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.

Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases

“…Several recent studies have reported gene-targeting frequencies of 1-18% HR events per 5 mammalian cell when the targeted DSB was introduced by natural or artificial endonucleases [2][3][4][5][6][7][8] . The ability to generate precise modifications of the genome -knockouts, mutations or corrections -within 1 in 100 to 1 in 5 cells presents this methodology as a potentially powerful tool for genetic studies, biotechnology and gene therapy.…”

Progress and prospects: oligonucleotide-directed gene modification in mouse embryonic stem cells: a route to therapeutic application

“…Homology-directed repair of the double-stranded DNA break using a linear double-stranded donor DNA fragment carrying the desired alteration may be accompanied by site-specific alteration of the genome. 5,6 Although this approach can be remarkably effective (single base changes have been obtained in±20% of exposed cells), an obvious obstacle is that zinc-finger nucleases need to be designed and constructed for every specific location to be modified. Zinc-finger nucleases-directed gene modification may therefore be particularly useful to generate a series of substitutions at the same location or, with a view on gene therapy, to repair a frequently occurring mutation.…”

Estrogen withdrawal, increased breast cancer risk and the KRAS-variant

“…We generated isogenic MCF10A lines with and without the KRAS-variant using the CompoZr TM custom designed zinc-finger nuclease (ZFN) targeted genome editing technology (SigmaAldrich), 35 per manufacturer's instructions. MCF10a cells are an immortalized, non-transformed mammary epithelial cell line derived from human fibrocystic mammary tissue and have a lack of tumorigenicity in nude mice and lack of anchorage independent growth.…”