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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


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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TS was supported by Grant-in-Aid for JSPS Research Fellow Grant Number 15J05689. SO was supported by the Basic Science and Platform Technology Program for Innovative Biological Medicine from Japan Agency for Medical Research and Development (AMED). TY was supported by the Friends of Garrett Cumming Research Chair Fund, the HM Toupin Neurological Science Research Chair Fund, Muscular Dystrophy Canada, the Canada Foundation for Innovation (CFI), Alberta Advanced Education and Technology (AET), Canadian Institutes of Health Research (CIHR), Jesse’s Journey – The Foundation for Gene and Cell Therapy, the University of Alberta Faculty of Medicine and Dentistry, and the Women and Children’s Health Research Institute (WCHRI).

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Correspondence to Satoshi Obika.

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Shimo, T., Hosoki, K., Nakatsuji, Y. et al. A novel human muscle cell model of Duchenne muscular dystrophy created by CRISPR/Cas9 and evaluation of antisense-mediated exon skipping. J Hum Genet 63, 365–375 (2018).

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