Bioengineering and Characterization of an Oncolytic Vaccinia Virus for Tumor Localized Lentivirus Production

Abstract

Oncolytic viruses (OVs) are multi-mechanistic, tumor-selective therapeutics that lyse cancer cells while stimulating anti-tumor immunity. However, one limitation of OVs as monotherapies is their inability to infect and eliminate every tumor cell. Since infected tumor cells ultimately undergo lysis, arming OVs with transgenes that promote bystander effects by targeting uninfected cells within the tumor microenvironment, including other tumor cells or immune cells, can enhance therapeutic efficacy. One extensively engineered OV platform is vaccinia virus (VV), owing to its large transgene capacity and favorable safety profile. In parallel, retrovirus-derived virus-like-particles (VLPs) and lentiviral vectors have emerged as versatile therapeutic delivery systems. VLPs transiently deliver proteins, whereas lentiviral vectors mediate stable gene delivery with clinical applications including immune cell engineering, such as CAR-T cell generation. My thesis focuses on the development of retrovirus-derived therapeutic delivery platforms that could be integrated with oncolytic VV to enable localized transient or stable manipulation within tumors. I optimized and established a VLP platform for transient protein delivery, with an emphasis on genome editing of oncogenes. Next, I engineered VV to encode the components required for production of a self-inactivating lentivirus vector, creating a “virus within a virus”. I inserted three transgenes: the lentivirus packaging component (Gag-Pol), the vesicular stomatitis virus glycoprotein pseudotyping envelope (VSV-G) and a lentiviral vector genome containing a gene-of-interest. Infection of producer cells with engineered VV enabled the production of transduction-competent lentivirus particles, thereby establishing a proof-of-concept for this hybrid system. The findings from this thesis establish retrovirus-derived therapeutic delivery platforms, including CRISPR-Cas9 loaded VLPs and a VV-lentiviral hybrid system, providing a versatile strategy for next-generation viral cancer therapies, with potential for direct tumor-targeting or localized in vivo immune cell engineering, including the generation and reprogramming of therapeutic immune cells, such as CAR-T cells, within the tumor microenvironment.