WO2010080953A1 - Modèle de souris transgénique du métabolisme de la lipoprotéine humaine, de l'hypercholestérolémie et d'une maladie cardiovasculaire - Google Patents

Modèle de souris transgénique du métabolisme de la lipoprotéine humaine, de l'hypercholestérolémie et d'une maladie cardiovasculaire Download PDF

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WO2010080953A1
WO2010080953A1 PCT/US2010/020435 US2010020435W WO2010080953A1 WO 2010080953 A1 WO2010080953 A1 WO 2010080953A1 US 2010020435 W US2010020435 W US 2010020435W WO 2010080953 A1 WO2010080953 A1 WO 2010080953A1
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hypercholesterolemia
cardiovascular disease
transgenic mouse
mice
tissue
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PCT/US2010/020435
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Adam Mullick
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Isis Pharmaceuticals, Inc.
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Publication of WO2010080953A1 publication Critical patent/WO2010080953A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A01K2217/077—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out heterozygous knock out animals displaying phenotype
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0375—Animal model for cardiovascular diseases

Definitions

  • 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.
  • 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.
  • mice 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.
  • 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.
  • 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.
  • NCEP National Cholesterol Education Program
  • 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).
  • VLDL very low density lipoprotein
  • 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.
  • LDLr -/- mice are an unfit model to test such common pharmacologic agents or novel pathways that modulate hepatic LDLr activity.
  • 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.
  • 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+/-).
  • huCETP+/- human cholesteryl ester transfer protein
  • huApoB+/- hemizygous copy of human apolipoprotein B
  • mLDLr+/- murine low-density lipoprotein receptor
  • 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.
  • a normal murine chow diet i.e. a low-fat, cholesterol-free diet.
  • a high fat (HF), high cholesterol (HC) diet the transgenic mouse develops a human-like hypercholesterolemia.
  • 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.
  • 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 e.g., via inhibition of a protease involved in LDLr degradation.
  • FIG. 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+/-).
  • FIG. 1 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.
  • VLDL very low-density lipoprotein
  • LDL low-density lipoprotein
  • HDL high-density lipoprotein
  • mice 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 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.
  • FIG. 1 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.
  • FIG. 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.
  • TD.94059 or TD.88137 as described in Example 1, infra.
  • FIG. 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.
  • 2'-O-methoxyethyl 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 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.
  • Atheromatous plaques 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.
  • 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.
  • 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.
  • 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.
  • a dose can be administered in two or more injections to minimize injection site reaction in an animal.
  • 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.
  • a dosage unit is a vial containing lyophilized antisense oligonucleotide.
  • 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.
  • LDLr+/- we describe the condition of LDLr+/- as heterozygous.
  • 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 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.
  • 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.
  • TPC total plasma cholesterol
  • LDL-C >130 mg/dL.
  • these values are TPC > 240 mg/dL or LDL-C>160 mg/dL (NCEP Adult Treatment Panel III).
  • Mode 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.
  • 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.
  • 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.
  • tissue or cells can be isolated from the mouse model.
  • Modulate means to change a factor.
  • 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 refers to an agent that ameliorates or treats a condition or disease.
  • 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.
  • a desired effect is reduction of a target nucleic acid.
  • a desired effect is reduction of CETP or ApoB mRNA.
  • Target nucleic acid means a nucleic acid capable of being targeted by an antisense compound.
  • 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.
  • huCETP human cholesteryl ester transfer protein
  • huApoB human apolipoprotein B
  • 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.
  • 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.
  • the transgenic mouse model provided herein comprises huCETP+ ⁇ , 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.
  • 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.
  • 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.
  • LDLr is a cell-surface receptor that recognizes apoBl 00 in LDL particles and mediates the endocytosis of LDL.
  • 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.
  • 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).
  • the transgenic mouse model provided herein exhibits a human-like lipoprotein metabolism and profile when fed a normal low-fat, cholesterol free chow diet.
  • 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.
  • the 3 Het mouse develops atherosclerosis after being fed a typical murine atherogenic diet.
  • 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.
  • 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.
  • decreasing LDL, TG, TPC and increasing HDL expression are markers for preventing or ameliorating hypercholesterolemia and/or cardiovascular disease.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the agent that reduces plasma CETP activity is an antisense oligonucleotide targeted to CETP.
  • 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.
  • parenteral routes of administration include, but is not limited to, topical, intraperitoneal, intravenous (i.v.), and subcutaneous (s.c).
  • 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).
  • tissues or cells derived from the transgenic mouse can be used.
  • 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.
  • the tissue or cell is derived from the aortic or other artery.
  • the tissue or cell is derived from the heart.
  • the tissue or cell is derived from the liver.
  • 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.
  • hepatocytes contacted with an antisense oligonucleotide targeting CETP is evaluated for the level of CETP mRNA, protein expression or protein activity.
  • hepatocytes contacted with an antisense oligonucleotide targeting a protease involved in LDLr degradation is evaluated for the level of protease mRNA or protein expression.
  • 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.
  • PCR competitive polymerase chain reaction
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • mice 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.
  • 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.
  • mice 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.
  • Transgenic mice developed in the breeding program described above were screened to confirm that they carried the desired genotype.
  • 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.
  • 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:
  • 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 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.
  • the plasma lipoprotein cholesterol profile (black) of chow-fed mice was analyzed on a HPLC utilizing size-exclusion chromatography.
  • 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 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%.
  • 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.
  • 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.
  • 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.
  • the addition of the huApoB transgene to such mice i.e. the 3 Het
  • 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)).
  • 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
  • mice 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.
  • mice 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).
  • NCEP National Cholesterol Education Program
  • Atorvastatin treatment was evaluated in Western diet-fed mice for plasma cholesterol lowering pharmacology.
  • 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).
  • NCEP National Cholesterol Education Program
  • Example 8 LDL Cholesterol-Lowering Treatment Results in a Decrease in Atherosclerotic Lesion Severity

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Abstract

L'invention porte sur une souris transgénique qui est un modèle du métabolisme de la lipoprotéine humaine, de l'hypercholestérolémie et/ou d'une maladie cardiovasculaire. L'invention porte également sur des procédés d'utilisation du modèle de souris transgénique pour étudier l'hypercholestérolémie et/ou une maladie cardiovasculaire et des traitements prophylactiques et/ou thérapeutiques associés à la prévention contre l'hypercholestérolémie et/ou une maladie cardiovasculaire.
PCT/US2010/020435 2009-01-08 2010-01-08 Modèle de souris transgénique du métabolisme de la lipoprotéine humaine, de l'hypercholestérolémie et d'une maladie cardiovasculaire WO2010080953A1 (fr)

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US9394333B2 (en) 2008-12-02 2016-07-19 Wave Life Sciences Japan Method for the synthesis of phosphorus atom modified nucleic acids
US10329318B2 (en) 2008-12-02 2019-06-25 Wave Life Sciences Ltd. Method for the synthesis of phosphorus atom modified nucleic acids
US10307434B2 (en) 2009-07-06 2019-06-04 Wave Life Sciences Ltd. Nucleic acid prodrugs and methods of use thereof
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