RNA interference: the next genetics revolution?

The central process of RNAi is the chopping of dsRNA into smaller pieces of a defined length by the appropriately named enzyme Dicer (Fig. 1). Dicer chops dsRNA into two classes of smaller RNAs — microRNAs (miRNAs) and small interfering RNAs (siRNAs) — that are around 21-23 nucleotides in length. Although miRNAs also stop protein production (and, as described in another article, the boundaries between siRNAs and miRNAs is becoming blurred), siRNAs are thought to be the main protagonists in RNAi. Dicer delivers these siRNAs to a group of proteins called the RNA-induced silencing complex (RISC), which uses the antisense strand of the siRNA to bind to and degrade the corresponding mRNA, resulting in gene silencing. RNAi is astonishingly efficient, because the RISC is an enzyme and catalyses multiple rounds of RNAi, perhaps hundreds or thousands in vivo. It also shows an amazing persistence in some organisms, such as worms, which suggests there could also be an amplification response (Fig. 2).

		Figure 1 | RNAi-mediated gene silencing. RNA interference (RNAi) is an apparently ancient defence mechanism against foreign double-stranded RNA (dsRNA). RNAs of just 22 nucleotides in length, called small interfering RNAs (siRNAs), are snipped from longer dsRNA chains by an enzyme called Dicer. The antisense strand of the siRNA is used by an RNA-induced silencing complex (RISC) to guide messenger RNA (mRNA) cleavage, so promoting mRNA degradation. Modified with permission from McManus, M. T. & Sharp, P. A. Gene silencing in mammals by small interfering RNAs. Nature Rev. Genet. 3, 737-747 (2002) © Macmillian Magazines Ltd. miRNA, microRNA; stRNA, small temporal RNA.

		Figure 2 | A model for the mechanism of RNAi. Silencing triggers in the form of double-stranded RNA can be presented in the cell as synthetic RNAs, replicating viruses or can be transcribed from nuclear genes. These are recognized and processed into small interfering RNAs (siRNAs) by Dicer. The double stranded siRNAs are passed to the RNA-induced silencing complex (RISC), and the complex becomes activated by unwinding of the duplex. Activated RISC complexes can regulate gene expression at many levels. Almost certainly, such complexes act by promoting RNA degradation with siRNAs and translational inhibition with microRNAs. However, similar complexes probably also target chromatin remodelling. In plants, amplification of the silencing signal can also occur. Modified with permission from Hannon, G. RNA interference. Nature 418, 244-251 (2002) © Macmillian Magazines Ltd.

Easy knockout
As RNAi relies on the sequence-specific interaction between siRNA and mRNA, siRNAs can be tailored to silence almost any gene. siRNAs that have been chemically synthesized or created by in vitro transcription systems can induce silencing in several systems, including mammalian cells. Transfected plasmids that produce dsRNA hairpins, called short hairpin RNAs (shRNAs), can also elicit RNAi effects. In the laboratory, the silencing of genes with RNAi seems to be rapid and convenient, and can be used to tackle many genes at the same time. RNAi can be achieved remarkably easily in C. elegans — gene silencing occurs by direct feeding of dsRNA feeding with bacteria that expressed dsRNA or even soaking worms in dsRNA, and the effect can also be transmitted to the next generation.

The relative ease with which genes can be silenced using RNAi has caused a minor revolution in molecular biology. Armed with data from genome sequencing projects, gene silencing with RNAi can be used on a breathtaking scale. For example, Julie Ahringer's group at the University of Cambridge has created a library of more than 16,000 cloned dsRNAs (around 86% of the C. elegans genome). By feeding these clones to worms, they have determined the function of 1,722 genes, most of which were previously unknown. Gary Ruvkun's group at Harvard Medical School has identified many hundreds of genes involved in fat storage. And Gregory Hannon's group at Cold Spring Harbor are looking to determine the functions of every gene in the human genome. The list of projects in this area is seemingly endless.

Therapeutic possibilities
Taking the DNA sequence of a gene and designing dsRNA that can specifically and effectively silence a disease-related gene is analogous to monoclonal antibody production. There was an initial problem in developing therapeutic approaches, as dsRNAs that are 30 nucleotides in length or longer can trigger non-sequence-specific interferon responses in mammalian cells. But this has been overcome by delivering siRNAs into mammalian cells in culture. There are many ways of doing this, such as direct transfection of siRNAs into cells, creating an expression construct in which a promoter drives the production of both the sense and antisense siRNAs which then hybridize in the cell to produce the double-stranded siRNA, and using viral vectors to infect cells with an expression construct. Because of this, designing RNAi-based therapeutics is beginning to progress rapidly in certain areas.

HIV. Several groups have shown that siRNAs can inhibit HIV replication effectively in culture. HIV infection can also be blocked by targeting either viral genes (for example, gag, rev, tat and env) or human genes (for example, CD4, the principal receptor for HIV) that are involved in the HIV life cycle. This is promising, as antiviral therapies that can attack multiple viral and cellular targets could circumvent genetic resistance of HIV. But these results have been achieved by transfecting the siRNAs into cells, and getting the siRNAs to function in vivo is likely to be a more difficult task. To get around the delivery problem, many groups have designed promoter systems that can express functional siRNAs when transfected into human cells. Early results have shown that this can decrease replication of HIV considerably and the group are currently working on inserting the system into a lentiviral vector to test its effectiveness in acute HIV-1 infections.

Hepatitis. This has provided the first tangible evidence for RNAi as a therapy for diseases in live animals. Early RNAi studies noted that RNA silencing was prominent in the liver, which made this organ an attractive target for therapeutic approaches. Many immune-related liver diseases are characterized by apoptosis, which is mediated by a protein called Fas. So Judy Lieberman's group injected siRNA targeting Fas intravenously into two models of autoimmune hepatitis in mice. This decreased Fas mRNA and protein levels in hepatocytes and protected the cells against liver injury from apoptosis, even when siRNA was administered after the induction of injury. Extending these findings to other liver diseases looks hopeful, but the authors concluded that other strategies, such as viral vectors, might be required to target organs in which RNA silencing is less effective than in the liver.

Cancer. Gregory Hannon and colleagues have used RNAi to silence expression of p53 — the 'guardian of the genome', which protects against any tumour-associated DNA damage — by introducing several p53-targeting shRNAs into stem cells and looking at the effect in mice. The shRNAs produced a wide range of clinical effects, ranging from benign to malignant tumours, the severity and type of which correlated with the extent to which the shRNA had silenced p53. As tumour suppressors such as p53 usually work as part of a complex and finely regulated network, the ability to dampen these networks to varying degrees in these libraries — which the authors term an epi-allelic series of hypomorphic mutations — will be of enormous value when it comes to investigating the early stages of disease. The success of these modified stem cells also gives hope that this could treat diseases in which stem cells can be modified ex vivo and then re-introduced into the affected individual.

Researchers at the charity Cancer Research UK and the Netherlands Cancer Institute have recently announced that they intend to generate a large library of human cells, each containing a silenced gene. They initially want to silence 300–8,000 cancer genes, and hope to eventually cover the entire human genome. Their aim is to uncover all the genes that become overexpressed in human cancers and to find out precisely what needs to be taken away from a cancerous cell in order to make it normal again.

Why RNAi?
It is thought that RNAi, at least in plants, might be an ancient defence mechanism that defends against RNA viruses, transposons and repetitive sequences. But recent work in yeast and the ciliate Tetrahymena has shown that RNAi might also influence transcription in the cell nucleus, partly by determining the form of the chromatin in which the DNA is wrapped (Fig. 2). So, which genes are transcribed and which remain wrapped up or hidden could be under the control of small RNAs, although how this mechanism works is currently unknown.

Many mysteries of RNAi have yet to unfold, but one thing that is certain is that these small RNAs have revolutinized the way scientists think about how DNA, RNA and proteins are controlled and function. This has caught the imagination of both big pharmaceutical companies, which are using RNAi as a target validation tool, and smaller companies, which are optimizing RNAi methodologies and looking at therapeutic avenues. With some of these companies predicting that the first RNAi-based drugs will begin clinical trials in the next 2-3 years, these might be early days, but they are incredibly exciting ones.

Further Reading
Carmell, M. A., Zhang, L., Conklin, D. S., Hannon, G. J. & Rosenquist, T. A. Germline transmission of RNAi in mice. Nature Struct. Biol. 10, 91-92 (2003).
| article |
Hammond, S. M., Caudy, A. A. & Hannon, G. J. Post-transcriptional gene silencing by double-stranded RNA. Nature Rev. Genet. 2, 110-119 (2001).
| article |
Hemann, M. T. et al. An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo. Nature Genet. 33, 396-400 (2003). | article |
McManus, M. T. & Sharp, P. A. Gene silencing in mammals by small interfering RNAs. Nature Rev. Genet. 3, 737-747 (2002). | article |
Song, E. et al. RNA interference targeting Fas protects mice from fulminant hepatitis. Nature Med. 9, 347-351 (2003). | article |
Zamore, P. D, Tuschl, T., Sharp, P. A., & Bartel, D. P. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25-33 (2000).