CRISPR-mediated Stxbp1 gene activation restores Munc18-1 levels in Stxbp1-haploinsufficient mice

Abstract

Mutations in the syntaxin-binding protein 1 (STXBP1) gene, which encodes the presynaptic protein Munc18-1, cause a spectrum of severe epileptic encephalopathies and neurodevelopmental disorders, including Ohtahara syndrome, for which no curative treatment is currently available. Because the disease pathomechanism is thought to be driven by haploinsufficiency, restoring expression of the wild-type allele to physiological levels could provide therapeutic benefit. Here, we evaluated CRISPR-mediated transcriptional activation (CRISPR-ON), based on a dCas9-VPR transcriptional activator, as a strategy to upregulate endogenous Stxbp1 expression in a Stxbp1-haploinsufficient (Stxbp1+/-) mouse model. Screening of guide RNAs (gRNAs) targeting the Stxbp1 promoter in Neuro2A cells identified a multiplexed four-gRNA cassette that elevated Stxbp1 mRNA approximately six-fold. AAV-PHP.eB vectors co-expressing this 4xgRNA cassette and Cre recombinase under the EF1a promoter were administered intracerebroventricularly to neonatal Stxbp1+/-/dCas9-VPRfl/+ mice. CRISPR-ON treatment raised brain Stxbp1 mRNA in Stxbp1+/- mice to 1.5-fold the level of wild-type controls, whereas Munc18-1 protein was restored to the wild-type level. In small exploratory cohorts, spike-wave discharge frequency and attack counts tended to be lower in CRISPR-ON-treated than in untreated Stxbp1+/- mice; the discharge data, from two animals per group, were not tested statistically, and the difference in attack counts was not significant, so efficacy cannot be judged from the present data. Locomotor activity was normal in untreated mutants and unaffected by treatment; reduced body weight was not rescued. Together, these findings establish that CRISPR-ON can correct Stxbp1/Munc18-1 expression in vivo, whereas its effect on disease phenotypes remains to be determined.