RNAi Therapies—Cure or Chimaera?

Dylan Duncan led a relatively normal life up until the age of 10. That’s when his aunt, Cindy, got sick and came to live with his family in Vermont. She had just had a liver transplant that would hopefully cure her disease, and she had to take masses of pills. Duncan remembers them spilling out onto the kitchen table. But they didn’t seem to make much of a difference. Cindy got sicker and sicker. His mom, Janet, took care of her. The women had dealt with terminal illness before. As teenagers they had cared for their mom before she died. But Duncan found this new reality strange and terrifying. Many nights he would camp out on his sister’s floor so that he wouldn’t have to be alone.

After Cindy died, Janet’s health began to decline. At first the symptoms were barely noticeable. She would step into a hot bath and not be able to feel the heat in her legs. But eventually the disease began to attack other parts of her body, just as it did to her sister. Janet couldn’t keep food down, and she lost control of her bowels. In 1992, she received a new liver, and for four or five months it seemed like the transplant had worked. “There was a lot of hope,” Duncan says. But then the disease returned with a vengeance and that hope all but vanished. Janet was so sick that Duncan found himself secretly wishing she would die. “It was that hard for her and for all of us,” he says.

In 1997, Janet did die. Duncan’s grandfather encouraged him to find out if he carried the same mutated gene that she did, but Duncan didn’t want to think about the disease. He had spent seven years surrounded by the paraphernalia of illness, pill bottles and adult diapers. Duncan was still in high school, and he wanted desperately to experience all the teenage things he’d missed while his mom was sick—dates, parties, and sports. “I wanted to concentrate on catching up and having fun,” he says.

Sticky proteins

Duncan’s family carries a genetic mutation, a misprint in the genetic code that causes them to make an abnormal version of a protein called transthyretin (TTR). Normally, this protein shuttles vitamin A around the body. But the defective version folds abnormally and can’t do its job. Worse yet, it tends to lodge itself in the nerves and heart, forming insoluble clumps called amyloid deposits.

The damage that ensues can cause a variety of symptoms. At first, patients might lose feeling in their limbs or experience prickling and burning sensations. As the disease progresses, the symptoms become more severe: “They begin to lose muscle power and become weaker and weaker to the point they can’t walk,” says Philip Hawkins, head of the National Amyloidosis Center at University College London. “Some become completely bed bound.” The amyloid can also damage the nerves that control automatic processes like digestion. That gives rise to “all manner of horrible symptoms,” Hawkins says, including difficulty eating, diarrhea, vomiting, constipation, and low blood pressure. People who have this hereditary disease, called TTR amyloidosis, have a 50% chance of passing the mutated gene on to their children.

In a lab in Cambridge, Massachusetts, researchers are racing to develop a cure for TTR amyloidosis.

In 1990, physicians began offering patients with the disease liver transplants. TTR is produced in the liver, and the thought was that a new, healthy liver would prevent production of the abnormal proteins and halt progression of the disease. “We had high expectations,” Hawkins says. “But it has not turned out to be such a good treatment.” Liver transplants are hard to come by, and patients like Cindy and Janet demonstrated that the strategy doesn’t always work. In recent years, researchers began looking at whether drugs might be able to stabilize the abnormal TTR and keep it from misfolding. Recent clinical studies found that two stabilizing drugs—diflunisal and tafimidis—do seem to help slow progression of the disease. But, Hawkins says, “it was not the cure we’re hoping for.”

Now researchers are pursuing a new class of medicines that aim to do what no therapy has done before—silence the TTR gene. This therapy won’t just address the symptoms of TTR amyloidosis, it will get at the root of the problem by blocking production of the protein. And the strategy holds promise for a wide variety of diseases.

Early days

The seeds of this new therapy took root in the 1980s, when researchers began to see some confusing results in the laboratory. Plant biologists, for example, noticed that when they introduced extra copies of a gene into purple petunias in an attempt to deepen the color of their petals, they ended up with white flowers. Petunias and other living things produce proteins by following the DNA instructions found in genes. Extra copies of a gene should have prompted the plants to produce more protein, but instead they seemed to be producing less. Somehow the genetic code wasn’t being translated. “It was a puzzling phenomenon,” says Phillip Zamore, co-director of the RNA Therapeutics Institute at the University of Massachusetts in Worcester.

In 1998, a team of researchers working with a tiny, transparent worm called C. elegans took a major step towards solving that puzzle. They found that double-stranded RNA could trigger this gene silencing. They called the process RNA interference, or RNAi. “That was a huge discovery,” says Michael Czech, a molecular biologist at the University of Massachusetts, a discovery that later earned two scientists the Nobel Prize.

How RNAi works inside a cell. To learn more about RNA, visit NOVA’s RNA Lab.

Over the next few years, researchers began to puzzle out how this cellular response works. To produce a protein, cells require messenger RNA. This molecule carries the instructions contained in the cell’s nucleus to protein-producing factories in the cell’s cytoplasm. But double-stranded RNA, which can occur naturally or be introduced into a cell, can interfere with this process. The first step involves an enzyme called Dicer, which snips up chunks of double-stranded RNA into smaller pieces about 20 nucleotides long. Next, these double-stranded bits, known as small interfering RNAs (siRNAs), bind to a class of proteins called Argonautes. The Argonautes can then seek out messenger RNA that carry a complementary sequence of nucleotides, the compounds that make up RNA. Once they find this messenger RNA, they slice the strand, rendering it useless. Without messenger RNA, genes can’t become proteins. The siRNA can bind again and again, so “one single molecule is able to lead to the degradation of hundreds of messenger RNA molecules inside a given cell,” says John Maraganore, CEO of Alnylam, one of the companies developing RNAi therapies.

These discoveries were exciting. But the real buzz began in 2001, when a team of researchers discovered that RNA interference also works in human cells. The technique gave scientists a powerful new way to switch off genes in the laboratory, but it also raised the possibility that RNAi could become a novel therapy. “The entirety of human disease is either created by too much of a bad protein or too little of a good protein,” Maraganore says. By introducing siRNAs into people, researchers might be able to stop the production of harmful proteins and address at least the first problem.

Phillip Sharp, a Nobel laureate and molecular biologist at the Massachusetts Institute of Technology in Cambridge, found all this research fascinating. The science was still young, but he could see the promise. Conventional drugs block the activity of proteins by binding to them. But many proteins lack a good binding site. Out of the 100,000 plus proteins that the body can produce, scientists have developed drugs to target only a few hundred of them. RNAi offered a way block all of them. “With RNAi we can stop a flood by turning off a faucet. Small molecules can only mop up the floor,” Maraganore says. In 2002, Sharp joined forces with Zamore and some of the other scientists who had been involved in the early discoveries and launched Alnylam, the first company aimed at developing RNAi therapies. “This was an opportunity to try to translate new science into innovation that would help people,” Sharp says.

Their first challenge involved figuring out how to get these snippets of RNA into cells. The bloodstream is full of enzymes whose sole purpose is to chop up RNA and other nucleic acids. A naked strand of RNA injected into the bloodstream won’t last long. And even if the siRNAs evade these molecules, they must penetrate the membranes that encase cells—no small feat. “These are monstrous molecules, not little small molecules like most drugs are,” Czech says. To facilitate this process, the researchers at Alnylam began experimenting with fatty substances called lipid nanoparticles, which are “sort of like little grease balls,” Maraganore says.

That delivery system seemed to work, and over the next couple of years, Alnylam made slow but steady progress. In 2004, they showed that they could inject siRNAs into mice and turn off a gene in the liver. By 2006, they had shown that they could accomplish the same goal in macaques. Using RNAi to treat human diseases, it seemed, was just around the corner.


For years, Duncan lived not knowing whether he carried the mutation that killed his aunt and mother. He wanted to focus on living, not on worrying about whether he was going to die. “It was so much a part of my life for so long,” he says. “I didn’t want to have anything to do with it.” But, one day in 2006, as he lay holding his girlfriend, his arm went numb. Duncan was only 26, but he worried that the numbness might be the first sign of his mother’s disease. He couldn’t take the uncertainty any longer, so he called his mom’s physician and asked her to run the test. Six weeks later, on his birthday, he got the results: He had the mutated gene. “I don’t remember if I cried,” he says. But his sister and grandfather did when they heard the news. “It was a hard day.”

Duncan had the gene, but it wasn’t yet clear whether he had the disease. Over the next year, however, he started to experience a variety of frightening symptoms. Most alarmingly, his limbs began to tingle. Was this a sign of nerve damage? Duncan couldn’t decide whether the pins and needles were a harbinger of amyloidosis or simply a side effect of his anxiety. So he went in for a biopsy. When the lab examined the specimen, they saw clear evidence of protein deposits. The news should have been devastating, but Duncan found the diagnosis oddly calming. “I seemed to stop worrying about my health,” he says. “I remember this relief. Like, OK, now I know.”

Alnylam, meanwhile, was dealing with its own setbacks. Enthusiasm for RNAi therapies had begun to wane. “I don’t know if it was our own fault, or if the industry got caught up in the initial excitement, but somehow people got the impression that this was all going to happen quickly,” Zamore says. When those therapies didn’t materialize, some figured that RNAi was a bust. In 2010, the pharmaceutical giant Roche, one of Alnylam’s financial backers, decided to stop pursuing RNAi therapies. “That’s the biggest vote of no-confidence yet for RNAi,” wrote Derek Lowe, a pharmaceutical industry organic chemist, on his blog In the Pipeline.

But Alnylam had already had some promising results in humans, and they managed to push ahead. Initially, the company hadn’t set out to treat TTR amyloidosis. They were thinking about viral infections and cancer, though that soon changed. They quickly realized that their delivery systems worked best at targeting proteins made in the liver. The company also decided to focus on rare diseases for which few treatments exist. TTR amyloidosis fit the bill.

In July 2010, Alnylam launched the first human study of its therapy for TTR amyloidosis, an siRNA wrapped in a lipid nanoparticle. Participants received just a single dose. The results were promising, and over the next couple of years the company tweaked the therapy and found even better results. In 2012 the company launched another study to examine the safety of multiple doses of medicine and its impact on production of TTR. The study showed that one dose of the medicine reduced levels of the TTR protein in the blood by as much as 94%. A month after receiving the medication, participants still had 80% reduction in protein levels.

And the drug appears to be relatively safe. The body sees non-native RNA as a sign of a viral infection, so “there has always been the fear of setting off a general immune response,” Lowe says. But Alnylam has tweaked the chemical composition of its siRNAs to prevent this, and so far the company hasn’t seen any serious immune reactions. “The adverse events that we see are largely related to the delivery technology,” not the siRNAs, Maraganore says. The most common adverse event was a mild reaction related to the infusion.

Last winter, Alnylam launched a phase three trial, typically the last hurdle before a drug is approved. The 18-month study will examine whether the medication has any impact on the participants’ nerve function. If the medication works, the researchers should be able to see a difference between the group receiving a saline solution and the group that is taking the drug, called patisiran. The goal of the drug is to block the production of TTR and halt or slow progression of the disease, but there’s also hope that patisiran could reverse the symptoms. In mice, the therapy helped clear old protein deposits.

Patisiran and other siRNA therapies that rely on lipid nanoparticles must be administered via an IV. It’s not an ideal way to take a drug—you have to visit a doctor’s office and sit in a chair while the compound slowly makes its way into your body. So the company has also launched studies to examine a new way of delivering their therapies that relies on sugar groups that are attached to the siRNA. These sugars bind to receptors found only on liver cells, making the therapy targeted. The new delivery system enables researchers to administer the therapy via a simple injection. “It’s been very powerful as a new approach,” Maraganore says. He expects to launch a phase three trial to test the new delivery system in patients with heart failure due to TTR amyloidosis by the end of the year.

Hope and Promise

Patisiran is the first systemic RNAi therapy to enter a phase three clinical trial. If all goes well, it may be the first RNAi therapy on the market, giving hope to patients like Duncan. But the promise of RNAi extends far beyond amyloidosis. Alnylam is currently developing medicines for seven other diseases, including hemophilia and high cholesterol. And other companies are working to develop siRNAs to treat everything from liver cancer to ebola. “Imagine [treating] a whole swath of human diseases that today can’t be approached,” Maraganore says. “It opens up the door to a whole new class of medicines for diseases where existing drugs really are inadequate.”

Lowe, who has been following the development of RNAi therapies closely, is more realistic. “It’s not like you’re going to be able to walk in and treat every disease,” he says. “The list of diseases that can be improved by shutting down a particular protein is long, but not infinite.” In addition, Alnylam has only shown that it can deliver siRNAs to the liver. “Everything that comes out of the gut goes into the hepatic portal vein and drains straight into the liver,” Lowe says. “The liver is the easiest place to deliver anything.” Whether Alnylam and other companies can find ways to deliver effective siRNA therapies to other parts of the body remains to be seen. Still, if Alnylam’s patisiran makes it to market, “that will really help validate the whole idea,” Lowe says. “I wish them luck.”

Even if patisiran succeeds, the therapy won’t be widely available for several years. Because so few treatments are available to treat TTR amyloidosis, the U.S. Food and Drug Administration had agreed to expedite its review of the medicine. But Alnylam won’t have the results from the phase three study until 2017.

For people like Duncan, the timing is crucial. The amyloid that causes so much damage accumulates slowly. From one day to the next, there isn’t a noticeable difference. “It’s almost like a light snowfall,” Duncan says. But even the lightest flurries can shutter a city if given enough time. Duncan has already started experiencing numbness in his hands. He has been taking one of the TTR-stabilizing drugs for the past five years, and it seems to be helping. But it’s unclear how long the effect will last.

When Duncan was a kid, his dad told him not to worry. “By the time you get to be the age your mother was when she got sick,” he had said, “they’ll have something to treat it.” Today Duncan is on the cusp of turning 34. That’s twelve years younger than his mother was when she died, but there’s no guarantee his disease will progress as slowly. His aunt died when she was just 36. Nevertheless, Duncan tries not to let dark thoughts weigh him down. He has never been one to plan for the future, but lately he’s started picturing what it would be like to have a wife and become a father. “I think I would be the best at that,” he says.