American Foundation for AIDS Research, February 2003
Gretchen Schmelz Armstrong

Introduction

How tomatoes grow, what determines a beetle’s eye color, and how to block HIV are very different questions. Yet molecular biologists are now reaching for the same genetic tool to find the answers. Small interfering RNA (siRNA) is a new technology that holds great promise in all these areas. siRNA provides a simple mechanism by which researchers, and perhaps even practicing physicians, can turn genes on and off at will.

Small interfering RNAs are components of a larger antiviral response called RNA interference (or RNAi), a cellular defense first uncovered in flatworms and plants. Certain viruses are composed of RNA in double-stranded form. The presence of such obviously foreign RNA in plants’ and invertebrates’ cells triggers a response led off by the “Dicer” enzyme, which chops up the offending RNA into small pieces. Other cell proteins collect around the strand, forming an RNA-induced Silencing Complex, coined RISC. RISC unwinds the RNA pieces and uses them as a guide to seek out and bind onto (or “silence”) any matching RNA sequences present in the cell. By blocking this RNA, the cell ensures that it cannot make viral products.

Cells of higher animals react to viruses differently than plants and worms. Although Dicer and the RISC proteins exist in these cells, they do not depend on the RNAi pathway to degrade unwanted double-stranded RNA. When confronted with long double-stranded RNA strands, animal cells activate a complex, interferon-based inflammatory response that shuts down all protein production.

Just over a year ago, Thomas Tuschl (of the Max Planck Institute for Biophysical Chemistry, Göttingen, Germany) found a way to trigger the RNA interference response in mammalian cells instead of the inflammatory one. “That was the surprising find,” said Tuschl, now considered a pioneer in the field. “You could uncouple an unspecific response from a specific response.” The trick was to supply the cells with short double-stranded pieces of RNA – the siRNAs. Because these RNA strands were so short (only 21 to 22 nucleotide pairs long), they slipped unnoticed past the inflammatory response, only to be taken up by the RNAi pathway. Almost immediately, scientists realized that they could exploit this response to block any gene they liked. All they had to do was introduce siRNA with a sequence derived from the target gene.

Instant Acceptance

Four research teams have now applied the technique to stopping HIV in cell culture. Last May, John Rossi (of the City of Hope’s Beckman Research Institute, Duarte, CA) reported successfully inhibiting HIV with siRNA derived from the rev gene. Duke’s Brian Cullen has employed the technique to silence tat and rev, shutting down HIV replication. Mario Stevenson (University of Massachusetts Medical School, Worcester, MA) suppressed HIV replication by silencing the vif and nef genes plus the long terminal repeat at the end of the HIV genome. And Philip Sharp at MIT effectively inhibited HIV by silencing the cellular gene for the CD4 receptor as well as HIV’s gag gene.

Animal studies are right on the heels of the cell culture experiments. Frederic Bushman (The Salk Institute, La Jolla, CA) moved the technology further in August, when he published results of silencing a retrovirus in chick embryos. Most recently, Beverly Davidson (University of Iowa College of Medicine, Iowa City, IA) successfully silenced genes after injecting siRNA into the brains of mice.

“Before, you could not turn off a human gene at will,” Tuschl remarked. “Now, in a very systematic, pragmatic way, you can turn off any genes you want to study. If you think there is a gene involved in HIV infection you can turn it off and see the effect.”

One immediate application relates to Michael Malim’s recent Nature article. Malim, a researcher at London’s Kings College, reported that the HIV Vif protein turns off an antiviral cell gene, CEM15. In Malim’s experiments, Vif-negative HIV mutants were able to replicate normally in cells that did not contain CEM15. But adding the CEM15 gene resulted in defective HIV virions that could not infect new cells.

Malim did not perform the opposite experiment, removing CEM15 in cells that normally express it to show that they would now permit an ongoing infection with Vif-negative HIV. He was reluctant to use the currently available ribozyme, antisense and gene knockout technology, citing inefficiency and low success rate. Mention RNAi, though, and the quick answer is, “We’re working on it.”

“I’ve been a scientist for 21 years, and I’ve seen all the new tools for regulating gene expression. I have never seen the kind of instant acceptance of a technology,” said Eric Lader at Qiagen, a biotech company based in Germany. In response to demand, biotech firms are scrambling to optimize delivery systems and sell easy-to-use kits and reagents. Lader’s rough estimate is that sales of RNA oligos (the made-to-order siRNA strands) went “from nothing to 15 million dollars” in a year. He credits the allure of siRNA to the fact that it works in such a specific, focused manner.

Technique or Treatment?

For Tuschl, siRNA’s real value is as a tool to understand HIV pathogenesis. “siRNA as a research tool will have a dramatic impact on developing HIV therapeutics—and the impact may be more dramatic than siRNA as a therapy in and of itself.” Applied researchers across disciplines, meanwhile, are looking at siRNA to potentially treat such diseases as polio, cancer, hepatitis, human papillomavirus and HIV.

In HIV therapy, RNAi faces the usual challenge – resistance. siRNAs have to be short to silence genes (21 or 22 nucleotide pairs long) and sequence-specific. “Change one nucleotide, and it’s useless,” Bushman remarked. Biologists have already identified two plant viruses that can escape RNA interference. Overcoming a single sequence would not be difficult for rapidly mutating HIV. To circumvent escape, researchers propose targeting very conserved regions of the virus, or including several siRNA sequences in a single delivery system – a new kind of combination therapy.

Harvard’s Judy Lieberman collaborated with Philip Sharp and envisions an alternative approach: silencing host genes critical for optimal HIV replication. “If you target host genes, then you are less likely to have to deal with viral sequence variation and mutation,” said Lieberman. “CD4 [one of the target genes in her study] admittedly is not an ideal host gene because if you interfere with CD4 expression you are going to mess up the immune response. But we know that people who have homozygous mutations [deleting the cellular CCR5 coreceptor for HIV] are completely normal immunologically. We think targeting CCR5 will be well tolerated, and as a host gene it won’t be constantly mutating.”

Rossi and his collaborators are gearing up to use RNAi therapy in humans. For many years they have been experimenting with gene therapy to introduce ribozymes (enzymes that cleave RNA) into people with HIV and lymphoma. Now, they are drawing on their gene therapy experience to formulate a combined siRNA-ribozyme anti-HIV regimen. Rossi’s goal is to remove blood precursor cells from people with HIV, genetically alter them, and transplant them back into the individuals from whom they came. The cells divide and mature into the various blood cells, including the ones attacked by HIV.

Rossi intends to insert siRNAs against both host and viral genes and anti-HIV ribozymes into the cells. By equipping these cells and their many progeny with anti-HIV genes, the researchers hope to protect the cells against HIV. Macaque monkey studies are slated for next year, and if safety can be established, Rossi wants to be testing the application in people within three years.

“I don’t have a lot of faith in gene therapy as an approach to infectious disease…it’s just too hard,” Bushman remarked. While many see gene therapy as the inevitable and rational therapeutic approach, Bushman’s enthusiasm stems from another aspect of RNA interference that, to date, remains documented only in plants and worms. This feature is “amplification,” the ability of the original siRNA to provoke the production of more siRNA each time it links up with its target RNA sequence and to spread from cell to cell.

Bushman said, “What really excites me about the RNAi business is the prospect that, once started, it can amplify and spread between cells as it does in these model organisms. You can induce RNAi in a plant and graft on a branch from another plant and see the RNAi effect spread into the new graft. You can soak a worm in RNAi or feed it RNAi and watch it start in the worm’s mouth and spread to other parts of the body. It can even be heritable between generations of worms. It’s not clear whether any of this will work in vertebrates, but it is that kind of thinking that made us want to work in the area. It’s just incredible what’s been seen in those systems.”

The RISCy Frontier

“This instant acceptance is a bit premature,” said Lader. “The problem is there has not been a lot of careful examination to make sure the effects of RNAi are specific.” Companies are now in the business of studying whole-genome expression. “These companies are mapping all the expression in the cell. So if you want to silence a particular gene, these guys will look at all the other 30,000 genes and ask, ‘Does what we are seeing make sense? Are we seeing the effects we expect to see after silencing a specific gene, or are there 400 other things going on that we can’t explain?’”

RNAi technology faces several challenges. Accidentally silencing essential genes is a “worst-case scenario” for therapeutic applications. There are inherent delivery problems unless RNAi can spread out from a few inoculated cells. Without amplification, durability is problematic, too. But no one has yet documented RNAi amplification in mammalian cells.

RNAi’s greatest potential – as therapy – is a long way off. “Clinical application of RNA interference is, with one possible exception, at least a decade away,” Sharp observed. The exception is siRNA-based gene therapy experiments like Rossi’s that would be able to render blood precursor cells resistant to HIV. “Any fundamental science – and this is the most new and fundamental of sciences – is a long way from any clinical application,” Sharp concluded.

siRNA-directed HIV inhibition

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