The Discovery and Characterization of Endosomal Escape Enhancing Compounds to Improve Protein Delivery Efficacy

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 thesis, I designed and implemented a screen to discover small molecules and 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, I discovered a 13-residue peptide, aurein 1.2, that substantially increased non-endosomal protein delivery by up to ~10-fold in cultured cells. Three independent assays for non-endosomal protein delivery confirmed 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. Collectively, these findings describe a genetically encodable, endosome escape-enhancing peptide that can greatly increase the cytoplasmic delivery of cationic proteins in vitro and in vivo.