Antisense-induced exon skipping and synthesis of dystrophin in the <i>mdx</i> mouse

TLDR

Duchenne muscular dystrophy results from dystrophin gene defects that prevent functional protein production, whereas Becker muscular dystrophy arises from in‑frame deletions allowing a shorter but functional protein, and current cell or gene replacement therapies have failed to restore dystrophin. The study proposes using 2′‑O‑methyl antisense oligoribonucleotides to alter dystrophin pre‑mRNA splicing in the mdx mouse model of DMD. By targeting splicing motifs, the antisense oligonucleotides induced exon 23 skipping and removed the mdx nonsense mutation without disrupting the reading frame, first optimized in vitro in mdx myoblasts and then applied in vivo. Immunohistochemistry after intramuscular delivery of antisense‑liposome complexes revealed dystrophin and γ‑sarcoglycan synthesis with correct subsarcolemmal localization, suggesting the strategy could lessen DMD severity by producing a Becker‑like protein.

Abstract

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease arising from defects in the dystrophin gene, typically nonsense or frameshift mutations, that preclude the synthesis of a functional protein. A milder, allelic version of the disease, Becker muscular dystrophy, generally arises from in-frame deletions that allow synthesis of a shorter but still semifunctional protein. Therapies to introduce functional dystrophin into dystrophic tissue through either cell or gene replacement have not been successful to date. We report an alternative approach where 2′- O -methyl antisense oligoribonucleotides have been used to modify processing of the dystrophin pre-mRNA in the mdx mouse model of DMD. By targeting 2′- O -methyl antisense oligoribonucleotides to block motifs involved in normal dystrophin pre-mRNA splicing, we induced excision of exon 23, and the mdx nonsense mutation, without disrupting the reading frame. Exon 23 skipping was first optimized in vitro in transfected H-2K b -tsA58 mdx myoblasts and then induced in vivo . Immunohistochemical staining demonstrated the synthesis and correct subsarcolemmal localization of dystrophin and γ-sarcoglycan in the mdx mouse after intramuscular delivery of antisense oligoribonucleotide:liposome complexes. This approach should reduce the severity of DMD by allowing a dystrophic gene transcript to be modified, such that it can be translated into a Becker-dystrophin-like protein.