Résumé :
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Mutations in the gene encoding dysferlin cause Limb Girdle Muscular Dystrophy Type 2B (LGMD2B) and Miyoshi Myopathy (MM). While LGMD2B leads to muscle weakness and atrophy in proximal muscles of the limb girdle regions, MM mainly affects posterior distal muscle groups. Identical mutations have been observed to cause either phenotype.Dysferlin is predominantly expressed in skeletal muscle and therein locates at the sarcolemma. It comprises seven cytoplasmatic C2 domains, which bind membrane phospholipids in a calcium-dependent manner. Together with AHNAK, affixin, annexin A1 and A2 it has been proposed to be involved in sarcolemma repair, which is disturbed in case of dysferlin-deficiency. As a response to membrane wounding in other cell types then muscle, dysferlin has been implicated in intercellular calcium signaling as well.Several hundred mutations have been detected in the 55-exon-spanning dysferlin sequence, though, they do not accumulate in a mutational hot spot and repair of the messenger by exon-skipping is not often appropriate in the case of dysferlin. Therefore, we apply the mRNA trans-splicing approach. We designed different pre-mRNA-trans-splicing molecules to exchange large regions of the messenger in order to heal a variety of mutations. As a test system we employ human immortalized dysferlin-deficient myoblasts. We immortalized and characterised primary human myoblasts retrieved from muscle biopsies at the muscular dystrophy outpatient clinic Berlin Buch in collaboration with the cell-culture platform in Paris. We deliver the pre-mRNA-trans-splicing molecule via lenti virus and plan to employ adeno-associated virus for the delivery in dysferlin-deficient mouse models. We assess efficiency by quantitative PCR and immunoblot. To prove functional recovery of the dysferlin protein we apply two cell membrane wounding assays, using atomic force and confocal laser microscopy.
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