) Although we have demonstrated that the strategy for gene therapy using E. coli PNP is feasible in vivo, we need to evaluate this strategy further in order to determine if it is suitable for clinical trials. This will be accomplished by (1) using Lentivirus cell lines that express high levels of E. coli PNP activity to define the limits of the bystander effect in vivo, (2) using germ-free mice to improve the activity of the prodrug/E. coli PNP combination, (3) evaluating state-of-the-art vectors for the delivery of E. coli PNP to tumors and subsequently their use with our three leading prodrugs (6-MeP-dR, 2-F-dAdo, and 2-F-araAMP), and (4) evaluating other prodrugs or enzyme/prodrug combinations. Lentivirus transduced cell lines that express high levels of E. coli PNP activity will be generated by Laboratory Program 1 (LP1) using several human tumor cell lines (e.g., PC-3 prostate, D54MG glioma, and SR 475 head and neck). We will first characterize the in vivo growth of these cell lines in nude mice. If suitable growth is observed for a particular cell line, then antitumor evaluations will be conducted using one of the three leading prodrugs and varying proportions of a parental tumor cell line and the corresponding Lentivirus transduced line (e.g., 10%, 1%, 0.1%, and 0.01% transduced line). LP1 will evaluate a method to reduce the normal gut flora in mice and thereby increase the MTD of the three leading prodrugs. If successful, then this core will conduct antitumor evaluations using higher dosages of the three leading prodrugs and an appropriate tumor model to determine if the absence of the gut flora will permit greater antitumor activity. Three state-of-the-art vectors (a non-replicating adenovirus, a replicating adenovirus, and a modified Salmonella), which will be furnished by LP1, will be evaluated in this core for the delivery of E. coli PNP to tumors. Varying amounts of these vectors containing E. coli PNP will be inoculated into sc implanted tumors and the tumors will be evaluated for PNP activity at various time points. Once the amount of vector and the time for optimal PNP activity are determined, then antitumor evaluations will conducted using one or more of our three leading prodrugs. If a new prodrug for E. coli PNP is deemed appropriate for evaluation, then the basic studies conducted for the three leading prodrugs will be performed (e.g., MTD determination, pharmacokinetics, antitumor evaluations, etc.). Similarly, if a new enzyme/prodrug combination (e.g., PNP M64V mutant enzyme and 5'-methyltallo-6-MeP-riboside) is deemed appropriate for evaluation, then the basic studies conducted for the 6-MeP-dR/E. coli PNP model will be performed.
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