Figure 1.
Intracellular reduction of targeted PCSK9 mRNA by LNA ASO.
Two human hepatic cell lines: HepG2 and HuH7 and a mouse insulinoma β-TC3 cell line; were transfected with LNA ASO, at concentrations of 10 and 25 nM. Total RNA was extracted at two different time points- 24 h and 48 h post transfection and QPCR analysis was performed using specific primers. The levels of PCSK9 mRNA were normalized to S14 mRNA for (A) HepG2 and (B) HuH7 cells; and (C) with S16 mRNA for β-TC3 cells.
Figure 2.
Effect of LNA ASO on mRNA expression of LDLR.
Human hepatic HepG2 and HuH7 and mouse insulinoma β-TC3 cells were transfected with LNA ASO at concentrations of 10 nM and 25 nM, and the levels of LDLR mRNA analyzed by QPCR after 24 h and 48 h. The levels of LDLR mRNA were normalized to (A and B) S14 mRNA and (C) S16 mRNA for human and mouse origin, respectively.
Figure 3.
QPCR analysis of HMGCoAR mRNA.
(A) HepG2 cells and (B) β-TC3 cells transfected with 10 and 25 nM LNA ASO and analyzed at 24 h and 48 h post-transfection. Human S14 and mouse S16 mRNAs were used for normalization.
Figure 4.
Reduction in the levels PCSK9 protein by LNA ASO is accompanied by an increase in LDLR protein expression.
HepG2 cells were transfected with 5, 10 and 25 nM LNA ASO (lanes 2–4). For mock, only water was used instead of LNA solution (lane 1). (A) 72h later, cell lysates were analyzed by Western blot for endogenous levels of human PCSK9 along with LDLR protein levels. The protein β-actin was used as a loading control. (B) Western blot analysis of overnight conditioned media of LNA ASO-transfected HepG2 cells, reflecting the levels of secreted PCSK9.
Figure 5.
Cell surface expression of LDLR in LNA ASO treated HepG2 cells.
Cells were transfected with 10 and 25 nM LNA ASO, and after 72 h FACS analysis was done to evaluate surface expression of LDLR using anti human LDLR as primary antibody and a suitable secondary antibody labeled with alexa 647.
Figure 6.
HepG2 cells were transfected for 4h with water for mock or 10 and 25 nM LNA ASO using Lipofectamine 2000 and incubated for 48h. The cells were then treated with Alamar blue reagent and analyzed on an Elisa plate reader for cell viability.
Figure 7.
Dose-dependent PCSK9 mRNA response in mice after a single i.v. injection at 5–40 mg/kg.
(A) Dose-response curve of PCSK9 mRNA levels in the liver (mean and SEM, n = 5), where the ED50 estimation is 9 mg/kg and maximum effect 95% reduction of the PCSK9 mRNA. (B) There is no change in mRNA levels of LDLR or Apobec. (C) LDLR protein is up-regulated 2–3 fold in the treated animals in the high dose groups. (Mean and SEM, n = 4–5).
Figure 8.
Duration of action after a single i.v. injection in mice.
A single injection of 20 mg/kg LNA ASO generated a significant reduction of PCSK9 mRNA lasting for more than two weeks (A), with concomitant increase in LDLR protein (B). Levels were normalized at day 32. (Mean and SEM, n = 4–5).
Figure 9.
Weekly repeated dosing at 5 mg/kg of the LNA ASO led to a stable reduction of PCSK9 mRNA (A), unchanged levels of LDLR mRNA (B) and 2–3 fold up-regulation of LDLR protein levels (C) (white bars: Saline, black bars: LNA ASO) (Mean and SD, n = 4–5).