Hearing loss is the most common sensory disorder worldwide. Approximately 15% of American adults report trouble hearing. Prevalence of hearing loss increases from 2% (age 45-54) to 50% (over age 75). Between 2 and 3 per 1000 live births present with hearing loss in one or both ears; 50% of hearing loss at birth is genetic in etiology. While the first cochlear gene therapy for hearing loss is underway, early results, as well as data from preclinical models, indicate a need for improved gene delivery vehicles for safe and efficient targeting of relevant cell types in the cochlea. In particular, outer hair cells (OHC) have been a challenging target for AAV. OHCs are important for auditory sensitivity and frequency selectivity, key parameters for complex and sophisticated hearing function in humans.Here, we evaluated several common AAV serotypes as well as Anc80L65, an in silico designed AAV based on evolutionary modeling, for its ability to target the neurosensory cells in the inner ear, particularly inner (IHC) and outer hair cells. Cochlear explants of both CBA/CaJ and C57Bl/6 mice were exposed to equal doses of iodixonal purified preparations of AAV1,2,6,8,9, and Anc80L65 that encoded eGFP. After 48h, explants were evaluated either immediately or after an additional 5 days of culture. In both animal model explants, Anc80L65 demonstrated highly efficient IHC and OHC transduction (60-100% at 48h, and 100% at 48h+5d). AAV2 in this setting was also efficient, although to a lesser extent. Most serotypes targeted supporting cells and the spiral limbus, to a moderate extent, with AAV8 being least efficient. GFP expression was present from apex to base, with moderately increased targeting for basal hair cells.Next, we tested Anc80L65 in vivo using a clinically relevant round window injection approach. C57Bl/6 mice received 1µl injection of vector at p0-p1 and cochlea were dissected at p6. Results demonstrate transduction from base to apex of IHC, OHC, and supporting cells at rates of close to 100% for IHC, and ranging from approximately 50-100% of OHCs, although at lesser fluorescent intensity. Animals injected at p1 retained similar levels of GFP hair cell positivity at p30 day of sacrifice. Animals in our study were monitored for function of single cells electrophysiogically, hearing function by Auditory Brainstem Response and Distortion Product Otoacoustic Emission measurements, ectopic expression, and immunological parameters. Ongoing studies are evaluating the transduction properties at later times of injection and disease models of hearing loss.In conclusion, Anc80L65's unique ability to target IHC as well as OHC following RW injection is highly desirable to restore higher level hearing function in genetic forms of deafness through gene therapy.