Alan Dove, PhD
Contributing Editor

Short interfering RNA, also known as siRNA or RNAi, has been a windfall for basic research and target validation. Will it also be useful in the clinic?

In less than a decade, short interfering RNA (siRNA) blossomed from an obscure laboratory finding in worms to a research technology nearly as useful and widespread as polymerase chain reaction. Now, siRNAs and their kin are attracting serious attention from drug developers, and many expect short RNA molecules to become a multi-billion-dollar therapeutic platform in the next few years.

The key to this revolution is a set of naturally occurring mechanisms that eukaryotic cells use to regulate gene expression. Though researchers are still dissecting the
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Eukaryotic cells process small double-stranded RNAs (dsRNA) into short interfering RNA (siRNA) via an enzyme called Dicer. siRNA binds its complementary mRNA and targets it for translational inhibition and/or destruction. The naturally occurring micro-RNA (miRNA) gene regulation pathway works similarly to exogenous siRNAs.
details, short double-stranded siRNAs can induce a cell to destroy complementary pieces of messenger RNA (mRNA), preventing the target message from being transcribed. In the laboratory, scientists now exploit this phenomenon, called RNA inhibition, or RNAi, to inhibit any gene of interest in nearly any kind of cell.

Some labs now use it to inhibit genes in whole animals as well. "It has a lot of advantages over so-called conditional knockout approaches," says Miles Wilkinson, PhD, professor of biochemistry at the M.D. Anderson Cancer Center, Houston, Texas. Wilkinson says that a transgenic RNAi-based knockout mouse can be produced in as little as four months, compared to a year or more for most conditional gene knockouts. Drug companies have taken notice, and many are already using RNAi-based techniques for faster target validation.

For drug developers, though, the biggest attraction of RNAi is at the next step: using the short RNAs themselves as drugs. Several startup companies have rushed into the field, and a handful of RNAi-based drugs have now reached clinical trials. But even boosters of the technology see some major hurdles ahead.

It’s all in the delivery
The field’s biggest challenge will be getting the therapeutic RNA to its target mRNAs. "Nature does not like to admit exogenous oligonucleotides to cells; that’s just the bottom line," says Cy Stein, MD, PhD, a professor of Medicine at Albert Einstein College of medicine, New York.

Patents Pending
Therapeutic RNAi has all the hallmarks of a new frontier in biotechnology: numerous small companies, considerable buzz among investors, and, of course, a gathering storm of patent lawsuits.

“The patent position in all this is an absolute mess . . . with everybody claiming they own everything, and it’s not clear who owns anything,” says Cy Stein, MD, PhD, a professor of medicine at Albert Einstein College of Medicine, New York, and a prominent RNAi researcher.

Most sources agree that Alnylam, Cambridge, Mass., is the field’s 800-pound gorilla, and the company is not shy about its position. “There are eight issued patents in the US and Europe that specifically relate to small interfering RNAs. We have all eight, and seven of the eight we have exclusively,” says John Maraganore, PhD, president and CEO of Alnylam.

But Todd Woolf, CEO of RXi Pharmaceuticals, Worcester, Mass., is one of many competitors questioning the importance of Alnylam’s claims. Citing the original patent filed by Craig Mello and Andrew Fire, who shared the Nobel prize for their discovery of RNAi, Woolf explains that “you can operate under the umbrella Fire-Mello patent, [and] a lot of this other IP won’t be relevant.”

Several lawsuits are already underway, and observers expect a lengthy struggle. “I think that the adjudication of the various patent claims is going to be somewhat difficult and arduous, and probably is going to take awhile,” says Stein.
In target validation and basic research studies, scientists can simply insert RNAi-encoding transgenes into an organism, or expose cultured cells to high enough concentrations of the short RNAs to guarantee that some will get in. Neither approach is practical in the clinic.

"My guess is you’re not going to find a delivery strategy that fits all indications. If you want to deliver to hepatocytes, you may need one strategy, [and] if you want to deliver to tumor cells, you may need another strategy," says Stein.

As a result, companies working on RNAi have focused much of their attention on devising novel delivery systems. While the strategies continue to multiply, most fall into one of three broad categories: DNA or viral vectors, localized injection, and synthetic modification or encapsulation.

The DNA-based strategies are promising, but even their developers agree that using a vector simply moves the delivery hurdle. "Knocking down a gene in tumor cells is great, but if you knock down a gene in some normal cells and it causes problems, in that case you have to make the delivery more specific," says Wilkinson.

Localized delivery of RNAi into a target tissue is the most direct solution, and it may be perfect for some diseases. Alnylam, Cambridge, Mass., for example, is now conducting phase I clinical trials on an inhaled treatment for respiratory syncytial virus (RSV). The therapy’s RNA molecule targets an essential gene in the virus. The company is also working on directly-delivered treatments for neurological and opthalmological conditions.

The vast majority of valuable disease targets are unlikely to yield to DNA-based or direct injection approaches, though, so Alnylam is also trying synthetic delivery systems. "We’ve used conjugates, we’ve used liposomes . . . we’ve used polymer-based systems, we’ve used peptides," says John Maraganore, PhD, president and CEO of Alnylam. Such cosmopolitan taste in delivery systems is unusual; most RNAi companies are focusing on their own proprietary delivery systems instead.

RXi Pharmaceuticals, Worcester, Mass., for example, uses special synthetic organic molecules that bind RNAi. "You just mix them with the RNAi, you don’t have to formulate lipsomes," says Todd Woolf, CEO of RXi. Once the RNAi is linked to these molecules, which the company calls nanotransporters, it can cross cell membranes to inactivate its target messenger RNA. "We can get one milligram per kilogram activity systemically in the liver with [nanotransporters]," says Woolf. RXi hopes to use the system to inhibit genes involved in diabetes and obesity.

Meanwhile, Intradigm, Palo Alto, Calif., has taken a page from viruses’ playbook. The company’s RNAi-delivery system is entirely synthetic, but bears a strong resemblance to a membrane-enveloped RNA virus. "With these polymers that we encapsulate the RNAi with, we can attach a targeting moiety that will attract these capsules to a certain tissue," explains Mohammad Azab, MD, president and CEO of Intradigm. The company’s current lead product is an anti-tumor RNAi in a capsule targeted to a tumor-specific receptor.

Antisense and sensibility
Though delivery is a major challenge for RNAi drug developers, it is not the only one. Because cells use small RNAs naturally as gene regulators, RNAi-based therapies could easily induce cross-talk in signaling pathways researchers are still struggling to understand.

Worse, the long coevolutionary history of RNA viruses and eukaryotes has left humans with a robust set of immunological responses against foreign RNA, especially small double-stranded pieces of it. Among other effects, RNAi can stimulate Toll-like receptors and the interferon response, possibly confounding the outcome measures in clinical trials or even causing harmful side-effects in vulnerable patients.

So far, RNAi companies are confident they can handle these problems. "We have found even short RNAis can induce interferon, but they can be made to not induce it by certain chemical modifications," says RXi’s Woolf.

For RNAi companies, perhaps the bigger concern with the interferon effect is that it could remind investors of the elephant in the room: antisense. Indeed, some experienced RNA researchers see disturbing parallels between the current state of RNAi drug development and the heady boom times of antisense DNA, a technology that largely failed to live up to its early hype.

"To me, this whole thing is deja vu all over again. It’s almost the same type of thinking now, with all the excitement about therapeutic uses of siRNAs, as there was about therapeutic uses of antisense," says Stein, who has worked on translational inhibition for more than 20 years.

The downfall of antisense stemmed largely from its off-target effects, especially the tendency of certain nucleic acid sequences to induce generalized immune responses, as well as the difficulty of delivering the therapies to the right cell types. As the magnitude of these problems became clear, most standalone antisense companies folded.

Besides delivery problems and the danger of side-effects, RNAi has other limitations. "There are lots of issues of tremendous costs that are involved [in RNA manufacturing], and inconvenience to patients because these obviously have to be administered intravenously," says Stein.

Most believers in RNAi take exception to the comparison with antisense, but concede that the field may face some tough terrain in the coming years. "All technologies will face their challenges, and all technologies will face their moments of promise and moments of reality setting in," says Alnylam’s Maraganore.

For now, investors are happy to support RNAi; CytRx, which owns RXi, gained $150 million in market capitalization when it announced that it was forming an RNAi subsidiary. Big pharmaceutical companies also seem to like the technology; in December, Merck bought RNAi developer Sirna Therapeutics, San Francisco, Calif., for $1.1 billion.

Nobody in the field expects the route to RNAi therapies to be quick or painless, but the fast target validation and easy synthesis of RNAi could ultimately make it a prime choice for drug developers. "In the next 20 to 30 years, I think this is going to be a multi-billion-dollar market," says Azab.

About the Author
Originally trained as a microbiologist, Alan Dove has been writing about science and its interfaces with industry and goverment for more than a decade.

This article was published in Drug Discovery & Development magazine: Vol. 10, No. 4, April, 2007, pp. 38-40.