WO2001023562A2 - Osteoprotegerin regulatory region - Google Patents
Osteoprotegerin regulatory region Download PDFInfo
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- WO2001023562A2 WO2001023562A2 PCT/US2000/026497 US0026497W WO0123562A2 WO 2001023562 A2 WO2001023562 A2 WO 2001023562A2 US 0026497 W US0026497 W US 0026497W WO 0123562 A2 WO0123562 A2 WO 0123562A2
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- osteoprotegerin
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- opg
- regulatory region
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to the fields of medical therapeutics and diagnostics. More particularly, the present invention relates to the development of therapeutic drugs, treatment methods, and diagnostic methods in the area of skeletal and other diseases, such as arterial and immune diseases, associated with the over- or under-expression of osteoprotegerin. Among its many aspects, the present invention provides assay methods useful in developing therapeutic drugs for the treatment of such diseases.
- Bone growth, development and maintenance in mammals is a highly regulated process.
- the level of bone mass is dependent on the balance of bone formation and resorption.
- this balance involves the coordinate regulation and interaction of its component cell types: bone forming cells, called osteoblasts, and bone-resorbing cells, called osteoclasts.
- Osteoblasts are derived from mesenchymal stem cells and produce bone matrix during development, after bone injury and during bone remodeling.
- Osteoclasts the only cells that resorb bone, are derived from hematopoietic precursors, most likely of the monocyte/macrophage series (CFU-GM) .
- CFU-GM monocyte/macrophage series
- Osteoprotegerin a member of the TNF receptor family, inhibits osteoclast formation at an early stage of development. See Tsuda et al . , Biochem . Biophys . Res . Comm . , 234:137-142 (1998); Simonet et al . , Cell , 89:309-319 (1997); Morinaga et al . , Eur . J. Biochem . , 254:685-691 (1998). Over- expression of OPG in transgenic mice inhibits osteoclast formation, causing osteopetrosis . Similarly, treatment of ovarectomized (OVX) rats with OPG prevents bone loss. See Simonet et al .
- Osteoprotegerin was identified independently by a second group and called osteoclast inhibitory factor (OCIF) .
- OPF osteoclast inhibitory factor
- the ligand for OPG called osteoclast differentiation factor (ODF) or OPG ligand (OPGL) , or rank ligand (RANKL)
- OPF osteoclast differentiation factor
- OPGL OPG ligand
- RNKL rank ligand
- Osteoprotegerin is a secreted glycoprotein of the TNF receptor superfamily that has been shown to inhibit the development, activity, and survival of osteoclasts in vi tro, and bone resorption in vivo (Bucay et al . , Genes & Development , 12:1260-1268 (1998); Hakeda et al . , Biochem . Biophys . Res . Comm . , 251:796-801 (1998); Simonet et al . , Cell , 89:309-319 (1997); Akatsu et al . , Bone, 23:495-498 (1998);
- RANKL RANK ligand
- ODF Osteoclast Differentiation Factor
- TRANCE TNF-related activation-induced cytokine
- OPGL Osteoclast Differentiation Factor
- TRAP + multinucleated osteoclasts that are capable of resorbing bone in dentine slices.
- OPG acts as a decoy receptor and blocks RANKL-RANK interaction, resulting in inhibition of osteoclast differentiation.
- a number of hormones, growth factors, and cytokines have long been known to affect osteoclast differentiation and bone resorption indirectly by acting on osteoblasts.
- osteotropic agents namely parathyroid hormone (PTH), parathyroid hormone related protein (PTHrP), 1,25- (OH) 2 vitamin D3, TGF- ⁇ , IL-1, IL-11, prostaglandin E 2 (PGE 2 ), estradiol, and dexamethasone
- PTH parathyroid hormone
- PTHrP parathyroid hormone related protein
- PGE 2 prostaglandin E 2
- dexamethasone Karl et al . , FASEB J. , 12:845-854 (1998); Yasuda et al . , Proc . Natl . Acad . Sci . USA, 95:3597-3602 (1998); Hofbauer et al . , Biochem . Biophys . Res . Comm . , 250:776-781 (1998); Horwood et al .
- TGF- ⁇ l (referred to hereafter as TGF- ⁇ ) , the founding member of the multifunctional transforming growth factor (TGF) family of growth and differentiation factors, is produced by a number of cell types, including osteoblasts and stromal cells, and plays a major role in the regulation of bone formation and resorption (Centrella et al . , Endocrine Rev. , 15:27-39 (1994)) . It is stored in abundant amounts in bone, and is released from the bone matrix during osteoclastic bone resorption. It has been shown to stimulate the proliferation and differentiation of osteoblasts and to increase mineralized bone formation (Noda et al . , Endocrinology, 124:2991-2994 (1989)) .
- TGF- ⁇ l treatment resulted in enhanced osteoclast differentiation in hematopoietic cell cultures that were stimulated with RANKL and M-CSF (Sells Galvin et al . , Biochem . Biophys . Res . Comm . , 265:233-239 (1999)) .
- TGF- ⁇ has been shown to be a potent inhibitor of osteoclast differentiation and osteoclast survival (Murakami et al . , Biochem . Biophys . Res . Comm . , 252:747-752 (1998); Takai et al . , J. Biol . Chem.
- Effective screening assays evaluate test compounds by comparing: (1) a compound's effect on expression and (2) a baseline which represents a normal level of expression. Screening for compounds which affect a level of expression that is not the norm, such as that stimulated by a partial regulatory region, is of little value.
- an effective screening assay for identifying physiologically- relevant ligands requires a gene's complete regulatory region. Therefore, there exists a need to fully characterize the transcriptional regulatory region of the opg gene.
- the present invention provides the complete regulatory region for the opg gene.
- the present invention provides methods of identifying compounds that affect osteoclast formation, bone resorption, and immune responsiveness. In another aspect, the present invention provides methods of diagnosing bone, immune, and arterial diseases in a patient .
- DNA sequence that represents the complete regulatory region of the opg gene.
- DNA constructs containing the OPG regulatory region also are provided.
- host cells comprising such constructs, which cells are in vivo or in vi tro, also are encompassed by the present invention.
- the present invention provides a method for identifying a compound that affects osteoclast formation and/or bone resorption, comprising:
- the present invention provides a method for identifying a compound that affects osteoclast formation and/or bone resorption, comprising:
- the present invention provides methods for identifying a compound that affects OPG expression, in vivo or in vi tro, by using a host cell or cell-free system comprising a reporter gene operably linked to an OPG regulatory region of the invention, and assaying for evidence of expression of the reporter gene.
- the present invention provides a method for diagnosing bone disease in a patient, comprising comparing the DNA from bone cells of the patient with the presently disclosed DNA sequence.
- methods for diagnosing immune and arterial diseases in a patient comprising comparing the DNA from bone cells of the patient with a DNA sequence of the present invention.
- the present invention provides a method of identifying in a patient susceptibility, receptiveness, or responsiveness to drug therapy, comprising comparing the DNA from bone cells of said patient with a DNA sequence of the present invention.
- the present invention provides methods of identifying in a patient a predisposition to developing bone, arterial, or immune disease, comprising comparing the
- methods of modulating bone resorption and immune responsiveness, and of preventing arterial disease, in a patient comprising administering to the patient a DNA construct of the present invention, wherein the construct comprises a polynucleotide encoding osteoprotegerin.
- the present invention provides methods of modulating bone resorption and immune responsiveness, and of preventing arterial disease, in a patient, comprising administering to the patient pharmaceutically effective amount (s) of one or more compounds identified using a screening assay of the present invention.
- pharmaceutically effective amounts of compounds for in vivo use can be routinely determined.
- the present invention provides an isolated nucleic acid fragment comprising the transcriptional regulatory region of the human opg gene, a subfragment thereof, or a functional variant of either, exhibiting human opg gene transcriptional regulatory activity, excluding the opg protein coding region.
- the isolated nucleic acid fragment or subfragment thereof can comprise a nucleotide sequence selected from the group consisting of SEQ ID NO:l through SEQ ID NO: 11, or the complement of any one of said nucleotide sequences .
- the present invention provides an isolated nucleic acid fragment that hybridizes to the complement of a nucleotide sequence selected from the group consisting of SEQ ID N0:1 through SEQ ID NO: 11 in IX phosphate buffer comprising 0.
- the present invention provides an isolated nucleic acid fragment having a sequence identity in the range of from about 85% to about 99% compared to a nucleotide sequence selected from the group consisting of SEQ ID N0:1 through SEQ ID NO: 11, wherein said fragment exhibits human opg gene regulatory region transcriptional regulatory activity, with the proviso that said fragment comprises a novel nucleotide sequence, previously unknown at the time of filing of this application.
- the present invention provides a recombinant DNA construct comprising any of the preceding isolated nucleic acid fragments, subfragments, or functional variants of either.
- the recombinant DNA construct can further comprise a polynucleotide encoding a protein of interest, and, optionally, at least one translational regulatory region required for expression of said polynucleotide, wherein said polynucleotide encoding said protein of interest is operably linked for expression to said isolated nucleic acid fragment, subfragment, or functional variant, and to said translational regulatory region.
- the recombinant DNA construct can be an expression cassette or an expression vector.
- the present invention provides a cultured host cell comprising any one of the foregoing recombinant DNA constructs.
- the present invention provides the use of any of the foregoing isolated nucleic acid fragments, subfragments , or functional variants thereof, in an assay to identify an agonist or antagonist of osteoprotegerin expression.
- the present invention provides the use of any one of the foregoing isolated nucleic acid fragments, subfragments, or functional variants thereof for the manufacture of a composition for the diagnosis of a human susceptible to, predisposed to, or at increased risk for developing a symptom, condition, or disease caused by over- or under-expression of osteoprotegerin.
- the present invention provides a composition, comprising any of the foregoing isolated nucleic acid fragments or sub ragments , or functional variants thereof, recombinant DNA constructs, or host cells, and a carrier, diluent, or excipient .
- the present invention provides a pharmaceutical composition, comprising any of the foregoing isolated nucleic acid fragments or subfragments, or functional variants thereof, recombinant DNA constructs, or host cells, and a pharmaceutically acceptable carrier, diluent, or excipient .
- the present invention provides a method of identifying a compound that modulates expression of osteoprotegerin, comprising: (a) contacting:
- a host cell in which osteoprotegerin is normally expressed and (ii) a test compound, wherein said host cell comprises a DNA expression construct comprising a nucleic acid fragment or subfragment selected from the group consisting of SEQ ID NO : 1 through SEQ ID NO: 11, or a functional variant thereof, and a reporter polynucleotide operably linked thereto, and wherein said reporter polynucleotide is expressed; (b) determining the level of expression of said reporter polynucleotide in said host cell of step (a) ;
- step (c) determining the level of expression of said reporter polynucleotide in a host cell identical to said host cell of step (a) , wherein said identical host cell is not contacted with said test compound;
- the host cell can be selected from the group consisting of an osteoclast progenitor cell, an osteoclast, an osteoblast, a stromal cell, a chrondrocyte, a T-cell, and a fibroblast .
- the present invention provides a method of identifying a compound that modulates expression of osteoprotegerin, comprising:
- a plasmid comprising a nucleic acid fragment or subfragment selected from the group consisting of SEQ ID NO : 1 through SEQ ID NO: 11, or a functional variant thereof, and a reporter polynucleotide operably linked for expression thereto, and
- an effector plasmid comprising a nucleotide sequence that codes on expression for a factor required for osteoprotegerin expression, wherein both said reporter polynucleotide and said factor required for osteoprotegerin expression are expressed;
- step (b) determining the level of expression of said reporter polynucleotide in said host cell of step (a) ;
- step (c) determining the level of expression of said reporter polynucleotide in a host cell identical to said host cell of step (a) , wherein said identical host cell is not contacted with said test compound; and
- step (d) comparing the level of expression of said reporter polynucleotide in step (b) with the level of expression of said reporter polynucleotide in step (c) , wherein an increase or decrease in the level of expression of said reporter polynucleotide in step (b) compared to the level of expression of said reporter polynucleotide in step (c) identifies said test compound as a compound that modulates osteoprotegerin expression.
- the factor required for osteoprotegerin expression can be osteoblast specific transcription factor 2, and the effector plasmid can be pEF/Cbfal/myc/cyto, encoding Cbfal (osteoblast specific transcription factor 2) .
- the host cell can be selected from the group consisting of CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3 , and WI38 cell lines.
- expression of the reporter polynucleotide can be determined by measuring activity of the expressed reporter polynucleotide product, which can be beta-galactosidase .
- an increase in expression of the reporter polynucleotide in step (b) compared to that in step (c) identifies the test compound as an agonist of osteoprotegerin expression
- a decrease in expression of the reporter polynucleotide in step (b) compared to that in step (c) identifies the test compound as an antagonist of osteoprotegerin expression.
- the present invention provides an agonist or antagonist of osteoprotegerin expression identified by any of the foregoing methods .
- the present invention provides the use of an agonist or antagonist identified by any of the foregoing methods in the manufacture of a medicament for the treatment of a disease in a human caused by under-expression or over- expression, respectively, of osteoprotegerin.
- the present invention provides the use of a compound that modulates expression of osteoprotegerin in the manufacture of a medicament for the treatment of a disease in a human caused by abnormal expression of osteoprotegerin.
- a disease can be bone disease, arthritis, arterial disease abnormal immune function, abnormal lymph node development, or abnormal T- or B-cell function caused by abnormal expression of osteoprotegerin.
- the bone disease can be malignant bone disease, rheumatoid arthritis, osteoarthritis , elevated bone resorption, osteoporosis, Paget's disease of bone, hypercalcemia of malignancy, expansile osteolysis, or periodontal disease, and the compound can be an agonist of osteoprotegerin expression.
- the arterial disease can be arterial calcification, and the compound can be an agonist of osteoprotegerin expression.
- the compound can be an antagonist of osteoprotegerin expression.
- the compound can be identified by any of the methods discussed above.
- the human in the use according to any one of these indications, the human can be diagnosed as having a polymorphism or mutation at one or more nucleotide positions in the osteoprotegerin regulatory region in DNA thereof.
- the present invention provides a composition, comprising an agonist or antagonist of osteoprotegerin expression, and a carrier, diluent, or excipient.
- Such agonist or antagonist is preferably a novel compound unknown prior to the time of filing of this application, and one other than a hormone, growth factor, or cytokine such as osteoclast differentiation factor, TGF- ⁇ l, parathyroid hormone, parathyroid hormone related protein, l ⁇ , 25-dihydroxyvitamin D 3 , IL-l ⁇ , bone morphogenetic protein 2, TNF- ⁇ , TNF- ⁇ , IL-11, prostaglandin E 2 , estradiol, or dexamethasone .
- the agonist or antagonist can be identified by any one of the methods discussed above.
- the present invention provides a pharmaceutical composition or pharmaceutical pack, comprising an agonist or antagonist of osteoprotegerin expression, and a pharmaceutically acceptable carrier, diluent, or excipient.
- agonist or antagonist is preferably a novel compound unknown prior to the time of filing of this application, and one other than a hormone, growth factor, or cytokine such as osteoclast differentiation factor, TGF- ⁇ l, parathyroid GTGGAGTGCCGCAACCAGCTGGACAGCTCCGCGGTCGCCCCCCAC V E C R N Q L D S S A V A P H 1441
- the comparison can be conducted using nucleotide sequence analysis or nucleic acid hybridization analysis.
- the present invention provides a method of identifying a human subject or patient at increased risk for having an altered susceptibility or predisposition to developing a bone disease, cartilage disease, immune disease, arterial disease, or other disease caused by abnormal osteoprotegerin expression, comprising comparing the nucleotide sequence of the osteoprotegerin regulatory region of the opg gene in DNA from said subject or patient with a nucleotide sequence selected from the group consisting of SEQ ID NO:l through SEQ ID NO: 11, wherein any difference in nucleotide sequence between said osteoprotegerin regulatory region DNA and said nucleotide sequence identifies a mutation or polymorphism in the osteoprotegerin regulatory region of said subject's or patient's DNA that places said subject or patient at increased risk for having an altered susceptibility or predisposition to developing said bone disease, cartilage disease, arterial disease, immune disease, or other disease.
- the present invention provides a method of identifying a human patient or subject at increased risk for having an altered susceptibility or receptiveness to treatment of a disease caused by abnormal osteoprotegerin expression with a compound that affects osteoprotegerin expression through an interaction with the osteoprotegerin gene regulatory region, comprising comparing the nucleotide sequence of the osteoprotegerin regulatory region of the opg gene from DNA of said subject or patient with a nucleotide sequence selected from the group consisting of SEQ ID N0:1 through SEQ ID NO: 11, wherein any difference in nucleotide sequence between said osteoprotegerin regulatory region DNA and said nucleotide sequence identifies a mutation or polymorphism in the osteoprotegerin regulatory region of said subject's or patient's DNA that places said subject or patient at increased risk for having an altered susceptibility or receptiveness to said treatment.
- the present invention provides a method of treating a human suffering from a symptom, condition, or disease caused by over-expression of osteoprotegerin, comprising administering to said human a pharmaceutically effective amount of an antagonist of osteoprotegerin expression.
- the antagonist can be identified by any of the methods discussed above.
- the present invention provides a method of treating a human suffering from a symptom, condition, or disease caused by under-expression of osteoprotegerin, comprising administering to said human a pharmaceutically effective amount of an agonist of osteoprotegerin expression.
- the agonist can be identified by any of the methods discussed above.
- the present invention provides a method of treating a human in need of treatment with an agonist of osteoprotegerin expression, comprising:
- the present invention provides a method of treating a human in need of treatment with an antagonist of osteoprotegerin expression, comprising: (a) determining whether a polymorphism or mutation exists at one or more nucleotide sites in the osteoprotegerin regulatory region in DNA of said human; and (b) if a polymorphism or mutation exists, administering to said human a pharmaceutically effective amount of an antagonist of osteoprotegerin expression.
- the human can be suffering from a symptom, condition, or disease caused by an abnormal level of expression of osteoprotegerin.
- the present invention provides a method of modulating bone resorption in a patient in need thereof, comprising administering to said patient a pharmaceutically effective amount of a DNA construct as discussed above, wherein the protein of interest is osteoprotegerin .
- the present invention provides a method of modulating bone resorption in a patient in need thereof, comprising administering to said patient a pharmaceutically effective amount of a compound identified by any of the methods discussed above.
- the present invention provides a method of modulating immune responsiveness in a patient in need thereof, comprising administering to said patient a pharmaceutically effective amount of a DNA construct as discussed above, wherein the protein of interest is osteoprotegerin .
- the present invention provides a method of modulating immune responsiveness in a patient in need thereof, comprising administering to said patient a pharmaceutically effective amount of a compound identified by any of the methods discussed above.
- the present invention provides a kit or package, comprising an isolated nucleic acid fragment comprising the transcriptional regulatory region of the human opg gene, a subfragment thereof, or functional variant of either exhibiting human opg gene transcriptional regulatory activity, wherein said fragment, subfragment, or functional variant thereof excludes the opg protein coding region.
- the isolated nucleic acid fragment or subfragment thereof can comprise a nucleotide sequence selected from the group consisting of SEQ ID N0:1 through SEQ ID NO: 11.
- the isolated nucleic acid fragment, subfragment thereof, or functional variant of either can be contained within an expression cassette.
- the isolated nucleic acid fragment, subfragment thereof, or functional variant of either can be (a) operatively linked within a vector to a polynucleotide encoding human osteoprotegerin, or (b) operatively linked within a vector to a polynucleotide encoding a heterologous reporter molecule.
- the vector can be contained within a vector-releasing cell.
- the vector of (a) can further comprise, operably linked to said polynucleotide encoding said human osteoprotegerin, at least one translational regulatory region required for expression of said human osteoprotegerin in said vector-releasing cell.
- the vector of (b) can further comprise, operably linked to said polynucleotide encoding said heterologous reporter molecule, at least one translational regulatory region required for expression of said heterologous reporter molecule in said vector-releasing cell.
- the present invention provides a computer readable medium having stored thereon the nucleotide sequence of a nucleic acid fragment encoding the transcriptional regulatory region of the human opg gene, a subfragment thereof, or a functional variant of either, exhibiting osteoprotegerin transcriptional regulatory region activity, wherein said fragment, subfragment thereof, or functional variant thereof excludes the opg protein coding region.
- the nucleotide sequence can be selected from the group consisting of SEQ ID NO : 1 through SEQ ID NO: 11. other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
- a nucleic acid molecule of the present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 and 3, or a complement of any of this nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein.
- TSRX nucleic acid sequences can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et ⁇ l., eds., MOLECULAR CLONING: A LABORATORY MANUAL 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et ⁇ l., eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)
- a nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
- the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
- oligonucleotides corresponding to TSRX nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
- oligonucleotide refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction.
- a short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
- Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length.
- an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at lease 6 contiguous nucleotides of SEQ ID NO: 1 and 3 , or a complement thereof. Oligonucleotides may be chemically synthesized and may be used as probes.
- an isolated nucleic acid molecule of the invention includes a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NO: 1 and 3.
- an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NO: 1 and 3, or a portion of this nucleotide sequence.
- Figure 2 shows the nucleotide sequence (SEQ ID NO: 12) of the human OPG regulatory region disclosed in Figure 2 of Morinaga et al . , Eur . J. Biochem . , 254:685-691 (1998). This sequence is 1175bp, and extends from -1105 to +70. Also highlighted is the TATA box (underlined) , the +1 start site (T; in bold, and underlined) , and two bases upstream of the +1 start site that differ from the bases in the corresponding positions in the OPG regulatory region disclosed herein (SEQ ID NO:l) (A and T; in bold, and italicized) .
- FIG 3 illustrates the basal expression achieved in ROS 17/2.8, SaOS-2 and UMR 106 cells when transiently transfected with the OPG Regulatory region/beta-galactosidase fusion gene.
- Figure 4 schematically depicts various OPG regulatory region 5' deletion constructs employed in the Examples herein.
- Figure 5 graphically depicts the basal expression achieved by various OPG regulatory region deletion constructs in UMR106 and COS1 cells.
- Figure 6 shows the effect of sequential 5 ' -deletions of the OPG regulatory region on baseline expression in BALC cells .
- Figure 7 shows the effect of parathyroid hormone (PTH) (panel a) and Vitamin D (panel b) on beta-galactosidase expression from pOPG5.9 ⁇ gal in ROS 17/2.8 cells transfected with the OPG regulatory region/beta-galactosidase fusion gene.
- PTH parathyroid hormone
- Vitamin D panel b
- Figure 8 illustrates the effect of TGF- ⁇ l on OPG regulatory region expression in UMR 106 cells.
- Panel A shows dose-dependent increase in OPG promoter activity;
- Panel B shows time-dependent increase in OPG promoter activity.
- Figure 9 illustrates the concentration- and time- dependent effects of PTH (panel A) and Vitamin D 3 (panel B) on OPG regulatory region expression in UMR 106 cells stably transfected with the pOPG5.9 ⁇ gal vector.
- Curves in panel A for PTH shown from top to bottom at 10 "7 M are: 8 hours (black circles) ; 12 hours (red circles) ; 24 hours (inverted green triangles) ; and 48 hours (inverted yellow triangles) .
- Curves in panel B for vitamin D 3 shown from top to bottom at 10 ⁇ 6 M are: 8 hours (black circles); 12 hours (red circles); 24 hours (inverted green triangles); and 48 hours (inverted yellow triangles) .
- Figure 10 shows the responsiveness of the OPG regulatory region deletion constructs to TGF ⁇ l, PTH, and Vitamin D treatment .
- Figure 11 shows the effect of 0sf2 on OPG regulatory region expression in COS1 cells.
- Figure 12 shows the effect of Osf2 on OPG regulatory region expression in BALC cells .
- Figure 13 shows the effect of substitution mutation and deletion of proximal OSE2 sites on Osf2 transactivation in COS1 cells.
- Figure 14 schematically depicts fragments of the OPG regulatory region cloned upstream of the osteocalcin minimal promoter .
- Figure 15 shows TGF- ⁇ stimulation of endogenous OPG gene expression in BALC stromal/osteoblastic cells and UMR106 osteosarcoma cells.
- the number of TRAP 4" multinucleated cells formed in the cultures were counted on day 6, and the data (mean of osteoclast number + standard error) from a representative experiment are shown. The dashed line on top represents the number of osteoclasts in a control culture to which no TGF- ⁇ was added.
- Panel (B) BALC cells were treated with lOng/ml TGF- ⁇ for the designated periods of time. Northern analysis was performed using 2 ⁇ g of poly A+ RNA in each lane that was probed with an OPG- , or ol gonuc eotide.
- the oligonucleotide typically comprises a region of nuc eot de sequence t at hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 or more consecutive sense strand nucleotide sequence of SEQ ID NO: 1 and 3; or an anti-sense strand nucleotide sequence of SEQ ID NO: 1 and 3; or of a naturally occurring mutant of SEQ ID NO: 1 and 3.
- Probes based on the human TSRX nucleotide sequence can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
- the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
- Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a TSRX protein, such as by measuring a level of a TSRX-encoding nucleic acid in a sample of cells from a subject e.g., detecting TSRX mRNA levels or determining whether a genomic TSRX gene has been mutated or deleted.
- a "polypeptide having a biologically active portion of TSRX” refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency.
- a nucleic acid fragment encoding a "biologically active portion of TSRX” can be prepared by isolating a portion of SEQ ID NO: 1 and 3 that encodes a polypeptide having a TSRX biological activity (biological activities of the TSRX proteins are described below), expressing the encoded portion of TSRX protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of TSRX.
- a nucleic acid fragment encoding a biologically active portion of TSRX can optionally include an ATP-binding domain.
- a nucleic acid fragment encoding a biologically active portion of TSRX includes one or more regions.
- the invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NO: 1 and 3 due to the degeneracy of the genetic code. These nucleic acids thus encode the same TSRX protein as that encoded by the nucleotide sequence shown in SEQ ID NO: 1 and 3, e.g. , the polypeptide of SEQ ID NO: 2 and 4.
- an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO: 2 and 4 expressed as percent change over control activity (serum free control, with no TGF- ⁇ addition) . The results represent the mean + standard error of 4-8 separate treatments.
- Figure 17 shows TGF- ⁇ l, - ⁇ 2 , and - ⁇ 3 , but not BMP-4, stimulation of OPG promoter activity.
- UMR106 stable cells were treated with increasing amounts of TGF- ⁇ l, - ⁇ 2, - ⁇ 3, or BMP-4 and incubated for 48 h.
- the ⁇ -gal activity (mean + standard error) measured in cell extracts from a representative experiment are shown.
- Figure 18 shows mapping of the region of the OPG promoter responsible for mediating TGF- ⁇ effects.
- Panel (B) Analysis of OPG deletion constructs for responsiveness to TGF- ⁇ .
- the deletion constructs were transiently transfected into UMR106 cells, followed by treatment with TGF- ⁇ for 48 hours.
- the ⁇ -gal activity in cell extracts (open circles) is expressed as percent increases over its own control.
- cells were also transfected with the promoterless ⁇ - gal vector, and its expression relative to each OPG deletion construct is shown by the shaded circles.
- Figure 19 shows the region between -372 and -190 nucleotides (183bp fragment; SEQ ID NO: 10) imparting TGF- ⁇ responsiveness to a heterologous (osteocalcin) minimal promoter.
- the 183bp fragment of the OPG promoter was fused to the -34/+13 fragment of the osteocalcin (minimal) promoter using PCR, and ligated upstream of ⁇ -gal coding sequence (-372 to -190 OC ⁇ -gal).
- This construct was transiently transfected into UMR106 cells that were subsequently treated with TGF- ⁇ .
- a construct containing the osteocalcin promoter fused to ⁇ -gal was used as a control. The fold-induction in ⁇ -gal activity from a representative experiment, done in triplicate wells, is shown.
- Figure 20 shows the functional analysis of the role of consensus DNA elements (OSE , API-like, and SBE) in mediating
- Panel (A) Schematic representation of the spatial arrangement of OSE 2 , API-like, and Smad-binding elements (SBE) in the region between -372 and -190 nucleotides in the OPG promoter. The nucleotide numbers represent the exact location of the elements corresponding to the transcription start site (+1) .
- Panel (B) Mutational analysis of the role of the elements in the context of the native (OPG) promoter. Substitution mutations in the elements were created (either individually or in combination) to disrupt the sequence of one or more of the three DNA elements. The wild-type and mutant constructs were transiently transfected into UMR106 cells that were then treated with TGF- ⁇ .
- Panel (C) Mutational analysis of the role of the elements in the context of a heterologous (osteocalcin) promoter. To assess the function of the elements in the context of the osteocalcin minimal promoter, the same substitution mutations were created in the (-372 to -190 OC ⁇ -gal) construct. The constructs were transfected into UMR106 cells that were then treated with TGF- ⁇ for 48 hours. The ⁇ -gal activity in cell extracts from one of three independent experiments performed in triplicate is shown.
- OPG promoter- ⁇ gal constructs were transfected into COS1 cells along with either pEF-Cbfal or pEF/myc/cyto, and the ⁇ -gal activity was measured in cell extracts 48 hours after transfection.
- the fold induction in ⁇ -gal activity directed by each of the reporter constructs in Cbfal transfected cells compared to that in empty vector transfected cells is shown on the right.
- Three independent transfection experiments were performed in triplicate wells, and the data (mean + std. error) from a representative experiment are shown.
- API activator protein-1
- BMP-4 bone morphogenetic protein-4
- Cbfal core binding factor al
- ELISA enzyme-linked immunosorbent assay
- EMSA electrophoretic mobility shift assay
- JNK c-Jun N-terminal kinase
- M-CSF macrophage colony stimulating factor
- NF-KB nuclear factor-KB
- the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 75% homologous to the amino acid sequence of SEQ ID NO: 2 and 4.
- the protein encoded by the nucleic acid is at least about 80% homologous to SEQ ID NO: 2 and 4, more preferably at least about 90%, 95%, 98%, and most preferably at least about 99% homologous to SEQ ID NO: 2 and 4.
- Mutations can be introduced into the nucleotide sequence of SEQ ID NO: 1 and 3 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
- conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
- a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
- amino acids with basic side chains e.g., lysine, arginine, histidine
- acidic side chains e.g., aspartic acid, glutamic acid
- uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
- nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
- beta-branched side chains e.g., threonine, valine, isoleucine
- aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
- a predicted nonessential amino acid residue in TSRX is replaced with another amino acid residue from the same side chain family.
- mutations can be introduced randomly along all or part of a TSRX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for TSRX biological activity to identify mutants that retain activity.
- the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.
- nucleic acid fragment excludes whole chromosomes or total chromosomal DNA from cells.
- nucleic acid fragments can comprise, consist essentially of, or consist of the specific nucleotide sequences described herein.
- the phrase “consisting essentially of” includes, but is not limited to, allelic variants (polymorphs) of the disclosed sequence, as well as in vi tro chemically or genetically modified versions thereof.
- an allelic variant is an alternate form of a polynucleotide sequence that may contain an addition, deletion, or substitution of one or more nucleotides.
- isolated nucleic acid fragment refers to a nucleic acid fragment, for example DNA, that has been removed from its native or naturally occurring environment. As noted above, such nucleic acid fragments do not include whole chromosomes, or the entire chromosomal DNA of a cell. For example, recombinant nucleic acid fragments or molecules contained or generated in culture, in a vector, and/or in a host cell are considered isolated for the purposes of the present invention.
- isolated nucleic acid fragments include recombinant nucleic acid molecules maintained in heterologous host cells, or purified (partially or substantially) nucleic acid molecules in solution.
- isolated nucleic acid fragments according to the present invention further include nucleic acid molecules produced synthetically, or purified from or provided in cells containing such synthetic nucleic acids, where the nucleic acid exists in other than a naturally occurring form, quantitatively or qualitatively.
- the disclosed OPG regulatory region, and fragments thereof provides an invaluable tool for regulating osteoclastogenesis.
- the disclosed OPG regulatory region, or fragments or variants thereof can be used in screening assays to identify drugs that regulate bone balance or bone loss, or which can be used to treat metabolic bone diseases, such as osteoporosis, osteopetrosis, Paget's disease, rheumatoid arthritis, periodontal disease, bone tumors and hypercalcemia of malignancy, and arterial related diseases, such as vascular calcification, or to regulate immune function, lymphocyte development, lymph node development, or T- and B-cell formation (note Kong et al .
- OPG regulatory region, fragments thereof, or functional variants of either are useful in drug screening assays
- diseases having immune system involvement such as autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus , and the spondyloarthropathies; adult and childhood leukemias; and various viral infections, such as hepatitis and HIV.
- mutations or polymorphisms in the OPG regulatory region, or fragments thereof can be used as prognostic diagnostic markers for bone, cartilage, immune, arterial, and the other diseases mentioned above, and for determining a patient's susceptibility to therapy.
- the presently disclosed OPG regulatory region, or fragments or variants thereof can be used in expression vectors to control the expression in vi tro or in vivo of a protein or reporter molecule of interest.
- the disclosed regulatory region, or fragments or variants thereof can be used to identify, isolate and clone cis-elements and interacting trans-factors. the major groove of the double helix.
- An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
- antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens.
- the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.
- vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
- the antisense nucleic acid molecule of the invention is an ⁇ -anomeric nucleic acid molecule.
- An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641).
- the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA -DNA analogue (Inoue et al. (1987) FEBS Lett 215: 327-330).
- modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
- an antisense nucleic acid of the invention is a ribozyme.
- Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as a mRNA, to which they have a complementary region.
- ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)
- a ribozyme having specificity for a TSRX-encoding nucleic acid can be designed based upon the nucleotide sequence of a TSRX DNA disclosed herein (i.e., SEQ ID NO: 1 and 3). For example, a derivative of a Tetrahymena L-19 IVS
- the second approach involved screening a conventional genomic library.
- a human PI library in pAdlOSacBII vector (Genome Systems, Inc., St. Louis, MO) was screened using the full length OPG cDNA (GenBank # U94332) . Three positive clones, each containing 70-100 kb of genomic DNA, were identified.
- each positive clone was digested with a panel of restriction enzymes and then Southern blotted with the 1,874 bp regulatory region fragment described above.
- the 1,874 bp OPG regulatory region fragment hybridized to a 5,936 bp _Jsfcl-_-C ⁇ 47III fragment.
- the 5,936 bp fragment was gel- purified, cloned, and sequenced using standard procedures (See Sambrook et al . , Molecular Cloning, A Laboratory Manual , Second Edition, Cold Spring Harbor Laboratory Press (1989) and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1987 and updates)).
- the nucleotide sequence of the 5,936 bp Sstl-Eco47III fragment is shown in SEQ ID NO : 2.
- functional variants exhibiting the activities noted above can be identified using nucleic acid hybridization assays.
- Functional variants for example fragments, analogs, or derivatives, can be identified by their ability to hybridize to the complement DNA sequence of the presently disclosed OPG regulatory region (SEQ ID N0:1), or the complement of fragments thereof, i.e., the complements of SEQ ID NO: 2, SEQ ID NO : 3 , SEQ ID NO : 4 , SEQ ID NO : 5 , SEQ ID NO: 6, SEQ ID NO : 7 , SEQ ID NO : 8 , SEQ ID NO : 9 , SEQ ID NO: 10, or SEQ ID NO: 11 under mild to stringent hybridization conditions.
- the following conditions illustrate one example of a mildly stringent hybridization condition:
- IX phosphate buffer (comprising 0. IM Na 2 HP0 4 , 0.5M NaCl, 0.0052 M EDTA) pH 7.0, and 1% Sarkosyl, at 45-65°C, preferably 55-65°C, more preferably 60-65°C, for approximately 2 hours to overnight;
- First Wash lmM Tris-HCl, pH 8.0, 1% sarkosyl at room temperature for approximately 10-15 minutes ;
- Second-Fifth Washes (if needed): lmM Tris-HCl, pH 8.0, for approximately 10-15 minutes each.
- Any ammo acid substitution, insertion, or deletion is encompassed by the invention In favorable circumstances, the substitution is a conservative substitution as defined above
- TSRX protems and biologically active portions thereof, or derivatives, fragments, analogs or homologs thereof
- polypeptide fragments suitable for use as immunogens to raise anti-TSRX antibodies can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques
- TSRX proteins are produced by recombinant DNA techniques
- a TSRX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques
- an “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the TSRX protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized
- the language “substantially free of cellular material” includes preparations of TSRX protein in which the protem is separated from cellular components of the cells from which it is isolated or recombinantly produced
- the language “substantially free of cellular material” includes preparations of TSRX protein having less than about 30% (by dry weight) of non-TSRX protein (also referred to herein as a "contaminating protein”), more preferably less than about 20% of non-TSRX protein, still more preferably less than about 10% of non-TSRX protein, and most preferably less than about 5% non-TSRX protein
- culture medium represents
- Preferred polynucleotides are those having at least about 50% sequence identity, more preferably at least about 55% sequence identity, more preferably at least about 60% sequence positions are then compared When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i e , as used herein ammo acid or nucleic acid "homology” is equivalent to ammo acid or nucleic acid “identity")
- the nucleic acid sequence homology may be determined as the degree of identity between two sequences
- the homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package See, Needleman and Wunsch 1970 J Mol Biol 48 443-453 Using GCG GAP software with the following settings for nucleic acid sequence comparison GAP creation penalty of 5 0 and GAP extension penalty of 0 3, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably
- sequence identity refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison
- percentage of sequence identity is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e g , A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison ( ⁇ e the window size), and multiplying the result by 100 to yield the percentage of sequence identity
- substantially identity denotes a charactenstic of a polynucleotide sequence, wherem the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually
- EP 0 784 093, EP 0 870 023, EP 0 980 432, and EP 1 029 038 are specifically excluded from the functional variants or hybridizing nucleic acid fragments encompassed by the present invention.
- the presently disclosed OPG regulatory region, a fragment thereof such as those disclosed herein, or a functional variant of either thereof is used in a method for identifying a compound that affects osteoclast formation, activity, or survival, or bone resorption.
- An example of such a method is a cell-based screening assay, wherein an expression cassette or vector form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the TSRX proteins.
- Variants of the TSRX protein that function as either TSRX agonists (mimetics) or as TSRX antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the TSRX protein for TSRX protein agonist or antagonist activity.
- a variegated library of TSRX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
- a variegated library of TSRX variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential TSRX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of TSRX sequences therein.
- a degenerate set of potential TSRX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of TSRX sequences therein.
- methods which can be used to produce libraries of potential TSRX variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector.
- degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential TSRX sequences.
- Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu Rev Biochem 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucl Acid Res 11 :477.
- libraries of fragments of the TSRX protein coding sequence can be used to generate a variegated population of TSRX fragments for screening and subsequent selection of variants of a TSRX protein.
- a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a TSRX coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with Si nuclease, and ligating the resulting fragment library into an expression vector.
- an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the TSRX protein. expressed product.
- Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and other gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of expressed foreign proteins. Accordingly, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and/or phosphorylation of the expressed product can be used. Examples of appropriate mammalian host cells for this purpose include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3 , and WI38 cell lines.
- test compounds include low molecular weight chemical compounds, for example having molecular weights less than about 1500 daltons, suitable as pharmaceutical or veterinary agents for human or animal use.
- Compounds may stimulate (agonists), inhibit (antagonists) , or have no effect on, expression of the reporter polynucleotide operably linked to the OPG regulatory region, fragment thereof, or functional variant of either due to their effect on transcription, including transcription initiation.
- reporter gene expression is measured in the presence of an agonist, with and without a second compound, which is the candidate agonist.
- Increasing amounts (or concentrations) of the second compound can be used to assess its antagonistic effect, if any, on the expression induced by a given amount amino acid sequence of the full length protein, such as an amino acid sequence shown in SEQ ID NO: 2 and 4, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope.
- the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues.
- Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.
- At least one epitope encompassed by the antigenic peptide is a region of TSRX-related protein that is located on the surface of the protein, e.g., a hydrophilic region.
- a hydrophobicity analysis of the huma TSRX-related protein sequence will indicate which regions of a TSRX-related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production.
- hydropathy plots showing regions of hydrophihcity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation.
- a protein of the invention may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
- the present invention can be cloned into the Sstl / Smal site of this vector.
- the OPG regulatory region construct can then be transiently transfected into osteoblast cells using FugeneTM 6 reagent (Boehringer Mannheim) , as recommended by the manufacturer. After transfection, the cells are plated in 96 well plates (50,000 cells/well). Four hours after plating, the cells are transferred to medium containing 0.1% fetal bovine serum, and incubated overnight. The cells are then treated with a test compound.
- the cells are lysed in lysis buffer, and a portion of the extracts, for example 1/3, is assayed for beta-galactosidase activity using a luminometer.
- a portion of the extracts for example 1/3
- a luminometer By comparing the levels of beta-galactosidase activity in those samples treated with the test compounds to those of a control sample, compounds that alter OPG expression can be identified.
- agonists and antagonists of OPG expression can be identified in such screening assays.
- Compounds that affect OPG expression include, but are not limited to, parathyroid hormone (PTH), Vitamin D, transforming growth factor ⁇ , and Osf2.
- a pharmaceutically effective amount of that compound can be determined using techniques that are well-known to the skilled artisan. Note, for example, Benet et al . , in Goodman & Gilman ' s The Pharmacological Basis of Therapeutics, Ninth Edition, Hardman et al., Eds., McGraw-Hill, New York (1996), Chapter 1, pp. 3- 27, and the references cited therein. Thus, the appropriate dose(s) range, and dosing regimens, of such a compound can be easily determined by routine methods.
- the screening assays described above also can be used to identify compounds that are useful for treating arterial diseases caused by over- or under-expression of OPG.
- Targeted deletion of OPG in mice results not only in severe osteoporosis, but also in calcification of the aorta and renal arteries. See Bucay et al . , Genes & Development, 12:1260-1268 (1998). Therefore, compounds that control the expression of OPG can influence the calcification of arteries.
- the screening assays described above can be used to identify compounds that can be used to regulate cartilage function, immune function, lymph node development, and T- and B-cell formation. Since osteoclasts are derived from hematopoietic precursors, alterations in osteoclast precursor proliferation/differentiation can affect immune modulation. Note, in this regard, Green et al . , J. Exp . Med. , 189(7) .1017-1020 (1999), and Bachmann et al . , J " . Exp . Med . 189 (7) .1025-1031 (1999).
- a compound may preferentially promote differentiation of common precursors down the osteoclast lineage, depleting formation of other lineage cells involved in immune function, for example macrophages .
- inhibition of the c-fos gene by knock out blocks osteoclast formation, and increases macrophage cell formation (Grigoriadi ⁇ et al . , Science, 266(5184) -.443-448 (1994).
- OPG osteoclast differentiation factor
- ODF osteoclast differentiation factor
- the presently disclosed OPG regulatory region, fragment thereof, or variant of either can be used in methods to diagnose the presence of, or in prognostic methods to assess the susceptibility to or predisposition to develop, bone, cartilage, immune, arterial, etc., diseases in a human patient. Altered levels of OPG can result in a variety of bone, cartilage, arterial, and immune response diseases. Gain of function or activating mutations in the OPG regulatory The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
- the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art.
- the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
- antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.
- the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium.
- the hybridoma cells can be grown in vivo as ascites in a mammal.
- the monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
- the monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
- DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
- the hybridoma cells of the invention serve as a preferred source of such DNA.
- the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
- host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
- the DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
- the initial step is to generate target DNA by amplifying DNA extracted from the cells of a clinical sample using the polymerase chain reaction (PCR) .
- PCR polymerase chain reaction
- the amplified DNA is then bound to a filter, which is placed into a hybridization tube containing a radiolabeled probe complementary to the genetic mutation of interest. After hybridization and appropriate washing, the filter is examined radiographically for binding of the probe to the target DNA.
- PCR polymerase chain reaction
- Hybridization is usually performed in two stages. First, in the "binding" stage, the probe is bound to the target under conditions favoring hybridization.
- a representative hybridization solution comprises 6X SSC, 0.5% SDS, 5X Denhardt ' s solution and lOO ⁇ g of non-specific carrier DNA. See Ausubel et al . , 1989, Current Protocols in Molecular Biology, section 2.9, supplement 27 (1994), Green Publishing Associates and Wiley Interscience, N.Y..
- a stock 20X SSC solution contains 3M sodium chloride, 0.3M sodium citrate, pH 7.0. Of course many different, yet functionally equivalent, buffer conditions are known. For high stringency, the temperature is between about 65°C and 70° C in a hybridization solution of 6X SSC, 0.5% SDS, 5X Denhardt ' s solution and lOO ⁇ g of non-specific carrier DNA.
- washing solutions typically contain lower salt concentrations.
- a medium stringency wash solution contains the equivalent ionic strength of 2X SSC and 0.1% SDS.
- a high stringency wash solution contains the equivalent ionic strength of less than about 0.2X SSC, with a preferred stringent solution containing about 0. IX SSC.
- the temperatures associated with various stringencies are the same as discussed above for "binding.”
- the washing solution is replaced a number of times during washing. For example, typical high stringency washing conditions comprise washing twice for 30 minutes at 55°C and three times for 15 minutes at 60°C.
- clinical samples are evaluated for genetic mutations using a so-called "gene chip” diagnostic platform.
- Gene chip Such platforms have been developed by, inter alia, Synteni and Affymetrix. Briefly, mutant-specific probes can be immobilized on a chip surface and used to identify mutations in targeted DNA via hybridization to the surface of the chip. Suitable chip technology is described for example, in Wodicka et al . , Nature Biotechnology, 15:1359 (1997) which is hereby incorporated by reference in its entirety, and references cited therein.
- sequencing analysis can be used to detect genetic mutations or polymorphisms. This is accomplished by sequencing the D ⁇ A extracted from target cell populations. Diagnosis is accomplished by comparing the sequenced target sample with the disclosed sequence (s) of the present invention.
- the presence or absence of variant nucleotides in a patient's or subject's OPG regulatory region can be detected by reference to the loss or gain of restriction endonuclease sites within the OPG regulatory region.
- RFLPs restriction fragment length polymorphisms
- the presently disclosed OPG regulatory region, or fragment thereof can be used in a method to diagnose a patient's predisposition, susceptibility, or risk of developing any of the bone, cartilage, arterial, immune, etc., diseases discussed herein.
- a predisposition WO 96/34096.
- This animal produces B cells which secrete fully human immunoglobulins.
- the antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.
- the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.
- U.S. Patent No. 5,939,598 An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.
- a method for producing an antibody of interest such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.
- the hybrid cell expresses an antibody containing the heavy chain and the light chain.
- techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Patent No. 4,946,778).
- methods can be adapted for the construction of F ab expression can be used as diagnostic or prognostic markers for susceptibility, receptiveness, or responsiveness to therapy in patients.
- a patient's OPG regulatory region, or fragment thereof can be evaluated by comparing DNA from the patient's bone cells or other cells with the disclosed regulatory sequence of the present invention by, for example, hybridization or sequencing analysis, using the techniques described above.
- the degree of similarity (sequence identity) of a patient's OPG regulatory region, or fragment thereof, to the present OPG regulatory region, or fragment thereof, is expected to be indicative of patient's susceptibility, receptiveness, or responsiveness to therapy: any deviations may influence the effect of therapeutic drugs that act directly, or indirectly through another molecule, through an interaction, e.g., binding, with the OPG regulatory region, to affect OPG expression and therefore the level of OPG in cells.
- Individuals who carry particular allelic variants of the OPG regulatory region disclosed herein may therefore exhibit differences in OPG levels under different physiological conditions, and thus altered abilities to react to different diseases.
- differences in OPG level resulting from allelic variation may directly affect an individual's response to drug therapy.
- OPG polymorphism may therefore have a significant effect on the efficacy of drugs designed to modulate the activity of OPG.
- the polymorphism ⁇ s) may also affect the response to agents acting on other biochemical pathways regulated by an OPG ligand.
- the diagnostic methods provided herein may therefore be useful both to predict the clinical response to such agents, and to determine therapeutic dose.
- Pharmacogenetics can also be used in pharmaceutical research to assist the drug development process.
- polymorphisms can also be used in mapping the human genome to elucidate the genetic component of diseases.
- Clinical trials have shown that patient response to drugs can be heterogeneous, creating a necessity for improved approaches to pharmaceutical design and therapy.
- the following references provide further information on pharmacogenetics and other uses of polymorphism detection: Linder et al . , Clin . Chem.
- the present invention provides a method for diagnosing at least one nucleotide polymorphism or mutation in the OPG regulatory region of a human, comprising determining the nucleotide sequence of the OPG regulatory region of the human, and determining the status of the human by referring to SEQ ID NO:l, or a fragment thereof as disclosed herein.
- the term "human” includes both a human having, or suspected of having, an OPG regulatory region-mediated disease, as well as an asymptomatic human who may be tested for predisposition, risk, or susceptibility to developing such disease. At each nucleotide position so- identified, the human may be homozygous for an allele, or the human may be a heterozygote .
- the present invention provides a method for diagnosing an OPG regulatory region-mediated disease, comprising:
- SNPs single nucleotide polymorphisms
- the diagnostic methods of the present invention can be used in developing new drug therapies that selectively target one or more allelic variants of the OPG regulatory region. Identification of a link between a particular allelic variant and predisposition to disease development or response to drug therapy can significantly impact the design of drugs intended for use in treating OPG -mediated diseases. Drugs can be specifically designed to regulate the expression of OPG driven by particular allelic variants in the OPG regulatory region, while minimizing effects on other variants or wild-type OPG regulatory regions .
- the OPG regulatory region, SEQ ID N0:1, and fragments thereof disclosed herein represent valuable information that can be used to identify further similar sequences, and to characterize individuals in terms of, for example, their identity, haplotype, and other sub-groupings, such as susceptibility, risk, or predisposition to developing symptoms, conditions, or diseases associated with OPG over- or under-expression, and susceptibility to treatment with particular drugs.
- Such approaches are facilitated by storing the sequence information in a computer readable medium, and then using the information in standard macromolecular structure programs or to search sequence databases using search tools such as GCG (Genetics Computer Group) , BlastX, BlastP, BlastN, FASTA (Altschul et al . , J. Mol . Biol . ,
- sequences provided herein are particularly useful as components in databases useful for searching for sequence identity, genome mapping, pharmacogenetics, and related search analyses.
- sequence information disclosed herein can be reduced to, converted into, or stored in a tangible medium, such as a computer disk, preferably in computer readable form.
- the present invention therefore also provides a computer readable medium having stored thereon a nucleotide sequence comprising, consisting essentially of, or consisting of the OPG regulatory region nucleic acid sequence shown in SEQ ID NO:l, or fragments thereof, useful for diagnostic purposes.
- the computer readable medium can be any composition of matter used to store information or data, including, for example, floppy disks, tapes, chips, compact disks, digital disks, video disks, punch cards, and hard drives.
- a computer based method for performing diagnosis comprising determining the nucleotide sequence of the OPG regulatory region of DNA from a human subject, and comparing this sequence to a nucleotide sequence comprising, consisting essentially of, or consisting of the OPG regulatory region nucleic acid sequence shown in SEQ ID N0:1, or fragment thereof useful for diagnostic purposes, in a computer readable medium to identify any polymorphism or mutation that may be present in said human subject's DNA.
- the present invention provides methods of modulating osteoclast formation and function, bone resorption, and the other diseases, symptoms, and conditions discussed herein.
- modulate or “affects” denotes an alteration, i.e., either an increase or a decrease.
- a compound that modulates bone resorption is one that either increases or decreases bone resorption.
- a compound that "affects" reporter gene expression is one that stimulates or inhibits reporter gene transcription or expression.
- agonists compounds that stimulate or increase OPG gene expression
- antagonists compounds that inhibit or decrease OPG gene expression
- Osteoclast formation and function, and therefore bone resorption can be modulated by administering to a patient one or more compounds identified by the methods described herein.
- a compound that increases reporter gene expression in a screening assay of the present invention employing a nucleic acid construct comprising the present OPG regulatory region, fragment thereof, or variant of either, is expected to increase OPG formation, and hence be a candidate for treatment of abnormal bone resorption; osteoporosis; arterial disease; metastatic bone disease such as that resulting from prostate cancer, breast cancer, multiple myeloma, humoral hypercalcemia of malignancy, and lung cancer; rheumatoid arthritis; osteoarthritis ; Paget's disease of bone; hypercalcemia of malignancy; osteolysis; and periodontal disease.
- a compound that inhibits reporter gene expression, and hence OPG expression, would be expected to be effective, for example, for treating or preventing osteopetrosis, a condition characterized by abnormal, increased hardening and thickening of bone.
- immune responsiveness or function including, for example, lymph node development, T- and B-cell development, T-cell activation, etc., can also be regulated by administering to a patient one or more compounds identified in the methods described herein.
- plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated
- viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome
- Certain vectors are capable of autonomous replication m a host cell mto which they are introduced (e g , bactenal vectors having a bacterial origin of replication and episomal mammalian vectors)
- Other vectors e g , non-episomal mammalian vectors
- certain vectors are capable of directing the expression of genes to which they are operatively-lmked Such vectors are referred to herein as "
- the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-hnked to the nucleic acid sequence to be expressed
- "operably-hnked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e g , in an in vitro transcnption/translation system or m a host cell when the vector is introduced into the host cell)
- the term "regulatory sequence” is intended to includes promoters, enhancers and other expression control elements (e g , polyadenylation signals) Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOG
- Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only m certam host cells (e g , tissue-specific regulatory sequences) It will be appreciated by those skilled m the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protem desired, etc
- the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e g , TSRX protems, mutant forms of TSRX protems, fusion protems, etc )
- TSRX proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY METHODS IN ENZYMOLOGY 185, Academic Press, San
- the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase
- Fusion vectors add a number of amino acids to a protein encoded therein, usually to the ammo terminus of the recombinant protein
- Such fusion vectors typically serve three purposes (i) to increase expression of recombinant protem, (u) to increase the solubility of the recombinant protem, and (in) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification
- a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protem
- enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokmase Typical
- suitable inducible non-fusion E coli expression vectors include pTrc (Amrann et al , (1988) Gene 69 301-315) and pET 1 Id (Studier et al , GENE EXPRESSION TECHNOLOGY METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif (1990) 60-89) nucleic acid delivery system, and a pharmaceutically acceptable carrier, diluent, or excipient.
- Such compositions can also be used in transfecting cells for in vi tro assays such as those described herein. Slow release matrices containing the nucleic acid delivery vehicle can also be employed.
- the delivery system can comprise a pharmaceutical composition comprising recombinant cells, and a pharmaceutically acceptable carrier, diluent, or excipient.
- the present invention also provides in one of its aspects a kit or package, in the form of a sterile- filled vial or ampoule, that contains a polynucleotide comprising SEQ ID NO : 1 , a fragment thereof, or a functional variant of either of the foregoing, or a vector containing SEQ ID NO : 1 , etc., operatively linked to the opg gene or a heterologous coding sequence such as a reporter gene or other polynucleotide, as well as instructions for use in these various methods.
- the vector can optionally be contained within a vector-releasing cell.
- the kit contains a polynucleotide vector containing an OPG regulatory region, fragment thereof, or functional variant thereof, operatively linked to an opg coding region as an administration-ready formulation, in either unit dose or multi-dose amounts, wherein the package incorporates a label or manual with instructions for use of its contents for the treatment of one or more of the symptoms, conditions, or diseases discussed herein.
- the package provides a sterile-filled vial or ampoule containing a vector- releasing cell or cell line.
- kits or packages can also contain media and reagents, such as reaction buffers, for carrying out appropriate methods as disclosed herein with the nucleic acids, recombinant constructs, vectors, or cells contained therein, as well as instructions therefor.
- media and reagents such as reaction buffers
- any prevention or alleviation of an undesirable symptom, condition, or disease as noted herein would be desirable.
- treatment or therapeutic use refer to any and all uses of the presently claimed compositions that remedy a disease state, condition, or symptoms, or which prevent, hinder, retard, or reverse the progression of symptoms, conditions, or diseases discussed herein.
- Effective amounts of OPG regulatory region constructs, delivery vehicles containing such constructs, agonists, and antagonists, and treatment protocols can be determined by conventional means.
- the medical practitioner can commence treatment with a low dose in a subject or patient in need thereof, and then increase the dosage, or systematically vary the dosage regimen, monitor the effects thereof on the patient or subject, and adjust the dosage or treatment regimen to maximize the desired therapeutic effect.
- Further discussion of optimization of dosage and treatment regimens can be found in Benet et al . , in Goodman & Gilman ' s The Pharmacological Basis of Therapeutics, Ninth Edition, Hardman et al . , Eds., McGraw-Hill, New York, (1996), Chapter 1, pp. 3-27, and L.A.
- beta-galactosidase activity was assayed using the beta-galactosidase reporter gene assay kit (Boehringer Mannheim) as recommended by the manufacturer.
- beta-galactosidase was assayed in a fixed a amount of the extracts (1/3 of the extracts) using the beta-galactosidase reporter gene assay kit. The results represent the mean ⁇ SEM of 12 separate wells.
- results, shown in 3, demonstrate that the disclosed regulatory region transiently directs expression of the reporter gene in all three osteoblast cell lines.
- the regulatory region was particularly effective at directing expression in the ROS17/2.8 cell line.
- Example 2 Characterization of the OPG Regulatory Region offspnng borne of this female foster animal will be a clone of the animal from which the cell (e g , the somatic cell) is isolated
- compositions suitable for administration typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable earner
- pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration Suitable earners are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text m the field, which is incorporated herein by reference Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin Liposomes and non-aqueous vehicles such as fixed oils may also be used The use
- Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al , Proc Natl Acad Sci USA, 82 3688 (1985), Hwang et al , Proc Natl Acad Sci USA, 77 4030 (1980), and U S Pat Nos 4,485,045 and 4,544,545 Liposomes with enhanced circulation time are disclosed in U S Patent No 5,013,556
- Particularly useful liposomes can be generated by the reverse-phase evaporation method with a hpid composition comprising phosphatidylchohne, cholesterol, and PEG- de ⁇ vatized phosphatidylethanolamme (PEG-PE)
- Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al , J Biol Chem , 257 286-288 (1982) via a disulf
- the ends were then blunted with T4 DNA polymerase, and the fragment was religated with T4 DNA ligase.
- the Smal- __co47III fragments (1874 bp, 1473 bp and 391bp) obtained from the GenomeWalker library described above were cloned into the Smal site of pGL3 to obtain pOPGl .9 (-1855 to +19), pOPGl .5 (-1454 to +19), and pOPGO .4 (-372 to +19), respectively.
- the vector pOPGO .9 (-872 to +19) was generated by cloning the 0.9 kb Xhol fragment of pOPGl .5 into the same site of pGL3 and by screening for the proper orientation.
- the vector pOPGO .2 (-188 to +19) was derived by digesting pOPG0.4 with BamHI/Bglll, and subcloning the 0.2kb insert into the Bglll site of pGL3.
- Osteotrophic hormones such as l ⁇ , 25-hydroxyvitamin D 3 (Vitamin D) and parathyroid hormone (PTH) modulate bone resorption by promoting the development of osteoclasts .
- Viamin D 25-hydroxyvitamin D 3
- PTH parathyroid hormone
- sustained-release preparations include semipermeable matnces of solid hydrophobic polymers containing the antibody, which matnces are in the form of shaped articles, e g., films, or microcapsules
- sustamed-release matrices include polyesters, hydrogels (for example, poly(2- hydroxyethyl-methacrylate), or poly(v ⁇ nylalcohol)), polylactides (U.S. Pat No.
- copolymers of L-glutamic acid and ethyl-L-glutamate non-degradable ethylene-vmyl acetate
- degradable lactic acid-glycohc acid copolymers such as the LUPRON DEPOT TM (mjectable microspheres composed of lactic acid-glycohc acid copolymer and leuprohde acetate)
- poly-D-(-)-3-hydroxybuty ⁇ c acid While polymers such as ethylene-vmyl acetate and lactic acid-glycohc acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods
- compositions can be included in a container, pack, or dispenser together with instructions for administration
- the isolated nucleic acid molecules of the invention can be used to express TSRX protem (e g , via a recombinant expression vector in a host cell in gene therapy applications), to detect TSRX mRNA (e g , in a biological sample) or a genetic lesion m a TSRX gene, and to modulate TSRX activity, as described further, below
- TSRX proteins can be used to screen drugs or compounds that modulate the TSRX protein activity or expression as well as to treat disorders characterized by insufficient or excessive production of TSRX protein or production of TSRX protein forms that have decreased or aberrant activity compared to TSRX wild-type protem
- the anti-TSRX antibodies of the invention can be used to detect and isolate TSRX proteins and modulate TSRX activity
- the invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as descnbed, supra
- the results shown in Figure 8 demonstrate that TGF ⁇ l induced a 5-fold increase in beta-galactosidase reporter gene expression. TGF ⁇ l effects on OPG transcription were time and concentration dependent, optimally 24-48 hours and lOng/ml, respectively.
- TGF- ⁇ stimulation of OPG gene expression is presented in Example 9, below.
- OPG regulatory region contains 13 osteoblast specific element (OSE) motifs, shown in bold in Figure 1, that function as binding sites for osteoblast specific transcription factor 2 (Osf2), the role of Osf2 in OPG expression was evaluated.
- OSE osteoblast specific element
- Osf2 The ability of Osf2 to transactivate OPG regulatory region constructs was evaluated in COS1 cells, a monkey kidney cell line lacking endogenous Osf2 protein, and in BALC cells, a spontaneously immortalized mouse calvaria-derived stromal cell line that supports osteoclastogenesis.
- the COS-1 cell line was obtained from the American Type Culture Collection, Bethesda, MD (ATCC CRL 1650) , and was grown in DMEM, supplemented with 10% fetal bovine serum (FBS) and antibiotics.
- FBS fetal bovine serum
- reporter plasmid OPG regulatory region constructs- linked to ⁇ -gal or p ⁇ gal-Basic
- effector plasmid pEF/Cbfal/myc/cyto plasmid coding Cbfal (Osf2) under the control of the human translation elongation factor EF-la) or the control vector pEF/myc/cyto, using FugeneTM6 transfection reagent (Boehringer Mannheim) .
- the Cbfal coding sequence was from mouse (Ducy et al . , Cell , 89 (5 ): 747-754 (1997); GenBank accession no. AF010284) .
- the constructs (1 ⁇ g each in a total volume of 20 ⁇ l in T.E. buffer, pH 8.0) were mixed with diluted FugeneTM6 reagent (194 ⁇ l serum-free medium + 6 ⁇ l Fugene) and incubated for 15 minutes at room temperature. The DNA-Fugene mix was then added drop-wise to the plates, and the cells were incubated for an additional 36-48 hours. Following transfection, the plates were washed twice with PBS (Gibco, BRL) , and then lysed with 100 ⁇ l of lysis buffer provided with the beta-galactosidase reporter gene assay kit (Boehringer Mannheim) .
- the cell extracts were centrifuged for 2 minutes at 14,000 rpm in a microfuge to precipitate cellular debris. Twenty microliters of the supernatant were transferred to white, opaque microtiter plates, and beta-galactosidase activity was measured using an automated injection MLX Luminometer (Dynex Corporation, Chantilly, VA) according to the manufacturer's instructions.
- the beta-galactosidase activity values represent the integral value of light emitted over a period of two seconds, and are expressed as fold induction over basal (control vector transfected) levels.
- Figures 11 and 12 show the effect of Osf2 on OPG transcription in COS1 and BALC cells, respectively.
- Cotransfection of an Osf2 expression construct along with the pOPG5.9 ⁇ gal reporter construct led to a 64-fold and 5.4-fold increase in beta-galactosidase activity in C0S1 and BALC cells, respectively.
- Sequential deletions of the regulatory region led to a progressive decrease in OPG regulatory region transcription activity.
- the removal of all putative 0sf2- binding elements led to a complete loss of transactivation. A complete loss of activity was observed in the most proximal regulatory region, pOPG0.2 ⁇ gal (-188 to +19; SEQ ID N0:8), which lacked an OSE.
- a fusion protem can be provided that adds a domain that allows one or both of the protems to be bound to a matnx
- GST-TSRX fusion protems or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St Louis, MO) or glutathione deriva
- TSRX protem or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin
- Biotinylated TSRX protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succmimide) using techniques well-known within the art (e g , biotmylation kit, Pierce Chemicals, Rockford, 111 ), and immobilized in the wells of streptavidm-coated 96 well plates (Pierce Chemical)
- antibodies reactive with TSRX protem or target molecules, but which do not interfere with binding of the TSRX protem to its target molecule can be derivatized to the wells of the plate, and unbound target oi TSRX protem trapped m the wells by antibody conjugation
- Methods for detecting such complexes include immunodetection of complex
- the 0.2 kb OPG regulatory region fragment was amplified using one of the forward primers and the p ⁇ gal-R reverse primer with pOPG0.2 ⁇ gal serving as template.
- the resultant PCR products were digested with Asp718 and Bglll, and ligated to a p ⁇ gal- Basic vector that was digested with the same enzymes, resulting in lxOSE-0.2kbOPG ⁇ gal and 3xOSE-0.2kbOPG ⁇ gal clones.
- the constructs were transfected into COS1 cells, and the beta- galactosidase activity measured as described above.
- Figure 13 shows that substituting or deleting the proximal OSE2 sites of the presently disclosed OPG regulatory region decreases Osf2 transactivation by 50 to 70%. This directly confirms a role for the proximal 0SE2 sites in Osf2 transactivation of OPG gene promoter. The finding that substitution or deletion of the proximal OSE2 sites did not completely abrogate Osf2 responsiveness suggests the presence of other novel Osf2 response sites in the proximal promoter region spanning nucleotides -372 to -190 (SEQ ID NO: 10) (compare 0.4 kb to 0.4kb OSE mutant in Figure 13).
- PCR amplification was performed using OPG392AspF (5'-ata ggt ace gcc cag ccc tec cac cgc tgg t-3 ' ; SEQ ID NO: 21) as forward primer, and 100OPGOG2 (5'-ata tag ate tga ctt gtc tgt tec tgc ace etc cag cat cca gta gca ttt ata teg ccc agg gag gtg ggg cgt ga-3'; SEQ ID NO: 24) as reverse primer (that contains a Bglll restriction site and the osteocalcin -34/+13 sequence at the 5' end).
- pOPG0.4 ⁇ gal served as template.
- the region between -293 and -190 was PCR amplified using OPG312AspF (5 '-ata ggt ace gga gac age age cgc ctt gtt- 3'; SEQ ID NO:25) as forward primer and 100OPGOG2 as reverse primer, with pOPGO .4 ⁇ gal or pOPGO .40SE mutant serving as template.
- the resultant PCR products were digested with Asp718 and Bglll , and ligated to p ⁇ gal-Basic vector that was digested with the same enzymes, to generate the (-372 to -190 OC ⁇ gal) and (-293 to -190 OC ⁇ gal) constructs.
- the -34/+13 OC ⁇ gal construct was created by annealing the 0G2For (5 '-tat agg tac ccg ata taa atg eta ctg gat get gga ggg tgc agg aac aga caa gtc aga tct ata t-3 ' ; SEQ ID NO: 26) and 0G2Rev (5 '-ata tag ate tga ctt gtc tgtgt tec tgc ace etc cag cat cca gta gca ttt ata teg ggt ace tat a-3'; SEQ ID NO: 27) oligonucleotides, digesting the double-stranded oligonucleotide with Asp718 and Bglll , and ligating them to p ⁇ gal-basic digested with the same enzymes.
- Bone marrow cells from the femora of male Balb/C mice were seeded into 24-well cluster dishes (Costar, Cambridge, MA) at a density of 5 x IO 4 mononuclear cells/cm 2 in growth media (RPMI 1640) (Life Technologies, Gaithersburg, MD) containing 5% heat-inactivated fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1% antibiotic/antimycotic solution (Life Technologies).
- FBS heat-inactivated fetal bovine serum
- BALC cells 1.5 x IO 4 cells/cm 2
- TRAP-positive cell quantitation At the termination of the experiments, the cultures were fixed with 3.7% formalin for 10 min., and stained for TRAP as described by John et al . ⁇ Endocrinology, 137:2457-2463 (1996)) . The number of TRAP-positive multinucleated cells (containing 3 or more nuclei), indicative of osteoclasts, was counted in each well.
- the 5.9kb fragment of the human OPG promoter (pOPG5.9 ⁇ gal) as well as sequential 5 ' -deletions of the promoter (pOPG3.6 ⁇ gal, pOPG1.9 ⁇ gal, pOPG1.5 ⁇ gal, pOPG0.9 ⁇ gal, pOPG0.4 ⁇ gal, and pOPG0.2 ⁇ gal) linked to the ⁇ -galactosidase ( ⁇ -gal) reporter gene in p ⁇ gal-Basic reporter vector (Clontech, Palo Alto, CA) , were prepared as described above in Example 2.
- proximal OSE 2 element Site-directed mutagenesis of the proximal OSE 2 element, the API-like element, and the Smad binding element (SBE) were performed using the two-step PCR strategy (Ausubel et al . , 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y., Chapter 8.5) .
- the proximal OSE 2 core element (AACCTCA, at position -309 to -303; SEQ ID NO: 28) in the pOPGO .9 ⁇ gal construct (in the p ⁇ gal-Basic vector backbone) was substituted with a random sequence of 6 nucleotides (AGATATC : EcoRV recognition site, underlined; SEQ ID NO: 29) .
- the API-like element (GGAGACA, at position -293 to -287; SEQ ID NO: 30) was substituted with a 6-nucleotide random sequence (CTCGAGA: Xhol recognition site, underlined; SEQ ID NO-.31), and the SBE element (CAGACA, at position -230 to -225; SEQ ID NO: 32) was substituted with another 6-nucleotide random sequence (GAATTC: EcoRI recognition site; SEQ ID NO: 33) .
- the mutant primers used for one of the two first-step PCR reactions were 5 '-etc ate aat gta tct tat gg-3 ' (p ⁇ gal-F: vector primer; SEQ ID NO: 17) with either 5 ' -get gtc tec gcg ggg etc gat ate ttc ccg gcc ect tec cgc c-3 ' (OSEmutRev; SEQ ID NO: 18), or 5 ' -gag gaa caa ggc ggc tgc tct cga ggc ggg get ctg agg ttt cc-3' (OPGAPlMr; SEQ ID NO:34), or 5 ' -gaggg tgg ggc ggt ggg aat teg ccc ctg gga gag cag ggg a-3' (SBEmutRev;
- PCR reaction was performed with flanking primers (p ⁇ gal-F and p ⁇ gal-R), using the two first-step PCR products as template.
- flanking primers p ⁇ gal-F and p ⁇ gal-R
- the second-step PCR product was digested with Asp718 and Bglll, and ligated to p ⁇ gal-Basic vector containing the same restriction ends.
- the 0.4kb region (-372 to +19; SEQ ID NO: 6) containing the mutation was PCR amplified from pOPG0.9OSEmut ⁇ gal, pOPGO .9APlmut ⁇ gal , and pOPGO .9SBEmut ⁇ gal, respectively, using the primers, 5 ' -ata ggt ace gcc cag ccc tec cac cgc tgg t-3' (OPG392AspF; SEQ ID NO: 21) and p ⁇ gal-R
- PCR amplification was performed using OPG392AspF as forward primer and 100OPGOG2 (5' -ata tag ate tga ctt gtc tgt tec tgc ace etc cag cat cca gta gca ttt ata teg ccc agg gag gtg ggg cgt ga-3'; SEQ ID NO:24) as reverse primer (containing a Bglll restriction site and the osteocalcin -34/+13 sequence at the 5' end) .
- pOPGO .4 ⁇ gal served as template.
- the resultant PCR products were digested with Asp718 and Bglll, and ligated to p ⁇ gal-Basic vector that was digested with the same enzymes, to generate the (-372 to -190 OC ⁇ gal) construct.
- Substitution mutations in the OSE2 , API-like, and SBE elements were created in (-372 to -190 OC ⁇ gal) by PCR amplification using the appropriate mutant pOPGO .4 ⁇ gal construct as template, and OPG392AspF (SEQ ID NO:21) and 100OPGOG2 (SEQ ID NO:24) as forward and reverse primers.
- the -34/+13 OC ⁇ gal construct was created by annealing the OG2For (5 '-tat agg tac ccg ata taa atg eta ctg gat get gga ggg tgc agg aac aga caa gtc aga tct ata t-3'; SEQ ID NO: 26) and OG2Rev (5 '-ata tag ate tga ctt gtc tgt tec tgc ace etc cag cat cca gta gca ttt ata teg ggt ace tat a-3 ' ; SEQ ID NO: 27) oligonucleotides, digesting the double-stranded oligonucleotide with Asp718 and Bglll, and ligating them to p ⁇ gal-basic digested with the same enzymes.
- Double mutants (OSE 2 and SBE) were created utilizing the same two-step PCR strategy, with a template containing 0SE 2 mutation and primers containing SBE mutation.
- pEF/myc/cyto control vector
- pEF-Cbfal containing the coding region for the Cbfal isoform starting with amino acids MASNS (SEQ ID NO: 38) and ending with VWRPY (SEQ ID NO: 39) (Osf2Met 69 ) (Thirunavukkarasu et al . , Mol . Cell Biol . , 18:4197-4208 (1998)) was generated as described in Thirunavukkarasu et al . , J. Biol . Chem. , 275 (33 ): 25163-25172 (2000) . The integrity of all plasmid constructs was confirmed by restriction mapping and automated DNA sequencing.
- the GCG Wisconsin Package (Genetics Computer Group, Inc., Madision, WI) was used to analyze the 5.9kb OPG promoter for the presence of consensus transcription factor binding sites, including API sites, Smad-binding elements (SBE), OSE 2 (Cbfal- binding element), and other elements.
- F-12 (3:1) (GIBCO BRL), supplemented with 10% fetal bovine serum, 50 mM Hepes, and 2 mM glutamine. All cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% C0 2 . Experiments were initiated when cells were approximately 70-80% confluent.
- Cells were then treated with either vehicle or TGF- ⁇ (lOng/ml), for the indicated times. After the treatments, cells were lysed in lOO ⁇ l of lysis buffer (Roche) , and ⁇ -gal activity was assayed in a fixed amount of the extracts (1/5 of the extracts) using the 5,493,531) can be used to score for the presence of specific mutations by development or oss of a ribozyme cleavage site.
- genetic mutations in TSRX can be identified by hybndizmg a sample and control nucleic acids, e g., DNA or RNA, to high-density anays containing hundreds or thousands of ohgonucleotides probes. See, e.g., Cronm, et al., 1996. Human Mutation 7: 244-255; Kozal, et al., 1996. Nat Med. 2: 753-759.
- genetic mutations in TSRX can be identified in two dimensional anays containing light-generated DNA probes as described in Cron , et al., supra Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations This is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
- any of a variety of sequencing reactions known in the art can be used to directly sequence the TSRX gene and detect mutations by comparing the sequence of the sample TSRX with the corresponding wild-type (control) sequence.
- Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977. Proc Natl Acad Sci USA 74 560 or Sanger, 1977 Proc Natl Acad Sci USA 74
- any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (see, e g , Naeve, et al , 1995 Biotechniques 19 448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No WO 94/16101 ; Cohen, et al, 1996. Adv Chromatography 36- 127-162; and Griffin, et al , 1993. Appl Biochem. Bwtechnol 38: 147-159).
- RNA/RNA or RNA/DNA heteroduplexes Other methods for detecting mutations in the TSRX gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes. See, e g , Myers, et al., 1985. Science 230: 1242.
- the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type TSRX sequence with potentially mutant RNA or DNA obtained from a tissue sample The double-stranded duplexes are treated analyzed using Student's t test, and probability (p) values of less than 0.05 were considered statistically significant.
- RNA Isolation and Northern blot analysis BALC and UMR106 cells were plated in T150 flasks and allowed to grow until the cells reached 70-80% confluence. They were serum starved overnight (medium containing 0.1 % serum), and then treated with TGF- ⁇ (lOng/ml) for the indicated periods of time. Cell culture samples were pooled into treated or control groups for each indicated time point after treatment. Total RNA was extracted from the cells using Ultraspec-IITM reagent as recommended by the manufacturer (Biotecx, Houston, TX) . Poly A + RNA was isolated from total RNA using Oligotex resin (Qiagen, Santa Clarita, CA) according to the manufacturer's protocol, and quantified by spectrophotometry .
- Oligotex resin Qiagen, Santa Clarita, CA
- OPG cDNA (Onyia et al . , J. Bone and Mineral Res . , 15 (5 ): 863-871 (2000)) and mouse RANK ligand cDNA were used to generate radioactive probes using the Random Primer DNA labeling kit (GIBCO BRL) .
- the mouse RANK ligand cDNA was amplified by RT-PCR from BALC cell mRNA using the primers 5 ' -ate aga aga cag cac tea ct-3 ' (0DF1; SEQ ID NO: 40) and 5 '-ate tag gac ate cat get aat gtt c-3' (ODF2; SEQ ID N0.41) .
- Rat GAPDH cDNA probe (Onyia et al . , J. Bone and Mineral Res . , 15 ( 5 ): 863-871 (2000)) was used as a control for RNA quantitation and integrity. Twenty-five nanograms of cDNA were labeled using 32 P-dCTP (Amersham) , and free nucleotides were removed by centrifugation through a Centricon-50 column (Amicon) . OPG and RANK ligand mRNA expression was analyzed by northern blotting. Prehybridization and hybridization were carried out at 48°C in NorthernMax buffers (Ambion, Inc.,
- OPG ELISA assay In order to quantify the amount of OPG secreted into the cell culture medium, BALC (30,000 cells/well in a 24-well plate) and UMR106 cells (40,000 cells/well in a 96-well plate) were treated with TGF- ⁇ (10 ng/ml) and incubated for 72 hours. The amount of OPG secreted into the culture medium was analyzed using a sandwich ELISA procedure, utilizing rabbit polyclonal antiserum directed against recombinant human OPG, as described in Onyia et al . , J. Bone and Mineral Res . , 15:863-871 (2000) and Thirunavukkarasu et al . , J " . Biol . Chem . , 275(33) :25163-25172 (2000).
- TGF- ⁇ stimulates the expression of endogenous OPG in stromal (BALC) and osteoblastic (UMR106) cell lines
- the methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits compnsing at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e g , in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a TSRX gene
- any cell type or tissue preferably penpheral blood leukocytes, in which TSRX is expressed may be utilized m the prognostic assays described herein
- any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells
- TSRX activity e g , TSRX gene expression
- a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (e g , , cancer, rheumatoid arthritis and ocular neovasulansation )
- the pharmacogenomics i e , the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug
- the pharmacogenomics of the individual permits the selection of effective agents (e g , drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype
- Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens Accordingly, the activity of TSRX protein, expression
- Pharmacogenomics deals with clinically significant hereditary vanations in the response to drugs due to altered drug disposition and abnormal action in affected persons See e g , Eichelbaum, 1996 Clin Exp Pharmacol Physiol , 23 983-985, Linder, 1997 Clin Chem , 43 254-266
- two types of pharmacogenetic conditions can be had either a low, medium, or high basal expression of ⁇ -gal activity.
- Treatment of these clones with TGF- ⁇ led to a dose- dependent (optimal 10 ng/ml) and time-dependent (optimal 24-48 hours) increase in OPG promoter activity in all three clones.
- TGF- ⁇ isoforms and members of the TGF- ⁇ superfamily on OPG promoter activity
- TGF- ⁇ TGF- ⁇
- BMP-4 Bone Morphogenetic Protein-4
- 0.4kb did not significantly decrease TGF- ⁇ responsiveness, while deletion of the region between 0.4 and 0.2kb (-372 to -190 nucleotides) completely abolished the response to TGF- ⁇ ( Figure 18B) . Therefore, there are proximal regions and a distal region in the 5.9kb OPG promoter that each contribute to a majority of TGF- ⁇ responsiveness. Furthermore, the proximal region is necessary for responsiveness. This indicates a role for both the distal ( ⁇ -1.9 kb to -1.5kb) and proximal (0.4 to 0.2kb) promoter regions in TGF- ⁇ regulation of OPG expression.
- the -372 to -190 nucleotide region of the OPG promoter confers TGF- ⁇ responsiveness to a heterologous minimal promoter
- the invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant TSRX expression or activity
- Disorders associated with aberrant TSRX expression of activity include for example, cancer, rheumatoid arthntis and ocular neovasulansation
- Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner
- Therapeutics that may be utilized include, but are not limited to (i) an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof, (n) antibodies to an aforementioned peptide, extended nucleic acids encoding an aforementioned peptide, (iv) administration of antisense nucleic acid and nucleic acids that are "dysfunctional" (.
- Therapeutics that increase (i.e., are agonists to) activity may be administered in a therapeutic or prophylactic manner.
- Therapeutics that may be utilized include, but are not limited to, an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability.
- Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of an aforementioned peptide).
- tissue sample e.g., from biopsy tissue
- assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of an aforementioned peptide).
- Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, and the like).
- immunoassays e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.
- hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, and the like).
- the invention provides a method for preventing, in a subject, a disease or condition associated with an abenant TSRX expression or activity, by administering to the subject an agent that modulates TSRX expression or at least one TSRX activity.
- Subjects at risk for a disease that is caused or contributed to by aberrant TSRX expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein.
- Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the TSRX abenancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
- a TSRX agonist or TSRX antagonist agent can be used for treating the subject.
- the appropriate agent can be determined based on screening assays described herein. The prophylactic methods of the invention are further discussed in the following subsections. factors binding to the 0SE 2 and SBE in the regulation of OPG promoter activity mediated by Cbfal and TGF- ⁇ .
- TGF- ⁇ treatment resulted in a dose- and time-dependent stimulation of OPG promoter activity.
- the effects were mimicked by two of the isoforms of TGF- ⁇ (TGF- ⁇ 2 and TGF- ⁇ 3) , but not by BMP-4.
- TGF- ⁇ 2 and TGF- ⁇ 3 TGF- ⁇ 2 and TGF- ⁇ 3
- BMP-4 BMP-4 could not induce OPG promoter activity, suggesting a TGF- ⁇ signal-specific effect.
- Smad 2 and 3 TGF- ⁇ signal-specific Smad proteins
- Smad4 common Smad
- Extensive deletion analysis of the 5.9kb OPG promoter facilitated identification of a proximal 183bp region (-372 to -190 nucleotides; SEQ ID NO: 10) that appears to be necessary for imparting TGF- ⁇ responsiveness to the promoter.
- the same region was sufficient to confer TGF- ⁇ responsiveness to the heterologous osteocalcin promoter.
- This region includes, among others, a Cbfal-binding element (OSE 2 ) , an API-like binding element (an API element is known to mediate TGF- ⁇ effects on the TGF- ⁇ and c-jun gene promoters (Kim et al . , Mol . Cell Biol .
- TGF- ⁇ could increase Cbfal expression in UMR106 cells that in turn would functionally interact with the Smad proteins and contribute to activation of the OPG promoter. Since TGF- ⁇ has been shown to regulate expression of various target genes through a variety of different response elements (Roberts, Mineral and Electrolyte Metabolism, 24:111-119 (1998)), it is also possible that other known or novel elements in the 183bp region, either alone or in combination with factors binding to the OSE 2 and SBE sites, could mediate TGF- ⁇ effects. Further studies are required to gain a more complete understanding of the molecular interactions involved in mediating TGF- ⁇ stimulation of OPG promoter activity.
- the preceding data provide evidence that TGF- ⁇ directly stimulates OPG promoter activity in the osteoblast- like osteosarcoma cell line UMR106, and that the effects of TGF- ⁇ are mediated by a 183bp proximal region in the promoter. Consensus DNA-binding sites for Cbfal, Smad proteins, and possibly other elements present in this region could potentially mediate the full complement of TGF- ⁇ stimulation of the promoter. Identification of factors that modulate OPG promoter activity and the cognate elements that mediate the effects can aid in the design of novel strategies to regulate bone resorption in pathological situations characterized by high bone turnover .
- PCR products having an approximate size of 1 kbp were isolated from agarose gel and ligated to pCR2.1 vector (Invitrogen, Carlsbad, CA). The cloned inserts were sequenced, using vector specific, Ml 3 Forward(-40) and Ml 3 Reverse primers as well as the gene specific primers.
- Thes primers include:
- 17897469 S4 GTC GCA GTG ACG GCT AGA (SEQ ID NO: 18) 17897469 S5 : TGC CGT CCA GAC ACG GTG (SEQ ID NO: 19) and
- cloning is described for a fragment of the 17897469.0.7 clone.
- Oligonucleotide primers were designed to PCR amplify a DNA fragment coding for residues 13-634 of clone 17897469.0.7.
- the forward primer includes an in frame BamHI restriction site and the reverse primer contains an in frame Xhol restriction site.
- the sequences of the PCR primers are the following: forward: GGATCCTCCATAAATGGAGCTTATTGGGAG (SEQ ID NO:21) and reverse:CTCGAGCAGGGCCTCCGTGCACTCGTGCGACGC (SEQ ID NO:22)
- PCR reactions were performed using a total of 5ng of a mixture containing equal amounts of cDNA derived from human fetal brain, human testis, human mammary and human skeletal muscle tissues, and 1 mM of each of the forward and reverse primers, 5 mM of dNTP (Clontech Laboratories, Palo Alto CA) and 1 mL of 50xAdvantage-HF 2 polymerase (Clontech Laboratories, Palo Alto CA) in 50 microliter volume.
- the reaction conditions described in EXAMPLE 1 were used , except that step (g) was extended to 3 minutes per cycle.
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Abstract
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Citations (6)
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EP0816380A1 (en) * | 1995-02-20 | 1998-01-07 | Snow Brand Milk Products Co., Ltd. | Novel protein and methods for the production of the same |
EP0874045A1 (en) * | 1996-08-19 | 1998-10-28 | Snow Brand Milk Products Co., Ltd. | Novel dnas and process for producing proteins by using the same |
WO2000042216A2 (en) * | 1999-01-18 | 2000-07-20 | Osteometer Biotech A/S | Genetic predisposition to abnormal calcification conditions |
WO2001004137A1 (en) * | 1999-07-09 | 2001-01-18 | Genaissance Pharmaceuticals, Inc. | Drug target isogenes: polymorphisms in the osteoclastogenesis inhibitory factor gene |
WO2001007611A2 (en) * | 1999-07-26 | 2001-02-01 | Genentech, Inc. | Novel polynucleotides and method for the use thereof |
WO2001023559A1 (en) * | 1999-09-27 | 2001-04-05 | Eli Lilly And Company | Osteoclast differentiation factor regulatory region |
-
2000
- 2000-09-26 AU AU77206/00A patent/AU7720600A/en not_active Abandoned
- 2000-09-26 WO PCT/US2000/026497 patent/WO2001023562A2/en active Application Filing
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
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EP0816380A1 (en) * | 1995-02-20 | 1998-01-07 | Snow Brand Milk Products Co., Ltd. | Novel protein and methods for the production of the same |
EP0874045A1 (en) * | 1996-08-19 | 1998-10-28 | Snow Brand Milk Products Co., Ltd. | Novel dnas and process for producing proteins by using the same |
WO2000042216A2 (en) * | 1999-01-18 | 2000-07-20 | Osteometer Biotech A/S | Genetic predisposition to abnormal calcification conditions |
WO2001004137A1 (en) * | 1999-07-09 | 2001-01-18 | Genaissance Pharmaceuticals, Inc. | Drug target isogenes: polymorphisms in the osteoclastogenesis inhibitory factor gene |
WO2001007611A2 (en) * | 1999-07-26 | 2001-02-01 | Genentech, Inc. | Novel polynucleotides and method for the use thereof |
WO2001023559A1 (en) * | 1999-09-27 | 2001-04-05 | Eli Lilly And Company | Osteoclast differentiation factor regulatory region |
Non-Patent Citations (7)
Title |
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BRANDSTROM H ET AL: "POLYMORPHISM IN THE PROMOTER REGION OF THE HUMAN GENE FOR OSTEOPROTEGERIN: CORRELATION WITH BONE MINERAL DENSITY" JOURNAL OF BONE AND MINERAL RESEARCH,NEW YORK, NY,US, vol. 14, no. SUPPL. 01, September 1999 (1999-09), page S334 XP000915449 ISSN: 0884-0431 * |
DATABASE EMBL SEQUENCE DATABSE [Online] Hinxton, GB; 6 August 1998 (1998-08-06) T. MORINAGA: "Homo sapiens gene for osteoclstogenesis inhibitory factor, exon 1" XP002169892 * |
HOFBAUER L C ET AL: "OSTEOPROTEGERIN AND ITS COGNATE LIGAND: A NEW PARADIGM OF OSTEOCLASTOGENESIS" EUROPEAN JOURNAL OF ENDOCRINOLOGY,NO,SCANDINAVIAN UNIVERSITY PRESS, vol. 139, no. 2, August 1998 (1998-08), pages 152-154-190, XP000876579 ISSN: 0804-4643 * |
LACEY D L ET AL: "Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation" CELL,US,CELL PRESS, CAMBRIDGE, NA, vol. 93, no. 2, 17 April 1998 (1998-04-17), pages 165-176, XP002117567 ISSN: 0092-8674 * |
MORINAGA TOMONORI ET AL: "Cloning and characterization of the gene encoding human osteoprotegerin/osteoclastogenesis-inhibit ory factor." EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 254, no. 3, June 1998 (1998-06), pages 685-691, XP002934581 ISSN: 0014-2956 * |
SIMONET W ET AL: "Osteoprotegerin: a novel secreted protein involved in the regulation of bone density" CELL,US,CELL PRESS, CAMBRIDGE, NA, no. 89, 18 April 1997 (1997-04-18), pages 309-319, XP002077048 ISSN: 0092-8674 * |
YASUDA HISATAKA ET AL: "Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): A mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro." ENDOCRINOLOGY, vol. 139, no. 3, March 1998 (1998-03), pages 1329-1337, XP002934584 ISSN: 0013-7227 * |
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