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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

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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.

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.

El sistema TK/GCV es, problamente, la estrategia suicida mejor caracterizada hasta el momento. No obstante, se desconocen muchos aspectos relacionados con su mecanismo de acción. Con el objetivo de indentificar condicionantes de la respuesta TK/GCV, realizamos un estudio comparativo de la expresión de genes y de las vías de señalización que se activan en células sensibles y en células resistentes al tratamiento. Así, pudimos asociar la actividad de la quinasa Chk1, y la expresión de genes involucrados en el control del ciclo celular, con una mayor respuesta al sistema suicida. Así mismo, determinamos que la combinación de TK/GCV con el inhibidor de Chk1 UCN-01 produce un efecto antagónico en las células sensibles a TK/GCV. Por otro lado, la terapia combinada capaz de lisar las células e inducir muerte celular por fosforilación de GCV, en un único agente (ICOVIR11), resultó en una potenciación de sus efectos citotóxicos, permitiendo la compensación de la pérdida de potencia secundaria al uso de un promotor selectivo de tumor. Más aún, la expresión de TK como gen tardío de ICOVIR11,permitió la monitorización in vivo y de manera no invasiva, de la actividad TK y la replicación viral. / Although extensively characterized, the paradigmatic suicide system TK/GCV conceals the details of its ultimate mechanism of action. In order to shed some light on this issue, we conducted a series of experiments with resistant and sensitive cell lines, allowing us to identify cell cyclerelated genes that are deregulated in cells with induced resistance to TK/GCV. In addition, the association of Chk1 activation with a greater sensitivity to TK/GCV, pointed out the relevance of the cell cycle status at the moment of receiving the treatment, and its control in response to genotoxic insults. Treatment with a Chk1 inhibitor induced, in sensitive cells, an antagonistic effect on TK/GCV cytotoxicity. On the other hand, single-agent combination therapy of TK/GCV with adenoviral lysis resulted in enhanced cytotoxicity. In this setting the expression of TK as a late gene in an oncolytic adenovirus minimized the loss of potency associated to the conditioning of viral replication. On top of that, TK expression allowed for in vivo, real time, non-invasive monitoring of viral replication in mice, and was used to analyze the effects of treatment schedule on treatment outcome.