THE ART OF SILENCING: Small interfering RNA molecules are incorporated into an RNA-induced silencing complex where they bind and degrade target messenger RNAs (yellow with red rings). Taking advantage of this natural RNA interference (RNAi) pathway, researchers are developing therapeutics for liver-based diseases, viral infections, cancer, and more.© MEDI-MATION LTD/SCIENCE SOURCE

Since its discovery 16 years ago, researchers have been eyeing RNA interference (RNAi)—a natural process of posttranscriptional silencing of genes by small fragments of the nucleic acid—for its potential in therapy, especially in treating forms of cancer and other diseases that are particularly hard to address with existing drugs. But the path of such RNAi therapies to the clinic has been nothing short of a pharmaceutical roller-coaster ride.

Andrew Fire and Craig Mello first demonstrated RNAi in C. elegans in 1998, a discovery recognized in 2006 when they won the Nobel Prize in Physiology or Medicine.1...

“RNAi rapidly became a workhorse technique for basic research,” says Douglas Fambrough, chief executive officer at Dicerna Pharmaceuticals in Watertown, Massachusetts. “It was really easy to get it to work, and it worked really well.”

At the same time, the scientific community began to develop a growing interest in RNAi therapies. Among its benefits, RNAi can prevent the proteins actually driving an illness from being translated, which avoids the need to attack the disease somewhere downstream in a molecular cascade, as small-molecule drugs and biologics often do, says Akshay Vaishnaw, chief medical officer at Alnylam Pharmaceuticals in Cambridge, Massachusetts. “Why not turn them off at their source?” he asks.

RNAi can provide greater target specificity than small molecules and inhibit the expression of proteins that lack the enzymatic pocket necessary for binding small-molecule drugs, says Mark Murray, president of Tekmira Pharmaceuticals in Vancouver, British Columbia. RNAi can also target proteins that can’t be reached directly by monoclonal antibodies because of their intracellular location.

Around 2005, the field entered what independent biotech consultant Dirk Haussecker refers to as an “era of irrational exuberance” surrounding the new approach. Major pharmaceutical companies invested several billion dollars in RNAi therapeutics “regardless of technical obstacles,” says Haussecker, who specializes in RNAi and other RNA-based therapies. Numerous biotechs jumped into RNAi therapeutics—especially for diseases with well-validated genetic targets not addressed by current treatments.

But early clinical trials generally failed to meet expectations. Most strikingly, Miami, Florida–based pharmaceutical and diagnostics company OPKO Health shut down its Phase 3 trial of an RNAi treatment for wet macular degeneration in 2009 after the intervention failed to meet the trial goals. Other RNAi-based drugs provoked strong innate immune reactions, failed to deliver patient benefit, or both.

The disappointments hung over the field for the next few years, a period Haussecker refers to as “the era of doubts and despair.” In the face of broader industry turmoil, RNAi programs often were among the first to get chopped. In 2010, Roche, which had invested about $500 million in RNAi, shut down its internal research program. The following year, Pfizer and Abbott also pulled out of in-house RNAi development, and Merck shuttered the RNAi laboratory it had acquired in 2006 with its $1.1 billion purchase of Sirna Therapeutics.

The good news is that the RNAi roller coaster has been on a fairly steady climb for the past few years, following refinements in RNAi targeting and delivery. The number of early clinical successes is climbing, and many in the field expect the next few years to see the approval from the US Food and Drug Administration (FDA) for numerous RNAi drugs now in the pipeline. “There are some who say that the RNAi cup is half empty,” says Alnylam’s Vaishnaw, “but the regulatory authorities are not in that group. Our early development work is extremely compelling to regulators and they partner vigorously because of that.” (The FDA declined to comment on its assessment of RNAi therapeutics as a new class of drugs.)

Speculation about future therapy approvals aside, there’s no question about the field’s recent scientific progress, says David Lewis, chief scientific officer at Arrowhead Research in Madison, Wisconsin. “The days of wondering whether RNAi will be effective in humans are behind us.”

Lodging in the liver

MOLECULAR SNIPERS: Therapies based on interference RNA (RNAi) deliver small interfering RNAs (siRNAs) to diseased cells, where the siRNA’s antisense strand is incorporated into an RNA-induced silencing complex and used as a template to identify and degrade target messenger RNA.
See full infographic: JPG | PDF
The process of RNAi has now been well-documented in mammals. Double-stranded RNA sequences of approximately 70 nucleotides, known as short hairpin RNAs (shRNAs), are exported to the cytoplasm, where the Dicer enzyme cleaves them into small interfering RNAs (siRNAs) about 21 nucleotides long. The siRNA’s antisense strand then incorporates into an RNA-induced silencing complex (RISC), which can attach to and degrade its complementary target messenger RNA, reducing or stopping the expression of proteins. (See illustration.) As a natural process, RNAi plays major roles in defending the genome against intruders and in aiding developmental processes. As a therapeutic, RNAi has the potential to wipe out proteins that drive disease.

Importantly, the phenomenon is highly conserved across mammals. “We’ve explored many species and can achieve RNAi reliably and consistently across all of them with the same siRNAs,” Vaishnaw says. “That has allowed us to move quickly across species with confidence that results will translate into humans and to define dose and regimen for humans very quickly.”

While unmodified siRNAs have been injected locally into the eye and other organs in early trials, those released directly into the bloodstream are degraded by enzymes and are unable to cross cell membranes. One strategy for smuggling siRNAs through the blood and into diseased cells is to embed them in lipid nanoparticles (LNPs). (See “Nanomedicine,” The Scientist, August 2014.) When scientists tested early siRNA LNPs in animal models, they found that the particles generally ended up in the liver. The liver is highly vascularized, Vaishnaw explains, and its endothelium is peppered with pores about 100 nanometers in diameter, wide enough for 70- to 80-nanometer LNPs to slip through en route to hepatocytes. Moreover, once the LNPs are released into the bloodstream, they are rapidly coated with apolipoprotein E (ApoE), which binds to receptors on hepatocytes and eases cell entry of the nanoparticles.

We’ve explored many species and can achieve RNAi reliably and consistently across all of them with the same siRNAs. That has allowed us to move quickly across species with confidence that results will translate into humans.—Akshay Vaishnaw,
Alnylam Pharmaceuticals

Exploiting these mechanisms, many of today’s promising RNAi drugs address liver-linked diseases. After targeting a range of illnesses in its early years, Alnylam now concentrates on liver-based diseases, with more than 15 RNAi therapies in clinical development. Most advanced is the LNP-formulated siRNA drug patisiran (ALN-TTR02), which is delivered via infusion to treat transthyretin-mediated (TTR) amyloidosis, an orphan liver disease that can lead to heart failure and has no approved treatments in the United States. In April, the company reported positive preliminary results of a Phase 2 trial, demonstrating that the drug could reduce blood levels of the TTR proteins that drive the disease by 80 percent or more. The firm is moving ahead with a Phase 3 trial and hopes to report results in 2017.

Another LNP-packaged siRNA drug, ALN-PCS02, lowers expression of the enzyme proprotein convertase subtilisin/kexin type 9 (PCSK9), which is involved in regulating blood cholesterol, to treat an inherited form of high cholesterol. In a Phase 1 trial of 32 volunteers with raised levels of low-density lipoprotein cholesterol (LDL-C), ALN-PCS02 lowered levels of LDL-C by as much as 57 percent.3

Alnylam researchers are also taking an alternate approach to delivering RNAi agents to target tissues, involving the covalent linkage of engineered siRNAs with the sugar N-acetylgalactosamine (GalNAc), for which hepatocytes have a membrane receptor. This technique is producing a new class of drugs for diverse liver diseases that can be administered as a shot in the arm rather than infused intravenously, as is done with LNP drugs. The company has applied this strategy to its ALN-PCS program, for example, developing a PCSK9-targeting GalNAc agent that Alnylam researchers hope to have in clinical trials by early next year.

An approved conjugated therapeutic would have to compete with PCSK9 monoclonal antibodies now in Phase 3 trials, but RNAi drugs may still grab a significant share of this market, says Haussecker, if they have milder side effects or work more consistently across patients. Unlike monoclonal antibodies, which do not work as well for individuals who have high levels of PCSK9, “RNAi doesn’t care about the baseline expression level of the target gene,” he explains.

Arrowhead and Dicerna also have clinical programs targeting liver-linked diseases. Importantly, the latest generation of siRNA therapies for liver illnesses typically display good safety profiles and are well-tolerated among patients. Some patients do suffer from flu-like symptoms as a result of an innate immune reaction known as the interferon response. “The issues that the field is wrestling with are all short-term reactions to therapy,” says Dicerna’s Fambrough.

But other than these reactions, today’s drugs generally “look quite innocuous,” Vaishnaw says. However, he emphasizes that “I don’t think we can be glib about this; human safety is human safety, and this is still an early technology.” Haussecker and other experts also emphasize that concerns remain that unexpected effects may crop up over the long term.

Going viral

Hepatitis C virus© JAMES CAVALLINI/SCIENCE SOURCERNAi is also showing promise in the treatment of viral infections. By conservative estimates, hepatitis B virus (HBV) chronically infects more than 300 million people worldwide, killing at least 780,000 annually through liver scarring (cirrhosis), liver failure, and liver cancer. The disease can be treated, but many patients don’t respond to standard therapy with an interferon drug, and newer oral nucleoside and nucleotide agents can inhibit HBV replication without curing the illness when viral DNA sequences have become integrated into patients’ DNA.

Even when current drugs stop HBV viral replication, “patients still produce a fair amount of viral surface antigen, and that production of antigen really dampens the immune system,” says Arrowhead’s Lewis. “The idea is that if you can decrease the level of viral antigen production, you’ll be able to reawaken the immune system so that it can actually clear the liver of the virus. We thought that would be a perfect opportunity to use RNAi.”

In March, Arrowhead researchers began dosing HBV patients in a Phase 2 clinical trial with the company’s ARC-520 RNAi agent. The drug combines two sequences of siRNA with Arrowhead’s so-called dynamic polyconjugate (DPC), which includes a polymer backbone along with chains of polyethylene glycol (PEG) that stabilize the compound in the blood and targeting ligands that direct the therapeutic to the cell type of interest. The company hopes to report the results of the study this year. Alnylam, Benitec Biopharma of Sydney, Australia, and Tekmira are following suit, readying HBV agents for clinical trials.

Meanwhile, Benitec is taking a different RNAi approach to targeting hepatitis C virus (HCV), which infects more than 150 million people worldwide and can lead to liver failure, liver cancer, and other life-threatening illnesses. The company’s RNAi therapeutic, called TT-034, uses an adeno-associated virus to deliver a plasmid carrying three shRNA-encoding genes to the host nucleus. (See illustration.) The genes are expressed in host cells to suppress production of HCV protein in hepatocytes.4 In May, an HCV-infected patient, who had failed to respond to previous therapies, was the first to receive an infusion of TT-034 in a Phase 1/2 trial. A total of 14 patients will receive increasing doses while being closely monitored.

“This is the first time a viral vector encoding for short hairpin RNA has gone directly into the patient,” says David Suhy, senior vice president for research and development at Benitec. “You’re essentially setting up shop within these cells, and transcription is running 24/7. It’s a slow-and-go study, because it’s nonwithdrawable; if there’s a serious adverse event we won’t be able to withdraw the compound.”

On the upside, the gene-therapy approach comes with a very large potential advantage over current HCV drugs, Suhy adds. “It doesn’t rely on patient compliance in taking their pills on a daily basis to achieve therapeutic efficacy. Patients come in, they get infused, they walk out, and it’s done.” This benefit may be particularly compelling among sub-Saharan and East Asian populations, which have the highest rates of chronic HCV infection and often lack access to health care.

Tekmira is targeting another virus that attacks the liver and can kill far more quickly: Ebola. The firm’s infused TKM-Ebola LNP delivers siRNAs that target multiple sites in the virus genome, inhibiting its replication.5 “We have been able to establish in animal models that we can rescue the animals from certain death following an Ebola infection,” says Murray.

Working under a contract with the US Department of Defense, Tekmira is testing TKM-Ebola under the FDA’s “animal rule,” which allows safety testing in healthy human volunteers while efficacy is probed in animal models. The company reported favorable first results for a Phase 1 human safety trial in May, but the FDA put the trial on hold in July, due to problematic immune responses among volunteers given the highest dose.

Interfering with cancer

From day one, RNAi seemed to offer a dream weapon against cancer, especially against currently undruggable oncogenes. MYC, for example, has long been known as a major oncogene, but “the pharmaceutical industry has never had a tool to deal with it,” says Dicerna’s Fambrough. “RNAi presents us with a tool.” Unlike small-molecule drugs and biologics, which inhibit the mutant proteins that drive a tumor’s growth, RNAi drugs can stop the proteins’ production with great specificity.

But while some early studies showed promise, no drug has moved into Phase 3 trials. In 2010, Arrowhead’s CALAA-01 was the first to exhibit mechanistic evidence of RNAi induced by an siRNA agent in humans in a Phase 1 trial testing the therapy’s effect on melanoma. CALAA-01, a nanoparticle built up from a polymer containing cyclodextrin sugar molecules, appeared to accumulate in solid tumors and to block translation of its target, RRM2, the M2 subunit of the enzyme ribonucleotide reductase, which plays a role in cell division.6 But questions cropped up about the therapy’s effectiveness and its side effects, and Arrowhead later quietly dropped CALAA-01 in favor of drugs based on the company’s DPC platform.

At least three other companies are now pursuing RNAi approaches to treat cancer, however, with early-stage trials now underway.7 This April, Dicerna began a Phase 1 study of its DCR-MYC, based on the company’s LNP technology, and the firm is enrolling patients with a broad range of MYC-driven solid tumors, as well as multiple myeloma and lymphomas.

The days of wondering whether RNAi will be effective in humans are behind us.David Lewis, Arrowhead Research—David Lewis,
Arrowhead Research


In May, Tekmira kicked off a Phase 1/2 trial for treating hepatocellular cancer with its TKM-PLK1, which inhibits expression of polo-like kinase 1 (PLK1), another established target in oncology. That study is running alongside an ongoing Phase 2 trial for gastrointestinal neuroendocrine tumors and adrenocortical carcinoma. “In all of these disease areas, there are very poor or in some cases no therapeutic alternatives,” Murray emphasizes.

Silenseed, a pharmaceutical company located in Jerusalem, has set its sights even higher—on the oncogene k-RAS, “almost a holy grail” in cancer research, company CEO Amotz Shemi says. For 30 years, people have been trying to target mutant k-RAS with small-molecule drugs, but it’s very difficult to inhibit without interfering with other proteins, Shemi says. Silenseed takes the approach of embedding siRNAs that target mutant k-RAS in a millimeter-scale matrix called LODER (Local Drug EluteR). Those LODERs are then injected into a tumor, where the siRNAs release over time.

Silenseed tested the concept in a Phase 1 trial among patients with locally advanced pancreatic cancer, a famously aggressive and difficult-to-treat disease. Injected with an endoscopic biopsy needle, LODERs were designed to release about half of the siRNAs in the first week or two, deliver the rest of their payload over four months, and then dissolve completely.8 Among 12 patients, none showed tumor progression when examined 8 to 12 weeks after a single treatment, and median overall survival among trial participants was about 15 months, whereas most patients with the disease die within a year of diagnosis. The company will follow up with a Phase 2/3 randomized trial expected to launch by early 2015.

New leads

Research on more targeted and more powerful RNAi agents charges ahead in academia as well as in pharmaceutical labs, with some instances highlighted at a summer symposium on RNA biology held in June at MIT’s Koch Institute for Integrative Cancer Research. MIT professor Sangheeta Bhatia, for example, presented on advances in a nanocomplex architecture in which siRNAs are wrapped inside peptides designed to penetrate tumors and pass through cell membranes. In its first incarnation, the nanocomplex not only validated inhibitor of DNA binding 4 (ID4) as a novel oncogene, but improved survival in mouse models of ovarian cancer by knocking down its expression.9 In another example, MIT associate professor Daniel Anderson described progress in generating intricately assembled LNPs that incorporate PEG-lipids, lipopeptides, phospholipids, cholesterol, and siRNAs, offering 100 times the potency of earlier designs.10

As developers refine RNAi targeting and delivery, Anderson points out, siRNAs offer the opportunity to do modular pharmacology, with drug designers swapping in targeting ligands aimed at certain cell types and RNA sequences aimed at specific mutations. Arrowhead, for example, is investigating a version of its DPC nanoparticle platform that accepts different targeting ligands and uses different chemistry to extend the agent’s circulation time, improving the drug’s ability to reach targets outside the liver, Lewis says.

“RNAi has come out of the experimental realm into the clinical realm,” Lewis says. “We’re in a hugely exciting time right now. It’s not often you get a chance to be part of a nascent field like this that will really make an impact on human medicine.” 

Eric Bender is a freelance science writer based in Newton, MA.


  1. A. Fire et al., “Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans,” Nature, 391:806-11, 1998.
  2. S.M. Elbashir et al., “Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells,” Nature, 411:494-98, 2001.
  3. K. Fitzgerald et al., “Effect of an RNA interference drug on the synthesis of proprotein convertase subtilisin/kexin type 9 (PCSK9) and the concentration of serum LDL cholesterol in healthy volunteers: a randomised, single-blind, placebo-controlled, phase 1 trial,” The Lancet, 383:60-68, 2014.
  4. H. Denise et al., “Deep sequencing insights in therapeutic shRNA processing and siRNA target cleavage precision,” Mol Ther Nucleic Acids, 3:e145, 2014.
  5. T.W. Geisbert et al., “Postexposure protection of non-human primates against a lethal Ebola virus challenge with RNA interference: a proof-of-concept study,” The Lancet, 375:1896-905, 2010.
  6. M.E. Davis et al., “Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles,” Nature, 464:1067-70, 2010.
  7. S.Y. Wu et al., “RNAi therapies: Drugging the undruggable,” Sci Transl Med, 6:240ps7, 2014.
  8. E. Zorde Khvalevsky et al., “Mutant KRAS is a druggable target for pancreatic cancer,” PNAS, 110:20723-28, 2013.
  9. Y. Ren et al., “Targeted tumor-penetrating siRNA nanocomplexes for credentialing the ovarian cancer oncogene ID4,” Sci Transl Med, 4:147ra112, 2012.
  10. Y. Dong et al., “Lipopeptide nanoparticles for potent and selective siRNA delivery in rodents and nonhuman primates,” PNAS, 111:3955-60, 2014.

Interested in reading more?

Magaizne Cover

Become a Member of

Receive full access to digital editions of The Scientist, as well as TS Digest, feature stories, more than 35 years of archives, and much more!
Already a member?