When Craig C Mello and Andrew Fire published their discovery of ribonucleic acid (RNA) interference on Nature in 1998, it was a gradual birth of a technology with immense potential in unravelling the secrets of life and a help to mankind to cope up with health and disorders. The research, which found out a potent gene silencing effect after injecting double stranded ribonucleic acid (RNA) into C elegant, in turn helped the scientists to bag nobel prize for Physiology or Medicine in 2006.
The task of specific gene knockdown in in vitro has been facilitated through the use of short interfering RNA (siRNA), which is now widely used for studying gene function and identifying and validating new drug targets.
Several groups explored the possibility of using siRNA for dissecting cellular pathways through siRNA mediated gene silencing followed by gene expression profiling and systematic pathway analysis. An approach, which combines the use of siRNA mediated gene silencing, mediated microarray screening and quantitative pathway analysis, can be used in functional genomics to elucidate the role of the target gene in intracellular pathways. The approach also holds significant promise for compound selection in drug discovery.
siRNA in biotherapeutic applications
RNA interference (RNAi) is a robust method of post-transcriptional silencing of genes using double-stranded RNA (dsRNA) with sequence homology driven specificity. The dsRNA is 21-23nt long and is converted to small interfering RNA (siRNA), which then mediates gene silencing by degradation or blocking of translation of the target messenger RNA (mRNA).
RNA interference is a simple, fast and cost effective alternative to existing gene targeting approaches both in in vitro and in vivo. The discovery of siRNAs that cause RNA interference in mammalian cells opened the door to the therapeutic use of siRNAs. Highly intense research efforts are now being carried out for developing siRNAs for therapeutic purposes.
Recent advances in the design and delivery of targeting molecules now allow efficient and highly specific gene silencing in mammalian systems. Synthetic siRNA libraries targeting thousands of mammalian genes are publicly available for high throughput genetic screens for target discovery and validation. The clinical potential of aptly designed siRNAs in various types of viral infections, cancer and renal and neurodegenerative disorders has proven worth in recent inventions.
Novel therapeutic strategies of siRNA technology, which is the latest development in nucleic acid based tools for knocking down gene expression, are now most promising and it has potential for silencing genes associated with various human diseases.
The biochemical and genetic approaches taken by several research groups have led to the current models of the RNAi mechanism. In these models, RNAi includes both initiation and effector steps.
The initiation step involves input of dsRNA, which is digested into 21-23 nucleotide small interfering RNAs. These siRNAs are produced when the enzyme Dicer (RNase III family member dsRNA-specific ribonucleases) processively cleaves dsRNA (introduced directly or via a transgene or virus) in an ATP-dependent, processive manner. Due to repeated cleavage the dsRNA is degraded into 19-21 bp duplexes (siRNAs), each with 2-nucleotide 3' overhangs.
Then comes the effector step, where the siRNA duplexes bind to a nuclease complex to form the RNA-induced silencing complex, or RISC. An ATP depending unwinding of the siRNA duplex is required for activation of the RISC. The active RISC then targets the homologous transcript by base pairing interactions and cleaves the mRNA ~12 nucleotides from the 3' terminus of the siRNA.
If an amplification step is included within RNAi pathway, it will lead to copying of the input dsRNAs, which would generate more siRNAs. This is the concept of siRNA mediated gene silencing event. siRNA is a very powerful gene silencing tool because of the enormous potential of RNA interference in silencing the production of any protein. RNAi is therefore a useful research tool to determine the function of genes. RNAi also takes the science of functional genomics out of model experimental systems, such as arabidopsis and mice, to allow scientists to study gene function in most plants and animals.
In plant research, RNA interference is used as a tool to obtain information about the function of individual genes through specific silencing. Scientists are focusing in particular on genes, which influence growth behaviour or stress resistance. One of the famous examples of antisense RNA technology was its utilisation in production of FlavrSavr tomato which was first launched in USA. It is no longer grown today.
Various approaches in safety research use RNA interference to prevent the spread of genetically modified plants, e.g. by inhibiting pollen development. In this approach, the pollen development is suppressed by using RNA interference to switch off a key enzyme for pollen ripening. As a result, the plants cannot reproduce and transgenic traits are not transferred. A current project in genetically modified fruits with apple plants aims to inhibit the formation of transgenic pollen by grafting non-transgenic apple plants to a transgenic rootstock. Starting from the transgenic rootstock, genes in the non-transgenic apple plants are to be switched off using RNA interference.
Recent advances in utilisation of siRNA had made impact in human health also. In fighting bird flu with siRNA, scientists at CSIRO's Australian Animal Health Laboratory (AAHL) in Geelong had launched a potentially revolutionary research project aimed at reducing the threat of the lethal H5N1 avian influenza virus. The therapeutic option involved in delivering small RNA molecules to chickens in their drinking water or via an aerosol spray, priming the birds' own natural RNAi defences to recognise and destroy the virus.
In another instance, Kylin Therapeutics is known to have collaborated with Fort Dodge Animal Health for an ambitious project that involved use of patented RNA nanoparticle technology called "pRNA" to explore the enormous potential of RNA interference (RNAi) and develop new RNA based therapeutics for the treatment of cancer. In return for its participation in this effort, Fort Dodge will receive an exclusive license to the pRNA/RNAi therapeutic compounds for the treatment of cancers in companion animals. Kylin will retain rights to the therapies for human use.
siRNAs research is also underway to address hepatitis. The power of small RNAs to shut down specific gene activities has been brought to bear on an animal model of this disease. Mice infused with siRNA against a cell death receptor leads to recovery of liver function after experimentally induced injury.
In fact, recent studies suggest that small interfering RNAs (siRNAs) hold promise as a therapeutic agent even without further engineering. Investigators therefore have provided in vivo evidence that infusion of siRNAs into an animal can alleviate hepatitis disease.
Apart, extensive research is underway in the area of anticancer siRNA therapy. At least two siRNA based anticancer therapies, both delivered to tumours in nanoparticles, have begun human clinical trials. Now, reports highlight the progress that researchers are making in developing broadly applicable, nanoparticle-enabled siRNA anticancer therapeutics.
New drugs typically take more than a decade to get from the lab to market. And before RNAi drug developers can make much progress, they must solve a daunting problem and that is getting gene-silencing medicines to where they are needed in the body.
Compounds used in test-tube studies trigger RNA interference break down in seconds in the bloodstream. Once that stability problem is solved, researchers still must keep devising ways to deliver the drugs to desired tissues in a form that penetrates cells.
Thus the challenge is broadening and no longer depends just on to the concept of siRNA and its ability to silence gene and alter protein production. It also extends to efficient methods of delivery, which remains a daunting task keeping the inherent instability of RNA molecules in mind.
(The author is CEO of Geneombio Technologies, Pune)