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Archive > Volume 30 Issue 5 > 2023,30(5):373-379. DOI:10.3872/j.issn.1007-385X.2023.05.002 Prev Next
Basic Research
CRISPR/Cas9-mediated endogenous TCR knockout enhances TCR-T cells targeted killing HPV16-positive cervical cancer cells
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Abstract:
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.
Keywords:
cervical cancer ; SiHa cell ; CRISPR/Cas9 ; TCR knockout ; TCR-T cell ; HPV16
