Abstracts

Rationale: Voltage-gated ion channels, including SCN2A and KCNB1, are responsible for maintaining neuronal excitability. Pathogenic variants in SCN2A and KCNB1 can result in a spectrum of phenotypes, ranging from zero seizures to early-onset epileptic encephalopathies. Electrophysiological characterization of epilepsy-associated ion channel variants also show a wide range of dysfunction. However, the in vitro to in vivo correlation is not straightforward. One goal of our Channelopathy-Associated Epilepsy Center Without Walls is to determine which in vitro characteristics best predict dysfunction and treatment response in an intact, fully developed brain. Therefore, we generated mouse models with pathogenic patient variants in Scn2a and Kcnb1 that represent major classes of channel dysfunction based on variant characterization in heterologous expression systems

Methods: Crispr/CAS9 genome editing generated Scn2a^K1422E, Scn2a^E430A, Kcnb1^G379R and Kcnb1^R306C mutations on C57BL/6J (Jackson, #000064). Founder and N1 generations were sequenced for desired and off-target events by Sanger sequencing. Scn2a phenotype comparisons were made between WT and heterozygous littermates, while Kcnb1 comparisons included homozygous littermates. Transcript and protein expression were performed by RT-ddPCR and immunoblotting (n≥6/sex/genotype). General health, spontaneous seizures and survival were monitored in home cages for ≤6 months. Thresholds to flurothyl-induced seizures were assessed at ~10 weeks old (n≥12/sex/genotype). Two-channel headmounts (Pinnacle Technology) were implanted (n≥5/sex/genotype) and mice were continuously video-EEG monitored for 7-14 days. Data was acquired using Sirena software (Pinnacle) and analyzed offline with LabChart (ADInstruments) by a blinded observer.

Results: Scn2a^K1422E, Scn2a^E430A, Kcnb1^G379R and Kcnb1^R306C mutations were generated without off-targets events. Scn2a^K1422E and Kcnb1^R306C lines showed no difference in transcript or protein expression between littermates. The Kcnb1^G379R line revealed a genotype-dependent reduction in protein (p < 0.0001). Spontaneous seizures were observed in Scn2a^E430A, Kcnb1^G379R and Kcnb1^R306C home cages, and were occasionally followed by sudden death. Scn2a^K1422E, Kcnb1^G379R and Kcnb1^R306C lines demonstrated altered thresholds to flurothyl-induced seizures (p < 0.05). EEG from Scn2a and Kcnb1 mutants depicted generalized tonic-clonic seizures, regionalized non-convulsive seizures and epileptiform spiking, including spike-wave discharges and isolated sharp waves.

Conclusions: Scn2a^K1422E, Scn2a^E430A, Kcnb1^G379R and Kcnb1^R306C mouse models demonstrated altered neuronal excitability, manifested as observable spontaneous seizure activity and/or altered thresholds to induced seizures. Complete characterization of each mouse line will direct comparisons of channel dysfunction between in vitro and in vivo model systems. Furthermore, these models can serve as a useful platform for evaluating response to therapeutic interventions.

Funding: Please list any funding that was received in support of this abstract.: R01 NS053792 (JAK); R21 OD025330 (JAK); U54 NS108874-7711 (JAK).