Induction of revertant fibres in the mdx Mouse using antisense oligonucleotides

Duchenne muscular dystrophy (DMD) is a fatal genetic disorder caused by dystrophin mutations that preclude synthesis of a functional protein. The mdx mouse model has a nonsense mutation in exon 23. Removal of this single defective exon induces an in-frame mRNA transcript that encodes a shortened but still functional dystrophin protein. Despite the primary dystrophin gene lesion, at least 50% of DMD patients, mdx mice and a canine model of DMD have rare dystrophin-positive or ‘revertant fibres’ which arise from some naturally occurring exon-skipping event. Immunostaining studies have shown that the majority of revertant fibres miss multiple exons flanking the DMD mutation. These revertant fibres could provide a template for more functional dystrophin design, rather than skipping of single exons. We aim to emulate these naturally occurring revertant fibres using either bi-functional or combination antisense oligonucleotides (AO) to induce multiple exon skipping. We have developed AO cocktails that consistently induce removal of exons 19–25 and 21–25, two revertant transcripts that have been detected in untreated dystrophic mouse muscle. We are investigating whether these ‘induced revertant’ transcripts generate a more functional dystrophin protein than the minimal exon 23 skip to by-pass the nonsense mutation