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Gene therapy with AAV vectors carrying genes for neuropeptide Y and its receptor Y2 has been shown to inhibit seizures in multiple animal models of epilepsy. It is however unknown how the AAV serotype or the sequence order of these two transgenes in the expression cassette affects the actual parenchymal gene expression levels and the seizure-suppressant efficacy. To address these questions, we compared three viral vector serotypes (AAV1, AAV2 and AAV8) and two transgene sequence orders (NPY-IRES-Y2 and Y2-IRES-NPY) in a rat model of acutely induced seizures. Wistar male rats were injected bilaterally with viral vectors and 3 weeks later acute seizures were induced by a subcutaneous injection of kainate. The latency until 1st motor seizure, time spent in motor seizure and latency to status epilepticus were measured to evaluate the seizure-suppressing efficacy of these vectors compared to an empty cassette control vector. Based on the results, the effect of the AAV1-NPY-IRES-Y2 vector was further investigated by in vitro electrophysiology, and its ability to achieve transgene overexpression in resected human hippocampal tissue was evaluated. The AAV1-NPY-IRES-Y2 proved to be better to any other serotype or gene sequence considering both transgene expression and ability to suppress induced seizures in rats. The vector also demonstrated transgene-induced decrease of glutamate release from excitatory neuron terminals and significantly increased both NPY and Y2 expression in resected human hippocampal tissue from patients with drug-resistant temporal lobe epilepsy. These results validate the feasibility of NPY/Y2 receptor gene therapy as a therapeutic opportunity in focal epilepsies.