Highly efficient endogenous human gene correction using designed zinc-finger nucleases

Fyodor D. Urnov, Jeffrey C. Miller, Ya-Li Lee, Christian M. Beausejour, Jeremy M. Rock, Sheldon Augustus, Andrew C. Jamieson, Matthew H. Porteus (), Philip D. Gregory and Michael C. Holmes ()
Additional contact information
Fyodor D. Urnov: Sangamo BioSciences, Inc. Pt. Richmond Tech Center 501
Jeffrey C. Miller: Sangamo BioSciences, Inc. Pt. Richmond Tech Center 501
Ya-Li Lee: Sangamo BioSciences, Inc. Pt. Richmond Tech Center 501
Christian M. Beausejour: Sangamo BioSciences, Inc. Pt. Richmond Tech Center 501
Jeremy M. Rock: Sangamo BioSciences, Inc. Pt. Richmond Tech Center 501
Sheldon Augustus: Sangamo BioSciences, Inc. Pt. Richmond Tech Center 501
Andrew C. Jamieson: Sangamo BioSciences, Inc. Pt. Richmond Tech Center 501
Matthew H. Porteus: University of Texas Southwestern Medical Center
Philip D. Gregory: Sangamo BioSciences, Inc. Pt. Richmond Tech Center 501
Michael C. Holmes: Sangamo BioSciences, Inc. Pt. Richmond Tech Center 501

Nature, 2005, vol. 435, issue 7042, 646-651

Abstract: Abstract Permanent modification of the human genome in vivo is impractical owing to the low frequency of homologous recombination in human cells, a fact that hampers biomedical research and progress towards safe and effective gene therapy. Here we report a general solution using two fundamental biological processes: DNA recognition by C2H2 zinc-finger proteins and homology-directed repair of DNA double-strand breaks. Zinc-finger proteins engineered to recognize a unique chromosomal site can be fused to a nuclease domain, and a double-strand break induced by the resulting zinc-finger nuclease can create specific sequence alterations by stimulating homologous recombination between the chromosome and an extrachromosomal DNA donor. We show that zinc-finger nucleases designed against an X-linked severe combined immune deficiency (SCID) mutation in the IL2Rγ gene yielded more than 18% gene-modified human cells without selection. Remarkably, about 7% of the cells acquired the desired genetic modification on both X chromosomes, with cell genotype accurately reflected at the messenger RNA and protein levels. We observe comparably high frequencies in human T cells, raising the possibility of strategies based on zinc-finger nucleases for the treatment of disease.

Date: 2005
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DOI: 10.1038/nature03556

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