PURPOSE: Suicide gene therapy has proved to be successful in enhancing the therapeutic index by sensitizing genetically modified tumor cells to prodrugs. Two of the most widely studied suicide genes, herpes simplex virus type 1 thymidine kinase and Escherichia coli cytosine deaminase, have proved effective at selectively eliminating malignant tumor cells. We previously demonstrated that transduced 9L glioma cells expressing E. coli cytosine deaminase and herpes simplex virus type 1 thymidine kinase concomitantly as a fusion protein exhibited greater levels of targeted cytotoxicity and radiosensitization than could be achieved by single suicide gene therapy. The present in vivo studies were carried out to determine whether double suicide gene therapy would enhance the tumor control rate of orthotopically implanted malignant glioma growing in the brain when coupled with radiotherapy. MATERIALS AND METHODS: Rat 9L gliosarcoma cells were transfected with retroviral vectors containing an E. coli cytosine deaminase and herpes simplex virus type 1 thymidine kinase fusion gene and maintained in Dulbecco's modified Eagle's medium. The antitumor response of 9L E. coli cytosine deaminase and herpes simplex virus type 1 thymidine kinase tumors growing in the brain of Fischer rats was evaluated with small tumors (6-day-old tumors) versus large tumors (14-day-old tumors) against single versus double prodrug treatments. In the large brain tumors, the therapeutic efficacy of the combined single and double prodrugs coupled with radiotherapy was evaluated. RESULTS: Double suicide gene therapy using two prodrugs, 5-fluorocytosine (500 mg/kg) and ganciclovir (30 mg/kg), was effective in achieving long-term tumor control (50% survival) against early-stage brain tumors (6 days after implantation) but was only marginally effective against advanced stage tumors (14 days old). However, when these prodrugs were combined with radiotherapy and double suicide gene therapy against advanced-stage tumors, more than 70% of the animals were cured, whereas radiotherapy alone (20 Gy) failed to achieve any cure at all. Combined radiotherapy and single prodrug therapy showed a moderate increase in the animal survival rate (17% and 40% for 5-fluorocytosine and ganciclovir, respectively) but was inferior to the combination therapy of radiation and double prodrugs. CONCLUSION: The present in vivo results indicate that double suicide gene therapy combined with radiotherapy may represent a new, effective approach to achieve a high tumor cure rate without producing any excessive normal tissue damage.
PURPOSE: Suicide gene therapy has proved to be successful in enhancing the therapeutic index by sensitizing genetically modified tumor cells to prodrugs. Two of the most widely studied suicide genes, herpes simplex virus type 1 thymidine kinase and Escherichia coli cytosine deaminase, have proved effective at selectively eliminating malignant tumor cells. We previously demonstrated that transduced 9L glioma cells expressing E. coli cytosine deaminase and herpes simplex virus type 1 thymidine kinase concomitantly as a fusion protein exhibited greater levels of targeted cytotoxicity and radiosensitization than could be achieved by single suicide gene therapy. The present in vivo studies were carried out to determine whether double suicide gene therapy would enhance the tumor control rate of orthotopically implanted malignant glioma growing in the brain when coupled with radiotherapy. MATERIALS AND METHODS:Rat 9L gliosarcoma cells were transfected with retroviral vectors containing an E. coli cytosine deaminase and herpes simplex virus type 1 thymidine kinase fusion gene and maintained in Dulbecco's modified Eagle's medium. The antitumor response of 9L E. coli cytosine deaminase and herpes simplex virus type 1 thymidine kinase tumors growing in the brain of Fischer rats was evaluated with small tumors (6-day-old tumors) versus large tumors (14-day-old tumors) against single versus double prodrug treatments. In the large brain tumors, the therapeutic efficacy of the combined single and double prodrugs coupled with radiotherapy was evaluated. RESULTS: Double suicide gene therapy using two prodrugs, 5-fluorocytosine (500 mg/kg) and ganciclovir (30 mg/kg), was effective in achieving long-term tumor control (50% survival) against early-stage brain tumors (6 days after implantation) but was only marginally effective against advanced stage tumors (14 days old). However, when these prodrugs were combined with radiotherapy and double suicide gene therapy against advanced-stage tumors, more than 70% of the animals were cured, whereas radiotherapy alone (20 Gy) failed to achieve any cure at all. Combined radiotherapy and single prodrug therapy showed a moderate increase in the animal survival rate (17% and 40% for 5-fluorocytosine and ganciclovir, respectively) but was inferior to the combination therapy of radiation and double prodrugs. CONCLUSION: The present in vivo results indicate that double suicide gene therapy combined with radiotherapy may represent a new, effective approach to achieve a high tumor cure rate without producing any excessive normal tissue damage.