Wall Street Journal - August 6, 2002
Antonio Regalado, Staff Reporter of The Wall Street Journal

Scientists working on the petunia mystery have uncovered what's shaping up to be a critical piece of cellular machinery, a process by which plant and animal cells seem to blot out the activity of particular genes. Scientists say the discovery may help explain a lot that had perplexed them about life's basic functions, and they are already applying it as a research tool in the hunt for new medicines. Venture capitalists also are betting that it will yield super drugs that act like molecular torpedoes aimed at HIV or cancer. Scores of companies and academic labs have joined the hunt.

An experiment at the Massachusetts Institute of Technology this year has shown that HIV infection can be slowed in a lab dish by using the new science to aim RNA molecules at the genetic code of the virus. Others are under way to see if the technique can do battle with cancer in laboratory mice.

Work on the new gene-silencing phenomenon, also known as "RNA interference," is helping fill a knowledge gap exposed by the Human Genome Project. When the human genetic code was published last year, scientists realized they only knew what about 2% of our DNA was for. Equally disconcerting, about half of human DNA seemed to be not human at all, but rather a junkyard of debris left by eons of invasions by viruses and other parasites.

The latest research on gene-silencing looks like it will explain some of that. In some organisms, the process appears to be acting like the genome's virus-protection software, erasing the effects of corrupted junk genes.

Last week, a Cambridge, Mass., start-up founded by scientists at MIT raised $15 million to develop treatments against hepatitis and cancer.

Among the other half-dozen entrants is Nucleonics Inc. of Malvern, Pa., a firm founded by former Wyeth immunologists who independently stumbled on the silencing effect while studying cancer in mice. German scientists have formed two companies to create gene-silencing drugs and are hoping to begin human tests within two years.

Antisense

The field of biotechnology is littered with the remains of technologies that looked exciting in early laboratory tests but proved difficult to translate into treatments for humans. The RNA interference story may just be a twist on the tale of a heavily hyped technology of the early 1990s known as antisense, which has been slow to develop into usable drugs.

But for now, drug companies are using RNA interference to help locate gene targets involved in cancer and other diseases. "The wave of interest has expanded because of the reproducibility of the basic claims," says Riccardo Cortese, who studies cancer for Merck & Co. in Italy.

At Exelixis Inc. in South San Francisco, Calif., 600 people carry out large-scale gene research for drug giants such as Pharmacia Corp. and Bristol-Myers Squibb Co. The company, whose labs are packed with jars of flies and worms used in such research, says nearly 80% of its gene studies now use the technology. "People have adopted this like wildfire from an experimental point of view," says CEO Geoffrey Duyk. "It's a very high-throughput method for turning genes off."

New Targets

Dr. Duyk says the technique recently yielded new drug targets for Pharmacia after the company used it to study proteins involved in Alzheimer's disease. Working with transparent worms known as C. elegans, the company used gene silencing to methodically shut down around 10,000 worm genes, tracking the effect on the formation of a protein known to be involved in Alzheimer's in humans.

After identifying all the worm genes linked to the process, they used computer databases to find the equivalent genes in the human genome. That information was passed to Pharmacia so that it can start testing drugs.

A cell's DNA sends commands out of the nucleus in the form of RNA, a closely related molecule that is also made up of genetic code. RNA serves as the blueprint from which the body makes proteins, completing a three-step relay biologists call the "The Central Dogma." But the dogma can't explain everything. With gene-silencing, it's now clear there's a new class of RNA molecules whose job isn't to help make protein at all, but to stop other RNA messages from doing so.

Following the petunia experiments in his lab at biotech company DNA Plant Technology Corp., Dr. Jorgensen, now at the University of Arizona, concluded that the extra copy of the pigment gene he'd added was somehow cueing the plants to shut off their purple color, sometimes only partially. One flower's pattern of purple and white looked like a man leaping. Dr. Jorgensen named it the "Cossack Dancer."

It wasn't clear why the pigment gene was shutting down, and it took another decade for someone to find the next major clue. In 1998, Andrew Fire of the Carnegie Institution, a nonprofit research laboratory in Baltimore, Md., and Craig Mello of the University of Massachusetts announced they had discovered how to design a double-stranded RNA molecule that would predictably silence any gene they chose. The effect, which they dubbed "RNA interference," appeared quite potent. Just a few molecules were enough to render a gene's activity all but undetectable in the worms they worked with.

Dr. Fire's paper set off a flurry of activity, as it dawned on scientists that if gene-silencing was working in plants, and now in worms, it might be a general phenomenon in all animals. If so, it was likely to have some deep and fundamental purpose.

At MIT, Phillip Sharp was gripped by the implications of Dr. Fire's work. Most basic mechanisms in the cell were believed to be already understood. "It was an almost retro process," says Dr. Sharp, a Nobel Prize-winning gene researcher who also heads a new $350 million brain institute on campus. "I was just dumbfounded that it hadn't been described before."

The next step was clear. RNA interference had to be made to work in a test-tube reaction so that it could be dissected piece by piece. There were bound to be enzymes in the cell that helped the strands target specific gene messages for destruction. Those needed to be discovered as quickly as possible to keep pushing the research forward. Along with another MIT professor, David Bartel, Dr. Sharp put two postdoctoral students on the job immediately.

The news was spreading fast. At the Cold Spring Harbor Laboratory, an independent research institute on Long Island, Gregory Hannon learned in a meeting that gene-silencing seemed to work in fly embryos as well. "I got on the phone with my lab and said, 'This is a general phenomenon, get the fly cells out of the freezer right now,' " he remembers.

Though Dr. Hannon had been working on cancer genes, he now dropped everything in order to mash fly cells to make the liquidy cell "extract" needed to start sifting through the new reactions' biochemical components.

It was also natural to wonder if the technique could be used in human cells. But there was a roadblock. The kind of molecules created by Dr. Fire -- long, double-stranded RNA molecules -- were known to be toxic to animal cells. The big molecules triggered the cells' sophisticated defenses against viral invaders, throwing them into a panic mode and causing them to commit cellular suicide.

But then, British plant scientists found a new clue -- tiny bits of double-stranded RNA floating in the cells of Dr. Jorgensen's petunias as well as other plants. It looked as if the big strands were being diced into tiny ones.

The next move was obvious to everyone: Both the MIT group and Dr. Hannon raced to search for these small strands of RNA in their fly extracts. Credit for such findings would go to whoever published first. Phil Zamore, one of the MIT students who had gone on to start his own lab at the University of Massachusetts, says he'd found the small RNAs already when he heard though the grapevine in late February 2000 that Dr. Hannon had similar results. "That was a huge race. I hope never to be that stressed out again," he says, recalling how he pulled together his manuscript in a week of 18-hour workdays.

They published within days of each other the following month, but Dr. Zamore told the more complete story. The small RNAs were guiding the silencing reaction. If their sequences were programmed to match a gene, it would shut it off almost completely.

Chopping RNA

The finding made sense. Big double-strands of RNA were being chopped into smaller ones, amplifying the effect. Dr. Hannon later identified the enzyme doing the chopping, which his lab dubbed "Dicer." Like a multiple warhead, the smaller segments were each homing in on messages being sent by the target gene, then calling in enzymes to destroy it.

That finding suggested it was possible to overcome the immune-response obstacle. "The small RNAs are critical, because if you inject those, they no longer induce the reaction. People are not going to keel over because of a massive viral response," says Dr. Fire.

This summer Dr. Sharp's lab showed that gene-silencing seemed to slow the growth of HIV in laboratory cultures. And with researchers at Stanford University, Dr. Hannon studied gene-silencing in mice whose livers had been engineered with a gene from a firefly -- lending a whitish glow to their organs when viewed through a special microscope. After designing small RNAs to target the gene, called luciferase, they reported that the mouse livers lost as much as 98% of their glow.

Last week, Dr. Sharp and his collaborators raised $15 million to form Alnylam Inc., which aims to develop treatments against hepatitis and cancer. Dr. Sharp in 1978 also helped found one of the world's first biotechnology companies, Biogen Inc.

But the commercial landscape is already becoming crowded, and with scores of patent applications being made, people in the industry predict that scientists will eventually have to haul out their lab notebooks to prove exactly who knew what and when.

Although teams such as Dr. Hannon's and the MIT researchers have been first to publish many of the most important results, there has also been much research going on behind the scenes. "They did a great job on the science, but with respect to the patent application, they are later than us," says Stephan Limmer, a Bavarian biochemist who has raised $4 million from the German government and investors to back Ribopharma AG, the company he and a colleague formed in 2000 to start working on drug treatments.

MIT says it thinks its own patents will stand up and is looking to resolve the situation amicably.

Meanwhile, the discovery of the molecular trigger for gene-silencing is starting to unleash major new insights into what the human genome is actually doing and tying together a number of loose threads in biology.

The biggest find now emerging is that the human genome appears to carry code for hundreds of small RNA molecules of its own. Dr. Zamore reported in Science last week that these molecules could use the cell's silencing machinery to shut off other genes. In doing so, they are probably controlling crucial aspects of embryonic development by, for instance, repressing the activity of genes responsible for the formation of the brain or limbs once their jobs are completed.

The process appears to have still other roles. In plants, it's known to protect against viruses, which hijack cells in order to carry out massive, unapproved copying of their own genes. Dr. Fire thinks gene-silencing is part of an ancient game of genetic hide-and-seek between cells and viruses, which carry their genes in the form of RNA. "Watching a toddler get virus after virus, you'd think this was working all the time," says Dr. Fire.

Many of these effects now seem to be at work in many forms of life, including plants, animals and fungi. "The fact that these have been conserved in evolution means they have very important roles," says MIT's Dr. Bartel. "It looks like small RNAs have been shaping gene expression since the beginning of multicellular life."

Write to Antonio Regalado at antonio.regalado@wsj.com
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