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

Angelo Lombardo, Pietro Genovese, Christian M. Beausejour, Silvia Colleoni, Ya Li Lee, Kenneth A. Kim, Dale Ando, Fyodor D. Urnov, Cesare Galli, Philip D. Gregory, Michael C. Holmes, Luigi Naldini

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)1298-1306
Number of pages9
JournalNature Biotechnology
Volume25
Issue number11
DOIs
Publication statusPublished - Nov 2007

ASJC Scopus subject areas

  • Microbiology

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    Lombardo, A., Genovese, P., Beausejour, C. M., Colleoni, S., Lee, Y. L., Kim, K. A., Ando, D., Urnov, F. D., Galli, C., Gregory, P. D., Holmes, M. C., & Naldini, L. (2007). Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nature Biotechnology, 25(11), 1298-1306. https://doi.org/10.1038/nbt1353