Abstracts

Developmental and epileptic encephalopathy 52 (DEE52), caused by biallelic, pathogenic variants in SCN1B, encoding voltage-gated sodium channel (Nav) b1 subunits, presents with severe pharmaco-resistant seizures of multiple etiologies, a markedly elevated risk of SUDEP, profound intellectual disability, developmental delays, ataxia, gastrointestinal disturbances, and failure to thrive. Given the absence of effective treatments for SCN1B-related disorders, our goal is to establish a gene replacement strategy using adeno-associated virus (AAV)-mediated delivery of the b1 subunit coding sequence, Navb1. We initiated this approach using Scn1b constitutive null mice.  However, because DEE52 patients are, in general, not null for SCN1B, we are now extending this work using knock-in mouse models expressing human pathogenic SCN1B variants as proof-of-concept for therapeutic efficacy and safety.

To evaluate the therapeutic potential of AAV-mediated Navβ1 delivery (AAV-Navβ1) in a knock-in mouse model of DEE52, we administered bilateral intracerebroventricular injections to wildtype (Scn1b^R89/R89) and homozygous Scn1b-p.R89C (Scn1b^C89/C89) mice at postnatal day (P) 2. The AAV-Navβ1 construct encoded an in-frame, C-terminal epitope-tagged β1 subunit driven by a modified Gad1 promoter, which we have shown previously to express ubiquitously across the brain in both excitatory and inhibitory neurons. To assess expression, brains were harvested at 14 and 28 days post-injection for Western blotting and immunocytochemistry to quantify β1 protein levels and neuronal cell type localization. Seizure phenotypes were assessed via continuous video monitoring for spontaneous events and via a controlled hyperthermia protocol at P15 in which core body temperature was heated step-wise to 42°C.

Our previous work in Scn1b null mice showed that AAV-Navβ1 treatment at P2 reduced spontaneous seizure severity and duration, prolonged lifespan, prevented hyperthermia-induced seizures, and restored cortical neuron excitability. AAV-Navβ1 administration to WT mice resulted in β1 protein overexpression in brain but no obvious adverse effects. Here, using the Scn1b-p.R89C loss-of-function knock-in model of DEE52, neonatal mice received AAV-Navβ1 at P2, and were subjected to hyperthermia-induced seizure testing at P15. Consistent with our previous work, 100% (11/11) of untreated Scn1b^C89/C89 mice had hyperthermia-induced seizures. In contrast, 90% (9/10) of Scn1b^C89/C89 tested exhibited no hyperthermia-induced seizures during the experimental recording period. Neither AAV-Navβ1 treated (n=5) nor untreated (n=12) wildtype Scn1b^R89/R89 mice displayed seizures under these conditions.

These results support the potential of AAV-mediated SCN1B gene replacement as a viable therapeutic strategy for DEE52. In the Scn1b-p.R89C model, AAV-Navβ1 treatment conferred robust protection against hyperthermia-induced seizures. Ongoing experiments in a second DEE52 knock-in model, Scn1b-p.C121W mice, will further elucidate whether this approach is broadly applicable across distinct pathogenic mechanisms associated with SCN1B variants.

Citizens United for Research in Epilepsy (CURE), AWD028955, to LLI.