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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| Format: | Article |
| Language: | English |
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BMC
2021-11-01
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| Series: | Stem Cell Research & Therapy |
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| Online Access: | https://doi.org/10.1186/s13287-021-02648-4 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-14T16:28:29Z |
| format | Article |
| id | doaj.art-d1ce988184354b54b4107b2f9529c336 |
| institution | Directory Open Access Journal |
| issn | 1757-6512 |
| language | English |
| last_indexed | 2024-12-14T16:28:29Z |
| publishDate | 2021-11-01 |
| publisher | BMC |
| record_format | Article |
| series | Stem Cell Research & Therapy |
| spelling | doaj.art-d1ce988184354b54b4107b2f9529c3362022-12-21T22:54:38ZengBMCStem Cell Research & Therapy1757-65122021-11-0112111310.1186/s13287-021-02648-4Targeted integration of EpCAM-specific CAR in human induced pluripotent stem cells and their differentiation into NK cellsShin Yi Tang0Shijun Zha1Zhicheng Du2Jieming Zeng3Detu Zhu4Yumei Luo5Shu Wang6Department of Biological Sciences, National University of SingaporeDepartment of Biological Sciences, National University of SingaporeDepartment of Biological Sciences, National University of SingaporeInstitute of Bioengineering and NanotechnologyKey Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical UniversityKey Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical UniversityDepartment of Biological Sciences, National University of SingaporeAbstract 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.https://doi.org/10.1186/s13287-021-02648-4Induced 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 |
| spellingShingle | 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 Stem Cell Research & Therapy 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 |
| title | 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_short | 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 |
| 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 |
| url | https://doi.org/10.1186/s13287-021-02648-4 |
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