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International audience; X-linked myotubular myopathy (XLMTM) is a severe congenital disorder in male infants that leads to generalized skeletal muscle weakness and is frequently associated with fatal respiratory failure. XLMTM is caused by loss-of-function mutations in the MTM1 gene, which encodes myotubularin, the founder member of a family of 15 homologous proteins in mammals. We recently demonstrated the therapeutic efficacy of intravenous delivery of rAAV vectors expressing MTM1 in animal models of myotubular myopathy. Here, we tested whether the closest homologues of MTM1, MTMR1, and MTMR2 (the latter being implicated in Charcot-Marie-Tooth neuropathy type 4B1) are functionally redundant and could represent a therapeutic target for XLMTM. Serotype 9 recombinant AAV vectors encoding either MTM1, MTMR1, or MTMR2 were injected into the tibialis anterior muscle of Mtm1-deficient knockout mice. Two weeks after vector delivery, a therapeutic effect was observed with Mtm1 and Mtmr2, but not Mtmr1; with Mtm1 being the most efficacious transgene. Furthermore, intravenous administration of a single dose of the rAAV9-Mtmr2 vector in XLMTM mice improved the motor activity and muscle strength and prolonged survival throughout a 3-month study. These results indicate that strategies aiming at increasing MTMR2 expression levels in skeletal muscle may be beneficial in the treatment of myotubular myopathy.

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.

International audience; Several approaches have been developed for gene therapy in RPE65-related Leber congenital amaurosis. To date, strategies that have reached the clinical stages rely on adeno-associated viral vectors and two of them documented limited long-term effect. We have developed a lentiviral-based strategy of RPE65 gene transfer that efficiently restored protein expression and cone function in RPE65-deficient mice. In this study, we evaluated the ocular and systemic tolerances of this lentiviral-based therapy (LV-RPE65) on healthy nonhuman primates (NHPs), without adjuvant systemic anti-inflammatory prophylaxis. For the first time, we describe the early kinetics of retinal detachment at 2, 4, and 7 days after subretinal injection using multimodal imaging in 5 NHPs. We revealed prolonged reattachment times in LV-RPE65-injected eyes compared to vehicle-injected eyes. Low- (n = 2) and high-dose (n = 2) LV-RPE65-injected eyes presented a reduction of the outer nuclear and photoreceptor outer segment layer thickness in the macula, that was more pronounced than in vehicle-injected eyes (n = 4). All LV-RPE65-injected eyes showed an initial perivascular reaction that resolved spontaneously within 14 days. Despite foveal structural changes, full-field electroretinography indicated that the overall retinal function was preserved over time and immunohistochemistry identified no difference in glial, microglial, or leucocyte ocular activation between low-dose, high-dose, and vehicle-injected eyes. Moreover, LV-RPE65-injected animals did not show signs of vector shedding or extraocular targeting, confirming the safe ocular restriction of the vector. Our results evidence a limited ocular tolerance to LV-RPE65 after subretinal injection without adjuvant anti-inflammatory prophylaxis, with complications linked to this route of administration necessitating to block this transient inflammatory event.

International audience

Body: Mutations in MTM1 result in X-linked myotubular myopathy (XLMTM), a rare neuromuscular disease that causes devastating muscular weakness in affected children, often fatal in the first years of life. A naturally occurring MTM1 mutation in Labrador retrievers provides unprecedented opportunity to assess MTM1 gene replacement strategies. Here, we report clinical and physiological outcomes more than 3 years after a single intravenous infusion of AAV8-MTM1 in two dogs. Readouts include neurological assessment, limb strength, gait, respiratory function, and histology. Results (Fig 1Fig 1) are shown in comparison to age-matched littermates, and indicate that a single infusion of AAV8-MTM1 can markedly prolong life and achieve near normal levels of neurological function, strength, gait, and respiratory function for more than 3 years in MTM1 mutant dogs.View Large Image | Download PowerPoint Slide

Myotonic dystrophy type 1 (DM1) is an autosomal dominant muscular disease caused by an expansion of the CTG trinucleotide repeat located in the 3’ untranslated region (3’ -UTR) of the DMPK gene. The size of the expansion correlates with disease severity and to date there is no effective treatment for patients. Mutated DMPK transcripts aberrantly interact with proteins in the nucleus and form stable complexes where splicing factors are sequestered. As a consequence, the alternative splicing of numerous transcripts is pathogenically misregulated.We aim at developing a strategy to delete the CTG expansion from the human DMPK locus by using the CRISPR/Cas9 system. We have generated several constructs which express a small size Cas9 nuclease and guide-RNAs (gRNAs), and show their ability to excise the targeted DNA region in various cell lines. We are currently producing adeno-associated vectors for in vivo delivery of these constructs in a DM1 mouse model.

X-linked severe combined immunodeficiency (SCID-X1, OMIM #300400) is caused by mutations in the gene encoding the interleukin-2 receptor γ-chain (IL2RG) resulting in a lack of response to common γ-chain-dependent cytokines, 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 decided to developed a SIN lentiviral-vector (LV) carrying a codon-optimized human IL2RG cDNA under the control of the human EF1α-S promoter, including a mutated WPRE. The vector was VSV-G-pseudotyped and produced by a new manufacturing process based on quadri-transfection of suspension-adapted 293T cells grown in bioreactors in serum-free conditions, purified by ion-exchange chromatography and concentrated by tangential-flow filtration. The activity of the LV was tested in vitro by transduction of the IL2RG-deficient EDR7 human T-cell line and cord blood-derived or peripheral blood mobilized CD34+ hematopoietic progenitors. The LV reconstituted normal levels of surface IL2RG in ED7R cells at an average VCN of 2 and 90% transduction efficiency. Transduction of CD34+ cells resulted in a VCN of 2.8±0.7 at a transduction efficiency of 82±9% by CFC assay with no impact on viability and cloning efficiency. Induction of aberrant splicing of genes harboring LV proviruses was assayed in EDR7 cell clones in which LV integrations were characterized by LM-PCR and sequencing. The LV caused abnormal splicing at low frequency between upstream exons and constitutive or cryptic proviral splice sites located in the LTR or at internal positions. To test the therapeutic efficacy and potential genotoxicity of the LV, we transplanted lethally-irradiated IL2RG-deficient mice with BM-derived, lin- cells transduced at 72±15% efficiency with a VCN of 2.5±1.1. Transplanted mice are being tested at 3 and 6 months post-transplantation for restoration of T- and B-cell function. Integration site analysis will be performed to monitor LV integration and analyze the clonal repertoire of transduced BM and peripheral blood cells. Potential genotoxicity is also assayed by an in vitro immortalization (IVIM) study on primary cells. These studies will enable a multicenter phase-I/II/registration clinical trial aimed at establishing the safety and clinical efficacy of lentiviral vector-mediated gene therapy for SCID-X1.

Myotonic dystrophy type 1 (DM1) belongs to the group of nucleotide repeat disorders. More specifically this autosomal form of muscular dystrophy is caused by the expansion of the CTG trinucleotide repeat located at the 3’ untranslated region (3’-UTR) of the DMPK gene. Elongated CUG repeats of the mutated DMPK mRNAs become sequestration sites for splicing factors, and induce the formation of stable ribonucleoprotein complexes visualized as foci. As a consequence, the alternative splicing of numerous transcripts is dysregulated, which leads to the DM1 pathological alterations affecting various tissues. We have developed a strategy to delete the CTG repeat expansion in the human DMPK locus by using the CRISPR/Cas9 system. For that purpose, we constructed different expression platforms for small size Cas9 nucleases under either a ubiquitous or a muscle-specific promoter and guide RNAs (sgRNAs) targeting the 3’-UTR of the DMPK gene. Co-transfection of these constructs in DM1 primary myoblasts resulted in the deletion of the CTG repeat. We are currently optimizing the delivery of these Cas9 and sgRNAs constructs in cells by their vectorization in lentiviral and adeno-associated vectors and will present data on in vitro (DM1 patient-derived cell lines) and in vivo (wild-type mice) assays.

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; We investigated the efficacy of liver-directed gene therapy using lentiviral vectors in a large animal model of hemophilia B and evaluated the risk of insertional mutagenesis in tumor-prone mouse models. We showed that gene therapy using lentiviral vectors targeting the expression of a canine factor IX transgene in hepatocytes was well tolerated and provided a stable long-term production of coagulation factor IX in dogs with hemophilia B. By exploiting three different mouse models designed to amplify the consequences of insertional mutagenesis, we showed that no genotoxicity was detected with these lentiviral vectors. Our findings suggest that lentiviral vectors may be an attractive candidate for gene therapy targeted to the liver and may be potentially useful for the treatment of hemophilia.

Myotubular myopathy (XLMTM) is a fatal pediatric disease of skeletal muscle due to mutations in the MTM1 gene. Patients with XLMTM typically present with generalized muscle weakness and respiratory failure. We have previously demonstrated the efficacy of AAV-mediated MTM1 gene therapy in animal models. In order to express MTM1 preferentially in skeletal muscles after systemic gene delivery, we have constructed AAV vectors that contain the Mtm1 cDNA under the potent desmin promoter and a miRNA-208a target sequence for cardiac detargeting. We show that intravenous delivery of this vector leads to myotubularin expression in skeletal muscles scattered throughout the body, including the diaphragm, and reduced expression in heart. We have selected one of these vectors for gene therapy in XLMTM mice and show the results in survival, pathology and contractile force. We also analyzed the long-term effect of the transgene in miRNA-208a level and gene expression in heart.

International audience; Abstract Human immunodeficiency virus type 1-derived lentiviral vectors (LVs) are becoming major tools for gene transfer approaches. Several gene therapy clinical studies involving LVs are currently ongoing. Industrial production of clinical-grade LVs is therefore an important challenge. Some improvements in LV production protocols have already been possible by acting on multiple steps of the production process like transfection, cell culture, or media optimizations. Yet, the effects of physicochemical parameters such as pH remain poorly studied. Mammalian cell cultures are generally performed at neutral pH, which may not be the optimal condition to produce high quantities of LVs with optimal infectious properties. In this study, we showed that lentiviral transient production in HEK293T cells is inversely dependent on the pH value of the harvesting medium. Infectious and physical titers of LVs pseudotyped with GALVTR or VSV-G glycoproteins are enhanced by two- to threefold at pH 6 compared with neutral conditions. pH 6-produced LVs are highly infectious on cell lines but also on relevant primary target cells like hCD34+ hematopoietic stem/progenitor cells. GALVTR-LV particles produced at pH 6 are highly stable at 37°C and resistant to multiple freeze-thaw cycles. Higher levels of expression of intracellular pr55gag polyproteins are observed within HEK293T producer cells cultured at pH 6. The positive effect of pH 6 conditions is also observed for moloney-derived retroviral vectors produced from NIH-3T3 fibroblasts, arguing that the mildly acidic pH effect is not limited to the lentivirus genus and is reproducible in various producer cell lines. This observation may help us in the design of more effective LV production protocols for clinical applications.

Crigler-Najjar syndrome (CN) is an autosomal recessive rare disorder caused by mutations in the UDP-glucuronosyltransferase 1 isotype A1 (UGT1A1) gene. In severe CN, lack or reduced activity of UGT1A1 results in high levels of serum unconjugated bilirubin (UCB), which can lead to brain damage and death. Treatment of CN consists of phototherapy for 10-12 hours per day to convert UCB into soluble photoisomers without the need of conjugation, which presents several limitations and has a major impact on life quality of the patients. Liver transplantation is the only curative option for CN. The limited therapeutic options available prompted us to develop a new therapy for CN based on the transfer of a corrected copy of the UGT1A1 gene to hepatocytes.We developed an AAV8 vector optimized for the liver expression of the hUGT1A1 transgene (AAV8-hUGT1A1). Safety and efficacy of correction of total serum bilirubin (TB) levels with AAV-hUGT1A1 were demonstrated in mouse and rat models of CN at vector doses as low as 5×10 11 vector genomes (vg)/kg, a result confirmed also by the detection of conjugated bilirubin in bile of treated rats. In juvenile CN rats, long-term correction of total bilirubin levels were observed for more than 8 months following AAV8-hUGT1A1 gene transfer. In neonate Ugt1a1–/– mice, intraperitoneal delivery of the vector at doses as low as 1×10 9 vg/mouse resulted in correction of TB at 4 weeks. However, vector delivery in P2 and P4 animals resulted in lower efficiency of correction when compared with P11-transduced mice, a finding likely due to the more advanced development of the liver at later time points.Based on these data, dose finding studies were performed in CN rats, which showed a profile of liver transduction with AAV8 vectors similar to humans, i.e. lower efficiency of transduction than mice. No differences in efficacy of correction of TB were observed in male vs. female rats.A scalable process for the production of AAV8-hUGT1A1 production was established based on a protocol of triple transfection of suspension HEK293 cells, leading to high yield vector preparations with excellent purity profile. In vitro transduction assays in human hepatocytes based on Western blot for hUGT1A1 protein and in vivo studies in CN rats demonstrated that vectors produced with the suspension process have potency characteristics undistinguishable from research grade vectors produced by triple transfection of adherent cells and purified by cesium chloride gradient centrifugation.In conclusion, our data demonstrate the safety and efficacy of gene transfer for Crigler-Najjar syndrome in two relevant animal models of the disease and provide tools and a strong rationale for the translation of these results in human subjects.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in the survival-motor-neuron 1 (SMN1) telomeric gene. Deficiencies in the ubiquitous SMN function affect multiple tissues and organs; however neuronal tissue is primarily sensitive, resulting in α-motor neuron degeneration in the ventral horn of the spinal cord with subsequent neuromuscular-junction dysfunction and proximal muscle weakness. The onset of disease and degree of severity are variable in patients and they are determined in part by multiple copies of the centromeric homologue SMN2 that inversely correlate with the phenotypic severity. Indeed, SMN2 gene mainly produces a truncated form SMNΔ7 by aberrant alternative splicing and a small amount (~10%) of the fully active full-length SMN, thus buffering the SMN deficiency. A potential strategy for treating SMA patients is to increase SMN levels in the affected tissues, hence gene therapy and modifiers of SMN2-alternative splicing have proved therapeutic efficacy in SMA animal models.In this study, we explored the possibility of applying targeted genome editing technology to the human SMN locus in order to revert the SMN2 sequence to a SMN1-like sequence that may undergo proper splicing under the the endogenous transcriptional control. The resulting correction would be permanent and lead to longlasting protein production in gene-edited cells. We used the streptococcus pyogenes Cas9-CRISPR system to target the SMN2 gene at different locations. Two main strategies were explored: i) SMN1_exon7 addition/correction by promoting homology-driven DNA repair, ii) SMN2_intron7_ intronic-splicing-silencer (ISS-N1)mutation and correction of SMN2 aberrant splicing, by exploiting the non-homologous end-joining (NHEJ) pathway. Plasmids encoding Cas9-GFP under the control of CMV promoter, and selected gRNAs downstream to the Pol-III U6 promoter (Addgene) were transfected in HEK-293T cell line and in immortalized myoblasts derived from either healthy donors or SMA patients. Transfection efficiency was estimated as percentage of GFP-expressing cells (20-50% and 1-10%, respectively) and nuclease activity detected by Surveyor assay and target site sequencing. In particular, in SMA patient-derived myoblasts we detected mutations (indels) at the level of the induced DNA double-strand break at ~30% frequency. Levels of SMN restoration will be investigated by qPCR of the different species of SMN transcripts and by western blotting of SMN protein. The goal of this study is to provide an in vitro proof of principle of effective gene correction in SMA patient-derived cells. In the context of a multisystemic, complicated disease such as SMA, targeted genome editing strategy could represent an additional therapeutic tool.

International audience; Loss-of-function mutations in the myotubularin gene (MTM1) cause X-linked myotubular myopathy (XLMTM), a fatal, congenital pediatric disease that affects the entire skeletal musculature. Systemic administration of a single dose of a recombinant serotype 8 adeno-associated virus (AAV8) vector expressing murine myotubularin to Mtm1-deficient knockout mice at the onset or at late stages of the disease resulted in robust improvement in motor activity and contractile force, corrected muscle pathology, and prolonged survival throughout a 6-month study. Similarly, single-dose intravascular delivery of a canine AAV8-MTM1 vector in XLMTM dogs markedly improved severe muscle weakness and respiratory impairment, and prolonged life span to more than 1 year in the absence of toxicity or a humoral or cell-mediated immune response. These results demonstrate the therapeutic efficacy of AAV-mediated gene therapy for myotubular myopathy in small- and large-animal models, and provide proof of concept for future clinical trials in XLMTM patients.

Myotubular myopathy (XLMTM) is a severe congenital disease that affects skeletal musculature, which is characterized by the presence of small myofibers with frequent occurrence of central nuclei. The disease is due to mutations in the MTM1 gene, encoding a phosphoinositide phosphatase named myotubularin, and specific treatment is currently unavailable. We have previously demonstrated the efficacy of local administration of AAV vectors carrying the Mtm1 cDNA to treat the disease, and have more recently extended these studies to the whole body. In order to express MTM1 preferentially in skeletal muscles after systemic gene delivery, we have constructed several AAV vectors that contain the Mtm1 cDNA under the potent desmin promoter and miRNA target sequences for cardiac detargeting. We show that intravenous delivery of these vectors leads to myotubularin expression in skeletal muscles scattered throughout the body, including the diaphragm, and reduced expression in heart. We have selected one of these vectors for gene therapy in XLMTM mice and show the results in survival, pathology and contractile force. We demonstrate that this strategy is very efficient to drive selected expression of transgenes in skeletal muscles, avoiding potential side effects in heart, and is useful for the treatment of disorders that mainly affect the skeletal musculature, such as myotubular myopathy.

International audience; Lentiviral vectors (LVs) are promising delivery systems for gene therapy. To enhance the efficiency of target cell transduction by LVs, protocols often include the addition of culture additives. In this study, the cationic amphipathic peptide LAH4-L1 (KKALLAHALHLLALLALHLAHALKKA), a DNA transfection agent, was evaluated for its capacity to improve LV infectivity in cell lines and primary cells. Results show that LAH4-L1 enhances infectivity of all LV pseudotypes tested, particularly GALVTR-LVs. More importantly, LAH4-L1 promotes the transduction of CD34+ hematopoietic stem cells with GALVTR-LVs as efficiently as Retronectin, a culture additive used in ex vivo clinical protocols involving LVs. The action of LAH4-L1 relies both on the GALVTR-LV adhesion and post-adhesion steps. LAH4-L1 represents a new and attractive transduction enhancer for hematopoietic gene therapy protocols.

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.

Patients with mutations in UNC13D gene, coding for Munc13-4 protein, suffer from type 3 Familial hemophagocytic lymphohistiocytosis (FHL3), a life-threatening disorder of the immune system which represents 25% of all FHLs. Munc13-4 controls docking of lytic granules before they fused with the plasma membrane in cytotoxic T and NK lymphocytes and it's defect results in defective cytotoxic function of these cells. Hematopoietic stem and progenitor cell (HSPC) transplantation, which is the only curative treatment for FHL3 to date, is partially successful even when a compatible donor is available because of the important inflammatory background of patients. In this context gene therapy could be a promising therapeutic option especially for those patients without any compatible donor. In this study, we took advantages from a murine model of FHL3, the Jinx mice, to investigate the feasibility of HSPC gene therapy for this pathology. Jinx mice do not spontaneously develop clinical features of hemophagocytic lymphohistiocytosis (HLH), but do so when infected with lymphocytic choriomeningitis virus (LCMV). We generated and used a self-inactivated lentiviral vector to complement HSC from Unc13d -/- (Jinx) mice and transplanted them back into the irradiated Jinx recipients. This transplantation led to the complete reconstitution of the immune system at levels comparable to that of control mice. The recipients were then challenged with LCMV. While Jinx mice reconstituted with GFP expressing HSPC developed leukopenia, anemia and body weight loss, characteristic of HLH in this murine model, gene corrected Jinx recipients developed only mild or no HLH manifestations. This reduction in HLH manifestation correlated with a significant reduction of virus titer in the liver and serum level of IFN-g and inflammatory cytokines. All these ameliorations might be explained by the restoration of cytotoxic function of CTLs as demonstrated in an in-vitro degranulation assay. Overall, this study provides data supporting the potential of HSC gene therapy in a FHL immune dysregulation such as UNC13D deficiency.

IPEX (Immunodysregulation Polyendocrinopathy Enteropathy X-linked) syndrome is the prototype of primary immunodeficiency with prevailing autoimmunity. The disease is caused by mutations in the gene encoding the transcription factor forkhead box P3 (FOXP3), which leads to the loss of function of thymus-derived CD4+CD25+ regulatory T (tTreg) cells. In IPEX patients, the absence of a functional Treg cell compartment leads to the development of multiple autoimmune manifestations (including severe enteropathy, type 1 diabetes and eczema) usually in the first months or years of life. The current treatments for IPEX syndrome include immunosuppressive, hormone replacement therapies. Unfortunately, immunosuppressive treatments are usually only partially effective and their dose is often limited because of the occurrence of infectious complications and toxicity. Currently, the only curative treatment for IPEX syndrome is allogeneic hematopoietic stem cell transplantation (HSCT). The absence of an HLA-compatible donor for all patients and their poor clinical condition particularly expose them to a risk of mortality when HLA partially compatible donors are used. For all these reasons, effective alternative therapeutic approaches are urgently needed. Various preclinical studies have shown that partial donor chimerism is sufficient for complete remission meaning that a small number of functional natural Treg is sufficient to restore immune tolerance. This suggests that T cell gene therapy approaches designed to selectively restore the repertoire of Treg cells is a promising potential cure for IPEX. We demonstrated that FOXP3 gene transfer into CD4+ T cells from IPEX patients enable the generation of potent and stable regulatory T cells. Indeed we showed the functional suppressive properties of the generated CD4IPEX-FOXP3 cells in an optimized flow-cytometry-based in vitro suppression assay. We are currently comparing the transcriptional profile of these regulatory CD4IPEX-FOXP3 cells in comparison to natural Treg by RNA-seq analysis. Therefore, the introduction of a functional copy of the FOXP3 gene into an IPEX patient's T cells may be enough to restore immune tolerance and thus avoid the complications of allogenic HSCT. We will also discuss the challenge of generating a large, homogenous and stable population of cells in vitro for adoptive transfer and whether it can ensure long-term disease correction without generating a context of generalized immunosuppression.

MTM1 gene mutations cause X-linked myotubular myopathy (XLMTM), a fatal congenital myopathy characterized by small myofibers with frequent central nuclei. XLMTM patients typically present with severe hypotonia, muscle weakness and respiratory failure. Previous intramuscular (IM) injection studies in Mtm1 KO mice demonstrated potential efficacy of gene therapy to treat the disease. We extended these results to a large animal model by testing IM delivery of an AAV8-canine MTM1 vector into the cranial tibialis muscle of dogs with XLMTM. We observed dramatic improvement in strength of treated limb muscles to levels approaching that of age-matched normal littermates. This response was detectable at 1 week post-injection and improvement continued until a terminal measurement at 6 weeks post-injection. Concomitantly, the AAV-injected XLMTM muscles showed substantial increase in mass and myofiber size, and decreases in pathological features. We also utilized the Mtm1 KO mouse to determine the response to systemic intravenous delivery of an AAV serotype 9 vector expressing myotubularin under the muscle-specific desmin promoter. Myotubularin was rapidly and persistently expressed in muscles scattered throughout the body and this translated into robust improvement of skeletal muscle pathology and contractile force, and normalized motor activity of treated mice. Importantly, the lifespans of treated mice, which normally survive ∼7–8 weeks, were prolonged to >6 months. We then evaluated the effects of a targeted protein-replacement agent (3E10Fv-MTM1) injected into the tibialis anterior muscle of Mtm1 KO mice. Injection of 3E10Fv-MTM1 over 2 weeks increased contractile function (p

A major impediment to the use of adeno-associated virus (AAV)-mediated gene delivery to muscle in clinical applications is the pre-existing immune responses against the vector. Pre-existing humoral response to different AAV serotypes is now well documented. In contrast, cellular responses to AAV capsid have not been analyzed in a systematic manner, despite the risk of T cell reactivation upon gene transfer. AAV1 has been widely used in humans to target muscle. In this study, we analyzed PBMCs and sera of healthy donors for the presence of AAV1 capsid-specific T cell responses and AAV1 neutralizing factors. Approximately 30% of donors presented AAV1 capsid-specific T cells, mainly effector memory CD8+ cells. IFN-γ–producing cells were also observed among effector memory CD4+ cells for two of these donors. Moreover, to our knowledge, this study shows for the first time on a large cohort that there was no correlation between AAV1-specific T cell and humoral responses. Indeed, most donors presenting specific Ig and neutralizing factors were negative for cellular response (and vice versa). These new data raise the question of prescreening patients not only for the humoral response, but also for the cellular response. Clearly, a better understanding of the natural immunology of AAV serotypes will allow us to improve AAV gene therapy and make it an efficient treatment for genetic disease.

Background Gene modified dendritic cells (DC) are able to modulate DC functions and induce therapeutic immunity or tolerance in an antigen-specific manner. Among the different DC subsets, plasmacytoid DC (pDC) are well known for their ability to recognize and respond to a variety of viruses by secreting high levels of type I interferon. Methods We analyzed here, the transduction efficiency of a pDC cell line, GEN2.2, and of pDC derived from CD34+ progenitors, using lentiviral vectors (LV) pseudotyped with different envelope glycoproteins such as the vesicular stomatitis virus envelope (VSVG), the gibbon ape leukaemia virus envelope (GaLV) or the feline endogenous virus envelope (RD114). At the same time, we evaluated transgene expression (E-GFP reporter gene) under the control of different promoters. Results We found that efficient gene transfer into pDC can be achieved with VSVG-pseudotyped lentiviral vectors (LV) under the control of phoshoglycerate kinase (PGK) and elongation factor-1 (EF1α) promoters (28% to 90% of E-GFP+ cells, respectively) in the absence of phenotypic and functional maturation. Surprisingly, promoters (desmin or synthetic C5–12) described as muscle-specific and which drive gene expression in single strand AAV vectors in gene therapy protocols were very highly active in pDC using VSVG-LV. Conclusion Taken together, our results indicate that LV vectors can serve to design pDC-based vaccines in humans, and they are also useful in vitro to evaluate the immunogenicity of the vector preparations, and the specificity and safety of given promoters used in gene therapy protocols.

International audience

International audience

International audience; Calpainopathy (limb-girdle muscular dystrophy type 2A, LGMD2A) is a recessive muscular disorder caused by deficiency in the calcium-dependent cysteine protease calpain 3. To date, no treatment exists for this disease. We evaluated the potential of recombinant adeno-associated virus (rAAV) vectors for gene therapy in a murine model for LGMD2A. To drive the expression of calpain 3, we used rAAV2/1 pseudotyped vectors and muscle-specific promoters to avoid calpain 3 cell toxicity. We report efficient and stable transgene expression in muscle with restoration of the proteolytic activity and without evident toxicity. In addition, calpain 3 was correctly targeted to the sarcomere. Moreover, its presence resulted in improvement of the histological features and in therapeutic efficacy at the physiological levels, including correction of atrophy and full rescue of the contractile force deficits. Our results establish the feasibility of AAV-mediated calpain 3 gene transfer as a therapeutic approach.

The murine Fv1 gene restricts infection by N- or B-tropic murine leukemia viruses at a postentry, preintegration stage. The Fv1- sensitive viruses previously used for the study of Fv1 encode an ecotropic envelope gene and thus only infect rodent cells. Consequently, the study of Fv1 restriction has been carried out solely in mice and murine cell lines. By infection with retroviral vectors containing N- or B-tropic core and pantropic vesicular stomatitis virus-G envelope protein, we now demonstrate that cell lines derived from various mammalian species, including humans, have an Fv1 -like retrovirus restriction function, preventing N-tropic vector infection. Like Fv1 , restriction is directed at amino acid 110 of the viral capsid protein. In contrast to Fv1 , the novel restriction is characterized by the absence of reverse-transcribed viral DNA. We speculate that these activities have been selected for by retroviral epidemics in the distant past.