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Checking exon-skipping events in candidates for clinical trials of morpholino.
MedLine Citation:
PMID:  21342350     Owner:  NLM     Status:  Publisher    
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.
Shiho Nakano; Shiro Ozasa; Kowashi Yoshioka; Isao Fujii; Kouichi Mitsui; Keiko Nomura; Hirofumi Kosuge; Fumio Endo; Makoto Matsukura; Shigemi Kimura
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2011-2-22
Journal Detail:
Title:  Pediatrics international : official journal of the Japan Pediatric Society     Volume:  -     ISSN:  1442-200X     ISO Abbreviation:  -     Publication Date:  2011 Feb 
Date Detail:
Created Date:  2011-2-23     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  100886002     Medline TA:  Pediatr Int     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
© 2011 The Authors. Pediatrics International © 2011 Japan Pediatric Society.
Department of Child Development, Kumamoto University Graduate School, Kumamoto, Japan Laboratory of Clinical Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan Department of Pediatrics, Kumamoto University Graduate School, Kumamoto, Japan.
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