Silence is golden

Now that decoding of the human genome is complete, attention is turning to RNA. Sarah Houlton considers the potential role of gene silencing in discovering and developing the drugs of the future

The decoding of the human genome has opened up new frontiers for drug discovery. Pharma's traditional focus on protein receptor targets has been supplemented by gene therapies that target DNA, made possible by the mapping of the human genome. The step between DNA and proteins, RNA, is being used as the starting point for discovering new drugs.DNA is nature's recipe book, containing all the information required to synthesise the proteins that organisms need to function. RNA is the inter-mediate messenger which translates the code of DNA into proteins, with each set of three nucleic acid bases coding for a different amino acid. By moving a step back up the chain from proteins to DNA, a whole new set of targets emerges that can be exploited to discover and develop novel medicines.

DNA is made up of just four different nucleic acid building blocks: adenine (A), cytosine (C), guanine (G) and thymine (T). In DNA's double helix, C always pairs up with G and A with T, so to reproduce, all the DNA has to do is unwind itself, splitting into two single strands, giving a pair of templates on which new second strands can be built. Similarly, to create the messenger RNA that codes for proteins, again the DNA first unwinds and splits to give a template. This time, nucleic acid bases attached to ribose rather than the sugar in DNA, 2-deoxyribose, build the second string, in the same pairings (except thymine is replaced with uracil, or U, in RNA). The two strands then separate again, leaving a single strand of mRNA ready to create the ribosomes that make proteins.

double strands

Now, scientists have found that they can create small pieces of RNA that have two strands instead of one, and these can be used to turn specific genes off. This has applications such as stopping cancers from growing and fighting off viruses. In this process, known as RNA interference, the base pairs of the double stranded RNA (dsRNA) match up perfectly with the mRNA being targeted, blocking it and, as a result, switching the gene off, or silencing it. This process happens in nature, and was first seen in plants more than a decade ago; it occurs in all species, including humans. However, in complex animals like humans, long pieces of dsRNA will be recognised as foreign, triggering cell death and the release of interferon to 'warn' neighbouring cells of the invasion. But it has now been found that short pieces of dsRNA, 21 to 25 base pairs long, do not trigger this response when they are introduced into cells. As a result, these short fragments of dsRNA, dubbed small interfering RNA or siRNA, have potential as treatments for diseases.

Once it enters cells, the siRNA forms a stable protein-RNA complex, or RNA-induced silencing complex (RISC) that is able to recognise and destroy the target mRNA. The complex is activated by ATP, unwinding the siRNA to incorporate into the mRNA. The matching up of base pairs means that the fragment is accurately targeted at a specific substrate.

In contrast to the older technique of antisense targeting, which introduced single strands of RNA with the intention of binding mRNA, the double strands of siRNA operate in a similar way to a natural process. Antisense technology has proved difficult to target precisely, and there are stability issues. Acting through RISC means that siRNA is using the body's own chemistry to shut off the gene.

In the five years since its significance in animals was recognised, RNAi has rapidly become an essential tool for probing the functions of genes. Its initial use in the nematode worm, Caenorhabditis elegans, and the fruit fly, Drosophila melanogaster, has since been extended to mammalian cells. As most drugs target specific proteins, eliminating, potentiating or altering their activity, the identification of these proteins is important. This often involves breeding 'knockouts', which are animals, usually mice, in which the protein has been eliminated as a target validation, showing that the postulated activity of the protein and its related pathways are correct.

The process is not quick - it can take at least a year. By introducing RNAi into a cell to silence genes, the time target validation takes can be slashed, as can the costs.

leading the way

Unsurprisingly, many companies are rushing to try and exploit the potential of RNAi, in the hope of creating safe, usable therapy options. The first company to demonstrate RNAi in mammalian and human cells was Australia's Benitec, which made the breakthrough in 1998. Last year, it succeeded in silencing a gene in a live mammal. The Queensland-based company is working on several areas, including Type II diabetes, a field in which it has a collaboration with the Garvan Institute of Medical Research; this deal was substantially widened earlier this year because of the initial success of the project.

One company heavily involved in RNAi technology is US-based Alnylam Pharmaceuticals, of Cambridge, MA. Among the therapeutic areas it is investigating with RNAi technology is Parkinson's disease, which affects neurons in the part of the brain that controls muscle movement. Parkinson's patients commonly suffer from trembling, difficulty walking, muscle rigidity and problems with balance and co-ordination. Most sufferers are aged over 50, but a small percentage of younger people are also affected.

possible treatments

Researchers at the Mayo Clinic in the US have discovered a causal pathway in Parkinson's, having pinpointed a specific gene, alpha synuclein, that is overexpressed in Parkinson's sufferers.

Alpha synuclein is found in various body tissues, but particularly the brain. It is believed it plays a key role in synaptic vesicle recycling, the method by which nerves transmit their signals. The Mayo researchers found that simple overexpression of normal, wild type alpha synuclein is enough to cause Parkinson's in a family with multiple affected members. If RNAi were able to prevent the gene being expressed, then it could have a dramatic effect on Parkinson's patients.

Alnylam is providing RNAi technology and funding research to Mayo, and it aims to identify, synthesise and provide RNAi-based drug compounds targeted to the expression of the alpha synuclein gene. Mayo will then test these compounds with in vitro and in vivo studies, in the hope that a successful treatment can be found.

Sirna Therapeutics, of Boulder, CO, is using RNAi to develop nucleic acid-based therapeutics. Its projects include a collaboration with the Wilmer Eye Institute at Johns Hopkins University, where it is working on a treatment for age related macular degeneration, one of the most common causes of blindness in those over 55. It is a result of the degeneration of the central retina, and one of the causes is choroidal neovascularisation, or CNV, which is an abnormal growth of blood vessels within the eye.

Along with Peter Campochiaro at Johns Hopkins, Sirna has used its proprietary chemically modified siRNAs in a mouse model of laser-induced CNV. The mice were treated with intraocular injections of siRNAs that targeted vascular endothelial growth factor 1 mRNA, which resulted in a significant reduction of CNV compared with controls. Currently still in the lead identification and optimisation stage of development, Sirna hopes to move the programme into clinical development in 2004.

Amyotrophic lateral sclerosis (ALS), better known as motor neurone disease or Lou Gehrig's disease, is one of the focuses of Los Angeles-based CytRx. ALS is currently incurable; its cause is unknown. The progressive neurodegenerative disease results in the degeneration of motor neurones in the central nervous system leading to paralysis. CytRx is funding a research programme at the University of Massachusetts Medical School to use its RNAi gene silencing technology to develop a treatment. The university's Zuoshang Xu hopes the RNAi will determine the underlying cause of ALS at the genetic level by first searching for and then silencing the disease-causing gene.

specific products

The university and CytRx are also collaborating on using RNAi to find treatments for obesity and Type II diabetes. Michael Czech at the university has found a way to apply siRNA gene silencing technology to cultured mouse fat cells. The technique allows the rapid depletion of one or more specific gene products to assess the function of the genes in fat cell metabolism, which is a key element in controlling appetite, energy expenditure and sugar levels in the blood.

Specific gene products in fat cells are likely to be important drug targets for both obesity and Type II diabetes, and RNAi technology means that the function of each gene candidate can be rapidly and directly tested. Czech has managed to deplete two related protein kinases, both individually and in combination in cultured fat cells.

This significantly lowered the insulin signalling to glucose transporters that allow the fat cells to take up sugar, indicating that these protein kinases do indeed play an essential role in signalling by the hormone insulin in fat cells. The discovery is now being developed into a high throughput screening method to look for potential drug targets for both indications.

library hopes

Another company heavily involved in the investigation of RNAi is Dharmacon, of Lafayette, CO, US. It specialises in RNA and RNAi research products, and has pioneered a custom siRNA design service that uses its SmartSelection algorithms to maximise the efficiency of gene silencing. It has entered into collaborations with several pharma companies. With Abbott Laboratories it is creating a siRNA library covering 4,000 genes. It is hoped that this will identify potential drug targets in Abbott's key therapeutic areas, including cancer, diabetes, pain management, inflammation and neurological disorders. The library will be used for high throughput functional genomic studies to identify and validate drug targets.

Dharmacon claims its technology overcomes some of the difficulties that have been experienced with RNAi by using its algorithms to select the best siRNA sequences for each application, removing the need for trial and error, and then pooling four candidates for guaranteed gene silencing.

Another recent collaboration set up by Dharmacon is with Rosetta Inpharmatics, a wholly owned subsidiary of Merck & Co. The aim is to develop a deeper understanding of factors that affect the potency and specificity of the siRNA reagents that are used for gene silencing. Selecting potent, highly specific siRNA for mammalian cells is challenging. Rosetta specialises in bioinformatics and gene expression profiling, and the two companies hope to be able to create robust and reliable assays for high throughput research.

Dharmacon is also collaborating with Odyssey Thera in a study of the effects of siRNA-induced gene silencing on cellular signal pathways. The aim is to combine Dharmacon's Smart gene silencing technologies with Odyssey Thera's protein fragment complementation process to quantify and analyse signalling events in living human cells. It is currently difficult to measure the cell signalling events that are critical to pathway activity. Odyssey Thera's PCA process allows these downstream effects to be measured, and also is able to identify off-pathway effects, which are a major concern as they could lead to side-effects.

The technique can be applied to a wide variety of medically relevant proteins, including receptors, kinases and transcription factors. Dharmacon is making siRNA reagents for targets selected by Odyssey Thera that have been associated with disease-related events such as angiogenesis, inflammation and apoptosis. Other information that can be pinpointed includes changes in localisation of signalling molecules, which would indicate, for example, the activation of a transcription factor or the desensitisation of a membrane receptor. The aim is to identify novel targets and nucleic acid therapeutics, as well as assays to be used in high throughput screening.

Benitec continues to push the frontiers of RNAi research. Its latest breakthrough is the simultaneous disabling of multiple genes by RNAi.

resistance implications

'We believe our "multiple warhead" capability will allow us to understand more clearly those conditions that result from multiple gene defects, such as Type II diabetes, autoimmune disorders and cardiovascular dysfunctions, and perhaps also provide viable treatment regimes,' explains ceo John McKinley. He believes that this approach could have implications in diseases that are prone to developing drug resistance, by simultaneously disabling multiple targets.

'The greater the number of genes that can be targeted, the lower the possibility that any one cell or virus would develop resistance to the treatment,' he claims. This is analogous to the concept of combination therapy, where several drugs are used together. This multi-hit approach could provide a single agent that could deliver potent, specific 'drugs' effectively against multiple targets. An additional bonus is that it eliminates the possibility of a cell inadvertently being exposed to just one of the drugs in a combination, and developing resistance as a result.

future promise

Finding fragments that knock out genes in experimental systems is one thing; actually being able to deliver them to cells is quite another. One company that has been addressing this problem is Intradigm, of Rockville, MD, US. It has applied its TargeTran targeted delivery nanoparticles technology to target siRNA agents to neovascalature in tumours, and other tissues where unwanted angiogenesis is taking place. It found its method was effective in delivering siRNAs that inhibit vascular endothelial growth factor pathway genes after intravenous administration to an animal model. The company now aims to develop siRNA-based anti-angiogenesis therapeutics.

Although still a very long way from providing successful, licensed medicines, the enormous interest in RNAi clearly shows its potential. Several companies, including Alnylam, have stated they hope to begin clinical trials as early as 2005.

It remains to be seen whether the current hype will indeed lead to the medicines of the future.