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PubMed · 15806097

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·Michael C Holmes
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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.

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AllelesCD4-Positive T-LymphocytesCell LineCells, CulturedChromosomes, Human, XDNADNA DamageDNA RepairEndodeoxyribonucleasesGene TargetingGenes, ReporterGenetic LinkageGenetic TherapyHumansRNA, MessengerReceptors, Interleukin-2Recombination, GeneticSequence Homology, Nucleic AcidSevere Combined ImmunodeficiencySubstrate SpecificityZinc FingersJournal Article

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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, Michael C Holmes. 2005-04-03. Highly efficient endogenous human gene correction using designed zinc-finger nucleases.. https://doi.org/10.1038/nature03556

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