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forum Working with ribonucleic acid (RNA)-based biotechnologies) Advances in the laboratory now indicate that ribonucleic acid (RNA)-based biotechnologies are fast approaching application in animals. The most likely area of use will be in disease prevention or mitigation. There are several RNA-based biotechnologies but, in general, they all aim to reduce the production of protein form a given gene. At present, RNA-based biotechnologies have most promise in combating virus infection in animals through reducing viral gene activity. RNA-based biotechnologies are likely to impact on animal health within the next ten years. RNA-based approaches to regulate gene activity have been practiced in the lab for some time. Significant technical advances and validation of strategies now mean they are likely to see application in animals in the field soon. There are a variety of RNA-based biotechnologies differing in technical aspects of the mode of action. However, they all aim to reduce the production of protein from a given gene. This article will introduce the different types of RNA–based biotechnologies, briefly indicate how they might be delivered to an animal and indicate possible applications. The focus will be only impact to animal health. RNA-interference (RNAi) Although less than a decade has passed since RNAi was shown to work in mammals, this term has come common interaction leads to the destruction of the messenger RNA, place in discussion about regulating gene expression. But preventing production of protein which should have been what is RNAi? In its simplest definition it is the use of RNA produced by that messenger RNA. This activity is often (ribonucleic acid) molecules to modulate the activity of a called ‘gene knockdown’. To give an example, if an RNAi target gene. When you delve deeper a multitude of terms molecule was designed to target a viral gene, then its appear – siRNA, shRNA, miRNA to name a few. These terms activity would prevent normal virus production will be expanded more later; but first how are genes activated. Genes within an animals’ genome are transcribed when 8 can bind directly to a target messenger RNA. This RNAi molecules come in different forms. As independent small double stranded RNA molecules called short-interfering RNA (siRNA) that can only survive the appropriate activation signals are present in a given cell. transiently in an animal. For prolonged an activity the The action of transcription results in the production of siRNA must be incorporated into an expression vector that messenger RNA. An RNA molecule comprises of a string of functions like a gene. In this case they are called short- nucleotides; the string of nucleotides in messenger RNAs hairpin RNA (shRNA), due to their physical structure, or encode for the proteins that make up an animals cell. It is microRNA (miRNA). The latter form are found naturally in against this messenger RNA that RNAi acts. The important animals and are now believed to play a major role in functional component of RNAi is a short single-stranded normal cell growth and survival. Synthetic miRNA can also RNA molecule of about 20 nucleotide bases in length which be constructed in the lab for delivery to an animal. 2007 • 4 forum Other RNA-based biotechnologies For both gene therapy delivery and transgenic strategies, Although currently most optimism for successful more elaborate RNAi molecule designs that incorporate application of RNA-based biotechnologies revolves around inducible gene knockdown activity are the use of RNAi, other types of RNA molecule exist that possible. In this case the RNAi molecule is could be applied to animals. These include RNA decoys to only produced in the presence of the block activity of an enzyme. A potential application would appropriate inducing factor. be to block the activity of a viral polymerase and thus The practicalities of prevent viral replication. For some time the prospect of delivery of RNA molecules to using antisense molecules or other oligonucleotide-type animals are likely to be molecules. common to those addressed Both antisense and RNAi molecules evoke normal during development of drug cellular enzymes to destroy the target RNA. There are treatment strategies and some RNA molecules that contain their own enzymatic vaccination. activity. Ribozymes, that specifically bind to and subsequently catalyze the cleavage of other target RNA molecules, and represent an interesting alternative to the RNAi. Unfortunately ribozyme design has proved difficult. It is likely that further RNA molecules will be identified. Applications of RNA-based biotechnologies in animal health For example, recently RNA molecules that can destroy Most applications that could be miRNA called antagomirs. To date, all RNA-based applied to animals are still in the biotechnologies function to reduce target gene activity. experimental phase. There are, however, several preclinical studies Delivering RNA-based molecules for applications in humans and it is How can RNA-based molecules be delivered to animals? reasonable to expect that these will Two different strategies are possible. First they could be accelerate the use of RNA- delivered in a gene-therapy or vaccination-type of based biotechnologies in approach. Depending on what type of RNA-based animals. A few current studies molecule was delivered the effect could be either a will be described to illustrate transient of stable change in gene activity. For example the the potential of RNA-based use of siRNA would result in a transient alteration in gene biotechnologies. activity somewhat similar to vaccination, except that the Perhaps the most likely timescale would most likely be considerably shorter that application of RNA-based the intended protection period offered by vaccination. This biotechnologies will be to interfere with timescale will impact on what you could use siRNA for. virus infection. Several projects are With a gene therapy delivery strategy the aim would be to focussing on the use of RNAi to prevent virus enable the change in gene activity to be present for as replication and thus combat virus induced long as cells carrying the RNA-based molecule survive in disease. These studies are most the animal. For most applications, actual delivery vehicles advanced for FMDV, EIAV and flu. remain to be evaluated. Commonly the target is the viral Alternatively, shRNA or miRNA could be delivered as a polymerase. Antisense-type transgene to generate genetically modified animals. This approaches are also being targeted approach would result in the stable inheritance of the against viruses. In one project the RNA molecule RNAi transgene by offspring and subsequent generations functions as a decoy, again aiming to prevent influenza of animals derived from the original founder animal. viral polymerase enzyme activity to intervene in virus replication. 2007 • 4 9 forum Alternative uses of RNA-based biotechnologies Another widely discussed concern relates to possible that can be envisaged for the future include inefficient eaffects on non-target genes. This is possible modulation of some aspect of the immune system through weak interaction of the RNA molecule to In this scenario the variation in gene activity seen in natural animal populations could be messenger RNAs transcribed form non-target genes. Obviously if this occurred in a living animal the effect mimicked through the knockdown afforded by could be quite severe. This is an area of intense RNAi. In a similar vein, some other aspect of animal investigation at the moment and the imminent human function, for example, growth could be modulated. clinical trials with RNA-based biotechnologies will be informative in this regard. Potential risks associated with RNA-based biotechnologies It is now widely acknowledged that RNAi can be an efficient way to reduce gene activity. However, as with most emerging technologies, some concerns remain to be overcome. The use of RNA-based technologies can provoke a evoke the internal immune response within an animal’s cell. In extreme situations this can lead to cell death. For those applications that aim to combat virus infection this may be an advantage. 10 2007 • 4 Bruce Whitelaw