A dual‐AAV approach restores fast exocytosis and partially rescues auditory function in deaf otoferlin knock‐out mice

Abstract Normal hearing and synaptic transmission at afferent auditory inner hair cell (IHC) synapses require otoferlin. Deafness DFNB9, caused by mutations in the OTOF gene encoding otoferlin, might be treated by transferring wild‐type otoferlin cDNA into IHCs, which is difficult due to the large size of this transgene. In this study, we generated two adeno‐associated viruses (AAVs), each containing half of the otoferlin cDNA. Co‐injecting these dual‐AAV2/6 half‐vectors into the cochleae of 6‐ to 7‐day‐old otoferlin knock‐out (Otof−/−) mice led to the expression of full‐length otoferlin in up to 50% of IHCs. In the cochlea, otoferlin was selectively expressed in auditory hair cells. Dual‐AAV transduction of Otof−/− IHCs fully restored fast exocytosis, while otoferlin‐dependent vesicle replenishment reached 35–50% of wild‐type levels. The loss of 40% of synaptic ribbons in these IHCs could not be prevented, indicating a role of otoferlin in early synapse maturation. Acoustic clicks evoked auditory brainstem responses with thresholds of 40–60 dB. Therefore, we propose that gene delivery mediated by dual‐AAV vectors might be suitable to treat deafness forms caused by mutations in large genes such as OTOF

Disruption of adaptor protein 2μ (AP‐2μ) in cochlear hair cells impairs vesicle reloading of synaptic release sites and hearing

Active zones (AZs) of inner hair cells (IHCs) indefatigably release hundreds of vesicles per second, requiring each release site to reload vesicles at tens per second. Here, we report that the endocytic adaptor protein 2μ (AP‐2μ) is required for release site replenishment and hearing. We show that hair cell‐specific disruption of AP‐2μ slows IHC exocytosis immediately after fusion of the readily releasable pool of vesicles, despite normal abundance of membrane‐proximal vesicles and intact endocytic membrane retrieval. Sound‐driven postsynaptic spiking was reduced in a use‐dependent manner, and the altered interspike interval statistics suggested a slowed reloading of release sites. Sustained strong stimulation led to accumulation of endosome‐like vacuoles, fewer clathrin‐coated endocytic intermediates, and vesicle depletion of the membrane‐distal synaptic ribbon in AP‐2μ‐deficient IHCs, indicating a further role of AP‐2μ in clathrin‐dependent vesicle reformation on a timescale of many seconds. Finally, we show that AP‐2 sorts its IHC‐cargo otoferlin. We propose that binding of AP‐2 to otoferlin facilitates replenishment of release sites, for example, via speeding AZ clearance of exocytosed material, in addition to a role of AP‐2 in synaptic vesicle reformation