This invention relates to morphogenic proteins which can induce tissue morphogenesis in mammals; to methods of identifying these proteins and obtaining them from natural sources or producing synthetic forms of these proteins by expressing recombinant DNA encoding the proteins; to the fabrication of tissue-specific acellular matrices; and to methods for promoting tissue stasis, repair and regeneration, and methods for increasing progenitor cell populations using these proteins.
Cell differentiation is the central characteristic of morphogenesis which initiates in the embryo, and continues to various degrees throughout the life of an organism in adult tissue repair and regeneration mechanisms. The degree of morphogenesis in adult tissue varies among different tissues and is related, among other things, to the degree of cell turnover in a given tissue. On this basis, tissues can be divided into three broad categories: (1) tissues with static cell populations such as nerve and skeletal muscle where there is no cell division and most of the cells formed during early development persist throughout adult life; (2) tissues containing conditionally renewing populations such as liver where there is generally little cell division but, in response to an appropriate stimulus, cells can divide to produce daughters of the same differentially defined type; and (3) tissues with permanently renewing populations including blood, testes and stratified squamous epithelia which are characterized by rapid and continuous cell turnover in the adult. Here, the terminally differentiated cells have a relatively short life span and are replaced through proliferation of a distinct subpopulation of cells, known as stem or progenitor cells.
The cellular and molecular events which govern the stimulus for differentiation of these cells is an area of intensive research. In the medical field, it is anticipated that the discovery of factor(s) which control cell differentiation and tissue morphogenesis will significantly advance medicine""s ability to repair and regenerate diseased or damaged mammalian tissues and organs. Particularly useful areas include reconstructive surgery and in the treatment of tissue degenerative diseases including arthritis, emphysema, osteoporosis, cardiomyopathy, cirrhosis, and degenerative nerve diseases.
A number of different factors have been isolated in recent years which appear to play a role in cell differentiation. Some of these factors are gene transcription activators such as the NOTCH gene, identified in Drosophila and the related XOTCH gene identified in Xenopus, as well as a number of transcription activators identified in Caenorhabditis elegans. 
The hemopoietic system, because of its continually renewing cell population, is an area of concentrated study. Factors identified in this system which may be involved in cell renewal include interleukin 3 (IL-3), erythropoietin, the CSFs (GM-CSF, G-CSF, M-CSF et al.) and various stem cell growth factors.
Other proteins thought to play a role in cell differentiation include proteins that are members of the family of insulin-like growth factors (IGF), members of the family of heparin-binding growth factors, (e.g., FGFxe2x80x94acidic and basic fibroblast growth factors, and ECDGFxe2x80x94embryonal carcinoma-derived growth factor) as well as several transforming oncogenes (hst and int-2, see for example, Heath et al., (1988), J. Cell Sci. Suppl. 10:256-256.) DIF (Differentiation Inducing Factor), identified in Dictyostelium discoideum, is another bioregulatory protein, directing prestock cell differentiation in that organism.
The structurally related proteins of the TGF-xcex2 superfamily of proteins also have been identified as involved in a variety of developmental events. For example, TGF-xcex2 and the polypeptides of the inhibin/activin group appear to play a role in the regulation of cell growth and differentiation. MIS (Mullerian Inhibiting Substance) causes regression of the Mullerian duct in development of the mammalian male embryo, and DPP, the gene product of the Drosophila decapentaplegic complex is required for appropriate dorsal-ventral specification. Similarly, Vg-1 is involved in mesoderm induction in Xenopus, and Vgr-1 has been identified in a variety of developing murine tissues.
Another source that has revealed a wealth of information is in the area of bone morphogenesis. The development and study of a bone model system has identified the developmental cascade of bone differentiation as consisting of chemotaxis of mesenchymal cells, proliferation of these progenitor cells, differentiation of these cells into cartilage, vascular invasion, bone formation, remodeling, and finally, marrow differentiation (Reddi (1981) Collagen Rel. Res. 1:209-206). Proteins capable of inducing endochondral bone formation in a mammal when implanted in association with a matrix now have been identified in a number of different mammalian, species, as have the genes encoding these proteins, (see, for example, U.S. Pat. No. 4,968,590; U.S. Pat. No. 5,011,691; and U.S. Ser. No. 07/599,543, filed Oct. 18, 1990, and subseqently abandoned in favor of U.S. Pat. No. 5,266,683). These proteins, which share significant amino acid sequence homology with one another as well as structural similarities with various members of the TGF-xcex2 super family of proteins, have been shown to induce endochondral bone formation and/or bone cartilage formation when implanted in a mammal in association with a suitably modified matrix. Proteins capable of inducing a similar developmental cascade of tissue morphogenesis of other tissues have not been identified.
It is an object of this invention to provide morphogenic proteins (xe2x80x9cmorphogensxe2x80x9d), and methods for identifying these proteins, which are capable of inducing the developmental cascade of tissue morphogenesis for a variety of tissues in mammals different from bone or bone cartilage. This morphogenic activity includes the ability to induce proliferation and differentiation of progenitor cells, and the ability to support and maintain the differentiated phenotype through the progression of events that results in the formation of adult tissue. Another object is to provide genes encoding these proteins as well as methods for the expression and isolation of these proteins, from either natural sources or biosynthetic sources, using recombinant DNA techniques. Still another object is to provide tissue-specific acellular matrices that may be used in combination with these proteins, and methods for their production. Other objects include providing methods for increasing a progenitor cell population in a mammal, methods for stimulating progenitor cells to differentiate in vivo or in vitro and maintain their differentiated phenotype, methods for inducing tissue-specific growth in vivo and methods for the replacement of diseased or damaged tissue in vivo. These and other objects and features of the invention will be apparent from the description, drawings, and claims which follow.
This invention provides morphogenic proteins (xe2x80x9cmorphogensxe2x80x9d) capable of inducing the developmental cascade of tissue morphogenesis in a mammal. In particular, these proteins are capable of inducing the proliferation of uncommitted progenitor cells, and inducing the differentiation of these stimulated progenitor cells in a tissue-specific manner under appropriate environmental conditions. In addition, the morphogens are capable of supporting the growth and maintenance of these differentiated cells. These morphogenic activities allow the proteins of this invention to initiate and maintain the developmental cascade of tissue morphogenesis in an appropriate, morphogenically permissive environment, stimulating stem cells to proliferate and differentiate in a tissue-specific manner, and inducing the progression of events that culminate in new tissue formation. These morphogenic activities also allow the proteins to stimulate the xe2x80x9credifferentiationxe2x80x9d of cells previously induced to stray from their differentiation path. Under appropriate environmental conditions it is anticipated that these morphogens also may stimulate the xe2x80x9cdedifferentiationxe2x80x9d of committed cells (see infra.)
In one aspect of the invention, the proteins and compositions of this invention are useful in the replacement of diseased or damaged tissue in a mammal, particularly when the damaged tissue interferes with normal tissue or organ function. Accordingly, it is anticipated that the proteins of this invention will be useful in the repair of damaged tissue such as, for example, damaged lung tissue resulting from emphysema, cirrhotic kidney or liver tissue, damaged heart or blood vessel tissue, as may result from cardiomyopathies and/or atherothrbmbotic or cardioembolic strokes, damaged stomach tissue resulting from ulceric perforations or their repair, damaged neural tissue as may result from physical injury, degenerative diseases such as Alzheimer""s disease or. multiple sclerosis or strokes, damaged dentin tissue as may result from disease or mechanical injury, and damaged cartilage and ligament tissue. When the proteins of this invention are provided to, or their expression stimulated at, a tissue-specific locus, the developmental cascade of tissue morphogenesis is induced (see infra). Cells stimulated ex vivo by contact with the proteins or agents capable of stimulating morphogen expression in these cells also may be provided to the tissue locus. In these cases the existing tissue provides the necessary matrix requirements, providing a suitable substratum for the proliferating and differentiating cells in a morphogenically permissive environment, as well as providing the necessary signals for directing the tissue-specificity of the developing tissue. Alternatively, the proteins or stimulated cells may be combined with a formulated matrix and implanted as a device at a locus in vivo. The formulated matrix should be a biocompatible, preferably biodegradable, appropriately modified tissue-specific acellular matrix having the characteristics described below.
In many instances, the loss of tissue function results from scar tissue, formed in response to an initial or repeated injury to the tissue. The degree of scar tissue formation generally depends on the regenerative properties of the injured tissue, and on the degree and type of injury. Thus, in another aspect, the invention includes morphogens that may be used to prevent or substantially inhibit the formation of scar tissue by providing the morphogens, or morphogen-stimulated cells, to a newly injured tissue loci (see infra).
The morphogens of this invention also may be used to increase or regenerate a progenitor or stem cell population in a mammal. For example, progenitor cells may be isolated from an individual""s bone marrow, stimulated ex vivo for a time and at a morphogen concentration sufficient to induce the cells to proliferate, and returned to the bone marrow. Other sources of progenitor cells that may be suitable include biocompatible cells obtained from a cultured cell line, stimulated in culture, and subsequently provided to the body. Alternatively, the morphogen may be provided systemically, or implanted, injected or otherwise provided to a progenitor cell population in an individual to induce its mitogenic activity in vivo. For example, an agent capable of stimulating morphogen-expression in the progenitor cell population of interest may be provided to the cells in vivo, for example systemically, to induce mitogenic activity. Similarly, a particular population of hemopoietic stem cells may be increased by the morphogens of this invention, for example by perfusing an individual""s blood to extract the cells of interest, stimulating these cells ex vivo, and returning the stimulated cells to the blood. It is anticipated that the ability to augment an individual""s progenitor cell population will significantly enhance existing methods for treating disorders resulting from a loss or reduction of a renewable cell population. Two particularly significant applications include the treatment of blood disorders and impairment or loss of immune function. Other cell populations whose proliferation may be exploited include the stem cells of the epidermis, which may be used in skin tissue regeneration, and the stem cells of the gastrointestinal lining for healing of ulcers.
In still another aspect of the invention, the morphogens also may be used to support the growth and maintenance of differentiated cells, inducing existing differentiated cells to continue expressing their phenotype. It is anticipated that this activity will be particularly useful in the treatment of tissue disorders where loss of function is caused by cells becoming senescent or quiescent, such as may occur in osteoporosis. Application of the protein directly to the cells to be treated, or providing it by systemic injection, can be used to stimulate these cells to continue expressing their phenotype, thereby significantly reversing the effects of the dysfunction (see infra). Alternatively, administration of an agent capable of stimulating morphogen expression in vivo also may be used. In addition, the morphogens of this invention also may be used in gene therapy protocols to stimulate the growth of quiescent cells, thereby potentially enhancing the ability of these cells to incorporate exogenous DNA.
In yet another aspect of the invention, the morphogens of this invention also may be used to induce xe2x80x9credifferentiationxe2x80x9dof cells that have strayed from their differentiation pathway, such as can occur during Tumorigenesis. It is anticipated that this activity of the proteins will be particularly useful in treatments to reduce or substantially inhibit the growth of neoplasms. The method also is anticipated to induce the de-and re-differentiation of these cells. As described supra, the proteins may be provided to the cells directly or systemically, or an agent capable of stimulating morphogen expression in vivo may be provided.
Finally, modulations of endogenous morphogen levels may be monitored as part of a method for detecting tissue dysfunction. Specifically, modulations in endogenous morphogen levels are anticipated to reflect changes in tissue or organ stasis, and can be followed by monitoring fluctuations in the body""s natural antibody titer to morphogens.
The morphogenic proteins and compositions of this invention can be isolated from a variety of naturally-occurring sources, or they may be constructed biosynthetically using conventional recombinant DNA technology. Similarly, the matrices may be derived from organ-specific tissue, or they may be formulated synthetically, as described below.
A key to these developments was the discovery and characterization of naturally-occurring osteogenic proteins followed by observation of their remarkable properties. These proteins, originally isolated from bone, are capable of inducing the full developmental cascade of bone formation, including vascularization, mineralization, and bone marrow differentiation, when implanted in a mammalian body in association with a suitably modified matrix. Native proteins capable of inducing this developmental cascade, as well as DNA sequences encoding these proteins now have been isolated and characterized for a number of different species (e.g., OP-1 (comprising sequences shown in Seq. ID No. 5 or Seq. ID No. 6), OP-2 (comprising sequences shown in Seq. ID No. 7 or Seq. ID No. 8), and CBMP-2 (comprising sequences shown in Seq. ID No. 9 or Seq. ID No. 10). See, for example, U.S. Pat. Nos. 4,968,590 and 5,011,691; U.S. Pat. No. 4,975,526 and U.S. Ser. Nos. 07/600,024 and 07/599,543, both filed Oct. 18, 1990 and both subsequently abandoned in favor of U.S. Pat. No. 5,266,683; Sampath et al. (1990) J. Bio. Chem 265:13198-13205 and Ozkaynak, et al. (1990) EMBO 9:2085-2 093). The mature forms of these proteins share substantial amino acid sequence homology, especially in the C-terminal regions of the mature proteins. In particular, the proteins share a conserved six or seven cysteine skeleton in this region (e.g., the linear arrangement of these C-terminal cysteine residues is essentially conserved in the different proteins, in addition to other, apparently required amino acids (see Table II, infra)).
Polypeptide chains not normally associated with bone or bone formation, but sharing substantial amino acid sequence homology with the C-terminus of the osteogenic proteins, including the conserved six or seven cysteine skeleton, also have been identified as competent for inducing bone in mammals. Among these are amino acid sequences identified in Drosophila and Xenopus, (e.g., DPP, comprising Seq. ID No. 11, and Vgl, comprising Seq. ID No. 12; see, for example, U.S. Pat. No. 5,011,691 and Table II, infra). In addition, non-native biosynthetic constructs designed based on extrapolation from these sequence homologies, including the conserved six or seven cysteine skeleton, have been shown to induce endochondral bone formation in mammals when implanted in association with an appropriate matrix (See Table III, infra and U.S. Pat. No. 5,011,691).
It has now been discovered that this xe2x80x9cfamilyxe2x80x9d of proteins sharing substantial amino acid sequence homology and the conserved six or seven cysteine skeleton are true morphogens, capable of inducing, in addition to bone and bone cartilage, tissue-specific morphogenesis for a variety of other organs and tissues. The proteins apparently bind to surface receptors or otherwise contact and interact with progenitor cells, predisposing or stimulating the cells to proliferate and differentiate in a morphogenically permissive environment. The morphogens are capable of inducing the developmental cascade of cellular and molecular events that culminate in the formation of new organ-specific tissue, including any vascularization, connective tissue formation, and nerve ennervation as required by the naturally occurring tissue.
It also has been discovered that the way in which the cells differentiate, whether, for example, they differentiate into bone-producing osteoblasts, hemopoietic cells, or liver cells, depends on the nature of their local environment (see infra). Thus, in addition to requiring a suitable substratum on which to anchor, the proliferating and differentiating cells also require appropriate signals to direct their tissue-specificity. These signals may take the form of cell surface markers. Thus, in a suitable, typically bone powder-derived matrix presented in a vascular supported environment, the morphogen-activated progenitor cells differentiate not only through the bone-producing cascade including transformation to chondrocytes and then to osteoblasts, including formation of the necessary associated vascular network.
When the morphogens (or progenitor cells stimulated by these morphogens) are provided at a tissue-specific locus (e.g., by systemic injection or by implantation or injection at a tissue-specific locus, or by administration of an agent capable of stimulating morphogen expression in vivo), the existing tissue at that locus, whether diseased or damaged, has the capacity of acting as a suitable matrix. Alternatively, a formulated matrix may be externally provided together with the stimulated progenitor cells or morphogen, as may be necessary when the extent of injury sustained by the damaged tissue is large. The matrix should be a biocompatible, suitably modified acellular matrix having dimensions such that it allows the influx, differentiation, and proliferation of migratory progenitor cells, and is capable of providing a morphogenically permissive environment (see infra). The matrix preferably is tissue-specific, and biodegradable.
Formulated matrices may be generated from dehydrated organ-specific tissue, prepared for example, by treating the tissue with solvents to substantially remove the non-structural components from the tissue. Alternatively, the matrix may be formulated synthetically using a biocompatible, preferably in vivo biodegradable, structural polymer such as collagen in association with suitable tissue-specific cell attachment factors. Currently preferred structural polymers comprise tissue-specific collagens. Currently preferred cell attachment factors include glycosaminoglycans and proteoglycans. The matrix further may be treated with an agent or agents to increase the number of pores and micropits on its surfaces, so as to enhance the influx, proliferation and differentiation of migratory progenitor cells from the body of the mammal.
Among the proteins useful in this invention are proteins originally identified as osteogenic proteins, such as the OP-1, OP-2 and (comprising sequences shown, respectively, in Seq. ID Nos. 5 and 6, 7 and 8, 9 and 10), as well as amino acid sequence related proteins such as DPP (from Drosophila; comprising a sequence shown in Seq. ID No. 11), Vgl (from Xenopus; comprising a sequence shown in Seq. ID No. 12), Vgr-1 (from mouse comprising a sequence shown in Seq. ID No. 13, and the recently identified GDF-1 protein (comprising a sequence shown in Seq. ID No. 14). The members of this family, which include members of the TGF-xcex2 super-family of proteins, share""substantial amino acid sequence homology in their C-terminal regions. Table I, below, describes the various morphogens identified to date, including their nomenclature as used herein, and Seq. ID references.
The OP-2 proteins have an additional cysteine residue in this region (position 41 of Seq. ID Nos. 7 and 8), in addition to the conserved cysteine skeleton in common with the other proteins in this family. The GDF-1 protein has a four amino acid insert within the conserved skeleton (residues 44-47 of Seq. ID No. 14) but this insert likely does not interfere with the relationship of the cysteines in the folded structure. In addition, the CBMP2 proteins (comprising Seq. ID Nos. 9 and 10) are missing one amino acid residue within the cysteine skeleton.
The morphogens are inactive when reduced, but are active as oxidized homodimers and as various oxidized heterodimers. Thus, as defined herein, a morphogen of this invention is a dimeric protein comprising a pair of polypeptide chains, wherein each polypeptide chain comprises at least the C-terminal six cysteine skeleton defined by residues 43-139 of Seq. ID No. 5, including functionally equivalent arrangements of these cysteines (e.g., amino acid insertions or deletions which alter the linear arrangement of the cysteines in the sequence but not their relationship in the folded structure), such that, when the polypeptide chains are folded, the dimeric protein species comprising the pair of polypeptide chains has the appropriate three-dimensional structure, including the appropriate intra- or inter-chain disulfide bonds such that the protein is capable of acting as a morphogen as defined herein. Specifically, the protein is capable of all of the following biological functions in a morphogenically permissive environment: stimulating proliferation of progenitor cells; stimulating the differentiation of progenitor cells; stimulating the proliferation of differentiated cells; and supporting the growth and maintenance of differentiated cells, including the xe2x80x9credifferentiationxe2x80x9d of these cells. In addition, it is also anticipated that the morphogens of this invention will be capable of inducing dedifferentiation of committed cells under appropriate environmental conditions.
In one preferred aspect, the morphogens of this invention comprise one of two species of generic amino acid sequences: Generic Sequence 1 (Seq. ID No. 1) or Generic Sequence 2 (Seq. ID No. 2); where each Xaa indicates one of the 20 naturally-occurring L-isomer, xcex1-amino acids or a derivative thereof. Generic Sequence 1 comprises the conserved six cysteine skeleton and Generic Sequence 2 comprises the conserved six cysteine skeleton plus the additional cysteine identified in OP-2 (see, e.g., Seq. ID Nos. 7 and 8). In another preferred aspect, these sequences further comprise the following sequence (Seq. ID No. 17) at their N-terminus:
Preferred amino acid sequences within the foregoing generic sequences include: Generic Sequence 3 (Seq. ID No. 3) and Generic Sequence 4 (Seq. ID No. 4), listed below, which accommodate the homologies shared among the various members of this morphogen family identified to date, as well as the amino acid sequence variation among them. Note that these generic sequences allow for an additional cysteine at position 41 or 46 in Generic Sequences 3 or 4, respectively (Seq. ID Nos. 3 and 4), providing an appropriate cysteine skeleton where inter- or intramolecular disulfide bonds can form, and contain certain critical amino acids which influence the tertiary structure of the proteins.
wherein each Xaa is independently selected from a group of one or more specified amino acids defined as follows: xe2x80x9cRes.xe2x80x9d means xe2x80x9cresiduexe2x80x9d and Xaa at res.4=(Ser, Arg, Asp or Glu); Xaa at res.6=(Arg, Gln, Ser or Lys); Xaa at res.7=(Asp or Glu); Xaa at res.8=(Leu or Val); Xaa at res.11=(Gln, Leu, Asp, His or Asn); Xaa at res.12=(Asp, Arg or Asn.); Xaa at res.14=(Ile or Val); Xaa at res.15=(Ile or Val); Xaa at res.18=(Glu, Gln, Leu, Lys, Pro or Arg); Xaa at res.20=(Tyr or Phe); Xaa at res.21=(Ala, Ser, Asp, Met, His, Leu or Gln); Xaa at res.23=(Tyr, Asn or Phe); Xaa at res.26=.(Glu, His, Tyr, Asp or Gln); Xaa at res.28=(Glu, Lys, Asp or Gln); Xaa at res.30=(Ala, Ser, Pro or Gln); Xaa at res.31=(Phe, Leu or Tyr); Xaa at res.33=(Leu or Val); Xaa at res.34=(Asn, Asp, Ala or Thr); Xaa at res.35=(Ser, Asp, Glu, Leu or Ala); Xaa at res.36=(Tyr, Cys, His, Ser or Ile); Xaa at res.37=(Met, Phe, Gly or Leu); Xaa at res.38=(Asn or Ser); Xaa at res.39=(Ala, Ser or Gly); Xaa at res.40=(Thr, Leu or Ser); Xaa at res.44=(Ile or Val); Xaa at res.45=(Val or Leu); Xaa at res.46=(Gln or Arg); Xaa at res.47=(Thr, Ala or Ser); Xaa at res.49=(Val or Met); Xaa at res.50=(His or Asn); Xaa at res.51=(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.52=(Ile, Met, Asn, Ala or Val); Xaa at res.53=(Asn, Lys, Ala or Glu); Xaa at res.54=(Pro or Ser); Xaa at res.55=(Glu, Asp, Asn, or Gly); Xaa at res.56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser or Ala); Xaa at res.57=(Val, Ala or Ile); Xaa at res.58=(Pro or Asp); Xaa at res.59=(Lys or Leu); Xaa at res.60=(Pro or Ala); Xaa at res.63=(Ala or Val); Xaa at res.65=(Thr or Ala); Xaa at res.66=(Gln, Lys, Arg or Glu); Xaa at res.67=(Leu, Met or Val); Xaa at res.68=(Asn, Ser or Asp); Xaa at res.69=(Ala, Pro or Ser); Xaa at res.70=(Ile, Thr or Val); Xaa at res.71=(Ser or Ala); Xaa at res.72=(Val or Met); Xaa at res.74=(Tyr or Phe); Xaa at res.75=(Phe, Tyr or Leu); Xaa at res.76=(Asp or Asn); Xaa at res.77=(Asp, Glu, Asn or Ser); Xaa at res.78=(Ser, Gln, Asn or Tyr); Xaa at res.79=(Ser, Asn, Asp or Glu); Xaa at res.80=(Asn, Thr or Lys); Xaa at res.82=(Ile or Val); Xaa at res.84=(Lys or Arg); Xaa at res.85=(Lys, Asn, Gln or His); Xaa at res.86=(Tyr, Ala or His); Xaa at res.87=(Arg, Gln or Glu).; Xaa at res.88=(Asn, Glu or Asp); Xaa at res.90=(Val, Thr or Ala); Xaa at res.92=(Arg, Lys, Val, Asp or Glu); Xaa at res.93=(Ala, Gly or Glu); and Xaa at res.97=(His or Arg); and Generic Seq. 4:
wherein each Xaa is independently selected from a group of one or more specified amino acids as defined by the following: xe2x80x9cRes.xe2x80x9d means xe2x80x9cresiduexe2x80x9d and Xaa at res.2=(Lys or Arg); Xaa at res.3=(Lys or Arg); Xaa at res.4=(His or Arg); Xaa at res.5=(Glu, Ser, His, Gly, Arg or Pro); Xaa at res.9=(Ser, Arg, Asp or Glu); Xaa at res.11=(Arg, Gln, Ser or Lys); Xaa at res.12=(Asp or Glu); Xaa at res.13=(Leu or Val); Xaa at res.16=(Gln, Leu, Asp, His or Asn); Xaa at res.17=(Asp, Arg, or Asn); Xaa at res.19=(Ile or Val); Xaa at res.20=(Ile or Val); Xaa at res.23=(Glu, Gln, Leu, Lys, Pro or Arg); Xaa at res.25=(Tyr or Phe); Xaa at res.26=(Ala, Ser, Asp, Met, His, Leu, or Gln); Xaa at res.28=(Tyr, Asn or Phe); Xaa at res.31=(Glu, His, Tyr, Asp or Gln); Xaa at res.33=Glu, Lys, Asp or Gln); Xaa at res.35=(Ala, Ser or Pro); Xaa at res.36=(Phe, Leu or Tyr); Xaa at res.38=(Leu or Val); Xaa at res.39=(Asn, Asp, Ala or Thr); Xaa at res.40=(Ser, Asp, Glu, Leu or Ala); Xaa at res.41=(Tyr, Cys, His, Ser or Ile); Xaa at res.42=(Met, Phe, Gly or Leu); Xaa at res.43=(Asn or Ser) Xaa at res.44=(Ala, Ser or Gly); Xaa at res.45=(Thr, Leu or Ser); Xaa at res.49=(Ile or Val); Xaa at res.50=(Val or Leu); Xaa at res.51=(Gln or Arg); Xaa at res.52=(Thr, Ala or Ser); Xaa at res.54=(Val or Met); Xaa at res.55=(His or Asn); Xaa at res.56=(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.57=(Ile, Met, Asn, Ala or Val); Xaa at res.58=(Asn, Lys, Ala or Glu); Xaa at res.59=(Pro or Ser); Xaa at res.60=(Glu, Asp, or Gly); Xaa at res.61=(Thr, Ala, Val, Lys, Asp, Tyr, Ser or Ala); Xaa at res.62=(Val, Ala or Ile); Xaa at res.63=(Pro or Asp); Xaa at res.64=(Lys or Leu); Xaa at res.65=(Pro or Ala); Xaa at res.68=(Ala or Val); Xaa at res.70=(Thr or Ala); Xaa at res.71=(Gln, Lys, Arg or Glu); Xaa at res.72=(Leu, Met or Val); Xaa at res.73=(Asn, Ser or Asp); Xaa at res.74=(Ala, Pro or Ser); Xaa at res.75=(Ile, Thr or Val); Xaa at res.76=(Ser or Ala); Xaa at res.77=(Val or Met); Xaa at res.79=(Tyr or Phe); Xaa at res.80=(Phe, Tyr or Leu); Xaa at res.81=(Asp or Asn); Xaa at res.82=(Asp, Glu, Asn or Ser); Xaa at res.83=(Ser, Gln, Asn or Tyr); Xaa at res.84=(Ser, Asn, Asp or Glu); Xaa at res.85=(Asn, Thr or Lys); Xaa at res.87=(Ile or Val); Xaa at res.89=(Lys or Arg); Xaa at res.90=(Lys, Asn, Gln or His); Xaa at res.91=(Tyr, Ala or His); Xaa at res.92=(Arg, Gln or Glu); Xaa at res.93=(Asn, Glu or Asp); Xaa at res.95=(Val, Thr or Ala); Xaa at res.97=(Arg, Lys, Val, Asp or Glu); Xaa at res.98=(Ala, Gly or Glu); and Xaa at res.102=(His or Arg).
Particularly useful sequences include the C-terminal seven-cysteine domains, shown in Table II, infra of Vgl (Seq. ID No. 12), Vgr-1 (Seq. ID No. 13), DPP (Seq. ID No. 11), OP-1 (residues 38-139 of Seq. ID Nos. 5 and 6), OP-2 (residues 38-139 of Seq. ID Nos. 7 and 8), CBMP-2A (Seq. ID No. 9), CBMP-2B (Seq. ID No. 10) and GDF-1 (Seq. ID No. 14). In addition, biosynthetic constructs designed from the generic sequences, such as COP-1, 3-5, 7, 16 (Seq. ID Nos. 18-23, respectfully (see U.S. Pat. No. 5,011,691 Table III, infra) also are useful. Others include CBMP3 and the inhibin/activin proteins. Accordingly, other useful sequences are those sharing at least 70% amino acid sequence homology, and preferably 80% homology with any of the sequences above. These are anticipated to include allelic and species variants and mutants, and biosynthetic muteins, as well as novel members of this morphogenic family of proteins.
The invention thus provides proteins comprising any of the polypeptide chains described above, whether isolated from naturally-occurring sources, or produced by recombinant DNA techniques, and includes allelic and species variants of these proteins, naturally-occurring or biosynthetic mutants thereof, as well as various truncated and fusion constructs. Deletion or addition mutants also are envisioned to be active (see infra), including those which may alter the conserved C-terminal cysteine skeleton, provided that the alteration does not functionally disrupt the relationship of these cysteines in the folded structure. Accordingly, such active forms are considered the equivalent of the specifically described constructs disclosed herein. The proteins may include forms having varying glycosylation patterns, varying N-termini, a family of related proteins having regions of amino acid sequence homology, and active truncated or mutated forms of native or biosynthetic proteins, produced by expression of recombinant DNA in host cells.
The morphogenic proteins can be expressed from intact or truncated CDNA or from synthetic DNAs in procaryotic or eucaryotic host cells, and purified, cleaved, refolded, and dimerized to form morphogenically active compositions. Currently preferred host cells include E. coli or mammalian cells, such as CHO, COS or BSC cells.
Thus, in view of this disclosure, skilled genetic engineers can isolate genes from CDNA or genomic libraries of various different 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 procaryotes and eucaryotes, to produce large quantities of active proteins capable of inducing tissue-specific cell differentiation and tissue morphogenesis in mammals including humans.
The invention thus further comprises these methods of inducing tissue-specific morphogenesis using the morphogenic proteins of this invention.