Abstracts

Rationale: Dravet syndrome (DS) is a severe developmental and 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 DS patients carry de novo mutations in SCN1A leading to haploinsufficiency of the voltage-gated sodium channel (VGSC) alpha subunit Nav1.1. We have developed a novel therapeutic approach to treat DS using antisense oligonucleotides (ASOs) to increase the endogenous expression of SCN1A mRNA and Nav1.1 protein by inhibiting generation of a splice variant transcript that contains a premature termination codon leading to degradation by nonsense mediated decay (NMD). We are testing this approach using the F1:129S-Scn1a+/- x C57BL/6J DS mouse model, that has been shown previously to recapitulate 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. Using this approach, we identified an ASO that can increase productive Scn1a transcript and NaV1.1 protein in mouse and human cells. We administered different dosage levels of optimized TANGO ASO to DS mice and wildtype littermates by single intracerebroventricular (ICV) injection at postnatal days (P) 2 or 14. ASO levels, pharmacodynamic effects in tissues, and efficacy in the mouse model were measured.  Results: A single ICV injection of 20 mg of ASO at P2 in Scn1a+/- mice resulted in a 98% survival rate (1 of 51 died) of mice tested, with a subset of mice monitored through ~90 days of development. In contrast, a 35% survival rate of littermate Scn1a+/- mice treated with vehicle was observed (48 of 74 died). There were no deleterious effects on 87 ASO-treated littermate Scn1a+/+ mice tested, with a subset of mice monitored through ~90 days of development. A single ICV injection of 30 mg of ASO at P14, around the time of disease onset, resulted in a 65% survival rate (14 of 40 died) of Scn1a+/- mice, with no deleterious effects on 25 ASO-treated Scn1a+/+ mice. Doubling the dose of the single ASO injection to 60 mg at P14 resulted in a 87% survival rate (6 of 45 died) of Scn1a+/- mice, with no deleterious effects on 40 ASO-treated Scn1a+/+ mice. Tissue analysis revealed significant ASO exposure up to ~90 days in the brains of animals dosed at P2 and P14. A more detailed evaluation of tissue ASO pharmacokinetics is ongoing, but T1/2 in mouse brain appears to be prolonged. ASO treatment at both P2 and P14 resulted in long-lasting, significant increases in Scn1a mRNA and NaV1.1 protein in mouse brain. EEG recordings from control and P2-treated DS mice are ongoing.  Conclusions: These results provide evidence that TANGO technology can be used to rescue the DS phenotype in a mouse model of Scn1a-linked DS and has the potential to provide a gene-specific, disease-modifying treatment to restore Nav1.1 to physiological levels to provide therapeutic benefits to DS patients.  Funding: Supported by a grant to LLI from Stoke Therapeutics.