title: Discovery and characterization of a peptide that enhances endosomal escape of delivered proteins in vitro and in vivo.
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.
authors.affiliation: Department of Chemistry & Chemical Biology, Harvard University , 12 Oxford Street, Cambridge, Massachusetts 02138, United States.
authors.forename: Margie
authors.initials: M
authors.lastname: Li
authors.affiliation: Department of Otolaryngology and Program in Neuroscience, Harvard Medical School and Eaton Peabody Laboratory, Massachusetts Eye and Ear Infirmary , Boston, Massachusetts 02114, United States.
authors.forename: Yong
authors.initials: Y
authors.lastname: Tao
authors.affiliation: Department of Otolaryngology and Program in Neuroscience, Harvard Medical School and Eaton Peabody Laboratory, Massachusetts Eye and Ear Infirmary , Boston, Massachusetts 02114, United States.|Department of Otology and Skull Base Survery, Eye, Ear, Nose and Throat Hospital, Shanghai Medical College, Fudan University , Shanghai 200031, China.|Key Laboratory of Hearing Medicine, Ministry of Health , Shanghai, 200031, China.
authors.forename: Yilai
authors.initials: Y
authors.lastname: Shu
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University , New Haven, Connecticut 06520-8107, United States.
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: LaRochelle
authors.affiliation: Department of Chemistry, Yale University , New Haven, Connecticut 06520-8107, United States.
authors.forename: Angela
authors.initials: A
authors.lastname: Steinauer
authors.affiliation: Department of Chemistry & Chemical Biology, Harvard University , 12 Oxford Street, Cambridge, Massachusetts 02138, United States.
authors.forename: David
authors.initials: D
authors.lastname: Thompson
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University , New Haven, Connecticut 06520-8107, United States.|Department of Chemistry, Yale University , New Haven, Connecticut 06520-8107, United States.
authors.forename: Alanna
authors.initials: A
authors.lastname: Schepartz
authors.affiliation: Department of Otolaryngology and Program in Neuroscience, Harvard Medical School and Eaton Peabody Laboratory, Massachusetts Eye and Ear Infirmary , Boston, Massachusetts 02114, United States.
authors.forename: Zheng-Yi
authors.initials: ZY
authors.lastname: Chen
authors.affiliation: Department of Chemistry & Chemical Biology, Harvard University , 12 Oxford Street, Cambridge, Massachusetts 02138, United States.|Howard Hughes Medical Institute, Harvard University , 12 Oxford Street, Cambridge, Massachusetts 02138, United States.
authors.forename: David R
authors.initials: DR
authors.lastname: Liu
mesh_terms: D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D016503:Drug Delivery Systems; D007758:Ear, Inner / Q000166:cytology* / Q000378:metabolism; D011992:Endosomes / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D019426:Integrases / Q000378:metabolism*; D051379:Mice
chemical_list: D023181:Antimicrobial Cationic Peptides; C506635:aurein 1.2 peptide; D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D019426:Integrases