Rationale:
Mutations in the four main voltage-gated sodium channels (VGSCs) active in the brain are leading causes of pediatric epilepsies, with mutations in SCN8A (encoding Nav1.6) causing up to 1% of all epilepsy diagnoses. No specific treatments are currently available for patients with SCN8A developmental epileptic encephalopathy (DEE), with most interventions focused on reducing seizures, but not correcting the disease or addressing the many other comorbidities these patients have. SCN8A undergoes developmentally regulated alternative splicing of exon 5 with mutually exclusive use of the neonatal (5N) and adult (5A) exons, with the 5A isoform having reduced Nav1.6 activity. For SCN8A, more than 40 patients have mutations in exon 5A or 5N. Inducing a switch in splicing to include the non-mutated exon and generate a normal Nav1.6 protein could offer a disease-correcting treatment for these patients. The goal of our work is to develop antisense oligonucleotides (ASOs) that can shift the splicing of SCN8A from mutated exon 5N (or 5A) toward the intact exon 5, to normalize NaV1.6 channel activity, with potential to reduce seizures and promote development.
Methods:
We screened 16mer ASOs with 2’MOEs and PS backbones in and around exons 5A and 5N in SCN8A, using the mouse neuroblastoma cell line ND7/23, the human neuroblastoma line SH-SY5Y and in primary cortical neurons from P0 mice using lipofectamine or free uptake and cultured for 24hrs. Exon 5 isoform inclusion was assessed via agarose gel post RNA isolation, reverse transcription, PCR and restriction enzyme digestion using an enzyme specific to either exon 5A or exon 5N.
Results:
We identified 6 ASOs that reduce inclusion of Scn8a exon 5N and increase inclusion of exon 5A, in both ND7/23 cells and primary neurons with no impact on the exon 5 alternative splicing of VGSC genes SCN2A, SCN3A, SCN5A and SCN9A. The ASOs also did not alter overall Scn8a mRNA transcript levels. We have also identified 3 ASOs that drive the opposite switch, reducing inclusion of the 5A exon and increasing inclusion of the 5N exon.
Conclusions: We have identified multiple splice-switching ASOs that target both the mouse and human SCN8A genes, that can induce a splice-switch of exon 5 in either direction. Next we plan to evaluate the effects of these ASOs on Nav 1.6 sodium channel activity
in vitro and
in vivo in healthy mice, IPS-derived neurons from a patient with an S217P 5N mutation, and mice with an S217P exon 5N mutation in
Scn8a.
Our work will assess whether ASO-driven exon switching to reduce mutated Nav1.6 protein could represent a disease-altering therapy with long-term anti-seizure and developmental benefits, an approach that could potentially be applied to DEEs driven by mutations in other VGSCs with similar alternative splicing.
Funding: This work is supported by a DP2CA271387 award from NIH and the Oudin Lab Tufts SCN8A Epilepsy Research Fund.