Introduction
RNA interference (RNAi) is a potent method using only a few double stranded RNA (dsRNA) molecule per cell to silence the expression of protein. Long ago scientists conducted gene knock out using antisense, dominant negative or knockout techniques which were ineffective, but the discovery of RNAi has enabled to knock out gene in any organism efficiently. RNA silencing was first discovered in transgenic plants, where it was termed co-suppression or post trans c reptional gene silencing (PTGS). Five years ago the evidence for RNA silencing emerged from experimental observation on Caenorhabditis elegans, It’s a phenomenon of gene silencing which offers a quick and easy way to determine the function of a gene. It’s a natural catalytic process and an intrinsic propaerty of every cell of every multicellular organism. And RNAI technique is 1,000-fold more effective than antisense. The new field of RNAi based genomics is increasingly being qualified as a fundamental paradigm shift for biomedical research and development and quite possibly the start of a veritable revolution for the development of modern therapeutics.
2 Definition
RNA silencing is a sequence specific RNA degradation process that is triggered by the formation of double stranded RNA that can be introduced by virus or transgenes. Duplexes 21- nucleotide (nt) RNAs with symmetric 2-nt 3’overhangs are introduced into the cell mediating the degradation of mRNA. (i.e) long double strand RNA don’t introduced in to the cell then fragmented ,but it is intrduced as a short double strand RNA of 21 pb is introduced and combined with DICER protein. According to central dogma of molecular biology, proteins are made in two steps. The first step, trans c ripion, copies genes from double stranded deoxyribonucleic acid (ds DNA) molecules to mobile, single- stranded ribonucleic acid (RNA) molecules called mRNA. In the second step, translation, the mRNA is converted to its functional protein form. Since there are two steps to making a protein, there are two ways of preventing one from being made. Scientists have made exciting progress in blocking the protein synthesis through the second step, translation. One way they have accomplished this is by inserting synthetic molecules that triggers a cellular process called RNA interference.
History
The revolutionary finding of RNAi was proceeded by reports of unexpected outcomes in experiments performed by plant scientists in the USA and The Netherlands. The goal was to produce petunia plants with improved flower colors. To achieve this goal, they introduced additional copies of a gene encoding a key enzyme for flower pigmentation into petunia plants. Surprisingly, many of the petunia plants carrying additional copies of this gene did not show the expected deep purple or deep red flowers but carried fully white or partially white flowers. When the scientists had a closer look they discovered that both types of genes, the endogenous and the newly introduced transgenes, had been turned off. Evidence was obtained for
post trans c riptional inhibition of gene expression that involved an increased rate of mRNA degradation. This phenomenon was called "co-suppression of gene expression", but the molecular mechanism remained unknown.
A few years later plant virologists made a similar observation. In their research they aimed towards improvement of resistance of plants against plant viruses. At that time it was known that plants expressing virus-specific proteins show enhanced tolerance or even resistance against virus infection. However, they also made the surprising observation that plants carrying only short regions of viral RNA sequences not coding for any viral protein showed the same effect. They concluded that viral RNA produced by transgenes can also attack incoming viruses and stop them from multiplying and spreading throughout the plant. They did the reverse experiment and put short pieces of plant gene sequences into plant viruses. Indeed, after infection of plants with these modified viruses the expression of the targeted plant gene was suppressed. They called this phenomenon “virus-induced gene silencing” or simply “VIGS”. These phenomena are collectively called post trans c riptional gene silencing.
After these initial observations in plants many laboratories around the world searched for the occurrence of this phenomenon in other organisms. Mello and Fire's 1998 Nature paper based on research conducted with their colleagues at the Carnegie Institution of Washington and the University of Massachusetts reported a potent gene silencing effect after injecting double stranded RNA into C. elegans. In investigating the regulation of muscle protein production, they observed that neither mRNA and antisense RNA injections had an effect on protein production, but double-stranded RNA successfully silenced the targeted gene. As a result of this work, they coined the term RNAi. The discovery of RNAi in C. elegans is particularly notable, as it represented the first identification of the causative agent (double stranded RNA) of this heretofore inexplicable phenomenon. Fire and Mello were awarded the Nobel Prize in Physiology or Medicine in 2006 for their work
Silencing by siRNA
RNAi mediated by the introduction of long dsRNA has been used as a method to investigate gene function in various organisms including plants, planaria, Trypanosomes, Drosophila, mosquitoes and mouse oocytes .
Long dsRNA enables the effective silencing of gene expression by presenting various siRNA sequences to the target RNA. The applicability of this approach is limited in mammals because the introduction of dsRNA longer than 30 nt induces a sequence-nonspecific interferon response. Interferon triggers the degradation of mRNA by inducing 2′-5′ OLIGOADENYLATE SYNTHASE,
which in turn activates RNASE L. In addition, interferon activates the protein kinase PKR, which phosphorylates the translation initiation factor eIF2α leading to a global inhibition of mRNA translation. To test whether siRNAs could mediate effective silencing of gene expression without inducing the interferon response, Tuschl and colleagues introduced chemically synthesized siRNA into mammalian cells .
First, they showed that the synthetic siRNAs were functional in vivo by co-transfecting Drosophila S2 ells with luciferase siRNA and a luciferase reporter construct. This resulted in a loss of luciferase activity comparable to that obtained with long dsRNA. More importantly, they showed that siRNA transfection resulted in the sequence-specific silencing of luciferase expression, as well as the endogenous nuclear envelope proteins lamin A/C, in several mammalian cell lines without activating nonspecific effects. These findings have led to the widespread use of this technology to study gene function including the targeted disruption of clinically relevant genes, alluding to the potential therapeutic applications of RNAi-based technologies.
To promote efficient gene silencing using an siRNA to a single site in the target mRNA, consideration of the siRNA sequence is crucial. Although the rules that govern efficient siRNA-directed gene silencing remain undefined, it is known that siRNAs that target different regions of the same gene vary markedly in their effectiveness. The base composition of the siRNA sequence is probably not the only determinant of how efficiently it will silence a gene. Other factors that are likely to have a role include the secondary structure of the mRNA target and the binding of RNA-binding proteins . In an attempt to optimize the siRNA sequences, several groups have used a SYNTHETIC OLIGODEOXYRIBONUCLEOTIDE/RNASE H METHOD to determine sites on the mRNA that are in a conformation that is susceptible to siRNA-directed silencing. These studies found that there was a significant correlation between the RNase-H-sensitive sites and sites that promote efficient siRNA-directed mRNA degradation. Vickers et al. found that placing the mRNA recognition site of a usually active siRNA into a highly structured RNA region abrogated its ability to inhibit gene expression.
Although this work indicates that there is an interplay between the effectiveness of the siRNA and the mRNA structure of the target region,more work is necessary to define this relationship precisely. Recently, several groups have used either Escherichia coli RNase III or recombinant human Dicer to cleave in vitro transcribed long dsRNA into siRNAs that can be transfected into mammalian cells. This approach allows for the presentation of siRNAs with multiple specificities to the target without activating an interferon response.
Biochemical characterization showed that siRNAs are 21–23-nt dsRNA duplexes with symmetric 2–3-nt 3′ overhangs and 5′-phosphate and 3′-hydroxyl groups (FIG. 2).
This structure is characteristic of an RNASE III-like enzymatic cleavage pattern, which led to the identification of the highly conserved Dicer family of RNase III enzymes as the mediators of the dsRNA cleavage15–17. Extensive biochemical and genetic evidence has provided a better understanding of how long dsRNAs could cause the degradation of the target messenger RNA (FIG. 3)
Several studies have shown that this process is restricted to the cytoplasm . In the first step,Dicer cleaves long dsRNA to produce siRNAs. These siRNAs are incorporated into a multiprotein RNA-inducing silencing complex (RISC). There is a strict requirement for the siRNA to be 5′ phosphorylated to enter into RISC, and siRNAs that lack a 5′ phosphate are rapidly phosphorylated by an endogenous kinase. The duplex siRNA is unwound, leaving the antisense strand to guide RISC to its homologous target mRNA for endonucleolytic cleavage. The target mRNA is cleaved at a single site in the centre of the duplex region between the guide siRNA and the target mRNA, 10 nt from the 5′ end of the siRNA. Interestingly, endogenously expressed siRNAs have not been found in mammals. However, the related micro (mi)RNAs have been cloned from various organisms and cell types. These short RNA species (~22 nt) are produced by Dicer cleavage of longer (~70 nt) endogenous precursors with imperfect hairpin RNA structures .
The miRNAs are believed to bind to sites that have partial sequence complementarity in the 3′ untranslated region (UTR) of their target mRNA, causing repression of translation and inhibition of protein synthesis. In addition to Dicer, other PAZ/PIWI DOMAIN PROTEINS (PPD), including eukaryotic translation initiation factor 2C 2 (eIF2C2), are likely to function in both pathways.
Non-specific and specific dsRNA silencing pathways :
The presence of extremely low levels of viral dsRNA triggers an interferon response (called acute-phase response) which resulting in RNAase L activation. This cascade induces a global non-specific suppression of translation, which in turn triggers apoptosis . Small dsRNA called siRNA specifically switched off genes in human cells without initiating the acute phase response. Thus these siRNAs are suitable for gene target validation and therapeutic applications in many species, including humans. The recent success in triggering the RNAi pathway in vertebrate systems now opens the door to direct use of dsRNA molecules as therapeutic agents with exquisitely controllable specificity to alleviate human disease. In addition to the promise of finally achieving truly personalized machines, this approach holds the potential for greatly accelerated and more cost-effective preclinical development while bypassing some of the key obstacles met by antisense therapeutics, for example, the instability of ssRNA molecule.
6 Applications of RNA interference
RNAi tools for functional genomics
A major challenge in the post-genome era of biology is to determine the functions of all the genes in the genome. A straightforward approach to this problem is to reduce or knock out expression of a gene with the hope of seeing a phenotype that is suggestive of its function. Insertional mutagenesis is a useful tool for this type of study, but it is limited by gene redundancy, lethal knock-outs, nontagged mutants and the inability to target the inserted element to a specific gene. RNA interference (RNAi) of organisms genes, using constructs encoding self-complementary ‘hairpin’ RNA, largely overcomes these problems. RNAi has been used very effectively in Caenorhabditis elegans functional genomics, and resources are currently being developed for the application of RNAi to high-throughput functional genomics.
One of the most exciting opportunities offered by RNAi is the facility to identify all the genes required for certain physiological processes using genome-wide RNAi screens. High-throughput screens to identify genes involved in development and carcinogenesis have been successfully carried out in C. elegans and
D. melanogaster and w eb databases that archive and distribute RNAi data for these organisms have been developed (www.RNAi.org and www.flyRNAi.org).The ability to extend such screens to mammalian systems is potentially very powerful, as most genes have now been identified to some level of accuracy, hence, it should be possible to define the role of genes in cell-based phenotypic assays by systematic inhibition of gene expression. Although synthetic siRNAs are compatible with high-throughput formats genome-scale libraries of chemically synthesized siRNA have not been reported, however, smaller collections for the screening of between 30 and 500 genes have been. An siRNA library that targeted > 8000 human genes based on an expression system in which siRNA duplexes are generated upon transfection into mammalian cells .
Most of the efforts in this area have concentrated on the use of vector-based shRNA libraries. Large-scale gene-knockdown studies using retroviral vectors to express shRNAs have been reported in mammalian cells. Both studies included “molecular bar codes” in the vectors so that individual shRNAs in the cells could be easily detected. The screens identified new members of the pathways analysed, however, the failure to detect certain known members of the pathways highlights the current limitations of these approaches. Adenoviral vectors have been used to generate an shRNA library aimed at knocking down nearly 5000 trans c ripts encoding “druggable” proteins. Potentially, genome-wide screens could be carried out for any process for which a tissue culture model exists. Furthermore, the development of transfected cell arrays in which si/shRNAs are printed onto a modified glass surface provides a way of reducing reagent usage and costs in such large screens. The large amounts of phenotypic data arising from such large-scale knockdown studies will require the development of bioinformatics tools to transform these complex datasets into “digitised” formats that can be readily mined 7 The use of RNA interference in Medicine
siRNA has been widely used in mammalian cells to define the functional roles of individual genes, particularly in disease. siRNAs have been extensively used in cell-based studies for pathway dissection and to knockdown the expression of genes involved in a range of cellular processes, including endocytosis, signal transduction , apoptosis , and the cell cycle , as well as genes relevant to neurodegenerative disease and xenotransplantation. In this section, we will concentrate on the use of RNAi to study disease-relevant genes in cancer, infection, and respiratory disease. It may be possible to exploit RNA interference in therapy. Although it is difficult to introduce long dsRNA strands into mammalian cells due to the interferon response, the use of short interfering RNA mimics has been more successful. The first applications to reach clinical trials were in the treatment of macular degeneration and respiratory syncytial virus, developed by Sirna Therapeutics and Alnylam Pharmaceuticals respectively. RNAi has also been shown effective in the reversal of induced liver failure in mouse models.
Other proposed clinical uses center on antiviral therapies, including the inhibition of viral gene expression in cancerous cells, the silencing of hepatitis A and hepatitis B genes, silencing of influenza gene expression, and inhibition of measles viral replication. Potential treatments for neurodegenerative diseases have also been proposed, with particular attention being paid to the polyglutamine diseases such as Huntington's disease. RNA interference is also often seen as a promising way to treat cancer by silencing genes differentially upregulated in tumor cells or genes involved in cell division. A key area of research in the use of RNAi for clinical applications is the development of a safe delivery method, which to date has involved mainly viral vector systems similar to those suggested for gene therapy.
Despite the proliferation of promising cell culture studies for RNAi-based drugs, some concern has been raised regarding the safety of RNA interference, especially the potential for "off-target" effects in which a gene with a coincidentally similar sequence to the targeted gene is also repressed. A computational genomics study estimated that the error rate of off-target interactions is about 10%. One major study of liver disease in mice led to high death rates in the experimental animals, suggested by researchers to be the result of "oversaturation" of the dsRNA pathway.
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8 Therapeutic potential of RNA interference against cancer
To develop siRNA for cancer therapy, several researchers have investigated siRNA in animal models.
To obtain efficient and long-lived gene silencing using RNAi, several groups have incorporated the siRNA expression cassettes into a variety of viral vectors. An adenovirus, despite its disadvantage in that immunogenicity of viral vector has precluded multiple administrations and resulted in toxicity limitations, is one of the most well-known viral vectors for gene delivery. past experience with AS-ODN and ribozymes suggest that most cationic lipid (liposome) delivery systems are too toxic when used in vivo, some companies (e.g. Nippon Shinyaku, Kyoto, Japan) have developed novel cationic liposomes that can be administered safely in vivo. Yano et al. have used such a liposome to demonstrate that anti-bcl-2 siRNA complexed with liposome had a strong antitumor activity when administered intravenously in the mouse model of liver me tastasis. In addition, Nogawa et al. reported that intravesical injection of polo-like kinase-1 (PLK-1) siRNA/ liposomes successfully prevented the growth of bladder cancer in an orthotopic mouse model. One attractive method is through delivery of siRNA using cancer cell-specific antibo dy. Song et al. showed that an antib ody against ErbB2 fused to a protamine fragment specifically and effectively delivers siRNA only to ErbB2-expressing breast cancer cells. they recently developed an atelocollagen-mediated siRNA delivery system in vivo. In the next section, advances using atelocollagen mediated gene delivery methods are introduced.
Other Therapeutics
The results obtained with animal models are very encouraging. Direct injection into the tail vein of mice of siRNA targeted against apolipoprotein B reduced the cholesterol levels by 60 to 80%. In addition to chemically increasing stability, as mentioned before, the sense strand was conjugated with cholesterol, increasing siRNA binding to human serum albumin and improving the pharmacokinetic properties when compared to unconjugated siRNA . It is important to emphasize that the anti-sense strand, which is responsible for the silencing effect, was not linked to cholesterol, thus making the sense strand a carrier for the antisense.
Clinical trials have already started in humans, targeting the VEGFR1 through intraocular injections of siRNAs to treat age-related macular degeneration, a deterioration of the retina that is the leading cause of blindness in the elderly .
The potential applications of RNAi therapy are extremely broad , including all cases in which a deletion or reduction of a defined gene is desired, i.e., genetic diseases, autoimmunity, viral infections, cancer, and many others. The main advances in the use of RNAi have being made in the antiviral and anticancer fields. Although producing efficient antiviral activity, shRNAs targeted to the HIV-2 genome lost their effect due to the emergence of viral variants harboring a point mutation in the shRNA target region. Therefore, as is the case with antiviral drug therapy, RNAi has to be targeted to several genes or 21mers in the same gene at the same time, preferentially in conserved regions of the viral genome . However, considering the ease of development of shRNA-expressing plasmids, the specific parts of the viral genome of patients could be sequenced, and the siRNA sequences adapted to the viral genome. This probably is the only technology able to advance faster than both the progression of the disease and the changes in the viral genome, therefore potentially allowing a completely personalized treatment.
Recent developments in the delivery of RNAi to the tumor are a new hope for a highly specific cancer treatment , since it theoretically allows the specific targeting of oncogenes present in a given cancer. Targeting oncogenes has already proven to be a very efficient way of fighting cancer since an inhibitor of the Bcr/Abl oncogene was able to dramatically increase survival of patients with chronic lymphomas. The development of this drug took years, and such a strategy may not even be applicable to all kinds of oncogenes, as indicated by the low efficiency of farnesyltransferase inhibitors in reducing the effects of the ras oncogene. RNAi, on the other hand, can be produced and tested for efficacy within weeks and can be applied to any gene, without exception. An example of RNAi already tested against oncogenes is the siRNA against the RasV12 oncogene, with an A to T change when compared to its proto-oncogene . This siRNA silenced the oncogene without affecting the expression of the proto-oncogene and efficiently blocked the growth of a pancreatic cancer cell line in vivo. As a proof of principle that not only mutations but also tran******ions can be targeted, a breakpoint-specific siRNA was capable of selectively inhibiting Bcr-Abl-dependent cell growth .
Methodological advances almost always produce changes in paradigms. The use of RNAi is already producing small changes in paradigms in several fields. For instance, the Akt kinase has been long known to be activated by the phosphorylation of two sites, Thr308 and Ser473. While the kinase that phosphorylates Thr308 has been known for about 10 years, the kinase that phosphorylates Ser473 has remained a mystery despite active research . Using mainly RNAi, Sarbassov et al. showed that mTor in complex with the protein Rictor is responsible for the phosphorylation of this site in Akt. mTor was never considered a candidate for the Akt Ser473 kinase because a very specific and efficient inhibitor of mTor, rapamycin, did not reduce the phosphorylation at this site. It turned out that rapamycin only inhibits the mTor/Raptor complex, without affecting the mTor/Rictor complex. RNAi against mTor reduced the phosphorylation of Ser473 of Akt since it eliminated mTor from both complexes, in contrast to rapamycin, which only affected the mTor/Raptor complex.
Science comes in waves of fashionable subjects and sometimes, after the fashion ends, little substance is left. It definitely seems that this is not the case for RNAi and that after the RNAi hype is over we will be left with two very important things: 1) the discovery and de******ion of a fundamental biological mechanism and 2) an easy, fast and reliable way of knocking down a specific gene. If this holds true, RNAi will very soon be part of our everyday lives.
والله عارفه اني مررررررررررررررره مزوداها بس والله حدعي للي تعملي هوا من كل قلبي :(