Creation of DMD Muscle Cell Model Using CRISPR-Cas9 Genome Editing to Test the Efficacy of Antisense-Mediated Exon Skipping
Duchenne muscular dystrophy (DMD) is a devastating muscle disorder caused by mutations in the DMD gene. Antisense-mediated exon skipping is a promising strategy to treat DMD. The approval of Exondys 51 (eteplirsen) targeting exon 51 was the most noteworthy accomplishment in 2016. To evaluate and optimize the sequence of antisense oligonucleotides (AOs), muscle cell lines with DMD mutations are useful tools. However, there are only several immortalized muscle cell lines with DMD mutations available that can be used to test the efficacy of exon skipping in vitro. In addition, an invasive muscle biopsy is required to obtain muscle cells from patients. Furthermore, many DMD mutations are very rare and it is hard to find a patient with a specific mutation for muscle biopsy in many cases. Here, we describe a novel approach to create an immortalized muscle cell line with a DMD deletion mutation using the human rhabdomyosarcoma (RD) cell line and the CRISPR/Cas9 system that can be used to test the efficacy of exon skipping.
Key wordsExon skipping/inclusion Antisense oligonucleotides (AOs) Splice switching oligonucleotides (SSOs) Duchenne/Becker muscular dystrophy (DMD/BMD) Clustered regularly interspaced short palindromic repeat/CRISPR associated protein 9 (CRISPR/Cas9)-mediated genome editing Phosphorodiamidate morpholino oligomers (PMOs) Golodirsen 2′-O-methyl RNA NS-065/NCNP-01 The human rhabdomyosarcoma (RD) cell line
This work is supported by the Muscular Dystrophy Canada, the Friends of Garrett Cumming Research Fund, the HM Toupin Neurological Science Research Fund, the Canadian Institutes of Health Research (CIHR), the Alberta Innovates: Health Solutions (AIHS), the Canada Foundation for Innovation (CFI), the Alberta Advanced Education and Technology, and the Women and Children’s Health Research Institute (WCHRI).
- 4.Nakamura A, Fueki N, Shiba N et al (2016) Deletion of exons 3-9 encompassing a mutational hot spot in the DMD gene presents an asymptomatic phenotype, indicating a target region for multiexon skipping therapy. J Hum Genet 61(7):663–667. https://doi.org/10.1038/jhg.2016.28 CrossRefPubMedGoogle Scholar
- 6.Nakamura A, Shiba N, Miyazaki D et al (2016) Comparison of the phenotypes of patients harboring in-frame deletions starting at exon 45 in the Duchenne muscular dystrophy gene indicates potential for the development of exon skipping therapy. J Hum Genet. https://doi.org/10.1038/jhg.2016.152 CrossRefGoogle Scholar
- 7.Echigoya Y, Nakamura A, Nagata T et al (2017) Effects of systemic multiexon skipping with peptide-conjugated morpholinos in the heart of a dog model of Duchenne muscular dystrophy. Proc Natl Acad Sci U S A 114(16):4213–4218. https://doi.org/10.1073/pnas.1613203114 CrossRefPubMedPubMedCentralGoogle Scholar
- 17.Komaki H, Nagata T, Saito T, Masuda S, Takeshita E, Sasaki M, Tachimori H, Nakamura H, Aoki Y, Takeda S’i (2018) Systemic administration of the antisense oligonucleotide NS-065/NCNP-01 for skipping of exon 53 in patients with Duchenne muscular dystrophy. Sci Transl Med 10(437):eaan0713CrossRefGoogle Scholar