Phd in CLINICAL EXPERIMENTAL NEUROSCIENCE AND PSYCHIATRY

Thesis title: Enhancing Natural Killer cell cytotoxicity to counteract Glioblastoma growth

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults, characterized by rapid proliferation, extensive infiltration, and profound therapeutic resistance. Despite advances in surgical, chemotherapeutic, and radiotherapeutic approaches, patient prognosis remains poor, with median survival of approximately 15 months. A major obstacle to effective therapy lies in the highly immunosuppressive tumor microenvironment (TME), which limits the efficacy of both innate and adaptive immune responses. In particular, Natural Killer (NK) cells, key effectors of innate immunity, exhibit impaired cytotoxicity within GBM due to the upregulation of inhibitory immune checkpoints such as NKG2A and PD-1. Overcoming this functional impairment of NK cells represents a promising therapeutic strategy to restore anti-tumor immunity. In this work, we investigated whether the silencing of nkg2a and pd-1 mRNA through small interfering RNA (siRNA) delivered via amphiphilic dendrimers (AD) complexes could enhance NK cell cytotoxicity against GBM. In vitro, D-siRNAs treatment reduced nkg2a and pd-1 mRNA expression in NK cells, leading to a significant increase in activation marker CD107 and cytotoxicity. To increase the effect of D-siRNAs in vivo in murine orthotopic GBM models, we co-administered D-siRNA complexes with the tumor-penetrating peptide iRGD, which enabled efficient gene knockdown and led to a pronounced reduction in tumor volume. Moreover, the treatment with these nanosystem modulated the TME, reducing the recruitment of myeloid cells without altering their phenotype and lessened astrogliosis. Overall, these findings demonstrate that silencing of nkg2a and pd-1 enhances NK cell cytotoxicity and reshapes the GBM microenvironment toward a less immunosuppressive state. The integration of RNA interference (RNAi) and nanotechnology-based delivery offers a promising platform for potentiating immune checkpoint blockade in GBM.