Endogenous expression of Killer Peptides as a novel strategy for targeted Cancer Cell Death

Cancer remains the second leading cause of death worldwide after cardiovascular diseases. Traditional therapies such as surgical interventions, radiotherapy, and chemotherapy are often limited in their effectiveness against cancer as they affect both cancerous and healthy cells. Given the complex mutational landscape of cancer, there is an increasing need for personalized therapies that selectively target cancer cells while preserving healthy tissue. This thesis aims to develop strategies to trigger selective cell death in mutated cells using suicide genetic elements through genome editing approaches. Initially, I attempted to exploit the microhomology-mediated end joining (MMEJ) pathway to insert a thymidine kinase suicide gene into mutated cells, using the Cas12a endonuclease. Testing this system in engineered HEK293T cells revealed low efficiency in thymidine kinase integration. For this reason, the system needs more improvements. Consequently, I sought to identify alternative strategies to trigger cell death. I investigated pro-apoptotic peptides, which are typically administered exogenously fused with translocator domains. My study examined the feasibility of inducing cell death through endogenous expression of these peptides. The killer peptides investigated included Killer B, Killer E, ABH-3, and KLAK – characterized by α-helical structures and lengths ranging from 8 to 18 amino acids – as well as other peptides with different characteristics. To assess their effects on cancer cells, I engineered expression constructs by fusing their coding sequences with a green fluorescent protein (eGFP) via a T2A self-cleaving peptide sequence. Transient transfection of these constructs into HEK293T and A375, and transduction into Jurkat cell lines demonstrated a significant increase in cell death. Our findings suggest potential applications for these expression cassettes as tools in gene therapy approaches against cancer.