<h3>Objective:</h3> The objective of this study was to determine if lowering the expression of the Glutamate Ionotropic Receptor Kainate Type Subunit 2 (GRIK2) using RNA interference can be a therapeutic strategy for temporal lobe epilepsy (TLE). <h3>Background:</h3> TLE is the most common form of focal epilepsy characterized by recurrent seizures generated in the hippocampus. Patients with TLE are often resistant to anti-seizure medications. Recurrent mossy fiber sprouting from dentate granule cells (DGCs) is a consistent anatomical hallmark of TLE leading to synaptic reorganization in an aberrant excitatory network. In TLE the DGCs operate via ectopic expression of GluK2/GluK5 receptors. GluK2/GluK5 plays a central role in the generation of seizure activity. <h3>Design/Methods:</h3> We created an AAV9-vector carrying two miRNAs under the control of a neuron-specific promotor targeting GRIK2 (AMT-260). The vector has been tested in a pilocarpine mouse model for TLE and assessed for safety in non-human primates (NHP). Additionally, hippocampal organotypic slices from TLE patients were treated with AMT-260. The efficacy of AMT-260 was measured in the mouse model using electroencephalogram (EEG) and behavior as readouts, and in hippocampal organotypic slices using electrophysiological recordings. The safety and biodistribution of AMT-260 was assessed in NHP after 1 month of treatment. <h3>Results:</h3> AMT-260 showed a dose-dependent decrease in electrical seizures and hyperlocomotion and improved health. A full biological effect was observed at 5.0E+09 vg per hippocampus. Organotypic slices from patients with TLE treated with AMT-260 showed a decrease in the number of electrical seizures. Convection enhanced, MRI guided intrahippocampal delivery in NHP resulted in local high copies of vector and miRNA expression with substantial knockdown of GRIK2, but with no adverse findings and limited peripheral exposure to the vector. <h3>Conclusions:</h3> AMT-260 shows an excellent preclinical and safety profile and will proceed into clinical testing to treat patients with refractory mesial temporal lobe epilepsy. <b>Disclosure:</b> Nick Pearson has nothing to disclose. Dr. Baudouin has received personal compensation for serving as an employee of Uniqure. Dr. Baudouin has stock in Uniqure. Dr. Wartel has received personal compensation for serving as an employee of uniQure biopharma B.V.. Dr. Wartel has stock in uniQure biopharma B.V.. Mr. Keskin has received personal compensation for serving as an employee of uniQure. Mr. Keskin has stock in uniQure. Mr. Keskin has a non-compensated relationship as a Technician with uniQure that is relevant to AAN interests or activities. Dr. Danos has received personal compensation for serving as an employee of REGENXBIO. Dr. Danos has received personal compensation in the range of $10,000-$49,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Vor Bio. Dr. Danos has received personal compensation in the range of $10,000-$49,999 for serving as an officer or member of the Board of Directors for Coave Therapeutics. Dr. Danos has stock in REGENXBIO. Dr. Danos has stock in Coave Therapeutics. Dr. Danos has stock in Rocket Therapeutics. Dr. Mulle has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Corlieve Therapeutics. Dr. Mulle has stock in Corlieve Therapeutics. Mr. Partouche has nothing to disclose. Dr. CREPEL has received personal compensation in the range of $10,000-$49,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Corlieve Therapeutics. Dr. CREPEL has stock in Corlieve Therapeutics. The institution of Dr. CREPEL has received research support from Corlieve Therapeutics. Dr. CREPEL has received intellectual property interests from a discovery or technology relating to health care. Dr. Borta has received personal compensation for serving as an employee of uniQure. Dr. Borta has stock in uniQure. Dr. Porter has received personal compensation for serving as an employee of Uniqure. Dr. Porter has stock in uniqure. Dr. Porter has stock in roche.