WO1998027995A1 - Traitement local du myocarde mammifere avec un morphogene, ou avec des cellules precurseurs myogeniques morphogeniquement traitees - Google Patents

Traitement local du myocarde mammifere avec un morphogene, ou avec des cellules precurseurs myogeniques morphogeniquement traitees Download PDF

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WO1998027995A1
WO1998027995A1 PCT/US1997/023611 US9723611W WO9827995A1 WO 1998027995 A1 WO1998027995 A1 WO 1998027995A1 US 9723611 W US9723611 W US 9723611W WO 9827995 A1 WO9827995 A1 WO 9827995A1
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Charles M. Cohen
Kuber T. Sampath
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Creative Biomolecules, Inc.
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Priority to EP97953356A priority Critical patent/EP0952845A1/fr
Priority to JP52899898A priority patent/JP2001507354A/ja
Priority to AU57119/98A priority patent/AU741350B2/en
Priority to CA002275436A priority patent/CA2275436A1/fr
Publication of WO1998027995A1 publication Critical patent/WO1998027995A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/02—Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
    • C12N5/0659—Satellite cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor

Definitions

  • the present invention relates generally to methods and preparations for the treatment of mammals, including humans, at risk of, or afflicted with, loss of or damage to myocardium.
  • the methods involve the implantation of mammalian myogenic precursor cells treated with certain morphogens, inducers of those morphogens, agonists of the corresponding morphogen receptors, or with small molecule morphogenic activators.
  • myocardium arises by end- to-end fusion of myogenic precursor cells to form branched myofibers in which individual cardiac myocytes are joined by intercalated disks.
  • the myogenic precursor cells which give rise to the myocardium are derived from the splanchic mesoderm, which is derived from the lateral mesodermal mesenchyme which, in turn, arises from the mesoderm formed after gastrulation.
  • mammalian skeletal muscle has much greater capacity for growth and regeneration, even in adulthood.
  • skeletal muscle has its first origins after the induction of the mesoderm.
  • the dorsal mesodermal mesenchyme differentiates to form myotomes which, in turn, differentiate to form the myogenic precursor cells which ultimately form skeletal muscle.
  • the skeletal muscle precursors fuse side-to-side to form unbranched, multinucleated myofibers.
  • some portion of the skeletal myogenic precursor cells do not differentiate into myocytes but, rather, attach to the plasmalemmas of the myocytes.
  • satellite cells may remain, throughout adulthood, as largely undifferentiated, quiescent skeletal muscle "satellite cells.”
  • these satellite cells Upon injury of a skeletal muscle, however, these satellite cells are revealed to be myogenic precursor cells, or muscle “stem cells,” which proliferate and differentiate into new and functional skeletal muscle. Even after injury, however, a portion of the proliferated satellite cells remain undifferentiated and attach to the newly formed myofibers.
  • the satellite cells of skeletal muscle provide a constant and renewable source of myogenic precursor cells which allows for skeletal muscle repair and regeneration throughout mammalian life.
  • the proliferation and differentiation of skeletal muscle satellite cells has been extensively studied in vitro.
  • a simple saline extract of skeletal muscle has been shown to cause satellite cells to proliferate in culture (Bischoff (1989) in Myoblast Transfer Therapy. Griggs and Karpati, eds., pp. 147-158).
  • chick embryo extract or the conditioned medium of differentiated myotubes from young mice exhibits a strong mitogenic effect on satellite cells, but that conditioned medium from older murine myotubes has a lesser effect (Mezzogiorno et al. (1993) Mech. Ageing & Develop. 70:35-44).
  • TGF- ⁇ i is widely believed to inhibit satellite cell proliferation, as does contact with the myofiber plasmalemma, but not the basal lamina (Bischoff (1989); but see Hathaway et al. (1991) J. Cell Physiol. 146:435-441).
  • skeletal muscle satellite cells proliferated in vitro, may be able to serve as a source of myogenic precursor cells for muscle restoration or regeneration therapy.
  • mouse fetal cardiomyocytes which are not terminally differentiated and retain the ability to divide, have been directly injected into the myocardium of a syngeneic adult mouse, and have been shown to form new and apparently functional myocardium (Soonpaa et al. (1994) Science 264:98-101).
  • a great many proteins have now been identified which appear to act as morphogenetic or growth factors, regulating cell proliferation and/or differentiation. Typically these growth factors exert their effects on specific subsets of cells and/or tissues. Thus, for example, epidermal growth factors, nerve growth factors, fibroblast growth factors, various hormones, and many other proteins inducing or inhibiting cell proliferation or differentiation have been identified and shown to affect some subset of cells or tissues.
  • morphogens includes members of the family of bone morphogenetic proteins (BMPs) which were initially identified by their ability to induce ectopic, endochondral bone morphogenesis Subsequent characterization of the nucleic acid and amino acid sequences of the BMPs has shown them to be a subgroup of the TGF ⁇ superfamily of growth and differentiation factors
  • BMP7 mammalian osteogenic proteinl
  • OP2 osteogenic protein2
  • OP3 osteogenic protein3
  • BMP2 also known as BMP2A or CBMP2A
  • BMP3, BMP4 also known as BMP2B or CBMP2B
  • BMP5 BMP6, Vgrl and GDF1
  • Members of this family encode secreted polypeptides that share common structural features
  • BMP-2 The members of the morphogen family of proteins are expressed naturally in a variety of tissues during development BMP-2 (l e , BMP-2 A), for example, is expressed in embryonic mouse hair follicles, cartilage and bone (Lyons et al (1989) Genes & Develop 3 1657-1668), BMP3 has been shown to be most highly expressed in human embryonic lung and kidney, highly expressed in intestinal mucosa and skeletal tissues such as the perichondrium and periosteum, expressed in brain, but undetectable in embryonic heart and liver (Vukicevic et al (1994) J_ Histochem Cytochem 42 869-875), BMP4 has been shown to be expressed in the developing limbs, heart, facial processes and condensed mesenchyme associated with early whisker follicles in embryonic mice (Jones, et al (1991) Development 111 531-542), and OPl (BMP7) has been shown immunohistochemically to be present in human embryos in
  • the present invention is directed to methods of treatment, and pharmaceutical preparations for use in the treatment, of mammalian subjects at risk of, or afflicted with, loss of or damage to myocardium
  • Such subjects include subjects already afflicted with the loss of myocardial tissue, such as those which have already suffered a myocardial infarction, physical trauma to the heart (e g , in an automobile accident, or those already suffering from congestive heart failure, as well as subjects reasonably expected to suffer from myocardial infarction or congestive heart failure Whether a particular subject is at risk is a determination which may routinely be made by one of ordinary skill in the relevant medical or veterinary art
  • myogenic precursor cells are implanted into a mammal at a site at risk of, or afflicted with, loss of or damage to myocardium, and the myogenic precursor cells are morphogemcally-treated prior to, simultaneously with, or subject to implantation
  • morphogemcally-treated mammalian myogenic precursor cells may be implanted into a mammalian heart at the site of a myocardial mfarct, or into the damaged or weakened myocardium of a subject with congestive heart failure
  • the mammalian myogenic precursor cells may be derived from skeletal muscle (e g , skeletal muscle satellite cells), from embryonic tissue (e g , embryonic mesodermal mesenchyme) or from a myogenic precursor cell line maintained in vitro
  • the myogenic precursor cells may be derived from a donor (e g , a tissue-type matched donor, sibling, identical twin, or fetus), may be derived
  • the morphogenic treatment of the implanted cells may include treatment of the cells with a morphogen, morphogen inducer, agonist of a morphogen receptor, or small molecule morphogenic activator prior to implantation, simultaneously with implantation, or subsequent to implantation.
  • the present invention is further directed to methods of promoting the proliferation and differentiation of mammalian myogenic precursor cells in vivo or in vitro.
  • myogenic precursor cells isolated from mammalian skeletal muscle tissue, embryonic myogenic precursor cells, or myogenic precursor cell lines may be stimulated to proliferate by treatment with a morphogen, an inducer of a morphogen, an agonist of a morphogen receptor, or a small molecule morphogenic activator.
  • mammalian myogenic precursor cells may be stimulated to differentiate into myocytes, particularly myocytes which express markers of myocardial tissue, in a morphogenically permissive environment.
  • the present invention is further directed to therapeutic preparations comprising isolated mammalian myogenic precursor cells and an amount of a morphogen, inducer of a morphogen, agonist of a morphogen receptor, or small molecule morphogenic activator sufficient to promote proliferation or differentiation of the myogenic precursor cells in a morphogenically permissive environment.
  • compositions of the present invention capitalize in part upon the fact that certain proteins of eukaryotic origin, defined herein as morphogens, may be used to treat myogenic precursor cells such that, when these morphogenically-treated myogenic precursor cells are present in a morphogenically permissive environment, they may migrate, proliferate and/or differentiate so as to form new and functional myocardium.
  • morphogens proteins of eukaryotic origin
  • the present invention is based in part upon the fact that treatment of myogenic precursor cells with these morphogens enhances or increases the probability, rate, or efficiency with which these cells migrate, proliferate and/or differentiate into new and functional myocardium in a morphogenically permissive environment.
  • morphogenically-treated myogenic precursor cells may be used to restore or regenerate lost or damaged myocardium in a mammal, or to prophylactically treat a mammal at risk of such loss or damage.
  • the present invention is novel in that myocardial tissue is believed to lack a sufficient number of myogenic precursor cells for adequate regeneration or repair of lost or damaged tissue and, therefore, the ability of the morphogens to promote the migration, proliferation and/or differentiation of myogenic precursor cells (e g , skeletal muscle satellite cells) into functional myocardium is unexpected
  • the morphogen is a dime ⁇ c protein comprising a pair of folded polypeptides, each having an ammo acid sequence that shares a defined relationship with an amino acid sequence of a reference morphogen
  • Preferred morphogen polypeptides share a defined relationship with a sequence present in morphogenically active human OP-1 (SEQ ID NO 4)
  • any one or more of the naturally occurring or biosynthetic sequences disclosed herein similarly could be used as a reference sequence
  • Preferred morphogen polypeptides share a defined relationship with at least the C-termmal six cysteine domain of human OP-1 (residues 43- 139 of SEQ ID NO 4)
  • morphogen polypeptides share a defined relationship with at least the C-terminal seven cysteine domain of human OP-1 (residues 38-139 of SEQ ID NO 4) That is, preferred morphogen polypeptides in a dime ⁇ c protein with morphogenic activity each comprise a sequence that corresponds to
  • Functionally equivalent sequences include functionally equivalent arrangements of cysteine residues disposed within the reference sequence, including amino acid insertions or deletions which alter the linear arrangement of these cysteines, but do not materially impair their relationship in the folded structure of the dime ⁇ c morphogen protein, including their ability to form such intra- or inter-chain disulfide bonds as may be necessary for morphogenic activity
  • Functionally equivalent sequences further include those wherein one or more amino acid residues differs from the corresponding residue of a reference morphogen sequence, e g , the C-terminal seven cysteine domain (also referred to herein as the conserved seven cysteine skeleton) of human OP-1, provided that this difference does not destroy morphogenic activity
  • conservative substitutions of corresponding amino acids in the reference sequence are preferred
  • Amino acid residues that are "conservative substitutions" for corresponding residues in a reference sequence are those that are physically or functionally similar to the corresponding reference residues, e g , that have similar size, shape, electric charge, chemical properties including the ability to form covalent or hydrogen bonds, or the like
  • Particularly preferred conservative substitutions are those fulfilling the criteria defined for an "accepted point mutation" m Dayhoff, et al (1978) Atlas of Protein Sequence and Structure, 5 Suppl 3, ch 22 (pp 354- 352), Natl Biomed Res Found , Washington, D C 20007, the teachings
  • a polypeptide suspected of being functionally equivalent to a reference morphogen polypeptide is aligned therewith using the method of Needleman, et al (1970) J Mol Biol 48 443-453, implemented conveniently by computer programs such as the Align program (DNAstar, Inc )
  • Align program DNAstar, Inc
  • internal gaps and ammo acid insertions in the candidate sequence are ignored for purposes of calculating the defined relationship, conventionally expressed as a level of amino acid sequence homology or identity, between the candidate and reference sequences
  • Amino acid sequence homology is understood herein to include both amino acid sequence identity and similarity Homologous sequences share identical and/or similar amino acid residues, where similar residues are conservative substitutions for, or "allowed point mutations" of, corresponding ammo acid residues in an aligned reference sequence
  • a candidate polypeptide sequence that shares 70% amino acid homology with a reference sequence is one in which any 70% of the aligned residues are either identical to, or are conservative substitutions of, the corresponding
  • morphogen inducer is a compound that stimulates the production (l e , transcription, translation, and/or secretion) of morphogen by a cell competent to produce and/or secrete a morphogen encoded within the genome of the cell Endogenous or administered morphogens can act as endocrine, parac ⁇ ne or autoc ⁇ ne factors Therefore, an inducer of a morphogen may stimulate endogenous morphogen by the cells in which the morphogenetic responses are induced, by neighboring cells in vivo or in vitro (e g , in tissue culture) or by cells of a distant tissue in vivo (in which case the secreted morphogen is transported to the site of morphogenesis, e g , by the individual's bloodstream) In preferred embodiments, the inducer stimulates expression and/or secretion of a morphogen so as to increase
  • an inducer of a morphogen may administered locally or systemically to induce morphogen production by the myogenic precursor cells themselves, or by neighboring or distant cells in a mammal's body.
  • an agent which acts as an agonist of a morphogen receptor may be administered instead of the morphogen itself.
  • An "agonist" of a receptor is a compound which binds to the receptor, and for which the result of such binding is similar to the result of binding the natural, endogenous ligand of the receptor. That is, the compound must, upon interaction with the receptor, produce the same or substantially similar transmembrane and/or intracellular effects as the endogenous ligand.
  • an agonist of a morphogen receptor binds to the receptor and such binding has the same or a functionally similar result as morphogen binding (e.g., induction of morphogenesis).
  • the activity or potency of an agonist can be less than that of the natural ligand, in which case the agonist is said to be a "partial agonist,” or it can be equal to or greater than that of the natural ligand, in which case it is said to be a "full agonist.”
  • a small peptide or other molecule which can mimic the activity of a morphogen in binding to and activating the morphogen's receptor may be employed as an equivalent of the morphogen.
  • the agonist is a full agonist, but partial morphogen receptor agonists may also be advantageously employed.
  • Methods of identifying such agonists are known in the art and include assays for compounds which induce morphogen-mediated responses (e.g., induction of differentiation of metanephric mesenchyme, induction of endochondral bone formation, and the like).
  • Such an agonist may also be referred to as a morphogen "mimic,” “mimetic,” or “analog.”
  • a small molecule morphogenic activator may be administered instead of the morphogen itself to promote the migration, proliferation, and/or differentiation of myogenic precursor cells by increasing the level of expression of proteins associated with myocardial phenotype.
  • Exemplary methods comprise introducing a small molecule morphogenic activator that regulates some portion or portions of a morphogen-induced regulatory pathway, resulting in an effective increase in expression or activity of myocardium- specific protein. This may result either from stimulating an increase in the endogenous expression of such protein or from a decrease in the inhibition of normal expression of such protein.
  • a small molecule morphogenic activator may act at the type I or type II morphogen receptor; or at the serine/threonine kinase, or other kinase domains of those receptors.
  • Another target of pathway activation is the Smad proteins, including the monomeric, dimeric (including heteromeric and homomeric complexes) or trimeric forms (including heteromeric and homomeric complexes).
  • a small molecule morphogenic activator may lead to activation of a transcription factor (for example, the X-protein shown in Figure 2) that causes phenotype-specific gene expression (i.e., expression of protein characteristic of myocardium).
  • the morphogens, morphogen inducers, agonists of morphogen receptors, or small molecule morphogenic activators are directly contacted with the myogenic precursor cells in solution either in vitro prior to implantation, in vivo at the time of implantation, or in vivo subsequent to implantation.
  • the morphogens, morphogen inducers, agonists of morphogen receptors may be administered by any route which is compatible with the selected agent, and may be formulated with any pharmaceutically acceptable carrier appropriate to the route of administration.
  • Preferred systemic routes of administration are parenteral and, in particular, intravenous and intraperitoneal.
  • the present invention provides pharmaceutical compositions comprising a morphogen, or morphogen inducer, agonist of a morphogen receptor, or small molecule morphogenic activator in combination with one or more of a "muscle extract,” conditioned medium from differentiated myotubes grown in culture, bFGF, IGF, PDGF, LIT, ACTH, MSH, or G-CSF.
  • a morphogen, or morphogen inducer, agonist of a morphogen receptor, or small molecule morphogenic activator in combination with one or more of a "muscle extract,” conditioned medium from differentiated myotubes grown in culture, bFGF, IGF, PDGF, LIT, ACTH, MSH, or G-CSF.
  • Panels 1-1 through 1-12 of this figure are a tabular alignment of the amino acid sequences of various naturally occurring morphogens with a preferred reference sequence of human OPl, residues 38-139 of SEQ ID NO: 4. Morphogen polypeptides shown in this figure also are identified in the Sequence Listing.
  • Figure 2 is a schematic representation of a morphogen-activated regulatory pathway for expression of a phenotype-specific gene.
  • a subject preferably a mammal, e.g., a human
  • a subject is said to be at risk of, or afflicted with, loss of or damage to myocardium, if the subject has suffered a loss of functional myocardial tissue which is clinically detectable in terms of reduced or altered cardiac function, or if the subject may reasonably be expected to suffer such a loss.
  • Subjects at risk of, or afflicted with, loss of or damage to myocardium include, but are not limited to, subjects which have already suffered a myocardial infarction, which have suffered a physical trauma to the heart (e.g., in an automobile accident) which has reduced cardiac function, or which have already been diagnosed with congestive heart failure; as well as subjects which can reasonably be expected to suffer a myocardial infarction or congestive heart failure. Whether a particular subject is at risk is a determination which may routinely be made by one of ordinary skill in the relevant medical or veterinary art.
  • Myogenic precursor cells refers to cells capable of myogenesis, or the process of proliferation and differentiation into new and functional muscle when present in a morphogenically permissive environment. Myogenic precursor cells are variously referred to in the literature as “myoblasts,” “muscle stem cells” or “satellite cells.”
  • Morphogenically permissive environment is an environment which allows or promotes the differentiation of cells into a specific cell type or types.
  • a “morphogenically permissive environment” is, therefore, sufficiently free of inhibitors of cell differentiation to allow or promote cell differentiation.
  • a morphogenically permissive environment is one which provides signals (e.g., through cell-cell contact, cell-extracellular matrix contact, or diffusible factors) which allow or promote a pluripotent cell to follow a particular morphogenic pathway.
  • a morphogenically permissive environment includes an environment of intact or damaged myocardial tissue which provides signals to myogenic precursor cells which allow or promote the differentiation of those cells into new and functional myocardium. It is known, for example, that myogenic precursor cells differentiate into myocytes at least partly in response to contact with the plasmalemma of a myofiber The presence of myofiber plasmalemmas, therefore, may be one element of a morphogenically permissive environment for myogenesis Similarly, electrical or biochemical stimuli from nerves, as well as a variety of growth factors (see below), appear to be elements of a morphogenically permissive environment for myogenesis Thus, a morphogenically permissive environment may include one or more of these elements
  • the present invention depends, in part, upon the surprising discovery that morphogenically-treated mammalian myogenic precursors cells, when implanted in vivo at a site of lost or damaged mammalian myocardium, undergo a process of proliferation and/or differentiation to produce new and functional mammalian myocardium, thereby restoring or regenerating the lost or damaged tissue in whole or in part
  • This result is particularly unexpected in light of the fact that mammalian myocardial tissue is believed to lack a sufficient number of myogenic precursor cells for adequate regeneration or repair of lost or damaged tissue and, therefore, mammalian myocardium previously has been believed to be a poor responder for functional restoration or regeneration after tissue loss or damage
  • the present invention depends, in part, upon the surprising discovery that non-myocardial cells, such as those obtained from mammalian skeletal muscle or embryonic myogenic precursor cells, may be induced to proliferate and differentiate into myocardium in a morphogenically permissive environment It
  • the morphogens, morphogen inducers, agonists of morphogen receptors, or small molecule morphogenic activators may promote the proliferation of myogenic precursor cells and render them more susceptible to differentiation into new and functional myocardium when implanted in a morphogenically permissive environment
  • the morphogens, morphogen inducers, agonists of morphogen receptors, or small molecule morphogenic activators may increase the plu ⁇ potentiality of these myogenic precursor cells, such that they may "switch fates" and, rather than differentiating only into smooth or skeletal muscle, they may proliferate and then differentiate into new and functional myocardium B Isolating and Culturmg Mammalian Myogenic Precursor Cells
  • myogenic precursor cells may be obtained, as further described in the examples below, by dissociation of skeletal muscle and subsequent culturmg of the satellite cells
  • myogenic precursor cells may be obtained from embryonic tissues, where they arise as fetal myoblasts from the myotomes of the somites, after induction of the mesoderm
  • Myogenic precursor cells may also be obtained from cell lines, such as a plu ⁇ potent mesodermal mesenchyme cell line or a partially dedifferentiated laboratory cell line, which may be induced to differentiate into myoblasts after implantation into a morphogenically permissive environment See, generally, Hathaway, et al (1991) J Cell Physiol 146 435-441, Mezzogiorno et al (1993) Mech Ageing & Develop 70 35-44, Alameddine and Fardeau (1989), Chiu et al (1995) Ann Thor
  • the myogenic precursor cells are obtained from skeletal muscle
  • the skeletal muscle donor is preferably the subject for myocardial treatment or an identical twin in order to avoid problems of histocompatibility and possible tissue rejection
  • other family members or histocompatible donors including transgenic mammals raised for organ transplantation purposes (e g , lacking MHC markers or expressing humanized MHC proteins), may be employed as donors of the skeletal muscle tissue
  • standard methods of immunosuppression may be needed in conjunction with the present invention to prevent rejection of the implanted cells
  • a sample of skeletal muscle is excised from one or more skeletal muscles of a subject under local or general anesthesia Any excessive connective tissue and fasciae are dissected away, the muscle is rinsed in sterile solution, and the muscle is dissociated by, for example, mincing with scissors or passage through a meat grinder until substantially homogeneous
  • the amount of muscle excised will depend, of course, upon the quantity of myogenic precursor cells required by the treatment, as well as the degree of myogenic precursor cell proliferation which is to be promoted in vitro. Typically, however, amounts of 1-100 grams, more preferably 10-50 grams, of skeletal muscle tissue are removed.
  • Such quantities may be excised conveniently from one or more of the larger, relatively superficial muscles of the limbs (e.g., biceps brachii, triceps brachii, brachialis, brachioradialis, rectus femoris, biceps femoris, semitendinosus, gracilis, vastus lateralis, gastrocnemius, tibialis anterior), chest and shoulders (e.g., pectoralis, deltoid), pelvis and hips (e.g., gluteus maxims, gluteus maximus), back (e.g., trapezius, latissimus dorsi) or abdomen (e.g., obliquus abdominis externus, rectus abdominis), but may be obtained from any available skeletal muscle.
  • the larger muscles of the limbs e.g., biceps brachii, triceps brachii, brachialis, bra
  • the dissociated muscle then is incubated with a proteolytic enzyme (e.g., pronase (Sigma, St. Louis, MO), collagenase (Sigma, St. Louis, MO), hyaluronidase (Sigma, St. Louis, MO), or trypsin (Difco Laboratories, Inc., Detroit, MI) at 37°C for 15 min to 1 hr to remove remaining connective tissue.
  • a proteolytic enzyme e.g., pronase (Sigma, St. Louis, MO), collagenase (Sigma, St. Louis, MO), hyaluronidase (Sigma, St. Louis, MO), or trypsin (Difco Laboratories, Inc., Detroit, MI) at 37°C for 15 min to 1 hr to remove remaining connective tissue.
  • the mass of digested muscle tissue optionally may be further dissociated by, for example, repeated pipetting or mixing.
  • the digested mass optionally may be washed, pellet
  • the cells are then suspended in a sterile buffer (e.g., phosphate buffered saline solution) and centrifuged at approximately 500-550 g for approximately 10 minutes to sediment the larger, multinucleated skeletal muscle fibers and myocytes, while leaving the satellite cells in the supernatant.
  • a sterile buffer e.g., phosphate buffered saline solution
  • serum such as fetal bovine serum (FBS, GIBCO BRL, Grand Island, NY)
  • FBS fetal bovine serum
  • satellite cells may be separated from fibroblasts and other remaining cells using a density centrifugation method (see, e.g., Yablonka-Reuveni and Nameroff (1987) Histochemistrv 87:27-38). 2. Isolating Myogenic Precursor Cells from Embryos
  • Myogenic precursors cells may be isolated from mammalian embryonic or fetal (together “embryonic”) tissues at various stages of development after induction of the mesoderm.
  • myogenic precursor cells may be obtained from the embryonic mesoderm prior to its further differentiation into dorsal, intermediate, and lateral mesodermal mesenchyme.
  • any mesodermal cells may be employed but, preferably, cells are employed which arise along the routes of differentiation toward skeletal or cardiac muscle
  • the dorsal mesodermal mesenchyme differentiates to form the myotomes which, in turn, differentiate to form both the skeletal muscles of the trunk and the limb buds
  • the mesodermal mesenchyme of the limb buds further differentiates to form the skeletal muscles of the appendages (as well as the appendicular skeleton )
  • the lateral mesodermal mesenchyme differentiates, in part, to form the splanchic mesoderm which, in turn, differentiates to form the myocardium and smooth muscles of the viscera (as well as the gonads, circulatory system and other primary elements of the viscera)
  • embryonic cells for use in the present invention (see, e g , Soonpaa et al (1994) Science 264 98-101
  • myogenic precursor cell lines including myogenic precursor cell lines, myoblast cell lines, or mesenchymal cell lines
  • established cell lines including myogenic precursor cell lines, myoblast cell lines, or mesenchymal cell lines
  • the established murine myoblast cell line C 2 C ⁇ 2 has been implanted into mouse hearts and shown to differentiate into functional myocardium and fuse with native myocardium
  • plu ⁇ potent mesodermal stem cell lines including primary dermal fibroblast lines, smooth muscle cell lines, or chondroblast lineages may be caused to differentiate into muscle cells
  • Myogenic precursor cells may be cultured on solid or in liquid media
  • the myogenic precursor cells may be suspended in a flask of liquid medium while maintaining mild or periodic agitation
  • the cells may be plated on a solid substrate and fed with a liquid medium
  • liquid media include, but are not limited to, McCoy's, Ml 99, Minimal Essential Medium (MEM), Dulbecco's Modified Eagle Medium (commercially available from, for example, GIBCO BRL, Grand Island, NY, or Sigma Chemical Company, St Louis, MO), and the like
  • MEM Minimal Essential Medium
  • Dulbecco's Modified Eagle Medium commercially available from, for example, GIBCO BRL, Grand Island, NY, or Sigma Chemical Company, St Louis, MO
  • additional buffers or nutrient solutions e g , 10% fetal bovine serum, 3% horse serum
  • antimycotics and/or antibiotics e g , 50-5,000 IU/ml penicillin, 50-5,000 ⁇ g/ml strepto
  • myogenic precursor cell proliferation has been shown to be inhibited by TGF- ⁇ (Allen and Boxhorn (1989) J Cell Phvsiol 138 311-315) and contact with myofiber plasmalemmas, (Bischoff (1989)), and has been shown to be promoted by a saline "muscle extract" (Bischoff (1986) Dev Biol 115 140), conditioned medium from differentiated myotubes grown in culture (Mezzogiorno et al (1993) Mech Ageing & Develop 70 35-44), basic fibroblast growth factor (bFGF) (Clegg et al (1987) J Cell Biol 105 949-56), insulin-like growth factors (IGF) (Ewton and Florim (1977) Endocrinology 106 577-587, Tollfsen et al (1989) Proc Nat Acad Sci (USA) 15
  • TGF- ⁇ Allen and Boxhorn (1989) J Cell Phvsiol 138 311-315
  • the cells may be grown in the presence of one or more of these factors, or other known mitogens
  • proliferation of such cells may be promoted by repeated passaging (e g , treatment with dilute trypsin to remove adhered cells from the culture plate and replating at a lower density every 2-3 days), growth in liquid culture, growth in the absence of enhancers of cell adhesion, growth in the presence of inhibitors of cell adhesion, and/or growth at densities below confluence
  • repeated passaging e g , treatment with dilute trypsin to remove adhered cells from the culture plate and replating at a lower density every 2-3 days
  • growth in liquid culture growth in the absence of enhancers of cell adhesion
  • growth in the presence of inhibitors of cell adhesion and/or growth at densities below confluence
  • Myogenic precursor cells may be harvested by brief trypsin treatment to remove any cells adhered to the culture plate or vessel, and centrifugation (e g , 10-15 min at 500-1000 g) The cells may then be resuspended in a physiologically acceptable buffer solution (e g , PBS, Ringer's saline) at an appropriate density (e g , 10 3 -10 7 cells/ml) - li
  • a physiologically acceptable buffer solution e g , PBS, Ringer's saline
  • morphogens, morphogen inducers, agonists of morphogen receptors, and small molecule morphogenic activators may be used to treat the myogenic precursor cells during culturing (if any) to aid in proliferation and/or subsequent differentiation.
  • the myogenic precursor cells may be treated with a morphogen, morphogen inducer, agonist of a morphogen receptor, or small molecule morphogenic activator either simultaneously with, or subsequent to, implantation.
  • the myogenic precursor cells may be co-cultured with auxiliary cells which respond to these morphogen inducers by producing morphogen.
  • the myogenic precursor cells then may be implanted along with these auxiliary cells, or may be isolated from the co-culture by standard cell separation techniques, which are known in the art, but which will vary with the type of auxiliary cells employed (e.g., density centrifugation separation, cell type specific cytotoxins).
  • auxiliary cells e.g., density centrifugation separation, cell type specific cytotoxins.
  • the myogenic precursor cells are implanted in a physiologically acceptable buffer solution.
  • the cells may be at a relatively high titer within this solution (e.g., 10 5 -10 7 cells/ml).
  • the solution may contain growth factors, as described above, to promote further proliferation of the myogenic precursor cells within the implant site, or may be free of such factors so as to promote differentiation into new and functional myocardium in the morphogenically permissive environment of the myocardial implant site.
  • the myogenic precursor cells may be implanted either simultaneously with a morphogen, morphogen inducer, agonist of a morphogen receptor, or small molecule morphogenic activator, or the morphogenic treatment may be subsequent to implantation.
  • a solution of myogenic precursor cells and a morphogen, morphogen inducer, agonist of a morphogen receptor, or small molecule morphogenic activator may be implanted at a site of myocardial infarction in essentially the following manner
  • a left thoracotomy is performed on a subject under general anesthesia in an intercostal space (e g , the sixth intercostal space) and the site of the infarct is determined by observation
  • the heart may or may not be stopped and systemic blood flow shunted to a heart-lung machine
  • Myogenic precursor cells then may be directly injected into one or more sites within the infarct using an intravenous catheter (e g , a 16-gauge Teflon catheter from C ⁇ ticon, Tampa, FL)
  • the initial ⁇ nject ⁇ on(s) may include a morphogen, morphogen inducer,
  • the treatment of chronically deteriorating mammalian myocardium may be performed similarly except that the implantation sites are chosen to correspond to areas of generalized myocardial deterioration and, therefore, may be more diffuse
  • the number of myogenic precursor cells implanted will vary according to the amount of myocardial tissue to be restored or regenerated
  • the volume of cells to be restored or regenerated may be ascertained by standard techniques of cardiac imaging Generally, it is expected that on the order of approximately 10 4 -10 5 myogenic precursor cells will be required to restore or regenerate 1 mg of myocardial tissue (see, e g , Alameddine and Fardeau (1989)) D Morphogens.
  • Morphogens useful in the present invention include eukaryotic proteins originally identified as osteogenic proteins (see U S Patent 5,011,691, incorporated herein by reference) such as the OPl, OP2, OP3, CBMP2A (BMP-2), CBMP-2B (BMP-4) and BMP3 proteins (SEQ ID NOs: 4-9, 15-22, 25-27), as well as amino acid sequence-related proteins such as DPP (SEQ ID NO: 10, from Drosophila). Vgl (SEQ ID NO: 11, from Xenopus).
  • Vgrl (SEQ ID NO: 12, from mouse), GDF1 (SEQ ID NOs: 13, 30 and 31, from humans, see Lee (1991), PNAS 88:4250-4254), 60A (SEQ ID NOs: 23 and 24, from Drosophila. see Wharton et al. (1991) PNAS 88:9214-9218), dorsalin-1 (from chick, see Basler et al. (1993) Cell 73:687-702 and GenBank accession number L12032) and GDF5 (from mouse, see Storm et al. (1994) Nature 368:639-643).
  • Additional useful morphogens include biosynthetic morphogen constructs disclosed in U.S. Pat. No.
  • Naturally occurring proteins identified and/or appreciated herein to be morphogens form a distinct subgroup within the loose evolutionary grouping of sequence-related proteins known as the TGF ⁇ superfamily or supergene family.
  • the naturally occurring morphogens share substantial amino acid sequence homology in their C-terminal regions (domains).
  • the above- mentioned naturally occurring morphogens are translated as a precursor, having an N-terminal signal peptide sequence, typically less than about 30 residues, followed by a "pro" domain that is cleaved to yield the mature C-terminal domain.
  • 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.
  • the pro domain typically is about three times larger than the fully processed mature C-terminal domain.
  • the "pro" form of a morphogen refers to a morphogen comprising a folded pair of polypeptides each comprising the pro and mature domains of a morphogen polypeptide.
  • the pro form of a morphogen is more soluble than the mature form under physiological conditions.
  • the pro form appears to be the primary form secreted from cultured mammalian host cells.
  • Table 1 summarizes various naturally occurring morphogens identified to date, including their nomenclature as used herein, their Sequence Listing references, and publication sources for the amino acid sequences for the full length proteins not included in the Sequence Listing.
  • Table 1 Each of the generic terms set forth in Table 1 is intended and should be understood to embrace morphogenically active proteins expressed from nucleic acids encoding the identified sequence mentioned below and set forth in the Sequence Listing, or a morphogenically active fragment or precursor thereof, including functional equivalents such as naturally occurring and biosynthetic variants thereof
  • Naturally occurring variants include allelic variant forms isolated from other individuals of a single biological species, and phylogenetic counterpart (species) variant forms (homologues) isolated from phylogenetically distinct biological species.
  • OPl refers gene ⁇ cally to morphogenically active proteins expressed from nucleic acids encoding OPl proteins, including at least the human OPl protein disclosed in SEQ ID NO 4 (“hOPl”), and the mouse OPl protein disclosed in SEQ ID NO 5
  • mOPl In each of human and mouse OPl proteins, the conserved seven cysteine skeleton is defined by residues 38 to 139 cDNA sequences and amino acid sequences encoded therein and corresponding to the full length proteins are provided in SEQ ID NOs 15 and 16 (hOPl) and SEQ ID NOs 17 and 18 (mOP- 1 ) The mature proteins are defined by residues 293-431 (hOPl) and 292-430
  • OP2 refers gene ⁇ cally to morphogenically active proteins expressed from nucleic acids encoding the OP2 proteins, including at least the human OP2 protein disclosed in
  • the mature proteins are defined essentially by residues 264- 402 (hOP2) and 261-399 (mOP2)
  • the "pro" regions of the proteins, cleaved to yield the mature, morphogenically active proteins are defined essentially by residues 18-263 (hOP2) and residues 18-260 (mOPl) "OP3 " Refers gene ⁇ cally to morphogenically active proteins expressed from nucleic acids encoding OP3 proteins, including at least the mouse OP3 protein disclosed in SEQ ID NO 26 (“mOP3")
  • the conserved seven cysteine domain is defined by residues 298 to 399 of SEQ ID NO 26, which shares greatei than 79% amino acid identity with the corresponding mOP2 and hOP2 sequences, and greater than 66% identity with the corresponding OPl sequences
  • a cDNA sequence encoding the above- mentioned ammo acid sequence is provided in SEQ ID NO 25 OP3 is unique among the morphogens identified to date in that the residue at position 9 in the conserved seven
  • CBMP2 refers gene ⁇ cally to morphogenically active proteins expressed from nucleic acids encoding the CBMP2 proteins, including at least the human CBMP2A protein disclosed in SEQ ID NO 8 (hCBMP2A) and the human CBMP2B protein disclosed in SEQ ID NO 9 (hCBMP2B)
  • the amino acid sequence for the full length proteins referred to in the literature as BMP2A and BMP2B, or BMP2 and
  • BMP4 appear in Wozney, et al (1988) Science 242 1528-1534
  • the pro domain for BMP2 (BMP2A) likely includes residues 25-248 of the published sequence, the mature protein, residues 249-396
  • the pro domain for BMP4 (BMP2B) likely includes residues 25-256 of the published sequence, the mature protein, residues 257-408
  • DPP Refers gene ⁇ cally to proteins encoded by the Drosophila DPP gene and defining at least the conserved seven cysteine skeleton of SEQ ID NO 10
  • the amino acid sequence for the full length protein appears in Padgett, et al (1987) Nature 325 81-84
  • the pro domain likely extends from the signal peptide cleavage site to residue 456 of the published sequence, the mature protein likely is defined by residues 457-588
  • Vgl Refers gene ⁇ cally to proteins encoded by the Xenopus Vgl gene and defining at least the conserved seven cysteine skeleton of SEQ ID NO 11 The amino acid sequence for the full length protein appears in Weeks (1987) Cell 51 861-867 The prodomain likely extends from the signal peptide cleavage site to residue 246 of the published sequence, the mature protein likely is defined by residues 247-360
  • Vgrl Refers generically to proteins encoded by the murine Vgrl gene and defining at least the conserved seven cysteine skeleton of SEQ ID NO 12
  • the amino acid sequence for the full length protein appears in Lyons, et al (1989) PNAS 86 4554- 4558
  • the prodomain likely extends from the signal peptide cleavage site to residue 299 of the published sequence, the mature protein likely is defined by residues 300-438
  • GDF1 Refers generically to proteins encoded by the human GDF1 gene and defining at least the conserved seven cysteine skeleton of SEQ ID NO 13
  • the cDNA and encoded amino sequence for the full length protein are provided in SEQ ID NOs 30 and 31
  • the prodomain likely extends from the signal peptide cleavage site to residue 214, the mature protein likely is defined by residues 215- 372
  • 60A Refers generically to morphogenically active proteins expressed from nucleic acid encoding 60A proteins or morphogenically active fragments thereof, including at least the Drosophila 60A protein disclosed in SEQ ID NO 24
  • a Drosophila 60A cDNA is disclosed in SEQ ID NO 23
  • the prodomain likely extends from the signal peptide cleavage site to residue 324, the mature protein likely is defined by residues 325-455
  • the active fragment of 60A protein likely is defined by the conserved seven cysteine skeleton of residues 354 to 455 of SEQ ID NO 24
  • the 60A protein is considered likely herein to be a phylogenetic counterpart variant of the human and mouse OPl genes, Sampath, et al (1993) PNAS 90 6004-6008
  • BMP3 Refers generically to proteins encoded by the human BMP3 gene and defining at least the conserved seven cysteine skeleton of SEQ ID NO 27
  • the amino acid sequence for the full length protein appears in Wozney, et al (1988) Science 242 1528-1534
  • the pro domain likely extends from the signal peptide cleavage site to residue 290 of the published sequence; the mature protein likely is defined by residues 291-472.
  • BMP5 Refers generically to proteins encoded by the human BMP5 gene and defining at least the conserved seven cysteine skeleton of SEQ ID NO: 28. The amino acid sequence for the full length protein appears in Celeste, et al. (1991) PNAS
  • the pro domain likely extends from the signal peptide cleavage site to residue 316 of the published sequence; the mature protein likely is defined by residues 317-454.
  • BMP6 Refers generically to proteins encoded by the human BMP6 gene and defining at least the conserved seven cysteine skeleton of SEQ ID NO: 29.
  • the amino acid sequence for the full length protein appears in Celeste, et al. (1990) PNAS 87:9843-5847.
  • the pro domain likely extends from the signal peptide cleavage site to residue 374 of the published sequence; the mature protein likely is defined by residues 375-513.
  • the OP2 and OP3 proteins have an additional cysteine residue in the conserved C-terminal region (e.g., see residue 41 of SEQ ID NOs: 6 and 7), in addition to the conserved cysteine skeleton or domain in common with the other known proteins in this family.
  • the GDF1 protein has a four amino acid insert within the conserved skeleton (residues 44-47 of SEQ ID NO: 13) but this insert likely does not interfere with the relationship of the cysteines in the folded structure. Further, the CBMP2 proteins are missing one amino acid residue within the cysteine skeleton. Thus, these morphogen polypeptides illustrate the principles of alignment used herein with respect to the preferred reference morphogen sequence of human OPl, residues 38- 139 of SEQ ID NO: 4.
  • morphogens useful herein include those in which the amino acid sequences of morphogen polypeptides comprise a sequence sharing at least 70%) amino acid sequence homology or "similarity", and preferably 80%> homology or similarity with a reference morphogen sequence selected from the foregoing sequences or naturally occurring morphogens.
  • the reference morphogen is human OP
  • the reference sequence thereof is the C-terminal seven cysteine domain present in morphogenically active forms of human OPl, residues 38-139 of SEQ ID NO: 4.
  • Certain particularly preferred morphogen polypeptides share at least 60% amino acid identity with the preferred reference sequence of human OPl, still more preferably at least 65%> amino acid identity therewith.
  • the family of morphogen polypeptides useful in the present invention, and members thereof are defined by a generic amino acid sequence.
  • Generic Sequence 7 (SEQ ID NO: 1) and Generic Sequence 8 (SEQ ID NO: 2) disclosed below, accommodate the homologies shared among preferred morphogen protein family members identified to date, including at least OPl, OP2, OP3, CBMP2A, CBMP2B, BMP3, BMP5, BMP6, DPP, Vgl, Vgrl, 60A, and GDF1.
  • the amino acid sequences for these proteins are described herein (see Sequence Listing) and/or in the art, as summarized above.
  • the generic sequences include both the amino acid identity shared by these sequences in the C-terminal domain, defined by the six and seven cysteine skeletons (Generic Sequences 7 and 8, respectively), as well as alternative residues for the variable positions within the sequence.
  • the generic sequences provide an appropriate cysteine skeleton where inter- or intramolecular disulfide bonds can form, and contain certain critical amino acids likely to influence the tertiary structure of the folded proteins.
  • the generic sequences allow for an additional cysteine at position 41 (Generic Sequence 7) or position 46 (Generic Sequence 8), thereby encompassing the morphogenically active sequences of OP2 and OP3.
  • Generic Sequence 8 (SEQ ID NO 2) includes all of Generic Sequence 7 and in addition includes the following sequence (SEQ ID NO 14) at its N-terminus
  • each "Xaa” in Generic Sequence 8 is a specified ammo acid defined as for Generic Sequence 7, with the distinction that each residue number described for Generic Sequence 7 is shifted by five in Generic Sequence 8
  • Xaa at res 2 (Lys, Arg, Ala or Gin)
  • Xaa at res 3 (Lys, Arg or Met)
  • Xaa at res 4 (His, Arg or Gin)
  • Xaa at res 5 (Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr)
  • useful morphogen polypeptide sequences useful in this invention have greater than 60% identity, preferably greater than 65% identity, with the amino acid sequence defining the conserved six or seven cysteine skeleton of hOPl (e g , residues 43-139 or 38-139 of SEQ ID NO 4)
  • These particularly preferred sequences include allelic and phylogenetic counterpart variants of the OPl and OP2 proteins, including the Drosophila 60A protein (SEQ ID NO 24)
  • useful morphogens include active proteins comprising pairs of polypeptide chains within the generic ammo acid sequence herein referred to as "OPX" (SEQ ID NO 3), which corresponds to the seven cysteine skeleton and accommodates the homologies between several identified variants of OPl and OP2 As described therein, each Xaa at a given position independently is selected from the residues occurring at the corresponding position in the C-terminal sequence of mouse or human OP 1 or OP2 (see S
  • useful morphogen polypeptides have amino acid sequences comprising a sequence encoded by a nucleic acid that hybridizes, under stringent hybridization conditions, to DNA or RNA encoding reference morphogen sequences, e g , C- terminal sequences defimng the conserved seven cysteine domains of OPl or OP2, e g , nucleotides 1036-1341 and nucleotides 1390-1695 of SEQ ID NO 15 and 19, respectively
  • stringent hybridization conditions are defined as hybridization according to known techniques in 40% formamide, 5 X SSPE, 5 X Denhardt's Solution, and 0 1% SDS at 37°C overnight, and washing in 0 1 X SSPE, 0 1% SDS at 50°C
  • morphogens useful in the present invention generally are dime ⁇ c proteins comprising a folded pair of the above polypeptides Morphogens are inactive when reduced, but are active as oxidized homodimers and when oxidized in combination with other morphogens of this invention to produce heterodimers
  • members of a folded pair of morphogen polypeptides in a morphogenically active protein can be selected independently from any of the specific morphogen polypeptides mentioned above
  • the morphogens useful in the methods, compositions and devices of this invention include proteins comprising any of the polypeptide chains described above, whether isolated from naturally-occurring sources, or produced by recombinant DNA or other synthetic techniques, and includes allelic and phylogenetic counterpart variants of these proteins, as well as biosynthetic variants (muteins) thereof, and various truncated and fusion constructs Deletion or addition mutants also are envisioned to be active, including those which may alter the conserved C- terminal six or seven cysteine domain, provided
  • the morphogenic proteins can be expressed from intact or truncated cDNA or from synthetic DNAs in prokaryotic or eukaryotic host cells, and purified, cleaved, refolded, and dimerized to form morphogenically active compositions.
  • Currently preferred host cells include R coli or mammalian cells, such as CHO, COS or BSC cells.
  • a detailed description of the morphogens useful in the methods, compositions and devices of this invention is disclosed in published application W092/15323, the disclosure of which is incorporated by reference herein.
  • skilled genetic engineers can isolate genes from cDNA or genomic libraries of various different biological species, which encode appropriate amino acid sequences, or construct DNAs from oligonucleotides, and then can express them in various types of host cells, including both prokaryotes and eukaryotes, to produce large quantities of active proteins capable of stimulating the morphogenesis of, and/or inhibiting damage or loss of, mammalian myocardial tissue.
  • a protein is morphogenic herein generally if it induces the developmental cascade of cellular and molecular events that culminate in the formation of new, organ-specific tissue.
  • a morphogen comprises a pair of polypeptides having a sequence that corresponds to or is functionally equivalent to at least the conserved C-terminal six or seven cysteine skeleton of human OPl, included in SEQ ID NO: 4.
  • the morphogens generally are competent to induce a cascade of events including all of the following, 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 morphogens useful in this invention first were identified, as well as a description on how to make, use and test them for morphogenic activity are disclosed in published application W092/15323.
  • the morphogens can be purified from naturally-sourced material or recombinantly produced from prokaryotic or eukaryotic host cells, using the genetic sequences disclosed therein. Alternatively, novel morphogenic sequences can be identified following the procedures disclosed therein.
  • Exemplary useful morphogens include naturally derived proteins comprising a pair of polypeptides, the ammo acid sequences of which comprise sequences selected from those disclosed in the Sequence Listing and Figure 1
  • Other useful sequences include those of the naturally derived morphogens dorsalin-1, SCREW, NODAL, UNIVIN and GDF5, discussed herein in connection with Table 1, as well as biosynthetic constructs disclosed in U S Pat
  • morphogens useful in the methods and compositions of this invention can be described as morphogenically active proteins having amino acid sequences sharing 70% or, preferably, 80% homology with a reference morphogen sequence described above, e g , residues 38-139 of SEQ ID NO 4, where "homology" is as defined herein above
  • morphogens useful in the methods and compositions disclosed herein fall withm the family of polypeptides described by Generic Sequence 7, SEQ ID NO 1, more preferably by Generic Sequence 8, SEQ ID NO 2 Figure 1 herein sets forth an alignment of the amino acid sequences of the active regions of exemplary naturally occurring proteins that have been identified or appreciated herein as morphogens, including human OPl (hOPl, SEQ ID NOs 4 and 15-16), mouse OPl (mOPl, SEQ ID NOs 5 and 17-18), human and mouse OP2 (SEQ ID NOs 6, 7, and 19-22), mouse OP3 (SEQ ID NOs 25-26), CBMP
  • SEQ ID NO 10 SEQ ID NO 10
  • Vgl from Xenopus.
  • SEQ ID NO 11 from mouse, SEQ ID NO 12
  • GDF1 from mouse and/or human, SEQ ID NOs 13, 30 and 31
  • 60A protein from Drosophila.
  • SEQ ID NOs 23 and 24 BMP5 (SEQ ID NO 28) and BMP6 (SEQ ID NO 29)
  • the sequences are aligned essentially following the method of Needleman, et al (1970) J Mol Biol .
  • an agent which acts as an agonist of a morphogen receptor may be administered instead of the morphogen itself
  • Such an agent may also be referred to an a morphogen "mimic,” “mimetic,” or “analog "
  • a small peptide or other molecule which can mimic the activity of a morphogen in binding to and activating the morphogen's receptor may be employed as an equivalent of the morphogen
  • the agonist is a full agonist, but partial morphogen receptor agonists may also be advantageously employed Methods of identifying such agonists are known in the art and include assays for compounds which induce morphogen-mediated responses (e.g., induction of differentiation of metanephric mesenchyme, induction of endochondral bone formation).
  • a small molecule morphogenic activator may be used for promoting the migration, proliferation, and/or differentiation of myogenic precursor cells by increasing the level of expression of proteins associated with myocardial phenotype.
  • Exemplary methods comprise introducing a small molecule morphogenic activator that regulates some portion or portions of a morphogen-induced regulatory pathway, resulting in an effective increase in expression or activity of myocardium-specific protein. This may result either from stimulating an increase in the endogenous expression of such protein or from a decrease in the inhibition of normal expression of such protein.
  • a small molecule morphogenic activator may act at the type I or type II morphogen receptor; or at the serine/threonine kinase, or other kinase domains of those receptors.
  • Another target of pathway activation is the Smad proteins, including the monomeric, dimeric (including heteromeric and homomeric complexes) or trimeric forms (including heteromeric and homomeric complexes).
  • the Smads have been characterized, and are known in the art. See, e.g., Baker, et al., Curr. Op. Genet. Develop., 7: 467-473 (1997), incorporated by reference herein.
  • a small molecule morphogenic activator may lead to activation of a transcription factor (for example, the X-protein shown in Figure 2) that causes phenotype-specific gene expression (i.e., expression of protein characteristic of myocardium).
  • a small molecule morphogenic activator may act to facilitate, mimic, or, if desired, prevent any one or several of the following: type I and/or type II receptor binding, phosphorylation of the type I receptor, phosphorylation of the Smad molecules, Smad complex formation, Smad translocation into the nucleus, nuclear accumulation of the Smad complex, or transcription modulation of the Smad complex.
  • a small molecule morphogenic activator may act on Smads or Smad complexes to alter tertiary structure, thereby to facilitate or inhibit interaction of the Smad or Smad complex with a receptor kinase domain, other Smads, DNA binding proteins, or DNA itself.
  • a small molecule morphogenic activator is contacted with myogenic precursor cells in vivo or in vitro, or is administered to a patient, wherein the small molecule morphogenic activator facilitates formation of Smad complexes, particularly complexes comprising molecules of Smadl, Smad2, Smad3, Smad4, Smad5 and/or Smad8 in order to induce myogenic precursor cells to migrate, proliferate and/or differentiate into cells expressing markers of a myocardial tissue phenotype.
  • methods comprise administering a small molecule morphogenic activator composition that activates a serine/threonine kinase domain associated with a morphogen type I or type II receptor, thereby to activate the pathway involved in morphogen-induced gene expression.
  • methods of the invention comprise activating Smad4 association with Smadl, thereby to induce morphogen-responsive phenotype.
  • Methods of the invention may also facilitate Smad interaction with specific nucleic acids, such as promoters of myocardial tissue phenotype- specific gene expression (i.e., expression of genes for a phenotypic protein; a protein associated with preservation, restoration, or enhancement of phenotype, including a protein which is critical for production of non-protein phenotypic markers, such as characteristic lipids or carbohydrates; a protein associated with performance of a phenotypic function or morphology; or a morphogen).
  • Such interaction may be, for example, in association with a transcription control factor that is capable of binding to a regulatory portion of a gene and, simultaneously, to one or more regulatory proteins such as a Smad complex (See Figure 2).
  • Morphogens are ligands for the type I and type II receptors. Following phosphorylation of the type I receptor by the type-II receptor, the type I receptor specifically phosphorylates Smadl homodimers. The type I receptor also specifically phosphorylates Smad5 homodimers. The homodimers then separate to form, in association with a phosphorylated Smad4 molecule, a phosphorylated heteromeric complex comprising at least a Smadl and a Smad4. A phosphorylated Smadl/Smad5/Smad4 heterotrimer may alternatively be formed. The heteromeric complex then translocates into the nucleus, and accumulates therein.
  • the Smad complex binds operative DNA, either alone or in association with a specific DNA binding protein (the X-protein in Figure 2), to initiate DNA transcription.
  • the "X-protein” acts as a DNA-binding protein, binding the Smad heteromeric complex to the DNA.
  • the Smadl, Smad2, Smad3 and Smad5 proteins consist of conserved amino- and carboxy-terminal domains linked by a region that is more divergent among the Smads
  • the carboxy-terminal domain has an effector function
  • the ammo-terminal domain interacts physically with the carboxy-terminal domain, inhibiting its effector and contributes to DNA binding Receptor-mediated phosphorylation of the se ⁇ ne residues at the end of the carboxy-terminal domain relieves the carboxy-terminal domain from the inhibitory action of the amino-terminal domain
  • Phosphorylated Smad molecules form a heteromeric complex with at least one other specific Smad family molecule
  • the resulting Smad complex then translocates into and accumulates in the cell nucleus There, the heteromeric Smad complexes regulate transcriptional responses either alone or by specific interaction with a DNA-binding protein, such as forkhead activin signal transducer-1 (FASTI)
  • FASTI forkhead activin signal transducer-1
  • a small molecule morphogenic activator for use in the invention is a compound that affects one or more intracellular pathways that normally are under morphogen regulation
  • Such small molecule morphogenic activators preferably have the ability to enter the cell and target one or more intracellular pathway components in order to stimulate or inhibit their activity
  • a small molecule morphogenic activator that promotes Smad complex formation between Smadl, Smad4, and Smad5 will stimulate pathways leading to expression of genes encoding phenotype-specific proteins
  • One way in which to identify a candidate small molecule morphogenic activator is to assay for the ability of the candidate to modulate the effective systemic or local concentration of a morphogen This may be done, for example, by incubating the candidate in a cell culture that produces the morphogen, and assaying the culture for a parameter indicative of a change in the production level of the morphogen according the methods of U S S N 08/451,953 and/or U S 5,650,276, the teachings of each of which are incorporated by
  • Candidates having the desired affect on protein production or pathway regulation are selected for use in methods of the invention If, for example, a composition up-regulates the production of OP-1 by a kidney cell line, it would then be desirable to test systemic administration of this compound in an animal model to determine if it up-regulates the production of OP-1 in vivo
  • the level of morphogen in the body may be a result of a wide range of physical conditions, e g , tissue degeneration such as occurs in diseases including arthritis, emphysema, osteoporosis, kidney diseases, lung diseases, cardiomyopathy, and cirrhosis of the liver
  • the decrease in level of morphogens in the body may also occur as a result of the normal process of aging
  • a composition selected by these screening methods is then used as a treatment or prophylactic
  • An appropriate test cell is any cell comprising DNA defining a morphogen-responsive transcription activating element operatively associated with a reporter gene encoding a detectable phenotype-specific gene product.
  • DNA can occur naturally in a test cell or can be a transfected DNA
  • the induced intracellular effect typically is characteristic of morphogenic biological activity, such as Smad activation, or activation of a cascade of biochemical events, such as described above, or involving, for example, cyclic nucleotides, diacylglycerol, and/or and other indicators of intracellular signal transduction such as activation or suppression of gene expression, including induction of mRNA resulting from gene transcription and/or induction of protein synthesis resulting from translation of mRNA transcripts indicative of tissue morphogenesis
  • Exemplary morphogen-responsive cells are preferably of mammalian origin and include, but are not limited to, osteogenic progenitor cells, calva ⁇ a-de ⁇ ved cells, osteoblasts, osteoclasts, osteos
  • a currently preferred reporter gene system is the firefly luciferase reporter system Gould, et al , Anal Biochem , 7 404-408 (1988), incorporated herein by reference
  • the luciferase assay is fast and sensitive
  • a lysate of the test cell is prepared and combined with ATP and the substrate lucife ⁇ n
  • the encoded enzyme luciferase catalyzes a rapid, ATP- dependent oxidation of the substrate to generate a light-emitting product
  • the total light output is measured and is proportional to the amount of luciferase present over a wide range of enzyme concentrations
  • CAT is another frequently used reporter gene system, a major advantage of this system is that it has been an extensively validated and is widely accepted as a measure of promoter activity Gorman , et al , Mol Cell Biol , 2 1044-1051 (1982), incorporated by reference herein
  • test cells are transfected with CAT expression vectors and incubated with
  • cell extracts are prepared.
  • the extracts are incubated with acetyl Co A and radioactive chloramphenicol.
  • acetylated chloramphenicol is separated from nonacetylated form by thin layer chromatography.
  • the degree of acetylation reflects the CAT gene activity with the particular promoter.
  • Another suitable reporter gene system is based on immunologic detection of hGH. This system is also quick and easy to use. Selden, et al., Mol. Cell. Biol. 6:3173-3179 (1986), incorporated by reference herein.
  • the hGH system is advantageous in that the expressed hGH polypeptide is assayed in the media, rather than in a cell extract. Thus, this system does not require the destruction of the test cells. It will be appreciated that the principle of this reporter gene system is not limited to hGH but rather adapted for use with any polypeptide for which an antibody of acceptable specificity is available or can be prepared.
  • a small molecule morphogenic activator composition may up-regulate a morphogen- activated pathway by acting at any one or more point.
  • small molecule morphogenic activator potentiation of the pathway may be initiated at the receptor level.
  • the transmembrane receptors may be type I and/or type II, or may be comprise variations on either type I or type II receptors.
  • OP-1 is capable of activating regulatory pathways comprising at least two variations of both type I and type II receptors (ActR- 1 and BMPR-1B, and ActRII and BMPR-II, respectively).
  • a small molecule morphogenic activator may stimulate the pathway by acting as a ligand and binding to any of the receptors, thereby inducing phosphorylation of type I receptors and/or Smad molecules.
  • a small molecule morphogenic activator may activate the regulatory pathway at the level of the serine/threonine kinase domain of the receptors, thereby stimulating phosphorylation of type I receptors and/or Smad molecules.
  • a small molecule morphogenic activator may activate the regulatory pathway at the level of Smad complex formation.
  • a small molecule morphogenic activator may stimulate the formation of Smad family homodimers, heterodimers, or other homomeric or heteromeric complexes.
  • a small molecule morphogenic activator may activate the pathway by interacting with a Smad molecule or Smad complex, thereby altering its tertiary structure.
  • a small molecule morphogenic activator may activate the regulatory pathway by facilitating translocation of a Smad molecule or Smad complex or accumulation of the Smad molecule or Smad complex within the nucleus of the cell.
  • a small molecule morphogenic activator may activate the regulatory pathway by increasing transcriptional activity caused by the Smad molecule or Smad complex.
  • a small molecule morphogenic activator can act to stimulate the regulatory pathway by interfering with an inhibitor of the pathway.
  • Smad ⁇ and Smad7 which are structurally different than Smadl, Smad2, Smad3 and Smad5, act as inhibitors of certain types of desirable phenotype-specific protein expression (e.g., by activating TGF- ⁇ to induce scar tissue formation).
  • Smad ⁇ forms a stable association with type I receptors and interferes with the phosphorylation of other Smad proteins, including Smad2 and Smad 1, and their subsequent heteromerization with Smad4.
  • Smad7 also forms a stable association with activated type I receptors and blocks access and phosphorylation of certain Smad molecules, thereby preventing formation of certain Smad heteromeric complexes.
  • Smad7 also inhibits nuclear accumulation of Smad heteromeric complexes.
  • a small molecule morphogenic activator may interfere with the inhibitory activity of these Smad proteins by, for example, tightly binding to either one or both proteins and rendering either protein incapable of stable association with type I receptors, or by competitively binding and stimulating the morphogen-activated transmembrane receptors.
  • a small molecule morphogenic activator may activate the inhibitory effects of these Smads in order to inhibit an undesirable effect (e.g., TGF ⁇ activity).
  • the methods of the present invention may be utilized for any mammalian subject at risk of, or afflicted with, loss of or damage to myocardium.
  • Mammalian subjects which may be treated according to the methods of the invention include, but are not limited to, human subjects or patients.
  • the invention may be employed in the treatment of domesticated mammals which are maintained as human companions (e.g., dogs, cats, horses), which have significant commercial value (e.g., dairy cows, beef cattle, sporting animals), which have significant scientific value (e.g., captive or free specimens of endangered species), or which otherwise have value.
  • the subjects for treatment with the methods of the present invention need not present indications for morphogen treatment other than those associated with loss of or damage to myocardium That is, the subjects for treatment generally are expected to be otherwise free of indications for morphogen treatment In some number of cases, however, the subjects may present with other symptoms (e g , osteoporosis, chronic renal failure) for which morphogen treatment also would be indicated In such cases, the morphogen treatment should be adjusted accordingly to avoid excessive dosing
  • a mammalian subject may be regarded as a subject at risk of, or afflicted with, loss of or damage to myocardium if that subject has already been diagnosed as at risk of, or afflicted with, loss of or damage to myocardium
  • Such subjects include, but are not limited to, those which have already suffered a myocardial infarction, which have suffered a physical trauma to the heart, or which have been diagnosed with congestive heart failure
  • the morphogens, morphogen inducers, agonists of morphogen receptors, or small molecule morphogenic activators of the present invention may be provided to myogenic precursor cells by any suitable means, preferably directly (e g , in vitro or locally after implantation, as by addition to culture medium, injection or topical administration to a tissue locus) or systemically (e g , parenterally or orally)
  • the morphogen, morphogen inducer, agonist of a morphogen receptor, or small morphogenic activators of the present invention may be provided
  • 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
  • the resultant solution preferably is vortexed extensively.
  • the morphogens, morphogen inducers, agonists of morphogen receptors, or small molecule morphogenic activators of the present invention may be administered by any route which is compatible with the particular morphogen, inducer, or agonist employed.
  • the agent preferably comprises part of an aqueous solution.
  • administration may be by periodic injections of a bolus of the morphogen, inducer, agonist, or small molecule morphogenic activator, or may be made more continuous by intravenous or intraperitoneal administration from a reservoir which is external (e.g., an i.v. bag) or internal (e.g., a bioerodable implant, or a colony of implanted, morphogen-producing cells).
  • a given morphogen or other agent may be made more soluble by association with a suitable molecule.
  • association of the mature morphogen dimer with the pro domain results in the pro form of the morphogen which typically is more soluble or dispersible in physiological solutions than the corresponding mature form.
  • endogenous morphogens are thought to be transported (e.g., secreted and circulated) in the mammalian body in this form.
  • This soluble form of the protein can be obtained from culture medium of morphogen-secreting mammalian cells, e.g., cells transfected with nucleic acid encoding and competent to express the morphogen.
  • a soluble species can be formulated by complexing the mature dimer (or an active fragment thereof) with a morphogen pro domain or a solubility-enhancing fragment thereof (described more fully below).
  • a solubility-enhancing fragment thereof 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 OPl by 80%>.
  • Other components found in milk and/or various serum proteins also may be useful.
  • Useful solutions for parenteral administration may be prepared by any of the methods well known in the pharmaceutical art, described, for example, in Remington's Pharmaceutical Sciences (Gennaro, A., ed.), Mack Pub., 1990.
  • Formulations of the therapeutic agents of the invention may include, for example, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like.
  • Formulations for direct administration in particular, may include glycerol and other compositions of high viscosity to help maintain the agent at the desired locus.
  • Biocompatible, preferably bioresorbable, polymers including, for example, hyaluronic acid, collagen, tricalcium phosphate, polybutyrate, lactide, and glycolide polymers and lactide/glycolide copolymers, may be useful excipients to control the release of the agent in vivo.
  • Other potentially useful parenteral delivery systems for these agents include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation administration 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 may also include glycocholate for buccal administration, methoxysalicylate for rectal administration, or cutric acid for vaginal administration.
  • Suppositories for rectal administration also may be prepared by mixing the morphogen, inducer, agonist, or small molecule morphogenic activator with a non-irritating excipient such as cocoa butter or other compositions which are solid at room temperature and liquid at body temperatures.
  • Formulations for local or topical administration to a tissue or skin surface may be prepared by dispersing the morphogen, inducer, agonist or small molecule morphogenic activator with an acceptable carrier such as a lotion, cream, ointment or soap. Particularly useful are carriers capable of forming a film or layer over the skin or tissue to localize application and inhibit removal.
  • the agent may be dispersed in a liquid tissue adhesive or other substance known to enhance adsorption to a tissue surface.
  • tissue-coating solutions such as pectin-containing formulations may be used.
  • the agents described herein may be administered orally.
  • Oral administration of proteins as therapeutics generally is not practiced as most proteins are readily 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, OPl has been identified in mammary gland extract, colostrum and 57-day milk.
  • the OP 1 purified from mammary gland extract is morphogenically active and also is detected in the bloodstream.
  • Maternal administration via ingested milk, may be a natural delivery route of TGF ⁇ superfamily proteins.
  • the morphogen form found in milk is readily soluble, probably by association of the mature, morphogenically active form with part or all of the pro domain of the intact sequence and/or by association with one or more milk components. Accordingly, the compounds provided herein also may be associated with molecules capable of enhancing their solubility in vitro or in vivo.
  • the compounds provided herein also may be associated with molecules capable of targeting the morphogen, inducer, agonist or small molecule morphogenic activator to the desired tissue.
  • molecules capable of targeting the morphogen, inducer, agonist or small molecule morphogenic activator to the desired tissue may be used.
  • Useful targeting molecules may be designed, for example, using the single chain binding site technology disclosed, for example, in U.S. Pat. No. 5,091,513.
  • Targeting molecules can be covalently or non- covalently associated with the morphogen, inducer, agonist, or small molecule morphogenic activator.
  • the formulated compositions contain therapeutically effective amounts of the morphogen, morphogen inducers, agonists of morphogen receptors, or small molecule morphogenic activators. That is, they contain amounts which provide appropriate concentrations of the agent to the mammalian myogenic precursor cells for a time sufficient to stimulate morphogenesis of new and functional myocardium, and/or to prevent, inhibit or delay further significant loss of myocardium or decline of myocardial function.
  • the concentration of the compounds described in a therapeutic composition of the present invention will vary depending upon a number of factors, including the biological efficacy of the selected agent, the chemical characteristics (e.g., hydrophobicity) of the compounds employed, the formulation of the compound excipients, the administration route, and the treatment envisioned, including whether the active ingredient will be administered directly to cells in vitro, directly into a tissue site, or systemically.
  • the preferred dosage to be administered also is likely to depend on such variables such as the condition of the diseased or damaged tissues, and the overall health status of the particular subject. As a general matter, for systemic administration, daily or weekly dosages of 0.00001-
  • a daily or weekly dosage of 0.01-1000 ⁇ g/kg body weight, more preferably 0.1-100 ⁇ g/kg body weight, may be advantageously employed. Dosages are preferably administered continuously, but daily, multi-weekly, weekly or monthly dosages may also be employed. In addition, in order to facilitate frequent infusions, implantation of a semipermanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular) may be advisable.
  • a semipermanent stent e.g., intravenous, intraperitoneal, intracisternal or intracapsular
  • the morphogens, inducers, agonists or small molecule morphogenic activators of the invention may, of course, be administered alone or in combination with other molecules known to be beneficial in the treatment of the conditions described herein.
  • the present invention provides pharmaceutical compositions in which a morphogen, morphogen inducer, agonist of a morphogen receptor, or small molecule morphogenic activator is combined with other agents which promote or enhance the proliferation and differentiation of myogenic precursor cells into new and functional myocardium.
  • the present invention provides pharmaceutical compositions comprising a morphogen, or morphogen inducer, or agonist of a morphogen receptor, or small molecule morphogenic activator, in combination with one or more of a "muscle extract," conditioned medium from differentiated myotubes grown in culture, bFGF, IGF, PDGF, LIF, ACTH, MSH, or G-CSF.
  • a "muscle extract” conditioned medium from differentiated myotubes grown in culture
  • bFGF, IGF, PDGF, LIF, ACTH, MSH, or G-CSF conditioned medium from differentiated myotubes grown in culture
  • the ratios or the morphogenic and mitogenic agents may be adjusted based upon their activities, as disclosed in the literature or as determined through simple experimentation, to provide a therapeutically effective dosage of each compound in a single unit dosage.
  • the morphogenic and mitogenic agents in such a composition each preferably comprise at least about 1%>, and more preferably more than 5% or 10%, of the dry weight of the composition.
  • the compositions may, however, include other pharmaceutical carriers and active agents, as described above and, generally, in Remington's Pharmaceutical Sciences (Gennaro, A., ed.), Mack Pub., 1990, and, therefore, the morphogenic and mitogenic agents may each comprise a small fraction of the final weight of the pharmaceutical composition.
  • a currently preferred form of the morphogen useful herein, having improved solubility in aqueous solutions is a dimeric morphogenic protein comprising at least the C-terminal seven cysteine domain characteristic of the morphogen family, complexed with a peptide comprising a pro region of a member of the morphogen family, or a solubility-enhancing fragment thereof, or an allelic, species or other sequence variant thereof.
  • the dimeric morphogenic protein is complexed with two pro region peptides.
  • the dimeric morphogenic protein preferably is noncovalently complexed with the pro region peptides.
  • the pro region peptides preferably comprise at least the N-terminal eighteen amino acids that define the pro domain of a given naturally occurring morphogen, or an allelic or phylogenetic counterpart variant thereof. In other preferred embodiments, peptides defining substantially the full length pro domain 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 noncovalently associated with a cleaved pro domain peptide.
  • 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 within the pro domain polypeptide.
  • possible pro sequences include sequences defined by residues 30-292 (full length form); 48-292; and 158-292.
  • pro region comprises the full length form rather than a truncated form, such as the residues 48-292 truncated form, in that residues 30-47 show sequence homology to the N-terminal portions of other morphogens, and currently are believed to have particular utility in enhancing complex stability for all morphogens.
  • pro domains include peptides comprising at least the N-terminal fragment, e.g., amino acid residues 30-47 of a naturally occurring morphogen pro domain, or a biosynthetic variant thereof that retains the solubility and/or stability enhancing properties of the naturally-occurring peptide.
  • useful sequences encoding the pro region can 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.
  • useful pro region peptides include polypeptide chains comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a DNA or RNA sequence encoding at least the N-terminal eighteen amino acids of the pro region sequence for OPl or OP2, e.g., nucleotides 136-192 and 152-211 of SEQ ID NOs: 15 and 19, respectively.
  • Morphogens are expressed from mammalian cells as soluble complexes. Typically, however the complex is disassociated during purification, generally by exposure to denaturants often added to the purification solutions, such as detergents, alcohols, organic solvents, chaotropic agents and compounds added to reduce the pH of the solution.
  • denaturants such as detergents, alcohols, organic solvents, chaotropic agents and compounds added to reduce the pH of the solution.
  • the method is rapid, reproducible and yields isolated soluble morphogen complexes in substantially pure form. Soluble morphogen complexes can be isolated from conditioned media using a simple, three step chromatographic protocol performed in the absence of denaturants.
  • 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 includes an immunoaffinity column, created using standard procedures and, for example, using antibody specific for a given morphogen pro domain (complexed, for example, to a protein A-conjugated Sepharose column). Protocols for developing immunoaffinity columns are well described in the art (see, for example, Guide to Protein Purification. M. Lieber, ed., Academic Press, San Diego, 1990, particularly sections VII and XI thereof).
  • OP 1 was expressed in mammalian (CHO, Chinese hamster ovary) cells as described in the art (see, for example, international application US90/05903 (WO91/05802).
  • the CHO cell conditioned media containing 0.5% FBS was initially purified using Immobilized Metal- Ion Affinity Chromatography (IMAC).
  • IMAC Immobilized Metal- Ion Affinity Chromatography
  • the soluble OPl complex from conditioned media binds very selectively to the Zn-IMAC resin and a high concentration of imidazole (50 mM imidazole, pH 8.0) is required for the effective elution of the bound complex.
  • the Zn-IMAC step separates the soluble OPl from the bulk of the contaminating serum proteins that elute in the flowthrough and 35 mM imidazole wash fractions.
  • the Zn-IMAC purified soluble OPl is next applied to an S- Sepharose cation-exchange column equilibrated in 20 mM NaP0 4 (pH 7.0) with 50 mM NaCl.
  • This S-Sepharose step serves to further purify and concentrate the soluble OPl 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 can be isolated from one or more body fluids, including serum, cerebrospinal fluid or peritoneal fluid.
  • IMAC was performed using Chelating- Sepharose (Pharmacia) that had been charged with three column volumes of 0 2 M ZnS0 4 The conditioned media was titrated to pH 7 0 and applied directly to the Zn-IMAC resin equilibrated in 20 mM HEPES (pH 7 0) with 500 mM NaCl The Zn-IMAC resin was loaded with 80 mL of starting conditioned media per mL of resin After loading, the column was washed with equilibration buffer and most of the contaminating proteins were eluted with 35 mM imidazole (pH 7 0) in equilibration buffer The soluble OPl complex then is eluted with 50 mM imidazole (pH 8 0) in 20 mM HEPES and 500 mM NaCl
  • the 50 mM imidazole eluate containing the soluble OPl complex was diluted with nine volumes of 20 mM NaP0 4 (pH 7 0) and applied to an S-Sepharose (Pharmacia) column equilibrated in 20 mM NaP0 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 NaP0 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
  • the soluble OPl complex elutes with an apparent molecular weight of 1 10 kDa This agrees well with the predicted composition of the soluble OPl complex with one mature OPl 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 ITPLC 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 standard procedures (see, for example, Guide to Protein Purification. M. Deutscher, ed., Academic Press, San Diego, 1990, particularly pp. 602-613), and the identity of the isolated 36 kDa, 39 kDa proteins confirmed as mature morphogen and isolated, cleaved pro domain, respectively.
  • N-terminal sequencing of the isolated pro domain from mammalian cell produced OPl revealed two 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 N-termini for the mature sequence, beginning at residues 293, 300, 313, 315, 316, and 318, of SEQ ID NO: 16, all of which are active, as demonstrated by the standard bone morphogenesis assay set forth in published application W092/15323 as incorporated herein by reference.
  • soluble complexes can be formulated from purified pro domains and mature dimeric species.
  • Successful complex formation apparently requires association of the components under denaturing conditions sufficient to relax the folded structure of these molecules, without affecting disulfide bonds.
  • 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.
  • the 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 developing a suitable renaturing protocol readily can be determined by one having ordinary skill in the art.
  • One useful text on the subject is Guide to Protein Purification, M. Lieber, ed., Academic Press, San Diego, 1990, particularly section V. Complex formation also may be aided by addition of one or more chaperone proteins.
  • the stability of the highly purified soluble morphogen complex in a physiological buffer can be enhanced by any of a number of means.
  • the currently preferred method is by means of a pro region that comprises at least the first 18 amino acids of the pro sequence (e.g., residues 30-47 of SEQ ID NO: 16 for OP- 1), and preferably is the full length pro region. Residues 30-47 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).
  • 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.
  • ADDRESSEE TESTA, HURWITZ & THIBEAULT, LLP
  • each Xaa is independently selected from a group of one or more specified amino acids as defined in the specification.
  • Asp Trp Xaa lie Ala Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly 20 25 30
  • ORGANISM Homo sapiens
  • TISSUE TYPE HIPPOCAMPUS
  • MOLECULE TYPE protein
  • ORGANISM HOMO SAPIENS
  • TISSUE TYPE hippocampus
  • MOLECULE TYPE protein
  • ORGANISM Homo sapiens
  • F TISSUE TYPE
  • CAG GTG CTC CAG GAG CAC TTG GGC AGG GAA TCG GAT CTC TTC CTG CTC 681
  • GAC AGC CGT ACC CTC TGG GCC TCG GAG GAG GGC TGG CTG GTG TTT GAC 729
  • CTGCAGCAAG TGACCTCGGG TCGTGGACCG CTGCCCTGCC CCCTCCGCTG CCACCTGGGG 60
  • AAGCATGTAA GGGTTCCAGA AACCTGAGCG TGCAGCAGCT GATGAGCGCC CTTTCCTTCT 1593
  • ORGANISM Homo sapiens
  • TISSUE TYPE HIPPOCAMPUS
  • GCCAGGCACA GGTGCGCCGT CTGGTCCTCC CCGTCTGGCG TCAGCCGAGC CCGACCAGCT 60
  • GAG CAC TCC AAC AGG GAG TCT GAC TTG TTC TTT TTG GAT CTT CAG ACG 641 Glu His Ser Asn Arg Glu Ser Asp Leu Phe Phe Leu Asp Leu Gin Thr 170 175 180
  • CTTTCCCAGT TCCTCTGTCC TTCATGGGGT TTCGGGGCTA TCACCCCGCC CTCTCCATCC 1499 TCCTACCCCA AGCATAGACT GAATGCACAC AGCATCCCAG AGCTATGCTA ACTGAGAGGT 1559
  • CTCTGCACCA TTCATTGTGG CAGTTGGGAC ATTTTTAGGT ATAACAGACA CATACACTTA 1739

Abstract

La présente invention fournit des procédés ainsi que des médicaments pour le traitement des sujets ayant perdu, ou risquent de perdre ou d'endommager leur tissu myocardite. Ces procédés impliquent l'administration de certains morphogènes, d'inducteurs de ces morphogènes, d'agonistes des récepteurs morphogéniques correspondants, ou bine d'activateurs morphogéniques de petites molécules; ou alors, recourir à l'implantation des cellules induites par ces agents. Les morphogènes utiles dans cette invention comprennent OP1, CBMP-2A (BMP-2), CBMP-2B (BMP-4), et d'autres membres de la famille des morphogènes appartenant à la superfamille des facteurs de croissance et de différentiation TGFβ.
PCT/US1997/023611 1996-12-20 1997-12-19 Traitement local du myocarde mammifere avec un morphogene, ou avec des cellules precurseurs myogeniques morphogeniquement traitees WO1998027995A1 (fr)

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EP97953356A EP0952845A1 (fr) 1996-12-20 1997-12-19 Traitement local du myocarde mammifere avec un morphogene, ou avec des cellules precurseurs myogeniques morphogeniquement traitees
JP52899898A JP2001507354A (ja) 1996-12-20 1997-12-19 局所的モルフォゲンまたは形態形成処理された筋形成前駆体細胞による哺乳動物心筋層の処置
AU57119/98A AU741350B2 (en) 1996-12-20 1997-12-19 Treatment of mammalian myocardium with morphogen locally, or with morphogenically-treated myogenic precursor cells
CA002275436A CA2275436A1 (fr) 1996-12-20 1997-12-19 Traitement local du myocarde mammifere avec un morphogene, ou avec des cellules precurseurs myogeniques morphogeniquement traitees

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WO2001007568A2 (fr) * 1999-07-23 2001-02-01 Diacrin, Inc. Cellules musculaires et leur utilisation dans la reparation cardiaque
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WO2005028616A2 (fr) * 2003-07-24 2005-03-31 Caritas St. Elizabeth's Medical Center Of Boston, Inc. Compositions morphogenes et leurs methodes d'utilisation pour le traitement de maladies cardiaques
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AU741350B2 (en) 2001-11-29
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AU5711998A (en) 1998-07-17
JP2001507354A (ja) 2001-06-05

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