EVALUATION OF HERPES SIMPLEX VIRUS TYPE 1-BASED ONCOLYTIC VIRUSES IN ADVANCED 3D MODELS

Glioblastoma, also known as glioblastoma multiforme (GBM), is the most aggressive and common type of primary malignant brain tumor in adults. Despite some progress with chimeric antigen receptor (CAR)-T cell therapies immunotherapy remains largely ineffective against GBM, due to the immunosuppressive tumor microenvironment, the target antigen heterogeneity, and the limited trafficking across the Blood-Brain Barrier (BBB), which often requires intraventricular administration or intracranial administration, relatively invasive methods of administration of therapeutics and not easily repeatable. Furthermore, the infiltrative nature of the disease and its tendency to recur in distant brain areas make localized therapy insufficient. In these experiments we focus on the production of cerebral organoids which represent an advanced 3D model composed of various types of cells including neurons, astrocytes, microglia-like cells, and oligodendrocytes precursor cells (OPCs) which can mature into myelin-producing oligodendrocytes, and others, to create an in-vitro environment that mimics the brain structures heterogeneity for testing oncolytic virotherapy. Oncolytic viruses (OVs) are naturally or genetically engineered viruses which aim to boost the anti-tumoral response and exhibit cell bursting via oncolysis – they are designed to specifically target tumor cells and spare healthy cells. The latter is in the core of the scope of the experiments, as we have tested one type of oHSV-1 oncolytic viruses (called oHSV-1-mCherry), and compared it to the wild type virus (called HSV-1 V-41) in its ability to infect healthy organoids. To prove that, via confocal/fluorescence microscopy we evaluated the infectivity of the viruses regarding the healthy cells, exploiting their reporter genes production. Furthermore, we confirmed the previous results through a viral titration experiment, that permitted the quantification of viral load in each infected organoid. Interestingly, the neuroattenuation of oHSV1-mCherry, obtained through γ34.5 and Us12 deletions and miR-124 target sequence recognition, significantly reduced HSV-1 ability to replicate inside healthy neurons, confirming its neuroattenuation.