CRISPR‐Based KCC2 Upregulation Attenuates Drug‐Resistant Seizure in Mouse Models of Epilepsy

Objective The precise intervention of K‐Cl cotransporter isoform 2 (KCC2) as a promising target for drug‐resistant epilepsy remains elusive. Methods Here, we used a CRISPRa system delivered by adeno‐associated viruses to specifically upregulate KCC2 in the subiculum to confirm its therapeutic potential in various in vivo epilepsy models. Calcium fiber photometry was used to reveal the role of KCC2 in the restoration of impaired GABAergic inhibition. Results CRISPRa system effectively upregulated KCC2 expression both in in vitro cell culture and in vivo brain region. Delivery of CRISPRa with adeno‐associated viruses resulted in upregulating the subicular KCC2 level, contributing to alleviating the severity of hippocampal seizure and facilitating the anti‐seizure effect of diazepam in a hippocampal kindling model. In a kainic acid‐induced epilepticus status model, KCC2 upregulation greatly increased the termination percentage of diazepam‐resistant epilepticus status with the broadened therapeutic window. More importantly, KCC2 upregulation attenuated valproate‐resistant spontaneous seizure in a kainic acid‐induced chronic epilepsy model. Finally, calcium fiber photometry showed CRISPRa‐mediated KCC2 upregulation partially restored the impaired GABA A ‐mediated inhibition in epilepsy. Interpretation These results showed the translational potential of adeno‐associated viruses‐mediated delivery of CRISPRa for treating neurological disorders by modulating abnormal gene expression that is directly associated with neuronal excitability, validating KCC2 as a promising therapeutic target for treating drug‐resistant epilepsy. ANN NEUROL 2023;94:91–105