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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.

Le cancer ovarien est la 1ère cause de mortalité parmi les femmes atteintes de cancers gynécologiques avec une survie à 5 ans de 45% tous stades confondus et de seulement 17% pour les stades avancés (plus de 50% des patientes) qui présentent un risque de plus de 80% de rechute, d’où l’importance de trouver de nouvelles options thérapeutiques. On sait que les cancers ovariens épithéliaux (90% des cas) présentent une forte expression de la petite guanosine triphosphatase (GTPase) Ran (Ras-related nuclear protein) associée à un mauvais pronostic qui s'améliore lorsqu'on cherche à diminuer son expression, or aucune thérapie ciblant Ran n’existe actuellement. Une étude en 1997 menée par Her et al. déclare que la protéine de matrice du virus de la stomatite vésiculaire (VSV) bloque Ran. De façon intéressante, il existe une souche oncolytique de VSV, VSVΔ51, utilisée en études cliniques. Les virus oncolytiques sont de puissants outils anticancer qui infectent et tuent spécifiquement les cellules tumorales et induisent une immunité antitumorale sans endommager les tissus sains. Basés sur ces données de littérature, nous avons voulu évaluer le potentiel de VSVΔ51 dans le cadre du cancer ovarien via sa capacité à bloquer Ran. Dans un premier article, nous avons étudié l’interaction entre Ran et VSVΔ51. Tandis que nous n’observons pas d’impact de VSVΔ51 à l’encontre de RanGTP, nous remarquons que Ran améliore l’infection par VSV. Cette fonction jusqu’alors inconnue offre de nouvelles possibilités concernant la virothérapie par VSV et d’autres virus oncolytiques, qui seraient d’autant plus efficaces chez les patientes présentant des cancers ovariens de haut grade connus pour surexprimer Ran. Étant donné que bloquer Ran ne semble pas la meilleure stratégie pour ces patientes, nous nous sommes focalisés, dans un second manuscrit, sur l’optimisation d’une nouvelle virothérapie oncolytique efficace dans le cancer ovarien. Nous avons utilisé la biobanque du CRCHUM nous donnant accès à 32 lignées cellulaires de cancer ovarien issues de patientes. De façon intéressante, près de 2/3 des lignées sont sensibles à la virothérapie par VSVΔ51. Afin d’améliorer la thérapie pour le tiers des modèles résistants à VSV, des combinaisons avec différentes drogues ont été testées. Bien qu’aucune des thérapies actuelles en cancer ovarien n’ait aidé la virothérapie, aucune de ces combinaisons n’a induit une diminution de l’infection virale. De plus, nous avons montré que les lignées résistantes à VSV sécrètent et répondent à l’interféron, et deviennent sensibles lorsque nous bloquons cette voie à l’aide d’inhibiteur de Janus kinase (JAK). Cette étude offre de nouvelles perspectives pour les patientes atteintes de cancers ovariens qu’elles soient sensibles ou résistantes à la virothérapie par VSV seule, afin de prolonger leur survie ainsi que leur qualité de vie. Ce projet de recherche montre que RanGTP, en plus d’être un marqueur pronostique, pourrait également servir de critère de sélection thérapeutique pour la virothérapie de patientes atteintes de cancer ovarien. De plus, on sait désormais que ces patientes pourraient bénéficier de la virothérapie par VSV en combinaison avec des inhibiteurs de JAK afin d’augmenter leurs chances de survie. / Ovarian cancer is the deadliest gynecologic cancer with a 5-year survival rate of only 45%. At diagnosis, more than 50% of patients present a high-grade cancer associated with a 5-year survival rate of only 17% as well as a probability of relapse of 80% hence the importance of finding new therapeutic options for these patients. Epithelial ovarian cancers (90% of the cases) present a high expression of the small guanosine triphosphatase (GTPase) Ran (Ras-related nuclear protein) which is associated with a poor prognosis. Inhibition of Ran leads to a better survival, however there is still no therapy targeting Ran. The study of Her et al. in 1997, claimed that the vesicular stomatitis virus (VSV)’s matrix protein inhibited Ran. Interestingly, VSVΔ51, an oncolytic variant of VSV, is largely studied in clinical studies. Oncolytic viruses, such as VSVΔ51, represent a promising immunotherapeutic option leading to infection and killing of tumour cells as well as the induction of an antitumour immune response, leaving healthy cells undamaged. Based on the literature, we sought to evaluate VSVΔ51’s therapeutic potential in ovarian cancer via its capacity to block Ran. In a first scientific paper, we studied the interplay between Ran and our model virus. Even though we did not find Ran to be inhibited by VSV, we discovered a previously unknown pro-viral function of Ran. This offers new possibilities for virotherapy using VSV, and other viruses, which are thought to be even more efficient in high grade ovarian cancers that are known to highly express Ran. Although the results of our first study show that VSV is a promising treatment for ovarian cancers, our initial hypothesis has not been confirmed and using VSV to block Ran is not a good strategy for patients with ovarian cancer. In a second paper, we focused on the optimization of a new effective oncolytic virotherapy for these patients. We took advantage of the CRCHUM’s ovarian cancer biobank giving us access to 32 cell lines derived from patients. Interestingly, nearly 2/3 of the cell lines are sensitive to VSVΔ51 virotherapy. In order to improve therapy for the third of patients not responding optimally to VSV alone, combinations with drugs have been tested. Although none of the combinations with current therapies in ovarian cancer improved VSV virotherapy, there was no decrease in viral infection. Moreover, we have shown that lines resistant to virotherapy secrete and respond to interferon and become sensitive upon interferon blockade using JAK inhibitors. This study offers new treatment possibilities for patients with ovarian cancers, whether they are sensitive or resistant to VSV virotherapy alone, in order to improve their survival and quality of life. This research project shows that RanGTP, in addition to being a prognostic marker, can also be used as a therapeutic selection criterion for virotherapy of patients with ovarian cancer. In addition, we now know that patients with ovarian cancer could benefit from VSV virotherapy in combination with JAK inhibitors as a new therapeutic option to increase their chances of survival.

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.

Neuroendocrine tumors (NETs), originally described as carcinoids, represent a rare and heterogeneous group of neoplasms associated with intensive secretion of hormones, bioactive peptides and amines. Most of the patients are diagnosed at a late stage of disease, often with liver metastases. Surgery remains the main treatment to control metastatic disease, but is not curative. Oncolytic virotherapy represents a promising approach to treat cancer and different strategies have been exploited to restrict viral replication to tumor cells. We developed an oncolytic adenovirus based on serotype 5, Ad5[CgA-E1A], where the chromogranin A (CgA) promoter controls expression of the E1A gene and thereby virus replication. We found that Ad5[CgA-E1A], selectively replicates in NET cells and it is able to suppress fast-growing human BON carcinoid tumors in nude mice. The activity of Ad5[CgA-E1A] was not completely blocked in liver cells. We further repressed virus replication in hepatocytes by targeting E1A with miR122, an miRNA specifically expressed in the liver. miRNAs bind to mRNA and induce its cleavage or translational blockage. By insertion of tandem repeats of miR122 target sequences in 3’UTR of E1A gene, we observed reduced E1A protein expression and replication arrest in miR122 expressing liver cells. The oncolytic potency of the miR122-targeted virus was not affected in NET cells. Since some NET and neuroblastoma cells express high levels of somatostatin receptors (SSTRs), we introduced in the virus fiber knob cyclic peptides, which contain four amino acids (FWKT) and mimic the binding site of somatostatin for SSTRs. The FWKT-modified Ad5 transduces midgut carcinoid cells from liver metastases about 3-4 times better than non-modified Ad5. Moreover, FWKT-modified Ad5 overcomes neutralization in an ex vivo human blood loop model to a greater extent than Ad5, indicating that the fiber knob modification may prolong the systemic circulation time. NETs represent a huge therapeutic challenge and novel diagnostic markers are needed for early detection and effective treatment of NETs. We have profiled primary tumors and liver metastases of ileocaceal NETs, using Affymetrix microarrays and advanced bioinformatics. We have identified six novel marker genes and show high similarity between primary lesions and liver metastases transcriptome by hierarchical clustering analysis.

No description available.

Poor drug penetration through tumor tissue has emerged as a fundamental obstacle to cancer therapy. The solid tumor microenvironment presents several physiological abnormalities which reduce the uptake of intravenously administered therapeutics, including leaky, irregularly spaced blood vessels, and a pressure gradient which resists transport of therapeutics from the bloodstream into the tumor. Because of these factors, a systemically administered anti-cancer agent is unlikely to reach 100% of cancer cells at therapeutic dosages, which is the efficacy required for curative treatment. The goal of this project is to use high-intensity focused ultrasound (HIFU) to enhance drug delivery via phenomena associated with acoustic cavitation. ‘Cavitation’ is the formation, oscillation, and collapse of bubbles in a sound field, and can be broadly divided into two types: ‘inertial’ and ‘stable’. Inertial cavitation involves violent bubble collapse and is associated with phenomena such as heating, fluid jetting, and broadband noise emission. Stable cavitation occurs at lower pressure amplitudes, and can generate liquid microstreaming in the bubble vicinity. It is the combination of fluid jetting and microstreaming which it is attempted to explore, control, and apply to the drug delivery problem in solid tumors. First, the potential for cavitation to enhance the convective transport of a model therapeutic into obstructed vasculature in a cell-free in vitro tumor model is evaluated. Transport is quantified using post-treatment image analysis of the distribution of a dye-labeled macromolecule, while cavitation activity is quantified by analyzing passively recorded acoustic emissions. The introduction of exogenous cavitation nuclei into the acoustic field is found to dramatically enhance both cavitation activity and convective transport. The strong correlation between inertial cavitation activity and drug delivery in this study suggested both a mechanism of action and the clinical potential for non-invasive treatment monitoring. Next, a flexible and efficient method to simulate numerically the microstreaming fields instigated by cavitating microbubbles is developed. The technique is applied to the problem of quantifying convective transport of a scalar quantity in the vicinity of acoustically cavitating microbubbles of various initial radii subject to a range of sonication parameters, yielding insight regarding treatment parameter choice. Finally, in vitro and in vivo models are used to explore the effect of HIFU on delivery and expression of a biologically active adenovirus. The role of cavitation in improving the distribution of adenovirus in porous media is established, as well as the critical role of certain sonication parameters in sustaining cavitation activity in vivo. It is shown that following intratumoral or intravenous co-injection of ultrasound contrast agents and adenovirus, both the distribution and expression of viral transgenes are enhanced in the presence of inertial cavitation. This ultrasound-based drug delivery system has the potential to be applied in conjunction with a broad range of macromolecular therapeutics to augment their bioavailability for cancer treatment. In order to reach this objective, further developmental work is recommended, directed towards improving therapeutic transducer design, using transducer arrays for treatment monitoring and mapping, and continuing the development of functionalized monodisperse cavitation nuclei.