RNA Interference and its therapeutic applications

RNAi is a potent method, requiring only a few molecules of dsRNA per cell to silence the expression. Long molecules of double stranded RNA (dsRNA) trigger the process. The dsRNA comes from virus and transposon activity in natural RNAi process, while it can be injected in the cells in experimental processes. The strand of the dsRNA that is identical in sequence to a region in target mRNA molecule is called the sense strand, and the other strand which is complimentary is termed the antisense strand. An enzyme complex called DICER thought to be similar to RNAase III then recognizes dsRNA, and cuts it into roughly 22nucleotide long fragments. These fragments termed siRNAs for “small interfering RNAs” remain in double stranded duplexes with very short 3' overhangs. However, only one of the two strands, known as the guide strand or antisense strand binds the argonaute protein of RNA-induced silencing complex (RISC) and target the complementary mRNA resulting gene silencing. The other anti-guide strand or passenger strand is degraded as a RISC substrate during the process of RISC activation. This form of RNAi is termed as post transcriptional gene silencing (PTGS); other forms are also thought to operate at the genomic or transcriptional level in some organisms. In mammals dsRNA longer than 30 base pairs induces a nonspecific antiviral response. This so-called interferon response results in a nonspecific arrest in translation and induction of apoptosis. This cascade induces a global non-specific suppression of translation, which in turn triggers apoptosis. Interestingly, dsRNAs less than 30 nt in length do not activate the antiviral response and 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.


Introduction
functional protein form.Since there are two steps to making a protein, there are two ways of preventing one RNA interference (RNAi) is a molecular from being made.Scientists have made exciting mechanism in which fragments of double-stranded progress in blocking the protein synthesis through the ribonucleic acid (dsRNA) interfere with the expression second step, translation. of a particular gene that shares a homologous sequence One way they have accomplished this is by with the dsRNA.In 2006, American scientists Andrew inserting synthetic molecules that triggers a cellular Fire and Craig C. Mello shared the Nobel Prize in process called RNA interference (Thakur, 2003).Physiology or Medicine for their work on RNA Before RNA interference was well charactinterference in the nematode worm Caenorhabditis erized, the phenomenon was known by other names, elegans.RNA silencing is a sequence specific RNA including post transcriptional gene silencing, degradation process that is triggered by the formation transgene silencing and quelling.Well before RNAi of double stranded RNA that can be introduced by was discovered, RNA was used to reduce gene expression virus or transgenes.Duplexes 21-nucleotide (nt) RNAs in plant genetics.Single-stranded antisense RNA was with symmetric 2-nt 3'overhangs are introduced into the introduced into plant cells and hybridized to the cell mediating the degradation of mRNA.According homologous single-stranded "sense" messenger RNA. to central dogma of molecular biology, proteins are It is now clear that the resulting dsRNA was responsible made in two steps.The first step, transcription, copies for reducing gene expression.( Lee et al., 1993).genes from double stranded deoxyribonucleic acid (ds The revolutionary finding of RNAi resulted DNA) molecules to mobile, single-stranded ribo-from the unexpected outcome of experiments performed by plant scientists in the Western World nucleic acid (RNA) molecules called mRNA.In the (Napoli et al., 1990;Jorgensen et al., 1994).While second step, translation, the mRNA is converted to its trying to increase the purple colour in Petunias, then unwound by a helicase activity associated with a scientists introduced a pigment producing gene under distinct multiprotein complex known as the RNAthe control of a powerful promoter.Instead of induced silencing complex (RISC).The strand of the expected deep purple colour, many of the flowers dsRNA that is identical in sequence to a region in appeared variegated or even white.This observed target mRNA molecule is called the sense strand, and phenomenon was named "cosuppression" since the the other strand which is complimentary is termed the expression of both the introduced gene and homologus antisense strand.An enzyme complex called DICER endogenous gene was suppressed.There are two that is similar to RNAase III then recognizes ds RNA, mechanisms through which silencing of both and cuts it into roughly 22-nucleotide long fragments.transgenes and endogenous loci could occur.In one These fragments termed siRNAs for "small interfering mechanism transgene induced silencing was RNAs" which remain in double stranded duplexes accompanied by heavy methylation of silenced loci, with very short 3' overhangs, then act as templates for leading to transcriptional gene silencing.In the other the RNAi inducing silencing complex to destroy the mechanism post transcriptional gene silencing homologous message, thus specifically suppressing (PTGS) occurred.. Transgene cosuppression is not its expression.This form of RNAi is termed as post only limited to plants but has also been shown in fungi, transcriptional gene silencing (PTGS), other forms are Drosophila, C. elegans and rodent fibroblasts.This also thought to operate at the genomic or transcripphenomenon has been well characterized in tional level in some organisms.Neurospora crassa, where it is known as "quelling"

Systemic Silencing in Animals
and it occurs at the post-transcriptional level.It was clear that co-suppression in plants, quelling in fungi Animals have a system for amplification and spread of silencing.This is quite evident in C. elegans.and RNAi in nematodes all shared a common mechanism.
If these animals are injected with dsRNA, or if they are A few years later plant virologists made a similar allowed to feed on bacteria that produce dsRNA there observation.In their research they aimed towards is systemic silencing of a corresponding endogenous improvement of resistance of plants against plant RNA.It is presumed that the signal molecule is viruses.At that time it was known that plants produced in the C. elegans cells that receive dsRNA.
This signal then moves and causes RNA silencing in expressing virus-specific proteins show enhanced other cells that become sources of secondary signal tolerance or even resistance against virus infection.
(Weinberg et al., 2006).However, they also made the surprising observation that plants carrying only short regions of viral RNA

RNAi in Mammalian Cells
sequences not coding for any viral protein showed the After its discovery, RNAi was rapidly employed same effect.They concluded that viral RNA produced as a powerful tool for large-scale reverse genetic by transgenes can also attack incoming viruses and screens in C elegans, where it can be easily induced by stop them from multiplying and spreading throughout direct injection of dsRNAs, by feeding worms with the plant.They did the reverse experiment and put bacteria engineered to express si-RNAs, or simply by short pieces of plant gene sequences into plant viruses.
soaking the animals in medium containing siRNAs.Indeed, after infection of plants with these modified Furthermore, in nematodes, RNAi acts systemically viruses the expression of the targeted plant gene was and is a long-lasting heritable event due to the suppressed.They called this phenomenon "viruspresence of RNA-dependent RNA polymerases induced gene silencing" (VIGS").
(RdRPs) which allow the amplification of the trigger

Mechanisma of RNAi
and the perpetuation of the silencing process.Long molecules of double stranded RNA In mammals, however, initial efforts to use (dsRNA) when introduced into the cell cytoplasm RNAi for specific gene silencing encountered more trigger the process.The dsRNA comes from virus and difficulties, especially due to the induction of transposon activity in natural RNAi process, while it nonspecific inhibition of gene expression resulting can be injected in the cells in experimental processes from the activation of the interferon (IFN) response (Elbashir et al., 2001).In the initiation step the pathway by dsRNAs longer than 30 bp.Hence, the "trigger" dsRNA molecule, usually several hundred RNAi pathway was thought to be nonfunctional in base pair long, is cleaved to form 21-23 bp double mammalian cells, where dsRNA longer than 30 base stranded fragments known as short interfering RNAs pairs induces a nonspecific antiviral response.This so-(siRNAs).In the effector step the duplex siRNA are called interferon response is characterized by the to induce silencing.One may have to test three or four activation of the RNA-dependent protein kinase, siRNAs in order to find one that results in more than leading to phosphorylation and there by inactivation 90% reduction in target gene expression. of the translation initiation factor eIF-2a which results This reduction in target gene expression may be in a nonspecific arrest in translation and induction of related to one or more of the following factors: incorporation of siRNA into RISC and stability of apoptosis.Moreover, the synthesis of 2'-5 ' 2001) showed be incorporated into the activated RISC.They found that siRNA, when directly introduced into mammalian that the absolute and relative stabilities of the base cells, does not trigger the RNA-dependent protein pairs at the 5'ends of the two siRNA strands determine kinase response but effectively elicits RNAi, the degree to which each strand participates in the presumably by directly associating with RISC.RNAi pathway.The strand with lower 5' end stability Targeted gene silencing in mammalian cells by the is preferred.As a consequence, a highly functional application of siRNA is well established.The high siRNA is characterized by lower internal stability at degree of sequence specificity inherent to the the 5'end of the antisense strand as compared with less technology is emphasized by several reports showing effective duplexes (Chiu and Rana (2003).that even a 1-2 nt mismatch in the siRNA sequence To comply with the rules promoting asymmetric hampers targeted gene silencing.The effectiveness of incorporation into RISC, the base pair at the 5' end of siRNAs varies -the most potent siRNAs result in the siRNA antisense (guide) strand should have a >90% reduction in target RNA and protein levels.The lower thermodynamic stability compared with the 3'most effective siRNAs turn out to be 21 nt dsRNAs end.In addition, is is recommended that each strand with 2 nt 3'overhangs.Sequence specificity of siRNA must have 2-nt 3' overhangs; A/U base pairing at the 5' is very stringent, as single base pair mismatches end of the antisense strand and G/C base pairing at the between the siRNA and its target mRNA dramatically 5' end of the sense strand; AU richness in the 5' reduce silencing.Unfortunately, not all siRNAs with terminal third of the antisense strand; avoidance of these characteristics are effective.The reasons for this introns , 5' and 3' untranslated regions, regions within are unclear but may be a result of positional effects.
75 bases of the start codon and sequences with >50% Guanine and Cytosine content; maximizing sequence

Selection and Designing of Small
divergence from related mRNA.Despite applying Interfering RNA these criteria, differences in silencing efficiencies Elbashir et a.l, (2002) reported a number of between siRNAs occur, and occurrence of offtarget guidelines for the design of siRNA molecules.Several siRNA activity has been recognized as an important design tools are also available from the internet.factor in siRNA experimental design (Reynolds et al., Although one can follow these guidelines it is still 2004).necessary to test several siRNAs, targeting distinct In vivo Systemic Delivery of siRNAs regions within the gene of interest, because there is Direct injection of siRNA into the blood would great variability in the capacity of an individual siRNA be ineffective because of rapid degradation of the RNA attempted by targeting genes linked to apoptosis control in the liver in two mice models of autoimmune by serum ribonucleases.However, it was recently hepatitis.siRNA was directed against caspase (Zender demonstrated that chemical modification can protect et al., 2003 and Fas (Song et a.l, 2003) and was the siRNA molecule from degradation and might even delivered hydrodynamically via the tail and portal vein prolong the silencing effect due to slower depletion respectively.In both the cases there was successful within the cell.Several groups reported different approaches for systemic in vivo delivery of siRNAs.reducing hepatocyte necrosis and inflammation, and Soutschek et al (2004) described intravenous injection protected the mice from future chronic fibrosis.Since in mice of chemically modified naked siRNAs then, several experiments using RNA interference to coupled to a cholesterol group chemically linked to the target respiratory viruses have been attempted.Intially terminal hydroxyl group of the sense strand to promote influenza virus was chosen due to its significant public entry into the cells.In vivo delivery of chemically health issues and lack of wholly effective vaccine modified siRNAs encapsulated into liposome (Tompkins et al., 2004).Proteins were targeted that are particles has been reported by Morrisey et al (2005).highly conserved across several sub types of influenza Song et al ( 2005) described an antibody-based and which are essential for viral replication.It was delivery system which could offer a possibility for found that combination of intranasal delivery and systemic, cell-type-specific siRNA delivery.Thus far, intravenous injection was most effective in inhibiting synthetic siRNAs have been applied in animals via the virus replication at the site of infection.Kumar et al hydrodynamic transfection (i.e. the intravenous (2006) described that promising results for the injection of a substantial dose of siRNA within a large therapeutic potential of RNAi in treating viral volume of liquid), resulting in a knockdown efficiency encephalitis, both virus specific and across species.up to 70-80%, at least in some organs, including liver, They induced RNAi in mice with either a lentivirally kidney, spleen, lung and pancreas.Using this method, expressed short hair pin RNA or a synthetic siRNA.the silencing of either Fas receptor or caspase-8 Kim et al, (2008) applied adenoviruses expressing resulted in a clearly measurable protection from siRNA both before and after foot and mouth disease severe Fas-induced liver damage.In vivo application virus (FMDV) infection in vitro and in vivo.Treatment of siRNA against genes of the hepatitis B virus also led after FMDV infection gave effective viral inhibition, to an effective inhibition of virus replication.This but a combination of treatment before and after FMDV method is of course not applicable to humans.It is also infection gave the best results in IBRS-2 cells.They limited by the fact that siRNA can only be delivered to obtained high survival rates in suckling mice by the a certain set of organs and it is not possible to target use of therapeutic injections following challenge.The specific organs or cells.Development of cell-specific results of their study suggest that treatment with or organ-specific delivery systems for siRNA, as is siRNA could enhance antiviral effects and may be required for broad in vivo application of this helpful in the control of FMDV in an outbreak.technique, is indeed a demanding task.One promising

Limitations of RNAi
avenue is the complexation of siRNAs with the polyethyleneimine (PEI) which efficiently stabilizes Despite the proliferation of promising cell siRNAs and upon systemic administration leads to the culture studies for RNAi-based drugs, some concern delivery of the intact siRNAs into different organs ( has been raised regarding the safety of RNA Aigner, 2006).
interference, especially the potential for "off-target" effects in which a gene with a coincidentally similar Therapeutic applications of Small RNAi sequence to the targeted gene is also repressed.A The most obvious clinical uses of RNAi are for computational genomics study estimated that the error diseases in which selective depletion of one or a few rate of off-target interactions is about 10%.One major study of liver disease in mice led to high death rates in specific proteins would be expected to slow or halt the the experimental animals, suggested by researchers to disease process in the affected cells.Ideally this would be the result of "oversaturation" of the dsRNA pathway.be accomplished with no or tolerable side effects.RNAi may provide new therapeutics for treating viral infections,