SCIENCE & TECHNOLOGY generally believe that RISC binds only to m R N A that is completely complementary to the siRNA strand. Once the two strands bind, the target R N A is cleaved, preventing protein synthesis or viral replication, depending on the nature of the target. In the short time since the discovery of t h e p h e n o m e n o n , R N A i has skyrocketed as a tool for functional genomics and as a potential therapeutic. CELIA M. HENRY, C&EN WASHINGTON At a conference sponsored by IBC Life Sciences held last month in Boston, representatives from academia and indusITH ANTISENSE MOLEplanted by the interferon system, but the try gathered to discuss the potential of cules and ribozymes, scimachinery is still there and still works. In the technology as a target validation tool entists have been trying the natural pathway, long stretches of and as a therapeutic. for more than a decade dsRNA are chopped by the enzyme dicer to turn RNA into a drug. into shorter pieces that are 21 to 23 base In his opening remarks as chairman of Neither of those strategies has worked pairs long, known as short interfering one of the sessions, John J. Rossi, a proas well as originally hoped. Now, the buzz RNA, or siRNA. The siRNA is incorfessor of molecular biology at the Beckis all about R N A interference. Is RNAi porated into a protein complex known man Institute at City of Hope in Duarte, the technology that will finally Calif., called RNAi "one of the turn RNA into a viable drug? Ac™. £ hottest things in target validacording to scientists and compation in the last few years." siRNAs nies that are developing the techHowever, Rossi pointed out nology as both a research tool that the "process of RNAi is and a p o t e n t i a l t h e r a p e u t i c , more complicated than most lilHI'illll·" RNAi is the real deal and all the dsRNA trigger people like to think," because hype surrounding it is justified. short RNAs are involved in mulRNasel In R N A i , double-stranded tiple pathways. In addition to RISC R N A (dsRNA) is used to supsiRNA, there are naturally ocsiRNAs RISC press the expression of targeted curring small RNAs known as / genes. The gene-silencing effects Selective gene silencing microRNAs. In the microRNA Endonucleolytic 0 were first discovered in plants, pathway, a single-stranded RNA where they serve as the plants' is incorporated into a complex antiviral defense system. Later, a that binds to the 3'-untranslated team led by Andrew Fire of the region of m R N A and blocks Cleaved mRNA Carnegie Institution of Washtranslation. The microRNApathington and Craig C Mello of the way requires less complementarRUNNING INTERFERENCE The RNAi pathway University of Massachusetts ity than RNAi, and the mRNAis uses double-stranded RNA (dsRNA) to silence gene Medical School discovered that not degraded. Also, in plants, expression. Long stretches of dsRNA such as from a dsRNA also interferes with gene small RNAs are involved in chrovirus can be cut by the enzyme dicer to shorter expression in the worm Caenomatin silencing and reorganizalengths, or synthetic short interfering RNA (siRNA) rhabditis elegans, indicating that tion. A similar role has yet to be can be added directly to the cell, bypassing dicer. it is a conserved eukaryotic demonstrated in animal cells. These siRNAs are incorporated into the RNAmechanism [Nature, 3 9 1 , 8 0 6 induced silencing complex (RISC), which binds and "There are multiple pathways (1998)}. They coined the term cleaves the target messenger RNA. that involve the same compoRNAi to describe the phenomnents," Rossi said. "If you make enon. Since then, RNAi has also enough small RNAs in the cell, been shown to work in mammalian cells they could be entering any of those as RISC, for RNA-induced silencing by Thomas Tuschl, now at Rockefeller pathways at the same time. It's not clear complex. Synthetic siRNA can also be University, and his coworkers [Nature, that any one of those pathways would added to the cell and incorporated into 411,494 (2001)]. be dominant." the RNAi pathway, bypassing dicer. One of the R N A strands is separated from These multiple pathways complicate The RNAi effect takes advantage of a siRNA, and the remaining strand binds the selection of R N A sequences for naturally occurring pathway. In animals, to the target messenger RNA. Biologists RNAi, but designing around them is posthe defense mechanism has been sup-
HIGH HOPES FOR RNAINTERFERENCE
Chemists have a role to play in turning this biological discovery into pharmaceutical reality
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sible. The first step is a careful computer search to make sure that the selected siRNA sequence is unique. However, Rossi pointed out, unique can mean that one or two other genes have only a couple of mismatches and could be targets for the microRNA pathway. Rossi and others also recommend using the lowest possible concentration of siRNA. "When you overexpress, you're saturating the RNAi machinery," Rossi said. "That means these RNAs could be directed—less efficiently, but directed— to nontargeted transcripts in the cell, as well as possibly to chromatin, where they operate at the level of chromatin reorganization and chromatin silencing." Unfortunately, RNAi may not be as specific as people assume. Aimee L.Jackson, a senior biologist at Rosetta Inpharmatics, a subsidiary of Merck in Kirkland, Wash., described research that shows significant "off-target" effects in RNAi. Using genome-wide expression profiling to investigate the specificity of siRNA, her team found that mRNA expression patterns were dictated by the siRNA rather than the target, meaning
that the siRNA was affecting the expression of more than just the target gene [Nat Biotechnoi, 21,635 (2003)}. THEY DESIGNED eight siRNAs to target the gene called MAPK14, which codes for mitogen-activated protein kinase 1. They compared the expression profiles generated by gene silencing with the different siRNAs. Surprisingly, although the siRNAs targeted the same gene, there was little overlap in the expression profiles. The different expression profiles appear to result from the siRNAs themselves rather than from the silencing of the target gene. Diluting the siRNAs did not eliminate the off-target effects. Jackson and her colleagues found that "strandedness" (which strand was incorporated into RISC) played a role in the off-target effects. They were able to improve the specificity of siRNA for the target gene by inactivating the siRNA sense strand. However, off-target effects could not be completely eliminated, she said. Companies are chemically modifying siRNAs to make them practical as ther-
apeutics. Unmodified RNAis rapidly degraded in cells and has poor cell permeation. Modifications can protect siRNAs from degradation by nucleases, thus making them last longer, and can improve their cell permeation. Chemists have a major role to play in making RNAi practical. Muthiah Manoharan, vice president for drug discovery at Alnylam Pharmaceuticals, an RNAi company in Cambridge, Mass., told C&EN, "Although RNAi is a biological process, making it practical is a chemistry-driven process. Of course it's a biological discovery, but it is not a biotechnology." Whether for therapeutic use or target validation purposes, he added, siRNA drug molecules have to be chemically synthesized. The place for chemists to start, Manoharan said, is in optimizing RNA synthesis for large-scale drug manufacturing. "The RNA synthesis protocols were developed about 25 or 30 years ago for small-scale synthesis," he noted. "Not much has gone [into] further improvements of this chemistry to enable us to make larger quantities. We don't have
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SCIENCE & TECHNOLOGY GETTING PHYSICAL Chemists must work on the physical properties of siRNAs like this one to improve their pharmacological properties. siRNAs are 21 to 23 nucleotides long, with a molecular weight of 12,000 to 15,000. They include 40 to 42 negative charges over two turns of the helix. They are hydrophilic and heavily hydrated. These structures were generated using published coordinates [Biochemistry, 38, U784 (1999)]. and antisense drugs may not be directly applicable to RJSTAi. However, modifications simply for the sake of modification should be avoided, he said. "Some chemical modifications are extremely synthetically challenging," he told C&EN. "If you need to use complex multiple steps just to build the monomer that goes into the RNA synthesis machine, that modification may not be useful in making a practically viable drug." Manoharan also believes that modifications should be tested in animal models as early as possible. "Chemists and biologists would both know what the next step is. The chemists can go back to the bench and modify their chemistries," he said. If suitable animal models are not available, chemists could end up making molecules that work in cell culture but fail in animals and humans. "You can't just take unmodified R N A
RNAi drugs will have to last at least as long between administrations as currently available drugs. However, chemically modifying siRNA won't be useful unless the activity is maintained, Chowrira said. Sirna has actually modified some siRNAs so that they no longer contain RNA because the 2 '-hydroxyl group has been removed. " % u don't need all-RNA to get RNA interference," Chowrira told C&EN. "The RISC mechanism actually recognizes our chemically modified oligos just fine. In fact, they are as potent as allRNA. From a manufacturing point of view, the cost of goods goes down dramatically" because coupling efficiencies are better during synthesis. Sirna calls these molecules short interfering nucleic (as opposed to ribonucleic) acids.
very efficient methods available today for manufacture." Because RNA synthesis requires protecting the 2'-hydroxyl group on the ribose, the coupling efficiency between individual nucleotides is not as high as manufacturers would like. "These are some of the synthetic challenges for the SOME CHEMICAL modifications can even R N A chemist, even before making modimprove the potency of siRNAs, as the ifications, just to optimize the RNA syncompany Dharmacon, located in Lafathesis," Manoharan said. yette, Colo., is discovering. At first, the company was just looking to imFurthermore, chemists need to prove the stability of siRNA for in make the siRNA molecules into vivo applications. "We were thinksuitable drugs by chemically moding that people are going to be ifying them to improve their stalooking at RNAi as a potential bility, cellular delivery, and biodistherapeutic or working with it in tribution. "Unmodified siRNA vivo," Stephen A. Scaringe, the molecules are not suitable to be firm's cochairman of the board and drug molecules," Manoharan said. chief scientific officer, told "They don't have intrinsic favorC&EN. "What we discovered is able biodistribution properties. If that we can actually incorporate you administer siRNA intravemodifications that enhance its acnously or subcutaneously, that moltivity as well." ecule will go to the kidney and be excreted fast." Scientists at Dharmacon were surprised that they could improve on Another way that chemists are the natural process. "In general, you able to advance RNAi is by explorthink this is a process that nature has ing how the RISC complex works evolved over millions or billions of to degrade the target mRNA as a LONG LASTING Fluonsscently labeled years. W h o are we to think that we function of the chemistry of the unmodified siRNAs (top r ow) and Dharmacon's can come in with modifications and siRNA. "Better understanding of modified siRNAs (bottom row) can be compared actually make it better?" Scaringe the mechanism will improve the after 48 hours (left colunin) and after seven asked. Usually the goal with modifipotency and reduce the frequency days (right column). The modified siRNAs cations is to improve stability withof dosing," Manoharan said. "We (shown in red) are abund ant at both time out too big a decrease in activity can design better drugs if we know points, demonstrating th eir improved stability. exactly how these modifications Dharmacon launched a new line work." of siRNA molecules incorporating these modifications last week. and expect it to work for a long time," Although M a n o h a r a n advocates Scaringe declined to identify the type of chemical modifications, he also believes Bharat M. Chowrira, vice president for modifications that Dharmacon is using. that care needs to be taken in selecting legal affairs, licensing, and patent counHowever, he did note that they are not those modifications. Chemistries that sel, at Sirna Therapeutics in Boulder, "exotic" modifications. "These are modwere developed in the past for ribozymes Colo., told C&EN. To be competitive,
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ifications weVe developed in-house that are amenable to high-throughput production," he said. Companies are putting together algorithms to help select the best siRNAs. Dharmacon's algorithm includes 25 criteria that are weighted differently, including such characteristics as guanine and cytosine content (between 30 and 50% is best), thermal binding energy profiles, and the preference for certain bases at particular locations. They apply this algorithm to every 19-base sequence in the gene of interest. The algorithm generates a score between 0 and 100, with the higher scoring sequences being more likely to be active siRNAs. Every sequence must have at least four mismatches with sequences in other genes. Some siRNAs are so active t h a t Scaringe describes them as "hyperfunctional."These sequences are categorized by the extent of silencing (90% or more of the target mRNA is knocked down), the concentration of siRNA required (less than 1 nM), and the duration of the silencing. Scaringe would eventually like to see hyperfunctional siRNA molecules
that work at concentrations below 500 p M or even 100 pM. Sirna actually started out with a focus on the catalytic RNA molecules known as ribozymes, when the company was known as Ribozyme Pharmaceuticals. The company worked for 10 years to stabilize synthetic ribozymes so they wouldn't be rapidly degraded in living systems, according to Chowrira. T h e company chose to tackle difficult diseases such as hepatitis C and cancer from the outset, and funding ran out. W h e n RNAi started becoming popular about two years ago, Sirna began investigating whether it had advantages over ribozymes. "Ribozymes were not as promising as we initially thought, and RNAi turned out to be easier to use and more potent than ribozymes, ,, Chowrira told C&EN. However, the work that Sirna put into ribozymes won't be wasted, and the company will apply the lessons it learned to RNAi. "We learned a lot about how to stabilize RNA for in vivo applications, ,, Chowrira said. "We also have a lot of experience with process development and
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manufacturing of RNA in large quantities," which he said will be necessary for RNAi to succeed as a therapeutic. SIRNA HAS SELECTED age-related macular degeneration (AMD) as its first disease target. "One of the failings of ribozymes was that we chose high visibility but at the same time high-risk indications for an unproven technology," Chowrira explained. "We didn't want to make that mistake again." A M D offers a localized application in which siRNAs can be injected directly into the eye, thus separating the issues of efficacy and delivery. "We're taking it in two stages—first prove the technology in vivo in humans and then go after o t h e r indications," Chowrira told C&EN. Sirna plans to file an investigational new drug application for its AMD drug with the Food & Drug Administration late next year. Research is also under way for hepatitis C and cancer. Alnylam has not yet decided which target it wants to pursue first, Manoharan said. The company is currently considering several targets for both local and
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SCIENCE & TECHNOLOGY systemic delivery, although Manoharan declined to specify what those targets are. "We have more than one target in both areas, and we haven't narrowed it down. We have very encouraging data in both areas—local delivery and systemic delivery—but we believe that local de livery offers the near-term path to the clinic." Rossi is concentrating on developing RNAi as a t r e a t m e n t for HIV. One approach he and his colleagues are using is to take Τ cells —the im mune cells that H I V in fects—from p a t i e n t s and genetically modify them to produce short hairpin R N A that tar gets the virus. (These hairpin RNAs are processed in vivo to form siRNAs.) The cell population would be expanded in culture and then put back into the patient. The disadvantage of this approach is that Τ cells are not long-lived. Alternatively, Rossi and colleagues are genetically modifying hematopoietic progenitor cells—the cells that differ entiate into the various cells in blood— so that they produce protected Τ cells and macrophages. T h e modified cells
mine how the bone marrow cells can be safely manipulated. In big pharma, the current focus for R N A i is as a validation tool. At t h e Boston meeting, William L. Wishart of the Novartis Institutes for Biomedical Research in Basel, Switzerland, described what his company is doing with RNAi. Wishart explained how RNAi was used to inhibit the expression of a receptor known as P2X 3 , which is believed to be involved in chron ic neuropathic pain. There is nearly unan imous agreement that delivery is one of the ma jor hurdles to turning siRNA into a therapeu tic. Some companies are looking at ways to ad dress that challenge. Martin C. Woodle, president and chief scientific officer of Intradigm Corp., in Rockville, Md., said that the biggest hur dles for siRNA delivery are extracellular stability, pharmacokinetic properties, and cellular uptake. Intradigm is working on a drug delivery technology called TargeTran to address those issues. TargeTran is based on self-assembling, layered nanoparticles that consist of a core, an in
Although RNAi is a biological procesSp making it practical is a chemistrydriven process/'
TAKING AIM Fluorescently tagged siRNAs with targeting peptides are delivered to hepatocytes in mice by injecting them into the tail vein using hydrodynamic delivery (left). The siRNA (red) accumulates in the hepatocytes (H) but not in sinusoidal cells (S). siRNA with a mutant peptide does not reach the hepatocytes (right). are put in the bone marrow. "I think that's more tractable as a way of deliv ering a therapeutic than systemic deliv ery, which means you'd have to inject the body with a virus carrying these genes or the siRNAs themselves in a packaged form. There's very low uptake efficien cy when you do something like that," Rossi told C&EN. They will be working with a rhesus monkey model to deter 36
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ert coating, and targeting ligands. The exposed ligands allow binding to recep tors on the cell surface. David Lewis, a senior scientist at M i r u s , in Madison, Wis., described methods for delivering nucleic acids. Naked nucleic acids (those that are not in a polymeric complex) can be deliv ered via hydrodynamic intravascular in jection, which involves rapid injection
of large volumes. A 2-mL injection rep resents 10% of the mouse's body weight. Lewis showed that siRNA could be de livered to the liver this way and could successfully knock down expression of a target gene. Such a method is good for target validation but not for a thera peutic, Lewis said. By using this method to deliver plasmids that express siRNA, knockdown of the target gene is stable over at least 11 weeks. Mirus is also working on "recharged" particles for siRNA delivery using the bloodstream. Nucleic acid particles are usually made by condensing the nucleic acid with cationic polymers. However, such positively charged particles tend to aggregate in serum and are effective on ly for very localized delivery To make the recharged particles, polyanions are added to the condensed siRNA particle. Com pared to cationic complexes, the re charged particles have increased serum stability and decreased toxicity. ALTHOUGH RNAi is still a relatively re cent discovery, scientists and companies have great hopes for its future. They firm ly believe that it is here to stay as a re search tool and that it shows promise as a therapeutic. "I've been in the RNA field for almost my entire scientific career. I have not seen an RNA-based mechanism that can be applied to gene analysis or therapeu tics that's as powerful as RNAi," Rossi told C&EN. "The fact that it's so multifaceted, that these small RNAs are do ing so many different things in the cells, makes it even more exciting. This is something that is a real breakthrough and that needs to be looked at at all lev els, from the science to the potential therapeutic applications." Chowrira agrees. "I've been working in nucleic acids for almost two decades. I haven't come across a nucleic acid technology that is as robust and potent as siRNAs. Until siRNAs came on the scene, there was no other technology that one could use as a tool to inhibit gene expression in a consistent and re liable manner. I think that's why it has really caught on in the functional ge nomics area," he said. "Whether these become therapeutics, that's a more dif ficult question. There's no reason to think that it's not going to work, based on its track record in the functional ge nomics area. We are seeing robust ac tivity in both animals and cell culture. Whether they behave the same way in humans, we have to wait and see." • HTTP://WWW.CEN-ONLINE.ORG
