Résumé :
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Dysferlinopathies are recessive muscular disorders caused by defects in dysferlin. Genetic mutations are responsible for two major phenotypes: Limb Girdle Muscular Dystrophy type 2B and Distal Miyoshi Myopathy. These skeletal muscle diseases are characterized by progressive loss of muscle integrity and strength. Recently, dysferlin was demonstrated to be involved in membrane repair process, providing a preliminary understanding of the pathophysiological mechanism in these diseases. Currently, no treatment is available. Considering the recessive nature of dysferlinopathies, a possible therapeutic strategy is gene transfer. To date, the best vector for gene transfer in muscle is Adeno Associated Virus (AAV). However, the human dysferlin cDNA size approximates 7 kb, preventing its direct incorporation into a single AAV vector, since the encapsidation limit is around 4.7 kb. In order to bypass this limitation, we set up a strategy taking advantage of the concatemerization ability of AAV vectors. The dysferlin cDNA was separated into two AAV2/1 vectors, one carrying a muscle specific promoter followed by the 5’ half of dysferlin cDNA and a 5’ splicing signal and another one carrying a 3’ splicing signal, the remaining dysferlin sequence and a polyadenylation signal. We demonstrated the ability of these vectors to produce a human full-length dysferlin mRNA in a cellular model. To test this approach in vivo, intramuscular injection of both vectors was performed into dysferlin deficient mice. Dysferlin mRNA was correctly spliced, expressed at a level close to the endogenous one and was stable for an entire year. At the protein level, dysferlin was detected at 237 kDa, the size of the expected full-length protein. The functionality of dysferlin was demonstrated by a membrane repair assay based on 2-photon injury on isolated muscle fibers. Taken together, all these data prove that gene transfer based on AAV concatemerization allows the expression of a full-length functional dysferlin.
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