Impact of stereopure chimeric backbone chemistries on the potency and durability of gene silencing by RNA interference

Figures

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  Figure 1.. Application of chiral control over PS modification to siRNA. (A) General design of siRNAs showing stereorandom reference format-1 (30) with GalNAc-modified sense strand (5’-end), with 2’-F and 2’-OMe ribose modifications and terminal stereorandom PS modification on each end (red brackets). Antisense (AS) strands have two terminal stereorandom PS modifications on each end (red brackets). Chemical structures for phosphodiester (PO) and phosphorothioate (PS) backbones are also shown along with legend for chemical modifications. (B) Application of PS stereochemistry to sense and antisense strands based on stereorandom reference format 1 in mouse primary hepatocytes. We generated 4 stereopure sense strands and 16 stereopure antisense strands with all possible combinations of Rp and Sp in the PS-modified positions shown in A. Forest plot showing summary of silencing data on 64 siRNAs (4 sense × 16 antisense) highlighting impact of PS stereochemistry in sense strand (bottom left) with SpSp configuration showing the highest mean activity independent of AS strand. All other configurations are shown with respect to activity of SpSp. Interaction between PS stereochemistry in 5'- and 3'-ends…

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  Figure 2.. Application of stereopure PN chemistry to siRNA and interrogation of the sense strand in vitro. (A) Chemical structures for phosphoryl-guanidine (PN) backbone with legend depicting how these stereopure backbone modifications are denoted in subsequent panels. (B) Heat map depicting percentage of mouse Ttr mRNA remaining relative to stereopure reference oligonucleotide with no PN backbone modifications in primary mouse hepatocytes treated with stereopure siRNAs composed of the depicted stereopure sense-1 strand and one of 88 antisense strands (Supplementary Table S2). The antisense strands were generated by walking either an Rp PN linkage or an Sp PN linkage through the backbone while fixing two Sp linkages on the 3’-end with an RpSp or an SpRp configuration on the 5’-end. Increasingly darker shades of blue correspond to increasing silencing; decreasing blue (increasing white) corresponds with decreasing silencing. (C) Models of stereopure siRNA interaction with Ago2 based on (55,56). View of antisense seed region and interactions with Ago2 for fully PO backbone (top left) and zoomed in views of backbone at position 3 in seed of antisense strand with PO linkage (lower left), Sp PN…

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  Figure 3.. Optimization of number, position, and stereochemistry of PN linkages in vivo. (A) Cartoons depicting siRNAs. (B) Percentage of Ttr mRNA expression in wild-type mice 1-week post-dose after treatment with the indicated siRNAs. Horizontal bars depict mean, and individual points represent one mouse. Stats compared with TTR-2: ** P < 0.01 **** P < 0.0001, Welch's One-way ANOVA with Dunnett post-hoc test comparisons to TTR-2. (C) Concentration of antisense strand in mouse liver 1-week post-dose (left). Stats: One-way ANOVA with Dunnett post-hoc test comparisons to TTR-2 ** P < 0.01, *** P < 0.001. Relative Ago2 loading for the indicated siRNAs (right) standardized to miR-122 and normalized to TTR-2 for the indicated oligonucleotide. Data represent mean ± sd, n = 5. Stats: Welch's one-way ANOVA with Dunnett post-hoc test comparisons to TTR-2 ** P < 0.01.

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  Figure 4.. Evaluation of alternate sense strand formats. (A) Cartoons illustrating stereorandom siRNA reference formats 1 and 2, showing double-stranded configurations. Alternative sense strand configurations (Sense-1 and Sense-2) are shown (in box) for stereorandom and stereopure designs. Assessment of sense strand impact on siRNA activity, with siRNAs based on stereorandom reference formats (TTR-2 and NTC) or composed of the illustrated stereopure antisense strand with either stereopure sense-1 or stereopure sense-2. Silencing activity is shown for increasing concentrations (150, 500, 1500 pM) of the indicated siRNAs in primary mouse hepatocytes with the same series of antisense strands (shown to left of graph). Data are shown as mean ± sd, n = 3 per concentration per siRNA. (B) Percentage Ttr serum protein in mice over time after treatment with the indicated siRNA. The reference compound (TTR-2–1) is in the stereorandom format-1. TTR-501 is tested in siRNAs containing sense-1 (green) or sense-2 (light blue) configurations. Data are presented as mean ± sd, n = 5 per group. Stats: * P < 0.05, *** P < 0.001, **** P < 0.0001 linear mixed effects ANOVA with post-hoc test comparing TTR-501–1…

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  Figure 5.. Application of stereopure PN chemistry and interrogation of the sense strand in vivo. (A) Cartoons of siRNAs evaluated in panels B–D, which contain sense-2 strand configuration and the indicated antisense strands. (B) Percentage of Ttr liver mRNA expression in mice 1-week after treatment with the indicated siRNA. Dosing regimen is shown, with green arrow indicating time of treatment and red arrow indicating time of tissue collection. Horizontal bar is mean, and each dot represents a different mouse, n = 5 per group. Stats: Two-way ANOVA with post-hoc comparison to stereorandom siRNA at the same dose. * P < 0.05, ** P < 0.01, **** P < 0.0001. (C). Relative Ago2 loading of antisense strand (AS) standardized to miR-122 and normalized to TTR-2–2 in livers treated with 2 mg/kg siRNAs as illustrated in panel B. Data are presented as mean ± sd, n = 5 per group. Stats: Welch's one-way ANOVA with post-hoc comparisons * P < 0.05, **** P < 0.0001. (D) Percentage Ttr serum protein in mice over time after treatment with 6 mg/kg of the indicated siRNA. Dosing regimen is shown, with green arrow indicating time of treatment and red arrow indicating time of serum collection. A pre-dose serum…

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  Figure 6.. Application of PN chemistry to stereorandom reference format-3 featuring reduced 2'-F content. (A) Schematic representation of Stereorandom reference format 3, designed based on (7). (B) Application of PN chemistry to reference format 3 in mice. Relative serum Ttr protein levels detected in mice treated with the indicated siRNA molecules. Data are presented as mean ± sem, n = 5 per group.

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  Figure 7.. Optimization of 2′-sugar chemistry pattern for PN linkages. (A) Schematic representation of stereorandom reference formats 1–3, highlighting differences in the 2'-ribose modification patterns on the antisense strands. (B) Optimization of PN chemistry and 2'-ribose modifications for the antisense strand in primary mouse hepatocytes. Schematics on the left illustrate ‘PN walk’ where 750 pM of siRNA containing antisense strand with Sp PN or Rp PN linkage was tested at each position of the oligonucleotide backbone (top), or an Sp PN with 2'-F or Rp PN with 2'-F was tested (bottom). All antisense strands are shown in Supplementary Tables S4 and S5. Low concentration (250 pM) data shown in Supplementary Figure S6A. Heat map (right) depicts Relative Ttr mRNA remaining for the stereopure siRNAs compared with TTR-871 or TTR-872 controls (750 pM), which lack PN linkages. Increasingly darker shades of blue correspond to increasing silencing; decreasing blue (increasing white) corresponds with decreasing silencing. The stereopure sense stand shown in panel C was used. The 5′-end PS pattern is indicated, as is PN chirality (red bars, Rp; blue bars, Sp), and sugar chemistry 3′ to the PN…

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  Figure 8.. Optimized siRNA format 3 shows improved silencing activity. (A) Schematic representation of siRNAs tested in the study (left). Dosing regimen and Ttr serum protein levels in mice treated with 0.5 mg/kg of the indicated siRNA. Serum Ttr levels (compared to PBS) over time for the indicated siRNAs. Data are presented as mean ± sd, n = 5 per group per time point. (B) Antisense strand Ago2 loading standardized to miR-122 and normalized to TTR-1194 for the indicated oligonucleotide at 1-week post-dose (left). Concentration of antisense strand in liver 1-week post-dose. Data are presented as mean ± sem. Stats: One-way ANOVA with post-hoc test comparisons to TTR-1194. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. (C) Gene expression changes detected based on RNA-seq analysis of primary mouse hepatocytes treated with 2 μM of the indicated siRNA compared with PBS. Log2 fold-change in expression is plotted with respect to the P value. Dotted lines indicate statistical significance: the horizontal line denotes P = 0.01, and the vertical lines denote >1-fold-change in expression, with decreased expression to the left and increased expression to the right.

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  Figure 9.. PN-based RNAi format has nominal impact on liver function or serum proteins in mice and is specific in primary hepatocytes. General design for siRNAs tested and dosing regimen for evaluation of liver function in wild-type mice. Graphs show liver enzyme function for alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), albumin and total serum proteins in CD-1 mice treated with the indicated siRNA or PBS. Each symbol represents one mouse and data are presented as mean ± sd, n = 5 per group.

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  Figure 10.. PN-based GalNAc-siRNA format translates to an additional liver target. (A) Schematic representation of siRNAs tested in the study. Dosing regimen for transgenic HSD17B13 mice, with dosing at day 0 (D0, green arrow) and mouse sacrifice and tissue evaluation at the indicated times (orange arrows) in weeks (W) post-dose. (B, C) HSD17B13 mRNA levels in liver of transgenic mice treated with the indicated siRNA 7-weeks post dose (B) or for the entire 14-week experiment (C). Data in B are presented as scatter plots with lines denoting mean mRNA levels. Data in C are presented as mean ± sem, n = 5. Stats: Two-way ANOVA with post-hoc comparison between HSD-1933 and HSD-1930 at each time point. ** P < 0.01, **** P < 0.0001. (D) Concentration of antisense strands in livers 2-, 7- or 14-weeks post-dose for the indicated treatment. Data are presented as mean ± sd, n ≥ 3. (E) Relative Ago2 loading for antisense strands in mouse livers over time with normalization to miR-122. Data are presented as mean ± sd, n = 5. Stats: Two-way ANOVA with post-hoc comparisons between HSD-1930 and HSD-1933 at each time point. (F) Fold-change Ago2 loading for HSD-1933 compared to HSD-1930 in mouse livers over…

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  Figure 11.. HSD17B13 silencing with low, single dose. (A) Dosing regimen for transgenic HSD17B13 mice, with dosing at day 0 (D0) and tissue evaluation at the indicated time in weeks (W) post-dose. HSD17B13 mRNA levels in liver of transgenic mice treated with the indicated siRNA at 1.5 or 3.0 mg/kg 12-weeks post-dose. Data are presented as scatter plots with lines denoting mean mRNA levels, n ≥ 4. Stats: One-way ANOVA with post-hoc test comparison to PBS ****P < 0.0001. (B) Concentration of antisense strands in livers 12-weeks post-dose for the indicated treatment. Data are presented as mean ± sd, n ≥ 4. (C) Relative Ago2 loading for antisense strands in mouse livers over time with normalization to miR-122. Data are presented as mean ± sd, n = 5. Stats: Unpaired, two-tailed t test * P < 0.05.

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  Figure 12.. Assessment of multiple PN backbone chemistries. (A) Chemical structures of PN backbone variants. (B) Mean Ttr expression in mouse primary hepatocytes after gymnotic treatment with the indicated siRNA. Data are presented as mean ± sd, n = 2 per siRNA per concentration.