Restoration of dystrophin expression in the MDX mouse using antisense oligonucleotides in a gene "knock-in" approach

Duchenne muscular dystrophy (DMD) is a severe, musclewasting disease arising from mutations in the massive dystrophin gene that preclude the synthesis of a functional protein. Affected boys show signs of weakness between the ages of 3 to 5 years, become restricted to a wheelchair by the age of 12 years and 90% will die from cardiac or respiratory complications before their third decade. DMD dystrophin gene defects are typically nonsense or frameshift mutations that lead to premature termination of translation. In-frame dystrophin gene mutations can result in a Becker muscular dystrophy (BMD), a milder, allelic form of DMD where the shortened dystrophin protein contributes to a milder phenotype. There is considerable variation in severity where some BMD patients become restricted to a wheelchair by the age of 15 years while others are almost asymtopmatic and have been diagnosed late in life (mid 60’s). The very mild BMD patients clearly demonstrate that the intact or full-length dystrophin protein is not essential for near normal function. Antisense oligonucleotides have been used to “knock-out” or suppress specific gene expression, either through the activation of a RNaseH activity or by blocking translation. We have used 2’-O-methyl antisense oligonucleotides (2’- O-Me AOs), which do not induce RnaseH activity, to “knock-in” a defective dystrophin gene so that a functional protein can be produced from a specifically modified transcript. These antisense oligonucleotides were directed to crucial splicing motifs in the dystrophin pre-mRNA, thereby blocking inclusion of specific exons in the mature dystrophin transcript. Such an approach would allow exons carrying nonsense mutations to be removed, or exons flanking a genomic deletion to be skipped to restore the reading frame. The mdx mouse model of DMD has a nonsense mutation in exon 23 of the dystrophin gene which results in premature termination of translation. By targeting antisense oligonucleotides to the 5’ splice site of intron 23 of the mouse dystrophin gene, it was possible to specifically induce skipping of exon 23 from the processed dystrophin mRNA. The induced skipping of dystrophin exon 23 removes the nonsense mutation without disrupting the reading frame so that a Becker-like protein could be produced. Delivery of 2’-O-Me AOs to the nucleus of cultured H-2K mdx cells was confirmed using an FITClabelled oligo, and induced exon skipping was assessed by an RT-PCR assay that revealed amplification of a dystrophin transcript missing exon 23. Transfection efficiency was improved by complexing 2’-O-Me AOs with commercially available liposomes. In vivo studies in the mdx mouse have shown synthesis and correct localisation of dystrophin and g-sarcoglycan after repeated i.m. injection of a 2’-O-Me AO:liposome complex. This approach offers an alternative to gene replacement or correction, and future work will assess in vivo toxicity, persistence of the protein and further refine delivery