Gene therapy offers the prospect of selectively introducing genes into cancer cells, leaving them susceptible to specific antitumor drugs. Current protocols to elicit tumor reduction utilize Herpes Simplex Virus type 1(HSV) thymidine kinase (TK) with the prodrug ganciclovir (GCV) or the E. coli cytosine deaminase with the prodrug 5-fluorocytosine (5FC). While the wild-type TK is functional as a suicide gene, a number of caveats restrict its full effectiveness. These include a poor Km or binding affinity for GCV (approximately 47muM) and the toxicity associated with high doses of GCV. Another prodrug, acyclovir (ACV), predominantly used as an anti-herpetic drug, does not demonstrate the immunosuppressive attributes of GCV, even at very high doses. However, the very high Km that HSV TK displays towards ACV (approximately 320 muM), precludes its use as a prodrug in ablative gene therapy. Similarly, there are caveats with cytosine deaminase and 5FC that preclude its potential as a safe and effective suicide gene. We seek to identify the optimal suicide gene and prodrug combination for the safest and most effective cancer gene therapy. Towards this end we seek 1) to understand the structure-function relationship of nucleoside metabolizing enzymes important to suicide gene therapy and 2) to manipulate HSV-1 thymidine kinase and cytosine deaminase genes for superior performance in ablative gene therapy settings. The goal of this work is to 1) create novel genes by mutagenesis, 2) evaluate mutant genes for improved tumor sensitivity to various prodrugs, 3) construct mini-pathways and 4) create fusion proteins with other genes to enhance prodrug activation and tumor ablation. Not only will the results from the project impact the choice of gene(s) used for cancer treatment, but it also has wide-reaching applications including graft versus host disease, restenosis, AIDS, tumor imaging, cell lineage ablation studies, in negative selection systems and selection against non-homologous recombination for the generation of transgenic mice. Furthermore, understanding the molecular basis of nucleoside metabolizing enzyme function and interaction with current drugs will have far-reaching ramifications in the design, development and use of novel antiviral, antifungal and antibacterial drugs.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA085939-01
Application #
6092901
Study Section
Experimental Therapeutics Subcommittee 1 (ET)
Program Officer
Wolpert, Mary K
Project Start
2000-05-11
Project End
2005-04-30
Budget Start
2000-05-11
Budget End
2001-04-30
Support Year
1
Fiscal Year
2000
Total Cost
$214,176
Indirect Cost
Name
Washington State University
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
041485301
City
Pullman
State
WA
Country
United States
Zip Code
99164
Ardiani, Andressa; Johnson, Adam J; Ruan, Hongmei et al. (2012) Enzymes to die for: exploiting nucleotide metabolizing enzymes for cancer gene therapy. Curr Gene Ther 12:77-91
Johnson, A J; Ardiani, A; Sanchez-Bonilla, M et al. (2011) Comparative analysis of enzyme and pathway engineering strategies for 5FC-mediated suicide gene therapy applications. Cancer Gene Ther 18:533-42
Serve, Kinta M; Darnell, Jennifer L; Takemoto, Jody K et al. (2010) Validation of an isocratic HPLC method to detect 2-fluoro-beta-alanine for the analysis of dihydropyrimidine dehydrogenase activity. J Chromatogr B Analyt Technol Biomed Life Sci 878:1889-92
Ardiani, A; Sanchez-Bonilla, M; Black, M E (2010) Fusion enzymes containing HSV-1 thymidine kinase mutants and guanylate kinase enhance prodrug sensitivity in vitro and in vivo. Cancer Gene Ther 17:86-96
Fuchita, Michi; Ardiani, Andressa; Zhao, Lei et al. (2009) Bacterial cytosine deaminase mutants created by molecular engineering show improved 5-fluorocytosine-mediated cell killing in vitro and in vivo. Cancer Res 69:4791-9
Ardiani, Andressa; Goyke, Amanda; Black, Margaret E (2009) Mutations at serine 37 in mouse guanylate kinase confer resistance to 6-thioguanine. Protein Eng Des Sel 22:225-32
Stolworthy, Tiffany S; Korkegian, Aaron M; Willmon, Candice L et al. (2008) Yeast cytosine deaminase mutants with increased thermostability impart sensitivity to 5-fluorocytosine. J Mol Biol 377:854-69
Willmon, C L; Krabbenhoft, E; Black, M E (2006) A guanylate kinase/HSV-1 thymidine kinase fusion protein enhances prodrug-mediated cell killing. Gene Ther 13:1309-12
Korkegian, Aaron; Black, Margaret E; Baker, David et al. (2005) Computational thermostabilization of an enzyme. Science 308:857-60
Mahan, Sheri D; Ireton, Greg C; Knoeber, Catherine et al. (2004) Random mutagenesis and selection of Escherichia coli cytosine deaminase for cancer gene therapy. Protein Eng Des Sel 17:625-33

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