By Ben Butkus
SpeeDx, an Australian biotech spun out last year from a Johnson & Johnson research subsidiary, has developed a class of enzymes that it claims provides significant advantages over current enzymes for multiplex qPCR applications.
The company is seeking to license the enzymes, called MNAzymes, to third parties for qPCR-based applications in the human in vitro diagnostic, veterinary, environmental, and food-testing markets; and is eyeing collaborations with undisclosed third parties to bring point-of-care instruments and assays based on the enzymes to the human IVD market, according to a company official.
In the meantime, SpeeDx is currently selling its first MNAzyme-based product — a kit to measure DNA extraction efficiency — in Australia and New Zealand through research tools firm Bioline, which also plans to launch the kits worldwide sometime this summer, the official said.
SpeeDx was founded by researchers at Johnson & Johnson Research, a Sydney-based entrepreneurial subsidiary of J&J. The researchers invented SpeeDx's core technology, called multi-component nucleic acid enzymes, or MNAzymes, while at J&J, Caroline Fuery, director of product development at SpeeDx, wrote in an e-mail to PCR Insider.
When it spun out of J&J, SpeeDx acquired an exclusive, worldwide license to the technology, Fuery said. The company is privately held and has received an undisclosed amount of funding from Australian venture capital firms, and last month moved into new office and laboratory space at the National Innovation Centre in Sydney.
At their core, MNAzymes are catalytic nucleic acid enzymes "that provide a suite of highly specific and cost-effective methods for detection of analytes," Fuery said.
"Catalytic nucleic acids are cheap, stable, chemically manufactured enzymes that can be designed to detect any nucleic acid target of interest," Fuery added. "This huge versatility and high specificity makes them ideal tools for robust diagnostics." The enzymes have applications in isothermal direct detection formats, and can be linked to existing target amplification technologies such as quantitative PCR, she said.
More specifically, MNAzymes are composed of multiple partial enzymes, or 'partzymes,' each of which comprises part of a catalytic core flanked by substrate arms and which only form in the presence of target analytes that serve as assembly facilitators, according to the company's website. When this happens, the MNAzymes are activated and can catalytically modify substrates, such as reporter probes, indicating the presence of a target while leaving it intact.
SpeeDx described MNAzymes and their potential uses, including as diagnostic biosensors, molecular computers, and nanoscale machines, in a paper published in January in the Journal of the American Chemical Society.
Also, at Cambridge Healthtech Institute's XGen Conference, held March 15-19 in San Diego, SpeeDx chief scientific officer and general manager Alison Todd gave a presentation describing what may be the most immediate promising application for MNAzymes: as catalysts in multiplexed qPCR.
In this application, primers amplify target nucleic acid sequences and produce amplicons that serve as templates for MNAzyme formation. Once the partzymes have assembled into MNAzymes, universal reporter probes such as fluorophore-quencher dye pairs bind to them and are enzymatically cleaved, resulting in measurable changes in fluorescence that can be used to detect or quantify the target nucleic acid in real-time.
Fuery explained in her e-mail that "a significant advantage of using MNAzymes as a real-time readout for PCR is that signal is produced by a cleavage of universal probes, not target-specific probes." In comparison, technologies such as TaqMan or molecular beacons require the design and use of specific probes for each new target.
This advantage, in turn, leads to faster assay development because researchers do not need to design and test a new probe for each new target, Fuery added.
Further, due to the requirement for the two MNAzyme partzymes to bind adjacent to each other, qPCR reactions using MNAzymes have enhanced specificity compared to other qPCR chemistries, Fuery said. Using two PCR primers produces an assay with four levels of specificity, she added.
This in turn makes the enzymes optimal for multiplex reactions, as "the four levels of specificity in MNAzyme qPCR assays means that there is no interference or cross talk between the MNAzymes themselves, and therefore no chance of false positive signals," Fuery said.
SpeeDx has used the probes successfully in duplex, triplex, quadruplex, and quintaplex reactions; and has demonstrated the use of MNAzyme qPCR in applications such as methylation analysis, SNP detection, rapid cycling, and long amplicon production.
The company has used the technology in house to detect a wide variety of nucleic acid targets such as fusion transcripts; viruses such as hepatitis C, HIV, and respiratory syncytial virus; bacteria; endogenous controls; and cancer targets from a wide variety of templates such as blood, plasma, serum, and nucleic acid extracted from sputum, urine, fresh frozen and paraffin-embedded tissue, and environmental samples.
At XGen, Todd was trumpeting the use of MNAzymes in qPCR-based IVDs, and cited data showing the method was more robust than other real-time chemistries during clinical validation for a Phase II HIV gene therapy trial being conducted at J&J.
SpeeDx is also developing a class of enzymes called DNAzymes, which it has licensed from the Scripps Research Institute. These enzymes, developed in the laboratory of Scripps researcher Gerald Joyce, are single strands of DNA that have catalytic activity and are being explored as therapeutic and diagnostic tools.
Patents covering both technologies are either granted or pending in various countries, and although they both are key to SpeeDx's development plans, the MNAzymes appear to be riper for the picking.
"Our short-term goals are to license MNAzymes for qPCR applications to third parties for human IVD, veterinary applications, environmental and food testing," Fuery said. SpeeDx's sole license so far is to Bioline, which through its Australian subsidiary is selling a DNA extraction control product in Australia and New Zealand and plans to launch similar kits worldwide in July.
However, SpeeDx continues to actively seek outlicensing opportunities for MNAzyme qPCR in the life sciences research market; and is looking to collaborate with third parties "to bring isothermal point of care instruments and associated assays to the human IVD market, with assays that will provide robust and fast diagnostics for time-critical situations," Fuery said.
"We would be interested in hearing from anyone who has an application where they think MNAzymes may be beneficial," she added.