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How RNAi Will Untangle Gene Function

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Some call it the most revolutionary biological tool since polymerase chain reaction. Nobel laureate and Biogen founder Phil Sharp says it will "fundamentally change how we do cell biology." And technology licensing officers at the Carnegie Institution and MIT, which sell commercial rights to two separate pools of patents for RNA-mediated-interference technologies, say that these could well be the most significant inventions, at least in biology, ever to cross their desks.

However you measure its importance, there's no question that RNAi has electrified the functional genomics field. Experimentally demonstrated in a model organism only five years ago and for the first time in a human cell in 2001, the knockout method is now touted as the single best way to study how genes work.

While investors go wild about the therapeutic possibilities for siRNAs (VCs have already poured more than $75 million into companies trying to figure out how to administer them directly to patients), a whole new business sector is emerging around the use of RNA interference for gene-function analysis and drug target validation.

Some pioneering scientists have already applied the technology to entire genomes. RNAi studies of all C. elegans and Drosophila genes are soon to be published. One company and at least three public-sector groups have announced plans to systematically screen the whole human genome with siRNAs. And Harvard Medical School awaits funding to open what would be the first RNAi core facility to provide Drosophila genome screening services.

Surprisingly, in a field that's been burned time and again by over-hyped technologies, this one seems to be without detractors. Explaining that because "the technology isn't 100 percent robust yet" and that "others are already doing" high-throughput genome scans, Christopher Austin, senior advisor to NHGRI's director for translational research, says in an e-mail to GT that the institute "decided not to do anything in siRNAs for now." But of more than 30 people interviewed for this article, none expressed doubts about the usefulness of the technology for gene function analysis, and several consider NHGRI's decision a mistake.

Adoption and Adaptation

Seldom has a technology been adopted so quickly by so many biologists. A PubMed search for "'siRNA' OR 'RNA interference'" brings up more than 750 papers published in the five years since a paper by Andrew Fire and Craig Mello ignited the industry in 1998.

According to Dmitry Samarsky, director of technology development for Sequitur, which sells custom siRNAs for target validation that he says are manufactured under the MIT license, "The technique has become so popular that people are trying to adjust their [research] to the technology ... asking, 'What should I knock down to use this?' It's fun to use and could help you to get good papers."

Phillip Zamore, who has three siRNA patent applications under his belt, cites two precedents for this sort of craze: "the use of bacterial enzymes to clone things in plasmid vectors and PCR." The reason RNAi has taken off? "'Cause it's so unbelievably cool," Zamore gasps.

What renders him and others so breathless is the first reliable, reproducible way to silence a gene and connect genotype to phenotype. Where microarrays allow a user to correlate an overexpressed gene and a tumor, siRNAs provide a causal link. They're far easier and quicker to use than that high-maintenance gene-silencing tool, the knockout mouse.

There's no new instrument or system required to do RNAi, and the reagents are readily available to researchers on tight budgets. (Chemically synthesized siRNAs cost upward of $250 per pair and a single gene experiment typically requires three to five pairs; a less costly option is to generate hairpin RNAs in vitro with plasmid- or virus-based DNA molecules.)

Sequence and Snafus

That's not to say that the art of RNA interference has been perfected. Far from it. It's simple enough to copy and paste a few 21-base sequences from GenBank into a vendor's e-mail order form, but consider some of the things that can go wrong in an siRNA experiment: Transfection - just getting the siRNA into cells - doesn't take; you induce toxicity by adding too much siRNA to a cell; because of SNPs or database errors, the GenBank gene sequence that you've designed your siRNA against is not the same one that exists in the sample in your dish (an obvious mistake, Zamore notes, "but even smart people forget it"); the 21-base segment you've selected is homologous to more than one gene and ends up silencing the wrong one; darn, you should have confirmed before buying that the sequence in your mouse tumor study has homology with a human gene; or, oops, you ordered your sequence from the vendor in a 3' to 5' format rather than the recommended 5' to 3' direction.

Other snafus remain to be explained, such as why high GC content can, but won't always, trip things up; why the rates of suppression vary among siRNAs; or why off-target effects, as one pharma team that will soon publish a paper on the topic claims, seem to be an inherent feature of short oligos.

There's plenty more to the RNAi story. For the complete article, which covers patent and licensing issues as well as more history and applications of the technology, see the April 2003 issue of Genome Technology.