MXPA01001979A - STABLE HYPOXIA INDUCIBLE FACTOR-1 alpha AND METHOD OF USE - Google Patents

Abstract

Substantially purified stable human hypoxia-inducible factor-1&agr;(sHIF-1alpha) proteins and polynucleotides encoding stable human hypoxia-inducible factor-1&agr;proteins are provided. A method is provided for treating a hypoxia-related tissue damage in a subject by administering to the subject a therapeutically effective amount of an sHIF-1alpha protein or a nucleic acid encoding a stable HIF-1alpha protein. Formulations are provided for the administration of stable human hypoxia inducible factor-1&agr;(HIF-1alpha) polypeptide or a polynucleotide encoding stable human hypoxia inducible factor-1alpha (HIF-1alpha) to a patient having or at risk of having hypoxia- or ischemia-related tissue damage.

Description

FACTOR-1 ALFA INDUCED BY HYPOXIA, STABLE, AND METHOD OF USE Research Statement Sponsored by the Federal Government of the United States This invention was made in part with funds from the National Heart, Lung, and Blood Institute, grant 1R01-HL55338. The government of the United States may have certain rights in this invention. Field of the Invention This invention relates generally to hypoxia-inducible DNA binding proteins, and more specifically, to DNA binding proteins that are modified such that they are stable under non-hypoxic as well as hypoxic conditions. Background of the Invention Mammals require molecular oxygen (02) for essential metabolic processes, including oxidative phosphorylation in the mud serves as an electron acceptor during the formation of ATP. Systemic, local and intracellular homeostatic responses elicited by hypoxia (the state where the demand for 02 exceeds supply) include erythropoie-sis by anemic individuals or at a high altitude (Jelkmann, Physiol Rev. 72: 449-489 , 1992), neovascularization in the ischemic myocardium (Hite et al., Circ Res. 71: 1490- 1500, 1992), and glycolysis in cultured cells with a reduced 02 tension (Olfe et al, Eur. J. Biochem.135: 405-412, 1983). These adaptive responses increase the supply of 02, or activate alternative metabolic pathways that do not require 02. Hypoxia-inducible gene products that participate in these responses include erythropoietin (EPO) (reviewed in Semenza, Hematol, Oncol. Amer..8: 863-884, 1994), vascular endothelial growth factor (VEGF) (Shweiki et al, Na ture 359: 843-845, 1992; Banai et al., Cardiovasc. Res. 28 .: 1116-1119 , 1994, Goldberg and Schneider, J. "Biol. Chem. 269: 4355-4359, 1994), and glycolytic enzymes (Firth et al., Proc. Nati, Acad. Sci. USA 51: 6496-6500, 1994; Semenza et al. collaborators, J. Biol. Chem. 2679: 23757-23763, 1994.) The molecular mechanisms that mediate genetic responses to hypoxia have been extensively investigated for the EPO gene, which encodes a growth factor that regulates erythropoiesis and, for therefore, the carrying capacity of 02 of the blood (Jelkmann, 1992, supra; Semenza, 1994, supra). The cis-acting DNA sequences required for the activation of transcription in response to hypoxia were identified in the 3 'flanking region of EPO, and a trans-acting factor that binds to the enhancer, inducible factor-1. Hypoxia (HIF-1) met the criteria for a physiological regulator of EPO transcription. In particular, the inducers of EPO expression (02 to 1 percent, Cobalt chloride [CoCl2], and deferrioxamine [DFX]) also induced DNA binding activity of HIF-1 with similar kinetics. In addition, inhibitors of EPO expression (actinomycin D, cycloheximide, and 2-aminopurine) blocked the induction of HIF-1 activity. In addition, mutations in the 3 'flanking region of EPO that eliminated the binding of HIF-1, also eliminated the enhancer function (Semenza, 1994, supra). These results support a signal transduction pathway that requires a continuous transcription, translation, and protein phosphorylation that participates in the induction of the DNA binding activity of HIF-1 and in the transcription of EPO in the epoxy cells (Semenza , 1994, supra). EPO expression is cell-type specific, but induction of HIF-1 activity was detected by 02 to 1 percent, CoCl2 or DFX in many mammalian cell lines (Wang and Semenza, Proc. Nati, Acad. Sci. USA 90: 4304-4308, 1993). The EPO enhancer directed the hypoxia-inducible transcription of reporter genes transfected into non-EPO-producing cells (Wang and Semenza, 1993, supra, Maxwell et al, Proc. Nati, Acad. Sci. USA 90: 2423-2421, 1993) . RNAs encoding various glycolytic enzymes were induced by 02 per cent, CoCl2 or DFX in EPO or HeLa non-EPO-producing Hep3B cells, whereas cycloheximide blocked their induction and the glycolytic genetic sequences containing HIF binding sites -1 mediated inducible transcription by hypoxia in transfection assays (Firth et al., 1994, supra; Semenza et al., 1994, supra). These experiments support the role of HIF-1 in the activation of homeostatic responses to hypoxia. Hypoxia-inducible factor-1 (HIF-1) is a mammalian transcription factor expressed exclusively in response to physiologically relevant levels of hypoxia (Wang, GL et al., Proc. Nati, Acad. Sci. USA 92: 5510-5514 , 1995, Wang, GL and Semenza, GL, J. "Biol. Chem. 270: 1230-1237, 1995; U.S. Patent No. 5,882,914.) HIF-1 is a basic helix protein of cyclo-helix it is linked to the elements that respond to cis-acting hypoxia of the hypoxia-induced genes (Wang, GL and Semenza, GL, Curr Opin, Hematol 3: 156-162, 1992; Jiang, BH et al., Biol. Chem. 272: 19253-19260, 1997). Genes that are activated by HIF-1 in cells subjected to hypoxia include EPO, vascular endothelial growth hormone (VEGF), heme oxygenase-1, inducible nitric oxide synthase, and the glycolytic enzymes aldolase A, enolase 1, lactate dehydrogenase A, phosphofructokinase I and phosphoglycerate kinase 1 (Semenza, GL et al., Kid. Int. 51: 553-555, 1997). The DNA binding activity of HIF-1, and the concentration of the HIF-1 protein increase exponentially as they are subjected to the cells at decreasing concentrations of 02 (Jiang, BH et al., Am. J. Physiol. : C1172-C1180, 1996).

HIF-1 also activates the transcription of the vascular endothelial growth factor gene in hypoxic cells (Forsythe et al., 1996, Iyer et al., 1998). When the cultured cells are transfected with the plasmid pCEP4 / HIF-lalfa under conditions that allow the expression of HIF-lalfa from a cytomegalovirus promoter and a reporter plasmid containing the hypoxia response element from the factor gene of vascular endothelial growth, the expression of the reporter gene in cells under non-hypoxic conditions is increased, and a dramatic superinduction occurs under hypoxic conditions that depends on the presence of an intact HIF-1 binding site (Forsythe et al., 1996 ). In embryonic stem cells from a sleeping mouse, which lack expression of HIF-lalfa, there is no expression of vascular endothelial growth factor mRNA in response to hypoxia (Iyer et al., 1998). HIF-1 is a heterodimer of two subunits, HIF-lalfa and HIF-lbeta. The HIF-lalfa subunit is unique to HIF-1, while HIF-lbeta (also known as the nuclear translocator of the aryl hydrocarbon receptor, ARNT) can be dimerized with other proteins. The concentration of the HIF-lalfa and HIF-lbeta RNA, and the HIF-lalfa and HIF-lbeta polypeptide are increased in cells exposed to hypoxic conditions (Wiener, C.M. et al., Biochem. Biophys., Res. Commun. 225: 485-488, 1996; Yu, A. Y. et al., Am J. Physiol. 275: L818-L826, 1998). The structural analysis of HIF-lalfa revealed that dimerization requires two domains, called HLH and PAS. The DNA linkage is mediated by a basic domain (Semenza, G.L. et al., Kid. Int. 51: 553-555, 1997). Two transactivation domains are contained in HIF-lalfa, located between amino acids 531 and 826. The minimal transactivation domains are in amino acid residues 531-575 and 786-826 (Jiang, BH et al., Supra; Semenza, GL and collaborators, 1997, supra). Amino acids 1-390 are required for optimal heterodimerization with HIF-lbeta (tRNA) and DNA binding. In addition, the deletion of the carboxyl terminus of HIF-lalf (amino acids 391-826) reduced the ability of HIF-1 to activate transcription. However, HIF-lalfa (1-390) was expressed at high levels in both hypoxic and non-hypoxic cells, in contrast to full-length HIF-lalfa (1-826) which was expressed at much higher levels in the hypoxic cells relative to non-hypoxic cells (Jiang, BH et al., Biol. Chem. 271: 11111-11118, 1996). Consequently, hypoxia has two independent effects on the activity of HIF-lalfa: (1) hypoxia increases the levels of continuous state of the HIF-lalfa protein by its stabilization (ie, reducing its degradation); and (2) hypoxia increases the specific transcription activity of the protein (ie, regardless of the concentration of the protein). SUMMARY OF THE INVENTION This invention is based on the discovery and isolation of unique variant forms of the HIF-lalfa polypeptide that are stable under hypoxic and non-hypoxic conditions. The invention further includes chimeric proteins having a DNA binding domain of HIF-lalpha and dimerization domains, and a heterologous transactivation domain. Given the structural and functional similarities between HIF-lalfa, HIF-2alpha (also known as EPAS1, HLF, HRF and MOP2), and HIF-3alpha (see Gu, YZ et al, Gene Expr. 7: 205-213, 1998) , it is understood that HIF-lalfa is described for illustrative purposes, but that all of these HIFs are included herein. A stable HIF-lalfa protein (sHIF-lalfa) of the invention includes the following properties: (1) sHIF-lalfa will contain the basic helix-cycle-helix-PAS domain of HIF-lalfa that mediates dimerization with HIF-lbeta ( RNAT) and the binding to the recognition sites of HIF-1 on the DNA, for example, the sequence 5 '-TACGTGCT-3' of the human EPO gene (which was originally used to purify HIF-1), or the sequence 51 -TACGTGGG-3 'from the human vascular endothelial growth factor gene (Forsythe et al., 1996; Semenza and Wang, Mol. Cell. Biol. 12: 5447-5454, 1992); (2) sHIF-lalfa will contain deletions or substitutions of amino acids that substantially increase their half-life in cells under non-stable conditions.

Hypfxicas, in such a way that the protein sHIF-lalfa accumulates to levels much higher than the wild-type HIF-lalfa protein under these conditions. There are many deletions and / or substitutions of different amino acids that will result in this effect; Multiple examples are provided, but these are not limiting; (3) sHIF-lalfa contains one or more transcription activation domains derived, either from HIF-lalfa, or from another eukaryotic or viral transcription factor. Depending on the activation domain used, the transcription activity of sHIF-lalfa can be regulated by oxygen concentration, or it can be constitutively active independently of the oxygen concentration. sHIF-lalfa mediates increased transcription of hypoxia-inducible genes normally regulated by HIF-1. In one embodiment, the invention includes an isolated nucleic acid sequence that encodes a stable HIF-alpha protein that is a chimeric transactivator. This chimeric transactivator includes: a) a nucleotide sequence encoding a DNA binding domain and a dimerization domain of a hypoxia-inducible factor (e.g., HIF-lalfa, HIF-2alpha or HIF-3alpha); and b) a nucleotide sequence encoding a transcription activation domain. The preferred hypoxia inducible factor of the invention is HIF-lalfa. In another embodiment, the invention provides stable non-chimeric HIF-lalfa polypeptides. These polypeptides include, but are not limited to, the amino acid residues of HIF-lalfa 1-391 and 521-826 of SEQ ID NO: 1; the amino acid residues 1-391 and 549-826 of SEQ ID NO: 1; amino acid residues 1-391 and 576-826 of SEQ ID NO: 1; amino acid residues 1-391 and 429-826 of SEQ ID NO: 1, where 551 is no longer serine, and 552 is not threonine; amino acid residues 1-391 and 469-826 of SEQ ID NO: 1, where 551 is no longer serine, and 552 is not threonine; amino acid residues 1-391 and 494-826 of SEQ ID NO: 1, where 551 is no longer serine, and 552 is not threonine; amino acid residues 1-391 and 508-826 of SEQ ID NO: 1, where 551 is no longer serine, and 552 is not threonine; amino acid residues 1-391 and 512-826 of SEQ ID NO.-1, where 551 is no longer serine, and 552 is not threonine; and amino acid residues 1-391 and 517-826 of SEQ ID NO: 1, where 551 is no longer serine, and 552 is not threonine. The invention further provides a method for providing constitutive expression of a hypoxia inducible factor in a cell, under hypoxic or non-hypoxic conditions. The method includes contacting the cell with a nucleic acid sequence encoding a chimeric transactivator protein as described herein, or a stable HIF-alpha as described herein, under conditions that allow the expression of the sequence of nucleic acid, thus providing the constitutive expression of an inducible factor by hypoxia The invention also provides a method for increasing the expression of a hypoxia-inducible gene in a cell. The method includes contacting the cell with an expression vector containing a polynucleotide encoding a stable HIF-alpha of the invention, or a chimeric transactivator protein as described herein, under conditions that allow the expression of the nucleic acid sequence contained in the vector, thereby providing greater expression of the hypoxia-inducible genes in the cell. These genes include, for example, vascular endothelial growth factor. In addition, the invention includes a method for reducing tissue damage related to hypoxia or ischemia in a subject who has or is at risk of having this damage. The method includes administering to the subject a therapeutically effective amount of a nucleic acid sequence encoding a chimeric transactivator protein as described herein, or a stable HIF-lalpha as described herein, in a pharmaceutically acceptable carrier, inducing this way the genetic expression that will reduce, or prevent, or repair, tissue damage. Examples of gene products whose expression is induced by sHIF-lalfa that result in a therapeutic effect include vascular endothelial growth factor and other mediators of angiogenesis, and insulin type 2 growth factor (IGF-2), and other factors that promote cell survival (Iyer et al., 1998; Feldser, D. et al., Cancer Res. 59: 3915, 1999). In another embodiment, the invention provides a method of providing prophylactic therapy for tissue in a subject in need, which comprises administering to the subject an amount of a polypeptide encoded by a polynucleotide that encodes a chimeric transactivator protein as described herein. , or a stable HIF-lalfa, as described herein, such that angiogenesis is induced at higher levels than before administration of the polypeptide, thereby providing prophylactic therapy. In one embodiment, the invention provides a substantially purified polypeptide having a sequence as set forth in SEQ ID NO: 1, wherein amino acids 392 to 428 are deleted therefrom, amino acid 551 is changed from one serine to any other amino acid, and the amino acid 552 is changed from a threonine to any other amino acid. Isolated polynucleotides encoding this polypeptide, as well as antibodies that preferentially bind to this polypeptide, are also provided in a particular embodiment, and serine 551 is changed to glycine, and threonine 552 to alanine. In one embodiment, a method is provided for the treatment of tissue damage related to hypoxia in a subject, by administering to the subject a therapeutically effective amount of a nucleotide sequence comprising an expression control sequence operably linked to a polynucleotide encoding a polypeptide having a sequence as set forth in SEQ ID NO: 1, where amino acids 392 to 428 are deleted from it, amino acid 551 is changed from a serine to any other amino acid, and amino acid 552 is changed from a threonine to any other amino acid. In another embodiment, the invention provides a method for treating tissue damage related to hypoxia in a subject, by administering to the subject a therapeutically effective amount of a polypeptide having a sequence as set forth in SEQ ID NO: 1, wherein amino acids 392 to 428 are deleted therefrom, amino acid 551 is changed from a serine to any other amino acid, and amino acid 552 is changed from a threonine to any other amino acid. In a further embodiment, the invention provides a formulation for the administration of the human-inducible hypoxia factor-inducible factor (HIF-lalfa) polypeptide to a patient having tissue damage related to hypoxia. The method includes a substantially pure polypeptide having a sequence as set forth in SEQ ID NO: 1, wherein amino acids 392 to 428 are deleted therefrom, the amino acid 551 from a serine to any other amino acid, and amino acid 552 is changed from a threonine to any other amino acid; and a pharmaceutically acceptable vehicle. The invention also provides a formulation for the administration of a polynucleotide encoding stable human hypoxia-inducible factor-lalfa (HIF-lalfa) to a patient having hypoxia-related tissue damage, including a therapeutically effective amount of an acid sequence. nucleic comprising an expression control sequence operably linked to a polynucleotide encoding a polypeptide having a sequence as set forth in SEQ ID NO: 1, wherein amino acids 392 to 428 are deleted from it, the amino acid is changed 551 from a serine to any other amino acid, and the amino acid 552 is changed from a threonine to any other amino acid; and a pharmaceutically acceptable vehicle. BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-H are the amino acid sequence (SEQ ID N0: 1) of wild type HIF-lalfa. Figure 2 shows an analysis of the effect of carboxyl-terminal deletions on the regulated expression of HIF-lalfa. Figure 3 shows an analysis of the effect of internal deletions on the regulated expression of the HIF-lalfa polypeptide. Oxygen regulation of the HIF-lalfa polypeptide which contains the indicated internal suppression, is shown in the "wt" column, where a "+" indicates that the polypeptide is regulated and, therefore, is unstable under non-hypoxic conditions. Each of the internal deletions indicated in the HIF-lalfa has been combined with a double-point mutation (a mutation of serine to glycine in amino acid 551, and a mutation of threonine to alanine in residue 552). The oxygen regulation of the polypeptide containing both the indicated internal suppression and the double point mutation is shown in the "mut" column, where a "+" indicates that the polypeptide is regulated and, therefore, is unstable under conditions not hypoxic Figure 4 shows a model of the regulated expression of HIF-lalfa. The putative regulatory sequences identified within the HIF-lalfa protein are indicated by suppression analysis. Potential interactions with regulatory proteins, such as a phosphatase, kinase or protease, are also shown. Figure 5 is a bar graph illustrating luciferase activity after co-transfection of human 293 cells with a reporter gene containing a hypoxic response element (including an HIF-1 binding site) with the vector of expression pCEP4 encoding (1) no protein; (2) Full length HIF-lalfa (amino acids 1-826); (3) HIF-lalfa (1-391 / 429-826, deletion only); (4) HIF-lalfaDP (deletion and a mutation of serine to glycine in the amino acid 551, and a mutation of threonine to alanine at residue 552). The expression of the reporter gene is shown in 1 percent (black bars) and in 20 percent of 02 (white bars). Figure 6 is a bar graph illustrating luciferase activity after co-transfection of Hep3B cells with a reporter gene containing a hypoxic response element (including an HIF-1 binding site), and with the pCEP4 expression vector encoding: (1) no protein; (2) HIF-lalfa; (3) HIF-lalfa (1-391 / 429-826, deletion only); (4) HIF-lalfaDP (deletion and a mutation of serine to glycine at amino acid 551, and a mutation of threonine to alanine at residue 552). The expression of the reporter gene is shown in 1 percent (black bars) and in 20 percent of 02 (white bars). Detailed Description of the Invention It should be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references, unless the context clearly indicates otherwise. Accordingly, for example, the reference to "one cell" includes a plurality of cells, and the reference to "the plasmid" includes reference to one or more plasmids and their equivalents known to those skilled in the art, etc. Unless defined otherwise, all the technical and scientific terms used in this have the same meaning as is commonly understood by an ordinary expert in the field to which this invention pertains. Although any methods, devices and materials similar or equivalent to those described herein can be used, in the practice or testing of the invention, preferred methods, devices and materials will now be described. All publications mentioned herein are hereby incorporated by reference in their entirety for the purpose of describing and disclosing the cell lines, vectors, and methodologies described in publications that may be used in connection with the presently described invention. The publications discussed above and throughout the text are provided exclusively by their disclosure prior to the filing date of the present application. Nothing contained herein should be construed as an admission that the inventors have no right to advance the date of this disclosure under the prior invention. The invention provides a substantially pure hypoxia-inducible factor-lalfa inducible protein (sHIF-lalfa), or mutein. Full-length, wild-type HIF-lalfa is expressed at lower levels in non-hypoxic cells, compared to hypoxic cells (Wang, GL et al, Proc. Nati, Acad. Sci. USA 92: 5510-5514 , 1995; Wang, GL and Semenza, GL, J. Biol. Chem. 27_0: 1230-1237, 1995; Jiang, BH and collaborators, J. Biol. Chem. 272: 19253-19260, 1997, incorporated herein by reference), while sHIF-lalfa is stable under non-hypoxic as well as hypoxic conditions. The wild-type HIF-lalfa and the sHIF-lalfa are characterized by being able to form heterodimers with the HIF-lbeta to form a DNA binding protein, hypoxia-inducible factor-1 (HIF-1), a factor of mammalian transcription. HIF-1 activates the transcription of multiple genes, including those that encode erythropoietin (EPO), vascular endothelial growth factor (VEGF), glucose transporters, and glycolytic enzymes. The term "mutein", as used herein, refers to a variant form of the HIF-lalfa polypeptide that is stable under hypoxic or non-hypoxic conditions. The HIF-lalfa polypeptide, after dimerization with HIF-lbeta, is a DNA binding protein, which is characterized by activating gene expression in which the promoter region of the target gene contains an HIF-1 binding site (Semenza , GL et al., Kid. Int. 51: 553-555, 1997; Iyer, NV et al., Genes Dev. 12: 149-162, 1998, both incorporated herein by reference). Examples of these structural genes include erythropoietin (EPO), vascular endothelial growth hormone (VEGF), and glycolytic genes. The HIF-lalfa migrates on electrophoresis in SDS-polyacrylamide gel with an apparent molecular mass of 120 kDa, and has essentially the amino acid sequence stipulated in SEQ ID N0: 1. The term HIF-lalfa includes the polypeptide stipulated in SEQ ID NO: 1, and the conservative variations of the polypeptide sequence. The term "conservative variant", as used herein, denotes the replacement of an amino acid residue by another biologically similar residue. Examples of conservative variations include the substitution of a hydrophobic residue, such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic acid for aspartic acid , or glutamine by asparagine, and the like. In a preferred embodiment, HIF-lalfa has the sequence stipulated in SEQ ID NO: l. HIF-lalfa is described in detail in the pending United States patent application Serial No. 08 / 480,473, incorporated herein by reference. In general, a mutein will have an amino acid sequence that differs from the native sequence by including substitutions, insertions, and / or deletions, for example. Muteins are easily prepared using modern cloning techniques, or they can be synthesized by solid state methods, by site-directed mutagenesis. A mutein may include dominant negative forms of a polypeptide. The invention provides a substantially pure hypoxia-inducible factor-lamellar mutein (sHIF-lalfa).

The sHIF-lalfa polypeptide has a sequence as stipulated in SEQ ID NO: 1, wherein amino acids 392 to 428 are deleted from it, amino acid 551 is changed from a serine to any other amino acid, and the amino acid is changed 552 from a threonine to any other amino acid. In one embodiment, amino acids 392 to 428 of SEQ ID NO: 1 are deleted, and amino acid 551 is changed from a serine to a glycine. In another embodiment, amino acids 392 to 428 of SEQ ID NO: 1 are deleted, and amino acid 552 is changed from a threonine to an alanine. In yet another embodiment, amino acids 392 to 428 of SEQ ID NO: 1 are deleted, and amino acid 551 is changed from a serine to a glycine, and amino acid 552 is changed from a threonine to an alanine. Without being bound by theory, we have identified two regions of full-length HIF-lalfa that are important for the stable expression of HIF-lalfa. The AB region is located from about amino acid 392 to amino acid 552. Within this region, two A and B sequences have been identified. In particular, sequence A is from amino acid 392 to amino acid 428 of SEQ ID NO: 1, and sequence B is at approximately amino acid 429 to 552 of SEQ ID NO: 1. Region C is located from about amino acid 703 to amino acid 726 of SEQ ID NO: 1. A "mutation" in SEQ ID NO: l refers to a deletion, insertion, mutation or substitution of one or more amino acids. Stable HIF-lalfa is it can comprise a mutation or deletion in both regions A and B. Alternatively, stable HIF-lalfa can be composed of a deletion in region C. For example, regions A and B can be deleted, regions A and B can be mutated, or region A can be mutated and region B can be deleted, region A can be deleted and region B can be mutated, or region C can be mutated, or region C can be deleted . In a non-limiting example, stable HIF-alpha is composed of a deletion of amino acid 392 to amino acid 520 of SEQ ID NO: 1. In another non-limiting example, stable HIF-alpha is composed of a deletion of amino acid 392 to 428 of SEQ ID NO: 1, combined with a point mutation of either amino acid 551 or 552, or combined with a point mutation of both amino acids. 551 and 552. Point mutations can be combined with a deletion of amino acids 392 through amino acid 428 of SEQ ID NO: 1, or point mutations can be combined with a deletion of amino acid 392 to any amino acid between amino acid 429 and amino acid 550 inclusive, of SEQ ID NO: l. In yet another non-limiting example, stable HIF-alpha is composed of a deletion of amino acid 704 to amino acid 826 of SEQ ID NO: 1. This deletion removes the transactivation domain (amino acid 786 to amino acid 826), and therefore, may result in a loss of biological activity. In one embodiment, stable HIF-lalfa can be formed by suppressing amino acid 704 to amino acid 826 of SEQ ID NO: 1, with the addition of a heterologous transactivation domain followed by amino acid 704. The "heterologous" transactivation domain is a transactivation domain derived from a polypeptide different from HIF-1I. In one embodiment, the activity of the heterologous transactivation domain is not affected by the concentration of oxygen. In a non-limiting example, the heterologous transactivation domain is from the VP16 protein of the herpes simplex virus (HSVC) (amino acids 413-490). In this embodiment, the deletion of amino acids 391 to 704 is combined with a deletion of amino acid 704 to amino acid 826. The transactivation domain from VP126 protein of herpes simplex virus is then fused to amino acids 1 to 390 of the HIF-lalfa polypeptide. In yet another modality, a transactivation domain is fused from HIF-lalfa (amino acids 786-826) with amino acids 1-390 (Jiang et al., 1997). Additional combinations of regions identified as significant for the formation of the sHIF-lalfa mutein will be readily apparent to one skilled in the art. A stable HIF-lalfa is a HIF-lalfa polypeptide that has a longer half-life, compared to wild-type HIF-lalfa under non-hypoxic conditions. In one embodiment, in a given cell, sHIF-lalfa has the same half-life under hypoxic or non-hypoxic conditions, and is present in the same concentration in cells exposed to non-hypoxic conditions, such as in cells exposed to hypoxic conditions. Hypoxia is a condition where the oxygen demand in a tissue exceeds the oxygen supply in that tissue. The terms "hypoxic" and "non-hypoxic" are understood as relative terms with respect to the concentration of oxygen typically observed in a particular tissue. The ability of wild-type HIF-lalfa to activate transcription is regulated by the concentration of oxygen, regardless of the effect of oxygen on the stability of the HIF-lalfa protein (Jiang et al., 1997, supra). The sHIF-lalpha region located at amino acids 576-785 is a negative regulatory domain that, when suppressed, results in increased transcription under non-hypoxic conditions (Jiang et al., J. Biol. Chem. 272: 19253, 1997), incorporated herein by reference). Therefore, without being bound by theory, the deletion of one or more amino acids in this sequence, such that the amino acid is replaced by a bond, results in a higher transcription activity, regardless of the half-life of the protein . Therefore, the suppression of amino acids 576-785 of HIF-lalfa can be combined with the deletion of amino acids 392-428, and the point mutation of amino acid 551 from a serine to a glycine, and the point mutation of amino acid 552 from a threonine to an alanine, to produce a stable HIF-lalfa polypeptide. The deletion of amino acid 576 to amino acid 785 of HIF-lalfa can also be combined with the deletion of amino acids 392 to 520, to produce a stable HIF-lalfa polypeptide. Alternatively, the deletion of amino acid 576 to amino acid 785 of HIF-lalfa can be combined with the deletion of amino acid 704 to amino acid 826 (resulting in the deletion of amino acids 576 to 826 of HIF-lalfa) to produce a stable HIF-lalfa polypeptide. These combinations will be readily apparent to an ordinary expert in this field. The term "substantially pure", as used herein, refers to HIF-lalfa that is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. One skilled in the art can purify HIF-lalfa using conventional techniques for protein purification, such as affinity chromatography of DNA (eg, Wang, GL and Semenza, J., J. "Biol. Chem. 270: 1230 -1237, 1995), and immuno-precipitation (for example, Jiang, BH for example, J. Biol. Chem. 271: 11111-11118, 1996.) The substantially pure polypeptide will produce a single band on a non-reducing polyacrylamide gel. The purity of the HIF-lalfa polypeptide can also be determined by amino-terminal amino acid sequence analysis.The HIF-lalfa protein includes functional fragments of the polypeptide, provided that the activity and stability remain under non-hypoxic conditions of sHIF-lalfa. Accordingly, smaller peptides containing the biological activity of sHIF-lalfa are included in the invention. Accordingly, smaller peptides containing the biological activity of sHIF-lalfa are included in the invention. The invention provides polynucleotide sequences encoding the sHIF-lalfa polypeptide, having a sequence as set forth in SEQ ID NO: 1, wherein amino acids 392 to 428 are deleted therefrom, amino acid 551 is changed from a serine to any other amino acid, and amino acid 552 is changed from a threonine to any other amino acid. These polynucleotides include DNA, cDNA and RNA sequences encoding sHIF-lalfa. It is also understood that all polynucleotides that encode all or a portion of the sHIF-lalfa are also included herein, as long as they encode a polypeptide with HIF-lalfa activity that is stable under hypoxic and non-hypoxic conditions. These polynucleotides include polynucleotides that occur naturally, synthetic, and intentionally manipulated. For example, the sHIF-lalfa polynucleotide can be subjected to site-directed mutagenesis. The polynucleotide sequence for sHIF-lalfa also includes anti-sense sequences. The polynucleotides of the invention include sequences that are degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Accordingly, all degenerate nucleotide sequences are included in the invention, provided that the amino acid sequence of the HIF-lalfa polypeptide is encoded by the nucleotide sequence and remains functionally unchanged. Minor modifications of the primary amino acid sequence of sHIF-lalfa can result in proteins that are stable under non-hypoxic conditions, and that have substantially equivalent activity, compared to the sHIF-lalfa polypeptide described herein. These minor modifications include minor differences found in the sequence of the HIF-lalfa polypeptide isolated from different species (e.g., human, mouse, and rat HIF-alphal polypeptide). These proteins include those defined by the term "having essentially the amino acid sequence" of the sHIF-lalpha of the invention. These modifications can be deliberate, such as through site-directed mutagenesis, or they can be spontaneous, such as those found in different species. All polypeptides produced by these modifications are included herein, provided that the biological activity of sHIF-lalfa still exists, and that the polypeptide is stable under non-hypoxic conditions, as compared to wild-type HIF-lalfa. In addition, deletions of one or more amino acids can also result in the modification of the structure of the resulting molecule, without altering a significantly its biological activity. This can lead to the development of a smaller active molecule that would have a wider utility. For example, amino- or carboxyl-terminal amino acids that are not required for the biological activity of sHIF-lalfa can be removed. A DNA sequence encoding the human sHIF-lalfa mutein is specifically disclosed herein. The invention provides polynucleotide sequences encoding the stable HIF-lalfa mutein having a sequence as set forth in SEQ ID NO: 1, wherein amino acids 392 to 428 are deleted therefrom, amino acid 551 is changed from a serine to any other amino acid, and amino acid 552 is changed from a threonine to any other amino acid. The wild-type HIF-lalfa contains an open reading frame that encodes a polypeptide of 826 amino acids in length. When amino acid 551 (serine) of SEQ ID NO: 1 is replaced by another amino acid, such as a glycine, or amino acid 552 (threonine) of SEQ ID NO: 1 it is replaced by another amino acid, such as alanine, and one or more of amino acid 392 to amino acid 429 of SEQ ID NO: l is replaced by a bond, the polynucleotide will encode a polypeptide having a reduced length by a corresponding number of amino acids. In another embodiment, the invention provides polynucleotides encoding sHIF-lalpha, as well as complementary nucleic acid sequences for the polynucleotides encoding sHIF-lalfa. The term "polynucleotide" or "nucleic acid sequence" refers to a polymeric form of nucleotides of at least 10 bases in length. An isolated polynucleotide means a polynucleotide that is not immediately contiguous with both coding sequences with which it is immediately adjacent (one on the 5 'end, and one on the 3' end) in the naturally occurring genome of the organism from which is derived Accordingly, the term includes, for example, a recombinant DNA that is incorporated into a vector; in a plasmid or self-replicating virus; or in the genomic DNA of a prokaryote or eukaryote, or that exists as a separate molecule (eg, a cDNA) independent of other sequences. The nucleotides of the invention may be ribonucleotides, deoxy-ribonucleotides, or modified forms of any nucleotide. The term includes the single-stranded and double-stranded forms of DNA. A complementary sequence may include an anti-sense nucleotide. When the sequence is RNA, deoxynucleotides A, G, C and T in the polynucleotide encoding HIF-lalfa are replaced by ribonucleotides A, G, C and U, respectively. Also included in the invention are fragments of the nucleic acid sequences identified above, which are at least 15 bases in length, which is sufficient to allow the fragment to selectively hybridize to the nucleic acid encoding sHIF-lalfa, but not the SEQ ID NO: l under physiological conditions. Specifically, the fragments should hybridize selectively to the nucleic acid encoding the sHIF-lalfa polypeptide. The term "selectively hybridize" refers to hybridization under moderately or highly stringent conditions, which excludes unrelated nucleotide sequences. In nucleic acid hybridization reactions, the conditions used to achieve a particular level of astringency will vary, depending on the nature of the nucleic acids that are hybridizing. For example, the length, the degree of complementarity, the composition of the nucleotide sequence (eg, GC content against AT), and the type of nucleic acid (eg, RNA against DNA) of the hybridization regions of the nucleic acids, can be considered in the selection of hybridization conditions. A further consideration is whether one of the nucleic acids is immobilized, for example, on a filter. An example of the progressively higher stringency conditions is as follows: SSC 2 x / 0.1% SDS at about room temperature (hybridization conditions); SSC 0.2 x / 0.1 percent SDS at approximately room temperature (low stringency conditions); SSC 0.2 x / 0.1% SDS at approximately 42 ° C (moderate stringency conditions); and SSC 0.1 x at approximately 68 ° C (high stringency conditions). It can be washed using only one of these conditions, for example, conditions of high stringency, or each of the conditions may be used, for example, for 10 to 15 minutes each, in the order listed above, repeating any or all of the steps mentioned. However, as mentioned above, optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically. When a specific probe of sHIF-lalfa is used, it may be necessary to amplify the nucleic acid prior to binding with a specific probe of sHIF-lalfa. Preferably, the polymerase chain reaction (PCR) is used; however, other nucleic acid amplification methods, such as ligase chain reaction (LCR), ligated activated transcription (LAT), and amplification based on nucleic acid sequence (NASBA) can be used. The sHIF-lalfa polynucleotide of the invention can be derived from a mammalian organism, and more preferably from a human. Tracing procedures that rely on nucleic acid hybridization make it possible to isolate any genetic sequence from any organism, provided that the appropriate probe is available. Oligonucleotide probes, which correspond to a part of the sequence encoding the protein in question, can be synthesized chemically. This requires knowing the short oligopeptide stretches of the amino acid sequences. The DNA sequence encoding the protein can be deduced from the genetic code; however, the degeneracy of the code must be taken into account. In a preferred embodiment, the probe can be delineated between sHIF-lalfa and wild-type HIF-lalfa. It is possible to perform a mixed addition reaction when the sequence is degenerate. This includes a heterogeneous mixture of denatured double-stranded DNA. For this screening, the hybridization is preferably carried out on the single-stranded DNA or the denatured double-stranded DNA. Hybridization is particularly useful in the detection of derived cDNA clones from sources where an extremely low amount of mRNA sequences related to the polypeptide of interest is present. In other words, by using the astringent hybridization conditions aimed at avoiding a non-specific binding, it is possible, for example, to allow the self-radiographic visualization of a specific cDNA clone by hybridizing the target DNA with that single probe in the mixture that is its complete complement (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Plainview, New York, United States, 1998). The development of specific DNA sequences that encode sHIF-lalfa can also be obtained through site-directed mutagenesis of a nucleic acid sequence encoding SEQ ID N0: 1, or the chemical manufacture of a DNA sequence to provide the necessary codons for the polypeptide of interest. The synthesis of DNA sequences is often the method of choice when the entire amino acid residue sequence of the desired polypeptide product is known. A cDNA expression library, such as in phage lambda gtll, can be screened indirectly to determine sHIF-lalfa peptides having at least one epitope, using antibodies specific for sHIF-lalfa. These antibodies can be derived polyclonally or monoclonally, and can be used to detect the expression product that indicates the presence of sHIF-lalfa cDNA. The DNA sequences encoding the sHIF-lalfa can be expressed in vi tro, by transfer of the DNA to a suitable host cell. The "host cells" are cells in which a vector can be propagated, and its DNA can be expressed. Host cells include both prokaryotic and eukaryotic cells. The term also includes any progeny of the host cell object. It is understood that all progeny may not be identical to the parental cell, because there may be mutations that occur during replication. However, this progeny is included when the term "host cell" is used. The methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, they are known in the art. The "modified" versions of the specific sHIF-lalfa can be designed to further improve the stability, biological activity, production, purification or yield of the expressed product. For example, the expression of a fusion protein, or of a dissociable fusion protein comprising sHIF-lalfa and a heterologous protein can be designed. This fusion protein can be easily isolated by affinity chromatography, for example, by immobilization on a column specific for the heterologous protein. When designing a dissociation site between the HIF-lalpha fraction and the heterologous protein, the HIF-lalfa polypeptide can be released from the chromatography column by treatment with an appropriate enzyme or agent that digests at the dissociation site (Booth and collaborators, Jnmunolol, Lett 19: 65-708, 1988, Gardella for example, J. Biol. Chem. 265: 15854-15859, 1990. The invention provides an isolated nucleic acid sequence encoding a fusion protein. The fusion protein is encoded by a nucleotide sequence encoding a DNA binding domain and a dimerization domain of a hypoxia inducible factor, preferably HIF-alpha, and a nucleotide sequence encoding a transcription activation domain. This "chimeric" transactivator is useful to affect the genetic expression of target genes, such as vascular endothelial growth factor, and neovascularization of ischemic tissue. The nucleotide sequence encoding a DNA binding domain and a dimerization domain of a hypoxia inducible factor is useful for providing constitutive activation of genes, regardless of the oxygen concentration in the surrounding environment. A chimeric transactivator of the invention provides specific activation of the expression of hypoxia-inducible genes that contain elements that respond to hypoxia (HREs), thus reaching high levels of gene expression. The elements that respond to hypoxia each contain a binding site for HIF-1, which is recognized by the chimeric transactivator due to the presence of HIF-lalpha dimerization and DNA binding domains. The chimeric transactivation proteins of the invention function in vertebrate cells, and may include transcriptional transcription proteins that occur naturally, or protein domains from eukaryotic cells, including vertebrate cells, viral transactivating proteins, or any synthetic amino acid sequence that can stimulate transcription from a vertebrate promoter. A transactivation domain of the chimeric transactivator is derived from transactivating proteins, including, but not limited to, VP16 herpes virus simplex, a heat shock factor, p53, fos, v-jun, factor EF-C, HIV tat, HPV E2, Ad E1A, Spl, API, CTF / NF1, E2F1, HAP1, HAP2, MCM1, PH02, GAL4 , GCN4 and GAL 11, and NFkB, and other heterologous proteins having this transactivating domain. One skilled in the art will recognize that a transcriptional activation domain for use in a composition of the invention can be from a protein that occurs naturally, or can be synthetic, for example, based on a sequence not contained in a protein. that occurs naturally. The identification of a transactivation domain can be determined by operatively linking a desired domain from a protein with an appropriate sequence, and assaying for the expression of a reporter sequence. A recombinant nucleic acid construct encoding a chimeric transactivating protein of the invention can be placed under the control of, or "can be operably linked to," a suitable promoter and / or other regulatory sequences of expression control. It may be desirable for the transactivator protein to be placed under the control of a constitutively active promoter sequence, although the transactivator protein may also be placed under the control of an inducible promoter, such as the metallothionein promoter, or a tissue-specific promoter. An inducible promoter allows the increase or controlled reduction of the expression of a particular gene, while the constitutive expression allows the expression continuous of a gene, for example, to produce a genetic product in culture, or in a transgenic animal. Other promoter sequences that are useful include, but are not limited to, the SV40 early promoter region; RSV or other retroviral LTRs; the herpes thymidine kinase promoter, the immediate promoter / enhancer of cytomegalovirus (CMV). Other promoters that are used for this purpose include the control region of the elastase 1 gene; the control region of the insulin gene; the control region of the immunoglobulin gene; the control region of the mouse mammary tumor virus; the control region of the albumin gene; the control region of the alpha-fetoprotein gene; the control region of the alpha-1-antitrypsin gene, and the control region of the beta-globin gene. The nucleic acid sequence encoding the DNA binding domain and the dimerization domain of HIF-lalfa, and the heterologous transactivation domain are operably linked, such that the structural and functional activities of each region are retained (i.e. , DNA binding, dimerization and transactivating activity). Figures 2 and 3 provide the results of different deletions in the HIF-lalfa, and the effects on the regulation of gene expression. Based on the results shown in the figures and in U.S. Patent No. 5,882,914, the chimeric transactivator of the invention may include a DNA binding and dimerization region encoding, for example, the amino acids of HIF-lalfa 1- 703 of SEQ ID NO: l; amino acids 1-681 of SEQ ID NO: 1; amino acids 1-608 of SEQ ID NO: 1; or amino acids 1-391 of SEQ ID NO: l. The invention also includes expression vectors containing a nucleic acid sequence encoding a chimeric transactivator as described herein. Vectors include adenovirus, AAV, lentivirus, herpes virus, vaccinia virus, baculovirus, retrovirus, bacteriophage, cosmids, plasmids, fosmides, fungal vectors and other vectors known in the art, which are used for expression in eukaryotic host cells and prokaryotes, and that can be used live for gene therapy, or in vi tro in cell culture, for example. The stable HIF-lalfa proteins of the invention also include, but are not limited to, the amino acid residues of HIF-lalpha 1-391 and 521-826 of SEQ ID N0: 1; the amino acid residues 1-391 and 549-826 of SEQ ID NO: 1; amino acid residues 1-391 and 576-826 of SEQ ID NO: 1; amino acid residues 1-391 and 429-826 of SEQ ID NO: 1; where 551 is no longer serine, and 552 is not threonine; amino acid residues 1-391 and 469-826 of SEQ ID NO: 1; where 551 is no longer serine, and 552 is not threonine; amino acid residues 1-391 and 494-826 of SEQ ID NO: 1; where 551 is no longer serine, and 552 is not threonine; amino acid residues 1-391 and 508-826 of SEQ ID NO: 1; where 551 is no longer serine, and 552 it is not threonine; amino acid residues 1-391 and 512-826 of SEQ ID NO: 1, where 551 is no longer serine, and 552 is not threonine; and amino acid residues 1-391 and 517-826 of SEQ ID NO: 1, where 551 is no longer serine, and 552 is not threonine; when serine 551 is changed, for example, amino acid residue 551 can be glycine. In addition, when threonine 552 is changed, amino acid residue 552 can be alanine. In addition to these polypeptides, the invention includes nucleic acid sequences encoding these polypeptides, and expression vectors containing these nucleic acid sequences. It should be understood that one skilled in the art can manipulate the amino acid or nucleic acid sequences provided herein, by deletion or addition of amino acid or nucleotide residues, respectively, provided the activity ascribed to the sequences is retained, by example, the properties of constitutive transactivation or stable HIF-lalfa, as described herein. One skilled in the art could use the teachings herein to test these activities (see Examples). In the present invention, the sHIF-lalfa polynucleotide sequences can be inserted into an expression vector. The term "expression vector" refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the sHIF-lalfa gene sequences. The polynucleotide sequence encoding the sHIF- lalpha can be operably linked to expression control sequences. "Operably linked" refers to a juxtaposition, wherein the components so described are in a relationship, allowing them to function in their intended manner. An expression control sequence operably linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. As used herein, the term "expression control sequences" refers to nucleic acid sequences that regulate the expression of a nucleic acid sequence with which it is operably linked. The expression control sequences are operably linked to a nucleic acid sequence when the expression control sequences control and regulate transcription, and, as appropriate, translation of the nucleic acid sequence. Accordingly, the expression control sequences may include appropriate promoters, enhancers, transcription terminators, such as the start codon (ie, ATG) in front of a gene encoding the protein, the splice signal for introns, the maintenance of the correct reading frame of that gene to allow proper translation of the mRNA, and stop codons. The term "control sequences" is intended to include, at a minimum, the components whose presence may influence the expression, and may also include additional components whose presence is convenient, for example, leader sequences and fusion component sequences. The expression control sequences may include a promoter. "Promoter" means the minimum sequence sufficient to direct the transcription. Also included in the invention are promoter elements that are sufficient to make promoter-dependent gene expression controllable for cell-type-specific, tissue-specific, or inducible by signals or external agents; these elements can be located in the 5 'or 3' regions of the gene. Both constitutive and inducible promoters are included in the invention (see, for example, Bitter et al., Methods in Enzymology 153: 516-544, 1987). For example, when cloning into bacterial systems, inducible promoters can be used, such as pL of bacteriophage Y, plac, ptrp, ptac (ptrp-lac hybrid promoter), and the like. When cloning into mammalian cell systems, promoters derived from the genome of mammalian cells (eg, metallothionein or elongation factor-lalpha promoter), or from mammalian virus (eg, repeat) may be used. long terminal of the retrovirus; the late adenovirus promoter; the 7.5K vaccine virus promoter; the cytomegalovirus promoter. Promoters produced by recombinant DNA or synthetic techniques can also be used to provide transcription of acid sequences nucleics of the invention. In the present invention, the polynucleotide encoding sHIF-lalfa can be inserted into an expression vector containing a promoter sequence that facilitates efficient transcription of the inserted host genetic sequence. The expression vector usually contains a replication origin, a promoter, as well as specific genes that allow the phenotypic selection of the transformed cells. Vectors suitable for use in the present invention include, but are not limited to, the T7-based expression vector for expression in bacteria (Rosenberg et al., Gene 55: 125, 1987), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J. Biol. Chem. 263: 3521, 1988), and baculovirus derived vectors for expression in insect cells. The DNA segment may be present in the vector operably linked to the regulatory elements, eg, a promoter (eg, CMV, T7, metallothionein I, or polyhedrin promoters). Mammalian expression systems using recombinant viruses or viral elements can be designed to direct expression. For example, when adenovirus expression vectors are used, the sHIF-lalfa coding sequence can be ligated with an adenovirus transcription / translation control complex, eg, the late promoter and tripartite leader sequence, or a promoter heterologous (for example, CMV) cloned in a replication-deficient adenovirus (Armentano, D., et al., Hum. Gene Ther.6: 1343-1353, 1995; Hehir, KM et al., J. Virol. 70: 8459-8467, 1996 ). Alternatively, the 7.5K vaccine virus promoter can be used (for example, see Mackett et al., Proc. Nati, Acad. Sci. USA 79: 7415-7419, 1982, Mackett et al., J ". Virol., 49: 857-864, 1984; Panicali et al., Proc. Nati, Acad. Sci. USA 79: 4927-4931, 1982.) Vectors based on bovine papilloma virus have the ability to replicate as extractive elements. chromosomes (Sarver et al., Mol.Cell. Biol. 1: 486, 1981) Shortly after the entry of this nucleic acid into mouse cells, the plasmid replicates up to about 100 to 200 copies per cell. of the inserted cDNA does not require integration of the plasmid into the host chromosome, thus producing a high level of expression.These vectors can be used for stable expression by including a selectable marker in the plasmid, such as, for example, example, the neo gene. Alternatively, the retroviral genome can be modified to be used as a vector capable of introducing and directing the expression of the sHIF-lalfa gene in the host cells (Cone and Muíligan, Proc. Nati Acad. Sci. USA 81: 6349-6353, 1984). High-level expression can also be achieved using inducible promoters, including, but not limited to, the metallothionein IIA promoter, and the shock promoters by hot. Depending on the vector used, the polynucleotide sequences encoding sHIF-lalfa can be expressed in prokaryotes or in eukaryotes. Hosts may include microbial, yeast, insect and mammalian organisms. Methods for expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expressing and replicating in a host are known in the art. These vectors are used to incorporate DNA sequences of the invention. For the long-term high yield production of recombinant proteins, stable expression is preferred. Instead of using expression vectors containing viral replication origins, the host cells can be transformed with the SHIF-lalfa cDNA controlled by the appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. The selectable marker in the recombinant plasmid confers resistance to selection, and allows the cells to stably integrate the plasmid into their chromosomes, and grow to form foci, which in turn can be cloned and expanded into cell lines. For example, immediately after the introduction of nucleic acid strange, the designed cells can be allowed to grow for one to two days in an enriched medium, and then they are changed to a selective medium. A number of selection systems can be used, including, but not limited to, the herpes simplex virus thymidine kinase gene (Wigler et al. Cell 11: 223, 1977), the hypoxanthine-guanine phosphoribosyl transferase gene.

(Szybalska and Szybalski Proc. Nati, Acad. Sci. USA 48: 2026, 1962), and adenosine p-phosphoribosyl transferase (Lowy et al., Cell 22: 817), which can be used in tk- cells, hgprt "or aprt-, respectively. Additionally, anti-metabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler et al., Nati, Acad. Sci. USA 22: 3561, 1980; O 'Hare et al., Proc. Nati, Acad. Sci. USA 78: 1527, 1981); the gpt gene, which confers resistance to mycophenolic acid (Mulligan and Berg, Proc. Nati, Acad. Sci. USA 78: 2072, 1981); the neo gene, which confers resistance to aminoglycoside G-418 (Colberre-Garapin et al., J. Mol. Biol. 150: 1, 1981); and the hygro gene, which confers resistance to hygromycin (Santerre et al., Gene 30 ,: 147, 1984). Recently, additional selectable genes have been described, i.e., trpB, which allows cells to use indole instead of tryptophan; hisD, which allows cells to use histinol in place of histidine (Hartman and Mulligan, Proc. Nati, Acad. Sci. USA 85_: 8047, 1988); and ODC (ornithine decarboxylase), which confers resistance to the ornithine decarboxylase inhibitor, 2- (difluoromethyl) -DL-ornithine, DFMO (McConlogue L., in: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory editors, 1987). "Transformation" means a genetic change induced in a cell following the incorporation of new DNA (i.e., DNA exogenous to the cell). When the cell is a mammalian cell, genetic change is usually achieved by introducing the DNA into the cell's genome (ie, stable). "Transformed cell" means a cell in which (or in an ancestor of which) a DNA molecule encoding sHIF-lalfa has been introduced by recombinant DNA techniques. The transformation of a host cell with recombinant DNA can be done by conventional techniques, as are well known to those skilled in the art. When the host is prokaryotic, such as E. Coli, competent cells can be prepared that are capable of recovering DNA from harvested cells after the exponential growth phase, and subsequently treated by the CaCl 2 method using well-known procedures in the technique. Alternatively, MgCl 2 or RbCl can be used. The transformation can also be performed after forming a protoplast of the host cell if desired. When the host is a eukaryote, they can be used these DNA transaction methods as calcium phosphate is co-precipitated, conventional mechanical methods, such as micro-injection, electro-incorporation, insertion of a plasmid enclosed in liposomes, or virus vectors. Eukaryotic cells can also be co-transformed with DNA sequences encoding the sHIF-lalpha of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40), adenovirus, or bovine papilloma virus, to transiently infect or transform eukaryotic cells, and express the protein (see, for example, Eukaryo-tic Viral). Vectors, Cold Spring Harbor Laboratory, Gluzman, ed., 1982). Isolation and purification of the expressed microbial polypeptide, or fragments thereof, provided by the invention, it can be carried out by conventional means, including preparation chromatography and immunological separations involving monoclonal or polyclonal antibodies. The HIF-lalfa polypeptides of the invention can also be used to produce antibodies that are immunoreactive, or selectively bind with epitopes of the sHIF-lalfa polypeptides. An anti-body that "binds selectively" with SHIF-lalfa is an anti-body that binds SHIF-lalfa with a higher affinity than the anti-body binds. body with wild-type sHIF-lalfa. Accordingly, the anti-bodies of the invention can be used to distinguish the presence of the sHIF-lalfa mutein from the wild-type HIF-lalfa polypeptide. Anti-bodies consisting essentially of monoclonal anti-bodies grouped with different epitopic specificities are provided, as well as different preparations of monoclonal anti-bodies. Monoclonal anti bodies are made from fragments containing protein antigen, by methods well known in the art (Kohler et al., Nature 256: 495. 1972; Current Protocols in Molecular Bioloqy, Ausubel et al., Editors, 1989). ). The term "anti-body", as used in this invention, includes intact molecules, as well as fragments thereof, such as Fab, F (ab ') 2 and Fv, which are capable of binding to the epitope determinant. These anti-body fragments retain some ability to selectively bind to their antigen or receptor, and are defined as follows: (1) Fab, the fragment containing a monovalent antigen binding fragment of an anti-body molecule, can be produce by digestion of the entire anti-body with the enzyme papain to produce an intact light chain, and a heavy chain portion; (2) Fab ', the fragment of an anti-body molecule can be obtained by treating the whole anti-body with pepsin, followed by reduction, to produce an intact light chain, and a portion of the heavy chain; two Fab1 fragments are obtained per anti-body molecule; (3) (Fab ') 2, the anti-body fragment that can be obtained by treating the whole anti-body with the enzyme pepsin without subsequent reduction; F (ab ') 2 is a dimer of two Fab1 fragments held together by two bisuifide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain, and the variable region of the heavy chain, expressed as two chains; and (5) Single chain anti-body ("SCA"), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker, such as a single chain genetically fused molecule. The methods for making these fragments are known in this field. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference. As used in this invention, the term "epitope" means any antigenic determinant on an antigen to which the paratope of an anti-body is linked. Epitopic determinants usually consist of surface groupings chemically active molecules, such as amino acids, or sugar side chains, and usually have specific three-dimensional structural characteristics, as well as specific loading characteristics. Anti-bodies that selectively bind to the SHIF-lalfa polypeptide of the invention can be prepared using an intact polypeptide, or fragments containing small peptides of interest, such as the immunizing antigen. The polypeptide or a peptide used to immunize an animal, can be derived from the translated cDNA, or by chemical synthesis, which can be conjugated with a carrier protein, if desired. These commonly used carriers, which are chemically coupled with the peptide, include orifice limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (, and tetanus toxoid. The coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit). If desired, the polyclonal or monoclonal anti-bodies can be further purified, for example, by their binding to, and elution from, a matrix to which the polypeptide is fixed or a peptide with which the antibodies were reproduced. . Those skilled in the art will know different techniques common in the immunology technique for the purification and / or concentration of polyclonal anti-bodies, as well as monoclonal anti-bodies. See, for example, Colligan et al., Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994, incorporated herein by reference. It is also possible to use anti-idiotype technology to produce monoclonal anti-bodies that mimic an epitope. For example, an anti-idiotypic monoclonal anti-body made for a first monoclonal anti-body, will have a binding domain in the hyper-variable region, which is the "image" of the epitope linked to the first anti-monoclonal body. For the purposes of the invention, an anti-body or nucleic acid probe specific for sHIF-lalfa can be used to detect the sHIF-lalfa polypeptide, or the polynucleotide in biological fluids, cultured cells or tissues. The anti-body that reacts with sHIF-lalfa or the nucleic acid probe, preferably is labeled with a compound that allows the detection of the binding with sHIF-lalfa. Any sample containing a detectable amount of antigen or polynucleotide can be used. The invention provides methods for the treatment of disorders mediated by HIF-1, including tissue damage related to hypoxia or ischemia, which are ameliorated or ameliorated by modulating the expression or activity of HIF-1. The term "modular" provides for the induction or increase of HIF-1 expression when appropriate. The term "lessen" denotes a reduction of the detrimental effect of the associated disease in the subject receiving therapy. When the expression or the Increased expression of HIF-1, the method of treatment includes administration of the substantially purified sHIF-lalfa polypeptide, or the polynucleotide encoding it. In accordance with the method of the invention, the substantially purified sHIF-lalfa mutein, or the sequence of the polynucleotide encoding sHIF-lalfa in an appropriate vector, is introduced into a human patient for the treatment or prevention of related tissue damage. with hypoxia / ischemia. Non-limiting examples include patients with coronary, cerebral or peripheral arterial disease, and patients with one or more wounds that do not heal. Relevant clinical conditions treated by the methods and compositions of the invention include ischemia due to cerebral, coronary or peripheral circulation disease. A therapeutic goal is to promote angiogenesis in ischemic tissue by over-expressing sHIF-lalfa, which would dimerize with the endogenous HIF-lbeta, bind to specific DNA sequences, and activate the transcription of the relevant hypoxia-inducible genes. for angiogenesis, such as, but not limited to, the gene encoding vascular endothelial growth factor (VEGF), a known HIF-1 target gene (JA Forsythe et al., Mol Cell Biol 16: 4604, 1996; Iyer et al., Genes Dev 12: 149, 1998). The regionalization to use HIF-lalfa is that, because it is a transcription factor that controls the expression of multiple genes involved in angiogenesis, will give a superior clinical result, compared with treatment with a single angiogenic factor, such as vascular endothelial growth factor. However, the method of delivering DNA to the tissue site is in no way affected by the identity of the particular gene being delivered. In addition, many patients with coronary artery disease do not have reduced blood flow to the myocardium or hypoxia at rest. It is only when they are active and require greater blood flow to the myocardium when they experience symptoms of angina resulting from myocardial ischemia. Alternatively, a narrow coronary vessel may become completely obstructed, either by a spasm or a clot, resulting in myocardial infarction (heart attack). Therefore, the goal of treatment with the stable form of HIF-lalfa is to induce angiogenesis in these patients, even when there is no hypoxia at the time, in order to prevent heart attacks. In accordance with the above, the stable HIF-lalfa compositions of the invention provide prophylactic as well as therapeutic regimens. The present invention provides the introduction of polynucleotides encoding sHIF-lalfa for the treatment of hypoxia-related disorders, which are ameliorated or ameliorated by expression of the HIF-lalfa polypeptide. This therapy would achieve its therapeutic effect by introducing the polynucleotide sHIF-lalfa in cells exposed to hypoxic conditions. HIF-lalfa, therefore, is expressed both in surrounding hypoxic and non-hypoxic tissues, such that it can be dimerized with HIF-lbeta (which is present in excess in hypoxic and non-hypoxic cells), and activate the transcription of the target genes downstream. Examples of genes that can be activated by HIF-1 are vascular endothelial growth factor, glucose transporters, glycolytic enzymes, and insulin-like growth factor 2. These genes mediate important adaptive responses to hypoxia , including angiogenesis and glycolysis, and the prevention of cell death. Based on the foregoing, the invention provides a method for increasing the expression of a hypoxia-inducible gene in a cell. The method includes contacting the cell with an expression vector that contains a polynucleotide that encodes a stable HIF-lalfa of the invention, or a chimeric transactivator protein as described herein, under conditions that allow the expression of the sequence of nucleic acid contained in the vector, thereby providing greater expression of a hypoxia-inducible gene in the cell. These genes include, for example, those encoding vascular endothelial growth factor, glucose transporters, glycolytic enzymes, IGF-2, IGF binding proteins and the like.

The invention further provides a method for providing constitutive expression of a hypoxia inducible factor in a cell, under hypoxic or non-hypoxic conditions. The method includes contacting the cell with a nucleic acid sequence encoding a chimeric transactivator protein as described herein, or a stable HIF-alpha as described herein, under conditions that allow the expression of the sequence of nucleic acid, thereby providing the constitutive expression of a hypoxia-inducible factor. In addition, the invention includes a method for reducing tissue damage related to hypoxia or ischemia in a subject who has, or is at risk of having, this damage. The method includes administering to the subject a therapeutically effective amount of a nucleic acid sequence encoding a chimeric transactivator protein as described herein, or a stable HIF-alpha as described herein, in a pharmaceutically acceptable carrier, reducing This way the tissue damage. In another embodiment, the invention provides a method of providing prophylactic therapy for tissue in a subject in need, which comprises administering to the subject an amount of a polypeptide encoded by a polynucleotide that encodes a chimeric transactivator protein as described herein. , or a stable HIF-lalfa as described herein, such that angiogenesis is induced at levels that are greater than before the administration of the polypeptide, thereby providing prophylactic therapy. The delivery of the polynucleotide can be achieved using a recombinant expression vector, such as a chimeric virus, or a colloidal dispersion system. For the therapeutic delivery of the sequences, the use of targeted liposomes is especially preferred. Different viral vectors that can be used for gene therapy as taught herein, include adenovirus, adeno-associated virus, herpes virus, vaccine, or preferably an RNA virus, such as a retrovirus. Preferably, the retroviral vector is a derivative of a murine or bird retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), murine sarcoma virus Harvey (HaMuSV), murine mammary tumor virus ( MuMTV), and Rous Sarcoma Virus (RSV). Preferably, when the subject is a human being, a vector is used, such as the gibbon monkey leukemia virus (GaLV). A number of additional retroviral vectors can incorporate multiple genes. All these vectors can transfer or incorporate a gene for a selectable marker, in such a way that transduced cells can be identified and generated. By inserting a The sHIF-lalpha sequence of interest in the viral vector, together with another gene encoding the ligand for a receptor on a specific target cell, for example, the vector is now target-specific. Retroviral vectors can be made specific to the target by binding, for example, a sugar, a glycolipid, or a protein. The preferred direction is made using an anti-body to direct the retroviral vector. Those skilled in the art will know of, or can readily assert without undue experimentation, specific polynucleotide sequences that can be inserted into the retroviral genome or attached to a viral envelope to allow specific delivery of the retroviral vector target containing the polynucleotide. sHIF-lalfa. Because the recombinant retroviruses are defective, they require assistance, in order to produce infectious vector particles. This assistance can be provided, for example, by the use of helper cell lines containing plasmids that encode all the structural genes of the retrovirus under the control of regulatory sequences within the LTR. These plasmids lack a nucleotide sequence that makes possible the packaging mechanism to recognize an RNA transcript for encapsidation. Auxiliary cell lines that have deletions of the packaging signal include, but are not limited to, 2, PA317 and PA12, for example. These cell lines produce empty virions, because no genome is packed. If a retroviral vector is introduced into these cells where the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged, and the vector virion can be produced. Alternatively, NIH 3T3 cells or other tissue culture cells can be transfected directly with plasmids encoding the retroviral structural genes gag, pol and env, by conventional transfection with calcium phosphate. Then these cells are transfected with the plasmid of the vector that contains the genes of interest. The resulting cells release the retroviral vector into the culture medium. Another targeted delivery system for HIF-1 polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include complexes of macromolecules, nanocapsules, microspheres, beads and lipid-based systems, including oil-in-water emulsions, mycelia, mixed mycelia, and liposomes. The preferred colloidal system of this invention is a liposome. Liposomes are artificial membrane vesicles that are useful as delivery vehicles in vi tro and in vivo. It has been shown that large unilamellar vesicles (LW), which have a size of 0.2 to 4.0 microns, can encapsulate a substantial percentage of an aqueous regulator containing large macromolecules. RNA, DNA and intact virions can be encapsulated inside the interior aqueous, and can be delivered to the cells in a biologically active form (Fraley et al., Trends Biochem, Sci. 6: 11, 1981). In addition to mammalian cells, liposomes have been used to deliver polynucleotides in plant, yeast and bacterial cells. In order for a liposome to be an efficient gene transfer vehicle, the following characteristics must be present: (1) encapsulation of the genes of interest with high efficiency, while not compromising its biological activity; (2) preferential and substantial linkage with a target cell compared to non-target cells; (3) supply of the aqueous content of the vesicle to the target cell cytoplasm with high efficiency; and (4) accurate and effective expression of the genetic information (Mannino et al., Biotechniques 6: 682, 1988). The composition of the liposome is usually a combination of phospholipids, particularly phospholipids of high phase transition temperature, usually in combination with sterols, especially cholesterol. Other phospholipids or other lipids can also be used. The physical characteristics of liposomes depend on the pH, the ionic concentration, and the presence of divalent cations. Examples of lipids useful in the production of liposomes include phosphatidyl compounds, such as phosphatidyl glycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosi-two. They are particularly diacylphosphatidyl glycerols, wherein the lipid fraction contains from 14 to 18 carbon atoms, particularly from 16 to 18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine. The direction of the liposomes can be classified based on anatomical and mechanical factors. The anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific. The mechanical direction can be distinguished based on whether it is passive or active. The passive direction uses the natural tendency of the liposomes to be distributed in the cells of the reticuloendothelial system (RES) in the organs that contain sinusoidal capillaries. On the other hand, active management involves the alteration of the liposome by coupling liposome with a specific ligand, such as a monoclonal anti-body, sugar, glycolipid, or protein, or changing the composition or size of the liposome in order to achieve the direction to the organs and cell types different from the localization sites that occur naturally. The surface of the targeted delivery system can be modified in a variety of ways. In the case of a liposomal targeted delivery system, the lipid groups they can be incorporated into the lipid bi-layer of the liposome, in order to maintain the targeting ligand in stable association with the liposomal bi-layer. Different linking groups can be used to link the lipid chains with the targeting ligand. The sHIF-lalfa polypeptide can be used in therapeutic administration. For this administration, the polypeptide must be sterile. Sterility is easily achieved by sterile filtration through membranes (for example, 0.2 microns). The compound of the invention will ordinarily be stored as single or multi-dose containers, for example, sealed vials or flasks, as an aqueous solution, because it is highly stable to thermal and oxidative denaturation. Freeze-dried formulations for reconstitution are also acceptable. The polypeptide will be administered as a pharmaceutical composition (see below). The invention also discloses a method for the treatment of a subject having a disorder related to hypoxia, by administering to the subject a therapeutically effective amount of cells expressing sHIF-lalfa. "Therapeutically effective", as used herein, refers to the amount of cells that is an amount sufficient to alleviate a symptom of the disease, or to lessen the disorder related to hypoxia. The effective amount gives as Resulting expression of biologically active stable HIF-lalfa over a period of time such that one or more symptoms of the disease / disorder are alleviated. These methods are useful for increasing or sustaining the expression of HIF-lalpha and / or hypoxia-inducible genes in tissues under hypoxic or non-hypoxic conditions. In some preferred embodiments of the methods of the invention described above, HIF-lalfa is administered locally (eg, inter-lesionally) and / or systemically. The term "local administration" refers to delivery to a defined area or region of the body, such as for wounds that do not heal, while the term "systemic administration" means that it encompasses delivery to the subject by the oral route, or by intramuscular, intravenous, intra-arterial, subcutaneous or intra-peritoneal injection. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The term "physiologically acceptable" refers to a non-toxic material that is compatible with a biological system, such as a cell, cell culture, tissue or organism. The sHIF-lalfa compositions of the invention can be used as part of a pharmaceutical composition, when combined with a physiologically and / or pharmaceutically acceptable carrier. The characteristics of the vehicle will depend of the route of administration. This composition may contain, in addition to the synthetic oligonucleotide and the carrier, diluents, fillers, salts, pH regulators, stabilizers, solubilizers and other materials well known in the art. The pharmaceutical composition of the invention may also contain other factors and / or active agents that improve expression, or that help stimulate angiogenesis. For example, sHIF-lalfa, in combination with vascular endothelial growth factor, can be used in the pharmaceutical compositions of the invention. The pharmaceutical composition of the invention may be in the form of a liposome, wherein the sHIF-lalfa compositions of the invention are combined, in addition with other pharmaceutically acceptable carriers, with amphipathic agents, such as lipids, that exist in cumulative form as mycelia, insoluble mono-layers, liquid crystals, or lamellar layers that are in an aqueous solution. Suitable lipids for the liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. A particularly useful lipid vehicle is lipofectin. The preparation of these liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Patent Nos. 4,235,871; 4,501,728; 4,837,028 and 4,737,323. The pharmaceutical composition of the invention may also include compounds such as cyclodextrins and the like, which improve the delivery of the nucleic acid molecules to the cells, or the slow release polymers. When a therapeutically effective amount of the composition of the invention is administered by intravenous, subcutaneous, intramuscular, intra-arterial, infra-ocular or intra-peritoneal injection, the composition will be in the form of an aqueous, pyrogen-free, parenterally acceptable solution. The preparation of these parenterally acceptable solutions, having due consideration of pH, isotonicity, stability, and the like, is within the skill in this field. A preferred pharmaceutical composition for intravenous, subcutaneous, intramuscular, intraperitoneal, or infraocular injection must contain, in addition to the sHIF-lalpha composition, an isotonic vehicle, such as Sodium Chloride Injection, Ringer's Injection, Injection Dextrose, Injection of Dextrose and Sodium Chloride, Lactated Ringer's Injection, or other vehicle known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, pH regulators, antioxidants or other additives known to those skilled in the art. The amount of the sHIF-lalfa composition, in the pharmaceutical composition of the present invention, will depend on the nature and severity of the condition being treated, and on the nature of the previous treatments to which it has been applied. submitted the patient. Finally, the attending physician will decide the amount of sHIF-lalfa composition, with which he will treat each individual patient. Initially, the attending physician will administer low doses of the sHIF-lalfa composition, and observe the patient's response. Higher doses of the sHIF-lalfa composition can be administered, until the optimal therapeutic effect is obtained for the patient, and at that point, the dosage is no longer increased. It is contemplated that the different pharmaceutical compositions used to practice the method of the present invention should contain from about 10 micrograms to about 20 milligrams of the sHIF-lalfa composition per kilogram of body or organ weight. The duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated, and the condition and potential idiosyncratic response of each individual patient. Finally, the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention. Cell transduction is performed in vi tro, usually with populations of isolated cells or cell lines. The cells may be xenogeneic, allogeneic, syngeneic or autologous, preferably autologous, with the object of reducing adverse immune responses. The cells can be administered in any physiologically acceptable medium, usually in an intravascular manner, although they can also be introduced into the surrounding tissue to a vessel or other convenient site, where the cells can find an appropriate site for their expansion and differentiation. . "Aminorate" refers to reducing or decreasing the detrimental effect of the disease or disorder on the patient receiving the therapy. Any of the transplantation or implant procedures known in the art can be used. For example, selected cells or cells of interest may be surgically implanted in the recipient or in the subject. The transplant or implant is usually by simple injection through a hypodermic needle having a sufficient orifice diameter to allow the passage of a suspension of cells therethrough without damaging the cells or the coating of the tissue. For the implant, typically encapsulated or coated cells are formulated as pharmaceutical compositions together with a pharmaceutically acceptable carrier. These compositions contain a sufficient number of coated transplant cells that can be injected into, or can be administered through a laparoscope to, a subject. Typically, at least about lx104 to lx105 cells, preferably lx106 or more, will be administered. The cells can be frozen at temperatures of liquid nitrogen, and can be stored for long periods of time, being used when thawing. Once thawed, the cells can expand. In addition, the cells can be administered in an encapsulated form or in an unencapsulated form. Preferably, the cells are encapsulated. Although not required, it may be desirable to administer an immunosuppressive agent to a recipient of the cells prior to, concurrent with, and / or after transplantation. In particular, an immunosuppressive agent can be used with xenogenetic or allogeneic cells expressing sHIF-lalfa. An agent such as Cyclosporin A (CsA) is preferable; however, other immunosuppressive agents can be used, such as rapamycin, deoxyspergualin, FK506 and the like. These agents are administered to cause an immunosuppressive effect in the subject, such that the transplanted cells are not rejected by the immune system of that subject. Normally, the immunosuppressive agent is administered continuously throughout the transplant treatment, typically over a period of days or weeks; for example, treatment with CsA is from about 2 to about 20 days in a dosage range of about 5 to 40 milligrams per kilogram of body weight per day. The agent can be administered by a variety of means, including parenteral, subcutaneous, intra-pulmonary, oral, intranasal administration and the like. From Preferably, the dosage is given by oral administration. Cells that express HIF-lalfa can also be encapsulated before transplantation. Although the cells are normally micro-encapsulated, they can be enclosed in different types of hollow fibers or in a block of encapsulating material. A variety of micro-encapsulation methods and compositions are known in the art. A number of micro-encapsulation methods for use in transplant therapy have focused on the use of alginate or agarose polymers to deliver the encapsulation compositions. Alginates are linear polymers of mannuronic and guluronic acid residues, which are formed into blocks of several adjacent guluronic acid residues, forming blocks of guluronate, and blocks of adjacent mannuronic acid residues forming mannuronate blocks, interspersed with mixed blocks or heterogeneous residues of alternating gururonic and mannuronic acid residues. In general, monovalent cation alginate salts are soluble, for example, Na alginate. Divalent cations, such as Ca ++, Ba ++ or Sr ++, tend to interact with the guluronate, and the cooperative bonding of these cations within the guluronate blocks provides the primary intra-molecular cross-linking responsible for the formation of matched ion alginate gels stable Alginate encapsulation methods generally take advantage of alginate gelation in the presence of these solutions. divalent cations. In particular, these methods involve the suspension of the material to be encapsulated, in a solution of monovalent cation alginate salt, for example, sodium. Then drops of the solution are generated in air, and collected in a solution of divalent cations, for example, CaCl2. The divalent cations interact with the alginate in the phase transition between the drop and the divalent cation solution, resulting in the formation of a stable alginate gel matrix. The generation of alginate drops has been previously performed by a number of methods. For example, droplets have been generated by extruding the alginate through a tube by gravitational flow, to a solution of divalent cations. In a similar manner, electrostatic droplet generators have been described that rely on the generation of an electrostatic differential between the alginate solution and the divalent cation solution. The electrostatic differential results in the solution of alginate being extracted through a tube, towards the solution of divalent cations. Methods have been described wherein drops are generated from a stream of the alginate solution, using a laminar air flow extrusion device. Specifically, this device comprises a capillary tube inside an external jacket. Air is blown through the outer jacket, and the flow of the polymer solution through the inner tube is regulated. The flow of air from the outer jacket breaks the fluid flowing from the capillary tube, in small droplets (see U.S. Patent No. 5,286,495). To see a general discussion of droplet generation in the encapsulation processes, see, for example, M.F.A. Goosen, Fundamentals of Animal Cell Encapsulation and Immobilization, Chapter 6, pages 114-142 (CRC Press, 1993). Attempts to transplant organ tissue into genetically distinct hosts without immuno-suppression are generally overridden by the host's immune system. In accordance with the foregoing, attempts have been made to provide other effective barrier barrier coatings, for example, by micro-encapsulation, to isolate the transplant tissues from the host immune system. Successful cell or tissue transplants generally require a coating that prevents their destruction by the immune system of a host, that prevents fibrosis, and that is permeable to, and allows the free diffusion of, the nutrients towards the coated transplant, and the removal of secretion and waste products from the coated transplant. Tissue and viable cells have been successfully immobilized in polylysine-coated alginate capsules (see above, and J. Pharm. Sci. 70: 351-354, 1981) .The development of transplants encapsulated in alginate capsules is also described. calcium that reacted with polylysine, for example, in U.S. Patent Nos. 4,673,566, 4,689,293, 4,789,550, 4,806,355 and 4,789,550. U.S. Patent No. 4,744,933 discloses encapsulation solutions containing biologically active materials in an interreacted alginate membrane and polyamino acid. U.S. Patent No. 4,696,286 reports a method for coating suitable transplants to be transplanted to genetically distinct individuals. The method involves coating the transplant with a surface-forming bonding bridge of a multi-functional material that is chemically fixed to a surface component of the transplant, which is wrapped in a biologically compatible semi-permeable layer of a polymer that is chemically fixed to the layer of the link bridge. In U.S. Patent No. 5,277,298, a method is disclosed for introducing a second alginate gel coating to cells already coated with polylysine alginate. Both the first and the second coatings of this method require stabilization by polylysine. The encapsulation methods applied to make these materials have included a procedure to form drops of the encapsulating medium and the biological material, and a method for solidifying the encapsulating medium. Encapsulated agarose materials have been formed by freezing an emulsion of agarose droplets containing biological materials, as shown by Nilsson et al., Nature 302: 629-630 (1983), and Nilsson et al., Eur. J. Appl. Microbiol. Biotechnol. 17: 319-326 (1983). The injection of polymer droplets containing biological materials into a refrigerant body, such as a concurrently liquid stream, has been reported by Gin et al., J. "Microencapsulation 4: 329-242 (1987) This invention involves administering to a subject , a therapeutically effective dose of a pharmaceutical composition containing the compositions of the present invention, and a pharmaceutically acceptable carrier The "administration" of the pharmaceutical composition of the present invention can be carried out by any means known to the skilled person. they are preferably prepared and administered in unit dosages Solid unit dosages are tablets, capsules and suppositories For the treatment of a patient, depending on the activity of the compound, the mode of administration, the nature and severity of the disorder, the patient's age and body weight are necessary Different daily sis. However, under certain circumstances, higher or lower daily doses may be appropriate. The administration of the daily dose can be performed either by a single administration in the form of an individual unit dose, or otherwise, several smaller unit doses, and also by the multiple administration of subdivided doses at specific intervals.

The pharmaceutical compositions according to the invention are generally administered topically, orally, intravenously, or by another parenteral route, or as implants, or rectal use is even possible in principle. Suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, tablets, coated tablets (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or an injectable solution in the form of an ampoule, and also preparations with prolonged release of the active compounds, in which excipients and preparation additives, and / or auxiliaries, such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used, as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of the present methods for drug delivery, see Langer, Science, 249: 1527-1533, 1990, which is incorporated herein by reference. For the delivery of the sHIF-lalfa mutein, the formulations are prepared by contacting the sHIF-lalfa mutein in a uniform and intimate manner with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, configuring the product in the desired formulation. Preferably, the vehicle is a parenteral vehicle, more preferred a solution that is isotonic with the recipient's blood. Examples of these vehicles include water, serum, Ringer's solution, dextrose solution, and 5 percent human serum albumin. Nonaqueous vehicles, such as fixed oils and ethyl oleate, as well as liposomes are also useful herein. In general, the vehicle may contain minor amounts of additives, such as substances that improve isotonicity and chemical stability, for example, pH regulators and preservatives, as well as low molecular weight polypeptides (less than about 10 residues), proteins, amino acids, carbohydrates, including glucose or dextrans, chelating agents, such as EDTA, or other excipients. The composition of the present is also suitably administered by sustained release systems. Suitable examples of sustained release compositions include semipermeable polymer matrices in the form of shaped articles, eg, films, microcapsules, or microspheres. Sustained-release matrices include, for example, polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and ethyl L-glutamate (Sidman et al., Biopolymers 22: 547-556, 1983), or either poly-D- (-) - 3-hydroxybutyric acid (European patent No. EP 133,988). Sustained release compositions also include one or more liposomally entrapped compounds of Formula I. These compositions are prepared by methods known per se, for example, as taught by Epstein et al., Proc. Nati Acad. Sci. USA 82: 3688-3692, 1985. Ordinarily, the liposomes are of the small unilamellar type (from 200 to 800 A), where the lipid content is greater than about 30 mole percent cholesterol, the selected proportion being adjusted to the optimal therapy. The pharmaceutical compositions according to the invention can be administered locally or systemically. A "therapeutically effective dose" means the amount of a compound according to the invention, necessary to prevent, cure, or at least partially arrest the symptoms of the disorder and its complications. Of course, the effective amounts for this use will depend on the severity of the disease and the weight and general condition of the patient. Normally, the dosages used in vi tro can provide useful guidance in amounts useful for the in-situ administration of the pharmaceutical composition, and animal models can be used to determine effective dosages for the treatment of particular disorders. Different considerations are described, for example, in Gilman et al., Editors, Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th edition, Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Co., Easton, Pennsylvania, United States, 1990; each of which is incorporated herein by reference.

The stable chimeric and HIF-lalfa transactivator compositions of the invention can also be delivered in the form of naked DNA, for example, by the methods described in U.S. Patent No. 5,589,466. The following examples are intended to illustrate, but not limit, the invention. Although typical of those that could be used, other methods known to those skilled in the art can be used alternatively. EXAMPLE 1 GENERATION OF A CONSTITUTIVELY EXPRESSED FORM OF H F-lalfa It has previously been demonstrated (Jiang et al., J. Biol. Chem 272: 19253, 1997; Pugh et al., J., Biol. Chem. 272: 11205) that a fusion protein consisting of the GAL4 DNA binding domain fused to the residues of HIF-lalfa 531-826, is a constitutively expressed protein that can activate the transcription of reporter genes containing GAL4 binding sites. GAL4 / HIF-lalfa does not activate the normal target genes regulated by HIF-1, and conversely, it was shown that the amino acids of HIF-lalfa 1-390 are sufficient for the dimerization of HIF-lalfa with HIF-lbeta, and the link with sequences of target DNA, but insufficient for optimal activation of genetic transcription (Jiang, BH et al., J. Biol. Chem. 271: 17771-17778, 1996; United States Patent No. 5,882,914) .To generate a constitutive form expressed entity of HIF-lalfa, two series of suppression constructions were produced, one where deletions started at the carboxyl-terminal end of the molecule (amino acid 826) and extended to the amino terminus, and one where deletions started at the amino acid 392 and they extended to the carboxyl terminus. Each of these constructs was expressed in mammalian cells under non-hypoxic conditions (20 percent of 02) 0 hypoxic (1 percent of 02), and the expression of the endogenous full-length HIF-lalfa and the deleted HIF-lalfa transfecta-do, was quantified by immunoblotting assay, using purified anti-HIF-lalfa anti-bodies by affinity. These studies revealed that the endogenous HIF-lalfa showed a regulated expression (more protein expressed in the cells with the 1 percent of 02 with cells of 20 percent of 02). In addition, the studies showed that C-terminal deletion up to amino acid 726, had no effect on the regulation of the expression of HIF-lalfa protein by the concentration of 02, while deletion to amino acid 703 or beyond, resulted in loss of regulation (ie, constitutive expression, see Figure 2). Internal deletions extending from amino acid 392 to 517 had no effect on expression, whereas deletion of amino acid 392 to amino acid 521 resulted in loss of regulation (see Figure 3). In addition, mutations in the wrong direction S551G / 552A (a substitution of serine to glycine and threonine to alanine in amino acids 551 and 552, respectively) resulted in the loss of regulation of internal suppression constructs that otherwise showed regulation (ie, deletions extending from amino acid 392 to any part between amino acids 429 and 517). These misdirected mutations alone did not cause a misregulated expression of full-length HIF-lalfa (amino acids 1-826, see Figure 3). The results suggested that there were two regions of HIF-lalfa that were required for regulated expression, such that suppression of any region resulted in poorly regulated expression (see Figure 4). The first of these regions is the AB region (amino acids 392-552). Within this internal region, two sequences (A and B) that seemed functionally redundant were identified, because the presence of any sequence was sufficient for regulation. One of these sequences (A) was identified by deletion 392-428, and the other sequence (B) was identified by deletion 392-520, or point mutations S551G / T552A. This latter result suggested that the serine and / or threonine residue was subject to phosphorylation / dephosphorylation, which could be altered by the deletion 392-520. Because the loss of serine / threonine sequence mimicked hypoxia, these results suggest phosphorylation of serine 551 and / or threonine 552 under nonhypoxic conditions, and dephosphorylation under hypoxic conditions. Based on the A and B redundancy, it is possible that a phosphatase is also fixed at site A, and dephosphorylates a nearby residue of serine or threonine. The C region is defined by the different effects of the deletions spanning amino acids 704 to 826, compared to the deletions spanning amino acids 727 to 826. The loss of region C is not redundant with the loss of region AB, and therefore, it is possible that this region is involved in some other function related to the regulation of the stability of HIF-lalfa. Without being bound by theory, it is possible that this region is involved in ubiquity or proteolysis. A powerful transactivation domain is located between amino acids 786 and 826. As a result, although HIF-lalfa (amino acids 1-703) is constitutively expressed, it is not as biologically active as full-length HIF-lalfa. In order to determine whether sHIF-lalf would demonstrate greater biological activity compared to full-length HIF-lalfa, co-transfection experiments were performed using the HIF-lalfa deletion / dot mutant (1-391 / 512-826). / S551G / T552A), a stable HIF-lalfa. 293 cells (see Figure 5), or Hep3B cells (see Figure 6) were co-transfected with a reporter gene that contained a hypoxia response element that includes an HIF-lalpha binding site, and with the mammalian expression vector pCEP4 (Invitro- gene) encoding (1) no protein, (2) HIF-lalfa (1-826), (3) HIF-lalfa (1-391 / 429-826) (deletion only), or (4) stable HIF-lalfa (HIF-lalfaDP, a form of sHIF-lalfa containing 1-391 / 512-826 / S551G / T552A). The endogenous HIF-lbeta is constitutively expressed in these cells at levels in excess of HIF-lalfa expression. In both cell types, HIF-lalfaDP (s'HIF-lalfa) measured a significantly higher expression of the reporter gene in cells exposed to 20 percent of 02, due to the presence of higher levels of biologically active HIF-lalfa. (Note that HIF-lalfa is usually expressed only with 1 percent of 02). These results demonstrate that a constitutively expressed and biologically active form of HIF-lalfa has been generated. Although the invention has been described with reference to the currently preferred embodiments, it should be understood that various modifications can be made without departing from the spirit of the invention. In accordance with the above, the invention is limited only by the following claims.

Claims (47)

1. CLAIMS 1. A polypeptide selected from the group comprising: amino acid residues 1-391 and 521-826 of SEQ ID NO: 1; amino acid residues 1-391 and 549-826 of SEQ ID NO: 1; amino acid residues 1-391 and 576-826 of SEQ ID NO: 1; amino acid residues 1-391 and 429-826 of SEQ ID NO: 1, where 551 is no longer serine and 552 is not threonine; amino acid residues 1-391 and 469-826 of SEQ ID NO: 1, where 551 is no longer serine and 552 is not threonine; amino acid residues 1-391 and 494-826 of SEQ ID NO: 1, where 551 is no longer serine and 552 is not threonine; amino acid residues 1-391 and 508-826 of SEQ ID NO: 1, where 551 is no longer serine and 552 is not threonine; amino acid residues 1-391 and 512-826 of SEQ ID NO: 1, where 551 is no longer serine and 552 is not threonine; and amino acid residues 1-391 and 517-826 of SEQ ID NO: 1, where 551 is no longer serine and 552 is not threonine.
2. 2. A polypeptide of claim 1, wherein the amino acid residue 551 is glycine.
3. 3. A polypeptide of claim 1, wherein the amino acid residue 552 is alanine.
4. 4. A nucleic acid sequence encoding a polypeptide of claim 1.
5. 5. An expression vector comprising a nucleic acid sequence of claim 1.
6. 6. A method for increasing the expression of a hypoxia-inducible gene in a cell, which comprises contacting the cell with an expression vector of claim 5 under conditions that allow the expression of the nucleic acid sequence contained in the vector, thereby providing for the increased expression of a hypoxia-inducible gene in the cell .
7. The method of claim 6, wherein the hypoxia inducible factor is HIF-lalfa.
8. 8. A method for providing constitutive expression of a hypoxia-inducible factor in a cell, comprising contacting the cell with a nucleic acid sequence of claim 2 under conditions that allow expression of the nucleic acid sequence, with this providing the constitutive expression of a hypoxia-inducible factor.
9. 9. A method for reducing or preventing tissue damage related to hypoxia or ischemia in a subject having or at risk for such damage, comprising administering to the subject a therapeutically effective amount of a nucleic acid sequence of claim 2 in a pharmaceutically carrier acceptable, thereby reducing tissue damage.
10. The method of claim 9, wherein the administration is in vivo.
11. The method of claim 9, wherein the administration is ex vivo.
12. 12. The method of claim 9, wherein the tissue damage related to hypoxia or ischemia is due to a disorder of cerebral, coronary or peripheral circulation.
13. A method for providing prophylactic therapy for tissues in a subject in need thereof, comprising administering to the subject an amount of a polypeptide of claim 1, such that angiogenesis is induced at levels that are greater than before administration of the polypeptide , thereby providing prophylactic therapy.
14. The method of claim 13, wherein the subject is at risk of coronary artery disease.
15. 15. The method of claim 13, wherein the subject is at risk of ischemic tissue damage.
16. 16. A stable, substantially purified form of hypoxia inducible alpha-l factor (HIF-lalfa), having a sequence as indicated in SEQ ID NO: 1, where amino acids 392 to 428 are deleted from it, the amino acid 551 is changed from a serine to any other amino acid, and amino acid 552 is changed from a threonine to any other amino acid.
17. 17. The stable form of hypoxia-inducible alpha-factor l of claim 16, wherein amino acid 551 is a glycine.
18. 18. The stable form of hypoxia-inducible alpha-l factor of claim 16, where amino acid 552 is an alanine.
19. 19. The stable form of hypoxia-inducible alpha-factor l of claim 16, further comprising a deletion of amino acids 576-785, or any portion thereof.
20. 20. A nucleic acid sequence comprising a polynucleotide encoding the stable form of hypoxia-inducible human alpha-l factor (HIF-alpha) of claim 16.
21. 21. The nucleic acid of claim 20, further comprising a sequence of expression control operably linked to it.
22. 22. The nucleic acid sequence of claim 21, wherein the expression control sequence is a promoter.
23. 23. The polynucleotide of claim 22, wherein the promoter is tissue specific.
24. 24. An expression vector containing the polynucleotide of claim 20.
25. 25. The vector of claim 24, wherein the vector is a plasmid.
26. 26. The vector of claim 24, wherein the vector is a viral vector.
27. 27. The vector of claim 26, wherein the vector is a retroviral vector.
28. 28. A host cell containing the vector of claim 24.
29. 29. A host cell of claim 28, wherein the cell is a eukaryotic cell.
30. 30. A host cell of claim 28, wherein the cell is a prokaryotic cell.
31. 31. An anti-body that selectively binds to the polypeptide of claim 16.
32. 32. The anti-body of claim 31, wherein the anti-body is monoclonal.
33. 33. The anti-body of claim 31, wherein the anti-body is polyclonal.
34. 34. A method of treating tissue damage related to hypoxia in a subject, comprising administering to said subject a therapeutically effective amount of a nucleotide sequence comprising an expression control sequence operably linked to a polynucleotide encoding a polypeptide having a sequence as indicated in SEQ ID NO: 1, where amino acids 392 to 428 are deleted from it, amino acid 551 is changed from a serine to any other amino acid, and amino acid 552 is changed from a threonine to any other amino acid.
35. 35. The method of claim 34, wherein amino acid 551 is a glycine.
36. 36. The method of claim 34, wherein amino acid 552 is an alanine.
37. 37. A method of treating tissue damage related to hypoxia in a subject, which comprises administering to said subject a therapeutically effective amount of a polypeptide having a nucleotide sequence as set forth in SEQ ID NO: 1, where it is deleted from the same amino acids 392 to 428, amino acid 551 is changed from a serious to any other amino acid, and amino acid 552 is changed from a threonine to any other amino acid.
38. 38. The method of claim 37, wherein amino acid 551 is a glycine.
39. 39. The method of claim 37, wherein amino acid 552 is an alanine.
40. 40. A formulation for administering stable, hypoxia-inducible, human alpha-L-factor polypeptide (HIF-alpha) to a patient having tissue damage related to hypoxia, comprising: (a) a therapeutically effective amount of a polypeptide substantially pure having a sequence as indicated in SEQ ID NO: 1, where amino acids 392 to 428 are deleted from it, amino acid 551 is changed from a any other amino acid, and amino acid 552 is changed from a threonine to any other amino acid; and (b) a pharmaceutically acceptable carrier.
41. 41. The formulation of claim 40, wherein the carrier is a liposome.
42. 42. The formulation of claim 40, wherein amino acid 551 is a glycine.
43. 43. The formulation of claim 40, wherein amino acid 552 is an alanine.
44. 44. A formulation for administration of a hypoxia-inducible, human-inducible, hypo-inducible, human alpha-factor alpha-1 polynucleotide (HIF-lalfa) to a patient having hypoxia-related tissue damage, comprising: (a) a therapeutically effective amount of a nucleic acid sequence comprising an expression control sequence operably linked to a polynucleotide encoding a polypeptide having a sequence as set forth in SEQ ID NO: 1, where amino acids 392 to 428, the amino acid, are deleted therefrom; 551 is changed from a serine to any other amino acid, and amino acid 552 is changed from a threonine to any other amino acid; and (b) a pharmaceutically acceptable carrier.
45. 45. The formulation of claim 44, wherein the carrier is a liposome.
46. 46. The formulation of claim 44, wherein the amino acid 551 is a glycine.
47. 47. The formulation of claim 44, wherein amino acid 552 is an alanine.