<p>Despite improvements in detection and treatment, cancer is the leading cause of death worldwide. Current treatment modalities have not been able to improve the mortality rates and significant toxicities limit efficacy. Therefore there is a need for development of novel therapeutics.</p> <p>Oncolytic viruses have the ability to efficiently replicate in and destroy tumours while leaving normal tissues unharmed. These treatment platforms have been gaining momentum in recent years due to pre-clinical and clinical successes. Oncolytic viruses are extremely safe with limited toxicity observed in phase I/II clinical trials, and objective responses have been observed in some patients treated with oncolytic viruses. However, there is still room to improve on these therapeutic platforms.</p> <p>Recently, the importance of the induction of anti-tumour immunity during oncolytic virotherapy has been realized and harnessing this immune response can be used to improve current oncolytic virus platforms. To this end we have conducted numerous studies assessing our ability to improve oncolytic VSV through the addition of transgenes to enhance the immunostimulatory properties of oncolytic VSV treatment. These studies showed that only the addition of a highly secreted form of human IL-15 was able to improve VSV therapy through enhanced anti-tumour immunity. However, expressing cell-autonomous transgenes from oncolytic VSV was unable to modify the therapeutic efficacy of VSV due to limited replication, both temporally and geographically within the tumour, and the indirect vascular shutdown induced by VSV infection of tumours. We believe that the drastic vascular shutdown observed following VSV therapy is an important component to the success of VSV and we have investigated which steps in this process are critical for induction of anti-tumour immunity.</p> <p>The research presented in this thesis further enforces the requirement for induction of anti-tumour immune responses in the success of OV therapy. Our findings also indicate that manipulating the tumour as a whole, rather than the virus, will lead to improved oncolytic therapeutics.</p> / Doctor of Philosophy (PhD)

Cell-free protein synthesis is an emerging technology that has many applications. The open nature of this system makes it a compelling technology that can be manipulated to answer many needs that are unavailable in other systems. This dissertation reports on engineering this technology for: 1) sense codon emancipation for incorporation of multiple unnatural amino acids; 2) expressing a hard-to-express anticancer biotherapeutic and introducing a just-add-water system; 3) a biosensing ligand that interacts with nuclear hormone receptors. Emancipating sense codons toward a minimized genetic code is of significant interest to science and engineering. A promising approach to sense codon emancipation is the targeted in vitro removal of native tRNA. Here we introduce a new in-vitro or "cell-free" approach to emancipate sense codons via efficient and affordable degradation of endogenous tRNA using RNase-coated superparamagnetic beads. The presented method removes greater than 99% of tRNA in cell lysates, while preserving cell-free protein synthesis activity. The resulting tRNA-depleted lysate is compatible with in vitro-transcribed synthetic tRNA for the production of peptides and proteins. Biotherapeutics have many promising applications, such as anti-cancer treatments, immune suppression, and vaccines. However, due to their biological nature, some biotherapeutics can be challenging to rapidly express and screen for activity through traditional recombinant methods. In this work, we demonstrate the use of cell-free systems for the expression and direct screening of the difficult-to-express cytotoxic protein onconase. Using cell-free systems, onconase can be rapidly expressed in soluble, active form. Furthermore, the open nature of the reaction environment allows for direct and immediate downstream characterization without the need of purification. Also, we report the ability of a "just-add-water" lyophilized cell-fee system to produce onconase. Here we introduce a Rapid Adaptable Portable In-vitro Detection biosensor platform (RAPID) for detecting ligands that interact with nuclear hormone receptors (NHRs). The biosensor is based on an engineered, allosterically-activated fusion protein, which contains the ligand binding domain from a target NHR. The presented RAPID biosensor platform is significantly faster and less labor intensive than commonly available technologies, making it a promising tool for detecting environmental EDC contamination and screening potential NHR-targeted pharmaceuticals.