EP0804573A1 - Procedes et compositions de modulation de l'expression de proteines morphogenetiques - Google Patents

Procedes et compositions de modulation de l'expression de proteines morphogenetiques

Info

Publication number
EP0804573A1
EP0804573A1 EP95923784A EP95923784A EP0804573A1 EP 0804573 A1 EP0804573 A1 EP 0804573A1 EP 95923784 A EP95923784 A EP 95923784A EP 95923784 A EP95923784 A EP 95923784A EP 0804573 A1 EP0804573 A1 EP 0804573A1
Authority
EP
European Patent Office
Prior art keywords
seq
cell
sequence
vector
reporter gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95923784A
Other languages
German (de)
English (en)
Inventor
Engin Ozkaynak
Hermann Oppermann
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Stryker Corp
Original Assignee
Creative Biomolecules Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Creative Biomolecules Inc filed Critical Creative Biomolecules Inc
Publication of EP0804573A1 publication Critical patent/EP0804573A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA

Definitions

  • the invention relates generally to the field of drug screening assays. More particularly, the invention relates to methods and compositions for identifying molecules that modulate production of true tissue morphogenic proteins.
  • a class of proteins recently has been identified, the members of which are true tissue morphogenic proteins.
  • the members of this class of proteins are characterized as competent for inducing the developmental cascade of cellular and molecular events that culminate in the formation of new organ-specific tissue, including any vascular and connective tissue formation as required by the naturally occurring tissue.
  • the morphogen ⁇ are competent for inducing all of the following biological functions in a morphogenically permissive environment: (1) stimulating proliferation of progenitor cells; (2) stimulating differentiation of progenitor cells; (3) stimulating the proliferation of differentiated cells and (4) supporting the growth and maintenance of differentiated cells.
  • the morphogenic proteins can induce the full developmental cascade of bone tissue morphogenesis, including the migration and proliferation of mesenchymal cells, proliferation and differentiation of chondrocytes, cartilage matrix formation and calcification, vascular invasion, osteoblast proliferation, bone formation, bone remodeling, and hematopoietic bone marrow differentiation. These proteins also have been shown to induce true tissue morphogenesis of non-chondrogenic tissue, including dentin, liver, and nerve tissue.
  • a particularly useful tissue morphogenic protein is human OP-1 (Osteogenic Protein-1) , described in U.S. 5,011,691; US Pat. No. 5,266,683 and Ozkaynak et al. (1990) EMBO J. 9 : 2085-2093.
  • Species homologues identified to date include mouse OP-1 (see US Pat. 5,266,683) and the Drosophila homologue 60A, described in Wharton et al. (1991) PNAS 88:9214-9218) .
  • Other closely related proteins include OP-2 (Ozkaynak (1992) J. Biol. Chem. 267:25220- 25227 and US Pat. No. 5,266,683); BMP5, BMP6 (Celeste et al. (1991) PNAS 87:9843-9847) and Vgr-1 (Lyons et al. (1989) . These disclosures are incorporated herein by reference.
  • tissue morphogens can be administered to an animal to regenerate lost or damaged tissue.
  • administering a molecule capable of modulating expression of the endogenous tissue morphogen as a means for providing morphogen to a site in vi vo.
  • the compounds thus identified have utility both in vi tro and in vi vo .
  • Useful compounds contemplated include those capable of stimulating transcription and/or translation of the OP-1 gene, as well as compounds capable of inhibiting transcription and/or translation of the OP-1 gene.
  • the invention features compositions and methods for screening candidate compounds for the ability to modulate the effective local or systemic quantity of endogenous OP-1 in an organism, and methods for producing the compounds identified.
  • the method is practiced by: (1) incubating one or more candidate compounds with cells transfected with a DNA sequence encoding, in operative association with reporter gene, a portion of an OP-1 non-coding DNA sequence that is competent to act on and affect expression of the associated receptor gene; (2) measuring the level of reporter gene expression in the transfected cell, and (3) comparing the level of reporter gene expressed in the presence of the candidate compound with the level of reporter gene expressed in the absence of the candidate compound.
  • the invention features the compound that is identified by use of the method of the invention.
  • the screening method of the invention provides a simple method of determining a change in the level of a reporter gene product expressed by a cell following exposure to one or more compound( ⁇ ) .
  • the level of an expressed reporter gene product in a given cell culture, or a change in that level resulting from exposure to one or more compound(s) indicates that application of the compound can modulate the level of the morphogen expressed and normally associated with the non-coding sequence.
  • an increase in the level of reporter gene expression is indicative of a candidate compound's ability to increase OP-1 expression in vivo .
  • a decrease in the level of reporter gene expression is indicative of a candidate compound's ability to decrease or otherwise interfere with OP-1 expression in vi vo .
  • the methods and compositions of the invention can be used to identify compounds showing promise as therapeutics for various in vivo and ex vi vo mammalian applications, as well as to identify compounds having numerous utilities.
  • morphogen expression inducing compounds can be used in vi vo to correct or alleviate a diseased condition, to regenerate lost or damaged tissue, to induce cell proliferation and differentiation, and/or to maintain cell and tissue viability and/or a differentiated phenotype in vivo or ex vivo.
  • the compounds also can be used to maintain the viability of, and the differentiated phenotype of, cells in culture.
  • the various in vivo, ex vivo, and in vi tro utilities and applications of the morphogenic proteins described herein are well documented in the art. See, for example, US 92/01968 (WO 94/03200), filed March 11, 1992; US 92/07358 (WO
  • Morphogen expression inhibiting compounds identified by the methods, kits and compositions described herein can be used to modulate the degree and/or timing of morphogen expression in a cell. Such compounds can be used both in vi tro and in vi vo to more closely regulate the production and/or available concentration of morphogen.
  • gene expression is understood to refer to the production of the protein product encoded by a DNA sequence of interest, including the transcription of the DNA sequence and translation of the mRNA transcript.
  • operative association is a fusion of the described DNA sequences with a reporter gene in such a reading frame as to be co-transcribed, or at such a relative positioning as to be competent to modulate expression of the reporter gene.
  • vector is understood to mean any nucleic acid comprising a nucleotide sequence of interest and competent to be incorporated into a host cell and recombining with and integrating into the host cell genome.
  • vectors include linear nucleic acids, plasmids, phagemids, cosmids, YAC'S (yeast artificial chromosomes) and the like.
  • non-coding sequence or “non-coding DNA” includes DNA sequences that are not transcribed into RNA sequence, and/or RNA sequences that are not translated into protein. This category of “non-coding sequence” has been defined for ease of reference in the application, and includes sequences occurring 5' to the ATG site which indicates the start codon and sequences 3' to the stop codon, as well as intervening intron sequences that occur within the coding region of the gene.
  • an "OPl-specific" non-coding sequence is understood to define a non- coding sequence that lies contiguous to OP1 specific coding sequence at an OP-1 gene locus under naturally-occurring condition ⁇ . The sequences may include 5', 3' and intron sequences.
  • allelic, species and other sequence variants thereof includes point mutations, insertions and deletions such as would be naturally occurring or which can genetically engineered into an OP-1 non-coding DNA sequence and which do not affect substantially the regulation of a reporter gene by the OP-1 non-coding sequence.
  • site directed mutagenesis to modify , as by deletion, for example, one or more of the OP-1 non-coding sequences described herein without substantially affecting the regulation of OP-1 or a reporter gene by the modification. Such modifications are considered to be within the scope of the disclosure provided herein.
  • a "Wt-l/Egr-1 consensus binding sequence” or Kt-l/Egr-1 consensus binding element” is a nine base sequence which has been shown to be bound by the DNA binding proteins Wt-1 and Egr-1.
  • the consensus sequence of the Wt-l/Egr-1 binding site has been determined by homology to be GN3NGGGNG, Seq. ID No. 4 (Rauscher et al. , Science 250:1259-1262 (1990), incorporated herein by reference) .
  • a "TCC binding sequence” or “TCC binding element” is an approximately 15 to 20 base sequence of DNA which contains at least three contiguous or non-contiguous repeats of the DNA sequence TCC.
  • the TCC binding sequence identified in human OP-1 genomic DNA is shown in Seq. ID No. 5, and the TCC binding sequence identified in murine OP-1 genomic DNA is shown in Seq. ID No. 6.
  • the TCC binding sequence has also been shown to be bound by the DNA binding proteins Wt-1 and Egr-1 (Wang et al., Proc. Natl. Acad. Sci. 90:8896-8900 (1993)'; Wang et al. , Biochem Biophys Res. Comm. , 188:433-439 (1992)) .
  • FTZ binding sequence or “FTZ binding element” is a Fushi-tarazu DNA sequence (FTZ) that has been shown to be bound by the DNA binding protein Fushi-tarazu (FTZ-F1) .
  • the FTZ binding sequence identified in human OP-1 genomic DNA is shown in Seq. ID No. 7.
  • the FTZ consensus sequence a consensus sequence for the nuclear hormone receptor ⁇ uperfamily, is YCAAGGYCR.
  • a " ⁇ teroid binding sequence” or “steroid binding element” is a DNA sequence that has been shown to be bound by one or more elements, in response to activating signal molecules.
  • Example ⁇ of such "activating signal molecules” include retinoids, Vitamin D, and also include steroids such as estrogen and progesterone. Useful elements are anticipated to include the FTZ-F1 protein, WT-1 and Egr-1.
  • Activating signal molecules of the nuclear receptor family have recently been ⁇ hown to bind to DNA a ⁇ homodimer ⁇ , heterodimers or as monomers (Parker, M.G., Curr. Op. Cell Biol., 1993, 5:499-504). The formation of heterodimers among the nuclear receptor family molecule ⁇ may significantly increase the diversity of binding elements which are recognized by these nuclear receptors, and provide for differential regulation of genes containing the specific binding sites.
  • nuclear receptors have been shown to interact with other accessory factors, such as transcription factors, to stimulate or repress transcription. These interactions, between the nuclear receptors and the nuclear receptors and acce ⁇ ory factors, indicate that there could be significant number of nuclear receptor/accessory factor interaction ⁇ which have widely different tran ⁇ criptional activitie ⁇ .
  • nucleic acids include, without limitation, CaP0 4 co-precipitation, electroporation, DEAE-dextran mediated uptake, protoplast fusion, microinjection and lipofusion.
  • a key to the invention is the DNA sequences with which the cell is transfected, rather than the mechanical or chemical process by which the DNA incorporation is accomplished.
  • U ⁇ eful reporter genes are characterized as being easy to transfect into a suitable host cell, easy to detect using an established assay protocol, and genes whose expression can be tightly regulated.
  • reporter genes contemplated to have utility include, without limitation, the luciferase gene, the Green Fluorescent Protein (GFP) gene, the chloramphenicol Acetyl Transferase gene (CAT) , human growth hormone, and beta- galactosidase.
  • GFP Green Fluorescent Protein
  • CAT chloramphenicol Acetyl Transferase gene
  • Additional useful reporter genes are any well characterized genes the expression of which is readily as ⁇ ayed, and examples of such reporter genes can be found in, for example, F.A. Ausubel et al., Eds., Current Protocols in Molecular Biology, John Wiley Sons, New York, (1989).
  • the listed reporter genes are only a few of the possible reporter genes, and it is only for ea ⁇ e of description that all available reporter genes are not listed.
  • a cell line is to be establi ⁇ hed, particularly where the transfected DNA i ⁇ to be incorporated into the cell' ⁇ genome, lines that can be immortalized are e ⁇ pecially desirable.
  • immortalized cell lines are viable for multiple .pas ⁇ ages (e.g., greater than 50 generations) without significant reduction in growth rate or protein production.
  • Useful cell types for the method and compositions according to the invention include any eukaryotic cell.
  • cell types known to express OP-1 include epithelial cells and cells of uro-genital cell origin, including renal (kidney or bladder) cells, as well as liver, bone, nerve, ovary, cardiac muscle and the like.
  • the cells may be derived from tissue or cultured from an established cell line. See, for example Ozkaynak et al. (1991) Bioche . BioPhys . Res. Com . 179 :116-123 for a detailed de ⁇ cription of tissues known to express OP-1.
  • Other useful cells include those known to exhibit a steroid receptor, including cells having an estrogen receptor and cells responsive to the FTZ-F1 protein.
  • CHO Chinese hamster ovary
  • MDCK canine kidney
  • NBT-2 rat bladder
  • Useful cell types can be obtained from the American Type Culture Collection (ATCC) , Rockville, MD or from the European Collection of Animal Cell Cultures, Portion Down, Salisbury
  • SP40JG U.K.
  • derived means the cells are from the cultured tissue itself, or are a cell line whose parent cells are of the tissue itself.
  • the invention features a vector having a reporter gene operatively associated with a portion of one or more OP-1 non-coding sequences.
  • the OP-1 non-coding sequence chosen is independently selected from the 5' (or "upstream") non-coding human or murine OP-1 sequence shown in Seq. ID Nos. 1 and 2, respectively, the 3' (or "downstream") non-coding human or murine OP-1 sequence shown in Seq. ID Nos. 1 or 3, and the human intron non-coding OP-1 sequences shown in Seq. ID No. 1. Also - 9 -
  • non-coding sequences e.g., 5', 3' and intron
  • portion of OP-1 sequence included in the vector can be a combination of two or more 5' non-coding, 3" non-coding and/or intron OP-1 sequences.
  • the vector can include a non-coding 0P1- specific sequence selected from at least one of the following sequence segments of Seq. ID No. 1 presented below, and which define human genomic OP-1 sequence comprising approximately 3.3 Kb of 5' non-coding sequence.
  • the start codon begins at po ⁇ ition 3318, and the up ⁇ tream ⁇ equence (bases 1 to 3317) is composed of untranscribed (1 to 2790) and untranslated (2791 to 3317) OPl- ⁇ pecific DNA; approximately 1 Kb of which is presented in Fig. 1 (bottom strand) .
  • Useful sequence segments include bases 2548-3317, representing 750 base ⁇ sharing significant (greater than 70% identity) between the mouse and human OP-i homologs (See Fig. 1), and base ⁇ 3170- 3317; 3020-3317; 2790-3317; 2548-2790 of Seq. ID No. 1, all shorter fragments of this region of the DNA.
  • base 2790 is the mRNA start site
  • other useful sequences include 2790-3317, representing transcribed but not translated 5' coding sequence and shorter fragments of this DNA region as noted above; upstream fragments of OPl-specific DNA, bases 2548-2790; 1549-2790; 1-2790 of Seq. ID No. 1.
  • u ⁇ eful ⁇ equence ⁇ egment ⁇ include the approximately 750 ba ⁇ es that have homology between the human and mou ⁇ e OP-1 sequences with additional upstream sequence ⁇ , 2300 to 3317,; 1300 to 3317; 1-3317; all fragments of the disclo ⁇ ed upstream OPl-specific DNA sequence ⁇ of Seq. ID No. 1.
  • sequences are defined by the non- coding sequences of the mouse OP-1 homolog, including the following 5 1 non-coding sequence ⁇ (Seq. ID No. 2): 2150-2296, 2000-2296, 1788-2296, and 1549-2296 all of which define the 750 base ⁇ ⁇ haring high sequence identity with the human homolog (See, Fig. 1); 800-2296; 1-2296; 1549-1788, 800-1788 and 1-1788.
  • 5 1 non-coding sequence ⁇ (Seq. ID No. 2): 2150-2296, 2000-2296, 1788-2296, and 1549-2296 all of which define the 750 base ⁇ ⁇ haring high sequence identity with the human homolog (See, Fig. 1); 800-2296; 1-2296; 1549-1788, 800-1788 and 1-1788.
  • Egr/Wt-1 sites 8 in hOP-1; 7 in mOP-1, known in the art to bind the regulatory elements Egr and Wt-1.
  • the invention contemplates a screening material for identifying co pounds which modulate OP-1 expression, the as ⁇ ay comprising the step of identifying compounds which bind Egr/Wt-1 site.
  • At least oneWt/Egr-1 element, preferably between 1-6 elements, or at least 6 Wt/Egr-1 element ⁇ are included in a sequence. The relative locations of these elements are indicated in Fig. 1 and at positions 3192-3200; 3143-3151; 3027-3035; 2956-2964; 2732-2740; 2697-2704 of Seq. ID No.
  • the length ⁇ of ba ⁇ e ⁇ within the ⁇ e 5' non-coding ⁇ equence ⁇ i ⁇ selected to include portions of the sequence of DNA which was determined to be homologous between murine and human genomic OP-1, separately and as a part of a larger sequence including non-homologous DNA.
  • the portion of OP-1 sequence selected can be a portion of the region of homology between murine and human OP-1 DNA sequences, bases 2548-2790 or 2548-3317 of Seq. ID No. 1, or bases 1549 to 1788 or 1549 to 2296 of Seq. ID No. 2, and/or at least one of an Wt-l/Egr-1 consensus binding sequence.
  • the portion of OP-1 sequence selected can include a TCC binding sequence, a FTZ binding sequence, a steroid binding sequence, or part or all of an OP-1 intron sequence.
  • the relative positions of the TCC and FTZ elements are indicated in Fig. 1 and at positions 2758-2778 (TCC); 2432-2441 (FTZ) of Seq. ID No. 1 and 1755-1769 (TCC) of Seq. ID No. 2.
  • the invention features a cell that has been transfected with a reporter gene in operative a ⁇ ociation with a portion of OP-1 non-coding DNA ⁇ equence.
  • the portion of OP-1 non ⁇ coding ⁇ equence is independently selected from the 5' (or upstream) non-coding human or murine OP-1 sequence shown in Seq. ID Nos. 1 and 2, the 3' (or downstream) non-coding murine OP-1 sequence shown in Seq. ID No. 3, and the human intron non-coding OP-1 sequence shown in Seq. ID No. 1.
  • the six human intron non ⁇ coding OP-1 sequences are at base ⁇ 3736 to 10700; bases 10897 to 11063; bases 11217 to 11424; feese ⁇ 11623 to 13358; bases 13440 to 10548; base ⁇ 15166 to 17250; all of Seq. ID No. 1.
  • the portion of OP-1 ⁇ equence ⁇ elected can be a combination of 5' non-coding, 3' non-coding and/or intron OP-1 ⁇ equence.
  • the cell can have been transfected with a reporter gene in operative association with a portion of 5' non-coding OP-1 genomic sequence that is independently selected from base ⁇ 3170 to 3317; 3020 to 3317; 2790 to 3317; 2548 to 3317; 2300 to 3317; 1300 to 3317; 1 to 3317; 2548 to 2790; 1549 to 2790; and 1 to 2790; all of Seq. ID No. 1 or ba ⁇ e ⁇ 2150 to 2296; 2000 to 2296; 1788 to 2296; 1549 to 2296; 800 to 2296; 1 to 2296; 1549 to 1788; 800 to 1788; 1 to 1788; all of Seq. ID No. 2.
  • the lengths of bases within these 5' non-coding sequences is selected to include portions of the sequence of DNA which was determined to be homologous between murine and human genomic OP-1, separately and as a part of a larger sequence including non-homologous DNA. Additionally, the portion of OP-1 sequence selected can be a portion of the region of homology between murine and human OP-1 DNA sequence ⁇ , such as bases 2548-2790 or 2548-3317 of Seq. ID No. 1, or bases 1549 to 1788 or 1549 to 2296 of Seq. ID No. 2, and at least one of an Wt- 1/Egr-l consensus binding sequence, a TCC binding sequence, a FTZ binding ⁇ equence, a steroid binding sequence, and an intron.
  • the portion of OP-1 ⁇ equence ⁇ elected can be a portion of the 5' non-coding human or murine OP-1 genomic DNA sequences, as stated above, and at least one Wt-l/Egr-1 consensus binding sequence alone or in combination with at least one of a TCC binding sequence, a FTZ binding sequence, a steroid binding ⁇ equence, and a human OP-1 intron DNA ⁇ equence.
  • more than one wt-l/Egr-1 element i ⁇ used for example, between 1-6, or at least six. These cells are suitable for use in the method of the invention.
  • part of the OP-1 coding region i ⁇ anticipated to have an expression regulatory function and also can be added to a vector for use in the screening assay described herein.
  • OP-1 protein i ⁇ translated as a precursor polypeptide having an N-terminal signal peptide ⁇ equence (the "pre pro" region) which i ⁇ typically le ⁇ than about 30 amino acid re ⁇ idues, followed by a "pro” region which is about 260 amino acid residues, followed by the additional amino acid residues which comprise the mature protein.
  • the pre pro and pro regions are cleaved from the primary translation sequence to yield the mature protein sequence.
  • the mature sequence comprises both a conserved C-terminal seven cysteine domain and an N-terminal sequence which varies significantly in sequence between the variou ⁇ morphogens .
  • the ature polypeptide chains dimerize and these dimers typically are stabilized by at least one interchain disulfide bond linking the two polypeptide chain subunits.
  • the pro domain After the pro domain is cleaved from the OP-1 protein it associate ⁇ noncovalently with the mature dimeric protein, pre ⁇ umably to enhance solubility and/or targeting properties of the mature species. See, for example, PCT/US93/07189, filed July 29, 1993.
  • the pro region represents the nucleotide sequence occurring approximately 87 bases downstream of the ATG start codon, and continues for about 980 bases.
  • the nucleotide sequence encoding the pro region is highly enriched in a "GC" sequence, which well may be competent to form a secondary ⁇ tructure (e.g., as part of the mRNA transcript) which itself may modulate OP-1 expres ⁇ ion. Accordingly, part or all of the nucleotide sequence encoding an OP-1 pro region, particularly that portion corresponding to a GC rich region, may be used, preferably in combination with one or more OP-1 non coding sequences, in the compositions and methods of the invention.
  • the method can be practiced using a cell known to express the OP-1 gene.
  • Suitable DNA sequence ⁇ for tran ⁇ fection are de ⁇ cribed below, as well as suitable cells containing transfected DNA sequences.
  • kits comprises a cell transfected with a DNA ⁇ equence compri ⁇ ing a reporter gene in operative association with a portion of OP-1 upstream DNA sequence and the reagents nece ⁇ ary for detecting expression of the reporter gene.
  • the portion of OP-1 upstream DNA chosen can be any of the various portions which have been described herein.
  • medium flux screen assays, and kits therefore, for identifying OP-1 expression modulating compounds are available. These compounds can be naturally occurring molecules, or they can be designed and biosynthetically created using a rational drug design and an established structure/function analysis methodology.
  • the compounds can be amino acid-ba ⁇ ed or can be compo ⁇ ed in part or whole of non-proteinaceou ⁇ ⁇ ynthetic organic molecules.
  • the OP-1 expression modulating compounds thus identified then can be produced in reasonable quantities using standard recombinant expres ⁇ ion or chemical synthesi ⁇ technology well known and characterized in the art and/or as described herein. For example, automated means for the chemical ⁇ ynthesis of nucleic and amino acid sequences are commercially available. Alternatively, promising candidates can be modified using standard biological or chemical methodologies to, for example, enhance the binding affinity of the compound for a DNA element and the preferred candidate derivative then can be produced in quantity.
  • a compound which upregulates (increases) the pro__ction of OP-1 in a kidney cell line is a candidate for systemic administration.
  • the candidate can be as ⁇ ayed in an animal model to determine the candidate molecule' ⁇ efficacy in vi vo .
  • the ability of a compound to upregulate level ⁇ of circulating OP-1 in vi vo can be u ⁇ ed to correct bone metabolism disea ⁇ e ⁇ ⁇ uch a ⁇ o ⁇ teoporo ⁇ i ⁇ (See, for example, PCT/US92/07932, supra).
  • Useful in vi vo animal models for systemic administration are disclosed in the art and below.
  • OP-1 i ⁇ differentially expres ⁇ ed in different cell types. Accordingly, it further is anticipated that a candidate compound will have utility a ⁇ an inducer of OP-1 expre ⁇ ion in one cell type but not in another. Thu ⁇ , the invention further contemplates testing a candidate compound for it ⁇ utility in modulating expression of OP-1 in different cells in vivo, including different cells known to express OP-1 under native physiological conditions. Thu ⁇ , in view of this disclosure, one of ordinary skill in recombinant DNA techniques can design and construct appropriate DNA vectors and transfect cells with appropriate DNA sequences for use in the method according to the invention to as ⁇ ay for compounds which modulate the expre ⁇ ion of OP-1. The ⁇ e identified compound ⁇ can be u ⁇ ed to modulate OP-1 production and it ⁇ available concentrations in both in vi vo and in vi tro contexts.
  • Fig. 1 shows the alignment of upstream sequences of the murine and human OP-1 gene.
  • the murine sequence i ⁇ present in the upper sequence lines and the human sequence is the lower sequence on all lines.
  • the murine sequence is numbered backwards, counting back from the first ATG of the translated sequence which is shown highlighted.
  • da ⁇ he ⁇ are introduced into the DNA sequence, and three portions of human DNA sequence have been cut from the sequence and placed underneath a gap, below a solid triangle;
  • Fig. 2 shows a time course of murine uterus OP-1 mRNA regulation by estrogen; and
  • Fig. 3a shows a schematic of the 2 kb and 4 kb OP-1 mRNA ⁇ , the hybridization locations of probes 1 through 7 (indicated by the bars under the schematic) .
  • the solid line indicates OP-1 mRNA
  • the * indicate potential poly A signals
  • the boxes indicate the translated portion of OP-1 mRNA. with the hatched box showing the TGF- ⁇ -like domain.
  • the dashed lines indicate genomic DNA sequences.
  • the arrows mark the locations of the cleavage site for OP-1 maturation.
  • Fig. 3b show ⁇ a Northern blot hybridization analy ⁇ i ⁇ of OP-1 specific 2 kb and 4 kb mRNAs in murine uterine tissue.
  • Lanes 1 through 7 correspond to probes 1 through 7 respectively.
  • the 2 kb and 4 kb mRNAs are indicated by the 4- and 2-on the left side of Fig. 3b, and a 0.24 to 9.49 kb RNA ⁇ ize ladder is indicated by dashes to the right of the figure.
  • OP-1 expression in vivo can be dependent both on cell type and on the statu ⁇ of the cell in a ti ⁇ sue.
  • OP-1 protein expres ⁇ ion i ⁇ differentially regulated in uterine ti ⁇ sue depending on the status of the uterine ti ⁇ ue.
  • OP-1 expression is dramatically down-regulated in uterine mouse tissue during pregnancy, whereas it is normally expres ⁇ ed in this ti ⁇ sue in virgin mice.
  • OP-1 expression in other tissues such as renal tis ⁇ ue apparently i ⁇ unaffected during pregnancy.
  • Admini ⁇ tration of estrogen to a virgin mouse is capable of duplicating this down-regulation of OP-1 gene expression.
  • Thi ⁇ ⁇ creen yielded four lambda clones which were analyzed by Southern blotting. All clones yielded a 1.5 kb XmnI fragment which was subcloned from lambda 071 into a Bluescript vector and sequenced. Three polyadenylation signal ⁇ (AATAAA) (Proudfoot et al, (1976) Nature, 263:211-214) were found in thi ⁇ genomic fragment, at 3.52-, 3.58-, and 3.59 kb (shown schematically in Fig. 3a by the *) .
  • AATAAA polyadenylation signal ⁇
  • Human upstream non-coding sequence and additional mouse upstream non-coding ⁇ equence were obtained by ⁇ creemng human and mou ⁇ e genomic libraries, HL1067J and ML1030J respectively
  • a schematic diagram of the 2- and 4 kb OP-1 mes ⁇ ages is ⁇ hown in Figure 3a with dashed lines indicating supplementing information derived from murine upstream and downstream genomic DNA.
  • Northern blot hybridization ⁇ were performed with probes made from seven non-overlapping DNA fragments (Fig. 3a; probes 1 through 7) specific to the 5' and 3' non-coding region, the protein coding sequence, and genomic regions upstream or downstream of the predicted mRNAs, respectively.
  • probe 7 which is specific to sequence ⁇ further down ⁇ tream of the fourth (last) polyadenylation signal, did not hybridize to any message.
  • the result ⁇ obtained with the ⁇ e probes confirm the two OP-1 mRNA structures and the approximate 5'- and 3 ⁇ -end boundaries of OP-1 transcript ⁇ ⁇ hown in Figure 3a.
  • Thi ⁇ demonstrates that the 2 kb and 4 kb mRNA's are from the same OP-1 genomic locu ⁇ rather than from multiple gene ⁇ .
  • the untranslated sequence itself may act therefore to influence mRNA stability.
  • the sequence may interact with another protein as has been described for transferrin receptor mRNA.
  • IRE-binding protein stabilizes the transferrin receptor mRNA by binding to the 3 ' -end of the mRNA (Standard et al. , 1990, Genes Dev. , l:2157-2168, incorporated herein by reference).
  • the 3 ' -end sequences may be interacting with the 5' -end sequence ⁇ thereby affecting initiation of protein ⁇ ynthe ⁇ i ⁇ or, the 3 ' -end sequence ⁇ may be ⁇ erving a ⁇ a binding ⁇ ite for other RNA ⁇ which can interfere with the binding of an expression in modulating molecule, including repressor molecule. (Klausner et al . , 1989, Science, 246:870-872; Kozak, 1992, Ann. Rev. Cell Biol., 8 :197-225, incorporated herein by reference) .
  • the cloning of the 5' non-coding genomic murine and human OP-1 DNA sequences demonstrated that a high degree of sequence homology exists between the human and murine 5' non-coding DNA sequences.
  • the homology extends from the base immediately upstream of the translation start site for the OP-1 morphogen protein to approximately 750 bases upstream of the translation start site, a ⁇ is shown in the shaded regions of Fig. 1, with the murine sequences being the upper lines and the human sequences being the lower lines.
  • the 5' nucleotide of the region of homology for the human OP-1 5' non-coding ⁇ equence i ⁇ base 2548 of Seq. ID No. 1 and for the murine OP-1 5' non-coding sequence is base 1549 of Seq. ID No. 2.
  • thi ⁇ region contain ⁇ ⁇ everal conserved DNA sequences which have been identified as the DNA binding sequences for two DNA binding proteins, Wt-1 and Egr-1, which both recognize these DNA sequences.
  • the DNA binding sequences for Wt-l/Egr-1 present in human and murine are marked in Fig. 1 with a single line.
  • the TCC binding sequence, a DNA binding sequence for Wt-1 and Egr-1 is marked in Fig. 1 by the - 19 -
  • WT-1 and Egr-1 proteins have also been implicated in the regulation of expression of several genes which are unrelated to OP-1.
  • the analysis also revealed, in the human upstream region, a pattern of seven TCC repeats, present at -561, immediately 3' of two Egr/Wt-1 sequences (at -624 and -587) (Figure 1, shown by double solid lines and at position 2758-2778 of Seq. ID No. 1) .
  • the mouse upstream region contains a similar, albeit les ⁇ obviou ⁇ ⁇ equence at -356 and at position 1755-1769 of Seq. ID No. 2.
  • Thi ⁇ TCC-repeat pattern is found in the promoters of PDGF-A and several other growth-related genes, and Wt-1 has been found to activate transcription when either of the ⁇ equences are present and to suppress it when both sequences are present.
  • estrogen receptor may exert its effect on OP-1 expres ⁇ ion in uteru ⁇ by upregulating Wt-1, either directly or indirectly.
  • other regulatory element ⁇ located further up ⁇ tream of the OP-1 gene may be involved in estrogen regulation.
  • the human 5' non-coding DNA sequence is shown to contain a Fushi-tarazu (FTZ) binding sequence which i ⁇ marked by carat ⁇ below the human DNA ⁇ equence.
  • a FTZ binding sequence i ⁇ bound by the Fushi-tarazu protein (FTZ-F1) which i ⁇ a member of the superfamily of nuclear receptors (Parker, (1993) Current opinion in Cell Biology, _5:499-504, ) .
  • the superfamily of nuclear receptor proteins include steroid hormones, retinoids, thyroid hormone, nerve growth factor and Fu ⁇ hi-tarazu, and are structurally related.
  • FTZ-F1 is likely to belong to a ⁇ ubfamily of nuclear receptor ⁇ that bind DNA as monomers.
  • the FTZ-F1 protein is a positive regulator at the fu ⁇ hi-tarazu gene in blastoderm stage embryos of Drosophila .
  • FTZ-F1 is closely related in the silkworm (Bombyx) BmFTZ-Fl protein and the mouse embryonal long terminal repeat binding protein (ELP) and all of them are members of the nuclear hormone receptor superfamily, which recognizes the same 9 base pair sequence, 5 ' -PyCAAGGPyCPu- 3'.
  • the FTZ binding sequence doe ⁇ not apparently have a direct or inverted repeat. In contrast, other members of the nuclear hormone receptor superfamily usually bind to repeated sequences.
  • the FTZ-F1, BmFTZ-Fl and ELP proteins have high affinities for the FTZ binding site DNA, indicating that the mechanism that the binding is somewhat different from that of other members of the nuclear hormone receptor superfamily.
  • the transcription initiation site for the human OP-1 gene is at ba ⁇ e 2790 of Seq. ID No. 1 and the analogou ⁇ ⁇ ite for murine i ⁇ at base 1788 of Seq. ID No. 2.
  • the tran ⁇ lation initiation site for the human OP-1 gene is at base 3318 of Seq. ID No. 1 and for murine it i ⁇ at ba ⁇ e 2296 of Seq. ID No. 2.
  • RNA from the various organ ⁇ of mice wa ⁇ prepared u ⁇ ing the acid-guanidine thiocyanate-phenol-chloroform method (Chomczynski et al., (1987) Anal. Biochem. 162:156-159) .
  • the RNA was di ⁇ olved in TES buffer (10 ml. Tris-HCl, 1 mM Na : -EDTA, 0.1% SDS, pH7.5) containing Proteinase K (Stratagene, La Jolla, CA; approx. 1 mg proteina ⁇ e /ml TES) and incubated at 37°C for 1 hr.
  • RNA total RNA obtained from 1 g of ti ⁇ ue wa ⁇ mixed with approximately 0.Ig of oligo(dT) -cellulo ⁇ e (in 11 ml TES containing 0.5 M NaCl) .
  • the tube ⁇ containing the RNA and oligo(dT) -cellulose were gently shaken for approx. 2 hrs. Thereafter, the oligo(dT)- cellulose was wa ⁇ hed twice in lx binding buffer and once in 0.5x binding buffer (0.25 M NaCl, 10 mM Tri ⁇ -HCl, 1 mM Na -EDTA, pH 7.4) and poly (A)+ RNA wa ⁇ eluted with water and precipitated with ethanol.
  • RNA Poly(A)+ RNA (5 mg per lane) wa ⁇ electrophoresed on 1.2% agarose-formaldehyde gels with 1 mg of 400 ⁇ g/ml ethidium bromide added to each sample prior to heat denaturation (Rosen et al. , (1990) Focus, _12:23-24). Electrophoresis was performed at 100 Volts with continuous circulation of the 1 x MOPS buffer (Ausubel et al. , eds., (1990) Current Protocols in Molecular Biology, John Wiley _. Sons, New York). Following electrophoresi ⁇ , the gels were photographed, rinsed briefly in water, and blotted overnight onto Nytran (Schleicher _. Schuell Inc., Keene, NH) or Duralon-UV
  • the 32 P-labeled probe was made from a murine OP-1 cDNA fragment (0.68 kb BstXI-BGlI frg.) by random hexanucleotide priming (Feinberg et al., (1984) Anal. Biochem. , 137:266-267) .
  • the hybridization ⁇ were done in 40% formamide, 5x SSPE, 5x Denhardt's, 0.1% SDS, pH 7.5 at 37°C overnight.
  • the non-specific counts were washed off by shaking in O.lx SSPE, 0.1% SDS at 50°C.
  • filters were stripped in 1 mM Tris-HCl, 1 mM Na 2 -EDTA, 0.1% SDS, pH 7.5 at 80° C for 10 min.
  • the high embryonal OP-1 expre ⁇ ion also is detected consistent with the relatively high levels of OP-1 mRNA, found in human placenta.
  • the level of OP-1 mRNA measured in the embryo is in the same range as that mea ⁇ ured in adult kidney or virgin uteru ⁇ tissue.
  • OP-1 plays a critical role in the development of the embryo which may require appropriate amounts of OP-1 at very specific stage ⁇ of ti ⁇ ue and organ morphogensis.
  • OP-1 expression in uterine tissue during pregnancy potentially could interfere with the level of OP-1 produced by the developing embryo, and thereby interfere with proper development of the embryo. Therefore, a shut-down or inhibition of uterine OP-1 expres ⁇ ion during pregnancy might be for the benefit of the fetus.
  • non-pregnant female mice were ⁇ ubcutaneously admini ⁇ tered 17 ⁇ -e ⁇ tradiol, or progesterone, or a combination of both.
  • the rapid increase in estrogen and progesterone levels during pregnancy was ⁇ imulated.
  • Non-pregnant mice were injected ⁇ ubcutaneou ⁇ ly on four con ⁇ ecutive days with increasing dose ⁇ , ⁇ tarting with 20 mg 17 ⁇ -e ⁇ tradiol, or 100 mg progesterone or the combination of both and doubling the dose on each following day.
  • the uterus has been identified as a major site of OP-1 expression.
  • the level of OP-1 expres ⁇ ion in uterine ti ⁇ sue is comparable to that observed in renal tis ⁇ ue.
  • the uterine OP-1 mRNA levels are reduced to the limit of detection.
  • the loss of OP-1 expres ⁇ ion corre ⁇ pond ⁇ withalso is rising levels of estrogen during this ⁇ ame time frame.
  • co-regulation by means of one or more accessory molecules that interact with estrogen or a related nuclear receptor molecule( ⁇ ) may allow for the independent regulation.
  • each of Wt-1 protein (which binds to the wt-l/Egr-1 element) and OP-1 protein are required for normal kidney development, and each are expres ⁇ ed at high level ⁇ during kidney ti ⁇ sue development.
  • the OP-1 promoter region contains Wt-1 con ⁇ ensu ⁇ binding element ⁇ .
  • Wt-1 protein al ⁇ o has been ⁇ hown to negatively regulate the tran ⁇ cription of the insulin growth factor II gene and the platelet-derived growth factor A chain gene. Kreidberg et al . , Cell, 1993, 74:679-691.
  • Wt-1 protein either alone or in combination with one or more molecules is involved in the expression of OP-1.
  • Wt-1 protein may act in concert with a nuclear hormone receptor element, including, for example,the estrogen receptor element.
  • Estrogen also has been shown to inhibit the uterine expression of calbindin-D 28k , a vitamin D dependent calcium binding protein, the ⁇ -subunit expression of the glycoprotein hormones, and other protein ⁇ involved in bone formation. Estrogen also ha ⁇ been shown to cause dramatic decreases in the steady state mRNA levels of the bone matrix proteins osteocalcin, prepro ⁇ 2(I) chain type I collagen, osteonectin, o ⁇ teopontin, and alkaline pho ⁇ phata ⁇ e in an ovariectomized rat, which i ⁇ a rat model for o ⁇ teoporosis.
  • E ⁇ trogen appears to mediate its beneficial effect on bone metaboli ⁇ m in the o ⁇ teoporotic model through inhibition of o ⁇ teoclasts. E ⁇ trogen doe ⁇ not reverse o ⁇ teoporosis.
  • OP-1 which is expre ⁇ ed in uterine, renal and bone ti ⁇ ues, is able to induce an increa ⁇ e in bone mass in the osteoporotic model. Thu ⁇ , the negative effect of e ⁇ trogen on OP-1 expre ⁇ sion in uterine tissue may seem unexpected in view of estrogen's effect on bone metabolism.
  • the other non-coding sequences such as a ⁇ intron ⁇ and 3 ' non-coding ⁇ equence ⁇ may be involved in the modulation of OP-1 protein expression.
  • This invention pre ⁇ ent ⁇ a method in which these non ⁇ coding sequences are assayed while in operative association with a reporter gene for their influence on the expression of OP-1.
  • Non- coding sequences which are involved in the modulation of OP-1 expres ⁇ ion will be identified by culturing cells transfected with the non-coding sequences, in operative association with a reporter gene, with one or more compound( ⁇ ), measuring the level of reporter gene expression, and comparing this level of expre ⁇ sion to the level of reporter gene expression in the absence of the compound(s) .
  • Any eukaryotic cell including an immortalized cell line suitable for long term culturing conditions i ⁇ contemplated to be u ⁇ eful for the method and cell of the invention.
  • Useful cells ⁇ hould be ea ⁇ y to tran ⁇ fect, are capable of ⁇ tably maintaining foreign DNA with an unrearranged ⁇ equence, and have the nece ⁇ ary cellular component ⁇ for efficient tran ⁇ cription and tran ⁇ lation of the protein, including any element ⁇ required for post- tran ⁇ lational modification and ⁇ ecretion, if necessary.
  • the cell genotype preferably is deficient for the endogenous selection gene.
  • the cell line also ha ⁇ simple media composition requirements, and rapid generation times.
  • Particularly useful cell lines are mammalian cell line ⁇ , including myeloma, HeLa, fibroblast, embryonic and variou ⁇ tis ⁇ ue cell line ⁇ , e.g., kidney, liver, lung and the like.
  • a large number of cell line ⁇ now are available through the American Type Culture Collection (Rockville, MD) or through the European Collection of Animal Cell Cultures (Porton Down, Salisbury, SP4 OJG, U.K.)
  • a ⁇ here, the expression of a reporter gene that is controlled by non-coding sequences of the morphogen OP-1 is to be analyzed, particularly u ⁇ eful cells and cell .
  • lines are envisioned to include eukaryotic, preferably mammalian cells of a tissue and cell type known to expres ⁇ OP-1 and/or closely related protein ⁇ .
  • Such cell ⁇ include, without limitation, cells of uro-genital cell origin, including kidney, bladder and ovary cel.'.s, lung, liver, mammary gland and cardiac cells, cells of gonadal origin, cells of ga ⁇ trointestinal origin, glial cell ⁇ and other cell line ⁇ known to express endogenous genes encoding morphogenic protein ⁇ .
  • Preferred cell lines are of epithelial origin.
  • Useful vectors for use in the invention include, but are not limited to cosmids, phagemids, yeast artificial chromosomes or other large vectors. Vectors that can be maintained within the nucleus or integrated into the genome by homologous recombination are also useful. For example a vector such a ⁇ PSV2CAT would be u ⁇ eful. Selected portions of non-coding OP-1 ⁇ equence can be cloned into a useful vector using standard molecular cloning techniques, as will be apparent to one of ordinary skill in the art. Restriction endonuclease sites will be utilized when possible, and can be engineered into the sequence when needed.
  • restriction endonuclease sites are needed to be engineered into the sequence, eight base recognition sites are preferable because they generally occur infrequently in DNA and will enhance a practitioners ability to obtain the sequence of interest. Restriction endonuclease sites can be engineered into the non-coding sequence using the common techniques such as site directed mutagenesis and PCR with primers including the desired restriction endonuclease site.
  • murine and human OP-1 sequences share a region of high homology covering approximately 750 base ⁇ upstream of the translation initiation site as shown by the shading in Fig. 1.
  • This region is positions 2548-3317 of Seq. ID No. 1 and positions 1549-2296 of Seq. ID No. 2.
  • the mRNA transcription initiation site lie ⁇ within this region at position 2790 of Seq. ID No. 1 and by analogy at po ⁇ ition 1788 of Seq. ID No. 2, ⁇ hown in Fig. 1 by the upward arrow. Thi ⁇ suggests that positions 2548- 2790 of Seq. ID No. 1 and 1549-1788 of Seq. ID No.
  • 3 ' non-coding ⁇ equence ⁇ and intron ⁇ equence ⁇ also can be fused in operative as ⁇ ociation with a reporter gene, either separately or in combination with each other or with 5' non-coding sequences.
  • a reporter gene either separately or in combination with each other or with 5' non-coding sequences.
  • the positions of the six introns are shown in Seq. ID No.
  • nucleic acid construct al ⁇ o could include intron ⁇ equence ⁇ and/or 3" non-coding ⁇ equences.
  • a range of u ⁇ eful 5' non-coding fragments has been provided, and as will be apparent to those of ordinary skill in the art, smaller fragments of OP-1 sequence al ⁇ o are useful.
  • Such smaller fragment ⁇ can be identified to deleting bases from one or both ends of the provided 5' non-coding fragments, using techniques that are well known in the art and testing the truncated constructs for their ability to modulate reporter gene expre ⁇ sion. In thi ⁇ way, the shorte ⁇ t modulating ⁇ equences can be identified.
  • any method for incorporating nucleic acids into cells of interest is contemplated in the method of the invention.
  • Calcium phosphate (CaPO , followed by glycerol shock i ⁇ a standard means used in the art for introducing vectors, particularly plasmid DNA into mammalian cells.
  • Other methods that may be used include electroporation, protoplast fusion, particularly useful in myeloma tran ⁇ fections, microinjections, lipofections and DEAE-dextran mediated uptake. Method ⁇ for these procedures are described in F.M.
  • DNA concentrations per transfection will vary according to the transfection protocol.
  • calcium pho ⁇ phate transfection for example, preferably 5-10 ⁇ g plasmid DNA per plasmid type is transfected.
  • the DNA to be transfected preferably is es ⁇ entially free of contaminant ⁇ that may interfere with DNA incorporation.
  • a ⁇ tandard means used in the art for purifying DNA is by ethidium bromide banding.
  • reporter system There are numerous reporter system ⁇ commercially available, which include, without limitation, the chloramphenicol acetyltransferase (CAT), lucifera ⁇ e, GAL4, and the human growth hormone (hGH) assay systems.
  • CAT chloramphenicol acetyltransferase
  • hGH human growth hormone
  • CAT is a well characterized and frequently used reporter system and a' major advantage of this ⁇ ystem is that it is an extensively validated and widely accepted measure of promoter activity. See, for example, Gorman, CM., Moffat, L.F., and Howard, B.H. (1982) Mol. Cell. Biol., 2:1044-1051 for a description of the reporter gene and general methodology.
  • cells are harvested 2-3 days after tran ⁇ fection with CAT expression vectors and extracts prepared. The extracts are incubated with acetyl CoA and radioactive chloramphenicol. Following the incubation acetylated chloramphenicol is ⁇ eparated from nonacetylated form by thin layer chro atography. In thi ⁇ assay the degree of acetylation reflects the CAT gene activity with the particular promoter.
  • Another well-recognized reporter system is the firefly luciferase reporter system. See, for example Gould, S.J., and
  • the luciferase a ⁇ ay is fast and has increased ⁇ en ⁇ itivity.
  • the ⁇ y ⁇ tem also is particularly useful in bulk transfections or if the promoter of interest is weak.
  • thi ⁇ assay transfected cells are grown under standard conditions, and when cultured under assay conditions both ATP and the substrate luciferin is added to the cell lysate.
  • the enzyme luciferase catalyzes a rapid, ATP dependent oxidation of the sub ⁇ trate which then emits light.
  • the total light output is mea ⁇ ured using a luminometer according to manufacturer' ⁇ in ⁇ tructions (e.g., Cromega) and is proportional to the amount of lucifera ⁇ e pre ⁇ ent over a wide range of enzyme concentrations.
  • a third reporter system is ba ⁇ ed on immunologic detection of hGH, it is quick and easy to use. (Selden, R., Burke-Howie, K. Rowe, M.E., Goodman, H.M., and Moore, D.D. (1986), Mol. Cell. Biol. , _6:3173-3179 incorporated herein by reference) .
  • hGH i ⁇ assayed in the media, rather than in cell extracts. This allows direct monitoring over by a single population of transfected cells over time.
  • the instant invention enables and discloses vector ⁇ , cell ⁇ and a method for screening co pound ⁇ to determine the capability of compound ⁇ to modulate the expre ⁇ ion of OP-1 via the non-coding sequences of the OP-1 genomic DNA.
  • Genomic OP-1 up ⁇ tream and promoter ⁇ equence ⁇ preferably 3000 to 5000 nucleotides in length, and which mediate the homologous recombination, are attached to the luciferase gene.
  • the OP-1 upstream sequence ⁇ down to the fir ⁇ t coding ATG can be attached at the ⁇ tart codon ATG of the lucifera ⁇ e coding ⁇ equence, u ⁇ ing a restriction ⁇ ite such as Ncol, which can be introduced by site directed mutagenesis into both the promoter and the lucifera ⁇ e sequence ⁇ .
  • Al ⁇ o included is a selective marker, preferably the neo gene, without its own promoter.
  • selectable marker (neo) is placed downstream of the reporter gene (luciferase), after an intercistronic sequence derived from the polibviru ⁇ genome and which allows translation of the sequence marker on the same transcript as the reporter gene transcripts.
  • a genetic arrangement of OP-1 promoter (as much genomic OP-1 up ⁇ tream sequence as possible, up to 10,000 bp) and reporter gene (without its original promoter but joined directly to the OP-1 ATG or in its vicinity) can al ⁇ o be introduced into cell ⁇ on standard eukaryotic expression vectors. These vectors carry selectable markers (neo, dhfr, etc.) and will typically be integrated into the ho ⁇ t genome with variable copy number ranging from one to ⁇ everal copie ⁇ without effort ⁇ at amplification. Al ⁇ o, if desired, the vector or gene copy number can be enhanced u ⁇ ing a well characterized amplifiable gene, ⁇ uch as dhfr in conjunction with methotrexate.
  • One source vector is the Episomal Expre ⁇ ion Epstein Barr Virus Vector (pREP, Invitrogen Corp., San Diego CA) .
  • Introns also can be tested for regulatory ⁇ equences as described hereinabove using the methods described herein.
  • One or more intron sequences derived from a genomic OP-1 locus preferably is introduced into proper mammalian cells using, for example, a yeast artificial chromosome (pYACneo, Clontech, Inc. Palo Alto, CA) (Ref. Albertson, H.M. et al.
  • Candidate compound(s) which may be admini ⁇ tered to affect the level of a given endogenou ⁇ morphogen, ⁇ uch as OP-1, or a reporter gene that is fused to OP-1 non-coding sequence may be found using the following screening assay, in which the level of reporter gene production by a cell type which produces measurable levels of the reporter gene expre ⁇ sion product by incubating the cell in culture with and without the candidate compound, in order to assess the effects of the compound on the cell. This can be accomplished by detection of the reporter expression product either at the protein or RNA level.
  • the protocol is based on a procedure for identifying compounds which alter endogenous levels of morphogen expres ⁇ ion, a detailed description also may be found in PCT US 92/07359.
  • Cultured cells are transfected with portions of OP-1 non- coding sequences in operative association with a reporter gene, and such transfected cells are maintained with the vector remaining as a pla ⁇ mid in the cell nucleus or the vector can be integrated into the host cell genome, preferably at the OP-1 genomic locus.
  • Cell ⁇ ample ⁇ for te ⁇ ting the level of reporter gene expre ⁇ ion are collected periodically and evaluated for reporter gene expre ⁇ ion u ⁇ ing the appropriate a ⁇ say for the given reporter gene as indicated in the section describing reporter gene as ⁇ ays, or, alternatively, a portion of the cell culture itself can be collected periodically and u ⁇ ed to prepare polyA(+) RNA for mRNA analysis.
  • candidate compounds can be produced in reasonable, u ⁇ eful quantitie ⁇ u ⁇ ing ⁇ tandard methodologies known in the art.
  • Amino acid-based molecules can be encoded by synthetic nucleic acid molecules, and expre ⁇ sed in a recombinant expres ⁇ ion system as described herein above or in the art.
  • such molecule ⁇ can be chemically synthesized, e.g., by means of an automated peptide ⁇ ynthe ⁇ izer, for example.
  • Non-amino acid-based molecule ⁇ can be produced by standard organic chemical synthe ⁇ is procedures.
  • CAT gene as the reporter gene and one or more mammalian cell line ⁇ known to expre ⁇ OP-1.
  • the example is non limiting, and other cells, reporter genes and OP-1 non-coding sequences are envisioned.
  • a DNA fragment containing the OP-1 promoter can be joined to a reporter gene for transfection into a cell line that expresses endogenous OP-1.
  • Suitable cell lines are selected by Northern blot hybridization to an OP-1 specific probe (by analyzing the cell extracts for OP-1 mRNA) .
  • OP-1 specific probe by analyzing the cell extracts for OP-1 mRNA.
  • An approximately 5 Kb EcoRI, BamHI genomic fragment containing approximately 4 Kb of upstream OP-1 ⁇ equences as well as part of the first intron is blunt-ended with T4 DNA polymerase and cloned into a polylinker of a pUC vector (p0146-l).
  • the -3.5kb fragment has blunt ends and contains mo ⁇ tly 5' non-coding ⁇ equences and al ⁇ o include ⁇ a ⁇ hort ⁇ tretch of 30 ba ⁇ es into the 0?-l gene.
  • This upstream fragment is of -3.5kb ligated to a 1.6 kb Hindlll-BamHI fragment from the CAT gene obtained from the vector SV2CAT by 5' Hindlll end blunted ligation.
  • the 1.6kb CAT gene fragment contains about 70 base ⁇ of upstream sequence ⁇ .
  • the ⁇ e ligated fragments are cloned into Bluescript KS(-) vector (Stratgene, La Jolla, CA) .
  • Thi ⁇ construct in turn is subjected to ⁇ ite specific mutagenesis to delete the extra ⁇ equence ⁇ (approximately 30 base ⁇ ) from the 3' end of the OP-l up ⁇ tream sequences and the adjacent 5' non-coding sequences (approximately 70 bases) from the CAT gene.
  • This mutagene ⁇ i ⁇ re ⁇ ult ⁇ in the elimination of any OP-l coding ⁇ equences from the promoter fragment as well as any non-coding sequence ⁇ upstream of the CAT gene.
  • the resulting con ⁇ truct is a fusion of OP-l up ⁇ tream ⁇ equences with the CAT gene sequences which encode the CAT protein.
  • Suitable cell lines include cell lines that have been shown to contain high levels of OP-l mRNA, indicating that the OP-l promoter is active in the cell ⁇ . Two of these cell lines are mou ⁇ e inner medullary collecting duct (IMCD) cell ⁇ , and the rat bladder carcinoma line (NBT II) . However other cell line ⁇ of the uro-genital system that produce high levels of the OP-l mes ⁇ age can be u ⁇ ed in addition to the many previously mentioned cell types and cell line ⁇ .
  • IMCD inner medullary collecting duct
  • NBT II rat bladder carcinoma line
  • tran ⁇ fection of thi ⁇ vector into an OP-l producing cell line is accomplished following standard techniques, i.e., transfection using calcium phosphate, lipo ⁇ ome mediated tran ⁇ fection, electroporation, or DEA.E-dextran tran ⁇ fection.
  • the tran ⁇ fected cells are harvested 48-72 hours after tran ⁇ fection with the CAT expre ⁇ ion vector and extracts are made by succe ⁇ ive freeze-thawing.
  • 20 ⁇ l of 4 mM acetyl CoA 32.5 ⁇ l of 1 M Tri ⁇ -HCl, pH 7.5, and 75.5 ⁇ l of water is added to 20 ml of cell extract, and incubated for 1 hour at 37 degree ⁇ Cel ⁇ ius.
  • 1 ml ethyl acetate is added to the reaction, microcentrifuged for 1 minute and the top layer i ⁇ removed.
  • the main con ⁇ truct can be deleted in sections to determine the regions that are responsible for the observed CAT activity.
  • the upstream sequences can be deleted unidirectionally, using an exonuclease such as Bal31, and the deletion product can be analyzed in the CAT activity assay.
  • This system can also be used in the method of the invention to screen compounds for their ability to modulate OP-l expression by dividing the cells into several groups, and culturing one group in the absence of any added compounds, and culturing the other groups with one or more candidate compound, and comparing the resulting levels of CAT activity.
  • OP-l coding sequence al ⁇ o may be used in the screening method of the invention.
  • the OP-l expression preferably is determined by an immunoas ⁇ ay or by Northern or dot blot or other means for measuring mRNA transcript. See, for example, WO 95/11983, published May 4, 1995 for a detailed description on as ⁇ aying changes in OP-l levels in a cell or fluid.
  • OP-l is expres ⁇ ed in a variety of different cell type ⁇ , including renal, bone, lung, heart, uterine, cardiac and neural tissue.
  • Candidate compounds can be identified which have a modulating effect on cells of one ti ⁇ ue type but not another, and/or wherein the effect i ⁇ modulated in the different cells.
  • the as ⁇ ay described belov; can be used to evaluate the effect of a candidate compound( ⁇ ) in a particular cell type known to express OP-l under physiological conditions.
  • kidneys may be explanted from neonatal or new born or young or adult rodents (mouse or rat) and used in organ culture as whole or ⁇ liced (1-4 mm) tissue ⁇ .
  • Primary ti ⁇ ue cultures and established cell lines, also derived from kidney, adrenals, urinary, bladder, brain, mammary, or other ti ⁇ ue ⁇ may be established in multiwell plates (6 well or 24 well) according to conventional cell culture techniques, and are cultured in the absence or presence of serum for a period of time (1-7 days) .
  • Cells may be cultured, for example, in Dulbecco's Modified Eagle medium (Gibco, Long Island, NY) containing serum (e.g., fetal calf serum at 1%-10%, Gibco) or in serum-deprived medium, as desired, or in defined medium (e.g., containing insulin, transferrin, glucose, albumin, or other growth factors) .
  • serum e.g., fetal calf serum at 1%-10%, Gibco
  • serum-deprived medium e.g., fetal calf serum at 1%-10%, Gibco
  • defined medium e.g., containing insulin, transferrin, glucose, albumin, or other growth factors
  • Samples for testing the level of OP-l production includes culture supernatants or cell ly ⁇ ate ⁇ , collected periodically and evaluated for OP-l production by immunoblot analysis (Sambrook et al., eds., 1989, Molecular Cloning, Cold Spring Harbor Press, Cold Spring Harbor, NY), or a portion of the cell culture itself, collected periodically and used to prepare polyA+ RNA for RNA analysi ⁇ .
  • OP-l ⁇ ynthe ⁇ i ⁇ some cultures are labeled according to conventional procedures with an 35 S- methionine/ 35 S-cy ⁇ teine mixture for 6-24 hour ⁇ and then evaluated to OP-l ⁇ ynthesi ⁇ by conventional im unoprecipitation method ⁇ .
  • osteoporotic rat model provides an in vivo model for evaluating the efficacy of a candidate modulating compound.
  • alkaline phosphate and o ⁇ teocalcin levels are mea ⁇ ured under conditions which promote o ⁇ teoporo ⁇ i ⁇ , e.g., wherein o ⁇ teoporo ⁇ is is induced by ovary removal in rats and in the presence and absence of a candidate modulating compound.
  • a compound competent to enhance or induce endogenous OP-l expression should result in increased osteocalcin and alkaline phosphate levels.
  • ovariectomized rats Forty Long-Evans rats (Charles River Laboratorie ⁇ , Wilmington) weighing about 200g each are ovariectomized (OVX) u ⁇ ing ⁇ tandard ⁇ urgical procedure ⁇ , and ten rat ⁇ are sham operated. The ovariectomization of the rats produces an osteoporotic condition within the rats as a result of decreased estrogen production. Food and water are provided ad libitum.
  • the rat ⁇ prepared as described above, are divided into three groups: (A) sham-operated rats; (B) ovariectomized rats receiving 1 ml of phosphate-buffered saline (PBS) i.v.
  • PBS phosphate-buffered saline
  • t ariectomized rats receiving various dose ranges of the candiate stimulating agent either by intravenous injection through the tail vein or direct administration to kidney tissue.
  • the effect of the candidate compound on in vivo bone formation can be determined by preparing sections of bone tissue from the ovariectomized rats. Each rat i ⁇ injected with 5 mg of tetracycline, which will stain the new bone (visualized as a yellow color by fluorescence) , on the 15th and 21st day of the study, and on day 22 the rats are sacrificed. The body weights, uterine weights, serum alkaline phosphate levels, serum calcium levels and serum osteocalcin levels then were determined for each rat.
  • Bone section ⁇ are prepared and the di ⁇ taance ⁇ eparating " each tetracycline straining i ⁇ mea ⁇ ured to determine the amount of new bone growth.
  • the level ⁇ of OP-l in ⁇ erum following injection of the candidate agent al ⁇ o can be monitered on a periodic ba ⁇ is using, for example, the immunoassay described in section ⁇ V and VII above.
  • OP-l may be detected u ⁇ ing a polyclonal antibody specific for OP-l in an ELISA, as follows. l ⁇ g/100 ⁇ l of affinity-purified polyclonal rabbit IgG specific for OP-l is added to each well of a 96-well plate and incubated at 37°C for an hour. The wells are washed four time ⁇ with 0.167M ⁇ odium borate buffer with 0.15 M NaCl (BSB) , pH 8.2, containing 0.1% Tween 20.
  • BBSB 0.15 M NaCl
  • the wells are blocked by filling completely with 1% bovine serum albumin (BSA) in BSB and incubating for 1 hour at 37°C.
  • BSA bovine serum albumin
  • the wells are then wa ⁇ hed four times with BSB containing 0.1% Tween 20.
  • biotinylated rabbit anti-OP-1 ⁇ erum ( ⁇ tock solution is about 1 mg/ml and diluted 1:400 in BSB containing 1% BSA before use) i ⁇ added to each well and incubated at 37°C for 30 min.
  • the well ⁇ are then wa ⁇ hed four time ⁇ with BSB containing 0.1% Tween 20.
  • 100 ⁇ l streptavidin-alkaline (Southern Biotechnology A ⁇ ociate ⁇ , Inc. Birmingham, Alabama, diluted 1:2000 in BSB containing 0.1% Tween 20 before u ⁇ e) i ⁇ added to each well and incubated at 37 C C for 30 min.
  • the plates are washed four times with 0.5M Tris buffered Saline (TBS), pH 7.2.
  • 50 ⁇ l substrate ELISA Amplification System Kit, Life Technologies, Inc., Bethesda, MD
  • 50 ⁇ l amplifier from the same amplification system kit
  • 50 ⁇ l amplifier i ⁇ added and incubated for another 15 min at room temperature.
  • the reaction is stopped by the addition of 50 ⁇ l 0.3 M sulphuric acid.
  • the OD at 490 nm of the solution in each well is recorded.
  • a OP-l standard curve is performed in parallel with the test samples.
  • Polyclonal antibody for OP-l protein may be prepared as follows. Each rabbit is given a primary immunization of 100 ⁇ g/500 ⁇ l E. coli produced OP-l monomer (amino acids 328-431 in SEQ ID NO:5) in 0.1% SDS mixed with 500 ⁇ l Complete Freund's Adjuvant. The antigen i ⁇ injected ⁇ ubcutaneously at multiple sites on the back and flanks of the animal. The rabbit is boo ⁇ ted after a month in the same manner using incomplete Freund's Adjuvant. Test bleeds are taken from the ear vein seven days later. Two additional boo ⁇ t ⁇ and test bleeds are performed at monthly interval ⁇ until antibody against OP-l is detected in the serum using an ELISA as ⁇ ay. Then, the rabbit i ⁇ boo ⁇ ted monthly with 100 ⁇ g of antigen and bled (15 ml per bleed) at days ⁇ even and ten after boosting.
  • Monoclonal antibody specific for OP-l protein may be prepared as follows. A mouse is given two injection ⁇ of E. coli produced OP-l monomer. The fir ⁇ t injection contain ⁇ lOO ⁇ g of OP-l in complete Freund' ⁇ adjuvant and i ⁇ given ⁇ ubcutaneou ⁇ ly. The ⁇ econd injection contain ⁇ 50 ⁇ g of OP-l in incomplete adjuvant and is given intraperitoneally. The mouse then receives a total of 230 ⁇ g of OP-l (amino acids 307-431 in SEQ ID NO:5) in four intraperitoneal injections at various times over an eight month period.
  • mice are boosted intraperitoneally with 100 ⁇ g of OP-l (307-431) and 30 ⁇ g of the N-terminal peptide (Ser 2.3 -Asn 3 o 9 -Cys) conjugated through the added cy ⁇ teine to bovine serum albumin with SMCC crosslinking agent.
  • the mou ⁇ e spleen cells are then fu ⁇ ed to myeloma (e.g., 653) cells at a ratio of 1:1 using PEG 1500 (Boeringer Mannheim) , and the cell fusion is plated and screened for OP-1-specific antibodies using OP-l (307-431) as antigen.
  • the cell fusion and monoclonal screening then are according to standard procedures well described in standard texts widely available in the art. VII. Exemplary Process for Detecting OP-l in Serum
  • OP-l may be detected in body fluids, including serum, and can be used in a protocol for evaluating the efficacy of an OP-l modulating compound in vi vo .
  • agarose- activated gel e.g., Affi-GelTM, from Bio-Rad Laboratories, Richmond, CA, prepared following manufacturer' ⁇ instructions
  • any commercially available preservation solution may be u ⁇ ed to advantage.
  • u ⁇ eful ⁇ olutions known in the art include Collins ⁇ olution, Wisconsin solution, Belzer solution, Eurocollins solution and lactated Ringer's ⁇ olution.
  • an organ pre ⁇ ervation ⁇ olution u ⁇ ually po ⁇ e ⁇ e s one or more of the following properties: (a) an osmotic pressure sub ⁇ tantially equal to that of the in ⁇ ide of a mammalian cell, (solutions typically are hyperosmolar and have K+ and/or Mg++ ions present in an amount sufficient to produce an osmotic pres ⁇ ure slightly higher than the inside of a mammalian cell); (b) the ⁇ olution typically is capable of maintaining sub ⁇ tantially normal ATP level ⁇ in the cells; and (c) the solution usually allows optimum maintenance of glucose metabolism in the cell ⁇ .
  • Organ pre ⁇ ervation ⁇ olution ⁇ al ⁇ o may contain anticoagulant ⁇ , energy ⁇ ources such as glucose, fructose and other ⁇ ugar ⁇ , metabolite ⁇ , heavy metal chelator ⁇ , glycerol and other material ⁇ of high viscosity to enhance survival at low temperatures, free oxygen radical inhibiting agents and a pH indicator.
  • energy ⁇ ources such as glucose, fructose and other ⁇ ugar ⁇ , metabolite ⁇ , heavy metal chelator ⁇ , glycerol and other material ⁇ of high viscosity to enhance survival at low temperatures
  • free oxygen radical inhibiting agents and a pH indicator.
  • the therapeutic agent may be provided by any suitable means, preferably directly (e.g., locally, as by injection to the tissue or organ locus) or ⁇ y ⁇ temically (e.g., parenterally or orally).
  • U ⁇ eful ⁇ olutions for parenteral administration may be prepared by any of the methods well known in the pharmaceutical art, described, for example, in Remington's Pharmaceutical Sciences (Gennaro, A., ed.), Mack Pub., 1990.
  • Formulations may include, for example, polyalkylene glycols such as polyethylene glycol, oils of vegetable original, hydrogenated naphthalene ⁇ , and the like.
  • Formulation ⁇ for direct administration may include glycerol and other compo ⁇ ition ⁇ of high vi ⁇ co ⁇ ity to help maintain the agent at the de ⁇ ired locus.
  • Biocompatible, preferably bioresorbable, polymer ⁇ including, for example, hyaluronic acid, collagen, tricalcium phosphate, polybutyrate, lactide and glycolide polymer ⁇ and lactide/glycolide copolymer ⁇ , may be u ⁇ eful excipients to control the release of the agent in vivo.
  • the concentration of the compound ⁇ de ⁇ cribed in a therapeutic compo ⁇ ition will vary depending upon a number of factor ⁇ , including the dosage of the drug to be administered, the chemical characteristics (e.g., hydrophobicity) of the compounds employed, and the route of administration.
  • the dosage likely will depend for example, on the size of the tissue or organ to be transplanted, the overall health status of the organ or tissue itself, the length of time between harvest and transplantation (e.g., the duration in ⁇ torage) , the frequency with which the preservation solution is changed, and the type of ⁇ torage anticipated, e.g., low temperature.
  • preferred ranges include a concentration range between about 0.1 ng to 100 ⁇ g/kg per tis ⁇ ue or organ weight per day.
  • the preferred dosage of drug to be administered al ⁇ o i ⁇ likely to depend on such variables as the type and extent of progression of the disease, the overall health status of the particular patient, the relative biological efficacy of the compound selected, the formulation of the compound excipients, and its route of administration.
  • a suitable compound of this invention may be provided in an aqueous physiological buffer solution containing about 0.001% to 10% w/v compound for parenteral admini ⁇ tration. Typical dose ranges are from about 10 ng/kg to about 1 g/kg of body weight per day; and preferred dose range is from about 0.1 ⁇ g/kg to 100 mg/kg of body weight per day.
  • MOLECULE TYPE DNA (genomic)
  • ORIGINAL SOURCE
  • CTCTGGCTCC CCTTTCTCCA CCCATGTGGC CCCTCAGGCT GCCATCTAGT CCAAAAGTCC 1380
  • GAGCCCCTCC CAGACAAGCG CCCCCGCTTC CCCAACCTCA GCCCTTCCCA GTTCATCCCA 1560
  • ACCTGTCCCC TCGTGGTGCG CCCGCCTTAG GCTACCGGCC GCTCCGAGCC TTGGGGCCCC 4260
  • GTGCGCGCAC ATTCTCCAGA CTTGCTCAAA CTAACCCCCC GGAGCAGCGC ACGGGCTGGG 4980
  • GCATGTGTGC TTGTATATCA TCGTGTCCTC CTGGAGGAAG ACACCAGGAA CTGGAGAGAG 5520
  • TCTCCTGCCT CAGCCTCCCG AGTAGCTGGG AATACAGGCG CCCGCACCAT GCGCGGCTAA 7140
  • ATCTTGAACT TCCCAGTCCC CAGAACCATG AGCTAAATAA ACCTTTTTTC TCTATAAATT 9180
  • GTCAGGTACC CACCAGGGCC ATGTGCAAAC TGAGATAATG GGGACATGGA ACAAGGGTAA 9600
  • CTCTGGGCCT CGGAGGAGGG CTGGCTGGTG TTTGACATCA CAGCCACCAG CAACCACTGG 11160 GTGGTCAATC CGCGGCACAA CCTGGGCCTG CAGCTCTCGG TGGAGACGCT GGATGGTGAG 11220 TCCCCCGCCA CTGCCAGTCC TAATGCAGCC TGTGCTCCTG GACTTCAGGA GGGTCTCAGC 11280 AGTGCTCATG CTTGCTTCAC TACAAACAGG CTTCCCCGCC CCTCCCAACC AGTACTCCAT 11340
  • ATCTCATCTA TCCCTTTCAT TTGATTCTGC TCTTTGAGGG CAGGGGTTTT TGTTTCTTTG 16020 TTTGTTTTTT TAAGTGTTGG TTTTCCAAAG CCCTTGCTCC CCTCCTCAAT TGAAACTTCA 16080
  • MOLECULE TYPE DNA (genomic)
  • AAACAATTTA TCCCCGTTTC TTGGTTTATT CTGACTTTGT AAACAGAAAA GCCGGGGCTG 540
  • CTGCCAGTCC CTCAGCCCCA ATTCCTGCCC CATGGTAGGA AATCCATGAG AAAAGCAAAG 960
  • CAGAGCCCCA GCTGCCCCAA TGGTTCCTAG CTTCAAATGC AGAGGGTTAA ACTGGCTGCC 1320
  • GCCCCCCACC TCCCCGGCAA GCTCAGGGCA GTGCTCATCT GGCTACATCG GTCTTTGAAG 2460
  • CAGGCTGAAG CCCAGAGCCA CAAGCCGGAG GGTCCAGATG TGGCCTCTCA GATGTGTGCC 2700
  • TTAGCCTCTC AACCCCACCC CCACCCCCAA CCCCAGTGAT GTTTACACAT CTTAAAAAAC 2760
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

Abstract

Des procédés et des compositions servent à tester la capacité de composés de moduler l'expression d'une protéine morphogénétique des tissus, notamment la protéine ostéogénétique-1, les homologues de la protéine ostéogénétique-1 et les protéines qui lui sont étroitement apparentés, au moyen d'une ou plusieurs séquences de nucléotides non codants spécifiques de la protéine ostéogénétique-1 et d'un gène reporteur approprié.
EP95923784A 1994-06-07 1995-06-07 Procedes et compositions de modulation de l'expression de proteines morphogenetiques Withdrawn EP0804573A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US25525094A 1994-06-07 1994-06-07
US255250 1994-06-07
PCT/US1995/007349 WO1995033831A1 (fr) 1994-06-07 1995-06-07 Procedes et compositions de modulation de l'expression de proteines morphogenetiques

Publications (1)

Publication Number Publication Date
EP0804573A1 true EP0804573A1 (fr) 1997-11-05

Family

ID=22967501

Family Applications (1)

Application Number Title Priority Date Filing Date
EP95923784A Withdrawn EP0804573A1 (fr) 1994-06-07 1995-06-07 Procedes et compositions de modulation de l'expression de proteines morphogenetiques

Country Status (5)

Country Link
EP (1) EP0804573A1 (fr)
JP (2) JPH10505223A (fr)
AU (1) AU703445B2 (fr)
CA (1) CA2191583C (fr)
WO (1) WO1995033831A1 (fr)

Families Citing this family (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6090544A (en) * 1995-07-26 2000-07-18 Creative Biomolecules, Inc. Methods and compositions for identifying morphogen analogs
US6103491A (en) * 1995-07-26 2000-08-15 Creative Biomolecules, Inc. Methods and compositions for identifying morphogen analogs
US5834188A (en) * 1995-07-26 1998-11-10 Creative Biomolecule, Inc. Methods and compositions for identifying morphogen analogs
US5932716A (en) * 1995-07-26 1999-08-03 Creative Biomolecules, Inc. Morphogen-responsive regulatory elements
US7306903B1 (en) 1995-07-26 2007-12-11 Curis, Inc. Methods and compositions for identifying morphogen analogs
EP0862651A2 (fr) * 1995-10-16 1998-09-09 Chiron Corporation Procede de criblage pour la recherche de facteurs qui modulent l'expression genique
JP2001509010A (ja) * 1996-11-22 2001-07-10 アクゾ・ノベル・エヌ・ベー Ｂｍｐ―４プロモーターならびに骨粗鬆症の予防および／または治療用の治療剤のスクリーニングにおけるその用途
WO1998054344A2 (fr) * 1997-05-29 1998-12-03 Creative Biomolecules, Inc. Modulateurs de l'expression de morphogenes et procedes d'identification correspondants
JPH11313675A (ja) * 1998-04-30 1999-11-16 Hoechst Marion Roussel Kk ヒトｂｍｐ−７プロモーターおよびこれを用いた骨関連物質の探索法
GB9828709D0 (en) * 1998-12-24 1999-02-17 Novartis Ag Assay
US7598079B2 (en) 1998-12-24 2009-10-06 Novation Pharmaceuticals, Inc. Assay for identifying compounds which affect stability of mRNA
US8426194B2 (en) 2003-01-21 2013-04-23 Ptc Therapeutics, Inc. Methods and agents for screening for compounds capable of modulating VEGF expression
CA2514184C (fr) 2003-01-21 2016-04-12 Ptc Therapeutics, Inc. Procedes pour identifier des composes qui modulent l'expression de genes dependants d'une region non traduite, et procedes d'utilisation correspondants
US9068234B2 (en) 2003-01-21 2015-06-30 Ptc Therapeutics, Inc. Methods and agents for screening for compounds capable of modulating gene expression
US8283115B1 (en) 2007-06-20 2012-10-09 Ptc Therapeutics, Inc. Methods of screening for compounds for treating muscular dystrophy using UTRN mRNA translation regulation
US8283116B1 (en) 2007-06-22 2012-10-09 Ptc Therapeutics, Inc. Methods of screening for compounds for treating spinal muscular atrophy using SMN mRNA translation regulation

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5266683A (en) * 1988-04-08 1993-11-30 Stryker Corporation Osteogenic proteins
WO1992021365A1 (fr) * 1991-06-05 1992-12-10 The Procter & Gamble Company Compositions therapeutiques d'osteoinduction
EP0601129B1 (fr) * 1991-08-30 2000-11-15 Creative Biomolecules, Inc. Procede de triage de proteines morphogeniques
GB9206874D0 (en) * 1992-03-30 1992-05-13 Connaught Lab Generation of improved inducible mammalian expression vectors
US5512483A (en) * 1993-05-21 1996-04-30 Mcgill University Expression vectors responsive to steroid hormones
US5585237A (en) * 1993-10-25 1996-12-17 Creative Biomolecules, Inc. Methods and compositions for high protein production from recombinant DNA
AU1180195A (en) * 1993-11-16 1995-06-06 Children's Medical Center Corporation Method of identifying a substance capable of inducing bone formation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9533831A1 *

Also Published As

Publication number Publication date
JP2006025796A (ja) 2006-02-02
AU703445B2 (en) 1999-03-25
JPH10505223A (ja) 1998-05-26
WO1995033831A1 (fr) 1995-12-14
AU2822395A (en) 1996-01-04
CA2191583A1 (fr) 1995-12-14
CA2191583C (fr) 2007-03-27

Similar Documents

Publication Publication Date Title
US6083690A (en) Methods and compositions for identifying osteogenic agents
EP0804573A1 (fr) Procedes et compositions de modulation de l'expression de proteines morphogenetiques
Ko et al. Murine and human T-lymphocyte GATA-3 factors mediate transcription through a cis-regulatory element within the human T-cell receptor δ gene enhancer
US5916763A (en) Promoter for VEGF receptor
Li et al. Steroid effects on osteogenesis through mesenchymal cell gene expression
US5712119A (en) Methods and compositions for high protein production form recombinant DNA
Mittanck et al. Essential promoter elements are located within the 5′ untranslated region of human insulin-like growth factor-I exon I
Tamura et al. Identification of a DNA sequence involved in osteoblast-specific gene expression via interaction with helix-loop-helix (HLH)-type transcription factors.
WO1996038590A9 (fr) Procedes et compositions pour identifier des agents osteogeniques
US6103466A (en) Double-muscling in mammals
EP1181056B1 (fr) Variants d'epissage de la proteine lim de mineralisation
US6071695A (en) Methods and products for identification of modulators of osteogenic protein-1 gene expression
US20050271637A1 (en) BMP-2 estrogen responsive element and methods of using the same
Bunyaratavej et al. Bone morphogenetic proteins secreted by breast cancer cells upregulate bone sialoprotein expression in preosteoblast cells
US6828095B1 (en) Modulators of morphogen expression and methods of identifying the same
US20080003675A1 (en) Methods and compositions for identifying morphogen analogs
AU773649B2 (en) Methods and compositions for modulating morphogenic protein expression
AU743061B2 (en) Methods and compositions for modulating morphogenic protein expression
US7741117B2 (en) Bone mineralization protein expression systems, and methods of studying intracellular signaling pathways induced thereby
AU9719101A (en) Modulators of morphogen expression and methods of identifying the same
Norton et al. A highly conserved region upstream of the fibronectin alternative exon EIIIA 3′ splice site interacts with cell-type-specific nuclear proteins
WO1998026047A1 (fr) Techniques et compositions permettant d'identifier des analogues de morphogene
Matsui et al. The mechanism of biogenesis and potential function of the two alternatively spliced mRNAs encoded by the murine Msx3 gene
Agarwal Regulation of alpha-1 (I) collagen gene transcription by TGF-beta

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19961231

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: LT;LV;SI

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: CURIS, INC.

17Q First examination report despatched

Effective date: 20020819

17Q First examination report despatched

Effective date: 20020819

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: STRYKER CORPORATION

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20081125