Unlocking the
Mysteries of
Extracellular RNA

Once thought to exist only inside cells, RNA is
known to travel outside of cells and play a role in newly
discovered mechanisms of cell-to-cell communication.

Our bodies are made up of trillions of cells that work together to keep us alive. A major challenge in their success is communication between cells in different parts of the body. Our cells have ingenious ways of overcoming this challenge, with exosomes emerging as key players. Exosomes are cell-derived vesicles that can carry cargo in the form of nucleic acids, lipids, or proteins from one cell to another. The sender cell packages cargo into an exosome, which then leaves the cell by being pinched off from the cell membrane. The exosome finds it way to a neighboring cell or into the bloodstream, from which it can be sent throughout the body. The exosome has signs on its surface that determine what cells can receive the cargo, so it only goes to the intended receiver. If a heart cell wants to talk to another heart cell, it puts markers on its exosomes that make them stick to other heart cells. When those exosomes are taken into the receiving cells, their cargo can bring about physiologic changes there.

Extracellular vesicles and their cargo. Source: BioProcess International.

Exosomes play a major role not only in our regular physiology but also in disease. One of the fields in which the role of exosomes is being uncovered is cardiovascular disease. For example, heart endothelial cells (cells that line the blood vessels) communicate with heart muscle cells via exosomes that contain microRNA, a kind of molecule that can decrease how many transcripts of a particular set of genes get made in the target cell. This process may play a role in the heart’s response to plaque formation. One can envision the possibility for engineering exosomes so that we can communicate with our bodies to treat or prevent disease. The lab of Dr. Susmita Sahoo at the Icahn School of Medicine at Mount Sinai is interested in doing just that.

Before talking about how Dr. Sahoo’s group is using exosomes in treating heart failure, let’s talk about a specific cause of heart failure: epitranscriptomics. You may or may not have heard of epigenetics, which is the study of heritable, chemical changes to DNA that do not change the sequence of the DNA. Epitranscriptomics is based on the exact same idea, but the change happens at the RNA level. One such epitranscriptomic modification is the addition or removal of methyl groups on adenosines within certain mRNAs in cells.

Structure of N6-Methyladenosine (m6A)

Heart muscle cells (cardiomyocytes) usually use electrical signals to interact and pulse in unison with a set rhythm. Work from Dr. Sahoo’s team suggests that decreasing levels of FTO, an enzyme that removes these methyl groups from RNA, leads to arrhythmia, a disturbance in that synchronized pulse. This finding is corroborated by the fact that failing hearts have low levels of FTO and elevated levels of mRNA methylation. Delivering exosomes with extra FTO to these cells might help them maintain healthy levels of FTO and decrease the chance of heart failure; this approach holds tremendous promise for the treatment of heart disease.

Dr. Sahoo’s research on exosomes is not limited to the failing heart. Recent work from her group suggests that a specific type of exosome, known to carry a marker called CD34 on its surface, improves angiogenesis, or formation of new blood vessels. Angiogenesis is a crucial step in healing after an injury. Dr. Sahoo’s group has shown that exosomes are able to improve healing in mice by providing microRNAs important for angiogenesis to cells near the site of injury. This work is not only important in helping patients after an injury, but it also teaches us about fundamental roles of microRNAs in angiogenesis and gene regulation.

We have outlined only some of the work going on in Dr. Sahoo’s lab. You can visit her website or watch her recent ERCC seminar to learn more about exosomes and her research on their role in cardiac medicine.

Therapeutic exosomes and Huntington’s disease
Extracellular vesicles, specifically exosomes, are currently being explored as therapeutic delivery systems for disease-targeting RNA molecules. In a talk at the ERCC9 conference, Reka A. Haraszti, M.D., a researcher in Dr. Anastasia Khvorova’s group at the University of Massachusetts Medical School, described how exosomes could be used to treat Huntington’s disease, a progressive neurodegenerative disorder. There are currently no effective therapies for this illness, which is caused by a mutation in the Huntingtin gene. Exosomes capable of transporting molecular payloads designed to silence the defective Huntingtin gene represent a potential therapy for this fatal disease.

Comparison of exosome production methods
Technical challenges in the large-scale production of exosomes currently limit their utility for disease treatment. To address this issue, Dr. Haraszti and Dr. Khvorova’s group teamed up with MassBiologics to develop and compare two different exosome production methods for yield and therapeutic efficacy of the exosomes. They utilized Tangential Flow Filtration (TFF) and ultracentrifugation to isolate exosomes from the conditioned media of cultured mesenchymal stem cells.

In TFF, conditioned media is continuously swept along the surface of a filter while a downward pressure is applied to force molecules through the filter. This process is like shaking a sifter to concentrate large particles blocking the holes in the filter, allowing smaller particles to pass through. In contrast, ultracentrifugation works by placing the conditioned media in a column of viscous fluid and spinning rapidly to separate extracellular vesicles in the media by their differing densities.

The researchers found that isolation by TFF resulted in 10-100 times more exosomes than ultracentrifugation. TFF-generated exosomes were also more heterogenous and contained 10 times more protein.

Exosomes produced by TFF and ultracentrifugation were also studied for their therapeutic potential in Huntington’s disease. After purification, exosomes were loaded with small interfering RNA (siRNA) molecules that could silence the expression of the mutant Huntingtin gene in target cells that take up the exosomes.

In cell cultures of primary neurons, TFF-generated exosomes showed greater inhibition of Huntingtin expression than those generated by ultracentrifugation. Moreover, TFF-generated exosomes infused into the brains of mice suppressed Huntingtin gene expression in vivo.

Future for therapies using exosomes isolated by Tangential Flow Filtration
The findings of Dr. Haraszti’s group indicate that Tangential Flow Filtration can generate a higher yield of exosomes for clinical use than older methods. Further, TFF-generated exosomes were effective gene therapy agents in experimental models of Huntington’s disease, and hold promise as delivery systems for clinical treatments.
Related Mini-conference

There is an upcoming mini-conference on EV manufacturing and isolation, a topic closely related to the research described here. The in-person conference is in Gainesville, Florida, but a webcast will also be available for those who want to participate remotely.
1. Haraszti RA, et al. Loading of extracellular vesicles with chemically stabilized hydrophobic siRNAs for the treatment of disease in the central nervous system. Bio-protocol (2017) 7: e2338. doi: 10.21769/BioProtoc.2338
2. Schwartz L., and Seeley K. Introduction to tangential flow filtration for laboratory and process development applications. Retrieved from https://laboratory.pall.com/content/dam/pall/laboratory/literature-library/non-gated/id-34212.pdf
3. Sunkara V, Woo HK, and Cho YK. Emerging techniques in the isolation and characterization of extracellular vesicles and their roles in cancer diagnostics and prognostics. Analyst (2016) 141: 371-81. doi: 10.1039/c5an01775k
4. Synder Filtration. “Characterization of polymeric, porous membranes: UF/MF & solute rejection measurements.” Retrieved from http://synderfiltration.com/learning-center/articles/membranes/characterization-of-polymeric-porous-membranes/

A recent study by Wei et al., 2017 catalogs the composition and characteristics of extracellular RNA (exRNA) secreted via three different routes from parent cells. The work provides novel insights into the biology of exRNA transport and intercellular communication, as well as the clinical potential of exRNA as a biomarker of disease.

The senior investigator of the study, Anna Krichevsky, Ph.D., at Brigham and Women’s Hospital and Harvard Medical School described the rationale for the study: “To understand the functions of exRNA complexes, we first have to define the exRNA repertoire with minimal bias.”

The exRNA Composition of Microvesicles, Exosomes, and Ribonucleoproteins (RNPs)
Dr. Krichevsky’s group sequenced RNA in microvesicles, exosomes, and extravesicular ribonucleoproteins (RNPs) isolated from glioma stem cells. They found that the majority of exRNA in all three fractions is noncoding. Although most exRNA studies focus on one class of noncoding RNA called microRNA (miRNA; 21-25 nucleotide molecules that repress gene expression), they reported that <10% of exRNA secreted by glioma stem cells is miRNA.

Comparing the profile of exRNA isolated from RNPs and extracellular vesicles (EVs) — including both exosomes and microvesicles, they found that RNPs contain higher amounts of noncoding cytoplasmic Y RNA and transfer RNA (tRNA) fragments.

Y RNA folds into a characteristic stem-loop structure and was originally found in protein-RNA complexes of individuals with autoimmune diseases. According to Dr. Krichevsky, “Despite abundant expression in all vertebrate cells, the physiological functions of Y RNA are only beginning to emerge.” Some evidence suggests that Y RNA plays a role in DNA replication and RNA quality control. Y RNA fragments may also be involved in cell death, ribosomal RNA maintenance, and histone gene expression.

tRNA is well known as a mediator of the translation of mRNA to protein. However, recent studies suggest that tRNA fragments found in exRNA are involved in regulating gene expression during cellular stress responses. Dr. Krichevsky discussed the implications of extracellular tRNA on cellular communication: “Based on the exRNA levels and biological functions of tRNA, we hypothesize that transferred tRNA transcripts can have a major impact on recipient cells.”

Along with noncoding RNA, a small proportion of gene-encoding messenger RNA (mRNA) was detected in extracellular vesicles and RNPs. Previous studies have also found extracellular mRNA; however, they did not determine if the mRNA transcripts were intact or fragmented. Dr. Krichevsky’s group was the first to show that short (<1000 nucleotides), endogenous full-length mRNAs can be packaged into exosomes, and microvesicles contain even longer mRNAs. Fragments of long mRNA transcripts were also present in exRNA.

Using exRNA as a Biomarker
Researchers are currently exploring the use of exRNAs as potential biomarkers for the diagnosis and monitoring of diseases. However, many exRNA biomarker studies have been limited in scope because they examined a heterogeneous pool of exRNA purified from an unfractionated collection of EV types.

By fractionating conditioned media from glioma stem cells into microvesicle, exosome, and RNP fractions, Dr. Krichevsky’s group was able to compare their RNA profiles with those of the parent cells. They found that the RNA content of microvesicles most closely resembled that of the parent cells, making this type of exRNA carrier a good candidate for disease biomarkers.

Dr. Krichevsky said, “We believe there is more intact mRNA in microvesicles, which we can consider for biomarkers. We can think about genes that are mutated. On the other hand, miRNAs are more enriched in the exosomes. It would be great if we could detect cancer mutations and non-coding RNA biomarkers in biofluids; then we would not need to do a biopsy.”

By developing novel experimental approaches to illustrate that exRNA composition differs by exRNA carrier, Krichevsky’s group has made significant contributions to exRNA research. Moreover, they highlighted that exRNA contains more than the well-studied miRNAs, including full-length mRNA molecules, Y RNA, and tRNA. Their data also indicate that the RNA profile of microvesicles is most similar to that of the cell of origin, including the presence of full-length mRNAs, making microvesicle exRNA a good candidate for some disease biomarkers.

In an interview, Dr. Krichevsky discussed the importance of this study: “Our work changed the way people thought about exRNA by showing them the exRNA in numbers, which helps appreciate their heterogeneity and the overall impact. The field is shifting from focusing on a specific extracellular miRNA to now considering that there are thousands of different RNAs present in extracellular complexes.”

Wei, Z. et al. Coding and noncoding landscape of extracellular RNA released by human glioma stem cells. Nature Communications (2017) 8:1145. doi:10.1038/s41467-017-01196-x