December 19, 2023 | A decade ago, the National Institutes of Health (NIH) launched a program designed to answer fundamental questions about extracellular RNA (exRNA), such as how it is used by cells and distant organs and if diseased and healthy cells produce different varieties of it. The program concludes at the end of this year but leaves science with technologies to address some of the biggest bottlenecks encountered by researchers in the niche field of study, according to Christine Happel, Ph.D., program officer for the Extracellular RNA Communication Program.
One of the biggest holdups has been a means to discriminate between exRNA subclasses and understand the heterogeneity of different exRNA carriers, she says. To that end, investigators at Vanderbilt University Medical Center (VUMC) have discovered so-called “supermeres,” functional nanoparticles that could potentially serve as disease biomarkers and therapeutic targets for various cancers as well as Alzheimer’s and cardiovascular disease (Nature Cell Biology, DOI: 10.1038/s41556-021-00805-8).
And when the COVID-19 pandemic struck, some of the technologies developed through the Extracellular RNA Communication Program could be repurposed for the public health crisis through the Rapid Acceleration of Diagnostics Radical (RADx-Rad) initiative because of the similarities between extracellular vesicles (EVs) transporting exRNA and SARS-CoV-2 variants (Viruses, DOI: 10.3390/v14051083), says Happel. Researchers at The Ohio State University are in the approval process with the Food and Drug Administration (FDA) on an Emergency Use Authorization (EUA) submission for the new diagnostic test.
The Extracellular RNA Communication Program is funded by the NIH Common Fund supporting bold scientific initiatives intended to catalyze discovery across biomedical and behavioral research as well as collaboration between multiple NIH institutes/centers and the broader scientific community, she says. “We once thought that RNA only existed within a cell, but now we know that RNA can be exported from cells and plays a role in cell-to-cell communication, but there are a lot of unknowns involved in how that works. It is still a bit of a Wild West.”
The Extracellular RNA Communications Program launched in 2013 and the second phase got underway in 2019, when the focus shifted to filling critical gaps in knowledge and technology related to separating and characterizing bulk populations of exRNA carriers as well as single EVs, says Happel. By the end of the first phase in 2018, it was clear progress was being stymied by inconsistencies in the research due to technical and biological variability.
Technologies developed during phase 2 of the program are starting to come to fruition now, she reports. The task has become disseminating and popularizing those tools within the wider scientific community so that some of the basic questions being tackled during phase 1 can be addressed.
In this final stage of the program (iScience, DOI: 10.1016/j.isci.2022.104653), two types of technologies are under investigation, continues Happel. One looks at highly complex biofluids and separates them into homogenous populations and the other examines single EVs, the most common type of exRNA carrier, and assesses their cargo. Many currently available tools for looking at single cells lack the ability to peer at these much smaller EVs, she notes.
The carrier separation group includes a technology to isolate and inventory the contents of distinct populations of small EVs and nanoparticles so that their cargo could be assigned to the correct carriers, which was used by the lab of Bob Coffey, M.D., at VUMC to discover supermeres (short for supernatant of exomeres). Supermeres are essentially non-vesicular nanoparticles that carry exRNA, the role of which remains a mystery, says Happel.
Coffey and his team were able to purify these supermeres using a specific, high-speed ultracentrifugation process. They are extremely tiny and distinct from many other exRNA carrier populations, she adds.
A dozen awards were made during phase 2 of the Extracellular Communication Program, five of which were devoted to carrier separation and six to single EVs. There was also a single data management and dissemination awardee that has been in the role for the entirety of the program.
At the end of phase 1, which had 32 total awardees looking at a wide breadth of different topics, Cell Press published a package of 18 papers highlighting some of the major discoveries. A second bundle of papers will be published covering data coming out of the more focused phase 2 of the program, says Happel.
Over the course of the program, the Extracellular RNA Communication Consortium Research Portal has been the go-to place for output from the work of awardees, she adds. It has been tapped by newcomers to the field looking for protocols and information to get started as well as veteran exRNA biologists who want to reevaluate previously published data or stay up to speed with the “latest and greatest” happenings.
Extracellular RNA has tremendous diagnostic and therapeutic potential because of the specificity and immunogenicity of EVs, says Happel. That makes them an attractive alternative to currently available cell therapy products.
Tissue-on-a-chip platforms are increasingly being applied to the study of EVs and the characterization of exRNA, Happel continues. EVs can also be used for regenerative medicine, as a growing body of evidence suggests they are an appropriate and hopeful new source for tissue repair and restoration of lost organ function. On a heart-on-a-chip model, EVs derived from stem cell sources have been found to induce cardioprotective effects during ischemia-reperfusion injury, highlighting their therapeutic potential (Science Translational Medicine, DOI: 10.1126/scitranslmed.aax8005).
From the perspective of the National Center for Advancing Translational Science (NCATS), where Happel is program officer in the Office of Special Initiatives, interest in EVs and exRNA is tied to its mission to translate knowledge into treatments for patients in real-world healthcare settings, she says.
Stumbling Into Supermeres
The Vanderbilt team stumbled into supermeres following a series of experiments initiated by the discovery that one of the growth factors that binds to the epidermal growth factor receptor (EGFR) gene is released in EV-housed cells, according to Coffey, professor of medicine at VUMC. The field, at the time, was a “bit tarnished” by extravagant claims being made about the high-speed pellets created by the specialized ultracentrifugation process such that results, in some instances, could not be reproduced. The pellets had been called exosomes but in fact consisted of other material.
Coffey and his team came up with a more rigorous approach to isolating EVs by spinning them in a bottom-loaded 12-36% OptiPrep gradients at a centrifugal force of 120,000 × g for 15 hours (Nature Protocols, DOI: 10.1038/s41596-023-00811-0). It was then possible to take individual fractions and load them onto a gel to identify the exosomal fractions as defined by those containing the tetraspanins CD9, CD63, and CD81.
Proteins co-migrating with these fractions qualified as “exosomal cargo,” says Coffey. However, many proteins didn’t track with the exosomal fractions but were instead in the so-called non-vesicular fraction.
To complicate matters further, he points out, exosomes are not the only small EVs out there. In addition, there are large EVs called microvesicles that bud from the cell surface.
A sophisticated tangential flow microfluidics device was subsequently used by David Lyden, M.D., Ph.D. (Weill Cornell Medicine), to isolate non-vesicular, non-membranous “collection of proteins” lacking the expected lipid biolayer, Coffey says. These were termed exomeres.
Since the device was unaffordable for Coffey and his team, they decided to just take the supernatant from the exosome pellet and spin it faster, he continues. Happily, the tactic succeeded in finding exomeres with nearly identical cargo that Lyden had found with the pricier piece of equipment.
On a second go-round, they spun the supernatant even faster to discover supermeres. These are also non-membranous collections of proteins, but atomic force microscopy has shown that they also have features making them distinct from exomeres.
“So, now we have three different particles [EVs, exomeres, and supermeres] that we are particularly interested in, and they all have different sets of cargo, and we think that they may be very useful in terms of diagnosis or possibly as predictive biomarkers, and a number of them have potential therapeutic targets we might be able to go after,” says Coffey.
While Lyden is a “lumper,” measuring whatever his isolation exercise turns up, Coffey’s lab is populated with “spliters” who see the value in looking at what is being carried by each type of nanoparticle. Their rationale is the possibility of assigning cargo, be it protein or RNA, to the correct carrier. “Sometimes you can’t measure an analyte in the whole serum or the whole plasma, but you can highly enrich for that protein if you... isolate those different fractions [by centrifuging the plasma].”
The Coffey lab is interested in learning what kind of information is carried by supermeres and how the nanoparticles impact other cells—T cells, macrophages, and fibroblasts in the tumor microenvironment, for example. The team has already discovered that when supermeres are added to macrophages they induce the release of immunosuppressant cytokines. “That may be a way that supermeres help promote a tumor, by releasing a protein that would prevent the immune system from coming in to help block tumor progression,” says Coffey, noting this is an active area of investigation.
Colon cancer has been a research priority, including experiments to determine why supermeres are able to confer resistance to the EGFR monoclonal antibody cetuximab, he continues. Candidates for the treatment are patients who don’t have a KRAS mutation.
But the therapeutic potential of supermeres extends well beyond colon cancer, says Jeff Franklin, Ph.D., research assistant professor in the Coffey lab. Much of the cargo of supermeres is in fact related to neurodegeneration.
The study that was published in Nature Cell Biology was led by Qin Zhang, Ph.D., VUMC research associate professor of medicine, who found that supermeres travel throughout the body and can cross the blood-brain barrier. It is speculated that some supermeres may be regulated by different disease processes, says Franklin, and might also be used as a delivery vehicle, as artificial nanoparticles are now, for therapeutic drugs.
Supermeres contain a lot of RNA as well as nucleic acids, making them potentially useful as couriers of nucleic acid drugs targeting disease at the genetic level, he adds. In terms of diagnostics, investigators are hoping to detect supermeres in the plasma of cancer patients to replicate their feat in the lab.
Exploring Clinical Utility
Most recently, the Coffey lab published a study in Cell (DOI: 10.1016/j.cell.2023.11.006) suggesting that it may be possible to expand the use of immunotherapies for treating microsatellite unstable colon cancer—representing the roughly 10% of cases where patients aren’t considered candidates for the approach because it doesn’t confer meaningful remissions. The team has found a four-gene signature in microsatellite stable colon cancer (the other 90% of patients) that correlates with immune exclusion, specifically the ability to exclude CD8+ T cells from the tumor proper.
“Three of those genes encode proteins that are highly upregulated in either EVs or supermeres,” reports Coffey, including a transmembrane protein called DDR1 carried by supermeres. The ectodomain shed by DDR1 can alter the microenvironment so T cells can’t get to the tumor.
Vanderbilt researchers have just partnered with Incendia Therapeutics to launch a trial testing the ability of DDR1 neutralizing antibody PRTH-101 to block that shed ectodomain, he notes. “The goal now is to deliver the antibody and then give immunotherapy.”
Other collaborative work is directed at finding ways to measure the cargo within non-membranous nanoparticles more easily, Coffey says. A colleague studying glioblastoma has found elevated levels of some of the same supermere proteins that are highly upregulated in colon cancer. Justus Ndukaife, assistant professor of electrical and computer engineering at Vanderbilt, has been leading efforts to better trap individual fluorescently-labeled supermeres to analyze their physical characteristics (Nano Letters, DOI: 10.1021/acs.nanolett.3c02014).
Development of the COVID-19 diagnostic test at The Ohio State University was under the leadership of Eduardo Reategui, Ph.D., associate professor of chemical and biomolecular engineering, and enabled by the RADx-Rad initiative. But the work started with the Extracellular RNA Communication Consortium (ERCC), specifically a project focused on new technologies for characterizing exRNA carriers at the single vesicle level, he says.
Initially, their approach had been “bulk analysis” of various exRNA in EVs, by lysing them to extract the RNA cargo, continues Reategui. While this was a good first step, it told them nothing about the particles’ tissue of origin or subpopulations. Platforms for analyzing them at the single vesicle level helps address that issue.
Reategui’s group is one of a handful of scientists developing this type of technology, he points out. The goal with the single EV approach is to analyze the RNA in situ without losing the identity of each vesicle or particle. That makes it possible to know not only if EVs came from, say, cancer cells, but also how a patient is evolving in response to therapy based on the analysis of a subpopulation of particles, including lipoproteins, which are another class of exRNA carrier particles.
The technology enables researchers to individually examine EVs and their RNA contents, including proteins inside and on their surface, Reategui says, noting that he and his team have had several studies published on this point (Journal of Extracellular Vesicles, DOI: 10.1002/jev2.12369). They pivoted to COVID-19 when the pandemic struck and NCATS started looking for exosome technologies that could be repurposed for SARS-CoV-2 detection.
EVs and envelope viruses are quite similar, he says, since they are both nanoparticles surrounded by a lipid membrane. “In the case of the EVs you are talking about RNA, DNA, and proteins, and in the case of the SARS-CoV-2 virus you just have the messenger RNA,” says Reategui.
It seemed conceivable that with a few tweaks their single EV platform could be used for SARS-CoV-2 detection—and, importantly, be done in situ without destroying the viral particles, he says. That is, they could test for the presence of surface antigens and viral RNA inside each particle at the same time.
The advantage of this diagnostic methodology is that it provides antigen and molecular testing in a single assay “but with much higher sensitivity because you are not doing any sort of amplification,” explains Reategui. Results of a study using the integrated detection tool, available as a preprint, indicate the high-throughput assay is more sensitive than the gold-standard PCR test.
The platform can target specific mutations of the SARS-CoV-2 virus, as Reategui and his team extensively demonstrated in validation studies with different cohorts and types of biofluids, including plasma, saliva, and nasopharyngeal swabs. It is now in the process of approval of EUA by the FDA, which when granted will make it a permanently marketable device.
While demand for COVID diagnostics has waned, the integrated antigenic and nucleic acid detection platform can be used as a panel for diagnosing multiple diseases at once, he points out. Since the technology analyzes nanoparticles in situ, it can also be used to investigate the EVs themselves. “For example, we can trace residual viral RNA in the EVs in patients who are probably already fully recovered from the initial infection but may go into what’s called long COVID.”
One of the big studies published by Reategui and his colleagues relates to their EV analysis specific to non-small cell lung cancer, which he likens to simultaneously doing a molecular (PCR) and ELISA test. The clinical need is not so much in terms of detection but prediction of patient response to immunotherapy, which is where their investigative sights are now set along with early detection enabled by the higher sensitivity of the dual approach (Journal of Extracellular Vesicles, DOI:10.1002/jev2.12258).
The technology has also been extensively used for testing in glioblastoma, where the objective is to characterize the number of microRNAs and messenger RNAs that serve as biomarkers of the largely fatal brain cancer, says Reategui. Even when diagnosed through imaging, once the tumor is resected it is difficult to tell the difference between pseudoprogression or tumor growing.
The largest cohorts will be needed to test the system for the diagnosis of different cancers across different stages, to identify the best biomarkers of disease progression, he adds. “Overall, what we are trying to prove is that our liquid biopsy approach has a lot of promise... [as] an alternative to an imaging or tissue biopsy method.”
The diagnostic possibilities extend well beyond cancer. In fact, Reategui is collaborating with faculty at The Ohio State Wexner Medical Center to use the platform for detecting the molecular markers of neurodegenerative diseases such as multiple sclerosis. “The proof of concept is all the data we have produced over the last three to four years that hopefully is going to carry us to the next set of studies,” he says. Being part of the ERCC has facilitated collaboration with outside groups so that the different technologies used for sample processing can be integrated into the workflow.