dCas9-Based Scn1a Gene Activation Restores Inhibitory Interneuron Excitability and Attenuates Seizures in Dravet Syndrome Mice

Abstract

Dravet syndrome (DS) is a severe epileptic encephalopathy caused mainly by heterozygous loss-of-function mutations of the SCN1A gene, indicating haploinsufficiency as the pathogenic mechanism. Here, we tested whether catalytically dead Cas9 (dCas9)-mediated Scn1a gene activation can rescue Scn1a haploinsufficiency in a mouse DS model and restore physiological levels of its gene product, the Na v 1.1 voltage-gated sodium channel. We screened single guide RNAs (sgRNAs) for their ability to stimulate Scn1a transcription in association with the dCas9 activation system. We identified a specific sgRNA that increases Scn1a gene expression levels in cell lines and primary neurons with high specificity. Na v 1.1 protein levels were augmented, as was the ability of wild-type immature GABAergic interneurons to fire action potentials. A similar enhancement of Scn1a transcription was achieved in mature DS interneurons, thus rescuing their ability to fire. To test the therapeutic potential of this approach, we delivered the Scn1a-dCas9 activation system to DS pups using adeno-associated viruses. Parvalbumin interneurons recover their firing ability and febrile seizures were significantly attenuated. Our results pave the way for exploiting dCas9-based gene activation as an effective and targeted approach in DS and other disorders resulting from altered gene dosage.

High-Capacity Adenoviral Vectors: Expanding the Scope of Gene Therapy

“…Therefore, alternative versions of CRISPR/Cas9 with nickase instead of nuclease activity are being developed [204]. Other approaches use the DNA binding domains of ZFN, TALEN, and CRISPR/Cas9 systems fused with other polypeptides to obtain site-specific transcriptional activators or repressors [205,206]. However, they often require persistent expression of the transcriptional modifier to maintain the effect.…”

Dead Cas Systems: Types, Principles, and Applications

“…CRISPRa can help treat haploinsufficiency-induced diseases by overexpressing an intact copy of the insufficient gene. This method has been utilized in vivo to treat obesity [80] and Dravet syndrome [81].…”

Gene Therapy in Models of Severe Epilepsy due to Sodium Channelopathy

“…As the genetic mechanism of Dravet syndrome is haploinsufficiency with heterozygous loss of function of the SCN1A gene, increasing the expression or activity of Nav1.1 subunit-containing Na + channels is a logical approach to therapy. Colasante et al 1 used a CRISPR/dCas9 system to screen for guide RNAs that bind to specific regions of the mouse Scn1a promoter and that increase Scn1a gene expression. Briefly, CRISPR/Cas9 is the gene-editing technique whereby DNA sequences called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) facilitate targeting and subsequent destruction of DNA by the Cas (CRISPR-associated) proteins.…”