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Rheonix Presents Data to Back Flagship MDx Platform; Maps Regulatory Timeline for Two Tests

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By Ben Butkus

Rheonix has disclosed the results of three research studies — two conducted in house and one in collaboration with a Chinese partnering organization — that the company said demonstrate the utility of its integrated sample prep and PCR testing platform and provide a preview of the diagnostic markets that it will pursue in coming months.

In addition, Rheonix reiterated its intent to submit its first test on the platform, a warfarin sensitivity genotyping test, for 510(k) clearance from the US Food and Drug Administration by the end of the year; and said that it will likely be followed by a submittal for regulatory approval in China of a human papillomavirus test using the technology.

Rheonix, based in Ithaca, NY, is an early-stage molecular diagnostics company whose flagship "chemistry and reagent device," or CARD, system integrates sample preparation, nucleic acid amplification, and endpoint detection all on a single microfluidics-based, point-of-care polymeric module a few inches thick and with an approximate footprint the size of a credit card.

In April the company came out of stealth mode by announcing the completion of a $12.6 million Series A financing and the disclosure that it was developing several in vitro diagnostic assays for the CARD platform, including tests for warfarin sensitivity, HPV, HIV, and sepsis (PCR Insider, 4/29/10).

At the time, Rheonix President Tony Eisenhut said that the warfarin sensitivity test would "probably be first to market" and that Rheonix planned to file a 510(k) application for the test before the end of this year.

This week, Eisenhut told PCR Insider that the company was still on track for such a submittal, likely in December. The company plans to submit as a single operating system the CARD-based test along with a benchtop workstation called Encompass MDx that uses software to control all aspects of the assay.

Several other molecular diagnostics tests for warfarin sensitivity have previously been cleared by the FDA.

"It's the nearest term for regulatory submission," Eisenhut said. "It has its place in the market, and yes, we think that the warfarin sensitivity diagnostic market is a good market. But also, one of the reasons we pursued this is that it really shows the breadth and power of our system to be able to perform that type of SNP analysis on a fully automated system without technician intervention."

To wit, last week at the American Association for Clinical Chemistry annual meeting in Anaheim, Calif., the company presented results from an in-house study using its CARD system for the rapid and automated determination of warfarin sensitivity SNP profiles for 21 individuals to demonstrate how such an assay could help guide individualized dosing of the drug.

To perform the test, technicians collected a buccal swab from test subjects' cheeks and applied them unadulterated to the CARD system, which performed cell lysis, multiplex PCR, biotinylated primer extension, and image analysis to detect SNPs in three genes — VKOR1:1173G>A, CYP2C9*2, and CYP2C9*3 — associated with warfarin dosing sensitivity.

The CARD assay called the correct genotype for each sample, and the genotypes were confirmed using bi-directional DNA sequencing, the company said. "The predicted outcomes were exactly what we got," Richard Montagna, Rheonix's senior vice president for corporate business development and scientific affairs, told PCR Insider.

In another study, conducted with researchers from an undisclosed Chinese joint venture partner, Rheonix reported the successful detection of 20 clinically relevant HPV types from 69 clinical samples using the CARD system.

The study involved collecting vaginal swabs from test subjects and, like in the warfarin sensitivity test, applying the samples directly to the CARD system, which automatically lysed cells, purified DNA, and subjected it to multiplex PCR in the presence of biotinylated primers to amplify any one of 20 target HPV types and the human beta-globin gene.

In addition, the system denatured the resulting amplicons, applied them to a DNA microarray, and hybridized them to specific capture probes for subsequent imaging and analysis to detect specific HPV types. "In that particular test we're using a reverse dot-blot detection modality," Eisenhut said. "We have a [reverse dot blot] filter paper that has a low-density array spotted with probes that we're able to — on chip — detect the various infections in that case."

Of the specimens analyzed, 48 were found to be HPV negative by both the Rheonix test and an FDA-approved comparison product, the Qiagen Digene HC2 HPV test. Also, 14 were determined to be positive by both tests, and six specimens found to be negative on the Digene test were positive on the Rheonix test.

The presence of HPV in the discordant samples was confirmed by amplicon sequencing, Rheonix said.

Montagna noted that one additional sample scored positive on the Digene test but negative on the Rheonix test, but that the sample was "right at their signal-to-noise threshold," calling into question the accuracy of that particular result. He also pointed out that the Digene tests were conducted by a clinical reference lab not affiliated with Rheonix.

Lastly, Rheonix also presented results from a study demonstrating that the CARD system could identify the presence of four sexually transmitted infections in a multiplex assay. In this study, 5 million C33A cells per mL were spiked with 10,000 copies per mL of genomic DNA from N. gonorrhoeae, C. trachomatis, T. palladium, and T. vaginalis either singly or in combination.

For each sample, 1 µL was applied to the CARD system, which performed automated sample prep, multiplex PCR, and DNA reverse dot-blot imaging to correctly identify the genomic DNA present.

Eisenhut said that following the warfarin sensitivity test, HPV and other STI tests will be the next priority.

However, "it doesn't necessarily mean that we're going to pursue regulatory approval in the US," he said. "In the HPV market, for instance … it will be our next submittal, but we're going to submit for approval in Asia, and China specifically."

In addition, Rheonix has a clinical collaboration in place with a National Institutes of Health-funded consortium comprising Lehigh University, New York University, Leiden University, and the University of Pennsylvania, focused on HIV detection; and is working in house on a sepsis assay for the CARD system.

"On the HIV front, there are some unique capabilities where the consortium has actually been able to integrate on our CARD system an immunoassay, as well as a molecular amplification diagnostic in parallel on the card from a single sample," Eisenhut said.

Rheonix continues to seek partnerships to explore the development of further tests on CARD. In addition, Eisenhut said that the company was confident that the $12.6 million it raised earlier this year will take it through at least its first regulatory submission.

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