Targeting Alternative Splicing of Sodium Channels to Treat Pediatric Developmental Epileptic Encephalopathies - Project Summary Epilepsy is a debilitating neurological condition affecting 3.4 million Americans, with 0.6% of children dealing with active epilepsy. Mutations in the four main voltage-gated sodium channels active in the brain are leading causes of pediatric epilepsies, with mutations in SCN8A accounting for up to 1% of all epilepsy diagnoses. Many patients do not respond to current anti-epileptic drugs and current treatments focus only on treating seizures, but not on correcting the disorder. There is a critical need for new treatments for patients with SCN8A epilepsy. Exon 5 in SCN8A undergoes a highly conserved and developmentally regulated alternative splicing during development, with initially higher usage of the 5N “neonatal” exon, shifting gradually to predominant splicing of the 5A “adult” exon in late childhood. Recent studies show that Nav1.6 encoded by 5N-containing cDNA is less active than Nav1.6 encoded by 5A-containing cDNA in neuroblastoma cells, and that inclusion of exon 5A or 5N in SCN8A mRNAs can impact the effect of pathogenic mutations in other regions of the gene on Nav1.6 function. There are over 40 patients with SCN8A epilepsy that have mutations in exon 5 of SCN8A, that could potentially benefit from a splice-switching ASO therapy as it would prevent inclusion of the mutated exon, switching to the intact exon, without altering Nav1.6 protein expression. Further, the mechanism which regulates this switch in splicing from 5N to 5A in SCN8A remain poorly understood. We have optimized ASOs that induce a switch in splicing between exon 5N and 5A in both directions and developed a novel mouse model of SCN8A epilepsy with a pathogenic mutation in exon 5N. The ASOs we have developed can be used to study the role of each exon in brain function, neuronal development and susceptibility to seizures in healthy mice and as a therapeutic approach for patients with mutations in exon 5. We hypothesize that the alternative splicing of Scn8a exon 5 controls Nav1.6 activity and seizure susceptibility in the brain, and that ASO-driven splice switching to replace mutated exons with healthy exons in Scn8a mRNAs can reduce or prevent epilepsy, improve development and extend survival. First, we will evaluate the role of Scn8a exon 5 alternative splicing on brain activity, neuronal excitability, and seizure susceptibility. Second, we will determine the efficacy of an Scn8a splice-switching ASO in correcting Nav1.6 function and reducing seizure activity in a mutant exon 5N Scn8a mouse model. Third, we will identify RNA elements and splicing factors that regulate the splicing of Scn8a exons 5N/5A and use this knowledge to design new ASOs with distinct regulatory effects. These studies will shed light on the regulation of Nav1.6 activity, findings that could be applied to the other sodium channels in the brain, which are highly conserved and undergo a similar pattern of splicing regulation. The development of a disease-modifying treatment for patients with mutations in exon 5 of SCN8A may not only reduce seizures, but also aid development, increase functional skills, improve quality of life and reduce mortality in patients with severe DEE.
