Résumé :
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Duchenne Muscular Dystrophy (DMD) is an X-linked recessive disorder due to mutations in the gene that encodes dystrophin. Most of these mutations consist in large genomic deletions, although their extent is not directly correlated with the severity of the phenotype. Out-of-frame deletions lead to abortion of translation, dystrophin deficiency and severe DMD phenotypes, while internal deletions that produce in frame mRNAs leading to shorter proteins are responsible for a milder myopathy known as Becker Muscular Dystrophy (BMD). About 80% of the out-of-frame mutations could be theoretically rescued after restoring the translational frame by using exon skipping strategies. Here we used gene transfer in a large animal model of DMD, the Golden Retriever Muscular Dystrophy (GRMD) dog, to achieve the precise skipping of multiple exons spaced over 125,000 bp of the dystrophin pre-mRNA and the re-expression of a functional protein. This led to sustained correction of the dystrophic phenotype in extended muscle areas and muscle strength recovery. Exon skipping was obtained with U7snRNAs (U7smOPT) carrying antisense sequences designed to mask determinants of exon 6 and 8 definition. These U7smOPT were introduced into skeletal muscle fibres by using Adeno Associated Viral (AAV2/1) vectors. After two months, levels of dystrophin were almost normal in transduced fibres. Histological examination revealed that the dystrophin glycoprotein complex was restored and that spontaneous muscle damages were stopped. Muscle architecture was fully corrected and fibres displayed the hallmarks of mature and functional units. Muscle force reflecting fibers functional integrity were improved. Our study documents for the first time the recovery of dystrophin at the scale of entire limbs in a large animal and thus represents a critical milestone for the development of clinical trials in human patients.
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