Résumé :
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Mutations in gene encoding dysferlin (DYSF, Chr. 2p13; 55 exons, mRNA 6,2kb) causes Limb Girdle Muscular Dystrophy type 2B and Miyoshi myopathy. Both diseases are autosomic recessive diseases with an age of onset at the early adulthood, associated with a slow progression and a high level of CPK. They could sometimes lead to the loss of walking abilities. Nowadays, there is no curative treatment but some therapeutics approaches are investigated.Dysferlin (2080 amino acids, 237 kDa) is composed of several homologous C2 domains and a C-terminal transmembrane domain. Even if, these C2 domains differ from each other by the interaction established with dysferlin partners, there are some evidences that some C2 domains could have redundant functions. Based on this observation, an exon-skipping strategy could be envisaged. Indeed, as for Duchenne Muscular Dystrophy (DMD), some deletions have been associated with moderate phenotype. One of them has been identified in a patient with an in-frame deletion of exon 32. Based on this observation we have postulated that this exon could be a target for an exon-skipping therapeutic strategy. Moreover, 4% of patients presenting with a dysferlinopathy have mutations in this exon. By bioinformatics predictions we have identified four essentials sites for the maintenance of exon 32, and we have developed Antisense OligoNucleotides (AON) directed against them. These experiments have been realized on patients cells (carrying mutations in exon 32), and control. Three of these AON have been very efficient for the skipping of exon 32 on RT-PCR. We then tested the functionality of this "quasi-dysferlin" resulting from the skipping of exon 32, in sarcolemma repair mechanisms and myotubes formation. This "quasi-dysferlin" is able to repair myotubes membrane lesions and allow a partial restoration of myotubes/myoblasts fusion process. We will go through the different steps of a pre-clinical trial using our AON on a murine model carrying a nonsense codon in exon 32. If these steps prove to be satisfactory a therapeutic strategy based on exon skipping for dysferlinopathies will be tested in patients.
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