Precise Correction of the Dystrophin Gene in Duchenne Muscular Dystrophy Patient Induced Pluripotent Stem Cells by TALEN and CRISPR-Cas9

Hongmei Lisa Li,Naoko Fujimoto,Noriko Sasakawa,Saya Shirai,Tokiko Ohkame,Tetsushi Sakuma,Michihiro Tanaka,Naoki Amano,Akira Watanabe,Hidetoshi Sakurai,Takashi Yamamoto,Shinya Yamanaka,Akitsu Hotta

doi:10.1016/j.stemcr.2014.10.013

Hongmei Lisa Li, Naoko Fujimoto + Show 11 more

Open Access

https://doi.org/10.1016/j.stemcr.2014.10.013

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Abstract

SummaryDuchenne 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.

Highlights

Duchenne muscular dystrophy (DMD) is a severe muscular degenerative disease caused by loss-of-function mutations in the dystrophin gene located on the X chromosome
Duchenne muscular dystrophy (DMD) is a severe muscle-degenerative disease caused by a mutation in the dystrophin gene
We further investigated the genomic integrity by karyotyping, copy number variation array, and exome sequencing to identify clones with a minimal mutation load

Summary

Introduction

Duchenne muscular dystrophy (DMD) is a severe muscular degenerative disease caused by loss-of-function mutations in the dystrophin gene located on the X chromosome. The dystrophin gene consists of 79 exons, and disruption of the protein reading frame by small deletions, exon duplications, or loss of exons leads to DMD (Pichavant et al, 2011). The delivery of truncated microdystrophin or microutrophin by an adeno-associated viral (AAV) vector (Okada and Takeda, 2013), lentiviral vector (Pichavant et al, 2011), or Sleeping Beauty transposon (Filareto et al, 2013) has been investigated for DMD gene therapy. Genomic correction using programmable nucleases is an ideal approach that can correct the mutated dystrophin gene

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