Study On The Function Of ?CD19-CAR-T By Silencing Of ACAT1 Gene

CAR-T therapy is the most promising tumor immunotherapy in the past 20 years,but the costs of treatment is too expensive for patients to afford.How to improve the anti-tumor efficacy of CAR-T cells has become the focus of many scientists.Cholesterol acyltransferases 1(ACAT1)can regulate the metabolism of cholesterol and affect the anti-tumor abbility of CD8~+T cells.In this study,the anti-tumor effect of?CD19 CAR-T was explored by silencing of ACAT1 through RNA interference technology.Aim to reduce the amount of novel CAR19-T cells resulting in reducing the costs.According to the ACAT1 gene sequence in GenBank,three ACAT1 specific shRNAs were designed and inserted into the pLL3.7-U6-EF1?-EGFP lentiviral vector.The recombinant lentiviral vector was co-transfected with the helper plasmid into 293T cells to package lentivirus carrying ACAT1-shRNA gene sequence.The interfering sequence was screened by transfecting the lentivirus to Jurkat cells and analyzing ACAT1 mRNA and protein expression.The letiviral vector pLL3.7-ACAT1-shRNA-?CD19-CAR was constructed and the lentiviruses were packaged.After Jurkat cell line or primary T cells were infected by the lentiviruses,the anti-tumor ability,activation,proliferation of shRNA-?CD19-CAR-Jurkat,ACAT1-shRNA-?CD19-CAR-T cells were studied.The sequencing results showed that the four synthesized ACAT1-shRNA sequences were successfully inserted into p LL3.7-U6-EF1?-EGFP vector.ACAT1-shRNA-1 has been proved to have the best interference efficiency,and the silencing effect was persistent.pLL3.7-ACAT1-sh RNA-?CD19-CAR vector was successfully constructed,and the titer of the concentrated viruses has reached 2×10~8 TU/m L.The infection effciency was up to 80%if Jurkat cells were infected with the lentiviruses.After co-culture of the modified Jurkat cells with target cells,ACAT1-shRNA-?CD19-CAR-Jurkat cells showed the enhanced antitumor ability,the upregulated activation level,more expression of IFN-?and granzyme B,and the increased degranulation were detected in comparison with?CD19-CAR-Jurkat cells.Similarly,ACAT1-shRNA-?CD19-CAR-T cells were prepared by infecting T lymphocytes with lentiviruses.Although the positive rate was about 25-50%,ACAT1-shRNA could knockdown the expression of ACAT1 in?CD19-CART cells and the interfering efficiency was 40%,the immunological functions of?CD19-CAR-T cells have been enhanced after silencing of ACAT1.This study demonstrated that knock down ACAT1 has effectively enhanced the anti-tumor ability of CAR19-T cells,which provided experimental basis for the research on the novel CAR-T cells.