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Pompe disease (PD) is a severe neuromuscular disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). PD is currently treated with enzyme replacement therapy (ERT) with intravenous infusions of recombinant human GAA (rhGAA). Although the introduction of ERT represents a breakthrough in the management of PD, the approach suffers from several shortcomings. Here, we developed a mouse model of PD to compare the efficacy of hepatic gene transfer with adeno-associated virus (AAV) vectors expressing secretable GAA with long-term ERT. Liver expression of GAA results in enhanced pharmacokinetics and uptake of the enzyme in peripheral tissues compared to ERT. Combination of gene transfer with pharmacological chaperones boosts GAA bioavailability, resulting in improved rescue of the PD phenotype. Scale-up of hepatic gene transfer to non-human primates also successfully results in enzyme secretion in blood and uptake in key target tissues, supporting the ongoing clinical translation of the approach., Pompe disease is currently treated with enzyme replacement therapy (ERT) with recombinant human acid alpha-glucosidase (GAA). Here, the authors show hepatic-directed gene therapy with AAV vectors enhances GAA bioavailability compared with ERT, resulting in improved rescue of the disease phenotype in mice and broad enzyme distribution in mice and non-human primates.

Pompe disease (PD) is a neuromuscular disorder caused by deficiency of acidalpha-glucosidase (GAA), leading to motor and respiratory dysfunctions. Available Gaa knock-out (KO) mouse models do not accurately mimic PD, particularly its highly impaired respiratory phenotype. Here we developed a new mouse model of PD crossing Gaa KOB6;129 with DBA2/J mice. We subsequently treated Gaa KODBA2/J mice with adeno-associated virus (AAV) vectors expressing a secretable form of GAA (secGAA). Male Gaa KODBA2/J mice present most of the key features of the human disease, including early lethality, severe respiratory impairment, cardiac hypertrophy and muscle weakness. Transcriptome analyses of Gaa KODBA2/J, compared to the parental Gaa KOB6;129 mice, revealed a profoundly impaired gene signature in the spinal cord and a similarly deregulated gene expression in skeletal muscle. Muscle and spinal cord transcriptome changes, biochemical defects, respiratory and muscle function in the Gaa KODBA2/J model were significantly improved upon gene therapy with AAV vectors expressing secGAA. These data show that the genetic background impacts on the severity of respiratory function and neuroglial spinal cord defects in the Gaa KO mouse model of PD. Our findings have implications for PD prognosis and treatment, show novel molecular pathophysiology mechanisms of the disease and provide a unique model to study PD respiratory defects, which majorly affect patients. This work was supported by Genethon, the French Muscular Dystrophy Association (AFM), the European Commission (grant nos. 667751, 617432, and 797144), and Spark Therapeutics. This work was supported by Genethon and the French Muscular Dystrophy Association (AFM, to F.M.). It was also supported by the European Union’s research and innovation program under grant agreement no. 667751 (to F.M.), the European Research Council Consolidator Grant under grant agreement no. 617432 (to F.M.), Marie Skodowska-Curie Actions Individual Fellowship (MSCA-IF) grant agreement no. 797144 (to U.C.), and by Spark Therapeutics under a sponsored research agreement. Sí

Pompe disease is a neuromuscular disorder caused by disease-associated variants in the gene encoding for the lysosomal enzyme acid α-glucosidase (GAA), which converts lysosomal glycogen to glucose. We previously reported full rescue of Pompe disease in symptomatic 4-month-old Gaa knockout (Gaa−/−) mice by adeno-associated virus (AAV) vector-mediated liver gene transfer of an engineered secretable form of GAA (secGAA). Here, we showed that hepatic expression of secGAA rescues the phenotype of 4-month-old Gaa−/− mice at vector doses at which the native form of GAA has little to no therapeutic effect. Based on these results, we then treated severely affected 9-month-old Gaa−/− mice with an AAV vector expressing secGAA and followed the animals for 9 months thereafter. AAV-treated Gaa−/− mice showed complete reversal of the Pompe phenotype, with rescue of glycogen accumulation in most tissues, including the central nervous system, and normalization of muscle strength. Transcriptomic profiling of skeletal muscle showed rescue of most altered pathways, including those involved in mitochondrial defects, a finding supported by structural and biochemical analyses, which also showed restoration of lysosomal function. Together, these results provide insight into the reversibility of advanced Pompe disease in the Gaa−/− mouse model via liver gene transfer of secGAA., Graphical Abstract, AAV vector-mediated hepatic expression of secretable GAA drives clearance of glycogen from multiple tissues, resulting in rescue of muscle function in Pompe mice with advanced pathology. Reversal of the Pompe phenotype in mice includes normalization of lysosomal parameters and mitochondrial homeostasis, which is also reflected in the muscle transcriptomics.

Sarcoglycanopathies are rare autosomic limb girdle muscular dystrophies caused by mutations in one of the genes coding for sarcoglycans. Sarcoglycans form a complex, which is an important part of the dystrophin-associated glycoprotein complex and which protects the sarcolemma against muscle contraction-induced damage. Absence of one of the sarcoglycans on the plasma membrane reduces the stability of the whole complex and perturbs muscle fiber membrane integrity. There is currently no curative treatment for any of the sarcoglycanopathies. A first clinical trial to evaluate the safety of a recombinant AAV2/1 vector expressing γ-sarcoglycan using an intramuscular route of administration showed limited expression of the transgene and good tolerance of the approach. In this report, we undertook a dose-effect study in mice to evaluate the efficiency of an AAV2/8-expressing γ-sarcoglycan controlled by a muscle-specific promoter with a systemic mode of administration. We observed a dose-related efficiency with a nearly complete restoration of gamma sarcoglycan (SGCG) expression, histological appearance, biomarker level, and whole-body strength at the highest dose tested. In addition, our data suggest that a high expression threshold level must be achieved for effective protection of the transduced muscle, while a suboptimal transgene expression level might be less protective in the context of mechanical stress. Keywords: LGMD2C, γ-sarcoglycanopathy, limb girdle muscular dystrophy, gene therapy, translational medicine, AAV, miRNA biomarker, mechanical stress

International audience; Unexpectedly, the synthetic antioxidant MnTBAP was found to cause a rapid and reversible downregulation of CD4 on T cells in vitro and in vivo. This effect resulted from the internalization of membrane CD4 T cell molecules into clathrin-coated pits and involved disruption of the CD4/p56(Lck) complex. The CD4 deprivation induced by MnTBAP had functional consequences on CD4-dependent infectious processes or immunological responses as shown in various models, including gene therapy. In cultured human T cells, MnTBAP-induced downregulation of CD4 functionally suppressed gp120- mediated lentiviral transduction in a model relevant for HIV infection. The injection of MnTBAP in mice reduced membrane CD4 on lymphocytes in vivo within 5 days of treatment, preventing OVA peptide T cell immunization while allowing subsequent immunization once treatment was stopped. In a mouse gene therapy model, MnTBAP treatment at the time of adenovirus-associated virus (AAV) vector administration, successfully controlled the induction of anti-transgene and anti-capsid immune responses mediated by CD4(+) T cells, enabling the redosing mice with the same vector. These functional data provide new avenues to develop alternative therapeutic immunomodulatory strategies based on temporary regulation of CD4. These could be particularly useful for AAV gene therapy in which novel strategies for redosing are needed.

Genetic deficiency of the nuclease DCLRE1C/Artemis causes radiosensitive severe combined immunodeficiency (RS-SCID) with lack of peripheral T and B cells and increased sensitivity to ionizing radiations. Gene therapy based on transplanting autologous gene-modified hematopoietic stem cells could significantly improve the health of patients with RS-SCID by correcting their immune system. A lentiviral vector expressing physiological levels of human ARTEMIS mRNA from an EF1a promoter without post-transcriptional regulation was developed as a safe clinically applicable candidate for RS-SCID gene therapy. The vector was purified in GMP-comparable conditions and was not toxic in vitro or in vivo. Long-term engraftment of vector-transduced hematopoietic cells was achieved in irradiated Artemis-deficient mice following primary and secondary transplantation (6 months each). Vector-treated mice displayed T and B lymphopoiesis and polyclonal T cells, had structured lymphoid tissues, and produced immunoglobulins. Benign signs of inflammation were noted following secondary transplants, likely a feature of the model. There was no evidence of transgene toxicity and no induction of hematopoietic malignancy. In vitro, the vector had low genotoxic potential on murine hematopoietic progenitor cells using an immortalization assay. Altogether, these preclinical data show safety and efficacy, and support further development of the vector for the gene therapy of RS-SCID. Keywords: gene therapy, SCID, primary immunodeficiency, Artemis, T cell, B cell, mouse, preclinical study

International audience; Glycogen storage disease type III (GSDIII) is an autosomal recessive disorder caused by a deficiency of glycogen-debranching enzyme (GDE), which results in profound liver metabolism impairment and muscle weakness. To date, no cure is available for GSDIII and current treatments are mostly based on diet. Here we describe the development of a mouse model of GSDIII, which faithfully recapitulates the main features of the human condition. We used this model to develop and test novel therapies based on adeno-associated virus (AAV) vector-mediated gene transfer. First, we showed that overexpression of the lysosomal enzyme alpha-acid glucosidase (GAA) with an AAV vector led to a decrease in liver glycogen content but failed to reverse the disease phenotype. Using dual overlapping AAV vectors expressing the GDE transgene in muscle, we showed functional rescue with no impact on glucose metabolism. Liver expression of GDE, conversely, had a direct impact on blood glucose levels. These results provide proof of concept of correction of GSDIII with AAV vectors, and they indicate that restoration of the enzyme deficiency in muscle and liver is necessary to address both the metabolic and neuromuscular manifestations of the disease.

Duchenne muscular dystrophy (DMD) is an incurable X-linked muscle-wasting disease caused by mutations in the dystrophin gene. Gene therapy using highly functional microdystrophin genes and recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to treat DMD. Here we show that locoregional and systemic delivery of a rAAV2/8 vector expressing a canine microdystrophin (cMD1) is effective in restoring dystrophin expression and stabilizing clinical symptoms in studies performed on a total of 12 treated golden retriever muscular dystrophy (GRMD) dogs. Locoregional delivery induces high levels of microdystrophin expression in limb musculature and significant amelioration of histological and functional parameters. Systemic intravenous administration without immunosuppression results in significant and sustained levels of microdystrophin in skeletal muscles and reduces dystrophic symptoms for over 2 years. No toxicity or adverse immune consequences of vector administration are observed. These studies indicate safety and efficacy of systemic rAAV-cMD1 delivery in a large animal model of DMD, and pave the way towards clinical trials of rAAV–microdystrophin gene therapy in DMD patients., Duchenne muscular dystrophy is a progressive degenerative disease of muscles caused by mutations in the dystrophin gene. Here the authors use AAV vectors to deliver microdystrophin to dogs with muscular dystrophy, and show restoration of dystrophin expression and reduction of symptoms up to 26 months of age.

Oculo-pharyngeal muscular dystrophy (OPMD) is characterised by progressive eyelid drooping (ptosis) and difficulties with swallowing (dysphagia). In order to determine the role of growth factors, cytokines and chemokines in the physiopathology of muscle disease we have compared the level of expression of 174 factors in both the affected (cricopharyngeal) and non-affected (sternocleidomastoid) muscles of 8 OPMD patients by means of antibody arrays. Despite an important inter-individual variability the expression of sixty factors was found to be persistently different between affected and non-affected muscles. Many of the differentially expressed factors in our study are known to be involved in the formation of fibrosis in both the liver and the lung, indicating the possibility that treatments such as those used in hepatic fibrosis may have a beneficial effect in OPMD patients.

International audience; Duchenne muscular dystrophy (DMD) is an X-linked inherited muscle-wasting disease primarily affecting young boys with a prevalence of 1:5000. The disease is caused by loss-of-function mutations in the gene encoding for the Dystrophin protein and is characterised by systemic, progressive, irreversible and severe loss of muscle function. Among vector systems that allow efficient in vivo gene transfer, recombinant Adeno Associated Virus vectors (rAAV) hold great promise and result in particular in very efficient transduction of skeletal and cardiac muscles.

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 codon-optimized 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 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.

International audience; Myotonic dystrophy is the most common adult muscle dystrophy. In view of emerging therapies, which use animal models as a proof of principle, the development of reliable outcome measures for in vivo longitudinal study of mouse skeletal muscle function is becoming crucial. To satisfy this need, we have developed a device to measure ankle dorsi- and plantarflexion torque in rodents. We present an in vivo 8-month longitudinal study of the contractile properties of the skeletal muscles of the DMSXL mouse model of myotonic dystrophy type 1. Between 4 and 12months of age, we observed a reduction in muscle strength in the ankle dorsi- and plantarflexors of DMSXL compared to control mice although the strength per muscle cross-section was normal. Mild steady myotonia but no abnormal muscle fatigue was also observed in the DMSXL mice. Magnetic resonance imaging and histological analysis performed at the end of the study showed respectively reduced muscle cross-section area and smaller muscle fibre diameter in DMSXL mice. In conclusion, our study demonstrates the feasibility of carrying out longitudinal in vivo studies of muscle function over several months in a mouse model of myotonic dystrophy confirming the feasibility of this method to test preclinical therapeutics.

Injured skeletal muscle degeneration comprises early microvascular changes and inflammatory cell infiltration, possibly under the control of several growth factors. We have studied the role of basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF1), and transforming growth factor beta-1 (TGF beta 1), by injecting specific anti-growth factor neutralizing antibodies into mouse extensor digitorum longus muscle at the time of injury (denervation and devascularization). Four days later, at the height of damaged myofiber phagocytosis, we assessed quantitatively revascularization, phagocytic activity, and inflammation. The immune neutralization of bFGF reduced the number of capillaries, macrophages and mast cells, and delayed necrotic myofiber phagocytosis. The immune neutralization of IGF1 or TFG beta 1 promoted muscle revascularization, macrophage infiltration and necrotic myofiber phagocytosis. While IGF1 neutralization reduced the number of mast cells and did not modify that of T-cells or neutrophils, TGF beta 1 neutralization increased the number of all of these cells. This study strongly suggests differing roles for bFGF, IGF1 and TFG beta 1 in angiogenic and inflammatory responses during muscle degeneration, apart from their known effects on the behaviour of myogenic cells.

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 lack of response to common γ-chain-dependent cytokines and profound defects in T-, B- and NK-cell functions. Previous gene therapy clinical trials based on autologous transplantation of hematopoietic stem/progenitor cells (HSPCs) genetically corrected with MLV-derived retroviral vectors expressing IL2RG in non-conditioned patients showed long-lasting restoration of T-cell immunity in most cases but resulted in vector-induced leukemia through enhancer-mediated insertional mutagenesis in 25% of patients. The use of an enhancer-less MLV vector to deliver the IL2RG gene caused no adverse events while retaining a significant clinical benefit. To increase the efficacy of gene therapy and reduce at the same time the probability of genotoxic insertional events, we developed a third-generation lentiviral vector carrying a codon-optimized human IL2RG cDNA under the control of the human EF1α-S promoter. The vector was VSV-G-pseudotyped and produced by transient transfection of 293T cells, followed by ion-exchange chromatography, tangential-flow filtration and formulation in HSC growth medium. Replacement of the native IL2RG open reading frame by a codon-optimized 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 performance of the vector was demonstrated in vitro by the restoration of a normal level of IL2RG mRNA or protein in a human IL2RG-deficient T-cell line at a VCN of 1 to 3, and by high efficiency (>80%) transduction of human mobilized CD34+ HSPCs and transgene expression with no impact on viability or clonogenic capacity. An in vitro immortalization assay (IVIM) showed a safe genotoxic profile, while the in vivo safety and efficacy of the vector was tested it in a preclinical model of SCID-X1 gene therapy based on transplantation of genetically corrected Lin- cells from IL2rg-/- donor mice into lethally-irradiated IL2rg-/--Rag2-/- recipients. The study showed biodistribution of the transgene in hematopoietic organs only, restoration of T, B and NK cell counts in bone marrow and peripheral blood, normalization of lymphoid organs (thymus and spleen) and a low frequency of hematopoietic malignancies, comparable to that of untreated animals, six months after transplantation. An extensive insertion site analysis was carried out in bone marrow, thymus and peripheral blood of individual or groups of animals to detect the presence of any clonal abnormality or skewing, and any deviation from a normal lentiviral integration pattern. The study showed the expected genomic integration profile and no signs of clonal dominance of transduced cells. Interestingly, analysis of >100,000 integration sites in pre- and post-transplant murine cells showed that lentiviral vectors target at high frequency a substantially different set of genes compared to human CD34+ cells, uncovering the need for appropriate controls and at the same time the limits in the predictive power of mouse-based genotoxicity studies, particularly those addressing proto-oncogene activation and 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. The clinical protocol is based on transplantation of autologous CD34+ HSPCs transduced with the EF1a-IL2RGco lentiviral vector in infants after non-myeloablative conditioning, and aims at stable and sustained restoration of both T- and B-cell immunity. Disclosures Williams: Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees. Gaspar:Orchard therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mavilio:Adverum Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Consultancy, Research Funding.

Duchenne Muscular Dystrophy (DMD) is a X-linked inherited muscle-wasting disease primarily affecting young boys with a prevalence of 1:5,000. The disease is caused by loss-of-function mutations in the gene encoding for the Dystrophin protein and is characterized by systemic, progressive, irreversible and severe loss of muscle function. Among vector systems that allow efficient in vivo gene transfer, recombinant Adeno-Associated Virus vectors (rAAV) hold great promise and allow very efficient transduction of skeletal and cardiac muscles. However, full-length dystrophin cDNA exceeds the packaging capacity for a single rAAV gene-delivery cassette. Therefore, truncated versions namely micro-dystrophins have been designed and optimized to contain few clinically important regions of the dystrophin protein. We have tested a rAAV2/8 vector encoding a sequence optimised canine micro-dystrophin transgene, driven by a muscle-synthetic Spc512 promoter (rAAV2/8-Spc512-µDys) in a total of 12 Golden Retriever Muscular Dystrophy (GRMD) dogs, the canine model of DMD. Isolated limb perfusion studies using a single administration of vector induced high levels of micro-dystrophin expression in the treated limb (up to 90% dystrophin positive fibres) with significant normalisation of histological, NMR imaging and spectroscopy parameters and muscle strength, without deleterious immune responses. Similarly, single-dose intravascular delivery of the same rAAV2/8-Spc512-µDys, in absence of immunosuppression, led to long-term transduction of distant muscle groups and extended lifespan (up to 2 years). Profound improvement of multiple clinical features was observed, including gait and respiratory parameters and no toxicity or deleterious humoral and/or cell-mediated immune responses were observed. This study demonstrates the safety and long term efficacy of rAAV2/8-Spc5.12-µDys gene therapy in a relevant large-animal models of DMD and paves the way towards human clinical gene therapy using systemic peripheral vein administration of vector, and applicable to all DMD patients regardless of their genotype.

Recombinant adeno-associated virus (rAAV)-based vectors are promising tools for the treatment of Duchenne muscular dystrophy (DMD) by gene therapy. Following rAAV injection in murine and canine models of DMD, several groups have reported significant phenotype improvements without notable toxicity, raising hope for future clinical translations. However, the long term maintenance of therapeutic benefits is an important, and yet unresolved issue. In previous studies conducted in DMD mice, we and others have demonstrated that rAAV-mediated transgene expression decreases progressively, even when clinically relevant vector doses are injected. Our team described several “restriction” factors having a negative impact on rAAV transduction, notably a loss of vector genomes resulting from muscle cell necrosis but also an oxidative damage affecting transgene mRNA. These first results support the fact that the tissue context in which rAAV vectors are delivered is of critical importance and can significantly affect their efficiency. In addition, they open new avenues for improvement since we can now consider counteracting these restriction phenotypes prior to rAAV injection. In the case of DMD, oxidative stress seems to occupy a central position in both muscle cell pathophysiology and rAAV transgene mRNA degradation. Therefore, we designed an innovative strategy using a relevant antioxidant agent routinely used in human medicine: N-acetylcysteine. DMD mice, pre-treated or not with this compound, were subsequently injected with a rAAV vector carrying a reporter transgene. The transduction efficiency, together with the expression and activity of the transgene, were carefully monitored and compared two months later. The outcome of this innovative approach will certainly pave the way for future combinatorial protocols using pharmacological agents and rAAV vectors in DMD muscles.

Like cellular transplantation, gene therapy is often limited by immune rejection of the newly expressed antigen. In a model of gene transfer in muscle, delivery of the influenza hemagglutinin (HA) membrane protein by adeno-associated virus (AAV) is impaired by a strong immune response that leads to a rapid rejection of the transduced fibers. We show here that injection of HA-specific CD4+CD25+ T cells from T-cell receptor (TCR)-transgenic animals, concomitant with gene transfer, down-regulates the anti-HA cytotoxic and B-lymphocyte responses and enables persistent HA expression in muscle. This demonstrates for the first time that adoptive transfer of antigen-specific CD4+CD25+ regulatory T cells can be used to induce sustained transgene engraftment in solid tissues. (Blood. 2003;102:4326-4328)

Wiskott-Aldrich Syndrome (WAS) is a life-threatening X-linked disease characterized by immunodeficiency, thrombocytopenia, autoimmunity, and malignancies. Gene therapy could represent a therapeutic option for patients lacking a suitable bone marrow (BM) donor. In this study, we analyzed the long-term outcome of WAS gene therapy mediated by a clinically compatible lentiviral vector (LV) in a large cohort of was(null) mice. We demonstrated stable and full donor engraftment and Wiskott-Aldrich Syndrome protein (WASP) expression in various hematopoietic lineages, up to 12 months after gene therapy. Importantly, we observed a selective advantage for T and B lymphocytes expressing transgenic WASP. T-cell receptor (TCR)-driven T-cell activation, as well as B-cell's ability to migrate in response to CXCL13, was fully restored. Safety was evaluated throughout the long-term follow-up of primary and secondary recipients of WAS gene therapy. WAS gene therapy did not affect the lifespan of treated animals. Both hematopoietic and non-hematopoietic tumors arose, but we excluded the association with gene therapy in all cases. Demonstration of long-term efficacy and safety of WAS gene therapy mediated by a clinically applicable LV is a key step toward the implementation of a gene therapy clinical trial for WAS. Wiskott-Aldrich Syndrome (WAS) is a life-threatening X-linked disease characterized by immunodeficiency, thrombocytopenia, autoimmunity, and malignancies. Gene therapy could represent a therapeutic option for patients lacking a suitable bone marrow (BM) donor. In this study, we analyzed the long-term outcome of WAS gene therapy mediated by a clinically compatible lentiviral vector (LV) in a large cohort of was(null) mice. We demonstrated stable and full donor engraftment and Wiskott-Aldrich Syndrome protein (WASP) expression in various hematopoietic lineages, up to 12 months after gene therapy. Importantly, we observed a selective advantage for T and B lymphocytes expressing transgenic WASP. T-cell receptor (TCR)-driven T-cell activation, as well as B-cell's ability to migrate in response to CXCL13, was fully restored. Safety was evaluated throughout the long-term follow-up of primary and secondary recipients of WAS gene therapy. WAS gene therapy did not affect the lifespan of treated animals. Both hematopoietic and non-hematopoietic tumors arose, but we excluded the association with gene therapy in all cases. Demonstration of long-term efficacy and safety of WAS gene therapy mediated by a clinically applicable LV is a key step toward the implementation of a gene therapy clinical trial for WAS.

Long-term correction of the disease phenotype following gene therapy in vivo with adeno-associated virus (AAV) vectors has been demonstrated in several preclinical and clinical studies. With clinical development, however, it has become obvious that the host immune system represents an important obstacle to safe and effective gene transfer with AAV vectors. Indeed T cell responses directed against the viral capsid can have a detrimental effect on the duration of transgene expression and can result in tissue-specific toxicity due to the clearance of vector infected cells. ELISpot and flow cytometry analysis of immune responses to the vector capsid in peripheral blood mononuclear cells (PBMCs) show that both CD4+ and CD8+ T cells reactive to the AAV capsid are detectable in peripheral blood following gene transfer in humans, however little is known about T cell clonality following gene transfer in PBMC vs. locally in the tissue in which the vector is delivered.Here we used TCRbeta deep sequencing of T cells infiltrating muscle injected with AAV vectors and correlated results with a time course of sequencing of TCRbeta performed in fractionated CD8+ and CD8- PBMC from subjects injected with an AAV1 vector expressing γ-sarcoglycan (AAV1-γ-sarco). PBMC and muscle samples from three subjects who received an intramuscular injection of 4.5×1010 vector genomes of AAV1-γ-sarco (Brain 2012; 135:483) were analyzed. In one of these subjects, AAV capsid IFN-γ ELISpot on PBMCs showed activation of T cells responses peaking at day 30 post gene delivery. Consistent with this finding, TCRbeta sequencing of sorted CD8+ T cells showed the de novo expansion of T cell clones in PBMC after gene transfer, which were detectable at day 30 and 180 but not at baseline before vector administration. Additional CD8+ T cell clones present at baselines also expanded between day 0 and day 180. Interestingly, CD8+ T cell clones that expanded in PBMC following gene transfer were not found in biopsies of injected muscle collected at day 30. Conversely, several T cell clones shared between muscle and more in PBMC CD8+ than CD8- population did not expand upon AAV vector injection, possibly representing the underlying inflammation of muscle characteristic of the disease. Finally, we found T cell clones over-represented in muscle following AAV injection compared to PBMC, suggesting specific differences in homing of T cells in different body tissues. For example in one subject, we observed a clear expansion of T cell clones in muscle at day 30 post vector administration compared to baseline; these clones were not detected in the CD8+ T cell compartment in peripheral blood.In conclusion, this work addresses the crucial issue of correlation of T cell responses to AAV in peripheral blood vs. the transduced tissue. Our results suggest that T cell reactivity in peripheral blood does not necessarily represent the state of inflammation in the injected tissue, at the site of antigen presentation.

The mouse mdr1a and mdr1b genes are expressed in skeletal muscle, though their precise role in muscle is unknown. Dystrophic muscle is characterized by repeated cycles of degeneration and regeneration. To explore the role of the mdr1 genes during muscle regeneration, we have created a triple knockout mouse lacking the mdr1a, mdr1b, and the dystrophin genes. The resulting ReX mice developed normally and were fertile. However, as adults, ReX had a higher proportion of degenerating muscle fibers and greater long-term loss of muscle mass than mdx. ReX muscles were also characterized by a reduced proportion of muscle side population (mSP) cells, of myogenic cells, and a reduced capacity for muscle regeneration. We found too that mSP cells derived from dystrophic muscle are more myogenic than those from normal muscle. Thus, in dystrophic muscle, the mdr1 gene plays an important role in the preservation of the mSP and of the myogenic regenerative potential. Moreover, our results suggest a hitherto unappreciated role of mdr1 in precursor cells of regenerating tissue; they therefore provide an important clue to the physiological significance of mdr1 expression in stem cells.

It has been suggested that anergic T cells may not be only inert cells but may rather play an active role, for example by regulating immune responses. We have previously reported the existence of “anergic” IL-10-producing CD4+T cells generatedin vivoby continuous antigenic stimulation. Using a gene transfer system where the antigen recognized by such T cells is expressed in skeletal muscle by two different DNA viral vectors, we show that these cells not only remain tolerant toward their cognate antigen but also can suppress the immune response of naïve T cells against the immunogenic adenoviral proteins. Furthermore, they can completely inhibit tissue destruction that takes place as a result of an immune response. The system presented here is unique in that the T cells have been anergizedin vivo, their antigen specificity and functional status are known, and the amount, form, and timing of antigen expression can be manipulated. This model will therefore permit us to carefully dissect the mechanisms by which these anergic T cells regulate the priming and/or effector function of naïve T cells.

Vectors derived from the adeno-associated virus (AAV) have been successfully used for the long-term expression of therapeutic genes in animal models and patients. One of the major advantages of these vectors is the absence of deleterious immune responses following gene transfer. However, AAV vectors, when used in vaccination studies, can result in efficient humoral and cellular responses against the transgene product. It is therefore important to understand the factors which influence the establishment of these immune responses in order to design safe and efficient procedures for AAV-based gene therapies. We have compared T-cell activation against a strongly immunogenic protein, the influenza virus hemagglutinin (HA), which is synthesized in skeletal muscle following gene transfer with an adenovirus (Ad) or an AAV vector. In both cases, cellular immune responses resulted in the elimination of transduced muscle fibers within 4 weeks. However, the kinetics of CD4 + T-cell activation were markedly delayed when AAV vectors were used. Upon recombinant Ad (rAd) gene transfer, T cells were activated both by direct transduction of dendritic cells and by cross-presentation of the transgene product, while upon rAAV gene transfer T cells were only activated by the latter mechanism. These results suggested that activation of the immune system by the transgene product following rAAV-mediated gene transfer might be easier to control than that following rAd-mediated gene transfer. Therefore, we tested protocols aimed at interfering with either antigen presentation by blocking the CD40/CD40L pathway or with the T-cell response by inducing transgene-specific tolerance. Long-term expression of the AAV-HA was achieved in both cases, whereas immune responses against Ad-HA could not be prevented. These data clearly underline the importance of understanding the mechanisms by which vector-encoded proteins are recognized by the immune system in order to specifically interfere with them and to achieve safe and stable gene transfer in clinical trials.

Molecular Therapy (2009); 17: 1073–1082. doi:10.1038/mt.2009.31 Following the publication of this article, the authors noted that Dr. Liabeuf's affiliation is incorrect. Her correct affiliation is Genethon, Evry, France.