The aim of this study was to study an antimicrobial peptide (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 postnatal day 1-2 (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 (tdT) expression was observed in outer hair cells (OHCs; 20.77%, 23.02%, 76.36%, and 92.47%, respectively) and inner hair cells (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 round window membrane (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. Aurein 1.2–+36 GFP has the potential to be developed as protein-based therapy in regeneration and genome editing in the mammalian inner ear.

这项研究的目的是研究一种抗菌肽（AMP），aurein 1.2，该蛋白可显着增加体内直接向多种哺乳动物内耳细胞类型中直接递送的蛋白质。将具有不同浓度的带有与Cre重组酶融合的带正电荷的GFP（+36 GFP）蛋白的金黄色素1.2通过各种递送方法递送至出生后的第1-2天（P1-2）和Cre报告基因转基因小鼠的成年耳蜗中。通过在不同浓度的金黄色素1.2– + 36 GFP（分别为1μM，5μM，22.5μM和50μM）中进行耳蜗切开术，在外毛细胞（OHCs）中观察到了tdTomato（tdT）表达； 20.77％，23.02％，耳蜗中的内毛细胞（分别为76.36％和92.47％）和内毛细胞（分别为14.90％，44.50％，89.59％和96.13％）。最佳浓度为22.5μM，具有最高的转染效率和最低的细胞毒性。在新生小鼠和成年小鼠中，在耳蜗支持细胞，尿道支持细胞，听觉神经和螺旋韧带中检测到广泛的tdT信号。与耳蜗切开术相比，通过圆窗膜（RWM）注射还产生了高效tdT +标记的细胞，细胞损失较少。总之，融合有+36 GFP的金黄色素1.2肽显着扩展了靶细胞，并提高了内耳直接蛋白递送的效率。Aurein 1.2– + 36 GFP具有发展为哺乳动物内耳再生和基因组编辑中基于蛋白质的疗法的潜力。