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 Kir_2.1 trafficking sequence (TS_Kir2.1) 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-TS_Kir2.1 to be a promising candidate for clinical translation of optogenetic neurostimulation such as by future optical cochlear implants.