ANTISENSE MEDIATED DYSTROPHIN READING FRAME RESTORATION

Exon skipping using antisense oligonucleotides (AONs) has successfully been used to \nreframe the mRNA in various DMD (Duchenne muscular dystrophy) patients carrying \ndeletions and in the mdx mouse model. This study can be devided in two parts: in the \nfirst part we have tested the feasibility of the exon skipping approach for patients with \nsmall mutations in in-frame exons, while in the second part a quantitative comparison of \nexon skipping revealing techniques is addressed. \nWe first identified 55 novel disease-causing point mutations. We selected 5 patients \nwith nonsense or frameshifting mutations in exons 10, 16, 26, 33 and 34. Wild type and \nmutation specific 2‟OMePS AONs were tested in cell-free splicing assays and in \ncultured cells derived from the selected patients. The results obtained confirm cell-free \nsplicing assay as an alternative system to test exon skipping propensity when patients‟ \ncells are unavailable. In myogenic cells, similar levels of exon skipping were observed \nfor wild type and mutation specific AONs for exons 16, 26 and 33, while for exon 10 and \nexon 34 the efficiency of the AONs was significantly different. Interestingly, in some \ncases skipping efficiencies for mutated exons were quite dissimilar compared to what \npreviously reported for the respective wild type exons. This behaviour may be related to \neffect of the mutations on exon skipping propensity and highlights the complexity of \nidentifying optimal AONs for skipping exons with small mutations. \nIn the second part we compared different techniques to reveal the exon skipping levels \nin the muscles of 7 different mdx mice. An absolute quantification of the dystrophin \ntranscript amount was possible using a digital array. Results underline the low \nexpression of the dytrophin gene and the amount needed to correctly quantify the exon \nskipping percentage.