Channelrhodopsin fluorescent tag replacement for clinical translation of optogenetic hearing restoration

Sensory restoration by optogenetic neurostimulation provides a promising future alternative to current electrical stimulation approaches. So far, channelrhodopsins (ChRs) typically contain a C-terminal fluorescent protein (FP) tag for visualization that potentially poses an additional risk for clinical translation. Previous work indicated a reduction of optogenetic stimulation efficacy upon FP removal. Here, we further optimized the fast-gating, red-light-activated ChR f-Chrimson to achieve efficient optogenetic stimulation in the absence of the C-terminal FP. Upon FP removal, we observed a massive amplitude reduction of photocurrents in transfected cells in vitro and of optogenetically evoked activity of the adeno-associated virus (AAV) vector-transduced auditory nerve in mice in vivo. Increasing the AAV vector dose restored optogenetically evoked auditory nerve activity but was confounded by neural loss. Of various C-terminal modifications, we found the replacement of the FP by the Kir2.1 trafficking sequence (TSKir2.1) to best restore both photocurrents and optogenetically evoked auditory nerve activity with only mild neural loss few months after dosing. In conclusion, we consider f-Chrimson-TSKir2.1 to be a promising candidate for clinical translation of optogenetic neurostimulation such as by future optical cochlear implants.