title: Exon-skipping events in candidates for clinical trials of morpholino.
abstract: 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.
  
  
  CONCLUSIONS
  An in vitro system was developed that could easily screen the effectiveness of antisense sequences and identify good candidates for therapy with morpholino.
authors.affiliation: Department of Child Development Pediatrics, Kumamoto University Graduate School, Kumamoto, Japan.
authors.forename: Shiho
authors.initials: S
authors.lastname: Nakano
authors.forename: Shiro
authors.initials: S
authors.lastname: Ozasa
authors.forename: Kowashi
authors.initials: K
authors.lastname: Yoshioka
authors.forename: Isao
authors.initials: I
authors.lastname: Fujii
authors.forename: Kouichi
authors.initials: K
authors.lastname: Mitsui
authors.forename: Keiko
authors.initials: K
authors.lastname: Nomura
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Kosuge
authors.forename: Fumio
authors.initials: F
authors.lastname: Endo
authors.forename: Makoto
authors.initials: M
authors.lastname: Matsukura
authors.forename: Shigemi
authors.initials: S
authors.lastname: Kimura
mesh_terms: D002986:Clinical Trials as Topic; D004247:DNA / Q000235:genetics; D005091:Exons; D005347:Fibroblasts; D015316:Genetic Therapy; D006801:Humans; D066298:In Vitro Techniques; D060172:Morpholinos / Q000627:therapeutic use*; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics / Q000628:therapy*; D016376:Oligonucleotides, Antisense / Q000627:therapeutic use*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D013873:Thionucleotides / Q000627:therapeutic use; D014162:Transfection
chemical_list: D060172:Morpholinos; D016376:Oligonucleotides, Antisense; D013873:Thionucleotides; D004247:DNA