© 2003, Elsevier Science
Small interfering RNAs (siRNAs) may be synthesized and then transfected into cells (A), or generated by the RNAase activity of Dicer (B) on short hairpin RNAs (shRNAs) transcribed in vivo (C). Transfection (D) and integration (E) of plasmid DNA containing a selectable marker and a promoter to drive shRNA expression are used to generate a stable cell line. Alternatively, DNA encoding shRNA may be introduced by viral-mediated transduction (D). siRNAs (21–22 nucleotides dsRNAs) bind to one or more proteins (F) to form the RNA induced silencing complex, RISC, which targets complementary mRNA for degradation. (From
Drug resistance stops cancer treatments in their tracks between 20% and 50% of the time. In hopes of defeating resistance, researchers have begun employing RNA interference. As various mutations accumulate during cancer's uncontrolled growth, many cells become resistant through overexpression of one protein, Pglycoprotein, which ferries drugs out of cells. Other cancer-drug targets become mutated or overexpressed to win out over therapeutics. Recent reports of RNAi used to stop or stall such overproduction and reverse malignant cell's multidrug resistance have added to the heap of applications for the technology.
RNAi's popularity has spread quickly through research laboratories in the past six years, and Big Pharma has taken notice and has started to seek approval for human clinical trials. The technique has generated excitement because it may be the most powerful, accurately targeted, and inexpensive means to shutting down specific genes. "It's the biggest advance since the discovery of recombinant DNA," says Bryan R. Cullen, professor of molecular genetics and microbiology at Duke University Medical Center in Durham, NC. But when it comes to medical applications, cancer included, considerable obstacles remain, especially an old problem: delivery. "I think it's going to be like gene therapy in some ways. We'll see very slow, incremental progress," Cullen adds.
A DIFFERENT TASK
Hitting worms or cells in a culture dish with RNA or DNA is quite a different task than injecting it systemically and hoping to affect translation at specific cells. The delivery problem has nearly undone previous technologies such as gene therapy and antisense.1 But RNAi seems to have some advantages over both techniques, including stronger gene-silencing effects and greater ease of use, some researchers say.
Injected into cells or whole organisms such as roundworms, double-stranded RNAs are cut by the Dicer enzyme into pieces 21 to 25 nucleotides long. Then an enzyme complex uses these small interfering RNAs (siRNAs) as templates to find and cleave complementary messenger RNAs. Through multiple organisms, researchers have used the technique to shut down various genes.
Several studies have examined RNAi in cancer. Most have involved forms of leukemia because of its accessibility, but researchers say RNAi could have wider application because they involve genes common to many cancers. P-glycoprotein causes multidrug resistance in not only in leukemia but also breast, lymphatic, colon, prostate, renal, and liver cancers, says Jin-Ming Yang, associate professor of pharmacology at Robert Wood Johnson Medical School, Piscataway, NJ. "Combining RNA-based agents with chemotherapy should be an effective approach," he says.
A team at Imperial College London studied chronic myelogenous leukemia cells exhibiting multidrug resistance. Plasmids designed to generate siRNAs that target the gene for P-glycoprotein were introduced into the cancer cells. This "almost completely to stop or stall such overproduction and reverse malig nant cell's multidrug resistance have added to the abolished" the protein's expression and restored the cells' drug sensitivity, the researchers reported.2
In other approaches to cancer, researchers target fusion proteins. Gene fusions occur when the DNA from two genes accidentally join, which can create abnormal proteins whose downstream effects can influence cell cycle or apoptosis. Fusion proteins have been implicated in certain solid tumors and in leukemias, including 95% of chronic myeloid leukemia cases. Some researchers are trying to block fusion protein production using siRNAs with base sequences matching common fusion sites. Harvard Medical School's Jing Chen and colleagues used siRNAs in hematopoietic cells with a common leukemia-associated tyrosine kinase fusion called TEL-PDGFβR. Although effective drugs targeting the abnormal protein exist, including imatinib (Gleevec), cells can develop resistance by increasing expression or mutating the proteins slightly.
Chen and colleagues found that RNAi cut expression of the abnormal protein by 90%, helping to resensitize drug-resistant cells and reduce the cells' malignant tendencies when injected in mice.3 Chen says his team plans further animal studies. "The technology is getting more and more mature," he says.
Another technique involves a newly identified RNAi pathway. Some siRNAs may be able to silence genes by methylating their promoters. University of Tokyo researchers reported that by exploiting this pathway they reduced mRNA levels of an oncogene,
Although delivering siRNAs into living organisms is complicated, researchers have engineered ways to at least get them into cultured cells. Most methods fall into one of two categories: introducing synthetic siRNAs into cells directly, or introducing a plasmid or virus encoding a gene sequence leading to production of appropriate siRNAs. The latter technique, versions of which Imperial College and Harvard Medical School teams have used, gives longer-lasting results because it makes cells produce siRNAs themselves. But it's unclear whether it would work in humans. And, the chill over using viral vectors remains due to setbacks in gene therapy trials. The Imperial College study is an important "proof of principle," says Chris Higgins, professor at Imperial College and a coauthor, but "is not going to be directly applicable to the clinic."
Despite the downsides, researchers cite reasons for optimism. Unlike gene therapy, RNAi might not require viral vectors or other exotic delivery methods; siRNAs can be introduced directly into tissues, though this entails its own hurdles. The body quickly degrades many siRNAs, and they must be chemically modified to enhance stability.
Companies are working on such modifications, says David Corey, professor of pharmacology at University of Texas Southwestern Medical Center at Dallas and consultant to Isis Pharmaceuticals in Carlsbad, Calif. They are finding the job easier thanks to an "enormously rich" repository of knowledge developed previously for delivering antisense RNA into cells. Researchers "can go and immediately apply [this experience] to siRNA," he says. "That's why I think we can expect rapid progress in this area in the next year." Businesses are investigating siRNA alterations, Corey explains, such as addition of phosphorothioate linkages (in which sulfur replaces an oxygen in the phosphate groups) and changes to the molecule's sugar moieties.
Researchers have also found other advantages of RNAi. It produces stronger gene expression inhibition than that found in antisense,1 and has gained greater acceptance because of its ease of use. Duplex RNA is more stable than single-stranded anti-sense molecules, says Corey, and requires less chemical tweaking to function, thus reducing toxicity.
But the need for progress on delivery strategies, Higgins remarks, is reflected in the fact that most studies of RNAi in cancer involve leukemia, which produces no solid tumors. "Getting expression of the RNAi molecule in the center of solid tumors is probably going to be pretty tricky," he says. "Maybe [clinicians] will still have to use surgery to get rid of the bulk, and RNAi could help clear up the residues."
HITTING THE MAINSTREAM
Though daunting, in vivo use of RNAi isn't without precedent; investigations are ongoing for several noncancer diseases. Encouraging results with RNAi for animal models of macular degeneration have prompted both Philadelphia-based Acuity and Boulder, Colo.-based Sirna Therapeutics to announce plans to commence Phase I trials this year. "We're coming pretty close to the first time siRNAs are going to be put into people," says Nassim Usman, Sirna's senior vice president and CEO. Sam Reich, Acuity's senior director of research and development, says its macular degeneration therapies target the VEGF family of angiogenesis proteins, which are also key cancer targets. "We've had a lot of interest and offers for our cancer portfolio from a number of firms," he says. Researchers have also reported successes using siRNA in mice to suppress hepatitis infections, and even polyglutamine-induced neurodegeneration.5
A CANCER GENE, QUIETED:
© 2004 Nature Publishing Group
University of Tokyo researchers used a tRNAVal expression system to produce short hairpin RNAs (shRNAs) targeted to 5 CpG islands in the
Liver ailments are sensible targets because "the liver is the body's garbage can," Cullen says. "siRNAs sent to bloodstream accumulate in liver." The polyglutamine work shows that brain is also a feasible target, says University of Iowa Professor and Sirna consultant Beverly Davidson. Before human studies begin, she says, "We need to confirm there are no untoward effects of RNAi in mammalian brain. The early signs are promising; the mice developed no overt signs of diseases."
All the work should be interpreted cautiously, Corey warns. "The history of the antisense field teaches us it's very easy to be deceived with results. When you put any molecule into a cell or animal, it's possible to get an effect on gene expression. That effect may have nothing to do with recognition of a specific gene." It took years to develop appropriate standards to avoid these errors for antisense, and researchers should adopt these for RNAi, he says. "The burden of proof is really on the experimenter."