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 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.

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Figure 1: Designed ZFNs enable correction of a chromosomal reporter gene in over 10% of the cells.
Figure 2: Design and optimization of ZFNs directed against the X-linked SCID mutation hotspot of IL2Rγ.
Figure 3: High-frequency HR at the endogenous IL2Rγ locus driven by designed ZFNs.
Figure 4: High-frequency HR at the endogenous IL2Rγ locus driven by designed ZFNs in primary human CD4+ T cells.
Figure 5: High-frequency monoallelic and biallelic alteration of IL2Rγ driven by designed ZFNs.
Figure 6: Serial modification of the IL2Rγ locus demonstrates the usefulness of ZFNs in somatic cell genetics and the potential for gene correction therapy.

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Acknowledgements

We are grateful to C. Case for his initial discussions with M.H.P. regarding this project. We thank Sangamo's production group for technical support; S. Brennan, C. Dent, D. Kohn, Y. Marahrens, T. Martin, C. Pabo and P. Sung for suggestions and discussions; and A. Klug for comments on the manuscript. We also thank E. Lanphier for encouragement and support.

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Correspondence to Michael C. Holmes.

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F.D.U., J.C.M., C.M.B., Y.-L.L., J.M.R., P.D.G. and M.C.H. are full-time employees of Sangamo BioSciences, Inc. S.A. and A.C.J. were employed during the course of this work.

Supplementary information

Supplementary Figures S1-S5

Contains Supplementary Figures and accompanying Supplementary Figure Legends (DOC 987 kb)

Supplementary Notes

Narrative for Supplementary Figures S2 and S3. (DOC 30 kb)

Supplementary Methods

Includes details of donor plasmid generation, tissue culture procedures, PCR-based assay for gene correction at γC and quantitative RT-PCR, and Western blot assays for γC. (DOC 23 kb)

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Urnov, F., Miller, J., Lee, Y. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646–651 (2005). https://doi.org/10.1038/nature03556

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