Glioblastoma multiforme (GBM) is the most prevalent deadly brain tumour and is currently incurable. Patients die from recurrent tumours that grow from cells surviving the first round of treatment. This is mainly due to the narrow therapeutic window of current drugs and to the low permeability of these drugs across the blood-brain barrier (BBB). Targeted non-viral delivery systems have shown great promise to decrease side effects of therapeutics and to enable the transport across biological barriers. The main objective in this proposal is to generate a dually targeted nanoparticle loaded with mRNA encoding a conditionally toxic protein to treat brain cancer. The nanoparticle will be modified with two ligands: an antibody for targeting tumour cells and a peptide that enables transport across the BBB. In addition, I will develop an acid-labile coating that enhances the stability of the polyplex in blood and is released upon internalization. This coating will present the ligands in a defined orientation using bioorthogonal chemistry, thereby maximising their targeting efficiency. The chemical handles will be introduced using genetic code expansion in the antibody and solid-phase synthesis in the peptide. A variety of formulations will be screened in vitro to evaluate their stability and transfection efficiency, and BBB permeability will be assessed in a human cell-based BBB model. Subsequently, the efficacy of the most promising candidates will be tested in a GBM mouse model. The combination of a polyplex having high transfection efficiency with a sheddable coating and oriented targeting ligands is designed to generate the first efficient non-viral gene delivery system for the systemic treatment of GBM, reaching all tumour cells to provide a more efficient treatment and potentially prevent recurrence.