WO1994006447A2 - Morphogenese induite par la proteine 60a - Google Patents

Morphogenese induite par la proteine 60a Download PDF

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WO1994006447A2
WO1994006447A2 PCT/US1993/008741 US9308741W WO9406447A2 WO 1994006447 A2 WO1994006447 A2 WO 1994006447A2 US 9308741 W US9308741 W US 9308741W WO 9406447 A2 WO9406447 A2 WO 9406447A2
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tissue
protein
val
cells
leu
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PCT/US1993/008741
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WO1994006447A3 (fr
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Thangavel Kuberasampath
Roy H. L. Pang
Hermann Oppermann
David C. Rueger
Charles M. Cohen
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Creative Biomolecules, Inc.
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Priority to AU51292/93A priority Critical patent/AU5129293A/en
Publication of WO1994006447A2 publication Critical patent/WO1994006447A2/fr
Publication of WO1994006447A3 publication Critical patent/WO1994006447A3/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
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2310/00—Prostheses classified in A61F2/28 or A61F2/30 - A61F2/44 being constructed from or coated with a particular material
    • A61F2310/00005—The prosthesis being constructed from a particular material
    • A61F2310/00365—Proteins; Polypeptides; Degradation products thereof
    • 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

Definitions

  • This invention relates to methods for the use of morphogenically active fragments of 60A protein to induce tissue morphogenesis in mammals, including methods for promoting tissue stasis, repair and
  • tissues can be divided into three broad categories: (1) tissues with static cell populations such as nerve and skeletal muscle where there is no cell division and most of the cells formed during early development persist throughout adult life; (2) tissues containing conditionally renewing populations such as liver where there is generally little cell division but, in response to an appropriate stimulus, cells can divide to produce daughters of the same differentially defined type; and (3) tissues with permanently renewing populations including blood, testes and stratified squamous epithelia which are characterized by rapid and
  • the terminally differentiated cells have a relatively short life span and are replaced through proliferation of a distinct subpopulation of cells, known as stem or progenitor cells.
  • tissue degenerative diseases including arthritis, emphysema, osteoporosis, cardiomyopathy, cirrhosis, and
  • TGF transforming growth factor
  • morphogenically active C-terminal domains including a conserved six or seven cysteine skeleton, are capable of inducing tissue-specific morphogenesis in a variety of organs and tissues, including bone, cartilage. liver, dentin, periodontal ligament, cementum, nerve tissue and the epithelial mucosa of the
  • the proteins apparently bind to surface receptors or otherwise contact and interact with progenitor cells, predisposing or stimulating the cells to proliferate and differentiate in a
  • the morphogens are capable of inducing the developmental cascade of cellular and molecular events that culminate in the formation of new organ-specific tissue, including any vascularization, connective tissue formation, and nerve ennervation as required by the naturally occurring tissue.
  • proteins useful in tissue morphogenesis are proteins originally identified as bone inductive proteins, such as the OP-1, OP-2 and the CBMP2
  • TGF- ⁇ superfamily members comprise a distinct subfamily of proteins different from other members of the TGF- ⁇ superfamily in that the family of morphogenic proteins are able to induce the full cascade of events that result in tissue
  • morphogenesis including stimulating cell proliferation and cell differentiation, supporting the growth and maintenance of differentiated cells and inducing the "redifferentiation" of transformed cells to display a morphology characteristic of untransformed cells.
  • the morphogenic proteins apparently act as endocrine factors rather than as local-acting growth factors like TGF- ⁇ .
  • the endogenous morphogens may be synthesized and secreted from a factor-producing tissue and can be transported to, and act on, a tissue at a distance, e.g., a tissue other than the tissue in which they are synthesized.
  • the morphogens are synthesized in the cell as a precursor molecule approximately three times larger than the mature protein that is processed to yield mature disulfide-linked dimers comprising the
  • the proteins are inactive when reduced, but are active as oxidized homodimeric species as well as when oxidized in combination with other morphogens to produce
  • morphogenesis typically require a suitable environment enabling cells to proliferate and differentiate in a tissue-specific manner into, e.g., bone-producing osteoblasts, hemopoietic cells, or liver cells,
  • the proliferation and differentiation of cells induced by the morphogenic proteins requires a suitable local environment including a suitable substratum on which the cells can anchor.
  • differentiating cells also require the presence of appropriate signals to direct their tissue-specificity, such as cell surface markers.
  • the morphogenic properties of 60A protein include the ability to induce proliferation and
  • morphogenically active fragment of 60A protein in a range of methods, including methods for increasing a progenitor cell population in a mammal; methods for stimulating progenitor cells to differentiate in vivo or in vitro and to maintain their differentiated phenotype; methods for inducing tissue-specific growth in vivo, and methods for the replacement of diseased or damaged tissue in vivo.
  • This invention provides methods for utilizing morphogenically active fragments of 60A protein to induce the developmental cascade of tissue
  • morphogenesis in a mammal. Specifically, methods are provided for utilizing a morphogenically active
  • fragment of 60A protein to induce the proliferation of uncommitted progenitor cells, to induce the
  • 60A protein can be utilized to initiate and maintain the developmental cascade of tissue morphogenesis in an appropriate, morphogenically permissive environment.
  • useful Protein 60A morphogens include proteins encoded by the DNA sequence provided in Seq. ID No. 1 and allelic variants thereof, as well as other naturally-occurring and biosynthetic mutants that are morphogenically active as defined herein.
  • Morphogenically active fragments is understood to include all proteins and protein fragments encoded by part or all of the sequence of Seq. ID No. 1, and which have morphogenic activity as defined herein.
  • a morphogen is a dimeric protein comprising a pair of polypeptide chains, wherein each polypeptide chain comprises at least the C-terminal six cysteine skeleton defined by residues 359-455 of Seq. ID No. 1 (or residues 43-139 of Seq. ID No. 3), including functionally equivalent arrangements of these cysteines (e.g., amino acid insertions or deletions which alter the linear
  • the dimeric protein species comprising the pair of polypeptide chains has the appropriate three-dimensional structure, including the appropriate intra- or inter-chain
  • the morphogens generally are capable of all of the following biological functions in a morphogenically permissive environment: stimulating proliferation of progenitor cells; stimulating the differentiation of progenitor cells; stimulating the proliferation of differentiated cells; and supporting the growth and maintenance of differentiated cells.
  • these morphogens are capable of inducing redifferentiation of committed cells under appropriate environmental conditions.
  • the morphogens of this invention comprise a morphogenically active dimeric species comprising a pair of polypeptide chains, wherein at least one of the polypeptide chains comprises the amino acid sequence defined by residues 359-455 of Seq. ID No. 1, including allelic and other mutant variants thereof.
  • at least one of the polypeptide chains comprises the amino acid sequence defined by residues 359-455 of Seq. ID No. 1, including allelic and other mutant variants thereof.
  • at least one of the polypeptide chains comprises the amino acid sequence defined by residues 359-455 of Seq. ID No. 1, including allelic and other mutant variants thereof.
  • at least one of the polypeptide chains comprises the amino acid sequence defined by residues 359-455 of Seq. ID No. 1, including allelic and other mutant variants thereof.
  • at least one of the polypeptide chains comprises the amino acid sequence defined by residues 359-455 of Seq. ID No. 1, including allelic and other mutant variants thereof.
  • at least one of the polypeptide chains comprises the amino
  • polypeptide chain comprises the sequence defined by residues 354-455 or residues 326-455 of Seq. ID No. 1. Alternatively, the amino acid sequence of both
  • polypeptide chains may be defined by part or all of the amino acid sequence of Seq. ID No. 1, including allelic and other mutant variants thereof.
  • the other polypeptide chain preferably comprises at least the sequence defining the C-terminal six cysteine skeleton of any of the other known morphogen family members, including OP1, OP2, CBMP2A, CBMP2B, BMP3, BMP6, Vg1, Vgr-1, DPP and GDF-1, (described in Seq. ID. Nos. 3-14), including allelic, species and other mutant variants thereof.
  • Other useful sequences include biosynthetic constructs, such as are described in U.S. Pat. No. 5,011,691.
  • 60A proteins are useful in the replacement of diseased or damaged tissue in a mammal, such as damaged lung tissue resulting from emphysema; cirrhotic kidney or liver tissue; damaged heart or blood vessel tissue, as may result from cardiomyopathies and/or
  • Atherothrombotic or cardioembolic strokes damaged stomach and other tissues of the gastrointestinal tract resulting from ulceric perforations or their repair; damaged nerve tissue as may result from physical injury, degenerative diseases such as Alzheimer's disease or multiple sclerosis, or strokes; damaged bone tissue as may result from metabolic bone diseases and other bone remodeling disorders; or damaged dentin, periodontal and/or cementum tissue as may result from disease or mechanical injury.
  • fragments of 60A protein are provided to a tissue-specific locus in vivo, to induce the developmental cascade of tissue morphogenesis at that site.
  • Cells stimulated ex vivo by contact with 60A protein also may be provided to the tissue locus.
  • the existing tissue provides the necessary matrix requirements, providing a suitable substratum for the proliferating and differentiating cells in a
  • the proteins or stimulated cells may be combined with a formulated matrix and implanted as a device at a locus in vivo.
  • the formulated matrix should be a biocompatible, preferably biodegradable, appropriately modified tissue-specific acellular matrix having the characteristics described below.
  • the loss of tissue function results from the tissue destructive effects and the subsequent formation of scar tissue associated with the body's immune/inflammatory response to an initial or repeated injury to the tissue.
  • the degree of scar tissue formation generally depends on the regenerative properties of the injured tissue, and on the degree and type of tissue damage.
  • morphogenically active fragments of 60A protein may be used to prevent or to substantially inhibit the
  • the protein also may be provided as a prophylactic, provided to a site in anticipation of tissue injury, such as part of a surgical or other clinical procedure likely to produce tissue damage, and to induce an inflammatory/immune response.
  • 60A protein also may be used to increase or
  • progenitor cells may be isolated from an individual's bone marrow, stimulated ex vivo with morphogenic 60A protein for a time and at a concentration sufficient to induce the cells to
  • progenitor cells proliferate, and returned to the bone marrow.
  • Other sources of progenitor cells include biocompatible cells obtained from a cultured cell line, stimulated in culture, and subsequently provided to the body.
  • 60A protein may be provided by systemic (e.g., oral or parenteral) administration, or it may be injected or otherwise provided to a progenitor cell population in an
  • 60A protein may be provided to the cells in vivo, e.g., by systemic injection, to induce mitogenic activity.
  • a particular population of hemopoietic stem cells may be increased by exposure to 60A protein, for example by plasmaphoresis of an individual's blood to extract the cells of interest, stimulating these cells ex vivo, and returning the stimulated cells to the blood.
  • morphogenic 60A protein may be used to support the growth and maintenance of differentiated cells, inducing existing differentiated cells to continue expressing their phenotype. It is anticipated that this activity will be particularly useful in the treatment of tissue disorders where loss of function is caused by reduced or lost metabolic function and cells become senescent or quiescent, such as may occur in aging cells and/or may be manifested in osteoporosis and a number of nerve degenerative diseases, including Alzheimer's disease.
  • Application of 60A protein directly to the cells to be treated, or providing it systemically, as by oral or parenteral administration, can stimulate these cells to continue expressing their phenotype, thereby
  • 60A protein also may be used in gene therapy protocols to stimulate the growth of quiescent cells, thereby potentially enhancing the ability of these cells to incorporate exogenous DNA.
  • morphogenically active fragment of 60A protein also may be used to induce "redifferentiation" of cells that have strayed from their differentiation pathway, such as can occur during tumorgenesis, inducing the cells now to exhibit a morphology characteristic of
  • a morphogenic 60A protein fragment may be provided to the cells directly or systemically.
  • 60A protein may be used to stimulate cell adhesion molecule (CAM) expression levels in a cell.
  • CAMs are molecules defined as carrying out cell-cell interactions
  • CAMs are believed to play a fundamental regulatory role in tissue
  • tissue boundary formation including embryonic induction and migration, and tissue
  • Altered CAM levels have been implicated in a number of tissue disorders, including congenital defects, neoplasias, and
  • N-CAM expression is associated with normal neuronal cell development and differentiation, including retinal formation, synaptogenesis, and nerve-muscle tissue adhesion. Inhibition of one or more of the N-CAM isoforms is known to prevent proper tissue development. Altered N-CAM expression levels also are associated with neoplasias, including neuroblastomas (see infra), as well as with a number of neuropathies, including normal pressure hydrocephalous and type II schizophrenia.
  • Application of the morphogen directly to the cells to be treated, or providing the morphogen to the mammal systemically, for example, parenterally, or indirectly by oral administration, may be used to induce cellular expression of one or more CAMs, particularly N-CAMs and L1. CAMs also have been postulated as part of a
  • morphogens described herein may act as the inducer of this pathway.
  • inventions may be derived from organ-specific tissue, or they may be formulated synthetically. In one
  • 60A protein is provided at a tissue-specific locus, e.g., by systemic administration, implantation or injection at a tissue-specific locus, the existing tissue at that locus, whether diseased or damaged, has the capacity of acting as a suitable matrix.
  • a formulated matrix may be provided externally together with the stimulated progenitor cells or morphogenically active 60A protein fragment, as may be necessary when the extent of injury sustained by the damaged tissue is large.
  • the matrix should be a biocompatible, suitably modified acellular matrix having dimensions such that it allows the influx, differentiation, and proliferation of migratory
  • progenitor cells and is capable of providing a
  • the matrix also preferably is tissue-specific, and biodegradable.
  • Formulated matrices may be generated from
  • dehydrated organ-specific tissue prepared for example, by treating the tissue with solvents to substantially remove the intracellular, non-structural components from the tissue.
  • the matrix may be formulated synthetically using a biocompatible
  • tissue-specific cell attachment factors preferably in vivo biodegradable, structural molecule in association with suitable tissue-specific cell attachment factors.
  • the molecule may be a naturally occurring one such as collagen, laminin or hyaluronic acid, or a synthetic polymer comprising, for example, polylactic acid, polybutyric acid, polyglycolic acid, and copolymers thereof.
  • preferred structural polymers comprise tissue-specific collagens.
  • preferred cell attachment factors include
  • the matrix further may be treated with an agent or agents to increase the number of pores and micropits on its surfaces, so as to enhance the influx, proliferation and differentiation of migratory progenitor cells from the body of the mammal.
  • the invention thus relates to compositions and methods for the use of morphogenically active fragments of 60A protein, a species variant of the generic family of morphogens disclosed in international application US 92/01968 (WO 92/15323) as a tissue morphogen.
  • Active 60A protein useful in the compositions and methods of this invention may include forms having varying
  • the 60A proteins can be expressed from intact or truncated cDNA or from synthetic DNAs in procaryotic or eucaryotic host cells, and purified, cleaved, refolded, and dimerized to form
  • Useful host cells include procaryotes, including E. coli, and eucaryotic cells, including mammalian cells, such as CHO, COS or BSC cells, or the insect/baculovirus system.
  • procaryotes including E. coli
  • eucaryotic cells including mammalian cells, such as CHO, COS or BSC cells, or the insect/baculovirus system.
  • the invention provides methods and compositions for inducing the developmental cascade of tissue
  • compositions provided herein may be utilized in a range of applications, including stimulating the proliferation and/or
  • morphogenic 60A proteins of the invention are a species variant of the family of morphogens disclosed in international application US 92/01968 (WO 92/15323) the disclosures of which are incorporated hereinabove by reference. As described herein, 60A protein may be isolated from natural sources or constructed
  • Morphogenically active fragments of 60A protein are useful for initiating and maintaining the tissue-specific developmental cascade in a variety of tissues, including bone, cartilage, dentin, neural tissue, liver, periodontal ligament, cementum, lung, heart, kidney and numerous tissues of the gastrointestinal tract.
  • morphogenically active 60A proteins can induce the proliferation and differentiation of these progenitor cells.
  • 60A proteins are capable of reproducing the cascade of cellular and molecular events that occur during embryonic development to yield functional tissue. For example, the protein can induce the
  • a protein is morphogenic if it is capable of inducing the developmental cascade of cellular and molecular events that culminate in the formation of new, organ-specific tissue and comprises at least the conserved C-terminal six cysteine skeleton or its functional equivalent (see supra).
  • the morphogens generally are capable of all of the following biological functions in a
  • morphogenically permissive environment stimulating proliferation of progenitor cells; stimulating the differentiation of progenitor cells; stimulating the proliferation of differentiated cells; and supporting the growth and maintenance of differentiated cells. Details of how the morphogen family of proteins
  • the morphogens may be purified from naturally-sourced material or recombinantly produced from
  • procaryotic or eucaryotic host cells preferably as described therein, using the genetic sequences
  • Particularly useful proteins identified to date include OP1, OP2, CBMP2A and CBMP2B (the
  • BMP2A and BMP2B morphogenically active domains of proteins referred to in the art as BMP2A and BMP2B, or BMP2 and BMP4, respectively
  • BMP3, BMP5, BMP6, GDF-1, Vgl, Vgr-1, and DPP including their allelic and species variants, as well as other mutant variants.
  • the amino acid sequence for either the mature or 7 cysteine forms of the proteins are presented in Seq. ID Nos. 3-14. Detailed descriptions of the proteins also may be found in, for example, international application PCT US 92/07432 (WO 93/05751). Morphogenically active biosynthetic
  • constructs such as those disclosed in U.S. Pat. No. 5,011,691, the disclosure of which is incorporated herein by reference (e.g., COP-1, COP-3, COP-4, COP-5, COP-7, and COP-16) also are envisioned to be useful.
  • COP-1, COP-3, COP-4, COP-5, COP-7, and COP-16 the gene encoding a novel member of the TGF- ⁇ superfamily of structurally related proteins was identified in the Drosophila genome and named "60A”.
  • the cDNA sequence and encoded amino acid sequence (“60A protein") are described in Wharton et al. (1991) Proc. Natl. Acad. Sci. USA, 88: 9214-9218, and in Seq. ID No. 1.
  • the Drosphila 60A gene encodes a protein ("60A") first expressed as an immature translation product that is 455 amino acids in length. This precursor form, referred to herein as the "prepro” form, (Seq. ID.
  • residues 1-455 includes an N-terminal signal peptide sequence, typically less than about
  • the "pro” form of the protein includes the pro domain and the mature domain, and forms a soluble species that appears to be the primary form secreted from cultured mammalian cells.
  • the signal peptide is cleaved rapidly upon translation, at a cleavage site that can be predicted in a given sequence using the method of Von Heijne ((1986) Nucleic Acids Research 14:4683-4691). By amino acid sequence homology with other, known morphogens, the pro domain likely is cleaved at
  • All morphogens comprise at least a conserved six cysteine skeleton in the amino acid sequence C-terminal domain.
  • the conserved six cysteine skeleton is defined in 60A protein by residues 359-455; the conserved seven cysteine skeleton is defined by residues 354-455.
  • the morphogenically active protein comprises a mature, processed sequence, including fragments thereof, appropriately dimerized and disulfide bonded.
  • the mature sequence of 60A protein shares
  • the seven cysteine skeleton shows approximately 70% amino acid identity with the corresponding hOP1 sequence.
  • the 60A protein seven cysteine skeleton also shares
  • 60A protein seven cysteine skeleton also shares about 59% identity with the corresponding sequence of another morphogen identified in Drosophila, DPP. Without being limited to a particular theory, based on amino acid homology, 60A protein likely may be the Drosophila homolog or species variant of OP-1.
  • amino acid sequence homology is understood to mean amino acid sequence similarity, and homologous sequences share identical or similar amino acids, where similar amino acids are conserved amino acids as defined by Dayoff et al., Atlas of Protein Sequence and Structure; vol.5, Suppl.3, pp.345-362 (M.O. Dayoff, ed., Nat'l BioMed. Research Fdn.,
  • a candidate sequence sharing 70% amino acid homology with a reference sequence requires that, following alignment of the candidate sequence with the reference sequence, 70% of the amino acids in the candidate sequence are identical to the corresponding amino acid in the reference sequence, or constitute a conserved amino acid change thereto.
  • Amino acid sequence identity is understood to require identical amino acids between two aligned sequences.
  • a candidate sequence sharing 60% amino acid identity with a reference sequence requires that, following alignment of the candidate sequence with the reference sequence, 60% of the amino acids in the candidate sequence are identical to the
  • homologies and identities calculated use OP-1 as the reference sequence. Also as used herein, sequences are aligned for homology and identity calculations using the method of Needleman et al. (1970) J.Mol. Biol. 48:443-453 and identities calculated by the Align program (DNAstar, Inc.) In all cases, internal gaps and amino acid insertions in the candidate sequence as aligned are ignored when making the homology/identity calculation.
  • Table I compares the C-terminal amino acid sequences defining the seven cysteine skeleton of 60A protein, native human OP-1 (hOP-1 Seq. ID No. 3), and DPP (from Drosophila), (Seq. ID. No. 9). In the table, three dots indicates that the amino acid in that position is the same as the amino acid in the corresponding position in 60A protein.
  • Table II the sequences of the morphogens OP2, CBMP2A, CBMP2B, BMP3, BMP5, BMP6, Vgl, Vgr-1, GDF-1, DPP and 60-A all are compared to OP-1.
  • sequences in this region comprise Asn-Ser (residues 58, 59), with CBMP-2A then comprising Lys and Ile, whereas CBMP-2B comprises Ser and Ile.
  • GDF-1 amino acid sequence between residues 43 and 44 of GDF-1 lies the amino acid sequence Gly-Gly-Pro-Pro.
  • significant amino acid changes can be made within the sequences while retaining the morphogenic activity.
  • GDF-1 protein sequence depicted in Table II shares only about 50% amino acid identity with the hOP-1 sequence
  • the GDF-1 sequence shares greater than 70% amino acid sequence homology (or "similarity") with the hOP-1 sequence, where "homology" or
  • the morphogens described herein may be provided to an individual by any suitable means, preferably
  • morphogen is to be provided directly (e.g., locally, as by injection, to a desired tissue site), or parenterally, such as by intravenous, subcutaneous, intramuscular, intraorbital, ophthalmic,
  • the morphogen preferably comprises part of an aqueous solution.
  • the solution is
  • the aqueous medium for the morphogen thus may comprise normal physiologic saline (0.85% NaCl, 0.15M), pH 7-7.4.
  • the aqueous solution containing the morphogen can be made, for example, by dissolving the protein in 50% ethanol, acetonitrile containing 0.1% trifluoroacetic acid (TFA) or 0.1% HCl, or equivalent solvents.
  • TFA trifluoroacetic acid
  • One volume of the resultant solution then is added, for example, to ten volumes of phosphate buffered saline (PBS), which further may include 0.1-0.2% human serum albumin (HSA).
  • PBS phosphate buffered saline
  • HSA human serum albumin
  • a given morphogen may be made more soluble by association with a suitable molecule.
  • the pro form of 60A protein comprises a species that is soluble in physiological solutions.
  • the endogenous protein is thought to be
  • This soluble form of the protein may be obtained from the culture medium of morphogen-secreting mammalian cells. Alternatively, a soluble species may be formulated by complexing the mature dimer (or an active fragment thereof) with part or all of a pro domain.
  • Another molecule capable of enhancing solubility and particularly useful for oral administrations, is casein. For example, addition of 0.2% casein increases solubility of the mature active form of OP-1 by 80%.
  • Other components found in milk and/or various serum proteins also may be useful.
  • administration may be prepared by any of the methods well known in the pharmaceutical art, described, for example, in Remington's Pharmaceutical Sciences,
  • Formulations may include, for example, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin,
  • Formulations for direct administration may include glycerol and other compositions of high viscosity.
  • Biocompatible, preferably bioresorbable polymers including, for example, hyaluronic acid, collagen, tricalcium phosphate, polybutyrate, polyglycolide, polylactide and lactide/glycolide copolymers, may be useful excipients to control the release of the
  • morphogen in vivo Other potentially useful parenteral delivery systems for these morphogens include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation administration may contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally.
  • Formulations for parenteral administration also may be particularly formulated for buccal administration, rectal administration, or vaginal administration.
  • the morphogens described herein may be administered orally.
  • proteins as therapeutics generally is not practiced as most proteins readily are degraded by digestive enzymes and acids in the mammalian digestive system before they can be absorbed into the bloodstream.
  • the morphogens described herein typically are acid-stable and protease-resistant (see, for example, U.S. Pat. No. 4,968,590.)
  • at least one morphogen, OP-1 has been identified in bovine mammary gland extract, colostrum and milk, as well as saliva.
  • the OP-1 purified from mammary gland extract is
  • this protein induces endochondral bone formation in mammals when implanted subcutaneously in association with a suitable matrix material, using a standard in vivo bone assay, such as is disclosed in U.S. Pat.No. 4,968,590.
  • endogenous morphogen also is detected in human serum.
  • the compounds provided herein also may be associated with molecules capable of enhancing their solubility in vitro or in vivo, including, for example, part or all of a morphogen pro domain as described herein below (see Section II.A), and casein, as described above.
  • the compounds provided herein also may be
  • tetracycline and diphosphonates are known to bind to bone mineral, particularly at zones of bone remodeling, when they are provided systemically in a mammal. Accordingly, these molecules may be included as useful agents for targeting 60A protein to bone tissue. Alternatively, an antibody or other binding protein that interacts specifically with a surface molecule on the desired target tissue cells also may be used. Such targeting molecules further may be
  • an acid labile bond such as an Asp-Pro
  • Useful targeting molecules may be designed, for example, using the single chain binding site technology disclosed, for example, in U.S. Pat. No.
  • the morphogen family members share significant sequence homology in the C-terminal active domains.
  • the sequences diverge significantly in the sequences which define the pro domain.
  • the pro domain may be morphogen-specific.
  • morphogen pro domains may serve as targeting molecules for the morphogens described herein.
  • the pro domains may interact specifically with one or more molecules at the target tissue to direct the morphogen associated with the pro domain to that tissue.
  • another useful targeting molecule for targeting 60A protein to bone tissue may include part or all of a morphogen pro domain
  • the pro domain of GDF-1 may be used to target morphogenic 60A protein to nerve tissue, particularly brain tissue where GDF-1 appears to be primarily expressed (see, for example, CRP070 and Lee(1991)PNAS:88:4250-4254, incorporated herein by reference).
  • morphogen species comprising the pro domain may be obtained from the culture medium of morphogen-secreting mammalian cells.
  • a tissue-targeting species may be formulated by complexing the mature dimer (or an active fragment thereof) with part or all of a pro domain.
  • the morphogenic 60A proteins provided herein may be administered alone or in combination with other molecules known to have a beneficial effect on tissue morphogenesis, including molecules capable of tissue repair and regeneration and/or inhibiting inflammation. Examples of useful cofactors for
  • individuals for example, include vitamin D 3 ,
  • compositions can be formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable nontoxic excipients and carriers.
  • pharmaceutically acceptable nontoxic excipients and carriers may be prepared for parenteral administration, particularly in the form of liquid solutions or suspensions; for oral administration, particularly in the form of tablets or capsules; or intranasally, particularly in the form of powders, nasal drops, or aerosols.
  • compositions can be formulated for parenteral or oral administration to humans or other mammals in therapeutically effective amounts, e.g., amounts which provide appropriate concentrations of a morphogenic 60A protein to target tissue for a time sufficient to induce morphogenesis, including particular steps thereof, as described above.
  • the concentration of the compounds described in a therapeutic composition will vary depending upon a number of factors, including the dosage of the drug to be administered, the chemical characteristics (e.g., hydrophobicity) of the compounds employed, and the route of administration.
  • the preferred dosage of drug to be administered also is likely to depend on such variables as the type and extent of tissue loss or defect, the overall health status of the particular patient, the relative biological efficacy of the compound selected, the formulation of the compound excipients, and its route of administration.
  • the compounds of this invention may be provided in an aqueous physiological buffer solution containing about 0.1 to 10% w/v compound for parenteral
  • Typical dose ranges are from about 10 ng/kg to about 1 g/kg of body weight per day; a
  • preferred dose range is from about 0.1 ⁇ g/kg to
  • the 60A protein dosage given in all cases is between 1-100 ⁇ g of protein per kilogram weight of the patient.
  • morphogen e.g., OP-1, 20 ⁇ g
  • 10 ⁇ g systemic injections of morphogen e.g., OP-1
  • OP-1 morphogen-1
  • a currently preferred form of the morphogen useful in therapeutic formulations, having improved solubility in aqueous solutions and consisting essentially of amino acids, is a dimeric morphogenic protein
  • the dimeric morphogenic protein is complexed with two peptides. Also, the dimeric morphogenic protein
  • the pro region peptides preferably is noncovalently complexed with the pro region peptide or peptides.
  • the pro region peptides also preferably comprise at least the N-terminal eighteen amino acids that define a given morphogen pro region. In a most preferred embodiment, peptides defining substantially the full length pro region are used.
  • soluble forms of morphogens include dimers of the uncleaved pro forms of these proteins, as well as "hemi-dimers" wherein one subunit of the dimer is an uncleaved pro form of the protein, and the other subunit comprises the mature form of the protein, including truncated forms thereof, preferably
  • useful pro domains include the full length pro regions, as well as various truncated forms hereof, particularly truncated forms cleaved at proteolytic Arg-Xaa-Xaa-Arg cleavage sites.
  • possible pro sequences include sequences defined by residues 30-292 (full length form); 48-292; and 158-292 (see, for example,
  • Soluble OP-1 complex stability is enhanced when the pro region comprises the full length form rather than a truncated form, such as the 48-292 truncated form, in that residues 30-47 show sequence homology to the
  • pro sequences are those encoding the full length form of the pro region for a given morphogen.
  • possible pro sequences include sequences defined by residues 20-281 or 20-326, and truncated sequences defined by residues 124-281 or 124-326, or 127-281 or 127-326 (see Seq ID No.1).
  • pro sequences contemplated to have utility include biosynthetic pro sequences, particularly those that incorporate a sequence derived from the N-terminal portion of one or more morphogen pro sequences.
  • useful sequences encoding the pro region may be obtained from genetic sequences encoding known morphogens.
  • chimeric pro regions can be constructed from the sequences of one or more known morphogens.
  • Still another option is to create a synthetic sequence variant of one or more known pro region sequences.
  • Morphogens are expressed from mammalian cells as soluble complexes. Typically, however the complex is disassociated during purification, generally by
  • soluble proteins from conditioned media (or, optionally, a body fluid such as serum, cerebro-spinal or peritoneal fluid), under non-denaturing conditions.
  • the method is rapid, reproducible and yields isolated soluble proteins
  • Soluble morphogen complexes in substantially pure form. Soluble morphogen complexes can be isolated from conditioned media using a simple, three step
  • the protocol involves running the media (or body fluid) over an affinity column, followed by ion exchange and gel filtration chromatographies.
  • the affinity column described below is a Zn-IMAC column.
  • the present protocol has general applicability to the purification of a variety of morphogens, all of which are anticipated to be isolatable using only minor modifications of the protocol described below.
  • An alternative protocol also envisioned to have utility an immunoaffinity column, created using standard
  • OP-1 was, expressed in mammalian CHO (Chinese hamster ovary) cells as described in the art (see, for example, international application
  • the Zn-IMAC step separates the soluble OP-1 from the bulk of the contaminating serum proteins that elute in the flow through and 35 mM imidazole wash fractions.
  • the Zn-IMAC purified soluble OP-1 is next applied to an S-Sepharose cation-exchange column equilibrated in 20 mM NaPO 4 (pH 7.0) with 50 mM NaCl.
  • This S-Sepharose step serves to further purify and concentrate the soluble OP-1 complex in preparation for the following gel filtration step.
  • the protein was applied to a Sephacryl S-200HR column equilibrated in TBS.
  • soluble morphogens also may be isolated from one or more body fluids, including serum, cerebro-spinal fluid or peritoneal fluid.
  • the 50 mM imidazole eluate containing the soluble OP-1 complex was diluted with nine volumes of 20 mM NaPO. (pH 7.0) and applied to an S-Sepharose (Pharmacia) column equilibrated in 20 mM NaPO 4 (pH 7.0) with 50 mM NaCl.
  • the S-Sepharose resin was loaded with an equivalent of 800 mL of starting conditioned media per mL of resin. After loading the S-Sepharose column was washed with equilibration buffer and eluted with 100 mM NaCl followed by 300 mM and 500 mM NaCl in 20 mM NaPO 4 (pH 7.0).
  • the 300 mM NaCl pool was further purified using gel filtration chromatography. Fifty mis of the 300 mm NaCl eluate was applied to a 5.0 X 90 cm Sephacryl S-200HR (Pharmacia) equilibrated in Tris buffered saline (TBS), 50 mM Tris, 150 mM NaCl
  • ADH alcohol dehydrogenase
  • BSA bovine serum albumin
  • CA carbonic anhydrase
  • cytochrome C cyt C, 12.5 kDa
  • the soluble OP-1 complex elutes with an apparent molecular weight of 110 kDa. This agrees well with the predicted composition of the soluble OP-1 complex with one mature OP-1 dimer (35-36 kDa) associated with two pro-domains (39 kDa each). Purity of the final complex can be verified by running the appropriate fraction in a reduced 15% polyacrylamide gel. The complex components can be verified by running the complex-containing fraction from the S-200 or S-200HR columns over a reverse phase C18 HPLC column and eluting in an acetonitrile gradient (in 0.1% TFA), using standard procedures. The complex is dissociated by this step, and the pro domain and mature species elute as separate species. These separate species then can be subjected to N-terminal sequencing using
  • N-terminal sequencing of the isolated pro domain from mammalian cell produced OP-1 revealed 2 forms of the pro region, the intact form (beginning at residue 30 of Seq. ID No. 16) and a truncated form, (beginning at residue 48 of Seq. ID No. 16.)
  • N-terminal sequencing of the polypeptide subunit of the isolated mature species reveals a range of
  • soluble complexes may be formulated from purified pro domains and mature dimeric species. Successful complex formation
  • the denaturing conditions mimic the environment of an intracellular vesicle sufficiently such that the cleaved pro domain has an opportunity to associate with the mature dimeric species under relaxed folding conditions.
  • concentration of denaturant in the solution then is decreased in a controlled, preferably step-wise manner, so as to allow proper refolding of the dimer and pro regions while maintaining the association of the pro domain with the dimer.
  • Useful denaturants include 4-6M urea or guanidine hydrochloride (GuHCl), in buffered solutions of pH 4-10, preferably pH 6-8.
  • the soluble complex then is formed by controlled dialysis or dilution into a solution having a final denaturant concentration of less than 0.1-2M urea or GuHCl, preferably 1-2 M urea of GuHCl, which then preferably can be diluted into a physiological buffer. Protein purification/renaturing procedures and considerations are well described in the art, and details for
  • the stability of the highly purified soluble morphogen complex in a physiological buffer can be enhanced by any of a number of means.
  • a physiological buffer e.g., tris-buffered saline (TBS) and phosphate-buffered saline (PBS)
  • TBS tris-buffered saline
  • PBS phosphate-buffered saline
  • a pro region that comprises at least the first 18 amino acids of the pro sequence, and preferably is the full length pro region.
  • These first 18 amino acid residues show sequence homology to the N-terminal portion of other morphogens and are believed to have particular utility in enhancing complex stability for all morphogens.
  • Other useful means for enhancing the stability of soluble morphogen complexes include three classes of additives.
  • additives include basic amino acids (e.g., L-arginine, lysine and betaine); nonionic detergents (e.g., Tween 80 or Nonldet P-120); and carrier proteins (e.g., serum albumin and casein).
  • basic amino acids e.g., L-arginine, lysine and betaine
  • nonionic detergents e.g., Tween 80 or Nonldet P-120
  • carrier proteins e.g., serum albumin and casein.
  • Useful concentrations of these additives include 1-100 mM, preferably 10-70 mM, including 50 mM, basic amino acid;, 0.01-1.0%, preferably 0.05-0.2%, including 0.1% (v/v) nonionic detergent;, and 0.01-1.0%, preferably 0.05-0.2%, including 0.1% (w/v) carrier protein.
  • a morphogenically active fragment of 60A protein may be implanted surgically, dispersed in a
  • the matrix also may provide signals capable of directing the tissue specificity of the differentiating cells, as well as providing a
  • the formulated matrix may be shaped as desired in anticipation of surgery or may be shaped by the physician or technician during surgery.
  • the material may be used in topical, subcutaneous, intraperitoneal, or intramuscular implants to repair tissue or to induce its growth de novo.
  • the matrix preferably is biodegradable in vivo, being slowly absorbed by the body and replaced by new tissue growth, in the shape or very nearly in the shape of the
  • Suitable biocompatible, in vivo biodegradable acellular matrices may be prepared from naturally-occurring tissue. The tissue is treated with suitable agents to substantially extract the cellular,
  • the agents also should be capable of extracting any growth
  • the resulting material is a porous, acellular matrix, substantially depleted in nonstructurally-associated components.
  • the matrix also may be further treated with agents that modify the matrix, increasing the number of pores and micropits on its surfaces.
  • agents that modify the matrix, increasing the number of pores and micropits on its surfaces.
  • Those skilled in the art will know how to determine which agents are best suited to the extraction of nonstructural components for different tissues. For example, soft tissues such as liver and lung may be thin-sectioned and exposed to a nonpolar solvent such as, for example, 100% ethanol, to destroy the cellular structure of the tissue and extract nonstructural components. The material then is dried and pulverized to yield nonadherent porous particles.
  • Structural tissues such as cartilage and dentin where collagen is the primary component may be demineralized and extracted with guanidine, essentially following the method of Sampath et al. (1983) PNAS 80:6591-6595. For example, pulverized and
  • demineralized dentin is extracted with five volumes of 4M guanidine-HCl, 50mM Tris-HCl, pH 7.0 for 16 hours at 4°C. The suspension then is filtered. The insoluble material that remains is collected and used to
  • the material is mostly
  • the matrix particles may further be treated with a collagen fibril-modifying agent that extracts potentially unwanted components from the matrix, and alters the surface structure of the matrix material.
  • Useful agents include acids, organic solvents or heated aqueous media. A detailed description of these matrix treatments are disclosed, for example, in U.S. Patent No. 4,975,526 and PCT publication US90/00912, published September 7, 1990 (WO90/10018).
  • the currently most preferred agent is a heated aqueous fibril-modifying medium such as water, to increase the matrix particle surface area and porosity.
  • the currently most preferred aqueous medium is an acidic aqueous medium having a pH of less than about 4.5, e.g., within the range of about pH 2 - pH 4 which may help to "swell" the collagen before heating. 0.1% acetic acid, which has a pH of about 3, currently is most preferred. 0.1 M acetic acid also may be used.
  • aqueous medium (1g matrix/30ml aqueous medium) under constant stirring in a water jacketed glass flask, and maintained at a given temperature for a predetermined period of time. Preferred treatment times are about one hour, although exposure times of between about 0.5 to two hours appear acceptable.
  • the temperature employed is held constant at a temperature within the range of about 37°C to 65°C.
  • the currently preferred heat treatment temperature is within the range of about 45°C to 60°C.
  • the matrix is filtered, washed, lyophilized and used for implant. Where an acidic aqueous medium is used, the matrix also is preferably neutralized prior to washing and
  • a currently preferred neutralization buffer is a 200mM sodium phosphate buffer, pH 7.0.
  • the matrix preferably first is allowed to cool following thermal treatment, the acidic aqueous medium (e.g., 0.1% acetic acid) then is removed and replaced with the neutralization buffer and the matrix agitated for about 30 minutes.
  • the acidic aqueous medium e.g. 0.1% acetic acid
  • neutralization buffer then may be removed and the matrix washed and lyophilized.
  • Other useful fibril-modifying treatments include acid treatments (e.g., trifluoroacetic acid and
  • the treated matrix may be washed to remove any extracted components, following a form of the procedure set forth below: 1. Suspend matrix preparation in TBS (Tris-buffered saline) 1g/200 ml and stir at 4°C for 2 hrs; or in 6 M urea, 50 mM Tris-HCl, 500 mM NaCl, pH 7.0 (UTBS) or water and stir at room temperature (RT) for 30 minutes (sufficient time to neutralize the pH);
  • TBS Tris-buffered saline
  • tissue-specific matrices may be formulated synthetically if appropriately modified.
  • porous biocompatible, in vivo biodegradable synthetic matrices are disclosed in PCT publication US91/03603, published December 12, 1991 (WO91/18558), the disclosure of which is hereby
  • collagen comprises a porous crosslinked structural polymer of biocompatible, biodegradable collagen and appropriate, tissue-specific glycosaminoglycans as tissue-specific cell attachment factors.
  • Collagen derived from a number of sources may be suitable for use in these synthetic matrices, including insoluble collagen, acid-soluble collagen, collagen soluble in neutral or basic aqueous solutions, as well as those collagens which are commercially available.
  • Glycosaminoglycans or mucopolysaccharides are hexosamine-containing polysaccharides of animal origin that have a tissue specific distribution, and therefore may be used to help determine the tissue specificity of the morphogen-stimulated differentiating cells. Reaction with the GAGs also provides collagen with another valuable property, i.e., inability to provoke an immune reaction (foreign body reaction) from an animal host.
  • GAGs are made up of residues of
  • hexosamines glycosidically bound and alternating in a more-or-less regular manner with either hexouronic acid or hexose moieties (see, e.g., Dodgson et al. in
  • GAGs include hyaluronic acid, heparin, heparin sulfate, chondroitin 6-sulfate, chondroitin 4-sulfate, dermatan sulfate, and keratin sulfate.
  • Other GAGs are suitable for forming the matrix described herein, and those skilled in the art will either know or be able to ascertain other suitable GAGs using no more than routine experimentation.
  • chondroitin-6-sulfate can be used where endochondral bone formation is desired.
  • Heparin sulfate may be used to formulate synthetic matrices for use in lung tissue repair.
  • Collagen can be reacted with a GAG in aqueous acidic solutions, preferably in diluted acetic acid solutions.
  • a GAG aqueous acidic solutions
  • coprecipitates of tangled collagen fibrils coated with GAG results.
  • This tangled mass of fibers then can be homogenized to form a homogeneous dispersion of fine fibers and then filtered and dried.
  • Insolubility of the collagen-GAG products can be raised to the desired degree by covalently cross-linking these materials, which also serves to raise the resistance to resorption of these materials.
  • any covalent cross-linking method suitable for cross-linking collagen also is suitable for cross-linking these composite materials, although
  • crosslinking by a dehydrothermal process is preferred.
  • the crosslinked particles are essentially spherical, with diameters of about 500 ⁇ m.
  • Scanning electron miscroscopy shows pores of about 20 ⁇ m on the surface and 40 ⁇ m on the interior.
  • the interior is made up of both fibrous and sheet-like structures, providing surfaces for cell attachment.
  • the voids interconnect, providing access to the cells throughout the interior of the particle.
  • the material appears to be roughly 99.5% void volume, making the material very efficient in terms of the potential cell mass that can be grown per gram of microcarrier.
  • the morphogenically active fragments of 60A protein described herein can be combined and dispersed in an appropriately modified tissue-specific matrix using any of the methods described below:
  • Matrix is added to the morphogen dissolved in guanidine-HCl. Samples are vortexed and incubated at a low temperature. Samples are then further vortexed. Cold absolute ethanol is added to the mixture which is then stirred and incubated. After centrifugation
  • ACN/TFA trifluroacetic acid
  • a preparation of a morphogenically active fragment of 60A protein in physiological saline also may be vortexed with the matrix and lyophilized to produce morphogenically active material.
  • Tissue morphogenesis requires a morphogenically permissive environment. Clearly, in fully-functioning healthy tissue that is not composed of a permanently renewing cell population, there must exist signals to prevent continued tissue growth. Thus, it is
  • the bone-derived carrier is not demineralized but rather is washed only in low salt, for example, induction of endochondral bone formation is inhibited, suggesting the presence of one or more inhibiting factors within the carrier.
  • compositions of this invention may be purified from natural sources or produced using standard recombinant methodology.
  • Drosophila S2 cell line a Drosophila melanogaster cell line derived from late embryonic stages, as described below.
  • selection sequences may be obtained commercially from, for example, from Clontech, Inc., Palo Alto, or the ATCC, Rockville, MD (e.g.,from plasmid #37148).
  • FCS fetal cal serum
  • MTX methotrexate
  • the 60A protein expressed in the S2 system cells is produced as a processed mature
  • the recombinantly produced 60A protein then was purified from the medium using two chromatography steps: S-sepharose (Sigma Chemical Co., St. Louis) and reverse phase HPLC (e.g., Aldrich Chemical Co.,
  • a typical purification utilized 50 ml of medium containing 5% fetal calf serum.
  • the media was diluted with 2 volumes of 9 M urea, 20 mM MES, pH 7.0 and applied to a 10 ml column of S-sepharose
  • step elution of bound protein was accomplished with the same buffer containing 100 and 300 mM NaCl 2 .
  • the 300 mM NaCl fraction then was sequentially dialysed against water and 30%
  • morphogen e.g., 60A protein
  • Immunoreactive fractions then were pooled and the purity of 60A protein determined by standard gel scanning methods.
  • concentration of protein was estimated by scanning the Coomassie-stained protein band in the gel at 580 nm in reference to a known amount of standard bovine serum albumin protein.
  • the purified protein is a processed mature
  • Drosophila DPP also was cloned and purified as described for 60A protein. DPP was expressed and secreted as a processed mature disulfide-linked dimer which then bound to and accumulated on the petri dish surface. The DPP protein that bound to plates was extracted with 200 mM CaCl 2 /l% Tween-20/20 mM MES buffer pH 7.2, and purified on an S-Sepharose and C-18 column as described for 60A protein.
  • E. coli produced human BMP-2 and BMP4, for BMP-2-specific antisera, or E. coli produced OP-1 (for OP-1-specific antibody), using standard immunology
  • 60A protein likely may be the Drosophila homolog or species variant of OP1, and DPP, the homolog or the species variant of BMP2.
  • the ability of 60A protein to induce proliferation of osteoblasts may be determined in vitro using the following assay.
  • rat osteoblast-enriched primary cultures preferably are used. Although these cultures are heterogeneous in that the individual cells are at different stages of differentiation, the culture is believed to more accurately reflect the metabolism and function of osteoblasts in vivo than osteoblast cultures obtained from established cell lines.
  • all chemicals referenced are standard, commercially available reagents, readily available from a number of sources, including Sigma Chemical, Co., St. Louis; Calbiochem, Corp., San Diego and Aldrich Chemical Co., Milwaukee.
  • Rat osteoblast-enriched primary cultures are prepared by sequential collagenase digestion of newborn suture-free rat calvaria (e.g., from 1-2 day-old animals, Long-Evans strain, Charles River Laboratories, Wilmington, MA), following standard procedures, such as are described, for example, in Wong et al., (1975) PNAS 72:3167-3171. Rat osteoblast single cell suspensions then are plated onto a multi-well plate (e.g., a
  • the cultured cells are divided into three groups: (1) wells which receive, for example, 0.1, 1.0, 10.0, 40 and 80.0 ng of 60A protein; (2) wells which receive 0.1, 1.0, 10.0 and 40 ng of a local-acting growth factor (e.g., TGF- ⁇ ); and (3) the control group, which receive no growth factors.
  • the cells then are divided into three groups: (1) wells which receive, for example, 0.1, 1.0, 10.0, 40 and 80.0 ng of 60A protein; (2) wells which receive 0.1, 1.0, 10.0 and 40 ng of a local-acting growth factor (e.g., TGF- ⁇ ); and (3) the control group, which receive no growth factors.
  • TGF- ⁇ a local-acting growth factor
  • the resulting cell lysates are harvested using standard means well known in the art, and the incorporation of 3 H-thymidine into cellular DNA determined by liquid scintillation as an indication of mitogenic activity of the cells.
  • 60A protein stimulates 3 H-thymidine incorporation into DNA, and thus promote osteoblast cell proliferation.
  • TGF- ⁇ is transient and biphasic. At high concentrations, TGF- ⁇ has no
  • 60A protein The in vitro effect of 60A protein on osteoblast proliferation also can be tested on human primary osteoblasts (obtained from bone tissue of a normal adult patient and prepared as described above) and on human osteosarcoma-derived cell lines. In all cases 60A protein is anticipated to induce cell proliferation in accordance with the morphogen's ability to induce endochondral bone formation (see Example 7, below).
  • Useful naive stem cells include pluripotential stem cells, which may be isolated from bone marrow or umbilical cord blood using conventional methodologies, (see, for example, Faradji et al., (1988) Vox Sang., 55
  • embryonic stem cells e.g., from a cultured mesodermal cell line
  • embryonic cells may be useful.
  • Another method for obtaining progenitor cells and for determining the ability of 60A protein fragments to stimulate cell proliferation is to capture progenitor cells from an in vivo source. For example, a
  • biocompatible matrix material able to allow the influx of migratory progenitor cells may be implanted at an in vivo site long enough to allow the influx of migratory progenitor cells.
  • a bone-derived, guanidine-extracted matrix formulated as disclosed for example in Sampath et al. ((1983) PNAS 80:6591-6595), or U.S. Patent No. 4,975,526, may be implanted into a rat at a subcutaneous site, essentially following the method of Sampath et al. After three days the implant is removed, and the progenitor cells associated with the matrix dispersed and cultured.
  • Progenitor cells are incubated in vitro with a morphogenic 60A protein fragment under standard cell culture conditions well described in the art and described hereinabove. In the absence of external stimuli, the progenitor cells do not, or only minimally, proliferate on their own in culture. However, progenitor cells cultured in the presence of a morphogenically active fragment of
  • 60A protein do proliferate.
  • Cell growth can be any cell growth.
  • Embryonic Mesenchyme Differentiation Morphogenically active fragments of 60A protein can be utilized to induce cell differentiation.
  • the ability of 60A protein to induce cell differentiation can be determined by culturing early mesenchymal cells in the presence of 60A protein and then studying the histology of the cultured cells by staining with toluidine blue using standard cell culturing and cell staining methodologies well described in the art.
  • rat mesenchymal cells destined to become mandibular bone when separated from the overlying epithelial cells at stage 11 and cultured in vitro under standard tissue culture conditions, e.g., in a chemically defined, serum-free medium, containing for example, 67% DMEM (Dulbecco's modified Eagle's medium), 22% F-12 medium, 10mM Hepes pH 7, 2mM glutamine, 50 ⁇ g/ml transferring, 25 ⁇ g/ml insulin, trace elements, 2mg/ml bovine serum albumin coupled to oleic acid, with HAT (0.1 mM hypoxanthine, 10 ⁇ M
  • aminopterin 12 ⁇ M thymidine
  • Stage 11 mesenchymal cells cultured in vitro in the presence of 60A protein, e.g., 10-100 ⁇ g/ml, will continue to differentiate in vitro to form chondrocytes just as they continue to differentiate in vitro if they are cultured with the cell products harvested from the overlying endodermal cells.
  • 60A protein e.g., 10-100 ⁇ g/ml
  • This experiment may be performed with different mesenchymal cells to assess the cell differentiation capability of different morphogenically active fragments of 60A protein.
  • the ability of 60A proteins to induce osteoblast differentiation may be evaluated in vitro using primary osteoblast cultures or osteoblast-like cells lines and assaying for a variety of bone cell markers that are specific markers for the
  • differentiated osteoblast phenotype e.g., alkaline phosphatase activity, parathyroid hormone-mediated cyclic AMP (cAMP) production, osteocalcin synthesis, and enhanced mineralization rates.
  • cAMP parathyroid hormone-mediated cyclic AMP
  • PNPP paranitrosophenylphosphate
  • the long term effect of 60A morphogen on the production of alkaline phosphatase by rat osteoblasts also may be demonstrated as follows.
  • Rat osteoblasts are prepared and cultured in multi-well plates as described above.
  • six sets of 24 well plates are plated with 50,000 rat osteoblasts per well.
  • Each plate then is incubated for different lengths of time: 0 hours (control time), 24 hours, 48 hours, 96 hours, 120 hours and 144 hours. After each incubation period, the cell layer is extracted with 0.5 ml of 1% Triton X-100. The resultant cell extract is centrifuged, and alkaline phospatase activity determined as for Example 3.1, using
  • 60A protein stimulates the production of alkaline phosphatase in osteoblasts in dose-dependent manner so that increasing doses of 60A further increase the level of alkaline phosphatase production, and moreover, the 60A-stimulated elevated levels of alkaline phosphatase in the treated osteoblasts is anticipated to last for an extended period of time.
  • PNPP paranitroso-phenylphosphate
  • Rat osteoblasts are prepared and cultured in a multiwell plate as described above.
  • the cultured cells then are divided into three groups: (1) wells which receive, for example, 1.0, 10.0 and 40.0 ng 60A protein ml medium); (2) wells which receive for example, TGF- ⁇ , at 0.1, 1.0, and 5.0 ng/ml medium); and (3) a control group which receives no growth factors.
  • the plate is then incubated for another 72 hours. At the end of the 72 hours the cells are treated with medium containing 0.5% bovine serum albumin (BSA) and 1mM 3-isobutyl-1-methylxanthine for 20 minutes followed by the addition into half of the wells of human recombinant parathyroid hormone (hPTH, Sigma, St. Louis) at a concentration of 200 ng/ml for 10 minutes.
  • BSA bovine serum albumin
  • hPTH human recombinant parathyroid hormone
  • the cAMP levels then are determined using a radioimmunoassay kit (e.g., Amersham, Arlington Heights, Illinois). 60A protein alone stimulates an increase in the PTH-mediated cAMP response, and thus promotes the growth and expression of the osteoblast differentiated phenotype.
  • a radioimmunoassay kit e.g., Amersham, Arlington Heights, Illinois. 60A protein alone stimulates an increase in the PTH-mediated cAMP response, and thus promotes the growth and expression of the osteoblast differentiated phenotype.
  • Osteocalcin is a bone-specific protein synthesized by osteoblasts which plays an integral role in the rate of bone mineralization in vivo. Circulating levels of osteocalcin in serum are used as a marker for
  • Rat osteoblasts are prepared and cultured in a multi-well plate as above.
  • the medium is supplemented with 10%FBS, and on day 2, cells are fed with fresh medium supplemented with fresh 10 mM ⁇ -glycerophosphate (Sigma, Inc.). Beginning on day 5 and twice weekly thereafter, cells are fed with a complete mineralization medium containing all of the above components plus fresh L(+)-ascorbate, at a final concentration of 50 ⁇ g/ml medium.
  • 60A protein then is added to the wells directly, e.g., in 50% acetonitrile (or 50% ethanol) containing 0.1% trifluoroacetic acid (TFA), at no more than 5 ⁇ l morphogen/ml medium.
  • Control wells receive solvent vehicle only.
  • the cells then are re-fed and the conditioned medium sample diluted 1:1 in standard radioimmunoassay buffer containing standard protease inhibitors and stored at -20° C until assayed for osteocalcin.
  • Osteocalcin synthesis is measured by standard radioimmunoassay using a commercially available osteocalcin-specific antibody.
  • 60A protein stimulates osteocalcin synthesis in osteoblast cultures.
  • osteoclacin synthesis in response to 60A protein is expected to be dose dependent and to show a significant increase over the basal level after 13 days of incubation.
  • the enhanced osteocalcin synthesis also can be confirmed by detecting the elevated osteocalcin mRNA message (20-fold increase) using a rat osteocalcin-specific probe.
  • the increase in osteoclacin synthesis is expected to correlate with increased mineralization in long term osteoblast cultures as determined by the appearance of mineral nodules 60A protein is expected to increase the initial mineralization rate about 20-fold compared to untreated cultures.
  • CAMs are atypically characterized by the following properties: atypically characterized by the following properties: atypically characterized by the following properties: atypically characterized by the following properties: atypically characterized by the following properties: atypically characterized by the following properties: atypically characterized by the following properties: atypically characterized by the following properties: atypically characterized by the following properties: atypically characterized by the following CAMs.
  • N-CAMs which comprise at least 3 isoforms (N-CAM-180, N-CAM-140 and N-CAM-120, where "180", “140” and “120” indicate the apparent molecular weights of the isoforms as measured by polyacrylamide gel electrophoresis) are expressed at least transiently in developing tissues, and
  • N-CAM-180 and N-CAM-140 isoforms are expressed in both developing and adult tissue.
  • the N-CAM-120 isoform is found only in adult tissue.
  • Another neural CAM is L1.
  • the ability of 60A proteins to stimulate CAM expression can be demonstrated using the following protocol, using NG108-15 cells. NG108-15 is a
  • transformed hybrid cell line (neuroblastoma x glioma, America Type Tissue Culture (ATCC), Rockville, MD), exhibiting a morphology characteristic of transformed embryonic neurons.
  • ATCC America Type Tissue Culture
  • NG108-15 cells exhibit a fibroblastic, or minimally differentiated, morphology and express only the 180 and 140 isoforms of N-CAM normally associated with a developing cell. Following morphogen treatment these cells exhibit a morphology characteristic of adult neurons and express enhanced levels of all three N-CAM isoforms.
  • NG108-15 cells are cultured for 4 days in the presence of increasing concentrations of 60A protein using standard culturing procedures, and standard Western blots performed on whole cell
  • N-CAM isoforms are detected with an antibody which crossreacts with all three isoforms, mAb
  • H28.123 obtained from Sigma Chemical Co., St. Louis, the different isoforms being distinguishable by their different mobilities on an electrophoresis gel.
  • Control NG108-15 cells express both the 140 kDa and the 180 kDa isoforms, but not the 120 kDa, as determined by Western blot analyses using up to 100 ⁇ g of protein.
  • Treatment of NG108-15 cells with 60A protein results in a dose-dependent increase in the expression of the 180 kDa and 140 kDa isoforms, as well as the induction of the 120 kDa isoform induced.
  • CAM expression correlates with cell
  • Morphogen treatment also induces expression of another neural CAM, L1.
  • the 60A protein morphogens described herein also can induce redifferentiation of transformed cells to a morphology characteristic of untransformed cells.
  • the examples provided below detail morphogen-induced redifferentiation of a transformed human cell line of neuronal origin (NG108-15); as well as mouse
  • neuroblastoma cells N1E-115
  • human embryo N1E-115
  • carcinoma cells to a morphology characteristic of untransformed cells.
  • NG108-15 is a transformed hybrid cell line produced by fusing neuroblastoma x glioma cells (obtained from ATTC, Rockville, MD), and exhibiting a morphology characteristic of transformed embryonic neurons, e.g., having a fibroblastic
  • the cells have polygonal cell bodies, short, spike-like processes and make few contacts with neighboring cells. Incubation of
  • NG108-15 cells cultured in a chemically defined, serum-free medium, with 0.1 to 300 ng/ml of morphogen (e.g; OP-1) for four hours induces an orderly, dose-dependent change in cell morphology.
  • morphogen e.g; OP-1
  • NG108-15 cells are subcultured on poly-L-lysine coated 6 well plates. Each well contains 40-50,000 cells in 2.5 ml of chemically defined medium. On the third day, 2.5 ⁇ l of morphogen (e.g., 60A protein) in 60% ethanol containing 0.025%
  • morphogen e.g., 60A protein
  • Morphogenic 60A protein of varying concentrations may be tested
  • Morphogenic 60A protein induces a dose-dependent redifferentiation of the transformed cells, including a rounding of the soma, an increase in phase brightness, extension of the short neurite processes, and other significant changes in the cellular ultrastructure. After several days treated cells will begin to form epithelioid sheets that then become highly packed, multi-layered aggregates, as determined visually by microscopic examination.
  • morphogen-induced redifferentiation occurs without any associated changes in DNA synthesis, cell division, or cell viability, making it unlikely that the morphologic changes are secondary to cell differentiation or a toxic effect of the morphogen.
  • the morphogen-induced redifferentiation does not inhibit cell division, as determined by
  • 3 H-thymidine uptake unlike other molecules which have been shown to stimulate differentiation of transformed cells, such as butyrate, DMSO, retanoic acid or
  • 60A protein maintains cell stability and viability after inducing redifferentiation.
  • the 60A protein morphogens described herein are described herein
  • neoplasias and neoplastic lesions of the nervous system particularly in the treatment of neuroblastomas, including retinoblastomas, and gliomas.
  • the effect of 60A protein fragments on human EC cells may be determined.
  • human EC cells epio carcinoma cells, e.g., NTERA-Z CL.D1, ATCC, Rockville, MD
  • these cells can be maintained as undifferentiated stem cells, and can be induced to grow in serum free media (SFM).
  • SFM serum free media
  • the cells proliferate rampantly and are anchorage-independent.
  • morphogen EC cells grow as flattened cells, becoming anchorage dependent and forming
  • the cells are induced to differentiate.
  • varying concentrations of 60A protein e.g., 0-300 ng/ml
  • cultured cells e.g., 40-50,000 cells in 2.5 ml chemically defined medium
  • 60A protein stimulates redifferentiation of these cells to a morphology characteristic of untransformed embryo cells.
  • Morphogenically active fragments of 60A protein also may be used to maintain a cell's differentiated phenotype. This application is particularly useful for inducing the continued expression of phenotype in senescent or quiescent cells. 5.1 In vitro Model for Phenotypic Maintenance
  • morphogens The phenotypic maintenance capability of morphogens is assessed readily. A number of differentiated cells become senescent or quiescent after multiple passages in vitro under standard tissue culture conditions such well described in the art, e.g.. Culture of Animal Cells: A Manual of Basic Techniques (R. Freshney, ed., Wiley, 1987). However, if these cells are cultivated in vitro in association with a morphogen such as 60A protein, cells are stimulated to maintain expression of their phenotype through multiple passages. For
  • the alkaline phosphatase activity of cultured osteoblasts is significantly reduced after multiple passages in vitro.
  • the cells are cultured osteosarcoma cells and calvaria cells.
  • alkaline phosphatase activity is maintained over extended periods of time.
  • osteoblasts are cultured as described in Example 2. The cells are divided into groups,
  • Osteoblasts cultured in the absence of 60A protein have a reduced alkaline phosphatase activity, as compared to 60A protein-treated cells.
  • Phenotypic maintenance capability also may be assessed in vivo, using a rat model for osteoporosis, as disclosed in international application US 92/07432 (WO 93/05751).
  • Long Evans female rats (Charles River Laboratories, Wilmington, MA) are ovariectomized using standard surgical techniques, to produce an osteoporotic condition resulting from decreased estrogen production.
  • Eight days after ovariectomy rats are systemically provided with phosphate buffered saline (PBS) or morphogen, (e.g., 60A protein, 2-20 ⁇ g) for 21 days.
  • PBS phosphate buffered saline
  • morphogen e.g., 60A protein, 2-20 ⁇ g
  • 60A protein is comparable to that of the sham-operated (e.g., nonovarectomized) rats.
  • Progenitor cells may be stimulated to proliferate in vivo or ex vivo.
  • the cells may be stimulated in vivo by injecting or otherwise providing a sterile preparation containing the morphogenically active fragment of 60A protein into the individual.
  • the hemopoietic pluripotential stem cell population of an individual may be stimulated to proliferate by injecting or otherwise providing an appropriate concentration of the morphogenically active fragment of 60A protein to the individual's bone marrow.
  • Progenitor cells may be stimulated ex vivo by contacting progenitor cells of the population to be enhanced with a morphogenically active fragment of 60A protein under sterile conditions at a concentration and for a time sufficient to stimulate proliferation of the cells. Suitable concentrations and stimulation times may be determined empirically, essentially following the procedure described in Example 2, above. A morphogen concentration of between about
  • the stimulated cells then are provided to the
  • biocompatible progenitor cells may be obtained by any of the methods known in the art or described hereinabove.
  • the morphogenically active fragments of Drosophila 60A protein may be used to repair diseased or damaged mammalian tissue.
  • the tissue to be repaired preferably is assessed first, and excess necrotic or interfering scar tissue removed as needed, e.g., by ablation or by surgical, chemical or other methods known in the medical arts.
  • the 60A protein then may be provided directly to the tissue locus as part of a sterile, biocompatible composition, either by surgical implantation or
  • the morphogen also may be provided systemically, as by oral or parenteral administration.
  • morphogenically active fragment of 60A protein may be provided to the tissue locus.
  • the existing tissue at the locus whether diseased or damaged, provides the appropriate matrix to allow the proliferation and tissue-specific differentiation of progenitor cells.
  • a damaged or diseased tissue locus particularly one that has been further assaulted by surgical means, provides a morphogenically permissive environment. Systemic provision of a morphogenically active fragment of 60A protein will be sufficient for certain applications (e.g., in the treatment of
  • osteoporosis and other disorders of the bone remodeling cycle are examples.
  • the tissue may not be capable of providing a sufficient matrix for cell influx and proliferation. In these instances, it may be necessary to provide the 60A protein or progenitor cells
  • the matrix preferably is in vivo biodegradable.
  • the matrix also may be tissue-specific and may comprise porous particles having dimensions within the range of 70-850 ⁇ m, most preferably 150-420 ⁇ m.
  • the morphogenic 60A protein also may be used to prevent or substantially inhibit immune/inflammatory response mediated tissue damage and scar tissue formation following an injury.
  • 60A protein is provided to a newly injured tissue locus, to induce tissue morphogenesis at the locus, preventing the aggregation of migrating fibroblasts into non-differentiated connective tissue.
  • the 60A protein fragment preferably is provided as a sterile pharmaceutical preparation injected into the tissue locus within five hours of the injury. Below are several examples, describing protocols for assessing 60A protein-induced tissue morphogenesis in bone, liver, nerve, dentin, cementum and periodontal ligament. 7.1 60A Protein-Induced Bone Morphogenesis
  • a particularly useful mammalian tissue model system for demonstrating and evaluating the morphogenic activity of a protein is the endochondral bone tissue morphogenesis model known in the art and described, for example, in U.S. Pat. No. 4,968,590 and incorporated herein by reference.
  • endochondral bone formation includes the ability to induce the proliferation and subsequent differentiation of progenitor cells into chondroblasts and osteoblasts, the ability to induce cartilage matrix formation, cartilage calcification, and bone remodeling, and the ability to induce formation of an appropriate vascular supply and hematopoeitic bone marrow differentiation.
  • the local environment in which the morphogenic material is placed is important for tissue
  • local environment is understood to include the tissue structural matrix and the environment surrounding the tissue. For example, in addition to needing an appropriate anchoring
  • the fragments and compositions may be injected or surgically implanted in a mammal, following any of a number of procedures well known in the art. For example, surgical implant bioassays may be performed essentially following the procedure of Sampath et al. (1983) PNAS 80:6591-6595 and U.S. Pat No. 4,968,590.
  • Histological sectioning and staining is preferred to determine the extent of morphogenesis in vivo, particularly in tissue repair procedures.
  • Excised implants are fixed in Bouins Solution, embedded in paraffin, and cut into 6-8 ⁇ m sections. Staining with toluidine blue or hemotoxylin/eosin demonstrates clearly the ultimate development of the new tissue. Twelve day implants are usually sufficient to determine whether the implants contain newly induced tissue. Successful implants exhibit a controlled
  • the stages include:
  • cartilage calcification on day eight cartilage calcification on day eight
  • appearance of osteoclast and the commencement of bone remodeling and dissolution of the implanted matrix on days twelve to eighteen hematopoietic bone marrow
  • tissue markers may be used as markers for tissue
  • Useful markers include tissue-specific enzymes whose activities may be assayed (e.g.,
  • alkaline phosphatase activity may be used as a marker for osteogenesis.
  • incorpororation of systemically provided morphogenic fragments of 60A protein may be followed using tagged fragments (e.g., radioactively labelled) and
  • the 60A protein also may be provided with a tissue-specific molecular tag, whose uptake may be monitored and correlated with the
  • concentration of the 60A protein fragment provided.
  • ovary removal in female rats results in reduced bone alkaline phosphatase activity and renders the rats predisposed to osteoporosis (as described in Example 5).
  • the female rats now are provided with a 60A protein a reduction in the systemic concentration of calcium may be seen, which correlates with the presence of the provided 60A protein and which is anticipated to correspond with increased alkaline phosphatase activity.
  • both Drosophila proteins, DPP and 60A protein can induce the formation of new cartilage, bone and bone marrow at non-bony sites in mammals.
  • DPP or 60A protein was reconstituted with rat collagen carrier by the 50% acetonitrile/0.1% TFA lyophilization method as described.
  • 25 mg of 4 M guanidine HCl-extracted demineralized rat collagenous matrix (rat collagen carrier) was added to varying concentrations of protein dissolved in 200 ⁇ l of 50% acetonitrile/0.1% TFA, vortexed and then
  • Rat collagen carrier alone was the negative control and intact demineralized bone matrix was the positive control.
  • the day of implantation was designated as day 0 of the assay. Implants were removed on day 12 and bone forming activity in the implants was monitored by the specific activity of alkaline phosphatase and calcium content as described above. Values are the average of four to six
  • Bone formation was calculated as described in U.S. Pat. No. 4,968,590. Specifically, one bone forming unit represents the amount of protein needed for half maximal bone forming activity of the implant on day 12. The bone forming activity elicited by intact bone matrix is considered to be the maximal bone differentiation activity for comparison purposes in
  • morphogenesis of substantially injured liver tissue following a partial hepatectomy utilizing a morphogenic 60A protein is presented. Variations on this general protocol may be used to test morphogen activity of
  • the general method involves excising an essentially
  • liver has a potential to regenerate upon injury during post-fetal life.
  • the morphogenic 60A protein e.g., 1 mg/ml, in a biocompatible solution, for example, (e.g., a purified recombinant mature form, is solubilized in 50% ethanol (or compatible solvent) containing 0.1% trifluoroacetic acid (or compatible acid).
  • the mature protein may be solubilized by association with a pro domain.
  • the injectable 60A protein solution is prepared, e.g., by diluting one volume of 60A protein solvent-acid stock solution with 9 volumes of 0.2% rat serum albumin in sterile PBS (phosphate-buffered saline).
  • 60A protein injected may be, e.g., 100 ⁇ g in 1000 ⁇ l of PBS/RSA (phosphate buffered saline/rat serum albumin) injection buffer. Placebo samples are injection buffer only. In experimental assays, five rats in each group preferably are used. The wound is closed and the rats are allowed to eat normal food and drink tap water.
  • PBS/RSA phosphate buffered saline/rat serum albumin
  • the 60A protein fragment-injected group shows, e.g., complete liver tissue regeneration with no sign remaining of any cut in the liver.
  • the control group into which only PBS is injected shows only minimal regeneration with the incision remaining in the sample.
  • morphogens e.g., OP-1
  • Cynomolgus monkeys are chosen as primate models as monkeys are presumed to be more indicative of human dental biology than models based on lower non-primate mammals.
  • Pulp treatments used may include: a
  • a carrier matrix e.g., demineralized, gunaidine-exracted pulverized bone collagen particles, prepared, for example, as described in U.S. Pat. No. 4,975,526); carrier matrix alone, and no treatment (PBS). Twelve teeth per animal (four for each
  • demineralized teeth and 60A protein into surgically prepared canine tooth sockets are anticipated to stimulate cementum and periodontal ligament formation, as well as new bone tissue.
  • 60A protein may be demonstrated using a rat brain stab model. Briefly, male Long Evans rats are anesthetized and the head area prepared for surgery. The calvariae is exposed using standard surgical procedures and a hole drilled toward the center of each lobe using a 0.035K wire, just piercing the calvariae. 25 ⁇ l solutions containing either morphogen (e.g., 60A protein, 25 ⁇ g in PBS) or PBS alone then is provided to each of the holes by Hamilton syringe. Solutions are delivered to a depth approximately 3 mm below the surface, into the underlying cortex, corpus callosum and hippocampus. The skin then is sutured and the animal allowed to recover.
  • morphogen e.g., 60A protein, 25 ⁇ g in PBS
  • glial fibrillary acidic protein a marker protein for glial scarring, to qualitatively determine the degree of scar formation. Sections also are probed with 60A protein-specific antibody to determine the presence of the protein. Reduced levels of glial fibrillary acidic protein are seen in the tissue sections of animals treated with 60A protein evidencing the ability of the morphogen to inhibit glial scar formation, thereby stimulating nerve regeneration.
  • 60A protein to stimulate peripheral nervous system axonal growth over extended distances may be demonstrated using the following model. Neurons of the peripheral nervous system can sprout new
  • morphogens e.g., OP-1
  • 60A protein stimulation of nerve regeneration is assessed using the rat sciatic nerve model.
  • the rat sciatic nerve can regenerate
  • avascular intermuscular plane between vastus lateralis and hamstring muscles are entered and followed to the loose fibroareolar tissue surrounding the sciatic nerve.
  • the loose tissue is divided longitudinally thereby freeing the sciatic nerve over its full extent without devascularizing any portion.
  • the sciatic nerves are transected with microscissors at mid-thigh and grafted with a 60A protein gel graft that separates the nerve stumps by 12 mm.
  • the graft region is encased in a silicone tube 20 mm in length with a 1.5 mm inner diameter, the interior of which is filled with the morphogen
  • the central 12 mm of the tube consists of an 60A protein gel prepared by mixing 1 to 5 ⁇ g of substantially pure recombinately produced 60A protein with approximately 100 ⁇ l of MATRIGELTM (from Collaborative Research, Inc., Bedford, MA), an
  • extracellular matrix extract derived from mouse sarcoma tissue, and containing solubilized tissue basement membrane, including laminin, type IV collagen, heparin sulfate, proteoglycan and entactin, in phosphate-buffered saline.
  • the morphogen-filled tube then is implanted directly into the defect site, allowing 4 mm on each end to insert the nerve stumps. Each stump is abutted against the morphogen gel and is secured in the silicone tube by three stitches of commercially

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Abstract

L'invention se rapporte à des procédés d'utilisation d'un fragment morphogéniquement actif de la protéine 60A pour induire la morphogénèse tissulaire, ainsi qu'à des procédés d'accroissement de la population des cellules souches chez un mammifère, à des procédés de stimulation des cellules souches pour différencier et maintenir leur phénotype différencié in vivo ou in vitro, à des procédés d'induction de la croissance spécifique des tissus in vivo, et à des procédés de remplacement des tissus malades ou endommagés in vivo.
PCT/US1993/008741 1992-09-15 1993-09-15 Morphogenese induite par la proteine 60a WO1994006447A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999047156A2 (fr) * 1998-03-14 1999-09-23 Creative Biomolecules, Inc. Compositions pour moduler la differentiation des cellules comprenant un lipide et un morphogene

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993004692A1 (fr) * 1991-08-30 1993-03-18 Creative Biomolecules, Inc. Modulation, induite par morphogene, de reaction inflammatoire
WO1993005751A2 (fr) * 1991-08-30 1993-04-01 Creative Biomolecules, Inc. Proteines osteogeniques pour le traitement de maladies osseuses
WO1994003200A1 (fr) * 1992-07-31 1994-02-17 Creative Biomolecules, Inc. Regeneration et reparation du systeme nerveux induites par morphogene

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993004692A1 (fr) * 1991-08-30 1993-03-18 Creative Biomolecules, Inc. Modulation, induite par morphogene, de reaction inflammatoire
WO1993005751A2 (fr) * 1991-08-30 1993-04-01 Creative Biomolecules, Inc. Proteines osteogeniques pour le traitement de maladies osseuses
WO1994003200A1 (fr) * 1992-07-31 1994-02-17 Creative Biomolecules, Inc. Regeneration et reparation du systeme nerveux induites par morphogene

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PROC. NATL. ACAD. SCI. USA vol. 88, no. 20 , 1991 pages 9214 - 9218 K.A.WHARTON 'Drosophila 60A gene, another transforming growth factor beta family member, is closely related to human bone morphogenetic proteins.' *
PROC. NATL. ACAD. SCI. USA vol. 90, no. 13 , July 1993 pages 6004 - 6008 T.K. SAMPATH 'Drosophila transforming growth factor beta superfamily proteins induce endochondral bone formation in mammals.' *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999047156A2 (fr) * 1998-03-14 1999-09-23 Creative Biomolecules, Inc. Compositions pour moduler la differentiation des cellules comprenant un lipide et un morphogene
WO1999047156A3 (fr) * 1998-03-14 1999-12-29 Creative Biomolecules Inc Compositions pour moduler la differentiation des cellules comprenant un lipide et un morphogene

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