AUTHOR=Sellon Jonathan B. , So Kathy S. , D'Arcangelo Andrew , Cancelarich Sarah , Drummond Meghan C. , Slade Peter G. , Pan Ning , Gibson Tyler M. , Yang Tian , Burns Joseph C. , Palermo Adam T. , Becker Lars 

TITLE=Recovery kinetics of dual AAV-mediated human otoferlin expression

JOURNAL=Frontiers in Molecular Neuroscience

VOLUME=17

YEAR=2024

URL=https://www.frontiersin.org/journals/molecular-neuroscience/articles/10.3389/fnmol.2024.1376128

DOI=10.3389/fnmol.2024.1376128

ISSN=1662-5099

ABSTRACT=<p>Deafness-causing deficiencies in <italic>otoferlin</italic> (<italic>OTOF</italic>) 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 <italic>ex vivo</italic> assay to determine the kinetics of dual-AAV mediated expression of <italic>OTOF</italic> in hair cells of the mouse utricle. We utilized two different recombinant vectors that comprise DB-OTO, one containing the 5′ portion of <italic>OTOF</italic> under the control of the hair cell-specific <italic>Myo15</italic> promoter, and the other the 3′ portion of <italic>OTOF</italic>. We explored specificity of the <italic>Myo15</italic> promoter in hair cells of the mouse utricle, established dose response characteristics of DB-OTO <italic>ex vivo</italic> 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 <italic>OTOF</italic>. Finally, we established a plateau in quantity of recombined <italic>OTOF</italic> mRNA and protein expression by 14 to 21 days <italic>ex vivo</italic> with comparable recovery timing to that <italic>in vivo</italic> model. These findings demonstrate the utility of an <italic>ex vivo</italic> model system for exploring expression kinetics and establish <italic>in vivo</italic> and <italic>ex vivo</italic> recovery timing of dual AAV-mediated <italic>OTOF</italic> expression.</p>