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Johns Hopkins, NanoView Biosciences Using EVs to ID HIV-Related Neurological Disorder Biomarkers

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NEW YORK – Backed by nearly $1 million from the National Institutes of Health, an international group spearheaded by researchers from Boston-based NanoView Biosciences and Johns Hopkins University seeks to apply extracellular vesicles (EVs) to detect HIV-associated neurological disorders, Alzheimer's disease, and Parkinson's disease. 

NanoView will run patients' blood plasma samples on its microparticle detection platform to develop custom assays that identify EVs containing biomarkers potentially linked to the neurological disorders. 

Founded in 2014 by Chief Operating Officer David Freedman and CSO George Daaboul, NanoView is commercializing an automated instrument called ExoView, which is based on imaging technology developed by researchers at Boston University. NanoView CEO Jerry Williamson explained that BU has licensed out the IP to the firm, which later spun out from the university's incubation lab in July 2018. The ExoView platform consists of an ExoView R100 reader, a consumable kit, as well as acquisition and analysis software.

A researcher using the platform begins by pipetting about 35 μl of a plasma sample in up to nine microchips. According to Daaboul, each chip can examine between 8 to 12 biomarkers in the plasma sample. After the chips incubate overnight, the researcher washes the samples off the chip and introduces labeling antibodies for single vesicle biomarker colocalization.  

Washing off the labeling antibodies, the researcher then places the sample on the R100 reader and starts the assay. The instrument uses an assay-specific file along with the kit to automatically read the chips without any user input, providing particle size analysis, EV count, EV phenotype, and biomarker colocalization. 

Daaboul noted that the platform requires about half a day to fully characterize EVs.  

NanoView has raised over $1.1 million in academic grants to develop the ExoView system, including a $750,000 National Science Foundation Phase II Small Business Innovation Research grant last September. NanoView also raised a total of $14 million through Series A and B financing rounds, which helped the firm launch ExoView for RUO applications in February. 

NanoView currently offers three RUO antibody-based assays — ExoView Human Tetraspanin Kit, ExoView Tetraspanin Plasma Kit, and ExoView Mouse Tetraspanin Kit — in addition to customized assays that it aims to develop for collaborations with groups like JHU.

According to Daaboul, JHU molecular and comparative pathobiology associate professor Kenneth Witwer participated in NanoView's early-access program. In the new initiative, Daaboul said that the JHU team wants to use ExoView to analyze subsets of EVs and examine their heterogeneity. 

"Basically, we're enabling [the Witwer lab] to make these multiplex arrays with different capture agents to isolate the different cell populations on a chip," Daaboul explained. 

Witwer noted that the researchers are partnering with NanoView on the new initiative because of their shared previous collaborations on HIV and HIV-related neurological research. 

"The platform has let us perform single vesicle analysis, since to really understand what's in the disease, you need to be cognizant that the EVs in circulation are coming from everywhere," Witwer said. By doing single vesicle analysis on EVs stemming from the brain, Witwer's team can potentially identify the markers on the vesicle's surface and its cellular source, or even analyze the molecular cargo inside the vesicle.  

Witwer explained that HIV-positive patients often exhibit neurological symptoms because the virus infects the central nervous system using EVs containing its genetic material. The disease can move across the blood-brain barrier and cause neuroinflammation and neurodegeneration, which can lead to a variety of neurological conditions referred to as HIV-associated neurocognitive disorders. Witwer's team has therefore studied how EVs could inhibit or enhance infection caused by retroviruses like HIV. 

"We're using some of those particles to see what's happening in HIV-related diseases, so that they could be used as biomarkers," Witwer explained. "We are then examining what they're doing functionally and how the virus interacts with other parties that might exacerbate or stop the disease." 

Witwer's team believes that if it can selectively target EVs in blood samples, the group can identify biosignatures that it hopes reflect the disease. Witwer said the platform may allow the researchers to achieve spatial resolution and trace EV cargo across multiple biomarkers, which the group expects could lead to diagnostic and prognostic approaches. 

To further support this work, Witwer's team is establishing an international partnership that is part of the Extracellular RNA Communication Consortium Stage 2 (ERCC2), working with researchers from across several different academic institutions, including the National Institute of Standards and technology, the University of California, Davis, and the Institut Pasteur de Montevideo.

Using ExoView, Witwer's team will filter a variety of different particles in the bloodstream that can contain nucleic acids, including EVs, exosomes, and lipoprotein molecules.

"The technology we're trying to develop will separate different classes of biomarker carriers in the patient's circulation, but we want to make sure what we're tracing back to its source are EVs, and not some big lipoprotein," Witwer said. "We think that this opportunity to discriminate between the different types of particles will really help focus our research in the future to [select] the specific particle types that are most informative in the disease." 

Witwer's team will collect blood plasma samples from patients suspected to have one of the three neurological disorders. While the group has worked with cerebral spinal fluid, urine, and saliva, Witwer noted that blood plasma "seems to be the sample type we know the most about and where we have a lot of access to archived samples." 

After receiving initial blood plasma samples from Witwer's team, NanoView will create custom EV panels on its chips using EvoView R100, relying on the samples containing the targeted antibodies. Witwer's group will then use the custom panels as part of a "secondary detection" of specific EVs that it believes stem from the neurological disorders. 

According to Daaboul, NanoView's chips will help Witwer's team understand the proportion of certain particles and the overall composition of the biofluids. Witwer believes the researchers will be able to better comprehend how different particles are enriched or unenriched. 

As ERRC2 starts collecting blood plasma samples, Witwer expects that his team may initially struggle with using the proper reagents to identify antibodies — or any type affinity reagent — that are specific only to the cancer cell that the group is trying to detect. 

"If we find a positive event, we need to [ensure that] it's coming from the right cell type," Witwer said. "The specificity of our antibodies, and the specificity of the markers for one cell type is a major assumption we might make, which might not always be true."

However, Witwer noted that his team also doesn't want to be too dogmatic when searching for antibodies and their sources.

"As long as [the signal] is reproducible, then the source doesn't matter, since if you can identify something in a patient, it probably doesn't matter exactly what cell it came from," Witwer said. "But that does become important as we try to understand the underlying biology, and that's where we want to be clear about how good our reagents are." 

If Witwer's team successfully identifies neurological disorder biomarkers, it will have the opportunity to receive up to an additional $2 million from the NIH as part of a Phase II study and potentially develop a targeted diagnostic tool. Witwer hopes to develop a customized assay based on the ExoView platform that might provide up to five times the purity or speed of vesicle separation, compared to standard techniques such as ultracentrifugation. 

University of Sussex glioblastoma researcher Thomas Simon, who is not a part of ECCR2, believes that having a centralized effort that meets twice a year will help the international collaboration develop EV diagnostic technology. His own team at the University of Sussex is researching EVs to identify RNA biomarkers that might be linked to glioblastoma. 

However, Simon emphasized that collecting enough EV samples that might potentially lead to producing actionable results is normally very complicated and time consuming. 

"We're trying to do something like [ERRC2] ourselves in the UK [by] working with other labs and hospitals interested in researching EVs and [cancer]," Simon noted. "By increasing the number of samples, we may eventually find data that is actionably significant and make a difference in EV research."  

Future opportunities

NanoView may also pursue additional opportunities as the firm improves the technology in future iterations. 

Witwer envisions moving past antibodies as a molecular reagent on the ExoView platform, and instead using other affinity reagents such as proteins or genetic material. He believes NanoView could also optimize some of the instrument's parameters for customized experiments. This could include offering improved optical concentrations; faster EV analysis; better washing methods to reduce background noise; minimizing incubation times; as well as seeing whether researchers could simultaneously look at high and low biomarker concentrations in the vesicles.

"If we can find biomarkers by capturing populations of EVs and using [ExoView's] fluorescent capabilities, we can look at three biomarkers on all those captured populations," Witwer said. "This would give us the possibility to move to something that is diagnostic and could be done in a clinical chemistry lab." 

In the future, Witwer envisions using a technology that allows his team to examine the interior of captured EV particles — rather than just the outside — for potential disease biomarkers. 

In addition to partnering with Witwer's team on HIV-related disorders, NanoView is using its technology in cancer and neurodegenerative disease research. Working with undisclosed collaborators to learn how EVs may reduce antitumor response in pancreatic cancer, the firm is building assays to identify PDL-1 positive EVs from plasma. NanoView is also attempting to build a panel for exosomes by using antibodies that might enrich for brain derived EVs.  

Overall, Daaboul hopes that NanoView will be able to leverage ExoView's capabilities to help inform the research field and better understand EVs' complex heterogeneity and composition. 

"If we learn something about the EVs' composition, we can make new assays to open up new applications, as there is clearly potential to develop them," Daaboul said. "Basically, if we're working with a group, we hope to have a panel that isolates particles from EVs coming from brain-derived [diseases]."

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