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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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References

  1. Sedivy, J. M. & Joyner, A. L. Gene Targeting (Oxford Univ. Press, Oxford, 1992)

    Google Scholar 

  2. Yanez, R. J. & Porter, A. C. Therapeutic gene targeting. Gene Ther. 5, 149–159 (1998)

    Article  CAS  PubMed  Google Scholar 

  3. Kohn, D. B., Sadelain, M. & Glorioso, J. C. Occurrence of leukaemia following gene therapy of X-linked SCID. Nature Rev. Cancer 3, 477–488 (2003)

    Article  CAS  Google Scholar 

  4. Persons, D. A. & Nienhuis, A. W. Gene therapy for the hemoglobin disorders. Curr. Hematol. Rep. 2, 348–355 (2003)

    PubMed  Google Scholar 

  5. Thomas, K. R., Folger, K. R. & Capecchi, M. R. High frequency targeting of genes to specific sites in the mammalian genome. Cell 44, 419–428 (1986)

    Article  CAS  PubMed  Google Scholar 

  6. Brown, J. P., Wei, W. & Sedivy, J. M. Bypass of senescence after disruption of p21CIP1/WAF1 gene in normal diploid human fibroblasts. Science 277, 831–834 (1997)

    Article  CAS  PubMed  Google Scholar 

  7. Bunz, F. et al. Targeted inactivation of p53 in human cells does not result in aneuploidy. Cancer Res. 62, 1129–1133 (2002)

    CAS  PubMed  Google Scholar 

  8. Miller, J., McLachlan, A. D. & Klug, A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes . EMBO J. 4, 1609–1614 (1985)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Tupler, R., Perini, G. & Green, M. R. Expressing the human genome. Nature 409, 832–833 (2001)

    Article  ADS  CAS  PubMed  Google Scholar 

  10. Pavletich, N. P. & Pabo, C. O. Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 Å. Science 252, 809–817 (1991)

    Article  ADS  CAS  PubMed  Google Scholar 

  11. Klug, A. Protein designs for the specific recognition of DNA. Gene 135, 83–92 (1993)

    Article  CAS  PubMed  Google Scholar 

  12. Choo, Y., Sanchez-Garcia, I. & Klug, A. In vivo repression by a site-specific DNA-binding protein designed against an oncogenic sequence. Nature 372, 642–645 (1994)

    Article  ADS  CAS  PubMed  Google Scholar 

  13. Pabo, C. O., Peisach, E. & Grant, R. A. Design and selection of novel Cys2his2 zinc finger proteins. Annu. Rev. Biochem. 70, 313–340 (2001)

    Article  CAS  PubMed  Google Scholar 

  14. Choo, Y. & Isalan, M. Advances in zinc finger engineering. Curr. Opin. Struct. Biol. 10, 411–416 (2000)

    Article  CAS  PubMed  Google Scholar 

  15. Jamieson, A. C., Miller, J. C. & Pabo, C. O. Drug discovery with engineered zinc-finger proteins. Nature Rev. Drug Discov. 2, 361–368 (2003)

    Article  CAS  Google Scholar 

  16. Kim, Y. G., Cha, J. & Chandrasegaran, S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl Acad. Sci. USA 93, 1156–1160 (1996)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  17. Smith, J., Berg, J. M. & Chandrasegaran, S. A detailed study of the substrate specificity of a chimeric restriction enzyme. Nucleic Acids Res. 27, 674–681 (1999)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. West, S. C. Molecular views of recombination proteins and their control. Nature Rev. Mol. Cell Biol. 4, 435–445 (2003)

    Article  CAS  Google Scholar 

  19. Symington, L. S. Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. Microbiol. Mol. Biol. Rev. 66, 630–670 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Paques, F. & Haber, J. E. Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae . Microbiol. Mol. Biol. Rev. 63, 349–404 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Rouet, P., Smih, F. & Jasin, M. Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. Proc. Natl Acad. Sci. USA 91, 6064–6068 (1994)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  22. Sargent, R. G., Brenneman, M. A. & Wilson, J. H. Repair of site-specific double-strand breaks in a mammalian chromosome by homologous and illegitimate recombination. Mol. Cell. Biol. 17, 267–277 (1997)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Choulika, A., Perrin, A., Dujon, B. & Nicolas, J. F. Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae . Mol. Cell. Biol. 15, 1968–1973 (1995)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Bibikova, M. et al. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol. Cell. Biol. 21, 289–297 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Bibikova, M., Beumer, K., Trautman, J. K. & Carroll, D. Enhancing gene targeting with designed zinc finger nucleases. Science 300, 764 (2003)

    Article  CAS  PubMed  Google Scholar 

  26. Porteus, M. H. & Baltimore, D. Chimeric nucleases stimulate gene targeting in human cells. Science 300, 763 (2003)

    Article  PubMed  Google Scholar 

  27. Cavazzana-Calvo, M. et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 288, 669–672 (2000)

    Article  ADS  CAS  PubMed  Google Scholar 

  28. Elliott, B., Richardson, C., Winderbaum, J., Nickoloff, J. A. & Jasin, M. Gene conversion tracts from double-strand break repair in mammalian cells. Mol. Cell. Biol. 18, 93–101 (1998)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Moore, M., Klug, A. & Choo, Y. Improved DNA binding specificity from polyzinc finger peptides by using strings of two-finger units. Proc. Natl Acad. Sci. USA 98, 1437–1441 (2001)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  30. Buckley, R. H. Primary immunodeficiency diseases due to defects in lymphocytes. N. Engl. J. Med. 343, 1313–1324 (2000)

    Article  CAS  PubMed  Google Scholar 

  31. Schell, T., Kulozik, A. E. & Hentze, M. W. Integration of splicing, transport and translation to achieve mRNA quality control by the nonsense-mediated decay pathway. Genome Biol. 3, 1–6 (2002)

    Article  Google Scholar 

  32. Isalan, M. & Choo, Y. Rapid, high-throughput engineering of sequence-specific zinc finger DNA- binding proteins. Methods Enzymol. 340, 593–609 (2001)

    Article  CAS  PubMed  Google Scholar 

  33. Wilson, J. H. Pointing fingers at the limiting step in gene targeting. Nature Biotechnol. 21, 759–760 (2003)

    Article  CAS  Google Scholar 

  34. Choo, Y. & Klug, A. Physical basis of a protein-DNA recognition code. Curr. Opin. Struct. Biol. 7, 117–125 (1997)

    Article  CAS  PubMed  Google Scholar 

  35. Bibikova, M., Golic, M., Golic, K. G. & Carroll, D. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161, 1169–1175 (2002)

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Zhang, L. et al. Synthetic zinc finger transcription factor action at an endogenous chromosomal site. Activation of the human erythropoietin gene. J. Biol. Chem. 275, 33850–33860 (2000)

    Article  CAS  PubMed  Google Scholar 

  37. Liu, P. Q. et al. Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions. Activation of vascular endothelial growth factor A. J. Biol. Chem. 276, 11323–11334 (2001)

    Article  CAS  PubMed  Google Scholar 

  38. Isalan, M., Klug, A. & Choo, Y. A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter. Nature Biotechnol. 19, 656–660 (2001)

    Article  CAS  Google Scholar 

  39. Tan, S. et al. Zinc-finger protein-targeted gene regulation: genomewide single-gene specificity. Proc. Natl Acad. Sci. USA 100, 11997–12002 (2003)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

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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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Competing interests

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, YL. 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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