CRISPR/dCas9-based Scn1a gene activation in inhibitory neurons ameliorates epileptic and behavioral phenotypes of Dravet syndrome model mice.

Dravet syndrome is a severe infantile-onset epileptic encephalopathy which begins with febrile seizures and is caused by heterozygous loss-of-function mutations of the voltage-gated sodium channel gene SCN1A. We designed a CRISPR-based gene therapy for Scn1a-haplodeficient mice using multiple guide RNAs (gRNAs) in the promoter regions together with the nuclease-deficient Cas9 fused to transcription activators (dCas9-VPR) to trigger the transcription of SCN1A or Scn1a in vitro. We tested the effect of this strategy in vivo using an adeno-associated virus (AAV) mediated system targeting inhibitory neurons and investigating febrile seizures and behavioral parameters. In both the human and mouse genes multiple guide RNAs (gRNAs) in the upstream, rather than downstream, promoter region showed high and synergistic activities to increase the transcription of SCN1A or Scn1a in cultured cells. Intravenous injections of AAV particles containing the optimal combination of 4 gRNAs into transgenic mice with Scn1a-haplodeficiency and inhibitory neuron-specific expression of dCas9-VPR at four weeks of age increased Nav1.1 expression in parvalbumin-positive GABAergic neurons, ameliorated their febrile seizures and improved their behavioral impairments. Although the usage of transgenic mice and rather modest improvements in seizures and abnormal behaviors hamper direct clinical application, our results indicate that the upregulation of Scn1a expression in the inhibitory neurons can significantly improve the phenotypes, even when applied after the juvenile stages. Our findings also suggest that the decrease in Nav1.1 is directly involved in the symptoms seen in adults with Dravet syndrome and open a way to improve this condition.