Antisense Oligonucleotide-induced Exon Skipping Across the Human Dystrophin Gene Transcript

Abstract

Protein-truncating mutations in the dystrophin gene lead to the most common childhood form of muscle wasting, Duchenne muscular dystrophy. Becker muscular dystrophy, a condition that typically arises from dystrophin gene lesions that do not disrupt the reading frame, clearly indicates that substantial domains of the dystrophin protein are not essential. Potential therapeutic intervention exists during pre-mRNA splicing, whereby selected exons are excised to either remove nonsense mutations or restore the reading frame around frame-shifting mutations from the mature mRNA. Appropriately designed antisense oligonucleotides (AOs), directed at amenable splicing motifs across the dystrophin gene transcript, block exon recognition and/or spliceosome assembly so that targeted exons are removed from the mature mRNA. We describe a panel of AOs designed to induce skipping of every exon within the human dystrophin gene transcript, with the exception of the first and last exons. Every exon targeted in vitro could be removed from the dystrophin mRNA, although some exons are more efficiently excluded than others. No single motif has emerged as a universal AO annealing site for redirection of dystrophin pre-mRNA processing, although the general trend is that the most efficient compounds are directed at motifs in the first half of the target exon.

Antisense-mediated exon skipping: A versatile tool with therapeutic and research applications

“…In general, only the targeted exon was skipped, but occasionally unexpected results were observed. A striking example is that AONs targeting exon 8 always induce skipping of both exon 8 and 9 in human and dog transcripts (Aartsma-Rus et al 2005;McClorey et al 2006b;Wilton et al 2007). The most likely explanation for this phenomenon is that the AONs somehow affect only the acceptor splice site but leave the donor splice site intact.…”

Comparative Analysis of Antisense Oligonucleotide Sequences for Targeted Skipping of Exon 51 During Dystrophin Pre-mRNA Splicing in Human Muscle

“…One strategy, based on previous results obtained in mouse dystrophin exon 23 (Graham et al, 2004), was that of designing an overlapping stepped array of 2-OMe AOs complementary to the 5Ј (donor) splice site of intron 51. The other strategies examined other potential splice motifs across exon 51, including the 3Ј (acceptor) splice site and the branch point, as well as exon-internal sequences (Aartsma-Rus et al, 2004b;Wilton et al, 2007). After initial characterization in each partner laboratory (Table 2) (Aartsma- Rus et al, 2002Rus et al, , 2005Harding et al, 2007), eight AOs, six targeting the 5Ј splice site designed in the first partner laboratory (RH) (C20 to H20; Fig.…”

In vitro evaluation of novel antisense oligonucleotides is predictive of in vivo exon skipping activity for Duchenne muscular dystrophy

“…Modulation of pre-mRNA splicing using short antisense oligonucleotides (AOs) can induce splice correction of aberrant diseaserelated transcripts to restore their function [1]. Such splice correcting AOs have been shown to correct out-offrame dystrophin transcripts via exon skipping of specific DMD exons, to yield truncated but functional dystrophin protein and, thus, such AOs have therapeutic potential [2][3][4][5][6][7][8][9].…”