Exon-skipping events in candidates for clinical trials of morpholino

Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by abnormalities in the DMD gene. The majority of DMD patients have out-of-frame deletion(s), which disrupt the reading frame; while some cases of DMD are caused by duplication or nonsense mutation(s). Most patients with BMD have in-frame deletion(s), which keep the reading frame. The phenotype of BMD is generally milder than that of DMD. Antisense morpholino-mediated exon-skipping, which changes out-of-frame deletions to in-frame deletions, is a promising therapeutic approach for DMD. However, it is necessary to confirm the exon-skipping event in cells of DMD patients before the clinical trial. Methods: Fibroblasts isolated from four DMD patients were induced to differentiate into the myogenic lineage by infection with Ad.CAGMyoD. Then, the cells were transfected with two types of morpholino. The exon-skipping event was analyzed by RT-PCR. Results: Morpholino B30, which is located at the splicing enhancer of exon 51 of the DMD gene, yielded the desired exon-51-skipping event in all deletion patterns of cells we tested. Morpholino I25, which is located at the exon donor, induced two different exon-skipping patterns, which are total or partial exon-51-skipping events. According to the sequence analysis, the unexpected un-skipped regions were the 95-bp section and the 188-bp section of exon 51, showing that the cryptic splicing donor was newly produced with I25. Unfortunately, these cryptic splicing donors gave rise to out-of-frame patterns. Based on these in vitro results, B30 would presumably be an effective therapy. Interestingly, the cocktail of B30 and I25 appeared to yield a more efficient exon-51-skipping event. Conclusion: We developed an in vitro system that could easily screen the effectiveness of antisense sequences and identify good candidates for therapy with morpholino.