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With RNA interference, gene silencing roars into labs

Christen Brownlee

Although you don't hear a thing, there is a raucous party going on inside each one of your cells. Each minute of every day, molecules are murmuring information from one to the next in an ancient version of the game of telephone.

DNA, the genetic party host, starts each round by whispering a message to its chemical cousin RNA. Keeping the game going, RNA passes this communication to the ribosomes, the cell's amino acid–linking machines. They, in turn, spit out a protein translation of the original message. The proteins that result from this string of chatter fulfill all the cell's vital functions—and keep it going for countless more rounds of telephone throughout its life.

But what would happen if someone put a molecular muzzle on one of these players, disrupting a round of the game? It would muffle the influence of a particular gene. Such a tool would be a tremendous boon to scientists looking to discover that gene's function. Researchers could infer, by omission, the gene's role in a cell.

Shushing unwanted messages could also provide medical researchers with a means to rid cells of proteins that cause diseases.

Scientists have spent decades searching for such a tool. One method, antisense RNA technology, proved to be unreliable. Another, engineering animals to lack a specific gene, can take at least a year. What's more, genes essential for life can't be studied with this knockout approach.

Eight years ago, scientists stumbled on a phenomenon called RNA interference (RNAi). They've now developed it into a technique that many researchers suspect might be more useful than the other tools available for inactivating genes. RNAi precisely targets a specific bit of RNA, effectively stifling its message before it makes a protein.

The tool, also known as gene silencing, doesn't actually turn the volume all the way down. Genes are muted at 90 to 95 percent efficiency, a state that scientists refer to as a knockdown.

For the past several years, the technique has been picking up steam. Scientists in thousands of labs are using RNAi to determine the roles of particular genes in a variety of organisms. Other teams are using RNAi to develop potential treatments for diseases including macular degeneration, AIDS, and Huntington's disease.

"There's almost no field not affected by the discovery of RNAi," says Phillip Zamore of the University of Massachusetts Medical School in Worcester, who studies the mechanisms by which RNAi works.

Molecular mute

RNAi got its humble start in the mid-1990s after researchers had witnessed some strange genetic behavior. Adding bits of DNA to some organisms, including fungi, plants, and worms, seemed to snuff out the activity of selected genes.

photo

STEP BY STEP. Researchers have identified several basic stages in the mechanism of RNA interference.

E. Roell

A second set of odd observations came from researchers trying to block certain genes with the antisense method. In this technique, a complementary strand of RNA, the antisense strand, binds to part of a cell's normal, single-stranded RNA. When ribosomes encounter the resulting double-stranded bumps on a strip of single-stranded RNA, they don't translate those regions into proteins.

This technique was applied to the roundworm Caenorhabditis elegans by several researchers, including Andrew Fire, then of the Carnegie Institution in Baltimore, Md., and now of the Stanford School of Medicine, and Craig Mello of the University of Massachusetts Medical School.

The scientists soon had a mystery on their hands. Working together, Fire and Mello performed tests with copies of a cell's single-stranded RNA, which shouldn't bind to the cell's RNA because this so-called sense RNA isn't complementary to it. But sense RNA seemed to work just as well as antisense RNA does at muzzling genes.

"We couldn't see a reason why sense and antisense would both work," Fire says.

After puzzling over the phenomenon for months, Fire and Mello hit on an explanation. Both the sense and antisense samples that they had been injecting into C. elegans were contaminated by tiny amounts of double-stranded RNA (dsRNA). The researchers next tailored bits of dsRNA to match particular gene sequences. When the scientists deliberately injected the worms with that RNA, they effectively shut down those genes. The RNAi technique was born.

Over the next 7 years, Fire, Mello, and other scientists began deciphering RNAi's basic mechanism. Interference starts when a piece of double-stranded RNA, either present naturally in a cell or injected by researchers, bumps into an enzyme called dicer, which circulates inside a cell's fluid contents. As its name suggests, dicer acts like a sword-wielding ninja. It chops the dsRNA into bite-size pieces of about 22 base pairs, the individual chemical blocks that make up RNA.

Each of these smaller pieces, called short interfering RNAs (siRNAs), then unzips into two RNA strands. One of these strands joins with a clump of several different proteins, the combination being dubbed an RNA-induced silencing complex (RISC). This complex then hunts down strands of RNA inside a cell that bind to, or complement, its siRNA strand. Once a complementary piece of RNA binds to RISC, several enzymes, including one named slicer, hack through and degrade it.

Since the viruses known as retroviruses often produce dsRNA while they're replicating, some researchers have proposed that dicer, slicer, and other components associated with RNAi acted in early animals as a defense mechanism. This primitive type of protection, the theory goes, is no longer necessary in mammals and other organisms with complex immune systems.

However, modern cells seem to use RNAi to control a wide variety of genes. Studies over the past few years have turned up dozens of RNAi-like mechanisms that affect, for example, embryonic development, stem cell activity, or virus assembly inside cells.

"We've discovered a way that nature turns genes off," says Zamore.

Can of worms

Scientists are still figuring out the details of how RNAi works. Zamore notes that a cadre of yet-unknown proteins, for example, seems to participate in each step of the process. But what's important, he says, is that RNAi does work. During the past few years, thousands of researchers have used RNAi to determine the functions of genes that were previously inaccessible to laboratory study.

photo

AS THE WORM TURNS. A normal planarian glides over a composite background of defects that scientists generated by applying the technique of RNA interference to other planarians.

Sanchez Alvarado

For example, one group is elucidating which genes are important for regeneration in planarian worms. These flatworms can fully regrow any amputated part of their bodies in just a few days, and an entire worm can regenerate from a tiny fragment of its body. A team led by Alejandro Sanchez Alvarado of the University of Utah Health Sciences Center in Salt Lake City recently used RNAi to examine this process. By discovering how these worms regenerate, researchers may eventually find tools to improve wound healing in people.

"Planarians have been around [in biological research] for about 200 years, and we've sliced and diced them in every conceivable fashion," says Sanchez Alvarado. However, the worms, which are frequently sterile, aren't suitable for traditional genetics studies, in which scientists breed organisms.

Sanchez Alvarado happened to be working at the Carnegie Institute when Fire and Mello made their pivotal RNAi discovery. "[Fire] surmised that I should be able to use these methods to make this genetically intractable organism tractable," Sanchez Alvarado notes. "It's worked extremely well."

The May Developmental Cell describes his team's most recent efforts using RNAi to find regeneration genes in planarians. Most of the worms' genome had already been sequenced. The researchers produced bacteria containing bits of dsRNA engineered to match an individual planarian gene and then fed that interfering RNA to a batch of worms. The RNA made its way into the worms' cells. The researchers repeated the process using each of 1,065 different bits of RNA and observed each bit's effect in silencing individual worm genes.

About 145 of the silenced genes affected regeneration. Sanchez Alvarado and his colleagues now plan to determine how each of these genes operates.

Sanchez Alvarado notes that 38 of the other genes tested are related to human genes associated with diseases, including cancer. Only 8 of those 38 genes are currently under study in knockout mice. So, researchers using planarians may learn about gene functions that can't currently be studied in knockout animals.

Muting genes with RNAi holds advantages over creating knockout animals. While attempts to engineer animals free of certain genes sometimes simply kill them, animals that have a gene that's knocked down usually survive long enough to provide information about the gene's function, says Bryan Cullen of Duke University in Durham, N.C.

Greg Hannon of Cold Spring Harbor (N.Y.) Laboratory says, "RNAi will never replace knockouts, but what it does is it hugely expands the questions you can ask and the speed with which you can ask them." Hannon explains that creating a knockout mouse can take well over a year, while knocking down a gene with RNAi takes only a few days.

Furthermore, Hannon notes that scientists don't plan on tinkering with people's genomes by knocking out genes. Instead, RNAi can silence a human gene in cells growing in lab dishes, enabling scientists to get a sense of the gene's role in the body.

To that end, Hannon and his colleagues have set up a library of RNAi sequences at Cold Spring Harbor (N.Y.) Laboratory that researchers can use to study gene function in mice and people. The library already has enough siRNA sequences to silence about two-thirds of the human genome and around half of the mouse genome.

Dicing disease

Applying the new, reliable way to reduce activity of genes in human cells, researchers are now developing RNAi-based drugs to quiet genes that cause diseases. The main prerequisite to developing an RNAi-based solution is to learn which gene is problematic, says Fire.

Another limiting factor, notes Howard Robin of San Francisco–based Sirna Therapeutics, is getting the bits of interfering RNA inside cells and making sure the bits don't degrade before they do their jobs. "That is the huge challenge of developing these drugs," he says.

Many researchers predict that macular degeneration will be the first disease successfully treated with RNAi. This currently incurable disease is a leading cause of blindness in older Americans. It obscures a person's straight-ahead vision when extra blood vessels grow, and then leak, in a central portion of the retina.

According to Robin, the retina is an ideal place for administering RNAi. The retina is self-contained, so a drug injected there would remain where it needs to work. Moreover, retina cells easily take up bits of siRNA on their own.

Several companies, including Sirna Therapeutics and Cambridge, Mass.–based Alnylam Pharmaceuticals, are currently developing RNAi-based drugs for macular degeneration. Sirna recently wrapped up its first phase of experiments of its top drug candidate. Each of 10 people with the disease received an RNAi-containing solution injected into the eye. This treatment halted progression of the disease with no notable side effects. Five of these patients also improved their ability to read letters, a significant advance in treating macular degeneration.

Clinical trials will also be under way next year for an RNAi-based drug to treat HIV, the virus that causes AIDS. John Rossi of City of Hope, a medical-research center in Duarte, Calif., is now testing the drug in mice. The treatment prompts immune cells to produce siRNA that shuts down critical viral genes. Without the activity of these genes, the virus can't replicate and spread through the body.

In experiments with human blood cells growing in the laboratory, the intervention "works like a charm," says Rossi. "It basically blasts away at the virus."

Researchers are making steady advances in work on several other diseases, including neurological disorders, although RNAi-based therapies there probably have a long way to go before they reach the clinic. For example, Beverly Davidson of the University of Iowa in Iowa City has developed a strategy to use RNAi to treat Huntington's disease, which chips away at a person's ability to walk, talk, and reason.

The fatal disease results from production of a mutated protein that's toxic to some types of brain cells. Davidson and her colleagues created genes that produce a type of RNAi that blocks production of the toxic protein. The researchers shuttled those genes into the brain cells of mice that develop a version of Huntington's.

The mice have since shown a dramatic improvement in symptoms. Davidson notes that these results are somewhat surprising because this intervention knocks down the mutant proteins by only about 60 percent, making the payoff remarkable. "We got a lot for a little," she says.

Even with these promising successes, many researchers don't expect RNAi to provide a quick fix for many health problems. Fire predicts years of ups and downs as scientists learn the potential of RNAi. "I think there will be failures and successes in trying to get these therapeutics to work," says Fire.

Nevertheless, says Robin, "I think we're making excellent progress." With so many researchers turning to RNAi, this field is now moving swiftly—and not so silently—ahead.

********

References:

Fire, A. … and C.C. Mello. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391(Feb. 19):806–811. Full Text.

Harper, S.Q. … and B.L. Davidson. 2005. RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model. Proceedings of the National Academy of Sciences 102(April 19):5820–5825. Abstract.

Paddison, P.J. … and G.J. Hannon. 2004. A resource for large-scale RNA-interference-based screens in mammals. Nature 428(March 25):427–431. Full Text.

Reddien, P.W. … and A. Sanchez Alvarado. 2005. Identification of genes needed for regeneration, stem cell function, and tissue homeostasis by systematic gene perturbation in planarians. Developmental Cell 8(May):635–649. Summary.

Unwalla, H.J. … and J.J. Rossi. 2004. Negative feedback inhibition of HIV-1 by TAT-inducible expression of siRNA. Nature Biotechnology 22(December):1573–1578. Abstract.

Sources:

Alejandro Sanchez Alvarado
Department of Neurobiology and Anatomy
University of Utah
School of Medicine
401 MREB, 20N 1900E
Salt Lake City, UT 84132

Bryan R. Cullen
Duke University Medical Center
Department of Molecular Genetics and Microbiology
426 CARL Building
Box 3025 DUMC
Durham, NC 27710

Beverly L. Davidson
Program in Gene Therapy
Departments of Internal Medicine and Neurology
University of Iowa
Iowa City, IA 52242

Andrew Fire
Carnegie Institution of Washington
Department of Embryology
115 West University Parkway
Baltimore, MD 21210

Gregory J. Hannon
Cold Spring Harbor Laboratory
Watson School of Biological Sciences
1 Bungtown Road
Cold Spring Harbor, NY 11724

Craig C. Mello
Program in Molecular Medicine
Department of Cell Biology
University of Massachusetts Cancer Center
Two Biotech Suite 213
373 Plantation Street
Worcester, MA 01605

Howard Rubin
Sirna Therapeutics, Inc.
Corporate Headquarters
185 Berry Street, Suite 6504
San Francisco, CA 94107

John J. Rossi
Division of Molecular Biology
Beckman Research Institute of The City of Hope
Duarte, CA 91010

Phillip Zamore
University of Massachusetts Medical School
Biochemistry & Molecular Pharmacology
364 Plantation Street
Worcester, MA 01605


From Science News, Volume 168, No. 1, July 2, 2005, p. 7.