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Pronota Shows Proof of Concept for Next-Gen Sequencing-Based Protein Biomarker Discovery Platform

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By Adam Bonislawski

Belgian protein biomarker firm Pronota announced recently that it has successfully completed a proof of concept study for a diagnostic platform employing next-generation sequencing in the identification and quantification of proteins.

The study combined the use of aptamers — short, selectively binding oligonucleotide sequences — with NGS technology, binding the aptamers to target proteins and then sequencing the protein-bound aptamers to quantify the proteins.

"We were inspired by the next-generation DNA and RNA sequencing abilities," Koen Kas, Pronota's founder and chief scientific officer, told ProteoMonitor. "It cost something like $3 billion to sequence the first human genome in 2003. Now we can do that for something like a few thousand dollars. So we wondered if it would be feasible to use next-generation sequencing to sequence proteins."

Because NGS technology works only with DNA and RNA, Kas's team needed an interface between the Illumina Genome Analyzer and the study's target protein, immunoglobulin E — a protein biomarker for various types of allergic responses. They selected aptamers, demonstrating in studies with immunoglobulin E that they could be sequenced using NGS machines and that they bound to the protein in a consistent ratio that allowed for quantitation via sequencing.

According to Kas, the development of this NGS-based technique could allow for significant streamlining of what is currently a cumbersome biomarker discovery process.

"You [presently] need three platforms," he said. "A discovery platform, which is mass spec-based; a verification platform, which in the past was antibody-based but now more people are turning to mass spec; and then you have to turn to a third platform, a final immunoassay."

"With what we have we believe we've built the cornerstone for a diagnostic device that can measure DNA, RNA, and protein and which you can use for discovery, validation, and clinical application as well," he said. "And you don't have to shift from platform to platform to platform."

Readout results from Pronota's NGS-based system correlate strongly with those from conventional ELISA assays, Kas said. The company, which presented these findings last month at the Knowledge for Growth life sciences convention in Ghent, Belgium, has filed two patent applications based on the technology.

A potential issue with the technology is the present lack of aptamers against many proteins. There are some 250 to 300 aptamers available in the public domain, Kas estimated, compared to more than 2,000 antibodies.

It also remains uncertain, he noted, whether it will be possible to generate aptamers for most of the human proteome.

"An aptamer is a short piece of DNA. You have four nucleotides, so in theory you could make, from a nucleotide of 20 bases, 420 different aptamers which all recognize different proteins," he said. "But maybe that assumption is wrong. Maybe different sequences quite often recognize the same protein. In theory there's a limitless number of DNA aptamers, but a limited number of aptamers that recognize different proteins."

The other possibility, he said, is that researchers simply aren't very good at aptamer discovery yet. To that end, Pronota is embarking on another study using NGS technology — attempting to generate novel aptamers by putting large numbers of random nucleotides together with target proteins and then sequencing to see if any specific sequences emerge more often than others.
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"It provides a pre-selection of what could be binding sequences for a given protein," Kas said.

While the library of available aptamers is a small one, it holds the promise of offering an alternative to the antibody development process.

"It's 2010 and you can't find more than 2,000 antibodies that are useful in clinical analysis," Kas said. "That's pretty poor. And then you have to think that the majority of our proteins don't exist in full-length fashion – they get processed, phosphorylated, cleaved. Also there we have a long way to travel."

The company also expects that coming advances in NGS technology will further streamline the aptamer-based technique. In particular, Kas said, new NGS technology in development at IBM will allow researchers to sequence aptamers without having to bind them with a ligand to the instrument's sequencing chip – cutting a step out of the process.

Last fall, the National Human Genome Research Institute awarded IBM Research $2.6 million over three years to develop an electrical device, called a DNA transistor, for controlling the translocation of DNA through a nanopore to enable single-molecule sequencing.

Pronota is presently focused on developing protein biomarkers for the detection of heart failure, kidney failure, preeclampsia, sepsis, and ovarian cancer. According to Kas, the company plans to continue developing conventional antibody assays for these biomarkers while working in parallel on assays using the new aptamer-based, NGS technique.

Pronota received €7.9 million ($9.6 million) in three closings of a Series B financing round that began last July (PM 07/09/2009) with the latest close coming in May.

The newly developed platform positions Pronota to take advantage of the coming clinical adoption of NGS technology, Kas said.

"Two years from now I believe you'll start to see the first next-generation sequencing applications that are FDA cleared," he said. "These will be DNA or RNA sequencing, but that will lead to an acceptance of these sequencing devices in clinical labs."

Already, he noted, the Mayo Clinic uses NGS to sequence cancer genes for its cancer patients. Other non-profit labs, like Emory University's Genetics Laboratory, are also using next-gen sequencing on clinical samples, while several companies, such as Sequenom, Ambry Genetics, and GeneDx, are developing sequencing-based tests for a number of conditions.

And Pronota isn't alone in its quest to adapt the technology for protein research. This week antibody company Affomix announced a collaboration with the University of Montreal's Pharmacogenomics Centre that will employ antibodies tagged for NGS in a study of protein biomarkers for cardiovascular disease (see related story this issue).

"First DNA, then RNA, and we believe that the next wave will be proteins," Kas said.

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