Journal article
Targeted, homology-driven gene insertion in stem cells by ZFN-loaded 'all-in-one' lentiviral vectors
Aarhus University1
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark2
Research Groups, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark3
CHO Core, Translational Management, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark4
iLoop, Translational Management, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark5
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 et, 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 ZEN-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.
Language: | English |
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Publisher: | eLife Sciences Publications, Ltd |
Year: | 2016 |
ISSN: | 2050084x |
Types: | Journal article |
DOI: | 10.7554/eLife.12213 |
ORCIDs: | 0000-0002-0007-7759 , 0000-0002-1322-3209 , 0000-0002-6449-6426 , 0000-0001-8847-9201 and Anderson, Mads Valdemar |
Biology (General) CD34+ cells Cells, Cultured DNA Repair Enzymes Deoxyribonucleases Gene Targeting Genetic Therapy Genetic Vectors Homologous Recombination Human Humans Lentivirus Medicine Mutagenesis, Insertional Q QH301-705.5 R Science Stem Cells Virus chromosomes developmental biology genes human iPSC lentiviral vector protein transduction stem cells virus zinc-finger nucleases