MicroRNAs Make Big Impression in Disease After Disease

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Science  28 Mar 2008:
Vol. 319, Issue 5871, pp. 1782-1784
DOI: 10.1126/science.319.5871.1782

Hunting for new ways to diagnose and treat common diseases, biologists and companies are racing to decipher the promise of these RNAs.

Hunting for new ways to diagnose and treat common diseases, biologists and companies are racing to decipher the promise of these RNAs

When it comes to RNA molecules, the deeper biologists dig the more they seem to uncover. The 2006 Nobel Prize honored the discovery of RNA interference, in which scientists use short strands of the chemical to silence specific genes, and RNAi has helped shed light on the fact that cells naturally use RNA molecules just 20 to 22 nucleotides long, dubbed microRNAs, to regulate gene expression. Lately, microRNAs are garnering attention on the biomedical front, startling researchers with ever-expanding roles in disease. A flood of studies show that microRNAs may offer a window into the development of various ailments, including cancer, diabetes, and heart failure, and provide a chance to strike disease targets that until now were unreachable.

New view of the body.

From the heart to the blood to the pancreas and beyond, scientists are finding tantalizing hints that microRNAs can help keep us healthy or make us sick. Biotech firms are springing up to convert these discoveries into new products that can diagnose, treat, or predict the course of disease. “People are essentially stunned that this whole level of regulation existed, and we just didn't know about it until a few years back,” says developmental biologist Frank Slack of Yale University, who has branched out from studying microRNAs in cancer to linking them to Alzheimer's disease and life span. When poorly regulated, the molecules appear to drive cancer and a host of other diseases, and this could make them useful in diagnosing disease very early. MicroRNAs made by viruses, meanwhile, may help pathogens gain a foothold in their host, which could suggest new targets for antiviral drugs.

Biologists who have made some of the early microRNA discoveries are eager to push toward new treatments for patients; hoping to also cash in, many are teaming up with biotechnology companies or establishing their own. Companies that formed several years ago to capitalize on different types of RNA molecules, notably ones used for RNAi or others known as antisense, are now expanding into the microRNA arena.

Two microRNA-based therapeutic strategies are being considered: delivering mimics of the molecules that could promote health or blunting the impact of ones that contribute to disease. As with any novel therapy, microRNAs come with their own set of challenges that must be overcome before testing begins in people. Delivering the molecules to the right cells is still a technological hurdle, as is identifying the genes microRNAs influence to ensure that modifying their expression won't have untoward effects. Many individual microRNAs home in on dozens or even hundreds of genes.

Adding a reason for caution, biologists conducting animal tests have been taken aback by the dramatic effects of slightly dialing up or down the dose of a single microRNA. Many believe microRNAs will lead to therapeutics but warn that it will take time. “The microRNA network is really subtle,” says geneticist John Rossi of City of Hope in Duarte, California, who is considering how microRNAs might modulate HIV infections as well as behavioral syndromes such as schizophrenia. He predicts that many correlations between microRNAs and disease “are going to be wrong in the end. But you have to start somewhere.”

Help for faltering hearts

Out of the 25,000 or so human genes, scientists have identified about 500 that yield microRNAs, and they continue to surprise. Consider an experiment that caused Deepak Srivastava, who directs the Gladstone Institute of Cardiovascular Diseases at the University of California, San Francisco, to do a double take. Srivastava studies cardiac development and gene pathways that, when disrupted, can lead to congenital or adult heart disease. When he learned of microRNAs, which were first discovered in 1993 in worms, he began hunting for ones expressed at high or low levels in the heart, first in mice and in fruit flies and later in people. After identifying about 10 such microRNAs, he pursued them one by one, testing whether changing expression levels led to changes in heart function or development. Last April, he and his colleagues described in Cell one of the first examples of mice engineered to lack a specific microRNA, called miR-1-2.

Although miR-1-2 is highly expressed in heart muscle, Srivastava's team wasn't expecting their mice to look much different from normal ones. That's because miR-1-2 has a twin, an identical DNA sequence on another chromosome that the researchers didn't delete. By knocking out one copy, they dialed down the dose of this microRNA by 50%. The effect was dramatic: Half the animals died of holes in the heart, and others were found to have fatal disruptions in cardiac rhythm (Science, 27 April 2007, p. 530).

“At the time, it was a really surprising result,” says Srivastava. Today, he believes, it “makes a little more sense.” That's because scientists are now learning that microRNAs are “the regulators of the master regulators,” as Srivastava puts it—potentially the controllers of entire pathways of genes. The cardiac defects seen in these mice resemble those that are among “the most common heart defects in humans,” says Srivastava, although no one has proven yet that the human defects are caused by a microRNA deficiency, too. Srivastava, who's considering helping establish a company, is now studying how microRNAs govern the development of the heart's four chambers, each of which displays a unique gene-expression pattern and has a distinct function.

Although Srivastava's work hints that problems with microRNAs could explain some heart defects seen at birth, these bits of nucleic acids may also affect the adult heart. At the University of Texas Southwestern Medical Center in Dallas, molecular biologist Eric Olson and his postdoctoral fellow Eva van Rooij are using mice to connect microRNAs to common heart diseases such as cardiac hypertrophy, in which the heart's walls thicken and the organ struggles to keep pumping. Eventually, the condition leads to heart failure; there are few effective treatments. The pair has identified a mouse microRNA, miR-208, whose DNA sequence is hidden within a gene that encodes the muscle protein myosin, which helps the heart contract and function under stress. More recently, they've found that many myosin genes contain microRNAs that regulate one another to keep heart muscle healthy—an interconnection among microRNAs that no other group has described, says van Rooij.

By manipulating microRNAs such as miR-208, “we can really start attacking disease in a whole different way,” predicts William Marshall, a biologist and chemist in Boulder, Colorado. Marshall, who has worked at various RNA-focused biotechnology companies, met Olson through a mutual friend. They recruited others to launch the company Miragen Therapeutics last August; Marshall is the president and chief executive officer. Van Rooij will be the director of research beginning in January 2009.

The cardiology field has an advantage over others: Its doctors have experience supplying drugs straight into the target organ, for example, by injection into the coronary arteries. It should be doable, some predict, to shoot extra microRNAs, or microRNA suppressors, directly into the heart. “I think we might see the first trials [of a microRNA-based therapy] in the cardiology field,” says Markus Stoffel, a molecular biologist at the Swiss Federal Institute of Technology in Zürich.

Target practice

Researchers such as Stoffel are hoping that microRNAs won't share the woes of a related technology that disappointed back in the 1990s, called antisense therapy. Like potential microRNA therapies, antisense sought to modulate gene expression but did so by targeting the messenger RNAs that translate genes into proteins, as a way of blocking protein synthesis. Researchers found that antisense “just doesn't work very well for inhibiting messenger RNAs,” says geneticist and molecular biologist Joshua Mendell of Johns Hopkins University in Baltimore, Maryland, who focuses on microRNAs and cancer. Only one antisense drug has received approval, in 1998.

MicroRNA hunter.

Molecular biologist Markus Stoffel is chasing early signs of a connection between the small RNAs and diabetes.


In some diseases, microRNAs are overabundant, and the goal will be to dial down their expression by injecting a complementary RNA sequence that binds to and disables the target microRNA. But in other ailments, such as certain cancers, microRNAs appear in lower concentrations than in normal tissue, suggesting that treatments will need to add microRNA “mimics.”

For the overabundance problem—which requires blunting microRNA expression—many scientists are now using a strategy designed by Stoffel in 2005. Stoffel studies microRNAs in diabetes and metabolism and realized that he needed a delivery system to get RNA sequences into cells where they could silence microRNAs. To accomplish this, Stoffel collaborated with Muthiah Manoharan of Alnylam Pharmaceuticals, an RNAi company in Cambridge, Massachusetts, to create “antagomirs,” so called because they antagonize the miR, or microRNA. Stoffel's antagomirs are RNA snippets linked to cholesterol molecules, which help slip the silencers into cells. After being injected into the tail veins of mice, antagomirs travel through the body; they have successfully modified microRNA expression in many organs. Antagomirs can't cross the blood-brain barrier, but scientists have injected them directly into the brain, where they penetrated brain cells. By the third day after an antagomir injection, Stoffel says, the microRNAs targeted disappear and stay silent for weeks. He believes that's because the antagomirs remain in the cells for some time, blunting any new microRNAs a cell produces.

Alnylam, of whose scientific advisory board Stoffel is a member, has acquired rights to the antagomirs. And in September, Alnylam and another company, Isis Pharmaceuticals, pooled their intellectual property to create a new microRNA-focused company called Regulus Therapeutics.

It's not clear yet whether antagomirs will remain the delivery vehicle of choice for microRNA silencers because the cholesterol they contain might harm the liver, says Srivastava. Although he uses them in animal experiments without apparent side effects, Srivastava suspects that they may not be acceptable for treating people.

The mirror image of these efforts—the attempt to boost rather than silence microRNAs for therapeutic purposes—may be just as big a challenge. When it comes to overexpressing a microRNA, “it's not as straightforward as I thought it would be,” says Stoffel. The favored approach involves supplementing a weak supply of a microRNA by administering synthetic precursors, which cells would take up and process into mature microRNAs. But how to get the precursors into the body's cells isn't clear. Developing traditional small-molecule drugs that boost the activity of genes that encode microRNAs is widely considered a long shot. “I don't hold out a lot of hope for it,” says Marshall.

From the pancreas to the liver

Stoffel has juggled his work on antagomirs with another pet project, identifying microRNAs that may play a role in diabetes. Stoffel screened insulin-producing beta cells derived from a mouse pancreas for highly expressed microRNAs. Ten showed up that had not been identified in other organs, and Stoffel's group has focused on miR-375, the one most strongly expressed by the beta cells. Unpublished work Stoffel has done in mice suggests that miR-375 helps islets in the pancreas adapt to certain stresses, such as pregnancy or obesity, which call on the body to produce extra insulin.

MicroRNA researchers are also considering how to beat back viral infections. Molecular virologist Bryan Cullen of Duke University in Durham, North Carolina, has puzzled over how viruses invading the human body interact with natural RNAi machinery. But recently, he began focusing on the microRNAs made by some viruses and whether they might give the invading pathogens an advantage against their host. Many viruses in the herpesvirus family, for example, “make a ton of microRNAs” themselves. “The champion at this point is Epstein-Barr virus, with 23,” he says.

Preliminary research by labs in France and Germany suggests that disabling some of these viral microRNA genes makes the viruses less harmful to mice. But at this point, “it's hard for us to tell what's going on,” says Cullen. He is collaborating with the company Regulus to determine just how important microRNAs are for the viruses that carry them. And viruses also appear to exploit certain human microRNAs: Regulus's first project will be blunting a microRNA in the human liver that seems to help the hepatitis C virus replicate, says John Maraganore, Alnylam's CEO.

Detecting cancer's first steps

The starting point for microRNAs' role in disease was cancer, and that line of inquiry remains arguably the most active. Broad screens of human tumor tissue have shown that microRNAs tend to be expressed differently in cancer cells compared with normal tissue of the same organ. Moreover, patterns of microRNA expression—certain ones overexpressed, others underexpressed—correlate with disease prognosis, according to retrospective studies of cancer patients.

In some cases in which a microRNA is underexpressed in a cancer, replenishing its supply in cancerous cells stops the disease from proliferating in animal models. But at least in petri dishes, extra amounts of certain microRNAs don't appear to affect normal cells, which already boast abundant supplies.

Fast track to cancer.

Overexpressing a particular microRNA in some of a mouse's immune cells leads to leukemia; in a mouse without extra microRNA (left), liver tissue (pink) is normal, but in one with boosted microRNA (right), leukemia cells (blue) infiltrate the liver.


In mice, certain microRNAs can be manipulated to drive cancer. Carlo Croce of Ohio State University, Columbus, one of the first in the cancer-microRNA arena, and others have found that artificially upregulating or down-regulating particular microRNAs can initiate or spur on the disease. Croce has founded a company, Crogen Pharmaceuticals, to tackle microRNA-based diagnostics, prognostics, and therapeutics in cancer. One of the first microRNA companies, Rosetta Genomics, was launched in Israel in 2000 and is also pouring resources into cancer.

To nail down the role of microRNAs in tumors, it's crucial to develop genetically engineered animals born without specific microRNAs, says Mendell of Johns Hopkins. “We need to do a better job of documenting their roles in cancer and documenting the mechanisms” by which they act.

That's tougher than it sounds. For example, Mendell has found that the protein produced by the oncogene myc, which is frequently active in cancer cells, downregulates dozens of microRNAs. But that's far from the whole story, for the microRNA-gene network is unimaginably complex. Although some proteins made by oncogenes home in on microRNAs, as Mendell describes, the reverse is also true, with other microRNAs controlling the activity of oncogenes. “The result is a series of interactions that can have a very potent effect” on cancer, says Mendell.

Beyond exploiting microRNA biology to treat cancer, many are eyeing potential diagnostics that would detect cancer at an early stage or diagnose it when standard approaches fail. One strategy is to examine microRNA expression patterns in metastatic tumors of unknown origin, a problem that occurs in 2% to 4% of cancers and presents treatment challenges. In 2005, a team led by Todd Golub at the Dana-Farber Cancer Institute in Boston described in Nature their effort to classify 17 tumors that couldn't be cataloged based on their appearance. They correctly identified the origins of 12 of the 17 based on microRNA patterns in tumors that vary slightly depending on where in the body the tumor originated. Traditional gene-expression signatures using messenger RNAs correctly identified just one.

Rosetta Genomics officials say they hope to begin selling three microRNA-based cancer diagnostic tests later this year; one will pin down tumors of unknown origin, and the others will help doctors distinguish between different lung-related cancers. In 2006, the company Asuragen in Austin, Texas, was launched and now focuses on microRNAs in body fluids as a cancer-diagnosis tool. Slack of Yale is one of the academics it's collaborating with. Company officials won't say much about their plans, beyond saying that Asuragen will concentrate on many major cancers, including lung, prostate, colon, breast, and stomach, and expects the development of microRNA-based diagnostics to take several years. Many microRNAs overlap among a number of cancers, which makes commercializing them less daunting. Recently, Asuragen closed on a second round of funding, garnering $18.5 million.

MicroRNAs in medicine “is a hot field,” agrees Bruce Booth, a venture capitalist at Atlas Venture in Waltham, Massachusetts, who helped found and fund Miragen. Still, no one knows whether targeting microRNAs will wind up helping people—but if they do, they may play a huge role in patient care. “We're willing to take on higher risk with such significant upsides,” says Booth. Like many others, he's betting big that his investment will pay off.

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