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Stem Cell Reports. 2015 Jan 13;4(1):143-54. doi: 10.1016/j.stemcr.2014.10.013. Epub 2014 Nov 26.

Precise correction of the dystrophin gene in duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR-Cas9.

Author information

1
Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan.
2
Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan; iCeMS, Kyoto University, Kyoto 606-8501, Japan.
3
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.
4
Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan; iCeMS, Kyoto University, Kyoto 606-8501, Japan; Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA.
5
Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan; iCeMS, Kyoto University, Kyoto 606-8501, Japan; PRESTO, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan. Electronic address: akitsu.hotta@cira.kyoto-u.ac.jp.

Abstract

Duchenne muscular dystrophy (DMD) is a severe muscle-degenerative disease caused by a mutation in the dystrophin gene. Genetic correction of patient-derived induced pluripotent stem cells (iPSCs) by TALENs or CRISPR-Cas9 holds promise for DMD gene therapy; however, the safety of such nuclease treatment must be determined. Using a unique k-mer database, we systematically identified a unique target region that reduces off-target sites. To restore the dystrophin protein, we performed three correction methods (exon skipping, frameshifting, and exon knockin) in DMD-patient-derived iPSCs, and found that exon knockin was the most effective approach. We further investigated the genomic integrity by karyotyping, copy number variation array, and exome sequencing to identify clones with a minimal mutation load. Finally, we differentiated the corrected iPSCs toward skeletal muscle cells and successfully detected the expression of full-length dystrophin protein. These results provide an important framework for developing iPSC-based gene therapy for genetic disorders using programmable nucleases.

PMID:
25434822
PMCID:
PMC4297888
DOI:
10.1016/j.stemcr.2014.10.013
[Indexed for MEDLINE]
Free PMC Article

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