Antisense oligonucleotide induced exon skipping and the dystrophin gene transcript: Cocktails and chemistries

Abstract

AOs have been used in vitro and in vivo to redirect dystrophin pre-mRNA processing in human and animal cells, thereby allowing protein-truncating mutations to be removed or bypassed, so that a shorter but still functional protein can be generated. Targeted exon skipping may be directed at the dystrophin gene transcript to remove nonsense or frame-shifting mutations that would otherwise have lead to Duchenne Muscular Dystrophy, the most common childhood cause of muscle wasting. Although many dystrophin exons can be excised using a single AO, several exons require two motifs to be masked for efficient or specific exon skipping. Some AOs were inactive when applied individually, yet pronounced exon excision was induced when used in combinations, clearly indicating synergistic rather than cumulative effects on splicing. We describe optimisation and excision of recalcitrant dystrophin exons from the mature mRNA using AO cocktails for enhanced efficiency and/or specificity. The use of either 2_-O-methyl modified bases on a phosphorothioate backbone (2OMeAOs) or phosphorodiamidate morpholino oligomers (PMOs) does not seem to influence exon skipping trends, indicating optimisation of AO design with 2OMe chemistry should be directly applicable to PMO