Hypoxia Inducible Factors (HIFs) are very important transcription factors that can respond to low oxygen concentrations in the cellular environment. Inhibition of HIF’s transcriptional activity represents a promising approach to new anticancer compounds. Herein, we describe the design and synthesis of a series of HIF-1 inhibitors. Evaluation of these inhibitors using a cell-based luciferase assay led to the discovery compounds with sub-micromolar potency.

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Replicating viruses for the treatment of cancer have a number of advantages over traditional therapeutic modalities. They are highly targeted, self-amplifying, and have the added potential to act as both gene-therapy delivery vehicles and oncolytic agents. ORFV, (Parapoxvirus ovis, or Orf virus) is the prototypic species of the Parapoxvirus genus, causing a benign disease in its natural ungulate host. ORFV possesses a number of unique properties that make it an ideal viral backbone for the development of a cancer therapeutic: it is safe in humans, has the ability to cause repeat infections even in the presence of antibody, and it induces a potent Th-1 dominated immune response. Here I show for the first time that live replicating ORFV induces an anti-tumour immune response in multiple syngeneic mouse models of cancer that is mediated largely by the potent activation of both cytokine-secreting, and tumouricidal natural killer (NK) cells. I have also highlighted the clinical potential of the virus by demonstration of human cancer cell oncolysis including efficacy in an A549 xenograft model of cancer. The mechanism of ORFV-mediated activation of NK cells has been explored, where I have demonstrated activation via direct ex vivo infection of NK cells. I have also highlighted ORFV-mediated activation of dendritic cells (DCs), both in vivo and by direct infection ex vivo. An in vivo DC depletion study demonstrated an indirect mechanism for ORFV NK cell activation, where in the absence of DCs, NK cell activation was diminished, as was the ability of ORFV to clear lung metastases. The ORFV innate immune stimulatory profile has been harnessed for therapeutic application in an experimental surgery model of cancer, where ORFV therapy at the time of surgery reduces the number of cancer metastases. These data highlight the clinical potential of a live, immune stimulating Parapoxvirus therapeutic.

This thesis includes two parts related to hydrogels as therapeutically useful constructs: a biomimetic hydrogel carrying stem cells for bone regeneration and an acid-sensitive hydrogel carrying drugs for cancer therapy. In tissue engineering, one of the biggest difficulties is the control of stem cell fate on scaffolds. A biodegradable and cell attachable cross-linker was synthesized by one-step Michael additional reaction, and was used to fabricate a novel hydrogel to control the stem cell fate. For anti-cancer therapy, releasing drug on tumor cells or organs while having low effects on health cells under physiological conditions is a critical requirement. Two nature polymers are modified to achieve loading anti-cancer drug while forming hydrogels which can selectively release the drug in tumor environment by acid-sensitive linkages.

Replicating viruses for the treatment of cancer have a number of advantages over traditional therapeutic modalities. They are highly targeted, self-amplifying, and have the added potential to act as both gene-therapy delivery vehicles and oncolytic agents. ORFV, (Parapoxvirus ovis, or Orf virus) is the prototypic species of the Parapoxvirus genus, causing a benign disease in its natural ungulate host. ORFV possesses a number of unique properties that make it an ideal viral backbone for the development of a cancer therapeutic: it is safe in humans, has the ability to cause repeat infections even in the presence of antibody, and it induces a potent Th-1 dominated immune response. Here I show for the first time that live replicating ORFV induces an anti-tumour immune response in multiple syngeneic mouse models of cancer that is mediated largely by the potent activation of both cytokine-secreting, and tumouricidal natural killer (NK) cells. I have also highlighted the clinical potential of the virus by demonstration of human cancer cell oncolysis including efficacy in an A549 xenograft model of cancer. The mechanism of ORFV-mediated activation of NK cells has been explored, where I have demonstrated activation via direct ex vivo infection of NK cells. I have also highlighted ORFV-mediated activation of dendritic cells (DCs), both in vivo and by direct infection ex vivo. An in vivo DC depletion study demonstrated an indirect mechanism for ORFV NK cell activation, where in the absence of DCs, NK cell activation was diminished, as was the ability of ORFV to clear lung metastases. The ORFV innate immune stimulatory profile has been harnessed for therapeutic application in an experimental surgery model of cancer, where ORFV therapy at the time of surgery reduces the number of cancer metastases. These data highlight the clinical potential of a live, immune stimulating Parapoxvirus therapeutic.

This thesis includes two parts related to hydrogels as therapeutically useful constructs: a biomimetic hydrogel carrying stem cells for bone regeneration and an acid-sensitive hydrogel carrying drugs for cancer therapy. In tissue engineering, one of the biggest difficulties is the control of stem cell fate on scaffolds. A biodegradable and cell attachable cross-linker was synthesized by one-step Michael additional reaction, and was used to fabricate a novel hydrogel to control the stem cell fate. For anti-cancer therapy, releasing drug on tumor cells or organs while having low effects on health cells under physiological conditions is a critical requirement. Two nature polymers are modified to achieve loading anti-cancer drug while forming hydrogels which can selectively release the drug in tumor environment by acid-sensitive linkages.

The phenomenon of multidrug resistance (MDR) in cancer cells is generally associated with P-glycoprotein (P-gp) expression and presents an obstacle to successful chemotherapy. Attempts to overcome P-gp-associated MDR using P-gp modulators, such as verapamil, have been hindered by their intrinsic in vivo toxicity. In 1991, however, Scala et al. demonstrated the alteration of P-gp function by interferon-alpha (IFN-α) in vitro at non-toxic in vivo concentrations, suggesting a basis for the use of IFN-α clinically in patients exhibiting P-gp-associated MDR. Drug resistance in B-CLL has been linked to the phenomenon of MDR, however, publications regarding this have been conflicting. The contrasting results prompted further investigation of the role of P-gp-associated anthracycline resistance and, using isolated β-lymphocytes from B-CLL patients, this investigation examined P-gp expression, function and IFN-α modulation in vitro. Optimum conditions for in vitro analysis of P-gp-associated anthracycline resistance were determined by examining the stability of the anthracycline, daunorubicin, in varying cell culture conditions. The resulting system balanced conditions affecting drug stability with those affecting cell survival. While other investigations have neglected the issue of drug stability, this study demonstrates that the instability of daunorubicin may be a critical variable determining the outcome of drug sensitivity studies. In RPMI + 2mM L-glutamine and 10% (v/v) FBS, loss of drug concentration is due to both adsorption and degradation and these experiments show that the presumed availability of drug may be over-estimated in in vitro studies. Furthermore, the degradation products might interfere with P-gp function and modulation. MDRl gene mRNA was detected in the B-cells of forty-three out of fifty B-CLL patients analysed, whereas P-gp expression, as measured by flow cytometry, resulted in only sixteen patients out of fifty-five being classed as positive (> 10% increase in staining as compared to the control). P-gp functionality and modulation studies on the B-cells of eleven patients confirmed the existence of an efflux mechanism with identical characteristics to P-gp using verapamil, the dye rhodamine 123 (rho123) and daunorubicin. Four patients were classed as functional low expressers (functional P-gp with low P-gp expression (7-10% increase in staining)), six were classed as functional high expressors (functional P-gp with high P-gp expression (20-57% increase in staining)) and one as a non-functional high expressor (non-functional P-gp with high P-gp expression (13.4% increase in staining)). Verapamil modulated rho123 efflux in all ten patients classed as P-gp functional expressors, and daunorubicin efflux in eight of these patients. However, IFN-α modulated rho123 and daunorubicin efflux in only two and one patients, respectively, even at concentrations higher than 500I.U./ml. In contrast to Scala et al. (1991), this finding suggests that at a well tolerated concentration IFN-α may not be suitable for use as a P-gp modulating agent in vivo in B-CLL, although conclusive evidence would require a larger study.

Gold nanoparticles exhibit an array of properties, both intrinsic (chemical) and extrinsic (photothermal), that can be exploited for their use in cancer therapeutics. Owing to their size and ease with which they can be functionalized with various ligands, gold nanoparticles represent a class of highly functional biomedically relevant nanostructures. Here, we explore the use of gold nanoparticles as intrinsic (chemical) antineoplastic agents, with their ability to cause DNA damage and cytokinesis arrest, to induce apoptosis in a metallic composition-dependent manner, as well as their ability to enhance sensitivity to chemotherapy by regulation of the cell cycle. The extrinsic (photothermal) properties of gold nanoparticles are also examined, in detail, through both theoretical and experimental assessment, for their use as photothermal contrast agents in vitro. Based on this assessment, the gold nanoparticles are tested in the plasmonic photothermal therapy of head and neck cancer in a mouse model.

Oncolytic Viruses (OVs) are often attenuated to increase their safety profile, however this can lead to reduced efficacy in heterogeneous malignancies and result in resistance to OV therapy. Our group utilizes small molecule enhancers of OV therapy termed viral sensitizers. These small molecules have been shown to enhance the replication and spread of oncolytic rhabdovirus VSVΔ51 in vitro and prolong survival in tumour-bearing mice. In this study, we evaluate the ef-fect of these viral sensitizers on the innate antiviral response in order to identify the mechanism of action responsible for their viral-sensitizing properties. Our previous data suggest that VSe1 and its structural analogues affect the type I IFN antiviral response and have the potential to af-fect cellular redox homeostasis. We hypothesized that VSe1 and its structural analogues potenti-ate VSV∆51 activity by inhibiting the type I IFN response via redox-mediated dysregulation. In this study, we demonstrate that the viral sensitizers inhibit the nuclear translocation and transcrip-tional activity of NFκB, which in turn dampens the expression of antiviral cytokines IFN-, TNFα and IL-6. We also provide evidence supporting the possibility that the NFκB inhibition may be a result of the formation of ROS intermediates by the viral sensitizers, which leads to re-duced nuclear translocation of NFκB subunits, thereby preventing NFκB-mediated cytokine production. Overall, this work contributes to the identification of the mechanism of action of our viral sensitizers and highlights the finding that oncolytic VSV infection can be enhanced through redox-mediated modulation of the innate antiviral response.

Replicating viruses for the treatment of cancer have a number of advantages over traditional therapeutic modalities. They are highly targeted, self-amplifying, and have the added potential to act as both gene-therapy delivery vehicles and oncolytic agents. ORFV, (Parapoxvirus ovis, or Orf virus) is the prototypic species of the Parapoxvirus genus, causing a benign disease in its natural ungulate host. ORFV possesses a number of unique properties that make it an ideal viral backbone for the development of a cancer therapeutic: it is safe in humans, has the ability to cause repeat infections even in the presence of antibody, and it induces a potent Th-1 dominated immune response. Here I show for the first time that live replicating ORFV induces an anti-tumour immune response in multiple syngeneic mouse models of cancer that is mediated largely by the potent activation of both cytokine-secreting, and tumouricidal natural killer (NK) cells. I have also highlighted the clinical potential of the virus by demonstration of human cancer cell oncolysis including efficacy in an A549 xenograft model of cancer. The mechanism of ORFV-mediated activation of NK cells has been explored, where I have demonstrated activation via direct ex vivo infection of NK cells. I have also highlighted ORFV-mediated activation of dendritic cells (DCs), both in vivo and by direct infection ex vivo. An in vivo DC depletion study demonstrated an indirect mechanism for ORFV NK cell activation, where in the absence of DCs, NK cell activation was diminished, as was the ability of ORFV to clear lung metastases. The ORFV innate immune stimulatory profile has been harnessed for therapeutic application in an experimental surgery model of cancer, where ORFV therapy at the time of surgery reduces the number of cancer metastases. These data highlight the clinical potential of a live, immune stimulating Parapoxvirus therapeutic.