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Antisense oligonucleotide-induced exon skipping restores dystrophin expression in vitro in a canine model of DMD

Gene Therapy volume 13, pages 1373–1381 (2006)Cite this article

Abstract

Manipulation of pre-mRNA splicing by antisense oligonucleotides (AOs) offers considerable potential for a number of genetic disorders. One of these is Duchenne muscular dystrophy (DMD), where mutations in the dystrophin gene typically result in premature termination of translation that causes a loss of functional protein. AOs can induce exon skipping such that the mutation is by-passed and the reading frame restored, producing an internally deleted protein similar to that found in the milder Becker muscular dystrophy. To date, this approach has been applied to the mdx mouse model in vitro and in vivo and in human myoblast cultures. Here, we report the application of AO-directed exon skipping to induce dystrophin expression in vitro in a canine model of DMD, golden retriever muscular dystrophy (GRMD). The efficacy of 2′-O-methyl phosphorothioate (2OMe), phosphorodiamidate morpholino oligomers (PMOs) and peptide-linked PMOs (PMO-Pep) to induce dystrophin expression was assessed. The 2OMe chemistry was only effective for short-term induction of corrected transcript and could not induce detectable dystrophin protein. The PMO chemistry generally induced limited exon skipping at only high concentrations; however, a low level of dystrophin protein was produced in treated cells. Use of the PMO-Pep, applied here for the first time to a DMD model, was able to induce high and sustained levels of exon skipping and induced the highest level of dystrophin expression with no apparent adverse effects upon the cells. The induction of dystrophin in the GRMD model offers the potential for further testing of AO delivery regimens in a larger animal model of DMD, in preparation for application in human clinical trials.

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Acknowledgements

We thank Russell Johnson for technical assistance. This work was funded by Parent Project Muscular Dystrophy, USA, National Institute of Health, USA, National Health and Medical Research Council, Australia, Muscular Dystrophy Association, USA and the Medical and Health Research Infrastructure Fund, Western Australia.

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Authors and Affiliations

  1. Experimental Molecular Medicine Group, Australian Neuromuscular Research Insitute, Centre for Neuromuscular and Neurological Disorders, University of Western Australia, QEII Medical Centre, Perth, Western Australia, Australia

    G McClorey, S Fletcher & S D Wilton

  2. AVI BioPharma Inc.,, Corvallis, OR, USA

    H M Moulton & P L Iversen

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  1. G McClorey
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  2. H M Moulton
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  3. P L Iversen
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  4. S Fletcher
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  5. S D Wilton
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Correspondence to S D Wilton.

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McClorey, G., Moulton, H., Iversen, P. et al. Antisense oligonucleotide-induced exon skipping restores dystrophin expression in vitro in a canine model of DMD. Gene Ther 13, 1373–1381 (2006). https://doi.org/10.1038/sj.gt.3302800

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