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Rewriting the Rules for RNA Interference
By Nancy Touchette

A promising new technique for silencing genes is creating a stir as several new studies have produced conflicting results about whether the technique affects genes other than the intended target. The technique, called RNA interference, has been used experimentally to turn off disease genes in human cells in culture and to inhibit the hepatitis virus in mice.

Microarray analysis of different siRNAs targeting the MAPK14 gene.

The question of whether or not RNA interference is specific is important to answer because the technique shows such promise as a new way to treat disease and to study gene function.

Researchers have capitalized on a system designed by nature that seeks out and destroys unwanted RNA. One new study now reports that molecules known as small interfering RNAs (siRNAs) can selectively bind to larger RNA molecules to trigger their destruction. But a second study claims that siRNAs are not nearly as specific as previously thought.

“It’s clear from these papers that some siRNAs can be highly specific,”says Andrew Z. Fire of the Carnegie Institution in Baltimore, Maryland, one of the first to describe RNA interference.

“For reasons we don’t yet understand, there certainly could be off-target effects,” he says. “We just need to figure out how to tweak the system to get an acceptable level of specificity.”

Microarray analysis of different siRNAs targeting the Rb gene.

In one of the recent studies, Stephen Fesik at Abbott laboratories in Abbott Park, Illinois, designed five sets of siRNAs to target four different genes and found that they activate a similar set of genes throughout the genome. These findings suggest that siRNAs can be designed that are highly specific.

But Steven R. Bartz, along with colleague Aimee L. Jackson, of Rosetta Inpharmatics in Kirkland, Washington, also created sets of siRNAs that target different regions of two other genes. They found that their siRNAs hit many unintended targets.

“Several studies have reported on the exquisite specificity of siRNA,” says Bartz. “But when we look at the whole genome, we see that these molecules are not specific at all.”

Some of the siRNA molecules tested by the Abbott researchers also affected unintended targets. But the researchers were able to come up with siRNAs that blocked RNA molecules of interest without affecting other RNAs.

The question to be asking may be not whether or not siRNAs are specific, but how they can be designed to be more specific.

“We need to be able to design drugs that are specific for their targets,” says Jackson. “We are trying to figure out the rules, but we don’t have an answer at the moment. It’s an open book.”

See related GNN article
»Gene Silencing Passes Test in Human Cells

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Jackson, A.L. et al. Expression profiling reveals off-target gene regulation by RNAi. Nature Biotechnol. 21, 635-637 (June 2003).
Semizarov, D. et al. Specificity of short interfering RNA determined through gene expression signatures. Proc Natl Acad Sci USA 100, 6347-6353 (May 27, 2003).

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