title: Staurosporine allows dystrophin expression by skipping of nonsense-encoding exon.
abstract: BACKGROUND
  Antisense oligonucleotides that induce exon skipping have been nominated as the most plausible treatment method for dystrophin expression in dystrophin-deficient Duchenne muscular dystrophy. Considering this therapeutic efficiency, small chemical compounds that can enable exon skipping have been highly awaited. In our previous report, a small chemical kinase inhibitor, TG003, was shown to enhance dystrophin expression by enhancing exon skipping.
  
  
  OBJECTIVE
  Staurosporine (STS), a small chemical broad kinase inhibitor, was examined for enhanced skipping of a nonsense-encoding dystrophin exon.
  
  
  METHODS
  STS was added to culture medium of HeLa cells transfected with minigenes expressing wild-type or mutated exon 31 with c.4303G>T (p.Glu1435X), and the resulting mRNAs were analyzed by RT-PCR amplification. Dystrophin mRNA and protein were analyzed in muscle cells treated with STS by RT-PCR and western blotting, respectively.
  
  
  RESULTS
  STS did not alter splicing of the wild-type minigene. In the mutated minigene, STS increased the exon 31-skipped product. A combination of STS and TG003 did not significantly increase the exon 31-skipped product. STS enhanced skipping of exon 4 of the CDC-like kinase 1 gene, whereas TG003 suppressed it. Two STS analogs with selective kinase inhibitory activity did not enhance the mutated exon 31 skipping. When immortalized muscle cells with c.4303G>T in the dystrophin gene were treated with STS, skipping of the mutated exon 31 and dystrophin expression was enhanced.
  
  
  CONCLUSIONS
  STS, a broad kinase inhibitor, was shown to enhance skipping of the mutated exon 31 and dystrophin expression, but selective kinase inhibitors did not.
authors.affiliation: Department of Physical Therapy, Faculty of Rehabilitation, Kobe Gakuin University, Japan; Biopharmaceutical Innovation Research Department, Research Institute, Research Division, JCR Pharmaceuticals Co. Ltd., Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nishida
authors.affiliation: Department of Physical Therapy, Faculty of Rehabilitation, Kobe Gakuin University, Japan; Department of Clinical Pharmacology, Kobe Pharmaceutical University, Japan.
authors.forename: Ayaka
authors.initials: A
authors.lastname: Oda
authors.affiliation: Department of Clinical Pharmacology, Kobe Pharmaceutical University, Japan.
authors.forename: Atsuko
authors.initials: A
authors.lastname: Takeuchi
authors.affiliation: Department of Pediatrics, Hyogo College of Medicine, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Lee
authors.affiliation: Department of Pediatrics, Kobe University Graduate School of Medicine, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Awano
authors.affiliation: Department of Regenerative Medicine, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Japan.
authors.forename: Naohiro
authors.initials: N
authors.lastname: Hashimoto
authors.affiliation: Department of Pediatrics, Hyogo College of Medicine, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Takeshima
authors.affiliation: Department of Physical Therapy, Faculty of Rehabilitation, Kobe Gakuin University, Japan. Electronic address: mmatsuo@reha.kobegakuin.ac.jp.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Matsuo
mesh_terms: D018389:Codon, Nonsense; D003893:Desmin / Q000378:metabolism; D016189:Dystrophin / Q000378:metabolism*; D005091:Exons; D006367:HeLa Cells; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D019311:Staurosporine / Q000378:metabolism*; D014162:Transfection
chemical_list: D018389:Codon, Nonsense; D003893:Desmin; D016189:Dystrophin; C066780:Clk dual-specificity kinases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019311:Staurosporine
keywords: Chemical; Dystrophin; Exon skipping; Nonsense mutation; Staurosporine