FROM THE SPINAL CORD TO THE MUSCLE: O.12 Midi-Dysferlin gene therapy for dysferlinopathies

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Dysferlinopathies are a group of muscular dystrophies caused by recessive mutations in the DYSF gene encoding the dysferlin protein. These muscular dystrophies, which affect at least 5,000 people in the world, are characterised by skeletal muscle degeneration and weakness. Dysferlin is a transmembrane protein located in the muscle fibers sarcolemma. This protein is involved in several muscle cell functions like T tubule formation, vesicle trafficking and membrane repair. In 2009, a study showed the presence of fourteen dysferlin transcripts from alternative splicing (Pramono Z.A.D. et al, 2009). We were particularly interested in dysferlin transcript 11 which contains the alternative exon 40a. Exon 40a encodes a protein region cleaved by calpains during the muscle membrane repair mechanism. This cleavage produces a mini-dysferlin (from exon 40a to exon 55) that is recruited to the injury site (Lek A. et al, 2013). Our previous work shows that dysferlin transcript 11 is essential in the membrane repair process. Moreover, dysferlin transcript 11 is able to participate in other muscle cell functions like vesicle trafficking and membrane protection from mechanical stress. These findings suggest that, as part of a therapeutic strategy, it is necessary to restore the protein sequence encoded by exon 40a in patients with dysferlinopathies. Noteworthy, dysferlin transcript is too large to be packaged into AAV. Therefore, we have synthesized a midi-dysferlin which contains the alternative exon 40a and the most essential domains for muscle cell functions (including the last C2 domains and the transmembrane segment). To explore the therapeutic effect of this midi-dysferlin, we performed membrane repair, osmotic shock and transferrin assays on muscle cells. We also carried out preliminary analyses on dysferlin deficient mouse model. The results obtained within this project will be shown during this congress.