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Cell-Free DNA, RNA May Hold Key to New Diagnostics, Chronix Biomedical Says


Chronix Biomedical is betting that profiles of circulating DNAs and RNAs can help detect chronic disease, including some cancers and neurologic and autoimmune diseases.

The company is making its projection at a time when several research groups have begun to use second-generation sequencing to analyze blood-borne cell-free nucleic acids that could be applied to diagnostic or prognostic use.

Over the last four months, the 10-person company, headquartered in San Jose, Calif., published proof-of-concept studies of the approach in two scientific papers and a conference abstract. In those studies, its team used Roche's 454 Life Sciences sequencing platform to characterize CNAs in the blood of healthy individuals, a cancer patient, and animals with prion disease.

After filing patent applications to protect the use of circulating nucleic acid biomarkers in several disease areas, Chronix is now in discussions with undisclosed companies to license its biomarker profiles, validate them in clinical studies, and develop them into diagnostic tests, which might use a different analysis platform than sequencing.

Up until now, the company, which has research labs in Göttingen, Germany, and a processing lab in Brookings, SD, has outsourced its 454-based sequencing needs to service providers, including Eurofins Medigenomix in Germany.

Chronix said its sequencing-based profiling approach is independent of a specific sequencing platform, and said that in the future it plans to test several other second-generation sequencers to see how longer or shorter read lengths would affect its results.

Recently, the company and colleagues from the University of Göttingen and the University of Milan in Italy studied the representation of different genomic regions of DNA, including repetitive elements and transposons, in circulating nucleic acids from 51 apparently healthy individuals. That study was published in January in Clinical Research.

While the 454 platform has so far "performed marvelously for us, we will be testing with these various [other types of] machines [to see] if you use [a shorter] read length, do you lose the identity of these transposable elements? And if you use longer read lengths, is it just too much information that doesn't get you any farther?" Chronix CEO and co-founder Howard Urnovitz told In Sequence.

One of the earliest applications of Chronix's results might be in the early diagnosis of prion diseases in animals, based on results of a study that used 454 sequencing to find specific blood-based CNA patterns in elk infected with chronic wasting disease and in cattle infected with BSE several months before clinical signs of the diseases appeared.

That research, which appeared last December in Nucleic Acids Research, was conducted with company researchers and their collaborators at the University of Calgary in Alberta, the University of Göttingen, and elsewhere. In it, the researchers wrote that the patterns "provide a starting point for the development of a relatively simple, cost-effective live animal test," for example based on PCR.

And last May, the company was awarded a US patent for a method to detect abnormal serum nucleic-acid profiles to assess the risk of a prion disease, such as BSE, and has several patents pending in this area.

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Chronix researchers and their collaborators also presented an abstract at the annual American Society of Hematology meeting in December 2008. The abstract described how they sequenced CNAs in a single myeloma patient multiple times during the course of the patient's treatment for more than a year.

"The details of associated DNA sequences provide insights to the underlying molecular pathophysiology linked to myeloma disease progression, response to successful therapy, as well as subsequent relapse," the authors noted in the abstract.

Earlier this month, Urnovitz told In Sequence that the company in the meantime has conducted similar studies on 38 breast cancer patients and 13 multiple sclerosis patients, and is about to submit a paper on the multiple sclerosis study.

He also said Chronix is currently starting a Series C round of investment to help it complete studies in four cancer types.

So far, the company has raised $18 million in funding, including $5.1 million in a Series A preferred stock round, led by PrairieGold Venture Partners, and $2.3 million in a Series B preferred stock found from Prairie and existing investors.

Chronix's team is not the only one pursuing cell-free DNA and RNA for potential diagnostic purposes. Last October, researchers from Stanford University, led by Stephen Quake, published a study in PNAS describing how they identified cases of different trisomies in fetuses by sequencing cell-free DNA from the plasma of pregnant women on Illumina's Genome Analyzer. Two months later, researchers from the Chinese University of Hong Kong published a similar study in PNAS describing how they detected trisomy 21 after using the Genome Analyzer to analyze maternal plasma DNA.

Dennis Lo, a professor of medicine and chemical pathology at the Chinese University of Hong Kong and a co-author of the second study, told In Sequence last week that circulating DNA in blood could have "exciting diagnostic potential for noninvasive prenatal diagnosis, cancer detection, and infectious diseases."

However, he said he believes that "cost is probably the major issue" preventing high-throughput sequencing from being used routinely in a clinical setting at the moment.

Other issues he cited are turnaround time, currently at least one week including sample prep and bioinformatics analysis.

He also said the bioinformatics infrastructure required to run today's second-generation sequencers is so extensive that "services would likely have to be provided from a few centralized facilities rather than through multiple small labs in many hospitals."

In the future, increased read lengths "might not be crucial for sequencing plasma/serum DNA because these are generally fragmented, with most of the order of 180 base pairs or less," he said. Rather, "capacity in terms of the number of molecules sequenced might be more important than how long a molecule one can sequence."

Lo said he believes that the current generation of instruments "have potential to allow them to move in the direction" that will allow them to be used routinely in the clinic, including cost reduction.