P.20.12 Gene therapy of Duchenne Muscular Dystrophy using rAAV vectors: Patterns of dystrophin expression and histological improvements

Abstract

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 AAV-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. All muscles of the limbs were observed about 14weeks later for dystrophin expression and histological patterns Depending on the injected dose and the injection protocol, we showed low to high (up to 80%) dystrophin expression levels in muscles of the injected legs depending of the vector dose. Improvement of the histological pattern was assessed by histomorphological analysis of the immunological detection of dystrophin expression, sclerosis (immunopexoxydase against Collagen I), regeneration (developmental Myosin Heavy Chain), calcium accumulation, anisocytosis, macrophagic infiltration, and lymphocytic infiltration. The dystrophin expression was characterized by a diffuse pattern with single or small groups of positive fibers in most fascicles. A significant decrease of sclerosis in muscles exhibiting more than 60% of dystrophin positive fibers and an important decrease of regeneration (in muscles containing up to 30% of dystrophin), was observed. These results show the efficiency of the rAAV8-U7 strategy to restrict the natural degradation of the dystrophic muscle. Supported by the Association Française contre les Myopathies and Advanced Diagnostics for New Therapeutic Approaches, a program dedicated to personalized medicine, coordinated by Institut Mérieux and supported by OSEO.