Journal
STEM CELL REPORTS
Volume 4, Issue 1, Pages 143-154Publisher
CELL PRESS
DOI: 10.1016/j.stemcr.2014.10.013
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Funding
- JSPS KAKENHI
- JST PRESTO
- JST Yamanaka iPS Cell Special Project
- JST Research Center Network for Realization of Regenerative Medicine
- JSPS DC1 fellowship
- Grants-in-Aid for Scientific Research [25890014] Funding Source: KAKEN
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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.
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