KR20020029651A - 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 - Google Patents

Abstract

The present invention relates to methods and compositions for increasing levels of high density lipoprotein (HDL) cholesterol and apolipoprotein AI in a patient and methods and compositions for reducing levels of ultra low density lipoprotein (VLDL) cholesterol and low density lipoprotein (LDL) cholesterol in a patient. will be. The present invention also relates to compositions and methods that affect the expression of, or affect the enzymatic activity of, the gene LIPG, which encodes a lipase enzyme that is a member of the triacylglycerol lipase family.

Description

Compositions and Methods for Effecting the Levels of High Density Lipoprotein (HDL) Cholesterol (HDL) Cholesterol and Apolipoprotein AI, Ultra Low Density Lipoprotein (LDL) Cholesterol and Low Density Lipoprotein (LDL) Cholesterol and apolipoprotein AI, very low density lipoprotein (VLDL) cholesterol and low density lipoprotein (LDL) cholesterol}

The present application is directed to U.S.C. US patent application Ser. No. 08 / filed Dec. 5,1997, claiming benefits of both US Provisional Application Nos. 60 / 032,254 and 60 / 032,783, filed December 6, 1996 under §119 (e). Partial Serial Application, which claims priority of US Patent Application Serial No. 09 / 277,401, filed March 26, 1999, filed March 26, 1999, the content of which is incorporated herein by reference in its entirety.

The present invention relates to methods and compositions for increasing levels of high density lipoprotein (HDL) cholesterol and apolipoprotein AI in a patient and to methods and compositions for reducing levels of ultra low density lipoprotein (VLDL) cholesterol and low density lipoprotein (LDL) cholesterol in a patient. will be. The present invention includes within its scope methods and compositions that reduce the expression of, or inhibit the activity of, the enzyme LIPG, which encodes a lipase enzyme that lowers the levels of HDL cholesterol and apolipoprotein AI. The present invention further encompasses methods and compositions that reduce the levels of VLDL and LDL cholesterol by increasing the expression of lipase enzymes or enhancing their activity within the scope thereof.

Geology

Lipids are water-insoluble organic biomolecules and are essential components for various biological functions, including storage, transport and metabolism of energy, and membrane structure and fluidity. Lipids are derived from two sources in humans and other animals: some lipids are ingested as fats and oils in food and others are biosynthesized by humans or animals. In mammals, at least 10% of body weight is lipids, most of which are in the form of triacylglycerols.

Triacylglycerols, also known as triglycerides and triacylglycerides, consist of three fatty acids esterified to glycerol. Food triacylglycerols are stored as energy sources in adipose tissue or hydrolyzed in the digestive tract by triacylglycerol lipases, the most important of which are pancreatic lipases. Triacylglycerols are transported in the form of lipoproteins between tissues.

Lipoproteins are micelle-like aggregates found in plasma that contain different types of lipids and proteins (called apoproteins) in various proportions. There are five main classes of plasma lipoproteins whose main function is lipid transport. These classes include kilomicrons, ultralow density lipoproteins (VLDL), medium density lipoproteins (IdL), low density lipoproteins (LDL), and high density lipoproteins (HDL) in order of increasing density. Although many types of lipids are found in combination with each lipoprotein class, each class carries mainly one type of lipid. The triacylglycerols described above are transported in kilomicrons, VLDL and IdL while the phospholipids and cholesterol esters are transported in HDL and LDL, respectively.

Phospholipids are di-fatty acid esters of glycerol phosphate with polar groups attached to phosphates. Phospholipids are important structural components of cell membranes. Phospholipids are hydrolyzed by an enzyme called phospholipase. Phosphatidylcholine, a representative phospholipid, is a major component of most eukaryotic cell membranes.

Cholesterol is a metabolic precursor of steroid hormones and bile acids and is also an essential component of cell membranes. In humans and other animals, cholesterol is consumed by food and also synthesized by the liver and other tissues. Food cholesterol is transported from the small intestine to the liver by large lipoprotein makeup in the blood. The liver secretes very low density lipoproteins (VLDL), which transport cholesterol and cholesterol esters and many other compounds into the bloodstream. VLDL is partially converted to low density lipoprotein (LDL) in adipose tissue. LDL transports both free and esterified cholesterol into body tissues. High density lipoprotein (HDL) transports cholesterol into the liver and cholesterol is broken down and secreted.

The membrane surrounds all living cells and acts as a barrier between intracellular and extracellular compartments. Membranes also perform special functions as in the myelin sheaths that surround the nuclei of eukaryotic cells, form vesicles, and enclose axons. Typical membranes contain about 40% lipids and 60% protein, but there are significant changes. The major lipid components are phospholipids (particularly phosphatidylcholine and phosphatidylethanolamine) and cholesterol. The physicochemical properties of the membrane, such as fluidity, can be varied by changing the fatty acid profile or cholesterol content of the phospholipid. Regulating the composition and composition of membrane lipids also leads to regulation of membrane-dependent cellular functions such as receptor activity, endocytosis and cholesterol efflux.

enzyme

Triacylglycerol lipases are a family of enzymes that play several pivotal roles in the metabolism of lipids in the body. Three members of the human triacylglycerol lipase family are known: pancreatic lipase, lipoprotein lipase and liver lipase [Goldberg, I.J., Le, N.-A., Ginsberg, H.N., Krauss, R.M., and Lindgren, F.T. (1988) J. Clin. Invest. 81, 561-568; Goldberg, I. J., Le, N., Paterniti J. R., Ginsberg, H. N., Lindgren, F. T., and Brown, W. V. (1982) J. Clin. Invest. 70, 1184-1192; Hide, W. A., Chan, L., and Li, W.-H. (1992) J. Lipid. Res. 33, 167-178. Pancreatic lipases are primarily responsible for the hydrolysis of food lipids. Variants of pancreatic lipase have been published but their physiological roles are unknown [Giller, T., Buchwald, P., Blum-Kaelin, D., and Hunziker, W. (1992) J. Biol. Chem. 267, 16509-16516. Lipoprotein lipases are the main enzymes responsible for the distribution and use of triglycerides in the body. Lipoprotein lipase hydrolyzes triglycerides in both kilomicrons and VLDL. Liver lipases hydrolyze triglycerides in IDL and HDL and are responsible for lipoprotein reconstitution. Liver lipases also act as phospholipases and hydrolyze phospholipids in HDL.

Phospholipases play an important role in catabolizing and reconstituting the phospholipids of lipoproteins and the phospholipids of membranes. Phospholipases also play a role in releasing arachidonic acid and subsequently forming prostaglandins, leukotriene and other lipids involved in various inflammatory processes.

The lipase is about 450 amino acids in length and has a leader signal peptide that promotes secretion. Lipase consists of two key domains (Winkler, K., D'Arcy, A. and Hunziker, W. (1990) Nature 343, 771-774). The amino terminal domains contain catalytic sites and the carboxy domains are thought to be responsible for substrate binding, cofactor binding and interaction with cellular 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., Davis, R.C., Thuren, T., Goers, J.W., Nikazy, J., Waite, M. and Schotz, M.C. (1994) J. Biol. Chem. 269, 10319-10323; Chappell, D.A., Inoue, I., Fry, G.L., Pladet, M.W., Bowen, S.L., Iverius, P.-H., Lalouel, J.-M. and Strickland, D.K. (1994) J. Biol. Chem. 269, 18001-18006. The overall level of amino acid homology between members of this family is 22-65% and the local region of high homology corresponds to structural homology associated with enzyme function.

Natural lipoprotein lipases are glycosylated. Glycosylation is required for LPL enzyme 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. Liver and lipoprotein lipase has two sites for N-linked glycosylation and one for pancreatic lipase. In addition, four sets of cysteine form disulfide crosslinks that are essential for maintaining structural preservation for enzyme activity [Lo, J.-Y., Smith, L.C. and Chan, L. (1995) Biochem. Biophys. Res. Commun. 206, 266-271; Brady, L., Brzozowski, AM, Derewenda, ZS, Dodson, E., Dodson G., Tolley, S., Turkenburg, JP, Christiansen, L., Huge-Jensen B., Norskov, L., Thim, L and Mengu, U. (1990) Nature 343, 767-770.

Members of the triacylglycerol lipase family share many conserved structural features. One such feature is the "GXSXG" motif, where the central serine residue is one of the three residues that make up the "catalyst 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 remainder of the catalyst triad. A short range of 19 to 23 amino acids ("lead region") forms an amphoteric helical structure and covers the catalytic pocket of the enzyme [Winkler, K., D'Arcy, A. and Hunziker, W. (1990) Nature 343, 771-774. This region varies considerably among members of the lipase family. This range has recently been determined to provide substrate specificity for enzymes [Dugi, K.A., Dichek H.L. and Santamarina-Fojo, S. (1995) J. Biol. Chem. 270 25396-25401. Comparison between liver and lipoprotein lipase demonstrated that the difference in triacylglycerol lipase and phospholipase activity of the enzyme is partially mediated by the lead region [Dugi, K.A., Dichek H.L. and Santamarina-Fojo, S. (1995) J. Biol. Chem. 270, 25396-25401.

Triacylglycerol lipases show heparin binding activity to varying degrees. Lipoprotein lipases have the highest affinity for heparin. This binding activity has been found to be derived from long charge residues extending in the amino terminal domain [Ma, Y., Henderson, HE, Liu, M.-S., Zhang, H., Forsythe, IJ, Clarke-Lewis, I., Hayden, MR and Brunzell, J.D. J. Lipid Res. 35, 2049-2059. Localization of lipoprotein lipase to the endothelial surface [Cheng, C.F., Oosta, G.M., Bensadoun, A. and Rosenberg, R.D. (1981) J. Biol. Chem. 256, 12893-12896] are mediated primarily through binding to surface proteoglycans [Shimada K., Gill, P.J., Silbert, J.E., Douglas, W.H.J. and Fanburg, B.L. (1981) J. Clin. Invest. 68, 995-1002; Saxena, U., Klein, M.G. and Goldberg, I.J. (1991) J. Biol. Chem. 266, 17516-17521; Eisenberg, S., Sehayek, E., Olivecrona, T. and Vlodavsky, I. (1992) J. Clin Invest. 90, 2013-2021]. It is this binding activity that promotes LDL uptake by acting as a bridge between LDL and the cell surface [Mulder, M., Lombardi, P., Jansen, H., van Berkel T.J., Frants R.R. and Havekes, L. M. (1992) Biochem. Biophys. Res. Comm. 185, 582-587; Rutledge, J.C. and Goldberg, I. J., (1994) J. Lipid Res. 35. 1152-1160; Tsuchiya, S., Yamabe, M., Yamaguchi, T., Kobayashi, Y., Konno, T. and Tada, K. (1980) Int. J. Cancer 26, 171-176.

Lipoprotein lipases and pancreatic lipases are both known to act in combination with coactivator proteins (apolipoprotein CII for lipoprotein lipases and collipase for pancreatic lipases).

Gene sequences encoding human pancreatic lipase, liver lipase and lipoprotein lipase are disclosed (Genbank Accession Nos. M93285, J03540 and M15856, respectively). The messenger RNAs of human liver and pancreatic lipases are about 1.7 and 1.8 kilobases each in length. The human lipoprotein lipase gene produces two mRNA transcripts of 3.6 and 3.2 kilobases. These two transcripts utilize another polyadenylation signal and differ in 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.

Physiological Process

Metabolism of lipids includes the interaction of lipids, apoproteins, lipoproteins and enzymes.

Hepatic lipases and lipoproteins Lipases are multifunctional proteins that mediate the binding, absorption, catabolism and reconstitution of lipoproteins and phospholipids. Lipoprotein lipases and hepatic lipases function by binding to the intraluminal surface and liver of endothelial cells in peripheral tissues, respectively. Both enzymes participate in reverse cholesterol transport, which transports cholesterol from peripheral tissues to the liver for secretion or recycling in vitro. Genetic defects in both liver and lipoprotein lipases are known to be responsible for familial diseases of lipoprotein metabolism. Metabolic defects of lipoproteins lead to severe metabolic diseases including hypercholesterolemia, hyperlipidemia and atherosclerosis.

Reported progress

Atherosclerosis is a complex multigenetic disease defined from the histological point of view by the deposition of lipids and other blood derivatives (lipid or fibrolipid plaques) in the vascular wall, especially the macro arteries (aorta, coronary arteries, carotid arteries). Depending on the progress of the atherosclerosis process, more or less calcified plaques can bind the lesion and are associated with the vascular accumulation of fatty deposits consisting essentially of cholesterol esters. These plaques are accompanied by thickening of the vascular wall, hypertrophy of smooth muscle, the appearance of foam cells (cells filled with lipids resulting from unregulated uptake of cholesterol by supplementary macrophages) and accumulation of fibrous tissue. Atheromatous plaques prominently protrude from the wall and impart stenosis to the wall, causing vascular obstruction, thrombosis or embolism by atherosclerosis in most patients. These lesions can cause severe cardiovascular diseases such as infarction, sudden death, heart failure and seizures.

High Density Lipoprotein (HDL) Cholesterol Levels and Atherosclerosis Disease

High-density lipoprotein (HDL) cholesterol levels are inversely proportional to the risk of atherosclerotic cardiovascular disease [Gordon et al., N. Engl. J. Med., 321, 1311-1316 (1989). More than 50% change 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). However, the genes that drive changes in HDL levels are not fully understood. Both members of triacylglycerol (TG), lipoprotein lipase (LPL) and liver lipase (HL), both affect HDL metabolism [Breslow; Murthy et al., Pharmacol. Ther., 70, 101-135 (1996); Goldberg, J. I., J. Lipid Res., 37, 693-707 (1996); Bensadoun et al., Curr. Opin. Lipidol., 7, 77-81 (1996)], the HL (LIPC) locus has been known to be associated with changes in HDL cholesterol levels in humans [Cohen et al., J. Clin. Invest., 94, 2377-2384 (1994); Guerra et al., Proc. Natl. Acad. Sci. USA, 94, 4532-4537 (1997). The normal range of HDL cholesterol is about 35-65 mg / dl and HDL levels should account for at least 25% of total cholesterol.

Very low density lipoprotein (VLDL) and low density lipoprotein (LDL) cholesterol levels and atherosclerosis disease

High levels of circulating LDL and VLDL cholesterol are associated with increased risk of atherosclerosis.

VLDL is a precursor of LDL. Therapies that lower plasma VLDL and LDL cholesterol levels are highly desirable because these lipid variables are very well known to be associated with coronary heart disease.

Epidemiological studies have demonstrated a strong relationship between increased LDL cholesterol and coronary heart disease (CHD) and other atherovascular diseases [Kannell, W.B., Am. J. Cardiol., 76, 69C-77C (1995)]. In three large-scale secondary prevention trials conducted with statins, a decrease in LDL cholesterol levels has been demonstrated to result in a significant decrease in the incidence of CHD and overall mortality [Scandinavian Simvastatin Survival Study Group, Lancet, 344, 1383-1389 (1994); Sacks et al., N. Engl. J. Med., 335, 1001-1009 (1996); Tonkin et al., N. Engl. J. Med., 339, 1349-1357 (1998); Grundy, S.M., Circulation, 1436-1439 (1998). Two large primary prevention trials conducted with statins have also demonstrated that the reduction of LDL cholesterol by statins provides a significant advantage of a reduction in cardiovascular incidence [Grundy; Shepherd et al., N. Engl. J. Med., 333, 1301-1307 (1995); Downs et al., JAMA, 279, 1615-1622 (1998). However, current therapies do not adequately reduce LDL cholesterol levels in all people. VLDL cholesterol levels have also been recognized to be associated with increased risk of CHD [Kannel, supra]. Current therapies do not have as much effect on reducing VLDL cholesterol as LDL cholesterol. Thus, new methods of reducing both LDL and VLDL cholesterol are needed.

Ideally, the range of VLDL cholesterol is about 1 to 30 mg / dL and the range of LDL cholesterol is about 60 to 160 mg / dL. The ratio of LDL to HDL is ideally less than 3.5.

Role of Triacylglycerol Lipase in Atherosclerosis Disease

The role of triacylglycerol lipase in vascular diseases such as atherosclerosis has been an important area of research [Olivecrona, G. and Olivecrona, T. (1995) Curr. Opin. Lipid. 6, 291-305]. In general, the action of lipoprotein lipase is believed to exhibit anti-arteriosclerosis because this enzyme lowers serum triacylglycerol levels and promotes HDL formation. Transgenic animals expressing human lipoprotein lipase reduced plasma triglyceride levels and increased levels of high density lipoprotein (HDL) [Shimada, M., Shimano, H., Gotoda, T., Yamamoto, K., Kawamura , M., Inaba, T., Yazaki, T. and Yamada, N. (1993) J. Biol. Chem. 268, 17924-17929; Liu, M.-S., Jirik, FR, LeBoeuf, RC, Henderson, H., Castellani, LW, Lusis, AJ, Ma, Y., Forsythe, IJ, Zhang, H., Kirk, E., Brunzell, JD and Hayden, M.R. (1994) J. Biol. Chem. 269, 11417-11424. Persons with genetic defects that cause reduced concentrations of lipoprotein lipase activity have been shown to exhibit hypertriglyceridemia but have no increased risk of coronary heart disease. This is reported to be because no medium-sized atherosclerotic lipoproteins are produced that can accumulate in the subcutaneous space [Zilversmit, D.B. (1973) Circ. Res. 33, 633-638.

In contrast to lipoprotein lipase (LPL), the physiological function of HL appears to be related to the metabolism of lipoprotein residues and HDL [Bensadoun et al., Curr. Opin. Lipidol., 7, 77-81 (1996)]. Genetic deficiency of HL has been described by humans [Hegele et al., Arterioscler. Thromb., 13, 720-728 (1993)] and mutant mice [Homanics et al., J. Biol. Chem., 270, 2974-2980 (1995)] and is associated with slightly increased levels of HDL cholesterol. Despite elevated plasma cholesterol levels, HL deficiency is associated with a reduction in atherosclerosis in apoE mutant mice [Mezdour et al., J. Biol. Chem., 272, 13570-13575 (1997). Transgenic animals overexpressing HL had reduced 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 shown to be associated with changes in plasma HDL cholesterol levels in humans [Cohen et al., J. Clin. Invest., 94, 2377-2384 (1984); Guerra et al., Proc. Natl. Acad. Sci. USA, 94 4532-4537 (1997)], which accounts for only a portion of the genetic reasons for changes in HDL cholesterol levels. There is one or more major loci that affect HDL cholesterol levels in humans that are distinct from the HL locus [Mahaney et al., Arterioscler. Thromb. Vasc. Biol., 15, 1730-1739 (1995).

At the localized site of atherosclerotic lesions, increased levels of lipase activity have been hypothesized to promote the atherosclerosis process [Zilversmit, D.B. (1995) Clin. Chem. 41, 153-158; Zambon, A., Torres, A., Bijvoet, S., Gagne, C., Moojani, S., Lupien, P.J., Hayden M.R., and Brunzell, J.D. (1993) Lancet 341, 1119-1121]. This may be due to increased binding and uptake of lipoproteins by lipase-mediated vascular tissues [Eisenberg, S., Sehayek, E., Olivecrona, T. Vlodavsky, I. (1992) J. Clin. Invest. 90, 2013-2021; Tabas, I., Li, I., Brocia R. W., Xu, S. W., Swenson T. L., Williams, K. J. (1993) J. Biol. Chem. 268, 20419-20432; Nordestgaard, B.G. and Nielsen, A.G. (1994) Curr. Opin. Lipid. 5, 252-257; Williams, K.J. and Tabas, I. (1995) Art. Thromb. And Vasc. Biol. 15, 551-561. In addition, local high levels of lipase activity can lead to cytotoxic levels of fatty acids and lysophosphatidylcholine produced as precursors of atherosclerotic lesions.

Despite an understanding of the role of lipase enzyme activity in regulating the levels of lipids and various plasma lipoproteins, not only does it increase the levels of HDL cholesterol, but also lowers the levels of VLDL and LDL cholesterol to reduce the risk of atherosclerotic cardiovascular disease. There is a need to develop therapies that can be reduced.

Summary of the Invention

The present invention provides compositions comprising antisense nucleic acids, including expression vectors comprising antisense nucleic acids, for reducing expression of LIPG genes in a patient. Examples of preferred expression vectors are retroviral vectors, adenovirus vectors, adeno-associated viral vectors, herpesvirus vectors and naked DNA vectors. Antisense nucleic acids can be, for example, oligonucleotides containing chemically modified bases.

In another aspect, the present invention includes an expression vector comprising a DNA sequence encoding a neutralizing antibody, wherein the present invention comprises a neutralizing antibody capable of binding to and decreasing its enzymatic activity and inhibiting the enzymatic activity of the LIPG polypeptide in a patient. Provided is a composition for lowering. Examples of preferred expression vectors include retroviral vectors, adenovirus vectors, adeno-associated viral vectors, herpesvirus vectors and naked DNA vectors.

In another aspect, the invention provides a composition for reducing the enzymatic activity of a LIPG polypeptide in a patient, including the intracellular binding protein, including an expression vector comprising a DNA sequence encoding the intracellular binding protein. Examples of preferred expression vectors include retroviral vectors, adenovirus vectors, adeno-associated viral vectors, herpesvirus vectors and naked DNA vectors.

In another aspect, the present invention provides a composition comprising (A) an inhibitor that can inhibit the enzymatic activity of LIPG polypeptide in a patient; (B) a composition comprising an inhibitor capable of lowering the expression of the LIPG gene in a patient; And (C) an expression vector comprising a DNA sequence that can reduce the expression of LIPG in a patient and that encodes a ribozyme. Examples of preferred expression vectors include retroviral vectors, adenovirus vectors, adeno-associated viral vectors, herpesvirus vectors and naked DNA vectors. Preferred ribozymes are hammerhead ribozymes.

The present invention also provides a composition comprising (D) an increase in the concentration of LIPG polypeptide in a patient and a composition comprising an enhancer capable of increasing the expression of the LIPG gene or an expression vector comprising the DNA sequence encoding the LIPG polypeptide and (E) Provided are compositions comprising enhancers that increase the enzymatic activity of a LIPG polypeptide in a patient and bind to and increase the enzymatic activity of the LIPG polypeptide.

In addition, the present invention, for example, by lowering the concentration of LIPG polypeptides in a patient, administering to the patient a composition that lowers the enzyme activity of LIPG in the patient, thereby increasing the level of high density lipoprotein (HDL) cholesterol and apolipoprotein AI in the patient. It provides a way to increase. In a preferred form, the methods of the present invention comprise the use of compositions comprising antisense nucleic acids, in particular those modified to increase the chemical stability of the nucleic acid. The method also includes a composition comprising a neutralizing antibody that can bind to and degrade its enzymatic activity, an inhibitor that inhibits the enzymatic activity of the LIPG polypeptide, eg, a composition comprising a compound that decreases the expression of the LIPG gene. , A composition comprising a ribozyme that degrades mRNA encoding LIPG or a composition comprising a DNA molecule and a liposome, for example a cationic liposome.

In a preferred form, the method also includes the administration of a composition capable of expressing apolipoprotein AI in the patient.

In another aspect, the invention can increase the enzymatic activity of LIPG in a patient, for example by using a composition comprising a LIPG polypeptide and a pharmaceutically acceptable carrier and preferably express the LIPG polypeptide. A method of lowering the level of ultra low density lipoprotein (VLDL) cholesterol in a patient is provided by administering to the patient a composition comprising an expression vector, more preferably a retroviral vector, an adenovirus vector or an adeno-associated viral vector. The method can be carried out using a composition comprising an enhancer that increases the enzymatic activity of the LIPG polypeptide or an enhancer that increases the expression of the LIPG gene.

In another aspect, the invention preferably uses an expression vector capable of expressing a LIPG polypeptide, for example by using a LIPG polypeptide, preferably a retroviral vector, an adenovirus vector or an adeno-associated viral vector. Thereby providing a method of lowering the level of low density lipoprotein (LDL) cholesterol in a patient by administering to the patient a composition that can increase the enzymatic activity of LIPG in the patient. The method involves the use of a composition comprising an enhancer that increases the enzymatic activity of the LIPG polypeptide or an enhancer that increases the expression of the LIPG gene.

In addition, the present invention lowers the level of LDL cholesterol in a patient by administering to the patient an enhancer that preferentially increases the enzymatic response between the LIPG polypeptide and LDL cholesterol as compared to the enzymatic reaction between the LIPG polypeptide and HDL cholesterol and apolipoprotein AI. It provides a method to make it.

In addition, the present invention lowers the level of VLDL cholesterol in a patient by administering to the patient an enhancer that preferentially increases the enzymatic response between the LIPG polypeptide and VLDL cholesterol as compared to the enzymatic reaction between the LIPG polypeptide and HDL cholesterol and apolipoprotein AI. It provides a method to make it.

In another aspect, the present invention relates to HDL cholesterol and apolipoprotein AI by taking a tissue sample from a patient and measuring the concentration of LIPG polypeptide in the sample, for example, by using blood tissue and using an immunoassay for the measurement. It provides a method of diagnosing predisposition for low levels. In another aspect of the invention, the level of LIPG polypeptide is measured by measuring the level of LIPG mRNA.

In a further aspect, the present invention provides a method for determining the level of HDL cholesterol and apolipoprotein AI in a first sample comprising (A) (1) HDL cholesterol and apolipoprotein AI, (2) LIPG polypeptide and (3) a test compound. (4) HDL cholesterol and apolipoprotein AI and (5) HDL cholesterol and apolipoprotein AI levels in other samples containing LIPG polypeptides, and (B) the first sample is at a higher level than HDL cholesterol. Test compounds for LIPG polypeptides, HDL cholesterol and apolipoproteins, including whether the test compound is effective in inhibiting enzymatic reactions between LIPG polypeptides, HDL cholesterol and apolipoproteins AI by observing It provides a method of determining whether it can inhibit the enzymatic reaction between AIs.

In addition, the present invention provides a method for preparing a level of VLDL cholesterol in a first sample comprising (A) (1) VLDL cholesterol, (2) LIPG polypeptide and (3) test compound, (4) VLDL cholesterol and (5) LIPG Enzymatic reaction between LIPG polypeptide and VLDL cholesterol by comparing the levels of VLDL cholesterol in other samples containing polypeptides and (B) whether the first sample has a lower level of VLDL cholesterol than other samples. Methods for determining whether a test compound can increase the enzymatic reaction between the LIPG polypeptide and the VLDL cholesterol, including confirming that it is effective in increasing the level of LIPG.

In another aspect, the present invention provides a method for determining the level of LDL cholesterol in a first sample comprising (A) (1) LDL cholesterol, (2) LIPG polypeptide, and (3) a test compound, (4) LDL cholesterol and ( 5) by comparing the level of LDL cholesterol in other samples containing the LIPG polypeptide and (B) observing whether the first sample has a lower level of LDL cholesterol than the other samples, the test compound is between LIPG polypeptide and LDL cholesterol. Provided are methods for determining whether a test compound can increase the enzymatic reaction between the LIPG polypeptide and LDL cholesterol, including identifying whether it is effective in increasing the enzymatic response of the enzyme.

1 shows the sequences (SEQ ID NOs: 17-31) of the primers used for the exemplified PCR amplification.

Figure 2 shows the nucleic acid sequence (SEQ ID NO: 1) and putative amino acid sequence (SEQ ID NO: 2) of the differential display RT-PCR product containing the LIPG gene cDNA. Sequences corresponding to the two primers used for amplification are underlined. Stop codons and polyadenylation signals are indicated by boxes. GAATTC motifs and flanking sequences are derived from pCRII vectors from which the product has been cloned.

3 shows the nucleic acid sequence (SEQ ID NO: 3) and putative amino acid sequence (SEQ ID NO: 4) of the 5'RACE extension of LIPG cDNA. Sequences corresponding to the two primers used for amplification are underlined. GAATTC motifs and flanking sequences are derived from pCRII vectors from which the product has been cloned.

4 shows the sequence of cDNA (SEQ ID NO: 7) containing the complete open reading frame of the LIPG gene LLGXL. The starting codon (ATG) and the ending codon (TGA) are boxed. The DraI site (TTTAAA) and SrfI site (GCCCGGGC) used in the construction of expression vectors are underlined.

5 shows the putative amino acid sequence of LLGXL protein (SEQ ID NO: 8). The estimated signal sequence is underlined.

Figure 6 shows the arrangement of protein sequences of members of the triacylglycerol lipase gene family (SEQ ID NOS: 13-15). The shaded residues are identical to the LLGXL protein (SEQ ID NO: 8). The putative amino acid sequence of human LIPG (EL) is described in the upper line and compared with other major members of the TG lipase family, LPL, HL and PL. The same EL residues as in one or more other members of the TG lipase family are shaded with the corresponding residues in the members of the other family. Numbers of amino acids are designated according to convention, beginning with the first residue of the secreted protein. Presumed sites for signal peptide cleavage are indicated by solid lines between amino acid residues. GXSXG lipase motifs containing active serine are indicated by boxes. Amino acids of the catalytic triad are indicated by asterisks. Conserved cysteine is shown in black circles. Potential N-linked glycosylation sites are indicated by arrowheads. The lead region is indicated by a thick line. A gap was introduced in the sequence to maximize the alignment value using the CLUSTAL program.

7 shows northern analysis of LIPG mRNA in THP-1 cells. Cells were stimulated with PMA or PMA and oxidized LDL (PMA + oxLDL). Numbers on the left indicate the location of RNA standards (kilobase units).

Figure 8 shows the results of Northern-blot analysis in comparison with LPL expression of LIPG mRNA in human tissue. Blots containing mRNA from indicated human tissues were incubated with radiolabeled LPL and β-actin (ACTB) probes as described.

9 shows the northern-blot analysis of cultured cell lines. The left panel (lanes 1 to 6) hybridized with the LIPG (EL) probe and the right panel (lanes 7 to 12) hybridized with the LPL probe. Lanes 1 and 7 are non-stimulated HUVECs, lanes 2 and 8 are PMA stimulated HUVECs, lanes 3 and 9 are thrombin-stimulated HUVECs, lanes 4 and 10 are non-stimulated HCAECs, lanes 5 and 11 are PMA-stimulated HCAECs , Lanes 6 and 12 were THP-1 stimulated with PMA.

10 shows the relationship between the sequence of the immunized peptide (SEQ ID NO: 16) and its LLGXL protein sequence. Peptides are shown as shaded boxes. Terminal cysteines were introduced to assist in linking the peptide to the carrier protein.

FIG. 11 shows the results obtained after immunoblot analysis of HUVEC and HCAEC cultures with rabbit anti-EL peptide antiserum. Lane 1 is unconjugated medium, lane 2 is unstimulated HUVEC, lane 3 is PMA stimulated HUVEC, lane 4 is non-stimulated HCAEC, lane 5 is PMA stimulated HCAEC.

Figure 12 shows the results of Western analysis of heparin-sepharose bound proteins in medium in which transiently transfected or mock COS-7 cells were cultured with expression vectors containing cDNA for LLGN or LLGXL. Proteins from PMA-stimulated endothelial cells (HCAEC + PMA) were included for size reference. The numbers on the left indicate the apparent molecular weight of the major immunoreactive protein determined in comparison to protein standards.

Figure 13 shows the arrangement of the sequence between the rabbit LIPG PCR product (RLLG. Sequence, SEQ ID NO: 12) and the corresponding sequence in the rabbit LIPG PCR product and human cDNA (LLG7742A). The same nucleotides are shaded.

FIG. 14 shows phospholipase A activity of human EL-AS, EL and LPL using phosphatidylcholine substrate. To perform the assay, 700 μl of conditioned medium collected from COS-7 cells transiently transfected with pcDNA3.0 / LIPG-AS, LIPG or LPL expression construct was repeated three times for phospholipase activity described below. Analyzed. After incubation at 37 ° C. for 2 hours, the reaction was terminated and the 14C labeled free fatty acid was extracted and counted to determine the amount of free fatty acid produced.

Figure 15 shows triacylglyceride lipase activity of human EL-AS, EL and LPL using triolein substrate. To perform the assay, 700 μl of conditioned medium collected from COS-7 cells transiently transfected with pcDNA3.0 / LIPG-AS, LIPG or LPL expression construct was repeated three times for triglyceride activity as described below. Analyzed. After incubation at 37 ° C. for 2 hours, the reaction was terminated and the 14C labeled free fatty acid was extracted and counted to determine the amount of free fatty acid produced.

FIG. 16 shows hybridization of LIPG probes and LPL probes to genomic DNA from various species.

17 shows expression of LIPG in the liver of wild-type mice 5 days after AdhEL injection. Lane 1, liver from Adnull injected mice; Lane 2, liver from AdhEL injected mice.

Figure 18 depicts plasma levels of HDL cholesterol in AdhEL and Adnull injected wild type mice.

FIG. 19 shows lipoprotein profiles before (left) and 14 days after injection (right) as seen in wild-type mice injected with AdhEL and Adnull.

Figure 20 depicts HDL cholesterol levels seen in human apoA-I mutant mice after injection of Adnull or AdhEL.

Figure 21 depicts ApoA-I levels seen in human apoA-1 mutant mice after injection of Adnull or AdhEL.

FIG. 22 shows the effect on VLDL / LDL cholesterol levels seen after AdhEL injection in LDL receptor deficient mice.

Figure 23 depicts the effect of AdhEL on HDL cholesterol levels on HDL receptor deficient mice.

24 is a Western blot analysis of LIPG (EL) demonstrating the effects of glycosidase and tunicamycin on EL. Lanes represent the following components from left to right: untreated EL control; EndoF-treated EL; EndoH-treated EL; Neuraminidase treated EL; Markers, plasma samples; EL untreated lysate; EL tunicamycin treated lysates; EL untreated pellets; EL tunicamycin treated pellets; EL untreated medium; EL tunicamycin treated medium, markers; plasma.

25 is a Western blot illustrating the effect on EL upon heparin administration. "Pre" lanes are blood samples taken from mice prior to heparin injection; A "post" lane is a blood sample taken after heparin injection. No EL was added to the "control virus"; "EL virus" is an adenovirus vector that expresses EL.

26A and 26B are bar graphs illustrating triglyceridase activity (FIG. 26A) and phospholipase activity (FIG. 26B) of LPL (lipoprotein lipase), HL (liver lipase) and EL (endothelial lipase).

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).

28A and 28B illustrate expression of EL at various viral doses. 28A is a Western blot illustrating EL present at various viral doses. 28B illustrates phospholipase activity at various virus doses.

The following detailed description sets forth the basics and definitions of the present invention. Definition Description The following describes the various compositions useful in the practice of the present invention and the methods used to lower or increase the LIPG activity concentration.

Enzymatic Activity of LIPG Gene Product

The present invention relates to a method of modulating the levels of HDL cholesterol and apolipoprotein AI, VLDL cholesterol and LDL cholesterol using methods and compositions for lowering or elevating the activity of LIPG lipase enzymes. In particular, the present invention is based, in part, on the enzymatic activity of polypeptide products of the LIPG gene against HDL cholesterol and apolipoprotein AI, VLDL cholesterol and LDL cholesterol. The polypeptide product of LIPG is a member of the triacylglycerol lipase family and comprises about 39 kD catalytic domains of the triacylglycerol lipase family, such as the sequence of SEQ ID NO: 10. This first discovered lipase was found to be synthesized by endothelial cells, which is a unique feature when compared to other members of the triacylglycerol lipase family, termed "endothelial lipase" (EL). The LIPG gene will be described fully below in section, and EL is hereinafter referred to as LIPG polypeptide for clarity. In general, LIPG polypeptides are observed in two main forms, referred to hereinafter as "LLGN polypeptide" and "LLGXL polypeptide". The LLGN polypeptide has 354 amino acids. The LLGXL polypeptide shows 43% similarity to human lipoprotein lipase and 37% similarity to human liver lipase and has 500 amino acids. As used herein, “LIPG polypeptide” or “LIPG protein” includes both LLGN and LLGXL.

The sequence of the LIPG polypeptide contains a characteristic GXSXG lipase motif, a conserved catalytic triad, a 19 residue residue region, conserved heparin and lipoprotein binding sites, and five potential N-linked glycosylation sites. Include. The region of greatest sequence divergence in the triacylglycerol lipase family is the lead domain, which forms an amphoteric helix occupying the catalytic pocket of this enzyme [Winkler et al., Nature, 343, 771-774 ( 1990); van Tilbeurgh et al. J. Biol. Chem., 269, 4626-4633 (1994)] confers substrate specificity for enzymes of this system [Dugi et al., J. Biol. Chem., 270, 25396-25401 (1995). The lead region of the 19 residues of LIPG is 3 residues shorter and less amphoteric than those found in lipoprotein lipases and liver lipases, consistent with different enzymatic profiles. The inferred molecular weight of the mature protein is about 55 kD; The 68 kD form is assumed to be a glycosylated form and the 40 kD form may be the product of specific proteolytic cleavage.

LIPG polypeptides have the property of lowering the levels of VLDL cholesterol and LDL cholesterol as well as the levels of HDL cholesterol and apolipoprotein AI. It is well known that lowered levels of HDL cholesterol increase susceptibility to atherosclerosis and increased levels of HDL cholesterol can significantly reduce susceptibility to atherosclerosis.

One physiological role of LIPG is to hydrolyze HDL phospholipids present in peripheral tissues and liver to facilitate selective uptake of HDL cholesteryl esters via HDL receptor SR-BI [Kozarsky et al., Nature , 387, 414-417 (1997). Another possible role is to facilitate the absorption of residual lipoproteins containing apoB, similar to the role of liver lipases (Mahley et al., J. Lipid Res., 40, 1-16 (1999)). In addition, LIPG is expressed in large amounts in the placenta, which may play a role in this enzyme in growth and provides the importance of lipid transport in fetal growth [Farese et al., Trends Genet., 14, 115-120 (1998). ].

Based on the beneficial properties of HDL cholesterol, it is desirable to increase the level of HDL cholesterol by lowering the enzymatic activity of LIPG. Accordingly, the present invention relates to methods and compositions for lowering the activity of LIPG present in the body by lowering the expression of the LIPG gene or lowering the enzymatic activity of the LIPG polypeptide.

It is desirable to lower the level of the compound in a patient if a study is provided that demonstrates the relationship between the properties of LIPG polypeptides that reduce levels of VLDL cholesterol and LDL cholesterol and the high concentrations of the compound and atherosclerosis disease. Accordingly, the present invention provides methods and compositions that increase the expression of the LIPG gene and increase the enzymatic activity of the LIPG polypeptide.

Hereinafter, definitions of terms used in the present specification and description of preferred specific examples of the present invention will be disclosed.

Justice

The following defined terms are used throughout this specification and will help to understand the scope and practice of the present invention.

A "polypeptide" is a polymeric compound consisting of covalently linked amino acid residues. Amino acids are classified into seven groups according to side chains: (1) aliphatic side chains, (2) side chains containing hydroxyl (OH) groups, (3) side chains containing sulfur atoms, (4) acidic groups or amide groups Proline which is a side chain containing (5), a side chain containing a basic group, (6) a side chain containing an aromatic ring, and (7) an imino acid in which the side chain is fused to an amino group.

A "protein" is a polypeptide that plays a structural or functional role in living cells.

The polypeptides and proteins of the invention may or may not be glycosylated.

"Homologousity" means similarity of sequence that reflects the common evolutionary origin. A polypeptide or protein is said to be homologous or similar when a significant number of amino acids have (1) the same or (2) chemically similar side chains. Nucleic acids are said to be homologous if many of the nucleotides are identical.

The term "isolated polypeptide" or "isolated protein" is a polypeptide or protein that is substantially free of compounds (eg, other proteins or polypeptides, nucleic acids, carbohydrates, lipids) that are generally associated in their natural state. "Isolated" does not interfere with artificial or synthetic mixtures with other compounds, or biological activities, and excludes the presence of incomplete tablets, addition of stabilizers or impurities that may be provided in the preparation of pharmaceutically acceptable formulations. It does not mean to make.

Molecules that can specifically interact with antigen recognition molecules of the immune system, such as immunoglobulins (antibodies) or T cell antigen receptors, are "antigenic". Antigenic polypeptides contain at least about 5, preferably at least about 10 amino acids. The antigenic portion of the molecule may be a portion that is immunodominant in antibody or T cell receptor recognition or may be a portion used to generate an antibody against the molecule by conjugating the antigenic portion to an immunizing carrier molecule. Antigenic molecules do not need to be immunogenic to themselves induce an immune response without a carrier.

"LLGN polypeptide" and "LLGN protein" refer to a polypeptide comprising the sequence of SEQ ID NO: 6, which polypeptide may be glycosylated or unglycosylated.

"LLGXL polypeptide" and LLGXL protein "are polypeptides comprising the sequence of SEQ ID NO: 8, and may be glycosylated or non-glycosylated.

"LIPG polypeptide" and "LIPG protein" are lipase enzymes encoded by the LIPG gene and generally refer to both LLGN polypeptides and LLGXL polypeptides.

"Endothelial lipase" or "EL" refers to a lipase enzyme encoded by the LIPG gene and has the same meaning as a LIPG polypeptide.

LIPG polypeptides or proteins of the invention include all analogs, fragments, derivatives or variants derived from LIPG polypeptides and retaining one or more biological properties of the LIPG polypeptide. Different variants of LIPG polypeptides exist in nature. Such variants may be allelic variants with differences in the nucleotide sequence of the structural gene encoding this protein, or may be different splicing or post-translational modifications. One skilled in the art can generate variants with single or multiple amino acid substitutions, deletions, additions or replacements. Such variants include, in particular, (a) variants in which one or more amino acids are replaced with conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to a LIPG polypeptide, (c) one or Variants wherein the more amino acids comprise substituents, 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 an amino acid residue of one species is replaced by a corresponding residue of another species in a conserved or non-conserved position. In another specific example, amino acid residues at non-conservative positions are substituted with conservative or non-conservative residues. Methods of obtaining such variants are well known to those skilled in the art, including genetic techniques (inhibition, deletion, mutations, etc.), chemical techniques, and enzymatic techniques.

The allelic variants, analogs, fragments, derivatives, variants and investigators, including alternative mRNA splicing forms and alternative post-translational modification forms, are derivatives of LIPG polypeptides possessing any biological properties of LIPG polypeptides. It is in the range of.

A "nucleic acid" is a polymeric compound consisting of covalently bonded subunits called nucleotides. Nucleic acids include polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), which may be single or double stranded. DNA includes cDNA, genomic DNA, synthetic DNA and semisynthetic DNA. The sequence of nucleotides encoding a protein is called a sense sequence.

An "antisense nucleic acid" is a nucleotide sequence that is complementary to a sense sequence. Antisense nucleic acids can be used to inhibit or control the expression of polypeptides encoded by the sense chain.

"Isolated nucleic acid" means a nucleic acid with few compounds that are generally bound in their natural state. "Isolated" refers to the presence of artificial or synthetic mixtures with other compounds or impurities that do not interfere with biological activity and which may be provided upon incomplete purification, addition of stabilizers or preparations into pharmaceutically acceptable formulations. It does not mean to exclude.

The term “nucleic acid that hybridizes under high stringent conditions” means that the hybridized nucleic acid can withstand washing under high stringent conditions. Examples of high stringent wash conditions for DNA-DNA hybrids are 0.1X SSC, 0.5% SDS at 68 ° C. Other conditions of high stringent washing are well known in the art.

"Regulatory region" refers to a nucleic acid sequence that controls the expression of a nucleic acid. Regulatory regions may include sequences that play a naturally important role in expressing a particular nucleic acid (homologous region) or sequences of other origin (sequences that play an important role in the expression of other proteins or even synthetic proteins). In particular, the sequence may be a sequence of or derived from a eukaryotic or viral gene that stimulates or inhibits transcription of the gene in a specific or nonspecific manner and in an inducible or non-inductive manner. Regulatory regions include replication origins, RNA splicing sites, enhancers, transcription termination sequences, signal sequences and promoters that direct polypeptides into the secretory pathway of target cells.

The regulatory region from a "heterologous source" is a regulatory region that is not naturally associated with the expressed nucleic acid. Among heterologous regulatory regions include regulatory regions from other species, regulatory regions from other genes, hybrid regulatory sequences, and regulatory sequences that do not occur in nature and are devised by those skilled in the art.

"Vector" is any means of delivering a nucleic acid described herein to a host cell. The term "vector" includes viral means and non-viral means for introducing nucleic acids into prokaryotic or eukaryotic cells in vitro, ex vivo or in vivo. Non-viral means include plasmids, liposomes, electrically charged lipids (cytofectin), DNA-protein complexes, and biopolymers. Viral vectors include retroviruses, adeno-associated viruses, fox, baculovirus, vaccinia, herpes simplex, Epstein Barr and adenovirus vectors. In addition to the nucleic acids described herein, the vector may comprise selectable markers useful for selecting, measuring, and monitoring one or more regulatory regions and / or nucleic acid delivery results (delivery to tissue, duration of expression, etc.).

"Recombinant cell" means a cell comprising a nucleic acid not originally present in the cell. "Recombinant cells" include higher eukaryotic cells such as mammalian cells, lower eukaryotic cells such as yeast cells, prokaryotic cells and primitive bacterial cells.

The term "pharmaceutically acceptable carrier" includes diluents and fillers which are pharmaceutically acceptable for the method of administration, sterile and may be aqueous or oily suspensions prepared using suitable dispersing or wetting agents and suspending agents. do. The specific pharmaceutically acceptable carrier and the ratio of active compound to carrier are determined by the solubility and chemical nature of the composition, the particular mode of administration, and typical pharmaceutical practice.

"Lipase" is a protein capable of enzymatic cleavage of lipid substrates.

A "phospholipase" is a protein capable of enzymatic cleavage of phospholipid structures.

"Triacylglycerol lipase" is a protein capable of enzymatic cleavage of triacylglyceride substrates.

"Phosphatidylcholine" is a glycerol phospholipid, also known as lecithin.

"Lipid profile" means a set of concentrations of cholesterol, triglycerides, lipoprotein cholesterol and other lipids present in the body of a human or other animal.

An "undesired lipid profile" is a condition where the level of cholesterol, triglycerides or lipoprotein cholesterol is outside the reference range adjusted for age and gender. Generally, the concentration of total cholesterol is greater than 200 mg / dL, the concentration of plasma triglycerides is greater than 200 mg / dL, the concentration of LDL cholesterol is greater than 130 mg / dL, the concentration of HDL cholesterol is less than 39 mg / dL or total cholesterol A ratio of greater than 4.0 to HDL cholesterol is considered an undesirable lipid profile. Undesirable lipid profiles are associated with various pathological conditions, including hyperlipidemia, diabetic hypercholesterolemia, atherosclerosis and other forms of coronary artery disease.

A "ribozyme" is an RNA molecule that can act as an enzyme.

A "neutralizing antibody" is an antibody that can bind to a LIPG polypeptide and can reduce or eliminate the enzymatic activity of the LIPG polypeptide. This antibody may be a monoclonal antibody or a polyclonal antibody. The present invention includes chimeric antibodies, single chain antibodies, humanized antibodies as well as products of Fab fragments and Fab expression libraries and products of Fv fragments and Fv expression libraries.

An "inhibitory molecule" or "inhibitor" is a molecule that lowers or eliminates the expression of a LIPG polypeptide or reduces or eliminates the enzymatic activity of a LIPG polypeptide.

An "enhancer molecule" or "enhancer" is a molecule that increases the expression of a LIPG polypeptide or increases the enzymatic activity of a LIPG polypeptide.

"Liposomes" are synthetic or naturally occurring phospholipid vesicles.

A "cationic liposome" is a liposome with a pure positive charge.

In the following, the components used in the methods and compositions claimed in the present invention and preferred specific examples thereof are described.

Polypeptide

The present invention utilizes polypeptides encoded by LIPG that are members of the triacylglycerol lipase family and comprise the 39 kD catalytic domain (eg, having the sequence of SEQ ID NO: 10) of the triacylglycerol lipase family. In some specific examples of the invention, isolated LIPG polypeptides are used that comprise the sequence of SEQ ID NO: 6 and have an apparent molecular weight of about 40 kD on a 10% SDS-PAGE gel. In another specific embodiment of the present invention an isolated LIPG polypeptide comprising the sequence of SEQ ID NO: 8 and having an apparent molecular weight of about 55 kD or 68 kD on a 10% SDS-PAGE gel is used. In another specific example, a polypeptide used in the present invention is a subfragment of said polypeptides. In another specific example, a polypeptide used in the present invention is an antibody capable of binding to a LIPG polypeptide.

The polypeptides and proteins used in the present invention are recombinant polypeptides, natural polypeptides or synthetic polypeptides and may be of human, rabbit or other animal origin. Such polypeptides are characterized by reproducible single molecular weight and / or plurality of molecular weights, chromatographic reaction and elution profiles, amino acid composition and sequence, and biological activity.

Polypeptides for use in the present invention can be isolated according to purification procedures known to those of skill in the art from natural sources such as placental extracts, human plasma or conditioned media derived from cultured cells such as macrophages or endothelial cells.

Alternatively, the polypeptide used in the present invention binds a nucleic acid encoding the polypeptide to a suitable vector, inserts the obtained vector into a suitable host cell, recovers and recovers the polypeptide produced by the obtained host cell. It can be prepared using recombinant DNA techniques comprising the step of purifying the polypeptide.

Nucleic acid

The present invention utilizes isolated nucleic acids encoding LIPG polypeptides.

The present invention also utilizes antisense nucleic acids that can be used to inhibit or control the expression of LIPG polypeptides in vitro, ex vivo or in vivo.

Recombinant DNA techniques are well known to those skilled in the art. General methods for cloning and expressing recombinant molecules are disclosed in Maniatis, Molecular Cloning, Cold Spring Harbor Laboratories, 1982, and Ausubel, Current Protocols in Molecular Biology, Wiley and Sons, 1987.

Nucleic acids of the invention may be associated with one or more regulatory regions. The selection of suitable regulatory regions is a common problem at the level of ordinary skill in the art. Regulatory regions include promoters, and may include enhancers, inhibitors, and the like.

Promoters that may be used in the present invention include constitutive promoters and modulated (inducible) promoters. These promoters may be prokaryotic or eukaryotic, depending on the host. Among the prokaryotic promoters useful in practicing the present invention are the lacI, lacZ, T3, T7, lambda P _r , P _l and trp promoters. Among the eukaryotic (including virus) promoters useful in the practice of the present invention are ubiquitous promoters (eg, HPRT, non-mentin, actin, tubulin), interventional filament promoters (eg, desmin, neurofilament, keratin, GFAP), treatment Enemy gene promoters (e.g., MDR type, CFTR, Factor VIII), tissue specific promoters (e.g., actin promoters in smooth muscle cells or Flt and Flk promoters active in endothelial cells), animals used for mutant animals and exhibiting tissue specificity Transcriptional regulatory regions; Elastase I gene regulatory regions active in acinar cells of the pancreas [Swift et al., 1984, Cell 38: 639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50: 399-409; MacDonald, 1987, Hepatology 7: 425-515; Active insulin gene regulatory region in beta cells of the pancreas [Hanahan, 1985, Nature 315: 115-122], immunoglobulin gene regulatory region active in lymphoid cells [Grosschedl et al., Cell 38: 647-658; Adames et al., 1985, Nature 318: 533-538; Alexander et al., 1987, Mol. Cell. Biol. 7: 1436-1444; Mouse breast cancer virus regulatory region active in testes, breast, lymphatic and adipocytes [Leder et al., 1986, Cell 45: 485-495], active albumin gene regulatory region in liver [Pinkert et al., 1987, Genes and Devel. 1: 268-276], an active α-fetoprotein gene regulatory region in the liver [Krumlauf et al., 1985, Mol. Cell. Biol. 5: 1639-1648; Hammer et al., 1987, Science 235: 53-58, α1-antitrypsin gene regulatory region active in the liver [Kelsey et al., 1987, Genes and Devel. 1: 161-171], an active β-globin gene regulatory region in myeloid cells [Mogram et al., 1985, Nature 315: 338-340; Kollias et al., 1986, Cell 46: 89-94], myelin basic protein gene regulatory regions that are active in hyperplasia neuroglial cells in the brain [Readhead et al., 1987, Cell 48: 703-712], active in skeletal muscle Myosin light chain-2 gene regulatory region [Sani, 1985, Nature 314: 283-286], and gonadotropin releasing hormone gene regulatory region active in the hypothalamus [Mason et al., 1986, Science 234: 1372-1378] It includes.

Other promoters that may be used in embodiments of the present invention include promoters that are primarily activated in dividing cells, promoters that respond to stimulation (eg, steroid hormone receptors, retinoic acid receptors), transcriptional regulators regulated by tetracycline, cytomegalo Viral (CMV) early expression promoter, retrovirus LTR, metallothionein, SV-40, E1a and MLP promoters. The transcriptional regulators and CMV promoters regulated by tetracycline are disclosed in WO 96/01313, US 5,168,062 and 5,385,839, which are incorporated herein by reference.

Virus vector system

The viral vector used in the gene therapy of the present invention is preferably a replication defective type, that is, a vector that cannot autonomously replicate in a target cell. In general, the genome of a replication defective viral vector used within the scope of the present invention lacks one or more regions necessary for replication of the virus in infected cells. This region should be removed (in whole or in part) or imparted non-functionality by any technique known to those skilled in the art. Such techniques include complete removal, substitution (substitution with other sequences, in particular inserted nucleic acids), partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed using in vitro (on isolated DNA) or in situ genetic engineering techniques or by treatment with mutagenesis agents.

Replication-defective viruses preferably have a genomic sequence that is essential for the encapsidation of viral particles.

Retroviruses are integrated viruses that infect dividing cells. The retroviral genome comprises two LTRs, capsidized sequences and three coding regions (gag, pol and env). For the construction of recombinant retroviral vectors, see, in particular, EP 453242, EP 178220, Bernstein et al., Genet. Eng. 7 (1985) 235; McCormick, BioTechnology 3 (1985) 689, and the like. In recombinant retroviral vectors the gag, pol and env genes are generally deleted in whole or in part and replaced with heterologous nucleic acid sequences of interest. This vector can be used for several types of retroviruses, such as MoMuLV ("rat Molecular Leukemia Virus", MSV ("rat Molecular Sarcoma Virus"), HaSV ("Havey Sarcoma Virus"), SNV ("Splenic Necrosis Virus"), RSV) ("Laux Sarcoma Virus") and Friend Viruses.Lentiviral vector systems can also be used in the practice of the present invention.Lentiviral genomes, as in all retroviruses, have three structural genes (gag, pol, env). ) Is a 9000 to 10000 base pair (+)-stranded polyadenylation RNA containing 5 'to 3'. Detailed reports on lentiviral systems are described in Fields Virology, Second Edition, Volume 2, Chapter 55, " Lentiviruses ", pp. 1571-1589, Raven Press, New York, 1990.

In general, to construct a recombinant retrovirus comprising a sequence encoding LIPG, a plasmid comprising an LTR, a capsidized sequence and a coding sequence is constructed. This construct is used to transfect packaging cell lines that can automatically supply the plasmid with missing retroviral function. In general, packaging cell lines can express gag, pol and env genes. Such packaging cell lines are disclosed in the prior art, for example cell line PA317 (US Pat. No. 4,861,719); PsiCRIP cell line [WO 90/02806] and GP ⁺ envAm ^- 12 cell line [WO 89/07150]. In addition, recombinant retroviral vectors may include modifications within the LTR to inhibit transcriptional activity as well as extended encapsulation sequences that may include some gag genes [Bender et al., J. Virol. 61 (1987) 1639. Recombinant retroviral vectors are purified by standard techniques known to those skilled in the art.

Adeno-associated virus (AAV) is a relatively small sized DNA virus that can integrate into the genome of infected cells in a stable and site specific manner. The virus can infect various cells without affecting the growth, morphology or differentiation of the cells and does not appear to be involved in human pathology. The AAV genome has been cloned, sequenced and characterized. An inverted terminal repeat (ITR) region of about 4700 bases and about 145 bases at each end serving as the origin of replication of the virus. The other part of this genome contains two essential regions that have capsidizing functions: the left side of the genome containing the rep gene involved in viral replication and expression of the viral gene and the genome containing the cap gene encoding the capsid protein of the virus It is divided into the right part of.

Vectors derived from AAV have already been disclosed for use in delivering genes in vitro and in vivo [WO 91/18088; WO 93/09239; US 4,797,368, US 5,139,941, EP 488 528]. These publications describe various AAV-derived constructs in which the rep and / or cap genes have been deleted and replaced with the gene of interest and the above for delivering genes of interest in vitro (to cultured cells) or in vivo (directly to the organism). The use of the construct is disclosed. Replication-defective recombinant AAV described herein uses plasmids that contain a plasmid in which the nucleic acid sequence of interest is adjacent to two AAV inverted terminal repeat (ITR) regions and an AAV capsidation gene (rep and cap genes). Cell lines infected with human helper viruses (eg, adenovirus) can be prepared by cotransfection. The AAV recombinants produced are then purified by standard techniques. The present invention also relates to a recombinant virus derived from AAV comprising a sequence encoding a LIPG polypeptide having an adjacent AAV ITR in its genome. The present invention also relates to a plasmid comprising a sequence encoding a LIPG polypeptide in which two ITRs derived from AAV are contiguous. Such plasmids can be used as is to deliver nucleic acid sequences and, if necessary, incorporated into liposome vectors (pseudoviruses).

In a preferred specific example, the vector is an adenovirus vector.

Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver nucleic acids of the invention to a variety of cell types.

Adenoviruses have a variety of serotypes. Of these serotypes, it is preferred to use type 2 or type 5 human adenoviruses (Ad2 or Ad5) or adenoviruses of animal origin (see WO 94/26914) within the scope of the present invention. Adenoviruses of animal origin that can be used within the scope of the present invention include dogs, cattle, mice (eg, Mav1, Beard et al., Virology 75 (1990) 81), sheep, pigs, birds, and monkeys (examples: SAV) adenoviruses of origin. Preferably the adenovirus of animal origin is canine adenovirus, more preferably CAV2 adenovirus (eg, Manhattan or A26 / 61 strain (ATCC VR-800)).

Preferably, the replication defective adenovirus vectors of the invention comprise an ITR, a capsidizing sequence and a nucleic acid of interest. More preferably, at least the El region of the adenovirus vector is nonfunctional. The deletion portion in the El region is preferably nucleotides 455 to 3329 in the sequence of the Ad5 adenovirus. In addition, other regions, in particular the E3 region [WO 95/02697], the E2 region [WO 94/28938], the E4 region [WO 94/28152, WO 94/12649 and WO 95/02697] Or any of the late genes L1 to L5. Defective retroviral vectors are disclosed in WO 95/02697.

In a preferred specific example, the adenovirus vector has deletions in the El and E4 regions. In another preferred embodiment, the adenovirus vector has a deletion in the E4 region and in the E1 region into which the sequence encoding the LLG is inserted (see FR 94 13355, incorporated herein by reference).

Replication-defective recombinant adenoviruses disclosed herein can be prepared by any technique known to those skilled in the art [Levrero et al., Gene 101 (1991) 15, EP 185 573; Graham, EMBO J. 3 (1984) 2917]. In particular, it can be prepared by homologous recombination between the plasmid carrying the DNA sequence of interest and adenovirus. This homologous recombination is carried out after cotransfection of the appropriate cell line with the adenovirus and plasmid. The cell line applied preferably should be able to (i) be transformed with the above components, (ii) complementary to the genome portion of the replication-deficient adenovirus, and preferably in an integrated form to avoid the risk of recombination. Must contain a sequence of phosphorus. Examples of cell lines that can be used include human embryonic kidney cell line 293 [Graham et al., J. Gen. Virol. 36 (1977) 59 and applications (WO 94/26914 and WO 95/02697) are cell lines that may be complementary to El and E4 functions. Recombinant adenoviruses are recovered and purified using standard molecular biological techniques well known to those skilled in the art.

Antisense nucleic acid

Inhibition regulation of gene expression using antisense nucleic acids can be carried out at the translational or transcriptional stage. The antisense nucleic acid of the present invention can specifically hybridize with all or a part of a nucleic acid encoding LIPG or a corresponding messenger RNA. Preferably, the dLT is a nucleic acid fragment. In addition, antisense nucleic acids have been devised or identified that reduce the expression of the LIPG gene by inhibiting splicing of primary transcripts. Such antisense nucleic acids can be prepared and tested for efficacy based on information on the structure and partial sequence of the LIPG gene.

The antisense nucleic acid is preferably an oligonucleotide consisting mostly of deoxyribonucleotides, modified deoxyribonucleotides or any combination of the two. Antisense nucleic acids can be synthetic oligonucleotides. This oligonucleotide can be chemically modified if necessary to improve stability and / or selectivity. Such modifications include, for example, the use of sulfur atoms to replace the free oxygen of the phosphodiester bonds since the oligonucleotides are sensitive to degradation by intracellular nucleases. This modification is also called phosphorothioate linkage. Phosphorothioate antisense oligonucleotides are water soluble, polyanionic and resistant to endogenous nucleases. In addition, when the phosphorothioate antisense oligonucleotide hybridizes to the target site, the RNA-DNA double strand activates the endogenous enzyme ribonuclease H, which cleaves the mRNA component of the hybrid molecule.

In addition, antisense oligonucleotides having phosphoramidite and polyamide (peptide) bonds can also be synthesized. This molecule must be very resistant to nuclease degradation. In addition, chemical groups may be added to the 2 ′ carbon of the sugar residue and to carbon 5 (C-5) of the pyrimidine to improve stability and facilitate binding of antisense oligonucleotides to the target site. Modifications may include 2'deoxy, O-pentoxy, O-propoxy, O-methoxy, fluoro, methoxyethoxy phosphorothioate, modified bases as well as other modifications known to those skilled in the art. Can be.

Antisense nucleic acids may also be DNA sequences that, when expressed in cells, produce RNA that is complementary to all or part of the sequence of LIPG mRNA. Antisense nucleic acids can be prepared by expressing 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, as described in EP 140308. The length of the antisense sequence is suitable for practicing the present invention in any length so long as it can inhibit, control or block the expression of LIPG. Preferably, the antisense sequence is 20 nucleotides in length or more. The preparation and use of antisense nucleic acids, DNA encoding antisense RNA, and the use of oligo and gene antisense are disclosed in WO 92/15680, which is incorporated herein by reference.

One method of determining the optimal fragment of LIPG for use in antisense nucleic acid treatment methods is to prepare random fragments of LIPG cDNA by mechanical shear, enzymatic treatment, and to clone the fragments into any vector system described herein. It includes. Each clone or clone collection is used to infect LIPG expressing cells and monitor LIPG expression at the RNA or protein level to identify effective antisense LIPG cDNA fragments.

The vector systems of the retroviruses, adeno-associated viruses and adenoviruses described above can all be used to introduce and express antisense nucleic acids into cells. Antisense synthetic oligonucleotides can be introduced according to a variety of methods, including those described below.

Ribozyme

Reduction of LIPG polypeptide concentrations can be accomplished using ribozymes. Ribozymes are catalytic RNA molecules (RNA enzymes) each having a catalytic domain and a substrate binding domain. The substrate binding sequence binds by nucleotide complementarity and possibly non-hydrogen binding interaction with the target sequence. The catalytic portion cleaves the target RNA at a specific site. The ribozyme's substrate domain can be made to drive ribozymes to specific mRNA sequences. Ribozymes recognize complementary base pairs and then bind to target mRNAs through the base pairs. When bound to the correct target site, ribozymes act enzymatically to cleave the target mRNA. Cleavage of LIPG mRNA by ribozymes destroys the properties that induce the synthesis of LIPG polypeptides. Ribozymes can be cleaved off the target sequence and then released to repeatedly bind and cleave other LIPG mRNAs.

In a preferred specific example of the invention, the ribozyme is formed in the form of a hammerhead motif. Other forms include hairpin motifs, delta hepatitis virus, group I introns or RnaseP RNA (coupled with an RNA guiding sequence) motif or neurospora VS RNA motif. Hammerhead motifs are disclosed in Rossi et al., 1992, Aids Research and Human Retroviruses, 8, 183. Hairpin motifs are disclosed in Hamel and Tritz, 1989, Biochemistry, 28, 4929, and Hampel et al., 1990, Nucleic Acids Res., 18, 299. For hepatitis delta virus motifs (Perrotta and Been, 1992, Biochemistry, 31, 16), for RnaseP motifs, Guerrier-Takada et al., 1983, Cell, 35, 849, neurospora VS For RNA ribozyme motifs, see 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, 32, 2795-2799, for Group I intron motifs are disclosed in Cech et al., US Pat. No. 4,987,071.

One method of preparing ribozymes is to chemically synthesize oligodeoxyribonucleotides having ribozyme catalytic domains (about 20 nucleotides) next to sequences that hybridize to the target LIPG mRNA after transcription. This oligodeoxyribonucleotide is amplified by using a substrate binding sequence as a primer. The amplification product is cloned into eukaryotic expression vectors.

Ribozymes bearing hammerhead structures or hairpin structures can be readily prepared because these catalytic RNA molecules can be expressed intracellularly by eukaryotic promoters (eg, 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. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992; Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990, Science, 247, 1222-1225. Ribozymes of the invention can be expressed in eukaryotic cells from suitable DNA vectors. If necessary, the second ribozyme can be used to enhance the activity of the ribozyme by dissociating it from the primary transcript [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.

One method of preparing ribozymes is to express ribozymes from transcription units inserted into DNA vectors, RNA vectors or viral vectors. Transcription of ribozyme sequences is induced by a promoter of eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II) or RNA polymerase III (pol III). Transcription by the pol II promoter or polIII promoter is expressed at high concentrations in all cells; The concentration of a given pol II promoter present in a given cell type depends on the contiguous gene regulatory sequence. In addition, a prokaryotic RNA polymerase promoter may be used as long as the prokaryotic RNA polymerase enzyme is expressed in a suitable cell [Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10, 4529-37. Ribozymes expressed from these promoters have been demonstrated to be able to function in mammalian cells [Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992, Proc. Natil. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992 Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8; Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 8000-4.

In one specific example of the invention, a transcription unit expressing a ribozyme that cleaves LIPG RNA is inserted into a plasmid DNA vector, a retroviral vector, an adenovirus DNA viral vector or an adeno-associated virus vector. This recombinant vector is preferably a DNA plasmid or adenovirus vector. However, vectors of other mammalian cells that induce the expression of RNA can also be used in this application. These vectors are delivered as recombinant viral particles. DNA can be delivered alone or in combination with various excipients. DNA, DNA / excipient complexes or recombinant viral particles are topically administered to the treatment site as described below. Recombinant vectors capable of expressing ribozymes are preferably delivered locally and remain in target cells as described below. Once expressed, ribozymes cleave the target LIPG mRNA.

Ribozyme can be administered to a patient in a variety of ways. Ribozymes can be administered directly to the target tissue, administered as complexes with cationic lipids, packaged into liposomes, or delivered to target cells by any other method known in the art. Topical administration to preferred tissues can be carried out using catheters, infusion pumps or stents with or without ribozyme in the biopolymer as described below. Alternative routes of administration include, but are not limited to, intravenous injection, intramuscular injection, transdermal injection, aerosol inhalation, oral (in tablet or pill form), topical, systemic, ophthalmic, intraperitoneal and / or intracapsular delivery. no. A more detailed description of ribozyme delivery and administration is provided in Sullivan et al., PCT WO 94/02595 and Draper et al., PCT WO 93/23569, which are incorporated herein by reference.

Non-virus delivery system

Some non-viral systems have been used in the art, which can easily introduce DNA encoding a LIPG polypeptide or antisense nucleic acid into a patient.

DNA vectors encoding preferred LIPG polypeptides or antisense sequences can be introduced in vivo by lipofection. In the past decade, there has been an increase in the use of liposomes to encapsulate and transfect nucleic acids in vitro. Synthetic cationic lipids designed to limit the difficulties and risks encountered in liposome-mediated transfection can be used to prepare liposomes for in vivo transfection of genes encoding markers [Felgner, et al., Proc. Natl. Acad. Sci. USA. 84: 7413-7417 (1987); Mackey, et al., Proc. Natl. Acad. Sci. USA. 85: 8027-8031 (1988); Ulmer et al., Science 259: 1745-1748 (1993). Cationic lipids used can promote encapsulation of negatively charged nucleic acids and fusion with negatively charged cell membranes (Felgner and Ringold, Nature 337: 387-388 (1989)). Particularly useful lipid compounds and compositions for delivering nucleic acids are disclosed in International Patent Publication Nos. WO 95/18863 and WO 96/17823, and US Pat. No. 5,459,127. Lipofection methods used to introduce exogenous genes into specific organs in vivo have particular practical advantages. One of the advantages is that the liposomes can be molecularly targeted to specific cells. Inducible transfection for certain cell types has been found to be particularly advantageous in tissues with cell heterogeneity, such as the pancreas, liver, kidney and brain. Lipids can be chemically bound to other molecules for targeting (Mackey et al., Supra). Targeted peptides such as hormones or neurotransmitters, and proteins such as antibodies or nonpeptidyl molecules can be chemically bound to liposomes.

In addition, other molecules capable of easily transfecting nucleic acids in vivo, such as cationic oligopeptides (eg International Patent Publication No. WO 95/21931), peptides derived from DNA binding proteins [eg, International Patent Publication No. WO 96/25508 or cationic polymers (eg WO 95/21931) may also be used.

DNA vectors encoding LIPG polypeptides or antisense sequences can also be introduced in vivo as naked DNA plasmids (see US Pat. Nos. 5,693,622, 5,589,466 and 5,580,859). Naked DNA vectors used in gene therapy can be introduced into preferred host cells by methods known in the art. Examples of such methods include transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, the use of gene guns or the use of DNA vector transporters. See, eg, 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 on March 13, 1990; Williams et al., Proc. Natl. Acad. Sci. USA 88: 2726-2730 (1991). Receptor-mediated DNA delivery can also be used [Curiel et al., Hum. Gene Ther. 3: 147-154 (1992); Wu and Wu, J. Biol. Chem. 262: 4429-4432 (1987).

Antibodies

The present invention provides antibodies to LIPG polypeptides. This antibody may be a monoclonal antibody or a polyclonal antibody. The present invention includes chimeric antibodies, single-chain antibodies and humanized antibodies as well as products of Fab fragments and Fab expression libraries, and products of Fv fragments and Fv expression libraries.

Polyclonal antibodies can be prepared for antigenic fragments of LIPG polypeptides as described in the Examples section below. Antibodies may also be generated against native LIPG proteins or polypeptides, or fragments, derivatives or epitopes of these proteins or polypeptides. Antibodies can be obtained after administration of the protein, polypeptide, fragment, derivative or epitope to an animal using techniques and procedures known in the art.

Monoclonal antibodies can be prepared using the methods disclosed in Mishell, B.B., et al., Selected Methods In Cellular Immunology, (W.H. Freeman, ed.) San Francisco (1980). Briefly, splenocytes of Balb / C mice are immunized using the polypeptides of the invention. Immunized spleen cells are fused with myeloma cells. Fusion cells containing the characteristics of splenocytes and myeloma cells are separated by growing in HAT medium, which is a medium in which both parent cells die and survive and grow only the fusion product.

The monoclonal antibodies of the invention may be "humanized" such that the host does not elicit an immune response against the antibody. "Humanized antibodies" refer to complementarity determining regions (CDRs) and / or other parts of the variable domain backbone of the light and / or heavy chains, while the rest are derived from non-human immunoglobulins, while the remaining parts are derived from one or more human immunoglobulins. It is. Humanized antibodies also include antibodies characterized by a humanized heavy chain that is associated with an unmodified light chain or chimeric light chain of a donor or receptor or vice versa. Humanization of antibodies can be accomplished according to methods known in the art [eg, G.E. Mark and E.A. Padlan, "Chapter 4. Humanization of Monoclonal Antibodies", The Handbook of Experimental Pharmacology Vol. 113, Springer-Verlag, New York, 1994]. In addition, transformed animals can be used to express humanized antibodies.

Techniques for producing single chain antibodies known in the art can be modified to produce single chain antibodies against immunogenic polypeptides and proteins of the invention.

In a preferred specific example, anti-LIPG antibodies are used to bind to LIPG in patients and inhibit the enzymatic activity of LIPG.

In addition, anti-LIPG antibodies are useful in assays that measure or quantify the concentration of LIPG. As one specific example, this assay can be used in methods for clinical diagnosis and evaluation of LIPG under various disease states, and for monitoring treatment efficacy. In addition, the anti-LIPG antibodies can be used to quantify LIPG in tissue samples to predict sensitivity to lowered levels of HDL cholesterol and apolipoprotein AI.

How to identify and use inhibitory and enhancer molecules

The present invention provides methods for selecting natural product sources or small molecule libraries of enhancers (agonists) or co-activators, including proteinaceous co-activators or inhibitors (antagonists) of LIPG activity. Potential enhancers or inhibitors are contacted with the LIPG protein and the substrate of the LIPG and the nature of the potential enhancer or inhibitor that enhances or inhibits LIPG activity is measured.

In addition, this screening method allows the compound to act as a substrate specific enhancer or inhibitor, i.e., the compound can enhance the enzymatic activity of LIPG against one substrate while lowering some level of enzymatic activity against another substrate, For example, the LIPG polypeptides of the present invention utilize HDL cholesterol as a substrate and also LDL cholesterol and VLDL cholesterol as substrates. In some specific examples, it may be desirable to isolate and identify substrate specific enhancers or inhibitors that enhance or enhance the enzymatic activity of LIPG polypeptides against LDL cholesterol or VLDL cholesterol while maintaining or decreasing normal levels of enzyme activity against HDL cholesterol. have.

LIPG proteins 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 through methotrexate amplification of these cells. In addition, LIPG proteins can be purified from natural sources such as conditioned media from human plasma, placental extract or endothelial cells, THP-1 cells or macrophage cultures.

Parameters of the assay, such as pH, ion concentration, temperature, substrate concentration and emulsification conditions, etc. can be empirically optimized by those skilled in the art.

Fatty acid substituents of the substrate may vary in length as well as in unsaturated and unsaturated positions. This substrate may be radiolabeled at any of several positions. Phospholipids such as phosphatidylcholine can be radiolabeled, for example, at the Sn-1 or Sn-2 fatty acid position or at glycerol, phosphate or polar head groups (choline in the case of phosphatidylcholine).

As an alternative to radiolabeled substrates, other classes of labeled substrates, such as fluorescent substrates or thio containing substrates, may be used in the selection method.

Fluorescent substrates are particularly useful in screening assays because they can continuously measure the catalysis of enzymes by measuring the fluorescence intensity without physically separating (extracting) the product from the substrate. An example of a fluorescent phosphatidylcholine substrate is C ₆ NBD-PC {1-acyl-2- [6- (nitro-2,1,3-benzoxadiazol-4-yl) amino] caproylphosphatidylcholine.

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.

In addition to inhibitory and enhancer molecules that act at enzymatically active concentrations, there are inhibitory and enhancer molecules that act at the expression concentration of the LIPG gene. One way to identify compounds that can enhance or inhibit the expression of LIPG is to use the reporter gene system. This system utilizes reporter gene expression vectors comprising a cloning site at which a given promoter can be cloned upstream of a “reporter gene” that can be easily detected and quantified. Those skilled in the art will be able to readily identify the promoter of the LIPG gene as well as other regulatory sequences and subclon it into a commercially available reporter gene expression vector. This expression vector is delivered to a host cell and the cell is exposed to the test compound (presumed inhibitor or enhancer molecule) to determine the effect of the test compound on the expression of the reporter gene product. Specifically, cells are analyzed for the presence of reporter gene product by directly measuring the amount of reporter mRNA, the amount of reporter protein or the enzymatic activity of the reporter protein. Reporter genes are not expressed endogenously in the cell type of interest and are ideally provided for sensitive quantitative rapid assays. Various reporter assay constructs are commercially available, various reporter genes and assays have been developed and can also be readily prepared by those skilled in the art. The most common systems for monitoring gene activity in eukaryotic cells include chloramphenicol acetyltransferase (CAT), β-galactosidase, firefly luciferase, growth hormone (GH), and β-glucururodase (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.

As mentioned above, reporter gene activity can be measured by analyzing reporter mRNA or reporter protein. Reporter mRNA can be measured by Northern blot analysis, ribonuclease blocking assay or RT-PCR. These assays are more direct than the measurement of protein expression. In contrast, many assays have been developed to measure the presence of reporter proteins rather than mRNA present in cells. Reporter proteins can be analyzed by spectroscopy or by measuring enzymatic activity. The concentration of reporter protein can also be determined by assays based on antibodies. In general, enzymatic assays are very sensitive and are a preferred method for monitoring reporter gene expression.

Composition

The present invention provides a composition comprising a polypeptide, nucleic acid, vector and antibody of the present invention as a biologically compatible (biocompatible) solution. Biologically compatible solutions are solutions in which the polypeptide, nucleic acid, vector or antibody of the invention is maintained in an active form, such as a form capable of exhibiting biological activity. For example, the polypeptide of the invention must have phospholipase activity, the nucleic acid must be able to replicate, decode the message or hybridize to complementary nucleic acid, the vector must be able to transfect the target cell, The antibody should be able to bind the polypeptide of the present invention. Generally, such biologically compatible solutions are aqueous buffers containing salt ions such as Tris, Phosphate or HEPES buffers. Typically, the concentration of salt ions is similar to the physiological level. In certain specific examples, the biocompatible solution is a pharmaceutically acceptable composition. Biologically compatible solutions may include stabilizers and preservatives.

Such compositions can be formulated for administration by topical, oral, parenteral, intranasal, transdermal and intraocular routes. Parenteral administration means intravenous injection, intramuscular injection, intraarterial injection or infusion technique. The compositions may be administered parenterally, if necessary, in unit doses of formulations containing known standard nontoxic physiologically acceptable carriers, adjuvants and excipients.

Preferred sterile injectables can be solutions or suspensions in nontoxic parenterally acceptable solvents or diluents. Examples of pharmaceutically acceptable carriers include saline, buffered saline, isotonic saline (e.g. monosodium phosphate or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride or mixtures of these salts), Ringer's solution, dextrose, water, Sterile water, glycerol, ethanol and combinations thereof. 1,3-butanediol and sterile immobilized oils are conveniently used as a solvent or suspending medium. Immobilized oils of all trade names can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid are also useful for the preparation of injectables.

In addition, the medium of the composition may be a hydrogel made from any biocompatible or noncytotoxic (homo or hetero) polymer, such as a hydrophilic polyacrylic acid polymer that can act as a drug absorbing sponge. Such polymers are disclosed, for example, in WO 93/08845, the entire contents of which are incorporated herein by reference. Some of these, such as in particular polymers obtained from ethylene oxide and / or propylene oxide, are commercially available. The hydrogel can be deposited directly on the surface of the tissue to be treated, for example during surgical procedures.

Another preferred specific example of the invention relates to pharmaceutical compositions containing replication defective recombinant virus and poloxamer. More specifically, the present invention relates to a composition comprising a replication defective recombinant virus and poloxamer comprising a nucleic acid encoding a LIPG polypeptide. Preferred poloxamers are commercially available from BASF, Parsippany, NJ, non-toxic biocompatible polyols and most preferred is Poloxamer 407. Poloxamers impregnated with the recombinant virus can be deposited directly on the surface of the tissue to be treated, for example, during surgical procedures. Poloxamers provide almost the same advantages as hydrogels with lower viscosity.

How to treat

The present invention provides a method of treatment comprising administering to a human or other animal an effective amount of a composition disclosed herein.

The effective amount may vary depending on age, type and extent of disease to be treated, weight, desired treatment duration, method of administration and other variables. The effective amount is determined by the attending physician or other qualified physician. Most doses can be adjusted to be maintained after the desired level of HDL cholesterol and apolipoprotein AI is achieved. Similarly, the dosage can be adjusted to lower the levels of VLDL cholesterol and LDL cholesterol to an acceptable level and to bring the ratio of HDL cholesterol to LDL cholesterol and VLDL cholesterol to a desired range.

The polypeptides disclosed herein generally range from about 0.01 mg / kg body weight to about 100 mg / kg, preferably from about 0.1 mg / kg to about 50 mg / kg, most preferably about 1 mg / kg per day. To a dose of about 10 mg / kg.

The neutralizing antibodies disclosed in the present invention may be delivered only as a pill, infused for a certain time, or administered as a pill and an infusion for a certain time. Although the dosage depends on the variables mentioned above, based on the binding of the antibody, it can be administered as a pill, with a dosage of 0.2 to 0.6 mg / kg, followed by an infusion time of 2 to 12 hours. Alternatively, a plurality of pill injections may be administered every other day or every three or four days if necessary. Dosage may be adjusted depending on the level of HDL cholesterol and / or the levels of VLDL and LDL cholesterol.

As mentioned above, recombinant viruses can be used to introduce DNA and antisense nucleic acids encoding LIPG and subfractions of LIPG. Recombinant virus according to the present invention is generally administered in a dosage form of about 10 ⁴ to about 10 ¹⁴ pfu. Preference is given to using doses of about 10 ⁶ to about 10 ¹¹ pfu for AAV and adenovirus. The term pfu (“plaque forming unit”) corresponds to the infectivity of virion suspensions and is determined by measuring the number of plaques formed after infecting a suitable cell culture. Techniques for measuring the pfu titer of viral solutions are well described in the prior art.

Ribozymes disclosed herein may be administered in an amount ranging from about 5 to about 50 mg / kg / day in a pharmaceutically acceptable carrier. Dosage may be adjusted depending on the therapeutic efficacy measured.

Appropriate concentrations of inhibitory or enhancer molecules can be determined using the parameters described above by a qualified physician.

The present invention provides compositions and methods that increase the levels of HDL cholesterol and apolipoprotein AI in patients and lower the levels of VLDL and LDL cholesterol. The present invention also provides a method for treating a human or other animal in which the LIPG polypeptide activity is abnormally high such that the lipid profile is undesirable.

Methods and compositions for lowering LIPG polypeptide activity levels

Antisense nucleic acids may be included to increase the levels of HDL cholesterol and apolipoprotein AI and to reduce the expression of LIPG polypeptides in order to treat diseases or disorders associated with undesirable lipid profiles due to LIPG polypeptide activity. A method of administering a composition, a method of administering a composition containing an intracellular binding protein such as an antibody, an inhibitory molecule that inhibits the enzymatic activity of LIPG, such as an LLGN polypeptide or a small molecule molecule that encodes a subfragment of LIPG There is a method of administering a composition comprising an expression vector, such as a method of administering a small molecular weight compound that inhibits and regulates LIPG expression at the transcriptional, translational or posttranslational stage, and a method of administering ribozyme cleaving mRNA encoding LIPG.

How to Use Antisense Nucleic Acids

As one specific example, a composition containing an antisense nucleic acid is used to block or inhibit the expression of LIPG. In one preferred embodiment, this nucleic acid encodes an antisense RNA molecule. In this specific example, the nucleic acid is operably linked to a signal capable of expressing the nucleic acid sequence and is preferably introduced into the cell using a recombinant vector construct, which vector construct is introduced into the cell. When introduced it expresses antisense nucleic acid. Examples of suitable vectors are plasmids, adenoviruses, adeno-associated viruses, retroviruses and herpesviruses. The vector is preferably adenovirus. Most preferably, the vector is a replication defective adenovirus containing deletions in the E1 and / or E3 regions of the virus.

In another specific example, the antisense nucleic acid may be synthesized and then chemically modified to be resistant to degradation by intracellular nucleases as described above. The synthesized antisense oligonucleotides can be introduced into cells using liposomes. Antisense oligonucleotides are taken up by cells when encapsulated in liposomes. Effective delivery systems allow the use of low concentrations of nontoxic antisense molecules to inhibit translation of target mRNA. In addition, liposomes conjugated to cell specific binding sites induce antisense oligonucleotides into specific tissues.

How to use neutralizing antibodies and other binding proteins

In another specific example, expression of LIPG is blocked or inhibited by expressing a nucleic acid sequence encoding an intracellular binding protein that can selectively interact with LIPG. WO 94/29446 and WO 94/02610, incorporated herein by reference, discloses transfection of cells with genes encoding intracellular binding proteins. Intracellular binding proteins include all proteins capable of neutralizing the function of bound LLG by selectively interacting or binding to LIPG in the cell in which it is expressed. Intracellular binding proteins are preferably neutralizing antibodies or fragments of neutralizing antibodies. More preferably, the intracellular binding protein is a single chain antibody.

WO 94/02610 discloses methods for preparing antibodies and methods for identifying nucleic acids encoding specific antibodies. Thus, certain monoclonal antibodies are prepared by techniques known in the art using LIPG or fragments thereof. Next, a vector containing a nucleic acid encoding an intracellular binding protein or part thereof and that can be expressed in a host cell is prepared and used in the method of the present invention.

Alternatively, administration of neutralizing antibodies into the circulation can block LIPG activity. Such neutralizing antibodies can be administered directly as a protein or expressed from a vector (retaining secretion signal).

Methods of using inhibitory molecules that inhibit the enzymatic activity of LIPG

In another specific example, a composition containing a subfragment of the LIPG polypeptide, such as LLGN, is administered to inhibit LIPG activity. The composition can be administered by convenient methods such as oral, topical, intravenous, intraperitoneal, intramuscular, transdermal, intranasal or intradermal routes. The composition may be administered directly or encapsulated (eg, in a lipid system, in amino acid microspheres or in spherical dendrimers). In some cases, the polypeptide may be attached to other polymers, such as serum albumin or polyvinyl pyrrolidone.

In another specific example, LIPG activity is inhibited through the use of gene therapy, ie by administering a composition comprising a nucleic acid encoding a subfragment of LIPG, such as LLGN, and inducing its expression.

In another specific example, LIPG activity is inhibited by using inhibitory molecules. These low molecular weight compounds interfere with the enzymatic properties of LIPG or prevent proper recognition of cell binding sites.

In certain specific examples, the LIPG polypeptides of the invention also have affinity for heparin. Thus, LIPG polypeptides can bind to extracellular heparin in the lumen of blood vessels and act as a bridge between LDL and extracellular heparin to accelerate LDL uptake. Localized areas of atherosclerotic lesions are thought to increase the concentration of lipase activity, thus accelerating the process of atherosclerosis [Zilversmit, D.B. (1995) Clin. Chem. 41, 153-158; Zambon, A., Torres, A., Bijvoet, S., Gagne, C., Moojani, S., Lupien, P.J., Hayden M.R., and Brunzell, J.D. (1993) Lancet 341, 1119-1121. This may be due to increased binding and uptake of lipoproteins into lipase-mediated vascular tissues [Eisenberg, S., Sehayek, E., Olivecrona, T. Vlodavsky, I. (1992) J. Clin. Invest. 90, 2013-2021; Tabas, I., Li. I., Brocia R. W., Xu, S. W., Swenson T. L. Williams, K.J. (1993) J. Biol. Chem. 268, 20419-20432; Nordestgaard, B.G. and Nielsen, A.G. (1994) Curr. Opin. Lipid. 5, 252-257; Williams, K.J., and Tabas, I. (1995) Art. Thromb. and Vasc. Biol. 15, 551-561. In addition, localized high levels of lipase activity produce cytotoxic concentrations of fatty acids and lysophosphatidylcholine produced in precursors of atherosclerotic lesions. Particular activity of these LLGs can provide excessive lipid concentrations in the subject, particularly due to dietary or genetic factors, to develop or progress atherosclerosis. Thus, the present invention can inhibit lipoprotein accumulation by inhibiting the expression of LIPG polypeptides or inhibiting binding to lipoproteins (eg, LDL).

Methods of using inhibitory molecules that block LIPG gene expression

In another specific example, inhibitory molecules, including small molecular weight compounds, can inhibit LIPG expression at the transcriptional, translational or posttranslational stages. The reporter gene system described above can be used to identify such inhibitory molecules. This inhibitory molecule may be mixed with a pharmaceutically acceptable carrier and administered according to conventional methods known in the art.

How to use Ribozyme

Ribozymes can be administered to cells via encapsulation in liposomes, iontophoresis, hydrogels, cyclodextrins, biodegradable nanocapsules and incorporation into bioadhesive microspheres, or various other methods described above. Ribozyme may be injected directly or delivered to target tissue using a catheter, infusion pump or stent. Alternative routes of delivery include intravenous injection, intramuscular injection, transdermal injection, aerosol inhalation, oral (in tablet or pill form), topical, systemic, ophthalmic, intraperitoneal and / or intravesicular delivery.

As a preferred specific example, the ribozyme coding sequence is cloned into a DNA expression vector. Transcription of ribozyme sequences is induced by eukaryotic RNA polymerase II (pol II) or RNA polymerase III (pol III) promoters. Expression vectors can be incorporated into a variety of vectors, including viral DNA vectors such as the adenovirus or adeno-associated virus vectors described above.

In a preferred embodiment of the invention, a transcription unit expressing a ribozyme that cleaves LIPG RNA is inserted into an adenovirus DNA viral vector. This vector is delivered as recombinant viral particles and topically administered to the treatment site via a catheter, stent or infusion pump.

Administration of Apolipoprotein AI

In another specific example, the aforementioned methods of lowering LIPG polypeptide activity levels are used in conjunction with administration of apolipoprotein AI or an expression system capable of expressing apolipoprotein AI in a patient (eg, incorporated herein by reference). See US Pat. No. 5,866,551].

Methods and compositions for increasing LIPG polypeptide activity concentration

Methods for increasing the expression or activity of LIPG polypeptides to lower the levels of VLDL and LDL cholesterol include administration of compositions containing LIPG polypeptides, administration of compositions containing expression vectors encoding LIPG polypeptides, LIPG poly Administration of a composition containing an enhancer molecule that increases the enzymatic activity of the peptide and administration of an enhancer molecule that increases expression of the LIPG gene.

How to Use LIPG Polypeptides

In one specific example, LIPG activity levels are increased through administration of a composition comprising a LIPG polypeptide. The composition can be administered by conventional methods such as oral, topical, intravenous, intraperitoneal, intramuscular, transdermal, intranasal or intradermal routes. The composition may be administered directly or encapsulated (eg in a lipid system, in amino acid microspheres or in a spherical dendrimer). In some cases, the polypeptide may be attached to other polymers, such as serum albumin or polyvinyl pyrrolidone.

How to use vectors expressing LIPG

In another specific example, the concentration of LIPG is increased through the use of gene therapy, i.e., through the administration of a composition containing a nucleic acid encoding and directing expression of the LIPG polypeptide. In this embodiment, the LIPG polypeptide is cloned into a suitable expression vector. The vector systems and promoters that can be used are as described above sufficiently. Expression vectors are delivered to target tissue using one of the vector delivery systems described above. Such delivery may be via an in vitro method of delivering a nucleic acid to a cell in a laboratory and then administering the modified cell to a human or other animal, or an in vivo method of directly delivering a nucleic acid to a cell in a human or other animal. As a preferred specific example, it is preferable to use an adenovirus vector system for delivery of expression vectors. If desired, tissue specific promoters may be used in the expression vectors described above.

Non-viral vectors are known in the art, including calcium phosphate co-precipitation, lipofection (synthetic anion and cationic liposomes), receptor mediated gene transfer, naked DNA injection, electroporation and bioballistic or particle acceleration. Any method can be used to deliver to the cell.

Methods of using enhancer molecules to increase the enzymatic activity of LIPG

In another specific example, the activity of LIPG is increased by enhancer molecules that increase the enzymatic activity of LIPG or increase appropriate recognition of the cell binding site. This enhancer molecule can be introduced in the same manner as described above for administration of the polypeptide.

How to use enhancer molecules to increase LIPG gene expression

In another specific example, the level of LIPG is increased through the use of small molecular weight compounds capable of upregulating LIPG expression at the transcriptional, translational or posttranslational stages. This compound may be administered by a method as described above for administration of the polypeptide.

Methods of treatment associated with impaired bile secretion

Hepatic cholestasis may be characterized by an increase in serum cholesterol and phospholipid levels. A recently published paloidine drug-induced hepatic cholestatic rat model demonstrated a significant increase in serum and cholesterol levels of phospholipids [Ishizaki, K., Kinbara, S., Miyazawa, N., Takeuchi, Y., Hirabayashi, N., Kasai, H., and Araki, T. (1997) Toxicol. Letters 90, 29-34. The products of the present invention can be used to treat hepatic cholestasis in patients with elevated serum cholesterol and / or phospholipids. In addition, this rat model showed a significant decrease in the rate of bile cholesterol secretion. LIPG polypeptides and nucleic acid products of the invention can be used to treat patients with an impaired bile secretion system.

Intrahepatic cholestasis is also characterized by impaired bile supply from the liver. Recently, loci of advanced familial hepatic cholestatic (PFIC or Byler disease) and benign recurrent hepatic cholestatic (BRIC) have been mapped to 18q21-q22 [Carlton, VEH, Knisely, AS, and Freimer, respectively] NB (1995) Hum. Mol. Genet. 4, 1049-1053; And Houwen, R.H., Baharloo, S., Blankenship, K., Raeymaekers, P., Juyn, J., Sandkuiji, L.A., and Freimer, N.B. (1994) Nature Genet. 8, 380-386. Thus, if the LLG gene is mapped in the chromosomal region of 18q21, the LLG gene or product of the invention can be used to treat patients suffering from intrahepatic cholestasis caused by mutation or deletion expression of the PFIC / BRIC disease gene.

In another specific example, the LLG gene or polypeptide product of the present invention may be used to treat patients suffering from hepatic cholestasis but not due to deficiencies in the PFIC / BRIC disease gene of 18q21-q22. Recent studies have suggested that other loci located outside the 18q21-q22 region can also yield a PFIC phenotype [Strautnieks, S.S., Kagalwalla, A.F., Tanner, M.S., Gardiner, R.M., and Thompson, R.J. (1996) J. Med. Genet. 33, 833-836. Nevertheless, direct administration of LLG polypeptides or through gene therapy can alleviate this form of disease.

Methods and compositions for diagnosing predisposition to low HDL levels

If the LIPG polypeptide has the property of lowering the levels of HDL cholesterol and apolipoprotein AI, the concentration of LIPG polypeptide in the body can be used to determine patients with low levels of HDL cholesterol and apolipoprotein AI. Specifically, a tissue sample is first taken from the patient. This tissue sample may be blood or one of the tissues that has been demonstrated to express LIPG as described through the Examples below. The determination of the LIPG concentration can be carried out by various methods known to those skilled in the art. As a preferred specific example, antibodies raised against LIPG polypeptides can be used to determine the concentration of LIPG in tissue samples.

The following examples illustrate the invention. This embodiment is illustrative only and is not intended to limit the scope of the invention.

Example 1 Identification of Differential Expressed cDNAs

RNA manufacturing

Human monocyte THP-1 cells [Smith, PK, Krohn, RI, Hermanson, GT, Mallia, AK, Gartner, FH Provenzano, MD, Fujimoto, EK, Goeke, NM, Olson, BJ and Klenk, DC (1985) Anal. Biochem. 150, 76-85] were cultured in RPMI-1640 medium (GIBCO) containing 25 mM HEPES, 10% fetal calf serum, 100 units / ml penicillin G sodium and 100 units / ml streptomycin sulfate. Cells were seeded in a 15 cm tissue culture dish at a concentration of 1.5 x 10 ⁷ cells / plate and treated with 40 ng / ml phorbol 12-myristate 13-acetate (Sigma) for 48 hours to induce cell differentiation. It was. Human low density lipoprotein (LDL) was purchased from Calbiochem and thoroughly dialyzed against PBS at 4 ° C. The LDL was then diluted to 500 μg / ml and dialyzed for 16 h at 37 ° C. against 5 μM CuSO ₄ in PBS. To stop oxidation, the LDL was thoroughly dialyzed against 150 mM NaCl, 0.3 mM EDTA and then filtered sterilized. Protein concentrations were determined by the BCA method [Schuh, J. Fairclough, GF, and Haschemeyer, RH (1978) Proc. Natl. Acad. Sci. USA 75, 3173-3177 (manufactured by Pierce). The degree of oxidation was between 25 and 30 nmol MDA equivalent / mg protein as measured by TBARS [Chomczynski, P. (1993) Biotechniques 15, 532-537]. Differentiated THP-1 cells were exposed to 50 μg / ml oxidized LDL or NaCl-EDTA buffer in RPMI medium with 10% lipoprotein-defected fetal calf serum (Sigma) for 24 hours. The plates were washed with 10 ml of PBS to collect RNA, and 14 ml of Trizol (Liang, P. and Pardee, AB (1992) Science 257, 967-971) (GIBCO) was prepared in each plate. Added. After the solution was mixed by pipetting several times, the same sample was stored in a centrifuge tube and 3 ml of chloroform per plate was added and mixed. The tube was centrifuged at 12000 xg for 15 minutes. After centrifugation, the supernatant was transferred to a new tube, 7.5 ml of isopropanol was added per plate and mixed. This tube was again centrifuged at 12000 xg for 20 minutes. The pellet was washed with ice-cold 70% ethanol and dried at room temperature. The pellet was suspended in 500 μl TE (Tris-EDTA) and treated with 200 units of RNase-depleted DNAseI and 200 units of RNasin placental RNase inhibitor (Promega) for 30 minutes at 37 ° C. RNA was purified by continuous extraction with phenol, phenol / chloroform / isoamyl alcohol (25: 24: 1) and chloroform / isoamyl alcohol (24: 1), followed by ethanol precipitation.

cDNA synthesis

cDNA synthesis and PCR amplification was performed using the protocol of the Differential Display Kit (Version 1.0, Display Systems Biotechnology, Inc.). This system was originally based on a technique disclosed by Liang and Paddy [Mead, DA, Pey, NK, Herrnstadt, C., Marcil, RA, and Smith, LM, (1991) Bio / Technology 9, 657-663]. will be. The primer pairs yielding cDNA fragments containing the first information of the lipase like gene were downstream primer 7 and upstream primer 15. CDNA for amplification was synthesized using RNA from PMA treated THP-1 cells exposed to buffer or oxidized LDL as follows: 3 μl of 25 μM downstream primer 7, and diethylpyrocarbonate (DEPC) -treated 7.5 μl of water was added to 300 ng (3.0 μl) of RNA from any of the above THP-1 RNA samples. It was heated to 70 ° C. for 10 minutes and then cooled on ice. This tube was treated with 3 μl of 5 × PCR buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl) (GIBCO), 3 μl 25 mM MgCl _2, 3 μl 0.1 M DTT, 1.2 μl 500 μM dNTP, 0.7 μl RNasin, and DEPC treated. 5.6 μl of water was added. The tube was incubated for 2 minutes at room temperature and then 1.5 μl (300 units) of Superscript II RNase H-reverse transcriptase (GIBCO) was added. The tube was incubated for 2 minutes at room temperature, 60 minutes at 37 ° C. and 5 minutes at 95 ° C. and then cooled on ice. PCR amplification was performed with 117 μl of 10 × PCR buffer (500 mM KCl, 100 mM Tris-HCl pH 8.3, 15 mM MgCl ₂ and 0.01% (w / v) gelatin), 70.2 μl 25 mM MgCl ₂ , α- ³² P dATP (10 m Ci / ml, Made from Main mixture containing 5.9 μl of DuPont NEN), 4.7 μl of 500 μM dNTP mixture, 11 μl of AmpliTaq DNA polymerase (5 units / μl, Perkin-Elmer) and 493.3 μl of DEPC treated water. For each reaction, 12 μl of the main mixture was added to 2 μl of downstream primer # 7, 1 μl of cDNA and 5 μl of upstream primer # 15. The reaction mixture was heated to 94 ° C. for 1 minute and then heat-circulated 40 times in a denaturation step at 94 ° C. for 15 seconds, annealing step at 40 ° C. for 1 minute, and elongation step at 72 ° C. for 30 seconds. After 40 cycles, the reaction was incubated at 72 ° C. for 15 minutes and then stored at 10 ° C. PCR reactions were carried out using a GeneAmp System 9600 thermocycler manufactured by Perkin-Elmer.

4 μl of amplification reaction was mixed with an equal amount of loading buffer (0.2% bromophenol blue, 0.2% xylene cyanol, 10 mM EDTA pH 8.0 and 20% glycerol). 4 μL of this mixture was run for 3 hours at 1200 volts (constant voltage) on a 6% unmodified acrylamide sequencing format gel. The gel was dried at 80 ° C. for 1.5 hours and then exposed to Kodak XAR film. Amplification products found only in reactions containing cDNA from THP-1 cells exposed to oxidized LDL were identified and cleaved from the gel. 100 μl of DEPC treated water was added to the microcentrifuge tube containing the cut gel fragments and incubated at room temperature for 30 minutes and then at 95 ° C. for 15 minutes.

To re-amplify the PCR product, 26.5 μl of eluted DNA contained 5 μl of 10 × PCR buffer, 3 μl of 25 mM MgCl ₂ , 5 μl of 500 μM dNTP, 5 μl of 2 μM downstream primer, 7.5 μl of upstream primer 15 and 0.5 μl of Amplitaq polymerase. Was added to the amplification reaction. PCR circulating variables and instruments are as described above. After amplification, 20 μl of reamplifier was analyzed on agarose gel and 4 μl of TA cloning system [Frohman, MA, Dush, MK, and Martin, GR (1988) Proc. Natl. Acad. Sci. USA 85, 8998-9002 (Invitrogen) was used to subclones PCR products into vector pCRII. After overnight linking at 14 ° C., the ligation product was used to form E. co. E. coli was transformed. The resulting transformant was collected and incubated overnight, and 3 µl of the culture was used for microplasmid micropreparation. Insert size was determined by digesting the plasmid with EcoRI, and clones containing the appropriately sized insert of the original PCR product were subjected to fluorescent dye-terminator reagents (Prism, Applied Biosystems) and Applied Biosystems 373 DNA sequencer. Was sequenced. The sequence of the PCR product is shown in FIG. 2. The sequence of the amplification primers is underlined.

5'RACE Reaction

CDNA identified via RT-PCR was stretched 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). This 5'RACE procedure used 1 μg of THP-1 RNA (oxidized LDL treated) used for the first differential display reaction:

1 μl (1 μg) of RNA was mixed with 3 μl of primer 2a (3 pmol) and 11 μl of DEPC treated water and heated to 70 ° C. for 10 minutes and then left on ice for 1 minute. 2.5 μl of 10 × reaction buffer (200 mM Tris-HCl, pH 8.4, 500 mM KCl), 3 μl of 25 mM MgCl ₂ , 1 μl of 10 mM dNTP mixture and 2.5 μl of 0.1M DTT were added. The mixture was incubated at 42 ° C. for 2 minutes, then 1 μl of Superscript II reverse transcriptase was added. The reaction was further incubated at 42 ° C. for 30 minutes, 70 ° C. for 15 minutes and on ice for 1 minute. 1 μl of RNase H (2 units) was added and the mixture was incubated at 55 ° C. for 10 minutes. cDNA was purified using GlassMax columns (Sambrook, J. Fritsch, EF, and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, Plainview, NY) contained in the kit. . From this column cDNA was eluted in 50 μl of dH ₂ O, lyophilized and resuspended in 21 μl of dH ₂ O. Tailing of cDNA was carried out in the following reactions: 7.5 μl of dH ₂ O, 2.5 μl of reaction buffer (200 mM Tris-HCl pH 8.4, 500 mM KCl), 1.5 μl of 25 mM MgCl _2, 2.5 μl of 2m dCTP and cDNA 10 ΜL was incubated for 3 min at 94 ° C. and then 1 min on ice. 1 μl (10 units) of terminal deoxynucleotide transferase was added and the mixture was incubated at 37 ° C. for 10 minutes. The enzyme was heat inactivated by incubation at 70 ° C. for 10 minutes and the mixture was left on ice. PCR amplification of cDNA was carried out in the following steps: 5 μl of tailed cDNA, 5 μl of 10 × PCR buffer (500 mM KCl, 100 mM Tris-HCl pH 8.3, 15 mM MgCl ₂ and 0.01% (w / v) gelatin), 1 μl of 10 mM dNTP mixture, 2 μl of anchor primer (10 pmol), 1 μl of primer 3a (20 pmol) and 35 μl of dH ₂ O were added to the reaction. The reaction was heated to 95 ° C. for 1 min, then 0.9 μl (4.5 units) of Amplitaq polymerase was added. The reaction was circulated 40 times under the following conditions: 5 seconds at 94 ° C, 20 seconds at 50 ° C and 30 seconds at 72 ° C. 1 μl of this reaction was used for nested reamplification to increase the concentration of the specific product to be subsequently separated. For reamplification, 1 μl of primary amplification, 5 μl of 10 × PCR buffer, 1 μl of 10 mM dNTP mixture, 2 μl of universal amplification primer (20 pmol), 2 μl of primer 4a (20 pmol) and 38 μl of dH ₂ O were used. The reaction was heated at 95 ° C. for 1 min, after which 0.7 μl (3.5 units) of Amplitaq polymerase was added. The reaction was circulated 40 times under the following conditions: 5 seconds at 94 ° C., 20 seconds at 50 ° C., 30 seconds at 72 ° C. Amplification products were analyzed via 0.8% agarose gel electrophoresis. About 1.2 kilobase pairs of main product were detected. 2 μl of this reaction product was cloned into a pCRII vector from TA Cloning Kit (Invitrogen) and incubated overnight at 14 ° C. This using a coupling product. E. coli was transformed. Insert size of the resulting transformants was measured after digestion with EcoRI. Clones containing nearly PCR product size inserts were sequenced using fluorescent dye-terminator reagent (Prism, Applied Biosystems) and Applied Biosystems 373 DNA sequencer. The sequence of the RACE product containing the EcoRI site from the TA vector is shown in FIG. 3. The sequence of the amplamer (complement to the universal amplification primers and the 5 'RACE primer 4a) is underlined.

Example 2 Cloning and Chromosome Positioning of LIPG Genes

cDNA library screening

Human placenta cDNA libraries (uplifted dT and randomly primed, Cat # 5014b, Lot # 52033) were obtained from the manufacturer (Clontech, Palo Alto, Calif.). Radiolabeled probes were obtained by cleaving the insert of the plasmid containing the 5'RACE reaction PCR product described above. The probes were radiolabeled using a random priming technique: DNA fragments (50-100 ng) were incubated with 1 μg of random hexamer (Gibco) at 95 ° C. for 10 minutes and then on ice for 1 minute. Then 3 μl, 0.5 mM dATP, dGTP, 3 μl dTTP, 100 μCi α- ³² PdCTP at ¹⁰ × Clenau buffer (100 mM Tris-HCl, pH 7.5, 50 mM MgCl ₂ , 57 mM Dithiothreitol; NewEngland Biolabs) (3000 Ci / mmol, New England Nuclear), and 1 μl of Klenow fragment of DNA polymerase I (5 units, Gibco) were added. The reaction was incubated at room temperature for 2-3 hours, the volume of the reaction was increased to 100 μl with TE pH 8.0 and EDTA was added to a final concentration of 1 mM to stop the reaction. The volume of the reaction was increased to 100 μl and passed over a G-50 spin column (Boehringer Mannheim) to remove unintegrated nucleotides. The inactivation of the probes obtained was at least 5 × 10 ⁸ cpm / μg DNA.

Libraries were probed by conventional methods (Walter, P., Gilmore, R., and Blobel, G. (1984) Cell 38,5-8). Briefly, the filter is 4.8X SSPE (20X SSPE = 3.6M NaCl, 0.2M NaH ₂ PO ₄ , 0.02M EDTA, pH 7.7), 20mM Tris-HCl pH 7.6, 1X Denhardt Solution (100X = 2% Picol 400 , 2% polyvinylpyrrolidone, 2% BSA), 10% dextran sulfate, 0.1% SDS, 100 μg / ml salmon sperm DNA and 1 × 10 ⁶ cpm / ml radiolabeled probe at 65 ° C. for 24 hours. Hybridization. The filter was then washed three times for 15 minutes at room temperature with 2X SSC (1X SSC = 150 mM NaCl, 15 mM sodium citrate pH 7.0), 0.1% sodium dodecyl sulfate (SDS), followed by 0.5X SSC, 0.1% SDS. Washed three times at 65 ° C. for 15 minutes each. Phage hybridizing to the probe was isolated and amplified. DNA was purified from the amplified phage using LambdaSorb reagent (Promega) according to the manufacturer's instructions. Inserts were digested with EcoRI and cleaved from phage DNA. Inserts were subcloned into the EcoRI site of the plasmid vector (Bluescript II SK, Stratagene). The sequence of the open reading frame contained in the 2.6 kb EcoRI fragment of cDNA was determined by automated sequencing as described above. The sequence is shown in FIG. 4. The amino acid sequence of the predicted protein encoded by this open reading frame is shown in FIG. 5 and named LLGXL. It is assumed that the first methionine is encoded by nucleotide pairs 252-254. The deduced protein is 500 amino acids in length. The first 18 amino acids form the characteristic sequence of secreted signal peptides (Higgins, DG, and Sharp, PM (1988) Gene 73, 237-244). The molecular weight of this propeptide is expected to be 56,800 daltons. Assuming the signal peptide is cleaved at position 18, the molecular weight of the unmodified mature protein is 54,724 daltons.

The overall similarity between this protein and other known proteins of the triacylglycerol lipase family is shown in Figure 6 and Table 1. In the alignment shown in Figure 6, LIPG is the polypeptide (SEQ ID NO: 6) encoded by the cDNA (SEQ ID NO: 5) described in Example 1, hereinafter referred to as LLGN. This protein has 345 residues at the amino terminus of the LLGXL protein. Nine unique residues are followed by a stop codon, producing a 39.3 kD propolypeptide and a 37.3 kD mature protein. The sequences common to LLGN and LLGXL are the nucleic acid sequence of SEQ ID NO: 9 and the amino acid sequence of SEQ ID NO: 10.

Interestingly, the position where the LLGN and LLGXL proteins differ is in a region known from the structure of the other lipase such that the position of the proteins between the amino and carboxy domains of these proteins. Thus, LLGN protein appears to consist of only one of the two domains of triacylglycerol lipase. This sequence contains a “GXSXG” lipase motif characteristic at positions 167-171, wherein the catalytic triad residue is conserved at Ser 169, Asp 193, and His 274. Conservation of cysteine residues (positions 64, 77, 252, 272, 297, 308, 311, 316, 463 and 483) involved in disulfide bonds in other lipases suggests that the LLGXL protein is structurally similar to other enzymes. There are five potential sites for N-linked glycosylation: amino acid positions 80, 136, 393, 469 and 491. The protein sequence used for comparison is human lipoprotein lipase (LPL; Genbank Accession No. M15856, SEQ ID NO: 13). ), Human liver lipase (HL; Genbank Accession No. J03540, SEQ ID NO: 14), human pancreatic lipase (PL; Genbank Accession No. M93285, SEQ ID NO: 15), human pancreatic lipase related protein-1 (PLRP-1; Genbank Accession No. M93283) and human pancreatic lipase related protein-2 (PLRP-2; Genebank Accession No. M93284).

Similarity of Triacylglycerol Lipase Gene Family LLGXL LPL HL PL PLRP1 PLRP2 LLGXL - 42.7 36.5 24.5 22.5 22.6 LPL 42.7 - 40.0 22.8 22.7 20.9 HL 36.5 40.0 - 22.8 24.0 22.0 PL 24.5 22.8 22.8 - 65.2 62.2 PLRP1 22.5 22.7 24.0 65.2 - 61.7 PLRP2 22.6 20.9 22.0 62.2 61.7 -

The similarity (%) is determined by the Cluster algorithm (Camps, L., Reina, M., Llobera, M., Vilaro, S., and Olivecrona, T. 1990, Am. J. Physiol. 258, C673-C681], based on paired alignments.

Chromosomal Positioning

DNA from a P1 clone containing genomic LLG DNA (Sternberg, N., Ruether, J. and DeRiel, K. The New Biologist 2: 151-62, 1990) was subjected to nick translation by digoxigenin UTP. Labeled. Labeled probes were combined with sheared human DNA and hybridized to PHA stimulated peripheral blood lymphocytes derived from male donors in a solution containing 50% formamide, 10% dextran sulfate and 2X SSC. Specific hybridization signals were measured by incubating the hybridized cells in fluorescein added antidigoxigenin antibodies followed by counterstaining with DAPI. The first experiment showed that the Group E chromosome was specifically labeled and was assumed to be chromosome 18 based on DAPI staining.

Secondary experiments were conducted in which a covalent hybrid of a biotin labeled probe specific for the chromosome 18 chromosome with the LLG probe. As a result of this experiment, the chromosome 18 isotopically labeled red, and the long arm (p) of chromosome 18 was specifically labeled green. Measurement of eleven specifically labeled and hybridized chromosomes 18 demonstrated that Flter of LLG corresponds to band 18q21 as 0.67 (Franke measurement 0.38). Several genetic diseases, including intrahepatic cholestasis, conical dystrophy and familial swelling osteomalacia, are thought to include defects in this chromosomal region.

Example 3-LIPG RNA Analysis

Expression of LIPG RNA in THP-1 Cells

cDNA-derived mRNA was analyzed by Northern analysis of THP-1 RNA. RNA of these cells was prepared as described above. mRNA was purified from total RNA using a Poly-dT-Magnetic Bead System (Promega). 3 μg of mRNA containing poly (A) was electrophoresed on a 1% agarose-formaldehyde gel. This gel was washed in dH ₂ O for 30 minutes. RNA was vacuum transferred to the nylon membrane using alkaline transfer buffer (3M NaCl, 8 mM NaOH, 2 mM Sarcosyl). After delivery, blots were neutralized by incubation for 5 minutes in 200 mM phosphate buffer (pH 6.8). RNA was crosslinked to the membrane using an ultraviolet crosslinker device (Stratagene).

Probes were prepared by cleaving inserts of plasmids containing the 5'RACE reaction PCR product described above. This probe was radiolabeled using the random priming technique described in Example 2.

Filters were prehybridized at QuikHyb rapid hybridization solution (Stratagene) at 65 ° C. for 30 minutes. Radiolabeled probes (1-2 × 10 ⁶ cpm / ml) and sonicated salmon sperm DNA (final concentration 100 μg / ml) were denatured by heating at 95 ° C. for 10 minutes and iced prior to addition to the filter in QuikHyb. Rapid cooling on phase. Hybridization was carried out at 65 ° C. for 3 hours. The unhybridized probe was removed by washing twice for 15 minutes at room temperature with 2X SSC, 0.1% sodium dodecyl sulfate and then washing twice for 15 minutes at 62 ° C. with 0.1X SSC, 0.1% SDS. After washing, the filter was briefly dried and then exposed at -80 ° C to a Kodak XAR-2 film with a light collecting screen. The results are shown in FIG. 7 and represent a major mRNA species of about 4.5 kb. In addition, small amounts of mRNA of 4.3 kb and 1.6 kb were also found. The expected size of LLGN cDNA is 1.6kb. The LLGXL sequence is assumed to be encoded by the primary mRNA species observed above.

Expression of LIPG RNA in Various Human Tissues

Commercially available filters containing 3 µg each of mRNA from human tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas) were obtained from the manufacturer (Clontech, Catalog # 7760-1). This filter was probed and treated as described above. After probed with radiolabeled LLG fragments and photographed by autoradiography, the probe was removed by washing twice with 15 × boiling 0.1X SSC, 0.1% SDS in a 65 ° C. incubator. The membrane was then probed with a pair of 1.4 kb DNA fragments encoding human lipoprotein lipase. This fragment was obtained by RT-PCR of THP-1 RNA (PMA and oxLDL treated) using the 5'LPL primer and 3'LPL primer shown in Figure 1, conditions of RT-PCR as described above. After autoradiography, the probe was removed from the membrane and reprobed with radiolabeled fragments of human β-actin cDNA to define RNA content. The results of this analysis are shown in FIG. 8. The highest concentration of LIPG message was measured in placental RNA and the lowest level was observed in RNA from lung, liver and kidney tissues. This has been described by other researchers in Verhoeven, A.J.M., Jansen, H. (1994) Biochem. Biophys. Consistent with Acta 1211, 121-124, lipoprotein lipase messages were observed in various tissues, with the highest levels observed in heart and skeletal muscle tissue. This analysis suggests that the tissue distribution of LIPG expression is very different from that of LPL. Expression patterns of LIPG were also described by other researchers [Wang, C.-S., and Hartsuck, J.A. (1993) Biochem. Biophys. Acta 1166, 1-19; Semenkovich, C.F., Chen, S.-W., Wims, M., Luo C.-C., Li, W.-H., and Chan, L. (1989) J. Lipid Res. 30, 423-431; Adams, M.D., Kerlavage, A.R., Fields, C., and Venter, C. (1993) Nature Genet. 4, 256-265, differing from the expression pattern of hepatic lipase or pancreatic lipase.

Another commercial membrane was probed with LLGXL cDNA to measure expression patterns in another human tissue. This dot blot (Human RNA Master Blot, manufactured by Clontech, Cat. # 7770-1) contains 100-500 ng of mRNA from 50 different tissues and has been specified for the corresponding housekeeping gene expression [Chen, L , and Morin, R. (1971) Biochim. Biophys. Acta 231, 194-197. The 1.6 kb DraI-SrfI fragment of LLGXL cDNA was labeled with ³² P dCTP using a random oligonucleotide priming system (Prime It II, Stratagene) according to the manufacturer's instructions. After 30 min prehybridization at 65 ° C., the probe was added to the QuikHyb hybridization solution at a concentration of 1.3 × 10 ⁶ cpm / ml. Hybridization was carried out at 65 ° C. for 2 hours. The unhybridized probe was removed by rinsing the filter twice with 15 × SSC, 0.1% sodium dodecyl sulfate at room temperature twice for 15 minutes and then twice with 15 × 0.1 ° SSC, 0.1% SDS at 62 ° C. for 15 minutes. After washing, the filter was simply dried and exposed to Kodak XAR-2 film with a light collecting screen at −80 ° C. for various hours. The resulting image was quantified with a photo densitometer. The results are shown in Table 2. The relative expression levels of tissues in the Northern blots of the various tissues and the dot blots of the various tissues were similar, with the highest levels being observed in the placenta and the lowest levels in the lungs, liver and kidneys. Liver, kidney and lung of the fetus also expressed almost the same levels as in adult tissues. Surprisingly, expression level in thyroid tissue was highest in all tissues presented, and 122% of expression level in placental tissue. There is precedent for lipase expression by placenta [Rothwell, JE, Elphick, MC (1982) J. Dev. Physiol. 4, 153-159; Verhoeven, AJM, Carling D., and Jansen H. (1994) J. Lipid Res. 35, 966-975; Burton, BK, Mueller, HW (1980) Biochim. Biophys. Acta 618, 449-460, the thyroid expressing lipase is not known in the prior art. These results indicate that LIPG expression may be involved in the maintenance of the placenta, where LIPG may act to release free fatty acids as energy sources from substrates such as phospholipids. LIPG expressed in the thyroid gland can provide a precursor to the synthesis of bioactive molecules by the thyroid gland.

Expression of LIPG mRNA in Various Human Tissues Whole brain N.D. Blackness N.D. Womb N.D. cable N.D. lungs 29 one-way N.D. Temporal lobe N.D. prostate 5 kidney 44 Agency 12 Unknown nucleus N.D. thalamus N.D. Camouflage N.D. liver 61 placenta 100 cerebellum 4 Hypothalamus N.D. Testicle 9 Intestine 6 Fetal brain 5 Brain cortex N.D. spinal cord N.D. ovary N.D. spleen N.D. Fetal heart N.D. Frontal lobe N.D. Heart N.D. Pancreas N.D. Thymus N.D. Fetal kidney 56 hippocampus N.D. artery N.D. pituitary N.D. Peripheral White Blood Cells N.D. Fetal liver 14 Soft water N.D. Skeletal muscle N.D. suprarenal body N.D. Imp clause N.D. Fetal spleen N.D. Occipital lobe N.D. colon 8 thyroid 122 marrow N.D. Fetal thymus N.D. Cockleshell N.D. bladder N.D. Line N.D. appendix 7 Fetal lung 8

The values presented above represent percent expression after random setting of levels in placental tissue to 100%. This value is the average of the values measured with a photo densitometer after two autoradiograph exposures. N.D. is an unmeasurable value.

Expression of LIPG RNA in Cultured Endothelial Cells

Human umbilical vein endothelial cells (HUVEC) and human coronary artery endothelial cells (HCAEC) were obtained from Clonetics. HUVECs are derived from cerebellar extracts [Maciag, T., Cerundolo, J., Ilsley, S., Kelley, P.R., and Forand, R. (1979) Proc. Natl. Acad. Sci. USA 76, 5674-5678] (Clonetics) is grown on commercially available endothelial cell growth medium (EGM, Clonetics) supplemented with 3 mg / ml and HCAEC is cerebellar extract 3 mg / ml and 3% fetal calf. Serum (final concentration 5%) was grown in supplemented EGM. Cells were propagated until confluent and the medium was exchanged with EGM without cerebellar extract added. Cultures were stimulated by adding 100ng / ml of phorbol myristate (Sigma). After incubation for 24 hours, RNA was extracted from the cells by the above-mentioned Trizol method. 20 μg total RNA was electrophoresed and delivered to the membrane for analysis. This membrane was probed with LIPG and LPL probes as described above. The results are shown in FIG. 20 μg of total RNA from THP-1 cells stimulated with PMA was run on a comparative blot. RNA hybridizing to LIPG probes was detected in unstimulated HUVEC cells and PMA stimulated HUVEC cells. In contrast, detectable levels of LIPG mRNA were only observed in HCAEC cultures after PMA stimulation. No detectable lipoprotein lipase mRNA could be observed in endothelial RNA, consistent with previous findings by other researchers [Verhoeven, A.J.M., Jansen, H. (1994) Biochem. Biophys. Acta 1211, 121-124.

Example 4-LIPG Protein Analysis

Antibody manufacturing

Antisera were generated for peptides with sequences corresponding to the putative protein regions encoded by the LIPG cDNA open reading frame. The peptide was chosen because of its high expected antigenicity index. Jameson B.A., and Wolf, H. (1988) Comput. Applic. in the Biosciences 4, 181-186]. The sequence of the immunized peptide was not found in any protein or translated DNA sequence in the GenBank database. The corresponding position in the LIPG protein is shown in FIG. 10. The carboxy terminal cysteines of this peptide did not correspond to residues present in the LIPG putative protein, but were introduced to facilitate coupling to the carrier protein. Peptides were synthesized on a peptide synthesizer (Applied Biosystems Model 433A). 2 mg of the peptide was coupled to 2 mg of maleimide activated keyhole limpet hemocyanin according to the protocol attached to the Immune Activated Immunogen Conjugation Kit (Pierce Chemical). After desalting, half the conjugate was emulsified with the same amount of Freund's complete adjuvant (Pierce). This emulsion was injected into New Zealand white rabbits. Four weeks after the first inoculation, the emulsion prepared as described above was inoculated twice except for using Freund's incomplete adjuvant (Pierce). Two weeks after boosting the test was bled and the titer of the specific antibody was measured by ELISA with immobilized peptide. Follow-up inoculations were then performed one month after the first booster.

Western analysis of medium obtained from endothelial cell culture

Cells were cultured and stimulated with PMA as described in Example 3C except that HUVEC and HCEAC cells were stimulated with PMA for 48 hours. Conditional medium samples (9 mL) were incubated with 500 μl of Heparin-Sepharose CL-6B in 50% slurry suspended in phosphate buffered saline (PBS, 150 mM sodium chloride, 100 mM sodium phosphate, pH 7.2). Heparin-Sepharose was selected for partial purification and concentration of LIPG proteins due to the retention of residues in the LLGXL sequence identified as important for heparin binding activity of LPL [Ma, Y, Henderson, HE, Liu, M.-S. Zhang, H., Forsythe, IJ, Clarke-Lewis, I., Hayden, MR, and Brunzell, JD J. Lipid Res. 35, 2049-2059 (FIG. 6). After spinning for 1 hour at 4 ° C., the samples were centrifuged at 150 × g for 5 minutes. The culture was aspirated and Sepharose was washed with 14 ml PBS. Pelletized heparin-sepharose after centrifugation and aspiration was 200 μl of 2 × SDS loading buffer (4% SDS, 20% glycerol, 2% β-mercaptoethanol, 0.002% bromophenol blue and 120 mM Tris pH 6.8) Suspended in. Samples were heated at 95 ° C. for 5 minutes and loaded with 40 μl on a 10% Tris-glycine SDS gel. After electrophoresis at 140 V for about 90 minutes, the protein was delivered to the nitrocellulose membrane via a Novex electroblotting device (210 V, 1 hour). This membrane was blocked for 30 minutes in blocking buffer (5% skim milk powder, 0.1% Tween 20, 150 mM sodium chloride, 25 mM Tris pH 7.2). Antipeptide antiserum and normal rabbit serum were diluted at a dilution of 1: 5000 with blocking buffer and incubated with the membrane overnight at 4 ° C. under slow shaking. The membrane was then washed four times for 15 minutes with TBST (0.1% Tween 20, 150 mM sodium chloride, 25 mM Tris pH 7.2). Goat anti-rabbit peroxidase conjugated antiserum (Boehringer Manheim) was diluted with blocking buffer at a ratio of 1: 5000 and incubated with the membrane for 1 hour under shaking. The membrane was washed as described above, reacted with Renaissance chemiluminescent reagent (DuPont NEN) and exposed to Kodak XAR-2 film. The result is shown in FIG. Two immunoreactive proteins were present in samples from unstimulated HUVEC and HCAEC cells. The concentration of immunoreactive protein in unstimulated HCAEC samples was much lower than that in the corresponding HUVEC samples. When stimulated with PMA, three immunoreactive proteins were secreted by endothelial cell culture. Upon PMA exposure the concentration of LIPG protein produced by HCAEC cultures was greatly increased. However, PMA induction of LLG protein did not show a sharp increase in HUVEC culture.

Example 5-Recombinant LIPG Protein Production

LIPG Expression Constructs

CDNA encoding LLGN and LLGXL proteins were cloned into mammalian expression vector pCDNA3 (Invitrogen). This vector uses cytomegalovirus major late promoters to express heterologous genes in many mammalian cells. The LLGN 5 'RACE product was cloned into the EcoRI site of pCDNA3. LLGXL cDNA was digested with DraI and SrfI to yield 1.55 kb cDNA (FIG. 4). The vector was digested with the restriction enzyme EcoRV and the vector and insert were linked according to the manufacturer's instructions with T4 DNA ligase and reagents of the Quick Link Kit (Boehringer Manheim). Using this linking product, the capacity of E. coli was transformed. The resulting colonies were selected by restriction enzyme analysis and sequenced to examine the presence and orientation of the inserts in the expression vector.

Transient Transfection of LIPG on COS-7 Cells

LIPG expression vectors were introduced into COS-7 cells using Lipofectamine cationic lipid reagent (GIBCO). 24 hours prior to transfection, COS-7 cells were plated at a concentration of 2 × 10 ⁵ cells / plate on a 60 mm tissue culture dish. The cells were grown in Dulbecco's modified Eagle's medium (DMEM; GIBCO) supplemented with 10% fetal calf serum, 100 U / ml penicillin, 100 μg / ml streptomycin. 1 μg of plasmid DNA was added to 300 μl of Optimem I serum-free medium from Gibco. 10 μl of lipofectamine reagent was diluted with 300 μl of Optimem I medium, which was combined with DNA solution and left at room temperature for 30 minutes. Cultures were harvested from the plates and cells were washed with 2 ml Optimem medium. DNA-lipopeptamine solution was added to the plate with 2.7 ml of Optimem medium and the plate was incubated at 37 ° C. for 5 hours. After incubation, serum-free medium was removed and replaced with DMEM supplemented with 2% FBS and antibiotics. After 12 hours of transfection, a portion of the culture was treated with the protease inhibitor 0.25 mM Pefabloc SC (Boehringer Manheim) or 10 U / ml heparin. Thirty minutes before harvesting, the heparinized samples were further treated with 40 U / ml heparin. The media was removed from the cells 60 hours after transfection. Heparin-Sepharose CL-4B (200 μl of 50% slurry in PBS pH 7.2) was added to 1 ml of medium and mixed at 4 ° C. for 1 hour. Sepharose was pelleted by low speed centrifugation and washed three times with 1 ml of ice-cold PBS. This Sepharose was pelleted and suspended in 100 μl of 2 × loading buffer. The sample was heated to 95 ° C. for 5 minutes. 40 μl of each sample was loaded on a 10% SDS-PAGE gel. Electrophoresis and Western analysis were performed using anti-LIPG antiserum as described above. The results are shown in FIG. Proteins from HCAEC conditioned media were included for size control. LLGN moved to about 40 kD, corresponding to the lowest band in HCAEC. Cultures derived from COS cells transfected with LLGXL cDNA included both 68 kD and 40 kD species. When these cells were treated with heparin, the amount of 68kD protein and 40kD protein recovered from the culture solution increased dramatically, suggesting the release of proteoglycan-bound protein from the cell surface or stabilization of the protein by heparin. When cells were treated with the protease inhibitor Pefabloc, the amount of 68kD protein was increased compared to the amount of 40kD protein. This suggests that the low molecular weight protein produced by these cells is a 68kD form of proteolytic product. The role of mRNA encoding a smaller 40kD protein species and confirmed via differential display is unknown. However, there have been reports of hepatic lipases in a selectively spliced form, with distinct tissue-specific expression and yielded truncated proteins.

Example 6-LIPG in Animal Species

Cloning of LIPG Rabbit Homolog

Fragments of rabbit homologs of the LIPG gene were isolated using a commercial lambda cDNA library from rabbit lung tissue (Clontech, Cat. # TL1010b). 5 μl of stock library was added to 45 μl of water and heated at 95 ° C. for 10 minutes. Then ₂₀₀ μM dNTP, 20 mM Tris-HCl pH 8.4, 50 mM KCl, 1.5 mM MgCl ₂ , 100 μM each primer dLIP774 and LLGgen2a, and 2.5 U Taq polymerase (GIBCO) were added to a final volume of 100 μL. The reaction was subjected to 35 cycles of reaction for 15 seconds at 94 ° C, 20 seconds at 50 ° C and 30 seconds at 72 ° C. 10 μl of this reaction was analyzed by agarose gel electrophoresis. About 300 base pairs of product were detected. A portion of the reaction mixture (4 μl) was used to clone the product through the TA cloning system. The insert of the resulting clone was sequenced (SEQ ID NO: 11). The deduced rabbit amino acid sequence (SEQ ID NO: 12) and the corresponding sequence of human cDNA are shown in FIG. 14. Of the total nucleotides of either amplification primers, there was 85.8% identity between the rabbit LLG sequence and the human LLG sequence. The inferred protein encoded by this rabbit cDNA shared 94.6% identity with the human protein and showed most nucleotide substitutions at the third or "wobble" position of the codon. In particular, this region extends to the “lid” sequence of the speculative LLG protein and is a variable domain present in the lipase gene family. This proves that the gene is highly interstitial conservative.

LIPG present in other species

To demonstrate the presence of the LLG gene in other species, genomic DNA obtained from various species was limited to EcoRI, isolated via agarose gel electrophoresis and blotted onto nitrocellulose membranes.

The membrane was randomly primed ³² P-LLG or ³² P-LPL (lipoprotein lipase) in a hybridization solution containing 6 × SSC, 10% dextran sulfate, 5 × Denhardt solution, 1% SDS and 5 μg / ml salmon sperm DNA. Hybridization with probe 2.5 × 10 ⁶ cpm / ml overnight at 65 ° C. The membrane was washed successively with 0.1 × SSC, 0.5% SDS for 10 minutes at room temperature, 40 ° C., 50 ° C. and 55 ° C. for 10 minutes. Automatic radiographs of the blots are shown in FIG. 16.

FIG. 16 shows the presence of the LLG gene and the LPL gene in all species examined except that no hybridization was observed by the LLG probe for rat DNA. Exceptional data in the rats indicate that the artifact is due to the generation of abnormally sized restriction fragments containing LLG sequences. These fragments may be outside the fractional range of the agarose gel or may be blotted inefficiently. Different bands detected by both probes suggest that LPL and LIPG are independent and evolutionarily conserved genes.

Example 7-Enzymatic Activity of LLGXL

Phospholipase activity

Phospholipase activity was assayed with conditioned media from COS-7 cells transiently expressing human lipoprotein lipase (LPL), LLGN or LLGXL. MEM containing 10% FBS (MEM) was used as a control, and conditioned medium from COS-7 cells transfected with antisense LLGXL plasmid (AS) was used as a negative control.

Phosphatidylcholine (PC) emulsions consisted of 10 μl of phosphatidylcholine (10 mM), 40 μl of ¹⁴ C-phosphatidylcholine, dipalmitoyl (2 μCi) and tris-TCNB (100 mM Tris, 1% Triton, 5 mM CaCl ₂ labeled with sn 1 and 2 positions) , 200 mM NaCl, 0.1% BSA) was used. This emulsion was evaporated for 10 minutes and then the final volume was made up to 1 ml with Tris-TCNB.

The reaction was carried out twice, and 50 µl of PC emulsion and 950 µl of medium were added. Samples were incubated for 2-4 hours at 37 ° C. in a shaking water bath. The reaction was terminated by addition of 1 mL of 1N HCl, followed by extraction with 4 mL of 2-propanol: hexane (1: 1). The upper 1.8 ml hexane layer was passed through a silica gel column and the released ¹⁴ C-free fatty acids contained in the effluent fraction were quantified with a scintillation counter. The results of this analysis are shown in FIG.

Triacylglycerol Lipase Activity

Triglycerol lipase activity was assayed with conditioned media from COS-7 cells transiently expressing human lipoprotein lipase (LPL), LLGN or LLGXL. MEM containing 10% FBS was used as a control, and conditioned medium from COS-7 cells transfected with antisense LLGXL plasmid (AS) was used as a negative control.

Substrate concentration is prepared in the anhydrous emulsion of the [9,10- ³ H (N)], and (a = 150㎎, 6.25 x 10 ⁸ cpm final total triolein) of unlabeled triolein labeled triolein, and 9 mg of lecithin in 100% glycerol was added to stabilize. 0.56 mL (0.28 mCi) of ³ H-triolein was mixed with 0.17 mL of unlabeled triolein and 90 μl (9 mg) of lecithin. This mixture was evaporated under nitrogen. The dried lipid mixture was emulsified in sonication (2.5 seconds of pulse level 2 followed by a cycle of 2 seconds of cooling for 5 minutes) in 2.5 ml of 100% glycerol.

Analytical substrates were prepared by diluting 1 volume of concentrated substrate to 4 volumes of 0.2M Tris-HCl buffer (pH 8.0) containing 3% w / v fatty acid depleted bovine serum albumin. The diluted substrate was vigorously vortexed for 5 seconds.

The reaction was repeated twice in a total volume of 0.2 ml containing 0.1 ml of assay substrate and 0.1 ml of the desired medium. The reaction was incubated at 37 ° C. for 90 minutes. The reaction was terminated by addition of 3.25 mL of methanol-chloroform-heptane 1.41: 1.25: 1 (v / v / v) followed by 1.05 mL of 0.1 M potassium carbonate-borate buffer (pH 10.5). After vigorously mixing for 15 seconds, the samples were centrifuged for 5 minutes at 1000 rpm. An amount of 1.0 ml in the upper aqueous phase was counted with a scintillation counter. The results of this analysis are shown in FIG. 15.

Example 8 Use of LIPG Polypeptides Used to Select Enhancers or Inhibitors

Recombinant LIPGs are produced in baculovirus infected insect cells or stably transfected CHO cells or other available mammalian host cells. Recombinant LIPG was purified from serum-containing or medium without serum by chromatography on heparin-sepharose followed by chromatography on cation exchange resin. Third chromatography or additional chromatography steps, such as molecular sieves, were used for purification of LIPG, if necessary. During purification, LIPG proteins were monitored using antipeptide antibodies and LIPG activity was measured using phospholipase assay.

In fluorescence assay, final assay conditions were about 10 mM Tris-HCl (pH 7.4), 100 mM KCl, 2 mM CaCl ₂ , 5 μΜ C ₆ NBD-PC {1-acyl-2- [6- (nitro-2,1,3- Benzoxadiazol-4-yl) amino] caproylphosphatidylcholine and LIPG protein (about 1-100 ng). The reaction was fluorescently excited at 470 nm, and enzyme activity was measured by fluorescence emission at 540 nm and continuously monitored. Compounds and / or substances tested for stimulation and / or inhibition of LIPG activity were added as 10-200 mM solutions in dimethylsulfoxide. Compounds that stimulate or inhibit LIPG activity were found to exhibit increased or decreased fluorescence emission at 540 nm.

In the thio assay, the final assay conditions are about 25 mM Tris-HCl (pH 8.5), 10 mM KCl, 10 mM CaCl ₂ , 4.24 mM Triton X-100, 0.5 mM 1,2-bis (hexanoylthio) -1,2-dide Oxy-sn-glycero-3-phosphorylcholine, 5 mM 4,4'-dithiobispyridine (from 50 mM stock in ethanol) and 1 to 100 ng of recombinant LIPG. Phospholipase activity was analyzed by measuring the increase in absorbance at 342 nm. Compounds and / or substances tested for stimulation and / or inhibition of LIPG activity were added as 10-200 mM solutions in dimethylsulfoxide. Compounds that stimulate or inhibit LIPG activity were found to exhibit increased or decreased absorbance at 342 nm.

Example 9 Transformed Mice Expressing Human LIPG

To further study the physiological role of LIPG, transformed mice expressing human LIPG were prepared.

The 1.53 kb DraI / SrfI restriction fragment encoding LLGXL was cloned downstream of the promoter for the ubiquitously expressed 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase gene of the plasmid vector (pHMG). It was. Transformed mice expressing different concentrations of human LLGXL were prepared using standard methods [eg, G.L. Tremp et al. Gene 156: 199-205, 1995]. These transformed mice were used to determine the effect of LLGXL overexpression on lipid profile, vascular pathology, growth rate and extent of atherosclerosis, and other physiological variables.

Example 10 Expression of LIPG in Atherosclerosis Tissue

LLGXL expression in atherosclerosis was investigated by performing reverse transcriptase-polymerase chain reaction (RT-PCR) using mRNA isolated by vascular biopsy from four patients with atherosclerosis. Tissue samples were obtained from the aortic wall (1 sample), iliac artery (2 samples), and carotid artery (1 sample).

Atherosclerosis biopsies were obtained from the Royal Hospital of England, and polyA + mRNA was prepared and resuspended in diethylpyrocarbonate (DEPC) treated water at a concentration of 0.5 μg / μL mRNA. Reverse transcriptase reactions were performed according to the manufacturer's protocol (GibcoBRL) (Superscript Preamplification System for First Chain cDNA Synthesis). Briefly, cDNA was synthesized as follows: 2 μl of each mRNA was added to 1 μl of oligo (dT) _12-18 primer and 9 μl of DEPC water. The tube was incubated at 70 ° C. for 10 minutes and left on ice for 1 minute. The following components were added to each tube: ₂ μl of ₁₀ × PCR buffer, 2 μl of 25 mM MgCl ₂ , 1 μl of 10 mM dNTP mixture and 2 μl of 0.1M DTT. After 5 minutes at 42 ° C., 1 μl (200 units) of Superscript II reverse transcriptase was added. The reaction was mixed at low speed and then incubated at 42 ° C. for 50 minutes. The reaction was incubated at 70 ° C. for 15 minutes and then left on ice to terminate the reaction. The remaining mRNA was destroyed by adding 1 μl of RNase H to each tube and incubating at 37 ° C. for 20 minutes.

PCR amplification was performed using 2 μl of cDNA reactant. The following components were added to each tube: 5 μl 10 × PCR buffer, 5 μl 2mM dNTP, 1 μl hllg-gsp1 primer (20 pmol / ml, see FIG. 1), hllg-gsp2a primer (20 pmol / ml, see FIG. 1). ) 1 μl, 50 μl MgCl ₂ , 0.5 μl Taq polymerase ( ₅ U / mL) and 34 μl water. After maintaining the reaction at 95 ° C. for 2 minutes, PCR was cycled 30 times under the following conditions: 15 seconds at 94 ° C., 20 seconds at 52 ° C. and 30 seconds at 72 ° C. The resulting reaction was maintained at 72 ° C. for 10 minutes and then analyzed by agarose gel electrophoresis. The hllg-gsp primer is specific for LIPG and produces a 300bp expected product. In parallel PCR to demonstrate successful cDNA synthesis reaction, primers specific for the housekeeping gene G3PDH (human glyceraldehyde 3-phosphate dehydrogenase) were used (1 μl each at a concentration of 20 pmol / ml). .

G3PDH primers (see FIG. 1) produced 983 bp of expected product in a total of four vascular biopsy samples. LIPG expression was detected in three of four samples, and no expression was observed in carotid artery samples.

Example 11-Differential Display, RT-PCR and cDNA Library Screening

To perform the experiments presented in Examples 12-16, the following procedure (based on the procedure outlined in Example 1) was used to obtain cDNA of LIPG. THP-1 cells were plated for 48 hours in the presence of phorbol 12-myristate 13-acetate (PMA, 40 ng / ml; Sigma). Differentiated THP-1 cells were exposed to oxLDL (50 μg / ml) or control medium for 24 hours. Total RNA was harvested and purified according to standard procedures. Poly (A) ⁺ RNA was purified from total RNA using a poly-dT magnetic bead system (Promega). cDNA synthesis and PCR amplification was performed using the protocol of differential display kit version 1.0 (Display Systems Biotechnology). The primer pairs that produced the primary cDNA fragment of EL are downstream primer 7 (5'-TTTTTTTTTTTGA-3 ') and upstream primer 15 (5'-GATCCAATCGC-3'). This amplification reaction was isolated on a 6% unmodified acrylamide sequencing gel and amplified products found only in the reaction containing cDNA from THP-1 cells exposed to oxLDL were cleaved from the gel. Reamplification using the same primers was performed and the product was cleaved and subcloned into pCRII vectors using a TA cloning system (Invitrogen). The size of the insert was determined by digesting the plasmid with EcoRI, and the fluorescent dye-terminator reagent (Prism, Applied Biosystems) and Applied Biosystems 373 cDNA with a clone containing the insert approximately the size of the original PCR product. Sequence analysis was performed using a sequencer. The cDNA sequence of the original cDNA cut in the gel was stretched using the 5'-RACE system (GIBCO). RNA (1 μg) derived from THP-1 cells first used in the differential display reaction was used to perform a 5′-RACE procedure using gene specific primers (5′-TAGGACATGCACAGTGTAATCTG-3 ′) of first strand cDNA synthesis. PCR amplification of this cDNA was performed using anchor primers and gene specific primer 2 (5'-GATTGTGCTGGCCACTTCTC-3 '). This reaction was used for nested reamplification using universal amplification primers (5'-CUACUACUACUAGGCCACGCGTCGACTAGTAC-3 ') and gene specific primer 3 (5'-GACACTCCAGGGACTGAAG-3') to determine the concentration of specific product for subsequent separation. Increased. The reaction product was cloned into the pCRII vector of the TA cloning kit and sequenced. Human placenta cDNA libraries (oligo dT and randomly primed) were obtained from Clontech and probed with 5′-RACE reaction PCR products. DNA from hybridization clones was purified using LambdaSorb reagent (Promega). Inserts were digested with EcoRI, cleaved from phage DNA, subcloned into EcoRI sites of the Bluescript II SK plasmid vector (Stratagene) and sequenced.

Example 12-Antibody Preparation

17 residue peptides (GPEGRLEDKLHKPKATC) corresponding to residues 8 to 23 of the LIPG gene product secreted with a peptide synthesizer (Model 433A, Applied Biosystems) were synthesized. 2 mg of the peptide was coupled to 2 mg of maleimide activated keyhole limpet hemocyanin according to the protocol attached to the Immune Activated Immunogen Conjugation Kit (Pierce Chemical). After desalting, half the conjugate was emulsified with the same amount of Freund's complete adjuvant (Pierce). This emulsion was injected into New Zealand white rabbits. Four weeks after the first inoculation, the emulsion prepared as described above was further inoculated, except for using Freund's incomplete adjuvant (Pierce). Two weeks after boosting the test was bled and the titer of the specific antibody was measured by ELISA with immobilized peptide.

Example 13 Gene Expression Studies

HUVECs were grown in commercial endothelial cell growth medium (EGM, Clonetics) supplemented with 3 mg / ml cerebellar extract (Clonetics), and HCAEC supplemented with 3 mg / ml cerebellar extract and 5% fetal calf serum. Were grown in EGM. 100 ng / ml PMA was added to stimulate culture. After incubation for 24 hours, RNA was extracted from the cells via Trizol method, electrophoresed on a 1% agarose-formaldehyde gel, followed by nitroran on Turboblotter (Schleicher and Schuell) Transfer to (Nytran) membrane and crosslink to membrane using Stratalinker UV crosslinker (Stratagene). The 5'-RACE reaction PCR product was radiolabeled by random priming technique. Radiolabeled probes (1-2 × 10 ⁶ cpm / ml) were denatured by heating at 95 ° C. for 10 minutes and then rapidly cooled on ice and then added to the filters in QuikHyb. Hybridization proceeded at 65 ° C. for 3 hours. The filter was exposed at -80 ° C to Kodak XAR-2 film with condensing screen. HUVEC conditioned medium and HCEAC conditioned medium were incubated with heparin-Sepharose CL-6B for 1 hour at 4 ° C. After centrifugation, the pelleted heparin-sepharose was suspended in SDS loading buffer, heated to 95 ° C. for 5 minutes and then loaded onto a 10% Tris-glycine SDS gel (NOVEX). After electrophoresis at 140 V for 90 minutes, the protein was delivered to the nitrocellulose membrane and goat anti-rabbit peroxidase conjugated antiserum (1: 5,000; Boehinger) as a second antibody with rabbit anti-LIPG peptide antiserum (1: 5,000). Was detected using. The membrane was reacted with a Renaissance chemiluminescent reagent from DuPont NEN and exposed to Kodak XAR-2 film. A commercial filter containing poly (A) ⁺ RNA (3 μg each) from human heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas (Clontech) was hybridized with radiolabeled fragments and described above. Treated as After autoradiography, the blots were separated by washing twice with boiling 0.1 × SSC and 0.1% SDS for 15 min at 65 ° C., and then probed as described above with 1.4 kb cDNA fragments encoding human LPL. This fragment was obtained by RT-PCR of THP-1 RNA (PMA and oxLDL treated) using 5'LPL primer (5'-ACCACCATGGAGAGCAAAGCCCTG-3 ') and 3'LPL primer (5'-CCAGTTTCAGCCTGACTTCTTATTC-3'). . After exposure to the film, the membranes were washed again and reprobed with radiolabeled fragments of human βactin cDNA to define RNA content.

Human umbilical vein endothelial cells (HUVEC) were negative in LPL mRNA expression as expected, but mRNA for the LIPG gene was observed to be constitutively expressed at high concentrations (FIG. 9).

Human coronary endothelial cells (HCAEC) were also observed to express more upregulated mRNA when treated with phorbol ester (FIG. 9).

Stimulated medium derived from HUVEC and HCAEC stimulated contained immunoreactive proteins of about 68 kD and 40 kD and smaller 55 kD bands (FIG. 11).

Northern blot analysis of a plurality of human tissues using probes for LIPG and LPL was performed to determine tissue sites of LIPG production in vivo. Large amounts of LIPG mRNA were found in lung, liver and kidney (FIG. 8) tissues, but low concentrations of LPL expression were observed. LIPG was also expressed in high concentrations in the placenta (Figure 8), suggesting that it is likely to play an important role in growth.

LIPG expression was not measured in tissues such as heart and skeletal muscle expressing the highest concentrations of LPL (Goldberg, J.I., J. Lipid Res., 37, 693-707 (1996)). This fact suggests that the tissue distribution of LIPG expression as well as those reported for HL and PL are very different from the tissue distribution of LPL. LIPG mRNA was not observed in the adrenal glands and ovaries, but very little LIPG mRNA was observed in the testicles (data not shown). In addition, HepG2 cells were observed to express LIPG mRNA and protein in vitro in an amount less than 10% of the amount expressed by HUVEC (data not shown).

Example 14-Lipase Analysis

The cDNA and 1.4-kb LPL cDNA were cloned into the EcoRV site of mammalian expression qrpxj pCDNA3 (Invitrogen). Antisense pCDNA 3 vector was used as a negative control. Recombinant expression vectors (3 μg) were mixed with lipofectamine (Life Technologies) and transfected four times with 1/2 coherent COS7 cells in a 60 mm dish. Using established methods, samples of conditioned media obtained from transfected COS7 cells were analyzed for TG lipase and phospholipase activity [Goldberg, JI, J. Lipid Res., 37, 693-707 (1996)]. . For TG lipase analysis 9,10- ³ H (N) - a triolein (250μCi; manufacturer: NEN) to an unlabeled triolein in (150㎎) and glycerol type IV-S-α of lecithin (9㎎; Manufacturer: Sigma). The mixture was evaporated under nitrogen, sonicated with a sonicator (Branson Sonifier 450) and emulsified in glycerol (2.5 mL). The assay substrate was mixed with 1 volume of emulsified substrate, 4 volumes of Tris-HCl (0.2M, pH 8.0) containing 3% (w / v) fatty acid depleted bovine albumin (BSA) and 1 volume of heat deactivated bovine serum. It was prepared by. The reaction was repeated three times under a total volume (0.2 mL) containing the assay substrate (0.1 mL) and the conditioned medium (0.1 mL). The reaction was incubated at 37 ° C. for 2 hours, methanol-chloroform-heptane (1.41: 1.25: 1; 3.25 mL) was added followed by the addition of potassium carbonate-borate buffer (1.05 mL; 0.1 M, pH 10.5). Was terminated. After vigorously mixing for 15 seconds, the samples were centrifuged at 1,000 rpm for 5 minutes and the upper aqueous phase (1.0 mL) was counted on a scintillation counter. For phospholipase analysis, ¹⁴ C-dipalmitoyl PC (2 μCi; NEN) and lecithin (10 μl) were added to Tris-TCNB (100 μl; 100 mM Tris-HCl pH 7.4, 1% Triton X-100, 5 mM CaCl ₂ , 200 mM NaCl, 0.1% BSA) was mixed to prepare a phosphatidylcholine (PC) emulsion. This mixture was vortexed for 2 minutes and then evaporated under nitrogen. The dried lipids were restored to TCNB (1 mL) and vortexed for 10 seconds. PC emulsion (50 μl), conditioned medium (600 μl) and MEM (350 μl) were added to the reaction and prepared three times. Samples were incubated at 37 ° C. for 2 hours, terminated by addition of HCl (1 mL) and extracted with 2-propanol: hexanes (1: 1; 4 mL). A sample of the upper hexane layer (1.8 ml) was passed through a silica gel column and the released ¹⁴ C-free fatty acids contained in the effluent fraction were quantified with a scintillation counter. For both assays, MEM containing 10% FBS was used as a control and a control medium derived from COS7 cells transfected with antisense plasmid (AS) was used as a negative control.

Example 15-Recombinant Adenovirus Construction and Animal Studies

Recombinant adenoviruses encoding human LIPG are described in Tsukamoto et al., J. Clin. Invest., 100, 107-114 (1997); Tsukamoto et al., J. Lipid Res., 38, 1869-1876 (1997). Briefly, full-length human cDNA was subcloned into shuttle plasmid vector pAdCMVLink1. After selecting the appropriate orientation by restriction analysis, the plasmid was linearized with NheI and co-transfected 293 cells with adenovirus DNA digested with ClaI. Cells were applied on agar and incubated at 37 ° C. for 15 days. Six plaques were taken and PCR screened; two plaques positive for cDNA were subjected to two plaque purification treatments. After confirming the presence of cDNA, recombinant adenovirus was propagated at 37 ° C. in 293 cells. Cell lysates were used to infect HeLa cells and expression of human LIPG was confirmed by western blot of the conditioned medium. Recombinant adenovirus (adhEL) was further propagated in 293 cells and purified by cesium chloride ultracentrifugation. A control adenovirus (Adnull) containing no cDNA insert was also plaque purified and purified as described above. Purified virus was stored at −80 ° C. in 10% glycerol / PBX. Wild-type C57BL / 6, human apoA-I mutant mice and LDL receptor mutant mice were obtained from the Jackson Laboratory. All mice were fed a Chinese diet. Wild-type and human apoA-I mutant mice were injected intravenously with AdhEL or Adnull 1 × ^{10 11} particles (about 2 × 10 ⁹ pfu) through the tail vein and 1 × ^{10 10} particles into LDLR-deficient mice. In all experiments, blood was obtained from the tracheal plexus 1 day before and at various time points after injection.

Intravenous injection of AdhEL into wild-type C57BL / 6 mice resulted in HDL cholesterol expression in the liver (FIG. 17) and decreased plasma concentrations and maintained significantly lower levels than control virus injected mice for at least 41 days after injection ( 18). Lipoproteins were separated by FPLC gel filtration, and no HDL was detected after 14 days of adenovirus injection (FIG. 19). Recombinant LIPG adenovirus was injected into human apoA-I mutant mice (having much higher concentrations of HDL cholesterol and apoA-I) resulting in a decrease in both levels of HDL cholesterol (FIG. 20) and apoA-I (FIG. 21). To measure the relative effect of LIPG expression on HDL compared to lipoproteins VLDL and LDL containing apoB, low-level LIPG adenoviruses were fed Chinese diets containing about 70% VLDL / LDL and about 30% HDL. Received LDL receptor defective mice were injected. As before, expression of LIPG decreased HLD cholesterol levels (FIG. 23). LIPG expression reduced VLDL / LDL cholesterol levels in the same mouse (FIG. 24), but the effect was proportionally less. Overexpression of LIPG reduces VLDL / LDL cholesterol and cannot rule out the role of LIPG in the regulation of apoB containing lipoproteins.

Example 16 Lipid / Lipoprotein Analysis

Plasma total cholesterol and HDL cholesterol levels were measured enzymatically on a diagnostic system using Sigma reagents (Cobas Fara, Roche Diagnotic systems). ApoA-I was quantified using Turbidity Assay (Sigma) on Cobas Fara. Plasma samples were collected and described in Tsukamoto et al., J. Clin. Invest., See description above, was analyzed by rapid protein liquid chromatography (FPLC) gel filtration (Pharmacia LKB Biotechnology) using two Superose 6 columns in series. Fractions (0.5 mL) were collected and cholesterol levels were measured using an enzyme assay (Wako Pure Chemical Industries).

Example 17 Identification of LIPG Inhibitors

Modulators of EL activity can be identified by the following method.

Recombinant LIPG can be purified from conditioned medium of stably transfected Chinese hamster ovary cells, baculovirus infected insect cells, yeast (Pichia pastoris, Kluveromyces Lactis) or other sources. Non-recombinant members of LIPG (eg, human plasma, endothelial cell conditioned medium, etc.) may also be used. One example of primary screening to explore modulators of LIPG activity is to use the soluble fluorescent substrate 4-methylumberriferyl hepatanoate. This method is continuous and homogeneous. Hydrolysis of the substrate by LIPG can be measured with a microplate fluorometer to produce highly fluorescent 4-methylumbelliferone. Other primary screening assays that can be used include scintillation proximity assays (Amersham) measuring phospholipase activity, radioactive phosphors of lower throughput than those described in Example 7 (also proposed below as secondary assays). Lipase assays or alternative phospholipase assays as shown in Example 8.

Like other TG lipases, the catalytic center of LIPG consists of the same catalytic triads as found in serine proteases (ser, his, asp). In fact, other TG lipases such as lipoprotein lipases are inhibited by serine protease inhibitors such as PMSF and DFP. Any one of these compounds can be used as a positive control for LIPG activity inhibitors.

Secondary Assay: Active compounds in the esterase assay or the alternative screening assay described above can be analyzed by a typical radiometric phospholipase A assay. This assay measures radiolabeled palmitic acid released from complex micelles containing [14C] -dipalmitoyl-phosphatidylcholine. Other assays that utilize fluorescent substrates and are easier to process larger amounts may also be used.

Selectivity Analysis: Compounds can also be analyzed for inhibition of the related enzymes lipoprotein lipase (LPL) and pancreatic lipase (PL). Human PL and cattle LPL are commercially available, and this assay can be easily performed. The phospholipase activity of PL is measured in the same manner as described above for the secondary assay of LIPG. Since LPL is originally a TG lipase, a secondary assay measures the release of radiolabeled fatty acids (oleic acid) from radiolabeled TG (triolein) substrates (see Example 7). This assay is similar in capacity and can be transformed into other assays that utilize fluorescent substrates and can analyze higher throughputs.

The phospholipase activity of LIPG can be tested in an in vitro assay for HDL, a substrate in vivo. Radiolabeled HDL can be made by exchange with radiolabeled phospholipids, which can then be used to measure LIPG phospholipase activity and the activity of the compounds obtained after selection.

In another assay, the effect of preculture of LIPG, HLD, +/- compounds on radiolabeled cholesterol release from cultured cells such as rat Fu5AH liver cancer cell line can be measured.

In vivo assays to evaluate compounds were performed with wild type mice, mice overexpressing LIPG and mice without LIPG as controls. If mutant mice show reduced HDL compared to control mice as in the case of adenoEL expression, treatment of mutant mice with LIPG inhibitory compounds is expected to elevate HDL to levels of control mice. Compounds can also be tested for LIPG inhibitory activity (elevation of HDL) in other animals such as LDLR-/-mice, apoA1 mutant mouse hamsters or rabbits. Compounds that elevate LIPG or LIPG activity are expected to elevate HDL in these or other animal models.

Example 18-Inhibitory Small Molecule Treatment Methods

With the small molecule identified in the screening method outlined in Example 17 as being capable of inhibiting LIPG polypeptide in vitro (hereinafter referred to as "inhibitory small molecule"), it was tested whether the LIPG polypeptide could be inhibited in vivo. Small molecules are investigated by oral (if orally bioavailable) or by intravenous injection to wild-type and LIPG mutant mice. The activity of the LIPG polypeptide is measured before and after injection of heparin (to release the enzyme from the bound site). In addition, cholesterol, VLDL, LDL and HDL cholesterol and apoA-I levels are monitored in animals administered with inhibitory small molecules. Finally, LDL receptor deficient mice are fed athero-induced diets and administered with inhibitory small molecules or placebo for 8 weeks. Atherosclerosis was quantified in the aorta of mice to determine whether administration of inhibitory small molecules reduced the progression or induced regression of atherosclerosis. Based on these preclinical data, hamsters, rabbits, or pigs, such as hamsters, rabbits, or pigs, act on inhibitory small molecules that increase HDL cholesterol levels, decrease VLDL and LDL cholesterol levels, and / or inhibit the progression of atherosclerosis. Additional animal models can be tested.

Inhibitory small molecules that have been found to exhibit desirable properties can be administered to a patient with a pharmaceutically acceptable carrier. Inhibitory small molecules can be administered to a patient in a variety of ways, such as oral administration and intravenous injection. Patient HDL, VLDL and LDL cholesterol levels are monitored to determine the efficacy of inhibitory small molecules and to optimize dosage and dosing protocol.

Example 19-Inhibitory Peptide Treatment Methods

The therapeutic peptide is confirmed by testing a fragment of the LIPG polypeptide, ie by measuring whether the fragment inhibits the activity of the LIPG polypeptide in vitro. Once identified, the "inhibitory peptide" is then tested for activity that inhibits the LIPG polypeptide in vivo. Inhibitory peptides are E. coli. It can be produced recombinantly in E. coli and purified by methods known to those skilled in the art. The effect of inhibitory peptides is investigated in wild-type mice and LIPG mutant mice by intravenous injection of inhibitory peptides. The activity of the LIPG polypeptide is measured with plasma before and after injection of heparin (to release the enzyme from the binding site). In addition, cholesterol, VLDL, LDL and HDL cholesterol and apoA-I levels are monitored in animals administered with inhibitory peptides. Finally, LDL receptor deficient mice are fed athero-induced diets and administered inhibitory peptides or placebo for 8 weeks. Atherosclerosis was quantified in the aorta of mice to determine whether administration of inhibitory peptides reduced the progression or induced regression of atherosclerosis. Based on these preclinical data, hamsters, rabbits, or pigs, such as hamsters, rabbits, or pigs, act on inhibitory small molecules that increase HDL cholesterol levels, decrease VLDL and LDL cholesterol levels, and / or inhibit the progression of atherosclerosis. Additional animal models can be tested.

Inhibitory peptides that have been found to exhibit desirable properties can be administered to a patient with a pharmaceutically acceptable carrier. Inhibitory peptides can be administered to a patient in a variety of ways, such as oral administration and intravenous injection. Patient HDL, VLDL and LDL cholesterol levels are monitored to measure the efficacy of inhibitory peptides and optimize dosage and dosing protocols.

Example 20 Antisense Treatment Methods

A series of antisense oligonucleotides each complementary to about 20 bases in the LIPG cDNA sequence are chemically synthesized by standard techniques. Each oligonucleotide is used to transfect each cell expressing the LIPG gene according to standard transfection protocols to determine the most effective oligonucleotide for use in therapy.

About 24 to 48 hours after transfection with oligonucleotides, LIPG mRNA concentrations in cells are measured by quantitative PCR, northern blots, RNAse blocking or other suitable methods. Alternatively, LIPG expression can be monitored with specific antibodies that can be used to select effective antisense oligonucleotides. Oligonucleotides that effectively reduce LIPG mRNA concentrations are formulated for in vivo delivery as a therapeutic agent.

Antisense LIPG sequences can be delivered to gene therapy vectors such as adenoviruses, adeno-associated viruses, retroviruses, naked DNA, or any other system described above. Such fragments can be used therapeutically when delivered to gene therapy vectors. Liver expression of such recombinant vectors is a preferred approach. Alternatively, synthetic antisense oligonucleotides may be formulated for in vivo delivery as a therapeutic agent, as described above.

Antisense oligonucleotides can be administered by the following routes: intravenous, transdermal, intradermal, pulmonary, oral, intraventricular, intravesicular, and topical routes. Routes of administration may include direct administration to the vessel wall (ie, endothelial and / or smooth muscle of the vessel). As one example, patients with low HDL-C may be administered by intravenous infusion of 0.5-2 mg / kg of effective antisense oligonucleotide every other day for up to 2-3 weeks. If LIPG is expressed in the liver, it is desirable to deliver antisense reagents to the blood circulation of the portal vein. This can be done by conjugating or complexing the oligonucleotide with a liver targeting component such as asialoglycoprotein. The therapeutic dosage and timing can vary depending on the efficacy of antisense delivery as well as variables such as the half-life, specificity and toxicology of antisense oligonucleotides.

The increase in HDL-C can be monitored using standard clinical laboratory procedures. Repeat the initial dosing regimen (as described above) as needed to maintain HDL-C greater than 35 mg / dl.

Example 21-Ribozyme Treatment Methods

Based on the LIPG cDNA sequence, a hammerhead ribozyme is prepared that effectively reduces LIPG mRNA concentration. This ribozyme consists of two "arms" consisting of 6 to 7 bases each of the nucleotide sequence complementary to LIPG mRNA, separated by a catalytic moiety. Examples of such hammerhead motifs are disclosed in Rossi et al., 1992, Aids Research and Human Retroviruses, 8, 183. This ribozyme is expressed in eukaryotic cells from suitable DNA vectors.

This ribozyme can be administered encapsulated in liposomes as described above.

Ribozyme / liposomal compositions are injected directly or delivered to the liver using a catheter, infusion pump, or stent. Routes of administration may include direct administration to the vascular wall (ie, endothelial and / or vascular smooth muscle). Patients are treated for up to 2 weeks with ribozyme 5-50 mg / kg / day in a pharmaceutically acceptable carrier. Increasing HDL-C and dosage regimen are monitored and measured as in antisense oligonucleotides.

Example 22-Neutralizing Antibody Treatment Methods

Chimeric antibodies consisting of anti-LIPG antibodies, antibody fragments or one or more LIPG binding moieties prepared as in Example 12 are used to inhibit LIPG activity in vivo. The antibody can be delivered only as a pill, infused for a period of time, or administered as a pill and infusion for a period of time. Generally, a dosage of 0.2 to 0.6 mg / kg can be given as a pill and then administered for an infusion time of 2 to 12 hours. Alternatively, a plurality of pill injections may be administered as needed to reduce LIPG and raise HDL-C every other day or every three or four days. Repeated doses are determined by measuring HDL-C levels. In addition, antibodies to LIPG can also be delivered through excipients in gene therapy to facilitate expression in vivo. The expression concentration of the antibody can be determined indirectly by measuring HDL-C levels or other vectors can be used if necessary.

Example 23 Use of Inhibitory Molecules or Enhancer Molecules

The binding competition to native LIPG can deliver a fragment of LIPG protein that can inhibit LIPG activity, the required coactivator molecule, cell surface receptor or binding protein as a therapeutic recombinant protein or via a vector of gene therapy.

As one example, LLGN polypeptides based on LIPG are described in Tsukamoto et al., J. Clin. Invest., 100, 107-114 (1997); Tsukamoto et al., J. Lipid Res., 38, 1869-1876 (1997), are cloned into recombinant adenovirus. LLGN cDNA was cloned into shuttle plasmid vector pAdCMVLink1. After selecting the appropriate orientation by restriction analysis, the plasmid was linearized with NheI and co-transfected 293 cells with adenovirus DNA digested with ClaI. Cells were layered on agar and incubated at 37 ° C. for 15 days. Plaques were taken and PCR screened; The cDNA positive plaques were subjected to two plaque purification treatments. After confirming the presence of cDNA, recombinant adenovirus was propagated at 37 ° C. in 293 cells. Cell lysates were used to infect HeLa cells and the expression of human EL was confirmed by western blot in conditioned medium. Recombinant adenovirus was further propagated in 293 cells and purified by cesium chloride ultracentrifugation. Purified virus was stored at −80 ° C. in 10% glycerol / PBX. Patients were injected intravenously with recombinant adenovirus 1 × ^{10 11} particles (about 2 × 10 ⁹ pfu).

Example 24-Method of Increasing LIPG Concentration in a Patient by Expression of LIPG from an Expression Vector

Recombinant adenovirus encoding human LIPG [Tsukamoto et al., J. Clin. Invest., 100, 107-114 (1997); Full length LIPG cDNA was cloned into Tsukamoto et al., J. Lipid Res., 38, 1869-1876 (1997). Full length human LIPG cDNA was cloned into shuttle plasmid vector pAdCMVLink1. After selecting the appropriate orientation by restriction analysis, the plasmid was linearized with NheI and co-transfected 293 cells with adenovirus DNA digested with ClaI. Cells were layered on agar and incubated at 37 ° C. for 15 days. Plaques were taken and PCR screened; cDNA positive plaques were treated twice with plaque purification. After confirming the presence of cDNA, recombinant adenovirus was propagated at 37 ° C. in 293 cells. Cell lysates were used to infect HeLa cells and expression of human LIPG was confirmed by western blot of the conditioned medium. Recombinant adenovirus (AdhEL) was further propagated in 293 cells and purified by cesium chloride ultracentrifugation. Purified virus was stored at −80 ° C. in 10% glycerol / PBX. Patients were injected intravenously with AdhEL or Adnull 1 × ^{10 11} particles (about 2 × 10 ⁹ pfu).

Example 25-Method of Increasing LIPG Activity Levels by Administration of Full-Length Wild-Type or Genetically Modified Recombinant LIPG Proteins

Wild-type LIPG proteins reduce VLDL and LDL cholesterol levels, and LIPG can be engineered to specifically act on VLDL and LDL cholesterol without affecting HDL cholesterol. Administration of wild-type or engineered recombinant LIPG can in some cases be used as a therapy to reduce the levels of VLDL and / or LDL cholesterol. Wild-type and / or synthesized LIPG proteins ("recombinant LIPG proteins") are E. coli. It can be produced recombinantly in E. coli and purified according to methods known in the art. Wild-type mice can also be studied by intravenous injection of recombinant LIPG protein. The activity of LIPG is measured in plasma. In addition, cholesterol, VLDL, LDL and HDL cholesterol and apoA-I levels are monitored in animals receiving recombinant LIPG protein. Finally, LDL receptor deficient mice are fed an atheroinduced diet and administered recombinant LIPG protein or placebo for 8 weeks. Atherosclerosis was quantified in the aorta of mice to determine whether administration of recombinant LIPG protein reduced the progression or induced regression of atherosclerosis. Based on these preclinical data, additional animal models, such as hamsters, rabbits, or pigs, may be used for the activity of recombinant LIPG proteins that reduce VLDL and LDL cholesterol levels (or) and inhibit the development of atherosclerosis. Can be tested Recombinant LIPG proteins that have been found to reduce VLDL and LDL cholesterol levels and / or exhibit desirable properties to inhibit the progression of atherosclerosis can be administered to a patient in combination with a pharmaceutically acceptable carrier. Recombinant LIPG polypeptides can be administered in a variety of ways, such as oral administration and intravenous injection.

All references presented herein are incorporated by reference.

Those skilled in the art who can easily understand the present invention can modify the present invention to carry out the above-described objects as well as to obtain the results and advantages as well as those set forth herein. The peptides, polynucleotides, methods, procedures, and techniques disclosed herein are presented by way of representative examples, but are illustrative and are not intended to limit the scope of the invention. Modifications and other uses of the invention, which are within the scope of the invention or defined by the appended claims, will be practiced by those skilled in the art.

Example 26-Demonstration of Glycosylation and Biological Activity of LIPG

A number of experiments were conducted to further characterize the LIPG. The following experiments show the biological activity of LIPG (named "Endothelial Lipase" or "EL" in this experiment and in the figures).

24 demonstrates that endothelial lipase is a glycoprotein. EL was treated with glycosidase EndoF, EndoH and neuraminidase. As a result, it was confirmed that the reduction of the EL band size through Western blot, which is consistent with the case where carbohydrates were removed from the polypeptide chain. Glycosylation was also inhibited with tunicamycin in EL expressing cells. As a result, a substantial reduction in the EL band size was observed. Glycosidase and tunicamycin experiments suggest that EL is a glycoprotein.

25 demonstrates that endothelial lipase is a heparin binding protein. Mice were injected with adenovirus vectors expressing EL AdhEL (Example 15). Blood was collected before and after injection of heparin. The intensity of the full-length 68kD EL band increased significantly after heparin injection, consistent with the presumption that EL is bound to heparin sulfate proteoglycans and released upon heparin administration.

26A and 26B and FIGS. 27A and 27B illustrate the lipolytic activity of endothelial lipase relative to lipoprotein lipase (LPL) and hepatic lipase (HL). This experiment is to determine whether endothelial lipase has triglyceride (TG) lipase activity (FIG. 27A). In multiple studies with different conditioned media, clear evidence of TG lipase activity was repeatedly observed. Phospholipase activity of endothelial lipase was analyzed in the same conditioned medium sample (FIG. 27B). The ratio of phospholipase activity to TG lipase activity was very constant as analyzed in various condition media. As an absolute value, the moles of fatty acids produced per conditioned medium volume were much higher by phospholipase activity than by TG lipase activity. It was then tested whether the serum affected endothelial lipase activity (FIGS. 27A and 27B). As expected, serum addition significantly increased lipoproteinlipase TG lipase activity in the absence of other apoC-II sources. Interestingly, the serum reproducibly substantially reduced the activity of endothelial lipase. This demonstrates that neither apoC-II nor other serum factors are needed as cofactors for endothelial lipase. It also suggests the presence of endogenous inhibitors of endothelial lipase in serum.

28A and 28B illustrate that expression of EL increases plasma phospholipase activity after heparin addition in a dose dependent manner. Injecting mice with three doses (1 × ^{10 11} , 3 × ^{10 10} or 1 × ^{10 10} particles) of AdhEL revealed the presence of EL protein in plasma after heparin injection. Main bands of 68kD and 40kD and subbands of 55kD were observed (FIG. 28A). The 68kD band is the full length glycosylated form of EL and 40kD is considered to be proteolytic fragment. The amount of EL protein in plasma measured by Western blot was proportional to the dose of injected vector (FIG. 28B). Expression of various concentrations of human EL significantly increased plasma-phospholipase activity after heparin injection compared to Adnull injected mice (9779 +/- 733, 6501 +/- 1299, 3963 + /, respectively). 796 nmol / ml / hr versus 952 +/- 258 nmol / ml / hr). Phospholipase activity following heparin injection was correlated with the concentrations of human EL protein corresponding to 68 kD (r = 0.98, P <0.01), 55 kD (r = 0.83, P <0.05) and 40 kD (r = 0.94, P <0.05) bands. Correlated.

Claims (65)

A composition comprising antisense nucleic acids to reduce expression of LIPG genes in a patient. The composition of claim 1 comprising an expression vector containing an antisense nucleic acid. The composition of claim 2, wherein the expression vector is selected from the group consisting of retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, and naked DNA vectors. The composition of claim 1, wherein the composition is a synthetic antisense nucleic acid. The composition of claim 4 wherein the antisense nucleic acid is an oligonucleotide. The composition of claim 5 wherein the oligonucleotide comprises a chemically modified base. A composition for lowering the enzymatic activity of a LIPG polypeptide in a patient, comprising a neutralizing antibody capable of binding to and reducing the enzymatic activity of the LIPG polypeptide. 8. The composition of claim 7, comprising an expression vector containing a DNA sequence encoding said antibody. The composition of claim 8, wherein the expression vector is selected from the group consisting of retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, and naked DNA vectors. A composition for containing an intracellular binding protein to lower the enzymatic activity of a LIPG polypeptide in a patient. The composition of claim 10 comprising an expression vector containing a DNA sequence encoding an intracellular binding protein. The composition of claim 11, wherein the expression vector is selected from the group consisting of retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, and naked DNA vectors. A composition containing an inhibitor capable of inhibiting the enzymatic activity of LIPG polypeptide in a patient. A composition containing an inhibitor capable of lowering the expression of the LIPG gene in a patient. A composition comprising ribozymes, capable of lowering the expression of LIPG in a patient. The composition of claim 15 comprising an expression vector containing a DNA sequence encoding a ribozyme. The composition of claim 16, wherein the expression vector is selected from the group consisting of retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, and naked DNA vectors. The composition of claim 14, wherein the ribozyme is a hammerhead type ribozyme. A composition for increasing the level of LIPG polypeptide in a patient, including an expression vector containing a DNA sequence encoding a LIPG polypeptide. A composition for increasing the level of LIPG polypeptide in a patient, comprising the LIPG polypeptide and a pharmaceutically acceptable carrier. A composition for increasing the level of LIPG polypeptide in a patient, comprising an enhancer capable of increasing expression of the LIPG gene. A composition for increasing the enzymatic activity of a LIPG polypeptide in a patient, comprising an enhancer that binds to and increases its enzymatic activity. A method of increasing levels of high density lipoprotein (HDL) cholesterol and apolipoprotein AI in a patient, comprising administering to the patient a composition that reduces the enzymatic activity of LIPG in the patient. The method of claim 23, wherein the composition lowers the level of LIPG polypeptide in the patient. The method of claim 23, wherein the composition contains antisense nucleic acid. The method of claim 25, wherein the antisense nucleic acid is a synthetic antisense nucleic acid. The method of claim 26, wherein the antisense nucleic acid is modified to increase the chemical stability of the nucleic acid. The method of claim 23, wherein the composition comprises neutralizing antibodies capable of binding to and lowering the enzymatic activity of the LIPG polypeptide. The method of claim 23, wherein the composition comprises an inhibitor that inhibits the enzymatic activity of the LIPG polypeptide. The method of claim 29, wherein the inhibitor comprises a compound that lowers the expression of the LIPG gene. The method of claim 23, wherein the composition comprises a ribozyme that cleaves mRNA encoding LIPG. The method of claim 23, wherein the composition comprises DNA molecules and liposomes. 33. The method of claim 32, wherein the liposomes are cationic liposomes. The method of claim 23, wherein the composition comprises DNA and a pharmaceutically acceptable carrier. The method of claim 23, wherein the composition comprises an expression vector. 36. The method of claim 35, wherein the expression vector is selected from the group consisting of retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, and naked DNA vectors. 36. The method of claim 35, wherein the expression vector comprises a DNA sequence encoding a ribozyme. The method of claim 36, wherein the expression vector is selected from the group consisting of retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, and naked DNA vectors. 36. The method of claim 35, wherein the expression vector comprises a DNA sequence encoding an antisense nucleic acid. The method of claim 35, wherein the expression vector comprises a DNA sequence encoding a neutralizing antibody that binds to LIPG. 36. The method of claim 35, wherein the expression vector comprises a DNA sequence encoding an intracellular binding protein capable of binding to and neutralizing LIPG. The method of claim 41, wherein the intracellular binding protein is an antibody. 36. The method of claim 35, wherein the expression vector comprises a DNA sequence encoding an inhibitory molecule that inhibits the enzymatic activity of LIPG. The method of claim 23, further comprising administering to the patient a composition capable of expressing apolipoprotein AI. A method of lowering ultra low density lipoprotein (VLDL) cholesterol in a patient, comprising administering to the patient a composition that can increase the enzymatic activity of LIPG in the patient. 46. The method of claim 45, wherein the composition comprises a LIPG polypeptide and a pharmaceutically acceptable carrier. The method of claim 46, wherein the composition is an expression vector capable of expressing a LIPG polypeptide. 48. The method of claim 47, wherein the expression vector is selected from the group consisting of retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors. 46. The method of claim 45, wherein the composition comprises an enhancer that increases the enzymatic activity of the LIPG polypeptide. 46. The method of claim 45, wherein the composition comprises an enhancer that increases the expression of the LIPG gene. A method of lowering low density lipoprotein (LDL) cholesterol in a patient, comprising administering to the patient a composition that can increase the enzymatic activity of LIPG in the patient. The method of claim 51, wherein the composition comprises a LIPG polypeptide and a pharmaceutically acceptable carrier. The method of claim 52, wherein the composition is an expression vector capable of expressing a LIPG polypeptide. 54. The method of claim 53, wherein the expression vector is selected from the group consisting of retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors. The method of claim 53, wherein the composition comprises an enhancer that increases the enzymatic activity of the LIPG polypeptide. The method of claim 53, wherein the composition comprises an enhancer that increases expression of the LIPG gene. Lowering the level of LDL cholesterol in the patient, including administering to the patient an enhancer that preferentially enhances the enzymatic reaction between the LIPG polypeptide and LDL cholesterol as compared to the enzymatic reaction between the LIPG polypeptide and HDL cholesterol and apolipoprotein AI. Way. Lowering the level of VLDL cholesterol in the patient, including administering to the patient an enhancer that preferentially enhances the enzymatic reaction between the LIPG polypeptide and VLDL cholesterol as compared to the enzymatic reaction between the LIPG polypeptide and HDL cholesterol and apolipoprotein AI. Way. A method for diagnosing predisposition to low levels of HDL cholesterol and apolipoprotein AI, comprising obtaining a tissue sample from a patient and measuring the level of LIPG polypeptide in the sample. 60. The method of claim 59, wherein the tissue is blood. 61. The method of claim 60, wherein the level of LIPG polypeptide in the sample is measured by immunoassay. 60. The method of claim 59, wherein the level of LIPG polypeptide is analyzed by measuring the level of LIPG mRNA. (A) the level of HDL cholesterol and apolipoprotein AI in a first sample comprising (1) HDL cholesterol and apolipoprotein AI, (2) LIPG polypeptide, and (3) a test compound. Compare the levels of HDL cholesterol and apolipoprotein AI in other samples containing protein AI and (5) LIPG polypeptides, and (B) the first sample has HDL cholesterol and apolipoprotein AI at a higher level than other samples. By observing whether the test compound is effective in inhibiting the enzymatic reaction between the LIPG polypeptide and HDL cholesterol and apolipoprotein AI, including that the test compound is enzymatic between the LIPG polypeptide and HDL cholesterol and apolipoprotein AI. How to determine if the reaction can be suppressed. A level of VLDL cholesterol in a first sample comprising (A) (1) VLDL cholesterol, (2) LIPG polypeptide and (3) test compound, (4) VLDL cholesterol and (5) LIPG polypeptide Compare the level of VLDL cholesterol in other samples and (B) increase the enzymatic reaction between the LIPG polypeptide and VLDL cholesterol by observing whether the first sample has VLDL cholesterol at a lower level than other samples. To determine whether the test compound can increase the enzymatic reaction between the LIPG polypeptide and the VLDL cholesterol, including confirming that it is effective in A level of LDL cholesterol in a first sample comprising (A) (1) LDL cholesterol, (2) LIPG polypeptide, and (3) a test compound, comprising: (4) LDL cholesterol and (5) LIPG polypeptide Compare the level of LDL cholesterol in other samples and (B) increase the enzymatic reaction between the LIPG polypeptide and LDL cholesterol by observing whether the first sample has LDL cholesterol at a lower level than the other samples. A method for determining whether a test compound can increase the enzymatic reaction between the LIPG polypeptide and LDL cholesterol, including confirming that it is effective in the treatment.