Reading Frame Correction by Targeted Genome Editing Restores Dystrophin Expression in Cells From Duchenne Muscular Dystrophy Patients

Genome editing with engineered nucleases has recently emerged as an approach to correct genetic mutations by enhancing homologous recombination with a DNA repair template. However, many genetic diseases, such as Duchenne muscular dystrophy (DMD), can be treated simply by correcting a disrupted readi...

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Bibliographic Details
Published in:Molecular therapy 2013-09, Vol.21 (9), p.1718-1726
Main Authors: Ousterout, David G, Perez-Pinera, Pablo, Thakore, Pratiksha I, Kabadi, Ami M, Brown, Matthew T, Qin, Xiaoxia, Fedrigo, Olivier, Mouly, Vincent, Tremblay, Jacques P, Gersbach, Charles A
Format: Article
Language:English
Subjects:
Biomedical engineering
Cytotoxicity
DNA repair
Dystrophin - biosynthesis
Dystrophin - genetics
Dystrophin - metabolism
Endonucleases - genetics
Endonucleases - metabolism
Exome
Gene Targeting
Genes
Genetic disorders
Genetic Therapy - methods
Genome, Human
Genomes
HEK293 Cells
Humans
Muscular dystrophy
Muscular Dystrophy, Duchenne - genetics
Muscular Dystrophy, Duchenne - metabolism
Muscular Dystrophy, Duchenne - therapy
Mutation
Original
Protein expression
Proteins
Reading
Reading Frames
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Summary:Genome editing with engineered nucleases has recently emerged as an approach to correct genetic mutations by enhancing homologous recombination with a DNA repair template. However, many genetic diseases, such as Duchenne muscular dystrophy (DMD), can be treated simply by correcting a disrupted reading frame. We show that genome editing with transcription activator-like effector nucleases (TALENs), without a repair template, can efficiently correct the reading frame and restore the expression of a functional dystrophin protein that is mutated in DMD. TALENs were engineered to mediate highly efficient gene editing at exon 51 of the dystrophin gene. This led to restoration of dystrophin protein expression in cells from Duchenne patients, including skeletal myoblasts and dermal fibroblasts that were reprogrammed to the myogenic lineage by MyoD. Finally, exome sequencing of cells with targeted modifications of the dystrophin locus showed no TALEN-mediated off-target changes to the protein-coding regions of the genome, as predicted by in silico target site analysis. This strategy integrates the rapid and robust assembly of active TALENs with an efficient gene-editing method for the correction of genetic diseases caused by mutations in non-essential coding regions that cause frameshifts or premature stop codons.
ISSN:1525-0016
1525-0024
DOI:10.1038/mt.2013.111