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WO2000006734A1 - Molecule induisant la chondrogenese et l'osteogenese - Google Patents

Molecule induisant la chondrogenese et l'osteogenese Download PDF

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Publication number
WO2000006734A1
WO2000006734A1 PCT/US1999/017342 US9917342W WO0006734A1 WO 2000006734 A1 WO2000006734 A1 WO 2000006734A1 US 9917342 W US9917342 W US 9917342W WO 0006734 A1 WO0006734 A1 WO 0006734A1
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Prior art keywords
chondrogenic
sequence
protein
gene
cells
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English (en)
Inventor
Arthur Veis
Denise R. Nebgen
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Northwestern University
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Northwestern University
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Priority to US09/744,128 priority Critical patent/US6677306B1/en
Priority to AU52468/99A priority patent/AU5246899A/en
Publication of WO2000006734A1 publication Critical patent/WO2000006734A1/fr
Anticipated expiration legal-status Critical
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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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to polypeptides with in vitro chondrogenic and in vivo osteogenic activity.
  • BMPs bone morphogenesis
  • BMPs were originally studied for their osteogenic inductive capabilities, following their cloning an explosion of developmental research revealed that the BMPs have a variety of regulatory functions throughout development, and at the embryonic level, in the transfer of signals during epithelial-mesenchymal interactions.
  • Bones and teeth are related tissues in that the bone and dentin matrices are comprised of type I collagen fibers that become mineralized by impregnation with crystals of carbonated calcium hydroxyapatite. It can thus be argued that bone and dentin mineralization follow similar pathways, although they do differ in detail. Most of the extracellular matrix proteins that have been found in bone have also been described in dentin. However, dentin contains several specific proteins that have not yet been found in bone.
  • epithelial cells differentiate into ameloblasts, which secrete the enamel, while ectomesenchymal cells derived from the neural crest differentiate into odontoblasts, which secrete the dentin.
  • These differentiation processes occur through a set of staged reciprocal interactions between the epithelial and mesenchymal cells, leading to the formation of the mature tooth (Lumsden, 1988).
  • Epigenetic signals passed between the cells appear to induce the appropriate cellular morphogenesis events.
  • BMP-4 has been localized within the tooth germ and has been proposed as one of the signals regulating the reciprocal transfer of information (Vainio et al., 1993), although BMP-4 alone cannot completely reproduce these changes, and other signals appear to be necessary.
  • Dentin matrix probably does contain the expressed BMPs.
  • the BMP proteins themselves have not been successfully purified from dentin.
  • Beesho et al. (1990, 1991) isolated proteins with BMP-like activity from rabbit and human dentin, but the ⁇ H 2 -terminal amino acid sequence for the human dentin-derived BMP did not resemble that of the known BMP family.
  • Amar et al. (1991) described the isolation from rat incisor dentin of a polypeptide exhibiting chondrogenic inducing activity (CIA) in cell culture assays (Koskinen et al. 1985, Veis et al., 1989). The amount of protein recovered from rat teeth was small and the NH 2 -terminal sequencing data was very limited. Nevertheless, as in the report of Bessho et al. (1991), it was clear that the CIA did not correspond to the known BMPs.
  • the present invention provide chondrogenic and osteogenic inducing molecules (CIM), also called chondrogenic inducing agents (CIA).
  • CIM chondrogenic and osteogenic inducing molecules
  • CIA chondrogenic inducing agents
  • the invention provides a chondrogenic and osteogenic inducing molecule having the nucleotide sequence SEQ ID NO: 16.
  • This polynucleotide corresponds to a splice product of the rat amelogenin gene encoded by rat amelogenin gene exons 2, 3 ,4, 5, 6 and 7.
  • the invention provides a chondrogenic and osteogenic inducing molecule, also referred to herein as "rA4", that has the nucleotide sequence SEQ ID NO: 1.
  • rA4 corresponds to a specific defined splice product of the rat amelogenin gene encoded by rat amelogenin gene exons 2, 3 ,4, 5, 6d and 7.
  • the invention provides a chondrogenic and osteogenic inducing molecule that has the nucleotide sequence SEQ ID NO: 17. This polynucleotide corresponds to a splice product of the rat amelogenin gene that includes exons 2, 3, 5, 6 and 7 of the rat amelogenin gene.
  • the invention provides a chondrogenic and osteogenic inducing molecule, also referred to herein as "r(A-4)" that has the nucleotide sequence SEQ ID NO:2.
  • r(A-4) corresponds to a specific defined splice product of the rat amelogenin gene that includes exons 2, 3, 5, 6d and 7 of the rat amelogenin gene.
  • Another aspect of the present invention is directed to nucleic acid sequences complementary to, or showing sequence similarity to, the DNA sequences identified in SEQ ID NOS: 1, 2, 16 and 17.
  • the present invention is also directed to those sequences which are at least 60%, preferably at least 80%, and most preferably at least 95%, especially 98%, identical thereto.
  • These molecules have surprisingly been found to be useful to induce differentiation of cells to the osteogenic and chondrogenic phenotypes and may be used in a composite cell construct for bone and cartilage regeneration.
  • the invention provides a composition and a method for enhancing osteogenic and or chondrogenic generation or growth.
  • the method comprises exposing cells to the chondrogenic/osteogenic polypeptides of the invention.
  • FIG. 1 Assays for the activity of the S-100 fractions in the EMF [ 35 S]- SO 4 -incorporation assay.
  • A The activity of Sephacryl S-100 fractions 1 and 2, as compared to positive (bBMP) and negative (PBS/BSA) controls.
  • 1- S-100, Fractionl, 1 mg/ml. 2 - S-100, Fraction 2, 1 mg/ml. Fractions 3-11 were comparable to the negative PBS control.
  • B A comparison of the concentration dependence of the activity of the combined S-100 (Fractions 1 & 2) with that of recombinant human BMP-2. Note that the rhBMP-2 was active at the 50-100 ng/ml level, while the S-100 stimulated sulfate incorporation to the same degree but at 100-1000 ⁇ g/ml levels.
  • FIG. 3 Electrophoresis of the low molecular weight fractions obtained by chromatography of the 4.0 M GuHCl/10.0 mM Tris-HCl extract on Sephacryl S- 100 denoted in Figure 1, on a 15% SDS-polyacrylamide gel. Mercaptoethanol was present in the sample buffer. Components with M r ⁇ 18000 were the most prominent in both fractions. Lane 1 - BioRad molecular weight standards . Lane 2 - S- 100 Fraction 1. Lane 3 - S- 100 Fraction 2.
  • EMF EMF upon culture in the continuous presence of the S-100 fraction, by Northern analysis.
  • One ⁇ g EMF mRNA was loaded at day 0 and day 6 and electrophoresed on an agarose-formaldehyde minigel, transferred to Hybond -N membrane and probed.
  • FIG. 1 Cell morphology and staining after monolayer culture of EMF with the putative chondrogenic agents for 49 days.
  • Row 1 unstained cells.
  • Row 2 cultures stained with Toluidine Blue (showing proteoglycan).
  • Row 3 cultures stained with Alizarin Red (showing calcification).
  • Column 1 control cultures, PBS/0.1% BSA, no factors added.
  • Column 2 Bovine BMP (Urist), 200 ⁇ g/cm 2 .
  • Figure 6 First stage purification of S-100 by reverse phase chromatography on a Vydac C-18 semi-preparative column.
  • the elution program covered 75 minutes. As shown by the solid straight lines, elution began with 15 min of 10% A-90% B, followed by a linear increase to 100% B over the next 35 min, followed by 100% B and return to 10% A-90% B as indicated.
  • the eluate in each fraction was recovered and taken to dryness. A number of identical runs were required to accumulate sufficient material for the EMF [ 35 S]- SO 4 -incorporation assay for the smaller peaks.
  • the inset shows the result of the assay, using identical amounts of the test fractions.
  • Fraction 5 was typical of the inactive peaks, equivalent to the PBS/0.1% BSA. Fraction 4, and 10 (data not shown) inhibited sulfate incorporation. Fraction 8, at 57.5 ⁇ g/ml was as active as rhBMP-2 at 100 ng/ml. Fraction 8 was the only active fraction. All of peak 8 was collected and combined from a number of runs, concentrated and run again under the same program. The major component was a single peak eluting at the same position. This was designated fraction 8b.
  • Lane 1- Molecular weight standards. Lane 2. Iodinated fraction S-100. Lane 3. Iodinated fraction 8b-5. The major component of 8b-5 is in the 6-10 K range of M r .
  • Figure 10 Identification of the sizes of the full length amelogenin splice products obtained from the rat incisor tooth cDNA library.
  • the amplified products are (A-4) (PCR200, Lane 2), A4 (PCR250, Lane 4), (B4) (PCR600, Lane 5) and B4 (PCR650, Lane 3).
  • the PCR products were run on a 1% agarose gel and visualized by staining with ethidium bromide. Lane 1 was loaded with a 1 kb DNA ladder. Arrows indicate the 0.5 and 1 kb bands.
  • Figure 11 The amelogenin gene alternative splice products detected in the rat incisor ⁇ gtl 1 cDNA library.
  • FIG 11 A The exon-intron distribution in the rat amelogenin gene.
  • the primers used initially to verify the presence of amelogenins in the library, P 1 and P2, and the primers used to determine the specific splice products present, P3 and P4, are indicated.
  • the exon designations follow the system of Simmer (1995).
  • the number of amino acids encoded by each exon is shown within the exon box.
  • the exon compositions of the PCR products, (B4), (B-4), (A4), (A-4), correspond to the bands shown in Figure 10, lanes 3, 5, 4 and 2, respectively.
  • Figure 11B The nucleotide and amino acid compositions of (A4) (SEQ ID
  • FIG. 12 HPLC purification of thrombin cleaved GST-fusion protein containing r(A-4).
  • the inset shows the Coomassie stained gel of the final r(A-4), peak obtained at 21.2 min. This homogeneous protein fraction was used for the bioassays.
  • the rA4 was purified in the same manner.
  • Figure 14 In vivo assay for osteogenic, mineralization activity of A4 and (A-4). Numbers 1, 2 and 3 were taken from r(A-4) treated implants; 4,5 and 6 were from rA4 treated implants; and 7 and 8 were BSA implants. Numbers 1, 4 and 7 were Von Kossa stained; 2, 5 and 8 were Alizarin Red stained. Numbers 3 and 6 were EGTA- treated sections of (A-4) and A4, respectively. These data show that the (A-4) implants were highly positive for deposition of mineral, comparable to BMP2 implants (Whang et al. 1998). A4 had some focal deposits of mineral, and the BSA implant controls were negative. Number 9 shows the radiograph of the (A-4) and A4 implants immediately after excision and before processing for histology. Note the heavier mineralization around the periphery of the (A-4) implant in contrast to the more punctate deposition of mineral within A4.
  • FIG. 15 Hematoxylin and Eosin stained sections of implants of r(A-4) and rA4 after 4 weeks.
  • the dense tissue at the upper portion of the micrograph is the connective tissue encapsulating the implant. Some of this tissue grows into the implant at the implant interface. The white, open areas are the implant scaffold. The intense vascularization of the implants is obvious. 2,3,4.
  • the scaffolds have been infiltrated by many cells, and an abundant network of capillaries. As seen in micrograph 2, a dense extracellular matrix has begun to form within the scaffold surrounding the capillaries. 5, 6.
  • Implants containing rA4 after 4 weeks The implants are vascularized but more sparsely than the r(A-4) implants. There is nevertheless, copious cellular infiltration.
  • the present invention relates to a polynucleotide that expresses a polypeptide having chondrogenic and osteogenic activity comprising the nucleotide sequence SEQ ID NO: 16.
  • This polynucleotide corresponds to a splice product of the rat amelogenin gene encoded by rat amelogenin gene comprising exons 2, 3 ,4,
  • Exon 6 of the rat amelogenin gene contains segments 6a, b, c and d.
  • the present invention provides a polynucleotide that expresses a polypeptide having chondrogenic and osteogenic activity wherein the polynucleotide encodes exons 2, 3, 4, 5, 6d and 7 in the rat amelogenin gene and has the sequence SEQ ID NO: 1.
  • the present invention further relates to a polynucleotide that expresses a polypeptide having chondrogenic and osteogenic activity comprising the nucleotide sequence SEQ ID NO: 17.
  • This polynucleotide corresponds to a splice product of the rat amelogenin gene comprising exons 2, 3, 5, 6 and 7 of the rat amelogenin gene.
  • the polynucleotide encodes exons 2, 3, 5, 6d and 7 in the rat amelogenin gene and has the sequence SEQ ID NO:2.
  • the present invention also provides polypeptide molecules having chondrogenic and osteogenic activity. These polypeptides are related to the amelogenin family of proteins. Applicants have found that splice products of the amelogenin gene have chondrogenic/osteogenic activity. Accordingly, the present invention provides polypeptides with in vitro chondrogenic and in vivo osteogenic activity.
  • the polypeptide rA4 has the amino acid sequence SEQ ID NO:6.
  • the polypeptide corresponds to the secreted form of a small alternative splice product of the rat amelogenin gene, with the amino acid sequence related to exons 2, 3, 4, 5, 6d and 7 in the rat amelogenin gene.
  • the present invention provides a polypeptide with in vitro chondrogenic and in vivo osteogenic activity, polypeptide r(A-4) which has the amino acid sequence SEQ ID NO:5.
  • the polypeptide corresponds to the secreted form of a small alternative splice product of the rat amelogenin gene, with the amino acid sequence related to exons 2, 3, 5, 6d and 7 in the rat amelogenin gene.
  • Another aspect of the present invention is directed to nucleic acid sequences complementary to the foregoing, or showing sequence similarity to, the DNA sequences identified in SEQ ID NOS: 1, 2, 16 and 17.
  • the present invention is also directed to those sequences which are at least 60%, preferably at least 80%, and most preferably at least 95%, especially 98%, identical thereto, and to DNA (or RNA) sequences encoding the same polypeptide as the sequences of SEQ ID NOS: 5 and 6, including fragments and portions thereof and, when derived from natural sources, includes alleles thereof.
  • Amelogenins belong to the family of extracellular matrix proteins in developing tooth enamel and exhibit a high degree of conservation at the amino acid sequence level for bovine, mouse, pig and human species (Salido et al. 1992). Amelogenins are produced by ameloblasts and have been thought to play a role in enamel mineralization. No morphogenetic activities have been previously described for these molecules.
  • the present invention also relates to a human polypeptide that has chondrogenic and osteogenic activity, such polypeptide being a splice product of the expression of the human amelogenin gene (Simmer 1995).
  • the polypeptide is encoded by regions of the human amelogenin gene which correspond to exons 2, 3, 5, 6d and 7.
  • the polypeptide is encoded by regions of the human amelogenin gene which correspond to exons 2, 3, 4, 5, 6d and 7.
  • the polypeptides can be used to induce chondrogenesis and osteogenesis.
  • the present invention also provides a polynucleotide that expresses in human a polypeptide having chondrogenic and osteogenic activity.
  • the polynucleotide includes exons 2, 3, 5, 6d and 7 in the human amelogenin gene.
  • the polynucleotide includes exons 2, 3,
  • the present invention is further directed to human amelogenin gene nucleic acid sequences complementary to the foregoing, or showing sequence similarity to, the DNA sequences that include exons 2, 3, 4, 5, 6d and 7 or 2, 3, 5, 6d and 7.
  • the present invention is also directed to those sequences which are at least 60%, preferably at least 80%, and most preferably at least 95%, especially 98%, identical thereto, and to DNA (or RNA) sequences encoding the human polypeptide corresponding to the sequences of SEQ ID NOS: 5 and 6, including fragments and portions thereof and, when derived from natural sources, includes alleles thereof.
  • the present invention also provides a polynucleotide that expresses a bovine polypeptide having chondrogenic and osteogenic activity.
  • the polynucleotide is encoded by exons 2, 3, 5, 6d and 7 in the bovine amelogenin gene which sequence has been described (Gibson et al., 1992).
  • the polynucleotide is encoded by exons 2, 3, 4, 5, 6d and 7 in the bovine amelogenin gene.
  • the invention also provides a bovine polypeptide which can be used to induce chondrogenesis and osteogenesis, said polypeptide encoded by the polynucleotide corresponding to exons 2, 3, 5, 6d and 7 of the bovine amelogenin gene.
  • the polypeptide is encoded by the polynucleotide corresponding to exons 2, 3, 4, 5, 6d and 7 in the bovine amelogenin gene.
  • the invention further relates to degradation polynucleotides of the foregoing polynucleotides.
  • the term “percent identity” or “percent identical,” when referring to a sequence, means that a sequence is compared to a claimed or described sequence after alignment of the sequence to be compared (the "Compared Sequence”) with the described or claimed sequence (the “Reference Sequence”).
  • Percent Identity 100 [1-(C/R)] wherein C is the number of differences between the Reference Sequence and the Compared Sequence over the length of alignment between the Reference Sequence and the Compared Sequence wherein (i) each base or amino acid in the Reference Sequence that does not have a corresponding aligned base or amino acid in the Compared Sequence and (ii) each gap in the Reference Sequence and (iii) each aligned base or amino acid in the Reference Sequence that is different from an aligned base or amino acid in the Compared Sequence, constitutes a difference; and R is the number of bases or amino acids in the Reference Sequence over the length of the alignment with the Compared Sequence with any gap created in the Reference Sequence also being counted as a base or amino acid.
  • the Compared Sequence has the specified minimum percent identity to the Reference Sequence even though alignments may exist in which the hereinabove calculated Percent Identity is less than the specified Percent Identity.
  • the polynucleotides 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 sequences which encodes the polypeptides may be identical to the sequences disclosed herein 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 polypeptide as the polynucleotide sequences of SEQ ID NOS: 1, 2, 16 and 17.
  • polynucleotide as used for the present invention encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequences.
  • the present invention further relates to variants of the hereinabove described polynucleotides which encode fragments, analogs and derivatives of the polypeptides having the amino acid sequences of SEQ ID NOS: 5 and 6.
  • Variants of the polynucleotide may be naturally occurring allelic variants of the polynucleotides or a non-naturally occurring variant of the polynucleotides.
  • 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 term "gene” means the segment of DNA (or DNA segment) involved in producing a polypeptide chain; it includes regions preceding and following the coding region (5'-and 3'- untranslated regions, or UTRs, also called leader and trailer sequences, regions, or segments) as well as intervening sequences (introns) between individual coding segments (exons), which intronic regions are typically removed during processing of post- transcriptional RNA to form the final translatable mRNA product. Of course, by their nature, cDNAs contain no intronic sequences.
  • the DNA and RNA sequences, and polypeptides, disclosed in accordance with the present invention may be in isolated form.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide, or DNA present in a living animal is not isolated, but the same polynucleotide or DNA, separated from some or all of the coexisting materials in the natural system, is isolated.
  • DNA could be part of a vector and/or such polynucleotide could be part of a composition, and still be isolated in that such vector or polynucleotide is not part of its natural environment.
  • the DNA and RNA sequences, and polypeptides, disclosed in accordance with the present invention may also be in "purified” form.
  • the term “purified” does not require absolute purity; rather, it is intended as a relative definition, and can include preparations that are highly purified or preparations that are only partially purified, as those terms are understood by those of skill in the relevant art.
  • Individual clones isolated from a cDNA library have been conventionally purified to electrophoretic homogeneity.
  • the cDNA clones are obtained via manipulation of a partially purified naturally occurring substance (messenger RNA). By conversion of mRNA into a cDNA library, pure individual cDNA clones can be isolated from the synthetic library by clonal selection.
  • creating a cDNA library from RNA and subsequently isolating individual clones from that library results in an approximately 10 6 fold purification of the native message.
  • Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
  • claimed polynucleotide which has a purity of preferably 0.001%, or at least 0.01% or 0.1%; and even desirably 1% by weight or greater is expressly contemplated.
  • coding region refers to that portion of a human gene which either naturally or normally codes for the expression product of that gene in its natural genomic environment, i.e., the region coding in vivo for the native expression product of the gene.
  • the coding region can be from a normal, mutated or altered gene, or can even be from a DNA sequence, or gene, wholly synthesized in the laboratory using methods well known to those of skill in the art of DNA synthesis.
  • nucleotide sequence refers to a heteropolymer of deoxyribonucleotides.
  • DNA segments encoding the proteins provided by this invention are assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon.
  • expression product means that polypeptide or protein that is the natural transcription product of the gene and any nucleic acid sequence coding equivalents resulting from genetic code degeneracy and thus coding for the same amino acid(s).
  • fragment when referring to a coding sequence means a portion of DNA comprising less than the complete human coding region whose expression product retains essentially the same biological function or activity as the expression product of the complete coding region.
  • portion when referring to a portion of a polypeptide, as used herein, the terms “portion,” “segment,” and “fragment,” refer to a continuous sequence of residues, such as amino acid residues, which sequence forms a subset of a larger sequence. For example, if a polypeptide were subjected to treatment with any of the common endopeptidases, such as trypsin or chymotrypsin, the oligopeptides resulting from such treatment would represent portions, segments or fragments of the starting polypeptide.
  • primer means a short nucleic acid sequence that is paired with one strand of DNA and provides a free 3 'OH end at which a DNA polymerase starts synthesis of a deoxyribonucleotide chain.
  • promoter means a region of DNA involved in binding of RNA polymerase to initiate transcription.
  • ORF open reading frame
  • reference to a DNA sequence includes both single stranded and double stranded DNA.
  • specific sequence unless the context indicates otherwise, refers to the single strand DNA of such sequence, the duplex of such sequence with its complement (double stranded DNA) and the complement of such sequence.
  • the polynucleotides of the present invention can be derived from natural sources or synthesized using known methods.
  • the sequences falling within the scope of the present invention are not limited to the specific sequences described, but include human allelic and species variations thereof. Allelic variations can be routinely determined by comparison of one sequence with a sequence from another individual of the same species.
  • the invention includes sequences coding for the same amino acid sequences as do the specific sequences disclosed herein. In other words, in a coding region, substitution of one codon for another which encodes the same amino acid is expressly contemplated. (Coding regions can be determined through routine sequence analysis.)
  • the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including for example, a promoter, operably linked to the sequence.
  • a promoter operably linked to the sequence.
  • Bacterial pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNHl ⁇ a, pNHl ⁇ a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).
  • Eukaryotic pWLneo, pSV2cat, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).
  • the present invention is not restricted to such constructs or sequences alone but also includes expression vehicles, which may include plasmids, viruses, or any other expression vectors, including cells and liposomes, containing any of the nucleic acids, nucleotide sequences, DNAs, RNAs, or fragments thereof, as disclosed according to the present invention.
  • expression vehicles which may include plasmids, viruses, or any other expression vectors, including cells and liposomes, containing any of the nucleic acids, nucleotide sequences, DNAs, RNAs, or fragments thereof, as disclosed according to the present invention.
  • a promoter region that may include a promoter different from that normally associated in vivo with the genes coding for the gene expression products and proteins disclosed according to the present invention.
  • Promoter regions can be selected from any desired gene using 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 , and trc.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • the present invention relates to host cells containing the above-described construct(s).
  • 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 procaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the host cell can be effected by calcium phosphate fransfection, DEAE, dextran mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, 1986)) .
  • the constructs in host cells can be used in a conventional manner to produce the gene product coded by the recombinant sequence.
  • the encoded polypeptide once the sequence is known from the cDNAs, or from isolation of the pure product, can be synthetically produced by conventional methods of peptide synthesis, either manual or automated.
  • conventional techniques in molecular biology can be used to obtain the polypeptide.
  • the present invention includes all polypeptides coded for by any and each of the DNA or RNA sequences disclosed herein, including fragments of said polypeptides, as well as derivatives and functional analogs thereof.
  • the amino acid sequence can be synthesized using commercially available peptide synthesizers. This is particularly useful in producing small peptides and fragments of larger polypeptides. (Fragments are useful, for example, in generating antibodies against the native polypeptide.)
  • the DNA encoding the desired polypeptide can be inserted into a host organism and expressed.
  • the organism can be a bacterium, yeast, cell line, or multicellular plant or animal.
  • the literature is replete with examples of suitable host organisms and expression techniques.
  • polynucleotide DNA or mRNA
  • This methodology can be used to deliver the polypeptide to the animal, or to generate an immune response against a foreign polypeptide.
  • the coding sequence can be inserted into a vector, which is then used to transfect a cell.
  • the cell (which may or may not be part of a larger organism) then expresses the polypeptide.
  • fragment when referring to the polypeptides disclosed herein also mean polypeptides that retain essentially the same biological function or activity as said polypeptides.
  • an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
  • fragments, derivatives and analogs must have sufficient similarity to the polypeptides disclosed herein so that activity of the native polypeptide is retained.
  • the polypeptides of the present invention may be recombinant polypeptides, natural polypeptides or synthetic polypeptides, preferably recombinant polypeptides.
  • Recombinant means that a protein is derived from recombinant
  • Microbial refers to recombinant proteins made in bacterial or fungal (e.g., yeast) expression systems.
  • "recombinant microbial” defines a protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation.
  • Protein expressed in most bacterial cultures e.g., E. coli, will be free of glycosylation modifications; protein expressed in yeast will have a glycosylation pattern different from that expressed in mammalian cells.
  • the fragment, derivative or analog of a polypeptide of SEQ ID NOS: 5 or 6 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 secretory sequence 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 abilities of those skilled in the art in view of the teachings herein.
  • polypeptides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity. When applied to polypeptides, the term "isolated" has its already stated meaning.
  • polypeptides of the present invention include the polypeptides of SEQ ID NOS: 5 and 6, as well as polypeptides which have at least 70% identity to these polypeptides, or which have, at least 90% identity to these polypeptides, still more preferably at least 95% identity to these polypeptides. 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 as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
  • the present invention also relates to vectors which include polynucleotides of the present invention, host cells 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 vectors of this invention which may be, for example, a cloning vector or an expression vector, either of which may be 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 genes of the present invention.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, 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; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • any other vector may be used as long as it is replicable and viable in the host.
  • an appropriate DNA sequence or segment may be inserted into the vector by a variety of procedures.
  • the DNA sequence is inserted into the appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
  • the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (for example, a promoter sequence) to direct mRNA synthesis.
  • an appropriate expression control sequence(s) for example, a promoter sequence
  • promoters there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda P L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • 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 resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in ⁇ . 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 host to permit the host to express the protein.
  • bacterial cells such as ⁇ . coli, Streptomyces, Salmonella typhimurium
  • fungal cells such as yeast
  • insect cells such as Drosophila S2 and Spodoptera Sf9
  • animal cells such as CHO, COS or Bowes melanoma
  • adenoviruses plant cells, etc.
  • Recombinant expression vehicle or vector refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence.
  • the expression vehicle can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences.
  • Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.
  • recombinant protein when expressed without a leader or transport sequence, it may include an N-terminal methionine residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.
  • Recombinant expression system means host cells which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit extra chromosomally.
  • the cells can be prokaryotic or eukaryotic.
  • Recombinant expression systems as defined herein will express heterologous protein upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed.
  • Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
  • Appropriate cloning and expression vectors for use with prokaryatic and eukaryotic hosts are described by Sambrook, et al, Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor, N.Y., 1989), Wu et al, Methods in Gene Biotechnology (CRC Press, New York, NY, 1997), and Recombinant Gene Expression Protocols, in Methods in Molecular Biology, Vol. 62, (Tuan, ed., Humana Press, Totowa, NJ, 1997), the disclosures of which are hereby incorporated by reference.
  • Enhancer sequence Transcription of the DNA encoding the polypeptides according to the present invention by higher eukarotes can be increased by insertion of an enhancer sequence into the vector.
  • enhancers have been known for some time and are usually cis-acting elements of DNA, usually anywhere from 10 to 300 bp that act on a promoter to increase transcription. Common examples include the SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer and the enhancers found in adenovirus.
  • recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S.
  • promoters derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
  • promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), ⁇ -factor, acid phosphatase, or heat shock proteins, among others.
  • PGK 3-phosphoglycerate kinase
  • the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.
  • the heterologous sequence can encode a fusion protein including an N- terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.
  • the vector will comprise one or more phenotypic selectable markers 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 Staphylococcus, 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 vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
  • the selected promoter is derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • appropriate means e.g., temperature shift or chemical induction
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • mammalian cell culture systems can also be employed to express recombinant protein.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
  • 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 sequences.
  • DNA sequences derived from the SV40 viral genome for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
  • Recombinant protein produced in bacterial culture is conveniently isolated by initial extraction from cell pellets, followed by one or more salting-out, aqueous ion exchange or size exclusion chromatography steps. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
  • the chondrogenic/osteogenic inducing molecule(CIM), also known as chondrogenic inducing agent (CIA), has been isolated from the 4.0 M guanidinium hydrochloride extract of deminerahzed, defatted, bovine dentin extracellular matrix. Dentin matrix is known to contain components capable of inducing chondrogenesis and osteogenesis at ectopic sites when implanted in vivo, and chondrogenesis in cultures of embryonic muscle-derived fibroblasts (EMF) in vitro. Following Sephacryl S-100 chromatography, activity (bCIA) was identified in fractions by assay for uptake of [ 35 S]-SO 4 into proteoglycan by the EMF after 24 hours in culture.
  • CCM chondrogenic/osteogenic inducing agent
  • the active S-100 fraction induced the EMF to produce type II collagen mRNA and decrease production of type I collagen mRNA after 5 days in culture.
  • the EMF cultures + bCIA for 4 to 7 weeks condensed into Toluidine Blue and Alizarin Red stainable nodules, changes indicative of chondrogenic induction.
  • In vivo implants in rat muscle with collagen carrier produced ectopic bone after 7 weeks.
  • the bCIA was brought to near homogeneity by several rounds of reverse phase high performance liquid chromatography, tested at each step by EMF [ 35 S]- SO 4 incorporation assays.
  • a striking feature of the isolated peptide fraction was the amino acid composition.
  • the peptide was proline and leucine rich and devoid of cysteine, ruling out the possibility that the peptide had been derived from the BMP/TGF- ⁇ family.
  • Amino terminal sequencing of the peptide by Edman degradation of tryptic fragments of the fraction 8b-5 led to the identification of the bioactive peptide(s) as arising from, or containing the amino-terminal domain of bovine amelogenin (Gibson et al. 1992).
  • SEQ ID NO:7 The peptides having sequences SEQ ID NO:7 and SEQ ID NO:8 (amino acids 1-5 and 25-30, respectively, of the amelogenin sequence) were recovered in the tryptic digest.
  • SEQ ID NO:7 was clearly at the N-terminal of the secreted form of the amelogenin, and SEQ ID NO:8 follows the first internal Lys residue of the 176 amino acid residue form of the bovine amelogenin.
  • the peptide sequences were used to generate PCR primers and full length reverse transcribed cDNAs for 2 amelogenins cloned from a fetal rat dentin library.
  • the cDNAs were transduced in E.coli and the two amelogenins produced in the BL21 expression system.
  • the amelogenin proteins were purified by HPLC.
  • amelogenins A4 and A-4 confirmed that the amelogenin peptides have specific biological activities, equivalent to rBMP-2, in directing the change in phenotype of the embryonic rat muscle fibroblasts in vitro and inducing the onset of osteogenesis in muscle implants in vivo.
  • These specific small amelogenin gene splice products can influence the differentiation of embryonic or stem cells and modulate aspects of their phenotypic expression.
  • the polypeptides may be employed as chondrogenic and osteogenic enhancing factors. Accordingly, the polypeptides may be used in a method for enhancing the differentiation of stem cells by contacting cells in vitro or in vivo. Alternatively the polypeptides may be placed in contact with areas of potential connective tissue growth or connective tissue damage to enhance or stimulate growth in vivo for regeneration and or repair. Using these methods synthesis of bone matrix or articular surfaces at or near implant sites may be enhanced and can be initiated in a controlled and uniform manner.
  • EMF Embryonic Muscle Fibroblast Tissue Culture Assay Embryonic Muscle Fibroblasts.
  • Rat EMF cells were derived from the thigh muscles of 20 day old Sprague Dawley rat embryos for use in primary tissue culture assays using a modification of the Koskinen harvesting technique (Koskinen et al., 1985), and grown in growth media (GM) [ ⁇ -Modified Eagle's Medium ( ⁇ MEM , Life Technologies, Gaithersburg, MD), containing 10% heat inactivated fetal bovine serum (FBS), 1% Penicillin/Streptomycin (P/S)] in 5% O 2 /95% air at 37°C. EMFs were frozen at passage 2 (P-2).
  • GM ⁇ -Modified Eagle's Medium
  • FBS heat inactivated fetal bovine serum
  • P/S Penicillin/Streptomycin
  • the EMFs were plated (day 0) at a concentration of 10 4 cells per well in a 96 well plate which had been precoated with 100 ⁇ l of a 50 ⁇ g/ml solution of type I Rat Tail Collagen (Collaborative Biomedical Products).
  • the cells were grown for 5 days in GM. On day 5, the cells were conditioned by removing the GM and adding conditioning media (CM) ( ⁇ MEM containing 0.5% FBS, 1 % P/S) for 24 hours. This media was replaced with 100 ⁇ l of fresh CM, just prior to the addition of the test factors (hour 0). Test factors were added to test wells in triplicate.
  • CM conditioning media
  • PBS/0.1% BSA alone served as a negative control.
  • rhBMP-2 (a gift from Genetics Institute, Boston, MA) and deminerahzed dentin, suspended in PBS/0.1% BSA, were used as positive controls.
  • the cells were labeled with 1 ⁇ Ci 35 S- SO 4 /10 ⁇ l sterile PBS/well/96 well plate for 20 hours.
  • the tube and filters were washed with buffer B 3 times. The filters were allowed to dry briefly, and were transferred to a scintillation vial. Then 5 ml scintillation cocktail was added per vial and the radioactive counts were measured in the scintillation counter.
  • mRNA was electrophoresed in a formaldehyde denaturing gel and then transferred to Nylon membrane. The membrane was hybridized with Type I and Type II collagen probes. Controls were run with 0.1 % BSA/PBS added to the cultures in place of the active fractions. Cultures were examined at day 0 and day 6.
  • the type I probe (pHF677 ( ⁇ l(I))
  • the type II probe (pi 377 ( ⁇ l(II))) (Kohno et al., 1984) contained a 550 bp clone coding for the amino terminal portion of the rat pro ⁇ l(II) chain. Bone Formation In Vivo. Implantation in Muscle For implantation, 25 ⁇ g S-100 or 35 ng rhBMP-2 in 200 ⁇ l of PBS/0.1%
  • BSA were absorbed onto 0.45 mg rat tail tendon collagen and lyophilized.
  • the coated collagen was sterilized, then placed into sterile 5 mm gelatin capsules.
  • Capsules were prepared with the collagen only as a negative control. Deminerahzed insoluble dentin matrix was used as a positive control.
  • a 1 cm incision was made and a small intramucscular pouch was created by blunt dissection into the thigh muscle of the hind limbs of 100 g (4 week old) Long-Evans rats.
  • the capsules were inserted, then the fascia was closed using 4-0 Vicryl sutures.
  • the skin was closed with 4-0 silk sutures.
  • EXAMPLE 1 CIM Isolation Bovine Teeth Isolation and Preparation. Heads were obtained at the abattoir (Chiappetti Packing House, Chicago) from 5 month old animals, and immediately placed on ice. All teeth were removed from the heads, cleaned of adherent bone, periodontal ligament, and soft tissue, and placed in a 15% NaCl wash solution containing 50.0 mM Tris.HCl, pH 7.5, 20.0 mM Ethylenediaminetetraacetic acid (EDTA), and protease inhibitors (Pis) including 2.5 mM Benzamidine HC1, 50.0 mM ⁇ -Amino-n-Caproic Acid, 0.5 mM N- Ethylmaleimide (NEM), and 0.3 mM Phenylmethylsulfonyl Fluoride (PMSF) at 4°C. The pulpal tissues were removed and the teeth were pulverized into small particles in a Spex 6700 Freezer/Mill at liquid nitrogen temperature,
  • the tooth particles were washed in 5.0 mM sodium azide (NaN 3 ) and deminerahzed in 0.6 N HC1 plus 1 ⁇ g/ml Pepstatin A at 4°C with continuous stirring for 5 days.
  • Buffer A was 99.9% dH 2 O; 0.1% TFA, Buffer B was 99.9% ACN; 0.1% TFA.
  • the samples were dissolved in a 90% A, 10% B solution. The samples were applied to the columns and run using varying programs resulting in sequentially more shallow gradient elution of the samples from the columns. The absorbance was monitored at 220nm. The semi-prep column was run at a flow rate of 5 ml/min and the analytical column at a flow rate of 1 ml/min, with 5 ml and 0.5 ml fractions collected, respectively.
  • the resulting HPLC fractions were pooled according to the distribution of peaks on the chromatographic profiles and lyophilized to dryness for activity testing using the EMF 35 S-SO 4 -incorporation assay. Characterization of Fractions. Protein Quantitation. The protein fractions were quantitated using the
  • Bio-Rad Protein Assay kit according to the manufacturers directions.
  • Protein fractions were analyzed for their amino acid compositions at the Northwestern University Biotechnology Facility, the
  • Amino Acid Sequencing NH 2 amino acid sequencing was performed by the Northwestern University Biotechnology facility. Internal tryptic digestion, and microsequencing was performed at the Harvard Microchemistry Facility, under the direction of Dr. William Lane, or at Osiris Therapeutics. Amino terminal sequencing using automated Edman degradation (Edman, 1950) was performed on an Applied Biosystems (model 477 A) sequencer through 20 cycles. The tryptic digestion was performed according to Stone et al. (1989).
  • Mass Spectral Analysis Samples were analyzed by Matrix Assisted Laser Desorption lonization (MALDI) and electrospray (ESI), either at The University of Illinois School of Chemical Sciences mass spectrometry laboratory under the supervision of Dr. Richard Milberg, or at Osiris Therapeutics.
  • MALDI Matrix Assisted Laser Desorption lonization
  • ESI electrospray
  • the unincorporated l25 I was removed by separation on a gel filtration column (MW cutoff 1,500) (Pharmacia disposable PD-10 columns prefilled with Sephadex G-15) that had been prewashed with 5 ml of 20 mg BSA/ml PBS, pH 7.4 and 100 ml of PBS minus BSA, pH 7.4.
  • the protein was eluted with PBS, pH 7.4, collecting 1 ml fractions.
  • the incorporation was measured in a gamma counter.
  • the iodinated proteins, which eluted in the 5th and 6th fractions, were pooled, electrophoresed on agarose gels, dried, and exposed to film for visualization.
  • fractions 1 and 2 The components of the shoulder beginning at the 17 K M r marker, labeled fractions 1 and 2, had all of the activity.
  • the sulfate incorporating activities of fractions 1 and 2 were in the same range as the activity of a crude preparation of bovine bone BMP, Figure 2A, but the concentration required for activity was 1000-fold higher when compared with the activity of rhBMP-2, Figure 2B .
  • the medium (CM+S-100) was changed at days 2 and 4.
  • the cells were harvested and the mRNA extracted.
  • the mRNA was extracted from a parallel EMF culture at day 0.
  • the message level for Type I collagen was detected at day 0 following addition of bCIA S-100 fraction into the media of the EMFs.
  • the expression of the type I collagen mRNA decreased significantly but was still evident.
  • no message could be detected for type II collagen, but the type II message was strongly expressed at day 6 ( Figure 4B). Two isoforms of the type II message were evident, at 5.0 and 5.3 kb, at approximately the same intensity.
  • EMF cultures were exposed to S-100 for culture periods up to 49 days. In this system, one had to use the GM throughout the culture period. The cells were therefore exposed to the S-100 according to the procedure of Iwata et al. (1993). The S-100, in PBS/0.1%) BSA was added to uncoated plastic, and then dried and insolubilized by exposure to UV light. T150 plates (150cm 2 area) were used. Preliminary studies in 12 well plates were used to determine the optimum amounts of S-100, and bBMP (crude, from Urist) for positive control. In this case, optimal was defined as that amount which permitted long-term viable culture. The optimal amount of S-100 was 100 ⁇ g/cm 2 , the bBMP optimum was 200 ⁇ g/cm 2 . Those were the amounts used for the final study.
  • Control cultures grown on PBS/0.1% BSA, reached confluence in 5 to 7 days and continued as a confluent monolayer of mostly spindle-shaped cells, evenly distributed over the plates through the first two weeks. Over the following two weeks the cell layers lifted off the plates and rolled up. New outgrowths then began to regrow the monolayer.
  • the cells grown on the S-100 and bBMP behaved differently. After reaching confluence in 5-7 days, cells in both culture systems began to increase in density, migrating into cellular accumulations or nodules with overlapping cells. The more elongated cells on the periphery of the nodular areas appeared to be polarized towards the nodule centers.
  • peak 8 material was combined from a number of semipreparative runs and then chromatogramed again over a more shallow gradient, but little further separation of peak 8 into sub-components was achieved. Nevertheless, the major component of rechromatogrammed peak 8 (8b) was submitted for Laser Desorption Mass Spectrometry. Peptides with masses 6,797, 10,607, and 22,421 D were prominent, plus smaller amounts of peptides at 14,660 and 29,240 D.
  • Peak 8b was separated into an alarming number of components. Peak 8b-5 contained the activity, but the broad domain labeled as "8b-6" also showed activity.
  • rhBMP-2 and hTGF- ⁇ l were chromatogramed using the same program. As shown in the superimposed chromatograms, rhBMP-2 elution corresponded to "6", while TGF- ⁇ 1 eluted between "5" and "6".
  • Bovine molar teeth in common with the teeth of other herbivores, have very complex structures in which the enamel is enfolded into the dentin. They also contain clefts that contain coronal cementum. Even when frozen in liquid N 2 and shattered, it is impossible to separate these components mechanically among the shards. Thus, the crushed, deminerahzed bovine tooth matrix might contain proteins from all of these sources.
  • Rat incisors have enamel on the labial side of the tooth and cementum on the lingual side. Since it is virtually impossible to remove all of these mineralized tissues prior to preparing dentin extracts, the extracts will always exhibit some degree of contamination.
  • the amount of mineralized dentin far exceeds the amount of cementum, and since the protein content of the enamel is so low, one can generally assume that the major protein components of a dentin extract originated in the dentin. This may not be the case in the present instance, where the proteins of interest appear to be present and active at the cytokine level.
  • the behavior of the S-100 fractions of the rat incisor (Amar et al., 1991) and bovine dentin extracts were comparable, however, in spite of the markedly enhanced ease of cleaning the rat incisors. Thus, we have retained language suggesting that the bCIA is a dentin component.
  • the S-100 fraction of the bovine dentin extract was a mixture of a number of low M r proteins (Figure 3). In culture, this fraction promoted enhanced sulfate incorporation into proteoglycans (Figure 2) and led to morphological changes in the appearance of embryonic muscle fibroblasts with the development of an acidic proteoglycan-rich extracellular matrix (Figure 5), and the expression of the mRNA for type II collagen and down regulation of type I collagen mRNA ( Figure 4). These changes in cell phenotype were seen within the period from day 1 to day 6 in culture. On long term culture (49 days), multilayered nodules rich in proteoglycan (Toluidine Blue stained) were formed.
  • the forward primer PI SEQ ID NO: 10
  • SEQ ID NO: 7 was based on the amelogenin amino terminal peptide sequence SEQ ID NO: 7
  • the reverse primer P2 SEQ ID NO: 11 corresponded to the tryptic peptide sequence SEQ ID NO: 8.
  • the PCR conditions were 95°C for 1 min., 55°C for 1 min., and 72°C for 1 min. for 25 cycles.
  • the PCR amplified bands were cloned in pGEMT vector and sequenced. Each of the amplified bands was used to screen the ⁇ gt 1 1 cDNA library prepared from rat incisor odontoblasts as described by George et al. (1993). Positive clones were picked and plaque purified through three successive rounds of screening. A single pure plaque was then amplified and the phage DNA was prepared according to established procedures (Maniatis et al, 1989). The phage DNA was digested with EcoRl, cloned into the EcoRl site of pBluescript KS and sequenced.
  • Forward primer P3 SEQ ID NO: 12 contained a unique EcoRl site and included the ATG start codon and first fifteen nucleotides.
  • the reverse primer P4 SEQ ID NO: 13 contained a unique Xhol site and included the last 15 nucleotides and the stop codon TAA.
  • the cloned amelogenins were expressed as GST- fusion proteins.
  • the inserts in pGEMT were re-amplified by PCR, using the primers P3 and P4 and conditions described above.
  • the PCR products were digested with EcoRl and Xhol, purified on a 1% agarose gel and cloned in frame into the EcoRl/Xhol site of the GST expression vector pGEXT4.
  • the resulting plasmid was introduced into the E. coli strain BL21 (DE3).
  • a single colony was inoculated into 10 ml of LB and grown overnight.
  • the thrombin released peptides were a heterogeneous mixture, containing some prematurely terminated peptides as well as full-length peptide. Therefore the thrombin cleaved, eluted protein was passed over a C-18 reverse phase column eluted by an increasing gradient of acetonitrile plus 1% trifluoroacetic acid as described in Example 1. for the final step of purification of the protein extracted from dentin matrix.
  • 35 S-SO 4 incorporated into secreted proteoglycan was determined by precipitation of the proteoglycan with cetyl pyridinium chloride, as described above, followed by scintillation counting of the precipitate. The cell layer was trypsinized and the number of cells counted. Incorporation is presented as counts per minute per cell x 10 3 .
  • Recombinant human BMP2 (A kind gift from the Genetics Institute, Boston, MA) and the bioactive crude SI 00 fractions from rat incisor dentin (Amar et al., 1991) and/or bovine dentin (see Example 1) were used as the positive controls.
  • Bovine serum albumin in phosphate buffered saline (PBS) was the negative control.
  • the recombinant proteins, Emdogain and controls were each included in a bioabsorbable polymer matrix implants.
  • the implants were prepared in poly (lactide)-poly(glycolide) scaffolds by the procedure of Whang et al. (1998) with 167 ⁇ g per implant. Implants were placed in the quadriceps of the hind legs of 100 g, 4 week old, male Long-Evans rats.Each animal received a negative control implant containing 167 ⁇ g of BSA in PBS. The implants were kept in place for 28 days. The implants were removed and radiographed, then they were fixed in 4% paraformaldehyde, dehydrated in graded ethanol and embedded in paraffin.
  • Sections (7 ⁇ thick) were cut and stained with either Von Kossa or Alizarin Red dyes, both of which can indicate the presence of mineralized deposits containing divalent cations. To assure that the stains seen were calcific deposits, serial sections were treated with 5% EGTA for 10 min before staining. Results
  • PCR1 was much more intense than PCR2, and corresponded to the expected complete amino terminal sequence from amelogenin gene exons 2, 3 and 5.
  • PCR2 included exon 4.
  • the presence of protein containing the 14 amino acid sequence corresponding to exon 4 in the rat has not been previously reported.
  • PCR1 and PCR2 would be expected to be present in all of the usual amelogenin gene splice products.
  • new PCR primers P3 and P4 were prepared.
  • Four PCR product bands were amplified from the template phage DNA, at approximate sizes 650, 600, 250 and 200 bp.
  • PCR600 and PCR200 were strong, PCR 650 and PCR250 were weak. All four bands were amplified, Figure 10, and cloned in pGEMT vector and sequenced.
  • PCR650 SEQ ID NO: 16 corresponding to exons 2, 3, 4, 5, 6 and 7
  • PCR600 SEQ ID NO: 17 corresponding to exons 2, 3, 5, 6 and 7
  • PCR250 SEQ ID NO:6 corresponding to exons 2, 3, 4, 5, 6d and 7
  • PCR200 SEQ ID NO:5 corresponding to exons 2, 3, 5, 6d and 7,.
  • the nucleotide and amino acid sequences for PCR250, which is rA4, are shown in Figure 1 IB.
  • the splice patterns for the four PCR products are also shown.
  • r(A-4) was active in stimulating production of 35 S-SO 4 -labeled proteoglycan by embryonic rat muscle fibroblasts in monolayer culture, at a concentration as low as 1 ng/ml, ⁇ 140 pM.
  • the rA4 was less active at equivalent concentrations, but was quite comparable to the effect of rhBMP-2 used as a positive control at 10 ng/ml.
  • the r(A-4) did not act as a growth factor. Even after 5 days in culture, after a 24 h exposure to r(A-4), the cell number did not increase as it did in the presence of rhBMP2 and rA4.
  • the r(A-4) implants were also shown to be positive for alkaline phosphatase, another marker of mineralizing systems. Recombinant full-length amelogenins, rB4 and r(B-4) were inactive. The r(A-4) and rA4 implants had become vascularized and filled with extracellular matrix within 4 weeks, Figure 15. The ingrowth of capillaries was more prominent in the r(A-4) implants, as was the formation of the extracellular matrix. However, islands of capillaries, and the formation of a surrounding extracellular matrix was also evident in the rA4 implants. Certain small splice products of the amelogenin gene are present in trace amounts in dentin.
  • the presence of the gene product was determined on the basis of a fractionation scheme focused on bioactivity.
  • chondrogenesis proceeds to osteogenesis.
  • Rat incisor dentin contains a factor stimulating rat muscle cells in vitro to incorporate sulfate into an altered proteoglycan. Connect Tissue Res 23: 135-144.
  • Rat incisor dentine contains a factor which alters the phenotypic expression and stimulates chondrogenesis in fibroblast-like cells in vitro Biomaterials 1135-37.

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Abstract

La présente invention se rapporte à des peptides amélogénine qui sont capables d'induire la chondrogénèse et l'ostéogénèse au niveau de sites ectopiques lorsqu'ils sont implantés in vivo, et la chondrogénèse dans des cultures de fibroblastes dérivés de muscle embryonnaire in vitro. On décrit également des compositions et des procédés utilisés pour améliorer la croissance osseuse et cartilagineuse à l'aide de ces mêmes peptides.
PCT/US1999/017342 1998-07-29 1999-07-29 Molecule induisant la chondrogenese et l'osteogenese Ceased WO2000006734A1 (fr)

Priority Applications (2)

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US09/744,128 US6677306B1 (en) 1998-07-29 1999-07-29 Chondrogenic and osteogenic inducing molecule
AU52468/99A AU5246899A (en) 1998-07-29 1999-07-29 Chondrogenic and osteogenic inducing molecule

Applications Claiming Priority (2)

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US9448998P 1998-07-29 1998-07-29
US60/094,489 1998-07-29

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WO2000006734A1 true WO2000006734A1 (fr) 2000-02-10

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6677306B1 (en) 1998-07-29 2004-01-13 Northwestern University Chondrogenic and osteogenic inducing molecule
WO2006064381A3 (fr) * 2004-12-10 2006-08-17 Straumann Holding Ag Nouvelle preparation de proteine
EP1862170A1 (fr) * 2004-12-10 2007-12-05 Straumann Holding AG Formulation de protéine
US7423013B2 (en) 2004-12-10 2008-09-09 Straumann Holding Ag Protein formulation
WO2012153333A1 (fr) 2011-05-09 2012-11-15 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Régénération et réparation de tissu mésenchymateux par utilisation d'amélogénine
WO2012166626A1 (fr) * 2011-05-27 2012-12-06 University Of Washington Through Its Center For Commercialization Réactifs et procédés pour traiter une maladie dentaire
US9957314B2 (en) 2011-05-09 2018-05-01 Hadasit Medical Research Services And Development Ltd. Regeneration and repair of mesenchymal tissue using amelogenin

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6677306B1 (en) 1998-07-29 2004-01-13 Northwestern University Chondrogenic and osteogenic inducing molecule
WO2006064381A3 (fr) * 2004-12-10 2006-08-17 Straumann Holding Ag Nouvelle preparation de proteine
EP1862170A1 (fr) * 2004-12-10 2007-12-05 Straumann Holding AG Formulation de protéine
US7423013B2 (en) 2004-12-10 2008-09-09 Straumann Holding Ag Protein formulation
US7985845B2 (en) 2004-12-10 2011-07-26 Straumann Holding Ag Protein formulation
US8029769B2 (en) 2004-12-10 2011-10-04 Straumann Holding Ag Protein formulation
WO2012153333A1 (fr) 2011-05-09 2012-11-15 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Régénération et réparation de tissu mésenchymateux par utilisation d'amélogénine
US9957314B2 (en) 2011-05-09 2018-05-01 Hadasit Medical Research Services And Development Ltd. Regeneration and repair of mesenchymal tissue using amelogenin
US11034753B2 (en) 2011-05-09 2021-06-15 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Regeneration and repair of mesenchymal tissue using amelogenin
WO2012166626A1 (fr) * 2011-05-27 2012-12-06 University Of Washington Through Its Center For Commercialization Réactifs et procédés pour traiter une maladie dentaire
JP2014517693A (ja) * 2011-05-27 2014-07-24 ザ ユニバーシティ オブ ワシントン スルー イッツ センター フォー コマーシャライゼーション 歯科疾患を治療するための試薬および方法
US9290555B2 (en) 2011-05-27 2016-03-22 University Of Washington Through Its Center For Commercialization Reagents and methods for treating dental disease
US9809633B2 (en) 2011-05-27 2017-11-07 University Of Washington Through Its Center For Commercialization Reagents and methods for treating dental disease

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