Oncolytic HSV-1 armed with P2G to disrupt CXCR4 pathway in Glioblastoma

Glioblastoma is an aggressive high-grade astrocytoma (WHO, grade IV). Current standard treatments consist of maximal surgical resection followed by chemo-/radio- therapy. Unfortunately, this treatment protocol is impaired by systematic tumour reformation promoted by CXCR4+ glioblastoma stem-like cells (GSCs). GSCs are able to migrate from the tumour mass to the sub ventricular zone through the corpus callosum (cc), following a CXCL12 gradient (CXCR4 sole ligand). Besides, GSCs are responsible, with M2-like macrophages and cancer-associated fibroblasts, for creating a particular protumoral microenvironment at the tumour site driven by CXCL12/CXCR4 pathway-related autocrine and paracrine stimulation. This CXCR4/CXCL12-driven microenvironment promotes tumour cells' selfrenewal, proliferation, migration, tumour angiogenesis, and pro-tumoral immune modulation. Thus, GSCs and CXCL12/CXCR4 pathway became targets for new therapies against GBM recurrence and protumoral microenvironment development. In this context, stereotaxic injection of attenuated oncolytic HSV-1 (oHSV) expressing a specific CXCL12/CXCR4 pathway inhibitors (oHSV-P2G) seems to be a potent therapeutic strategy. Through infection and continuous production of inhibitors at the tumour site, such a virus would have the potential to cause GSCs lysis and inhibit their survival and functions. This way, oHSV-P2G will disrupt the pro-tumoral microenvironment and trigger inflammation against virus and tumour antigens. Preliminary in vitro results realized on human and murine primary cells and GSCs neurospheres showed the successful arming of oHSV and the inhibition of the CXCL12/CXCR4 pathway by oHSV-P2G. Indeed, glioblastoma cells' self-renewal and migration abilities, as well as the expression of neural stem markers, were decreased upon infection by oHSV-P2G compared to oHSV or upon treatment with conditioned media containing P2G. Inhibition of invasion abilities was also assessed in vivo - in an orthotopic immunodeficient nude mice model - by light-sheet microscopy 3D imaging of brains and tumours 50 days post-injection (d.p.i.) of 105 GB138 human glioblastoma cells. Analyses of these images on a small number of mice showed a decrease in the invasion of tumour cells in the cc in the group treated with 106 pfu oHSV-P2G 20 d.p.i. compared to the oHSV and PBS treated controls. These results are currently being repeated on larger groups. More in vivo assays on a syngeneic orthotopic immunocompetent C57/Bl6 mouse model injected with GL261 N4 or CT2A N+ murine glioblastoma cells will focus on the impact of oHSV-P2G on the protumoral microenvironment and innate immune response. Toxicity issues and efficacy of viral replication will also be addressed in this model, which is more relevant knowing the importance of the immune response in virotherapy.