CRISPRa-Mediated Upregulation of scn1laa During Early Development Causes Epileptiform Activity and dCas9-Associated Toxicity.

Dravet syndrome (DS) is a monogenic epileptic encephalopathy caused by loss-of-function mutations in the voltage-gated sodium channel (VGSC) gene SCN1A. DS has an age of onset within the first year of life and severe disease prognosis. In the past years, it has been shown that upregulation of endogenous SCN1A can be beneficial in animal models for DS, but a complete rescue was not observed. We hypothesized that upregulation during early development that precedes onset of first symptoms might improve disease outcome. To test this hypothesis, we first evaluated the CRISPR activating method for early upregulation of voltage gated sodium channels during early development. We injected CRISPRa components, which target the proximal or distal promoter region of the VGSC gene scn1Laa in the yolk of one-cell stage zebrafish embryos. The effect of both dCas9-VPR and dCas9-VP64 was evaluated. Both CRISPRa fusions showed toxicity in the majority of embryos, with or without guide RNAs. The few embryos that survived developed normally, and dCas9-VPR induces an upregulation of scn1Laa mRNA until 24 hours after fertilization. At 5 days post fertilization, CRISPRa-injected embryos showed an epileptic phenotype, including locomotor burst movements, hyperactivity, and epileptiform activity originating from the brain. In addition to previously published scn1Laa and scn1Lab loss-of-function models, we conclude that gain of scn1Laa function can have an equally severe phenotype. Upregulation of scn1Laa in the current zebrafish model for DS, scn1Lab-KO, aggravated the disease phenotype, highlighting that early-stage upregulation using CRISPRa can lead to both toxicity and a worsening of the disease phenotype.

NaV1.1 and NaV1.6 selective compounds reduce the behavior phenotype and epileptiform activity in a novel zebrafish model for Dravet Syndrome.

Dravet syndrome is caused by dominant loss-of-function mutations in SCN1A which cause reduced activity of Nav1.1 leading to lack of neuronal inhibition. On the other hand, gain-of-function mutations in SCN8A can lead to a severe epileptic encephalopathy subtype by over activating NaV1.6 channels. These observations suggest that Nav1.1 and Nav1.6 represent two opposing sides of the neuronal balance between inhibition and activation. Here, we hypothesize that Dravet syndrome may be treated by either enhancing Nav1.1 or reducing Nav1.6 activity. To test this hypothesis we generated and characterized a novel DS zebrafish model and tested new compounds that selectively activate or inhibit the human NaV1.1 or NaV1.6 channel respectively. We used CRISPR/Cas9 to generate two separate Scn1Lab knockout lines as an alternative to previous zebrafish models generated by random mutagenesis or morpholino oligomers. Using an optimized locomotor assay, spontaneous burst movements were detected that were unique to Scn1Lab knockouts and disappear when introducing human SCN1A mRNA. Besides the behavioral phenotype, Scn1Lab knockouts show sudden, electrical discharges in the brain that indicate epileptic seizures in zebrafish. Scn1Lab knockouts showed increased sensitivity to the GABA antagonist pentylenetetrazole and a reduction in whole organism GABA levels. Drug screenings further validated a Dravet syndrome phenotype. We tested the NaV1.1 activator AA43279 and two novel NaV1.6 inhibitors MV1369 and MV1312 in the Scn1Lab knockouts. Both type of compounds significantly reduced the number of spontaneous burst movements and seizure activity. Our results show that selective inhibition of NaV1.6 could be just as efficient as selective activation of NaV1.1 and these approaches could prove to be novel potential treatment strategies for Dravet syndrome and other (genetic) epilepsies. Compounds tested in zebrafish however, should always be further validated in other model systems for efficacy in mammals and to screen for potential side effects.