Targeted, homology-driven gene insertion in stem cells by ZFN-loaded 'all-in-one' lentiviral vectors

Elife. 2016 Jun 9;5:e12213. doi: 10.7554/eLife.12213.


Biased integration remains a key challenge for gene therapy based on lentiviral vector technologies. Engineering of next-generation lentiviral vectors targeting safe genomic harbors for insertion is therefore of high relevance. In a previous paper (Cai et al., 2014a), we showed the use of integrase-defective lentiviral vectors (IDLVs) as carriers of complete gene repair kits consisting of zinc-finger nuclease (ZFN) proteins and repair sequences, allowing gene correction by homologous recombination (HR). Here, we follow this strategy to engineer ZFN-loaded IDLVs that insert transgenes by a homology-driven mechanism into safe loci. This insertion mechanism is driven by time-restricted exposure of treated cells to ZFNs. We show targeted gene integration in human stem cells, including CD34(+) hematopoietic progenitors and induced pluripotent stem cells (iPSCs). Notably, targeted insertions are identified in 89% of transduced iPSCs. Our findings demonstrate the applicability of nuclease-loaded 'all-in-one' IDLVs for site-directed gene insertion in stem cell-based gene therapies.

Keywords: CD34+ cells; chromosomes; developmental biology; genes; human; iPSC; lentiviral vector; protein transduction; stem cells; virus; zinc-finger nucleases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cells, Cultured
  • DNA Repair Enzymes / genetics
  • DNA Repair Enzymes / metabolism
  • Deoxyribonucleases / genetics
  • Deoxyribonucleases / metabolism
  • Gene Targeting / methods*
  • Genetic Therapy / methods*
  • Genetic Vectors*
  • Homologous Recombination*
  • Humans
  • Lentivirus / genetics*
  • Mutagenesis, Insertional*
  • Stem Cells / physiology*


  • Deoxyribonucleases
  • DNA Repair Enzymes

Grant support

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.