RESTORING <EM>SCN1A</EM> ACTIVITY IN DRAVET SYNDROME THROUGH TARGETED GAIN-OF-FUNCTION (GOF) VARIANT INSERTION

Dravet syndrome (DS) is a severe developmental epileptic encephalopathy characterized by drug-resistant seizures, and cognitive and behavioral deficits. It is caused by heterozygous Loss-of-Function (LoF) mutations in SCN1A encoding the voltage-gated sodium channel α-subunit Na_V1.1, predominantly expressed in inhibitory interneurons.
A recent study identified a SCN1A variant in a DS patient; interestingly, this variant was also found in asymptomatic relatives carrying a GoF variant on the other SCN1A allele, suggesting that it might exert protective effect. To investigate this hypothesis, different LoF variants and the described GoF were introduced into a tetrodoxin-resistant SCN1A coding sequence fused to a fluorescent reporter to determine the effect on excitability of transduced neurons by whole-cell patch-clamp recordings. We next aim to introduce the selected GoF mutation into the endogenous Scn1a locus using gene-editing and evaluate its impact on the phenotype of a DS mouse model.
Whole-cell patch-clamp recordings of SCN1A LoF transduced neurons showed dramatically impaired action potential firing (mean maximum AP=3 vs. wt=18), whereas GoF channels ensured sustained activity under high-intensity stimulation compared to wild-type (wt) channels (mean AP at 500 mA: wt=7, GoF=16). To evaluate potential interactions between variants, we are co-expressing different LoF mutations with the GoF variant and measuring their impact on neuronal activity.
We are currently working on gene-editing strategy to introduce the GoF variant into the endogenous Scn1a gene to assess its effect on Nav1.1 channel function at physiological expression levels.