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Two Pore Guys Developing Assays for Autoimmune, Infectious Diseases on POC Nanopore Platform

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NEW YORK (360Dx) – Two Pore Guys is looking to expand its presence in the diagnostics space with the development of a nanopore platform for use in doctors' clinics to test for autoimmune conditions and infectious diseases.

A clinical assay for these applications could take about three years before it hits the market, but the firm anticipates eventually releasing a sensitive test for type 1 diabetes that would cost far less than a laboratory-based test, Two Pore Guys Cofounder and Chief Scientific Officer Trevor Morin said in an interview.

To count and characterize molecules based on specific signatures that are indicative of disease conditions, the firm's diagnostic platform uses a reader that notes the amount of time it takes for specific molecules to translocate through nanometer-size holes in silicon chips and measures how much they interfere with a trickle of electric current running through the holes. The process is the basis of a platform being developed for applications at the point of care, including clinical diagnostics, agriculture, food safety, and environmental monitoring, Morin said.

The firm currently is working with Bayer "to complete development of the nanopore sensor instrument and assay for biomolecule detection, and we are developing the same platform with assays for autoimmune and infectious diseases," Morin said.

The firm's collaboration with Bayer's Monstanto business, inked in October last year and involving development and evaluation of assays for detection of certain biomolecules in crops, pests, and pathogens, could be a stepping stone for more long-term development of clinical diagnostic assays and the applications in other industries. Two Pore Guys is simultaneously testing its technology in studies done in collaboration with the University of California San Francisco for analysis of cell-free DNA to detect cancers.

For clinical diagnostics, the firm has plans to move its platform and assays along a commercial path, seeking US Food and Drug Administration clearance and scaling up manufacturing. All of that is likely to take a few years. However, according to Morin, it should be worth the wait because the level of accuracy and the versatility of the platform for point-of-care applications, potentially could rival that of assays used in laboratories, including radiation-based ELISAs used for diabetes testing and droplet digital platforms used to test for cell free DNA in blood.

Both types of laboratory assays take far longer to provide diagnostic results and are far more expensive to purchase as systems than the nanopore platform, he said.

The firm also has funding to support its commercial efforts. In April last year, it closed a $24.5 million Series A financing round led by Khosla Ventures.

Two Pore Guys is continuing development of technology that would enable mapping and sequencing of long DNA, Morin said, but it doesn't yet have a projected timeline for deployment.

Its executives published a paper in May 2016 related to use of the platform to detect a cystic fibrosis mutation, and it has also considered developing a saliva-based HIV antibody detection assay. However, the company doesn't have plans to commercialize those tests.

"We now have a proof-of-concept platform and want to make the next iteration closer to the final product," Morin said. "That iteration needs to be kicked around internally, put through its paces, and moved into beta testing," during which users can provide feedback to Two Pore Guys for further refine the design.

Following that, the company anticipates locking down the design and preparing an application for the FDA for 510(k) clearance "if that's what's needed for humans," and sending it to Monsanto to implement in its application, Morin said.

The platform's design differs significantly from most others being developed or already in use for point-of-care applications in that it uses solid-state, silicon nanopores as sensors in detecting molecules, but has no optical components, Morin said.

For sample-to-answer testing, point-of-care technologies tend to rely on optics that can make them much more expensive and complex, Dale Yuzuki, senior global business development manager at Pillar Biosciences, wrote recently in his publication Next Generation Technologist. "The nanopore sensor at [Two Pore Guys] and their mechanism to detect DNA amplicons electronically could be used downstream of existing sample prep technology in a much cheaper, portable, and more rapid manner."

"We carve out a two by two-millimeter silicon chip and drill a hole in it, and that's our sensor," Morin said. The firm packages the chip in a disposable cartridge that's rugged and durable, which is important for portable applications.

The nanosized pores in the silicon chip have a relatively large tolerance for dimensional variations, so the firm doesn't need overly precise drilling, Morin said. That enables it to make thousands of chips during one manufacturing run, drill them all at once, and then dice the wafer into the silicon chips used in the disposable cassette.

Using this design, the system can detect and differentiate molecules in a solution, such as blood or urine, and it can count molecule by molecule, differentiating long strand DNA from short strand DNA, for example. "The test results are extremely accurate because unlike aggregate fluorescence from a PCR-based test or an immunoassay where you see color change, and you are wondering how much of a change occurred, the platform gives a quantitative digital count," Morin said. "You can see a molecule go through and you can bin that as the target molecule, so you are literally counting molecules one by one to get positives results."

For testing, the platform applies a voltage so that a trickle of current runs through silicon pores in the chip residing inside the disposable cartridge. A salt buffer above and below the chip is connected only by the electrical path running through the nanopores. The system measures open-channel conductance; when there is no molecule in the nanopore, current is stable, but when a molecule blocks the current, the current level dips until the molecule passes through. That electrical event reflects the molecule's properties. "If you are looking for a particular size of DNA, you get a signal that characterizes that," Morin said.

With Bayer, Two Pore Guys is measuring crop traits. "We can do very fast DNA extraction [from a component of the crop] and then amplify the genetic region that aligns with a trait of interest," Morin said. "If the trait is present, we get a long DNA molecule, say 300 base pairs, and if it is not present, we get a short DNA molecule, say 100 based-pairs, and we send those molecules through our nanopores."

In so doing, the platform collects data on just less than a few hundred molecules in seconds. The system calculates the ratio of the long versus short molecules, and the result reflects whether the trait of interest is present.

Two Pore Guys uses the same platform in its collaboration with UCSF in the development of a liquid biopsy test to detect mutant KRAS DNA floating in blood or urine from patients undergoing treatment in clinics. The firm extracts cell-free DNA from samples and amplifies KRAS DNA within the sample. The platform is looking to identify the KRAS G12D mutation, which is prevalent in pancreatic and colorectal cancer, Morin said.

In a first UCSF study launched in 2017, the platform "accurately measured the amount of circulating KRAS mutant DNA from blood and urine compared to a droplet digital PCR predicate platform from 10 patient samples," Morin said. The company is starting a second study with UCSF to measure circulating KRAS tumor DNA in about 30 patients and in a longitudinal fashion — 48 hours after therapy, two weeks later, and two- to four-months later. The goal is to ascertain whether ctDNA can predict therapy efficacy at earlier timepoints then imaging, such as CT scanning, Morin said.

The collaborators aim to enable clinicians to run frequent liquid biopsy tests that reflect the kinetics of the circulating tumor DNA in order  to give them a very early indication of whether they are giving a patient a drug that is responding as expected, so that they can take quick corrective action and improve the patient's clinical outcome, he said.

For testing of cell-free DNA, Two Pore Guys compares the performance of its test with that of lab-based droplet digital PCR platforms that can cost around $100,000 and require trained laboratorians to operate them. Its platform also is likely to be available at a more palatable price, and avoid concerns over getting accurate test results stemming from the transportation of samples, Morin said.

In its process, Two Pore Guys uses a benchtop technique to extract cell-free DNA. Thus far, its data from clinical samples measures up well with that of laboratory-based droplet digital PCR testing, Morin said. "Now we can say that the assay is good, and the data are good," he said. The firm is, therefore, focusing on developing either an automated or semi-automated sample preparation process for the test.

"Automating cell-free DNA extraction from a tube to generating a result from whatever kind of detector you are using is not a small feat," Morin said. "That involves significant technology development, and we are working toward it."

Two Pore Guys in the nearer term is developing alternate applications. Testing for infectious disease or autoimmune antibodies is interesting because the tests use an agglutination PCR-based approach, Morin said. "If a target antibody is present, it kicks off a PCR amplification reaction, and then you measure the output of the PCR amplification reaction using our nanopores," Morin said.

These PCR-based tests using the nanopore platform have low femtomolar sensitivities and can match the performance of the most sensitive radio immunoassays that can cost $1,000 per test because it's so specialized, Morin said.

Two Pore Guys anticipates matching the performance of the RIA test and significantly reducing the cost of testing for type 1 diabetes, Morin said. "We are trying to open the door for pediatricians to test their patients and enable testing for type 1 diabetes of all children along with their yearly physical," he said.