Реферат
@#Objective: To prepare human peripheral blood T lymphocytes with TIM3 (T cell immunologlobulin and mucin-3) gene knockout by using CRISPR-Cas9 gene editing technique and plasmid electrotransfection system, and to discuss whether the knockout of TIM3 gene could enhance the immune response and anti-tumor efficacy of T cells. Methods: Double plasmids hTIM3 sgRNA/Cas9 were transfected into human peripheral blood T lymphocytes of EBV positive gastric cancer patients by using electrotransfection system. The transfection efficiency was examined 24 h later by flow cytometry and fluorescence microscope. The proliferation activity of the T cells after gene knockout was observed during in vitro culture, and the knockout efficiency and phenotypes of the modified T cells were evaluated by flow cytometry. Furthermore, tumor antigen peptide was used to activate T cells, and the level of modified T cells secreting cytokines and its cytotoxicity against gastric cancer AGS-EBV cells were evaluated. Results: Electrotransfection system could successfully transfect hTIM3 sgRNA/Cas9 double plasmids into human peripheral blood T lymphocytes with an average transfection efficiency of (41.5±3.6)%, and the gene knockout efficiency fluctuated between 40.0% and 50.0% (all P<0.01). The proliferation of the modified T cells was not significantly changed in the TIM3 gene knockout group even after the prolonged co-culturing with tumor antigenic peptide;and for the activated molecules, only HLA-DR exhibited significant elevation as compared with control group (P<0.05). Remarkably, T cells with TIM3 gene knockout showed significantly elevated secretion of TNF-α and IFN-γ (P<0.01 or P<0.05), as well as obviously enhanced in vitro cytotoxicity against gastric cancer AGS-EBV cells (P<0.05). Conclusion: It’s simple and feasible of CRISPR-Cas9 gene editing technique and plasmid electrotransfection system to prepare T lymphocytes with engineered TIM3 gene knockout. The expression level of TIM3 was down-regulated in in vitro culture. More importantly, the modified T cells performed superior immune response and cytotoxicity, which may provide a new idea for gene engineering cell immunotherapy.