doses strongly promoted the transduction of human cell lines (up to 10-fold) and of hCD34+ HSPCs (2-fold) with numerous LVs pseudotypes. The clonogenic integrity and differentiation of HSPCs in vitro were not affected by TB1. In vivo, TB1-treated-HSPCs efficiently engrafted in the bone marrow of humanized mice (NSG) enabling human immune system reconstitution comparable to controls. TB1 did not trigger any autophagy on HSPCs as measured by LC3-based assays. In the LV viral cycle, TB1 was found to act on the adhesion and fusion steps, through a molecular mechanism which is independent of extracellular viral aggregation but which may involve the endosomal pathway through the GAPR-1 protein, a TB1 peptide target. In conclusion, our data suggest that beclin-1 regulates the early steps of the HIV-1 life cycle. As a tool, TB1 appears to relieve a newly-identified restriction block for retrovirus-based vectors in human cells.
: San Raffaele Telethon Institute for Gene Therapy (SR-Tiget)
Genetically engineered HSC hold great promise as cell therapy for genetic diseases, with the current limits that ex-vivo engineering may be inefficient and reduce engraftment capacity. We found that PGE2 doubles lentiviral vector (LV) copy number (VCN) in HSPC by acting on early steps of transduction, allowing reduction of culture time while maintaining transduction efficiency with clinical-grade vectors. Shortening ex-vivo culture increased engraftment in NSG mice. Unexpectedly, this was most evident for CD38int progenitors rather than CD34+CD38- HSC. We then optimized a GMP-compatible selection procedure to separate mobilized peripheral blood (mPB) samples into a CD34+CD38- stem and CD38+ progenitor cell fraction, allowing their independent manipulation and dosing. We reached high purity (84+/-7.5% CD34+) and recovery of CD34+CD38- cells (38+/-11%), making their isolation clinically feasible. Bead-selected CD34+CD38- cells showed higher engraftment potential than equivalent numbers of FACS-sorted cells. Single-hit transduction of healthy donor CD34+CD38- cells with a clinical-grade b-globin LV yielded a mean VCN of 5 that was stable over 18 weeks in a xenograft. Co-infusion of unmanipulated (culture-sensitive) CD38+ supporter cells with genetically-engineered CD34+CD38- cells may be a good strategy to accelerate hematologic recovery. We show in co-infusion experiments that, after 3 months, most of these short-term repopulating progenitors were replaced by gene-marked HSPC deriving from CD34+CD38- cells. Finally, we show by limiting-dilution xenotransplantation assay that CD34+CD38- cells from mPB can be expanded ex-vivo in the presence of SR1 and/or UM171, compounds developed for the expansion of cord blood cells, underlining that LT-HSC from mPB can tolerate prolonged ex-vivo culture.
: Institute of Experimental Hematology, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany
Mendelian Susceptibility to Mycobacterial Disease (MSMD) is a rare primary immunodeficiency, characterized by recurrent severe infections caused by otherwise weakly virulent mycobacteria. Specifically, mutations in the interferon-gamma- (IFNγ)-receptor-1 (IFNγR1) gene result in a life-threatening disease phenotype. Here, we introduce a novel hematopoietic stem cell (HSC) gene therapy approach for IFNγR1 deficiency and have designed a SIN lentiviral vector expressing Ifnγr1 from SFFV promoter (Lv.SFFV .Ifnγr1.iGFP). Transduction of murine Ifnγr1-/- HSCs showed Ifnγr1 expression by FACS and qRT-PCR with no abnormalities in clonogenic growth. Moreover, differentiation of transduced cells towards macrophages (MΦ) by M-CSF was normal as determined by morphology on cytospins and surface marker expression of CD11b/CD200R/CD115/CD45/F4/80. When stimulated with IFNγ, corrected MΦ were able to clear IFNγ from the medium comparable to WT cells. Furthermore, Lv.SFFV.Ifnγr1.iGFP-transduced MΦ revealed restored up-regulation of HLA-DR and CD86. IFNγ-dependent T-cell activation demonstrated that WT-MΦ, in contrast to Ifnγr1-/–MΦ, were unable to activate T-cells in the presence of IFNγ and ovalbumin, whereas corrected MΦ induced only slight T-cell proliferation. This observation was accompanied by the induction of indoleamine-2,3-dioxygenase (IDO) in WT and corrected MΦ, suggesting IDO-induced tryptophan depletion from the medium interfering with T-cell proliferation. In addition, IFNγR1 downstream signaling showed restored phosphorylation of STAT1 and induction of iNos and Irf1 upon stimulation with IFNγ in corrected MΦ. Most importantly, corrected MΦ showed significantly improved anti-mycobacterial activity in killing of Mycobacterium avium and Bacille Calmette Guérin (BCG). Thus, we here introduce a novel gene therapy approach for MSMD in the context of Ifnγr1 deficiency.
: San Raffaele Telethon Insitute for Gene Therapy (SR-Tiget)
Shortening ex-vivo culture and lowering vector doses remain a high priority goal for hematopoietic stem and progenitor cell (HSPC) gene therapy. We previously showed that Cyclosporin A (CsA) and Rapamycin (Rapa) significantly improve lentiviral vector (LV) transduction efficiency in long-term repopulating human CD34+ HSPC. We have assessed here their efficacy and safety in clinical ex-vivo culture conditions using bone-marrow (BM)-derived HSPC and two different clinical-grade LVs. Both protocols yielded VCN/human genome comparable to the two-hit gold standard in vitro and in the BM of transplanted mice long-term. The shorter 36-hour ex-vivo culture period per se improved HSPC engraftment as significantly higher percentages of human CD45+ cells were measured in the periphery and in the BM of the mice for all shorter protocols. Interestingly, the highest engraftment levels were observed for HSPC transduced in presence of CsA. In agreement, exposure of HSPC to CsA significantly reduced their proliferation, preserving the quiescent G0 fraction and the more primitive CD34+CD38-CD45RA-CD90+ stem and CD34+CD38-CD45RA-CD90- multipotent progenitors. Importantly, no alterations in the vector integration profiles could be detected between CsA and control transduced HSPC. Limiting dilution experiments are on-going to better determine the potential of CsA to preserve the stem cell compartment during ex vivo gene transfer. Overall, these transduction protocols represent a potential alternative over the current clinical gold standard. In particular, CsA allows improved HSPC preservation and engraftment while achieving efficient and safe transduction with a single LV hit in clinically relevant settings.
: Fred Hutchinson Cancer Research Center
We developed an optimized protocol for lentivirus (LV)-mediated FANCA gene transfer. Our protocol uses BM harvest to collect unmanipulated HSCs and does not include conditioning. Patient 1 was a 22-year old male, a total of 3.2 × 107 CD34+ cells were present in 1.1L of BM, but only 9.4 × 107 total CD34+ cells were successfully isolated owing to dim CD34 expression. LV transduction at 10 infectious units (IU)/cell resulted in 18.4% gene transfer in colony-forming cells. Patient 2 was a 10-year old male, a total of 400mL of BM was collected, which contained a total of 30.6 × 106 CD34+ cells. Based on the very dim expression of CD34 on FA marrow cells and thus poor recovery after CD34 selection, we omitted the CD34 purification and the red blood cell depleted BM product was subjected to LV transduction at 10 IU/cell. Gene transfer was higher with 43% in colony-forming cells. Both patients displayed declining levels of transduced cells in peripheral blood after infusion and have maintained stable blood cell counts since gene therapy. Patient 1 has displayed a stably increased HCT value for >1 year since treatment. These data demonstrate that LV gene therapy in FA patients is safe but suggest that CD34 selection does not allow recovery and maintenance of adequate numbers of CD34+ HSCs. Thus we are currently developing a lineage depletion strategy that would allow us to maximize the recovery and maintenance of FA HSCs with adequate overall cell reduction to permit efficient gene transfer.
: San Raffaele Telethon Institute for Gene Therapy (SR-Tiget)
Mucopolysaccharidosis type-I (MPS-I) is a lysosomal storage disorder caused by the deficiency of alpha-L-iduronidase (IDUA) activity. The available treatments are enzyme-replacement therapy (ERT) and hematopoietic stem cell (HSC) transplantation. An alternative therapeutic option is HSC gene therapy, and preclinical studies performed in naïve-MPS-I mice demonstrated its potentiality. However, many MPS-I patients undergo ERT that induces anti-IDUA IgG in 90% of treated patients, which may jeopardize treatment efficacy. We investigated the impact of experimentally-induced anti-IDUA immunity on HSC gene therapy in the preclinical model of the disease. We injected MPS-I mice with IDUA and adjuvant and we showed a consistent induction of anti-IDUA antibodies and cytotoxic CD8+ T cells. Immunized MPS-I mice were lethally irradiated and transplanted with gene-corrected HSC. After hematopoietic reconstitution, mice were sacrificed to quantify engraftment efficiency and anti-IDUA immunity. We showed that the level of engraftment inversely correlated with the strength of the anti-IDUA response: in this experimental model, 100% of IDUA-immunized mice experienced the complete eradication of transplanted cells, while naïve-MPS-I mice were fully reconstituted with gene-corrected cells. We are currently evaluating the relative impact of humoral or cellular anti-IDUA responses on engraftment of gene-corrected HSC. Overall these data demonstrate, for the first time, the relevance of pre-existing anti-transgene immunity on ex vivo gene therapy and illustrate the potential need to adapt the immunosuppressive component of the conditioning regimen in future gene therapy trials for MPS-I.
: Kings College London
: Généthon
Sickle cell disease is an inherited blood disorder caused by a single amino acid substitution in the β-globin chain. Gene therapy could be a therapeutic alternative, particularly in patients lacking an allogeneic bone marrow donor. Designing an efficient vector that combines high-level globin expression with high infectivity remains a formidable challenge. We directly compared two anti-sickling lentiviral vector carrying a modified β-globin transgene (AS3): the Lenti/βAS3-FB (Romero, Urbinati, JCI 2013), and Globe_AS3 (Miccio, PNAS 2008). Comparable titers on HT-29 cells (2×10^8 TU/ml) and p24 values (6×10^4 ng/ml) were obtained for the two vectors. BM CD34+ cells from healthy donors or SCD patients were transduced to an average VCN of 1 and 3 with Lenti/βAS3-FB and Globe_AS3 respectively, (%PCR+ CFU average 35% and 69%, respectively). The higher transduction efficiency for the Globe_AS3 vector was also maintained in vivo in transplantation studies using the NSG mouse model (average VCN 0.3 vs. 0.6, in BM). Despite a higher transduction efficiency, βAS3-globin transgene expression per vector copy was 15–20% lower from Globe_AS3 than by Lenti/βAS3-FB in human erythrocytes, both in vitro and in RBC differentiated from the in vivo NSG mouse model. In addition, we will present data on in vivo repopulation activity in NSG mice and vector integration profiles, as well as comparison of globin expression and hematologic correction of the SCD phenotype in an in vivo SCD mouse model. This study provides a comprehensive analysis of two globin lentiviral vectors, useful to determine the optimal candidate for gene therapy of SCD.
: University Vita-Salute San Raffaele, Milan, Italy
Clinical application of lentiviral vector (LV)-based hematopoietic stem and progenitor cells (HSPC) gene therapy is rapidly becoming a reality. Nevertheless, LV-induced signaling and its potential functional consequences on HSPC biology remain poorly understood. We have performed a transcriptome analysis on human HSPC exposed to VSV-g pseudotyped LV at a high multiplicity of infection, matching current clinical vector dose requirements. We unravel a remarkably limited impact of LV on the HSPC transcriptome. LV efficiently escaped innate immune sensing that instead led to robust IFN responses upon transduction with a gamma-retroviral vector. However, LV transduction did trigger DNA damage responses in human HSPC. In particular, p53 signaling was among the most significantly altered pathways (p < 1.03×10-12) and induction of several key players, including an 8-fold increase in p21 mRNA, was further confirmed by Taqman. LV-mediated triggering of p53 depended on efficient nuclear import of reverse-transcribed viral DNA but did not require integration as it occurred also using an integrase-defective LV and with a non-integrating Adeno-associated vector. Functionally speaking, LV-mediated signaling led to a slight delay in HSPC proliferation, increased apoptosis in culture and reduced engraftment capacity in vivo. Blocking LV-induced signaling partially rescued both apoptosis and in vivo engraftment, highlighting a novel strategy to further dampen the impact of ex vivo gene transfer on HSPC. Overall, our results shed light on viral vector sensing in HSPC and provide critical insight for the development of more stealth gene therapy strategies.
: CIRI, INSERM U1111, Lyon, France
T cells represent valuable tools for treatment of cancers, infectious and inherited diseases. T-cell based therapies would strongly benefit from gene transfer into live-long persisting T cells. Therefore, we compared the gene transfer efficiency into more immature naive T cell with a new lentiviral vector (LV) pseudotyped with the baboon retrovirus envelope (BaEV) to other LV pseudotypes such as VSV-G-LVs. These BaEV-LVs outperformed other LV pseudotypes by far for transduction (80–90%) of naïve adult T cells and cord blood recent thymic emigrants upon IL-7 stimulation, allowing to conserve their naive phenotype. Equivalent transduction levels were revealed for BaEV-LVs in freshly isolated human early thymic progenitors (ETP), progenitor and pre T cells reaching again 80% transduction. Therefore we evaluated BaEV-LV for transduction of ETPs and progenitor T cells, differentiated from CD34+-cells by culture on the notch Ligand, DL-4. Of utmost importance, these ETPs and T progenitors were transduced up to 80–90% and allowed efficient reconstitution of NOD/SCID, gc-/- mice in vivo, maintaining these high transduction levels in all derived T cell lineages. Moreover, they permitted an accelerated T-cell lineage reconstitution as compared to HSCs this mouse model. These results indicate that BaEV-LVs are valuable tools for genetic modification of naïve and early T cell lineages, essential targets for gene therapy application where long-lived T cell persistence is important for durable treatment/correction of patients. Additionally, coinjection of LV-corrected autologous T cell progenitors and HSCs might accelerate T cell reconstitution in patients as compared to solely injecting HSCs avoiding a gap in immune reconstitution.
: UCL Institute of Child Health
Chronic Granulomatous Disease (CGD) is an inherited primary immunodeficiency disorder caused by defective components of the NADPH oxidase, the phagocytic enzyme responsible for pathogen killing. CGD patients are susceptible to recurrent infections and hyper inflammation. A phase I/II clinical trial of lentiviral gene therapy is currently underway for the most common form of the disease, X-linked CGD. We propose to use a similar strategy to tackle p47phox CGD, the second most common form of the disease, caused by mutations in p47phox, a cytosolic component of the NADPH oxidase. To this aim, we have made a lentiviral vector, pCCLChimp47phox, that contains the chimeric cathepsin G/c-fes myeloid promoter and a codon optimized version of the human p47phox gene. We have introduced the lentiviral vector in a myeloid leukemia cell line deficient for p47phox, in primary monocytes-derived macrophages taken from p47phox CGD patients and in a mouse model of p47phox CGD. The results show that the lentiviral gene therapy efficiently restores p47phox expression and NAPDH oxidase function in all models tested. Of note, p47phox-/- mice transplanted with gene corrected stem cells (bearing ∼1 copy of the vector) contained an average of ∼85% functional granulocytes, with levels of oxidase activity comparable to that of the wild type. Overall our study shows that the pCCLChimp47phox vector is a promising tool for the clinical application of p47phoxCGD gene therapy.
: Telethon Institute of Gene Therapy (SR-Tiget)
Chronic Granulomatous Disease (CGD) is caused by defective NADPH oxidase function in phagocytes causing increased susceptibility to fungal and bacterial infections. Gene therapy (GT) with hematopoietic stem cells (HSC) may represent an alternative to conventional transplantation and to this aim we developed a GT approach based on regulated lentiviral vectors (LVs) to target gp91phox expression to the differentiated myeloid compartment while sparing primitive HSC, in order to reduce the risk of genotoxicity. We previously showed that our LVs were able to restore gp91phox expression and function in human XCGD myeloid cell lines, primary monocytes, and differentiated myeloid cells. Importantly, XCGD mice treated with GT were protected from S. aureus pneumonia and related inflammatory responses. We performed detailed immune phenotype analyses of bone marrow (BM) cells from two pediatric XCGD patients and we found a decreased frequency of HSC and an increased frequency of granulocyte-macrophage progenitors (GMP) as compared to pediatric controls. LV transduced BM CD34+ cells from these patients were transduced with LV in the presence of cytokines and PGE2 (vector copy number 0.3-1.4). Transduced CD34+ cells from these XCGD patients were able to engraft in NSG immunodeficient mice showing restored expression of gp91phox. Thirteen weeks after engraftment, BM CD34+ cells from primary NSG mice (Pt1) were then transplanted into secondary 3GS NSG mice (which favour myeloid differentiation). This resulted in more terminally differentiated human myeloid cells and restored gp91phox expression in the GT group. In conclusion, this regulated LV construct represents a promising candidate for further clinical development.
: Bruce Lefroy Centre, Murdoch Childrens Research Institute (MCRI), Parkville VIC, 3052, Australia
Friedreich ataxia (FRDA) is a debilitating neurodegenerative disorder that typically begins around 10 years of age and is characterised by progressive gait ataxia. Affected individuals become wheelchair-dependent within 15 years of disease onset and have a markedly shorter lifespan of approximately 40 years, with cardiomyopathy being the main cause of death. With no current treatment which can cure or slow the neurodegeneration inherent to FRDA, treatments that slow FRDA disease progression are urgently needed. FRDA is most often caused by homozygous GAA repeat expansions in intron 1 of FXN. As a GAA expansion decreases the encoded frataxin protein, but does not alter the protein sequence or structure, increasing frataxin will likely be beneficial. Using cell and animal models, this project aims to increase frataxin to alleviate the FRDA phenotype via cell and gene therapy. Lethally irradiated FRDA mice have been transplanted with either GFP-positive wild-type bone marrow (BM; corrected) or FRDA BM (non-corrected). Reconstitution of the haematopoietic system demonstrated successful engraftment in corrected and non-corrected mice. In corrected recipients, GFP-positive cells were observed in the spinal cord and dorsal root ganglia (DRG), key tissues of neuropathology. Both DRG neuronal marking, particularly proprioceptive neurons, and frataxin levels increased. Corrected mice also exhibited significant improvement in motor coordination post-bone marrow transplant (BMT). Toward developing autologous gene therapy via BMT, we constructed a lentiviral vector that expresses frataxin. These data together illustrate the corrective potential of BMT to treat FRDA and provide an avenue for delivering therapeutic viral vectors for autologous gene therapy.
: San Raffaele Telethon Institute for Gene Therapy (SR-Tiget)
The analysis of the viral vector genomic integration sites (IS) in hematopoietic stem cell-based (HSC) gene therapy applications supports the assessment of safety and efficacy of the treatment and basic research. In our ongoing clinical trial for metachromatic leukodystrophy (MLD) with a self-inactivating lentiviral vector (LV), we retrieved a polyclonal repertoire of >10.000 vector marked clones for each patient. In terms of safety assessment, clonal abundance analyses showed no sustained clonal dominance in any patient. Statistical analysis of frequently targeted genomic regions indicated that integrations in these regions were the product of an intrinsic LV integration bias rather than of insertional mutagenesis. For long term efficacy assessment, we estimated an average clonal HSC population of ∼10.000 active stem cells from 9 months post treatment. The reconstitution of the hematopoietic system in terms of population diversity showed similar pattern for all patients, with an initial fluctuation period in clonal diversity, 1–9 months, followed by a stabilization period from 12–18 months. These findings suggest HSPC dynamics in terms of cell proliferation and differentiation. Moreover, many clones observed during fluctuations were not recaptured in steady-state, underlying a selection process. Using shared IS among HSPC and differentiated cells, we observed a progressive increase in the relative percentage of IS with multi lineage potential until 12–18 months after transplant that then decreased up to a stabilization at ∼18 months in favor of the increase in lineage committed cells, reminiscent to the recently described mechanism of homeostatic maintenance of hematopoiesis by committed progenitors.
: Department of Chemical and Biomolecular Engineering, Yonsei University
The patterned fibrous matrices are highly valuable for spatially orchestrating hierarchical cellular constructs. Especially for neural tissue engineering, the regulation of cell structures in terms of striated alignment or directional growth of axons is a key for the functional recovery of damaged nervous systems. Therefore, understanding the structural elements of patterned fibrous scaffolds can provide crucial clues for designing advanced tissue engineering scaffolds for the promotion of neural regeneration. In this study, the salt-induced electrospun patterned fiber bundles (i.e., SiEP-bundles) which consist of the longitudinally stacked multiple fibers were successfully fabricated and characterized, and by comparision with the conventionally fabricated 2-dimensional fibrous sheet of randomly oriented fibers and individually aligned fibers, their capabilities of spatially regulating the responses of neural cells, including PC12 cells, chick dorsal root ganglia (DRG), and human neural stem cells (hNSCs) were analyzed. The SiEP- bundles possessed significantly distinctive morphological and topographical characteristics with multi-complexed structures of nano- and micro-scale fibers, rough surfaces, and soft mechanical properties. Furthermore, the SiEP-bundles induced cellular elongations and robust neurite extensions corresponding to the fiber directions. Importantly, the neuronal differentiation of the hNSCs on the topographically rough grooves of the SiEP-bundles were boosted. These findings can offer crucial insights for designing the advanced fibrous scaffolds that can spatially regulate cellular responses and potentially provide powerful strategies for the functional recovery of the damaged nervous system.
: GENyO- Centro de Genomica e Investigacion Oncologica: Pfizer/Universidad de Granada/Junta de Andalucia
Wiskott-Aldrich syndrome(WAS) is an X-linked immunodeficiency caused by mutations in WAS gene which is expressed exclusively in the hematopoietic lineage. Gene therapy (GT) is a promising alternative to treat the disease since recent clinical trials using Lentiviral vectors (LVs) have shown impressive results in terms of immune reconstitution. However, these clinical trials, have not achieved a complete restoration of platelets (Plts) numbers nor the associated bleeding disorder. We hypothesized that, in order to achieve good plts restoration, we need to mimic the expression levels during megacariopoiesis. Consequently, we aimed to study WASP expression pattern during megakaryopoiesis and thrombopoiesis, and to improve the therapeutic LVs to mimic this pattern. We analyzed the WASP expression profile during megakaryopoyesis and thrombopoyesis by promoting megacaryocitic differentiation in immortalized cell lines (K562 and MEG-01) and in hematopoietic stem cells (HSCs). We could observe that WASP expression decrease during megakaryopoyesis in vitro. LVs expressing eGFP under different fragments of the WAS endogenous promoter (only the proximal promoter or a combination of the proximal and the alternative promoters) in the absence or presence of insulators. We could observe that all our lentiviral vectors mimicked the downregulation of WASP expression during megacaryopoiesis with minimal differences between them. Finally, we tested the WW1.6kb (expressing WASP through the proximal promoter, provided by Adrian Thrasher) and the AW (expressing WASP through the proximal and the alternative promoters) LVs on HSCs from a WAS patient. Both LVs mimic the WASP endogeneous pattern but the AW had a slight increased expression on PLts.
: Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
Human embryonic stem cell (hESC)-derived endodermal cells are of interest for the development of cellular therapies to treat disorders such as liver failure and diabetes. The soluble form of activin A (Act) has been widely used as an in vitro inducer of definitive endoderm (DE). However, the localized delivery of differentiation inducers through covalent immobilization may be advantageous for enhancing stability and efficacy of the delivery. Therefore, we have developed a nanofibrous poly (ɛ-caprolactone) substrate, biofunctionalized with Act, for directed differentiation of hESCs into DE. Bioconjugation of Act on nanofibrous meshes was confirmed by enzyme-linked immunosorbent assay (ELISA) and immunostaining. In order to investigate the bioactivity of immobilized Act (iAct), hESCs were cultivated on the Act-conjugated nanofibers for five days. The nanofibers with covalent iAct significantly increased expression levels of the endodermal markers SOX17, FOXA2 and CXCR4, compared to physically adsorbed Act (aAct) or without Act (noAct). In addition, iAct retained its bioactivity after storage at 37°C for five days in the absence of cell seeding. The capability of cultivated cells to generate the DE-derived lineage was evaluated through further differentiation of seeded cells into hepatocyte-like cells (HLCs). Interestingly, the iAct sample showed a higher level of hepatic markers compared to the aAct sample. We also demonstrated that iAct in the presence of soluble Act (sAct) could improve the conventional protocol used to generate HLCs from hESCs. The results of this study can be a step towards creating more stable and defined methods for DE differentiation of hESC.
: Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, 28040 Madrid, Spain
VLA-4 (CD49d/CD29) is an integrin expressed in hematopoietic stem cells (HSCs) and is a key adhesion molecule for the homing of transplanted HSCs in recipient bone marrow. This process is mainly mediated by the interaction of VLA-4 with its ligands, such as fibronectin (FN) in the extracellular matrix and the endothelial VCAM-1. Therefore, modulation of VLA-4 levels may have a major impact on the ability of transplanted HSCs to home into recipient bone marrow. The fibronectin fragment CH-296 (RetroNectin) is widely used in gene therapy protocols to enhance the transduction of HSCs with gamma-retroviral or lentiviral vectors. This molecule facilitates the co-localization of the HSCs with the viral particles through the interaction of the viral particles with the H-domain of RetroNectin, and through the interaction of VLA-4 and VLA-5 expressed in the HSCs with the RetroNectin CS-1 and C-domain sites, respectively. In our experiments we have observed that when cord blood CD34+ cells are pre-stimulated with hematopoietic growth factors in the absence of RetroNectin, an up-regulated expression of VLA-4 takes place. This effect was, however, prevented when RetroNectin-coated plates were used. This effect implied a reduction in the adhesion of pre-stimulated CD34+ cells either to other plate with RetroNectin or to HUVEC cells expressing VCAM-1. Transplantation experiments of cord blood CD34+ cells pre-stimulated in RetroNectin-coated or control BSA-coated plates have been recently conducted to investigate potential effects upon the engraftment of CD34+ cells.
: Novartis Pharma AG
Cell therapy products pose new challenges for analytical quality control (QC) testing strategies when compared to traditional biological product development. Scientific understanding and extensive characterization of the different product attributes which determine safety, purity and potency of the cell therapy product are important to ensure adequate product quality and safety. Here, the approach to developing a comprehensive analytical testing strategy for the allogeneic stem cell therapy HSC835 which is currently in Phase I/II clinical trials is presented. The current analytical control strategy includes monitoring of known critical quality attributes like cell viability and sterility. In addition, several other product characteristics are routinely assessed during manufacturing. Analysis of these data and correlation to clinical efficacy has confirmed the criticality of key attributes such as number of CD34+ cells, but also identified a number of attributes for which the impact is not yet fully clear. For example, the contribution to efficacy of both the committed precursor cells and the non-hematopoietic stem cells (HSC) in the HSC835 product remains only partly understood. Increasing product understanding and larger datasets obtained during ongoing clinical development will help to further determine key product attributes predictive for product safety and clinical efficacy. This knowledge will be then used to further refine critical quality attributes and develop the analytical control strategy for commercial quality control of a cell therapy product.
: Inserm
The entry of lentiviral vectors (LV) into target cells is mediated by cellular receptors which may be regulated through pre-activation steps to facilitate transduction. The low density lipoprotein receptor (LDLR) enables entry of vesicular stomatitis virus glycoprotein G-pseudotyped LV (VSVG-LV). Recent litterature showed that LDLR expression must be induced on human B cells to transduce them efficiently with VSVG-LV. It is not known if this is the case with murine B cells. Human and mouse LDLR are only 78% homologous and co-receptors exist. Also, the effects of various B cell activation protocols on LDLR have not been reported. This prompted us to examine the effects of various B cell-activating cytokines and mitogens on levels of LDLR and transduction in mice. Around 5% of spleen CD19+B220+ cells constitutively expressed low levels of LDLR by FACS. Contrary to humans, murine B cells could be transduced without prestimulation (about 50% of transgene-positive cells after 3 days). Overnight stimulation with either IL-4, PWM, LPS or IL-4+LPS significantly but differentially increased LDLR surface levels on CD19+B220+ cells without changing that of heparan sulfate, another co-receptor of VSVG. The highest levels of LDLR were induced by LPS+IL-4 (60% LDLR++ cells; n = 5 experiments). Pre-activation with LPS+IL-4 enabled the transduction of up to 90% of murine B cells. All treatments considered, there was a positive correlation between levels of LDLR on murine B cells and transduction. This suggests that transduction protocols for VSVG-LV could be optimized based on the modulation of LDLR levels on target cells.
: GlaxoSmithKline
Manufacture of GSK's gene therapy products currently allow for a short shelf life for transduced haematopoietic stem and progenitor cells (HSPCs). Ways of extending this through hypothermic storage or cryopreservation are being investigated. Increased reactive oxygen species are the main cause of cold-induced cell injury. Adding antioxidants to the formulation (0.9% NaCl) may therefore maintain cell viability. Cells were held at 4°C for 7 days, at a concentration of 1×10^6 cells/ml. NaCl alone showed a viability of below 70% after 7 days, and no clonogenic potential after 72 hours. In contrast, 100% foetal bovine serum (FBS), 2 μM 2-methylaminochroman (2-MAC), 25 mM deferoxamine (DFO) and 100 μM 2,6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) all resulted in a viability above 85%. DFO resulted in lower clonogenic potential than NaCl alone; 2-MAC maintained clonogenic potential only up to day 3, while FBS and Trolox maintained clonogenic potential up to 7 days, suggesting that these supplements are able to maintain HSPC viability and function. Cryopreservation causes cell damage through ice formation or hyperosmotic shock. Dimethyl sulfoxide (DMSO) is a commonly used cryoprotectant for HSPCs but is associated with adverse events and cell toxicity. Therefore alternatives were investigated: 1,2-propanediol; FreezIS (proprietary DMSO-free); BamBanker and Stem Cell Banker (proprietary reduced DMSO). Cells were frozen at a concentration of 1×10^6 cells/ml at a rate of −1°C/min, and held at −196°C for 28 days. Only DMSO and Stem Cell Banker were able to maintain HSPC viability and function. These studies suggest that increasing the shelf life of HSPCs is possible through formulation improvements.
: GlaxoSmithKline
The colony forming unit (CFU) assay measures differentiation potential of transduced haematopoietic stem and progenitor cells (HSPCs). However, it requires 10–14 days to obtain results; it is subjective and cannot easily be standardised; and does not measure proliferation. Therefore an alternative characterisation method is desirable. We have evaluated the Hemogenix HALO® assay: an instrument-based, high-throughput alternative to the CFU assay, which relies on the correlation between intracellular ATP (iATP) concentration and proliferative capacity of the cells. iATP is released after cell lysis and acts as a substrate for a luciferin/luciferase reaction which produces measurable bioluminescence. iATP concentration can be calibrated against an external standard, allowing standardisation between assays. Results can be obtained in 5 days. Two assays were tested: the PCAEq assay, which measures ATP in a single cell population; and the PMT assay, which analyses several different stem and progenitor cell populations at a range of cell concentrations, providing additional information about the differentiation potential of the cells. Initial experiments evaluated the HALO-PCAEq in parallel with the CFU assay in bone marrow, mononuclear cells, and CD34+ and CD34- fractions. A strong correlation was found between the two assays. Subsequent experiments evaluated the HALO-PMT assay, using flow cytometry to confirm proliferation of the different cell subsets. Again, a strong correlation between assays was observed. The HALO assay provides a fast, standardised result which correlates well with the results of the CFU assay, suggesting that it could prove a useful alternative for analysing potency of CD34+ cells.
: Yousei University
In the field of central nervous system (CNS) regenerations, the transplantation of neural stem cells (NSCs) is studied to be effective treatment for rescuing and repairing serious disease such as spinal cord injury (SCI) and hypoxic-ischemic encephalopathy (HIE). For increasing the therapeutic effect, our group has been studying the adeno-associated viral vector (AAV) based cell-gene therapy. In this study, the 3-dimensional cell culture system showed the unique environment for hNSCs culture. These culture conditions are provided reinforced intracellular interactions. And this 3D culture conditions showed the increased transduction efficiency and transgene expression with short exposure time. This attractive 3D culture system has high impact as a platform technology for effective cell-gene therapy.
: University of Lausanne
In Zebrafish, Müller glia cells participate to the regeneration of cone photoreceptor when the retina is injured. Such phenomenon was shown to not occur in physiological conditions in the mammalian retina. In the present work, we studied whether proteins of the polycomb group influence the neurogenic and gliogenic capacity of mammalian Müller cells. In Bmi1-KO mice, no gross retina morphology changes were observed. When these mice are crossed with a mouse model of retinal degeneration, the Rd1mouse, Müller glia loose the expression of the p27 tumor suppressor, express the progenitor marker Pax6, start DNA duplication and migrate into the photoreceptor layers. To trace Müller cells, lentiviral vectors (LV), pseudotyped with the Mokola envelope coding for a siRNA against Bmi1 followed by an EGFP sequence, were injected into the eye vitreous of young (PN14) WT and Rd10 retina to specifically target Müller cells. Control groups were transduced with a LV-EGFP. In control groups, around 90% of the EGFP-positive cells presented a Müller cell morphology one week after the injection. In Rd10 retina injected with the Bmi1-siRNA, 90% of the EGFP cells had a photoreceptor morphology. Such observations were obtained only in the peripheral retina where neurogenesis stopped at PN12, and not in the central retina where neuron production stopped earlier. These data suggest that BMI1 transiently controls retina neurogenesis by blocking Pax6 expression and by stimulating p27. The control of this pathway, in combination with other actors, may serve to stimulate Müller cells for retinal regeneration.
: Ospedale San Raffaele, via Olgettina 58, Milan, Italy
Current gene-therapy approaches are limited to loss-of-function recessive disorders to restore gene function. However, these approaches are not applicable to dominant genetic disorders, such as the P23H allele, causative of a dominant form of RP. Specific inactivation of the mutant, but not wild-type is required for an effective disease treatment. We explored the feasibility of this paradigm using a knock-in mice model of RP that faithfully reproduce the human disease both genetically and phenotypically. We designed a CRISPR/Cas9 strategy to specifically inactivate the P23H mutant, while preserving the wild-type allele. Analysis in Cas9 targeted wild-type or RhoP23H/P23H fibroblasts confirmed specific targeting of the mutant allele, but not of wild-type allele. Since the retina is a confined and easily accessible tissue retinal disorders are well suited for gene-therapy approaches. We, therefore, translated this approach in vivo by delivering directly in the Rho+/P23H retinae the CRISPR/Cas9 components by electroporation. Eletroporated cells were marked with GFP to be then FACS-sorted and processed for molecular analysis. Interestingly, direct Sanger sequencing showed that 80–90% of the P23H Rho allele presented indel mutations, among which >70% inactivated the gene product. On contrary, the wild-type Rho allele resulted mostly spared by our treatment maintaining its full functionality. Experiments are in progress to evaluate rescue of rod degeneration by this approach. These results indicate that the CRISPR/Cas9 system can be tailored to exclusively target the mutant gene allele causing a human dominant genetic disorder.
: Institut de la Vision
Human pluripotent stem cells are an unlimited and unique source for cell replacement, tissue engineering and in vitro disease modeling. Given their differentiation capacity they can be used to generate photoreceptors among other retinal phenotypes. Despite the advances in the recent years of numerous groups to obtain mature and functional photoreceptors for cell replacement strategies, up to date no functional outer segments have been well described. Therefore new technologies are necessary to address this issue. Optogenetics uses light to control cells that have been modified to express light sensitive ion channels or pumps. Here, we have developed a protocol of differentiation to obtain mature photoreceptors, which have been genetically engineered to carry a hyperpolarizing chloride pump that renders them responsive to light even in the absence of outer segments. In accordance with recent publications, demonstrating that development can be recapitulated better in 3D conditions, we have generated a 3D retinal organoid system. Our retinal organoids efficiently differentiate towards photoreceptor cells, expressing all the major specific markers for this population such as: RCVN, CAR, OTX2 and CRX. Furthermore they can be modified to express a light-sensitive protein at their membrane, a pump hyperpolarizing the cells upon light stimulation. The colocalization studies demonstrated this was specific of cones. Using a reporter system we corroborated the functionality of our approach by targeting the engineered cones, and by using whole cell patch-clamp recording we observed that they responded to light in a fast and robust manner.
: Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands
Diseases that affect the light-sensitive cells in our eye, the rods and cones, are the most common causes of familial vision impairment. Mutations in one of ∼170 genes that control the development or normal function of these and other retinal cells can give rise to retinal diseases (RD). The most frequently mutated RD gene is the ABCA4 gene, associated with retinal disease in almost all persons with autosomal recessive Stargardt disease, 25% with autosomal recessive cone-rod dystrophy, and 5% with autosomal recessive retinitis pigmentosa. Variants in the ABCA4 gene give rise to recessive diseases, and affected persons should carry causal variants in both copies of the gene. About 25% of persons with STGD1 carry only one or no ABCA4 variant; one plausible explanation for the ‘missing mutations’ is that they are located outside the protein-coding parts of the gene. To identify deep-intronic ABCA4 variants, Haloplex-based locus sequencing was performed in 35 mono-allelic maculopathy cases. The effect of putative splice variants was tested in vitro by cloning genomic DNA fragments carrying putative disease variants into minigenes, which were transfected into HEK393T cells. Fibroblasts of 11 cases were reprogrammed into induced pluripotent stem cells, which were differentiated into photoreceptor progenitor cells (PPCs). The effects of a frequent non-canonical splice variant, c.5461-10T>C, and deep-intronic variants on RNA splicing were analysed by reverse transcription PCR of PPC mRNA and by in vitro assays with minigene constructs. Antisense-oligonucleotides treatment of some of the variants allowed us to restore the correct splicing in otherwise truncated transcripts.
: Benitec Biopharma
Choroidal neovascularization (CNV), the root cause of a number of human diseases including age related macular degeneration (AMD), results in the infiltration of new blood vessel into the retina and leads to significant loss of vision. Although Ranibizumab or Aflibercept, commercial therapies that “mop up” secreted factors that stimulate CNV, have been useful at controlling the progression of the disease, the relatively short half-life of these proteins hampers long term use by requiring repeat intravitreal injections as frequently as every month or bimonthly. Here, we describe a gene therapy approach based upon RNA interference in which we generated a recombinant AAV expression construct that produces three short hairpin RNA (shRNA) to target VEGF-a, VEGF-b and PlGF, three clinically validated gene targets with etiology to AMD. This construct is efficient at dose dependent inhibition of the angiogenic proteins at levels as high as 95% in vitro and can be directly correlated with the levels of shRNA that are produced. Because RNAi requires the transduction of large numbers of cells to be effective, therapeutic evolution is being employed to identify AAV capsids which are capable of transducing cells throughout the retina following intravitreal injection. Screening was performed directly in primates to ensure that novel capsids have the ability to traverse the inner limiting membrane, a dense ocular tissue generally impermissive to passage of viral particles. Completion of multiple rounds of screening has revealed a number of candidate AAV capsids that transduce deep retinal layers including the RPE and photoreceptors following intravitreal injection.
: University of Geneva
Personalized medicine demands customized control strategies to ensure patient safety. TargetAMD aims at completion of a clinical trial for the personalized treatment of neovascular age-related macular degeneration (nAMD). Within one surgical intervention, autologous iris pigment epithelial (IPE) cells will be transfected with the gene for pigment epithelium derived factor (rPEDF) and transplanted subretinally in the same patient to suppress choroidal neovascularization by decreasing the expression of vascular endothelial growth factor. Rabbit IPE cells were transfected with the PEDF or the Venus reporter gene using the SB100X/pFAR4 miniplasmid technology and transfection efficiency determined by image-based cytometry. To demonstrate the feasibility of isolating, transfecting and transplanting IPE cells in one hour, cells were isolated, transfected and transplanted in 4 rabbits. Biodistribution was examined by transplanting subretinally in rabbits 20’000 PEDF-transfected homologous IPE cells and from 1 to 90 days, 43 organs were analyzed macroscopically and histologically for abnormalities and by qRT-PCR for the presence of the vector or rPEDF. Cell isolation time was 30 minutes (75 min of trypsination, unnecessary for human cell isolation), transfection plus delivery to operating room was 20 minutes and transplantation 10 minutes, for a total of ∼60 minutes. Transfection efficiency was 31.2 ± 5.9%. No gross or histological abnormalities were found after transplantation of transfected IPE cells and no systemic trafficking of transfected cells was observed. It is evident from the results above that cell isolation, transfection and transplantation within one hour is feasible and safe, since organ integrity is not affected and transfected cells are not trafficked systemically.
: Università di Modena e Reggio Emilia
Many progresses have been made in understanding the genetic basis for Retinitis Pigmentosa (RP), however therapeutic interventions are still lagging behind. Rhodopsin (RHO) mutations represent a common cause of RP, accounting for 25% of autosomal dominant RP and 8–10% of all RP (Hartong et al., 2006) with more than 100 different mutations identified so far. Here we show the application of CRISPR-Cas9 technology to knock out the RHO defective alleles by introducing double strand breaks into the target gene. We designed single or double gRNAs to knock-down mutant RHO expression by targeting exon 1 of the RHO gene carrying the P23H dominant mutation. The two gRNAs were tested singularly or together in vitro in HeLa clones stably expressing P23H RHO. Cel I assay, TIDE and sequencing analyses demonstrated insertions or deletions (indels) in the genomic DNA specifically in the RHO gene, which caused strong reduction of RHO expression up to 90%. The higher effect was obtained with two gRNAs together. The CRISPR/Cas9 plasmid expressing two gRNAs were then in vivo tested in P23H RHO transgenic mice by sub-retinal electroporation, together with EGFP expressing plasmids. Analysis of indels in FACS-sorted EGFP+ cells demonstrated up to 30% of in vivo genome editing of the human P23H RHO gene, without targeting of the murine Rho allele. We also detected reduction of RHO at mRNA and protein levels. Thus, successful in vivo application of the CRISPR/Cas9 system confirms its efficacy as a genetic engineering tool and its potential use in gene therapy.
: Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, Paris, France
GS010, a rAAV2/2-ND4 vector administered via intravitreal injection (IVT) is currently being assessed in Phase 3. Despite the eye's immune privilege and because AAVs are known to trigger host immune responses, we reviewed the relationship between serum levels of anti-AAV2 neutralizing antibodies (NAbs) and ocular inflammation in monkeys (n = 36) and LHON-ND4 patients (n = 15) following GS010 injection. At baseline, monkeys had undetectable NAbs titers (55%) or low titers (45%, <1:400). All animals showed an increase in NAbs up to a maximum of 1:12800 starting at week 2 post-injection. This remained stable up to 6 months. Ocular inflammation was observed in up to 80% of animals without deleterious effects on the retina. Among the 15 patients of the dose escalation study, 7 had undetectable NAbs levels at baseline. NAbs increased up to a maximum of 39 times their baseline level in 10 patients 2–8 weeks post injection without obvious correlation with the dose. Thirteen patients experienced ocular inflammation (mostly mild) without apparent correlation with the humoral response. Aqueous humor was sampled at baseline (n = 8) for NAbs and all were negative despite the status of serum NAbs. In patients and monkeys, GS010 injection induced a reversible ocular and a humoral serum response starting 2 weeks after injection. There was no consistent correlation between the occurrence or extent of ocular inflammation and immune response profile. Differences between monkeys and patients were noted with regard to the proportions developing an immune response, the greater magnitude of the immune response and longer persistence of NAbs in monkeys.
: Oxford BioMedica
Neovascular age-related macular degeneration (NVAMD) is a prevalent cause of vision loss. VEGF-neutralizing proteins provide benefit but many patients require frequent injections for a prolonged period; new, sustained delivery approaches are therefore needed. We tested the safety and feasibility of a lentiviral vector platform to provide persistent expression of endostatin and angiostatin in eyes of advanced NVAMD patients. This is the first time in Man lentiviral vector has been administered to the eye. Twenty-one patients in three dose groups (n = 3/group) and final cohort at highest tolerated dose (n = 12)) with active CNV at baseline were treated. All patients had advanced stage disease with central subretinal fibrosis and poor anti-VEGF response. Subretinal injection of OXB-201 (formerly known as RetinoStat®) was well-tolerated at all doses with little or no ocular inflammation. There was one serious adverse event (macular hole) associated with the surgery. Vascular leakage, present in all subjects at baseline, was absent in the majority of patients at 6/12 months. Visual outcome showed little change in these advanced patients. Endostatin and angiostatin in aqueous fluid increased dose-dependently and has continued into the longterm follow-up study (>4 years in some patients). This is the first demonstration of direct quantitation of a therapeutic transgene following ocular gene therapy. The study met its primary endpoint: OXB-201 was safe and well tolerated. The data demonstrates the LentiVector® gene therapy platform safely and efficiently delivers genes to the retina resulting in stable, long-term gene expression.
: Adverum Biotechnologies, Inc
AAV-mediated gene therapy using sub-retinal (SR) injections is promising for some ocular diseases. Intravitreal (IVT) administration of current AAV serotypes, while less invasive, is far less efficient due to the inner limiting membrane (ILM), which is a profound barrier in the primate retina. It would be beneficial to develop AAV vectors that can effectively transduce target cells following IVT injection for diseases where SR injection is not a preferred method. 2.5T, a chimeric vector with VP1 from AAV2 and VP2 and VP3 from AAV5 has a better neutralizing antibody profile compared to AAV2, which could be advantageous for IVT delivery. It transduces photoreceptors when injected SR, but is unable to transduce the outer retina post IVT injection, which may be due to its complete inability to bind heparan sulfate proteoglycan (HSPG) receptors, which are abundant on the ILM. We attempted to create 2.5T variants with enhanced HSPG-binding capability for IVT delivery. We engineered variants by either substituting two positively charged resides, a short region comprising of 6 binding residues, or a longer region consisting of the binding domain and flanking residues in the receptor-binding region of 2.5T, from AAV2. A majority of the swap variants were successfully packaged and demonstrated varying levels of HSPG-binding compared to the parental 2.5T capsid. An interesting observation in pig explants was a trend in altered tropism for specific cell types. Efficacy of candidate variants is currently being tested in non-human primates.
: Uskudar University-Istanbul
: Kyushu University
: Université de Lausanne - Jules Gonin Eye Hospital
The retinal pigmented epithelium (RPE) is a monolayer of pigmented cell located between the retina and the choroid. RPE contributions to the visual process are the recycling of the chromophore required for phototransduction, the phagocytosis of shed photoreceptor outer segments and the regulation of fluid and nutrient. The neural retina activity relies on RPE functions and its deficiency give rise to several diseases, of which most of them result in visual impairments or blindness. The ability to generate hRPE for disease modelling, drug screening or transplantation is particularly worth to answer these important challenges. Starting from iPSCs, we established a three-step protocol able to induce pigmented foci as early as 16 days in differentiation. RPE cells are characterized by their pigmentation and cobblestone-like morphology, the expression of mRNA markers such as RPE65 (visual cycle), MERTK (phagocytosis), ZO-1 (junctions) or MITF (transcription factor). Protein presence of some of these markers was assessed by immunohistochemistry and phagocytosis ability was investigated by western blotting after photoreceptor segment integration. RPE cells polarization was verified by electron microscopy whereas specific factors secretion was quantified by ELISA assays. Finally, to test the robustness of the differentiation protocol, we exposed our iPSCs-derived hRPE to several lentiviral constructs in order to challenge the specificity of different promoter activities. In conclusion, we developed a consistent method to generate hRPE from iPSCs that will be combined to a CRISPR approach to produce in vitro model for RPE deficient-induced retinal diseases.
: Aarhus University
Programmable nucleases are powerful tools in gene editing studies allowing site-specific genome engineering. We will use the CRISPR/Cas9 system for intraocular gene editing of the vascular endothelial growth factor (VEGF) gene. Over-expression of VEGF in RPE cells cause serious eye diseases resulting in vision loss. We have designed 6 pairs of ‘VEGF guide strands’ and cloned them into a lentivirus (LV)-CRISPR/Cas vector. Following LV-transduction of retinal cells, including RPE cells, specific cutting in the VEGF gene was demonstrated. Next, we assessed the possibility to use the LV-CRISPR/Cas vector for intraocular editing following subretinal injections in mice. We observe expression of the GFP-marker simultaneously expressed from the LV-CRISPR/Cas vector, suggesting efficient delivery and expression of the CRISPR/Cas expression cassette. Ocular gene editing is assessed in RPE cells isolated from injected eyes using FACS and the TIDE analysis.
: Department of Ophthalmology, Nippon Medical School
: University of Geneva
Side-effects, the high costs of current treatments for neovascular age-related macular degeneration (nAMD), the need for life-long, repeated intravitreal injections of anti-VEGFs (Vascular Endothelial Growth Factor), has stimulated the search for innovative treatments. The European-funded TargetAMD project aims at the development of reagents, devices and protocols for a once-in-a-lifetime gene-therapeutic treatment for nAMD by transplanting subretinally autologous PEDF-transfected pigment epithelial cells. Autologous iris pigment epithelial (IPE) cells will be isolated from a patient's iris biopsy, and transfected with the PEDF gene using the non-viral Sleeping Beauty (SB100X) transposon system followed by subretinal transplantation in the same patient within one surgical session lasting approximately one hour. The PEDF gene and the SB100X transposase will be encoded by pFAR4 miniplasmids, and delivered to IPE cells ex vivo by electroporation. A clinically approved electroporation device and a newly developed buffer have made possible the efficient transfection of freshly isolated IPE cells from human donor eyes under GMP conditions. Evaluation of safety and efficacy of nAMD treatment with PEDF-transfected pigment epithelial cells has been shown to be safe while producing a significant reduction of choroidal neovascularization (CNV) in a rat model. Significant and essential progress has been made toward the performance of a phase Ib/IIa clinical trial to treat neovascular AMD by additive gene therapy. Here we have shown that the approach is not only feasible, but it is effective as shown by the reduced neovascularization in a laser-induced rat model of CNV.
: Group “Gene therapies and animal models for neurodegenerative diseases”, UPMC-INSERM-CNRS-Institut de la Vision, 17 rue Moreau, 75012 Paris, France
Cone photoreceptors are the primary target cells for gene therapy aiming to treat achromatopsia and cone-rod dystrophies. AAV gene therapy has been successful in the retina, however one current limitation is the lack of specific vectors for targeting cones using an innocuous intravitreal injection. Clinical trials have shown that AAV is useful for targeting deep retinal layers when injected subretinally, but these injections are associated with risks of tissue damage. The development of more efficient and specific vectors for cell-type specific targeting from the vitreous remains a critical issue. The aim of this study was to test the feasibility of restricting gene expression to cones using specific promoters in combination with intravitreally delivered AAVs. We tested the ability of an engineered AAV variant, AAV2-7m8, to transduce exclusively mouse cones when combined with several cone specific promoters including the newly described human red opsin promoter pR1.7. The same vector was also tested for its ability to transduce human cones in retinal organoids derived from induced pluripotent stem cells (iPSCs), and in human retinal explants. Remarkably, cone-specific gene delivery was achieved in both rodent and human tissue using the same vector-promoter combination. Our data show, for the first time, that cones can be transduced specifically using a non-invasive administration route. This finding is highly relevant for future clinical applications in humans for targeting cones located in the fovea – the most delicate part of the retina where mechanical damage due to surgical procedures needs to be avoided.
: Tigem, Telethon Institute of Genetics and Medicine
Rod and cone photoreceptors (PRs) convert light stimuli in information signaling for visual perception. Specific gene mutations primarily affecting both cone and rod PRs types lead to retinal dystrophies. The project aims at investigating rod and cone transcription regulation controlling their identities in adult pig retina. Retrieving transcription factor sets has biological relevance and may be instrumental to design gene-based and/or regenerative therapeutic strategies. We developed a method to isolate rods and cones from the same retinal samples using AAV vectors with an expression cassette encoding both mCherry and eGFP under the control of rod and cone GRK1 and GNAT1 rod-specific promoter, respectively. Histological analysis, 15 days after subretinal injection, showed expression of both fluorophores in rods, whereas cones showed exclusive expression of mCherry. Immunofluorescence with cone arrestin showed co-localization with mCherry signal. qRTPCR on FACS sorted cells confirmed photoreceptor specific identities: RHO, NRL, GNAT1 in rods; S-opsin, M-opsin, GNAT2 in cones and the absence of cross-contaminations. RNA-seq differential expression gene analysis showed a total of 905 differentially expressed genes in rods compared with cones, in which 127 genes were up and 778 genes down regulated. Furthermore, 25 TFs were found differentially expressed in rods and 60 in cones. We showed that the “double fluo” provided a robust method to isolate retinal rod and cone specific regulatory programs in a relevant large animal model such as adult pig retina. These data support the use of TF sets to manipulate their expression via AAV vectors somatic gene transfer for therapeutic applications.
: Department of Ophthalmology, University Hospital RWTH Aachen, Aachen, Germany
Age-related macular degeneration (AMD) is the most common cause of severe vision loss in the elderly. Neovascular AMD (nAMD) is characterized by choroidal blood vessels growing into the subretinal space, which results from overexpression of the vascular endothelial growth factor and decreased expression of the pigment epithelium-derived factor (PEDF). We have developed an approach, which involves the transplantation of genetically modified pigment epithelial (PE) cells that stably overexpress PEDF. The PEDF transgene is incorporated in the genome of PE cells by the enhanced Sleeping Beauty (SB100X) transposon system delivered by electroporation of non-viral miniplasmids free of antibiotic resistance markers (pFAR). PE cells from human donor eyes were transfected immediately after isolation by using as few as 2×104 cells. The amount of secreted PEDF (quantified by immunoblotting and ELISA) was 0.6 ng/h/1×104 cells, and thus 6-fold higher compared to non-transfected cells. Analysis of total PEDF gene expression via quantitative RT-PCR revealed a 60-fold increase compared to the endogenous level. In sum, SB100X-mediated transfection of freshly isolated PE cells resulted in increased PEDF secretion and PEDF gene expression. The pFAR technology improves biosafety of non-viral gene transfer, as it allows for plasmid propagation in the absence of antibiotics, which is an crucial safety issue for human trials. The transfection of freshly isolated PE cells is an important step towards the ultimate goal of the TargetAMD project, which is efficient and safe delivery of the PEDF transgene to autologous cells ex vivo, followed by the transplantation to the subretinal space of nAMD patients.
: Aarhus University
Lenti (LV)- and adeno-associated viral (AAV) vectors are powerful tools for gene delivery to the retinal cells for treatment of autosomal dominant retinopathies caused by mutations in single genes. Intraocular vascular diseases such as neovascular age-related macular degeneration and proliferative diabetic retinopathy are multifactorial diseases, and therefore current therapy targeting single cellular pathways are often insufficient. Due to size limitations, packaging of multiple genes and/or inhibitors in the same vector have until now proved to be inefficient. We have designed multigenic vectors for combined expression of multiple anti-VEGF targeting miRNAs and the multifunctional protein PEDF. Moreover, expression is limited to target cells by a tissue-specific promoter. These expression cassettes have been packaged as LV and AAV particles. We observe persistent expression of the GFP-marker concomitantly with PEDF expressed from the viral vectors for >2 month after subretinal injection in mice. Furthermore, expression is limited to the retinal pigment epithelium when the expression is driven by the VMD2 promoter. The anti-angiogenic effect is evaluated in the laser-induced choroidal neovascularization mouse model by immunostaining of choroidal/RPE flatmounts. Our preliminary data demonstrates packaging of large, multigenic contructs not only in LV but also AAV particles, and suggest efficient delivery and expression of our anti-angiogenic molecules in target cells.
: Instituto Universitario de Oftalmobiología Aplicada - Universidad de Valladolid (IOBA -UVa)
: University of Bern
In order to quantify retinal homing and possible rescue effects of endogenously mobilized bone marrow-derived stem cells (BMSC) an SDF-1-inhibiting L-RNA aptamer (NOX-A12) was introduced in combination with an increased SDF-1 gradient towards the damaged retina. Retinal degeneration was induced by an i.v. injection of sodium iodate (NaIO3) in GFP-chimera mice. BMSC mobilization and intra-retinal attraction were triggered by an i.v. injection of NOX-A12 three days after NaIO3 and intraocular injection of SDF-1 four hours afterwards. The number and type of GFP+ cells in the retina was quantified at day 7 after BMSC mobilization by immunohistochemistry (IHC). The sections were also stained for BMSC, microglia and macrophage markers. Retina-specific markers were visualized to study possible differentiation of migrated BMSC. Outer nuclear layer (ONL) thickness was quantified after mobilization in H&E stained paraffin sections. At different time points visual function was measured using the optokinetic reflex. NOX-A12 led to migration of GFP+ BM-derived cells into the NaIO3-altered sensory retina and this was dose-dependently increased by intraocular SDF-1 injection. Injection of SDF-1 did also lead to an increased number of GFP+ BMSC in the retina. Furthermore, a trend towards less activated microglia was observed. The number of macrophages in the retina remained unchanged. No co-staining of GFP+ BMSC with retina-specific markers was observed. A gain in ONL thickness could not be found after NOX-A12 treatment. However, a significant increase in visual acuity was found after NOX-A12 + SDF-1 injection compared to NOX-A12 + BSS as well as to SDF-1 only treated animals.
: Oxford BioMedica
Due to both the avascularity of the cornea and relatively immune-privileged status of the eye corneal transplantation is one of the most successful clinical transplant procedures. However in high risk patients, which account for >20% of the 100,000 transplants carried out worldwide each year, the rejection rate is higher due to vascularization of the recipient cornea. Neovascularisation is thus an attractive target to prevent corneal graft rejection. OXB-202 is a human donor cornea which, prior to transplantation, is genetically modifed ex vivo with genes encoding secretable forms of the angiostatic human proteins, endostatin and angiostatin. This is achieved using a lentiviral vector derived from the Equine Infectious Anaemia Virus (EIAV) called pONYK1EiA, which subsequently prevents rejection by suppressing neovascularization in the grafted cornea. We will present vector biodistribution, histopathogy and immunology data from a 3-month GLP toxicology and safety study in a rabbit corneal transplant model. The GLP study employed the use of clinically relevant in-life assessments that included regular slit-lamp ophthalmic examinations, evaluation of corneal thickness and endothelial cell density using pachymetry and specular microscopy respectively, data from which will also be shown. The summary of results of the GLP study will be presented and will show that there are no safety issues. The GLP safety study data supports the evaluation of OXB-202 corneas in a First-in-Man clinical study. The clinical study will start in the first half of 2017 at the Moorfields Eye Hospital. The project has been supported by the UK Technology Strategy Board (Innovate UK).
: Instituto Murciano de Investigación Biosanitaria Virgen de la Arrixaca (IMIB-Arrixaca), Universidad de Murcia, Spain
Traumatic injury to mammalian central nervous system (CNS) neurons causes an immediate and irrecoverable loss of function and the death of the wounded neurons. Optic nerve axotomy triggers the specific death of retinal ganglion cells (RGCs). Using this very well characterized model of CNS trauma, we have analyzed in vivo the neuroprotective properties of mesenchymal stem cells isolated from the Wharton's jelly of the human umbilical cord (hWJ-MSCs) on axotomized rat RGCs. hWJ-MSCs were isolated from 3 different human umbilical cords and characterized regarding its differentiation capacity, immunophenotype, and immunomodulatory properties as stated by the International Society for Cellular Therapy (ISCT). Then, they were administered into the vitreous chamber of adult albino rats. Three animal groups were done: i/ intravitreal administration of hWJ-MSCs into intact eyes, to study toxicity, ii/ optic nerve crush (ONC) and intravitreal administration of hWJ-MSCs, to study RGC neuroprotection and, iii/ ONC plus intravitreal administration of vehicle, as the control group. Our data show that hWJ-MSCs administered intravitreally are neither toxic nor tumorigenic. In injured retinas, RGC survival was significantly higher in the hWJ-MSCs-treated groups compared to vehicle ones (twofold at 7 days and threefold at 14 days post lesion). Furthermore, there was no difference in the neuroprotection conferred by hWJ-MSCs isolated from different umbilical cords. All these data indicate that mesenchymal stem cells isolated from human umbilical cord are a promising therapy to treat the injured mammalian CNS.
: UniQure
Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the first exon of the HTT gene. Lowering the expression of mutant HTT (gene silencing) and thereby reducing downstream toxic effects is hypothesized to be therapeutically beneficial. Gene therapy using viral vectors can induce long-term HTT silencing following single administration. The gene silencing therapy developed by uniQure is based on a microRNA targeting human HTT (miHTT). The DNA expression cassette for the miHTT is delivered using adeno-associated viral vector serotype 5 (AAV5-miHTT). Proof-of-concept (PoC) studies showed that local injection of AAV5-miHTT in the striatum of a HD rat model resulted in reduced number of HTT aggregates and subsequently prevention of striatal neuronal dysfunction. In the humanized (Hu)128/21 mouse model sustained target engagement was shown with up to 80% HTT silencing seven months post intra-striatal injection. No acute toxicity or deleterious effects of HTT silencing have been observed at 7 months p.i. in Hu18/18 control mice. Currently, long-term therapeutic PoC studies to examine mitigation of HD phenotype after AAV5-miHTT administration are ongoing in various rodent HD models and transgenic HD mini pigs. AAV5-GFP administration into the central nervous system of large-brain animals (mini pigs and non-human primates) has demonstrated wide vector distribution. Efficacy, safety and tolerability of AAV5-miHTT are being evaluated in NHPs. The combination of widespread vector distribution, great target engagement, long-term expression and good safety profile allowed us to select AAV5-miHTT as lead candidate for HD gene therapy development.
: Benitec Biopharma
BB-HB-331 is a recombinant adeno-associated virus serotype 8 vector designed to treat chronic HBV infection using RNAi. This self-complementary vector expresses three short hairpin RNAs that simultaneously target three well-conserved sequences in the Core, S-antigen and X protein regions on the viral RNAs. Human primary hepatocytes (PHs) isolated from chimeric PhoenixBio (PXB) mice were infected in vitro with HBV genotype C for 12 days before treatment with BB-HB-331. Adenovirus was used to deliver the recombinant BB-HB-331 DNA (Ad-BB-HB-331) because AAV does not efficiently transduce PHs in vitro. Increasing doses of Ad-BB-HB-331 were applied to HBV-infected PHs for 16 days and demonstrated dose-dependent expression of the anti-HBV shRNAs and corresponding inhibition of the HBV viral RNAs. At an MOI = 3, the extracellular levels of HBsAg, HBeAg, and HBcrAg were reduced by 87% or more. Intracellular and extracellular HBV DNA quantities were reduced by 89%, and cccDNA levels were reduced by 70% as assessed by QPCR. In vivo studies showed that a single administration of BB-HB-331 into HBV infected PXB mice resulted in reduction in serum HBV DNA by 1.83 logs, equivalent to 98.5% reduction. Continual decreases were noted through the Day-56 pre-determined endpoint and suggests further reduction may be observed in a longer study. These data correlated with reduced intracellular liver HBV DNA by 95% while HBsAg and HBeAg decreased by 97% and 92%, respectively. Lastly, significant reductions in the levels of HBV viral RNA and cccDNA were observed. Collectively, these data provide encouraging data for continued clinical development of HB-BB-331.
: San Raffale Telethon Institute for Gene Therapy (SR-Tiget)
Transposable elements (TEs) are able to move throughout the genome, representing both drivers of evolution and mutagenic agents. The host genome has evolved powerful mechanisms to permanently block TEs mobilization, including nucleosome compaction and de novo DNA methylation triggered by the KRAB Zinc-Finger proteins and DNMTs, respectively. We have exploited this knowledge to develop Engineered Transcriptional Repressors (ETRs), chimeric proteins in which the KRAB domain, the catalytic domain of DNMT3A and DNMT3L are fused to custom-made DNA binding domains able to recognize human gene promoters. When transiently co-delivered into the cells, these ETRs were able to instruct repressive histone marks and de novo DNA methylation, ensuring effective (up to 90%) and long-term stable (up to 180 cell generations) silencing of multiple endogenous genes. Importantly, silencing was resistant to several activation stimuli and relieved only by targeted DNA demethylation. To identify endogenous players involved in the processes of permanent gene silencing induced by the ETRs, we are performing genome-wide loss-of-function studies in the ETR-silenced cells. To this end, we have generated drug-inducible Cas9-expressing cell lines, and transduced these cells with sgRNA-LV libraries against genes either with unknown functions or encoding for chromatin regulators and nuclear proteins. Preliminary data showed that, upon Cas9 induction, a significant fraction of the transduced cells reactivated expression of the ETR-silenced gene. We are currently enriching these cells to identify the sgRNA-targeted genes. We will then dissect the function of the identified genes and assess their potential involvement in physiological and pathological processes of gene silencing.
: TIGEM
Emerging principles are showing that the genetic bases of biological complexity lies on the regulatory genome (RG). Indeed, species identities and their cell specificities arise from the RG orchestration of relative low number of genes. In the laboratory we are developing a series of natural and synthetic regulatory elements (Abstracts: Transcriptional silencing via synthetic DNA binding protein lacking canonical repressor domains as a potent tool to generate therapeutics, Salvatore Botta; Retrieving rod and cone photoreceptors genetic regulatory identities to generate therapeutics, Nicola de Prisco) in which by hijacking Transcription Factors principles we modulate RG with purpose to generate novel therapeutics. Recently we showed that in vivo delivery of a synthetic DNA-binding protein (ZF6-DB) uncoupled from the repressor domain entails complete and gene-specific transcriptional silencing of the Rhodopsin (Rho) gene(Rhodopsin targeted transcriptional silencing by DNA-binding. Botta S et al. Elife. 2016 Mar 14;5. pii: e12242. doi: 10.7554/eLife.12242). To further evaluate the consequence of ZF6-DB delivery on rod photoreceptor Gene Regulatory Networks (GRNs), we carried out RNA-seq at single cell resolution. Single-rod transciptome analysis showed the precision and potency of ZF6-DB. In addition, we applied systems biology methods (co-espression analysis via inter-individual rod cells variability assessment) to determine the biology of rod GRN with and without its key protein Rhodopsin.
: Institute for Cell and Gene Therapy, Medical Center - University of Freiburg
The human immunodeficiency virus (HIV) is a major global health burden which has claimed over 25 million lives in the past 30 years. The CCR5 co-receptor is necessary for HIV entry into host cells and individuals homozygous for inactivating mutations in the CCR5 gene are largely protected from infection. Therefore, this receptor has gained interest as a possible target for gene therapy against HIV infection. We hypothesize that transcriptional repression via epigenetic modification of the CCR5 promoter may provide a safer alternative to inactivate CCR5 expression compared to genome editing since the genomic sequence remains unchanged and the severity of off-target effects may be reduced. We have generated a panel of transcription activator-like effector-based repressors (rTALEs) and TALE-based designer epigenome modifiers (T-DEMs) and tested their activity in a CCR5-EGFP reporter cell line. While both platforms were able to efficiently modulate reporter gene expression, delivery of CCR5-specific T-DEMs resulted in more efficient and stable EGFP silencing in up to 80% of the target cells for over three weeks. Functionality of the T-DEMs was also tested in CD4+ primary T cells and we observed a significant reduction in CCR5 gene expression following delivery of a T-DEM encoding mRNA. Investigations into the extent of DNA methylation via bisulfite sequencing and transcriptome-wide changes including off-target effects via RNA-seq are currently ongoing. This study aims to dissect the efficacy and safety of epigenome editing as a novel therapeutic strategy based on gene inactivation and provides a leap towards establishing a safer therapy for HIV infection.
: Department of Molecular Biology, Research Institute of Advanced Omics, Dankook University, Yongin 16890, Republic of Korea
The objective of this study was to develop anti-hepatitis C virus (HCV) genetic agents that can inhibit chronic liver injury and fibrosis by HCV. To this end, we first determined the molecular mechanisms underlying the liver diseases caused by HCV. This study revealed that miR-192 expression was induced by HCV infection without influencing viral replication. However, the viral-induced miR-192 up-regulated TGF-β1 expression in liver cells at transcriptional level. TGF-β1 stimulation by HCV-induced miR-192 was triggered through ZEB1 down-regulation and TGF-β1 increased miR-192 level via positive feedback pathway. Increase in miR-192 expression by HCV infection was due to HCV core protein released and/or expressed by viral infection. Taken together, HCV infection resulted in increased TGF-β1 transcription in hepatocytes through ZEB1 down-regulation by HCV core-mediated miR-192 stimulation. Moreover, we observed that expression of fibrogenic genes was enhanced in hepatic stellate cell(HSC)-LX2 cells when co-cultured with JFH-1 HCV stable cells, compared to when co-cultured with naïve Huh-7 cells. Interestingly, supernatant from miR-192-transfected Huh-7 cells elicited expression of fibrogenic genes in the HSC-LX2 cells. We observed that these influences could occur through exosome-mediated transfer of the miRNA. These results indicated that HCV-induced miR-192 could contribute to HCV-mediated fibrogenesis through exosome-mediated activation of hepatic stellate cells. Importantly, miR-192 inhibition with anti-miR-192 rescued ZEB1 expression down-regulated by HCV infection, thus down-regulating TGF-β1 expression induced by HCV infection in hepatocytes and inhibiting fibrogenic gene expression through HSC deactivation. Therefore, miR-192 would be a diagnostic marker for HCV-induced fibrosis and a critical target for attenuating the viral pathogenesis.
: Korea Research Institute of Bioscience & Biotechnology
Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent a potential source of megakaryocytes and platelets for transfusion therapies. However, most current ES/iPS cell differentiation protocols are limited by low yields of platelet-releasing megakaryocytes. Mutations in the mouse and human genes encoding transcription factor GATA1 cause accumulation of proliferating, developmentally arrested megakaryocytes. Previously, we hypothesized that the suppression of GATA1 in ES cell differentiation cultures would expand megakaryocyte precursors. We recently reported that the engineered murine ES cells to express doxycycline (dox)-inducible shRNA against Gata1 mRNA generated a self-renewing hematopoietic progenitor, termed G1ME2 (Gata1-null Meg-Erythroid Progenitor, version 2) cells. Upon dox withdrawal, Gata1 expression was restored and the precursors developed into mature, platelet-producing megakaryocytes that are functional. In order to translate this strategy to human ES/iPS cells, we took advantage of the CRISPR/Cas9 technology to develop GATA1 knockout (KO) human ES cell lines, as a proof-of-principle. We targeted exon 3 of GATA1 gene in wild type (WT) male hESCs. Two out of 22 colonies represented frameshift mutations and one colony got expanded. The full length of GATA1 protein was not detected in the KO during hematopoietic differentiation. Importantly, the CD235+ erythroid precursors were not generated in the KO, indicating the loss-of-function in GATA1. We are trying to expand the megakaryocyte precursors in the KO with the different hematopoietic differentiation protocols. Meanwhile, development of more knockout cell lines using the cutting edge genome editing technologies will provide us a better understanding on the mechanisms of action in the future.
: CNRS-Inserm-Univ Paris Descartes-Chimie ParisTech
Small interfering RNAs (siRNA) with a capacity to inhibit expression of pathologic proteins in vivo represent a promising approach to the treatment of various diseases. However, even though RNA interference is a highly specific technique and has the potential to selectively silence the expression of any gene, the systemic or local delivery of RNAi molecules remains a challenge. Recently, we developed efficient cationic lipid-based siRNA vectors obtained by self-assembly of cationic lipid and a mixture of siRNA and anionic polymer. We have designed these adjuvant-supplemented siRNA lipoplexes to solidify the obtained particles and hypothesized that anionic polymer, bearing negative charges, would be capable to mix with siRNA and take part in the auto-assembly of particles. We found that the obtained particles were more efficient for gene silencing and more stable than lipoplexes prepared with no anionic polymer. We also extensively studied the structural and physico-chemical characteristics of adjuvant-supplemented siRNA lipoplexes as compared to classical siRNA lipoplexes using Transmission Electron Microscopy, Atomic Force Microscopy, Small-Angle X-ray Scattering and Thermogravimetry. We found significant differences between both types of particles in terms of morphology, surface properties and structure. We also demonstrated that adjuvant supplemented-siRNA lipoplex were suitable and efficient for in vivo gene silencing in mouse disease model or for elucidating gene function in whole organism.
: Paul Ehrlich Institute
Induced pluripotent stem cells (iPSC) are an ideal source for the generation of blood cells for patient-specific therapy. We are mainly interested in the differentiation of platelets from iPS cells for transfusion. Platelets were successfully generated via embryoid body formation followed by co-culture on OP-9 cells supplemented with thrombopoietin. Megakaryocyte differentiation, maturation and platelet generation were demonstrated by cytomorphology and cell surface phenotype. However, the overall recovered platelets number was low. To improve differentiation, supporting factors such as essential transcription factors may be expressed in iPS cells during the culture at well-defined time points and durations. This can be achieved using tetracycline (Tet)-inducible all-in-one retroviral vectors. Hereby transgene expression is tightly controlled by the tet-responsive T11 promoter responding in the on-state to the rtTA-M2 transactivator expressed by the PGK promoter (Heinz et al., Hum Gene Ther, 2011). However, vector silencing is a major drawback in pluripotent cells. To overcome this, we inserted different sized chromatin opening elements from the human HNRPA2B1-CBX3 locus (UCOE, 1.5kb, 1kb, 670bp, 630bp) into the gammaretroviral all-in-one tet-inducible vector. Incorporation of UCOEs did not hamper vector titers. Elements in sense-orientation in general showed a higher fold-induction compared to the corresponding anti-sense elements. Repeated induction/repression by application and withdrawal of doxycycline was most faithful when using the 670bp element of the CBX3 5’ region/gene (sense orientation), which also showed the highest fold-induction. Highest anti-silencing effects in P19 cells also resulted from the 670bp element making the vector an interesting tool for regulated expression in pluripotent cells
: Benitec Biopharma
CAR T-Cell therapy has been an exciting advancement in the field of oncology by providing the ability to modify a subject's own immune cells to be able to treat their cancer. Although the autologous adoptive cell transfer approach has been successfully employed in the clinic, an allogeneic approach has the potential to significantly streamline the manufacturing and thus might provide more accessible options to patients as well as enhance safety by reducing the possibility of graft-versus-host-disease. Restricting expression of the T-Cell Receptor (TCR) on the modified T-Cells helps eliminate the ability to recognize major and minor histocompatibility antigens in the recipient. In the approach described, we have generated a recombinant expression construct to produce short hairpin RNA (shRNA) against multiple subunits comprising the TCR complex. Individually, each of the highly selective shRNA were shown to inhibit protein and mRNA expression by up to 93% of the endogenous levels, albeit potentially insufficiently for therapeutic benefit. Yet, when multiple shRNA against the different subunits were concomitantly expressed from the same vector, we observed a nearly complete depletion of the TCR complex from the cell surface (>99%) as measured by FACS analyses. Furthermore, TCR functionality was inhibited when treated cells were stimulated with either CD3 or in B cell co-cultures with Staphylococcal enterotoxins. IL-2 secretion was inhibited to undetectable levels by ELISA by the multi-shRNA treatment and >98% by qPCR. Taken altogether, these data point to a viable approach towards generating allogeneic T-Cells for immunotherapies against certain cancers.
: Adnan Menderes University
: Inönü University, Faculty of Pharmacy, Department of Pharmaceutical Biotechnology
Mesangioproliferative glomerulonephritis (MPGN) is a disease characterized by glomerular mesangial cell proliferation and matrix accumulation. PDGF-B via PDGFR-β induces mesangial cell activation and proliferation and enhances synthesis of extracellular matrix. Instead of affecting the activity of signalling pathways, silencing of genes in those pathways by siRNA offers a new therapeutic approach. The present study is the first in vivo study that examine the therapeutic efficiency of chitosan nanoplexes containing siRNA targeting to PDGF-B and PDGFR-β in MPGN. siPDGF-B and siPDGFR-β were complexed with chitosan and the characterization of these nanoplexes (size, surface charge) were done. Chitosan/siPDGF-B+siPDGFR-β nanoplexes were injected intravenously to rats forming MPGN by anti-Thy1.1 antibody. PDGF-B and PDGFR-β mRNA and protein expression levels during the experiment were determined by qRT-PCR, immunohistochemistry and western blotting. Apoptosis was studied by TUNEL method. The size and zeta potential of nanoplexes were 341.0 nm and +22.3 mV. PDGF-B and PDGFR-β mRNA expressions were reduced approximately 65% and 80% by chitosan nanoplexes containing siPDGF-B and siPDGFR-β. In TUNEL study, apoptotic index was decrased in treated group (2.6 ± 1.5) compared to untreated control group (11 ± 1.4). Western blot analysis showed decrease in PDGF-B protein level in the nanoplex group. These results showed that the chitosan/siRNA nanoplexes targeting to PDGF-B and PDGFR-β have a highest suppressive effect on protein and mRNA expressions. In additon, chitosan, which is a reliable and efficient biopolymer, can be used as siRNA delivery system in kidney drug targeting.
: Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Biotechnology, 34668, Istanbul, TURKEY
Cancer can be treated by targeting genes that cause tumor formation and development, and gene therapy has come into question as a new approach in the treatment of cancer. siRNA, inducer of RNA interference (RNAi) mechanism that is an important tool of gene therapy, is used to suppress gene expression. Breast cancer is common type of cancer among women. Vascular endothelial growth factor (VEGF) is a major pro-angiogenic factor in development and metastasis of the breast cancer. The aim of this study is to investigate and compare inhibition of VEGF gene on various breast cancer cell lines using 35 and 21 bp siRNAs containing chitosan nanoparticles. For this purpose, nanoparticle formulations were prepared using chitosan and tripolyphosphate (TPP) with ionic gelation method. Afterwards, the physicochemical properties such as zeta-potential, particle size, serum stability and morphologic features of these formulations were measured. Release study of the nanoparticles was performed for a month. Transfection efficiency was observed with fluorescence microscopy and the amount of VEGF was assayed using ELISA after performing transfection to different breast cancer cell lines. In conclusion, gene delivery with 35 bp siVEGF/chitosan nanoparticle is performed successfully and similar results were obtained for the both siVEGF (21 bp and 35 bp) in different breast cancer cell lines. In the future, the data were shown 35 bp siVEGF/chitosan nanoparticle can be investigated in vivo studies of VEGF inhibition and therapy in breast cancer model.
: Cell Therapy Program, Center for Applied Medical Research (CIMA), University of Navarra. Instituto de Investigación Sanitaria de Navarra, IdiSNA, Pamplona, Spain
Primary hyperoxaluria type I (PH1) is a rare, autosomal recessive inherited metabolic disorder of the glyoxylate metabolism cause by mutations in the hepatic enzyme alanine-glyoxylate aminotransferase (AGT) coded by Agxt gene. Combined liver-kidney transplantation is the only curative treatment for this disease, thus there is an urgent need for new and safe therapies. In this work we have evaluated the therapeutic potential of Agxt gene correction using a universal HDR-mediated knock-in strategy by CRISPR/Cas9-mediated in vivo gene editing in a mouse model of PH1. This strategy allows the correction of any Agxt-associated mutation inserting a corrected copy of the Agxt gene at the endogenous genomic locus in order to restore proper AGT expression and regulation. First we have evaluated the cut efficiency and indel formation of different gRNAs in cell culture by cell transfection as well as in vivo, in the liver of mouse, delivering the CRISPR/Cas9 using an AAV8 vector. After gRNA selection both CRISPR/Cas9 and an AAV carrying the DNA template for genetic correction was administered in PH1 mice. Successful in vivo HDR has been achieved allowing Agxt gene expression and the therapeutic efficacy is being analyzed. This strategy would represent a promising therapeutic approach to treat metabolic diseases like PH1.
: Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, 28040 Madrid, Spain
Gene editing has emerged as a new approach for the treatment of patients with inherited diseases such as Fanconi Anemia (FA). Our previous results have demonstrated that gene editing by homologous directed repair (HDR) is feasible in hematopoietic stem and progenitor cells (HSPCs) from FA patients. However, because the frequency of homologous recombination (HR) in HSPCs is much lower than non-homologous end joining (NHEJ) and given that mild HR defects have been reported in FA cells, we have now explored the NHEJ DNA repair pathway to generate compensatory mutations that could result in functional FA proteins in hematopoietic cells from FA patients, resembling what happens in mosaic patients (Waisfisz et al.,1999). For this purpose we selected the CRISPR/Cas9 system and designed two different guide RNAs (gRNAs) targeting the most common mutation described in FA-A Spanish patients, that leads to a stop codon in exon 4 (c.295C>T). Experiments conducted in FA-A lymphoblastic cells demonstrated that both gRNAs target the FANCA locus with efficiencies around 20%. Furthermore, Next Generation Sequencing revealed that some of the repair events removed the pathogenic mutation and restored the FANCA open reading frame. Notably, NHEJ-mediated gene editing conferred a marked in vitro proliferation advantage, reverted the characteristic MMC sensitivity and restored FANCD2 foci formation in FA-LCLs. Finally, western-blot analysis also confirmed the stable expression of FANCA in FA-A reverted clones. Altogether our results demonstrate for the first time the possibility of correcting the phenotype of FA cells using a NHEJ-mediated gene editing approach.
: GENyO- Centro de Genomica e Investigacion Oncologica: Pfizer/Universidad de Granada / Junta de Andalucia
Specific nucleases (SNs), ZFNs, TALENs and CRISPR, are powerful tools for genome editing. Primary immunodeficiencies (PID) are a main target for gene editing strategies since a small number of corrected cells could cure patients. However, their efficiency and safety should be analyzed in detail before translation into clinic. Our final aim is the development of a gene edition tool for efficient genetic rescue of human hematopoietic stem cells (hHSCs) from Wiskott-Aldrich syndrome (WAS) patients. In this work, we have designed different WAS-specific CRISPR/Cas9 systems and compared their efficacy and specificity with homodimeric and heterodimeric WAS-specific ZFNs using nucleofection and integrative-deficient Lentiviral Vectors (IDLVs) as delivery systems. Our results showed that, although CRISPR/Cas9 system can be as efficient as the best ZFNs for gene disruption, the outcome depends on the delivery system. Indeed, while both systems achieved similar results using nucleofection, IDLV delivery of ZFNs outperformed CRISPR/Cas9 systems. We next analyzed the efficacy and specificity of the different systems for genome edition of the WAS locus by homogologous recombination. Interestingly, the heterodimeric ZFNs showed the best results in terms of specificity (around 83% in target) followed by the CRISPR/Cas9 systems (near 75% in target). The homodimeric ZFNs showed the worst specificity, showing up to 30 % donor insertions in non-target loci. In summary, our data indicates that optimized ZFNs could be a better alternative for therapeutic intervention of the WAS locus when compared to the home-designed CRISPR/Cas9 systems.
: Intellia Therapeutics
Effective and safe delivery of CRISPR/Cas9 components, whether based on viral or non-viral delivery vehicles, would require specific targeting of a tissue or cell type; and brief half-life in order to minimize potential off-target activity and innate and humoral immune responses. In addition, the ability to re-administer the therapy to attain stable, therapeutically relevant levels of gene editing would be an advantage. With these requirements in mind, we have explored the use of lipid nanoparticles (LNPs) for delivery of CRISPR/Cas9 components to the liver to mediate editing of target DNA within hepatocytes. Cas9 mRNA and chemically synthesized gRNA specific to the mouse transthyretin gene (Ttr) were co-formulated into LNPs, and administered to mice via intravenous tail vein injection. Various parameters were explored, including the nature of the guide RNA (sgRNA vs. dgRNA & chemical modification), the dosing regimen, and molecular strategy (single target site vs. two-target site micro-deletion). We found that the best results were obtained with a chemically modified single guide co-formulated with Cas9 mRNA. We were able to achieve a median dose-dependent editing of up to 55% of the gene copies in liver biopsies. A corresponding dose-dependent reduction of serum transthyretin protein levels was seen, with a decrease of up to 80%. Notably, the DNA repair patterns in liver were distinctly different from those seen in cell lines using the same Ttr-specific gRNA. These results demonstrate that therapeutically meaningful levels of in vivo CRISPR/Cas9-mediated gene editing can be obtained with a completely synthetic and scalable single-agent system.
: Chulalongkorn University
Wiskott-Aldrich syndrome is a rare severe X-linked disorder characterized by microthrombocytopenia, immune deficiency, recurrent infection, and a high risk of developing autoimmunity and cancer. The disease is caused by mutation in WAS gene which codes WASP protein, an important cytoskeletal regulator in many hematopoietic cells. We previously reported that megakaryocytes generated from induced pluripotent stem cells (iPSCs) derived from WAS patients exhibited defects in proplatelet formation and produced small size platelets. Here we demonstrate that correction of mutation in WAS iPSCs using zinc-finger nuclease (zfn) and Transcription activator-like effector nuclease (TALEN) mediated homologous recombination restored WASP expression in WAS-iPSC derived hematopoietic lineages to wild-type level. Megakaryocytes derived from gene-corrected WAS iPSCs improved proplatelet extension and produced bigger platelets than isogenic uncorrected lines. Gene-corrected WAS iPSCs- derived macrophage, NK cells and dendritic cells demonstrated improved cell migration and phagocytic activity than uncorrected-WAS iPSC derived cells. These results support therapeutic potentials of gene-corrected WAS-iPSCs
: CIBIO, University of Trento
Viral vectors are powerful tools to deliver exogenous factors in mammalian cells, including those cells that are refractory to any delivery technique (chemical and mechanical transfection techniques). Therefore, viral vectors have been widely used to transduce genome editing tools (ZFN, Talens and CRISPR/Cas9). Nevertheless, this technique of delivery is still strongly limited by two main aspects: 1) permanent expression of the genome editing tool which correlates with increased off target activity, 2) transfer of viral genetic material to target cells, correlating with pathogenic effects. We have developed two lentiviral (HIV-1) based delivery systems, consisting of viral like particles to transport CRISPR/Cas9 components, which has the potential to overcome the limitation mentioned above. The VLPs, were developed using two main approaches to incorporate Cas9 into the viral like particles. In addition, a dedicated strategy to express sgRNAs in the cytoplasm to optimize their incorporation into VLPs has been developed. The VLPs have been proven to efficiently induce NHEJ as well as HR of specific targeted genes in mammalian cells. We will present data proving in vivo editing with VLPs in the heart of mice. Being composed of ribonucleoprotein complexes, free from genomic materials, the VLPs are cleared from targeted cells after genome editing, minimizing the off-target activities and pathogenic potential correlating with viral genomes, thus paving the way for a safer genome editing platform for in vivo applications.
: Intellia Therapeutics
Gene editing approaches all require the introduction of double strand breaks (DSBs) in DNA, as the substrate for the repair and editing process. As for any pharmacologically active agent, off-target activity is a natural concern, especially due to permanent nature of genome editing. Numerous methods to measure off-target activity have been described, but unbiased, cell-based genome-wide methods are considered state-of-the-art. Oligo-based DSB capture in living cells, followed by next-generation sequencing and computational deconvolution, is a popular method, but is usually performed without reference to any recurrent DSBs that occur in the host, in the absence of editing agents. Here we describe new computational methods that first extract the background DSB model from the host, before asking what additional sites have been introduced. We find that in some transformed cell lines, there are thousands of genomic sites with recurrent DSBs, some of which occur as frequently as DSBs induced by nucleases such as Cas9. Thus, searching for sites that match a guide without subtraction of the background model can identify false positive sites due to a fortuitous match between guide sequence and site. Finally, guides that were initially selected to have relatively unique genomic sequences and then subjected to our new analysis method consistently showed very low off target activity.
: Institute for Cell and Gene Therapy, Medical Center - University of Freiburg, 79106, Germany
Patients with autosomal dominant hyper-IgE syndrome (AD-HIES) due to dominant-negative mutations in STAT3 suffer from different immunological and non-immunological features. While allogeneic haematopoetic stem cell transplantation is generally not recommended due to potential severe side effects, transplantation of autologous cells in which the mutated STAT3 allele has been specifically inactivated may represent a promising alternative. Here, we have generated allele-specific RNA-guided nucleases (RGNs) to target the most common STAT3 mutations (H58Y, C328_P330dup, V463del, R382W and V637M). To this end, we have used various Cas proteins and fine-tuned different guide RNA (gRNA) parameters, such as lengths and number of mismatched nucleotides between spacer and protospacer. Allele-specificity was initially tested in episomal reporter systems in which the normal or mutated STAT3 genes were fused to E2Crimson or EGFP, respectively. The most efficient allele-specific RGNs were then assessed for their ability to discriminate between chromosomal variants of normal and mutant STAT3 alleles in HEK293T cells. For the five mutations tested, allele-specific disruption frequencies ranged from 15% to 30%, leaving the normal STAT3 allele unaltered. Our results show that allele-specific gene disruption can be efficiently achieved using the RGN system. Applying these tools in patient-derived haematopoetic stem cells will represent a promising strategy to be explored for future clinical translation to treat AD-HIES and other diseases caused by dominant genetic defects.
: Murdoch Childrens Research Institute, Parkville, 3052, Australia
Human induced pluripotent stem cells (iPSCs) can be derived by the in vitro reprogramming of somatic cells and resemble embryonic stem cells (hESCs) derived from the inner cell mass of the pre-implantation blastocyst stage embryo. In vitro, pluripotent stem cells (PSCs) can be directed to differentiate towards specific lineages and therefore represent a tractable platform for the study of important events during the specification of early human cell types. The ability to reliably express either fluorescent reporters or other genes of interest is important for using pluripotent stem cells as a platform for investigating cell fates and gene gene function. We describe a simple expression system, designated GAPTrap (GT) in which reporter genes, including GFP, mCherry, mTagBFP2, luc2, Gluc, and lacZ have been inserted into the ubiquitously expressed GAPDH locus. Independent PSC clones harbouring variations of the GT vector express remarkably consistent levels of the associated reporter gene. In vitro differentiation experiments showed that reporters were maintained at robust levels in hematopoietic cells and cardiac mesoderm, CXCR4+EPCAM+ definitive endoderm and tyrosine hydoxylase+ neurons. Similarly, analysis of teratomas derived from hPSCs harbouring a GT-LacZ gene showed that beta-galactosidase expression was maintained in a spectrum of different cell types, including cartilage, pigmented epithelium and mesenchyme. Thus, the GAPTrap vectors represent a robust and straightforward tagging system that complements existing methods for gene over-expression in pluripotent stem cells and their differentiated derivatives.
: Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Australia
Gene correction through targeted genome editing would be the optimal treatment for patients with monogenic disorders. The recent adaptation of the CRISPR/Cas9 nuclease system for site-directed genome editing in mammalian cells provides an ideal tool for this therapeutic approach. However, precise gene correction relies on the activity of the cellular homology-directed repair (HDR) pathway, which is often overshadowed by the non-homologous end joining (NHEJ) pathway, a process associated with disruptive nucleotide deletions and insertions (‘indels’). In order to identify interventions that bias genome editing towards HDR, we have developed an assay that simultaneously detects HDR and NHEJ frequencies by targeting the fluorescent reporter GFP. We show that successful substitution of a single nucleotide via HDR converts GFP to BFP, resulting in a shift in the fluorescence excitation and emission spectra as detected by flow cytometry. Conversely, nucleotide indels as a consequence of NHEJ destroy the function of the reporter, which is detected by the loss of GFP fluorescence. In addition, we demonstrate that further modifications in the HDR template can increase the frequency of HDR as well as BFP fluorescence intensity. In two cell lines (K562 and in HEK293T) stably expressing GFP, we show targeted modification of GFP. HDR occurred at a frequency of up to 5.8% in K562 cells and 21.5% in HEK293T cells, corresponding to ∼18% and ∼40% of NHEJ frequency, respectively. We propose, that this novel cellular assay system will serve to facilitate the identification of compounds and genes that can enhance HDR in a high-throughput screen.
: Reprogramming and Gene Therapy, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany
Pulmonary Alveolar Proteinosis (PAP) constitutes a severe lung disease caused by the functional insufficiency of alveolar macrophages due to a deficient GM-CSF receptor. As treatment options for PAP are rare, we here evaluated the suitability of genome engineering to restore GM-CSF-mediated signalling in iPSC-derived monocyte/macrophages (M/M). PAP-iPSC were generated from CD34+-bone marrow cells of a GM-CSF receptor α-chain (CSF2RA, CD116) deficient PAP-patient (c.199C>T in exon 7). TALE-nucleases (TALENs) and RNA-guided nucleases (RGNs) were generated and stimulation of homology directed repair (HDR) in exon 7 evaluated in a HEK293T- reporter cell line. HDR efficiency was up to 100-fold higher in nuclease-treated samples as compared to controls. Alternatively to the direct repair of the disease-causing mutation, we integrated a codon-optimized CSF2RA gene into the AAVS1 safe harbor site of patient-derived PAP-iPSC. This strategy resulted in 100% targeted cells after puromycin selection with 95% mono-allelic integration determined by PCR. Moreover, detailed integration analysis by Southern blot in 6 selected clones revealed 50% of the correctly targeted clones, without additional vector integrations. Hematopoietic differentiation of PAP-iPSC clones yielded M/Ms of typical morphology and phenotype (CD14+, CD11b+, CD45+), capable of performing GM-CSF dependent functions (CD11b activation, GM-CSF uptake, and STAT-5 phosphorylation). These were profoundly impaired in M/Ms derived from uncorrected clones. Of note, M/M derived from AAVS1-corrected PAP-iPSC showed restored expression of CD116. In summary, we demonstrate the feasibility of using designer nucleases to restore CSF2RA expression in PAP-iPSC and highlight M/Ms differentiated from uncorrected PAP-iPSC clones as a functional disease model for hereditary PAP.
: Center for Regenerative Medicine-University of Modena
Engineered nucleases are extensively used for in situ correction of genetic defect by homologous recombination (HR) into the native locus. The genetic defect we are addressing is the junctional epidermolysis bullosa (JEB), a skin adhesion disorders due to mutations in the LAMB3 gene coding for the laminin-332. This project aims at in situ correction of the LAMB3 gene in primary JEB keratinocytes. We designed TALENs specific for intron II of LAMB3 gene and a HR cassette including a splicible LAMB3 cDNA. Immortalized JEB (iJEB) keratinocytes were treated with TALEN mRNAs and IDLV vector carrying the HR cassette. The in situ gene correction was evaluated by molecular analyses of single-cell clones isolated from the TALENs/IDLV-treated iJEB population. Gene-corrected clones hosted the HR cassette correctly integrated into the native locus, expressed the corrected LAMB3 gene and produced the laminin-332 protein. Based on the advantages of in vitro adhesion of LAMB3-corrected keratinocytes, we executed a “selective adhesion strategy” on iJEB bulk population transfected with TALENs mRNA and transduced with IDLV donor. A single round of enrichment resulted in 15-fold higher expression respect to untreated iJEB. The enriched iJEB-corrected bulk showed correct HR events and produced laminin-332 protein. To translate the knock-in targeting platform to primary JEB keratinocytes we developed an Adenovector carrying the CRISPR-Cas9 (AdCas9gRNA) nuclease targeting the same locus of TALENs. Co-infection of JEB keratinocytes with AdCas9gRNA vector and IDLV donor vector resulted in correct HR of the splicible LAMB3 cDNA only in the bulk that underwent “selective adhesion strategy”.
: Editas Medicine
Transplantation of genetically modified autologous hematopoietic stem/progenitor cells (HSPCs) is an effective treatment for many hematologic diseases; however, there remain a number of disorders that are not amenable to gene augmentation approaches. The ability to effect targeted genetic changes in HSPCs could provide a therapeutic approach to these otherwise untreatable diseases. Here we demonstrate precise and efficient genetic modification in human HSPCs by CRISPR/Cas9. Human CD34+ cells from 20 donors were electroporated with Cas9 ribonucleoprotein (RNP) targeting HBB, AAVS1, or CXCR4. Sequence analysis demonstrated up to 80% editing in cord blood CD34+ cells (mean ± s.d: 62% ± 9%) and up to 57% in adult CD34+ cells (39% ± 13%). Co-delivery of Cas9 RNP with single-stranded oligodeoxynucleotide donor (ssODN) led to up to 12% donor-mediated homology directed repair (HDR) and increased total editing (HDR+NHEJ) by 20% (RNP: 48% ± 15%; RNP+ssODN: 69% ± 8%). RNP-edited and control CD34+ cells reconstituted human hematopoiesis in primary and secondary recipient immunodeficient mice, with ∼85% human CD45+ cell peripheral blood reconstitution 4 months after primary transplantation. Human B and T lymphoid, myeloid, and erythroid cells were detected in hematopoietic organs, with 20% CD34+ cell engraftment in the marrow of both RNP-edited or control CD34+ cells. The level of editing in human myeloid, erythroid, and CD34+ cells recovered from primary recipients (∼50%) was similar to editing in the pre-infusion product (∼60%). These findings show that Cas9 gene-edited HSPCs retain long-term engraftment potential and support multilineage blood reconstitution in vivo, thus supporting further investigation of therapeutic strategies comprising CRISPR/Cas9-mediated gene editing of HSPCs.
: Molecular Imaging Research Center (MIRCen), Institut d'Imagerie Biomédicale (I2BM), Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA), Fontenay-aux-Roses, 92260, France
Huntington's disease (HD) is an autosomal dominant genetic disorder caused by a CAG repeat expansion mutation located in the first exon of the Huntingtin gene (HTT). We use spliceosome mediated RNA trans-splicing (SMaRT) to develop an innovative strategy of gene therapy that will significantly reduce or eliminate the expression of the mutant protein while restoring a physiological level of normal HTT. This process is achieved using an artificial RNA, termed pre-trans-splicing molecules (PTMs), which has the capacity to exchange the mutant exon with a mutation-free exon. In the case of a dominant mutation, therapeutic benefits can only result from high rate of corrections. Therefore, our goal is to achieve the most efficient trans-splicing reaction possible by implementing a high content screening in order to select HTT-targeting PTMs, efficient enough to trigger a therapeutic benefit for the affected neuronal population. Our screening strategy is based on a fluorescent reporter system which results in the production of two fluorescent proteins with different emission spectra indicative of cis- and trans-splicing events, suitable with an easy detection and quantification. This strategy requires the construction of PTM libraries and the development of human cell lines allowing the assessment of their efficiency. Pre-screening validation, that confirmed the reliability of this system and the possibility of identifying the best PTMs as well, will be presented. Selected PTMs will subsequently be vectorized in a gene transfer vector and validated in term of therapeutic efficacy in relevant cellular models, such as HD patient-derived iPS cells demonstrating mutation-mediated cellular dysfunctions.
: Universidade Federal do Rio Grande do Sul
: EB House Austria, Research Programme for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, 5020, Austria
The inherited skin disease epidermolysis bullosa (EB) is characterized by severe blistering of the skin and mucous membranes caused by minor mechanical trauma. Dominant negative mutations within KRT14 encoding keratin 14 are responsible for EB simplex (EBS) and lead to loss of integrity of the intermediate filament network within basal keratinocytes. In order to correct a dominant mutation in an EBS patient cell line, the spliceosome-mediated RNA trans-splicing (SMaRT) technology was exploited to recombine two different RNA molecules by trans-splicing to create a new chimeric gene product. RNA trans-splicing molecules (RTMs) bind to the target pre-mRNA via a complementary binding domain (BD) carrying the necessary core splicing signals to facilitate the splicing reaction as well as the wild type coding sequence for correction. In order to increase the repair efficiency of a previously created KRT14-specific RTM, antisense RNAs (asRNAs) binding the KRT14 target pre-mRNA region were randomly generated and tested via a fluorescence-based split-GFP screening system. Reconstitution of the GFP signal serves as a readout for accurate trans-splicing between the target region and the RTM. We analyzed the functionality of 76 asRNAs varying in length and binding position in triple-transfection experiments in HEK293 cells and achieved an increase in trans-splicing efficiency of up to 7-fold as monitored by flow cytometry. This enhancement was also confirmed by sqRT-PCR analysis. Our results suggest that the inclusion of asRNAs can increase the trans-splicing efficiency to levels that may be sufficient to overcome the EBS phenotype.
: Leiden University Medical Center
RNA-guided nucleases (RGNs) composed of single-guide RNAs (sgRNAs) and Cas9 nucleases derived from prokaryotic CRISPR/Cas9 adaptive immune systems have become preeminent tools for modifying specific genomic sequences in cells of higher eukaryotes. Importantly, various strategies aiming at improving the specificity of RGNs are being devised for minimizing collateral off-target DNA cleavage. These strategies include the use of paired RGN nickases, truncated sgRNAs (tru-sgRNAs) and Cas9 variants with enhanced DNA targeting specificities. The impact that higher-order chromatin structures have on these high-specificity gene editing approaches is, however, presently unknown. Therefore, in this study, we sought to investigate the interaction between distinct chromatin conformations and high-specificity gene editing tools by using validated quantitative cellular models in which the euchromatic and heterochromatic statuses of isogenic target DNA sequences are stringently controlled by small-molecule drug availability (Chen et al. Nucleic Acids Res. doi: 10.1093/nar/gkw524). Our experiments revealed that targeted mutagenesis induced by pairs of nicking RGNs, containing Cas9.D10A or Cas9.H840A, or by cleaving RGNs harbouring tru-gRNAs and wild-type Cas9, are significantly hindered at heterochromatic DNA sequences. The extent to which the levels of targeted mutagenesis are reduced at epigenetically closed DNA sequences varied from about 2-fold to over 10-fold. Moreover, when compared to RGNs containing full-length sgRNAs, RGN complexes harbouring high-specificity Cas9 variants and tru-sgRNAs displayed impaired site-specific DNA cleaving activities. We conclude that the epigenetic environment in which the primary RGN target sequences are embedded is an important parameter to consider during the selection and conception of efficient and specific gene editing protocols.
: Josep Carreras Leukemia Research Institute
Coenzyme Q10 (CoQ10) plays a crucial role in mitochondria as an electron carrier within the mitochondrial respiratory chain (MRC), and is an essential antioxidant. Mutations in genes responsible for CoQ10 biosynthesis (COQ genes) cause primary CoQ10 deficiency, which is a rare and heterogeneous mitochondrial disorder with no clear genotype-phenotype association, mainly affecting tissues with high-energy demand including brain and skeletal muscle (SkM). Here, we report a 4-year old girl diagnosed with modest mental retardation and lethal rhabdomyolysis harboring a heterozygous mutation (c.483G>C (E161D)) in COQ4. The patient's fibroblasts showed a severe decrease in [CoQ10], CoQ10 biosynthesis and MRC activity affecting complexes I/II+III. Bona fide iPSC carrying the COQ4 mutation (CQ4-iPSC) were generated, characterized and genetically edited using the CRISPR-Cas9 system (CQ4ed-iPSC). Extensive differentiation and metabolic assays of control-iPSC, CQ4-iPSC and CQ4ed-iPSC demonstrated a genotype-phenotype association, faithfully reproducing the disease features. Similar to the patient`s symptoms, the COQ4 mutation in iPSC was associated with CoQ10 deficiency, metabolic dysfunction and impaired differentiation into SkM; however, it did not impair iPSC differentiation into dopaminergic or motor neurons. This study offers an unprecedented iPSC model recapitulating CoQ10 deficiency-associated functional and metabolic phenotypes caused by COQ4 mutation.
: San Raffaele Telethon Institute for Gene Therapy (SR-Tiget)
We are developing a gene editing strategy in HSPC to correct the IL2RG mutations causing SCID-X1 disease. To model SCID-X1 gene correction, we developed a mouse model carrying the human IL2RG including a disease-causing mutation in place of murine Il2rg. To evaluate efficacy and safety of the hematopoietic reconstitution from a limited number of corrected HSPC we performed competitive transplantation with wild-type and IL2RG-/- HSPC and found that 1% of WT cells are sufficient to partially reconstitute the lymphoid compartments and that the administration of a conditioning regimen before HSPC infusion is required to avoid lymphoma development from the transplanted progenitors. We then tested gene correction of the SCID-X1 Lin- HSPC by the delivery of donor template by IDLVs followed by transfection of ZFN mRNAs. This protocol yielded high on-target nuclease activity (40%) and ∼6% HDR integration, but also high cytotoxicity. Upon transplant, only the gene corrected cells were able to generate functional B and T lymphoid lineages, showing clear selective advantage over uncorrected cells. Whereas editing (by NHEJ and HDR) was nearly undetectable in the engrafted HSC, corrected lymphoid cells persisted long-term in the mice, indicating successful editing of 1% progenitors able to sustain lymphopoiesis and partially correct the disease phenotype. We finally developed a new protocol based on CRISPR/Cas9 that enabled substantial levels of targeted DNA repair by NHEJ (70%) and HDR (∼25%) with minimal cytotoxicity and provided stable engraftment of the edited cells in transplanted mice. We are currently evaluating long-term phenotype correction by this improved protocol.
: bluebird bio
Genetic disruption of immunoregulatory genes via nuclease-mediated genome editing may improve adoptive T cell immunotherapy outcomes, provided potency enhancement can be accomplished without compromising safety. Beyond the challenge of striking an appropriate potency/safety balance, therapeutic genome editing must also be achieved with sufficient specificity to minimize off-target editing events. Multiplexed gene editing magnifies this challenge, highlighting the need for highly selective enzymes which act in an orthogonal manner to reduce cumulative off-target activity. MegaTALs, derived by fusing a TALE DNA binding array to an engineered meganuclease, can be iteratively refined in response to off-target detection, achieving a genome-wide increase in on- versus off-target discrimination. To illustrate this, we generated a panel of megaTALs targeting several immunotherapy-related genes. Specifically, we reprogrammed a megaTAL to target the TCRα gene and identified a dominant off-target site. Optimization of this megaTAL using a combination of protein engineering approaches, exploiting the depth and modularity of our meganuclease variant databases, resulted in greatly improved on- to off-target ratios with the primary off-target site eliminated. Importantly, multiplexed gene editing in primary T cells resulted in the simultaneous disruption of multiple receptor gene targets while achieving the desired resistance to ligand stimulation, highlighting the orthogonal behavior of the megaTAL reagents. These data demonstrate that the megaTAL architecture facilitates the iterative refinement of nuclease specificity and activity, generating exquisitely selective, single-component reagents. Additionally, multiplexed editing may enable T cell anti-tumor responses to be tuned to reduce the impact of immunosuppressive external stimuli, producing a potentially more efficacious immunotherapy product.
: San Raffaele Telethon Institute for Gene Therapy (SR-Tiget)
Targeted genome editing has brought gene correction within the reach of gene therapy. We recently showed that editing in the most primitive hematopoietic stem/progenitor cells (HSPC) is constrained by gene transfer efficiency and a bias against the use of homology directed repair. By coupling tailored HSPC culture conditions with optimal methods for transient but sustained delivery of donor template and engineered nucleases, we overcame in part these barriers and provided evidence of targeted integration (TI) in long-term repopulating HSPC. Here, in order to improve the tolerability of the procedure and establish a transferable-to-the-clinic gene correction protocol we further optimized reagents and scaled-up the gene editing procedure. We developed new ZFNs targeting an upstream intron of the IL2RG gene to correct the majority of SCID-X1 mutations with only one ZFN/donor set. We compared the performance of IDLV and AAV6 as donor vehicles and found a similar rate of HDR, allowing up to 20% TI in bulk HSPC. We found that inclusion of modified nucleotides during mRNA production and clinical grade purification of IDLV allow decreasing cellular innate response and electroporation toxicity, respectively, and increased the fraction of edited cells. To optimize ex-vivo HSPC manipulation we tested pyrimidoindole derivatives added to the culture and found improved HSPC expansion in conditions that preserve their primitive phenotype, thus increasing the yield of edited cells that are able to repopulate NSG mice. Currently, we are producing large-scale lots of gene-corrected cells using high volume electroporators and have successfully treated up to 40 million HSPCs.
: Center for Stem Cell and Regenerative Medicine, Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX, 77030, USA
Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disease. It is caused by mutations in the WAS gene (WAS) which encodes for the WAS protein (WASp), exclusively expressed in hematopoietic cells and required for proper lymphoid cell function. We investigated restoration of T- and NK-cell functionality following a zinc-finger nuclease (ZFN)- mediated genome editing strategy for correction of WAS. We generated induced pluripotent stem cells (iPSC) from skin fibroblasts of a WAS patient carrying an insertional frame-shift mutation. Subsequently, a WAS-2A-eGFP transgene was targeted at the endogenous chromosomal location by homology-directed repair using ZFN, thereby correcting the gene defect and creating a GFP reporter for WASp expression. Hematopoietic progenitor cells were generated from WAS iPSC and gene-corrected iPSC (cWAS). GFP expression was pronounced in all CD43+ hematopoietic lineages. Hematopoietic precursors were cultured on OP9-DL1 to generate T and NK cells. NK cells were readily obtained from cWAS and hESC (WA01/WA09) progenitors, but to a far more limited extent from WAS progenitors. WAS-derived NK cells were unable to generate IFN-γ or TNF-α upon stimulation with K562. Cytokine production was restored in cWAS- derived NK cells. Interestingly, in T cell generation, although WAS, cWAS, and WA01 lines were able to generate CD5+CD7+ T cell precursors, only low numbers of CD3+ TCRαβ and TCRγδ cells were obtained with WAS. Significantly, T cell generation was restored in cWAS. Taken together these results indicate that targeted correction of the WAS gene results in restoration of the lymphoid developmental and functional defects observed for WAS-iPSC.
: CIEMAT/CIBERER
Fanconi anemia (FA) is an inherited disease associated with bone marrow failure and cancer predisposition. This disease is caused by mutations in any of the 19 FANC genes that belong to FA/BRCA DNA repair pathway. In this study, we have conducted a gene editing strategy aiming at the insertion of a reporter EGFP and a therapeutic hFANCA donor into the Mbs85 safe harbor locus, ortholog of the human AAVS1 locus. To achieve our goal, Mbs85 TALEN were nucleofected together with the different donor templates in Fanca-/- (FA-A) MEFs and also in mouse HSPCs from WT and FA-A mice. In these experiments, we demonstrated the cleavage activity of designed TALEN in the Mbs85 locus of different mouse cell types. Additionally, targeted integration (TI), as well as hFANCA expression and also phenotypic correction was demonstrated in gene-edited FA-A MEFs, as deduced from the reversion of their characteristic mitomycin C (MMC) hypersensitivity. Gene editing experiments with WT and FA-A HSPCs also allowed us to demonstrate TI in these cell types. As observed in FA MEFs evidence of phenotypic correction was observed in FA hematopoietic progenitor cells, although a significant toxicity was associated to the DNA nucleofection of these cells. Our results demonstrate for the first time the feasibility of performing a therapeutic targeted integration strategy in a safe harbor locus in cells from a mouse model of FA.
: KU Leuven
To fulfill a productive infection cycle, HIV co-opts cellular proteins, coined host-cell co-factors. Interference with these co-factors hold great promise in protecting cells against HIV infection. CRISPR/Cas9 has been successfully implemented to inhibit viral entry and infection by disrupting CCR5 and/or CXCR4, a receptor and co-receptor for the HIV-1 infection. LEDGF/p75 is the cellular co-factor, encoded by the PSIP1 gene, that binds HIV integrase (IN) and tethers the pre-integration complex of HIV to the host cell chromatin. Depletion of LEDGF/p75 in human CD4+ T-cells renders cells refractory to HIV replication, opening perspectives for gene therapy. However, LEDGF/p75 also serves as a docking hub for endogenous proteins, and thus complete disruption may be detrimental if applied to hematopoietic stem cells. Here, we describe a PSIP1 editing approach that exclusively disrupts the interaction with HIV IN while retaining its cellular function. First, we demonstrated the functionality of the designed gRNAs by CRISPR/Cas9-based indel formation in PSIP1 in T-cell lines. Next, we introduced a site-specific mutation, replacing the aspartic acid residue that supports HIV-IN binding (D366N) via homology directed repair. The resulting cell line showed successful disruption of the LEDGF/p75-IN interaction while retaining the potential to bind cellular binding partners. In our hands, the LEDGF D366N cells do not support HIV replication. These results underscore the potential of site-directed knock-in mediated by CRISPR/Cas9 to render cells resistant to HIV infection, and provide a new strategy to protect patient derived T cells against HIV-1 infection.
: Human Stem Cells Institute, Moscow, Russia
Mutations in DYSF gene lead to limb girdle muscular dystrophy type 2B and Miyoshi myopathy, which are among the most prevalent muscular dystrophies. There is no effective treatment for these diseases yet. We have chosen artificial trans-splicing (AT) to correct mutations in this gene. AT allows to reprogram RNA splicing process to substitute a corrupt exon to a normal via pre-trans-splicing molecules (PTMs). PTMs are complex molecules consisting of 3 domains: binding domain, targeted to an exon of interest; splicing domain, initiating the splicing process on a PTM; and a coding domain, a sequence, coding a new exon. We will assess two types of AT: 5’-trans-splicing for the exon 3 of the DYSF gene and internal exon replacement for the exon 26. We have immortalized patients’ skin and gum fibroblasts for the exon 26 mutation and patients monocytes for the exon 3 mutation. All cell lines will proceed DYSF gene activation by a CRISPR/Cas9 SAM tool, DYSF gene activation will be confermed by RNA-seq. Afterwards cells will be transfected by AAV8 carrying PTMs targeted to corresponding exons. Changes in DYSF gene expression will be evaluated by PCR, restriction analysis and RNA-seq. After AT performed gum fibroblasts will be trans-differentiated to myoblasts and myotubes to assess ultrastructural changes by electron microscopy and to perform a comparison with myotubes derived from healthy patients.
: INSERM UMR1193
Familial hypercholesterolemia type IIa (FH) is a liver genetic disease causing early cardiovascular disorders due to mutations in the Low Density Lipoprotein receptor (LDLR) which removes cholesterol from the circulation. As hepatocytes are the only cells able to metabolize cholesterol into bile acids, they are therefore the target for FH cell/gene therapy approaches. We have generated FH-induced pluripotent stem cells (FH-iPSCs) from a homozygous patient with an early stop codon in the LDLR sequence and showed that they display the disease phenotype. We have evidenced a role of LDLR in VSV-G pseudotyped lentiviral vector entry on these cells. We have improved a stepwise protocol mimicking liver organogenesis to differentiate these FH-iPSCs into hepatocytes expressing transcription factors (HNF4α, HNF3β), specific proteins (albumin, α-1 antitrypsin) and Hepatitis C Virus (HCV) receptors (claudin1, CD81, SRB1). We have shown that Ataluren, a small-molecule drug used for CFTR treatment, suppressed the premature stop codon and promoted LDLR read-through translation. We have also genetically corrected the FH-iPSCs using CRISPR/Cas9 technology and a therapeutic cassette containing the normal LDLR cDNA controlled by Apolipoprotein A2 promoter. Half of the 33 isolated clones showed accurate monoallelic integration at the targeted AAVS1 safe harbor locus. We differentiated one of them into hepatocytes that expressed LDLR and showed functional correction by internalization of Dil-LDL fluorescent ligand. Both corrected and non-corrected FH-iPSCs will now be used to study the regulation of cholesterol pathways and to further understand the still debated role of LDLR in HCV entry.
: MolMed S.p.A.
Targeted gene correction is achieved by the sequential delivering of first an integration-defective lentiviral vector (IDLV) carrying the corrective cDNA template sequence and then a pair of Zinc Finger Nucleases (ZFN) mRNAs specific for the mutated gene into target cells. ZFN mRNA is normally transcribed in vitro starting from cDNA plasmid template. A major limitation of this procedure is the generation of residual contaminants, i.e. short RNAs and double stranded RNAs that affects spectrophotometric quantification of the product and leads to risky activation of unwanted innate immune response. To improve nuclease expression, decrease mRNA immunogenicity and thus lengthen modified cells persistence in vivo, we combined different strategies. (i) the inclusion of UTRs and polyA tails in the plasmid template; (ii) the use of modified nucleotides for mRNA production and (iii) the purification of the mRNAs by dHPLC Wave System (Transgenomic); (iv) the electroporation of primary cells by the 4D-Nucleofector (Lonza). We produced up to 500 μg of mRNA using pseudo-UTP, 5-Methyl-CTP modified-nucleotides and 3’ O-Me-m7G(5’)ppp(5’)G RNA Cap in a single reaction and dHPLC purified up to 500 μg RNA in a single injection for each preparative run with an average 60% purification yield. The level of residual organic purification solvent is compliant to regulatory indication and the efficiency of ZFN mRNA nucleofection is 50% (NHEJ %) in up to 1 × 108 primary T lymphocytes without observing any cell toxicity. In conclusion, the developed process represents a feasible and reproducible protocol applicable in any gene editing clinical protocol.
: Inserm UMR1193
Hemophilia B (HB) is a genetic disorder characterized by a reduced activity of circulating clotting factor IX (FIX), synthesized by hepatocytes. Current treatment based on regular intravenous injections of FIX is very restrictive, costly and only palliative. Gene therapy trials show promising results but their long-term efficacy is still unknown. It is therefore important to explore other treatment strategies. To demonstrate the feasibility of a gene/cell therapy approach using patient's iPSCs, we reprogrammed skin fibroblasts from a severe hemophilia B patient (FIX activity <1%). One iPSC clone was deeply characterized by conventional techniques demonstrating self-renewal and pluripotency. Karyotype of the iPSCs was normal and DNA sequencing confirmed the presence of the patient's F9 mutation. We then targeted, using the CRISPR/Cas9 technology, the genomic insertion of a therapeutic cassette including the apolipoprotein AII (APOAII) promoter driving the expression of a F9 mini-gene bearing the Padua mutation to enhance FIX specific activity. One third of the amplified clones showed accurate monoallelic integration at the targeted AAVS1 safe harbor locus. The corrected or non-corrected iPSCs were differentiated into hepatocytes expressing specific markers like HNF4α and albumin. Due to the promoter used, the FIX mRNA expressed by the therapeutic cassette is detected during the early stages of differentiation whereas the RNA of the non-corrected clone is only detected at late stages. The therapeutic efficacy of the correction in vivo is being assessed by transplantation of hepatocytes derived from corrected iPSCs into the liver of newborn HB mouse model.
: Faculty of Animal Science and Food Engineering (FZEA)/University of Sao Paulo
The Duchenne muscular dystrophy (DMD) is a genetic disease that causes progressive degeneration of the skeletal muscles, affects 1 in every 3.500 born boys. Preclinical studies in dogs (Golden Retriever Muscular Dystrophy – GRMD) may better predict the potential for success of a treatment in humans. This study aims to generate iPS (induced pluripotent stem cells) from Duchenne muscular dystrophy affected dogs, to characterize these cells and to correct the mutation by precise gene editing trough CRISPR-Cas9 technology. Two affected animals were selected after clinical evaluation and skin samples were collected according to previously described methods for isolation of fibroblasts. All experiments were performed by comparison with control animal (fibroblasts established from healthy dog) and approved by Ethic Committee (CEUA 8048210116). The fibroblast cells were molecular characterized for a mutation in a dystrophy gene by PCR and restriction enzyme and used for reprogramming process (STEMCCA lentivirus vector - human and/or murine factors), with different protocols (FGF2, LIF) for subsequent correction (CRISPR-Cas9 editing methodology). For editing gene, the mutated region was sequenced, and the product used as template for plasmid construction. After cloning, the plasmids will be transfected with lipofectamine. The colonies will be sequenced after correction by CRISPR-Cas9 to confirm the knock-in. The genetic correction of GRMD into iPS cells expands a newly explored field, allowing the correction in the Golden Retriever model, screening this gene and mutation site and establishment an important disease model. Basing future research in humans, enabling the correction of a degenerative genetic disease.
: Leiden University Medical Center
Mutations disrupting the reading frame of the ∼2.4 Mb dystrophin-encoding DMD gene cause a fatal X-linked muscle-wasting disorder called Duchenne muscular dystrophy (DMD). Genome editing based on paired RNA-guided nucleases (RGNs) from CRISPR/Cas9 systems has been proposed as a therapeutic approach for permanently repairing faulty DMD loci. However, amongst other research, such multiplexing strategies warrant developing and testing delivery systems for introducing gene editing tools into target cells. We have investigated the suitability of adenoviral vectors (AdVs) for multiplexed DMD editing by packaging in single vector particles expression units encoding the Streptococcus pyogenes Cas9 nuclease and sequence-specific gRNA pairs. These RGN components were customized to trigger short- and long-range intragenic DMD segments encompassing reading frame-disrupting mutations. Following “all-in-one” AdV transductions of patient-derived muscle progenitor cells, we demonstrate that both of these gene editing strategies result in the correction of over 10% of defective DMD alleles readily rescuing dystrophin synthesis in unselected muscle cell populations. Moreover, we report that AdV-based gene editing can be tailored for removing mutations spanning the over 500-kb major DMD mutational hotspot. Hence, this single DMD editing strategy can in principle tackle a broad spectrum of mutations present in more than 60% of patients with DMD. Towards potential clinical translation, “all-in-one” AdVs will serve as robust and versatile tools for facilitating the testing of different DMD editing reagents and strategies in human myogenic cells harbouring mutations which are not represented in the currently available animal models.
: Cell Therapy Program, Center for Applied Medical Research (CIMA), University of Navarra. Instituto de Investigación Sanitaria de Navarra, IdiSNA, Pamplona, Spain
The CRISPR/Cas9 system has emerged as an editing tool that allows quick and easy modifications of the genome, including NHEJ-mediated gene knock-down or HDR-mediated correction of genetic diseases. In particular, in vivo genome editing is a promising approach for the development of new treatments for inherited metabolic rare diseases, like primary hyperoxalurias (PH1), where current treatments are very limited. PH1 is a rare autosomal recessive inherited metabolic disorder of the glyoxylate metabolism, where the excessive oxalate production leads to calcium oxalate stone formation and renal parenchyma damage, which results in progressive deterioration of renal function and, eventually, end-stage renal disease. The combined liver-kidney transplantation is the only curative treatment. Substrate reduction therapies (SRT) try to prevent the production of oxalate precursors like glyoxylate. In this work we have evaluated the therapeutic potential of CRISPR/Cas9-mediated in vivo gene editing as SRT strategy in a mouse model of PH1. In particular, we have explored the efficacy of knocking-down the hydroxyacid oxidase 1 (Hao1) gene, which encodes glycolate oxidase (GO), to reduce the hepatic conversion of glycolate to glyoxylate. This approach efficiently reduced GO protein levels in the livers of mice, normalizing the urine oxalate levels. These studies provide convincing evidence of the efficacy of in vivo gene editing as promising therapeutic approach for metabolic diseases like PH1.
: Cell Differentiation and Cytometry Unit-Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, 28040 Madrid, Spain
Pyruvate Kinase Deficiency (PKD) is an autosomal recessive disease caused by mutations in the PKLR gene. PKD produces chronic non-spherocytic hemolytic anemia, which can be fatal during early childhood. The only curative treatment is allogeneic bone marrow transplantation, making PKD a good scenario for gene therapy. We developed a gene therapy strategy in a PKD mouse model using a lentiviral vector (LV) carrying a codon-optimized version of the PKLR cDNA (coRPK). This vector has been recently designated as Orphan Drug for the treatment of PKD by the EMA and FDA. Due to difficulties obtaining primary CD34+ cells from PKD patients to test the efficacy of the therapeutic LV, an alternative source of human PKD-hematopoietic progenitors is needed. Thus, we have generated CRISPR/Cas9 system tools to knock-out RPK expression. Up to six different gRNAs were specifically developed to cleave the wild type PKLR gene. All gRNAs contain at least 3 mismatches with the coRPK to avoid the cleavage of the therapeutic transgene. Two gRNAs showed the capability to generate double strand breaks in the PKLR gene in 293T cells and in primary CD34+ cells. In order to identify and select edited cells, Cas9-gRNAs components were cloned into a plasmid Cas9-2A-ZsGreen1. Cord Blood CD34+ cells were electroporated, sorted and differentiated in vitro along the erythroid lineage. Final maturation of RBC is impaired due the targeted gene editing, validating this approach as a model for PKD that can be used to investigate the mechanism of action of this mutation and potential therapeutic interventions.
: Généthon, R&D department, INTEGRARE research unit, Evry F-91002, France
Autologous transplantation of genetically corrected hematopoietic stem cells (HSC) is an attractive therapeutic alternative for patients with genetic blood diseases lacking a compatible HSC donor. This study aims at developing an efficient genome editing-based targeted therapy for β-hemoglobinopathies. The CRISPR/Cas9 system is a suitable tool for reproducing naturally occurring large deletions (Hereditary Persistence of Fetal Hemoglobin) in the β-globin locus, which result in beneficial expression of fetal hemoglobin (HbF). Here, we aimed at optimizing and comparing the efficiency of RNA-, ribonucleoprotein particle (RNP)- and lentiviral (LV)-based methods to deliver the CRISPR/Cas9 system into hematopoietic stem/progenitor cells (HSPC). Specific gRNAs generating targeted deletions in the HBB locus were selected to this purpose. We optimized the RNA and RNP nucleofection protocols to achieve a high genome editing rate coupled with minimal toxicity. We tested several parameters, such as nucleofection program, cell concentration and RNA/RNP amount, and we ameliorated cell culture conditions to maximize CRISPR-Cas9 delivery and HSPC viability. In parallel, we explored the use of lentiviral vectors to deliver the CRISPR/Cas9 system in hematopoietic cells. We designed LVs expressing: (i) gRNA pairs generating the targeted deletions and a GFP reporter transgene; (ii) Cas9 nuclease and the blasticidin resistance gene. Genome editing efficiency was evaluated in HSPC as frequency of targeted deletion and InDels events at each gRNA target site. HbF re-activation was observed by RTqPCR and FACS analyses in HSPC-derived erythroid precursors upon targeted disruption of potential HbF silencers.
: Intellia Therapeutics
The programmable CRISPR/Cas9 nuclease emerged in 2012 into an environment well-poised to capitalize on its therapeutic potential. However, in order to bring CRISPR/Cas9 technology to the clinic, a number of technological hurdles needed to be addressed. We have been creating a platform that addresses these hurdles, to allow the efficient identification of safe & efficacious CRISPR/Cas9-based agents for both the ex vivo and in vivo delivery setting. For in vivo delivery, we have focused on the use of biodegradable lipid nanoparticles (LNPs) targeting the liver, and have developed a completely synthetic and scalable system where a single LNP administration can achieve up to 55% liver editing and 80% serum protein knockdown. To better evaluate specificity, we have been exploring the background rates of DNA double strand breaks in primary cells and cell lines, and have shown that guides with low to no off-target activity are readily found. One key finding from this data set as a whole is that the pattern and frequency of repair events is locus- and cell type-specific, although very reproducible in a given setting, implying that screening of guides has to be conducted with the ultimate target cell in mind.
: UCL Institute of Child Health
XLA is an inherited immunodeficiency caused by mutations in the gene encoding the Bruton Tyrosine kinase (BTK) that impair B cell development and lead to the absence of mature B lymphocytes in peripheral blood and lymphoid tissues. Consequently, XLA patients suffer from recurrent and often life threatening bacterial infections, partially ameliorated by a life-long immunoglobulin therapy. A promising strategy for a definitive treatment of this disease is the replacement of the faulty BTK gene with a correct copy via Cas 9 RNA guided endonucleases (RGNs)-mediated Homology Directed Repair (HDR). To this aim we have designed and tested in a B cell line, four RGNs targeting BTK exon 1 or 2. Using TIDE analysis we found indel rates in the BTK locus of up to ∼64% upon ribonucleoprotein delivery. Via single cell cloning we generated a BTK deficient B cell line in which ablation of BTK was confirmed by DNA sequencing and protein readouts. To test a potential therapeutic strategy for XLA patients, the two best performing RGNs have been further tested in human CD34+ cells. Both RGNs were able to disrupt BTK in CD34+ cells, albeit to a lesser extent as compared to B cell lines. HDR is currently being attempted in our BTK deficient cell line and in human CD34+ cells by co delivery of RGNs ribonucleoproteins and AAV6 donor template containing a GFP reporter cassette flanked by ∼800bp BTK homology arms. AAV6 transduction rates of up to 50% in CD34+ cells show the feasibility of our approach.
: University of California, San Francisco
Cystic fibrosis (CF) is the most common inherited disease in the Caucasian population with significant multiorgan damage, caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The approach of gene and cell therapies makes it possible to develop personalized and mutation specific therapeutic strategy for CF reported >2,000 CFTR mutations. The studies here describe the generation of iPSCs from a CF patient homozygous for the W1282X, Class I CFTR mutation, and its seamless correction via Homology Directed Repair (HDR) and via plasmid donor DNA insertion. By following the common seamless gene editing technique, we first performed a drug selection-based and HDR-depended gene correction using wtCFTR PiggyBac donor DNA carrying puromycin resistance gene with CRISPR/Cas9 nickases. While the donor DNA alone did not catalyze site-directed HDR in clones exhibiting puromycin resistance, co-transfection of the donor DNA with the optimized CRISPR/Cas9n pairs gave a targeting efficiency of 4.76% (1/21), 5.7% (2/35) and 28.6% (12/42) (colonies with HDR/puromycin resistant colonies) in independent experiments. The drug-resistance cassette in the clones was excised by overexpressing PiggyBac transposase resulting in seamless correction of the CF-iPSCs. Interestingly, as the separated event above, we also confirmed plasmid donor DNA insertion resulting tandem duplication of targeting sequence with the frequency of 57.1% (20/35) and 19.0% (8/42) in independent experiments. And we also demonstrated the excision of this insertion to obtain seamless corrected CF-iPSCs. Here we show seamless gene correction in CF-iPSCs using a novel technique through donor DNA insertion as well as PiggyBac-depended HDR.
: MEL, Agency for Science Technology and Research
Down Syndrome (DS) is one of the most frequent genetic causes of intellectual disability characterized by several pathological phenotypes; among which muscular dystrophy and cardiac defects are key features. To understand the pathogenesis of DS, we have preprogrammed amniotic fluid (AF) from woman carrying DS pregnancy into DS iPSC and compared it to normal AF derived iPSC. These DS iPSC have in turn been characterized and differentiated to form motor neurons and cardiomyocytes. In parallel, we have carried out a targeted removal of the trisomy via CRISPR technology to create isogenic, disomic lines of the iPSC derived from DS AF cells. These DS iPSCs derived from the AF would further allow for us to study the role of DS specific biomarker genes, by comparing it with healthy closely matched controls. The use of CRISPR on these DS iPSC lines have allowed us create targeted knockouts of DS specific candidate genes and subsequently, we hope to study the effect of inhibiting these genes on cellular phenotypes derived from DS iPSC; using the reverted disomic lines as control to provide futher novel mechanistic insights into AD pathogenesis. This study will lay the groundwork for understanding the effect of aneuploidy in the early development of DS and establishing DS cellular phenotypes in culture. Furthermore, it would establish the generation of efficient techniques in deriving iPSC from fetal stem cells as well as identifying possible drug targets for DS therapeutics.
: University of Helsinki
Hemoglobinopathies, like sickle cell disease and beta thalassemias are inherited diseases affecting tens of millions of families and causing approximately 100,000 deaths yearly. These recessive diseases are caused by mutations in the hemoglobin (Hb) beta gene (HBB). The production of the beta subunit of Hb changes during human development genetically from expression of the embryonal (HBE) to fetal (HBG) to adult (HBB) gene. One possible treatment modality is to increase the production of fetal hemoglobin from the HBG gene, but pharmacological agents like hydroxyurea have only a limited effect in patients. An alternative possibility is the transcriptional activation of the HBG gene, which is normally silenced at birth. Some individuals retain a persistent HBG production due to inherited genetic variants, known as the Hereditary Persistence of Fetal Hemoglobin syndrome (HPFH). These described variants vary from point mutations to copy number variations. To test the effects of different HPFH genetic variants, we developed an in vitro model using a reporter cell line in which we can measure the production of HbF in differentiated hematopoietic cells. Using CRISPR/Cas9, we knocked-in an EmGFP reporter in the HBG2 coding region in HUDEP-2 cells. This hematopoietic cell line grows undifferentiated when stimulated with doxycycline and can be directed to differentiate into erythroid lineage and produce mature erythrocytes. We will genome edit in this reporter cell line the different genetic variants found in the HPFH patients to determine their effects in HBG gene regulation.
: University of Helsinki
Human pluripotent stem cells (hPSC) can be efficiently differentiated into monohormonal pancreatic endocrine cells that are functionally immature. We have recently developed an inducible system to activate transcription of endogenous genes based on the use of the catalytically inactive Cas9 (dCas9) protein fused with artificial transcriptional activators. Differentiation of the hPSC resulted in the generation of islet-like cell clusters that contain endocrine cells (10–20% single INS+; 5–10% double INS+GCG+ cells). In vitro static glucose stimulated insulin secretion showed that these cells are not responsive to glucose alone, but they do respond when treated with Forskolin, Tolbutamide, KCl or when other metabolic fuels, leucine, glutamine or pyruvate, are used. While the levels of some beta cell markers like PDX1, NKX6.1 and MAFB are similar to adult human islet controls, the levels of INS, and mature beta cell markers MAFA and GCK are significantly lower, explaining why these cells are not yet glucose responsive. After transplantation, the cells undergo functional maturation in vivo and start to exhibit glucose stimulated insulin secretion. Our results suggest that the stem cell-derived beta cells are metabolically immature and require final maturation steps to become fully functional in vitro. We have generated hPSC-lines containing the CRISPR/dCas9 activator system where we can control the transcription of mature beta cell genes MAFA, GCK and UCN3. Characterisation of the functional outcome is ongoing. The combination of CRISPR/Cas9 activation systems with stem cell differentiation is a promising approach to elucidate the relevant genes involved beta-cell maturation.
: University of Helsinki
Permanent neonatal diabetes (PNDM) is caused by mutations impairing the pancreatic beta cell development or function. Activating mutations in STAT3 were recently identified as a cause of PNDM. The patient with the most activating mutation K392R presented with hypoplastic pancreas and high beta-cell autoantibody levels already at birth. We used patient-derived iPS cells to study whether mutation may cause pancreatic developmental failure. Patient's skin fibroblasts were reprogrammed to iPSC, and differentiated into pancreatic progenitors with a 17-days protocol. Expression levels of pancreatic progenitor markers PDX1 and NKX6.1 did not differ between STAT3K392R cells and healthy controls. Instead, NEUROG3 expression was upregulated prematurely together with significantly higher NKX2.2 (4-5 fold,) INS (10-fold) and GCG (5-fold) levels. The STAT3 mutation was corrected using CRISPR/Cas9. Corrected isogenic cells differentiated similarly to control cells, showing a complete reversal of the disease phenotype. STAT3K392R activating properties are not explained by an increase in DNA-binding affinity or its phosphorylation status. Instead, protein proximity assay and quantitative immunocytochemistry revealed increased nuclear translocation of STAT3 and increased transcriptional activity in STAT3K392R overexpressing cells Our results suggest that increased STAT3 localization in the nucleus of STAT3K392R cells leads to abnormally early upregulation of NEUROG3 and premature endocrine differentiation, resulting in reduction of the pancreatic progenitor pool, consistent with the pancreatic hypoplasia. Patient-specific iPSC in combination with CRISPR-based genome editing are valuable tools for recapitulating pancreatic developmental defects, enabling the study of pathogenic mechanisms leading to monogenic diabetes.
: Molecular, cellular and genomic biomedicine group - Medical Research Institute Hospital La Fe (IIS La Fe), Valencia, Spain
Usher syndrome is a rare autosomal recessive disease causing sensorineural hearing loss, retinitis pigmentosa and, sometimes, vestibular dysfunction. It is a clinically and genetically heterogeneous disorder, categorised into three subtypes (Usher I, II and III) and with more than 10 associated genes, being USH2A the most common mutated gene. To date, there is no medical treatment, except for the hearing aids or cochlear implants for the hearing impairment. Therefore, the repair of specific mutations by gene editing is an interesting strategy that can be performed using the CRISPR technology, a method based on a nuclease (Cas9) that, once introduced in a cell, cleaves the double DNA strand at a specific site via an RNA fragment that acts as a guide to the target locus. Upon cleavage, the target locus undergoes one of two pathways for damage repair, both of which can be used for the editing process: the error-prone non-homologous end-joining (NHEJ) or the high fidelity homologous recombination (HDR) pathway, in case a DNA template with homology arms is ectopically introduced. The present study shows the positive edition results of this gene targeting project based on CRISPR/Cas9 technology that we have implemented for two prevalent mutations responsible for Usher syndrome, namely c.2999delG and p.C759F, which are both located on exon 13 of USH2A gene.
: University of California San Francisco
Sickle cell anemia (SCA) in the US affects approximately 70,000 to 100,000 individuals, mostly in the African American and Hispanic communities. Whereas clinical management of SCA, and related hematopathies, have extended the lifespan and quality of life for patients, a lasting cure is now forseeable through the transplant of gene-corrected hematopoietic stem cells. One potential approach to treatment of SCA utilizes patient-derived iPSCs for correction of the underlying disease-causing exon 1 mutation prior to differentiation into engraftable progenitor cells. In these studies, we report the homology directed repair of the HBs exon 1 SCA A > T transversion mutation in human sickle cell iPSCs using sequence-specific TALENs with human hemoglobin beta gene-targeting vectors to generate gene corrections. Two classes of gene correction events were observed. A novel class of products consistent with vector insertion events resulting in partial duplication of human hemoglobin beta gene sequences were observed in addition to vector replacement events. We will discuss use of cell lines with vector insertion events for deriving cell lines with footprint-free gene corrections. These studies were supported by NIH Program Project Grant (PPG) DK088760.
: DKFZ/NCT Heidelberg
Adoptive transfer of T-cells with transgenic high avidity T-cell receptors (TCR) is a promising therapeutic approach. However, endogenous and transferred TCR-chains compete for surface expression and may pair inappropriately, potentially leading to autoimmunity. This can be prevented by designer nucleases such as transcription activator-like effector nucleases (TALEN) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) system. In order to disrupt endogenous TCR expression we assembled a set of TALEN and guide RNA (gRNA) targeting the constant regions of the TCR α-chain (TRAC) and TCR β-chain (TRBC1 and TRBC2), respectively. Here we show the induction of targeted DNA double strand breaks (DSB) by TALEN and CRISPR/Cas and the successful elimination of surface CD3 expression in up to 76% of T-cells. The nuclease specificity was examined by localizing integrase-deficient lentiviral vector (IDLV)-marked DSB using LAM-PCR and deep sequencing. Clustered IDLV integration sites (CLIS) that indicate nuclease activity were detected at all nuclease target sites. Only one CLIS for one gRNA and two CLIS for each TALEN mRNA were mapped at off-target positions, indicating a high level of specificity. To confirm and quantify off-target cleavage, DSB-derived indel frequency at each potential off-target site was determined by deep sequencing. Furthermore, using the presented TALEN, HDR-mediated targeted integration of a GFP expression cassette into the TRAC- and TRBC1 locus was successfully accomplished in up to 5% of primary T-cells. To sum up, here we present highly efficient and specific TALEN and CRISPR/Cas and their utility for T-cell engineering.
: Intellia Therapeutics
CRISPR/Cas9 is an RNA-guided nuclease which can be directed to specific sites in the mammalian genome to catalyze double stranded breaks. The spectrum of repair events both insertion and deletions of one or more nucleotides in genomic DNA after cleavage by CRISPR/Cas9 in a population of mammalian cells is not random, but is a function of the target site of the guide RNA, the micro-homology in or around that site of cleavage and the active DNA repair machinery in a particular cell. We report on a comparison of patterns of repair seen in various human derived tissue culture cell lines with the repair structures seen in human primary cells (hepatocytes and CD34+ cells) using next-generation sequencing. We also demonstrate that primary hepatocytes are more predictive of the repair patterns seen in vivo in mouse liver than are immortalized cell lines. In immortalized tissue culture cell lines, the predominant pathway for repair appears to be micro-homology-mediated end joining (MMEJ), resulting in deletions of various sizes dependent upon the sequence in proximity to the cut-site. In contrast, in primary hepatocytes, the predominant repair pathway appears to be classic non-homologous end joining (C-NHEJ) mediated resulting in one to two base insertions or deletions. In addition, down-regulation of the critical repair protein POLQ or LIG4 from MMEJ or C-NHEJ pathway respectively, alter the repair pattern seen by biasing the DNA repair to one pathway or another. These observations are critical for the selection of guide RNAs for potential human therapeutics.
: SISSA, Trieste, Italy
Artificial transactivation of endogenous genes ad libitum is a desirable goal for a number of basic and applied research purposes. We recently developed a fully synthetic ribonucleoproteic transactivator prototype. Kept together via an MS2 coat protein/RNA interface, it includes a fixed, polypeptidic transactivating domain and a variable RNA domain that recognizes the desired gene. Thanks to this device, we specifically upregulated five genes, in cell lines and primary cultures of murine pallial precursors. Even if gene upregulation is small, however it is sufficient to inhibit neuronal differentiation. Our transactivator activity was restricted to cells in which the target gene is normally transcribed. These features make our prototype a promising tool for clean rescue of gene haploinsufficiencies, since it could lead to a specific overstimulation of the spared gene allele in its physiological expression domain. On the other hand, we are interested in stimulating transcription of endogenous genes by small activating RNAs (saRNAs). These are miRNA/siRNA-like molecules, supposed to destabilize transcription-inhibiting ncRNAs or facilitate the recruitment of transcriptional complexes to chromatin. The transcription gain they elicit is small; however it may be sufficient to influence the behaviour of cells in a robust way. Moreover, silent genes generally do not respond to them. As such, also saRNAs are a promising tool for therapeutic stimulation of gene transcription. We selected a number of saRNAs able to stimulate haploinsufficient genes involved in CNS morphogenesis and physiology. We are studying their mechanism of action and exploiting saRNAs for in vivo correction of gene haploinsufficiency.
: EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria
Mutations within the COL7A1 gene lead to the dystrophic form of epidermolysis bullosa (DEB) characterized by disturbed expression of type VII collagen and blister formation below the lamina densa. We exploited the recently discovered CRISPR/Cas9 system to correct a homozygous mutation within exon 80 of the COL7A1 gene by homology-directed repair (HDR). We have predicted protospacer adjacent motifs (PAMs) within intron 80 of the COL7A1 gene, enabling the specific cleavage of the DNA via the CRISPR/Cas9 system at the desired location. The guide RNA (gRNA) for the predicted PAM sequence was cloned either into a wild-type Cas9 dual vector system, aiming to induce double strand DNA breaks, or a D10A Cas9 dual vector system, aiming to cause single strand DNA breaks within intron 80. Homology COL7A1 arms for HDR were cloned into a donor vector, including a ruby/puro selection cassette. Transfected patient keratinocytes were selected either via antibiotic selection or fluorescent-activated cell sorting (FACS). PCR and sequence analysis of genomic DNA, isolated from puromycin selected cell populations and single cell clones, showed the genetic correction of the COL7A1 mutation. Restriction enzyme digestion of PCR products, employing a newly introduced restriction site, revealed the presence of 26% reverted alleles. Additionally, type VII collagen restoration was confirmed via Western blot analysis and immunofluorescence staining of puromycin selected cell populations and single cell clones. Our data suggest that genome editing using the CRISPR/Cas9 system can be used to repair genes involved in the severe skin disease epidermolysis bullosa.
: EB House Austria
Epidermolytic ichthyosis (EI) is an inherited skin fragility disorder caused by dominant-negative mutations in either the keratin 1 (KRT1) or keratin 10 (KRT10) genes. As EI is difficult to treat and currently lacks a cure, there is an acute need for novel therapies. Keratin pairs specifically polymerise to build the intermediate filament cytoskeleton of epithelial cells. Dominant-negative mutant keratins integrate into this, resulting in fragility and collapse upon mild stress. This leads to cytolysis and blistering of the skin. Elimination of these mutant keratins is essential for curation of the disease. Our group previously demonstrated that zinc finger nucleases (ZFNs) can inactivate an EGFP transgene in murine keratinocyte stem cells efficiently without impairment of stem cell properties. Transcription activator-like effector nucleases (TALENs) are a new generation of highly sequence specific designer nucleases. They require few design parameters, while off-target effects are rare. We are developing an optimised ex vivo gene therapy for EI, using TALENs to knockout mutant KRT10 alleles in keratinocyte stem cells (KSCs). Optimised TALENs targeting KRT10 have been constructed. These cleave efficiently at the target site and induce frame-shift mutations known to knockout KRT10. TALEN-treated clones were seeded and screened, with 54% displaying successful modification without selection. A number of those identified were expanded and are being used in further biochemical studies to confirm phenotypic rescue. We aim to take a skin biopsy from an EI patient, isolate, grow and treat KSCs with TALENs to phenotypically cells prior to grafting these onto the patient's skin.
: Kazan (Volga region) Federal University. Kazan, 420008, Russia
Dysferlin is a membrane protein encoded by gene DYSF, it promotes membrane repair in striated muscle fibers (MF). Mutations in DYSF lead to loss of dysferlin, impaired membrane repair in muscle fibres and its destruction clinically manifesting as limb-girdle muscle dystrophy 2B. Preclinical studies of cell and gene therapy aiming to restore impaired muscle regeneration require well characterized small animal model. Gastrocnemius muscle of 5-months old dysferlin-deficient A/J and C57Bl/6 mice was injected by 100 μl of 5mg/ml bupivacaine (myotoxic agent). Paraffin sections of injected and contralateral calf muscles obtained 2, 4, 10 days after injection were stained with H&E, immunohistochemically with antibodies against macrophage antigen, myogenin, Ki-67. Necrotic MF with macrophage infiltration were found up to 10 days after injection in A/J mice, in C57Bl/6 mice on 2nd day after injection only, percentage of necrotic MF in A/J mice was significantly higher (17,3 ± 12,1% vs 9,5 ± 3,3% in C57Bl/6 mice), reflecting more severe and continuous muscle damage in dysferlin deficiency. In C57Bl/6 mice injected muscle restores normal structure by 10th day after injection. Percentage of centrinucleated MF and proliferating myosatellites were significantly higher in all time points in A/J mice with maximum on 10th day after injection (34,7 ± 14,1% vs 5,5 ± 3.0% and 18,6 ± 12,9% vs 3,3 ± 1,6% in C57Bl/6 mice, respectively) showing activated but incomplete MF regeneration associated with necrosis of newly formed MF. Modulation of regenerative response in dysferlin-deficient skeletal muscle is potential point of application for gene and cell therapy. This work was funded by a Russian Science Foundation grant (14-15-00916).
: CIBIO-University of Trento
In vivo application of the CRISPR/Cas9 technology is still limited by unwanted Cas9 genomic cleavages. Long term expression of Cas9 increases the number of genomic loci non-specifically cleaved by the nuclease. We developed a Self-Limiting Cas9 circuitry for Enhanced Safety and specificity (SLiCES) which consists of an expression unit for the Streptococcus pyogenes Cas9 (SpCas9), a self-targeting sgRNA and a second sgRNA targeting a chosen genomic locus. The self limiting circuit by controlling Cas9 levels results in increased genome editing specificity. For its in vivo utilization, we next integrated SLiCES into a lentiviral delivery system (lentiSLiCES) via circuit inhibition to achieve viral particle production. Following its delivery into target cells, the lentiSLiCES circuit is switched on to edit the intended genomic locus while simultaneously stepping up its own neutralization through SpCas9 inactivation. By preserving target cells from residual nuclease activity, our hit and go system increases safety margins for genome editing.
: San Raffale Telethon Institute for Gene Therapy (SR-Tiget)
It has been shown that HIV-1 insertions targeting transcription factors like BACH2 and MKL2 are enriched and persist for decades in patients under Anti-Retroviral-Therapy (ART), indicating that insertional mutagenesis provided a selective advantage to these cell clones. We identified that chimeric mRNA transcripts containing viral HIV-1 sequences fused by splicing to the first protein-coding exon of STAT5B or BACH2 are present in the hematopoietic cells of 30/87 patients under ART. These chimeric mRNAs, putatively encoding for the wild type forms of BACH2 or STAT5B, were found to be specifically enriched (p < 0.001) in T-regulatory (Treg) cells in all patients tested (N = 9), as the result of a viral driven selection mechanism known as promoter insertional mutagensis. LV-mediated overexpression of STAT5B and BACH2 in Treg cells purified from healthy donors significantly increased their proliferation rate (p < 0.001) without impacting on their immune-suppressive function as observed from specific in vitro and in vivo assay. These data suggest that HIV-mediated deregulation of STAT5B and BACH2 favor the maintenance and expansion of Treg cells in infected patients promoting long-term viral persistence. Beside its implication in HIV biology, the proliferative effect conferred by the STAT5B and/or BACH2 overexpression in Treg cells could represent a novel suitable approach for adoptive immunotherapy clinical application. Indeed, high number of cells are required for such clinical purposes, and this approach should led to higher yield of Treg cells over other protocols of expansion and should promote the persistence of the transduced Treg cells in vivo.
: Experimental Hematology Unit, Division of Immunology, Tranplantation and Infectious diseases, San Raffaele Scientific Institute, Milan, Italy
Memory Stem T cells (TSCM) are long living self-renewing memory T cells with long-term persistence capacity, which play a relevant role in immunological memory and protection against infectious diseases and cancer. The aim of this work is to investigate the potential role of TSCM as a reservoir of arthritogenic T cells in Rheumatoid arthritis (RA). We analysed the dynamics of TSCM (here identified as CD45RA+ CD62L+ CD95+ T cells) and other memory T-cell subpopulations by multiparametric 11-color flow cytometry in 27 patients with active RA and in 14 of them also during treatment with anti-TNFα biological agents (Etanercept). We found a significant expansion of CD4+ TSCM in patients with active RA both in terms of frequency and absolute numbers. Notably, CD4+ TSCM significantly contracted upon anti-TNFα treatment, suggesting a role of TNFα in TSCM accumulation. Furthermore, CD4+ TSCM in RA patients displayed enrichment in the TH17 phenotype, largely implied in autoimmune disorders. Phenotypic analysis of T-cell subpopulations revealed a significantly increased expression of IL7Rα on TSCM that correlated with clinical outcome. At the antigen-specific level, we were able to trace, in patients carrying the RA-susceptibility allele HLA-DRB1*04:01, CD4+ T cells specific for the arthritogenic citrullinated-vimentine peptide. Of notice, citrullinated-vimentin specific CD4+ T cells, including TSCM, contracted during anti-TNFα treatment. Overall, these results suggest that TSCM, by representing a long-term reservoir of undesired specificities, might play a non redundant role in sustaining RA and possibly other T cell mediated disorders, thus representing novel biomarkers as well as therapeutic targets.
: San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), Milan, Italy
Type 1 diabetes (T1D) is an autoimmune disease resulting in the complete destruction of insulin-producing pancreatic beta-cells by auto-reactive T cells targeting islet-associated antigens. Induction of antigen (Ag)-specific tolerance represents a potential therapeutic option. We showed that systemic administration of LV.ET.InsB9-23.142T (LV.InsB), enabling stable expression of insulin B chain 9-23 (InsB9-23) in hepatocytes, arrests beta-cell destruction in NOD mice at advanced pre-diabetic stage, by generating InsB9-23-specific FoxP3+ T regulatory cells (Tregs). Furthermore, LV.InsB combined with a suboptimal dose (1X 5μg) of anti-CD3 mAb in NOD mice reverts overt diabetes. We tested the LV.InsB/anti-CD3 combined-therapy to prevent recurrence of autoimmunity and maintain insulin-independence after pancreatic islet transplantation. Pancreatic islets from NOD-scid were transplanted in diabetic NOD mice (glucose >350mg/dL), and treated with LV.InsB/anti-CD3; anti-CD3 mAb (1X 5μg) alone or left untreated as control. Mice receiving anti-CD3 mAb alone or untreated controls returned hyperglycemic due to recurrence of T1D, while LV.InsB/anti-CD3 combined-therapy allowed stable normoglycemia up to 250 days. Auto-Ags stimulation of splenocytes isolated from transplanted mice treated with LV.InsB/anti-CD3 showed persistence of autoreactive T cells and increased frequency of FoxP3+ Tregs in renal- and pancreatic-lymph nodes, indicating that LV.InsB/anti-CD3 treatment induces a mechanism of active suppression of autoreactive responses at target sites of autoimmunity. Moreover, combined-therapy controlled recurrence of diabetes in 50% of NOD mice transplanted with allogeneic-islets. These studies suggest that induction of Ag-specific tolerance by combining autoAg expression in hepatocytes and tolerogenic compound such as anti-CD3 mAb may represent an innovative therapy for autoimmunity and transplantation tolerance.
: Graduate School of Pharmaceutical Sciences, Osaka University
The development of vaccines that realize both mucosal and systemic protection against various pathogens is a high priority in global health. Recently, it has been reported that intramuscular (i.m.) vaccination of an adenovirus vector (Adv) can induce Ag-specific CTLs in both systemic and gut mucosal compartments. Although an Adv vaccine is expected to be the next generation mucosal vaccine, the molecular mechanisms underlying the induction of mucosal immunity have not been fully elucidated. Surprisingly, by using IFNAR2 KO mice, we previously found that type I IFN signaling is required for the induction of gut-mucosal CTLs, but not systemic CTLs, following the i.m. Adv vaccination. In this study, we discuss the physiological roles of type I IFN signaling in the induction of gut-mucosal CTLs following the i.m. Adv vaccination. Briefly, we found that Th17-polarizing cytokine expressions and inflammatory DCs (Inf DCs) were induced through type I IFN signaling in the inguinal lymph nodes (iLNs). Since it has been reported that Inf DCs are involved in the differentiation of Th17 and that Th17 promotes the proliferation and activation of CTLs, we next checked Th17 induction following the vaccination. We found that in the gut mucosa of KO mice, Th17 was reduced compared with WT mice. These suggested that type I IFN signaling was important for the Th17 induction through Inf DC recruitment in the iLNs, resulting in the positive regulation of gut-mucosal CTLs by Th17. Our findings should lead to the development of more efficient and safer mucosal Adv vaccines.
: Department of Pediatrics, Jichi Medical University
: Universita del Piemonte Orientale
We studied the activity of F8 promoter (pF8) sequence to drive transgene expression in a LV construct to verify the feasibility of expressing FVIII under its natural promoter for gene therapy approaches. In silico analysis of Transcriptional Factors (TF) consensus sequences predicted the presence of several myeloid-specific TF, in addition to hepatocytes- and endothelial-specific TF. After LV.pF8.GFP injection in C57Bl/6 mice, GFP expression was restricted to liver sinusoidal endothelial (LSEC). Instead, in spleen and bone marrow, GFP was in myeloid cells. We injected LV.pF8.hFVIII in C57/BL6 hemophilic mice and aPTT assay demonstrated FVIII activity in therapeutic range (up to 10% of normal FVIII activity) without antibodies formation up to 1 year and consistent blood loss reduction in gene-corrected hemophilic mice. To improve FVIII levels we generated LVs in which BDD-FVIII was substitute with more active forms of FVIII such as FVIII.RH and FVIII.N6 producing up to two fold more FVIII activity in hemophilic mice. Further, we transplanted LV-pF8.FVIII transduced human cord-blood CD34+ cells in busulfan-treated NOD/SCID gamma-null HA-mice. Human cells engrafted in mice showing a chimerism around 30% up to 4 months later. aPTT assay on plasma of LV-pF8-FVIII-CD34+ transplanted mice showed therapeutic levels of FVIII activity up to 10% of normal meanwhile transplantation of untransduced CD34+ cells reached 2% of activity. Our results demonstrate that pF8 is differentially active in cell-subpopulations of several organs contributing to identify the FVIII producing cells and targeting FVIII expression in these cells by LV produced FVIII in therapeutic range in hemophilic mice.
: University of Manchester
Somatic cells can be de-differentiated towards pluripotency in vivo by ectopic expression of reprogramming transcription factors. Sustained expression of such cues triggers tumorigenesis, but strategies relying on transient in vivo reprogramming induce pluripotency and proliferation only temporarily, without teratoma formation. Here, we sought to achieve transient reprogramming within mouse skeletal muscle with a localized injection of naked plasmid DNA (pDNA) encoding reprogramming factors. We hypothesized that such approach would enhance the regeneration capability of the tissue after a severe and clinically-relevant injury. A single intramuscular administration of the reprogramming pDNA rapidly triggered the expression of pluripotency genes (Nanog, Ecat1, Rex1) and a marker of myogenic progenitors (Pax3) in the healthy gastrocnemius of various mouse strains, but significant levels were only maintained for 2–4 days. In agreement, distinct clusters of mononucleated cells expressing pluripotency and early-myogenesis markers appeared among the myofibers soon after injection, and proliferated only transiently. While no dysplastic lesions were found, morphometric analysis suggested that the reprogrammed cells might have eventually fused to neighboring myofibers. Nanog was also significantly upregulated in the gastrocnemius when reprogramming pDNA was administered 7 days after surgical laceration of its medial head. Regeneration was enhanced in reprogrammed tissues, as shown by the rapid appearance of centronucleated myofibers, reduced fibrosis and modest improvement in the recovery of muscle force. These results could pave the way for the development of new gene therapy strategies in the treatment of clinically-relevant muscle injuries via generation of transiently-proliferative, pluripotent-like intermediate cells by in vivo reprogramming.
: GENETHON
Crigler-Najjar (CN) syndrome is a rare metabolic disorder of the liver caused by mutations in the UDP-glucuronosyltransferase-1-isotype-A1 (UGT1A1) gene. The absence or the reduced activity of UGT1A1 enzyme in patients results in the accumulation of unconjugated bilirubin (UCB) in serum. If untreated, this leads to brain damage and death. Treatment of CN consists of phototherapy for 10–12 hours per day, which presents several limitations and has a major impact on the quality of life of patients. Liver transplantation remains the only curative treatment for the disease. The limited therapeutic options available for CN syndrome prompted us to develop a strategy based on liver gene transfer with an adeno-associated virus vector expressing UGT1A1 (AAV-UGT1A1). For this purpose, we tested multiple codon-optimized UGT1A1 sequences and we also optimized noncoding sequences in the expression cassette. Our results indicated that codon-optimization can improve efficacy of gene transfer but needs to be carefully tested in vitro and in vivo. Additionally, while inclusion of introns can enhance gene expression, their optimization, and in particular removal of cryptic ATGs, is an important maneuver to enhance safety and efficacy of gene transfer. Finally, we demonstrated that a single intravenous administration of an optimized AAV-UGT1A1 prevented lethality in CN mice and was sufficient for the long-term (>1 year) correction of hyperbilirubinemia in affected rats, resulting in UCB levels undistinguishable from wild-type animals. Based on these encouraging results, a multicenter clinical trial for AAV-UGT1A1 vector-mediated liver gene transfer in CN patients will open for enrollment in the near future.
: Osaka University
Replication-incompetent adenovirus (Ad) vectors are widely used in gene therapy studies due to their various advantages as a gene delivery vehicle; however, a major limitation of gene therapy using Ad vectors is inhibition of Ad vector-mediated transduction by anti-Ad neutralizing antibodies (AdNAbs). Interaction between Ad hexon and blood coagulation factor X (FX) is crucial for Ad vector-mediated transduction in the liver. It is generally considered that AdNAbs prevent FX-mediated transduction with an Ad vector; however, it has not been fully understood how AdNAbs inhibit Ad vector-mediated transduction in the liver. In particular, it remains to be clarified which Ad capsid protein-specific antibodies most efficiently inhibit Ad vector-mediated in vivo transduction. In this study, we examined which Ad capsid protein-specific antibodies are important to inhibit in vivo transduction with Ad vectors. First, in order to prepare mice possessing antibodies recognizing each Ad capsid protein, mice were immunized by plasmids expressing each Ad major capsid protein, including the fiber knob, hexon, and penton base (PB). After the immunization, significant levels of Ad capsid protein-specific antibodies were detected in the serum. Next, transduction efficiencies of a luciferase-expressing Ad vector in the liver of the pre-immunized mice were determined following intravenous administration. Luciferase expression levels in the livers of mice possessing anti-fiber and anti-PB antibody were significantly reduced, compared with those in mice possessing anti-hexon antibody and non-immunized mice. Now we are examining the mechanisms of anti-fiber and anti-PB antibody-mediated inhibition of in vivo transduction with Ad vectors.
: University of Tokyo
Osteoarthritis (OA) is a chronic degenerative joint disease and a major health problem in the elderly population. No disease-modifying osteoarthritis drug has been made available for clinical use. For direct and effective disease-modifying therapy of OA, we introduced mRNA encoding transcription factors that initiate a transcriptional cascade into the target cells in situ in the joint. Despite the fact that mRNA delivered in vivo are susceptible to highly active RNases that are ubiquitous in the extracellular space, we have established a DDS using polyplex nanomicelle to deliver mRNA into target cells by preventing its degradation. When injecting luciferase mRNA into mouse joints using the nanomicelles, luciferase signal was induced in the joints at as early as one day after the injection, and sustained for up to 4 days. Using EGFP mRNA, GFP proteins were expressed diffusively in the superficial and middle zones of the articular cartilage. Then, we injected mRNA encoding a cartilage-anabolic transcriptional factor, RUNX1, into surgically-created OA joints using nanomicelles once every 3 days for one month. The RUNX1-injected group showed a clear trend toward suppression of the OA phenotypes. By evaluating OA severity using OARSI scoring, the mRNA significantly suppressed the OA progression compared with the controls. The expression of SOX9 and Type II collagen was increased in the articular chondrocytes. No obvious inflammatory symptoms, such as outgrowth of the synovial membrane or infiltration of inflammatory cells, were observed. Thus, this study provides a proof of concept of mRNA-based therapy using therapeutic transcription factors for degenerative diseases.
: Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Naples, Italy
In view of a gene therapy clinical trial for MPS VI, we performed GLP-compliant non-clinical studies to assess the safety and biodistribution of AAV2/8.TBG.hARSB, a recombinant AAV2/8 vector encoding human ARSB (hARSB) under the control of the thyroxine-binding globulin promoter (TBG), in transgenic C57/BL6-TgARSBC91S mice that are immune tolerant to hARSB. Mice were treated with either AAV2/8.TBG.hARSB or the vehicle alone, as control. Toxicity was evaluated on day 15 (D15) and 180 (D180) after systemic injection of 2×1013 gc/kg (10× the highest dose proposed for the clinical study). No mortality or signs of toxicity were observed in mice treated with AAV vector. Histopathology revealed thyroid epithelial hypertrophy in AAV-treated mice. AAV2/8.TBG.hARSB biodistribution and expression was evaluated on D15 and D180 at the dose of 2×1012 gc/kg (1× the highest dose proposed for the clinical study). Although vector DNA was present in all organs on D15, it was sequestered mainly in liver. Vector DNA declined on D180, but remained high in liver. Accordingly, hARSB was mainly expressed stably in liver. Vector DNA was found in gonads of both sexes, although a robust reduction of vector DNA was observed on D180. In situ hybridization in ovaries showed AAV localization in oocytes nuclei, and a breeding study is ongoing to elucidate the risk of germline transmission in females. Finally, AAV DNA was only transiently present in mice body fluids. In conclusion, these studies show a safe profile of intravenous administrations of AAV2/8.TBG.hARSB and pave the way for a phase I/II clinical trial.
: The University of Tokyo
Achondroplasia is the most common type of short limb dwarfism, caused by graded activation of fibroblast growth factor receptor 3 (FGFR3). The excess FGFR3 signal inhibits proliferation and differentiation of chondrocytes, leading to abnormal bone growth with short stature with arms and legs, a large head with frontal bossing, and characteristic facial features. Because C-type Natriuretic Peptide (CNP) modulates FGFR3 signal, treatment with CNP peptide was tried, however, due to the very short blood half-life of CNP, it was difficult to obtain long-lasting effect in the body. In this study, we established pDNAs for expressing alternative peptides of CNP for sustained regulation of FGFR3 signal. We chose BMN 111, an analog of CNP with long blood half-life, and the soluble FGFR3 to act as a dominant negative inhibitor of the signal. By in vitro evaluation, both peptides secreted from the transfected cells by the pDNAs induced significant upregulation of chondrogenic markers in chondrocyte cell line (ATDC5). For in vivo evaluation using 3-day old WT mice, the bone growth of femur and tibia was significantly accelerated by subcutaneously administering the pDNAs every three days for 3 weeks. Finally, using a mouse model of achondroplasia possessing a point mutation (G380R), the bone growth was also enhanced compared with non-treated controls without any side effects. Between the two pDNAs, sFGFR3-expressing pDNA exerted the higher effect. This strategy is promising for a gene therapy aiming at radical cure of achondroplasia.
: Shire
: San Raffaele Telethon Institute for Gene Therapy (SR-Tiget)
Sandhoff disease (SD) is a fatal neurodegenerative GM2-gangliosidosis caused by genetic defects in β-N-hexosaminidase (Hex) activity. SD shares with leukodystrophies the early onset, severe progression and unexpected precocious myelin defects. Thus, we hypothesized that the combined strategy based on neonatal neural stem cell transplantation (NSCT) coupled to bone marrow transplant (BMT) that we have shown to benefit GLD mice (Ricca et al, 2015) could be similarly effective in SD mice (Hexb-/-) that resemble infantile SD. NΣXT (NSC ισoλατɛδ φρoμ ωτ λιττɛρματɛσ, ɛϖɛντυαλλψ ɛνγινɛɛρɛδ τo overexpress Hex) was πɛρφoρμɛδ ατ ΠNΔ2. BMT (τoταλ BM ισoλατɛδ φρoμ ωτ τγΓΦΠ μιχɛ) was πɛρfoρμɛδ ατ ΠNΔ7. Mιχɛ τρɛατɛδ ωιτη τηe single approaches served ασ χoντρoλσ. Hematopoietic cell engraftment was 30%-100%. Preliminary data indicate stable engraftment of NSC (in CNS) and BM-derived myeloid progeny (in CNS and periphery). Hex reconstitution was ≈10% of normal activity in CNS (all treatments) and 50–100% of normal activity in the periphery (BMT/combined treatment), with restored Hex isoenzyme composition. Lifespan increased (+30 days) in SD mice treated with BMT and BMT+NSCT as compared to untreated and NSC-treated littermates. We are currently monitoring large cohorts of mice that will be analyzed to evaluate δισɛασɛ−αssociated pathological features, τηɛιρ πρoγρɛσσιoν ανδ τηɛ ɛξτɛντ τo which they can be reversed by treatments. Besides assessing the therapeutic potential of the proposed strategies, this study will clarify the input of CNS and periphery to the disease, and the relative contribution of treatments to the therapeutic outcome, with important implications for designing treatment strategies for GM2-gangliosidosis.
: Medgenics Medical Israel, Ltd.
We have previously reported on our TARGT™ system (Transduced Autologous Restorative Gene Therapy), an innovative ex-vivo gene therapy approach, which allows sustained delivery of therapeutic proteins. Pre-clinical studies using this novel technology demonstrate in-vitro secretion of proteins, peptides, enzymes and mAb at levels of tens μg/day. SCID mice implanted with human TARGT exhibited sustained and long term in-vivo secretion of therapeutic proteins. More importantly, we have demonstrated stable long-term secretion of erythropoietin and amelioration of anemia in renal failure patients implanted with TARGT. Herein we extend our observations in ex-vivo HDAd transduced fibroblasts, implanted SQ or in the CNS, produce functional active enzymes and mAbs. Hunter Syndrome (MPS II) is a lysosomal storage disease caused by iduronate-2-sulfatase (IDS) enzyme deficiency. Enzyme replacement therapy, with frequent injections of the recombinant IDS enzyme (Elaprase®), aiming to stop glycosaminoglycans (GAG) accumulation, showed good clinical outcome, however, failed to stop disease progression in the CNS. A single intrathecal implantation of fibroblast cells transduced ex-vivo with our HDAd viral system, into MPS II mice reduced brain GAG accumulation. Rheumatoid Arthritis (RA) is an autoimmune inflammatory disorder affecting mainly the joints. The mAb adalimumab (Humira®) is extensively used to treat this disorder. A single subcutaneous implantation of fibroblasts cells embedded in Matrigel and transduced ex-vivo with our HDAd viral system to secret adalimumab, significantly reduced the RA score in a mouse RA model. The results presented here demonstrate the capability of our system to produce and deliver functional active enzymes and mAbs into circulation and CNS.
: Institut de Myologie-U974
CD8+ T cell responses directed to foreign transgenes represent an important hurdle after recombinant adeno-associated virus (rAAV) vector-mediated gene transfer, as it can lead to the elimination of gene-modified cells. Direct transduction of DC had initially been suggested to be a key driver of adaptive responses after gene transfer and strategies have successfully been developed to prevent expression in antigen presenting cells (APC) through the use of tissue-specific promoters or miRNA-based regulation of transgene expression in the hematopoietic system. While efficient at preventing direct presentation of transgene-derived antigens, these strategies, however, cannot prevent the uptake and cross-presentation of such antigens by non-transduced APC patrolling in the target tissue. Using a model miR142-3p-regulated ovalbumin transgene, we now demonstrate that strong cross-presentation of the transgene product occurs in the lymph nodes draining the transduced muscle. Potent cross-priming leading to muscle fibers rejection, however, required the presence of either a strong MHC class II epitope or antibodies-mediated recognition of the transgene product. In this context, a cytosolic form of the ovalbumin transgene lacking strong MHC II epitope remained permanently expressed in muscle, despite an ongoing anti-transgene CD8+ T cell response. This response showed clear characteristics of T cell exhaustion and was further associated with the expansion of regulatory T cells in muscle-draining lymph nodes which migrated to the transduced muscle. A better understanding of the mechanisms regulating the induction of cross-tolerance to miR-142-3p-regulated transgene should help us define novel immunomodulatory strategies to extend the domain of application of AAV-miR-142-3p vectors for muscle transduction.
: Biogen
Ex vivo gene therapy with lentiviral vectors (LV) for gene replacement has demonstrated clinical efficacy and currently safe toxicity profile on multiple indications, with multi-year follow up in treated patients not showing evidence of tumorigenesis. Here we assessed the feasibility of in vivo gene therapy with LV for hemophilia A using a FVIII deficient (HemA) mouse model. FVIII variants was placed under a hepatocyte-specific promoter and built into a LV system that contains multiple copies of microRNA-142 target sequences for preventing FVIII expression in antigen presenting cells. LV-FVIII variants were evaluated in 2–14 days old mice to reduce FVIII antibody response. After intravenous administration of LV-FVIII, circulating FVIII level was monitored by activity and antigen assays, and LV transduction efficiency in the liver was assessed by QPCR and RNA in situ hybridization. Persisting FVIII expression was observed for all LV-FVIII variants post LV treatment; at 1.5E10/kg dose level, LV encoding codon optimized FVIII (LV-coFVIII) drove significantly higher level of FVIII in circulation than that of wild type FVIII (LV-wt-FVIII), while the vector copy number in liver cells and % of FVIII positive cells were comparable in all tested groups. Combination of codon optimization with XTEN (LV-coFVIII-XTEN), a hydrophilic peptide that presumably improves the circulating half-life by increasing the hydrodynamic size of the payload, resulted in a 50 IU/ml FVIII activity in plasma, representing a 50-fold increase from the normal circulating FVIII level. Our result supports further development of LV-FVIII for in vivo gene therapy of hemophilia A.
: TIGEM
Vectors based on AAV are the most promising for retinal gene therapy. We have shown that AAV cargo capacity limited to 5kb can be expanded to 9kb by dual AAV vector where a large transgene expression cassette is split in two halves which are independently packaged in two single AAV vectors. Their co-administration results in AAV genome intermolecular recombination and full-length transgene reconstitution which is however greatly reduced compared to a regular single AAV. As AAV intracellular trafficking and transduction are negatively affected by phosphorylation of capsid residues, we set to identify kinase inhibitors that are able to increase dual AAV vector transduction. We performed high throughput screening of kinase inhibitors that enhance transduction mediated by dual AAV vectors which express EGFP. Two automated rounds of selection reduced the number of compounds from 273 to 12. Positive hits were further reduced to 7 by a Western blot validation step which showed an increment over a proteasome inhibitor used as positive control. We are currently validating by siRNA-mediated knock down the specific kinases targeted by the 7 identified compounds as well as their ability to enhance dual AAV-mediated mouse retinal transduction.
: Department of Physiology and Regenerative Medicine Institute, School of Medicine, National University of Ireland Galway, Ireland
Duchenne muscular dystrophy (DMD) is progressive muscle degenerative disorder due to mutations in the dystrophin gene resulting in loss of the dystrophin protein. Dystrophin and a complex of proteins maintains the integrity of the sarcolemma particularly during contractile stress. Disruption in the sarcolemma results in Ca2+ dysregulation via direct Ca2+ influx and/or calcium leakage from the sarcoplasmic reticulum (SR). Recently, transgenic developmental overexpression of the sarco/endoplasmic reticulum Ca2+ATPase (SERCA1a) was reported to ameliorate the dystrophic phenotype and improve muscle function in different mouse models of muscular dystrophy, although the mechanism is unknown. Here, we examine the effect of viral overexpression of the SERCA2a gene in adult hindlimb muscles of the mdx mouse model of DMD. We used the Adeno-Associated Virus (AAV) serotype 9 as reported to be the most efficient gene delivery system for infecting muscle. Morphological indexes of the dystrophic phenotype were improved in the mdx treated mice compared to controls including a decrease in both fibre size heterogeneity and central nucleated fibres, 6 weeks after a single muscle injection. To gain an insight into the mechanism we examined the allosteric regulators of SERCA including sarcolipin (SLN), phospholamban (PLN) and recently identified myoregulin (MLN) and Dwarf. In conclusion, gene targeting of SERCA regulation maybe a form of compensatory therapy for muscular dystrophic disorders.
: TIGEM
Alström syndrome (AS) is a rare monogenic disease inherited as autosomal recessive and characterised by retinitis pigmentosa (RP), among other features. AS is caused by mutations in ALMS1, which has a coding sequence (CDS) of 12.5 kb and encodes a large ciliary protein. Gene therapy with adeno-associated viral (AAV) vectors holds great promise for the treatment of many inherited retinal diseases including AS. The main obstacle for AS gene therapy is that AAV cargo capacity is limited to ∼5 kb. To overcome this, we split the ALMS1 expression cassette in 3 parts separately packaged in 3 independent AAV vectors that reconstitute ALMS1 upon co-administration and intermolecular recombination. We show that triple AAV ALMS1 vectors reconstitute full length ALMS1 protein expression both in HEK 293 cells and mouse photoreceptors (PR). We also observed that subretinal administration of 3 independent AAV vectors separately carrying the reporter eGFP, DsRed and EBFP2 transgenes results in about 10% PR co-transduction. Finally, we show that the retina of the MVR102 mouse model of AS undergoes progressive outer nuclear layer thinning and reduction of electrical responses to light. We conclude that triple AAV vectors expand AAV transfer capacity in the retina up to 13Kb and can be tested for treatment of the AS mouse model RP.
: Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Centre, Austrian Cluster for Tissue Regeneration, European Institute of Excellence on Tissue Engineering and Regenerative Medicine Research (Expertissues EEIG), Vienna-Branch, Vienna, Austria
Gene-activated matrix (GAM)-based therapeutics for tissue regeneration are limited by efficacy, the lack of spatiotemporal control and availability of target cells, all of which impact negatively on their translation to the clinic. Here we describe an advanced ultrasound-responsive GAM containing target cells that facilitates matrix-assisted sonoporation (MAS) to induce osteogenic differentiation. Ultrasound-responsive GAMs consisting of fibrin/collagen hybrid-matrices containing microbubbles, bone morphogenetic protein BMP2/7 co-expression plasmids together with C2C12 cells were treated with ultrasound either in vitro or following parenteral intramuscular implantation in vivo. Using direct measurement for alkaline phosphatase activity, von Kossa staining and immuno-histochemical analysis for osteocalcin expression, MAS-stimulated osteogenic differentiation was confirmed in the GAMs in vitro 7 days post treatment with ultrasound. At day 30 post-treatment with ultrasound, ectopic osteogenic differentiation was confirmed in vivo using X-ray microcomputed tomography (μCT) and histological analysis. Osteogenic differentiation was indicated by the presence of ectopic bone structures in all animals treated with MAS when compared with controls. In addition, bone volumes in this group were statistically greater than those in the control groups. This novel approach of incorporating a MAS capability into GAMs could provide a minimally invasive means of stimulating in situ transgene delivery with enhanced spatiotemporal control for osteoinductive gene-based therapies.
: Myology Centre for Research, Sorbonne Universités UPMC Univ Paris 06 – Inserm – CNRS – Institut de Myologie, Paris, France
Due to mutations in the DMD gene that lead to the absence of dystrophin protein, Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease causing a progressive muscle wasting with dysfunction of cardiac and respiratory muscles. AAV-based therapies aiming to restore the dystrophin in the muscles, either by exon skipping via U7 snRNA or by microdystrophin expression are very promising. However, the absence of dystrophin in dystrophic skeletal muscles induces cellular and muscular perturbations that can hinder AAV therapy efficiency. We have recently shown that AAV genomes are not maintained in dystrophic muscles certainly due to the ongoing cycles of necrosis-regeneration. We now focus on the impact of the myofiber centronucleation, one of the major pathological changes commonly observed in dystrophic muscles, on AAV transduction efficiency. We generated centronucleated myofibers by cardiotoxin injection, a protein kinase C inhibitor triggering muscular fiber regeneration, prior to AAV injections. This model of muscular centronucleation exhibits a drastic decrease of AAV transgene expression with normal vector genome content in the myofibers but a low transcription and mRNA instability. This study will help to define the impact of dystrophic features on AAV transduction, allowing the design of more efficient therapeutic protocol to treat muscular dystrophies.
: Neuroscience Research Center, Babol University of Medical Sciences
Alzheimer's disease (AD) is one neurodegenerative disorders that is more prevalent in adolescents. Recent studies have shown the possibility of converting the astrocyte to neuron in vivo. Our previous study also showed that astrocytes can convert to neuron by miR-302/367 cluster in healthy brain. Despite of recent studies, there is no report regarding positive effects of induced neurons on brain injury repair and behavioral improvement. Therefore, in this study, the role of induced cells on behavioral improvement and brain repair after induction of Alzheimer's disease model will be investigated. AD model induced by injection of Amyloid beta. Barnes and Y-maze tests used to assess spatial memory and working memory respectively. MiR-302/367 lentiviral particles injected to dentate gyrus 7 days post injection of amyloid beta and behavioral tests performed. Then, animals brain investigated for immunostaining and patch clamp recording. Our results showed that miR-302/367 can enhance memory parameters and immunostaining indicate that some transfected cells expressed mature neuronal markers. Patch clamp recording analysis showed that electrophysiological properties of these induced neurons were endogenous neurons. Taken together, miR-302/367 convert astrocytes to neurons and these induced cells can enhance hippocampal repair in AD animal model.
: Ludwig-Maximilians-Universitat München
Craniofacial bone defects are challenging problems for maxillofacial surgeons over the years. With the development of cell and molecular biology, gene therapy is a new technology with the aim of regenerating tissues by acting as a delivery system for therapeutic genes in the craniofacial region rather than treating genetic disorders. A systematic review was conducted summarizing the articles reporting gene therapy in maxillofacial surgery to answer the question: Was gene therapy successfully applied to regenerate bone in the maxillofacial region? Electronic searching of online databases was performed in addition to hand-search of the references of the included articles. No language or time restrictions were enforced. Meta analysis was done to assess significant bone formation after delivery of gene material in the surgically induced maxillofacial defects. The search identified 2081 articles of which 57 were included with 1726 animals. Bone morphogenetic proteins (BMPs) were commonly used proteins for gene therapy. Viral vectors were the universally used vectors. Sprague-Dawley rats were the frequently used animal model in experimental studies. The quality of the articles ranged from excellent to average. Meta-analysis results performed on 21 articles showed that defects favoured bone formation by gene therapy. Funnel plot showed symmetry with the absence of publication bias. Gene therapy is on the top list of innovative strategies that developed in the last 10 years with the hope of developing a simple chair-side protocol in the near future combining improvement of gene delivery as well as knowledge of the molecular basis of oral and maxillofacial structures.
: University of Massachusetts Medical School
Alpha-one antitrypsin (AAT) deficiency is a genetic disease causing lung disease due to the lack of anti-protease activity in the lung. On-going human trials use intra-muscular delivery of adeno-associated virus (rAAV1), allowing myofibers to produce and secrete normal AAT protein. In the Phase IIa trial patients in the highest dose cohort (6x10^12vg/kg) were given 100 intra-muscular (IM) injections of undiluted vector, with serum AAT levels still below target serum levels. Previous work has shown that delivering rAAV vector to the musculature via limb perfusion leads to widespread gene expression in myofibers. We hypothesize that widespread delivery would result in an overall increase in serum AAT levels with the same dose of AAV gene therapy vector and allow for increased volume and thereby dose of vector. In macaques, similar or modestly higher (573.0 ng/ml versus 562.5 ng/nl) serum AAT levels were seen using vascular delivery when compared to IM delivery at the same total vg dose with either rAAV1 or rAAV8, while not being limited to a small volume of IM injection. These data prove the concept that a 30-fold expanded volume of rAAV-AAT could be delivered to myofibers using limb perfusion without loss of potency on a per vg basis, thereby potentially enabling achievement of therapeutic levels.
: IFMB K(P)FU Kazan 420015 Russia
In patients with diabetes mellitus trophic ulcers develop due to damage of microvessels and loss of innervation. The aim of the study was to evaluate the effect of the injection of the pCMV-VEGF165 plasmid in skin wound in rats with experimental diabetes mellitus. After 21st day of pharmacologically induced hyperglycemia in the interscapular region of Wistar rats 8 × 8 mm skin wound was applied. Along the edges of the wound solution of 60 μg pCMV-VEGF165 plasmid in 200 μl water for injections or 200 μl water for injections was injected intradermally. After 10 days the euthanasia was performed. Paraffin sections were stained with hematoxylin and eosin, immunohistochemically with antibodies against CD31 (cluster of differentiation 31) and α-SMA (α-smooth muscle actin). Analysis of sections showed decreased length of the site without epithelium in the experimental group (0.92 ± 0.92 mm) compared to the control group (3.7mm). The α-SMA-positive area in granulation tissue is larger in the experimental group (32% ± 2.6%) compared to the control (25% ± 14%). In experimental animals the number of vessels in the center of the scar is higher (11 ± 3.1 vessels) than in the control group (3 ± 1.1 vessels). At the periphery of the scar vessels amount is higher in the experimental group (8.3 ± 3.3 vessels) than in control group (5 ± 3.1 vessels). Thus, injection of the pCMV-VEGF165 plasmid accelerates wound epithelialization and increases the number of vessels in the granulation tissue. Due to the small sample size these results should be considered as preliminary. This work was funded by a Russian Science Foundation grant (14-45-00018).
: Shire
: Akdeniz University Center for Gene and Cell Therapy
TNF-Related Apoptosis-Inducing Ligand (TRAIL) is a TNF superfamily member, highly homologous to CD95L/FasL and TNF-alpha. Studies aiming to clear out the role of TRAIL in diabetes development suggest a protective role for this molecule in diabetes. In fact, soluble TRAIL (sTRAIL) application was implicated in attenuation of metabolic abnormalities in high-fat-fed mice. We aimed to test the possible protective and/or therapeutic effect of lentiviral vector-mediated TRAIL transfer with or without sTRAIL application in high-fat-fed C57BL6 obesity and type 2 diabetic models. Animals were fed ad libitum with chow (5–10% fat) and water until 8 weeks of age. Experimental groups were then shifted to high fat diet (HFD, 60% fat) for at least 8 weeks. β-cell loss was induced in type 2 diabetes models via a single dose (150 mg/kg) intraperitoneal STZ application. Follow-up studies included weekly measurements of weight and non-fasting blood glucose, and periodic applications of intraperitoneal glucose tolerance (IPGTT) and insulin resistance tests (IPITT). IL6 levels were measured via ELISA method. TRAIL-encoding 3rd generation lentiviral vectors were produced for intraperitoneal applications. Both lentiviral- and sTRAIL-mediated applications improved glucose tolerance and insulin resistance in obesity and type 2 diabetic models, the highest protective/therapeutic potential appearing in lentiviral- together with sTRAIL treatments. Systemic inflammation appeared lower in the experimental groups. Lentiviral vector-mediated TRAIL application is particularly significant for long-term protective/therapeutic benefits in both disease models. Although further investigation is required, these findings support a possible protective plus therapeutic effect for TRAIL in obesity and diabetes. (Grant support: TUBITAK 112S450).
: Universita del Piemonte Orientale
Recent studies have demonstrated that FVIII is not secreted by hepatocytes, but it is mainly, although not exclusively, secreted by endothelial cells (ECs) and myeloid cells. We investigated whether the injection of Lentiviral Vectors (LVs), containing GFP or FVIII genes, under the control of endothelial- or myeloid-specific promoters (PGK: ubiquitous; CD11b: myeloid; VEC: endothelial) could induce the expression of the transgenes (GFP or FVIII) in the corresponding target cells and allow their long-term expression in WT and hemophilic mice. Furthermore, to restrict transgene expression to desired cell types we included miRNA target sequences (miRTs), i.e. miRT-122, miRT-142-3p or miRT-126, to silence expression in hepatocytes, hematopoietic and endothelial cells, respectively. The resulting vectors revealed cell-specific and long-term gene expression in wild type C57Bl/6 and hemophilia A mice. Interestingly, LV.CD11b.FVIII-126 and LV.VEC-FVIII ±122-142-injected hemophilic mice reached phenotypic correction with an average of 5% FVIII activity without presenting anti-FVIII antibodies. Injection of LV.VEC-FVIII ±122-142 induced tolerance in previously FVIII-immunized mice resulting in anti-FVIII antibody titer reversion, while regulatory T cell depletion reduced FVIII activity and induced anti-FVIII antibodies mainly in the IgG1 and IgG2b classes in LV-injected mice. Moreover, injection of LV.CD11b-FVIII-126 prevented an immune response to FVIII by detargeting transgene expression in plasmacytoid dendritic cells allowing long-term expression. In conclusion, cell-specific FVIII-expression was able to limit the immune response to transgene providing long-term phenotypic correction in hemophilic mice.
: Shire
Adeno-associated virus (AAV)-based gene therapy holds great promise for the treatment of hemophilia. Data from several clinical phase 1/2 hemophilia B trials indicated that therapeutically relevant FIX levels can be achieved for an extended period of time. These studies also revealed the importance of developing high expressing vectors. In hemophilia A, efficient expression cassettes are even more relevant considering the many challenges associated with FVIII expression. The large size of the FVIII coding sequence precludes packaging of full-length FVIII into AAV and the transgene is poorly translated and secreted. We have designed vectors harboring expression cassettes that confer liver-specific expression of a B-domain deleted variant of FVIII. Due to the known importance of vector optimization a screening program for improved FVIII coding sequences using a combination of available codon-optimization algorithms and manual sequence editing was conducted. Altogether 44 codon-optimized BDD FVIII sequences were packaged into AAV2/8 vectors and screened in FVIII knock-out mice for FVIII expression. This approach resulted in identification of a lead construct [AAV8.BDD-FVIII (Orth 04)] that expressed 74-fold higher levels of FVIII than a vector harboring the corresponding wild-type nucleotide sequence. Importantly, the plasmid encoding the Orth04 FVIII variant also gave rise to more efficient virion packaging and virus production making this vector a promising drug candidate for Shire's FVIII gene therapy program. With this construct, we expect to sustainably transform the bleeding phenotype of patients with hemophilia from severe into mild to moderate by a single treatment.
: Kazan (Volga region) Federal University, Kazan, 420008, Russia
Nowadays an active search for new methods of liver diseases treatment using stem cells and gene-cell therapy is held. In this work we studied rat liver regeneration on the model of partial hepatectomy (PH) after transplantation of the hepatic stellate cells (HSC), transduced by adenovirus with tracer gene (red fluorescent protein (RFP)) (group 1) or in combination with therapeutic genes of hepatocyte growth factor (hgf) and fibroblast growth factor 4 (fgf4) (group 2). HSC were isolated from the rat livers. Transduced cells were injected into rat portal vein immediately after PH. In group 1 on first day after transplantation in liver RFP+ hepatocyte-like cells appeared. There were no HGF+ or FGF4+ cells. On the first day some α–FP+ hepatoblasts were found, after 3rd day their number decreased. In group 2 in the early stages only small round HGF+ cells were detected, but at 28th day there were numerous HGF+ hepatocytes. FGF4 appears on the 1st day in rounded cells, after 2 days - in hepatocytes. There were a lot of RFP+ cells already at the early stages (up to 20-25 cells/portal tract), after the 7th day their number decreased. From the 14th day there was a strong increase in number of α-FP+ hepatoblasts. At the same time, a significant part of hepatocytes expressed stem cells marker c-kit. The results showed that transduction of HSC with hgf and fgf4 genes facilitates HSC engraftment, their hepatic differentiation and liver regeneration. This work was funded by Russian Basic Science Foundation grant 12-04-97088-p.
: Kazan (Volga region) Federal University, Kazan, Russia
The study aims to investigate the impact of gene therapy on healing of skin defect after autodermoplasty. Autodermoplasty was performed on Wistar rats by transplantation of full-thickness 2 × 2 cm skin graft. Animals were injected intradermally with 1 ml of 0.3 mg pCMV-VEGF165 plasmid solution (experimental group) or saline (control) immediately after surgery. The results were evaluated on 3rd, 6th, 9th, 12th and 18th day by macroscopic assessment, laser Doppler flowmetry (LDF), histologically. In the experimental group the complete necrosis of the skin graft was marked by day 9, 6 days later than in the control group. LDF data of the experimental group had no statistically significant difference compared to control. Histologically epidermis necrosis in both cases was observed. Morphometric measurements indicated the acceleration of epithelization, the size of the defect in the experimental group by 12th day was 1.7 ± 1.7 mm, by 18th day 5.83 ± 1.73 mm, in control group - 11.28 mm and 8.29 mm respectively. The number of vessels in the granulation tissue in the center under the graft in the experimental group was 26 ± 2.9, on the edges - 27 ± 3.4, in the skin muscle - 21.2 ± 3.9 that was significantly higher compared to control (under the graft - 20 ± 8.0, on the edges - 12 ± 3.9, in the skin muscle - 12.4 ± 3.6, P = 0.03) Direct gene therapy with pCMV-VEGF165 has positive effect on healing of skin defect after autodermoplasty. There is an acceleration of wound epithelization and increase of vessels numbers in autotransplant. This work was funded by a Russian Science Foundation grant (14-45-00018).
: The University of Oxford
Persistent transgene expression is crucial for many gene therapy applications. We developed the hCEF enhancer/promoter comprising CpG-free versions of the human CMV enhancer (hC) and the human Elongation Factor 1a (EF) promoter, directing persistent luciferase expression in mouse lung (Hyde et al, 2008, Nat Biotech 26:549). Persistent expression requires use of a CpG-free transgene cassette in plasmids and minicircles (Bazzani et al, 2016, Biomaterials 93:20), and is inflammation-free when all CpGs are removed. Deletion variants of the hC enhancer were generated to investigate the utility of hCEF. Complete deletion of the 302bp hC enhancer from hCEF resulted in 65-fold lower luciferase expression. When hC was replaced with the equivalent, 39% identical, murine enhancer (mC), high-level expression fell to background by day 7 (p < 0.001). When the hC enhancer was progressively deleted, the distal 93bp was found to be both necessary and sufficient for persistent expression in the lung, although this expression profile was not recapitulated in the liver. In silico analysis revealed five predicted transcription factor binding sites uniquely present in the minimal 93bp hC, but not the mC, enhancer, including SATB1.01 identified as a component of the nuclear matrix and highly expressed in mouse lung. Interestingly, expression from lentiviral vectors utilising hCEF was also higher and more persistent than conventional CMV and EF1a promoters in mouse nose and lungs (Alton et al, 2016 Thorax, In Press). Together, these data support the exploitation of hCEF in both viral and non-viral vectors for lung gene therapy applications.
: Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Amsterdam, The Netherlands
Clinical success has demonstrated the feasibility of AAV mediated liver directed gene therapy via the systemic circulation. However, upon loss of correction presence of neutralizing antibodies (Nabs) renders re-treatment ineffective. Because AAV does not integrate into the host genome, this limits its use in disorders that need to be treated shortly after birth when the liver is growing rapidly. Replication-defective recombinant SV40 (rSV40) vectors do not induce Nabs and are therefore more suitable for treating these diseases. Unlike AAV in which only the region located between both ITRs is replicated and packaged, while the bacterial plasmid backbone is excluded, production of rSV40 requires removal of the bacterial backbone from the plasmid and plasmid re-circularization. This is time consuming and difficult to comply with GMP standards. We aim to develop an efficient GMP-compliant rSV40 vector production system using Cre-recombinase mediated removal of the bacterial backbone from rSV40 vector plasmids. An rSV40 plasmid encoding the human UGT1A1 gene (pSVUGT1A1) in which the bacterial backbone DNA is located between two loxP recombination sites was used in these experiments. Co-transfection of Cos-1 producer cells with pSVUGT1A1 and a Cre-recombinase-expressing plasmid, resulted in an efficient high-titer production of SVUGT1A1 vector particles, whereas co-transfection of pSVUGT1A1 and a luciferase-expressing plasmid did not. Sequence analysis confirmed Cre-mediated deletion of the bacterial backbone DNA from the pSVUGT1A1 plasmid. To further improve the efficiency of the rSV40 production process, producer cell lines with inducible Cre expression are being developed.
: Institut de Myologie-U974
Adeno-associated virus (AAV) are of particular interest as vectors used in gene therapy for Duchenne muscular dystrophy (DMD). In DMD, dystrophin deficiency results in secondary alterations as modifications of the microtubule network that is essential for efficient endocytic trafficking of AAV vectors. The pathophysiological muscular status should impact on crucial steps for AAV effectiveness as conformational changes which occurs in endosomal compartments, uncoating, nuclear entry and transgene expression. In this study we explored the endocytic trafficking in muscle cells of DMD patients and in mdx mice and showed that the dystrophic cellular status could affect the endocytic system and subsequently the intracellular trafficking of AAV vectors.
: Human Gene and Cell Therapy Center of Akdeniz University
Diabetes is a disease characterized by insulin deficiency leading to hyperglycemia. The disease, itself, does not result from a single genetic defect, but rather from the functional loss of pancreatic beta cells or insulin resistance through multifactorial mechanisms. Despite the development of several insulin analogs, recent meta-analysis indicated that rapid and long acting insulin analogs provide little benefit compared to conventional insulins regarding to glycemic control. Therefore, gene therapy methods are investigated to develop a novel treatment approach to reconstitute a natural endogenous insulin expression profile in diabetic patients. In addition, in contrast to human insulin and insulin analogues, insulin gene therapy targets to supplement patients not only with insulin but also with C-peptide. So far, insulin gene therapy has had a limited success because of delayed and/or transient gene expression in ectopic tissues. Different routes of gene delivery (systemic injection with transient liver blockade, intraperitoneal injection and retrograde pancreatic intraductal delivery) should be tested to reveal the best way to enhance insulin gene expression in pancreas. Complementary gene delivery methods involving Vasoactive Intestinal Peptide might be essential to block autoreactive T cell assault on pancreatic beta cells and to induce immune tolerance for the ultimate cure of T1DM. Grant support: TUBITAK-215S820 and Akdeniz University 1- Sanlioglu AD, Altunbas HA, Balci MK, Griffith TS, Sanlioglu S: Insulin gene therapy from design to beta cell generation. Expert Rev Mol Med. 2012 Oct 15;14:e18. doi: 10.1017/erm.2012.12.
: Akdeniz University Center for Gene and Cell Therapy
Since liraglutide and exenatide (well known Glucagon-like peptide-1 receptor agonists) treatments can increase beta-cell proliferation, and inhibit beta-cell apoptosis as shown by previous preclinical studies, we wanted to know the outcome of Glucagon like peptide-1 (GLP1) gene delivery on pancreatic beta cell mass in an animal model of Type 2 diabetes (T2DM).1 For this purpose, Sprague Dawley rats (SD) were fed with a high fat diet prior to induction of diabetes by intraperitoneal (IP) STZ administration. Then, LentiGLP1 vectors were injected into diabetic obese SD rats. Animals were sacrified following insulin sensitivity and glucose tolerance tests. Insulin positive (+) beta cell areas on pancreatic sections were determined via immunohistochemical analysis using antibodies developed against insülin, GLP1 and GLP-1 receptor (GLP1R). Diabetic obese rats exhibited hyperglycemia (≥400 mg/dl), insulin resistance and glucose intolerance. 96% loss of beta cell mass with a drastic decrease in GLP1 and GLP1R expression were detected in diabetic obese rats. On the contrary, LentiGLP1 administrated rats exhibited %45 insulin(+) beta cell mass following STZ administration without any alteration in GLP1 and GLP1R expression compared to the control group. Moreover, small insulin(+) cell clusters were observed in acinar and ductal regions of pancreatic regions. Further immunohistochemical analyses are underway to clarify the origin and nature of these pancreatic cell clusters. Grant support: TUBITAK-112S114 and Akdeniz University 1. Tasyurek, M., et al., GLP-1-mediated gene therapy approaches for diabetes treatment. Expert Rev Mol Med, 2014. 16: p. e7.
: Akdeniz University Center for Gene and Cell Therapy
Most of the previous experimental gene therapy modalities concerning insulin gene therapy involved virus mediated insulin gene delivery to tissues (such as liver or lung) other than pancreas in diabetic subjects. Since controlled transcription and translation of proinsulin, presence of a regulated secretory pathway and inductive secretion are the major notable features of the pancreatic beta cells, none of the tissues tested turned out to be ideal to replace pancreatic beta cell function for timely expression and secretion of insulin in response to varying blood glucose levels. In order to solve this problem, a novel lentiviral vector with insulin promoter hooked up to proinsulin gene sequence (LentiINS) was designed to provide diabetics not only with basal insulin coverage but also with glucose-responsive insulin secretion to manage postprandial hyperglycemia. To accomplish this, a promotor entry vector was constructed carrying human minimal insulin promoter synthesized in vitro. Multisite gateway cloning strategy involving insulin promotor, insulin gene and a destination vector with lentivirus vector backbone was employed to generate the gene delivery vehicle. Diagnostic restriction enzyme analysis and DNA sequencing were employed to confirm the sequence and the orientation. Gene expression was detected using insulin ELISA. 1- Sanlioglu AD, Altunbas HA, Balci MK, Griffith TS, Sanlioglu S: Insulin gene therapy from design to beta cell generation. Expert Rev Mol Med. 2012 Oct 15;14:e18. doi: 10.1017/erm.2012.12. Sources of Grant Support: Akdeniz University Scientific Research Administration Division and the Scientific and Technological Research Council of Turkey (TUBITAK-215S820).
: UCL Cancer Institute
Lentiviral (LV) vectors have utility in gene therapy as delivery vehicles since they can stably transduce both dividing and non-dividing cells in vivo and in vitro. LV vectors are labile and complex structures. Consequently, current LV purification methods are time consuming, cumbersome and yield low recoveries (typically 20–40% yield with 20 to 2000-fold concentration). The objective of this work was to develop a simple and efficient affinity chromatography method yielding high recovery of LV particles with reduced levels of impurities. We genetically engineered 293T cells to express high levels of cell surface anchored biotin mimicking peptides on their surface. We compared several of these peptides and anchor strategies, identifying a disulphide-constrained peptide (ECHPQGPPCIEGRK) called CTAG, displayed on a CD8 stalk to be the most promising. We hypothesized that LV vectors generated from these 293T cells would incorporate CTAG peptides during budding. Notably, CTAG binds streptavidin in the micromolar range, which should allow elution from streptavidin matrix in physiological conditions by biotin. Experimentation determined that our hypothesis was correct, resulting in the capture of 100% of VSV-G and RDpro pseudotyped LV particles using streptavidin magnetic beads. Furthermore, the addition of 0.5 mM biotin resulted in the recovery of routinely >50% of infectious viral particles in the elution medium. These results were also repeatable at large scale resulting in >30-fold concentration of eluted titer. In conclusion, this novel single-step LV purification process allows efficient and competitive viral titer recoveries and will be of use in large-scale lentiviral production processes.
: MolMed S.p.A.
The efficacy of several adoptive T cell therapy (ACT) approaches for leukemia and lymphoma treatment can be compromised by the insurgence of unfavorable events as graft-versus host disease (GvHD) secondary to bone marrow transplantation. Ex-vivo manipulation of transplantable T cells through introduction of drug-induced suicide genes (e.g. the herpes simplex virus thymidine kinase, HSV-TK), represents a valid approach to control gene-modified cell proliferation and survival overtime. Transplanted modified cells are selectively deleted in vivo by specific activation of an otherwise inactive prodrug in those cells. Despite the well-documented utility of this approach, some limitations arose in terms of poor prodrug activation kinetics, escape from drug selection and immunological rejection. Thus, new engineered variants targeting the active site of the human deoxyCytidine Kinase (dCK) suicide enzyme were described, being able to activate a large variety of nucleoside analogue-based prodrugs with low immunogenicity. We demonstrated that lentiviral vector (LV)-mediated delivery of the triple mutant dCK.R104M.D133A.S74E (dCK.DM.S74E) fused to the truncated low density nerve growth factor receptor (LNGFR) marker renders human cells highly sensitive to the prodrug bromovinyl-deoxyuridine (BVdU), via replication block and induction of caspase mediated apoptosis. We developed RD114-TR pseudotyped self-inactivating LV RD-MolPack-trLNGFR/dCK.DM.S74E producer cells which produced unconcentrated LV with titer above 105 TU/106 cells/day. These cells were successfully adapted for serum-free and suspension growth. Our data suggest that the human suicide gene trLNGFR/dCK.DM.S74E can be constitutively produced in LV stable packaging cells without toxicity and its use can be considered a valid option in the scenario of prodrug convertase enzymes.
: Department of Cardiovascular Surgery & INSERM U970
Extracellular Vesicles (EV) are small membrane vesicles encapsulating cell-derived factors that can be used as diagnostic tools or as innovative therapeutic agents. EV's intrinsic therapeutic activity displays a tissue-repairing capacity, the specificity of which depends on the parent cells they are derived from. Our laboratory is developing a scalable process for clinical grade EV production from adipose tissue-derived stem cells (ADSC) as an alternative to cells for the treatment of chronic limb ischemia. In this indication, EV's have been identified as the active paracrine component possibly responsible for the therapeutic benefit of these cells. Here we report the successful transfer of EV's production process from T-flask to iCELLis® single-use fixed-bed bioreactor, with productivities similar to benchmark flask cultures of ADSC. iCELLis® bio-reactor is therefore a promising single-use platform for large scale production of EV's, since these bio-reactors develop surfaces ranging from 0.53 to 500 m2 with linear scalability and are already successfully used in the field for vaccines, proteins and viral vector production at manufacturing scale. Traditionally, EV isolation and purification methods relies on ultra-centrifugation steps which are time- and labor-intensive, involve aseptic operations in open phase and as such are not suited for clinically-translatable large scale manufacturing. Filtration devices are promising alternative tools for efficient and low-cost EV's purification at either small (clinical manufacturing for pilot trials) or large (commercial manufacturing for widespread clinical applications) scale.
: Swansea University
: Dimension Therapeutics, Inc.
The use of recombinant adeno-associated virus (rAAV) vectors in gene therapy for preclinical studies and clinical trials is increasing, as these vectors have been shown to be safe and to mediate persistent transgene expression in vivo. Improvement in rAAV manufacturing processes has paralleled this evolution to meet the needs for larger vector batches, higher vector titer, and improved vector quality. Stable producer cell lines containing both the rep and cap genes, and rAVV vectors can be infected with a helper virus to provide efficient production of rAAV harvest. Compared with transient transfection technology, stable producer cell line technology has minimal scale limitations, more batch-to-batch consistency, and has become the preferred system for large scale production of rAAVs for clinical applications. However, producer cell line development is commonly based on random genome integration of rAAV expressing genes and requires approximately 6–8 months to screen large numbers of cell clones to identify stable, high producer clones. To accelerate cell line development, we developed a three-tier clone selection process with tier specific screening models, and predictive markers placed at each round. This selection process is capable of quickly funneling down the numbers of clones for selection and predicting manufacturing behavior at a very early stage. We were able to establish a process from transfection to bank of high producing clones in less than three months. The selection process also streamlines the cell line, cell culture media and process development, and allows us to explore the synergies between cell line and process development.
: Fluidigm Corporation
Recent advances in the stem cell field opened many opportunities for disease modeling and cell-based therapies. However, to define the optimal condition for cell expansion, differentiation, and reprogramming remains labor-intensive and costly. To facilitate the exploratory processes, we have developed CallistoTM, an automated cell culture system for cell manipulation. The system consists of an integrated fluidic circuit (IFC), an electropneumatic controller instrument, experimental designer software and automated run time control software. Each IFC has 32 culture microchambers and 16 reagent inlets. Each microchamber can be dosed separately with different combinations and ratios of up to 16 reagents at various predefined time points. Using this system, we have developed a protocol for reprogramming human somatic cells into pluripotent cells by viral infection. Gene expression analysis demonstrates efficient reprogramming of multiple cell types on IFC, similar to the results on standard well plates. Reprogrammed cells can be exported live from individual chambers and replated onto standard plates for expansion. Furthermore, on-IFC differentiation assays will be used to confirm pluripotency of the reprogrammed cells. We have demonstrated that human induced pluripotent stem cells (iPSCs) can be directed into lineages of all three germ layers on one IFC. Multiple commercially available differentiation kits are also tested and validated. In summary, the automated microfluidic platform employs precise control of the microenvironment of cells, facilitates studies of multifactorial combinations, and enables development of robust, reproducible, and chemically defined cell culture and manipulation.
: Memorial Sloan Kettering Cancer Center
Parkinson's disease (PD) is the second most common neurodegenerative disease affecting around 5 million people worldwide. Most of the debilitating symptoms occur due to the progressive and irreversible loss of midbrain dopamine neurons. Here, we present our experience in manufacturing a human embryonic stem cell-derived cell product (MSK-DA01) intended to replace lost midbrain dopamine neurons in patients. A research-grade protocol was adapted to clinically compatible raw materials and methods were derived to cryopreserve neurons. We have now manufactured billions of cells in a GMP facility that were cryopreserved in hundreds of vials per run, with four lots manufactured at scale. One lot will be used for all IND enabling studies and for 10 patients if safety and efficacy can be demonstrated and the FDA allows our progression to the clinic. Our timeline to finish the preclinical studies and approach the FDA is early 2018. This work is funded by NYSTEM (C028503), New York State's publicly funded program to advance stem cell biology.
: State Institute of Genetic and Regenerative Medicine NAMSU, Kiev, Ukraine
: PSNResearch
Regulation (EC) No 1394/2007 states that ATMPs manufacture should be in compliance with GMP principles set out in Commission Directive 2003/94/EC providing the development of specific GMP Guidelines for ATMPs in its Article 5. In 2015, a consultation on this topic was launched by EMA to stakeholders involved in ATMPs who were invited to provide their views on the GMP requirements that should apply for ATMPs including those used in clinical trials. It was taken into consideration that early phases of research may take place in a hospital setting operating under a quality system different from the quality system typical of the pharmaceutical sector. There was a very strong support for the application of risk-based approach and it was demanded to recognize the quality systems established under Directive 2004/23/EC2 and/or JACIE accreditation system. The possibility to accept the use of a clean room of C/D grade was strongly supported by academia, as required flexibility beyond early phases of clinical trials. However, there was a common objection to the principle that the Guideline would not apply to the hospital exemption and to the principle that concurrent manufacture of different viral gene therapy vectors in the same area is not acceptable. The Commission services have developed draft Guidelines and a new consultation has been launched from June to September 2016 with the purpose to finalize the GMP Guidelines for ATMPs. Under this process underlies the essential of having a correct and well documented process for manufacturing ATMPs to comply with recent legislation.
: National Measurement Institute
There is a recognised need for standardisation of protocols for vector genome analysis in vector manufacturing, biodistribution studies, to establish dosage and detect gene doping in sport. Typically such analysis is performed by qPCR using plasmid-based calibrants incorporating transgenic sequences. These often undergo limited characterisation and differ between manufacturers, potentially leading to inaccurate quantification, inconsistent inter-laboratory results and affecting clinical outcomes. Importantly, contamination of negative samples with such calibrants could cause false positive results. We developed a unique design strategy for synthetic DNA reference materials (RMs) with modified transgenic sequences to prevent false positives due to cross-contamination. Such RMs were amplified in PCR with the same efficiency as a vector; yet, the amplicons from the RM and the transgene were distinguishable based on size and sequence. Using this strategy, we generated a RM for analysis of four human transgenes for IGF1, GH1, GHRH and FST, and characterised it for quantity, purity, homogeneity and stability according to ISO Guide 35. Finally, we validated the RM in analysis of nonviral vectors carrying any of the four transgenes in vitro and ex vivo in blood using PCR. The generated RM can be used in gene therapy or detection of doping with one of four genes used in this study. Incorporation of multiple transgenic sequences into one RM reduces the number of RMs needed for analysis of different vectors or transgenes. The design strategy can be used for production of RMs for other transgenes or transcripts for applications requiring standardised, accurate and reliable analysis.
: Swiss Stem Cell Foundation, 6925 Gentilino (Switzerland)
The use of adipose-derived stem/stromal cells (ASCs) as a cell therapy for several diseases has shown promising results in early-phase clinical trials. However, current manufacturing methods in flasks or cell factories are labor-intensive and involve a large number of open procedures. For the manufacturing of clinical-grade ASCs for use in later phase studies, production must be cost-effective, safe and reproducible. We evaluated an automated hollow-fiber bioreactor for the expansion of ASCs from stromal vascular fraction or pre-selected ASCs and compared the results with a flask-based method. Comparison involved cell yield (doubling time) and quality, including immunophenotypic profile, colony-forming unit fibroblast (CFU-F) assay, tri-lineage differentiation potential (adipogenic, osteogenic, chondrogenic), and expression of genes involved in DNA damage, repair, apoptosis and cell cycle. Cell yield from the hollow-fiber bioreactor was variable and donor-dependent. Doubling times for flask-expanded ASCs was lower than for the hollow-fiber bioreactor. After supplementation of the culture medium with basic fibroblast growth factor (bFGF), doubling times became similar. For both expansion methods, ASCs were comparable in terms of immunophenotypic profile, CFU-F frequency, tri-lineage differentiation ability and expression of genes involved in DNA damage, repair, apoptosis and cell cycle. ASCs can be expanded efficiently in an automated hollow-fiber bioreactor to clinically relevant numbers in a culture medium supplemented with bFGF. The expansion takes place with less need for operator time and in a closed system, rendering it easier to upgrade in a GMP-environment. Last but not least the quality of these cells is comparable to cells grown in flask-based methods.
: Alfa Wassermann BV
Adeno-associated virus (AAV) vectors have gained more and more attention in the field of gene therapy research. So far AAV vectors have usually been purified through either density gradient ultracentrifugation in small volumes centrifuge tubes or column chromatography. Though these purification methods have their unique benefits, there is still a need for technology that can process large volume of lysate with high AAV recovery rate. We reasoned that continuous flow ultracentrifugation could meet these requirements. We tested the Alfa Wassermann's AW Promatix 1000TM, a research scale continuous flow ultracentrifuge, as a proof of concept for AAV vector purification. In the initial experiments, we tested cesium chloride (CsCl) solution as density gradient media for AAV vector purification but found out that CsCl solution was not stable enough to form a linear gradient even when sucrose was added to increase its viscosity for AAV purification. We then tested iodixanol solution as density gradient media and got satisfactory purification of AAV vectors. Our results indicate that we can obtain near-purified AAV vectors in a single-step of centrifugation with AAV recovery rate exceeding 50%. Further experiments indicate that minor impurities associated with the purified AAV vectors could be removed by adding salts to the iodixanol solution such as CsCl to increase the ionic strength of the density gradient. The data presented here indicate that continuous flow centrifugation can be used for large scale purification of AAV vectors and it should provide an additional tool to facilitate the translation from research to the clinic.
: Miltenyi Biotec GmbH
Transduction of primary human T cells with gammaretroviral vectors pseudotyped with GALV or RD114 envelope glycoproteins is less effective than with lentiviral vectors pseudotyped with VSVG. This deficit can be compensated by using a higher multiplicity of infection (MOI), an increased viral vector concentration or transduction enhancement reagents. Polycationic reagents such as polybrene and protamine sulphate or bridging molecules such as recombinant fibronectin can be combined with centrifugation to enhance transduction. We have assessed Vectofusin-1®, a histidine-rich, cationic amphipathic peptide, as an alternative transduction enhancer. Vectofusin-1 is a short peptide of 26 amino acids which can easily be synthesised to high purity for clinical use. Unlike recombinant fibronectin, it is a soluble reagent which does not have to be precoated on cell culture surfaces, which makes automation of transduction processes for future clinical application less cumbersome. We compared the transduction performance of Vectofusin-1 and recombinant fibronectin on primary human T cells that had been magnetically enriched from peripheral blood. After 2 days of activation with TransAct Reagent, the CD4+CD8+ T cells were transduced with GALV or RD114 pseudotyped gammaretroviral vectors encoding GFP at an MOI = 1 or 2 respectively. Vectofusin-1 consistently showed a comparable transduction performance (RD114, 35–45% GFP + T cells; GALV, >60% GFP + T cells). T cells were fully functional. The reagent is compatible with both static and spinoculation protocols, which will allow easy integration into automated workflows such as the T cell transduction process on the CliniMACS Prodigy®.
: Miltenyi Biotec GmbH
Genetically engineered T cells redirected against cancer are showing tremendous clinical potential. However, adoptive immunotherapy still faces several challenges in the complexity associated with the current clinical manufacturing methods. Most commonly used protocols for the preparation of autologous gene-modified T cells employ lenti- or gamma-retroviral vectors to obtain a stable expression of the transgene (e.g. chimeric antigen receptors). Conventionally, the protocols comprise many (open) handling steps, are labor intensive and are not adapted for commercial manufacturing. For lentiviral transduction and expansion of selected T cells, we recently developed and released a highly automated manufacturing process based on the CliniMACS Prodigy® platform, the T Cell Transduction (TCT) Process. Now we further developed our process to include spinoculation and enable gamma-retroviral transduction within the single-use closed system. CD4+ and CD8+ T cells were magnetically enriched and activated with TransAct Reagent before transduction with gamma-retroviral vectors pseudotyped with GALV or RD114. Rather than using a surface coated with recombinant fibronectin, the transduction efficiency was further increased by adding the soluble transduction enhancer, Vectofusin-1®. Spinoculation as well as Vectofusin reduce the amount and cost of retroviral vector needed per patient. Transduction efficiencies comparable to small scale controls (24-well) were reached using the automated system. Genetically modified T cells were expanded for 8–13 days to clinically relevant numbers and showed in vitro functionality. The flexibility and ease of use associated with this process will enable treatment of large patient groups and make economic commercial-scale manufacturing possible.
: A.I.Virtanen Institute
Lentiviral vectors (LVs) have recently emerged as a promising vector type for gene therapy, mainly due to their versatility and ability to induce long-term transgene expression. Clinical applications require large-scale production methods capable of producing large quantities of high-quality vector. LV processing, however, remains challenging due to the fragile nature of the vector. In this study, we developed an optimized and scalable downstream method for processing recombinant lentiviral vectors. Our downstream process consisted of clarification with continuous 0.6 – 1.2 μm depth filtration, and subsequent concentration and diafiltration into chromatography buffer with 100 kDa flat sheet tangential flow filtration. Different filter types and operation conditions were tested to optimize the process for highest recovery and straightforward scalability. The effect of storage conditions, including temperature and buffer, was also assessed. Samples were analyzed with flow cytometry for infective titer, ELISA for total vector particle concentration, and BCA method for total protein concentration, after each downstream step. Purification optimization with anion exchange chromatography (AIEX) utilizing fast performance liquid chromatography (FPLC) is in progress. Different chromatographic supports and columns, as well as the effects of pH and ionic strength on this downstream step will be evaluated. Following clarification, we have obtained full infective particle recovery of the product, and achieved up to 85% infective particle recovery after optimization of concentration and diafiltration. The results of this study show a high recovery of LVs from a production-scale of approximately 5–10 liters, and confirm efficient scalability of LV downstream processing.
: Dimension Therapeutics, Inc.
: Pall Life Sciences
Adeno-Associated Viral vectors (AAV) are powerful tools for in vivo gene delivery to cure congenital diseases. AAV can be efficiently produced by transient transfection of HEK293 cultured in static 2-D multi-tray system (MT) and this method is widely used at small scale for preclinical and clinical studies purpose. However, industrial transposition (>200L) of such process, in compliance with current Good Manufacturing Practice (cGMP), leads to very high manufacturing costs challenging the commercialization of these therapies. Cost modeling tools should then be used as early as possible in the process development to ensure that retained solution will be transposable at large scale at acceptable investment cost. In this study we compare AAV manufacturing costs between 200L and 1000L for three Upstream process (USP) technologies: static MT, suspension bioreactor and fixed-bed bioreactor, using transient transfection in HEK293 cells in serum-free media as expression system. Utilizing the BIOSOLVE Software for modeling entire processes in cGMP conditions, cost structure is determined and sensitivity analysis is used to identify parameters that can leverage the cost of goods (CoGs). While scale-out of MT leads to CoGs highly dependent on cost of work we show that suspension and fixed-bed USP CoGs are efficiently driven down during scale-up and rely mainly on clinical grade transfection plasmid price. By introducing in the model both literature and experimental data, further scenarios are explored to take into account specificities of fixed-bed technology.
: Department of Ophthalmology, University Hospital RWTH Aachen, 52074 Aachen, Germany
The TargetAMD consortium aims to develop a non-viral therapeutic approach to treat patients with neovascular age-related macular degeneration (nAMD). Contrary to the current medication of intravitreal injections of vascular endothelial growth factor (VEGF) antibodies, TargetAMD has developed a protocol for subretinal transplantation of autologous pigment epithelial cells genetically modified ex vivo. Cells will be transfected with the anti-angiogenic pigment epithelium-derived factor (PEDF) gene tending to prevent choroidal neovascularization. Stable gene transfer is mediated by electroporation and the hyperactive Sleeping Beauty (SB100X) transposon system, cloned into miniplasmids free of antibiotic resistance markers (pFAR4). Here, we report the establishment of a protocol for the isolation and subsequent transfection of iris pigment epithelial (IPE) cells harvested from patients’ iridectomies, which mimics the surgical procedure. Patients’ iridectomies were received 3 to 24 hours after surgery from patients 28 to 86 years of age. Measurement of the stroma resulted in a mean value of 1.84 × 1.37 mm2 (N = 26). Separated, isolated IPE cells were transfected using 30 ng of pFAR4-SB100X transposase miniplasmid and 470 ng of pFAR4-PEDF or the pFAR4-PEDF-His transposon miniplasmids. Transfected IPE cells showed PEDF secretion during the entire cultivation time of up to 150 days. The PEDF secretion level was 3-fold higher compared to control cells as analyzed and quantified by immunoblotting. Further analysis are ongoing. The described protocol is important for standard operating procedures to manufacture the investigational medicinal product and indicates the feasibility of the gene therapeutic approach.
: Cobra Biologics
Adeno-Associated Virus (AAV) based viral vectors have become the delivery vehicle of choice for use in a wide range of gene therapy indications. Currently, 25% of all viral vectors in development are AAV-based and are used in 60% (80 out of 133) of Gene Therapy trials for monogenetic diseases [Roots Anal. 2015]. However, a major challenge preventing further exploitation of AAV viral vectors, is the lack of an affordable scalable production platform to generate sufficient quantities of AAV to support treatment of larger indication groups and/or late Phase clinical trials. In collaboration, Cobra Biologics (CDMO) and Tecrea are investigating whether the application of the novel transfection reagent, Nanocin™, in combination with hollow fibre bioreactors can be used for helper free transient production of AAV-based viral vectors; such an approach would reduce operational costs and the manufacturing facility footprint required for AAV production, compared to the current standard scale-out production processes that utilise large volumes of plastic ware, labour and production space.
: Division of Neuroscience San Raffaele Hospital, Milan
Human neural/stem precursor cells (hNPCs) from fetal human Central Nervous System (CNS) can establish stable, transplantable non-immortalized cell lines in vitro. We aimed to demonstrate the feasibility and quality assessment of a hNPCs-derived Master Cell Bank (MCB) in view of treating patients with progressive Multiple Sclerosis in a Phase I clinical trial. Primary human neural progenitors were derived from a single fetus at 10–12 weeks age from therapeutic pregnancy interruption. Cells were cultured in stationary conditions. Permission to use of human fetal CNS tissue was granted by the ethical committee of the San Raffaele Hospital. Cryopreserved primary cells were seeded and scaled up for a total of 6 passages. A total of about 1.9×109 cells were recovered and stored in liquid nitrogen vapour phase (forty-one vials with 46×106 cells each). Fold increase was 2.16 ± 0.37, and cell viability was 84.17% ± 6.82%. MCB was then tested for identity and safety (Charles River, GmbH) and it resulted fully compliant with all established specifications. Next we assessed the Working Cell Bank (WCB) process validation by the expansion of MCB (two consecutive runs). Cells were plated at 35.000 ± 5.000 cells/cm2, and neurospheres were dissociated when they reached approximately 100 - 150 μm in diameter. After 6 steps cells were harvested, washed and stored as for MCB. Fold increase (mean±SD of 6 passages) was 1.85 ± 0.68 and 2.32 ± 0.96, and cell viability was 77.69% ± 3.90% and 76.99% ± 5.16% for the two lots, respectively. We demonstrated feasibility of the GMP manufacturing of hNPCs for clinical use in an academic setting.
: FinVector Vision Therapies Oy
Lentiviral vectors (LVV) are promising tools for gene therapy due to their unusual property of efficiently transduce both dividing as well as non-dividing cells. In addition, with LVV it is possible to achieve a stable transgene expression. During the last decade, use of LVV in Clinical Trials has been increasing. We have optimized the production of third generation self-inactivating LVVs expressing green fluorescence protein (GFP) under human phosphoglycerate kinase promoter in adherent HEK293T cells using iCELLis Nano bioreactor (PALL Life Sciences). Two different bioreactor bed sizes, 2.67 m2 with low compaction bed and 4 m2 with high compaction bed, were tested. Transfection was performed four days after inoculation using calcium phosphate precipitation or Polyethyleneimine (PEI), and LVV were harvested from 24 h to 72 h post-transfection by perfusion. Use of different mediums, effect of pH, various transfection protocols, and perfusion rates were tested. Samples were taken from bioreactor and perfusate at 24 h, 48 h and 72 h time points after transfection. Both the viral particle titer based on p24 ELISA and transductive titer based on GFP expression in transduced cells, were analyzed from the samples taken. In most optimal runs using PEI transfection, total of over 1.0E+13 viral particles and 1.0E+10 transductive units (TU) were obtained. LVVs can be produced in iCELLis Nano with high yields, and based on the optimized parameters in smaller scale it is possible to proceed to larger scale using iCELLis 500 system providing up to 500 m2 cell culturing area.
: Université Laval, Chemical Engineering Department, Quebec, Canada
Lentiviral vectors (LV) manufacturing must be improved to increase the yield, facilitate the scale-up and satisfy to health regulatory agencies. We have developed and optimized a LV production process in serum-free medium using an inducible HEK293 producer cell line which grows in suspension culture. By adding two inducing molecules (cumate and doxycycline), this cell line produces LV pseudotyped with the G protein of the vesicular stomatitis virus without the need of any transfection. Our tested LV carried an expression cassette for GFP to facilitate LV quantification. To optimize the process, a design of experiment (DoE) was prepared. It included the study of different culture media, high cell density production using six commercial cell boosts, inducers concentration, pH, temperature and components known to improve production of viral vectors as sodium butyrate, caffeine and valproic acid. We found that the SFM4TransFx-293 media supplemented with Hyclone's cell boost five produced highest titers at high cell density. The optimized conditions were at a cell concentration of 4 × 106 cells/mL; pH of 6.85; doxycycline and cumate concentrations of 0.3 μg/mL and 21 μg/mL, respectively. A LV carrying the cDNA for a mini-version of dystrophin (transgene of 5.8 kb vs 0.7 kb for GFP) was also constructed. We will evaluate if optimized conditions for LV-GFP production is applicable to LV-mini-dystrophin. The later LV could be used for gene and cell therapy of Duchenne Muscular Dystrophy.
: University of Oxford
Development of lentiviral-based therapeutics is hindered by high costs of cGMP manufacturing, a particular concern for in vivo delivery where high titres and volumes are typically required. The key cost component of viral-vector manufacturing is production titre, which is typically several log-orders lower for lentiviral vectors than non-enveloped vectors such as rAd or rAAV. Efforts to increase lentiviral production have largely focused on optimisation and scale-up of transient transfection-based processes. Conversely, little attention has been paid to the optimisation of the mammalian host cell, with nearly all reported processes relying on HEK293T derivatives. We hypothesised that HEK293T cells were not necessarily optimal for lentiviral production and have embarked on a rational design process to establish new cell lines with enhanced manufacturing properties. We identified 130 cellular factors active in the late phase of the lentiviral life cycle that are potentially relevant to lentiviral vector production. We evaluated the effect of siRNA knock-down of these factors (3 days production, 2e5cells/well, 100nM siRNA/well in quadruplicate) on lentiviral vector production titre (3 days transduction, 5e4cells/well). We identified 9 host factors, the knockdown of which significantly increased lentiviral production by 1.4 to 2.1-fold (threshold: >2 SD over control, p < 0.05, power >80%). CRISPR/Cas9-mediated genetic disruption of these host factors has yielded novel cell lines that support 2-fold greater lentiviral production following transient transfection of producer plasmids. We anticipate that combinatorial disruption will yield further lines with even greater ability to support higher production titres; offering reduced manufacturing costs and thus increasing the speed of clinical development.
: Division of Hematology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
The applications of human hepatocytes in biomedical research and the treatment of end-stage liver diseases is limited by the shortage of hepatocyte donors. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) hold great potential as an unlimited source of hepatocytes. In this study, we have successfully established hepatocyte-like cells from both human ESC and iPSC using a feeder-free, three-step protocol. The hepatocyte-like cells derived from both sources exhibited typical hepatic morphology and expressed high levels of several hepatocyte-specific markers, including hepatocyte nuclear factor 4α (HNF4α) and α-fetoprotein (AFP) and albumin (ALB). Moreover, these hepatocytes also expressed cytochrome P450 (CYP7A1) which is important for drug metabolism and possessed an ability to store glycogen. In conclusion, the differentiation system used in our study could be effectively used to induce the differentiation of both ESC and iPSC toward functional hepatocytes which expressed high levels of hepatocyte-specific genes and proteins. The pluripotent stem cells-derived hepatocytes established in our study could potentially be used as renewable sources of hepatocytes for biomedical researches and clinical applications.
: iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal
Viral vectors are the most used delivery system in gene therapy clinical trials since they provide efficient cell entry and expression of the therapeutic gene. Nevertheless, its manufacture is still challenging, mainly for envelope viral vectors such as recombinant retrovirus (RV). The success of immune therapies and the approval of gene therapy products increased the demand for robust and high-yielding production hosts. Metabolic engineering of producer cell lines emerges as a tool to address this challenge. Previously, our group identified glutathione metabolism to be important for the production of RV. Herein, we engineered this pathway by over-expressing, at different levels, seven candidate genes in a human producer cell line. This approach unveiled the influence of each gene as well as the interplay between their expression levels and RV productivity. Resulting populations were characterized for cell growth, productivity, viral components and metabolic gene expression. Different dynamics in the producer state were induced - from downswing to upswing - depending of the metabolic gene and its over-expression level. In this context, RV productivity was raised up to 10-fold. High-titer yielding manipulations were associated to substantial amplification of viral components expression, now under investigation. These results demonstrate glutathione metabolism is a good engineering target to improve RV manufacture and reinforce the need of fine-tune host gene expression to achieve hyperproducing phenotypes. This work contributes for the gene therapy field by not only debottlenecking RV manufacture, but also, by providing tools to elucidate and engineer better RV and other viral vector producer cells.
: Adverum Biotechnologies, Inc.
Adeno-associated virus (AAV) based clinical gene therapy has been increasingly utilized owing both to an enhanced molecular understanding of human diseases, and to the progressive improvement of structural biology and gene expression technologies. One major challenge is to develop a robust, scalable, and economical production process for both clinical and commercial manufacturing of AAVs. The implementation of AAV manufacturing technology requires the optimization of a number of variables, including (1) cell lines capable of synthesizing the required AAV at high productivities to ensure low operating costs; (2) culture media and bioreactor culture conditions that achieve the requisite productivity (3) scalable downstream purification methods and unit operations that can meet product quality specifications (4) robust formulation strategies that can achieve high dose requirements. We have developed a proprietary, completely single-use production platform for the manufacturing of various different serotypes of AAV based on the sf9/baculovirus system that has optimized all of these variables. This production platform is scalable from shake flask to 10L, 50L, and 200L bioreactors. Moreover, all critical product quality attributes were found to be indistinguishable from vectors manufactured by plasmid transfection in HEK293 cells in in vitro qualified assays and in in vivo comparability assessments.
: 4MEDi-CBTD, Ostrava, Czech Republic
: Centre for Regenerative Medicine, University of Bath
Human Embryonic Stem Cells (hESCs) show significant therapeutic potential in treating degenerative disorders with their ability to produce a limitless supply of starting cells and their potential to differentiate into more than 200 different cell types. Our current aim is to generate a robust protocol for the differentiation of hESCs to respiratory epithelial cells. These cells could then be used either for transplantation studies or, as an in vitro model for drug toxicity testing. We differentiated hESCs into the Definitive Endoderm (DE) lineage using a two stage protocol based on culture with the novel Glycogen Synthase Kinase-3 (GSK-3) inhibitor (termed 1m), along with Activin A. We confirmed the status of the cells by a combination of immunostaining and PCR. We show loss of pluripotency markers (Sox2 and Oct3/4) and gain of DE markers (Sox17, FoxA2 and CXCR4). After the induction of DE, we then treated the cells with transforming growth factor (TGF)-β and bone morphogenic protein (BMP) pathway inhibitors (SB431542 and Noggin respectively). This combinatorial treatment resulted in the differentiation into the Anterior Foregut Endoderm (AFE) lineage based on expression of Pax9+FoxA2+ plus the up-regulation of Sox2. Further differentiation of AFE derivatives into Lung Progenitor Cells via treatment with a cocktail of trophic factors (BMP4, EGF, all-trans Retinoic Acid, FGF10, KGF and Wnt3a; BEAFKW) yielded a population of Nkx2.1+FoxA2+ cells that potentially corresponded to the lung lineage. Further studies are required to confirm these observations and to refine the differentiation protocol for production of homogenous lung epithelial cell types.
: City of Hope Medical Center, Duarte, CA, USA
The promise of stem cell-based therapies to a myriad of clinical applications, including cell replacement, tissue regeneration, enzyme replacement, and cancer elimination is undoubtedly pivotal in this era. As we move from early clinical trials toward potential commercialization, there exists a great challenge to scale-up production of adherent cells, as well as toward off-the-shelf products that can be readily distributed and prepared at multiple sites. We first adopted an off-the shelf product profile, using a freeze/thaw method accompanied by a pre-packaged cell preparation kit, and pre-qualifying our cell bank in advance; cutting our NSC product preparation time to only 1 hour prior to patient administration. Currently, we are conducting two first-in-human phase 1 studies using genetically modified, allogeneic neural stem cells (NSCs) that activate prodrugs to chemotherapeutic agents selectively at brain tumor sites. As our trials progressed to multiple treatment rounds, current production methods became insufficient in yield, time-consuming and costly. We therefore developed a novel method of NSC scale-up and adenoviral transduction using the hollow fiber Quantum® Cell Expansion System. Seeding of 4 × 107 NSCs in one unit generated a production yield of up to 3 × 109 cells within 10 days. NSCs characterized for genetic and functional stability demonstrated equivalent results to NSCs expanded in conventional cell culture flasks. We then simultenously used 7 bioreactors to propagate the first GMP grade NSCs for clinical use with 50% reduction in production cost and time. Considerations to improve product stability and production processes are imperative for product commercialization.
: iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal
Lentiviral vectors (LV) are efficient tools to mediate gene transfer, allowing long-term expression of the therapeutic transgene after integration on target cell genome. In the last 10 years their use in gene therapy clinical trials has grown due to their specific ability to transduce both dividing and non-dividing cells and its safer integration pattern. Nowadays, the major obstacle to the use of LV in gene therapy is their production at high titers. Despite recent improvements in transient LV productions at higher scales, those are still limited to a short production period, are expensive and raise safety issues. A continuous production system where a stable producer cell line constitutively produces LV may overcome those disadvantages. However, its development has being very laborious and time-consuming due the toxicity of some viral components and the difficult screening of a clone with high expression of all LV components. Here we present a new approach for LV cell line development. In this work we started by optimizing the expression cassettes (3rd generation LV packaging system) and the viral components, testing it in transient productions. After optimization, stable populations were established by transfection of 293T cells with those, a non-toxic envelope glycopoteins and a transgene. In the end the populations are screened in order to indentify the best LV producer clones. This work shows a new approach for stable LV producer cell line generation, without using transduction and avoiding inducible systems, increasing the safety standards of LV production.
: Dimension Therapeutics, Inc.
The development and commercialization of a biological pharmaceutical, such as recombinant adeno-associated virus (rAAV), requires rigorous quantitative and qualitative testing. An accurate particle titer measurement is important for the development of purification processes to support commercial scale. Current methods for titering rAAV include qPCR, for measuring genome copy titer, and ELISA, for measuring particle titer. Additionally, methods such as dynamic light scattering (DLS) have been considered for assessment of particle size and aggregation but with limited success for rAAV. Here, a novel method is described, called Nanoparticle Tracking Analysis (NTA), to provide rAAV titer (concentration) within minutes, as opposed to hours or days required by traditional techniques. NTA characterization was enabled by a gold labeling technique to increase the light scattered from rAAVs. With a purified and gold labeled rAAV sample, NTA results indicated a monodisperse size distribution and a measured rAAV particle titer equivalent to the concentration based on genome copies by qPCR. Particle titer determination can also be relevant for assessment of product stability. NTA was used to assess the stability of rAAV over a 3 month thermal stress test. The NTA method enabled the detection of a reduction of the primary rAAV particle peak within 1 month, as well as further reductions over time. These initial results demonstrate the potential uses of NTA for product characterization and correlate well with orthogonal stability indicating methods.
: Institute of Child Health, University College London, London, UK
Lentiviral vectors (LV) are being widely applied to deliver therapeutic genes to human cells. High yields of vector stocks are required for most therapeutic applications, driving the need to develop more efficient manufacturing platforms. We hypothesized that HIV-1 derived LV may trigger innate immune responses in producer cells during vector production and that this might be rate-limiting for large scale campaigns. Manipulation of such pathways could unlock more efficient, higher yield vector manufacturing platforms. We measured the production of inflammatory cytokines and the induction of NF-kB/IFNb/IFIT-1-dependent reporter gene expression as surrogates for innate immune activation in HEK293T. Overexpression or depletion of innate signaling molecules was used to manipulate the innate response in producer cells. We measured LV titres with and without innate immune pathway manipulation to assess the effect of intracellular innate immunity on LV production. We show that the commonly used vector packaging cell line HEK293T is capable of producing a type I interferon response after activation of DNA sensing by expression of DNA sensor cGAS and adaptor STING. Furthermore, inflammatory cytokines were detected in response to transfection of packaging constructs, and varied depending on envelope and genome sequences. Nonetheless, vector titres were independent of type I interferon responses, and were not influenced by either inhibition or activation of innate immune pathways. Indeed, neither adding exogenous interferon beta, nor activating innate inflammatory responses significantly impaired CAR19 therapeutic vector production. Understanding how vector production is accommodated, despite triggering of innate responses, is now a key question under further investigation.
: GENETHON
With the objective of producing AAV vectors at industrial scale, we adapted the HEK293 cell line to grow in suspension culture in chemically defined culture medium. Upon Poly Ethylene Imine -mediated triple transfection, our current process consistently delivers >1E10 vg/mL of AAV8 or AAV9 vectors in 2L, 10L, 50L and 200L bioreactors. To further boost the productivity and/or to secure the medium supply, we wanted to evaluate alternative culture media. We had the opportunity to test media prototypes developed by Irvine Scientific. One of the prototypes allowed direct adaptation of our suspension 293 cells. This medium, named BalanCD HEK293, supports cell growth and does not induce cell aggregation even though it does not contain anti-clumping agents traditionally known for inhibiting transfection. As a result, the cells displayed a satisfying transfectability and a potent production of AAV vectors. The experiments in 2L and 10L showed a productivity >1e10 vg/mL. Interestingly, the culture medium was not depleted for essential nutrients at the end of the production process which what not the case for our reference medium. This new culture medium appears as an advantageous raw material for the production of Gene Therapy vectors. Particularly, as a serum free, chemically defined and animal origin free product, this medium fulfills the current requirements for biological risks management in the biotech industry. Our next goal is to confirm the process performance at large scale. In addition, preliminary results using 293T cells showed that the medium also supports potent production of Lentivirus vectors.
: Cellular Dynamics Intl, a FUJIFILM company
The fast pace progress in the human induced pluripotent stem (iPS) cell) technology area offers new opportunities in therapeutics applications. CDI's proprietary episomal, non-integrating, reprogramming method together with CDI's process for mass producing human cells, enable the potential use of iPS cell-derived tissue cells in a clinical setting. Allogeneic transplants have low or poor matching qualities between the donated tissue and the patient, thus resulting, too often, in rejection. Current alternatives for tissue or organ transplants include autologous and partial HLA match, or HLA superdonor, options. HLAs allow the immune system to distinguish “self” from “non-self” therefore play a key part in in determining tissue compatibility and immune rejection. CDI has manufactured the first two HLA superdonor iPS cell lines in the world under current Good Manufacturing Practices (cGMP), using blood samples collected from eligible consented donors. These lines are homozygous at certain HLA loci providing a partial HLA match to 19% of the U.S. population. These HLA superdonor iPS cell lines can be used to manufacture tissue cells for potential cell therapy and organ rehabilitation. CDI has already expanded this master stem cell bank by reprogramming 5 donor samples that provide a partial HLA match to 35% of the U.S. population. CDI is now planning on collecting blood samples from 12 donors possessing haplotypes that offer a partial match to 50% of the U.S. population, with the goal to include additional donors to reach a 95% coverage of the U.S. population.
: Oxford BioMedica
Large-scale production of gene therapeutics comprising lentiviral vectors (LV) would benefit from the development of producer cell lines (PCL) enabling the generation of large quantities of vector; which remains a challenge using current transient transfection processes. To generate PCLs, clone isolation by limiting dilution cloning (LDC) in selective media is required. Current manual processes are time consuming and labour intensive, limiting the number of clones that can be processed (typically ∼200). By screening a greater number of clones the probability of isolating a clone that can produce high titre LV would be increased. Oxford BioMedica has developed a bespoke Automated Cell Screening System (ACSS) which uses state-of-the-art automation capable of isolating, screening and processing up to 3000 clones at a time. The ACSS can also perform routine passage as well as high-throughput screening of clones for LV productivity. The ACSS is comprised of several integrated components, all controlled by a single piece of software. The equipment is contained within a class II containment workcell, and consists of a liquid handler, liquid dispenser, incubator and a barcode scanner (facilitates traceability). Plates are transferred between these components with the aid of a robotic arm, and clones are analysed via an automated microscope imaging system. The latter can be used to indicate monoclonality, as well as clone productivity. Automating the various stages of LV PCL development in a single bespoke technology greatly increases the number of clones that can be isolated and screened, while accelerating overall cell line development timelines.
: GENETHON
Recombinant baculoviruses are extensively used to express heterologous genes in cultured insect cells. The baculovirus expression vector system (BEVS) is particularly expedient for large-scale applications, such as the production of recombinant adeno-associated virus (rAAV). To date, the most advanced system is based on the use of two different viruses providing the AAV helper functions rep-cap and the recombinant AAV vector sequence flanked by the ITRs to infect Sf9 cells at low MOI (multiplicity of infection). This study deals with the kinetics of infection of Sf9 cells by several baculoviruses that takes place during the production process. Baculoviruses expressing genes coding for different fluorescent proteins were used to co-infect Sf9 cells and the resulting fluorescence was monitored by spectral flow cytometry. Thus, the kinetics of co-infection have given an insight on the cells being infected by either a single or several baculoviruses. Moreover, we have determined the impact of uncertainty/variability in the titration process of infectious baculovirus particles and the potential disequilibrium between both baculoviruses in the case of co-infection at low MOIs. Nevertheless, even in the case of using exactly identical low MOIs (i.e. 0.05 per baculovirus species, equilibrated MOI for both baculoviruses) for two different baculoviruses it could be established that only 60–70% of all cells have been infected by both baculoviruses and 30–40% only with one baculovirus species. This information is critical to understand and improve the production process of rAAV. Moreover, these results have led us to work on the development of the monobaculovirus system.
: Laboratorio di Terapia Cellulare e Genica Stefano Verri, ASST-Monza, Ospedale San Gerardo, Monza (MB)
Bone marrow (BM)-derived human mesenchymal stromal cells (hMSC) show efficacy in the treatment of steroid-resistant severe acute graft-versus-host disease (aGvHD) patients. hMSC were generated in the context of a clinical protocol (EudraCT#2008-007869-23) for patients exhibiting acute or chronic, steroid-resistant GvHD. The manufacturing process was performed under a GMP conditions in an academic cell factory. hMSCs were isolated and ex vivo expanded as previously described (Capelli C et al, Bone Marrow Transplantation 2007). hMSC were supplied from 4 healthy donors (median age 11.5 years). An intermediate product was obtained from each donor after a first expansion run (P1) consisting of four lots (118 single vials −106 hMSCs per vial). A total of 34.33 × 106 intermediate product hMSCs were used to produce 13 batches of drug product hMSC providing 8,073.75 × 106 total cells (317 single vials – 25×106 hMSCs per vial). Median duration of drug product hMSC was 12 days, whereas median Fold Increase and Doubling Time were 238.14 (range 135.2 – 341.88) and 7.9 (range 7.08 – 8.42) respectively. All 13 batches of hMSC were released for clinical use being fully compliant for safety, identity and potency parameters. In the frame of the clinical protocol, 17 pediatric patients were treated with hMSC with a total of 111 single ready-to-use vials infused trough 77 single administrations procedures (median 4 vials per patient). The production process of hMSC was robust, reproducible, and sufficient to sustain the clinical needs of the protocol allowing rapid administration on demand.
: Cobra Biologics
The increased demand for the production of AAV and Lenti viral vectors through transient expression approaches for use in both regenerative and immunotherapy clinical studies, has created a need for innovative approaches to be taken for the production of the starting plasmid DNA vectors used as their production. The production of these vectors creates a number of challenges for drug developers with regards to the cost, time and quality standards. Plasmids used in virus production have been designated as being a GMP Starting material by the EU Regulatory authorities. In response to these demands Cobra has developed a High Quality (HQ) plasmid DNA offering which has streamlined manufacturing approach based on an existing GMP platform approach. This allows for plasmid manufacturing to be performed in under 8 weeks to produce fully compliant plasmid DNA that can be used to support early phase clinical virus production within the EU.
: Swansea University
: Universitat Autònoma de Barcelona (UAB)
Viral vectors can be used safely in vitro and in vivo, applying standard procedures in a controlled setting. Producing the vectors however, requires the application of specialized techniques, access to expensive equipment and biological safety laboratories. The Vector Production Unit (UPV) is a technological platform at the Universitat Autònoma de Barcelona. It has Biological Safety Level 2 and 3 facilities, and it is staffed by experienced and highly qualified personnel. Since its opening in 2003 it has been dedicated to the design, development, production and purification of hundreds of viral vectors for basic research and gene therapy pre-clinical studies for both public and private research laboratories. Key products and services: - AAV vectors: Concentrations starting at 2x10E12 vg/mL, although titres higher than 1x10E13 vg/mL are frequently obtained. - AAV pseudotypes routinely produced are: AAV1, AAV2, AAV6, AAV8, AAV9 and AAV rh10. Contact us to request information for AAV3, AAV4, AAV5 and AAV7 vectors. - Adenovirus vectors: Concentrations starting at 1x10E12 pp/mL. Serotypes produced are: Ad5, Ad5/40, Ad5/52, CAV2. - Vector generation, amplification, purification and QC tests, usually in 6–10 weeks. - Discounts are offered for large-scale productions. - The UPV has been authorized by the Spanish “Ministerio de Agricultura, Alimentación y Medio Ambiente” to work with Biosafety level 2 GMOs. ID: A/ES/14/I-29. Funding: UPV is partially supported by TECNIO (ACCIÓ; TECDTP15-1-0007) which is sponsored by the Generalitat de Catalunya in Spain. JP is partially supported by the “Personal Técnico de Apoyo” programme (#PTA-09357) of the “Ministerio de Economía y Competitividad”.
: PlasmidFactory GmbH & Co. KG
Amongst other applications, our plasmid DNA is used in the GMP-compliant production of recombinant viruses, antibodies and RNA, where these are the active pharmaceutical ingredients (API) used in clinical trials. Here, it is not always necessary to produce the plasmid DNA under GMP as well, in order to use it for such applications. An alternative is the so-called High Quality Grade plasmid DNA which is highly purified and well-characterised and, hence, meeting the requirements of most regulating agencies. High Quality Grade plasmid DNA is produced in our facility based on a research cell bank (RCB) and the very effective, patented ccc Grade DNA technology. A number of quality controls, both to the cell bank and to the plasmid DNA product, ensure that the final result is a product designed especially for the intended application and that complies with the appropriate regulatory standards. PlasmidFactory's new facility for the production of this high purity plasmid DNA is now operating successfully in new, modern labs where plasmid DNA of the highest quality is being produced in accordance with the EMEA guideline CHMP/BWP/2458/03. To ensure product safety, substances of animal origin are not used at any stage of the entire process, guaranteeing maximum possible product purity by reliable exclusion of contaminants such as bacterial chromosomal DNA or damaged plasmids. Only one plasmid is produced in each area - different plasmids are not produced in parallel in the same lab.
: University Medical Center Johannes Gutenberg Univerity Mainz
We have recently shown that expression of Sox2 and Ascl1 can lineage-convert pericytes from the adult human brain into induced neurons (Karow et al., Cell Stem Cell 2012). Here we addressed whether the functional synergism of Sox2 and Ascl1 in pericyte-to-neuron reprogramming is based on a molecular synergism. Toward this we performed transcriptome analyses of pericytes expressing Sox2, Ascl1 or both factors. This revealed that Ascl1, postulated to act as pioneer factor in fibroblast reprogramming, fails to transactivate many of its targets in pericytes. While Sox2 alone did not cause massive alterations in gene expression, co-expression of both factors resulted in the significant transactivation of neurogenesis genes, including many known Ascl1 direct targets. Consistent with a GABAergic phenotype, co-expression of these reprogramming factors resulted in the induction of master regulators of interneuron fate. Finally, single cell transcriptome interrogation showed that pericytes expressing Ascl1 alone can adopt an alternative myocyte-like fate, as desribed previously for fiborblasts (Treutlein et al, Nature 2016), while Sox2 and Ascl1 co-expression promotes a neuronal identity. This data suggest that Sox2 co-expression has a massive influence on Ascl1-dependent gene expression.
: Memorial Sloan Kettering Cancer Center
We delineated a regulatory network involving the p53 tumor suppressor family and the Wnt pathway, acting together with the TGF-β pathway, to drive mouse and human mesendoderm differentiation. Knockout of all three members, p53, p63 and p73, shows that the family is essential for mesendoderm specification as cells exit pluripotency in vivo and in culture. Wnt3 and its receptor Fzd1 are among a small set of p53 target genes that are specifically activated in this context. Induction of Wnt signaling by p53 is critical for activation of mesendoderm differentiation genes. Globally, we show that Wnt3-activated Tcf3 and nodal-activated Smad2/3 transcription factors depend on each other for co-occupancy of target enhancer elements in master differentiation loci. Our results reveal a selective interdependence between signal-activated Tcf and Smad transcription factors. Thus, the p53 family governs a regulatory network that integrates essential Wnt-Tcf and nodal-Smad inputs for mesendoderm differentiation in the early embryo.
: Università degli Studi di Napoli “Federico II”
The exit of mouse embryonic stem cells (ESCs) from the pluripotent ground state requires crucial changes in gene expression. We have demonstrated that the chromatin-associated protein Hmga2 is necessary for gene expression changes that allow the transition from ESCs into epiblast-like stem cells (EpiSCs). We have found that lack of Hmga2 in induced pluripotent stem cells (iPS) cells incapacitates these cells to exit from the pluripotent ground state. This function of Hmga2 is due to its cooperation with Otx2 in the engagement of new enhancers and thus in a substantial modification of gene expression. In turn, Otx2 activates Hmga2 expression soon after the induction of ESC differentiation. In Hmga2 null cells we have observed that Otx2 fails to induce the proper expression of its targets cause of the direct involvement of Hmga2 in the Otx2-DNA interaction. Our findings reveal a novel mechanism necessary for the exit of ESCs from the pluripotent ground state: Hmga2 gene is activated by Otx2 and Hmga2 protein binds to the enhancers targeted by Otx2, thus facilitating the engagement and/or the stable association of Otx2. Moreover, we have found that this cooperation between Otx2 and Hmga2 is crucial for the induction of the RNA binding protein Lin28. On the other hand, Lin28 controls the proper level of Hmga2 during differentiation generating a feedback loop. Therefore, our results demonstrate that Hmga2 is a key element of the regulatory network that governs the exit of ESCs from the pluripotent ground state.
: Massachusetts Institute of Technology
Stem cell derived 3D liver models hold great promise for modeling human diseases in a patient specific manner. However, most cell-culture models suffer from limited functionality due to incomplete differentiation of human stem cells, or rapid loss of functionality of primary cells in culture. We identified a small molecule that enhances hepatocyte functions in vitro and promotes the differentiation of induced pluripotent stem cell– derived hepatocytes toward a more mature phenotype. In addition, the molecule enhanced maturation of iPSC derived cardiac tissue. Furthermore, small molecule treatment enhanced liver size during zebrafish development and enhanced survival following fatal doses of acetaminophen, in embryos and as well as adult animals. In order to provide insight into the molecule's mechanism of action, we performed L1000 expression profiling. Additionally, we synthesized close analogues to study the structure activity relationship and generated a bait molecule for affinity-based target identification. We performed affinity purification-mass spectrometry combined with network analysis and found several interacting proteins involved in alternative splicing. Identifying the targets associated with this small molecule is essential for further optimization, can lead to better understanding of the maturation process of iPS cells down multiple lineages and eventually help to create better, more predictive patient specific models of liver disease.
: Department of Obstetrics and Gynecology, Reproductive Endocrinology and Infertility unit, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Thailand
This study investigated a novel isolation protocol of human amniotic fluid stem cells (hAFSCs) from amniotic fluid and developed the method for differentiation hAFSCs to be the functional beta like cells in vitro. The human amniotic fluid is a perinatal source of multipotent stem cells derived from the amniotic membranes and fetus may be useful for clinical applications. Because of the highly heterogeneous cell morphology within the adherent culture we applied the optimum viable clonal isolation protocol for hAFSCs selection based on live immunostaining with the embryonic surface antigens. The selected hAFSCs not only expressed pluripotency-related genes Oct4, Sox2, Klf4, Nanog and Rex1 but also development foregut endoderm markers Hlxb9, Cdx2, Tcf2 and Hes1. Lentiviral vector delivered human PAX4 and PDX1 beta-cell transcription factors into the hAFSCs. After transduction, hAFSCs were sequentially treated with the combination of extrinsic factors and showed the induced expression of the downstream beta cell markers NeuroD1, INS and Glut2. More importantly, C-peptide ELISA results confirmed the de novo insulin secretion in a glucose-dependent manner, demonstrating in vitro functionality.
: National Institute of Biological Standards and Controls
CRISPR/Cas9 has become a mainstream technology allowing for highly specific and efficient genome editing, and is revolutionising human gene therapy with the potential of giving rise to an entire new class of therapeutics. The use of Adeno-associated viral vectors (AAV) for gene therapy has been most promising because of their safety profile; however, current production methods limit desirable titres of AAV vectors achieved. As a result, significant efforts have been made to improve AAV production systems, including the optimisation of vector expression cassettes and the regulation of producer cell factors. In this study, we endeavoured to alter the producer cell gene expression using CRISPR/Cas9 technology, as a number of studies have shown that host cell proteins play important roles in AAV assembly and production. In addition, our recent results have revealed that down-regulation of host cell protein YB1 significantly enhanced AAV production by up to 45-fold; however, shRNA down-regulation of YB1 displayed a transient gene regulation and the observed enhancement of AAV production was progressively lost overtime. Using CRISPR/Cas9 methodology, we have established YB1 knockout cells from 293T producer cell line that may provide a permanent high titre production system for AAV vectors.
: School of Dentistry, Seoul National University
Mineralized tissues in vertebrate are found in bone and tooth (enamel, dentin, and cementum). Bone marrow mesenchymal stem cells (BMSCs) have the capacity to differentiate into osteoblasts. Dental epithelial stem cells (DESCs), which will give rise to ameloblasts, and dental mesenchymal stem cells (MSC) will form the odontoblasts and cementoblasts. Our previous studies indicate that NFI-C is required for tooth root development and bone formation. The purpose of this study was performed to uncover the underlying mechanism of NFI-C function in mineralized tissue forming cells. To investigate the role of NFI-C in DESCs of mouse incisor, we examined the incisors from Nfic-/- mice by micro-CT and histology. Expression of Sox2 and Nfic was evaluated by IHC, RT-PCR, and western blot. Micro-CT analysis showed that the Nfic-/- mice developed short mandible incisors compared to WT mice. Nfic-/- mice displayed rudimentary cervical loop and decreased epithelial Sox2 expression in cervical loop. PCNA positive cells were significantly diminished in developing cervical loop epithelium of Nfic-/- mice compared to WT. Sox2 expression was decreased in primary dental epithelial cells of Nfic-/- mice, but it was significantly increased by Nfic overexpression in ALCs. NFI-C directly regulates Sox2 expression through the FGF-8 signaling pathway. In dental pulp cells (DPCs) and BMSCs, expression of Sox2 and stemness-related genes such as Nanog and Lin 28 was significantly increased by Nfic overexpression. These results indicate that NFI-C plays an important role in the maintenance of self-renewal in mineralized tissue forming cells.
: Laboratorio Andaluz de ReprogramaciÃ, 3n Celular (LARCEL)/FundaciÃ, 3n Pðblica Andaluza Progreso y Salud (FPS)
iPSCs offer an invaluable source of patient-specific pluripotent stem cells for disease modeling, drug screening, toxicology tests and importantly for regenerative medicine, and already have been employed to unmask novel insights into human diseases While iPSCs can be consistently generated through overexpression of the four Yamanaka Factors OCT4, SOX2, KLF4 and c-MYC (OSKM), reprogrammed cells present worrisome differences with embryonic stem cells in transcriptional and epigenetic profiles, as well as developmental potential and difficulties in cell culturing. A thorough mechanistic understanding of the reprogramming process is critical to overcoming these barriers to the clinical use of iPSC. We have recently published a novel factor combination based on molecules specifically enriched in the metaphase II human oocyte. We have shown that just the overexpression of histone-remodeling chaperone ASF1A and OCT4 in hADFs previously exposed to the oocyte-specific paracrine growth factor GDF9 can reprogram hADFs into pluripotent cells (AO9-iPSCs). Our study contributed to the understanding of the molecular pathways governing somatic cell reprogramming. Here we go deeper in the reprogramming mechanisms showing the importance of somatic cell origin for reprogramming efficiency, and analyzing the transcriptional and epigenetic characteristics of AO9-iPSCs. Our data suggest that these cells could have better culture properties and differentiation potential than OSKM iPSCs.
: Laboratorio di Terapia Cellulare e Genica Stefano Verri, ASST-Monza, Ospedale San Gerardo, Monza (MB)
Operational tolerance is an alternative to lifelong immunosuppression after transplantation. One strategy to achieve tolerance is by T regulatory cells. Safety and feasibility of a T regulatory type 1 (Tr1)-cell–based therapy to prevent graft versus host disease in patients with hematological malignancies has been already proven. We are now planning to perform a Tr1-cell–based therapy after kidney transplantation. Aim of the current work was to develop a protocol to generate clinical-grade donor-specific Tr1 cells and test method feasibility and reproducibility in an academic-based GMP site. Tolerogenic donor-derived dendritic cells (DC-10), required for the generation of donor specific Tr1 cells, were generated by leukapheresis upon selection of CD14+ monocytes with CliniMacs™ (mean±SD purity: 99.08% ± 0.65%, recovery: 82.3% ± 21.75%, viability: 97.26% ± 3.32% n = 3). Donor monocytes were cultured in bags for 7 days in the presence of IL-10, GM-CSF and IL-4. Tolerogenic DC-10 (CD14+CD16+CD86+ cells), were successfully generated. Recipient CD4+ T cells, purified from leukapheresis with CliniMacs™ (purity: 97.38% ± 1.84%, recovery: 70.96% ± 16.79%, viability: 98.94% ± 0.53%), were cultured with irradiated donor DC-10 and an average of 1568.42x106 ± 624.78 Tr1 cells with a donor-specific anergy of 83.83% ± 8.02% were generated. This cell-recovery is compliant with the planned cell number to be infused (2x106 Tr1 cells/kg). The final cell product was constituted of 98.79% ± 0.16% CD4+ T cells; the remaining non-CD4+ T cells were donor-derived DC-10 cells that were irradiated and therefore dead or prone to die. These data demonstrate the successful translation of a research-grade protocol to an academic-based GMP-cell factory and provide solid ground for proceeding with the clinical trial.
: University of Wroclaw
Muscle directed gene therapy vectors do not provide sufficient therapeutic level due to low-level, short-term or unspecific expression. This can be changed by insertion of optimal regulatory elements. The promoters utilized until now either are strong and unspecific (giving rise to immune response or silencing) or specific but not efficient enough. We developed a novel, Muscle Hybrid (MH) promoter, highly active in muscle cells, composed of mammalian regulatory elements only. Functional elements of promoter were chosen using in silico analysis, then optimized and combined in order to obtain a modular structure. Afterwards a designed minimal intronic sequence was also inserted as a functional element structure of promoter. The intron highly enhances expression level in myoblasts and myotubes in vitro and increases construct's stability and specificity. We showed that MH promoter used for delivery of reporter gene GFP with AAV-9 vector is more efficient in vivo than CMV or desmin promoters in both skeletal muscle and heart tissues. MH promoter also provides high expression level in vitro of lamin A, which causes Emery-Dreifuss muscular dystrophy (EDMD2) when its gene is mutated. MH promoter is a promising tool for gene therapy of muscle related genetic disorders. Its specificity should decrease immune response to therapeutic genes which is major limiting property of unspecific, highly active promoters such as CMV or EF1α promoters. Our MH promoter together with the powerful intronic element should provide specific, stable, long term expression at sufficiently high level of therapeutic genes in muscle tissues and facilitate effective gene therapy.
: The Johns Hopkins School of Medicine
The derivation and maintenance of human pluripotent stem cells (hPSC) in stable naïve pluripotent states has wide impact in human developmental biology. However, hPSC are unstable in classical naïve mouse ESC WNT and MEK/ERK signal inhibition (2i) culture. We show that a broad repertoire of conventional human embryonic stem cell (hESC) and transgene-independent hiPSC lines could be reverted to stable human preimplantation ICM-like naïve states with only WNT, MEK/ERK, and tankyrase inhibition (LIF-3i). LIF-3i-reverted hPSC retained normal karyotypes and attained defining mouse ESC-like functional features including high clonal self-renewal, independence from MEK-ERK signalling, dependence on JAK-STAT3 and BMP4 signaling, and naïve-specific transcriptional and epigenetic configurations. Tankyrase inhibition promoted a stable acquisition of a preimplantation ICM-like ground state via modulation of WNT signalling, and was most efficacious in efficiently reprogrammed conventional hiPSC. Importantly, naïve reversion of a broad repertoire of conventional hiPSC reduced lineage-primed gene expression, and significantly improved their multi-lineage differentiation capacities. LIF-3i reversion of multiple independently-derived hiPSC significantly improved differentiation efficiency to endodermal (e.g., FOXA2+ and CXCR4+SOX17+), ectodermal neural progenitor (e.g., SOX1+Nestin+; PAX6+ Nestin+), and mesodermal vascular-pericytic (e.g., CD31+CD146+; KDR+CD73+ progenitor) populations. LIF-3i-reverted hPSC lines differentiated more efficiently, with more rapid kinetics, and with less interline variability than their conventional primed states. Altogether, these data suggested that naïve reversion produced a more homogenous pluripotent stem cell population with reduced lineage-primed gene variability, and increased functional pluripotency. Stable naïve hPSC with reduced genetic variability and improved functional pluripotency will have great utility in regenerative medicine and human disease modeling.
: Department of Oral Biology, School of Dentistry, University of Leeds, Leeds, UK
Regeneration of tissue interfaces requires delivery modalities that can provide multiple instructive morphogenic cues in a spatially controlled manner. In order to provide a smart biomaterial that is capable of generating gradients of multiple morphogenic cues we aimed at designing hydrogel based gene-activated matrices (GAMs) for the nonviral delivery of multiple therapeutic genes in a spatially controlled manner. Hydrogels were loaded with multiple plasmid DNAs using a novel method for spatially controlled synthesis of DNA/nanoparticle co-precipitates in different areas of the gels. Nanoparticles were analyzed by scanning electron microscopy and composition confirmed by energy dispersive Xray spectroscopy (EDX). Transfection efficacies and cell compatibility were analyzed in vitro using cell lines and subcutaneous in vivo implantation via bioluminescence imaging. Characterization of the agarose GAMs showed pDNA/nanoparticle (size <200 nm) formation in the gel in distinct areas, confirming spatial control of loading. EDX confirmed these particles as nanocarrier/plasmid DNA coprecipitates. In vitro testing showed no substantial toxicity and confirmed enhanced transfection compared to naked DNA loaded matrices. In vivo results demonstrated gene delivery and persistent gene expression for more than 2 weeks and indicated improved gene expression using the complexation strategy. In conclusion this initial study demonstrated the generation of therapeutic gene/nanoparticle gradients within a hydrogel biomaterial for spatially controlled nonviral gene delivery in vitro and in vivo. The developed strategy will be applied in developmental engineering approaches in the future using geneencoded morphogenic gradients to induce the formation of complex tissue interfaces.
: Luxembourg Center for Systems Biomedicine
Cellular phenotypes are characterized by stable gene expression states determined by underlying gene regulatory networks (GRNs), particularly by functionally important sub-networks (i.e., GRN motifs). A classical GRN motif, the toggle switch, constitutes a molecular mechanism that determines cell fate decisions, and provides stability to transcriptional programs of binary cell fate choices. However, in general, more complex GRN motifs can underlie this mechanism. Here, we propose a general model of stem cell differentiation, in which a general class of GRN motifs, known as the feedback loops (a toggle switch is a special case) that contain lineage specifiers, are responsible for determining cellular identity. In addition, we further develop a computational method, which relies on the integration of epigenetics information (chromatin marks and gene accessibility) and gene expression data in order to systematically predict known and novel lineage specifiers, and their key GRN motifs in various stem cell differentiation systems. Moreover, experimental validation of novel predicted lineage specifiers confirmed that they induce neuronal and astrocyte differentiation of mouse neural stem cells. Taken together, the proposed computational method is, to our knowledge, the first to systematically predict lineage specifiers and their key GRN motifs relying solely on transcriptome data, without prior knowledge of potential candidate genes, pathways and gene ontology. Importantly, it can be applied to systems where differentiation is incomplete and opposing lineages are phenotypically very similar to each other. Further, our method would be useful for assisting researchers in the stem cell and regenerative medicine fields in designing experimental strategies.
: I.I.S. Biodonostia
The dermal Panniculus carnosus (PC) muscle is important for wound contraction in lower mammals and might represent an interesting model of muscle regeneration, due to its unusually high cell turnover. The resident satellite cells (the bona fide muscle stem cells) remain poorly characterized. We here analyzed PC satellite cells with regard developmental origin and purported function. Lineage tracing shows they originate in Myf5+, Pax3/Pax7+ cell populations. To understand their putative role in wound contraction, we performed full thickness wounding of the skin that included the underlying PC muscle. Skin and muscle wounding increased PC myofiber turnover, with the satellite cell progeny being involved in muscle regeneration but with no detectable contribution to the wound bed myofibroblasts. Since hematopoietic stem cells fuse to PC myofibers in the absence of injury, we also studied the contribution of bone marrow-derived cells to the PC satellite cell compartment, demonstrating that cells of donor origin are capable of repopulating the PC muscle stem cell niche after irradiation and bone marrow transplantation, but may not fully acquire the relevant myogenic commitment. Additionally, since precursor cells with the in vitro capacity to generate striated muscle fibers have been isolated from murine skin, being largely attributed to culture-induced cellular plasticity, we pursued to identify and characterize the myogenic cell population present in dermis-derived sphere cultures. We conclusively demonstrate that dermis-derived myogenesis originates from the PC satellite cell population.
: MC Toxicology Consulting GmbH
The novelty and uniqueness of highly innovative cell-based, gene and gene editing therapies hold significant challenges for both, product developers and regulators. In particular a whole slew of gene editing drug candidates, mostly based on the CRISPR/Cas9 technology is expected in the regulatory arena soon. All heading for investigational new drug applications (INDs) and/or clinical trial applications (CTAs), thereby stumbling into an increasing misalignment between scientific development rationale and standard regulatory requirements. Therefore, strict compliance with formal regulatory recommendations bears the significant risk to substantially slow down programs and generate artificial or even useless data. As it stands many preclinical programs in this field have to rely on scientific approaches often including non-standardized, non-validated methodologies in support of non-clinical pharmacological and toxicological profiling. Key challenges among others include potency testing, in vivo proof-of concept, migration and toxicology (mostly gene-toxicity), as well as batch-to-batch comparability. We provide an overview on the regulatory framework for the non-clinical development of ATMPs in Europe and CGTPs in the US and illustrate, that despite the availability of numerous guidance documents, only a product-tailored and sound scientific-based approach including a thorough risk assessment will lead to a successful development of candidate drugs. Our poster depicts examples of scientific approaches, which were accepted in the past by regulatory authorities. It is concluded that there is a clear need for early authority interactions (both in Europe and the US) to seek support for innovative preclinical development strategies at least in parts deviating from classical guidance.
: Urology & Nephrology Center, Mansoura, Egypt
We could produce insulin-producing cells (IPCs) from human mesenchyme stem cells (MSCs) by directed differentiation. Although the yield of the formed IPCs was modest, yet transplantation of these cells in diabetic mice resulted in their cure. We have tried to provide an explanation for this observation. Differentiated MSCs were transplanted under the renal capsule of diabetic mice. The kidneys were harvested after 1, 2, 4 and 12 weeks. IPCs were counted at each period. The proportion of IPCs increased to reach a maximum of ∼20% at 4 weeks. To study the efficiency of these cells in treatment of larger animals and identify their functional longevity, we have induced diabetes in 6 dogs (15 – 20 Kg) by a mixture of alloxan and streptozotocin. Differentiated human cells (5 million/kg) were encapsulated and transplanted beneath the rectus sheath. Six dogs are currently under follow up. Three had completed a 6 months follow up. Two became euglycemic with normal glucose tolerance curve. The third is on the hyperglycemic side although the profile of its glucose tolerance resembles a normal one. A harvested capsule after 6 months form transplantation was examined. By immunoflourescence, IPCs were seen and co-expression with c-peptide was confirmed. The proportion of IPCs was again in the range of ∼20%. In conclusion, IPCs can be formed by directed differentiation from of MSCs. These cells undergo further differentiation in vivo. Evidence was provided that these cells can cure chemically induced diabetes in small as well as large animals.
: European Molecular Biology Laboratory - European Bioinformatics Institute
The European Bank for Induced Pluripotent Stem Cells (EBiSC) launched a public catalogue of iPS cell lines in 2016 (https://cells .ebisc.org). EBiSC aims to be the largest European catalogue of human iPS cell lines, and it makes available cell lines generated by multiple institutes from across the continent. We describe here the Information Management System (IMS) underlying the operations of the EBiSC bank. The IMS joins together existing specialized European services: hPSCreg (
: Osaka University
The adenovirus (Ad) serotype 5 genome encodes two noncoding small RNAs (virus-associated RNAs: VA-RNA I and II), which are approximately 160nt-long noncoding RNAs transcribed by RNA polymerase III. It is well-known that VA-RNA I supports Ad replication via inhibition of double-stranded RNA-dependent protein kinase (PKR), which recognizes double-stranded RNA and acts as an antiviral system. In addition, recent studies revealed that although VA-RNA I is processed by Dicer into VA-RNA I-derived miRNA (mivaRNA I), mivaRNA I does not promote Ad replication. On the other hand, roles of VA-RNA II and VA-RNA II-derived miRNA (mivaRNA II) in Ad replication have remained to be clarified. In this study, we examined mechanism of VA-RNA II-mediated promotion of Ad replication. Overexpression of VA-RNA II by a VA-RNAII-expressing plasmid significantly promoted Ad replication, whereas mutation in mivaRNA II-coding sequence of the VA-RNA II-expressing plasmid canceled the promotion of Ad replication. Transfection with chemically synthesized mivaRNA II-138, one of the most abundant mivaRNAII, significantly enhanced Ad replication, while other species of mivaRNAII which contain seed sequences different from mivaRNA II-138, did not. These data suggest that mivaRNA II-138 promoted Ad replication by suppressing the expression of its specific target genes in a miRNA-like manner. We identified 8 putative target genes of mivaRNA II-138 by microarray analysis and in silico analysis. Among 8 target genes of mivaRNA II-138, knockdown of 3 genes significantly enhanced Ad replication. These data indicate that VA-RNA II promotes Ad replication by knockdown of the target genes via posttranscriptional gene silencing.
: Technische Universität-Berlin
Coxsackievirus B3 (CVB3) is a standard virus for investigation of experimental virus-induced myocarditis in mice. However, CVB3 also infects the pancreas and destroy it. This leads to strong pain and stress for the animals. The aim of this project is to improve the CVB3 mice-myocarditis model by development of a pancreas-attenuated CVB3. For this we inserted target sites (miR-TS) of a pancreas-specific expressed microRNA into the CVB3 genom to achieve pancreas specific degradation of the virus genome.
: Genome Institute of Singapore
The core pluripotency transcriptional network (PTN), comprising Nanog, Oct4, and Sox2, endows the mouse embryonic stem cells (ESCs) their ability to self-renew or differentiate to all adult tissues, including the germ line. A defined media supplemented with two inhibitors, MEK inhibitor PD0325901 and GSK3 inhibitor CHIR99021 (herein called 2i) was shown to maintain ES cell self-renewal. While the MEK inhibitor checks on the fibroblast growth factor 4 (FGF4) mediated ES cell differentiation, the GSK3 inhibitor stabilizes β-catenin, which associates with TCF7L1 (TCF3) and abrogates the latter's repressive effect on PTN. However, the interaction of stabilized β-catenin with TCF7 (TCF1), activator of β-catenin mediated transcription, is shown to up-regulate differentiation promoting genes. Therefore the 2i culture of mESCs appears to balance the requirement of β-catenin to maintain stemness over differentiation, which is evident from the relative abundance of a set of lineage specific early pro-differentiation genes expressed at a higher level in 2i grown cells. In this study we report the development of a novel culture media, that we call the “new-2i” or the n2i, which includes the addition of PD0325901 and inhibitor of β-Catenin Responsive Transcription 3 (iCRT3), that improves long-term self-renewal and enhances functional pluripotency of mESC by delaying exit from pluripotency. We further show that the n2i media defines a new ‘ground state’ for pluripotency and efficient germline transmission reminiscent of the 2i ground state. Therefore we propose that the n2i provides a better and alternative culture method for mESCs.
: CIEMAT/CIBERER
Disease models are essential to understand the molecular mechanisms that drive pathogenesis of metabolic diseases, where target cells are often inaccessible, and enable the development of novel therapies. In particular, cell reprogramming offers a valuable tool to develop patient-specific disease models. Primary hiperoxaluria type 1 (PH1) is an inherited autosomal rare metabolic disease, caused by a deficiency in the alanine:glyoxylate aminotransferase (AGT), due to mutations in AGXT gene. Its activity in humans is restricted to hepatic peroxisomes. In this work we describe two different strategies to develop in vitro models of PH1 by cell reprogramming of dermal fibroblasts and peripheral blood mononuclear cells (PBMCs) obtained from a PH1 patient, homozygous for the p.I244T mutation, highly prevalent in Canary Islands due to a founder effect. In the first strategy we have obtained induced hepatocytes (iHeps) directly from human fibroblasts by overexpression of hepatocyte specific transcription factors and a hepatocyte defined culture media. On the other hand, we have generated patient-specific induced pluripotent stem cells (iPSCs) from PBMCs and dermal fibroblasts from the same patient. In both approaches we have obtained PH1 deficient cells expressing hepatocyte markers, including albumin expression, glycogen storage and others. Cells obtained from either strategy are being used to study the biology of PH1 in vitro and will be used in the future to develop strategies for the genetic correction of the disease.
: The University of Tokyo
As many studies of Regenerative Medicine (RM) are getting close in on their clinical trial phase, trial subjects are about to be faced to make their own decision to take participate in clinical trials. There is a certain number of previous studies and theories on Medical Decision Making (MDM) which focus on the participations in clinical trials. According to them, we must take into account of various factors (such as explanation of risk-benefit balance, attention for therapeutic misconception, and so on) to obtain Informed Consent from research subjects. So, to help research subjects to gain knowledge about the clinical trials and make his/her decision, some Decision Aids (DAs) for providing medical information or health education have been produced. However, MDM is a decision not only information-based, but also his/her values-based. The conventional DAs are useful for research subjects to get the information, whereas little thought is given to its values or social significance of the clinical trial. Therefore, it is important to build a framework that research subjects can recognize the social significance of clinical trials to make their decision. Through conducting literature searching and taking a cue from the theory of Collaborative Decision Making, our presentation will propose one of the ideal frameworks that research subjects can recognize the social significance of clinical trials and feel as if he/she was one of the member of the research teams. Funding Source: This study was funded by the Japan Agency for Medical Research and Development (AMED).
: San Raffaele Scientific Institute
The embryonic spinal cord has provided a suitable model to reveal genetic codes that define the identity of neural cells in the vertebrate CNS. The pathways of neuronal specification have been well established for spinal motor neurons (MNs), the cells that are lost in neurodegenerative diseases including ALS. Uncovering these developmental rules is useful to devise strategies to obtain a replenishable source of MNs from stem cells. However, there is limited understanding of how the inductive signals of motor identity are integrated with the molecular programs that control cellular morphogenesis. In order to unmask novel determinants of motor differentiation, we use reporter mice to gene profile MNs, and manipulate their molecular signatures in embryo models. A putative member of the RhoGAP family, ARHGAP36, is one of the most expressed genes in MNs and has recently been found up-regulated in a subset of human medulloblastomas. Forced expression of ARHGAP36 in spinal cord neural progenitors induces dorsal ectopic activation of MN markers in domains that normally give rise to interneuron classes. Moreover, a marker of the ventral-most progenitor domain becomes aberrantly expressed in the dorsal spinal cord. These homeotic changes are consistent with a role of ARHGAP36 in interpreting morphogen signals that emanate from the ventral (Shh) and dorsal (BMP, WNT) spinal cord. In conclusion, ARHGAP36 functions as a determinant of MN identity. We are performing a structure-function analysis to identify protein domains required for expansion of the MN pool. A mouse mutant lacking ARHGAP36 has being generated using Crispr/Cas9 technique.
: University of Tokyo
In February 2016, the International Society for Stem Cell Research published a statement titled “ISSCR ENDORSES FETAL TISSUE RESEARCH AS ESSENTIAL.” The statement characterizes fetal tissue as “an essential gold-standard resource” to investigate the mechanism of human tissue and organ development in laboratories, and emphasizes the importance of fetal tissue research (FTR) and its benefits. Contrary to this position, Japan is a country that has placed a moratorium on FTR although Japan has strongly promoted stem cell research. Therefore, the purpose of our study is to reflect on the ethical, legal, and social issues that were deliberated on in deciding on the moratorium, in order to discuss what steps should be taken to promote further discussion of FTR. Our methodology was a literature review, focusing on the meeting minutes of the Health Science Council of the Ministry of Health, Labor, and Welfare as the council developed its “Guidelines on clinical research using human stem cells,” which was abolished in 2014, because the moratorium was discussed in the minutes. Until August 2004, the panel was drafting the guidelines to promote FTR. In July 2004, however, news was reported sensationally that aborted fetuses were cut into pieces and dumped as garbage at an obstetrics and gynecology clinic. Then in 2005, the panel drastically changed direction: they decided to place a moratorium on FTR and promote other stem cell research first, although they still recognized the importance of FTR. Our poster provides a detailed discussion of this issue.
: Institute of Cytology of The Russian Academy of Science
Embryonic stem cells (ESCs) are characterized by a high rate of self-renewal and are capable of differentiating into all cell types of the adult organism. Due to these unique features ESC are a promising tool for cell-based therapies, including xenotransplantation and modeling of various diseases. However, ESCs often display spontaneous differentiation in culture thus making the process of expanding these cells highly inefficient. Resveratrol has exhibited a broad range of beneficial properties in experimental settings (anti-oxidant, anti-tumor, cardioprotective, neuroprotective, anti-inflammatory effects). All these resveratrol properties make it possible to assume that resveratrol could positively regulate mESCs capacity to self-renewal and maintain pluripotency. We have explored the effect of resveratrol on mouse ESCs (mESCs) as a model for the progenitor cell source of normal tissues.
: European Commission
The 8th European Union (EU) programme for Research and Innovation, Horizon 2020 (2014-2020), supports the gene and cell therapy field by publishing calls for proposals for (clinical) collaborative research on chronic or rare diseases, in regenerative medicine, or for new technological developments, amongst other. Small- and medium-size entreprises (SMEs) in the field can apply, even as single partner, via a dedicated SME instrument. US partners are welcome throughout the Health research programme and can be funded as well. Other funding opportunities for researchers, such as the Marie Sklodowska-Curie actions (training), the European Research Council grants (individual frontier research), the Innovative Medicines Initiative projects (public-private partnership with the European Federation of the Pharmaceutical Industries and Associations), are also available on regular basis. The first Horizon 2020 projects dealing with gene and/or cell therapy supported in 2014–2016 will be presented as well as the trends for the future Health programme. Participant portal:
: UCL Institute of Child Health
Perforin deficiency is caused by mutations in the PRF1 gene and accounts for up to 50% of familial haemophagocytic lymphohistiocytosis syndromes. A deficient cytolytic secretory pathway leads to hypercytokinaemia and hyperactivation of notably CD8 T cells with subsequently inflammation of various organs. The application of autologous gene corrected T cells could reduce disease activity, alleviate HLH symptoms and potentially bridge for a desired HSCT. We developed a gammaretroviral vector expressing perforin to transduce CD8 cells and its subsets in the prf-/- mouse model. Furthermore, we followed different strategies to transduce CD8 subsets individually. We see efficient engraftment and functional reconstitution of cytotoxicity after intravenous administration of gene corrected prf-/- CD8 cells into prf-/- mice. In a second step to verify further the functional correction of prf-/- T cells in vivo, a tumour model using an LCMV epitope transfected murine lung carcinoma cell has been evaluated in the perforin deficient mouse. We transferred gene corrected T cells and eliminated the tumour as efficient as by the transplant of wild type T cells. In-vivo experiments to evaluate the efficacy after inducing HLH with LCMV infection are ongoing. Finally, we show the correction of cytotoxicity in human T cells of a perforin deficient patient after transduction with a PRF encoding lentiviral vector. These pre clinical data demonstrate the potential application of T cell gene therapy for perforin deficient HLH.
: State Institute of Genetic and Regenerative Medicine NAMS of Ukraine, Kiev, 04114, Ukraine
During the embryogenesis the skull bones arise from two sources – mesodermal mesenchyme and neural crest-derived ectomesenchyme. The healing of calvarial bone defects are of significant clinical challenge, since the used osteoplastic materials and auto-bone transplantation techniques have disadvantages. Bone tissue engineering proposes an alternative and promising approach but require a choice of optimal cell source.
: University of North Carolina at Chapel Hill Gene Therapy Center
BAX 335 (AAV8.sc-TTR-FIXR338Lopt) is an AAV vector incorporating the increased specific activity FIXR338L mutation. The comparative risk of factor IX neutralizing antibody (“inhibitor”) formation following AAV expression of mutant FIXR338L compared to wild type factor IX (WT FIX) is unknown and was studied in novel strains of humanized hemophilia B (huFIX-/-) mice. huFIX-/- mice carry no mouse major histocompatibility complex II but instead express a common human MHC II haplotype (HLA DRB1*1501), associated with inhibitor formation in hemophilia A. Following subcutaneous immunization with human FIX and complete Freund's adjuvant (CFA), all huFIX-/-and WT C57Bl/6 (B6FIX-/-, murine HLA H2b) hemophilia B mice developed FIX inhibitors. When inhibitors were screened against a FIX peptide library, none of the immunodominant FIX epitopes included amino acid 338. B6FIX-/- and huFIX-/- mice were treated intravenously (I.V.) with BAX 335 (huFIX-/- n = 22; B6FIX-/-n = 88) or vector encoding human WT FIX (huFIX-/- n = 22; B6FIX-/-, n = 15). Neither vector led to FIX inhibitors in either strain. In contrast, inhibitors developed after intramuscular AAV.FIX injection in huFIX-/- and B6FIX-/- mice, as expected. There was, however, no difference in the titers, IgG subtype, or persistence of inhibitors that developed from FIXR338L or WT FIX-expressing vectors. Additionally, huFIX-/- mice treated I.V. with BAX 335 maintained WT FIX tolerance and maintained plasma FIXR338L expression without inhibitors despite subsequent subcutaneous immunization with human FIX + CFA. Using transgenic mice derived for FIX inhibitor evaluation, no increase in comparative risk of inhibitor formation was observed following gene therapy employing the increased specific activity FIXR338L mutant.
: Oncology and Molecular Pathology Laboratory, School of Medicine and Surgery, Milano-Bicocca University, Monza, 20900, Italy
The existence and the potential localization of adult renal stem cells are still a matter of debate, as well as their possible use as a tool for cell therapy approaches in kidney diseases. We isolated by sphere forming assay a population of adult renal stem cells (RSC), able to self-renew and differentiate into multiple renal lineages. We described within the nephrospheres cells with a PKHhigh/CD133+/CD24- phenotype as those with stem properties. To better characterize the PKHhigh status we performed a transcriptomic and bioinformatic analysis of stem cells, progenitors and differentiated cells, obtaining a RSC signature. We identified differentially expressed genes (DEGs) and exclusive genes for each differentiation step, using MultiExperiment Viewer as statistical tool for microarray data analysis. To determine the signature of PKHhigh/CD133+/CD24- cells we crossed DEGs with fold change ≥1.5 and p-value ≤0.05 with exclusive genes. Based on fold change value and the rare expression in adult renal tissue, we found, in addition to CD133, potential markers, which we are now validating at protein level by immunofluorescence. Furthermore, the transcriptomic profile allowed us to identify specific transcription factors useful for attempts of direct reprogramming of terminally differentiated cells to adult RSC. The previously assessed RSC signature will help us to check the achievement of cell reprogramming. This approach will permit to obtain reprogrammed RSC, potentially useful for regenerative medicine in kidney diseases.
: National Cheng Kung University Medical College
Prothymosin α (ProT) is involved in oxidative stress, inflammation, cell proliferation, and apoptosis. Increased oxidative stress and chronic inflammation participate in diabetes pathogenesis. ProT was reported to be a ligand of toll-like receptor 4 (TLR4), which plays an important role in the development of insulin resistance. However, its physiological role remains poorly understood. Here we investigated whether ProT contributes to the development of insulin resistance. A total of 185 human subjects were recruited and classified into nondiabetes (n = 95) and newly diagnosed diabetes (n = 90) groups. Transgenic mice overexpressing ProT were used to investigate the role of ProT in the development of insulin resistance. Lentiviral vectors carrying shRNA specific for ProT were delivered via the portal vein to silence hepatic ProT expression in mice with high-fat diet-induced insulin resistance. Our results show that the serum ProT levels of patients with type 2 diabetes were significantly higher than those of normal individuals (mean + SEM, 419.8 + 46.47 vs. 246.4 + 27.89 pg/mL; P < 0.001). ProT, diabetes, body mass index (BMI), and hypertriglyceridemia were independently associated with homeostasis model assessment for insulin resistance (HOMA-IR). Furthermore, ProT transgenic mice exhibited an insulin-resistant phenotype. ProT induced insulin resistance through a TLR4-NF-κB-dependent pathway. Notably, knockdown of ProT expression via lentivirus-mediated hepatic delivery of ProT shRNA ameliorated high-fat diet-induced insulin resistance in mice. Collectively, our results support the role for ProT in the development of insulin resistance and suggest ProT as a potential novel therapeutic target for type 2 diabetes.
: Hannover Medical School
Mutations in non-coding regions of a gene may result in identical phenotypes as exonic mutations. In addition, they might be underrepresented in state-of-the-art exome sequencing approaches. Loss-of-function mutations in the gene encoding factor IX lead to hemophilia B. Here, we dissected the consequences and the pathomechanism of a point mutation in the factor IX 3’UTR. Using plasmid-derived factor 9 minigenes we revealed that the mutation leads to reduced factor IX RNA levels due to the de-novo creation of a 5’ splice site (SS). This 5’SS binds the spliceosomal component U1 snRNP, which is able to suppress the nearby factor 9 poly(A) site. Blockade of U1 snRNP by either antisense morpholinos or LNAs rescued mutant RNA expression. In order to observe factor 9 secretion and to monitor clotting activity, we replaced the promoter/5’UTR and incorporated a truncated intron I resulting in an ∼30-fold higher factor 9 RNA expression. However, the phenotype persisted indicating a high penetrance and tight regulation. Using these minigenes we demonstrated that the reduced RNA levels in the context of the mutation finally translate into a lower clotting activity as observed in the patients. Finally, we used the vice versa approach and introduced an antisense oligonucleotide shielding the mutant site from recognition by U1 snRNP resulting again in a rescue of factor 9 RNA expression. This strategy may also be applicable to other 3’UTR mutations.
: Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
Muscle satellite cells (mSC) are key players in postnatal muscle growth. Previously we showed that heme oxygenase-1 (Hmox1), a rate-limiting enzyme in a heme degradation, induces proliferation while decreases differentiation and apoptosis of muscle myoblasts. Here we investigated the role of Hmox1 in mSC in animal model of Duchenne Muscular Dystrophy (mdx mice). In gastrocnemius muscles of WT mice the expression of Hmox1 was strongly induced at day 1 after cardiotoxin (CTX) injection. Lack of Hmox1 was associated with higher muscle degeneration and inflammation. Population of activated mSC (CD45-CD31-Sca1-a7i+CD34-) was decreased on the 3rd day in Hmox1-/- mice. Less mSC were also observed in Hmox1-/- mice after triple CTX injury and in 2-year-old animals. Although in whole skeletal muscle of mdx animals expression of Hmox1 is elevated starting from 8 week of age, in mSC cells it is downregulated and negatively correlated to increased miR-206. Double knockout Hmox1-/-mdx mice have more severe dystrophic phenotype. The RNA sequencing showed a strong effect of dystrophin deficiency on mSC transcriptome when compared to WT cells suggesting that cells of mdx animals are more activated (upregulated cell cycle, DNA replication, while downregulated calcium and cGMP signaling pathways). In accordance, mSC of mdx and Hmox1-/-mdx show significantly stronger differentiation in comparison to WT and Hmox1-/- cells, respectively, whereas Hmox1-/- cells tend to differentiate more than WT mSC. Precocious rounds of activation can lead to the premature exhaustion of mSC in Hmox1-/- mice. Decreased Hmox1 in mSC may additionally contribute to pathology of DMD.
: Dulbecco Telethon Institute, M. Tettamanti Research Center, Department of Pediatrics, University of Milano-Bicocca, Monza, 20900, Italy
Hematopoietic stem cell transplantation (HSCT) represents a powerful therapeutic opportunity for inborn errors of metabolism (IEMs). Nonetheless, considering current experimental and clinical data, we identified two factors that could allow a better outcome: the precociousness of the transplant and the use of umbilical cord blood (UCB) as a source (Boelens, 2013). We are thus testing the combination of an early (neonatal) intervention and the use of UCB in the murine model of Mucopolysaccharidosis type I (MPS-I), a lysosomal disease caused by mutations in the IDUA gene, with glycosaminoglycans accumulation in tissues leading to multi-organ dysfunctions (Clarke, 1997). We collected UCB from murine fetuses aged 18 days (1.9x10^5 ± 8.0x10^4 cells from each, n = 371). We characterized UCB in comparison with bone marrow (BM) cells by FACS analysis and CFC-assay on methylcellulose. The transplantation of lethally-irradiated adult wild-type mice with 1x10^5 UCB-cells led to high levels of donor engraftment at +4 months (94.5 ± 1.9% in peripheral blood, n = 3). UCB-derived cells in the recipients’ BM contained myeloid and lymphoid progenitors and hematopoietic stem cells that could reconstitute secondary recipients. We conditioned 1-day-old MPS-I pups with busulfan (20mg/kg) and intravenously injected 2x10^5 UCB-cells/pup 24 hours later. Twenty weeks after transplant, MPS-I mice with donor engraftment ≥50% showed improvements in organ pathology and clinical signs compared to untreated controls. Indeed, IDUA activity was increased in multiple organs, and glycosaminoglycan storage reduced. This study could help in setting up transplantation of UCB in newborn babies, and investigates new potential cell and gene therapy approaches for IEMs.
: Medicine Department of UNIFESP
We investigated the effects of mesenchymal stem cells (MSCs), your conditioned medium (CM) or exosomes (EXOs) in a LPS-induced nephrotoxicity and the role of renal stem progenitor cells (RPCs). Rats received i.v.: LPS (10 mg/B.W.) or PBS (CTL) with MSCs (1x106), CM (500 μl) or its EXOs (100 μg/ml) from MSCs incubated or not for 12 hours with cytochalasin B (CB; 1 μM) or actinomycin D (AD; 2.6 μM) given in 1 or 3 doses and sacrificed after 72 hours. Blood, urine samples were collected for creatinine (sCr), urea (sU) and FENa. Kidneys were analyzed for HE, KI67, caspase 3, RPCs as Wnt1, BrDU, PAX2, EXOs as CD9, CD63. Y chromosome, IL6, TNF-α, INF-γ and IL10 were evaluated. It was observed increases in sCr, sU, FENa, caspase 3 marking, proinflammatory cytokines and reduction of KI67 with lesions in proximal tubules induced by LPS. However, these parameters were ameliorated with MSCs, CM or EXOs treatments. LPS and LPS+EXOs increased BrDU, Wnt1, PAX2 and CD63 expressions indicating activation of RPCs. CB and AD inhibited the protective effect of EXOs. The effect of 3 times administration of MSCs or CM or EXOs decreased the mortality in LPS. Therefore, results support that the MSCs and its CM and EXOs protected from LPS-nephrotoxicity. It is reasonable to suggest that the mediation by EXOs is, at least in part, by stimulating the expression of RPCs in this cascade of events and those EXOs alone could be employed in order to ameliorate LPS cute kidney injury (AKI).
: Faculty of Pharmacy, Osaka Ohtani University
One in 11 adults has diabetes mellitus, and every six seconds a person dies from diabetes in the world (IDF diabetes atlas-7th edition). Therefore, innovative therapeutic and preventive approaches for diabetes mellitus are needed. Recent genome-wide association studies identified susceptibility loci for type 2 diabetes mellitus. The zinc finger AN1-type domain gene is one of the type 2 diabetes mellitus-susceptibility genes. However, the contribution of this gene to type 2 diabetes mellitus remains unknown. In this study, to overexpress the zinc finger AN1-type domain gene in primary mouse hepatocytes, we generated a zinc finger AN1-type domain gene-expressing adenovirus (Ad) vector using a novel Ad vector exhibiting significantly lower hepatotoxicity (Ad-E4-122aT, Shimizu K. et al., Mol. Ther. Methods. Clin. Dev. 2014). Primary mouse hepatocytes isolated from C57BL/6 mice were transduced with zinc finger AN1-type domain gene-expressing Ad vector to thoroughly explore the impact of the zinc finger AN1-type domain gene on glucose metabolism. The gene expression level of phosphoenolpyruvate carboxykinase, one of the key gluconeogenic enzyme, was significantly suppressed in primary mouse hepatocytes treated with zinc finger AN1-type domain gene-expressing Ad vector compared to those in control Ad vector-transduced primary mouse hepatocytes. Hepatocyte nuclear factor-4 mRNA expression in primary mouse hepatocytes was also suppressed by zinc finger AN1-type domain gene overexpression. These results suggested that zinc finger AN1-type domain gene overexpression attenuated hepatic gluconeogenesis, which could potentially lead to improvement of type 2 diabetes mellitus.
: Department of Integrated Life Sciences, Research Institute of Advanced Omics, Dankook University, Yongin 16890, Korea
Hepatitis C virus (HCV) is the major causative agent of progressive liver disease such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. In this study, we reported an RNA aptamer (R16) which acted as potent decoys to competitively impede replicase-catalyzed RNA synthesis activity of the HCV non-structural protein 5B. Cytoplasmic expression of R16 efficiently inhibited HCV replication and suppressed HCV infectious virus particle formation through specific interaction with, and sequestration of, the target protein in a cell culture system. To develop this aptamer as clinically relevant HCV inhibitor, we optimized and minimized through determining the structural element responsible for interacting with NS5B protein with structure probing and footprinting analyses, and identified two stem-loop regions, indicated as X and Y. SPR analysis showed that each X and Y domain had a 10-fold higher KD value when compared with full-length aptamer. Anti-HCV efficacy of minimized aptamer, R16-X and R16-Y or mutant form of R16, which have mutation at X (mt1) or Y (mt2) domain, was observed in HCV-replicon cells at the level comparable to that of full-length R16 aptamer. Through colony forming assay, we observed that minimized R16-Y and R16-mt1 significantly reduced appearance of escape mutant of HCV, compared to full-length aptamer. We speculate that Y domain of aptamer is essential for binding to HCV replicase substrate binding domain which is deleterious to HCV replication when mutated. Therefore, minimized R16-Y aptamer could be a potent anti-HCV device beyond the full-length R16 aptamer in terms of both efficacy and safety.
: Vall d'Hebron Research Institute (VHIR)
In this work we generated a cellular model of the royal disease (RD), a form of hemophilia B (HB) that affected several European royal families during the 19th and 20th centuries. The model was based on the identification of a HB patient bearing the RD mutation in the factor IX gene (F9) who was not related to the royal family, and subsequent generation of induced pluripotent stem cells (iPSCs) for both the patient and his carrier mother. These iPSCs were differentiated into hepatocyte-like cells (HLCs) that were used to characterize the RD mutation at the mRNA level using next-generation sequencing on amplified cDNA fragments. These analyses confirmed the generation of an aberrant splicing site, resulting in a frameshift and the appearance of a premature termination codon, as previously predicted. We also found that, even though the mutated F9 mRNA probably undergoes nonsense-mediated decay in patient's HLCs, a small proportion of F9 mRNA transcripts are correctly spliced, suggesting residual factor IX (FIX) production. Our work reports the first cellular model of hemophilia B (HB) that is based on patient-specific iPSCs and which allowed the characterization of mutated FIX mRNA in iPSC-derived HLCs.
: Meyer Children's hospital
Kidney disorders are a major global health issue and new tools are needed to expand therapeutic options. In complex organs like the kidney, one of main stem cell-based regenerative strategies relies on the possibility of modulating the regenerative capacity of the resident progenitor populations. CD133+/CD24+ human renal progenitor cells (RPC) can be easily cultured in vitro and we propose their use as a platform for first-step screening of chemicals for their potential effects on the regeneration of differentiated renal cells, such as podocytes, and also for the evaluation of the drug safety of new and existing medical products. The screening of a library of 50 small compounds for their potential to promote RPC differentiation into podocytes has been based on the analysis of levels of expression of the podocyte marker nephrin by qRT-PCR after exposure to all-trans retinoic acid (RA), which is known to be a powerful inducer of RPC differentiation into podocytes in culture. The Z-factor calculated in a pilot screen was >0.5. Ten compounds induced an increase in nephrin expression equal or 20% higher than RA alone. Three of these compounds were inhibitors of the GSK3 pathway. MTT assay was used to evaluate cell viability and proliferation. The results demonstrated the involvement of the Shh and of the TGF-beta superfamily pathways in RPC proliferation. Thus, in vitro cultures of RPCs are a good platform with which to perform first-step screening of many synthetic chemicals for their cytotoxicity and for their potential effects on the generation of podocytes.
: Hanyang University
Acute lung injury (ALI) is an umbrella term for inflammatory conditions of the lung due to various causes, such as infection, sepsis, multiple transfusions, and ischemia/reperfusion. The only clinical option for ALI treatment is oxygen supplementation by intratracheal intubation. In this study, a RAGE binding peptide (RBP) was designed, based on the RAGE binding site of high mobility group box-1 (HMGB-1). The recombinant RBP was produced and purified in the bacterial overexpression system. We hypothesized that RBP would bind to RAGE on the surface of the cells and induce endocytosis, resulting in inhibition of the interaction between RAGE and its ligands. To confirm this hypothesis, the down-regulation of RAGE mediated inflammation reaction was evaluated in in vitro and in vivo ALI models. The 264.7-macrophage cells were activated by lipopolysaccharide (LPS) and then, treated with RBP. The results showed that pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β. Intratracheal administration of RBP into the ALI animal models reduced the inflammation reaction, reducing pro-inflammatory cytokines. The results suggest that RBP may be useful for the treatment of inflammatory diseases such as ALI. *This work was supported by the grant of BK21plus program from the Ministry of Education in Korea.
: Izmir International Biomedicine and Genome Institute, Dokuz Eylul University, Balcova/Izmir, 35340, TURKEY
Human induced pluripotent stem cells (iPSCs) hold great promise for advancements in cell-based therapy and modeling of human disease. Here, we examined the use of human iPSCs for modeling Citrullinemia type I, results from deficiency of the enzyme argininosuccinate synthase (ASS), an inherited urea cycle disorders of the liver. Patients suffering from citrullinmenia present in early infancy with life-threatening hyperammonemia that can be fatal or result in permanent neurologic damage. Despite the progress in pharmacologic treatment, long-term survival is poor especially for severe cases and hepatocyte transplantation is promising approach, providing enzymatic activity. In this study, dermal fibroblasts from a citrullinemia patient was used to generate patient-specific human iPSCs via episomal reprogramming and differentiated into hepatocyte using a novel 3-step differentiated protocol in chemically defined conditions. The resulting cells exhibited properties of mature hepatocytes functions, such as albumin secretion, cytochrome P450 metabolism, LDL uptake, glycogen storage as well as gene expression. Moreover, hepatocytes generated from patients distinctly have ability to secrete ammonia to medium in comparison to the ones generated from normal human fibroblast as defined as disease pathology. Therefore, we firstly defined a platform for hepatocyte generation from patient-specific human iPSCs for citrullinemia disease. Keywords: Induced pluripotent stem cells, In-Vitro disease modeling, Hepatocyte differentiation.
: University of A Coruña
: Shire, Inc
Gene therapy (GT) is a promising technology to cure hemophilia B. The BAX 335 (AAV8.sc-TTR-FIXR338Lopt) vector contains the hyperactive F9 transgene in an adeno-associated virus 8 (AAV8) capsid. Vector shedding in body fluids is an important safety consideration. The purpose of this evaluation was to describe vector genome (vg) biodistribution in blood and other body fluids after BAX 335 treatment. Eight adult males with hemophilia B (FIX ≤2%) received a single dose of BAX 335 in 3 ascending cohorts: cohort 1 (2E11 vg/kg), cohort 2 (1E12 vg/kg), and cohort 3 (3E12 vg/kg). Quantitative real time polymerase chain reaction (qPCR) was used to detect BAX 335 vgs in blood, saliva, semen, urine, and stool on day 1 post-treatment and at weekly intervals until 2 consecutive samples were below limit of detection. Peak vg values in blood were detected between days 1–14. In cohort 1, both subjects were negative by month 5. In cohort 2, four subjects became undetectable between months 6–9. In cohort 3, both subjects had detectable vgs at 12 months, corresponding to the highest peak Factor IX (FIX) activity. In semen, vg detection peaked between days 1–14 and became negative by week 4. Stool, saliva, and urine reached peak values at days 1–7 and were negative by week 5. In conclusion, peak FIX activity from BAX 335 transgene expression correlates with duration of detectable vgs in blood, while vgs are not detectable after 4–5 weeks in other fluids. These findings have implications for safety in future GT trials.
: Imagine Institut
Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare and severe genetic skin disease responsible for blistering of the skin and mucosa. RDEB is caused by a wide variety of mutations in COL7A1 encoding type VII collagen, the major component of anchoring fibrils which are key attachment structures for dermal-epidermal adhesion. We have generated Induced Pluripotent Stem Cells (IPSCs) from two RDEB patients’ fibroblasts harboring homozygous recurrent COL7A1 null mutations in the purpose of the correction of causative mutations by gene editing. We established transgene-free iPSCs derived from RDEB fibroblasts using the single-stranded RNA Sendai virus. Three vector preparations encoding for a polycistronic Klf4–Oct3/4–Sox2, cMyc and Klf4 transgene, were used to generate RDEB IPSCs. No cytotoxicity was observed after transduction. IPSCs colonies were stained with alkaline phosphatase confirming the undifferentiated state of cells with the potential to self-renew. The pluripotent state of generated IPSCs was confirmed by gene and protein expression of stem cell markers by RT-PCR and immunocytochemistry. Embryoid body formation confirmed their capacity to differentiate into cell types of all three germ layers in vitro. Moreover, we also confirmed the differentiation capabilities of these iPSCs into keratinocytes, fibroblasts and mesenchymal stem cells, which are relevant cell types for the treatment of RDEB. Next steps will aim at correcting RDEB IPSCs through CRISPR/Cas9-mediated Homologous Recombination and differentiating them into cells of interest for clinical translation.
: Medicine Department of UNIFESP
RPCs were characterized by (CD24, PAX2 or CD133 and were preconditioned (PC) with LPS (10μg/ml) or hypoxia (12 hours). Mices (C57BL6J) received PBS or LPS 10mg/BW i.p., after 24 hours received EXOs (100μg/ml) into caudal vein, and after 72 hours were killed and kidney were used to HE (μm2), NGAL, IL18 or KIM 1 (%), creatinine (Cr), urea (U) (mg/dl) evaluations. RPCs were positive for PAX2, CD24 and CD133. Animals treated with LPS showed increase in Cr and U vs. CTL group (0.34 ± 0.02 vs.0.8 ± 0.03; 69 ± 3.4 vs.12 ± 1.9, p < 0.05; respectively). Animals of LPS+EXOs groups, the Cr and U were lower vs. LPS (0.54 ± 0.03 vs.0.8 ± 0.03; 24.8±x vs. 69 ± 3.4, p < 0.05, respectively). ATN (%) were higher and labeling intensity of NGAL, KIM1 and IL18 in LPS vs. CTL, respectively (14 ± 2.1 vs. 6.4 ± 0.8; 26.7 ± 5.7 vs.1 ± 0.07; 37.3 ± 6.2 vs.0.5; 9.9 ± 2.3 vs.0.8 ± 0.03; p < 0.05). These parameters were minimized with EXOs (8.0 ± 0.9; 11 ± 2.1; 11.3 ± 0.9; 6.9 ± 0.07;
: Celixir Limited
Celixir Limited has developed a novel topical gel (Tendoncel) for the treatment of lateral epicondylitis (also know as tennis elbow). Tendoncel is a proprietary regenerative allogeneic gel that incorporates a unique combination of platelet growth factors, including PDGF-BB, VEGF, PDGF-AA, thrombospondin and angiopoietin, within a cellulose-derived gel. We investigated Tendoncel in a double blind placebo controlled Phase II clinical trial in lateral epicondylitis. Patients were randomized to receive either treatment (Tendoncel and physiotherapy) or placebo (placebo gel and physiotherapy) once daily for 21 days with follow up over a period of 3 months. The Tendoncel treated patients showed a statistically and clinically significant improvement in all measurements taken (Disabilities of the Arm, Shoulder and Hand (DASH) score, Patient-rated Tennis Elbow Evaluation (PTREE) score and PTREE pain score) at all time points (two of which are shown below). DASH Treatment was 52% of placebo at 4 weeks (p < 0.05) Treatment was 60% of placebo at 3 months (p < 0.05) PTREE Treatment was 48% of placebo at 4 weeks (p < 0.05) Treatment was 52% of placebo at 3 months (p < 0.05) PTREE pain Treatment was 60% of placebo at 4 weeks (p < 0.01) Treatment was 69% of placebo at 3 months (p < 0.05) These results clearly show that Tendoncel is both efficacious and safe in the treatment of lateral epicondylitis.
: KU Leuven
Gene therapy trials using integrating retroviral vectors has shown outspoken therapeutic benefits in the clinic. However, occurrence of insertional mutagenesis in a small subset of patients due to vector integration in promoter regions and subsequent upregulation of oncogenes cause severe adverse events. We identified bromodomain and extraterminal domain (BET) proteins as cellular cofactors that tether MLV pre-integration complex to the host chromatin thereby dictating the typical gammaretroviral integration profile. We mutated the MLV integrase (IN) to disrupt the interaction with BET proteins. The resulting BET-independent (Bin)MLV vectors integrate about two-fold less frequent near promoter regions and CpG islands. To achieve an even more dispersed integration profile we engineered next-generation BinMLV vectors by fusing alternative chromatin-binding peptides to BinMLV IN. Resulting vectors transduce and integrate in line with wild-type MLV, with an integration profile that is detargeted even more from traditional MLV markers (about 4-fold less integration near TSS). Bioinformatic assessment of the safety profile shows an increase in the fraction of potentially safe integrations for the next-generation BinMLV vectors compared to WT. In addition, initial experiments in an in vitro genotoxicity assay (IVIM) show that the oncogenic potential of these vectors may be diminished. Together, these findings show the potential of engineering next-generation BinMLV vectors with a reduced risk of insertional mutagenesis without compromising transduction efficiency. As such, next-generation BinMLV vectors can improve the safety of gammaretroviral vector gene therapy.
: Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, Krakow, Poland
Antiviral response of fibroblasts to vectors introducing reprogramming factors has been described to affect generation of iPSCs, however direct comparison of different reprogramming methods and their influence on immunomodulatory and cytoprotective factors, such as heme oxygenase-1 (HO-1) has not been investigated. Therefore here we utilized policistronic STEMCCA lentiviral and Sendai and evaluated initial phase of reprogramming as well as differentiation potential of generated iPSCs. Transduction of human BJ fibroblasts with Sendai vectors resulted in potent immune response observed as elevated expression of anti-viral factors such as DDX58 which senses viral infection and induces type I interferons and ISG20, an interferon-induced exoribonuclease that acts on single-stranded RNA. Cytotoxicity of the vectors was further confirmed with altered cell morphology and increased level of LDH release. Importantly, similar effects were not observed with STEMCCA vectors, which provided higher infection efficiency, examined by OCT4 expression in transduced fibroblasts without signs of cell death. Interestingly, changes in HO-1 expression were inversely correlated with p53 level which was elevated with different kinetics after transduction with Sendai and STEMCCA vectors. Further analysis revealed that downregulation of HO-1 in human fibroblasts resulted in increased p53 expression in these cells. Interestingly, also HO-1-deficient murine fibroblasts demonstrated higher p53 level and decreased reprogramming efficiency in comparison to their control counterparts. Additionally, stimulation of cells with cobalt protoporphyrin IX (CoPPIX) which induces HO-1 expression increased the number of iPSCs colonies generated from murine fibroblasts indicating that regulation of HO-1 can optimize the reprogramming process.
: CureVac AG, Tübingen, Germany
The delivery of genetic information has emerged as a promising alternative to overcome substantial drawbacks associated with the use of recombinant proteins when applied in protein therapies. Until recently, these approaches mainly pursued the development of DNA-based gene therapies whereas RNA was not considered as an appropriate genetic carrier due to its transient and unstable nature. Against all odds it could be demonstrated that RNA, besides its remarkable potential to act as a vaccine, can promote a strong and robust protein expression and thus be utilized in gene therapy approaches. In this line, we recently demonstrated that the application of RNA can boost the serum levels of erythropoietin and consistently leads to long-lasting biologically-relevant effects, even in large animals. An important field of protein therapies is passive immunization with recombinant antibodies against threats such as infectious diseases or intoxication. In the present study we asked whether mRNA can fulfil its promise and be utilized even in scenarios where a prompt and strong response is crucial. In animals, a single injection of mRNA immediately leads to high neutralizing antibody titers, sufficient to provide fast protection in lethal challenge situations. We postulate that the mRNA technology provides a potent alternative/means for passive immunization.
: NCT DKFZ Heidelberg
Lentiviral vectors have been successfully employed as a therapeutic gene delivery tool for targeting hematopoietic stem cells and to treat specific inherited immune deficiencies, including severe combined immune deficiencies (SCID). To assess the safety and efficacy of lentiviral vector mediated gene therapy for Adenosine deaminase (ADA) deficient SCID, autologous CD34+ cells from 10 ADA deficient patients have been modified using a lentiviral vector encoding a codon optimized version of the human ADA cDNA under the transcriptional control of the EF1αS promoter. Gene modified cells have been returned to patients after non-myeloablative conditioning and cessation of PEG-ADA enzyme replacement therapy. We performed linear amplification mediated PCR (LAM-PCR) combined with high throughput sequencing to analyze the clonal inventory of the gene-corrected hematopoiesis up to 36 months post gene therapy. Integration site (IS) analysis allowed us to retrieve between 2.500 and 20.000 unique IS per patient. Chromosomal distribution of the SINLV-EF1aS-ADA vector revealed a typical lentiviral integration pattern. High resolution gel electrophoresis and Diversity Measurements by Poly-Monoclonal Distance Index (PMD Index, unpublished data) showed a polyclonal reconstitution of the hematopoiesis in all patients. No common integration sites (CIS) associated to know proto- /oncogene loci have been identified and no persistent clonal dominance was observed till the last observation time point (up to M36 post GT) in all subjects. The clinical and molecular follow-up of these patients will further allow clarifying the risk/benefit ratio, and will help us to yield deep insights into clonal dynamics and repopulation kinetics of the blood forming system.
: University Hospital Olomouc
MicroRNAs (miRNAs) are small non-coding RNA molecules. They regulate gene expression at the post-transtranscriptional level by mechanism RNA interference (RNAi). They are involved in the management of different biological processes and their dysregulation can be observed during pathogenesis of many diseases. Expression pattern of miRNA molecules is time and tissue dependent. The aim of this study was to quantify miR-126 level in different regions of musculus tibialis anterior in health and ischemic rabbit hindlimb. MiR-126 was chosen because it considerably participates in angiogenesis. We separately homogenised up to 250 mg of frozen tissue samples from 4 different regions of musculus tibialis anterior from each rabbit hindlimb. Then we homogenised the rest of tissue and took approximately the same amount of tissue mass. We isolated total RNA from all 10 samples using mirVana™ miRNA Isolation Kit. After preamplification we relatively quantified miR-126 and miR-126* level by TaqMan® MicroRNA Assays according to manufacturer protocols (Thermo Fisher Scientific) using LightCycler® 480 System (Roche). RNU6B was used as an endogenous control. We have found that the difference in Ct values of individual measured miRNAs is ±1 Ct. According to the common rules for relative quantification by PCR this range of Ct values should not mean the different expression level. We can conclude that the expression level of analysed miRNAs in different regions of rabbit hindlimb is quite homogenous. It indicates that there is no need for processing whole musculus tibialis anterior but it can be taken a representative piece of tissue for miR-126 quantification.
: EB Haus Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, Austria
Mutations within the keratin 14 gene underlie generalized-severe epidermolysis bullosa simplex (gen-sev EBS), characterized by severe skin blistering even upon minor trauma. In contrast to other EB-subtypes, it is mainly inherited in an autosomal dominant way, which requires gene therapy approaches with high rates of gene correction. Spliceosome mediated RNA trans-splicing (SMaRT) provides a powerful tool to correct mutations at pre-mRNA level, leading to a restoration of wildtype K14. In this study, patient keratinocytes harboring a K14 mutation were corrected using the SMaRT technology to replace the complete coding region spanning exons 1-7. To investigate whether restoration of K14 increases epidermal stability and reduces blister formation in vivo, skin equivalents of corrected patient cells were generated in the lab and transplanted onto the back of mice. In contrast to corrected skin equivalents, which resembled wild type skin, those derived from non- corrected gen-sev EBS keratinocytes developed both spontaneous and mechanically induced blisters. Transmission electron microscopy (TEM) and HE- staining both confirmed the human origin of the differentiated skin equivalents and allowed a clear distinction from the surrounding murine skin. Immunofluorescence staining of various epidermal markers showed correct differentiation of skin equivalents. In addition we were able to trace the pre- mRNA trans-splicing molecule (PTM) 8 weeks post transplantation. Taken together, we were able to stabilize the epidermal basal layer in skin equivalent grafts from EBS patients bearing a K14 mutation using the SMaRT technology. We suggest this technology as a potential tool to correct dominantly inherited mutations.
: Department of Integrated Life Sciences, Research Institute of Advanced Omics, Dankook University, Yongin 16890, Korea
In this study, we describe a novel genetic approach against hepatitis C virus (HCV), which is based upon trans-splicing ribozyme that can selectively induce interferon α2 (IFN-α2) only in HCV-infected cells through replacement of HCV genomes and can remove the viral genome by cleavage activity at the same time. We developed group I intron-based ribozymes targeting the highly conserved HCV core coding region (362th nucleotide from 5′ end of the viral genome) with high efficiency, accuracy, and specificity. These ribozymes were designed to trans-splice the 3′ tagging sequence that is IFN-α2 coding sequence to the targeted HCV genome. These ribozymes were found to efficiently and precisely trans-splice the targeted HCV RNA in vitro. Moreover, specific ribozymes with ∼230 mer antisense sequences against genotype 1b HCV-replicon RNA or genotype 2a JFH-1 RNA selectively triggered expression of IFN-α2 only in HCV genomic and/or sub-genomic RNA replicating cells and efficiently inhibited HCV replication through efficient and accurate trans-splicing reaction. These results suggest that IFN-α2 induction by the HCV core targeting trans-splicing ribozyme could be a potent anti-HCV approach through regulating anti-viral signaling pathways, specifically and selectively in HCV-infected cells.
: The University of Tokyo
Gene therapy is promising for treating apoptosis-related diseases by introducing anti-apoptotic factors into cells in a sustained manner. However, the introduction using DNA is likely unacceptable due to the risk of random genomic integration of the delivered gene. mRNA is a good alternative that can avoid the risk. Here we examined the use of mRNA for introducing an anti-apoptotic factor, Bcl-2, to treat model mice of fulminant hepatitis. A synthesized polymer-based carrier, polyplex nanomicelle, was used for the hydrodynamic intravenous injection of mRNA into the liver. By analyses on the transgene expression profiles in the liver using GFP mRNA, almost 100% of liver cells were GFP-positive after the hydrodynamic injection. In contrast, plasmid DNA (pDNA) injection provided a smaller percentage of GFP-expressing cells, although their expression level were higher than that by mRNA. Analyses using Cy5-labelled mRNA and pDNA revealed that the diffusive manner of transgene expression by mRNA was attributed to a simple intracellular mechanism, without the need for nuclear entry. Consistent with this observation, Bcl-2 mRNA was more effective on reducing apoptosis in the liver of mice with fulminant hepatitis than Bcl-2 pDNA. Therefore, mRNA-based therapeutics combined with an effective delivery system such as polyplex nanomicelles is a promising treatment for intractable diseases associated with excessive apoptosis.
: Kyoto University
We reported on our Borna disease virus (BoDV) vector as a novel RNA virus-based episomal vector system. BoDV vector is amenable for long-term transgene expression in stem cells without host gene modification, however, the vector production efficiency is very low due to the low titer of virus particles in the culture supernatants. Here in order to improve the production of BoDV vector, we identified host factor involved in the regulation of BoDV production. The screening of shRNA library based on the viral titer revealed the knockdown (KD) of insulin-like growth factor 2 (IGF2) promoted the BoDV production. IGF2 KD also increased the viral polymerase activity in both BoDV minigenome assay and recombinant BoDV-infected cells. Overexpression of IGF2 impaired the polymerase activity of BoDV. We also found that the expression level of endogenous IGF2 was involved in the susceptibility to BoDV of human cell lines. Immunoprecipitation assay revealed that IGF2 interacted with GP1 of BoDV envelope glycoprotein (G). The amount of BoDV-G was strongly increased by IGF2 KD in both BoDV-infected cells and viral particles. These results indicate that IGF2 is involved in the suppression of BoDV production through the regulation of G expression. We estimated the efficiency of BoDV vector production in the IGF2 KD cells. In the BoDV producer and expanding cells, IGF2 KD promoted virus production and virus spread at approx. 2- and 5-fold higher than control cells. These data suggest that the IGF2 KD cells could be a powerful tool for the efficient production of BoDV vector.