Résumé :
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Duchenne Muscular Dystrophy (DMD) is the most severe form of dystrophinopathy, in which nul mutations in the DMD gene (mostly frameshifting deletions, and nonsense point mutations) result in the complete absence of dystrophin. A milder phenotype of the disease, Becker Muscular Dystrophy (BMD), generally arises from in-frame deletions allowing the synthesis of a shorter but still quasi-functional protein. Based on these observations, therapeutic approaches to restore the reading frame by exon skipping have been developed. We and others demonstrated the use of exon skipping to restore the expression of a fully functional quasi-dystrophin. In our experiments, the targeted pre mRNA sequences are carried by an engineered U7 small nuclear RNA which is delivered to the myoblasts by a lentiviral vector. When getting patients muscle biopsies is unavailable or impossible, we used skin fibroblasts (CD90+, CD56-) easily obtained. After having cultured control fibroblasts, they are transfected with a lentivirus carrying MyoD under the tet on promoter to induce myogenic differentiation (63% after 3 days are CD56+). Control fibroblast-derived myoblasts expressed myogenic protein e.g. myogenin, desmin and dystrophin. Using DMD patients fibroblast-derived myoblasts, we obtained the similar myogenic differentiation but no dystrophin. On these cells, using the same U7 lentivirus, skipping either exon 45 or exon 51 restored a dystrophin open reading frame mRNA detected by nested RT PCR and a quasi dystrophin detected by Western Blot. Conclusions: We validated efficient target sequences to successfully skip exon 45 or 51 in the dystrophin pre messenger RNA. Moreover, we showed that the exon skipping method worked on skin biopsies fibroblast-derived myoblasts, providing an alternative to muscular biopsy.
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