Those pursuing the development of drugs through RNAi have largely focused their efforts on a small number of disease areas, including hepatitis, macular degeneration, and cancer. One group of scientists at Harvard, how- ever, is setting its sights on HIV, and finding out that the problem of delivery extends into all areas of research.
Heading up much of this work is Judy Lieberman, a senior investigator with Harvard’s Center for Blood Research and an associate professor of pediatrics at Harvard Medical School. Lieberman started out primarily as an AIDS researcher, she told RNAi News. But, in the wake of Tom Tuschl’s success with siRNAs in mammals, MIT’s Carl Novina approached her about using the gene silencing technology, and she incorporated RNAi into her work.
Since then, she has published several papers on the topic, including one in Nature Medicine last July demonstrating that siRNAs inhibit the production of HIV by targeting the mRNAs for either the HIV-1 cellular receptor CD-4, the viral structural Gag protein p24, or green fluorescence protein substituted for the Nef regulatory protein. (This paper was written with colleagues including Harvard’s Premlata Shankar and MIT’s Phil Sharp.)
Lieberman, Shankar, Sharp, and colleagues followed up this article with a paper in the Journal of Virology this July, which showed that long-term suppression of HIV replication in macrophages (an important reservoir for HIV) was possible using siRNAs against CCR5, a major HIV-1 co-receptor, and Gag.
This March, Nature Medicine published an article in which Lieberman and her co-authors reported that intravenous injection of Fas siRNA specifically reduced Fas mRNA levels and expression of Fas protein in mouse hepatocytes. This effect — which lasted without diminution for 10 days — protected mice from liver failure and fibrosis in two models of autoimmune hepatitis.
The gist of these papers is basically that siRNAs effectively silence target genes in vivo and can specifically be used to fight HIV. But while getting a desired biological response, or halting a particular response, is an important piece of the RNAi-as-therapeutics puzzle, the need for effective delivery is once again central to solving the puzzle altogether.
“In my mind, delivery is the real problem for siRNA and that’s really where our focus is right now,” Lieberman said.
Lieberman and colleagues have experimented with both viral and non-viral delivery of siRNAs, she said. And while “we have lentiviruses that … work ... I want to avoid viral delivery because it opens up a hornet’s nest of problems that all gene therapy will encounter in terms of where the thing integrates, possibilities of recombination with endogenous retroviral sequences, possibilities of integrating in a transforming mode.
“For HIV … the recombination problem could be even more serious because you have all this integrated HIV already there.”
Lieberman said that part of her lab’s current work involves investigating “whether you really need hydrodynamic injection to get good transfection in vivo.” Perhaps surprisingly, she added, “the answer is: maybe not.”
“[In this effort] we’re using the Fas model because it’s a very nice readout for whether something is working or not,” she said, noting that “we also have another model we’re working on” but declining to provide specifics given that data from these ongoing experiments is still unpublished.
“Hydrodynamic delivery is incredibly difficult even to perform in mice,” she said, let alone in humans, where delivery of such a relatively large volume of siRNA intravenously would be costly and probably dangerous. A more viable alternative might be to use regional catheterization to locally deliver a high volume of siRNA to a particular organ, she said.
In a much earlier-stage project, the Lieberman lab is also working to develop a topical RNAi-based microbicide for HIV prophylaxis.
“My notion is that the delivery problems will be less difficult to deal with for local situations where you want to treat a local area, say, the vagina to prevent sexual transmission of HIV,” she said. Aside from harnessing the highly specific knockdown effects of RNAi, such a drug would benefit from the technology’s duration.
“What’s very attractive to me is that because the silencing lasts for weeks, you might be able to make a product that wouldn’t have to be taken before every time you had sex, which is the main barrier for a microbicide; people don’t reliably take it in advance of having sex and therefore they have unpro- tected sex.”
Beyond HIV, Liberman’s lab is also working on more general RNAi problems.
“We’re also looking at more basic science questions, like ‘how does virus infection interact with RNA interference,” and recently secured a $12 million grant from the National Institute of Allergy and Infectious Diseases to study how RNAi might be used to combat bioterror (see RNAi News, 9/10/03).
Additionally, the researchers are helping Sirna Therapeutics figure out how to develop its RNA interference-based drugs under a sponsored research arrangement.
“We’re looking at stabilization, as well as delviery ... using the animal models that we have” under the Sirna deal, Lieberman said, adding that, despite Sirna’s focus on stabalization, just keeping siRNAs in circulation appears to be the real challenge.
According to Sirna CSO Nassim Usman, the company forged its partnership with Lieberman in June. Under the arrangement, he said, Lieberman is conducting research on a number of undisclosed targets for the company.