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

Authors: 
Anwar S, Roshmi RR, Woo S, Haque US, Arthur Lee JJ, Duddy WJ, Bigot A, Maruyama R, Yokota T
Citation: 
Mol Ther Nucleic Acids. 2024 Dec 21;36(1):102443. doi: 10.1016/j.omtn.2024.102443. PMID: 39967852; PMCID: PMC11834094
Abstract: 
Dysferlinopathies are debilitating autosomal recessive muscular dystrophies caused by mutations in the DYSF 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 DYSF intron 26, causing exon 26 exclusion, an out-of-frame transcript, and no detectable dysferlin protein. We hypothesized that skipping DYSF exon 27 could restore the reading frame and membrane repair function. Using an in-house in silico 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 DYSF exon 27 skipping restores functional dysferlin in patient-derived cells, paving the way for future in vivo and clinical studies.
Epub: 
Not Epub
Organism or Cell Type: 
patient-derived myoblasts and differentiated myotubes
Delivery Method: 
Endo-Porter