Induction of revertant fibres in the mdx mouse using antisense oligonucleotides

Abbie M Fall,Pat Iversen,Susan Fletcher,Russell Johnsen,Kaite Honeyman,Stephen D Wilton

doi:10.1186/1479-0556-4-3

Abbie M Fall, Pat Iversen + Show 4 more

Open Access

https://doi.org/10.1186/1479-0556-4-3

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Abstract

BackgroundDuchenne muscular dystrophy is a fatal genetic disorder caused by dystrophin gene mutations that result in premature termination of translation and the absence of functional protein. Despite the primary dystrophin gene lesion, immunostaining studies have shown that at least 50% of DMD patients, mdx mice and a canine model of DMD have rare dystrophin-positive or 'revertant' fibres. Fine epitope mapping has shown that the majority of transcripts responsible for revertant fibres exclude multiple exons, one of which includes the dystrophin mutation.MethodsThe mdx mouse model of muscular dystrophy has a nonsense mutation in exon 23 of the dystrophin gene. We have shown that antisense oligonucleotides (AOs) can induce the removal of this exon, resulting in an in-frame mRNA transcript encoding a shortened but functional dystrophin protein. To emulate one exonic combination associated with revertant fibres, we target multiple exons for removal by the application of a group of AOs combined as a "cocktail".ResultsExons 19–25 were consistently excluded from the dystrophin gene transcript using a cocktail of AOs. This corresponds to an alternatively processed gene transcript that has been sporadically detected in untreated dystrophic mouse muscle, and is presumed to give rise to a revertant dystrophin isoform. The transcript and the resultant correctly localised smaller protein were confirmed by RT-PCR, immunohistochemistry and western blot analysis.ConclusionThis work demonstrates the feasibility of AO cocktails to by-pass dystrophin mutation hotspots through multi-exon skipping. Multi-exon skipping could be important in expediting an exon skipping therapy to treat DMD, so that the same AO formulations may be applied to several different mutations within particular domains of the dystrophin gene.

Highlights

Duchenne muscular dystrophy is a fatal genetic disorder caused by dystrophin gene mutations that result in premature termination of translation and the absence of functional protein
In this report we describe the in vitro evaluation and optimisation of an antisense oligonucleotides (AOs) cocktail designed to remove exons 19– 25, first using 2'-O-methyl phosphorothioate (2OMe) AOs and subsequently phosphorodiamidite morpholino oligonucleotides (PMOs), to induce multiple dystrophin exon skipping in the mdx mouse
The likelihood of successful exon skipping after targeting any particular exonic splicing enhancer (ESE) region increased when more than one serine/arginine-rich (SR) binding site was covered by the AO

Summary

Introduction

Duchenne muscular dystrophy is a fatal genetic disorder caused by dystrophin gene mutations that result in premature termination of translation and the absence of functional protein. Despite the primary dystrophin gene lesion, immunostaining studies have shown that at least 50% of DMD patients, mdx mice and a canine model of DMD have rare dystrophin-positive or 'revertant' fibres. Fine epitope mapping has shown that the majority of transcripts responsible for revertant fibres exclude multiple exons, one of which includes the dystrophin mutation. Duchenne muscular dystrophy (DMD), the most wellknown of the nine major types of muscular dystrophy, is a severe muscle-wasting disease that arises from mutations in the dystrophin gene (Xp21.2) (review [1,2]). Revertant fibres tend to increase in frequency with age [6,13] in both human and animal models of DMD, possibly indicating a selective advantage over dystrophin negative fibres

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