Figure 3

Exon skipping in myotube cultures derived from an exon 44 duplicated patient. A. Schematic overview of the mutation. Original and duplicated exons are shown in blue and red, respectively. Restoration of the normal transcript can be achieved by skipping of either the original or mutated exon 44 (upper panel) but skipping of both exons 44 results in out-of-frame transcripts (middle panel). Skipping exon 43 and both exons 44 will restore the reading frame (lower panel). B. RT-PCR analysis. Low levels of single exon 44 skipping were observed in the non treated sample (NT). After treatment with exon 44 specific AONs (44) both copies of exon 44 were skipped in the majority of the transcripts, generating an out-of-frame product. Using a combination of AONs targeting exon 43 and 44 (43, 44) it was feasible to induce an in-frame transcript where exon 43 is skipped in addition to both copies of exon 44. In-frame and out-of-frame transcripts are depicted in green and red, respectively. Duplicated exons are shaded in blue. M is 100 bp size standard, -RT is negative control. C. Western blot analysis. Dystrophin signals were detected in protein isolated 2 and 5 days (2d and 5d) after AON treatment and not in an untreated sample (NT). The control sample (HC) was diluted 10 times to prevent overexposure. Myosin staining was used to confirm equal sample loading (myo). D. Immuno-histochemical analysis. No dystrophin signal was detected in non-treated myosin-positive myotubes with either the Dy8 (data not shown) or the MANDYS1 antibody. After AON treatment over 70% of myosin positive cells stained positive for dystrophin as detected with Dy8 (data not shown) and MANDYS1.