Enhanced in vitro and in vivo Bystander Effect by Double Transfer of Herpes Simplex Virus Thymidine Kinase Gene

ABSTRACT

OBJECTIVE: The bystander effect, the phenomenon that non-transduced tumor cells can be killed along with the transduced cells, enables suicidal gene therapy feasible in spite of low efficiency of gene transfer at present time. Increment of thymidine kinase activity in the cells through double copy insertion of HSVtk gene may lead to increased bystander effect in vitro and in vivo, therefore enhancing the therapeutic potential of suicidal gene therapy. To examnine this hypothesis, we did an experiment to improve bystander effect by using double transfer of HSVtk gene into 9L tuomr cells. METHODS: We transduced 9L glioblastoma cells which had one copy or two copies of HSVtk gene using retroviral vector. Two different retroviral vector plasmids containing HSVtk gene were made employing pBabePuro or LXSN plasmid whose selection markers were puromycin and G418, respectively(LtkSP and LtkSN). Recombinant retrovirus was produced from ecotrophic PA317 packaging cells. Infection of the 9L cells with LtkSP recombinant retrovirus and selection with puromycin was done in vitro to make 9L/LtkSP(9LtkS). These cells were infected again with LtkSN recombinant retrovirus and selected under G418 to establish cells containing two copies of HSVtk gene, 9L/LtkSP/ LtkSN(9LtkD). RESULTS: By measuring the intracellular amount of phosphorylated 3H-GCV, 9LtkD cell lines showed significantly increased HSVtk activity, being 70% higher than that of 9LtkS cell lines. The sensitivity to GCV was also markedly increased. In vitro bystander effect, examined by coculturing with HSVtk gene transduced cells and 9L cells, was significantly increased on 9LtkD cell lines. To evaluate the in vivo bystander effect, 9LtkD/9L(20%80%) cells or 9LtkS/9L (20%80%) cells were implanted into the brains of Fisher 344 inbred rats, followed by administration of ganciclovir. Rats implanted with 9LtkD/9L cells showed better tumor regression and longer survival than those of 9LtkS/9L after treatment with ganciclovir. CONCLUSION: These results suggest that increasing herpes simplex virus thymidine kinase activity by introducing double copy of HSVtk gene into tumor cells would improve in vitro and in vivo bystander effect, and lead to enhanced efficacy of suicidal gene therapy.