RNAi Solution Knocks Down Herpes Infection

Microbicide Effective in Animal Study, Next Target HIV

Ever since RNA interference hit the scene a few years ago as a way to selectively turn off gene expression, researchers have been investigating whether these small but powerful bits of RNA can be used as therapies. The problem has been delivery—how to get the RNA into the cell where it can silence gene expression. A study in the Nov. 23 Nature showcases a novel therapeutic approach for RNAi—as a vaginal microbicide that can reduce genital herpesvirus infection in mice. The result of a collaboration between the labs of Judy Lieberman and David Knipe, the study shows that small interfering RNAs (siRNAs) can be readily taken up by mucosal surfaces of the body and reduce viral infection.

Photo by Graham Ramsay

Deborah Palliser was surprised at how easily RNAi could be delivered as a vaginal microbicide. “It was taken up all through the tissue,” she said. “Nobody knows how that happens.”

Lieberman, HMS professor of pediatrics at the CBR Institute for Biomedical Research, has been working with RNAi as a potential therapy for HIV infection. She suspected that siRNAs might be delivered more easily to the vaginal surface than to other tissues as part of a microbicide for HIV. Deborah Palliser, HMS instructor in pediatrics at Children’s Hospital Boston and CBR, began working with Lieberman to investigate the usefulness of an siRNA-based microbicide. First they tested the concept using a mouse that expresses green fluorescent protein (GFP) in each of its cells. When Palliser treated the mice intravaginally with GFP siRNA, the treatment turned off GFP at the surface of the genital tissue and even into the supportive stromal layer beneath the vaginal lining. “What we found very surprising is the degree or the depth to which it was taken up,” Palliser said.

Ready Model
Though their ultimate goal is to target HIV, Palliser and Lieberman decided to test the approach on herpesvirus-2 (HSV-2) infection, which can be studied more easily in mice. They teamed up with Knipe, the Higgins professor of microbiology and molecular genetics at HMS, and Qing-Yin Wang, a postdoctoral fellow in his lab, who had already developed a mouse model of herpes infection. Knipe identified viral genes that are necessary for the virus to replicate in cells and that would make good targets for siRNA silencing. After testing the effects of the siRNAs in cultured cells, the team chose two siRNAs to test in vivo, one coding for a protein on the virus’s outer envelope and the other an essential DNA-binding protein.

The mouse model of herpes mimics the clinical symptoms of human disease, but unlike in most humans, the infection is lethal in mice. The team delivered the microbicide just before and just after challenging the mice with HSV-2 at two times the dose that would normally cause half the mice to die. In mice that were treated with the siRNA for the DNA-binding protein, “we found that about 75 percent of the control mice died, but about 75 percent of the treated mice survived,” Lieberman said. The siRNA for the viral envelope was less effective, with 60 percent surviving. The team also found that if they combined the two siRNAs, they could promote survival even if they administered the treatment a few hours after the virus.

The study was “a merger of two areas of expertise,” Knipe said. “It was an exciting opportunity to test the potential of siRNA in a viral infection in vivo.” He said that while this first microbicide was not as effective in mice as experimental herpes vaccines that he has tested, “in some ways it’s complementary to vaccine approaches,” because it could be used at the time of exposure, whereas vaccines take time to build immunity. Presumably, the siRNA works by silencing viral genes in infected cells at a very early stage of infection, but Knipe said that further research is needed to clarify the exact mechanism.

RNA Uptake
Phillip Sharp, institute professor at MIT, said, “The results are quite striking, particularly the effective uptake of siRNAs by the mucosal layer.” Lieberman’s lab has been working to develop tricks for getting siRNA into cells, including an approach using antibodies (see Focus, June 10, 2005). “Most people believe the main therapeutic obstacle for using RNA interference is how to get it into the cells where you want it to work,” Lieberman said. In this case, the researchers simply delivered siRNAs within a lipid-based solution that integrates with the lipids at cell membranes. Its performance bodes well for other therapies that deliver siRNAs to mucosal surfaces of the body. While there are many potential applications for this approach, Sharp said, “there would have to be a lot of clinical development before the potential could be turned into fact.” A practical product would need to be effective, be retained in the vagina, and be inexpensive to produce.

One of the study’s surprises is that the siRNA silencing seems to persist in the cells for more than a week. “That’s an attractive feature if that assumption can be validated,” said John Moore, professor of microbiology and immunology at Weill Medical College at Cornell University, who has worked on microbicides for HIV. A microbicide would normally be used at every sexual encounter, but a treatment that persists for longer might allow a grace period or only require weekly applications.

Palliser and Lieberman will be moving forward to test this concept on HIV infection in primates. HIV mutates much more rapidly than HSV-2, but Palliser said they hope to get around that hurdle by creating a cocktail of different siRNAs, to identify viral genes that are highly conserved and less likely to mutate, and to target a host gene needed for sexual transmission of HIV.

After languishing on the margins of HIV therapy development for years, microbicides are now receiving more attention and funding. Many people hope that an effective microbicide for HIV could help protect women who are unable to insist that their partners wear condoms. Although the current push is toward devising HIV treatments, Lieberman said it would be ideal to develop a therapy that simultaneously works against other sexually transmitted viruses like HSV-2 and human papillomavirus.

Still, Moore said, a microbicide is not yet a reality but “an experimental concept,” and those in clinical trials now are relatively nonspecific and expected to show little benefit. Nevertheless, several labs are pursuing the next generation of strategies, which are more sophisticated and have a better chance of realizing a microbicide for HIV that is clinically effective.

—Courtney Humphries

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