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NuProbe Developing DNA Probe For Highly Sensitive Liquid Biopsy Tests


NEW YORK (GenomeWeb) – Boston and Shanghai-based NuProbe Global is targeting multiplexed enrichment of rare DNA variants associated with a broad range of medical conditions, including cancers and infectious diseases.

According to Peng Yin, NuProbe's cofounder, the firm's toehold probe platform enables highly sensitive, multiplexed molecular diagnostic and next-generation-sequencing tests. By flagging the most useful, rare DNA variants in a liquid sample, its technology can detect a diverse range of signatures suitable for uncovering the early stages of disease, added Yin, who also is a professor of systems biology at Harvard’s Wyss Institute of Biologically Inspired Engineering,

The firm recently announced that it had raised $11 million to continue development of an ultra-sensitive, noninvasive blood test for the early detection of cancers and infectious diseases.

Sequoia China and Serica Partners led the Series A funding that included additional investors such as WuXi AppTec Corporate Ventures. NuProbe said that it will use the funds to grow its staff in Cambridge, Massachusetts and Shanghai, China, and to continue technology development and commercialization.

According to NuProbe, its technology has been verified with blood plasma samples from cancer patients and has shown that it improves the sensitivity of qPCR mutation detection one hundredfold and achieves sensitivities down to 0.01 percent. The firm said that its variant enrichment capabilities also enable greater sensitivity and higher sample throughput for next-generation sequencing.

NuProbe's approach has a unique feature in that "it leverages thermodynamic principles to facilitate detection of mutant DNA sequences at high specificity," Maxmilian Diehn, assistant professor of radiation oncology at Stanford, said in an interview. Diehn and his colleagues recently developed an error correction method for a circulating tumor DNA assay that enabled them to detect mutant alleles at a frequency of .004 percent.

He said that potential clinical applications of the NuProbe technology includes diagnosis of infectious diseases and detection of low frequency mutations in tumor samples or plasma of cancer patients.

Abhijit Patel, an associate professor in the department of therapeutic radiology at Yale University School of Medicine, said that NuProbe's technology enables ultra-low-frequency variant detection across a highly-multiplexed set of genomic targets with very little sequencing depth. Patel is a co-author of a paper published in Nature Biomedical Engineering with the Harvard researchers, but he doesn't work with NuProbe.

He said that the technology uses "standard, unmodified oligonucleotides, which reduces the cost and complexity of designing new, customized enrichment panels."

In 2012, in a paper published in Nature Chemistry, Yin and his team described the early development of their toehold probe technology. In September 2017, in the Nature Biomedical Engineering paper, they described the application of the toehold probe to rare allele PCR enrichment and detection.

First products

NuProbe expects that its first product will be a ctDNA liquid biopsy platform that uses its toehold probe technology and PCR to detect non-small-cell lung cancer. The firm expects to launch the test in the Chinese market in two to three years, and is evaluating its strategy for entering the US markets. The company plans to apply for CE registration in Europe, Yin said.

The firm's toehold technology has potential to not only improve detection but also help guide treatment decisions that may increase survival for patients, said Yin, who is also a cofounder of Ultivue, a developer of technology for biomarker detection and analysis in tissue.

Among NuProbe's goals is to make next-generation sequencing more clinically compatible and eventually suitable for use by hospitals for clinical applications.

"This is not specific to just cancers," Yin said. "We are also looking at infectious diseases and antibiotic resistance and any diseases where genetics are a causal factor of deviations from a healthy state."

Yin developed NuProbe's technology with NuProbe Cofounder David Zhang, an assistant professor of bioengineering at Rice University. Victor Shi, former founding president of Qiagen Asia Pacific, is also a cofounder and serves as CEO of NuProbe. The firm recently inked a global licensing agreement for use of the technology with Harvard’s Office of Technology Development.

Yin said that the NuProbe technology detects rare DNA variants using two types of strands — a probe strand and a protector strand — that operate using thermodynamic principles.

The toehold probes detect not only DNA but also RNA that's based on specifically engineered nucleic acid sequences and combines high sequence selectivity and specificity with high temperature robustness to enable the identification of multiple variants, the firm said.

Toeholds are short sequences at the ends of a strand of DNA called the probe strand that are either complementary to a target's DNA sequence or to a second strand of DNA called the protector strand. Within a toehold probe, the probe and protector strands are hybridized because their nucleotide sequences are complementary.

The short-sequence toeholds initiate two exchange reactions. In one, the protector strand releases from the probe strand and lets the probe strand bind to a DNA target, allowing its detection. In the second exchange reaction, the probe strand engages with the protector strand and leaves the target DNA behind.

The two competing exchange reactions lead to an equilibrium that is highly predictable and highly sensitive to perturbations, NuProbe said. It contributes to the ability to separate out only the most important diseased targets for analysis, Yin said.

In the context of toehold probe hybridization, the system seeks to capture the wildtype target and then eliminate it, while leaving the mutant target that carries a disease signature untouched, Yin said. In the context of PCR diagnostics, the mutant sequencing will be specifically amplified and thus enriched using similar principles, he said.

Emerging technologies

Last year, liquid biopsy technologies gained one of their first solid footholds in the clinical care of cancer patients. Clinicians began to demonstrate an acceptance of liquid biopsy in the care of lung cancer patients, establishing this niche as a proving ground for the technology.

In 2016, a PCR-based assay for EGFR mutations developed by Roche became the first liquid biopsy test approved by the US Food and Drug Administration, when the agency approved the Cobas EGFR Mutation Test v2 as a companion diagnostic for Genentech's Tarceva (erlotinib). A few months later, the FDA expanded the test label, adding an indication as a companion diagnostic for non-small cell lung cancer patients considering treatment with AstraZeneca's Tagrisso (osimertinib). Last month, the FDA granted premarket approval for expanded use of the test as a companion diagnostic test with AstraZeneca's Tagrisso (osimertinib) for non-small cell lung cancer patients, covering tissue and liquid biopsy applications.

While there are many other existing technologies in the ctDNA liquid biopsy space, NuProbe offers "a very simple workflow, since the NGS library is derived from PCR amplicons rather than ligated and hybrid-captured DNA fragments," Patel said. "Also, because it requires very little sequence depth, [the technology] permits many samples and targets to be multiplexed using lower-throughput, benchtop sequencers, which would be more affordable for smaller research and clinical labs."

Diehn said that a key question going forward with respect to the commercialization of NuProbe's technology is whether it will be possible to apply the approach to larger genomic regions in the range of hundreds of kilobases to megabases. "This would be required for many clinically relevant applications," he said.