The potential of exon skipping for treatment for Duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy is mainly caused by mutations that disrupt the generation of a translatable mRNA transcript. Most such mutations occur in parts of the gene that are not essential for its function and thus might be eliminated from the transcript to permit translation of a partially functional protein that would convert the disease to a milder clinical form. Two such antisense oligonucleotides of different backbone chemistries have been successful when tested on the mdx mouse, targeting exon 23, containing the nonsense mutation. Subsequently, the morpholino, the more effective of these, has been tested on the dystrophic dog, where it is necessary to skip 2 exons, again with beneficial results. Currently, results of 2 human trials targeting exon 51 have also yielded promising preliminary results.

Figure 1

A. Diagram of the configuration of exon boundaries in the region of exon 45 of the normal dystrophin gene as represented in the pre-spliced RNA transcript. Flat ends represent coincidence of exon and codon boundaries. Small or large blocks at the boundary denote respectively one or 2 nucleotides beyond the last complete codon. In the normal gene, the bondaries of adjacent exons complement one another to maintain the open reading frame B. The most common Duchenne mutation, deletion of exon 45, puts 2 incompatible boundaries together in the spliced mRNA, resulting in a shift of open reading frame that generates a downstream nonsense sequence, resulting in premature termination of translation. The consequent lack of dystrophin produces a severe Duchenne pathology. C. When the deletion includes exon 46, the splicing of exon 44 to 47 leaves the open reading frame undisturbed and so that the mRNA can be translated into a slightly shortened but partly functional protein, resulting in the milder Becker muscular dystrophy phenotype. In some instances, boys with exon 45 deletions spontaneously skip exon 46 from a proportion of the transcripts, resulting in an unexpectedly mild clinical phenotype

Figure 2

A. The mdx mouse model of Duchenne muscular dystrophy is attributable to a nonsense mutation in exon 23 that blocks translation beyond this point B. Because exon 23 is not a frame-shifting exon, it can be skipped to generate a translatable transcript that produces dystrophin.

Figure 3

A. In the golden retriever muscular dystrophy dog, a single base mutation in the acceptor splice site of exon 7 prevents this exon from being spliced into the mRNA transcript. The resulting apposition of exons 6 and 8 produces a frame shift that prevents its productive translation. B. To restore open reading frame to this mutation, a skip is required of at least 2 exons, 6 and 8. When this is provoked by use of oligonucleotides directed against sequences involved in the splicing of these 2 exons, exon 9 is also lost from the transcript. Since this is not a frame-shifting exon, the resulting mRNA, lacking exons 5–10, is translatable into a partially functional dystrophin protein.