





Turning off genes

Jan. 2, 2005

Turning off genes

Big in the lab, ready for the clinic?



Staff Writer

A new way to selectively turn off genes has taken the biologylaboratory by storm in the last few years. The increasingly populartechnique, called RNA interference, helps scientists to probe thefunction of a gene and could potentially be used to treat a varietyof diseases.

RNA interference resembles antisense, a technique thatCarlsbad-based Isis Pharmaceuticals has painstakingly developedsince its founding in 1989. Antisense uses synthetic bits ofgenetic material to block the function of a specific gene insidethe body.

In principle, antisense could be used against inherited diseases,chronic conditions such as heart disease or autoimmune disorders,or even viruses such as hard-to-treat hepatitis B or C. In the caseof an inherited disease such as Huntington’s or Lou Gehrig’s, thetarget would be the faulty copy of the gene to blame.


Scientists would just need to figure out which of the human body’s30,000 genes to home in on, and how to get the antisense drugs intothe right cells ---- two daunting tasks.

Despite exciting investors and attracting millions of researchdollars during the last two decades, antisense technology hasproduced just one FDA-approved drug, Vitravene, sold by Isis andinjected in the eye to combat an AIDS-related viralinfection.

“I think everyone believes (RNA interference) has incrediblepotential,” said Mark Kay, a pediatrician and geneticist atStanford University, who is studying ways to use RNA interferenceto fight hepatitis viruses.

“Compared with previous antisense techniques, there’s no doubt thatthis is the most effective,” he said. “But we need to move forwardvery cautiously.”


Yet momentum is building behind research on using RNA interferenceas a therapeutic technique. Researchers at one of Isis’ partners,Alnylam Pharmaceuticals in Massachusetts, demonstrated recently inthe journal Nature that they could lower cholesterol in mice byusing an RNA interference-based treatment.

The first clinical trial that uses an RNA interference-based drugbegan in October. Philadelphia-based Acuity Pharmaceuticals isaiming to treat macular degeneration, a major cause of adultblindness.

But many researchers say there are still significant barriers towider use of RNA interference clinically, rather than in universityand industry labs, where the technique has had most of its successso far.

How it works


RNA acts as the messenger between DNA, the material of the geneticcode, and proteins, the tools that do the work of the cell. Eachgene can be read out into an RNA message to make a specificprotein, but not every gene gets turned on in a given cell.

Both antisense and RNA interference cause messenger RNA to getchewed up by proteins in the cell, preventing the message frombeing acted upon.

Antisense uses DNA that reads like the complement ---- or antisense---- of the targeted message and binds to it. The RNA-DNA hybridstructure gets recognized by a special enzyme that eats itup.

RNA interference is really an extension of antisense, said FrankBennett, vice president of antisense research at Isis. Thedifference is that RNA interference uses double-stranded RNA, orpart of a gene read into RNA and its complement stuck together inthe same molecule.


RNA interference appears to be more potent than previous versionsof antisense because it hijacks some of the cell’s internalregulatory machinery that determines whether genes get turned on oroff. This special potency was discovered in 1998 by scientists atthe Carnegie Institution of Washington working on worms, but itworks in plants, fruit flies and humans, as well.

The machines that RNA interference take over appear to be part ofan ancient evolutionary defense mechanism against viruses, whoseperverted genes sometimes come in a double-stranded RNA form.

“It’s part of how creatures learned to resist viruses before thereever was an immune system with antibodies,” said Mario Stevenson, avirologist at University of Massachusetts Medical School.

There are hints that RNA interference machinery still has ananti-viral function because influenza virus, for example, tries toknock it out when it invades cells, he said.


Big business ---- in the lab

RNA interference quickly gained popularity among universitybiologists who wanted a way to shut off specific genes. In 2002, itwas proclaimed “Breakthrough of the Year” by Sciencemagazine.

Carlsbad-based Invitrogen, which sells tools to biologyresearchers, has jumped into the field in a big way, becoming oneof the leading suppliers of RNA interference products.

“In the past year, it’s really gained acceptance as a standardmethod in the lab,” said Marilyn Datta, vice president of RNAi forInvitrogen. The earliest adopters of the technique were universityscientists, but an increasing number of industrial researchers areusing it, she said.


Invitrogen can make synthetic RNA for scientists to use on cellsgrowing in dishes, as well as more elaborate tools for introducingRNA interference agents into animals.

Consulting firm Frost & Sullivan predicted this year that RNAinterference sales revenues will grow by 30 percent a year, growingto $328 million in 2010 from around $48 million in 2003.

The largest part of that money would come from pharmaceutical firmsusing RNA interference for “target validation,” the firm projected.Target validation means figuring out whether a potential drug acompany’s scientists are studying will turn genes on and off in theway they want.

Some venture capital firms such as Boston’s Oxford Bioscience haveinvested in start-up companies that use RNA interference, such asAlnylam and Sirna Therapeutics in Colorado. But Forward Ventures,San Diego’s premier biotech venture capital firm, has stayed clearso far.


“It’s too soon,” said Ivor Royston, a partner at Forward Ventures."I don’t think the current RNA interference companies are going togenerate viable products soon enough to reward our investors.”

Barriers to clinical use

Antisense and RNA interference both face similar obstacles beforethey can be effective ways to fight disease.

“It’s all about delivery,” said Stevenson with the University ofMassachusetts.


It’s no coincidence that the first RNA interference drug tested ina clinical trial gets injected into the eye, the same site ofaction of the first antisense drug, he said.

“It’s the easiest place to go; the delivery problems aren’t therebecause you’re delivering the RNA to a specific place,” hesaid.

The authors of Alnylam’s recent Nature paper used a clever trick bychemically attaching the RNA to a molecule of cholesterol, whichgets taken up by the liver.

“It’s frustrating,” he said. “Most of any RNA agent gets taken upby the liver, anyway.”


Stevenson is working on using RNA interference to fight HIV; in hiscase, the task is to deliver a drug specifically to the lymphnode.

In addition to the specificity problem, unprotected RNA lasts onlyminutes in the blood because enzymes break it down. In highquantities, double-stranded RNA also can activate a generalanti-viral defense system called the interferon response.

Isis has spent years developing chemical modifications that protectRNA from the body’s enzymes, Frank Bennett said. The modificationseven enhance the inhibitory effects, he said.

Other companies, such as Rosetta Inpharmatics in Seattle, have alsodetected glitches where RNA interference can shut off genes thatare similar to, but not the same as, the target gene.


“It will take some time and effort to overcome the hurdles,"Bennett said. “But we’re making a bet that it will be an excitingarea in the future.”