Overcoming cellular barriers for RNA therapeutics

Journal name:
Nature Biotechnology
Year published:
Published online


RNA-based therapeutics, such as small-interfering (siRNAs), microRNAs (miRNAs), antisense oligonucleotides (ASOs), aptamers, synthetic mRNAs and CRISPR–Cas9, have great potential to target a large part of the currently undruggable genes and gene products and to generate entirely new therapeutic paradigms in disease, ranging from cancer to pandemic influenza to Alzheimer's disease. However, for these RNA modalities to reach their full potential, they first need to overcome a billion years of evolutionary defenses that have kept RNAs on the outside of cells from invading the inside of cells. Overcoming the lipid bilayer to deliver RNA into cells has remained the major problem to solve for widespread development of RNA therapeutics, but recent chemistry advances have begun to penetrate this evolutionary armor.

At a glance


  1. The four-billion-year-old lipid bilayer protects cells from invading RNAs.
    Figure 1: The four-billion-year-old lipid bilayer protects cells from invading RNAs.

    Unlike small-molecule drugs that can slip across the lipid bilayer, with the exception of some single-stranded phosphorothioate ASOs that can productively enter cells, the vast majority of RNA-based therapeutics are too charged and/or too large to enter cells, and require a delivery agent.

  2. Common ASO and siRNA modifications.
    Figure 2: Common ASO and siRNA modifications.

    (a) Phosphate backbone modifications: native, anionic charged phosphodiester (achiral phosphorus atom); charged phosphorothioate (phosphorus atom is chiral); neutral phosphotriester (phosphorus atom is chiral, but becomes achiral after intracellular conversion to charged phosphodiester); neutral morpholino backbone (PMO) and peptide nucleic acid (PNA) backbones align nucleobases with native mRNA nucleobase spacing. (b) Common 2′ modifications of the sugar: native 2′-hydroxyl (OH), 2′-fluoro (F), 2′-hydroxymethyl (O-Me), 2′-methoxyethyl (MOE) and 2′,4′-bicyclics that contain O-methylene bridge or locked nucleic acid (LNA).

  3. The numerology of endosomal escape.
    Figure 3: The numerology of endosomal escape.

    Tris-GalNAc binding to liver ASGPR (~106/hepatocyte) induces endocytosis (~15 min) where a small fraction of the siRNA or ASO cargo escapes into the cytoplasm to induce selective RNA drug responses. In contrast, targeting non-hepatic cell surface receptors (104–105) that have a much slower rate of endocytosis (~90 min) has proven extremely difficult. Assuming there is no endosomal escape advantage in ASGPR endosomes, ASGPR brings in ~100-fold more siRNAs/ASOs into hepatocytes than is mathematically possible in any other ligand–receptor pair. Consequently, development of next-generation RNA-based therapeutics needs to incorporate new chemistries, materials and/or mechanisms of enhancing endosomal escape ~100-fold.

  4. Endosomal escape agents.
    Figure 4: Endosomal escape agents.

    Protonation of small-molecule chloroquine (CQ) traps it in the endosome, resulting in a dramatic increase in its concentration and lysis of the endosome (left panel). Pore-forming melittin peptide from bee venom contains pH-sensitive protecting groups that are removed as the endosomal pH drops resulting in endosomal lysis (middle panel). Influenza virus contains a pH-sensitive fusogenic hemagglutinin-2 protein domain (HA2) that inserts into the endosomal membrane to locally destabilize it in a non-toxic manner to facilitate virus entry into the cytoplasm (right panel).


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  1. Department of Cellular and Molecular Medicine, University of California, San Diego, School of Medicine, La Jolla, California, USA.

    • Steven F Dowdy

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S.F.D. is a member of the board of directors of Solstice Biologics, and a member of the scientific advisory board of Verndari.

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