Interneuron-specific dual-AAV gene replacement corrects epileptic phenotypes in mouse models of Dravet syndrome.

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 , which encodes the voltage-gated sodium channel (Na)1.1 alpha subunit and has been associated with inhibitory neuron dysfunction. Here, we generated a split-intein form of 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-. The split-intein vectors produced full-length Na1.1 protein, and functional sodium channels were recorded in HEK293 cells in vitro. Administration of dual DLX2.0- AAVs to wild-type mice produced full-length, reconstituted human protein by Western blot and telencephalic interneuron-specific and dose-dependent Na1.1 expression by immunohistochemistry. These vectors also conferred strong dose-dependent protection against postnatal mortality and seizures in and DS mouse models. Injection of single or dual DLX2.0- AAVs into wild-type mice did not result in increased mortality, weight loss, or gliosis as measured by immunohistochemistry. In contrast, expression of in all neurons driven by the human 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 gene replacement can rescue DS phenotypes in mouse models and suggest that it could be a therapeutic approach for patients with DS.