Exploring Novel Methods to Express Cytotoxic Proteins from Oncolytic Adenoviral Vectors

Abstract

Oncolytic viruses (OVs) are promising cancer therapeutics because they can selectively infect and kill tumor cells while also stimulating anti-tumor immune responses. However, the efficacy of many OVs in solid tumors is limited by poor intratumoral spread, often resulting in incomplete tumor destruction. Conditionally replicating adenoviruses (CRAds) are a well-characterized OV platform that can be engineered to selectively replicate in tumor cells and express therapeutic transgenes. One strategy to improve OV spread and local tumor cell killing is to arm vectors with fusogenic proteins, which promote cell-cell fusion and extend cytopathic effects beyond initially infected cells. Fusion-associated small transmembrane (FAST) proteins are small, non-structural fusogenic proteins encoded by fusogenic reoviruses and represent attractive candidates for incorporation into human adenovirus (HAdV)-based vectors. However, their fusogenic activity can also complicate vector production by inducing premature syncytium formation and producer cell toxicity. Therefore, identifying an optimal FAST protein and developing strategies to regulate transgene expression during vector production are important steps toward improving FAST-armed adenoviral platforms. In this thesis, I first compared the fusogenic capacity and cytotoxic effects of three FAST proteins, p10, p14, and p15, in vitro. FAST proteins were expressed in HEK293 cells by either plasmid transfection or HAdV infection, and their effects were assessed using fluorescence microscopy, image-based fusion quantification, and metabolic activity assays. Among the FAST proteins tested, an untagged version of the p14 FAST protein induced the highest level of syncytium formation and the strongest reduction in metabolic activity, whereas p10 showed limited fusogenic activity and p15 produced an intermediate phenotype. I next evaluated two regulatory strategies to suppress transgene expression from HAdV vectors. A miRNA-based system was tested using HAdV vectors expressing reporter genes targeted by a corresponding miRNA, while a LacI/LacO-based system was tested using LacI-expressing producer cells and HAdV vectors containing LacO sequences within the transgene expression cassette. These two systems successfully reduced transgene expression from HAdV; however, further optimization is required to minimize their effects on vector yield. Together, these studies identify untagged p14 FAST as the most fusogenic candidate among the FAST proteins tested and provide foundational data on two regulatory strategies that may be adapted to improve the production of cytotoxic transgene-armed HAdV vectors.