1. Sangamo BioSciences, Inc. Pt. Richmond Tech Center 501, Canal Blvd, Suite A100 Richmond, California 94804, USA
2. Department of Pediatrics and Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, Texas 75390, USA

Correspondence to: Michael C. Holmes¹ Correspondence should be addressed to M.C.H. (Email: mholmes@sangamo.com) or M.H.P. (Email: matthew.porteus@UTSouthwestern.edu); requests for materials should be addressed to M.C.H.

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 IL2R 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.

1. Sangamo BioSciences, Inc. Pt. Richmond Tech Center 501, Canal Blvd, Suite A100 Richmond, California 94804, USA
2. Department of Pediatrics and Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, Texas 75390, USA

Correspondence to: Michael C. Holmes¹ Correspondence should be addressed to M.C.H. (Email: mholmes@sangamo.com) or M.H.P. (Email: matthew.porteus@UTSouthwestern.edu); requests for materials should be addressed to M.C.H.

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