Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates

Abstract

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low density lipoprotein receptor (LDLR) protein levels and function. Loss of PCSK9 increases LDLR levels in liver and reduces plasma LDL cholesterol (LDLc), whereas excess PCSK9 activity decreases liver LDLR levels and increases plasma LDLc. Here, we have developed active, cross-species, small interfering RNAs (siRNAs) capable of targeting murine, rat, nonhuman primate (NHP), and human PCSK9. For in vivo studies, PCSK9 and control siRNAs were formulated in a lipidoid nanoparticle (LNP). Liver-specific siRNA silencing of PCSK9 in mice and rats reduced PCSK9 mRNA levels by 50-70%. The reduction in PCSK9 transcript was associated with up to a 60% reduction in plasma cholesterol concentrations. These effects were shown to be mediated by an RNAi mechanism, using 5'-RACE. In transgenic mice expressing human PCSK9, siRNAs silenced the human PCSK9 transcript by >70% and significantly reduced PCSK9 plasma protein levels. In NHP, a single dose of siRNA targeting PCSK9 resulted in a rapid, durable, and reversible lowering of plasma PCSK9, apolipoprotein B, and LDLc, without measurable effects on either HDL cholesterol (HDLc) or triglycerides (TGs). The effects of PCSK9 silencing lasted for 3 weeks after a single bolus i.v. administration. These results validate PCSK9 targeting with RNAi therapeutics as an approach to specifically lower LDLc, paving the way for the development of PCSK9-lowering agents as a future strategy for treatment of hypercholesterolemia.

Fig. 1.

Lipidoid formulation and effects of PCSK9 silencing in wild-type mice. (A) Cationic lipidoid component structure of the formulation. (B) Dose-dependent decrease in hepatic PCSK9 mRNA (relative to controls) 2 days after dose (n = 6 per group). (C) Liver PCSK9 mRNA and total serum cholesterol levels in mice (n = 5 per group) 3 days after a dose of 5 mg/kg LNP-PCS-A2 or PBS. (D) Duration of hepatic PCSK9 transcript silencing in mice (n = 5 per group) after a single injection of 5 mg/kg or 7.5 mg/kg LNP-PCS-A2. (B) one-way ANOVA with Student's t test; (C and D) two-way ANOVA with Bonferroni test. (B–D) Each value is the group mean ± STDEV. Asterisks represent statistical difference between PBS and PCSK9 siRNA treated groups. **, P ≤ 0.01; ***, P ≤ 0.001

Fig. 2.

Hepatic PCSK9 silencing, hepatic TGs, and LDLR levels in rats. (A) LNP-PCS-A2 mediated dose-dependent lowering of hepatic PCKS9 mRNA and total serum cholesterol 3 days after dose (n = 6 per group). Each value is the group mean ± STDEV. One-way ANOVA with Student's t test. Asterisks represent statistical difference between PBS- and PCSK9 siRNA-treated groups *, P ≤ 0.05; **, P ≤ 0.01. (B) Total serum cholesterol lowering of LNP-PCS-A2 treated rats (n = 6 per group) is maximal (≈60%) by 2 days after dose and returns to baseline over ≈21 days. PCSK9-treated groups are statistically significant (until approximately day 16) compared with PBS and LNP-Crtl groups (one-way ANOVA, Student's t test, with P values of ≤ 0.05). (C) Liver TGs and cholesterol contents from treated and control animals (same as in A). There were no significant differences in the liver TGs (ANOVA: P = 0.824 for cholesterol content ANOVA on ranks; P = 0.935 for LNP-PCS-A2-treated animals vs. the LNP-Crtl- or PBS-treated control groups for TG levels). (D) Immunoblot of liver extracts from LNP-PCS-A2-, LNP-Crtl-, and PBS-treated rats (same as in A). Transferrin receptor (TFR) levels were used to normalize for protein loading. (Note that treated animal lane 15 was a noticeable outlier that did not up-regulate LDLR and on close examination also did not lower PCSK9 levels, possibly because of a misinjection). †, relative to PBS.

Fig. 3.

siRNA-mediated cleavage of PCSK9 mRNA in rats (5′-RACE). Rats (n = 4 per group) were administered with 4 mg/kg LNP-PCS-A2, LNP-Crtl, or PBS and killed 4 days later. 5′-RACE detects the predicted mRNA cleavage product in LNP-PCS-A2- and not in LNP-Crtl- or PBS-treated animals. Eighty-seven percent of clones from the circled bands mapped to the predicted siRNA specific cleavage site.

Fig. 4.

Silencing of human PCSK9 mRNA and protein reduction in PCSK9 humanized mice. (A) Transgenic mice (n = 4 per group) expressing the human PCSK9 full-length cDNA under the apoE promoter were dosed with LNP-PCS-A2 and LNP-PCS-C2, and LNP-Crtl or PBS. Both LNP-PCS-C2 and LNP-PCS-A2 significantly lowered the human transcript as measured by quantitative PCR 3 days after dose. Each value is the group mean ± STDEV. (One-way ANOVA, Student's t test, P ≤ 0.001 between PBS- and PCSK9 siRNA-treated groups). (B) Circulating human PCSK9 protein levels were reduced in treated vs. control transgenic mice (n = 4 per group) as measured by ELISA. Each value represents group mean ± STDEV. All time points for the PCSK9 siRNA treated groups are statistically significant compared with PBS (Two-way ANOVA, Bonferroni test, P ≤ 0.01).

Fig. 5.

Pharmacology of PCSK9 silencing in NHPs. (A) Direct LDLc measurements of serum from cynomolgus monkeys treated with 5 mg/kg LNP-PCS-A2 (n = 5 per group), LNP-PCS-B2 (n = 4 per group), or PBS (n = 3 per group). (B) Total cholesterol/HDL ratios as measured in the samples described in A. (C) Plasma samples from the treatment groups in A were analyzed for their levels of PCSK9 protein by ELISA. Values for LDLc, Tc/HDLc, and PCSK9 concentrations were graphed as a ratio of the average values of after dose compared with predose values within an animal. Those values were then combined into group averages. (A–C) LNP-Crtl behaved similarly on d4 and d7 as PBS (data not shown). PBS values are the mean of the groups ± STDEV averaged over days 3–14. Each value represents the mean of the group ± STDEV. Two-way ANOVA with Bonferroni test was used. Asterisks represent statistical difference between PBS- and PCSK9 siRNA-treated groups. **, P ≤ 0.01; ***, P ≤ 0.001.