Bernard Gjata

Successful Interference with Cellular Immune Responses to Immunogenic Proteins Encoded by Recombinant Viral Vectors

ABSTRACT 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.

Regulatory function of in vivo anergized CD4+ T 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 generated in vivo by 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 anergized in 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.

A translationally optimized AAV-UGT1A1 vector drives safe and long-lasting correction of Crigler-Najjar syndrome

Crigler-Najjar syndrome is a severe metabolic disease of the liver due to a reduced activity of the UDP Glucuronosyltransferase 1A1 (UGT1A1) enzyme. In an effort to translate to the clinic an adeno-associated virus vector mediated liver gene transfer approach to treat Crigler-Najjar syndrome, we developed and optimized a vector expressing the UGT1A1 transgene. For this purpose, we designed and tested in vitro and in vivo multiple codon-optimized UGT1A1 transgene cDNAs. We also optimized noncoding sequences in the transgene expression cassette. Our results indicate that transgene codon-optimization is a strategy that can improve efficacy of gene transfer but needs to be carefully tested in vitro and in vivo. Additionally, while inclusion of introns can enhance gene expression, optimization of these introns, and in particular removal of cryptic ATGs and splice sites, is an important maneuver to enhance safety and efficacy of gene transfer. Finally, using a translationally optimized adeno-associated virus vector expressing the UGT1A1 transgene, we demonstrated rescue of the phenotype of Crigler-Najjar syndrome in two animal models of the disease, Gunn rats and Ugt1a1-/- mice. We also showed long-term (>1 year) correction of the disease in Gunn rats. These results support further translation of the approach to humans.

Efficacy and biodistribution analysis of intracerebroventricular administration of an optimized scAAV9-SMN1 vector in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive disease of variable severity caused by mutations in the SMN1 gene. Deficiency of the ubiquitous SMN function results in spinal cord α-motor neuron degeneration and proximal muscle weakness. Gene replacement therapy with recombinant adeno-associated viral (AAV) vectors showed therapeutic efficacy in several animal models of SMA. Here, we report a study aimed at analyzing the efficacy and biodistribution of a serotype-9, self-complementary AAV vector expressing a codon-optimized human SMN1 coding sequence (coSMN1) under the control of the constitutive phosphoglycerate kinase (PGK) promoter in neonatal SMNΔ7 mice, a severe animal model of the disease. We administered the scAAV9-coSMN1 vector in the intracerebroventricular (ICV) space in a dose-escalating mode, and analyzed survival, vector biodistribution and SMN protein expression in the spinal cord and peripheral tissues. All treated mice showed a significant, dose-dependent rescue of lifespan and growth with a median survival of 346 days. Additional administration of vector by an intravenous route (ICV+IV) did not improve survival, and vector biodistribution analysis 90 days postinjection indicated that diffusion from the cerebrospinal fluid to the periphery was sufficient to rescue the SMA phenotype. These results support the preclinical development of SMN1 gene therapy by CSF vector delivery.

Longitudinal in vivo muscle function analysis of the DMSXL mouse model of myotonic dystrophy type 1

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 12 months 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.

Analysis of growth factor expression in affected and unaffected muscles of oculo-pharyngeal muscular dystrophy (OPMD) patients: A pilot study

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.

503. Adeno-Associated Virus Vector (AAV) Microdystrophin Gene Therapy Prolongs Survival and Restores Muscle Function in the Canine Model of Duchenne Muscular Dystrophy (DMD)

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.

690. Development of a Clinical Lentiviral Vector for Gene Therapy of SCID-X1

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.

390. Impact of a Treatment with Antioxidant on Gene Transfer Efficiency After Recombinant Adeno-Associated Vector Injection in a Mouse Model of Duchenne Muscular Dystrophy

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.