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Quanterix, Partners Develop PCR-Free miRNA Detection Method for Liver Toxicity Testing

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NEW YORK (GenomeWeb) – A team of investigators from Quanterix, Destina Genomics, and the University of Edinburgh have developed a new approach for the amplification-free detection of microRNA-122, a biomarker used for diagnosing liver toxicity.

The success of the method, described this month in the journal PLoS ONE, has multiple ramifications for the parties involved. For Lexington, Massachusetts-based Quanterix, which to date has deployed assays for protein analysis on its Single Molecule Array, or Simoa, platform, the approach demonstrates the applicability of its technology in nucleic acid detection, and miRNA detection in particular, as it prepares to launch a new system.

For Edinburgh, UK-based Destina Genomics, meantime, the study showcases the use of its chemistry for the PCR-free detection of miRNA on a commercial system. And for scientists at the University of Edinburgh, it offers the hope of introducing miRNA testing that is faster than PCR and less expensive than next-generation sequencing.

"What we are measuring is PCR, which is fine, but it's time consuming," said James Dear, a reader in pharmacology at the University of Edinburgh and paper coauthor, whose work is related to investigating the use of miRNA biomarkers, such as miRNA-122, that are associated with liver toxicity, particularly those resulting from the overuse of acetaminophen. Roughly 100,000 people are admitted to emergency departments in the UK annually, he said, and obtaining information about liver toxicity is crucial to prescribing treatment that can quickly mediate the drug's effects.

Clinicians currently rely on another PCR-based test for the alanine transaminase (ALT) enzyme to diagnose liver toxicity, noted Dear. Yet miRNA-122 is seen by some as a more sensitive biomarker that has not yet progressed in clinical research due to the challenges associated with detecting it using conventional PCR-based assays.

"That's the main driver of this work with Quanterix," said Dear. "PCR takes time, so if we take a sample, it will take most of the day to get the result." By partnering with Quanterix and Destina, Dear and his colleagues aimed to produce an assay for miRNA-122 that has the potential to give a more rapid result.

"This system, because it is amplification free, can give a result very quickly, which can inform whether someone is treatable or not, and can inform drug development," said Dear.

Founded by University of Edinburgh researchers in 2010, Destina's core technology is based on pairing its aldehyde-modified Smart Nucleobases with peptide nucleic acid capture probes containing an abasic position that can be made to complement any nucleic acid system. The result is a dynamic chemistry approach that relies on a chemical, rather than enzymatic, method for nucleic acid testing.

According to Juan Diaz Mochon, cofounder and CSO of Destina, the company evaluated several platforms for Dear's project, looking at alternatives to "time-consuming PCR," and ultimately decided on using the Quanterix platform, given the ability of the Simoa system to provide digital results at single-molecule resolution without a need for amplification.

"It's very disruptive from our view because if we can do direct detection with single-molecule resolution, this opens up option of doing microRNAs, which is the obvious choice," said Diaz Mochon.

As detailed in the new paper, by pairing the Destina chemistry with Quanterix's platform, the investigators were able to develop a single-probe method for detecting miRNA from human serum using Quanterix's single-molecule arrays, which had the advantages of reading sequence specificity down to a single base without the need for amplification.

For the application, Destina designed an abasic peptide nucleic acid probe for detecting miRNA with a reactive amine in lieu of a specific nucleic acid sequence. The probe was coupled to Quanterix's superparamagnetic beads, and the beads were then incubated with a sample containing miRNA, a reactive nucleobase that complemented the missing base in the probe sequence, and a reducing agent.

Following hybridization with the target molecule, the beads were labeled and suspended in a fluorogenic enzyme substrate, loaded into Quanterix's array, and imaged to determine which beads were associated with single enzymes, as well as the number of enzymes per bead.

According to the researchers, the assay had a limit of detection of 500 femtometers, leading the authors to claim it was about "500 times more sensitive" than an unnamed analog, bead-based assay. They also demonstrated its ability to measure miRNA-122. All patients with liver injury had higher levels of miRNA-122 in their blood compared to controls, and the concentrations of the marker were correlated with real-time quantitative PCR.

The results led the authors to write that the approach allowed the "rapid quantification of circulating miRNA with single-base specificity and a limit of quantification suitable for clinical use."

How rapid? According to Destina's Diaz Mochon, the team was able to complete the analysis of 24 samples on the Simoa platform, including instrument set up and run time, in three and a half hours. For PCR, he said, the amount of time dedicated to the analysis of the same amount of samples would be about five hours.

"Pretty much as soon as we tried it, it worked," said David Duffy, CTO of Quanterix. He said that Quanterix had been intrigued by the idea of using its system for nucleic acid detection, especially the detection of miRNAs.

"We were hearing about people struggling with PCR in regards to miRNA," said Duffy. "People are actually turning to next-generation sequencing for miRNAs because PCR wasn't doing it for them," he said. "It seemed like the right kind of molecule to go after."

Quanterix has worked mainly in the protein space since it was founded in 2007, pursuing opportunities in testing for a number of indications, such as traumatic brain injury. Last year, a paper in Analytical Chemistry detailed how investigators were able to use the Simoa platform to quantify proteins from single cells.

"Pretty much everything we have done has been in sensitive detection of proteins," said Duffy. "Yet we have a detection platform that isolates single molecules and can be applied to any kind of molecule you can find a binding partner for," he said. "All along we have been thinking about other molecules. Nucleic acids have been there."

He noted that PCR and next-generation sequencing are the "gorillas in the room" the company would have to compete against in the nucleic acid testing space, but given the limitations of PCR and the cost of sequencing, the firm might have an opportunity given the right application. "We have a goal to come up with an alternative to PCR, which is 30 years old," said Duffy. "We have always felt that Simoa can offer something."

The miRNA space is where Quanterix is "more confident" that customers might switch platforms, choosing Simoa over PCR and sequencing.

The firm is also interested in commercializing an assay for miRNA-122. "We are definitely looking at the commercial opportunities," said Duffy. "A lot of drug companies are looking at this marker," he noted.

Quanterix will continue to work with Dear's group at the University of Edinburgh, he said, while it looks into pairing Destina's chemistry with the Simoa platform as part of a combined offering. "It is definitely something we are looking at seriously, a complement to what we have going with protein," said Duffy.

As it explores its relationship with Destina, Quanterix is also hoping to enable miRNA analysis using its technology. Duffy said the firm will soon introduce a new reader that support various assays for nucleic acid detection. Jeremy Lambert, director of marketing at Quanterix, said that the company will most likely make the new system, which relies on the same core technology as its HD1-Analyzer, available to users later this year, followed by a full commercial launch in early 2018.

"Our instrument [the HD1-Analyzer], was really designed for proteins [in terms of the] liquid handling, the buffers we used, temperature control," said Duffy. With the new system, Quanterix can offer "much more flexibility on the sample preparation," supporting diverse assays.

"What you see is a lot of different pieces of the puzzle falling into place," he added. "I am pretty confident that it's going to bear some fruit for us."

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