Targeted Inhibition of Nuclear Factor-κB Pathway Using Recombinant Adeno-Associated Virus 5 in 2D and 3D Glioblastoma Models.

PURPOSE: Glioblastoma (GBM) is a primary brain tumor with pronounced heterogeneity, aggressive nature and resistance to standard therapies. The dysregulation of nuclear factor kappa B (NF-κB) is central to glioblastoma pathology. But the limitations of existing NF-κB inhibitors have underscored the need for gene therapy approaches capable of achieving controlled and sustained pathway suppression. In this study, a previously engineered bicistronic recombinant AAV5 (rAAV5) vector co-expressing a dominant-negative mutant of IκBα (IκBα-M) alongside a luciferase reporter was packaged into viral particles. The functionality of rAAV5 viral particles was evaluated in both two-dimensional (2D) U87-MG cells and three-dimensional (3D) glioblastoma spheroids. METHODS: A bicistronic rAAV5 vector co-expressing IκBα-M and luciferase was packaged and transduced into U87-MG cells at a multiplicity of infection of 10,000 vg/cell. Transduction efficiency was assessed using a rAAV5 GFP reporter virus and evaluated by flow cytometry and confocal imaging. NF-κB activity was evaluated using luciferase reporter assay, immunofluorescence, qPCR, Western blot analysis and ELISA. Apoptosis and cell viability were measured using Annexin V/PI and Live/Dead imaging respectively. ATP-based viability assay was evaluated in both 2D culture and 3D spheroids. RESULTS: U87-MG cells transduced with rAAV5 GFP reporter virus exhibited high transduction efficiency with 82% GFP positive cells in 2D and efficiently transduced 3D spheroids. IκBα-M overexpression resulted in cytoplasmic retention of NF-κB and significant reduction in luciferase activity. NF-κB and hypoxia-inducible factor 1 subunit alpha (HIF1A) gene expression levels were downregulated and protein analysis confirmed increased NF-κB levels in the cytoplasmic protein. Additionally, cells transduced with rAAV5 virus induced apoptosis and reduced cell viability in GBM spheroids. CONCLUSION: The findings of this study establish that rAAV5-mediated NF-κB responsive delivery of IκBα-M enables effective and targeted suppression of aberrant NF-κB signaling, supporting its potential for further preclinical development in GBM gene therapy.