Abstract
The term ‘extracellular vesicles’ refers to a heterogeneous population of vesicular bodies of cellular origin that derive either from the endosomal compartment (exosomes) or as a result of shedding from the plasma membrane (microvesicles, oncosomes and apoptotic bodies). Extracellular vesicles carry a variety of cargo, including RNAs, proteins, lipids and DNA, which can be taken up by other cells, both in the direct vicinity of the source cell and at distant sites in the body via biofluids, and elicit a variety of phenotypic responses. Owing to their unique biology and roles in cell–cell communication, extracellular vesicles have attracted strong interest, which is further enhanced by their potential clinical utility. Because extracellular vesicles derive their cargo from the contents of the cells that produce them, they are attractive sources of biomarkers for a variety of diseases. Furthermore, studies demonstrating phenotypic effects of specific extracellular vesicle-associated cargo on target cells have stoked interest in extracellular vesicles as therapeutic vehicles. There is particularly strong evidence that the RNA cargo of extracellular vesicles can alter recipient cell gene expression and function. During the past decade, extracellular vesicles and their RNA cargo have become better defined, but many aspects of extracellular vesicle biology remain to be elucidated. These include selective cargo loading resulting in substantial differences between the composition of extracellular vesicles and source cells; heterogeneity in extracellular vesicle size and composition; and undefined mechanisms for the uptake of extracellular vesicles into recipient cells and the fates of their cargo. Further progress in unravelling the basic mechanisms of extracellular vesicle biogenesis, transport, and cargo delivery and function is needed for successful clinical implementation. This Review focuses on the current state of knowledge pertaining to packaging, transport and function of RNAs in extracellular vesicles and outlines the progress made thus far towards their clinical applications.
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Acknowledgements
The Breakefield laboratory acknowledges grant support from the US National Institutes of Health National Cancer Institute (P01 CA069246 and R35 CA232103). S.U. acknowledges financial support from Associazione Italiana per la Ricerca sul Cancro (grant IG 20210) to S. Giordano. L.C.L. is supported by US National Institutes of Health grant U01HL126494. The authors thank M. Brennan for insights on mesenchymal stem cells and S. McDavitt for skilled editorial assistance.
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The authors contributed equally to all aspects of the article, with K.O’B. and K.B. doing the major draft writing.
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ClinicalTrials.gov: https://www.clinicaltrials.gov/
exRNA Atlas: https://exrna-atlas.org/
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Glossary
- Astrocytes
-
Glial cells of the central nervous system.
- Large oncosomes
-
Extracellular vesicles between 1,000 nm and 10,000 nm in diameter derived from tumour cells that contain abnormal and transforming macromolecules, in addition to other cargo.
- Lipoproteins
-
Macromolecular aggregates of proteins (apoproteins) and different classes of hydrophobic and amphipathic biomolecules that range in size from 10 nm to 1 μm. These aggregates have the function of collecting and transporting lipids, triglycerides, free cholesterol (unesterified cholesterol) and esterified cholesterol in blood plasma.
- Ribonucleoproteins
-
Proteins structurally associated with nucleic acids, the prosthetic group (nucleic acid) of which contains ribose.
- High-resolution density gradient centrifugation
-
A method in which extracellular vesicles and particles are centrifuged in a gradient medium (for example, iodixanol) and separated primarily on the basis of their density.
- Size-exclusion chromatography
-
A technique in which a porous stationary phase is used to sort extracellular vesicles from proteins on the basis of the larger size of the former.
- Asymmetric field-flow fractionation
-
A technique that separates differentially sized particles ranging from 1 nm to 100 μm in a fluid suspension where the rate of laminar flow is not uniform. A force is applied in a perpendicular direction, facilitating the separation of particles on the basis of size.
- Immunoaffinity purification
-
A method of isolation of extracellular vesicles using antibodies, typically conjugated to magnetic beads, that recognize proteins exposed on the surface of extracellular vesicles.
- Antisense oligonucleotides
-
Sequences of DNA or RNA roughly 15–25 nucleotides in length that hybridize to complementary RNA targets, resulting in altered splicing or degradation of the targeted RNA.
- Guide RNAs
-
RNAs with a specific sequence that recognize and aid nucleases, such as Cas9, in cutting the genomic DNA region of interest for targeted genome engineering.
- Self-amplifying RNAs
-
Engineered viral genomes encoding proteins involved in the replication mechanism and therefore capable of directing their own replication.
- Small nuclear RNAs
-
Short sequences (about 150 nucleotides) of ribonucleic acid, usually very rich in uracil, involved in the maturation of mRNA in the cell nucleus.
- Small nucleolar RNAs
-
(snoRNAs). RNA species consisting of 60–300 nucleotides capable of promoting certain chemical modifications and maturation of many cellular RNAs.
- PIWI-interacting RNAs
-
(piRNAs). RNAs approximately 21–35 nucleotides long representing a class of RNAs that form protein–RNA complexes by interacting with PIWI proteins. These piRNA complexes have been linked to the transcriptional silencing of retrotransposons and other genetic elements in germ line cells.
- Y RNAs
-
Small non-coding RNAs essential for the initiation of chromosomal DNA replication. When bound to the RO60, they are involved in RNA stability and cellular responses to stress.
- Vault RNAs
-
(vtRNAs). Small RNA components of the vault ribonucleoprotein complex. Approximately 100 nucleotides in length, they function as the intracellular and nucleocytoplasmic transporters.
- Dicer
-
The RNase III known as Dicer is involved in the cleavage of double-stranded RNA and microRNA precursor molecules forming short double-stranded fragments called ‘small interfering RNAs’ or ‘microRNAs’.
- Argonaute
-
The Argonaute protein family has a critical role in the RNA-induced silencing complex. These proteins form complexes with microRNAs, small interfering RNAs and PIWI-interacting RNAs, interfering with mRNA translation and/or degrading target mRNAs.
- Circular RNAs
-
Single-stranded, non-coding RNAs that may play a role in transcriptional regulation and in mediating protein interactions. They are characterized by a covalently closed loop feature without 5′-end caps or 3′ poly(A) tails.
- tRNA fragments
-
Transfer RNA (tRNA)-derived fragments are cleaved from either mature or precursor tRNAs. Functionally, they can act through RNA interference pathways, participate in the formation of stress granules, move mRNA from RNA-binding proteins and/or inhibit translation.
- Multivesicular bodies
-
(MVBs). Represent a late endosomal compartment, which contains luminal vesicles. Vesicles within the MVBs either can be degraded when MVBs fuse with lysosomes or can be secreted as exosomes when MVBs fuse with the plasma membrane.
- Lipid rafts
-
Subdomains of the plasma membrane represented by accumulations of proteins and lipids, with high concentrations of cholesterol and glycosphingolipids.
- Blebbing
-
A bulging of the plasma membrane accompanied by membrane decoupling from the underlying cytoskeleton, which has the potential to release vesicles surrounded by the plasma membrane.
- Secretory autophagy
-
The transportation of proteins via the autophagosome to the plasma membrane, multivesicular bodies or secretory lysosomes resulting in extracellular release of proteins.
- Macropinocytosis
-
An endocytic process that results in the cellular uptake of fluid and particles less than 0.2 μm in diameter via invaginated membranes that then form intracellular vesicles.
- Tunnelling nanotubes
-
Dynamic actin-driven protrusions from the plasma membrane of cells that form connections between cells more than 100 μm away to facilitate intercellular communication.
- Proteoglycans
-
Highly glycosylated proteins that form the major component of the extracellular matrix. Glycosaminoglycans of proteoglycans are divided into seven main classes: hyaluronic acid, chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, keratan sulfate I, keratan sulfate II and heparin. Proteoglycans act as molecular filters, regulating movement of macromolecules, such as albumin and immunoglobulins, throughout the matrix.
- Liposomes
-
Small spherical nanoparticles that contain an aqueous core solution and at least one phospholipid bilayer. They can be prepared from biological membranes and are often used for drug delivery.
- Amyloid precursor protein
-
A transmembrane protein that is a precursor of β-amyloid, a protein involved in the pathology of Alzheimer disease.
- Mesenchymal stem cells
-
Multipotent stromal progenitor cells found in a number of different tissues that are capable of self-renewal and differentiation into a variety of cell types.
- Cre recombinase
-
A type I topoisomerase from bacteriophage P1 that catalyses cleavage and ligation of DNA at specific loxP recognition sites.
- RIG-I-like receptors
-
Intracellular pattern recognition receptors that belong to the RNA box helicase family that function as cytoplasmic sensors of pathogen-associated molecular patterns elicited by viral RNA.
- NOD-like receptor family
-
Cytoplasmic receptors expressed by innate immune and other cells. They are part of the family of intracellular pattern recognition receptors and can recognize structures associated with pathogenic microorganisms (pathogen-associated molecular patterns) or with cell damage (damage-associated molecular patterns), thereby activating the innate immune response and inflammation.
- Type I interferon
-
A large subgroup of cytokines that help to regulate the activity of the immune system. They act to degrade viral DNA and proteins in infected cells and provide protection to surrounding cells through interaction with and signalling from interferon receptors.
- Toll-like receptors
-
(TLRs). A class of transmembrane proteins involved in the defence of the organism through innate immunity. They are mainly expressed on the membrane of sentinel cells, such as macrophages and dendritic cells, and recognize certain typical structures of pathogens and microorganisms.
- Lysosome-associated membrane protein 2B
-
A particular isoform of LAMP2 — a protein that associates with the membrane of multivesicular bodies and lysosomes.
- Tetraspanin
-
A family of membrane proteins consisting of four transmembrane domains, with intracellular N and C terminals and two extracellular domains. Tetraspanins are present in the membranes of extracellular vesicles.
- ‘Zipcode’ sequences
-
Sequences typically found in the 3′ untranslated regions of mRNA transcripts that can be recognized by specific RNA-binding proteins.
- Lipofectamine
-
A transfection reagent, composed of a cationic tail and a lipid region. Lipofectamine binds exogenous DNA and RNA, creating liposomes that, once in contact with the plasma membrane of the cells, merge with it and facilitate entry of nucleic acids into the cytoplasm.
- V-ATPase
-
Vacuolar-type H-ATPase. A transmembrane protein found mainly in intracellular organelles and the plasma membrane. It catalyses the hydrolysis of ATP, allowing transport of solutes and acidification of organelles.
- Heparanase
-
Heparanases are a group of enzymes that act both at the cell surface and within the extracellular matrix to degrade polymeric heparan sulfate molecules into shorter chain length oligosaccharides.
- Gene therapy
-
A variety of methods resulting in alterations in the genome and/or transcriptome of cells in order to correct deficiency or dominant negative effects caused by genetic mutations or infections.
- RNA aptamers
-
Small oligonucleotides that bind to target molecules with high affinity and specificity through their 3D structures.
- Major histocompatibility complexes
-
A group of genes encoding plasma membrane proteins that facilitate recognition of foreign peptides by the adaptive immune system.
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O’Brien, K., Breyne, K., Ughetto, S. et al. RNA delivery by extracellular vesicles in mammalian cells and its applications. Nat Rev Mol Cell Biol 21, 585–606 (2020). https://doi.org/10.1038/s41580-020-0251-y
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DOI: https://doi.org/10.1038/s41580-020-0251-y
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