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

Abstract

ABSTRACT: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 non-endosomal 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 non-endosomal 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 from +36-GFP-Cre without aurein 1.2. Collectively, these findings describe a genetically encodable, endosome escape-enhancing peptide that can substantially increase the cytoplasmic delivery of cationic proteins in vitro and in vivo.

Enhancing Endosomal Escape for Intracellular Delivery of Macromolecular Biologic Therapeutics

“…Using a systematic approach, we then narrowed down the optimal EED composition to containing two indole rings or one indole ring and two phenyl groups in either a FWF or WW composition. Interestingly, Li et al identified a highly charged Aurein 1.2 peptide that enhances endosomal escape 5-fold29.…”

Glycosylated Triterpenoids as Endosomal Escape Enhancers in Targeted Tumor Therapies

“…This demonstrates that the process of lysosomal degradation is the most probable basis for resistance and emphasizes the need for the discovery of novel endosomal escape enhancers [13,57,93]. As a result of lysosomal degradation, relatively high concentrations of the targeted toxin are usually needed for modest cytosolic or nuclear delivery, leading to severe side-effects also for off-target cells [94]. Consequently, to increase the therapeutic window for tumor targeted toxins, the efficacy of delivery has to be enhanced and the following characteristics should be fulfilled: low immunogenicity and toxicity, minimized unspecific off-target effects, high efficacy and specificity as well as ease of use and production [13,57,95].…”

Interaction of the Antimicrobial Peptide Aurein 1.2 and Charged Lipid Bilayer

“…It also shows a moderate anti-cancer activity on 52 out of the 54 cancer cells in the NCI testing program, at the concentrations that can kill bacterial and cancer cells without harming mammalian cells 2 . Even more interestingly, it substantially enhances the endosomal escape of protein when conjugated with a cargo cationic protein 4 . This short yet effective membrane-active peptide is of great interest for a better understanding of the interaction between antimicrobial peptides and membranes.…”