The discovery of the short interfering RNA was a eureka moment. Long, double-stranded RNAs had triggered fatal interferon responses in mammals, but siRNAs succeeded in silencing genes without inducing unwanted effects. The technology suddenly had a future as a tool for discovering human gene function and attacking the roots of disease.
But recent research suggests that siRNAs might not be so specific. Bryan Williams, whose own siRNA research appears in the September issue of Nature Cell Biology, says, “I suspect, and can certainly identify, a number of papers in the literature where results are described that are probably not due to the specific knockdown of the targets.”
Williams and colleagues at the Cleveland Clinic Foundation’s Lerner Research Institute have shown that siRNAs could in fact activate some genes associated with the interferon system. Postulating that long, double-stranded RNAs did not work in mammalian cells due to the upregulation of dsRNA response proteins, PKR and RNAse L, the team tested for sequence-specific suppression of reporter luciferase activity using vectors that produce 500-bp inverted repeat dsRNA molecules complimentary to GL3 or RL mRNA in mouse cell lines with PKR and RNAse L deletions. Williams says he went “along with the dogma” that siRNAs don’t cause the non-specific effects seen with long dsRNA. But a by-product of their research was the discovery that various 21-mer siRNAs resulted in interferon-mediated activation of the Jak-Stat signaling pathway and upregulation of a number of interferon-stimulated genes.
Says Williams: “We had already modeled … double-stranded RNA onto the PKR double-stranded RNA binding domain. So we know how [the domains] fold, we know the structure of those domains, and we know they recognize 16-base pairs. These are 21-base pairs, and I thought: ‘Well, maybe they’re not going to activate … when you add [the siRNAs] to a cell. It turns out, of course, they do activate.”
What this means is that in cells where the interferon-response pathway is functional, the phenotypes measured by researchers conducting gene expression research might not be due to the specific knockdown of a target but instead to the non-specific upregulation of ISGs.
Williams also cautions that siRNAs being developed as drugs are likely to have non-specific effects. “Almost all humans have an intact interferon-response pathway, that’s how we survive virus infections … and so this is going to lead to major problems in the development of siRNAs as therapeutics.”
But Williams sees his discoveries as roadblocks for RNAi, not dead ends. In some cases, non-specific effects won’t be a major issue, he says. In a number of cell lines, such as certain tumor cell lines, he says, the interferon pathway is defective. In such cases, “researchers can be pretty certain that the effects of siRNA are due to the knockdown of their target.” And toxicities in humans might be addressed with chemical modifications to siRNA or the development of stable, single-stranded siRNA.
Cenix BioScience CEO Chris Echeverri cautions against overreacting to the new study: “Let’s remember that RNAi is still quite a young field. We’re still learning exactly how the pathway works, and we’re going to get some surprises. We should expect some things will be not exactly as we planned.”
For functional genomics and target validation, Echeverri says, “The lesson to be learned is to design your experiments carefully with the proper controls. And one of the key things you can do to protect yourself against the chance that the phenotype you’re seeing is not specific to your target is to use multiple siRNAs against that target.”
He adds, “Last year we went through the problem that RNAi was over-hyped. This year we’re going … in the other direction. The reality will probably [be somewhere] in the middle.”
A longer version of this article, including comments from Alnylam executive Tom Ulich about what Williams’ research means for siRNA therapeutic development, appeared in the September 5 edition of RNAi News.
Doug Macron is the editor of RNAi News, a new weekly newsletter from GenomeWeb at www. rnainews.com. He can be reached at dmacron @genomeweb.com.