title: 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 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.
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy, via Olgettina, 58, 20132 Milan, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Lombardo
authors.forename: Pietro
authors.initials: P
authors.lastname: Genovese
authors.forename: Christian M
authors.initials: CM
authors.lastname: Beausejour
authors.forename: Silvia
authors.initials: S
authors.lastname: Colleoni
authors.forename: Ya-Li
authors.initials: YL
authors.lastname: Lee
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Kim
authors.forename: Dale
authors.initials: D
authors.lastname: Ando
authors.forename: Fyodor D
authors.initials: FD
authors.lastname: Urnov
authors.forename: Cesare
authors.initials: C
authors.lastname: Galli
authors.forename: Philip D
authors.initials: PD
authors.lastname: Gregory
authors.forename: Michael C
authors.initials: MC
authors.lastname: Holmes
authors.forename: Luigi
authors.initials: L
authors.lastname: Naldini
mesh_terms: D004260:DNA Repair; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000201:enzymology*; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D019426:Integrases / Q000235:genetics; D053631:Interleukin Receptor Common gamma Subunit / Q000235:genetics; D016086:Lentivirus / Q000201:enzymology / Q000235:genetics*; D017354:Point Mutation; D013698:Templates, Genetic; D019076:Transgenes; D016662:Virus Integration / Q000235:genetics; D016335:Zinc Fingers
chemical_list: C507941:IL2RG protein, human; D053631:Interleukin Receptor Common gamma Subunit; D019426:Integrases; D015252:Deoxyribonucleases, Type II Site-Specific