Development of an integrated platform for in vitro expansion and CRISPR-Cas9 gene editing of umbilical cord blood NK cells
10.16352/j.issn.1001-6325.2025.05.0608
- VernacularTitle:脐带血NK细胞的体外扩增与CRISPR/Cas9基因编辑平台的构建
- Author:
Xiaolin CHI
1
;
Shaowei YUN
;
Yao YAO
;
Shuquan RAO
Author Information
1. 中国医学科学院血液病医院(中国医学科学院血液学研究所)血液与健康全国重点实验室 国家血液系统疾病临床医学研究中心 细胞生态海河实验室,天津30020;天津医学健康研究院,天津 301600
- Keywords:
natural killer cells;
gene editing;
CRISPR-Cas9 non-viral delivery
- From:
Basic & Clinical Medicine
2025;45(5):608-615
- CountryChina
- Language:Chinese
-
Abstract:
;Objective To establish an integrated feeder-free platform for in vitro expansion and gene editing to tack-le 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 op-timization of culture medium and nucleofection conditions.Cell phenotype(CD56+CD3-),viability,editing effi-ciency,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 tech-nical platform for clinical application of NK cell immunotherapy and potentially encourage application of multiplex gene editing in cancer therapy.
