Article abstract
Nature Biotechnology 25, 1298 - 1306 (2007)
Published online: 28 October 2007 | doi:10.1038/nbt1353
Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery
Angelo Lombardo1,2, Pietro Genovese1,2, Christian M Beausejour3,6, Silvia Colleoni4, Ya-Li Lee3, Kenneth A Kim3, Dale Ando3, Fyodor D Urnov3, Cesare Galli4,5, Philip D Gregory3,7, Michael C Holmes3,7 & Luigi Naldini1,2,7
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 
-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.
- San Raffaele Telethon Institute for Gene Therapy, via Olgettina, 58, 20132 Milan, Italy.
- "Vita Salute San Raffaele" University, via Olgettina, 58, 20132 Milan, Italy.
- Sangamo BioSciences, Inc., Pt. Richmond Tech Center, 501 Canal Blvd., Suite A100 Richmond, California 94804, USA.
- Reproductive Technologies Laboratory, CIZ, Istituto Sperimentale Italiano Lazzaro Spallanzani, via Porcellasco 7/F, 26100 Cremona, Italy.
- Dipartimento Clinico Veterinario, University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia, Italy.
- Present address: Département de Pharmacologie, Centre de Recherche, CHU Ste-Justine 3175, Côte Ste-Catherine, Montréal, Quebec H3T 1C5, Canada.
- These authors contributed equally to this work.
Correspondence to: Luigi Naldini1,2,7 e-mail: naldini.luigi@hsr.it
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