Dharmacon is seeking to set standards for siRNA function.
At his presentation to the Beyond Genome conference at San Diego's civic center June 16, Dharmacon executive vice president of R&D Bill Marshall outlined these standards.
First, the company is recommending that researchers use 100 nanomols of siRNA. "We've selected 100 nanomols because what we've found is by the 100 nanomol point, you have reached a plateau of the inherent functionality of any siRNA sequence," Marshall told GenomeWeb.
Secondly, the company is recommending that researchers indicate the percentage of gene knockdown that a particular siRNA effects, using a functionality, or F-standard: So, for example, an F50 would mean 50 percent knockdown, whereas F80 would mean 80 percent knockdown. This measurement is to be taken at the 24 hour time point after the siRNA is introduced into the target cells, and Dharmacon recommends that the amount of knockdown be quantitated using QT-PCR or branch DNA.
Dharmacon itself uses Branch DNA, Marshall said, because of "the robustness of the method" and because it does not require any isolation of mRNA. While some researchers have measured downregulation of proteins using antibodies, Marshall said these latter methods can be complicated by the fact that different proteins have different lifetimes in cells.
Meanwhile, in a follow-up to discussion of the off-target effects that siRNA can have, Marshall pointed out that the Rosetta group which reported this off-target effect in the June issue of Nature Biotechnology did not use Dharmacon's design tools to design their siRNAs. "We've designed in the bioinformatics solutions up front to avoid unfortuitous matches to other genes," Marshall said. "We ensure that any siRNA sequence has to have at least three mismatches to any other gene product." The company, he said, has developed its own Blast routine to identify close mismatches.
Other significant talks at the Beyond Genome RNAi track this week indicated that big pharma and biotech are really using RNAi, and are already working toward the sort of standardization that Marshall discussed.
Kathrin Heermeier, a senior scientist at Aventis, presented on the unique siRNA solution her group has developed: siRNA molecules of 21 to 23 nucleotides with 2'5' overhangs, instead of the 3'5' overhangs that are a standard feature of the siRNA pioneered by Tuschl and colleagues. The group found that these 2'5' overhangs, which do not cause binding between the sugars, work just as well as the 3'5' variety (and don't require a license to the Tuschl patents). They also tried to stabilize the molecules by introducing phosphorothioates, but found that the non-modified molecules worked slightly better than the non-modified ones, and that the modified ones had toxic side effects.
Interestingly, Heermeier's group has, like Dharmacon, found that 100 nanomols of oligos is optimal. The group is using these siRNAs to knock down active genes in chondrocytes, cartilage cells that play an important role in osteoarthritis.
Dimitri Samarsky from Sequitur also discussed his company's efforts at designing an alternative to the standard 21-25 base-pair siRNA. "Not all siRNAs are as innocent" as they seem, he warned. "Some siRNA causes an interferon response." The company's stealth RNAi, he asserted, is longer than siRNA by a few bases, and also produces a stable knockdown of 68 percent (that would be F68 in Dharmacon's language) after 24 hours. The secret? Well, true to the "stealth" brand name, Samarsky wouldn't reveal it to GenomeWeb. But he said the secret was in a form of chemical modification.
A further alternative to standard siRNA, Benitec's ddRNAi, is moving more directly into the US market: Promega, which holds a sublicense to the recently granted patent around Benitec's ddRNAi for target validation purposes, is initiating a program to further sublicense this technology to various parties. The company does not expect too many pharmas to be lining up yet: but once they begin to develop the drug-like potential of ddRNAi, a DNA molecule that causes siRNA to be produced in the cell, "those companies will come to us to commercially license" the technology, said Richard Schiffereen, director of technology and market development for Promega.