WO1996039431A1 - Proteine morphogenique osseuse (bmp-10) - Google Patents

Proteine morphogenique osseuse (bmp-10) Download PDF

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Publication number
WO1996039431A1
WO1996039431A1 PCT/US1995/007915 US9507915W WO9639431A1 WO 1996039431 A1 WO1996039431 A1 WO 1996039431A1 US 9507915 W US9507915 W US 9507915W WO 9639431 A1 WO9639431 A1 WO 9639431A1
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Prior art keywords
polypeptide
polynucleotide
dna
amino acid
receptor
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PCT/US1995/007915
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English (en)
Inventor
Wei Wu He
Timothy A. Coleman
Gregg A. Hastings
Craig A. Rosen
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Human Genome Sciences, Inc.
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Publication date
Application filed by Human Genome Sciences, Inc. filed Critical Human Genome Sciences, Inc.
Priority to PCT/US1995/007915 priority Critical patent/WO1996039431A1/fr
Priority to AU29473/95A priority patent/AU2947395A/en
Publication of WO1996039431A1 publication Critical patent/WO1996039431A1/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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus

Definitions

  • This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is Bone Morphogenic Protein-10 "BMP-10". The invention also relates to inhibiting the action of such polypeptides.
  • BMP may be used to induce bone and/or cartilage formation and is therefore useful in wound healing and tissue repair.
  • BMP may be used for treating a number of bone defects and periodontal disease and various types of wounds.
  • a 32-36 kDa osteogenic protein purified from bovine bone matrix is composed of dimers of two members of the transforming growth factor-beta super family, the bovine equivalent of human osteogenic protein-1 and bone morphogenic protein-2a. It is reported that recombinant human osteogenic protein-1 (HOP-1) induces new bone formation in vivo with a specific activity compatible with natural bovine osteogenic protein and stimulates osteobla ⁇ t proliferation and differentiation in vitro (Sampath, T. ., et al., j. Biol. Chem. , 267:20352-62 (1992)).
  • HOP-1 human osteogenic protein-1
  • mammalian cells The recombinant human osteogenic protein-1 (HOP-1) was produced in mammalian cells as a processed mature disulfide-linked homodimer with an apparent molecular weight of 36,000.
  • the evaluation of HOP-l effects on cell growths and collagen synthesis in rat osteobla ⁇ t-enriched bone cell cultures showed that both cell proliferation and collagen synthesis were stimulated in a dose-dependent manner and increased three-fold in response to 40ng of HOP-l/ml.
  • DVR-4 (Bone Morphogenetic Protein-4) induces amphibian embryos to develop with an overall posterior and/or ventral character, and that DVR-4 induces ventral types of mesoderm in animal explants. DVR-4 is therefore the first molecule reported both to induce posteroventral mesoderm and to counteract dorsalizing signals such as activin, (Jones, CM. et al, Development, 115:639-47 (1992)).
  • polypeptide of the present invention is of human origin.
  • polynucleotides (DNA or RNA) which encode such polypeptides.
  • agonists which mimic the polypeptide of the present invention and bind to the receptors.
  • nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to a nucleic acid sequence of the present invention.
  • diagnostic assays for detecting diseases or susceptibility to diseases related to mutations in the nucleic acid sequences encoding a polypeptide of the present invention.
  • FIG. l depicts the cDNA and corresponding deduced amino acid sequence of the mature BMP-10 polypeptide.
  • the amino acid sequence is represented by the standard three letter code for amino acids.
  • the polypeptide of the present invention has a putative leader sequence comprising the initial 33 amino acids.
  • the active portion of the protein comprises from amino acid 344 to amino acid 478 of Figure 1 (SEQ ID NO:2) .
  • nucleic acid which encodes for the mature polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. on June 2, 1995.
  • a polynucleotide encoding a polypeptide of the present invention was discovered in a fetal lung cDNA library. It contains an open reading frame encoding a mature polypeptide of 378 amino acids and shows 80 % sequence identity to the BMP-3a gene product.
  • the polypeptide is a member of the bone morphogenic protein family which is a subfamily of the transforming growth factor Beta (TGF-jS) superfamily.
  • the polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
  • the DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand.
  • the coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 (SEQ ID NO:l) or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same, mature polypeptide as the DNA of Figure 1 (SEQ ID NO:l) or the deposited cDNA.
  • the polynucleotide which encodes for the mature polypeptide of Figure 1 (SEQ ID NO:2) or for the mature polypeptide encoded by the deposited cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.
  • polynucleotide encoding a polypeptide encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
  • the present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) or the polypeptide encoded by the cDNA of the deposited clone.
  • the variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non- naturally occurring variant of the polynucleotide.
  • the present invention includes polynucleotide ⁇ encoding the same mature polypeptide as shown in Figure 1 (SEQ ID NO:2) or the same mature polypeptide encoded by the cDNA of the deposited clone as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID NO:2) or the polypeptide encoded by the cDNA of the deposited clone.
  • nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
  • the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figure 1 (SEQ ID N0:1) or of the coding sequence of the deposited clone.
  • an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.
  • the present invention also includes polynucleotides, wherein the coding sequence for the mature polypeptide may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell.
  • the polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide.
  • the polynucleotides may also encode for a proprotein which is the mature protein plus additional 5' amino acid residues.
  • a mature protein having a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved an active mature protein remains.
  • the polynucleotide of the present invention may encode for a mature protein, or for a protein having a prosequence or for a protein having both a prosequence and a presequence (leader sequence) .
  • the polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention.
  • the marker sequence may be a hexa- histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).
  • gene means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons) .
  • Fragments of the full length gene of--,the present invention may be used as a hybridization probe for a cDNA library to isolate the full length cDNA and to isolate other cDNAs which have a high sequence similarity to the gene or similar biological activity.
  • Probes of this type preferably have at least 30 bases and may contain, for example, 50 or more bases.
  • the probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promotor regions, exons, and introns.
  • An example of a screen comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
  • the present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 80%, preferably at least 90%, and more preferably at least 95% identity between the sequences.
  • the present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides.
  • stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • polypeptides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which either retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of Figure 1 (SEQ ID NO:l) or the deposited cDNA(s) .
  • the polynucleotide may have at least 20 bases, preferably 30 bases, and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which has an identity thereto, as hereinabove described, and which may or may not retain activity.
  • such polynucleotides may be employed as probes for the polynucleotide of SEQ ID NO:l, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.
  • the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 90% and more preferably at least a 95% identity to a polynucleotide which encodes the polypeptide of SEQ ID NO:2 as well as fragments thereof, which fragments have at least 30 bases and preferably at least 50 bases and to polypeptides encoded by such polynucleotides.
  • the present invention further relates to a BMP-10 polypeptide which has the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) or which has the amino acid sequence encoded by the depo ⁇ ited cDNA, as well as fragments, analogs and derivatives of ⁇ uch polypeptide.
  • fragment when referring to the polypeptide of Figure 1 (SEQ ID NO:2) or that encoded by the depo ⁇ ited cDNA, means a polypeptide which retains essentially the same biological function or activity as such polypeptide.
  • an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
  • the fragment, derivative or analog of the polypeptide of Figure la or that encoded by the deposited cDNA may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol) , or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or ⁇ ecretory ⁇ equence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence.
  • Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein
  • polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it i ⁇ naturally occurring) .
  • a naturally- occurring polynucleotide or polypeptide pre ⁇ ent in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural sy ⁇ tem, i ⁇ isolated.
  • Such polynucleotides could be part of a vector and/or ⁇ uch polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of it ⁇ natural environment.
  • polypeptides of the present invention include the polypeptide of SEQ ID NO:2 (in particular the mature polypeptide) as well as polypeptides which have at least 80% similarity (preferably at least 80% identity) to the polypeptide of SEQ ID NO:2 and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID NO:2 and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of SEQ ID NO:2 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
  • Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed a ⁇ intermediate ⁇ for producing the full-length polypeptide ⁇ .
  • Fragment ⁇ or portion ⁇ of the polynucleotide ⁇ of the pre ⁇ ent invention may be u ⁇ ed to ⁇ ynthe ⁇ ize full-length polynucleotide ⁇ of the pre ⁇ ent invention.
  • the pre ⁇ ent invention al ⁇ o relate ⁇ to vectors which include polynucleotides of the pre ⁇ ent invention, ho ⁇ t cell ⁇ which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • Host cells are genetically engineered (transduced or transformed or transfected) with the vector ⁇ of thi ⁇ invention which may be, for example, a cloning vector or an expres ⁇ ion vector.
  • the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the BMP-10 genes.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expres ⁇ ion, and will be apparent to the ordinarily skilled artisan.
  • the polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques.
  • the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide.
  • Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vector ⁇ derived from combinations of plasmid ⁇ and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • any other vector may be used as long as it i ⁇ replicable and viable in the ho ⁇ t.
  • the appropriate DNA ⁇ equence may be inserted into the vector by a variety of procedures.
  • the DNA sequence is in ⁇ erted into an appropriate re ⁇ triction endonuclease site( ⁇ ) by procedure ⁇ known in the art.
  • procedure ⁇ known in the art.
  • the DNA sequence in the expres ⁇ ion vector is operatively linked to an appropriate expression control ⁇ equence( ⁇ ) (promoter) to direct mRNA ⁇ ynthe ⁇ i ⁇ .
  • ⁇ equence( ⁇ ) promoter
  • a ⁇ repre ⁇ entative example ⁇ of ⁇ uch promoter ⁇ there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda P promoter and other promoter ⁇ known to control expre ⁇ sion of genes in prokaryotic or eukaryotic cells or their viruse ⁇ .
  • the expression vector also contains a ribosome binding ⁇ ite for tran ⁇ lation initiation and a transcription terminator.
  • the vector may also include appropriate sequence ⁇ for amplifying expre ⁇ ion.
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin re ⁇ i ⁇ tance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • the vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate ho ⁇ t to permit the host to express the protein.
  • bacterial cells such as E. coli, Streptomyces. Salmonella tvphimurium
  • fungal cell ⁇ such as yeast
  • insect cells such as Drosophila and ____
  • animal cells such as CHO, COS or Bowes melanoma
  • plant cells etc.
  • the selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
  • the present invention also includes recombinant constructs comprising one or more of the sequence ⁇ a ⁇ broadly described above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a ⁇ equence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequence ⁇ , including, for example, a promoter, operably linked to the sequence.
  • regulatory sequence ⁇ including, for example, a promoter, operably linked to the sequence.
  • Bacterial pQE70, pQE60, pQE-9 (Qiagen) , pbs, pDlO, phage ⁇ cript, p ⁇ iX174, pblue ⁇ cript SK, pb ⁇ k ⁇ , pNH ⁇ A, pNH16a, pNH18A, pNH46A (Stratagene) ; ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia) .
  • Eukaryotic pWLNEO, PSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3 ⁇ pBPV, pMSG, pSVL (Pharmacia) .
  • any other pla ⁇ mid or vector may be used as long as they are replicable and viable in the host.
  • Promoter regions can be selected from any desired gene u ⁇ ing CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • Two appropriate vectors are PKK232-8 and PCM7.
  • Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P R , P L and trp.
  • Eukaryotic promoters include GMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter i ⁇ well within the level of ordinary ⁇ kill in the art.
  • the pre ⁇ ent invention relate ⁇ to host cells containing the above-described constructs.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated transfection, or electroporation. (Davis, L., Dibner, M. , Battey, I., Basic Methods in Molecular Biology, (1986) ) .
  • the construct ⁇ in ho ⁇ t cell ⁇ can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides of the invention can be ⁇ ynthetically produced by conventional peptide ⁇ ynthesizers.
  • Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cell ⁇ under the control of appropriate promoters.
  • Cell-free translation system ⁇ can also be employed to produce such proteins using RNA ⁇ derived from the DNA con ⁇ truct ⁇ of the present invention.
  • Appropriate cloning and expres ⁇ ion vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al. , Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late ⁇ ide of the replication origin, and adenoviru ⁇ enhancer ⁇ .
  • recombinant expre ⁇ ion vector ⁇ will include origin ⁇ of replication and ⁇ electable marker ⁇ permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
  • promoters can be derived from operon ⁇ encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK) , ⁇ -factor, acid phosphatase, or heat shock proteins, among others.
  • the heterologous ⁇ tructural sequence is a ⁇ sembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of tran ⁇ lated protein into the peripla ⁇ mic space or extracellular medium.
  • the heterologous sequence can encode a fu ⁇ ion protein including an N-terminal identification peptide imparting de ⁇ ired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • Useful expression vectors for bacterial u ⁇ e are con ⁇ tructed by in ⁇ erting a ⁇ tructural DNA sequence encoding a desired protein together with suitable tran ⁇ lation initiation and termination ⁇ ignals in operable reading phase with a functional promoter.
  • the vector will comprise one or more phenotypic ⁇ electable marker ⁇ and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis. Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcu ⁇ , although others may also be employed as a matter of choice.
  • useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017) .
  • cloning vector pBR322 ATCC 37017
  • Such commercial vector ⁇ include, for example, pKK223-3 (Pharmacia Fine Chemical ⁇ , Uppsala, Sweden) and GEMl (Promega Biotec, Madison, WI, USA) .
  • pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
  • the ⁇ elected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • Cells are typically harvested by centrifugation, disrupted by phy ⁇ ical or chemical mean ⁇ , and the resulting crude extract retained for further purification.
  • Microbial cells employed in expre ⁇ ion of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.
  • mammalian cell culture sy ⁇ tem ⁇ can also be employed to express recombinant protein.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibrobla ⁇ ts, de ⁇ cribed by Gluzman, Cell, 23:175
  • Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequence ⁇ .
  • DNA sequences derived from the SV40 splice, and polyadenylation site ⁇ may be u ⁇ ed to provide the required nontran ⁇ cribed genetic element ⁇ .
  • the polypeptide ⁇ can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography and lectin chromatography. It is preferred to have low concentrations (approximately 0.15-5 mM) of calcium ion present during purification. (Price et al., J. Biol. Chem. , 244:917 (1969)). Protein refolding step ⁇ can be u ⁇ ed, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification step ⁇ .
  • HPLC high performance liquid chromatography
  • the polypeptide ⁇ of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, in ⁇ ect and mammalian cell ⁇ in culture) .
  • a prokaryotic or eukaryotic host for example, by bacterial, yeast, higher plant, in ⁇ ect and mammalian cell ⁇ in culture
  • the polypeptide ⁇ of the pre ⁇ ent invention may be glyco ⁇ ylated or may be non-glyco ⁇ ylated.
  • Polypeptide ⁇ of the invention may al ⁇ o include an initial methionine amino acid re ⁇ idue.
  • the BMP-10 may be employed to promote de novo bone formation which may be u ⁇ ed in the treatment of periodontal di ⁇ ea ⁇ e and other bone defect ⁇ of the oral cavity.
  • BMP-10 may al ⁇ o be used during surgical insertion of protheses. In hip replacements, knee replacements and other surgical insertion of prothese ⁇ , the prothesis is held in place by surgical cement. The cement eventually loosens, however, making it necessary to perform another surgery. This second surgery is a much more difficult procedure and is responsible for surgeons reluctantance to insert protheses in young people.
  • BMP-10 may, therefore, be used to coat the prothesis before insertion which results in bone formation around the prothesis, making a stronger union and allowing for the use of les ⁇ cement.
  • BMP-10 may al ⁇ o be employed in the treatment of o ⁇ teoporo ⁇ i ⁇ , which i ⁇ characterized by exce ⁇ ive bone re ⁇ orption resulting in thin and brittle bones. BMP-10 would stimulate bone formation to help alleviate this condition.
  • This invention provides a method for identification of the receptor for the polypeptide of the present invention.
  • the gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al., Current Protocols in Immun. , 1(2), Chapter 5, (1991)).
  • expression cloning is employed wherein polyadenylated RNA i ⁇ prepared from a cell responsive to the polypeptide of the pre ⁇ ent invention, and a cDNA library created from this RNA is divided into pools and u ⁇ ed to transfect COS cells or other cells that are not re ⁇ pon ⁇ ive to the polypeptide of the pre ⁇ ent invention.
  • BMP-10 can be labeled by a variety of means including iodination or inclu ⁇ ion of a recognition ⁇ ite for a ⁇ ite- ⁇ pecific protein kina ⁇ e.
  • the ⁇ lide ⁇ are ⁇ ubjected to auto-radiographic analy ⁇ i ⁇ .
  • Po ⁇ itive pool ⁇ are identified and ⁇ ub-pool ⁇ are prepared and re-tran ⁇ fected u ⁇ ing an iterative ⁇ ub-pooling and re-screening proces ⁇ , eventually yielding a ⁇ ingle clone that encodes the putative receptor.
  • labeled ligand can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule.
  • Cross-linked material is resolved by PAGE and exposed to X- ray film.
  • the labeled complex containing the ligand-receptor can be excised, resolved into peptide fragments, and subjected to protein microsequencing.
  • the amino acid sequence obtained from microsequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor.
  • This invention also provides a method for detecting compounds which mimic the activity of the polypeptide of the present invention.
  • the method for determining whether a ligand can bind to the BMP-10 receptor comprise ⁇ tran ⁇ fecting a cell population (one presumed not to contain the receptor) with the appropriate vector expres ⁇ ing the receptor, ⁇ uch that the cell will now express the receptor.
  • a suitable response system is obtained by tran ⁇ fection of the DNA into a suitable host containing the de ⁇ ired second messenger pathways including cAMP, ion channels, phosphoinositide kinase, or calcium response.
  • Such a transfection system provides a response sy ⁇ tem to analyze the activity of variou ⁇ ligand ⁇ exposed to the cell.
  • Ligands cho ⁇ en could be identified through a rational approach by taking known ligand ⁇ that interact with ⁇ imilar type ⁇ of receptor ⁇ or u ⁇ ing ⁇ mall molecule ⁇ , cell supernatants or extracts or natural products.
  • a mammalian cell or membrane preparation expressing the receptor i ⁇ incubated with the compound expressing the receptor i ⁇ incubated with the compound.
  • the ability of the compound to stimulate the re ⁇ pon ⁇ e of a known ⁇ econd me ⁇ enger ⁇ y ⁇ tem following interaction of compound and receptor would be measured.
  • second messenger systems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.
  • polypeptides and agonists which are polypeptides may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which i ⁇ often referred to a ⁇ "gene therapy.”
  • cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide.
  • a polynucleotide DNA or RNA
  • cell ⁇ may be engineered by procedure ⁇ known in the art by u ⁇ e of a retroviral particle containing RNA encoding a polypeptide of the present invention.
  • cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art.
  • a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo.
  • the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be u ⁇ ed to engineer cell ⁇ in vivo after combination with a ⁇ uitable delivery vehicle.
  • Retroviruses from which the retroviral pla ⁇ mid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruse ⁇ such as Rou ⁇ Sarcoma Viru ⁇ , Harvey Sarcoma Viru ⁇ , avian leukosi ⁇ viru ⁇ , gibbon ape leukemia viru ⁇ , human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
  • the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.
  • the vector includes one or more promoters.
  • Suitable promoter ⁇ which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegaloviru ⁇ (CMV) promoter described in Miller, et al., Biotechniques. Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoter ⁇ ⁇ uch as eukaryotic cellular promoters including, but not limited to, the hi ⁇ tone, pol III, and /3-actin promoters) .
  • CMV human cytomegaloviru ⁇
  • viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoter ⁇ .
  • TK thymidine kinase
  • B19 parvovirus promoter ⁇ The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
  • Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or hetorologou ⁇ promoter ⁇ , ⁇ uch a ⁇ the cytomegaloviru ⁇ (CMV) promoter; the respiratory syncytial viru ⁇ (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such a ⁇ the Herpe ⁇ Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described) ; the jS-actin promoter; and human growth hormone promoters.
  • the promoter include, but are not limited to, adenoviral promoters, such as the adenoviral
  • the retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines.
  • packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ⁇ -2 , ⁇ -AM, PA12, T19-14X, VT-19-17-H2, ⁇ CRE , ⁇ CRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy. Vol. l, pgs. 5-14 (1990), which is incorporated herein by reference in its entirety.
  • the vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP0 4 precipitation.
  • the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a ho ⁇ t.
  • the producer cell line generates infectious retroviral vector particle ⁇ which include the nucleic acid sequence(s) encoding the polypeptides.
  • retroviral vector particle ⁇ then may be employed, to transduce eukaryotic cells, either in vitro or in vivo.
  • the tran ⁇ duced eukaryotic cells will express the nucleic acid sequence( ⁇ ) encoding the polypeptide.
  • Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cell ⁇ , as well as hematopoietic stem cells, hepatocytes, fibrobla ⁇ t ⁇ , myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.
  • the polypeptide ⁇ and agoni ⁇ ts of the present invention may be employed in combination with a suitable pharmaceutical carrier.
  • a suitable pharmaceutical carrier include ⁇ but is not limited to ⁇ aline, buffered saline, dextrose, water, glycerol, ethanol, and combination ⁇ thereof.
  • a carrier include ⁇ but is not limited to ⁇ aline, buffered saline, dextrose, water, glycerol, ethanol, and combination ⁇ thereof.
  • the formulation ⁇ hould suit the mode of administration.
  • the invention al ⁇ o provide ⁇ a pharmaceutical pack or kit compri ⁇ ing one or more container ⁇ filled with one or more of the ingredient ⁇ of the pharmaceutical compo ⁇ ition ⁇ of the invention.
  • a ⁇ ociated with such container(s) can be a notice in the form pre ⁇ cribed by a governmental agency regulating the manufacture, u ⁇ e or ⁇ ale of pharmaceuticals or biological product ⁇ , which notice reflect ⁇ approval by the agency of manufacture, use or sale for human administration.
  • the polypeptides and agonist ⁇ of the present invention may be employed in conjunction with other therapeutic compounds.
  • BMP-10 is preferably used topically, however, when it is used systemically, the pharmaceutical composition ⁇ may be admini ⁇ tered in a convenient manner ⁇ uch a ⁇ by the oral, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, or intradermal routes.
  • the amounts and dosage regimens of pharmaceutical compositions of BMP-10 administered to a subject will depend on a number of factors such a ⁇ the mode of administration, the nature of the condition being treated, the body weight of the subject being treated and the judgment of the prescribing physician.
  • compositions of BMP-10 are given, for example, in appropriate doses of at least about 10 ⁇ g kQ body weight and in most case ⁇ will not be administered in an amount in exces ⁇ of about 8 mg/kg body weight, and preferably is given in dose ⁇ of about 10 ⁇ g/kg body weight to about 1 mg/kg daily, taking into account the route ⁇ of admini ⁇ tration, ⁇ ymptoms, etc.
  • BMP-10 may be prepared as a gel matrix formulation and administered, for example, ectopically, which is preferably administered at the site of bone fracture at a dosage of 50 mg, by applying the matrix directly to the site of the fracture. This matrix may also be applied to protheses before insertion.
  • This invention is also related to the use of the gene of the present invention as a diagnostic. Detection of a mutated form of the gene will allow a diagnosis of a disease or a susceptibility to a disease which results from underexpres ⁇ ion of BMP-10.
  • Nucleic acids for diagnosis may be obtained from a patient's cells, including but not limited to blood, urine, saliva, tissue biopsy and autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analysis.
  • RNA or cDNA may also be used for the same purpose.
  • PCR primers complementary to the nucleic acid encoding BMP-10 can be used to identify and analyze mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA or alternatively, radiolabeled antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.
  • Sequence differences between the reference gene and genes having mutations may be revealed by the direct DNA sequencing method.
  • cloned DNA segments may be employed as probes to detect ⁇ pecific DNA ⁇ egment ⁇ .
  • the sensitivity of this method is greatly enhanced when combined with PCR.
  • a sequencing primer is u ⁇ ed with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedure ⁇ with fluore ⁇ cent-tag ⁇ .
  • DNA fragment ⁇ of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al . , Science, 230:1242 (1985)) .
  • Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (e.g., Cotton et al . , PNAS, USA, 85:4397-4401 (1985)) .
  • nuclease protection assays such as RNase and SI protection or the chemical cleavage method (e.g., Cotton et al . , PNAS, USA, 85:4397-4401 (1985)) .
  • the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g., Restriction Fragment Length Polymorphism ⁇ (RFLP) ) and Southern blotting of genomic DNA.
  • restriction enzymes e.g., Restriction Fragment Length Polymorphism ⁇ (RFLP)
  • mutation ⁇ can also be detected by in situ analysi ⁇ .
  • the present invention also relates to a diagnostic assay for detecting altered levels of the polypeptide of the present invention in various tissues since an over-expression of the proteins compared to normal control tis ⁇ ue samples can detect the presence of lung cancer.
  • Assays used to detect levels of the polypeptide of the pre ⁇ ent invention in a sample derived from a host are well-known to those of skill in the art and include radioimmunoassays, competitive-binding a ⁇ ay ⁇ , We ⁇ tern Blot analy ⁇ i ⁇ and preferably an ELISA assay.
  • a reporter antibody is prepared against the monoclonal antibody.
  • a detectable reagent such as radioactivity, fluorescence or in this example a horseradish peroxidase enzyme.
  • a sample is now removed from a ho ⁇ t and incubated on a ⁇ olid ⁇ upport, e.g. a poly ⁇ tyrene di ⁇ h, that bind ⁇ the protein ⁇ in the ⁇ ample. Any free protein binding ⁇ ites on the di ⁇ h are then covered by incubating with a non- ⁇ pecific protein such as bovine serum albumin.
  • the monoclonal antibody i ⁇ incubated in the di ⁇ h during which time the monoclonal antibodie ⁇ attached to any of the polypeptide of the present invention attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer.
  • the reporter antibody linked to horseradish peroxidase is now placed in the di ⁇ h resulting in binding of the reporter antibody to any monoclonal antibody bound to the polypeptide of the present invention. Unattached reporter antibody is then washed out.
  • Peroxidase substrates are then added to the dish and the amount of color developed in a given time period i ⁇ a measurement of the amount of the polypeptide of the present invention present in a given volume of patient sample when compared against a standard curve.
  • a competition assay may be employed wherein antibodies specific to the polypeptide of the present invention are attached to a solid support and labeled and a sample derived from the host are passed over the solid support and the amount of label detected attached to the solid support can be correlated to a quantity of the polypeptide of the present invention in the ⁇ ample.
  • the ⁇ equence ⁇ of the pre ⁇ ent invention are al ⁇ o valuable for chromo ⁇ ome identification.
  • the ⁇ equence i ⁇ ⁇ pecifically targeted to and can hybridize with a particular location on an individual human chromo ⁇ ome.
  • there i ⁇ a current need for identifying particular ⁇ ite ⁇ on the chromo ⁇ ome.
  • Few chromo ⁇ ome marking reagent ⁇ ba ⁇ ed on actual sequence data (repeat polymorphisms) are pre ⁇ ently available for marking chromo ⁇ omal location.
  • the mapping of DNA ⁇ to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
  • sequence ⁇ can be mapped to chromo ⁇ ome ⁇ by preparing PCR primer ⁇ (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region of the gene is used to rapidly select primer ⁇ that do not ⁇ pan more than one exon in the genomic DNA, thus complicating the amplification proces ⁇ .
  • the ⁇ e primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
  • PCR mapping of somatic cell hybrids is a rapid procedure for as ⁇ igning a particular DNA to a particular chromosome.
  • sublocalization can be achieved with panel ⁇ of fragment ⁇ from specific chromosomes or pools of large genomic clones in an analogous manner.
  • Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
  • Fluorescence in si tu hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromo ⁇ omal location in one step.
  • FISH Fluorescence in si tu hybridization
  • a cDNA precisely localized to a chromosomal region as ⁇ ociated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb) .
  • the polypeptides, their fragments or other derivatives, or analogs thereof, or cells expres ⁇ ing them can be u ⁇ ed a ⁇ an immunogen to produce antibodies thereto.
  • These antibodies can be, for example, polyclonal or monoclonal antibodies.
  • the present invention also includes chimeric, single chain, and humanized antibodies, a ⁇ well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
  • Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides it ⁇ elf. In thi ⁇ manner, even a ⁇ equence encoding only a fragment of the polypeptides can be used to generate antibodie ⁇ binding the whole native polypeptide ⁇ . Such antibodie ⁇ can then be used to isolate the polypeptide from tissue expressing that polypeptide.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodie ⁇ and' Cancer Therapy, Alan R. Li ⁇ , Inc., pp. 77-96) .
  • Plasmid ⁇ are de ⁇ ignated by a lower case p preceded and/or followed by capital letters and/or numbers.
  • the starting plasmids herein are either commercially available, publicly available on an unrestricted ba ⁇ i ⁇ , or can be constructed from available plasmid ⁇ in accord with published procedure ⁇ .
  • equivalent pla ⁇ mid ⁇ to those described are known in the art and will be apparent to the ordinarily skilled artisan.
  • “Digestion” of DNA refers to catalytic cleavage of the DNA with a re ⁇ triction enzyme that act ⁇ only at certain ⁇ equence ⁇ in the DNA.
  • the variou ⁇ restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirement ⁇ were used a ⁇ would be known to the ordinarily ⁇ killed arti ⁇ an.
  • For analytical purpo ⁇ es typically 1 ⁇ g of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
  • isolating DNA fragments for pla ⁇ mid construction typically 5 to 50 ⁇ g of DNA are digested with 20 to 250 units of enzyme in a larger volume.
  • buffer ⁇ and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37 * C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction i ⁇ electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
  • Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res., 8:4057 (1980).
  • Oligonucleotides refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strand ⁇ which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
  • Ligase refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragment ⁇ (Maniati ⁇ , T. , et al., Id., p. 146). Unle ⁇ otherwi ⁇ e provided, ligation may be accomplished using known buffers and conditions with 10 unit ⁇ to T4 DNA ligase ("ligase”) per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragments to be ligated.
  • ligase DNA ligase
  • the DNA sequence encoding for human BMP-10 is initially amplified using PCR oligonucleotide primers corresponding to the 5' and 3' end of the DNA sequence to synthesize insertion fragments.
  • the 5' oligonucleotide primer 5'- GATCGGATCCAAAGCCCGGAGGAAGCAG-3' contains a Bam HI restriction enzyme ⁇ ite followed by 18 nucleotide ⁇ of BMP-lO- coding sequence starting from amino acid 4 (Lys) ; the 3' ⁇ equence 5' -GTACTCTAGATCACCGGCAGGCACAGGTG-3' contain ⁇ complementary sequences to an Xba I site, a translation stop codon and the last 16 nucleotides of BMP-10 coding sequence.
  • the plasmid vector encodes antibiotic resi ⁇ tance (Amp r ) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter/operator (P/O) , a ribosome binding site (RBS) , a 6- histidine tag (6-His) and restriction enzyme cloning sites.
  • the pQE-9 vector was digested with Bam HI and Xba I and the insertion fragment ⁇ were then ligated into the pQE-9 vector maintaining the reading frame initiated at the bacterial RBS.
  • the ligation mixture was then used to transform the E. coli strain M15/rep4 available from Qiagen under the trademark ml5/rep4.
  • M15/rep4 contains multiple copies of the plasmid pREP4, which expresse ⁇ the lad repre ⁇ sor and also confers kanamycin resi ⁇ tance (Kan r ) . Tran ⁇ formant ⁇ are identified by their ability to grow on LB plates containing both Amp and Kan.
  • RNAzolTM B system Biotecx Laboratories, Inc., 6023 South Loop East, Houston, TX 77033
  • About 10 ug of total RNA isolated from each human tissue specified was separated on 1% agarose gel and blotted onto a nylon filter (Sambrook, J. et al.. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989)).
  • the labeling reaction was done according to the Stratagene Prime- It kit with 50 ng DNA fragment.
  • the labeled DNA wa ⁇ purified with a Select-G-50 column from 5' Prime -- 3 Prime, Inc., 5603 Arapahoe Road, Boulder, CO 80303.
  • the filter was then hybridized with radioactive labeled full length BMP-10 gene at 1,000,000 cpm/ml in 0.5 M NaPo 4 and 7% SDS overnight at 65'C.
  • the filters were washed twice at room temperature and twice at 60°C with 0.5 x SSC, 0.1% SDS, the filters were then exposed at -70 * C overnight with inten ⁇ ifying screen.
  • Fibroblast ⁇ are obtained from a subject by skin biopsy.
  • the resulting tis ⁇ ue i ⁇ placed in ti ⁇ ue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask.
  • the flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunk ⁇ of ti ⁇ sue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin, is added. Thi ⁇ i ⁇ then incubated at 37°C for approximately one week.
  • fresh media e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin
  • the cDNA encoding a polypeptide of the present invention is amplified using PCR primers which correspond to the 5' and 3' end sequences respectively.
  • the 5' primer contains an EcoRI site and the 3' primer include ⁇ a Hindlll site.
  • Equal quantities of the Moloney murine sarcoma virus linear backbone and the amplified EcoRI and Hindlll fragment are added together, in the presence of T4 DNA ligase.
  • the resulting mixture is maintained under conditions appropriate for ligation of the two fragments.
  • the ligation mixture is used to transform bacteria HB101, which are then plated onto agar-containing kanamycin for the purpose of confirming that the vector had the gene of intere ⁇ t properly in ⁇ erted.
  • the amphotropic pA317 or GP+aml2 packaging cell ⁇ are grown in tissue culture to confluent density in Dulbecco' ⁇ Modified Eagles Medium (DMEM) with 10% calf serum (CS) , penicillin and ⁇ treptomycin.
  • DMEM Dulbecco' ⁇ Modified Eagles Medium
  • CS calf serum
  • penicillin and ⁇ treptomycin The MSV vector containing the gene is then added to the media and the packaging cells are transduced with the vector.
  • the packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells) .
  • Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cell ⁇ .
  • the spent media containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cell ⁇ and this media i ⁇ then used to infect fibroblast cells.
  • Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cell ⁇ . This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it i ⁇ nece ⁇ sary to use a retroviral vector that has a selectable marker, such as neo or his.
  • the engineered fibroblast ⁇ are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads.
  • the fibroblasts now produce the protein product.
  • Plasmid pN346 is used for the expression of the BMP- 10 protein.
  • Plasmid pN346 is a derivative of the plasmid pSV2-dhfr [ATCC Acce ⁇ ion No. 37146] . Both pla ⁇ mid ⁇ contain the mouse dhfr gene under control of the SV40 early promoter. Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with the ⁇ e pla ⁇ mid ⁇ can be selected by growing the cells in a selective medium (alpha minus MEM, Lift Technologies) supplemented with the chemotherapeutic agent methotrexate.
  • a selective medium alpha minus MEM, Lift Technologies
  • DHFR target enzyme
  • a second gene is linked to the dhfr gene it is usually co-amplified and over- expressed. It is state of the art to develop cell lines carrying more than 1,000 copies of the gene ⁇ . Subsequently, when the methotrexate is withdrawn, cell lines contain the amplified gene integrated into the chromosome(s) .
  • Plasmid pN346 contains for the expression of the gene of interest a strong promoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus (Cullen, et al., Molecular and Cellular Biology, March 1985, 438-447) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegaloviru ⁇ (CMV) (Boshart et al., Cell 41:521-530, 1985) . Downstream of the promoter are the following ⁇ ingle restriction enzyme cleavage sites that allow the integration of the genes: Ba HI, Pvull, and Nrul.
  • LTR long terminal repeat
  • CMV cytomegaloviru ⁇
  • the pla ⁇ mid contain ⁇ tran ⁇ lational ⁇ top codon ⁇ in all three reading frame ⁇ ollowed by the 3' intron and the polyadenylation ⁇ ite of the rat preproin ⁇ ulin gene.
  • Other high efficient promoters can al ⁇ o be used for the expres ⁇ ion, e.g., the human ⁇ -actin promoter, the SV40 early or late promoter ⁇ or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI.
  • the polyadenylation of the mRNA other ⁇ ignal ⁇ e.g., from the human growth hormone or globin genes can be used as well.
  • Stable cell lines carrying a gene of interest integrated into the chromosome can also be selected upon co-transfection with a selectable marker such a ⁇ gpt, G418 or hygromycin. It i ⁇ advantageous to u ⁇ e more than one ⁇ electable marker in the beginning, e.g. G418 plu ⁇ methotrexate.
  • the plasmid pN346 was digested with the restriction enzyme BamHI and then depho ⁇ phorylated u ⁇ ing calf intestinal phosphatase by procedures known in the art. The vector was then isolated from a 1% agarose gel.
  • the DNA sequence encoding BMP-10, ATCC # was amplified u ⁇ ing PCR oligonucleotide primers corresponding to the 5' and 3' sequence ⁇ of the gene:
  • the 5' primer has the sequence 5' GCGGATCCGCCATGGCTCATGT CCCC 3' and contains a BamHI re ⁇ triction enzyme site (in bold) followed by 18 nucleotides re ⁇ embling an efficient signal for tran ⁇ lation (Kozak, M., ⁇ upra) plus the first 12 nucleotides of the gene (the initiation codon for tran ⁇ lation "ATG" i ⁇ underlined.).
  • the 3' primer has the sequence 5' GCGGATCCTCAGGCGCA GGCTGTCCA 3' and contains the cleavage site for the restriction endonuclease BamHI and 18 nucleotides complementary to the 3' non-translated sequence of the gene.
  • amplified fragments were isolated from a 1% agarose gel as described above and then digested with the endonuclease Bglll and then purified again on a 1% agarose gel.
  • Chinese hamster ovary cells lacking an active DHFR enzyme were used for tran ⁇ fection.
  • 5 ⁇ g of the expre ⁇ ion pla ⁇ mid N346 were cotran ⁇ fected with 0.5 ⁇ g of the pla ⁇ mid pSVneo u ⁇ ing the lipofectin method (Feigner et al., ⁇ upra).
  • the plasmid pSV2-neo contains a dominant selectable marker, the gene neo from Tn5 encoding an enzyme that confers resi ⁇ tance to a group of antibiotics including G418.
  • the cell ⁇ were seeded in alpha minus MEM supplemented' with 1 mg/ml G418.
  • the cells were trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) and cultivated from 10-14 day ⁇ . After this period, single clones were trypsinized and then seeded in 6-well petri dishes using different concentrations of methotrexate (25, 50 nm, 100 nm, 200 nm, 400 nm) . Clones growing at the highest concentrations of methotrexate were then transferred to new 6-well plates containing even higher concentrations of methotrexate (500 nM, 1 ⁇ M, 2 ⁇ M, 5 ⁇ M) . The same procedure was repeated until clones grew at a concentration of 100 ⁇ M.
  • the expression of the desired gene product was analyzed by Western blot analy ⁇ i ⁇ and SDS-PAGE.

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Abstract

L'invention concerne un polypeptide humain, la protéine BMP-10, l'ADN (ARN) codant ce polypeptide ainsi qu'une procédure de production d'un tel polypeptide par des techniques de recombinaison. L'invention concerne également des procédés d'utilisation d'un tel polypeptide dans le but de stimuler la synthèse osseuse de novo. L'invention concerne en outre des tests diagnostiques servant à l'identification de mutations dans la séquence d'acides nucléiques codant un polypeptide selon l'invention et permettant de déceler une modification des concentrations dudit polypeptide, révélatrice d'une maladie telle que le cancer.
PCT/US1995/007915 1995-06-06 1995-06-06 Proteine morphogenique osseuse (bmp-10) WO1996039431A1 (fr)

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WO1999011664A1 (fr) * 1997-09-05 1999-03-11 Genetics Institute, Inc. Cellules genetiquement manipulees qui expriment des proteines morphogenetiques d'os
EP1095159A1 (fr) * 1998-07-15 2001-05-02 Human Genome Sciences, Inc. Proteine morphogenetique osseuse
WO2002068962A2 (fr) * 2001-02-21 2002-09-06 Stryker Corporation Methodes d'utilisation de proteines morphogeniques osseuses comme biomarqueurs dans la determination de la degenerescence et le vieillissement du cartilage
WO2005113590A2 (fr) * 2004-05-12 2005-12-01 Acceleron Pharma Inc. Propeptides de bmp10 et procédés correspondants
US7465706B2 (en) 2004-06-24 2008-12-16 Acceleron Pharma Inc. GDF3 propeptides and related methods
US7625867B2 (en) 2004-03-26 2009-12-01 Acceleron Pharma Inc. BMP-3 propeptides and related methods
US10336800B2 (en) 2014-07-10 2019-07-02 Cambridge Enterprise Limited Therapeutic use of bone morphogenetic proteins

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US5011691A (en) * 1988-08-15 1991-04-30 Stryker Corporation Osteogenic devices

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WO1999011664A1 (fr) * 1997-09-05 1999-03-11 Genetics Institute, Inc. Cellules genetiquement manipulees qui expriment des proteines morphogenetiques d'os
EP1095159A1 (fr) * 1998-07-15 2001-05-02 Human Genome Sciences, Inc. Proteine morphogenetique osseuse
EP1095159A4 (fr) * 1998-07-15 2003-01-02 Human Genome Sciences Inc Proteine morphogenetique osseuse
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WO2002068962A3 (fr) * 2001-02-21 2003-11-27 Stryker Corp Methodes d'utilisation de proteines morphogeniques osseuses comme biomarqueurs dans la determination de la degenerescence et le vieillissement du cartilage
US7625867B2 (en) 2004-03-26 2009-12-01 Acceleron Pharma Inc. BMP-3 propeptides and related methods
WO2005113590A3 (fr) * 2004-05-12 2006-03-02 Acceleron Pharma Inc Propeptides de bmp10 et procédés correspondants
WO2005113590A2 (fr) * 2004-05-12 2005-12-01 Acceleron Pharma Inc. Propeptides de bmp10 et procédés correspondants
US7741284B2 (en) 2004-05-12 2010-06-22 Acceleron Pharma Inc. BMP10 propeptides and related methods
US8287868B2 (en) 2004-05-12 2012-10-16 Acceleron Pharma Inc. BMP10 antibodies and related methods
US8772234B2 (en) 2004-05-12 2014-07-08 Acceleron Pharma, Inc. BMP10 propeptides and related methods
US7465706B2 (en) 2004-06-24 2008-12-16 Acceleron Pharma Inc. GDF3 propeptides and related methods
US8293238B2 (en) 2004-06-24 2012-10-23 Acceleron Pharma Inc. GDF3 antibodies and related methods
US8765670B2 (en) 2004-06-24 2014-07-01 Acceleron Pharma Inc. GDF3 propeptides and related methods
US10336800B2 (en) 2014-07-10 2019-07-02 Cambridge Enterprise Limited Therapeutic use of bone morphogenetic proteins
EP3669886A1 (fr) 2014-07-10 2020-06-24 Cambridge Enterprise, Ltd. Protéines morphogénétiques osseuses
EP3906936A1 (fr) 2014-07-10 2021-11-10 Cambridge Enterprise, Ltd. Protéines morphogénétiques osseuses
US11572396B2 (en) 2014-07-10 2023-02-07 Cambridge Enterprise Limited Therapeutic use of bone morphogenetic proteins

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