T.P.21 Exon skipping for dysferlinopathies

Abstract

Abstract Mutations in the dysferlin encoding DYSF gene have been reported for limb-girdle muscular dystrophy 2B, miyoshi myopathy and distal myopathy with anterior tibial onset patients. Most patients have small mutations within exons, inducing premature stops or amino acid substitutions, which lead to protein instability or mislocalization. The dysferlin protein has a suggested function in membrane repair and vesicle trafficking. It contains six or seven calcium-dependent C2 lipid binding (C2) domains that probably mediate vesicle fusion. Based on reports of mild patients lacking one or more C2 domains, it has been suggested some of the C2 domains are redundant. The exon skipping approach employs antisense oligonucleotides (AONs) to hide exons from the splicing machinery during pre-mRNA splicing. This allows reframing of transcripts (as is currently tested in phase 3 clinical trials for Duchenne muscular dystrophy) but might also be a viable therapeutic approach for dysferlinopathies. Here the mutated exon is targeted to bypass the mutation. For in-frame exons the open reading frame will be maintained, allowing the production of an internally deleted but hopefully partially functional dysferlin. For out-of-frame exons, skipping of additional flanking exons would be needed. We here further explored this approach for dysferlinopathies by testing antisense oligonucleotides (AONs) to induce the skipping of exons 8, 9, 20, 21, 33, 38 and 43 in control myogenic cells. In addition, we have tested AONs targeting exon 30 in myoblast cultures from a patient with a mutation in exon 30, to confirm dysferlin restoration, and proper localization. Finally, we have tested AONs targeting exon 30 in mice to obtain in vivo proof of concept.