(228et) A Comprehensive Mutagenesis Study to Improve Yeast Cytosine Deaminase for Enzyme/Prodrug Therapy

Yeast cytosine deaminase (yCD) is an enzyme commonly used in gene directed enzyme/prodrug therapy (GDEPT) for cancer. GDEPT is a two-step process whereby the gene encoding a prodrug-converting enzyme is selectively delivered to tumor cells, followed by systemic administration of a nontoxic prodrug. Consequently, only cells that express the prodrug-converting enzyme (i.e. tumor cells) will be capable of converting the prodrug to its toxic form and induce cell death. One challenge of GDEPT is the inefficient transduction of tumor cells to carry the gene encoding the prodrug-converting enzyme, which limits the efficacy of this therapy. This work aims to address this issue by improving yCD activity against its prodrug, 5-fluorocytosine (5-FC), thus increasing cytotoxin production. While several studies have examined the effects of mutations at specific residues in yCD, a yCD variant with significantly improved activity against 5-FC has yet to be discovered. We performed a comprehensive mutagenesis study on yCD to improve its activity against 5-FC. Using inverse PCR, we created a comprehensive site-saturation mutagenesis library comprised of approximately 3000 yCD mutants. These mutants were subjected to selection for retained catalytic activity and subsequently screened for increased toxicity of 5-FC to E. coli. Mutants (and combinations thereof) that conferred increased 5-FC toxicity in our assays were further characterized to determine the mechanism for the increased sensitivity.