T lymphocyte transduction with herpes simplex virus-thymidine kinase (HSV-tk) gene: Comparison of four different infection protocols

In this study, we assessed the efficiency of T lymphocyte transduction with a retroviral vector carrying the herpes simplex virus thymidine kinase (HS V-tk);and neomycin phosphotransferase (neo) genes by four different protoco ls: standard supernatant infection, supernatant infection plus centrifugati on steps, supernatant infection on fibronectin fragment-coated wells, and c ocultivation. After retrovirus-mediated gene transfer of tk-neo in PHA/IL-2 -stimulated primary T lymphocytes and G418 selection, T cells retained thei r proliferative activity, alloresponsiveness, ability to produce and to res pond to IL-2, and ganciclovir (gcv)-specific sensitivity. When the four dif ferent transduction techniques were compared, no significant differences we re seen in terms of cellular viability, proliferation capacity, and immunop henotyping. fk gene expression was the same in all transduced selected popu lations, as indicated by gcv sensitivity. Transduction efficiency was evalu ated by semiquantitative PCR. Using the standard supernatant infection meth od, the rate of infection was extremely low (<5%). After adding the centrif ugation step or performing supernatant infection on fibronectin fragment-co ated wells, PCR analysis showed a 30%-40% rate of transduced cells. After i nfection by cocultivation, the rate of transduced cells was 30%-40%. These results demonstrate that supernatant infection plus centrifugation, superna tant infection on fibronectin fragment-coated wells, and cocultivation meth ods provide equivalent rates of transduced cells. The lack of reproducibili ty and safety indicates that cocultivation is not suitable for clinical stu dies. In our view, supernatant infection plus centrifugation is easier to p erform than using fibronectin fragments, and it is currently the optimal me thod for clinical studies when large quantities of T lymphocytes are being processed.