WO2001023559A1 - Zone de regulation de facteurs de differenciation des osteoclastes - Google Patents

Zone de regulation de facteurs de differenciation des osteoclastes Download PDF

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Publication number
WO2001023559A1
WO2001023559A1 PCT/US2000/026407 US0026407W WO0123559A1 WO 2001023559 A1 WO2001023559 A1 WO 2001023559A1 US 0026407 W US0026407 W US 0026407W WO 0123559 A1 WO0123559 A1 WO 0123559A1
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Prior art keywords
expression
differentiation factor
osteoclast differentiation
human
regulatory region
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PCT/US2000/026407
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English (en)
Inventor
Srinivasan Chandrasekhar
David Lloyd Halladay
Thomas John Martin
Rebecca Ruth Miles
Jude Emeka Onyia
Kannan Thirunavukkarasu
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Eli Lilly And Company
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Priority to AU76156/00A priority Critical patent/AU7615600A/en
Publication of WO2001023559A1 publication Critical patent/WO2001023559A1/fr

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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
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154

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 osteoclast differentiation factor. 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); and Morinaga et al . , Eur. J. Biochem . , 254:685-691 (1998). Overexpression of OPG in transgenic mice inhibits osteoclast formation, causing osteop ⁇ trosis . Similarly, treatment of ovarectomized (OVX) rats with OPG prevents bone loss. See
  • ODF osteoclast differentiation factor
  • ODF expression is regulated in osteoblast/stromal cells by factors that increase osteoclast formation. See Tsukii et al . , Biochem . Biophys . Res . Comm . , 246:337-341 (1998).
  • Three other research groups independently cloned a molecule identical to ODF. The first group named the molecule TRANCE (TNF-related activation induced cytokine) , and demonstrated that it regulated T-cell-dependent immune responses and activated c-jun N-terminal protein kinases (JNK) . See Wong et al . , J. Exper . Med. , 186:2075-2080 (1997).
  • the second group identified the molecule in T-cells as a ligand for receptor activation of NF-KB (RANK) , a TNFR family protein expressed in dendritic cells.
  • RANK NF-KB
  • a third group cloned the same molecule and called it OPG ligand (OPGL) .
  • OPG OPG ligand
  • OPGL OPG ligand
  • ODF osteoclast formation, function, and, therefore, bone resorption. Consequently, compounds that alter ODF expression have significant therapeutic value.
  • Therapeutic compounds can be identified via in vi tro screening assays. While the regulatory region of murine ODF has been partially characterized (Kodaira et al . , Gene 230:121-127 (1999); Kitazawa et al . , Biochimica et Biophysica Acta, 1445:134-141 (1999)), this sequence is undesirable for use in screening assays used to identify compounds that alter human ODF expression.
  • Other publications disclosing aspects of ODF gene structure/expression include Lacey et al .
  • Effective screening assays evaluate test compounds by comparing: (1) a compound's effect on expression and (2) a baseline that represents a normal level of expression. Screening for compounds that affect a level of expression that is not the norm, such as that stimulated by a partial regulatory region derived from another species, is of little value in human pharmaceutical development. In this regard, an effective screening assay for identifying physiologically-relevant ligands preferably requires a gene's complete regulatory region. Therefore, there exists a need to fully characterize the transcriptional and translational regulatory region of the human odf gene.
  • the present invention provides the nucleotide sequence of the complete regulatory region for the human odf gene.
  • the present invention provides a method of identifying compounds that affect osteoclast formation, bone resorption, or immune responsiveness.
  • the present invention provides methods of diagnosing bone, immune, and arterial diseases in a patient .
  • a DNA sequence that represents the complete regulatory region of the human odf gene there is provided, in accordance with one aspect of the present invention, a DNA sequence that represents the complete regulatory region of the human odf gene. DNA constructs containing the ODF regulatory region also are provided.
  • a method for identifying a compound that affects osteoclast formation and/or bone resorption comprising:
  • DNA from bone cells of the patient with the presently disclosed DNA sequence there are also provided methods for diagnosing immune and arterial diseases in a patient, comprising comparing the DNA from bone cells of the patient with the presently disclosed DNA sequence.
  • 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 the presently disclosed DNA sequence.
  • methods of identifying in a patient a predisposition to developing bone or immune disease comprising comparing the DNA from bone cells of said patient with the presently disclosed DNA sequence .
  • methods of modulating bone resorption or immune responsiveness in a patient comprising administering to the patient a DNA construct of the present invention, wherein the construct comprises a polynucleotide encoding osteoclast differentiation factor.
  • methods of modulating bone resorption or immune responsiveness in a patient comprising administering to the patient one or more compounds identified using the methods of the presently disclosed invention.
  • methods of modulating ODF expression in a cell, in vi tro or in vivo using one or more compounds identified using a screening assay of the present invention.
  • Pharmaceutically effective amounts of compounds for in vivo use can be determined by routine methods well known the pharmaceutical arts, such as by establishing dose-response relationships in subjects.
  • the present invention provides an isolated nucleic acid fragment comprising the transcriptional regulatory region of the human odf gene, a subfragment thereof, or a functional variant of either, exhibiting human odf gene transcriptional regulatory activity, excluding the odf 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, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, 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 NO:l, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 in IX phosphate buffer comprising 0. IM Na 2 HP0 4 , 0.5M NaCl, 0.0052 M EDTA, pH 7.0, and
  • 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 NO:l, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, wherein said fragment exhibits human odf 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 osteoclast differentiation factor 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 osteoclast differentiation factor.
  • the present invention provides a composition, comprising any of the foregoing isolated nucleic acid fragments or subfragments, 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 osteoclast differentiation factor, comprising:
  • 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 N0:1, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, or a functional variant thereof, and a reporter polynucleotide operably linked thereto, and wherein said reporter polynucleotide is 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;
  • 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 osteoclast differentiation factor, comprising: (a) contacting a test compound, and a host cell comprising:
  • a plasmid comprising a nucleic acid fragment or subfragment selected from the group consisting of SEQ ID NO : 1 , SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO : 14 , and SEQ ID NO: 15, 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 osteoclast differentiation factor expression, wherein both said reporter polynucleotide and said factor required for osteoclast differentiation factor expression are 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 factor required for osteoclast differentiation factor expression can be osteoblast specific transcription factor 2
  • 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 osteoclast differentiation factor 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 osteoclast differentiation factor expression.
  • the present invention provides an agonist or antagonist of osteoclast differentiation factor 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 osteoclast differentiation factor .
  • the present invention provides the use of a compound that modulates expression of osteoclast differentiation factor in the manufacture of a medicament for the treatment of a disease in a human caused by abnormal expression of osteoclast differentiation factor.
  • 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 osteoclast differentiation factor.
  • 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 antagonist of osteoclast differentiation factor expression.
  • the arterial disease can be arterial calcification, and the compound can be an antagonist of osteoclast differentiation factor expression.
  • the bone disease is osteopetrosis
  • the compound can be an agonist of osteoclast differentiation factor expression.
  • the human can be diagnosed as having a polymorphism or mutation at one or more nucleotide positions in the osteoclast differentiation factor regulatory region in DNA thereof .
  • the present invention provides a composition, comprising an agonist or antagonist of osteoclast differentiation factor expression, and a 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 bone morphogenetic protein 2, l ⁇ , 25-dihydroxy vitamin D 3 , dibutyryl cyclic AMP, dexamethasone, IL-11, IL-17, prostaglandin E 2 , parathyroid hormone, or the molecules disclosed in PCT International Publication WO 00/15807.
  • 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 osteoclast differentiation factor 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 bone morphogenetic protein 2, l ⁇ , 25-dihydroxy vitamin D 3 , dibutyryl cyclic AMP, dexamethasone, IL-11, IL-17, prostaglandin E 2 , parathyroid hormone, or the molecules disclosed in PCT International Publication WO 00/15807.
  • the agonist or antagonist can be identified by any one of the methods discussed above.
  • the pharmaceutical pack can comprise instructions for administration of the agonist or antagonist to a human.
  • the agonist or antagonist can be identified by any of the methods discussed above.
  • the human can be diagnostically tested for a polymorphism or mutation at one or more nucleotide positions in the osteoclast differentiation factor regulatory region in DNA thereof.
  • the present invention provides a process for making an agonist or antagonist of osteoclast differentiation factor expression, comprising:
  • the present invention provides a method of preparing a medicament for the treatment of a bone disease, arthritis, arterial disease, abnormal immune function, abnormal lymph node development, abnormal T- or B- cell function, or other disease in a human caused by abnormal osteoclast differentiation factor expression, comprising: (a) identifying an agonist or antagonist of osteoclast differentiation factor expression by any of the methods discussed above; and (b) formulating said agonist or antagonist as a medicament .
  • the present invention provides a method of identifying a mutation or polymorphism in the osteoclast differentiation factor regulatory region of a human subject's or patient's odf gene, comprising comparing the nucleotide sequence of the osteoclast differentiation factor regulatory region of the odf gene in DNA from said subject or patient with a nucleotide sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, wherein any difference in nucleotide sequence between said osteoclast differentiation factor regulatory region DNA and said nucleotide sequence identifies a mutation or polymorphism in the osteoclast differentiation factor regulatory region of said subject's or patient's DNA.
  • 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 osteoclast differentiation factor expression, comprising comparing the nucleotide sequence of the osteoclast differentiation factor regulatory region of the odf gene in DNA from said subject or patient with a nucleotide sequence selected from the group consisting of SEQ ID N0:1, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, wherein any difference in nucleotide sequence between said osteoclast differentiation factor regulatory region DNA and said nucleotide sequence identifies a mutation or polymorphism in the osteoclast differentiation factor 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 osteoclast differentiation factor expression with a compound that affects osteoclast differentiation factor expression through an interaction with the osteoclast differentiation factor gene regulatory region, comprising comparing the nucleotide sequence of the osteoclast differentiation factor regulatory region of the odf gene from DNA of said subject or patient with a nucleotide sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO: 11, SEQ ID NO : 12 , SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, wherein any difference in nucleotide sequence between said osteoclast differentiation factor regulatory region DNA and said nucleotide sequence identifies a mutation or polymorphism in the osteoclast differentiation factor 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 osteoclast differentiation factor, comprising administering to said human a pharmaceutically effective amount of an antagonist of osteoclast differentiation factor 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 osteoclast differentiation factor, comprising administering to said human a pharmaceutically effective amount of an agonist of osteoclast differentiation factor 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 osteoclast differentiation factor expression, comprising: (a) determining whether a polymorphism or mutation exists at one or more nucleotide sites in the osteoclast differentiation factor 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 agonist of osteoclast differentiation factor expression.
  • the present invention provides a method of treating a human in need of treatment with an antagonist of osteoclast differentiation factor expression, comprising:
  • the human can be suffering from a symptom, condition, or disease caused by an abnormal level of expression of osteoclast differentiation factor.
  • 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 osteoclast differentiation factor.
  • 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 osteoclast differentiation factor.
  • 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 odf gene, a subfragment thereof, or functional variant of either exhibiting human odf gene transcriptional regulatory activity, wherein said fragment, subfragment, or functional variant thereof excludes the odf 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, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.
  • 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 osteoclast differentiation factor, 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 osteoclast differentiation factor, at least one translational regulatory region required for expression of said human osteoclast differentiation factor 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 odf gene, a subfragment thereof, or a functional variant of either, exhibiting osteoclast differentiation factor transcriptional regulatory region activity, wherein said fragment, subfragment thereof, or functional variant thereof excludes the odf protein coding region.
  • the nucleotide sequence can be selected from the group consisting of SEQ ID NO:l, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.
  • the present invention provides a diagnostic method, comprising determining the nucleotide sequence of the osteoclast differentiation factor transcriptional regulatory region in DNA from a human, or a diagnostically useful fragment thereof, and comparing said nucleotide sequence to a nucleotide sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 provided in a computer readable medium, thereby identifying any polymorphism or mutation in said osteoclast differentiation factor transcriptional regulatory region in said DNA from said human.
  • Figure 1 provides the nucleotide sequence of the human ODF regulatory region.
  • the entire sequence shown in Figure 1 is 4.628 kilobases, and starts proximally at the ATG translational start site (in bold face) at the 3' end of the sequence.
  • the initial 31 nucleotides shown in bold at the 5' end of the nucleotide sequence shown in Figure 1 are from the PI vector (pAdlOSacBII) component of the human PI library, discussed below.
  • the nucleotide sequence of the human ODF regulatory region per se is 4.597 kilobases, and has the following sequence (SEQ ID NO:l):
  • TATAA box in bold face, italicized, and underlined
  • +1 site the G residue 49 nucleotides downstream of the TATAA box, shown in bold, and underlined
  • potential OSE-2 sites binding sites for osteoblast specific factor 2, Osf2/CBFAl
  • Sstl restriction endonuclease site underlined
  • the cloned ODF regulatory region sequence excluded the sequence 5 ' -CGAAGCGAGAGGGCCGAGCGCCATG-3 ' (SEQ ID NO: 2) after the Sstl site, containing the ODF ATG start codon.
  • Figure 2 schematically depicts the ODF regulatory region 5' deletion constructs of Example 1.
  • Figure 3 graphically depicts basal expression achieved by the ODF regulatory region deletion constructs of Example 1 in UMR106 cells.
  • Figure 4 shows the effect of Osf2 on ODF regulatory region expression in COS1 cells using the regulatory region deletion constructs of Example 1.
  • the present invention provides a nucleic acid fragment containing the complete transcription regulatory region of the human odf gene.
  • the novel 4.6 kb regulatory region contains several regulatory elements that are utilized by a variety of transcription factors to influence ODF expression.
  • Potential OSE-2 sites binding sites for osteoblast specific transcription factor 2 (Osf2; CBFA1) are shown in bold face and underlined in Figure 1. Table 1 lists these sequences. Table 1 .
  • the presently disclosed ODF regulatory region may contain other transcriptional regulatory elements and one or more translational regulatory elements .
  • ODF regulatory region refers to the DNA region (or fragments thereof) upstream of, and which regulates the transcription of, the odf gene structural nucleic acid sequence that codes on expression for the ODF protein. This region can include the ATG start codon. Thus, these terms exclude the structural nucleic acid sequence (exons) encoding the ODF protein, or fragments thereof, except for the ATG start codon. Also as used herein, the terms “nucleic acid fragment” or “fragment” exclude 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. Further examples of 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 presently disclosed ODF regulatory region, fragments thereof such as those described in Example 1 below, and functional variants of either, provide invaluable tools for regulating osteoclastogenesis .
  • the disclosed ODF regulatory region, fragments thereof, or functional variants of either can be used in screening assays to identify drugs that regulate bone balance, bone loss, or which can be used to treat metabolic bone diseases, such as osteoporosis, osteopetrosis, Paget's disease, rheumatoid arthritis, osteoarthritis, 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., Nature, 397:315-323, (1999)).
  • ODF 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 ODF 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 ODF regulatory region, fragments thereof, or functional variants of either can be used in expression vectors to control the expression in vi tro or in vivo of a protein or reporter of interest.
  • the disclosed regulatory element, fragments thereof, or functional variants of either can be used to identify, isolate, and clone cis-elements and interacting trans-factors.
  • transcription regulatory region and “regulatory region” refer to the section of DNA located upstream of the sequence encoding a protein and which regulates gene transcription.
  • a regulatory region may include a variety of cis-acting elements, including, but not limited to, promoters, enhancers, and hormone response elements.
  • the first approach utilized the GenomeWalker kit (Clonetech, Palo Alto, CA) .
  • the polymerase chain reaction (PCR) was used to "walk" upstream of known DNA sequences.
  • the following gene-specific primers were designed from the published ODF cDNA sequence (GenBank #AF019047) :
  • primers specific to the GenomeWalker library adaptor were used in combination with primers specific to the GenomeWalker library adaptor to amplify a 1 kb fragment of DNA upstream from the human odf gene. This fragment was subcloned into a pCR2.1 vector (Invitrogen, Carlsbad, CA) , and designated pODF5.
  • 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 ODF cDNA (#AF019047) .
  • the 4603 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 4603 bp fragment was cloned into the pSPORTl vector (Life Technologies, Inc., Rockville, MD) .
  • the nucleotide sequence of the 4603 bp Not I/Ss l fragment is provided in Figure 1 (SEQ ID NO:l) .
  • 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 ODF regulatory region (SEQ ID NO:l), or the complement of fragments thereof, i.e., the complements of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, under mild to stringent hybridization conditions.
  • SEQ ID NO:l ODF regulatory region
  • the following conditions illustrate one example of a mildly stringent hybridization condition: Hybridization: IX phosphate buffer, (comprising 0. IM
  • Second-Fifth Washes (if needed): ImM Tris-HCI, pH 8.0, for approximately 10-15 minutes each.
  • nucleic acid sequences of the present invention identified by nucleic acid hybridization can hybridize fully, i.e., along their entire length, to SEQ ID NO: 1
  • Functional variants can also be identified in silico by comparing their structural similarity, or sequence homology (sequence identity), to the presently disclosed ODF regulatory region or fragments thereof.
  • 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 identity, more preferably at least about 65% sequence identity, more preferably at least about 70 % sequence identity, and even more preferably about, or at least about, 75% sequence identity to SEQ ID N0:1 using the Smith-Waterman algorithm. More preferred variant polynucleotides have at least about 80% sequence identity, more preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, more preferably at least about 95% sequence identity, more preferably at least, or at least about 98% sequence identity, and even more preferably at least about 99% sequence identity to SEQ ID N0:1, or fragments thereof disclosed herein.
  • the polynucleotide comprises DNA having at least about 50% sequence identity, preferably at least about 51% sequence identity, more preferably at least about 52% sequence identity, yet more preferably at least about 53% sequence identity, yet more preferably at least about 54% sequence identity, yet more preferably at least about 55% sequence identity, yet more preferably at least about 56% sequence identity, yet more preferably at least about 57% sequence identity, yet more preferably at least about 58% sequence identity, yet more preferably at least about 59% sequence identity, yet more preferably at least about 60% sequence identity, yet more preferably at least about 61% sequence identity, yet more preferably at least about 62% sequence identity, yet more preferably at least about 63% sequence identity, yet more preferably at least about 64% sequence identity, yet more preferably at least about 65% sequence identity, yet more preferably at least about 66% sequence identity, yet more preferably at least about 67% sequence identity, yet more preferably at least about 68% sequence identity, yet more preferably at least about 69% sequence identity, yet more preferably at least about
  • the presently disclosed ODF regulatory region, or a fragment thereof such as those disclosed in Example 1, below, or a functional variant of either 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 comprising the presently disclosed ODF regulatory region, fragment thereof, or functional variant of either, and an operably linked polynucleotide encoding a protein of interest heterologous to the ODF regulatory region, e.g., a reporter polynucleotide encoding a reporter molecule, are transfected into an appropriate cell line.
  • An appropriate cell line is one that is "competent,” i.e., one in which ODF expression can or does occur naturally, for example one which naturally expresses factors, such as transcription factors, required for ODF expression, for example osteoblast specific transcription factor 2 (Osf2) or other bone-specific transcription factors.
  • factors such as transcription factors, required for ODF expression, for example osteoblast specific transcription factor 2 (Osf2) or other bone-specific transcription factors.
  • An example is a bone cell line.
  • suitable bone cell lines include osteoblasts, for example UMR106 cells, osteoclasts, osteocytes, fibroblasts, stromal cells, chondrocytes, T-cells, and other cells of similar origin. Screens can also be performed using non-competent cells that are engineered to be competent for ODF expression. This can be achieved by, for example, introducing an "effector plasmid," i.e., a plasmid that expresses a factor that binds to one or more expression regulatory elements necessary for
  • an effector plasmid is one that comprises a nucleotide sequence which codes on expression for Osf2, operably linked to transcriptional and translational regulatory elements required for expression.
  • an effector plasmid such as pEF/Cbfal/myc/cyto, encoding Cbfal
  • Osf2 osteoblast specific transcription factor 2
  • a vector comprising the presently disclosed ODF regulatory region, fragment thereof, or functional variant of either, and an operably linked reporter polynucleotide
  • a host cell strain that modulates the expression of the reporter molecule, or modifies and processes the reporter polynucleotide expression product in the specific fashion desired, can be used.
  • modifications for example, glycosylation, and processing, for example cleavage, of protein products may be important for the function of the 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.
  • 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.
  • 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 ODF 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 antagonist. 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 (or concentration) of agonist.
  • expression of the reporter gene is measured in cells exposed to a test compound, and compared to expression in identical control cells that have not been contacted with the test compound.
  • screening assays are useful for identifying compounds that promote or inhibit the synthesis of ODF. Since ODF is known to promote osteoclast formation, a compound that increases ODF expression is expected to increase osteoclast formation, and lead to more bone resorption. Conversely, a compound that reduces ODF expression should decrease osteoclast formation, and therefore decrease the amount of bone resorption.
  • reporter polynucleotides and genes are known in the art. Non-limiting examples include acid phosphatase, alkaline phosphatase, chloramphenicol acetyltransferase, aequorin, firefly luciferase, and ⁇ -glucuronidase . Expression of reporter polynucleotides can be carried out using techniques that are well-known in the art. See, for example, Alam et al . , Anal . Biochem . , 188:245-254 (1990), Bronstein et al . , Anal . Biochem . , 219:169-181 (1994), and Bronstein et al . , Clin . Chem .
  • reporter polynucleotide or gene transcription, translation, or activity of the expressed product.
  • reporter mRNA, protein levels, or protein activity can be measured.
  • a reporter polynucleotide is that which encodes the enzyme beta-galactosidase .
  • An ODF regulatory region/beta-galactosidase construct can be created by excising the regulatory region fragment from the pSPORTl vector, supra , with the restriction enzymes SnaBl and Sail, and then subcloning it into the Smal and Xhol sites of the p ⁇ GAL-Basic vector (CLONTECH, Palo Alto, CA) .
  • the ODF regulatory region/p ⁇ GAL-Basic vector 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
  • the cells are transferred to medium containing 0.1% fetal bovine serum, and incubated overnight. The cells are then treated with a test compound. After a sufficient period of time, usually 4 to 24 hours, 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. 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 ODF expression can be identified. As noted above, agonists and antagonists can be identified in such screening assays.
  • 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 ODF, for example arterial calcification.
  • ODF a non-membrane decoy receptor
  • 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 (Grigoriadis et al . , Sci ence, 266 (5184) :443-448 (1994) .
  • ODF has been shown to be instrumental in lymph-node organogenesis, T- and B-cell maturation, T-cell activation, and formation of normal growth plate (cartilage). See Kong et al . , Nature, 397:315-323 (1999) .
  • the presently disclosed ODF 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 ODF can result in a variety of bone, cartilage, and immune response diseases.
  • Gain of function or activating mutations in the ODF regulatory region resulting in increased ODF expression or function, or loss of function or inactivating mutations therein resulting in decreased ODF expression or loss of function can induce or cause, or may be associated with, a variety of disease states, such as osteoporosis, osteopetrosis , expansile osteolysis, rheumatoid arthritis, osteoarthritis, metastatic bone disease, hypercalcemia, humoral hypercalcemia of malignancy, and
  • Paget ' s disease of bone due to the effect of such mutations on the level of ODF expression.
  • These mutations can therefore serve as diagnostic markers for patients at risk for developing bone or cartilage disease, arterial disease, altered immune response, etc., due to abnormally elevated or depressed levels of ODF.
  • Mutations or polymorphisms in genes related to ODF are associated with familial expansile osteolysis (Hughes et al . , ature Genetics, 24:45-48 (2000), while altered expression of ODF has been demonstrated in cancer cells responsible for humoral hypercalcemia of malignancy (Nagai et al . , Biochem . Biophys . Res . Comm . , 269:532-536 (2000) .
  • a wide variety of methods can be used to detect genetic mutations and polymorphisms. Examples thereof, and the use of such methods in medical diagnostics, are discussed in U.S. Patent 6,083,698 and PCT International Publication WO 00/06767, herein incorporated by reference in their entirety.
  • “Mutation” refers to an altered genetic sequence that can result in altered gene expression or function. A deleterious mutation can be associated with pathology, or the potential for pathology.
  • Polymorphism refers to a sequence variation in a gene that is not necessarily associated with pathology. Genetic mutations and polymorphisms can be detected, for example, by nucleic acid hybridization and nucleic acid sequence analysis, which can be facilitated by PCR amplification of the ODF regulatory region. In either case, the subject's ODF regulatory sequence can be compared to that disclosed herein to determine if any differences exist.
  • 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.
  • the filter is examined radiographically for binding of the probe to the target DNA. See, for example, Sambrook et al . , Molecular Cloning, A Laboratory Manual , 2 nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al . , Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1987 and updates) .
  • 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 ,
  • 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 DNA 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 ODF regulatory region can be detected by reference to the loss or gain of restriction endonuclease sites within the ODF regulatory region.
  • RFLPs restriction fragment length polymorphisms
  • the presently disclosed ODF 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, susceptibility, or increased risk of developing any of these diseases can be determined by comparing DNA from cells of a patient or subject, for example from the patient's or subject's bone cells, or from a sample of blood, bronchoalveolar lavage fluid, sputum, urine, or other body fluid or tissue obtained from an individual, with the ODF regulatory sequence (s) disclosed herein.
  • All or part of the subject's ODF regulatory region can first be amplified using any convenient technique, for example PCR, prior to analysis of sequence variation. Using the techniques discussed above, DNA sequences can be compared by hybridization or sequencing analysis. Any deviation in sequence of a patient's ODF regulatory region compared to the sequence of the ODF regulatory region disclosed herein could result in alteration (either an increase or decrease) in the level of ODF expression, or ODF function, and can therefore result in a predisposition to developing any of the diseases or conditions discussed herein.
  • the presently disclosed ODF regulatory region, or fragment thereof can be used in a method to determine a patient's susceptibility, receptiveness, or responsiveness to a particular therapy for treating bone-, cartilage-, arterial-, or immune-related diseases related to ODF over- or under- expression.
  • Evaluation of the nucleotide sequence of a patient's ODF regulatory region can indicate whether a proposed therapy with a compound that alters ODF expression (or function) by acting, directly or indirectly, at a discrete location (s) within the ODF regulatory region, will be effective.
  • Deviations in ODF regulatory region sequence especially in regions required for drug interaction/response, can be used as diagnostic or prognostic markers for susceptibility, receptiveness, or responsiveness to therapy in patients.
  • a patient's ODF 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 ODF regulatory region, or fragment thereof, to the present ODF 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 ODF regulatory region, to affect ODF expression and therefore the level of ODF in cells.
  • Individuals who carry particular allelic variants of the ODF regulatory region disclosed herein may therefore exhibit differences in ODF levels under different physiological conditions, and thus altered abilities to react to different diseases.
  • differences in ODF level resulting from allelic variation may directly affect an individual's response to drug therapy.
  • ODF polymorphism may therefore have a significant effect on the efficacy of drugs designed to modulate the activity of ODF.
  • the polymorphism(s) may also affect the response to agents acting on other biochemical pathways regulated by an ODF 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 ODF regulatory region of a human, comprising determining the nucleotide sequence of the ODF 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.
  • human includes both a human having, or suspected of having, an ODF 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 ODF regulatory region-mediated disease, comprising:
  • the "normal" nucleotide residue at a particular position is identified by it being the most common residue found at that position among the individuals tested, and is to some extent an arbitrary designation.
  • polymorphisms or mutations associated with certain clinical features, such as adverse or abnormal events are likely to occur at low frequency within the population, low frequency single nucleotide polymorphisms ("SNPs") can be particularly useful in identifying these mutations.
  • 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 ODF 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 ODF-mediated diseases. Drugs can be specifically designed to regulate the expression of ODF driven by particular allelic variants in the ODF regulatory region, while minimizing effects on other variants or wild-type ODF regulatory regions .
  • the ODF regulatory region, SEQ ID NO:l, 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 ODF 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 .
  • 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 ODF regulatory region nucleic acid sequence shown in SEQ ID N0:1, 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 ODF regulatory region of DNA from a human subject, and comparing this sequence to a nucleotide sequence comprising, consisting essentially of, or consisting of the ODF 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.
  • 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.
  • compounds that stimulate or increase ODF gene expression are referred to as "agonists,” while those that inhibit or decrease ODF gene expression are referred to as "antagonists.” 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 decreases reporter gene expression in a screening assay of the present invention employing a nucleic acid construct comprising the present ODF regulatory region, fragment thereof, or functional variant of either is expected to 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 ODF expression, would be expected to be effective, for example, for treating or preventing osteoporosis, a condition characterized by decrease in bone mass with decreased bone density, mineral content, and connectivity, producing porosity and fragility; tumor metastasis to bone; and rheumatoid arthritis.
  • a compound that stimulates or increases reporter gene expression in a screening assay would be expected to increase ODF expression, and hence be effective for preventing or treating osteopetrosis , a condition characterized by abnormal thickening and hardening of bone.
  • immune responsiveness or function including, for example, lymph node development, T- and B-cell development, T-cell activation, etc.
  • immune responsiveness or function can be regulated by administering to a patient one or more compounds identified in the methods described herein.
  • alterations in osteoclast precursor proliferation or differentiation can directly or indirectly affect immune modulation, lymph node development, and T- and B-cell formation.
  • gene therapy can be utilized by administering to a patient a pharmaceutical composition comprising a recombinant DNA construct comprising the ODF regulatory region disclosed herein, a fragment thereof, or a functional variant thereof, operably linked to the odf gene.
  • the literature teaches a variety of different methods for introducing exogenous genes into cells ex vivo and in vivo; vectors for delivering nucleic acids can be viral, non-viral, or physical. See, for example, Rosenberg et al . , Science, 242:1575-1578 (1988), and Wolff et al . , Proc . Natl . Acad . Sci . USA, 86:9011-9014 (1989) .
  • Recent reviews discussing methods and compositions for use in gene therapy include Eck et al . , in Goodman & Gilman ' s The Pharmacological Basis of
  • U.S. Patent No. 6,080,728 also provides a discussion of a wide variety of gene delivery methods and compositions.
  • the routes of delivery include, for example, systemic administration and administration in si tu .
  • Well-known viral delivery techniques include the use of adenovirus, retrovirus, lentivirus, foamy virus, herpes simplex virus, and adeno-associated virus vectors.
  • Exemplary non-viral techniques include the use of naked DNA; DNA complexed with cationic Iipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles comprising DNA condensed with cationic polymers such as heterogeneous polylysine, defined-length oligopeptides, and polyethylene imine, in some cases contained in liposomes; and the use of ternary complexes comprising a virus and polylysine-DNA.
  • Physical methods include the use of needle-free injectors, such as "gene gun” devices and devices using liquid under high pressure for delivery into interstitial spaces, and electroporation .
  • Administration of pharmaceutical preparations comprising the present ODF regulatory region or fragments thereof disclosed herein, or functional variants thereof, can be systemic, such as with liposomes, by, for example, intravenous injection.
  • Specific expression of constructs in target cells can occur predominantly from the specificity conferred by the cell type-specific expression due to the ODF regulatory sequence, or this regulatory sequence in combination with the nucleic acid delivery vehicle targeting particular cell types.
  • Administra-tion can also be in si tu, such as with viral vectors. Delivery of recombinant constructs can be limited by localized introduction, for example by catheter (see U.S. Patent No. 5,328,470), local injection, or by stereotactic injection (Chen et al . , Proc . Natl . Acad . Sci . USA, 91:3054- 3057 (1994) ) .
  • Suitable vectors can be constructed by any of the methods well known in the art. See, for example, Sambrook et al . , Molecular Cloning, a Laboratory Manual , Second Edition, Cold Spring Harbor Press (1989), and Ausubel et al . , eds., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1987 and updates) .
  • the use of cationic liposomes, such as the DC- Chol/DOPE liposome has been widely documented as an appropriate vehicle to deliver DNA to a wide range of tissues through intravenous injection of DNA/cationic liposome complexes. See Caplen et al . , Nature Med .
  • Liposomes transfer genes to target cells by fusing with the plasma membrane. Examples of the successful application of liposome complexes include those of Lesson-Wood et al . , Human Gene Therapy, 6:395-405 (1995), and Xu et al . , Molecular Genetics and Metabolism, 63:103-109 (1998).
  • compositions for gene therapy comprising ODF regulatory region constructs can comprise the desired 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. Where desirable or necessary, 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:l, a fragment thereof, or a functional variant thereof, or a vector containing SEQ ID NO:l, etc., operatively linked to the odf 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 ODF regulatory region, fragment thereof, or functional variant thereof, operatively linked to an odf 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.
  • Such 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.
  • treatment or “therapeutic use” as used herein 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 ODF 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.
  • Viral vector-mediated gene transfer has been used successfully in mouse models and human clinical trials. See Fujiwara et al . , Cancer Research, 54:2287-2291 (1994), and Roth et al., Nature Medicine, 2:985-991 (1996). Mountain, TIBTECH, 18:119-128 (2000) discusses recent examples of gene therapy with clinical benefit progressing to Phase II clinical studies using cationic Iipids, adenovirus, retrovirus, and adeno-associated virus vectors, as well as naked DNA. Cavazzana-Calvo et al .
  • ODF regulatory region 5' deletion constructs were prepared. Each construct contained the reporter gene beta-galactosidase. See Figure 2.
  • the 4603 bp ODF regulatory region fragment was excised from the pSPORTl vector, supra , with SnaBI and Sail, vector restriction sites which flank the ⁇ Votl and Sstl sites of the insert.
  • the fragment, SEQ ID NO: 11 was then subcloned into the Sma I and Xho I sites of p ⁇ GAL-Basic (Clontech, Palo Alto, CA) , and designated pODF4.6 ⁇ GAL (-4467 to +105).
  • the pODF4.6 ⁇ GAL vector was digested with Xbal (one site is in the vector), releasing 1.2 KB of the 5' end of the ODF regulatory region, SEQ ID NO: 12, and religated with T4 DNA ligase, forming pODF3.4 ⁇ GAL (-3283 to +105).
  • the pODF4.6 ⁇ GAL vector was also digested with Xbal and EcoRI , releasing 2.1 kb of the 5' end of the ODF regulatory region, SEQ ID NO: 13.
  • the fragment was treated with T4 DNA polymerase to blunt the ends and religated with T4 DNA ligase to form the vector pODF2.5 ⁇ GAL (-2382 to +105).
  • the vector pODFl.O ⁇ GAL (-835 to +100) was derived from the pODF5 vector.
  • pODF5 was obtained by ligating the 1 kb fragment of the ODF regulatory region obtained via the Genome Walker Kit into a pCR2.1 vector.
  • the pODF5 vector was digested with Smal and Sstl, releasing the 1 kb insert, SEQ ID NO: 14, treated with T4 DNA polymerase, blunting the Sstl site on the 3 ' end, and subcloned into the Smal site of p ⁇ GAL-Basic.
  • the pODFl.O ⁇ GAL vector was digested with PvuII and Bglll to release a fragment containing 356 bp of the 3' end of the ODF regulatory region, SEQ ID NO: 15. The fragment was then subcloned into the Smal /Bglll site of p ⁇ GAL-Basic to form pODF0.4 ⁇ GAL (-256 to +100).
  • SEQ ID Nos: 11-15 are shown in the 5' to 3 ' direction.
  • the ODF regulatory region deletion constructs positive control plasmid pcDNA3.
  • l/V5-HIS/lacz Invitrogen, Carlsbad, CA
  • l/V5-HIS/lacz containing the beta-galactosidase gene under the control of the CMV promoter
  • negative control plasmid p ⁇ GAL-basic were transiently transfected into a confluent T150 flask of UMR 106 cells using FugeneTM 6 reagent (Boehringer Mannheim, Indianapolis IN) as recommended by the manufacturer. After transfection, the cells were plated in 96 well plates (Becton Dickinson Labware, Franklin Lakes, NJ) (50,000 cells/well) .
  • the UMR 106 cells were maintained in DMEM/Ham's F-12 (3:1) (GIBCO BRL, Grand Island, NY) containing 10% fetal bovine serum (FBS) and glutamine (GIBCO BRL) , and incubated at 37°C in a humidified atmosphere of 95% air and 5% C0 2 - Four hours after plating, the cells were transferred to medium containing 0.1% fetal bovine serum and incubated overnight at 37°C in a humidified atmosphere of 95% air and 5% C0 2 .
  • the cells were lysed in 60 ⁇ l of lysis buffer provided by the manufacturer, and beta-galactosidase activity was assayed in a fixed amount of the extracts (1/3 of the extracts) using the beta-galactosidase reporter gene assay kit (Boehringer Mannheim) . Twenty microliters of the supernatant were transferred to white, opaque microtiter plates (Dynex, Franklin, MA) , and beta-galactosidase activity was measured using an automated injection MLX Luminometer (Dynex
  • Beta-galactosidase enzyme activity was determined and expressed as relative light units. The results, shown graphically in Figure 3, represent the mean ⁇ SEM of 4-12 separate wells.
  • ODF regulatory region of the present invention contains approximately 10 osteoblast specific element (OSE 2 ) motifs, shown in bold face and underlined in Figure 1, that function as binding sites for osteoblast specific transcription factor 2 (0sf2) , the role of Osf2 in ODF expression was evaluated.
  • the ability of Osf2 to transactivate regulatory region constructs was evaluated in C0S1 cells, a monkey kidney cell line lacking endogenous 0sf2 protein.
  • 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
  • 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 in DMEM supplemented with 10% FBS.
  • the plates were washed twice with phosphate- buffered saline (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 (Dynex, Franklin, MA) , 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. The results are shown in Figure 4.

Abstract

La présente invention présente la zone de régulation de transcription complète du gène humain odf. On peut utiliser les séquences présentées, des fragments de celles-ci, et des variantes fonctionnelles de celles-ci, dans des procédés servant à réguler l'ostéoclastogénèse et à traiter des maladies des os et d'autres maladies causées par une surexpression ou une sousexpression du facteur de différenciation des ostéoclastes. Ces séquences sont également utiles pour diagnostiquer la sensibilité d'un patient à développer des maladies des os, des cartilages, du système immunitaire, et des artères, liées à des facteurs de différenciation des ostéoclastes (ODF), et pour diagnostiquer la réceptivité d'un patient à un traitement à base de médicaments destinés à combattre ces maladies. Cette invention concerne également des procédés servant à identifier des composés qui modulent la formation d'ostéoclastes, la résorption des os, et d'autres maladies des os, des cartilages, du système immunitaire, et des artères, liées à des facteurs de différenciation des ostéoclastes (ODF).
PCT/US2000/026407 1999-09-27 2000-09-26 Zone de regulation de facteurs de differenciation des osteoclastes WO2001023559A1 (fr)

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WO2001023562A2 (fr) * 1999-09-27 2001-04-05 Eli Lilly And Company Region regulatoire d'osteoprotegerine
WO2002072842A1 (fr) * 2001-03-13 2002-09-19 Oscotec Inc. Lignee cellulaire transfectee pouvant etre utilisee dans le depistage de masse de l'expression du facteur de transcription runx2 specifique de l'osteoblaste et utilisation de ladite lignee cellulaire
US7700574B2 (en) 2003-09-17 2010-04-20 Isis Pharmaceuticals, Inc. Modulation of RANKL expression

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001023562A2 (fr) * 1999-09-27 2001-04-05 Eli Lilly And Company Region regulatoire d'osteoprotegerine
WO2001023562A3 (fr) * 1999-09-27 2002-01-24 Lilly Co Eli Region regulatoire d'osteoprotegerine
WO2002072842A1 (fr) * 2001-03-13 2002-09-19 Oscotec Inc. Lignee cellulaire transfectee pouvant etre utilisee dans le depistage de masse de l'expression du facteur de transcription runx2 specifique de l'osteoblaste et utilisation de ladite lignee cellulaire
US7700574B2 (en) 2003-09-17 2010-04-20 Isis Pharmaceuticals, Inc. Modulation of RANKL expression

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