title: Highly efficient endogenous human gene correction using designed zinc-finger nucleases.
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 IL2Rgamma 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.
authors.affiliation: Sangamo BioSciences, Inc., Pt. Richmond Tech Center 501, Canal Blvd, Suite A100 Richmond, California 94804, USA.
authors.forename: Fyodor D
authors.initials: FD
authors.lastname: Urnov
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Miller
authors.forename: Ya-Li
authors.initials: YL
authors.lastname: Lee
authors.forename: Christian M
authors.initials: CM
authors.lastname: Beausejour
authors.forename: Jeremy M
authors.initials: JM
authors.lastname: Rock
authors.forename: Sheldon
authors.initials: S
authors.lastname: Augustus
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Jamieson
authors.forename: Matthew H
authors.initials: MH
authors.lastname: Porteus
authors.forename: Philip D
authors.initials: PD
authors.lastname: Gregory
authors.forename: Michael C
authors.initials: MC
authors.lastname: Holmes
mesh_terms: D000483:Alleles; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D041321:Chromosomes, Human, X / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter / Q000235:genetics; D008040:Genetic Linkage / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015375:Receptors, Interleukin-2 / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D016511:Severe Combined Immunodeficiency / Q000235:genetics* / Q000628:therapy; D013379:Substrate Specificity; D016335:Zinc Fingers
chemical_list: D012333:RNA, Messenger; D015375:Receptors, Interleukin-2; D004247:DNA; D004706:Endodeoxyribonucleases