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In 1989, Sidney Altman and Thomas Cech received a Nobel Prize in Chemistry for the discovery of RNA as a biocatalyst, in addition to being the molecule of heredity. While announcing the award, the Royal Swedish Academy of Sciences made a perceptive comment: "future use of gene shears will require that we learn more about the molecular mechanisms of RNA". The discovery of an evolutionarily conserved mechanism of RNA-interference (RNAi) accentuates this objective.
RNAi is an effective post-transcriptional mechanism involving direct messenger-RNA (mRNA) degradation or inhibition of protein translation via double-stranded RNA in a sequence specific manner. Present in all eukaryotes, from yeast to mammals, this system helps determine the active genes and their functioning within the living cells. This method of gene silencing is essentially adapted for both gene expression and regulation of cell growth, and provides considerable defence to host and its genome against parasitic viruses and transposons.
It is believed that the process of RNAi evolved over time as a cellular defence mechanism against foreign invaders like RNA viruses, which temporarily exist in the host cell in a double-stranded form once replicated. This intermediate form triggers RNAi in the host cell, causing inactivation of the virus' genes and thereby preventing an infection. Likewise, RNAi also aids in combating the spread of DNA segments like transposons, which jump along the entire genome causing mutations that lead to cancer. Similar to RNAi, transposons take up the double-stranded intermediate form that activates RNAi within the host cell.
RNAi was first observed in the flower petunias by plant biologists in an attempt to deepen the flowers' purple colour. The introduction of the pigment-producing gene within the flower, suppressed the colour, rather than intensifying it, thereby forming white patches over the flowers. Nevertheless, the actual discovery of RNAi was fairly accidental, when the American scientists Andrew Fire and Craig Mello were performing experiments on the anti-sense RNA. They were awarded the Nobel Prize for Physiology or Medicine soon after their work was published in 1998. Likewise, the genes responsible for RNAi mechanism were discovered in 2000 by scientists Brenda Bass and her colleagues while working on genetically altered strains of roundworm C. elegans. It was also determined that 3 of the 7 genes involved in RNAi process belong to smg gene family, which led the molecular biologists to utilize this mechanism as an experimental tool for degradation of mRNA in the living cells of C. elegans and Drosophila. Ever since, there have been numerous experiments on plants and mammalian cell cultures involving the implementation of exogenous double-stranded RNAs (dsRNAs). These being complimentary to the targeted mRNAs, cause the attenuation or suppression of gene expression within the cells. Consequently, these experiments serve as the basis for the study of reverse genetics, research and are enormously beneficial if utilized in drug target validation. With the current knowledge on the genetic etiology of many diseases, RNAi has been targeted as a potential therapeutic tool, both in vivo and in vitro. Several human diseases in animal models have been successfully treated using the RNAi technique. Moreover, a recent report by Grimm et al not only declares an affirmative use of the RNAi technique but also serves as a sombre warning that the effectiveness of the RNAi techniques depends on the absolute understanding of the molecular mechanisms involved.
This dissertation aims to elucidate the primary mechanism of RNA interference and discusses its potential in therapeutic sector by addressing specific examples of a few significant diseases. The recent advancements in the RNAi technology, its delivery in vivo of a diseased host and its definitive limitations will also be addressed.
2.0 Cellular mechanism of RNA Interference
MicroRNAs (miRNAs) and the small interfering RNAs (siRNAs) are most vital in the process of RNAi. The RNA silencing pathway comprises of two main phases; the first phase involves the recognition and cleavage of long dsRNA molecules by RNase-III enzyme called Dicer in the cytoplasm. The dsRNAs utilized for this process could either be adapted from an endogenous source such as pre-miRNAs from RNA-encoding genes or a synthetic exogenous source such as infection from foreign viruses. However, when micro-RNAs are utilized, they are first subjected to post-transcriptional modification with the help of a long RNA coding gene called pri-miRNA. This serves as a primary transcript to the miRNA and is processed in the nucleus to form a stem-loop structure of 70 nucleotides called the pre-miRNA. The pre-miRNA comprises of RNase III enzyme called Drosha and dsRNA-binding protein called Pasha. When acted upon by the enzyme Dicer, the dsRNA portion of the pre-miRNA is cleaved. The resultant short interfering RNAs (siRNAs) formed are made up of 21-28 nucleotide duplexes with symmetric 2-3 nucleotide 3' overhangs and forms a part of a 'silencing complex'.
In the second phase, one of the two strands of each siRNA molecule; the guide strand, is assembled into a multiprotein RNA-induced silencing complex (RISC) with the help of an Adenosine triphosphate (ATP) independent activity of the protein components of RISC. After the activation of the RISC complex, the siRNAs direct them towards the homologous target mRNAs via their unwound antisense strand, known as the anti-guide strand. This consecutively triggers the endonucleolytic cleavage of the mRNA by a Slicer enzyme called Argonaute-2 which is a catalytic component of RISC. This protein is localised in specific regions in the cytoplasm called the P-bodies which have acutely high rates of mRNA decay. Additionally, the process of translation of the target mRNA strand is functional to RNAi, since RNAi can be more effective against non translated targets.
The cleavage of the target mRNA begins at a single site in the middle of the duplex region of the guide siRNA strand and the target mRNA, about 10 nucleotides upstream of the 5' end of the siRNA. This results in target mRNA degradation and subsequently causes gene silencing. Since the anti-guide strand of the siRNA is conserved within the RISC complex, it is further utilized as a catalyst for the degradation of additional copies of mRNA. Furthermore, RNAi does not necessarily require a complete sequence homology for the target mRNA, with as few as seven consecutive complementary base pairs being adequate enough to bypass RNAi-mediated silencing. As a result, this process is highly effective and advantageous in mammalian cells.
Translation of RNAi technique to Gene Therapy
To exploit the use of RNAi technique for its application in gene therapy, chemically synthesized siRNAs are used. Other forms of RNA produced from the inserted DNA molecule such as short-hairpin RNA or small-hairpin RNA (shRNA) can also be utilized, although they have proven to be more toxic than siRNAs. The most standardized and efficient way to deliver RNAi systemically into the host cells is via packaging inside nanoparticles. These particles include artificially produced lipidoids which are lipid-like molecules, and their variations could be customized for distinct RNAi therapies and drug development. It is possible to silence upto five different genes at the same time with the help of these RNAi injections, whose effects lasts up to four weeks. This improves the possibility of treating diseases with multiple genes.
The siRNA delivery can be made possible for a wide range of diseases such as cancer, viral infections, neurodegenerative disorders etc. The siRNA delivery to infected lungs can block the destructive action of respiratory syncytial virus. Moreover, when siRNAs are targeted to immune cells such as cutaneous dendritic cells or macrophages, they may aid in preventing allergic skin diseases. The recent development in the research sector for therapeutics related to various diseases is uncovered in the subsequent sections.
4.0 Applications of RNAi in biomedical research for drug development and Gene therapies for different diseases
RNAi has become a very powerful tool which has enormous potential impact on medicine and biomedical research. For that reason, there have been extensive investigations made to understand the role of RNAi in normal as well as diseased cells, and to exploit the mechanism for medical therapeutics.
A straightforward and practical application of RNAi in research is the knock down of the expression of the target genes and monitoring its consequences. Prior to the discovery of RNAi, this process was extremely laborious and time consuming. The use of RNAi can accelerate the evaluation of these target genes for the drug development, since it allows the rapid and effective suppression of any protein expression within nearly all cell types. RNAi can also be potentially useful if applied to screen large sets of gene families and target their cell kinases, ion channels or G protein-coupled receptors, in order to locate the starting point for the development of a new drug.
4.1 RNAi in Cancer
The main targets of the RNAi-based therapies are those diseases that can be treated by the knock down of one or several genes involved. For instance, cancer is often caused by the overactivity of genes whose suppression could productively halt the disease. At present, various pharmaceutical companies are testing RNAi-based therapies for different forms of cancer. Another new RNAi based research concept for the development of cancer drugs is the genotype-specific drug target. It primarily targets those proteins in the body, whose inactivation causes toxicity only to those cells that carry a cancer-specific genetic lesion. Hypothetically, these drugs would target for the cancerous cells more effectively than the ordinary cytotoxic drugs, due to their prevailing specificity towards cancer lesions. RNAi technique can also be particularly used to expose the synthetic lethal interactions; a combination of two non-lethal interactions resulting in cell death within the mammalian cells, with its first screenings recently described in the research sector. However, due to the varied nature of tumours, it has proved to be a rather complicated approach for clinical trials.
4.2 RNAi in Viral Infections
Yet other potential targets for RNAi-based therapies are viral infections. The activity of crucial viral genes can be reduced which in turn would weaken the viruses, and this forms the basis of several studies that hint towards treating viral infections via RNAi. Recent experiments have already made it possible to halt the growth of viruses such as Human Immuno-deficiency virus (HIV), polio and hepatitis C in laboratory-grown human cells. The components of RNAi pathway form a concrete basis for the treatment of such viruses. Like for instance, the siRNA oligonucleotides can be of versatile aid to target genes for suppression as compared to the expensive and prolonged procedure of small-molecule drugs. The main issue that plagues the oligonucleotide based therapy, is how to deliver siRNAs to specific cellular or tissue targets. In the experiments performed by Song and colleagues, they engineered mouse melanoma cells to express GP160 cell surface antigen protein of HIV-1 and implanted these into the host mice. This was followed by the injection of cells consisting of a mixture of siRNA and protamine, a protein that binds DNA, which was designed to target genes regulating the cell cycle (c-myc), apoptosis (mdm2) and angiogenesis (vegf). This mixture inhibited the establishment of tumours expressing the surface antigen GP160, but was ineffective against the remainder. In this case, the antibody either binds to the appropriate antigen or a cell surface receptor ligand. Consequently, this technique is believed to soon enter clinical trials as long as the appropriate target cell and its expressed antigen or receptor is selected.
4.3 RNAi in Huntington's Disease (HD)
Huntington's disease (HD) is a neurogenerative genetic disorder, which affects muscle co-ordination and some cognitive functions. The persons suffering from this disease may have two different alleles; one normal and other HD allele whose protein forms clumps or aggregates to disrupt the normal functioning of the nerve cells. RNAi technique can potentially be used to differentiate the HD allele proteins from the normal ones, and target them for gene silencing with the use of single nucleotide polymorphisms (SNPs). Hence, by generating siRNA templates complementary only to the HD mRNA, the disruptive protein can be degraded.
4.4 RNAi targeting multidrug resistance (MDR) genes and molecules related to DNA repair mechanisms
It is observed that the resistance of cells towards chemo- and radiotherapy often hampers the treatment of diseases like cancer, and the cellular mechanism towards resistance is typically ambiguous. In this case, RNAi is capable of identifying genes involved in multidrug resistance (MDR) by simply knocking down genes from drug-sensitive cells in vitro, exposing them to the appropriate drugs and then evaluating their survival rate. This uncovers the MDR genes, and with the help of clinical trials, the pathways and mechanisms of MDR genes can be authenticated.
When the cancerous cells are under stressed conditions such as chemo- or radiotherapy, they tend to overexpress the proteins of the DNA repair mechanisms for the restoration of therapy-induced DNA damage within the cells. In this case, RNAi technology can potentially be used for the downregulation of the DNA repair genes, thereby increasing the sensitivity of cancer cells towards chemo- or radiotherapy. This could be done by the transfection of siRNAs targeting DNA repair proteins for their suppression. As a result, these molecules are also capable of serving as potential therapeutic targets in conjunction with existing chemotherapy and irradiation.
5.0 Current limitations in RNAi technology
RNAi is still far-fetched in terms of utilizing its powerful potential for treating various genetic disorders. The first and foremost obstacle for converting RNAi technology from an efficient research tool into a practical therapeutic strategy is the effective delivery of the small RNA molecules to its target cell type in vivo. Despite the use of chemical modifications to improve the stability of siRNAs, their systemic delivery still requires further improvement which is limited by their transitory gene silencing effects. It has been reported that after the introduction of siRNAs in the host cell, their extracellular degradation is at the peak at around 36-48 hours, and gradually decreases after 96 hours. Also, due to the considerable inter-animal variation and the differences in the levels of siRNA uptake by the target cells, the degree of silencing is not absolute. Moreover, the differentiated host cells observe longer duration of gene silencing which can last up to several weeks, while the rapidly dividing host cells have a relatively short lived effect of RNAi lasting approximately 5-6 days. This may be accountable to the increasing dilution of siRNA with repetitive cell division and the simultaneous enzymatic degradation within the host cells. For these reasons, the only promising RNAi technology in the short term appears to be the delivery of siRNA to confined compartments, such as the eye, since it bypasses most of the problems associated with systemic delivery.
Sometimes, despite the high specificity of RNAi, it may lead to a sequence-independent interferon response, if the dsRNAs or siRNAs present are 21 base-pair or longer. Additionally, as reported, high concentrations of vector-based or synthetic siRNAs can trigger this response in sensitive cell lines. The interferon triggers the degradation of mRNA by activating RNase and dsRNA-dependent protein kinase (PKR), whose phosphorylation eventually leads to inhibition of mRNA translation. This response, causing an obstruction in the RNAi technique by degrading the siRNAs needs to be resolved with greater understanding of the interactions between the siRNAs and the target gene. Likewise, it is also reported that siRNAs, when recognised by toll like receptors, can activate cells of the immune system such as dendritic cells, and transmit a danger signal to trigger a proinflammatory response. This provides an evidence that RNAi technology may trigger autoimmune diseases, in vivo.
RNAi silencing based on nucleic acid molecules may also have adverse effects on off-target genes due to the similarities in nucleic acid sequences. Therefore, when siRNAs are not carefully selected, it may either cause mRNA degradation in those molecules which are partially complementary, or the silencing of the off-target molecules. Remarkably, from the studies in primitive organisms, it has been observed that complete dsRNAs instead of synthetic siRNAs cease the off-target gene effects. Various algorithms and tools have been designed to select appropriate siRNA target sequences with low off-target effects, although the process is still in an impending phase.
For the treatment of neurogenerative diseases in humans, RNAi is yet at a primary research level. The RNAi technology is still facing complications due to challenges in delivering the vector into the nerve cells in the brain for therapy, and the testing of similar viruses in animal models. Although it promises 70% accuracy, it could prove potentially deleterious to the off-target cells.
Lastly, the problem of potential toxic side-effects on the host cells must be resolved. Like for instance, the use of large doses of siRNAs are believed to have incredibly low toxic effects in comparison with miRNA products and are therefore, easier and safer to operate. However, the use of synthetic siRNAs arise a bunch of issues including the aforementioned in vivo systemic delivery and responses of immune stimulation.
RNAi is an attractive mechanism that has successfully evolved from a significant research tool used to explicate the function of novel genes, to a potential therapeutic agent in the field of scientific technology. While there have been numerous host targets discovered with complex cellular pathways for which gene silencing in vitro and in animal models has been successful, their translation in vivo to a more complex arena is ever-challenging. However, the use of RNAi therapy in conjunction with chemotherapy or irradiation may provide improved desirable effects, although by following multiple dosing treatments. Gradually, with the ongoing research, we can closely relate the regulation of gene expression with the RNAi technique and some day wish to overcome the obstacles existing in this technology, thus exploiting this potentially powerful tool in therapeutics.