MODULATING THE GLIOBLASTOMA IMMUNE LANDSCAPE THROUGH RNA INTERFERENCE AND CYTOKINE AND CHEMOKINE-BASED NANOMEDICINE

Glioblastoma (GBM) remains one of the most aggressive and treatment-resistant tumors of the central nervous system, in part due to the profoundly immunosuppressive microenvironment that limits the effectiveness of current therapies. The European RAIN project was established to explore a novel therapeutic approach based on Radiotherapy-Activated Immunizing Niches (RAIN), a sustained-release platform designed to deliver cmRNAs encoding immunostimulatory factors (IL-12 and CXCL9) in combination with siRNAs targeting putative immunosuppression-related genes (Lgals1, Lgals9, and Vsir) in myeloid cells. This combinatorial strategy was hypothesized to enhance immune activation while reducing suppressive pathways, thereby improving the potential efficacy of radiotherapy in GBM. As part of this consortium, this PhD thesis focused on evaluating the immunomodulatory potential of zwitterionic polymeric nanoparticles (Ze-NPs) carrying either cmRNAs or siRNAs cargoes using in vitro and in vivo models to characterize their biological effects. Intratumoral delivery of mIL-12 and hCXCL9-encoding cmRNA promoted the infiltration of inflammatory monocytes in tumor-bearing rodents, although the effect was transient, highlighting the need for a slow-release system such as RAIN. In combination with radiotherapy, cmRNA-Ze-NPs embedded within the hydrogel showed signs of shifting the immune environment toward a more inflammatory, M1-like phenotype. In parallel, siRNA-loaded Ze-NPs achieved efficient downregulation of Lgals9 and Vsir in microglia. Although evidence for functional reprogramming of myeloid cells remains preliminary, these findings suggest that combining immune-stimulatory factors with the silencing of genes potentially involved in immunosuppression may represent a promising strategy to counteract myeloid-driven immunosuppression in GBM. In conclusion, this thesis contributes to the preclinical evaluation of localized RNA-based immunotherapies for GBM, supporting the rationale for further investigation of RAIN as part of a multimodal treatment strategy. While additional studies are needed to validate efficacy and clarify mechanisms, the integrative approach explored here highlights potential avenues for overcoming the immunological barriers that limit current therapies.