Renal failure can occur as a complication of trauma, shock, poisoning, acute pancreatitis, septicemia, chronic exposure to certain drugs, poisoning, and other causes. Acute tubular necrosis (ATN), the most common cause of acute renal failure, usually occurs after a period of inadequate blood flow to the peripheral organs. Anoxia or poisoning leads to death of tubular epithelial cells and progression to acute renal failure. Chronic analgesic nephritis, which results from prolonged exposure to combinations of phenacetin, aspirin, and acetominophen, may also be due to necrosis of tubular epithelial cells. See, Robbins et al., Basic Pathology, Third Edition, W. B. Saunders Co., Philadelphia, 1981, 421-456.
Ischemia- and nephrotoxin-induced renal damages are the main causes of acute renal failure and are characterized by structural and functional damages to renal tubular epithelial cells, predominantly to the proximal tubuli (Oliver et al., J. Clin. Invest. 30:1307-1439, 1951). Damage to the proximal tubular epithelium is repaired by a complex regeneration process. After cell desquamation, dedifferentiated proximal tubular cells proliferate and migrate into the denuded area of the basement membrane to establish a new epithelium (Wallin et al., Lab Invest. 66:474-484, 1992). In many respects, this nephrogenic repair process resembles the late stage of the development of nephrons, when the embryonic mesenchyme converts to a tubular epithelium (Wallin et al., ibid.; Hammermann et al., A. J. Physiol. 262:F523-532, 1992).
While a functional tubular epithelium may be regenerated in as little as 2 weeks, the clinical course of ATN is prolonged in many patients, and treatment consists of supportive care, including dialysis. Without adequate treatment, ATN results in death.
There remains a need in the art for compositions and methods for stimulating the proliferation of kidney tubule epithelial cells in vivo, and thereby improving kidney function.
The present invention provides materials and methods for improving kidney function or enhancing proliferation or survival of kidney tubule epithelial cells or epithelial cell precursors in a mammal.
Within one aspect of the invention there is provided a method of improving kidney function in a mammal in need thereof, comprising administering to the mammal a composition comprising a therapeutically effective amount of a zvegf4 protein or a zvegf4 protein-encoding polynucleotide in combination with a pharmaceutically acceptable delivery vehicle.
Within a second aspect of the invention there is provided a method of enhancing proliferation or survival of kidney tubule epithelial cells or epithelial cell precursors in a mammal, comprising administering to the mammal a composition comprising a therapeutically effective amount of a zvegf4 protein or a zvegf4 protein-encoding polynucleotide in combination with a pharmaceutically acceptable delivery vehicle.
Within certain embodiments of the above-disclosed methods, a zvegf4 protein is administered to the mammal. Within selected embodiments, the zvegf4 protein is a disulfide-bonded dimer of two polypeptide chains, each of the chains comprising residues 258-370 of SEQ ID NO:2, residues 250-370 of SEQ ID NO:2, or residues 246-370 of SEQ ID NO:2. Within other embodiments the zvegf4 protein is a disulfide-bonded dimer of two polypeptide chains, each of the chains consisting of residues X to 370 of SEQ ID NO:2, wherein X is an integer from 246 to 258, inclusive, and wherein the protein is optionally glycosylated.
Within other embodiments of the above-disclosed methods, a zvegf4 protein-encoding polynucleotide is administered to the mammal. Within selected embodiments, the polynucleotide encodes a polypeptide comprising residues 258-370 of SEQ ID NO:2, residues 19-370 of SEQ ID NO:2, or residues 1-370 of SEQ ID NO:2. Within other embodiments, the polynucleotide is a viral vector or plasmid.
Within other embodiments of the invention, the zvegf4 protein is a disulfide-bonded dimer of two polypeptide chains, each of the chains consisting of residues x-y of SEQ ID NO:2, inclusive, wherein the protein is optionally glycosylated, and wherein x is selected from the group consisting of 16, 17, 18, 19, 20, 21, 22, 24, 25, 35, 52, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 246, 250, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, and 263; and y is selected from the group consisting of 365, 366, 367, 368, 369, and 370.
Within other embodiments of the above-disclosed methods, the mammal is suffering from acute tubular necrosis.
These and other aspects of the invention will become evident upon reference to the following detailed description of the invention and the accompanying Figure.
The Figure is a Hopp/Woods hydrophilicity profile of the amino acid sequence shown in SEQ ID NO:2. The profile is based on a sliding six-residue window. Buried G, S, and T residues and exposed H, Y, and W residues were ignored. These residues are indicated in the figure by lower case letters.
xe2x80x9cConservative amino acid substitutionsxe2x80x9d are defined by the BLOSUM62 scoring matrix of Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992, an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins. As used herein, the term xe2x80x9cconservative amino acid substitutionxe2x80x9d refers to a substitution represented by a BLOSUM62 value of greater thanxe2x88x921. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. Preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
A xe2x80x9cpolypeptidexe2x80x9d is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as xe2x80x9cpeptidesxe2x80x9d.
A xe2x80x9cproteinxe2x80x9d is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless. Thus, a protein xe2x80x9cconsisting ofxe2x80x9d, for example, from 15 to 1500 amino acid residues may further contain one or more carbohydrate chains.
The terms xe2x80x9ctreatxe2x80x9d and xe2x80x9ctreatmentxe2x80x9d are used broadly to denote therapeutic and prophylactic interventions that favorably alter a pathological state, including alleviating symptoms thereof. Treatments include procedures that moderate or reverse the progression of, reduce the severity of, prevent, or cure a disease.
The term xe2x80x9czvegf4 proteinxe2x80x9d is used herein to denote a protein comprising the growth factor domain of a zvegf4 polypeptide (e.g., residues 258-370 of human zvegf4 (SEQ ID NO:2) or mouse zvegf4 (SEQ ID NO:4)), wherein said protein or a proteolytically activated form thereof is mitogenic for cells expressing cell-surface PDGF xcex1- and/or xcex2-receptor subunit. Zvegf4 has been found to activate the xcex1xcex1, xcex1xcex2, and xcex2xcex2 isoforms of PDGF receptor. Zvegf4 proteins include homodimers and heterodimers as disclosed below. Using methods known in the art, zvegf4 proteins can be prepared in a variety of forms, including glycosylated or non-glycosylated, pegylated or non-pegylated, with or without an initial methionine residue, and as fusion proteins as disclosed in more detail below.
A xe2x80x9czvegf4 protein-encoding polynucleotidexe2x80x9d is a polynucleotide that encodes, upon expression by a host cell, a zvegf4 polypeptide that is post-translationally processed to yield a dimeric zvegf4 protein as defined above. Post-translational processing events include, without limitation, disulfide bond formation, proteolysis (including secretory peptide removal), and carbohydrate addition. Those skilled in the art will recognize that the primary translation product of a zvegf4 protein-encoding polynucleotide will ordinarily differ in structure from the final protein. In addition, zvegf4 protein-encoding polynucleotides may include operably linked transcription promoters, terminators, and other genetic elements that provide for expression and/or maintenance of the polynucleotide within the host cell or delivery into the host cell.
All references cited herein are incorporated by reference in their entirety.
The present invention provides methods for improving kidney function in a patient using zvegf4. Zvegf4 is a protein that is structurally related to platelet-derived growth factor (PDGF) and the vascular endothelial growth factors (VEGF). This protein is also referred to as xe2x80x9cPDGF-Dxe2x80x9d (WIPO Publication WO 00/27879). Zvegf4 is a multi-domain protein with significant homology to the PDGF/VEGF family of growth factors.
Structural predictions based on the zvegf4 sequence and its homology to other growth factors suggests that the polypeptide can form homomultimers or heteromultimers that act on tissues by modulating cell proliferation, migration, differentiation, or metabolism. Experimental evidence supports these predictions. Zvegf4 heteromultimers may comprise a polypeptide from another member of the PDGF/VEGF family of proteins, including VEGF, VEGF-B, VEGF-C, VEGF-D, zvegf3/ PDGF-C (WO 00/34474), PlGF (Maglione et al., Proc. Natl. Acad. Sci. USA 88:9267-9271, 1991), PDGF-A (Murray et al., U.S. Pat. No. 4,899,919; Heldin et al., U.S. Pat. No. 5,219,759), or PDGF-B (Chiu et al., Cell 37:123-129, 1984; Johnsson et al., EMBO J. 3:921-928, 1984).
The zvegf4 polypeptide chain comprises a growth factor domain and a CUB domain. The growth factor domain is characterized by an arrangement of cysteine residues and beta strands that is characteristic of the xe2x80x9ccystine knotxe2x80x9d structure of the PDGF family. The CUB domain shows sequence homology to CUB domains in the neuropilins (Takagi et al., Neuron 7:295-307, 1991; Soker et al., Cell 92:735-745, 1998), human bone morphogenetic protein-1 (Wozney et al., Science 242:1528-1534, 1988), porcine seminal plasma protein and bovine acidic seminal fluid protein (Romero et al., Nat. Struct. Biol. 4:783-788, 1997), and X. laevis tolloid-like protein (Lin et al., Dev. Growth Differ. 39:43-51, 1997).
A representative human zvegf4 polypeptide sequence is shown in SEQ ID NO:2, and a representative mouse zvegf4 polypeptide sequence is shown in SEQ ID NO:4. DNAs encoding these polypeptides are shown in SEQ ID NOS:1 and 3, respectively. Analysis of the amino acid sequence shown in SEQ ID NO:2 indicates that residues 1 to 18 form a secretory peptide. The CUB domain extends from residue 52 to residue 179. A propeptide-like sequence extends from residue 180 to either residue 245, residue 249 or residue 257, and includes four potential cleavage sites at its carboxyl terminus, monobasic sites at residue 245 and residue 249, a dibasic site at residues 254-255, and a target site for furin or a furin-like protease at residues 254-257. Protein produced in a baculovirus expression system showed cleavage between residues 249 and 250, as well as longer species with amino termini at residues 19 and 35. The growth factor domain extends from residue 258 to residue 370, and may include additional residues at the N-terminus (e.g., residues 250 to 257 or residues 246 to 257). Those skilled in the art will recognize that domain boundaries are somewhat imprecise and can be expected to vary by up to xc2x15 residues from the specified positions. Corresponding domains in mouse and other non-human zvegf4s can be determined by those of ordinary skill in the art from sequence alignments. Cleavage of full-length human zvegf4 with plasmin resulted in activation of the zvegf4 polypeptide. By Western analysis, a band migrating at approximately the same size as the growth factor domain was observed.
Signal peptide cleavage is predicted to occur in human zvegf4 after residue 18 (xc2x13 residues). Upon comparison of human and mouse zvegf4 sequences, alternative signal peptide cleavage sites are predicted after residue 23 and/or residue 24. This analysis suggests that the zvegf4 polypeptide chain may be cleaved to produce a plurality of monomeric species, some of which are shown in Table 1. In certain host cells, cleavage after Lys-255 is expected to result in subsequent removal of residues 254-255, although polypeptides with a carboxyl terminus at residue 255 may also be prepared. Cleavage after Lys-257 is expected to result in subsequent removal of residue 257. Actual cleavage patterns are expected to vary among host cells.
Zvegf4 can thus be prepared in a variety of multimeric forms comprising a zvegf4 polypeptide as disclosed above. These zvegf4 polypeptides include zvegf419-370, zvegf452-370, zvegf4246-370, zvegf4250-370, and zvegf4258-370. Variants and derivatives of these polypeptides can also be prepared as disclosed herein.
Expression of a zvegf4 polynucleotide in cultured mammalian cells results in the production of a disulfide-bonded, dimeric protein that may be proteolytically processed. The mitogenically active protein is generated upon proteolytic processing to remove the CUB and interdomain regions. An active growth factor domain dimer can be produced directly by expressing a truncated polynucleotide.
Zvegf4 proteins can be prepared as fusion proteins comprising amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, an affinity tag, or a targetting polypeptide. For example, a zvegf4 protein can be prepared as a fusion with an affinity tag to facilitate purification. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include, for example, a poly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase (Smith and Johnson, Gene 67:31, 1988), a Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985), substance P, FLAG(trademark) peptide (Hopp et al., Biotechnology 6:1204-1210, 1988), streptavidin binding peptide, maltose binding protein (Guan et al., Gene 67:21-30, 1987), cellulose binding protein, thioredoxin, ubiquitin, T7 polymerase, or other antigenic epitope or binding domain. Fusion of zvegf4 to, for example, maltose binding protein or glutatione S transferase can be used to improve yield in bacterial expression systems. In these instances the non-zvegf4 portion of the fusion protein ordinarily will be removed prior to use. Separation of the zvegf4 and non-zvegf4 portions of the fusion protein is facilitated by providing a specific cleavage site between the two portions. Such methods are well known in the art. Zvegf4 can also be fused to a targetting peptide, such as an antibody (including polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(abxe2x80x2)2 and Fab fragments, single chain antibodies, and the like) or other peptidic moiety that binds to a target tissue.
Variations can be made in the zvegf4 amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:4 to provide biologically active varaints of zvegf4 proteins. Such variations include amino acid substitutions, deletions, and insertions. In general, conservative amino acid substitutions are preferred. While not wishing to be bound by theory, it is believed that residues within regions 273-295 and 307-317 of human zveg4 (SEQ ID NO:2) may be involved in ligand-receptor interactions. The effects of amino acid sequence changes at specific positions in zvegf4 proteins can be assessed using procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244, 1081-1085, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991). Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989). Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204), region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988), and DNA shuffling as disclosed by Stemmer (Nature 370:389-391, 1994) and Stemmer (Proc. Natl. Acad. Sci. USA 91:10747-10751, 1994). The resultant mutant molecules are tested for receptor binding, mitogenic activity, or other properties (e.g., stimulation of growth factor production) to identify amino acid residues that are critical to these functions. Mutagenesis can be combined with high volume or high-throughput screening methods to detect biological activity of zvegf4 variant polypeptides.
Zvegf4 variants can be analyzed for receptor binding activity by a variety of methods well known in the art, including receptor competition assays (Bowen-Pope and Ross, Methods Enzymol. 109:69-100, 1985) and through the use of soluble receptors, including receptors produced as IgG fusion proteins (U.S. Pat. No. 5,750,375). Receptor binding assays can be performed on cell lines that contain known cell-surface receptors for evaluation. The receptors can be naturally present in the cell, or can be recombinant receptors expressed by genetically engineered cells.
Activity of zvegf4 variants can be measured in vitro using cultured cells. For example, mitogenic activity can be measured using known assays, including 3H-thymidine incorporation assays (as disclosed by, e.g., Raines and Ross, Methods Enzymol. 109:749-773, 1985 and Wahl et al., Mol. Cell Biol. 8:5016-5025, 1988), dye incorporation assays (as disclosed by, for example, Mosman, J. Immunol. Meth. 65:55-63, 1983 and Raz et al., Acta Trop. 68:139-147, 1997) or cell counts. Suitable mitogenesis assays measure incorporation of 3H-thymidine into (1) 20% confluent cultures to look for the ability of zvegf4 proteins to further stimulate proliferating cells, and (2) quiescent cells held at confluence for 48 hours to look for the ability of zvegf4 proteins to overcome contact-induced growth inhibition. Suitable dye incorporation assays include measurement of the incorporation of the dye Alamar blue (Raz et al., ibid.) into target cells. See also, Gospodarowicz et al., J. Cell. Biol. 70:395-405, 1976; Ewton and Florini, Endocrinol. 106:577-583, 1980; and Gospodarowicz et al., Proc. Natl. Acad. Sci. USA 86:7311-7315, 1989. Activity can also be assayed by measuring metabolic changes in target cells, such as changes in production of other proteins (including other growth factors) by immunological assays.
The biological activities of zvegf4 variants can be studied in non-human animals by administration of exogenous protein or by expression of zvegf4 variant polynucleotides. Viral delivery systems (disclosed below) can be employed. Zvegf4 variants can be administered or expressed individually, in combination with other zvegf4 proteins, or in combination other compounds, including other growth factors. Test animals are monitored for changes in such parameters as clinical signs, body weight, blood cell counts, clinical chemistry, histopathology, and the like.
Zvegf4 proteins, including full-length polypeptides, variant polypeptides, biologically active fragments, and fusion proteins, can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells (including cultured cells of multicellular organisms). Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al., eds., Current Protocols in Molecular Biology, Green and Wiley and Sons, NY, 1993. In general, a DNA sequence encoding a zvegf4 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers. See, for example, WO 00/34474. Exemplary expression systems include yeasts, such as Saccharomyces cerevisiae (see, e.g., U.S. Pat. No. 5,527,668) or Pichia methanolica (U.S. Pat. Nos. 5,716,808, 5,736,383, 5,854,039, and 5,955,349); mammalian cells, such as baby hamster kidney (BHK) cells (ATCC(trademark) No. CRL 1632 or No. CRL 10314), COS-1 cells (ATCCT(trademark) No. CRL 1650), COS-7 cells (ATCC(trademark) No. CRL 1651), 293 cells (ATCC(trademark) No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) or Chinese hamster ovary cells (e.g. CHO-K1, ATCC(trademark) No. CCL 61; or CHO DG44, Chasm et al., Som. Cell. Molec. Genet. 12:555, 1986); baculovirus (Luckow et al., J. Virol. 67:4566-4579, 1993; available in kit form BAC-TO-BAC(trademark) kit; LIFE TECHNOLOGIES(trademark), Rockville, Md.)); and bacterial cells (e.g., E. coli). Suitable cell lines are known in the art and available from public depositories such as the AMERICAN TYPE CULTURE COLLECT(trademark), Manassas, Va.
Zvegf4 proteins can comprise non-naturally occurring amino acid residues. Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-809, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-10149, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271:19991-19998, 1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).
Zvegf4 polypeptides or fragments thereof can also be prepared through chemical synthesis according to methods known in the art, including exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid Phase Peptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, Ill., 1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford, 1989.
Zvegf4 proteins are purified by conventional protein purification methods, typically by a combination of chromatographic techniques. See, in general, Affinity Chromatography: Principles and Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988; and Scopes, Protein Purification: Principles and Practice, Springer-Verlag, N.Y., 1994. Proteins comprising a polyhistidine affinity tag (typically about 6 histidine residues) are purified by affinity chromatography on a nickel chelate resin. See, for example, Houchuli et al., Bio/Technol. 6: 1321-1325, 1988. Proteins comprising a glu-glu tag can be purified by immunoaffinity chromatography according to conventional procedures. See, for example, Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985. Maltose binding protein fusions are purified on an amylose column according to methods known in the art.
Zvegf4 is highly expressed in the kidney as shown by Northern blotting and PCR analysis. As shown in more detail in the examples that follow, over-expression of zvegf4 in mice by injection of an adenovirus vector encoding zvegf4 elicits tubular epithelial cell proliferation in the kidney. Tubular generation in the treated animals was characterized by the presence of tubular epithelial cells with increased basophilia. These changes were not observed in animals that were exposed to a control adenovirus expressing an unrelated protein. These findings indicate that an increase in zvegf4 protein can modify the function of, and the interactions among, mesangial cells (a type of myofibroblast; see, Powell et al., Am. J. Physiol. 277 (Cell Physiol. 46):C1-C19, 1999), epithelial cells, endothelial cells, smooth muscle cells, and interstitial cells, which are all key players in glomerular and vascular diseases of the kidney. Furthermore, zvegf4 has been found to affect cell proliferation in at least some of these cells in vitro. Experiments have also shown that the activity of zvegf4 is mediated by two PDGF receptor subunits, alpha and beta (PDGF-xcex1R and PDGF-xcex2R). These receptor subunits are widely expressed in most renal cell types, and their expression is upregulated in a number of kidney pathologies (e.g., lida et al., Proc. Natl. Acad. Sci. USA 88:6560-6564, 1991). The experiments summarized above and disclosed in more detail herein suggest that zvegf4 proteins have a positive effect on renal tubule viability, regeneration, and/or function. These results indicate that zvegf4 may be useful in reversing certain forms of renal failure, such as acute tubular necrosis or chronic analgesic nephritis. In this context, zvegf4 protein may be delivered directly to a mammal or may be produced in situ following delivery to a mammal of a zvegf4 protein-encoding polynucleotide.
While not wishing to be bound by theory, the generative effects of zvegf4 on renal tubules may be due to direct or indirect effects on epithelial cells and/or epithelial cell precursors. xe2x80x9cIndirect effectsxe2x80x9d include the stimulation of production of other factors that act directly on the affected cells. Zvegf4 may stimulate cell proliferation, enhance cell survival, or stimulate the production of other factors that exert these effects on epithelial cells or epithelial cell precursors. Myofibroblasts, for example, are known to secrete cytokines and growth factors that stimulate proliferation, differentiation, and migration of epithelial cells, and to play key roles in organogenesis and wound healing. See, for example, Powell et al., ibid.; Nakagawa et al., Am. J. Pathol. 155:1689-1699, 1999; and Matsumoto and Nakamura, Kidney Int. 59:2023-2038, 2001.
The growth factor domain of zvegf4 has been found to be the active species of the molecule. Proteolytic processing to remove the N-terminal portion of the molecule is required for activation. Within the present invention zvegf protein may be provided as the active growth factor domain alone or as a precursor requiring activation in vivo. Exemplary precursors include, without limitation, zvegf419-370 and zvegf452-370. Fusion proteins and other biologically active zvegf4 variants can also be employed.
For pharmaceutical use, zvegf4 proteins are formulated according to conventional methods. Conventional routes of delivery for pharmaceutical proteins will be employed. Because patients suffering from acute renal failure will ordinarily be undergoing treatment involving intravenous infusion, catheterization, or dialysis, the protein may be administered through existing intravenous lines, catheters, or shunts. Other routes of administration include intravenous, intramuscular, and subcutaneous injection. In general, pharmaceutical formulations will include a zvegf4 protein in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water, or the like. Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc. Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, Pa., 19th ed., 1995. A xe2x80x9ctherapeutically effective amountxe2x80x9d of a composition is an amount that produces a statistically significant effect, such as a statistically significant reduction in disease progression or a statistically significant improvement in organ function. Within the context of acute renal failure, improvement in organ function is indicated by one or more of decreased uremia, increased creatinine or inulin clearance, restoration of electrolyte balance, and increased urine production. Zvegf4 will commonly be used in a concentration of about 10 to 100 xcexcg/ml of total volume, although concentrations in the range of 1 ng/ml to 1000 xcexcg/ml may be used. The exact dose will be determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc.; determination of dose is within the level of ordinary skill in the art. Because acute renal failure is a life-threatening condition, large doses may be employed. The therapeutic formulations will generally be administered over the period required to achieve a beneficial effect, commonly several hours to several weeks. Dosing is continuous or intermittent over the period of treatment. Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours. Sustained release formulations can also be employed.
Zvegf4 therapy may be combined with other agents or clinical techniques appropriate to the restoration of kidney function. For example, zvegf4 may be administered in combination with a vasodilator or in combination with angioplasty to restore circulation in renal arteries.
Gene therapy may be used to provide zvegf4 to a patient. To facilitate expression of zvegf4 in the kidney, a transcription promoter from a gene that is highly expressed in kidney (e.g., erythropoietin gene) may be employed. Therapeutic polynucleotides can be delivered to patients or test animals by way of viral delivery systems. Exemplary viruses for this purpose include adenovirus, herpesvirus, retroviruses, vaccinia virus, and adeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acids. For review, see Becker et al., Meth. Cell Biol. 43:161-89, 1994 and Douglas and Curiel, Science and Medicine 4:44-53, 1997. The adenovirus system offers several advantages. Adenovirus can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters. Because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.
By deleting portions of the adenovirus genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid. When intravenously administered to intact animals, adenovirus primarily targets the liver. If the adenoviral delivery system has an E1 gene deletion, the virus cannot replicate in the host cells. However, the host""s tissue (e.g., liver) will express and process (and, if a signal sequence is present, secrete) the heterologous protein.
An alternative method of gene delivery comprises removing cells from the body and introducing a vector into the cells as a naked DNA plasmid. The transformed cells are then re-implanted in the body. Naked DNA vectors are introduced into host cells by methods known in the art, including transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter. See, Wu et al., J. Biol. Chem. 263:14621-14624, 1988; Wu et al., J. Biol. Chem. 267:963-967, 1992; and Johnston and Tang, Meth. Cell Biol. 43:353-365, 1994.
The invention is further illustrated by the following non-limiting examples.