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.
Trang 1Staying on message: design principles for controlling
nonspecific responses to siRNA
Shirley Samuel-Abraham1and Joshua N Leonard1,2
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.
Trang 2initiated – 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 IjB,
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
Trang 3siRNA 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 factor-and IL-6 induction in periperhfactor-al blood mononuclefactor-ar
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(N9)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 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.
Trang 4studies 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 ducom-plex 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
Trang 52¢-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 vitrofrom 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
Trang 6methylsulfate [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
Trang 7[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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