The discovery of RNA interference (RNAi), the endogenous regulatory system that employs small double-stranded RNA molecules to silence gene expression, has generated enthusiasm for using small interfering RNAbased molecules (siRNA) to treat a variety of diseases. It didn't take long for investigators to dream up several strategies for siRNA to target cancer. And recent studies offer tantalizing clues that RNAi is not only a potential therapeutic measureit can also act as an intrinsic part of the oncogenic process.
Some of the enthusiasm surrounding RNAi is based on the assumption that since RNAi taps into a fundamental regulatory process, it should be well tolerated and generate few side effects.
"RNAi works by tapping into a natural process that has evolved to regulate gene expression," said Cold Spring Harbor Laboratory's Greg Hannon, Ph.D., a pioneer in RNAi. "The cells use this already; all we are doing is altering the programming. We are not trying to force a cell to do something that it normally doesn't do. With RNAi you have an entire machinery that has evolved to find complementary targets. ... I think that in the end we are going to find that tapping into this natural mechanism is going to be a lot more effective [than other types of nucleic acidbased therapy]."
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"We now have 75% or 80% of the equation for making a pretty good siRNA," said Hannon. "The more natural your trigger becomes, the more effectively it is incorporated in the RISC complex."
Challenges for RNAi-based Drugs
But despite all that has been learned about the RNAi pathway, obstacles to effective RNAi-based drugs remain. "I think you are really looking at different delivery modalities for different diseases," Hannon said. "But there are things that all of these strategies share, which is the problem of getting high-efficiency delivery of nucleic acid. This is a problem that people have been working on for a long time."
The body has effective strategies for eliminating foreign nucleic acids, particularly double-stranded RNA, which cells associate with viral pathogens. Reports of nonspecific gene activation and induction of the innate immune system have somewhat tempered enthusiasm for RNAi-based drugs, but as investigators learn more about what triggers an immune response (in general, double-stranded RNA longer than 30 base pairs), they have begun designing siRNA to reduce an immune reaction.
"I think if you can get the appropriate ligands onto a carrier, you can get into compartments of the body where naked RNA just isn't going to do it," said Rossi. But he also points out that relatively little is known about how RNA is taken up by the cell.
Rossi agrees that it should be possible to design an siRNA that will avoid off-target effects, but he says that the side effect to watch out for, "which not everyone is talking about, is the perturbation of the microRNA (miRNA) pathway. To me that may be more of a problem than some of these off-targeting effects. By throwing in large amounts of siRNA in the cell, those siRNAs are going to be competing for the proteins that the microRNAs are ordinarily using. ... In a long-term clinical setting this could be very bad," he said.
Still, Rossi and his collaborator Mark Davis, Ph.D., of the California Institute of Technology, were confident enough in RNAi's potential to form a biotechnology company, Calando Inc., which has leased Davis' nanoparticle technology to target several diseases (see sidebar, p. 627).
Link to Cancer
While researchers in several disciplines figure out the best way to harness RNAi for treatments, a recently published study was the first to show a direct link between miRNA and cancer and is likely to set off a flurry of interest in the role RNAi plays in the oncogenic process and its potential to stop or perhaps reverse that process. In the March 11 issue of Cell, Frank Slack, Ph.D., and his colleagues at Yale University and Ambion Inc., an Austin, Texas, supplier of tools for RNAi, showed that an miRNA called lethal-7 (let-7) can act as a tumor suppressor that directly regulates the RAS oncogene.
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Slack and his colleagues combined studies in C. elegans with an examination of human lung tumor samples and used bioinformatics to show that human RAS genes contain let-7complementary sites, regions in the 3' untranslated region of the RAS mRNA that serve as targets for miRNA regulation. The study demonstrated that let-7 downregulates RAS in C. elegans and that in human lung cancer let-7 levels are inversely correlated with RAS levels.
"The same RAS mutations that cause cancer in humans also cause vulval tumors in C. elegans," said Slack. "If you give those worms that have those vulval tumors extra let-7 you can repress those tumors; you can cure those tumors. So that gives us some hope that you should be able to do the same in mouse and eventually humans. But the real test is going to be can we get it into humans and do it safely."
Some of the answers about safety of RNAi-based therapeutics could come quickly, as the first human clinical trials wrap up later this year. In October, Acuity Pharmaceuticals began testing an siRNA that targets vascular endothelial growth factor (VEGF) mRNA for age-related macular degeneration. VEGF promotes the blood vessel growth that leads to vision loss. The siRNA-containing solution is injected directly into the vitreous humor in the eye, in the hopes that the localized delivery will keep toxicity low.
A better test of RNAi's safety could come later this year, as investigators try more systemic approaches. Rossi and his colleagues at the City of Hope are collaborating with the Australia-based RNAi company Benitec Ltd. to treat AIDS-related lymphoma with siRNA. Rossi has developed a lentivirus vector that directs production of siRNA against human immunodeficiency virus (HIV). The vector will be inserted into patients' own blood stem cells, which will then be reintroduced by bone marrow transplantation. A second trial, funded by the National Institutes of Health, will attempt to halt HIV replication in patients who have not responded to antiretroviral therapy. It will use a similar lentiviral vector to transfect T cells with anti-HIV siRNA in the hope of stopping HIV replication in those cells. Rossi says he is planning to seek FDA approval for both trials later this year.
"One of the things we are learning about cancer is that it may be a stem cell disease," said Rossi. "Well, microRNAs are very involved in regulating early development. I think understanding the natural microRNA regulatory networks is in my opinion going to be one of the more exciting forms of anticancer therapy in the future."
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