In vivo screening of modified siRNAs for non-specific antiviral effect in a small fish model: number and localization in the strands are important.

1.6 MB·English

by  Schyth, Brian Dall

Published online 28 January 2012 Nucleic Acids Research, 2012, Vol. 40, No. 10 4653–4665
doi:10.1093/nar/gks033
In vivo screening of modified siRNAs for
non-specific antiviral effect in a small fish
model: number and localization in the strands
are important
Brian Dall Schyth1,*, Jesper Bertram Bramsen2,3, Malgorzata Maria Pakula2,3,
Sekar Larashati1, Jørgen Kjems2,3, Jesper Wengel4 and Niels Lorenzen1
1Department of Poultry, Fish and Fur Animals, National Veterinary Institute, Technical University of Denmark,
Hangøvej 2, DK-8200 Aarhus N, 2Department of Molecular Biology, University of Aarhus, C.F. Møllers Alle´,
Aarhus University, DK-8000 Aarhus C, 3Interdisciplinary Nanoscience Center, Aarhus University, DK-8000
Aarhus C and 4Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark,
Campusvej 55, DK-5230 Odense M
Received January 30, 2011; Revised January 5, 2012; Accepted January 6, 2012
ABSTRACT
INTRODUCTION
Small interfering RNAs (siRNAs) are promising new
Small interfering RNAs (siRNAs) program specific gene
active compounds in gene medicine but the induc-
transcripts for destruction via the cellular RNA interfer-
tion of non-specific immune responses following ence pathway (1). SiRNAs typically consist of two 21-nt
their delivery continues to be a serious problem. long strands annealed to generate 19bp with a 2-nt
Withthepurposeofavoidingsucheffectschemical- overhang at each 30-end (2,3). Upon delivery to the cell
lymodifiedsiRNAsaretestedinscreeningassaybut cytoplasm, siRNA duplexes are taken up by the RNA
often only examining the expression of specific im- induced silencing complex (RISC) (4–6) in which the
munologically relevant genes in selected cell popu- Argonaute 2 component, cleaves one strand by its endo-
nucleaseactivityleavingtheantisensestrand(AS)tobind
lations typically blood cells from treated animals or
and induce cleavage of the complementary mRNA tran-
humans.Assaysusingarelevantphysiologicalstate
scripts (7–10). As this enables down-regulation of specific
in biological models as read-out are not common.
genes (11) siRNAs are both regarded as valuable tools in
Here we use a fish model where the innate anti-
functionalgenomicsaswellaspotentialtherapeuticagents
viral effect of siRNAs is functionally monitored as
(12). However, the occurrence of cellular immune recep-
reduced mortality in challenge studies involving an tors capable of recognizing single and double-stranded
interferon sensitive virus. Modifications with locked RNA species (13–22) complicates the interpretation of
nucleic acid (LNA), altritol nucleic acid (ANA) and siRNA experiments (23–25) as these receptors activate
hexitol nucleic acid (HNA) reduced the antiviral pro- innateimmunedefencemechanismswhichcanpotentially
tection in this model indicative of altered immuno- lead to physiological changes of the biological system
genicity. For LNA modified siRNAs, the number and under investigation (26–28). Chemical modifications of
siRNA duplexes have been shown to affect the regulation
localization of modifications in the single strands
of genes involved in innate immunological response using
was found to be important and a correlation
either cell cultures or specific cell types from treated or
between antiviral protection and the thermal stabil-
non-treatedmiceorhumanbloodcellstypicallyperipheral
ity of siRNAs was found. The previously published
blood mononuclear cells (PBMC’s) or levels of immuno-
sisiRNAwillinsomesequences,butnotall,increase
logically relevant proteins in serum (18,29–37) but still
theantiviraleffectofsiRNAs.Theappliedfishmodel little is known about the functional effects and conse-
represents a potent tool for conducting fast but quences of such regulation in animal model assays where
statistically and scientifically relevant evaluations chemically modified siRNAs are applied. In this study,
of chemically optimized siRNAs with respect to we test a range of modified siRNAs for their ability
non-specific antiviral effects in vivo. to induce a non-specific antiviral response in a small
*To whom correspondence should be addressed. Tel:[phone]; Fax:[phone]; Email: [email protected]
(cid:2)TheAuthor(s)2012.PublishedbyOxfordUniversityPress.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http://creativecommons.org/licenses/
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4654 NucleicAcidsResearch,2012,Vol.40,No.10
vertebratemodel.Weexploitourpreviousfindingthatthe with VHSV (27). Briefly, 1mg siRNA was mixed with
activation of a functional innate immune mechanisms by 2mg DOTAP in 0.9% NaCl (physiological saline,
delivered siRNAs can delay and reduce the mortality of Nycomed, Denmark). Fish were anaesthetized in 0.01%
small juvenile rainbow trout challenged with the fish benzocaine (ethyl p-aminobenzoate, Sigma, Brøndby,
pathogenic rhabdovirus Viral Haemorrhagic Septicaemia Denmark) and injected intraperitoneally with 20ml of
Virus (VHSV) (27). Our data indicate that the induced the formulated siRNAs using a 27G needled syringe
immune effect of modified siRNAs in vivo is determined before adding them to the respective aquaria’s containing
by several factors such as nucleotide sequence of the 8l of running tap water. A total of 2(cid:2)30 fish were
siRNA,modificationchemistries,numberandlocalization included in each group. The water flow was stopped 24h
of modifications and probably, as our results seem to post-siRNA injection followed by addition of VHSV
indicate, the thermal stability of RNA bindings. isolate DK-3592B (45) to each of the experimental
aquaria giving an approximate titre of 105 TCID /ml in
50
the water of each aquarium. After 2h of exposure the
MATERIALS AND METHODS water flow was restarted. For the next 2 weeks, diseased
fish were daily registered and removed from each
Synthesis, purification and annealing of duplexes
aquarium, examined for external signs of the disease and
The synthesis of non-modified and chemically modified frozen down for later verification of VHSV infection.
siRNAs was performed as described in Bramsen et al. Verification was carried out by testing material of
(38,39) using previously published sequences designed homogenized whole fish (without tail fin) by a VHSV
totargetthereportergeneenhancedgreenfluorescentpro- specific ELISA (46). Relative percent survival (RPS) at
tein (EGFP) and the housekeeping gene Glyceraldehyde Day 10–12 post-viral challenge was calculated from the
3-phosphate dehydrogenase (GAPDH) from mouse established mortality curves (Figures 2, 3 and 4) as RPS
(39; appendix 2). Locked nucleic acid (LNA; 40) (%)=100*[1(cid:3)(mortalityfish/mortalitypositivecontrol
modified oligonucleotides were obtained by using com- fish)]. The respective time points post-challenge for
mercially available LNA phosphoramidite building calculating RPS were chosen because they corresponded
blocks (www.exiqon.com). The chemical synthesis of the with nearly full mortality of non-treated positive control
remaining modified phosphoramidites have previously fish challenged with virus as well as with near linearity of
been described: HM (40-C-hydroxymethyl-DNA) (41), the rest of the mortality curves.
OMe (20-O-methyl) (41), ANA (altritol nucleic acid)
(42), HNA (hexitol nucleic acid) (43) and AEM Estimating the binding energy of annealed duplexes
(20-aminoethoxymethyl) (44). Their structures are shown
The thermal stability of annealed siRNA duplexes was
inFigure1.Followingannealingofstrandstheformation
estimated by analysis of dissociation curves generated in
of double-stranded siRNAs was verified by 12% PAGE.
a Stratagene Mx3000p RealTime PCR machine as has
been described for annealed strands of DNA and RNA
In vivo screening for the antiviral potential of duplexes
(47). Briefly 5ml of the duplex dilution was added to a
Duplexes were formulated in the liposome-formulated mixture containing 19ml physiological saline (0.9%
polycationic transfection agent 1, 2-dioleoyl-3-trimethyl- NaCl) and 1ml SYBR Green (Molecular Probes,
ammonium propane (DOTAP; Roche Diagnostics, Invitrogen, Denmark) pre-diluted 1:1000. A dissociation
Basel, Switzerland), injected into the intraperitoneum curvewasrecordedwhileincreasingthetemperaturefrom
(IP) of 1g large rainbow trout followed by challenge 25(cid:4)C to 95(cid:4)C at a temperature change of 1.8(cid:4)C/min.
O
O O
CCHH bbase
o2 CHo2 base CHo2 base
O OH O OH O
Non-modified ANA HNA
RNA nucleotide nucleotide nucleotide
O O O O
CHoo2 base CHoo2 base CHo2 base CHo2 base
HOHC
O OCH3 O O 2 O O OCH2OCH2CH2NH3+
2´OMe LNA HM AEM
nucleotide nucleotide nucleotide nucleotide
Figure 1. Chemical structures of the oligonucleotides used to modify siRNA strands in this study. See text for explanation of acronyms. RNA,
ANA, HNA, 20-OMe, LNA, HM and AEM.
NucleicAcidsResearch,2012,Vol.40,No.10 4655
(a)
(b) (c)
(d)
Figure 2. The fish-virusmodelisabletodistinguishbetweenthelevelsofantiviralprotection inducedbychemically modifiedsiRNAs.Variantsof
one siRNA targeting the VHS virus irrelevant reporter gene EGFP were formulated in DOTAP and IP injected into small rainbow trout prior to
challenge with the fish pathogenic and interferon sensitive VHS virus (a). Sequences and modifications of strands used to generate the injected
siRNAs can be seen in Supplementary Table S1, but are shown here diagrammatically (b). All strands were chemically modified variants of the
siRNAsequenceseenintheupperpanel.Strandnumbersusedthroughoutthetextareplacednexttothediagrammaticrepresentationsofstrands.
ThechemistriesusedtomodifyRNAstrandsareshowninFigure1(themeaningofwhite,greyandblackballshapesaswellasthelightningsymbol
isexplainedinthediagram).Inordertogeneratemortalitycurves(c)thedeadfishfromthechallengestudywerecountedandmonitoredasmean
accumulated mortality±SD(errorbars)oftreatedgroups.Eachgroupwasruninduplicatewith30fishperreplicate. (d)Knock-downactivityof
themodifiedsiRNAswasexaminedbytheuseofaninvitroluciferaseassayasexplainedin‘MaterialsandMethods’section.Thecolourcodingused
for the various modified siRNAsin (b)is equal tothe one used in themortality curves (c) andin the figure onthe in vitro knock-downeffect (d).
4656 NucleicAcidsResearch,2012,Vol.40,No.10
(a) EGFP targeting siRNA
5´-ACUUGUGGCCGUUUACGUC GCU-´3
3´-UCU UGAACACCGGCAAAUGCAG-´5
= RNA = LNA modified
100%
PosC no siRNA W209 : W181
+2 +8 +2
yy 8800%% WW220099:: SS
alit +4 +2
mort 60% AS : W181
c. +2 +4
c 40%
a
% W006 : W181
+2 +4 +2
20%
siEGFPAS : SS W006 : SS
00%% ++22
1 3 5 7 9 11 13 15 17 19 21
days post challenge
(b)
GAPDH targeting siRNA
5´-AAUACGACCAAAUCCGUUG UU-´3
3´-UUUUAUGCUGGUUUAGGCAAC -´5
100%
PosC no siRNA
id1715 : W204
+2 +8 +2
80%
yy iidd11771155::SS
alit +4 +2
rt 60%
o
m AS : W204
c. 40% +2 +4
c
a
% W203 : W204
20% +2 +4 +2
siGAPDH AS : SS
W203 : SS
0%
++22
11 33 55 77 99 1111 1133 1155 1177 1199 2211
days post challenge
Figure 3. The number and localization of LNA modifications in the siRNAs seems to determine their antiviral effect in the fish-virus model.
Screening of LNA modified variants of one siRNA-targeting EGFP (a) and of one targeting GAPDH (b) using the same pattern of modification
showedthatthereisalsoaneffectofsequence.Rightpanel:diagrammaticrepresentationsofsiRNAswithwhiteballshapesasRNAnucleotidesand
black ball shapes as LNA. Counts below the siRNA diagrams are counts of LNA modifications inside the duplex part and in the overhangs
respectively.ChallengeexperimentsandmortalitycurvesweregeneratedlikeexplainedforFigure2.Colourcodeswereusedtodifferentiatebetween
siRNAs. Detailed sequence information with annotation of modified bases can be found in Supplementary Table S2.
Emitted fluorescence from SYBR Green bound to thermal stabilities of siRNAs were correlated with the
duplexes was recorded every 0.5s. The FAM filter set RPS of fish treated with these siRNAs in the challenge
with excitation wavelength at 492nm and emission wave- studies (see ‘Materials and Methods’ section above on
length at 516nm was used. MxPro software (Stratagene, the in vivo screening of antiviral potential of duplexes).
La Jolla, CA) was used for determining T by the –R0(T)
m
methods, where Tm is defined as the temperature where TNFa regulation in human PBMC treated with siRNAs
the drop in fluorescence is maximal. As melting is per- PBMCs from healthy volunteers (200000/well) were
formed relatively fast we cannot expect equilibrium treated with 100nM siRNA-targeting EGFP mRNA,
between bound and unbound strands to be reached again using DOTAP as the delivery reagent. After 9, 12
during measurement which is why we do not regard and 18h the supernatant from the cells was collected and
measured melting temperatures to be exact values but frozenin(cid:3)80(cid:4)Cuntiluse.Thesampleswerethenassayed
rather relative approximations of thermal stability on humanTNF-aELISA MaxTMDeluxe Sets (Biolegend
between differently modified siRNAs. The approximated #430205).
NucleicAcidsResearch,2012,Vol.40,No.10 4657
(a) EGFP targeting siRNA 5´-ACUUGUGGCCGUUUACGUC GCU-´3
33´´-UUCCUU UUGGAAAACCAACCCCGGGGCCAAAAAAUUGGCCAAGG-´´55
100% W209 : W181
PosC no siRNA
W209 : W178 / W179
80%
y
alit
ort 60% AS : W181
m AS:W178/W179
c.
c 40%
a
%
20%
W006 : W181
siEGFP AS : SS
W006 : W178 / W179
0%
1 3 5 7 9 11 13 15 17 19 21
days post challenge
(b) GAPDH targeting siRNA
5´-AAUACGACCAAAUCCGUUG UU-´3
3´-UUUUAUGCUGGUUUAGGCAAC -´5
100% id1715 : W204
PosC no siRNA
id1715 : W214 / W216
80%
y
ortalit 60% AASS :: WW220144 / W216
m
c. 40%
cc
a
% 20% W203 : W204
siGAPDH AS : SS W203 : W214 / W216
0%
1 3 5 7 9 11 13 15 17 19 21
days post challenge
Figure 4. A knick introduced in the SS increases antiviral effect in the fish-virus model. (a) Screening of the LNA modified siRNAs W209:W181,
AS:W181 and W006:W181 (from Figure 3a) against versions of the same siRNAs where the W181 strand was substituted with the two smaller
strandsW178andW179.(b)InthesamewaytheLNAmodifiedsiRNAsfromFigure3b:id1715:W204,AS:204andW203:W204werecomparedto
siRNAswhereW204hadbeensubstitutedwiththetwostrandsW214andW216.Challengeexperimentsandmortalitycurvesweregeneratedusing
two duplicate aquaria of 30 fish per treatment group. Mean mortalities±SD. Colour codes were used to differentiate between siRNAs. Sequence
informationforthesesiRNAstrandscanbefoundinSupplementaryTableS2.AnickintheSSisnotedbyalighteningsymbolinthediagrammatic
representation of strands.
Test of RNAi functionality of modified siRNAs in vitro RPMI per well. After 4h, the transfection mixture was
substituted with growth medium containing serum. Cell
Test for siRNA silencing activity was done using a previ-
lysates were harvested after 48h and luciferase assays
ously established protocol (48). Briefly, the human lung
were performed using the ‘Dual-luciferase reporter assay
cancer cell line H1299 was grown in RPMI-1640 contain-
system’ (Promega) according to the manufacturer’s
ing 10% FBS, 1% penicillin/streptomycin. A stable
protocol on a FLUOstar luminometer (BMG labtech);
luciferasereporterH1299celllinewascreatedbytransfec-
Renilla luciferase signals (sample) were normalized to
tion with a psiCHECK2 vector (Promega) modified to
the firefly luciferase signals (transfection control).
contain a perfect target site for the EGFP specific
siRNA in the multiple cloning site (MCS) located in the
30-UTRoftheRenillaluciferasegene.Apuromycinresist- RESULTS
ance cassette in the vector allowed for the selection of
Chemical modifications of siRNAs modulate their
stably transfected cells (1mg puromycin/ml medium).
antiviral effects in a viral challenge model
Evaluation of knock-down efficiency was performed
using this cell line and transfections with EGFP specific In the VHS virus challenge model, rainbow trout injected
siRNAs at 10nm concentrations in triplicates. with a non-modified siRNA targeting the virus-irrelevant
Lipofectamine 2000 reagent (Invitrogen) was used in a reporter gene EGFP (RNA/RNA-targeting eGFP duplex
96-well format with 15000 cells in 100ml serum-free in Figure 2) experienced a lower degree of virus induced
4658 NucleicAcidsResearch,2012,Vol.40,No.10
mortality compared to buffer injected control fish (PosC their antiviral induction to different levels seen as varying
no siRNA in Figure 2c). Initial experiments using 1–2mg degreesofreductioninthemortalitycurve.Duplexescon-
siRNA did not show signs of concentration dependence taining an AS strand with only 30-end LNA modification
upon mortality why we are probably working with a (W006:W181, W006:SS and W203:W204, W203:SS in
saturated system (data not shown). We have previously Figure 3a–b) showed no abrogation of antiviral response
shown indirect evidence that the non-specific reduction compared to the siRNA composed only of RNA. This
of mortality was due to induction of an interferon was also seen when LNA substitutions where placed
response (27). In order to determine whether chemical in both overhangs (W208:W194 in Figure 2) and
modification could influence the antiviral effect, we did not seem to be related to whether the overhang was
screened a range of modified versions of this siRNA 3- or 2-nt long.
(Figure 2b) for their ability to protect fish from viral
induced mortality (Figure 2c). Most of the modified
A knick in the SS (sisiRNA) increases the antiviral effect
siRNAs induced a high antiviral protection equal to or
in some siRNAs
in some cases even stronger than that induced by the
non-modified siRNA indicative of a potent interferon As LNA nucleotide substitutions are known to increase
response. However, three of the modified siRNAs, con- the thermal stability of RNA and DNA duplexes (40,49)
taining LNA, HNA or AEM nucleotides respectively we checked whether a reduction of duplex stability
exhibited a reduced antiviral effect as revealed by an through introduction of a destabilizing break in the pas-
increased and more acute mortality in the challenge senger strands would be able to increase their antiviral
model (lighter red, orange and yellow curves in effect (Figure 4a and b). This was the case, but the effect
Figure 2c). Especially the sisiRNA (40), which was LNA was most pronounced for the GAPDH siRNA where the
modified in the stem part and where the passenger strand sisiRNA design led to decreased mortality for all modifi-
[sense strand (SS)] was replaced by two shorter 10-nt and cation patterns.
12-nt strands, showed a dramatically reduced antiviral
effect compared to the non modified RNA/RNA duplex.
In contrast, the siRNA which was LNA modified only in A high LNA load reduces knock-down efficiency but can
the overhangs (W208:W194), showed no abrogation of be partly restored by the sisiRNA design
antiviral response in our model. Except from the AEM We checked the knock-down efficiency of our siRNAs in
modified version, all the siRNAs retained knock-down
an in vitro assay using human cells and found that all
functionality in vitro (Figure 2d), although the sisiRNA
worked as potent inhibitors except for the highly LNA
seemed less potent. Accordingly, our initial observations
modified W209:W181 double-strand (Figure 5). On the
show that it is possible to strongly reduce induction of
other hand, the sisiRNA design of this duplex
innate mechanisms while still retaining a significant
(W209:W178/W179) increased the knock-down efficiency
knock-downeffect.Interestinglywedidfindastronganti-
from 0%to65%, which showsthat we can,aspreviously
viral effect of our 20OMe nucleotide modified siRNA
postulated, increase the knock-down efficiency of these
despite that 20OMe has previously been shown to be a
modified siRNAs by pre-cleaving the passenger strand
potent antagonist of immunostimulatory RNA in the
(39). The interesting finding here was that whereas this
mammalian systems where it was tested (32,35).
cleaving in many cases resulted in higher antiviral effect
(lowermortalityinthechallengestudy)suchaneffectwas
LNA modifications in the siRNA stem reduces not significant for the W209:W178/W179 duplex
antiviral effect (Figure 4a) indicating that it might be possible in some
The effect of introducing LNAs into siRNAs was cases to design LNA modified siRNAs causing none or
investigatedbyvaryingthenumberandpositionsofmodi- only insignificant side effects while still retaining their
fications in the EGFP-targeting siRNA (Figure 3a). In knock-down efficiency.
order to control for sequence related effects we also
included non-modified and modified versions of a previ-
Discrepancies between the fish model and a mammalian
ouslypublishedGAPDH-targetingsiRNAusingthesame
in vitro system
modification patterns as for the EGFP-targeting siRNA
(Figure 3b). The setup included native strands, strands We investigated whether the innate immune response
with four LNAs in the stem and two LNAs in the to the siRNA variants, as observed in the fish model,
overhang and finally strands with only two LNAs in the could be recapitulated in human PBMCs using the level
overhang (only AS). Pairing of AS and SS containing six of the proinflammatory molecule TNF-a as read-out
LNAsinbothstrandsstronglyreducedtheantiviraleffect (Supplementary Figure S1). We saw the tendency of
of both the EGFP and the GAPDH specific siRNA seen decreased immune stimulation with increased LNA load,
as a higher mortality compared to the non-modified but whereas the immune stimulatory effect of siRNAs
siRNAs (compare the siRNAs W209:W181 and containing LNA modifications in the overhang only
id1715:W204 with the non-modified siRNAs called (siRNA harbouring the W006 strand) resembled that of
AS:SS in Figure 3a and b respectively). When shifting theunmodifiedRNAduplexinthefishmodelthiswasnot
strands to ones containing a lower number of LNA as clear in the human PBMC’s where only the W006:SS
residues in the stem part of siRNAs these again resumed duplex was highly stimulatory.
NucleicAcidsResearch,2012,Vol.40,No.10 4659
using TNF-a as a measurement of immune induction we
found nearly similar results (Supplementary Figure S2).
Single strands induce the antiviral response which can be
abrogated by LNA modification
We tested whether the single strands used to generate our
siRNA duplexes could themselves induce the antiviral
response in the fish model as has previous been shown
in mammalian cells (18,21,33). This was also the case in
the fish model where our results indicated that the anti-
viraleffectoftheduplexescouldbeexplainedbytheeffect
of the strongest inducer of the two strands [Figure 8a–b;
AS from the AS:SS (non-modified RNA duplex), W006
from W006:W181 and SS from W006:SS]. This indicated
strand competition for receptors rather than an additive
Figure 5. Highly LNA modified siRNAs show low knock-down effi- effect of strands although this should be further
ciency which is possible to regain by introducing a knick in the SS. investigated by using a larger set of sequences. The same
Suchfindingshavepreviouslybeenshown(29)butwetesteditagainon
was seen when using two strands of the same orientation
thespecificstrandsusedinthisstudy.Thecellcultureassayisdescribed
in‘MaterialsandMethods’section.Dataaremeanvaluesfortriplicate which are not expected to hybridize (Figure 8c;
wells and error bars represent SDs. All values were normalized to the Combinations of the AS, which is the non-modified
meansignalfromcellstreatedwithamismatchedsiRNAnotexpected RNA AS, the W006 strand which was only end modified
totargettheluciferaseexpression.Anexpressionvalueof1isequalto
by LNA and the W010 strand containing six LNA
notargetreduction(100%luciferasesignal).Thestrandnumbersinthe
leftpanelarethesameasusedthroughoutthetextandFigures3and4. modifications).
DISCUSSION
Antiviral effects of LNA modified siRNAs in the fish
Viraldiseasesofrainbowtroutarewellstudiedduetothe
model are correlated with their thermal stability
use of this species in aquaculture. Good infection models
The suggested relationship between duplex stability and existmakingthisaninterestinganimalmodelforthestudy
immune stimulatory properties was further analysed by of antiviral medicines. Recently, the use of siRNA knock
plotting estimated melting temperatures of siRNAs with down for targeting viral diseases has been tested in
their corresponding effect on mortality in the infection rainbowtrout(27),but giventhestrong non-specific anti-
trials. For LNA modified siRNAs (presented in viral effect, which are under some circumstances induced
Figures 3 and 4) we found a positive correlation between by siRNAs, it can, like in the mammalian studies (23), be
the relative thermal stabilities of LNA modified siRNAs difficult to get a relevant read-out of the specific effect
and their antiviral effect seen in our infection model (26,27). In rainbow trout we have previously shown that
(Figure 6b–c). Such a relationship should probably not IP injected siRNAs, when formulated by the effective
be expected for all types of modifications as indicated by liposomal delivery reagent DOTAP, are first taken up by
our initial screening experiments where thermal stability macrophage-like cells in the intraperitoneum followed by
couldnotexplaindifferencesinimmunestimulationacross an antiviral immune response which is able to protect the
different types of modifications (Figure 6a; siRNAs from fish against VHS virus induced mortality (27). Previously
screening shown in Figure 2). IP injected mice were shown to take up DOTAP
formulated siRNAs by the same route, but unfortunately
LNAs in the siRNA stem can abrogate the effect of a
the absence of an interferon response in this model was
previously published immune stimulating motif
onlystudiedinintravenously(IV)injectedmice(50).Ithas
Toinvestigatethegeneralityofourobservationsinthefish previously been reported that introduction of chemical
model, we tested an siRNA previously found to contain modifications into siRNAs were able to reduce their in-
a mammalian immune stimulatory motif (‘GUCCUU duction of innate immune mechanisms in mammalian
CAA’) in the SS and compared this to a modified systems (17,18,22,23,28–35). Here we used the fish-virus
siRNA containing four LNA substitutions inside the model to screen for types of modifications which reduce
motif as a means of abrogating the stimulatory effect theabilityofsiRNAstoactsasantiviralstimulantsinvivo
(18). Using our in vivo model, we found that four LNA- without compromising their knock-down efficiency as
modifications within the immune stimulatory motif evaluated in vitro in a mammalian system. The initial
reduced the antiviral effects significantly (Figure 7a) in screenings clearly demonstrated that the fish-virus model
agreement with the mammalian study (18). Notably, a could differentiate between different modified versions of
similar reduction in antiviral effect was seen when the thesiRNAbasedontheirantiviraleffecttowardstheVHS
LNA substitutions were placed in the AS of the region virus. Notably, the antiviral effect of the LNA modified
complementary to the immune stimulatory motif. For sisiRNA, the HNA and the AEM modified siRNAs were
these siRNAs melting temperature and %RPS was also significantly reduced compared to the effect seen when
negatively correlated (Figure 7b). In the human PBMCs using the non-modified siRNA (Figure 2c), but only the
4660 NucleicAcidsResearch,2012,Vol.40,No.10
(a) siRNAstargetingEGFP (variousmodificationsfrom Fig. 2)
Nonmodified
RNA AS + SS
Highantiviral 2nts in overhang LNA mod.
90
protection (W208:W194)
0 70 2´OMe
1
y ANA
a AEM
d 50
S HM
P HNA
R 30
% Lowantiviral sisiRNAhighLNA load
10 protection (W209 + W178 / W179)
45 55 65 75 85 95
(b)
siRNAstargetingEGFP (LNA mod. from Fig.3-4 )
W006 : SS
W006 : W181
W006 : W178 / W179
90
AS+SS
0 AS : W181
1 70
y AS : W178 / W179
a
d 50 W209 : SS
S
P sisiRNAhighLNA load
R 30
% (W209 : W178 / W179)
10 siRNAhighLNA load
(W209 : W181)
45 55 65 75 85 95
(c) siRNAstargetingGAPDH (LNA mod. from Fig.3-4 )
W203 : SS
90
W203 : W204
AS + SS
12 70 W203 : W214 / W216
y
a
S d 50 AS : W204
P ID1715 : SS AS : W214 / W216
R 30
% sisiRNAhighLNAload
10 (id1715 + W214 / W216)
siRNAhighLNA load
45 55 65 75 85 95 (id1715 + W204)
Relative thermal stability of
siRNAs
in fysiological saline
Figure 6. Antiviral effect of siRNAs in the fish-virus model showed a negative correlation with the estimated thermal stabilities for the LNA
modified siRNAs (b–c) but not for the assembly of modified siRNAs screened in Figure 2 (a). RPS was calculated from the mortality curves
shown in Figure 2 (6a) and 3–4 (6b–c) respectively using the procedure described in ‘Materials and Methods’ section and correlated with relative
thermalstability ofsiRNAs estimated asthe meltingtemperature foundbydissociation curve analysis. Strandandmodification namesrefer tothe
ones used in Figures 1, 2, 3, 4 and the Supplementary Tables S1 and S2.
LNA modified sisiRNA and the HNA modified siRNA we were not able to show the same general effect of
withoutabrogatingtheknock-downpotential(Figure2d). including the W006 strand in our human PBMC cell cul-
We further investigated the role of LNA modification. tureexperimentswhereonlytheW006:SSsiRNAstrongly
Our results indicated the importance of the modification induced the expression of TNFa (Supplementary
pattern in our siRNAs. Introduction of LNAs inside the Figure S1). Discrepancies between the results from the
stempartgenerallyreducedtheantiviralstimulationinthe mammalian in vitro models including our own and our
fishascomparedtomodificationsintheoverhangs.Arole fish in vivo model systems could result from the former
of LNA modifications in siRNA overhangs on cellular using human PBMC’s in in vitro systems and only
stress has previously only been described with respect to assayingtheregulationofaspecificstressorimmunologic-
reduced viability in HeLa cells (38; Here using the same ally relevant gene. One other interesting difference
strands W006 and W203 as we used in our study). between the innate response in our model and previous
This could potentially be due to the innate response we mammalian models included the absence of an effect by
sawasa functional antiviralprotection inour model.But our 20-OMe modified siRNA. As we only included one
NucleicAcidsResearch,2012,Vol.40,No.10 4661
(a)
100% 5´-UUGAAGGACAGGUUAAGCU dTdT-´3
PosC no siRNA 3´-dTdTAACUUCCUGUC CAAUUCGA -´5
80% Immune stim. Motif (Hornung et al, 2004)
y
rtalit 60% 3´-dT5d´T-UAUACGTAUACGCGUAGCUACG GCAUAUUAUACGGCAU -d´5TdT-´3
o
mm
c. 40% 5´-UTGAAGGACAGGUUAAGCU dTdT-´3
ac 3´-dTdTAACUUCCUGUC CAAUUCGA -´5
%
20%
5´-UTGAAGGACAGGUUAAGCU dTdT-´3
0% 3´-dTdTAACTUCCUGUC CAAUUCGA -´5
1 3 5 7 9 11 13 15 17 19 21
days post VHSV challenge Underlined = LNA
(b) 90
70
0
1
y
a
d 50
S
P
RR
% 30
10
75 80 85 90
Relative thermal stability of
siRNAs
in fysiological saline
Figure 7. The relation between thermal stability and antiviral effect in vivo was also found using LNA modifications of a previously published
siRNA containing an immune stimulatory motif (13). Challenge experiments (a) and estimation of siRNA thermal stability was carried out as
described in the text. The immune stimulating motif is boxed in grey in the sequence diagrams in (a). The first siRNA from the top is the
non-modified siRNA. The second from the top is also a sequence from the Hornung et al. study where the immune stimulatory effect of the
siRNAwasreducedbysubstitutingfourRNAnucleotideswithLNAnucleotidesinthemotif(LNAresiduesareunderlined).ThenextsiRNAwas
our own design and included LNA modifications only in the strand opposite to the proposed motif. In the last siRNA (lower row) modifications
wereintroducedintobothstrands.(b)WeshowthatRPSandthermalstabilityofthesesiRNAsarenegativelycorrelated.Methodsareasdescribed
in the ‘‘Materials and Methods’ section’ section as well as in the text for Figure 5. Mortality curves were made from mean values for duplicate
groups of fish with the same treatment. Error bars represent SDs of duplicates.
20-OMe modified duplex in our initial experiments we Furthermore, we found a correlation between thermal
cannot safely say whether this is a general finding in the stability and antiviral effect of a previously published
fish model or whether a different pattern of modification siRNAs harbouring animmunostimulatory motif (18).
would have behaved different. One possible explanation for the influence of thermal
LNAmodificationsinthestempartofdouble-stranded stability of siRNAs upon antiviral activation when de-
nucleic acids are known to increase their thermal stability livered by DOTAP comes from recent findings that this
which could change the siRNAs ability to react with cationic liposome based delivery reagent promotes effi-
cellular receptors. For our in vivo model we found indica- cient delivery via uptake into endosome compartments
tions that the thermal stability of LNA modified siRNAs of the vertebrate cell (21,51). Here they allow for the rec-
could be a determinator of the degree of antiviral effect ognition of both double-stranded RNA and single-
induced. First, we demonstrated that fish injected with stranded RNA species by TLR receptors 3, 7 and 8 all
siRNAs containing more LNA modifications in the stem signalling the initiation of the interferon response
experienced a higher mortality following viral infection (19; 51). At least in isolated mouse leucocytes the single
indicative of a lower innate antiviral stimulation. strands of siRNAs have been shown to act as ligand for
Secondly, the introduction of destabilizing nicks in the TLR-7 and 8 (18). This could explain the correlation
stem part (the sisiRNA design) had the opposite effect. between thermodynamic stability and antiviral effect in
Thirdly, the relative thermal stability between strands as our in vivo model. Importantly, fish also have TLR3, 7
estimatedbythedissociationcurvemethod(47)wasnega- and 8 although two forms of TLR8 are present in the
tivelycorrelatedwiththeRPSintheviralchallengestudies. rainbow trout (52–54).
4662 NucleicAcidsResearch,2012,Vol.40,No.10
(a) 100% PosC
AS : SS
80%
y
rtalit 60% AS
o
mm SSSS
c. 40%
c
a W010 : W181
%
20%
W010
0%
W181
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29
days post challenge
(b)
100%
PosC
80%
y WW000066+:SSSS
alit 60%
rt
o WW000066+:WW118811
m
c. 4400%%
ac W006
% 20%
SS
0%
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 W181
days post challenge
(c) 100% PosC
+
80% W006 + AS
60% WW000066 +/ WW001100 +
4400%%
WW000066
20%
AS
0%
W010
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29
Figure 8. LNAmodificationisabletoreducetheantiviraleffectinducedbysingleRNAstrandsinthefishmodel.Theantiviraleffectofthesingle
strandsseemstodeterminetheeffectofthecorrespondingsiRNAduplexinanon-additivemanner.StrandsscreenedweretheAS,SS,W010,W181
and W006 (siRNA-targeting EGFP; Figures 3a, 4a and Supplementary Figure S2) including combinations of these which were formulated in
DOTAP and injected into rainbow trout subsequently infected with VHSV as described in ‘‘Materials and Methods’ section’. Mortality curves
anderrorbarswheremadeasdescribedinpreviousfigures.TheW010strandresemblestheW209strandusedthroughoutthisstudy,butcontainsan
extraUinthe30-end.Thethreegraphsa,bandcrepresentsdatafromthesameexperimentalset-upwhere2groupsof30fishwereusedtotesteach
strand or strand combination respectively.
In line with this hypothesis single strands were able to innate induction by LNA nucleotide chemistry on the
induce the innate mechanisms and explain the antiviral one side and higher thermal stability introduced through
effects of siRNAs in the fish. But, our finding that LNA LNA modification on the other side. But, this does not
modification of single strands could also reduce the anti- explain why breaking the passenger strand as in the
viral effect outside a duplex context shows that the effect sisiRNA design also increased the antiviral effect of
of LNA can at least not exclusively be assigned to the siRNAs. Breaking one of the strands in a duplex would
strengthening of the siRNA duplex. As the number of mean that three new strands would be responsible for
LNAs in nucleic acid duplexes is strongly correlated increasing the antiviral effect and we have no evidence
with their stability, the correlation we initially found of smaller strands being better at stimulating cellular re-
between thermal stability and antiviral effect could ceptors for inducing an interferon response. In fact the
partly be due to a secondary correlation between lower opposite is usually proposed (17,55).