Antisense Mediated Dystrophin Reading Frame Restoration

Exon skipping using antisense oligonucleotides (AONs) has been used to reframe the mRNA in patients derived cell cultures and in vivo in the mdxmouse model. The performed studies can be divided into two parts: in the first part we have tested the feasibility of the exon skipping approach for patients with small mutations in in-frame exons, while in the second part a quantitative comparison of exon skipping revealing techniques is addressed. We first identified 55 novel DMD causing small mutations. We selected 5 patients with nonsense or frameshifting mutations in exons 10, 16, 26, 33 and 34. Antisense oligonucleotides targeting the control sequence of these exons and mutation specific AONs were tested in cell-free splicing assays and in patients derived cultured cells. The obtained results confirm cell-free splicing assay as an alternative system to test exon skipping propensity when patients cells are unavailable. In myogenic cells, comparable levels of exon skipping were observed for exons 16, 26 and 33 for AONs targeting the mutation or the adjacent sequences. For exon 10 the mutation specific AON was more effective, while for exon 34 the AON targeting the adjacent sequence was more effective. Interestingly, in some cases skipping efficiencies for mutated exons were quite dissimilar compared to what previously reported for the respective wild type exons. This behaviour may be related to effect of the mutations on exon skipping propensity and highlights the complexity of identifying optimal AONs for skipping exons with small mutations. In the second part we compared different methods to quantify exon skipping in vivo in mdxmice. Absolute quantification using a digital array allowed to provide an estimate of the copies of dystrophin mRNA per ng of total RNA in muscle in vivo.