Comparative Analysis of Enzyme and Pathway Engineering Strategies for 5FC-Mediated Suicide Gene Therapy Applications

摘要

Bacterial and yeast encoded cytosine deaminases (bCD and yCD, respectively) are widely investigated suicide enzymes used in combination with the prodrug 5-fluorocytosine (5FC) to achieve localized cytotoxicity. Yet characteristics such as poor turnover rates of 5FC (bCD) and enzyme thermolability (yCD) preclude their full therapeutic potential. We previously applied regio-specific random mutagenesis and computational design to create novel bCD and yCD variants with altered substrate preference (bCD1525) or increased thermostability (yCDdouble, yCDtriple) to aid in overcoming these limitations. Others have utilized pathway engineering in which the microbial enzyme uracil phosphoribosyltransferase (UPRT) is fused with its respective CD, creating bCD/bUPRT or yCD/yUPRT. In this study, we evaluated whether the overlay of CD mutants onto their respective CD/UPRT fusion construct would further enhance 5FC activation, cancer cell prodrug sensitivity and bystander activity in vitro and in vivo. We show that all mutant fusion enzymes allowed for significant reductions in IC50 values relative to their mutant CD counterparts. However, in vivo the CD mutants displayed enhanced tumor growth inhibition capacity relative to the mutant fusions, with bCD1525 displaying the greatest tumor growth inhibition and bystander activity. In summary, mutant bCD1525 appears to be the most effective of all bacterial or yeast CD or CD/UPRT enzymes examined and as such is likely to be the best choice to significantly improve the clinical outcome of CD/5FC suicide gene therapy applications.