Investigating the effect of DMD non-sequential splicing on exon skipping strategies

Duchenne muscular dystrophy is one of the most common myopathies, with an incidence of 1:5000 in new-born boys. The root cause of the disease are mutations in the dystrophin (DMD) gene which disrupt the reading frame. The use of antisense oligonucleotides (AON) to induce exon skipping is a therapy aimed at restoring the dystrophin reading frame to form a partially functional protein. Two AONs have been approved by the Food and Drug Administration (FDA, USA). DMD is the longest gene in the human genome, spanning over 2 megabases on chromosome X. The large size of the DMD muscle transcript (Dp427m) is largely explained by the size of its 78 introns, which can be up to 250 kilobases in length. Introns are removed from the transcript by splicing, ultimately joining together the exons to generate a messenger RNA that is translated into protein. Previous work from our lab has determined that of the 78 DMD introns, almost half are not spliced sequentially. Exons can be flanked by introns which are spliced out rapidly ("fast"), or are retained in the transcript longer ("slow"). This means that certain introns are only removed from the transcript after a downstream intron has already been spliced out. This has implications for the skippability of dystrophin exons, where exons flanked by slowly spliced introns should be easier to skip than those flanked by quickly spliced introns. To test this hypothesis, we have selected a series of DMD exons preceded and/or followed by either slow- or fast-splicing introns. We will use a large set of unique phosphorodiamidate morpholino oligomers (PMOs) to assess whether there is a correlation between the speed of intron removal and the efficiency of exon skipping in in vitro cultured myocytes. Data obtained from this approach will further our understanding of the dynamics of the DMD transcript, and prove valuable for selecting the next promising exon to target for treatment of DMD.