Abstracts

Rationale: Adenoassociated viral (AAV) vectors have emerged as a promising gene therapy modality with transformative clinical potential in severe pediatric neurological disease. AAV-based gene therapy is a one-time treatment that has the potential to provide lasting benefit for genetic epilepsies by targeting the underlying disease mechanism, thus altering the disease course not only to reduce seizures, but also to improve developmental outcomes. However, the complex molecular underpinnings of many genetic epilepsies necessitate the development of new tools to target AAV to specific neuronal subtypes and, in some cases, modulate the expression of large, complex genes. One such example is the SCN1A gene, which is associated with multiple seizure-related disorders, including Dravet syndrome (DS). Loss-of-function mutations in SCN1A result in a 50% reduction in NaV1.1 protein function and a dramatic impairment in GABAergic transmission. Therefore, a therapy that restores SCN1A expression and selectively targets GABAergic interneurons is highly desirable.  Methods: We propose a modified gene therapy approach that addresses two limitations of standard gene replacement therapy for the treatment of SCN1A+ epilepsy. Traditional gene therapy technology drives constitutive expression of a delivered gene across all cell types infected, and AAV has packaging constraints that prevent the delivery of large transgenes such as SCN1A. To address these limitations, we are proposing an AAV-based gene therapy approach capable of (1) driving cell-type selective expression within GABAergic inhibitory interneurons and (2) upregulating endogenous SCN1A- expression. The potential benefits of this innovative treatment on clinical and developmental outcomes can be examined via standardized longitudinal assessments. Results: Our approach leverages human regulatory sequences to target vector expression to GABAergic interneurons and to upregulate endogenous SCN1A expression levels. Gene expression is driven by a GABA-selective regulatory element (REGABA) that was derived from a combination of human promoter and enhancer sequences. REGABA has demonstrated high selectivity for GABAergic inhibitory interneurons in mouse and nonhuman primate central nervous systems. For endogenous SCN1A upregulation, we utilized an engineered transcription factor (eTFSCN1A) that recognizes a highly conserved noncoding regulatory sequence upstream of the SCN1A locus and promotes increased SCN1A transcription. By incorporating these elements into a clinically validated AAV capsid, we successfully generated a viral gene therapy vector that moves beyond the technical limitations of standard gene replacement. Furthermore, in a validated mouse model of DS, this engineered viral vector has been shown to dramatically reduce hyperthermic and spontaneous seizures and to rescue mice from early mortality; these results will be presented separately at this congress.  Conclusions: Our gene regulation approach permits the selective expression of genes in targeted cell types and circumvents the delivery of large transgenes. By overcoming these challenges, our approach offers the potential to improve clinical outcomes for SCN1A+ epilepsy across multiple domains, including seizures; cognitive, behavioral, and motor function development; and quality of life. We propose that gene regulation can shift the treatment paradigm of SCN1A+ epilepsy from symptomatic management toward lasting disease modification. Funding: This work was supported by Encoded Therapeutics, Inc.