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 or yeast cytosine deaminase (CD) with the prodrug 5-fluorocytosine (5FC). While functional as suicide genes, a number of caveats restrict their full effectiveness. These include a poor Km or binding affinity for prodrugs and toxic side effects associated with the high prodrug doses necessary to elicit tumor response. We seek to identify the optimal suicide gene and prodrug combination for the safest and most effective cancer gene therapy.
The specific aims of this project are to optimize three separate suicide gene systems [cytosine deaminase, guanylate kinase/TK (pathway engineering) and CD/TK (converging pathway engineering)] using mutagenesis strategies and to test the efficacy of enzyme variants in tumor cell lines and animal models. This research endeavors to overcome the kinetic limitations found in current suicide gene therapy strategies and will address and compare: 1) increasing production of activated prodrugs and the impact on tumor cell killing; 2) enhancing the bystander effect as it relates to increased cytotoxin production; 3) reducing prodrug doses for therapeutic efficacy to offset toxic side effects and; 4) augmenting synergy of the dual suicide gene approach (converging pathway engineering). Not only will the results from this project impact the choice of gene(s) used for cancer treatment but they also have broad application elsewhere including for graft versus host disease, restenosis, AIDS, in noninvasive tumor imaging, cell lineage ablation studies, in negative selection systems and selection against non-homologous recombination for the generation of transgenic mice.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA085939-09
Application #
7414487
Study Section
Special Emphasis Panel (ZRG1-ONC-Q (01))
Program Officer
Arya, Suresh
Project Start
2000-05-11
Project End
2010-02-28
Budget Start
2008-03-01
Budget End
2009-02-28
Support Year
9
Fiscal Year
2008
Total Cost
$263,260
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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