Abstracts

Dravet syndrome (DS) is a devastating developmental and epileptic encephalopathy marked by treatment- resistant seizures and a high rate of premature death.  In over 80% of patients with DS, the disease is caused by heterozygous loss-of-function mutations in SCN1A, the gene that encodes the Na_V 1.1 channels. Such mutations lead to impaired excitability specifically in GABAergic interneurons and key DS phenotypes are recapitulated when these mutations are specifically introduced into GABAergic interneurons in mouse models. These observations suggest that interneurons constitute a promising target for precision gene therapy in DS.

We designed a novel strategy for AAV-mediated gene replacement therapy using segmental gene-delivery, cell-type specific enhancers, and protein trans-splicing techniques to (1) circumvent AAV cargo capacity limitations, (2) specifically target forebrain GABAergic interneurons, and (3) reconstitute the full therapeutic gene product. SCN1A gene was split in half and DNA constructs of the two gene segments were built and expressed in HEK 293 cells. Western blot studies were conducted to evaluate the reconstitution of full length Na_V 1.1 channels and patch-clamp electrophysiology to functionally characterize the expressed channels. Constructs were packaged into AAV vectors and administered by intracerebroventricular (ICV) injections in DS mice carrying a heterozygous knockout (KO) allele of Scn1a at postnatal day zero to three. Immunohistochemistry was performed to assess transgene expression. Sudden unexpected death in epilepsy (SUDEP) and thermal seizure susceptibility were examined in treated DS mice.

The split-SCN1A genes expressed and produced Na_V 1.1 polypeptides that reconstituted full-length proteins and yielded robust sodium currents comparable to unsplit gene in HEK293 cells. When administered in DS mice, the dual AAV vectors led to a strong cell-type specific and dose-dependent expression of the therapeutic cargo in GABAergic interneurons of different forebrain regions.  Remarkably, these vectors conferred a strong protection against SUDEP and thermally induced seizures in a dose-dependent manner. These findings were also observed at a second independent research site in a second model of DS harboring a nonsense Scn1a mutation, thus providing additional evidence of treatment potency. Vectors with pan-neuronal expression of SCN1A led to weaker protection from DS symptoms and showed major side effects including preweaning lethality. Together, these findings suggest that cell class specific gene replacement therapy might not only be efficacious but also safer than pan-neuronal strategies.

Together these findings provide a strong proof-of-concept that interneuron specific gene therapy mediated by dual AAV gene delivery of SNC1A could serve as an effective precision therapy for DS.