The background to the discovery that won a Nobel Prize...
RNA interference is the newest kid on the genetic block. By allowing scientists to selectively turn off genes it promises to set the scientific world alight with its therapeutic potential and wide-ranging applications including onions that canít make you cry and new treatments for degenerative diseases. But how does it work?
The process stems from an important series of experiments carried out in the 1990s by the American biologists Craig Mello and Andrew Fire. They were injecting worms with a short piece of RNA that was the genetic mirror image of a muscle gene called unc-22. The results were worms that twitched, just like another family of worms from which the unc-22 gene had been removed altogether. This told Mello and Fire that the RNA they had injected must have shut off or ďsilencedĒ the muscle gene in the injected animals.
Building upon their initial worm work, the two scientists then slowly pieced together the molecular machinery behind this "RNA-interference" process, revealing as they did so what looks set to become one of the most powerful instruments in the molecular biologist's toolkit.
Evolutionary Protection Mechanism
The basis of RNA interference dates from the 1950s when scientists first discovered that RNA can also exist in a double stranded form (dsRNA) a bit like its relative, DNA. dsRNAs can crop up naturally when cells are infected by certain pathogens such as viruses like HIV, and by jumping pieces of genetic material called transposons, which can damage a cellís DNA. To deal with these threats cells have evolved a mechanism for degrading any dsRNAs that occur in order to block the replication of the offending agent.
Destruction of the dsRNA is achieved by an appropriately-named enzyme called Dicer, which operates in partnership with a second enzyme called Argonaute (randomly named after its resemblance to a family of octopuses, which were in turn Christened after the sailors in Greek mythology who, led by Jason aboard the Argo, searched for the Golden Fleece).
Together these enzymes initiate the interference process by locating the dsRNA and slicing it up, so that the gene it represents cannot be translated into a protein sequence, effectively "silencing" the gene. The same process is at work in a wide variety of organisms from fungi and plants through to worms, mice and humans.
But how can we turn it to our advantage? The basic idea is to repeat what Mello and Fire did with their worms and switch off trouble-making genes linked to certain diseases. The RNAi genes could be delivered to vulnerable cells using modified viruses, or just injected into specific cells as naked RNA.
Difficult as it sounds, researchers are already making significant progress in turning this concept into reality. In 2006 Williams et al. published a study in which they explored the use of RNAi to stop sperm from fertilising eggs. They used the technique to silence a gene encoding a sperm-binding protein which is normally present on the surfaces of human or mouse ova (eggs). The resulting eggs, which lacked the protein, were unable to interact with sperm and so remained unfertilised. Since current contraceptives involve the manipulation of female hormones or placing copper devices in the uterus, both of which can cause problems for some individuals, this alternative method of temporarily switching off a gene thatís essential for fertilisation is a very attractive prospect as a future birth-control strategy.
On a lighter note, albeit slightly "tearier", researchers at New Zealand Institute for Crop and Food Research Limited have been using the same technique to silence a key gene thatís responsible for the tear-jerking effect of slicing an onion.
The tear-factor is a sulphur compound called syn-propanethial-S-oxide, which irritates the front of the eye and triggers increased tear production. Itís produced by a chemical pathway involving several steps catalysed by enzymes that turn pungent chemicals in the onion, called amino acid sulphoxides, into the active crying chemical.
Cutting into the onion flesh frees these enzymes, which include one called allinase and another called lacrimatory factor synthase, from the special compartments where they are normally sequestered inside an onionís cells.
Itís this step that the New Zealand team have targeted with RNAi. Theyíve inserted a short piece of genetic material which is the mirror image of the lacrimatory factor synthase gene. When this mirror image pairs up with the gene the two form a segment of dsRNA, which the cell degrades, preventing the gene from being expressed.
Now, instead of converting the sulphur compounds into the tearing agent, the onion instead diverts them down an alternative metabolic pathway that produces flavourant molecules. As a result they excite the taste buds rather than the lacrimal glands.
Dr Colin Eady, who is heading the project, is confident about the potential of his 'tearless onion'. "They are such a versatile and nutritious vegetable, and the onion is one of the main sources of dietary fibre for many countries, if we can manage to get more people cooking and eating fresh onions, then that has got to be a positive outcome", he says. And who could disagree?
But more important than onions that canít make you cry is the prospect of using RNAi to tackle certain human genetic diseases. Researcher Beverly Davidson and her team at the University of Iowa have recently shown that conditions like Huntingtonís Disease may be amenable to an RNA interference-based therapy (Nature Medicine doi:10.1038/nm1076).
Working with mice programmed to develop a degenerative condition called spinocerebellar ataxia, the Iowa team have successfully silenced the culprit gene in five-week-old mice so that the toxic protein it encodes is not produced and the degenerative disease is prevented. After six months of this gene therapy the mice in the study displayed normal movement and showed no signs of brain damage compared with untreated mice, which all developed symptoms.
Despite the positive results of this study, which, as Davidson points out, is the "first example of targeted gene silencing of a disease gene in the brains of live animals [suggesting] that this approach may eventually be useful for human therapies," there remains some scepticism on the possible future success of treatments like this.
For a start, the gene involved in Huntingtonís is active in a different part of the brain to the gene examined in this study. Accessing the Huntingtonís gene with the viral vectors used in Davidson's study would not be a trivial matter. However, in other conditions many of the genes being targeted are active mainly during embryonic development, so silencing them in later life, with minimal side effects, seems feasible.
All the same, age might turn out to be a problem, warns Thomas Tuschl, a researcher based at the Max Planck Institute in Germany and leading player in the gene silencing field. "Davidsonís group have shown they can prevent the disease if they started the therapy early, before symptoms were apparent. They have not shown whether treating a diseased 10-week old animal would improve the symptoms. For most medical applications, the patients already have symptoms when they are diagnosed."
Nevertheless, this study is not an isolated example of investigating how RNA interference and the silencing of genes could improve medical therapies in future. The possibilities seem endless and the treatment, at least, appears to be developing fast. This field is both exciting and progressing at a pace where we could be reaping the benefits of gene therapy in the near rather than distant future.