P.20.13 Gene therapy of Duchenne Muscular Dystrophy using rAAV vectors: Exon skipping and microdystrophin approaches in GRMD dogs

Abstract

The Duchenne Muscular Dystrophy (DMD) is caused by mutations in the gene encoding dystrophin. About 60% of the affected boys are treatable by exon skipping, i.e. the selective removal of exons flanking an out-of frame mutation in the dystrophin messenger, which results in shorter in-frame transcripts that are translated into functionally active dystrophin. Recombinant adeno-associated virus (rAAV) derived vectors carrying modified U7snRNA sequences are promising tools to allow an efficient exon skipping. In order to prepare a clinical trial in non-ambulant DMD patients using a rAAV8-U7 vector injected in the forearm, we injected in one forelimb a total of 21 Golden Retriever Muscular Dystrophy (GRMD) dogs to determine the therapeutic dose of the product and establish safety of the treatment. Results demonstrated high dystrophin expression levels (up to 80%) in muscles of dogs injected with 2.5E13 vg/kg. Strength improvement and correction of histological, NMR imaging and spectroscopy indices were demonstrated in muscles with more than 40% dystrophin expression. Finally, since a proportion of DMD patients are not amenable to treatment by exon skipping, we also evaluated a therapy based on the transfer of a rAAV8 vector encoding a sequence-optimized microdystrophin (MD1), which could eventually benefit all DMD patients. Three GRMD dogs were injected in one forelimb with this vector, exactly as in the U7-GRMD dogs. Treated limbs of these dogs exhibited consistent MD1 expression, which correlated with local correction of the pathology and strength improvement. The complete results obtained during these two pre-clinical trials will be presented and a comparison of these two therapeutic approaches will be discussed. Supported by the Association Francaise contre les Myopathies, the UK Muscular Dystrophy Campaign and by Advanced Diagnostics for New Therapeutic Approaches, a program dedicated to personalized medicine, coordinated by Institut Merieux and supported by OSEO.