The second in a two-part series on RNAi
Carolyn Riley Chapman, PhD
Contributing Editor
RNAi therapeutics are in phase I and II clinical trials, but systemic delivery remains the primary challenge for harnessing their full potential.
In 1998, scientists discovered that RNA not only acts as a simple intermediary between genes and proteins, but that it also carries out regulatory activities of its own. The
 click to enlarge Alnylam Pharmaceuticals is developing RNAi therapeutics that can be delivered by direct application or local administration, thus minimizing the need to address the challenges posed by systemic delivery. (Source: Alnylam Pharmaceuticals) |
discovery of RNA interference (RNAi) by Fire and Mello was breathtaking for two reasons: first, researchers hadn’t known about it before, and second, it had amazing potential as a research tool and even as a new class of therapeutics. With the sequence of the human genome in hand, RNAi essentially allows scientists to knock down the activity of any protein at will by causing mRNA degradation.
Indeed, RNAi has become a standard technique for basic and drug discovery research (see July DD&D). Today, it seems probable that RNAi drugs will eventually make it to the market. While there is no question the mechanism works, “it is not yet a clinically validated technology,” cautions Paul Johnson, PhD, senior vice president of research and development and chief scientific officer at Nastech Pharmaceutical Co., Bothell, Wash.
To receive approval from the FDA, small interfering RNA (siRNA) drugs still need to demonstrate efficacy and safety in humans, even those that take advantage of local administration and delivery paradigms. But for RNAi to become a broader platform applicable to all therapeutic areas, the systemic delivery challenge, or getting siRNAs into appropriate cells within humans, must be solved.
A small group of biotech companies, both public and private, is actively pursuing the discovery and development of RNAi therapeutics, and some pharmaceutical companies have allied with them. For example, Novartis, Basel, Switzerland, and Merck, Whitehouse Station, N.J., have partnered with Alnylam Pharmaceuticals, Cambridge, Mass. And Eli Lilly, Indianapolis, Ind., and GlaxoSmithKline, London, are working with Sirna Therapeutics, San Francisco. But while there is a great deal of work going on, perhaps the biggest barrier to new entrants in the field is the intellectual property associated with RNAi technologies, an area of considerable complexity. “Usually those issues don’t get resolved until a drug comes to market,” says Steven Kriegsman, president and chief executive officer of CytRx Corp., Los Angeles.
Despite the intellectual property issues, there is now substantial progress in the development of RNAi therapeutics. There are currently three RNAi drug candidates in the clinic. One is Bevasiranib, Cand5, from Acuity Pharmaceuticals, Philadelphia, which is in phase II trials for age-related macular degeneration (AMD) and diabetic macular edema. In addition, there are Sirna-027 from Sirna, which is also for macular degeneration, and Alnylam’s ALN-RSV01 for pediatric RSV. Acuity’s Bevasiranib, which targets vascular endothelial growth factor (VEGF), was the first siRNA to enter both phase I and II clinical trials. “The challenge of being first is like being the first person in a bike race, or the first person breaking through snow if you’re hiking through snow,” says Sam Reich, co-founder and vice president of research and development at Acuity.
Targeting VEGF
Dale Pfost, PhD, Acuity’s chairman, president and chief executive officer, says his company’s approach was to use the breakthrough technology of siRNA to silence a clinically-validated target. “VEGF, the protein, is central to the angiogenesis and the leakage that causes these diseases. We have seen time and again now that an anti-
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VEGF approach is a fruitful one to pursue. So, therefore, translating that into an siRNA strategy was really quite appropriate,” says Pfost. For example, Macugen and Lucentis, drugs for AMD from OSI Pharmaceuticals, Melville, N.Y., and Genentech, San Francisco, respectively, are both VEGF antagonists. Macugen is a pegylated aptamer and Lucentis is a humanized therapeutic antibody. In that respect, it is important to recognize that siRNA drugs will compete not only with other siRNA drugs, but with many different types of therapies, including small molecules and antibodies.
So far, results with Bevasiranib have been promising. In phase I trials, more than 100 patients were exposed to the drug, and Reich points out that the approval of two phase II protocols is validation of the safety findings from phase I. But he adds that “one of the safety advantages we have is lack of systemic exposure to a potent VEGF inhibitor.” Bevasiranib is administered by intravitreal injection, and without modifications to increase its stability, it quickly degrades in serum.
Sirna, in collaboration with Allergan, Irvine, Calif., is also pursuing a siRNA drug, Sirna-027, for the treatment of AMD, and it is now in phase I clinical trials. But Roberto Guerciolini, MD, the company’s chief medical officer, expresses even more excitement about Sirna-034, “because we switch now from local administration to systemic administration.” Sirna expects to file an IND for intravenous administration of Sirna-034 for the treatment of chronic hepatitis C by the end of 2006. Guerciolini believes Sirna-034 meets the chemical stabilization, modification, and delivery challenges associated with a systemic RNAi therapy. He says Sirna-034 combines two different RNA sequences in a proprietary nanoparticle formulation that preferentially delivers RNA into hepatocytes.
One way Sirna improves the stability of its siRNA drugs is by removing ribose from the molecules so they become “siNAs” or small interfering nucleic acids instead of ribonucleic acids. Sirna-034 has been significantly modified to maintain stability in extracellular and intracellular environments, says Guerciolini, but it still retains some riboses
| Designing siRNA Therapeutics Since siRNAs are now routinely used by academic, pharmaceutical, and biotech investigators for basic and drug discovery research, it’s interesting to note that there are big differences in how companies go about designing siRNA therapeutics versus siRNA tools. “You certainly can’t just buy an off-the-shelf siRNA and expect it to be therapeutically relevant. It won’t be; it will degrade in seconds,” says Rebecca Robinson, senior director of corporate strategy, Sirna Therapeutics, San Francisco. “It’s a sexy area right now. Folks are interested in it, but it’s also a challenging one, and it takes a lot of expertise and understanding.” siRNAs, particularly ones that will be administered systemically, are modified and formulated to enhance their drug-like properties. Nagesh Mahanthappa, PhD, senior director of business development and strategy at Alnylam Therapeutics, points out fundamental distinctions between siRNA design for therapeutic use and research use. “If you’re involved in genome-wide screening, obviously you’re up against an economic barrier. You can’t make hundreds of different siRNAs for every gene in the human genome. For that reason, you’re quite reliant on algorithms that will maximize specificity and potency.” In contrast, Alnylam relies heavily on brute force, wet lab testing to empirically identify siRNAs for select therapeutic targets of interest, he says, and has not become overly tied to algorithms for siRNA design because of their inherent limitations. Mahanthappa characterizes Alnylam’s siRNA design and selection process as a funnel with attrition, pointing out that Alnylam doesn’t run all assays on all siRNAs. They start off by synthesizing a set of siRNAs that pass two major filters. First, they eliminate any sequences that are identical to sequences in off-target genes. Second, they aim to maximize sequence identity not only to the human gene target, but also to its orthologues in species used for nonclinical studies. These siRNAs are then assayed for activity against the intended target. Alnylam then tests the most active siRNAs for interferon induction by incubating them with primary blood cells and performing enzyme-linked immunosorbent assays. “If we find an siRNA that we thought was potent and useful, but it induces interferon, we eliminate it from further work,” says Mahanthappa. Other secondary assays are employed to characterize toxicity profiles. |
so it is not a complete siNA molecule. He says Sirna has also been able to chemically modify their oligonucleotides in such a way as to avoid triggering an immune response. Guerciolini says Sirna’s success with targeting Sirna-034 to hepatocytes will allow the company to evaluate a variety of endogenous targets in the liver for future systemic siRNA therapeutics. The next systemic program at Sirna may be aimed at phosphatase 1B, a major regulator of insulin signaling. “That is probably the one that will be evaluated in animal models of disease in the next few months.”
In contrast to Sirna, Alnylam is focusing its near-term efforts on developing siRNAs that can be delivered by direct application or local administration, thereby minimizing the need to address the challenges posed by systemic siRNA delivery, according to Barry Greene, the company’s chief operating officer. “We could, through inhalation, target drugs to the respiratory system; through injection, target drugs to the eyes for ocular disease; and through interthecal or infusion pump technology, target drugs to the CNS [central nervous system].” Alnylam has published on systemic delivery of cholesterol-conjugated and liposome-encapsulated siRNAs in rodents and primates, respectively, but Nagesh Mahanthappa, PhD, senior director of business development and strategy, emphasizes that “siRNA technology today is ready to go for these local administration paradigms” in humans.
Alnylam’s lead clinical candidate, ALN-RSV01, is an siRNA drug targeting the conserved N protein of the RSV genome. It has completed phase I trials in the United States and Europe. Alnylam’s goal with ALN-RSV01 is to develop a drug that will be administered directly to the lungs of a patient using a nebulizer. “siRNAs, for reasons that are not clear, when administered directly to the airway epithelium, are taken up very effectively, even in the absence of complicated formulation approaches,” Mahanthappa, says.
RNAi for flu
Based on their experience developing ALN-RSV01, Alnylam’s next program seeks to develop a drug to treat pandemic flu. Like ALN-RSV01, an RNAi therapeutic against flu would target a gene or genes required for viral replication, and would be delivered to the lungs via inhalation. “We have an opportunity to actually develop a drug targeting a sequence within a gene required for viral replication that’s consistent across flu viruses. We can develop a drug before the actual virus that becomes a pandemic emerges,” says Greene. Alnylam will partner with Novartis to move candidates to and through clinical development.
Nastech is also aiming to develop RNAi therapeutics for influenza. This February, the company announced its acquisition of the RNAi assets of Galenea Corp., Cambridge,
| Antagomirs: Antagonizing miRNA miRNAs regulate the expression of one or many messenger RNAs (mRNA) through suppression of protein translation or degradation of their target mRNAs. Human miRNAs are thought to affect the expression of many genes, playing important roles in regulating cellular proliferation and development. The mis-expression of specific miRNAs has been correlated with human diseases such as cancer. Last year, researchers at Alnylam Pharmaceuticals, Cambridge, Mass., and Rockefeller University and New York University in New York, demonstrated that they could silence miRNAs in vivo with a new class of chemically modified, cholesterol-conjugated single-stranded RNA analogues complementary to miRNAs in a Nature paper. They named the chemically-engineered oligonucleotides “antagomirs” since they antagonize miRNA. So, antagomir is to miRNA as siRNA is to mRNA. Get the picture? While there is a great deal of excitement about designing antagomir therapeutics which modulate the activity of miRNAs, Alnylam chief operating officer Barry Greene cautions that this is a long-term proposition. “The therapeutic application of microRNAs, we envision in a four- or five-year time frame. We’re taking steps now to ensure future optionality.” |
Mass., in the areas of respiratory viral infections, including influenza, rhinovirus, and other respiratory diseases. Galenea’s G00101 represents one of four lead compounds for influenza, which Johnson expects Nastech to bring to the clinic in 2007.
Johnson says their lead influenza siRNAs have a spectrum across all avian and human sequences, and are particularly insensitive to mutational change. He believes Galenea’s intellectual property relating to the use of RNAi against flu is strong, and also cites Nastech’s expertise in solving drug delivery problems for macromolecules as one of the strengths of their RNAi program. “siRNAs are not easy to deliver to the sites and cells of action and get them into cells in high enough amounts so that they have a strong therapeutic effect, and that’s our specialty,” says Johnson.
Like others, Johnson believes full clinical validation of RNAi therapeutics rests on overcoming delivery issues. Nastech is investigating several different agents that may help siRNAs be delivered to and into cells, including peptides, lipids, and lipid-peptide combinations. Like Alnylam, Nastech is initially focusing on indications that allow
 click to enlarge RNAi Molecules in Clinic |
therapeutics to be administered by inhalation through the lung or nose, but Johnson believes that siRNAs need a delivery agent to get into cells efficiently. Nastech has a “pretty major effort on the identification of small, cost-effective peptides with properties that help siRNA be delivered into cells,” he says, adding that peptides can also have a stabilizing effect on the siRNAs. Johnson also notes that studies performed at Nastech have shown that peptide delivery agents can have lower toxicity and off-target profiles as compared to lipid-based delivery agents.
Calando Pharmaceuticals, Duarte, Calif., is another siRNA company that has been built “on the delivery vehicle as opposed to the siRNA technology per se,” says John Rossi, PhD, co-founder of Calando and chair and professor of the division of molecular biology at the Beckham Research Institute of the City of Hope. Calando’s technology incorporates two components, a linear, cyclodextrin-containing polycation and an siRNA. When the two are mixed together, they self-assemble into nanoparticles that are less than 100 nanometers in diameter.
Rossi says siRNAs delivered with cyclodextrins do not activate the interferon immune response in the same way that lipid-delivered siRNAs do. Because of the way they enter and exit the cell, Rossi says, the cyclodextrin siRNAs escape toll-like receptor encounters. Another advantage is that ligands can be added to the carriers to direct tissue-specific or cell-specific uptake. Calando’s goal is to move a lead candidate into clinical trials for cancer in 2007. Earlier this year, Calando announced it will collaborate with the National Cancer Institute to develop RNAi therapeutics for pediatric neuroblastoma.
About the Author
Carolyn Riley Chapman, PhD is a freelance writer based in Harrison, N.Y.
This article was published in Drug Discovery & Development magazine: Vol. 9, No. 8, August, 2006, pp. 38-46.
Filed Under: Drug Discovery
