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RNAi Roundup: Off-Target Gene Silencing Issues; Why Exelixis Chose Dharmacon to Provide 600 siRNAs; and Sequitur s Stealth RNAi

NEW YORK, June 13 - One would think the recent evidence that short interfering RNA has off-target gene silencing effects might put a damper on the burgeoning business of siRNA companies.


In these findings, published in the June issue of Nature Biotechnology, Aimee  Jackson and colleagues at Merck subsidiary Rosetta Inpharmatics used chemically synthesized siRNAs provided by Dharmacon, of Lafayette, Colo., in two separate experiments to silence the the exogenous genes  luciferase and MAPK14 in human HeLa cells. In both cases, when they performed gene expression analysis using microarrays, they identified off-target silencing effects.


But in the eyes of Dharmacon, findings on off-target gene silencing only provide further reason for researchers to choose its bioinformatically designed pools of siRNA.


"One of the key developments lately has been that an excess amount of siRNA in the cell can lead to nonspecific effects," said Dharmacon marketing vice president Mike Deines. "All of a sudden there's a realization that you do need to be a little bit careful" in the amount of siRNA you introduce.


"But one of the key advantages [of Dharmacon's siRNA]  is that you can deliver a known quantity to the cells, whereas with [an] in vitro transcription-based system, once it gets into the cell, you have no control over how much of the siRNA is expressed," Deines said.


Jackson pointed out to GenomeWeb that her group did titrate down 1000-fold the amount of siRNA introduced into the cells (it's in Figure 1c) and still found that there was a small off-target effect.  Since the paper was published, she said, the group has tried this experiment with dozens of other genes, and found that there was off-target gene silencing in every other case. "We are working hard now to improve on siRNA design to reduce the off-target effect," while maintaining the gene silencing, she added.


Exelixis, meanwhile, seems to have had better luck with Dharmacon siRNA. The company chose Dharmacon as the manufacturer and provider of a library of 600 siRNAs for kinases and other drug target genes, in a deal announced earlier this week.


Exelixis' decision to choose Dharmacon as an siRNA provider for the project emerged out of the relationship that the two companies built as Exelixis sought to identify a reliable manufacturer of siRNA, said Geoffrey Duyk, the company's CSO and president of R&D. "In working together to validate the siRNA, they proved to us that they are committed to this area and are going to be a survivor." While Exelixis used its own bioinformatics to figure out which genes to target, Dharmacon provided effective algorithms for design of effective siRNA, according to Duyk.


Dharmacon claims that it is able to achieve at least 70 percent gene silencing for any gene using its Smartselection algorithm, which uses 34 different selection guidelines, along with its technique of providing a pool of four siRNAs for each gene, synthesized with 2'-ACE(R) RNA synthesis chemistry. (Jackson said the Rosetta group did not use the pools of four siRNAs that Dharmacon ordinarily provides, but rather individual ones targeted at the genes in the experiment.)


While Duyk said that off-target effects and other unintended perturbations do occur "from time to time," he emphasized that this issue is not specific to Dharmacon's siRNA. "What everyone has to realize is that you have to think of RNAi like any other drug-like molecule. It does in fact usurp an internal biological process. You just have to be careful."


In the Exelixis deal, Dharmacon will exclusively provide the drug discovery company with siRNAs for about 100 target genes, which will then be used in functional genomics studies, Deines said. Dharmacon will also include siRNAs for 500 publicly available genes in the kinase and other families, which it will then also be able to sell as "catalog" siRNAs to other customers. 


The company expects this library of 500 siRNAs to be available by the end of the third quarter. Although pricing has not been firmed up yet, Deines said he expects these siRNAs to be priced comparably to the company's current offerings of "catalog" siRNAs in the tyrosine kinase family: the starting price is $295 for a pool of four siRNAs designed to silence a single gene.


Meanwhile, early next week Dharmacon is planning to announce next week another deal with a biotech or pharma that is similar to the Exelixis collaboration, Deines said.


The company is also readying to establish a minimum standard for gene silencing, according to Deines. "We're going to be pushing more, getting companies to be more upfront when they say siRNA works or doesn't work," he said " If it's not 50 or 70 percent, to say that it works is too vague of a term."


Bill Marshall, head of R&D at Dharmacon, is likely to touch on this topic when he speaks next week at Beyond Genome in San Diego, Deines said.


Marshall's talk is one among many in an RNA Interference track at the conference, which is being organized by Cambridge Healthtech Institute and runs from June 16 through 19 at the San Diego convention center.


The RNA Interference track, which takes place on June 16 and 17, includes talks on topics ranging from the usual issues - effective knockdown for target validation, synthesis, and delivery for drug development - as well as more specific addresses on use of RNAi to characterize brain disease-related genes and to "investigate mechanisms of hormone-regulated growth of breast cancer cells."


On the product front, Sequitur, of Natick, Mass., has just released a new technology, stealth RNAi, which it says is "distinct from [ordinary] RNAi." The company claims that Stealth RNAi offers increased activity, is more stable in cells than RNAi, does not induce the interferon reaction or PKR stimulation, is less toxic than RNAi, and is stable in serum.


The company also said that it has renewed multi-year deals to provide RNAi to Bristol-Myers Squibb and Procter & Gamble, and that its paper comparing RNAi vs. antisense, on stability, efficacy, toxicity, and hit rates is scheduled for July publication in Nature Biotechnology. 

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