Epilepsy, characterized by recurrent spontaneous seizures, affects over 50 million people worldwide and is one of the most common neurological disorders. Heterozygous loss-of-function mutations in the voltage-gated sodium channel ? subunit gene, SCN1A (encoding the protein Nav1.1), are associated with multiple forms of treatment-resistant epilepsy, including Dravet syndrome (DS), a catastrophic, early-life encephalopathy. As 70- 80% of cases of DS can be attributed to a reduction in functional Nav1.1, strategies that can increase SCN1A transcription from the wild-type allele are predicted to improve patient outcomes. Despite the important role of SCN1A in epilepsy and, more broadly, in the normal regulation of neuronal excitability, little is currently known about the machinery underlying the regulation of SCN1A transcription. Therefore, we are limited in our ability to develop strategies to increase SCN1A transcription. We identified three predicted Functional Genomic Elements (FGEs) within the SCN1A locus that significantly increased reporter activity in a dual luciferase assay: FGE2, FGE17, and FGE23.
In Aim 1, we will test the hypothesis that FGE2, FGE17, and FGE23 endogenously act as transcriptional enhancer elements for SCN1A. We will use the Synergistic Activation Mediator (dCas9SAM) system to localize transcriptional activators to these FGEs in neuronal cell culture. Previous studies have shown that localizing transcriptional activators to gene promoters or enhancers can elicit an increase in gene expression, making dCas9SAM a powerful tool for evaluating putative regulatory elements. Additionally, we will perform ATAC-seq on GABAergic interneurons, which preferentially express SCN1A, in an effort to identify additional FGEs that contribute to SCN1A transcriptional regulation. Our preliminary data also show that the minor allele of the epilepsy-associated SNP, rs6732655, located in SCN1A intron 16, significantly decreases reporter activity in a dual luciferase assay, compared to the major allele.
In Aim 2, we will test the hypothesis that the rs6732655 minor allele introduces a silencer element, thereby reducing SCN1A expression. We will examine SNP allele-dependent protein-binding in order to identify candidate factors that bind to the minor allele. We will also knock-in the minor allele of this SNP to SH-SY5Y human neuroblastoma cells to evaluate the endogenous impact on SCN1A expression. Our long-term goal is to translate these findings into clinically-relevant strategies for increasing Nav1.1 levels, thereby providing new therapeutic options.
Over 50 million people worldwide are diagnosed with epilepsy, making it one of the most common neurological disorders. We study Dravet syndrome, a severe form of treatment-resistant childhood epilepsy, which is caused by mutations in the gene SCN1A that significantly reduce functional levels of the protein Nav1.1. This project will attempt to characterize several elements that regulate SCN1A transcription, a critical step in ultimately designing approaches to increase SCN1A expression (and Nav1.1 levels) as a therapy for patients with Dravet syndrome.
