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Multiple exon skipping in the dystrophin gene: A more effective therapy?

Errington, S.J., Mann, C.J., Fletcher, S. and Wilton, S.D. (2003) Multiple exon skipping in the dystrophin gene: A more effective therapy? In: 3rd Meeting of Australasian Gene Therapy Society, 30 April - 2 May 2003, Queensland Institute of Medical Research, Brisbane.


Duchenne muscular dystrophy (DMD) is an X-linked recessive muscle wasting disorder characterised by the absence of the protein dystrophin. Rare dystrophin-positive revertant fibres exist in DMD muscle, arising from a naturally occurring phenomenon where the splicing machinery has been re-directed to exclude the DMD mutation. Furthermore, protein and mRNA transcript studies have indicated that revertant fibres in the mouse model of muscular dystrophy involved skipping of at least 5 exons. The most common revertant fibres were missing more than 20 exons. Alternatively processed dystrophin gene transcripts missing only exon 23, the minimum rearrangement to by-pass the mutation, have never been detected in untreated mdx muscle. Insitu hybridisation studies indicated that there were no gross genomic rearrangements and suggested that the revertant exon skipping arose through alternative splicing.

Antisense oligonucleotides have been used to re-direct dystrophin pre-mRNA processing by blocking sequences crucial to pre-mRNA splicing, thereby inducing skipping of target exons. We wished to determine if AO targeting of multiple exons in the dystrophin gene transcript was possible and if we could induce a revertant-like transcript. Based on the observation that revertant fibres increase with age, there may be some selective advantage to dystrophin missing the protein encoded by several exons. The revertant dystrophins may be more biologically functional than a protein missing only 71 amino acids encoded by exon 23.

Antisense oligonucleotides were directed at motifs involved in pre-mRNA splicing of dystrophin exons 19-25. Cultured mdx mouse myotubes were transfected with either single antisense oligonucleotides or combinations. RT-PCR studies were undertaken to determine both specificity and sensitivity of induced multiple-exon skipping. Singly, each antisense oligonucleotide was able to induce the skipping of the target dystrophin exon at which it was directed. Selected combinations of antisense oligonucleotides were able to induce the removal of the targeted exons. No other regions of the dystrophin message were altered in the final transcript as a consequence of these antisense oligonucleotide treatments. The ability to manipulate the outcome of the final message provides real potential for a genetic therapy, as these modifications may emulate naturally occurring processes within the dystrophic muscles of Duchenne muscular dystrophy patients.

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