PerspectivesAre you interested in submitting a Perspective Article? Be sure to read The Science Advisory Board's Editorial Guides for Perspective Articles. Click here. RNA Interferences by Wim D'Haeze, Ph.D. This year’s Nobel Prize in Physiology or Medicine was awarded on October 2, 2006 to the duo Andrew Z. Fire, currently Professor of Pathology and Genetics at Stanford University School of Medicine, and Craig C. Mello, currently Professor of Molecular Medicine and Howard Hughes Medical Investigator, Program in Molecular Medicine, at the University of Massachusetts Medical School. Fire and Mello were awarded the Nobel Prize for their discovery of an efficient and widely applicable approach to selectively turn off the expression of any gene of interest in the nematode worm Caenorhabditis elegans, a discovery that was first published in 1998 in the journal Nature (Fire et al., Nature 1998;391:806–811). The method is known as RNA interference (RNAi) and was ever since shown to be an effective method for turning off the expression of genes in a wide variety of plants and animals, including humans. The RNAi technique is now widely used in academic and industrial research laboratories worldwide to study the function of genes and plans are underway to develop RNAi-based therapeutic treatments for a wide variety of diseases, but also to prevent major crop losses in agricultural plants. According to the central dogma of molecular biology, information contained in a DNA sequence is transcribed into mRNA which is then translated into proteins. The key observation of Fire and Mello was that neither sense nor antisense single-stranded RNA encoding a muscle protein exhibited an effect after injection into wild-type C. elegans, an observation which was in stark contrast to the injection of double-stranded RNA which caused wild-type C. elegans to behave as worms carrying the defective muscle gene as demonstrated by significant twitching. In other words, the presence in the cell of double-stranded RNA encoding a given gene appears to affect the expression of the given gene. Many years of investigations followed which allowed to decipher the biochemistry behind this remarkable process. It was proven that double-stranded RNA binds to a protein called Dicer which cleaves the double-stranded RNA into smaller fragments that then bind to the protein complex RISC. The single-stranded RNA bound to RISC links RISC to the complementary mRNA molecules by basepairing, followed by the degradation of the respective mRNA molecule and consequently blocking protein production. In other words, double-stranded RNA activates biochemical machinery which degrades those mRNA molecules that carry a genetic code identical to that of the double stranded RNA which ultimately leads to gene silencing. Interestingly, this RNA interference process does not appear to be an artificial situation observed in a laboratory setting, but is as a matter of fact a common process used in the cell for various purposes. For example, when an RNA virus infects the cell, its genome composed of double-stranded RNA is injected in the cell where RNAi assures the degradation of the viral double-stranded RNA preventing the generation of new viruses. In addition, the synthesis of many proteins is controlled by genes encoding microRNA, which, after processing, prevents the translation of mRNA to protein. It is needless to mention that the discovery of RNAi is recognized globally as a major development in eukaryotic research and for the pharmaceutical and biotechnology industry with plenty of applications that are currently being evaluated and that yet have to be engineered, some of which are mentioned hereafter. First, it is well known that viruses develop resistance against currently available therapies. Although in vivo experiments in larger animals still need to be performed as well as the development of efficient delivery methods, data based on in vitro and in vivo experiments are promising and convincingly illustrated the effectiveness of RNAi in inhibiting many viruses that cause severe health and economical problems. Second, preclinical data suggest that protein kinase C α (PKCα) plays a crucial role in tumor development in multiple tumor types, including breast cancer. Antisense oligonucleotides have been designed that bind to an mRNA encoding PKCα. As mentioned above, binding to mRNA creates double-stranded RNA which is subsequently degraded thereby blocking the production of PKCα. This mechanism offers the potential to inhibit tumor growth as demonstrated by encouraging phase II/III clinical trials. Third, Huntington disease is caused by an inherited mutation in a specific gene, i.e. the HD gene, that when mutated encodes for a truncated derivative that exhibits abnormal properties as it misfolds which is toxic to brain cells and leads to Huntington disease. It is thought that RNAi directed at the HD gene would turn off its expression and thus prevent the production of the toxic protein if this can be achieved in a effective and specific matter. Fourth and currently a hot topic related to the use of RNAi is to target prion diseases. It has been shown that mice infected with scrapie, a model which resembles Creutzfeldt-Jakob disease in human for which no treatment is currently available, live longer after exposure to the appropriate RNAi molecules. The downside is that a high proportion of cells (i.e., about 65% of prion genes need to be turned off) must be treated with RNAi before a curing effect could be observed. When these observations are extrapolated to Creutzfeldt-Jakob patients, it is obvious that the approach is still a long way from a cure for prion diseases in humans. Unfortunately, it would have been too good to be true if RNAi-based treatments would not be accompanied by side effects that need general attention. RNA molecules appear to be not so stable in the human body and are not easily absorbed by cells. To circumvent this problem, viruses such as the adeno-associated virus that does not cause disease in human, are employed to hijack their human cell-infection apparatus to facilitate the uptake of RNAi molecules by the cell. Alternatively, the desired RNAi molecules could be chemically modified without affecting the desired RNA binding properties in a manner that when injected in the bloodstream, they effectively enter the target cells. It has also been demonstrated that in some cases a single-base mismatch between an RNAi molecule and its target could block the response completely. In addition, off-target suppression of genes similar but not identical to an RNAi molecule might occur which may have severe consequences. Furthermore, a recent publication reports that mice died from liver toxicity after being treated with high doses of a certain type of RNA. This observation suggests that toxicity issues related to the exposure of RNAi molecules need to be considered. The latter effect was likely due to the high dose and not to the RNA sequence as reducing the number of RNAi molecules substantially reduced toxicity. However, in case of prion diseases in human, it might be madatory to expose human cells to massive amounts of RNAi molecules in order to switch off the expression of relatively high numbers of distinct genes involved in prion disease. RNAi is without any doubt a very powerful approach that has potential to ameliorate a large number of diseases in the near future. Although it is not necessarily true that RNAi molecules that are currently being tested in the clinic will exhibit the considerable side effects as described above, it is important to investigate time and resources in the nearly complete understanding of how a particular RNAi molecule silences a gene of interest in the genetic and biochemical background of a human body and how this RNAi molecule will behave in the short and the long run in a human body that might be exposed to other environmental conditions such as the intake of other medications or the presence of hereditary mutations. Wim D’Haeze is Bio-Engineer in Chemistry and received his Ph.D. in Biotechnology at Ghent University (Belgium) in June 2001. His doctoral thesis work was focused on the understanding of several early steps of the symbiotic interaction between the Gram-negative soil bacterium Azorhizobium caulinodans and the tropical legume Sesbania rostrata. The initial steps require the production of bacterial compounds including signal molecules and complex surface polysaccharides that are pivotal for invasion of the plant tissue and the formation of new organ tissues. In the three subsequent years, he performed post-doctoral research at the Complex Carbohydrate Research Center at the University of Georgia (Athens, GA) dealing in part with the structural and functional characterization of azorhizobial extracellular polysaccharides. Currently, Wim D’Haeze is employed as Science Writer and focuses on a new horizon regarding the molecular basis of devastative neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases, in order to screen for and develop new therapeutics. In addition, he is also a freelance Medical Editor. E-mail: wim.dhaeze@sbcglobal.net. ### << Previous Next >> [ View All Perspectives ] |
|