The role of WNT signaling in inhibiting the oncolytic virus VSV-GP

Cancer is one of the leading causes of death worldwide. Unfortunately, to date, existing therapies are unable to effectively treat advanced stage tumours, guaranteeing only in some cases an increase in life expectancy and life quality. One of the emerging therapies is the use of viruses, either natural or genetically modified, that can selectively attack tumour cells and kill them, leaving normal cells unharmed. One of these viruses is VSV-GP (vesicular stomatitis virus-GP), a chimeric form of VSV in which the G protein has been replaced by the GP protein of lymphocytic choriomeningitis virus. This virus is not able to kill healthy cells with a physiological interferon (IFN) antiviral pathway, however, in many tumours IFN activity is aberrant and in these cells the virus is able to replicate and cause lysis. IFN non-responsive cancer cells were highly susceptible for VSV-GP infection in vitro, however some tumours formed with the same cell lines showed VSV-GP resistance in vivo. This thesis focused on IFN independent virus resistance mechanism ac-tive in vivo but no effect in vitro, in particular, on Wnt pathway antiviral effects. Wnt path-way is a well-known pathway which is involved in cellular proliferation, differentiation and migration; all recognized as hallmarks of cancer. Wnt pathway also showed an antiviral effect against several virus. The project had two main objectives: objective 1 was understanding the role of β-catenin in antiviral effect by Wnt pathway, objective 2 was characterizing VSV-GP expressing Wnt in-hibitor and optimizing the Wnt inhibitor itself. For Objective 1 to study role of β-catenin in Wnt pathway related antiviral effect, cell lines expressing constitutive activation β-catenin and shRNA mediated inhibition of β-catenin activity, a downstream effector of Wnt pathway, were produced. Prior to this study VSV-GP encoding a Wnt inhibitor I had been produced. The cargo was able to extracellularly inhibit Wnt ligand so blocking Wnt pathway selectively in the surround-ings of infected cells. For Objective 2 to improve the cargo production a new virus based on VSV-GP was produced presenting the cargo, Wnt inhibitor I, in first position, instead of fifth position, so increasing cargo production. A second virus presenting the cargo Wnt inhibitor II, a recombinant form of Wnt inhibitor I with a retention peptide putatively increasing cargo half-life in the tumour microenvironment, was also produced. After generation of the cell lines, their β-Catenin activity was validated with a luciferase assay and its expression was characterized via western blot. Antiviral activity was studied infecting cells with a recombinant VSV-GP expressing GFP and observing the cells under fluorescence microscope. Cells supernatants were tested with TCID50 assay to study viral production. New recombinant viruses were then produced and their cargo production was studied using western blot. Replication kinetics was studied by a single step growth curve. Western blots showed the efficacy of the shRNA in reducing active β-catenin. Fluorescence microscopy and TCID50 demonstrated that activating β-catenin reduced viral activity and replication while inhibiting it rescued them. Newly generated 1st position cargo viruses showed an increase of cargo presence via western blot in supernatant compared to the VSV-GP with the cargo in fifth position. The β-catenin-modified cell lines confirmed the hypothesis of the antiviral activity of Wnt pathway on VSV-GP. Cells overexpressing β-catenin led to inhibition of viral replication. shRNA mediated β-catenin inhibition led to a rescue of viral replication. Both the new virus proved to be superior to the old one with cargo in 5th position in terms of cargo production. More studies are required to complete the cell line characterizations and more in vitro and in vivo studies are required to confirm the efficacy of the WNT-inhibiting virus variants.