siRNA-induced liver ApoB knockdown lowers serum LDL-cholesterol in a mouse model with human-like serum lipids

Coronary atherosclerosis is the most prevalent disease in industrialized societies. Although numerous advances have been made in understanding the underlying causes of atherosclerosis and treatment thereof, this condition still remains the leading cause of death in the Western world. The most important risk factor for atherosclerosis is hyperlipidemia (1). Development of atherosclerosis correlates with high levels of low density lipoprotein cholesterol (LDL). As a result, several therapies have been developed for management of LDL levels. Among these, statins are most widely used (2). However, there is a range of statin response in humans, and a subset of familial hyperlipidemia patients is unresponsive to statins, prompting the development of additional therapies. Mouse models have been widely used for studies of atherosclerosis and hyperlipidemia. Since wild-type mice have very high anti-atherogenic high density lipoprotein (HDL) and low pro-atherogenic LDL lipoprotein levels, genetic manipulations have been necessary for development of mouse models of coronary atherosclerosis. Development of these animal models started with the discovery of human mutations in individuals with lipoprotein disorders. The best-known examples include mutations in the LDL receptor (LDLr) in association with familial hypercholesterolemia (FH) and mutations in ApoE (including ApoE Leiden) in cases of Type III hyperlipoproteinemia (HLP) (3–5). As a result, LDLr–/–, Apo E–/–, and ApoE3-Leiden transgenic mice have been widely used as mouse models of atherosclerosis (6–9). These animals show a range of changes in serum lipids in response to different diets and exhibit atherosclerotic lesions very similar to the lesions found in humans. However, these animals have serum lipid profiles that are very different from those of normal healthy humans that make them less than optimal for use in studies of lipid homeostasis. Here we describe the development of a mouse model with a human-like lipid profile for use in preclinical studies of coronary atherosclerosis. These mice were genetically engineered to contain one copy of human cholesteryl ester transfer protein (CETP+/–) and are hemizygous for a LDL receptor mutation (Ldlr+/–). Ldlr CETP+/– hemizygous mice have serum lipid levels very similar to those of healthy humans, which make them suitable for investigation of lipid changes in response to different treatment regimens, and are very similar in lipid composition to ApoE3-Leiden/CETP transgenic mice (10, 11). We have used Ldlr CETP+/– mice to explore the effect of targeting ApoB mRNA in the liver using chemically modified siRNAs. ApoB is the main lipoprotein required for synthesis and secretion of VLDL particles from the liver (12). Levels of ApoB protein, LDL, and total cholesterol (TC) are highly correlated with increased risk for atherosclerosis. Patients with FH, who show reduced uptake of apoB-bound LDL from the circulation, are at high risk for development of coronary heart disease and atherosclerosis (13, 14). Contrarily, humans with very low levels of plasma apoB reported in particular cases of familial hypobetalipoproteinemia (FHBL) are at a reduced risk for coronary atherosclerosis (15). Because targeting of ApoB has proven difficult with conventional small molecule approaches, it presents an attractive target for development of a putative RNAi-based therapeutic. RNA interference (RNAi) is a regulatory sequence-dependent RNA silencing mechanism that uses small double-stranded RNA (dsRNA) molecules to direct gene silencing in a homology-based manner (16). These molecules, also known as short-interfering RNAs (siRNA), recruit a RNA-induced silencing complex (RISC) to the target mRNA and eventually lead to site-specific cleavage of the target mRNA and its subsequent degradation (17). RNAi-mediated gene silencing has been extensively used for target validation as it enables fast and relatively inexpensive screens without the need to generate knockout (KO) animals. We used chemically modified siRNAs in a mouse model with a human-like lipid profile to interrogate ApoB pathways. We demonstrated that LNP-formulated siRNAs can be successfully used in Ldlr CETP+/– hemizygous mice to achieve hepatic ApoB mRNA knockdown and that this reduction in ApoB mRNA levels results in significant reductions in serum ApoB protein, changes in genes in the lipid and fatty acid pathways, significant and prolonged reductions in serum total cholesterol, triglycerides, and LDL levels, as well as correlative hepatic steatosis.