Development of an integrated platform for in vitro expansion and CRISPR-Cas9 gene editing of umbilical cord blood NK cells
Objective To establish an integrated feeder-free platform for in vitro expansion and gene editing to tackle the major challenges in clinical applications of cryopreserved primary human natural killer (NK) cells in terms of low expansion efficiency, technical difficulty in genetic modification and sa...
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| Format: | Article |
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Institute of Basic Medical Sciences and Peking Union Medical College Hospital, Chinese Academy of Medical Sciences / Peking Union Medical College.
2025-05-01
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| Series: | Jichu yixue yu linchuang |
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| Online Access: | https://journal11.magtechjournal.com/Jwk_jcyxylc/fileup/1001-6325/PDF/1001-6325-2025-45-5-608.pdf |
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| author | CHI Xiaolin, YUN Shaowei, YAO Yao, RAO Shuquan |
| author_facet | CHI Xiaolin, YUN Shaowei, YAO Yao, RAO Shuquan |
| author_sort | CHI Xiaolin, YUN Shaowei, YAO Yao, RAO Shuquan |
| collection | DOAJ |
| description | Objective To establish an integrated feeder-free platform for in vitro expansion and gene editing to tackle the major challenges in clinical applications of cryopreserved primary human natural killer (NK) cells in terms of low expansion efficiency, technical difficulty in genetic modification and safety concerns. Methods A non-viral CRISPR-Cas9 ribonucleoprotein (RNP)-based multiplex gene editing system was developed through systematic optimization of culture medium and nucleofection conditions. Cell phenotype (CD56+CD3-), viability, editing efficiency, and tumor-killing activity were evaluated via flow cytometry and cytotoxicity assays. Results The number of NK cells achieved 5 000-fold expansion over 25 days while maintaining high purity (CD56+CD3- ＞95%) and viability (＞90%).Post-thawing viability (＞80%) and tumor-killing capacity were preserved.Cas9 RNP delivery enabled efficient dual knockout of NKG2A and CISH immune checkpoint genes (＞80%), significantly enhanced cytotoxicity against K562 tumor cells (P＜0.05). Conclusions Compared to viral vectors, the non-viral strategy eliminates genomic integration risks and reduces off-target effects. This result may provide a safe and efficient technical platform for clinical application of NK cell immunotherapy and potentially encourage application of multiplex gene editing in cancer therapy. |
| format | Article |
| id | doaj-art-a08d941db4e94c2ebf3855d8b7467f16 |
| institution | Kabale University |
| issn | 1001-6325 |
| language | zho |
| publishDate | 2025-05-01 |
| publisher | Institute of Basic Medical Sciences and Peking Union Medical College Hospital, Chinese Academy of Medical Sciences / Peking Union Medical College. |
| record_format | Article |
| series | Jichu yixue yu linchuang |
| spelling | doaj-art-a08d941db4e94c2ebf3855d8b7467f162025-08-20T03:29:57ZzhoInstitute of Basic Medical Sciences and Peking Union Medical College Hospital, Chinese Academy of Medical Sciences / Peking Union Medical College.Jichu yixue yu linchuang1001-63252025-05-0145560861510.16352/j.issn.1001-6325.2025.05.0608Development of an integrated platform for in vitro expansion and CRISPR-Cas9 gene editing of umbilical cord blood NK cellsCHI Xiaolin, YUN Shaowei, YAO Yao, RAO Shuquan01. State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, CAMS & PUMC, Tianjin 300020;;2. Tianjin Institutes of Health Sciences, Tianjin 301600, ChinaObjective To establish an integrated feeder-free platform for in vitro expansion and gene editing to tackle the major challenges in clinical applications of cryopreserved primary human natural killer (NK) cells in terms of low expansion efficiency, technical difficulty in genetic modification and safety concerns. Methods A non-viral CRISPR-Cas9 ribonucleoprotein (RNP)-based multiplex gene editing system was developed through systematic optimization of culture medium and nucleofection conditions. Cell phenotype (CD56+CD3-), viability, editing efficiency, and tumor-killing activity were evaluated via flow cytometry and cytotoxicity assays. Results The number of NK cells achieved 5 000-fold expansion over 25 days while maintaining high purity (CD56+CD3- ＞95%) and viability (＞90%).Post-thawing viability (＞80%) and tumor-killing capacity were preserved.Cas9 RNP delivery enabled efficient dual knockout of NKG2A and CISH immune checkpoint genes (＞80%), significantly enhanced cytotoxicity against K562 tumor cells (P＜0.05). Conclusions Compared to viral vectors, the non-viral strategy eliminates genomic integration risks and reduces off-target effects. This result may provide a safe and efficient technical platform for clinical application of NK cell immunotherapy and potentially encourage application of multiplex gene editing in cancer therapy.https://journal11.magtechjournal.com/Jwk_jcyxylc/fileup/1001-6325/PDF/1001-6325-2025-45-5-608.pdfnatural killer cells|gene editing|crispr-cas9 non-viral delivery |
| spellingShingle | CHI Xiaolin, YUN Shaowei, YAO Yao, RAO Shuquan Development of an integrated platform for in vitro expansion and CRISPR-Cas9 gene editing of umbilical cord blood NK cells Jichu yixue yu linchuang natural killer cells|gene editing|crispr-cas9 non-viral delivery |
| title | Development of an integrated platform for in vitro expansion and CRISPR-Cas9 gene editing of umbilical cord blood NK cells |
| title_full | Development of an integrated platform for in vitro expansion and CRISPR-Cas9 gene editing of umbilical cord blood NK cells |
| title_fullStr | Development of an integrated platform for in vitro expansion and CRISPR-Cas9 gene editing of umbilical cord blood NK cells |
| title_full_unstemmed | Development of an integrated platform for in vitro expansion and CRISPR-Cas9 gene editing of umbilical cord blood NK cells |
| title_short | Development of an integrated platform for in vitro expansion and CRISPR-Cas9 gene editing of umbilical cord blood NK cells |
| title_sort | development of an integrated platform for in vitro expansion and crispr cas9 gene editing of umbilical cord blood nk cells |
| topic | natural killer cells|gene editing|crispr-cas9 non-viral delivery |
| url | https://journal11.magtechjournal.com/Jwk_jcyxylc/fileup/1001-6325/PDF/1001-6325-2025-45-5-608.pdf |
| work_keys_str_mv | AT chixiaolinyunshaoweiyaoyaoraoshuquan developmentofanintegratedplatformforinvitroexpansionandcrisprcas9geneeditingofumbilicalcordbloodnkcells |