The unifying goal of cancer therapy is to selectively eradicate cancer cells while sparing normal tissue from damage. As many current anticancer therapies lack specificity and cause unwanted side effects there is an unmet medical need for the development of clinical strategies able to overcome these obstacles. Suicide gene therapy (SGT) is an anticancer strategy with promise to reach this goal. Nevertheless, limitations such as poor prodrug activation and suicide enzyme immunogenicity hinder its applicability in a clinical setting. The work herein describes efforts to overcome these limitations.
We evaluated whether the combination of enzyme and pathway engineering strategies would augment prodrug activation for the cytosine deaminase/5-fluorocytosine (CD/5FC) SGT system and demonstrated that the bacterial CD mutant bCD1525 showed a superior combination of tumor cytotoxicity and bystander activity in vitro and in vivo relative to all wild-type and mutant constructs examined. Results suggest that the use of pathway engineering in combination with our previously identified CD mutants negatively influences bystander activity in vivo and diminishes the therapeutic value of the CD/5FC system. Therefore, bCD1525 may be the most promising enzyme for CD/5FC SGT clinical applications.
To address immunogenicity and help diminish the likelihood of a patient eliciting an immune response during therapy, human enzymes were tested for use in SGT. In effort to enhance gemcitabine (dFdC) cytotoxicity, we created a bifunctional fusion enzyme that combines the enzymatic activities of human deoxycytidine kinase (dCK) and uridine/cytidine monophosphate kinase (UCMK). While kinetic analyses revealed the UCMK/dCK fusion possesses native activities, the fusion rendered cells sensitive to dFdC at the same level as dCK expression alone. Results suggest that UCMK activity is ratelimiting in dFdC-mediated cytotoxicity and may warrant the implementation of studies aimed at engineering UCMK variants with improved activity. In a separate study, we performed structure/function studies on human deoxyguanosine kinase (dGK) to identify the impact of specific active site residues on the substrate binding profile of dGK. This body of work will influence the design and add to the arsenal of future SGT applications and aid in the development of new clinical strategies able to enhance anticancer treatment selectivity.
|Advisor:||Black, Margaret E.|
|Commitee:||Davis, William B., Her, Chengtao, Magnuson, Nancy S., Reeves, Raymond|
|School:||Washington State University|
|School Location:||United States -- Washington|
|Source:||DAI-B 73/06, Dissertation Abstracts International|
|Subjects:||Molecular biology, Biochemistry, Oncology|
|Keywords:||Cytosine deaminase, Deoxycytidine kinase, Deoxyguanosine kinase, Enzyme engineering, Fluorocytosine, Suicide gene therapy|
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