Oncolytic measles virus is a promising cancer therapeutic in clinical trials which possesses multiple characteristics that are advantageous over traditional therapies. Currently, clinical oncolytic measles virus vectors are unmodified or express reporter transgenes that benefit its therapeutic efficacy. The next phase in its development will see genetically engineered vectors encoding transgenes that enhance its antineoplastic effects. To this end, preclinical research has focused on studying novel transgenes which favour viral replication, cytotoxicity, and the anti-cancer immune response. We sought to encode artificial micoRNAs targeting RIG-I as a strategy to interfere with innate immunity. Silencing RIG-I with multiple siRNAs yielded one which promotes measles virus syncytia formation through a mechanism that appears to be independent of RIG-I. The mechanism caused by the siRNA leads to enhanced measles virus cell-cell fusion and has peculiar characteristics which are not fully understood.

Adenovirus-mediated gene therapy is a potential complement to standard cancer treatments. Advantages are that vectors can be used to target tumors and that replicating viruses lead to increased therapeutic dosage. In this thesis, an oncolytic serotype 5 adenovirus (Ad5), Ad[i/PPT-E1A, E3], was developed where viral replication is controlled by the insulator-shielded (i) prostate-specific PPT promoter. The adenoviral E3 region was inserted for its immune regulatory and lysis functions. Ad[i/PPT-E1A, E3] had improved cytotoxic abilities both in vitro and in a prostate cancer xenograft mouse model compared to a virus lacking the E3 region. To further improve adenoviral vectors, the histone deacetylase inhibitor (HDACi) FK228 was studied. FK228 has been proposed to enhance the effect of adenoviral therapy by upregulation of CAR, the primary receptor for Ad5 infection. In the present study, we observed that FK228 promotes transgene expression even better when administered after viral transduction, indicating a post-transductional enhancement of transgene expression. Another interesting finding was that FK228 reduced transgene expression from the PPT promoter in the prostate cancer cell line LNCaP. This is explained by the fact that different HDACi have the ability to provoke a neuroendocrine phenotype of LNCaP. A potential drawback with adenoviral gene therapy is the rapid clearance of the virus from the circulation. Viral particles have been coated with polyethylene glycol (PEG) to evade immune recognition, a strategy that works well in mouse models. However, less is known about the effects of adenoviral PEGylation in human blood. We have studied cell interactions and immune responses to PEGylated and uncoated Ad5 vectors in human whole blood using a blood loop model with constant blood flow. Limited effects of PEGylation were observed in human blood, which were associated with the neutralizing ability of the donor blood. An important finding that donors with high neutralizing ability in whole blood do not necessarily have neutralizing antibodies against the virus strongly implies that neutralization should be measured in whole blood.

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.

Dans l’approche expérimentale de l’immunothérapie des tumeurs solides, les modèles murins sont utilisés pour des raisons de rapidité et de reproductibilité. Le plus souvent, les modèles tumoraux murins sont ectopiques ce qui constitue un modèle artificiel qui ne reflète qu’une partie de la réalité biologique de tumeurs provenant de diverses origines.Mon projet de thèse a consisté à mettre au point de nouveaux modèles pré-cliniques murins permettant de mieux mimer les situations pathologiques des tumeurs solides chez l’homme. Pour cela, je me suis notamment intéressée à un modèle orthotopique de cancer du rein (implantation de cellules RenCa ou RenCa-MUC1 dans la capsule rénale) et à un modèle spontané de cancer du sein (souris MMTV-PyMT). Ces modèles ont ensuite permis l’évaluation de trois différentes approches d’immunothérapie à savoir la thérapie virale oncolytique, la vectorisation d’antigènes tumoraux au moyen d’un vecteur viral ainsi que la thérapie via un anticorps monoclonal. / In experimental approaches to immunotherapy of cancer, mouse tumor models are used for reasons of speed and reproducibility. Ectopic mouse tumor models are the most often used, but they constitute artificial models that reflect only a part of the biological reality of tumors from various origins.The aim of my thesis project was to develop new mouse preclinical tumor models to better mimic the pathological situations of solid tumors in humans. First, I developed an orthotopic model of kidney cancer (subcapsular kidney implantation of a renal carcinoma cell line which either expressed or did not express the human xeno-antigen, MUC1). In addition to this, I also studied a spontaneous model of breast cancer (MMTV-PyMT).These models enabled us to evaluate the efficacy of three different immunotherapy approaches namely oncolytic virus strategy, tumor antigen vectorization by using a viral vector, and monoclonal antibody.

Due to the inevitable reality that most patients diagnosed with cancer will eventually relapse, modern oncology research has been forced to tackle this outcome primitively using combination therapies. Adoptive T-cell transfer with Oncolytic Virus Vaccination represents a new class of combination therapies that can facilitate the crosstalk of multiple aspects of the immune system such that they work in concert to prevent this outcome for many types of cancer. Despite this, immunosuppressive systems like those characterized in the B16F10-gp33 melanoma model pose a new problem for this approach. Typically, this model has total regression but is subsequently followed by relapse. Previous work from the Wan lab has suggested that this may be an outcome of total target gene deletion. Here we present two approaches to tackle this through the targeting of DNA repair pathways of the host cell. Our data can show that both VSV and Vaccinia infection/ propagation does lead to the generation of DNA damage but in the case of VSV this leads to incomplete cell lysis, and ultimately target gene loss via double-stranded DNA repair mechanisms. We were able to tackle the phenomenon following VSV administration by adding DNA repair inhibitors to the mix and showed that the proportion of cells that escaped after the loss of the target antigen was decreased by half when compared to the standard procedures. Additionally, this work also gave a preliminary understanding of how Vaccinia may achieve a similar outcome to this via its unique cytoplasmic replication mechanisms. / Thesis / Master of Science (MSc)

No description available.

Le cancer est une maladie qui touche des millions de personnes et ne discrimine pas. La forme de cancer la plus répandue chez les femmes au Canada est le cancer du sein et la deuxième cause de décès par le cancer chez cette population. Les traitements dépendent de plusieurs facteurs dont le stade du cancer, la ménopause, le statut des récepteurs hormonaux et du récepteur HER2 du cancer, etc. Les traitements qui existent sont la chirurgie suivie par la radio- et/ou chimiothérapie et l’hormonothérapie. Malgré les nombreuses études et les avancées dans les traitements pour différents cancers, plusieurs patients ont des cancers du sein qui sont réfractaires aux traitements disponibles. Une alternative naissante est l’utilisation de virus oncolytiques, c’est-à-dire des virus qui ciblent spécifiquement les cellules cancéreuses et laissent intact les cellules saines. Malheureusement, certains cancers demeurent réfractaires aux traitements avec virus oncolytiques. Ceci nous amène donc à regarder plus en détail des facteurs de l’environnement tumoral qui pourraient prédire la susceptibilité virale et engendrer des résultats positifs. C’est dans cette perspective que nous avons découvert que l'estrogène, précisément l’estradiol, rend les cellules cancéreuses qui en expriment le récepteur plus sensible au virus oncolytique VSV (virus de la stomatite vésiculaire). Cependant, nous ignorons toujours si d’autres hormones peuvent également moduler l’action de VOs. Nous émettons donc l’hypothèse que, comme l’estrogène, d’autres hormones vont affecter l’efficacité des VOs et qu’il serait possible de manipuler ces interactions pour améliorer la réponse au traitement. Notre étude nous permettra de concevoir des stratégies thérapeutiques améliorées pour les patients atteints du cancer du sein. L’importance de cette étude est que jusqu’à présent l’impact des hormones sur l’efficacité des virus oncolytiques reste un sujet inexploré. Nous allons déterminer l’effet de différents niveaux d’hormones sur la réplication et l’effet oncolytique de VSV. Ceci nous donnera ainsi la possibilité et les connaissances d’améliorer la sélection des patients pour le traitement et la conception d’une nouvelle génération de virus oncolytiques perfectionnés. / Cancer is a disease that affects millions of people across the world. The most common cancer in Canadian women is breast cancer and it also represents the second cause of death by cancer in this same group. The treatment depends on multiple factors including the stage of the cancer, menopause status, hormone receptor status, HER2 receptor status, etc. The available treatments for breast cancer are surgery followed by either radiation or chemotherapy as well as endocrine therapy. Despite numerous studies and advances in the treatment of different cancers, many patients’ cancer still remains refractory to these treatments. An exciting new alternative treatment is the use of oncolytic viruses. An oncolytic virus is a virus that can specifically target cancer cells all while leaving healthy normal cells intact. However, many cancers remain refractory to treatment with oncolytic viruses. There was thus a need to investigate different factors or the tumor microenvironment that may predict viral susceptibility and obtain positive outcomes. In this vein, it was found that estrogen (specifically estradiol), a hormone found in the body, can render cancer cells that express its receptor more sensitive to oncolytic virus infection by VSV (vesicular stomatitis virus). In spite of that, we are unaware if there are other hormones capable of modulating the actions of oncolytic viruses. Our hypothesis is that, like estrogen, other hormones will affect the efficacy of oncolytic viruses and that it will be possible to manipulate these interactions with the goal to improve treatment response. Our research will allow the conception of enhanced therapeutic strategies for patients with breast cancer. The importance of this study is that as of now the interplay between hormones and oncolytic viruses remains unexplored. We will determine the effects of hormone levels on viral replication and oncolytic ability of VSV. This knowledge will allow for a greater selection of patients for which oncolytic virus treatment will have a positive outcome. Additionally, it will allow for the development of a new generation of perfected oncolytic virus platforms.

This project combines oncolytic Vaccinia virus (VV) with irinotecan (CPT-11) for the treatment of cancer. VV can infect, replicate in and destroy cancer cells, yet leave healthy cells relatively unaffected. CPT-11 is a chemotherapeutic of which ~5% is converted to the more active chemotherapeutic SN-38 by endogenous carboxylesterase (CE) enzymes. SN-38 is a topoisomerase I inhibitor that induces DNA double strand breaks, leading to growth arrest and apoptosis. Consequently, VV has been engineered to express a more effective isoform of the CE enzyme. The virus’ tumour tropism should restrict enhanced conversion of CPT-11 to the tumour. Neither CPT-11 nor SN-38 interfered with VV replication or spread. Engineered recombinants expressed CE enzyme which, when combined with CPT-11, produced DNA double strand breaks and cancer cell death. In vitro, the combination of CE-virus and CPT-11 killed more K-562 cancer cells than its non-CE counterpart and CPT-11.

This project combines oncolytic Vaccinia virus (VV) with irinotecan (CPT-11) for the treatment of cancer. VV can infect, replicate in and destroy cancer cells, yet leave healthy cells relatively unaffected. CPT-11 is a chemotherapeutic of which ~5% is converted to the more active chemotherapeutic SN-38 by endogenous carboxylesterase (CE) enzymes. SN-38 is a topoisomerase I inhibitor that induces DNA double strand breaks, leading to growth arrest and apoptosis. Consequently, VV has been engineered to express a more effective isoform of the CE enzyme. The virus’ tumour tropism should restrict enhanced conversion of CPT-11 to the tumour. Neither CPT-11 nor SN-38 interfered with VV replication or spread. Engineered recombinants expressed CE enzyme which, when combined with CPT-11, produced DNA double strand breaks and cancer cell death. In vitro, the combination of CE-virus and CPT-11 killed more K-562 cancer cells than its non-CE counterpart and CPT-11.

This project combines oncolytic Vaccinia virus (VV) with irinotecan (CPT-11) for the treatment of cancer. VV can infect, replicate in and destroy cancer cells, yet leave healthy cells relatively unaffected. CPT-11 is a chemotherapeutic of which ~5% is converted to the more active chemotherapeutic SN-38 by endogenous carboxylesterase (CE) enzymes. SN-38 is a topoisomerase I inhibitor that induces DNA double strand breaks, leading to growth arrest and apoptosis. Consequently, VV has been engineered to express a more effective isoform of the CE enzyme. The virus’ tumour tropism should restrict enhanced conversion of CPT-11 to the tumour. Neither CPT-11 nor SN-38 interfered with VV replication or spread. Engineered recombinants expressed CE enzyme which, when combined with CPT-11, produced DNA double strand breaks and cancer cell death. In vitro, the combination of CE-virus and CPT-11 killed more K-562 cancer cells than its non-CE counterpart and CPT-11.