Targeted integration of EpCAM-specific CAR in human induced pluripotent stem cells and their differentiation into NK cells
Tang, Shin Yi Zha, Shijun Du, Zhicheng Zeng, Jieming Zhu, Detu Luo, Yumei Wang, Shu
Zeng, Jieming
Zhu, Detu
Luo, Yumei
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Alternative Title
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).
Keywords
Adeno-associated virus integration site 1 (AAVS1), Chimeric antigen receptors (CAR), Epithelial cell adhesion molecule (EpCAM), Genetic engineering, Immunotherapy, Induced pluripotent stem cells (iPSC), Natural killer cells (NK), NK differentiation, Targeted integration, Zinc finger nuclease (ZFN)
Source Title
Stem Cell Research and Therapy
Publisher
BioMed Central Ltd
Series/Report No.
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Rights
Attribution 4.0 International
Date
2021-11-21
DOI
10.1186/s13287-021-02648-4
Type
Article