Identification Of a Therapeutic Threshold For AAV-STXBP1 Gene Therapy in a Rodent Model of STXBP1 Developmental & Epileptic Encephalopathy

STXBP1-related developmental and epileptic encephalopathy (STXBP1-DEE) is a debilitating genetic epilepsy disorder caused by heterozygous loss of function mutations in the STXBP1 gene. Gene supplementation therapy using adeno-associated virus (AAV) technology is an attractive approach to restore functional STXBP1 expression in the central nervous system (CNS) and improve STXBP1-DEE symptoms. Currently, there is no clear clinical data or human evidence defining a therapeutic threshold of AAV-mediated STXBP1 expression. Here, we describe an approach to define this therapeutic threshold using Stxbp1+/- mice and correlating neuronal transduction with phenotypic improvement. Our AAV-STXBP1 vectors dose-dependently rescued disease-associated phenotypes, with ≥44% cortical neuronal transduction needed for phenotypic improvement, setting a potential therapeutic threshold. Long term analysis demonstrated that STXBP1 transgene expression in the brain is durable up to one-year. Finally, a mass spectrometry assay capable of simultaneous detection of STXBP1 and syntaxin-1 (STX1) in human cerebrospinal fluid (CSF) is presented that may enable translation into a clinical grade assay. Altogether, our results identify a potential therapeutic threshold for gene therapy expression in key brain regions for STXBP1-DEE and advance the possible utility of STXBP1 and STX1 in CSF as key protein biomarkers, thereby supporting further development of AAV-based gene supplementation strategies for this disorder.