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.