PROTEIN ENGINEERING IMPROVEMENTS TO ENZYME/PRODRUG THERAPY USING YEAST CYTOSINE DEAMINASE
Warren, Tiana D
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Gene-directed enzyme prodrug therapy (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) can convert the prodrug to its toxic form and induce cell death. One challenge of GDEPT is the inefficient transduction of tumor cells with the gene encoding the prodrug-converting enzyme, which limits the efficacy of this therapy. Using the yeast cytosine deaminase (CD)/5-fluorocytosine (5-FC) GDEPT system, I employ two separate strategies to mediate the challenges associated with targeted gene delivery. In the first approach, I performed mutagenesis studies to engineer a yCD mutant that increases the sensitivity of E. coli and mammalian cells to 5-fluorocytosine. An improved yCD variant should convert more 5-FC to 5-FU thus increasing cancer cell toxicity. Furthermore, increased 5-FU concentrations could potentially promote the bystander effect, which occurs when nearby, non-transduced tumor cells take up the 5-FU released by transduced tumor cells. In the second strategy, I fused yCD to HIF-1alpha, a well-established cancer marker in hypoxic tumors, to promote the degradation of the enzyme only in healthy cells experiencing normoxia, but allow its accumulation in hypoxia. In normoxia, select yCD-HIF-1alpha fusions were degraded via HIF-1alpha’s degradation pathway and were therefore present in negligible quantities. However, in mock-hypoxic cancer cells, where HIF-1alpha is not degraded, the yCD-HIF-1alpha fusion proteins accumulated to high levels and, upon administration of 5-FC, converted 5-FC to 5-FU within the cancer cell to induce apoptosis; therefore, increasing the therapeutic difference between normoxic and hypoxic cancer cells. This thesis discusses the progress, challenges, and future directions of my work.