Adeno associated vector-based gene therapy strategy for type 3 glycogen storage disease

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Abstracts 2017 / Neuromuscular Disorders 27 (2017) S96–S249

describe an AAV-based “silence and replace” strategy in a mouse model of OPMD where combination of complete knockdown of the endogenous PABPN1 and its replacement with a sequence optimized, wildtype PABPN1 significantly reduced the amount of INIs, decreased fibrosis, counteracted muscle atrophy, restored muscle strength and normalized the muscle transcriptome. These results support the application of a gene therapy approach as a novel treatment for OPMD in humans. http://dx.doi.org/10.1016/j.nmd.2017.06.541

TH.O.16 A new AAV10-mediated gene therapy for SOD1-linked ALS M. Biferi 1, M. Cohen-Tannoudji 1, A. Cappelletto 1, B. Giroux 1, M. Roda 1, S. Astord 1, T. Marais 1, A. Ferry 1, T. Voit 2, M. Barkats 1 1 Center of Research in Myology, Paris, France; 2 NIHR GOSH Biomedical Research Centre, UCL, London, UK Our research is devoted to the identification of efficient strategies to target the central nervous system (CNS) and to the development of novel therapies for motor neuron disorders. In particular, using the unique therapeutic potential of self-complementary adeno-associated virus (AAV) vectors, we recently elaborated a new gene therapy strategy for a genetic form of Amyotrophic Lateral Sclerosis (ALS), a lethal disease with limited therapeutic options. Approximately 20% of familial ALS cases are caused by mutations in the Superoxide Dismutase 1 (SOD1) gene and toxic gain of function of the mutant protein. Indeed, hSOD1 suppression has proven its therapeutic efficacy in several studies on transgenic animals overexpressing mutant forms of human SOD1. We tested a new method for SOD1 silencing, by inducing the skipping of a constitutive hSOD1 exon using antisense oligonucleotides (ASOs) complementary to splicing regulatory elements on the primary transcript. The resulting deleted mRNA, containing a premature stop codon, is then degraded by the endogenous cellular surveillance non-sense mediated decay pathway. We demonstrated the therapeutic potential of this exon skipping strategy in both newborn (P1) and adult (P50) SOD1G93A mice, by the administration of ASOs against hSOD1 embedded in a modified U7 small-nuclear RNA and vehiculated by AAV vectors serotype 10 (AAV10). The results obtained in this study offer new hope for treatment of this devastating genetic disease and open perspectives for a clinical development. http://dx.doi.org/10.1016/j.nmd.2017.06.542

TH.O.17 Whole-body rescue of Pompe disease with AAV liver delivery of engineered secretable GAA transgenes P. Colella 1, F. Puzzo 1, M. Biferi 2, D. Bali 3, N. Paulk 4, P. Vidal 1, F. Collaud 1, M. Simon-Sola 1, S. Charles 1, R. Hardet 2, C. Leborgne 1, P. Sellier 1, L. van Wittenberghe 1, F. Boisgerault 1, M. Barkats 2, P. Laforêt 5, M. Kay 4, D. Koeberl 3, G. Ronzitti 1, F. Mingozzi 6 1 Genethon and INSERM U951, Evry, France; 2 University Pierre and Marie Curie Paris 6 and INSERM U974, Paris, France; 3 Duke University School of Medicine, Durham, USA; 4 Stanford University, Standford, USA; 5 Paris-Est neuromuscular center, Pitié-Salpêtrière Hospital, Paris, France; 6 Genethon and INSERM U951, UPMC and INSERM U974, Evry, Paris, France Pompe disease is a severe neuromuscular disorder caused by mutations in the lysosomal enzyme acid a-glucosidase (GAA), which result in the pathological accumulation of glycogen in all tissues. Enzyme replacement therapy (ERT) is available for Pompe disease, however it has limited efficacy, high immunogenicity, and fails to correct nervous tissue and muscle groups refractory to cross-correction. Using bioinformatics analysis and protein engineering, we developed secretable GAA transgenes for enhanced crosscorrection of Pompe disease via adeno-associated virus (AAV) vector liver gene transfer. Pompe mice were treated with AAV vectors optimized for hepatic expression of secretable GAA transgenes and followed for up to 10 months.

Gene transfer resulted in dose- and time- dependent whole-body correction of biochemical and functional defects in muscle, central nervous system and spinal cord, with normalization of cardiac hypertrophy, muscle and respiratory function, and survival undistinguishable from wild-type littermates. In these experiments, secretable GAA transgenes showed superior therapeutic efficacy and markedly low immunogenicity compared with their native GAA counterpart. Scale-up to non-human primates, and modeling of GAA expression in primary hepatocytes using hepatotropic AAV vector serotypes, demonstrate the therapeutic potential of AAV vector-mediated liver expression of secretable GAA transgenes, supporting the feasibility of the approach in Pompe patients. http://dx.doi.org/10.1016/j.nmd.2017.06.543

TH.O.18 Adeno associated vector-based gene therapy strategy for type 3 glycogen storage disease P. Vidal 1, G. Ronzitti 1, F. Collaud 1, M. Simon Sola 2, P. Collela 1, F. Puzzo 1, H. Costa Verdera 2, S. Charles 1, A. Vignaud 1, L. Van Wittenberghe 1, B. Gjata 1, M. Gjorgjieva 1, P. Laforêt 3, F. Rajas 4, E. Malfatti 5, G. Comi 6, F. Mingozzi 7 1 Genethon, Evry, France; 2 UPMC, Paris, France; 3 Institut de Myologie, Paris, France; 4 Université de Lyon, Lyon, France; 5 Hopital Pitié Salpêtrière, Paris, France; 6 University of Milan, Milan, France; 7 INSERM U951, Paris, France Glycogen storage disease type III (GSDIII) is a recessive disorder due to mutations in the glycogen debranching enzyme (GDE), the enzyme involved in the linearization of cytosolic glycogen. The lack of GDE leads to glycogen accumulation in all tissues. During childhood, GSDIII is mainly a metabolic disease characterized by hepatomegaly and fasting hypoglycemia. Recurrent hypoglycemia is managed by uncooked cornstarch supplied in a strict dietary regimen. During adolescence, the metabolic manifestation becomes less prominent and muscle weakness appears. At present no curative treatment exists for GSDIII. Here, we derived a new KO mouse model of GSDIII (GDE-/-). Like in humans, the absence of GDE in mice leads to the accumulation of glycogen in muscles, liver and brain, hypoglycemia and muscle weakness. Adeno associated virus (AAV) are the vectors of choice for in vivo gene therapy. One of the limitations in the development of an AAV-based gene therapy for GSDIII is their limited packaging capacity (~5 Kb). Being the coding sequence of GDE 4.6 Kb, this transgene hardly fits into a single AAV vector. To overcome this limitation, and based on promising in vitro results, we first tested overexpression of the lysosomal enzyme acid alpha-glucosidase in GSDIII mice. However, this approach resulted only in the correction of liver glycogen accumulation. Dual vectors have been proposed to rescue muscular diseases. Following this approach, transgenes larger than 5 Kb are expressed using two different vectors each containing one portion of the transgene and a recombination region. After infection, recombination occurring between the two vectors reconstitutes the full-size transgene. Dual AAV vectors expressing GDE completely rescued the disease phenotype in GDE-/- mice. In conclusion, our work represents the first proof-of-concept study of successful gene therapy in the mouse model of GSDIII. http://dx.doi.org/10.1016/j.nmd.2017.06.544

TH.O.19 Tamoxifen increases survival, improves motor function and reduces levels of BIN1 and DNM2 in a mouse model of X-linked centronuclear (myotubular) myopathy E. Gayi 1, H. Ismail 1, B. Cowling 2, L. Neff 1, J. Laporte 2, L. Scapozza 1, O. Dorchies 1 1 University of Geneva, Geneva, Switzerland; 2 IGBMC – INSERM, CNRS, University of Strasbourg, Illkirch, France