Morpholino-Mediated Exons 28-29 Skipping of Dysferlin and Characterization of Multiexon-skipped Dysferlin using RT-PCR, Immunoblotting, and Membrane Wounding Assay.

Dysferlinopathies are a group of disabling muscular dystrophies  that includes limb girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy, and distal myopathy with anterior tibial onset (DMAT) as the main phenotypes. They are associated with molecular defects in DYSF, which encodes dysferlin, a key player in sarcolemmal homeostasis. Previous investigations have suggested that exon skipping may be a promising therapy for many patients with dysferlinopathies. It was reported that exons 28-29 of DYSF are dispensable for dysferlin functions. Here, we present a method for multiexon skipping of DYSF exons 28-29 using a cocktail of two phosphorodiamidate morpholino oligomers (PMOs) on cells derived from a dystrophinopathy patient. Also, we describe assays to characterize the multiexon skipped dysferlin at several levels by using one-step RT-PCR, immunoblotting, and a membrane wounding assay.
(© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)