Discovery and Characterization of a Peptide That Enhances Endosomal Escape of Delivered Proteins in Vitro and in Vivo

作者：Margie Li, Yong Tao, Yilai Shu, Jonathan R. LaRochelle, Angela Steinauer, David B. Thompson, Alanna Schepartz, Zheng‐Yi Chen, David R. Liu · 发表于：Journal of the American Chemical Society · 年份：2015 · DOI：10.1021/jacs.5b05694 · 被引用次数：137 · 研究领域：RNA Interference and Gene Delivery、Advanced biosensing and bioanalysis techniques、Antimicrobial Peptides and Activities

The inefficient delivery of proteins into mammalian cells remains a major barrier to realizing the therapeutic potential of many proteins. We and others have previously shown that superpositively charged proteins are efficiently endocytosed and can bring associated proteins and nucleic acids into cells. The vast majority of cargo delivered in this manner, however, remains in endosomes and does not reach the cytosol. In this study we designed and implemented a screen to discover peptides that enhance the endosomal escape of proteins fused to superpositively charged GFP (+36 GFP). From a screen of peptides previously reported to disrupt microbial membranes without known mammalian cell toxicity, we discovered a 13-residue peptide, aurein 1.2, that substantially increases cytosolic protein delivery by up to ∼5-fold in a cytosolic fractionation assay in cultured cells. Four additional independent assays for nonendosomal protein delivery collectively suggest that aurein 1.2 enhances endosomal escape of associated endocytosed protein cargo. Structure-function studies clarified peptide sequence and protein conjugation requirements for endosomal escape activity. When applied to the in vivo delivery of +36 GFP-Cre recombinase fusions into the inner ear of live mice, fusion with aurein 1.2 dramatically increased nonendosomal Cre recombinase delivery potency, resulting in up to 100% recombined inner hair cells and 96% recombined outer hair cells, compared to 0-4% recombined hair cells fr...