Báo cáo khoa học: Staying on message: design principles for controlling nonspecific responses to siRNA pdf

MINIREVIEW Staying on message: design principles for controlling nonspecific responses to siRNA Shirley Samuel-Abraham 1 and Joshua N. Leonard 1,2 1 Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA 2 Member, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL, USA Introduction In the decade since RNA interference (RNAi) was initially discovered in Caenorhabditis elegans [1] and shown to be inducible in mammalian cells [2,3], technologies for harnessing this mechanism to induce targeted gene silencing have become routine laboratory tools and, increasingly, are making their way into clinical trials (reviewed in Castanotto & Rossi [4]). Over this same period, however, it has become clear that the short interfering RNA (siRNA) commonly delivered to induce RNAi can also induce multiple nonspecific effects. A poignant example comes from the first system for which clinical trials of RNAi were Keywords innate immunity; OAS1; off-target; RIG-I; RNA interference; RNAi; short interfering RNA; siRNA; TLR; Toll-like receptors Correspondence J. N. Leonard, Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Rd, Room E-136, Evanston, IL 60208 USA Fax: +1 847 491 3728 Tel: +1 847 491 7455 E-mail: j-leonard@northwestern.edu (Received 7 July 2010, accepted 26 August 2010) doi:10.1111/j.1742-4658.2010.07905.x Short interfering RNAs (siRNA) are routinely used in the laboratory to induce targeted gene silencing by RNA interference, and increasingly, this technology is being translated to the clinic. However, there are multiple mechanisms by which siRNA may be recognized by receptors of the innate immune system, including both endosomal Toll-like receptors and cytoplas- mic receptors. Signaling through these receptors may induce multiple non- specific effects, including general reductions in gene expression and the production of type I interferons and inflammatory cytokines, which can lead to systemic inflammation in vivo. The pattern of immune activation varies depending upon the types of cells and receptors that are stimulated by a particular siRNA. Although we are still discovering the mechanisms by which these recognition events occur, our current understanding pro- vides useful guidelines for avoiding immune activation. In this minireview, we present a design-based approach for developing siRNA-based experi- ments and therapies that evade innate immune recognition and control nonspecific effects. We describe strategies and trade-offs related to siRNA design considerations including the choice of siRNA target sequence, chem- ical modifications to the RNA backbone and the influence of the delivery method on immune activation. Finally, we provide suggestions for conduct- ing appropriate controls for siRNA experiments, because some commonly employed strategies do not adequately account for known nonspecific effects and can lead to misinterpretation of the data. By incorporating these principles into siRNA design, it is generally possible to control nonspecific effects, and doing so will help to best utilize this powerful technology for both basic science and therapeutics. Abbreviations dsRNA, double-stranded RNA; GFP, greem fluorescent protein; IFN, interferon; IL, interleukin; OAS1, 2¢-5¢-oligoadenylate synthetase; PKR, protein kinase R; RIG-I, retinoic acid-inducible gene I; RISC, RNA-induced silencing complex; RNAi, RNA interference; siRNA, short interfering RNA; ssRNA, single-stranded RNA; TLR, Toll-like receptor. 4828 FEBS Journal 277 (2010) 4828–4836 ª 2010 The Authors Journal compilation ª 2010 FEBS initiated – intravitreous injection of siRNA against vascular endothelial growth factor to block angiogenesis in patients with blinding choroidal neovascularization [4]. Recent data from animal models of choroidal neo- vascularization indicate that the therapeutic benefits of this treatment are mediated in large part by nonspecific mechanisms involving recognition of siRNA by the innate immune system [5,6]. Nonetheless, definitive proof that siRNA can also induce RNAi-mediated spe- cific gene silencing in human patients was recently reported in a clinical trial for nanoparticle-mediated siRNA delivery for melanoma treatment [7]. In addi- tion, some clinical strategies are now being designed to harness both the specific and nonspecific effects of siRNA therapeutics [8,9], although the relative contri- butions of each mechanism remain somewhat unclear. Given the complexity and potential subtlety of these nonspecific effects, siRNA-based experiments and pre-clinical studies should incorporate our growing knowledge of the molecular features that give rise to innate immune recognition. This review presents a design-oriented approach for controlling innate immune system-mediated interactions when developing siRNA-based therapeutics. In higher animals, RNAi constitutes one arm of an arsenal of innate defenses against viral infections. Consequently, the same molecules that induce targeted gene silencing through RNAi [including double- stranded RNA (dsRNA) and siRNA] also induce nonspecific antiviral responses through these overlap- ping mechanisms. Two cytoplasmic receptors that have long been known to recognize long dsRNA include protein kinase R (PKR) and 2¢-5¢-oligoadeny- late synthetase (OAS1). Upon binding to dsRNA, PKR catalyzes the phosphorylation of eIF2a and I jB, which induces a general inhibition of translation and drives the production of type I interferons (e.g. IFN-a and IFN-b) through NF-jB [10,11]. Most siRNA are shorter than the 30 bp minimum dsRNA length required to potently activate PKR [12], and although some reports indicate that detectable PKR activation can be induced by siRNA, it is not yet clear whether this low-level of activation induces biologically rele- vant responses [13,14]. OAS1 is activated by binding to dsRNA and induces sequence-independent degrada- tion of viral and cellular single-stranded RNA (ssRNA) by activating RNaseL [15]. OAS1 also plays an important role in the amplification of innate immune responses, because OAS1 expression is upreg- ulated by type I interferon, and the small dsRNA products of RNaseL-digested cellular or viral mRNA can activate innate immune receptors in neighboring cells [16]. Some evidence indicates that certain dsRNA of only 19 bp in length can activate OAS1 directly [17]. Our current understanding is that the most potent nonspecific siRNA-induced effects are mediated by more recently characterized receptors located in dis- tinct subcellular compartments. The nucleic acid- responsive Toll-like receptors (TLRs) interact with pathogen-associated molecules in endosomal vesicles, and TLR3 [5,6,18], TLR7 [19–21] and TLR8 [19,22] have each been implicated in the response to siRNA. Of these, TLR7 and TLR8 are thought to mediate the dominant immune response to siRNA in vivo, and each responds even more robustly to the single- stranded RNA constituents of an siRNA duplex [23]. TLR7 and TLR8 signal through the MyD88 pathway and induce the production of type I interferons and inflammatory cytokines [24]. However, the overall immune responses induced through these receptors dif- fer because of their unique patterns of expression – TLR7 is expressed by plasmacytoid dendritic cells and B cells and mediates interferon-dominated responses, whereas TLR8 is expressed on myeloid dendritic cells, monocytes and macrophages, and mediates inflamma- tory cytokine-dominated responses [25]. TLR3 signals through a unique adapter called TRIF and is an espe- cially potent inducer of IFN-b and inflammatory cyto- kines such as interleukin (IL)-6 and tumor necrosis factor-a [24,26]. In the cytosol, retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 recog- nize dsRNA and play central but distinct roles in antiviral defense [27]. However, only RIG-I is known to be activated by siRNA [28], and this mechanism is thought to explain many observations of nonspecific changes in gene expression and interferon production induced by cytoplasmically localized siRNA. Each of these innate immune receptors recognizes defined siRNA molecular features, and some features are rec- ognized by multiple receptors. The following sections summarize our current understanding of these recogni- tion events and how siRNA might be designed to control immune recognition (Fig. 1). siRNA design considerations siRNA sequence When selecting an siRNA sequence, potency is often the first consideration, and strategies for selecting a potent siRNA sequence for a given target mRNA are discussed in a companion minireview in this issue by Walton et al. [29]. However, the siRNA sequence also plays an important role in determining whether a given S. Samuel-Abraham and J. N. Leonard Controlling nonspecific responses to siRNA FEBS Journal 277 (2010) 4828–4836 ª 2010 The Authors Journal compilation ª 2010 FEBS 4829 siRNA duplex will induce an innate immune response. The rules for predicting this property are not entirely known, but they clearly vary between innate immune receptors. Activation of TLR7 and TLR8 is particu- larly dependent on siRNA sequence. Characteristic features of RNA such as the presence of uridine resi- dues and the ribose sugar backbone are necessary for recognition of RNA by TLR7, and short single- stranded RNA need only contain several uridines in close proximity to effectively activate TLR7 [30]. Certain sequence motifs, such as GUCCUUCAA, may be particularly immunostimulatory [20]. This property also depends on the overall length of the ligand, because 19-bp siRNAs containing this motif were more potent inducers of cytokine production in plasmacy- toid dendritic cells than were 12- or 16-bp siRNAs that contain the same motif. Modification of immunostimu- latory sequences modulates immune stimulation in a context-dependent manner. In one such example, sub- stitution of U with A abrogated tumor necrosis factor- a and IL-6 induction in periperhal blood mononuclear cells, whereas substituting G with A abrogated only the induction of IFN-a in plasmacytoid dendritic cells without affecting the induction of tumor necrosis fac- tor-a, IL-6 and IL-12 in peripheral blood mononuclear cells [19]. The overall sequence composition of an siRNA can also influence its immunostimulatory prop- erties. Single-stranded RNAs that are GU-rich are potent ligands for human TLR7 and TLR8, whereas AU-rich motifs preferentially activate TLR8 [19,22]. However, these features are not necessarily required, because some siRNAs also activate TLR7 independent of GU content [20], and sequences that lack G and U nucleotides can still trigger an immune response [31]. Other innate receptors, such as OAS1, also exhibit dsRNA sequence-dependent activation [17]. In this study, synthetic dsRNAs of 19 bp in length were eval- uated for their capacity to activate OAS1. All OAS1- stimulating dsRNAs contained the consensus motif, NNWW(N 9 )WGN (W indicates an A or U), and mutational analysis confirmed that this motif is required for activation. The consensus is only 16 nucle- otides long, because it occurred at various positions along the 19-nucleotide sequences tested. Interestingly, these 19-bp dsRNAs are substantially shorter than the oligonucleotides typically thought to activate OAS1, which suggests that siRNA might also directly activate OAS1 by a similar mechanism. The length of an siRNA is generally an impor- tant determinant of innate immune activation. Initial Backbone chemistry siRNA sequence End features Key Fig. 1. Design considerations for controlling nonspecific responses to siRNA. This figure summarizes known recognition interactions between innate immune receptors (green ovals) and siRNA molecular features (blue rectangles), grouped by category of design consider- ation, and strategies that can be employed to overcome such recognition (red octagons, with interruption of a recognition interaction indi- cated by red lines). Backbone chemistry modifications at the 2¢ position of ribose moities include deoxy (-H), fluoro (-F) and O-methyl (-O-Me) substitutions. These substitutions can generally be limited to a subset of sites within the sense strand to balance suppression of im- munostimulation with retention of capacity to induce RNAi. To some extent, one can select siRNA target sequences that avoid known im- munostimulatory motifs (the list shown here is representative but not exhaustive). Choosing end features such as 3¢ overhangs and avoiding 5¢ triphosphates reduce immune stimulation by both RIG-I and unknown receptors (i.e. ‘???’). The siRNA image is modified from PDB struc- ture 2F8S. Controlling nonspecific responses to siRNA S. Samuel-Abraham and J. N. Leonard 4830 FEBS Journal 277 (2010) 4828–4836 ª 2010 The Authors Journal compilation ª 2010 FEBS studies indicated that siRNAs shorter than 30 bp could evade the immune system and thus avoid any off- target activity [3]. However, subsequent studies indicated that dsRNA molecules longer than 21 bp can lead to a sequence-independent interferon response [32]. In some studies, even 19-bp molecules provided the minimal length required for immune stimulation [20]. The length of dsRNA required to activate TLR3 also remains somewhat uncertain. Our in vitro studies using TLR3 reporter cells and biophysical measurements using recombinant TLR3 protein indicated that ligands shorter than 30 bp neither bind nor activate human TLR3 [33]. However, shorter siRNAs have been shown to induce TLR3-dependent inflammation [5,6]. In each of these cases, it is likely that recognition of siRNA by multiple receptors may explain some apparent conflicts between these observations, although this can only be resolved by elucidating the molecular mechanisms of siRNA recognition by each receptor. To date, the rules governing the relationship between siRNA sequence and the capacity to stimulate an innate immune response are not yet clear. There- fore, in practice, controlling innate immune responses to siRNA still requires a systematic characterization of the immunostimulatory properties of multiple alterna- tive siRNA sequences for each given target, or the implementation of additional strategies for suppressing immune stimulation. Backbone chemistry Naturally occurring nucleoside modifications in mam- malian RNA appear to provide a mechanism by which the innate immune system discriminates self-oligonu- cleotides from those of viral origin [34]. Similarly, some immune recognition of siRNA may be abrogated by altering the chemistry of the RNA backbone. To implement this strategy, one must decide whether to modify every base in a strand or only selected bases, and whether to modify just one strand or both strands in a duplex. Backbone modification choices are guided in large part by the mechanism through which an siRNA par- ticipates in RNAi via the RNA-induced silencing com- plex (RISC). Only one strand of the siRNA duplex is incorporated into RISC, and in order to direct RISC to cleave a target mRNA sequence, the antisense strand must be incorporated to serve as a template. Modifications to the backbone chemistry of a strand may impair its incorporation into RISC, so siRNA-induced silencing is best maintained if modifi- cations are confined to the sense strand [35,36]. However, modifications at position 9 of the sense strand (immediately upstream of the cleavage site) may inhibit sense strand cleavage, which reduces the efficiency of RISC assembly and therefore gene silenc- ing [37]. Although some alterations to the antisense strand abrogate gene silencing, certain antisense modi- fications seem to preserve functionality [8,38]. At this point, the rules for predicting which site and type of modifications one should use on the antisense strand to preserve its functionality are not clear [20,36,38]. Furthermore, some immunostimulatory antisense strands can be made nonstimulatory by modifying the backbone chemistry of the cognate sense strand (and only the sense strand) in a duplex [39,40]. This trans-inhibition of immune activation may indicate that the receptor involved recognizes the duplex rather than the component single strands. An additional advantage to modifying the backbone chemistry of the sense strand is that by impairing the incorporation of this strand into RISC, one avoids off-target gene silencing of mRNAs that are complementary to the siRNA sense strand. A variety of siRNA backbone modification chemis- tries have been investigated for their capacity to sup- press immune activation while maintaining gene silencing activity. Because of the requirement of ribose-containing nucleotides for many types of immune stimulation [30], one common strategy is to replace the 2¢-hydroxyl group of the ribose backbone with 2¢-fluoro, 2¢-deoxy or 2¢-O-methyl groups [23]. In particular, making such substitutions at uridine resi- dues often reduces the immunostimulatory capacity of siRNA [23]. Although strand-wide modifications have also been investigated for their capacity to block immune activation [8], such extensive changes are probably not required. For example, incorporation of only two 2¢-O-methyl guanosine or uridine residues in the sense strands of highly immunostimulatory siRNA molecules was sufficient to abrogate siRNA-mediated interferon and inflammatory cytokine induction in human peripheral blood mononuclear cells and in mice in vivo [39]. In this example, such modifications repre- sented  5% of the native 2¢-hydroxyl positions in the siRNA duplex, and no other modifications were required. Notably, 2¢-O-methyl modification of cyti- dines was not as effective as the other substitutions in abrogating the immune response. For dsRNAs that activate OAS1, 2¢-O-methyl substitution of residues in the stimulatory motif of the sense strand abolished OAS1 activation (these positions are presumed to inter- act with OAS1), whereas similar substitutions on the opposite strand preserved stimulation of OAS1 [17]. Certain backbone modifications necessitate consider- ations unique to their particular chemistry. Making S. Samuel-Abraham and J. N. Leonard Controlling nonspecific responses to siRNA FEBS Journal 277 (2010) 4828–4836 ª 2010 The Authors Journal compilation ª 2010 FEBS 4831 2¢-deoxy substitutions is equivalent to including DNA bases in siRNA molecules (except in the case of 2¢-deoxy uridine, which remains distinct from thymi- dine), and this substitution has been reported to increase silencing activity [41]. However, it is also pos- sible that these ligands might activate TLR9, especially if they contain CpG motifs [42]. A distinct type of modification is the use of locked nucleic acids, wherein the ribose contains a 2¢-O, 4¢-C methylene bridge. This modification renders oligonucleotides resistant to nuc- leases and may also reduce the immunostimulatory activity of siRNA [20]. Locked nucleic acid modifica- tions at the 3¢-termini or both the 3¢- and 5¢-termini of the sense strand of an siRNA duplex block immune stimulation but have very little effect on the capacity of the siRNA to induce RNAi. Conversely, locked nucleic acid modifications at the termini of the anti- sense strand do not affect immune stimulation, but RNAi induction may be impaired or even abrogated (in the case in which both 5¢- and 3¢-termini of the antisense strand are modified). Overall, these findings suggest several general strate- gies that reduce immune stimulation and preserve functionality, such as modifying the sense strand of an siRNA duplex (only) at the 2¢ positions of several ribose moieties. However, no one strategy is yet uni- versally applicable. For example, 2¢-O-methyl substitu- tion of uridines did not prevent siRNA-mediated activation of TLR3 [5]. For now, some systematic investigation of possible backbone modifications (or at least sites to be modified) is required to find the opti- mal balance between maintaining siRNA efficacy and preventing nonspecific effects. End features The termini (ends) of an siRNA are major determi- nants of immune recognition. In the context of viral infections, RIG-I detects viral RNA by binding to its uncapped 5¢ triphosphate terminus [28]. Maximal acti- vation of RIG-I requires that the 5¢ triphosphate end of the dsRNA be blunt [43]. Not suprisingly, siRNAs that share either or both of these features are also im- munostimulatory. For this reason, siRNAs transcribed in vitro from phage polymerases are particularly immu- nostimulatory unless they are processed to remove 5¢ triphosphates (or the initially transcribed nucleotides to which these moieties are attached) [44]. Mimicking the 3 ¢ overhangs that result when DICER processes long dsRNA into siRNA seems to improve siRNA properties in several ways. When compared with blunt- ended oligonucleotides, siRNA with 3¢ overhangs more efficiently induce gene silencing in vivo [37], and by adding 3¢ overhangs, otherwise immunostimulatory 27- bp siRNA can evade immune recognition [13]. Given our current understanding of these features, end chem- istry-mediated immune stimulation can generally be avoided. Delivery vehicles and strategies The use of siRNA delivery vehicles is essential for practical siRNA-mediated silencing because naked siRNA face rapid degradation in the extracellular environment and are not efficiently internalized into cells [45,46]. Various strategies for efficiently delivering siRNA are discussed in the companion minireview in this issue by Shim & Kwon [47]. The choice of delivery strategy also impacts whether an siRNA will induce innate immune activation. In trafficking from the extracellular environment, through endosomal compartments and to the cyto- plasm, there exist multiple points at which recognition of siRNA by the innate immune system may occur. TLR-mediated recognition of siRNA takes place in en- dosomes. Receptor–ligand interactions are thought to require this acidic milieu because inhibitors of endoso- mal maturation, such as bafilomycin, block immune activation by siRNA via TLR7 and TLR8 [48]. Conju- gation of siRNAs to cholesterol may enhance cytoplas- mic delivery, and to some extent, such complexes may bypass the endosomes without activating endosomal receptors [46]. Experimentally, direct delivery of siRNA to the cytoplasm by electroporation may also suppress an immune response [48]. However, because siRNA must be released into the cytoplasm in order for them to be incorporated into RISC, any siRNA motifs that activate cytoplasmic receptors would still induce immune activation regardless of the choice of delivery vehicle. When siRNA are systemically admin- istered, targeting these molecules to specific cellular subsets may also reduce stimulation of the innate immune response in nontargeted cells. For example, a protamine–antibody fusion protein was designed to deliver siRNA specifically to tumor cells expressing the ErbB2 antigen [49]. Although no interferon-induced gene expression was observed when delivering an anti- green fluorescent protein (GFP) control siRNA to cells via a protamine–antibody fusion, is it not possible to conclude from these experiments that targeting facili- tated immune evasion. An siRNA may be immunologically inert when deliv- ered as a naked siRNA but will stimulate immunity when complexed with a delivery vehicle. Such effects have been observed using cationic lipids, such as N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium Controlling nonspecific responses to siRNA S. Samuel-Abraham and J. N. Leonard 4832 FEBS Journal 277 (2010) 4828–4836 ª 2010 The Authors Journal compilation ª 2010 FEBS methylsulfate [48] and lipofectamine [35], cationic polymers such as poly(ethyleneimine) or poly(l-lysine) [35] and stable nucleic acid–lipid particles [39]. When interpreting these results, it is important to remember that complexing siRNA with a delivery vehicle may have several effects. Because naked siRNAs are not efficiently taken into cells, some of the enhanced immune stimulation observed may be due to enhanced trafficking of siRNA to endosomes and the cytoplasm, and therefore to enhanced interaction with endosomal and cytoplasmic receptors. It is also possible that presentation of siRNA in a large polyvalent complex makes these ligands more immunostimulatory (to either endosomal or cytoplasmic receptors) than the free siRNA would be alone. An additional consideration is that the subcellular location at which immune activation occurs determines the type of immune response that is induced. For example, ligand-mediated activation of TLR7 (or TLR9) in endosomal compartments induces type I interferon production via IRF-7, whereas activation of TLR7 or TLR9 in lysosomal compartments may induce inflammatory cytokine secretion via IRF-5 [50,51]. This may be related to the observation that siRNA complexed with lipofectamine or poly(l-lysine) (which form large complexes) induces a response domi- nated by inflammatory cytokines, whereas siRNA complexed with poly(ethyleneimine) or stable nucleic acid lipid particles induces a response dominated by interferon production [35]. It is possible that differ- ences in intracellular trafficking might explain the dis- tinct biological effects conferred by these vectors. Overall, no delivery vehicle is sufficient to confer full and general protection against siRNA-induced immune activation, particularly that which is mediated by cyto- plasmic receptors. It is likely that any delivery vehicle will need to be paired with other strategies for evading immune activation. Concluding remarks For most mRNA targets, it should be possible to gen- erate multiple siRNA that induces specific gene knock- down without inducing nonspecific inhibition of nontargeted genes. Some strategies can be employed generally, such as avoiding terminal 5¢ triphosphates and including 3¢ overhangs [13,28,37,43,44]. Choices of siRNA sequence are specific to the mRNA targeted, and although it may be prudent to avoid potent immu- nostimulatory motifs (such as those known to activate TLR7, TLR8, and OAS1 [17,20,30]), it may also be possible to overcome this activation through judicious modifications to the siRNA backbone [20,23,39]. In particular, making 2¢-hydroxy substitutions in several ribose moieties in the siRNA sense strand (such as 2¢- O-methyl and 2¢-fluoro) may suffice to block recogni- tion of potentially stimulatory motifs by innate immune receptors while retaining the capacity to func- tionally induce RNAi. In practice, selecting these sites currently requires both avoiding known trouble spots (i.e. position 9, immediately upstream of the RISC cleavage site [37]) and experimentally evaluating possi- ble combinations of backbone modifications. Many delivery vehicles may enhance immune stimulation by siRNA [35,39,48], and although others may suppress some mechanisms of immune activation [46], one can- not rely upon vehicle choice alone, particularly because free siRNAs are eventually released into the cyto- plasm, where they may interact with cytoplasmic receptors. Strategies that seek to intentionally induce specific types of immune activation are more challeng- ing, because in many cases the precise nature of the immune recognition event is unknown. In general, this gap in knowledge underlies current limitations on our ability to predict the immunostimulatory capacity of a given siRNA design. In particular, we know little about the mechanisms by which siRNAs are recognized by the TLRs known to play central roles in nonspecific responses to siRNA in vivo. TLR7 and TLR8 are thought to mediate the majority of both inflammatory cytokine and inter- feron-dominated immune responses to siRNA in vivo [25,52], yet we do not know how these receptors recog- nize siRNA, ssRNA, dsRNA or their commonly used nucleoside analog ligands. Similarly, TLR3 plays an important role in siRNA-mediated nonspecific immune activation [5,6], yet the mechanism by which recogni- tion of siRNA occurs is also unclear. For example, some evidence suggests that siRNA-mediated activa- tion of TLR3 occurs at the cell surface [5,6], yet it is not clear how the receptor would interact with dsRNA in this neutral pH milieu. When recognizing longer dsRNA, an acidic milieu is required so that histidine residues on TLR3 become positively charged and interact electrostatically with the negatively charged backbone of the dsRNA ligand [53–55]. Thus siRNA- mediated activation might occur via a coreceptor or via a mechanism distinct from that by which longer dsRNA is recognized. It is also possible that, at least in some cases, TLR3-dependent immune activation by siRNA occurs by indirect mechanisms. For example, activation of RNaseL by OAS1 (which may itself be induced by siRNA-mediated production of interferon through other receptors) produces a pool of self- derived dsRNA ligands, some of which fall into size ranges that may activate TLR3 on neighboring cells S. Samuel-Abraham and J. N. Leonard Controlling nonspecific responses to siRNA FEBS Journal 277 (2010) 4828–4836 ª 2010 The Authors Journal compilation ª 2010 FEBS 4833 [16,33]. In this way, TLR3 would be an important part of an siRNA-induced feedback loop even if TLR3 did not recognize siRNA directly. Another complication is that cell lines transfected to overexpress TLR3 exhibit generally enhanced interferon-induced responses [18], so overexpression of TLR3 may also enhance cytoplas- mic receptor-mediated responses to siRNA. Further investigations are required to elucidate the mechanisms of these recognition events in order to enhance our ability to predict, a priori, whether a given siRNA will activate these potent immune responses. Given our understanding of the various mechanisms by which siRNAs induce nonspecific immune responses, it is essential that appropriate experimental controls be designed accordingly. Traditionally, control siRNAs have included target sequences derived from GFP or luciferase, a random sequence, or a scrambled form of the test siRNA target sequence. Failing to account for the nonspecific effects of either the control or the test siRNA can lead to misinterpretation of experimental results. This was recently demonstrated in a murine model of influenza, in which an anti-influenza siRNA conferred greater antiviral protection than did an anti- GFP control siRNA [56]. However, this protection was conferred by nonspecific immune activation, which appeared to be specific only because the anti-GFP con- trol siRNA was particularly nonimmunostimulatory. For these reasons, it is necessary to include experimental controls that make it possible to differentiate between the specific and nonspecific effects of a given test siRNA. For example, in an in vivo model for hepatitis B virus infection, an unmodified inverted siRNA control was found to nonspecifically inhibit viral replication [8]. Therefore, both unmodified (potentially immunostimu- latory) and chemically modified (nonimmunostimulato- ry) versions of both anti-hepatitis B virus and control siRNA were tested to evaluate the relative contributions of specific and nonspecific antiviral effects. Finally, experiments evaluating whether a particular siRNA (or siRNA-delivery technology, for that matter) is immuno- stimulatory must be designed considering that expres- sion patterns of the innate immune receptors that recognize siRNA vary between cell types (especially between immune and non-immune cells), and that rec- ognition by different receptors and different cells results in distinct patterns of innate immune responses (e.g. interferon vs. inflammatory cytokine production). Con- sidering all known mechanisms of innate immune stimu- lation by siRNA and working to further advance our understanding of these recognition events are each of paramount importance as we continue to design and interpret siRNA-based experiments and tap the enor- mous potential of siRNA-based therapeutics. Acknowledgements This work was supported with funding from North- western University and the Robert R. McCormick School of Engineering and Applied Science. 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