MRNA transfection by a Xentry-protamine cell-penetrating peptide is enhanced by TLR antagonist E6446.

Glenn D Bell,Yi Yang,Euphemia Leung,Geoffrey W Krissansen,Valentin Ceña

doi:10.1371/journal.pone.0201464

Abstract

Messenger RNA (mRNA) transfection is a developing field that has applications in research and gene therapy. Potentially, mRNA transfection can be mediated efficiently by cell-penetrating peptides (CPPs) as they may be modified to target specific tissues. However, whilst CPPs are well-documented to transfect oligonucleotides and plasmids, mRNA transfection by CPPs has barely been explored. Here we report that peptides, including a truncated form of protamine and the same peptide fused to the CPP Xentry (Xentry-protamine; XP), can transfect mRNAs encoding reporter genes into human cells. Further, this transfection is enhanced by the anti-malarial chloroquine (CQ) and the toll-like receptor antagonist E6446 (6-[3-(pyrrolidin-1-yl)propoxy)-2-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl]benzo[d]oxazole), with E6446 being >5-fold more potent than CQ at enhancing this transfection. Finally, E6446 facilitated the transfection by XP of mRNA encoding the cystic fibrosis transmembrane regulator, the protein mutated in cystic fibrosis. As such, these findings introduce E6446 as a novel transfection enhancer and may be of practical relevance to researchers seeking to improve the mRNA transfection efficiency of their preferred CPP.

Highlights

Messenger RNA has potential advantages over DNA as an alternative for use in gene therapy [1,2,3]
We reviewed the literature for clues to alternative agents to use to enhance Messenger RNA (mRNA) transfection by Xentry protamine fusion peptide (XP)
The review identified a promising candidate in the form of E6446, a small molecule which antagonizes nucleic acidsensing toll-like receptors (TLRs) more potently than CQ or HCQ and is less toxic than CQ when administered to mice [33,34,35]

Summary

Introduction

Messenger RNA (mRNA) has potential advantages over DNA as an alternative for use in gene therapy [1,2,3]. MRNA transcripts are smaller and simpler to engineer than DNA, as there is no need for promoter and terminator sequences, and mRNA’s transient nature may allow improved control over protein expression kinetics. Together, these attributes could make gene therapy safer, cheaper, and quicker to enter into clinical testing [1,2,3].

Methods

Results

Conclusion