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Tufts Research Looks to RNAi for Help Answering Questions About Blood Parasite

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RNA interference has proven to be such a powerful technology that researchers from a variety of disciplines have been finding ways to incorporate it into their work. One of these scientists, Tufts University’s Patrick Skelly, has recently begun a project to see if RNAi can be used to find out more about the human platyhelminth parasites Schistosoma.

According to Skelly, who is an assistant professor in the department of biomedical sciences at Tuft’s school of veterinary medicine, schistosomes infect approximately 200 million people worldwide. In their adult form, the parasites live in water and can infect vertebrates by passing through unbroken skin, he explained.

“They’ve got a whole battery of proteases and other molecules that they release on our skin, and they’ve got propulsive tails that push them beneath the outer layers of the skin,” Skelly told RNAi News. People infected with schistosomes are often asymptomatic, he said, but others experience liver fibrosis and other liver pathologies that can be fatal.

“It’s an important human pathogen,” Skelly said. “But the knowledge we have about this organism lags behind the knowledge that we have of lots of other pathogens. I think one of the reasons [for this] is that we have never been able to study it at the molecular level — we don’t have many techniques available that we can use to address hypotheses,” he added.

Although he had not previously worked with RNAi, Skelly said that he was aware of the gene silencing technology and thought that “RNAi might be a good way for us to start to get in and answer important questions about this organism.”

In preliminary experiments, Skelly said he was able to use double-stranded RNA to knockdown expression of a cathepsin gene in schistosomes by simply soaking the parasites in dsRNA and medium.

“I measured enzyme activity, which was way down, [and] I did some immunofluorescence, because I had an antibody against this protease, and found that the population structure had changed so that most of the parasites … treated with the double-stranded RNA didn’t stain for the enzyme [while] most of the controls stained very brightly,” he said. “Lastly … I tried to purify RNA from those parasites and looked for my specific message — I effectively couldn’t find it in the treated ones.”

With these data, Skelly was able to secure a five-year National Institutes of Health grant, worth about $250,000 a year in direct costs, to further support his work with RNAi in schistosomes.

The first part of the grant project will focus on investigating whether RNAi is effective in the parasite at any stage of its life cycle, while also optimizing techniques for using the technology.

“There’s lots of different ways to have the [gene] suppression [from RNAi] work now, so [we’ll] try things like siRNAs,” Skelly said. He added that he will also examine “how long … you need to treat [schistosomes with RNAi molecules]; what sorts of amounts of RNA do you need to … introduce to get an effect; [and whether] it’s better to try to microinject, to [use] a gene gun to shoot these things in, or [if] soaking is perfectly fine.”

The other part of the grant will involve using the RNAi protocols developed to test hypotheses about the involvement of proteases in schistosome-associated hemoglobin degradation and nutrient acquisition.

“[The parasites] are sitting in the bloodstream, they are ingesting red blood cells, degrading hemoglobin — the precise biochemistry of this pathway has been of interest to many people,” Skelly said. “A number of hypotheses have been generated as to which enzymes act when and which enzymes activate other enzymes, but we’ve never really been able to address these questions before.

“It seems to me that RNAi would be a great way to allow us to address these questions,” he said.

The grant project began on March 15 and runs through February 2009.

— DM

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