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

G. Colasante, G. Lignani, S. Brusco, C. Di Berardino, J. Carpenter, S. Giannelli, N. Valassina, S. Bido, R. Ricci, V. Castoldi, S. Marenna, T. Church, L. Massimino, G. Morabito, F. Benfenati, S. Schorge, L. Leocani, D.M. Kullmann, V. Broccoli

Research output: Contribution to journalArticlepeer-review

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 Nav1.1 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. Nav1.1 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, 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 recovered 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 to DS and other disorders resulting from altered gene dosage. © 2019 The American Society of Gene and Cell Therapy Colasante et al. exploit an activatory CRISPR-targeting Scn1a gene promoter as a therapeutic strategy to rescue Scn1a haploinsufficiency in a mouse model of Dravet syndrome and restore physiological levels of its gene product, the Nav1.1 voltage-gated sodium channel. © 2019 The American Society of Gene and Cell Therapy
Original languageEnglish
Pages (from-to)235-253
JournalMol. Ther.
DOIs
Publication statusPublished - 2020

Keywords

  • activatory CRISPR
  • Dravet syndrome
  • epileptic encephalopathy
  • gene therapy
  • 4 aminobutyric acid receptor
  • Scn1a protein, mouse
  • sodium channel Nav1.1
  • action potential
  • animal
  • C57BL mouse
  • cytology
  • disease model
  • embryology
  • female
  • genetics
  • hippocampus
  • interneuron
  • metabolism
  • mouse
  • myoclonus epilepsy
  • procedures
  • seizure
  • transcription initiation
  • transgenic mouse
  • treatment outcome
  • tumor cell line
  • Action Potentials
  • Animals
  • Cell Line, Tumor
  • CRISPR-Associated Protein 9
  • Disease Models, Animal
  • Epilepsies, Myoclonic
  • Female
  • GABAergic Neurons
  • Genetic Therapy
  • Hippocampus
  • Interneurons
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • NAV1.1 Voltage-Gated Sodium Channel
  • Seizures
  • Transcriptional Activation
  • Treatment Outcome

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