Résumé :
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Duchenne Muscular Dystrophy (DMD) is the most severe form of dystrophinopathy, in which null 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. Deletions and duplications in the DMD gene represent 60% to 70% of the abnormalities involved in dystrophinopathies and their identification usually relies on targeted approaches such as QF-PCR and MLPA. We recently developed a new oligonucleotide-based CGH-array approach for high-throughput detection of exonic deletions and duplications and showed the efficiency of this approach through detection of exonic deletions/duplications in the DMD gene. Since this approach is completely scalable, this new molecular tool will provide the means for molecular biologists to screen optimised combinations of genes involved in a particular group of clinically and genetically heterogeneous disorders such as muscular dystrophies, brain malformations, and mental retardation as well as common disorders with genetic susceptibility. In the emerging field of therapeutic development of dystrophinopathies, therapeutic approaches to restore the reading frame of DMD transcripts by exon skipping have been developed and validated, both ex-vivo and in-vivo, by obtaining the desired in-frame mRNA and ensuing dystrophin synthesis. Because of the high diversity of mutational defects in DMD patients, many allele-specific correcting patterns must be designed and tested. Ideally this must be carried out in cultured myoblasts derived from patient muscle specimens. Since in most cases no fresh muscle is available, we have tried to use primary cultures of patient's skin-fibroblasts that have been induced into myoblasts by a lentiviral vector expressing the muscle transcription factor myoD. The myoconverted cells from distinct DMD patients were subsequently exposed to lentiviral vectors expressing U7 snRNA-linked antisense oligonucleotides designed to induce efficient skipping of exon 51 or 45. In these cells, the expected skipped mRNA, and the resulting quasi-dystrophin were obtained. This protocol is a useful alternative for the evaluation of each construct both in terms of frame restoration, and of biological relevance of the resulting protein. (1) Institut Cochin, INSERM Unité 567, UMR 8104, Université Paris Descartes, et Laboratoire de Biochimie et Génétique Moléculaire, Hôpital Cochin, Paris, France. (2) UMR S 787, Inserm/UPMC-Paris 6, Institut de Myologie, Paris, France.
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