title: Targeted gene addition in human epithelial stem cells by zinc-finger nuclease-mediated homologous recombination.
abstract: Preclinical and clinical studies showed that autologous transplantation of epidermis derived from genetically modified epithelial stem cells (EpSCs) leads to long-term correction of inherited skin adhesion defects. These studies were based on potentially genotoxic retroviral vectors. We developed an alternative gene transfer strategy aimed at targeting a "safe harbor" locus, the adeno-associated virus integration site 1 (AAVS1), by zinc-finger nuclease (ZFN)-induced homologous recombination (HR). Delivery of AAVS1-specific ZFNs and a GFP-expressing HR cassette by integration-defective lentiviral (LV) vectors (IDLVs) or adenoviral (Ad) vectors resulted in targeted gene addition with an efficiency of > 20% in a human keratinocyte cell line, > 10% in immortalized keratinocytes, and < 1% in primary keratinocytes. Deep sequencing of the AAVS1 locus showed that ZFN-induced double-strand breaks are mostly repaired by nonhomologous end joining (NHEJ) in primary cells, indicating that poor induction of the HR-dependent DNA repair pathway may be a significant limitation for targeted gene integration. Skin equivalents derived from unselected keratinocyte cultures coinfected with a GFP-IDLV and a ZFN-Ad vector were grafted onto immunodeficient mice. GFP-positive clones were observed in all grafts up to 18 weeks post-transplantation. By histological and molecular analysis, we were able to demonstrate highly efficient targeting of the AAVS1 locus in human repopulating EpSCs.
authors.affiliation: Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Coluccio
authors.forename: Francesca
authors.initials: F
authors.lastname: Miselli
authors.forename: Angelo
authors.initials: A
authors.lastname: Lombardo
authors.forename: Alessandra
authors.initials: A
authors.lastname: Marconi
authors.forename: Guidantonio
authors.initials: G
authors.lastname: Malagoli Tagliazucchi
authors.forename: Manuel A
authors.initials: MA
authors.lastname: Gonçalves
authors.forename: Carlo
authors.initials: C
authors.lastname: Pincelli
authors.forename: Giulietta
authors.initials: G
authors.lastname: Maruggi
authors.forename: Marcela
authors.initials: M
authors.lastname: Del Rio
authors.forename: Luigi
authors.initials: L
authors.lastname: Naldini
authors.forename: Fernando
authors.initials: F
authors.lastname: Larcher
authors.forename: Fulvio
authors.initials: F
authors.lastname: Mavilio
authors.forename: Alessandra
authors.initials: A
authors.lastname: Recchia
mesh_terms: D000818:Animals; D002460:Cell Line; D017690:Cell Transplantation; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D018390:Gene Targeting; D005822:Genetic Vectors; D059765:Homologous Recombination; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D051379:Mice; D013234:Stem Cells / Q000378:metabolism*; D014161:Transduction, Genetic; D016662:Virus Integration; D016335:Zinc Fingers
chemical_list: D004720:Endonucleases