Neuron-specific WWOX gene therapy produces dose-dependent, durable rescue in a model of WWOX-related epileptic encephalopathy

Bi-allelic WWOX loss-of-function mutations underlie a spectrum of developmental and epileptic encephalopathies, including the severe WOREE syndrome and the milder SCAR12, for which no effective therapies exist. Gene replacement represents a compelling strategy; however, critical parameters for clinically translatable delivery remain undefined. Here, we establish a rationally optimized WWOX gene therapy framework using a severe Wwox-null mouse model. Systematic evaluation of promoter selection, cellular targeting, and vector configuration, dose, and timing identified neuron-restricted expression driven by the human synapsin I promoter as the most effective approach, yielding robust and durable phenotypic rescue compared to non-specific or oligodendrocyte-directed strategies. Removal of the WPRE element enabled controlled transgene expression and facilitated dose calibration within a clinically relevant range. An optimal AAV9-hSynI-WWOX dose restored survival, growth, metabolic function, behavior, and fertility to near wild-type levels. Therapeutic efficacy was associated with sustained reconstitution of WWOX across central and peripheral neural tissues, absence of off-target hepatic expression, enhanced myelination, and reduced neuroinflammation. Early neuronal hyperexcitability was suppressed following treatment. Importantly, efficacy was shown in an early postnatal therapeutic window (P1–P5), supporting durable rescue. Collectively, these findings define key design and dosing principles for translation of WWOX gene therapy to patients with developmental and epileptic encephalopathies.