基础医学与临床2025，Vol.45Issue(5)：608-615,8.DOI:10.16352/j.issn.1001-6325.2025.05.0608
脐带血NK细胞的体外扩增与CRISPR/Cas9基因编辑平台的构建
Development of an integrated platform for in vitro expansion and CRISPR-Cas9 gene editing of umbilical cord blood NK cells
摘要
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.关键词
自然杀伤细胞/基因编辑/CRISPR/Cas9非病毒递送Key words
natural killer cells/gene editing/CRISPR-Cas9 non-viral delivery分类
基础医学引用本文复制引用
迟晓琳,云少伟,姚瑶,饶书权..脐带血NK细胞的体外扩增与CRISPR/Cas9基因编辑平台的构建[J].基础医学与临床,2025,45(5):608-615,8.基金项目
国家自然科学基金(82370118) （82370118）
中国医学科学院医学与健康科技创新工程(2024-I2M-ZH-015,2023-I2M-2-007) （2024-I2M-ZH-015,2023-I2M-2-007）
