Cellular uptake and intracellular trafficking of oligonucleotides

doi:10.1016/j.addr.2015.04.005

Advanced Drug Delivery Reviews

Volume 87, 29 June 2015, Pages 35-45

https://doi.org/10.1016/j.addr.2015.04.005 Get rights and content

Abstract

Oligonucleotides manifest much promise as potential therapeutic agents. However, understanding of how oligonucleotides function within living organisms is still rather limited. A major concern in this regard is the mechanisms of cellular uptake and intracellular trafficking of both ‘free’ oligonucleotides and oligonucleotides associated with various polymeric or nanocarrier delivery systems. Here we review basic aspects of the mechanisms of endocytosis and intracellular trafficking and how insights from these processes can be used to understand oligonucleotide delivery. In particular we discuss opportunities for escape of oligonucleotides from endomembrane compartments and describe recent studies using small molecules to enhance oligonucleotide effects.

Graphical abstract

Introduction

Realization of the potential oligonucleotides as therapeutic agents began more than three decades ago with the discovery of antisense molecules [1]. The discovery of RNA interference [2], [3] increased enthusiasm and was further reinforced by insights into the complex roles of non-coding RNAs in regulating genome function [4]. Many aspects of oligonucleotide therapeutics are covered in this theme issue. Here we will stress a key basic aspect of oligonucleotide behavior that underlies all potential therapeutic utilization, namely the cellular uptake and intracellular trafficking of these molecules.

Despite FDA approval of the first antisense drug [5] and the advent of multiple clinical trials in cancer and other diseases [6], [7], [8], [9], [10], oligonucleotide therapeutics has progressed slowly. A major issue has been the poor efficacy of oligonucleotides. In large part this is because effective delivery of such large, polar molecules to their sites of action within tissues is a very challenging problem [11], [12], [13]. This review will primarily emphasize the behavior of ‘free’ oligonucleotides and of molecular-scale oligonucleotide conjugates. Oligonucleotides associated with nanocarriers are more fully discussed elsewhere in this theme issue. The emphasis here will be on processes at the cellular and subcellular level rather than on classical pharmacokinetics and biodistribution. A key theme for this article is the observation that the processes that govern intracellular traffic of internalized molecules are enormously complex and that understanding these processes is vital for the future of oligonucleotide therapeutics.

Section snippets

Endocytotic and trafficking pathways

Oligonucleotides usually enter cells via endocytosis; however, it is important to realize the complexities associated with the multiple pathways of internalization and subsequent trafficking. These pathways are regulated by a plethora of unique proteins and lipids that underlie all aspects of internalization and trafficking. An illustration of some of the pathways is given in Fig. 1.

Overview

There are thousands of publications on the pharmacokinetics, biodistribution, cellular uptake and biological effects of oligonucleotides as individual molecules or associated with various carriers. To provide an overview of this complex literature, this theme issue contains timely reviews on oligonucleotide pharmacokinetics and biodistribution by Geary and colleagues, on the behavior of oligonucleotide conjugates by Ming and colleagues, and on delivery using lipoplexes or cell-penetrating

Conclusions

The therapeutic development of antisense, siRNA, SSOs and other types of oligonucleotides has, to some degree, outstripped our fundamental knowledge of how these molecules behave in cells and in the body. Thus although there is much information regarding the overall pharmacokinetics and biodistribution of oligonucleotides, there is not an equivalent depth of knowledge about their behavior at the cellular and intracellular levels. Recent work has uncovered some interesting and surprising

Acknowledgments

This work was supported by NIH grant RO1 CA151964 to RLJ.

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^☆: This review is part of the Advanced Drug Delivery Reviews theme issue on “Oligonucleotide therapeutics”.

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Cellular uptake and intracellular trafficking of oligonucleotides☆

Abstract

Graphical abstract

Introduction

Section snippets

Endocytotic and trafficking pathways

Overview

Conclusions

Acknowledgments

Lancet Neurol.

Lancet

J. Control. Release

Int. Rev. Cell Mol. Biol.

Curr. Opin. Cell Biol.

Curr. Opin. Cell Biol.

Virology

Semin. Cell Dev. Biol.

Semin. Cell Dev. Biol.

Semin. Cell Dev. Biol.

Curr. Opin. Cell Biol.

Trends Cell Biol.

Curr. Biol.

Dev. Cell

Adv. Enzym. Regul.

Dev. Cell

Curr. Opin. Cell Biol.

Curr. Opin. Cell Biol.

Curr. Opin. Cell Biol.

Curr. Opin. Cell Biol.

Curr. Opin. Cell Biol.

Adv. Drug Deliv. Rev.

Curr. Opin. Cell Biol.

Cell

Adv. Drug Deliv. Rev.

Adv. Drug Deliv. Rev.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Mol. Ther.

Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide

Proc. Natl. Acad. Sci. U. S. A.

Revealing the world of RNA interference

Nature

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells

Nature

Small RNAs in transcriptional gene silencing and genome defence

Nature

Regulatory watch: antisense approval provides boost to field

Nat. Rev. Drug Discov.

Safety and efficacy of RNAi therapy for transthyretin amyloidosis

N. Engl. J. Med.

Clinical trials using antisense oligonucleotides in Duchenne muscular dystrophy

Hum. Gene Ther.

First-in-human Phase I study of EZN-4176, a locked nucleic acid antisense oligonucleotide to exon 4 of the androgen receptor mRNA in patients with castration-resistant prostate cancer

Br. J. Cancer

Biological barriers to therapy with antisense and siRNA oligonucleotides

Mol. Pharm.

Knocking down barriers: advances in siRNA delivery

Nat. Rev. Drug Discov.

Molecular structure, function, and dynamics of clathrin-mediated membrane traffic

Cold Spring Harb. Perspect. Biol.

Mechanisms of endocytosis

Annu. Rev. Biochem.

Clathrin-independent pathways of endocytosis

Cold Spring Harb. Perspect. Biol.

Dissecting dynamin's role in clathrin-mediated endocytosis

Biochem. Soc. Trans.

Retromer and sorting nexins in endosomal sorting

Biochem. Soc, Trans.

Clathrin-dependent mechanisms of G protein-coupled receptor endocytosis

Traffic

Caveolae as plasma membrane sensors, protectors and organizers

Nat. Rev. Mol. Cell Biol.

Lipid rafts as a membrane-organizing principle

Science

Endocytosis and signalling: intertwining molecular networks

Nat. Rev. Mol. Cell Biol.

Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells

Nat. Cell Biol.

Defining macropinocytosis

Traffic