An antisense method for efficient exon skipping and its application to Duchenne muscular dystrophy

Antisense-mediated exon skipping has been used to treat Duchenne muscular dystrophy (DMD) by restoring the DMD reading frame to express partially functional dystrophin. However, difficulty in designing effective antisense oligonucleotides (ASOs) has hindered the development of clinically effective therapies. Here we describe a robust bipartite ASO design, termed 5' splice site decoy (5D)-ASO, in which a short tail sequence is employed to enhance the splicing repression exerted by an antisense moiety. The tail carried by the antisense moiety to an exon of interest in a target pre-mRNA interferes with the recognition of the exon's 5' splice site by U1 snRNA and markedly enhances exon skipping, compared to tail-less ASOs. 5D-ASO has broad applicability, based on multiple genes tested. Particularly, an 8-nt tail, when appended to sequences targeting DMD exon 51, elicited a pronounced increase in exon skipping in mouse models, restored dystrophin expression in muscle tissues and improved the phenotype, without obvious signs of toxicity. The lead ASO further demonstrated a marked exon-skipping effect and an overall safe profile in monkeys. Our data establish a valuable platform technology for RNA-targeted therapeutics.