Study On ShRNA-loaded CAR-T Lentiviral Vector For Silencing PD-1 Gene Expression

This topic is based on CAR-T technology,centered on cell programmed death receptor(PD-1)and PD-1 ligand(PD-L1).PD-1 is mainly expressed on activated T cell surface,but the expression of it in a large number of tumor cells significantly increases.PD-L1 combined with PD-1 of T cells,which inhibits the activation of T cells and induces apoptosis of them.The goal of the project is to infect T cells by lentivirus carrying the anti-PD-1 shRNA gene sequence so that the PD-1 expression of the cell surface was greatly reduced while the CAR-T cells were constructed.The inhibition of tumor cell surface ligand interacts with PD-1 can improve the anticancer effect of CAR-T cells.The silencing of PD-1 gene and the inhibition of PD-1 expression on cell surface were achieved by RNA interference.In this paper,the experimental method is expected to design the corresponding small RNA interference sequence for the PD-1 gene sequence.The double stranded DNA was cloned into plasmid vectors and 293T cells were then transfected with three plasmid system.Preparation of multiple lentiviral vectors which can induce PD-1 gene silencing in T cell and then screening the lentivirus vector which has the best efficiency of PD-1 gene silencing through in vitro cell experiment.The experimental results showed that compared with the negative control,the effect of the lentivirus which silences PD-1 gene was significantly different.It effectively reduced the expression of PD-1 gene on the cell surface.Infecting T lymphocytes with the lentivirus can be used to prepare the new form of CAR-T cells which the production of PD-1 expression suppressed for subsequent scientific research.