Researchers at the University of Texas Medical Branch Galveston's School of Medicine are conducting animal studies to test the ability of their SELDI-based thioaptamer chip to diagnose an individual's immune response to certain diseases. The group hopes to work with Ciphergen to begin commercializing the chip in one to two years.
"We've shown proof of principle that it's working," said David Gorenstein, a professor of biological sciences at UTMB who is leading the studies. "We've shown that [the chip] can detect low-fentomolar ranges of protein, and we're using guinea pigs and mice that are models of Lhasa hemorrhagic fever to monitor how well the chip works in detecting infection."
Thioaptamers are DNA or RNA fragments with sulfur interspersed into the molecules' backbones. When interspersed effectively, the sulfur enables the aptamers to bind more effectively, and to have greater specificity and nuclease resistance, said Gorenstein.
"We've shown that with thiophosphate substitutions, especially dithiophosphate substitutions, where you replace both non-bridging oxygens with sulfurs, you get a molecule that is stickier than normal nucleic acids," Gorenstein explained. "The trick is to sprinkle the sulfur in at appropriate positions in the nucleic acid backbone. If you put all the thiosphoshates in there, it becomes too sticky."
"What we want the chip to be able to do is not so much say specifically that 'This has to be a West Nile virus, or influenza infection,' but to say that there is an effective immune response."
Gorenstein believes that thioaptamers are going to be effective substitutes for antibodies. They are more stable than antibodies at room temperature, and therefore easier to maintain on a chip, he said.
In 2003, Gorenstein formed a company, AptaMed, to develop thioaptamers, which have been patented by UTMB, into diagnostics or therapeutics. Gorenstein also established a partnership with Ciphergen in 2002 to co-develop thioaptamer chip-based diagnostics.
Lee Lomas, the director of research and development at Ciphergen, said that Ciphergen would probably begin commercializing the thioaptamer chip in about two years, but the chip is not likely to be placed in non-research type settings so soon.
"If you're talking about commercialization in terms of running research-grade tests, that's in the near future," he said. "But in terms of putting the product in a reference, or non-research type setting, that has more implications in terms of regulation, so it's a longer-term goal."
The thioaptamer chip that is currently furthest along in development contains thioaptamers that target proteins involved in the immune inflammatory response.
"What we want the chip to be able to do is not so much say specifically, that 'This has to be a West Nile virus, or influenza infection,' but to say that there is an effective immune response," said Gorenstein. "A lot of times, like in hemorrhagic fevers, the patients die not so much from the virus itself, but from an over-response to the infection. There's a cytokine storm, the inflammatory response goes awry, and the people die of shock. So it's very important to be able to monitor at an early stage the immune response."
Currently, there are a battery of blood tests for monitoring how a patient's immune system is handling an infection or disease, but those tests are not very specific for telling at what stage the immune response is in, and what type of infection is present, Gorenstein said.
"The current tests look at just a few proteins, essentially. This [chip] would be much more specific in giving biosignatures for infection," he said.
Though Gorenstein's research currently is not meant to distinguish between pathogens, it could potentially help diagnose infection by bioterrorist agents such as anthrax, botulism, small pox, viral hemorrhagic fever, or SARS. As a result, he has received much of his funding from the National Institutes of Health's biodefense budget and the Defense Advanced Research Projects Agency (see ProteoMonitor 8/1/2003).
Gorenstein recently received the fourth installment of a five-year, $6.3 million grant from the NIH. The installment was for $1.2 million. Gorenstein said he plans to develop more specific chips in the future that would distinguish one pathogen from another by identifying their "biosignature patterns."
"The current chip would probably give a clue through its biosignature as to what pathogen it is not, but the jury's still out on whether it would be highly specific," said Gorenstein. "Certainly, you would be able to distinguish between, for example, exposure to anthrax, Ebola, or hemorrhagic fever through their predicted type of immune response."
Gorenstein's research group has been developing thioaptamers since the mid-1990s. The researchers have developed a bead-based approach for selecting aptamers: They synthesize bead libraries of thioaptamers, with one unique thioaptamer on each bead, and then use flow cytometry to isolate beads that have the most target protein bound to them.
"That's the newest way. It's very efficient and very fast," said Gorenstein.
The work on hemorrhagic fever animal models is done within Biosafety Level 2 laboratories at UTMB, Gorenstein said. Work on Lhasa fever is done in the university's BSL 4 laboratories.
Aside from developing a thioaptamer chip for diagnosing immune responses, Gorenstein's research group is also working on developing a thioaptamer chip that can be used to help diagnose asthma. That work is being funded by the National Heart, Lung, and Blood Institute.
"With that project, again, we've shown proof of principle that it works, and we've also developed some high-throughput methods for selecting out thioaptamers," said Gorenstein. "There'll be animal models and humans, but we haven't gotten into them yet."
Tien-Shun Lee ([email protected])