Construction Of HSV-TK/CD20 Double Suicide Gene Vector And Its Killing Effect On T Cells

As a new type of cell-drugs,TCR-T and CAR-T cells have been widely used in clinical trials of tumor adoptive cell therapy?ACT?.T cells of the patients were genetically modified in vitro,made it to become autologous cell-drugs with new characteristics and functions.And then the genetically edited cell-drugs were transferred to the patients to achieve specific killing of tumor cells.However,there are still some side effects in the treatment of TCR-T and CAR-T.At present,adverse effects have been observed in the treatment process,such as cytokine release syndrome?CRS?and on-target/off-tumor toxicity.Unlike traditional drugs which can be metabolized in vivo,cell-drugs are endowed with the ability to proliferate in vivo,which resulted in the uncertainty of adverse effects of cell drugs.We intended to enhance the safety and controllability of cell-drugs by means of self-elimination.HSV-TK is a herpes simplex virus thymidine kinase.The triphosphate produced by the combination of ganciclovir and HSV-TK can inhibit DNA replication,and then induce cells apoptosis.However,it took 72 hours for HSV-TK to eliminate cells,and the use of GCV has limitations.HSV-TK is an intracellular protein,which can not be selected directly after being transfected into cells.Therefore,it's necessary to ligate an extracellular protein as a select marker.RQR8 is a small synthetic transmembrane protein containing a CD20 epitope and a CD34 epitope tag.It contains both selecting markers which can be recognized by clinical CD34 sorting system and CD20 epitope which can be targeted by the clinical drug rituximab.Rituximab results in CD20 cell death through complement-dependent cytotoxicity.CD20 suicide system only need 2 hours to eliminate cells,but the therapeutic effect is affected by complement state of the patients.HSV-TK and CD20 can achieve cell self-cleaning through different ways,which can complement each other in the time and mode of action.This study aim to link RQR8 and HSV-TK through T2A,to construct a pLV-RQR8-T2A-TK lentivirus plasmid.Then,establish a HSV-TK/CD20 double suicide gene system which can be selected by CD34 beads.And then detect its self-cleaning ability and selecting ability in Jurkat cells and T cells.In this study,the pLV-RQR8-T2A-TK lentivirus plasmid was constructed by in-fusion cloning technique.The enzymes digestion and DNA sequencing confirmed the construction was correct.LV-RQR8-T2A-TK lentivirus was obtained by lentivirus packaging technique.The virus was used to infect Jurkat cells and T cells.The expression of CD34 epitopes in RQR8 was detected by anti-CD34 antibody.After CD34 magnetic beads selected,the purity of RQR8⁺TK⁺Jurkat cells was 99.5%and the purity of RQR8⁺TK⁺T cells was 92.46%.These results suggested that the CD34 epitopes in RQR8 can be used as a selecting site,and the T2A linker has no effect on the expression of RQR8 gene.Then,two kinds of cells were treated with rituximab,after 20 h the number of living cells was measured by flow cytometry.The results showed that when the concentration of RTX was increased to 30?90?180?g/ml,the cell survival rate was significantly lower than control group?P<0.05?,and the survival rate showed dose-dependence.RTX can cause apoptosis in RQR8 cells through CDC,which proved the expression of CD20 epitopes in RQR8 was correct and the function of RQR8 was complete.After treated with GCV for 72 h,the proliferation of RQR8⁺TK⁺Jurkat cells and RQR8⁺TK⁺T cells was inhibited significantly.The IC₅₀ of GCV on RQR8⁺TK⁺Jurkat cells was 20.66?M and RQR8⁺TK⁺T cells was 2.14?M.The apoptosis rate of RQR8⁺TK⁺Jurkat cells and RQR8⁺TK⁺T cells were significantly higher than control group?P<0.01?.These results showed that the expression of HSV-TK was normal,and T2A has no effect on the function of HSV-TK.It was concluded that the pLV-RQR8-T2A-TK lentivirus co-expression plasmid was constructed successfully.CD34 epitope in RQR8 can be used as a marker in T cells sorting,which ensure all clinical infusion cell-drugs containing double suicide plasmid.HSV-TK/CD20 double suicide gene system has been constructed successfully.These two suicide genes can induce apoptosis through different ways,which can provide an alternative option for clinical treatment,and then further ensure the safety of treatment.