Humanized PCSK9 mice as a resource for lipid management studies

Introduction: mitigating dyslipidemia to prevent cardiovascular disease

Dyslipidemia is a metabolic derangement that clinically presents with abnormal levels of lipids (cholesterol, triglycerides, and/or phospholipids) in the blood. Specifically, the condition is marked by high levels of low-density lipoprotein (LDL) and low levels of high-density lipoprotein (HDL) cholesterol. Of note, high levels of LDL cholesterol are a major risk factor for cardiovascular disease, due to its ability to accumulate in the walls of arteries, leading to plaque formation and atherosclerosis. Genetic predisposition, as well as poor diet, lack of exercise, obesity, and certain medical conditions can cause dyslipidemia. Early detection and management of dyslipidemia, such as lifestyle modifications and statins, are important for preventing the development and progression of cardiovascular disease.

PCSK9: a key target that prevents the removal of LDL from circulation

In recent years, an emerging target in the field of lipid-lowering drug development is PCSK9, due to its ability to regulate lipid metabolism in vivo and subsequently affect the occurrence and development of cardiovascular disease. Under physiological conditions, LDL is internalized via clathrin-mediated endocytosis after binding to the LDL receptor (LDLR) on the cell surface of hepatocytes1. Following LDL dissociation from LDLR, due to the acidic environment of the early endosome, LDL particles are subsequently trafficked to the lysosome for degradation1. While a majority of LDLR is recycled back to the plasma membrane, mechanistic studies have revealed two independent routes of PCSK9-induced lysosomal-mediated LDLR degradation1,2. Either intracellular LDLR can be directed from the trans-Golgi to lysosomes for degradation after binding to nascent PCSK9, or secreted PCSK9 binds LDLR at the cell surface, is internalized, which then prohibits endocytic recycling of LDLR, ultimately leading to lysosomal degradation of PCSK9 and LDLR2. Collectively, these studies have shown that PCSK9 blocks the removal of LDL from circulation via LDLR degradation. 

Following the discovery of PCSK9’s role in LDL metabolism, intense therapeutic interest has focused on identifying modulators capable of inhibiting PCSK9 function in order to reduce LDL levels and prevent subsequent major cardiovascular events. Currently, three FDA-approved pharmaceutical products targeting PCSK9 are available: alirocumab and evolocumab, which are fully humanized monoclonal antibodies, and inclisiran, which blocks intracellular synthesis of PCSK9 via small interfering RNA3. To advance PCSK9 research and drug development, BioMice has independently developed a humanized B-hPCSK9 mouse model, which provides a robust preclinical model for in vivo efficacy evaluation of novel anti-human PCSK9 drugs. 

B-hPCSK9 mice: a humanized model for testing human PCSK9 therapeutics

Model information

PCSK9 expression analysis in humanized B-hPCSK9 mice

Species-specific PCSK9 protein expression analysis in wild-type and humanized B-hPCSK9 mice. Sera was collected from wild-type C57BL/6 (+/+) and homozygous B-hPCSK9 (H/H) mice and analyzed using species-specific PCSK9 ELISA kits. Murine PCSK9 protein was detected in wild-type mice, while human PCSK9 protein was detected in B-hPCSK9 mice.  

Analysis of basal lipid metabolism in humanized PCSK9 mice

Lipid metabolism analysis in wild-type and humanized B-hPCSK9 mice. Plasma concentrations of TG, TC, LDL-C, and HDL-C in wild-type C57BL/6 and humanized B-hPCSK9 mice (n = 36, 6 weeks) were analyzed by Hitachi automatic biochemical analyzer 3110. Levels were similar between wild-type and B-hPCSK9 mice. TG: triglycerides; TC: total cholesterol; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol. 

In vivo efficacy of anti-human PCSK9 antibodies in western diet-induced B-hPCSK9 mice

Anti-human PCSK9 antibodies improved lipid metabolism in western diet-induced B-hPCSK9 mice. (A) Humanized B-hPCSK9 mice fed a western-diet, consisting of 40% fat 43% carbohydrates and 1.5% cholesterol, were treated with either alirocumab (internal), evolocumab (internal) or an isotype control antibody (single dose, s.c.) (n=8, male mice).  Blood was collected on days –5, 1, 3, 5 and 8 and analyzed by Hitachi automatic biochemical analyzer 3110. WD-induced B-hPCSK9 mice treated with alirocumab or evolocumab showed reduced (B) LDL-C and (B) TC levels compared to mice treated with an isotype control, indicating that anti-human PCSK9 antibodies were effective in lowering lipid levels in B-hPCSK9 male mice. Values are expressed as averages ± SEMs. TC: total cholesterol; LDL-C: LDL cholesterol; WD: Western diet.

Anti-human PCSK9 antibodies increased LDLR levels of western diet-induced B-hPCSK9 mice. Humanized B-hPCSK9 mice fed a western-diet were treated with either alirocumab (internal), evolocumab (internal) or an isotype control antibody (single dose, s.c.) (n=6, male mice). Liver tissue was collected on day 8 for ELISA analysis. LDLR levels increased in WD-induced B-hPCSK9 mice treated with anti-human PCSK9 antibodies compared to the isotype control group. Values are expressed as averages ±SEM. LDLR: LDL-cholesterol receptor; WD: Western diet.

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References:

1Islam, M. M., Hlushchenko, I., and Pfisterer, S. G. (2022). Low-Density Lipoprotein Internalization, Degradation and Receptor Recycling Along Membrane Contact Sites. Front. Cell Dev. Biol. 10 (826379). doi:10.3389/fcell.2022.826379

2Lagace, T. A. (2014). PCSK9 and LDLR degradation: regulatory mechanisms in circulation and in cells. Curr. Opin. Lipidol. 25 (5), 387-393. Doi:10.1097/MOL.0000000000000114

3Pokhrel, B., Yuet, W. C., and Levine, S. N. (2022). PCSK9 Inhibitors. In StatPearls. StatPearls Publishing.