Targeted gene addition in human epithelial stem cells by zinc-finger nuclease-mediated homologous recombination Articles uri icon

authors

  • Coluccio, Andrea
  • Miselli, Francesca
  • Lombardo, Angelo
  • Marconi, Alessandra
  • Malagoli Tagliazucchi, Guidantonio
  • Pincelli, Carlo
  • Maruggi, Giulietta
  • RIO NECHAEVSKY, MARCELA ANDREA DEL
  • Naldini, Luigi
  • LARCHER LAGUZZI, FERNANDO
  • MAVILIO, FULVIO
  • Recchia, Alessandra

publication date

  • September 2013

start page

  • 1695

end page

  • 1704

issue

  • 9

volume

  • 21

International Standard Serial Number (ISSN)

  • 1525-0016

Electronic International Standard Serial Number (EISSN)

  • 1525-0024

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.