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

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.

Relevant aspects of golden retriever muscular dystrophy for the study of Duchenne muscular dystrophy in humans

“…CRISPRCas9 technology has already been used in the correction of the mutant dystrophin gene in mice, avoiding the development of muscular dystrophy, and also in the repair of cystic fibrosis transmembrane receptor locus by homologous recombination in cultured intestinal stem cells from human patients with this disease (WU et al, 2013). LI et al (2015) performed the genetic correction of the gene that causes muscular dystrophy in humans and obtained promising results with three different techniques, demonstrating that the CRISPR technique was Table 3 -Summary of muscular dystrophy therapies used in dogs. equally effective in the TALEN technique and that both obtained minimal mutagenic effects outside the initial target when directed to a single sequence region.…”

Mor på en ny måte

“…Systemet har vist seg effektivt i korreksjon av sykdomsfremkallende genvarianter i iPS-celler isolert og laget fra pasienter med Duchenne muskeldystrofi 210 og β-thalassemi 211 . Genmodifiserte iPSC har ennå ikke vaert prøvet ut i klinikken.…”

Excitation et crispations autour de CRISPR : lorsque la réalité dépasse la science-fiction

“…C'est pourquoi les protocoles d'essais cliniques de thérapie génique conventionnelle par transfert de gène exigent d'exclure tout risque de contamination des lignées germinales. Aussi, lorsque la révolution CRISPR a fait son irruption, les seules éditions pratiquées sur du matériel humain l'ont été ex vivo sur le génome somatique de cellules en culture, et en particulier des cellules souches pluripotentes induites (en anglais Induced pluripotent stem cells ou hiPSC) 43,44,45 . Or, le tabou du génome germinal humain a été transgressé en Chine au printemps 2015 avec une expé-rience d'édition du génome d'embryons humains non viables et non suivie de réimplantation utérine, la cible étant le gène de la bêta-globine 46 .…”