Browsing by Author "Balaz, Melanie"

Gene editing holds tremendous potential for treating a variety of diseases, but concerns about safety, particularly the risk of edited cells becoming cancerous, must be addressed. This thesis explores a safety mechanism to prevent unwanted cell proliferation and tumor formation in induced pluripotent stem cells that have been edited for use in gene therapy. The mechanism bases on the genetic disruption (knockout) of the thymidylate synthase gene (TYMS), the only enzyme in charge of synthesizing deoxythymidine monophosphate (dTMP), an essential building block of DNA. Without dTMP, cells cannot successfully proliferate, while RNA synthesis remains unaffected. Through RNA sequencing analysis, we investigate the early response of TYMS knockout cells to dTMP withdrawal and find evidence of the activation of apoptosis and stress pathways, as well as differentiation and changes in the cell cycle. In addition, we demonstrate the effectiveness of the TYMS knockout mechanism in preventing proliferation of cancerous cells in a laboratory setting.