Antisense oligonucleotide-mediated exon 27 skipping restores dysferlin function in dysferlinopathy patient-derived muscle cells.

Dysferlinopathies are debilitating autosomal recessive muscular dystrophies caused by mutations in the gene, encoding dysferlin, a protein crucial for sarcolemmal homeostasis and membrane resealing. Currently, no therapies exist for dysferlinopathies. Dysferlin features a modular structure with multiple calcium-dependent C2 lipid-binding domains. Clinical reports of mild, late-onset phenotypes suggest partial retention of functionality despite missing C2 domains, supporting exon-skipping therapies using antisense oligonucleotides (ASOs). In this study, we identified a patient-derived muscle cell line with a splice site mutation in intron 26, causing exon 26 exclusion, an out-of-frame transcript, and no detectable dysferlin protein. We hypothesized that skipping exon 27 could restore the reading frame and membrane repair function. Using an in-house tool, we designed ASOs targeting exon 27. Treatment resulted in 65%-92% exon 27 skipping in myoblasts and myotubes, leading to a 39%-51% rescue of normal dysferlin expression, demonstrating robust efficacy of our designed ASOs. Two-photon laser-based assays indicated functional membrane repair. Additionally, we observed improved myotube fusion, cell vitality, and reduced apoptosis levels post-treatment. These findings provide proof of concept that exon 27 skipping restores functional dysferlin in patient-derived cells, paving the way for future and clinical studies.