Oncolytic virotherapy with the adenovirus mutant dl 1520 in combination with chemotherapy has shown clinical response. Approaches to cancer gene therapy involving delivery of enzymes to activate the prodrugs CB 1954 and 5-FC are currently being tested in clinical trials. We hypothesised that the combination of an adenoviral vector equivalent to dl1520 with activation ofCB 1954or 5-FC and the combination of CB 1954 activation with 5-FU may further improve the antitumour effects for colorectal cancer therapy. The initial in vitro data showed that the combination of dl 1520 with CB 1954 activation or 5-FU (metabolite of 5-FC activation) and the combination of CB 1954 activation with 5-FU led to an additive or synergistic cytotoxicity. Subsequent data showed that the incorporation of Ntr or CD-UPRT genes into replicating oncolytic adenoviruses (ROAds) resulted in enhanced Ntr expression or CD-UPRT activity and augmented cytotoxic effects in tissue culture, surpassing the levels and cytotoxic effects mediated by the corresponding replication-defective vectors. When tested in subcutaneous human colon cancer xenografis, Ntr expression mediated by the ROAd was apparently higher than the level mediated by replication-defective CTLI02. Importantly, the antitumoural efficacy of CB 1954 activation mediated by ROAd is significantly superior to that mediated by CTLI 02 (p = 0.01). The ROAds displayed viral replication and oncolysis in vitro and in vivo and these attributes can contribute to the increased gene expression level and enhanced efficacy. Overall, the data suggested that the use of ROAds improved Ntr or CD-UPRT expression and antitumoural efficacy in the presence of corresponding prodrugs and may have the potential to achieve clinical significance in the treatment for colorectal cancer.

Oncolytic virotherapy is emerging as a new treatment option for cancer patients. At present, there are relatively few oncolytic virus clinical trials that are underway or have been conducted, however one virus that shows promise in pre-clinical models is Vesicular Stomatitis Virus (VSV). VSV is a naturally occurring oncolytic rhabdovirus that has the ability to preferentially replicate in and kill malignant versus normal cells. VSV also has a low seroprevalence, minimal associated morbidity and mortality in humans, and simple non-integrating genome that can be genetically manipulated, making it an optimal oncolytic vector. Currently, many labs are using a variety of different strategies including inserting trans genes that can modulate the innate and adaptive immune response. VSV can also be retargeted by altering its surface glycoprotein (G) or be made replication incompetent by deleting the G protein. Currently, our lab has engineered a series of new recombinant VSVs, incorporating either the murine p53 (mp53), IPS-1, or TRIF transgene. mp53, IPS-1 and TRIF were incorporated into the normal VSV-XN2 genome and mp53 was also incorporated into the mutated VSV-ΔM vector generating VSV-mp53, VSV-IPS-1, VSV-TRIF and VSV-ΔM-mp53. Our data using these new viruses indicate that these viruses preferentially replicate in and kill transformed versus non-transformed cells and efficiently express the transgene. However, despite the ability for VSV-IPS-1 and VSV-TRIF to induce a robust type 1 IFN response, VSV-ΔM-mp53 was the only construct that had reduced toxicity and elicited an increased anti-tumor response against a syngeneic metastatic mammary tumor model. VSV- ΔM-mp53 treatment lead to a reduction in IL-6 and IP-10 production, an increase in tumor specific CD8+ T cells, and immunologic memory against the tumor. Collectively these studies highlight the necessity for additional VSV construct development and the generation of new clinically relevant treatment schema.

The use of intraperitoneal (i.p.) adenovirus virotherapy of ovarian cancer is currently limited by insufficient efficacy and high toxicity. Both factors are associated with adenovirus serotype 5 (Ad5) in this setting and may be serotype-specific. Low levels of uptake receptors (CAR and αV integrins) on ovarian tumour cells and widespread immunity against Ad5 among patients appear to restrict efficacy and intraperitoneal inflammatory responses against Ad5 were among the reasons for the termination of a phase II/III clinical trial in ovarian cancer. This thesis sought to overcome these obstacles by investigating the alternative adenovirus serotypes Ad3 and Ad11. For these viruses lower pre-existing antiviral immunity and utilisation of different uptake receptors have been reported. Furthermore, virus cloaking with novel polymers which could impart enhanced protection from neutralisation was examined. In vitro, wild-type Ad3, Ad5 and Ad11 displayed differential oncolytic activity in a panel of ovarian cancer cell lines which partly correlated to uptake receptor expression and virus internalisation. However, some cell lines displayed lysis resistance in a serotype-specific manner. While the inflammatory response six hours after i.p. administration of Ad11 in CD46-transgenic mice did not differ from Ad5, in long-term studies of repeated administration Ad5 induced significantly more severe pathologic effects in the form of adhesions and liver toxicity than Ad11 or mock-treatment. Oncolysis inhibition assays using malignant exudate samples demonstrated greater neutralisation of Ad3 and Ad5 in comparison to Ad11 at low concentrations of samples. Notably, 10-fold less Ad11 than Ad5 was required for oncolytic efficacy at a sample concentration of 10%. In an ex vivo model of ascites from ovarian cancer patients Ad5 modified with novel polymer formulations achieved at least 50% cell kill in six of eight samples, in contrast to two of eight samples for non-modified Ad5. These data suggest that virotherapy using Ad11 might be advantageous over Ad3 or Ad5. The lack of strong inflammation and the possibility to decrease treatment doses due to less neutralisation of Ad11 might result in considerably improved patient safety. Chemical modification of Ad with novel polymers presents an exciting advancement in overcoming treatment neutralisation in adenovirus virotherapy.

Les virus oncolytiques sont une nouvelle classe d’agents thérapeutiques pouvant être une alternative au traitement des cancers. Plusieurs virus oncolytiques sont actuellement développés en clinique, néanmoins de nombreuses améliorations sont à apporter afin de créer une nouvelle classe de virus plus efficaces et moins toxiques. Le premier objectif de cette thèse a été d’améliorer la spécificité tumorale du virus de la vaccine via le ciblage de l’antigène MUC1 présenté à la surface des cellules tumorales. Pour cela un virus recombinant présentant à sa surface un fragment d’anticorps (scFv) dirigé contre l’antigène tumoral MUC1 a été construit et produit. Les tests in vitro n’ont toutefois pas permis de mettre en évidence un ciblage spécifique du virus recombinant. Un deuxième aspect de cette thèse a été de tester le potentiel oncolytique de virus de la famille des Poxviridae. Durant ce travail de thèse, les capacités oncolytiques de douze poxvirus, appartenant à 8 genres différents, ont été étudiés. Leurs effets sur la prolifération de cellules cancéreuses humaines ont été évalués. Les virus caractérisés par un effet oncolytique élevé ont été, par la suite, modifiés et armés par ingénierie virale afin d’augmenter leur efficacité. La dernière partie de cette thèse a été consacrée à la génération et la sélection de virus chimériques basées sur la méthode d’évolution dirigée. Cette méthode est utilisée pour mimer le processus naturel de sélection évolutif. Appliqué à la virothérapie oncolytique, ce procédé nous a permis de générer un nouveau virus oncolytique chimérique caractérisé par un potentiel anti-cancéreux amélioré. En résumé, cette thèse a permis, par des techniques d’ingénierie virale, par un criblage de nouveaux virus et par la méthode d’évolution dirigée, de créer et de sélectionner une nouvelle génération de poxvirus oncolytiques présentant une activité thérapeutique accrue avec un profil de toxicité atténué et pouvant être utilisés dans diverses indications thérapeutiques. / Oncolytiques viruses are a new class of therpeutic agents which could be an alternative for cancer treatment. Currently, several oncolytic viruses are evaluated in clinical trial, nevertheless improvements are needed to create a new class of more efficiente and less toxic viruses. The first objective of this thesis was to improved the vaccinia virus specificity through the targeting of the tumor-associated antigen MUC1. To address this goal, a recombinant virus expressing an scFv targeting the MUC1-protein was engineered and produced. However, in vitro, the demonstration of a specific targeting by the recombinant virus was not possible. A second aspect of this thesis work was to evaluate the oncolytic potential of Poxviridae family viruses. Oncolytic capacities of twelve viruses, belonging to eight genera, were evaluated. Their impact on human cancer cells was tested. In order to increase their efficacity, viruses with the highest oncolytic capacities were then modified and armed by genetic engineering. The third part of this work was devoted to the generation of chimeric viruses based on directed evolution process. This methodology is used to mimic the natural process of evolutionary selection. Applied to oncolytic virotherapy, this technique allowed the generation of a new chimeric oncolytic virus caracterised by an enhanced antitumoral potential. In summary, this thesis has allowed, through viral engineering, poxviruses screening and directed evolution methodology, the creation and selection of a new generation of oncolytic poviruses. These viruses demonstrate an increased therpeutic activity and greatest safety profil enabling their application in several therapeutic indication.

abstract: Since the molecular biology revolution in the 1980s, ease of gene editing had led to the resurgence of Oncolytic Virotherapy. Countless viruses have been engineered yet only three are approved for clinical use worldwide, with only one being approved by the U.S Food and Drug Administration (FDA). Vaccinia virus (VACV) has a large genome, contains many immune evasion genes and has been thoroughly studied, making it a popular candidate for an oncolytic platform. VACV mutants with deletions in the E3 immune evasion protein have been shown to have oncolytic efficacy but the mechanism of tumor selectivity has not been fully elucidated. These mutants have been shown to be regulated by the necroptosis pathway, a pathway that has been shown to be deficient in certain cancers. Using a pan-cancer screening method that combines dye exclusion assays, western blot analysis, and viral growth curve, the role of necroptosis in regulating VACV replication and oncolytic efficacy in cancer was further characterized. Results demonstrate a preliminary correlation between necroptosis, viral replication, and oncolytic efficacy. This correlation is clearest in breast cancer and melanomas yet may apply to other cancer subgroups. This data was also used to guide the development of a receptor-interacting protein kinase 3 (RIP3) matched pair mouse model in the E0771 mouse breast cancer line which can be used to further study the role of necroptosis and oncolytic efficacy in vivo. Understanding the contribution necroptosis plays in oncolytic efficacy can guide to design enhance the design of clinical trials to test VACV E3L mutants and may lead to better efficacy in humans and an improvement in clinical oncology. / Dissertation/Thesis / Masters Thesis Biology 2020

No description available.

The poor stability of most therapeutic viruses in the human bloodstream is a major obstacle in the field of cancer virotherapy, preventing systemic intravenous delivery to treat tumour metastases. Delivery is typically limited by inactivation of virus particles by blood components and rapid scavenging by hepatic phagocytes. Members of the Alphavirus family are exposed to blood during natural infections; as such, we hypothesised that evolutionary pressure may have led to blood stability and clearance kinetics superior to those of other viruses currently in development for use as oncolytic agents. Sindbis virus is a member of the Alphavirus family that has shown promising anti-cancer activity in pre-clinical models. A concern for the clinical use of Sindbis virus as an anticancer agent is its pathology in humans, known as Pogosta disease. The symptoms of Pogosta disease may be a result of Sindbis virus replication in neuronal, muscle or haematopoietic tissues. Inhibiting virus replication in these tissues could, therefore, alleviate such potential side effects of virotherapy treatment. Introduction of microRNA response elements, perfectly complementary to microRNAs specifically expressed in liver (miR122), neuronal (miR124), muscle (miR133 and miR206) and haematopoietic (miR142-3p) cells, successfully attenuated SV replication in these tissues. In contrast to all other viruses studied, data presented in this thesis show that Sindbis virus infectivity in vitro is not significantly inhibited by incubation with neat, whole naïve human blood. Despite full infectivity in naïve mouse blood, virus particles were rapidly cleared from the circulation of mice in vivo by the liver. An attempt to decrease the clearance rate by depletion of Kupffer cells through pre-treatment of mice with clodronate liposomes was ineffective. We also explored the use of Sindbis virus packaged in mosquito cells to more closely mimic virus particles exposed to blood in the wild during mosquito mediated transmission, but this also failed to improve virus circulation kinetics in mice. Despite rapid clearance from the circulation, intravenous administration of Sindbis virus had significant anti-cancer efficacy in C57BL/6 mice bearing syngeneic B16F10 metastatic melanomas. Overall, data presented support our proposed use of Sindbis virus as a systemically delivered oncolytic agent and suggest decreasing the rate of clearance by the liver could dramatically enhance therapeutic outcomes. In addition it is shown that microRNA targeting of Sindbis virus provides a means of alleviating potential side effects of the administration of large virus doses.

Prostate cancer (CaP) is the second leading cause of cancer related deaths in men in the United States. Currently, androgen depletion is an essential strategy for CaP combined with surgery, chemotherapy and radiation. Hormone independent cancer stem cells escaping conventional therapy present a major therapeutic challenge. The available treatment regimens for hormone resistant CaP are only palliative and marginally increase survival. Therefore, novel strategies to eradicate CaP including stem cells are imperative. Oncolytic virus (OV) therapy is a novel approach that overcomes the limitations posed by radiation and chemotherapy. Oncolytic virotherapy of cancer is based on the use of replication competent, tumor selective viruses with limited toxicity. Newcastle Disease Virus (NDV), an avian paramyxovirus, is a safe and promising OV successfully used in many clinical trials. NDV is inherently tumor selective and cytotoxic but replication restricted in normal cells. But, systemically delivered NDV fails to reach solid tumors in therapeutic concentrations and also spreads poorly within the tumors due to barriers including complement, innate immunity and extracellular matrix. Overcoming these hurdles is paramount to realize the exceptional oncolytic efficacy of NDV. Therefore, we engineered the fusion (F) glycoprotein of NDV and generated a recombinant NDV (rNDV) cleavable exclusively by prostate specific antigen (PSA). The rNDV replicated efficiently and specifically only in prostate cancer (CaP) cells but failed to replicate in the absence of PSA. Further, PSA-cleavable rNDV caused specific lysis of androgen independent and dependent/responsive CaP cells with a mean effective concentration (EC50) ranging from 0.01 to 0.1 multiplicity of infection (MOI). PSA retargeted rNDV efficiently lysed three-dimensional prostaspheres, suggesting efficacy in vivo. Also, PSA-cleavable NDV failed to replicate in chicken embryos, indicating absence of pathogenicity to its natural host, chickens. Prostaspheres generated from DU-145 CaP cell line derived xenografts showed self-renewal, proliferative and clonogenic potential in vitro, and exhibited increased tumorigenicity in vivo. Embryonic stem and progenitor cell markers like Nanog, Nestin and CD44 were overexpressed in spheres as compared to the cell line suggesting prostaspheres comprise tumor-initiating cells from CaP. Xenograft and cell line derived prostaspheres were permissive for rNDV replication, when the fusion protein was activated by exogenous PSA. The EC50 against tumor initiating cells was 0.11-0.14 MOI, suggesting an excellent therapeutic margin for in vivo studies. PSA retargeting is likely to enhance the therapeutic index of rNDV owing to tumor restricted replication and enhanced fusogenicity. Our results suggest PSA retargeted rNDV selectively replicates and lyse PSA producing CaP cells including tumor-initiating cells and is a promising candidate for immediate Phase I/II clinical trials. / Ph. D.

This thesis is concerned with mathematical modelling of oncolytic virotherapy: the use of genetically modified viruses to selectively spread, replicate and destroy cancerous cells in solid tumours. Traditional spatially-dependent modelling approaches have previously assumed that virus spread is due to viral diffusion in solid tumours, and also neglect the time delay introduced by the lytic cycle for viral replication within host cells. A deterministic, age-structured reaction-diffusion model is developed for the spatially-dependent interactions of uninfected cells, infected cells and virus particles, with the spread of virus particles facilitated by infected cell motility and delay. Evidence of travelling wave behaviour is shown, and an asymptotic approximation for the wave speed is derived as a function of key parameters. Next, the same physical assumptions as in the continuum model are used to develop an equivalent discrete, probabilistic model for that is valid in the limit of low particle concentrations. This mesoscopic, compartment-based model is then validated against known test cases, and it is shown that the localised nature of infected cell bursts leads to inconsistencies between the discrete and continuum models. The qualitative behaviour of this stochastic model is then analysed for a range of key experimentally-controllable parameters. Two-dimensional simulations of in vivo and in vitro therapies are then analysed to determine the effects of virus burst size, length of lytic cycle, infected cell motility, and initial viral distribution on the wave speed, consistency of results and overall success of therapy. Finally, the experimental difficulty of measuring the effective motility of cells is addressed by considering effective medium approximations of diffusion through heterogeneous tumours. Considering an idealised tumour consisting of periodic obstacles in free space, a two-scale homogenisation technique is used to show the effects of obstacle shape on the effective diffusivity. A novel method for calculating the effective continuum behaviour of random walks on lattices is then developed for the limiting case where microscopic interactions are discrete.