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Circulomics Developing miRNA Profiling Kits to Fill Multiplex Analysis Niche

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With a handful of recently awarded grants, startup Circulomics is kicking off development of its technology as a tool for lower cost and more accessible multiplex analysis of disease-associated microRNAs, and is planning to launch its first kit, a 30-plex, semi-customizable cancer panel, in 2014.

The company announced this month that it had received two $400,000 Small Business Innovative Research awards from the National Institutes of Health to support the development of its Ligo-miR assay technology and PicoSep single-molecule analysis platform.

In addition, the company also received $75,000 this year from the Maryland Technology Development Corporation to back the development of its first commercial product, Ligo-miR EZ, which will be a multiplex, 30-miRNA cancer kit combining a fixed set of general cancer-associated miRNAs with several slots for researchers to choose their own content.

Kelvin Liu, who founded Circulomics to advance technology he developed at Johns Hopkins University, told Gene Silencing News this week that the company hopes Ligo-miR will serve researchers who want to do mid-range multiplex analyses without a need for expensive instrumentation.

"People have identified smaller panels of miRNAs that are relevant to them, so maybe there are 30 for breast cancer, maybe 10 to 20 for diabetes, [and] 20 for cardiovascular diseases, for example," Liu said. "In order to effectively analyze these panels, you need a method that is efficient for a targeted panel of 20 or 30.”

Liu noted that the most common multiplex methods for miRNA analysis are microarray based. "Those methods are really good for analyzing 500 or 1,000 miRNAs in really large surveys," he said. "But if you want to use them to analyze these panels of 20, there is really a lot of overkill and in terms of access for users many of these multiplex methods require you buy an instrument first."

As a result, he said, most labs resort to PCR. "Everyone has a thermocycler, so even though it's tedious, you're doing individual PCR reactions. That's OK for 10 or so samples, but we need a more efficient way to screen these panels of 20 or 30 microRNAs as the market matures and people start testing larger numbers of samples."

"We tried to design an approach that uses basically the same equipment and workflow as PCR but you get multiplex data," Liu said.

Toward this aim, Circulomics is working to develop the Ligo-miR approach and then adapt it to create a number of targeted assays for multiplex analysis of panels of 30 or so miRNAs, starting first in cancer, but then potentially expanding to other disease areas.

Ligo-miR is a PCR-free, multiplex ligation approach. In the grant abstract for the company's SBIR award, Liu describes the system as involving ligation of locked nucleic acid probes to generate miRNA-specific ligation products encoded by length.

Liu said the plan is to make the system adaptable to work using a variety of different sample inputs and detection/readout approaches. Ligo-miR "uses a two-step ligation process to generate encoded miRNA signatures that you can then analyze using any number of types of equipment," he explained.

In the planned Ligo-miR EZ kit, for example, the technology will work with total RNA as an input and researchers can hook the system up to a variety of detection techniques. "Using a plain PAGE gel you can get a sensitivity about 100-fold higher than microarray, and then if you want a step beyond that you can run it on a capillary electrophoresis machine," said Liu.

"Then a step beyond that you can run it on our single-molecule separation platform," he said.

Liu is working to develop this add-on instrument for single-molecule separation, called PicoSep, with the company's second SBIR grant.

According to the description of the project in the company's grant abstract, adding PicoSep to the Ligo-miR kit will enable PCR-free detection with sensitivity to less than 20 copies and sample volume below 10 pL.

"If researchers want to get their feet wet, they can buy the simple assay. And then if they want to step up, if they want PCR-level sensitivity, they could buy our instrument and run the same sample on the instrument," Liu said.

Liu said the company plans to use the grant funding to support development of both systems up to a planned commercial launch of the EZ assay, hopefully in early 2014.

"Right now we have a few collaborations with investigators studying miRNA in esophageal and pancreatic cancer here at Johns Hopkins," he said. "So they will be doing the benchmarking for us."

Liu added that when Circulomics gets to the point of a commercial launch, he expects to need additional equity investment, and he plans to start actively looking for that in about six months.

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