Targeted gene therapy alleviates epilepsy and hyperexcitability in models of SCN2A deficiency

Abstract Mutations in the voltage-gated sodium channel gene SCN2A , which encodes the Na V 1.2 channel, cause severe epileptic seizures. Patients with SCN2A loss-of-function (LoF) mutations, such as protein-truncating mutations, often experience later-onset and drug-resistant epilepsy, highlighting an urgent unmet clinical need for new therapies. We previously developed a gene-trap Scn2a ( Scn2a gt/gt ) mouse model with a global Na V 1.2 reduction in the widely used C57BL/6N (B6) strain. Although these mice display multiple behavioral abnormalities, EEG recordings indicated only mild epileptiform discharges, possibly attributable to the seizure-resistant characteristics associated with the B6 strain. To enhance the epileptic phenotype, we derived congenic Scn2a gt/gt mice in the seizure-susceptible DBA/2J (D2J) strain. Notably, we found that these mice exhibit prominent spontaneous absence seizures, marked by both short and long spike-wave discharges (SWDs). Restoring Na V 1.2 expression in adult mice substantially reduced their SWDs, suggesting the possibility of SCN2A gene replacement therapy during adulthood. RNA sequencing revealed significant alterations in gene expression in the Scn2a gt/gt mice, in particular a broad downregulation of voltage-gated potassium channel (K V ) genes, including K V 1.1. The reduction of K V 1.1 expression was further validated in human cerebral organoids with SCN2A deficiency, highlighting K V 1.1 as a promising therapeutic target for refractory seizures associated with SCN2A dysfunction. Importantly, delivery of exogenous human K V 1.1 expression via adeno-associated virus (AAV) in D2J Scn2a gt/gt mice substantially reduced absence seizures. Together, these findings underscore the influence of mouse strain on seizure severity and highlight the potential of targeted gene therapies for treating SCN2A deficiency-related epilepsies. Graphical abstract In brief Scn2a deficiency leads to absence seizures in D2J mice and neuronal hyperexcitability with compensatory K V reduction; restoring Na V 1.2 or introducing human K V 1.1 reduces seizure burden. Highlights Scn2a deficiency induces robust absence seizures in the DBA/2J but not the C57BL/6N strain. Cortical neurons in adult DBA/2J mice with Scn2a deficiency exhibit intrinsic hyperexcitability. Severe Scn2a deficiency leads to downregulation of multiple potassium channel genes. Genetic restoration of Na V 1.2 expression alleviates spike-wave discharges (SWDs). AAV-mediated human K V 1.1 delivery substantially reduced absence seizures, demonstrating the therapeutic potential of targeted gene therapy.