Abstract
Duchenne Muscular dystrophy (DMD), a severe neuromuscular disorder, is caused by nonsense or frameshift mutations in dystrophin gene that results in an absence of functional dystrophin. Loss of functional dystrophin renders muscle fibres vulnerable to membrane damage during contraction. Antisense oligonucleotide (AO) induced exon skipping has been used to induce specific exon removal and by-pass the disease-causing mutation. Some dystrophin mutations will require removal of more than one exon. We are investigating multi-exon skipping in the B6Ros.Cg-Dmdmdx−4Cv/J (4cv) muscular dystrophy mouse model, which has a nonsense mutation in exon 53 of the dystrophin gene. This area is of relevance to the human dystrophin gene since this area corresponds to the major deletion hot spot of the human dystrophin gene. To restore the reading frame of the 4cv dystrophin mRNA, both exons 52 and 53 must be excised from the mature dystrophin gene transcript. 2_-O-Methyl AOs on a phosphorothioate backbone (2OMe) have been designed to mask motifs involved in splicing to remove these exons during pre-mRNA processing. We describe the removal of single exon and multi-exons in cell culture and in vivo. The exploration of exon skipping events in the 4CV mouse model will provide additional information in AO design, and will be a molecular model relevant to many DMD cases.