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. 2012 May;40(10):4653-65.
doi: 10.1093/nar/gks033. Epub 2012 Jan 28.

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 Schyth  1 , Jesper Bertram Bramsen, Malgorzata Maria Pakula, Sekar Larashati, Jørgen Kjems, Jesper Wengel, Niels Lorenzen
Affiliations

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 Schyth et al. Nucleic Acids Res. 2012 May.

Abstract

Small interfering RNAs (siRNAs) are promising new active compounds in gene medicine but the induction of non-specific immune responses following their delivery continues to be a serious problem. With the purpose of avoiding such effects chemically modified siRNAs are tested in screening assay but often only examining the expression of specific immunologically relevant genes in selected cell populations typically blood cells from treated animals or humans. Assays using a relevant physiological state in biological models as read-out are not common. Here we use a fish model where the innate antiviral effect of siRNAs is functionally monitored as reduced mortality in challenge studies involving an interferon sensitive virus. Modifications with locked nucleic acid (LNA), altritol nucleic acid (ANA) and hexitol nucleic acid (HNA) reduced the antiviral protection in this model indicative of altered immunogenicity. For LNA modified siRNAs, the number and localization of modifications in the single strands was found to be important and a correlation between antiviral protection and the thermal stability of siRNAs was found. The previously published sisiRNA will in some sequences, but not all, increase the antiviral effect of siRNAs. The applied fish model represents a potent tool for conducting fast but statistically and scientifically relevant evaluations of chemically optimized siRNAs with respect to non-specific antiviral effects in vivo.

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Figures

Figure 1.
Figure 1.
Chemical structures of the oligonucleotides used to modify siRNA strands in this study. See text for explanation of acronyms. RNA, ANA, HNA, 2′-OMe, LNA, HM and AEM.
Figure 2.
Figure 2.
The fish-virus model is able to distinguish between the levels of antiviral protection induced by chemically modified siRNAs. Variants of 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 siRNA sequence seen in the upper panel. Strand numbers used throughout the text are placed next to the diagrammatic representations of strands. The chemistries used to modify RNA strands are shown in Figure 1 (the meaning of white, grey and black ball shapes as well as the lightning symbol is explained in the diagram). In order to generate mortality curves (c) the dead fish from the challenge study were counted and monitored as mean accumulated mortality ± SD (error bars) of treated groups. Each group was run in duplicate with 30 fish per replicate. (d) Knock-down activity of the modified siRNAs was examined by the use of an in vitro luciferase assay as explained in ‘Materials and Methods’ section. The colour coding used for the various modified siRNAs in (b) is equal to the one used in the mortality curves (c) and in the figure on the in vitro knock-down effect (d).
Figure 3.
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 showed that there is also an effect of sequence. Right panel: diagrammatic representations of siRNAs with white ball shapes as RNA nucleotides and black ball shapes as LNA. Counts below the siRNA diagrams are counts of LNA modifications inside the duplex part and in the overhangs respectively. Challenge experiments and mortality curves were generated like explained for Figure 2. Colour codes were used to differentiate between siRNAs. Detailed sequence information with annotation of modified bases can be found in Supplementary Table S2.
Figure 4.
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 strands W178 and W179. (b) In the same way the LNA modified siRNAs from Figure 3b: id1715:W204, AS:204 and W203:W204 were compared to siRNAs where W204 had been substituted with the two strands W214 and W216. Challenge experiments and mortality curves were generated using two duplicate aquaria of 30 fish per treatment group. Mean mortalities ± SD. Colour codes were used to differentiate between siRNAs. Sequence information for these siRNA strands can be found in Supplementary Table S2. A nick in the SS is noted by a lightening symbol in the diagrammatic representation of strands.
Figure 5.
Figure 5.
Highly LNA modified siRNAs show low knock-down efficiency which is possible to regain by introducing a knick in the SS. Such findings have previously been shown (29) but we tested it again on the specific strands used in this study. The cell culture assay is described in ‘Materials and Methods’ section. Data are mean values for triplicate wells and error bars represent SDs. All values were normalized to the mean signal from cells treated with a mismatched siRNA not expected to target the luciferase expression. An expression value of 1 is equal to no target reduction (100% luciferase signal). The strand numbers in the left panel are the same as used throughout the text and Figures 3 and 4.
Figure 6.
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 thermal stability of siRNAs estimated as the melting temperature found by dissociation curve analysis. Strand and modification names refer to the ones used in Figures 1, 2, 3, 4 and the Supplementary Tables S1 and S2.
Figure 7.
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 siRNA was reduced by substituting four RNA nucleotides with LNA nucleotides in the motif (LNA residues are underlined). The next siRNA was our own design and included LNA modifications only in the strand opposite to the proposed motif. In the last siRNA (lower row) modifications were introduced into both strands. (b) We show that RPS and thermal stability of these siRNAs are negatively correlated. Methods are as described 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.
Figure 8.
Figure 8.
LNA modification is able to reduce the antiviral effect induced by single RNA strands in the fish model. The antiviral effect of the single strands seems to determine the effect of the corresponding siRNA duplex in a non-additive manner. Strands screened were the AS, 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 and error bars where made as described in previous figures. The W010 strand resembles the W209 strand used throughout this study, but contains an extra U in the 3′-end. The three graphs a, b and c represents data from the same experimental set-up where 2 groups of 30 fish were used to test each strand or strand combination respectively.
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