TRANSGENIC MURINE MODEL OF HUMAN LIPOPROTEIN METABOLISM, HYPERCHOLESTEROLEMIA AND CARDIOVASCULAR DISEASE

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format.

The Sequence Listing is provided as a file entitled BIOL0108WOSEQ.txt, created 12/30/2009, which is 1.4 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides a transgenic mouse model of hypercholesterolemia and cardiovascular disease and methods of using the model. Such models are useful for screening for prophylactic and/or therapeutic drugs for cardiovascular disease.

BACKGROUND OF THE INVENTION

Although some inbred strains of mice are susceptible to atherosclerosis, the duration of exposure and nature of the injurious diets required to produce lesions compromises the use of such mice as models of cardiovascular disease. For example, the mouse strain C57B1/6, which is a relatively atherosclerosis-susceptible inbred strain, requires several months of feeding an inflammatory hepatotoxic cholic acid-enriched high fat/high cholesterol diet before induction of detectable lesions (Variation in susceptibility to atherosclerosis among inbred strains of mice.

Paigen B, Morrow A, Brandon C, Mitchell D, Holmes P. Atherosclerosis. 1985 Oct;57(l):65-

73.). The technology of manipulating the mouse genome thereby creating transgenic and knockout mice transformed the use of mice as models of cardiovascular disease (CVD).

Currently available transgenic and knockout murine models have been instrumental in understanding the pathophysiology of hypercholesterolemia and cardiovascular disease such as atherosclerosis. The most widely used models of atherosclerosis, low-density lipoprotein receptor deficient (LDLr -/-) and apolipoprotein E deficient (apoE -/-) mice, have allowed researchers to study lesion progression in a relatively short study time period of 10 - 20 weeks on diets that are not hepatotoxic. The ability to induce atherosclerotic lesion development in these mice models is critically dependent upon plasma levels of cholesterol. A survey of atherosclerosis studies using either LDLr -/- or apoE -/- mice indicates typical levels of total plasma cholesterol to be greater than 1,000 mg/dL, in many cases as high as 2,000 mg/dL (G. S. Getz, C. A. Reardon, Arterioscler Thromb Vase Biol 26, 242 (Feb, 2006).). The severity of this level of plasma cholesterol is underscored by the classification of high total cholesterol of >240 mg/dL by the National Cholesterol Education Program (NCEP) in patients at risk (National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). Circulation. 2002 Dec 17;106(25):3143-421). The utility of mouse models with such severe hypercholesterolemia lies in the rapid development of atherosclerosis in these models, however, such supraphysiologic levels of plasma cholesterol can obscure many other atherogenic processes as well as create pathophysiological processes not observed in humans. Thus, an atherosclerosis-susceptible model with moderate hypercholesterolemia would be desirable. Other specific limitations of LDLr -/- or apoE -/- mouse models of atherosclerosis include total loss of LDLr activity (in LDLr -/- mice) and an accumulation of very low density lipoprotein (VLDL) and chylomicron remnants (in apoE -/- mice). In fact, a major critique of the apoE -/- mouse is that it has a lipoprotein profile not typically observed in hypercholesterolemic patients (S. Zadelaar, R. Kleemann, L. Verschuren, J. de Vries-Van der Weij, J. van der Hoora, H. M. Princen, and T. Kooistra Mouse Models for Atherosclerosis and Pharmaceutical

Modifiers. Arterioscler. Thromb. Vase. Biol., August 1, 2007; 27(8): 1706 - 1721). Additionally, apoE has pleiotropic effects that impact processes beyond cholesterol metabolism, such as immune function, inflammation and proliferation. Therefore, the use of apoE -/- mice, or any other model of genetically altered apoE, to study chronic inflammatory processes like atherosclerosis has limitations (Apolipoprotein E and atherosclerosis: beyond lipid effect. Davignon J. Arterioscler Thromb Vase Biol. 2005 Feb;25(2):267-9 AND Apolipoprotein E promotes the regression of atherosclerosis independently of lowering plasma cholesterol levels. Raffai RL, Loeb SM, Weisgraber KH. Arterioscler Thromb Vase Biol. 2005 Feb;25(2):436-41). Because of the pleiotropic effects of apoE on various biological processes, it would be desirable to develop a mouse model having native, unaltered apoE. Although LDLr -/- mice have the genetic deficiency seen in patients with homozygous familial hypercholesterolemia (HoFH), this condition is quite rare (1 in 1 million). Additionally, because the common mechanistic basis of cholesterol-lowering for the common hypercholesterolemic drugs like statins and bile acid sequestrants is an upregulation of hepatic LDLr activity, LDLr -/- mice are an unfit model to test such common pharmacologic agents or novel pathways that modulate hepatic LDLr activity. Interestingly, the most frequent cause of hypercholesterolemia and CVD resulting from a single gene defect is intact, but compromised hepatic LDLr activity (Molecular medicine. The cholesterol quartet. Goldstein JL, Brown MS. Science. 2001 May 18;292(5520): 1310-2.). Accordingly, it would be desirable to develop a mouse model having intact, but compromised, hepatic LDLr activity.

In short, there is a need for an atherosclerotic-susceptible mouse model that can better approximate human lipoprotein metabolism, hypercholesterolemia and cardiovascular disease.

SUMMARY OF THE INVENTION Provided herein is a transgenic mouse model of cardiovascular disease and methods of using the model. The mouse model comprises a hemizygous copy of human cholesteryl ester transfer protein (huCETP+/-), a hemizygous copy of human apolipoprotein B (huApoB+/-) and has a partial deficiency of murine low-density lipoprotein receptor (mLDLr+/-).

The transgenic mouse exhibits a human-like lipoprotein phenotypic profile when fed a normal murine chow diet i.e. a low-fat, cholesterol-free diet. When fed a high fat (HF), high cholesterol (HC) diet, the transgenic mouse develops a human-like hypercholesterolemia. Additionally, the transgenic mouse develops atherosclerosis within four months of being fed a typical murine atherogenic diet.

The mouse model provided herein is useful for screening prophylactic and/or therapeutic agents for hypercholesterolemia and/or cardiovascular diseases such as atherosclerosis and heart disease; testing the efficacy of prophylactic and/or therapeutic agents for hypercholesterolemia and/or cardiovascular diseases; comparing the efficacies of prophylactic and/or therapeutic agents for hypercholesterolemia and/or cardiovascular diseases; studying molecular and cellular aspects associated with hypercholesterolemia and/or cardiovascular disease; and identifying potential therapeutic targets useful for treating or preventing hypercholesterolemia and/or cardiovascular disease. Agents that can be tested in the mouse model of the invention include, but are not limited to, specific diets, exercise regimens or hypocholesterolemia drugs. Examples of hypocholesterolemia drugs that decrease the level of cholesterol in a subject include, but are not limited to, nicotinic acid, agents that reduce plasma CETP activity, the statins, ezetimibe, fenofibrate, novel agents that reduce plasma apolipoprotein C-III (apoC-III) or novel agents that increase LDLr activity e.g., via inhibition of a protease involved in LDLr degradation.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Diagram of the breeding scheme to generate the "triple het" mouse model as described in Example 1 , infra. The mouse model is hemizygous for the human CETP transgene (huCETP+/-), hemizygous for the human apoB transgene (huApoB+/-) and contains one working copy of murine LDLr (mLDLr+/-).

Figure 2. Mouse plasma lipoprotein cholesterol profile comparisons. Animal models bred to create the 3 Het genotype demonstrate the transformation of a murine lipoprotein cholesterol profile to one resembling a human profile as described in Example 1 , infra. All mice were chow- fed and their plasma lipoprotein cholesterol profile (black) was analyzed on a HPLC utilizing size-exclusion chromatography. In gray is a reference lipoprotein profile used to define the very low-density lipoprotein (VLDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) peaks. This reference was loaded at the same concentration in each chromatogram to allow for comparisons of profiles. The addition of the huCETP transgene alone (B) or with a partial deficiency of mLDLr (C) results in a decrease in the HDL peak and an increase in the LDL peak relative to the background strain of these mice (A). The addition of the huApoB transgene in animals expressing huCETP and partially deficient in mLDLr (D) results in a further decrease in HDL and increase in LDL. Importantly, the magnitude of the LDL peak now eclipses the HDL peak, a common feature observed in human plasma. All mouse plasma samples were loaded undiluted at the same total volume. For clarification purposes, only the genetic perturbation of each animal model has been shown above each panel. C57B1/6 is the background strain for all mice used.

Figure 3. Plasma Lipid Parameters: Comparison of chow-fed 3 Het mice to LDLr+/- mice lacking either the huCETP (II) or huApoB (III) transgenes as described in Example 1, infra. The addition of both huApoB and huCETP in LDLr+/- mice (I) creates a human-like lipoprotein profile.

Figure 4. Plasma Lipid Parameters: Comparison of hypercholesterolemic huCETP+/-, LDLr+/- mice without the huApoB transgene as described in Example 1, infra. Diet-induced hypercholesterolemia is achieved in 3 Het, but not huCETP+/-LDLr+/- mice fed TD.94059.

Figure 5. Tolerability and Long Term Plasma Lipid Changes in the 3 Het mice fed either

TD.94059 or TD.88137 as described in Example 1, infra. The 3 het mice fed either the hypercholesterolemic diet TD.94059 or TD.88137 (Harlan Teklad, Madison, WI, USA) for up to

15 weeks exhibited sustained hypercholesterolemia without any increases in ALT. Both diets are commonly used in murine atherosclerosis studies.

Figure 6. En Face Aortic Atherosclerosis in 3 Het mice fed TD.88137 for 15 wks. Aortic tissue sections from 3 het transgenic mice observed to contain atheromatous plaques after 15 weeks on an atherosclerosis inducing diet as described in Example 8, infra.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms, such as "includes" and "included", is not limiting.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.

Definitions

Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Where permitted, all patents, applications, published applications and other publications, GENBANK Accession Numbers and associated sequence information obtainable through databases such as National Center for Biotechnology Information (NCBI) and other data referred to throughout in the disclosure herein are incorporated by reference in their entirety. Unless otherwise indicated, the following terms have the following meanings:

"2'-O-methoxyethyl" (also 2'-MOE and 2'-O(CH ₂ ) ₂ -OCH ₃ ) refers to an O-methoxy-ethyl modification of the 2' position of a furosyl ring. A 2'-O-methoxyethyl modified sugar is a modified sugar.

"3 het" or "triple het" refers to the mouse model provided herein with hemizygous huCETP, hemizygous huApoB and heterozygous mLDLr (huCETP+/-, huApoB+/-, mLDLr +/-).

"5-methylcytosine" means a cytosine modified with a methyl group attached to the 5' position. A 5-methylcytosine is a modified nucleobase.

"Administering" or "administration" means providing an agent to the 3 het mouse model or tissues or cells derived from the mouse model. "Antisense compound" means an oligomeric compound that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.

"Antisense inhibition" means reduction of a target nucleic acid levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels in the absence of the antisense compound. "Antisense oligonucleotide" or "ASO" means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.

"Atherosclerosis" refers to a thickening and hardening of the walls of arteries as a result of fat deposits (atheromatous plaques containing cholesterol and lipids) on their inner lining. "Atherogenic" refers to the process of forming atheromatous plaques in the inner lining of arteries i.e. the process of developing and progressing atherosclerosis.

"Atherosclerotic diet" or "atherogenic diet" refers to the diet fed to the mouse model that induces atherosclerosis in the mouse. Atherogenic diets are commercially available from Harlan Teklad (Madison, WI, USA). Examples of atherogenic diets include a "Western" Diet (Catalog #TD.88137) and other diets with added cholesterol (Catalog #TD.94O59). "Cardiovascular disease" refers to any disease of the heart and blood vessel system. Examples of blood vessels diseases include, but are not limited to, atherosclerosis, arteriosclerosis, arterial stenosis and peripheral artery occlusive disease. Examples of heart diseases include, but are not limited to, myocardial infarction and congestive heart failure. "Chimeric antisense compounds" means an antisense compounds that have at least two chemically distinct regions, each position having a plurality of subunits. A "gapmer" means an antisense compound in which an internal position having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having one or more nucleotides that are chemically distinct from the nucleosides of the internal region. A "gap segment" or "gap" means the plurality of nucleotides that make up the internal region of a gapmer. A "wing segment" or "wing" means the external region of a gapmer. For example, a "3-14-3" gapmer has three nucleotides at the wing segments and fourteen nucleotides at the gap segment and a "5-10-5" gapmer has five nucleotides at the wing segments and ten nucleotides at the gap segment. "Chow" refers to a normal a low-fat, cholesterol-free diet fed to laboratory animals. Chow may be obtained from Purina Test Diets (e.g., Labdiet 5001).

"Typical murine hypercholesterolemic diets" refers to high fat, cholesterol-enriched murine diets like TD.94059 and TD.88137 from Harlan Teklad. These diets are commonly used in murine atherosclerosis studies. Both lack the addition of cholic acid, an inflammatory hepatotoxic ingredient. "Dose" means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose can be administered in two or more boluses, tablets, or injections. For example, in certain embodiments, where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection. In such embodiments, two or more injections can be used to achieve the desired dose, hi certain embodiments, a dose can be administered in two or more injections to minimize injection site reaction in an animal. In other embodiments, the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses can be stated as the amount of pharmaceutical agent per hour, day, week or month. "Dosage unit" means a form in which a pharmaceutical agent is provided, e.g. pill, tablet, or other dosage unit known in the art. In certain embodiments, a dosage unit is a vial containing lyophilized antisense oligonucleotide. In certain embodiments, a dosage unit is a vial containing reconstituted antisense oligonucleotide.

"Hemizygous" describes having a functional copy of a gene that was inherited from only one parent. Since the copy number of a transgene is not fixed, a hemizygous transgenic mouse may have multiple copies of the transgene, but all of these copies were inherited from only one parent.

"Heterozygous" describes the condition of having two different alleles at a locus. Herein, we describe the condition of LDLr+/- as heterozygous. In this case, there are two different alleles at the LDLr; one is the intact LDLr gene and the other is a disrupted LDLr gene that encodes a truncated non- functional protein that does not bind LDL (Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery. Ishibashi S, Brown MS, Goldstein JL, Gerard RD, Hammer RE, Herz J. J Clin Invest. 1993 Aug;92(2):883-93).

"High fat" and "high cholesterol" diet (i.e. HF/HC diet) refers to the diet that when fed to the mouse model will induce hypercholesterolemia in the mouse. High fat and high cholesterol diets are commercially available from Harlan Teklad. The diets used here are TD.88137 and TD.94059 from Harlan Teklad. TD.88137 has 21.2% fat (wt/wt) and 0.2% cholesterol. TD.94059 has 15.8% fat (wt/wt) and 1.25% cholesterol.

"Homozygous" means to have two functional copies of a particular gene. "Human-like hypercholesterolemia" refers to the hypercholesterolemia profile of the mouse model when fed a high fat, high cholesterol diet. For example, a hypercholesterolemia profile for individuals considered 'borderline high' for risk of CVD is total plasma cholesterol (TPC) >200 mg/dL or LDL-C >130 mg/dL. For individuals considered at 'high' risk of CVD, these values are TPC > 240 mg/dL or LDL-C>160 mg/dL (NCEP Adult Treatment Panel III). "Moderate hypercholesterolemia" refers to the hypercholesterolemic level of the mouse model compared to typical murine models of atherosclerosis, such as LDLr -/- or apoE -/- mice, when fed common murine hypercholesterolemic diets. The 3 Het mice fed diets commonly used in murine atherosclerosis studies, such as TD.94059 and TD.88137, result in total plasma cholesterol levels to be 400 - 800 mg/dL. By comparison, in LDLr -/- or apoE -/- mice, these diets result in total plasma cholesterol levels to be 1 ,000 - 2,500 mg/dL. "Severe hypercholesterolemia" refers to the hypercholesterolemia observed in commonly used atherosclerotic-susceptible mice, such as LDLr -/- or apoE -/- mice fed a Western diet (TD.88137). These diets result in total plasma cholesterol levels to be > 1,000 mg/dL.

"Human-like lipoprotein profile" and/or "Human-like lipoprotein metabolism" refers to the lipoprotein levels in a non-human animal that are similar to human. For example, a human lipoprotein profile of an individual defined to be at low risk of CVD can include one or more of TPC < 200 mg/dL, LDL-C < 130 mg/dL, HDL-C > 40 mg/dL and HDL/TPC = 20 - 40%.

"Hypocholesterolemia drugs" refers to drugs that decrease the level of cholesterol in a subject. Examples of hypocholesterolemia drugs include, but are not limited to, nicotinic acid, agents that reduce plasma CETP activity, the statins, ezetimibe, fenofibrate or novel agents that increase LDLr activity e.g., via inhibition of a protease involved in LDLr degradation.

"Isolated" means altered "by the hand of man" from its natural state i.e. that, if it occurs in nature, it has been changed and/or removed from its original environment. For example, tissue or cells can be isolated from the mouse model. "Modulate" means to change a factor. For example, to modulate lipid metabolism means to change, by increasing or decreasing, lipid metabolism in a subject such as the 3 het mouse model.

"Phosphorothioate linkage" means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom.

"Prophylactic and/or therapeutic agent" or "agent" refers to an agent that ameliorates or treats a condition or disease. For example, prophylactic and/or therapeutic agents for ameliorating or treating atherosclerosis include, but are not limited to, diet, exercise or hypocholesterolemia drugs. "Targeted" or "targeted to" means having a nucleobase sequence that will allow specific hybridization of an antisense compound to a target nucleic acid to induce a desired effect. In certain embodiments, a desired effect is reduction of a target nucleic acid. In certain such embodiments, a desired effect is reduction of CETP or ApoB mRNA.

"Target nucleic acid," "target RNA," "target RNA transcript" and "nucleic acid target" all mean a nucleic acid capable of being targeted by an antisense compound. Certain Embodiments of the Invention

Described herein is a transgenic mouse model with three genetic perturbations incorporated into its genome comprising: (a) a human cholesteryl ester transfer protein (huCETP) transgene, (b) a human apolipoprotein B (huApoB) transgene, and (c) a single copy of murine LDLr.

The genetic perturbations of the transgenic mouse can be expressed in the liver and can modulate plasma lipid metabolism resulting in cardiovascular disease.

The cardiovascular disease can be atherosclerosis and/or heart disease.

The transgenic mouse can exhibit a human-like lipoprotein profile when fed a normal low-fat, cholesterol-free murine chow diet.

The transgenic mouse can develop a human-like hypercholesterolemia when fed a high fat, high cholesterol diet.

The transgenic mouse can develop atherosclerosis within four months of being fed an atherosclerotic diet. The transgenic mouse can be used in a method of screening for a prophylactic and/or therapeutic agent for preventing or ameliorating hypercholesterolemia and/or cardiovascular disease comprising administering a test compound to the transgenic mouse, or a tissue or cell thereof, and evaluating the effect of the test compound on hypercholesterolemia and/or cardiovascular disease in the transgenic mouse, or tissue or cell thereof, in order to screen for a prophylactic and/or therapeutic agent for preventing or ameliorating hypercholesterolemia and/or cardiovascular disease.

The transgenic mouse can be used in a method of testing the efficacy of a prophylactic and/or therapeutic agent for preventing or ameliorating hypercholesterolemia and/or cardiovascular disease comprising administering a test compound to the transgenic mouse, or a tissue or cell thereof, and evaluating the effect of the test compound on hypercholesterolemia and/or cardiovascular disease in the transgenic mouse, or tissue or cell thereof, in order to test the efficacy of a prophylactic and/or therapeutic agent for preventing or ameliorating hypercholesterolemia and/or cardiovascular disease.

The transgenic mouse can be used in a method of comparing the efficacies of potential prophylactic and/or therapeutic agents for preventing or ameliorating hypercholesterolemia and/or cardiovascular disease comprising a. administering a first test compound to a first transgenic mouse of the invention, or a tissue or cell thereof, b. administering a second test compound to a second transgenic mouse of the invention, or a tissue or cell thereof, c. evaluating the effect of the test compounds on hypercholesterolemia and/or cardiovascular disease in the transgenic mice, or tissue or cell thereof, and d. comparing the effects of the test compounds on hypercholesterolemia and/or cardiovascular disease in the transgenic mice, or tissue or cell thereof, so as to compare the efficacies of prophylactic and/or therapeutic agents for preventing or ameliorating hypercholesterolemia and/or cardiovascular disease in the transgenic mice.

The transgenic mouse can be used in a method of studying molecular and cellular aspects associated with hypercholesterolemia and/or cardiovascular disease comprising administering a test compound to the transgenic mouse, or a tissue or cell thereof, and evaluating the effect of the test compound on hypercholesterolemia and/or cardiovascular disease in the transgenic mouse, or tissue or cell thereof, in order to study the molecular and cellular aspects associated with hypercholesterolemia and/or cardiovascular disease.

The transgenic mouse can be used in a method of identifying a potential therapeutic target useful for treating or preventing hypercholesterolemia and/or cardiovascular disease comprising administering a test compound to the transgenic mouse, or a tissue or cell thereof, and evaluating the effect of the test compound on hypercholesterolemia and/or cardiovascular disease in the transgenic mouse, or tissue or cell thereof, in order to identify a potential therapeutic target for treating or preventing hypercholesterolemia and/or cardiovascular disease.

The agent used in the methods of the invention can be diet, exercise and/or a hypocholesterolemic drug. The hypocholesterolemic drug can be nicotinic acid, agents that reduce plasma CETP activity, statins, ezetimibe, fenofibrate, novel agents that reduce plasma apolipoprotein C-III (apoC-III) or novel agents that increase LDLr activity.

The agent that increases LDLr activity can be an inhibitor of LDLr degradation. The inhibitor of LDLr degradation can be an inhibitor of a protease involved in LDLr degradation.

3 Het Transgenic Mouse Model The transgenic mouse model provided herein comprises huCETP+Λ, huApoB+/- and mLDLr+/- genes.

CETP is a plasma protein that facilitates the transport of cholesteryl esters and triglycerides between lipoproteins, for example, by collecting triglycerides from VLDL or LDL and exchanging the triglyerides for cholesteryl esters from HDL. Wildtype mice lack plasma CETP activity. The copy of huCETP in the transgenic mouse of the invention in combination with the other genetic alterations in the mouse creates a human-like lipoprotein metabolism. In one embodiment, the human CETP gene has the sequence set forth in GENBANK Accession No. NG_008952 (X C Jiang, L B Agellon, A Walsh, J L Breslow, and A Tall. Dietary cholesterol increases transcription of the human cholesteryl ester transfer protein gene in transgenic mice. Dependence on natural flanking sequences. J Clin Invest. 1992 October; 90(4): 1290-1295., incorporated by reference herein).

ApoB is the primary apolipoprotein component of LDL. The copy of human apoB in the transgenic mouse of the invention in combination with the other genetic alterations in the mouse creates a human-like lipoprotein metabolism in the mouse. In one embodiment, the human ApoB gene has the sequence set forth in GENBANK Accession No. NC_000002 (Linton, M. F., R. V. Farese, Jr., G. Chiesa, D. S. Grass, P. Chin, R. E. Hammer, H. H. Hobbs, and S. G. Young. 1993. Transgenic mice expressing high plasma concentrations of human apolipoprotein BlOO and lipoprotein(a). J. Clin. Invest. 92: 3029-3037, incorporated by reference herein). LDLr is a cell-surface receptor that recognizes apoBl 00 in LDL particles and mediates the endocytosis of LDL. As discussed supra, LDLr -/- mice are an unfit model to test pharmacologic agents or novel pathways that modulate hepatic LDLr activity. The LDLr in the transgenic mouse of the invention provides an intact, although compromised, LDLr for studying agents or pathways that modulate LDLr activity. In one embodiment, the mLDLr gene has the sequence set forth in GENBANK Accession No. NC_000075 (Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery. Ishibashi S, Brown MS, Goldstein JL, Gerard RD, Hammer RE, Herz J. J Clin Invest. 1993 Aug;92(2):883-93 incorporated by reference herein).

In one embodiment, the transgenic mouse model provided herein exhibits a human-like lipoprotein metabolism and profile when fed a normal low-fat, cholesterol free chow diet. For example, chow fed 3 Het mice have human-like lipoprotein profiles that can include one or more of the following: TPC = 180 - 220 mg/dL, LDL-C = 110 - 130 mg/dL, HDL-C = 40 - 60 mg/dL and HDL/TPC ~ 25% as shown in Figure 3. By comparison, a chow-fed wildtype C57B1/6 mouse has the majority of its plasma cholesterol in HDL with TPC = 90 - 110 mg/dL, LDL-C = 10 - 20 mg/dL and HDL-C = 50 - 70 mg/dL and HDL/TPC ~ 60%. In another embodiment, the transgenic mouse model develops a human-like hypercholesterolemia when fed a high fat, high cholesterol (HF/HC) diet. The hypercholesterolemia in the transgenic mice can be expressed as elevated levels of LDL, TG, TPC and/or lowered HDL as described supra.

In yet another embodiment, the 3 Het mouse develops atherosclerosis after being fed a typical murine atherogenic diet. Usually, the mouse model develops atherosclerosis after about four (4) months of being fed an atherogenic diet. Indications that the transgenic mouse model is developing or has developed atherosclerosis include development of fatty streaks and atheromatous plaques at lesion-prone sites in the vasculature, such as the aortic arch.

Assays Using the 3 Het Mouse Model

Prophylactic and/or therapeutic agents can be tested in the 3 Het mouse model to assess their ability to produce phenotypic changes, such as decreasing the amount of LDL, TG, TPC and increasing the amount of HDL expressed in the transgenic mouse. In one embodiment, decreasing LDL, TG, TPC and increasing HDL expression are markers for preventing or ameliorating hypercholesterolemia and/or cardiovascular disease.

In an embodiment, the present invention provides a transgenic mouse model useful in a method for screening for a prophylactic and/or therapeutic agent for preventing or ameliorating hypercholesterolemia and/or cardiovascular disease. The screening method comprises administering a test compound to the transgenic mouse model, or a tissue or cell thereof, and evaluating the effect of the test compound on cardiovascular disease in the transgenic mouse, or tissue or cell thereof, in order to identify a prophylactic and/or therapeutic agent useful for preventing or ameliorating hypercholesterolemia and/or cardiovascular disease.

In another embodiment, the present invention provides a transgenic mouse model useful in a method for testing the efficacy of a prophylactic and/or therapeutic agent for preventing or ameliorating hypercholesterolemia and/or cardiovascular disease. The testing method comprises administering a test compound to the transgenic mouse model, or a tissue or cell thereof, and evaluating the effect of the test compound on hypercholesterolemia and/or cardiovascular disease in the transgenic mouse, or a tissue or cell thereof, in order to test the efficacy of a prophylactic and/or therapeutic agent for preventing or ameliorating hypercholesterolemia and/or cardiovascular disease. In yet another embodiment, the present invention provides a transgenic mouse model useful in a method for comparing the efficacies of prophylactic and/or therapeutic agents for preventing or ameliorating hypercholesterolemia and/or cardiovascular disease. The comparison method comprises: a) administering a first test compound to a first transgenic mouse, or a tissue or cell thereof; b) administering a second test compound to a second transgenic mouse, or a tissue or cell thereof; c) evaluating the effect of the test compounds on hypercholesterolemia and/or cardiovascular disease in the transgenic mice, or tissue or cell thereof; and d) comparing the effects of the test compounds on preventing or ameliorating hypercholesterolemia and/or cardiovascular disease in the transgenic mice. Accordingly, the efficacies of prophylactic and/or therapeutic agents for preventing or ameliorating hypercholesterolemia and/or cardiovascular disease in the transgenic mice are compared.

In a further embodiment, the present invention provides a transgenic mouse model useful in a method for studying molecular and cellular aspects associated with hypercholesterolemia and/or cardiovascular disease. The study method comprises administering a test compound to the transgenic mouse, or a tissue or cell thereof, and evaluating the effect of the test compound on hypercholesterolemia and/or cardiovascular disease in the transgenic mouse, or tissue or cell thereof, in order to study molecular and cellular aspects associated with hypercholesterolemia and/or cardiovascular disease.

In an additional embodiment, the present invention provides a transgenic mouse model useful in a method for identifying a potential therapeutic target useful for treating or preventing hypercholesterolemia and/or cardiovascular disease. The identification method comprises administering a test compound to the transgenic mouse, or a tissue or cell thereof, and evaluating the effect of the test compound on hypercholesterolemia and/or cardiovascular disease in the transgenic mouse, or tissue or cell thereof, in order to identify a potential therapeutic target for treating or preventing hypercholesterolemia and/or cardiovascular disease. Prophylactic and/or therapeutic agents that can be tested in the transgenic mouse model include, but are not limited to, specific diets, exercise regimens, hypocholesterolemia drugs or other as yet to be determined agents. Examples of hypocholesterolemia drugs that decrease the level of hypercholesterolemia and/or cardiovascular disease markers in a subject include, but are not limited to, nicotinic acid, agents that reduce plasma CETP activity, the statins, ezetimibe, fenofibrate or novel agents that increase LDLr activity e.g., via inhibition of a protease involved in LDLr degradation. In one embodiment the agent that reduces plasma CETP activity is an antisense oligonucleotide targeted to CETP. In another embodiment, the inhibitor of the protease involved in LDLr degradation is an antisense oligonucleotide targeted to the protease.

Preventing or ameliorating hypercholesterolemia and/or cardiovascular disease can be monitored by assaying various cardiovascular disease markers such as high cholesterol levels, high triglyceride levels, high LDL levels, low HDL levels and other markers.

Administration of an agent(s) to the transgenic mouse of the invention can be administered by a variety of routes including oral and/or parenteral routes. Examples of parenteral routes of administration include, but is not limited to, topical, intraperitoneal, intravenous (i.v.), and subcutaneous (s.c). In one embodiment, the agent is administered by injection.

Doses of an agent administered to the transgenic mouse of the invention will vary depending on the route of administration. Techniques for formulation and administration can be found in the latest edition of Remington's Pharmaceutical Sciences (Ed. Mack Publishing Co, Easton, Pa. or Lippincott Williams & Wilkins, Philadelphia, PA).

In Vitro Assays Derived from the 3 Het Mouse Model

In addition to using the 3 Het transgenic mouse model directly in the studies described supra, tissues or cells derived from the transgenic mouse can be used. For example, prophylactic and/or therapeutic agents can be assayed in vitro in a variety of tissues or cell types isolated from the transgenic mouse model to determine the efficacy of the agents in preventing or ameliorating cardiovascular disease. In one embodiment, the tissue or cell is derived from the aortic or other artery. In another embodiment, the tissue or cell is derived from the heart. In yet another embodiment, the tissue or cell is derived from the liver.

In general, tissue or cells derived from the 3 Het mouse model are contacted with an agent to be tested. The tissue or cells contacted with the agent are then evaluated to determine if they differ from tissue or cells from the same source which were not contacted with the agent. In one embodiment, hepatocytes contacted with an antisense oligonucleotide targeting CETP is evaluated for the level of CETP mRNA, protein expression or protein activity. In another embodiment, hepatocytes contacted with an antisense oligonucleotide targeting a protease involved in LDLr degradation is evaluated for the level of protease mRNA or protein expression. Techniques for evaluating mRNA or protein levels are well known in the art and can be carried out as described in standard laboratory manuals, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Methods to evaluate mRNA levels include, but are not limited to, Northern blot analysis, competitive polymerase chain reaction (PCR), or RT-PCR. Methods to evaluate protein expression levels include, but are not limited to, immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (for example, CETP activity assays), immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS).

Assaying Combinations of Prophylactic and/or Therapeutic Agents

In certain embodiments, two or more prophylactic and/or therapeutic agents can be administered to the 3 Het mouse, or tissues or cells thereof, in order to determine the efficacy of a combination of agents in preventing and/or ameliorating cardiovascular disease. The agents can administered at the same time or at different times. The agents can be prepared together in a single formulation or prepared separately.

ADVANTAGES OF THE INVENTION

The transgenic mouse described herein exhibits characteristics desirable in a mouse model for human lipoprotein metabolism, hypercholesterolemia and cardiovascular disease. The mouse model exhibits human-like lipoprotein cholesterol metabolism that develops moderate hypercholesterolemia (relative to typical murine models of atherosclerosis) when fed typical murine atherosclerotic diets. Additionally, the model has intact LDLr activity, has not had any genetic alteration in the apoE gene, develops atherosclerosis within four months when fed a typical high fat and high cholesterol murine diet, and responds to common and novel hypocholesterolemic agents in a predictable way. In summary, the 3 Het mouse is a unique model for human lipoprotein metabolism, hypercholesterolemia and cardiovascular disease due to (1) having intact LDLr activity, (2) susceptibility to diet-induced hypercholesterolemia and atherosclerosis using diets not containing cholic acid (3) having unaltered apoE and (4) responding to a broad set of hypocholesterolemic agents in a predictable fashion.

EXAMPLES

Nonlimiting disclosure and incorporation by reference

While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references recited in the present application is incorporated herein by reference in its entirety.

Example 1: Development of the Transgenic 3 Het Mouse Model

Production of the 3 Het Mice A breeding scheme to produce the "3 Het" (or "triple het") mouse model is shown in

Figure IA.

Mice hemizygous for human CETP were obtained from the Jackson Laboratory (JAX ^R strain B6.CB Tg(CETP)5203Tall/J, Stock #003904). The huCETP hemizygous mice were inbred at ISIS to obtain mice homozygous for huCETP (huCETP+/+, mLDLr+/+). The mice were confirmed to have two copies of human CETP by plasma huCETP mass via ELISA (ALPCO Immunoassays) and/or activity (Roar Biomedical, Inc.). Homozygous mice have approximately twice the plasma concentration or activity of huCETP relative to heterozygous mice. Additionally, backcrossing homozygous huCETP mice to wildtype mice produces 100% hemizygous huCETP mice; thus the presence of progeny mice with a complete deficiency of huCETP signified that the parent was not homozygous. Mice homozygous for huApoB with mLDLr deficiency (HuApoB+/+, mLDLr-/-) were generously provided by the laboratory of Larry L. Rudel and maintained at ISIS. These mice were developed at the Rudel lab by crossing LDLr deficient (mLDLr-/-) mice obtained from The Jackson Laboratory (strain B6.129S7-Ldlr ^tmlHer /J; stock #002207) to mice homozygous for huApoB developed in the laboratories of Helen Hobbs and Stephen Young (Linton, M. F., R. V. Farese, Jr., G. Chiesa, D. S. Grass, P. Chin, R. E. Hammer, H. H. Hobbs, and S. G. Young. 1993. Transgenic mice expressing high plasma concentrations of human apolipoprotein BlOO and lipoprotein(a). J. Clin. Invest. 92: 3029-3037; commercially available from Taconic (strain B6.SJL-Tg(APOB)l 102Sgy N20+?, model #001004-T-F or M) and inbreeding the progeny. Homozygosity of huApoB mice was verified by backcrossing homozygous huApoB mice to wildtype mice; any resulting progeny with a complete deficiency of huApoB signified that the parent was not homozygous.

The huCETP+/+, mLDLr+/+ mice were crossed with the huApoB+/+, mLDLr-/- mice to obtain the 3 Het mouse model i.e. mice heterozygous in three genes with a huApoB+/-, huCETP+/-, mLDLr+/- genotype as shown in Figure IA.

Confirmation of the 3 Het Mice

Transgenic mice developed in the breeding program described above were screened to confirm that they carried the desired genotype. As previously described, identifying homozygous huCETP or huApoB transgenic mice was performed by backcrossing prospective homozygous (from the cross of heterozygous mice) transgenic mice to wildtype mice and genotyping the progeny. The presence of progeny mice lacking the transgene signifies that the parent was not homozygous. Additionally, plasma huCETP mass or activity in homozygous huCETP transgenic mice are approximately twice the amount measured in heterozygous huCETP transgenic mice. PCR genotyping of heterogyous huCETP or huApoB transgenic mice was performed with the following PCR primers:

huCETP Forward Primer 5'- GAA TGT CTC AGA GGA CCT CCC -3' [SEQ ID NO:1] huCETP Reverse Primer 5'- CTT GAA CTC GTC TCC CAT CAG -3' [SEQ ID NO:2] huApoB Forward Primer 5 '- GCT TCT GGC TTG CTA ACC TCT CTG -3 ' [SEQ ID NO:3] huApoB Forward Primer 5'- CCT TGT CCT TCC ACT CTT GGT AGG -3' [SEQ ID NO:4] Murine LDLr heterozygosity was confirmed by PCR as described below. The primers had the following sequences:

Primer 1 : 5'- AAT CCA TCT TGT TCA ATG GCC GAT C -3' [SEQ ID NO:5]

Primer 2: 5'- CCA TAT GCA TCC CCA GTC TT -3' [SEQ ID NO:6] Primer 3: 5 ^? - GCG ATG GAT ACA CTC ACT GC -3' [SEQ ID NO:7]

Primers 2 and 3 amplify the mLDLr gene and produce a 167 bp fragment, whereas primers 1 and 2 produce a 350 bp fragment in mice that have the mutant allele (i.e. lack LDLr). Therefore, DNA samples in LDLr -/+ mice produce both a 167 and 350 bp DNA fragments.

The 3 Het Mouse Exhibits a Human-Like Phenotype

The animal models bred to create the 3 Het genotype demonstrate the transformation of a murine lipoprotein cholesterol profile to one resembling a human profile as shown in Figure 2 or 3.

As shown if Figure 2, the plasma lipoprotein cholesterol profile (black) of chow-fed mice was analyzed on a HPLC utilizing size-exclusion chromatography. In gray is a reference lipoprotein profile used to define the VLDL, LDL and HDL peaks. This reference was loaded at the same concentration in each chromatogram to allow for comparisons of profiles. The addition of the huCETP transgene alone (B) or with a partial deficiency of mLDLr (C) results in a decrease in the HDL peak and an increase in the LDL peak relative to the background strain of these mice (A). The addition of the huApoB transgene in animals expressing huCETP and partially deficient in mLDLr (D) results in a further decrease in HDL and increase in LDL. Importantly, the magnitude of the LDL peak now eclipses the HDL peak, a common feature observed in human plasma. All mouse plasma samples were loaded undiluted at the same total volume.

As shown in Figure 3, the addition of the huCETP transgene in huApoB+/-, LDLr+/- mice (compare I and II) or the addition of the huApoB transgene in huCETP+/-, LDLr+/- (compare I and III) mice increases LDL-C and decreases HDL-C in chow-fed mice to levels more typical in humans. Such changes provide a human-like lipoprotein profile such that LDL-C levels are greater than HDL-C levels and the fraction of total cholesterol that is within the HDL fraction is 20 - 40%. Additionally, unlike wildype murine plasma, the 3 Het mice have plasma CETP activity (data not shown) and therefore metabolize HDL and apoB-containing lipoproteins in a more similar fashion to humans than wildtype mice. Importantly, from Figures 2 and 3, the genetic additions of either huCETP or huApoB in LDLr+/- mice are not sufficient in creating a human-like lipoprotein cholesterol profile in mice; only the combination of all three genetic perturbations achieves this profile in chow-fed mice.

As shown in Figure 4, the huApoB transgene is critically important in creating hypercholesterolemia. HuCETP+/-, LDLr+/- fed a typical murine hypercholesterolemic diet has total plasma cholesterol levels that are below 300 mg/dL. Such results underscore the difficulty in generating a model of hypercholesterolemia in mice with intact LDLr activity. Therefore, the addition of the huApoB transgene to such mice (i.e. the 3 Het) allows for a model of hypercholesterolemia with intact LDLr activity. It should be noted that diet-induced hypercholesterolemia can be achieved in mice with intact LDLr activity using hepatotoxic cholic acid-containing diets (G. S. Getz, C. A. Reardon, Arterioscler Thromb Vase Biol 26, 242 (Feb, 2006)).

As shown in Figure 5, 3 het mice fed either TD.94059 or TD.88137 (Harlan Teklad, Madison, WI, USA) for up to 15 weeks exhibited sustained hypercholesterolemia without any increases in ALT. Both diets are commonly used in murine atherosclerosis studies.

Example 2: Antisense Inhibition of a Protease Involved in LDLr Degradation to decrease LDL-C in the 3 Het Mouse Model

Overview: A novel inhibitor of the protease involved in LDLr degradation was tested in the 3 Het mouse model. The compound was evaluated for its ability to reduce LDL-C and total plasma cholesterol.

Material and Methods: 8 week old 3 Het female mice fed TD.88137 (a hypercholesterolemic diet) and administered vehicle or the compound for 3 weeks via twice weekly s.c. injections of 25 mg/kg. Mice were fasted 6 hours and retro-orbital eye bleeds were performed at the initiation and after the dosing period.

Results: Relative to vehicle-treated 3 Het mice, the novel protease inhibitor decreased LDL-C and total plasma cholesterol by 54% and 51%, respectively. All treatments were well tolerated. The LDL-C and total plasma cholesterol lowering effects of this compound are absent in LDLr-/- mice (data not shown). These results demonstrate that functional LDLr activity is critical for the cholesterol-lowering pharmacology of the protease inhibitor and that the 3 Het mouse has functional hepatic LDLr activity subject to modulation by compounds that target hepatic LDLr protein homeostasis.

Plasma concentrations ± SEM

ALT (IU/L) LDL (mg/dL) TPC (mg/dL)

Control 25 ± 3 362 ± 13 468 ± 17

ISIS 394816 19 ± 1 166 ± 11 229 ± 15

Example 3: Inhibition of HuCETP in the 3 Het Mouse Model

Overview: A novel inhibitor of CETP expression was tested in the 3 Het mouse model. The compound was evaluated for its ability to increase HDL-C, decrease LDL-C and increase the fraction of total plasma cholesterol contained with HDL-C.

Material and Methods: 10 week old 3 Het male chow-fed mice were administered vehicle or the novel CETP expression inhibitor 3 weeks via twice weekly s.c. injections of 25 mg/kg. Mice were fasted 6 hours and retro-orbital eye bleeds were performed at the initiation and end of the dosing period.

Results: Relative to vehicle-treated mice, the novel CETP expression inhibitor increased HDL-C by 27%, decreased LDL-C by 41% and increased the ratio of HDL:Total Cholesterol by 61%. AU treatments were well tolerated. As shown in Figure 3, the genetic addition or absence of the huCETP transgene in huApoB+Λ, LDLr+/- mice modulates HDL-C and HDL:Total Cholesterol ratio in a similar fashion to that of the novel CETP expression inhibitor. The HDL-C raising effects of this compound is absent in huApoB+/- huCETP-/- mLDLr +/- mice (data not shown). Accordingly, these data demonstrate that the 3 Het mouse has plasma CETP activity and huCETP is a critical component of the three genetic alterations in the 3 Het mouse model responsible for creating a human-like lipoprotein metabolism.

Example 4: Inhibition of Apolipoprotein C-III (apoC-III) in the 3 Het Mouse Model

Overview: A novel inhibitor of apoC-III expression was tested in the 3 Het. The compound was evaluated for its ability to decrease total plasma cholesterol and triglycerides.

Material and Methods: 8 week old 3 Het female mice were fed a Western diet (TD.88137) and administered vehicle or the novel apoC-III inhibitor for 3 weeks via twice weekly s.c. injections of 25 mg/kg. Mice were fasted 6 hours and retro-orbital eye bleeds were performed after 3 weeks of treatment.

Results: Relative to vehicle- treated mice, the novel apoC-III expression inhibitor decreased LDL-C by 29%, decreased total plasma cholesterol by 32% and decreased plasma triglycerides by 41%. All treatments were well tolerated. These data are comparable to the effects of fenofibrate in humans, which has been shown to also decrease apoC-III expression (National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). Circulation. 2002 Dec 17;106(25):3143-421).

Example 5: HDL-Raising with Dietary Nicotinic Acid Supplementation

Overview: Dietary nicotinic acid (also known as niacin) supplementation was evaluated for HDL-raising pharmacology in the 3 Het mouse model. Prior studies (Melba Hernandez, Samuel D. Wright, Tian-Quan Cai, Critical role of cholesterol ester transfer protein in nicotinic acid- mediated HDL elevation in mice, Biochemical and Biophysical Research Communications, Volume 355, Issue 4, 20 April 2007, Pages 1075-1080, ISSN 0006-291X, DOI: 10.1016/j.bbrc.2007.02.079; Jose W.A. van der Hoorn, Willeke de Haan, Jimmy F.P. Berbee, Louis M. Havekes, J. Wouter Jukema, Patrick CN. Rensen, and Hans M.G. Princen. Niacin Increases HDL by Reducing Hepatic Expression and Plasma Levels of Cholesteryl Ester Transfer Protein in APOE*3Leiden.CETP Mice. Arterioscler. Thromb. Vase. Biol. 2008 28: 2016-2022) have established that huCETP expression is essential for the HDL-raising effects of nicotinic acid in mice. The aim of this study was to verify that this mouse model has a similar response to dietary nicotinic acid supplementation and can therefore model the lipid effects of nicotinic acid observed in humans.

Material and Methods: 10 week old 3 Het male mice were fed a chow diet supplemented with 1.0% nicotinic acid. Mice were fasted 6 hours and retro-orbital eye bleeds were performed at the initiation and after 3 weeks of dietary feeding.

Results: Relative to chow-fed mice, nicotinic acid supplementation increased HDL-C by 6%, decreased LDL-C by 30%, increased the ratio of HDL:Total Cholesterol by 36% and decreased plasma triglycerides by 32%. All treatments were well tolerated. Accordingly, the 3 Het mouse model responds to nicotinic acid in a manner similar to that described in other huCETP transgenic mouse models (see above references) as well as humans (National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). Circulation. 2002 Dec 17;106(25):3143-421).

Example 6: LDL Cholesterol-Lowering with Ezetimibe

Overview: Ezetimibe treatment was evaluated in Western diet-fed mice for plasma cholesterol lowering pharmacology.

Material and Methods: 8 week old 3 Het female mice were fed a Western diet (TD.88137) supplemented with 0.005% ezetimibe (wt/wt). Control mice were fed the Western diet without ezetimibe. Mice were fasted 6 hours and retro-orbital eye bleeds were performed after 3 weeks of treatment.

Results: Relative to control mice, ezetimibe decreased LDL-C by 50% and decreased total plasma cholesterol by 46%. All treatments were well tolerated. Accordingly, the 3 Het mouse model responds to ezetimibe in a manner similar to previous studies in other mouse models of hypercholesterolemia (Delineation of molecular changes in intrahepatic cholesterol metabolism resulting from diminished cholesterol absorption. Repa JJ, Turley SD, Quan G, Dietschy JM. J Lipid Res. 2005 Apr;46(4):779-89; Ezetimibe, a potent cholesterol absorption inhibitor, inhibits the development of atherosclerosis in ApoE knockout mice. Davis HR Jr, Compton DS, Hoos L, Tetzloff G. Arterioscler Thromb Vase Biol. 2001 Dec;21(12):2032-8).

Example 7: LDL Cholesterol-Lowering with Atorvastatin

Overview: Atorvastatin treatment was evaluated in Western diet-fed mice for plasma cholesterol lowering pharmacology.

Material and Methods: 8 week old 3 Het female mice were fed a Western diet (TD.88137) supplemented with 0.01% atorvastatin (wt/wt). Control mice were fed the Western diet without atorvastatin. Mice were fasted 6 hours and retro-orbital eye bleeds were performed after 3 weeks of treatment.

Results: Relative to control mice, atorvastatin decreased LDL-C by 26%, decreased total plasma cholesterol by 24% and decreased plasma triglycerides by 9%. All treatments were well tolerated. Accordingly, the 3 Het mouse model responds to atorvastatin in a manner similar to human subjects (National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). Circulation. 2002 Dec 17;106(25):3143-421).

Additionally, to our knowledge, this is the first demonstration of cholesterol-lowering activity of atorvastatin in a hypercholesterolemic mouse model that has not had its apoE gene modified (S.

Zadelaar, R. Kleemann, L. Verschuren, J. de Vries-Van der Weij, J. van der Hoorn, H. M.

Princen, and T. Kooistra Mouse Models for Atherosclerosis and Pharmaceutical Modifiers.

Arterioscler. Thromb. Vase. Biol., August 1, 2007; 27(8): 1706 - 1721). This underscores the utility of the 3 Het mouse as a model of human lipoprotein metabolism and human hypercholesterolemia that is suitable to test hypocholesterolemic agents. Plasma concentrations ± SEM

ALT (IVfL) LDL (mg/dL) TPC (mg/dL) TG (mg/dL)

Control 30 ± 3 379 ± 9 589 ± 20 87 ± 14

Atorvastatin 26 ± 2 281 ± 17 447 ± 22 79 ± 9

Example 8: LDL Cholesterol-Lowering Treatment Results in a Decrease in Atherosclerotic Lesion Severity

Overview: A novel inhibitor of the protease involved in LDLr degradation (as described in Example 2) was tested in the 3 Het for its ability to reduce aortic atherosclerosis.

Material and Methods: 8 week old 3 Het female mice fed TD.88137 (a hypercholesterolemic diet) and administered vehicle or the compound for 12 weeks via twice weekly s.c. injections of 25 mg/kg. Aortic arch atherosclerosis was assessed by en face oil red O staining of pinned aorta.

Results: Relative to vehicle-treated mice, the novel protease inhibitor decreased aortic arch atherosclerosis by 60% as shown in Figure 6. Western blot analysis of liver tissue demonstrated an increase in LDLr protein expression in mice treated with the inhibitor (data not shown). All treatments were well tolerated. Coupled with the results of Example 2, these data demonstrate that functional LDLr activity is critical for the atherosclerosis-lowering pharmacology of the protease inhibitor and that the 3 Het mouse is a suitable model for screening agents that increase hepatic LDLr activity thereby reducing atherosclerosis.