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

作　　者：迟晓琳 云少伟 姚瑶 饶书权 CHI Xiaolin;YUN Shaowei;YAO Yao;RAO Shuquan(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;Tianjin Institutes of Health Sciences,Tianjin 301600,China)

机构地区：[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 [2]天津医学健康研究院,天津301600

出　　处：《基础医学与临床》2025年第5期608-615,共8页Basic and Clinical Medicine

基　　金：国家自然科学基金(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)。

摘　　要：目的构建一种无饲养层依赖的体外NK细胞扩增与基因编辑整合的平台,以解决冷冻保存原代人类自然杀伤(NK)细胞在临床应用中面临的扩增效率低、基因编辑困难及安全性不足等关键问题。方法通过系统性优化培养基及CRISPR/Cas9核糖核蛋白(RNP)的核转染条件,建立基于非病毒载体的多重基因编辑体系。通过流式细胞测量术及细胞毒性实验评估细胞表型(CD56^(+)CD3^(-))、活力、编辑效率及肿瘤杀伤活性。结果NK细胞数可在25 d内扩增5000倍的同时保持高纯度(CD56^(+)CD3^(-)>95%)和细胞活力(>90%)。扩增后的细胞经冻存复苏后仍维持高活力(>80%)及肿瘤杀伤活性。利用Cas9-RNP递送系统,成功实现NKG2A和CISH双重免疫检查点的高效敲除(>80%),显著增强NK细胞对K562肿瘤细胞的毒性(P<0.05)。结论与病毒载体相比,非病毒递送策略避免了基因组整合风险,并降低脱靶效应。为临床级NK细胞免疫治疗提供了安全、高效的技术平台,推动了多重基因编辑在肿瘤治疗中的应用。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 5000-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.

关 键 词：自然杀伤细胞 基因编辑 CRISPR/Cas9非病毒递送 

分 类 号：R34[医药卫生—基础医学]