目的：基于CRISPR/Cas9基因编辑技术制备无内源TCR的TCR-T细胞并鉴定其在体外杀伤HPV16阳性宫颈癌SiHa细胞的功能。方法：培养健康志愿者外周血CD8+ T细胞和Jurkat细胞，CRISPR/Cas9基因编辑技术敲除CD8+ T、Jurkat细胞的TCR基因，制备过表达转基因TCR的重组慢病毒，在敲除内源性TCR的CD8+ T和Jurkat细胞中用慢病毒过表达转基因TCR制备TCR-T细胞，多色FCM检测TCR-T细胞中TCR和CD3的表达水平，荧光素酶活性实验检测TCR-T细胞对HPV16阳性SiHa细胞的杀伤效率。结果：CRIPSR/Cas9基因编辑技术高效地敲除了外周血CD8+ T细胞和Jurkat细胞中的TRAC和TRBC基因，敲除效率分别为（81.4±4.5）%（、98.5±0.07）%，制备的无内源TCR的TCR-T细胞高效表达转基因TCR，在外周血CD8+ T和Jurkat细胞中表达率为（66.0±17.8）%、（97.3±2.6）%，敲除内源TRAC和TRBC 基因有效增强CD8+ T 和Jurkat细胞膜表达转基因TCR（均P<0.01），敲除内源TCR增强TCR-T细胞特异性杀伤HPV16阳性的SiHa细胞[（71.4±1.0）% vs（35.1±2.0）%，P<0.01）]。结论：无内源TCR的TCR-T细胞显著增强转基因TCR的表达和对HPV16阳性宫颈癌SiHa细胞的靶向杀伤能力，为提高TCR-T细胞的临床疗效提供了实验依据。

Objective: To develop TCR-T cells with endogenous TCR knockout based on CRISPR/Cas9 gene editing technology, and to identify their cytotoxicity against cancer cells in vitro. Methods: Jurkat cells and CD8+ T cells from the peripheral blood of healthy volunteers were cultured in vitro, and endogenous TCR of the CD8+ T and Jurkat cells was knocked out by CRISPR/Cas9 gene editing technique. Transgenic TCR overexpression lentivirus was prepared and transfected into the CD8+ T and Jurkat cells with endogenous TCR knockout to prepare TCR-T cells. The expression levels of TCR and CD3 in TCR-T cells were detected by multi-color FCM. The killing efficiency of TCR-T cells against HPV16 positive SiHa cells was determined by luciferase activity assay. Results: CRIPSR/Cas9 gene editing technique effectively knocked out TRAC and TRBC genes in the peripheral blood CD8+ T and Jurkat cells, with a knockout efficiency of (81.4±4.5)% , (98.5±0.07)% , respectively. The obtained TCR-T cells after transfections efficiently expressed transgenic TCR, with an expression rate up to (66.0±17.8)% , (97.3±2.6)% . Knockout of endogenous TRAC and TRBC genes effectively enhanced transgenic TCR expression on cell membrane of CD8+ T and Jurkat cells (both P<0.01). Knockout of endogenous TCR enhanced the specific killing of TCR-T cells against HPV16-positive target cells ([71.4±1.0]% vs [35.1±2.0]% , P<0.01).Conclusion: The expression of transgenic TCR in TCR-T cells without endogenous TCR expression was significantly increased, and the targeted killing ability of HPV16-positive cervical cancer SiHa cells was significantly enhanced, which provides experimental basis for improving the clinical therapeutic effect of TCR-T cells.

国家自然科学基金青年科学基金（No. 82101931）；重庆市自然科学基金（No.cstc2020jcyj-msxmX0086）；中央高校基本科研业务基金（No.2021CDJYGRH-013）