The present invention relates to novel ABC1 polypeptides and nucleic acid molecules encoding the same. The invention also relates to recombinant vectors, host cells, and compositions comprising ABC1 polynucleotides, as well as to methods for producing ABC1 polypeptides. The invention also relates to antibodies that bind specifically to ABC1 polypeptides. In addition, the invention relates to methods for increasing cholesterol efflux as well as to methods for increasing ABC1 expression and activity. The present invention further relates to methods for identifying compounds that modulate the expression of ABC1 and methods for detecting the comparative level of ABC1 polypeptides and polynucleotides in a mammalian subject. The present invention also provides kits and compositions suitable for screening compounds to determine the ABC1 expression modulating activity of the compound, as well as kits and compositions suitable to determine whether a compound modulates ABC1-dependent cholesterol efflux.
Circulating lipids in human plasma or lymphatic fluid consist of cholesterol, cholesteryl esters, triglycerides and phospholipids. These lipids are transported in large molecular complexes called lipoproteins, which consist of a core of cholesteryl esters and/or triglycerides, an envelope of phospholipids and free cholesterol, and apolipoproteins (Scriver et al., Eds., The Metabolic and Molecular Basis of Inherited Disease, 7th Ed., p. 1841-1851 (McGraw-Hill, New York 1995)). Apolipoproteins are involved in the assembly and secretion of the lipoprotein, as well as the activation of lipoprotein modifying enzymes, such as lecithin cholesterol acyl transferase (LCAT). In addition, apolipoproteins provide structural integrity and are ligands for a large spectrum of receptors and membrane docking proteins. The plasma lipoproteins are categorized into five types according to size: chyloricrons (largest in size and lowest in density), very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL) and high density lipoprotein (HDL).
Chylomicrons, VLDLs, IDLs, and LDLs transport exogenous and endogenous cholesterol and triacylglycerols to peripheral sites, where the lipids play a role in various metabolic pathways and serve as a major constituent of cell membranes. Chylomicrons are assembled in the intestinal mucosa as a means to transport dietary cholesterol and triacylglycerols to various tissues. VLDLs are formed in the liver to transport endogenous cholesterol and triacylglycerols synthesized by the liver to extra-hepatic tissues, such as muscle and adipose tissue. In fasting serum, VLDLs contain 10-15% of the total serum cholesterol and most of the triglyceride. In circulation, VLDLs are converted to LDLs through the action of lipoprotein lipase. LDLs are the primary plasma carriers of cholesterol for delivery to all tissues, typically containing 60-70% of the total fasting serum cholesterol.
In contrast, HDLs are involved in xe2x80x9creverse cholesterol transportxe2x80x9d, the pathway by which excess cholesterol is transported from peripheral sites back to the liver, where it is excreted in the form of bile salts (Glomset, J. A., J. Lipid Res., 9, 155-167 (1968)). Nascent HDLs are synthesized de novo in the liver and small intestine, as protein-rich disc-shaped particles devoid of cholesterol and cholesterol esters. In fact, a major function of HDLs is to act as circulating stores of apolipoproteins, primarily apo C-I, apo C-II, and apoE. The nascent or protein-rich HDLs are converted into spherical lipoprotein particles through the accumulation of cholesteryl esters obtained from cellular sources. The HDL normally contain 20-30% of the total fasting serum cholesterol.
According to current theories, the reverse efflux of cellular cholesterol to HDL is mediated through two mechanisms: an aqueous diffusion pathway and an apolipoprotein-mediated pathway. The relative importance of these distinguishable mechanisms depends on the cell type and metabolic state (Oram et al., J. Lipid Res., 37:2743-2491 (1996); Rothblat et al., J. Lipid Res., 40:781-796 (1999); Stein et al., Atherosclerosis, 144:285-301 (1999)). For many cells, the aqueous diffusion pathway is the principle pathway through which cholesterol efflux occurs (Johnson et al., Biochim. Biophys. Acta, 1085:273-298 (1991)). This pathway involves the bidirectional exchange of cholesterol between cell membranes and a lipoprotein acceptor, such as HDL, in the extracellular space through a process of passive transport (Remaley et al., Arterioscler. Thromb. Vasc. Biol., 17:1813-1821 (1997); Rothblat et al., J. Lip. Res., 40:781-796 (1999)). The exchange may occur primarily at surface microdomains known as caveolae (Fielding et al., Biochemistry, 34:14288-14292 91995)). Net efflux can be driven by conversion of cholesterol in the exteracellular compartment to cholesteryl ester by the action of LCAT.
Alternatively, in macrophage and fibroblast cells, cholesterol and phospholipid efflux is primarily mediated through apolipoproteins, such as apo A-I, apo A-II, and Apo E (Remaley, supra (1997); Francis, et al., J. Clin. Invest., 96:78-87 (1995); Vega et al., J. Intern. Med., 226:5-15 (1989); Sakar et al., Biochim. Biophys. Acta, 1438: 85-98 (1999); Hara et al., J. Biol. Chem., 266:3080-3086 (1991); Fielding et al., J. Lipid Res., 38, 1503-1521 (1997); Oram et al., J. Lipid Res., 37, 2743-2491 (1996)). The process of apolipoprotein-mediated lipid efflux particularly dig dominates in macrophages and other scavenger cells when they are cholesterol-loaded and/or growth-arrested. Apolipoprotein-mediated efflux is an active transport process that requires the direct interaction of the apolipoprotein with the cell surface, the lipidation of the apolipoprotein, and the subsequent dissociation of the lipid-apolipoprotein particle from the cell (Oram, supra (1996); Mendez, A. J., J. Lipid Res., 38, 1807-1821 (1997); Remaley, supra (1997); Mendez, A. J., J. Lipid Res., 37, 2510-2524 (1996)). Once removed from the cell, the cholesterol-rich HDL particles are transported to the liver and removed from the body as described.
Abnormal lipoprotein function and/or metabolism resulting from genetic defect or as a secondary effect of another disorder can have serious biological consequences. In addition to dietary influences, disorders such as diabetes, hypothyroidism, and liver disease can result in elevated plasma levels of LDL-cholesterol and triglycerides. Elevated levels of LDL-cholesterol and triglycerides have been identified as major risk factors associated with the incidence of coronary heart disease, which is the primary cause of death in the United States and other industrialized nations (Hokanson et al., J. Cardiovasc. Risk., 3:213-219 (1996); The Expert Panel, JAMA, 269:3015-3023 (1993)). The accumulation of excess LDL-cholesterol on arterial walls can lead to the formation of atherosclerotic plaques, which play a major role in the development of heart disease. A plaque is believed to form when free radicals released from arterial walls oxidize LDL. According to theory, the oxidized form of LDL triggers an inflammatory response, attracting circulating cells to the site which contribute to the formation of a lipid plaque. Among these are macrophages and other cells that contain scavenger receptors that accumulate cholesterol in an unregulated manner (Brown et al., Ann. Rev. Biochem., 52:223-261 (1986)). Vast stores of internal cholesterol result in conversion to a foam cell phenotype, which is believed to be a major contributor to the development of vascular lesions. As the plaque builds up, the arterial walls constrict, reducing blood flow to the heart.
Interestingly, however, an estimated 60% of heart attacks occur in persons who do not have elevated blood levels of LDL-cholesterol. Of these, an estimated 45% are associated with below average blood levels of HDL-cholesterol, indicating that low HDL-cholesterol level is a significant risk factor for coronary heart disease. In fact, recent studies have indicated that a decreased HDL-cholesterol level is the most common lipoprotein abnormality seen in patients with premature coronary artery disease (Genest J., Circulation, 85:2025-2033 (1992); Genest et al., Arterioscler. Thromb., 13:1728-1737 (1993)). Although the basis for the inverse association between HDL-cholesterol and coronary heart disease is not well understood, it has been suggested that the cardioprotective role of HDL may stem from its activity relating to the promotion of cholesterol efflux from macrophage foam cells in atherosclerotic lesions.
One example of cardiovascular disease associated with low HDL is Tangier disease (TD), a rare genetic disorder characterized by a near or complete absence of circulating HDL. In addition to near zero plasma levels of HDL, patients with TD have a massive deposition and accumulation of cholesteryl esters in several tissues, including tonsils, lymph nodes, liver, a spleen, thymus, intestine, and Schwann cells (Fredrickson, D. S., J. Clin. Invest., 43, 228-236 (1964); Assmann et al., The Metabolic Basis of Inherited Disease, (McGraw-Hill, New York, 1995)). Although the cellular mechanisms have not been previously identified, recent studies have shown that cells from subjects with TD are defective in the process of apolipoprotein-mediated removal of cholesterol and phospholipids (Remaley et al., Arterioscler. Thromb. Vasc. Biol., 17, 1813-1821 (1997); Francis et al., J. Clin. Invest., 96, 78-87 (1995); Rogler et,al., Arterioscler. Thromb. Vasc. Biol., 15, 683-690 (1995)). These results have led to the proposal that the severe HDL deficiency in TD patients stems from the inability of nascent apo A-I to acquire lipids. Because they do not mature into lipid-rich particles, the nascent HDL in TD patients is rapidly catabolized and removed from the plasma, resulting in the near zero levels of circulating HDL (Remaley, supra (1997); Francis, supra (1995); Horowitz et al., J. Clin. Invest., 91, 1743-1752 (1993); Schaefer et al., J. Lip. Res., 22:217-228 (1981)).
Other disorders associated with severe premature atherosclerosis and high risk for coronary heart disease resulting from diminished HDL-cholesterol levels are hypoalphalipoproteinemia and familial HDL deficiency syndrome (FHA). Persons with these disorders often have normal LDL-cholesterol and triglyceride levels. In addition, disorders such as diabetes, alcoholism, hypothyroidism, liver disease, and elevated blood pressure can result in diminished plasma levels of HDL-cholesterol, although many of these disorders are also accompanied by elevated LDL-cholesterol and triglceride levels.
Current treatments for coronary heart disease have focused primarily on diet manipulations and/or drug therapies aimed at lowering the plasma level of LDL-cholesterol by inhibiting LDL secretion or promoting LDL turnover. Derivatives of fibric acid, such as clofibrate, gemfibrozil, and fenofibrate, promote rapid VLDL turnover by activating lipoprotein lipase. Nicotinic acid reduces plasma levels of VLDL and LDL by inhibiting hepatic VLDL secretion. In addition, HMG-CoaA reductase inhibitors, such as mevinolin, mevastatin, pravastatin, simvastatin, fluvastatin, and lovastatin reduce plasma LDL levels by inhibiting the intracellular synthesis of cholesterol, which causes an increase in the cellular uptake of LDL. In addition, bile acid-binding resins, such as cholestyrine, colestipol and probucol decrease the level of LDL-cholesterol by increasing the catabolism of LDL-cholesterol in the liver.
However, many of these therapies are associated with low efficacy and/or side effects that may prevent long-term use. For example, use of HMG-CoaA reductase inhibitors carry a significant risk of toxicity because they inhibit the synthesis of mevalonate, which is required for the synthesis of other important isoprenoid compounds in addition to cholesterol. Also, gemfibrozil and nicotinic acid are associated with serious adverse effects, including renal injury, myopathy, myoglobinuria and intolerable skin flushing and itching. In addition, the role of probucol in treating patients with coronary heart disease is uncertain because its administration results in lower HDL-cholesterol levels as a side effect of reducing LDL-cholesterol.
Furthermore, treating patients who have isolated low HDL-cholesterol levels provides a particularly difficult therapeutic challenge. For instance, patients with Tangier disease exhibit a 4- to 6-fold increase in cardiovascular disease even though their LDL levels are already reduced by about 50%. While there is some evidence that gemfibrozil and nicotinic acid may simultaneously elevate HDL levels, in general, therapies aimed at lowering plasma LDL-cholesterol levels are not effective for Tangier patients who suffer from coronary heart disease as a result of diminished HDL levels. Likewise, patients with hypoalphalipoproteinemia, familial HDL deficiency syndrome, or other cardiovascular disease resulting from low levels of HDL will not benefit from therapies aimed at lowering the level of plasma LDL.
The problems associated with current therapies for cardiovascular disease stem partially from the fact that the biology involved in the movement of cholesterol in and out of cells is not fully understood. Furthermore, the proteins that play a role in cholesterol movement are not fully known. Therefore, there remains a need for a better understanding of cholesterol cell biology, as well as new methods for treating humans suffering from cardiovascular disease and other disorders associated with hypercholesterolemia. Additionally, there remains a need for new methods of diagnosing cardiovascular disease and new methods of screening patients to identify those at high risk for developing cardiovascular disease.
The identification of genes and proteins involved in cholesterol transport would be useful in the development of pharmaceutical agents for the treatment of heart disease and other disorders associated with hypercholesterolemia and atherosclerosis. In addition, the identification of such genes would be useful in the development of screening assays to screen for compounds that regulate the expression of genes associated with cholesterol transport. The identification of such regulatory compounds would be useful in the development of further therapeutic agents. Furthermore, the identification of genes and proteins involved in cholesterol transport would be useful as diagnostic indicators of cardiovascular disease and other disorders associated with hypercholesterolemia.
The present invention provides novel polypeptides and polynucleotides involved in cholesterol efflux. Specifically, the present invention provides novel ATP-Binding Cassette (ABC1) polypeptides and novel polynucleotides that encode ABC1 polypeptides. The terms xe2x80x9cABC1xe2x80x9d and xe2x80x9cABCA1xe2x80x9d are alternative names for the same ATP-Binding Cassette protein and gene. The invention provides ABC1 polypeptides, polypeptide fragments, and polypeptide variants. In one preferred embodiment, the present invention provides an isolated polypeptide comprising SEQ ID NO: 2. In another preferred embodiment, the present invention provides an isolated polypeptide comprising an amino acid sequence that has at least 98% identity to SEQ ID NO: 2. The present invention also provides ABC1 polypeptides from Tangier disease patients. In one preferred embodiment, the present invention provides an isolated polypeptide comprising SEQ ID NO: 8. In another preferred embodiment, the present invention provides an isolated polypeptide comprising SEQ ID NO: 10.
In addition, the present invention provides ABC1 polynucleotides, polynucleotide fragments, and polynucleotide variants. In one preferred embodiment, the present invention provides an isolated polynucleotide that encodes the polypeptide comprising SEQ ID NO: 2. In another preferred embodiment, the invention provides an isolated polynucleotide that encodes a polypeptide comprising an amino acid sequence that has at least 98% identity to SEQ ID NO: 2. Also, in other preferred embodiments, the invention provides an isolated polynucleotide comprising a nucleotide sequence that is complementary to a polynucleotide encoding the polypeptide comprising SEQ ID NO: 2 or an isolated polynucleotide comprising a nucleotide sequence that is complementary to a polynucleotide encoding the polypeptide comprising an amino acid sequence that has at least 98% identity to SEQ ID NO: 2.
In another preferred embodiment, the present invention provides an isolated ABC1 polynucleotide comprising SEQ ID NO: 1. In a further preferred embodiment, the present invention provides an isolated polynucleotide comprising nucleotides 291-7074 of SEQ ID NO: 1. In yet another preferred embodiment, the invention provides a polynucleotide comprising a nucleotide sequence that has at least 90% identity with SEQ ID NO: 1. More preferably, the polynucleotide comprises a nucleotide sequence that has at least 95% identity with SEQ ID NO: 1. In other more preferred embodiments, the polynucleotide comprises a nucleotide sequence that has at least 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1. Also, in other preferred embodiments, the present invention provides an isolated polynucleotide comprising a nucleotide sequence that is complementary to the polynucleotide comprising SEQ ID NO: 1, an isolated polynucleotide comprising a nucleotide sequence that is complementary to a polynucleotide comprising nucleotides 291-7074 of SEQ ID NO: 1, and an isolated polynucleotide that is complementary to a polynucleotide comprising a nucleotide sequence that has at least 90% identity with SEQ ID NO: 1.
The present invention also provides ABC1 polynucleotides corresponding to the 5xe2x80x2 flanking region of the ABC1 gene. In one preferred embodiment, the invention provides an isolated polynucleotide comprising SEQ ID NO: 3. In other preferred embodiments, the invention provides an isolated polynucleotide comprising nucleotides 1-1532, 1080-1643, 1181-1643, 1292-1643, or 1394-1532 of SEQ ID NO: 3. Preferably, the isolated polynucleotide comprises nucleotides 1394-1532 of SEQ ID NO: 3. In another preferred embodiment, the invention provides an isolated polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising SEQ ID NO: 3. Also, in other preferred embodiments, the present invention provides an isolated polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising nucleotides 1-1532, 1080-1643, 1181-1643, 1292-1643, or 1394-1532 of SEQ ID NO: 3. In yet another preferred embodiment of the present invention, an isolated polynucleotide that has at least 80% identity to a polynucleotide comprising SEQ ID NO: 3 is provided. More preferably, the polynucleotide has at least 90% identity to a polynucleotide comprising SEQ ID NO: 3. Even more preferably, the polynucleotide has at least 95% identity to a polynucleotide comprising SEQ ID NO: 3. Also provided in preferred embodiments is an isolated polynucleotide that has at least 80% identity to a polynucleotide comprising nucleotides 1-1532, 1080-1643, 1181-1643, 1292-1643, or 1394-1532 of SEQ ID NO: 3. More preferably, the polynucleotide has at least 90% identity, and even more preferably at least 95% identity, to a polynucleotide comprising nucleotides 1-1532, 1080-1643, 1181-1643, 1292-1643, or 1394-1532 of SEQ ID NO: 3. In addition, the present invention provides an isolated polynucleotide comprising a nucleotide sequence that is complementary to the above described 5xe2x80x2 flanking ABC1 polynucleotides. In one preferred embodiment, the invention provides an isolated polynucleotide comprising a nucleotide sequence that is complementary to a polynucleotide comprising SEQ ID NO: 3. In another preferred embodiment, the present invention provides an isolated polynucleotide comprising a nucleotide sequence that is complementary to a polynucleotide comprising nucleotides 1-1532, 1080-1643, 1181-1643, 1292-1643, or 1394-1532 of SEQ ID NO: 3.
The present invention also provides ABC1 polynucleotides corresponding to the 3xe2x80x2 flanking region of the ABC1 gene. In preferred embodiments, the invention provides an isolated polynucleotide comprising SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, and the complementary sequences thereof. In other preferred embodiments, the invention provides an isolated polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, and the complementary sequences thereof. In still other preferred embodiments, the invention provides an isolated polynucleotide that has at least 80% identity to a polynucleotide comprising SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, and the complementary sequence thereof. More preferably, the polynucleotide has at least 90% identity to a polynucleotide comprising SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. Even more preferably, the polynucleotide has at least 95% identity to a polynucleotide comprising SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.
In addition, the present invention also provides ABC1 polynucleotides from Tangier disease patients. In one preferred embodiment, the present invention provides an isolated polynucleotide encoding the polypeptide comprising SEQ ID NO: 8. In another preferred embodiment, the present invention provides an isolated polynucleotide comprising SEQ ID NO: 7. In yet another embodiment, the present invention provides an isolated polynucleotide encoding the polypeptide comprising SEQ ID NO: 10. In still another preferred embodiment, the present invention provides an isolated polynucleotide comprising SEQ ID NO: 9. The present invention further provides an isolated polynucleotide comprising a nucleotide sequence that is complementary to the described polynucleotides.
In another aspect, the present invention provides a composition comprising any of the above described polynucleotides and a suitable carrier. In one preferred embodiment, the present invention provides a composition comprising an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO: 2, a polynucleotide comprising SEQ ID NO: 1, a polynucleotide comprising nucleotides 291-7074 of SEQ ID NO: 1, or a polynucleotide encoding a polypeptide comprising an amino acid sequence that has at least 98% identity to SEQ ID NO: 2, and a suitable carrier. In another preferred embodiment, the composition comprises an isolated polynucleotide comprising a nucleotide sequence that has at least 90% identity with a polynucleotide comprising SEQ ID NO: 1 and a suitable carrier. In other preferred embodiments, the composition comprises an isolated polynucleotide comprising SEQ ID NO: 3 or an isolated polynucleotide comprising nucleotides 1-1532, 1080-1643, 1181-1643, 1292-1643, or 1394-1532 of SEQ ID NO: 3 and a suitable carrier. In still other preferred embodiments, the invention provides a composition comprising a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising SEQ ID NO: 3, or a polynucleotide comprising nucleotides 1-1532, 1080-1643, 1181-1643, 1292-1643, or 1394-1532 of SEQ ID NO: 3, as well as a composition comprising a polynucleotide that has at least 80% identity to a polynucleotide comprising SEQ ID NO: 3, or a polynucleotide comprising nucleotides 1-1532, 1080-1643, 1181-1643, 1292-1643, or 1394-1532 of SEQ ID NO: 3 and a suitable carrier. Also provided by the present invention is a composition comprising an isolated polynucleotide comprising a nucleotide sequence that is complementary to any of the described polynucleotides and a suitable carrier.
In addition, the present invention provides recombinant vectors and host cells comprising any of the described ABC1 polynucleotide sequences. In one preferred embodiment, the present invention provides a recombinant vector comprising an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO: 2, an isolated polynucleotide comprising SEQ ID NO: 1, an isolated polynucleotide comprising nucleotides 291-7074 of SEQ ID NO: 1, or an isolated polynucleotide encoding the polypeptide comprising an amino acid sequence that has at least 98% identity to SEQ ID NO: 2. In another preferred embodiment, the recombinant vector comprises an isolated polynucleotide comprising a nucleotide sequence that has at least 90% identity, and more preferably at least 95% identity, with a polynucleotide comprising SEQ ID NO: 1. In still another preferred embodiment, the recombinant vector comprises an isolated polynucleotide comprising SEQ ID NO: 7 or SEQ ID NO: 9. The present invention further provides a recombinant vector comprising an isolated polynucleotide comprising a nucleotide sequence that is complementary to any of the described polynucleotides. In yet another preferred embodiment, the recombinant vector comprises any of the described polynucleotides and further comprises a heterologous promoter polynucleotide. One suitable heterologous promoter is a cytomegalovirus promoter. In a particularly preferred embodiment, the recombinant vector is pCEPhABC1.
The present invention also provides a recombinant vector comprising an isolated polynucleotide comprising an ABC1 5xe2x80x2 flanking sequence. In one preferred embodiment, the invention provides a recombinant vector comprising an isolated polynucleotide comprising SEQ ID NO: 3 or an isolated polynucleotide comprising nucleotides 1-1532, 1080-1643, 1181-1643, 1292-1643, or 1394-1532 of SEQ ID NO: 3. In still other preferred embodiments, the invention provides a recombinant vector comprising a polynucleotide that hybridizes under stringent conditions to the polynucleotide of SEQ ID NO: 3, or a polynucleotide comprising nucleotides 1-1532, 1080-1643, 1181-1643, 1292-1643, or 1394-1532 of SEQ ID NO: 3, as well as a recombinant vector comprising a polynucleotide that has at least 80% identity to these polynucleotides. The present invention further provides a recombinant vector comprising an isolated polynucleotide comprising a nucleotide sequence that is complementary to any of the described polynucleotides. In yet another preferred embodiment, the recombinant vector comprises any of the described polynucleotides and further comprises at least one polynucleotide encoding a heterologous polypeptide. Suitable heterologous polypeptides include luciferase, xcex2-galactosidase, chloramphenicol acetyl transferase transferase, and green fluorescent proteins. Preferably, the heterologous polypeptide is a luciferase protein. In a particularly preferred embodiment, the recombinant vector is pAPR1.
In addition, the present invention provides host cells comprising any of the described recombinant vectors. The present invention further provides compositions comprising any of the described recombinant vectors and a suitable carrier.
The present invention also provides methods for producing the ABC1 protein in a mammalian host cell as well as methods for expressing the ABC1 protein in a mammalian subject. The method for producing an ABC1 protein in a mammalian host cell comprises the steps of: (a) transfecting the mammalian host cell with a recombinant expression vector comprising a polynucleotide encoding ABC1 in an amount sufficient to produce a detectable level of ABC1 protein, and (b) purifying the produced ABC1 protein. The method for expressing ABC1 protein in a mammalian subject comprises the step of administering to a mammalian subject a recombinant expression vector comprising a polynucleotide encoding ABC1 in an amount sufficient to express ABC1 protein in the mammalian subject.
In addition, the present invention provides compositions and methods suitable for increasing cholesterol efflux from cells of a mammalian subject. In one preferred embodiment, the method comprises administering to the mammalian subject a recombinant expression vector comprising a polynucleotide encoding ABC1 in an amount sufficient to increase cholesterol efflux from the cells. Suitable recombinant expression vectors include vectors comprising an isolated polynucleotide encoding a polypeptide comprising SEQ ID NO: 2, an isolated polynucleotide comprising SEQ ID NO: 1, an isolated polynucleotide comprising nucleotides 291-7074 of SEQ ID NO: 1, and an isolated polynucleotide encoding the polypeptide comprising an amino acid sequence that has at least 98% identity to SEQ ID NO: 2. Preferred expression vectors include viral vectors, especially adenoviral vectors and lentiviral vectors. In other embodiments, the invention provides non-viral delivery systems, including DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct injection of DNA, CaPO4 precipitation, gene gun techniques, electroporation, liposomes and lipofection.
In another preferred embodiment, the method for increasing cholesterol efflux from cells of a mammalian subject comprises administering to the mammalian subject a therapeutic amount of a compound that increases the expression of ABC1 in the cells. One suitable method comprises administering to the mammalian subject a cAMP analogue. Suitable cAMP analogues include 8-bromo cAMP, N6-benzoyl cAMP, and 8-thiomethyl cAMP. Another suitable method comprises administering to the mammalian subject a compound that increases the synthesis of cAMP, e.g. forskolin. Yet another suitable method comprises administering to the mammalian subject a compound that inhibits the degradation of cAMP, such as a phosphodiesterase inhibitor. Suitable phosphodiesterase inhibitors include rolipram, theophylline, 3-isobutyl-1-methylxanthine, R020-1724, vinpocetine, zaprinast, dipyridamole, milrinone, amninone, pimobendan, cilostamide, enoximone, peroximone, and vesnarinone.
In addition, another suitable method for increasing cholesterol efflux from cells of a mammalian subject comprises administering to the mammalian subject a least one ligand for a nuclear receptor in an amount sufficient to increase cholesterol efflux. Suitable ligands include LXR, RXR, FXR, SXR and PPAR ligands. In one preferred embodiment, the method comprises administering to a mammalian subject a ligand for an LXR nuclear receptor. Suitable LXR ligands include 20(S) hydroxycholesterol, 22(R) hydroxycholesterol, 24(S) hydroxycholesterol, 25-hydroxycholesterol, and 24(S), 25 epoxycholesterol. Preferably, the LXR ligand is 20(S) hydroxycholesterol. In another preferred embodiment, the method comprises administering to a mammalian subject a ligand for an RXR nuclear receptor. Suitable RXR ligands include 9-cis retinoic acid, retinol, retinal, all-trans retinoic acid, 13-cis retinoic acid, acitretin, fenretinide, etretinate, CD 495, CD564, TTNN, TTNNPB, TFAB, and LGD 1069. Preferably, the RXR ligand is 9-cis retinoic acid. In another preferred embodiment, the method comprises administering to a mammalian subject a ligand for a PPAR nuclear receptor. One suitable ligand is a ligand selected from the class of thiazolidinediones. In yet another preferred embodiment, the method comprises administering at least two ligands for a nuclear receptor. In a particularly preferred embodiment, the ligands are 20(S) hydroxycholesterol and 9-cis retinoic acid.
In addition, another suitable method for increasing cholesterol efflux from cells of a mammalian subject comprises administering to the mammalian subject an eicosanoid in an amount sufficient to increase cholesterol efflux. Suitable eicosanoids include prostaglandin E2, prostaglandin J2, and prostacyclin (prostaglandin I2).
In another embodiment, the present invention provides a method for increasing cholesterol efflux from cells of a mammalian subject comprising administering to the mammalian subject a compound that increases ABC1 activity in an amount sufficient to increase cholesterol efflux from the cells.
The present invention also provides methods suitable for increasing the gene expression of ABC1 in a mammalian subject. In one preferred embodiment, the method comprises administering to the mammalian subject at least one ligand for a nuclear receptor in an amount sufficient to increase the gene expression of ABC1. Suitable ligands include ligands for LXR, RXR, FXR, SXR, and PPAR nuclear receptors. In another preferred embodiment, the method comprises administering to the mammalian subject a cAMP analogue in an amount sufficient to increase the gene expression of ABC1. In yet another preferred embodiment, the method comprises administering to the mammalian subject a compound that increases the synthesis of cAMP in an amount sufficient to increase the gene expression of ABC1.
In addition, the present invention provides a method for screening a test compound for ABC1 expression modulating activity comprising the steps of: (a) operatively linking a reporter cDNA with an expression modulating portion of the mammalian ABC1 gene to produce a recombinant reporter construct; (b) transfecting the recombinant reporter construct into a population of host cells; (c) assaying the level of reporter gene expression in a sample of the host cells; (d) contacting the host cells with the test compound being screened; (e) assaying the level of reporter gene expression in a sample of the host cells after contact with the test compound; and (f) comparing the relative change in the level of reporter gene expression caused by exposure to the test compound, thereby determining the ABC1 expression modulating activity. The recombinant reporter construct comprises a reporter gene operatively linked to an expression modulating portion of the mammalian ABC1 gene, such as any of the ABC1 5xe2x80x2 flanking region sequences provided by the present invention. In one preferred embodiment, the expression modulating portion of the ABC1 gene comprises SEQ ID NO: 3. In another preferred embodiment, the expression modulating portion of the ABC1 gene comprises nucleotides 1-1532, 1080-1643, 1181-1643, 1292-1643, 1394-1643, or 1394-1532 of SEQ ID NO: 3. Suitable reporter cDNAs include luciferase, xcex2-galactosidase, chloramphenicol acetyl transferase, and green fluorescent protein cDNA. Preferably, the host cell is a mammalian cell. In a particularly preferred embodiment of the method, the recombinant reporter construct is pAPR1.
Also provided by the present invention is a method for screening a test compound to determine whether the test compound promotes ABC1-mediated cholesterol efflux from cells in culture comprising the steps of: (a) assaying the level of cholesterol efflux in a sample of mammalian cells maintained in culture to determine a control level of cholesterol efflux; (b) contacting the cells with the test compound being screened; (c) assaying the level of cholesterol efflux in a sample of cells after contact with the test compound; (d) assaying the level of ABC1-mediated cholesterol efflux in a sample of cells after contact with the test compound, thereby determining whether the test compound promotes ABC1-mediated cholesterol efflux from cells in culture. The cells can be derived from primary cultures or a cell line. Suitable cells for screening the test compound include fibroblast, macrophage, hepatic, and intestinal cell lines. Preferably, the cell line is RAW 264.7. In one preferred embodiment, the ABC1-mediated cholesterol efflux is measured using an anti-ABC1 antibody that inhibits the activity of ABC1 upon binding. In another preferred embodiment, the ABC1-mediated cholesterol efflux is measured using an antisense ABC1 polynucleotide. In a particularly preferred embodiment, the antisense polynucleotide comprises SEQ ID NO: 57.
In addition, the present invention provides methods for detecting the comparative level of ABC1 expression in cells of a mammalian subject. Such methods can be used to determine the susceptibility of a subject to coronary heart disease. A method for detecting the comparative level of ABC1 expression in cells of a mammalian subject is provided which comprises (a) obtaining a cell sample from the mammalian subject, (b) assaying the level of ABC1 mRNA expression in the cell sample; and (c) comparing the level of ABC1 mRNA expression in the cell sample with a pre-determined standard level of ABC1 mRNA expression, thereby detecting the comparative level of ABC1 gene expression in the cells of a mammalian subject. Suitable methods for measuring the level of ABC1 mRNA expression include, for example, RT-PCR, northern blot, and RNAse protection assay.
The present invention also provides methods for detecting the comparative level of ABC1 protein in cells of a mammalian subject. Such methods can be used to determine the susceptibility of a subject to coronary heart disease. A method for detecting the comparative to amount of ABC1 protein in the cells of a mammalian subject is provided which comprises (a) obtaining a cell sample from the mammalian subject, (b) assaying the amount of ABC1 protein in the cell sample, and (c) comparing the amount of ABC1 protein in the cell sample with a predetermined standard amount of ABC1 protein, thereby detecting the comparative level of ABC1 protein in the cells of the mammalian subject. The amount of ABC1 protein can be determined using various immunoassays available in the art. For example, the amount of ABC1 protein can be determined by (a) contacting the cell sample with a population of anti-ABC1 antibodies and (b) detecting the specific-binding ABC1 antibodies associated with the sample. Suitable methods for detecting ABC1 antibodies include western blotting, immunoprecipitation, and FACS.
In another aspect, the present invention provides antibodies that bind specifically to the described ABC1 polypeptides. In one preferred embodiment, the present invention provides an isolated antibody that binds specifically to an isolated polypeptide comprising SEQ ID NO: 2. In another preferred embodiment, the invention provides an isolated antibody that bind specifically to an isolated polypeptide comprising an amino acid sequence that has at least 98% identity with SEQ ID NO: 2. The antibody can be a monoclonal antibody or the antibody can be a polyclonal antibody. In yet another embodiment, the antibody, upon binding to an ABC1 polypeptide, inhibits the cholesterol transport activity of the ABC1 polypeptide.
In addition, the present invention provides kits suitable for screening a compound to determine the ABC1 expression modulating activity of the compound comprising a reporter cDNA operatively linked to an expression modulating portion of the mammalian ABC1 gene in an amount sufficient for at least one assay and instructions for use. In one preferred embodiment, the kit further comprises means for detecting the reporter gene. In another preferred embodiment, the expression modulating portion of the mammalian ABC1 gene comprises SEQ ID NO: 3. In yet another preferred embodiment, the expression modulating portion of the mammalian ABC1 gene comprises nucleotides 1-1532, 1080-1643, 1181-1643, 1292-1643, 1394-1643, or 1394-1532 of SEQ ID NO: 3. Suitable reporter cDNAs include luciferase, xcex2-galactosidase, chloramphenicol acetyl transferase, and green fluorescent protein cDNA. Preferably, the reporter cDNA is luciferase. In a particularly preferred embodiment of the method, the recombinant reporter construct is pAPR1.
The present invention also provides kits suitable for screening a compound to determine whether the compound modulates ABC1-dependent cholesterol efflux. In one preferred embodiment, the kit comprises an inactivating anti-ABC1 antibody in an amount sufficient for at least one assay and instructions for use. In another preferred embodiment, the kit comprises an antisense ABC1 oligonucleotide in an amount sufficient for at least one assay and instructions for use. In a particularly preferred embodiment, the antisense ABC1 oligonucleotide comprises SEQ ID NO: 53.
USPTO Backgrounds
USPTO Backgrounds