- Email: [email protected]
690. Development of a Clinical Lentiviral Vector for Gene Therapy of SCID-X1
Hematologic & Immunologic Diseases II and health related costs for the patient. We have previously shown that hemophilia B mice with pathogenic inhibitory antibodies are effectively treated with a novel immune tolerance induction protocol using a liver directed adeno-associated virus expressing the gene encoding for human factor IX protein (AAV8-F9). AAV8-F9 ITI rapidly eliminates pre-existing inhibitors and provided sustained therapeutic levels of FIX protein even following repeated exposure to recombinant FIX protein. Based on our success with hemophilia B, we sought to develop a complementary protocol for hemophilia A mice with pre-existing inhibitors. Our initial studies were conducted using a codon-optimized F8 (coF8) gene, which has been shown to enhance the levels of transcribed FVIII protein. In the first set of experiments we tested two different vector doses (1x1011 and 1x1012 vg) of an AAV8-cohF8 vector for factor VIII expression levels and tolerance induction in hemophilia A mice on two different genetic backgrounds (BALB/c-F8-/Y and 129/BL6-F8-/Y). Since BALB/c-F8/Y mice develop lower titer FVIII inhibitors following intravenous hFVIII protein challenge compared to 129/BL6-F8-/Y mice we expected better outcomes with AAV8-cohF8 vector treated BALB/cF8-/Y mice. Surprisingly BALB/c-F8-/Y mice spontaneously developed FVIII inhibitors approximately 4 weeks following vector delivery. In contrast,129/BL6-F8-/Y mice expressed vector dose dependent therapeutic levels FVIII protein without inhibitors and 75% of the mice remained inhibitor free following four weekly IV FVIII protein challenges. Subsequent studies for AAV8-cohF8 ITI were conducted in 129/BL6-F8-/Y mice. We generated a group of inhibitor mice and treated a subset of these mice with 1x1012 vg AAV8-coF8. AAV8cohF8 ITI alone was not sufficient to eliminate inhibitors. Therefore we designed two additional studies to determine if 1x1012 vg AAV8coF8 ITI would be more effective if combined with transient immune suppression using oral rapamycin (4mg/kg) or anti-CD20 (10mg/kg) mediated B cell depletion. While rapamycin adjunct therapy failed to lower inhibitors, preliminary data show that anti-CD20 combined with AAV8-coF8 ITI leads to a sharp reduction in high titer inhibitors. Given that FVIII protein invokes a stronger immune response in mice and patients, our data suggests that successful gene therapy for hemophilia A may require supraphysiological levels of FVIII protein to prevent inhibitor formation and transient immune suppression to be effective as an ITI therapy.
689. The Content of the More Immature Hematopoietic Stem Cells (HSCs) Is Dependent from the Underlying Genetic Diseases: Consequence on Transduction Efficiencies
Laure Caccavelli1, Cécile Roudaut1, Maeva Joigneaux1, Johanna Blondeau1, Fabien Touzot1, Salima Hacein-Bey-Abina2, Marina Cavazzana3 1 Biotherapy, Hôpital Necker-Enfants Malades, AP-HP, Paris, France, 2Immunologie Biologique, Groupe HospitalierUniversitaire Paris-Sud, AP-HP, Le Kremlin-Bicêtre, France, Paris, France, 3CIC- Biotherapy, Groupe Hospitalier Universitaire Ouest, INSERM/AP-HP, Paris, France Ex vivo gene transfer into hematopoietic CD34+ cells provides therapeutic benefits in primary immunodeficiencies. However, the CD34+ cell population is heterogeneous and is mainly composed of progenitors (CD34+lin-CD38+) and multilineage progenitors (CD34+lin-CD38-). This latter population can be divided in 3 subtypes regarding to the CD45RA and CD90 expression. CD45RA+ define cells with lymphoid potential, CD45RA-CD90- defined cells with myeloid potential and CD45RA-CD90+ define cells with long term repopulating capability (HSCs) which constitutes the relevant target for gene correction. We found that in bone marrow (BM) CD34+linneg, addition of CD133 helps to delimitate CD38neg population and we have analysed HSC, MLP and MPP population in Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
BM from SCID-X1 (n=4), Wiskott Aldrich Syndrome (WAS) (n=2), Artemis (n=2), Chronic Granulomatous Disease (CGD) patients (n=3), sickle cell disease (SCD) (n=3) and age matched healthy controls (n=6).As expected, mean percentage of HSC represent 1% of CD34+ in healthy donors and similar results are obtained in CD34+ from SCID-X1 (1.76% ± 0.72), Artemis (1.98% ± 0.15), and SCD (2.16% ± 0.6) patients. On the contrary HSC percentage are lower in CD34+ from WAS (0.35% ± 0.15) and nearly undetectable in CGD patients. Moreover in multilineage progenitors, proportion of HSC, MLP and MPP varies from one pathology to another as shown in table1. Table 1: Mean percentage of HSC, MLP and MPP in the linnegCD133+CD38neg CD34+ cells HSCs MLP Healthy Donor 42% 33% SCIDX1 37% 45% WAS 35% 35% Artemis 30% 48% SCD 36% 19%
MPP 24% 28% 30% 20% 44%
Our aim was then to determine the ability of vectors used for gene therapy to transduce these different progenitors. We first analysed transduction of bulk CD34+ and HSCs by SIN retroviral vector for IL2RG in 2 SCIDX-1 patients enrolled in the clinical trial. After transduction, the 3 cell types were purified by flow cytometry and transduction determined in CFU after 14 days of culture in methyl cellulose in order to avoid detection of non-integrated vector. We show that in both patients, transduction efficiency was lower in HSCs than in bulk CD34+ cells. We were not able to recover enough CFU to determine VCN from MPP fraction and as expected, no CFU were obtained from MLP fraction. These results will be extended to transduction of CD34+ from WAS and SCD patients and efforts will be concentrated on transduction protocols in order to improve transduction of HSCs. Nevertheless low HSCs contents observed in CD34+ from CGD patients also represents a major difficulty to obtain long term reconstitution after gene therapy.
690. Development of a Clinical Lentiviral Vector for Gene Therapy of SCID-X1
Sabine Charrier1, Valentina Poletti1, Samia Martin1, Bernard Gjata1, Matthias Hebben1, Alban Vignaud1, Fang Zhang2, Karen Buckland2, Michael Rothe3, Axel Schambach3, Bobby Gaspar2, Adrian Thrasher2, Fulvio Mavilio1 1 Genethon, Evry, France, 2University College London, London, United Kingdom, 3Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the gene encoding the interleukin-2 receptor γ chain (IL2RG), and is characterized by profound immunological defects caused by a partial or complete absence of T and NK cells and the presence of non-functional B cells. To overcome the safety issues raised by the use of MLV-based retroviral vectors in previous gene therapy clinical trials, we designed a SIN lentiviral vector (LV) carrying the codon-optimized human IL2RG cDNA under the control of the human EF1αS promoter and a mutated WPRE. Replacement of the native IL2RG open reading frame by a codonoptimized sequence resulted in a 3-fold increase in mRNA expression and a 1.5-fold increase in IL2RG protein expression per integrated vector copy. The vector was VSV-G-pseudotyped and produced by a new manufacturing process based on quadri-transfection of suspension-adapted 293T cells grown in serum-free conditions in 50- to 200-L bioreactors, purified by ion-exchange chromatography and concentrated by tangential-flow filtration. The efficacy of this vector was demonstrated in vitro by the restoration of a normal level S273
Immunological Aspects of Gene Therapy II: AAV Vectors of IL2RG mRNA or protein in a human IL2RG-deficient T-cell line at a VCN of 1 to 3 and by high efficiency (81±7%) transduction of human mobilized CD34+ hematopoietic stem/progenitor cells with no impact on viability or clonogenic capacity. A biosafety evaluation study of the IL2RG LV in the murine model of the disease showed biodistribution of the transgene in hematopoietic organs only, restoration of T, B and NK cell counts, normalization of lymphoid organs (thymus and spleen) and a low frequency of hematopoietic malignancies, comparable to that of untreated animals. An in vitro assay (IVIM) showed a safe genotoxic profile, while insertion site analysis in transplanted mice revealed a standard lentiviral integration profile and no signs of clonal dominance. These studies will enable a multicenter phase-I/II clinical trial aimed at establishing the safety and clinical efficacy of lentiviral vector-mediated gene therapy for SCID-X1.
691.
Abstract Withdrawn
692. Genome Editing for Personalized Gene Therapy of IVSI-110 Beta-Thalassemia
Petros Patsali1,2, Claudio Mussolino3, Constantinos Loucari1,4, Coralea Stephanou1,2, Michael Antoniou2, Toni Cathomen3, Carsten W. Lederer1,4, Marina Kleanthous1,4 1 Molecular Genetics Thalassemia, Cyprus Institute of Genetics and Neurology, Nicosia, Cyprus, 2King’s College London, London, United Kingdom, 3Institute for Cell and Gene Therapy, University Medical Center Freiburg, Freiburg, Germany, 4Cyprus School of Molecular Medicine, Nicosia, Cyprus Thalassemia is amongst the commonest single-gene disorders worldwide, caused by deficient production of α- or β-globin. Of particular clinical relevance is β-thalassemia, which as a severe monogenic disease of the hematopoietic system is an ideal target for gene therapy, either by gene addition or gene correction. Problems inherent to the gene-augmentation approach for β-thalassemia, including insertional mutagenesis or low expression of the therapeutic transgene, may be avoided using targeted gene correction of mutations with site-specific designer nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 or transcription activator-like effector nucleases (TALENs). Our study is focused on the development of a personalized gene-correction therapy of the common β-thalassemia mutation, HBBIVS1-110, which in most Mediterranean and many Western countries has a frequency of above 20% (with 80% on the island of Cyprus) amongst β-thalassemia carriers. HBBIVS1-110 creates an abnormal splice-acceptor site in intron I of the β-globin gene, leading to a pre-mature in-frame stop codon in the aberrantly spliced mRNA and to early transfusion dependence of homozygotes. We have developed and evaluated designer nucleases targeting the site of the HBBIVS1-110 β-thalassemia mutation, including direct comparison of targeted disruption efficiency and toxicity for CRISPR/Cas9 and TALEN tools in human embryonic kidney cells and in murine erythroleukemia (MEL) cells carrying a HBBIVS1-110mutant transgene based on the GLOBE (MA821) vector. Using the latter, we have assessed the therapeutic efficiency and cleavage properties of these designer nucleases. Analyses included immunoblots, absolute quantification of correctly and aberrantly spliced HBB mRNAs using multiplex RT-qPCR, sequencing and characterization of the resulting genome-editing events. We noted superior performance of several TALEN pairs compared to a single CRISPR/Cas9 nuclease suitable for the target site. Moreover, we demonstrate a significant increase of correct splicing and β-globin chain synthesis for genome-edited transgenic MEL MA821 HBBIVS1-110 cells. For the two most efficient TALEN pairs, β-globin protein levels in pools of edited cells reach over 20% of that detected in MEL cells harbouring a wild-type MA821
S274
HBB control, up from 1% for the mock-treated MA821 HBBIVS1-110 control. Our findings validate our approach and indicate its suitability also for the correction of other intronic disease-causing mutations.
693. Single Cell Dynamics Causes Pareto-Like Effect in Stimulated T Cell Populations
Alice Moussy1, Jérémie Cosette2, Fanny Onodi3, Adrien AuffretCariou3, Thi My Anh Neildez-Nguyen1, Andras Paldi1, Daniel Stockholm1 1 Genethon/INSERM UMR_S951/EPHE, Evry, France, 2Genethon, Evry, France, 3Genethon/INSERM UMR_S951, Evry, France The host’s immune response is a frequent obstacle to successful gene therapy. The suppressive activity of regulatory T cells can be used to get round the problem. To do this, understanding the mechanism of proliferation and differentiation of CD4 + T cells is necessary. Here, we used time-lapse microscopy of individual murine CD4 + T cells to investigate the dynamics of proliferation and fate commitment. We observed highly heterogeneous division and death rates between individual clones resulting in a Pareto-like dominance of a few clones at the end of the experiment. Commitment to the regulatory T (Treg) fate was monitored using the expression of a GFP reporter gene under the control of the endogenous Foxp3 promoter. All possible combinations of proliferation and differentiation were observed and resulted in exclusively GFP-, GFP+ or mixed phenotype clones of very different population sizes. We simulated the process of proliferation and differentiation using a simple mathematical model of stochastic decision-making based on the experimentally observed parameters. The simulations show that a stochastic scenario is fully compatible with the observed Pareto-like imbalance in the final population. These observations may help to develop new strategies for the amplification of Treg cells for therapeutic use.
Immunological Aspects of Gene Therapy II: AAV Vectors 694. Anti-Transgene Cellular Immune Repsonses Can Be Induced by Subretinal Gene Transfer with rAAV in a Dose-Dependent Manner
Julie M. Vendomele1, Quentin Khebizi1, Mirella Mormin2, Sabrina Donnou1, Catherine Poinsignon1, Anne Galy1, Sylvain Fisson1 1 Généthon, UMR_S951, Université d’Evry Val d’Essonne (UEVE), Evry, France, 2Généthon, UMR_S951, Evry, France From animal experiments to the first human clinical trials in 2007, recombinant adeno-associated virus (rAAV)-mediated ocular gene therapy has shown successful results which have been attributed in part to the immune-privileged situation of the eye. Recently, some ocular gene therapy clinical trials have reported that visual acuity returned to baseline 6 to 12 months after therapy. The involvement of anti-transgene immune responses may lead to loss of therapeutic efficacy. This prompted us to evaluate in a murine model if rAAV gene transfer leads to the expected immune ignorance of the transgene or perhaps to an immuno-modulatory mechanism already described with subretinal peptide injection (eg. Anterior Chamber Associated Immune Deviation / ACAID), or to anti-transgene immunization. In this study, we characterized the CD4 and CD8 T cell responses specifically directed toward the transgene product in a murine model following rAAV2/8-mediated subretinal gene transfer. An rAAV2/8 encoding for the GFP-HY fusion protein under the phosphoglycerate kinase (PGK) promoter was used. The transgene expresses the HY male antigen which contains MHC class I and MHC class II-restricted T cell epitopes (UTY and DBY, respectively) that are immunodominant in female mice. For the study, 2µL of endotoxin-free, Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
689. The Content of the More Immature Hematopoietic Stem Cells (HSCs) Is Dependent from the Underlying Genetic Diseases: Consequence on Transduction Efficiencies
Laure Caccavelli1, Cécile Roudaut1, Maeva Joigneaux1, Johanna Blondeau1, Fabien Touzot1, Salima Hacein-Bey-Abina2, Marina Cavazzana3 1 Biotherapy, Hôpital Necker-Enfants Malades, AP-HP, Paris, France, 2Immunologie Biologique, Groupe HospitalierUniversitaire Paris-Sud, AP-HP, Le Kremlin-Bicêtre, France, Paris, France, 3CIC- Biotherapy, Groupe Hospitalier Universitaire Ouest, INSERM/AP-HP, Paris, France Ex vivo gene transfer into hematopoietic CD34+ cells provides therapeutic benefits in primary immunodeficiencies. However, the CD34+ cell population is heterogeneous and is mainly composed of progenitors (CD34+lin-CD38+) and multilineage progenitors (CD34+lin-CD38-). This latter population can be divided in 3 subtypes regarding to the CD45RA and CD90 expression. CD45RA+ define cells with lymphoid potential, CD45RA-CD90- defined cells with myeloid potential and CD45RA-CD90+ define cells with long term repopulating capability (HSCs) which constitutes the relevant target for gene correction. We found that in bone marrow (BM) CD34+linneg, addition of CD133 helps to delimitate CD38neg population and we have analysed HSC, MLP and MPP population in Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
BM from SCID-X1 (n=4), Wiskott Aldrich Syndrome (WAS) (n=2), Artemis (n=2), Chronic Granulomatous Disease (CGD) patients (n=3), sickle cell disease (SCD) (n=3) and age matched healthy controls (n=6).As expected, mean percentage of HSC represent 1% of CD34+ in healthy donors and similar results are obtained in CD34+ from SCID-X1 (1.76% ± 0.72), Artemis (1.98% ± 0.15), and SCD (2.16% ± 0.6) patients. On the contrary HSC percentage are lower in CD34+ from WAS (0.35% ± 0.15) and nearly undetectable in CGD patients. Moreover in multilineage progenitors, proportion of HSC, MLP and MPP varies from one pathology to another as shown in table1. Table 1: Mean percentage of HSC, MLP and MPP in the linnegCD133+CD38neg CD34+ cells HSCs MLP Healthy Donor 42% 33% SCIDX1 37% 45% WAS 35% 35% Artemis 30% 48% SCD 36% 19%
MPP 24% 28% 30% 20% 44%
Our aim was then to determine the ability of vectors used for gene therapy to transduce these different progenitors. We first analysed transduction of bulk CD34+ and HSCs by SIN retroviral vector for IL2RG in 2 SCIDX-1 patients enrolled in the clinical trial. After transduction, the 3 cell types were purified by flow cytometry and transduction determined in CFU after 14 days of culture in methyl cellulose in order to avoid detection of non-integrated vector. We show that in both patients, transduction efficiency was lower in HSCs than in bulk CD34+ cells. We were not able to recover enough CFU to determine VCN from MPP fraction and as expected, no CFU were obtained from MLP fraction. These results will be extended to transduction of CD34+ from WAS and SCD patients and efforts will be concentrated on transduction protocols in order to improve transduction of HSCs. Nevertheless low HSCs contents observed in CD34+ from CGD patients also represents a major difficulty to obtain long term reconstitution after gene therapy.
690. Development of a Clinical Lentiviral Vector for Gene Therapy of SCID-X1
Sabine Charrier1, Valentina Poletti1, Samia Martin1, Bernard Gjata1, Matthias Hebben1, Alban Vignaud1, Fang Zhang2, Karen Buckland2, Michael Rothe3, Axel Schambach3, Bobby Gaspar2, Adrian Thrasher2, Fulvio Mavilio1 1 Genethon, Evry, France, 2University College London, London, United Kingdom, 3Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the gene encoding the interleukin-2 receptor γ chain (IL2RG), and is characterized by profound immunological defects caused by a partial or complete absence of T and NK cells and the presence of non-functional B cells. To overcome the safety issues raised by the use of MLV-based retroviral vectors in previous gene therapy clinical trials, we designed a SIN lentiviral vector (LV) carrying the codon-optimized human IL2RG cDNA under the control of the human EF1αS promoter and a mutated WPRE. Replacement of the native IL2RG open reading frame by a codonoptimized sequence resulted in a 3-fold increase in mRNA expression and a 1.5-fold increase in IL2RG protein expression per integrated vector copy. The vector was VSV-G-pseudotyped and produced by a new manufacturing process based on quadri-transfection of suspension-adapted 293T cells grown in serum-free conditions in 50- to 200-L bioreactors, purified by ion-exchange chromatography and concentrated by tangential-flow filtration. The efficacy of this vector was demonstrated in vitro by the restoration of a normal level S273
Immunological Aspects of Gene Therapy II: AAV Vectors of IL2RG mRNA or protein in a human IL2RG-deficient T-cell line at a VCN of 1 to 3 and by high efficiency (81±7%) transduction of human mobilized CD34+ hematopoietic stem/progenitor cells with no impact on viability or clonogenic capacity. A biosafety evaluation study of the IL2RG LV in the murine model of the disease showed biodistribution of the transgene in hematopoietic organs only, restoration of T, B and NK cell counts, normalization of lymphoid organs (thymus and spleen) and a low frequency of hematopoietic malignancies, comparable to that of untreated animals. An in vitro assay (IVIM) showed a safe genotoxic profile, while insertion site analysis in transplanted mice revealed a standard lentiviral integration profile and no signs of clonal dominance. These studies will enable a multicenter phase-I/II clinical trial aimed at establishing the safety and clinical efficacy of lentiviral vector-mediated gene therapy for SCID-X1.
691.
Abstract Withdrawn
692. Genome Editing for Personalized Gene Therapy of IVSI-110 Beta-Thalassemia
Petros Patsali1,2, Claudio Mussolino3, Constantinos Loucari1,4, Coralea Stephanou1,2, Michael Antoniou2, Toni Cathomen3, Carsten W. Lederer1,4, Marina Kleanthous1,4 1 Molecular Genetics Thalassemia, Cyprus Institute of Genetics and Neurology, Nicosia, Cyprus, 2King’s College London, London, United Kingdom, 3Institute for Cell and Gene Therapy, University Medical Center Freiburg, Freiburg, Germany, 4Cyprus School of Molecular Medicine, Nicosia, Cyprus Thalassemia is amongst the commonest single-gene disorders worldwide, caused by deficient production of α- or β-globin. Of particular clinical relevance is β-thalassemia, which as a severe monogenic disease of the hematopoietic system is an ideal target for gene therapy, either by gene addition or gene correction. Problems inherent to the gene-augmentation approach for β-thalassemia, including insertional mutagenesis or low expression of the therapeutic transgene, may be avoided using targeted gene correction of mutations with site-specific designer nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 or transcription activator-like effector nucleases (TALENs). Our study is focused on the development of a personalized gene-correction therapy of the common β-thalassemia mutation, HBBIVS1-110, which in most Mediterranean and many Western countries has a frequency of above 20% (with 80% on the island of Cyprus) amongst β-thalassemia carriers. HBBIVS1-110 creates an abnormal splice-acceptor site in intron I of the β-globin gene, leading to a pre-mature in-frame stop codon in the aberrantly spliced mRNA and to early transfusion dependence of homozygotes. We have developed and evaluated designer nucleases targeting the site of the HBBIVS1-110 β-thalassemia mutation, including direct comparison of targeted disruption efficiency and toxicity for CRISPR/Cas9 and TALEN tools in human embryonic kidney cells and in murine erythroleukemia (MEL) cells carrying a HBBIVS1-110mutant transgene based on the GLOBE (MA821) vector. Using the latter, we have assessed the therapeutic efficiency and cleavage properties of these designer nucleases. Analyses included immunoblots, absolute quantification of correctly and aberrantly spliced HBB mRNAs using multiplex RT-qPCR, sequencing and characterization of the resulting genome-editing events. We noted superior performance of several TALEN pairs compared to a single CRISPR/Cas9 nuclease suitable for the target site. Moreover, we demonstrate a significant increase of correct splicing and β-globin chain synthesis for genome-edited transgenic MEL MA821 HBBIVS1-110 cells. For the two most efficient TALEN pairs, β-globin protein levels in pools of edited cells reach over 20% of that detected in MEL cells harbouring a wild-type MA821
S274
HBB control, up from 1% for the mock-treated MA821 HBBIVS1-110 control. Our findings validate our approach and indicate its suitability also for the correction of other intronic disease-causing mutations.
693. Single Cell Dynamics Causes Pareto-Like Effect in Stimulated T Cell Populations
Alice Moussy1, Jérémie Cosette2, Fanny Onodi3, Adrien AuffretCariou3, Thi My Anh Neildez-Nguyen1, Andras Paldi1, Daniel Stockholm1 1 Genethon/INSERM UMR_S951/EPHE, Evry, France, 2Genethon, Evry, France, 3Genethon/INSERM UMR_S951, Evry, France The host’s immune response is a frequent obstacle to successful gene therapy. The suppressive activity of regulatory T cells can be used to get round the problem. To do this, understanding the mechanism of proliferation and differentiation of CD4 + T cells is necessary. Here, we used time-lapse microscopy of individual murine CD4 + T cells to investigate the dynamics of proliferation and fate commitment. We observed highly heterogeneous division and death rates between individual clones resulting in a Pareto-like dominance of a few clones at the end of the experiment. Commitment to the regulatory T (Treg) fate was monitored using the expression of a GFP reporter gene under the control of the endogenous Foxp3 promoter. All possible combinations of proliferation and differentiation were observed and resulted in exclusively GFP-, GFP+ or mixed phenotype clones of very different population sizes. We simulated the process of proliferation and differentiation using a simple mathematical model of stochastic decision-making based on the experimentally observed parameters. The simulations show that a stochastic scenario is fully compatible with the observed Pareto-like imbalance in the final population. These observations may help to develop new strategies for the amplification of Treg cells for therapeutic use.
Immunological Aspects of Gene Therapy II: AAV Vectors 694. Anti-Transgene Cellular Immune Repsonses Can Be Induced by Subretinal Gene Transfer with rAAV in a Dose-Dependent Manner
Julie M. Vendomele1, Quentin Khebizi1, Mirella Mormin2, Sabrina Donnou1, Catherine Poinsignon1, Anne Galy1, Sylvain Fisson1 1 Généthon, UMR_S951, Université d’Evry Val d’Essonne (UEVE), Evry, France, 2Généthon, UMR_S951, Evry, France From animal experiments to the first human clinical trials in 2007, recombinant adeno-associated virus (rAAV)-mediated ocular gene therapy has shown successful results which have been attributed in part to the immune-privileged situation of the eye. Recently, some ocular gene therapy clinical trials have reported that visual acuity returned to baseline 6 to 12 months after therapy. The involvement of anti-transgene immune responses may lead to loss of therapeutic efficacy. This prompted us to evaluate in a murine model if rAAV gene transfer leads to the expected immune ignorance of the transgene or perhaps to an immuno-modulatory mechanism already described with subretinal peptide injection (eg. Anterior Chamber Associated Immune Deviation / ACAID), or to anti-transgene immunization. In this study, we characterized the CD4 and CD8 T cell responses specifically directed toward the transgene product in a murine model following rAAV2/8-mediated subretinal gene transfer. An rAAV2/8 encoding for the GFP-HY fusion protein under the phosphoglycerate kinase (PGK) promoter was used. The transgene expresses the HY male antigen which contains MHC class I and MHC class II-restricted T cell epitopes (UTY and DBY, respectively) that are immunodominant in female mice. For the study, 2µL of endotoxin-free, Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy