Targeting ANGPTL3 by GalNAc-conjugated siRNA ANGsiR10 lowers blood lipids with long-lasting and potent efficacy in mice and monkeys

Figures

• 

  Figure 1. Screening and validation of siRNAs targeting ANGPTL3 (A and B) The siRNAs were screened in vitro by luciferase assay in HEK293 cells (A) and inhibition assay in Huh7 cells (B). (C–E) The effect of ANGPTL3 silencing was further evaluated in C57BL/6 mice (C), ob/ob mice (D), and hApoC3-Tg mice (E) by measuring mRNA levels of ANGPTL3 in the liver by real-time PCR. (F) The serum ANGPTL3 level was measured by ELISA in hApoC3-Tg mice. Data are represented as mean ± SEM. NC, negative control; PC1/PC, positive control. Unpaired t test was performed comparing the ANGsiR10 group with the negative control. ∗p < 0.05; ∗∗p < 0.01.

• 

  Figure 2. ANGsiR10 significantly reduced the plasma TG and cholesterol levels in hApoC3-Tg mice hApoC3-Tg mice was treated with a single subcutaneous injection of ANGsiR10 (1 or 3 mg/kg). (A–D) TG (A), total cholesterol (B), and HDL-c (C) levels were monitored up to 14 weeks, while non-HDL-c (D) was calculated as the level of the total cholesterol minus that of the HDL-c. Multiple-dose regimens were performed using hApoC3-Tg mice. ANGsiR10 was administered in three regimens, e.g., 9 mg/kg × 1, 3 mg/kg qw × 3, and 3 mg/kg q4w × 3. (E and F) Both TG (E) and total cholesterol (F) levels were monitored up to 19 weeks. NC, negative control; PC, positive control. Data are represented as mean ± SEM of the percentage of baseline levels. Paired t test was performed compared with the baseline level. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

• 

  Figure 3. Percentage change in plasma lipids in dyslipidemic monkeys after the treatment of ANGsiR10 Twelve monkeys were divided into two groups and administered ANGsiR10 (9 mg/kg) or scramble siRNA as a negative control. (A–G) Blood samples were collected before and after treatment (weekly up to 5 weeks and then biweekly) and analyzed for TG (A), total cholesterol (B), non-HDL-c (C), HDL-c (D), LDL-c (E), ApoA (F), and ApoB (G). (H) The ApoA/ApoB ratio was calculated. Data are represented as mean ± SEM of the percentage of baseline levels. For each parameter, paired t test was performed comparing the value after treatment with the average baseline level. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

• 

  Figure 4. The expression of ANGPTL3 was inhibited in the monkeys treated with ANGsiR10 Before and after treatment with ANGsiR10 or scramble siRNA as a negative control, liver biopsy and blood sampling were performed and analyzed for the expression of ANGPTL3 by real-time PCR for mRNA and ELISA for protein. (A and B) The relative mRNA levels in the liver (A) and the protein levels in the serum (B) were significantly decreased in the ANGsiR10-treated group. (C) No sign of inflammation and/or tissue damage was observed in H&E-stained sections of livers in either the control or treatment group. (D and E) The ALT (D) and AST (E) levels in plasma were measured. Data are represented as mean ± SEM. Paired t test was performed compared with the baseline level. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

• 

  Figure 5. The effect of ANGsiR10 on physiological parameters was investigated in monkeys The body weight (BW) (A); waist (B); systolic/diastolic blood pressure (SBP/DBP) (C and D); fasting plasma glucose (FPG) (E); insulin (F); white blood cell (WBC) count (G); and high-sensitivity C-reactive protein (hsCRP) (H) were measured at baseline and 4 and 15 weeks after the dosing. Scramble siRNA was used as a negative control. Data are represented as mean ± SEM. Paired t test was performed compared with the baseline level. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.