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Endosomal escape of RNA therapeutics: How do we solve this rate-limiting problem?

Dowdy, Steven F.·2023·RNA
DOI 10.1261/rna.079507.122PMC PMC10019367
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Abstract

With over 15 FDA approved drugs on the market and numerous ongoing clinical trials, RNA therapeutics, such as small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs), have shown great potential to treat human disease. Their mechanism of action is based entirely on the sequence of validated disease-causing genes without the prerequisite knowledge of protein structure, activity or cellular location. In contrast to small molecule therapeutics that passively diffuse across the cell membrane's lipid bilayer, RNA therapeutics are too large, too charged, and/or too hydrophilic to passively diffuse across the cellular membrane and instead are taken up into cells by endocytosis. However, endosomes are also composed of a lipid bilayer barrier that results in endosomal capture and retention of 99% of RNA therapeutics with 1% or less entering the cytoplasm. Although this very low level of endosomal escape has proven sufficient for liver and some CNS disorders, it is insufficient for the vast majority of extra-hepatic diseases. Unfortunately, there are currently no acceptable solutions to the endosomal escape problem. Consequently, before RNA therapeutics can be used to treat widespread human disease, the rate-limiting delivery problem of endosomal escape must be solved in a nontoxic manner.

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Crossref · 10.1261/rna.079507.122

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FIGURE 1. Where do RNA therapeutics escape from endosomes? Unlike small molecule drugs, RNA therapeutics (siRNAs, ASOs, and PMOs) are too large, too charged and/or too hydrophilic to passively diffuse across the cell membrane lipid bilayer, but instead are taken up into cells by various forms of endocytosis. Endosomes are also composed of a lipid bilayer barrier that prevents the vast majority (∼99%) of RNA therapeutics from entering the cytoplasm. Following endocytosis, early endosomes mature into late endosomes and then fuse with lysosomes. Although it remains unclear where RNA therapeutics escape from endosomes, they likely escape from multiple types of endosomes. Depot endosomes occur in vivo, but their origins and composition are uncharacterized. RNA therapeutics escaping from depot endosomes results in long duration of responses (3 to 6 mo or more). Increasing the efficiency of endosomal escape can be thought of as two generalized approaches: (i) endosomal rupture; or (ii) enhanced endosomal escape.
FIGURE 1. Where do RNA therapeutics escape from endosomes? Unlike small molecule drugs, RNA therapeutics (siRNAs, ASOs, and PMOs) are too large, too charged and/or too hydrophilic to passively diffuse across the cell membrane lipid bilayer, but instead are taken up into cells by various forms of endocytosis. Endosomes are also composed of a lipid bilayer barrier that prevents the vast majority (∼99%) of RNA therapeutics from entering the cytoplasm. Following endocytosis, early endosomes mature into late endosomes and then fuse with lysosomes. Although it remains unclear where RNA therapeutics escape from endosomes, they likely escape from multiple types of endosomes. Depot endosomes occur in vivo, but their origins and composition are uncharacterized. RNA therapeutics escaping from depot endosomes results in long duration of responses (3 to 6 mo or more). Increasing the efficiency of endosomal escape can be thought of as two generalized approaches: (i) endosomal rupture; or (ii) enhanced endosomal escape.CC BY-NC · PubMed Central
FIGURE 2. Enveloped virus mechanism of endosomal escape. Viruses also have an endosomal escape problem. Enveloped viruses, including influenza, have evolved a similar approach to escape endosomes that involves selectively converting a parental trimeric protein, hemagglutinin (HA), into two domains inside of endosomes. In plasma, the outer shell HA1 domain contains the receptor binding domain (RBD) and serves to mask the hydrophobic HA2 endosomal escape fusion domain. After stimulating endocytosis, due to low pH and endosomal proteolytic activity, HA1 is shed to expose HA2 that buries its hydrophobic fusion peptide into the hydrophobic interior of the endosomal lipid bilayer membrane, resulting in a highly efficient insertion of the viral capsid into the cytoplasm.
FIGURE 2. Enveloped virus mechanism of endosomal escape. Viruses also have an endosomal escape problem. Enveloped viruses, including influenza, have evolved a similar approach to escape endosomes that involves selectively converting a parental trimeric protein, hemagglutinin (HA), into two domains inside of endosomes. In plasma, the outer shell HA1 domain contains the receptor binding domain (RBD) and serves to mask the hydrophobic HA2 endosomal escape fusion domain. After stimulating endocytosis, due to low pH and endosomal proteolytic activity, HA1 is shed to expose HA2 that buries its hydrophobic fusion peptide into the hydrophobic interior of the endosomal lipid bilayer membrane, resulting in a highly efficient insertion of the viral capsid into the cytoplasm.CC BY-NC · PubMed Central

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Research works printed but not linked: 25

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Metadata

Authors
Dowdy, S. F.
Venue
RNA
Publisher
Cold Spring Harbor Laboratory
Published
2023-01-20
Volume
29
Issue
4
Pages
396-401
ISSN
  • 1355-8382
  • 1469-9001
DOI
10.1261/rna.079507.122
PMC
PMC10019367
Cited by
171
References
5
Language
en
Funders
  • ASAP
  • National Institutes of Health
  • an Epstein Family Alzheimer’ Research Collaboration Award

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