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Mol Ther. 2015 Mar;23(3):523-32. doi: 10.1038/mt.2014.234. Epub 2014 Dec 10.

Correction of dystrophin expression in cells from Duchenne muscular dystrophy patients through genomic excision of exon 51 by zinc finger nucleases.

Author information

1
Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA.
2
Program in Computational Biology and Bioinformatics, Duke University, Durham, North Carolina, USA.
3
1] Center for Genomic and Computational Biology, Duke University, Durham, North Carolina, USA [2] Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, North Carolina, USA.
4
1] Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA [2] Center for Genomic and Computational Biology, Duke University, Durham, North Carolina, USA [3] Department of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina, USA.

Abstract

Duchenne muscular dystrophy (DMD) is caused by genetic mutations that result in the absence of dystrophin protein expression. Oligonucleotide-induced exon skipping can restore the dystrophin reading frame and protein production. However, this requires continuous drug administration and may not generate complete skipping of the targeted exon. In this study, we apply genome editing with zinc finger nucleases (ZFNs) to permanently remove essential splicing sequences in exon 51 of the dystrophin gene and thereby exclude exon 51 from the resulting dystrophin transcript. This approach can restore the dystrophin reading frame in ~13% of DMD patient mutations. Transfection of two ZFNs targeted to sites flanking the exon 51 splice acceptor into DMD patient myoblasts led to deletion of this genomic sequence. A clonal population was isolated with this deletion and following differentiation we confirmed loss of exon 51 from the dystrophin mRNA transcript and restoration of dystrophin protein expression. Furthermore, transplantation of corrected cells into immunodeficient mice resulted in human dystrophin expression localized to the sarcolemmal membrane. Finally, we quantified ZFN toxicity in human cells and mutagenesis at predicted off-target sites. This study demonstrates a powerful method to restore the dystrophin reading frame and protein expression by permanently deleting exons.

PMID:
25492562
PMCID:
PMC4351462
DOI:
10.1038/mt.2014.234
[Indexed for MEDLINE]
Free PMC Article

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