De novo heterozygous variants in the neuronal STXBP1 gene cause severe, early-onset developmental and epileptic encephalopathy. Adeno-associated virus (AAV)-based gene replacement therapy offers the potential for a one-time, disease-modifying approach for STXBP1-related disorders. However, off-target overexpression in the liver and in the dorsal root ganglion (DRG) are known potential toxicities of AAV vectors. In addition, while loss of STXBP1 in GABAergic interneurons contributes to disease pathogenesis, typical gene therapy promoters do not express well in these cell populations. We engineered novel promoter cassettes to drive potent, selective STXBP1 expression across both excitatory and inhibitory neurons, and a 3' UTR regulatory element to detarget expression in DRG. Bilateral intracerebroventricular (ICV) injection of these promoter candidates achieved robust neuronal expression of STXBP1 and rescued key behavioral phenotypes in Stxbp1+/- haploinsufficient mice. In nonhuman primates, widespread vector biodistribution and transgene expression were observed in the central nervous system after unilateral ICV administration of AAV9-STXBP1 vectors. The vectors were well tolerated, and addition of the detargeting regulatory element significantly reduced expression in DRG, while ameliorating histopathologic effects and functional nerve conduction alterations. Taken together, these data support the feasibility of a one-time AAV-based therapeutic approach for STXBP1-related disorders.