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Systematic Screening By RNAi

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Part Four in a Multi-part GT Series on RNA Interference

A New Whole-Genome Race Heats Up

 

By John S. MacNeil

Konrad Basler at the University of Z rich knows that the field of RNA interference is a hot one. The 42-year-old professor of molecular biology picked up on the technology two years ago, and it didn’t take him long to figure out he could systematically screen whole genomes with RNAi as a swift and effective means of determining which genes in Drosophila are involved in certain signal transduction pathways. Basler knows another thing too: he’d better act quickly to perform his RNAi experiments before a competing colleague beats him to the punch and publishes first.

“There are probably many more labs looking to study similar systems,” Basler says. “Most labs want to find genes for their particular problem, and if they know someone else is interested in that problem, they should probably hurry up.”

As a scientist, Basler is not alone in hoping to publish results before his competitors. But lately, scientists engaged in large-scale RNAi screens have felt the pressure a little more than usual. Given the growing interest in RNAi and its sheer power to uncover gene function, many scientists are scrambling to sift through entire genomes — model organisms and the human — to be the first to say they understand how sets of genes contribute to the function of biological pathways.

The rush is not confined to academia. Pharmaceutical companies including Novartis, Bristol-Myers Squibb, and partners such as Exelixis that have invested heavily to make RNAi part of their genomics technology arsenal are also moving quickly to consolidate their understanding of disease pathways with the help of double-stranded and short interfering RNA reagents. Startup RNAi companies such as Cenix BioScience in Dresden, Germany, are even more under the gun to capture intellectual property rights to the discoveries they make as a means of building value in their companies.

“Obviously people are keeping their cards reasonably close to their chests, because everyone wants to be the first to do certain screens,” says Chris Echeverri, Cenix’s CEO. “The race could be over next year, and this is partly why we’ve tried to push ahead rapidly,” he adds.

There are, however, those who discount the notion that RNAi’s rise in popularity is triggering a “land grab” for IP, or even just bragging rights. Science is competitive, they say, and who knows if courts will ever uphold some of the broad claims to gene function that many companies hope to secure. Greg Hannon, an RNAi specialist at Cold Spring Harbor Laboratory, says there are so many different ways to use RNAi to study a particular pathway that many groups could find unique ways to study the same system. “There’s no universal way to do a certain assay, and there’s no universal cell line in which you would do that assay,” he says. “These kinds of tools are going to be applied in a very individual way.”

Nevertheless, several groups of researchers in the US and Europe have developed various approaches to comprehensively screen genomes to determine functions of individual genes. Among others, René Bernards and Julian Downward are involved in one such effort in the UK and Europe, while Hannon and Norbert Perrimon at Harvard Medical School are separately engaged in similar work in the US. The researchers declined to disclose details on the assays they’re using to study gene function, but they did discuss their general strategies and what they might gain from their ambitious endeavors.

Creating a Core RNAi Screening Lab

At Harvard, developmental biologist Perrimon is gearing up to perform a series of genome-wide screening experiments in Drosophila. With at least $3 million in funding from NIGMS, his group opened the world’s first centralized RNAi screening facility in June, complete with robotics and seven staff running Drosophila cell-based assays submitted by collaborators. Perrimon says his goal is not only to provide genome-wide screening services to investigators (the NIGMS grant pays for the cost of the experiments), but also to generate a database of results useful for optimizing RNAi probes and comparing results from functional genomics experiments.

Perrimon points out several advantages to centralizing RNAi experiments. Acquiring the expertise to perform genome-wide RNAi screens takes time and money, he says, so it makes sense to consolidate that effort in one lab. “Overall the technology is quite involved, it’s quite expensive, and it takes some people who are really trained quite well,” he says. “It’s cost effective to allow people to have access to a center where those kinds of screens are being done.”

A specialized genome-wide RNAi lab could also eliminate some problems with comparing data across platforms. As has happened with DNA microarrays, researchers can’t always compare large RNAi screens generated at different labs because of variations in experimental conditions and protocols. Some researchers are advocating for a library of RNAi reagents that they could access to run assays in their own labs, “but what you lose when you do this is basically a lot of the quality control,” Perrimon says. By centralizing the screens, his lab should be able to produce data that researchers can use to make more definitive comparisons, he says, and also to learn more about what makes certain probes better than others.

Although Perrimon advocates a similar approach to performing genome-wide RNAi screens in mammalian cells, the task isn’t quite so simple given the competing methods for designing siRNAs. In the fruitfly community, researchers have coalesced around one method for designing probes: by synthesizing dsRNAs that unambiguously tie up and cleave their complementary mRNA transcripts. But several groups applying RNAi to mammalian cells are jockeying to prove their probe designs are the best. “There is going to be a lot of politics involved,” Perrimon says. “There are quite a few different efforts going on, and I’m not sure those people are talking to each other right now.”

Transnational Team

Bernards, at the Netherlands Cancer Institute in Amsterdam, is leading one of those efforts along with Downward at Cancer Research UK in London. Bernards’ group has so far managed to design RNAi vectors for 8,000 human genes, which he plans to study in collaboration with researchers at Cancer Research. Partly with funding from an undisclosed US-based large pharmaceutical company, Bernard’s group designed three separate siRNA probes for each of the 8,000 under investigation, and after completing experiments of their own, will send the reagents to the UK, where Downward’s group will begin performing other assays in September.

Bernards has devised assays to determine whether the genes are involved in a number of major tumor suppressor pathways, such as those involving the Rb and p53 genes, he says. And because Bernards uses the siRNA probes soon after synthesizing them, he says he has already completed several experiments, the results of which he expected to be published in Nature in July. Bernards and Downward are moving quickly, he adds, because “there’s a short window in which to capitalize [on new technology], and researchers should take advantage of that. It would be stupid not to.”

In addition to performing large-scale RNAi screens, Bernards says his project also includes assays that target certain sets of genes known to be druggable, such as those that express kinases and proteases. The subject of one of the group’s upcoming papers is a family of deubiquitinase enzymes that they designed siRNA probes to inactivate as a means of identifying which members of the family participate in certain cancer-related pathways.

Hannon’s Hairpins

Hannon at Cold Spring Harbor has made similar strides in developing a library of siRNAs for identifying genes relevant to cancer and other drug targets. Hannon’s technique for designing probes relies on making hairpin siRNAs from standard DNA oligos, which he has shown are effective for screening large numbers of genes. With $1 million from NCI and $4 million from DOD, he has already studied 5,000 human genes with three homemade hairpin constructs apiece. Hannon says he should be able to screen a total of 9,300 genes before his funding runs out.

Although Hannon admits that the pharmaceutical industry is one driving force behind the rush to perform large-scale RNAi screens, he adds that the goals for his RNAi experiments reach beyond drug development. “Our motivation for the last seven or eight years has been to try to develop tools that allow us to do genetics in mammals,” he says. “The nice thing about this kind of approach is that it really for the first time lets us harvest a natural process to do loss-of-function genetics.”

Many researchers using RNAi say the power of the technique is ultimately dependent on the range of assays researchers can develop. Performing genome-wide assays is one challenge, they say, but the real biological insights will come from innovative assays. Some will no doubt target pathways that, because of their relevance to cancer and other diseases, are popular with many scientists. But Hannon and others stress that there can be many different RNAi approaches to the same problem. “Right now, the issue is just to develop powerful assays,” says Perrimon. “It really comes down to what you are screening for.”

And just as sequencing the human genome has helped RNAi practitioners design effective probes and systematically investigate gene function, scientists believe genome-wide RNAi screens will enable researchers to develop a next generation of tools for exploring biology. “This is akin to developing a genetical model system — in a mammal,” says Hannon. And as data from Bernards and others’ large-scale RNAi screens start to pop up in journals, Hannon adds that pharma and biotech will start to pay even more attention. “They’ll be a lot more interested once they see how these [initial screens] begin to shape up, how they turn out,” he says.

In the meantime, the pressure is on for Bernards and his peers to publish the results of their experiments before others muscle into the ever-more-popular field. And, Basler admits, the pressure to work quickly should reap advantages as well. “The field is generally competitive, which is good, because it makes science much faster.”

 

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