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Pitt Researcher Begins Project to Establish Procedure for RNAi as Tool for Brain Research

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The National Institute of Diabetes and Digestive and Kidney Diseases has recently awarded a two-year grant, worth up to $200,000, to University of Pittsburgh researcher SuJean Choi in support of a project that uses RNAi to knock down the neuropeptide galanin and the galanin receptor-1 — an effort that may help establish a method of studying the effect of specific genes in specific brain cells.

The project, Choi told RNAi News, involves injecting dsRNAs targeting GAL and GAL-R1 — which play a role in appetite — directly into the hypothalamus of a male rat. After a recovery period, the rat’s feeding behavior will be evaluated.

Choi said that “the biggest sections of the grant are the parametric studies — to look at how long [silencing] lasts, how far … it spreads,” she said. “To see the spread, we need to use a gene that is expressed a little more evenly throughout an area, so that we can see exactly where the borders [of suppression] are,” and galanin fit the bill, Choi explained

The parametric studies will also examine the concentration of dsRNA needed to achieve adequate galanin silencing and the volumes in which the dsRNA can be delivered, as well as when the RNAi effect begins and ends “because this is not a permanent thing,” Choi said. “We’d like to know how long this will last before it comes back.”

Choi said that preliminary studies indicate that protein suppression for between five to seven days is achievable, but that actual duration of gene knockdown in vivo remains uncertain. These estimations are based on “cell-culture data … which gives us an idea, but we can’t know [for sure],” she noted. “It will depend on each protein; their turnover rate and their own synthesis rate … could vary, so it’s not an easy thing to predict.”

Choi said that she is fairly confident that she and her colleagues can achieve knock down for at least a week, “but we would like to continue that out to 10 to 14 days.” Even if the researchers can’t extend the silencing effect, however, “seven days is a pretty nice timeline to test something” and gives enough time to allow the effects of surgery and anesthesia on the animal to wear off before experimentation begins.

The ultimate goal of the project is to develop a “procedure for using RNAi [that is] reliable as a highly selective, neurochemically specific lesioning method” and offer “evidence of its utility in studying physiological functions of particular neuronal subpopulations in the brain,” according to the grant’s abstract.

“It’s for the basic researcher,” Choi said. “We’re interested in using this as a method of making a genetic lesion. This way we can suppress gene expression within a single cell,” she said, without “affecting gene expression of other genes in that same cell. We haven’t destroyed the cell, we haven’t damaged the cell, nor have we prevented the cell from functioning normally aside from the one gene that we have altered,” she said.

“This will give an opportunity to distinctly identify the role of [a particular protein] in any given function,” Choi said. Additionally, by locally delivering the dsRNA, “we can find out the effect of a single protein in a single area, but not necessarily in another area of the brain.”

All this, Choi said, makes the method a “very potent anatomical tool that could be used in a myriad of ways down the road.”

Choi stressed that she and her colleagues are using dsRNAs in this project, not siRNAs, noting that selecting an appropriate siRNA can be challenging.

“There are quite a lot of elaborate algorithms being put out to predict which part of the coding region is the best,” she said. “Since we didn’t want to deal with that, we inserted double-stranded RNA and let the Dicer enzymes choose where they prefer to make the cuts themselves.”

This apparently works, Choi said, although she and her colleagues are well aware of the potential for side effects. “You’re going to get immune responses with almost anything you do,” even simply injecting saline into brain tissue. She has not run into any physiological difficulties with test animals yet, but warned that “this may not be true for everybody else. It should all be taken in with a degree of caution that you’re going to have to look into it on a case-by-case basis.”

Choi is conducting her research in collaboration with the University of California, San Francisco’s Aditi Bhargava.

—DM

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