EP1171078A2 - Compositions et procedes agissant sur les taux de cholesterol des lipoproteines de haute densite (hdl) et des apolipoproteines ai, sur les taux de cholesterol des lipoproteines de tres faible densite (vldl) et sur le taux de cholesterol des lipoproteines de faible densite (ldl) - Google Patents

Compositions et procedes agissant sur les taux de cholesterol des lipoproteines de haute densite (hdl) et des apolipoproteines ai, sur les taux de cholesterol des lipoproteines de tres faible densite (vldl) et sur le taux de cholesterol des lipoproteines de faible densite (ldl)

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Publication number
EP1171078A2
EP1171078A2 EP00918362A EP00918362A EP1171078A2 EP 1171078 A2 EP1171078 A2 EP 1171078A2 EP 00918362 A EP00918362 A EP 00918362A EP 00918362 A EP00918362 A EP 00918362A EP 1171078 A2 EP1171078 A2 EP 1171078A2
Authority
EP
European Patent Office
Prior art keywords
lipg
leu
gly
cholesterol
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP00918362A
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German (de)
English (en)
Other versions
EP1171078A4 (fr
Inventor
Michael Jaye
Kevin J. Lynch
Dilip V. Amin
Kim-Anh Thi Doan
Dawn Marchadier
Cyrille Maugeais
Daniel J. Rader
John A. Krawiec
Victoria J. South
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Aventis Pharmaceuticals Inc
University of Pennsylvania Penn
Original Assignee
Aventis Pharmaceuticals Inc
University of Pennsylvania Penn
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Priority claimed from US09/277,401 external-priority patent/US7008776B1/en
Application filed by Aventis Pharmaceuticals Inc, University of Pennsylvania Penn filed Critical Aventis Pharmaceuticals Inc
Publication of EP1171078A2 publication Critical patent/EP1171078A2/fr
Publication of EP1171078A4 publication Critical patent/EP1171078A4/fr
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • This invention relates to methods and compositions for increasing the level of high density lipoprotein (HDL) cholesterol and apolipoprotein Al in a patient and to methods and compositions for lowering the levels of very low density lipoprotein (VLDL) cholesterol, and low density lipoprotein (LDL) cholesterol in a patient.
  • the invention includes within its scope methods and compositions which lower the expression of, or inhibit the activity of, a gene, LIPG, which encodes a lipase enzyme that lowers the levels of HDL cholesterol and apolipoprotein Al.
  • the invention additionally includes within its scope methods and compositions to increase the expression of, or enhance the activity of, the lipase enzyme, resulting in lower levels of VLDL and LDL cholesterol.
  • LJPids LJPids
  • Lipids are water-insoluble organic biomolecules, which are essential components of diverse biological functions, including the storage, transport, and metabolism of energy, and membrane structure and fluidity. Lipids are derived from two sources in man and other animals: some lipids are ingested as dietary fats and oils and other lipids are biosynthesized by the human or animal. In mammals, at least 10% of the body weight is lipid, the bulk of which is in the form of triacylglycerols.
  • Triacylglycerols also known as triglycerides and triacylglycerides, are made up of three fatty acids esterified to glycerol. Dietary triacylglycerols are stored in adipose tissues as a source of energy, or hydrolyzed in the digestive tract by triacylglycerol lipases, the most important of which is pancreatic lipase. Triacylglycerols are transported between tissues in the form of lipoproteins.
  • Lipoproteins are micelle-like assemblies found in plasma which contain varying proportions of different types of lipids and proteins (called apoproteins) .
  • apoproteins There are five main classes of plasma lipoproteins, the major function of which is lipid transport. These classes are, in order of increasing density: chylomicrons; very low density lipoproteins (VLDL) ; intermediate-density lipoproteins (IDL) ; low density lipoproteins (LDL) ; and high density lipoproteins (HDL) .
  • each lipoprotein class transports predominantly one type of lipid: triacylglycerols described above are transported in chylomicrons, VLDL, and IDL; whereas phospholipids and cholesterol esters are transported in HDL and LDL respectively.
  • Phospholipids are di-fatty acid esters of glycerol phosphate which contain a polar group coupled to the phosphate. Phospholipids are important structural components of cellular membranes. Phospholipids are hydrolyzed by enzymes called phospholipases. Phosphatidylcholine, an exemplary phospholipid, is a major component of most eukaryotic cell membranes.
  • Cholesterol is the metabolic precursor of steroid hormones and bile acids as well as an essential constituent of cell membranes. In man and other animals, cholesterol is ingested in the diet and is synthesized also by the liver and other tissues. Dietary cholesterol is transported from the intestine to the liver by large lipoprotein molecules in the blood. The liver secretes Very Low Density Lipoprotein
  • VLDL which transports cholesterol and cholesterol ester and various other compounds into the bloodstream.
  • VLDL is partially converted in adipose tissue to Low Density Lipoprotein (LDL) .
  • LDL transports both free and esterified cholesterol to body tissues.
  • High Density Lipoprotein (HDL) transports cholesterol to the liver to be broken down and excreted.
  • Membranes surround every living cell and serve as a barrier between the intracellular and extracellular compartments. Membranes also enclose the eukaryotic nucleus, make up the endoplas ic reticulum, and serve specialized functions such as in the myelin sheath that surrounds axons.
  • a typical membrane contains about 40% lipid and 60% protein, but there is considerable variation.
  • the major lipid components are phospholipids, specifically phosphatidylcholine and phosphatidylethanolamine, and cholesterol.
  • the physicochemical properties of membranes, such as fluidity, can be changed by modification of either the fatty acid profiles of the phospholipids or the cholesterol content. Modulating the composition and organization of membrane lipids also modulates membrane- dependent cellular functions, such as receptor activity, endocytosis, and cholesterol flux.
  • the triacylglycerol lipases are a family of enzymes which play several pivotal roles in the metabolism of lipids in the body. Three members of the human triacylglycerol lipase family have been described: pancreatic lipase, lipoprotein lipase, and hepatic lipase (Goldberg, I.J., Le, N.-A., Ginsberg, H.N. , Krauss, R.M. , and Lindgren, F.T. (1988) ⁇ 7. Clin . Invest . 81,561-568; Goldberg, I.J., Le, N. , Paterniti J.R. , Ginsberg, H.N.
  • Pancreatic lipase is primarily responsible for the hydrolysis of dietary lipids. Variants of pancreatic lipase have been described, but their physiological role has not been determined (Giller, T., Buchwald, P., Blum-Kaelin, D., and Hunziker, W. (1992) J " . Biol . Chem . 267,16509-16516).
  • Lipoprotein lipase is the major enzyme responsible for the distribution and utilization of triglycerides in the body. Lipoprotein lipase hydrolyzes triglycerides in both chylomicrons and VLDL. Hepatic lipase hydrolyzes triglycerides in IDL and HDL and is responsible for lipoprotein remodeling. Hepatic lipase also functions as a phospholipase and hydrolyzes phospholipids in HDL.
  • Phospholipases play important roles in the catabolism and remodeling of the phospholipid component of lipoproteins and the phospholipids of membranes. Phospholipases also play a role in the release of arachidonic acid and the subsequent formation of prostaglandins, leukotrienes, and other lipids which are involved in a variety of inflammatory processes.
  • the aforementioned lipases are approximately 450 amino acids in length and have leader signal peptides to facilitate secretion.
  • the lipases are comprised of two principal domains ( nomer, K. , D'Arcy, A., and Hunziker, . (1990)
  • the amino terminal domain contains the catalytic site while the carboxyl domain is believed to be responsible for substrate binding, cofactor association, and interaction with cell receptors (Wong, H. , Davis, R.C., Nikazy, J. , Seebart, K.E., and Schotz, M.C. (1991) Proc . Natl . Acad . Sci . USA 88,11290-11294; van Tilbeurgh, H. , Roussel, A., Lalouel, J.-M., and Cambillau, C. (1994) J. Biol . Chem . 269,4626-4633; Wong, H.
  • the naturally occurring lipoprotein lipase is glycosylated. Glycosylation is necessary for LPL enzymatic activity (Semenkovich, C.F., Luo, C.-C, Nakanishi, M.K., Chen, S.-H., Smith, L C. , and Chan L. (1990) J. Biol . Chem . 265, 5429-5433) . There are two sites for N-linked glycosylation in hepatic and lipoprotein lipase and one in pancreatic lipase. Additionally, four sets of cysteines form disulfide bridges which are essential in maintaining structural integrity for enzymatic activity (Lo, J.-Y., Smith, L.C., and Chan, L.
  • GXSXG triacylglycerol lipase family share a number of conserved structural features.
  • One such feature is the "GXSXG” motif, in which the central serine residue is one of the three residues comprising the "catalytic triad" (Winkler, K. , D'Arcy, A., and Hunziker, W. (1990) Nature 343, 771-774; Faustinella, F. , Smith, L.C., and Chan, L. (1992) Biochemistry 31,7219-7223).
  • conserved aspartate and histidine residues make up the balance of the catalytic triad.
  • a short, span of 19-23 amino acids forms an amphipathic helix structure and covers the catalytic pocket of the enzyme (Winkler, K. , D'Arcy, A., and Hunziker, W. (1990) Nature 343, 771-774). This region diverges significantly between members of the family. It has been determined recently that the span confers substrate specificity to the enzymes (Dugi, K.A. , Dichek H.L., and Santamarina-Fojo, S. (1995) J. Biol . Chem . 270, 25396-25401).
  • the triacylglycerol lipases possess varying degrees of heparin binding activity. Lipoprotein lipase has the highest affinity for heparin. This binding activity has been mapped to stretches of positively charged residues in the amino terminal domain (Ma, Y., Henderson, H.E., Liu, M.-S., Zhang, H., Forsythe, I.J., Clarke-Lewis, I., Hayden, M.R., and Brunzell, J.D. J. Lipid Res . 35, 2049-2059) .
  • Lipoprotein lipase and pancreatic lipase are both known to function in conjunction with co-activator proteins: apolipoprotein CII for lipoprotein lipase; and colipase for pancreatic lipase.
  • the genetic sequences encoding human pancreatic lipase, hepatic lipase and lipoprotein lipase have been reported (Genbank accession #M93285, #J03540, and #M15856 respectively) .
  • the messenger RNAs of human hepatic lipase and pancreatic lipase are approximately 1.7 and 1.8 kilobases in length respectively.
  • Two mRNA transcripts of 3.6 and 3.2 kilobases are produced from the human lipoprotein lipase gene. These two transcripts utilize alternate poiyadenylation signals and differ in their translational efficiency (Ranganathan, G. , Ong, J.M. , Yukht, A., Saghizadeh, M. , Simsolo, R.B., Pauer, A., and Kern, P.A. (1995) J. Biol . Chem . 270, 7149-7155).
  • the metabolism of lipids involves the interaction of lipids, apoproteins, lipoproteins, and enzymes.
  • Hepatic lipase and lipoprotein lipase are multifunctional proteins which mediate the binding, uptake, catabolism, and remodeling of lipoproteins and phospholipids.
  • Lipoprotein lipase and hepatic lipase function while bound to the luminal surface of endothelial cells in peripheral tissues and the liver respectively. Both enzymes participate in reverse cholesterol transport, which is the movement of cholesterol from peripheral tissues to the liver either for excretion from the body or for recycling.
  • Genetic defects in both hepatic lipase and lipoprotein lipase are known to be the cause of familial disorders of lipoprotein metabolism. Defects in the metabolism of lipoproteins result in serious metabolic disorders, including hypercholesterolemia, hyperlipidemia, and atherosclerosis.
  • Atherosclerosis is a complex, polygenic disease which is defined in histological terms by deposits (lipid or fibrolipid plaques) of lipids and of other blood derivatives in blood vessel walls, especially the large arteries (aorta, coronary arteries, carotid) .
  • These plaques which are more or less calcified according to the degree of progression of the atherosclerotic process, may be coupled with lesions and are associated with the accumulation in the vessels of fatty deposits consisting essentially of cholesterol esters.
  • These plaques are accompanied by a thickening of the vessel wall, hypertrophy of the smooth muscle, appearance of foam cells (lipid-laden cells resulting from uncontrolled uptake of cholesterol by recruited macrophages) and accumulation of fibrous tissue.
  • the atheromatous plaque protrudes markedly from the wall, endowing it with a stenosing character responsible for vascular occlusions by atheroma, thrombosis or embolism, which occur in those patients who are most affected. These lesions can lead to serious cardiovascular pathologies such as infarction, sudden death, cardiac insufficiency, and stroke.
  • HDL High density lipoprotein
  • High density lipoprotein (HDL) cholesterol levels are inversely associated with risk of atherosclerotic cardiovascular disease (Gordon et al., N. Engl . J. Med . , 321, 1311-1316 (1989)). At least 50% of the variation in HDL cholesterol levels is genetically determined (Breslow, J.L., The Metabolic Basis of Inhereited Disease , 2031-2052, McGraw- Hill, New York (1995); Heller et al., N. Engl . J . Med . , 328, 1150-1156 (1993)), but the genes responsible for variation in HDL levels have not been fully elucidated.
  • LPL lipoprotein lipase
  • HL hepatic lipase
  • TG triacylglycerol
  • the normal range for HDL cholesterol is about 35 to 65 mg/dL, and the HDL level should account for more than 25% of the total cholesterol.
  • VLDL Very low density lipoprotein
  • LDL low density lipoprotein
  • VLDL are the precursors of LDL.
  • Therapeutic agents that lower plasma VLDL and LDL cholesterol levels are highly desirable because of the known strong association between these lipid parameters and coronary heart disease.
  • the range for VLDL cholesterol is about 1 to 30 mg/dL and the range for LDL cholesterol is about 60 to 160 mg/dL.
  • the LDL to HDL ratio is ideally less than 3.5.
  • HL lipoprotein lipase
  • the physiologic function of HL appears to be related to the metabolism of lipoprotein remnants and HDL (Bensadoun et al., Curr. Opin . Lipidol . , 7 , 77-81 (1996)).
  • Genetic deficiency of HL is associated with modestly increased levels of remnants and HDL cholesterol in humans (Hegele et al., Arterioscler . Thromb . , 13, 720-728 (1993)) and mutant mice (Homanics et al., J. Biol . Chem . , 270, 2974-2980 (3995)).
  • HL deficiency is associated with reduced atherosclerosis in apoE mutant mice (Mezdour et al., J. Biol . Chem . , 272, 13570-13575 (1997)).
  • Transgenic animals overexpressing HL have decreased HDL (Busch et al., J . Biol . Chem . , 269, 16376-16382 (1994); Fan et al., Proc . Natl . Acad . Sci . USA, 91, 8724-8728 (1994)).
  • Increased HL activity in humans is associated with low HDL cholesterol.
  • the HL locus on chromosome 15q21 has been associated with variation in plasma HDL cholesterol levels in humans (Cohen et al., J. Clin . Invest . , 94, 2377-2384 (1994); Guerra et al. , Proc.
  • a high local level of lipase activity may result in cytotoxic levels of fatty acids and lysophosphatidylcholine being produced in precursors of atherosclerotic lesions.
  • compositions for lowering the expression of the LIPG gene in a patient comprising an antisense nucleic acid, including for example, an expression vector which includes said antisense nucleic acid.
  • an expression vector which includes said antisense nucleic acid examples include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, herpesviral vectors, and naked DNA vectors.
  • the antisense nucleic acid can be, for example, an oligonucleotide which contains chemically modified bases.
  • compositions for lowering the enzymatic activity of the LIPG polypeptide in a patient comprising a neutralizing antibody capable of binding to the LIPG polypeptide and lowering its enzymatic activity, including, for example, an expression vector which includes a DNA sequence encoding said antibody.
  • preferred expression vectors are retroviral vectors, adenoviral vectors, adeno-associated viral vectors, herpesviral vectors, and naked DNA vectors.
  • Still another aspect of the present invention is the provision of a composition for lowering the enzymatic activity of the LIPG polypeptide in a patient comprising an intracellular binding protein, including, for example, an expression vector which includes a DNA sequence encoding said intracellular binding protein.
  • an expression vector which includes a DNA sequence encoding said intracellular binding protein.
  • preferred expression vectors are retroviral vectors, adenoviral vectors, adeno-associated viral vectors, herpesviral vectors, and naked DNA vectors.
  • compositions which comprises an inhibitor that is capable of inhibiting the enzymatic activity of the LIPG polypeptide in a patient (B) a composition which comprises an inhibitor that is capable of lowering the expression of the LIPG gene in a patient; and (C) a composition which is capable of lowering the expression of LIPG in a patient and which comprises a ribozyme, including, for example, an expression vector which includes a DNA sequence encoding said ribozyme.
  • preferred expression vectors are retroviral vectors, adenoviral vectors, adeno-associated viral vectors, herpesviral vectors, and naked DNA vectors.
  • a preferred ribozyme is a hammerhead ribozyme.
  • the present invention provides also: (D) a composition which increases the level of LIPG polypeptide in a patient and which comprises an expression vector that includes a DNA sequence encoding the LIPG polypeptide or an enhancer that is capable of increasing the expression of the LIPG gene; and (E) a composition which increases the enzymatic activity of LIPG polypeptide in a patient which comprises an enhancer that binds to and enhances the enzymatic activity of the LIPG polypeptide.
  • the present invention provides a method for raising the level of high density lipoprotein (HDL) cholesterol and apolipoprotein Al in a patient by administering to the patient a composition which lowers the enzymatic activity of LIPG in said patient, for example, by lowering the level of LIPG polypeptide in the patient.
  • the method involves the use of a composition which comprises an antisense nucleic acid, particularly one that is modified to increase the chemical stability of the nucleic acid.
  • the aforementioned method can be practiced also by use of a composition which comprises a neutralizing antibody capable of binding to the LIPG polypeptide and lowering its enzymatic activity or a composition which comprises an inhibitor which inhibits the enzymatic activity of LIPG polypeptide, for example, a compound which lowers the expression of the LIPG gene or a composition which comprises a ribozyme that cleaves mRNA encoding LIPG, or a composition which comprises a DNA molecule and a liposo e, for example, a cationic liposome.
  • the aforementioned method comprises also the administration of a composition which is capable of expressing apolipoprotein Al in said patient.
  • Another aspect of the present invention is the provision of a method for lowering the level of very low density lipoprotein (VLDL) cholesterol in a patient by administering to the patient a composition which is capable of increasing the enzymatic activity of LIPG in said patient, for example, by use of a composition which comprises an LIPG polypeptide and a pharmaceutically acceptable carrier and which includes preferably an expression vector that is capable of expressing an LIPG polypeptide, preferably a retroviral vector, an adenoviral vector, or an adeno-associated viral vector.
  • the aforementioned method can be practiced by use of a composition which comprises an enhancer that enhances the enzymatic activity of LIPG polypeptide or an enhancer that increases expression of the LIPG gene.
  • Still another aspect of the present invention is the provision of a method for lowering the level of low density lipoprotein (LDL) cholesterol in a patient by administering to the patient a composition which is capable of increasing the enzymatic activity of LIPG in the patient, preferably by use of an LIPG polypeptide, for example, by use of an expression vector that is capable of expressing the LIPG polypeptide, preferably by use of a retroviral vector, an adenoviral vector, or an adeno-associated viral vector.
  • the aforementioned method includes preferably the use of a composition which comprises an enhancer that enhances the enzymatic activity of LIPG polypeptide or an enhancer which increases the expression of the LIPG gene.
  • the present invention provides also a method for lowering the level of LDL cholesterol in a patient by administering to the patient an enhancer which preferentially enhances the enzymatic reactions between LIPG polypeptide and LDL cholesterol relative to the enzymatic reactions between LIPG polypeptide and HDL cholesterol and apolipoprotein Al.
  • the present invention provides a method for lowering the level of VLDL cholesterol in a patient by administering to the patient an enhancer which preferentially enhances the enzymatic reactions between LIPG polypeptide and VLDL cholesterol relative to the enzymatic reactions between LIPG polypeptide and HDL cholesterol and apolipoprotein Al .
  • Still another aspect of the present invention is the provision of a method for diagnosing a predisposition to low HDL cholesterol and apolipoprotein Al levels by obtaining a tissue sample from a patient and measuring the level of LIPG polypeptide in the sample, for example, by use of blood tissue and the use of an immunoassay for measurement.
  • the levels of LIPG polypeptide are measured by measuring the levels of LIPG mRNA.
  • An additional aspect of the present invention is the provision of a method for determining whether a test compound can inhibit the enzymatic reaction between the LIPG polypeptide and HDL cholesterol and apolipoprotein Al comprising: (A) comparing the level of HDL cholesterol and apolipoprotein Al in a first sample comprising: (1) HDL cholesterol and apolipoprotein Al, (2) LIPG polypeptide, and (3) the test compound with the level of HDL cholesterol and apolipoprotein Al in another sample comprising: (4) HDL cholesterol and apolipoprotein Al, and (5) LIPG polypeptide; and (B) identifying whether or not the test compound is effective in inhibiting the enzymatic reaction between the LIPG polypeptide and HDL cholesterol and apolipoprotein Al by observing whether or not the first sample has a higher level of HDL cholesterol and apolipoprotein Al than that of said other sample.
  • the present invention provides also a method for determining whether a test compound can enhance the enzymatic reaction between the LIPG polypeptide and VLDL cholesterol comprising: (A) comparing the level of VLDL cholesterol in a first sample comprising: (1) VLDL cholesterol, (2) LIPG polypeptide, and (3) the test compound with the level of VLDL cholesterol in another sample comprising: (4) VLDL cholesterol, and (5) LIPG polypeptide; and (B) identifying whether or not the test compound is effective in enhancing the enzymatic reaction between the LIPG polypeptide and VLDL cholesterol by observing whether or not the first sample has a lower level of VLDL cholesterol than that of said other sample.
  • Still another aspect of the present invention is the provision of a method for determining whether a test compound can enhance the enzymatic reaction between the LIPG polypeptide and LDL cholesterol comprising: (A) comparing the level of LDL cholesterol in a first sample comprising: (1) LDL cholesterol, (2) LIPG polypeptide, and (3) the test compound with the level of LDL cholesterol in another sample comprising: (4) LDL cholesterol, and (5) LIPG polypeptide; and (B) identifying whether or not the test compound is effective in enhancing the enzymatic reaction between the LIPG polypeptide and LDL cholesterol by observing whether or not the first sample has a lower level of LDL cholesterol than that of said other sample.
  • Figure 1 shows the sequences (SEQ ID Nos: 17-31) of the primers used in the exemplified PCR amplifications.
  • Figure 2 shows the nucleic acid sequence (SEQ ID NO: 1) and the deduced amino acid sequence (SEQ ID NO: 2) of the differential display RT-PCR product containing the LIPG gene cDNA.
  • the sequences corresponding to the two primers used in the amplification are underlined.
  • the termination codon and poiyadenylation signal are boxed.
  • the GAATTC motifs and flanking sequence are from the pCRII vector into which the product was cloned.
  • Figure 3 shows the nucleic acid sequence (SEQ ID NO: 3) and the deduced amino acid sequence (SEQ ID NO: 4) of the 5 'RACE extension of the LIPG cDNA.
  • the sequences corresponding to the two primers used in the amplification are underlined.
  • the GAATTC motifs and flanking sequence are from the pCRII vector into which the product was cloned.
  • Figure 4 shows the sequence (SEQ ID NO: 7) of the cDNA containing the complete open reading frame of the LIPG gene, LLGXL.
  • the start codon (ATG) and termination codon (TGA) are boxed.
  • the Dral site (TTTAAA) and Srfl site (GCCCGGGC) used in the construction of the expression vectors are underlined.
  • Figure 5 shows the deduced amino acid sequence (SEQ ID NO: 8) of the LLGXL protein. The predicted signal sequence is underlined.
  • Figure 6 shows a protein sequence alignment of the members of the triacylglycerol lipase gene family (SEQ ID Nos: 13-15) . Shaded residues are identical to the LLGXL protein (SEQ ID NO: 8) .
  • the deduced amino acid sequence of human LIPG(EL) is provided on the top line and is compared with the other major members of the TG lipase family, LPL, HL and PL.
  • EL residues identical to those in at least one other member of the family are shaded as well as the corresponding residue in the other family member. Amino acids are numbered according to convention beginning with the initial residue of the secreted protein. The predicted sites of signal peptide cleavage are marked with a solid line between amino acid residues.
  • the GXSXG lipase motif containing the active serine is boxed.
  • the amino acids of the catalytic triad are marked with an asterisk.
  • the conserved cysteines are marked with filled circles.
  • Potential N-linked glycosylation sites are marked with arrowheads.
  • the lid region is indicated by a bold line. Gaps were introduced into the sequences to maximize the alignment values using the CLUSTAL program.
  • Figure 7 shows a northern analysis of LIPG mRNA in THP-1 cells.
  • Cells were stimulated with either PMA or PMA and oxidized LDL (PMA + oxLDL) .
  • Numbers at the left indicate the positions of RNA standards (in kilobases) .
  • Figure 8 shows a northern-blot analysis of expression of LIPG mRNA compared with LPL in human tissues. A blot containing mRNA from the indicated human tissues was incubated with radiolabelled LPL and ⁇ -actin (ACTB) probes as described.
  • ACTB ⁇ -actin
  • Figure 9 shows a Northern-blot analysis of cultured cell lines.
  • the panel on the left (lanes 1-6) was hybridized with the LIPG(EL) probe and that on the right (lanes 7-12) with the LPL probe.
  • Figure 10 shows the sequence of the immunizing peptide (SEQ ID NO: 16) and its relation to the LLGXL protein sequence.
  • the peptide is shown in the shaded box.
  • the terminal cysteine was introduced to aid coupling of the peptide to the carrier protein.
  • Figure 11 shows the results obtained when conditioned media from HUVEC and HCAEC were subjected to immunoblot analysis with rabbit anti-EL peptide antiserum.
  • Lane 1 unconditioned media; lane 2, unstimulated HUVEC; lane 3, HUVEC stimulated with PMA; lane 4, unstimulated HCAEC; lane 5, HCAEC stimulated with PMA.
  • Figure 12 shows a western analysis of heparin-Sepharose bound proteins in conditioned medium from COS-7 cells transiently transfected with an expression vector containing a cDNA for LLGN or LLGXL or no DNA (Mock) .
  • Proteins from PMA-stimulated endothelial cells (HCAEC + PMA) were included for size reference. Numbers to the left indicate the apparent molecular weight of the major immunoreactive proteins as determined by a comparison to protein standards.
  • Figure 13 shows the sequence of the rabbit LIPG PCR product (RLLG.SEQ, SEQ ID NO: 12) and the sequence alignment between the rabbit LIPG PCR product and the corresponding sequence in the human cDNA (LLG7742A) . Identical nucleotides are shaded.
  • Figure 14 shows the phospholipase A activity of human EL-AS, EL and LPL using a phosphatidylcholine substrate.
  • 700 ⁇ l of conditioned medium harvested from COS-7 cells transiently transfected with either pcDNA3.0/LIPG-AS, LIPG, or LPL expression constructs were assayed in triplicate for phospholipase activities as described below. Following a two hour incubation at 37°C, reactions were terminated, and 14C labeled free fatty-acid was extracted, and counted to determine the amount of free fatty-acid produced.
  • Figure 15 shows the triacylglyceride lipase activity of human EL-AS, EL and LPL using a triolein substrate.
  • 700 ⁇ l of conditioned medium harvested from COS-7 cells transiently transfected with either pcDNA3.0/LIPG-AS, LIPG, or LPL expression constructs was assayed in triplicate for triglyceride activities described below. Following a two hour incubation at 37°C, reactions were terminated, and 14C labeled free fatty-acid was extracted, and counted to determine the amount of free fatty- acid produced.
  • Figure 16 shows the hybridization of LIPG and LPL probes to genomic DNAs from different species.
  • Figure 17 shows expression of LIPG in the liver of a wild-type mouse 5 days after AdhEL injection.
  • Lane 1 liver from mouse injected with Adnuli; lane 2, liver from mouse injected with AdhEL.
  • Figure 18 shows plasma levels of HDL cholesterol in AdhEL- and Adnull-injected wild-type mice.
  • Figure 19 shows lipoprotein profiles in wild-type mice injected with AdhEL and Adnuli at baseline before injection (left) and 14 days after injection (right) .
  • Figure 20 shows HDL cholesterol levels in human apoA-I transgenic mice after injection with Adnull or AdhEL.
  • Figure 21 shows ApoA-I levels in human apoA-1 transgenic mice after injection with Adnull or AdhEL.
  • Figure 22 shows the effect of injection of AdhEL in LDL receptor-deficient mice on VLDL/LDL cholesterol levels.
  • Figure 23 shows the effect of AdhEL on HDL receptor- deficient mice on HDL cholesterol levels.
  • Figure 24 is a western blot analysis of LIPG (EL) demonstrating the effect of glycosidase and tunicamycin on EL.
  • the lanes are: untreated EL control; EndoF-treated EL; EndoH-treated EL; neuraminidase- treated EL; marker, plasma sample; EL untreated lysate; EL tunicamycin-treated lysate; EL untreated pellet; EL tunicamycin-treated pellet; EL untreated medium; EL tunicamycin-treated medium, marker; plasma.
  • Figure 25 is a western blot illustrating the effect on EL of heparin administration.
  • Pre lanes are blood samples taken from mice before heparin injection;
  • post lanes are blood samples taken after heparin injection.
  • Control virus did not include EL;
  • EL virus was an adenoviral vector expressing EL.
  • Figures 26A and 26B are bar graphs illustrating the triglyceridase activity (Fig. 26A) and the phospholipase activity (Fig. 26B) of LPL (lipoprotein lipase) , HL (hepatic lipase) and EL (endothelial lipase) .
  • Figures 27A and 27B are graphs illustrating the effect of human serum on lipoprotein lipase triglyceridase activity (Fig. 27A) and endothelial lipase triglyceridase activity (Fig. 27B) .
  • Figures 28A and 28B illustrate expression of EL at different virus doses.
  • Figure 28A is a western blot illustrating the EL present at the different viral doses.
  • Figure 28B illustrates phospholipase activity at the different viral dosages.
  • the present invention relates to methods for regulating the levels of HDL cholesterol and apolipoprotein Al, VLDL cholesterol and LDL cholesterol utilizing methods and compositions which lower or raise the activity of the LIPG lipase enzyme.
  • the present invention is based in part on the discovery of the enzymatic activity of the polypeptide products of the LIPG gene on HDL cholesterol and apolipoprotein Al, VLDL cholesterol and LDL cholesterol.
  • the polypeptide products of LIPG are members of the triacylglycerol lipase family and comprise an approximately 39 kD catalytic domain of the triacylglycerol lipase family, e,g., having the sequence SEQ ID NO: 10.
  • EL endothelial lipase
  • LLGXL polypeptide has 500 amino acids and exhibits 43% similarity to human lipoprotein lipase and 37% similarity to human hepatic lipase.
  • LIPG polypeptide or "LIPG protein” encompasses both LLGN and LLGXL.
  • the sequence of the LIPG polypeptide contains the characteristic GXSXG lipase motif, a conserved catalytic triad, a 19-residue lid region, conserved heparin and lipoprotein binding sites and 5 potential N-linked glycosylation sites.
  • the region with the greatest sequence divergence in the triacylglycerol lipase family is the lid domain, which forms an amphipathic helix covering the catalytic pocket of the enzyme (Winkler et al., Nature , 343, 771-774 (1990); van Tilbeurgh et al., J. Biol . Chem .
  • the 19-residue lid region of LIPG is three residues shorter and less amphipathic than those found in lipoprotein lipase and hepatic lipase, consistent with a different enzymatic profile.
  • the predicted molecule weight of the mature protein is approximately 55 kD; a 68 kD form is likely to be a glycosylated form, whereas a 40 kD form may be the product of a specific proteolytic cleavage.
  • the LIPG polypeptide has the ability to lower the levels of HDL cholesterol and apolipoprotein Al as well as the levels of VLDL cholesterol and LDL cholesterol. It is well established that lowered HDL cholesterol levels result in increased susceptibility to atherosclerosis and increased levels of HDL cholesterol can dramatically reduce susceptibility to atherosclerosis.
  • LIPG One physiologic role of LIPG may be to hydrolyse HDL phospholipid in peripheral tissues and in liver to facilitate selective uptake of HDL cholesteryl ester via the HDL receptor SR-BI (Kozarsky et al., Nature , 387, 414-417
  • the present invention is directed to methods and compositions which lower the activity of LIPG in the body by lowering the expression of the LIPG gene or lowering the enzymatic activity of the LIPG polypeptide.
  • the present invention additionally provides methods and compositions for increasing the expression of the LIPG gene and increasing the enzymatic activity of the LIPG polypeptides.
  • a ⁇ "polypeptide” is a polymeric compound comprised of covalently linked amino acid residues.
  • Amino acids are classified into seven groups on the basis of the side chain: (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group.
  • a "protein” is a polypeptide which plays a structural or functional role in a living cell.
  • polypeptides and proteins of the invention may be glycosylated or unglycosylated.
  • Homology means similarity of sequence reflecting a common evolutionary origin. Polypeptides or proteins are said to have homology, or similarity, if a substantial number of their amino acids are either (1) identical, or (2) have a chemically similar side chain. Nucleic acids are said to have homology if a substantial number of their nucleotides are identical.
  • isolated polypeptide or “isolated protein” is a polypeptide or protein which is substantially free of those compounds that are normally associated therewith in its natural state (e.g., other proteins or polypeptides, nucleic acids, carbohydrates, lipids) . "Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with biological activity, and which may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into a pharmaceutically acceptable preparation.
  • a molecule is "antigenic" when it is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor.
  • An antigenic polypeptide contains at least about 5, and preferably at least about 10, amino acids.
  • An antigenic portion of a molecule can be that portion that is immunodo inant for antibody or T cell receptor recognition, or it can be a portion used to generate an antibody to the molecule by conjugating the antigenic portion to a carrier molecule for immunization.
  • a molecule that is antigenic need not be itself immunogenic, i.e., capable of eliciting an immune response without a carrier.
  • "LLGN polypeptide” and "LLGN protein” mean a polypeptide including the sequence SEQ ID NO: 6, said polypeptide being glycosylated or non-glycosylated.
  • LLCXL polypeptide and "LLGXL protein” mean a polypeptide including the sequence SEQ ID NO: 8, said polypeptide being glycosylated or non-glycosylated.
  • LIPG polypeptide and "LIPG protein” describe the lipase enzyme encoded by the LIPG gene and generically describes both the LLGN polypeptide and the LLGXL polypeptide.
  • Endothelial lipase refers to the lipase enzyme encoded by the LIPG gene and is equivalent to the term LIPG polypeptide.
  • the LIPG polypeptide or protein of the invention includes any analogue, fragment, derivative, or mutant which is derived from an LIPG polypeptide and which retains at least one biological property of the LIPG polypeptide.
  • Different variants of the LIPG polypeptide exist in nature. These variants may be allelic variations characterized by differences in the nucleotide sequences of the structural gene coding for the protein, or may involve differential splicing or post-translational modification. The skilled artisan can produce variants having single or multiple amino acid substitutions, deletions, additions, or replacements.
  • variants may include, inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to the LIPG polypeptide, (c) variants in which one or more of the amino acids includes a substituent group, and (d) variants in which the LIPG polypeptide is fused with another polypeptide such as serum albumin.
  • Other LIPG polypeptides of the invention include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at conserved or non-conserved positions. In another embodiment, amino acid residues at non-conserved positions are substituted with conservative or non-conservative residues.
  • the techniques for obtaining these variants including genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques, are known to persons having ordinary skill in the art.
  • allelic variations, analogues, fragments, derivatives, mutants, and modifications, including alternative mRNA splicing forms and alternative post- translational modification forms result in derivatives of the LIPG polypeptide which retain any of the biological properties of the LIPG polypeptide, they are included within the scope of this invention.
  • nucleic acid is a polymeric compound comprised of covalently linked subunits called nucleotides.
  • Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA) , both of which may be single-stranded or double- stranded.
  • DNA includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA. The sequence of nucleotides that encodes a protein is called the sense sequence.
  • an “antisense nucleic acid” is a sequence of nucleotides that is complementary to the sense sequence. Antisense nucleic acids can be used to down regulate or block the expression of the polypeptide encoded by the sense strand.
  • isolated nucleic acid means a nucleic acid which is substantially free of those compounds that are normally associated therewith in its natural state. "Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with biological activity, and which may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into a pharmaceutically acceptable preparation.
  • a nucleic acid which hybridizes at high stringency means that the hybridized nucleic acids are able to withstand a washing under high stringency conditions.
  • An example of high stringency washing conditions for DNA-DNA hybrids is 0.1X SSC, 0.5% SDS at 68°C. Other conditions of high stringency washing are known to persons having ordinary skill in the art.
  • regulatory region means a nucleic acid sequence which regulates the expression of a nucleic acid.
  • a regulatory region may include sequences which are naturally responsible for expressing a particular nucleic acid (a homologous region) or may include sequences of a different origin (responsible for expressing different proteins or even synthetic proteins) .
  • the sequences can be sequences of eukaryotic or viral genes or derived sequences which stimulate or repress transcription of a gene in a specific or non-specific manner and in an inducible or non- inducible manner.
  • Regulatory regions include origins of replication, RNA splice sites, enhancers, transcriptional termination sequences, signal sequences which direct the polypeptide into the secretory pathways of the target cell, and promoters.
  • a regulatory region from a "heterologous source” is a regulatory region which is not naturally associated with the expressed nucleic acid. Included among the heterologous regulatory regions are regulatory regions from a different species, regulatory regions from a different gene, hybrid regulatory sequences, and regulatory sequences which do not occur in nature, but which are designed by one having ordinary skill in the art.
  • a “vector” is any means for the transfer of a nucleic acid according to the invention into a host cell.
  • the term “vector” includes both viral and nonviral means for introducing the nucleic acid into a prokaryotic or eukaryotic cell in vitro, ex vivo or in vivo .
  • Non-viral vectors include plasmids, liposomes, electrically charged lipids
  • Viral vectors include retrovirus, adeno-associated virus, pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr and adenovirus vectors.
  • a vector may also contain one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc. ) .
  • a “recombinant cell” is a cell which contains a nucleic acid which is not naturally present in the cell.
  • Recombinant cell includes higher eukaryotic cells such as mammalian cells, lower eukaryotic cells such as yeast cells, prokaryotic cells, and archaebacterial cells.
  • “Pharmaceutically acceptable carrier” includes diluents and fillers which are pharmaceutically acceptable for methods of administration, are sterile, and may be aqueous or oleaginous suspensions formulated using suitable dispersing or wetting agents and suspending agents.
  • the particular pharmaceutically acceptable carrier and the ratio of active compound to carrier are determined by the solubility and chemical properties of the composition, the particular mode of administration, and standard pharmaceutical practice.
  • a “lipase” is a protein which can enzymatically cleave a lipid substrate.
  • a "phospholipase” is a protein which can enzymatically cleave a phospholipid substrate.
  • triacylglycerol lipase is a protein which can enzymatically cleave a triacylglyceride substrate.
  • Phosphatidylcholine is a glycerol phospholipid. Phosphatidylcholine is also known as lecithin.
  • Lipid profile means the set of concentrations of cholesterol, triglyceride, lipoprotein cholesterol and other lipids in the body of a human or other animal.
  • An "undesirable lipid profile” is the condition in which the concentrations of cholesterol, triglyceride, or lipoprotein cholesterol are outside of the age- and gender- adjusted reference ranges.
  • a concentration of total cholesterol > 200 mg/dl, of plasma triglycerides > 200 mg/dl, of LDL cholesterol > 130 mg/dl, of HDL cholesterol ⁇ 39 mg/dl, or a ratio of total cholesterol to HDL cholesterol > 4.0 is considered to be an undesirable lipid profile.
  • An undesirable lipid profile is associated with a variety of pathological conditions, including hyperlipidaemias, diabetes hypercholesterolaemia, atherosclerosis, and other forms of coronary artery disease.
  • a "ribozyme” is an RNA molecule which can function as an enzyme.
  • a “neutralizing antibody” is an antibody which can bind to an LIPG polypeptide and lower or eliminate the enzymatic activity of the LIPG polypeptide. These antibodies may be monoclonal antibodies or polyclonal antibodies.
  • the present invention includes chimeric, single chain, and humanized antibodies, as well as Fab fragments and the products of an Fab expression library, and Fv fragments and the products of an Fv expression library.
  • an “inhibitory molecule” or “inhibitor” is a molecule which lowers or eliminates the expression of the LIPG polypeptide or which lowers or eliminates the enzymatic activity of the LIPG polypeptide.
  • an “enhancer molecule” or “enhancer” is a molecule which increases the expression of the LIPG polypeptide or which increases the enzymatic activity of the LIPG polypeptide.
  • a “liposome” is is an artificial or naturally-occurring phospholipid vesicle.
  • a “cationic liposome” is a liposome having a net positive electrical charge.
  • the present invention utilizes polypeptides encoded by LIPG which are members of the triacylglycerol lipase family, and which comprise a 39 kD catalytic domain of the triacylglycerol lipase family, e.g., having the sequence SEQ ID NO: 10.
  • an isolated LIPG polypeptide comprising the sequence SEQ ID NO: 6 and having an apparent molecular weight of about 40 kD on a 10% SDS-PAGE gel is utilized.
  • an isolated LIPG polypeptide comprising the sequence SEQ ID NO: 8 and having an apparent molecular weight of about 55 kD or 68 kD on a 10% SDS-PAGE gel is utilized.
  • the polypeptides utilized in the present invention are subfrag ents of these polypeptides.
  • the polypeptides used in the present invention are antibodies capable of binding to an LIPG polypeptide.
  • polypeptides and proteins utilized in the present invention may be recombinant polypeptides, natural polypeptides, or synthetic polypeptides, and may be of human, rabbit, or other animal origin.
  • the polypeptides are characterized by a reproducible single molecular weight and/or multiple set of molecular weights, chromatographic response and elution profiles, amino acid composition and sequence, and biological activity.
  • polypeptides utilized in the present invention may be isolated from natural sources, such as placental extracts, human. plasma, or conditioned media from cultured cells such as macrophages or endothelial cells, by using the purification procedures known to one of skill in the art.
  • the polypeptides utilized in the present invention may be prepared utilizing recombinant DNA technology, which comprises combining a nucleic acid encoding the polypeptide thereof in a suitable vector, inserting the resulting vector into a suitable host cell, recovering the polypeptide produced by the resulting host cell, and purifying the polypeptide recovered.
  • the present invention utilizes isolated nucleic acids which encode LIPG polypeptides.
  • the present invention also utilizes antisense nucleic acids which can be used to down regulate or block the expression of LIPG polypeptides in vitro , ex vivo or in vivo .
  • nucleic acids of the present invention may be linked to one or more regulatory regions. Selection of the appropriate regulatory region or regions is a routine matter, within the level of ordinary skill in the art. Regulatory regions include promoters, and may include enhancers, suppressors, etc..
  • Promoters that may be used in the present invention include both constituitive promoters and regulated (inducible) promoters.
  • the promoters may be prokaryotic or eukaryotic depending on the host.
  • prokaryotic (including bacteriophage) promoters useful for practice of this invention are lacl, lacZ, T3, T7, lambda P r , P x , and trp promoters.
  • eukaryotic (including viral) promoters useful for practice of this invention are ubiquitous promoters (e.g. HPRT, vimentin, actin, tubulin) , intermediate filament promoters (e.g.
  • tissue-specific promoters e.g. actin promoter in smooth muscle cells, or Fit and Flk promoters active in endothelial cells
  • animal transcriptional control regions which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant . Biol .
  • mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel . 1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol . Cell . Biol . , 5:1639-1648; Hammer et al., 1987, Science 235:53-58), alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al., 1987, Genes and Devel .
  • beta-globin gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94), myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712), myosin light chain- 2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).
  • promoters which may be used in the practice of the invention include promoters which are preferentially activated in dividing cells, promoters which respond to a stimulus (e.g. steroid hormone receptor, retinoic acid receptor) , tetracycline-regulated transcriptional modulators, cytomegalovirus immediate-early, retroviral LTR, metallothionein, SV-40, Ela, and MLP promoters.
  • a stimulus e.g. steroid hormone receptor, retinoic acid receptor
  • tetracycline-regulated transcriptional modulators cytomegalovirus immediate-early, retroviral LTR, metallothionein, SV-40, Ela
  • Tetracycline-regulated transcriptional modulators and CMV promoters are described in WO 96/01313, US 5,168,062 and 5,385,839, the contents of which are incorporated herein by reference.
  • the viral vectors used in the gene therapy methods of the present invention are replication defective, that is, they are unable to replicate autonomously in the target cell.
  • the genome of the replication defective viral vectors which are used within the scope of the present invention lack at. least one region which is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part) , or be rendered non-functional by any technique known to a person skilled in the art. These techniques include the total removal, substitution (by other sequences, in particular by the inserted nucleic acid) , partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed in vitro (on the isolated DNA) or in situ , using the techniques of genetic manipulation or by treatment with mutagenic agents.
  • the replication defective virus retains the sequences of its genome which are necessary for encapsidating the viral particles.
  • the retroviruses are integrating viruses which infect dividing cells.
  • the retrovirus genome includes two LTRs, an encapsidation sequence and three coding regions (gag, pol and env) .
  • the construction of recombinant retroviral vectors has been described: see, in particular, EP 453242, EP178220, Bernstein et al. Genet. Eng. 7 (1985) 235; McCormick,
  • retroviral vectors the gag, pol and env genes are generally deleted, in whole or in part, and replaced with a heterologous nucleic acid sequence of interest.
  • retrovirus such as, MoMuLV ("murine Moloney leukaemia virus” MSV ("murine Moloney sarcoma virus") , HaSV ("Harvey sarcoma virus”) ; SNV (“spleen necrosis virus”) ; RSV ("Rous sarcoma virus”) and Friend virus.
  • Lentivirus vector systems may also be used in the practice of the present invention.
  • the lentiviral genome is a positive- strand polyadenylated RNA of 9,000 to 10,000 base pairs containing three structural genes organized 5' to 3' (gag, pol, env) , typical of all retroviruses.
  • lentiviral systems see Fields Virology, Second Edition, Volume 2, Chapter 55, "Lentiviruses, " pp. 1571-1589, Raven Press, New York, 1990.
  • a plasmid which contains the LTRs, the encapsidation sequence and the coding sequence.
  • This construct is used to transfect a packaging cell line, which cell line is able to supply in trans the retroviral functions which are deficient in the plasmid.
  • the packaging cell lines are thus able to express the gag, pol and env genes.
  • Such packaging cell lines have been .described in the prior art, in particular the cell line PA317 (US4,861, 719) ; the PsiCRIP cell line (WO90/02806) and the GP+envAm-12 cell line (WO89/07150) .
  • the recombinant retroviral vectors can contain modifications within the LTRs for suppressing transcriptional activity as well as extensive encapsidation sequences which may include a part of the gag gene (Bender et al., J. Virol. 61 (1987) 1639). Recombinant retroviral vectors are purified by standard techniques known to those having ordinary skill in the art.
  • the adeno-associated viruses are DNA viruses of relatively small size which can integrate, in a stable and site-specific manner, into the genome of the cells which they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies.
  • the AAV genome has been cloned, sequenced and characterized. It encompasses approximately 4700 bases and contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as an origin of replication for the virus.
  • ITR inverted terminal repeat
  • the remainder of the genome is divided into two essential regions which carry the encapsidation functions: the left-hand part of the genome, which contains the rep gene involved in viral replication and expression of the viral genes; and the right-hand part of the genome, which contains the cap gene encoding the capsid proteins of the virus.
  • the replication defective recombinant AAVs utilized in the present invention can be prepared by cotransfecting a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes (rep and cap genes) , into a cell line which is infected with a human helper virus (for example an adenovirus) .
  • a human helper virus for example an adenovirus
  • the AAV recombinants which are produced are then purified by standard techniques.
  • the invention also relates, therefore, to an AAV- derived recombinant virus whose genome encompasses a sequence encoding an LIPG polypeptide flanked by the AAV ITRs.
  • the invention also relates to a plasmid encompassing a sequence encoding an LIPG polypeptide flanked by two ITRs from an AAV.
  • a plasmid can be used as it is for transferring the LIPG sequence, with the plasmid, where appropriate, being incorporated into a liposomal vector (pseudo-virus) .
  • the vector utilized in the present invention is an adenovirus vector.
  • Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid to a variety of cell types.
  • adenoviruses of animal origin preference is given, within the scope of the present invention, to using type 2 or type 5 human adenoviruses (Ad 2 or Ad 5) or adenoviruses of animal origin (see W094/26914) .
  • Those adenoviruses of animal origin which can be used within the scope of the present invention include adenoviruses of canine, bovine, murine (example: Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian, and simian (example: SAV) origin.
  • the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus (e.g. Manhattan or A26/61 strain (ATCC VR-800) , for example) .
  • the replication defective adenoviral vectors comprise the ITRs, an encapsidation sequence and the nucleic acid of interest.
  • at least the El region of the adenoviral vector is non-functional.
  • the deletion in the El region preferably extends from nucleotides 455 to 3329 in the sequence of the Ad5 adenovirus.
  • Other regions may also be modified, in particular the E3 region (WO95/02697) , the E2 region (W094/28938) , the E4 region
  • the adenoviral vector has a deletion in the El and E4 regions. In another preferred embodiment, the adenoviral vector has a deletion in the El region into which the E4 region and the sequence encoding LLG are inserted (see FR94 13355) .
  • the replication defective recombinant adenoviruses can be prepared by any technique known to the person skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3 (1984) 2917) . In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid which carries, inter alia, the DNA sequence of interest.
  • the homologous recombination is effected following cotransfection of the said adenovirus and plasmid into an appropriate cell line.
  • the cell line which is employed should preferably (i) be transformable by the said elements, and (ii) contain the sequences which are able to complement the part of the genome of the replication defective adenovirus, preferably in integrated form in order to avoid the risks of recombination.
  • Examples of cell lines which may be used are the human embryonic kidney cell line 293 (Graham et al., J. Gen. Virol.
  • Antisense nucleic acids of the invention are preferably nucleic acid fragments capable of specifically hybridizing with all or part of a nucleic acid encoding LIPG or the corresponding messenger RNA.
  • antisense nucleic acids may be designed or identified which decrease expression of the LIPG gene by inhibiting splicing of its primary transcript. With knowledge of the structure and partial sequence of the LIPG gene, such antisense nucleic acids can be designed and tested for efficacy.
  • the antisense nucleic acids are preferably oligonucleotides and may consist entirely of deoxyribo- nucleotides, modified deoxyribonucleotides, or some combination of both.
  • the antisense nucleic acids can be synthetic oligonucleotides.
  • the oligonucleotides may be chemically modified, if desired, to improve stability and/or selectivity. Since oligonucleotides are susceptible to degradation by intracellular nucleases, the modifications can include, for example, the use of a sulfur group to replace the free oxygen of the phosphodiester bond. This modification is called a phosphorothioate linkage.
  • Phosphorothioate antisense oligonucleotides are water soluble, polyanionic, and resistant to endogenous nucleases.
  • the RNA-DNA duplex activates the endogenous enzyme ribonuclease (Rnase) H, which cleaves the mRNA component of the hybrid molecule.
  • Rnase ribonuclease
  • antisense oligonucleotides with phosphoramidite and polyamide (peptide) linkages can be synthesized. These molecules should be very resistant to nuclease degradation.
  • chemical groups can be added to the 2 • carbon of the sugar moiety and the 5 carbon (C-5) of pyrimidines to enhance stability and facilitate the binding of the antisense oligonucleotide to its target site. Modifications may include 2' deoxy, O-pentoxy, O-propoxy, O- methoxy, fluoro, methoxyethoxy phosphoro-thioates, modified bases, as well as other modifications known to those of skill in the art.
  • the antisense nucleic acids can also be DNA sequences whose expression in the cell produces RNA complementary to all or part of the LIPG mRNA.
  • Antisense nucleic acids can be prepared by expression of all or part of a sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 7, or SEQ ID No. 11, in the opposite orientation, as described in EP 140308. Any length of antisense sequence is suitable for practice of the invention so long as it is capable of down-regulating or blocking expression of LIPG. Preferably, the antisense sequence is at least 20 nucleotides in length.
  • the preparation and use of antisense nucleic acids, DNA encoding antisense RNAs and the use of oligo and genetic antisense is disclosed in W092/15680, the contents of which are incorporated herein by reference.
  • One approach to determining the optimum fragment of LIPG to use in an antisense nucleic acid treatment method involves preparing random fragments of LIPG cDNA by mechanical shearing, enzymatic treatment, and cloning the fragment into any of the vector systems described herein. Individual clones or pools of clones are used to infect LIPG-expressing cells, and effective antisense LIPG cDNA fragments are identified by monitoring LIPG expression at the RNA or protein level.
  • retroviral, adeno-associated viral, and adenoviral vector systems discussed hereinabove may all be used to introduce and express antisense nucleic acids in cells.
  • Antisense synthetic oligonucleotides may be introduced in a variety of ways, including the methods discussed hereinbelow.
  • Ribozymes are catalytic RNA molecules (RNA enzymes) that have separate catalytic and substrate binding domains.
  • the substrate binding sequence combines by nucleotide complementarity and, possibly, nonhydrogen bond interactions with its target sequence.
  • the catalytic portion cleaves the target RNA at a specific site.
  • the substrate domain of a ribozyme can be engineered to direct it to a specified mRNA sequence.
  • the ribozyme recognizes and then binds a target mRNA through complementary base-pairing. Once it is bound to the correct target site, the ribozyme acts enzymatically to cut the target mRNA.
  • Cleavage of the LIPG mRNA by a ribozyme destroys its ability to direct synthesis of LIPG polypeptide. Once the ribozyme has cleaved its target sequence, it is released and can repeatedly bind and cleave at other LIPG mRNAs.
  • the ribozyme is formed in a hammerhead motif.
  • Other forms include a hairpin motif, a hepatitis delta virus, group I intron or RnaseP RNA (in association with an RNA guide sequence) motif or Neurospora VS RNA motif. Hammerhead motifs are described by Rossi et al., 1992, Aids Research and Human Retroviruses , 8, 183.
  • Hairpin motifs are described in Hampel and Tritz, 1989, Biochemistry, 28, 4929, and Hampel et al., 1990, Nucleic Acids Res . , 18, 299.
  • the hepatitis delta virus motif is described by Perrotta and Been, 1992, Biochemistry, 31,
  • the RnaseP motif is described by Guerrier-Takada et al., 1983, Cell , 35, 849
  • the Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990, Cell , 61, 685-696; Saville and Collins, 1991, Proc . Natl . Acad . Sci . USA, 88, 8826-8830; Collins and Olive, 1993, Biochemistry,
  • One approach in preparing a ribozyme is to chemically synthesize an oligodeoxyribonucleotide with a ribozyme catalytic domain (-20 nucleotides) flanked by sequences that hybridize to the target LIPG mRNA after transcription.
  • the oligodeoxyribonucleotide is amplified by using the substrate binding sequences as primers.
  • the amplification product is cloned into a eukaryotic expression vector.
  • Ribozymes possessing a hammerhead or hairpin structure are readily prepared since these catalytic RNA molecules can be expressed within cells from eukaryotic promoters (e.g., Scanlon et al., 1991, Proc . Natl . Acad . Sci . USA, 88, 10591- 5; Kashani-Sabet et al., 1992, Antisense Res . Dev. , 2, 3-15; Dropulic et al., 1992, J. Virol . , 66, 1432-41; Weerasinghe et al., 1991, J. Virol . , 65, 5531-4; Ojwang et al., 1992, Proc . Natl .
  • eukaryotic promoters e.g., Scanlon et al., 1991, Proc . Natl . Acad . Sci . USA, 88, 10591- 5; Kashani-Sabet
  • a ribozyme of the present invention can be expressed in eukaryotic cells from the appropriate DNA vector. If desired, the activity of the ribozyme may be augmented by its release from the primary transcript by a second ribozyme (Ohkawa et al., 1992, Nucleic Acids Symp. Ser . , 27, 15-6; Taira et al., 1991, Nucleic Acids Res . , 19, 5125-30; Ventura et al. , 1993, Nucleic Acids Res . , 21, 3249- 55) .
  • ribozymes are expressed from transcription units inserted into DNA, RNA, or viral vectors. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol (I) , RNA polymerase II (pol II) , or RNA polymerase III (pol III) . Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on nearby gene regulatory sequences. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells
  • a transcription unit expressing a ribozyme that cleaves LIPG RNA is inserted into a plasmid DNA vector, a retrovirus vector, an adenovirus DNA viral vector or an adeno-associated virus vector.
  • the recombinant vectors are preferably DNA plasmids or adenovirus vectors. However, other mammalian cell vectors that direct the expression of RNA may be used for this purpose.
  • the vectors are delivered as recombinant viral particles. DNA may be delivered alone or complexed with various vehicles.
  • the DNA, DNA/vehicle complexes, or the recombinant virus particles are locally administered to the site of treatment, as discussed below.
  • recombinant vectors capable of expressing the ribozymes are locally delivered as described below, and persist in target cells. Once expressed, the ribozymes cleave the target LIPG mRNA.
  • Ribozymes may be administered to a patient by a variety of methods. They may be added directly to target tissues, complexed with cationic lipids, packaged within liposomes, or delivered to target cells by other methods known in the art. Localized administration to the desired tissues may be done by catheter, infusion pump or stent, with or without incorporation of the ribozyme in biopolymers as discussed hereinbelow. Alternative routes of delivery include, but are not limited to, intravenous injection, intramuscular injection, subcutaneous injection, aerosol inhalation, oral (tablet or pill form) , topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Sullivan et al., PCT WO94/02595 and Draper et al., PCT W093/23569, which are incorporated by reference herein.
  • Certain non-viral systems have been used in the art and can facilitate introduction of DNA encoding the LIPG polypeptides or antisense nucleic acids into a patient.
  • a DNA vector encoding a desired LIPG polypeptide or antisense sequence can be introduced in vivo by lipofection.
  • liposomes for encapsulation and transfection of nucleic acids in vitro .
  • Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker [Feigner, et. al., Proc . Natl . Acad . Sci . U. S .A . 84:7413-7417 (1987); see
  • cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes [Feigner and Ringold, Nature 337:387-388 (1989)].
  • Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications W095/18863 and W096/17823, and in U.S. Patent No. 5,459,127.
  • lipofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages.
  • Molecular targeting of liposomes to specific cells represents one area of benefit. It is clear that directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, for example, pancreas, liver, kidney, and the brain.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting [see Mackey, et. al., supra ] .
  • Targeted peptides, e . g . hormones or neurotransraitters, and proteins for example, antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • a nucleic acid in vivo for example, a cationic oligopeptide (e.g., International Patent Publication W095/21931) , peptides derived from DNA binding proteins ⁇ e . g. , International Patent Publication WO96/25508) , or a cationic polymer (e . g. , International Patent Publication W095/21931) .
  • a cationic oligopeptide e.g., International Patent Publication W095/21931
  • peptides derived from DNA binding proteins ⁇ e . g. , International Patent Publication WO96/25508 e.g., International Patent Publication W095/21931
  • a cationic polymer e.g., International Patent Publication W095/21931
  • DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e . g . , transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter [see, e . g . , Wilson et al., J " . Biol . Chem . 267:963-967 (1992); Wu and Wu, J. Biol . Chem . 263:14621-14624 (1988); Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15, 1990; Williams et al., Proc .
  • the present invention provides antibodies against the LIPG polypeptide. These antibodies may be monoclonal antibodies or polyclonal antibodies.
  • the present invention includes chimeric, single chain, and humanized antibodies, as well as Fab fragments and the products of an Fab expression library, and Fv fragments and the products of an Fv expression library.
  • Polyclonal antibodies may be prepared against an antigenic fragment of an LIPG polypeptide, as described in the Examples section hereinbelow. Antibodies may also be generated against the intact LIPG protein or polypeptide, or against a fragment, derivative, or epitope of the protein or polypeptide. Antibodies may be obtained following the administration of the protein, polypeptide, fragment, derivative, or epitope to an animal, using the techniques and procedures known in the art.
  • Monoclonal antibodies may be prepared using the method of Mishell, B. B. , et al. , Selected Methods In Cellular Immunology, (W.H. Freeman, ed.) San Francisco (1980). Briefly, a polypeptide of the present invention is used to immunize spleen cells of Balb/C mice. The immunized spleen cells are fused with myeloma cells. Fused cells containing spleen and myeloma cell characteristics are isolated by growth in HAT medium, a medium which kills both parental cells, but allows the fused products to survive and grow.
  • the monoclonal antibodies of the present invention may be "humanized” to prevent the host from mounting an immune response to the antibodies.
  • a “humanized antibody” is one in which the complementarity determining regions (CDRs) and/or other portions of the light and/or heavy variable domain framework are derived from a non-human immunoglobulin, but the remaining portions of the molecule are derived from one or more human immunoglobulins.
  • Humanized antibodies also include antibodies characterized by a humanized heavy chain associated with a donor or acceptor unmodified light chain or a chimeric light chain, or vice versa. The humanization of antibodies may be accomplished by methods known in the art (see, e.g. G.E. Mark and E.A. Padlan, "Chapter 4.
  • Transgenic animals may be used to express humanized antibodies.
  • an anti-LIPG antibody is used to bind to and inhibit the enzymatic activity of LIPG in a patient.
  • the anti-LIPG antibodies are also useful in assays for detecting or quantitating levels of LIPG.
  • these assays provide a clinical diagnosis and assessment of LIPG in various disease states and a method for monitoring treatment efficacy.
  • These anti-LIPG antibodies may additionally be used to quantitate LIPG in a tissue sample in order to predict further susceptibility to lowered levels of HDL cholesterol and apolipoprotein Al.
  • the present invention provides methods of screening small molecule libraries or natural product sources for enhancers (agonists) or co-activators including proteinaceous co-activators or inhibitors (antagonists) of LIPG activity.
  • a potential enhancer or inhibitor is contacted with LIPG protein and a substrate of LIPG, and the ability of the potential enhancer or inhibitor to enhance or inhibit LIPG activity is measured.
  • these screening methods may also be used to determine if a compound can function as a substrate specific enhancer or inhibitor, that is, whether a compound can enhance the enzymatic activity of LIPG toward one substrate while lowering or maintaining a given level of enzymatic activity for a different substrate
  • the LIPG polypeptide of the present invention utilizes HDL cholesterol as a substrate and also utilizes LDL cholesterol and VLDL cholesterol as substrates.
  • the LIPG protein used in these methods can be produced recombinantly in a variety of host cells, including mammalian cells, baculovirus-infected insect cells, yeast, and bacteria. LIPG expression in stably transfected CHO cells can be optimized by methotrexate amplification of the cells. LIPG protein can also be purified from natural sources such as human plasma, placenta1 extracts, or conditioned media from cultured endothelial cells, THP-1 cells, or macrophages.
  • assay parameters including pH, ion concentrations, temperature, concentration of substrate, and emulsification conditions are determined empirically by one having ordinary skill in the art.
  • the fatty acid substituents of the substrates may vary in chain length as well as in degree and position of unsaturation.
  • the substrates may be radiolabelled in any of several positions.
  • Phospholipid substrates such as phosphatidylcholine can be radiolabelled, for example, in the Sn-1 or Sn-2 fatty acid position, or in the glycerol, phosphate, or polar head group (choline in the case of phosphatidylcholine) .
  • radiolabeled substrates As an alternative to radiolabeled substrates, other classes of labeled substrates, such as fluorescent substrates or thio-containing substrates, can also be used in the screening methods.
  • fluorescent substrates As an alternative to radiolabeled substrates, other classes of labeled substrates, such as fluorescent substrates or thio-containing substrates, can also be used in the screening methods.
  • Fluorescent substrates are particularly useful in screening assays because enzymatic catalysis can be measured continuously by measuring fluorescence intensity, without the physical separation (extraction) of the products from the substrates.
  • An example of a fluorescent phosphatidylcholine substrate is C 6 NBD-PC ⁇ l-acyl-2-[6- (nitro-2, 1, 3-benzoxadiazol- 4-yl) amino] caproylphosphatidylcholine.
  • the thio-containing substrates include 1,2- bis (hexanoylthio) -1, 2-dideoxy-sn-glycero-3-phosphorylcholine (L.J. Reynolds, W.N. Washburn, R.A. Deems, and E.A. Dennis, 1991. Methods in Enzymology 197: 3-23; L. Yu and E.A. Dennis, 1991. Methods in Enzymology 197: 65-75; L.A. Wittenauer, K. Shirai, R.L. Jackson, and J.D. Johnson, 1984. Biochem. Biophys. Res. Commun. 118: 894-901).
  • inhibitory and enhancer molecules which operate at the level of enzymatic activity
  • inhibitory and enhancer molecules which operate at the level of expression of the LIPG gene.
  • One method for identifying compounds which are able to enhance or inhibit the expression of LIPG is to use a reporter gene system. These systems utilize reporter gene expression vectors which include a cloning site into which a given promoter may be cloned upstream of a "reporter gene" which can be easily detected and quantified.
  • reporter gene expression vectors which include a cloning site into which a given promoter may be cloned upstream of a "reporter gene" which can be easily detected and quantified.
  • reporter gene expression vectors which include a cloning site into which a given promoter may be cloned upstream of a "reporter gene" which can be easily detected and quantified.
  • reporter gene expression vectors which include a cloning site into which a given promoter may be cloned upstream of a
  • the expression vector is transferred into host cells and the cells are exposed to a test compound (a putative inhibitor or enhancer molecule) to determine the effect of the test compound on expression of the reporter gene product.
  • a test compound a putative inhibitor or enhancer molecule
  • the cells are assayed for the presence of the reporter gene product by directly measuring the amount of reporter mRNA, the reporter protein itself or the enzymatic activity of the reporter protein.
  • the reporter gene is not endogenously expressed in the cell type of interest and lends itself to sensitive, quantitative and rapid assays.
  • a variety of reporter assay constructs are commercially available and several reporter genes and assays have been developed and can be readily prepared by those of skill in the art.
  • the most popular systems for monitoring genetic activity in eukaryotic cells include the chloramphenicol acetyltransferase (CAT) , ⁇ - galactosidase, firefly luciferase, growth hormone (GH) , ⁇ - glucurorudase (GUS) , alkaline phosphatase (AP) , green fluorescent protein (GFP) and Renilla luciferase.
  • Reporter assay constructs can be purchased from a variety of sources including Promega and Invitrogen.
  • reporter gene activity can be detected by assaying for the reporter mRNA or the reporter protein.
  • the reporter mRNA can be detected by northern blot analysis, ribonuclease protection assays or RT-PCR. While these assays are more direct than measuring protein expression, many assays have been developed to measure the presence of the reporter protein rather than the mRNA present in a cell. Reporter proteins can be assayed by spectrophotometry or by detecting enzymatic activity. Reporter protein levels may also be measured with antibody- based assays. In general, the enzymatic assays are very sensitive and are a preferred method of monitoring reporter gene expression.
  • the present invention provides compositions in a biologically compatible (biocompatible) solution, comprising the polypeptides, nucleic acids, vectors, and antibodies of the invention.
  • a biologically compatible solution is a solution in which the polypeptide, nucleic acid, vector, or antibody of the invention is maintained in an active form, e.g., in a form able to effect a biological activity.
  • a polypeptide of the invention would have phospholipase activity; a nucleic acid would be able to replicate, translate a message, or hybridize to a complementary nucleic acid; a vector would be able to transfect a target cell; an antibody would bind a polypeptide of the invention.
  • such a biologically compatible solution will be an aqueous buffer, e.g., Tris, phosphate, or HEPES buffer, containing salt ions. Usually the concentration of salt ions will be similar to physiological levels.
  • the biocompatible solution is a pharmaceutically acceptable composition.
  • Biologically compatible solutions may include stabilizing agents and preservatives.
  • compositions can be formulated for administration by topical, oral, parenteral, intranasal, subcutaneous, and intraocular, routes.
  • Parenteral administration is meant to include intravenous injection, intramuscular injection, intraarterial injection or infusion techniques.
  • the composition may be administered parenterally in dosage unit formulations containing standard, well known nontoxic physiologically acceptable carriers, adjuvants and vehicles as desired.
  • the preferred sterile in ectable preparations can be a solution or suspension in a nontoxic parenterally acceptable solvent or diluent.
  • pharmaceutically acceptable carriers are saline, buffered saline, isotonic saline (e.g. monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, or mixtures of such salts), Ringer's solution, dextrose, water, sterile water, glycerol, ethanol, and combinations thereof.
  • 1, 3-butanediol and sterile fixed oils are conveniently employed as solvents or suspending media. Any bland fixed oil can be employed including synthetic mono- or di-glycerides.
  • the composition medium can also be a hydrogel which is prepared from any biocompatible or non-cytotoxic (homo or hetero) polymer, such as a hydrophilic polyacrylic acid polymer that can act as a drug absorbing sponge.
  • a hydrogel can be deposited directly onto the surface of the tissue to be treated, for example during surgical intervention.
  • Another preferred embodiment of the present invention relates to a pharmaceutical composition comprising a replication defective recombinant virus and poloxamer. More specifically, the invention relates to a composition comprising a replication defective recombinant virus comprising a nucleic acid encoding an LIPG polypeptide and poloxamer.
  • a preferred poloxamer is Poloxamer 407, which is commercially available (BASF, Parsippany, NJ) and is a non- toxic, biocompatible polyol, and is most preferred.
  • a poloxamer impregnated with recombinant viruses may be deposited directly on the surface of the tissue to be treated, for example during a surgical intervention. Poloxamer possesses essentially the same advantages as hydrogel while having a lower viscosity.
  • the present invention provides methods of treatment which comprise the administration to a human or other animal of an effective amount of a composition of the invention.
  • Effective amounts may vary, depending on the age, type and severity of the condition to be treated, body weight, desired duration of treatment, method of administration, and other parameters. Effective amounts are determined by a physician or other qualified medical professional. In most cases, the dosage levels may be adjusted so that the desired levels of HDL cholesterol and apolipoprotein Al are achieved and maintained. Similarly, the dosage levels may be adjusted to lower the VLDL cholesterol and LDL cholesterol levels to acceptable levels and bring the ratio HDL cholesterol to LDL cholesterol and VLDL cholesterol to within desirable levels.
  • Polypeptides according to the invention are generally administered in doses of about 0.01 mg/kg to about 100 mg/kg, preferably about 0.1 mg/kg to about 50 mg/kg, and most preferably about 1 mg/kg to about 10 mg/kg of body weight per day.
  • Neutralizing antibodies according to the invention may be delivered as a bolus only, infused over time or both administered as a bolus and infused over time. Although the dosage amount will vary based on the parameters above, and on the binding ability of the antibody, a dose 0.2 to 0.6 mg/kg may be given as a bolus followed by a 2 to 12 hour infusion period. Alternatively, multiple bolus injections are administered every other day or every third or fourth day as needed. Dosage levels may be adjusted as determined by HDL cholesterol levels and/or VLDL and LDL cholesterol levels.
  • recombinant viruses may be used to introduce both DNA encoding LIPG and subfragments of LIPG as well as antisense nucleic acids.
  • Recombinant viruses according to the invention are generally formulated and administered in the form of doses of between about 10 4 and about 10 14 pfu. In the case of AAVs and adenoviruses, doses of from about 10° to about 10 11 pfu are preferably used.
  • pfu (“plaque-forming unit”) corresponds to the infective power of a suspension of virions and is determined by infecting an appropriate cell culture and measuring the number of plaques formed. The techniques for determining the pfu titre of a viral solution are well documented in the prior art.
  • Ribozymes according to the present invention may be administered in amounts ranging from about 5 to about 50 mg/kg/day in a pharmaceutically acceptable carrier. Dosage levels may be adjusted based on the measured therapeutic efficacy.
  • inhibitor or enhancer molecules may be determined by qualified medical personnel using the parameters discussed above.
  • the present invention provides compositions and methods for increasing the level of HDL cholesterol and apolipoprotein Al and lowering the levels of VLDL and LDL cholesterol in a patient.
  • the present invention further provides methods of treating a human or other animal having an undesirable lipid profile, wherein said undesirable lipid profile is the result of abnormally high expression of LIPG polypeptide activity.
  • the methods for decreasing the expression of LIPG polypeptide in order to increase the levels of HDL cholesterol and apolipoprotein Al and correct those conditions in which LIPG polypeptide activity contributes to a disease or disorder associated with an undesirable lipid profile include but are not limited to administration of a composition comprising an antisense nucleic acid, administration of a composition comprising an intracellular binding protein such as an antibody, administration of an inhibitory molecule which inhibits the enzymatic activity of LIPG, for example, a composition comprising an expression vector encoding a subfragment of LIPG, for example, LLGN polypeptide or a small molecular weight molecule, including administration of a small molecular weight compound which down regulates LIPG expression at the level of transcription, translation or post-translation, and administration of a ribozyme which cleaves mRNA encoding LIPG.
  • a composition comprising an antisense nucleic acid is used to down-regulate or block the expression of LIPG.
  • the nucleic acid encodes antisense RNA molecules.
  • the nucleic acid is operably linked to signals enabling expression of the nucleic acid sequence and is introduced into a cell utilizing, preferably, recombinant vector constructs, which will express the antisense nucleic acid once the vector is introduced into the cell.
  • suitable vectors includes plasmids, adenoviruses, adeno- associated viruses, retroviruses, and herpes viruses.
  • the vector is an adenovirus.
  • the vector is a replication defective adenovirus comprising a deletion in the El and/or E3 regions of the virus.
  • the antisense nucleic acid is synthesized and may be chemically modified to resist degradation by intracellular nucleases, as discussed above.
  • Synthetic antisense oligonucleotides can be introduced to a cell using liposomes. Cellular uptake occurs when an antisense oligonucleotide is encapsulated within a liposome. With an effective delivery system, low, non-toxic concentrations of the antisense molecule can be used to inhibit translation of the target mRNA.
  • liposomes that are conjugated with cell-specific binding sites direct an antisense oligonucleotide to a particular tissue.
  • the expression of LIPG is down- regulated or blocked by the expression of a nucleic acid sequence encoding an intracellular binding protein which is capable of selectively interacting with LIPG.
  • WO 94/29446 and WO 94/02610 disclose cellular transfection with genes encoding an intracellular binding protein.
  • An intracellular binding protein includes any protein capable of selectively interacting, or binding, with LIPG in the cell in which it is expressed and of neutralizing the function of bound LLG.
  • the intracellular binding protein is a neutralizing antibody or a fragment of a neutralizing antibody. More preferably, the intracellular binding protein is a single chain antibody.
  • WO 94/02610 discloses preparation of antibodies and identification of the nucleic acid encoding a particular antibody. Using LIPG or a fragment thereof, a specific monoclonal antibody is prepared by techniques known to those skilled in the art. A vector comprising the nucleic acid encoding an intracellular binding protein, or a portion thereof, and capable of expression in a host cell is subsequently prepared for use in the method of this invention.
  • LIPG activity can be blocked by administration of a neutralizing antibody into the circulation.
  • a neutralizing antibody can be administered directly as a protein, or it can be expressed from a vector (with a secretory signal) .
  • LIPG activity is inhibited by the administration of a composition comprising a subfragment of LIPG polypeptide, for example, LLGN.
  • This composition may be administered in a convenient manner, such as by the oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, or intradermal routes.
  • the composition may be administered directly or it may be encapsulated (e.g. in a lipid system, in amino acid microspheres, or in globular dendrimers) .
  • the polypeptide may, in some cases, be attached to another polymer such as serum albumin or polyvinyl pyrrolidone.
  • LIPG activity is inhibited through the use of gene therapy, that is, through the administration of a composition comprising a nucleic acid which encodes and directs the expression of a subfragment of LIPG, for example, LLGN.
  • LIPG activity is inhibited through the use of inhibitory molecules. These low molecular weight compounds interfere with LIPG ' s enzymatic properties or prevent its appropriate recognition by cellular binding sites.
  • the LIPG polypeptide of the present invention also has an affinity for heparin. LIPG polypeptide binding to extracellular heparin in the lumen of blood vessels would permit LIPG to bind to and accelerate LDL uptake by acting as a bridge between LDL and the extracellular heparin. In the localized area of an atherosclerotic lesion, an increased level of lipase activity is hypothesized to accelerate the atherogenic process (Zilversmit, D.B. (1995) Clin. Chem. 41,153-158; Zambon, A., Torres, A., Bijvoet, S. , Gagne, C. , Moojani, S.
  • a high local level of lipase activity may result in cytotoxic levels of fatty acids and lysophosphatidylcholine being produced in precursors of atherosclerotic lesions.
  • This particular activity of LLG may contribute to the development or progression of atherosclerosis, particularly in the context of excessive lipid levels in a subject due to dietary or genetic factors.
  • the present invention permits inhibition of lipoprotein accumulation by inhibiting LIPG polypeptide expression or binding to lipoprotein (e.g., LDL).
  • inhibitory molecules are able to down regulate LIPG expression at the level of transcription, translation or post-translation.
  • the reporter gene systems described above may be used.
  • These inhibitory molecules may be combined with a pharmaceutically acceptable carrier and administered using conventional methods known in the art.
  • Ribozymes may be administered to cells by encapsulation in liposomes, by iontophoresis, by incorporation into hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres or by any of a variety of other methods dicussed above.
  • the ribozyme may be delivered to a target tissue by direct injection or by use of a catheter, infusion pump or stent.
  • Alternative routes of delivery include intravenous injection, intramuscular injection, subcutaneous injection, aerosol inhalation, oral (tablet or pill form) , topical, systemic, ocular, intraperitoneal and/or intrathecal delivery.
  • a ribozyme-encoding sequence is cloned into a DNA expression vector. Transcription of the ribozyme sequence is driven from an eukaryotic RNA polymerase II (pol II) , or RNA polymerase III (pol III) promoter.
  • the expression vector can be incorporated into a variety of vectors including the viral DNA vectors such as adenovirus or adeno-associated virus vectors discussed above.
  • a transcription unit expressing a ribozyme that cleaves LIPG RNA is inserted into an adenovirus DNA viral vector.
  • the vector is delivered as recombinant viral particles and is locally administered to the site of treatment, through the use of a catheter, stent or infusion pump.
  • any of the methods discussed above for lowering the levels of LIPG polypeptide activity are utilized in combination with administration of apolipoprotein Al or an expression system capable of expressing apolipoprotein Al in a patient (see, for example, U.S. Patent No. 5,866,551, which is incorporated herein by reference) .
  • the methods for increasing the expression or activity of LIPG polypeptide to lower the levels of VLDL and LDL cholesterol include, but are not limited to, administration of a composition comprising the LIPG polypeptide, administration of a composition comprising an expression vector which encodes the LIPG polypeptide, administration of a composition comprising an enhancer molecule which enhances the enzymatic activity of the LIPG polypeptide and administration of an enhancer molecule which increases expression of the LIPG gene.
  • the level of LIPG activity is increased through the administration of a composition comprising the LIPG polypeptide.
  • This composition may be administered in a convenient manner, such as by the oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, or intradermal routes.
  • the composition may be administered directly or it may be encapsulated (e.g. in a lipid system, in amino acid microspheres, or in globular dendrimers) .
  • the polypeptide may, in some cases, be attached to another polymer such as serum albumin or polyvinyl pyrrolidone.
  • the level of LIPG is increased through the use of gene therapy, that is, through the • administration of composition comprising a nucleic acid which encodes and directs the expression of the LIPG polypeptide.
  • the LIPG polypeptide is cloned into an appropriate expression vector. Possible vector systems and promoters are extensively discussed above.
  • the expression vector is transferred into the target tissue using one of the vector delivery systems discussed above. This transfer is carried out either ex vivo in a procedure in which the nucleic acid is transferred to cells in the laboratory and the modified cells are then administered to the human or other animal, or in vivo in a procedure in which the nucleic acid is transferred directly to cells within the human or other animal.
  • an adenoviral vector system is used to deliver the expression vector.
  • a tissue specific promoter is utilized in the expression vector as described above.
  • Non-viral vectors may be transferred into cells using any of the methods known in the art, including calcium phosphate coprecipitation, lipofection (synthetic anionic and cationic liposomes) , receptor-mediated gene delivery, naked DNA injection, electroporation and bioballistic or particle acceleration.
  • the activity of LIPG is enhanced by enhancer molecules that increase the enzymatic activity of LIPG or increase its appropriate recognition by cellular binding sites.
  • enhancer molecules may be introduced by the same methods discussed above for the administration of polypeptides.
  • the level of LIPG is increased through the use of small molecular weight compounds, which can upregulate LIPG expression at the level of transcription, translation, or post-translation. These compounds may be administered by the same methods discussed above for the administration of polypeptides.
  • Intrahepatic cholestasis can be characterized by increased serum cholesterol and phospholipid levels.
  • a recently described, phalloidin drug-induced intrahepatic cholestasis model in rats demonstrated significant increases in the serum levels of cholesterol and phospholipid (Ishizaki, K. , Kinbara, S., Miyazawa, N. , Takeuchi, Y., Hirabayashi, N. , Kasai, H. , and Araki, T. (1997) Toxicol. Letters 90, 29-34) .
  • the products of this invention may be used to treat intrahepatic cholestasis in patients that have increased serum cholesterol and/or phospholipid.
  • this rat model also exhibited a severe decrease in biliary cholesterol excretion rates.
  • the LIPG polypeptide and nucleic acid products of this invention may be used to treat patients with an impaired biliary excretion system.
  • Intrahepatic cholestasis is also characterized by impaired bile flow from the liver. Recently ⁇ the loci for progressive familial intrahepatic cholestasis (PFIC or Byler disease) and benign recurrent intrahepatic cholestasis (BRIC) were mapped to 18q21-q22 (Carlton, V.E.H., Knisely, A.S., and Freimer, N.B. (1995) Hum. Mol. Genet. 4, 1049-1053 and Houwen, R.H., Baharloo, S., Blankenship, K. , Raeymaekers, P., Juyn, J., Sandkuijl, L.A. , and Freimer, N.B.
  • PFIC or Byler disease progressive familial intrahepatic cholestasis
  • BRIC benign recurrent intrahepatic cholestasis
  • the LLG gene or products of this invention may be used to treat patients with intrahepatic cholestasis that is caused by a mutation or defective expression of the PFIC/BRIC disease gene(s).
  • the LLG gene or polypeptide products of this invention may be used to treat patients with intrahepatic cholestasis that is not due to a defect in the PFIC/BRIC disease gene(s) at 18q21-q22.
  • administration of LLG polypeptide, either directly or via gene therapy may alleviate this form of the condition.
  • the level of LIPG polypeptide in the body may be used to determine whether an individual is predisposed to low levels of HDL cholesterol and apolipoprotein Al.
  • a tissue sample is taken from the patient.
  • the tissue may be blood or one of the tissues which has been demonstrated to express LIPG as discussed in the Examples section.
  • Measurement of the level of LIPG may be performed by a variety of methods known to those of skill in the art.
  • an antibody directed against LIPG polypeptide may be used to measure the level of LIPG in a tissue sample.
  • LDL Human low density lipoproteins
  • the degree of oxidation was determined by TBARS (Chomczynski, P. (1993) Biotechniques 15,532-537), and was between 25-30 nmol MDA equivalents/mg protein.
  • the differentiated THP-1 cells were exposed for 24 hours to either 50 ⁇ g/ml oxidized LDL or NaCl-EDTA buffer in RPMI medium with 10% lipoprotein- deficient fetal bovine serum (Sigma) . To harvest the RNA, the plates were rinsed with 10 ml of PBS, then 14 ml of
  • TRIZOL Liang, P. and Pardee, A.B. (1992) Science 257,967- 971) (GIBCO) were added to each plate. The solution was pipetted several times to mix, then like samples were pooled into centrifuge tubes and 3 ml chloroform per plate were added and mixed. The tubes were centrifuged for 15 minutes at 12000 x g. After centrifugation the upper layer was transferred to a new tube and 7.5 ml isopropanol per plate was added and mixed. The tubes were centrifuged at 12000 x g for 20 minutes. The pellet was rinsed with ice-cold 70% ethanol and dried at room temperature.
  • cDNA Synthesis cDNA synthesis and PCR amplification were accomplished using protocols from the Differential Display Kit, version 1.0 (Display Systems Biotechnology, Inc.) This system is based on the technique originally described by Liang and Pardee (Mead, D.A., Pey, N.K. , Herrnstadt, C. , Marcil, R.A. , and Smith, L.M. , (1991) Bio/Technology 9,657-663).
  • the primer pairs which yielded the cDNA fragment containing the first information of the lipase like gene were downstream primer 7 and upstream primer 15.
  • the cDNA for the amplification was synthesized as follows, using RNA derived from PMA treated THP-1 cells exposed to either buffer or oxidized LDL: 3 ⁇ l of 25 ⁇ M downstream primer 7 and 7.5 ⁇ l of diethylpyrocarbonate (DEPC) -treated water were added to 300 ng (3.0 ⁇ l) RNA from either sample of THP-1 RNA. This was heated to 70°C for 10 minutes then chilled on ice. To this tube were added 3 ⁇ l of 5x PCR buffer (250 mM Tris-HCl pH 8.3, 375 mM KCI) (GIBCO) , 3 ⁇ l 25 mM MgCl 2 , 3 ⁇ l 0.1M DTT,
  • 5x PCR buffer 250 mM Tris-HCl pH 8.3, 375 mM KCI
  • PCR amplification was performed using a master mix containing 117 ⁇ l lOx PCR buffer (500 mM KCI, 100 mM Tris-HCl pH 8.3, 15 M MgCl 2 , and 0.01% (w/v) gelatin), 70.2 ⁇ l 25 mM MgCl 2 , 5.9 ⁇ l alpha- 33 P dATP (10m Ci/ml, DuPont NEN) , 4.7 ⁇ l 500 ⁇ M dNTP mix, 11 ⁇ l AmpliTaq DNA polymerase (5 units/ ⁇ l, Perkin-Elmer) , and 493.3 ⁇ l DEPC-treated water.
  • 117 ⁇ l lOx PCR buffer 500 mM KCI, 100 mM Tris-HCl pH 8.3, 15 M MgCl 2 , and 0.01% (w/v) gelatin
  • 70.2 ⁇ l 25 mM MgCl 2 5.9 ⁇ l alpha- 33 P dATP (10m Ci/ml,
  • amplification reaction that also included 5 ⁇ l lOx PCR buffer, 3 ⁇ l 25 mM MgCl 2 , 5 ⁇ l 500 ⁇ M dNTPs, 5 ⁇ l 2 ⁇ M downstream primer 7, 7.5 ⁇ l upstream primer
  • Insert sizes were determined using EcoRI digestions of the plasmids and clones containing inserts of the approximate size of the original PCR product were sequenced using fluorecent dye-terminator reagents (Prism, Applied Biosystems) and an Applied
  • Biosystems 373 DNA sequencer The sequence of the PCR product is shown in Figure 2. The sequence of the amplification primers is underlined.
  • 5 'RACE Reaction Extension of the cDNA identified through RT-PCR was accomplished using the 5'RACE system (Loh, E.Y., Eliot, J.F., Cwirla, S. , Lanier, L.L., and Davis, M.M. (1989) Science 243, 217-219; Simms, D. , Guan, N. , and Sitaraman, K., (1991) Focus 13, 99-100) (GIBCO).
  • THP-1 RNA oxidized LDL treated
  • the mix was incubated at 42°C for 2 minutes, then 1 ⁇ l Superscript II reverse transcriptase was added. The reaction was incubated for an additional 30 minutes at 42°C, 15 minutes at 70°C, and on ice for 1 minute. One microliter of RNase H (2 units) was added and the mixture was incubated at 55°C for 10 minutes.
  • the cDNA was purified using the GlassMax columns (Sambrook, J. Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, Plainview, NY) included in the kit. The cDNA was eluted from the column in
  • Tailing of the cDNA was accomplished in the following reaction: 7.5 ⁇ l dH 2 0, 2.5 ⁇ l reaction buffer (200 mM Tris- HCl pH 8.4, 500 mM KCI), 1.5 ⁇ l 25 mM MgCl 2 , 2.5 ⁇ l 2 mM dCTP, and 10 ⁇ l of the cDNA were incubated at 94°C for 3 minutes, then 1 minute on ice. 1 ⁇ l (10 units) of terminal deoxynucleotidyl transferase was added and the mixture was incubated for 10 minutes at 37°C.
  • PCR amplification of the cDNA was performed in the following steps: 5 ⁇ l of the tailed cDNA was included in a reaction which also contained 5 ⁇ l lOx PCR buffer (500 mM KCI, 100 mM Tris-HCl pH 8.3 , 15 mM MgCl 2 , and 0.01% (w/v) gelatin), 1 ⁇ l 10 mM dNTP mix, 2 ⁇ l (10 pmol) anchor primer, 1 ⁇ l (20 pmol) primer 3a, and 35 ⁇ l dH 2 0.
  • 5 ⁇ l of the tailed cDNA was included in a reaction which also contained 5 ⁇ l lOx PCR buffer (500 mM KCI, 100 mM Tris-HCl pH 8.3 , 15 mM MgCl 2 , and 0.01% (w/v) gelatin), 1 ⁇ l 10 mM dNTP mix, 2 ⁇ l (10 pmol) anchor primer, 1 ⁇ l (20
  • the reaction was heated to 95°C for 1 minute, then 0.9 ⁇ l (4.5 units) Amplitaq polymerase was added.
  • the reaction was cycled 40 times under the following conditions: 94°C for 5 seconds, 50°C for 20 seconds, and 72°C for 30 seconds.
  • One microliter of this reaction was used in a nested reamplification to increase levels of specific product for subsequent isolation.
  • the reamplification included: 1 ⁇ l primary amplification, 5 ⁇ l lOx PCR buffer, 1 ⁇ l 10 mM dNTP mix, 2 ⁇ l (20 pmol) universal amplification primer, 2 ⁇ l (20 pmol) primer 4a, and 38 ⁇ l dH 2 0.
  • the reaction was heated to
  • a human placental cDNA library (Oligo dT and random primed, Cat #5014b, Lot #52033) was obtained from Clontech (Palo Alto, CA) .
  • a radiolabeled probe was created by excising the insert of a plasmid containing the 5 'RACE reaction PCR product described above. The probe was radiolabeled using the random priming technique: the DNA fragment (50-100 ng) was incubated with 1 ⁇ g of random hexamers (Gibco) at 95°C for 10 minutes followed by 1 minute on ice.
  • the resulting probes had a specific activity greater than 5xl0 8 cpm/ ⁇ g DNA.
  • the sequence of the open reading frame contained within the 2.6 kb EcoRI fragment of the cDNA was determined by automated sequencing as described above. The sequence is shown in Figure 4.
  • the amino acid sequence of the predicted protein encoded by the open reading frame is shown in Figure 5 and has been termed LLGXL.
  • the first methionine is predicted to be encoded by nucleotide pairs 252-254.
  • the predicted protein is 500 amino acids in length.
  • the first 18 amino acids form a sequence characteristic of a secretory signal peptide (Higgins, D.G., and Sharp, P.M. (1988) Gene 73, 237-244).
  • the propeptide is predicted to have a molecular weight of 56,800 Daltons. Assuming cleavage of the signal peptide at position 18, the unmodified mature protein has a molecular weight of 54,724 Daltons.
  • LIPG is the polypeptide (SEQ ID NO: 6) encoded by the cDNA (SEQ ID NO: 5) described in Example 1, and hereafter referred to as LLGN.
  • This protein is identical with the LLGXL protein in the amino terminal 345 residues.
  • Nine unique residues are followed by a termination codon, producing a propolypepti.de of 39.3 kD and a mature protein of 37.3 kD.
  • LLGXL are nucleic acid sequence SEQ ID NO: 9 and amino acid sequence SEQ ID NO: 10.
  • the position at which the LLGN and LLGXL proteins diverge is at a region known from the structure of the other lipase to be between the amino and carboxy domains of the proteins. Therefore, the LLGN protein appears to consist of only one of the two domains of triaclyglycerol lipases. This sequence contains the characteristic "GXSXG" lipase motif at positions 167-171 as well as conservation of the catalytic triad residues at Ser 169, Asp 193, and His 274.
  • the protein sequences used in the comparisons are human lipoprotein lipase (LPL; Genbank accession #M15856, SEQ ID NO: 13), Human hepatic lipase (HL; Genbank accession #J03540, SEQ ID NO: 14) , human pancreatic lipase (PL; Genbank accession # M93285, SEQ ID NO: 15) , human pancreatic lipase related protein-1 (PLRP-1; Genbank accession # M93283) , and human pancreatic lipase related protein-2 (PLRP-2; Genbank accession # M93284) .
  • Percent similarity was based on pairwise alignment using the Clustal algorithm (Camps, L. , Reina, M. , Llobera, M. , Vilaro, S., and Olivecrona, T. (1990) Am. J. Physiol. 258,C673-C681) in the Megalign program of the Lasergene Biocomputing Software Suite (Dnastar) .
  • DNA from a PI clone (Sternberg, N. , Ruether, J. and DeRiel, K. The New Biologist 2:151-62, 1990) containing genomic LLG DNA was labelled with digoxigenin UTP by nick translation. Labelled probe was combined with sheared human DNA and hybridized to PHA stimulated peripheral blood lymphocytes from a male donor in a solution containing 50% forma ide, 10% dextran sulfate, and 2X SSC. Specific hybridization signals were detected by incubating the hybridized cells in fluoresceinated antidigoxigenin antibodies followed by counterstaining with DAPI . This initial experiment resulted in specific labeling of a group E chromosome, which was believed to be chromosome 18 on the basis of DAPI staining.
  • RNA from these cells was prepared as described above.
  • the mRNA was purified from the total RNA through the use of a poly-dT- magnetic bead system (Polyattract system, Promega) .
  • Three micrograms of poly (A) -containing mRNA was electrophoresed on a 1% agarose-formaldehyde gel. The gel was washed for 30 minutes in dH 2 0.
  • RNAs were vacuum transferred to a nylon membrane using alkaline transfer buffer (3M NaCl, 8 mM NaOH, 2 mM sarkosyl) . After transfer, the blot was neutralized by incubation for 5 minutes in 200 mM phosphate buffer pH 6.8.
  • the RNA was crosslinked to the membrane using an ultraviolet crosslinker apparatus (Stratagene) .
  • a probe was made by excising the insert of a plasmid containing the 5'RACE reaction PCR product described above.
  • the probe was radiolabeled using the random priming technique described in Example 2.
  • the filters were prehybridized in QuikHyb rapid hybridization solution (Stratagene) for 30 minutes at 65°C.
  • the radiolabeled probe (1-2 x 10 6 cpm/ml) and sonicated salmon sperm DNA (final concentration 100 ⁇ g/ml) were denatured by heating to 95°C for 10 minutes and quick-chilled on ice before adding to the filter in QuikHyb. Hybridization was for 3 hours at 65°C.
  • the unhybridized probe was removed by washing the filters two times for 15 minutes with 2X SSC, 0.1 % sodium dodecyl sulfate at room temperature followed by two times for 15 minutes in 0.1X SSC, 0.1% SDS at 62°C. Following the washes, the filters were allowed to dry briefly and then exposed to Kodak XAR-2 film with intensifying screens at -80°C. The results are shown in Figure 7, which shov/s a major mRNA species of approximately 4.5 kilobases. Minor species of 4.3 and 1.6 kilobases are also present. The expected size of the LLGN cDNA is 1.6 kb.
  • the LLGXL sequence is likely to be encoded by the major species of mRNA detected.
  • This fragment was obtained by RT-PCR of the THP-1 RNA (PMA and oxLDL treated) using the 5 'LPL and 3 'LPL primers described in Figure 1. and the RT-PCR conditions described above. After autoradiography, the membranes were stripped again and reprobed with a radiolabeled fragment of the human beta actin cDNA to normalize for RNA content. The results of these analyses are shown in Figure 8. The highest levels of LIPG message were detected in placental RNA, with lower levels found in RNAs derived from lung, liver, and kidney tissue. In agreement with previous studies by others (Verhoeven, A.J.M., Jansen, H. (1994) Biochem. Biophys.
  • LLGXL cDNA contains 100-500 ng mRNA from 50 different tissues and is normalized for equivalent housekeeping gene expression (Chen, L. , and Morin, R. (1971) Biochim. Biophys. Acta 231,194-197).
  • a 1.6 kb Dral-Srfl fragment of the LLGXL cDNA was labeled with 32 PdCTP using a random oligonucleotide priming system (Prime It II, Stratagene) according to the manufacturer's instructions.
  • the probe was added to QuikHyb hybridization solution at 1.3xl0 6 cpm/ml. Hybridization was for 2 hours at 65°C .
  • the unhybridized probe was removed by washing the filters two times for 15 minutes with 2X SSC, 0.1 % sodium dodecyl sulfate at room temperature followed by two times for 15 minutes in 0.1X SSC, 0.1% SDS at 62 ⁇ C. Following the washes, the filters were allowed to dry briefly and then exposed to Kodak XAR-2 film with intensifying screens at -80°C. for varying amounts of time. The resulting images were quantitated b densitometry. The results are shown in Table 2.
  • the thyroid was not previously known to express any lipase.
  • LIPG expression may be involved in maintenance of the placenta, where LIPG may serve to liberate free fatty acids from substrates such as phospholipids as a source of energy.
  • the LIPG expressed in the thyroid may provide precursors for the synthesis of bioactive molecules by that gland.
  • HUVEC Human umbilical vein endothelial cells
  • HCAEC human coronary arterial endothelial cells
  • EGM endothelial cell growth medium
  • HCAECs were propagated in EGM supplemented with 3 mg/ml bovine brain extract and 3% fetal bovine serum (5% final concentration)
  • EGM endothelial cell growth medium
  • HCAECs were propagated in EGM supplemented with 3 mg/ml bovine brain extract and 3% fetal bovine serum (5% final concentration) .
  • RNAs were extracted from the cells via the Trizol method described above. Twenty micrograms of total RNA was electrophoresed and transferred to the membrane for analysis. The membranes were probed with LIPG and LPL probes as described above. The results are shown in Figure 9. Twenty micrograms of total RNA from THP-1 cells stimulated with PMA was run on the blot for comparison. RNA hybridizing to the LIPG probe was detected in unstimulated and PMA stimulated HUVEC cells.
  • Antisera were generated to peptides with sequences corresponding to a region of the predicted protein encoded by the LIPG cDNA open reading frame. This peptide was chosen because of its high predicted antigenicity index
  • HUVEC and HCEAC cells were cultured and stimulated with PMA as described in Example 3C, except that the cells were stimulated with PMA for 48 hours.
  • Samples of conditioned medium (9 ml) were incubated with 500 ⁇ l of a 50% slurry of heparin-Sepharose CL-6B in phosphate buffered saline (PBS, 150 mM sodium chloride, 100 M sodium phosphate, pH 7.2).
  • PBS phosphate buffered saline
  • Heparin-Sepharose was chosen to partially purify and concentrate the LIPG proteins because of the conservation of residues in the LLGXL sequence which have been identified as critical for the heparin-binding activity of LPL (Ma, Y., Henderson, H.E., Liu, M.-S., Zhang, H. , Forsythe, I.J., Clarke-Lewis, I., Hayden, M.R., and Brunzell, J.D. J. Lipid Res. 35, 2049-2059; and Fig. 6.). After rotation at 4°C for 1 hour, the samples were centrifuged for 5 minutes at 150 x g. The medium was aspirated and the Sepharose was washed with 14 ml PBS.
  • the pelleted heparin-Sepharose was suspended in 200 ⁇ l 2x SDS loading buffer (4% SDS, 20% glycerol, 2% ⁇ -mercaptoethanol, 0.002% bro phenol blue, and 120 mM Tris pH 6.8). The samples were heated to 95°C for 5 minutes and 40 ⁇ l was loaded onto a 10% Tris-Glycine SDS gel. After electrophoresis at 140 V for approximately 90 minutes, the proteins were transferred to nitrocellulose membranes via a Novex electroblotting apparatus (210 V, 1 hour) .
  • the membranes were blocked for 30 minutes in blocking buffer (5% nonfat dried milk, 0.1% Tween 20, 150 mM sodium chloride, 25 mM Tris pH 7.2).
  • Antipeptide antisera and normal rabbit serum was diluted 1:5000 in blocking buffer and was incubated with the membranes overnight at 4°C with gentle agitation. The membranes were then washed 4x 15 minutes with TBST (0.1% Tween 20, 150 mM sodium chloride, 25 mM Tris pH 7.2).
  • Goat anti-rabbit peroxidase conjugated antisera (Boehringer Mannheim) was diluted 1:5000 in blocking buffer and incubated with the membrane for 1 hour with agitation.
  • EXAMPLE 5 - Recombinant LIPG Protein Production LIPG expression constructs The cDNAs encoding the LLGN and LLGXL proteins were cloned into the mammalian expression vector pCDNA3 (Invitrogen) . This vector allows expression of foreign genes in many mammalian cells through the use of the cytomegalovirus major late promoter.
  • the LLGN 5 'RACE product was cloned into the EcoRI site of pCDNA3.
  • the LLGXL cDNA was digested with Dral and Srfl to yield a 1.55 kb cDNA (see Figure 4. ) .
  • the vector was digested with the restriction enzyme EcoRV and the vector and insert were ligated using T4 DNA ligase and reagents from the Rapid Ligation Kit (Boehringer Mannheim) according to the manufacturers . instructions.
  • the ligation products were used to transform competent E_-_ coli . Resultant colonies were screened by restriction analysis and sequencing for the presence and orientation of the insert in the expression vector.
  • the LIPG expression vectors were introduced into COS-7 cells through the use of Lipofectamine cationic lipid reagent (GIBCO) . Twenty-four hours before the transfection, COS-7 cells were plated onto 60 mm tissue culture dishes at a density of 2xl0 5 cells/plate. The cells were propagated in Dulbecco's modified Eagle's medium (DMEM; GIBCO) supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin. One microgra of plasmid DNA was added to 300 ⁇ l of Optimem I serum-free medium (Gibco) .
  • DMEM Dulbecco's modified Eagle's medium
  • Lipofectamine reagent Ten microliters of Lipofectamine reagent were diluted into 300 ⁇ l of Optimem I medium and this was combined with the DNA solution and allowed to sit at room temperature for 30 minutes. The medium was removed from the plates and the cells were rinsed with 2 ml of Optimem medium. The DNA- Lipofectamine solution was added to the plates along with 2.7 ml Optimem medium and the plates were incubated for 5 hours at 37°C. After the incubation, the serum free medium was removed and replaced with DMEM supplemented with 2% FBS and antibiotics. Twelve hours post-transfection, some of the cultures were treated with either 0.25 mM Pefabloc SC (Boehringer Mannheim) , a protease inhibitor, or 10 U/ml heparin.
  • Pefabloc SC Boehringer Mannheim
  • the heparin treated samples were treated with an additional 40 U/ml heparin.
  • the medium was removed from the cells 60 hours after transfection.
  • Heparin-Sepharose CL-4B 200 ⁇ l of a 50% slurry in PBS pH 7.2
  • the Sepharose was pelleted by low speed centrifugation and was washed three times with 1 ml cold PBS.
  • the Sepharose was pelleted and suspended in 100 ⁇ l 2x loading buffer. The samples were heated to 95°C for 5 minutes. 40 ⁇ l of each sample was loaded onto a 10% SDS- PAGE gel.
  • Electrophoresis and western analysis was performed using the anti-LIPG antiserum as described above. The results are shown in Figure 12. Proteins from HCAEC conditioned medium were included for size references. LLGN migrates at approximately 40 kD, corresponding to the lowest band in HCAEC. The medium from COS cells transfected with LLGXL cDNA contains both 68 kD and 40 kD species. When these cells were treated with heparin, the amount of both 68 kD and 40 kD proteins recovered from the medium increased dramatically, indicating either the release of proteoglycan- bound protein from the cell surface or stabilization of the proteins by heparin.
  • genomic DNAs from various species were restriction digested with EcoRI, separated by electrophoresis in agarose gels, and blotted onto nitrocellulose membranes.
  • the membranes were hybridized overnight at 65°C with 2.5 x 10 6 cpm/ml of random primed 32 P-LLG or 32 P-LPL (lipoprotein lipase) probe in a hybridization solution of 6X SSC, 10% dextran sulfate, 5 X Dendardt's solution, 1% SDS, and 5 ⁇ g/ml salmon sperm DNA.
  • 6X SSC 10% dextran sulfate
  • 5 X Dendardt's solution 1% SDS
  • 5 ⁇ g/ml salmon sperm DNA The membranes were washed with 0.1X SSC, 0.5% SDS for ten minutes at room temperature, then sequentially for ten minutes at 40°C, 50°C, and 55°C. Autoradiograms of the blots are shown in Figure 16.
  • Figure 16 shows the presence of LLG and LPL genes in all species examined, with the exception that no hybridization was observed with the LLG probe against rat DNA.
  • the exceptional data from rat may represent an artifact caused by generation of abnormally sized restriction fragments containing LLG sequences. Such fragments may be outside of the fractionation range of the agarose gel or may blot inefficiently.
  • the different bands detected by the two probes indicate that LPL and LIPG are separate, evolutionarily conserved genes.
  • EXAMPLE 7 Enzymatic Activity of LLGXL Phospholipase Activity
  • COS-7 cells transiently expressing human lipoprotein lipase (LPL) , LLGN, or LLGXL were assayed for phospholipase activity.
  • MEM containing 10% FBS (MEM) was used as the blank, and conditioned media from COS-7 cells transfected with an antisense LLGXL plasmid (AS) was used as a negative control.
  • a phosphatidylcholine (PC) emulsion was made up using 10 ⁇ l phosphatidylcholine (10 mM) , 40 ⁇ l 14 C- phosphatidylcholine, dipalmitoyl (2 ⁇ Ci) , labeled at the sn 1 and 2 positions, and 100 ⁇ l Tris-TCNB [100 mM Tris, 1% Triton, 5 mM CaCl 2 , 200 mM NaCl, 0.1% BSA). The emulsion was evaporated for 10 minutes, then brought to a final volume of 1 ml in Tris-TCNB.
  • Conditioned media from COS-7 cells transiently expressing human lipoprotein lipase (LPL) , LLGN, or LLGXL were assayed for triglycerol lipase activity.
  • MEM containing 10% FBS was used as the blank, and conditioned media from COS-7 cells transfected with an antisense LLGXL plasmid (AS) was used as a negative control.
  • the assay substrate was prepared by dilution of 1 volume of concentrated substrate with 4 volumes of 0.2M Tris-HCl buffer (pH 8.0) containing 3% w/v fatty acid free bovine serum albumin. The diluted substrate was vortexed vigorously for 5 seconds.
  • Reactions were performed in duplicate in a total volume of 0.2 ml containing 0.1 ml of assay substrate and 0.1 ml of the indicated conditioned media. The reactions were incubated for 90 minutes at 37°C. The reactions were terminated by adding 3.25 ml of methanol-chloroform-heptane 1.41:1.25:1 (v/v/v) followed by 1.05 ml of 0.1M potassium carbonate-borate buffer (pH 10.5). After vigorous mixing for 15 seconds, the samples were centrifuged for 5 minutes at 1000 rpm. A 1.0 ml aliquot of the upper aqueous phase was counted in a scintillation counter. The results of these assays are shown in Figure 15.
  • Recombinant LIPG is produced in baculovirus-infected insect cells or stably transfected CHO cells or other acceptable mammalian host cells.
  • Recombinant LIPG is purified from the serum-containing or serum-free conditioned medium by chromatography on heparin-Sepharose, followed by chromatography on a cation exchange resin.
  • a third chromatographic or further chromatographic steps, such as molecular sieving, is used in the purification of LIPG if needed.
  • anti-peptide antibodies are used to monitor LIPG protein and the phospholipase assay is used to follow LIPG activity.
  • the final assay conditions are approximately 10 mM Tris-HCl (pH 7.4), 100 mM KCI, 2 mM CaCl 2
  • the final assay conditions are approximately 25 mM Tris-HCl (pH 8.5), 100 mM KCI, 10 mM CaCl 2 , 4.24 mM Triton X-100, 0.5 mM 1, 2-bis (hexanoylthio) - 1, 2-dideoxy-sn-glycero-3-phosphorylcholine, 5 mM 4,4'- dithiobispyridine (from a 50 mM stock solution in ethanol) , and 1-100 ng recombinant LIPG.
  • Phospholipase activity is determined by measuring the increase in absorption at 342 nm.
  • Compounds and/or substances to be tested for stimulation and/or inhibition of LIPG activity are added as 10-200 mM solutions in dimethylsulfoxide.
  • Compounds which stimulate or inhibit LIPG activity are identified as causing an increased or decreased absorption at 342 nm.
  • the 1.53 kb Dral/Srfl restriction fragment encoding LLGXL was cloned into a plasmid vector (pHMG) downstream of the promoter for the ubiquitously expressed 3- hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase gene.
  • pHMG plasmid vector downstream of the promoter for the ubiquitously expressed 3- hydroxy-3-methylglutaryl coenzyme A reductase gene.
  • Transgenic mice expressing different levels of human LLGXL are generated using standard methods (see, e.g., G.L. Tremp et al. Gene 156:199-205, 1995). The transgenic mice are used to determine the impact of LLGXL overexpression on lipid profile, vascular pathology, rate of development and severity of atherosclerosis, and other physiological parameters.
  • LLGXL expression in atherosclerosis was examined by performing a reverse transcription-polymerase chain reaction (RT-PCR) using mRNA isolated from vascular biopsies from four patients with atherosclerosis.
  • the tissue samples were from the aortic wall (one sample) , the iliac artery (two samples) , and the carotid artery (one sample) .
  • Atherosclerosis biopsies were received from Gloucestershire Royal Hospital, England, and polyA+ mRNA was prepared and resuspended in diethylpyrocarbonate (DEPC) treated water at a concentration of 0.5 ⁇ g/ ⁇ l mRNA.
  • Reverse transcriptase reactions were performed according to the GibcoBRL protocol for Superscript Preamplification System for First Strand cDNA Synthesis. Briefly, the cDNA was synthesized as follows: 2 ⁇ l of each mRNA was added to 1 ⁇ l oligo (dT) 12 . 18 primer and 9 ⁇ l of DEPC water. The tubes were incubated at 70 °C for 10 minutes and put on ice for 1 minute. To each tube, the following components were added: 2 ⁇ l 10X PCR buffer, 2 ⁇ l 25 mM MgCl 2 , 1 ⁇ l 10 mM dNTP mix and 2 ⁇ l
  • PCR amplifications were performed using 2 ⁇ l of the cDNA reactions. To each tube the following were added: 5 ⁇ l 10X PCR buffer, 5 ⁇ l 2 mM dNTPs, 1 ⁇ l hllg-gspl primer (20 pmol/ml, see Figure 1) , 1 ⁇ l hllg-gsp2a primer (20 pmol/ml, see Figure 1), 1.5 ⁇ l 50 mM MgCl 2 , 0.5 ⁇ l Taq polymerase (5U/ml) and 34 ⁇ l water. After holding the reactions at 95°C for 2 minutes, thirty cycles of PCR were performed as follows: 15 seconds at 94°C, 20 seconds at 52°C, and 30 seconds at 72°C.
  • the finished reactions were held for 10 minutes at 72°C before analysis by agarose gel electrophoresis.
  • the hllg-gsp primers are specific for LIPG and yield an expected product of 300 bp.
  • primers specific for the housekeeping gene, G3PDH human glyceraldehyde 3-phosphate dehydrogenase were used (1 ⁇ l each at 20 pmol/ml) .
  • the G3PDH primers yielded the expected product of 983 bp in all four vascular biopsy samples. LIPG expression was detected in three of the four samples, with no expression being detected in the carotid artery sample.
  • THP-1 cells were plated in the presence of phorbol 12-myristate 13- acetate (PMA, 40 ng/ml; Sigma) for 48 hours. The differentiated THP-1 cells were exposed for 24 hours to either oxLDL (50 ⁇ g/ml) or control medium.
  • Total RNAs were collected and purified using standard procedures. Poly (A) + RNA was purified from total RNA using a poly-dT magnetic bead system (Promega) .
  • cDNA synthesis and PCR amplification were accomplished using protocols from the Differential Display kit, version 1.0 (Display Systems Biotechnology).
  • the primer pairs that yielded the initial cDNA fragment of EL were downstream primer 7 (5 ' -TTTTTTTTTTTGA-3 • ) and upstream primer 15 (5'-GATCCAATCGC-3 • ) .
  • the amplification reaction was fractionated on a 6% nondenaturing acrylamide sequencing format gel and an amplification product found only in the reaction containing cDNA from THP-1 cells exposed to oxLDL was identified and excised from the gel. A reamplification using the same primers was performed and the product was excised and subcloned into the pCRII vector using the TA cloning system (Invitrogen) .
  • Insert sizes were determined using EcoRI digestions of the plasmids, and clones containing inserts of the approximate size of the original PCR product were sequenced using fluorescent dye-terminator reagents (Prism, Applied Biosystems) and an Applied Biosystems 373 DNA sequencer.
  • RNA (1 ⁇ g) from the THP-1 cells used initially in the differential display reactions was used in the 5 ' -RACE procedure using a gene-specific primer (5 '-TAGGACATGCACAGTGTAATCTG-3 ' ) for first strand cDNA synthesis.
  • the DNA from hybridizing clones was purified using LambdaSorb reagent (Promega) . Inserts were excised from the phage DNA by digestion with EcoRI , subcloned into the EcoRI site of the Bluescript II SK plasmid vector (Stratagene) and sequenced.
  • a 17-residue peptide (GPEGRLEDKLHKPKATC) was synthesized corresponding to residues 8-23 of the secreted LIPG gene product on a Model 433A peptide synthesizer (Applied Biosystems) .
  • Peptide (2 mg) was coupled to maleimide- activated keyhole limpet haemocyanin (2 mg) following the protocols included in the Imject Activated Immunogen Conjugation kit (Pierce Chemical) . After desalting, one-half of the conjugate was emulsified with an equal volume of Freund's complete adjuvant (Pierce) and injected into a New Zealand White rabbit.
  • HUVECs were propagated in a commercially prepared endothelial cell growth medium (EGM, Clonetics) supplemented with bovine brain extract (3 mg/ml; Clonetics) , whereas HCAECs were propagated in EGM with bovine grain extract (3 mg/ml) and 5% fetal bovine serum. Cultures were stimulated by addition of PMA (100 ng/ml) . After 24 hours incubation, RNA was extracted from the cells via the Trizol method, electrophoresed on a 1% agarose-formaldehyde gel, transferred to Nytran membrane on a Turboblotter apparatus (Schleicher and Schuell) and crosslinked to the membrane using a Stratalinker ultraviolet crosslinker (Stratagene).
  • EGM endothelial cell growth medium
  • HCAECs were propagated in EGM with bovine grain extract (3 mg/ml) and 5% fetal bovine serum. Cultures were stimulated by addition of PMA (100 ng/ml) . After
  • the 5*- RACE reaction PCR product was radiolabelled using the random priming technique.
  • the radiolabelled probe (l-2xlO b cpm/ml) was denatured by heating to 95 °C for 10 minutes and quick- chilled on ice before adding to the filter in QuikHyb. Hybridization was allowed to proceed for 3 hours at 65 °C. Filters were exposed to Kodak XAR-2 film with intensifying screens at -80 °C. We incubated HUVEC- and HCEAC-conditioned medium with heparin-Sepharose CL-6B at 4 °C for 1 hour.
  • the pelleted heparin-Sepharose was suspended in SDS loading buffer, heated to 95 °C for 5 minutes and loaded onto a 10% Tris-Glycine SDS gel (NOVEX) . After electrophoresis at 140 V for 90 minutes, the proteins were transferred to nitrocellulose membranes and detected with rabbit anti-LIPG peptide antisera (1:5,000), with goat anti-rabbit peroxidase conjugated antisera (1:5,000; Boehringer) as the secondary antibody. The membranes were reacted with Renaissance chemiluminescent reagent (DuPont NEN) and exposed to Kodak XAR-2 film.
  • DuPont NEN Renaissance chemiluminescent reagent
  • a commercially prepared filter containing poly (A) + RNAs (3 ⁇ g each) from human heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas (Clontech) was hybridized with a radiolabelled fragment and processed as described above. Following autoradiography, the blot was stripped by washing in boiling O.lxSSC, 0.1% SDS for 2x15 minutes at 65 °C and then probed as described above with a 1.4-kb cDNA fragment encoding human LPL.
  • This fragment was obtained by RT-PCR of the THP-1 RNA (PMA and oxLDL treated) using the 5 • LPL and 3 ' LPL primers 5 ' -ACCACCATGGAGAGCAAAGCCCTG-3 ' and 5 ' - CCAGTTTCAGCCTGACTTCTTATTC-3 ' , respectively. After exposure to film, the membranes were stripped again and reprobed with a radiolabelled fragment of human ⁇ actin cDNA to normalize to RNA content.
  • Human umbilical vein endothelial cells were negative for LPL mRNA expression as expected, but were found to constitutively express a high level of mRNA for the LIPG gene ( Figure 9) .
  • HCAEC Human coronary artery endothelial cells
  • Conditional medium from stimulated HUVEC and HCAEC contained immunoreactive proteins of approximately 68 kD and 40 kD, as well as a less prominent band of 55 kD ( Figure 11) .
  • the cDNA and the 1.4-kb LPL cDNA were cloned into the EcoRV site of the mammalian expression vector pCDNA3 (Invitrogen) .
  • An antisense pCDNA3 vector was used as negative control.
  • the recombinant expression vectors (3 ⁇ g) were mixed with lipofectamine (Life Technologies) and transfected in quadruplicate into semiconfluent COS7 cells in 60-mm dishes. Established methods were used to assay samples of conditioned media from transfected COS7 cells for TG lipase and phospholipase activities (Goldberg, J.I., J . Lipid Res . , 37, 693-707 (1996)).
  • 9,10- 3 H(N) -triolein 250 ⁇ Ci; NEN
  • unlabeled triolein 150 mg
  • type IV-S- lecithin 9 mg
  • Sigma type IV-S- lecithin
  • the assay substrate was prepared by combining one volume of the emulsified substrate, four volumes of TrisHCl (0.2 M, pH 8.0) containing 3% (w/v) fatty acid-free bovine serum albumin (BSA) and one volume of heat-inactivated bovine serum.
  • BSA bovine serum albumin
  • Tris-TCNB 100 ⁇ l; 100 mM Tris-HCl pH 7.4, 1% Triton X-100, 5 mM CaCl 2 , 200 mM NaCl, 0.1% BSA.
  • the mixture was vortexed for 2 minutes and then evaporated under nitrogen.
  • the dried lipid was reconstituted with TCNB (1 ml) and vortexed for 10 seconds. Reactions were performed in triplicate and contained PC emulsion (50 ⁇ l) , conditioned media (600 ⁇ l) and MEM (350 ⁇ l) .
  • a recombinant adenovirus encoding human LIPG was constructed as described (Tsukamoto et al., J. Clin . Invest . , 100, 107-114 (1997); Tsukamoto et al., J . Lipid Res . , 38, 1869-1876 (1997)).
  • the full-length human cDNA was subcloned into the shuttle plasmid vector pAdCMVLinkl. After screening for the appropriate orientation by restriction analysis, the plasmid was linearized with Nhel and cotransfected into 293 cells along with adenoviral DNA digested with Clal . Cells were overlaid with agar and incubated at 37°C for 15 days.
  • plaque purification Six plaques were picked and screened by PCR; two plaques positive for cDNA were subjected to a second round of plaque purification. After confirmation of the presence of cDNA, the recombinant adenovirus was expanded in 293 cells at 37 °C. Cell lysates were used to infect HeLa cells for confirmation of the expression of human LIPG by western blot of conditioned media. The recombinant adenovirus (AdhEL) was further expanded in 293 cells and purified by cesium chloride ultracentrifugation. Control adenovirus containing no cDNA insert (Adnull) was also subjected to plaque purification and purified as described above.
  • the purified viruses were stored in 10% glycerol/PBX at -80 °C.
  • Wild-type C57BL/6, human apoA-I transgenic and LDL receptor mutant mice were obtained from Jackson Laboratory. All mice were fed chow diets. Wild-type and human apoA-I transgenic mice were injected intravenously via the tail vein with AdhEL or Adnull lxlO 11 particles (approximately 2xl0 9 pfu) and LDLR-deficient mice were injected with lxlO 10 particles. In all experiments, blood was obtained from the retro-orbital plexus 1 day before injection and at multiple time points following injection.
  • the plasma total cholesterol and HDL cholesterol levels were measured enzymatically on a Cobas Fara (Roche Diagnostic Systems) using Sigma reagents. ApoA-I was quantitated using a turbidometric assay (Sigma) on a Cobas Fara. Pooled plasma samples were subjected to fast protein liquid chromatography (FPLC) gel filtration (Pharmacia LKB Biotechnology) using two Superose 6 columns in series as described (Tsukamoto et al., J. Clin . Invest . , supra) . Fractions (0.5 ml) were collected, and cholesterol concentrations were determined using an enzymatic assay (Wako Pure Chemical Industries) .
  • FPLC fast protein liquid chromatography
  • Modulators of EL activity may be found using the following method:
  • Recombinant LIPG would be purified from the conditioned medium of stably transfected Chinese hamster ovary cells, from baculovirus infected insect cells, yeast (Pichia pastoris, Kluveromyces Lactis) or other sources.
  • Non- recombinant sources of LIPG such as human plasma, endothelial cell conditioned media, etc.
  • An example of a primary screen to look for modulators of LIPG activity would utilize the soluble fluorescent substrate 4-methylumberiferyl hepatanoate. This assay is continuous and homogeneous. Hydrolysis of this substrate by LIPG results in the production of highly fluorescent 4-methylumbelliferone that can be measured in a microplate fluorimeter.
  • the catalytic center of LIPG like other TG lipases, consists of the same catalytic triad (ser, his, asp) found in serine proteases. Indeed, other TG lipases, such as lipoprotein lipase, are inhibited by serine protease inhibitors such as PMSF and DFP. Either one of these compounds may serve as a positive control for inhibitors of LIPG activity.
  • LPL lipoprotein lipase
  • PL pancreatic lipase
  • Human PL and bovine LPL are commercially available and assays could be readily implemented.
  • the phospholipase activity of PL is measured in exactly the same way as described above for the secondary assay of LIPG. Since LPL is primarily a TG lipase, the secondary assay would measure radiolabelled fatty acid (oleic acid) release from a radiolabelled TG (triolein) substrate
  • This assay has a similar capacity and may be adapted to other assay formats which utilize fluorescent substrates and which would be amenable to a greater throughput.
  • Phospholipase activity of LIPG would be tested on its in vivo substrate, HDL, in an in vitro assay. Radiolabelled HDL could be generated by exchange with a radiolabelled phospholipid, and then used to measure LIPG phosphospholipase activity and the activity of compounds emerging from the screens.
  • An additional assay could measure the impact of preincubation of LIPG, HDL, +/- compounds on radiolabelled cholesterol efflux from cultured cells such as the rat Fu5AH hepatoma line.
  • In vivo assays for assessment of compounds can be run in wild-type, LIPG-overexpressing, and as control, LIPG null mice. If, as in the case of adenoEL expression, the transgenic mice exhibit decreased HDL relative to control mice, then treatment of transgenic mice with LIPG inhibitory compounds would be expected to raise HDL to the levels of control mice. It is also possible that compounds could be tested for their LIPG inhibitory activity (elevation of HDL) in other animals such as the LDLR-/- mouse, apoAl transgenic mice hamsters, or rabbits. Compounds which elevated LIPG or LIPG activity would be expected to raise HDL in these or other animal models.
  • a small molecule (hereafter an "inhibitory small molecule") identified in the screening outlined in Example 17 as able to inhibit the LIPG polypeptide in vitro is tested for its ability to inhibit the LIPG polypeptide in vivo .
  • Wild-type and LIPG transgenic mice will be studied by administering the small molecule orally (if orally bioavailable) or by intravenous injection.
  • Activity of the LIPG polypeptide will be measured in plasma before and after heparin injection (to release the enzyme from bound sites) .
  • cholesterol, VLDL, LDL and HDL cholesterol and apoA-I levels will be monitored in animals receiving the inhibitory small molecule.
  • LDL receptor deficient mice will be fed an atherogenic diet and administered the inhibitory small molecule or placebo for a period of 8 weeks.
  • Atherosclerosis will be quantitated in the aortas of the mice in order to determine whether administration of the inhibitory small molecule recudes the progression or induces regression of atherosclerosis.
  • additional animal models such as hamsters, rabbits, or pigs will be studied for the ability of the inhibitory small molecule to raise HDL cholesterol levels, reduce VLDL and LDL cholesterol levels, and/or inhibit the progression of atherosclerosis.
  • Those inhibitory small molecules found to have the desired properties will be administered to patients in combination with pharmaceutically acceptable carriers.
  • the inhibitory small molecules may be administered in a variety of ways, including oral administration and intravenous injection.
  • the patients' HDL, VLDL and LDL cholesterol levels will be monitored to determine efficacy of the inhibitory small molecule and to optimize dosage and administration protocols.
  • Therapeutic peptides are identified by testing fragments of the LIPG polypeptide to determine which of these fragments inhibit LIPG polypeptide activity in vitro . Once identified, an "inhibitory peptide" is then tested for its ability to inhibit the LIPG polypeptide in vivo . Inhibitory peptides will be produced recombinantly in E. coli and purified by methods known in the art. The effect of the inhibitory peptides will be studied in wild-type and LIPG transgenic mice by administering the inhibitory peptide by intravenous injection. Activity of the LIPG polypeptide will be measured in plasma before and after heparin injection (to release the enzyme from bound sites) .
  • mice will be fed an atherogenic diet and administered the inhibitory peptide or placebo for a period of 8 weeks.
  • Atherosclerosis will be quantitated in the aortas of the mice in order to determine whether administration of the inhibitory peptide reduces the progression or induces regression of atherosclerosis.
  • additional animal models such as hamsters, rabbits or pigs will be studied for the ability of the inhibitory small molecule to raise HDL cholesterol levels, reduce VLDL and LDL cholesterol levels, and/or inhibit the progression of atherosclerosis.
  • inhibitory peptides found to have the desired properties will be administered to patients in combination with pharmaceutically acceptable carriers.
  • the inhibitory peptides may be administered in a variety of ways, including oral administration and intravenous injection.
  • the patients' HDL, VLDL and LDL cholesterol levels will be monitored to determine efficacy of the inhibitory peptides and to optimize dosage and administration protocols.
  • a series of antisense oligonucleotides, each complementary to about 20 bases of the LIPG cDNA sequence are chemically synthesized by standard techniques. To determine the most efficient oligonucleotide to use therapeutically, each oligonucleotide is individually transfected into cells expressing the LIPG gene, using standard transfection protocols.
  • the LIPG mRNA level in cells is determined by quantitative PCR, northern blot, RNAse protection, or other appropriate methods.
  • LIPG expression may be monitored with specific antibodies, which can be used to screen for effective antisense oligonucleotides.
  • Oligonucleotides which effectively reduce LIPG mRNA levels are then formulated for in vivo delivery as therapeutics.
  • Antisense LIPG sequences may be delivered in a gene therapy vector, such as adenovirus, adeno-associated virus, retrovirus, naked DNA, or other systems discussed in the detailed description. Such fragments can be used therapeutically when delivered in gene therapy vectors. Hepatic expression of such recombinant vectors is a preferred approach. Alternatively, synthetic antisense oligonucleotides may be formulated for in vivo delivery as therapeutics as described above.
  • Antisense oligonucleotides may be administered by the following routes: intravenous, subcutaneous, introdermal, pulmonary, oral, intraventricular, intrathecal, and topical.
  • the route of administration may include direct administration to vessel walls (i.e., endothelium and/or vascular smooth muscle) .
  • vessels walls i.e., endothelium and/or vascular smooth muscle
  • patients with low HDL-C could receive a dose of 0.5-2 mg/kg of an effective antisense oligonucleotide, infused intravenously, every other day for up to 2-3 weeks.
  • LIPG is expressed in the liver, it may be desireable to deliver antisense reagents to the portal circulation.
  • oligonucleotide conjugates or complexing the oligonucleotide with a liver-targeting moiety, such as asialoglycoprotein.
  • Dose and timing of therapy would depend on efficiency of antisense delivery, as well as parameters such as half life, specificity and toxicology of the antisense oligonucleotide.
  • Increase in HDL-C can be monitored using standard clinical laboratory procedures.
  • the original dosing schedule (such as that described above) is repeated as often as required to maintain HDL-C above 35 mg/dL.
  • hammerhead ribozymes which effectively reduce LIPG mRNA levels are prepared. These consist of two “arms” of 6-7 bases each of nucleotide sequence complementary to LIPG mRNA, separated by the catalytic moiety of the ribozyme. Examples of such hammerhead motifs are described by Rossi et al., 1992, Aids Research and Human Retroviruses , 8, 183. The ribozymes are expressed in eukaryotic cells from an appropriate DNA vector.
  • the ribozymes may be administered encapsulated in liposomes, as discussed above.
  • the ribozyme/ liposome composition is delivered to the liver by direct injection or by use of a catheter, infusion pump or stent.
  • the route of administration may include direct administration to vessel walls (i.e., endothelium and/or vascular smooth muscle) .
  • Patients are treated for up to 2 weeks with 5-50 mg/kg/day ribozyme in a pharmaceutically effective carrier. Increase in HDL-C and dosing regimen are monitored and determined as for antisense oligonucleotides.
  • Anti-LIPG antibodies, antibody fragments, or chimeric antibodies consisting of at least one LIPG-binding moiety, prepared as described in Example 12, are used to inhibit LIPG activity in vivo.
  • the antibodies may be delivered as a bolus only, infused over time, or both. Typically a dose of 0.2-0.6 mg/kg is given as bolus, followed by a 2 to 12-hour infusion. Alternatively, multiple bolus injections are administered every other day, or every third of fourth day, as required to reduce LIPG and raise HDL-C. Repeat dosing is performed as determined by measurement of HDL-C levels.
  • Antibodies to LIPG may also be delivered in a gene therapy vehicle to facilitate expression in vivo . The level of expression of the antibody is determined indirectly by measuring HDL-C levels and additional vectors may be introduced as needed.
  • Fragments of LIPG protein which can inhibit LIPG activity by competing for binding to intact LIPG, required coactivator molecules, cell surface receptors or binding proteins, may be delivered as therapeutic recombinant proteins or from gene therapy vectors.
  • the LLGN polypeptide based on LIPG is cloned into a recombinant adenovirus as described (Tsukamoto et al., J. Clin . Invest . , 100, 107-114 (1997); Tsukamoto et al., J. Lipid Res . , 38, 1869-1876 (1997)).
  • the LLGN cDNA is cloned into the shuttle plasmid vector pAdCMVLinkl. After screening for the appropriate orientation by restriction analysis, the plasmid is linearized with Nhel and cotransfected into 293 cells along with adenoviral DNA digested with Clal.
  • adenovirus is expanded in 293 cells at 37°C.
  • Cell lysates are used to infect HeLa cells for confirmation of the expression of human EL by western blot of conditioned media.
  • the recombinant adenovirus is further expanded in 293 cells and purified by cesium chloride ultracentrifugation. The purified viruses are stored in 10% glycerol/PBX at -80°C.
  • the patient is injected intravenously with the recombinant adenovirus lxlO 11 particles (approximately 2xl0 9 pfu) .
  • the full length LIPG cDNA is cloned into a recombinant adenovirus (Tsukamoto et al., J. Clin . Invest . , 100, 107-114 (1997); Tsukamoto et al., J. Lipid Res . , 38, 1869-1876 (1997)) encoding human LIPG.
  • the full-length human LIPG cDNA is cloned into the shuttle plasmid vector pAdCMVLinkl . After screening for the appropriate orientation by restriction analysis, the plasmid is linearized with Nhel and cotransfected into 293 cells along with adenoviral DNA digested with Clal.
  • adenovirus is expanded in 293 cells at 37°C. Cell lysates are used to infect HeLa cells for confirmation of the expression of human LIPG polypeptide by western blot of conditioned media.
  • the recombinant adenovirus (AdhEL) is further expanded in 293 cells and purified by cesium chloride ultracentrifugation. • the purified viruses are stored in 10% glycerol/PBX at -80°C. Patients are injected intravenously with AdhEL or Adnull lxlO 11 particles (approximately 2xl0 9 pfu) .
  • Example 25 Methods of Increasing the Level of LIPG Activity by Administration of a Full-Length Wild-Type or Engineered Recombinant LIPG Protein
  • the wild-type LIPG protein reduces VLDL and LDL cholesterol levels and LIPG could be engineered to act specifically on VLDL and LDL cholesterol without having effects on HDL cholesterol.
  • Administration of wild-type or engineered recombinant LIPG could in certain circumstances be used as a therapy for reducing VLDL and/or LDL cholesterol levels.
  • Wild-type and/or engineered LIPG protein (“recombinant LIPG protein") will be produced recombinantly in E. coli and purified using methods known in the art. Wild- type mice will be studied by administering the recombinant LIPG protein by intravenous injection. Activity of LIPG will be measured in plasma.
  • mice receiving the recombinant LIPG protein will be fed an atherogenic diet and administered the recombinant LIPG protein or placebo for a period of 8 weeks.
  • Atherosclerosis will be quantitated in the aortas of the mice in order to determine whether adminsitration of the recombinant LIPG protein reduces the progression or induces regression of atherosclerosis.
  • additional animal models such as hamsters, rabbits, or pigs will be studied for the ability of the recombinant LIPG protein to reduce VLDL and LDL cholesterol levels and/or inhibit the progression of atherosclerosis.
  • Those recombinant LIPG proteins found to have the desired ability to reduce VLDL and LDL cholesterol levels and/or inhibit the progression of atherosclerosis will be combined with a pharmaceutically acceptable carrier and administered to patients.
  • the recombinant LIPG polypeptides may be administered in a variety of ways, including oral administration and intravenous injection.
  • FIG. 24 demonstrates that endothelial lipase is a glycoprotein.
  • EL was treated with the glycosidases EndoF, EndoH, and neuraminidase. This resulted in reductions in the size of the EL band on western blotting, consistent with the removal of carbohydrate from the polypeptide chain. Glycosylation was also inhibited with tunicamycin in EL- expressing cells. This resulted in a substantial reduction in the size of the EL band. The glycosidase and tunicamycin experiments indicate that EL is a glycoprotein.
  • Figure 25 demonstrates that endothelial lipase is a heparin binding protein.
  • An adenoviral vector expressing EL AdhEL (see Example 15) was injected into mice. Blood was drawn before (pre) and after (post) heparin injection. The intensity of the full-length 68 kD EL band greatly increased after heparin injection consistent with EL bound to heparin sulfate proteoglycans and released by the administration of heparin.
  • FIGs 26A and 26B and 27A and 27B illustrate the lipolytic activity of endothelial lipase relative to lipoprotein lipase (LPL) and hepatic lipase (HL) .
  • LPL lipoprotein lipase
  • HL hepatic lipase
  • Figures 28A and 28B illustrate that expression of EL increased post-heparin plasma phospholipase activity in a dose dependent fashion.
  • Injection of three different doses (lxlO 11 , 3xl0 10 or lxlO 10 particles) of AdhEL into mice resulted in the presence of EL protein in the post-heparin plasma.
  • Major bands of 68 kD and 40 kD and a minor band of 55 kD were observed (Fig. 28A) .
  • the 68 kD band is believed to be the full-length glycosylated form of EL and the 40 kD band a proteolytic fragment.
  • the amount of EL protein in plasma assessed by western blot is proportional to the dose of vector injected (Fig. 28B) .
  • TTCTTAATAC CATGCTGCAG AGCAGGGCAC ATCCTAGCCC AGGAGAAGTG GCCAGCACAA 260 TCCAATCAAA TCGTTGCAAA TCAGATTACA CTGTGCATGT CCTAGGAAAG GGAATCTTTA 320 CAAAATAAAC AGTGTGGACC CCTCAAAAAA AAAAAAAAGC CGAATTC 367
  • MOLECULE TYPE protein
  • Val Ala Gly Tyr Ala Gly Asn Phe Val Lys Gly Thr Val Gly Arg lie
  • 130 135 140 lie Ala Arg Met Leu Asp Trp Leu Gin Glu Lys Asp Asp Phe Ser Leu 145 150 155 160
  • Lys Gly lie Cys Leu Ser Cys Arg Lys Asn Arg Cys Asn Ser lie Gly 305 310 315 320
  • GAG AAA GAC GCC AAT GTA GTT GTG GTT GAC TGG CTC CCC CTG GCC CAC 384
  • MOLECULE TYPE protein
  • MOLECULE TYPE protein
  • GAG AAA GAC GCC AAT GTA GTT GTG GTT GAC TGG CTC CCC CTG GCC CAC 384 Glu Lys Asp Ala Asn Val Val Val Val Asp Trp Leu Pro Leu Ala His
  • MOLECULE TYPE protein
  • MOLECULE TYPE protein

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Abstract

La présente invention concerne des compositions et des procédés permettant d'élever le taux de cholestérol des HDL et des apolipoprotéines AI chez un patient et de réduire le taux de cholestérol des VLDL et des LDL chez un patient. Ces compositions et ces procédés agissent sur l'expression d'un gène, LIPG, qui code une lipase qui fait partie de la famille des triacylglycériol lipases ou qui agit sur l'activité enzymatique de cette enzyme.
EP00918362A 1999-03-26 2000-03-24 Compositions et procedes agissant sur les taux de cholesterol des lipoproteines de haute densite (hdl) et des apolipoproteines ai, sur les taux de cholesterol des lipoproteines de tres faible densite (vldl) et sur le taux de cholesterol des lipoproteines de faible densite (ldl) Ceased EP1171078A4 (fr)

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US09/277,401 US7008776B1 (en) 1996-12-06 1999-03-26 Compositions and methods for effecting the levels of high density lipoprotein (HDL) cholesterol and apolipoprotein AI very low density lipoprotein (VLDL) cholesterol and low density lipoprotein (LDL) cholesterol
PCT/US2000/007870 WO2000057837A2 (fr) 1999-03-26 2000-03-24 Compositions et procedes agissant sur les taux de cholesterol des lipoproteines de haute densite (hdl) et des apolipoproteines ai, sur les taux de cholesterol des lipoproteines de tres faible densite (vldl) et sur le taux de cholesterol des lipoproteines de faible densite (ldl)

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CA2363486C (fr) 2012-12-18
BR0009333A (pt) 2002-01-08
NZ514350A (en) 2004-12-24
WO2000057837A3 (fr) 2001-01-25
WO2000057837A2 (fr) 2000-10-05
NZ531180A (en) 2005-06-24
KR100887164B1 (ko) 2009-03-10
NO20100214L (no) 2001-11-21
WO2000057837A9 (fr) 2001-10-18
IL145526A0 (en) 2002-06-30
AU776684B2 (en) 2004-09-16
NO331779B1 (no) 2012-03-26
AU3918700A (en) 2000-10-16
IL145526A (en) 2010-11-30
EP1171078A4 (fr) 2002-11-06
WO2000057837A8 (fr) 2001-09-27
HK1043309A1 (zh) 2002-09-13
NO20014657L (no) 2001-11-21
MXPA01009727A (es) 2002-07-22
KR20020029651A (ko) 2002-04-19
CA2363486A1 (fr) 2000-10-05
KR20050044812A (ko) 2005-05-12
NO331784B1 (no) 2012-03-26
JP2002540127A (ja) 2002-11-26
ZA200107598B (en) 2003-05-28

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