Basic Studies Of Novel Suicide Gene Systems For Modifying Donor T Cells For Controlling Graft-versus-host-disease

Allogenic hematopoietic stem cell transplantation (alloHSCT) is the mostefficient treatment for hematological malignancies and some solid tumors.Allogenic donor T lynphocytes are also used for treatment of relapse orlymphoma associated with Epstein-Barr virus infection post transplantation. Butthe graft versus host disease (GVHD) induced by allogenic immune cells is one ofthe major causes of mortality in allo-HSCT.Specific and conditional in vivo ablation of alloreactive donor T cells afterallo-HSCT could significantly contribute to preventing and treating GVHD. Theuse of donor T cells expressing a "suicide" gene has the potential of achievingsuch a goal. Herpes simplex virus 1 thymidine kinase (HSV-TK) has been usedfor that purpose. Since the pro-drug ganciclovir (GCV) is widely used forprevetion and treatment of cytomeglovirus (CMV) infection, which is a commonand fetal complication in allo-HSCT, it will be an embarrassing situation whenusing prodrug GCV in patients with CMV infection who was givenHSV-TK-expressing T cells. New suicide gene systems should be exploredaccordingly. Yeast cytosine deaminase/5-fluorocytosine (YCD/5-FC) is suchnovel suicide gene systems investigated in this project.To reach the goal, the ex vivo highly efficient retroviral-mediated genetransfer into human T cells as well as pro-drug sensitivity of such gene-modifiedT cells should be established. Further more, the suicide gene modified human Tcells should retain full immune function and can be expanded to be enough innumber for clinical use.Objective: 1. To establish efficient technologies to introduce foreign genes into human Tcells and to quickly-select positive cells out. 2. To transfer YCD into human T cells, and to determine if the novel suicidegene system does work in vitro.. Methods and results: 1.We established the ways to get high-titer RV by using theliposome-mediated method. By pseudotyping with VSV-G, the infection abilitywas highly improved. 2.YCD gene transduction into target cells and function study. Mononuclearisolated from mobilized PB were cultured with CD3 monoclonal antibody (mAb)plus CD28 mAb and IL-2 for 3 days and then transferred to flasks coated withCH-296.Then the supermatant of retrovirus was added into the wells.Thetransfected efficiency was determined by PCR,and the function of transgeneic Tcells was studied. Combined with concentrated RV by high-speed centrifugationat the speed of 20,000g×4h, the fibronectin fragment CH-296 coating andcentrifugation infection, the efficiency of RV transfection reached 38.2% ofhuman T cells. The integration of YCD was detected in transgeneic T-YCD cellsby PCR. The system worked well in vitro. The YCD gene modification did notchanged ablities of T cells expansion, cytotoxic abilities to K562 leukemia cellsand functions of cytokine secretion. T-YCD can be killed by 38μmol/L 5-FC invitro. Conclusion: We well established the protocol of introducing foreign genes by RV intohuman T cell. Human T(hT-YCD) were efficiently transfected by using the...