Targeted, homology-driven gene insertion in stem cells by ZFN-loaded 'all-in-one' lentiviral vectors.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 27278774.
- Also identified by DOI 10.7554/eLife.12213 and PMC identifier 4900802.
- Licence recorded as CC BY.
- The licence permits redistribution, so the abstract is shown in full and the full text is available from the publisher.
Abstract
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.
Medical subject headings
- Gene Targeting
- Genetic Therapy
- Genetic Vectors
- Homologous Recombination
- Lentivirus
- Mutagenesis, Insertional
- Stem Cells