Deafness-causing deficiencies in () have been addressed preclinically using dual adeno-associated virus (AAV)-based approaches. However, timing of transduction, recombination of mRNA, and protein expression with dual hybrid AAV methods methods have not previously been characterized. Here, we have established an assay to determine the kinetics of dual-AAV mediated expression of in hair cells of the mouse utricle. We utilized two different recombinant vectors that comprise DB-OTO, one containing the 5' portion of under the control of the hair cell-specific promoter, and the other the 3' portion of . We explored specificity of the promoter in hair cells of the mouse utricle, established dose response characteristics of DB-OTO in an OTOF-deficient mouse model, and demonstrated tolerability of AAV1 in utricular hair cells. Furthermore, we established deviations from a one-to-one ratio of 5' to 3' vectors with little impact on recombined . Finally, we established a plateau in quantity of recombined mRNA and protein expression by 14 to 21?days with comparable recovery timing to that model. These findings demonstrate the utility of an model system for exploring expression kinetics and establish and recovery timing of dual AAV-mediated expression.