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Shoot The Messenger
In days of yore, if someone wanted to stop the news from getting through, they killed the messenger. In human society, it's a misplaced action, certainly, and a temporary solution, at best. As a means to prevent or treat disease, however, researchers are betting that the method will be more permanent. The news (say an oncogene, in this case) is still there, but by killing (or at least incapacitating) its messenger RNA (mRNA), it should be possible to prevent the cell from ever receiving the information (in the form of a cancer-causing protein) and acting on it.
The antisense drug developers hit on this approach about two decades ago. Yet, as they discovered, turning successful lab experiments into effective drugs was anything but straightforward. The first-generation compounds elicited a wide range of undesirable side effects that sent scientists back to the benches. Over the years, the handful of biotech companies that have made antisense their business have learned how to overcome these drawbacks, and though there is still but one antisense drug on the market -- Isis Pharmaceuticals Inc.'s Vitravene for treating cytomegalovirus-induced retinitis in AIDS patients -- there are more than 20 in the clinic. (If you'd like to read more about the history of antisense drug development, please refer to the Signals article, "Antisense: Poised To Strike.")
But while antisense technology -- in which a single stranded oligonucleotide is designed to bind to a gene's mRNA, thus inhibiting protein translation -- continues to be an important drug discovery tool -- especially for functional genomics and target validation -- a newer approach has reportedly revolutionized the practice of molecular biology.
Not surprisingly, new companies are popping up all over the place to capitalize on the technology: Many are focused on supplying excited researchers with the lab reagents they need to carry out the experiments. A few have set their sights on the ultimate goal -- to discover and develop new drugs.
This wildly popular method -- called RNA interference (RNAi) or gene silencing (a more general term) -- also relies on messing with the messenger. But in this case, double-stranded RNA (dsRNA) does the job. And the injected molecule doesn't just block the mRNA, it destroys it. Importantly, the underlying mechanisms are an inherent part of a cell's repertoire. More importantly, RNAi appears to be present in all cells, including mammals, vastly enhancing its use as a drug discovery tool. RNA interference has turned out to be a very quick and easy way to knock out gene expression, meaning it can be used to ascertain the function of an unknown gene, as long as the sequence is at hand.
Gene silencing was first discovered in petunias more than a decade ago, but it wasn't until 1998 that researchers began to understand the phenomenon. Not that they have it completely deciphered yet. Those in the know say that this field of inquiry is moving extremely quickly, and as a result it's full of conflicting data.
Still, according to current thought, after dsRNA, constructed to match a gene sequence of interest, is introduced into the target cell (or organism), it is digested into small interfering RNAs (siRNAs, 19-21 base pair duplexes) by an enzyme dubbed Dicer (a member of the RNase III family of dsRNA-specific ribonucleases). These siRNAs then bind to a nuclease complex to form an RNA-induced silencing complex, which, once activated, targets the homologous transcript by base pairing and cleaves the messenger RNA.
Not even this elegant method is perfect, it seems. In mammalian cells, for instance, gene expression is not completely eliminated; the effectiveness of different siRNAs varies; some cell types work better than others; success seems to depend on the level of expression of the target gene; and silencing eventually diminishes. Nonetheless, researchers are hooked.
It's rare to find a basic experimental technique that can be applied across the board with virtually no modifications -- and this certainly applies to RNA interference. So, as you might expect, researchers and reagent suppliers alike continue to devise numerous variations aimed at improving the efficiency, reliability and reproducibility of this powerful technology.
For instance, there are a number of ways to introduce dsRNA into the target cell (by electroporation, direct injection, transfection or expression vectors). But RNA oligos are expensive to synthesize and easily degraded. That's why Allele Biotechnology & Pharmaceuticals Inc., a small San Diego firm, has turned to DNA, which is not only cheaper to make and more stable, but also is able to generate a long-lasting response in mammalian cells. Allele has devised a linear cassette technology that uses DNA to encode for the siRNA.
The DNA template, which can be transfected in either linear or circular form, is used by the cell's endogenous transcription machinery to produce the desired siRNA, according to Jiwu Wang, Allele Biotechnology's president and co-CEO. "We use eukaryotic RNA polymerase III [Pol III] promoters to prompt the cells to produce their own dsRNA endogenously." Because efficient RNAi requires a perfect match between the interfering RNA and its target sequence, the company's engineered Pol III promoters are coupled with either terminators or a run off mechanism which ensures the precise start and end of the interfering RNAs, he added. Wang claims that his company's gene silencing cassette is capable of making "permanent cell lines," which could be used for toxicity testing, target validation and screening. As well, the cassette can be introduced into animals to generate genetically modified model organisms.
Like Allele Biotechnology, Australian firm Benitec Ltd. has patented a method that uses DNA constructs to induce RNA interference. Benitec's constructs, however, contain inverted repeat sequences, which when transcribed within the cell give rise to a linear RNA molecule that is half "sense" and half "antisense." But not for long: As the complementary base pairs seek each other out, the linear molecule bends back on itself to form a doubled stranded RNA structure called a hairpin loop. This dsRNA is attacked by the Dicer enzyme to form siRNAs, which trigger the series of events leading to destruction of the target mRNA.
Benitec claims that its technology platform can be used for high throughput target validation, functional analysis of anonymous genes and even therapeutic development. According to executive chairman and CEO John McKinley, "All evidence in mice (from our work and that of others) suggests that it is possible to get a therapeutic response [via RNA interference]."
That's exactly the premise that the founders of Alnylam Pharmaceuticals intend to prove. Based in Cambridge, MA, Alnylam was formed earlier this year by Nobel Laureate Phillip Sharp, National Academy of Sciences member Paul Schimmel and three academic pioneers in RNAi research -- Dave Bartel of MIT, Tom Tuschl of the Max Planck Institute and Phil Zamore of UMass Medical School -- whose ground-breaking findings were instrumental in propelling the field forward.
Backed by $17 million in new venture financing, and privy to key intellectual property in the field, the company intends to turn its energies to developing RNAi-based therapeutics for treating a wide range of diseases.
As such, Alnylam joins a still-small, but rapidly growing list of companies with a therapeutic focus, according to Christoph Westphal, Alnylam's start-up CEO and general partner at Polaris Venture Partners. "This is an incredibly important space, and we're probably just on the front edge of important new developments."
In fact, new proof that RNA interference actually works in living systems emerged just this week. Researchers at start-up company Intradigm Corp. presented scientific results at the American Chemical Society (ACS) meeting in Boston that they claim demonstrate RNAi's ability to silence endogenous genes, down-regulate the encoded protein and inhibit tumor growth in tumor-bearing animal models.
The company, based in Rockville, MD, was created in June 2001 to capitalize on synthetic vectors for gene delivery developed by Genetic Therapy Inc., a subsidiary of Novartis and one of the biotech sector's first gene therapy companies. In fact, Novartis Venture Fund was one of Intradigm's initial investors.
According to the company, the experiments reported at the ACS meeting involved knocking down expression of secreted vascular endothelial growth factor (VEGF) produced by human tumor cells in xenograft animal models. Additionally, company scientists demonstrated that they could knock down expression of the growth factor's receptor produced by the murine cells responsible for making the new blood vessels that would feed the tumor.
"We demonstrated in a number of proof-of-concept studies that we can quite rapidly discriminate genes that enhance or reduce tumor growth from those that have no effect," explained Casey Eitner, Intradigm's chief business officer. And the results presented at the ACS meeting mark the first time that anyone has reported disease-specific efficacy in vivo, he said.
That's a significant step forward. But RNA interference is both a complex and rapidly evolving field, with results continuing to pour forth almost daily. If Intradigm is, indeed, the first to achieve this milestone, gene-silencing research's rapid-fire pace almost guarantees that the firm won't hold that title for long.
originally published 08/22/2002 |