JP2003520780A - Compositions and methods for regulating HDL cholesterol and triglyceride levels - Google Patents

Abstract

The present invention features a method of treating a patient suffering from low HDL, higher than normal triglyceride levels, or cardiovascular disease by administering to the patient a compound that modulates ABC1 expression or activity. .

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION

Low HDL cholesterol (HDL-C), or low alpha lipoproteinosis, is a blood lipid disorder that is associated with high-risk cardiovascular disease (CVD), especially coronary artery disease (CAD), as well as cerebrovascular disease and coronary artery revascularization. Correlates with stenosis and peripheral vascular disease. HDL-C levels are affected by both environmental and genetic factors.

Epidemiological studies have continually suggested that plasma HDL-C levels are inversely correlated with the incidence of CAD. HDL-C levels are a strongly graded and independent cardiovascular risk factor. The protective effect of high HDL-C lasts up to 80 years. Low HDL-C is normal (<
5.2 mmol / l) Even at total plasma cholesterol levels are associated with an increased risk of CAD. The risk of coronary artery disease is 1 mg / dL of HDL-C (0.026 m
For each decrease in mol / l, there was a 2% increase in men and a 3% increase in women, and in the majority of studies this relationship is still statistically significant after adjustment for other lipid and non-lipid risk factors. Reduced HDL-C levels are the most common lipoprotein abnormalities found in patients with early onset CAD. 4% of patients with early onset CAD have the isolated form of reduced HDL-C levels without other lipoprotein abnormalities, while 25% have low HDL levels with concomitant hypertriglyceridemia.

At first glance, other dyslipidemias or secondary factors, HDL-C is also an important predictor of CAD. In a cohort of diabetic patients, isolated low HDL
Those with -C had a 65% increase in mortality compared to diabetic patients with normal HDL-C levels (> 0.9 mmol / l). Moreover, even in high-risk populations such as those with familial hypercholesterolemia, HDL-C levels are an important predictor of CAD. Low HDL-C levels have thus been shown to represent a major and independent risk for CAD.

These findings have raised interest in HDL-C levels as a focus for treatment,
Recommendations for the National Cholesterol Education Program will be made. These guidelines suggest that HDL-C values below 0.9 mmol / l pose a significant risk for men and women. Thus, approximately half of patients with CAD will have low HDL-C. Therefore, it is important to better understand the factors that contribute to this phenotype. Given that drug interventions with low HDL-C levels have so far proved unsatisfactory, an understanding of the factors that regulate these levels in the blood circulation is an understanding of this understanding as a new therapeutic target. Is important to clarify.

Absolute levels of HDL-C do not always predict the risk of CAD. C
In the case of ETP deficiency, individuals show an increased risk of CAD growth despite an increase in HDL-C levels. What is considered important in this case is the functional activity of the reverse cholesterol transport pathway, by which intracellular cholesterol travels from the cell to receptor proteins such as ApoAI or HDL. Another important genetic determinant of HDL-C levels, and its inverse relationship with CAD, may be in the processes leading to HDL formation and the passage and efflux of intercellular cholesterol. To date, this process is poorly understood, but also clearly not all components of this pathway have been identified. Thus, deficiencies that prevent proper HDL-mediated cholesterol efflux would be important predictors of CAD. Therefore, identifying and understanding novel genes involved in intracellular cholesterol trafficking and efflux pathways is extremely important.

[0006] HDL particles are central to the process of reverse cholesterol transport and thus central to the maintenance of tissue cholesterol homeostasis. This process is multi-step: binding of HDL to cell surface components, acquisition of cholesterol by passive absorption, LCAT (reticin cholesterol acyl transferase).
Esterification of this cholesterol by cholesterol and subsequent VLDL of esterified cholesterol by CETP (cholesterol ester transfer protein)
Ultra-low density lipoproteins) and chylomicron remnants for liver uptake. Each of these steps is known to strongly influence the plasma concentration of HDL.

Genetic alterations of ApoAI-CIII, lipoprotein lipase, CETP, hepatic lipase and LCAT all contribute to the determination of human HDL-C levels.
One rare form of genetic HDL deficiency was diagnosed in only about 40 people worldwide,
Also (listed in OMIM as autosomal recessive trait (OMIM205400
)) Tangier disease (TD) associated with almost complete lack of HDL-C levels.
These patients have very low levels of HDL-C and ApoAI, which is due to delayed acquisition of lipids and consequent failure to convert to mature HDL in nascent HDL and Apo.
It is attributed to the deterioration of AI lipid transport and hypercatabolism. TD patients accumulate cholesterol esters in some tissues, and as a result show unique properties such as widespread yellow tonsils, corneal opacities, hepatosplenomegaly, peripheral neuropathy, and cholesterol ester deposition in rectal mucosa. Removal of Cellular Cholesterol and Phospholipid Defects by ApoAI Similarly, a labeling defect in HDL-mediated efflux of intercellular cholesterol was shown in TD fibroblasts. Even if this is a rare disease, determining its molecular basis could identify relevant pathways for cholesterol regulation in the entire population. The reduced availability of free cholesterol efflux at the surface membranes of cells of Tangier patients appears to be due to defects in cellular lipid metabolism or passage. About 45% of patients with Tangier disease show signs of premature CAD, which results in reduced cholesterol efflux, low H
It suggests that there is a strong relationship between DL-C and CAD. Since increased cholesterol is observed in the rectal mucosa of people with TD, the molecular mechanism responsible for TD may also regulate cholesterol adsorption from the gastrointestinal (GJ) tract.

A more common form of genetic HDL deficiency usually has low plasma HDL-C below the 5th percentile of age and sex, but in patients with a deficient clinical presentation specific to Tangier disease. Occurrence (Marcil et al., Arterios Clerosis Thrombosis Vascular Biology, 19: 159-169, 1999; Marcil et al.
Arteriosclerosis Thrombosis Vascular Biology, Vol. 15, 1015-1024, 1995). These patients do not have overt environmental factors associated with this lipid phenotype and also have severe HDL deficiency disease (ApoAI, L
He did not have severe hypertriglyceridemia known to cause CAT, or mutations in LPL deficiency), nor was he diabetic. The mode of inheritance of this abnormality is most consistent with the Mendelian dominant trait (OMIM 10768).

The development of drugs that regulate cholesterol metabolism has so far been slow. Thus, a better understanding of the genetic components of the cholesterol efflux pathway is needed. The newly discovered components can then serve as targets for drug design.

[0010]

[Outline of the Invention]

In a first aspect, the invention features a method of treating a patient diagnosed with a below-normal HDL cholesterol level or above-normal triglyceride level. The method comprises administering to the patient a compound that modulates LXR-mediated transcriptional activity. Desirably, the compound is administered to a patient with a pharmaceutically acceptable carrier. The compound is, for example, 24- (S), 25-epoxycholesterol; 24 (S) -hydroxycholesterol; 22- (R) -hydroxycholesterol; 24 (R), 25.
-Epoxycholesterol; 22 (R) -hydroxy-24 (S), 25-epoxycholesterol; 22 (S) -hydroxy-24 (R), 25 epoxycholesterol; 24- (S), 25-iminocholesterol; methyl- 38-Hydroxy colonnade; N, N-dimethyl-3β-hydroxy coronamide; 24 (R
) -Hydroxycholesterol; 22 (S) -hydroxycholesterol; 22
(R), 24 (S) -dihydroxycholesterol; 25-hydroxycholesterol; 22 (R) -hydroxycholesterol; 22 (S) -hydroxycholesterol; 24 (S) 25-dihydroxycholesterol; 24 (R), 25
-Dihydroxycholesterol; 24,25-dehydroxycholesterol; 2
5-epoxy-22 (R) -hydroxycholesterol; 20 (S) -hydroxycholesterol; (20R, 22R) -cholest-5-ene-3β, 20,
22-triol; 4,4-dimethyl-5-α-cholesta-8,14,24-trien-3-β-ol; 7α-hydroxy-24 (S), 25-epoxycholesterol; 7β-hydroxy-24 ( S), 25-epoxycholesterol;
Selected from the group consisting of 7-oxo-24 (S) -epoxycholesterol; 7α-hydroxycholesterol; 7-oxocholesterol; and desmosterol. In a preferred embodiment, the compound is oxysterol.

In a second aspect, the invention features one method of treating a patient diagnosed with a subnormal HDL cholesterol level or a supranormal triglyceride level. The method comprises administering to the patient a compound that modulates RXR-mediated transcriptional activity. In this regard, RXR modulating compounds are ethylene derivatives / tricyclic retinoids; triene retinoids; benzocycloalkenylalkyls; diene or trienoic acid derivatives, tricyclic aromatic compounds and their derivatives; bicyclylmethylallylic acid derivatives; phenylmethyl heterocycles. Formula compounds; Tetrahydronaphthyl compounds; Allylthio-tetrahydro-naphthalene derivatives and heterocyclic analogs; 2,4-Pentadienoic acid derivatives; Tetralin-based compounds; Nonatetraenoic acid derivatives; SR
11237; dexamethasone; hydroxy, epoxy, and carboxy derivatives of mesoprene; bicyclic benzyl, pyridinyl, thiophene, furanyl, and pyrrole derivatives; benzofuran acrylic acid derivatives; allyl substitution and allyl and (3
-Oxo-1-propenyl) substituted benzopyran, benzothiopyran, 1,2-dihydroquinoline, and 5,6-dihydronaphthalene derivatives; vitamin D3 (1
, 25-dihydroxyvitamin D3) and analogs; 24-hydroxylase inhibitors; mono- or polyenecarboxylic acid derivatives; tetrahydroquinolin-2-one-6 or 7-yl and related derivatives; tetrahydronaphthalene; oximinoalkanoic acid derivatives LG 100268; and LGD 1069;

In a third aspect, the invention provides a nucleic acid molecule comprising (a) an ABC1 regulatory sequence or promoter operably linked to a reporter gene, (b) contacting the nucleic acid molecule with a candidate compound, (C) measuring the expression of the reporter gene,
Particularly directed to a method of determining whether a candidate compound regulates ABC1 expression by performing steps, wherein the mutated reporter gene expression is compared to a control not exposed to the compound, wherein the candidate compound regulates ABC1 expression. Show. In various preferred embodiments, the regulatory region is nucleotides 5854 to 6694, 7756 to 8318, 10479 to 10825, 15214 to 16068, 21636 to 22111, 27898 to 28721, 3295 of SEQ ID NO: 1.
1 to 33743, 36065 to 36847, 39730 to 40577, 4
It contains 50 or more contiguous amino acids selected from 543 to 5287, or 45081 to 55639. In another preferred embodiment, the regulatory region is nucleotides 1-28,707 or 29,011-53 of SEQ ID NO: 1.
, 228 of 50 or more contiguous amino acids. Desirably the regulatory regions are LXRs, RXRs, RORs, SREBPs, and PPARs.
It contains a binding site for a transcription factor selected from the group consisting of:

In a fourth aspect, the invention relates specifically to a method of determining whether a human has an altered risk of showing signs of cardiovascular disease. This method involves testing the human ABC1 gene for polymorphisms or mutations. The presence of polymorphisms or mutations associated with cardiovascular disease indicates that humans have an altered risk of showing signs of cardiovascular disease.

[0014] In a related aspect, the present invention provides that A modifies human response to a drug.
It particularly relates to a method of predicting human response to a drug by determining whether it has a polymorphism in the BC1 gene. The desired polymorphism is shown in FIG. In preferred embodiments of the fifth and sixth aspects, the polymorphism is in the 5'regulatory region of ABC1.

In a sixth aspect, the invention provides the nucleotide sequence AGATCANNNNAGG.
Specifically for the virtually purified LXR response element containing TCA, where each N is independently C, T, G or A (SEQ ID NO: 231). Desirably L
The XR response element has the sequence AGATCACTTTGAGGTCA (SEQ ID NO: 232). More preferably still, the LXR response element consists essentially of the nucleotide sequence AGATCANNNNAGGGTCA, where each N is independently C, T, G or A (SEQ ID NO: 231).

In a seventh aspect, the invention provides at least 50,100,150,300,500 of contiguous nucleotides selected from the following nucleotides of SEQ ID NO: 1:
Of particular reference to a virtually pure nucleic acid molecule consisting essentially of regions that are virtually identical to 750, 1000, 2000, 3000, 4000, 5000 or all, the nucleotides of which are 5854 to 6694, 7756 to 8318, 10479 to 10825. , 15214 to 16068, 21636 to 22111, 2789
8 to 28721, 32951 to 33743, 36065 to 36847, 3
9730 to 40577, 45081 to 55639, 4543 to 5287,
59188 to 60306, 60689 to 63548, 63574 to 651
10,65030 to 68312, 68605 to 73375, 73395 to 74692, 75586 to 77103, 74774 to 74920, 7751
9 to 87679, 87651 to 94160, 96916 to 97634, 9
4408 to 96595, 97807 to 98989, 100369 to 107
171, 107179 to 107983, 108039 to 108998, 10
9222 to 118212, 118612 to 123911, 124586 to 138185, 137773 to 138393, 147497 to 148051
, 158490 to 159118, 123718 to 125077, 13777.
3 to 138912, 139304 to 139699, 139351 to 14
6359, 146867 to 147637, 147733 to 149404, 1
49858 to 152699, 153064 to 153916, 153978 to 158516, 158719 to 160272, 160375 to 16445
8, 165279 to 169814, 164215 to 164592, 1647
86 to 165133, 165125 to 165429, 169882 to 17
0189, 170067 to 174018, 176845 to 178875, 1
79113 to 180606, and 181723 to 183284. In a related aspect, the invention relates specifically to a substantially pure nucleic acid molecule having a region that is virtually identical to the following nucleotides of SEQ ID NO: 1, which nucleotides are 5854 to 6694, 7756 to 8318, 10479 to 108.
25, 15214 to 16068, 21636 to 22111, 27898 to 28721, 32951 to 33743, 36065 to 36847, 3973
0 to 40577, 45081 to 55639, 4543 to 5287, 591
88 to 60306, 60689 to 63548, 63574 to 65110,
65030 to 68312, 68605 to 73375, 73395 to 746
92, 75586 to 77103, 74774 to 74920, 77519 to 87679, 87651 to 94160, 96916 to 97634, 9440
8 to 96595, 97807 to 98989, 100369 to 107171
, 107179 to 107983, 108039 to 108998, 10922
2 to 118212, 118612 to 123911, 124586 to 138
185, 137733 to 138393, 147497 to 148051, 15
8490 to 159118, 123718 to 125077, 137773 to 138912, 139304 to 139699, 139351 to 14635
9, 146867 to 147637, 147733 to 149404, 1498
58 to 152699, 153064 to 153916, 153978 to 15
8516,158719 to 160272,160375 to 164458,1
65279 to 169814, 164215 to 164592, 164786 to 165133, 165125 to 165429, 169882 to 17018
9, 170067 to 174018, 176845 to 178875, 1791
13 to 180606, or 181723 to 183284. Preferred nucleic acid molecules have regions that are virtually identical or exactly identical to nucleotides 1 to 28,707 of SEQ ID NO: 1 or nucleotides 29,011 to 53,228 of SEQ ID NO: 1.

In an eighth aspect, the invention is particularly directed to a method of treating humans with subnormal HDL cholesterol levels, supranormal triglyceride levels, or cardiovascular disease, which comprises the ABC1 polypeptide, or its cholesterol-or Comprising administering to a human a nucleic acid molecule encoding a triglyceride-regulating fragment, or ABC1 polypeptide, or cholesterol, triglyceride modulating fragment thereof. In a preferred embodiment, humans have lower cholesterol or higher triglyceride levels than normal. Desirably, the ABC1 polypeptide is wild-type ABC1 or has a mutation that increases its stability or its biological activity. Desirably, the nucleic acid molecule is operably linked to a promoter and included in an expression vector. Preferred mutations include the R → K mutation at position 219 of ABC1 and the V → A mutation at position 399. The desired biological activity is improved regulation of cholesterol transport.

In a ninth aspect, the invention relates specifically to a method of treating or preventing subnormal HDL cholesterol levels, supranormal triglyceride levels, or cardiovascular disease, which comprises wild type ABC1, R219K ABC1, or V399A A.
Compounds that mimic the activity of BC1 or modulate the biological activity of ABC1 are
(Eg human).

One desirable cardiovascular disease that can be treated using the methods of the invention is coronary artery disease. Others include cerebrovascular disease and peripheral vascular disease.

The finding that the ABC1 gene and protein are associated with cholesterol transport that affects serum HDL levels has been found to increase HDL, reduce triglycerides, or CV.
Allows the use of the ABC1 protein and gene in various diagnostic tests and assays for identification of D inhibitors. In one such assay, A that binds ATP
The domain capacity of the BC1 protein is exploited. Thus, compounds that enhance this binding are potent HDL-increasing or triglyceride-lowering drugs. At the same time, the anion transporting ability and pore forming function of cell membranes can be used for drug screening.

In a tenth aspect, ABC1 expression may further serve as a diagnostic tool for subnormal HDL cholesterol levels, higher than normal triglyceride levels, or CVD. Determining genotyping of the ABC1 gene sequence results in lower than normal HD
Can be used to subtype individuals or families with lower than normal HDL levels or higher than normal triglyceride levels to determine whether an L or higher than normal triglyceride phenotype is associated with ABC1 function . This diagnostic process can lead to tailoring drug treatment based on the patient's genotype, including predicting the patient's response, such as increased or decreased efficiency upon administration of the compound or drug, or undesired side effects.

Antibodies to the ABC1 polypeptide can be used in both therapeutic and diagnostic methods.
Antibodies are produced by immunologically attacking a B cell-containing biological system, eg, an animal such as a mouse, with an ABC1 polypeptide that stimulates the production of anti-ABC1 protein in B cells, and then isolating the antibody from the biological system. . Such an antibody can be used to measure ABC1 polypeptide in a sample by contacting a biological sample such as serum with the antibody and then measuring the immune complex as a measure of ABC1 polypeptide in the sample. Can also be used as a therapeutic agent for modulation of ABC1 biological activity.

Thus, in an eleventh aspect, the invention particularly relates to a purified antibody that specifically binds ABC1. In one preferred embodiment, the antibody modulates cholesterol or triglyceride when administered to a mammal.

[0024] In a twelfth aspect, the invention is particularly directed to a method of determining whether a candidate compound is useful in modulating cholesterol or triglyceride levels, the method comprising: (a) providing an ABC1 polypeptide. , (B) contacting the ABC1 polypeptide with a candidate compound, and (c) measuring the binding of the ABC1 polypeptide, wherein the binding of the ABC1 polypeptide is useful for the candidate compound to regulate cholesterol or triglyceride levels. It indicates that there is.

In the thirteenth example, the invention relates specifically to a method of determining whether a candidate compound is useful in the treatment of subnormal HDL cholesterol levels, supranormal triglyceride levels, or cardiovascular disease. . This method provides (a) an ABC transporter (eg, ABC1), (b) contacting the transporter with a candidate compound, and (
c) comprising measuring the biological activity of the ABC transporter, wherein the increased ABC transporter biological activity as compared to the transporter not contacted with the compound is that the candidate compound has lower than normal cholesterol levels, higher than normal triglyceride levels. , Or have been shown to be useful in the treatment of cardiovascular disease. Desirably, the ABC transporter is in a cell or cell-free assay system.

[0026] In a fourteenth aspect, the invention relates specifically to a method of determining whether a candidate compound is useful in modulating cholesterol or triglyceride levels. This method provides (a) a nucleic acid molecule comprising an ABC transporter promoter operably linked to a reporter gene, (b) contacting the nucleic acid molecule with a candidate compound, and (c) measuring the expression of the reporter gene. , Where increased expression of the reporter gene relative to the nucleic acid molecule not exposed to the compound indicates that the candidate compound is useful in regulating cholesterol or triglyceride levels.

In a fifteenth aspect, the invention particularly relates to non-human mammals having a transgene comprising a nucleic acid molecule encoding a mutant ABC1 polypeptide. In one example, the mutation is a dominant negative mutation, such as the M → T mutation at position 1091 of ABC1.

In a sixteenth aspect, the invention particularly relates to expression vectors, cells, or non-human mammals containing the ABC1 nucleic acid molecules of the invention.

In a related aspect, the invention particularly relates to cells from non-human mammals that carry a transgene containing a nucleic acid molecule of the invention.

In a seventeenth aspect, the invention particularly relates to methods of determining whether a candidate compound reduces inhibition of a dominant negative ABC1 polypeptide. This method is (
a) providing a cell expressing a dominant negative ABC1 polypeptide, (b) contacting the cell with a candidate compound, and (c) measuring the ABC1 biological activity of the cell, wherein the cell is not contacted with the compound An increase in ABC1 biological activity, in comparison, indicates that the candidate compound reduces inhibition of the dominant negative ABC1 polypeptide. A preferred dominant negative ABC1 polypeptide is M1091T ABC
It is 1.

In an eighteenth aspect, the invention particularly relates to a method of determining within a subject a propensity for a disease or disorder selected from the group consisting of subnormal HDL levels, supranormal triglyceride levels, and cardiovascular disease. . This method involves determining the presence or absence of at least one ABC1 polymorphism in a polynucleotide obtained from the ABC1 regulatory region, promoter, or coding sequence, or in the amino acid sequence of the ABC1 protein, within a region obtained from the subject, Here the presence or absence of the ABC1 polymorphism indicates a risk of disease or disorder. Desirably, the method further comprises at least 5 ABCs in the polynucleotide or amino acid sequence.
Including analysis of one polymorphic site.

In a nineteenth aspect, the invention provides that the ABC1 polymorphism has lower than normal HDL levels,
Specifically, it relates to a method of determining whether a subject is at risk for a disease or condition selected from the group consisting of higher than normal triglyceride levels and cardiovascular disease. The method (a) determines whether the prevalence of the disease or disorder in the first subject or set of subjects is different from the prevalence of the disease or disorder in the second subject or set of subjects, and (b) the first Analyzing the polynucleotide sequence of the ABC1 regulatory region, promoter or coding region or the amino acid sequence of the ABC1 protein in a subject or set of subjects and a sample obtained from a second subject or set of subjects, and (c) the first subject or subject Determining whether at least one polymorphism differs between the group of and the second subject or set of subjects, where AB
The presence or absence of the C1 polymorphism correlates with the prevalence of the disease or disorder, which results in A
Including determining whether the BC1 polymorphism is at risk. Desirably, the method further comprises analyzing at least 5 ABC1 polymorphic sites in the ABC1 regulatory region, promoter, or coding sequence or amino acid sequence of ABC1.

In a twentieth aspect, the present invention correlates with a record of a predisposition or prevalence of a disease or disorder selected from the group consisting of subnormal HDL levels, above normal triglyceride levels, and cardiovascular disease. Provide an electronic database with multiple sequence records of typology.

In a twenty-first aspect, the invention relates specifically to a method of selecting a desired therapeutic regimen for modulating ABC1 activity or expression in a subject. The method comprises (a) determining the presence or absence of at least one polymorphism in the polynucleotide sequence of the ABC1 regulatory region, promoter or coding region or the amino acid sequence of the ABC1 protein in a sample obtained from the subject, wherein The presence or absence of the ABC1 polymorphism indicates the safety or efficacy of at least one treatment for modulating ABC1 expression or activity, and (b) the desired treatment for modulating ABC1 expression or activity in a subject. Including determining the law. Preferably this method is AB
Analyzing at least 5 ABC1 polymorphic sites in the polynucleotide sequence of the C1 regulatory region, promoter or coding region or the amino acid sequence of the ABC1 protein.

In a twenty-second aspect, the present invention determines whether a candidate compound is useful in the treatment of a disease or disorder selected from the group consisting of subnormal HDL levels, supranormal triglyceride levels, and cardiovascular disease. Provides a way to make a decision. This method provides (a) an assay system with a measurable ABC1 biological activity, (b) contacting the assay system with a candidate compound, and (c) an ABC1 biological activity or A
Including measuring BC1 phosphorylation. Modulation of ABC1 biological activity or ABC1 phosphorylation in this assay system relative to ABC1 biological activity or ABC1 phosphorylation in a corresponding control assay system not exposed to the candidate compound is that the candidate compound is useful for disease or disorder Is shown. In the preferred embodiment, the assay system is a cell-based system or a cell-free system. Desirably, the candidate compound modulates both ABC1 protein phosphorylation and ABC1 activity.

[0036] In a twenty-third aspect, the invention provides compounds wherein the candidate compound is tested for the ability to ameliorate a disease or condition selected from the group consisting of subnormal HDL levels, supranormal triglyceride levels, and cardiovascular disease. Provide a way to confirm. The method comprises (a) contacting a subject or cell with a candidate compound, and (b) measuring ABC1 expression, activity, or protein phosphorylation in the subject or cell. Altered ABC1 expression, activity, or protein phosphorylation in this subject or cell, as compared to ABC1 expression, activity, or protein phosphorylation in a corresponding control subject or cell that is not contacted with the candidate compound, results in the candidate compound being diseased or abnormal. The ability to improve is identified as the compound tested. Desirably, the candidate compound modulates both ABC1 protein phosphorylation and ABC1 activity.

In a twenty-fourth aspect, is the invention useful for a candidate compound to modulate a disease or disorder selected from the group consisting of subnormal HDL levels, supranormal triglyceride levels, and cardiovascular disease? Provide a way to decide. This method provides (a) a cell expressing the ABC1 gene or a fragment thereof,
b) contacting the cells with a candidate compound and (c) measuring the ABC1 activity of the cells. The altered ABC1 activity in this cell relative to the corresponding ABC1 activity in the control cell that is not contacted with the compound indicates that the candidate compound is useful in modulating a disease or disorder.

[0038] In a twenty-fifth aspect, is the present invention useful for candidate compounds to modulate a disease or condition selected from the group consisting of subnormal HDL levels, above normal triglyceride levels, and cardiovascular disease? Provide a way to decide. This method involves (a) contacting cells expressing the ABC1 protein with a candidate compound and (b) measuring phosphorylation of the ABC1 protein. It is shown that altered ABC1 protein phosphorylation in this control cell as compared to ABC1 protein phosphorylation in the corresponding control cells not contacted with the candidate compound is useful in regulating the disease or disorder.

In a twenty sixth aspect, the invention provides compounds useful in the treatment of a disease or disorder selected from the group consisting of subnormal HDL levels, supranormal triglyceride levels, and cardiovascular disease. This compound modulates ABC1 biological activity and is also identified by (a) providing an assay system with measurable biological activity, (b) contacting the assay system with the compound, and (c) measuring ABC1 biological activity. ABC1 in the corresponding control assay system, which was exposed to no compound
Modulation of ABC1 biological activity relative to biological activity indicates that the compound is useful in treating a disease or disorder.

In a twenty-seventh aspect, the invention provides compounds useful in the treatment of a disease or disorder selected from the group consisting of subnormal HDL levels, supranormal triglyceride levels, and cardiovascular disease. This compound induces a change in ABC1 biological activity that mimics the change in ABC1 biological activity induced by the R219K ABC1 mutation.

In a twenty-eighth aspect, the invention provides compounds useful in the treatment of a disease or disorder selected from the group consisting of subnormal HDL levels, supranormal triglyceride levels, and cardiovascular disease. This compound binds or interacts with residue R219 of ABC1, thereby mimicking a mutation in ABC1 activity induced by the R219K ABC1 mutation.

In a twenty-ninth aspect, the invention provides compounds useful in the treatment of a disease or disorder selected from the group consisting of subnormal HDL levels, supranormal triglyceride levels, and cardiovascular disease. This compound induces a change in ABC1 biological activity that mimics a change in ABC1 biological activity induced by the V339A ABC1 mutation.

In a thirtieth aspect, the present invention provides compounds useful in the treatment of a disease or disorder selected from the group consisting of subnormal HDL levels, supranormal triglyceride levels, and cardiovascular disease. This compound binds or interacts with residue V399 of ABC1 and thereby mimics the change in ABC1 activity induced by the V399A ABC1 mutation.

In a thirty-first aspect, the invention provides compounds that modulate ABC1 activity and bind or interact with amino acids of ABC1, wherein the amino acids are amino acids 119-319 of ABC1 (SEQ ID NO: 5) or It is a residue selected from amino acids 299 to 499 of ABC1 (SEQ ID NO: 5).

In a thirty second aspect, the present invention determines whether a candidate compound is useful in the treatment of a disease or disorder selected from the group consisting of subnormal HDL levels, supranormal triglyceride levels, and cardiovascular disease. Provides a way to make a decision. This method involves (a) providing an assay system having a measurable LXR biological activity, (b) contacting the assay system with a candidate compound, and (c) measuring the LXR biological activity, wherein Modulation of LXR biological activity relative to LXR biological activity in the corresponding untouched control assay system indicates that the candidate compound is useful in treating a disease or disorder.

In a thirty-third aspect, the invention provides a method of determining whether a candidate compound is useful in modulating ABC1 biological activity. This method involves (a) providing an assay system having measurable LXR biological activity, (b) contacting the assay system with a candidate compound, and (c) measuring the LXR biological activity, wherein the candidate compound is LX compared to LXR bioactivity in the corresponding control assay system not contacted with LX
Modulation of R biological activity indicates that the candidate compound is useful in modulating ABC1 biological activity. Desirably, the LXR biological activity is modulation of ABC1 expression.

In a thirty-fourth aspect, the invention provides methods for identifying compounds that test for their ability to modulate ABC1 biological activity. This method involves contacting (a) a subject or cell with a candidate compound, and (b) assaying the activity of the LXR gene product in the subject or cell, wherein a corresponding control subject or cell not contacted with the candidate compound. Modulation of activity relative to that of A. coli is identified as a compound that tests the ability of a candidate compound to modulate the biological activity of ABC1.

[0048] In a thirty-fifth aspect, the invention provides a LXR in an assay that identifies compounds useful in the treatment of a disease or condition selected from the group consisting of subnormal HDL levels, supranormal triglyceride levels, and cardiovascular disease. Provide use of the gene product.

In the thirty sixth aspect, the invention provides the activity or expression of the LXR gene product for the treatment of a disease or disorder selected from the group consisting of subnormal HDL levels, supranormal triglyceride levels, and cardiovascular disease. It particularly relates to the use of compounds that modulate

In the thirty-seventh aspect, the invention identifies compounds that are tested for their ability to treat a disease or condition selected from the group consisting of subnormal HDL levels, above normal triglyceride levels, and cardiovascular disease. Provide a way. This method involves (a) providing an assay system having measurable LXR biological activity, (b) contacting the assay system with a candidate compound, and (c) measuring LXR biological activity.
Here, modulation of LXR biological activity, as compared to LXR biological activity in a corresponding control assay system that is not contacted with the candidate compound, confirms the ability of the candidate compound to treat a disease or disorder as the compound being tested.

In a thirty eighth aspect, the present invention screens candidate LXR agonists for their ability to treat a disease or condition selected from the group consisting of subnormal HDL levels, supranormal triglyceride levels, and cardiovascular disease. Provide a way to do. This method comprises: (a) contacting the assay system with a candidate LXR agonist,
(B) Accompanying the measurement of the cholesterol efflux activity of cells, in which the candidate L
Cholesterol efflux activity in cells, as compared to cholesterol efflux in corresponding control cells not exposed to the XR agonist, indicates that the candidate LXR agonist is useful in treating a disease or disorder.

[0052] In a thirty-ninth aspect, the invention screens candidate LXR modulating compounds for the ability to treat a disease or condition selected from the group consisting of subnormal HDL levels, supranormal triglyceride levels, and cardiovascular disease. Provide a way. This method involves contacting (a) cells with a candidate LXR modulating compound, and (b)
) Involves measuring the ABC1 activity of the cells, where the LXR modulating compound is not contacted with, and the increase of the ABC1 biological activity in the cell is compared to the ABC1 biological activity in the corresponding control cell where the LXR modulating compound causes the disease or disorder. It is shown to be useful in treating.

In one aspect, the invention provides methods for determining whether a candidate compound is useful in modulating triglyceride levels. This method provides (a) a cell comprising an ABC1 polypeptide comprising amino acids 1-60 of SEQ ID NO: 5,
(B) contacting the cell with a candidate compound and (c) measuring the half-life of the ABC1 polypeptide, wherein the half-life of said half-life is compared to that of the corresponding control cell not exposed to the compound. The increase indicates that the candidate compound is useful in regulating triglyceride levels.

In a related aspect, the invention relates specifically to a method of determining whether a candidate compound mimics ABC1 biological activity. This method provides (a) a cell that does not express an ABC1 polypeptide, (b) contacting the cell with a candidate compound, and (c)
Altering ABC1 bioactivity indicates that the candidate compound modulates ABC1 bioactivity, as compared to the corresponding control cells not contacted with the compound, which involves measuring the ABC1 bioactivity of the cells. Desirably, the cells carry the ABC1 null mutation. In one preferred embodiment, the cells are in a mouse or chicken (eg, WHAM chicken) whose ABC1 gene is mutated.

In a preferred embodiment of the screening method of the invention, the cells are in an animal. The desired biological activity is the transport of cholesterol (eg HDL cholesterol or LDL cholesterol) or interleukin-1, or the binding or hydrolysis of ATP by the ABC1 polypeptide. Desirably, the ABC1 polypeptide used in the screening method comprises amino acids 1-60 of SEQ ID NO: 5. Optionally, the ABC1 polypeptide can include a region encoded by a nucleotide sequence that hybridizes under high stringency conditions to nucleotides 75-254 of SEQ ID NO: 6. Desirably the subject is a human. Desirably the cell or assay system is SEQ ID NO: 94, SEQ ID NO: 9.
It has an exogenous feed replication of LXRE selected from the group consisting of 2 and an LXRE consensus motif at nucleotide 7670 at the 3'end of intron 1. For the various methods of the invention, the desired LXR bioactivity is modulation of ABC1 expression. A preferred LXR gene product is an ABC1 nucleic acid molecule or protein.

Additional sequences for the ABC1 regulatory region were labeled with 4I8,31 using the method described herein.
J20, 47O19, or 179G21 Research Genetics RPCI-
It must also be considered that it is determined by sequencing the rest of the 11 BACs. At least 50,100,150,300,50 of these areas
A substantially pure nucleic acid containing a region of substantially identical 0,750,1000,2000,3000,4000,5000 contiguous nucleotides is used in the methods of the invention.

By “polypeptide” is meant any chain of two or more amino acids despite post-translational modifications such as glycation or phosphorylation.

By “reporter gene” is meant any gene that encodes a product whose expression is detectable and / or quantifiable by physical, immunological, chemical, biochemical, or biological assays. To do. Reporter gene products may include, for example: It can have one of the attributes (eg ricin A), or the ability to be specifically bound by a second molecule (eg biotin or a detectably labeled antibody) without restriction. Genetically engineered variants of any of the reporter genes that are well available to those of skill in the art are also included without limitation in the above definitions.

By “operably linked” is meant that the gene and regulatory sequences are linked to allow expression of the gene product under the control of the regulatory sequences. A promoter also means that expression of the gene product is operably linked to the gene under the control of the promoter.

By “regulatory region” is meant a region capable of regulating the expression of a gene from a promoter when genetically linked to a promoter and a gene (eg, a reporter gene). Regulatory sequences include nuclear hormone transcription factor binding sites such as those found in the intron sequences described herein.

By “promoter” is meant a minimal sequence sufficient to direct the transcription of a genetically engineered binding gene.

By “substantially identical” is meant a polypeptide or nucleic acid that exhibits at least 50%, desirably 85%, more desirably 90%, and most desirably 95% identity to a reference amino acid or nucleic acid sequence. . For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably 25 amino acids, and most preferably 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides,
Desirably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably at least 110 nucleotides. One sequence contains 20% or less of additions or deletions (ie gaps) when compared to the second sequence. The optimal alignment of sequences is, for example, Gish and States (Nature Genetics, 3: 266-279: 1993), Altsal et al. (Journal of Molecular Biology, 215: 403-410, (1990). , Madden et al. (Methhemoglobin Enzymology, 266: 131-141, 1996) Altsal et al. (Nucleic Acid Research, 25: 3389-3402, 1997), or Zan et al. (Genome Research, 7 volumes). : 649-656, 1997).

Sequence identity is typically determined by sequence analysis software with the default parameters specified therein (eg, Sequence Analysis Software Package of Genetics Computer Group, 53705 Wisconsin, Madison, University Avenue 1710, University. Of Wisconsin Biotechnology Center). This software program matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, and phenylalanine, tyrosine.

By “substantially pure nucleic acid” is meant a nucleic acid that lacks the genes that flank the nucleic acid in the naturally occurring genome of the organism from which it is derived. The term thus includes, for example, a vector, an autonomously replicating plasmid or virus, integrated into a genomic nucleic acid of prokaryotic or eukaryotic cells, or another molecule independent of other sequences (eg, PC).
R or a recombinant nucleic acid present as a cDNA or genomic or cDNA fragment produced by restriction endonuclease digestion. It further includes a recombinant nucleic acid which is part of a hybrid gene encoding additional polypeptide sequence.

By “high stringency conditions” is meant hybridization at 2 × SSC, 40 ° C. with a DNA probe length of at least 40 nucleotides. For other definitions of high stringency conditions, see F. F., incorporated herein by reference. See Ausbel et al., Current Protocols in Molecular Biology, pages 6.3.1-6.3.6, John Wiley and Sons, New York, New York, 1994.

“Modulate” means increase or decrease. Desirably, LXR mediated transcription,
Compounds that modulate RXR-mediated transcription, ABC1 gene expression, HDL-C levels, or triglyceride levels are at least 5%, more desirably at least 10%,
Also most desirably, adjust by at least 25%, or even at least 50%.

By “purified antibody” is meant an antibody that lacks at least 60% protein by weight and naturally occurring organic molecules with which the protein is naturally associated. Desirably, the preparation is at least 75% by weight, more desirably 90%, and most desirably at least 99% by weight antibody. Purified antibodies can be obtained, for example, by affinity chromatography using recombinantly produced proteins or conserved motif peptides and standard techniques.

“Specifically binds” means, for example, that it recognizes and binds human ABC1 polypeptide, but does not effectively recognize other non-ABC1 molecules, eg, in a sample, eg, a biological sample that naturally contains proteins. An antibody that neither binds nor binds. The preferred antibody binds to the ABC1 polypeptide sequence of Figure 2A (SEQ ID NO: 5).

“Polymorphism” means that a nucleotide or nucleotide region is characterized as occurring in several different forms. "Sudden alteration" is a polymorphic form in which the expression level, stability, function or biological activity of the encoded protein is effectively altered.

“LXR” means the desired LXRs meaning the nuclear receptors LXRα and LXRβ.
Is human LXRα (GenBank, accession number Q13133) and human LXRβ (
Genbank, Access No. P55055) (Apfer et al., Molecular Cell Biology, 14: 7025-7035, 1994: Wiley et al., Gene Incidence, 9: 1033-1045, 1995: and Song See also National Academy of Sciences Bulletin, 91: 10809-10813, 1995. Each of these is incorporated herein by reference.

“RXR” means the nuclear receptors RXRα, RXRβ and RXRγ.
Desirable RXRs is human RXRα (GenBank, access number Q13133)
Human RXRβ (GenBank, access number S37781), and human RXR
γ (GenBank access number Q13133) is included.

By “ABC transporter” or “ABC polypeptide” is meant any transporter that hydrolyzes ATP and transports a substance across a membrane. Desirably, the ABC transporter polypeptide comprises an ATB binding cassette and a transmembrane region. Examples of ABC transporters include, but are not necessarily limited to, ABC1, ABC2, ABCR and ABC8.
including.

“ABC1 polypeptide” means ABC having the amino acid sequence of SEQ ID NO: 5.
By a polypeptide is meant a polypeptide that is virtually identical to one polypeptide.

“ABC biological activity” or “ABC1 biological activity” means hydrolysis or binding of ATP, transport of a compound (eg cholesterol, interleukin 1) or ion across a membrane, or cholesterol or phospholipid levels (eg. HD
(Either by increasing or decreasing L-cholesterol or LDL-cholesterol levels).

The present invention provides a method of treating a patient with low HDL-C and or higher than normal triglyceride levels by administering a compound that modulates ABC1 biological activity or expression. For example, the compounds modulate the transcriptional activity of LXR / RXR heterodimers. Many compounds that modulate LXR transcription activity or RXR transcription activity are known in the prior art. The preferred compound of the present invention is osixosterol.
Additional compounds are described herein.

The present invention provides screening procedures to identify therapeutic compounds (cholesterol modulators, triglyceride modulators, or anti-CVD drugs) that can be used in human patients. Compounds that modulate ABC1 biological activity or expression are considered useful in the present invention as compounds that modulate ABC concentration, protein stability, controlled catabolism, or the ability to bind other proteins or factors. Generally, the screening methods of the invention involve screening a number of compounds as therapeutically active agents by employing any number of in vitro or in vivo experimental systems. Representative methods useful for identifying such compounds are described below.

The method of the present invention simplifies the evaluation, identification and development of low HDL, higher than normal triglyceride levels, and active agents for the treatment and prevention of CVD. In general, screening methods provide a convenient means for selecting natural product extracts or compounds of interest from a large population that are further evaluated and enriched for some activity and selection material. The components of this pool are then their HDL elevation, triglyceride reduction,
Purified and evaluated with the methods of the invention to determine anti-CVD activity, or a combination of these.

Other characteristics and advantages of the invention will be apparent from the following specification of the preferred embodiments below.

We have previously discovered that the human ABC1 (also known as ABCA1) genomic region contains consensus binding sites for transcription factors such as LXRs, RXRs, PPARs, SREBPs and RORs. In the present invention, we also report the sequence of the ABC1 regulatory region containing the consensus binding site for transcription factors. We also found that heterozygotes for the ABC1 mutation had a risk of age-related loss of HDL, increased triglyceride levels, and significantly increased CAD. Furthermore, this phenotype is highly correlated with efflux, clearly indicating that impairment of reverse cholesterol transport is associated with decreased plasma HDL cholesterol, increased triglyceride levels, and increased atherogenesis. Therefore, the present invention is AB
Of particular interest are screening methods that identify therapeutics that increase C1 function, resulting in increased plasma HDL cholesterol, decreased triglyceride levels, protection against atherogenesis, or a combination of these effects.

Genes play an important role in affecting HDL levels. Tangier disease (TD) was the first reported genetic HDL deficiency. Until recently, the molecular principal component of TD was unknown, but now a mutation of ABC1 (as described below) has been confirmed in TD patients. For example, we identified two additional probands and their families, confirmed linkage,
The loci for a limited genomic region were refined. Mutations in the ABC1 gene that account for all four alleles of these two families were detected. More causes of low HDL levels are different diseases, familial HDL deficiency (FHA)
Is. On the basis of independent linkage, meiotic recombinants and haplotype-related diseases, FHA has been localized to a small genomic region surrounding the ABC1 gene. Mutations within conserved residues were separated by FHA. Antisense meiosis of ABC1 transcripts in fibroblasts was associated with a marked decrease in cholesterol efflux.

Cholesterol is normally assembled and secreted by intracellular lipids, but in TD this process is reversed and cholesterol is broken down into lysosomes. Intracellular cholesterol ester accumulation is associated with lysosomal morphological changes and Golgi apparatus and intracellular cholesterol ester accumulation associated with cholesterol ester accumulation in histiocytes, schwann cells, smooth muscle cells, mast cells and fibroblasts. Will increase.

The clinical and biochemical heterogeneity of patients with TD leads to the possibility that genetic heterogeneity also underlies this disease. With this in mind, we first set out different ancestors (TD-
1 is Dutch, TD-2 is British; Florich et al., Clin. Inve
st. Med. 10; 377-382, 1987), performed linkage analysis of these two families and found that the genetic mutations underlying TD in these families were the same 9q.
It was confirmed that a large family with TD localized in 31 regions was identified (Last et al., Nature Genetics, 20, 96-98,
1998). Detailed haplotype analysis revealed the localization of this gene with the construction of a physical map. A mutation in the ABC1 gene was found in TD.

FHA is generally more common than TD, although its exact frequency is unknown.
While TD has been described to date in only 40 families, we have identified 40 FHA families in the Netherlands and Quebec alone. After the first suggestion of the chain to 9q31,
Thirteen polymorphic markers spanning approximately 10 cM in this region were categorized with the highest LOD score at D9S277. Analysis of the marker homozygosity in the TD-2 proband, which was expected to be homozygous for markers close to TD because of their parents' relatives, placed the TD gene at a gene distal to D95127. It was Combined genetic data from the TD and FHA families was directed to the same genomic segment spanning approximately 1,000 kilobases between D9S127 and D9S1866. The ABC1 transporter gene was contained within the minimal genomic region. RT-PCR analysis of one family showed a deletion of leucine at residue 693 (Δ693) in the first transmembrane region of ABC1, which was segregated with an HDL deletion phenotype in this family.

ABC1 is part of the ATP-binding cassette (ABC transporter) superfamily, which involves energy-dependent transport of a wide variety of substrates across the membrane (Dean et al., Curr.
． Opin. Gen. Dev. 5: 779-785, 1995). These proteins have unique motifs that are conserved throughout evolution that distinguish this class of proteins from other ATP-binding proteins. In humans, these genes basically encode two ATP binding segments and two transmembrane domains (Dean et al., Curr. Opin. Gen. Dev. 5, 779-785, 1995). We will now show that the ABC1 transporter is important for intracellular cholesterol transport.

We showed that meiosis of the ABC1 transcript using the oligonucleotide antisense approach reduced efflux, clearly showing a link between the gene and alterations in its functional function. TD and FHA are now on the growing list of genetic disorders due to a deficiency in the ABC group of the protein, which is cystic fibrosis (Zielensky et al., Ann. Rev. Genet., 29, 777-807). , 1995), adrenoleukodystrophy (Mosser et al., Nature, 361).
Vol. 726-730, 1993). Zellweger Syndrome (Gertner et al. Nat. Genet. Vol. 1, 23, 1992), progressive familial intrahepatic cholestasis (Bull et al. Nat. Genet. Vol. 18, pp. 219-224, 199).
8 years), and different eye disorders including Stargardt's disease, (Arikmets et al.,
Nat. Genet. 15, 236-246, 1997), autosomal retinitis pigmentosa retinitis (Arikmets et al., Science, 277, 1805-18).
07, 1997) and cone dystrophy (Kremers et al., H.
um. Mol. Genet. 7, 355-362, 1998).

Patients with TD have Tangier disease as an autosomal degenerative disease (OMIM 20540)
In contrast, FHA has been distinguished from patients with FHA by being inherited as an autosomal dominant trait (OMIM 10768). Furthermore, patients with TD have clear evidence of intracellular cholesterol accumulation that is not seen in FHA patients. It is clear that TD heterozygotes strongly reduce HDL levels and that the same mechanism underlies the HDL deficiency and cholesterol efflux deficits found in FHA heterozygotes as well as TD. Moreover, the more severe phenotype in TD represents loss of function from both alleles of the ABC1 gene.

ABC1 is activated by protein kinases, possibly via phosphorylation, which also provides one explanation for the fundamental role of protein kinase C in promoting cholesterol efflux (Drovnik et al., Arterioscle.
r. Thromb. Vasc. Biol. Volume 15, pp. 1369-1377,
1995). Brefeldin, which blocks the passage between the endoplasmic reticulum and the Golgi, is probably
Through the inhibition of BC1 biological activity, it essentially recreates the effects of mutations within ABC1 and significantly inhibits cholesterol efflux. This finding is important for understanding the mechanisms leading to premature atherosclerosis. TD homozygotes develop immature coronary artery disease as seen in probands of TD-I (III-01) with 38 years of signs of coronary artery disease. It is worth noting that, in addition to presenting significantly reduced HDL, TD patients also have low LDL cholesterol and yet nevertheless develop atherosclerosis. This highlights the importance of HDL intracellular trafficking as an important mechanism of atherogenesis. There is also significant evidence that heterozygotes for TD further increase the risk of premature vascular disease (Schafer et al., Ann. Int. Med. 93, 261-266, 1980; Serfati-Lacrossnier et al., Atherome. Curing, vol. 107, pages 85-98,
1994). In addition, there is preliminary evidence of immature atherosclerosis in some probands with FHA. For example, a proband of FHA-2 (III-01) underwent coronary artery bypass grafting at age 46, while a proband of FHA-3 showed evidence of CAD around age 50. TD-1 probands had more severe outflow defects than TD-2 probands. Interestingly, the TD-2 proband showed no signs of CAD when he died of unrelated causes at age 62, with a degree of cholesterol efflux (partially mediated by the nature of the mutation). It provided preliminary evidence for a relationship between the likelihood of atherosclerosis.

The ABC1 gene plays a crucial role in cholesterol transport, especially intracellular cholesterol transport in monocytes and fibroblasts. It also plays an important role in other tissues such as the nervous system, gastrointestinal tract, cornea. Completely defective intracellular cholesterol transport results in peripheral neuropathy, corneal opacification, and cholesterol ester attachment to the rectal mucosa.

HDL deficiency is heterogeneous in nature. A brief description of the genetic basis of TD and FHA raises importance of this special pathway in intracellular cholesterol and transport, and its role in the pathogenesis of atherosclerosis. Elucidation of the molecular basis of TD and FHA could define a major step in the poorly defined pathway of cholesterol efflux from cells and could lead to a new approach to the treatment of patients with HDL deficiency in the total population. It was

HDL has been implicated in many other biological processes. It includes, but is not necessarily limited to, prevention of lipoprotein oxidation, absorption of endotoxin, protection from Bruce trypanosoma infection, regulation of endothelial cells, prevention of platelet aggregation (Genest et al., J. Invest. Med. 47: Pages 31-42, 1999
The year, here incorporated by reference. Any compound that modulates HDL levels is useful in modulating one or more of the above processes. The present discovery that ABC1 functions to regulate HDL binds ABC1 to the process for the first time.

By identifying the ABC1 protein as a major initiator of the efflux pathway, it has become possible to directly investigate the relationship between efflux, HDL, triglyceride levels, and CAD. We have characterized the phenotype of heterozygotes for some mutations in the ABC1 gene in a large cohort when the diagnosis is made by mutation identification. In addition, the heterozygous phenotype was compared to the unaffected family member phenotype, allowing results to be limited, at least in part, to other genetic and environmental influences. To the contrary, previous studies in true heterozygotes were limited to a small number, often with single-family zygotes, thus limiting their ability to analyze phenotypic expression in multiple mutations over age. It was

A cohort of 77 individuals heterozygous for multiple mutations in the ABC1 gene was identified, allowing characterization of 13 ABC1 mutations in 11 families (5TD
, 6FHA). ABC1 heterozygotes show about a 50% reduction in HDL cholesterol and apoAI, and to a lesser extent, apoAII. In addition, ABC1 heterozygotes have increased triglycerides, but almost no significant changes in LDL cholesterol as opposed to TD patients. HDL, apoA
Changes in I, and triglycerides are gene dose dependent, suggesting that they are directly associated with ABC1 function. Furthermore, heterozygotes carry an increased risk of more than 3-fold in mean age of onset with progressive CAD compared to unaffected individuals. Furthermore, CA is the heterozygote with the most severe defect in the outflow.
The frequency and incidence of D was higher. Interestingly, the incidence of phenotypes observed in heterozygotes appeared to be mutation-dependent, but there was no apparent relationship between mutation site and phenotype. Carriers of severe mutations that produce truncated or null alleles tended to be lower HDL than carriers of missense mutations. One notable exception is the M1091T missense mutation, which has the most severe phenotype that significantly reduces HDL cholesterol and efflux in affected family members, which acts as a dominant negative mutant. It suggests down-regulating the function of type alleles. Another interesting finding is a small cluster of mutations in the very C-terminal region of the protein, indicating that this region is important for ABC1 function.

Severe HDL deficiency in ABC1 heterozygotes results in residual cholesterol efflux
It is suggested to be a major determinant of DL cholesterol levels. Here we have 8% each in comparative efflux between cholesterol efflux and HDL cholesterol levels
Is expected to be associated with an increase of 0.1 mmol / L in HDL cholesterol levels. For example, in order to carry out a 30% increase in HDL cholesterol in a 40 year old man, a 50% increase in ABC1-mediated cholesterol efflux would be required. Although these numbers do not extrapolate directly to those observed in the general population in which other genetic and environmental factors are unregulated, nonetheless, these data show that relative to ABC1 function. It is suggested that small changes have a significant effect on plasma HDL cholesterol levels. Furthermore, the data presented here demonstrate that alterations in efflux due to alterations in ABC1 function directly affect plasma HDL cholesterol levels as well as triglyceride levels and CAD susceptibility, thus demonstrating the reverse cholesterol transport hypothesis and HDL cholesterol. To provide a demonstration of ABC1 as a therapeutic target that raises and lowers triglyceride levels and protects against atherogenesis.

The ABC1 heterozygous phenotype is further age-regulated. From a 20 year old member of the control cohort, there is a definite increase in HDL with aging, which is very low, but apparently not present in heterozygotes. One explanation for this finding is
There is usually an increase in ABC1 function associated with age, which is absent in heterozygotes but is probably a residual function-executing allele that is uniquely maximally upregulated in the increase in intercellular cholesterol. It will be because. ABC1 in heterozygotes
The lack of an age-related increase in function may be to exaggerate the difference in HDL levels between heterozygotes and control individuals in the aged group. There is some evidence for increased age-related regulation of ABC transporter expression (Gupta, Drug Aging, 7: 19-2.
0 page, 1995). Further evidence for potential increased age control in ABC1 function is:
ApoA found in the pre-β subfraction of HDL, an excellent cholesterol receptor
It follows from the observation that the rate of I decreases with age, suggesting an increase in the formation of mature α-migrating HDL with age.

In a degenerate growth assessment stainin study, it was discovered that carriers of the R219K ABC1 mutation had significantly lower triglyceride levels that were individualized without this mutation. This result binds near Arg219 in wild-type ABC1 or R2
Compounds that mimic the function provided by Lys219 in the 19K ABC1 variant reduce triglyceride levels and thus reduce the risk of CAD. In addition, carriers of the V399A ABC1 variant had higher HDL levels and fewer coronary events than individuals without this variant. Thus binding near Va1399 in wild type ABC1, or V339A ABC1
Compounds that mimic the function provided by A1a399 in variants increase cholesterol levels and reduce the risk of CAD. R219K or V3 in individual body
Determining the presence or absence of a 99A ABC1 variant is useful in selecting a treatment regimen for these subjects, such as HDL lowering, triglyceride raising, or anti-CAD.

The following examples illustrate the invention. It is not meant to limit the invention in any way.

[0097]

BEST MODE FOR CARRYING OUT THE INVENTION

Analysis of the TD Family Cholesterol Efflux Studies Both probands had a significant defect in cholesterol efflux similar to that previously shown in TD patients. TD-1 is of Dutch descent, while TD
-2 is of British descent.

Linkage Analysis and Demonstration of Physical Maps Multiple DNA markers were genotyped in the 9q31 region where linkage to TD was described (Last et al., Nat. Genet. 20, 96-98 (1998). )
. Two-point linkage analysis gave the greatest peak with a LOD score of 6.49 for D9S1832 with significant evidence of linkage for all labels at intervals up to 10 cM. Recombination with the closest marker D9S1690 was found in II-09 of the TD-1 family, providing a centromere boundary for the disease gene. Multipoint linkage analysis of these data did not enhance the accuracy of localization of the disease trait loci.

A physical map spanning approximately 10 cM in this region was established with expression of the YAC contig. In addition, 22 other polymorphic polyallelic markers spanning this special region were mapped to contigs, and these subsets were used to construct haplotypes (allotypes) for further analysis.

Although the Dutch family did not show any consanguinity, the proband of TD-2 was a descendant of a consanguineous marriage. We therefore hypothesized that the proband was a homozygous mutation, whereas the proband of the Dutch family appeared to be a complex heterozygote. Dutch probands showed mutations with completely different haplotypes, supporting this hypothesis.

The TD-2 proband was distal to D9S127 and was homozygous for all the tags tested, but heterozygous for D9S127 and its centromere DNA marker. This suggested that the gene for TD appeared to occupy a genomic region that is telomeric at D9S127 and was incorporated by a homozygous marker.

Detection of Mutations Based on defects in intracellular cholesterol transport in patients with TD, we searched the EST database for genes in this region that are associated with a role in this process.

The ABC1 transporter gene has previously been mapped to 9q31, but its exact physical location has not been determined (Lucani et al. Genomics, 21: 150-1.
59, 1994). The ABC1 gene is a member of the ATP-binding cassette transporter, which represents a superfamily of highly conserved proteins with membrane transport of different substrates including the amino acids, peptides, vitamins, and steroid hormones (Luchani et al., Genomics, 2
Volume 1: 150-159, 1994. Dean et al., Curr. Opin.
Gen. Dev. 5: 779-785, 1995). 3 of this gene
The primer for the'UTR was mapped to the YAC spanning D9S306 (887-B2 and 930-D3), which matches it because it is a strong candidate for TD. We spread about 800 km base around sign D9S306 B
Large-scale genome sequencing of AC has begun. (Research Genetics RDCI-1
1 BACs: 418, 31J20, 47O19, and 179G21, these are United States, 35801 Alabama, Huntsville, Memorial Parkway 21.
Officially available from Research Genetics at 30) BACs 4I8
, 31520, 47O19, and 179G21 are each the previously described BACs.
s 269, 274, 279 and 291 (U.S.S.N. 09.
/ 526,193; U. S. S. N. 60/124, 702; S. S. N.
60 / 138,048; S. S. N. 60 / 139,600; S. S.
N. 60 / 151,977; Brooks-Wilson et al. , Nat. Genet
． 22: 336-345, 1999). The ABC1 gene was revealed to contain 49 exons and a genomic sequence of at least 75 kilobases.
Given the potential function of this family of genes as cholesterol transporters and their expression in fibroblasts and localization of the minimal genomic segment underlying TD, we formally identified ABC1 as a candidate.

Patient and control whole fibroblasts were used for Northern blot analysis and RT-PCR and sequence analysis. RT-PCR and sequence analysis of TD-1 reveals a heterozygous T to C substitution in the TD-1 proband, resulting in a arginine cysteine substitution at a conserved residue between mouse and human. Will. Exon 3 of ABC1 gene
This mutation, confirmed by sequencing 1, showed a complete phenotypic segregation in one aspect of this family. This substitution created an HgaI site, allowing RFLP analysis of amplified genomic DNA and confirmation of mutations. A point mutation in exon 31 was not found in 200 normal chromosomes from humans unaffected by the Dutch pedigree and 250 chromosomes in the Western European descent, suggesting that it is polymorphic. There is. Northern blot analysis of fibroblast RNA from this patient using a cDNA containing exons 2 to 50 of the gene revealed a transcript of approximately 8 kilobases in normal size, as well as control RNA or other patients with HDL deficiency. Revealed truncated mutant transcripts that were invisible to RNA. In addition, Northern blot analysis using clones containing discrete regions of cDNA showed that mutant transcripts were detected in cDNAs containing exons 2-50, 2-42, 2-23, and were much less clear with probes spanning exons 24-30. Yes, and was not seen with probes containing exons 31-43 or with probes spanning exons 31-50. This is a control RNA, H
Repeated with multiple filters of RNA from other patients with DL deficiency and other TD probands, truncated transcripts were observed only in TD-1. Sequence analysis of the coding region did not reveal any sequence changes that could explain this finding. Furthermore, DNA analysis by Southern blot showed no necessary reconditioning. Completion of exon sequencing in genomic DNA indicates that this mutation is a G to C transversion mutation at position (+1) of intron 24, affecting the splice donor site and causing aberrant splicing. Indicated.

R of the fibroblast RNA encoding the ABC1 gene from the proband of TD-2
T-PCR analysis revealed a homozygous N-to-G nucleotide change at nucleotide 1864 of exon 14, which was the first predicted ABC1 residue conserved in the mouse and C. elegans (soil nematode) homologs. The substitution of arginine for glutamine occurred at residue 597, occurring just proximal to the transmembrane domain of. This mutation creates a second AciI site within exon 14. Segregation analysis of mutations in this family revealed a perfect match between mutations and a low HDL phenotype, as expected. TD-2
Probands of are homozygous for this mutation, consistent with our hope of causing the mutation in this consanguineous family.

Analysis of the FHA Family Linkage linkage and improvement of the minimal genomic region containing the gene for FHA Data from microsatellite assortments of individual families from four French Canadian strains were analyzed. A maximum LOD score of 9.67 with a recombinant fraction of 0.0 was detected at D9S277 on chromosome 9q31. Twenty-two markers were then categorized in these families with a region spanning 10 cm around this locus. The frequency of these markers was evaluated from a sample of unrelated and unaffected subjects of French descent.

TD and FHA have thus far been imitated to be distinct with some clinical and biochemical properties. Even if the genes for these diseases were located in the same region, it was uncertain whether FHA and TD were due to mutations in the same gene or, alternatively, genes in similar regions. FH
Modification of the region containing the A gene was possible by verification of haplotype sharing and identification of important recombination events. Seven distinct meiotic recombination events were found in these families, demonstrating that the minimal genomic region containing the potential disease gene is a region of approximately 4.4 cM genomic DNA spanned by the markers D9S1690 and D9S1866. did. This region reveals the maximum LOD score with the markers D9S277 and D9S306 and is consistent with the results of a two-point linkage analysis that essentially excludes the regions that are centromeres at D9S1690 and telomeres at D9S1866. The eighth meiotic recombination event further refined the FHA region distal to D9S277.

As described herein, the ABC1 gene occupied a position within this interval. Overlapping gene data strongly indicated that FHA is virtually allelic to TD. The use of a series of genetic data from FHA and TD is based on the homozygous data for TD-2 and telomere boundaries at D9S1866 (meiotic recombination) and D9S.
The centromere label at 127 was provided. This is D9S127 and D9S1866
The locus was improved to about 1 Mb. The ABC1 gene was located within this minimal region.

Mutation Detection in FHA Mutation evaluation of the ABC1 gene was performed in FHA1. We used RT-PCR analysis with primers that extended the overlapping segments of the mRNA and gave mutation analysis to these fragments. Deletion of 3 nucleotides results in FHA-1 III. 0
The RT-PCR sequence of 1 reveals a deletion of nucleotides 2151-2153 and a deletion of leucine (ΔL693) at amino acid 693. This leucine is preserved in mice and sea elegans. Changes were detected in the RT-PCR product as well as in the genomic sequence from exon 15 specific amplification. This mutation is EarI
The restriction site is lost. Analysis of genomic DNA from the family showed that the mutation was completely segregated with the HDL deletion phenotype. Deletion of the EarI site produces a larger fragment that remains heterozygous for this mutation. This mutation maps to the first putative transmembrane region of ABC1 and was not found on chromosome 130 in people of French Canadian descent or over 400 in people of other European descent.

Mutations were also found in the patient genomic DNA of strain FHA-3 from Quebec.
A 6 base pair deletion of nucleotides 5752-5757 in exon 42 results in amino acid 1
This results in a deletion of 893 (Glu) and 1894 (Asp). This deletion was detected as a double superimposed sequence starting from the point of deletion and detected by bidirectional sequence reads. Detection can be done on a 3% agarose or 10% polyacrylamide gel and separates with FHA-3 disease. It was not found on French Canadian 128 chromosomes or other 434 control chromosomes. Amino acids 1893 and 189
4 is conserved among humans, mice, and sea elegans, implying that it is functionally important.

Additional mutations were found in the patient genomic DNA of strain FHA-2 from Quebec. The change from the C to the T transition at position 6504 is due to position 2 of SEQ ID NO: 1.
At 144, the arginine was converted to the STOP codon, and the last 1 of the ABC1 protein was converted.
Causes an 18 amino acid truncation. This change separates the family FHA-2 disease.

Functional Relationship Between Changes in ABC1 Transcript Levels and Cholesterol Efflux An antisense approach was performed to reduce ABC1 transcripts and assess the effects of transcript alterations on intracellular cholesterol transport. The use of antisense primer to the 5'end of ABC1 resulted in about a 50% reduction in normal RNA levels.
This is expected to partially mimic the loss of function due to mutations at alleles similar to those found in heterozygotes in patients with TD and FHA. Importantly, reduction of mRNA to the ABC1 gene causes a significant reduction in cellular cholesterol efflux, further establishing the role of this protein in reverse cholesterol transport, and the detected mutations constituting loss of functional mutations. It also provided evidence that it would be. Furthermore, these data support the functional importance of the first 60 amino acids of the protein. The antisense oligonucleotide DAN-6 directs the novel start codon 5'to that shown in AJ012376.1, and this antisense oligonucleotide effectively suppresses efflux.

ABC1 5'Regulatory Sequences and Polymorphisms in the 5'UTR Several polymorphisms were identified in the human ABC1 5'regulatory sequence (SEQ ID NO: 1) (Figure 4). Because of their location, it is probable that ABC1 gene expression will differ between humans with different promoter polymorphisms, and that these individuals will also respond differently to the same drug treatment. Thus, these newly identified polymorphisms can be used to tailor drug treatment depending on which polymorphism is present in the patient. Presence or absence of a particular ABC1 polymorphism is a further ongoing CV
It could be used to determine an individual's predisposition to D.

[0114]   The method of the present invention is carried out using the materials and methods described below.

Biochemical Studies Extracted from blood into EDTA-containing tubes for analysis of plasma lipids, lipoprotein cholesterol, ApoAI, and triglycerides and for storage at −80 ° C. White blood cells are separated from the buffy coat for DNA extraction.

Lipoprotein measurements are performed on fresh plasma as described elsewhere (Rogler et al., Ar.
terioscler. Thromb. Vasc. Biol. Volume 15: 683-
690 pages, 1995). Lipid, cholesterol and triglyceride levels are measured before and after precipitation with dextran manganese in whole plasma (obtained in a preparative ultracentrifuge) and plasma at a density of d <1.006 g / mL. Apolipoprotein measurement is performed by nephelometry for ApoB and ApoAI.

Genome clone assembly and physical map construction of 9q31 region The target genetic marker at 9q31 was identified in the YAC contig using the Whitehead Institute / MIT Center for Genome Research Map as a reference. Was done. Additional labels located at approximately 9q31 intervals from public databases and literature were then tested on YAC clones by PCR and hybridization analysis. Based on haplotype analysis, the region between D9S227 and D9S306 was targeted for higher order physical mapping studies using bacterial artificial chromosomes (BACs). BACs in the target region are DNA labeled probes and RPCs.
Isolated by hybridization of whole YAC to a high density filter containing clones from the I-11 human BAC library.

Sequence Search and Alignment The human ABC1 mRNA sequence is GenBank Accession No. AJ012376.
Retrieved from GenBank using the Entre Nucleotide query in 1 (Baxsevanis et al., Practical Guide for Gene and Protein Analysis, AD Baksevanis, BF Kellet, Ed., 98: 120). , 1998). The version of the protein sequence we used as wild type (normal) is CAA10005.
It was 1.

We identified an additional 60 amino acids within the belief frame of the starting methionine (FIG. 9A). Bioinformatic analysis of additional amino acids shows the presence of a short stretch of basic amino acid residues, followed by a hydrophobic stretch, and a few more polar residues. This represents the leader sequence, or also the transmembrane or membrane associated region of one ABC1 protein. To differentiate among the above possibilities, antibodies directed to the region of amino acids 1-60 are taken up and used to determine the physical relationship of amino acids 1-60 associated with the cell membrane. Other standard methods such as expression of fusion proteins and cell division can also be used.

The mouse ABC1 sequence used has the accession number X75926. This mouse sequence appears to be sufficiently incomplete due to the lack of the additional 60 amino acids described here in human ABC1.

Clustal W version 1.7 was used for multiple sequence alignment in BOXSHADE to enhance graphically with default parameters (h
http: // www. isrec. isb-sib. ch: 8080 / soft
ware / BOX. form. html). The C. elegans ABC1 orthologue was identified in BLAST (version 2.08) using CAA1005.1 (see above) as a default parameter query, except using a biofilter for C. elegans. Selected protein sequences scored 375 and 1
It was AAC 69223.1 with an E value of 03.

Genomic DNA Sequencing BAC DNA was extracted from bacterial cultures using the Nucleo Bond Plasmid Maxi Kit (Clontech, Palo Alto, Calif.). D
For NA sequencing, a sublibrary was first constructed from each BAC DNAs (Lowen et al., Automated DNA Sequencing and Analysis, MD Adams, C. Fields, J. C. Venters, 1994). In summary, BAC DNA was isolated and randomly trimmed by nebulization. The trimmed DNA was then size-fractionated by agarose gel electrophoresis, and fragments larger than 2 kilobases were collected.
It was treated with nuclease and then cloned into SmaI digested M13mp19 treated with T4 DNA polymerase and Klehau enzyme to ensure blunt ends. Random clones were sequenced with ABI 373 or 377 sequencers and fluorescently labeled primers (Applied Biosystems, Foster City, Calif.). DNAStar software was used for gel trace analysis and contig assembly. All DNA sequences were verified against public databases available using BLASTn primarily as a repeat masker (University of Washington). The sequences of each of the assembled contigs are shown in Figures 1A-D.

Reverse transcription (RT) -PCR amplification and sequence analysis Total RNA was isolated from cultured fibroblasts of TD and FHA patients (Zan et al., J. Chem.
Biol. Chem. 27: 17776-1883, 1996) as described in Superscript II Reverse Transcriptase (Maryland, Rockville,
Life Technologies, Inc.) 250 units using oligo d
It was reverse transcribed with a CDS primer containing (T) 18. cDNA is the published human ABC1 cDNA sequence (Lucani et al., Genomics, 21: 150-15.
Page 9, 1944) and amplified with Taq DNA polymerase. Detected 6 DNA fragments designed to amplify each cDNA against 6 sets of primers, which is one of the full length human ABC1 cDNAs.
There was continuous overlap over 35 to 7014 base pairs. Nucleotides are numbered below according to the order of the published human cDNA sequence (AJ012376.1). Primer pair (1): 135-158 (f) and 1183-1
199 (r); (2): 1080-1107 (f) and 2247-2273 (
r); (3): 2171-2197 (f) and 3376-3404 (r); (
4): 3323-3353 (f) and 4587-4617 (r); (5) 45.
15-4539 (f) and 5782-5811 (r); (6) 5742-57.
69 (f) and 6985-7014 (r). The RT-PCR product was purified on a Qiagen spin column. Sequencing was performed using the Taq dideoxy terminator cycle sequencing and Big Dye Kit model 37 according to the manufacturer's protocol.
Performed on a 3A automated DNA sequencer (Applied Biosystems).

Northern Blot Analysis Northern transfer and hybridization was performed essentially as described (Zan et al., J. Biol. Chem. 27: 1776-1783, 1996). In summary, 20 μg of total fibroblast RNA sample contained 7% formaldehyde.
Denatured agarose (1.2%, w / v) in the presence of lysates were electrophoresed in gels and transferred to nylon membranes. Filters are 32P-labeled human ABC1 c as directed.
Probed with DNA. Pre-hybridization and hybridization was performed with ExpressHyb solution (Clontech) at 68 ° C. according to the manufacturer's protocol.

Cell Culture Dermal fibroblast cultures were established from 3.0 mm punch biopsies of the forearm of FHD patients and healthy control subjects as described (Marcil et al., Arterioscler).
． Thromb. Vasc. Biol. 19: 159-169, 1999).

Cellular Cholesterol Labeling and Loading Further described in detail in the protocol for cellular cholesterol efflux experiments (Marcil et al., Arterioscler. Thromb. Vasc. Biol. 19: 1).
59-169, 1999). The cells are labeled with 3H-cholesterol during growth,
Free cholesterol was loaded during growth arrest.

Cholesterol Efflux Studies Efflux studies were performed in the presence of purified ApoAI (10 μg protein / mL medium) for 0-24 hours. Efflux was determined by the percentage of free cholesterol in the medium after the cells had been incubated for a certain period of time. All experiments were performed in triplicate in the presence of cells from one control test and cells from the study test being tested.

Determination of the genomic structure of the ABC1 gene Most splice site sequences were determined from the genomic sequence generated from a BAC clone spanning the ABC1 gene. Over 160 kilobases of genomic sequence have been generated. The genomic sequence was aligned with the cDNA sequence to identify intron / exon boundaries. In some cases, long range PCR between adjacent exons can
It was used to amplify intron / exon boundaries with sequence-designed amplification primers. The genomic sequence of human ABC1 is shown in Figures 1A-D.

Analysis of ABC1 Heterozygotes Identification of Subjects Subjects who are heterozygous for a mutation in the ABC1 gene have previously described TD and FH.
They were individuals from 7 families of A (Brooks Wilson et al., Supra: Marcil et al., Supra). In addition, zygotic individuals from the new Danjiema disease 3 family (TD3-5) and the new FHA1 relative FHA6 were included. The second mutation is a member of the TD family (T
It was not identified in D4). However, the label immediately adjacent to ABC1 also cosegregates with a low HDL phenotype. Individuals with affected haplotypes were considered heterozygotes. The presence or absence of mutations identified by the proband's genomic sequence from each family is subsequently determined to determine heterozygous and unaffected individuals, respectively.
Confirmed by restriction fragment length polymorphism (RFLP).

The control cohort consisted of members of 11 unaffected families. These individuals share a heterozygous genetic background, and environmental factors are expected to be similar among family members. Thus many additional factors that influence HDL are regulated and the phenotype between heterozygotes and unaffected individuals is largely attributed to altered ABC1 gene activity.

All subjects gave informed consent to participate in this study,
The genetic analysis protocol was approved by the Ethics Committees of the University of British Columbia, Academic Medical Center in Amsterdam, and Clinical Research Institute of Montreal (IRCM).

Lipid and Cholesterol Efflux Assays Lipid levels of ABCA1 heterozygotes were measured at standardized lipid clinics in Vancouver, Montreal and Amsterdam as previously described (Brooks-Wilson et al., Supra; Marcil et al., Supra). LDL is Friedevalde et al.
lin. Chem. 18: 499-502, 1972),
Modified to account for lipid measurements in mmol / L.

Cellular cholesterol efflux from fibroblast cultures was measured as previously described (Brooks Wilson et al., Supra; Marcil et al., Supra). Each experiment was performed in triplicate wells and averaged. The measurements were reported as percent efflux in each subject relative to the average of at least 2 healthy controls included within the same experiment. Separate experiments were repeated at least twice and the average relative efflux over all experiments was used.

Statistics. In the heterozygous analysis, differences in mean baseline demographics and lipid levels within groups were compared to student t-tests. Comparisons of frequencies between males and females or distributions across various percentile ranges were performed using the chi-square test. Analysis of potential interactions between affected states and either sex or BMI was performed using a general linear model. Statistical analysis was performed using Prism (GraphPad software, version 3.00) or Cystat (SPSS Incorporated, version 8.0). All values were reported as mean ± standard deviation.

Reduced HDL cholesterol and increased CAD risk in ABC1 heterozygotes An analyzed cohort of 77 individuals from 11 families was heterozygous for a mutation in the ABC1 gene. A comparison of the average lipid levels of heterozygotes with the average levels of all unaffected family members (n = 156) is shown in FIG. Heterozygotes are about 40-in HDL and apoA-I when compared to unaffected family members.
There was a 45% decrease and apoA-II showed a slight (about 10%) decrease. Mean triglyceride levels (TG) were increased by approximately 40% compared to unaffected family members and were further increased in TD patients. Unlike in TD patients, there was no significant reduction in either total or LDL cholesterol in heterozygotes, and apo
The B level was heterozygous and did not differ much from the control. Mean HDL levels in each carrier of mutations were also approximately 40-50 compared to unaffected family members.
% (FIG. 9).

The heterozygous phenotype was examined by calculating the percentage of age- and sex-specific percentiles within a certain range based on the LRC criteria (Hiss et al., Supra; Heiss et al., Blood Circulation. , 61: 302-315, 1980). Many changes were apparent in the heterozygous phenotype. As shown in FIG. 5A, a significantly higher proportion of heterozygotes had less than the 5th percentile of HDL cholesterol for age and sex compared to unaffected controls (65% vs. 5%, P <0.0001)
, 5% of heterozygotes had HDL higher than the 20th percentile in age and sex, and HDL reached the 31st percentile in age and sex. Thus, in some individuals, the low HDL phenotype is clearly less severe. A broad distribution of triglyceride (TG) levels was also apparent (Fig. 5B). A significantly lower proportion of heterozygous individuals have TGs below the 20th percentile compared to age and sex unaffected family members (p = 0.03).
), And a significantly larger proportion was TG in the 80th percentile and above (p = 0.00).
5) but there was considerable overlap between the two distributions.

[0137] Another important question is whether individuals who are heterozygous for the ABC1 mutation have an increased risk of developing coronary artery disease (CAD). In our large cohort, symptomatic vascular disease was more than three times more frequent than adult heterozygotes in unaffected family members (Figure 6). The morphology of vascular disease was generally more severe in heterozygotes than in unaffected family members (Figure 7). Heterozygotes (5,
In unaffected individuals with severe vascular disease requiring multiple interventions, one with fatal) myocardial infarction, CAD was 2 in angina and another transient ischemic attack at 80 years of age Was obvious. Furthermore, mean age of onset was 10 years younger in heterozygotes compared to unaffected controls (FIG. 6).

Cholesterol Efflux, HDL Levels, and CAD in ABC1 Heterozygotes We have previously shown that individuals heterozygous for the ABC1 mutation reduced cholesterol efflux. The study further evaluated the relationship between cholesterol efflux levels, HDL cholesterol levels, and CAD. The relative cholesterol efflux of individuals heterozygous for the ABC1 mutation was plotted against the mean HDL cholesterol levels observed in carriers of the mutation and expressed as a percentage of unaffected members within the family (Figure 8). Cholesterol efflux levels associated with each mutation strongly predicted the corresponding HDL cholesterol levels in the family, accounting for 82% of changes in HDL cholesterol (r ² = 0.82, p = 0.005). In (FHA2) efflux was measured in 3 independent heterozygotes, r ² when plasma HDL levels here were plotted against efflux measurements here.
A value of 0.81 was obtained. The relationship of expected change between ABC1 efflux activity and HDL levels was estimated using the degeneration equation of the mean HDL level of heterozygotes at the outflow level of heterozygous carriers (p = 0.02). Based on this analysis, we predict that each 8% change in efflux level will be associated with a 0.1 mmol / L change in DHL cholesterol.

Relative cholesterol efflux levels are further associated with CAD within the family. Immature CAD
The family with the most obvious evidence of had an individual with minimal cholesterol efflux (bold in Figures 8 and 9). These data indicate that the level of residual ABC1 function is an important determinant of both HDL cholesterol levels and susceptibility to CAD.

Comparison of the type and location of mutations with respect to phenotypic severity in individuals heterozygous for the ABC1 mutation. We have so far demonstrated that the phenotypic representation of FHA heterozygotes is much more stringent than that of our heterozygotes. It is described that it is a thing. Furthermore, we are more FH than the TD family
It was also noted that there are more deletions of A-family proteins and immature truncations. Thus, as residual ABC1 activity is an important predictor of phenotypic severity, the effect of the nature of the mutation on the phenotypic expression of the mutation in the ABC1 gene was investigated. The severe mutations expected to occur in non-functional alleles were defined as deletions or mutations that caused premature truncations of the protein (frameshift and nonsense mutations) or disrupted the natural splicing of the protein. Missense mutations, on the other hand, occur with changes in only a single amino acid and still occur in protein products that remain partially active.

Lipid levels were compared in severe and missense mutation heterozygous carriers. On the other hand, carriers of severe mutations tended to have lower HDL levels than missense mutations, and this tendency did not reach significance (Fig. 10). A range of HDL levels was observed with individual missense and severe mutations (Figure 9). The M1091T missense mutation was the most severe mutation with respect to efflux effects and HDL levels and had a more severe phenotype than the early truncations of the protein (eg R909X).

Mutation sites within the ABC1 protein (eg N-terminal or C-terminal) did not affect the phenotype (FIG. 11). The presence of CAD is found in carriers of mutations in some domains of the protein. Patients with mutations in both alleles show signs of splenomegaly alone or associated with CAD (TD1). Thus, the phenotype is mutation-specific and probably resembles the residual ABC1 function of the wild-type allele and the residual function of the mutant allele, similar to what has been shown for mutations in ABCCR, a close homologue of ABC1. It appears to be dependent (Van Driel et al., Ophthalmology, 19: 117-122, 1998).

Relationship Between ABC1 Gene and Mutation Phenotype at Age One factor affecting phenotypic expression revealed in the family was age. We first characterized the effect of age on HDL levels of individuals in two previously reported families (Marcil et al., Supra) (Figures 12A and 12B). In the FHA3 family, all II and III generation heterozygous individuals were HD and below the 5th percentile in age and sex.
While having L-cholesterol levels, generation IV had a much more variable phenotype and H
DL cholesterol levels ranged to the 20th percentile. The same pattern was observed in the FHA1 family.

The distribution of individuals under the age of 30 across the HDL percentile range was compared to the distribution of corresponding individuals aged 30-70 across the HDL percentile (FIG. 13). 30-70
A significantly larger proportion of the year-old individuals had HDL cholesterol below the 5th percentile compared to the proportion of individuals below the age of 30 who had HDL cholesterol below the 5th percentile. Mean HDL is reduced in heterozygotes older than 30 compared to those younger than 30. On the contrary, no significant changes were observed in the unaffected controls (
(Fig. 14). Similar results were seen in men and women separately and in both premenopausal and postmenopausal age in women (FIGS. 15A and 15B). Triglyceride levels increase with age in both heterozygotes and unaffected family members.

Evaluation of the effects of sex and BMI on the phenotypic expression of the ABC1 mutation Females are known to have higher HDL and lower triglycerides than males. Thus, the phenotype of ABC1 heterozygotes was analyzed to determine if the phenotype was affected by sex. HDL cholesterol was significantly lower in both heterozygous men and women than in unaffected controls (0.70 ± 0.24 vs 1.2 respectively)
1 + 0.29, P <0.0001; 0.76 ± 0.25 vs 1.41 ± 0.38,
P <0.0001). This is a decrease in apoAI (0.9 for male and female respectively)
2 ± 0.27 vs 1.36 ± 0.22, P <0.0001; 0.92 ± 0.36 vs 1.49 ± 0.28, P <0.0001) Male compared to unaffected family members Both women and women tend to have a gradual decrease in apoAII (0.35 ± 0.
08 vs 0.40 ± 0.09, p = 0.08; 0.35 ± 0.09 vs 0.39 ± 0
． 07, p = 0.06). Triglycerides were male (2.07 ± 2.16 vs 1.30 ± 1.30, p = 0.02) and female (1.34 ± 0.86 vs 1.09 ± 0. 63, p = 0.08), which were all high in the heterozygotes. HDL difference between men and women is control (p = 0.11)
Heterozygotes decreased compared to the control, while the difference in triglycerides was lower in the controls (p = 0.1
It increased compared to 3).

One factor known to affect HDL and triglycerides is BMI.
Is. All cohorts are divided into 3 groups of BMI (tertile)
Mean HDL and triglyceride levels of heterozygotes and unaffected individuals by I tertile are shown in Figures 16A and 16B. BMI was compared to heterozygotes (P <0
． Both 0001) had a significant effect on both HDL and triglycerides. BM
Effect of I on HDL-C and triglyceride levels depends on the lower BMI of heterozygotes
It was more severe with the ABC1 heterozygotes than controls, as evidenced by (mid-tile). As posed, BMI was clearly associated with changes in HDL and triglyceride levels in heterozygotes compared to controls. However, neither effect reached statistical significance. HDL is all BMI ter
decreased in heterozygotes compared to controls in etile (P <0 in each tertile
． 0001). While triglyceride levels were increased in heterozygous BMI tertiles compared to unaffected family members, this difference was due to intermediate intermediate BMI ter
It was only significant at tile (p = 0.09).

Analysis of ABC1 SNPs from the REGRESS Study Identification of SNPs SNPs of the ABC1 gene were identified during the complete genomic sequencing of 14 unrelated probands with low HDL-C (Brooks Wilson et al., Supra 1999). Year;
Marcil et al., Supra, 1999). Variants identified in the low HDL family that did not co-segregate with the low HDL phenotype and were observed in unaffected individuals were presumed to be SNPs. Based on the sequencing of BAC clones spanning the entire ABC1 region (described above), sites different from those identified as heterozygotes or found in the sequenced individuals were also identified as polymorphisms. Sequence data was available from at least one control individual at all variant coding sites. SNPs are numbered from the nucleotides described as position 1 (Pallinger et al., Biochemical and Biophysical Research Communications, 271: 451-455, 2000) and named the first exon number 1. Was given. As a standard nomenclature for all variants, the "wild-type" allele (which appears more frequently in the REGRESS population) is designated A, while the (infrequent) variant allele is designated B.

Subjects We have previously described in detail to evaluate the effect of these SNPs on lipid levels and CAD (Jukema et al., Blood Circulation, 91: 2528-2540,
We studied a cohort of 804 people with documented coronary artery disease who cooperated with the Degenerate Growth Assessment Statin Study (REGRESS) described in (1995). In summary,
Collaborators evaluated at least one coronary artery with stenosis of 50% or more as assessed by coronary angiography, plasma cholesterol concentration of 4 to 8 mmol / L (155 to 310 m8 / dL), and 4 mmol / L (350 mg / dL) or less Was required to have a plasma triglyceride concentration of. The phenotypic effects of cSNPs were tested in relation to baseline lipid parameters.

Patients were randomized to receive pravastatin (Prabacol, Bristol-Myers Squibb, Princeton, NJ) or placebo for 2 years. Computer aided coronary angiography was previously described (Jukema et al., Supra (
1995)) as the beginning and end of the study. Baseline values and changes in mean segment diameter (MSD), which is a measure of mean non-occluded diameter along the vessel and at the smallest occluded diameter (MOD), which is a measure of the smallest unoccluded segment, were used as the primary means of CAD. MSD reflects diffuse changes in atherosclerosis and MOD reflects changes in focal atherosclerosis. Large MSD and MOD measurements reflect less vessel occlusion, and a decrease in these parameters reflects the progression of coronary atherosclerosis. In addition, coronary events leading to death such as myocardial infarction, unscheduled coronary angiography or bypass surgery (PTCA, CABG), or myocardial attack / transient cerebral ischemic attack were considered.

Blood was collected from each patient at baseline and DNA was extracted according to standard procedures.
Several subsequent genetic studies have been conducted in this cohort (Reimer et al., Nature Genetics, 10: 28-34, 1995; Jukema et al.,
Blood Circulation, 9: 1913-1918, 1996; Creuttmans et al., Blood Circulation, 96: 2573-2577, 1997; Casteline et al., Clinical Genetics, 53: 27-33, 1998;
Quivanhofen et al., New England of Journal of Medicine, 338: 86-93, 1998). REGRESS and its DN
A Secondary research was approved by all seven institutional review boards of the Cooperation Center and its Medical Ethnic Committee.

Additional Dutch subjects with low HDL and early-onset coronary artery disease were obtained from the procedure (Kuivanhofen et al., Atherosclerosis, Thrombosis and Vascular Biology, 1
7: 560-568, 1997; Barhoff et al., Atherosclerosis, 14
Volume 1: 161-166, 1998; Franco et al., British Journal of Hematology, 102: 1172-1175, 1998.
Year; Bitte Cork et al., Atherosclerosis, 146, 271-279, 19
1999; Franco et al., British Journal of Hematology, 10
Volume 4: 50-54, 1999). Dutch control subjects were taken from a large population-based study designed to assess the effects of various risk factors on CAD (Sidel et al., International Journal of Obiesity, 1).
9: 924-927, 1995; Quivanhofen et al., Atherosclerosis, Thrombosis and Vascular Biology, 17: 595-599, 1997).
The French Canadian subject was a random sample of individuals. The South African Black and Cantonese cohorts have been previously described (Ehrenbori et al., Atherosclerosis, Thrombosis and Vascular Biology, 17: 2672-2678, 1997). All subjects received informed consent.

CSNP Screening For each mutant, the restriction enzyme whose cleavage pattern is altered by the mutant is RFL
The degree of P-test was confirmed. If no suitable enzyme is found, a mismatch strategy is adopted whereby a single nucleotide mismatch is incorporated into the PCR primer and combined with either the wild type or mutant allele to create a restriction site. The specific conditions for all assays are listed in Figure 17. All PCR reactions are 1
X 5 μL volume in the presence of PCR buffer and 1.5 μM MgCl ₂ (Life Technologies). Thermocycling parameters for all assays were as follows: 95 ° C for 3 minutes; 95 ° C for 10 seconds for 35 cycles of denaturation,
Annealing for 30 seconds at the temperature specified in Figure 17, and stretching for 30 seconds at 72 ° C, and finally for 10 minutes at 72 ° C. All digests (15-20 μL, PC
R product) was run in the manufacturer's buffer (New England Biolabs) for 2 hours at the temperature specified by the manufacturer. As an example, the digestion result of R219K is shown in FIG.
Indicated by. The 177 base pair fragment with the A allele is not cleaved with EcoNI, while the B allele is digested to produce the 107 fragment and 70 base pairs. Heterozygous individuals thus all show three bands (177, 107, and 70 base pairs).

Genotyping in the TaqMan (r) Assay To facilitate mass screening of several mutants, the TaqMan Bayest Polymerase Chain Reaction (PCR) assay (Holland et al., Bulletin of the National Academy of Sciences, Vol. 88, No. 16). , 7276-7280, 1991; Lee et al., Nucleic Acids Research, 21 (16): 3761-3766, 1993) was developed for the detection of polymorphisms in the ABC1 gene. In this one-tube assay, two different fluorogenic hybrid trait probes (one for each allele) were 5 '
A different fluorescent reporter dye (FAM or TET) at the end, also 3 '
The end is labeled with a common quencher dye (TAMRA). These probes are cleaved by the 5'nuclease activity of the Taq enzyme during PCR amplification. This cleavage separates the reporter from the quencher dye, producing an increase in reporter fluorescence. By using two different reporter dyes, cleavage of the allele-specific probe can be detected in a single PCR. The difference in measured fluorescence intensity between the two TaqMan probes allows accurate allele calling.

Two TaqMan probes (25 nM each) and 4.5 mM MgCl ₂
PCR amplification with the flanking set of primers (300 nM) in the presence of was carried out using the following thermocycling protocol: 96 ° C. 10 min initial denaturation, followed by 96 ° C. 30 sec, 63 ° C. 1 min. And 39 cycles of 15 seconds at 72 ° C,
It was followed by a final extension of 10 minutes at 72 ° C. Each plate also contained controls of known genotype as well as controls (no DNA template). Fluorescence measurement and genotyping are perkin. Performed on an Elmer LS50B or ABI Prism 7700 Sequence Detector. The fluorescence from each reaction was normalized to the signal from the no template control.

Cellular Cholesterol Efflux Studies Cellular cholesterol efflux from fibroblast cell cultures was measured as described above in the "ABC1 Heterozygote Analysis" section.

Within the statistical REGRESS population, the baseline characteristics of the three genotypes (AA, AB, BB) were compared using a one-way analysis of variability and, if appropriate, the Kini test. If the BB genotype was rare, the AA group was compared to the AA + BB combination group. The cumulative incidence of coronary artery events in these genotype groups was compared using the log-rank test. The relationship between age and HDL levels was investigated using a linear regression model, and the differences between genotypes with respect to the slope of this regression line were tested using analysis of covariance. In addition to this degeneration analysis, the R219K genotype was compared between age-specific subgroups. Random extractions into placebo and pravas titanium were evaluated by the Kaini power analysis and were comparable in all genotype groups to all variants except the R1587K variant. In this variant, a low proportion of carriers was randomly selected for treatment with pravastatin. In addition, changes in MOD, MSD, and prevalence of coronary events (three variables measured during the trial and during subsequent randomization) were analyzed separately for the placebo and pravastatin subgroups. . Similar, genotypic effects of each of the variants were observed in the treatment subgroups (ie, pravastatin and placebo controls).

All lipid levels are reported in mmol / L. All values are reported as mean ± standard deviation.

Relationship between the R219K Polymorphism and Reduced Triglyceride Levels and Reduced CAD Risk The common R219 polymorphism results from the substitution of lysine for arginine at amino acid 219 of the ABC1 protein. The allele frequency of the variant, or "B" allele, was 46.3% and is shown in FIG.

The relationship between this polymorphism and CAD was investigated. The B allele of R219K was associated with reduced baseline CAD. Both MSD and MOD are AA to AB or BB
It was significantly increased in an allele dose-dependent manner (Fig. 20).

Angiographic data were parallel with differences in clinical coronary event rates. A smaller proportion of individuals, more rarely heterozygous for the B allele (BB), had myocardial infarction (MI) prior to the start of the trial. Carriers of the B allele showed a strong trend towards increasing event-free prevalence throughout the institution (Figure 21, p = 0.07).

The relationship between R219K polymorphisms and reduced CAD was associated with CAD-selected REGRES
It was further dictated by the frequency of reduction of this allele in the S cohort. In fact, the genotype frequencies observed in this mutant are inconsistent with Hardy-Weinberg equilibrium (
p = 0.004). There are fewer BB individuals and more AB individuals than expected (424AA, 330AB, and 36BB individuals were observed compared to the expected 439AA, 300AB, and 51BB individuals). REGR
The lack of Hardy-Weinberg equilibrium was a desirable choice for BB individuals, as the ESS cohort was selected in men with CAD, suggesting that it was consistent with the reduction in CAD observed in this group. There is.

There were no apparent differences in mean HDL-C levels between genotypes across groups (FIG. 22), but triglycerides were significantly lower in carriers of the B allele. This suggests that ABC1 function further directly affects plasma triglyceride levels and that this variant may be associated with gain of ABC1 function. These findings are further consistent with the reduced CAD observed in carriers, in which ABC1 regulation of triglyceride levels is also a mechanism by which ABC1 activity is reduced to CAD.
Suggests that it affects the risk of.

To further explore the apparent lack of action of this mutant on HDL-C, the relationship between R219K genotype and HDL at various ages was investigated. In younger individuals, carriers of the B allele had increased HDL-C compared to non-carriers (FIG. 23). Furthermore, HDL-C increased with age in AA individuals, whereas heterozygous carriers showed a much more modest increase, and HDL cholesterol decreased with age in heterozygous carriers (Figure 24). HDL cholesterol was positively correlated with age in AA individuals (P <0.001). On the contrary, this relationship was not apparent in individuals heterozygous for this mutant, and HDL cholesterol was not correlated with age in BB walks or zygotes. However, the correlation of either AB or BB individuals did not statistically differ from zero (Figure 25A). H of AA individuals
An age-related increase in DL-C is not maintained in carriers of the B allele (P value comparison slope = 0.04). Thus, reduced CAD in carriers of R219K may further be related to the fact that carriers continued to have increased HDL-C compared to non-carriers for most of their lifespan.

Changes in HDL-C with age for the various R219K genotypes reflect a similar trend of changes in cholesterol efflux with age (FIG. 25B). We genotyped this mutant to all individuals that we measured efflux and did not carry the ABC1 mutation. In AA individuals (n = 30), cholesterol efflux increased with age, while A
In B and BB individuals (n = 24), runoff decreases with age (P-value comparison slope = 0
． 15). Thus, the specific age-related changes in HDL found in different R219K genes are consistent with similar functional changes in ABC1 activity.

From FIG. 23, triglyceride levels generally decrease with age, a finding found in all R219K genotypes. The percent reduction in triglyceride levels was almost half (9.3%) in carriers compared to non-carriers (17.3%, p = 0.07). Differences in genotype are also observed in MSD and MOD. In non-carriers, MSD and MOD measurements decreased significantly with age, reflecting increased atherosclerosis in older individuals (Figures 23 and 26). In contrast, in carriers of the R219K mutant, these measurements did not change significantly with age. Thus vascular disease progresses much more slowly with age in carriers of the R219K mutant compared to non-carriers.

Populations of Asian and African origin have been shown to have increased HDL-C, reduced triglyceride levels, and reduced CAD risk compared to Caucasian populations (Tylor et al.,
Blood Circulation, Volume 62 (Appendix IV): IV-99-IV-107, 1980; Tao et al., International Journal of Epidemiology, Volume 21 (5): 893-903, 1992; Brown. Atherosclerosis and thrombosis, 13: 1139-1159, 1993; Adedege, Tropical and Geographical Medicine, 46 (1): 23-26, 1994; Simon et al. , American Journal of Public Health, Volume 85 (12): 1698.
-1702, 1995; Morrison et al., Metabolism, 47 (5): 514-.
521, 1998). This finding is similar to the phenotypic effects of this mutant.
Thus, because SNP frequencies often differ in different racial groups, variants within these two population groups were tested (Figure 27). This variant is more commonly found in individuals of either Cantonese or South African black offspring, where it is the dominant allele. This indicates that the increased frequency of this variant is increased HDL, decreased triglyceride levels and decreased C observed in these populations compared to the Caucasian population.
It is suggested to partially explain AD.

Effects of other cSNPs on plasma lipid levels and risk of CAD Lipids in T774P (n = 4), K776N (n = 3) or E1172D mutants (n = 34) compared to corresponding non-carriers No significant difference in level or CAD was observed. Carriers of V825I (n = 103 AB, 4BB) showed no apparent difference in lipid levels or baseline MSD or MOD. But V82
Carriers of the 5I mutant significantly increased events during the trial (44% vs. 33% .p).
= 0.001).

No carrier for the S1731C mutant was found in the REGRESS population,
This variant was detected in one of the FHA families (FHA2). In individuals heterozygous for the ABC1 mutation, this mutant was associated with significantly reduced HDL-C (
0.16 ± 0.04, n = 2 vs. 0.64 ± 0.14, n = 10; carrier vs. noncarrier p = 0.0009). For unaffected family members, however, S1731C
Carriers had lower HDL-C than non-carriers (1.03 ± 0.22)
V. 1.09 ± 0.23), which is not statistically significant. Control individuals in which this variant was also seen (FIG. 19) had low plasma HDL-C (0.72 mmol / L).

Similar to the carrier of R219K, carriers of the V771M mutant showed no difference in HDL-C compared to non-carriers. However, there was a marginally significant interaction between age and genotype at HDL-C levels (p = 0.05). All but two carriers of the V771M mutant are carriers of the R219K mutant. The interaction between age and genotype for HDL-C was marginally significant when regulated by the R219K genotype (p = 0.11), thus this variant was independent of age attributable to R219K. Will have an effect. CAD tends to decrease (MO
An increase in D) was observed in carriers of this mutant compared to non-carriers (1.89 ± 0).
． 38 vs 1.76 ± 0.35, p = 0.13).

At I883M cSNP, homozygous BB individuals (n = 14) increased progression in MOD (mean change of 0.53 ± 0.79 vs. 0.11 ± 0.25, P <0.
． 001). BB individuals had twice the event rate as those of AA individuals (21
． 4% vs. 10.6%), which was not statistically significant (p = 0.19).
. No difference was observed in mean lipid levels between the I883M genotypes. Furthermore, there were significantly more BB individuals than expected in the Hardy-Weinberg equilibrium (14BB, 86A compared to the expected 8BB, 98AB, and 314AA individuals).
B, and 320AA individuals were observed). This cohort was selected as an individual associated with CAD, suggesting that it preferentially includes that of the BB genotype. The association of this variant with CAD is further supported by the significantly increased frequency of this variant in the immature CAD population (CAD odds ratio of carriers of this variant = 0.43, 95% confidence interval 0). 22-0.85, p = 0.01) (FIG. 19).
These findings differ from the most recent reports suggesting that homozygous carriers of this cSNP increase HDL-C (Wan et al., Atherosclerosis, Thrombosis and Vascular Biology, 20: 1983-1989, 2000).

Carriers of V399A (AB, n = 9) were individuals who had AA at this site (n = 4).
0) tended towards higher HDL-C (1.03 ± 0.28 vs 0.92 ± 0.23, p = 0.15). In the AB group (14% of AA's,
No events were observed (compared to p = NS) and carriers had half the prevalence in the family history of CAD (22.2% vs 49.4%, p = 0.18). ). Furthermore, consistent with this data, carriers increased baseline MOD during the trial period (1.92 ± 0.3).
2 vs 1.73 ± 0.35, p = 0.13) and less progress in MSD (2.
-0.05 ± 0.10 vs. 0.08 ± 0.19, p = 0.16). However, it is not possible to draw a firm conclusion that this variant increases HDL-C and decreases CAD, because the number of carriers of Ikansen is too small.

Carriers of the R1587K mutant (AB, BB) tended to have an allele dose-dependent increase in HDL-C compared to non-carriers (0.86 for BB, AB, and AA, respectively). ± 0.16, 0.91 ± 0.23 and 0.94 ± 0.23, p = 0
． 03). No significant interaction with age was observed (p = 0.32). Furthermore, the R1587K genotype is an important predictor of HDL-C, based on multiple regressions including age, BMI, smoking, and triglyceride levels as covariants. However, there were no significant differences in CAD or events among carriers compared to non-carriers during the trial.

[0173] ABC1 gene variants and summary polymorphism following correlation study HDL levels or cardiovascular diseases were examined for their effects on predisposition of cholesterol modulating and cardiovascular disease. These polymorphisms are numbered from the nucleotide described as position 1 (Parinja et al., Supra) and are designated first exon number 1.

Substitution of G for A at nucleotide 1051 (R219K). Carriers of this mutant had decreased triglyceride levels, increased HDL cholesterol levels (
, Especially in young individuals), and with reduced CAD.

Substitution of T for C at nucleotide 1591 (V399A). This variant was associated with an increased propensity for HDL cholesterol in carriers.

Substitution of G for A at nucleotide 2706 (V771M). Carriers of this mutant were shown to have reduced CAD.

Substitution of A for C at nucleotide 2715 (T774P). This variant was not found more often in individuals with low HDL cholesterol levels or CAD than controls.

Substitution of G for C at nucleotide 2723 (K776N). This variant was found at a lower frequency (0.54% vs. 1.89%) in the coronary artery disease population relative to a similar background control population in the Netherlands.

Substitution of G for C at nucleotide 3911 (E1172D). This variant is found less frequently in individuals with low HDL and in some populations with early coronary artery disease.

Substitution of G for A at nucleotide 5155 (R1587K). This variant is associated with reduced HDL cholesterol levels in carriers.

Substitution of C for G at nucleotide 5587 (S1731C). Two FHA individuals carrying this variant on other alleles had much lower HDL cholesterol (0.155 ± 0.025) than FHA individuals from families not carrying this variant on other alleles. Have (0.64 ± 0.14, p = 0.000
9). This variant is also found in: One general population of French Canadian controls (0.92) with HDL in the 8th percentile, and also selected for low HDL cholesterol levels and coronary artery disease. It is one French-Canadian individual (0.72) from the identified population.

Substitution of A for G at nucleotide 2723 (I883M). This variant was found more frequently in individuals with early coronary artery disease of Dutch ancestry. In addition, homozygous carriers of this variant have significantly increased CAD progression compared to non-carriers.

Substitution of G for A at nucleotide 2868 (V825I). Carriers of this mutant had significantly more CAD events than individuals without this mutant.

Substitution of G for C at nucleotide -191. A homozygous carrier of this variant had a 3-fold increase in coronary event frequency (33.3% vs 11.2%, p = 0.00).
3) and almost twice the frequency of a clear family history of CAD (73.3% vs. 47.7%).
, P = 0.01).

Substitution of C for G at nucleotide-17. Carriers of this variant had significantly reduced coronary events (12.3% vs. 18.2%, p = 0.04) and significantly reduced incidence of myocardial infarction (heart attack, 43.6% vs. 52.8%, p = 0.02
) Has.

Substitution of C for T at nucleotide 69. Carriers of this variant have increased CAD progression compared to non-carriers.

Substitution of C for G at nucleotide 127. Carriers of this variant have a tendency towards a reduced progression of CAD compared to non-carriers.

Insertion of CCCT at nucleotide-1163 of intron 1. Carriers of this variant have a tendency to reduce HDL cholesterol levels.

Substitution of A for G at nucleotide-1095 of intron 1. Homozygous carriers of this variant have a tendency towards decreased HDL cholesterol levels and increased triglyceride levels compared to non-carriers.

Substitution of G for A at nucleotide-1027 of intron 1. Carriers of this variant are also carriers of G (-720) A. Thus, the effects due to the mutation may also be due to carriers of this mutant.

Substitution of G for A at nucleotide-720 of intron 1. Homozygous carriers of this mutant tended to have an increased frequency of overt familial history of myocardial infarction.

Substitution of A for C at nucleotide-461 of intron 1. The carrier of this mutant is also the carrier of A (-362) G. Thus, the effects due to the mutation may also be due to carriers of this mutant.

Substitution of A for G at nucleotide -362 of intron 1. Carriers of this variant have reduced triglyceride levels compared to non-carriers.

Insertion of G at nucleotide 319. Carriers of this variant have increased CAD compared to non-carriers.

Substitution of C for G at nucleotide 378. Carriers of this variant also have I
He is also a carrier of nsG319. Thus, the effects due to the variant may also be due to carriers of this variant.

Functional Role of LXRE Binding Sites in the ABC1 Genome Sequence The functional role of the three sites of the LXR consensus binding site identified in the ABC1 genome sequence of SEQ ID NO: 1 was determined using standard gel shift experiments. confirmed. In summary, -4389 and -1641 positions of the promoter region,
The sequence of the LXRE consensus binding site present at +4 of exon 1 and at −7670 and −7188 of 3 ′ intron 1 (FIG. 3) was described as a positive regulation by cyp7LXRE (Lehman et al., Journal of. Biology and Chemistry, 272: 3137-3140, 1
997) and tested by gel shift method.

In the first gel shift method, binding of the labeled cyp7LXRE probe was carried out with a 400-fold excess of unlabeled cyp7 probe, −4389 probe, −1641 probe, +4.
The probe or -7670 probe was used for competition. The signal disappeared completely in all cases.

For subsequent assays, the -4389 probe, -1641 probe, +4 probe, -7670 probe and -7188 probe are labeled to give LXR-R.
The binding to the XR complex was tested. A signal similar to the signal of positive regulation was observed for each of the ABC1 LXRE probes, but for the +4 probe, the signal was slightly stronger than those.

Competitive assays also used smaller amounts of each unlabeled probe, ie,
This was done using 5, 25 and 50 times more unlabeled probe than labeled cyp7 probe. The signal decreased in a dose-dependent manner for each probe. This decrease was more pronounced with the +4 and -7670 probes than the others. Furthermore, the signal was unchanged by competition with unlabeled Dr2-like probe. This suggests that the competition is indeed specific.

Therefore, each of the potential LXRE binding sites tested was in vitro LXR-.
It is thought to bind to the RXR heterodimer. +4 LXR in exon 1
The E binding site appears to have the greatest affinity, followed immediately by the -7660 LXRE binding site in 3'intron 1.

Agonists and Antagonists Useful therapeutic compounds include compounds that modulate ABC1 expression, activity or stability. To isolate such compounds, the expression, biological activity or regulated metabolism of ABC1 is measured after adding the candidate compound to the culture medium of ABC1-expressing cells. Alternatively, a candidate compound can be directly administered to an animal (eg, mouse, pig or chicken) and used to screen its effect on ABC1 expression.

In addition to its role in the regulation of cholesterol, ABC1 also binds ABC1
It is involved in various other biological processes in which the development of modulators is useful. In one example, ABC1 crosses the cell membrane and from outside the cell interleukin-.
Transports 1β (IL-1β). IL-1β is a precursor to the inflammatory response, so inhibitors or antagonists of ABC1 expression or biological activity include, but are not necessarily limited to, rheumatoid arthritis, systemic lupus erythematosus (SL).
E), may be useful in the treatment of any inflammatory disease including hypothyroidism or hyperthyroidism, inflammatory bowel disease and diabetes. In yet another example, macrophage-expressed ABC1 has been shown to be involved in dead cell encapsulation and clearance. The ability of macrophages to take up these apoptotic bodies is impaired after ABC1 is antibody-mediated blocked. Therefore, compounds that modulate ABC1 expression, stability or biological activity are useful for treating these diseases.

Expression of ABC1 can be achieved, for example, by the nucleic acid sequence of ABC1 (or a fragment thereof).
Is used as a hybrid format probe, or by Western blot using anti-ABC1 antibody and standard techniques. The expression level of ABC1 in the presence of the candidate molecule is compared to the level measured for the same cells in the same culture medium or in parallel groups of test animals, but in the absence of the candidate molecule. ABC1 activity can also be measured using a cholesterol efflux assay.

Transcriptional regulation of ABC1 expression ABC1 mRNA is increased approximately 8-fold when cholesterol is loaded. This increase is thought to be regulated at the transcription level. Transcription factors that bind to 5'regulatory sequences using the genomic sequences described herein are described in, for example, gel shift, DN.
It can be identified by performing an Ase protection assay or a reporter genotype assay in vitro or in vivo. The identified transcription factors, by themselves, drug compounds can be used for the regulation of HDL, the regulation of triglycerides, the prevention of atherosclerosis, and the treatment of cardiovascular diseases. For example, ABC
It is expected that the use of compounds that inhibit the transcription factor that regulates 1 results in upregulation of ABC1, and thus upregulation of HDL cholesterol levels and downregulation of triglyceride levels. In another example, a compound that increases expression or activity of a transcription factor is also
It is believed to increase ABC1 expression, increase HDL levels and decrease triglyceride levels.

Of particular relevance are the transcription factors known to regulate other genes in the regulation of the apolipoprotein gene or other cholesterol or lipid regulated genes. Such factors include, but are not necessarily limited to, steroid response element binding proteins (SREBP-1 and SREBP-2), and transcription factors for PPAR (peroxisome proliferative response receptor), RXR and LXR. Several consensus sites for a particular element
It is located in the sequenced region 5'to the ABC1 gene (Fig. 3), and therefore AB
It is considered that the expression of C1 is regulated. For example, LXR alters ABC1 transcription by various mechanisms, including forming heterodimers with various retinoid X receptors (RXRs) and then binding to various specific response elements (LXREs). Can be made. Examples of such LXRs include LXRα and LX
Rβ is included. Compounds that regulate transcriptional activity mediated by LXR are AB
It is believed that the expression of the C1 gene can be regulated and is therefore useful for regulating HDL cholesterol and triglyceride levels. Janowsky et al. (Bulletin of National Academy of Sciences, 96: 266-271, 199)
9) reported the role of naturally occurring oxysterols in promoter-mediated LXR-dependent transactivation of cholesterol 7a-hydroxylase (Cyp7a), the rate-limiting enzyme in bile acid synthesis. Janowsky further revealed that oxysterols bind directly to various LXRs. Regiospecific mono-oxidation of sterol side chains is required for high affinity binding and activation of LXR. Enhanced binding can be achieved through the use of 24-oxo ligands. The oxygen present at more than one carbon in the side chain of cholesterol reduced binding and activation of LXR compared to the monooxygenated analog. LXR
Has been found to require a stereoselective enzyme in the sterol side chain that functions as a hydrogen acceptor. Introduction of dimethylamide showed the greatest binding and activation compared to ester or carbonyl groups.

Compounds known to modulate LXR activity are not necessarily limited to 24
-(S), 25-epoxycholesterol; 24 (S) -hydroxycholesterol; 22- (R) -hydroxycholesterol; 24 (R), 25-epoxycholesterol; 22 (R) -hydroxy-24 (S), 25 -Epoxycholesterol; 22 (S) -hydroxy-24 (R), 25-epoxycholesterol; 24- (S), 25-iminocholesterol; methyl-38-hydroxycoronate; N, N-dimethyl-3β-hydroxycolon Amide; 24 (R) -hydroxycholesterol; 22 (S) -hydroxycholesterol; 22 (R)
, 24 (S) -dihydroxycholesterol; 25-hydroxycholesterol; 22 (R) -hydroxycholesterol; 22 (S) -hydroxycholesterol; 24 (S), 25-dihydroxycholesterol; 24 (R), 25-dihydroxycholesterol; 24,25-dehydrocholesterol; 25-epoxy-22 (R) -hydroxycholesterol; 20 (S) -hydroxycholesterol; (20R, 22R) -cholest-5-ene-3β, 20,22-triol; 4,4 -Dimethyl-5-α-cholesta-8,14,24-triene-
3-β-ol; 7α-hydroxy-24 (S), 25-epoxycholesterol; 7β-hydroxy-24 (S), 25-epoxycholesterol; 7-oxo-24 (S), 25-epoxycholesterol; 7α- Includes hydroxycholesterol; 7-oxocholesterol; and demosterol.

Additional LXR modulating compounds are described, for example, in Janowski et al., Nature, 383.
Volume: 728-731, 1996; Lehmann et al., Journal of Biology and Chemistry, 272: 3137-3140, 19
1997; See also Janowsky et al., Bulletin of the National Academy of Sciences, Volume 96: 299-2.
P. 71, 1998, each of which is incorporated herein by reference. In addition, one of skill in the art will recognize that synthetic sterols with LXR modulating activity can be readily identified using screening methods known in the art (eg, Janowsky et al., Bulletin of the National Academy of Sciences, 96: 266).
-271, 1998). RIP140 protein, LXR
Non-steroidal agonists such as (monoclonal or polyclonal) antibodies specific for α or LXRβ; tetradecyclooxy-flunacarboxylic acid (T
OFA); tetradecylthioacetic acid; also other fatty acids (eg Tobin et al., Mo
lec. Endocrin. 14: 741-752, 2000), etc. are also useful LXR modulators.

Additional transcription factors that further regulate ABC1 gene expression, thereby affecting HDL levels, triglyceride levels, atherosclerosis, and CAD risk are REV.
-ERBα, SREBP-1 and 2, ADD-1, EBPα, CREB binding protein, P300, HNF4, RAR, and RORα. Typical binding sites are shown in Figure 3. Additional binding sites for these factors are eg SEQ ID NO: 1
Can be found through examination of the sequence of

RXR heterodimerizes at many nuclear receptors, including LXR, helping to transtranscribe activation of target genes. Thus, a compound that regulates RXR-mediated transcriptional activity will further regulate ABC1 expression. A large number of RXR-modulating compounds (retinoid compounds) are known in the prior art, for example, heteroethylene derivatives / tricyclic retinoids; triene retinoids; benzocycloalkenylalkyls; diene or trienoic acid derivatives, tricyclic aromatic compounds and the like. Derivatives; Bicrylmethyl allyl acid derivatives; Phenylmethyl heterocyclic compounds; Tetrahydronaphthyl compounds; Allylthio-tetrahydro-naphthalene derivatives and heterocyclic analogues; 2,4
-Pentadienoic acid derivatives; Tetralin-based compounds; Nonatetraenoic acid derivatives; SR11237; Dexamethasone; Mesoprene hydroxy, epoxy, and carboxy derivatives; Bicyclic benzyl, pyridinyl, thiophene, furanyl, and pyrrole derivatives; Benzofuran acrylic acid derivatives; Allyl substitution and Allyl and (3-oxo-1-propenyl) -substituted benzopyrans, benzothiopyrans, 1,
2-dihydroquinoline and 5,6-dihydronaphthalene derivatives; vitamin D
3 (1,25-dihydroxyvitamin D3) and analogs; 24-hydroxylase inhibitors; mono- or polyenecarboxylic acid derivatives; tetrahydroquinoline-
2-One-6 or 7-yl and related derivatives; tetrahydronaphthalene; oxyiminoalkane acid derivatives; LG 100268; and LG D 1069.
including. Additional compounds are BRL 49653; troglitazone; pioglitazone;
WAY-120; Englitazone; AD5075; and Dalglitazone.

PPARs are ABC by a mechanism involving heterodimerization at retinoid X receptors (RXRs) and then binding to specific proliferative response elements (PPREs).
Change the transcription of 1. Examples of such PPARs are PPARs α, β, γ and δ.
including. These different PPARs have been shown to have transcriptional regulatory effects on different genes. PPARα is mainly expressed in liver and PPARγ is mainly expressed in adipocytes. PPARα and PPARγ are found in coronary and carotid atherosclerotic plaques and in endothelial cells, smooth muscle, monocytes and monocyte-derived macrophages.
Activation of PPARα is associated with lipoprotein lipase (LPL), apolipoprotein CIII (apoIII) and apolipoprotein AI (apoAI) and AII.
It produces denatured lipoprotein metabolism through the action of PPARα on genes such as (Apo II). PPARα activation results in overexpression of LPL and apoA-I as well as apoA-II, but inhibits apoCIII expression. PPARα activation further inhibits therapy, stimulates lipid oxidation, results in liver uptake and esterification of free fatty acids (FFAs). The activity of PPARα and PPARγ inhibits nitric oxide (NO) synthase in macrophages, and IL-6 interleukin-1 (I
L-1) inducible expression and cyclooxygenase-2 (COX-2) and NF-KB
Prevents activation of the thrombin-induced endothelin-1 expression and protein 1 signaling pathways secondary to the negative transcriptional regulation of A. It was also shown that PPARα induces monocyte-induced macrophage apoptosis through inhibition of NF-KB activity.

Activation of PPARα can be achieved with compounds such as fibrates, β-estradiol, arachidonic acid derivatives, WY-14, 643 and LTB4 or 8 (s) HETE. Activation of PPARγ is a thiozolidinedione antidiabetic, 9-
This can be achieved through compounds such as HODE and 13-HODE. Additional compounds such as nicotinic acid or HMG CoA reductase inhibitors also alter the activity of PPARs.

Compounds that alter the activity of any PPARs (eg, PPARα or PPARγ) affect ABC1 expression and therefore HDL levels, triglyceride levels,
Can affect the risk of atherosclerosis and CAD. PPARs are further regulated by fatty acids (including modified fatty acids such as 3 thia fatty acids), leukotrienes such as leukotriene B4, and prostaglandin J2, a naturally active compound / ligand of PPARγ. Agents that regulate PPARs therefore have important effects on regulated lipid levels (including HDL and triglyceride levels) and altered CAD risk. This effect could be achieved through regulation of ABC1 gene expression. The drug also acts indirectly on PPARs via other transcription factors such as adipocyte differentiation determinant 1 (ADD-1) and sterol regulatory element binding proteins 1 and 2 (SREBP-1, SREBP-2). , ABC1 gene expression and thus acts on HDL and triglyceride levels. Agents that combine PPARα and PPARγ agonist activity given in combination with the Examples, or PPARα and PPARγ agonists given in combination with the Examples will further increase HDL levels or reduce triglyceride levels.

The PPAR binding site (PPRE element) is found 5'of the ABC1 gene (Figure 3). Similar to the PPRE element found in the C-ACS, HD, CYP4A6 and ApoAI genes, this PPRE site is a trimer associated with the PPRE consensus sequence. This element among others appears to be of great physiological relevance for the regulation of the ABC1 gene, due in part to the similarities in number and sequence of repeats at this PPAR binding site.

ABC1 Polypeptides: Nucleic Acids, and Additional Utility of Modulators ABC1 is a toxic protein or protein fragment (eg, APP) from cells.
Acts as a transporter of. Thus, ABC1 agonists / upregulators are useful in the treatment of other disease areas including Alzheimer's disease, Niemann-Pick disease and Huntington's chorea.

It has been shown that the ABC transporter increases the uptake of long chain fatty acids from the cytosol to the peroxisomes and plays a role in β-oxidation of even longer chain fatty acids. Importantly, fatty acid metabolism in X-linked adrenoleukodystrophy (ALD) is abnormal due to defects in the peroxisomal ABC transporter. Any agent that upregulates ABC transporter expression or biological activity would therefore be useful in the treatment of ALD or any other lipid disorder.

ABC1 is expressed in macrophages and requires subsidence of cells that undergo programmed cell death. The apoptotic process itself and its regulation have important implications for diseases such as cancer, which is one of the mechanisms of cell damage that properly undergoes cell death. ABC1 promotes apoptosis and thus represents an intervention point in cancer treatment. Upregulation of ABC1 expression or activity, or ABC1 by either method, enhances apoptosis, thus potentially reducing aberrant cell proliferation characterized by this disease, thus building a therapeutic approach against cancer. Conversely, down-regulation of ABC1 by either method reduces apoptosis and allows increased cell proliferation in conditions where cell growth is restricted. Such disorders include, but are not necessarily limited to, nerve defects and degeneration, and developmental disorders. Therefore A
BC1 can potentially be used as a method for the identification of compounds used in the treatment of cancer, or the treatment of degenerative diseases.

Agents shown to inhibit ABC1 include, for example, the diabetes drugs glibenclamide and gluburide, flufenamic acid, diphenylamine-2-carboxylic acid, sulfobromophthalein, and DIDS.

Agents that upregulate ABC1 expression or biological activity include, but are not necessarily limited to, protein kinase A, protein kinase C, vanadate, okadaic acid, and IBMX1.

Those skilled in the art will recognize that other compounds can also modulate ABC1 biological activity, which other compounds are also within the spirit of this invention.

Drug Screens Based on the ABC1 Gene or Protein The ABC1 protein and genes can be used in screening assays to identify compounds that are potential drugs that regulate their activity and also regulate cholesterol or triglyceride levels. ABC15 'regulatory sequences and other regulatory sequences (
For example exon 1 and exon 2) regulate ABC1 expression and also for example HDL
It can be used in screening assays to identify compounds that are potential agents that regulate lipid levels, including -C, LDL-C, and triglycerides. A
A drug screen that identifies compounds that modulate BC1 expression employs an ABC1 regulatory region operably contiguous with ABC1. However, desirably, the regulatory region is operably contiguous with a reporter gene (eg, a gene encoding GFP, chloramphenicol transferase, or beta-galactosidase).

Useful ABC1 proteins include wild-type and mutant ABC1 proteins or protein fragments in recombinant form or expressed endogenously. Drug screens that identify compounds that act on ABC1 expression products take advantage of any functional properties of the protein. In one example, phosphorylation function or other post-translational repair is also monitored as a measure of ABC1 bioactivity. ABC1 has an ATP binding site and thus the assay tests, in whole or in part, the ability of ABC1 to bind ATP in whole or in part or to display adenosine triphosphatase (ATPase) activity. ABC1 is considered to be able to form a channel-like structure by analogy with similar proteins. Drug screening assays are based on assaying the ability of a protein to form a channel, or on the basis of the ability to transport cholesterol or also one molecule, or of other proteins that are bound or regulated by ABC1 to form a channel. Could be based on ability. Alternatively, phospholipids or lipid transport can also be used as a measure of ABC1 bioactivity.

In addition to its role as a modulator of cholesterol levels, there is evidence that ABC1 also transports anions. Functional assays are based on this property, and for example (but not necessarily limited to) the ability of various dyes to change color in response to changes in specific ion concentrations in such assays, in vesicles such as ribosomes. Drug screening techniques can be employed that can be performed or adapted to use whole cells.

Drug screening assays can also be based on the ability of ABC1 or other ABC transporters to interact with other proteins. Such interacting proteins can be identified by various conventionally known methods including, for example, radioimmunoprecipitation method, coimmunoprecipitation method, copurification method, and enzyme two-hybrid screening method. Such interactions can be further assayed by, but not necessarily limited to, fluorescence polarization or scintillation proximity. The drug screen also shows the function of ABC1 protein estimated by X-ray crystallography of the protein, and part 3
It can be based on comparison with that of proteins for which the function of the D structure is known. Such a crystal structure was determined to be HisP, a histidine permease, which is a member of the prokaryotic AB family. Drug screens can be based on the apparent function or properties for the generation of transgenic or knockout mice, or overexpression of proteins or protein fragments in mammalian cells in vitro. Moreover, expression of mammalian (eg, human) ABC1 in yeast or C. elegans screens candidate compounds in wild-type and mutant backgrounds, as well as screens for mutations that enhance or suppress the ABC1-dependent phenotype. to enable. The modification screen can also be performed in transgenic mice or knockout mice.

In addition, drug screening assays can be based on ABC1 function inferred from antisense interference in gene function. The effects of changes in ABC1 subcellular localization or protein subcellular localization can be used as assays for drug screening. Immunocytochemistry will be used to determine the precise location of the ABC1 protein.

Human and murine ABC1 proteins can be used as antigen-enhancing antibodies, including monoclonal antibodies. Such antibodies are useful for a variety of purposes including, but not necessarily limited to, functional research and development of drug screening assays and diagnostics. Monitoring the effects of agonists (eg, drug compounds, etc.) on ABC1 expression or biological activity can be applied to clinical trials as well as basic drug screening. For example, the effect of an agonist, as determined by the screening assays described herein to increase ABC1 gene expression, protein level, or biological activity, is in clinical trials in subjects exhibiting altered ABC1 gene expression, protein level, or biological activity. You can monitor. Alternatively, the effect of the agonist, as determined by a screening assay that regulates ABC1 gene expression, protein level, or biological activity, can be monitored in clinical trials in subjects who exhibit altered ABC1 gene expression, protein level, or biological activity. . In such clinical trials, for example, the expression or activity of ABC1 and preferably other genes involved in cardiovascular disease can be used to ascertain the effect of a particular drug.

For example, and without limitation, by treatment with an agent (eg, compound, drug or small molecule) that modulates ABC1 biological activity (eg, confirmed in a screening assay as described herein). It is possible to identify genes including ABC1 that are regulated in cells. Thus, in order to study the effects of agonists on cholesterol levels, triglyceride levels or cardiovascular disease, eg in clinical trials, cells were isolated, RNA regulated and expression levels of ABC1 and other genes associated with disease were analyzed. . The level of gene expression can be determined by Northern blot analysis or RT-
It can be quantified by PCR or by measuring the amount of protein produced as an option, by one of a number of conventionally known techniques, or by measuring the level of biological activity of ABC1 or other genes. . Thus gene expression can serve as a marker for the physiological response of cells to agonists.
Thus, this response function is determined prior to treatment of the individual with the agonist and at each time point during treatment.

In a preferred embodiment, the present invention employs agonists (eg, agonists, antagonists, peptidomimetics, proteins, peptides, nucleic acids, small molecules, or drug candidates identified in the screening assays described herein). A method of monitoring the effect of treatment of a subject, which comprises the following steps: (i) securing a pre-dose sample from the subject prior to administration of the agonist, and (ii) AB of the pre-dose sample.
The expression level of C1 protein, mRNA or genomic DNA is detected, and (ii
i) obtaining one or more samples after administration from a subject, (iv) detecting the expression level or activity level of ABC1 protein, mRNA, or genomic DNA in the sample after administration, and (v) ABC1 in the sample before administration. ABC1 protein in a sample after administration of expression or activity levels of protein, mRNA or genomic DNA,
comparing to that of the mRNA, or genomic DNA, and (vi) thus altering the administration of the agent to the subject. For example, increasing doses of agonist are desirable to increase ABC1 expression or activity at levels higher than that detected, ie, increase the effect of the agonist. As an option, a reduced dose of agonist is desirable to reduce the expression or activity of ABC1 at low levels below that detected.

The ABC1 gene or fragments thereof can be used as a tool to express the protein in appropriate cells in vitro or in vivo (gene therapy), or the large amount of ABC1 protein used in in vitro assays for drug screening can be used. It can be cloned into an expression vector that can be used to produce. Expression systems employed include eukaryotic systems such as baculovirus, herpes virus, adenovirus, adeno-associated virus, bacterial systems and CHO cells. Naked DNA
And DNA liposome complexes can also be used.

Assays for ABC1 activity include binding to intracellular interacting proteins, interacting with proteins that upregulate ABC1 activity, interacting with HDL particles or constituents, interacting with HDL or its constituents. Includes measurements of facilitating interactions with other proteins, and cholesterol efflux. Furthermore, the assay is based on the molecular dynamics of macromolecules, metabolism and ions with fluorescent protein biosensors. Alternatively, the effect of candidate modulators on expression or activity is at the level of ABC1 protein production using the same general approach combined with standard immunodetection methods such as Western blotting with ABC1-specific antibodies or immunoprecipitation. To be measured. Also useful cholesterol modulatory, triglyceride modulatory or anti-CVD therapeutic modulators are identified as producing alterations in ABC1 polypeptide production. The agonists also influence the ABC1 activity without any effect on the expression level.

Candidate modulators are components of a purified (or in effect purified) molecule or mixture of compounds (eg, extracts or supernatants obtained from cells). In a mixed compound assay, ABC1 expression was pooled of candidate compounds (eg, produced by standard purification techniques, eg, Ausubel et al., HPLC or FPLC) until a single compound or minimal compound mixture was shown to modulate ABC1 expression. Are tested on a progressively smaller subset of.

It has been discovered that agonists, antagonists or mimetics that are effective in modulating the level of cellular ABC1 expression or activity are useful in animal models (eg mouse, pig, rabbit or chicken). For example, the compounds improve low HDL levels of low alpha lipoproteinosis in mice or chickens or reduce triglyceride levels in animal models.

Compounds that promote increased ABC1 expression or activity are considered particularly useful in the invention. Such molecules are used, for example, as therapeutic agents to increase the level or activity of native cell ABC1 and thereby treat low HDL conditions in animals (eg humans). If desired, treatment with an agonist of the invention is combined with any other HDL-increasing, triglyceride-lowering or anti-CVD therapy.

One way to increase ABC biological activity is to increase the stability of the ABC protein or prevent its degradation. Thus, it would be useful to identify mutations in ABC polypeptides (eg, ABC1) that lead to increased protein stability. These mutations can be incorporated into any protein or gene therapy that is taken to address the abnormalities resulting from low HDL-C or any other loss of ABC1 biological activity. Similarly, compounds that increase the stability of a wild-type ABC polypeptide or reduce its catabolic activity are also useful in treating disorders resulting from low HDL-C or any other loss of ABC1 biological activity. Such mutations and compounds can be identified using the methods described herein.

In one example, cells expressing an ABC polypeptide having a mutation are transitionally metabolically labeled during translation and the half-life of the ABC polypeptide is determined using standard techniques. A mutation that increases the half-life of an ABC polypeptide is a mutation that increases the stability of the ABC protein. These mutations can then be evaluated for ABC biological activity. They can also be used to identify proteins that affect the stability of ABC1 mRNA or protein. Compounds that act on these factors or the ability of these factors to bind to ABC1 can then be assayed.

In another embodiment, cells expressing wild-type ABC polypeptide are metabolically labeled transitionally during translation and contacted with a candidate compound, and the half-life of the ABC polypeptide is determined using standard techniques. It is determined. Compounds that increase the half-life of ABC polypeptides are useful compounds in the present invention.

While ABC1 is the desired ABC transporter for the drug screens described herein, it is understood that other ABC transporters can also be used. ABC
Replacing 1 with one ABC transporter would mean ABC2, ABCR, or ABC
This is possible because 8 etc. will have similar regulatory mechanisms.

[0237]   Representative assays are described in particular detail below.

Protein-based Assays ABC1 polypeptides (purified or unpurified) also have the ability to bind one protein (including but not necessarily limited to proteins found to specifically interact with ABC1). Can be used in a test to determine The effect of the compound on its binding is then determined.

Protein Interaction Assays ABC1 protein (or polypeptide fragments or epitope-tagged forms or fragments thereof) can be immunoprecipitated from a suitable source (eg, from prokaryotic expression systems, eukaryotic cells, cell-free systems, or from ABC1 expressing cells). Harvested). The ABC1 polypeptide is then bound to a suitable support, such as nitrocellulose or an antibody or metal agarose column in the case of his labeled form of ABC1. Binding to the support material is preferably performed under conditions such that the protein associated with the ABC1 polypeptide remains associated with it. Such conditions include the use of buffers that minimize interference with protein interactions. The binding step can be performed in the presence or absence of the compound whose ability to interfere with the interaction between ABC1 and other molecules is tested. If desired, other proteins (eg, cell lysates) are added, allowing time for binding to the ABC polypeptide. The immobilized ABC1 polypeptide is then washed to remove proteins or other cells that bind non-specifically to the polypeptide or support material.
The immobilized ABC1 polypeptide is then dissociated from the support material so that the protein bound to it is released (eg by heating), or alternatively the bound protein is released from ABC1 without releasing the ABC1 polypeptide from the support material. Is released. Released proteins and other cellular constituents can be analyzed by, for example, SDS-PAGE gel electrophoresis, Western blotting and detection with specific antibodies, phosphoamino acid analysis, protease digestion, protein sequencing, or isoelectric focusing. . Normal and mutant forms of ABC1 can be employed in these assays to gain additional information as to which part of ABC1 a given factor binds. In addition, when incompletely purified polypeptide is employed, comparison of normal and mutant forms of the protein can be used to help distinguish true binding proteins.

The assays described above can be performed using purified or semi-purified or other molecules known to interact with ABC1. This assay includes the following steps: 1. Harvest the ABC1 protein and attach an appropriate fluorescent label to it. The interacting protein (or other molecule) is labeled with a second different fluorescent label. Different extinction patterns when the dyes are very close to each other and when they are physically separated (ie dyes that quench each other when the dyes are close to each other and not when they are not close) 2. using a dye that produces 3. exposing the interacting molecule to immobilized ABC1 in the presence or presence of the compound tested for its ability to interfere with the interaction between these two; Collecting fluorescence readout data is a step.

Another assay is the fluorescence resonance energy transfer (FRET) assay. This assay can be performed as follows.

1. Providing ABC1 protein or suitable polypeptide fragment thereof, suitable F
1. attaching a RET donor (eg nitrobenzoxadiazole (NBD)) to it, 2. labeling the interacting protein (or other molecule) with a FRET receptor (eg rhodamine); 3. exposing the acceptor labeled interacting molecule to the donor labeled ABC1 in the presence or absence of the compound tested for its ability to interfere with the interaction between the two; Fluorescence resonance energy transfer is measured.

The quenching assay and the FRET assay are related. Both are applicable when given depending on which fluorophore (fluorescent label) pair was used in the assay.

Membrane Permeability Assay ABC1 protein can be further tested for its effect on membrane permeability. Beyond its putative capacity to replace lipids, for example, ABC1 affects membrane permeability to ions. Other membrane associated proteins, the most prominent ones, cystic fibrosis transmembrane conductance regulator and sulfonylurea receptors, associate with and regulate ion channels.

ABC1 or a fragment of ABC1 is incorporated into synthetic vesicles, or, optionally, expressed in cells and vesicles, or other cellular substructures containing ABC1 are isolated. ABC1-containing vesicles or cells are loaded with a reporter molecule that can detect ions (to observe outward movement), such as a fluorescent ion supporter that alters its fluorescent properties when it binds to a special ion. Or as an option,
The external medium is loaded with such molecules (to observe the inward movement). Molecules that exhibit different properties when the molecule is inside the vesicle are desirable than when the molecule is outside the vesicle. For example, molecules with quenching properties that do not exist when the molecule is at high concentration, but also at one low concentration, would be suitable. The movement of loaded molecules (either the ability to move or the kinematics of movement) can be determined in the presence or absence of the tested chemical for their ability to affect the process.

In another assay, membrane permeability is determined electrophysiologically by measuring ABC1 mediated or regulated ion influx or efflux by standard electrophysiological techniques. Appropriate controls (eg TD cells or cell lines with very low endogenous ABC1 expression) can be used as controls in assays to determine whether the observed effect is specific to cells expressing ABC1. .

Furthermore, the uptake of radioisotope into or out of the vesicles can be measured in one assay. The vesicles are separated from the extravesicular medium and the radioactivity within and within the vesicles is quantified and compared.

Nucleic acid based assay The ABC1 nucleic acid is used in an assay based on binding of factors required for ABC1 gene transcription. The binding between ABC1 DNA and binding agent is assessed by any system that discriminates between protein binding and protein binding DNA (eg gel shift assay). The effect of compounds on the binding of factor ABC1 DNA is assessed by such an assay. In addition to the in vitro binding assay, an in vivo assay in which the regulatory region of the ABC1 gene is linked to the reporter gene is performed as well.

Assays for measuring ABC1 stability Cell-based or cell-free systems can be used to screen for compounds based on their effect on the half-life of ABC1 mRNA or ABC1 protein.
This assay employs labeled mRNA or protein. ABC1 as an option
mRNA is detected by specifically hybridizing probes or quantitative PCR assays. The protein is quantified, for example, by the fluorescent antibody basting method.

In vitro mRNA stability assay 1. 1. isolation or production of a suitable amount of ABC1 mRNA by in vitro transcription; 2. labeling ABC1 mRNA, 3. Exposing aliquots of mRNA to cell lysates in the presence or absence of compounds tested for their ability to modulate ABC1 mRNA stability, The intact state of residual mRNA is assessed at appropriate time points.

In vitro protein stability assay 1. 1. expressing an appropriate amount of ABC1 protein, 2. labeling the protein, 3. exposing an aliquot of the labeled protein to cell lysates in the presence or absence of the compound tested for its ability to modulate ABC1 protein stability, The intact state of residual mRNA is assessed at appropriate time points.

In vivo mRNA or protein stability assay 1. Cells expressing ABC1 mRNA or protein with tracer (radiolabeled ribonucleotide or radiolabeled amino acid, respectively) in a very short time (eg within 5 minutes) in the presence or absence of a compound whose action on mRNA or protein is regulated Keep warm, 2. 2. Incubate with unlabeled ribonucleotides or amino acids, ABC1 mRNA or protein radioactivity is quantified at time intervals beginning at the start of step 2 until the time when the radioactivity of ABC1 mRNA or protein is reduced by about 80%. Intact or almost intact mRN from radioactive degradation products by means such as gel electrophoresis for quantifying mRNA or protein
It is desirable to separate A or protein.

Assay variants measuring the inhibition of preferential negative activity The mutant ABC1 polypeptides have preferential negative activity (ie wild type ABC1).
Seems to have activity that interferes with function). Assays for compounds capable of interfering with such a variant are based on any method of quantifying normal ABC1 activity in the presence of the variant. For example, normal ABC1 will promote cholesterol efflux and preferential negative mutants will interfere with this effect. The ability of the compound to interfere with the action of the preferred negative mutant is based on cellular cholesterol efflux or on other normal activities of wild-type ABC1 that were made repressible by the mutant.

Assay for Measuring Phosphorylation Glu89 of the wild-type chicken ABC1 polypeptide appears to be part of the phosphorylation motif, thus this by E → K ABC1 in WHAM chickens (discussed further below). Elimination of the phosphorylation motif is due to WHAM chicken AB
Caused by a decrease in the biological activity of C1. Thus, compounds that regulate the phosphorylation state of ABC1 are considered to be clinically relevant modulators of human ABC1 activity.

The effect of compounds on ABC1 phosphorylation can be assayed by the method of measuring phosphate for assessing the phosphorylation status of proteins or specific residues of ABC1. Such methods include, but are not necessarily limited to ³² P labeling and immunoprecipitation, detection with anti-phosphoamino acid antibodies (eg anti-phosphoserine antibodies),
Includes phosphoamino acid analysis on two-dimensional TLC plates and protease digestion fingerprinting of proteins, followed by detection of ³² P-labeled fragments.

Assays for Measuring Other Post- Translational Modifications The effects of compounds on post-translational modifications of ABC1 are based on any method by which a particular modification can be quantitated. For example, the effect of compounds on glycosylation is assayed by treating ABC1 with glycosylase and measuring the amount and nature of the released charcoal compound.

Assay for Measuring ATP Binding The ability of ABC1 to bind ATP provides one assay that screens for compounds that affect ABC1. ATP binding can be measured as follows.

1. The ABC1 protein is provided at the appropriate purity level and it is reconstituted into lipid vesicles.

2. Vesicles are exposed to ATP analogs (eg gamma 35S-ATP) that are labeled but not hydrolyzed in the presence or absence of compounds tested for their effect on ATP binding. The azido ATP analog allows covalent attachment of the azido ATP protein (by ultraviolet light), making it easier to quantify the amount of ATP bound to the protein.

[0260]   3. The amount of ATP analog that binds ABC1 is quantified.

Assay for Measuring ATPase Activity A quantification of ATPase activity of ABC1 can be assayed for the effect of the compound on ABC1. This desirably causes ABC1 to intracellularly bind to many other ATP
It is performed in a cell-free assay similar to separating from ase. The ATPase assay is performed in the presence or absence of a membrane and with or without the incorporation of ABC1 protein into the membrane. If performed in a vesicle-based assay, the ATP hydrolyzate produced or hydrolyzed ATP is measured either intravesicularly or extravesicularly, or both. Such assays are based on the disappearance of ATP or the appearance of ATP hydrolysis products.

For high throughput screening, a binding ATPase assay is desirable. For example, a reaction mixture containing pyruvate kinase and lactate dehydrogenase can be used. The mixture comprises phosphoenolpyruvate (PEP), nicotinamide adenine dinucleotide (NAD ⁺ ), and ATP. ABC1 ATP
The ase activity purifies ADP from ATP. ADP is then returned to ATP as part of the pyruvate kinase reaction. The latter reaction produces dyed quinone (NADH) from the colorless substrate (NAD ⁺ ), and the whole reaction can be monitored by detection of a color change in the formation of NADH. Since ADP limits pyruvate kinase, this binding system accurately monitors the ATPase activity of ABC1.

Assays for Measuring Cholesterol Efflux Transport-based assays can be performed in vivo or in vitro. For example, the assay is based on some of the reverse cholesterol transport processes that can be easily recreated in culture, such as cholesterol or phospholipid efflux. As an option, the assay is based on whole-body net cholesterol transport assessed by labeling substances (such as cholesterol).

For high throughput fluorescent lipids can be used to measure ABC1 catalytic efflux. For phosphoric acid lipids, the fluorescent precursor C6-NBD-phosphatidic acid can be used. This lipid is dephosphorylated by phosphatidic acid phosphohydrolase taken up by cells. This product, NBD-diglyceride, then becomes the precursor for the synthesis of glycerophospholipids such as phosphatidylcholine (lecithin). NBD-phosphatidylcholine efflux can be monitored by detection of fluorescence resonance energy transfer (FRET) of NBD to appropriate receptors in cell culture medium.
This receptor can be rhodamine labeled phosphatidylethanolamine, a phospholipid that is not readily taken up by cells. The use of short chain precursors obviates the need for phospholipid transport proteins in the medium. NB for cholesterol
D-cholesterol ester can be reconstituted into LDL. LDL can effectively transport this lipid to cells through the LDL-receptive pathway. NBD Cholesterol ester is hydrolyzed to lysosomes and returned to the plasma membrane to allow NB outflow.
It produces D-cholesterol. Efflux can be monitored by the FRET assay described above in which the NBD transfers its fluorescence resonance energy to the rhodamine phosphatidyl ethanoline receptor.

Animal Model Systems Compounds identified as having activity in any of the above assays are then screened in any available animal model system including, but not necessarily limited to, pig, rabbit, WHAM chicken. . Test compounds are administered to these animals according to standard methods. The test compound is further tested in mice carrying a mutation in the ABC1 gene. Furthermore, the compounds are ABC1, ApoAI, ApoA
II, or their ability to enhance interactions between any ADL particles that make up ApoE, etc. is screened.

Cholesterol Efflux Assay as a Drug Screen The Cholesterol Efflux Assay measures the ability of cells to carry cholesterol to extracellular receptor molecules and is dependent on ABC1 function. In this procedure, cells are loaded with radiolabeled cholesterol by any of several biochemical pathways (Marcil et al., Ar.
terioscler. Thromb. Vasc. Biol. Volume 19: 159-
169 pages. 1999). Cholesterol efflux is then measured after incubation for various times (typically 0-24 hours) in the presence of HDL3 or purified ApoAI. Cholesterol efflux is measured as a percentage of total cholesterol in the culture medium after various incubation times. ABC1 expression levels and / or bioactivity are associated with increased efflux, while decreased levels of ABC1 are associated with decreased cholesterol efflux.

This assay can be readily adapted to the format used for drug screening, which consists of a multi-well (eg 96 well) format. Modifications of the assay that optimize it for drug screening include reducing and simplifying the procedure, modifying labeling methods, using different cholesterol receptors, changing incubation times, and changing cholesterol calculation methods. In all of these cases, the cholesterol efflux assay remains the same in its concept, although it modifies the experiment.
Transgenic mice overexpressing ABC1 are expected to have higher than normal HLD levels.

Knockout Mouse Models Animals such as mice with one or both ABC1 alleles inactivated (eg, by homologous recombination) have low HDL-C levels and higher than normal triglyceride levels. It appears to be a desirable animal model for screening compounds that appear to thus increase HDL-C levels and lower triglyceride levels. Such animals can be produced by standard methods. In addition to initial screening of test compounds, animals carrying the mutant ABC1 gene can be further tested for efficacy and stability of the first identified drug or agonist using one of the other screening methods described herein. Useful for. Cells removed from the animal and placed in culture can also be exposed to the test compound. HDL-C and triglyceride levels can be measured using standard techniques as described herein.

WHAM Chickens: Animal Model for Low HDL Cholesterol Wisconsin Low Alpha Mutant (WHAM) chickens were generated by spontaneous mutation in a closed group. Mutant chickens attracted attention through the Z-linked white shank and white beak phenotype, referred to as "recessive white skin" (McGibbon, 1981), and were subsequently found to be largely devoid of HDL (
Penama et al., 1990).

This chicken low HDL locus (Y) is Z-linked or sex-linked (in birds, ZW for females and ZZ for males). On the genetic map, the Y locus is the Z chromosome proximal to the ID locus (Bitgood, 1988) (Bitgood, 1985).
It was placed on the long arm of. Chicken Genome Mapping Project, Chick Map (retained at Roslin Institute; http: //www.ri.bbsrc.
ac. uk / chickmap / ChickMapHomePage. html
) Present mapping data showed that the region of synteny with human chromosome 9 is in the long arm of chicken Z chromosome (Zq) proximal to the ID locus. Evidence for this region of synteny is due to the chicken aldolase B locus (ALD) within this region.
OB) position. The human ALDOB locus is located on chromosome 9q22.3 not far from the position of human ABC1 (The Genome Database, http: ///
gdbwwv. gdb. org. /). This map is compared with the chicken ALDOB
It has been shown that the nearby chicken Zq region and the human 9q region near human ALDOB represent the synteny region between human and chicken.

Since the low HDL loci are located in the 9q position in humans and in the Zq region in chickens, these HDL loci are mostly located within the synteny region. Thus we have predicted that ABC1 is mutated in WHAM chickens. In support of this, we have previously identified an E⇒K mutation at a position corresponding to amino acid 89 of human ABC1. This non-conservative substitution is in a conserved position between human, mouse, and chicken, indicating that it is in a region of the protein that appears to have functional importance.

The discovery of a WHAM mutation in the amino-terminal portion of the ABC1 protein also demonstrates the importance of the amino-terminal region. This region is important because it is associated with cholesterol efflux or other proteins required to carry the associated task. It is an important regulatory region (the phosphorylation site for casein kinase near the mutated residue), or it helps direct the precise phase relationship with the cell membrane (
The N-terminal 60 amino acid region contains a putative membrane span or membrane-associated segment).

The amino acid terminal region of the protein (up to the first 6-TM region at about amino acid 639) is an ideal tool for screening factors that affect ABC1 activity. To test the interference of normal ABC1 function with truncated proteins, it is
C1 wild type cells can be expressed as any truncated protein. If the fragment were to act in a dominant negative manner, it could be used in immunoprecipitation to identify proteins that compete away from normal endogenous proteins.

The C-terminus is also amenable to experiments similar to those performed by the intracellular portion of the molecule, expressed in fragments or labels in the absence of the transmembrane region or as a fusion protein.

Because it is possible that there are several genes in the human genome that affect cholesterol efflux, any animal model used for human genetic disease represents a homologous locus in that animal and may perform similar functions. It is important to prove that they do not represent the different loci they carry. The above evidence establishes homology between the chicken Y locus and the human chromosome 9 low HDL locus. WHAM chickens thus represent an important animal for the identification of drugs that regulate cholesterol efflux.

However, the HDL-deficiency syndrome of WHAM chickens reflects the short lifespan of chickens. We propose the WHAM chicken as a model of human low HDL for the development and testing of agents that enhance human HDL. Such models may be employed in several forms through the use of these chicken cells or other derivatives, or by using the chicken itself for testing drug efficacy, toxicity, and other drug development purposes. did it.

Therapy Without necessarily limited to ABC1 polypeptides, ABC1 nucleic acids, other ABC transporters, LXR modulating compounds, RXR modulating compounds, and ABC1 organisms identified using any of the methods disclosed herein. Any therapeutic agent that modulates activity or expression is administered in a unit dosage form with a pharmaceutically acceptable diluent, carrier, or excipient. Conventional pharmaceutical practice is employed to provide suitable formulations or compositions to administer such compositions to patients. Any suitable route of administration, such as intravenous, parenteral, subcutaneous, intramuscular, skull, intraorbital, intraocular, intraventricular, intracapsular, intrathecal, intrathecal, intraperitoneal, intranasal , Aerosol or oral administration is employed. The therapeutic formulation is in the form of an aqueous solution or suspension. For oral administration, the formulation is in the form of tablets or capsules. In nasal formulations it is a powder, nasal drops or aerosol.

Well-known methods of preparing prescriptions are described in, for example, Remington: Science and Practice of Pharmacy (19th Edition). A. R. Genero edition. Found in the Mack Publishing Company, Pennsylvania, Easton, 1995. The formulation for parenteral administration is
Examples include excipients, sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalene. Biocompatible, biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyelelene-polyoxypropylene copolymers are used to control the release of compounds. Other potentially useful parenteral delivery systems for the agents of this invention include ethylene vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation include, for example, excipients such as lactose, or aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or in the form of drops for the nasal cavity or as a gel. It is an oily solution to administer.

Compounds In general, novel agents for the treatment of abnormal lipid levels and / or CVD are identified from large libraries, both natural products or synthetic (or semi-synthetic) extracts or chemical libraries, based on previously known methods. To be done. One skilled in the art or drug discovery and development will understand that the exact source of the test extract or compound is not critical to the screening procedure of the invention. Thus, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not necessarily limited to, plant-based extracts, fungal-based extracts, prokaryotic-based or animal-based extracts, fermented broth, and synthetic compounds, as well as modifications of existing compounds. Including things. Numerous methods are also, but not necessarily limited to,
Used to create random (or semi-synthetic or total) synthesis of any number of chemical compounds, including sugar-based compounds, lipid-based compounds, peptide-based compounds, and nucleic acid-based compounds . Synthetic compound libraries are commercially available from Brandon Associates (New Hampshire, Merrimack) and Aldrich Chemical (Wisconsin, Milwaukee). Optional libraries of compounds are Biotics (Sussex, UK), Zenova (Slough, UK), Harbor Branch Ocean Graphics Institute (Fort Pierce, Florida) and Farmer, U.S.A.
S. A. Commercially available from a number of sources including (Massachusetts, Cambridge). In addition, natural and synthetic output libraries are produced, if desired, according to methods known in the art, such as standard extraction and fractional distillation methods. Furthermore, if desired, any library or compound can be readily modified using standard chemical, physics or biochemical methods.

In addition, one of ordinary skill in the art of drug discovery and development will recognize replication loss (eg, taxonomic loss of replication, loss of biological replication, and loss of chemical replication, or any combination thereof). It is readily understood that the method must be employed or wherever HDL-elevating, triglyceride-lowering, or anti-CVD activity, the duplication or repeated removal of substances already known can be employed.

When a crude extract is found to have ABC1 gene expression, ABC1 biological activity, or a combination thereof, a more positive major fraction of the extract is the chemical constituent responsible for the observed effect. Need to be isolated. Thus the goals of the extract, fractional distillation, and purification process are to increase HDL, reduce triglyceride, or anti-CVD activity, A
Careful characterization and identification of the entire chemical composition within the crude extract with the ability to regulate BC gene expression, or a combination thereof. The same in vivo and in vitro assays described herein for detecting activity in a mixture of compounds can be used to purify the active ingredient and test its derivatives. Fractionation and purification of such heterogeneous extracts are known in the art. If desired, compounds shown to be useful agents for the treatment of pathogenicity are chemically modified according to methods known in the art. Compounds identified as having therapeutic value are then analyzed using standard animal models of either diabetes or obesity known in the art.

It will be appreciated that compounds that modulate ABC1 or modulate the activity of proteins regulated by ABC1 are useful compounds for modulating cholesterol and triglyceride levels. Exemplary compounds are provided herein, others are known.

Compounds that are structurally related to cholesterol or that mimic ApoAI or related apolipoproteins and enhance ABC1 biological activity are particularly useful compounds of the invention. Other compounds known to act on MDR proteins are used and further derivatized to enhance ABC1 biological activity and assayed for their potency. Typical MDR modulators are PSC833, bromocriptine, and cyclosporin A. Examples of other compounds that are tested for their ability to enhance ABC1 biological activity are oxysterols and their derivatives.

Screening Patients With Low HDL-C or Anti-Triglyceride Levels ABC1 expression, bioactivity and mutation analysis can serve as diagnostic tools for low HDL or higher than normal triglyceride levels, respectively. Thus, confirmation of a genetic subtyping of the ABC1 gene sequence is useful for subdividing individuals or families with low HDL or higher than normal triglycerides to determine whether a lower HDL or higher than normal triglyceride phenotype is associated with ABC1 function. Can be used to typify. This diagnostic process can lead to tailoring of drug treatment based on the patient's genotype (hereinafter referred to as pharmacogenomics), including the prediction of side effects upon administration of HDL-increasing or triglyceride-lowering drugs. Pharmacogenomics is an agent (eg, drug) for the therapeutic or prophylactic treatment of an individual based on the genotype of the individual (eg, an individual's genotype is tested to determine an individual's ability to respond to a particular agent). Allows the selection of.

Agents or modulators that have a stimulatory or inhibitory effect on ABC1 biological activity or gene expression are associated with diseases associated with abnormal ABC1 activity (eg cardiovascular disease, low HD).
L cholesterol or higher than normal triglyceride levels) can be administered to an individual. In connection with such treatment, the pharmacogenomics of the individual (ie a study of the relationship between the individual's genotype and the individual's response to external compounds or agents) is considered. Differences in therapeutic efficacy can lead to severe toxicity or treatment failure by altering the relationship between the dose of the pharmacologically active agent and its blood concentration. Thus, the pharmacogenomics of an individual allows for the selection of effective agents (eg, drugs) for prophylactic or therapeutic treatments based on consideration of the individual's genotype. Such pharmacogenomics can be used to further determine appropriate dosages and treatment regimens. Therefore, the activity of ABC1 protein, expression of ABC1 nucleic acid, or AB in an individual.
The mutated capacity of the C1 gene can thereby be determined to select the appropriate agent for therapeutic or prophylactic treatment of the individual.

Pharmacogenomics addresses clinically significant genetic alterations in the response to drugs resulting from altered drug preparation and abnormal behavior in affected individuals (M. Eikerbaum, Clin. Exp. Pharmacol. Physiol. 23: 983-
985, 1996; W. Linder, Clin. Chem. Volume 43,
254-266, 1997). In general, it can be differentiated into two types of pharmacogenetic conditions. It is a genetic condition that is transmitted as a single factor that modifies the way a drug acts on the body (altered drug action), and a genetic condition that is transmitted as a single factor that modifies the way a body acts on a drug (altered). Drug metabolism). The altered drug effect occurs in patients with polymorphisms (eg, single nucleotide polymorphisms or SNPs) within the promoter, intron or exon sequences of ABC1. Thus, determining the presence of a polymorphism and its prevalence can predict a patient's response to a particular therapeutic agent. Among other things, polymorphisms in the promoter region are important in determining risk of HDL deficiency, higher than normal triglyceride levels and CVD.

In addition to the mutations in the ABC1 gene described here, we used human ABC1
The polymorphism of the gene was detected (Fig. 4). These polymorphisms are located in the ABC1 promoter, intron, and exon sequences. Low HD using standard methods such as direct sequencing, PCR, SSCP, or any other polymorphism detection system.
L, triglyceride level above normal, cardiovascular disease, or any other ABC
Prior to establishing a drug treatment regimen for patients with 1-mediated pathology, it was possible to easily confirm whether these polymorphisms were present in the patient. It is possible that some of these polymorphisms are effectively weak mutations. Individuals carrying such mutations are at high risk of cardiovascular disease. Thus, these polymorphisms are also useful in making diagnostic decisions.

Other Examples All publications mentioned herein are cited within the same scope as if each individual independent publication was specifically or individually indicated to be incorporated by reference. Incorporated here as an example.

Although the present invention has been described in connection with this specific example, it will also be appreciated that it can be further modified. This application is generally based on the principles of this invention within its well-known and customary practice within the prior art, to which the present invention pertains and which is applied to the basic features heretofore set up, and deviations from this disclosure. All changes, uses, including
Or intended to handle applications.

[Sequence list]

[Brief description of drawings]

FIG. 1 (1) is a diagram showing the genomic sequence of human ABC1 containing exons 1-50 (SEQ ID NO: 1). Uppercase letters indicate exon sequences, lowercase letters indicate 5'regulatory sequences or intron sequences. "Z" indicates any nucleotide or others that do not contain nucleotides. The numbering used here for nucleotides in SEQ ID NO: 1 assumes that no nucleotide is present at the position indicated by "z". However, it will be readily apparent to one of skill in the art that the nucleotide numbering will vary if these sequences are present at some or all positions designated "z". "K" indicates nucleotide G or T. "Y" is nucleotide A
Or indicates C. "S" indicates nucleotide C or G. "H" indicates nucleotides A, C, or T. "B" indicates nucleotide C, G, or T. Because of the identification of the ABC1 exon upstream of exon 0α that we have previously disclosed, A
The numbering referred to herein for the BC1 exon is increased by one compared to the numbering we have used so far (U.S.S.N. 09 / 526,193).
U .; S. S. N. 60/124, 702; S. S. N. 60 / 138,0
48; U. S. S. N. 60 / 139,600; S. S. N. 60/151
, 977). For example, exon 0 described so far is referred to herein as exon 0 as exon 1.

[FIG. 1 (2)] Same as FIG. 1 (1)

[FIG. 1 (3)] Same as FIG. 1 (1)

[FIG. 1 (4)] Same as FIG. 1 (1)

[FIG. 1 (5)] Same as FIG. 1 (1)

[FIG. 1 (6)] Same as FIG. 1 (1)

[FIG. 1 (7)] Same as FIG. 1 (1)

[FIG. 1 (8)] Same as FIG. 1 (1)

[Fig. 1 (9)] Same as Fig. 1 (1)

[FIG. 1 (10)] Same as FIG. 1 (1)

[FIG. 1 (11)] Same as FIG. 1 (1)

[FIG. 1 (12)] Same as FIG. 1 (1)

[FIG. 1 (13)] Same as FIG. 1 (1)

[FIG. 1 (14)] Same as FIG. 1 (1)

[FIG. 1 (15)] Same as FIG. 1 (1)

[FIG. 1 (16)] Same as FIG. 1 (1)

[FIG. 1 (17)] Same as FIG. 1 (1)

[FIG. 1 (18)] Same as FIG. 1 (1)

[FIG. 1 (19)] Same as FIG. 1 (1)

[FIG. 1 (20)] Same as FIG. 1 (1)

[FIG. 1 (21)] Same as FIG. 1 (1)

[FIG. 1 (22)] Same as FIG. 1 (1)

[FIG. 1 (23)] Same as FIG. 1 (1)

[FIG. 1 (24)] Same as FIG. 1 (1)

[FIG. 1 (25)] Same as FIG. 1 (1)

[FIG. 1 (26)] Same as FIG. 1 (1)

FIG. 1 (27) Same as FIG. 1 (1)

[Fig. 1 (28)] Same as Fig. 1 (1)

FIG. 1 (29) Same as FIG. 1 (1)

[FIG. 1 (30)] Same as FIG. 1 (1)

[FIG. 1 (31)] Same as FIG. 1 (1)

[FIG. 1 (32)] Same as FIG. 1 (1)

[FIG. 1 (33)] Same as FIG. 1 (1)

[FIG. 1 (34)] Same as FIG. 1 (1)

[FIG. 1 (35)] Same as FIG. 1 (1)

[FIG. 1 (36)] Same as FIG. 1 (1)

[Fig. 1 (37)] Same as Fig. 1 (1)

[FIG. 1 (38)] Same as FIG. 1 (1)

[FIG. 1 (39)] Same as FIG. 1 (1)

[Fig. 1 (40)] Same as Fig. 1 (1)

FIG. 1 (41) Same as FIG. 1 (1)

[Fig. 1 (42)] Same as Fig. 1 (1)

[Fig. 1 (43)] Same as Fig. 1 (1)

FIG. 1 (44) Same as FIG. 1 (1)

FIG. 1 (45) Same as FIG. 1 (1)

FIG. 2A shows the amino acid sequence of human ABC1 protein (SEQ ID NO: 5).

FIG. 2B (1) shows the nucleotide sequence of human ABC1 cDNA (
Sequence identification number 6)

FIG. 2B (2) shows the nucleotide sequence of human ABC1 cDNA (
Sequence identification number 6)

FIG. 2B (3) shows the nucleotide sequence of human ABC1 cDNA (
Sequence identification number 6)

FIG. 2B (4) shows the nucleotide sequence of human ABC1 cDNA (
Sequence identification number 6)

FIG. 3 (1) Schematic representation of the location of consensus transcription factors within the human ABC1 5 ′ regulatory sequence. Abbreviations are as follows: PPRE = peroxisome proliferator-responsive receptor. SREBP = Steroid response element binding protein site. R
OR = RAR-related orphan receptors. The numbering used herein at the position of the transcription factor binding site assumes that no nucleotide is present at the position designated "z" in SEQ ID NO: 1. For polymorphisms in the promoter region, the numbering is based on the first base of the promoter at nucleotide number -1. In exon 1,
The numbering is based on the first base of exon 1 at nucleotide number +1. 5 '
In the terminal intron, the numbering is based on the first position of intron 1 at +1. At the 3'end of intron 1, the numbering is 1st base 5 of nucleotide number -1
Build from'to the starting point of Exon 2.

[FIG. 3 (2)] Same as FIG. 3 (1)

[Fig. 3 (3)] Same as Fig. 3 (1)

[FIG. 3 (4)] Same as FIG. 3 (1)

[FIG. 3 (5)] Same as FIG. 3 (1)

[Fig. 3 (6)] Same as Fig. 3 (1)

FIG. 4 (1): Table summarizing polymorphisms in the genomic ABC1 sequence.

FIG. 4 (2): Table summarizing polymorphisms in the genomic ABC1 sequence.

FIG. 4 (3) is a table summarizing polymorphisms in the genomic ABC1 sequence.

FIG. 4 (4) is a table summarizing polymorphisms in genomic ABC1 sequences.

FIG. 4 (5): A table summarizing polymorphisms in the genomic ABC1 sequence.

FIG. 4 (6): Table summarizing polymorphisms in genomic ABC1 sequences.

FIG. 5A is a bar graph showing the percentage of HDL (FIG. 5A) and triglyceride (TG) (FIG. 5B) heterozygotes and unaffected family members within a certain percentile of age and sex based on LRC criteria (Hysce. Others, Blood Circulation, Volume 62: IV-116-
IV-136, 1980). A wide distribution of HDL levels was observed in heterozygotes,
Spreads to the 31st percentile of age and sex. Although there is overlap in the distribution of triglycerides between heterozygotes and uninfected family members, most heterozygotes have triglyceride levels above the 80th percentile in age and sex.

5B Same as FIG. 5A

FIG. 6: Table characterizing TD patients, ABC1 heterozygotes, and unaffected family members.

FIG. 7: Table summarizing the incidence of CAD in ABC1 heterozygotes.

FIG. 8 is a graph showing the mean HDL level vs. efflux level in heterozygotes for each mutation measured in heterozygous carriers for each mutation. HDL levels are expressed as a percentage of the average HDL levels of unaffected members of the family. The efflux level correlates closely with the level of HDL cholesterol and is associated with 82% of the change in HDL cholesterol level.

FIG. 9 is a table summarizing HDL levels and the presence or absence of CAD in ABC1 heterozygotes. In the R2144X and R909X ABC1 mutations, the codon encoding Arg2133 or Arg909 is mutated to the STOP codon, resulting in a truncated codon. In the "Del E, D 1893,94" mutation, Gln189
The codons encoding 3 and Asp1894 are deleted. "Invs25 + 1
The G ...> C "" mutation causes the first nucleotide of intron 25 from "G" to "C".
To remove the splice site. "Del C 6825 ...> 214
For the 5X mutation, the deletion of C6825 in the nucleotide sequence results in a STOP codon at the codon corresponding to amino acid 2145 of the encoded protein. "CTC
In the 6952-4TT ...> 2203X mutation, "CTC" is replaced by "TT" in the nucleotide sequence, resulting in the conversion of the codon encoding amino acid 2203 into a stop codon.

FIG. 10: Table comparing mean lipid levels in ABC1 heterozygotes with family members unaffected by either missense or severe mutations.

FIG. 11. Schematic representation of the ABC1 protein illustrating the location of mutations and the presence or absence of CAD in carriers of mutations. The number (n) of heterozygotes 40 years or older is listed.

FIG. 12A is a diagram showing the strains of FHA3 and FHA1, which are relatives of FHA (Marcil et al., Lancet 354: 1341-1346, 1999)
Year). Males are indicated by a square symbol and females are indicated by a yen symbol. Heterozygous individuals with the mutation are given a half-shadow and the custodian is indicated by an arrow. The diagonal lines indicate the deceased individual. Both individuals have a higher percentile range of HDL cholesterol than older generations of humans.

12B is the same as FIG. 12A.

FIG. 13: 3 at HDL cholesterol levels in the given percentile range
A bar graph showing the proportion of individuals aged 0 or younger and 30 to 70 or older. Both individuals have a much broader distribution of HDL cholesterol levels, clearly indicating that the effect of ABC1 on HDL levels is affected by age.

FIG. 14: Table summarizing HDL and TG levels in different age groups of ABC1 heterozygotes and unaffected family members.

Figure 15A: Heterozygous males (Figure 15A) and females (Figure 15A) in the 10 year old group (plotted at the midpoint) compared to the 10th percentile distribution in the LRC population.
FIG. 15B) is a graph showing the average HDL level (Hiss et al., Supra). The error bar graph shows the standard deviation value of the written average value. The number of individuals in each group is shown at each data point below. At age 30 and older, mean HDL levels in heterozygotes are much lower than the 10th percentile distribution. On the contrary, the average H of heterozygotes under the age of 30
DL cholesterol levels approach the 10th percentile distribution.

15B is the same as FIG. 15A.

FIG. 16A is a graph showing mean HDL (FIG. 16A) and triglyceride levels (FIG. 18B) of heterozygotes and unaffected family members within each of the three groups of BMI. The three groups of BMI match the following values: (1) BMI <21.4; (
2) 21.4 <BMI <25.1; (3) BMI> 25.1

16B is the same as FIG. 16A

FIG. 17 (1) is a table showing the reaction conditions and oligonucleotides used for RFLP screening of ABC1 polymorphism.

FIG. 17 (2) is a table showing reaction conditions and oligonucleotides used for RFLP screening of ABC1 polymorphism.

FIG. 18 is a gel image showing the RFLP genotype of the R219K mutant. 177
The base pair PCR product does not digest the A allele, or the B allele digests 1
It produces 07 and 70 base pairs.

FIG. 19 is a table showing allele frequencies of polymorphisms in the ABC1 gene.

FIG. 20 is a table comparing MSD, MOD, and coronary events with R219K ABC1 compared to controls.

FIG. 21 is a graph showing event-free survival curves for carriers (AB + BB) and non-carriers (AA) of R219K ABC1 mutants. Mutant carriers increased event-free survival by 29% compared to non-carriers over a 2-year trial.

FIG. 22 is a table showing baseline demographics and lipid levels in the Degenerate Growth Assessment Status Study (REGRESS) with the R219K ABC1 genotype.

FIG. 23 is a table showing lipid levels and CAD above and below mean age in R219K ABC1 carriers and controls.

FIG. 24 is a bar graph showing the percentage difference in HDL cholesterol levels between people above and below the median age (56.7 years) for each R219K genotype.

FIG. 25A. Age-related HDL cholesterol (at R219K genotype
Graph showing correlation between FIG. 25A) and outflow (FIG. 25B)

25B is the same as FIG. 25A

FIG. 26A is a graph showing changes in MSD (FIG. 26A) and MOD (FIG. 26B) with median age of carrier (AB + BB) and non-carrier (AA) of R219K ABC1 mutant.

26B is the same as FIG. 26A.

FIG. 27. Table showing racial distribution of R219K ABC1 variants.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 48/00 A61P 3/06 A61P 3/06 9/00 9/00 9/10 9/10 43/00 111 43/00 111 C12Q 1/02 C12N 5/10 1/68 A 15/09 Z C12Q 1/02 A61K 37/02 1/68 C12N 15/00 A 5/00 B (31) Priority claim number 60 / 213,958 (32) Priority date June 23, 2000 (June 23, 2000) (33) Priority claiming country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, E, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE , GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ , VN, YU, ZA, ZW (72) Inventor Haydon, Michael, Earl. V6, Canada, 1 W9, British Columbia, Vancouver, West Seventh Avenue 4484 (72) Inventor Brooks-Wilson, Angela, All. Canada, V7 C4 Bee2 British Columbia, Richmond, Langton Road 7100 (72) Inventor Pimstone, Simon, N. Canada, Vee 6 Tee 1 Sea 5, British Columbia, Vancouver, West Sixth Avenue 4746 (72) Inventor Cree, Susan, M. Canada, V7 N1 M8 British Columbia, North Bankover, E. Osborne Road 627 F-term (reference) 4B024 AA01 AA11 CA04 CA09 DA02 HA14 4B063 QA12 QA18 QA19 QQ43 QR38 QR55 QR80 QS34 4B065 AA90 AB01 CA44 CA46 4C084 AA02 AA07 AA13 AA17 BA01 BA08 BA23 DC50 NA14 ZA362 ZC022 ZC332 4C086 AA01 AA02 AA03 DA08 EA16 MA01 MA04 NA14 ZA36 ZC02 ZC33

Claims (79)

[Claims] 1. A method of treating a patient diagnosed with HDL cholesterol levels below normal, triglyceride levels above normal, or cardiovascular disease, said methods comprising LXR-mediated transcriptional activity. Administering to the patient a compound that modulates 2. The method of claim 1, wherein the compound is oxysterol. 3. The method of claim 1, wherein the compound is the following compound: 24- (S), 25-epoxycholesterol; 24 (S) -hydroxycholesterol; 22- (R). -Hydroxycholesterol; 24 (R), 25-epoxycholesterol; 22 (R) -hydroxy-24 (S), 25-epoxycholesterol; 22 (S) -hydroxy-24 (R), 25-epoxycholesterol; 24- (S), 25-iminocholesterol; methyl-38-hydroxycoronate; N, N-dimethyl-3β-hydroxycolonamide; 24 (R)
-Hydroxycholesterol; 22 (S) -hydroxycholesterol; 22 (
R), 24 (S) -dihydroxycholesterol; 25-hydroxycholesterol; 22 (R) -hydroxycholesterol; 22 (S) -hydroxycholesterol; 24 (S), 25-dihydroxycholesterol; 24 (R), 25
-Dihydroxycholesterol; 24,25-dehydrocholesterol; 25-
Epoxy-22 (R) -hydroxycholesterol; 20 (S) -hydroxycholesterol; (20R, 22R) -cholest-5-ene-3β, 20,22
-Triol; 4,4-dimethyl-5-α-cholesta-8,14,24-trien-3-β-ol; 7α-hydroxy-24 (S), 25-epoxycholesterol; 7β-hydroxy-24 (S ), 25-epoxycholesterol; 7-
Oxo-24 (S), 25-epoxycholesterol; 7α-hydroxycholesterol; 7-oxocholesterol; and demosterol. 4. The method of claim 1, wherein the LXR is LXRα.
A method characterized by being. 5. A method of treating a patient diagnosed with HDL cholesterol levels below normal, triglyceride levels above normal, or cardiovascular disease, said method comprising RXR-mediated transcriptional activity. Administering to the patient a compound which modulates 6. The method of claim 5, wherein the RXR is RXRα.
A method characterized by being. 7. A method of determining whether a candidate compound regulates ABC1 expression, wherein the method provides (a) a nucleic acid molecule comprising an ABC1 regulatory region or promoter linked to a reporter gene, (B) contacting said nucleic acid molecule with said candidate compound, and (c) measuring the expression of said reporter gene, wherein said reporter gene expression of a corresponding control nucleic acid molecule not contacted with said compound. In contrast, modified reporter gene expression indicates that the candidate compound regulates ABC1 expression. 8. The method of claim 7, wherein the regulatory region is nucleotides 5854 to 6694, 7756 to 8318, 104 of SEQ ID NO: 1.
79 to 10825, 15214 to 16068, 21636 to 22111,
27898 to 28721, 32951 to 33743, 36065 to 368
47, 39730 to 40577, 4543 to 5287, and 45081 to 55639, comprising 50 consecutive nucleotides. 9. The method of claim 7, wherein the regulatory region is LXR.
s, RXRs, RORs, SREBPs, and PPARs, comprising a binding site for a transcription factor. 10. A substantially pure nucleic acid molecule comprising nucleotides 5854 to 6694, 7756 to 8318, 10479 to 1 of SEQ ID NO: 1.
0825, 15214 to 16068, 21636 to 22111, 27898
Through 28721, 32951 through 33743, 36065 through 36847, 39
730 to 40577, 4543 to 5287, or 45081 to 5563
9. A method comprising a region that is virtually identical to at least 50 contiguous nucleotides of 9. 11. A substantially pure nucleic acid molecule comprising a region that is virtually identical to nucleotides 1-28,707 of SEQ ID NO: 1. 12. Nucleotides 29,011 to 53,2 of SEQ ID NO: 1
One virtually pure nucleic acid molecule, characterized in that it comprises a region which is virtually identical to 28. 13. A cell expressing the nucleic acid molecule according to claim 10. 14. A non-human mammal expressing the nucleic acid molecule of claim 10. 15. A method of treating a human with higher than normal triglyceride levels, the method comprising administering to said human an ABC1 polypeptide, or a triglyceride-regulating fragment thereof. . 16. The method of claim 15, wherein the ABC1 polypeptide has the sequence of SEQ ID NO: 5. 17. The method of claim 15, wherein the ABC1 polypeptide comprises an R → K mutation at position 219 or a V → A mutation at position 399. 18. The method of claim 15, wherein the ABC1 polypeptide comprises a mutation that increases its stability. 19. The method of claim 15, wherein the ABC1 polypeptide comprises a mutation that increases biological activity. 20. A method of treating a human with higher than normal triglyceride levels, said method comprising administering to said human a nucleic acid molecule encoding an ABC1 polypeptide or triglyceride regulatory fragment. And how to. 21. The method of claim 20, wherein the ABC1 polypeptide has the amino acid sequence of SEQ ID NO: 5. 22. The method of claim 20, wherein the ABC1 polypeptide comprises an R → K mutation at position 219 or a V → A mutation at position 399. 23. The method of claim 20, wherein the ABC1 polypeptide comprises a mutation that increases its stability. 24. The method of claim 20, wherein the ABC1 polypeptide comprises a mutation that increases biological activity. 25. The method of claim 20, wherein the biological activity is cholesterol modulation. 26. The method of claim 20, wherein the human has below normal HDL cholesterol levels. 27. A method of treating a human with higher than normal triglyceride levels, wherein said method increases ABC1 biological activity or increases the activity of wild-type ABC1, R219K ABC1, or V399A ABC1 in said human. A method comprising administering a mimetic compound. 28. One non-human mammal comprising a transgene comprising a nucleic acid molecule encoding a dominant negative ABC1 polypeptide, characterized in that said dominant negative polypeptide comprises an M → T mutation at position 1091. Non-human mammal. 29. One method of determining whether a candidate compound reduces the inhibition of a dominant negative ABC1 polypeptide, wherein said dominant negative polypeptide comprises an M → T mutation at position 1091. ) Providing a cell expressing a dominant negative ABC1 polypeptide, (b) contacting said cell with said candidate compound, and (c) measuring ABC1 biological activity of said cell, wherein said compound A in the corresponding control cells not exposed to
The method wherein the increase in the ABC1 biological activity relative to the BC1 biological activity indicates that the candidate compound reduces the inhibition of the dominant negative ABC1 polypeptide. 30. A method of predicting human response to a triglyceride reducing drug, comprising determining whether the ABC1 gene, promoter or regulatory sequence that modifies the human response to the drug is polymorphic. A method characterized by. 31. A method of determining whether a candidate compound is useful in modulating triglyceride levels, said method comprising: (a) providing a chicken comprising a mutation in the ABC1 gene; (b) said Administering a candidate compound to the chicken, and (c) measuring the ABC1 biological activity of the chicken, wherein the altered ABC1 biological activity is compared to the biological activity of a corresponding control chicken not exposed to the compound. Wherein said candidate compound is useful in modulating triglyceride levels. 32. The method of claim 31, wherein the ABC1 biological activity is cholesterol transport. 33. A method of determining whether a candidate compound is useful in modulating triglyceride levels, the method comprising: (a) an ABC1 polypeptide comprising amino acids 1-60 of SEQ ID NO: 5. Corresponding cells that are not exposed to the compound, wherein (b) contacting the cells with the candidate compound and (c) measuring the ABC1 biological activity of the cells, wherein ABC1
The method characterized in that the altered ABC1 biological activity, as compared to the biological activity, indicates that the candidate compound is useful in modulating triglyceride levels. 34. A method of determining whether a candidate compound is useful in modulating triglyceride levels, said method comprising: (a) providing a cell expressing the ABC1 gene or a fragment thereof, (b) Contacting the cells with the candidate compound, and (c) measuring ABC1 expression in the cells, wherein the altered ABC1 expression is compared to the ABC1 expression in corresponding control cells not exposed to the candidate compound. Wherein said candidate compound is useful in modulating triglyceride levels. 35. A method of determining whether a candidate compound is useful in modulating triglyceride levels, the method comprising: (a) an ABC1 polypeptide comprising amino acids 1-60 of SEQ ID NO: 1. And (b) contacting the polypeptide with the candidate compound and (c) measuring ABC1 biological activity, wherein AB of the corresponding control ABC1 polypeptide not contacted with the candidate compound.
The method wherein the modified ABC1 biological activity is useful in modulating the triglyceride level of the candidate compound as compared to the C1 biological activity. 36. A method of determining whether a candidate compound is useful in modulating triglyceride levels, the method comprising: (a) an ABC1 polypeptide comprising amino acids 1-60 of SEQ ID NO: 5. And (b) contacting the polypeptide with the candidate compound and (c) measuring the expression of the ABC1 polypeptide, wherein the corresponding control ABC1 polypeptide not contacted with the compound The method wherein the altered expression of the ABC1 polypeptide relative to the expression is indicative of the candidate compound being useful in modulating triglyceride levels. 37. A method of determining whether a candidate compound is useful in modulating triglyceride levels, the method comprising: (a) an ABC1 polypeptide comprising amino acids 1-60 of SEQ ID NO: 5. And (b) contacting the polypeptide with the candidate compound and (c) measuring the binding of the candidate compound to the ABC1 polypeptide, wherein the candidate compound of the ABC1 polypeptide is provided. Binding to a candidate compound is shown to be useful in modulating triglyceride levels. 38. A method of determining whether a candidate compound is useful for modulating triglyceride levels, said method comprising (a) (i) amino acids 1-60 of SEQ ID NO: 5. Providing an ABC1 polypeptide, and (ii) a second polypeptide that interacts with the ABC1 polypeptide, (b) contacting the polypeptide with a candidate compound, and (c) the ABC1 polypeptide and the second polypeptide. Measuring the interaction with a peptide, wherein altering the interaction of said polypeptide with said second polypeptide is indicative that said candidate compound is useful in modulating triglyceride levels. A method characterized by. 39. A method of determining whether a candidate compound is useful in modulating triglyceride levels, the method comprising: (a) an ABC1 polypeptide comprising amino acids 1-60 of SEQ ID NO: 5. Providing a cell comprising: (b) contacting the cell with the candidate compound, and (c) measuring the half-life of the ABC1 polypeptide, wherein the corresponding control cell not exposed to the compound is The method wherein the increase in half-life relative to the half-life indicates that the candidate compound is useful in modulating triglyceride levels. 40. A method of determining whether a candidate compound is useful in modulating triglyceride levels, said method comprising: (a) providing an ABC1 polypeptide in a lipid membrane; (b) said Contacting the polypeptide with the candidate compound and (c) measuring ABC1-mediated lipid transport across the lipid membrane, wherein the lipid transport of the corresponding control ABC1 polypeptide not contacted with the compound A change in lipid transport relative to that indicates that the candidate compound is useful in modulating triglyceride levels. 41. The method of claims 35-38 or 40, wherein the ABC1 polypeptide is in a cell-free system. 42. The method according to claim 35 or 38 or 40,
The method wherein the ABC1 polypeptide is in a cell. 43. The method of claim 42, wherein the cells are WHA.
A method characterized in that it is from M chicken. 44. The method of claim 42, wherein the cell is in a human or non-human mammal. 45. The method of claim 44, wherein the animal is WHA.
A method characterized by being an M chicken. 46. The method of claim 31, 33, or 35, wherein the biological activity is lipid or interleukin-1 transport. 47. The method of claim 46, wherein the lipid is cholesterol. 48. The method of claim 47, wherein the cholesterol is HDL cholesterol. 49. The method of claim 31, 33, or 35, wherein the biological activity is binding or hydrolysis of ATP by an ABC1 polypeptide. 50. A method of determining the propensity of a disease or condition in a subject, wherein the disease or condition is selected from the group consisting of subnormal HDL levels, supranormal triglyceride levels, and cardiovascular disease. And the method determines the presence or absence of at least one ABC1 polymorphism in the polynucleotide sequence of the ABC1 regulatory region, promoter, or coding sequence or in the amino acid sequence of the ABC1 protein in a sample obtained from the subject. And the presence or absence of said polymorphism indicates a risk of said disease or disorder. 51. The method of claim 50, further comprising analyzing at least 5 ABC1 polymorphic sites in the polynucleotide sequence or the amino acid sequence. 52. A method of determining whether an ABC1 polymorphism is indicative of a risk of a disease or disorder in a subject, wherein the disease or disorder is lower than normal HDL levels, higher than normal triglyceride levels. And a cardiovascular disease, wherein the method comprises: (a) a prevalence of the disease or condition in a first subject or set of subjects, and a prevalence of the disease or condition in a second subject or set of subjects. Determining whether it is different from morbidity; (b) a polynucleotide sequence of an ABC1 regulatory region, a promoter, or a coding sequence in a sample obtained from said first subject or set of subjects and said second subject or set of subjects; Alternatively, the amino acid sequence of the ABC1 protein is analyzed, and (c) at least one polymorphism is present in the first subject. Or a set of subjects and the second
Differing between subjects or sets of subjects, wherein the presence or absence of said ABC1 polymorphism correlates with said prevalence of said disease or disorder, whereby said ABC1 polymorphism indicates said risk A method characterized by determining whether or not. 53. The method of claim 52, further comprising analyzing at least 5 ABC1 polymorphic sites in the polynucleotide sequence or the amino acid sequence. 54. Multiple sequences of ABC1 polymorphisms that correlate with recordings of a predisposition or prevalence of a disease or condition selected from the group consisting of subnormal HDL cholesterol levels, above normal triglyceride levels, and cardiovascular disease. An electronic database consisting of records. 55. A method of selecting a desired treatment for modulating ABC1 activity or expression in a subject, said method comprising: (a) an ABC1 regulatory region, a promoter, or a sample in a sample obtained from said subject. Determining the presence or absence of at least one ABC1 polymorphism in the polynucleotide sequence of the coding sequence, or the amino acid sequence of the ABC1 protein, wherein the presence or absence of said ABC1 polymorphism regulates ABC1 expression or activity Determining the desired treatment that demonstrates the safety and efficacy of at least one treatment and (b) modulates ABC1 expression or activity in said subject. 56. The method of claim 55, further comprising analyzing at least 5 ABC1 polymorphic sites in the polynucleotide sequence or the amino acid sequence. 57. The candidate compound has a lower than normal HDL cholesterol level,
One method of determining whether it is useful in the treatment of a disease or disorder selected from the group consisting of higher than normal triglyceride levels and cardiovascular disease, said method comprising: (a) a measurable ABC1 biological activity. An assay system comprising: (b) contacting the assay system with the candidate compound, and (c) measuring ABC1 biological activity or ABC1 phosphorylation, wherein a response not exposed to the candidate compound is provided. ABC in a controlled assay system
1 bioactivity or ABC1 phosphorylation compared to ABC1 bioactivity or ABC1
Modulating phosphorylation indicates that the candidate compound is useful in treating the disease or disorder. 58. The method of claim 57, wherein the assay system is a cell-based system. 59. The method of claim 57, wherein the system is a cell-free system. 60. A method of identifying a compound that tests for the ability to ameliorate a disease or condition selected from the group consisting of subnormal HDL cholesterol levels, above normal triglyceride levels, and cardiovascular disease, Wherein the method comprises the steps of: (a) contacting a subject or cell with a candidate compound, and (b) measuring ABC1 expression, activity or protein phosphorylation in the subject or cell, wherein the contacting with the candidate compound A in the corresponding control subject or cells not
Altered ABC1 expression, activity, or protein phosphorylation, as compared to BC1 expression, activity, or protein phosphorylation, confirms said candidate compound as a compound to test for its ability to ameliorate said disease or disorder. . 61. The method of claim 57 or 60, wherein the candidate compound modulates the ABC1 protein phosphorylation and the ABC1 activity. 62. For determining whether a candidate compound is useful in modulating a disease or disorder selected from the group consisting of subnormal HDL cholesterol levels, above normal triglyceride levels, and cardiovascular disease. One method comprises: (a) providing a cell expressing the ABC1 gene or a fragment thereof, (b) contacting the cell with the candidate compound, and (c) determining the ABC1 activity of the cell. Determining the altered ABC1 activity relative to the ABC1 activity in corresponding control cells not exposed to the compound, wherein the candidate ABC compound is useful for modulating the disease or disorder. A method characterized by showing. 63. For determining whether a candidate compound is useful in modulating a disease or disorder selected from the group consisting of subnormal HDL cholesterol levels, above normal triglyceride levels, and cardiovascular disease. One of the methods, the method comprising the steps of: (a) contacting cells expressing the ABC1 protein with the candidate compound, and (b) measuring phosphorylation of the ABC1 protein, wherein the compound is The method wherein the altered ABC1 protein phosphorylation is shown to be useful in modulating the disease or disorder as compared to the ABC1 protein phosphorylation in the corresponding untouched control cells. 64. A compound useful in the treatment of a disease or condition selected from the group consisting of subnormal HDL cholesterol levels, above normal triglyceride levels, and cardiovascular disease, wherein the compound is ABC1. Modulating biological activity, where the compound (a) provides an assay system with measurable ABC1 biological activity, (b) contacting the assay system with the compound, and (c) measuring ABC1 biological activity. The modulation of ABC1 biological activity, as compared to the ABC1 biological activity in corresponding control cells not exposed to the compound, is useful for the candidate compound to modulate the disease or disorder. A method characterized by indicating that there is. 65. A compound useful in the treatment of a disease or disorder selected from the group consisting of subnormal HDL cholesterol levels, above normal triglyceride levels, and cardiovascular disease, wherein said compound is R219K.
A compound, which induces a change in ABC1 biological activity that mimics a change in ABC1 biological activity induced by an ABC1 mutation. 66. A compound useful in the treatment of a disease or disorder selected from the group consisting of subnormal HDL cholesterol levels, above normal triglyceride levels, and cardiovascular disease, wherein the compound is ABC1. A compound characterized by binding to or interacting with residue R219 of claim 1, which mimics a change in ABC1 activity induced by the R219K ABC1 mutation. 67. A compound useful in the treatment of a disease or condition selected from the group consisting of subnormal HDL cholesterol levels, above normal triglyceride levels, and cardiovascular disease, wherein said compound is V339A.
A compound, which induces a change in ABC1 biological activity that mimics a change in ABC1 biological activity induced by an ABC1 mutation. 68. A compound useful in the treatment of a disease or condition selected from the group consisting of subnormal HDL cholesterol levels, above normal triglyceride levels, and cardiovascular disease, wherein the compound is ABC1. A compound which binds or interacts with the residue V399 of the above, thereby mimicking a change in ABC1 activity due to the V399A ABC1 mutation. 69. A compound which modulates ABC1 activity and binds or interacts with an amino acid of ABC1 wherein said amino acid is amino acids 119-319 of ABC1 (SEQ ID NO: 5) or ABC1 (SEQ ID NO: 5). A compound which is a residue selected from amino acids 299 to 499 of 5). 70. A method of determining whether a candidate compound is useful in treating a disease or disorder selected from the group consisting of subnormal HDL cholesterol levels, above normal triglyceride levels, and cardiovascular disease. hand,
The method comprises the steps of: (a) providing an assay system having measurable LXR biological activity; (b) contacting the assay system with the candidate compound; and (c) measuring LXR biological activity. The LXR in a corresponding control assay system not exposed to the candidate compound at
A method wherein modulation of LXR biological activity relative to biological activity indicates that said candidate compound is useful in treating said disease or disorder. 71. A method of determining whether a candidate compound is useful in modulating ABC1 biological activity, said method comprising: (a) providing an assay system with measurable LXR biological activity. And (b) contacting the assay system with the candidate compound and (c) measuring the LXR biological activity, wherein the LXR in a corresponding control assay system not contacted with the candidate compound.
Modulating LXR biological activity relative to biological activity, wherein said candidate compound is useful for modulating ABC1 biological activity. 72. The method of claim 71, wherein the LXR biological activity is modulation of ABC1 expression. 73. One method for identifying a compound to be tested for its ability to modulate ABC1 biological activity, said method comprising: (a) contacting a subject or cell with a candidate compound, and (b) said Assaying the activity of the LXR gene product in a subject or cell, wherein modulation of the activity of the candidate compound is compared to ABC1 in the corresponding control subject or cell not exposed to the compound. Confirming the ability to modulate the biological activity of the compound tested. 74. Of the LXR gene product in an assay that identifies one compound useful in the treatment of a disease or condition selected from the group consisting of subnormal HDL cholesterol levels, above normal triglyceride levels, and cardiovascular disease. use. 75. Modulating LXR gene product activity or expression for the treatment of a disease or disorder selected from the group consisting of subnormal HDL cholesterol levels, above normal triglyceride levels, and cardiovascular disease. Use of the compound. 76. A method of identifying a compound that is assayed for the ability to treat a disease or condition selected from the group consisting of subnormal HDL cholesterol levels, above normal triglyceride levels, and cardiovascular disease. , The method comprises: (a) providing an assay system having a measurable LXR biological activity, (b) contacting the assay system with a candidate compound, and (c) measuring the LXR biological activity, wherein: Modulation of the LXR biological activity relative to the LXR biological activity in a corresponding assay system not contacted with the candidate compound confirms the ability of the candidate compound to treat the disease or disorder as the compound being tested. How to characterize. 77. One method of screening candidate LXR agonists for their ability to treat a disease or condition selected from the group consisting of subnormal HDL cholesterol levels, subnormal triglyceride levels, and cardiovascular disease. Wherein the method comprises the steps of: (a) contacting the cells with the candidate LXR agonist and (b) measuring efflux activity of the cells with cholesterol. An increase in said cholesterol efflux activity in said cells as compared to said control cells is indicated to indicate that said candidate LXR agonist is useful in treating said disease or disorder. 78. A method of screening candidate LXR modulating compounds for the ability to treat a disease or condition selected from the group consisting of subnormal HDL cholesterol levels, subnormal triglyceride levels, and cardiovascular disease. Wherein the method comprises the steps of: (a) contacting the cell with the candidate LXR modulating compound and (b) measuring the ABC1 biological activity of the cell, wherein the corresponding control not contacted with the LXR modulating compound. ABC1 in cells
The method, wherein an increase in ABC1 biological activity in the cell relative to the biological activity is indicated to be useful for the treatment of the disease or disorder with the LXR modulating compound. 79. A method according to any one of claims 71 to 78,
Where the cell or assay system is SEQ ID NO: 94, SEQ ID NO: 92,
And an exogenous feed replication of LXRE selected from the group consisting of LXRE consensus motifs at about nucleotide 7670 at the 3'end of intron 1.