Abstracts

Rationale: Dravet syndrome is a severe pediatric epileptic encephalopathy characterized by high seizure frequency and severity, intellectual disability, and a high risk of sudden unexpected death in epilepsy (SUDEP). The majority of Dravet syndrome patients carry de novo mutations in SCN1A leading to haploinsufficiency of the voltage-gated sodium channel a subunit Nav1.1. Currently, there are no disease-modifying, targeted therapeutics to treat Dravet syndrome. We hypothesized that restoration of Nav1.1 physiological levels in patients should reduce or prevent seizures, decrease the risk of SUDEP, and potentially improve cognitive development. Here, we test a novel therapeutic approach using antisense oligonucleotides (ASOs), an established and FDA-approved therapeutic modality, to increase the endogenous expression of Scn1a in a Scn1a^+/- Dravet syndrome mouse model. The model recapitulates many patient phenotypes, including severe seizures, developmental delay, ataxia, sleep disorders, and SUDEP. Methods: We employed Targeted Augmentation of Nuclear Gene Output (TANGO), which exploits naturally-occurring non-productive splicing events to increase target protein expression via modulation of splicing. TANGO operates in a mutation-independent manner and does not alter protein coding splicing isoforms to achieve its goal. We identified an alternatively spliced exon conserved in human and mouse SCN1A that leads to the incorporation of a premature termination codon and the generation of a non-productive mRNA. We designed ASOs to target this non-productive alternatively spliced exon and tested them in vitro and in vivo. Results: Identified ASOs significantly increased the expression of SCN1A in cultured human neural-progenitor cells and in differentiated neurons with no effect on the expression of other voltage-gated sodium channel genes. This increase in SCN1A expression resulted from a gene-specific reduction in non-productive mRNA and an increase in productive mRNA. Intracerebroventricular (ICV) injection of the lead ASO in neonate and adult C57Bl/6 wild-type mice yielded a substantial, dose-dependent increase in Scn1a mRNA as well as Nav1.1 protein. Time course experiments in neonate and adult wild-type mice showed a sustained increase in Scn1a expression from a single, bolus ICV injection of the lead ASO monitored through 80 days. A single ICV injection of the lead ASO at postnatal day (P) 2 in F1:129 x C57Bl/6 Scn1a^+/- Dravet syndrome mice prevented generalized seizures and SUDEP in 99% (79 of 80) of Scn1a^+/- mice tested, with a subset of mice monitored through ~90 days of development. In contrast, approximately 50% of littermate Scn1a^+/- mice treated with a control ASO seized and died.  There were no deleterious effects on 101 ASO-treated littermate Scn1a^+/+ mice tested, with a subset of mice monitored through ~90 days of development.  A smaller cohort of Dravet syndrome mice (5 Scn1a^+/- and 1 Scn1a^+/+) received a single ICV injection of the lead ASO at P14. We have monitored these animals through ~45 days and found that generalized seizures and lethality were rescued in 100% of the Scn1a^+/- mice with no deleterious effects on the Scn1a^+/+ mouse. A larger cohort of mice injected at P14 is being monitored. Conclusions: These results indicate that TANGO technology can be used to rescue a mouse model of Scn1a-linked Dravet syndrome and may provide a gene-specific, disease-modifying approach to restore physiological Nav1.1 levels to prevent seizures and SUDEP in patients. Funding: Supported by a grant from Stoke Therapeutics to LLI.