Transduction of the herpes thymidine kinase gene into premalignant murine mammary epithelial cells renders subsequent breast cancers responsive to ganciclovir therapy.

Drug sensitivity ("suicide") genes can sensitize cancer cells to chemotherapy, but therapeutic use of these genes is limited by difficulties in delivering them to all areas of established cancers. An alternative strategy entails preemptive introduction of suicide genes into tissues at risk for cancer, thereby imparting drug sensitivity as a clonal property to cancers arising from sensitized cells. To test the preemptive approach, a retroviral vector was used to transduce the herpes thymidine kinase gene into the TM4 line of preneoplastic murine mammary epithelial cells to yield a clonal subline sensitized to the guanosine analog ganciclovir. Ganciclovir therapy of tumors that arose from the transduced cells retarded tumor growth and induced durable regressions in 7/20 mice; ganciclovir was ineffective against control tumors. The results imply the possibility of reducing cancer lethality by actions taken before cancers arise.

Escherichia coli gpt gene sensitizes rat glioma cells to killing by 6-thioxanthine or 6-thioguanine.

Genes that encode enzymes that convert inactive "prodrugs" into anticancer metabolites may be therapeutically useful against brain tumors. Unlike other genes tested to date in brain tumor models, the Escherichia coli gpt gene is unique in that it not only sensitizes cells to the prodrug 6-thioxanthine (6TX) but also encodes resistance to a different regimen (mycophenolic acid, xanthine, and hypoxanthine), thus providing a means to select for gpt-positive cells. In the present study, rat C6 glioma cells were infected with a retrovirus vector that transduces this gene. A clonal line (C6GPT-7) was derived that exhibited significant 6TX susceptibility in vitro with an ID50 of 2.5 mumol/L, whereas 50% growth inhibition of parental C6 cells was not achieved at concentrations tested (up to 50 mumol/L). This line also exhibited significant sensitivity to 6-thioguanine (6TG), with an ID50 of 0.05 mumol/L, whereas 50% growth inhibition of parental C6 cells was achieved at 0.5 mumol/L. In a "bystander" assay, C6GPT-7 tumor cells efficiently transferred 6TX sensitivity to C6 cells at ratios as low as 1:9 (C6GPT-7:C6). This in vitro bystander effect was abrogated when C6GPT-7 and C6 cells were separated by a microporous membrane, suggesting that it was not mediated by highly diffusible metabolites. In vivo both 6TX and 6TG significantly inhibited the growth of subcutaneously transplanted C6GPT-7 cells but not that of C6 cells in athymic mice. In an intracerebral model, both 6TX and 6TG exhibited significant antiproliferative effects against tumors formed by C6GPT-7 cells. These findings provide a basis for exploring further gene therapy strategies based on in vivo transfer of the E coli gpt gene to provide chemosensitivity against 6TX and 6TG.

Retrovirally transduced Escherichia coli gpt genes combine selectability with chemosensitivity capable of mediating tumor eradication.

"Suicide genes" encoding exceptional sensitivity to chemotherapeutic agents can potentially improve the selectivity of cancer therapy. As a component of a retroviral gene therapy vector, a suicide gene might also improve the safety of gene therapy by permitting the subsequent ablation of transduced cells exhibiting neoplastic or other aberrant behavior. An extra gene, however, cannot easily be added to vectors already carrying a therapeutic gene and a selectable drug resistance marker without compromising gene expression. To circumvent this obstacle, we have investigated a retrovirally transduced Escherichia coli gpt gene on the basis of evidence that it might subserve a dual sensitivity/resistance function. A gpt vector was used to transduce the gene into murine K3T3 sarcoma cells. In vitro, gpt-positive K3T3 clones could be selected on the basis of resistance to a regimen containing mycophenolic acid and xanthine; the same clones were 18 to 86 times as sensitive to 6-thioxanthine (6TX) as their gpt-negative counterparts. In mice, systemic 6TX therapy induced durable regressions in 19/20 gpt-positive K3T3 sarcomas without affecting gpt-negative tumors. These results indicate that selectability and in vivo chemosensitivity can be expressed in the same cell population from a single retrovirally transduced gene and imply the additional possibility of fusing the gpt gene with a therapeutic gene to create vectors expressing three important functions from a single gene.

Multiple transduction as a means of preserving ganciclovir chemosensitivity in sarcoma cells carrying retrovirally transduced herpes thymidine kinase genes.

The potential value of retroviral gene transfer as a means of targeting therapeutic genes to neoplastic cells is threatened by the tendency of occasional cells to lose transduced genes or their expression. To determine whether this threat could be reduced by transducing multiple copies of a therapeutic gene, we compared 8 murine sarcoma sublines carrying from 1 to 6 copies of a retrovirally transduced herpes simplex virus thymidine kinase gene, which sensitizes cells to ganciclovir (GCV). When variability consequent to differences in vector integration site was excluded, increased copy number was associated with an increase in GCV sensitivity and a major reduction in the frequency of GCV-resistant mutants. The results suggest a potential means of preserving the efficacy of future antineoplastic gene therapy strategies.