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GENETICS RNA Interference Cuts Hepatitis Down to Size First Animal Study to Demonstrate the Technique's Therapeutic Punch For the first time in an animal model, researchers have shown the therapeutic possibilities of the powerful gene-silencing technique RNA interference. Premlata Shankar, Sang-Kyung Lee, Erwei Song, and Judy Lieberman (clockwise from left) and colleagues used RNA interference to quash expression of a gene that mediates cell death, thereby treating or preventing liver failure in two mouse autoimmune models of hepatitis. (Photo by Steve Gilbert) In the March Nature Medicine (online Feb. 10), researchers report that small pieces of RNA can prevent liver injury and death in mice with chronic and severe autoimmune hepatitis. "This is the paper everyone has been waiting for," said Phillip Zamore, associate professor of biochemistry and molecular pharmacology at the University of Massachusetts Medical School. "It is really the first paper that says clinicians should be thinking about siRNAs [small interfering RNAs] and hoping that they will prove to be suitable for use in humans in some form." "This opens up so many avenues if it really works, and it seems to be working. It takes the whole siRNA field one step further." --Premlata Shankar In the mice, the results were startling. In the acute model, "all the mice died in the control groups, and 80 percent of the treated mice lived," said Judy Lieberman, HMS associate professor of pediatrics at the Center for Blood Research. RNA interference is a field that took off with the 1998 discovery that post-translational RNA gene-silencing is triggered by double-stranded RNA. Mechanistic details are still fuzzy and may vary, but RNA interference severely reduces the level of messenger RNA and markedly reduces gene expression. In only five years, research on siRNA has progressed from plants to worms and fruit flies to human cells and mice. Interfering with HIV Lieberman and her colleagues became involved in the fast-moving field of RNA interference about a year ago, when MIT postdoctoral fellow Carl Novina approached them for their expertise with the AIDS virus. In a project led by senior author Premlata Shankar, HMS assistant professor of pediatrics at Children's Hospital and the Center for Blood Research, the collaborators put siRNAs into human cells to inhibit HIV-1 sequences and cellular RNAs involved in viral infection. Those results were reported last year in Nature Medicine (see Focus, June 21, 2002) and followed up with preliminary results at more clinically relevant targets at this year's retrovirus meeting in Boston (see sidebar). Meanwhile, postdoctoral fellows Erwei Song, a liver surgeon from China, and Sang-Kyung Lee from South Korea became interested in observations from other labs that siRNAs seemed to accumulate mostly in the liver when delivered through a high-pressure injection, pumping the liquid equivalent of 10 percent of blood volume into mice. Small RNAs Protect Macrophages Against HIV In cell cultures of macrophages from healthy people, small RNAs provided a high, sustained protection against AIDS virus infection. The new studies build on a proof-of-principle paper published in the June 2002 Nature Medicine. In the earlier work, small RNAs targeted to the CD4 receptor and the HIV-1 gene sequence inhibited viral infection in cell lines and T cells for a short time (see Research Briefs, Focus, June 21, 2002). Despite the promising results, CD4's importance for a range of immune functions means it is not a good therapeutic target. Also, the antiviral effect is short-lived in dividing cells because of dilution of the small RNAs over time. Macrophages, on the other hand, do not divide and are an important reservoir of HIV that stubbornly resists antiretroviral drugs, said senior author Premlata Shankar, of the HMS Division of AIDS and an HMS assistant professor of pediatrics at Children's Hospital and the Center for Blood Research. She and her colleagues targeted the HIV co-receptor CCR5 in macrophages because the 1 percent of the Caucasian population missing the CCR5 gene have relatively normal immune functions and are relatively more resistant to HIV infection. Nonetheless, the researchers were surprised by how well small RNAs prevented infection of macrophages and also prevented viral replication in infected macrophages. The effect lasted as long as 20 days, the length of the experiments. A main stumbling block remains finding a way to deliver the genes safely. The work was presented at the 10th Congress on Retroviruses and Opportunistic Infections in Boston on Feb. 11 by Shankar and Judy Lieberman, a member of the AIDS Division and an HMS associate professor of pediatrics at the Center for Blood Research. The project was led by postdoctoral fellows Erwei Song and Sang-Kyung Lee. "Viral hepatitis is pervasive in both their countries. About 10 to 15 percent of the population chronically infected or carriers of hepatitis B, Song said, and almost all of the end-stage liver diseases in China are due to HBV infection." Apoptosis, accompanied by inflammation, is believed to play an important role in liver damage and recovery, no matter what the cause--viruses, autoimmunity, alcohol, or transplant rejection. To test the effect of siRNA on hepatic apoptosis, the postdocs teamed up with researchers at Song's alma mater, the Guangzhou Medical Institute and affiliated Sun-Yat-Sen Memorial Hospital. Lee and Song began their work online with the mouse and human genome databases. They manually worked through the target gene to find 21-nucleotide-long pieces specific only to that gene in order to find and design several potential siRNAs, some of which worked while others did not. "One base-pair mismatch abrogates the whole silencing effect," Song said. A New Target They spent two months unsuccessfully trying to knock down caspase 3. Then Lieberman suggested targeting Fas, upstream of the caspase apoptosis pathway and highly expressed in liver cells. Soon after, they sent a set of redesigned siRNAs to China for the animal experiments. Shortly, surprising observations and data began coming back in daily e-mails. In a chronic autoimmune hepatitis model induced by injecting concanavalin A weekly for 6 weeks, liver fibrosis was prevented even when Fas-siRNA injections were deferred until after the second and fourth injections. Treatment of an ongoing inflammatory process is a more relevant clinical scenario than treatment before a noxious insult has been delivered. In a more aggressive hepatitis model, 40 mice died, most within 24 hours of being injected with a Fas-specific antibody. But 33 of 40 mice pretreated with three of the most effective siRNA sequences (which had silenced Fas expression by 81 to 86 percent in preliminary tests) survived for the 10 days of observation. Fatalities in the treated group were from hemorrhage caused by liver failure. Control experiments used other related and nonspecific siRNAs and saline. "I was very skeptical when we started," Shankar said. "This opens up so many avenues if it really works, and it seems to be working. It takes the whole siRNA field one step further. People have been focused on a vector-driven delivery. But in hepatitis, you don't want stable RNA; you want it to go there, do its job, and go away." In this study, siRNA worked like a drug, not a gene therapy, with transient gene silencing. Long-term suppression of Fas, however, can cause cancer and other problems. Another concern is whether siRNA delivered by viral vectors can be passed through the germ line, as one mouse study reported. Lieberman and her colleagues are working on more clinically relevant delivery methods for siRNAs. Ultimately, their long-term goal is to develop siRNAs as a weapon in the fight against AIDS. --Carol Cruzan Morton