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The revolution in RNA interference has galvanized basic research. Now, some biopharmas are pushing the technology from the laboratory to the clinic.

By Nancy Weil

December 15, 2004 | "I have frequently said that RNA interference, as far as I'm concerned, is the most important discovery of the past decade," says MIT's Phillip A. Sharp. As co-founder of Biogen and Alnylam Pharmaceuticals, and Nobel laureate for his discovery of gene splicing, Sharp knows of what he speaks.

His small office at the MIT Center for Cancer Research is crammed with stacks, albeit neat ones, of journals and papers. Since his September announcement to step down as director of the McGovern Institute for Brain Research at MIT, Sharp can once again devote his considerable energies to lab research — and evangelizing RNA interference (RNAi).

"RNAi may be the most important discovery for a number of decades," Sharp continues. "It is among the discoveries that shift the way we understand the paradigms of biology as well as a new approach to therapeutics. It's very rare that you have an advance in the field that really changes the way we understand all biological systems, and as well suggests new means of therapeutic intervention." (See "Sharpest Knife in the Drawer," page 30.)

Even if RNAi never leads to a new class of drugs targeting myriad diseases, as many believe it will, it remains a fascinating and important biomedical breakthrough. Its rapid acceptance as an indispensable laboratory tool is helping scientists dissect gene function and unlock cellular processes.

Phillip A. Sharp, MIT professor and Nobel laureate, says there is a "platform of science for us to move forward in drug discovery."
While Sharp's views are widely shared by scientists, venture capitalists, and company executives, they recognize the challenges that must be overcome for RNAi therapeutics to become a reality.

RNAi is a natural process that evolved eons ago to help organisms ward off viruses — which makes its recent discovery all the more exciting and its applications all the more promising. From yeast to mammals, RNAi is a complex cellular reaction that not only fends off foreign invaders but also regulates aspects of cell development and death.

But while armies of scientists dedicate themselves to understanding how RNAi works at the molecular level, a handful of biotechs intend to apply RNAi to the creation of therapeutics for a host of diseases, including macular degeneration, hepatitis, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, cancer, and HIV.

Mystery Solved 
For several years scientists puzzled over the RNAi phenomenon they observed in various plants, fungi, and other organisms (see "Silence Is Golden," July 2003 Bio·IT World, page 26). More than a decade ago, plant biologists trying to increase gene activity in petunias found they were inadvertently producing white or variegated flowers instead of dark purple. They dubbed the phenomenon "co-suppression" because the transgene and endogenous gene were mutated.

RNA RICHES: Alnylam's John Maraganore sees no end of potential drug targets and potential applications.
The breakthrough came in 1998, in studies on the nematode Caenorhabditis elegans. Andrew Fire, now at Stanford University School of Medicine, and Craig Mello, at University of Massachusetts Medical School, were exploring "why some things were working much better than they should," Fire recalls. Su Guo, then a student at Cornell University, had found that preparations of antisense or sense RNA could inhibit gene expression in C. elegans. Even more surprising, Mello's lab found that this "interference" spread into the germ line even if the injection missed the germ line.

Fire and Mello subsequently found that double-stranded RNAs (dsRNAs) caused the gene silencing, and published a seminal paper on RNAi in Nature in 1998. A few years later, Rockefeller University's Thomas Tuschl determined that small interfering RNAs (siRNAs) could do the same thing in mammalian cells.

Recent studies have demonstrated that RNAi and microRNAs control the expression of hundreds of known genes — perhaps as much as 10 percent of the genome. "Some of those genes appear to be critical for development, some seem to be important for cell death control, and some for growth, so it's touching every aspect of biology," Sharp says.

Scientists didn't take long to realize that this major new tool in genomics research also had profound implications for drug discovery and development. "Not only can one contemplate using RNAi to cure a disease, we can use it now to understand how a disease works," Fire says.

"There's a lot that's happened in a very short time, and there's certainly a lot more that's going to happen," Fire says, noting that RNAi is used as a tool in hundreds of studies a week. "That base of users will encourage the use of RNAi as a tool, and that will benefit the clinical work."

RNA Eye 
This summer, two companies — Acuity Pharmaceuticals, a small Philadelphia-based startup, and Sirna Therapeutics in Boulder, Colo. — filed investigational new drug (IND) applications with the FDA to begin human RNAi trials for treatment of the "wet" form of age-related macular degeneration (AMD), the leading cause of age-related irreversible vision loss in developing countries.

Wet Age-Related Macular Degeneration RNAi Therapy

Download the pdf file of the diagram.
The procedure targets the vascular endothelial growth factor (VEGF) pathway, by suppressing the growth of new blood vessels and causing existing vessels to atrophy. In wet AMD, an abnormal number of blood vessels form behind the retina and leak, destroying cells in the central part of the retina (see "Wet Age-Related Macular Degeneration RNAi Therapy").

"RNAi offers the ideal pharmacological mechanism to inhibit the protein that is causing the vision loss and wet AMD," says Sam Reich, Acuity's co-founder and senior director of R&D. "The eye is an ideal organ to move forward with revolutionary technologies because it is easily accessed."

Acuity recently signed a manufacturing agreement with Avecia Biotechnology to receive supplies of Avecia's siRNA drug, Cand5, which is in Phase I trials with AMD patients. These are the first human clinical trials for pharmaceutical siRNA.

At Sirna, pre-clinical work went so well that the company beat its planned IND filing date by about three months, says Nassim Usman, chief operating officer and senior vice president. The Sirna-027 Phase I trial, targeting the VEGF receptor, began in November. Both trials for AMD will involve the injection of RNAi-based therapeutics directly into the eyes of patients at their doctor's offices, with close monitoring to determine efficacy and toxicity.

Alnylam, which says it is the first company formed specifically to develop RNAi therapies, is on target to apply for regulatory approval for clinical trials in macular degeneration in 2005, as well as for pre-clinical testing by the end of this year on therapies for Parkinson's disease. President and CEO John Maraganore says the Acuity and Sirna applications were taken in stride. "We're actually encouraged to see the field advance," he says, adding that clinical trials by any company will mean that the benefits of chemically modified, stable siRNAs can be studied and established, which has to happen for the market to progress.

While the AMD trials involve direct application of RNAi-based drugs to the sites where they are needed, systemic delivery of RNAi may follow if direct delivery to target tissues is a success. This could open the way for treatment of viral diseases such as HIV or influenza, which could be an important RNAi target given the expectation of another global pandemic and shortage of drugs to treat the flu. "That's where I think the biggest challenges are," says Cold Spring Harbor Laboratory's Gregory Hannon of systemic delivery.

Solving Stability 
But before systemic delivery of RNAi drugs becomes feasible, researchers must clear other hurdles. Stability is another problem. RNA molecules are large and don't pass readily through cell membranes. They are also subject to degradation, excretion, and are fragile to work with in the laboratory. Nevertheless, researchers are confident that both delivery and stability issues will be licked, and that synthetically produced RNAi will behave the same way as the naturally occurring process.

"We're well on the way to solving these challenges for different cell types, though it's different for each cell," says Sirna CEO Howard Robin. "Once we understand how to make these stable to deliver them in a more broad sense, if you know the sequence of the RNA you have a drug," he says. "That's not around the corner, but that's where we're headed."

Although some are skeptical, Robin believes it is "very reasonable" to think that if delivery and stability are solved, companies could be on the way to robust pipelines in the next five years. Depending on the disease and how patients respond, regular injections would likely be required for some duration, perhaps permanently. "We're not going to take a pill of RNAi," Sharp says.

In the chemical approach taken by Sirna, Alnylam, and Acuity, dosages can be altered to diminish negative side effects, or the therapy can be terminated if necessary. Researchers are reasonably confident there won't be long-lasting side effects, but that is not yet proven.

Commercial RNAi ventures are also pondering what Alnylam's Maraganore terms "an embarrassment of riches." There are so many potential drug targets and so many potential applications for RNAi-based therapies that it is difficult to stay focused on specific diseases. This is particularly true with areas of unmet medical need, which those in the RNAi industry widely agree should be the overarching initial emphasis for such a novel approach.

"How do you bring your research and everything you do to focus on a valuable target that is going to work clinically?" Sharp asks. "That's the real challenge."

The strongest immediate focus among most RNAi therapeutic companies is on diseases for which there currently are few or no effective treatments. When Fire was at Johns Hopkins University, he found, in conversations with those working with HIV-positive patients, that they "felt they could treat someone with HIV, and they didn't feel that there was ... an impetus to find revolutionary new treatments that have substantial risks associated with them."

Property Rights 
Today, many scientists feel compelled to help develop therapies for diseases such as AMD and Parkinson's, which, in the absence of a cure, leave patients willing to try novel therapies that might have greater risks. There is also a sense that the FDA is more likely to approve novel therapies for untreatable diseases, even allowing for the greater risks involved.

Big Pharma is also pursuing RNAi research and therapies, while leaving much of the heavy lifting to biotechs. Eli Lilly has a deal with Sirna to work on cancer therapeutics, and Merck has a pact with Alnylam.

Sharpest Knife in the Drawer
Phil Sharp shared the 1993 Nobel Prize in physiology or medicine with Richard J. Roberts for RNA splicing, a seminal discovery that not only transformed scientists’ understanding of gene regulation but also helped give rise to biotechnology.

Read More 
The chances of Alnylam and Sirna surviving among others enjoying long-lasting success depends largely on intellectual property rights. Alnylam protected its IP position by loading its employee roll with leaders in the field. The company was founded by Sharp and Tuschl, who teamed with RNA experts Paul Schimmel, David Bartel, and Phillip Zamore.

Alnylam further has a key deal with antisense leader Isis Pharmaceuticals to license all of Isis' IP estate and important chemistry work, which is a cornerstone of RNAi research, in return for Isis investing in Alnylam. When it hasn't been able to license or develop the IP it needs, Alnylam has bought it — acquiring Ribopharma of Germany to obtain an important patent.

Although some competitors grumble about Alnylam's aggressive approach to IP, Maraganore says, "we don't want to use our IP to block development of these important new drugs," and emphasizes that the company is amenable to licensing its IP.

Both Sirna and Alnylam develop synthetic dsRNAs, but not all companies take that approach. Australian biotech Benitec Ltd. introduces constructs that lead to creation of "hairpin" RNAs that fold up to make a double-stranded molecule. The idea is to induce the body to produce the RNAi mechanism, without the need for ongoing drugs.

Benitec is working with the City of Hope, in Duarte, Calif., to study that method in HIV patients suffering from a specific type of lymphoma. To qualify for the clinical trials, patients cannot have developed full-blown AIDS. However, they represent "an extreme case" in that they will have qualified for bone marrow transplants to treat lymphoma, says John Rossi, chair of molecular biology with the Beckman Research Institute at the City of Hope. Their own stem cells will be genetically modified to make them HIV resistant, and they will be placed back into their bodies with the hope that the modified stem cells will re-populate.

Special report on RNAi

Identifying RNAi Drug Candidates
David A. Bumcrot, director, preclinical, Alnylam Pharmaceuticals

Applications of RNAi
William Marshall, vice president of research and development, Dharmacon

An Unusual Path for RNAi
Dmitry A. Samarsky and Peter J. Welch, Invitrogen
"Theoretically, this is a great way of doing it," Rossi says. "The virus would have a very difficult time establishing resistance." HIV-based vectors will be used to deliver the modified genes. "It's an interesting turn of events that we could take the type of virus that is making patients sick and potentially make them well," he says.

Even here, however, patent challenges are looming. Pennsylvania's Nucleonics recently challenged the validity of a key Benitec patent, charging that prior art abolishes the novelty of the patented technology. Benitec has aggressively protected its IP, helped by the experience of CEO John McKinley, who was a U.K. lawyer specializing in patent law before he jumped into biotech.

Beyond the companies developing RNAi-based therapeutics, the booming market also has led to the emergence of vendors for laboratory hardware and software. For instance, Ambion, in Austin, Texas, specializes in products for stabilizing, synthesizing, handling, isolating, storing, detecting, and measuring RNA.

Qiagen, with corporate offices in the Netherlands and Germany; Promega in Madison, Wis.; and Dharmacon in Lafayette, Colo., are among established life science companies that expanded their portfolios to include RNAi as that market became hot. OligoEngine, in Seattle, was formed in 2001 to offer siRNA sequence-design software, while Open Biosystems, founded in 2002 in Huntsville, Ala., has added to the resources it offers short hairpin RNA (shRNA) expression vectors developed at Hannon's Cold Spring Harbor lab.

RNAi research is moving ahead so quickly that Hannon admits it is difficult to keep up. "You always wonder what you're going to read in the journals," he says. RNAi is, for once, a technology that actually warrants the hype and attention it has received. "There are a lot of great biological discoveries, and this is certainly a fun one to have been involved in," Fire says. "It's been an exciting ride."


For reprints and/or copyright permission, please contact  Jay Mulhern, (781) 972-1359,