Age outweighs route for AAV-PHP.eB transgene distribution via inner ear delivery.

Adeno-associated virus (AAV)-based gene therapy has demonstrated transformative potential in treating hereditary hearing loss (HHL). Investigating factors that influence AAV transduction in target and non-target organs is critical for improving therapeutic precision and safety. Given the postnatal maturation of the murine inner ear and compartmentalized structure of the cochlea, we evaluated whether developmental age or delivery routes affect AAV-PHP.eB tropism in cochlear hair cells (HCs) and adjacent brain regions. Following round window membrane (RWM), posterior semicircular canal (PSCC), or utricle delivery, neonatal mice showed robust OHC transduction (vs. minimal in juveniles), while IHC transduction remained consistently high across age groups. Across all three delivery routes, brain AAV transduction was significantly higher in neonates than in juveniles. Despite this, AAV genome copies were more highly enriched in the injected inner ear than in the brain at both ages. Dye-tracing experiments demonstrated distinct spatial distribution patterns following three inner ear delivery routes, with the cochlear aqueduct (CA) identified as the primary conduit for intracranial spread post-injection. These findings provide guidance for the design of studies in mouse models of deafness, particularly with respect to cochlear hair cell subtype targeting, therapeutic timing, and safety assessment of AAV-PHP.eB-based therapies for hearing loss.