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

Abstract

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.

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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.

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Acknowledgements

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.

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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.

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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.

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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). https://doi.org/10.1038/nbt1353

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