Induction of Sustained Hypercholesterolemia by Single Adeno-Associated Virus–Mediated Gene Transfer of Mutant hPCSK9

Abstract

Objectives-Patients with mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene have hypercholesterolemia and are at high risk of adverse cardiovascular events. We aimed to stably express the pathological human D374Y gain-of-function mutant form of PCSK9 (PCSK9 DY ) in adult wild-type mice to generate a hyperlipidemic and proatherogenic animal model, achieved with a single systemic injection with adeno-associated virus (AAV). Approach and Results-We constructed an AAV-based vector to support targeted transfer of the PCSK9 DY gene to liver. After injection with 3.5×10 10 viral particles, mice in the C57BL/6J, 129/SvPasCrlf, or FVB/NCrl backgrounds developed long-term hyperlipidemia with a strong increase in serum low-density lipoprotein. Macroscopic and histological analysis showed atherosclerotic lesions in the aortas of AAV-PCSK9 DY mice fed a high-fat-diet. Advanced lesions in these highfat-diet-fed mice also showed evidence of macrophage infiltration and fibrous cap formation. Hepatic AAV-PCSK9 DY infection did not result in liver damage or signs of immunologic response. We further tested the use of AAV-PCSK9DY to study potential genetic interaction with the ApoE gene. Histological analysis of ApoE −/− AAV-PCSK9 DY mice showed a synergistic response to ApoE deficiency, with aortic lesions twice as extensive in ApoE −/− AAV-PCSK9 DY-transexpressing mice as in ApoE −/− AAV-Luc controls without altering serum cholesterol levels. Conclusions-Single intravenous AAV-PCSK9DY injection is a fast, easy, and cost-effective approach, resulting in rapid and long-term sustained hyperlipidemia and atherosclerosis. We demonstrate as a proof of concept the synergy between PCSK9 DY gain-of-function and ApoE deficiency. This methodology could allow testing of the genetic interaction of several mutations without the need for complex and time-consuming backcrosses.

Low-Density Lipoprotein Receptor–Dependent and Low-Density Lipoprotein Receptor–Independent Mechanisms of Cyclosporin A–Induced Dyslipidemia

“…Interestingly, PCSK9 was predicted to be a direct binding partner for CsA in a human structural proteome-wide characterization of CsA targets. 34 Recently, induction of a gain-of-function mutant of PCSK9 increased VLDL-triglyceride and IDL/LDL levels in Ldlr −/− mice 35 and supports the possibility of elevated PCSK9 levels contributing to CsA-induced hyperlipidemia in our Ldlr −/− mice. Blood CsA levels achieved in our study were 711±91 ng/ mL after 4 weeks of treatment which are slightly higher than levels achieved in humans (100-400 ng/mL) 36 but are consistent with other animal studies, which report CsA levels of ≈1300 ng/mL at administered dose of 50 mg/kg per d 37 and between 360 and 1066 ng/mL CsA at 20 mg/kg per d. 14,38 Published literature indicates that blood CsA levels can correlate with plasma lipid levels 13,39 and may be relevant to some of our findings.…”

Recommendation on Design, Execution, and Reporting of Animal Atherosclerosis Studies: A Scientific Statement From the American Heart Association

“…[145][146][147] Gainof-function mutations of PCSK9 lead to hypercholesterolemia. 148,149 Therefore, an adenovirus-associated vector technique has been applied to introduce a gain-of-function mutation of human PCSK9D374Y or its mouse equivalent, D377Y, in mice, [150][151][152][153] which results in profound increases of plasma cholesterol concentration with lipoprotein distribution comparable to that of Ldlr −/− mice fed a Western diet. This approach is rapid and efficient and mimics the Ldlr-deficient condition, augmenting atherosclerosis within a short period in C57BL/6 mice.…”

“…This approach is rapid and efficient and mimics the Ldlr-deficient condition, augmenting atherosclerosis within a short period in C57BL/6 mice. [150][151][152]154 However, the C57BL/6 mouse strain, but not other normolipidemic mouse strains such as FVB, 129, and BALB/C, responds to enhanced expression of PCSK9 activity. 151,152 There is also evidence that PCSK9 has effects independent of LDL receptor regulation.…”

“…[150][151][152]154 However, the C57BL/6 mouse strain, but not other normolipidemic mouse strains such as FVB, 129, and BALB/C, responds to enhanced expression of PCSK9 activity. 151,152 There is also evidence that PCSK9 has effects independent of LDL receptor regulation. 155 Therefore, use of an adenovirus-associated vector approach for enhancing PCSK9 activity should take into account both the mouse strain and potential LDL receptor-independent effects.…”

Recent Advances in the Genetics of Atherothrombotic Disease and Its Determinants

“…76 Epidemiological links between dyslipidemia and clinical end points emphasize the need to continuously clarify its underlying mechanistic basis, particularly in animal models. Roche et al 77 used a novel approach in generating a stable hyperlipidemic, atherosclerotic mouse model by injecting a gain-of-function PCSK9 mutation into the liver using adenoassociated virus. Interestingly, parallel experiments in the severely hyperlipidemic ApoE-null mouse showed an even greater elevation of LDL-C levels and a more severe atherosclerotic phenotype, emphasizing synergism between these 2 mechanisms of raising atherogenic lipoproteins.…”