Fig 1: Single PS2 internucleotide linkages stabilize siRNA conjugates at their termini and increase activity in vitro(A) Tritosome stability of siRNA conjugates with 2 PS, 1 PS, or 1 PS2 at the indicated siRNA termini. PS2-stabilized siRNA conjugates were tested with a PS-containing GalNAc cluster and with a PO-containing GalNAc cluster. Data for siRNA-1 are identical in (A), (C), and Figure 3C; all tritosome stability analyses used for these figures were run in the same experiment with the same siRNA-1 data as baseline control. (B) Functional tolerance for PS2 at the individual termini was analyzed by receptor-mediated uptake of the siRNA conjugates in mouse primary hepatocytes and analysis of target gene repression. AT3 expression was normalized to ApoB and actin. Data represent the geometric mean (SD) of three technical replicates. (C) Tritosome stability of siRNA conjugates with combinations of VP, PS2, and PO-containing GalNAc moiety as indicated. (D) The in vitro activity of siRNA conjugates with the indicated combinations of VP, PS2, and PO-containing GalNAc moiety was analyzed by receptor-mediated uptake in mouse primary hepatocytes. AT3 expression was normalized to ApoB and actin. Data represent the geometric mean (SD) of three technical replicates. UT, untreated sample; NTC, non-targeting control.
Fig 2: In vitro serum and tritosome stability and in vitro efficacy of PS2 GalNAc-cluster variants(A) Cartoon representations of GalNAc-cluster variants. Half circles depict PS and full circles PS2. PAGE analysis after (B) serum and (C) tritosome stability assays run for 0, 24, and 96 h. (D) Knockdown efficacy in mouse primary hepatocytes after 24 h. AT3 expression was normalized to ApoB and actin. Data represent the geometric mean (SD) of three technical replicates. UT, untreated sample; NTC, non-targeting control.
Fig 3: PK-PD of PS2 siRNA conjugates in liver and kidney(A) Study design: male C57BL/6 mice were injected subcutaneously with 1 mg/kg GalNAc-conjugated siRNA. n = 4/group for all data except for explorative PK analysis during the first 4 h after injection (n = 2). (B) Plasma AT3 levels were normalized to individual pre-dose level as well as to the mean of PBS-treated animals at the respective time points. Data represent the mean (SD). One-way ANOVA was performed by comparing the mean rank of each treatment cohort against that of siRNA-1 by Kruskal-Wallis test with Dunn’s multiple comparison at respective time points. Asterisks indicate the number of digits after the decimals for p values. AT3 antisense strands were quantified in liver (C) and kidney (D). Data represent the mean (SD). One-way ANOVA was performed comparing the mean values of each treatment group with the mean of siRNA-1 cohort at the respective time point by Brown-Forsythe ANOVA test and Welch’s ANOVA test with Dunnett’s T3 multiple comparisons test. Asterisks indicate the number of digits after the decimals for p values.
Fig 4: In vivo siRNA efficacy in liver and siRNA content analysis in liver and kidney(A) In vivo study design. Male C57BL/6 mice (n = 4/group) were injected subcutaneously with 0.3 mg/kg GalNAc-siRNA conjugates. Plasma AT3 and liver and kidney siRNA levels were analyzed as indicated. (B) Plasma AT3 levels were normalized to individual pre-dose as well as to the mean of PBS-treated animals at the respective time point. Data represent the mean (SD). One-way ANOVA statistical analysis was performed comparing the mean rank of siRNA-2 against PBS by Kruskal-Wallis test with Dunn’s multiple comparison at respective time points. Asterisks indicate number of digits after the decimals for p values. The levels of the siRNA antisense strand in (C) liver and (D) kidney lysates were determined by stem-loop RT-qPCR 6 weeks after treatment. Shown are medians in box and whiskers. One-way ANOVA statistical analysis was performed by comparing the mean rank of each treatment cohort against every other by Kruskal-Wallis test with Dunn’s multiple comparison at respective time points. Asterisks indicate digits after the decimals for p values.
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