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siRNA: Utility
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siRNA: Utility D M Dykxhoorn, Harvard Medical School, Boston, MA, USA ã 2009 Elsevier Ltd. All rights reserved.
RNA interference (RNAi) is a highly conserved, naturally occurring cellular process that mediates the sequence-specific silencing of gene expression. The discovery of this process has challenged the long-held idea that the flow of genetic information occurs in a unidirectional manner from DNA to RNA to protein. In fact, it is now clear that RNA can act bidirectionally, with certain types of RNA being translated into proteins, whereas other forms can direct the regulation of gene expression. Regulatory RNAs have been shown to regulate a variety of processes, including transcription, splicing, and translation. In addition, the discovery of these regulatory RNAs has helped to begin to explain why the genomes of complex organisms retain large amounts of DNA that lack protein coding genes, referred to as intergenic regions. Analysis of the total RNA content, the so-called transcriptome, of a variety of organisms has shown that the transcriptional output of many organisms’ genomes is much greater than the number of protein coding genes and that the intergenic regions of the genome are rich sources for noncoding RNAs, the majority of which are probably involved in various regulatory processes.
Small Regulatory RNAs The small regulatory RNAs involved in RNAi have garnered a great deal of attention, both for their biological role as endogenous regulators of gene expression and as research tools for the selective suppression of gene expression. These small RNA molecules, ranging in size from approximately 19 to 26 nucleotides (nt), are highly conserved across diverse organisms from plants to worms and mammals and have been found to be involved in the regulation of a variety of cellular processes, including development and differentiation, metabolism, and the suppression of mobile genetic elements (e.g., transposons and retrotransposons) and viruses. Although micro (mi)RNAs were the first class of small regulatory RNA molecules identified, with the discovery that let-7 and lin-4 were noncoding RNAs involved in the regulation of genes responsible for proper developmental timing in the nematode Caenorhabditis elegans, the impact of this discovery was not immediately appreciated. It was the demonstration that the introduction of long double-stranded RNA (dsRNA) molecules into C. elegans led to the sequence-specific
suppression of gene expression that signaled the beginning of a new era in molecular biology. It was for this discovery, termed ‘RNA interference,’ that Drs. Andrew Fire and Craig Mello were awarded the 2006 Nobel Prize in Physiology or Medicine. In rapid succession, it was shown that these long dsRNAs were rapidly cleaved into small duplexed RNA molecules of 19–21 nt, referred to as small interfering (si)RNAs, and that these siRNAs were the effector molecules that guided the degradation of the homologous mRNA. It soon became clear that RNAi was related to a variety of silencing phenomena, including quelling in fungus and posttranscriptional gene silencing in plants. Since these initial discoveries, an increasing number of small regulatory RNAs have been identified using biochemical, genetic, and computational approaches. These small RNA species include miRNAs, siRNAs, repeat-associated siRNAs (rasiRNAs), and piwi-interacting RNAs. Both miRNAs and siRNAs are processed from dsRNA precursor molecules – long hairpin RNAs for miRNAs and long dsRNAs for siRNAs – by RNase III-type endonucleases (Figure 1). miRNAs are expressed as long transcripts, termed primary miRNAs (pri-miRNAs), from RNA polymerase II or III promoters. These highly structured pri-miRNAs are recognized by the nuclear localized RNase III-type enzyme Drosha that cleaves the pri-miRNA into an approximately 60–70 nt precursor miRNA (premiRNA) stemloop containing both the mature miRNA and the accompanying passenger strand separated by a noncomplementary loop region. This truncated stemloop is recognized by exportin-5 and translocated from the nucleus to the cytoplasm. In the cytoplasm, it is cleaved by the RNase III-type endonuclease Dicer into a short-lived small (21 nt) duplexed RNA containing 2 or 3 nt 30 overhangs characteristic of an RNase III-type cleavage reaction. In addition to producing miRNAs, Dicer is also able to cleave long dsRNAs into siRNAs. In fact, Dicer was originally identified based on its ability to process long dsRNAs. Additional dsRNA-binding proteins help Dicer to distinguish between these two precursor molecules. Interestingly, in plants, the pre-miRNA processing and long dsRNA cleavage activities of Dicer have been segregated into two RNase III family members, Dicer-like 1 (DCL-1) and DCL-2, respectively. In each case, the small duplexed RNAs, either the siRNAs or the miRNA, are loaded into an effector complex whereupon the passenger strand is dissociated from the active silencing strand (guide strand). Both siRNAs and miRNAs have an asymmetric nature that helps to distinguish the guide strand from the passenger strand. This asymmetric nature is determined by the relative thermodynamic
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Figure 1 RNA interference pathways. (a) Biochemical structure of siRNAs. siRNAs have the characteristic structure of an RNase III cleavage product with 50 phosphorylated ends, a 19 nt duplexed region, and 2–3 nt overhangs at the 30 termini. (b) The ‘classic’ RNAi pathway. Long dsRNAs were the first ‘trigger’ molecules associated with RNAi. These long dsRNAs were processed by the RNase III family member Dicer into siRNAs that serve as the effector molecules that guide the sequence-specific silencing of gene expression. Although Dicer can cleave the long dsRNAs in the absence of additional factors, the dsRNA-binding protein R2D2 helps to identify the end of the siRNA that has the lowest thermodynamic stability, a mechanism used to differentiate the active guide strand from the passenger strand. The duplexed siRNA enters a multiprotein complex, termed the RNA-induced silencing complex, where the passenger strand is cleaved and released, leaving the guide strand to identify the target mRNA sequences. Although the structure of RISC is not well understood, the key component of this complex is the Argonaute protein, Ago2, which is responsible for holding the siRNA (the 50 phosphorylated termini of the siRNA binding to a cleft in the Piwi domain and the 30 termini interacting with the PAZ domain) in the proper conformation to facilitate its interaction with target mRNA. In addition, Ago2 contains the endonuclease activity, located within the Piwi domain, responsible for cleaving the target mRNA. This pathway can be harnessed for the experimental silencing of gene expression by providing either the long dsRNA trigger molecules, a technique commonly used in C. elegans and Drosophila, or by directly transducing cells with the siRNA effector molecules. Although the RNAi machinery is highly conserved from plants to mammals, endogenous siRNAs, which are present in lower eukaryotes, have not been identified in mammalian cells. (c) The microRNA pathway. miRNAs, the most sequence-diverse class of small RNA regulatory molecules, are encoded on long, highly structured primary transcripts (pri-miRNA). These transcripts can be produced by RNA polymerase II or III and can encode a single miRNA or a cluster of miRNAs. These pri-miRNAs are processed by the nuclear localized RNase III family member Drosha, which recognizes the structure of the miRNA-encoding RNA hairpin and not the specific miRNA sequence. The Drosha-mediated cleavage reaction releases a short hairpin RNA, termed the precursor (pre)miRNA. This approximately 60–70 nt pre-miRNA is translocated to the cytoplasm by Exportin 5, where it is cleaved by Dicer into a dsRNA duplex containing the mature miRNA and the usually short-lived passenger strand (miRNA*). Similar to the long dsRNA-directed RNAi pathway, Dicer is associated with a dsRNA-binding protein called loquacious in Drosophila or TRBP (HIV-1 TAR RNA-binding protein) in mammals. The duplexed miRNA molecule is taken up by the effector complex, variously termed the
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properties of each of the 50 termini, with the end having the lower stability being preferentially maintained in the effector complex and used to guide the silencing reaction. Although this effector complex, variously termed the RNA-induced silencing complex for siRNAs or the miRNA-containing RNA-induced silencing complex (miRISC) for miRNAs, is poorly defined, it contains at its core a member of the Argonaute (Ago) family of proteins. The guide strand of the siRNA or the mature miRNA provides the specificity determinant for the effector complex by binding to complementary sites on the target mRNA. Gene silencing can take place by one of two mechanisms, either the inhibition of translation or the endonucleolytic cleavage of the mRNA. The method of silencing used for any target depends largely, but not entirely, on the degree of sequence complementarity between the siRNA or miRNA and the cognate mRNA. Those interactions with complete or nearly complete homology lead to mRNA cleavage, whereas interactions with a lower level of homology induce translation inhibition. The analysis of these silencing mechanisms has not only allowed for a better understanding of the role that small RNA molecules play in the regulation of many important cellular processes but also facilitated the development of technologies for the targeted silencing of gene expression. Although long dsRNAs had been used for silencing gene expression in C. elegans, this technology was not amenable for use in higher eukaryotes due to innate immune responses that have evolved in mammalian cells to recognize long dsRNA species and induce the activation of cell death pathways. In normal circumstances, the interferon response serves to protect cells from viral pathogens, many of which produce long dsRNAs as intermediates in their life cycle. Since the interferon response is efficiently triggered by dsRNAs more than 30 nt in length, it was believed that siRNAs, which fall below this size threshold, would be unable to activate this response. This was borne out by experiments that showed that the treatment of mammalian cells with chemically synthesized siRNAs could effectively silence gene expression without any untoward toxicity. This discovery led to the development of RNAi-based technologies allowing reverse genetic experiments to be performed in organisms that were intractable to more traditional gene-silencing approaches and laid the groundwork for the application of RNAi-based
silencing approaches as novel therapeutic modalities. In fact, the use of siRNAs for the localized silencing of gene expression has already entered clinical trials for the treatment of age-related macular degeneration and Rous sarcoma virus infections.
siRNAs as a Gene-Silencing Tool There are several features that make the use of siRNAs an attractive approach for the targeted silencing of gene expression. Foremost among these features is the fact that these silencing technologies take advantage of an endogenous gene-silencing mechanism that is conserved in organisms as diverse as plants, C. elegans, Drosophila, mice, and humans. In addition, unlike antisense approaches, potential target sequences can be readily identified. Initial studies showed that siRNA-mediated gene silencing was highly specific, being able to distinguish between genes that differed by as little as a single nucleotide, with no apparent toxicity. siRNA Sequence
A better understanding of the characteristics that distinguish highly potent siRNAs from nonfunctional siRNAs will facilitate the optimization of siRNA design to maximize silencing potential and minimize off-target effects. There are a number of traits associated with highly active siRNAs, including low internal stability, moderate to low GC content (optimally between 30% and 52%), lack of secondary structures within the siRNA sequence, and low thermodynamic stability at the 50 end of the guide strand. In addition, siRNA sequences that have homology with unintended targets or that contain immunostimulatory sequence motifs should be avoided to reduce the potential off-target effects. Many of these criteria have been incorporated into computer algorithms designed to aid in the identification of effective siRNA sequences targeting a particular gene of interest. Although these algorithms can improve the selection of potential siRNA sequences, experimental testing is necessary to determine the optimal siRNA sequence for any given gene. siRNA Specificity
Early studies using siRNAs to mediate gene silencing suggested that siRNAs, by virtue of their action,
miRNA-containing RISC (miRISC) or the miRNA nucleoprotein (miRNP) complex; the passenger strand (miRNA*) is dissociated; and the mature miRNA guides the complex to the target mRNA. The binding of the miRNA to target sites that have partial complementarity leads to the induction of translational repression. Although these pathways represent the classic views of the RNA interference pathways that induce posttranscriptional gene silencing, in actuality, siRNAs and miRNAs have interchangeable effector functions and either molecule can induce translational repression or mRNA cleavage depending on the degree of target site complementarity.
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required full complementarity and that a single nucleotide difference between the siRNA and the target binding site would be sufficient to abrogate silencing. In fact, it appears that mismatches between the siRNA and the target site can be accommodated without the complete loss of silencing. The degree to which mismatches can be tolerated depends on a number of factors, including the position and nucleotide composition of the mismatch. For example, the central nucleotides of the siRNA, around the site where cleavage of the mRNA occurs, are more sensitive to mismatches than either the 50 or 30 ends of the molecule. This is presumably due to steric constraints imposed by the active site of the Ago2 endonuclease, the core enzymatic component of RISC. It is not simply the presence of mismatched nucleotides that lends specificity but, more important, it is the nature of the mismatched nucleotides. That is, the juxtaposition of two purines within the central region of the siRNA:mRNA binding site will interfere with silencing more readily than the juxtaposition of two pyrmidines or a non-base-pairing purine:pyrmidine pair. However, by judiciously choosing the site and type of mismatch, it is possible to selectively silence genes that differ by as little as a single nucleotide. This has permitted the development of silencing tools that can distinguish between disease-causing alleles of a gene that differ by a single nucleotide from the ‘normal’ allele. This has been applied to disease causing single nucleotide polymorphisms (SNPs) involved in the development of cancer (e.g., RasV12), sickle cell anemia, and several dominantly inherited neurodegenerative disorders including superoxide dismutase 1 in amyotrophic lateral sclerosis and tau in frontotemporal dementia. Even for diseases whose phenotype is not directly attributable to an SNP, selective targeting of the disease can be accomplished by targeting SNPs that are linked to the disease allele. This has been used for the preclinical development of tools for the silencing of MJD1 (ataxin-3) in Machado–Joseph disease by targeting an SNP that tightly segregates with the pathogenic allele. Off-Target Effects
Although the initial studies using siRNAs for the targeted silencing of gene expression showed a high level of specificity and a lack of toxicity, as the technology has become more widely applied, the potential for off-target effects has become more apparent. The off-target effects of siRNAs are classified into two categories: (1) the silencing of unintended targets that have partial complementarity to the siRNA and (2) the induction of cellular responses that nonspecifically silence gene expression. The analysis of mRNA levels in cells treated with specific siRNAs has shown
changes to a large number of genes. Although the majority of these changes are small, a few genes can be severely affected. These nonspecific effects have been found for targets that share as few as 15 complementary nucleotides with as few as 11 contiguous nucleotides. These nonspecific effects can be generated from either strand of the duplexed siRNA. Microarray analysis can effectively measure changes in the amount of mRNA but fails to represent the changes that may occur due to translational inhibition, the common form of RNAi-mediated gene silencing induced by miRNAs in animal cells. Since miRNAs require a much lower level of complementarity, the potential off-targets could be significantly higher. It is difficult to evaluate these changes in protein levels since high-throughput proteomic approaches are much more difficult. Since effective translational repression mediated by miRNAs requires the concerted action of several miRNAs, the overall impact of these miRNA-style off-target effects may be minimal. As part of the innate antiviral response, mammalian cells have mechanisms for the recognition of dsRNA species, a common intermediate found in the life cycle of many viruses. This response induces the global inhibition of gene expression by impairing the cellular translation machinery. Although initial studies using siRNA for gene silencing found no induction of the interferon response with dsRNA smaller than 30 nt, subsequent studies using highly sensitive microarray analysis found that in certain circumstances expression of a subset of interferonresponsive genes was upregulated. This induction of the interferon response was found for only certain siRNA sequences and only when cells were treated with high concentrations of siRNAs. The treatment of cells with different siRNA sequences led to an increase in the expression of different subsets of interferon genes. Since no cytotoxicty was associated with any of these siRNA treatments, it appears that only a partial interferon response was induced. Experiments have suggested that the induction of the interferon genes may not be through the dsRNA-activated protein kinase; instead, interferon gene expression may be indirectly induced by the activation of Toll-like receptors (TLRs), TLR7 and TLR8. These TLRs can recognize specific immunostimulatory sequence motifs, particularly GU-rich sequences. The mode of administration of the siRNA affected the ability of the siRNA to stimulate an interferon response, suggesting that the method of cellular uptake plays a role in determining the immunostimulatory potential of an siRNA. For example, transduction of the cell by lipid-based transfection reagents resulted in a pronounced enhancement of off-target gene expression compared to delivery by electroporation.
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Since these off-target effects are concentration dependent, many of them can be avoided by minimizing the siRNA dosage. This can be accomplished by identifying highly potent siRNAs that can efficiently silence target gene expression at doses below the threshold necessary to induce off-target effect. Potential off-target effects can be minimized by careful siRNA sequence selection. Important criteria to reduce the potential for off-target effects include the avoidance of immunostimulatory sequences and designing the siRNA to minimize uptake of the passenger strand into RISC. In addition, all the potential siRNA sequences should be compared to the mRNA in the human genome to minimize the potential off-target binding sites. Although it may be impossible to avoid short stretches of homology, siRNAs that have extensive homology with unintended targets should not be used. In addition, the incorporation of chemical modifications within the siRNA can mitigate the immunostimulatory effects of the siRNA while retaining its silencing efficiency.
RNAi-Based Silencing Technologies Two basic approaches have been used for the targeted silencing of gene expression by siRNAs: (1) the introduction of chemically synthesized siRNAs into cells and (2) the introduction of DNA vectors expressing siRNA precursors that are processed intracellularly into active siRNAs (Figure 2). Each of these methods has distinct advantages and disadvantages. Chemically Synthesized siRNAs
The demonstration that chemically synthesized siRNAs could induce the silencing of a target gene of interest without activating an innate immune response has revolutionized biological research, providing a powerful tool for gene function analysis and opening up the possibility for the therapeutic application of these potent technologies. The transfection of synthetic siRNAs into tissue culture cells remains the most widely used method for gene silencing because of its specificity, ease of use, and adaptability to a variety of experimental conditions and cell types. An increasing number of lipid-based transfection reagents have been developed to effectively deliver the siRNAs into the cytoplasm of target cells. There are a number of factors that have limited the use of this technology, principal among these are the transient nature of the siRNA-based silencing response in actively dividing cells and the inability of traditional transfection reagents to transduce many important cell types. In most dividing cell lines, the silencing phenotype peaks at approximately day 3 posttransfection and subsides
approximately 5–7 days posttransfection. This loss of silencing over time appears to be due to the diluting out of the siRNAs during cell division until the intracellular concentration of the siRNA falls below a critical threshold needed to maintain the silencing phenotype. Long-lasting silencing (up to 3 weeks) has been achieved in cells that are nondividing, as demonstrated by in vitro experiments in postmitotic, terminal differentiated primary macrophage cells and in vivo delivery to hepatocytes. The degree to which a specific protein can be silenced depends on the halflife of the protein, the effectiveness of the siRNA, and the rate of cell doubling. Since the silencing phenotype is mediated by the targeted degradation of mRNA and not by degrading the protein, it is possible to effectively deplete the cell of a specific mRNA without altering the levels of the protein. This is particularly the case for proteins that are long-lived. Although the transfection of a wide variety of tissue culture cells is possible with the judicious choice of transfection reagents, there are a large number of cells that are refractory to lipid-based transfection protocols, including many clinically important primary cell types. In addition, the inherent toxicity of many of these lipid-based transfection reagents limits their usefulness for in vivo applications. Therefore, a variety of alternative transduction mechanisms have been developed (Figure 2(a)). One approach for the in vitro delivery of siRNAs to cell types that are difficult to transfect has been the combination of electroporation technologies with lipid-based transfection reagents, termed nucleofection. Although this approach can have some untoward toxicity, it has been effectively used for the silencing of gene expression in a wide range of cell types, including many primary cells. Another approach that has been proved to be effective for the delivery of siRNAs both in vitro (in tissue culture cell experiments) and in vivo has been the conjugation of the siRNA with cholesterol. Not only did this lead to the efficient uptake of the siRNA while retaining the siRNA’s silencing activity but also it improved the pharmacological properties of the siRNA by increasing the retention of the siRNA within the circulatory system. Since cholesterol receptors are relatively ubiquitously expressed, the cholesterol-conjugated siRNAs can enter a wide variety of cells. The broad spectrum of cells that can be effectively transduced by cholesterol-conjugated siRNAs makes this technology widely applicable. However, when cholesterol-conjugated siRNAs are given in vivo, they will be delivered to a wide variety of cells and tissues, only some of which are the intended target. This increases the risk of silencing gene expression in unintended cell types and could increase off-target effects. In addition, this nonspecific uptake increases the amount of the
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Figure 2 Methods for siRNA delivery. (a) Methods for the delivery of chemically synthesized siRNAs into cells. Although the use of lipid-based transfection reagents remains the most common method for the transduction of siRNAs into tissue culture cells, several methods have been developed that allow for the efficient delivery of siRNAs into cells for both in vitro and in vivo gene-silencing experiments. The covalent coupling of the siRNA, through the passenger strand, with either a cholesterol moiety (i) or an RNA aptamer (ii) has been shown to increase the pharmacological properties of the siRNAs (e.g., increasing the retention time of the siRNA within the circulatory system) while facilitating the uptake of the siRNA and maintaining its silencing capacity. Cholesterol permits the binding of the siRNA to cellular low-density lipoprotein receptors, which are expressed in a wide variety of cell types, and the uptake of the siRNA. Alternatively, the passenger strand of the siRNA can be incorporated into the termini of an aptamer, a structured RNA molecule that has been selected in vitro for specific properties, such as high-affinity binding to a distinct molecule. Using this approach, one can achieve cell type-specific delivery by choosing an aptamer that has a high affinity for a particular cell surface molecule. Similarly, cell type-specific delivery has been achieved using a recombinant protein containing the recognition domain of a single chain antibody or a fragment antibody fused to a nucleic acid binding motif (e.g., protamine) (iii). In this case, the siRNA interacts with the nucleic acid binding motif and is directed to the cell type of interest by the affinity of the antibody for a specific cell surface molecule. Although the majority of lipid-based transfection reagents have associated toxicities that prevent their use for in vivo applications, there are some lipid formulations that can be used for efficient delivery in vivo with negligible toxicity (iv). This usually involves the encapsulation of the siRNA into a lipid nanoparticle composed of several different types of lipids to provide it with the appropriate stability while allowing for the uptake and release of the siRNA into the cytoplasm of target cells. These lipid particles can be made to target specific cell types by the incorporation of specific moieties (e.g., antibody fragments) on their surface. (b) Stable silencing using DNA-based siRNA expression systems. Long-term silencing of gene expression can be achieved by expressing siRNA precursors, either (i) short hairpin (sh)RNAs or (ii) long hairpin RNAs based on the structure of endogenous miRNAs. These hairpin RNAs are processed by the endogenous RNAi machinery producing active siRNAs. These siRNA expression cassettes have been incorporated into a variety of DNA-based expression systems, including viral systems such as retroviral (oncoretroviral or lentiviral) and adeno-associated viral vectors. Typically, shRNAs have been expressed from RNA polymerase III (pol III) promoters that are uniquely suited for the production of short RNA species, whereas longer hairpin RNAs can be expressed from either Pol III or RNA polymerase II (pol II) promoters. Unlike pol III promoters that are ubiquitously expressed, pol II promoter-driven siRNA expression systems can be developed that permit the tissue-specific or inducible expression of the siRNA.
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siRNA needed to effectively silence expression of the target gene in the intended tissue or cell type. Therefore, delivery vehicles that could deliver siRNAs to specific cell types would be beneficial, particularly for therapeutic siRNA-based gene-silencing technologies. A number of technologies have been developed for the cell type-specific delivery of siRNAs. One such approach uses a recombinant bifunctional protein that contains a peptide that can recognize a specific cell-surface antigen (e.g., a single chain antibody or a fragment antibody against a cellular receptor or a ligand for a specific receptor) fused to a nucleic acid binding domain. Unlike the cholesterol-conjugated siRNAs, the siRNAs bind noncovalently to the recombinant protein and can be delivered to the cytoplasm of only those cells that express the specific protein on the cell surface. Therefore, the same deliver vehicle can be used to deliver any siRNA of interest for delivery. Cell type-specific delivery has also been achieved by conjugating the siRNA to an aptamer. Aptamers are structured nucleic acids that have been selected in vitro for specific characteristics, such as the ability to bind to specific proteins or to have a specific catalytic activity. The conjugation of an RNA aptamer that recognizes a specific cell surface protein to an siRNA can effectively deliver the siRNA to the cell type or tissue of interest. In order to retain the activity of the siRNA, the protein or RNA moiety is conjugated to the passenger strand of the siRNA, leaving the guide strand to perform its silencing function unimpaired. The mechanism by which the siRNAs are taken up and delivered to the cytoplasm to silence gene expression remains an area of active research. It remains to be determined how broadly applicable these technologies will be for the delivery of siRNAs. Despite these unknowns, these approaches have taken the therapeutic application of siRNAs one step closer to becoming a reality. Vector-Mediated RNA Interference
To overcome some of the intrinsic limitations of siRNA transfections, the transient nature of the silencing phenotype in actively dividing cells, and the difficulty of efficiently transducing certain cell types that are refractory to traditional lipid-based transfection protocols, siRNA-expressing DNA vectors have been developed (Figure 2(b)). Although Dicer was originally identified as an endonuclease that is capable of cleaving long dsRNA into siRNAs, it was soon realized that Dicer was also capable of processing the hairpin precursor molecule of miRNAs. By mimicking the structure of endogenously produced miRNA precursors, short hairpin RNAs were designed that could be efficiently expressed from DNA vectors and processed into active siRNAs. The majority of these expression systems were based on expression from
RNA polymerase III (pol III) promoters, either the U6 or the H1 promoter, which are uniquely suited for the expression of small RNA species since all of the necessary sites for activation of transcription are present upstream of the transcription start site and a run of uridine residues is sufficient to terminate transcription. The initial attempts to develop RNA pol II-based siRNA expression systems were unsuccessful. Although the transcripts were expressed, they were not processed by Dicer into functional siRNAs. It was only after the mapping of several miRNA transcripts that it was demonstrated that miRNA was encoded on primary transcripts that were highly structured hairpin RNAs containing frequent bulges and mismatched base-paired interactions. Based on these longer hairpin RNA structures, successful expression and processing of siRNAs was achieved. Unlike the ubiquitously expressing pol III promoters, pol II promoters that allow inducible, tissue- and cell type-specific transcription can be used for the regulated expression of siRNAs. In all these cases, the promoter drives transcription of a hairpin RNA containing the siRNA sequence and the accompanying passenger strand separated by a short noncomplementary loop sequence. These hairpin RNAs are processed by the endogenous RNAi machinery producing active siRNAs that enters RISC and direct the silencing of gene expression. The siRNA expression cassettes have been incorporated into an assortment of plasmid and viral vectors for the transduction of cells. Unlike synthetic siRNAs, plasmid vectors can be used for the transient delivery of siRNAs and, if the vector contains a suitable selectable marker, the stable delivery of siRNAs into many cell lines. However, their use is limited, in the same way as synthetic siRNAs, to cells that can be readily transfected. In addition, the low efficiency of generating stable silencing clones further limits their use in primary cells to transient siRNA expression. On the other hand, recombinant viral vectors have been developed which incorporate the siRNA expression cassette into the viral genome that is packaged into viral particles allowing for the infection of susceptible cell types. A variety of viral vectors have been adapted for use as siRNA delivery vehicles, including retroviral- and adeno-associated viral-based vectors. Since these viruses possess the necessary factors to facilitate uptake into cells, they are not limited to cells that can be readily transfected. Each of these systems has unique cell tropisms and life cycles, allowing them to productively deliver the siRNA expression cassette to specific subsets of cells. However, by changing the characteristic of the viral surface proteins involved in cellular recognition and uptake, it is possible to broaden the variety of cell types that can be infected. For example, HIV-1-based lentiviral constructs that utilize the HIV-1 viral
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envelope glycoprotein will infect only human cells that express the CD4 molecule on their surface. However, if the viral envelope glycoprotein is replaced by the glycoprotein from the vesicular stomatitis virus, which has a broad cell tropism, the lentiviral construct can be used to infect a variety of cell types from many different organisms, including human, mouse, rat, and even avian cells. Retroviral-based systems, derived from either lentiviruses or oncoretroviruses, have been used extensively for siRNA delivery. In these recombinant retroviral vectors, the genes necessary for productive infection have been deleted to ensure that the virus is unable to replicate in the target cells. Retroviruses are particularly well suited for the stable, long-term expression of siRNAs since they become integrated into the host genome and are replicated along with the host DNA. In addition, vectors based on lentiviral genomes, a class of retroviruses that include HIV-1, feline immunodeficiency virus, and equine infectious anemia virus, can infect postmitotic, nondividing cell types (e.g., terminal differentiated macrophage cells and neuron). Despite these advantages, retroviral vectors have the potential to cause insertional mutagenesis, leading to oncogenic transformation and clonal expansion of affected cells. Since these effects were seen in cells that were already actively replicating, it may be that this insertional mutagenesis will not be a significant barrier to the use of retroviral vectors in quiescent cells, such as those found in most tissues including the brain. Adeno-associated viral vectors have also been used successfully for in vivo siRNA delivery. Unlike retroviral vectors, adeno-associated vectors do not integrate into the host genome but can readily mediate long-term siRNA expression, particularly in nondividing tissues. In addition, adeno-associated viral vectors lack expression of viral proteins and as a result induce a limited immune response. Although these viral-based siRNA delivery systems have been shown to be effective both in vitro and in vivo, the packaging and purification of the recombinant viral particles can be a laborious process. For in vivo applications, siRNA expression from DNA-based vector systems is usually limited to the injection site and delivery requires a local regional administration (injection) or surgical procedure.
of the RNAi machinery, this has made it possible to silence any gene in the genomes of humans and other organisms. These approaches have been extensively applied in C. elegans and Drosophila cells to identify unknown genes involved in a wide array of cellular functions. Although large-scale screens in mammalian cells have presented a greater technical challenge, the refinement of RNAi-based technologies (siRNA transfection protocols and shRNA-based expression systems) has made these screens increasingly popular. It took very little time after RNAi was discovered in mammalian cells for researchers to recognize the potential of harnessing these pathways for therapeutic gene silencing. RNAi-based approaches have been extensively used for the silencing of pathogenic states ranging from viral infections to tumor development and metabolic and neurological disorders. The ease of design and synthesis, the low cost of production relative to other therapeutic agents such as antibodies and recombinant growth factors, the specificity of silencing, and the potency of silencing make siRNAs an attractive candidate for therapeutic applications. Delivery remains the major challenge for the successful therapeutic application of RNAi-based gene-silencing approaches. This is particularly the case for the systemic administration of siRNAs since unmodified siRNAs are sensitive to serum RNases and their small size results in their rapid clearance from the circulatory system. Despite these challenges, localized delivery of siRNAs has entered clinical trials for the treatment of age-related macular degeneration and respiratory syncytial virus infection. In a very short period of time, RNAi has gone from a novel silencing phenomenon in C. elegans to a highly conserved and ubiquitous gene regulatory network involved in many important cellular processes and, along the way, has been harnessed, providing an indispensable research tool and potential novel therapeutic modality.
Applications of RNAi-Based Technologies
Alisky JM and Davidson BL (2004) Towards therapy using RNA interference. American Journal of Pharmacogenomics 4: 45–51. Aravin A, Gaidatzis D, Pfeffer S, et al. (2006) A novel class of small RNAs bind to MILI protein in mouse testes. Nature 442: 203–207. Bartel DP (2004) MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 116: 281–297. Boudreau RL and Davidson BL (2006) RNAi therapy for neurodegenerative diseases. Current Topics in Developmental Biology 75: 73–92.
RNAi-mediated gene silencing has been widely applied as a research tool for reverse genetic experiments, which determine the function of a gene based on its disruption. The majority of these experiments have been done in a focused manner, examining the function of a single gene of interest at a time as well as other genomes, coupled with the ubiquitous nature
See also: Axonal mRNA Transport and Functions; Dendritic RNA Transport: Dynamic Spatio-Temporal Control of Neuronal Gene Expression; RNA Binding Protein Methods; Rodent Behavior: Approaches.
Further Reading
siRNA: Utility 885 Chu TC, Twu KY, Ellington AD, et al. (2006) Aptamer mediated siRNA delivery. Nucleic Acids Research 34: e73. Davidson BL and Harper SQ (2005) Viral delivery of recombinant short hairpin RNAs. Methods in Enzymology 392: 145–173. Dorsett Y and Tuschl T (2004) siRNAs: Applications in functional genomics and potential as therapeutics. Nature Reviews Drug Discovery 3: 318–329. Dykxhoorn DM and Lieberman J (2005) The silent revolution: RNA interference as basic biology, research tool, and therapeutic. Annual Review of Medicine 56: 401–423. Dykxhoorn DM, Palliser D, and Lieberman J (2006) The silent treatment: siRNAs as small molecule drugs. Gene Therapy 13: 541–552. Elbashir SM, Harborth J, Lendeckel W, et al. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494–498. Elbashir SM, Lendeckel W, and Tuschl T (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes & Development 15: 188–200. Fire A, Xu S, Montgomery MK, et al. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806–811. Girard A, Sachidanandam R, Hannon GJ, et al. (2006) A germlinespecific class of small RNAs binds mammalian Piwi proteins. Nature 442: 199–202. Jackson AL and Linsley PS (2004) Noise amidst the silence: Offtarget effects of siRNAs? Trends in Genetics 20: 521–524. Judge AD, Sood V, Shaw JR, et al. (2005) Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nature Biotechnology 23: 457–462. Khvorova A, Reynolds A, and Jayasena SD (2003) Functional siRNAs and miRNAs exhibit strand bias. Cell 115: 209–216. Kim DH and Rossi JJ (2007) Strategies for silencing human disease using RNA interference. Nature Reviews Genetics 8: 173–184.
Kim VN (2005) MicroRNA biogenesis: Coordinated cropping and dicing. Nature Reviews Molecular Cell Biology 6: 376–385. Liu J, Carmell MA, Rivas FV, et al. (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science 305: 1437–1441. Lu C, Tej SS, Luo S, et al. (2005) Elucidation of the small RNA component of the transcriptome. Science 309: 1567–1569. McNamara JO 2nd, Andrechek ER, Wang Y, et al. (2006) Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras. Nature Biotechnology 24: 1005–1015. Morrissey DV, Lockridge JA, Shaw L, et al. (2005) Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nature Biotechnology 23: 1002–1007. Pasquinelli AE, Hunter S, and Bracht J (2005) MicroRNAs: A developing story. Current Opinion in Genetics & Development 15: 200–205. Reynolds A, Leake D, Boese Q, et al. (2004) Rational siRNA design for RNA interference. Nature Biotechnology 22: 326–330. Schwarz DS, Hutvagner G, Du T, et al. (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell 115: 199–208. Song E, Zhu P, Lee SK, et al. (2005) Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nature Biotechnology 23: 709–717. Soutschek J, Akinc A, Bramlage B, et al. (2004) Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432: 173–178. Stewart SA, Dykxhoorn DM, Palliser D, et al. (2003) Lentivirusdelivered stable gene silencing by RNAi in primary cells. RNA 9: 493–501. Tomari Y and Zamore PD (2005) MicroRNA biogenesis: Drosha can’t cut it without a partner. Current Biology 15: R61–R64. Zamore PD, Tuschl T, Sharp PA, et al. (2000) RNAi: Doublestranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101: 25–33. Zaratiegui M, Irvine DV, and Martienssen RA (2007) Noncoding RNAs and gene silencing. Cell 128: 763–776.
siRNA: Utility D M Dykxhoorn, Harvard Medical School, Boston, MA, USA ã 2009 Elsevier Ltd. All rights reserved.
RNA interference (RNAi) is a highly conserved, naturally occurring cellular process that mediates the sequence-specific silencing of gene expression. The discovery of this process has challenged the long-held idea that the flow of genetic information occurs in a unidirectional manner from DNA to RNA to protein. In fact, it is now clear that RNA can act bidirectionally, with certain types of RNA being translated into proteins, whereas other forms can direct the regulation of gene expression. Regulatory RNAs have been shown to regulate a variety of processes, including transcription, splicing, and translation. In addition, the discovery of these regulatory RNAs has helped to begin to explain why the genomes of complex organisms retain large amounts of DNA that lack protein coding genes, referred to as intergenic regions. Analysis of the total RNA content, the so-called transcriptome, of a variety of organisms has shown that the transcriptional output of many organisms’ genomes is much greater than the number of protein coding genes and that the intergenic regions of the genome are rich sources for noncoding RNAs, the majority of which are probably involved in various regulatory processes.
Small Regulatory RNAs The small regulatory RNAs involved in RNAi have garnered a great deal of attention, both for their biological role as endogenous regulators of gene expression and as research tools for the selective suppression of gene expression. These small RNA molecules, ranging in size from approximately 19 to 26 nucleotides (nt), are highly conserved across diverse organisms from plants to worms and mammals and have been found to be involved in the regulation of a variety of cellular processes, including development and differentiation, metabolism, and the suppression of mobile genetic elements (e.g., transposons and retrotransposons) and viruses. Although micro (mi)RNAs were the first class of small regulatory RNA molecules identified, with the discovery that let-7 and lin-4 were noncoding RNAs involved in the regulation of genes responsible for proper developmental timing in the nematode Caenorhabditis elegans, the impact of this discovery was not immediately appreciated. It was the demonstration that the introduction of long double-stranded RNA (dsRNA) molecules into C. elegans led to the sequence-specific
suppression of gene expression that signaled the beginning of a new era in molecular biology. It was for this discovery, termed ‘RNA interference,’ that Drs. Andrew Fire and Craig Mello were awarded the 2006 Nobel Prize in Physiology or Medicine. In rapid succession, it was shown that these long dsRNAs were rapidly cleaved into small duplexed RNA molecules of 19–21 nt, referred to as small interfering (si)RNAs, and that these siRNAs were the effector molecules that guided the degradation of the homologous mRNA. It soon became clear that RNAi was related to a variety of silencing phenomena, including quelling in fungus and posttranscriptional gene silencing in plants. Since these initial discoveries, an increasing number of small regulatory RNAs have been identified using biochemical, genetic, and computational approaches. These small RNA species include miRNAs, siRNAs, repeat-associated siRNAs (rasiRNAs), and piwi-interacting RNAs. Both miRNAs and siRNAs are processed from dsRNA precursor molecules – long hairpin RNAs for miRNAs and long dsRNAs for siRNAs – by RNase III-type endonucleases (Figure 1). miRNAs are expressed as long transcripts, termed primary miRNAs (pri-miRNAs), from RNA polymerase II or III promoters. These highly structured pri-miRNAs are recognized by the nuclear localized RNase III-type enzyme Drosha that cleaves the pri-miRNA into an approximately 60–70 nt precursor miRNA (premiRNA) stemloop containing both the mature miRNA and the accompanying passenger strand separated by a noncomplementary loop region. This truncated stemloop is recognized by exportin-5 and translocated from the nucleus to the cytoplasm. In the cytoplasm, it is cleaved by the RNase III-type endonuclease Dicer into a short-lived small (21 nt) duplexed RNA containing 2 or 3 nt 30 overhangs characteristic of an RNase III-type cleavage reaction. In addition to producing miRNAs, Dicer is also able to cleave long dsRNAs into siRNAs. In fact, Dicer was originally identified based on its ability to process long dsRNAs. Additional dsRNA-binding proteins help Dicer to distinguish between these two precursor molecules. Interestingly, in plants, the pre-miRNA processing and long dsRNA cleavage activities of Dicer have been segregated into two RNase III family members, Dicer-like 1 (DCL-1) and DCL-2, respectively. In each case, the small duplexed RNAs, either the siRNAs or the miRNA, are loaded into an effector complex whereupon the passenger strand is dissociated from the active silencing strand (guide strand). Both siRNAs and miRNAs have an asymmetric nature that helps to distinguish the guide strand from the passenger strand. This asymmetric nature is determined by the relative thermodynamic
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Figure 1 RNA interference pathways. (a) Biochemical structure of siRNAs. siRNAs have the characteristic structure of an RNase III cleavage product with 50 phosphorylated ends, a 19 nt duplexed region, and 2–3 nt overhangs at the 30 termini. (b) The ‘classic’ RNAi pathway. Long dsRNAs were the first ‘trigger’ molecules associated with RNAi. These long dsRNAs were processed by the RNase III family member Dicer into siRNAs that serve as the effector molecules that guide the sequence-specific silencing of gene expression. Although Dicer can cleave the long dsRNAs in the absence of additional factors, the dsRNA-binding protein R2D2 helps to identify the end of the siRNA that has the lowest thermodynamic stability, a mechanism used to differentiate the active guide strand from the passenger strand. The duplexed siRNA enters a multiprotein complex, termed the RNA-induced silencing complex, where the passenger strand is cleaved and released, leaving the guide strand to identify the target mRNA sequences. Although the structure of RISC is not well understood, the key component of this complex is the Argonaute protein, Ago2, which is responsible for holding the siRNA (the 50 phosphorylated termini of the siRNA binding to a cleft in the Piwi domain and the 30 termini interacting with the PAZ domain) in the proper conformation to facilitate its interaction with target mRNA. In addition, Ago2 contains the endonuclease activity, located within the Piwi domain, responsible for cleaving the target mRNA. This pathway can be harnessed for the experimental silencing of gene expression by providing either the long dsRNA trigger molecules, a technique commonly used in C. elegans and Drosophila, or by directly transducing cells with the siRNA effector molecules. Although the RNAi machinery is highly conserved from plants to mammals, endogenous siRNAs, which are present in lower eukaryotes, have not been identified in mammalian cells. (c) The microRNA pathway. miRNAs, the most sequence-diverse class of small RNA regulatory molecules, are encoded on long, highly structured primary transcripts (pri-miRNA). These transcripts can be produced by RNA polymerase II or III and can encode a single miRNA or a cluster of miRNAs. These pri-miRNAs are processed by the nuclear localized RNase III family member Drosha, which recognizes the structure of the miRNA-encoding RNA hairpin and not the specific miRNA sequence. The Drosha-mediated cleavage reaction releases a short hairpin RNA, termed the precursor (pre)miRNA. This approximately 60–70 nt pre-miRNA is translocated to the cytoplasm by Exportin 5, where it is cleaved by Dicer into a dsRNA duplex containing the mature miRNA and the usually short-lived passenger strand (miRNA*). Similar to the long dsRNA-directed RNAi pathway, Dicer is associated with a dsRNA-binding protein called loquacious in Drosophila or TRBP (HIV-1 TAR RNA-binding protein) in mammals. The duplexed miRNA molecule is taken up by the effector complex, variously termed the
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properties of each of the 50 termini, with the end having the lower stability being preferentially maintained in the effector complex and used to guide the silencing reaction. Although this effector complex, variously termed the RNA-induced silencing complex for siRNAs or the miRNA-containing RNA-induced silencing complex (miRISC) for miRNAs, is poorly defined, it contains at its core a member of the Argonaute (Ago) family of proteins. The guide strand of the siRNA or the mature miRNA provides the specificity determinant for the effector complex by binding to complementary sites on the target mRNA. Gene silencing can take place by one of two mechanisms, either the inhibition of translation or the endonucleolytic cleavage of the mRNA. The method of silencing used for any target depends largely, but not entirely, on the degree of sequence complementarity between the siRNA or miRNA and the cognate mRNA. Those interactions with complete or nearly complete homology lead to mRNA cleavage, whereas interactions with a lower level of homology induce translation inhibition. The analysis of these silencing mechanisms has not only allowed for a better understanding of the role that small RNA molecules play in the regulation of many important cellular processes but also facilitated the development of technologies for the targeted silencing of gene expression. Although long dsRNAs had been used for silencing gene expression in C. elegans, this technology was not amenable for use in higher eukaryotes due to innate immune responses that have evolved in mammalian cells to recognize long dsRNA species and induce the activation of cell death pathways. In normal circumstances, the interferon response serves to protect cells from viral pathogens, many of which produce long dsRNAs as intermediates in their life cycle. Since the interferon response is efficiently triggered by dsRNAs more than 30 nt in length, it was believed that siRNAs, which fall below this size threshold, would be unable to activate this response. This was borne out by experiments that showed that the treatment of mammalian cells with chemically synthesized siRNAs could effectively silence gene expression without any untoward toxicity. This discovery led to the development of RNAi-based technologies allowing reverse genetic experiments to be performed in organisms that were intractable to more traditional gene-silencing approaches and laid the groundwork for the application of RNAi-based
silencing approaches as novel therapeutic modalities. In fact, the use of siRNAs for the localized silencing of gene expression has already entered clinical trials for the treatment of age-related macular degeneration and Rous sarcoma virus infections.
siRNAs as a Gene-Silencing Tool There are several features that make the use of siRNAs an attractive approach for the targeted silencing of gene expression. Foremost among these features is the fact that these silencing technologies take advantage of an endogenous gene-silencing mechanism that is conserved in organisms as diverse as plants, C. elegans, Drosophila, mice, and humans. In addition, unlike antisense approaches, potential target sequences can be readily identified. Initial studies showed that siRNA-mediated gene silencing was highly specific, being able to distinguish between genes that differed by as little as a single nucleotide, with no apparent toxicity. siRNA Sequence
A better understanding of the characteristics that distinguish highly potent siRNAs from nonfunctional siRNAs will facilitate the optimization of siRNA design to maximize silencing potential and minimize off-target effects. There are a number of traits associated with highly active siRNAs, including low internal stability, moderate to low GC content (optimally between 30% and 52%), lack of secondary structures within the siRNA sequence, and low thermodynamic stability at the 50 end of the guide strand. In addition, siRNA sequences that have homology with unintended targets or that contain immunostimulatory sequence motifs should be avoided to reduce the potential off-target effects. Many of these criteria have been incorporated into computer algorithms designed to aid in the identification of effective siRNA sequences targeting a particular gene of interest. Although these algorithms can improve the selection of potential siRNA sequences, experimental testing is necessary to determine the optimal siRNA sequence for any given gene. siRNA Specificity
Early studies using siRNAs to mediate gene silencing suggested that siRNAs, by virtue of their action,
miRNA-containing RISC (miRISC) or the miRNA nucleoprotein (miRNP) complex; the passenger strand (miRNA*) is dissociated; and the mature miRNA guides the complex to the target mRNA. The binding of the miRNA to target sites that have partial complementarity leads to the induction of translational repression. Although these pathways represent the classic views of the RNA interference pathways that induce posttranscriptional gene silencing, in actuality, siRNAs and miRNAs have interchangeable effector functions and either molecule can induce translational repression or mRNA cleavage depending on the degree of target site complementarity.
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required full complementarity and that a single nucleotide difference between the siRNA and the target binding site would be sufficient to abrogate silencing. In fact, it appears that mismatches between the siRNA and the target site can be accommodated without the complete loss of silencing. The degree to which mismatches can be tolerated depends on a number of factors, including the position and nucleotide composition of the mismatch. For example, the central nucleotides of the siRNA, around the site where cleavage of the mRNA occurs, are more sensitive to mismatches than either the 50 or 30 ends of the molecule. This is presumably due to steric constraints imposed by the active site of the Ago2 endonuclease, the core enzymatic component of RISC. It is not simply the presence of mismatched nucleotides that lends specificity but, more important, it is the nature of the mismatched nucleotides. That is, the juxtaposition of two purines within the central region of the siRNA:mRNA binding site will interfere with silencing more readily than the juxtaposition of two pyrmidines or a non-base-pairing purine:pyrmidine pair. However, by judiciously choosing the site and type of mismatch, it is possible to selectively silence genes that differ by as little as a single nucleotide. This has permitted the development of silencing tools that can distinguish between disease-causing alleles of a gene that differ by a single nucleotide from the ‘normal’ allele. This has been applied to disease causing single nucleotide polymorphisms (SNPs) involved in the development of cancer (e.g., RasV12), sickle cell anemia, and several dominantly inherited neurodegenerative disorders including superoxide dismutase 1 in amyotrophic lateral sclerosis and tau in frontotemporal dementia. Even for diseases whose phenotype is not directly attributable to an SNP, selective targeting of the disease can be accomplished by targeting SNPs that are linked to the disease allele. This has been used for the preclinical development of tools for the silencing of MJD1 (ataxin-3) in Machado–Joseph disease by targeting an SNP that tightly segregates with the pathogenic allele. Off-Target Effects
Although the initial studies using siRNAs for the targeted silencing of gene expression showed a high level of specificity and a lack of toxicity, as the technology has become more widely applied, the potential for off-target effects has become more apparent. The off-target effects of siRNAs are classified into two categories: (1) the silencing of unintended targets that have partial complementarity to the siRNA and (2) the induction of cellular responses that nonspecifically silence gene expression. The analysis of mRNA levels in cells treated with specific siRNAs has shown
changes to a large number of genes. Although the majority of these changes are small, a few genes can be severely affected. These nonspecific effects have been found for targets that share as few as 15 complementary nucleotides with as few as 11 contiguous nucleotides. These nonspecific effects can be generated from either strand of the duplexed siRNA. Microarray analysis can effectively measure changes in the amount of mRNA but fails to represent the changes that may occur due to translational inhibition, the common form of RNAi-mediated gene silencing induced by miRNAs in animal cells. Since miRNAs require a much lower level of complementarity, the potential off-targets could be significantly higher. It is difficult to evaluate these changes in protein levels since high-throughput proteomic approaches are much more difficult. Since effective translational repression mediated by miRNAs requires the concerted action of several miRNAs, the overall impact of these miRNA-style off-target effects may be minimal. As part of the innate antiviral response, mammalian cells have mechanisms for the recognition of dsRNA species, a common intermediate found in the life cycle of many viruses. This response induces the global inhibition of gene expression by impairing the cellular translation machinery. Although initial studies using siRNA for gene silencing found no induction of the interferon response with dsRNA smaller than 30 nt, subsequent studies using highly sensitive microarray analysis found that in certain circumstances expression of a subset of interferonresponsive genes was upregulated. This induction of the interferon response was found for only certain siRNA sequences and only when cells were treated with high concentrations of siRNAs. The treatment of cells with different siRNA sequences led to an increase in the expression of different subsets of interferon genes. Since no cytotoxicty was associated with any of these siRNA treatments, it appears that only a partial interferon response was induced. Experiments have suggested that the induction of the interferon genes may not be through the dsRNA-activated protein kinase; instead, interferon gene expression may be indirectly induced by the activation of Toll-like receptors (TLRs), TLR7 and TLR8. These TLRs can recognize specific immunostimulatory sequence motifs, particularly GU-rich sequences. The mode of administration of the siRNA affected the ability of the siRNA to stimulate an interferon response, suggesting that the method of cellular uptake plays a role in determining the immunostimulatory potential of an siRNA. For example, transduction of the cell by lipid-based transfection reagents resulted in a pronounced enhancement of off-target gene expression compared to delivery by electroporation.
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Since these off-target effects are concentration dependent, many of them can be avoided by minimizing the siRNA dosage. This can be accomplished by identifying highly potent siRNAs that can efficiently silence target gene expression at doses below the threshold necessary to induce off-target effect. Potential off-target effects can be minimized by careful siRNA sequence selection. Important criteria to reduce the potential for off-target effects include the avoidance of immunostimulatory sequences and designing the siRNA to minimize uptake of the passenger strand into RISC. In addition, all the potential siRNA sequences should be compared to the mRNA in the human genome to minimize the potential off-target binding sites. Although it may be impossible to avoid short stretches of homology, siRNAs that have extensive homology with unintended targets should not be used. In addition, the incorporation of chemical modifications within the siRNA can mitigate the immunostimulatory effects of the siRNA while retaining its silencing efficiency.
RNAi-Based Silencing Technologies Two basic approaches have been used for the targeted silencing of gene expression by siRNAs: (1) the introduction of chemically synthesized siRNAs into cells and (2) the introduction of DNA vectors expressing siRNA precursors that are processed intracellularly into active siRNAs (Figure 2). Each of these methods has distinct advantages and disadvantages. Chemically Synthesized siRNAs
The demonstration that chemically synthesized siRNAs could induce the silencing of a target gene of interest without activating an innate immune response has revolutionized biological research, providing a powerful tool for gene function analysis and opening up the possibility for the therapeutic application of these potent technologies. The transfection of synthetic siRNAs into tissue culture cells remains the most widely used method for gene silencing because of its specificity, ease of use, and adaptability to a variety of experimental conditions and cell types. An increasing number of lipid-based transfection reagents have been developed to effectively deliver the siRNAs into the cytoplasm of target cells. There are a number of factors that have limited the use of this technology, principal among these are the transient nature of the siRNA-based silencing response in actively dividing cells and the inability of traditional transfection reagents to transduce many important cell types. In most dividing cell lines, the silencing phenotype peaks at approximately day 3 posttransfection and subsides
approximately 5–7 days posttransfection. This loss of silencing over time appears to be due to the diluting out of the siRNAs during cell division until the intracellular concentration of the siRNA falls below a critical threshold needed to maintain the silencing phenotype. Long-lasting silencing (up to 3 weeks) has been achieved in cells that are nondividing, as demonstrated by in vitro experiments in postmitotic, terminal differentiated primary macrophage cells and in vivo delivery to hepatocytes. The degree to which a specific protein can be silenced depends on the halflife of the protein, the effectiveness of the siRNA, and the rate of cell doubling. Since the silencing phenotype is mediated by the targeted degradation of mRNA and not by degrading the protein, it is possible to effectively deplete the cell of a specific mRNA without altering the levels of the protein. This is particularly the case for proteins that are long-lived. Although the transfection of a wide variety of tissue culture cells is possible with the judicious choice of transfection reagents, there are a large number of cells that are refractory to lipid-based transfection protocols, including many clinically important primary cell types. In addition, the inherent toxicity of many of these lipid-based transfection reagents limits their usefulness for in vivo applications. Therefore, a variety of alternative transduction mechanisms have been developed (Figure 2(a)). One approach for the in vitro delivery of siRNAs to cell types that are difficult to transfect has been the combination of electroporation technologies with lipid-based transfection reagents, termed nucleofection. Although this approach can have some untoward toxicity, it has been effectively used for the silencing of gene expression in a wide range of cell types, including many primary cells. Another approach that has been proved to be effective for the delivery of siRNAs both in vitro (in tissue culture cell experiments) and in vivo has been the conjugation of the siRNA with cholesterol. Not only did this lead to the efficient uptake of the siRNA while retaining the siRNA’s silencing activity but also it improved the pharmacological properties of the siRNA by increasing the retention of the siRNA within the circulatory system. Since cholesterol receptors are relatively ubiquitously expressed, the cholesterol-conjugated siRNAs can enter a wide variety of cells. The broad spectrum of cells that can be effectively transduced by cholesterol-conjugated siRNAs makes this technology widely applicable. However, when cholesterol-conjugated siRNAs are given in vivo, they will be delivered to a wide variety of cells and tissues, only some of which are the intended target. This increases the risk of silencing gene expression in unintended cell types and could increase off-target effects. In addition, this nonspecific uptake increases the amount of the
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Figure 2 Methods for siRNA delivery. (a) Methods for the delivery of chemically synthesized siRNAs into cells. Although the use of lipid-based transfection reagents remains the most common method for the transduction of siRNAs into tissue culture cells, several methods have been developed that allow for the efficient delivery of siRNAs into cells for both in vitro and in vivo gene-silencing experiments. The covalent coupling of the siRNA, through the passenger strand, with either a cholesterol moiety (i) or an RNA aptamer (ii) has been shown to increase the pharmacological properties of the siRNAs (e.g., increasing the retention time of the siRNA within the circulatory system) while facilitating the uptake of the siRNA and maintaining its silencing capacity. Cholesterol permits the binding of the siRNA to cellular low-density lipoprotein receptors, which are expressed in a wide variety of cell types, and the uptake of the siRNA. Alternatively, the passenger strand of the siRNA can be incorporated into the termini of an aptamer, a structured RNA molecule that has been selected in vitro for specific properties, such as high-affinity binding to a distinct molecule. Using this approach, one can achieve cell type-specific delivery by choosing an aptamer that has a high affinity for a particular cell surface molecule. Similarly, cell type-specific delivery has been achieved using a recombinant protein containing the recognition domain of a single chain antibody or a fragment antibody fused to a nucleic acid binding motif (e.g., protamine) (iii). In this case, the siRNA interacts with the nucleic acid binding motif and is directed to the cell type of interest by the affinity of the antibody for a specific cell surface molecule. Although the majority of lipid-based transfection reagents have associated toxicities that prevent their use for in vivo applications, there are some lipid formulations that can be used for efficient delivery in vivo with negligible toxicity (iv). This usually involves the encapsulation of the siRNA into a lipid nanoparticle composed of several different types of lipids to provide it with the appropriate stability while allowing for the uptake and release of the siRNA into the cytoplasm of target cells. These lipid particles can be made to target specific cell types by the incorporation of specific moieties (e.g., antibody fragments) on their surface. (b) Stable silencing using DNA-based siRNA expression systems. Long-term silencing of gene expression can be achieved by expressing siRNA precursors, either (i) short hairpin (sh)RNAs or (ii) long hairpin RNAs based on the structure of endogenous miRNAs. These hairpin RNAs are processed by the endogenous RNAi machinery producing active siRNAs. These siRNA expression cassettes have been incorporated into a variety of DNA-based expression systems, including viral systems such as retroviral (oncoretroviral or lentiviral) and adeno-associated viral vectors. Typically, shRNAs have been expressed from RNA polymerase III (pol III) promoters that are uniquely suited for the production of short RNA species, whereas longer hairpin RNAs can be expressed from either Pol III or RNA polymerase II (pol II) promoters. Unlike pol III promoters that are ubiquitously expressed, pol II promoter-driven siRNA expression systems can be developed that permit the tissue-specific or inducible expression of the siRNA.
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siRNA needed to effectively silence expression of the target gene in the intended tissue or cell type. Therefore, delivery vehicles that could deliver siRNAs to specific cell types would be beneficial, particularly for therapeutic siRNA-based gene-silencing technologies. A number of technologies have been developed for the cell type-specific delivery of siRNAs. One such approach uses a recombinant bifunctional protein that contains a peptide that can recognize a specific cell-surface antigen (e.g., a single chain antibody or a fragment antibody against a cellular receptor or a ligand for a specific receptor) fused to a nucleic acid binding domain. Unlike the cholesterol-conjugated siRNAs, the siRNAs bind noncovalently to the recombinant protein and can be delivered to the cytoplasm of only those cells that express the specific protein on the cell surface. Therefore, the same deliver vehicle can be used to deliver any siRNA of interest for delivery. Cell type-specific delivery has also been achieved by conjugating the siRNA to an aptamer. Aptamers are structured nucleic acids that have been selected in vitro for specific characteristics, such as the ability to bind to specific proteins or to have a specific catalytic activity. The conjugation of an RNA aptamer that recognizes a specific cell surface protein to an siRNA can effectively deliver the siRNA to the cell type or tissue of interest. In order to retain the activity of the siRNA, the protein or RNA moiety is conjugated to the passenger strand of the siRNA, leaving the guide strand to perform its silencing function unimpaired. The mechanism by which the siRNAs are taken up and delivered to the cytoplasm to silence gene expression remains an area of active research. It remains to be determined how broadly applicable these technologies will be for the delivery of siRNAs. Despite these unknowns, these approaches have taken the therapeutic application of siRNAs one step closer to becoming a reality. Vector-Mediated RNA Interference
To overcome some of the intrinsic limitations of siRNA transfections, the transient nature of the silencing phenotype in actively dividing cells, and the difficulty of efficiently transducing certain cell types that are refractory to traditional lipid-based transfection protocols, siRNA-expressing DNA vectors have been developed (Figure 2(b)). Although Dicer was originally identified as an endonuclease that is capable of cleaving long dsRNA into siRNAs, it was soon realized that Dicer was also capable of processing the hairpin precursor molecule of miRNAs. By mimicking the structure of endogenously produced miRNA precursors, short hairpin RNAs were designed that could be efficiently expressed from DNA vectors and processed into active siRNAs. The majority of these expression systems were based on expression from
RNA polymerase III (pol III) promoters, either the U6 or the H1 promoter, which are uniquely suited for the expression of small RNA species since all of the necessary sites for activation of transcription are present upstream of the transcription start site and a run of uridine residues is sufficient to terminate transcription. The initial attempts to develop RNA pol II-based siRNA expression systems were unsuccessful. Although the transcripts were expressed, they were not processed by Dicer into functional siRNAs. It was only after the mapping of several miRNA transcripts that it was demonstrated that miRNA was encoded on primary transcripts that were highly structured hairpin RNAs containing frequent bulges and mismatched base-paired interactions. Based on these longer hairpin RNA structures, successful expression and processing of siRNAs was achieved. Unlike the ubiquitously expressing pol III promoters, pol II promoters that allow inducible, tissue- and cell type-specific transcription can be used for the regulated expression of siRNAs. In all these cases, the promoter drives transcription of a hairpin RNA containing the siRNA sequence and the accompanying passenger strand separated by a short noncomplementary loop sequence. These hairpin RNAs are processed by the endogenous RNAi machinery producing active siRNAs that enters RISC and direct the silencing of gene expression. The siRNA expression cassettes have been incorporated into an assortment of plasmid and viral vectors for the transduction of cells. Unlike synthetic siRNAs, plasmid vectors can be used for the transient delivery of siRNAs and, if the vector contains a suitable selectable marker, the stable delivery of siRNAs into many cell lines. However, their use is limited, in the same way as synthetic siRNAs, to cells that can be readily transfected. In addition, the low efficiency of generating stable silencing clones further limits their use in primary cells to transient siRNA expression. On the other hand, recombinant viral vectors have been developed which incorporate the siRNA expression cassette into the viral genome that is packaged into viral particles allowing for the infection of susceptible cell types. A variety of viral vectors have been adapted for use as siRNA delivery vehicles, including retroviral- and adeno-associated viral-based vectors. Since these viruses possess the necessary factors to facilitate uptake into cells, they are not limited to cells that can be readily transfected. Each of these systems has unique cell tropisms and life cycles, allowing them to productively deliver the siRNA expression cassette to specific subsets of cells. However, by changing the characteristic of the viral surface proteins involved in cellular recognition and uptake, it is possible to broaden the variety of cell types that can be infected. For example, HIV-1-based lentiviral constructs that utilize the HIV-1 viral
884 siRNA: Utility
envelope glycoprotein will infect only human cells that express the CD4 molecule on their surface. However, if the viral envelope glycoprotein is replaced by the glycoprotein from the vesicular stomatitis virus, which has a broad cell tropism, the lentiviral construct can be used to infect a variety of cell types from many different organisms, including human, mouse, rat, and even avian cells. Retroviral-based systems, derived from either lentiviruses or oncoretroviruses, have been used extensively for siRNA delivery. In these recombinant retroviral vectors, the genes necessary for productive infection have been deleted to ensure that the virus is unable to replicate in the target cells. Retroviruses are particularly well suited for the stable, long-term expression of siRNAs since they become integrated into the host genome and are replicated along with the host DNA. In addition, vectors based on lentiviral genomes, a class of retroviruses that include HIV-1, feline immunodeficiency virus, and equine infectious anemia virus, can infect postmitotic, nondividing cell types (e.g., terminal differentiated macrophage cells and neuron). Despite these advantages, retroviral vectors have the potential to cause insertional mutagenesis, leading to oncogenic transformation and clonal expansion of affected cells. Since these effects were seen in cells that were already actively replicating, it may be that this insertional mutagenesis will not be a significant barrier to the use of retroviral vectors in quiescent cells, such as those found in most tissues including the brain. Adeno-associated viral vectors have also been used successfully for in vivo siRNA delivery. Unlike retroviral vectors, adeno-associated vectors do not integrate into the host genome but can readily mediate long-term siRNA expression, particularly in nondividing tissues. In addition, adeno-associated viral vectors lack expression of viral proteins and as a result induce a limited immune response. Although these viral-based siRNA delivery systems have been shown to be effective both in vitro and in vivo, the packaging and purification of the recombinant viral particles can be a laborious process. For in vivo applications, siRNA expression from DNA-based vector systems is usually limited to the injection site and delivery requires a local regional administration (injection) or surgical procedure.
of the RNAi machinery, this has made it possible to silence any gene in the genomes of humans and other organisms. These approaches have been extensively applied in C. elegans and Drosophila cells to identify unknown genes involved in a wide array of cellular functions. Although large-scale screens in mammalian cells have presented a greater technical challenge, the refinement of RNAi-based technologies (siRNA transfection protocols and shRNA-based expression systems) has made these screens increasingly popular. It took very little time after RNAi was discovered in mammalian cells for researchers to recognize the potential of harnessing these pathways for therapeutic gene silencing. RNAi-based approaches have been extensively used for the silencing of pathogenic states ranging from viral infections to tumor development and metabolic and neurological disorders. The ease of design and synthesis, the low cost of production relative to other therapeutic agents such as antibodies and recombinant growth factors, the specificity of silencing, and the potency of silencing make siRNAs an attractive candidate for therapeutic applications. Delivery remains the major challenge for the successful therapeutic application of RNAi-based gene-silencing approaches. This is particularly the case for the systemic administration of siRNAs since unmodified siRNAs are sensitive to serum RNases and their small size results in their rapid clearance from the circulatory system. Despite these challenges, localized delivery of siRNAs has entered clinical trials for the treatment of age-related macular degeneration and respiratory syncytial virus infection. In a very short period of time, RNAi has gone from a novel silencing phenomenon in C. elegans to a highly conserved and ubiquitous gene regulatory network involved in many important cellular processes and, along the way, has been harnessed, providing an indispensable research tool and potential novel therapeutic modality.
Applications of RNAi-Based Technologies
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RNAi-mediated gene silencing has been widely applied as a research tool for reverse genetic experiments, which determine the function of a gene based on its disruption. The majority of these experiments have been done in a focused manner, examining the function of a single gene of interest at a time as well as other genomes, coupled with the ubiquitous nature
See also: Axonal mRNA Transport and Functions; Dendritic RNA Transport: Dynamic Spatio-Temporal Control of Neuronal Gene Expression; RNA Binding Protein Methods; Rodent Behavior: Approaches.
Further Reading
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