dCas9-based Scn1a gene activation restores inhibitory interneuron excitability and restrains epileptic crises in Dravet syndrome mice

Dravet syndrome (DS) is a severe epileptic encephalopathy caused by heterozygous loss-of-function mutations in the SCN1A gene, indicating a haploinsufficient genetic mechanism underlining this pathology. Here, we tested whether dCas9-mediated Scn1a gene activation could rescue Scn1a haploinsufficiency and restore physiological levels of its gene product, the Nav1.1 voltage-gated sodium channel. We screeened sgRNAs for their ability to stimulate Scn1a gene transcription in association with the dCas9 activation system. Interestingly, we identified one single sgRNA able to significantly increase Scn1a gene expression levels in cell lines as well as in primary neurons, with high specificity. Accordingly, levels of Nav1.1 protein were sufficiently augmented to potentiate firing ability of wild-type immature GABAergic interneurons. A similar effect in activating the Scn1a transcription was elicited in Dravet GABAergic interneurons rescuing their dysfunctional properties. To determine whether this approach could have therapeutic effect, we packaged adeno-associated viruses with the Scn1a-dCas9 activation system and showed their ability to ameliorate the febrile epileptic crises in DS mice. Our results pave the way for exploiting the dCas9-based gene activation as an effective and targeted approach to DS and other similar disorders resulting from altered gene dosage. 3 neuronal cultures overexpressing Scn1a sgRNA, and 3 control cultures