Abstracts

Rationale: Dravet Syndrome (DS) is a severe developmental epileptic encephalopathy predominantly caused by heterozygous loss-of-function (LOF) mutations in the SCN1A gene, leading to interneuron dysfunction. DS is marked by treatment resistant seizures and increased risk of premature mortality primarily due to sudden unexpected death in epilepsy (SUDEP).

We recently developed a novel AAV-based gene replacement therapy by precisely targeting interneurons, the cell population affected in DS. Since SCN1A exceeds the limited carrying capacity of AAVs, the gene was split in two and the gene segments were delivered using two separate AAVs. Then, the full-length channel protein was reconstructed post-translationally using the intein fusion protein technology. We observed a striking protection of this therapeutic approach against seizures at P25-35 and against premature mortality through P70. In this study, we investigated 1) the durability of efficacy through up to 1.5 years and 2) the dose response to this treatment.

Methods: DS mice were generated by crossing Scn1a^fl/fl with MeoxCre⁺ parents, which express Cre across the whole body. At postnatal day (P) 0-3, pups were injected with 5µL total volume of the dual AAV gene replacement therapy delivered bilaterally via intracerebroventricular (ICV) route. Mice were injected with a dose of 0, 0.1x10¹⁰, 1x10¹⁰, or 3x10¹⁰ viral genome copies (gc) of each vector. We conducted thermally induced seizure testing, elevating body temperature up to 42°C, at P30-35 and monitored premature mortality through P70. To examine therapy durability, mice were monitored for mortality through 1.5 years of life and submitted to a thermal seizure test at 1 and 1.5 years of age. Then, brain tissue was harvested to assess therapeutic transgene long-term expression by immunohistochemistry.

Results: Thermal seizure tests showed moderate protection against myoclonic (MC) seizure and near complete protection against generalized tonic clonic (GTC) seizure in treated compared to control mice up to 1.5 years post treatment administration. At 1 year post AAV administration, 100% of mice treated with 3x10¹⁰ gc of the vector remained GTC-free, compared to only 7% of the untreated counterparts. This protection against seizures was maintained up to 1.5 years of age. Immunodetection after 1.5 years of age showed strong expression of both halves of the dual AAV construct with coexpression in GABAergic interneurons.

Examination of dose dependency of treatment at P30-35 showed moderate protection against MC seizures and strong protection against GTCs correlated with the dosage level. Additionally, we saw a similar correlation between protection against mortality and treatment dosage. Interestingly, these therapeutic responses were observed even at the lowest dose tested (0.1x10¹⁰ gc).

Conclusions: These results indicate that our gene replacement therapy confers lasting protection, across a large dose range, against seizures and premature mortality. These findings suggest that this treatment may be an efficacious one-shot treatment for DS.

Funding: This work was supported by the following grant: NIH UG3/UH3.