B6-hPCSK9 Mice

Catalog Number: C001617

Strain Name: C57BL/6NCya-Pcsk9tm1(hPCSK9)/Cya

Genetic Background: C57BL/6NCya

Reproduction: Homozygote x Homozygote

 

Strain Description

Proprotein convertase subtilisin/kexin 9 (PCSK9) is a serine protease primarily produced in the liver but expressed in other tissues, including the intestine, heart, and neurons. The N-terminal domain of the PCSK9 protein is responsible for protein localization and stability, while the C-terminal domain is responsible for protein enzymatic activity [1]. The Low-density lipoprotein receptor (LDLR) is a receptor that is responsible for clearing low-density lipoprotein cholesterol (LDL-C) from the blood. PCSK9 cleaves the intracellular domain of LDLR on the cell surface, causing it to detach from the cell membrane and be transported to the lysosome for degradation, promoting LDLR degradation, and increasing plasma LDL-C. Overexpression or gain-of-function mutations of the PCSK9 gene can lead to LDL-C accumulation by reducing LDLR levels. This can cause hypercholesterolemia, which increases the risk of cardiovascular diseases, such as atherosclerosis and coronary heart disease, and neurodegenerative diseases, such as Alzheimer's disease [2]. PCSK9 has become an important target for the development of lipid-lowering drugs. Several PCSK9-targeted antibodies or small nucleic acid drugs have been approved for marketing worldwide, including evolocumab from Amgen, alirocumab from Sanofi and Regeneron, and inclisiran from Novartis. These drugs primarily work by inhibiting PCSK9 activity or preventing PCSK9 protein from binding to LDLR, lowering LDL-C levels in the blood to treat hypercholesterolemia [3-4]. In addition, PCSK9 can promote tumor growth and development by regulating cell proliferation, migration, and invasion. It can also regulate the expression of inflammatory factors that contribute to inflammation. Therefore, targeting the expression of PCSK9 has been investigated in tumor immunotherapy and autoimmune disease therapy [5-6].

B6-hPCSK9 mice are a humanized model generated by gene editing technology to replace the mouse Pcsk9 gene with the human PCSK9 gene sequence. These mice express human PCSK9 protein and can be used for research on various metabolic diseases, neurodegenerative diseases, tumor development, autoimmune disease mechanisms, and for the preclinical pharmacological evaluation of PCSK9-targeted drugs. In addition, Cyagen has developed a similar model, the B6-hPCSK9(CDS) mouse (PCSK9 coding sequence humanized model, Catalog Number: I001222). Compared to the B6-hPCSK9 mouse model, the B6-hPCSK9(CDS) mouse expresses higher levels of human PCSK9 and exhibits LDLR protein expression closer to physiological levels. It is recommended to choose the appropriate model based on the type of drug or research direction.

Strain Strategy


Figure 1. Gene editing strategy of B6-hPCSK9 mice. The mouse Pcsk9 gene sequence, including ~35.5 kb upstream and ~7 kb downstream sequences, was replaced with the corresponding sequences in the human PCSK9 gene, including the UTR regions.

Applications

  • Development and screening of PCSK9-targeted therapies;
  • Preclinical pharmacological and efficacy evaluation of PCSK9-targeted therapies;
  • Research on metabolic diseases such as hypercholesterolemia, atherosclerosis, and coronary heart disease;
  • Research on neurodegenerative diseases such as stroke and Alzheimer's disease.

 

Validation Data

1. Expression of human PCSK9 gene and mouse Pcsk9 gene

Figure 2. Expression of human PCSK9 and mouse Pcsk9 genes in the liver and colon of 6-week-old homozygous B6-hPCSK9 and wild-type (WT) mice (n=4). RT-qPCR analysis revealed significant expression of the human PCSK9 gene in the liver and colon of homozygous B6-hPCSK9 mice, with no detectable expression of the mouse Pcsk9 gene. In wild-type mice, only the mouse Pcsk9 gene was expressed. Furthermore, the expression of the human PCSK9 gene was higher in the liver compared to the colon of B6-hPCSK9 mice. Notably, female B6-hPCSK9 mice exhibited higher expression levels of the human PCSK9 gene in both the liver and colon than their male counterparts. (ND: Not detected)

2. Expression of human PCSK9 protein

a. Western Blots


Figure 3. Expression of human PCSK9 protein (hPCSK9) in the liver and colon tissues of wild-type (WT) and B6-hPCSK9 mice. Human-specific antibodies were employed in Western Blot analyses to detect the expression of human PCSK9 protein in the liver and colon of WT and B6-hPCSK9 mice. During synthesis, human PCSK9 initially forms a precursor protein, which undergoes autocatalytic cleavage to yield the mature protein subsequently secreted extracellularly. The antibody can detect both the precursor and mature forms of human PCSK9 protein. The results demonstrate that B6-hPCSK9 mice successfully express both forms of human PCSK9 protein.

*The antibody used is specific to the human PCSK9 protein. The bands observed in WT mice may be non-specific bands due to cross-reactivity.

b. ELISA


Figure 4. Expression of human PCSK9 protein in wild-type (WT) and B6-hPCSK9 mice serum. Human-specific antibodies were utilized in ELISA assays to detect the presence of human PCSK9 protein in the serum of WT and B6-hPCSK9 mice. The results revealed significant expression of human PCSK9 protein in the serum of B6-hPCSK9 mice, whereas no expression was detected in WT mice. (6 weeks old; homozygous; n=3; mice were not fasted prior to blood collection; data presented as mean ± SD)

3. Stable expression of human PCSK9 protein


Figure 5. ELISA of human PCSK9 protein in serum of B6-hPCSK9 mice at different age stages (n=5). The results showed that B6-hPCSK9 mice maintained stable expression of human PCSK9 protein for six consecutive weeks between 6 and 11 weeks of age. (Normal chow diet; mice were not fasted before blood collection; Bars represent mean ± SD)

4. Blood Biochemistry


Figure 6. Blood biochemical assay results of 11-week-old homozygous B6-hPCSK9 mice and wild-type (WT) mice (n=5). The results showed that the levels of T-CHO, HDL-C, LDL-C, and ALT were lower in B6-hPCSK9 mice than in WT mice, while the levels of TG and AST were not significantly different. (Mice were not fasted before blood collection; *p<0.05;**p<0.01;***p<0.001; Bars represent mean ± SEM)

* TG: Triglycerides; T-CHO: Total cholesterol; HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase.

5. Expression of the mouse Ldlr gene

Figure 7. Expression of the mouse Ldlr gene in the liver of 6-week-old homozygous B6-hPCSK9 and wild-type (WT) mice (n=4). RT-qPCR results show that the expression level of the mouse Ldlr gene in the liver of B6-hPCSK9 mice is higher than that in wild-type mice.

6. Expression of the mouse LDLR protein


Figure 8. Western Blot analysis of LDLR protein expression in the livers of 6-week-old homozygous B6-hPCSK9 mice and wild-type (WT) mice. The results from Western Blotting indicated that LDLR protein expression levels in the livers of B6-hPCSK9 mice were slightly elevated compared to wild-type mice. No significant difference in hepatic LDLR protein expression was observed between female and male B6-hPCSK9 mice.

7. Drug Efficacy Validation

(1) High-Fat, High-Cholesterol Diet Feeding

Figure 9. Body weight changes in B6-hPCSK9 and wild-type (WT) male mice following a high-fat, high-cholesterol diet (WD). Mice were acclimated to a high-fat, high-cholesterol diet for 1 week starting at 5 weeks of age, followed by a switch to a formal WD diet. WD feeding resulted in a significant increase in body weight in both B6-hPCSK9 and WT mice (Bars represent mean ± SD, n≥8).

(2) Evolocumab Efficacy Validation

Figure 10. Evolocumab efficacy validation study design and results.

  1. Efficacy validation study design: Mice were acclimated to a high-fat, high-cholesterol diet (WD) for 1 week starting at 5 weeks of age. From 6 weeks of age, mice were placed on a formal high-fat, high-cholesterol diet for 12 weeks. After 12 weeks on the formal WD diet, a single dose of Evolocumab (20mg/kg) was administered via tail vein injection for therapeutic intervention. To assess efficacy, blood samples were collected via retro-orbital bleeding under non-fasting conditions at 9 days prior to, and 2, 5, 8, 12, and 19 days post-Evolocumab administration. Serum was then harvested for subsequent analysis.
  2. Serum LDL-C levels: Following Evolocumab administration, serum LDL-C levels in B6-hPCSK9+WD mice were significantly reduced compared to WT+WD mice, while no significant alteration in LDL-C levels was observed in WT+WD mice (Bars represent mean ± SEM, n≥4).
  3. Serum PCSK9 levels: At 5 days post-Evolocumab injection, serum human PCSK9 levels in B6-hPCSK9+WD mice did not exhibit a significant decrease. This observation aligns with the anticipated mechanism of action for Evolocumab (Bars represent mean ± SEM, n=3).

Drug Information: Evolocumab functions by specifically binding to PCSK9 protein, thereby sterically hindering its interaction with LDLR. This blockade increases the quantity of LDLR receptors on the hepatocyte surface available for clearing LDL-C from the circulation, ultimately leading to a reduction in LDL-C levels. Consequently, Evolocumab has a relatively minor impact on overall PCSK9 protein levels [7].

(3) Inclisiran Efficacy Validation

Figure 11. Inclisiran efficacy validation study design and results.

  1. Efficacy validation study design: Mice were acclimated to a high-fat, high-cholesterol diet (WD) for 1 week commencing at 5 weeks of age. From 6 weeks of age, mice were maintained on a formal high-fat, high-cholesterol diet for 12 weeks. Following 12 weeks of formal WD feeding, a single dose of Inclisiran (3mg/kg) was administered via subcutaneous injection to initiate therapeutic intervention. To evaluate drug efficacy, blood samples were collected via retro-orbital bleeding under non-fasting conditions at 9 days prior to, and 5, 12, 19, and 26 days post-Inclisiran administration. Serum was then harvested for downstream analyses. Furthermore, at 19 days post-injection, livers from a subset of mice were harvested for Western Blot and RT-qPCR analyses.
  2. Serum LDL-C levels: The data revealed that following Inclisiran administration, serum LDL-C levels in B6-hPCSK9+WD mice exhibited a reduction compared to WT+WD mice, whereas no marked change in LDL-C levels was observed in WT+WD mice (Bars represent mean ± SEM, n≥4).
  3. Serum PCSK9 levels: At 19 days following Inclisiran injection, serum human PCSK9 levels in B6-hPCSK9+WD-treated mice were significantly decreased, indicating that Inclisiran effectively suppressed the synthesis of human PCSK9 protein (Bars represent mean ± SEM, n≥4).
  4. Liver PCSK9 levels: Liver PCSK9 levels in B6-hPCSK9+WD-treated mice were markedly reduced at 19 days post-Inclisiran injection, confirming that Inclisiran effectively inhibited hepatic PCSK9 protein synthesis (the band at approximately 70KD represents a non-specific band).

Drug Information: Inclisiran is a long-acting RNA interference therapeutic that functions by targeting human PCSK9 mRNA, thereby diminishing hepatic PCSK9 protein synthesis. This reduction in PCSK9 protein levels in vivo ultimately enhances the liver's capacity to clear LDL-C cholesterol [8].

 

References
[1]Melendez QM, Krishnaji ST, Wooten CJ, Lopez D. Hypercholesterolemia: The role of PCSK9. Arch Biochem Biophys. 2017 Jul 1;625-626:39-53.
[2]Seidah NG, Awan Z, Chrétien M, Mbikay M. PCSK9: a key modulator of cardiovascular health. Circ Res. 2014 Mar 14;114(6):1022-36.
[3]Pasta A, Cremonini AL, Pisciotta L, Buscaglia A, Porto I, Barra F, Ferrero S, Brunelli C, Rosa GM. PCSK9 inhibitors for treating hypercholesterolemia. Expert Opin Pharmacother. 2020 Feb;21(3):353-363.
[4]Sabatine MS. PCSK9 inhibitors: clinical evidence and implementation. Nat Rev Cardiol. 2019 Mar;16(3):155-165.
[5]Ding Z, Pothineni NVK, Goel A, Lüscher TF, Mehta JL. PCSK9 and inflammation: role of shear stress, pro-inflammatory cytokines, and LOX-1. Cardiovasc Res. 2020 Apr 1;116(5):908-915.
[6]Liu X, Bao X, Hu M, Chang H, Jiao M, Cheng J, Xie L, Huang Q, Li F, Li CY. Inhibition of PCSK9 potentiates immune checkpoint therapy for cancer. Nature. 2020 Dec;588(7839):693-698.
[7]Kasichayanula S, Grover A, Emery MG, Gibbs MA, Somaratne R, Wasserman SM, Gibbs JP. Clinical Pharmacokinetics and Pharmacodynamics of Evolocumab, a PCSK9 Inhibitor. Clin Pharmacokinet. 2018 Jul;57(7):769-779.
[8]Lamb YN. Inclisiran: First Approval. Drugs. 2021 Feb;81(3):389-395.