Silence Could Be Golden

Some gazers see a glow on biotech's horizon. Named technology of the year in 2002 by Science and generally heaped with praise, RNAi--or RNA interference--is a powerful regulating mechanism in cells and one that a number of companies are harnessing for drug discovery purposes.

An intriguing curiosity only a few years ago, RNAi is advancing as a target validation tool in drug discovery. Some new drug development companies have even higher hopes as they explore RNAi chemistry, invest, and tap eager investors in their quest for targeted therapeutics with low toxicity.

It all started with flowers. Extra copies of pigment genes added to plants in the early 1990s led not to a flower of an expected deeper color, but to white. The genes had been silenced, as Andrew Z. Fire of the Carnegie Institution in Washington, D.C., and Craig Mello at the University of Massachusetts Medical School (UMMS) eventually figured out in 1998.

Successive studies at a number of research institutions have begun to shed light on the mechanism through which mRNA (messenger RNA) is suppressed by RNA fragments called siRNA, for small interfering RNA (C&EN, July 5, page 16). The gene goes quiet because the information and manufacturing path between a given gene and protein for which it codes is interrupted.

Rather than look at genes and their functional effects one-by-one, drug developers would like a way to help them perform gene suppression in a high-throughput fashion. RNAi is that kind of gene-knockout tool. The RNA of a virus could be intercepted. A disease-causing gene could be silenced.

"What interests us is the potential to create a whole new therapeutic class of molecules," says Peter Barrett, senior investment principal at Atlas Venture. Not many opportunities of this kind come along, he says, that offer a chance to create a broader pipeline. Barrett says, "What is great about this approach is that you will be able to design the molecule for the target that you are trying to intervene with."

Atlas, Polaris Venture Partners, and Abingworth BioVentures were early backers of the Cambridge, Mass.-based RNAi company Alnylam (pronounced al-nigh-lam), which takes its name from a bright star in the constellation Orion. The company was founded in 2002 with a list of scientific advisory board members that includes many RNAi pioneers.

In July 2003, Alnylam acquired Ribopharma, a German company founded by scientists at the University of Bayreuth that holds some early patents in RNAi. Then in June, Alnylam went public, raising $30 million and becoming the first dedicated RNAi firm to be traded on the stock market.

As Chief Operating Officer (COO) Barry Greene explains, Alnylam's post-initial public offering days do not differ greatly from the pre-IPO phase because it "had already implemented operating discipline." He says the company is converting RNAi science into a "product engine" that uses chemical modifications to make drugs out of siRNAs. The company has identified initial areas of pragmatic emphasis for RNAi therapeutic development. "We call them Direct RNAi, and that's the eye, the brain, and the lungs," he says. "These are areas where we can deliver RNAi directly to the site of disease; because they are sequestered sites, we avoid other potential systemic issues."

LATER THERAPEUTIC developments, Greene says, will involve metabolic diseases and cancer. For now, the company believes it is ready to move some current advances toward the clinic. The power of RNAi, he says, may arise with those genes implicated in disease with well-validated targets but intractable to both small molecules and monoclonal antibodies. "RNAi may be the only way to silence them," he says.

One day last summer, Alnylam got an important phone call: Merck was on the line. "They have targets they very much believe in but cannot move forward with conventional mechanisms," Greene says. A deal was struck. Upon signature, $7 million was paid and another $7 million promised if milestones are reached. Under the terms of the deal, Merck offers targets, and Alnylam will advance them to the toxicology study stage while retaining the right to 50% of potential U.S. revenues down the road.

A second Merck partnership was launched in June to jointly develop RNAi therapeutics for ocular diseases where VEGF, or vascular endothelial growth factor, plays an important role. "Merck has a significant ocular franchise, and we had been going after VEGF," Greene says. Interfering with signaling in this pathway and thus inhibiting blood vessel growth and leakage has been shown to help in a type of age-related macular degeneration (AMD), which can lead to blindness. In AMD, the retina suffers as its nourishing arteries harden. In so-called wet AMD, the retina deteriorates because leaky new blood vessels form in an attempt to nourish the retina.

"They appreciate our progress in VEGF," Greene says. The Merck deal involves $19.5 million in up-front payments and milestones in the preclinical phase of the collaboration. Alnylam hopes to advance its RNAi therapeutic for AMD into the clinic in the second half of 2005. Alnylam is also collaborating in Parkinson's disease with the Mayo Foundation for Medical Education & Research. Preclinical testing will begin later this year.

Sirna Therapeutics, another RNAi company, is also targeting AMD by building on its own unique RNA expertise. Nassim Usman, the firm's COO, sees a distinct role for chemistry in the field. He is an organic chemist who set out to study biological problems and then left his academic post at Massachusetts Institute of Technology for the lure of Ribozyme Pharmaceuticals, an RNA company in Colorado.

Founded in 1992, Ribozyme synthesized and manufactured nucleic acids. As Usman explains, when RNAi began to emerge, the company--and venture capitalists like Oxford Bioscience--saw an opportunity. The company "reinvented itself" in April 2003 as Sirna to leverage its RNA expertise and intellectual property. "We got a jump on the competition in the field because of that long experience with RNA chemistry," Usman says. That knowledge--controlling synthesis, dealing with stability issues, doing quality control--will help in drug discovery because using unmodified siRNA for drugs will not work. "That would be hopelessly very naive," he says.

Although sparse on details, Usman is willing to share that the chemical modifications involve the ends of the RNA molecule and pertain to how delivery reagents are attached. "All of these modifications and formulations convey stability and delivery characteristics to the molecules," Usman says. The chemistries and formulations are tailored to the targeted tissue. "If you use a chemistry or formulation that is liver-centric, that won't work very well in a tumor model," he says. "We have that capability to get it where we want it to go."

In a given cell, one mRNA and a few ribosomes can crank out thousands of proteins. The general idea in RNAi therapeutics is to shut down protein production in a targeted way by picking specific siRNA sequences to go after specific mRNAs. That is unlike using small molecules to chase proteins that have already been produced. Interfering at the mRNA level, as RNAi can, is upstream from that protein factory.

"We think this will be much more potent than the other approaches," Usman says. Alnylam's Greene uses a household analogy and says using RNAi "turns off the faucet rather than mopping up the floor after the damage has been done."

IDEAS DIFFER on how to get RNAi into the cell as well as how to maintain gene silencing. Usman says a single dose of modified siRNA has been shown to silence genes for three weeks. Without modification, the effect lasts only a few days. "The chemistry is key," he says.

In February, Sirna entered into an 18-month, $2.2 million research collaboration in cancer with Eli Lilly. In an animal model and with Lilly's tumor targets, Sirna wants to show how and with which kind of chemical modifications and delivery specifications siRNAs could be useful in cancer.

The company has also entered a license agreement with the University of Iowa for intellectual property relating to RNAi technology covering siRNAs to target neurological diseases such as Huntington's and Alzheimer's.

Sirna-027, the company's own drug candidate, targets VEGF receptor-1 for therapeutic development to treat wet AMD. The company is confident enough about the receptor being targeted with siRNAs that it is planning to file an Investigational New Drug Application with the Food & Drug Administration later this year or early next year, to be followed by clinical trials. Sirna has its own current Good Manufacturing Practices manufacturing plant with which it generates some cash flow.

CytRx, a biopharmaceutical company based in Los Angeles with a subsidiary in Worcester, Mass., is plotting another kind of course in RNAi. For one, it does not call itself an RNAi company. "We let the therapeutics guide us into RNAi rather than let RNAi guide us into therapeutics," says Jack R. Barber, the firm's senior vice president of drug development. Its therapeutic focus areas are obesity, diabetes, ALS (amyotrophic lateral sclerosis) or Lou Gehrig's disease, and cytomegalovirus (CMV). The company is also pursuing small-molecule development to avoid, as Barber says, putting all of its proverbial eggs in one basket.

Although CytRx's approach differs from Sirna's, Barber shares the assessment about chemists' role in RNAi. "We need them right now--basically nucleotide chemists," he says. People will be crucial if they understand the biology of RNA enough to chemically tweak it so it is more druglike and yet still retains its gene-silencing traits, he says.

The firm did some inner rearrangements after merging with Global Genomics in 2002. It has formed a broad alliance with UMMS, where there is much RNAi expertise, Barber explains. At the school are RNAi pioneers Mello, Philip Zamore, and others who co-own important patent positions in RNAi along with the university. The school agreed to disclose new technology developed pertaining to RNAi, obesity, type 2 diabetes, CMV, and ALS to CytRx. In exchange for a payment, the company will have the option to license this technology. CytRx's minimum commitment is $750,000 per year.

For the longer term, Barber is banking on obesity and diabetes therapeutics, although the preclinical phase is still two to three years away. Despite long-standing work, diabetes is poorly understood, says Michael P. Czech, chair of molecular medicine and professor of biochemistry and molecular pharmacology at UMMS. He cofounded the Worcester subsidiary of CytRx, called CytRx Laboratories, and is chairman of the scientific advisory board and consultant to the company.

In type 2 diabetes, cells react sluggishly to insulin and sugar is not absorbed from the blood. "RNAi is changing the way this problem can be attacked," Czech says. As he looks at the molecular underpinnings of this disease, he is partial to a glucose transporter protein called GLUT4. It catalyzes glucose uptake into muscle and adipose tissue and is found only in those two tissues. "It is necessary for whole-body glucose homeostasis and controls the linkages between adipose and muscle tissue," he says. In response to insulin, GLUT4 moves from a sequestered compartment inside the cell to the cell surface, where it then catalyzes glucose uptake. One approach to diabetic therapy involves increasing insulin sensitivity by enhancing GLUT4 trafficking.

Czech has been working with adipocytes, or fat cells, which are both tricky and fragile. He has figured out how siRNA can silence genes in fat cells and has miniaturized the work for a 96-well screening assay. He and his colleagues picked 2,000 genes with activity involving insulin sensitivity. Using RNAi, they are knocking these genes down. Traditional methods permit this kind of work at a speed of around 10 genes a year. "We are getting through 30 to 50 genes a week with a relatively small group," Czech says. Work on 500 genes is done and has generated novel targets for the small-molecule development by newly hired CytRx chemists.

Half the targets are druggable--or amenable for small molecules--and the other half are not. "But they will be very amenable to knocking them down with RNAi in vivo," Czech says.

Where small molecules won't do the trick, the hope is to deliver RNAi in a stable, efficient way to silence those genes in question. Working with UMMS scientists Mello and Tariq Rana on the chemistry of RNAi, CytRx seeks to make RNAi more stable and accessible to delivery in a live animal and, potentially, a human.

For the near term, CytRx is interested in applying RNAi for ALS and to address CMV retinitis, an infection that AIDS patients often face. "In the case of ALS, because there is no treatment, we feel we will get expedited review," Barber says. ALS is a fatal disease of unknown origin in which, over time, motor neurons--the nerves controlling muscles--are destroyed.

UMMS biochemist and molecular pharmacologist Zuoshang Xu is applying RNAi to this disease. In some cases of ALS, a gene called SOD-1 (Cu,Zn superoxide dismutase 1) is mutated such that the protein it produces causes nerve-cell death. Patients with familial ALS have a normal copy of the gene and a mutated one. The cure hypothesis: Silence the mutated gene.

In collaboration with Zamore, Xu was able, at least in a mouse, to do just that. Xu did not use chemically synthesized siRNA but rather shRNA, or short hairpin RNAs. "You make a gene with a promoter, and the promoter is a stretch of DNA sequence that can be recognized by proteins in the cell that can direct RNA synthesis," Xu says. Inside the cell, shRNAs are processed into siRNAs.

"We still have a lot of work to do," Xu cautions, but he agrees that RNAi has huge potential for many diseases. "If we can find a way to deliver specific gene silencing in vivo, the principle can be applied to almost any disease," he says.