Osteoarthritis (OA) is a chronic joint disease characterized by the progressive degradation of articular cartilage. Delivering functional genes to chondrocytes to modulate the inflammatory environment offers a promising approach to treating OA. However, the dense extracellular matrix (ECM) in the OA microenvironment and the rapid clearance of naked nucleic acids from synovial fluid present significant challenges. Here we report the development of highly branched poly(β-amino ester)s (HPAEs) for effective delivery of F-box protein 6 (FBXO6) gene to treat OA. Four HPAEs were synthesized using an "A2 + B4 + C2" Michael addition strategy. By optimizing the chemical compositions and topological structures, the optimal HMDA-2 was identified to exhibit superior transfection efficiency, outperforming the commercial reagents Lipofectamine 3000 and branched polyethyleneimine (PEI). HMDA-2 was further employed to deliver FBXO6 plasmid to effectively regulate H