a variety of well - documented approaches have been developed for 
 inhibiting gene expression . these include the use of antisense 
 rnas , oligodeoxynucleotides ( odns ) , ribozymes , and rna 
 interference ( rnai ) [ 13 ] . among these approaches , 
 rnai - mediated mrna degradation is emerging as the most highly 
 effective antigene strategies [ 2 , 3 ] . although the same 
 phenomenon was described in transgenic plants in the late 1980s 
 , only in 1998 fire and colleagues have demonstrated that 
 ds rna introduced into caenorhabditis elegans can silence 
 the expression of homologous target gene by directing degradation 
 of its mrna . during rnai , the precursor double - stranded ( ds ) rna is processed by the dicer endonuclease into short 2124 
 nucleotides ds sirnas containing 2-base 3-overhangs . subsequently , the sirna duplexes are then incorporated into a 
 multiprotein complex , the rna - induced silencing complex ( risc ) , 
 which mediates the degradation of mrnas with sequence homologous 
 to the sirna . recent studies have showed that nucleotides 28 of 
 the sirna antisense strand form a seed sequence that 
 directs target mrna recognition . some of the 
 protein components of risc have been identified and assigned 
 functions . argonaute 2 ( ago2 ) is the rna endonuclease that cleaves 
 target mrna . despite the demonstration of rnai in plants and worms , researchers 
 have been pessimistic about using rnai in mammalian cells due to 
 the induction of the interferon ( inf ) pathway by long ds rnas 
 ( > 30 nucleotides ) , leading to nonspecific inhibition of protein 
 synthesis and degradation of mrnas . however , tuschl and colleagues found that sirna duplexes mimicking the sirnas 
 generated by dicer cleavage in lower organisms could trigger rnai 
 without inducing the ifn pathway , which is observed with long 
 double - stranded rna in most cell types . this discovery has paved the way for the study of gene function in mammalian 
 cells . in contrast to invertebrates , mammalian cells do not have 
 transitive rnai because of the absence of rna - dependent rna 
 polymerase [ 2 , 3 ] . consequently , the number of sirna molecules 
 introduced into a cell limits gene silencing induced by synthetic 
 sirnas . to circumvent this potential problem , several groups have 
 developed expression vectors that direct the synthesis of rna 
 duplexes or short hairpin rnas ( shrnas ) in mammalian cells , 
 leading to sustained production of sirnas . one approach for silencing relies on the expression of shrnas under the control 
 of h1 or u6 pol iii promoter , which is active in most mammalian 
 cell types . during the last 5 years , sirna - mediated gene knockdown has become 
 a standard method for studying gene function and drug target 
 validation . however , despite the success that has been achieved , 
 some serious concerns such as the activation of innate immunity , 
 inhibition of unintended target mrnas , and potential interference 
 with regulatory functions of endogenous micrornas need to be 
 resolved prior therapeutic applications in humans 
 [ 2 , 1012 ] . the gradual maturation of rnai technology from the laboratory to 
 the clinic involves several major challenges , many of which still 
 need to be resolved . these include delivery to target cells , 
 intracellular stability , and specificity [ 2 , 3 ] . it should be 
 noted that the introduction of synthetic sirnas into mammalian 
 cells in culture is relatively simple by transfection reagents 
 . sirnas can be chemically synthesized , or produced by in vitro transcription or by digestion of long dsrnas by recombinant 
 rnase iii or dicer [ 2 , 3 ] . most of the techniques that have been used for antisense delivery can be applied to synthetic sirnas and to dna constructs engineered to express shrnas . these 
 include electroporation , cell microinjection , and lipophilic 
 transfection . although sirnas were initially thought to be small enough to avoid 
 the activation of the ifn pathway , recent studies showed they could activate innate immunity in mammalian cells 
 [ 10 , 1416 ] . in this respect , sledz and colleagues reported that sirna could activate pkr , and the effects were sequence 
 independent and do not occur with the sense or the antisense rna 
 used to prepare the sirna duplexes . in contrast , kariko and colleagues found that sirna could activate cytokine and 
 interferon production via tlr3 , a receptor known to bind viral ds 
 rnas . however , we and others recently have demonstrated that pkr and 
 tlr-3 do not represent the major mechanism by which chemically 
 synthesized sirna activate innate immunity [ 1719 ] . in 
 addition , internalization of ds sirnas or ss sirnas is required 
 for immune activation . indeed , inhibitors of endosomal 
 maturation / acidification like bafilomycin a1 , a drug that inhibit 
 endosomal h+-proton pumps , blocked immune activation , indicating the involvement of endosomal tlr7 and tlr8 . consistent 
 with the role of endosomal tlrs in sirna signaling , tlr7 knockout 
 mice did not mount immune activation in response to sirnas 
 . taken together , the available data indicate that immune cells such as monocytes and dendritic cells recognize ds sirnas 
 and ss sirnas through tlr7 and tlr8 , leading to the production of 
 inflammatory cytokines and type i interferons . we also found that ss sirnas are more effective than ds sirna in 
 triggering tlr7 and tlr8 responses [ 17 , 21 ] . in addition , we 
 have found that human bone marrow hematopoietic cd34 + progenitor 
 cells express tlr7 and tlr8 . interestingly , incubation of these 
 cells with either immunostimulatory synthetic sirnas or r848 , a 
 specific ligand for tlr7 and tlr8 , induced their differentiation 
 into the myeloid lineage ( sioud et al in preparation ) . thus , the 
 interaction of viral rnas with cd34 + progenitor cells may 
 increase the pool of innate immune cells . as mentioned above mammalian cells notably , the immune system 
 uses a set of germ line encoded receptors called pattern 
 recognition receptors ( prrs ) to recognize common microbial 
 structures known as pathogen associated molecular patterns 
 . whereas several tlrs are expressed in the cell surface , tlr3 , tlr7 , tlr8 , and tlr9 , traffic between the endoplasmic 
 reticulum and intracellular compartment such as the endosomes and 
 the lysosomes . an obvious function of these trafficking pathways is to scan for viral and/or bacterial nucleic acids , thus 
 playing a central role in innate antiviral responses . therefore 
 sirnas internalized via endocytosis are more likely to activate 
 endosomal tlrs . it should be noted that cytoplasmic delivery of 
 immunostimulatoty synthetic sirnas or shrna did not induce immune 
 response in human blood cells [ 17 , 21 ] , suggesting that they 
 are not recognized by cytolasmic sensors of ds rnas . in this 
 respect , a recent study has showed that endogenously expressed 
 shrnas are not immunostimulatory in human cells . interestingly , williams and colleagues demonstrated that the 
 presence of 2-base 3-overhangs in synthetic sirnas or in dicer 
 processed shrnas blocks the activation of rig-1 , a major 
 cytolasmic sensor for viral ds rnas . initial experiments indicate that some types of secondary 
 structures and/or specific nucleotides are responsible for the 
 activation of nf-b signaling pathway by sirnas in adherent 
 pbmc , an enriched monocyte population . monocytes are circulating peripheral blood cells that can be differentiated by 
 cytokines into macrophages of different phenotypes as well as into 
 dendritic cells . as mentioned above , sirna effects are sequence 
 dependent and can occur with ds sirnas and ss sirnas . thus , what is the nature of ifn - inducing motif present in one 
 sequence but absent in another ? although gu dinucleotides were 
 found to trigger tlr7 and tlr8 activation , their absolute requirement in sirna activation of innate immunity is 
 still not clear . judge and colleagues identified one rna motif and its immunostimulatory effect seems to depend on the gu 
 content . however , hornung and colleagues identified a second rna motif that is recognized by tlr7 in the context of 
 sirna duplexes and the activity does not depend on gu content 
 . it is worth noting that several sirna sequences without gu nucleotides activated the immune system [ 21 , 26 ] . thus , it 
 is likely that in addition to gu dinucleotides other 
 characteristics such as rna structure , base position , and base 
 composition of the sirna flanking sequences may be involved . the identification of the immunostimulatory sequence that can 
 activate innate immunity will allow the design of sirnas to 
 minimize activation or to increase the immune response for 
 combating infections and tumor cells . considering the simplicity 
 of the immunostimulatory motifs and their high frequency in human 
 rnas , it is desirable to find strategies that evade immune 
 recognition of sirnas . at least two basic strategies can be used 
 to block immune recognition of sirna . one is to use delivery 
 agents that avoid the delivery and/or retention of sirna within 
 the endosomes . regarding the first strategy , song and colleagues described an 
 antibody - based delivery strategy that can result in gene silencing 
 without immune activation . the second strategy relies in the use of modified nucleotides , which are essential to protect 
 sirnas from nuclease degradation and ameliorate their 
 pharmacokinetic parameters in vivo . however , the chemical modification that blocks immune activation must 
 be chosen carefully so as not to inhibit sirna silencing activity . thus , finding the appropriate chemical modifications for 
 inhibiting sirna immune activation will be important for exploring 
 their therapeutic applications . in this respect , replacement of 
 the 2-hydroxyl uridines with either 2-fluoro , 2-deoxy , or 
 2-o - methyl uridines abrogated immune recognition of sirnas by 
 tlrs . thus , endosomal tlrs can distinguish between modified and unmodified rnas ( figure 1 ) . in order to distinguish between self and nonself rnas , collectively , these recent findings 
 offer the possibility of choosing the appropriate modifications 
 that evade immune activation without reducing sirna - silencing 
 potency . of note , sirna with either 2-fluoro uridines or 
 2-deoxy uridines maintained silencing activity [ 26 , 28 ] . another potential source of toxicity is the destruction of 
 cellular mrnas that share partial homology to the sirna sequences . indeed , recent studies demonstrated that both sirnas and micrornas 
 could interact with undesired target mrnas via base pairing of 
 only few nucleotides , leading to inhibition of gene expression 
 [ 12 , 29 ] . interestingly , we have found that chemical 
 modifications of sirnas not only evade immune activation but also 
 reduce the ss sirnas and ds sirna  off - target effects  
 . although the evading mechanism is not known , it is probable that the interaction of ss sirnas or ds sirnas with 
 unintended cellular mrnas is affected by chemical modifications . it is generally accepted that initiation of a 
 specific immune response requires activation of innate immunity 
 resulting in a proinflammatory response . the produced cytokines 
 and chemokines assist in activating and directing the adaptive 
 immune responses . therefore , a vaccine has to induce a 
 proinflammatory response to be effective . as discussed above , 
 sirna sequences containing immunostimulatory motifs induced 
 interferons , chemokines , proinflammatory cytokines , monocyte 
 differentiation , and dendritic cell maturation . 
 activated dcs produce high levels of il-12 , inf- , and 
 proinflammatory cytokines such as il-6 and tnf-. 
 ifn- triggers not only innate immune defense such as the 
 activation of nk cells , but also adaptive th-1 responses , which 
 are important for killing tumor cells and virus infected cells 
 . engagement of endosomal tlr7 and/or tlr8 with sirnas causes activation of at least three key transcription factors , 
 nf-b , ifn regulatory factor ( irf)-3 , and irf-7 , which are 
 important immune responses . previous studies have shown that the activation of tlr9 can improve both cell - mediated and 
 humoral responses to antigens . notably , endosomal tlr9 recognize unmethylated 2-deoxyribo 
 ( cytidine - phosphate - guanosine ) cpg motifs that are more commonly 
 found in bacterial and viral genomes . most , but not all , cpg dinucleotides are methylated in the genomic dna of 
 vertebrates . although much remains to be revealed regarding the 
 adjuvant potency of rna oligonucleotides , the reported data 
 indicate that ss sirna- and ds sirnas - containing immunostimulatory 
 rna motifs might improve cancer and viral vaccines . as with any new agent , issues of delivery and specificity are 
 major obstacles before sirnas can be used in patients . similar to 
 antisense oligonucleotides , certain synthetic sirnas activated 
 innate immunity via tlrs , in particular tlr7 and tlr8 . therefore , 
 there is a need to examine the immunostimulatory effects of 
 any potential therapeutic sirna in human immune cells prior to 
 clinical applications . the recent findings clearly show that the 
 2-hydroxyl uridines are absolutely essential for tlr7 and/or 
 tlr8 recognition and signaling . indeed , replacement of uridines 
 with 2-fluoro , 2-deoxy , or 2-o - methyl modified 
 counterparts abrogated immune activation by ss sirna and sirna 
 duplexes . alternatively , the presence of 2-modified uridines 
 might protect sirnas for being sensed by the immune system . also , 
 most of the other bystander effects not related to immune 
 activation were also inhibited by chemical modifications . these 
 findings will enable the rational design of sirnas that avoid the 
 induction of bystander effects . unmodified sirna 
 duplexes and single - stranded sirnas ( sense or antisense strand ) 
 activate innate immunity through tlr7 and tlr8 resulting in 
 cytokines and type i interferon production . modified ss sirnas did not compete with unmodified sirnas to activate tlr signaling , indicating that 
 2 modifications of uridines block the binding to tlrs ( unpublished data ) .
small interfering rnas ( sirnas ) represent a new class of antigene 
 agents , which has emerged as a powerful tool for functional 
 genomics and might serve as a potent therapeutic approach . 
 however , several studies have showed that they could trigger 
 several bystander effects , including immune activation and 
 inhibition of unintended target genes . 
 although activation of 
 innate immunity by sirnas might be beneficial for therapy in some 
 instances , uncontrolled activation can be toxic , and is therefore 
 a major challenging problem . 
 interestingly , replacement of 
 uridines in sirna sequences with their 2-modified counterparts 
 abrogated sirna bystander effects . here 
 we highlight these 
 important findings that are expected to facilitate the rational 
 design of sirnas that avoid the induction of bystander effects .