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A novel human muscle cell model of Duchenne muscular dystrophy created by CRISPR/Cas9 and evaluation of antisense-mediated exon skipping

Authors
  • Shimo, Takenori1, 2
  • Hosoki, Kana2
  • Nakatsuji, Yusuke1
  • Yokota, Toshifumi2, 3
  • Obika, Satoshi1
  • 1 Osaka University, Graduate School of Pharmaceutical Sciences, 1–6 Yamadaoka, Suita, Osaka, 565–0871, Japan , Suita (Japan)
  • 2 University of Alberta, Department of Medical Genetics, Faculty of Medicine and Dentistry, 8-31 Medical Science Building, Edmonton, AB, T6G 2H7, Canada , Edmonton (Canada)
  • 3 University of Alberta, Muscular Dystrophy Canada Research Chair, Edmonton, AB, T6G 2H7, Canada , Edmonton (Canada)
Type
Published Article
Journal
Journal of Human Genetics
Publisher
Springer Nature
Publication Date
Jan 16, 2018
Volume
63
Issue
3
Pages
365–375
Identifiers
DOI: 10.1038/s10038-017-0400-0
Source
Springer Nature
License
Yellow

Abstract

Oligonucleotide-mediated splicing modulation is a promising therapeutic approach for Duchenne muscular dystrophy (DMD). Recently, eteplirsen, a phosphorodiamidate morpholino oligomer-based splice-switching oligonucleotide (SSO) targeting DMD exon 51, was approved by the U.S. Food and Drug Administration as the first antisense-based drug for DMD patients. For further exploring SSOs targeting other exons in the DMD gene, the efficacy of exon skipping and protein rescue with each SSO sequence needs evaluations in vitro. However, only a few immortalized muscle cell lines derived from DMD patients have been reported and are available to test the efficacy of exon skipping in vitro. To solve this problem, we generated a novel immortalized DMD muscle cell line from the human rhabdomyosarcoma (RD) cell line. We removed DMD exons 51–57 (~0.3 Mb) in the RD cell line using the CRISPR/Cas9 system. Additionally, in this DMD model cell line, we evaluated the exon 50 skipping activity of previously reported SSOs at both the mRNA and protein levels. CRISPR/Cas9-mediated gene editing of the DMD gene in the RD cell line will allow for assessment of SSOs targeting most of the rare mutations in the DMD gene.

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