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

• DOI: 10.1016/j.bcmd.2007.10.064
• Publication Date: 2008-02-13T15:15:23Z
• Authors: A. Lombardo; P. Genovese; C.M. Beausejour; S. Colleoni; Y.-L. Lee; K.A. Kim; D. Ando; F. Urnov; C. Galli; P.D. Gregory; M.C. Holmes; L. Naldini
• Keywords: 0301 basic medicine; DNA Repair; Integrases; Virus Integration; Genetic Vectors; Lentivirus; Gene Transfer Techniques; Zinc Fingers; Templates, Genetic; 03 medical and health sciences; Humans; Point Mutation; Transgenes; Deoxyribonucleases, Type II Site-Specific; Genetic Engineering; Embryonic Stem Cells; Interleukin Receptor Common gamma Subunit

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 gamma-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.