Shhh: Silencing Genes with RNA Interference

 TECHNICAL KNOCKOUT: A Cy3-labeled siRNA targeting B-actin was transfected into HeLa cells and protein expression was analyzed 96-hours later. Red, Cy3-labeled siRNA; Blue: DAPI-stained nuclei; Green, B-actin protein. (siRNA was prepared and labeled using Ambion's Silencer siRNA construction kit and labeling kit, respectively). RNA interference, or RNAi, is all the rage these days. According to the Web of Science database (ISI, Philadelphia), the number of articles on the topic jumped fr

By | April 7, 2003

 TECHNICAL KNOCKOUT: A Cy3-labeled siRNA targeting B-actin was transfected into HeLa cells and protein expression was analyzed 96-hours later. Red, Cy3-labeled siRNA; Blue: DAPI-stained nuclei; Green, B-actin protein. (siRNA was prepared and labeled using Ambion's Silencer siRNA construction kit and labeling kit, respectively).

RNA interference, or RNAi, is all the rage these days. According to the Web of Science database (ISI, Philadelphia), the number of articles on the topic jumped from nine in 1998 to 229 in 2002. Why all the fuss? Because RNAi, or more broadly, posttranscriptional gene silencing, provides a simple way to knock out genes in vivo in organisms as varied as plants, worms, flies, and mice (see explanatory box).

Nobel laureate Phillip Sharp, professor and director of the McGovern Institute for Brain Research, Massachusetts Institute of Technology (MIT), calls RNAi the most important breakthrough in the past decade. At the National Cancer Institute of the National Institutes of Health in Bethesda, Md., Bruce Paterson, chief, biochemistry of gene expression, is more cautious, given the checkered history of other highly touted technological developments such as antisense RNA.

At the heart of these disparate viewpoints is an information void: Researchers still don't know all there is to know about RNAi. For instance, not every siRNA--the short double-stranded RNAs that actually cause RNAi--can effectively downregulate a gene. David Lewis, senior scientist at Madison, Wis.-based Mirus, estimates that between 25% and 75% of siRNAs are effective. "Some RNAi's have a 10-fold effect, some have seen a 50-fold effect," Sharp notes. And some don't work at all.

On a practical level, this may not really matter. "We haven't found a gene we can't put down [with RNAi]," Sharp says. And scientists have already demonstrated RNAi's utility in functional genomics research. "We've searched for decades for a way to knock out genes effectively," Sharp says. "We have about 35,000 different genes and have determined the function of only about 500 of them using an incredibly painful, expensive knockout technology. We won't ever understand the remaining functions with the existing technology."

RNAi, he and others predict, can fill this void,1 and dozens of companies are eagerly developing RNAi assays and delivery systems, banking on the promise of emerging findings. These same companies along with many academic researchers also are betting that, with optimization, RNAi could provide a simple and efficient way to cure diseases in humans, too.

GENOME MANIA Most researchers are enamored of RNAi because of the ease with which they can use it to produce gene knockouts. Coupled with newly available genome sequences, researchers are now able to apply RNAi on a genomic scale. Earlier this year, Julie Ahringer of the University of Cambridge, UK, and colleagues described a library of 16,757 unique, cloned double-stranded RNAs (dsRNA), representing about 86% of all Caenorhabditis elegans genes.2 Using this resource, which they administered by feeding the bacteria to the animals, these authors determined the function of 1,722 genes, most of which previously had no function associated with them.

Several groups have adopted this library for similar genome-wide studies. Gary Ruvkin's team at Harvard University identified several hundred genes involved in fat storage,3 while Marcel Tijsterman, Ronald Plasterk, and colleagues at the Center for Biomedical Genetics, Utrecht, Netherlands, used it to identify genes that protect the worm genome from mutations and instability.4

But RNAi isn't just for nematodes; researchers are becoming ever more adroit at inducing RNAi in vertebrates, too. One recent study used RNAi to monitor central nervous system development in chicken embryos.5 On the murine front, Gregory Hannon's group at Cold Spring Harbor Laboratory, NY, demonstrated they could stably block gene expression in mice either by introducing siRNA precursors into hematopoietic stem cells and using those cells to repopulate irradiated mice,6 or by germ-line transmission following stable integration of siRNA-producing cassettes in embryonic stem cells.7

In a vertebrate effort to parallel the C. elegans studies, Hannon is developing a genome-wide set of RNAi constructs for use in mammalian systems. He estimates that the complete library will reach 90,000 transcripts. Similarly, Dresden, Germany-based Cenix BioScience and Ambion of Austin, Texas, are codeveloping an siRNA library for the entire human genome, slated for release in mid-2003.

DELIVERY ISSUES One way to induce RNAi in cells is to introduce siRNAs directly, using microinjection or electroporation of synthetic RNAs, for example. Several companies offer specialized siRNA-delivery reagents.But many researchers employ some sort of expression construct instead. Some use an RNA polymerase III (Pol III) promoter to drive expression of both the sense and antisense strands separately, which then hybridize in vivo to make the siRNA. Others use Pol III to drive expression of short "hairpin" RNAs (shRNA), individual transcripts that adopt stem-loop structures, which are processed into siRNAs by the RNAi machinery.



EYE ON RNAi: Double-stranded RNAs (dsRNAs) enter the RNAi pathway in several different ways, for example by exogenous introduction or by their synthesis from genomic loci. Dicer recognizes and processes these into ~21-23-nt siRNAs or miRNAs, which then enter an effector complex, RISC, that suppresses gene expression through mRNA degradation or prevention of protein synthesis.
Click for full version (33K)

Yet another method makes use of viral vectors to infect cells with the dsRNA-expression construct. Some researchers exploit retroviral vectors,6 but recently, Luk Van Parijs' team at MIT,8 and Inder Verma's group at the Salk Institute9 independently described lentiviral-based systems. This approach enabled these investigators to deliver shRNAs to primary cells, stem cells, and noncycling cells, all of which are traditionally hard to transfect. By infecting zygotes, both groups made transgenic mice in which RNAi-directed gene downregulation occurs stably throughout the animal.

Delivering siRNAs directly to whole vertebrate animals is more problematic than it is for invertebrates or cell lines. "Animals can't absorb [the siRNA] through the skin," Mirus' Lewis explains, "and simply injecting it into the bloodstream has been ineffective." Last year, Hannon in collaboration with Mark Kay at Stanford University,10 and Lewis and colleagues,11 independently employed a "hydrodynamic transfection method" to deliver naked siRNAs to mice via tail-vein injection. These authors observed downregulation of a reporter gene by 80%-90% in the liver, kidney, spleen, lung, and pancreas, but the effect is relatively short-lived, lasting only a few days.

FLOCKING TO RNAi Like academicians, biotech companies are flocking to RNAi. Compugen, headquartered in Tel Aviv, Israel, for example, is developing a large-scale RNAi platform in collaboration with Basel, Switzerland-based Novartis. The goal is to design gene-specific and transcript-specific inhibitors for gene function evaluation and target validation.

"We have a good understanding of the transcriptome and can properly design siRNAs to silence specific mRNAs," says Alon Amit, executive director of technical marketing at Compugen. The company develops silencing probes that allow Novartis "to silence human genes of splice variants one by one and see what they affect in various contexts," he says.

Back in Madison, Wis., Mirus is using RNAi for target validation in the drug-discovery process. From thousands of genes screened, "we knock down the 50 or 60 most likely candidates to find the one or two that are causative," Lewis explains.



RNA Interference Explained
RNA interference, or RNAi, is an endogenous, efficient, and potent gene-specific silencing technique that uses double-stranded RNAs (dsRNA) to mark a particular transcript for degradation in vivo.1 First discovered in the nematode Caenorhabditis elegans, it has since been found to operate in a wide range of organisms. These creatures didn't evolve RNAi for the benefit of modern biologists, of course. RNAi probably exists to help fend off pathogens and to control gene expression. Evidence also exists to suggest the RNAi machinery can act at the DNA level, too. RNAi was recently linked to heterochromatin stability in fission yeast and to massive genomic rearrangements in Tetrahymena, for instance.2,3

Key to the technique are dsRNAs 21-25 nucleotides long, called short interfering RNAs (siRNA). Produced by degradation of long dsRNA molecules by an RNAse III-related nuclease called Dicer, siRNAs also can be introduced into a cell exogenously, or by transcription off an expression construct. Once formed, the siRNAs associate with a multiprotein complex called RISC, the RNA-induced silencing complex, which targets the homologous RNA by Watson-Crick base-pairing for degradation.

References
1. G.J. Hannon, "RNA interference," Nature, 418:244-51, 2002.

2. T.A. Volpe et al., "Regulation of heterochromatin silencing and histone H3 lysine-9 methylation by RNAi," Science, 297:1833-7, 2002.

3. K. Mochizuki et al., "Analysis of a piwi-related gene implicates small RNAs in genome rearrangement," Cell, 110:689-99, 2002.
 

Other companies that are focused on RNAi include Genetica of Cambridge, Mass.; Devgen of Ghent, Belgium; Cenix BioScience; Sequitur of Natick, Mass.; and Alnylam Pharmaceuticals of Cambridge, Mass. These companies are investigating RNAi's role not only in drug target identification, but also as a therapeutic agent itself.

Researchers have already demonstrated RNAi's efficacy in blocking both viruses and certain disease states. These investigators have, for instance, countered hepatitis C virus,10 poliovirus,12 and HIV13 using RNAi. Researcher Judy Lieberman of Harvard Medical School and colleagues recently reported that RNAi can successfully prevent liver injury and death in a mouse hepatitis model.14

RNAi, says Alnylam CEO John Maraganore, "may represent the broadest new class of human therapeutics since recombinant proteins and monoclonal antibodies," affording a unique way to target both viruses and disease states in which the overexpression of a particular gene causes pathology. Founded last year by a collection of RNA luminaries, including Sharp, Philip Zamore from the University of Massachusetts Medical School, Dave Bartel from MIT, Paul Schimmel from the Scripps Research Institute, and Thomas Tuschl from Rockefeller University, the company is currently developing siRNAs to target cancer, viruses, inflammatory diseases, and metabolic diseases.

To make such drugs effective, however, they first must be stable in biological fluids. Towards that goal, Alnylam hired a new head of drug discovery with 15 years experience in oligonucleotide chemistry. Maraganore expects to see RNAi-based drugs in clinical trials within two years.

Gail Dutton (gaildutton@gi-wireless.net) is a freelance writer in British Columbia.

Jeffrey M. Perkel can be contacted at jperkel@the-scientist.com.

References
1. M.T.M. Manus, P.A. Sharp, "Gene silencing in mammals by small interfering RNAs," Nat Rev Genet, 3:737-47, October 2002.

2. R.S. Kamath et al., "Systematic functional analysis of the Caenorhabditis elegans genome using RNAi," Nature, 421:231-7, Jan. 16, 2003.

3. K. Ashrafi et al., "Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes," Nature, 421:268-72, Jan. 16, 2003.

4. J. Pothof et al., "Identification of genes that protect the C. elegans genome against mutations by genome-wide RNAi," Genes Dev, 17:443-8, Feb. 15, 2003.

5. V. Pekarik et al., "Screening for gene function in chicken embryo using RNAi and electroporation," Nat Biotech, 21:93-6, January 2003.

6. M.T. Hemann et al., "An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo," Nat Genet, 33:396-400, March 2003.

7. M. Carmell et al., "Germline transmission of RNAi in mice," Nat Struct Biol, 10:91-2, February 2003.

8. D.A. Rubinson et al., "A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells, and transgenic mice by RNA interference," Nat Genet, 33:401-6, March 2003.

9. G. Tiscornia et al., "A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA," Proc Natl Acad Sci, 100:1844-8, Feb. 18, 2003.

10. A.P. McCaffrey et al., "Gene expression: RNA interference in adult mice," Nature, 418:38-9, 2002.

11. D.L. Lewis et al., "Efficient delivery of siRNA for inhibition of gene expression in postnatal mice," Nat Genet, 32:107-8, 2002.

12. L. Gitlin et al., "Short interfering RNA confers intracellular antiviral immunity in human cells," Nature, 418:430-4, 2002.

13. M.A. Martinez et al., "RNA interference of HIV infection," Trends Immunol, 23:559-61, December 2002.

14. E. Song et al., "RNA interference targeting Fas protects mice from fulminant hepatitis," Nat Med, 9:347-51, March 2003.
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