U.S. patent application number 11/254195 was filed with the patent office on 2006-04-27 for compositions and methods for effecting the levels of high density lipoprotein (hdl) cholesterol and apolipoprotein al, very low density lipoprotein (vldl) cholesterol and low density lipoprotein (ldl) cholesterol.
This patent application is currently assigned to Aventis Pharmaceuticals Inc.. Invention is credited to Dilip V. Amin, Kim-Anh Thi Doan, Michael Jaye, John A. Krawiec, Kevin J. Lynch, Dawn Marchadier, Cyrille Maugeais, Daniel J. Rader, Victoria J. South.
Application Number | 20060088504 11/254195 |
Document ID | / |
Family ID | 35966201 |
Filed Date | 2006-04-27 |
United States Patent
Application |
20060088504 |
Kind Code |
A1 |
Jaye; Michael ; et
al. |
April 27, 2006 |
Compositions and methods for effecting the levels of high density
lipoprotein (HDL) cholesterol and apolipoprotein Al, very low
density lipoprotein (VLDL) cholesterol and low density lipoprotein
(LDL) cholesterol
Abstract
Compositions and methods for raising the level of HDL
cholesterol and apolipoprotein AI in a patient and for lowering the
levels of VLDL cholesterol and LDL cholesterol in a patient,
including compositions and methods which effect the expression of a
gene, LIPG, which encodes a lipase enzyme that is a member of the
triacylglycerol lipase family or which effect the enzymatic
activity of the enzyme.
Inventors: |
Jaye; Michael; (Glensdale,
PA) ; Doan; Kim-Anh Thi; (Spring City, PA) ;
Krawiec; John A.; (Gulph Mills, PA) ; Lynch; Kevin
J.; (Gurnee, IL) ; Amin; Dilip V.; (Lansdale,
PA) ; South; Victoria J.; (Collegeville, PA) ;
Marchadier; Dawn; (Sicklerville, NJ) ; Maugeais;
Cyrille; (Philadelphia, PA) ; Rader; Daniel J.;
(Collegeville, PA) |
Correspondence
Address: |
SYNNESTVEDT & LECHNER, LLP
2600 ARAMARK TOWER
1101 MARKET STREET
PHILADELPHIA
PA
191072950
US
|
Assignee: |
Aventis Pharmaceuticals
Inc.
Bridgewater
NJ
|
Family ID: |
35966201 |
Appl. No.: |
11/254195 |
Filed: |
October 19, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09277401 |
Mar 26, 1999 |
7008776 |
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11254195 |
Oct 19, 2005 |
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08985492 |
Dec 5, 1997 |
6395530 |
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09277401 |
Mar 26, 1999 |
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60032254 |
Dec 6, 1996 |
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60032783 |
Dec 6, 1996 |
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Current U.S.
Class: |
424/93.2 ;
424/450; 435/456; 435/458; 435/6.18; 514/44R |
Current CPC
Class: |
C12N 9/18 20130101; G01N
2333/918 20130101; C12N 9/20 20130101; G01N 33/573 20130101 |
Class at
Publication: |
424/093.2 ;
514/044; 424/450; 435/456; 435/458; 435/006 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; A61K 9/127 20060101 A61K009/127; A61K 48/00 20060101
A61K048/00; C12N 15/86 20060101 C12N015/86; C12N 15/88 20060101
C12N015/88 |
Claims
1-21. (canceled)
22. A composition for increasing the enzymatic activity of LIPG
polypeptide in a patient comprising an enhancer which binds to and
enhances the enzymatic activity of the LIPG polypeptide.
23-56. (canceled)
57. A method for lowering the level of LDL cholesterol in a patient
comprising administering to the patient an enhancer which
preferentially enhances the enzymatic reactions between LIPG
polypeptide and LDL cholesterol relative to the enzymatic reactions
between LIPG polypeptide and HDL cholesterol and apolipoprotein
AI.
58. A method for lowering the level of VLDL cholesterol in a
patient comprising administering to the patient an enhancer which
preferentially enhances the enzymatic reactions between LIPG
polypeptide and VLDL cholesterol relative to the enzymatic
reactions between LIPG polypeptide and HDL cholesterol and
apolipoprotein AI.
59-62. (canceled)
63. A method for determining whether a test compound can inhibit
the enzymatic reaction between the LIPG polypeptide and HDL
cholesterol and apolipoprotein AI comprising: (A) comparing the
level of HDL cholesterol and apolipoprotein AI in a first sample
comprising: (1) HDL cholesterol and apolipoprotein AI, (2) LIPG
polypeptide, and (3) said test compound with the level of HDL
cholesterol and apolipoprotein AI in another sample comprising: (4)
HDL cholesterol and apolipoprotein AI, and (5) LIPG polypeptide;
and (B) identifying whether or not said test compound is effective
in inhibiting the enzymatic reaction between the LIPG polypeptide
and HDL cholesterol and apolipoprotein AI by observing whether or
not the first sample has a higher level of HDL cholesterol and
apolipoprotein AI than that of said other sample.
64. A method for determining whether a test compound can enhance
the enzymatic reaction between the LIPG polypeptide and VLDL
cholesterol comprising: (A) comparing the level of VLDL cholesterol
in a first sample comprising: (1) VLDL cholesterol, (2) LIPG
polypeptide, and (3) said test compound with the level of VLDL
cholesterol in another sample comprising: (4) VLDL cholesterol, and
(5) LIPG polypeptide; and (B) identifying whether or not said test
compound is effective in enhancing the enzymatic reaction between
the LIPG polypeptide and VLDL cholesterol by observing whether or
not the first sample has a lower level of VLDL cholesterol than
that of said other sample.
65. A method for determining whether a test compound can enhance
the enzymatic reaction between the LIPG polypeptide and LDL
cholesterol comprising: (A) comparing the level of LDL cholesterol
in a first sample comprising: (1) LDL cholesterol, (2) LIPG
polypeptide, and (3) said test compound with the level of LDL
cholesterol in another sample comprising: (4) LDL cholesterol, and
(5) LIPG polypeptide; and (B) identifying whether or not said test
compound is effective in enhancing the enzymatic reaction between
the LIPG polypeptide and LDL cholesterol by observing whether or
not the first sample has a lower level of LDL cholesterol than that
of said other sample.
Description
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 08/985,492, filed Dec. 5, 1997, which claims
the benefit of provisional applications under 35 U.S.C. .sctn.
119(e), 60/032,254 and 601032,783, both of which were filed Dec. 6,
1996, the disclosures of which are incorporated herein by reference
in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates to methods and compositions for
increasing the level of high density lipoprotein (HDL) cholesterol
and apolipoprotein AI in a patient and to methods and compositions
for lowering the levels of very low density lipoprotein (VLDL)
cholesterol, and low density lipoprotein (LDL) cholesterol in a
patient. The invention includes within its scope methods and
compositions which lower the expression of, or inhibit the activity
of, a gene, LIPG, which encodes a lipase enzyme that lowers the
levels of HDL cholesterol and apolipoprotein AI. The invention
additionally includes within its scope methods and compositions to
increase the expression of, or enhance the activity of, the lipase
enzyme, resulting in lower levels of VLDL and LDL cholesterol.
Lipids
[0003] Lipids are water-insoluble organic biomolecules, which are
essential components of diverse biological functions, including the
storage, transport, and metabolism of energy, and membrane
structure and fluidity. Lipids are derived from two sources in man
and other animals: some lipids are ingested as dietary fats and
oils and other lipids are biosynthesized by the human or animal. In
mammals, at least 10% of the body weight is lipid, the bulk of
which is in the form of triacylglycerols.
[0004] Triacylglycerols, also known as triglycerides and
triacylglycerides, are made up of three fatty acids esterified to
glycerol. Dietary triacylglycerols are stored in adipose tissues as
a source of energy, or hydrolyzed in the digestive tract by
triacylglycerol lipases, the most important of which is pancreatic
lipase. Triacylglycerols are transported between tissues in the
form of lipoproteins.
[0005] Lipoproteins are micelle-like assemblies found in plasma
which contain varying proportions of different types of lipids and
proteins (called apoproteins). There are five main classes of
plasma lipoproteins, the major function of which is lipid
transport. These classes are, in order of increasing density:
chylomicrons; very low density lipoproteins (VLDL);
intermediate-density lipoproteins (IDL); low density lipoproteins
(LDL); and high density lipoproteins (HDL). Although many types of
lipid are found associated with each lipoprotein class, each class
transports predominantly one type of lipid: triacylglycerols
described above are transported in chylomicrons, VLDL, and IDL;
whereas phospholipids and cholesterol esters are transported in HDL
and LDL respectively.
[0006] Phospholipids are di-fatty acid esters of glycerol phosphate
which contain a polar group coupled to the phosphate. Phospholipids
are important structural components of cellular membranes.
Phospholipids are hydrolyzed by enzymes called phospholipases.
Phosphatidylcholine, an exemplary phospholipid, is a major
component of most eukaryotic cell membranes.
[0007] Cholesterol is the metabolic precursor of steroid hormones
and bile acids as well as an essential constituent of cell
membranes. In man and other animals, cholesterol is ingested in the
diet and is synthesized also by the liver and other tissues.
Dietary cholesterol is transported from the intestine to the liver
by large lipoprotein molecules in the blood. The liver secretes
Very Low Density Lipoprotein (VLDL) which transports cholesterol
and cholesterol ester and various other compounds into the
bloodstream. VLDL is partially converted in adipose tissue to Low
Density Lipoprotein (LDL). LDL transports both free and esterified
cholesterol to body tissues. High Density Lipoprotein (HDL)
transports cholesterol to the liver to be broken down and
excreted.
[0008] Membranes surround every living cell and serve as a barrier
between the intracellular and extracellular compartments. Membranes
also enclose the eukaryotic nucleus, make up the endoplasmic
reticulum, and serve specialized functions such as in the myelin
sheath that surrounds axons. A typical membrane contains about 40%
lipid and 60% protein, but there is considerable variation. The
major lipid components are phospholipids, specifically
phosphatidylcholine and phosphatidylethanolamine, and cholesterol.
The physicochemical properties of membranes, such as fluidity, can
be changed by modification of either the fatty acid profiles of the
phospholipids or the cholesterol content. Modulating the
composition and organization of membrane lipids also modulates
membrane-dependent cellular functions, such as receptor activity,
endocytosis, and cholesterol flux.
Enzymes
[0009] The triacylglycerol lipases are a family of enzymes which
play several pivotal roles in the metabolism of lipids in the body.
Three members of the human triacylglycerol lipase family have been
described: pancreatic lipase, lipoprotein lipase, and hepatic
lipase (Goldberg, I. J., Le, N.-A., Ginsberg, H. N., Krauss, R. M.,
and Lindgren, F. T. (1988) 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 lipase is primarily responsible for the hydrolysis of
dietary lipids. Variants of pancreatic lipase have been described,
but their physiological role has not been determined (Giller, T.,
Buchwald, P., Blum-Kaelin, D., and Hunziker, W. (1992) J. Biol.
Chem. 267,16509-16516). Lipoprotein lipase is the major enzyme
responsible for the distribution and utilization of triglycerides
in the body. Lipoprotein lipase hydrolyzes triglycerides in both
chylomicrons and VLDL. Hepatic lipase hydrolyzes triglycerides in
IDL and HDL and is responsible for lipoprotein remodeling. Hepatic
lipase also functions as a phospholipase and hydrolyzes
phospholipids in HDL.
[0010] Phospholipases play important roles in the catabolism and
remodeling of the phospholipid component of lipoproteins and the
phospholipids of membranes. Phospholipases also play a role in the
release of arachidonic acid and the subsequent formation of
prostaglandins, leukotrienes, and other lipids which are involved
in a variety of inflammatory processes.
[0011] The aforementioned lipases are approximately 450 amino acids
in length and have leader signal peptides to facilitate secretion.
The lipases are comprised of two principal domains (Winkler, K.,
D'Arcy, A., and Hunziker, W. (1990) Nature 343, 771-774). The amino
terminal domain contains the catalytic site while the carboxyl
domain is believed to be responsible for substrate binding,
cofactor association, and interaction with cell receptors (Wong,
H., Davis, R. C., Nikazy, J., Seebart, K. E., and Schotz, M.
C.-(1991) Proc. Natl. Acad. Sci. USA 88, 11290-11294; van
Tilbeurgh, H., Roussel, A., Lalouel, J.-M., and Cambillau, C.
(1994) J. Biol. Chem. 269,4626-4633; Wong, H., 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 the family is 22-65%, with local regions of high
homology corresponding to structural homologies which are linked to
enzymatic function.
[0012] The naturally occurring lipoprotein lipase is glycosylated.
Glycosylation is necessary for LPL enzymatic activity (Semenkovich,
C. F., Luo, C.-C., Nakanishi, M. K., Chen, S.-H., Smith, L C., and
Chan L. (1990) J. Biol. Chem. 265, 5429-5433). There are two sites
for N-linked glycosylation in hepatic and lipoprotein lipase and
one in pancreatic lipase. Additionally, four sets of cysteines form
disulfide bridges which are essential in maintaining structural
integrity for enzymatic activity (Lo, J.-Y., Smith, L.C., and Chan,
L. (1995) Biochem. Biophys. Res. Commun. 206, 266-271; Brady, L.,
Brzozowski, A. M., Derewenda, Z. S., Dodson, E., Dodson G., Tolley,
S., Turkenburg, J. P., Christiansen, L., Huge-Jensen B., Norskov,
L., Thim, L., and Menge, U. (1990) Nature 343, 767-770).
[0013] Members of the triacylglycerol lipase family share a number
of conserved structural features. One such feature is the "GXSXG"
motif, in which the central serine residue is one of the three
residues comprising the "catalytic triad" (Winkler, K., D'Arcy, A.,
and Hunziker, W. (1990) Nature 343, 771-774; Faustinella, F.,
Smith, L. C., and Chan, L. (1992) Biochemistry 31, 7219-7223).
Conserved aspartate and histidine residues make up the balance of
the catalytic triad. A short span of 19-23 amino acids (the "lid
region") forms an amphipathic helix structure and covers the
catalytic pocket of the enzyme (Winkler, K., D.'Arcy, A., and
Hunziker, W. (1990) Nature 343, 771-774). This region diverges
significantly between members of the family. It has been determined
recently that the span confers substrate specificity to the enzymes
(Dugi, K. A., Dichek H. L., and Santamarina-Fojo, S. (1995) J.
Biol. Chem. 270, 25396-25401). Comparisons between hepatic and
lipoprotein lipase have demonstrated that differences in
triacylglycerol lipase and phospholipase activities of the enzymes
are in part mediated by this lid region (Dugi, K. A., Dichek H. L.,
and Santamarina-Fojo, S. (1995) J. Biol. Chem. 270,
25396-25401).
[0014] The triacylglycerol lipases possess varying degrees of
heparin binding activity. Lipoprotein lipase has the highest
affinity for heparin. This binding activity has been mapped to
stretches of positively charged residues in the amino terminal
domain (Ma, Y., Henderson, H. E., Liu, M.-S., Zhang, H., Forsythe,
I. J., Clarke-Lewis, I., Hayden, M. R., and Brunzell, J. D. J.
Lipid Res. 35, 2049-2059). The 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) is
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 which allows the enzyme to accelerate LDL uptake
by acting as a bridge between LDL and the cell surface (Mulder, M.,
Lombardi, P., Jansen, H., vanBerkel 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).
[0015] Lipoprotein lipase and pancreatic lipase are both known to
function in conjunction with co-activator proteins: apolipoprotein
CII for lipoprotein lipase; and colipase for pancreatic lipase.
[0016] The genetic sequences encoding human pancreatic lipase,
hepatic lipase and lipoprotein lipase have been reported (Genbank
accession #M93285, #J03540, and #M15856 respectively). The
messenger RNAs of human hepatic lipase and pancreatic lipase are
approximately 1.7 and 1.8 kilobases in length respectively. Two
mRNA transcripts of 3.6 and 3.2 kilobases are produced from the
human lipoprotein lipase gene. These two transcripts utilize
alternate polyadenylation signals and differ in their translational
efficiency (Ranganathan, G., Ong, J. M., Yukht, A., Saghizadeh, M.,
Simsolo, R. B., Pauer, A., and Kern, P. A. (1995) J. Biol. Chem.
270, 7149-7155).
Physiological Processes
[0017] The metabolism of lipids involves the interaction of lipids,
apoproteins, lipoproteins, and enzymes.
[0018] Hepatic lipase and lipoprotein lipase are multifunctional
proteins which mediate the binding, uptake, catabolism, and
remodeling of lipoproteins and phospholipids. Lipoprotein lipase
and hepatic lipase function while bound to the luminal surface of
endothelial cells in peripheral tissues and the liver respectively.
Both enzymes participate in reverse cholesterol transport, which is
the movement of cholesterol from peripheral tissues to the liver
either for excretion from the body or for recycling. Genetic
defects in both hepatic lipase and lipoprotein-lipase are known to
be the cause of familial disorders of lipoprotein metabolism.
Defects in the metabolism of lipoproteins result in serious
metabolic disorders, including hypercholesterolemia,
hyperlipidemia, and atherosclerosis.
REPORTED DEVELOPMENTS
[0019] Atherosclerosis is a complex, polygenic disease which is
defined in histological terms by deposits (lipid or fibrolipid
plaques) of lipids and of other blood derivatives in blood vessel
walls, especially the large arteries (aorta, coronary arteries,
carotid). These plaques, which are more or less calcified according
to the degree of progression of the atherosclerotic process, may be
coupled with lesions and are associated with the accumulation in
the vessels of fatty deposits consisting essentially of cholesterol
esters. These plaques are accompanied by a thickening of the vessel
wall, hypertrophy of the smooth muscle, appearance of foam cells
(lipid-laden cells resulting from uncontrolled uptake of
cholesterol by recruited macrophages) and accumulation of fibrous
tissue. The atheromatous plaque protrudes markedly from the wall,
endowing it with a stenosing character responsible for vascular
occlusions by atheroma, thrombosis or embolism, which occur in
those patients who are most affected. These lesions can lead to
serious cardiovascular pathologies such as infarction, sudden
death, cardiac insufficiency, and stroke.
High Density Lipoprotein (HDL) Cholesterol Levels and
Atherosclerotic Diseases
[0020] High density lipoprotein (HDL) cholesterol levels are
inversely associated with risk of atherosclerotic cardiovascular
disease (Gordon et al., N. Engl. J. Med., 321, 1311-1316 (1989)).
At least 50% of the variation in HDL cholesterol levels is
genetically determined (Breslow, J. L., The Metabolic Basis of
Inhereited Disease, 2031-2052, McGraw-Hill, New York (1995); Heller
et al., N. Engl. J. Med., 328, 1150-1156 (1993)), but the genes
responsible for variation in HDL levels have not been fully
elucidated. Lipoprotein lipase (LPL) and hepatic lipase (HL), two
members of the triacylglycerol (TG) lipase family, both influence
HDL metabolism (Breslow, supra; 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))
and the HL (LIPC) locus has been associated with variation 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 for HDL cholesterol is about 35
to 65 mg/dL, and the HDL level should account for more than 25% of
the total cholesterol.
Very Low Density Lipoprotein (VLDL) and Low Density Lipoprotein
(LDL) Cholesterol Levels and Atherosclerotic Diseases
[0021] High levels of circulating LDL and VLDL cholesterol are
associated with increased risk of atherosclerosis.
[0022] VLDL are the precursors of LDL. Therapeutic agents that
lower plasma VLDL and LDL cholesterol levels are highly desirable
because of the known strong association between these lipid
parameters and coronary heart disease.
[0023] Epidemiologic studies have demonstrated a strong
relationship between elevated LDL cholesterol and coronary heart
disease (CHD) and other atherosclerotic vascular diseases (Kannell,
W. B., Am. J. Cardiol., 76, 69C-77C (1995)). Three major secondary
prevention trials performed with statins have demonstrated that
reduction of LDL cholesterol levels result in significant reduction
in CHD events and total 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., Editorial, 1436-1439
(1998)). Two large primary prevention trials with statins have also
demonstrated significant benefit of LDL cholesterol reduction with
statins in reducing cardiovascular events (Grundy, supra; Shepherd
et al., N. Engl. J. Med., 33, 1301-1307 (1995); Downs et al., JAMA,
279, 1615-1622 (1998)). However, current therapies do not
adequately reduce LDL cholesterol levels in all persons. 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 in reducing VLDL cholesterol as LDL
cholesterol. Therefore, new approaches to reducing both LDL
cholesterol and VLDL cholesterol are still needed.
[0024] Ideally, the range for VLDL cholesterol is about 1 to 30
mg/dL and the range for LDL cholesterol is about 60 to 160 mg/dL.
The LDL to HDL ratio is ideally less than 3.5.
The Role of Triacylglycerol Lipases in Atherosclerotic Diseases
[0025] The role of triacylglycerol lipases in vascular pathologies
such as atherosclerosis has been an area of intense study (reviewed
in Olivecrona, G., and Olivecrona, T. (1995) Curr. Opin. Lipid. 6,
291-305). Generally, the action of the lipoprotein lipase is
believed to be antiatherogenic because this enzyme lowers serum
triacylglycerol levels and promote HDL formation. Transgenic
animals expressing human lipoprotein lipase have decreased levels
of plasma triglycerides and an increased level 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, F.-R., LeBoeuf, R.
C., Henderson, H., Castellani, L. W., Lusis, A. J., ma, Y.,
Forsythe, I. J., Zhang, H., Kirk, E., Brunzell, J. D., and Hayden,
M. R. (1994) J. Biol. Chem. 269, 11417-11424). Humans with genetic
defects resulting in decreased levels of lipoprotein lipase
activity have been found to have hypertriglyceridemia, but no
increased risk of coronary heart disease. This is reported to be
due to the lack of production of intermediate-sized, atherogenic
lipoproteins which could accumulate within the subendothelial space
(Zilversmit, D. B. (1973) Circ. Res. 33, 633-638).
[0026] In contrast to lipoprotein lipase (LPL), the physiologic
function of HL appears to be related to the metabolism of
lipoprotein remnants and HDL (Bensadoun et al., Curr. Opin.
Lipidol., 7, 77-81 (1996)). Genetic deficiency of HL is associated
with modestly increased levels of remnants and HDL cholesterol in
humans (Hegele et al., Arterioscler. Thromb., 13, 720-728 (1993))
and mutant mice (Homanics et al., J. Biol. Chem., 270, 2974-2980
(1995)). Despite increased plasma cholesterol levels, HL deficiency
is associated with reduced atherosclerosis in apoE mutant mice
(Mezdour et al., J. Biol. Chem., 272, 13570-13575 (1997)).
Transgenic animals overexpressing HL have decreased HDL (Busch et
al., J. Biol. Chem., 269, 16376-16382 (1994); Fan et al., Proc.
Natl. Acad. Sci. USA, 91, 8724-8728 (1994)). Increased HL activity
in humans is associated with low HDL cholesterol. The HL locus on
chromosome 15q21 has been associated with variation in plasma HDL
cholesterol levels in humans (Cohen et al., J. Clin. Invest., 94,
2377-2384 (1994); Guerra et al., Proc. Natl. Acad. Sci. USA, 94
4532-4537 (1997)), but accounts for only a portion of the genetic
contribution to variation in HDL cholesterol levels. There is at
least one major locus influencing HDL cholesterol levels in humans
that is distinct from the HL locus (Mahaney et al., Arterioscler.
Thromb. Vasc. Biol., 15, 1730-1739 (1995)).
[0027] In the localized area of an atherosclerotic lesion, the
increased level of lipase activity is hypothesized to accelerate
the atherogenic process (Zilversmit, D. B. (1995) Clin. Chem.
41,153-158; Zambon, A., Torres, A., Bijvoet, S., Gagne, C.,
Moojani, S., Lupien, P. J., Hayden M. R., and Brunzell, J. D.
(1993) Lancet 341, 1119-1121). This may be due to an increase in
the binding and uptake of lipoproteins by vascular tissue mediated
by lipases (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). Additionally,
a high local level of lipase activity may result in cytotoxic
levels of fatty acids and lysophosphatidylcholine being produced in
precursors of atherosclerotic lesions.
[0028] Despite the understanding that has evolved regarding the
role of lipase enzyme activity in regulating the levels of lipids
and the various plasma lipoproteins, there is a need to identify
and develop therapies which can increase the levels of HDL
cholesterol, as well as lower the levels of VLDL and LDL
cholesterol to reduce the risk of developing atherosclerotic
cardiovascular diseases.
SUMMARY OF THE INVENTION
[0029] In accordance with the present invention, there is provided
a composition for lowering the expression of the LIPG gene in a
patient comprising an antisense nucleic acid, including for
example, an expression vector which includes said antisense nucleic
acid. Examples of preferred expression vectors are retroviral
vectors, adenoviral vectors, adeno-associated viral vectors,
herpesviral vectors, and naked DNA vectors. The antisense nucleic
acid can be, for example, an oligonucleotide which contains
chemically modified bases.
[0030] Another aspect of the present invention is the provision of
a composition for lowering the enzymatic activity of the LIPG
polypeptide in a patient comprising a neutralizing antibody capable
of binding to the LIPG polypeptide and lowering its enzymatic
activity, including, for example, an expression vector which
includes a DNA sequence encoding said antibody. Examples of
preferred expression vectors are retroviral vectors, adenoviral
vectors, adeno-associated viral vectors, herpesviral vectors, and
naked DNA vectors.
[0031] Still another aspect of the present invention is the
provision of a composition for lowering the enzymatic activity of
the LIPG polypeptide in a patient comprising an intracellular
binding protein, including, for example, an expression vector which
includes a DNA sequence encoding said intracellular binding
protein. Examples of preferred expression vectors are retroviral
vectors, adenoviral vectors, adeno-associated viral vectors,
herpesviral vectors, and naked DNA vectors.
[0032] Yet other aspects of the present invention are the provision
of: (A) a composition which comprises an inhibitor that is capable
of inhibiting the enzymatic activity of the LIPG polypeptide in a
patient; (B) a composition which comprises an inhibitor that is
capable of lowering the expression of the LIPG gene in a patient;
and (C) a composition which is capable of lowering the expression
of LIPG in a patient and which comprises a ribozyme, including, for
example, an expression vector which includes a DNA sequence
encoding said ribozyme. Examples of preferred expression vectors
are retroviral vectors, adenoviral vectors, adeno-associated viral
vectors, herpesviral vectors, and naked DNA vectors. A preferred
ribozyme is a hammerhead ribozyme.
[0033] The present invention provides also: (D) a composition which
increases the level of LIPG polypeptide in a patient and which
comprises an expression vector that includes a DNA sequence
encoding the LIPG polypeptide or an enhancer that is capable of
increasing the expression of the LIPG gene; and (E) a composition
which increases the enzymatic activity of LIPG polypeptide in a
patient which comprises an enhancer that binds to and enhances the
enzymatic activity of the LIPG polypeptide.
[0034] In addition, the present invention provides a method for
raising the level of high density lipoprotein (HDL) cholesterol and
apolipoprotein AI in a patient by administering to the patient a
composition which lowers the enzymatic activity of LIPG in said
patient, for example, by lowering the level of LIPG polypeptide in
the patient. In preferred form, the method involves the use of a
composition which comprises an antisense nucleic acid, particularly
one that is modified to increase the chemical stability of the
nucleic acid. The aforementioned method can be practiced also by
use of a composition which comprises a neutralizing
antibody-capable of binding to the LIPG polypeptide and lowering
its enzymatic activity or a composition which comprises an
inhibitor which inhibits the enzymatic activity of LIPG
polypeptide, for example, a compound which lowers the expression of
the LIPG gene or a composition which comprises a ribozyme that
cleaves mRNA encoding LIPG, or a composition which comprises a DNA
molecule and a liposome, for example, a cationic liposome.
[0035] In preferred form, the aforementioned method comprises also
the administration of a composition which is capable of expressing
apolipoprotein AI in said patient.
[0036] Another aspect of the present invention is the provision of
a method for lowering the level of very low density lipoprotein
(VLDL) cholesterol in a patient by administering to the patient a
composition which is capable of increasing the enzymatic activity
of LIPG in said patient, for example, by use of a composition which
comprises an LIPG polypeptide and a pharmaceutically acceptable
carrier and which includes preferably an expression vector that is
capable of expressing an LIPG polypeptide, preferably a retroviral
vector, an adenoviral vector, or an adeno-associated viral vector.
The aforementioned method can be practiced by use of a composition
which comprises an enhancer that enhances the enzymatic activity of
LIPG polypeptide or an enhancer that increases expression of the
LIPG gene.
[0037] Still another aspect of the present invention is the
provision of a method for lowering the level of low density
lipoprotein (LDL) cholesterol in a patient by administering to the
patient a composition which is capable of increasing the enzymatic
activity of LIPG in the patient, preferably by use of an LIPG
polypeptide, for example, by use of an expression vector that is
capable of expressing the LIPG polypeptide, preferably by use of a
retroviral vector, an adenoviral vector, or an adeno-associated
viral vector. The aforementioned method includes preferably the use
of a composition which comprises an enhancer that enhances the
enzymatic activity of LIPG polypeptide or an enhancer which
increases the expression of the LIPG gene.
[0038] The present invention provides also a method for lowering
the level of LDL cholesterol in a patient by administering to the
patient an enhancer which preferentially enhances the enzymatic
reactions between LIPG polypeptide and LDL cholesterol relative to
the enzymatic reactions between LIPG polypeptide and HDL
cholesterol and apolipoprotein AI.
[0039] In addition, the present invention provides a method for
lowering the level of VLDL cholesterol in a patient by
administering to the patient an enhancer which preferentially
enhances the enzymatic reactions between LIPG polypeptide and VLDL
cholesterol relative to the enzymatic reactions between LIPG
polypeptide and HDL cholesterol and apolipoprotein AI.
[0040] Still another aspect of the present invention is the
provision of a method for diagnosing a predisposition to low HDL
cholesterol and apolipoprotein AI levels by obtaining a tissue
sample from a patient and measuring the level of LIPG polypeptide
in the sample, for example, by use of blood tissue and the use of
an immunoassay for measurement. In another aspect of the present
invention, the levels of LIPG polypeptide are measured by measuring
the levels of LIPG mRNA.
[0041] An additional aspect of the present invention is the
provision of a method for determining whether a test compound can
inhibit the enzymatic reaction between the LIPG polypeptide and HDL
cholesterol and apolipoprotein AI comprising: (A) comparing the
level of HDL cholesterol and apolipoprotein AI in a first sample
comprising: (1) HDL cholesterol and apolipoprotein AI, (2) LIPG
polypeptide, and (3) the test compound with the level of HDL
cholesterol and apolipoprotein AI in another sample comprising: (4)
HDL cholesterol and apolipoprotein AI, and (5) LIPG polypeptide;
and (B) identifying whether or not the test compound is effective
in inhibiting the enzymatic reaction between the LIPG polypeptide
and HDL cholesterol and apolipoprotein AI by observing whether or
not the first sample has a higher level of HDL cholesterol and
apolipoprotein AI than that of said other sample.
[0042] The present invention provides also a method for determining
whether a test compound can enhance the enzymatic reaction between
the LIPG polypeptide and VLDL cholesterol comprising: (A) comparing
the level of VLDL cholesterol in a first sample comprising: (1)
VLDL cholesterol, (2) LIPG polypeptide, and (3) the test compound
with the level of VLDL cholesterol in another sample comprising:
(4) VLDL cholesterol, and (5) LIPG polypeptide; and (B) identifying
whether or not the test compound is effective in enhancing the
enzymatic reaction between the LIPG polypeptide and VLDL
cholesterol by observing whether or not the first sample has a
lower level of VLDL cholesterol than that of said other sample.
[0043] Still another aspect of the present invention is the
provision of a method for determining whether a test compound can
enhance the enzymatic reaction between the LIPG polypeptide and LDL
cholesterol comprising: (A) comparing the level of LDL cholesterol
in a first sample comprising: (1) LDL cholesterol, (2) LIPG
polypeptide, and (3) the test compound with the level of LDL
cholesterol in another sample comprising: (4) LDL cholesterol, and
(5) LIPG polypeptide; and (B) identifying whether or not the test
compound is effective in enhancing the enzymatic reaction between
the LIPG polypeptide and LDL cholesterol by observing whether or
not the first sample has a lower level of LDL cholesterol than that
of said other sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] FIG. 1 shows the sequences (SEQ ID Nos: 17-31) of the
primers used in the exemplified PCR amplifications.
[0045] FIG. 2 shows the nucleic acid sequence (SEQ ID NO: 1) and
the deduced amino acid sequence (SEQ ID NO: 2) of the differential
display RT-PCR product containing the LIPG gene cDNA. The sequences
corresponding to the two primers used in the amplification are
underlined. The termination codon and polyadenylation signal are
boxed. The GAATTC motifs and flanking sequence are from the pCRII
vector into which the product was cloned.
[0046] FIG. 3 shows the nucleic acid sequence (SEQ ID NO: 3) and
the deduced amino acid sequence (SEQ ID NO: 4) of the 5' RACE
extension of the LIPG cDNA. The sequences corresponding to the two
primers used in the amplification are underlined. The GAATTC motifs
and flanking sequence are from the pCRII vector into which the
product was cloned.
[0047] FIG. 4 shows the sequence (SEQ ID NO: 7) of the cDNA
containing the complete open reading frame of the LIPG gene, LLGXL.
The start codon (ATG) and termination codon (TGA) are boxed. The
DraI site (TTTAAA) and SrfI site (GCCCGGGC) used in the
construction of the expression vectors are underlined.
[0048] FIG. 5 shows the deduced amino acid sequence (SEQ ID NO: 8:)
of the LLGXL protein. The predicted signal sequence is
underlined.
[0049] FIG. 6 shows a protein sequence alignment of the members of
the triacylglycerol lipase gene family (SEQ ID Nos: 13-15). Shaded
residues are identical to the LLGXL protein (SEQ ID NO: 8). The
deduced amino acid sequence of human LIPG(EL) is provided on the
top line and is compared with the other major members of the TG
lipase family, LPL, HL and PL. EL residues identical to those in at
least one other member of the family are shaded as well as the
corresponding residue in the other family member. Amino acids are
numbered according to convention beginning with the initial residue
of the secreted protein. The predicted sites of signal peptide
cleavage are marked with a solid line between amino acid residues.
The GXSXG lipase motif containing the active serine is boxed. The
amino acids of the catalytic triad are marked with an asterisk. The
conserved cysteines are marked with filled circles. Potential
N-linked glycosylation sites are marked with arrowheads. The lid
region is indicated by a bold line. Gaps were introduced into the
sequences to maximize the alignment values using the CLUSTAL
program.
[0050] FIG. 7 shows a northern analysis of LIPG mRNA in THP-1
cells. Cells were stimulated with either PMA or PMA and oxidized
LDL (PMA+oxLDL). Numbers at the left indicate the positions of RNA
standards (in kilobases).
[0051] FIG. 8 shows a northern-blot analysis of expression of LIPG
mRNA compared with LPL in human tissues. A blot containing mRNA
from the indicated human tissues was incubated with radiolabelled
LPL and .beta.-actin (ACTB) probes as described.
[0052] FIG. 9 shows a Northern-blot analysis of cultured cell
lines. The panel on the left (lanes 1-6) was hybridized with the
LIPG(EL) probe and that on the right (lanes 7-12) with the LPL
probe. Lanes 1, 7, unstimulated HUVEC; lanes 2, 8, HUVEC stimulated
with PMA; lanes 3, 9, HUVEC stimulated with thrombin; lanes 4, 10,
unstimulated HCAEC; lanes 5, 11, HCAEC stimulated with PMA; lanes
6, 12, THP-1 stimulated with PMA.
[0053] FIG. 10 shows the sequence of the immunizing peptide (SEQ ID
NO: 16) and its relation to the LLGXL protein sequence. The peptide
is shown in the shaded box. The terminal cysteine was introduced to
aid coupling of the peptide to the carrier protein.
[0054] FIG. 11 shows the results obtained when conditioned media
from HUVEC and HCAEC were subjected to immunoblot analysis with
rabbit anti-EL peptide antiserum. Lane 1, unconditioned media; lane
2, unstimulated HUVEC; lane 3, HUVEC stimulated with PMA; lane 4,
unstimulated HCAEC; lane 5, HCAEC stimulated with PMA.
[0055] FIG. 12 shows a western analysis of heparin-Sepharose bound
proteins in conditioned medium from COS-7 cells transiently
transfected with an expression vector containing a cDNA for LLGN or
LLGXL or no DNA (Mock). Proteins from PMA-stimulated endothelial
cells (HCAEC+PMA) were included for size reference. Numbers to the
left indicate the apparent molecular weight of the major
immunoreactive proteins as determined by a comparison to protein
standards.
[0056] FIG. 13 shows the sequence of the rabbit LIPG PCR product
(RLLG.SEQ, SEQ ID NO: 12) and the sequence alignment between the
rabbit LIPG PCR product and the corresponding sequence in the human
cDNA (LLG7742A). Identical nucleotides are shaded.
[0057] FIG. 14 shows the phospholipase A activity of human EL-AS,
EL and LPL using a phosphatidylcholine substrate. To perform the
assay 700 .mu.l of conditioned medium harvested from COS-7 cells
transiently transfected with either pcDNA3.0/LIPG-AS, LIPG, or LPL
expression constructs were assayed in triplicate for phospholipase
activities as described below. Following a two hour incubation at
37.degree. C., reactions were terminated, and 14C labeled free
fatty-acid was extracted, and counted to determine the amount of
free fatty-acid produced.
[0058] FIG. 15 shows the triacylglyceride lipase activity of human
EL-AS, EL and LPL using a triolein substrate. To perform the assay
700 .mu.l of conditioned medium harvested from COS-7 cells
transiently transfected with either pcDNA3.0/LIPG-AS, LIPG, or LPL
expression constructs was assayed in triplicate for triglyceride
activities described below. Following a two hour incubation at
37.degree. C., "reactions" were terminated, and 14C labeled free
fatty-acid was extracted, and counted to determine the amount of
free fatty-acid produced.
[0059] FIG. 16 shows the hybridization of LIPG and LPL probes to
genomic DNAs from different species.
[0060] FIG. 17 shows expression of LIPG in the liver of a wild-type
mouse 5 days after AdhEL injection. Lane 1, liver from mouse
injected with Adnull; lane 2, liver from mouse injected with
AdhEL.
[0061] FIG. 18 shows plasma levels of HDL cholesterol in AdhEL- and
Adnull-injected wild-type mice.
[0062] FIG. 19 shows lipoprotein profiles in wild-type mice
injected with AdhEL and Adnull at baseline before injection (left)
and 14 days after injection (right).
[0063] FIG. 20 shows HDL cholesterol levels in human apoA-I
transgenic mice after injection with Adnull or AdhEL.
[0064] FIG. 21 shows ApoA-I levels in human apoA-1 transgenic mice
after injection with Adnull or AdhEL.
[0065] FIG. 22 shows the effect of injection of AdhEL in LDL
receptor-deficient mice on VLDL/LDL cholesterol levels.
[0066] FIG. 23 shows the effect of AdhEL on HDL receptor-deficient
mice on HDL cholesterol levels.
DETAILED DESCRIPTION OF THE INVENTION
[0067] The detailed description which follows sets forth the basis
for the present invention, followed by a definitions section.
Following the definitions section, the various compositions useful
in the practice of the invention are discussed, followed by a
discussion of the methods used to lower or raise the levels of LIPG
activity.
The Enzymatic Activity of the LIPG Gene Product
[0068] The present invention relates to methods for regulating the
levels of HDL cholesterol and apolipoprotein AI, VLDL cholesterol
and LDL cholesterol utilizing methods and compositions which lower
or raise the activity of the LIPG lipase enzyme. In particular, the
present invention is based in part on the discovery of the
enzymatic activity of the polypeptide products of the LIPG gene on
HDL cholesterol and apolipoprotein AI, VLDL cholesterol and
LDL-cholesterol. The polypeptide products of LIPG are members of
the triacylglycerol lipase family and comprise an approximately 39
kD catalytic domain of the triacylglycerol lipase family, e,g.,
having the sequence SEQ ID NO: 10. Because this newly discovered
lipase was found to be synthesized by endothelial cells and this is
a unique feature compared with other members of the triacylglycerol
lipase family, this lipase has been named "endothelial lipase"
(EL). Because the LIPG gene will be discussed extensively in the
sections which follow, EL will be hereinafter referred to as LIPG
polypeptide, for the purposes of clarity. In general, the LIPG
polypeptide is found in two major forms, referred to hereinafter as
"the LLGN polypeptide" and "the LLGXL polypeptide." The LLGN
polypeptide, has 354 amino acids. The LLGXL polypeptide has 500
amino acids and exhibits 43% similarity to human lipoprotein lipase
and 37% similarity to human hepatic lipase. As used herein, the
term "LIPG polypeptide" or "LIPG protein" encompasses both LLGN and
LLGXL.
[0069] The sequence of the LIPG polypeptide contains the
characteristic GXSXG-lipase motif, a conserved catalytic triad, a
19-residue lid region, conserved heparin and lipoprotein binding
sites and 5 potential N-linked glycosylation sites. The region with
the greatest sequence divergence in the triacylglycerol lipase
family is the lid domain, which forms an amphipathic helix covering
the catalytic pocket of the enzyme (Winkler et al., Nature 343,
771-774 (1990); van Tilbeurgh et al., J. Biol. Chem., 269,
4626-4633 (1994)) and confers substrate specificity to the enzymes
of this family (Dugi et al., J. Biol.. Chem., 270, 25396-25401
(1995)). The 19-residue lid region of LIPG is three residues
shorter and less amphipathic than those found in lipoprotein lipase
and hepatic lipase, consistent with a different enzymatic profile.
The predicted molecule weight of the mature protein is
approximately 55 kD; a 68 kD form is likely to be a glycosylated
form, whereas a 40 kD form may be, the product of a specific
proteolytic cleavage.
[0070] The LIPG polypeptide has the ability to lower the levels of
HDL cholesterol and apolipoprotein AI as well as the levels of VLDL
cholesterol and LDL cholesterol. It is well established that
lowered HDL cholesterol levels result in increased susceptibility
to atherosclerosis and increased levels of HDL cholesterol can
dramatically reduce susceptibility to atherosclerosis.
[0071] One physiologic role of LIPG may be to hydrolyse HDL
phospholipid in peripheral tissues and in liver to facilitate
selective uptake of HDL cholesteryl ester via the HDL receptor
SR-BI (Kozarsky et al., Nature, 387, 414-417 (1997)). Another
possible role is the facilitation of apoB-containing remnant
lipoprotein uptake, similar to the role of hepatic lipase (Mahley
et al., J. Lipid Res., 40, 1-16 (1999)). In addition, LIPG is
abundantly expressed in the placenta, and a role for this enzyme in
development is possible, given the importance of lipid transport in
fetal development (Farese et al., Trends Genet., 14, 115-120
(1998)).
[0072] Based on HDL cholesterol's beneficial properties, it is
desirable to raise HDL cholesterol levels by lowering the enzymatic
activity of LIPG. Accordingly, the present invention is directed to
methods and compositions which lower the activity of LIPG in the
body by lowering the expression of the LIPG gene or lowering the
enzymatic activity of the LIPG polypeptide.
[0073] Given the ability of the LIPG polypeptide to reduce the
levels of VLDL cholesterol and LDL cholesterol and the studies
demonstrating the correlation between high levels of these
compounds and atherosclerotic diseases, it is desirable to lower
the level of these compounds in a patient. Accordingly, the present
invention additionally provides methods and compositions for
increasing the expression of the LIPG gene and increasing the
enzymatic activity of the LIPG polypeptides.
[0074] There are set forth hereafter definitions of terms used
herein and descriptions of preferred embodiments of the present
invention.
Definitions
[0075] The following defined terms are used throughout the present
specification and should be helpful in understanding the scope and
practice of the present invention.
[0076] A "polypeptide" is a polymeric compound comprised of
covalently linked amino acid residues. Amino acids are classified
into seven groups on the basis of the side chain: (1) aliphatic
side chains, (2) side chains containing a hydroxylic (OH) group,
(3) side chains containing sulfur atoms, (4) side chains containing
an acidic or amide group, (5) side chains containing a basic group,
(6) side chains containing an aromatic ring, and (7) proline, an
imino acid in which the side chain is fused to the amino group.
[0077] A "protein" is a polypeptide which plays a structural or
functional role in a living cell.
[0078] The polypeptides and proteins of the invention may be
glycosylated or unglycosylated.
[0079] "Homology" means similarity of sequence reflecting a common
evolutionary origin. Polypeptides or proteins are said to have
homology, or similarity, if a substantial number of their amino
acids are either (1) identical, or (2) have a chemically similar
side chain. Nucleic acids are said to have homology if a
substantial number of their nucleotides are identical.
[0080] "Isolated polypeptide" or "isolated protein" is a
polypeptide or protein which is substantially free of those
compounds that are normally associated therewith in its natural
state (e.g., other proteins or polypeptides, nucleic acids,
carbohydrates, lipids). "Isolated" is not meant to exclude
artificial or synthetic mixtures with other compounds, or the
presence of impurities which do not interfere with biological
activity, and which may be present, for example, due to incomplete
purification, addition of stabilizers, or compounding into a
pharmaceutically acceptable preparation.
[0081] A molecule is "antigenic" when it is capable of specifically
interacting with an antigen recognition molecule of the immune
system, such as an immunoglobulin (antibody) or T cell antigen
receptor. An antigenic polypeptide contains at least about 5, and
preferably at least about 10, amino acids. An antigenic portion of
a molecule can be that portion that is immunodominant for antibody
or T cell receptor recognition, or it can be a portion used to
generate an antibody to the molecule by conjugating the antigenic
portion to a carrier molecule for immunization. A molecule that is
antigenic need not be itself immunogenic, i.e., capable of
eliciting an immune response without a carrier.
[0082] "LLGN polypeptide" and "LLGN protein" mean a polypeptide
including the sequence SEQ ID NO: 6, said polypeptide being
glycosylated or non-glycosylated.
[0083] "LLGXL polypeptide" and "LLGXL protein" mean a polypeptide
including the sequence SEQ ID NO: 8, said polypeptide being
glycosylated or non-glycosylated.
[0084] "LIPG polypeptide" and "LIPG protein" describe the lipase
enzyme encoded by the LIPG gene and generically describes both the
LLGN polypeptide and the LLGXL polypeptide.
[0085] "Endothelial lipase," or "EL", refer to the lipase enzyme
encoded by the LIPG gene and is equivalent to the term LIPG
polypeptide.
[0086] The LIPG polypeptide or protein of the invention includes
any analogue, fragment, derivative, or mutant which is derived from
an LIPG polypeptide and which retains at least one biological
property of the LIPG polypeptide. Different variants of the LIPG
polypeptide exist in nature. These variants may be allelic
variations characterized by differences in the nucleotide sequences
of the structural gene coding for the protein, or may involve
differential splicing or post-translational modification. The
skilled artisan can produce variants having single or multiple
amino acid substitutions, deletions, additions, or replacements.
These variants may include, inter alia: (a) variants in which one
or more amino acid residues are substituted with conservative or
non-conservative amino acids, (b) variants in which one or more
amino acids are added to the LIPG polypeptide, (c) variants in
which one or more of the amino acids includes a substituent group,
and (d) variants in which the LIPG polypeptide is fused with
another polypeptide such as serum albumin. Other LIPG polypeptides
of the invention include variants in which amino acid residues from
one species are substituted for the corresponding residue in
another species, either at conserved or non-conserved positions. In
another embodiment, amino acid residues at non-conserved positions
are substituted with conservative or non-conservative residues. The
techniques for obtaining these variants, including genetic
(suppressions, deletions, mutations, etc.), chemical, and enzymatic
techniques, are known to persons having ordinary skill in the
art.
[0087] If such allelic variations, analogues, fragments,
derivatives, mutants, and modifications, including alternative mRNA
splicing forms and alternative post-translational modification
forms result in derivatives of the LIPG polypeptide which retain
any of the biological properties of the LIPG polypeptide, they are
included within the scope of this invention.
[0088] A "nucleic acid" is a polymeric compound comprised of
covalently linked subunits called nucleotides. Nucleic acid
includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid
(DNA), both of which may be single-stranded or double-stranded. DNA
includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA.
The sequence of nucleotides that encodes a protein is called the
sense sequence.
[0089] An "antisense nucleic acid" is a sequence of nucleotides
that is complementary to the sense sequence. Antisense nucleic
acids can be used to down regulate or block the expression of the
polypeptide encoded by the sense strand.
[0090] "Isolated nucleic acid" means a nucleic acid which is
substantially free of those compounds that are normally associated
therewith in its natural state. "Isolated" is not meant to exclude
artificial or synthetic mixtures with other compounds, or the
presence of impurities which do not interfere with biological
activity, and which may be present, for example, due to incomplete
purification, addition of stabilizers, or compounding into a
pharmaceutically acceptable preparation.
[0091] The phrase "a nucleic acid which hybridizes at high
stringency" means that the hybridized nucleic acids are able to
withstand a washing under high stringency conditions. An example of
high stringency washing conditions for DNA-DNA hybrids is
0.1.times.SSC, 0.5% SDS at 68.degree. C. Other conditions of high
stringency washing are known to persons having ordinary skill in
the art.
[0092] "Regulatory region" means a nucleic acid sequence which
regulates the expression of a nucleic acid. A regulatory region may
include sequences which are naturally responsible for expressing a
particular nucleic acid (a homologous region) or may include
sequences of a different origin (responsible for expressing
different proteins or even synthetic proteins). In particular, the
sequences can be sequences of eukaryotic or viral genes or derived
sequences which stimulate or repress transcription of a gene in a
specific or non-specific manner and in an inducible or
non-inducible manner. Regulatory regions include origins of
replication, RNA splice sites, enhancers, transcriptional
termination sequences, signal sequences which direct the
polypeptide into the secretory pathways of the target cell, and
promoters.
[0093] A regulatory region from a "heterologous source" is a
regulatory region which is not naturally associated with the
expressed nucleic acid. Included among the heterologous regulatory
regions are regulatory regions from a different species, regulatory
regions from a different gene, hybrid regulatory sequences, and
regulatory sequences which do not occur in nature, but which are
designed by one having ordinary skill in the art.
[0094] A "vector" is any means for the transfer of a nucleic acid
according to the invention into a host cell. The term "vector"
includes both viral and nonviral means for introducing the nucleic
acid into a prokaryotic or eukaryotic cell in vitro, ex vivo or in
vivo. Non-viral vectors include plasmids, liposomes, electrically
charged lipids (cytofectins), DNA-protein complexes, and
biopolymers. Viral vectors include retrovirus, adeno-associated
virus, pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr and
adenovirus vectors. In addition to nucleic acid according to the
invention, a vector may also contain one or more regulatory
regions, and/or selectable markers useful in selecting, measuring,
and monitoring nucleic acid transfer results (transfer to which
tissues, duration of expression, etc.).
[0095] A "recombinant cell" is a cell which contains a nucleic acid
which is not naturally present in the cell. "Recombinant cell"
includes higher eukaryotic cells such as mammalian cells, lower
eukaryotic cells such as yeast cells, prokaryotic cells, and
archaebacterial cells.
[0096] "Pharmaceutically acceptable carrier" includes diluents and
fillers which are pharmaceutically acceptable for methods of
administration, are sterile, and may be aqueous or oleaginous
suspensions formulated using suitable dispersing or wetting agents
and suspending agents. The particular pharmaceutically acceptable
carrier and the ratio of active compound to carrier are determined
by the solubility and chemical properties of the composition, the
particular mode of administration, and standard pharmaceutical
practice.
[0097] A "lipase" is a protein which can enzymatically cleave a
lipid substrate.
[0098] A "phospholipase" is a protein which can enzymatically
cleave a phospholipid substrate.
[0099] A "triacylglycerol lipase" is a protein which can
enzymatically cleave a triacylglyceride substrate.
[0100] "Phosphatidylcholine" is a glycerol phospholipid.
Phosphatidylcholine is also known as lecithin.
[0101] "Lipid profile" means the set of concentrations of
cholesterol, triglyceride, lipoprotein cholesterol and other lipids
in the body of a human or other animal.
[0102] An "undesirable lipid profile" is the condition in which the
concentrations of cholesterol, triglyceride, or lipoprotein
cholesterol are outside of the age- and gender-adjusted reference
ranges. Generally, a concentration of total cholesterol>200
mg/dl, of plasma triglycerides>200 mg/dl, of LDL
cholesterol>130 mg/dl, of HDL cholesterol<39 mg/dl, or a
ratio of total cholesterol to HDL cholesterol>4.0 is considered
to be an undesirable lipid profile. An undesirable lipid profile is
associated with a variety of pathological conditions, including
hyperlipidaemias, diabetes hypercholesterolaemia, atherosclerosis,
and other forms of coronary artery disease.
[0103] A "ribozyme" is an RNA molecule which can function as an
enzyme.
[0104] A "neutralizing antibody" is an antibody which can bind to
an LIPG polypeptide and lower or eliminate the enzymatic activity
of the LIPG polypeptide. These antibodies may be monoclonal
antibodies or polyclonal antibodies. The present invention includes
chimeric, single chain, and humanized antibodies, as well as Fab
fragments and the products of an Fab expression library, and Fv
fragments and the products of an Fv expression library.
[0105] An "inhibitory molecule" or "inhibitor" is a molecule which
lowers or eliminates the expression of the LIPG polypeptide or
which lowers or eliminates the enzymatic activity of the LIPG
polypeptide.
[0106] An "enhancer molecule" or "enhancer" is a molecule which
increases the expression of the LIPG polypeptide or which increases
the enzymatic activity of the LIPG polypeptide.
[0107] A "liposome" is is an artificial or naturally-occurring
phospholipid vesicle.
[0108] A "cationic liposome" is a liposome having a net positive
electrical charge.
[0109] The sections which follow discuss the elements used in the
claimed methods and compositions and the preferred embodiments of
these elements.
Polypeptides
[0110] The present invention utilizes polypeptides encoded by LIPG
which are members of the triacylglycerol lipase family, and which
comprise a 39 kD catalytic domain of the triacylglycerol lipase
family, e.g., having the sequence SEQ ID NO: 10. In certain
embodiments of the present invention, an isolated LIPG polypeptide
comprising the sequence SEQ ID NO: 6 and having an apparent
molecular weight of about 40 kD on a 10% SDS-PAGE gel is utilized.
In another embodiment of the present invention, an isolated LIPG
polypeptide comprising the sequence SEQ ID NO: 8 and having an
apparent molecular weight of about 55 kD or 68 kD on a 10% SDS-PAGE
gel is utilized. In yet another embodiment, the polypeptides
utilized in the present invention are subfragments of these
"polypeptides. In still yet another embodiment, the polypeptides
used in the present invention are antibodies capable of binding to
an LIPG polypeptide.
[0111] The polypeptides and proteins utilized in the present
invention may be recombinant polypeptides, natural polypeptides, or
synthetic polypeptides, and may be of human, rabbit, or other
animal origin. The polypeptides are characterized by a reproducible
single molecular weight and/or multiple set of molecular weights,
chromatographic response and elution-profiles, amino acid
composition and sequence, and biological activity.
[0112] The polypeptides utilized in the present invention may be
isolated from natural sources, such as placental extracts, human
plasma, or conditioned media from cultured cells such as
macrophages or endothelial cells, by using the purification
procedures known to one of skill in the art.
[0113] Alternatively, the polypeptides utilized in the present
invention may be prepared utilizing recombinant DNA technology,
which comprises combining a nucleic acid encoding the polypeptide
thereof in a suitable vector, inserting the resulting vector into a
suitable host cell, recovering the polypeptide produced by the
resulting host cell, and purifying the polypeptide recovered.
Nucleic Acids
[0114] The present invention utilizes isolated nucleic acids which
encode LIPG polypeptides.
[0115] The present invention also utilizes antisense nucleic acids
which can be used to down regulate or block the expression of LIPG
polypeptides in vitro, ex vivo or in vivo.
[0116] The techniques of recombinant DNA technology are known to
those of ordinary skill in the art. General methods for the cloning
and; expression of recombinant molecules are described in Maniatis
(Molecular Cloning, Cold Spring Harbor Laboratories, 1982), and in
Ausubel (Current Protocols in Molecular Biology, Wiley and Sons,
1987), which are incorporated by reference.
[0117] The nucleic acids of the present invention may be linked to
one or more regulatory regions. Selection of the appropriate
regulatory region or regions is a routine matter, within the level
of ordinary skill in the art. Regulatory regions include promoters,
and may include enhancers, suppressors, etc.
[0118] Promoters that may be used in the present invention include
both constituitive promoters and regulated (inducible) promoters.
The promoters may be prokaryotic or eukaryotic depending on the
host. Among the prokaryotic (including bacteriophage) promoters
useful for practice of this invention are lacI, lacZ, T3, T7,
lambda P.sub.r, P.sub.l, and trp promoters. Among the eukaryotic
(including viral) promoters useful for practice of this invention
are ubiquitous promoters (e.g. HPRT, vimentin, actin, tubulin),
intermediate filament promoters (e.g. desmin, neurofilaments,
keratin, GFAP), therapeutic gene promoters (e.g. MDR type, CFTR,
factor VIII), tissue-specific promoters (e.g. actin promoter in
smooth muscle cells, or Flt and Flk promoters active in endothelial
cells), including animal transcriptional control regions, which
exhibit tissue specificity and have been utilized in transgenic
animals: elastase I gene control region which is active in
pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646;
Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol.,
50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene
control region which is active in pancreatic beta cells (Hanahan,
1985, Nature 315:115-122), immunoglobulin gene control region which
is active in lymphoid cells (Grosschedl et al., 1984, Cell
38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et
al., 1987, Mol. Cell. Biol., 7:1436-1444), mouse mammary tumor
virus control region which is active in testicular, breast,
lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495),
albumin gene control region which is active in liver (Pinkert et
al., 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene
control region which is active in liver (Krumlauf et al., 1985,
Mol. Cell. Biol., 5:1639-1648; Hammer et al., 1987, Science
235:53-58), alpha 1-antitrypsin gene control region which is active
in the liver (Kelsey et al., 1987, Genes and Devel., 1:161-171),
beta-globin gene control region which is active in myeloid cells
(Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986,
Cell 46:89-94), myelin basic protein gene control region which is
active in oligodendrocyte cells in the brain (Readhead et al.,
1987, Cell 48:703-712), myosin light chain-2 gene control region
which is active in skeletal muscle (Sani, 1985, Nature
314:283-286), and gonadotropic releasing hormone gene control
region which is active in the hypothalamus (Mason et al., 1986,
Science 234:1372-1378).
[0119] Other promoters which may be used in the practice of the
invention include promoters which are preferentially activated in
dividing cells, promoters which respond to a stimulus (e.g. steroid
hormone receptor, retinoic acid receptor), tetracycline-regulated
transcriptional modulators, cytomegalovirus immediate-early,
retroviral LTR, metallothionein, SV-40, E1a, and MLP promoters.
Tetracycline-regulated transcriptional modulators and CMV promoters
are described in WO 96/01313, U.S. Pat. Nos. 5,168,062 and
5,385,839, the contents of which are incorporated herein by
reference.
Viral Vector Systems
[0120] Preferably, the viral vectors used in the gene therapy
methods of the present invention are replication defective, that
is, they are unable to replicate autonomously in the target cell.
In general, the genome of the replication defective viral vectors
which are used within the scope of the present invention lack at
least one region which is necessary for the replication of the
virus in the infected cell. These regions can either be eliminated
(in whole or in part), or be rendered non-functional by any
technique known to a person skilled in the art. These techniques
include the total removal, substitution (by other sequences, in
particular by the inserted nucleic acid), partial deletion or
addition of one or more bases to an essential (for replication)
region. Such techniques may be performed in vitro (on the isolated
DNA) or in situ, using the techniques of genetic manipulation or by
treatment with mutagenic agents.
[0121] Preferably, the replication defective virus retains the
sequences of its genome which are necessary for encapsidating the
viral particles.
[0122] The retroviruses are integrating viruses which infect
dividing cells. The retrovirus genome includes two LTRs, an
encapsidation sequence and three coding regions (gag, pol and env).
The construction of recombinant retroviral vectors has been
described: see, in particular, EP 453242, EP178220, Bernstein et
al. Genet. Eng. 7 (1985) 235; McCormick, BioTechnology 3 (1985)
689, etc. In recombinant retroviral vectors, the gag, pol and env
genes are generally deleted, in whole or in part, and replaced with
a heterologous nucleic acid sequence of interest. These vectors can
be constructed from different types of retrovirus, such as, MoMuLV
("murine Moloney leukaemia virus" MSV ("murine Moloney sarcoma
virus"), HaSV ("Harvey sarcoma virus"); SNV ("spleen necrosis
virus"); RSV ("Rous sarcoma virus") and Friend virus. Lentivirus
vector systems may also be used in the practice of the present
invention. The lentiviral genome is a positive-strand
polyadenylated RNA of 9,000 to 10,000 base pairs containing three
structural genes organized 5' to 3' (gag, pol, env), typical of all
retroviruses. For an extensive review of lentiviral systems, see
Fields Virology, Second Edition, Volume 2, Chapter 55,
"Lentiviruses," pp. 1571-1589, Raven Press, New York, 1990.
[0123] In general, in order to construct recombinant retroviruses
containing a sequence encoding LIPG, a plasmid is constructed which
contains the LTRs, the encapsidation sequence and the coding
sequence. This construct is used to transfect a packaging cell
line, which cell line is able to supply in trans the retroviral
functions which are deficient in the plasmid. In general, the
packaging cell lines are thus able to express the gag, pol and env
genes. Such packaging cell lines have been described in the prior
art, in particular the cell line PA317 (US4,861,719); the PsiCRIP
cell line (WO90/02806) and the GP+envAm-12 cell line (WO89/07150).
In addition, the recombinant retroviral vectors can contain
modifications within the LTRs for suppressing transcriptional
activity as well as extensive encapsidation sequences which may
include a part of the gag gene (Bender et al., J. Virol. 61 (1987)
1639). Recombinant retroviral vectors are purified by standard
techniques known to those having ordinary skill in the art.
[0124] The adeno-associated viruses (AAV) are DNA viruses of
relatively small size which can integrate, in a stable and
site-specific manner, into the genome of the cells which they
infect. They are able to infect a wide spectrum of cells without
inducing any effects on cellular growth, morphology or
differentiation, and they do not appear to be involved in human
pathologies. The AAV genome has been cloned, sequenced and
characterized. It encompasses approximately 4700 bases and contains
an inverted terminal repeat (ITR) region of approximately 145 bases
at each end, which serves as an origin of replication for the
virus. The remainder of the genome is divided into two essential
regions which carry the encapsidation functions: the left-hand part
of the genome, which contains the rep gene involved in viral
replication and expression of the viral genes; and the right-hand
part of the genome, which contains the cap gene encoding the capsid
proteins of the virus.
[0125] The use of vectors derived from the AAVs for transferring
genes in vitro and in vivo has been described (see WO 91/18088; WO
93/09239; U.S. Pat. No. 4,797,368, U.S. Pat. No. 5,139,941, EP 488
528). These publications describe various AAV-derived constructs in
which the rep and/or cap genes are deleted and replaced by a gene
of interest, and the use of these constructs for transferring the
said gene of interest in vitro (into cultured cells) or in vivo,
(directly into an organism). The replication defective recombinant
AAVs utilized in the present invention can be prepared by
cotransfecting a plasmid containing the nucleic acid sequence of
interest flanked by two AAV inverted terminal repeat (ITR) regions,
and a plasmid carrying the AAV encapsidation genes (rep and cap
genes), into a cell line which is infected with a human helper
virus (for example an adenovirus). The AAV recombinants which are
produced are then purified by standard techniques. The invention
also relates, therefore, to an AAV-derived recombinant virus whose
genome encompasses a sequence encoding an LIPG polypeptide flanked
by the AAV ITRs. The invention also relates to a plasmid
encompassing a sequence encoding an LIPG polypeptide flanked by two
ITRs from an AAV. Such a plasmid can be used as it is for
transferring the LIPG sequence, with the plasmid, where
appropriate, being incorporated into a liposomal vector
(pseudo-virus).
[0126] In a preferred embodiment, the vector utilized in the
present invention is an adenovirus vector.
[0127] Adenoviruses are eukaryotic DNA viruses that can be modified
to efficiently deliver a nucleic acid to a variety of cell
types.
[0128] Various serotypes of adenovirus exist. Of these serotypes,
preference is given, within the scope of the present invention, to
using type 2 or type 5 human adenoviruses (Ad 2 or Ad 5) or
adenoviruses of animal origin (see WO94/26914). Those adenoviruses
of animal origin which can be used within the scope of the present
invention include adenoviruses of canine, bovine, murine (example:
Mav1, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian,
and simian (example: SAV) origin. Preferably, the adenovirus of
animal origin is a canine adenovirus, more preferably a CAV2
adenovirus (e.g. Manhattan or A26/61 strain (ATCC VR-800), for
example).
[0129] Preferably, the replication defective adenoviral vectors
comprise the ITRs, an encapsidation sequence and the nucleic acid
of interest. Still more preferably, at least the E1 region of the
adenoviral vector is non-functional. The deletion in the E1 region
preferably extends from nucleotides 455 to 3329 in the sequence of
the Ad5 adenovirus. Other regions may also be modified, in
particular the E3 region. (WO95/02697), the E2 region (WO94/28938),
the E4 region (WO94/28152, WO94/12649 and WO95/02697), or in any of
the late genes L1-L5. Defective retroviral vectors are disclosed in
WO95/02697.
[0130] In a preferred embodiment, the adenoviral vector has a
deletion in the E1 and E4 regions. In another preferred embodiment,
the adenoviral vector has a deletion in the E1 region into which
the E4 region and the sequence encoding LLG are inserted (see FR94
13355).
[0131] The replication defective recombinant adenoviruses can be
prepared by any technique known to the person skilled in the art
(Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3
(1984) 2917). In particular, they can be prepared by homologous
recombination between an adenovirus and a plasmid which carries,
inter alia, the DNA sequence of interest. The homologous
recombination is effected following cotransfection of the said
adenovirus and plasmid into an appropriate cell line. The cell line
which is employed should preferably (i) be transformable by the
said elements, and (ii) contain the sequences which are able to
complement the part of the genome of the replication defective
adenovirus, preferably in integrated form in order to avoid the
risks of recombination. Examples of cell lines which may be used
are the human embryonic kidney cell line 293 (Graham et al., J.
Gen. Virol. 36 (1977) 59) which contains the left-hand portion of
the genome of an Ad5 adenovirus (12%) integrated into its genome,
and cell lines which are able to complement the E1 and E4
functions, as described in applications WO94/26914 and WO95/02697.
Recombinant adenoviruses are recovered and purified using standard
molecular biological techniques, which are well known to one of
ordinary skill in the art.
Antisense Nucleic Acids
[0132] The down regulation of gene expression using antisense
nucleic acids can be achieved at the translational or
transcriptional level. Antisense nucleic acids of the invention are
preferably nucleic acid fragments capable of specifically
hybridizing with all or part of a nucleic acid encoding LIPG or the
corresponding messenger RNA. In addition, antisense nucleic acids
may be designed or identified which decrease expression of the LIPG
gene by inhibiting splicing of its primary transcript. With
knowledge of the structure and partial sequence of the LIPG gene,
such antisense nucleic acids can be designed and tested for
efficacy.
[0133] The antisense nucleic acids are preferably oligonucleotides
and may consist entirely of deoxyribonucleotides, modified
deoxyribonucleotides, or some combination of both. The antisense
nucleic acids can be synthetic oligonucleotides. The
oligonucleotides may be chemically modified, if desired, to improve
stability and/or selectivity. Since oligonucleotides are
susceptible to degradation by intracellular nucleases, the
modifications can include, for example, the use of a sulfur group
to replace the free oxygen of the phosphodiester bond. This
modification is called a phosphorothioate linkage. Phosphorothioate
antisense oligonucleotides are water soluble, polyanionic, and
resistant to endogenous nucleases. In addition, when a
phosphorothioate antisense oligonucleotide hybridizes to its target
site, the RNA-DNA duplex activates the endogenous enzyme
ribonuclease (Rnase) H, which cleaves the mRNA component of the
hybrid molecule.
[0134] In addition, antisense oligonucleotides with phosphoramidite
and polyamide (peptide) linkages can be synthesized. These
molecules should be very resistant to nuclease degradation.
Furthermore, chemical groups can be added to the 2' carbon of the
sugar moiety and the 5 carbon (C-5) of pyrimidines to enhance
stability and facilitate the binding of the antisense
oligonucleotide to its target site. Modifications may include 2'
deoxy, O-pentoxy, O-propoxy, O-methoxy, fluoro, methoxyethoxy
phosphoro-thioates, modified bases, as well as other modifications
known to those of skill in the art.
[0135] The antisense nucleic acids can also be DNA sequences whose
expression in the cell produces RNA complementary to all or part of
the LIPG mRNA. -Antisense nucleic acids can be prepared by
expression of all or part of a sequence selected from the group
consisting of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 7, or SEQ ID
No. 11, in the opposite orientation, as described in EP 140308. Any
length of antisense sequence is suitable for practice of the
invention so long as it is capable of down-regulating or blocking
expression of LIPG. Preferably, the antisense sequence is at least
20 nucleotides in length. The preparation and use of antisense
nucleic acids, DNA encoding antisense RNAs and the use of oligo and
genetic antisense is disclosed in WO92/15680, the contents of which
are incorporated herein by reference.
[0136] One approach to determining the optimum fragment of LIPG to
use in an antisense nucleic acid treatment method involves
preparing random fragments of LIPG cDNA by mechanical shearing,
enzymatic treatment, and cloning the fragment into any of the
vector systems described herein. Individual clones or pools of
clones are used to infect LIPG-expressing cells, and effective
antisense LIPG cDNA fragments are identified by monitoring LIPG
expression at the RNA or protein level.
[0137] The retroviral, adeno-associated viral, and adenoviral
vector systems discussed hereinabove may all be used to introduce
and express antisense nucleic acids in cells. Antisense synthetic
oligonucleotides may be introduced in a variety of ways, including
the methods discussed hereinbelow.
Ribozymes
[0138] Reductions in the levels of LIPG polypeptide may be
accomplished using ribozymes. Ribozymes are catalytic RNA molecules
(RNA enzymes) that have separate catalytic and substrate binding
domains. The substrate binding sequence combines by nucleotide
complementarity and, possibly, nonhydrogen bond interactions with
its target sequence. The catalytic portion cleaves the target RNA
at a specific site. The substrate domain of a ribozyme can be
engineered to direct it to a specified mRNA sequence. The ribozyme
recognizes and then binds a target mRNA through complementary
base-pairing. Once it is bound to the correct target site, the
ribozyme acts enzymatically to cut the target mRNA. Cleavage of the
LIPG mRNA by a ribozyme destroys its ability to direct synthesis of
LIPG polypeptide. Once the ribozyme has cleaved its target
sequence, it is released and can repeatedly bind and cleave at
other LIPG mRNAs.
[0139] In preferred embodiments of this invention, the ribozyme is
formed in a hammerhead motif. Other forms include a hairpin motif,
a hepatitis delta virus, group I intron or RnaseP RNA (in
association with an RNA guide sequence) motif or Neurospora VS RNA
motif. Hammerhead motifs are described by Rossi et al., 1992, Aids
Research and Human Retroviruses, 8, 183. Hairpin motifs are
described in Hampel and Tritz, 1989, Biochemistry, 28, 4929, and
Hampel et al., 1990, Nucleic Acids Res., 18, 299. The hepatitis
delta virus motif is described by Perrotta and Been, 1992,
Biochemistry, 31, 16, the RnaseP motif is described by
Guerrier-Takada et al., 1983, Cell, 35, 849, the Neurospora VS RNA
ribozyme motif is described by Collins (Saville and Collins, 1990,
Cell, 61, 685-696; Saville and Collins, 1991, Proc. Natl. Acad.
Sci. USA, 88, 8826-8830; Collins and Olive, 1993, Biochemistry, 32,
2795-2799) the Group I intron motif is described by Cech et al.,
U.S. Pat. No. 4,987,071.
[0140] One approach in preparing a ribozyme is to chemically
synthesize an oligodeoxyribonucleotide with a ribozyme catalytic
domain (.about.20 nucleotides) flanked by sequences that hybridize
to the target LIPG mRNA after transcription. The
oligodeoxyribonucleotide is amplified by using the substrate
binding sequences as primers. The amplification product is cloned
into a eukaryotic expression vector.
[0141] Ribozymes possessing a hammerhead or hairpin structure are
readily prepared since these catalytic RNA molecules can be
expressed within cells from eukaryotic promoters (e.g., Scanlon et
al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet
et al., 1992, Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992,
J. Virol., 66, 1432-41; Weerasinghe et al., 1991, J. Virol., 65,
5531-4; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89,
10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver
et al., 1990, Science, 247, 1222-1225)). A ribozyme of the present
invention can be expressed in eukaryotic cells from the appropriate
DNA vector. If desired, the activity of the ribozyme may be
augmented by its release from the primary transcript by a second
ribozyme (Ohkawa et al., 1992, Nucleic Acids Symp. Ser., 27, 15-6;
Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et
al., 1993, Nucleic Acids Res., 21, 3249-55).
[0142] In one approach to preparing ribozymes, ribozymes are
expressed from transcription units inserted into DNA, RNA, or viral
vectors. Transcription of the ribozyme sequences are driven from a
promoter for eukaryotic RNA polymerase I (pol (I), RNA polymerase
II (pol II), or RNA polymerase III (pol III). Transcripts from pol
II or pol III promoters will be expressed at high levels in all
cells; the levels of a given pol II promoter in a given cell type
will depend on nearby gene regulatory sequences. Prokaryotic RNA
polymerase promoters are also used, providing-that the prokaryotic
RNA polymerase enzyme is expressed in the appropriate cells
(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). It has been demonstrated that ribozymes
expressed from these promoters can 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).
[0143] In one embodiment of the present invention, a transcription
unit expressing a ribozyme that cleaves LIPG RNA is inserted into a
plasmid DNA vector, a retrovirus vector, an adenovirus DNA viral
vector or an adeno-associated virus vector. The recombinant vectors
are preferably DNA plasmids or adenovirus vectors. However, other
mammalian cell vectors that direct the expression of RNA may be
used for this, purpose. The vectors are delivered as recombinant
viral particles. DNA may be delivered alone or complexed with
various vehicles. The DNA, DNA/vehicle complexes, or the
recombinant virus particles are locally administered to the site of
treatment, as discussed below. Preferably, recombinant vectors
capable of expressing the ribozymes are locally delivered as
described below, and persist in target cells. Once expressed, the
ribozymes cleave the target LIPG mRNA.
[0144] Ribozymes may be administered to a patient by a variety of
methods. They may be added directly to target tissues, complexed
with cationic lipids, packaged within liposomes, or delivered to
target cells by other methods known in the art. Localized
administration to the desired tissues may be done by catheter,
infusion pump or stent, with or without incorporation of the
ribozyme in biopolymers as discussed hereinbelow. Alternative
routes of delivery include, but are not limited to, intravenous
injection, intramuscular injection, subcutaneous injection, aerosol
inhalation, oral (tablet or pill form), topical, systemic, ocular,
intraperitoneal and/or intrathecal delivery. More detailed
descriptions of ribozyme delivery and administration are provided
in Sullivan et al., PCT WO94/02595 and Draper et al., PCT
WO93/23569, which are incorporated by reference herein.
Non-Viral Delivery Systems
[0145] Certain non-viral systems have been used in the art and can
facilitate introduction of DNA encoding the LIPG polypeptides or
antisense nucleic acids into a patient.
[0146] A DNA vector encoding a desired LIPG polypeptide or
antisense sequence can be introduced in vivo by lipofection. For
the past decade, there has been increasing use of liposomes for
encapsulation and transfection of nucleic acids in vitro. Synthetic
cationic lipids designed to limit the difficulties and dangers
encountered with liposome mediated transfection can be used to
prepare liposomes for in vivo transfection of a gene encoding a
marker [Felgner, et. al., Proc. Natl. Acad. Sci. U.S.A.
84:7413-7417 (1987); see Mackey, et al., Proc. Natl. Acad. Sci.
U.S.A. 85:8027-8031 (1988); Ulmer et al., Science 259:1745-1748
(1993)]. The use of cationic lipids may promote encapsulation of
negatively charged nucleic acids, and also promote fusion with
negatively charged cell membranes [Felgner and Ringold, Science
337:387-388 (1989)]. Particularly useful lipid compounds and
compositions for transfer of nucleic acids are described in
International Patent Publications WO95/18863 and WO96/17823, and in
U.S. Pat. No. 5,459,127. The use of lipofection to introduce
exogenous genes into the specific organs in vivo has certain
practical advantages. Molecular targeting of liposomes to specific
cells represents one area of benefit. It is clear that directing
transfection to particular cell types would be particularly
advantageous in a tissue with cellular heterogeneity, for example,
pancreas, liver, kidney, and the brain. Lipids may be chemically
coupled to other molecules for the purpose of targeting [see
Mackey, et. al., supra]. Targeted peptides, e.g., hormones or
neurotransmitters, and proteins for example, antibodies, or
non-peptide molecules could be coupled to liposomes chemically.
[0147] Other molecules are also useful for facilitating
transfection of a nucleic acid in vivo, for example, a cationic
oligopeptide (e.g., International Patent Publication WO95/21931),
peptides derived from DNA binding proteins (e.g., International
Patent Publication WO96/25508), or a cationic polymer (e.g.,
International Patent Publication WO95/21931).
[0148] It is also possible to introduce A DNA vector encoding a
LIPG polypeptide or antisense sequence in vivo as a naked DNA
plasmid (see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859).
Naked DNA vectors for gene therapy can be introduced into the
desired host cells by methods known in the art, e.g., transfection,
electroporation, microinjection, transduction, cell fusion, DEAE
dextran, calcium phosphate precipitation, use of a gene gun, or use
of a DNA vector transporter [see, e.g., Wu 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 Mar. 15, 1990; Williams et al., Proc. Natl. Acad. Sci. USA
88:2726-2730 (1991)]. Receptor-mediated DNA delivery approaches 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
[0149] The present invention provides antibodies against the LIPG
polypeptide. These antibodies may be monoclonal antibodies or
polyclonal antibodies. The present invention includes chimeric,
single chain, and humanized antibodies, as well as Fab fragments
and the products of an Fab expression library, and Fv fragments and
the products of an Fv expression library.
[0150] Polyclonal antibodies may be prepared against an antigenic
fragment of an LIPG polypeptide, as described in the Examples
section hereinbelow. Antibodies may also be generated against the
intact LIPG protein or polypeptide, or against a fragment,
derivative, or epitope of the protein or polypeptide. Antibodies
may be obtained following the administration of the protein,
polypeptide, fragment, derivative, or epitope to an animal, using
the techniques and procedures known in the art.
[0151] Monoclonal antibodies may be prepared using the method of
Mishell, B. B., et al., Selected Methods In Cellular Immunology,
(W. H. Freeman, ed.) San Francisco (1980). Briefly, a polypeptide
of the present invention is used to immunize spleen cells of Balb/C
mice. The immunized spleen cells are fused with myeloma cells.
Fused cells containing spleen and myeloma cell characteristics are
isolated by growth in HAT medium, a medium which kills both
parental cells, but allows the fused products to survive and
grow.
[0152] The monoclonal antibodies of the present invention may be
"humanized" to prevent the host from mounting an immune response to
the antibodies. A "humanized antibody" is one in which the
complementarity determining regions (CDRs) and/or other portions of
the light and/or heavy variable domain framework are derived from a
non-human immunoglobulin, but the remaining portions of the
molecule are derived from one or more human immunoglobulins.
Humanized antibodies also include antibodies characterized by a
humanized heavy chain associated with a donor or acceptor
unmodified light chain or a chimeric light chain, or vice versa.
The humanization of antibodies may be accomplished by methods known
in the art (see, e.g. G. E. Mark and E. A. Padlan, "Chapter 4.
Humanization of Monoclonal Antibodies", The Handbook of
Experimental Pharmacology Vol. 113, Springer-Verlag, New York,
1994). Transgenic animals may be used to express humanized
antibodies.
[0153] Techniques known in the art for the production of single
chain antibodies can be adapted to produce single chain antibodies
to the immunogenic polypeptides and proteins of the present
invention.
[0154] In a preferred embodiment, an anti-LIPG antibody is used to
bind to and inhibit the enzymatic activity of LIPG in a
patient.
[0155] The anti-LIPG antibodies are also useful in assays for
detecting or quantitating levels of LIPG. In one embodiment, these
assays provide a clinical diagnosis and assessment of LIPG in
various disease states and a method for monitoring treatment
efficacy. These anti-LIPG antibodies may additionally be used to
quantitate LIPG in a tissue sample in order to predict further
susceptibility to lowered levels of HDL cholesterol and
apolipoprotein AI.
Methods of Identifying and Utilizing Inhibitory Molecules and
Enhancer Molecules
[0156] The present invention provides methods of screening small
molecule libraries or natural product sources for enhancers
(agonists) or co-activators including proteinaceous co-activators
or inhibitors (antagonists) of LIPG activity. A potential enhancer
or inhibitor is contacted with LIPG protein and a substrate of
LIPG, and the ability of the potential enhancer or inhibitor to
enhance or inhibit LIPG activity is measured.
[0157] These screening methods may also be used to determine if a
compound can function as a substrate specific enhancer or
inhibitor, that is, whether a compound can enhance the enzymatic
activity of LIPG toward one substrate while lowering or maintaining
a given level of enzymatic, activity for a different substrate, for
example, the LIPG polypeptide of the present invention utilizes HDL
cholesterol as a substrate and also utilizes LDL cholesterol and
VLDL cholesterol as substrates. In certain embodiments, it is
desirable to isolate and identify substrate specific enhancers or
inhibitors which enhance the enzymatic activity of the LIPG
polypeptide towards LDL cholesterol or VLDL cholesterol while
lowering or maintaining the normal level of enzymatic activity for
HDL cholesterol.
[0158] The LIPG protein used in these methods can be produced
recombinantly in a variety of host cells, including mammalian
cells, baculovirus-infected insect cells, yeast, and bacteria. LIPG
expression in stably transfected CHO cells can be optimized by
methotrexate amplification of the cells. LIPG protein can also be
purified from natural sources such as human plasma, placental
extracts, or conditioned media from cultured endothelial cells,
THP-1 cells, or macrophages.
[0159] The optimization of assay parameters including pH, ion
concentrations, temperature, concentration of substrate, and
emulsification conditions are determined empirically by one having
ordinary skill in the art.
[0160] The fatty acid substituents of the substrates may vary in
chain length as well as in degree and position of unsaturation. The
substrates may be radiolabelled in any of several positions.
Phospholipid substrates such as phosphatidylcholine can be
radiolabelled, for example, in the Sn-1 or Sn-2 fatty acid
position, or in the glycerol, phosphate, or polar head group
(choline in the case of phosphatidylcholine).
[0161] As an alternative to radiolabeled substrates, other classes
of labeled substrates, such as fluorescent substrates or
thio-containing substrates, can also be used in the screening
methods.
[0162] Fluorescent substrates are particularly useful in screening
assays because enzymatic catalysis can be measured continuously by
measuring fluorescence intensity, without the physical separation
(extraction) of the products from the substrates. An example of a
fluorescent phosphatidylcholine substrate is
C.sub.6NBD-PC{1-acyl-2-(6-(nitro-2,1,3-benzoxadiazol-4-yl)amino]caproylph-
osphatidylcholine.
[0163] The thio-containing substrates include
1,2-bis(hexanoylthio)-1,2-dideoxy-sn-glycero-3-phosphorylcholine
(L. J. Reynolds, W. N. Washburn, R. A. Deems, and E. A. Dennis,
1991. Methods in Enzymology 197: 3-23; L. Yu and E. A. Dennis,
1991. Methods in Enzymology 197: 65-75; L. A. Wittenauer, K.
Shirai, R. L. Jackson, and J. D. Johnson, 1984. Biochem. Biophys.
Res. Commun. 118: 894-901).
[0164] In addition to inhibitory and enhancer molecules which
operate at the level of enzymatic activity, there are inhibitory
and enhancer molecules which operate at the level of expression of
the LIPG gene. One method for identifying compounds which are able
to enhance or inhibit the expression of LIPG is to use a reporter
gene system. These systems utilize reporter gene expression vectors
which include a cloning site into which a given promoter may be
cloned upstream of a "reporter gene" which can be easily detected
and quantified. One of skill in the art could readily identify and
subclone the promoter for the LIPG gene as well as other control
sequences into a commercially available reporter gene expression
vector. The expression vector is transferred into host cells and
the cells are exposed to a test compound (a putative inhibitor or
enhancer molecule) to determine the effect of the test compound on
expression of the reporter gene product. In particular, the cells
are assayed for the presence of the reporter gene product by
directly measuring the amount of reporter mRNA, the reporter
protein itself or the enzymatic activity of the reporter protein.
Ideally, the reporter gene is not endogenously expressed in the
cell type of interest and lends itself to sensitive, quantitative
and rapid assays. A variety of reporter assay constructs are
commercially available and several reporter genes and assays have
been developed and can be readily prepared by those of skill in the
art. The most popular systems for monitoring genetic activity in
eukaryotic cells include the chloramphenicol acetyltransferase
(CAT), .beta.-galactosidase, firefly luciferase, growth hormone
(GH), .beta.-glucurorudase (GUS), alkaline phosphatase (AP), green
fluorescent protein (GFP) and Renilla luciferase. Reporter assay
constructs can be purchased from a variety of sources including
Promega and Invitrogen.
[0165] As mentioned above, reporter gene activity can be detected
by assaying for the reporter mRNA or the reporter protein. The
reporter mRNA can be detected by northern blot analysis,
ribonuclease protection assays or RT-PCR. While these assays are
more direct than measuring protein expression, many assays have
been developed to measure the presence of the reporter protein
rather than the mRNA present in a cell. Reporter proteins can be
assayed by spectrophotometry or by detecting enzymatic activity.
Reporter protein levels may also be measured with antibody-based
assays. In general, the enzymatic assays are very sensitive and are
a preferred method of monitoring reporter gene expression.
Compositions
[0166] The present invention provides compositions in a
biologically compatible (biocompatible) solution, comprising the
polypeptides, nucleic acids, vectors, and antibodies of the
invention. A biologically compatible solution is a solution in
which the polypeptide, nucleic acid, vector, or antibody of the
invention is maintained in an active form, e.g., in a form able to
effect a biological activity. For example, a polypeptide of the
invention would have phospholipase activity; a nucleic acid would
be able to replicate, translate a message, or hybridize to a
complementary nucleic acid; a vector would be able to transfect a
target cell; an antibody would bind a polypeptide of the invention.
Generally, such a biologically compatible solution will be an
aqueous buffer, e.g., Tris, phosphate, or HEPES buffer, containing
salt ions. Usually the concentration of salt ions will be similar
to physiological levels. In a specific embodiment, the
biocompatible solution is a pharmaceutically acceptable
composition. Biologically compatible solutions may include
stabilizing agents and preservatives.
[0167] Such compositions can be formulated for administration by
topical, oral, parenteral, intranasal, subcutaneous, and
intraocular, routes. Parenteral administration is meant to include
intravenous injection, intramuscular injection, intraarterial
injection or infusion techniques. The composition may be
administered parenterally in dosage unit formulations containing
standard, well known nontoxic physiologically acceptable carriers,
adjuvants and vehicles as desired.
[0168] The preferred sterile injectable preparations can be a
solution or suspension in a nontoxic parenterally acceptable
solvent or diluent. Examples of pharmaceutically acceptable
carriers are saline, buffered saline, isotonic saline (e.g.
monosodium or disodium phosphate, sodium, potassium, calcium or
magnesium chloride, or mixtures of such salts), Ringer's solution,
dextrose, water, sterile water, glycerol, ethanol, and combinations
thereof. 1,3-butanediol and sterile fixed oils are conveniently
employed as solvents or suspending media. Any bland fixed oil can
be employed including synthetic mono- or di-glycerides. Fatty acids
such as oleic acid also find use in the preparation of
injectables.
[0169] The composition medium can also be a hydrogel which is
prepared from any biocompatible or non-cytotoxic (homo or hetero)
polymer, such as a hydrophilic polyacrylic acid polymer that can
act as a drug absorbing sponge. Such polymers have been described,
for example, in application WO93/08845, the entire contents of
which are hereby incorporated by reference. Certain of them, such
as, in particular, those obtained from ethylene and/or propylene
oxide are commercially available. A hydrogel can be deposited
directly onto the surface of the tissue to be treated, for example
during surgical intervention.
[0170] Another preferred embodiment of the present invention
relates to a pharmaceutical composition comprising a replication
defective recombinant virus and poloxamer. More specifically, the
invention relates to a composition comprising a replication
defective recombinant virus comprising a nucleic acid encoding an
LIPG polypeptide and poloxamer. A preferred poloxamer is Poloxamer
407, which is commercially available (BASF, Parsippany, N.J.) and
is a non-toxic, biocompatible polyol, and is most preferred. A
poloxamer impregnated with recombinant viruses may be deposited
directly on the surface of the tissue to be treated, for example
during a surgical intervention. Poloxamer possesses essentially the
same advantages as hydrogel while having a lower viscosity.
Methods of Treatment
[0171] The present invention provides methods of treatment which
comprise the administration to a human or other animal of an
effective amount of a composition of the invention.
[0172] Effective amounts may vary, depending on the age, type and
severity of the condition to be treated, body weight, desired
duration of treatment, method of administration, and other
parameters. Effective amounts are determined by a physician or
other qualified medical professional. In most cases, the dosage
levels may be adjusted so that the desired levels of HDL
cholesterol and apolipoprotein AI are achieved and maintained.
Similarly, the dosage levels may be adjusted to lower the VLDL
cholesterol and LDL cholesterol levels to acceptable levels and
bring the ratio HDL cholesterol to LDL cholesterol and VLDL
cholesterol to within desirable levels.
[0173] Polypeptides according to the invention are generally
administered in doses of about 0.01 mg/kg to about 100 mg/kg,
preferably about 0.1 mg/kg to about 50 mg/kg, and most preferably
about 1 mg/kg to about 10 mg/kg of body weight per day.
[0174] Neutralizing antibodies according to the invention may be
delivered as a bolus only, infused over time or both administered
as a bolus and infused over time. Although the dosage amount will
vary based on the parameters above, and on the binding ability of
the antibody, a dose 0.2 to 0.6 mg/kg may be given as a bolus
followed by a 2 to 12 hour infusion period. Alternatively, multiple
bolus injections are administered every other day or every third or
fourth day as needed. Dosage levels may be adjusted as determined
by HDL cholesterol levels and/or VLDL and LDL cholesterol
levels.
[0175] As discussed hereinabove, recombinant viruses may be used to
introduce both DNA encoding LIPG and subfragments of LIPG as well
as antisense nucleic acids. Recombinant viruses according to the
invention are generally formulated and administered in the form of
doses of between about 104 and about 10.sup.14 pfu. In the case of
AAVs and adenoviruses, doses of from about 10.sup.6 to about
10.sup.11 pfu are preferably used. The term pfu ("plaque-forming
unit") corresponds to the infective power of a suspension of
virions and is determined by infecting an appropriate cell culture
and measuring the number of plaques formed. The techniques for
determining the pfu titre of a viral solution are well documented
in the prior art.
[0176] Ribozymes according to the present invention may be
administered in amounts ranging from about 5 to about 50 mg/kg/day
in a pharmaceutically acceptable carrier. Dosage levels may be
adjusted based on the measured therapeutic efficacy.
[0177] Appropriate levels of inhibitor or enhancer molecules may be
determined by qualified medical personnel using the parameters
discussed above.
[0178] The present invention provides compositions and methods for
increasing the level of HDL cholesterol and apolipoprotein AI and
lowering the levels of VLDL and LDL cholesterol in a patient. The
present invention further provides methods of treating a human or
other animal having an undesirable lipid profile, wherein said
undesirable lipid profile is the result of abnormally high
expression of LIPG polypeptide activity.
[0179] Methods and Compositions for Lowering Levels of LIPG
Polypeptide Activity
[0180] The methods for decreasing the expression of LIPG
polypeptide in order to increase the levels of HDL cholesterol and
apolipoprotein AI and correct those conditions in which LIPG
polypeptide activity contributes to a disease or disorder
associated with an undesirable lipid profile include but are not
limited to administration of a composition comprising an antisense
nucleic acid, administration of a composition comprising an
intracellular binding protein such as an antibody, administration
of an inhibitory molecule which inhibits the enzymatic activity of
LIPG, for example, a composition comprising an expression vector
encoding a subfragment of LIPG, for example, LLGN polypeptide or a
small molecular weight molecule, including administration of a
small molecular weight compound which down regulates LIPG
expression at the level of transcription, translation or
post-translation, and administration of a ribozyme which cleaves
mRNA encoding LIPG.
[0181] Methods Utilizing Antisense Nucleic Acids
[0182] In one embodiment, a composition comprising an antisense
nucleic acid is used to down-regulate or block the expression of
LIPG. In one preferred embodiment, the nucleic acid encodes
antisense RNA molecules. In this embodiment, the nucleic acid is
operably linked to signals enabling expression of the nucleic acid
sequence and is introduced into a cell utilizing, preferably,
recombinant vector constructs, which will express the antisense
nucleic acid once the vector is introduced into the cell. Examples
of suitable vectors includes plasmids, adenoviruses,
adeno-associated viruses, retroviruses, and herpes viruses.
Preferably, the vector is an adenovirus. Most preferably, the
vector is a replication defective adenovirus comprising a deletion
in the E1 and/or E3 regions of the virus.
[0183] In another embodiment, the antisense nucleic acid is
synthesized and may be chemically modified to resist degradation by
intracellular nucleases, as discussed above. Synthetic antisense
oligonucleotides can be introduced to a cell using liposomes.
Cellular uptake occurs when an antisense oligonucleotide is
encapsulated within a liposome. With an effective delivery system,
low, non-toxic concentrations of the antisense molecule can be used
to inhibit translation of the target mRNA. Moreover, liposomes that
are conjugated with cell-specific binding sites direct an antisense
oligonucleotide to a particular tissue.
[0184] Methods Utilizing Neutralizing Antibodies and Other Binding
Proteins
[0185] In another embodiment, the expression of LIPG is
down-regulated or blocked by the expression of a nucleic acid
sequence encoding an intracellular binding protein which is capable
of selectively interacting with LIPG. WO 94/29446 and WO 94/02610,
the contents of which are incorporated herein by reference,
disclose cellular transfection with genes encoding an intracellular
binding protein. An intracellular binding protein includes any
protein capable of selectively interacting, or binding, with LIPG
in the cell in which it is expressed and of neutralizing the
function of bound LLG. Preferably, the intracellular binding
protein is a neutralizing antibody or a fragment of a neutralizing
antibody. More preferably, the intracellular binding protein is a
single chain antibody.
[0186] WO 94/02610 discloses preparation of antibodies and
identification of the nucleic acid encoding a particular antibody.
Using LIPG or a fragment thereof, a specific monoclonal antibody is
prepared by techniques known to those skilled in the art. A vector
comprising the nucleic acid encoding an intracellular binding
protein, or a portion thereof, and capable of expression in a host
cell is subsequently prepared for use in the method of this
invention.
[0187] Alternatively, LIPG activity can be blocked by
administration of a neutralizing antibody into the circulation.
Such a neutralizing antibody can be administered directly as a
protein, or it can be expressed from a vector (with a secretory
signal).
[0188] Methods Utilizing an Inhibitory Molecule Which Inhibits the
Enzymatic Activity of LIPG
[0189] In another embodiment, LIPG activity is inhibited by the
administration of a composition comprising a subfragment of LIPG
polypeptide, for example, LLGN. This composition may be
administered in a convenient manner, such as by the oral, topical,
intravenous, intraperitoneal, intramuscular, subcutaneous,
intranasal, or intradermal routes. The composition may be
administered directly or it may be encapsulated (e.g. in a lipid
system, in amino acid microspheres, or in globular dendrimers). The
polypeptide may, in some cases, be attached to another polymer such
as serum albumin or polyvinyl pyrrolidone.
[0190] In another embodiment, LIPG activity is inhibited through
the use of gene therapy, that is, through the administration of a
composition comprising a nucleic acid which encodes and directs the
expression of a subfragment of LIPG, for example, LLGN.
[0191] In another embodiment, LIPG activity is inhibited through
the use of inhibitory molecules. These low molecular weight
compounds interfere with LIPG's enzymatic properties or prevent its
appropriate recognition by cellular binding sites.
[0192] In a specific embodiment, the LIPG polypeptide of the
present invention also has an affinity for heparin. LIPG
polypeptide binding to extracellular heparin in the lumen of blood
vessels would permit LIPG to bind to and accelerate LDL uptake by
acting as a bridge between LDL and the extracellular heparin. In
the localized area of an atherosclerotic lesion, an increased level
of lipase activity is hypothesized to accelerate the atherogenic
process (Zilversmit, D. B. (1995) Clin. Chem. 41,153-158; Zambon,
A., Torres, A., Bijvoet, S., Gagne, C., Moojani, S., Lupien, P. J.,
Hayden M. R., and Brunzell, J. D. (1993) Lancet 341, 1119-1121).
This may be due to an increase in the binding and uptake of
lipoproteins by vascular tissue mediated by lipases (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). Additionally, a high local level of
lipase activity may result in cytotoxic levels of fatty acids and
lysophosphatidylcholine being produced in precursors of
atherosclerotic lesions. This particular activity of LLG may
contribute to the development or progression of atherosclerosis,
particularly in the context of excessive lipid levels in a subject
due to dietary or genetic factors. Thus, the present invention
permits inhibition of lipoprotein accumulation by inhibiting LIPG
polypeptide expression or binding to lipoprotein (e.g., LDL).
[0193] Methods Utilizing an Inhibitory Molecule Which Prevents LIPG
Gene Expression
[0194] In another embodiment, inhibitory molecules, including small
molecular weight compounds, are able to down regulate LIPG
expression at the level of transcription, translation or
post-translation. In order to identify such inhibitory molecules,
the reporter gene systems described above may be used. These
inhibitory molecules may be combined with a pharmaceutically
acceptable carrier and administered using conventional methods
known in the art.
[0195] Methods Utilizing Ribozymes
[0196] Ribozymes may be administered to cells by encapsulation in
liposomes, by iontophoresis, by incorporation into hydrogels,
cyclodextrins, biodegradable nanocapsules, and bioadhesive
microspheres or by any of a variety of other methods dicussed
above. The ribozyme may be delivered to a target tissue by direct
injection or by use of a catheter, infusion pump or stent.
Alternative routes of delivery include intravenous injection,
intramuscular injection, subcutaneous injection, aerosol
inhalation, oral (tablet or pill form), topical, systemic, ocular,
intraperitoneal and/or intrathecal delivery.
[0197] In preferred embodiments, a ribozyme-encoding sequence is
cloned into a DNA expression vector. Transcription of the ribozyme
sequence is driven from an eukaryotic RNA polymerase II (pol II),
or RNA polymerase III (pol III) promoter. The expression vector can
be incorporated into a variety of vectors including the viral DNA
vectors such as adenovirus or adeno-associated virus vectors
discussed above.
[0198] 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. The vector is delivered as
recombinant viral particles and is locally administered to the site
of treatment, through the use of a catheter, stent or infusion
pump.
[0199] Administration of Apolipoprotein AI
[0200] In another embodiment, any of the methods discussed above
for lowering the levels of LIPG polypeptide activity are utilized
in combination with administration of apolipoprotein AI or an
expression system capable of expressing apolipoprotein AI in a
patient (see, for example, U.S. Pat. No. 5,866,551, which is
incorporated herein by reference).
[0201] Methods and Compositions for Increasing Levels of LIPG
Polypeptide Activity
[0202] The methods for increasing the expression or activity of
LIPG polypeptide to lower the levels of VLDL and LDL cholesterol
include, but are not limited to, administration of a composition
comprising the LIPG polypeptide, administration of a composition
comprising an expression vector which encodes the LIPG polypeptide,
administration of a composition comprising an enhancer molecule
which enhances the enzymatic activity of the LIPG polypeptide and
administration of an enhancer molecule which increases expression
of the LIPG gene.
[0203] Methods Utilizing LIPG Polypeptides
[0204] In one embodiment, the level of LIPG activity is increased
through the administration of a composition comprising the LIPG
polypeptide. This composition may be administered in a convenient
manner, such as by the oral, topical, intravenous, intraperitoneal,
intramuscular, subcutaneous, intranasal, or intradermal routes. The
composition may be administered directly or it may be encapsulated
(e.g. in a lipid system, in amino acid microspheres, or in globular
dendrimers). The polypeptide may; in some cases, be attached to
another polymer such as serum albumin or polyvinyl pyrrolidone.
[0205] Methods Utilizing Vectors that Express LIPG
[0206] In another embodiment, the level of LIPG is increased
through the use of gene therapy, that is, through the
administration of composition comprising a nucleic acid which
encodes and directs the expression of the LIPG polypeptide. In this
embodiment, the LIPG polypeptide is cloned into an appropriate
expression vector. Possible vector systems and promoters are
extensively discussed above. The expression vector is transferred
into the target tissue using one of the vector delivery systems
discussed above. This transfer is carried out either ex vivo in a
procedure in which the nucleic acid is transferred to cells in the
laboratory and the modified cells are then administered to the
human or other animal, or in vivo in a procedure in which the
nucleic acid is transferred directly to cells within the human or
other animal. In preferred embodiments, an adenoviral vector system
is used to deliver the expression vector. If desired, a tissue
specific promoter is utilized in the expression vector as described
above.
[0207] Non-viral vectors may be transferred into cells using any of
the methods known in the art, including calcium phosphate
coprecipitation, lipofection (synthetic anionic and cationic
liposomes), receptor-mediated gene delivery, naked DNA injection,
electroporation and bioballistic or particle acceleration.
[0208] Methods Utilizing an Enhancer Molecule Which Enhances the
Enzymatic Activity of LIPG
[0209] In another embodiment, the activity of LIPG is enhanced by
enhancer molecules that increase the enzymatic activity of LIPG or
increase its appropriate recognition by cellular binding sites.
These enhancer molecules may be introduced by the same methods
discussed above for the administration of polypeptides.
[0210] Methods Utilizing an Enhancer Molecule Which Increases LIPG
Gene Expression
[0211] In another embodiment, the level of LIPG is increased
through the use of small molecular weight compounds, which can
upregulate LIPG expression at the level of transcription,
translation, or post-translation. These compounds may be
administered by the same methods discussed above for the
administration of polypeptides.
[0212] Treatment Methods Relating to Impaired Biliary Excretion
[0213] Intrahepatic cholestasis can be characterized by increased
serum cholesterol and phospholipid levels. A recently described,
phalloidin drug-induced intrahepatic cholestasis model in rats
demonstrated significant increases in the serum levels of
cholesterol and phospholipid (Ishizaki, K., Kinbara, S., Miyazawa,
N., Takeuchi, Y., Hirabayashi, N., Kasai, H., and Araki, T. (1997)
Toxicol. Letters 90, 29-34). The products of this invention may be
used to treat intrahepatic cholestasis in patients that have
increased serum cholesterol and/or phospholipid. In addition, this
rat model also exhibited a severe decrease in biliary cholesterol
excretion rates. The LIPG polypeptide and nucleic acid products of
this invention may be used to treat patients with an impaired
biliary excretion system.
[0214] Intrahepatic cholestasis is also characterized by impaired
bile flow from the liver. Recently, the loci for progressive
familial intrahepatic cholestasis (PFIC or Byler disease) and
benign recurrent intrahepatic cholestasis (BRIC) were mapped to
18q21-q22 (Carlton, V. E. H., Knisely, A. S., and Freimer, N. B.
(1995) Hum. Mol. Genet. 4, 1049-1053 and Houwen, R. H., Baharloo,
S., Blankenship, K., Raeymaekers, P., Juyn, J., Sandkuijl, L. A.,
and Freimer, N. B. (1994) Nature Genet. 8, 380-386, respectively).
As LLG gene maps within this chromosomal region at 18q21, the LLG
gene or products of this invention may be used to treat patients
with intrahepatic cholestasis that is caused by a mutation or
defective expression of the PFIC/BRIC disease gene(s).
[0215] In another embodiment, the LLG gene or polypeptide: products
of this invention may be used to treat patients with intrahepatic
cholestasis that is not due to a defect in the PFIC/BRIC disease
gene(s) at 18q21-q22. A recent study suggested that another locus,
located outside of the 18q21-q22 region may also produce the 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, administration of LLG polypeptide, either
directly or via gene therapy, may alleviate this form of the
condition.
[0216] Methods and Compositions for Diagnosing a Predisposition to
Low HDL Levels
[0217] Given the ability of LIPG polypeptide to lower the levels of
HDL cholesterol and apolipoprotein AI, the level of LIPG
polypeptide in the body may be used to determine whether an
individual is predisposed to low levels of HDL cholesterol and
apolipoprotein AI. In this method, a tissue sample is taken from
the patient. The tissue may be blood or one of the tissues which
has been demonstrated to express LIPG as discussed in the Examples
section. Measurement of the level of LIPG may be performed by a
variety of methods known to those of skill in the art. In preferred
embodiments, an antibody directed against LIPG polypeptide may be
used to measure the level of LIPG in a tissue sample.
EXAMPLES
[0218] The following examples illustrate the invention. These
examples are illustrative only, and do not limit the scope of the
invention.
Example 1
Identification of a Differentially Expressed cDNA
[0219] RNA Preparation
[0220] Human monocytic THP-1 cells (Smith, P. K., Krohn, R. I.,
Hermanson, G. T., Mallia, A. K., Gartner, F. H. Provenzano, M. D.,
Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C.
(1985) Anal. Biochem. 150, 76-85) were cultured in RPMI-1640 medium
(GIBCO) with 25 mM HEPES, 10% fetal bovine serum, 100 units/ml
penicillin G sodium and 100 units/ml streptomycin sulfate. Cells
were plated onto 15 cm tissue culture dishes at 1.5.times.10.sup.7
cells/plate, and treated with 40 ng/ml phorbol 12-myristate
13-acetate (Sigma) for 48 hours to induce differentiation of the
cells. Human low density lipoproteins (LDL) were purchased from
Calbiochem, and were dialyzed exhaustively versus PBS at 4.degree.
C. The LDL was then diluted to 500 .mu.g/ml and dialyzed versus 5
.mu.M CuSO.sub.4 in PBS at 37.degree. C. for 16 hours. To stop
oxidation, the LDL was dialyzed exhaustively versus 150 mM NaCl,
0.3 mM EDTA, then filter sterilized. Protein concentration was
determined by the BCA method (Schuh, J. Fairclough, G. F., and
Haschemeyer, R. H. (1978) Proc. Natl. Acad. Sci. USA 75, 3173-3177)
(Pierce). The degree of oxidation was determined by TBARS
(Chomczynski, P. (1993) Biotechniques 15, 532-537), and was between
25-30 nmol MDA equivalents/mg protein. The differentiated THP-1
cells were exposed for 24 hours to either 50 .mu.g/ml oxidized LDL
or NaCl-EDTA buffer in RPMI medium with 10% lipoprotein-deficient
fetal bovine serum (Sigma). To harvest the RNA, the plates were
rinsed with 10 ml of PBS, then 14 ml of TRIZOL (Liang, P. and
Pardee, A. B. (1992) Science 257,967-971) (GIBCO) were added to
each plate. The solution was pipetted several times to mix, then
like samples were pooled into centrifuge tubes and 3 ml chloroform
per plate were added and mixed. The tubes were centrifuged for 15
minutes at 12000.times.g. After centrifugation the upper layer was
transferred to a new tube and 7.5 ml isopropanol per plate was
added and mixed. The tubes were centrifuged at 12000.times.g for 20
minutes. The pellet was rinsed with ice-cold 70% ethanol and dried
at room temperature. The pellets were suspended in 500 .mu.l TE
(Tris-EDTA) and treated with 200 units RNase-free DNAse I and 200
units RNasin placental RNase inhibitor (Promega) for 30 minutes at
37.degree. C. The RNA was purified by sequential extractions with
phenol, phenol/chloroform/isoamyl alcohol (25:24:1), and
chloroform/isoamyl alcohol (24:1) followed by ethanol
precipitation.
[0221] cDNA Synthesis
[0222] cDNA synthesis and PCR amplification were accomplished using
protocols from the Differential Display Kit, version 1.0 (Display
Systems Biotechnology, Inc.) This system is based on the technique
originally described by Liang and Pardee (Mead, D. A., Pey, N. K.,
Herrnstadt, C., Marcil, R. A., and Smith, L. M., (1991)
Bio/Technology 9,657-663). The primer pairs which yielded the cDNA
fragment containing the first information of the lipase like gene
were downstream primer 7 and upstream primer 15. The cDNA for the
amplification was synthesized as follows, using RNA derived from
PMA treated THP-1 cells exposed to either buffer or oxidized LDL: 3
.mu.l of 25 .mu.M downstream primer 7 and 7.5 .mu.l of
diethylpyrocarbonate (DEPC)-treated water were added to 300 ng (3.0
.mu.l) RNA from either sample of THP-1 RNA. This was heated to
70.degree. C. for 10 minutes then chilled on ice. To this tube were
added 3 .mu.l of 5.times. PCR buffer (250 mM Tris-HCl pH 8.3, 375
mM KCl) (GIBCO), 3 .mu.l 25 mM MgCl.sub.2, 3 .mu.l 0.1M DTT, 1.2
.mu.l 500 .mu.M dNTPs, 0.7 .mu.l RNasin, and 5.6 .mu.l DEPC-treated
water. The tubes were incubated for 2 minutes at room temperature,
after which 1.5 .mu.l (300 units) Superscript II RNase H-reverse
transcriptase (GIBCO) were added. The tubes were incubated
sequentially at room temperature for 2 minutes, 60 minutes at
37.degree. C., and 5 minutes at 95.degree. C., followed by chilling
on ice. PCR amplification was performed using a master mix
containing 117 .mu.l 10.times. PCR buffer (500 mM KCl, 100 mM
Tris-HCl pH 8.3, 15 mM MgCl.sub.2, and 0.01% (w/v) gelatin), 70.2
.mu.l 25 MM MgCl.sub.2, 5.9 .mu.l alpha-.sup.33P dATP (10 m Ci/ml,
DuPont NEN), 4.7 .mu.l 500 .mu.M dNTP mix, 11 .mu.l AmpliTaq DNA
polymerase (5 units/.mu.l, Perkin-Elmer), and 493.3 .mu.l
DEPC-treated water. For each reaction, 12 .mu.l of the master mix
was added to 2 .mu.l downstream primer #7, 1 .mu.l of cDNA, and 5
.mu.l of upstream primer #15. The reaction mixes were heated to
94.degree. C. for 1 minute, then thermocycled 40 times with a
denaturing step of 94.degree. C. for 15 seconds, annealing step of
40.degree. C. for 1 minute, and an extension step of 72.degree. C.
for 30 seconds. Following the 40 cycles, the reactions were
incubated at 72.degree. C. for 5 minutes and stored at 10.degree.
C. The PCR reactions were performed in a Perkin-Elmer GeneAmp
System 9600 thermocycler.
[0223] Four microliters of the amplification reaction were mixed
with an equal volume of loading buffer (0.2% bromphenol blue, 0.2%
Xylene cyanol, 10 mM EDTA pH 8.0, and 20% glycerol). Four
microliters of this mix was run on a 6% nondenaturing acrylamide
sequencing format gel for 3 hours at 1200 volts (constant voltage).
The gel was dried at 80.degree. C. for 1.5 hours and exposed to
Kodak XAR film. An amplification product found only in the reaction
containing cDNA from THP-1 cells exposed to oxidized LDL was
identified and excised from the gel. 100 .mu.l of DEPC-treated
water was added to a microcentrifuge tube containing the excised
gel fragment and was incubated for 30 minutes at room temperature
followed by 15 minutes at 95.degree. C.
[0224] To reamplify the PCR product, 26.5 microliters of the eluted
DNA were used in a amplification reaction that also included 5
.mu.l 10.times. PCR buffer, 3 .mu.l 25 mM MgCl.sub.2, 5 .mu.l 500
.mu.M dNTPs, 5 .mu.l 2 .mu.M downstream primer 7, 7.5 .mu.l
upstream primer 15, and 0.5 .mu.l Amplitaq polymerase. The PCR
cycling parameters and instrument were as described above.
Following amplification, 20 .mu.l of the reamplification was
analyzed on an agarose gel and 4 .mu.l was used to subclone the PCR
products into the vector pCRII using the TA cloning system
(Frohman, M. A., Dush, M. K., and Martin, G. R. (1988) Proc. Natl.
Acad. Sci. USA 85, 8998-9002) (Invitrogen). Following an overnight
ligation at 14.degree. C., the ligation products were used to
transform E. coli. Resulting transformants were picked and 3 ml
overnight cultures were used in plasmid minipreparations. Insert
sizes were determined using EcoRI digestions of the plasmids and
clones containing inserts of the approximate size of the original
PCR product were sequenced using fluorecent dye-terminator reagents
(Prism, Applied Biosystems) and an Applied Biosystems 373 DNA
sequencer. The sequence of the PCR product is shown in FIG. 2. The
sequence of the amplification primers is underlined.
[0225] 5' RACE Reaction
[0226] Extension of the cDNA identified through RT-PCR was
accomplished using the 5' RACE system (Loh, E. Y., Eliot, J. F.,
Cwirla, S., Lanier, L. L., and Davis, M. M. (1989) Science 243,
217-219; Simms, D., Guan, N., and Sitaraman, K., (1991) Focus
13,99) (GIBCO). One microgram of the THP-1 RNA (oxidized LDL
treated) used initially in the differential display reactions was
utilized in the 5' RACE procedure:
[0227] 1 .mu.l (1 .mu.g) of RNA was combined with 3 .mu.l (3 pmol)
primer 2a and 11 .mu.l DEPC-treated water and heated to 70.degree.
C. for 10 minutes followed by 1 minute on ice. 2.5 .mu.l 10.times.
reaction buffer (200 mM Tris-HCl pH 8.4, 500 mM KCl), 3 .mu.l 25 mM
MgCl.sub.2, 1 .mu.l 10 mM dNTP mix, and 2.5 .mu.l 0.1 M DTT were
added. The mix was incubated at 42.degree. C. for 2 minutes, then 1
.mu.l Superscript II reverse transcriptase was added. The reaction
was incubated for an additional 30 minutes at 42.degree. C., 15
minutes at 70.degree. C., and on ice for 1 minute. One microliter
of RNase H (2 units) was added and the mixture was incubated at
55.degree. C. for 10 minutes. The cDNA was purified using the
GlassMax columns (Sambrook, J. Fritsch, E. F., and Maniatis, T.
(1989) Molecular Cloning: A Laboratory Manual, second edition, Cold
Spring Harbor Laboratory Press, Plainview, N.Y.) included in the
kit. The cDNA was eluted from the column in 50 .mu.l dH.sub.2O,
lyophilized, and resuspended in 21 .mu.l dH.sub.2O. Tailing of the
cDNA was accomplished in the following reaction: 7.5 .eta.l
dH.sub.2O, 2.5 .mu.l reaction buffer (200 mM Tris-HCl pH 8.4, 500
mM KCl), 1.5 .mu.l 25 mM MgCl.sub.2, 2.5 .mu.l 2 mM dCTP, and 10
.mu.l of the cDNA were incubated at 94.degree. C. for 3 minutes,
then 1 minute on ice. 1 .mu.l (10 units) of terminal
deoxynucleotidyl transferase was added and the mixture was
incubated for 10 minutes at 37.degree. C. The enzyme was heat
inactivated by incubation at 70.degree. C. for 10 minutes and the
mixture was placed on ice. PCR amplification of the cDNA was
performed in the following steps: 5 .mu.l of the tailed cDNA was
included in a reaction which also contained 5 .mu.l 10.times. PCR
buffer (500 mM KCl, 100 mM Tris-HCl pH 8.3, 15 mM MgCl.sub.2, and
0.01% (w/v) gelatin), 1 .mu.l 10 mM dNTP mix, 2 .mu.l (10 pmol)
anchor primer, 1 .mu.l (20 pmol) primer 3a, and 35 .mu.l dH.sub.2O.
The reaction was heated to 95.degree. C. for 1 minute, then 0.9
.mu.l (4.5 units) Amplitaq polymerase was added. The reaction was
cycled 40 times under the following conditions: 94.degree. C. for 5
seconds, 50.degree. C. for 20 seconds, and 72.degree. C. for 30
seconds. One microliter of this reaction was used in a nested
reamplification to increase levels of specific product for
subsequent isolation. The reamplification included: 1 .mu.l primary
amplification, 5 .mu.l 10.times. PCR buffer, 1 .mu.l 10 mM dNTP
mix, 2 .mu.l (20 pmol) universal amplification primer, 2 .mu.l (20
pmol) primer 4a, and 38 .mu.l dH.sub.2O. The reaction was heated to
95.degree. C. for 1 minute, then 0.7 .mu.l (3.5 units) Amplitaq
polymerase was added. The reaction was cycled 40 times under these
conditions; 94.degree. C. for 5 seconds, 50.degree. C. for 20
seconds, and 72.degree. C. for 30 seconds. The amplification
products were analyzed via 0.8% agarose gel electrophoresis. A
predominant product of approximately 1.2 kilobase pairs was
detected. Two microliters of the reaction products were cloned into
the pCRII vector from the TA cloning kit (Invitrogen) and incubated
at 14.degree. C. overnight. The ligation products were used to
transform E. coli. The insert sizes of the resulting transformants
were determined following EcoRI digestion. Clones containing
inserts of the approximate size of the PCR product were sequenced
using fluorescent dye-terminator reagents (Prism, Applied
Biosystems) and an Applied Biosystems 373 DNA sequencer. The
sequence of the RACE product including the EcoRI sites from the TA
vector are shown in FIG. 3. The sequences of the amplimers
(universal amplification primer and the complement to 5' RACE
primer 4a) are underlined.
Example 2
Cloning and Chromosomal Localization of the LIPG Gene
[0228] cDNA library screening
[0229] A human placental cDNA library (Oligo dT and random primed,
Cat #5014b, Lot #52033) was obtained from Clontech (Palo Alto,
Calif.). A radiolabeled probe was created by excising the insert of
a plasmid containing the 5' RACE reaction PCR product described
above. The probe was radiolabeled using the random priming
technique: the DNA fragment (50-100 ng) was incubated with 1 .mu.g
of random hexamers (Gibco) at 95.degree. C. for 10 minutes followed
by 1 minute on ice. At room temperature the following were added: 3
.mu.l 10.times. Klenow buffer (100 mM Tris-HCl pH 7.5, 50 mM
MgCL.sub.2, 57 mM dithiothreitol; New England Biolabs), 3 .mu.l 0.5
mM dATP, dGTP, dTTP), 100 .mu.Ci .alpha.-.sup.32PdCTP (3000
Ci/mmol, New England Nuclear), and 1 .mu.l Klenow fragment-of DNA
polymerase I (5 units, Gibco). The reaction was incubated for 2-3
hours at room temperature and the reaction was then stopped by
increasing the volume to 100 .mu.l with TE pH 8.0 and adding EDTA
to a final concentration of 1 mM. The unincorporated nucleotides
were removed by raising the reaction volume to 100 .mu.l and
passing over a G-50 spin column (Boehringer Mannheim). The
resulting probes had a specific activity greater than
5.times.10.sup.8 cpm/.mu.g DNA.
[0230] The library was probed using established methods (Walter,
P., Gilmore, R., and Blobel, G. (1984) Cell 38,5-8). Briefly, the
filters were hybridized for 24 hours at 65.degree. C. in 4.8.times.
SSPE (20.times. SSPE=3.6 M NaCl, 0.2 M NaH.sub.2PO.sub.4, 0.02 M
EDTA, pH 7.7), 20 mM Tris-HCl pH 7.6, 1.times. Denhardt's solution
(100.times.=2% Ficoll 400, 2% polyvinylpyrrolidone, 2% BSA), 10%
dextran sulfate, 0.1% SDS, 100 .mu.g/ml salmon sperm DNA, and
1.times.10.sup.6 cpm/ml radiolabelled probe. Filters were then
washed three times for 15 minutes at room temperature in
2.times.SSC (1.times.SSC=150 mM NaCl, 15 mM sodium citrate pH 7.0),
0.1% sodium dodecyl sulfate (SDS) followed by three washes for 15
minutes each at 65.degree. C. in 0.5.times.SSC, 0.1% SDS. Phage
which hybridized to the probe were isolated and amplified. DNA was
purified from the amplified phage using LambdaSorb reagent
(Promega) according to the manufacturer's instructions. The inserts
were excised from the phage DNA by digestion with EcoRI. The
inserts were subcloned into the EcoRI site of a plasmid vector
(Bluescript II SK, Stratagene). The sequence of the open reading
frame contained within the 2.6 kb EcoRI fragment of the cDNA was
determined by automated sequencing as described above. The sequence
is shown in FIG. 4. The amino acid sequence of the predicted
protein encoded by the open reading frame is shown in FIG. 5 and
has been termed LLGXL. The first methionine is predicted to be
encoded by nucleotide pairs 252-254. The predicted protein is 500
amino acids in length. The first 18 amino acids form a sequence
characteristic of a secretory signal peptide (Higgins, D. G., and
Sharp, P. M. (1988) Gene 73, 237-244). The propeptide is predicted
to have a molecular weight of 56,800 Daltons. Assuming cleavage of
the signal peptide at position 18, the unmodified mature protein
has a molecular weight of 54,724 Daltons.
[0231] The overall similarities between This protein and the other
known members of the triacylglycerol lipase family is illustrated
in FIG. 6 and Table 1. In the alignment shown in FIG. 6, LIPG is
the polypeptide (SEQ ID NO: 6) encoded by the cDNA (SEQ ID NO: 5)
described in Example 1, and hereafter referred to as LLGN. This
protein is identical with the LLGXL protein in the amino terminal
345 residues. Nine unique residues are followed by a termination
codon, producing a propolypeptide of 39.3 kD and a mature protein
of 37.3 kD. The sequences which are common to LLGN and LLGXL are
nucleic acid sequence SEQ ID NO: 9 and amino acid sequence SEQ ID
NO: 10.
[0232] Interestingly, the position at which the LLGN and LLGXL
proteins diverge is at a region known from the structure of the
other lipase to be between the amino and carboxy domains of the
proteins. Therefore, the LLGN protein appears to consist of only
one of the two domains of triaclyglycerol lipases. This sequence
contains the characteristic "GXSXG" lipase motif at positions
167-171 as well as conservation of the catalytic triad residues at
Ser 169, Asp 193, and His 274. Conservation of cysteine residues
(positions 64, 77, 252, 272, 297, 308, 311, 316, 463, and 483)
which have been implicated in disulfide linkage in the other
lipases suggests that the LLGXL protein has structural similarities
to the other enzymes. There are five predicted sites for N-linked
glycosylation; at amino acid positions 80, 136, 393, 469, and 491.
The protein sequences used in the comparisons are human lipoprotein
lipase (LPL; Genbank accession #M15856, SEQ ID NO: 13), Human
hepatic lipase (HL; Genbank accession #J03540, SEQ ID NO: 14),
human pancreatic lipase (PL; Genbank accession # M93285, SEQ ID NO:
15), human pancreatic lipase related protein-1 (PLRP-1; Genbank
accession # M93283), and human pancreatic lipase related protein-2
(PLRP-2; Genbank accession # M93284). TABLE-US-00001 TABLE 1
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 PRLP2 22.6 20.9 22.0 62.2
61.7 --
Percent similarity was based on pairwise alignment using the
Clustal algorithm (Camps, L., Reina, M., Llobera, M., Vilaro, S.,
and Olivecrona, T. (1990) Am. J. Physiol. 258, C673-C681) in the
Megalign program of the Lasergene Biocomputing Software Suite
(Dnastar).
[0233] Chromosomal Localization
[0234] DNA from a P1 clone (Sternberg, N., Ruether, J. and DeRiel,
K. The New Biologist 2:151-62, 1990) containing genomic LLG DNA was
labelled with digoxigenin UTP by nick translation. Labelled probe
was combined with sheared human DNA and hybridized to PHA
stimulated peripheral blood lymphocytes from a male donor in a
solution containing 50% formamide, 10% dextran sulfate, and
2.times.SSC. Specific hybridization signals were detected by
incubating the hybridized cells in fluoresceinated antidigoxigenin
antibodies followed by counterstaining with DAPI. This initial
experiment resulted in specific labeling of a group E chromosome,
which was believed to be chromosome 18 on the basis of DAPI
staining.
[0235] A second experiment was conducted in which a biotin labelled
probe specific for the centromere of chromosome 18 was cohybridized
with the LLG probe. This experiment resulted in the specific
labeling of the chromosome 18 centromere in red and the long arm of
chromosome 18 in green. Measurements of 11 specifically labelled
hybridized chromosomes 18 demonstrated that LLG has a Flter of 0.67
(Franke measurement of 0.38), which corresponds to band 18q21.
Several genetic diseases, including intrahepatic cholestasis, cone
rod dystrophy, and familial expansile osteolysis, are believed to
involve defects in this chromosomal region.
Example 3
LIPG RNA Analysis
[0236] Expression of LIPG RNA in THP-1 cells
[0237] Analysis of the mRNA from which the cDNA was derived was
performed by northern analysis of THP-1 RNA. RNA from these cells
was prepared as described above. The mRNA was purified from the
total RNA through the use of a poly-dT-magnetic bead system
(Polyattract system, Promega). Three micrograms of poly
(A)-containing mRNA was electrophoresed on a 1%
agarose-formaldehyde gel. The gel was washed for 30 minutes in
dH.sub.2O. RNAs were vacuum transferred to a nylon membrane using
alkaline transfer buffer (3M NaCl, 8 mM NaOH, 2 mM sarkosyl). After
transfer, the blot was neutralized by incubation for 5 minutes in
200 mM phosphate buffer pH 6.8. The RNA was crosslinked to the
membrane using an ultraviolet crosslinker apparatus
(Stratagene).
[0238] A probe was made by excising the insert of a plasmid
containing the 5' RACE reaction PCR product described above. The
probe was radiolabeled using the random priming technique described
in Example 2.
[0239] The filters were prehybridized in QuikHyb rapid
hybridization solution (Stratagene) for 30 minutes at 65.degree. C.
The radiolabeled probe (1-2.times.10.sup.6 cpm/ml) and sonicated
salmon sperm DNA (final concentration 100 .mu.g/ml) were denatured
by heating to 95.degree. C. for 10 minutes and quick-chilled on ice
before adding to the filter in QuikHyb. Hybridization was for 3
hours at 65.degree. C. The unhybridized probe was removed by
washing the filters two times for 15 minutes with 2.times.SSC, 0.1%
sodium dodecyl sulfate at room temperature followed by two times
for 15 minutes in 0.1.times.SSC, 0.1% SDS at 62.degree. C.
Following the washes, the filters were allowed to dry briefly and
then exposed to Kodak XAR-2 film with intensifying screens at
-80.degree. C. The results are shown in FIG. 7, which shows a major
mRNA species of approximately 4.5 kilobases. Minor species of 4.3
and 1.6 kilobases are also present. The expected size of the LLGN
cDNA is 1.6 kb. The LLGXL sequence is likely to be encoded by the
major species of mRNA detected.
[0240] Expression of LIPG RNA in Various Human Tissues
[0241] A commercially prepared filter containing 3 .mu.g each of
mRNAs from human tissues (heart, brain, placenta, lung, liver,
skeletal muscle, kidney, and pancreas) was obtained from Clontech
(Catalog #7760-1). This filter was probed and processed as
described above. After probing with the radiolabeled LLG fragment
and autoradiography, the probe was stripped by washing in boiling
0.1.times.SSC, 0.1% SDS for 2.times.15 min. in a 65.degree. C.
incubator. The membranes were then probed with a 1.4 kilobase pair
DNA fragment encoding human lipoprotein lipase. This fragment was
obtained by RT-PCR of the THP-1 RNA (PMA and oxLDL treated) using
the 5' LPL and 3' LPL primers described in FIG. 1. and the RT-PCR
conditions described above. After autoradiography, the membranes
were stripped again and reprobed with a radiolabeled fragment of
the human beta actin cDNA to normalize for RNA content. The results
of these analyses are shown in FIG. 8. The highest levels of LIPG
message were detected in placental RNA, with lower levels found in
RNAs derived from lung, liver, and kidney tissue. In agreement with
previous studies by others (Verhoeven, A. J. M., Jansen, H. (1994)
Biochem. Biophys. Acta 1211, 121-124), lipoprotein lipase message
was found in many tissues, with highest levels found in heart and
skeletal muscle tissue. Results of this analysis indicates that the
tissue distribution of LIPG expression is very different from that
of LPL. The pattern of LIPG expression is also different from that
of either hepatic lipase or pancreatic lipase, as reported by
others (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).
[0242] To determine the expression pattern in additional human
tissues, another commercially prepared membrane was probed with
LLGXL cDNA. This dot blot (Human RNA Master Blot, Clontech Cat. #
7770-1) contains 100-500 ng mRNA from 50 different tissues and is
normalized for equivalent housekeeping gene expression (Chen, L.,
and Morin, R. (1971) Biochim. Biophys. Acta 231, 194-197). A 1.6 kb
DraI-SrfI fragment of the LLGXL cDNA was labeled with .sup.32PdCTP
using a random oligonucleotide priming system (Prime It II,
Stratagene) according to the manufacturer's instructions. After 30
minutes prehybridization at 65.degree. C., the probe was added to
QuikHyb hybridization solution at 1.3.times.10.sup.6 cpm/ml.
Hybridization was for 2 hours at 65.degree. C. The unhybridized
probe was removed by washing the filters two times for 15 minutes
with 2.times.SSC, 0.1% sodium dodecyl sulfate at room temperature
followed by two times for 15 minutes in 0.1.times.SSC, 0.1% SDS at
62.degree. C. Following the washes, the filters were allowed to dry
briefly and then exposed to Kodak XAR-2 film with intensifying
screens at -80.degree. C. for varying amounts of time. The
resulting images were quantitated by densitometry. The results are
shown in Table 2. The relative expression levels of tissues
represented in both the multiple tissue northern and the multiple
tissue dot blot are similar, with highest levels in placenta, and
lower levels in lung, liver and kidney. Fetal liver, kidney, and
lung also express roughly the same levels as the adult tissues.
Surprisingly, thyroid tissue expression levels were the highest of
all tissues represented, with expression of 122% of that in
placental tissue. While there is precedence for lipase expression
by the placenta (Rothwell, J. E., Elphick, M. C. (1982) J. Dev.
Physiol. 4, 153-159; Verhoeven, A. J. M., Carling D., and Jansen H.
(1994) J. Lipid Res. 35, 966-975; Burton, B. K., Mueller, H. W.
(1980) Biochim. Biophys. Acta 618, 449-460), the thyroid was not
previously known to express any lipase. These results suggest that
LIPG expression may be involved in maintenance of the placenta,
where LIPG may serve to liberate free fatty acids from substrates
such as phospholipids as a source of energy. The LIPG expressed in
the thyroid may provide precursors for the synthesis of bioactive
molecules by that gland. TABLE-US-00002 TABLE 2 Expression of LIPG
mRNA in various human tissues whole brain N.D. substantial N.D.
uterus N.D. mammary N.D. lung 29 nigra gland amygdala N.D. temporal
N.D. prostate 5 kidney 44 trachea 12 lobe caudate N.D. thalamus
N.D. stomach N.D. liver 61 placenta 100 nucleus cerebellum 4
sub-thalamic N.D. testes 9 small 6 fetal brain 5 nucleus intestine
cerebral N.D. spinal cord N.D. ovary N.D. spleen N.D. fetal heart
N.D. cortex frontal lobe N.D. heart N.D. pancreas N.D. thymus N.D.
fetal kidney 56 hippocampus N.D. aorta N.D. pituitary N.D.
peripheral N.D. fetal liver 14 gland leukocyte medulla N.D.
skeletal N.D. adrenal gland N.D. lymph node N.D. fetal spleen N.D.
oblongata muscle occipital lobe N.D. colon 8 thyroid gland 122 bone
marrow N.D. fetal thymus N.D. putamen N.D. bladder N.D. salivary
N.D. appendix 7 fetal lung 8 gland
Values given are percentage of expression with levels in placental
tissue arbitrarily set at 100%. Values are average of densitometric
measurements from two autoradiographic exposures. N.D.=not
detectable.
[0243] Expression of LIPG RNA in cultured endothelial cells
[0244] Human umbilical vein endothelial cells (HUVEC) and human
coronary arterial endothelial cells (HCAEC) were obtained from
Clonetics. HUVECs were propagated in a commercially prepared
endothelial cell growth medium (EGM, Clonetics) supplemented with 3
mg/ml bovine brain extract (Maciag, T., Cerundolo, J., Ilsley, S.,
Kelley, P. R., and Forand, R. (1979) Proc. Natl. Acad. Sci. USA 76,
5674-5678), Clonetics), while HCAECs were propagated in EGM
supplemented with 3 mg/ml, bovine brain extract and 3% fetal bovine
serum (5% final concentration). Cells were grown to confluence,
then the medium was changed to EGM without bovine brain extract.
Cultures were stimulated by adding 100 ng/ml of phorbol myristate
(Sigma). After 24 hours incubation, the RNAs were extracted from
the cells via the Trizol method described above. Twenty micrograms
of total RNA was electrophoresed and transferred to the membrane
for analysis. The membranes were probed with LIPG and LPL probes as
described above. The results are shown in FIG. 9. Twenty micrograms
of total RNA from THP-1 cells stimulated with PMA was run on the
blot for comparison. RNA hybridizing to the LIPG probe was detected
in unstimulated and PMA stimulated HUVEC cells. In contrast,
detectable levels of LIPG mRNA were only found in HCAEC cultures
after stimulation with PMA. In agreement with previous studies of
others, no detectable lipoprotein lipase mRNA was detected in any
of the endothelial RNAs (Verhoeven, A. J. M., Jansen, H. (1994)
Biochem. Biophys. Acta 1211, 121-124).
Example 4
LIPG Protein Analysis
[0245] Antibody Preparation
[0246] Antisera were generated to peptides with sequences
corresponding to a region of the predicted protein encoded by the
LIPG cDNA open reading frame. This peptide was chosen because of
its high predicted antigenicity index (Jameson B. A., and Wolf, H.
(1988) Comput. Applic. in the Biosciences 4, 181-186). The sequence
of the immunizing peptide was not found in any protein or
translated DNA sequence in the Genbank database. Its corresponding
position in the LIPG protein is shown in FIG. 10. The carboxy
terminal cysteine of the peptide does not correspond to the residue
in the LIPG putative protein, but was introduced to facilitate
coupling to the carrier protein. The peptide was synthesized on a
Applied Biosystems Model 433A peptide synthesizer. Two milligrams
of peptide was coupled to two milligrams of maleimide-activated
keyhole limpet hemocyanin following the protocols included in the
Imject Activated Immunogen Conjugation Kit (Pierce Chemical). After
desalting, one-half of the conjugate was emulsified with an equal
volume of Freund's complete adjuvant (Pierce). This emulsification
was injected into a New Zealand White rabbit. Four weeks after the
initial inoculation, a booster inoculation was made with an
emulsification made exactly as described above except. Freund's
incomplete adjuvant (Pierce) was used. Two weeks after the boost, a
test bleed was made and titers of specific antibodies were
determined via ELISA using immobilized peptide. A subsequent boost
was made one month after the first boost.
[0247] Western Analysis of Medium from Endothelial Cell
Cultures
[0248] HUVEC and HCEAC cells were cultured and stimulated with PMA
as described in Example 3C, except that the cells were stimulated
with PMA for 48 hours. Samples of conditioned medium (9 ml) were
incubated with 500 .mu.l of a 50% slurry of heparin-Sepharose CL-6B
in phosphate buffered saline (PBS, 150 mM sodium chloride, 100 mM
sodium phosphate, pH 7.2). Heparin-Sepharose was chosen to
partially purify and concentrate the LIPG proteins because of the
conservation of residues in the LLGXL sequence which have been
identified as critical for the heparin-binding activity of. LPL
(Ma, Y., Henderson, H. E., Liu, M.-S., Zhang, H., Forsythe, I. J.,
Clarke-Lewis, I., Hayden, M. R., and Brunzell, J. D. J. Lipid Res.
35, 2049-2059; and FIG. 6.). After rotation at 4.degree. C. for 1
hour, the samples were centrifuged for 5 minutes at 150.times.g.
The medium was aspirated and the Sepharose was washed with 14 ml
PBS. After centrifugation and aspiration, the pelleted
heparin-Sepharose was suspended in 200 .mu.l 2.times. SDS loading
buffer (4% SDS, 20% glycerol, 2% .beta.-mercaptoethanol, 0.002%
bromphenol blue, and 120 mM Tris pH 6.8). The samples were heated
to 95.degree. C. for 5 minutes and 40 .mu.l was loaded onto a 10%
Tris-Glycine SDS gel. After electrophoresis at 140 V for
approximately 90 minutes, the proteins were transferred to
nitrocellulose membranes via a Novex electroblotting apparatus (210
V, 1 hour). The membranes were blocked for 30 minutes in blocking
buffer (5% nonfat dried milk, 0.1% Tween 20, 150 mM sodium
chloride, 25 mM Tris pH 7.2). Antipeptide antisera and normal
rabbit serum was diluted 1:5000 in blocking buffer and was
incubated with the membranes overnight at 4.degree. C. with gentle
agitation. The membranes were then washed 4.times.15 minutes with
TBST (0.1% Tween 20, 150 mM sodium chloride, 25 mM Tris pH 7.2).
Goat anti-rabbit peroxidase conjugated antisera (Boehringer
Mannheim) was diluted 1:5000 in blocking buffer and incubated with
the membrane for 1 hour with agitation. The membranes were washed
as above, reacted with Renaissance chemiluminescent reagent (DuPont
NEN), and exposed to Kodak XAR-2 film. The results are shown in
FIG. 11. Two species of immunoreactive proteins are present in the
samples from unstimulated HUVEC and HCAEC cells. Levels of
immunoreactive protein in the unstimulated HCAEC samples are much
lower than the corresponding HUVEC sample. Upon stimulation with
PMA, three immunoreactive proteins are secreted by the endothelial
cell cultures. PMA exposure greatly increased the level of LIPG
proteins produced by the HCAEC cultures. PMA induction of LLG
proteins was not as dramatic in the HUVEC cultures.
Example 5
Recombinant LIPG Protein Production
[0249] LIPG Expression Constructs
[0250] The cDNAs encoding the LLGN and LLGXL proteins were cloned
into the mammalian expression vector pcDNA3 (Invitrogen). This
vector allows expression of foreign genes in many mammalian cells
through the use of the cytomegalovirus major late promoter. The
LLGN 5' RACE product was cloned into the EcoRI site of pcDNA3. The
LLGXL cDNA was digested with DraI and SrfI to yield a 1.55 kb cDNA
(see FIG. 4.). The vector was digested with the restriction enzyme
EcoRV and the vector and insert were ligated using T4 DNA ligase
and reagents from the Rapid Ligation Kit (Boehringer Mannheim)
according to the manufacturers instructions. The ligation products
were used to transform competent E. coli. Resultant colonies were
screened by restriction analysis and sequencing for the presence
and orientation of the insert in the expression vector.
[0251] Transient Transfection of LIPG in COS-7 Cells
[0252] The LIPG expression vectors were introduced into COS-7 cells
through the use of Lipofectamine cationic lipid reagent (GIBCO).
Twenty-four hours before the transfection, COS-7 cells were plated
onto 60 mm tissue culture dishes at a density of 2.times.10.sup.5
cells/plate. The cells were propagated in Dulbecco's modified
Eagle's medium (DMEM; GIBCO) supplemented with 10% fetal calf
serum, 100 U/ml penicillin, 100 .mu.g/ml streptomycin. One
microgram of plasmid DNA was added to 300 .mu.l of Optimem I
serum-free medium (Gibco). Ten microliters of Lipofectamine reagent
were diluted into 300 .mu.l of Optimem I medium and this was
combined with the DNA solution and allowed to sit at room
temperature for 30 minutes. The medium was removed from the plates
and the cells were rinsed with 2 ml of Optimem medium. The
DNA-Lipofectamine solution was added to the plates along with 2.7
ml Optimem medium and the plates were incubated for 5 hours at
37.degree. C. After the incubation, the serum free medium was
removed and replaced with DMEM supplemented with 2% FBS and
antibiotics. Twelve hours post-transfection, some of the cultures
were treated with either 0.25 mM Pefabloc SC (Boehringer Mannheim),
a protease inhibitor, or 10 U/ml heparin. Thirty minutes before
harvest, the heparin treated samples were treated with an
additional 40 U/ml heparin. The medium was removed from the cells
60 hours after transfection. Heparin-Sepharose CL-4B (200 .mu.l of
a 50% slurry in PBS pH 7.2) was added to 1 ml of medium and was
mixed at 4.degree. C. for 1 hour. The Sepharose was pelleted by low
speed centrifugation and was washed three times with 1 ml cold PBS.
The Sepharose was pelleted and suspended in 100 .mu.l 2.times.
loading buffer. The samples were heated to 95.degree. C. for 5
minutes. 40 .mu.l of each sample was loaded onto a 10% SDS-PAGE
gel. Electrophoresis and western analysis was performed using the
anti-LIPG antiserum as described above. The results are shown in
FIG. 12. Proteins from HCAEC conditioned medium were included for
size references. LLGN migrates at approximately 40 kD,
corresponding to the lowest band in HCAEC. The medium from COS
cells transfected with LLGXL cDNA contains both 68 kD and 40 kD
species. When these cells were treated with heparin, the amount of
both 68 kD and 40 kD proteins recovered from the medium increased
dramatically, indicating either the release of proteoglycan-bound
protein from the cell surface or stabilization of the proteins by
heparin. When the cells were treated with the protease inhibitor
Pefabloc, the amount of 68 kD protein increased relative to that of
the 40 kD species. This suggests that the lower molecular weight
protein produced by these cells is a proteolysis product of the
larger 68 kD form. The role of the mRNA identified through
differential display which encodes a shorter, 40 kD species is not
known. There has, however, been a report of an alternately-spliced
form of hepatic lipase which apparently is expressed in a
tissue-specific manner and would create a truncated protein.
Example 6
LIPG in Animal Species
[0253] Cloning the Rabbit Homolog of LIPG
[0254] A commercially available lambda cDNA library derived from
rabbit lung tissue (Clontech, Cat. # TL1010b) was used to isolate a
fragment of the rabbit homolog of the LIPG gene. Five microliters
of the stock library were added to 45 .mu.l water and heated to
95.degree. C. for 10 minutes. The following were added in a final
volume of 100 .mu.l: 200 .mu.M dNTPs, 20 mM Tris-HCl pH 8.4, 50 mM
KCl, 1.5 mM MgCl.sub.2, 100 .mu.M each primer DLIP774 and LLGgen2a,
and 2.5 U Taq polymerase (GIBCO). The reaction was thermocycled 35
times with the parameters of: 15 seconds at 94.degree. C., 20
seconds at 50.degree. C. and 30 seconds at 72.degree. C. Ten
microliters of the reaction was analyzed via agarose gel
electrophoresis. A product of approximately 300 basepairs was
detected. A portion (4 .mu.l) of the reaction mix was used to clone
the product via the TA cloning system. The insert of a resulting
clone was sequenced (SEQ ID NO: 11). An alignment between the
deduced rabbit amino acid sequence (SEQ ID NO: 12) and the
corresponding sequence of the human cDNA is also shown in FIG. 14.
Of the nucleotides not part of either amplification primer, there
is an 85.8% identity between the rabbit and human LLG sequences.
The predicted protein encoded by this rabbit cDNA shares 94.6%
identity with that of the human protein, with most of the
nucleotide substitutions in the third or "wobble" positions of the
codons. Notably, this region spans the "lid" sequence of the
predicted LLG proteins and is a variable domain in the lipase gene
family. This is evidence that there is a high degree of
conservation of this gene between species.
[0255] LIPG in Other Species
[0256] To demonstrate the presence of LLG genes in other species,
genomic DNAs from various species were restriction digested with
EcoRI, separated by electrophoresis in agarose gels, and blotted
onto nitrocellulose membranes.
[0257] The membranes were hybridized overnight at 65.degree. C.
with 2.5.times.10.sup.6 cpm/ml of random primed .sup.32P-LLG or
.sup.32P-LPL (lipoprotein lipase) probe in a hybridization solution
of 6.times.SSC, 10% dextran sulfate, 5.times. Dendardt's solution,
1% SDS, and 5 .mu.g/ml salmon sperm DNA. The membranes were washed
with 0.1.times.SSC, 0.5% SDS for ten minutes at room temperature,
then sequentially for ten minutes at 40.degree. C., 50.degree. C.,
and 55.degree. C. Autoradiograms of the blots are shown in FIG.
16.
[0258] FIG. 16 shows the presence of LLG and LPL genes in all
species examined, with the exception that no hybridization was
observed with the LLG probe against rat DNA. The exceptional data
from rat may represent an artifact caused by generation of
abnormally sized restriction fragments containing LLG sequences.
Such fragments may be outside of the fractionation range of the
agarose gel or may blot inefficiently. The different bands detected
by the two probes indicate that LPL and LIPG are separate,
evolutionarily conserved genes.
Example 7
Enzymatic Activity of LLGXL
[0259] Phospholipase Activity
[0260] Conditioned media from COS-7 cells transiently expressing
human lipoprotein lipase (LPL), LLGN, or LLGXL were assayed for
phospholipase activity. MEM containing 10% FBS (MEM) was used as
the blank, and conditioned media from COS-7 cells transfected with
an antisense LLGXL plasmid (AS) was used as a negative control.
[0261] A phosphatidylcholine (PC) emulsion was made up using 10
.mu.l phosphatidylcholine (10 mM), 40 .mu.l
.sup.14C-phosphatidylcholine, dipalmitoyl (2 .mu.Ci), labeled at
the sn 1 and 2 positions, and 100 .mu.l Tris-TCNB [100 mM Tris, 1%
Triton, 5 mM CaCl.sub.2, 200 mM NaCl, 0.1% BSA). The emulsion was
evaporated for 10 minutes, then brought to a final volume of 1 ml
in Tris-TCNB.
[0262] Reactions were performed in duplicate and contained 50 .mu.l
PC emulsion and 950 .mu.l medium. Samples were incubated in a
shaking water bath for 2-4 hours at 37.degree. C. The reactions
were terminated by adding 1 ml 1N HCl, then extracted 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 liberated .sup.14C-free fatty
acids contained in the flow-thru fraction were quantitated in a
scintillation counter. The results of these assays are shown in
FIG. 14.
[0263] Triacylglycerol Lipase Activity
[0264] Conditioned media from COS-7 cells transiently expressing
human lipoprotein lipase (LPL), LLGN, or LLGXL were assayed for
triglycerol lipase activity. MEM containing 10% FBS was used as the
blank, and conditioned media from COS-7 cells transfected with an
antisense LLGXL plasmid (AS), was used as a negative control.
[0265] A concentrated substrate was prepared as an anhydrous
emulsion of labeled triolein, [9,10-.sup.3H(N)] and unlabeled
triolein (final total triolein=150 mg with 6.25.times.10.sup.8
cpm), which was stabilized by adding 9 mg of lecithin in 100%
glycerol. 0.56 ml of .sup.3H-triolein, (0.28 mCi) was mixed with
0.17 ml of unlabeled triolein and 90 .mu.l of lecithin (9 mg). The
mixture was evaporated under a stream of nitrogen. The dried lipid
mixture was emulsified in 2.5 ml 100% glycerol by sonication (30
second pulse level 2 followed by 2 second chill cycles over 5
minutes].
[0266] The assay substrate was prepared by dilution of 1 volume of
concentrated substrate with 4 volumes of 0.2M Tris-HCl buffer (pH
8.0) containing 3% w/v fatty acid free bovine serum albumin. The
diluted substrate was vortexed vigorously for 5 seconds.
[0267] Reactions were performed in duplicate in a total volume of
0.2 ml containing 0.1 ml of assay substrate and 0.1 ml of the
indicated conditioned media. The reactions were incubated for 90
minutes at 37.degree. C. The reactions were terminated by adding
3.25 ml of methanol-chloroform-heptane 1.41:1.25:1 (v/v/v) followed
by 1.05 ml of 0.1M potassium carbonate-borate buffer (pH 10.5).
After vigorous mixing for 15 seconds, the samples were centrifuged
for 5 minutes at 1000 rpm. A 1.0 ml aliquot of the upper aqueous
phase was counted in a scintillation counter. The results of these
assays are shown in FIG. 15.
Example 8
Use of LIPG Polypeptide to Screen for Enhancers or Inhibitors
[0268] Recombinant LIPG is produced in baculovirus-infected insect
cells or stably transfected CHO cells or other acceptable mammalian
host cells. Recombinant LIPG is purified from the serum-containing
or serum-free conditioned medium by chromatography on
heparin-Sepharose, followed by chromatography on a cation exchange
resin. A third chromatographic or further chromatographic steps,
such as molecular sieving, is used in the purification of LIPG if
needed. During purification, anti-peptide antibodies are used to
monitor LIPG protein and the phospholipase assay is used to follow
LIPG activity.
[0269] In the fluorescent assay, the final assay conditions are
approximately 10 mM Tris-HCl (pH 7.4), 100 mM KCl, 2 mM CaCl.sub.2,
5 .mu.M
C.sub.6NBD-PC{1-acyl-2-[6-(nitro-2,1,3-benzoxadiazol-4-yl)amino]cap-
roylphosphatidylcholine, and LIPG protein (approx. 1-100 ng). The
reaction is subjected to fluorescence excitation at 470 nm, and
enzyme activity, as measured by the fluorescence emission at 540 nm
is continuously monitored. Compounds and/or substances to be tested
for stimulation and/or inhibition of LIPG activity are added as
10-200 mM solutions in dimethylsulfoxide. Compounds which stimulate
or inhibit LIPG activity are identified as causing an increased or
decreased fluorescence emission at 540 nm.
[0270] In the thio assay, the final assay conditions are
approximately 25 mM Tris-HCl (pH 8.5), 100 mM KCl, 10 mM
CaCl.sub.2, 4.24 mM Triton X-100, 0.5 mM
1,2-bis(hexanoylthio)-1,2-dideoxy-sn-glycero-3-phosphorylcholine, 5
mM 4,4'-dithiobispyridine (from a 50 mM stock solution in ethanol),
and 1-100 ng recombinant LIPG. Phospholipase activity is determined
by measuring the increase in absorption at 342 nm. Compounds and/or
substances to be tested for stimulation and/or inhibition of LIPG
activity are added as 10-200 mM solutions in dimethylsulfoxide.
Compounds which stimulate or inhibit LIPG activity are identified
as causing an increased or decreased absorption at 342 nm.
Example 9
Transgenic Mice Expressing Human LIPG
[0271] To further study the physiological role of LIPG, transgenic
mice expressing human LIPG are generated.
[0272] The 1.53 kb DraI/SrfI restriction fragment encoding LLGXL
(see FIG. 4) was cloned into a plasmid vector (pHMG) downstream of
the promoter for the ubiquitously expressed
3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase gene.
Transgenic mice expressing different levels of human LLGXL are
generated using standard methods (see, e.g., G. L. Tromp et al.
Gene 1565:199-205, 1995). The transgenic mice are used to determine
the impact of LLGXL overexpression on lipid profile, vascular
pathology, rate of development and severity of atherosclerosis, and
other physiological parameters.
Example 10
Expression of LIPG in Atherosclerotic Tissues
[0273] LLGXL expression in atherosclerosis was examined by
performing a reverse transcription-polymerase chain reaction
(RT-PCR) using mRNA isolated from vascular biopsies from four
patients with atherosclerosis. The tissue samples were from the
aortic wall (one sample), the iliac artery (two samples), and the
carotid artery (one sample).
[0274] Atherosclerosis biopsies were received from Gloucestershire
Royal Hospital, England, and polyA+ mRNA was prepared and
resuspended in diethylpyrocarbonate (DEPC) treated water at a
concentration of 0.5 .mu.g/.mu.l mRNA. Reverse transcriptase
reactions were performed according to the GibcoBRL protocol for
Superscript Preamplification System for First Strand cDNA
Synthesis. Briefly, the cDNA was synthesized as follows: 2 .mu.l of
each mRNA was added to 1 .mu.l oligo (dT).sub.12-18 primer and 9
.mu.l of DEPC water. The tubes were incubated at 70.degree. C. for
10 minutes and put on ice for 1 minute. To each tube, the following
components were added: 2 .mu.l 10.times. PCR buffer, 2 .mu.l 25 MM
MgCl.sub.2, 1 .mu.l 10 mM dNTP mix and 2 .mu.l 0.1M DTT. After 5
minutes at 42.degree. C., 1 .mu.l (200 units) of Super Script II
reverse transcriptase was added. The reactions were mixed gently,
then incubated at 42.degree. C. for 50 minutes. The reactions were
terminated by incubation at 70.degree. C. for 15 minutes then put
on ice. The remaining mRNA was destroyed by the addition of 1 .mu.l
of RNase H to each tube and incubated for 20 minutes at 37.degree.
C.
[0275] PCR amplifications were performed using 2 .mu.l of the cDNA
reactions. To each tube the following were added: 5 .mu.l 10.times.
PCR buffer, 5 .mu.l 2 mM dNTPs, 1 .mu.l hllg-gsp1 primer (20
pmol/ml, see FIG. 1), 1 .mu.l hllg-gsp2a primer (20 pmol/ml, see
FIG. 1), 1.5 .mu.l 50 Mm MgCl.sub.2, 0.5 .mu.l Taq polymerase (5
U/ml) and 34 .mu.l water. After holding the reactions at 95.degree.
C. for 2 minutes, thirty cycles of PCR were performed as follows:
15 seconds at 94.degree. C., 20 seconds at 52.degree. C., and 30
seconds at 72.degree. C. The finished reactions were held for 10
minutes at 72.degree. C. before analysis by agarose gel
electrophoresis. The hllg-gsp primers are specific for LIPG and
yield an expected product of 300 bp. In a parallel PCR to show that
the cDNA synthesis reactions had been successful, primers specific
for the housekeeping gene, G3PDH (human glyceraldehyde 3-phosphate
dehydrogenase) were used (1 .mu.l each at 20 pmol/ml).
[0276] The G3PDH primers (see FIG. 1) yielded the expected product
of 983 bp in all four vascular biopsy samples. LIPG expression was
detected in three of the four samples, with no expression being
detected in the carotid artery sample.
Example 11
Differential Display, RT-PCR and cDNA Library Screening
[0277] To perform the experiments discussed in Examples 12 to 16,
the following procedure (based on the procedure outlined in Example
1) was used to obtain the cDNA for LIPG. THP-1 cells were plated in
the presence of phorbol 12-myristate 13-acetate (PMA, 40 ng/ml;
Sigma) for 48 hours. The differentiated THP-1 cells were exposed
for 24 hours to either oxLDL (50 .mu.g/ml) or control medium. Total
RNAs were collected and purified using standard procedures.
Poly(A).sup.+ RNA was purified from total RNA using a poly-dT
magnetic bead system (Promega). cDNA synthesis and PCR
amplification were accomplished using protocols from the
Differential Display kit, version 1.0 (Display Systems
Biotechnology). The primer pairs that yielded the initial cDNA
fragment of EL were downstream primer 7 (5'-TTTTTTTTTTTGA-3') and
upstream primer 15 (5'-GATCCAATCGC-3'). The amplification reaction
was fractionated on a 6% nondenaturing acrylamide sequencing format
gel and an amplification product found only in the reaction
containing cDNA from THP-1 cells exposed to oxLDL was identified
and excised from the gel. A reamplification using the same primers
was performed and the product was excised and subcloned into the
pCRII vector using the TA cloning system (Invitrogen). Insert sizes
were determined using EcoRI digestions of the plasmids, and clones
containing inserts of the approximate size of the original PCR
product were sequenced using fluorescent dye-terminator reagents
(Prism, Applied Biosystems) and an Applied Biosystems 373 DNA
sequencer. We extended the cDNA sequence of the original,
gel-excised cDNA using the 5'-RACE system (GIBCO). RNA (1 .mu.g)
from the THP-1 cells used initially in the differential display
reactions was used in the 5'-RACE procedure using a gene-specific
primer (5'-TAGGACATGCACAGTGTAATCTG-3') for first strand cDNA
synthesis. We performed PCR amplification of the cDNA using an
anchor primer and gene-specific primer 2
(5'-GATTGTGCTGGCCACTTCTC-3'). This reaction (1 .mu.l) was used in a
nested re-amplification using the universal amplification primer
(5'-CUACUACUACUAGGCCACGCGTCGACTAGTAC-3') and the gene-specific
primer 3 (5'-GACACTCCAGGGACTGAAG-3') to increase levels of specific
product for subsequent isolation. The reaction products were cloned
into the pCRII vector from the TA cloning kit and determined the
sequence. A human placental cDNA library (oligo dT and random
primed) was obtained from Clontech and probed with the 5'-RACE
reaction PCR product. The DNA from hybridizing clones was purified
using LambdaSorb reagent (Promega). Inserts were excised from the
phage DNA by digestion with EcoRI, subcloned into the EcoRI site of
the Bluescript II SK plasmid vector (Stratagene) and sequenced.
Example 12
Antibody Preparation
[0278] A 17-residue peptide (GPEGRLEDKLHKPKATC) was synthesized
corresponding to residues 8-23 of the secreted LIPG gene product on
a Model 433A peptide synthesizer (Applied Biosystems). Peptide (2
mg) was coupled to maleimide-activated keyhole limpet haemocyanin
(2 mg) following the protocols included in the Imject Activated
Immunogen Conjugation kit (Pierce Chemical). After desalting,
one-half of the conjugate was emulsified with an equal volume of
Freund's complete adjuvant (Pierce) and injected into a New Zealand
White rabbit. Four weeks after the initial inoculation, a booster
inoculation was administered with an emulsification made exactly as
described above except for the use of Freund's incomplete adjuvant
(Pierce). Two weeks after the boost, the titres of specific
antibodies were determined in a test bleed via ELISA using
immobilized peptide.
Example 13
Gene Expression Studies
[0279] HUVECs were propagated in a commercially prepared
endothelial cell growth medium (EGM, Clonetics) supplemented with
bovine brain extract (3 mg/ml; Clonetics), whereas HCAECs were
propagated in EGM with bovine grain extract (3 mg/ml) and 5% fetal
bovine serum. Cultures were stimulated by addition of PMA (100
ng/ml). After 24 hours incubation, RNA was extracted from the cells
via the Trizol method, electrophoresed on a 1% agarose-formaldehyde
gel, transferred to Nytran membrane on a Turboblotter apparatus
(Schleicher and Schuell) and crosslinked to the membrane using a
Stratalinker ultraviolet crosslinker (Stratagene). The 5'-RACE
reaction PCR product was radiolabelled using the random priming
technique. The radiolabelled probe (1-2.times.10.sup.6 cpm/ml) was
denatured by heating to 95.degree. C. for 10 minutes and
quick-chilled on ice before adding to the filter in QuikHyb.
Hybridization was allowed to proceed for 3 hours at 65.degree. C.
Filters were exposed to Kodak XAR-2 film with intensifying screens
at -80 .degree. C. We incubated HUVEC- and HCEAC-conditioned medium
with heparin-Sepharose CL-6B at 4.degree. C. for 1 hour. After
centrifugation, the pelleted heparin-Sepharose was suspended in SDS
loading buffer, heated to 95.degree. C. for 5 minutes and loaded
onto a 10% Tris-Glycine SDS gel (NOVEX). After electrophoresis at
140 V for 90 minutes, the proteins were transferred to
nitrocellulose membranes and detected with rabbit anti-LIPG peptide
antisera (1:5,000), with goat anti-rabbit peroxidase conjugated
antisera (1:5,000; Boehringer) as the secondary antibody. The
membranes were reacted with Renaissance chemiluminescent reagent
(DuPont NEN) and exposed to Kodak XAR-2 film. A commercially
prepared filter containing poly(A).sup.+ RNAs (3 .mu.g each) from
human heart, brain, placenta, lung, liver, skeletal muscle, kidney
and pancreas (Clontech) was hybridized with a radiolabelled
fragment and processed as described above. Following
autoradiography, the blot was stripped by washing in boiling
0.1.times.SSC, 0.1% SDS for 2.times.15 minutes at 65.degree. C. and
then probed as described above with a 1.4-kb cDNA fragment encoding
human LPL. This fragment was obtained by RT-PCR of the THP-1 RNA
(PMA and oxLDL treated) using the 5' LPL and 3' LPL primers
5'-ACCACCATGGAGAGCAAAGCCCTG-3' and 5'-CCAGTTTCAGCCTGACTTCTTATTC-3',
respectively. After exposure to film, the membranes were stripped
again and reprobed with a radiolabelled fragment of human .beta.
actin cDNA to normalize to RNA content.
[0280] Human umbilical vein endothelial cells (HUVEC) were negative
for LPL mRNA expression as expected, but were found to
constitutively express a high level of mRNA for the LIPG gene (FIG.
9).
[0281] Human coronary artery endothelial cells (HCAEC) were also
found to express the mRNA which was further upregulated on
treatment of these cells with phorbol ester (FIG. 9).
[0282] Conditional medium from stimulated HUVEC and HCAEC contained
immunoreactive proteins of approximately 68 kD and 40 kD, as well
as a less prominent band of 55 kD (FIG. 11).
[0283] To determine the tissue sites of LIPG production in vivo, a
multiple human tissue northern blot analysis with probes for both
LIPG and LPL was performed. Abundant levels of LIPG mRNA were found
in lung, liver and kidney (FIG. 8) tissues, which showed low levels
of LPL expression. LIPG was also expressed at high levels in the
placenta (FIG. 8), suggesting the potential for a role in
development.
[0284] In tissues such as heart and skeletal muscle, which
expressed the highest amount of LPL (confirming previous reports,
Goldberg, J. I., J. Lipid Res., 37, 693-707 (1996)), we did not
detect LIPG expression. This analysis indicated that the tissue
distribution of LIPG expression is very different from that of LPL,
as well as that reported for HL and PL. We found no LIPG mRNA in
adrenals or ovaries, but did find a very low level of LIPG mRNA in
the testes (data not shown). We also found that HepG2 cells express
LIPG mRNA and protein in vitro (data not shown), but at levels less
than 10% of that expressed by HUVECS.
Example 14
Lipase Assays
[0285] The cDNA and the 1.4-kb LPL cDNA were cloned into the EcoRV
site of the mammalian expression vector pcDNA3 (Invitrogen). An
antisense pcDNA3 vector was used as negative control. The
recombinant expression vectors (3 .mu.g) were mixed with
lipofectamine (Life Technologies) and transfected in quadruplicate
into semiconfluent COS7 cells in 60-mm dishes. Established methods
were used to assay samples of conditioned media from transfected
COS7 cells for TG lipase and phospholipase activities (Goldberg, J.
I., J. Lipid Res., 37, 693-707 (1996)). for the TG lipase assay,
9,10-.sup.3H(N)-triolein (250 .mu.Ci; NEN) was mixed with unlabeled
triolein (150 mg) and type IV-S-.alpha. lecithin (9 mg; Sigma) in
glycerol. The mixture was evaporated under nitrogen and emulsified
in glycerol (2.5 ml) by sonication with a Branson Sonifier 450. The
assay substrate was prepared by combining one volume of the
emulsified substrate, four volumes of Tris-HCl (0.2 M, pH 8.0)
containing 3% (w/v) fatty acid-free bovine serum albumin (BSA) and
one volume of heat-inactivated bovine serum. Reactions were
performed in triplicate in a total volume (0.2 ml) containing assay
substrate (0.1 ml) and conditioned media (0.1 ml). The reactions
were incubated for 2 hours at 37.degree. C. and terminated by
adding methanol-chloroform-heptane (1.41:1.25:1; 3.25 ml) followed
by potassium carbonate-borate buffer (1.05 ml; 0.1 M, pH 10.5).
After vigorous mixing for 15 seconds, the samples were centrifuged
for 5 minutes at 1,000 rpm and the upper aqueous phase (1.0 ml) was
counted in a scintillation counter. For the phospholipase assay, a
phosphatidylcholine (PC) emulsion was made by combining
.sup.14C-dipalmitoyl PC (2 .mu.Ci; NEN) and lecithin (10 .mu.l)with
Tris-TCNB (100 .mu.l; 100 mM Tris-HCl pH 7.4, 1% Triton X-100, 5 mM
CaCl.sub.2, 200 mM NaCl, 0.1% BSA). The mixture was vortexed for 2
minutes and then evaporated under nitrogen. The dried lipid was
reconstituted with TCNB (1 ml) and vortexed for 10 seconds.
Reactions were performed in triplicate and contained PC emulsion
(50 .mu.l ), conditioned media (600 .mu.l) and MEM (350 .mu.l).
Samples were incubated at 37.degree. C. for 2 hours, terminated by
addition of HCl (1 ml) and extracted with 2-propanol:hexane (1:1; 4
ml). A sample (1.8 ml) of the upper hexane layer was passed through
a silica gel column, and the liberated .sup.14C-free fatty acids
contained in the flow-through fraction were quantitated in a
scintillation counter. For both assays, MEM containing 10% FBS was
used as a blank and conditioned media from COS7 cells transfected
with an antisense plasmid (AS) was used as a negative control.
Example 15
Recombinant Adenovirus Construction and Animal Studies
[0286] A recombinant adenovirus encoding human LIPG was constructed
as described (Tsukamoto et al., J. Clin. Invest., 100, 107-114
(1997); Tsukamoto et al., J. Lipid Res., 38, 1869-1876 (1997)). In
brief, the full-length human cDNA was subcloned into the shuttle
plasmid vector pAdCMVLink1. After screening for the appropriate
orientation by restriction analysis, the plasmid was linearized
with NheI and cotransfected into 293 cells along with adenoviral
DNA digested with ClaI. Cells were overlaid with agar and incubated
at 37.degree. C. for 15 days. Six plaques were picked and screened
by PCR; two plaques positive for cDNA were subjected to a second
round of plaque purification. After confirmation of the presence of
cDNA, the recombinant adenovirus was expanded in 293 cells at
37.degree. C. Cell lysates were used to infect HeLa cells for
confirmation of the expression of human LIPG by western blot of
conditioned media. The recombinant adenovirus (AdhEL) was further
expanded in 293 cells and purified by cesium chloride
ultracentrifugation. Control adenovirus containing no cDNA insert
(Adnull) was also subjected to plaque purification and purified as
described above. The purified viruses were stored in 10%
glycerol/PBX at -80.degree. C. Wild-type C57BL/6, human apoA-I
transgenic and LDL receptor mutant mice were obtained from Jackson
Laboratory. All mice were fed chow diets. Wild-type and human
apoA-I transgenic mice were injected intravenously via the tail
vein with AdhEL or Adnull 1.times.10.sup.11 particles
(approximately 2.times.10.sup.9 pfu) and LDLR-deficient mice were
injected with 1.times.10.sup.10 particles. In all experiments,
blood was obtained from the retro-orbital plexus 1 day before
injection and at multiple time points following injection.
[0287] Intravenous injection of AdhEL into wild-type C57BL/6 mice
resulted in expression in the liver (FIG. 17) and reduction of
plasma levels of HDL cholesterol that remained significantly lower
than control virus-injected mice through at least 41 days
post-injection (FIG. 18). Lipoproteins were separated by FPLC gel
filtration, demonstrating that HDL was undetectable 14 days after
adenovirus injection (FIG. 19). Injection of recombinant LIPG
adenovirus into human, apoA-I transgenic mice (which have much
higher levels of HDL cholesterol and apoA-I) reduced both HDL
cholesterol (FIG. 20) and apoA-I (FIG. 21) levels. To determine the
relative effects of LIPG expression on HDL compared with the
apoB-containing lipoproteins VLDL and LDL, we injected a lower dose
of the LIPG adenovirus into chow-fed LDL receptor-deficient mice,
which have approximately 70% of cholesterol in VLDL/LDL and
approximately 30% in HDL. As before, expression of LIPG reduced HDL
cholesterol levels (FIG. 23). Although LIPG expression reduced
VLDL/LDL cholesterol levels in the same mice (FIG. 24), the effect
was proportionately less. Overexpression of LIPG reduced VLDL/LDL
cholesterol, therefore a role of LIPG in the modulation of
apoB-containing lipoproteins cannot be excluded.
Example 16
Lipid/Lipoprotein Analyses
[0288] The plasma total cholesterol and HDL cholesterol levels were
measured enzymatically on a Cobas Fara (Roche Diagnostic Systems)
using Sigma reagents. ApoA-I was quantitated using a turbidometric
assay (Sigma) on a Cobas Fara. Pooled plasma samples were subjected
to fast protein liquid chromatography (FPLC) gel filtration
(Pharmacia LKB Biotechnology) using two Superose 6 columns in
series as described (Tsukamoto et al., J. Clin. Invest., supra).
Fractions (0.5 ml) were collected, and cholesterol concentrations
were determined using an enzymatic assay (Wako Pure Chemical
Industries).
Example 17
Identification of Inhibitors of LIPG
[0289] Modulators of EL activity may be found using the following
method:
[0290] Recombinant LIPG would be purified from the conditioned
medium of stably transfected Chinese hamster ovary cells, from
baculovirus infected insect cells, yeast (Pichia pastoris,
Kluveromyces Lactis) or other sources. Non-recombinant sources of
LIPG (such as human plasma, endothelial cell conditioned media,
etc.) could also be employed. An example of a primary screen to
look for modulators of LIPG activity would utilize the soluble
fluorescent substrate 4-methylumberiferyl hepatanoate. This assay
is continuous and homogeneous. Hydrolysis of this substrate by LIPG
results in the production of highly fluorescent
4-methylumbelliferone that can be measured in a microplate
fluorimeter. Other primary screening assay formats that could be
used are a scintillation proximity assay (Amersham) that measures
phospholipase activity, the lower-throughput radiometric
phospholipase assay described in Example 7 (and proposed below as a
secondary assay), or the alternative phospholipase assays described
in Example 8.
[0291] The catalytic center of LIPG, like other TG lipases,
consists of the same catalytic triad (ser, his, asp) found in
serine proteases. Indeed, other TG lipases, such as lipoprotein
lipase, are inhibited by serine protease inhibitors such as PMSF
and DFP. Either one of these compounds may serve as a positive
control for inhibitors of LIPG activity.
[0292] Secondary assay: Compounds active in the esterase assay or
alternative screening assays described above will be assayed in a
standard, radiometric phospholipase A assay. This assay measures
the release of radiolabelled palmitic acid from mixed micelles
containing [14C]-dipalmitoyl-phosphatidylcholine. Other assay
formats could be envisioned which utilize fluorescent substrates
and which would be amenable to a greater throughput.
[0293] Selectivity assays: Compounds would be assayed for
inhibition of the related enzymes lipoprotein lipase (LPL) and
pancreatic lipase (PL). Human PL and bovine LPL are commercially
available and assays could be readily implemented. The
phospholipase activity of PL is measured in exactly the same way as
described above for the secondary assay of LIPG. Since LPL is
primarily a TG lipase, the secondary assay would measure
radiolabelled fatty acid (oleic acid) release from a radiolabelled
TG (triolein) substrate (described in Example 7). This assay has a
similar capacity and may be adapted to other assay formats which
utilize fluorescent substrates and which would be amenable to a
greater throughput.
[0294] Phospholipase activity of LIPG would be tested on its in
vivo substrate, HDL, in an in vitro assay. Radiolabelled HDL could
be generated by exchange with a radiolabelled phospholipid, and
then used to measure LIPG phosphospholipase activity and the
activity of compounds emerging from the screens.
[0295] An additional assay could measure the impact of
preincubation of LIPG, HDL, .+-.compounds on radiolabelled
cholesterol efflux from cultured cells such as the rat Fu5AH
hepatoma line.
[0296] In vivo assays for assessment of compounds can be run in
wild-type, LIPG-overexpressing, and as control, LIPG null mice. If,
as in the case of adenoEL expression, the transgenic mice exhibit
decreased HDL relative to control mice, then treatment of
transgenic mice with LIPG inhibitory compounds would be expected to
raise HDL to the levels of control mice. It is also possible that
compounds could be tested for their LIPG inhibitory activity
(elevation of HDL) in other animals such as the LDLR-/- mouse,
apoA1 transgenic mice hamsters, or rabbits. Compounds which
elevated LIPG or LIPG activity would be expected to raise HDL in
these or other animal models.
Example 18
Inhibitory Small Molecule Treatment Method
[0297] A small molecule (hereafter an "inhibitory small molecule")
identified in the screening outlined in Example 17 as able to
inhibit the LIPG polypeptide in vitro is tested for its ability to
inhibit the LIPG polypeptide in vivo. Wild-type and LIPG transgenic
mice will be studied by administering the small molecule orally (if
orally bioavailable) or by intravenous injection. Activity of the
LIPG polypeptide will be measured in plasma before and after
heparin injection (to release the enzyme from bound sites). In
addition, cholesterol, VLDL, LDL and HDL cholesterol and apoA-I
levels will be monitored in animals receiving the inhibitory small
molecule. Finally, LDL receptor deficient mice will be fed an
atherogenic diet and administered the inhibitory small molecule or
placebo for a period of 8 weeks. Atherosclerosis will be
quantitated in the aortas of the mice in order to determine whether
administration of the inhibitory small molecule recudes the
progression or induces regression of atherosclerosis. Based on
these preclinical data, additional animal models such as hamsters,
rabbits, or pigs will be studied for the ability of the inhibitory
small molecule to raise HDL cholesterol levels, reduce VLDL and LDL
cholesterol levels, and/or inhibit the progression of
atherosclerosis.
[0298] Those inhibitory small molecules found to have the desired
properties will be administered to patients in combination with
pharmaceutically acceptable carriers. The inhibitory small
molecules may be administered in a variety of ways, including oral
administration and intravenous injection. The patients' HDL, VLDL
and LDL cholesterol levels will be monitored to determine efficacy
of the inhibitory small-molecule and to optimize dosage and
administration protocols.
Example 19
Inhibitory Peptide Treatment Method
[0299] Therapeutic peptides are identified by testing fragments of
the LIPG polypeptide to determine which of these fragments inhibit
LIPG polypeptide activity in vitro. Once identified, an "inhibitory
peptide" is then tested for its ability to inhibit the LIPG
polypeptide in vivo. Inhibitory peptides will be produced
recombinantly in E.coli and purified by methods known in the art.
The effect of the inhibitory peptides will be studied in wild-type
and LIPG transgenic mice by administering the inhibitory peptide by
intravenous injection. Activity of the LIPG polypeptide will be
measured in plasma before and after heparin injection (to release
the enzyme from bound sites). In addition, cholesterol, VLDL, LDL
and HDL cholesterol and apoA-I levels will be monitored in animals
receiving the inhibitory peptide. Finally, LDL receptor deficient
mice will be fed an atherogenic diet and administered the
inhibitory peptide or placebo for a period of 8 weeks.
Atherosclerosis will be quantitated in the aortas of the mice in
order to determine whether administration of the inhibitory peptide
reduces the progression or induces regression of atherosclerosis.
Based on these preclinical data, additional animal models such as
hamsters, rabbits or pigs will be studied for the ability of the
inhibitory small molecule to raise HDL cholesterol levels, reduce
VLDL and LDL cholesterol levels, and/or inhibit the progression of
atherosclerosis.
[0300] Those inhibitory peptides found to have the desired
properties will be administered to patients in combination with
pharmaceutically acceptable carriers. The inhibitory peptides may
be administered in a variety of ways, including oral administration
and intravenous injection. The patients' HDL, VLDL and LDL
cholesterol levels will be monitored to determine efficacy of the
inhibitory peptides and to optimize dosage and administration
protocols.
Example 20
Antisense Treatment Method
[0301] A series of antisense oligonucleotides, each complementary
to about 20 bases of the LIPG cDNA sequence are chemically
synthesized by standard techniques. To determine the most efficient
oligonucleotide to use therapeutically, each oligonucleotide is
individually transfected into cells expressing the LIPG gene, using
standard transfection protocols.
[0302] At about 24-48 hours following transfection of the
oligonucleotides, the LIPG mRNA level in cells is determined by
quantitative PCR, northern blots RNAse protection, or other
appropriate methods. Alternatively, LIPG expression may be
monitored with specific antibodies, which can be used to screen for
effective antisense oligonucleotides. Oligonucleotides which
effectively reduce LIPG mRNA levels are then formulated for in vivo
delivery as therapeutics.
[0303] Antisense LIPG sequences may be delivered in a gene therapy
vector, such as adenovirus, adeno-associated virus, retrovirus,
naked DNA, or other systems discussed in the detailed description.
Such fragments can be used therapeutically when delivered in gene
therapy vectors. Hepatic expression of such recombinant vectors is
a preferred approach. Alternatively, synthetic antisense
oligonucleotides may be formulated for in vivo delivery as
therapeutics as described above.
[0304] Antisense oligonucleotides may be administered by the
following routes: intravenous, subcutaneous, introdermal,
pulmonary, oral, intraventricular, intrathecal, and topical. The
route of administration may include direct administration to vessel
walls (i.e., endothelium and/or vascular smooth muscle). As an
example, patients with low HDL-C could receive a dose of 0.5-2
mg/kg of an effective antisense oligonucleotide, infused
intravenously, every other day for up to 2-3 weeks. As LIPG is
expressed in the liver, it may be desireable to deliver antisense
reagents to the portal circulation. This may be accomplished by
conjugating or complexing the oligonucleotide with a
liver-targeting moiety, such as asialoglycoprotein. Dose and timing
of therapy would depend on efficiency of antisense delivery, as
well as parameters such as half life, specificity and toxicology of
the antisense oligonucleotide.
[0305] Increase in HDL-C can be monitored using standard clinical
laboratory procedures. The original dosing schedule (such as that
described above) is repeated as often as required to maintain HDL-C
above 35 mg/dL.
Example 21
Ribozyme Treatment Method
[0306] Based on the LIPG cDNA sequence, hammerhead ribozymes which
effectively reduce LIPG mRNA levels are prepared. These consist of
two "arms" of 6-7 bases each of nucleotide sequence complementary
to LIPG mRNA, separated by the catalytic moiety of the ribozyme.
Examples of such hammerhead motifs are described by Rossi et al.,
1992, Aids Research and Human Retroviruses, 8, 183. The ribozymes
are expressed in eukaryotic cells from an appropriate DNA
vector.
[0307] The ribozymes may be administered encapsulated in liposomes,
as discussed above.
[0308] The ribozyme/liposome composition is delivered to the liver
by direct injection or by use of a catheter, infusion pump or
stent. The route of administration may include direct
administration to vessel walls (i.e., endothelium and/or vascular
smooth muscle). Patients are treated for up to 2 weeks with 5-50
mg/kg/day ribozyme in a pharmaceutically effective carrier.
Increase in HDL-C and dosing regimen are monitored and determined
as for antisense oligonucleotides.
Example 22
Neutralizing Antibody Treatment Method
[0309] Anti-LIPG antibodies, antibody fragments, or chimeric
antibodies consisting of at least one LIPG-binding moiety, prepared
as described in Example 12, are used to inhibit LIPG activity in
vivo. The antibodies may be delivered as a bolus only, infused over
time, or both. Typically a dose of 0.2-0.6 mg/kg is given as bolus,
followed by a 2 to 12-hour infusion. Alternatively, multiple bolus
injections are administered every other day, or every third of
fourth day, as required to reduce LIPG and raise HDL-C. Repeat
dosing is performed as determined by measurement of HDL-C levels.
Antibodies to LIPG may also be delivered in a gene therapy vehicle
to facilitate expression in vivo. The level of expression of the
antibody is determined indirectly by measuring HDL-C levels and
additional vectors may be introduced as needed.
Example 23
Use of Inhibitory Molecules or Enhancer Molecules
[0310] Fragments of LIPG protein, which can inhibit LIPG activity
by competing for binding to intact LIPG, required coactivator
molecules, cell surface receptors or binding proteins, may be
delivered as therapeutic recombinant proteins or from gene therapy
vectors.
[0311] As an example, the LLGN polypeptide based on LIPG is cloned
into a recombinant adenovirus as described (Tsukamoto et al., J.
Clin. Invest., 100, 107-114 (1997); Tsukamoto et al., J. Lipid
Res., 38, 1869-1876 (1997)). The LLGN cDNA is cloned into the
shuttle plasmid vector pAdCMVLink1. After screening for the
appropriate orientation by restriction analysis, the plasmid is
linearized with NheI and cotransfected into 293 cells along with
adenoviral DNA digested with ClaI. Cells are then overlaid with
agar and incubated at 37.degree. C. for 15 days. Plaques are picked
and screened by PCR; plaques positive for cDNA are subjected to a
second round of plaque purification. After confirmation of the
presence of cDNA, the recombinant adenovirus is expanded in 293
cells at 37.degree. C. Cell lysates are used to infect HeLa cells
for confirmation of the expression of human EL by western blot of
conditioned media. The recombinant adenovirus is further expanded
in 293 cells and purified by cesium chloride ultracentrifugation.
The purified viruses are stored in 10% glycerol/PBX at -80.degree.
C. The patient is injected intravenously with the recombinant
adenovirus 1.times.10.sup.11 particles (approximately
2.times.10.sup.9 pfu).
Example 24
Methods of Increasing the Level of LIPG in a Patient by Expression
of LIPG from an Expression Vector
[0312] The full length LIPG cDNA is cloned into a recombinant
adenovirus (Tsukamoto et al., J. Clin. Invest., 100, 107-114
(1997); Tsukamoto et al., J. Lipid Res., 38, 1869-1876 (1997))
encoding human LIPG. The full-length human LIPG cDNA is cloned into
the shuttle plasmid vector pAdCMVLink1. After screening for the
appropriate orientation by restriction analysis, the plasmid is
linearized with NheI and cotransfected into 293 cells along with
adenoviral DNA digested with ClaI. Cells are overlaid with agar and
incubated at 37.degree. C. for 15 days. Plaques are picked and
screened by PCR; plaques positive for cDNA are subjected to a
second round of plaque purification. After confirmation of the
presence of cDNA, the recombinant adenovirus is expanded in 293
cells at 37.degree. C. Cell lysates are used to infect HeLa cells
for confirmation of the expression of human LIPG polypeptide by
western blot of conditioned media. The recombinant adenovirus
(AdhEL) is further expanded in 293 cells and purified by cesium
chloride ultracentrifugation. The purified viruses are stored in
10% glycerol/PBX at -80.degree. C. Patients are injected
intravenously with AdhEL or Adnull 1.times.10.sup.11 particles
(approximately 2.times.10.sup.9 pfu).
Example 25
Methods of Increasing the Level of LIPG Activity by Administration
of a Full-Length Wild-Type or Engineered Recombinant LIPG
Protein
[0313] The wild-type LIPG protein reduces VLDL and LDL cholesterol
levels and LIPG could be engineered to act specifically on VLDL and
LDL cholesterol without having effects on HDL cholesterol.
Administration of wild-type or engineered recombinant LIPG could in
certain circumstances be used as a therapy for reducing VLDL and/or
LDL cholesterol levels. Wild-type and/or engineered LIPG protein
("recombinant LIPG protein") will be produced recombinantly in
E.coli and purified using methods known in the art. Wild-type mice
will be studied by administering the recombinant LIPG protein by
intravenous injection. Activity of LIPG will be measured in plasma.
In addition, cholesterol, VLDL, LDL and HDL cholesterol and apoA-I
levels will be monitored in animals receiving the recombinant LIPG
protein. Finally, LDL receptor deficient mice will be fed an
atherogenic diet and administered the recombinant LIPG protein or
placebo for a period of 8 weeks. Atherosclerosis will be
quantitated in the aortas of the mice in order to determine whether
adminsitration of the recombinant LIPG protein reduces the
progression or induces regression of atherosclerosis. Based on
these preclinical data, additional animal models such as hamsters,
rabbits, or pigs will be studied for the ability of the recombinant
LIPG protein to reduce VLDL and LDL cholesterol levels and/or
inhibit the progression of atherosclerosis. Those recombinant LIPG
proteins found to have the desired ability to reduce VLDL and LDL
cholesterol levels and/or inhibit the progression of
atherosclerosis will be combined with a pharmaceutically acceptable
carrier and administered to patients. The recombinant LIPG
polypeptides may be administered in a variety of ways, including
oral administration and intravenous injection.
[0314] All the references discussed herein are incorporated by
reference.
[0315] One skilled in the art will readily appreciate the present
invention is well adapted to carry out the objects and obtain the
ends and advantages mentioned, as well as those inherent therein.
The peptides, polynucleotides, methods, procedures and techniques
described herein are presented as representative of the preferred
embodiments, and intended to be exemplary and not intended as
limitations on the scope of the present invention. Changes therein
and other uses will occur to those of skill in the art which are
encompassed within the spirit of the invention or defined by the
scope of the appended claims.
Sequence CWU 1
1
* * * * *