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dc.contributor.advisorBlack, Margaret E.
dc.creatorJohnson, Adam J.
dc.date.accessioned2012-04-27T17:26:44Z
dc.date.available2012-04-27T17:26:44Z
dc.date.issued2011
dc.identifier.urihttp://hdl.handle.net/2376/3483
dc.descriptionThesis (Ph.D.), School of Molecular Biosciences, Washington State Universityen_US
dc.description.abstractThe 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 rate-limiting 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.en_US
dc.description.sponsorshipDepartment of Molecular Biosciences, Washington State Universityen_US
dc.language.isoEnglishen_US
dc.rightsIn copyright
dc.rightsPublicly accessible
dc.rightsopenAccess
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/
dc.rights.urihttp://www.ndltd.org/standards/metadata
dc.rights.urihttp://purl.org/eprint/accessRights/OpenAccess
dc.subjectMolecular biologyen_US
dc.subjectBiochemistryen_US
dc.subjectOncologyen_US
dc.subject5-fluorocytosineen_US
dc.subjectcytosine deaminaseen_US
dc.subjectdeoxycytidine kinaseen_US
dc.subjectdeoxyguanosine kinaseen_US
dc.subjectenzyme engineeringen_US
dc.subjectsuicide gene therapyen_US
dc.titleOptimizing the Enzyme/Prodrug Axis of Suicide Gene Therapy Using Protein Engineering Strategies
dc.typeElectronic Thesis or Dissertation


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