Résumé :
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In Duchenne Muscular Dystrophy (DMD) the selective removal by exon skipping of exons flanking an out-of frame mutation in the dystrophin messenger can result in in-frame mRNA transcripts that are translated into shorter but functionally active dystrophin. The goal of our project was to determine in the dog model of DMD, the GRMD, the effective dose of our therapeutic product defined as a recombinant Adeno-Associated Virus serotype 8 (rAAV8) expressing a modified U7 snRNA specific for the skipping of exons 5 to 10 of the GRMD dystrophin transcript. The mode of delivery was the locoregional high pressure intravenous (IV) injection of a forelimb. Five groups of 2-3 GRMD dogs were exposed to different rAAV8-U7snRNA doses (from 2.5 1012 vg/kg to 2.5 1013 vg/kg diluted in two different volumes) + three dogs injected with same volume of ringer-lactate. Each dog was followed .3 months after injection until sacrifice and full autopsy. The primary outcomes were the restoration of dystrophin expression and the improvement of the tissue pathology in the injected limb and in muscles behind the tourniquet. Results demonstrate high level (> 80%) of dystrophin expression in dogs injected with 2.5 1013 vg/kg, not only in the injected limb, but also in lower quantity in other muscles behind the tourniquet. Lower levels of dystrophin expression (~30-40%) and (~10%) were observed in dogs injected with a dose 5 or 10 times lower, respectively. An excellent correlation was found between the viral copy number/diploid genome and the dystrophin amount. Strength improvement up to 50% was demonstrated in muscles with more than 45% dystrophin expression. In addition, we observed a dose dependant correction of various RMN pathological indexes. This demonstrates the feasibility and the dose-dependant effect of locoregional rAAV8 U7snRNA approach in a large animal model of DMD and open the way for a human trial in upper limb of non ambulatory patients. This project is supported by AFM (Association Francaise contre les Myopathies) and byADNA (Advanced Diagnostics for New Therapeutic Approaches), a program dedicated to personalized medicine, coordinated by Institut Merieux and supported by research and innovation aid from the French public agency, OSEO.
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