Abstracts

This abstract has been invited to present during the Basic Science Poster Highlights poster session.

Rationale: Dravet syndrome (DS) is caused by loss-of-function mutations in SCN1A with characteristic febrile seizures beginning in infancy. Genetic therapies currently under development have been most successful when initiated prior to seizure onset, however treatment at early timepoints in human patients may not be possible in all cases. DS mice are a reliable mimic of human DS progression, and we have developed a novel mouse model utilizing cre-lox mouse genetics to reliably correct Scn1a gene expression at any age point. These experiments aim to validate our new mouse model and induce gene recovery embryonically and in adulthood. 

Methods: Our novel recovery mouse model of DS (Scn1a^GTS/+) was generated using a gene-trap strategy (GTS) to replace the intronic region between exons 24 and 25 of Scn1a with a loxP flanked stop cassette. Following cre-recombinase expression the stop cassette is excised and normal gene expression restored (Scn1a^RES/+) which can be visualized as a shift in gene product size via PCR. Embryonic gene restoration was made by crossing Scn1a^GTS/+ mice with  an early embryonic expressing Cre-recombinase animal (Meox-Cre) and the resulting animals were followed for survival and tested for thermally evoked seizures in adulthood. A globally expressing cre-recombinase coupled to a mutated estrogen receptor that is activated following binding of tamoxifen (ERT2-cre) was crossed with our Scn1a^GTS/+ mice to achieve timed recovery. Tamoxifen (TAM) or vehicle (VEH) was administered at > P90 in ERT2-cre: Scn1a^GTS/+ mice (TAM, N=9; VEH, N=4). Two weeks after TAM treatment, thermal sensitivity to seizure was assayed by elevating each animal’s core body temperature in 0.5°C steps until 41.0°C was achieved or the animal experienced a seizure.

Results: Scn1a^GTS/+ mice have impaired survival (premature death rate of 39.6% before P90, 21/53, Scn1a ^GTS/+) and 19/19 animals had thermally evoked seizures (38.6 ± 0.21°C) at temperatures comparable to global DS mice. Animals with embryonic gene restoration (Scn1a^RES/+) were phenotypically indistinguishable from wildtype (Scn1a^+/+) littermates, with normal survival (Scn1a^RES/+, N=32; Scn1a^+/+, N=63) and insensitivity to thermally evoked seizures up to 41°C (Scn1a^RES/+, N=16; Scn1a^+/+, N=24). Following Tamoxifen treatment at P90, 2/9 treated ERT2-Cre:Scn1a^RES/+animals had seizures prior to 41°C (40.75 ± 0.15 C), as compared to 4/4 vehicle treated animals (39.1 ± 0.46 C) (p= 0.04, t-test).

Conclusions: Cre mediated gene rearrangement in our novel Scn1a^GTS/+ mouse efficiently restores impaired Scn1a expression. Adult (P90) restoration significantly increases the threshold for seizure initiation via thermal induction. These results validate our novel mouse model and provide a platform on which to determine the optimal timing for gene recovery and the contributions of seizures and gene mutation to comorbidity development.  

Funding: This work was supported by grants from the National Institutes of Health and the Dravet Syndrome Foundation.