AAV-mediated GPR173 gene therapy attenuates long-term refractory epilepsy by enhancing synaptic GABA_A receptor expression

Approximately 30% of epilepsy patients are drug-resistant, and sustained seizure activity often reduces responsiveness to conventional therapies. This study aimed to evaluate the therapeutic efficacy and safety of adeno-associated virus (AAV)-mediated GPR173 gene therapy in two refractory epilepsy mouse models, including delayed-treatment epilepsy (DTE) and drug-resistant epilepsy (DRE), and to investigate its underlying molecular mechanisms. AAV vectors encoding GPR173 under the CaMKII promoter were delivered in the DTE model (after 6 months of epileptogenesis) and the DRE model (following failure of two anti-seizure drugs). The seizure frequency was monitored for a 10-week period respectively before and after the gene therapy. GPR173-targeted gene therapy significantly reduced chronic seizure burden in both refractory TLE models. During the final week of monitoring, no seizures were detected in 25% of DTE mice and 29% of DRE mice. Notably, among mice that were responsive to anti-seizure drugs (ASDs), the subsequent addition of GPR173‑based gene therapy increased such proportion to 83%. Besides, safety was evaluated in healthy mice using brain and peripheral tissue histology and hepatotoxicity tests after CaMKII-driven GPR173 overexpression. To validate promoter selection, additional safety tests with CMV-driven GPR173 expression were included as a positive control for non-specific and high-level expression. No significant neural or peripheral abnormalities were observed after CaMKII-driven AAV delivery compared to CMV-driven expression. Furthermore, mechanistic studies showed that GPR173 upregulation may enhance cortical inhibitory control via increased expression of GABA_A receptors (GABA_ARs). Overall, this study indicated that CaMKII-driven GPR173 upregulation is an effective and safe gene therapy strategy for refractory epilepsy. Our findings also provide valuable insights into promoter selection for targeted gene therapy and reveal a potential mechanistic link between GPR173 signaling and strengthened cortical inhibition, with implications for clinical translation.