Targeted, homology-driven gene insertion in stem cells by ZFN-loaded 'all-in-one' lentiviral vectors.
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.
Department of Biomedicine, Aarhus University, Aarhus, Denmark.
Yujia
Y
Cai
Department of Biomedicine, Aarhus University, Aarhus, Denmark.
Anders
A
Laustsen
Department of Biomedicine, Aarhus University, Aarhus, Denmark.
Yan
Y
Zhou
Department of Biomedicine, Aarhus University, Aarhus, Denmark.
Chenglong
C
Sun
Department of Biomedicine, Aarhus University, Aarhus, Denmark.|Technical University of Denmark, Lyngby, Denmark.
Mads Valdemar
MV
Anderson
Department of Biomedicine, Aarhus University, Aarhus, Denmark.|Beijing Genomics Institute, Shenzhen, China.
Shengting
S
Li
Department of Clinical Medicine, Aarhus University, Aarhus, Denmark.
Niels
N
Uldbjerg
Department of Biomedicine, Aarhus University, Aarhus, Denmark.
Yonglun
0000-0002-0007-7759
Y
Luo
Department of Biomedicine, Aarhus University, Aarhus, Denmark.
Martin R
MR
Jakobsen
Department of Biomedicine, Aarhus University, Aarhus, Denmark.
Jacob Giehm
0000-0002-1322-3209
JG
Mikkelsen
D002478:Cells, Cultured; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D059765:Homologous Recombination; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D016254:Mutagenesis, Insertional; D013234:Stem Cells / Q000502:physiology*
D003851:Deoxyribonucleases; D045643:DNA Repair Enzymes
CD34+ cells; chromosomes; developmental biology; genes; human; iPSC; lentiviral vector; protein transduction; stem cells; virus; zinc-finger nucleases