484 Utilizing Convection Enhanced Delivery of PBAE Nanoparticles for Genetic Reprogramming in Glioblastoma

INTRODUCTION: Glioblastoma (GBM) is an aggressive brain cancer that evades immune detection by altering the immune microenvironment. Nanoparticle-mediated gene therapy offers a potentially safe targeted strategy. METHODS: PBAE nanoparticles were synthesized and characterized using dynamic light scattering. In vitro, CT2A mouse glioblastoma cells were transfected with nanoparticles containing GFP plasmid. Cell viability and GFP transfection efficiency were assessed using an MTS assay and flow cytometry. ELISA was used to measure IFNG production in a CT2A co-culture with human PBMCs. In vivo transfection was confirmed using bioluminescence-based imaging after the injection of luciferase-tagged nanoparticles into CT2A subcutaneous flank tumors in C57BL/6 mice. Impact on survival was tested in a CT2A flank model and a CT2A orthotopic model comparing four groups: control nanoparticles, 4-1BBL/IL12 nanoparticles, anti-PD1 therapy, and 4-1BBL/IL12 nanoparticles with anti-PD1 therapy. RESULTS: Nanoparticle 5-3-49 demonstrated the highest GFP transfection efficacy and the lowest cytotoxicity in CT2A cells in vitro. An IFNG ELISA validated the induction of a favorable inflammatory response by 4-1BBL/IL12 nanoparticles compared to control. For in vivo transfection validation, bioluminescence-imaging confirmed strong luciferase expression 12 hours post-injection of 5-3-49 nanoparticles harboring luciferase DNA into flank tumors. Survival analysis in the flank model (n = 32) revealed a statistically significant increase in survival for mice treated with the combined therapy, 4-1BBL/IL12 nanoparticles and anti-PD1, compared to monotherapies and control (p = 0.02). In the orthotopic model (n = 64), a significant survival advantage was observed, with the combination therapy group showing nearly doubled median survival (p = 0.003). Remarkably, 25% of mice in this group experienced complete tumor resolution. CONCLUSIONS: PBAE nanoparticles effectively delivered targeted gene therapy for GBM, demonstrating notable improvements in median survival and tumor resolution.