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Duke Researcher to Use RNAi to Hunt for Genes That May Be Used Against a Potential Bioweapon

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With the increased focus on terrorist attacks in the United States, researchers have been applying a variety of new technologies, including RNAi, toward dealing with the threat of bioterrorism.

Now a Duke University researcher, Donald Cook, has joined the list by starting work on an effort that will use RNAi to track down genes that could be used as therapeutic targets for the bacteria Francisella tularensis.

F. tularensis “is extremely pathogenic, and … it could conceivably be used as a biological weapon,” Cook told RNAi News last week. The bacteria can be transmitted through the air but can also be ingested, making the contamination of water supplies another bioterror possibility, he noted.

Tularemia, the disease caused by F. tularensis, is characterized by fever, headache, and nausea, among other symptoms. If untreated, the disease can lead to sepsis, respiratory distress, renal failure, disseminated intravascular coagulation, and meningitis, according to the New York City Department of Health.

While F. tularensis can be found in soil, and naturally occurring outbreaks of tularemia have occurred in the past in the United States, the disease is for the most part rare, Cook said. However, its potential as a weapon is well-known: Both the Soviet Union and the United States have weaponized tularensis in the past as part of their biological warfare programs.

There are antibiotic treatments for tularemia, but Cook noted that the possibility of using plasmids to encode drug resistance into F. tularensis makes the need for new therapies a priority. To help speed the development of such therapies, Cook is “trying to find genes that participate in naturally clearing the organism,” he said. “Once identified, the proteins encoded by those genes could be targeted by therapies aimed at increasing the [body’s immunological] natural response.”

Cook said his project is starting off by identifying the loci that contain genes that confer differential susceptibility.

“What we do is take two different strains of mice, one of which is resistant [to tularemia] and one of which is susceptible to the disease, and we scramble their genes by interbreeding them,” he explained. The resultant F1 generation of mice would then be interbred with each other.

The mice of the second generation “would differ from one another in terms of the assortment of … alleles they inherited from the original starting two strains,” Cook said, adding that some of these different alleles will affect the relative resistance of individual mice to the bacteria.

To identify the gene variants associated with resistance, Cook said he and his colleagues will make two different pools of DNA from susceptible mice and resistance mice of the F2 generation. “If a gene is not involved in the resistance to this organism, you’d expect alleles of it would be represented at 50 percent from each of those two parental strains — half the alleles would come from one parental strain and half from the other,” Cook said, noting that the percentage of DNA from the two strains can be determined using the DNA pools.

But for genes involved in the animals’ response to F. tularensis, “the parental strain contribution would not be 50 percent,” he said. “For example, we would expect that the pool of DNA from susceptible F2 mice would have more alleles from the susceptible parental strain than from the resistant parental strain.

“We won’t know which genes [are involved in the response] right away, but we can find the general location of these genes by testing each DNA pool for the abundance of strain-specific alleles at known markers along the chromosome,” Cook said. “If the abundance of the alleles is very far from the expected 50 percent, we know that that region contains a gene that affects the response to the bacteria.”

He said that this would give “a broad region on a chromosome that contains a gene that affects the response” to F. tularensis. But since that region could include hundreds of genes, “the problem is how you go from identifying this region that contains a gene to the gene itself,” he said.

In order to narrow down the number of genes that might play a role in F. tularensis response, Cook said that he and his colleagues start off by assuming “that the gene [being sought] is different somehow between these two strains … either because it’s differentially expressed … or [because it has] sequence differences in the coding region of the gene.”

Differential expression can be measured using microarrays, he noted, while differences in coding regions can be identified using genomic sequence databases.

“We’ve got two ways of looking for genes that are different between the strains [of mice] and also within the region [of the chromosome] we’ve identified,” Cook said. “Say we started off with 500 genes, and of those only 25 had coding region differences or are differentially expressed; this is where the RNAi comes in.”

Cook said that the function of the narrower set of genes is determined by knocking them down in an in vitro RNAi assay.

“The goal [would be] to take a macrophage cell line and infect the macrophages with” F. tularensis, he said. “We would introduce an RNAi molecule corresponding to each of these genes that have emerged as our candidate genes, and then measure the bacterial growth in these lines.”

Ultimately, Cook said, he plans to attempt to disrupt the function of genes identified as playing a role in responses to F. tularensis in vivo, but “whether that’s done through gene targeting technology or RNAi, we’ll have to see. Personally, I have more experience in generating gene-targeted mice than using RNAi in vivo, but it might also depend on how well the RNAi works.”

Cook’s work is being funded by a $500,000, two-year grant from the National Institute of Allergy and Infectious Diseases.

— DM

 

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