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Australia's SpeeDx Demonstrates Multiplexed MNAzyme qPCR; Looks to Move Tech to MDx Market

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Australian molecular diagnostics and research tools firm SpeeDx has published research demonstrating how its proprietary class of nucleic acid enzymes, called MNAzymes, can be used in combination with standard qPCR reagents to enable easy, inexpensive development of highly sensitive and specific multiplexed assays.

SpeeDx is using the combined methodology to develop its first in-house molecular diagnostic test, a multi-marker assay for meningitis-related bacteria, a company executive said this week. In addition, the company is developing multiplexed genotyping assays for oncology applications for an undisclosed European platform and reagent manufacturer.

PCR Insider has also learned that SpeeDx's MNAzyme technology forms the basis of Illumina's NuPCR reagents for research use, which Illumina launched earlier this year primarily to compete with real-time PCR products offered by rival Life Technologies.

SpeeDx has also licensed its MNAzyme technology to other partners for use in applied markets, such as veterinary, forensic, and agricultural testing; as well as for specialized applications such as electrochemical detection and qPCR extraction controls.

However, according to Alison Todd, chief scientific officer and general manager for SpeeDx, the company is now shifting its gaze away from research applications and toward diagnostic uses for the MNAzyme technology.

"Now that we've got some products out in the research market, we're working toward multiplexed assays for clinical targets," Todd said. "We are seeking partners definitely for MNAzyme [in vitro diagnostics] — not many more in research, but mostly in clinical diagnostics [using] MNAzyme qPCR."

SpeeDx is a spinoff of Johnson & Johnson Research, a Sydney-based entrepreneurial subsidiary of J&J. Prior to spinning off SpeeDx in 2009, J&J Research had obtained a license from the laboratory of Gerald Joyce at Scripps Research Institute to a class of single-stranded catalytic DNA enzymes called DNAzymes.

While at J&J Research, Todd and other eventual SpeeDx co-founders developed a modified version of DNAzymes called MNAzymes, which are nucleic acid enzymes formed from multiple partial enzymes, or "partzymes." These multiple partial enzymes can form a catalytic core flanked by substrate arms in the presence of target analytes that serve as assembly facilitators. When this happens, the MNAzymes are activated and can catalytically modify substrates, such as fluorescent reporter probes, indicating the presence of a target while leaving it intact.

MNAzymes have been shown to be able to detect a wide variety of nucleic acid targets with a high degree of specificity and sensitivity, since they form an intact catalysis agent only in the presence of target. What's more, the probes can be linked to a number of existing nucleic acid amplification technologies such as isothermal amplification and qPCR.

When used in conjunction with qPCR, MNAzymes become universal probes – that is, the same probe can be used for any target – as opposed to the target-specific probes required in standard TaqMan-based qPCR, for example. The upshot is that MNAzymes make multiplexed qPCR assay design much more straightforward and inexpensive, according to the company.

When SpeeDx branched off from J&J Research, it obtained intellectual property for DNAzymes and MNAzymes. Todd said that the DNAzyme technology still has utility and, in fact, the company is exploring its use as a signal amplification tool in R&D and various diagnostic applications. In addition, "there are a number of clinical trials underway … trying to develop the first DNAzymes as a therapeutic agent. But the jury is still out on that," Todd said.

However, multiplexed molecular diagnostics are increasingly in demand from the clinical market, and SpeeDx's MNAzymes combined with qPCR provide a useful tool for such applications.

In a paper published earlier this month in Clinical Chemistry, Todd and other SpeeDx scientists described multiplexed MNAzyme qPCR for the first time. More specifically, the researchers showed that using the method to detect three targets in single-plex and triplex formats yielded the same limit of detection and amplification efficiency; and that they could convert a single-target assay for 11 transcripts of interest to triplex assays containing two reference transcripts without having to optimize or modify conditions.

In addition, the researchers designed a quintuplex qPCR assay that simultaneously quantified five transcripts with five universal probes, producing high amplification efficiencies for all transcripts, and producing no false positives despite the large number of oligonucleotides in the reactions. Lastly, Todd and colleagues designed a quadruplex assay that combined MNAzymes with methylation-specific PCR to measure epigenetic biomarkers of prostate cancer. The assay was able to detect a single methylated DNA allele in a background of 100 to 10,000 unmethylated alleles, demonstrating the clinical diagnostic utility of the combined approach.

"There are not that many papers out there that can do five-plex reactions that can easily be adapted [to different targets]," Todd said. "This is an advance in multiplexing."

In the three years since its launch, SpeeDx has made significant progress in developing and licensing its DNAzyme and MNAzyme technology. The company had previously disclosed an agreement with Bioline, which sells an MNAzyme-based kit to measure DNA extraction efficiency (PCR Insider, 4/1/2010); and a partnership with Australia's Universal Biosensors to develop low-cost, point-of-care molecular diagnostic assays leveraging MNAzymes and electrochemical detection (PCR Insider, 9/22/2011).

More recently, SpeeDx disclosed that MNAzymes form the basis of Illumina's NuPCR reagent portfolio. Todd declined to provide additional details about the licensing agreement between the firms, and Illumina did not respond to requests for comment.

When Illumina launched its NuPCR reagents in July, it said that it would offer custom-designed probe-based assays for gene expression analysis and that it expected to compete with existing vendors primarily on price, offering its assays at about half the cost of competing probe-based assays (PCR Insider, 7/26/2012). An Illumina executive also said at the time that although Illumina did not invent the technology underlying NuPCR, it improved upon it "significantly," result in better sensitivity, specificity, and ease of multiplexing.

Todd said that SpeeDx has two other MNAzyme licensing agreements in place: one that involves developing "multiplexed oncology assays … for a European platform and reagent manufacturer;" and another about which she declined to provide any details.

SpeeDx is seeking more IVD development partners, but in the meantime, it is working on its own MNAzyme qPCR-based test for meningitis-related bacteria. Specifically, the company is working on a two-multiplex, eight-pathogen test and is aiming to release it as a CE IVD marked product next year.

"We are developing stuff with partners, but this is the first we're developing ourselves, though we'll no doubt partner with someone else a little bit further down the line to get it to market," Todd said. "But we're doing the assay development ourselves."

Todd noted that SpeeDx has also licensed a proprietary tuberculosis marker from an unnamed entity, and that a test for the pathogen will be next in the pipeline.

Lastly, SpeeDx has also recently developed an isothermal signal amplification cascade technology called EzyAmp, which uses a combination of MNAzymes and restriction enzymes and promises to be an ideal format for rapid, quantitative, inexpensive, point-of-care assays.

"I think we're basically going to try and keep that in house to grow our company and grow our presence" in the molecular diagnostics space, Todd said. Already, the company has grown from four employees in 2009 to 13 full-time and three part-time staff members today.

"We're very busy working on a number of different fronts," Todd said. "This is a kind of very flexible format, and that's evident from the breadth of the licensing we have done."

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