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SBIR Funds in Hand, DxTerity Developing Radiation Toxicity Predictor Using Chemical Ligation Probes

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Molecular diagnostics company DxTerity Diagnostics was recently awarded a $280,000 Small Business Innovation Research contract from the National Cancer Institute to develop a test for susceptibility to radiation sickness during cancer treatment.

The gene expression-based system aims to identify patients likely to develop acute radiation side effects.

The test, as it is now envisioned, will involve ex vivo irradiation of patient blood samples using radiation sources usually used to treat cancer. DxTerity will then screen the blood for genetic biomarkers that indicate an over-reaction to ionizing radiation, company Founder and CEO Robert Terbrueggen told PCR Insider in an interview.

This test will expand on the company's growing molecular diagnostics portfolio, which includes a panel of biomarkers that act as a dosimeter to gauge radiation exposure. This diagnostic, called Redi-Dx, measures normal gene expression changes in reaction to poisonously high doses of radiation and is currently slated for US Food and Drug Administration submission in 2015.

For radiological or nuclear emergencies, such as the 2011 Fukushima Daiichi nuclear disaster, where "everyone thinks they're exposed and is panicky," Terbrueggen explained, "it's critical to know the dose someone received, so you can separate 'worried-well' from individuals that need different treatment" in a triage situation.

DxTerity’s panel of 22 biomarkers can estimate, or call, radiation dose using 20 markers for a seven-days-after exposure sample, but only requires five genes to accurately call a one-day-after sample, said Terbrueggen.

Recently, NATO led a runoff between tests purporting to measure how much irradiation a person (or, in this case, a blood sample) actually received. Blood was drawn from one healthy 29-year-old male volunteer, aliquots were irradiated with up to 6.5 Grey of radiation, followed by a variable waiting period at 37 degrees to simulate time post-disaster. Then labs received 10 blinded samples each to call the dose of radiation.

Redi-Dx was the most accurate when compared to other gene-based dosage measures, according to the study. It was also among the most rapid turnaround times compared to other available testing methods.

According to the NATO study, published in September in the journal Radiation Research, the gold standard in biodosimetry remains the dicentric chromosome assay, but it suggests that for "situations where speed and throughput are more important than ultimate accuracy, the emerging rapid molecular assays have the potential to become useful triage tools." Among these types of assays, DxTerity's had superior accuracy in calling dose.

According to Terbrueggen, the NATO runoff shows DxTerity's ability to "take a finger stick collected blood sample and from a very small volume determine radiation exposure post event," adding, "we've demonstrated that we can, with comparable accuracy to the gold standard assay, do dose calling, but 10 times faster and at a much higher capacity." This speed could be particularly useful, according to the NATO study, in large-scale, mass casualty events.

One underlying reason for DxTerity's speed at biomarker testing is the company's unusual methods. Most other labs in the genetic assay category of the NATO comparison used qRT-PCR, requiring RNA isolation from the blood sample via QIAamp RNA blood mini kit or Ambion total RNA extraction kit prior to testing. DxTerity, meanwhile, used a chemical ligation probe assay that basically allowed it to skip this step and "to test directly in dirty samples without doing any purification," claimed Terbrueggen.

This chemical ligation is at the heart of all of the firm's molecular diagnostic products. Traditionally, since the enzymes that are used in PCR don't work on RNA, it must be converted to cDNA for PCR. "We figured out a way to eliminate that process of having to go from RNA to cDNA," said Terbrueggen. The technology uses labeled probes complimentary to the RNA of interest.

“After you add these probes to your sample they ligate together to go from two half probes to one full-length piece of material," he explained. "Because you've made it full length, the two primers are on the same piece of DNA and now it's amplifiable by PCR. When they get amplified each one will be a unique size — gene one will be 100, gene two will be 105, gene three 110 base pairs long." All of these probes are labeled with the same color, and, "that's why we use capillary electrophoresis to separate them, because I can separate on the basis of their difference in size."

For the FDA version of the Redi-Dx test, Terbrueggen told PCR Insider, "it's designed to run on the Life Technologies 3500 Genetic Analyzer, which is their newest capillary electrophoresis instrument." The SBIR-funded test currently in development will also use this platform, said Terbrueggen.

The SBIR award also continues DxTerity’s collaboration with researchers at the University of California, Los Angeles, in order to confirm the pilot data for the test in a clinical setting. For this radiation sickness predictor, the researchers have estimated a normal response to cancer-treatment levels of radiation in the blood, including "cell control markers as well as others that are related to the processing of cell reproduction or replication." Their ongoing experiments will focus on gastrointestinal side effects caused by radiation treatment of prostate and colon cancers, which are cases where acute radiation sickness leads to bowel obstruction and can be fatal.

The chemical ligation technology may also lend itself to other gene expression assays that can be done on blood, and DxTerity is working on a possible line of disease-monitoring tests, beginning with one for rheumatoid arthritis. They believe this line of tests will help calibrate treatment (and lower medication cost) when patients are in a remission state from a relapsing-remitting autoimmune disease.

"We have a platform that allows us to do very inexpensive testing from a drop of blood," said Terbrueggen.

As reported by PCR Insider earlier this year, the firm can also use this technology on formalin-fixed, paraffin-embedded samples, and has demonstrated its use in measuring HER2 gene expression levels in FFPE breast cancer samples. However, Terbrueggen said the HER2 assay "was really developed for more of a demonstrator," or proof of principal for the firm's chemical ligation methods.

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