Abstracts

Rationale: Mutations in SCN1A can produce a mild seizure phenotype or result in Dravet
syndrome, a developmental and epileptic encephalopathy (DEE) characterized by various
seizures with developmental delays. Genetic epilepsies within families often exhibit multiple
disease severities even though members have an identical sodium channel mutation. This
suggests that other factors, including genetic, modify the primary mutation and alter disease
severity. The genetic basis of epilepsy can be studied using mouse models by changing the
background strain the mutation is maintained on. The Scn1a +/- (KO/+) mouse model has a strain-dependent epilepsy phenotype. KO/+ on the 129S6/SvEvTac (129) strain exhibit normal
behaviors and longevity while F1[129xC57BL/6J].KO/+ mice experience spontaneous
generalized tonic-clonic seizures and high mortality. Previous mapping identified several
modifier loci responsible for the strain-dependent survival difference. One loci of interest, Dsm3
located on chr 8, was investigated and identified a candidate modifier gene, Psd3. To evaluate
Psd3 as candidate modifier gene, we both decreased and increased Psd3 expression in
F1.KO/+ mice through ASO-mediated knockdown and CRISPRa, respectively, and monitored
survival.

Methods: To reduced Psd3 expression, an ASO targeting Psd3 (5′-GTATTAATACTCTCTC-3′)
was utilized. Intracerebroventricular (ICV) injection delivered 10 ug of ASO or vehicle (dPBS)
into postnatal day (P) 0-1 F1.KO/+ mice. To increase Psd3 expression, a CRISPRa strategy
was utilized by crossing B6.dCAS9 (Jackson Labs, #031645) with 129.KO/+. We used the
CRISPick webtool to design gRNAs targeting Psd3 and selected top performers based on in
vitro testing. F1.KO/+;Tg/+ mice were ICV injected at P0-1 with 5.0x10 9 viral genome copies of
the promotor-targeting Psd3-AAV or non-target-AAV control (NTC). At P18-20, ASO and AAV
treated mice were subjected to a single hyperthermia priming seizure induced by elevating core
body temperature until seizure onset. Mice were then monitored for survival until P31.

Results: Treatment of F1.KO/+ mice with Psd3-ASO resulted in no changes in seizure threshold
in the hyperthermia assay. F1.KO/+ mice treated with dPBS or Psd3-ASO had average seizure
thresholds of 42.4±0.1 and 42.4±0.08 °C, respectively (p >0.99; n=8-10). Psd3-ASO treatment resulted in a trending improvement in survival (p >0.06; n=8-10), with dPBS treated mice surviving a median of 21 days, while Psd3-ASO treated survived 33 days. Treatment of
F1.KO/+;Tg/+ mice with Psd3-AAV resulted in significantly elevated seizure thresholds, with an
an average seizure threshold of 42.4±0.11°C, while treatment with NTC-AAV had an average
threshold of 41.6±0.09°C (p< 0.0004; n=5-7). Treatment of with Psd3-AAV resulted in no