Exon-skipping events in candidates for clinical trials of morpholino
Article first published online: 18 AUG 2011
© 2011 The Authors. Pediatrics International © 2011 Japan Pediatric Society
Volume 53, Issue 4, pages 524–529, August 2011
How to Cite
Nakano, S., Ozasa, S., Yoshioka, K., Fujii, I., Mitsui, K., Nomura, K., Kosuge, H., Endo, F., Matsukura, M. and Kimura, S. (2011), Exon-skipping events in candidates for clinical trials of morpholino. Pediatrics International, 53: 524–529. doi: 10.1111/j.1442-200X.2011.03330.x
- Issue published online: 18 AUG 2011
- Article first published online: 18 AUG 2011
- Accepted manuscript online: 22 FEB 2011 03:06PM EST
- Received 9 April 2010; revised 5 November 2010; accepted 30 November 2010.
- Duchenne muscular dystrophy;
- exon skipping;
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 preserve 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. It is necessary, however, 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. The cells were then transfected with two types of morpholino. The exon-skipping event was analyzed on reverse transcription–polymerase chain reaction.
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 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 unskipped 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: An in vitro system was developed that could easily screen the effectiveness of antisense sequences and identify good candidates for therapy with morpholino.