Delivery of RNA Therapeutics: The Great Endosomal Escape!

Abstract

RNA therapeutics, including siRNAs, antisense oligonucleotides, and other oligonucleotides, have great potential to selectively treat a multitude of human diseases, from cancer to COVID to Parkinson's disease. RNA therapeutic activity is mechanistically driven by Watson-Crick base pairing to the target gene RNA without the requirement of prior knowledge of the protein structure, function, or cellular location. However, before widespread use of RNA therapeutics becomes a reality, we must overcome a billion years of evolutionary defenses designed to keep invading RNAs from entering cells. Unlike small-molecule therapeutics that are designed to passively diffuse across the cell membrane, macromolecular RNA therapeutics are too large, too charged, and/or too hydrophilic to passively diffuse across the cellular membrane and are instead taken up into cells by endocytosis. However, similar to the cell membrane, endosomes comprise a lipid bilayer that entraps 99% or more of RNA therapeutics, even in semipermissive tissues such as the liver, central nervous system, and muscle. Consequently, before RNA therapeutics can achieve their ultimate clinical potential to treat widespread human disease, the rate-limiting delivery problem of endosomal escape must be solved in a clinically acceptable manner.

Keywords: ASO; RNA therapeutics; delivery; endosomal escape; siRNA.

FIG. 1.

RNA therapeutics, including PS ASOs, siRNAs, PMO ASOs, PNA ASOs, and other oligonucleotides, are too large, too charged, and/or too hydrophilic to passively diffuse across the billion-year-old cell membrane lipid bilayer, but instead require a TD to drive uptake by endocytosis. However, endosomes also comprise a lipid bilayer, and 1% or less of endocytosed RNA therapeutics escape the endosome. Thus, endosomal escape is the rate-limiting delivery step. TD, targeting domain; PS, phosphorothioate; ASO, antisense oligonucleotide.

FIG. 2.

Endosomal escape approaches. (A) Small-molecule endolytic agents, such as chloroquine (CQ), are membrane permeable, but become protonated, positively charged (CQ+), and trapped inside the low pH environment of an endosome, resulting in a 1,000-fold or more concentration increase that leads to endosomal bursting. (B) Endolytic peptides and polymers are often codelivered with RNA therapeutics in 10- to 30-fold molar excess and ultimately disrupt the endosomal lipid bilayer membrane, leading to endosomal bursting. (C) Enveloped viruses contain fusogenic peptide domains that are selectively activated in the low pH of endosomes, causing localized endosomal membrane disruption (not endosomal bursting). Adapted from the study by Dowdy [4].