Direct functional protein delivery with a peptide into neonatal and adult mammalian inner ear in vivo

Abstract The aim of this study was to study an AMP, aurein 1.2, which substantially increased protein delivery directly into multiple mammalian inner ear cell types in vivo. Different concentrations of aurein 1.2 with superpositively charged GFP (+36-GFP) protein fused with Cre recombinase were delivered to P1-2 and adult cochleae of Cre reporter transgenic mice with various delivery methods. By cochleostomy at different concentrations of aurein 1.2-+36-GFP (1 μM, 5 μM, 22.5 μM, and 50 μM respectively), the tdTomato expression was observed in OHCs (20.77%, 23.02%, 76.36% and 92.47%, respectively), IHCs (14.90%, 44.50%, 89.59% and 96.13%, respectively) in the cochlea. The optimal concentration was 22.5 μM with the highest transfection efficiency and the lowest cytotoxicity. Wide-spread tdT signals were detected in the cochlear supporting cells, utricular supporting cells, auditory nerve and spiral ligament in neonatal and adult mice. Compared to cochleostomy, injection through the RWM also produced highly efficient tdT+ labeled cells with less cell loss. In summary, the peptide aurein 1.2 fused with +36-GFP dramatically expanded the target cells with increased efficiency in direct protein delivery in the inner ear. Aurein1.2-+36 GFP has the potential to be developed as protein-based therapy in regeneration and genome editing in mammalian inner ear.