Antisense-mediated Multiple Exon Skipping Therapy in Dystrophic Mice

Toshifumi Yokota, Yusuke Echigoya, Bailey Miskew

Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry

Duchenne muscular dystrophy (DMD) is the most common lethal genetic disorder worldwide, characterized by progressive muscle deterioration. Many cases result from a frame shift mutation in the dystrophin (DMD) gene, within the mutation hot spot between exons 45-55. Antisense-mediated exon skipping is able to restore the reading frame, and thus is a potential therapeutic treatment for DMD. Exon skipping employs short DNA-like molecules that target mutated parts of the gene, and produce truncated but functional proteins. Remaining challenges include the limited applicability and unclear stability/function of resulting short dystrophins. Multiple exon skipping of exons 45-55 would address both issues. This strategy can treat approximately 60% of DMD patients with deletion mutations. In addition, this specific deletion mostly leads to normal individuals or remarkably mild symptoms, due to its very functional protein structure. To test the feasibility of exons 45-55 skipping, mice harboring a deletion mutation of exon 52 (mdx52) were injected systemically with an antisense vivo-phosphorodiamidate morpholino oligomer (vPMO) cocktail, which contains cell-penetrating moiety, targeting all of exons 45-55. Central nucleation (an indicative of muscle degeneration/regeneration), Western blotting, RT-PCR, immunohistochemistry, grip strength, and creatine kinase levels were used to evaluate the effectiveness of the treatment. 10 vPMO treated mice showed significant decreases in centrally nucleated fibers, increased grip strength, and increased dystrophin levels compared to non-treated mdx52 mice. RT-PCR demonstrated efficient exon skipping of exons 45-55 in the treated mice. Based on blood serum levels, vPMO at this dose has no toxic effect. With efficient exon skipping and symptom improvement in the 10 vPMO treated mdx52 mice, multi-exon skipping therapy targeting exons 45-55 is a promising therapeutic approach for DMD.