A platform for discovery of functional cell-penetrating peptides for efficient multi-cargo intracellular delivery

Katrin Hoffmann,Shane R Stone,Richard W Francis,Marie N Scobie,Paul M Watt,Clinton M Hall,R.m Hopkins ,Danie Champain,Robert E Dewhurst,Yew Foon Tan ,Volker Morath,Nadia Milech,Arne Skerra,Livia C Hool,Brooke Allison Cattell Longville ,Ferrer Ong,Laura Flórez ,Maria Kerfoot,Helena M Viola,A.m Adams ,Suzy M Juraja,Mark Anastasas,Heique Marlis Bogdawa ,Scott Winslow,Tatjana Heinrich,Gregory A Weiss,Karen M Kroeger ,S Fletcher ,Paula Cunningham ,Theresa Connor

doi:10.1038/s41598-018-30790-2

Abstract

Cell penetrating peptides (CPPs) offer great potential to deliver therapeutic molecules to previously inaccessible intracellular targets. However, many CPPs are inefficient and often leave their attached cargo stranded in the cell’s endosome. We report a versatile platform for the isolation of peptides delivering a wide range of cargos into the cytoplasm of cells. We used this screening platform to identify multiple “Phylomer” CPPs, derived from bacterial and viral genomes. These peptides are amenable to conventional sequence optimization and engineering approaches for cell targeting and half-life extension. We demonstrate potent, functional delivery of protein, peptide, and nucleic acid analog cargos into cells using Phylomer CPPs. We validate in vivo activity in the cytoplasm, through successful transport of an oligonucleotide therapeutic fused to a Phylomer CPP in a disease model for Duchenne’s muscular dystrophy. This report thus establishes a discovery platform for identifying novel, functional CPPs to expand the delivery landscape of druggable intracellular targets for biological therapeutics.

Highlights

Despite identification of over one thousand unique Cell penetrating peptides (CPPs) to date[6,7], few CPP-linked drugs have entered the clinic[8,9]
This expectation motivated the development and application of a new CPP discovery platform that selects and evolves CPPs based on successful, functional delivery into the cytoplasm of cells
We have developed a phage-based screening platform (Fig. 1) to identify CPPs that internalize and enter the cellular cytoplasm

Summary

Introduction

Despite identification of over one thousand unique CPPs to date[6,7], few CPP-linked drugs have entered the clinic[8,9]. Alternative approaches to improve uptake potency have included dimerization of TAT22, cyclization[23], the addition of cell binding peptides[24], and the use of synthetic endosomal escape domains[25] or adaptors[26] These approaches can improve delivery into the cytoplasm to varying degrees. New CPPs must be compatible with standard optimization approaches to enhance drug-like properties of biologics, such as the addition of moieties to increase half-life or confer tissue targeting We address this challenge using Phylomer peptide libraries[27,28]. The efficiency of Phylomer CPPs to deliver biologics offers a new path to improved therapeutic potency and reduced dosing thresholds Both the approach and the peptides reported here have great potential to expand the intracellular landscape of druggable targets

Methods

Results

Conclusion