Résumé :
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Mutations in gene encoding Dysferlin are involved in Limb-gird Muscular Dystrophy type 2B (LGMD-2B) and and Miyoshi myopathy (MM), both diseases are characterized by progressive weakness and wasting of skeletal muscles. Dysferlin is abundantly expressed in skeletal and cardiac muscles where its main function reported is membrane repair. So far, no treatment is available for these diseases but new hopes are coming from stem cell therapy. Recently, we found a subpopulation of human muscle or blood derived stem cells expressing the CD133 antigen which are able to efficiently participate in muscle regeneration in vivo. A successful strategy associating exon-skipping strategy and the use of CD133 + stem cell may be tested in the dysferlinopathies. For this reason, we characterized precisely dysferlin expression in normal human CD133+ stem cells obtained from both circulating blood and muscle, either alone or in co-culture experiments with normal or dysferlin null mouse-derived myotubes. In these experiments, we found human dysferlin expression both by RT-PCR and immunostaining experiments. In order to combine exon skipping and stem cell therapy, we designed oligonucleotides anti-sens nucleotides (AONs) targeting consensus regions important to direct splicing machinery, for patients carrying relevant mutations in dysferlin exons. The efficiency of skipping was tested by RT-PCR on CD133 + stem cell isolated from LGMD-2B or MM patients. By this approach we plan to test function of re-expressed dysferlin, after engraffing of human skipped CD133+ cells in a mouse model of dysferlinopathy (Dysf -/-) crossed with Scid immunodepressed mice. We believe that combined exon skipping and CD133+ stem cell therapy is a very promising approach that has to be deeply explored in the context of dysferlinopathies.
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