Final Paper 2008
Dr. J. Noveron
Impact of siRNA in Medicine
How the administration and use of siRNA can be effective in guarding against genetic diseases and its use in other genetic fields
By: Eithan Kotkowski
Out of the countless of breakthroughs in modern medicine through biotechnology, gene mapping, proteomics, and much more comes what might be considered the most important advancement in medicine in year,[ siRNA|http://en.wikipedia.org/wiki/Small_interfering_RNA]. Expanded as: small interfering RNA (ribonucleic acid), siRNA is a kind of RNA interference, or[ RNAi|http://en.wikipedia.org/wiki/Rnai], molecule that is used in genetic research studies to bind and interfere with a specific gene in an organism's genome, essentially blocking the gene expression. Furthermore, siRNA also acts alongside RNAi in various other processes such as in antiviral defense or in chromatin structure organization. It therefore comes as no surprise, due to the complexity of these functions and processes, that countless of studies have had, and continue, to be carried out in order to uncover the mystery of siRNA. In other words, Andrew Fire and Craig C. Mello's efforts in siRNA research became so significant in the world of medicine that they were awarded the Nobel Prize for Medicine in 2006.
An siRNA molecule is essentially a double-stranded RNA molecule composed of 20-21 complementary nucleotides created by the breakdown of a larger dsRNA molecule. These dsRNA molecules could be either endogenous, as in two RNA molecules transcribed from the original DNA and arranged in pairs, or exogenous, mimicking the process by which viruses reproduce (i.e. Mad Cow disease). The [enzyme responsible for the processing of dsRNA molecules into siRNAs is known as Dicer, a cytoplasmic enzyme from the RNAse III family, which arranges dsRNAs into smaller fragments with a 5' phosphate group and a 3' end with two free nucleotides and a hydroxyl (-OH) group (Gene Link, 2003).
SiRNAs suppress active gene expressions by inherently splitting its corresponding messenger RNA, mRNA, in two by way of the interaction of the double-stranded siRNA with the [RNA\- !worddav8a6e411902589137faf7be41387e4195.png|height=288,width=288! induced silencing complex]. Both halves of the mRNA degrade and are digested by the cytoplasmic lysosomes, concluding the basic process by which siRNA is able to block and suppress gene expression. On the other hand, siRNAs also promote DNA modification, in turn facilitating chromatin silencing since siRNA favors heterochromatin expansion through the [RNA\- !worddav8a6e411902589137faf7be41387e4195.png|height=288,width=288! induced silencing complex], RISC (Chiurazzi & Neri, 2003).
In order to understand the siRNA suppressing process it is also important to look into the background of the RISC complex. Basically, siRNAs generated by the enzyme Dicer become assembled in the RISC complex, which itself contains different cellular factors (Hammond et al, 2001). The active component of RISC is a protein from the endonuclease family known as Argonautes, which function by splitting the strands of mRNA directly associated to the siRNA and RISC complex (Daneholt, 2006). Since the fragments produced by Dicer are double-stranded, in theory, both strands could potentially produce one functional siRNA. Yet only one of these two strands, specifically the leading strand, binds to the Argonaute protein and conducts the complementary mRNA's degradation process. The other strand, or lagging strand, also degrades during the RISC activation process (Caudy et al, 2002). Although it was originally thought that ATP-dependent helicase was responsible for separating both strands (Freeman, 2004), in reality the process is actually ATP independent and is achieved directly by proteins that comprise the RISC complex (Matranga et al, 2005). The selected strand's 5' leading end tends to pair itself less with its complementary strand (Shwarz et al, 2003), although the selection of the leading strand is not affected by the direction through which Dicer splits the dsRNA before its incorporation into the RISC complex (Preall et al, 2006). Nevertheless, the R2D2 protein could serve as the identifying factor by binding the to the more stable 5' end of the lagging strand (Tomari et al, 2004).
Although there are various different proteins in the Argonaute family, only Ago2 is capable of forming RISC complexes capable of fragmenting the target mRNA (Liu et al, 2004). In the same study, it was demonstrated that Ago2 is capable of splitting the target mRNA in vitro in the presence of an siRNA but not a dsRNA, considering that the reaction is ATP independent. As a result two Ago2 residues necessary for enzyme activity were identified, D669 and D597. Despite these discoveries it is still unclear how the activated RISC complex localizes mRNAs in the cell's interior. It has been proposed that the process of fragmentation could be tied to the genetic process of [translation, the target mRNA translation is not essential for the degradation mediated by RNAi (Sen et al, 2005). In fact, the RNAi process could be more effective against non-translated target mRNAs (Gu & Rossi 2004).
How siRNA Functions
RNAi's mechanism is initiated when a cell receives a long dsRNA that could generate itself through a transgenic exogen, either a viral intervention or a self-genetic element. These long dsRNAs fragment themselves into siRNAs through the enzyme known as Dicer. A silencing complex induced by RNA, the RISC complex, selects between both siRNA strands, where the lagging strand degrades and the leading strand is used in the localization of the complementary mRNA, and could have distinct results depending on the organism (Novina & Sharp, 2004).
In experiments conducted with Drosophila (a small fruit fly used extensively in genetic research) and in mammals, the antisense strand is incorporated directly into the RISC complex in order to identify the complementary mRNA, which is later destroyed. In the absence of siRNAs, the RISC complex lacks the specificity to unite to any mRNA molecule. However when the leading siRNA strand binds to the RISC complex, once activated, it can essentially take part in repeated degradation cycles specified by the target mRNA, silencing the corresponding gene expression. In most cases, the gene's suppression is not always complete; this produces a reduction in the gene expression, a downregulation, known as a knockdown (Dillin, 2003).
In plants and nematodes, the process, although basically identical, serves another kind of purpose. After the Dicer has processed the original dsRNA, the antisense strand from the primary siRNAs is used in amplification, upregulation processes, as opposed to the mammal and Drosophila downregulation. The antisense strand, bound to an RNA-dependent RNA polymerase, can appear with a complementary mRNA, and act as a starting point for the synthesis of a new long dsRNA molecule. Dicer then acts on this dsRNA, fragmenting it to generate new secondary siRNA molecules, specified by different sequences for the same mRNA, hence generating an amplification effect. Therefore, the mRNA bound to an siRNA is destroyed, creating a knockdown for the corresponding gene. This amplification in plants and nematodes allows the extension of the silencing effect produced by RNAi throughout the somatic tissues by transferring the dsRNAs directly from cell to cell, generating a profuse resistance to viral infections (Shanfa et al, 2004).
Laboratory uses of siRNA
The types of tools that could be used to obtain RNAi within the laboratory vary depending on the organisms. For example with nematodes such as C. elegans, long dsRNAs are used, which can present themselves in the nematodes. This could be carried out by microinjection, either in the gonads,[ head|http://en.wikipedia.org/wiki/Head], or intestine, or by submerging the nematodes in a solution containing dsRNAs. In the Drosophila flies, long dsRNAs are injected directly into the embryos, or in the case of Drosophila cultivated cells, the dsRNAs could be added directly into the cultivation medium, where it is absorbed directly by the cells. In mammals, synthetic siRNA molecules can be injected or transfected into the embryos, oocytes, or differentiated cells in vivo cell cultivation. Finally, in plants, overexpressed simple-stranded RNAs could, through a transgene or virus, provoke a co-suppression (Applied Biosystems, 2008).
Considering that siRNA could be used for countless of purposes to knock out genes in various species, it is necessary to focus on a specific class, in this case mammals, since siRNA would one day play a key role in the lives of humans. Although dsRNA molecules could induce specific RNAi in young rat embryos as well as rat oocytes, dsRNAs in mammal somatic cells could also activate different cellular routs including the development of [apoptosis (McManus & Sharp, 2002).
The protein RNA-dependent kinase, PKR, is an important antiviral detector, which could activate upon binding to long dsRNAs, like those produced by viruses. Once the protein is activated it inhibits the translation of proteins through the phosphorylation of one of the subunits that initiated the translation, in this case EIF2. The phosphorylation of EIF2 produces a general suppression of protein synthesis that directs the cell towards apoptosis. The RNA-dependent kinase additionally activates the enzymes 2'5'-OligoA synthetase and RNAse L, which are the main central components in an antiviral pathway. When dsRNAs bind, the 2'5'-OligoA synthetase becomes activated, generating oligoadenylates, which in succession activate RNAse L, and at the same time degrade the RNA. The dsRNAs could also induce an[ interferon|http://en.wikipedia.org/wiki/Interferon] expression, which, in conjunction with other signals, could stimulate apoptosis (Langlois 2005).
At any rate, in 2001, Thomas Tuschl and his colleagues demonstrated that small synthetic siRNAs are capable of inducing RNAi in mammal cells without activating the RNA-dependent kinase protein and interferon, as published in Nature (Elbashir et al, 2001). This discovery subscribed a great interest in the technique used with RNAi in research with biomedics and in the development of new medications. A development which has sparked interest in the treatment of various genetically predisposed diseases such as cancer, Huntington's, and others.
Real World Applications of siRNA
The specificity and robust effect of RNAi through genetic expressions makes this mechanism an incredibly valuable tool in research, both in cell cultures and in living organisms, considering that dsRNAs or synthetic or transgenic siRNAs could be selectively induced through the cell so as to selectively silence specific genes. This has allowed an incredible advancement in biological cell development since RNAi is a fast and simple tool in understanding genetic functions. However, this is true despite the fact that overall it is crucial to develop a knockout organism in order to assure the consistency of the results. The technique used with RNAi in the laboratory could also be used to systematically silence each gene in a cell, as a genomic analysis, something that could help identify necessary components for a particular cellular process, such as cell division or signaling pathways (Dykxhoorn et al, 2003). In fact, there are large-scale collections of mammal RNAi that are available to the public, a tool that could be used by major corporations as well as in research and academics (Pei & Tuschl, 2006).
In gene silencing, for the development of knockdown or knockout organisms, the RNAi rout is consistently used in molecular and cellular biology in order to study the function of genes in cell cultures as well as in vivo organism models (Daneholt, 2006). For this, dsRNA, or siRNAs in mammals, must be synthesized with one complementary sequence of the gene of interest and then introduced into a cell or organism, where it is recognized as alien genetic material, activating the RNAi pathway. By using this mechanism, researches could produce a drastic reduction in the target gene's expression and by studying the effects of this diminishment, as in the physiological functions of the gene's product, the corresponding protein (Harborth et al, 2001). Since RNAi cannot completely block a gene's expression, this technique is commonly referred to as "knockdown," in comparison to the term "knockout" in which the gene's expression is completely suppressed.
Most of RNAi's applications in genomic functions in animals have been conducted in C. elegans and Drosophila, since these are the model organisms in which RNAi is most effective. The nematode C. elegans is particularly useful in RNAi research for two reasons: one, the silencing effect on the genes is usually heritable, and second, because the administration of dsRNA is extremely simple (Kamath & Ahringer, 2003). Although the administration is more complicated in other organisms, there are continuous efforts in research to come up with large-scale genomic studies in mammalian cells (Cullen & Arndt, 2005). The approximations for the design of RNAi genomic libraries could require much more sophistication than the design of one single siRNA for a defined set of experimental conditions. Artificial neuronal webs are frequently utilized in designing siRNA libraries and in predicting the probability of its efficiency in gene silencing (Ge et al, 2005).
Massive genomic screening is seen generally as a promising method to advance genomic data recording and has sparked developments in methods and screening to a larger scale with the help of microarrays (Janitz et al 2006). Conclusively, the usefulness of these screens and the possibility of applying the techniques developed in model organisms, as well as related species are questionable. An example of this would be to take into consideration the relationship of C. elegans to related nematode[ parasites|http://en.wikipedia.org/wiki/Parasite] (Geldhof et al, 2005). The functional genomic expressions used by RNAi is one particularly helpful technique in mapping and recording genomes in plants, since many plants are polyploidies, which represents a significant challenge for traditional methods for studying genetics. For example, RNAi has been successfully used in functional genomic studies in hexaploidy wheat, as well as other common plants such as maize (Travella et al, 2006).
When it comes to uses in medicine, one very interesting possibility is the use of RNAi's mechanism in therapy. Although it is difficult to administer long dsRNA molecules in mammal cells due to the unleashing of an interferon response, the use of synthetic siRNAs has had some success. Between the first implementation in clinical trials, the first treatment for macular degeneration and respiratory syncytial virus has been discovered (Sah, 2006). RNAi has also shown effectiveness in the reversal of hepatic malfunctioning induced in rat models. Other proposed clinical uses are centered around [antiviral !worddavd4560e83641baec705217a4c5c8e6fc9.png|height=316,width=240! therapies], including the inhibition of genetic viral expression as well as cancerous cells (Jiang & Milner, 2002). Other uses include the silencing of receptors and co-receptors of HIV within the host, the silencing of the hepatitis A and B virus, the cold virus, and even the inhibition of the measles virus' replication gene (Hu et al, 2005).
There have also been potential treatment ideas for neurodegenerative diseases, in particular those involving polyglutamines such as Huntington's disease (Raoul et al, 2006). RNAi has also been considered as an alternative for cancer treatment, through the differential silencing of overexpressed genes within tumor cells, or genes involved in cell division, apoptosis, and angiogenesis (Izquierdo, 2004). Specific articles relating to RNAi therapeutic strategy techniques summarizing different research articles could be found in the journal known as Molecular Therapy from the Nature Review[ (Li et al, 2008).|http://www.nature.com/mt/journal/v16/n5/abs/mt200851a.html] One key research area for RNAi use in clinical trials lies in the development of a safe delivery, which implies the uses of all viral vector systems similar to those used in genetic therapy (Takeshita & Oshya, 2006).
In spite of the expanse of studies in cell cultures intended on developing drugs based on RNAi there is still some anxiety with regards to the safety of RNAi, specifically due to "off-target" effects, in which a gene with a similar sequence to the target gene could also be silenced (Tong et al, 2005). One study has shown that the probability of mistakes due to "off-target" effects is approximately 10 to 1 (Qiu et al, 2005). Another important study, with regards to hepatic diseases in mice, produced a high mortality rate in the experimented animals due to an oversaturation of the RNAi pathway, according to researchers (Grimm et al, 2006). All of these and more are deep concerns and are currently under intense research so as to minimize the impact of the therapeutic use of RNAi.
The interference of RNA has also been heavily used in biotechnology applications, particularly in the design of[ edible plants|http://en.wikipedia.org/wiki/Edible_plants] that produce low levels of natural toxins. These techniques take advantage of the stable and heritable phenotypes of RNAi within plants. For example, cottonseeds are rich in nutritious proteins, but contain a natural[ toxin|http://en.wikipedia.org/wiki/Toxins] known as gossypol, making humans unable to consume the seed. RNAi was sued to produce cotton plants whose seeds contain reduced[ toxin|http://en.wikipedia.org/wiki/Toxins] levels through the inhibition of cadinene synthetase, a key enzyme in the production of the[ toxin|http://en.wikipedia.org/wiki/Toxins], without affecting its production in other parts of the plant where[ gossypol|http://en.wikipedia.org/wiki/Gossypol] is important in preventing infections and other disease causing agents in the plant (Sunilkumar et al, 2006). Similar efforts have led to the reduction of natural carcinogenic products in yucca plants (Siritunga & Sayre, 2004).
Although no food product that has undergone genetic engineering based on RNAi has passed the experimental phase, efforts in the development of these products have effectively reduced allergen levels in plants such as the tomato (Lorenz et al, 2006) and have minimized carcinogenic precursors in tobacco plants (Gavilano et al, 2006). Some other plant characteristics that have been modified in the lab include the natural production of non-narcotic opium poppy (Allen et al, 2004), viral resistance of common plants, and the increased production of antioxidants within tomato plants (Martin et al, 2004).
In conclusion, it could be agreed upon that the use of siRNA through RNAi is, in fact, one of the most important advancement in[ medicine|http://en.wikipedia.org/wiki/Medicine] to date. This small interfering RNA, used in genetic research studies by binding and interfering with specific genes within an organism owns itself to countless benefits that will undoubtedly arise the future. Genetically engineered food that is high in antioxidants and low toxicity, cancer treatment, HIV treatment, a cure for Huntington's, etc. will all play an important role in our future. It therefore comes as no surprise, due to the complexity of these functions and processes, that countless of studies have had, and continue, to be carried out in order to uncover the mystery of siRNA. Although there is still plenty of areas within the realm of RNA still to discover, it comes as a comfort to know that siRNA has taken a huge leap into modern research, and is moving fast.