hPCSK9(4)

Fig.1 Detection of PCSK9 expression in liver by RT-PCR. Human PCSK9 mRNA (138 bp) was detectable only in homozygous hPCSK9(4) knockin mice but not in wild-type mice. Liver RNA was extracted from 7-week-old male wild-type C57BL/6 (WT)(n=3) and homozygous hPCSK9(4) knockin mice (HO) (n=3), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mRNA primers. Mouse Pcsk9 mRNA (154 bp) was detectable only in wild-type C57BL/6 mice. 

Abbr. M, marker; HO, homozygous; WT, wild type.

Fig.2 Detection of human PCSK9 (A) expression in serum of WT C57BL/6 mice and HE hPCSK9(4) mice by ELISA.  Human PCSK9 was exclusively detectable in heterozygous hPCSK9(4) knockin mice. Serum were collected from wild-type C57BL/6 mice (n=2, male, 12 weeks old) and HE hPCSK9(4) mice (n=3, male, 14 weeks old), and analyzed by ELISA. 

Abbr.  HE, heterozygous; WT, wild type.

Fig.3 Detection of human PCSK9 (A) and mouse PCSK9(B) expression in serum of WT C57BL/6 mice and HO hPCSK9(4) mice by ELISA.  Serum were collected from wild-type C57BL/6 mice (n=3, male, 6 weeks old) and HO hPCSK9(4) mice (n=3, male, 6 weeks old), and analyzed by ELISA.

Abbr.  HO, homozygous; WT, wild type; N.D. not dected.

Fig.4 Blood lipid profiles of WT C57BL/6 mice and homozygous hPCSK9(4) knock-in mice. There were no significant differences in serum TG, T-CHO, HDL-C and LDL-C levels between 8 to 9-week-old male WT C57BL/6 mice and hPCSK9(4) knock-in mice. 

Serum samples of 8 to 9-week-old male WT C57BL/6 mice and homozygous hPCSK9(4) knock-in mice were collected following a 6-hour fast at different ages to measure levels of (A) triglycerides (TG), (B) total cholesterol (T-CHO), (C) high-density lipoprotein cholesterol (HDL-C) and (D) low-density lipoprotein cholesterol (LDL-C) (n=5/group, Mean ± SEM, t-test, ns: no significance). 

Abbr. WT, wild-type; HO, homozygous.

◆ In vivo efficacy of Inclisiran in hPCSK9(4) mice 

Fig.5 Blood lipid profiles of hPCSK9(4) mice after Inclisiran treatment under CD conditions. Following Inclisiran administration, serum T-CHO and HDL-C were significantly reduced and serum LDL-C showed a tendency to decline.

11-week-old, male WT C57BL/6 mice and homozygous hPCSK9(4) knock-in mice were treated with saline or Inclisiran by s.c. injection. Serum samples were collected following a 6-hour fast at different ages to measure levels of (A) triglycerides (TG), (B) total cholesterol (T-CHO), (C) high-density lipoprotein cholesterol (HDL-C) and (D) low-density lipoprotein cholesterol (LDL-C). (E-H) The normalized indices were calculated (n=5/group, Mean ± SEM, t-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). 

Abbr. WT, wild-type; HO, homozygous.

Fig.6 Serum hPCSK9 levels of hPCSK9(4) mice after Inclisiran treatment under CD conditions. Following Inclisiran administration, serum hPCSK9 levels were significantly reduced on Day 7 and 14 post-dosing.

11-week-old, male WT C57BL/6 mice and homozygous hPCSK9(4) knock-in mice were treated with saline or Inclisiran by s.c. injection. Serum samples were collected following a 6-hour fast to measure levels of serum hPCSK9 using an ELISA kit (n=5/group, Mean ± SEM, t-test, *p<0.05, **p<0.01, ***p<0.001). 

Abbr. WT, wild-type; HO, homozygous.

Fig.7 Liver LDLR levels of hPCSK9(4) mice after Inclisiran treatment under CD conditions. No significant difference was observed in liver LDLR levels between saline- and Inclisiran- treated hPCSK9(4) knock-in mice. 11-week-old, male WT C57BL/6 mice and homozygous hPCSK9(4) knock-in mice were treated with saline or Inclisiran by s.c. injection. On Day 32 post-injection, liver tissues were collected following a 6-hour fast to measure mouse LDLR concentrations using an ELISA kit (n=5/group, Mean ± SEM, t-test, ns: no significance). 

Abbr. WT, wild-type; HO, homozygous.

◆ In vivo efficacy of Rosuvastatin in hPCSK9(4) mice

Fig.8 Blood lipid profiles of hPCSK9(4) mice after Inclisiran treatment under CD conditions. Serum T-CHO, HDL-C and LDL-C were significantly reduced during Rosuvastatin administration, but returned toward baseline levels after dosing was discontinued.

11-week-old, male WT C57BL/6 mice and homozygous hPCSK9(4) knock-in mice were treated with saline or Rosuvastatin by oral gavage. Serum samples were collected following a 6-hour fast at different ages to measure levels of (A) triglycerides (TG), (B) total cholesterol (T-CHO), (C) high-density lipoprotein cholesterol (HDL-C) and (D) low-density lipoprotein cholesterol (LDL-C). (E-H) The normalized indices were calculated (n=5/group, Mean ± SEM, t-test, *p<0.05, ***p<0.001). 

Abbr. WT, wild-type; HO, homozygous.

Fig.9 Serum hPCSK9 levels of hPCSK9(4) mice after Rosuvastatin treatment under CD conditions. Following Rosuvastatin administration, serum hPCSK9 levels showed a tendency to increase.

11-week-old, male WT C57BL/6 mice and homozygous hPCSK9(4) knock-in mice were treated with vehicle or Rosuvastatin by oral gavage. Serum samples were collected following a 6-hour fast to measure levels of serum hPCSK9 using an ELISA kit (n=5/group, Mean ± SEM, t-test). 

Abbr. WT, wild-type; HO, homozygous.

Fig.10 Liver LDLR levels of hPCSK9(4) mice after Rosuvastatin treatment under CD conditions. 

No significant difference was observed in liver LDLR levels between saline- and Rosuvastatin-treated hPCSK9(4) knock-in mice. 

11-week-old, male WT C57BL/6 mice and homozygous hPCSK9(4) knock-in mice were treated with vehicle or Rosuvastatin by oral gavage. On Day 32 post-dosing, liver tissues were collected following a 6-hour fast to measure mouse LDLR concentrations using an ELISA kit (n=5/group, Mean ± SEM, t-test, ns: no significance). 

Abbr. WT, wild-type; HO, homozygous.