Novel Approach to Isolating and Quantifying EV Cargo for Disease Diagnosis

Parkinson's disease (PD) and Alzheimer's disease (AD) are examples of brain illnesses that begin to develop in patients far earlier than the onset of their initial clinical symptoms. There is currently no technique to diagnose brain illnesses at such pre-symptomatic phases, yet treating individuals at these early stages could slow or even stop their sickness.

For instance, brain biopsies, which are only available posthumously, are now the only way to identify the precise brain lesions brought on by Parkinson's disease.

The novel idea of “liquid biopsies,” which entails the simple extraction of blood or other bodily fluids using non-invasive techniques and their analysis for molecules coming from the brain and other solid tissues, has been pursued by researchers to get around this crucial bottleneck.

Extracellular vesicles (EVs), which are small, membrane-bound sacs released into bodily fluids by the brain and other cells, are a particularly interesting target in bodily fluids. These sacs may also include protected biomarkers for the early start of Parkinson's disease and other brain disorders since they contain a range of compounds that are specific to the cell types that make them, such as the brain.

EV specialists have not been able to address the issue of whether specific biomarker molecules they measured in separated EVs are non-specifically linked to their surface or strictly contained inside EVs, despite recent advancements. This difficulty has hindered their ability to draw clear findings regarding cargo molecules in EVs from a variety of tissues.

 By adding a critical step to a previously verified ultra-sensitive technique, a team led by David Walt, Ph.D. of Brigham and Women's Hospital (BWH) in Boston and the Wyss Institute at Harvard University has now resolved this issue. They were able to precisely target cargo that was protected inside EVs while removing general “contaminations” by enzymatically breaking down all surface-bound proteins from a purified EV population.

For the first time, scientists were able to precisely identify the minuscule percentage of any protein stored within EVs compared to the amount of it present free in total blood plasma using their improved procedure to assess the PD biomarker ⍺-synuclein in blood.

Crucially, they combined this development with a recently created ultra-sensitive detection assay for a kind of α-synuclein that becomes more phosphorylated as Parkinson's disease (PD) and its associated disorder, Lewy Body Dementia, progresses.

They were able to identify an enrichment of the pathogenic ⍺-synuclein protein among EVs in comparison to total plasma by analyzing a cohort of patient samples. The results are released in PNAS.

Research on EVs in our and other groups over the last few decades has steadily advanced our understanding of their complex biology and molecular composition. Yet, the isolation of pure tissue-specific EVs from body fluids like blood or the cerebrospinal fluid surrounding the central nervous system, including the brain, and validating and quantifying their true contents with precise measurements still present formidable technical challenges.”

Hansjörg Wyss, Professor, Biologically Inspired Engineering, Wyss Institute  

Hansjörg Wyss adds, “Our recent work is providing a solution to help fill this technological gap, and gets us closer to being able to obtain EVs free from contamination to use them as rich sources for clinical biomarkers, as we show with the example of phosphorylated ⍺-synuclein.”

Walt is also a faculty lead of the Wyss Institute’s Diagnostic Accelerator, is also the Hansjörg Wyss Professor of Biologically Inspired Engineering at Harvard Medical School (HMS), a Professor of Pathology at Brigham and Women’s Hospital, and a Howard Hughes Medical Institute Professor.

From Blood to EVs to Biomarkers to Diagnosis

The Walt group has been methodically adding crucial pieces to this technical jigsaw puzzle, especially inspired by the diagnostic promise of EVs for the early identification of PD, AD, and other brain illnesses.

 They previously created a technical framework for measuring EVs and utilized this quantification to assess EV extraction techniques from bodily fluids, with philanthropic sponsorship from Good Ventures, the Chan Zuckerberg Initiative, and most recently the Michael J. Fox Foundation.

Their approach combines ultra-sensitive “Simoa assays” that enable them to count individual protein molecules linked to EVs that they have captured and seen using certain antibodies with a separation technique called size exclusion chromatography (SEC) to recover the majority of EVs from biofluids.

To identify brain-specific EVs, the team has now developed Simoa assays for several EV-specific biomarkers and, crucially, eliminated L1CAM, a commonly used candidate surface protein, as a target. This has given the field a significant course correction.

To answer the conceptually simple but technically challenging question of what percentage of a given protein (such as ⍺-synuclein) present in plasma is inside of EVs relative to outside, we used SEC isolation methods that we previously developed to isolate most EVs from plasma together with an optimized ‘proteinase protection assay’ where we use an enzyme to gently but efficiently chew all proteins off the surface of isolated EVs while leaving the membrane-enclosed EV interior intact.”

Dima Ter-Ovanesyan, Study Co-First Author and Senior Scientist, Wyss Institute

Dima Ter-Ovanesyan also spearheads the EV project with Co-First Author and Postdoctoral Fellow Tal Gilboa, Ph.D.

Additionally, Gilboa, Postdoctoral Fellow Gina Wang, Ph.D., and Wyss Research Assistant Sara Whiteman in the Walt lab created a Simoa assay for ⍺-synuclein that is significantly more sensitive than any previously published assay to quantify ⍺-synuclein at very low levels.

By applying this test in their methodology, the researchers were able to ascertain that less than 5% of the total blood plasma ⍺-synuclein was present in EVs separated using their SEC protocol, and that the majority of the ⍺-synuclein in these EVs was protected.

Since EVs originating from a particular organ, such as the brain, are predicted to be rare in comparison to EVs from blood cells, where ⍺-synuclein is also expressed, knowing this number is especially crucial for the ultimate goal of quantifying ⍺-synuclein in neuron-derived EVs.

 Crucially, they have created an assay that can detect ⍺-synuclein that becomes phosphorylated at a specific point (pSer129) throughout the evolution of Parkinson's disease (PD), in addition to their ultra-sensitive Simoa assay that allowed them to detect the normal, unmodified ⍺-synuclein protein.

“When we applied our advanced methodology to a cohort of blood samples obtained from patients with PD and Lewy Body Dementia as well as healthy control donors, we found that the ratio of phosphorylated ⍺-synuclein relative to total ⍺-synuclein was two to three-fold higher inside EVs relative to the outside of EVs,” said Gilboa.

Gilboa said, “This was extremely exciting because it suggests that EVs may protect the phosphorylation state of proteins from circulating phosphatases that would otherwise erase this highly informative mark.” 

The team is currently investigating further if these assays could be used to distinguish between individuals with Parkinson's disease and those who do not.

The work by David Walt’s team presents a technological tour-de-force that brings us closer and closer to a next-generation diagnostic platform with extraordinary potential. At this point, we are not far from using these extremely rich and telling cell-derived vesicles as a window to peak into the brains of patients without requiring surgery.”

Donald Ingber, M.D., Ph.D., Founding Director, Wyss Institute

Donald Ingber is also the Judah Folkman Professor of Vascular Biology at HMS and Boston Children’s Hospital and the Hansjörg Wyss Professor of Biologically Inspired Engineering at Harvard’s John A. Paulson School of Engineering and Applied Sciences.