ANTISENSE MEDIATED DYSTROPHIN READING FRAME RESTORATION

Exon skipping using antisense oligonucleotides (AONs) has successfully been used to reframe the mRNA in various DMD (Duchenne muscular dystrophy) patients carrying deletions and in the mdx mouse model. This study can be devided in 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 disease-causing point mutations. We selected 5 patients with nonsense or frameshifting mutations in exons 10, 16, 26, 33 and 34. Wild type and mutation specific 2‟OMePS AONs were tested in cell-free splicing assays and in cultured cells derived from the selected patients. The results obtained confirm cell-free splicing assay as an alternative system to test exon skipping propensity when patients‟ cells are unavailable. In myogenic cells, similar levels of exon skipping were observed for wild type and mutation specific AONs for exons 16, 26 and 33, while for exon 10 and exon 34 the efficiency of the AONs was significantly different. 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 techniques to reveal the exon skipping levels in the muscles of 7 different mdx mice. An absolute quantification of the dystrophin transcript amount was possible using a digital array. Results underline the low expression of the dytrophin gene and the amount needed to correctly quantify the exon skipping percentage.