Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery


Achieving the full potential of zinc-finger nucleases (ZFNs) for genome engineering in human cells requires their efficient delivery to the relevant cell types. Here we exploited the infectivity of integrase-defective lentiviral vectors (IDLV) to express ZFNs and provide the template DNA for gene correction in different cell types. IDLV-mediated delivery supported high rates (13–39%) of editing at the IL-2 receptor common γ-chain gene (IL2RG) across different cell types. IDLVs also mediated site-specific gene addition by a process that required ZFN cleavage and homologous template DNA, thus establishing a platform that can target the insertion of transgenes into a predetermined genomic site. Using IDLV delivery and ZFNs targeting distinct loci, we observed high levels of gene addition (up to 50%) in a panel of human cell lines, as well as human embryonic stem cells (5%), allowing rapid, selection-free isolation of clonogenic cells with the desired genetic modification.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Editing of the endogenous IL2RG gene by IDLV delivery of ZFNs and donor DNA.
Figure 2: IL2RG gene editing in human lymphoblastoid cells.
Figure 3: Targeted transgene addition into IL2RG.
Figure 4: Targeted gene addition into the IL2RG locus in human lymphoblastoid cells.
Figure 5: Site-specific gene addition into CCR5.
Figure 6: Targeted gene addition in human stem cells.


  1. 1

    Capecchi, M.R. Generating mice with targeted mutations. Nat. Med. 7, 1086–1090 (2001).

    CAS  Article  Google Scholar 

  2. 2

    Chen, J.M., Cooper, D.N., Chuzhanova, N., Ferec, C. & Patrinos, G.P. Gene conversion: mechanisms, evolution and human disease. Nat. Rev. Genet. 8, 762–775 (2007).

    CAS  Article  Google Scholar 

  3. 3

    Hatada, S., Nikkuni, K., Bentley, S.A., Kirby, S. & Smithies, O. Gene correction in hematopoietic progenitor cells by homologous recombination. Proc. Natl. Acad. Sci. USA 97, 13807–13811 (2000).

    CAS  Article  Google Scholar 

  4. 4

    Zwaka, T.P. & Thomson, J.A. Homologous recombination in human embryonic stem cells. Nat. Biotechnol. 21, 319–321 (2003).

    CAS  Article  Google Scholar 

  5. 5

    Evans, M.J. The cultural mouse. Nat. Med. 7, 1081–1083 (2001).

    CAS  Article  Google Scholar 

  6. 6

    Smithies, O. Forty years with homologous recombination. Nat. Med. 7, 1083–1086 (2001).

    CAS  Article  Google Scholar 

  7. 7

    Hendrie, P.C. & Russell, D.W. Gene targeting with viral vectors. Mol. Ther. 12, 9–17 (2005).

    CAS  Article  Google Scholar 

  8. 8

    Baum, C. et al. Chance or necessity? Insertional mutagenesis in gene therapy and its consequences. Mol. Ther. 9, 5–13 (2004).

    CAS  Article  Google Scholar 

  9. 9

    Bushman, F. et al. Genome-wide analysis of retroviral DNA integration. Nat. Rev. Microbiol. 3, 848–858 (2005).

    CAS  Article  Google Scholar 

  10. 10

    Montini, E. et al. Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration. Nat. Biotechnol. 24, 687–696 (2006).

    CAS  Article  Google Scholar 

  11. 11

    Nienhuis, A.W., Dunbar, C.E. & Sorrentino, B.P. Genotoxicity of retroviral integration in hematopoietic cells. Mol. Ther. 13, 1031–1049 (2006).

    CAS  Article  Google Scholar 

  12. 12

    Aiuti, A. et al. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science 296, 2410–2413 (2002).

    CAS  Article  Google Scholar 

  13. 13

    Gaspar, H.B. et al. Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector. Lancet 364, 2181–2187 (2004).

    CAS  Article  Google Scholar 

  14. 14

    Cavazzana-Calvo, M., Lagresle, C., Hacein-Bey-Abina, S. & Fischer, A. Gene therapy for severe combined immunodeficiency. Annu. Rev. Med. 56, 585–602 (2005).

    CAS  Article  Google Scholar 

  15. 15

    Ott, M.G. et al. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1–EVI1, PRDM16 or SETBP1. Nat. Med. 12, 401–409 (2006).

    CAS  Article  Google Scholar 

  16. 16

    Hacein-Bey-Abina, S. et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302, 415–419 (2003).

    CAS  Article  Google Scholar 

  17. 17

    Woods, N.B., Bottero, V., Schmidt, M., von Kalle, C. & Verma, I.M. Gene therapy: therapeutic gene causing lymphoma. Nature 440, 1123 (2006).

    CAS  Article  Google Scholar 

  18. 18

    Thrasher, A.J. et al. Gene therapy: X-SCID transgene leukaemogenicity. Nature 443, E5; discussion E6–7 (2006).

    CAS  Article  Google Scholar 

  19. 19

    Goyenvalle, A. et al. Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping. Science 306, 1796–1799 (2004).

    CAS  Article  Google Scholar 

  20. 20

    Tahara, M. et al. Trans-splicing repair of CD40 ligand deficiency results in naturally regulated correction of a mouse model of hyper-IgM X-linked immunodeficiency. Nat. Med. 10, 835–841 (2004).

    CAS  Article  Google Scholar 

  21. 21

    Chamberlain, J.R. et al. Gene targeting in stem cells from individuals with osteogenesis imperfecta. Science 303, 1198–1201 (2004).

    CAS  Article  Google Scholar 

  22. 22

    Miller, D.G. et al. Gene targeting in vivo by adeno-associated virus vectors. Nat. Biotechnol. 24, 1022–1026 (2006).

    CAS  Article  Google Scholar 

  23. 23

    Calos, M.P. The phiC31 integrase system for gene therapy. Curr. Gene Ther. 6, 633–645 (2006).

    CAS  Article  Google Scholar 

  24. 24

    Porteus, M.H. & Carroll, D. Gene targeting using zinc finger nucleases. Nat. Biotechnol. 23, 967–973 (2005).

    CAS  Article  Google Scholar 

  25. 25

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

    Article  Google Scholar 

  26. 26

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

    CAS  Article  Google Scholar 

  27. 27

    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).

    CAS  Article  Google Scholar 

  28. 28

    Pabo, C.O., Peisach, E. & Grant, R.A. Design and selection of novel Cys2His2 zinc finger proteins. Annu. Rev. Biochem. 70, 313–340 (2001).

    CAS  Article  Google Scholar 

  29. 29

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

    CAS  Article  Google Scholar 

  30. 30

    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).

    CAS  Article  Google Scholar 

  31. 31

    Bitinaite, J., Wah, D.A., Aggarwal, A.K. & Schildkraut, I. FokI dimerization is required for DNA cleavage. Proc. Natl. Acad. Sci. USA 95, 10570–10575 (1998).

    CAS  Article  Google Scholar 

  32. 32

    O'Driscoll, M. & Jeggo, P.A. The role of double-strand break repair - insights from human genetics. Nat. Rev. Genet. 7, 45–54 (2006).

    CAS  Article  Google Scholar 

  33. 33

    Urnov, F.D. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646–651 (2005).

    CAS  Article  Google Scholar 

  34. 34

    Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267 (1996).

    CAS  Article  Google Scholar 

  35. 35

    Vargas, J. Jr., Gusella, G.L., Najfeld, V., Klotman, M.E. & Cara, A. Novel integrase-defective lentiviral episomal vectors for gene transfer. Hum. Gene Ther. 15, 361–372 (2004).

    CAS  Article  Google Scholar 

  36. 36

    Nightingale, S.J. et al. Transient gene expression by nonintegrating lentiviral vectors. Mol. Ther. 13, 1121–1132 (2006).

    CAS  Article  Google Scholar 

  37. 37

    Yanez-Munoz, R.J. et al. Effective gene therapy with nonintegrating lentiviral vectors. Nat. Med. 12, 348–353 (2006).

    CAS  Article  Google Scholar 

  38. 38

    Philippe, S. et al. Lentiviral vectors with a defective integrase allow efficient and sustained transgene expression in vitro and in vivo. Proc. Natl. Acad. Sci. USA 103, 17684–17689 (2006).

    CAS  Article  Google Scholar 

  39. 39

    Leavitt, A.D., Robles, G., Alesandro, N. & Varmus, H.E. Human immunodeficiency virus type 1 integrase mutants retain in vitro integrase activity yet fail to integrate viral DNA efficiently during infection. J. Virol. 70, 721–728 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    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).

    CAS  Article  Google Scholar 

  41. 41

    Buckley, R.H. Molecular defects in human severe combined immunodeficiency and approaches to immune reconstitution. Annu. Rev. Immunol. 22, 625–655 (2004).

    CAS  Article  Google Scholar 

  42. 42

    Lim, J.K., Glass, W.G., McDermott, D.H. & Murphy, P.M. CCR5: no longer a “good for nothing” gene–chemokine control of West Nile virus infection. Trends Immunol. 27, 308–312 (2006).

    CAS  Article  Google Scholar 

  43. 43

    Cowan, C.A. et al. Derivation of embryonic stem-cell lines from human blastocysts. N. Engl. J. Med. 350, 1353–1356 (2004).

    CAS  Article  Google Scholar 

  44. 44

    Tan, W., Dong, Z., Wilkinson, T.A., Barbas, C.F. III & Chow, S.A. Human immunodeficiency virus type 1 incorporated with fusion proteins consisting of integrase and the designed polydactyl zinc finger protein E2C can bias integration of viral DNA into a predetermined chromosomal region in human cells. J. Virol. 80, 1939–1948 (2006).

    CAS  Article  Google Scholar 

  45. 45

    Bushman, F.D. & Miller, M.D. Tethering human immunodeficiency virus type 1 preintegration complexes to target DNA promotes integration at nearby sites. J. Virol. 71, 458–464 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Ciuffi, A., Diamond, T.L., Hwang, Y., Marshall, H.M. & Bushman, F.D. Modulating target site selection during human immunodeficiency virus DNA integration in vitro with an engineered tethering factor. Hum. Gene Ther. 17, 960–967 (2006).

    CAS  Article  Google Scholar 

  47. 47

    Fletcher, T.M. III et al. Complementation of integrase function in HIV-1 virions. EMBO J. 16, 5123–5138 (1997).

    CAS  Article  Google Scholar 

  48. 48

    Sung, P. & Klein, H. Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat. Rev. Mol. Cell Biol. 7, 739–750 (2006).

    CAS  Article  Google Scholar 

  49. 49

    Birney, E. et al. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 447, 799–816 (2007).

    CAS  Article  Google Scholar 

  50. 50

    Miller, J.C. et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat. Biotechnol. 25, 778–785 (2007).

    CAS  Article  Google Scholar 

  51. 51

    Santoni de Sio, F.R., Cascio, P., Zingale, A., Gasparini, M. & Naldini, L. Proteasome activity restricts lentiviral gene transfer into hematopoietic stem cells and is down-regulated by cytokines that enhance transduction. Blood 107, 4257–4265 (2006).

    CAS  Article  Google Scholar 

  52. 52

    Follenzi, A. & Naldini, L. Generation of HIV-1 derived lentiviral vectors. Methods Enzymol. 346, 454–465 (2002).

    CAS  Article  Google Scholar 

  53. 53

    Brown, B.D., Venneri, M.A., Zingale, A., Sergi Sergi, L. & Naldini, L. Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer. Nat. Med. 12, 585–591 (2006).

    CAS  Article  Google Scholar 

Download references


We are grateful to Lucia Sergi Sergi and Anna Zingale for technical help, Giovanna Lazzari for advice with HUES cultures, Rosa Bacchetta and Alessandro Aiuti for providing lymphoblastoid cells, Brian Brown and Bernhard Gentner for helpful discussion. We also thank Russell DeKelver, Jianbin Wang, Aleida Perez and Anna Lam for donor DNA construct generation, Jeffrey Miller, Victor Bartsevich, Dmitry Guschin, Igor Rupniewski, Yanhong Kong, Edward Rebar, Lei Zhang, Adam Waite, Deng Xia, Sarah Hinkley and members of the Sangamo production group for the design and generation of the ZFNs used in this study, and Sean Brennan for reading the manuscript. Research was supported by grants from Telethon (TIGET), National Institutes of Health (2 P01 HL053750-11 CFDA No. 93.839), EU (CONSERT, LSHB-CT-2004-005242) and Sangamo BioSciences to L.N., and European Science Foundation (EUROCORES Programme, EuroSTELLS) to C.G. HUES were kindly provided by D. Melton from Harvard Stem Cell Institute, under specific Materials Transfer Agreement to C.G.

Author information




A.L. designed, performed experiments and wrote the paper; P.G., C.M.B., Y.-L.L. and K.A.K. performed experiments; S.C. performed ES cell cultures; D.A. and F.D.U. designed experiments; C.G. coordinated ES cell work; P.D.G. and M.C.H. designed experiments and wrote the paper; and L.N. coordinated the project, designed experiments and wrote the paper.

Corresponding author

Correspondence to Luigi Naldini.

Ethics declarations

Competing interests

C.M.B., Y.-L.L., K.A.K., D.A., F.U., P.D.G. and M.C.H. are current or former employees of Sangamo BioSciences, Inc. Work in L.N.'s laboratory was supported in part by a research collaboration agreement with Sangamo BioSciences.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9; Supplementary Methods (PDF 783 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lombardo, A., Genovese, P., Beausejour, C. et al. Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat Biotechnol 25, 1298–1306 (2007).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing