Atsushi Nishida1, Ayaka Oda2, Atsuko Takeuchi3, Tomoko Lee4, Hiroyuki Awano5, Naohiro Hashimoto6, Yasuhiro Takeshima4, Masafumi Matsuo7. 1. Department of Physical Therapy, Faculty of Rehabilitation, Kobe Gakuin University, Japan; Biopharmaceutical Innovation Research Department, Research Institute, Research Division, JCR Pharmaceuticals Co. Ltd., Japan. 2. Department of Physical Therapy, Faculty of Rehabilitation, Kobe Gakuin University, Japan; Department of Clinical Pharmacology, Kobe Pharmaceutical University, Japan. 3. Department of Clinical Pharmacology, Kobe Pharmaceutical University, Japan. 4. Department of Pediatrics, Hyogo College of Medicine, Japan. 5. Department of Pediatrics, Kobe University Graduate School of Medicine, Japan. 6. Department of Regenerative Medicine, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Japan. 7. Department of Physical Therapy, Faculty of Rehabilitation, Kobe Gakuin University, Japan. Electronic address: mmatsuo@reha.kobegakuin.ac.jp.
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. PURPOSE: 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.
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. PURPOSE: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.