RNA interference (RNAi), dubbed the "breakthrough of the year" for 2002 by the journal Science, is "the most exciting insight in biology in the past decade or two," said Nobel Prize winner Phillip Sharp, Ph.D., of the Massachusetts Institute of Technology, Cambridge. Hundreds of academic laboratories have eagerly adopted RNAi as the answer to their gene knockout problems. And the scientists who performed the pioneering RNAi mechanistic studies could very well be in line for their own Nobel Prize.
The buzz over RNAi stems from the almost mystical ability of double-stranded RNA to efficiently and potently silence any gene in plants, worms, fruit flies, or (most recently) mice. In 2001, a group led by Tom Tuschl, Ph.D., at the Max Planck Institute in Germany, demonstrated that very short interfering RNAs, or "siRNAs," could silence genes containing complementary sequences in human cells. This discovery instantly made RNAi-based gene silencing practical in mammalian systems as a replacement for tedious or unreliable gene knockout techniques. The Tuschl work also immediately raised the possibility of using RNAi itself as a drug.
Cancer is one obvious application for RNAi therapeutics, because many oncogenes and cell-signaling proteins present enticing drug target possibilities. RNAi, potent and specific, couldin theoryknock out oncogenes and potentially shrink tumors. "Can it be used against cancer cells?" asked Greg Hannon, Ph.D., of Cold Spring Harbor Laboratory at last years meeting of the American Association for Cancer Research. "If it were seven oclock in the evening, we could have beers and dream up a dozen ways to do that."
In the year since Hannon made those remarks, RNAi-based cancer therapy has taken a big leap. No longer a barroom fantasy, it is now a major research effort. Companies are spending millions of dollars to develop such therapies. "Theres no reason, at this point, to say it will not work," said Reuven Agami, Ph.D., of the Netherlands Cancer Institute in Amsterdam. Ribopharma, a German biotechnology company, plans to have an RNAi cancer therapy in the clinic next year.
But the technology is so new, argue some experts, that its problems have yet to even be identified, much less solved. "There will be an inevitable cooling off period," predicted Alan Gewirtz, M.D., of the University of Pennsylvania, Philadelphia. "When people attempt to do RNAi therapy, theyre going to see that this is going to be a very difficult task."
Evolution of a Revolution
RNAi has only recently become high-profile science, but the first hints appeared back in 1990. That year Rich Jorgensen, Ph.D., then a biologist at DNA Plant Technology Corp., tried to make purple petunias more purple. Jorgensen inserted a second copy of the gene that controls production of purple pigment, but to his surprise got white petunias instead. He called this paradoxical effect "cosuppression," but no one understood why adding more of a gene turned that gene off. (It is now known that this process triggers RNAi.)
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In 1998, Carnegie Institution biochemist Andy Fire, Ph.D., and University of Massachusetts geneticist Craig Mello, Ph.D., solved the mystery. Injecting both sense and antisense RNA into worms, they got an astonishingly potent silencing effect when the two strands mixed. After demonstrating that double-stranded RNA was the real silencing agent, Fire and Mello coined the term "RNA interference," and a new field was born. It has since been shown that RNAi works by causing an enzyme complex to chop up the homologous messenger RNA. This effectively silences the gene.
The Tuschl group, by extending the technique to mammalian cells, made RNAi what it is today: a laboratory necessity. "Nowadays, its ... pretty difficult to find a pharmaceutical company or a biotech company thats not using RNAi in mammalian cells," said University of Cambridge cell biologist David Glover, Ph.D.
The impact on cancer research has already been dramatic. "Anybody thats in cancer research thats working in the basic cell biology of oncogenesis [and] working on a specific gene, Im sure theyre considering RNA interference," said University of Michigan pathologist Arul Chinnaiyan, M.D., Ph.D. In what has become routine practice, Chinnaiyan last year used siRNA to silence a genein Chinnaiyans case, a candidate marker gene in a prostate cancer cell lineto see if the cells stopped growing. (They did.) "You can just basically target any gene you want and see the effect it has in vitro," said Chinnaiyan.
Not everyone has the same luck getting siRNA to work, cautioned Gewirtz. "Its no more or less amazing than any of these other hybridization-based techniques," he said. Still, he acknowledged, "a lot more people have been successful silencing genes and getting knockdowns with siRNA than they have been with other approaches."
RNA interference also makes genome-wide knockouts in human cells practical for the first time. Cancer Research U.K., for example, in February launched such a project, aimed initially at permanently knocking out single genes, and eventually the entire genome, in 10,000 otherwise identical cell lines. "Thats something thats been lacking in mammalian tissue culture until now," said Glover. "It really opens up a whole new avenue of carrying out genetics, to understand all kinds of problems relating to cell biology."
Silencing Oncogenes
But a big question remains: Can RNAi itself be a drug? Last year two groups demonstrated that siRNA worked in mice to silence reporter genes, and in February, Harvard Universitys Judy Lieberman, M.D., Ph.D., reported (in Nature Medicine) using siRNA to silence the Fas gene in mice, protecting them from liver failure in two models of autoimmune hepatitis. All these groups, however, delivered siRNAs to the mouse via high-pressure injection through the tail veina brute force method unsuitable for humans.
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Alnylam is the best-pedigreed RNA therapeutics company to date. Co-founded last year by Sharp, Tuschl, and two others, it has raised more than $17 million and is targeting both HIV and cancer with RNAi. In cancer, "were thinking both solid tumors and hematological malignancies," said Mahanthappa. Particularly attractive potential targets, he said, are cancers arising out of chromosomal translocations, because the resulting fusion proteins can be targeted with siRNAs without, in theory, affecting the wild-type proteins. Proof-of-concept work in mouse xenograft models will be the first step.
Companies such as Alnylam must decide whether to inject structurally modified siRNAs directly into patients, or to use a gene therapy approach to deliver a DNA construct designed to express siRNA once inside the cell. Agami, who invented one such siRNA expression vector, favors the gene therapy approach. Last year, in the journal Cell, he reported using this vector to knock out oncogenic K-ras in human pancreatic cancer cells while leaving normal ras intact. When the modified cells were injected into mice, they did not grow or form tumors. Now Agami is trying his system in mouse xenograft models, delivering his siRNA-coding DNA with retroviral vectors and hoping to shrink the tumors. "Whether this will happen or not, we dont know, of course," he said. "We are waiting for results in the next few months."
Gene therapys great advantage is DNA integration with long-term expression. The disadvantage is unexpected effects, causing the death of Jesse Gelsinger 3 years ago at the University of Pennsylvania and, more recently, two cases of leukemia in children with severe combined immunodeficiency treated with gene therapy in France.
"We want to stay away from the baggage associated with the gene therapy approaches," said Alnylams Mahanthappa. Instead, he is looking at making structural modifications to siRNA, similar or identical to what has already been done for antisense RNA, to give the RNA stability and to aid its entry into cells. Alnylam will also try lipid or polymer "carriers" to protect the RNAs passage through the bloodstream.
Minefields Ahead
Why should this approach work better for siRNA than it has for antisense RNA, which has been a disappointment in the clinic? Because "siRNAs are just much, much more potent than ribozymes and antisense constructsorders of magnitude more potent," argued Mahanthappa. Glover agrees. RNAi "is a much more amenable and robust technique," he said.
But Gewirtz said he thinks that the delivery problem will be hard to solve, especially for solid tumors. Agami and Mahanthappa admitted that, even with efficient delivery, it will not be possible to get siRNA into all cancer cells, but they believe that combining RNAi with other cancer therapies will ultimately prove effective.
Combination therapy should also address the issue of "escape mutants," cancer cells that evade the effects of gene silencing through genetic instability. "Cocktails" of siRNAs targeting different gene segments of a single gene, say Agami and Mahanthappa, are another possible solution.
Even if these problems are solved, still other issues loom. There is potential for siRNA to unintentionally harness the bodys natural "microRNA" gene regulatory system, blocking translation of the wrong proteins; theres a risk of activating innate immunity, because siRNAs may interact with Toll-like receptors, powerful immune system triggers; and siRNA may trigger chromatin remodeling, with unpredictable effects on gene expression.
Alnylam is already plotting solutions. Mahanthappa said that the specificity problem can be avoided by applying informatics filters, and innate immune system activation can be assayed for and avoided, or engineered around. As for siRNA-induced chromatin remodelingif it happens in mammals at allkeeping the RNA out of the cell nucleus is one solution, he said.
Despite the rapturous reception RNAi has enjoyed to date, Gewirtz warned that other unexpected problems will inevitably arise. The delivery problem alone, he stressed, remains a major obstacle to getting siRNA to work in people. RNA interference "could turn out to be the most unbelievable thing that ever happened in terms of targeted therapeutics," Gewirtz said. "[But] it would be good ... to inject just a mild cautionary note. And to at least have people go into this realistically. There are fundamental issues that have to be solved."
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