For more than ten years, the mainstay of pancreatic cancer chemotherapy has been gemcitabine [1], but its efficacy is still very limited with a response rate of 5-15% and median survival of 6 months [2]. To improve therapeutic efficacy, various combinations of anti-cancer agents have been tested, but none of them have shown dramatically better outcomes when compared to single-agent gemcitabine [3]. However, it has been recently reported that the combination of oral fluorouracil with gemcitabine may have beneficial effects [4]. This suggests that fluorouracil optimally delivered to tumor tissue may be effective in combination with gemcitabine. Prodrug enzyme therapy is a promising cancer therapy that allows localized delivery of a chemotherapeutic agent to tumor cells [5]. In this therapy, an exogenous enzyme is specifically expressed in tumor tissue where it converts a less toxic prodrug into a potent cytotoxic agent. The major problem here is achieving localized expression of the enzyme. Oncolytic viruses are mutant viruses that specifically replicate in tumor cells while sparing normal cells [6]. Because of their tumor-selective replication, they may serve as an ideal vector for prodrug enzyme therapy. HSV mutants are one of the most well-studied oncolytic viruses and several oncolytic HSV vectors have already been used in clinical trials [7]. In this grant proposal, the hypothesis is tested that tumor-targeted delivery of two different prodrug converting enzymes, yeast cytosine deaminase and uracil phosphoribosyltransferase fusion protein (yCD-UPRT) and rabbit carboxylesterase (rCE), by an oncolytic HSV vector and subsequent treatment with two prodrugs, 5-fluorocytosine (5-FC) and irinotecan (CPT-11), enhances the efficacy of gemcitabine chemotherapy against pancreatic cancer. yCD-UPRT converts 5-FC to fluorouracil and CE converts CPT-11 to SN-38, and gemcitabine and 5-FU have been reported to act synergistically with CPT-11 (or SN-38) [8, 9]. To test this hypothesis, an oncolytic HSV vector carrying yCD-UPRT and rCE gene (termed bMP6-CUCE) will be created. In the first part of the project, it will be tested if the bMP6-CUCE vector is more potent against pancreatic cancer cell lines in vitro than non-armed vector in the presence of gemcitabine, 5-FC and CPT-11. Synergism between gemcitabine and bMP6-CUCE vector in the presence of 5-FC and CPT-11 will be analyzed using the Chou-Talalay analysis [10]. To further analyze sequence-dependent synergism, the Chou-Talalay analysis will be also performed in groups in which the drugs are added sequentially. In the second part of the project, it will be tested if bMP6-CUCE in combination with the three drugs has a significantly enhanced efficacy in vivo compared to gemcitabine alone or bMP6-CUCE plus 5-FC and CPT-11 using the mouse pancreatic cancer xenograft model. These experiments will reveal the potential of 5-FC and CPT-11 prodrug enzyme therapy mediated by an oncolytic HSV vector to enhance the efficacy of gemcitabine against pancreatic cancer. PUBLIC HEALTH RELEVANCE: The aim of this proposal is to develop a new treatment for advanced pancreatic cancer by using a genetically-engineered herpes simplex virus vector carrying two prodrug converting enzyme genes. As this virus replicates selectively in tumor cells, it will deliver the genes of the enzymes specifically to tumor tissue causing potent anti-cancer agents to be produced locally from less toxic prodrugs. This therapy may potentially enhance the efficacy of existing pancreatic cancer chemotherapy using gemcitabine and ultimately prolong the survival of pancreatic cancer patients.