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Title: Targeted integration of EpCAM-specific CAR in human induced pluripotent stem cells and their differentiation into NK cells
Authors: Tang, Shin Yi 
Zha, Shijun 
Du, Zhicheng 
Zeng, Jieming
Zhu, Detu
Luo, Yumei
Wang, Shu 
Keywords: Adeno-associated virus integration site 1 (AAVS1)
Chimeric antigen receptors (CAR)
Epithelial cell adhesion molecule (EpCAM)
Genetic engineering
Induced pluripotent stem cells (iPSC)
Natural killer cells (NK)
NK differentiation
Targeted integration
Zinc finger nuclease (ZFN)
Issue Date: 21-Nov-2021
Publisher: BioMed Central Ltd
Citation: Tang, Shin Yi, Zha, Shijun, Du, Zhicheng, Zeng, Jieming, Zhu, Detu, Luo, Yumei, Wang, Shu (2021-11-21). Targeted integration of EpCAM-specific CAR in human induced pluripotent stem cells and their differentiation into NK cells. Stem Cell Research and Therapy 12 (1) : 580. ScholarBank@NUS Repository.
Rights: Attribution 4.0 International
Abstract: Background: Redirection of natural killer (NK) cells with chimeric antigen receptors (CAR) is attractive in developing off-the-shelf CAR therapeutics for cancer treatment. However, the site-specific integration of a CAR gene into NK cells remains challenging. Methods: In the present study, we genetically modified human induced pluripotent stem cells (iPSCs) with a zinc finger nuclease (ZFN) technology to introduce a cDNA encoding an anti-EpCAM CAR into the adeno-associated virus integration site 1, a “safe harbour” for transgene insertion into human genome, and next differentiated the modified iPSCs into CAR-expressing iNK cells. Results: We detected the targeted integration in 4 out of 5 selected iPSC clones, 3 of which were biallelically modified. Southern blotting analysis revealed no random integration events. iNK cells were successfully derived from the modified iPSCs with a 47-day protocol, which were morphologically similar to peripheral blood NK cells, displayed NK phenotype (CD56+CD3-), and expressed NK receptors. The CAR expression of the iPSC-derived NK cells was confirmed with RT-PCR and flow cytometry analysis. In vitro cytotoxicity assay further confirmed their lytic activity against NK cell-resistant, EpCAM-positive cancer cells, but not to EpCAM-positive normal cells, demonstrating the retained tolerability of the CAR-iNK cells towards normal cells. Conclusion: Looking ahead, the modified iPSCs generated in the current study hold a great potential as a practically unlimited source to generate anti-EpCAM CAR iNK cells. © 2021, The Author(s).
Source Title: Stem Cell Research and Therapy
ISSN: 1757-6512
DOI: 10.1186/s13287-021-02648-4
Rights: Attribution 4.0 International
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