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 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 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.
Highlights
We previously reported the discovery of superpositively-Proteins that bind extracellular targets, including monoclonal charged proteins, a class of engineered and naturally occurring antibodies1, Fc fusions2, and cytokines3, have served as important proteins that have abnormally high net positive charge, and their therapeutics4
To test this hypeptide that enhances the endosomal escape of a variety of cargo pothesis, we performed a screen of Antimicrobial peptides (AMPs) for their ability to infused to +36 GFP
We previously showed that +36 GFP–Cre alone can are consistent with the results of the glucocorticoid receptor (GR) translocation assay, and be delivered to mouse retina,15 albeit resulting in only modest levels further suggest that aurein 1.2 enhances the endosomal escape of of recombination consistent with inefficient endosomal escape
Summary
Proteins that bind extracellular targets, including monoclonal charged proteins, a class of engineered and naturally occurring antibodies, Fc fusions, and cytokines, have served as important proteins that have abnormally high net positive charge, and their therapeutics. A major challenge to developing agents that enhance endosobeen reported to be endosomolytic22 Many of these mal escape is the lack of well-established assays that can distinguish peptides, including HA2, are cytotoxic at concentrations required proteins trapped in the endosomes from proteins released into the for protein delivery. We discovered aurein 1.2 (GLFDIIKKIAESF) as a mammalian cell toxicity at useful concentrations To test this hypeptide that enhances the endosomal escape of a variety of cargo pothesis, we performed a screen of AMPs for their ability to infused to +36 GFP. Lysosomal degradation tency when fused with aurein 1.2 than in the absence of the peptide These results together provide a simple molecular strategy for enhancing the cytosolic delivery of proteins in cell culture and in endosomes vivo that is genetically encoded, localized to cargo molecules, and Membrane recycling does not require global treatment with cytotoxic small molecules
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
