Targeted integration of EpCAM-specific CAR in human induced pluripotent stem cells and their differentiation into NK cells

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...

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Autores principales:	Shin Yi Tang, Shijun Zha, Zhicheng Du, Jieming Zeng, Detu Zhu, Yumei Luo, Shu Wang
Formato:	article
Lenguaje:	EN
Publicado:	BMC 2021
Materias:	Induced pluripotent stem cells (iPSC) Natural killer cells (NK) Chimeric antigen receptors (CAR) Adeno-associated virus integration site 1 (AAVS1) Zinc finger nuclease (ZFN) Genetic engineering Medicine (General) R5-920 Biochemistry QD415-436
Acceso en línea:	https://doaj.org/article/d1ce988184354b54b4107b2f9529c336
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spelling	oai:doaj.org-article:d1ce988184354b54b4107b2f9529c3362021-11-28T12:06:24ZTargeted integration of EpCAM-specific CAR in human induced pluripotent stem cells and their differentiation into NK cells10.1186/s13287-021-02648-41757-6512https://doaj.org/article/d1ce988184354b54b4107b2f9529c3362021-11-01T00:00:00Zhttps://doi.org/10.1186/s13287-021-02648-4https://doaj.org/toc/1757-6512Abstract 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.Shin Yi TangShijun ZhaZhicheng DuJieming ZengDetu ZhuYumei LuoShu WangBMCarticleInduced pluripotent stem cells (iPSC)Natural killer cells (NK)Chimeric antigen receptors (CAR)Adeno-associated virus integration site 1 (AAVS1)Zinc finger nuclease (ZFN)Genetic engineeringMedicine (General)R5-920BiochemistryQD415-436ENStem Cell Research & Therapy, Vol 12, Iss 1, Pp 1-13 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Induced pluripotent stem cells (iPSC) Natural killer cells (NK) Chimeric antigen receptors (CAR) Adeno-associated virus integration site 1 (AAVS1) Zinc finger nuclease (ZFN) Genetic engineering Medicine (General) R5-920 Biochemistry QD415-436
spellingShingle	Induced pluripotent stem cells (iPSC) Natural killer cells (NK) Chimeric antigen receptors (CAR) Adeno-associated virus integration site 1 (AAVS1) Zinc finger nuclease (ZFN) Genetic engineering Medicine (General) R5-920 Biochemistry QD415-436 Shin Yi Tang Shijun Zha Zhicheng Du Jieming Zeng Detu Zhu Yumei Luo Shu Wang Targeted integration of EpCAM-specific CAR in human induced pluripotent stem cells and their differentiation into NK cells
description	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.
format	article
author	Shin Yi Tang Shijun Zha Zhicheng Du Jieming Zeng Detu Zhu Yumei Luo Shu Wang
author_facet	Shin Yi Tang Shijun Zha Zhicheng Du Jieming Zeng Detu Zhu Yumei Luo Shu Wang
author_sort	Shin Yi Tang
title	Targeted integration of EpCAM-specific CAR in human induced pluripotent stem cells and their differentiation into NK cells
title_short	Targeted integration of EpCAM-specific CAR in human induced pluripotent stem cells and their differentiation into NK cells
title_full	Targeted integration of EpCAM-specific CAR in human induced pluripotent stem cells and their differentiation into NK cells
title_fullStr	Targeted integration of EpCAM-specific CAR in human induced pluripotent stem cells and their differentiation into NK cells
title_full_unstemmed	Targeted integration of EpCAM-specific CAR in human induced pluripotent stem cells and their differentiation into NK cells
title_sort	targeted integration of epcam-specific car in human induced pluripotent stem cells and their differentiation into nk cells
publisher	BMC
publishDate	2021
url	https://doaj.org/article/d1ce988184354b54b4107b2f9529c336
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Targeted integration of EpCAM-specific CAR in human induced pluripotent stem cells and their differentiation into NK cells

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