By Deborah Borfitz
June 8, 2023 | A technique for isolating urinary extracellular vesicles (EVs) released by cells across the body has enabled the discovery of previously undetectable biomarkers for Parkinson’s disease (PD) in the protein cargo of the nanoparticles. EVtrap—short for extracellular vesicles total recovery and purification—is now the foundation of Purdue University spinoff Tymora Analytical Operations, co-founded by W. Andy Tao, professor of biochemistry, and Anton Iliuk, the company’s president and chief technology officer who received his doctorate in biochemistry from Purdue.
This opens the possibility of a liquid biopsy test for PD and other neurodegenerative diseases which, unlike cancer, are not genetically driven, says Tao. The G2019S mutation in the LRKK2 (leucine-rich repeat kinase 2) gene, the most dominant one in PD, only applies to about 1% to 2% of all Parkinson’s disease patients.
An exhaustive analysis of EVs found in urine samples from 138 individuals pointed to a unique panel of proteins in people with PD with and without that mutation relative to matched groups of healthy individuals, including people who are non-manifesting carriers of the mutation, he explains. Multiple proteins elevated in the EVs of those in the PD groups are known to be involved in important pathways of the disease, and a panel of six candidate biomarkers were partially validated, as reported recently in Communications Medicine (DOI: 10.1038/s43856-023-00294-w).
Diagnosing PD is currently a difficult proposition involving cognitive and movement testing that can take many months to confirm, delaying detection, says Iliuk. Finding disease earlier from a biofluid, via routine screening, would be groundbreaking because treatment could begin sooner when therapies are most effective.
EVtrap is based on magnetic beads coated with molecules that have strong affinity to EVs and pulls them out of urine or blood samples for analysis by mass spectrometry or any other method of choice. The nanobubbles carry not only proteins but just about every other molecule found in cells, including lipids, metabolites, RNA, and, according to some reports, DNA. “It’s a rich source of information,” Iliuk says.
Tymora Analytical can identify over 1,000 unique proteins from EVs using less than a drop of biofluid, he reports. The company was founded in 2010 to develop nanoscale platform technologies for unmet needs in analysis of protein phosphorylation related to the onset of numerous diseases, most notably cancer.
The first large studies on EVs by their group, in 2017, were for breast cancer, says Tao. In a breakthrough study that was published in Proceedings of the National Academy of Sciences (DOI: 10.1073/pnas.1618088114), researchers succeeded in identifying thousands of phosphorylated proteins in EVs from human plasma with their approach. Centrifuges were used to separate plasma from red blood cells, and high-speed and ultra-high-speed centrifuges to further separate microvesicles and exosomes. Subsequently EVtrap was introduced to make the isolation clinically friendly.
The Michael J. Fox Foundation approached the pair about implementing the same approach in individuals with PD to tease out a biosignature of the disease. Unlike cancer, liquid biopsy methods such as circulating tumor cells and circulating tumor DNA do not work for PD detection, Tao says. “For neurodegeneration studies, you had to go through the brain barrier to get to the samples [via lumbar puncture].”
As the latest study has shown, changes in urinary EV proteins can be a proxy for changes in brain proteins seen in PD disease. An earlier paper drew a connection between LRRK2 and brain proteins in urine samples. “The putative signature that we have detected is generalized to all Parkinson’s disease patients,” notes Iliuk.
Urine samples came from the LRRK2 Biobanking Study at Columbia University. The sample sizes used in the study with EVtrap—including about 80 subjects for the main experiment and another 60 to substantiate the biomarkers using machine learning—were too small to claim any true biomarker discovery, Tao says. “Because of this limitation, the proteins we identified still need a lot of further study.”
Importantly, many of the candidate biomarkers identified in the study were previously found to have a potential relationship with the neurodegeneration seen with PD. But the proteins were previously detected in animal studies or from patient tissue samples rather than noninvasively from human biofluid—making it a “game-changer,” says Iliuk.
Some of the markers, while relatively new, are part of pathways that can lead to neurodegeneration as well as neuroinflammation, which is now suspected to be a major reason patients are robbed of their cognitive abilities, he adds. It may seem counterintuitive that urine, produced by an organ so distal from the brain, would be a great source of these markers “but it actually is tremendously impactful.”
EV nanobubbles carry a range of nucleic acids and proteins that enable cell-to-cell communication via interaction on the cells surface, says Iliuk, which he describes as “the internet of the body.” They trigger body responses that differ depending on whether the signal is coming from a healthy or diseased cell.
Given that EVs are released by every type of organ, a major mission of Tymora Analytical Operations is to see this “universal technology” employed in the discovery of the biosignature of multiple diseases beyond neurodegenerative diseases and cancer, Tao says. But those are the current focus of researchers.
Of particular interest are kidney, endometrial, breast, and prostate cancers, all areas where EVs have shown diagnostic promise, says Iliuk. A major Alzheimer’s disease project is also underway using blood for EV detection.
A couple studies specific to diabetes have already published, one finding a heightened risk of severe COVID-19 in patients with comorbid type 2 diabetes (Diabetes Research and Clinical Practice, DOI: 10.1016/j.diabres.2023.110565) and another describing how the EVs can provide insights into the pathobiology of diabetes and alterations in EVs can occur early in prediabetes (International Journal of Molecular Sciences, DOI: 10.3390/ijms23105779). EVtrap has even been deployed for the discovery of snake venom biomarkers to aid the search for potential treatments for bite victims (Toxins, DOI: 10.3390/toxins13090654).
EVs were discovered several decades ago, but the field of study exploded after it was found that their cargo could be used diagnostically to identify disease, says Iliuk. The group behind the study started Exosomes Diagnostics, the most successful diagnostic company working in the EV space. Its ExoDx Prostate test is now covered by Medicare for high-risk men with a prior negative biopsy that are considering a repeat biopsy.
Bio-Techne acquired the test in 2018 through a $575 million deal for Exosome Diagnostics. A year later, the ExoDx Prostate test became the first liquid biopsy test to receive breakthrough status from the Food and Drug Administration for targeting exosomes. It is included in guidelines from the National Comprehensive Cancer Network for early detection of prostate cancer and has been a major revenue generator for the company, Iliuk says.
Getting at the cause of Parkinson’s disease, and how best to treat it, could take a good bit longer, says Tao, in talking about the many current avenues of exploration. Recent theories have implicated a common gut bacterium as a causal factor and suggested that oral hygiene could mediate some of the symptoms. “There are a lot of things we still don’t understand, but diagnosis for early detection is a highly important part of the research.”