Gene therapy constitutes an attractive approach for curing a variety of CNS diseases, including glioblastoma. Multiple studies have been carried out focusing on improving the total level and distribution of gene expression throughout the brain tissue. Viral gene delivery, though relatively efficient, has been limited by immunogenicity, low packaging capacity and difficulties in scale-up. Non-viral gene vectors offer an alternative strategy but are limited in their ability to generate high-level, widespread therapeutic effect. The nanoporous and electrostatically charged extracellular space found between healthy or tumor cells constitutes a steric and adhesive barrier, which hampers widespread distribution of gene vectors in the brain, regardless of administration method, thereby limiting gene transfer to target cells [1]. Indeed, following convention enhanced delivery (CED) conventional gene vectors are confined to the point of administration, limiting the clinical success of gene therapy for glioblastoma [2]. We developed a strategy to formulate small, stable and densely coated cationic polymer-based gene vectors that have the physicochemical characteristics required to overcome this ‘brain tissue barrier’. Using high resolution multiple particle tracking, we assessed the diffusion rates of gene vectors in freshly extracted ex vivo healthy and tumor rodent brain tissues. We thoroughly characterized their toxicity, cell uptake and transfection efficacy in vitro. Finally, we analyzed the ability of these vectors to rapidly penetrate and distribute throughout the brain tissue in vivo, thus reaching and transfecting cells over a large volume following CED. 1.Nance, E.A., et al., Sci Transl Med, 2012. 2.Voges, J., et al., Ann Neurol, 2003.