Neuron-Specific WWOX Gene Therapy Produces Dose-Dependent, Durable Rescue in a Model of WWOX-Related Epileptic Encephalopathy
Biallelic loss-of-function mutations in WWOX cause a spectrum of neurodevelopmental disorders, including the severe, early-onset WOREE syndrome, frequently associated with intractable epilepsy and premature mortality, and the milder SCAR12, characterized by subtler neurological manifestations. While neuronal replacement of WWOX has emerged as a potential therapeutic strategy, the parameters required for safe, durable, and clinically translatable gene delivery remain undefined. Here, we systematically delineate the determinants of effective WWOX gene therapy by evaluating promoter selection, cellular targeting, vector configuration, dose, and developmental timing in a severe Wwox-null mouse model. Neuron-restricted expression driven by the human Synapsin I promoter uniquely enabled sustained phenotypic correction, whereas ubiquitous or oligodendrocyte-restricted expression failed to confer durable benefit. To better regulate transgene expression, we generated a vector lacking the WPRE element, enabling dose calibration within a clinically relevant range. Comparative dose-response analyses identified an optimal therapeutic dose of AAV9-hSynI-WWOX that produced robust, dose-dependent rescue of survival and growth. Moreover, the rescued mice displayed glucose, behavioral function and fertility indistinguishable from WT mice, accompanied by long-term restoration of WWOX DNA, transcript, and protein across central and peripheral neural tissues without detectable hepatic expression. Neuronal WWOX reconstitution promoted widespread myelination and attenuated neuroinflammatory responses, including astrogliosis and microglial activation to levels indistinguishable from WT. Continuous electrocorticographic monitoring uncovered early postnatal neuronal hyperexcitability in Wwox-null mice, which was effectively suppressed by therapeutic WWOX delivery. Finally, our data define an early postnatal therapeutic window in Wwox-null mice, showing that treatment initiated between postnatal days 1 and 5 supports durable rescue. Together, these findings define a rigorously optimized, neuron-targeted AAV9-WWOX gene therapy framework and establish critical design and timing principles for translational treatment of WWOX-associated developmental and epileptic encephalopathies.