Combining gene and stem cell therapy in the treatment of disferlinopaties

Mutations in gene encoding for dysferlin are involved in two main phenotypes of autosomal recessive muscular dystrophies: Miyoshi myopathy and Limb-Girdle Muscular Dystrophy type 2B. Both diseases are characterized by progressive weakness and wasting of skeletal muscles. No treatment is available for these diseases and development of effective therapies remains a big challenge. Dysferlin is expressed in skeletal and cardiac muscles where its function is membrane repair. Dysferlin contains calcium-dependent C2 lipid binding domains and an essential transmembrane domain. However, mildly affected patients in whom one or a large number of DYSF exons were missing have been described, suggesting that internally deleted dysferlin proteins can be functional. These evidences encourage to apply the exon skipping approach in dysferlinopathies. In a LGMD-2B patient carrying a deletion of the exon 32. Aim of this work was to extend the feasibility of exon skipping approach on the treatment of a patient carrying a deletion in exon 22 found at heterozygous composite state with a large deletion (Δ25-29) predicted to be in-frame. In this work we tested different antisense oligonucleotides (AONs) in order to induce the skipping of four DYSF exons and in the same time, we tested complete dysferlin protein delivery by lentivirus in CD133+ patient’s blood cells and injected in scid/blAJ model. The LV-DYSF vector allowed dysferlin expression both in vitro, in mononuclear cells from our patients, and in vivo after intramuscular injection of transduced CD133+ stem cells in the scid/blAJ mouse model. We strongly believe that the combination of exon skipping strategy with lentivirus transduction of patient stem cell represents a useful tool for the development of new therapeutic approaches in dysferlinopathies. Moreover, lentivirus carrying complete dysferlin can be useful to bypass all unskippable mutations, as those located in transmembrane domains or other essential parts of the gene.