WO2003072744A2 - Dna construct for inducible expression of angiogenic protein and use thereof - Google Patents

Abstract

Disclosed is a nucleic acid construct that includes a DNA molecule encoding an angiogenic protein or polypeptide; a polyadenylation sequence operably coupled 3’ of the DNA molecule;.and a glucocorticoid-inducible promoter region operably coupled 5’ of the DNA molecule. Expression vectors and pharmaceutical compositions containing the nucleic acid construct, including liposomal compositions, are also disclosed. The nucleic acid construct, expression vectors, and compositions are intended for use in accordance with various methods of promoting neovascularization of tissues and treating ischemic conditions in patients.

Description

DNA CONSTRUCT FOR INDUCIBLE EXPRESSION OF ANGIOGENIC PROTEIN AND USE THEREOF

This application claims the benefit of U.S. Provisional Patent Application Serial No. 60/359,613, filed February 25, 2002, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION The present invention relates generally to a recombinant nucleic acid construct that, upon introduction into a host cell, expresses an angiogenic protein following induction by an inducing agent, as well as use of the recombinant nucleic acid construct for therapeutic purposes, such as promoting neovascularization and treating ischemic conditions.

BACKGROUND OF THE INVENTION

Development of regulated vectors for gene transfer is imperative for successful clinical application of gene-based therapeutics. Constitutive or long-lived expression of a biologically potent transgene may be deleterious to the viability and/or function of the cell expressing that factor. Great effort has been expended to create vector systems that exhibit negligible basal expression during the "off state and robust expression in the activated state. To that end, several such transcriptionally regulated vectors have been developed, including tetracycline, rapamycin, and steroid receptor-based gene switches (Lee et al., "Glucocorticoids Regulate Expression of Dihydrofolate Reductase cDNA in Mouse Mammary Tumour Virus Chimaeric Plasmids,"Nαtwre 294:228-232 (1981); Gossen & Bujard, "Tight Control of Gene Expression in Mammalian Cells by Tetracycline-responsive Promoters," Proc. Natl. Acad. Sci. U.S.A. 89:5547-5551 (1992); Gossen et al., "Transcriptional Activation by Tetracyclines in Mammalian Cells," Science 268:1766-1769 (1995); Ho et al., "Dimeric Ligands Define a Role for Transcriptional Activation Domains in Reinitiation," Nature 382:822-826 (1996); Mader & White, "A Steroid-inducible Promoter for the Controlled Overexpression of Cloned Genes in Eukaryotic Cells," Proc. Natl. Acad. Sci. U.S.A. 90:5603-5607 (1993); Wang et al., "A Regulatory System for Use in Gene Transfer," Proc. Natl. Acad. Sci. U.S.A. 91:8180-8184 (1994)). Individually, these systems have been utilized successfully in vitro and/or in vivo where controlled levels of transgene product are warranted. It is believed that the steroid-regulated system described by Mader & White has not been employed in vivo. One clinical application for gene transfer that appears to require strict regulation of transgene expression is that of angiogenic factor-based therapies.

Results from numerous studies have demonstrated that expression of the 165 amino acid isoform of Vascular Endothelial Growth Factor (VEGFι₆s), an endothelium- specific mitogen, via a naked DNA plasmid can promote the formation of new blood vessels in ischemic limb muscle. Successful therapeutic angiogenesis has been reported with intra-arterial (Takeshita et al. "Therapeutic Angiogenesis: A single Intraarterial Bolus of Vascular Endothelial Growth Factor Augments Revascularization in a Rabbit Ischemic Hind Limb Model," J. Clin. Invest. 93:662- 670 (1994); Takeshita et al., "Therapeutic Angiogenesis Following Arterial Gene Transfer of Vascular Endothelial Growth Factor in a Rabbit Model of Hindlimb Ischemia," Biochem. Biophys. Res. Commun. 227:628-635 (1996)) as well as intramuscular (Tsurumi et al., "Direct Intramuscular Gene Transfer of Naked DNA Encoding Vascular Endothelial Growth Factor Augments Collateral Development and Tissue Perfusion," Circulation 94:3281-3290 (1996); Tsurumi et al., "Treatment of Acute Limb Ischemia by Intramuscular Injection of Vascular Endothelial Growth Factor Gene," Circulation 96:11-382-388 (1997)) injections of VEGF-expressing plasmid DNA. Transgene-induced angiogenesis in animal models was associated with functional improvement in limb perfusion as measured by ankle brachial index, doppler flow measurements, and angiographic analyses (Tsurumi et al., "Direct Intramuscular Gene Transfer of Naked DNA Encoding Vascular Endothelial Growth Factor Augments Collateral Development and Tissue Perfusion," Circulation

94:3281-3290 (1996)). Additionally, patients enrolled in Phase 1 trials where intra- arterial and intramuscular injections of VEGF-expressing plasmid were administered exhibited clinical improvement (Isner et al., "Clinical Evidence of Angiogenesis After Arterial Gene Transfer of phVEGF165 in Patient with Ischaemic Limb," Lancet 348:370-374 (1996); Baumgartner et al., "Constitutive Expression of phVEGF165 After Intramuscular Gene Transfer Promotes Collateral Vessel Development in Patients with Critical Limb Ischemia," Circulation 97:1114-1123 (1998)). In all of these studies, the vectors that were utilized possessed constitutively active promoters that exacted significant but unregulated gene expression. High constitutively expressed levels of VEGF have been shown to lead to capillary leak and interstitial edema (Baumgartner et al., "Constitutive Expression of phVEGF165 After Intramuscular Gene Transfer Promotes Collateral Vessel Development in Patients with Critical Limb Ischemia," Circulation 97:1114-1123 (1998); Murohara, T., et al. (1998). "Vascular Endothelial Growth Factor Vascular Permeability Factor Enhances Vascular Permeability Via Nitric Oxide and Prostacyclin," Circulation 97:99-107 (1998)). Moreover, the nucleic acid constructs described in these studies is characterized by cessation of gene expression after about four weeks, which Baumgartner et al. consider an inherent safety feature of those systems. Nevertheless, long term expression of this potent mitogenic factor may produce even more deleterious effects that are presently unforeseen. For example, the consequence of repeated upregulation of the angiogenic protein remains unknown. It would be desirable, therefore, to identify recombinant DNA constructs that can reliably express angiogenic agents for purposes of gene transfer therapy against ischemia, yet avoid unwanted side effects prevalent with prior gene transfer approaches for the expression of angiogenic agents.

The present invention is directed to overcoming these and other deficiencies in the art.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a nucleic acid construct that includes: a DNA molecule encoding an angiogenic protein or polypeptide; a polyadenylation sequence operably coupled 3' of the DNA molecule; and a glucocorticoid-inducible promoter region operably coupled 5' of the DNA molecule.

A second aspect of the present invention relates to an expression vector into which is inserted the nucleic acid construct of the present invention.

A third aspect of the present invention relates to a pharmaceutical composition that includes a pharmaceutically acceptable carrier and an expression vector of the present invention.

A fourth aspect of the present invention relates to a liposomal composition that includes: a pharmaceutically acceptable carrier; a plurality of liposomes suspended in the pharmaceutically acceptable carrier, each including a lipid vesicle and an aqueous phase retained within the lipid vesicle; and one or more nucleic acid constructs of the present invention that are present within the aqueous phase of the liposomes. A fifth aspect of the present invention relates to a method of promoting neovascularization that includes: introducing an effective amount of a nucleic acid construct of the present invention into a tissue of a patient; and administering an amount of an inducing agent to the patient under conditions effective to upregulate expression of the angiogenic protein or polypeptide, which induces angiogenic development of blood vessels in the tissue.

A sixth aspect of the present invention relates to a method of treating an ischemic condition in a patient that includes: performing said method of promoting neovascularization in accordance with the present invention, wherein the tissue into which the nucleic acid construct is introduced is ischemic tissue and the resulting neovascularization occurs in the ischemic tissue, thereby treating the ischemic condition.

Because the nucleic acid constructs and expression vectors of the present invention do not afford constitutive expression of an angiogenic protein or polypeptide in patient tissues, the present invention minimizes that likelihood of patient harm that can be caused by long term exposure of such tissues to the angiogenic protein or polypeptide, such as capillary leak and interstitial edema. Moreover, the present invention allows for modifying expression levels of the angiogenic protein or polypeptide during the time course of therapy, by administering or withholding (either in whole or in part) additional doses of glucocorticoid inducing agent after monitoring patient tissue neovascularization. This allows for greater control over the nature and extent of neovascularization that can occur in ischemic tissues being treated in accordance with the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic representation of a glucocorticoid-regulated vector expressing VEGF₁₆₅. The pNGVL-hAP/GRE₅-vegf-pA plasmid contains five tandem repeats of the glucocorticoid-responsive element, the adeno virus minimal late promoter, VEGF cDNA, and SV40 polyadenylation site. A second transcription unit is present that expresses the human placental alkaline phosphatase gene under the transcriptional control of the CMV immediate-early promoter.

Figure 2 illustrates the results of an in vitro demonstration of VEGF_]65 regulation using plasmid DNA of Figure 1 transiently transfected into BHK cells. Following transfection, cells were treated with 0, 1, or 100 nM dexamethasone, and supernatants were harvested 48 h later. VEGF content within each media sample was assessed using a VEGF-specific ELISA kit (R&D Systems, Inc.). The levels of secreted VEGF were enhanced with increasing concentrations of exogenously supplied dexamethasone. Error bars represent standard deviation.

Figure 3 illustrated the results of dexamethasone-mediated regulation of VEGF₁₆₅ expression in mouse muscle. IM injections of 200 μg pNGVL- hAP/GREs-vegf-pA plasmid DNA were administered to the right hind limbs of C57BL/6 mice on Experimental Day 1. On Days 2 and 4 following plasmid inoculation, a subset of mice received either IP injections of 200 μg dexamethasone in saline (Mice #1 and 2) or saline vehicle alone (Mice #3 and 4). Animals were sacrificed on Day 5 and lysates prepared from muscle biopsies isolated from both right and left hind limbs. Induction of VEGF expression was measured at the protein level by Western Blot analysis. A VEGFι₆₅-specific band was only observed in protein samples prepared from the right limbs of Mice #1 and 2, indicating that dexamethasone-specific induction of VEGF expression following plasmid inoculation can be achieved in vivo.

Figures 4A-C illustrate dexamethasone-regulated VEGF expression and concomitant vascularization in a rabbit model of hindlimb ischemia. Male New Zealand white rabbits underwent hindlimb surgery to induce ischemia 10 days prior to receiving unilateral intramuscular inoculations (500 μg total) of pNGVL-hAP/GRE₅- vegf-pA plasmid DNA (Figure 4A). Dexamethasone (0.5mg/kg) was administered on Days 2, 6, 21, and 26 following plasmid injection and biopsies were taken on Days 7, 20, and 27. Biopsies were analyzed both qualitatively and by Western blots for VEGF expression. Western blot analysis revealed VEGF expression only in Biopsies #1 and 3, but not in Biopsy #2 (Figure 4B). No signal was detected in a biopsy sample processed from the contralateral limb. Portions of the biopsied material taken from pNGVL-hAP/GRE₅-vegf-pA injected limbs of rabbits undergoing hindlimb ischemia surgery were lysed and analyzed via VEGF-specific ELISA (Figure 4C). Tissue from the non-injected contralateral limb of each rabbit was also analyzed. Robust VEGF expression was only detected in biopsies excised soon after DEX treatment (Biopsies #1 and 3). Error bars represent standard deviation. These data further demonstrate the existence of tight transcriptional regulation within this plasmid.

Figures 5A-D illustrate the quantitation of capillary density and capillaries per muscle fiber in pNGVL-hAP/GREs-vegf-pA plasmid-treated and control animals. Images acquired from histochemical analyses of biopsy sections were utilized to enumerate capillary density and capillaries per muscle fiber. Five random fields from 10 sections for each biopsy were measured for capillary density (Figures 5A-B) and capillary/fiber ratio (Figures 5C-D). Capillary density/mm² (5 A) and capillary/fiber ratio (5C) measures were greatly enhanced in Biopsies #2 and 3 of ischemic rabbits that had received pNGVL-hAP/GRE₅-vegf-pA. Conversely, those measures did not change in ischemic animals receiving saline vehicle (5A or 5B and 5C or 5D) or in non-ischemic control rabbits (5B and 5D). Error bars represent standard deviation. Asterisk indicates statistical significance (.PO.001) over saline control as determined by single-factor ANOVA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a nucleic acid construct (DNA or RNA, but preferably DNA) designed for inducible expression of angiogenic proteins or polypeptides and its use for inducing expression of angiogenic proteins in vivo for purposes of promoting neovascularization and treating ischemia. The resulting expression of the angiogenic protein or polypeptide results in increased blood vessel formation throughout the ischemic tissue, thereby alleviating the symptoms or side- effects of ischemia. According to one embodiment, the nucleic acid construct includes a

DNA molecule encoding an angiogenic protein or polypeptide; a polyadenylation sequence operably coupled 3' of the DNA molecule; and a glucocorticoid-inducible promoter region operably coupled 5' of the DNA molecule. - / -

As used herein, angiogenic protein or polypeptide refers to proteins or polypeptides capable of inducing, directly or indirectly, angiogenesis, i.e., the formation of new blood vessels or neovascularization (see Folkman et al., "Angiogenic Factors," Science 235:442-447 (1987), which is hereby incorporated by reference in its entirety). Preferred angiogenic proteins or polypeptides are those capable of inducing angiogenesis following their introduction or expression in ischemic tissues. Suitable angiogenic proteins or polypeptides include, without limitation, acidic and basic fibroblast growth factors (FGF), vascular endothelial growth factors (VEGF), epidermal growth factor (EGF), transforming growth factor α and β (TGF-α and TFG-β), platelet-derived endothelial cell growth factor (PD- ECGF), platelet-derived growth factor (PDGF), tumor necrosis factor α (TNF-α), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), erythropoietin (EPO), colony stimulating factors such as macrophage-colony stimulating factor (M- CSF) or granulocyte/macrophage-colony stimulating factor (GM-CSF), and nitric oxidesynthase (NOS) (see Klagsbrun et al., "Regulators of Angiogenesis," Annu. Rev. Physiol. 53:217-239 (1991); Folkman et al., "Angiogenesis," J. Biol. Chem. 267:10931-10934 (1992); Symes et al., "Angiogenesis: Potential Therapy for Ischaemic Disease," Curr. Opin. Lipidology 5:305-312 (1994), each of which is hereby incorporated by reference in its entirety), as well as any isoforms, fragments, or variants of such angiogenic proteins or polypeptides which are capable of inducing angiogenesis. Determining whether a particular isoform, fragment, or variant induces angiogenesis can be performed according to any of a variety of known screening assays. Preferably, the angiogenic protein or polypeptide contains a secretory signal sequence allowing for secretion thereof by in vivo transformed cells. Preferred angiogenic proteins or polypeptides include VEGF-A and its various angiogenic fragments or isoforms, most preferably VEGF₁₆₅. Two features distinguish VEGF-A from other angiogenic growth factors. First, the amino terminus of VEGF is preceded by a typical signal sequence; therefore, unlike several other angiogenic proteins, VEGF can be secreted by intact cells. Second, its high-affinity binding sites, shown to include the tyrosine kinase receptors Flt-1 and Flt-1/KDR, are present on endothelial cells. Ferrara et al., "Pituitary Follicular Cells Secrete a Novel Heparin-Binding Growth Factor Specific for Vascular Endothelial Cells," Biochem. Biophys. Res. Commun. 161 :851- 855 (1989); Conn et al., "Purification of a Glycoprotein Vascular Endothelial Cell Mitogen from a Rat Glioma-derived Cell Line," Proc. Natl. Acad. Sci. U.S.A. 87:1323-1327 (1990), each of which is hereby incorporated by reference in its entirety. The angiogenic protein or polypeptide can be from any source organism, but preferably a mammal. To minimize immune responses, it is desirable to express in vivo the angiogenic protein or polypeptide from the same source mammal, e.g., human VEGF for human patients, mouse VEGF for mice, etc.

The angiogenic protein or polypeptide can also be a chimeric fusion protein including, e.g., a secretory signal and an angiogenic domain of any of the above-listed angiogenic proteins or polypeptides. Suitable secretory signals are known in the art, e.g., VEGF secretory signal, and construction of chimeric genes (for expression of the fusion proteins) can be carried out using an in-frame gene fusion prepared by standard recombinant techniques as described hereinafter. DNA (and thus RNA) encoding human angiogenic proteins and their isoforms are known in the art and have been reported in GenBank as follows: FGF (GenBank Accessions NM_000800, NM_033136, and NM_033137, each of which is hereby incorporated by reference in its entirety); VEGF types A-D (GenBank Accessions NM_003376, NM_003377, NM_005429, and NM_004469, each of which is hereby incorporated by reference in its entirety); EGF (GenBank Accession NM_001963, which is hereby incorporated by reference in its entirety); TGF-α (GenBank Accession M31172, which is hereby incorporated by reference in its entirety); TFG-β (GenBank Accessions Ml 9154 and M60315, each of which is hereby incorporated by reference in its entirety); PD-ECGF (GenBank Accession NM^001953, which is hereby incorporated by reference in its entirety); PDGF types A-D (GenBank Accessions NM_002607, NM_033023, NM_033016, NM_002608, AF260738, and AF336376, each of which is hereby incorporated by reference in its entirety); TNF-α (GenBank Accession Ml 6441, which is hereby incorporated by reference in its entirety); HGF (GenBank Accession M60718, which is hereby incorporated by reference in its entirety); IGF (GenBank Accessions S85346 and M27544, each of which is hereby incorporated by reference in its entirety); EPO (GenBank Accession Ml 1319, which is hereby incorporated by reference in its entirety); M-CSF (GenBank Accessions M27087 and M64592, each of which is hereby incorporated by reference in its entirety); GM-CSF (GenBank Accession Ml 3207, which is hereby incorporated by reference in its entirety); and NOS (GenBank Accession AF068236, which is hereby incorporated by reference in its entirety).

The glucocorticoid-inducible promoter region includes a promoter- effective DNA molecule and one or more glucocorticoid-responsive elements (GREs) that have a nucleotide sequence according to SEQ ID NO: 1 as follows:

agaacannntgttct 15

(where 'N' is any nucleotide). Generally, the more GRE's present, the higher levels of expression that can be induced. For example, two GREs will induce greater expression than a single GRE, three GREs will induce greater expression than two or fewer GREs, four GREs will induce greater expression than three or fewer GREs, and five GREs will induce greater expression than four or fewer GREs. Suitable promoter-effective DNA molecules are known in the art and include, without limitation, the adenovirus minimal late promoter, herpes thymidine kinase promoter, minimal cytomegalovirus promoter, any of a variety of cellular promoters. A preferred glucocorticoid-inducible promoter region contains five

GREs upstream (5') of the adenovirus minimal late promoter (Mader & White, "A Steroid-inducible Promoter for the Controlled Overexpression of Cloned Genes in Eukaryotic Cells," Proc. Natl. Acad. Sci. U.S.A. 90:5603-5607 (1993), which is hereby incorporated by reference in its entirety). Although this preferred glucocorticoid-inducible promoter region has previously been demonstrated to function in cultured cells in vitro, until the present invention it had not been demonstrated to be appropriately regulated in vivo in mammalian tissues. Gene transfer in vivo is not always predicted from cell culture experiments. In this case the availability and activity of glucocorticoid receptors in vivo could not have been prejudged to be sufficient to provide for highly regulated gene expression.

Any suitable polyadenylation signal can be employed in the expression vector of the present invention. Suitable polyadenylation signals include, without limitation, SV40 polyadenylation signal, CMV 35S polyadenylation signal, and any cellular mRNA poly A signal of the aauaaa (SEQ ID NO: 2) or alternate sequence type.

Once the nucleic acid construct has been prepared, it is preferably inserted into an expression vector for subsequent introduction into cells that are to be transformed. Construction of the recombinant expression vectors can be carried out according to known recombinant DNA techniques, including the use of restriction enzyme cleavage and ligation with DNA ligase. See U.S. Patent No. 4,237,224 to Cohen and Boyer; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, New York (1989), each of which is hereby incorporated by reference in its entirety.

Suitable expression vectors can include, without limitation, replication- defective viral vectors, such as adenoviral vectors, lentiviral vectors, adeno-associated vectors, baculovirus vectors, pox virus vectors, sendai virus vectors, herpes simplex virus vectors, etc.; and plasmid vectors. Adenovirus vectors can be readily prepared and utilized given the above-identified procedures and the disclosure provided in Berkner, "Development of Adenovirus Vectors for the Expression of Heterologous Genes," Biotechniques 6:616- 627 (1988) and Rosenfeld et al., "Adenovirus-mediated Transfer of a Recombinant alpha 1-Antitrypsin Gene to the Lung Epithelium in vivo," Science 252:431-434 (1991), WO 93/07283, WO 93/06223, and WO 93/07282, each of which is hereby incorporated by reference in its entirety. In vivo use of adenoviral vehicles is also described in Flotte et al., "Stable in vivo Expression of the Cystic Fibrosis Transmembrane Conductance Regulator with an Adeno-associated Virus Vector," Proc. Natl. Acad. Sci. U.S.A. 90:10613-10617 (1993); and Kaplitt et al., "Long-term Gene Expression and Phenotypic Correction Using Adeno-associated Virus Vectors in the Mammalian Brain," Nature Genet. 8:148-153 (1994), each of which is hereby incorporated by reference in its entirety. Additional types of adenovirus vectors are described in U.S. Patent No. 6,057,155 to Wickham et al.; U.S. Patent No. 6,033,908 to Bout et al.; U.S. Patent No. 6,001,557 to Wilson et al.; U.S. Patent No. 5,994,132 to Chamberlain et al.; U.S. Patent No. 5,981,225 to Kochanek et al.; and U.S. Patent No. 5,885,808 to Spooner et al.; and U.S. Patent No. 5,871,727 to Curiel, each of which is hereby incorporated by reference in its entirety). Retroviral vectors can be readily prepared and utilized given the above-identified procedures and the disclosure provided in U.S. Patent No. 5,849,586 to Kriegler et al., which is hereby incorporated by reference in its entirety.

Lentiviral can be readily prepared and utilized given the above- identified procedures and the disclosure provided in U.S. Patent No. 6,498,033 to Dropulic et al., U.S. Patent No. 6,428,953 to Naldini et al., U.S. Patent No. 6,277,633 to Olsen, U.S. Patent No. 6,235,522 to Kingsman, U.S. Patent No. 6,207,455 to Chang, and U.S. Patent No. 6,165,782 to Naldini et al., each of which is hereby incorporated by reference in its entirety. Adeno-associated vectors can be readily prepared and utilized given the above-identified procedures and the disclosure provided in U.S. Patent No. 6,309,634 to Bankiewicz et al. and U.S. Patent No. 6,221,349 to Couto et al., each of which is hereby incorporated by reference in its entirety.

Baculovirus vectors can be readily prepared and utilized given the above-identified procedures and the disclosure provided in U.S. Patent No. 5,516,657 to Murphy et al. and U.S. Patent No. 5,147,788 to Page et al., each of which is hereby incorporated by reference in its entirety.

Pox virus vectors can be readily prepared and utilized given the above- identified procedures and the disclosure provided in U.S. Patent No. 6,265,189 to Paoletti et al., which is hereby incorporated by reference in its entirety.

Sendai virus vectors can be readily prepared and utilized given the above-identified procedures and the disclosure provided in U.S. Patent No. 6,514,728 to Kai et al., which is hereby incorporated by reference in its entirety.

Herpes simplex virus vectors can be readily prepared and utilized given the above-identified procedures and the disclosure provided in U.S. Patent No. 6,379,674 to Rabkin et al., U.S. Patent No. 6,344,445 to Boursnell et al., PCT Publication WO 01/89304 to Federoff et al., and PCT Publication WO 02/056828 to Federoff et al., each of which is hereby incorporated by reference in its entirety.

As described hereinafter, for introduction into muscle cells, plasmid vectors are preferred. Exemplary plasmid vectors include, without limitation, pUCl 18, pBR322, or other known plasmid vectors, that include, for example, an E. coli origin of replication. The expression vector can also include a selectable marker such as the β-lactamase gene for ampicillin resistance or an alkaline phosphatase, provided that the marker polypeptide does not adversely effect the metabolism of the organism being treated. Referring to Figure 1 , an expression vector according to one preferred embodiment of the present invention includes, within a single transcriptional unit, five tandem repeats of a glucocorticoid-responsive element, an adenovirus minimal late promoter, VEGF cDNA, and an SV40 polyadenylation site. A second transcription unit is present that expresses the human placental alkaline phosphatase gene under the transcriptional control of the CMV immediate-early promoter.

A vector according to one preferred embodiment has a nucleotide sequence according to SEQ ID NO: 3 as follows:

tggccattgc atacgttgta tccatatcat aatatgtaca tttatattgg ctcatgtcca 60 acattaccgc catgttgaca ttgattattg actagttatt aatagtaatc aattacgggg 120 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg 180 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata 240 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc 300 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac 360 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg 420 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc 480 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc 540 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc 600 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct 660 cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga 720 agacaccggg accgatccag cctccgcggc cgggaacggt gcattggaac gcggattccc 780 cgtgccaaga gtgacgtaag taccgcctat agagtctata ggcccacccc cttggcttct 840 tatgcatgct atactgtttt tggcttgggg tctatacacc cccgcttcct catgttatag 900 gtgatggtat agcttagcct ataggtgtgg gttattgacc attattgacc actccaacgg 960 tggagggcag tgtagtctga gcagtactcg ttgctgccgc gcgcgccacc agacataata 1020 gctgacagac taacagactg ttcctttcca tgggtctttt ctgcagtcac cgtcgtcgac 1080 ggtatcgata agcttgatat cgaattcctg cctcgccact gtcctgctgc cctccagaca 1140 tgctggggcc ctgcatgctg ctgctgctgc tgctgctggg cctgaggcta cagctctccc 1200 tgggcatcat cccagttgag gaggagaacc cggacttctg gaaccgcgag gcagccgagg 1260 ccctgggtgc cgccaagaag ctgcagcctg cacagacagc cgccaagaac ctcatcatct 1320 tcctgggcga tgggatgggg gtgtctacgg tgacagctgc caggatccta aaagggcaga 1380 agaaggacaa actggggcct gagatacccc tggccatgga ccgcttccca tatgtggctc 1440 tgtccaagac atacaatgta gacaaacatg tgccagacag tggagccaca gccacggcct 1500 acctgtgcgg ggtcaagggc aacttccaga ccattggctt gagtgcagcc gcccgcttta 1560 accagtgcaa cacgacacgc ggcaacgagg tcatctccgt gatgaatcgg gccaagaaag 1620 cagggaagtc agtgggagtg gtaaccacca cacgagtgca gcacgcctcg ccagccggca 1680 cctacgccca cacggtgaac cgcaactggt actcggacgc cgacgtgcct gcctcggccc 1740 gccaggaggg gtgccaggac atcgctacgc agctcatctc caacatggac attgacgtga 1800 tcctaggtgg aggccgaaag tacatgtttc ccatgggaac cccagaccct gagtacccag 1860 atgactacag ccaaggtggg accaggctgg acgggaagaa tctggtgcag gaatggctgg 1920 cgaagcgcca gggtgcccgg tatgtgtgga accgcactga gctcatgcag gcttccctgg 1980 acccgtctgt gacccatctc atgggtctct ttgagcctgg agacatgaaa tacgagatcc 2040 accgagactc cacactggac ccctccctga tggagatgac agaggctgcc ctgcgcctgc 2100 tgagcaggaa cccccgcggc ttcttcctct tcgtggaggg tggtcgcatc gaccatggtc 2160 atcatgaaag cagggcttac cgggcactga ctgagacgat catgttcgac gacgccattg 2220 agagggcggg ccagctcacc agcgaggagg acacgctgag cctcgtcact gccgaccact 2280 cccacgtctt ctccttcgga ggctaccccc tgcgagggag ctccatcttc gggctggccc 2340 ctggcaaggc ccgggacagg aaggcctaca cggtcctcct atacggaaac ggtccaggct 2400 atgtgctcaa ggacggcgcc cggccggatg ttaccgagag cgagagcggg agccccgagt 2460 atcggcagca gtcagcagtg cccctggacg aagagaccca cgcaggcgag gacgtggcgg 2520 tgttcgcgcg cggcccgcag gcgcacctgg ttcacggcgt gcaggagcag accttcatag 2580 cgcacgtcat ggccttcgcc gcctgcctgg agccctacac cgcctgcgac ctggcgcccc 2640 ccgccggcac caccgacgcc gcgcacccgg ggcggtccgt ggtccccgcg ttgcttcctc 2700 tgctggccgg gaccctgctg ctgctggaga cggccactgc tccctgagtg tcccgtccct 2760 ggggctcctg cttccccatc ccggagttct cctgctcccc acctcctgtc gtcctgcctg 2820 gcctccagcc cgagtcgtca tccccggagt ccctatacag aggtcctgcc atggaacctt 2880 cccctccccg tgcgctctgg ggactgagcc catgacacca aacctgcccc ttggctgctc 2940 tcggactccc taccccaacc ccagggactg caggttgtgc cctgtggctg cctgcacccc 3000 aggaaaggag ggggctcagg ccatccagcc accacctaca gcccagtggg gtcgacagat 3060 ccagatccag atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag 3120 catctgactt ctggctaata aaggaaattt atttcattgc aatagtgtgt tggaattttt 3180 tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat cagaatcagt 3240 atttggttta gagtttggca acatatgcca ttcttccgct tcctcgctca ctgactcgct 3300 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 3360 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 3420 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 3480 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 3540 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 3600 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg 3660 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 3720 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 3780 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 3840 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt 3900 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 3960 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 4020 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 4080 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 4140 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac 4200 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt 4260 tcgttcatcc atagttgcct gactcggggg gggggggcgc tgaggtctgc ctcgtgaaga 4320 aggtgttgct gactcatacc aggcctgaat cgccccatca tccagccaga aagtgaggga 4380 gccacggttg atgagagctt tgttgtaggt ggaccagttg gtgattttga acttttgctt 4440 tgccacggaa cggtctgcgt tgtcgggaag atgcgtgatc tgatccttca actcagcaaa 4500 agttcgattt attcaacaaa gccgccgtcc cgtcaagtca gcgtaatgct ctgccagtgt 4560 tacaaccaat taaccaattc tgattagaaa aactcatcga gcatcaaatg aaactgcaat 4620 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga 4680 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg 4740 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt 4800 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagctt atgcatttct 4860 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc 4920 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa 4980 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca 5040 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc 5100 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga 5160 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg 5220 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag 5280 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca 5340 tccatgttgg aatttaatcg cggcctcgag caagacgttt cccgttgaat atggctcata 5400 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt 5460 ttatcttgtg caatgtaaca tcagagattt tgagacacaa cgtggctttc cccccccccc 5520 cattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 5580 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc 5640 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt 5700 cgtcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac 5760 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc 5820 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag 5880 agtgctagag gatccagaca tgataagata oattgatgag tttggacaaa ccacaactag 5940 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac 6000 cattataagc tgcaataaac aagttcctgg tgagagatct ggttcccgaa acgctgaggg 6060 aggctccttc ctcctgcccg gctcaccgcc tcggcttqtc acatctgcaa qtacgttcgt 6120 ttaactcaag ctgcctcgcc ttgcaacgcg agtctgtgtt tttgcaggaa catttacacg 6180 tctgcggatc ttgtacaaac aaatgctttc tccgctctga gcaaggccca cagggatttt 6240 cttgtcttgc tctatctttc tttggtctgc attcacattt gttgtgctqt aggaagctca 6300 tctctcctat gtgctggcct tqgtgaggtt tgatccgcat aatctgcatg gtgatgttgg 6360 actcctcagt gggcacacac tccaggccct cgtcattgca gcagcccccg catcgcatca 6420 qgggcacaca ggatggcttg aagatgtact cgatctcatc agggtactcc tggaagatgt 6480 ccaccagggt ctcgattgga tqgcagtagc tgcgctgata gacatccatg aacttcacca 6540 cttcgtgatg attctgccct cctccttctg ccatgggtgc agcctgggac cacttggcat 6600 ggtggaggta gagcagcaag gcaaggctcc aatgcaccca agacagcaga aagttcatgg 6660 tttcggaggc ccgaaagctt agatcttttg ccaaaatgat gagacagcac aataaccagc 6720 acgttgccca ggagctgtag gaaaaagaag aaggcatgaa catggttagc agaggggccc 6780 ggtttggact cagagtattt tatcctcatc tcaaacagtg tatatcattg taaccataaa 6840 gagaaaggca ggatgatgac caggatgtag ttgtttctac caataagaat atttccacgc 6900 cagccagaat ttatatgcag aaatattcta ccttatcatt taattataac aattgttctc 6960 taaaactgtg ctgaagtaca atataatata ccctgattgc cttgaaaaaa aagtgattag 7020 agaaagtact tacaatctga caaataaaca aaagtgaatt taaaaattcg ttacaaatgc 7080 aagctaaagt ttaacgaaaa agttacagaa aatgaaaaga aaataagagg agacaatggt 7140 tgtcaacaga gtagaaagtg aaagaaacaa aattatcatg agggtccatg gtgatacaag 7200 ggacatcttc ccattctaaa caacaccctg aaaactttgc cccctccata taacatgaat 7260 tttacaatag cgaaaaagaa agaacaatca agggtcccca aactcaccct gaagttctca 7320 ggatcctggc cctcgcagac agcgatgcgg aagagagtga ggacgaacgc gcccccaccc 7380 ccttttatag ccaaacctgg tttttgaccc cgaacggcca ggagatcgat ctaataaagt 7440 agctagaaca tcctgtacac ggatctaata aagtagctag aacatcctgt acagcggatc 7500 taataaagta gctagaacat cctgtacagc ggatctaata aagtagctag aacatcctgt 7560 acagcggatc taataaagta gctagaacat cctgtacagc ggatcttcta gacaccatat 7620 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagat tggctat 7677

where the pNGVL-hAP vector backbone spans nt 1-5889 and 7613-7677, the glucocorticoid-inducible promoter region (containing adenovirus minimum late promoter and five tandem repeats of GRE) spans nt 6681-7612 (italicized), the VEGF coding region spans nt 6026-6680 (underlined), and the SV40 polyadenylation signal spans nt 5890-6025 (bold).

Having prepared an expression vector of the present invention, it is capable of being administered to a patient for cellular uptake in targeted cells within a patient and subsequent induction of angiogenic protein or polypeptide expression following administration of an inducing agent. Thus, a further aspect of the present invention relates to methods of promoting neovascularization and treating ischemia in a patient. The methods of the present invention may be used to treat any ischemic tissue, i.e., a tissue having a deficiency in blood as the result of an ischemic disease or condition. Such tissues can include, without limitation, skeletal muscle, cardiac muscle, diaphragmatic muscle, brain, kidney and lung. Ischemic diseases or conditions can include without limitation, cerebrovascular or cranial ischemia, renal ischemia, pulmonary ischemia, limb ischemia, ischemic cardiomyopathy, and myocardial ischemia. Administration of the nucleic acid construct (or expression vector containing the same) to a patient can be achieved via administering naked DNA or by administering a liposomal delivery vehicle that includes the nucleic acid construct or the expression vector.

Liposomes are vesicles comprised of one or more concentrically ordered lipid bilayers which encapsulate an aqueous phase. They are normally not leaky, but can become leaky if a hole or pore occurs in the membrane, if the membrane is dissolved or degrades, or if the membrane temperature is increased to the phase transition temperature. Current methods of liposomal delivery require the liposome carrier to become permeable and release the encapsulated nucleic acid at the target site. This can be accomplished, for example, in a passive manner wherein the liposome bilayer degrades over time through the action of various agents in the body. Every liposome composition will have a characteristic half-life in the circulation or at other sites in the body and, thus, by controlling the half-life of the liposome composition, the rate at which the bilayer degrades can be somewhat regulated. In contrast to passive release, active release involves using an agent to induce a permeability change in the liposome vesicle. Liposome membranes can be constructed so that they become destabilized when the environment becomes acidic near the liposome membrane (see, e.g., Wang et al., "pH-sensitive Immunoliposomes Mediate Target-cell-specific Delivery and Controlled Expression of a Foreign Gene in Mouse," Proc. Natl. Acad. Sci. U.S.A. 84:7851 -7855 (1987); Wang et al., "Highly Efficient DNA Delivery Mediated by pH-sensitive Immunoliposomes," Biochemistry 28:9508-9514 (1989), each of which is hereby incorporated by reference in their entirety). When liposomes are endocytosed by cells in a targeted tissue, for example, they can be routed to acidic endosomes which will destabilize the liposome and result in nucleic acid release.

Different types of liposomes can be prepared according to Bangham et al., "Diffusion of Univalent Ions Across the Lamellae of Swollen Phospholipids," J. Mol. Biol. 13:238-252 (1965); U.S. Patent No. 5,653,996 to Hsu et al.; U.S. Patent No. 5,643,599 to Lee et al.; U.S. Patent No. 5,885,613 to Holland et al.; U.S. Patent No. 5,631,237 to Dzau et al.; and U.S. Patent No. 5,059,421 to Loughrey et al., each of which is hereby incorporated by reference in their entirety.

Regardless of the material introduced (i.e., naked DNA or liposomal vehicle), administration can be carried out intramuscularly, intraperitoneally, subcutaneously, transdermally, intravenously, or intracranially. The nucleic acid construct (or expression vector containing the same) can be delivered to more than one site within the ischemic tissue. Moreover, nucleic acids encoding different angiogenic proteins or polypeptides may be used separately or simultaneously. To facilitate injection, the nucleic acid construct is preferably formulated with a pharmaceutically acceptable carrier, such as saline, albumin, dextrose, or sterile water. The nucleic acid is injected into the ischemic tissue using standard injection techniques by use of, for example, a hypodermic needle. Hypodermic needle sizes between no. 29 to no. 16 are preferred. The nucleic acid may also be injected by an externally applied local injection apparatus, such as that used to inject antigens for allergy testing; or a transcutaneous patch capable of delivery to subcutaneous muscle.

The amount of nucleic acid to be delivered should be effective to produce an adequate level of the angiogenic protein, i.e., levels capable of inducing angiogenesis. Thus, the important aspect is the level of angiogenic protein or polypeptide expressed. There are several variables that influence the level of angiogenic protein or polypeptide that is expressed: the amount of nucleic acid administered to the patient, the number of GREs present in the promoter region of the administered nucleic acid, and the amount of inducing agent administered to the patient.

The effective dose of the nucleic acid will be a function of the particular expressed angiogenic protein or polypeptide, the target tissue, the patient and his or her clinical condition. Effective amounts of DNA are typically between about 1 μg and about 4000 μg, more preferably about 200 μg and about 4000 μg, most preferably between about 1000 μg and about 4000 μg.

Administration of the inducing agent to a patient can be achieved orally, topically (transdermally), parentally, intraperitoneally, subcutaneously, via intravenous delivery, via intra-arterial delivery, via inhalation, via intranasal instillation, intravaginally, or intrathecally.

Suitable inducing agents include, without limitation, glucocorticoids, preferably cortisone, cortisol, prednisone, methylprednisone, or dexamethasone. The effective dose of the inducing agent will be a function of the responsiveness of the inducible promoter (i.e., the number of GREs present), and the desired level of expression for the angiogenic protein or polypeptide. Generally, suitable initial dosage levels can be between about 0.01 and about 100 mg/kg, more preferably between about 0.1 and about 10 mg/kg. Secondary dosages will depend upon the nature of the patient response to the initial dose. Thus, initial dosages can be either higher or lower than the secondary dosages, as needed. One of skill in the art can measure the level of neovascularization in the tissue and, based on those levels, increase or decrease the secondary dosages accordingly.

As indicated above, multiple doses of the inducing agent can be delivered to achieve the desired degree of neovascularization of a targeted tissue. Typically, the level of neovascularization in the ischemic tissue will be monitored following the first dosage of the inducing agent and, if desired, additional doses of the inducing agent can be delivered to again upregulate expression of the angiogenic protein or polypeptide and promote further neovascularization within the targeted tissue. To measure the extent of neovascularization in the targeted tissue, cell morphometry, magnetic resonance spectroscopy, angiography, and/or Doppler flow studies can be employed.

Because the vectors containing the nucleic acid of interest are not normally incorporated into the genome of the cells, expression of the protein of interest takes place for only a limited time. However, it is believed that the expression vectors of the present invention are more stable than prior art expression vectors containing constitutive promoters, thereby affording expression of the angiogenic protein or polypeptide more than four weeks following initial transformation. EXAMPLES

The following Examples are intended to be illustrative and in no way are intended to limit the scope of the present invention.

Materials & Methods

Murine Plasmid Injections: Eight to ten week-old male C57BL/6 mice (~27 g; obtained from Jackson Laboratories) were handled in accordance with approved University of Rochester animal use guidelines. Mice were anesthetized with 0.7 ml Avertin (1.25% 2,2,2-tribromoethanol and 0.8% tert-amyl alcohol in dH₂0) and received intramuscular injections of 200 μg (1 μg/μl in saline) pNGVL-hAP/GRE₅- vegf-pA plasmid DNA in the right hindlimb. One and three days later, animals were administered IP injections of 200 μg DEX in saline or saline vehicle alone. On Day 5 post-plasmid injection, mice were sacrificed with lethal injection of pentobarbital, muscle biopsies of the injection sites obtained, protein lysates prepared, and Western blot analysis performed to assess levels of VEGF protein expression.

Rabbit Hindlimb Model of Ischemia: Male New Zealand White rabbits (4 kg) were pre-medicated with midazolam and glycopyrolate prior to isoflurane inhalation induction and maintenance. Rabbits were maintained at 3% isoflurane with 1 liter of oxygen during surgical manipulation. A skin incision was placed over the medial aspect of the right thigh, and the femoral artery dissected free from the inguinal ligament to its bifurcation into the saphenous and popliteal arteries. The artery was ligated at its origin from the external iliac and at the bifurcation into the popliteal and saphenous branches. All branches of the femoral artery were ligated and the vessel excised. This has been shown to render the thigh musculature dependent on collateral flow from the internal iliac artery (Takeshita et al. "Therapeutic Angiogenesis: A single Intraarterial Bolus of Vascular Endothelial Growth Factor Augments Revascularization in a Rabbit Ischemic Hind Limb Model," J. Clin. Invest. 93:662- 670 (1994); Pu et al., "A Persistent Hindlimb Ischemia Model in the Rabbit," J. Invest. Surg. 7:49-60 (1994), each of which is hereby incorporated by reference in its entirety). Animals were treated post-operatively with buprenorphine (0.025 mg/kg) for 5 days following ischemia surgery. All surgical interventions, pre- and post- operative care were performed according to University of Rochester animal care facility-approved guidelines. Ten days following surgery, one experimental group (n=4) received three separate injections of 500 μg pNGVL-hAP/GRE₅-vegf-pA DNA into their right gracillis muscle (Figure 4A). The other group (n=4) received saline injections. On Days 2 and 6 after injection each rabbit received 0.5 mg/kg of dexamethasone sodium phosphate (DEX; American Reagent Laboratories) intramuscularly to induce vector-derived VEGF expression. On Day 7 following DEX treatment, one injected site was biopsied (Biopsy #1) to measure VEGF expression. DEX was not delivered for 14 days, at which point another injection site was biopsied (Biopsy #2) to measure the decay of VEGF expression. Animals were then re-injected with DEX (0.5 mg/kg) 1 and 5 days following the second biopsy to re-induce VEGF expression. Six days after the last DEX injection (Day 26 following DNA injection) the final injection site was biopsied (Biopsy #3). Biopsies were also obtained from the contralateral gracillis muscle (non-ischemic, non-injected). Animals were sacrificed by lethal injection of pentobarbital in accordance with University of Rochester animal care guidelines.

Western Blotting andELISA: Protein homogenates were prepared from muscle biopsies by homogenizing a fresh piece of muscle in a solution containing (250 raM sucrose, 300 niM HEPES, 2 mM EGTA, 40 raM NaCl, 2 mM PMSF, pH 7.4). All samples were then centrifuged at 300,000 x g for 60 min at 4°C to remove fibrous material. Protein from the supernatants of homogenates was normalized via a Bradford assay prior to electrophoresis. Ten μg of each sample was run on a 15% Tris-HCl denaturing polyacrylamide gel. Proteins were transferred to nitrocellulose (Immobilon, Millipore) for a period of 1 hr at 100 volts. Membrane was blocked in lx PBS, 0.5% SDS, 5% non-fat dried milk (Blotto) for 1 hr at room temperature prior to primary antibody incubation. The blots were incubated in lx PBS, 0.5% Tween at 1 : 1,000 (goat anti-VEGF primary antibody, Chemicon) for 2 hrs at room temperature (23°C). Following incubation with primary antibody the blot underwent washing for 60 min in lx PBS, 0.5% Tween. A secondary antibody solution containing a 1:10,000 anti-goat horseradish peroxidase (HRP)-conjugated Ab was added to the blot for lhr at 23°C. Following secondary antibody incubation the blot was washed for 60 min in lx PBS, 0.5%) Tween. Chemiluminescent detection was performed as per the - Zϋ -

manufacturer's protocol (ECL kit, Amersham). Blots were exposed to film for 15 sec and developed in an X-OMAT processor (Kodak). Separate aliquots of protein homogenate prepared from these rabbits were analyzed for VEGF content using an ELISA-based assay (R&D Systems), that was performed according to the manufacturer's instructions.

Capillary Staining: Muscle biopsies from treated rabbits were immediately frozen in isopentane. Five-micrometer transverse sections were cut (Leica CM 1900) and slides were stored at -80°C prior to processing. Slides were incubated in acetone (4°C; 5 min) and air-dried prior to staining. An indoxyl-tetrazolium-based method (Baffour et al., "Enhanced Angiogenesis and Growth of Collaterals by in vivo Administration of Recombinant Basic Fibroblast Growth Factor in a Rabbit Model of Acute Lower Limb Ischemia: Dose-response Effect of Basic Fibroblast Growth Factor," J. Vase. Surg. 16:181-191 (1992); Ziada et al., "The Effect of Long-term Vasodilatation on Capillary Growth and Performance in Rabbit Heart and Skeletal Muscle,"

Cardiovasc. Res. 18:724-732 (1984), each of which is hereby incorporated by reference in its entirety) was used for detecting endogenous rabbit alkaline phosphatase. Slides were subsequently counterstained in eosin for 15 sec following alkaline phosphatase staining.

Capillary Quantitation and Image Analysis: Five random fields from 10 sections for each biopsy were measured for capillary density and capillary/fiber ratio. Capillaries were counted under a 20x objective following digital acquisition (SPOT RT camera,

Diagnostic Instruments, McHenry, IL) and analysis completed in an unbiased fashion using Image Pro-Plus (Media Cybernetics, Silver Spring, MD). Capillaries were scored as positive based on a macro written to distinguish between the size and color of alkaline phosphatase-positive capillaries. A total of 50 different fields were analyzed from each biopsy. Measuring the area of individual eosin-stained muscle fibers throughout each section assessed overestimation of capillary number (due to muscle atrophy) or underestimation (due to interstitial edema). The protocol for counting capillary density and capillary/muscle fiber ratio were completed using the identical macro within the Image Pro Plus program. Basically, a region of the tissue area is bounded and the number of immunohistochemically detected structures in the region is enumerated.

Example 1 - Construction of Plasmid Containing VEGF Coding Sequence Under Glucocorticoid-Regulated Promoter

A DNA fragment containing the open reading frame for human vascular endothelial growth factor (VEGF) was removed from a previously described pHSVhvegf amplicon vector (Mesri et al., "Expression of Vascular Endothelial Growth Factor from a Defective Herpes Simplex Virus Type 1 Amplicon Vector Induces Angiogenesis in Mice," Circ. Res. 76:161-167 (1995), which is hereby incorporated by reference in its entirety) with H/«dIII and .Yføl(blunted). The 650-bp VEGF insert was cloned into a HmdIII/Λ7*oI(blunted)-cut pGRE5-2 vector to create pGRE₅-vegf. The pGRE₅-vegf vector contained five tandem copies of the rat aminotransferase glucocorticoid regulatory element, a minimal adenoviral late promoter, and an SV40 polyadenylation signal to provide glucocorticoid-regulated expression of VEGF (Figure 1). The GRE₅-vegf-pA transcriptional unit was subsequently removed and inserted into a blunted Apalλ site of the pNGVL-hAP vector as a blunted Xbal/Kpnl fragment to create pNGVL-hAP/GRE₅-vegf-pA. The NGVL-hAP parent vector (kindly provided by the National Gene Vector Laboratory, Univ. Michigan) contained a cytomegalovirus (CMV) promoter-driven human placental alkaline phosphatase (hPLAP) expression unit and a kanamycin resistance marker. Endotoxin-free plasmid DNA stocks used for in vivo injections were produced under GLP conditions by Puresyn, Inc. (Malvern, PA). Introduction of this plasmid, pNGVL-hAP/GRE₅-vegf-pA, into BΗK cells via lipofection resulted in high levels of VEGF expression and secretion only when cells were incubated in the presence of DEX (Figure 2). The concentration of DEX (100 nM) required to induce VEGF expression from this vector was similar to that observed previously with a DEX-regulated, human growth hormone-expressing ΗSV amplicon vector (Lu and Federoff, "Herpes Simplex Virus Type 1 Amplicon Vectors With Glucocorticoid-inducible Gene Expression," Hum. Gene. Ther. 6:419- 428 (1995), which is hereby incorporated by reference in its entirety). - 11 -

Example 2 - Dexamethasone-regulated in vivo Expression of VEGF in Mice Transfected with pNGVL-hAP/GRE₅-vegf-pA Plasmid

The pNGVL-hAP/GRE₅-vegf-pA plasmid was injected into the right hindlimb muscle of C57BL/6 mice for analysis of gene regulation in vivo. Mice received 200 μg of vector DNA and on Days 2 and 4 post-gene transfer animals received intraperitoneal (IP) injections of 200 μg DEX in saline or saline vehicle alone. Biopsies were excised from injected and contralateral limbs on Experimental Day 5 and subjected to Western blot analysis to determine levels of VEGF₎₆₅ expression. A VEGFι₆₅-specific band was detected only in samples taken from the right hindlimbs of animals receiving DEX (Figure 3). In separate experiments, mice receiving the parental pNGVL-hAP plasmid did not exhibit any detectable expression of VEGF in either the absence or presence of DEX. These results demonstrate the glucocorticoid responsiveness of the pNGVL-hAP/GRE₅-vegf-pA vector was rapid and robust, and that the transgene-derived growth factor principally localized to the plasmid-injected limb.

Example 3 - Treatment of Ischemia in Rabbits Using pNGVL-hAP/GRE₅-vegf- pA Plasmid Transfection and Dexamethasone-Induced VEGF Expression

Previously, other investigators demonstrated that delivery of VEGF as a constitutively expressed transgene or as a protein in a rabbit model of hindlimb ischemia led to increased vascularization of the gracillis muscle (Takeshita et al., "Therapeutic Angiogenesis Following Arterial Gene Transfer of Vascular Endothelial Growth Factor in a Rabbit Model of Hindlimb Ischemia," Biochem. Biophys. Res. Commun. 227:628-635 (1996); Tsurumi et al., "Direct Intramuscular Gene Transfer of Naked DNA Encoding Vascular Endothelial Growth Factor Augments Collateral Development and Tissue Perfusion," Circulation 94:3281-3290 (1996); Tsurumi et al., "Treatment of Acute Limb Ischemia by Intramuscular Injection of Vascular

Endothelial Growth Factor Gene," Circulation 96:11-382-388 (1997), each of which is hereby incorporated by reference in its entirety). To examine the in vivo bioactivity of the pNGVL-hAP/GRE₅-vegf-pA vector, plasmid DNA was utilized in this well- characterized ischemia paradigm. - 15 -

Ten days prior to plasmid delivery, male New Zealand White rabbits underwent ligation of their distal external iliac arteries to induce ischemia (Figure 4A). Plasmid delivery occurred on Day 1. Thereafter, animals received IP injections of 0.5 mg/kg DEX on Days 2, 6, 21, and 26. Biopsies were taken of hindlimb muscle from both injected and non-injected limbs on Days 7, 20, and 27. Segments of biopsies were removed, lysed, and analyzed for VEGF expression using Western blot and ELISA assays. Finally, the remaining portions of biopsies were frozen in isopentane, sectioned, and histochemically stained to determine muscle-associated capillary density. Assessment of VEGF protein by Western blot and ELISA analyses indicated the therapeutic transgene product was present only following DEX treatment (Figures 4B and 4C). Non-ischemic contralateral leg muscle did not show any detectable VEGF immunoreactivity. The sensitivity of the Western blot assay was confirmed by a set of standards that exhibited a lower detectable limit of 3 ng, while the ELISA assay sensitivity was determined to be a lower detectable limit of 0.5 pg/ml. Therefore, regulation of transgene-derived VEGF was similar to that observed in muscle of pNGVL-hAP/GRE₅-vegf-pA injected mice.

Histological sections prepared from muscle biopsies were stained for alkaline phosphatase to detect capillaries and counterstained with eosin to visualize cellular structures. Capillary density was subsequently quantified as capillaries per mm² or per muscle fiber at a final magnification of 200x. Seven days following pNGVL-hAP/GRE -vegf-pA injection and DEX induction in ischemic muscle, there was no significant increase in capillary density as compared to muscle taken from saline-treated, ischemic control animals. However, Biopsy #2 derived from pNGVL- hAP/GREs-vegf-pA injected rabbits revealed a statistically significant 75% increase in capillary density and a 140% increase in capillary/fiber ratio (Figures 5 A and 5C) when compared to saline-treated, ischemic controls (Figures 5B and 5D). Following re-induction of plasmid-derived VEGF via DEX treatment, Biopsy #3 revealed a 178%) significant increase in capillary density and a 220% in capillary/fiber ratio (Figures 5A and 5C) when compared to saline-treated, ischemic controls (Figures 5B and 5D). All comparisons were statistically significant (p<0.001) as determined by a Student T-test. Untreated non-ischemic controls exhibited a significantly higher capillary density than untreated ischemic controls, as would be expected. Interestingly, an increase in capillary density was noticed in non-ischemic muscle from the time of the Biopsy #1 until Biopsy #3. This increase in density trended towards but did not achieve statistical significance (Figures 5B and 5D).

Discussion of Examples 1-3

Development of regulated gene therapeutic vectors is imperative for the safe and effective implementation of gene transfer modalities in the clinical arena. The above results demonstrate successful utilization of a glucocorticoid-regulated system (Mader & White, "A Steroid-inducible Promoter for the Controlled Overexpression of Cloned Genes in Eukaryotic Cells," Proc. Natl. Acad. Sci. U.S.A. 90:5603-5607 (1993), which is hereby incorporated by reference in its entirety) to achieve controlled transgene expression. This transcriptional cassette was shown previously in the context of a plasmid and HSV-1 amplicon vector to be tightly regulated in vitro in eukaryotic cells by the synthetic glucocorticoid dexamethasone (DEX). Applicants believe the present invention is the first demonstration of in vivo utility of this regulated transcription unit. In the context of a clinically relevant application, the biological impact of regulated vascular endothelial growth factor (VEGF) delivery in an animal model of hindlimb ischemia was assessed.

Glucocorticoids mediate their biologic action(s) by interacting with glucocorticoid receptor (GR) monomers predominantly localized within the cell cytoplasm. Upon ligand binding, activated GRs translocate to the nucleus where dimerization takes place, which results in interaction with consensus DNA sequences, termed glucocorticoid response elements (GREs). The consensus binding site is the palindromic 15-bp sequence of SEQ ID NO: 1 and in nature are found within the transcriptional regulatory regions of glucocorticoid-responsive genes. It has been demonstrated that the magnitude of steroid responsiveness directly correlates with the number of GREs located in a given gene promoter.

This study describes the use of a GRE5-driven plasmid expressing the potent angiogenic factor, VEGF, to achieve pharmacologically regulated transgene expression and angiogenesis in mice and an in vivo model of hindlimb ischemia. As evidenced by Western blot analysis, mice receiving pNGVL-hAP/GREs-vegf-pA plasmid DNA only expressed VEGF when administered DEX (Figure 3). The rabbit model of limb ischemia utilized mimics chronic severe lower extremity arterial insufficiency (Pu et al., "A Persistent Hindlimb Ischemia Model in the Rabbit," J Invest. Surg. 7:49-60 (1994), which is hereby incorporated by reference in its entirety). Consistent with previous reports, distinct paucity of capillaries was evident in saline-treated ischemic muscle (Figures 5A-D; Takeshita et al. "Therapeutic Angiogenesis: A single Intraarterial Bolus of Vascular Endothelial Growth Factor Augments Revascularization in a Rabbit Ischemic Hind Limb Model," J Clin. Invest. 93:662-670 (1994), which is hereby incorporated by reference in its entirety). Capillary density, whether measured as capillaries per unit area or per myofibril, did not allow for discrimination between saline-treated and pNGVL-hAP/GRE₅-vegf-pA treated ischemic muscle until the second biopsy (18 days following plasmid administration). Therefore, peak angiogenesis was temporally delayed as compared to peak VEGF induction.

The goal of the present study was to determine if episodic administration of DEX would lead to re-induction of VEGF expression, which in turn, would result in progressive angiogenesis. The selected dose was sufficient to induce VEGF expression and subsequently angiogenesis. Interestingly, the angiogenic effect of the initial VEGF induction was readily observable even when plasmid-derived VEGF expression had diminished to undetectable levels at 18 days, suggesting that the initial DEX-mediated enhancement of VEGF expression was sufficient to establish stable capillary formation. DEX administered just prior to the final biopsy, further enhanced capillary density in pNGVL-hAP/GRE₅-vegf-pA treated ischemic muscle as compared to saline-treated ischemic muscle (Figures 5A-B). It is unclear whether this was the result of an accelerated cellular response to VEGF upon re- induction or whether it was the cumulative effect of the initial VEGF expression peak. Clarification of this point could be achieved by following capillary density at protracted time points following an initial DEX treatment in the absence of a subsequent DEX re-induction.

Biopsies derived from non-ischemic, untreated muscle from contralateral legs exhibited an increase in capillary density as a function of time was noted but this enhancement did not reach statistical significance (Figures 5A-D).

There was no detectable increase in endogenous VEGF protein levels accompanying the increase in capillary density in those animals. However, the angiogenic response detected in untreated control limbs could still be the result of endogenous rabbit VEGF induction due to limitations in ELISA sensitivity, which could warrant reassessment by more sensitive quantitative assays such as real-time quantitative RT- PCR.

Patients with such severe arterial occlusive disease who are not amenable to successful bypass procedures have limited therapeutic options that often culminate in amputation. The application of VEGF gene therapy to the treatment of peripheral vascular ischemia has shown promise of limb salvage both in animal models (Tsurumi et al., "Direct Intramuscular Gene Transfer of Naked DNA Encoding Vascular Endothelial Growth Factor Augments Collateral Development and Tissue Perfusion," Circulation 94:3281-3290 (1996); Rivard et al., "Rescue of

Diabetes-Related Impairment of Angiogenesis by Intramuscular Gene Therapy with Adeno-VEGF," m. J. Pathol. 154:355-363 (1999); Mack et al. "Salvage Angiogenesis Induced by Adenovirus-Mediated Gene Transfer of Vascular Endothelial Growth Factor Protects Against Ischemic Vascular Occlusion," J. Vase. Surg. 27:699-709 (1998), each of which is hereby incorporated by reference in its entirety) and in early human trials (Isner et al., "Clinical Evidence of Angiogenesis After Arterial Gene Transfer of phVEGF165 in Patient with Ischaemic Limb," Lancet 348:370-374 (1996); Baumgartner et al., "Constitutive Expression of phVEGF165 After Intramuscular Gene Transfer Promotes Collateral Vessel Development in Patients with Critical Limb Ischemia," Circulation 97:1114-1123 (1998); Isner et al., "Arterial Gene Therapy for Therapeutic Angiogenesis in Patients With Peripheral Artery Disease," Circulation 91 :2687-2692 (1995), each of which is hereby incorporated by reference in its entirety). The molecular and cellular mechanisms responsible for the transition of endothelial cell proliferation to capillary formation to resultant vasculogenesis have been extensively studied. VEGF is known to induce endothelial cell proliferation, as do members of the angiopoetin family (Holash et al., "Vessel Cooption, Regression, and Growth in Tumors Mediated by Angiopoietins and VEGF," Science 284:1994-1998 (1999), which is hereby incorporated by reference in its entirety). While the goal of therapeutic angiogenesis is to produce increased collateral formation and to restore adequate blood flow, uncontrolled angiogenic therapy utilizing these potent factors can lead to untoward effects as evidenced by previous reports. Peripheral edema was reported with patients exhibiting critical limb ischemia that had received intramuscular injections of a plasmid construct constitutively expressing VEGF (Baumgartner et al., "Constitutive Expression of phVEGF165 After Intramuscular Gene Transfer Promotes Collateral Vessel Development in Patients with Critical Limb Ischemia," Circulation 97:1114-1123 (1998), which is hereby incorporated by reference in its entirety). Increased vascular permeability has also been demonstrated in guinea pigs following administration of recombinant VEGF (Murohara et al., "Vascular Endothelial Growth Factor/Vascular Permeability Factor Enhances Vascular Permeability Via Nitric Oxide and Prostacyclin," Circulation 97:99-107 (1998), which is hereby incorporated by reference in its entirety). Additionally, implantation of engineered myoblasts expressing high levels of VEGF has been shown to induce hemangioma formation (Springer et al., "VEGF Gene Delivery to Muscle: Potential Role for Vasculogenesis in Adults," Mol. Cell. 2:549-558 (1998), which is hereby incorporated by reference in its entirety). These side effects highlight the importance of developing regulated gene therapies, especially given that long-term effects of constitutive angiogenic factor expression in muscle have not yet been established.

Several other pharmacologically regulated gene therapy systems have been utilized in animal models. Rapamycin-activated vectors are well described, and have been demonstrated in vivo to be highly inducible (Ye et al., "Regulated Delivery of Therapeutic Proteins After in vivo Somatic Cell Gene Transfer," Science 283:88-91 (1999), which is hereby incorporated by reference in its entirety). However, this is a complex system that requires introduction of two vectors; one possessing a rapamycin-induced transcription factor, and the other containing the transgene whose cognate promoter is responsive to the induced transcription factor (Harvey and Caskey, "Inducible Control of Gene Expression: Prospects for Gene Therapy," Curr. Opin. Chem. Biol. 2:512-518 (1998), which is hereby incorporated by reference in its entirety). Expression of this transcription factor, comprised of non-native polypeptide sequences, introduces the potential for an immune response. In addition, rapamycin is a potent molecule that exhibits significant renal toxicity. Therefore, careful dosing and blood level monitoring are essential for safe in vivo application of the rapamycin system. Another well-studied regulation system involves the implementation of tetracycline-mediated transcriptional control (Gossen & Bujard, "Tight Control of Gene Expression in Mammalian Cells by Tetracycline-responsive Promoters," Proc. Natl. Acad. Sci. U.S.A. 89:5547-5551 (1992); Dhawan et al., "Tetracycline-regulated Gene Expression Following Direct Gene Transfer into Mouse Skeletal Muscle," Somat. CellMol. Genet. 21 :233-240 (1995); Bohl and Heard, "Transcriptional Modulation of Foreign Gene Expression in Engineered Somatic Tissues," Cell Biol. Toxicol. 14:83-94 (1998), each of which is hereby incorporated by reference in its entirety). In this model, the administration of tetracycline either induces or inhibits the transcriptional activity of a tetracycline-sensitive promoter. While induction and inhibition is fully titratable using varying doses of tetracycline, the system unfortunately involves the ectopic expression of transcriptional regulators that have the potential to be immunogenic. Additionally, long-term administration of tetracycline derivatives can lead to bone-mediated uptake of these compounds, which may undermine the ability to tightly regulate gene expression due to complex pharmacokinetics.

There exist a number of advantages for utilization of nuclear steroid receptor-based regulation systems. First, ligand activation of steroid receptor- mediated transcription is specific and dose dependent. Second, steroids are lipophilic molecules that can be administered by a variety of routes - orally, topically, and parentally - making such an approach attractive for in vivo applications. Third, the structure and function of steroid receptor systems are well understood and the components are interchangeable. Fourth, the system utilizes endogenous GRs to mediate the effect on transcription, thus obviating the use of co-expressed, potentially immunogenic, transcriptional activator proteins.

Although the invention has been described in detail for the purposes of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

Claims

What Is Claimed:

1. A nucleic acid construct comprising: a DNA molecule encoding an angiogenic protein or polypeptide; a polyadenylation sequence operably coupled 3' of the DNA molecule; and a glucocorticoid-inducible promoter region operably coupled 5' of the DNA molecule.

2. The nucleic acid construct according to claim 1 wherein the angiogenic protein or polypeptide is a mammalian angiogenic protein or polypeptide.

3. The nucleic acid construct according to claim 2 wherein the mammalian angiogenic protein or polypeptide is an acidic or basic fibroblast growth factor, a vascular endothelial growth factor, an epidermal growth factor, a transforming growth factor α or β, a platelet-derived endothelial cell growth factor, a platelet-derived growth factor, a tumor necrosis factor α, a hepatocyte growth factor, an insulin-like growth factor, an erythropoietin, a colony stimulating factor, a nitric oxidesynthase, or a combination thereof.

4. The nucleic acid construct according to claim 2 wherein the mammalian angiogenic protein or polypeptide is a human angiogenic protein or polypeptide.

5. The nucleic acid construct according to claim 1 wherein the glucocorticoid-inducible promoter region comprises a promoter and a glucocorticoid response element operably coupled 5' of the promoter.

6. The nucleic acid construct according to claim 5 wherein the promoter is an adenovirus minimal late promoter, a herpes thymidine kinase promoter, a minimal cytomegalovirus promoter, or a eukaryotic cellular promoter.

7. The nucleic acid construct according to claim 5 wherein the glucocorticoid-inducible promoter region comprises five tandem repeats of the glucocorticoid response element.

8. The nucleic acid construct according to claim 1 having the nucleotide sequence according to SEQ ID NO: 2.

9. An expression vector into which is inserted the nucleic acid construct according to claim 1.

10. The expression vector according to claim 9 wherein the expression vector is a plasmid vector.

11. The expression vector according to claim 9 wherein the expression vector is a viral vector.

12. The expression vector according to claim 11 wherein the viral vector is an adenoviral vector, a lentiviral vector, adeno-associated vector, baculovirus vector, pox virus vector, sendai virus vector, or herpes simplex virus vector.

13. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the expression vector according to claim 9.

14. The pharmaceutical composition according to claim 13 wherein the pharmaceutically acceptable carrier is saline, albumin, dextrose, or sterile water.

15. A liposomal composition comprising: a pharmaceutically acceptable carrier; a plurality of liposomes suspended in the pharmaceutically acceptable carrier, each comprising a lipid vesicle and an aqueous phase retained within the lipid vesicle; and one or more nucleic acid constructs according to claim 1 present within the aqueous phase of the liposomes.

16. The liposomal composition according to claim 15 wherein each of the one or more nucleic acid constructs is present in an expression vector.

17. A method of promoting neovascularization comprising: introducing an effective amount of the nucleic acid construct according to claim 1 into a tissue of a patient; administering an amount of an inducing agent to the patient under conditions effective to upregulate expression of the angiogenic protein or polypeptide, which induces angiogenic development of blood vessels in the tissue.

18. The method according to claim 17, wherein the tissue is skeletal muscle, cardiac muscle, diaphragmatic muscle, brain, kidney, or lung.

19. The method according to claim 17, wherein the tissue is ischemic tissue.

20. The method according to claim 17 wherein said introducing comprises delivering the nucleic acid construct intramuscularly, intraperitoneally, subcutaneously, transdermally, intravenously, or intracranially.

21. The method according to claim 17 wherein the effective amount of the nucleic acid construct is between about 1 μg and about 4000 μg.

22. The method according to claim 17 wherein said administering is carried out orally, topically, parentally, intraperitoneally, subcutaneously, via intravenous delivery, via intra-arterial delivery, via inhalation, via intranasal instillation, intravaginally, or intrathecally.

23. The method according to claim 17 wherein the effective amount of the inducing agent is between about 0.01 and about 100 mg/kg.

24. The method according to claim 17 wherein the inducing agent is a glucocorticoid.

25. The method according to claim 24 wherein the glucocorticoid is cortisone, cortisol, prednisone, methylprednisone, or dexamethasone.

26. The method according to claim 17 further comprising: measuring the extent of neovascularization in the tissue following said administering.

27. The method according to claim 26 further comprising: administering a second amount of the inducing agent to the patient under conditions effective to again upregulate expression of the angiogenic protein or polypeptide, which induces further angiogenic development of blood vessels in the tissue.

28. The method according to claim 17 wherein the angiogenic protein or polypeptide is a mammalian angiogenic protein or polypeptide.

29. The method according to claim 28 wherein the mammalian angiogenic protein or polypeptide is an acidic or basic fibroblast growth factor, a vascular endothelial growth factor, an epidermal growth factor, a transforming growth factor α or β, a platelet-derived endothelial cell growth factor, a platelet-derived growth factor, a tumor necrosis factor α, a hepatocyte growth factor, an insulin-like growth factor, an erythropoietin, a colony stimulating factor, a nitric oxidesynthase, or a combination thereof.

30. The method according to claim 28 wherein the mammalian angiogenic protein or polypeptide is a human angiogenic protein or polypeptide.

31. A method of treating an ischemic condition in a patient comprising: performing said method of promoting neovascularization according to claim 17, wherein the tissue into which the nucleic acid construct is introduced is ischemic tissue and the resulting neovascularization occurs in the ischemic tissue, thereby treating the ischemic condition.

32. The method according to claim 31 wherein the ischemic tissue is ischemic skeletal muscle, ischemic cardiac muscle, ischemic diaphragmatic muscle, ischemic brain, ischemic kidney, or ischemic lung.

33. The method according to claim 31 wherein the ischemic condition is cerebrovascular or cranial ischemia, renal ischemia, pulmonary ischemia, limb ischemia, ischemic cardiomyopathy, or myocardial ischemia.

34. The method according to claim 31 further comprising: measuring the extent of neovascularization in the ischemic tissue.

35. The method according to claim 34 wherein said measuring is carried out by cell morphometry, magnetic resonance spectroscopy, angiography, or

Doppler flow studies.

36. The method according to claim 31 further comprising: administering a second amount of the inducing agent to the patient under conditions effective to again upregulate expression of the angiogenic protein or polypeptide, which induces further angiogenic development of blood vessels in the ischemic tissue.

37. The method according to claim 31 wherein the angiogenic protein or polypeptide is a mammalian angiogenic protein or polypeptide.

38. The method according to claim 37 wherein the mammalian angiogenic protein or polypeptide is an acidic or basic fibroblast growth factor, a vascular endothelial growth factor, an epidermal growth factor, a transforming growth factor α or β, a platelet-derived endothelial cell growth factor, a platelet-derived growth factor, a tumor necrosis factor α, a hepatocyte growth factor, an insulin-like growth factor, an erythropoietin, a colony stimulating factor, a nitric oxidesynthase, or a combination thereof.

39. The method according to claim 37 wherein the mammalian angiogenic protein or polypeptide is a human angiogenic protein or polypeptide.