Compositions and methods for increasing cholesterol efflux and raising HDL using ATP binding cassette transporter protein ABC1

Abstract

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.

1 . An isolated polypeptide comprising SEQ ID NO: 2.  
     
     
         2 . An isolated polypeptide comprising an amino acid sequence that has at least 98% identity with the amino acid sequence of SEQ ID NO: 2.  
     
     
         3 . An isolated polynucleotide selected from the group consisting of a polynucleotide encoding the polypeptide of  claim 1 , a polynucleotide comprising SEQ ID NO: 1, a polynucleotide comprising nucleotides 291-7074 of SEQ ID NO: 1, and a polynucleotide encoding the polypeptide of  claim 2 .  
     
     
         4 . An isolated polynucleotide comprising a nucleotide sequence that is complementary to the polynucleotide of  claim 3 .  
     
     
         5 . An isolated polynucleotide comprising a nucleotide sequence that has at least 90% identity with the polynucleotide comprising SEQ ID NO: 1.  
     
     
         6 . An isolated polynucleotide comprising a nucleotide sequence that has at least 95% identity with the polynucleotide comprising SEQ ID NO: 1.  
     
     
         7 . An isolated polynucleotide comprising a nucleotide sequence that is complementary to the polynucleotide of  claim 5 .  
     
     
         8 . A composition comprising the polynucleotide of  claim 3  and a suitable carrier.  
     
     
         9 . A composition comprising the polynucleotide of  claim 4  and a suitable carrier.  
     
     
         10 . A composition comprising the polynucleotide of  claim 5  and a suitable carrier.  
     
     
         11 . A recombinant vector comprising the polynucleotide of  claim 3 .  
     
     
         12 . A recombinant vector comprising the polynucleotide of  claim 4 .  
     
     
         13 . A recombinant vector comprising the polynucleotide of  claim 5 .  
     
     
         14 . The recombinant vector of  claim 11 , further comprising a heterologous promoter polynucleotide.  
     
     
         15 . The recombinant vector of  claim 12 , further comprising a heterologous promoter polynucleotide.  
     
     
         16 . The recombinant vector of  claim 13 , further comprising a heterologous promoter polynucleotide.  
     
     
         17 . The recombinant vector of  claim 14 , wherein said heterologous promoter is a cytomegalovirus promoter.  
     
     
         18 . The recombinant vector of  claim 17 , wherein said vector is pCEPhABC1.  
     
     
         19 . A composition comprising the recombinant vector of  claim 11 .  
     
     
         20 . A composition comprising the recombinant vector of  claim 13 .  
     
     
         21 . A composition comprising the recombinant vector of  claim 14 .  
     
     
         22 . A host cell comprising the recombinant vector of  claim 11 .  
     
     
         23 . A host cell comprising the recombinant vector of  claim 13 .  
     
     
         24 . A host cell comprising the recombinant vector of  claim 14 .  
     
     
         25 . An isolated polypeptide comprising SEQ ID NO: 8.  
     
     
         26 . An isolated polynucleotide encoding the polypeptide of  claim 25 .  
     
     
         27 . An isolated polynucleotide comprising SEQ ID NO: 7.  
     
     
         28 . An isolated polypeptide comprising SEQ ID NO: 10.  
     
     
         29 . An isolated polynucleotide encoding the polypeptide of  claim 28 .  
     
     
         30 . An isolated polynucleotide comprising SEQ ID NO: 9.  
     
     
         31 . A recombinant vector comprising the polynucleotide of  claim 27 .  
     
     
         32 . A recombinant vector comprising the polynucleotide of  claim 30 .  
     
     
         33 . A method for producing an ABC1 protein in a mammalian host cell comprising 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.    
     
     
         34 . The method of  claim 33 , wherein the recombinant expression vector comprises the polynucleotide of  claim 3 .  
     
     
         35 . A method for expressing ABC1 in the cells of a mammalian subject comprising the step of administering to a mammalian subject a recombinant expression vector comprising a polynucleotide encoding ABC1 in an amount sufficient to express ABC 1 in the cells of a mammalian subject.  
     
     
         36 . The method of  claim 35 , wherein the recombinant expression vector comprises the polynucleotide of  claim 3 .  
     
     
         37 . A method suitable for increasing cholesterol efflux from cells of a mammalian subject comprising administering to the mammalian subject a recombinant expression vector comprising a polynucleotide encoding ABC1 in an amount sufficient to increase cholesterol efflux from said cells.  
     
     
         38 . The method of  claim 37 , wherein the recombinant expression vector comprises the polynucleotide of  claim 3 .  
     
     
         39 . The method of  claim 37 , wherein the recombinant expression vector is a viral delivery vector.  
     
     
         40 . The method of  claim 39 , wherein the viral delivery vector is an adenoviral vector.  
     
     
         41 . The method of  claim 39 , wherein the viral vector is a lentiviral vector.  
     
     
         42 . The method of  claim 37 , wherein the recombinant expression vector is a non-viral delivery vector.  
     
     
         43 . A method suitable for increasing cholesterol efflux from cells of a mammalian subject comprising administering to the mammalian subject via a non-viral delivery system a polynucleotide encoding ABC1 in an amount sufficient to increase cholesterol efflux from said cells.  
     
     
         44 . The method of  claim 43 , wherein the polynucleotide encoding ABC1 comprises the polynucleotide of  claim 3 .  
     
     
         45 . The method of  claim 43 , wherein the non-viral delivery system is selected from the group consisting of DNA-ligand complexes, adenovirus-ligand-DNA complexes, adeno-associated virus-ligand-DNA complexes, direct injection of DNA, CaPO 4  precipitation, gene gun techniques, electroporation, liposomes, and lipofection.  
     
     
         46 . A method suitable for increasing cholesterol efflux from cells of a mammalian subject comprising the step of administering to the mammalian subject a therapeutic amount of a compound that increases the expression of ABC1 in said cells.  
     
     
         47 . The method of  claim 46 , wherein said compound is a cAMP analogue.  
     
     
         48 . The method of  claim 47 , wherein said compound is selected from the group consisting of 8-bromo cAMP, N6-benzoyl cAMP, and 8-thiomethyl cAMP.  
     
     
         49 . The method of  claim 46 , wherein said compound increases the synthesis of cAMP.  
     
     
         50 . The method of  claim 49 , wherein said compound is forskolin.  
     
     
         51 . The method of  claim 46 , wherein said compound is a phosphodiesterase inhibitor.  
     
     
         52 . The method of  claim 51 , wherein said compound is selected from the group consisting of rolipram, theophylline, 3-isobutyl-1-methylxanthine, R020-1724, vinpocetine, zaprinast, dipyridamole, milrinone, amrinone, pimobendan, cilostamide, enoximone, peroximone, and vesnarinone.  
     
     
         53 . A method suitable for increasing the gene expression of ABC1 in the cells of a mammalian subject comprising the step of administering to the mammalian subject a cAMP analogue in an amount sufficient to increase the expression of ABC1 in said cells.  
     
     
         54 . A method suitable for increasing cholesterol efflux of the cells of a mammalian subject by administering to the mammalian subject a compound that increases ABC1 activity in an amount sufficient to increase cholesterol efflux.  
     
     
         55 . 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: 
 assaying the level of cholesterol efflux in a sample of mammalian cells maintained in culture to determine a control level of cholesterol efflux;    contacting the cells with the test compound being screened;    assaying the level of cholesterol efflux in a sample of cells after contact with the test compound; and    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.    
     
     
         56 . The method of  claim 55 , wherein the cultured cells are derived from a cell line.  
     
     
         57 . The method of  claim 56 , wherein the cell line is selected from the group consisting of fibroblast, macrophage, hepatic, and intestinal cell lines.  
     
     
         58 . The method of  claim 57 , wherein the cell line is RAW 264.7.  
     
     
         59 . The method of  claim 55 , wherein the level of ABC1-dependent cholesterol efflux is assayed using an anti-ABC1 antibody that inhibits the activity of ABC1 upon binding.  
     
     
         60 . The method of  claim 55 , wherein the level of ABC1-dependent cholesterol efflux is assayed using an anti-sense ABC1 polynucleotide.  
     
     
         61 . The method of  claim 60 , wherein the polynucleotide comprises SEQ ID NO: 57.  
     
     
         62 . A method for detecting the comparative level of ABC1 expression in the cells of a mammalian subject comprising the steps of: 
 obtaining a cell sample from the mammalian subject;    assaying the level of ABC1 mRNA expression in the cell sample; and    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.    
     
     
         63 . The method of  claim 62 , wherein detection of the comparative level of ABC1 expression in cells of a mammalian subject is used to determine a susceptibility to coronary heart disease of the mammalian subject.  
     
     
         64 . The method of  claim 62 , wherein the level of ABC1 mRNA expression is assayed by reverse transcription polymerase chain reaction, northern blot, or RNAse protection assay.  
     
     
         65 . A method for detecting the comparative level of ABC1 protein in the cells of a mammalian subject comprising the steps of: 
 obtaining a cell sample from the mammalian subject;    assaying the amount of ABC1 protein in the cell sample; and    comparing the amount of ABC1 protein in the cell sample with a pre-determined standard amount of ABC1 protein, thereby detecting the comparative level of ABC1 protein in the cells of the mammalian subject.    
     
     
         66 . The method of  claim 65 , wherein detection of the comparative level of ABC1 protein in cells of a mammalian subject is used to determine a susceptibility to coronary heart disease of the mammalian subject.  
     
     
         67 . The method of  claim 65 , wherein the assay to determine the amount of ABC1 protein comprises an immunoassay.  
     
     
         68 . The method of  claim 67 , wherein the amount of ABC1 protein is 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 cell sample.  
     
     
         69 . The method of  claim 68 , wherein the ABC1 antibodies are detected by western blotting, immunoprecipitation, or FACS.  
     
     
         70 . An isolated antibody that binds specifically to the isolated polypeptide of  claim 1  or  claim 2 .  
     
     
         71 . The antibody of  claim 70  wherein the antibody is a monoclonal antibody.  
     
     
         72 . The antibody of  claim 70  wherein the antibody is a polyclonal antibody.  
     
     
         73 . The antibody of  claim 70  wherein the antibody, upon binding to an ABC1 polypeptide, inhibits the cholesterol transport activity of the ABC1 polypeptide.  
     
     
         74 . A kit suitable for screening a compound to determine whether the compound modulates ABC1-dependent cholesterol efflux comprising an inactivating anti-ABC1 antibody in an amount sufficient for at least one assay and instructions for use.  
     
     
         75 . A kit suitable for screening a compound to determine whether the compound modulates ABC1-dependent cholesterol efflux comprising an antisense ABC1 oligonucleotide in an amount sufficient for at least one assay and instructions for use.  
     
     
         76 . The kit of  claim 75 , wherein the antisense ABC1 oligonucleotide comprises SEQ ID NO: 53.