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

Stem Cell Reports. 2015 Jan 13;4(1):143-154. doi: 10.1016/j.stemcr.2014.10.013. Epub 2014 Nov 26.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • CRISPR-Cas Systems / genetics*
  • DNA Copy Number Variations
  • Dystrophin / genetics*
  • Dystrophin / metabolism
  • Exons
  • Gene Order
  • Gene Targeting
  • Genetic Loci
  • Genetic Therapy
  • Humans
  • Induced Pluripotent Stem Cells / cytology*
  • Induced Pluripotent Stem Cells / metabolism*
  • Karyotype
  • Molecular Sequence Data
  • Muscle Fibers, Skeletal / metabolism
  • Muscular Dystrophy, Duchenne / genetics*
  • Muscular Dystrophy, Duchenne / metabolism
  • Muscular Dystrophy, Duchenne / therapy
  • Mutagenesis, Insertional
  • Mutation
  • Reading Frames
  • Sequence Deletion

Substances

  • Dystrophin