Interneuron-specific dual-AAV <i>SCN1A</i> gene replacement corrects epileptic phenotypes in mouse models of Dravet syndrome

Abstract

Dravet syndrome (DS) is a severe developmental epileptic encephalopathy marked by treatment-resistant seizures, developmental delay, intellectual disability, motor deficits, and a 10 to 20% rate of premature death. Most patients with DS harbor loss-of-function mutations in one copy of <i>SCN1A</i>, which encodes the voltage-gated sodium channel (Na_V)1.1 alpha subunit and has been associated with inhibitory neuron dysfunction. Here, we generated a split-intein form of <i>SCN1A</i> and used a dual-vector delivery approach to circumvent adeno-associated virus (AAV) packaging limitations. In addition, we applied previously developed enhancer technology to produce an interneuron-specific gene replacement therapy for DS, called DLX2.0-<i>SCN1A</i>. The split-intein <i>SCN1A</i> vectors produced full-length Na_V1.1 protein, and functional sodium channels were recorded in HEK293 cells in vitro. Administration of dual DLX2.0-<i>SCN1A</i> AAVs to wild-type mice produced full-length, reconstituted human protein by Western blot and telencephalic interneuron-specific and dose-dependent Na_V1.1 expression by immunohistochemistry. These vectors also conferred strong dose-dependent protection against postnatal mortality and seizures in <i>Scn1a^fl/+;Meox2-Cre</i> and <i>Scn1a^+/R613X</i> DS mouse models. Injection of single or dual DLX2.0-<i>SCN1A</i> AAVs into wild-type mice did not result in increased mortality, weight loss, or gliosis as measured by immunohistochemistry. In contrast, expression of <i>SCN1A</i> in all neurons driven by the human <i>SYNAPSIN I</i> promoter caused an adverse effect marked by increased mortality in the preweaning period, before disease onset. These findings demonstrate proof of concept that interneuron-specific AAV-mediated <i>SCN1A</i> gene replacement can rescue DS phenotypes in mouse models and suggest that it could be a therapeutic approach for patients with DS.