BUSINESS SILENCED Xu, here posing at UMMS, is using RNAi to silence the gene that causes Lou Gehrig's disease.
SILENCE COULD BE GOLDEN Gene silencing with RNAi might offer novel therapeutics and help with nondruggable targets V I V I E N MARX P C & E N N O R T H E A S T N E W S B U R E A U
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biotech's horizon. Named technology of the year in 2002 by Science and generally heaped with praise, RNAi—or RNAinterference—is apowerful 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 CUMMS) eventually figured out in 1998.
Successive studies at a number ofresearch 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&ENJuly5,page 16).The gene goes quiet because the information and manufacturingpath between agivengene 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. R N A i 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-nighlam), 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 2 0 0 3 , 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 maybe 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
"What interests us is the potential to create a whole new therapeutic class of molecules." 18
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THE NEAR TERM
RNAi Is Getting Use In Target Validation
E
ven though therapeutic applications of RNAi (RNA interference) are far in the future, there is already an siRNA (small interfering RNA) market in target validation work. Navigant Consult ing reports in a recent study that RNAi can potentially meet the need for highthroughput gene suppression. The tech nology, as Navigant senior consultant Alek Bituin says, is maturing. Navigant sees the siRNA market, esti mated at $60 million for 2004, as both rapidly growing and evolving. By 2009, siRNA technology might garner $246 million in sales. The leading technology categories involve the molecules them selves and the vehicles used to get them into cells: • RNA oligonucleotides, which are siRNA molecules that are synthesized chemically; • DNA-based siRNA, in which DNA templates are ferried into cells and siRNAs are produced in vivo; • Transfection reagents that permit nucleic material to be absorbed by cells. Currently, the siRNA market for tar get validation is "relatively immature and volatile," according to the Navigant. RNA oligonucleotides—chains of nucleic acid building blocks or nucleotides that can be chemically synthesized—dominate the market, but both DNA-based siRNA and transfection reagents are likely to encroach on oligonucleotides. Bituin says that, although the debate over the efficacy of DNA-based vectors versus oligonucleotides is not over, the market is shifting toward vectors as the technology becomes more validated. Those products are less expensive and, at times, easier to work with. The idea companies pursue is to integrate RNAi
milestones are reached. Under the terms of the deal, Merck offers targets, and Alnylam will advance them to the toxicologystudy 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 fac tor, plays an important role. "Merck has a significant ocular franchise, and we had been going after VEGF," Greene says. In terfering with signaling in this pathway and HTTP://WWW.CEN-ONLINE.ORG
into a target validation package for their customers. For example, Dharmacon founder Stephen Scaringe, together with Marvin Caruthers of the University of Colorado, Boulder, developed 2'-ACE RNA chem istry as a new way to synthesize RNA. The company began shipping in 1996 and was positioned for RNAi in the late 1990s. Fisher Scientific acquired Dhar macon earlier this year for $80 million. The company offers reagents as well as designed siRNA duplexes targeting hu man genes. One customer this year has been Bayer. Dharmacon has also just aligned itself with Genospectra to offer a new kind of assay that not only silences genes but also measures the degree of silencing achieved. Qiagen offers siRNA synthesized with proprietary chemistry during which the monomers are protected and then deprotected at the end of synthesis. Ambion just launched an RNAi library for the Drosophila genome together with Cenix, another RNAi company. Ambion was founded by Matt Winkler from the Univer sity of Texas, Austin, to fill his and others' need for reagents for RNA applications. Navigant's Bituin believes that the technology is moving toward highthroughput applications. "Everyone wants to ramp it up, to apply it to various assay and array technologies," he says. "That is what is going to be driving the market in the future." For now, Dharma con holds a leading 27% market share, but Navigant predicts that Ambion and Qiagen will increase their presence by 2009. "Qiagen and Ambion are poised for this kind of high-throughput application better than Dharmacon and some of the others are," Bituin says.
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 hard en. In so-called wet AMD, the retina dete riorates 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 mile stones in the preclinical phase of the col laboration. Alnylam hopes to advance its
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BUSINESS controlling synthesis, dealing with RNAi therapeutic for A M D into stability issues, doing quality conthe clinic in the second half of 2005. trol—will help in drug discovery beAlnylam is also collaborating in cause using unmodified siRNA for Parkinson's disease with the Mayo drugs will not work. "That would Foundation for Medical Education be hopelessly very naive," he says. & Research. Preclinical testing will Although sparse on details, Usbegin later this year. man is willing to share that the Sirna Therapeutics, another chemical modifications involve the RNAi company, is also targeting ends of the RNAmolecule and perAMD by building on its own unique tain to how delivery reagents are RNA expertise. Nassim Usman, the attached. 'All of these modificafirm's COO, sees a distinct role for tions and formulations convey stachemistry in the field. He is an orbility and delivery characteristics ganic chemist who set out to study to the molecules," Usman says. The biological problems and then left chemistries and formulations are his academic post at Massachusetts tailored to the targeted tissue. "If Institute of Technology for the lure you use a chemistry or formulation of Ribozyme Pharmaceuticals, an that is liver-centric, that won't work RNA company in Colorado. very well in a tumor model," he says. Founded in 1992, Ribozyme syn"We have that capability to get it thesized and manufactured nucleic where we want it to go." acids. As Usman explains, when STARTERS Alnylam's founders-•Thomas Tuschl In a given cell, one mRNA and RNAi began to emerge, the com- (from left), Phillip Sharp, David Bartel, and Zamore— a few ribosomes can crank out pany—and venture capitalists like have high hopes for RNAi. thousands of proteins. The generOxford Bioscience—saw an opporal idea in RNAi therapeutics is to shut tunity The company "reinvented itself" in jump on the competition in the field bedown protein production in a targeted way April 2003 as Sirna to leverage its RNA excause of that long experience with RNA by picking specific siRNA sequences to go pertise and intellectual property "WEî got a chemistry" Usman says. That knowledge—
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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 18month, $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
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," saysJack 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, al-
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though 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
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