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United States Patent6048850
Young , ; et al.April 11, 2000
Title
Method of inhibiting prostaglandin synthesis in a human host
Abstract
The invention relates to the gene encoding the mammalian prostaglandin H synthase-2 and its product. More specifically, the invention relates to the diagnosis of aberrant PGHS-2 gene or gene product; the identification, production, and use of compounds which modulate PGHS-2 gene expression or the activity of the PGHS-2 gene product including but not limited to nucleic acid encoding PGHS-12 and homologues, analogues, and deletions thereof, as well as antisense, ribozyme, triple helix, antibody, and polypeptide molecules as well as small inorganic molecules; and pharmaceutical formulations and routes of administration for such compounds.
Inventors:Young; Donald A. (Rochester, NY), O'Banion; Michael K.  (Pittsford, NY), Winn; Virginia D.  (Rochester, NY)
Appl. No.:487744
Filed:June 7, 1995
Current U.S. Class:514/183 514/241 514/326 514/601 435/183 435/189 435/25 435/28 435/29 514/1 
Field of Search:514/44,1,825,886,885,183,605,601 536/24.5 935/34,36 435/25,29,28,189,183
U.S. Patent Documents
4950599August 1990Bertling
4980281December 1990Housey
5087572February 1992Castellino et al.
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Primary Examiner: Myers; Carla J.
Attorney, Agent or Firm:Pennie & Edmonds LLP
Government Interests:

This invention was made with government support under grant number DK
16177, awarded by the National Institutes of Health. The government has
certain rights in the invention.
Parent Case Text


This is a division, of application Ser. No. 08/487,752, filed Jun. 7, 1995, with Attorney Docket No. 3996-009, which is a continuation-in-part of Ser. No. 08/231,456, filed Apr. 20, 1994 now abandoned, which is a continuation-in-part of Ser. No. 08/054,364, filed Apr. 28, 1993 now abandoned, which is a continuation-in-part of Ser. No. 07/983,835, filed Dec. 1, 1992 (abandoned), which is a continuation-in-part of Ser. No. 07/949,780, filed Sep. 22, 1992 (abandoned); and is also a continuation-in-part of Ser. No. 08/034,143, filed Mar. 22, 1993 now abandoned, which is a continuation of Ser. No. 07/949,780, filed Sep. 22, 1992 now abandoned, each which is incorporated by reference in its entirety.
Claims

What is claimed is:
1. A method for selectively inhibiting PGHS-2 activity in a human host, comprising administering a non-steroidal compound that selectively inhibits activity of the PGHS-2 gene product to a human host in need of such treatment.

2. The method of claim 1 in which the compound inhibits the enzymatic activity of the PGHS-2 gene product, and has minimal effect on enzymatic activity of PGHS-1.

3. The method of claim 1 in which the activity of PGHS-1 is not inhibited.

4. The method of claim 3 in which the compound is a non-steroid anti-inflammatory drug.

5. A method for selectively inhibiting PGHS-2 activity in a human host, comprising administering a non-steroidal compound that selectively inhibits activity of the PGHS-2 gene product in a human host in need of such treatment, wherein the activity of the non-steroidal compound does not result in significant toxic side effects in the human host.

6. A method for selectively inhibiting PGHS-2 activity in a human host, comprising administering a non-steroidal compound that selectively inhibits activity of the PGHS-2 gene product in a human host in need of such treatment, wherein the ability of the non-steroidal compound to selectively inhibit the activity of the PGHS-2 gene product is determined by:
a) contacting a genetically engineered cell that expresses human PGHS-2, and not human PGHS-1, with the compound for 30 minutes, and exposing the cell to a pre-determined- amount of arachidonic acid;
b) contacting a genetically engineered cell that expresses human PGHS-1, and not human PGHS-2, with the compound for 30 minutes, and exposing the cell to a pre-determined amount of arachidonic acid;
c) measuring the conversion of arachidonic acid to its prostaglandin metabolite; and
d) comparing the amount of the converted arachidonic acid converted by each cell exposed to the compound to the amount of the arachidonic acid converted by control cells that were not exposed to the compound, so that the compounds that inhibit PGHS-2 and not PGHS-1 activity are identified.

7. The method of claims 1, 3 or 4, which is used to treat inflammation.

8. The method of claims 1, 3, or 4, in which the inhibition of prostaglandin synthesis has anti-inflammatory action in the human host.
Description
1. INTRODUCTION

The present invention relates to the gene encoding the mammalian prostaglandin H synthase-2, hereinafter "PGHS-2," and its product. Mammalian prostaglandin H synthase-1, hereinafter "PGHS-1," is responsible for the constitutive prostaglandin synthesis in mammalian physiology. PGHS-2 was discovered to be responsible for the increased prostaglandin synthesis associated with inflammation. The invention relates to PGHS-2 and to compounds which specifically modulate the expression of PGHS-2 and not PGHS-1 including but not limited to nucleic acid encoding PGHS-2 and homologues, analogues, and deletions thereof, as well as antisense, ribozyme, triple helix, antibody, and polypeptide molecules and small inorganic molecules. The invention further relates to methods of diagnosing an aberrant PGHS-2 gene and gene product as well as gene therapies for use as a remedy for such aberrant PGHS-2 gene or gene product. In addition, the invention relates to pharmaceutical formulations and routes of administration for such remedies.

2. BACKGROUND OF THE INVENTION

Prostaglandins (which include PGE.sub.2, PGD.sub.2, PGF.sub.2a, PGI.sub.2 and other related compounds) represent a diverse group of autocrine and paracrine hormones that are derived from the metabolism of fatty acids. They belong to a family of naturally occurring eicosanoids (prostaglandins, thromboxanes and leukotrienes) which are not stored as such in cells, but are biosynthesized on demand from arachidonic acid, a 20-carbon fatty acid that is derived from the breakdown of cell-membrane phospholipids. Under normal circumstances, the eicosanoids are produced at low levels to serve as important mediators of many and diverse cellular functions which can be very different in different types of cells. However, the prostaglandins also play critical roles in pathophysiology. In particular, inflammation is both initiated and maintained, at least in part, by the overproduction of prostaglandins in injured cells. The central role that prostaglandins play in inflammation is underscored by the fact that those aspirin-like non-steroidal anti-inflammatory drugs (NSAIDS) that are most effective in the therapy of many pathological inflammatory states all act by inhibiting prostaglandin synthesis. Unfortunately, the use of these drugs is often limited by the side effects (gastrointestinal bleeding, ulcers, renal failure, and others) that result from the undesirable reduction in prostaglandins in normal cells that now suffer from a lack of those autocrine and paracrine functions that are required for the maintenance of normal physiology. The development of new agents that will act more specifically by achieving a reduction in prostaglandins in inflamed cells without altering prostaglandin production in other cells is one of the major goals for future medicinal therapy.

The cyclooxygenase reaction is the first step in the prostaglandin synthetic pathway; an enzyme (PGHS) with prostaglandin G/H synthetic activity converts arachidonic acid into the endoperoxide PGG.sub.2, which then breaks down to PGH.sub.2 (the two reactions are carried out by a single enzyme). PGH.sub.2 is in turn metabolized by one or more prostaglandin synthase (PGE.sub.2 synthase, PGD.sub.2 synthase etc.) to generate the final "2-series" prostaglandins, PGE.sub.2, PGD.sub.2, PGF.sub.2a, PGI.sub.2 and others which include the thromboxanes, TXA.sub.2. The first step (PGHS) is the one that is rate-limiting for prostaglandin synthesis. As such, the PGHS-mediated reaction is the principal target for anti-inflammatory drug action; and it is inhibition of PGHS activity that accounts for the activity of the NSAIDS (aspirin, acetominophen, ibuprofen, naproxen, indomethacin) and others that limit the overproduction of prostaglandins in inflammation (the desired therapeutic goal) and reduce the normal production of prostaglandins in uninflamed cells (which produces the undesirable side effects).

In addition to the abnormal changes associated with inflammation, multiple other factors are known to influence prostaglandin production under experimental conditions. These include growth factors, cAMP, tumor promoters, src activation and interleukins 1 and 2, all of which increase overall cellular PGHS activity. The adrenal glucocorticoid hormones and related synthetic anti-inflammatory steroids also inhibit prostaglandin synthesis, but their metabolic site of action is not well defined.

Human, ovine, and murine cDNAs have been cloned for PGHS-1. All show similar sequences and hybridize with 2.8-3.0-kb mRNAs on Northern blots. However, several research groups have recently identified and predicted the sequence of a protein reported to be related to PGHS-1 in some manner. In 1990, Han et al., 1990, Proc. Nat'l. Acad. Sci. USA, 87:3373-3377 reported changes in protein synthesis caused by the polypeptide pp60.sup.v-src, following infection of BALB/c 3T3 fibroblasts by Rous sarcoma virus temperature-sensitive mutant strain LA90. Giant two-dimensional gel electrophoresis detected induction of a 72-74 kDa protein doublet that is recognized by anticyclooxygenase antibodies. Synthesis of this doublet was also transiently increased by exposure to platelet-derived growth factor and inhibited by dexamethasone treatment. These changes in protein synthesis were strongly correlated with changes in cyclooxygenase activity. The protein doublet was also seen in mouse C127
fibroblasts where its synthesis was found to be regulated by serum and dexamethasone and correlated with cyclooxygenase activity. See O'Banion et al., 1991, J. Biol. Chem., 266:23261-23267.

Xie et al., 1991, Proc. Nat'l. Acad. Sci. USA, 2692-2696 followed Han's et al. earlier report with the isolation of a set of cDNAs corresponding to pp60v-arc inducible form "miPGHS.sub.ch ", for mitogen-inducible PGHS.sub.chicken. Although Xie et al. speculated that prostaglandin synthesis may play a role in src product-mediated cellular transformation, their experiments did not permit them to discriminate between miPGHS.sub.ch as a second cyclooxygenase or simply as the chicken homolog of sheep PGHS-1, "PGHS.sub.ov ".

In a separate set of experiments, Kujubu et al., 1991 J. Biol. Chem., 266:12866-12872 reported that one of the primary response genes cloned from mitogen-responding Swiss 3T3 cells (TIS10) has a long 3'-untranslated region and encodes a "predicted" 66 kDa protein which is about 60% identical to mouse PGHS-1. The sequence of this putative protein was essentially identical to that derived by Xie et al. On the basis of sequence similarities, Kujubu et al. speculated that the enzymatic activity of the protein encoded by the TIS10 gene would be likely to be "similar" to enzymatic activity of other types of mammalian PGHS-1. They concluded that "[p]roof of this conjecture, however, awaits the heterologous expression of this gene production from an expressible plasmid and the direct measurement of cyclooxygenase activity in transfected cells and/or purified preparations of the TIS10 protein."

There is increasing emphasis on the development of methods for the modulation and evaluation of the activity of the prostaglandin synthetic pathway. As noted above, nonsteroidal anti-inflammatory agents, such as aspirin and indomethacin, inhibit the cyclooxygenase which converts arachidonic acid into PGG.sub.2 and PGH.sub.2. Therefore, there is a need for improved methods to study the effectiveness of existing anti-inflammatory drugs and to evaluate the effectiveness of potential anti-inflammatory agents, at the molecular level, as well as for reagents for use in such methods.

3. SUMMARY OF THE INVENTION

The invention relates to the gene encoding the mammalian prostaglandin H synthase-2 and its product. The invention is based, in part, on the discovery that there are two PGHS genes; one constituitively expressed and termed herein PGHS-1, and a second which is responsive to regulatory control and termed herein PGHS-2. More specifically, the invention relates to the diagnosis of an aberrant PGHS-2 gene or gene product; the identification, production, and use of compounds which modulate PGHS-2
gene expression or the activity of the PGHS-2 gene product including but not limited to nucleic acid encoding PGHS-2 and homologues, analogues, and deletions thereof, as well as antisense, ribozyme, triple helix, antibody, and polypeptide molecules and small inorganic molecules; and pharmaceutical formulations and routes of administration for such compounds. The invention also relates to the identification of naturally occurring cells and the creation of cells that express PGHS-1 or PGHS-2 exclusively and the use of such cells in drug screening.

In the examples described infra, it is shown that a second PGHS gene, PGHS-2, has been identified in mouse and in human cells which is distinct from the PGHS-1 gene. It is further shown that PGHS-2 expression is responsive to regulatory control while PGHS-1 expression is constitutive. An assay employing PGHS-2 transfectants was used to successfully identify compounds which modulate the expression of the PGHS-2 gene. Assays for the activity of the PGHS-2 gene product are also described. In addition assays employing PGHS-2 and PGHS-1 transfectants are described for use in identifying compounds which modulate the expression of the PGHS-2 gene and not the PGHS-1 gene.

3.1. DEFINITIONS

As used herein, the following terms and abbreviations shall have the meanings indicated below:

______________________________________ base pair(s) bp complementary DNA cDNA counts per minute cpm deoxyribonucleic acid DNA kilobase pairs kb kilodation kDa micrograms .mu.g micrometer .mu.m nanograms ng nanometer nm nucleotide nt polyacrylamide gel electrophoresis PAGE polymerase chain reaction PCR prostaglandin H synthase PGHS radioimmunoassay RIA ribonucleic acid RNA sodium dodecyl sulfate SDS units u ______________________________________

As used herein, the word "modulate" shall have its usual meaning, but shall also encompass the meanings of the words enhance, inhibit, and mimic. In addition, as used herein, the word "expression" when used in connection with a gene such as PGHS-2 shall have its usual meaning, but shall also encompass the transcription of the gene, the longevity of functional mRNA transcribed from the gene, the translation of that MRNA, and the activity of the gene product.

4. DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the cDNA (SEQ ID NO:1) and predicted amino acid sequence (SEQ ID NO:2) of murine griPGHS ("PGHS-2"). The standard one letter code for amino acids is used. Based on a transcription start site determined by primer extension at -24, the numbering of this sequence starts at 25. A predicted signal peptide cleavage site between amino acids 17 and 18 is marked with an arrowhead. The position of the putative aspirin-modified serine is indicated by a circle, and potential N-glycosylation sites are double underlined.

FIG. 2 is a schematic depiction comparing the cDNA and protein sequences for the murine 2.8- and 4.1 kb RNA-encoded cyclooxygenases. cDNA structures for the 4.1 kb cDNA cloned from C127 cells and the murine 2.8 kb cDNA are drawn as the thick lines at top and bottom. The numbering of the 4.1 kb cDNA is based on primer extension data. Since the 5' end of the 2.8 kb mouse mRNA has not been determined, no numbers have been assigned to the translation start and stop sites. Alternative polyadenylation sites established from other CDNA clones are indicated with "A" and the 5'-AUUU.sub.n A-3' motifs are identified by dots underneath the sequence. These motifs are not found in the 2.8 kb cDNA. Deduced protein sequences are drawn colinearly with gaps (17 aa at the amino-terminal end of the 4.1 kb MRNA product, and 18 aa at the carboxy-terminal end of the 2.8 kb MRNA product) indicated by connecting lines. The 26 aa leader sequence for the 2.8 kb PGHS is indicated. Although its extent has not been precisely defined, a shorter, nonhomologous leader appears to exist for griPGHS with a mature N-terminal end at amino acid 18. The positions of potential N-glycosylation sites (NXS/T, "N") and the conserved aspirin modified serines are noted on each molecule. The hatched areas near the center of each molecule denote presumed axial (TIWLREHNRV, identical between the two molecules) and distal (KALGH/RGLGH) heme-binding sites as suggested by DeWitt et al., 1990, J. Biol. Chem.
265:5192-5198. Interestingly, the RGLGH sequence in griPGHS fits the consensus RXXXH (SEQ ID NO:18) distal heme-binding site described for other peroxidases, Kimura and Ikeda-Saito, 1988, Prot. Struc. Func. Genetics 3, 113-120, and supports the previous suggestion that KALGH serves the same purpose in the 2.8 kb gene product, DeWitt et al., 1990, J. Biol. Chem. 265-5192-5198. The bar at the bottom of the figure represents the similarities between the two mouse PGHS proteins (omitting the nonconserved N- and C-termini) as the percentage of identical residues for groups of 20 amino acids with increasing shading indicating 40-55% (no shading), 60-75%, 80-95%, and 100% identity. The overall identity is 64% and with conservative changes the similarity index is 79%.

FIGS. 3A-3B are a photographic depiction of autoradiographies obtained by Northern blotting monitoring the expression of the genes encoding griPGHS and the constitutive PGHS-1, as expressed in human monocytes, in response to interleukin-1
treatment, a known mediator of inflammation. Adherent human monocytes isolated from healthy donors were suspended in medium without serum at 1.times.10.sup.6 cells/ml. One ml aliquots in 5 ml polypropylene tubes were incubated with loosened caps in 5% CO.sub.2 at 37.degree. C. with occasional shaking. FIGS. 3A-3B are more fully described as follows:

FIG. 3A: Monocytes were incubated for 4 h in the presence or absence of dexamethasone (1 .mu.M) prior to total RNA isolation. Five .mu.g was subjected to Northern blot analysis with the indicated probes.

FIG. 3B: Monocytes were treated with dexamethasone (1 .mu.M), 1L-1.beta. (10 half-maximal units, Collaborative Research), or both for the indicated times prior to RNA isolation. Cycloheximide (25 .mu.M) was added to one set of incubations 15
min prior to he addition of cytokine or hormone.

FIG. 4 is a schematic depiction of griPGHS expression vector construction. griPGHS was prepared for directional subcloning into the pRC/CMV expression vector (Invitrogen) by digestion with Acc I, Klenow fill-in, and digestion with Not I. This fragment, extending from the Not I site 50 bases upstream of the cDNA end to nt 1947 of the cDNA, contains the full-coding region truncated immediately before any 5'-AUUUA-3' mRNA destabilizing regions, O'Banion et al., 1992, Proc. Nat'l. Acad. Sci. USA, 89:4888-4892. The pRc/CMV vector DNA was digested with Xba I, filled in with Klenow, then digested with Not I. The dots in the 3' untranslated region of griPGHS indicate the locations of 5'-AUUUA'-3'mRNA destabilizing sequences. "A" represents alternative polyadenylation sites, "N" represents potential glycosylation sites, and "SER 516" marks the location of the aspirin-acetylated serine.

FIGS. 5A-5D are a graphic depiction of the inhibition of murine griPGHS activity in stable transfected mammalian cell lines by preselected amounts of several non-steroidal anti-inflammatory drugs. FIGS. 5A-5D are more fully described as follows:

FIG. 5A: Acetominophen.

FIG. 5B: Ibuprofen.

FIG. 5C: Naproxen.

FIG. 5D: Indomethacin.

FIGS. 6A-6B depict the nucleotide sequence of the human PGHS-2 gene (SEQ ID NO:3). FIGS. 6A-6B are more fully described as follows:

FIG. 6A: Nucleotides 90-1049.

FIG. 6B: Nucleotides 1050-1923.

FIG. 7 depicts a comparison between the amino acid sequence of human PGHS-2 of the present invention (upper sequence) (SEQ ID NO:4) and the amino acid sequence published by H1a et al. (lower sequence) (SEQ ID NO:5). The sequences are given in standard single letter code.

FIGS. 8A-8D are a graphical depiction of the inhibition of human PGHS-2 activity in stably transformed COS cells by four non-steroidal anti-inflammatory drugs (NSAID): Acetominophen; Ibuprofen; Naproxen; and Indomethacin. FIGS. 8A-8D are more fully described as follows:

FIG. 8A: Acetominophen.

FIG. 8B: Ibuprofen.

FIG. 8C: Naproxen.

FIG. 8D: Indomethacin.

FIGS. 9A-9D are a graphical depiction of the inhibition of human PGHS-1 activity in stably transformed COS cells by four NSAID: Acetominophen; Ibuprofen; Naproxen; and Indomethacin. FIGS. 9A-9D are more fully described as follows:

FIG. 9A: Acetominophen.

FIG. 9B: Ibuprofen.

FIG. 9C: Naproxen.

FIG. 9D: Indomethacin.

FIGS. 10A-10D show a nucleic acid sequence comparison between the coding regions of human PGHS-2 (SEQ ID NO:14) and PGHS-1(SEQ ID NO:6). Solid-lined-boxes indicate regions where the sequence of PGHS-2 is least homologous to that of PGHS-1. Dashed-lined-boxes indicate regions where the sequence of PGHS-2 is most homologous to that of PGHS-1. FIGS. 10-10D are more fully described as follows:

FIG. 10A: PGHS-2 nucleotides 1-467.

FIG. 10B: PGHS-2 nucleotides 469-1004.

FIG. 10C: PGHS-2 nucleotides 1006-1537.

FIG. 10D: PGHS-2 nucleotides 1540-1834.

FIGS. 11A-11C show the nucleic acid sequence of the 5' promoter region of human PGHS-2 (SEQ ID NO:5) as compared with that of PGHS-1. Dashed-lined-boxes indicate the regions where the sequence of the PGHS-2 5' region is most homologous to that of PGHS-1. FIGS. 11A-11C are more fully described as follows:

FIG. 11A: PGHS-2 promoter nucleotides 1-950.

FIG. 11B: PGHS-2 promoter nucleotides 951-1900.

FIG. 11C: PGHS-2 promoter nucleotides 1901-2400.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a mammalian cell line which contains a chromosomally integrated, recombinant DNA sequence, which DNA sequence expresses mammalian, preferably human, glucocorticoid-regulated inflammatory PGHS, and which cell line does not significantly express autologous PGHS-1 or PGHS-2 activity. For brevity, glucocorticoid-regulated inflammatory PGHS will hereinafter be referred to as "griPGHS" or "PGHS-2", and the art-recognized mammalian PGHS encoded by the 2.8-3.0 kb mRNA (EC 1.14.99.1) will be referred to as -constitutive cyclooxygenase," or "constitutive PGHS," or "PGHS-1." The recitation that there is no "autologous PGHS-1 or PGHS-2 activity" relates to the inability of the cell line to express PGHS activity apart from that expressed by the recombinant DNA sequence. Autologous PGHS activity may also be referred to as "endogenous" PGHS activity in the art.

This invention is a result, in part, of the discovery that the 72-74 kDa cyclooxygenase reported by Han et al., the miPGHS.sub.ch reported by Xie et al., and the TIS10 protein reported by Kujubu et al. are essentially identical and represent a second cyclooxygenase, which second form is the primary target for inhibition by glucocorticoids and is also a target for inhibition by non-steroidal anti-inflammatory agents.

The synthesis of a 70 kilodalton (kDa) protein in C127 mouse fibroblasts, via a mouse 4 kilobase (kb) mRNA, and the derived amino acid sequence was reported. The protein encoded by the 4-kb mRNA shows 80% amino acid identify with the previously known mouse PGHS-1 protein product in a sequenced 240 base region. See O'Banion et al., 1991, J. Biol. Chem., 35:23261-23267.

The 70 kDa protein, designated griPGHS or PGHS-2 herein, was determined to be a discrete form of cyclooxygenase by several assays. The protein was precipitated by anti-PGHS serum, its synthesis and concomitant cyclooxygenase levels are rapidly induced by serum, and the induction is inhibited by dexamethasone. The regulation of PGHS-2 synthesis was found not to arise from alterations in the level of the 2.8-kb PGHS-1 mRNA, but resulted from changes in the level of a 4-kb mRNA species. This latter species is barely detectable with a 2.8-kb PGHS-1 DNA probes in cells treated with serum, but accumulates to significant levels in cells treated with cycloheximide or calcium ionophores. In contrast, there was no change in the level of, the
2.8-kb mRNA which encodes PGHS-1 or "constitutive PGHS" as observed following treatment with serum, dexamethasone or cycloheximide. Finally, by hybridization analysis, it was shown that the 4-Kb mRNA represented the product of a gene that is distinct from the gene giving rise to the 2.8-Kb mRNA.

These observations indicated that there are two cyclooxygenase genes; one constitutively expressed as a 2.8-kb mRNA, and a second giving rise to a growth factor and glucocorticoid-regulated 4-kb mRNA which encodes PGHS-2. It is believed that expression of the latter 4-kb RNA and concomitantly increased PGHS-2 levels are primarily, if not entirely, responsible for the enhanced prostaglandin synthesis that is responsible, directly or indirectly, for many of the adverse effects of inflammation.

The primary and perhaps sole action of most non-steroidal anti-inflammatory agents is to inhibit the enzyme prostaglandin G/H synthase, also known as cyclooxygenase, which serves as the first committed step in the biosynthesis of prostaglandins. PGHS-2 is a unique isoform of cyclooxygenase, which in contrast to the previously cloned, constitutively expressed enzyme, is dramatically up-regulated by growth factors, tissue injury, and proinflammatory cytokines, and down-regulated by glucocorticoids (O'Banion et al., 1991, J. Biol. Chem., 266:23261-23267; O'Banion et al., 1992, Proc. Nat'l. Acad. Sci. USA, 89:4888-4892: Pritchard et al., 1994, J. Biol. Chem., 269:8504-8509). Recent studies utilizing specific pharmacological inhibitors of PGHS-2 confirm that it plays a major role in peripheral inflammation (Futaki et al., 1993, J. Pharm. Pharmacol., 45:753-755; Masferrer et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 3228-3232; Vane et al., 1994, Proc. Nat'l. Acad. Sci. USA,
91:2046-2050).

The present invention also comprises an isolated DNA sequence (gene) encoding biologically active human PGHS-2; antisense and ribozyme molecules specific for the PGHS-2 transcript; polynucleotide molecules which form a triple helix at the 5' region of the PGHS-2 gene and thereby prevent or reduce transcription of the gene; the isolated, essentially pure human PGHS-2 gene product; antibodies to the gene product; continuous cell lines engineered to stably express PGHS-2; assays for screening compounds, including peptides, polynucleotides, and small organic molecules to identify those that inhibit the expression or activity of the PGHS-2 gene product; and methods of treating diseases characterized by aberrant PGHS-2 activity using such compounds.

5.1. DNA ENCODING MAMMALIAN PGHS-2

The screening of a murine cDNA library enriched in the 4 kb mRNA of O'Banion et al., 1991, J. Biol. Chem., 35:2326-23267 with a radiolabelled portion of the 2.8 kb PGHS cDNA revealed a 4.1 kb sequence (FIG. 1). Comparison of the 4.1 kb sequence with that of the previously cloned mouse 2.8 kb PGHS cDNA revealed a single open reading frame with 64% amino acid identity to the protein encoded by the 2.8 kb PGHS cDNA, O'Banion et al., 1992, Proc. Nat'l. Acad. Sci. USA, 89:4888-4892 . This 4.1
kb sequence is designated PGHS-2, and the 2.8 kb sequence is designated PGHS-1. The reduced amino acid sequences are colinear except that PGHS-2 has a shorter amino-terminus and longer carboxy-terminus than PGHS-1.

Three of four potential N-glycosylation sites are conserved between the two molecules and there is particularly high similarity in the regions surrounding a putative axial heme-binding domain (amino acids 273-342) and the region around the presumed aspirin modified-serine.sup.516 (amino acids 504-550). By far the largest difference in the two cDNAs is the presence of a 2.1 kb 3' untranslated region in the 4.1 kb cDNA. This region is rich in 5'-AUUUA-3' motifs that are associated with the decreased stability of many cytokine and protooncogene mRNAs. The presence of these motifs is consistent with the profound superinducibility of the 4.1 kb mRNA by cycloheximide, which is not observed for the 2.8 kb mRNA.

FIG. 2 schematically compares cDNA and protein sequences for the murine 2.8 and 4.1 kb mRNA-encoded cyclooxygenases. cDNA structures for the 4.1 kb cDNA cloned from murine C127 cells and the murine 2.8 kb cDNA (DeWitt et al., 1990, J. Biol. Chem., 265:5192-5198 are drawn as the thick lines at top and bottom. The numbering of the 4.1 kb cDNA is based on primer extension data. Since the 5' end of the 2.8 kb mouse mRNA has not been determined, no numbers have been assigned to the translation start and stop sites. Alternative polyadenylation sites established from other cDNA clones are indicated with "A" and the 5'-AUUUnA-3' motifs are identified by dots underneath the sequence. These motifs are not found in the 2.8 kb cDNA. Deduced protein sequences are drawn colinearly with gaps (17 aa at the amino-terminal end of the 4.1 kb mRNA product, and 18 aa at the carboxy-terminal end of the 2.8 kb mRNA product) indicated by connected lines. The 26 amino acid (aa) leader sequence for the
2.8 kb PGHS is indicated. Although its extent has not been precisely defined, a shorter, nonhomologous leader appears to exist for griPGHS with a mature N-terminal end at amino acid 18. The positions of potential N-glycosylation sites (NXS/T, "N") and the conserved aspirin modified serines are noted on each molecule. The hatched areas near the center of each molecule denote presumed axial (TIWLREHNRV (SEQ ID NO:7), identical between the two molecules) and distal (KALGH (SEQ ID NO:8)/RGLGH (SEQ ID NO:9)) heme-binding sites as suggested by DeWitt et al., cited above. The bar in the middle of the figure represents the similarities between the two mouse PGHS proteins (omitting the nonconserved N- and C-termini) as the percentage of identical residues for groups of 20 amino acids with increasing shading indicating 40-55% (no shading), 60-75%, 80-95%, and 100% identity. The overall identity is 64% and with conservative changes the similarity index is 79%.

Another specific embodiment of the invention is the human PGHS-2 gene and its product. The human PGHS-2 sequence differs from the human PGHS-2 sequence disclosed by H1a & Neilson, 1992, Proc. Nat'l. Acad. Sci. USA, 89:7384-7388, due to a glutamic acid (E) rather than a glycine (w) at amino acid position 165 of the PGHS-2 gene product (FIG. 7). The sequence for the PGHS-2 gene was confirmed by establishing the identity of the sequences of two other hPGHS-2 clones obtained from separate PCR runs, which demonstrates that the difference observed is not a PCR artifact. Furthermore, as shown in FIG. 1, mouse PGHS-2 also has a glutamic acid at this position. While the human PGHS-2 nucleotide sequence is similar to that of the mouse, there are regions of substantial divergence. These divergent regions in the nucleotide sequence of the human PGHS-2 (FIGS. 6A-6B) include, but are not limited to:

TCCACCCGCAGTACAGAAAGTATCACAGGCT 1375-----------------------------1405 GTGTTCCAGATCCAGAGCTCATTAAAACAGT 1797-----------------------------1827

PGHS-1 clones were similarly screened and the sequences of the PGHS-1 gene and enzyme confirmed to be identical to that shown in FIG. 2 (SEQ ID NO:6) in Yokahama and Tanabe, 1984 Biochem. Biophys. Res. Commun., 165:888-894; see also, H1a,
1986, Prostaglandins, 32:829-845.

Fragments of the PGHS-2 DNA are also included within the scope of the invention. In a further embodiment of the invention, the PGHS-2 DNA or a modified sequence thereof may be ligated to a heterologous sequence to encode a fusion protein. For example, for screening peptide libraries it may be useful to encode a chimeric PGHS-2 protein expressing a heterologous epitope that is recognized by a commercially available antibody. A fusion protein may also be engineered to contain a cleavage site located between the PGHS-2 sequence and the heterologous protein sequence, so that the PGHS-2 protein or protein fragment can be cleaved away from the heterologous moiety. In another embodiment, DNA sequences encoding a fusion protein comprising all or a portion of the PGHS-2 protein fused to another protein with a desired activity are within the scope of the invention; e.g., enzymes such as GUS (.beta.-glucuronidase), .beta.-galactosidase, luciferase, etc.

In another embodiment, DNAs that encode mutant forms of PGHS-2 are also included within the scope of the invention. Such mutant PGHS-2 DNA sequences encompass deletions, 30 additions and/or substitutions of nucleotide residues, or of regions coding for domains within the PGHS-2 protein. These mutated PGHS-2 DNAs may encode gene products that are functionally equivalent or which display properties very different from the native forms of PGHS-2.

The invention contemplates, in addition to the DNA sequences disclosed herein, 1) any DNA sequence that encodes the same amino acid sequence as encoded by the DNA sequences shown in FIGS. 1 and 6A-6B; 2) any DNA sequence that hybridizes to the complement of the coding sequences disclosed herein (see FIGS. 1 and 6A-6B) under highly stringent conditions, e.g., washing in 0.1.times.SSC/0.1% SDS at 68.degree. C. (Ausubel, et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & sons, Inc., New York, at p. 2.10.3) and encodes a functionally equivalent gene product; and/or 3) any DNA sequence that hybridizes to the complement of the coding sequences disclosed herein (see FIGS. 1 and
6) under less stringent conditions, such as moderately stringent conditions, e.g., washing in 0.2.times.SSC/0.1% SDS at 42.degree. C. (Ausubel, et al., 1989, supra), yet which still encodes a functionally equivalent gene product.

The invention also encompasses 1) DNA vectors that contain any of the coding sequences disclosed herein (see FIGS. 1 and 6), and/or their complements (i.e., antisense); 2) DNA expression vectors that contain any of the coding sequences disclosed herein (see FIGS. 1 and 6), and/or their complements (i.e., antisense), operatively associated with a regulatory element that directs the expression of the coding and/or antisense sequences; and 3) genetically engineered host cells that contain any of the coding sequences disclosed herein (see FIGS. 1 and 6), and/or their complements (i.e., antisense), operatively associated with a regulatory element that directs the expression of the coding and/or antisense sequences in the host cell. Regulatory element includes but is not limited to inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression. The invention includes fragments of any of the DNA sequences disclosed herein.

PGHS-2 sequence can be obtained from a variety of sources including cDNA libraries. For example, appropriate cDNA libraries which are good sources of PGHS-2 can be obtained from (Clonetech (Palo Alto, Calif.), Stratagene (La Jolla, Calif.) the ATCC Repository (Rockville, Md.). In addition, cDNA libraries may be prepared from mRNA pools collected from mammalian cells which express PGHS-2 either constitutively or inducibly. By way of example but not by way of limitation, such cells include C127 mouse fibroblasts and W138 human fibroblasts. The collection of mRNA pools and construction of cDNA libraries from these cells are set forth more fully in the examples described infra.

Any of the cDNA libraries described above may be screened by hybridization or. PCR using the PGHS-2 sequences described herein as oligonucleotide probes. Screening can be performed using those portions of the PGHS-2 sequence which are not in PGHS-1, see FIGS. 10A-10D. These sequences include the following regions in the nucleotide sequence of PGHS-2:

171-254

299-340

486-512

602-623

1214-1250

1283-1346

1521-1580

1718-1834

In addition to cDNA libraries, partial PGHS-2 sequence can be obtained from any genomic library by library screening or from genomic DNA by PCR. Full cDNA sequences can be obtained by PCR of total RNA isolated from any cell or tissue that expresses PGHS-2 including, but not limited to, brain, heart and lung (where PGHS-2 is expressed without apparent inflammation), as well as in many inflamed tissues such as synovial biopsies from rheumatoid arthritis. Cellular sources include, but are not limited to, primary and established cultures of fibroblasts, macrophages, endothelial cells, synoviocytes, vascular smooth muscle cells and astrocytes treated with growth factors, serum, inflammatory cytokines, calcium ionophores, or oncogenes, particularly if cycloheximide is included.

Alternatively, the cDNA libraries described above can be used to construct expression libraries in a cell line such as COS A2 which contains little or no autologous cyclooxygenase activity. These expression libraries can then be screened using antibodies which are specific to PGHS-2 and do not bind PGHS-1. Expression libraries for antibody screening may also be made in bacteria, such as E. coli, using phage vectors, such as lambda. Antibodies with specificity to PGHS-2 are commercially available through Cayman Chemical (Ann Arbor, Mich.), Oxford Biomedical Research, Inc. (Oxford, Mich.), and Transduction Laboratories (Lexington, Ky). These expression libraries may also be screened for PGHS-2 enzyme activity as set forth in the examples which are described in more detail infra.

5.2. EXPRESSING THE PGHS-2 GENE PRODUCT

In order to express a biologically active PGHS-2, the coding sequence for the enzyme, a function equivalent, or a modified sequence, as described in Section 5.1., supra, is inserted into an appropriate eukaryotic expression vector, i.e., a vector which contains the necessary elements for transcription and translation of the inserted coding sequence in appropriate eukaryotic host cells which possess the cellular machinery and elements for the proper processing, i.e., signal cleavage, glycosylation, phosphorylation, sialylation, and protein sorting. Mammalian host cell expression systems are preferred for the expression of biologically active enzymes that are properly folded and processed. When administered in humans such expression products may also exhibit tissue targeting.

The invention also encompasses peptide fragments of the PGHS-2 gene product. The PGHS-2 gene product or peptide fragments thereof, can be linked to a heterologous peptide or protein as a fusion protein. In addition, chimeric PGHS-2 expressing a heterologous epitope that is recognized by a commercially available antibody is also included in the invention. A durable fusion protein may also be engineered; i.e., a fusion protein which has a cleavage site located between the PGHS-2 sequence and the heterologous protein sequence, so that the PGHS-2 gene product, or fragment thereof, can be cleaved away from the heterologous moiety. For example, a collagenase cleavage recognition consensus sequence may be engineered between the PGHS-2 gene product, or fragment thereof, the heterologous peptide or protein. The PGHS-2 domain can be released from this fusion protein by treatment with collagenase.

CONSTRUCTION OF EXPRESSION VECTORS AND PREPARATION OF TRANSFECTANTS

Methods which are well-known to those skilled in the art can be used to construct expression vectors containing the PGHS-2 coding sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombination/genetic recombination. See, for example, the techniques described in Sambook et al., 1987, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, New York, Chapter 12.

Human PGHS-1 or PGHS-2 proteins produced by these methods would be useful for in vitro studies on the mechanism of action of the human forms of PGHS-1 and PGHS-2 and particularly for further studies on the mechanism of action of any inhibitors that are selective for PGHS-2 or PGHS-1 that are identified by drug screening with the stably expressing PGHS-2 or PGHS-1 cell lines, as infra, or for investigating the mechanism of action of existing drugs or of inhibitors that may be identified by other means. The purified human PGHS-2 or PGHS-1 proteins would also be useful for the production of crystals suitable for X-ray crystallography. Such crystals would be extremely beneficial for the rational design of drugs based on molecular structure. Although the crystal structure for ovine PGHS-1 has been obtained, this information is not yet available for either human PGHS-1 or PGHS-2. Expression of these chimeric DNA constructs in a baculovirus or yeast system and subsequent crystallization of the proteins would yield such data.

A variety of eukaryotic host-expression systems may be used to express the PGHS-2 coding sequence. Although prokaryotic systems offer the distinct advantage of ease of manipulation and low cost of scale-up, their major drawback in the expression of PGHS-2 is their lack of proper post-translational modifications of expressed mammalian proteins. Eukaryotic systems, and preferably mammalian expression systems, allow for proper modification to occur. Eukaryotic cells which possess the cellular machinery for proper processing of the primary transcript glycosylation, phosphorylation, and, advantageously secretion of the gene product should be used as host cells for the expression of PGHS-2. Mammalian cell lines are preferred. Such host cell lines may include but are not limited to CHO, VERO, BHK, HeLa, COS, MDWCK, -293, WI38, etc. Alternatively, eukaryotic host cells which possess some but not all of the cellular machinery required for optional processing of the primary transcript and/or post-translational processing and/or secretion of the gene product may be modified to enhance the host cell's processing capabilities. For example, a recombinant nucleotide sequence encoding a peptide product that performs a processing function the host cell had not previously been capable of performing, may be engineered into the host cell line. Such a sequence may either be co-transfected into the host cell along with the gene of interest, or included in the recombinant construct encoding the gene of interest. Alternatively, cell lines containing this sequence may be produced which are then transfected with the gene of interest.

Appropriate eukaryotic expression vectors should be utilized to direct the expression of PGHS-2 in the host cell chosen. For example, at least two basic approaches may be followed for the design of vectors based on SV40. The first is to replace the SV40 early region with the gene of interest while the second is to replace the late region (Hammarskjold, et al., 1986, Gene, 43:41-50. Early and late region replacement vectors can also be complemented in vitro by the appropriate SV40 mutant lacking the early or late region. Such complementation will produce recombinants which are packaged into infectious capsids and which contain the PGHS-2 gene. A permissive cell line can then be infected to produce the recombinant protein. SV40-based vectors can also be used in transient expression studies, where best results are obtained when they are introduced into COS (CV-1, origin of SV40) cells, a derivative of CV-1 (green monkey kidney cells) which contain a single copy of an origin defective SV40 genome integrated into the chromosome. These cells actively synthesize large T antigen (SV40), thus initiating replication from any plasmid containing an SV40 origin of replication.

In addition to SV40, almost every molecularly cloned virus or retrovirus may be used as a cloning or expression vehicle. Viral vectors based on a number of retroviruses (avian and murine), adenoviruses, vaccinia virus (Cochran, et al., 1985, Proc. Natl. Acad. Sci. USA, 82:19-23) and polyoma virus may be used for expression. Other cloned viruses, such as J C (Howley, et al., 1980, J. Virol, 36:878-882), BK and the human papilloma viruses (Heilmsan, et al., 1980, J. Virol, 36:395-407), offer the potential of being used as eukaryotic expression vectors. For example, when using adenovirus expression vectors the PGHS-2 coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the human enzyme in infected hosts (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA, 81:3655-3659). Alternatively, the vaccinia virus 7.5 K promoter may be used. (e.g., see, Hackett et al., 1982, Proc. Natl. Acad. Sci. USA, 79:7415-7419; Hackett et al., 1994, J. Virol. 49:857-864, Panicali et al., 1982, Proc. Natl. Acad. Sci. USA, 79:4927-4931). Of particular interest are vectors based on bovine papilloma virus (Sarver, et al., 1981, Mol. Cell. Biol., 1:486-496), or Semliki Forest Virus, which provides large quantities of active protein in induced cells (Olkkohnen et al., 1994, Meth. Cell. Biol., 43 part A:43-53; Lundstrum et al., 1994, Eur. J. Biochem., 224:917-921). These vectors have the ability to replicate as extrachromosomal elements. Shortly after entry of this DNA into mouse cells, the plasmid replicates to about 100 to 200 copies per cell. Transcription of the inserted cDNA does not require integration of the plasmid into the host's chromosome, thereby yielding a high level of expression. These vectors can be used for stable expression by including a selectable marker in the plasmid, such as the neo gene. High level expression may also be achieved using inducible promoters such as the metallothionine IIA promoter, heat shock promoters, etc.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days an enriched media, and then are switched to a selective media. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with the PGHS-2 DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell, 11:223-232), hypoxanthine-guanine phosphoribosylatransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA, 48:2026), and adenine phosphoribosyltransferase (Lowy, et al., 1980, Cell, 22:817-823) genes can be employed in tk.sup.-, hgprt.sup.- or aprt.sup.- cells respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77:3567-3570; O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA
78:1527-1531); ygpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA, 78:2072-2076); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol., 150:1-14); and hygro, which confers resistance to hygromycim (Santerre, et al., 1994, Gene, 30:147-156) genes. Recently, additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, 1988, Proc. Natl. Acad. Sci. USA, 85:8047-8051), and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluromethyl)-DL-ornithine, DFMO (McConlogue L., 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.).

Alternative eukaryotic expression systems which may be used to express the PGHS-2 enzymes are yeast transformed with recombinant yeast expression vectors containing the PGHS-2 coding sequence; insect cell system infected with recombinant virus expression vectors (e.g., baculovirus) containing the PGHS-2 coding sequence; or plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the PGHS-2 coding sequence.

In yeast, a number of vectors containing constitutive or inducible promoters may be used. For a review see, Current Protocols in Molecular Biology, Vol. 2, 1988, Ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant et al., 1987, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 31987, Acad. Press, New York, Vol. 153, pp. 516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Washington, D.C., Ch. 3; Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel Acad. Press, New York, Vol. 152, pp. 673-694; and The Molecular Biology of the Yeast Saccharomyces, 1982, Eds. Strathem et al., Cold Spring Harbor Press, Vols. I and II. For complementation assays in yeast, cDNAs for PGHS-2 may be cloned into yeast episomal plasmids (YEp) which replicate autonomously in yeast due to the presence of the yeast 2.mu. circle. The cDNA may be cloned behind either a constitutive yeast promoter such as ADH or LEU2 or an inducible promoter such as GAL (Cloning in Yeast, Chpt. 3, R. Rothstein In: DNA Cloning Vol. 11, A Practical Approach, Ed. DM Glover, 1986, IRL Press, Washington, D.C.). Constructs may contain the 5' and 3' non-translated regions of the cognate PGHS-2 mRNA or those corresponding to a yeast gene. YEp plasmids transform at high efficiency and the plasmids are extremely stable. Alternatively, vectors may be used which promote integration of foreign DNA sequences into the yeast chromosome.

Alternately, active, post-translationally modified human PGHS-1 and PGHS-2 proteins can be obtained using a yeast expression system such as the Pichia pastoris expression system marketed by Invitrogen (Pichia pastoris is owned and licensed by Research Corporation Technologies, Tucson, Ariz.; however, all components are available from Invitrogen, San Diego, Calif.). In this example, cDNAs encoding human PGHS-2 and PGHS-1 are independently cloned into the pHIL-D2 Pichia expression vector. After linearization with a restriction endonuclease, these constructs are transfected into spheroblasts of the his4 Pichia pastoris strain, GS115, and recombinant yeast carrying the cloned PGHS-1 or PGHS-2 DNA sequences are identified by screening for yeast clones that grow in the absence of histidine (now supplied by the recombinant vector), but do not efficiently utilize methanol as the sole carbon source (due to the presence of PGHS-1 or PGHS-2 in the place of AOXI gene sequence coding for methanol utilization). After expansion of such clones in the presence of an alternative carbon source such as glycerol, large quantities of cells would be transferred to liquid media containing methanol where replication ceases. However, cells remain viable for many days during which time human PGHS-1 or PGHS-2 proteins are specifically expressed at high levels under control of the AOXI promoter. The advantages of this system include very high protein yields and lower expense in the production and maintenance of cultures.

In cases where plant expression vectors are used, the expression of the PGHS-2 coding sequence may be driven by any of a number of promoters. For example, viral promoters such as the 355 RNA and 19S RNA promoters of CaMV (Brisson et al., 1984, Nature, 310:511-514), or the coat protein promoter of TMV (Takamatsu et al., 1987, EMBO J., 6:307-311) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al., 1994, EMBO J., 3:1671-1680; Broglie et al., 1984, Science, 224:838-843); or heat shock promoters, eg., soybean hsp 17.5-E or hsp 17.3-B (Gurley et al., 1986, Mol. Cell. Biol., 6:559-565) may be used. These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors; direct DNA transformation; microinjection, electroporation, etc. For reviews of such techniques see, for example, Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, New York, Section VIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9.

An alternative expression system which could be used to express PGHS-2 is an insect system. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The PGHS-2 sequence may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of the coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (e.g., see Smith et al., 1983, J. Virol., 46:584, Smith, U.S. Pat. No. 4,215,051).

In a specific embodiment of an insect system, the DNA encoding human PGHS-2 or PGHS-1 can be independently cloned into the pBlueBacIII recombinant transfer vector (Invitrogen, San Diego, Calif.) downstream of the polyhedrin promoter and transfected into Sf9 insect cells (derived from Spodoptera frugiperda ovarian cells, available from Invitrogen, San Diego, Calif.) to generate recombinant virus containing human PGHS-1 or PGHS-2. After plaque purification of the recombinant virus high-titer viral stocks are prepared that in turn would be used to infect Sf9 or High Five.TM. (BTI-TN-5B1-4 cells derived from Trichoplusia ni egg cell homogenates; available from Invitrogen, San Diego, Calif.) insect cells, to produce large quantities of appropriately post-translationally modified PGHS-1 or PGHS-2 proteins. Although it is possible that these cells themselves could be directly useful for drug assays, the PGHS-1 or PGHS-2 proteins prepared by this method can be used for in vitro assays of drug potency and selectivity.

5.2.2. IDENTIFICATION OF TRANSFECTANTS OR TRANSFORMANTS EXPRESSING THE PGHS-2 GENE PRODUCT

The host cells which contain the PGHS-2 coding sequence and which express the biologically active gene product may be identified by at least four general approaches: (a) DNA--DNA or DNA-RNA hybridization; (b) the presence or absence of "marker" gene functions; (c) assessing the level of transcription as measured by the expression of PGHS-2 mRNA transcripts in the host cell; and (d) detection of the gene product as measured by immunoassay or by its biological activity.

In the first approach, the presence of the PGHS-2 coding sequence inserted in the expression vector can be detected by DNA-DNA or DNA-RNA hybridization or PCR using probes comprising nucleotide sequences that are homologous to the mouse PGHS-2
coding sequence [SEQ ID NO:1] or human PGHS-2 coding sequence [SEQ ID NO:3] substantially as shown in FIGS. 1 and 6A-6B, or portions or derivatives thereof.

In the second approach, the recombinant expression vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.). For example, if the PGHS-2 coding sequence is within a marker gene sequence of the vector, recombinants containing the PGHS-2 coding sequence can be identified by the absence of the marker gene function. Alternatively, a marker gene can be placed in tandem with the PGHS-2 sequence under the control of the same or different promoter used to control the expression of the PGHS-2 coding sequence. Expression of the marker in response to induction or selection indicates expression of the PGHS-2 coding sequence. In addition, the marker gene may be identified by DNA-DNA or DNA-RNA hybridization or PCR.

In the third approach, transcriptional activity for the PGHS-2 coding region can be assessed by hybridization or PCR assays. For example, RNA can be isolated and analyzed by Northern blot using a probe homologous to the PGHS-2 coding sequence or particular portions thereof substantially as shown in FIG. 1 (murine, [SEQ ID NO:1]) or FIGS. 6A-6B (human, SEQ ID NO:3]). Alternatively, total nucleic acids of the host cell may be extracted and assayed for hybridization to such probes.

In the fourth approach, the expression of the PGHS-2 protein product can be assessed immunologically, for example by Western blots, immunoassays such as radioimmuno-precipitation, enzyme-linked immunoassays and the like. The ultimate test of the success of the expression system, however, involves the detection of the biologically active PGHS-2 gene product. Where the host cell secretes the gene product, the cell free media obtained from the cultured transfectant host cell may be assayed for PGHS-2 activity. Where the gene product is not secreted, cell lysates may be assayed for such activity. In either case, a number of assays can be used to detect PGHS-2 activity including but not limited to the following: cyclooxygenase activity may be determined in the culture medium by the addition of exogenous arachidonic acid substrate (30 .mu.M for 15 min. at 37.degree. C.) followed by conversion of the prostaglandin E.sub.2 product to a methyl oximate form. This derivative may then be quantitated by radioimmunoassay (kit from Amersham Corp.)

5.2.3. CELL LINES EXPRESSING PGHS-1 OR PGHS-2

The present invention also relates to cell lines containing recombinant DNA sequence, preferably a chromosomally integrated recombinant DNA sequence, which comprises a gene encoding the regulated inflammatory cyclooxygenase griPGHS or "PGHS-2" which cell lines further do not express autologous PGHS-1 or PGHS-2, apart from that encoded by the recombinant DNA sequence. The recombinant DNA also does not encode constitutive PGHS-1 (EC 1.14.99.1).

A specific embodiment of the present invention is an engineered mammalian cell line which contains a chromosomally integrated, genetically-engineered ("recombinant") DNA sequence, which DNA sequence expresses mammalian, preferably human, PGHS-2, but does not express constitutive mammalian PGHS-1, and wherein said cell line also does not express autologous PGHS-1 or PGHS-2. The cell line is preferably of human or primate origin, such as the exemplified monkey kidney COS cell line, but cell lines derived from other species may be employed, including chicken, hamster, murine, ovine and the like; the CHO (Chinese hamster ovary) cell line for example, may be particularly preferred for large scale production.

Any cell or cell line, the genotype of which has been altered by the presence of a recombinant DNA sequence is encompassed by the invention. The recombinant DNA sequence may also be referred to herein as "heterologous DNA," "exogenous DNA," "genetically engineered" or "foreign DNA," indicating that the DNA was introduced into the genotype or genome of the cell or cell line by a process of genetic engineering.

The invention includes, but is not limited to, a cell or cell line wherein the native PGHS-2 DNA sequence has been removed or replaced as a result of interaction with a recombinant DNA sequence. Such cells are called PGHS-2 knockouts, herein, if the resulting cell is left without a native DNA that encodes a functional PGHS-2 gene product.

As used herein, the term "recombinant DNA sequence" refers to a DNA sequence that has been derived or isolated from any source, that may be subsequently chemically altered, and later introduced into mammalian cells. An example of a recombinant DNA sequence "derived" from a source, would be a DNA sequence that is identified as a useful fragment within a given organism, and which is then chemically synthesized in essentially pure form. An example of such DNA sequence "isolated" from a source would be a DNA sequence that is excised or removed from said source by chemical means, e.g., by the use of restriction endonucleases, so that it can be further manipulated, e.g., amplified, for use in the invention, by the methodology of genetic engineering.

Therefore, "recombinant DNA sequence" includes completely synthetic DNA, semi-synthetic DNA, DNA isolated from biological sources, and DNA derived from introduced RNA. Generally, the recombinant DNA sequence is not originally resident in the genotype which is the recipient of the DNA sequence, or it is resident in the genotype but is not expressed.

The isolated recombinant DNA sequence used for transformation herein may be circular or linear, double-stranded or single-stranded. Generally, the DNA sequence is chimeric linear DNA, or is a plasmid or viral expression vector, that can also contain coding regions flanked by regulatory sequences which promote the expression of the recombinant DNA present in the resultant cell line. For example, the recombinant DNA sequence may itself comprise or consist of a promoter that is active in mammalian cells, or may utilize a promoter already present in the genotype that is the transformation target. Such promoters include the CMV promoter depicted in FIG. 4, as well as the SV40 late promoter and retroviral LTRs (long terminal repeat elements).

The general methods for constructing recombinant DNA which can transform target cells are well known to those skilled in the art, and the same compositions and methods of construction may be utilized to produce the DNA useful herein. For example, J. Sambrook et al., Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Laboratory Press (2d ed., 1989), provides suitable methods of construction.

Aside from recombinant DNA sequence that serve as transcription units for PGHS-1, PGHS-2 or other portions thereof, a portion of the recombinant DNA may be untranscribed, serving a regulatory or a structural function.

The recombinant DNA sequence to be introduced into the cells further will generally contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of transformed cells. Alternatively, the selectable marker may be carried on a separate piece of DNA and used in a co-transformation procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in mammalian cells. Useful selectable markers are well known in the art and include, for example, anti-biotic and herbicide resistance genes.

Sources of DNA sequences useful in the present invention include Poly-A RNA from mammalian cells, from which the about 4 kb mRNA encoding PGHS-2 can be derived and used for the synthesis of the corresponding cDNA by methods known to the art. Such sources include the lambda ZAP II (Stratagene) library of size fractionated poly-A RNA isolated from C127 murine fibroblasts treated with serum and cycloheximide as described by O'Banion et al., 1991, J. Biol. Chem., 266:23261-23267. Xie et al. obtained mRNA encoding chicken PGHS-2 as described in 1991, Proc. Nat'l. Acad. Sci. USA, 88:2692-2696. Sources of human mRNA encoding PGHS-2 include RNA from human monocytes treated with interleukin-1 and cycloheximide, in accord with O'Banion et al., 1992, Proc. Nat'l. Acad. Sci. USA, 89:4888-4892. Sources of human mRNA encoding PGHS-1 are also well known to the art.

Selectable marker genes encoding enzymes which impart resistance to biocidal compounds are listed in Table 1, below.

TABLE 1 ______________________________________ Selectable Marker Genes Resistance Confers Gene or Enzyme Resistance to: Reference ______________________________________ Neomycin phospho- G-418, neomycin, Southern et transferase (neo) kanamycin al., 1982, J. Mol. Appl. Gen., 1:327-341 Hygromycin Hygromycin B Shimizu et al., 1986, phosphotrans- Mol. Cell Biol., ferase (hpt or 6:1074-1087 hyg) Dihydrofolate Methotrexate Kwok et al., 1986, reductase (dhfr) Proc. Nat'l. Acad. Sci. USA, 4552-4555 Phosphinothricin Phosphinothricin DeBlock et al., 1987, acetyltransferase EMBO J., 6:2513-2518 (bar) 2,2-Dichloropro- 2-2,Dichloropro- Buchanan-Wollaston et pionic acid pionic acid al., 1989, J. Cell. dehalogenase (Dalapon) Biochem., Supp. 13D, 330 Acetohydroxyacid Sulfonylurea, Anderson et al. (U.S. synthase imidazolinone and Pat. No. triazolopyrimidine 4,761,373); G.W. herbicides Haughn et al., 1988 Mol. Gen. Genet., 211:266-271 5-Enolpyruvyl- Glyphosate Comai et al., 1985 shikimatephos- Nature, 317:741-744 phate synthase (aroA) Haloarylnitrilase Bromoxynil Stalker et al., published PCT appln. W087/04181 Acetyl-coenzyme A Sethoxydim, Parker et al., 1990 carboxylase haloxyfop Plant Physiol., 92:1220 Dihydropteroate Sulfonamide Guerineau et al., synthase (sul I) herbicides 1990, Plant Molec. Biol., 15:127-136 32 kD photosystem Triazine Hirschberg et al., II polypeptide herbicides 1983, Science, (psbA) 222:1346-1349 Anthranilate 5-Methyltryptophan Hibberd et al. (U.S. synthase Pat. No. 4,581,847) Dihydrodipicolin- Aminoethyl Glassman et al., ic acid synthase cysteine published PCT (dap A) application No. W089/11789 ______________________________________

Reporter genes are used for identifying potentially transformed cells and for evaluating the functionality of regulatory sequences. Reporter genes which encode for easily assayable marker proteins are well known in the art. In general, a reporter gene is a gene which is not present in or expressed by the recipient organism or tissue and which encodes a protein whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Preferred genes includes the chloramphenicol acetyl transferase gene (cat) from Tn9 of E. coli, the beta-galactosidase gene of E. coli, the beta-glucuronidase gene (gus) of the uidA locus of E. coli, and the luciferase gene from firefly Photinus pyralis. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.

Other elements such as introns, enhancers, polyadenylation sequences and the like, may also be a part of the recombinant DNA sequence. Such elements may or may not be necessary for the function of the DNA, but may provide improved expression of the DNA by affecting transcription, stability of the mRNA, or the like. Such elements may be included in the DNA as desired to obtain the optimal performance of the transforming DNA in the cell.

The recombinant DNA sequence can be readily introduced into the target cells by transfection with an expression vector, such as a viral expression vector, comprising cDNA encoding PGHS-2 or PGHS-1 by the modified calcium phosphate precipitation procedure of Chen et al., 1987, Mol. Cell. Biol., 7:2745-2752. Transfection can also be accomplished by other methods, including lipofection, using commercially available kits, e.g., provided by Life Technologies.

In a preferred embodiment of the invention, the cell lines of the invention are able to express a stable PGHS-2 gene product or analog, homologue, or deletion thereof after several passages through cell culture. While the instability of the PGHS-2 gene product has been hypothesized to be attributable to the 3' non-coding region of the PGHS-2 mRNA, it has been found that even cell lines which do not include this 3' region are often unable to express a stable PGHS-2 gene product for more than approximately five (5) passages in cell culture. The cell lines of the invention, however, are able to continue to produce a stable PGHS-2 gene product even after at least 5, 10, 15, or 20 passages through cell culture. The cell lines of the invention were selected by the single cell cloning of those cells which were able to continue to stably produce PGHS-2 even after the mere five passages through cell culture which defined the expressing limit of the cells of the prior art.

5.2.4. PURIFICATION OF THE PGHS-2 GENE PRODUCT

Once a cell that produces high levels of biologically active PGHS-2 is identified, the cell may be clonally expanded and used to produce large quantities of the enzyme, which may be purified using techniques well-known in the art including, but not limited to, immunoaffinity purification, chromatographic methods including high performance liquid chromatography and the like. Where the enzyme is secreted by the cultured cells, PGHS-2 may be readily recovered from the culture medium.

Where the PGHS-2 coding sequence, or fragment thereof, has been engineered to encode a cleavable fusion protein, the purification of the PGHS-2 gene product, or fragment thereof, may be readily accomplished using affinity purification techniques. For example, an antibody specific for the heterologous peptide or protein can be used to capture the durable fusion protein; for example, on a solid surface, a column etc. The PGHS-2 moiety can be released by treatment with the appropriate enzyme that cleaves the linkage site. cDNA construction using the polymerase chain reaction accompanied by transfection and purification of the expressed protein permits the isolation of sufficient quantities of PGHS-2 for characterization of the enzyme's physical and kinetic properties. Using site-directed mutagenesis or naturally occurring mutant sequences, this system provides a reasonable approach to determine the effects of the altered primary structure on the function of the protein. Fusion constructs of the PGHS-2 protein domain with the marker peptide preceding the amino terminus of PGHS-2 or following the carboxy terminus of PGHS-2 may also be engineered to evaluate which fusion construct will interfere the least, if at all, with the protein's biologic function and the ability to be purified.

Using this aspect of the invention, any cleavage site or enzyme cleavage substrate may be engineered between the PGHS-2 sequence and a second peptide or protein that has a binding partner which could be used for purification, e.g, any antigen for which an immunoaffinity column can be prepared.

5.3. ANTIBODIES TO THE PGHS-2 GENE PRODUCT

For the production of antibodies, various host animals may be immunized by injection with the PGHS-2 gene product, or a portion thereof including, but not limited to, portions of the PGHS-2 gene product in a recombinant protein. Such host animals may include but are not limited to rabbits, mice, and rats, to name but a few. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.

Monoclonal antibodies may be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein, 1975, Nature, 256:495-497, the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today, 4:72, Cote et al., 1983, Proc. Natl. Acad. Sci., 80:2026-2030) and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci., 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda et al., 1985, Nature, 314:452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies specific to one of the binding partners.

Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab').sub.2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab').sub.2 fragments. Alternatively, Fab expression libraries may be constructed (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

5.4. DIAGNOSTICS

The DNA of the invention encoding the PGHS-2 gene or homologues, analogues, or fragments thereof may be used in accordance with the invention to diagnose disease states which are phenotypic of an aberrant PGHS-2 genotype or of aberrant PGHS-2
expression.

For example, but not by way of limitation, in pulmonary fibrosis from radiation or chronic pulmonary disease, and in the skin disorder scleroderma, only a small percentage of those afflicted respond to glucocorticoids, McCune et al., 1994, Curr. Opin. Rheum., 6(3):262-272; Muir and Benhamou, 1994, [French] Annales de Med. Intern., 145 (Suppl):34-36; Labrune and Huchon, 1991, [French] Revue du Praticien, 41(14):1275-1277. These two disorders have been associated, Steen et al., 1994, Arthritis & Rheum., 37(9):1290-1296; Wells et al., 1994, Am. J. Resp. & Crit. Care Med., 149(6) 1583-1590. Therefore, both these disorders may be characterized by a constitute over expression of PGHS-2 or by excessive longevity of the PGHS-2 message which, in either case, is not diminished by glucocorticoid.

By way of another example, but not by way of limitation, many tumors may be characterized by a lack of, or excess of, PGHS-2 activity which may stem from mutations in the PGHS-2 coding or regulatory sequence.

In both of the examples above, afflicted cells, tissue sections, or biopsy specimens may be screened with the PGHS-2 DNA sequences of the invention and isolated PGHS-2 sequenced to determine which mutations in PGHS-2 are associated with the diseases. The DNAs of the invention may also be used to determine whether an individual carries an aberrant PGHS-2 gene.

In a specific embodiment of the invention, the detection of the aberrant PGHS-2 DNA is conducted by PCR amplification from a small tissue sample. Detection may also be via in situ hybridization or immunocytochemistry of pathology or biopsy specimens.

5.5. GENE THERAPIES BASED ON THE PGHS-2 GENE

A variety of gene therapy approaches may be used in accordance with the invention to modulate expression of the PGHS-2 gene in vivo. For example, antisense DNA molecules may be engineered and used to block translation of PGHS-2 mRNA in vivo. Alternatively, ribozyme molecules may be designed to cleave and destroy the PGHS-2 mRNAs in vivo. In another alternative, oligonucleotides designed to hybridize to the 5' region of the PGHS-2 gene (including the region upstream of the coding sequence) and form triple helix structures may be used to block or reduce transcription of the PGHS-2 gene. In yet another alternative, nucleic acid encoding the full length wild-type PGHS-2 message may be introduced in vivo into cells which otherwise would be unable to produce the wild-type PGHS-2 gene product in sufficient quantities or at all.

In a preferred embodiment, the antisense, ribozyme and triple helix nucleotides are designed to inhibit the translation or transcription of PGHS-2 with minimal effects on the expression of PGHS-1. To accomplish this, the oligonucleotides used should be designed on the basis of relevant sequences unique to PGHS-2; i.e., those sequences found in PGHS-2 and not in PGHS-1.

For example, and not by way of limitation, the oligonucleotides should not fall within those region where the nucleotide sequence of PGHS-2 is most homologous to that of PGHS-1 (see FIGS. 10A-10D), or the PGHS-2 sequence which is shown in FIG. 10
to be identically conserved between PGHS-1 and PGHS-2. These sequences include the following regions in the nucleotide sequence of PGHS-2:

427-457

555-601

624-646

822-901

975-997

1116-1154

1251-1282

1596-1634

Instead, it is preferred that the oligonucleotides fall ithin the following regions of PGHS-2, which are shown in FIGS. 10A-10D to diverge from the sequence of PGHS-1. These sequences include the following regions in the nucleotide sequence of PGHS-2:

171-254

299-340

486-512

602-623

1214-1250

1283-1346

1521-1580

1718-1834

In the case of antisense molecules, it is preferred that the sequence be chosen from the list above. It is also preferred that the sequence be at least 18 nucleotides in length in order to achieve sufficiently strong annealing to the target mRNA sequence to prevent translation of the sequence. Izant and Weintraub, 1984, Cell, 36:1007-1015; Rosenberg et al., 1985, Nature, 313:703-706.

In the case of the "hammerhead" type of ribozymes, it is also preferred that the target sequences of the ribozymes be chosen from the list above. Ribozymes are RNA molecules which possess highly specific endoribonuclease activity. Hammerhead ribozymes comprise a hybridizing region which is complementary in nucleotide sequence to at least part of the target RNA, and a catalytic region which is adapted to cleave the target RNA. The hybridizing region contains nine (9) or more nucleotides. Therefore, the hammerhead ribozymes of the present invention have a hybridizing region which is complementary to the sequences listed above and is at least nine nucleotides in length. The construction and production of such ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, 1988, Nature, 334:585-591.

The ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes") such as the one which occurs naturally in Tetrahymena Thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature, 324:429-433; published International patent application No. WO 88/04300 by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216). The Cech endoribonucleases have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place. The invention encompasses those Cech-type ribozymes which target eight base-pair active site sequences that are present in PGHS-2 but not PGHS-1.

In the case of oligonucleotides that hybridize to and form triple helix structures at the 5' terminus of the PGHS-2 gene and can be used to block transcription, it is preferred that they be complementary to those sequences in the 5' terminus of PGHS-2 which are not present in PGHS-1 (see FIGS. 11A-11C). Because of the lack of homology between these regions of PGHS-2 and PGHS-1, any sequence sufficiently long to hybridize to the PGHS-2 promoter will not hybridize to the promoter of PGHS-1. However, it is preferred that the sequences not include those regions of the PGHS-2 promoter which are even slighly homologous to that of PGHS-1. These slightly homologous sequences include the following regions in the nucleotide sequence of the PGHS-2
promoter:

382-438

669-696

797-826

856-885

980-1008

1142-1170

1204-1252

1863-1898

2013-2101

2126-2175

2356-2396

The foregoing compounds can be administered by a variety of methods which are known in the art including, but not limited to the use of liposomes as a delivery vehicle. Naked DNA or RNA molecules may also be used where they are in a form which is resistant to degradation such as by modification of the ends, by the formation of circular molecules, or by the use of alternate bonds including phosphothionate and thiophosphoryl modified bonds. In addition, the delivery of nucleic acid may be by facilitated transport where the nucleic acid molecules are conjugated to poly-lysine or transferrin. Nucleic acid may also be transported into cells by any of the various viral carriers, including but not limited to, retrovirus, vaccinia, AAV, and adenovirus.

Alternatively, a recombinant nucleic acid molecule which encodes, or is, such antisense, ribozyme, triple helix, or PGHS-2 molecule can be constructed. This nucleic acid molecule may be either RNA or DNA. If the nucleic acid encodes an RNA, it is preferred that the sequence be operatively attached to a regulatory element so that sufficient copies of the desired RNA product are produced. The regulatory element may permit either constitutive or regulated transcription of the sequence. In vivo, that is, within the cells or cells of an organism, a transfer vector such as a bacterial plasmid or viral RNA or DNA, encoding one or more of the RNAs, may be transfected into cells e.g. (Llewellyn et al., 1987, J. Mol. Biol., 195:115-123; Hanahan et al.
1983, J. Mol. Biol., 166:557-580). Once inside the cell, the transfer vector may replicate, and be transcribed by cellular polymerases to produce the RNA or it may be integrated into the genome of the host cell. Alternatively, a transfer vector containing sequences encoding one or more of the RNAs may be transfected into cells or introduced into cells by way of micromanipulation techniques such as microinjection, such that the transfer vector or a part thereof becomes integrated into the genome of the host cell.

5.6. DRUG SCREENING ASSAYS

The present invention provides a simple in vitro system for the screening of drug actions on both the constitutive and the inflammatory cyclooxygenase, which will be useful for the development of drugs that selectively inhibit inflammation without producing the side effects due to inhibition of constitutive prostaglandin production. Assays can be performed on living mammalian cells, which more closely approximate the effects of a particular serum level of drug in the body, or on microsomal extracts prepared from the cultured cell lines. Studies using microsomal extracts offer the possibility of a more rigorous determination of direct drug/enzyme interactions.

The PGHS-2-synthesizing cell lines are useful for evaluating the activity of potential bioactive agents on the inflammatory cyclooxygenase, since the elevated levels of prostaglandins that are a primary hallmark of inflammation and account for much of the adverse effects of inflammation, result from increases in the level of PGHS-2, rather than in changes in constitutively expressed cyclooxygenase, PGHS-1.

The present invention also provides a second mammalian cell line which contains a chromosomally integrated, recombinant DNA sequence, wherein said DNA sequence expresses mammalian, preferably human, PGHS-1, and wherein said DNA sequence does not express PGHS-2, and wherein said cell line also preferably does not express autologous PGHS-1 or PGHS-2 activity. This second cell line is also preferably a primate, murine or human cell line.

Thus, the present invention also provides a method to evaluate the relative inhibitory activity of a compound to selectively inhibit PGHS-2 versus PGHS-1, and thus to specifically inhibit the elevated prostaglandin synthesis that occurs in inflamed mammalian tissues, preferably human tissues, or in other physiological or pathological conditions in a mammalian host, preferably a human host, in which the PGHS-2 is elevated and the constitutive PGHS-1 is not. This assay comprises contacting the present PGHS-2-expressing transgenic cell line or a microsomal extract thereof with a preselected amount of the compound in a suitable culture medium or buffer, adding arachidonic acid to the mixture, and measuring the level of synthesis of a PGHS-mediated arachidonic acid metabolite, i.e., thromboxane synthesis, prostaglandin synthesis, e.g., the synthesis of PGE.sub.2, or the synthesis of any other metabolite unique to the cyclooxygenase pathway, by said cell line, or said microsomal extract, as compared to a control cell line or portion of microsomal extract in the absence of said compound. The compound can be evaluated for its ability to selectively inhibit PGHS-1 or PGHS-2 by performing a second assay employing the above-described steps, but substituting the PGHS-1-expressing transgenic cell line for the PGHS-2-expressing cell line of the invention.

More specifically, the present-invention provides a method of determining the ability of a compound to inhibit prostaglandin,synthesis catalyzed by PGHS-2 or PGHS-1 in mammalian cells comprising:

(a) adding a first preselected amount of said compound to a first transgenic mammalian cell line in culture medium, which cell line contains a chromosomally integrated, recombinant DNA sequence, wherein said DNA sequence expresses mammalian PGHS-2, and wherein said DNA sequence does not express PGHS-1, and wherein said cell line does not express autologous PGHS-1 or PGHS-2 activity;

(b) adding arachidonic acid to said culture medium;

(c) measuring the level of a PGHS-mediated arachidonic acid metabolite synthesized by said first cell line;

(d) comparing said level with the level of said metabolite synthesized by said first cell line in the absence of said compound;

(e) adding a second preselected amount of said compound to a second transgenic mammalian cell line in culture medium, which cell line contains chromosomally integrated, recombinant DNA sequence, wherein said DNA sequence expresses mammalian PGHS-1, and wherein said DNA sequence does not express PGHS-2, and wherein said cell line does not express autologous PGHS-1 or PGHS-2 activity;

(f) adding arachidonic acid to said culture medium of step (e);

(g) measuring the level of a PGHS-mediated arachidonic acid metabolite synthesized by said second cell line; and

(h) comparing said level with the level of said metabolite synthesized by said second cell line in the absence of said compound.

The invention also relates to methods for the identification of genes, termed "pathway genes", which are associated with the PGHS-2 gene product or with the biochemical pathways which extend therefrom. "Pathway gene", as used herein, refers to a gene whose gene product exhibits the ability to interact with the PGHS-2 gene product.

Any method suitable for detecting protein-protein interactions may be employed for identifying pathway gene products by identifying interactions between gene products and the PGHS-2 gene product. Such known gene products may be cellular or extracellular proteins. Those gene products which interact with such known gene products represent pathway gene products and the genes which encode them represent pathway genes.

Among the traditional methods which may be employed are co-immunoprecipitation, crosslinking and co-purification through gradients or chromatographic columns. Utilizing procedures such as these allows for the identification of pathway gene products. Once identified, a pathway gene product may be used, in conjunction with standard techniques, to identify its corresponding pathway gene. For example, at least a portion of the amino acid sequence of the pathway gene product may be ascertained using techniques well known to those of skill in the art, such as via the Edman degradation technique (see, e.g., Creighton, 1983, Proteins: Structures and Molecular Principles, W. H. Freeman & Co., New York, pp.34-49). The amino acid sequence obtained may be used as a guide for the generation of oligonucleotide mixtures that can be used to screen for pathway gene sequences. Screening made be accomplished, for example by standard hybridization or PCR techniques. Techniques for the generation of oligonucleotide mixtures and screening are well-known. (See, e.g., Ausubel et al., eds., 1987-1993, Current Protocols in Molecular Biology, John Wiley & Sons, Inc. New York, and PCR Protocols: A Guide to Methods and Applications, 1990, Innis, M. et al., eds. Academic Press, Inc., New York).

Additionally, methods may be employed which result in the simultaneous identification of pathway genes which encode the protein interacting with the PGHS-2 gene product. These methods include, for example, probing expression libraries with labeled protein known or suggested to be involved in cardiovascular disease, using this protein in a manner similar to the well known technique of antibody probing of .lambda.gt11 libraries.

One such method which detects protein interactions in vivo, the two-hybrid system, is described in detail for illustration only and not by way of limitation. One version of this system has been described (Chien et al., 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582) and is commercially available from Clontech (Palo Alto, Calif.).

Briefly, utilizing such a system, plasmids are constructed that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein fused to a known protein, and the other consists of the activator protein's activation domain fused to an unknown protein that is encoded by a cDNA which has been recombined into this plasmid as part of a cDNA library. The plasmids are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., lacZ) whose regulatory region contains the activator's binding sites. Either hybrid protein alone cannot activate transcription of the reporter gene: the DNA-binding domain hybrid because it does not provide activation function and the activation domain hybrid because it cannot localize to the activator's binding sites. Interaction of the two proteins reconstitutes the functional activator protein and results in expression of the reporter gene, which is detected by an assay for the reporter gene product.

The two-hybrid system or related methodology may be used to screen activation domain libraries for proteins that interact with the PGHS-2 gene product, herein also called the known "bait" gene protein. Total genomic or cDNA sequences may be fused to the DNA encoding an activation domain. Such a library and a plasmid encoding a hybrid of the bait gene protein fused to the DNA-binding domain may be cotransformed into a yeast reporter strain, and the resulting transformants may be screened for those that express the reporter gene. These colonies may be purified and the library plasmids responsible for reporter gene expression may be isolated. DNA sequencing may then be used to identify the proteins encoded by the library plasmids.

For example, and not by way of limitation, the bait gene may be cloned into a vector such that it is translationally fused to the DNA encoding the DNA-binding domain of the GAL4 protein.

A cDNA library of the cell line from which proteins that interact with bait gene are to be detected can be made using methods routinely practiced in the art. According to the particular system described herein, for example, the cDNA fragments may be inserted into a vector such that they are translationally fused to the activation domain of GAL4. This library may be co-transformed along with the bait gene-GAL4 fusion plasmid into a yeast strain which contains a lacZ gene driven by a promoter which contains the GAL4 activation sequence. A cDNA encoded protein, fused to the GAL4 activation domain, that interacts with bait gene will reconstitute an active GAL4 protein and thereby drive expression of the lacZ gene. Colonies which express lacZ may be detected by their blue color in the presence of X-gal. The cDNA may then be purified from these strains, and used to produce and isolate the bait gene-interacting protein using techniques routinely practiced in the art.

Once a pathway gene has been identified and isolated, it may be further characterized as, for example, discussed herein.

The proteins identified as products of pathway genes may be used to modulate PGHS-2 gene expression, as defined herein, or may themselves be targets for modulation to in turn modulate symptoms associated with PGHS-2 expression.

5.7. COMPOUNDS IDENTIFIED IN THE SCREENS

The compounds identified in the screen will demonstrate the ability to selectively modulate the expression of PGHS-2. These compounds include but are not limited to nucleic acid encoding PGHS-2 and homologues, analogues, and deletions thereof, as well as antisense, ribozyme, triple helix, antibody, and polypeptide molecules and small inorganic molecules.

5.8. PHARMACEUTICAL FORMULATIONS AND ROUTES OF ADMINISTRATION

Any of the identified compounds can be administered to an animal host, including a human patient, by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) at doses therapeutically effective to treat or ameliorate a variety of disorders, including those characterized by insufficient, aberrant, or excessive PGHS-2 activity. A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms associated with such disorders. Techniques for formulation and administration of the compounds of the instant application may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition.

A number of disorders in addition to inflammation have been characterized by insufficient, aberrant, or excessive PGHS-2 activity. In addition, several physiological states which may, from time to time be considered undesired, are also associated with PGHS-2 activity. By way of example, but not by way of limitation, such disorders and physiological states which may be treated with the compounds of the invention include but are not limited to neurologic disorders such as Alzheimer's disease, stroke, and acute head injury; colorectal carcinoma; ovulation; preterm labor; endometriosis; implantation; and pulmonary fibrosis.

Pathological features of Alzheimer's Disease (AD) include neuritic amyloid plaques, neurofibrillary tangles, neuronal cell loss, loss of synapses, and marked gliosis. Because they are unique features of the disease, many investigators have focused on the etiology and effects of amyloid plaques and neurofibrillary tangles. However, the significant gains made in understanding these neuropathologic markers have provided few clues regarding treatment of AD. In contrast, recent findings suggest that the "inflammatory processes" associated with gliosis represent a potential target for therapeutic intervention in the disease. In particular, Joe Rogers and colleagues have presented both retrospective and prospective evidence that non-steroidal anti-inflammatory agents can significantly slow the progress of AD (McGeer and Rogers, 1992, Neurology, 42:447-449; Rogers et al., 1993, Neurology, 43:1609-1611). Indeed, these results have prompted the initiation of anti-inflammatory therapy trials for AD.

Evidence for an "inflammatory component" to gliosis in AD includes increased expression of proinflammatory cytokines such as IL-1.beta. and TNF.alpha. (Griffin et al., 1989, Proc. Nat'l. Acad. Sci. USA, 88:7611-7615; Dickson et al., 1993, Glia, 7:75-83; Lapchak and Araujo, 1993, Soc. Neurosci. Abstr., 19:191) and the presence of activated complement components (McGeer et al., 1989, Neurosci. Let., 107: 341-346; Johnson et al., 1992, Neurobiol. Aging, 13:641-648; Walker and McGeer,
1992 Mol. Brain Res., 14:109-116). It should be noted that gliosis and the presence of proinflammatory cytokines with the potential to activate PGHS-2 are not limited to AD. Rather, they are a feature of many insults to and disease of the central nervous system including (but not limited to) acute head injury, stroke, spinal cord injury, multiple sclerosis, HIV infection of the brain and other viral encephalopathies, and most neurodegenerative disorders (e.g. Huntington's disease, Parkinson's disease, and amyotrophic lateral sclerosis).

PGHS-2 is expressed in cultured murine and rat astrocytes, and is strongly up-regulated by treatment with proinflammatory cytokines including IL-1.beta. and TNF.alpha. (O'Banion et al., 1994, Soc. Neurosci. Abstr.). The induction of PGHS-2
is rapid with mRNA levels peaking at 2 h. Concomitant increases in prostaglandin production are also observed. The fact that induced cyclooxgenase activity is blocked by NS-398, a specific inhibitor of PGHS-2, confirms that induction of PGHS-2 is responsible for increased prostaglandin production in cytokine-treated astrocytes. As in other cell types, glucocorticoid hormones suppress the induction of PGHS-2 by IL-1.beta..

Other investigators have confirmed that PGHS-2 is expressed in the brain (Yamagata et al., 1993, Neuron, 11:371-386). In these studies, the brains of rats subjected to electroconvulsive shock showed dramatic increases in the levels of PGHS-2
expression in neurons of the cerebral cortex and hippocampus. The authors further demonstrated that synaptic activation led to induction of PGHS-2 mRNA, suggesting that expression of this molecule plays a significant role (as yet undefined) in neuronal communication and/or function. In preliminary in situ hybridization studies it has been confirmed that PGHS-2 is expressed in human brain neurons (Chang et al., 1995, Soc. Neurosci. Ann. Mtg. San Diego, Submitted).

Similar to their proven therapeutic benefits in peripheral inflammation, it is proposed that the efficacy of nonsteroidal anti-inflammatory therapy in the treatment of AD is due to the inhibition of PGHS-2 activity in "inflamed" brain tissue. This therapeutic approach has the potential to benefit a multitude of neurological diseases and injuries with a prominent degree of glial activation. Development of selective inhibitors of human PGHS-2 which specifically target the central nervous system (i.e. that are designed to easily cross the blood-brain barrier and even accumulate in the brain) may prove much more efficacious than current NSAIDS for the treatment of AD and other neurologic disorders.

Colorectal carcinoma is a leading cause of death in westernized countries. Prostaglandins have been correlated with carcinogenesis in general and more specifically with colorectal cancer, Marnett, 1992, Cancer Research, 52:5575-5589. In several clinical trials, aspirin use was associated with decreased colon tumor growth and death, Thun et al., 1991, N. Engl. J. Med., 325:1593-6; Kune, et al., 1988, Cancer Res., 48:439-404. Sulindac, another cyclooxygenase inhibitor, has been demonstrated to cause colon polyp regression in patients with familial polyposis, Waddell and Loughry, 1983, J. Surg. Oncol., 24:83-87. These NSAIDS are able to inhibit both PGHS-1 and -2. Discovery of the gene for PGHS-2 makes clarification of the relative contribution or role in colon cancer possible. PGHS-2 is an immediate early gene suggesting its likely participation in regulating growth. The decreased tumor growth by aspirin is likely through action on PGHS-2. If PGHS-2 is directly implicated then specific inhibition of this enzyme may result in tumor suppression. Discovery of the PGHS-2 gene allows for further clarification of this contribution. Additionally, if inhibition is therapeutic then specific drugs that inhibit PGHS-2 can be obtained that would be ingested and directly act at the mucosal and have limited systemic absorption. In the case of familial polyposis, gene therapy may play an important therapeutic role.

Ovulation has in a broad sense can be viewed as an inflammatory process initiated by the LH surge during the menstrual cycle, Espey, 1980, Biol. Reprod, 22:73-106. NSAIDs have been shown to inhibit ovulation in a number of model systems, Espey,
1982, Prostaglandin, 23:329-335. By inhibiting prostaglandin formulation and interrupting the inflammatory response ovulation is halted. It has been demonstrated that PGHS-2 is specifically stimulated by LH in granulosa cells at the time of ovulation and likely the target of NSAIDs that results in inhibition of ovulation, Sirois and Richards, 1992, J. Biol. Chem., 267:6382-6388. Knowing the gene sequence and protein product not only provides the ability to further study this process but provides a specific target for contraception. PGHS-2 specific drugs would allow inhibition without effecting the prostaglandin production by PGHS-1 which is protective to GI mucosa as well as involved with kidney function and many other homeostatic mechanisms.

Preterm labor is a significant clinical problem. Current available drugs (tocolytics) are able to postpone labor but often are not able to stop labor definitively. Prostaglandins play an important role in induction of labor although their exact contribution and mechanism are yet to be clearly defined, Kelly, 1994, Endocrine Reviews, 15(5):684-706. With the discovery of PGHS-2 a better understanding of prostaglandin regulation in the fetus and uterus can be understood. Current medications used for preterm labor (tocolytics) work by blocking Ca flux thereby interfering with myometrium contraction. Common tocolytics include magnesium sulfate, .beta.-adrenergic receptor agonists, calcium channel blockers and oxytocin antagonists. Indomethacin has also been used effectively but raises concern with premature closure of the ductus arteriosus of the fetus. Closer examination of PGHS-1 and PGHS-2 in these roles may provide opportunities for specific intervention.

Recognition of preterm labor prior to cervical changes is difficult but also the point at which tocolytic agents are most effective. It is known that prostaglandins are intimately involved in myometrium contraction of normal labor, Williams Obstetrics, Cunningham, MacDonald, Gant, Leveno, and Gilstrap (eds) Williams Obstetrics 19th Ed. Appleton and Lange, Norwalk Conn., 1993. It may be possible to evaluate increased PGHS-2 expression and true labor prior to cervical changes. If safe sampling of the site of expression can be done then PCR methods may be able to provide a timely answer to whether the painful uterine contractions are Braxton-Hicks or true labor.

Dysmenorrhea and endometriosis are common, painful problematic conditions for women. It is well known that NSAIDs are extremely effective at treating dysmenorrhea and endometriosis pain by inhibiting prostaglandin production. It is highly likely that the hormones responsible for the cycle of dysmenorrhea and endometriosis also regulates PGHS-2 expression. Inhibition at the protein or genetic level could enhance specific treatment for dysmenorrhea and endometriosis.

Prostaglandin formation is also part of implantation. Manipulation of PGHS-2 expression may provide a means for induction of abortion.

PGHS-2 may play an important role in the lung pathology of cystic fibrosis. It has been demonstrated that high-dose ibuprofen slows the progression of lung disease in this patient population, Konstan, et al., 1995, N. Engl. J. Med., 332
(13):848-854. Lung disease results more from the inflammatory response than by the colonization of bacteria. Utilization of inhalers can directly deliver medication to the site of inflammation. This may provide a logical disease process to attempt anti-sense, ribozyme or triple helix gene therapy aimed at inhibiting PGHS-2 expression.

Besides attempts to inhibit cell growth by inhibiting PGHS-2 there may be certain circumstances whereby growth stimulation is desired as in tissue repair. Determination of the tissue specific regulation of PGHS-2 (studies which require gene sequence information) may lead to the ability to specifically up regulate PGHS-2 in particular cell types (i.e. fibroblasts, neurons). Additionally genetic constructs which will only be activated in particular cell types because of promoter construction could be developed.

Other options may include direct delivery of enzyme which has been produced and purified by genetic means using the cloned gene. Recombinant protein would also greatly facilitate investigation into the distinctions between the enzymes (PGHS-1
and PGHS-2) and the byproducts they produce.

Other isoforms may exist and may be cloned utilizing PGHS-2 sequence.

The compounds of the invention may be designed or administered for tissue specificity. If the compound comprises a nucleic acid molecule, including those comprising an expression vector, it may be linked to a regulatory sequence which is specific for the target tissue, such as the brain, skin, joints, bladder, kidney, liver, ovary, etc. by methods which are known in the art including those set forth in Hart, 1994, Ann. Oncol., 5 Suppl 4: 59-65; Dahler et al., 1994, Gene, 145: 305-310; DiMaio et al., 1994, Surgery, 116:205-213; Weichselbaum et al., Cancer Res., 54:4266-4269; Harris et al., 1994, Cancer, 74 (Suppl. 3):1021-1025; Rettinger et al., Proc. Nat'l. Acad. Sci. USA, 91:1460-1464; and Xu et al, Exp. Hematol., 22:223-230; Brigham et al., 1994, Prog. Clin. Biol. Res., 388:361-365. The compounds of the invention may be targeted to specific sites of inflammation by direct injection to those sites, such as joints, in the case of arthritis. Compounds designed for use in the central nervous system should be able to cross the b