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Updated: Jun 28, 2026
Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy
Published on: May 24, 2016
By-passing the nonsense mutation in the 4 CV mouse model of muscular dystrophy by induced exon skipping
Chalermchai Mitrpant1, Sue Fletcher, Patrick L Iversen
1Centre for Neuromuscular and Neurological Disorders, University of Western Australia, QE II Medical Centre, Nedlands, Australia.
Background:
Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder, is caused by protein-truncating mutations in the dystrophin gene. Absence of functional dystrophin renders muscle fibres more vulnerable to damage and necrosis. We report antisense oligomer (AO) induced exon skipping in the B6Ros.Cg-Dmd(mdx-4Cv)/J (4(CV)) mouse, a muscular dystrophy model arising from a nonsense mutation in dystrophin exon 53. Both exons 52 and 53 must be excised to remove the mutation and maintain the reading frame.
Methods:
A series of 2'-O-methyl modified oligomers on a phosphorothioate backbone (2OMeAOs) were designed and evaluated for the removal of each exon, and the most effective compounds were then combined to induce dual exon skipping in both myoblast cultures and in vivo. Exon skipping efficiency of 2OMeAOs and phosphorodiamidate morpholino oligomers (PMOs) was evaluated both in vitro and in vivo at the RNA and protein levels.
Results:
Compared to the original mdx mouse studies, induction of exon skipping from the 4(CV) dystrophin mRNA was far more challenging. PMO cocktails could restore synthesis of near-full length dystrophin protein in cultured 4(CV) myogenic cells and in vivo, after a single intramuscular injection.
Conclusions:
By-passing the protein-truncating mutation in the 4(CV) mouse model of muscular dystrophy could not be achieved with single oligomers targeting both exons and was only achieved after the application of AO cocktails to remove exons 52 and 53. As in previous studies, the stability and efficiency of PMOs proved superior to 2OMeAOs for consistent and sustained protein induction in vivo.
Insights
Antisense oligomers (AOs) were used to induce exon skipping in a mouse model of Duchenne muscular dystrophy (DMD). AO cocktails successfully restored dystrophin protein synthesis by removing specific exons, offering a potential therapeutic strategy for DMD.
Area of Science:
- Biochemistry
- Genetics
- Molecular Biology
Background:
- Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by mutations in the dystrophin gene.
- Absence of functional dystrophin leads to muscle fiber damage and necrosis.
- The 4(CV) mouse model exhibits a nonsense mutation in dystrophin exon 53, necessitating the removal of both exons 52 and 53 to restore the reading frame.
Purpose of the Study:
- To investigate antisense oligomer (AO)-induced exon skipping in the 4(CV) mouse model of DMD.
- To evaluate the efficacy of different AO chemistries (2OMeAOs and PMOs) for dual exon skipping.
- To assess the restoration of dystrophin protein synthesis in vitro and in vivo.
Main Methods:
- Design and evaluation of 2'-O-methyl modified oligomers on a phosphorothioate backbone (2OMeAOs) for exon removal.
- Combination of effective AOs to induce dual exon skipping (exons 52 and 53).
- Assessment of exon skipping efficiency using 2OMeAOs and phosphorodiamidate morpholino oligomers (PMOs) in myoblast cultures and in vivo at RNA and protein levels.
Main Results:
- Inducing exon skipping in the 4(CV) dystrophin mRNA was more challenging than in previous mdx mouse studies.
- PMO cocktails successfully restored near-full-length dystrophin protein synthesis in cultured 4(CV) myogenic cells and in vivo after a single intramuscular injection.
- Single oligomers targeting both exons were ineffective; AO cocktails were required for successful exon removal.
Conclusions:
- Bypassing the protein-truncating mutation in the 4(CV) mouse model requires AO cocktails targeting both exons 52 and 53.
- Phosphorodiamidate morpholino oligomers (PMOs) demonstrated superior stability and efficiency for sustained dystrophin protein induction in vivo compared to 2OMeAOs.
- This study highlights the potential of AO-mediated exon skipping as a therapeutic strategy for DMD, with PMOs showing promise for clinical application.
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