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

作者：Yujia Cai, Anders Laustsen, Yan Zhou, Chenglong Sun, Mads Valdemar Anderson, Shengting Li, Niels Uldbjerg, Yonglun Luo, Martin R. Jakobsen, Jacob Giehm Mikkelsen · 发表于：eLife · 年份：2016 · DOI：10.7554/elife.12213 · 被引用次数：23 · 研究领域：Virus-based gene therapy research、CRISPR and Genetic Engineering、RNA Interference and Gene Delivery

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.