EP0977854A2 - Truncated vegf-related proteins - Google Patents

Truncated vegf-related proteins

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Publication number
EP0977854A2
EP0977854A2 EP98919794A EP98919794A EP0977854A2 EP 0977854 A2 EP0977854 A2 EP 0977854A2 EP 98919794 A EP98919794 A EP 98919794A EP 98919794 A EP98919794 A EP 98919794A EP 0977854 A2 EP0977854 A2 EP 0977854A2
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Prior art keywords
vrp
truncated
pro
arg
cys
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English (en)
French (fr)
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Peter Bohlen
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Collateral Therapeutics Inc
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Collateral Therapeutics Inc
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/02—Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

  • the present invention relates to novel truncated forms of vascular endothelial growth factor (VEGF) -related proteins. More particularly, the invention relates to N-terminally truncated VEGF-related proteins that are substantially free of other proteins. Such truncated VEGF-related proteins may be used to stimulate angiogenesis in vivo and in vitro.
  • the invention also relates to nucleic acids encoding such novel truncated VEGF-related proteins, cells, tissues and animals containing such nucleic acids; methods of treatment using such nucleic acids; and methods relating to all of the foregoing.
  • VEGFs Vascular endothelial growth factors
  • VPFs vascular permeability factors
  • VEGFs When tested in cell culture, VEGFs are potently mitogenic
  • VEGFs induce plasminogen activator, plasminogen activator inhibitor, and plasminogen activator receptor (Mandriota et al., J. Biol. Chem. 270:9709-16, 1995; Pepper et al., 181: 902-06, 1991), as well as collagenases (Unemori et al., J. Cell. Physiol. 153:557-62, 1992), enzyme systems that regulate invasion of growing capillaries into tissues.
  • VEGFs also stimulate the formation of tube-like structures by endothelial cells, an in vitro example of angiogenesis (Nicosia et al., Am. J. Pathol., 145:1023-29, 1994).
  • VEGFs induce angiogenesis (Leung et al., Science 246:1306-09, 1989) and increase vascular permeability (Senger et al., Science 219:983-85, 1983).
  • VEGFs are now known as important physiological regulators of capillary blood vessel formation. They are involved in the normal formation of new capillaries during organ growth, including fetal growth (Peters et al., Proc. Natl. Acad. Sci. USA 90:8915-19, 1993), tissue repair (Brown et al., J. Exp. Med.
  • VEGFs appear to play an essential role in the de novo formation of blood vessels from blood islands (Risau & Flamme, Ann. Rev. Cell. Dev. Biol. 11:73-92, 1995), as evidenced by abnormal blood vessel development and lethality in embryos lacking a single VEGF allele (Carmeliet et al .
  • VEGFs are strongly implicated in the pathological blood vessel growth characteristic of many diseases, including solid tumors (Potgens et al., Biol. Chem. Hoppe-Seyler 376:57-70, 1995), retinopathies (Miller et al., Am. J. Pathol. 145:574-84, 1994; Aiello et al . , N. Engl . J. Med. 331:1480-87, 1994; Adamis et al., Am. J. Ophthalmol. 118:445-50, 1994), psoriasis (Detmar et al . , J. Exp. Med. 180:1141-46, 1994), and rheumatoid arthritis (Fava et al., J. Exp. Med. 180:341-46, 1994).
  • VEGF expression is regulated by hormones (Schweiki et al., J. Clin. Invest. 91:2235-43, 1993) growth factors (Thomas, J Biol. Chem. 271:603-06, 1996), and by hypoxia (Schweiki et al., Nature 359:843-45, 1992, Levy et al . , J. Biol. Chem. 271:2746- 53, 1996) .
  • Upregulation of VEGFs by hypoxic conditions is of particular importance as a compensatory mechanism by which tissues increase oxygenation through induction of additional capillary vessel formation and resulting increased blood flow. This mechanism is thought to contribute to pathological angiogenesis in tumors and in retinopathies .
  • VEGF expression after hypoxia is also essential in tissue repair, e.g., in dermal wound healing (Frank et al., J. Biol. Chem. 270:12607-613, 1995), and in coronary ischemia (Banai et al., Cardiovasc. Res. 28:1176-79, 1994; Hashimoto et al., Am. J. Physiol. 267 :H1948-H1954 , 1994).
  • VEGF vascular endothelial growth factor
  • vascular ischemia The potential of VEGF to pharmacologically induce angiogenesis in animal models of vascular ischemia has been shown in the rabbit chronic limb ischemia model by demonstrating that repeated intramuscular injection or a single intra-arterial bolus of VEGF can augment collateral blood vessel formation as evidenced by blood flow measurement in the ischemic hindlimb (Pu, et al., Circulation 88:208-15, 1993; Bauters et al., Am. J. Physiol. 267 : H1263-71, 1994; Takeshita et al., Circulation 90 [part 2], 11-228-34, 1994; Bauters et al., J. Vase. Surg.
  • VEGF has also been shown to act synergistically with basic FGF to ameliorate ischemia (Asahara et al., Circulation 92:[suppl 2], 11-365-71, 1995) .
  • VEGF was also reported to accelerate the repair of balloon-injured rat carotid artery endothelium thereby inhibiting pathological thickening of the underlying smooth muscle layers, and thus maintaining lumen diameter and blood flow (Asahara et al., Circulation 91:2793-2801, 1995).
  • VEGF has also been shown to induce EDRF (Endothelium-Derived Relaxing Factor (nitric oxide) ) -dependent relaxation in canine coronary arteries, thus potentially contributing to increased blood flow to ischemic areas via a secondary mechanism not related to angiogenesis (Ku et al . , Am. J. Physiol. 265:H586- H592, 1993) . Together, these data provide compelling evidence for a potential therapeutic role of VEGFs in wound healing, ischemic diseases and restenosis.
  • EDRF Endothelium-Derived Relaxing Factor (nitric oxide)
  • the VEGF family of proteins is comprised of at least 4 members VEGF-121, VEGF-165, VEGF-189, and VEGF-206.
  • the originally characterized VEGF is a 34-45 kDa glycosylated protein which consists of 2 identical subunits of 165 amino acid residues (Tischer et al., Biochem. Biophys. Res. Commun. 165:1198-1206, 1989).
  • the VEGF-165 cDNA encodes a 191-residue amino acid sequence consisting of a 26-residue secretory signal peptide sequence, which is cleaved upon secretion of the protein from cells, and the 165-residue mature protein subunit.
  • VEGF-165 binds strongly to heparin for which the strongly basic sequence between residues 115-159 is thought to be responsible
  • Fig. 1 Thimas, J. Biol. Chem., 271:603-06 (1996).
  • the other members of the VEGF family are homodimeric proteins with shorter or longer subunits of 121, 189 and 206 residues (VEGF- 121, VEGF-189, and VEGF-206, respectively) (Tischer et al., J. Biol. Chem. 266:11947-54, 1991; Park et al., Mol Biol Cell 4:1317-26 (1993)).
  • VEGF vascular endothelial growth factor
  • VEGF-121 exons 1- 5,8; VEGF-165, exons 1-5,7,8; VEGF-189, exons 1-5, 6a, 7, 8; VEGF-206, exons 1-5, 6b, 7, 8 (exon 6a and 6b refer to 2 alternatively spliced forms of the same exon)
  • VEGF sequences contain eight conserved disulfide-forming core cysteine residues. All VEGF genes encode signal peptides that direct the protein into the secretory pathway.
  • VEGF-121 and -165 are found to be readily secreted by cultured cells whereas VEGF-189 and -206 remain associated with the extracellular matrix.
  • These VEGF forms possess an additional highly basic sequence, corresponding to residues 115-139 in VEGF-189 and -206 (matrix- targeting sequence) , which confers high affinity to acidic components of the extracellular matrix (Thomas, J. Biol. Chem. 271:603-06 (1996) ) .
  • Mitogenic activity of the various VEGF isoforms varies depending on each isoform. For example, VEGF-121 and VEGF-165 have very similar mitogenic activity for endothelial cells.
  • VEGF-189 and VEGF-206 are only weakly mitogenic (Ferrara et al., Endocr. Rev. 13:18-32, 1992). The reduced activity of these isoforms is attributed to their strong association with cells and matrix, as evidenced by the normal mitogenic activity of a mutant of VEGF-206 which lacks the 24- residue "matrix targeting" sequence common to VEGF-189 and VEGF-206 (residues 115-139 in Fig. 1) (Ferrara et al . , Endocr. Rev. 13:18-32, 1992) .
  • VEGF-165 generated by plasmin (VEGF (1-110) ) bound with the same affinity to the KDR receptor as VEGF-165 and VEGF-121 whereas the C-terminal VEGF-fragment (111-165) had no binding activity (Keyt et al . , J. Biol. Chem. 271:7788-95, 1996).
  • the mitogenic activity of VEGF-121 and VEGF-110 was reduced by approximately 110-fold as compared to VEGF-165, suggesting a potential role of the C-terminal domain of VEGF-165 in the biological potency of VEGF isoforms.
  • VEGFs are related to platelet-derived growth factor (PDGF) (Andersson et al., Growth Factors 12:159-64, 1995). VEGFs are also related to the family of proteins derived from the Placenta Growth Factor (P1GF) gene, P1GF-129 and P1GF-150 (Maglione et al., Proc. Natl. Acad. Sci. USA 88:9267-71, 1991; Oncogene 8:925-31, 1993). More recently several additional VEGF-related genes have been identified and termed VEGF-B (also called VEGF-related factor VRF-1) (Grimmond et al . , Genome Res.
  • VEGF-B also called VEGF-related factor VRF-1
  • VRF-2 Grimmond et al., Genome Res. 6:122-29, 1996)
  • VEGF-C Joukov et al., EMBO J. 15:290-98, 1996; Lee et al., Proc. Natl. Acad. Sci. USA 93:1988-92, 1996)
  • VEGF-3 PCT Application No. PCT/US95/07283, published on December 12, 1996 as W096/39421) .
  • two virally encoded VEGF-related sequences have been identified, poxvirus ORF-l and ORF-2
  • VRPs VEGF-related proteins
  • VEGF vascular endothelial growth factor
  • P1GF P1GF subunits
  • VEGF-165/VEGF-B heterodimers were found to form after transfection of cells with both genes (Olofsson et al., Proc. Natl. Acad. Sci. U.S.A. 93:2567-81, 1996).
  • VEGFs interact with two receptors present on endothelial cells, KDR/flk-1 (Terman et al., Biochem. Biophys. Res. Commun.
  • VRPs are known to bind to one or more of three different endothelial cell receptors, each of which is a single transmembrane protein with a large extracellular portion comprised of 7 immunoglobulin-type domains and a cytoplasmic portion that functions as a tyrosine kinase.
  • These receptors are KDR/flk-1 (Terman et al., Biochem. Biophys. Res. Commun. 187:1579-86, 1992), flt-1 (De Vries et al., Science 255:989-91, 1992), and flt-4 (Pajusola et al . , Cancer Res. 52:5738-43, 1992) .
  • VEGF binds to KDR and fltl (Terman et al., Growth Factors 11:187-95, 1994) but not flt4 (Joukov et al., EMBO J. 15:290-98, 1996)
  • P1GF binds to fit 1 but not KDR (Terman et al., Growth Factors 11:187-95, 1994) and flt4 (Joukov et al., EMBO J. 15:290-98, 1996)
  • VEGF-C binds to flt-4 (Joukov et al., EMBO J. 15:290-98, 1996) but it is controversial whether it also binds to KDR
  • VRPs is not presently known.
  • VEGF-B stimulates endothelial cell proliferation (Olofsson et al., Proc. Natl. Acad. Sci. U.S.A. 93:2567-81, 1996) it may be speculated that VEGF-B can bind to KDR because KDR is thought to be primarily responsible for the angiogenic response of endothelial cells to VEGF-like growth factors (Gitay-Goren et al., J. Biol. Chem. 271:5519-23 (1996)).
  • VRPs have been shown to activate the KDR receptor which is thought to make endothelial cells "angiogenesis-competent .
  • Evidence for such activity has been presented for VEGF-B which stimulates endothelial cell proliferation (Olofsson et al., Proc. Natl. Acad. Sci. U.S.A. 93:2567-81, 1996), VEGF-C which stimulates endothelial cell migration and proliferation (Joukov et al., EMBO J. 15:290-98, 1996; Lee et al . , Proc. Natl. Acad. Sci.
  • VEGFs are expressed in many different tissues. Similarly, VRP genes are also expressed in multiple tissues but it is of particular interest that VEGF-B and to a lesser extent VRF-2 are strongly expressed in human heart and skeletal muscle
  • VEGF-B is expressed considerably more strongly in mouse heart tissue than VEGF (Olofsson et al., Proc. Natl. Acad. Sci. U.S.A. 93:2567-81, 1996).
  • VEGF-C is also strongly expressed in several human tissues, most notably in heart and skeletal muscle (Joukov et al., EMBO J. 15:290-98, 1996). This expression pattern, and the extraordinar specificity of VRPs for endothelial cells, suggest that these factors play a physiological role in angiogenesis in these tissues. This is thought to be relevant in pathological situations such as coronary ischemia where collateral angiogenesis is required to provide the heart muscle with an adequate capillary blood vessel supply.
  • VEGF mRNA in the rat or pig heart in vivo and hypoxia induces VEGF mRNA in cardiac myocytes and smooth muscle cells in vitro (Hashimoto et al., Am. J. Physiol 267, H1948- H1954, 1994; Banai, et al., Cardiovac. Res. 28:1176-79, 1994; Circulation 90, 649-52, 1994) .
  • the strong expression of VEGF and VRPs in the heart may help to ensure a redundant and competent regulatory system capable of inducing new blood vessel formation when it is needed.
  • VEGF is upregulated in epidermal keratinocytes during skin wound healing (Brown et al., J. Exp. Med. 176:1375-79, 1992).
  • therapy of various ischemic conditions such as cardiac infarction, chronic coronary ischemia, chronic lower limb ischemia, wound healing and stroke with VRPs may be potentially clinically beneficial.
  • the present invention is directed to novel truncated forms of VEGF-related proteins (VRPs), preferably human VRPs.
  • VRPs VEGF-related proteins
  • the preferred use of the truncated VRPs and nucleic acid molecule compositions of the invention is to use such compositions to aid in the treatment of patients with heart disease, wounds, or other ischemic conditions by stimulating angiogenesis in such patients.
  • the amino acid sequences of VRPs include eight disulfide-forming cysteine residues that are conserved between VRPs and VEGF proteins (core cysteines) .
  • VRPs include, but are not limited to, VEGF-B, VEGF-C, VRF-2, ORF-1, ORF-2, and PIGFs.
  • a first aspect of the invention provides for a truncated VRP having a deletion of at least one of the amino acid residues N-terminal to the first cysteine of the core sequence of said subunit.
  • Such compositions would be substantially free of other proteins.
  • the truncations range from truncating minimally the N-terminal residue of the mature protein subunit only (not including the signal sequence) and maximally all N-terminal amino acids of the mature protein up to and including the residue N-terminal to (prior to) the first core cysteine residue.
  • all of the amino acid residues N-terminal to the first cysteine of the core sequence, except the 1 to 5 amino acid residues immediately N-terminal to said first cysteine are deleted.
  • the amino acid deletions may consist of deletions of non-adjacent amino acid residues in the N-terminal sequence, it is preferred that the deletions be of consecutive amino acid residues.
  • the invention comprises human VRPs that have deletions of amino acid residue sequences of increasing lengths from the N-terminus of the N- terminal sequence up to the first cysteine of the core sequence of the VRP subunit sequence.
  • the invention provides for truncated versions of the VRPs VEGF-B, VRF-2, VEGF-C, VEGF-3, P1GF, poxvirus ORF-1, and poxvirus ORF-2.
  • each subunit may independently have a deletion of at least one of the amino acid residues N-terminal to the first cysteine of the core sequence of said subunit, or only one of the subunits may have such a deletion.
  • the truncated VRP subunit comprises a VRP subunit wherein various numbers of amino acid residues N-terminal to the first cysteine of the core sequence are deleted.
  • the remaining N-terminal residues consist of consecutive amino acid residues derived from the N- terminal sequence. These consecutive N-terminal residues may be derived from any location in the N-terminal sequence, however, a consecutive sequence starting from the N-terminus of the N-terminal sequence is preferred, and a sequence consisting of consecutive amino acid residues immediately N-terminal to the first cysteine of the core sequence of the VRP subunit is most preferred. Examples of such most preferred embodiments are depicted in Figure 2.
  • amino acid residues N-terminal to the first cysteine of the core sequence of the truncated VRPs of the invention are a randomly selected amino acid sequence, in yet other embodiments, these amino acid residues are derived from the N-terminal sequence of the full length VRP sequence, but are not necessarily consecutive amino acids from the full length VRP sequence.
  • the invention provides a truncated VRP subunit wherein the amino acid residues N- terminal to the first cysteine of the core sequence of said subunit are deleted.
  • the invention provides a truncated VRP subunit wherein the amino acid sequence N-terminal to the core sequence comprises 11 to 20, more preferably 11 to 15, more preferably 6 to 10, and most preferably 2 to 5 amino acid residues.
  • the amino acid sequence N-terminal to the core sequence comprises the consecutive amino acid residues immediately N-terminal to the first cysteine of the core sequence of said VRP subunit.
  • the truncated VRP comprises the core sequence, the necessary C-terminal sequence to the core sequence, and further comprises at the region N-terminal to the first cysteine of the core sequence, the 11 to 20, more preferably the 11 to 15, more preferably the 6 to 10, and most preferably the 2 to 5 consecutive amino acid residues of the amino acid sequence that is immediately N-terminal to the first cysteine of the core sequence of the full length VRP sequence.
  • a truncated VRP subunit comprises, for example, (X) amino acids N-terminal to the first cysteine of the core sequence
  • a truncated VRP subunit is one where the corresponding full length VRP subunit comprises (X + 1) amino acids N- terminal to the first cysteine of the core sequence.
  • the truncated VRPs of the invention include truncated VRP homodimers comprising two truncated VRP subunits of the invention, wherein the two truncated VRP subunits have the same amino acid sequence, and also include truncated VRP heterodimers comprising two truncated VRP subunits of the invention wherein the two subunits have different amino acid sequences from each other.
  • the term N-NN encompasses a deletion of anywhere from N to NN of the first amino acids after the signal sequence.
  • the truncated VRP subunit comprises a truncated hVEGFB protein subunit wherein the first 10-15 amino acids are deleted; more preferably, the first 15-20 amino acids are deleted; more preferably, the first 20-25 amino acids are deleted; and most preferably, the first 23-24 amino acids are deleted.
  • the truncated VRP subunit comprises a truncated hVRF2 protein subunit wherein the first 10-15 amino acids are deleted; more preferably, the first 15-20 amino acids are deleted; more preferably, the first 20-25 amino acids are deleted; and most preferably, the first 23-24 amino acids are deleted.
  • the truncated VRP subunit comprises a truncated hVEGFC protein subunit wherein the first 95-100 amino acids are deleted; more preferably, the first 100- 105 amino acids are deleted; more preferably, the first 105-110 amino acids are deleted; and most preferably, the first 108-109 amino acids are deleted.
  • the truncated VRP subunit comprises a truncated hPlGF protein subunit wherein the first 16-21 amino acids are deleted; more preferably, the first 21-26 amino acids are deleted; more preferably, the first 26-31 amino acids are deleted; and most preferably, the first 29-30 amino acids are deleted.
  • the truncated VRP subunit comprises a truncated hVEGF3 protein subunit wherein the first 10-15 amino acids are deleted; more preferably, the first 15-20 amino acids are deleted; more preferably, the first 20-25 amino acids are deleted; and most preferably, the first 23-24 amino acids are deleted.
  • the truncated VRP subunit comprises a truncated pvORFl protein subunit wherein the first 20-25 amino acids are deleted; more preferably, the first 25-30 amino acids are deleted; more preferably, the first 30-35 amino acids are deleted; and most preferably, the first 33-34 amino acids are deleted.
  • the truncated VRP subunit comprises a truncated pvORF2 protein subunit wherein the first 30-35 amino acids are deleted; more preferably, the first 35-40 amino acids are deleted; more preferably, the first 40-45 amino acids are deleted; and most preferably, the first 43-44 amino acids are deleted.
  • the sequences of some exemplary preferred truncated VRP subunits are set out in Figure 2.
  • the invention also provides for nucleic acid molecules coding for the truncated VRP subunits described herein.
  • the nucleic acid molecules may be, for example, DNA, cDNA or RNA.
  • the invention also provides for recombinant DNA vectors comprising the nucleic acid molecules encoding the truncated VRPs, and host cells transformed with such recombinant DNA vectors, wherein such vectors direct the synthesis of a truncated VRP subunit such as those described herein.
  • the invention further provides for nucleic acid molecules encoding biosynthetic precursor forms of N-terminally truncated subunits of VRPs for the purpose of facilitating the expression in suitable host systems.
  • nucleic acid molecules are comprised of DNA encoding a signal peptide that precedes the truncated subunits at their N-termini.
  • the signal sequences of VEGF or VRPs would be used to construct appropriate signal peptide-containing truncated forms of VRPs.
  • the human VEGF signal peptide is as follows: mnfllswvhwslalllylhhakwsqa (I) — [SEQ I.D. NO. 40] —
  • the signal peptides shown in Figure 1 may be used.
  • the signal peptide specific for the truncated VRP is used in the construct.
  • an appropriate nucleic acid molecule would be comprised of DNA encoding the signal sequence of VEGF-B, optionally followed by a codon for proline (the first residue of mature VEGF-B) , optionally followed by a codon for valine (the second residue of mature VEGF-B) , and followed by DNA encoding the N- terminally truncated VEGF-B.
  • the invention also provides for other appropriate signal peptide fusion constructs, best suitable for non-mammalian hosts, as known by those skilled in the art.
  • the signal peptides should optionally include residues needed for facilitation of signal peptide cleavage in mammalian cells for the various truncated VRP subunits of the present invention.
  • the present invention provides for recombinant DNA expression vectors wherein the 5' end of the nucleic acid molecule coding for the truncated VRP subunit is operably linked to a DNA sequence that codes for a signal peptide.
  • the signal peptide may be a human VRP signal peptide.
  • the DNA sequence coding for said signal peptide may be operably linked at the 3' end of said DNA sequence to DNA coding for the first amino acid residue of the mature non-truncated VRP subunit, and wherein the 3' end of said DNA coding for said residue is operably linked to the nucleic acid molecule coding for said truncated VRP subunit.
  • the DNA sequence coding for said signal peptide is operably linked at the 3 1 end of said DNA sequence to DNA coding for the first two amino acid residues of the mature non-truncated VRP subunits, and wherein the 3' end of said DNA coding for said two residues is operably linked to said nucleic acid molecule coding for said truncated VRP subunit.
  • the invention also provides a truncated VRP subunit of the invention as described above, further comprising at the N- terminus of said truncated VRP subunit, the first one or two amino acid residues of the mature non-truncated VRP subunit.
  • truncated VRP subunits of the invention include those wherein the final number of amino acids N-terminal to the first cysteine of the core sequence (including the additional one or two amino acids that may facilitate signal peptide cleavage) is at least one less than the number of amino acids N-terminal to the first cysteine of the core sequence of the corresponding full length VRP.
  • the invention provides truncated VRP homodimers or heterodimers comprising two truncated VRP subunits wherein said truncated VRP subunits comprise at the N-terminus of said truncated VRP subunits, the first one or two amino acid residues of the mature non- truncated VRP subunit.
  • the recombinant nucleic acid molecule coding for a truncated VRP subunit of the invention is operably linked to control sequences operable in a host cell transformed with said vector.
  • the present invention also provides for transformed or transfected host cells comprising the recombinant DNA vectors of the invention.
  • the present invention also includes delivery vectors which comprise nucleic acid molecules coding for the truncated VRPs of the invention.
  • delivery vectors may be, for example, viral vectors.
  • viral vectors may be, for example, adenovirus vectors or adenovirus-associated virus vectors.
  • an adenovirus vector comprising a nucleic acid molecule coding for a truncated VRP of the invention operably linked at the 5 1 end of the nucleic acid molecule to a DNA sequence that codes for a signal peptide.
  • the signal peptide is selected from the group consisting of VEGF signal peptide, VEGF-B signal peptide, VRF-2 signal peptide, VEGF-C signal peptide, P1GF signal peptide, VEGF-3 signal peptide, poxvirus ORF-1 signal peptide, and poxvirus ORF-2 signal peptide.
  • said signal peptide is VEGF-B signal peptide.
  • the DNA sequence coding for the signal peptide is operably linked at the 3' end of the DNA sequence to DNA coding for the first amino acid residue of the mature non-truncated VRP subunit, and wherein the 3' end of said DNA coding for said residue is operably linked to the nucleic acid molecule coding for said truncated VRPs.
  • the adenovirus vector comprises a nucleic acid molecule which codes for a truncated VRP subunit of Figure 2.
  • a filtered-injectable adenovirus vector preparation comprising a recombinant adenoviral vector, said vector containing no wild-type virus and comprising: a partial adenoviral sequence from which the E1A/E1B genes have been deleted, and a transgene coding for a truncated VRP subunit, driven by a promoter flanked by the partial adenovirus sequence; and a pharmaceutically acceptable carrier.
  • the preparation has been filtered through a 30 micron filter.
  • the truncated VEGF subunit is a truncated VEGF subunit of Figure 2.
  • the injectable adenoviral vector preparation comprises a promoter selected from the group consisting of a CMV promoter, a ventricular myocyte-specific promoter, and a myosin heavy chain promoter.
  • the invention provides a method of producing a truncated VRP polypeptide comprising growing, under suitable conditions, a host cell transformed or transfected with the recombinant DNA expression vector of the invention in a manner allowing expression of said polypeptide, and isolating said polypeptide from the host cell. Suitable conditions are then provided for the truncated VRP peptide to fold into a truncated VRP subunit.
  • suitable conditions are then provided for the truncated VRP peptide to fold into a truncated VRP subunit.
  • mammalian cells such conditions should be naturally provided by the cell.
  • appropriate pH, isotonicity, and reducing conditions must be provided, such as those described in, for example, Example 2.
  • the invention provides a method of producing a truncated VRP wherein suitable conditions are provided for said truncated VRP subunit to dimerize with a second VRP subunit selected from the group consisting of VRP subunits and truncated VRP subunits.
  • suitable conditions are provided for said truncated VRP subunit to dimerize with a second VRP subunit selected from the group consisting of VRP subunits and truncated VRP subunits.
  • methods of producing a truncated VRP homodimer comprising two truncated VRP subunits having the same amino acid sequence.
  • Such heterodimers may consist of one truncated VRP subunit and one non-truncated VRP subunit, or both VRP subunits may be truncated.
  • the two subunits may be derived from different VRPs.
  • the heterodimer may consist of one VEGF-B subunit and one truncated VEGF-C subunit, or both subunits may be truncated.
  • the present invention provides pharmaceutical compositions comprising a truncated VRP subunit of the present invention, in a suitable carrier.
  • the invention includes methods of stimulating blood vessel formation comprising administering to a patient such a pharmaceutical composition.
  • Methods are provided using the compounds of the present invention to stimulate endothelial cell growth or endothelial cell migration in vitro comprising treating said endothelial cells with truncated VRPs.
  • the present invention also provides methods of treating a patient suffering from a heart disease comprising administering to said patient a nucleic acid molecule coding for at least one truncated VRP subunit, said nucleic acid molecule capable of expressing the truncated VRP subunit in said patient.
  • methods are provided of stimulating angiogenesis in a patient comprising administering a therapeutic amount of a pharmaceutical composition comprising a truncated VRP of the present invention.
  • the pharmaceutical composition is in a therapeutically suitable delivery system.
  • a potentiating agent is administered to potentiate the angiogenic effect of said truncated VRP.
  • agents include, for example, basic Fibroblast Growth Factor (bFGF) (FGF-2) , acidic FGF (aFGF) (FGF-1) , FGF-4, FGF-5, FGF-6, or any FGF or other angiogenic factor that stimulates endothelial cells.
  • the pharmaceutical compositions may also be used to treat patients suffering from ischemic conditions such as cardiac infarction, chronic coronary ischemia, chronic lower limb ischemia, stroke, and peripheral vascular disease. Methods are also provided using the pharmaceutical compositions of the present invention to treat wounds, such as dermal or intestinal wounds.
  • ischemic conditions such as cardiac infarction, chronic coronary ischemia, chronic lower limb ischemia, stroke, and peripheral vascular disease.
  • Methods are also provided using the pharmaceutical compositions of the present invention to treat wounds, such as dermal or intestinal wounds.
  • methods are provided of stimulating angiogenesis in a patient comprising delivering a delivery vector to the myocardium of the patient by intracoronary injection directly into one or both coronary arteries, said vector comprising a nucleic acid molecule coding for at least one truncated VRP subunit, wherein said vector is capable of expressing the truncated VRP subunit in the myocardium.
  • the method may be used for stimulating coronary collateral vessel development.
  • a method for stimulating vessel development in a patient having peripheral vascular disease comprising delivering a delivery vector to the peripheral vascular system of the patient by intra-femoral artery injection directly into one or both femoral arteries, said vector comprising a transgene coding for a truncated VRP subunit, and capable of expressing the truncated VRP subunit in the peripheral vascular system, thereby promoting peripheral vascular development.
  • the delivery vector used in the invention is a viral delivery vector.
  • the delivery vector is a replication-deficient adenovirus vector.
  • the delivery vector is an adeno- associated virus vector.
  • Figure 1 depicts the amino acid sequences of VEGF-B [SEQ ID NO: 1]
  • VRF-2 [SEQ I.D. NO. 2]
  • VEGF-C [SEQ I.D. NO. 3]
  • P1GF human P1GF-2 [SEQ I.D. NO. 4] [SEQ I.D. NO. 4], VEGF-3 [SEQ I.D. NO. 5], poxvirus ORF-1 [SEQ I.D. NO. 6], and poxvirus ORF-2 [SEQ I.D.
  • Figure 2a-2f depicts examples of truncated VRP amino acid sequences below the corresponding full length (F/L) VRP sequence.
  • the amino acid sequences of each truncation are listed as follows:
  • the invention features a truncated VRP comprising at least one truncated VRP subunit.
  • truncated VRP subunit it is meant a VRP subunit having an amino acid sequence substantially similar to one of the VRPs, for example, but not limited to, one of the sequences shown in Figure 1, or an analog or derivative thereof, wherein at least one of the N- terminal amino-acid residues N-terminal to the first cysteine of the core sequence of the mature subunit is deleted.
  • a sequence that is "substantially similar" to a VRP comprises an amino acid sequence that is at least 25% homologous to the 8 cysteine core sequence of VEGF-B, comprises all of the essential conserved cysteine residues of said core sequence, and retains VRP activity.
  • truncated VRP subunit is also meant a VRP subunit wherein at least one of the N-terminal amino acid residues N-terminal to the first cysteine of the VEGF core sequence is deleted, and, at least one of the cysteines of the core sequence is deleted, wherein said cysteine is non-essential.
  • a non-essential cysteine is one that is not required to retain VRP activity.
  • Such non- essential cysteines have been described in connection with PDGF. (Potgens, et al . J. Biol. Chem. 269:32879-85 (1994)).
  • identity is meant a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved and have deletions, additions, or replacements may have a lower degree of identity. In calculating sequence identity, the two sequences are compared starting at the carboxy terminus of the N-terminal deletion. Those skilled in the art will recognize that several computer programs are available for determining sequence identity.
  • Analogs of a truncated VRP polypeptide or subunit are functional equivalents having similar amino acid sequence and retaining, to some extent, one or more activities of the related truncated VRP polypeptide or subunit.
  • functional equivalent is meant the analog has an activity that can be substituted for one or more activities of a particular truncated VRP polypeptide or subunit.
  • Preferred functional equivalents retain all of the activities of a particular truncated VRP polypeptide or subunit, however, the functional equivalent may have an activity that, when measured quantitatively, is stronger or weaker, as measured in VRP functional assays, for example, such as those disclosed herein.
  • truncated VRP polypeptides or subunits must be incorporated into a truncated VRP dimer in order to measure functional activity.
  • Preferred functional equivalents have activities that are within 1% to 10,000% of the activity of the related truncated VRP polypeptide or subunit, more preferably between 10% to 1000%, and more preferably within 50% to 200%.
  • a derivative to retain some activity can be measured using techniques described herein.
  • Derivatives include modification occurring during or after translation, for example, by phosphorylation, glycosylation, crosslinking, acylation, proteolytic cleavage, linkage to an antibody molecule, membrane molecule or other ligand (see Ferguson et al . , 1988, Annu . Rev. Biochem . 57:285-320) .
  • derivatives or analogs also include amino acid alterations such as deletions, substitutions, additions, and amino acid modifications.
  • a “deletion” refers to the absence of one or more amino acid residue (s) in the related polypeptide.
  • An “addition” refers to the presence of one or more amino acid residue (s) in the related polypeptide. Additions and deletions to a polypeptide may be at the amino terminus, the carboxy terminus, and/or internal.
  • Amino acid "modification” refers to the alteration of a naturally occurring amino acid to produce a non-naturally occurring amino acid.
  • a “substitution” refers to the replacement of one or more amino acid residue (s) by another amino acid residue (s) in the polypeptide.
  • Derivatives can contain different combinations of alterations including more than one alteration and different types of alterations.
  • the substituted amino acid is from the same group as the amino acid being replaced.
  • amino acids which are interchangeable: the basic amino acids lysine, arginine, and histidine; the acidic amino acids aspartic and glutamic acids; the neutral polar amino acids serine, threonine, cysteine, glutamine, asparagine and, to a lesser extent, methionine; the nonpolar aliphatic amino acids glycine, alanine, valine, isoleucine, and leucine (however, because of size, glycine and alanine are more closely related and valine, isoleucine and leucine are more closely related) ; and the aromatic amino acids phenylalanine, tryptophan, and tyrosine.
  • alanine, glycine, and serine seem to be interchangeable to some extent, and cysteine additionally fits into this group, or may be classified with the polar neutral amino acids.
  • Preferred derivatives have one or more amino acid alteration (s) which do not significantly affect the activity of the related truncated VRP polypeptide or subunit.
  • amino acids may be deleted, added or substituted with less risk of affecting activity.
  • amino acid alterations are less preferred as there is a greater risk of affecting VRP activity. Such alterations should be conservative alterations.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent.
  • conserved regions tend to be more important for protein activity than non-conserved regions. Standard procedures can be used to determine the conserved and non-conserved regions important for VRP activity using in vi tro mutagenesis techniques or deletion analyses and measuring VRP activity as described by the present disclosure.
  • Derivatives can be produced using standard chemical techniques and recombinant nucleic acid molecule techniques. Modifications to a specific polypeptide may be deliberate, as through site-directed mutagenesis and amino acid substitution during solid-phase synthesis, or may be accidental such as through mutations in hosts which produce the polypeptide. Polypeptides including derivatives can be obtained using standard techniques such as those described in Sambrook et al . , Molecular Cloning, Cold Spring Harbor Laboratory Press (1989) . For example, Chapter 15 of Sambrook describes procedures for site-directed mutagenesis of cloned DNA.
  • truncated VRP polypeptide is meant a polypeptide comprising the amino acid sequence of a truncated VRP subunit of the invention, or a functional analog or derivative thereof as described herein.
  • the term “truncated VRP polypeptide” also includes a truncated VRP subunit; the term subunit generally referring to a peptide that has been folded into an active three-dimensional structure.
  • truncated VRP is meant a dimer of two VRP subunits.
  • the two subunits may be derived from two different VRPs where both subunits are truncated VRP subunits.
  • One or both of the subunits may be truncated; the two subunits may also have different N-terminal deletions.
  • the truncated VRP, truncated VRP subunit, or truncated VRP polypeptide be enriched or purified.
  • enriched in this context is meant that the specific amino acid sequence constitutes a significantly higher fraction (2 - 5 fold) of the total of amino acid sequences present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other amino acid sequences present, or by a preferential increase in the amount of the specific amino acid sequence of interest, or by a combination of the two.
  • enriched does not imply that there are no other amino acid sequences present, just that the relative amount of the sequence of interest has been significantly increased.
  • the term "significant” here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other amino acid sequences of about at least 2 fold, more preferably at least 5 to 10 fold or even more.
  • the term also does not imply that there is no amino acid sequence from other sources.
  • the other source amino acid sequence may, for example, comprise amino acid encoded by a yeast or bacterial genome, or a cloning vector such as pUC19. The term is meant to cover only those situations in which man has intervened to elevate the proportion of the desired amino acid sequence.
  • an amino acid sequence be in purified form.
  • purified in reference to a polypeptide does not require absolute purity (such as a homogeneous preparation) ; instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level this level should be at least 10 fold greater, e.g., in terms of mg/ml).
  • Purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
  • the substance is preferably free of contamination at a functionally significant level, for example 90%, 95%, or 99% pure.
  • the invention features a nucleic acid molecule encoding a truncated VRP polypeptide or subunit.
  • nucleic acid molecule it is desirable for such nucleic acid molecule to be enriched or purified.
  • enriched in reference to nucleic acid molecule is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2 - 5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two.
  • enriched does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased.
  • the term significant here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other nucleic acids of about at least 2 fold, more preferably at least 5 to 10 fold or even more.
  • the term also does not imply that there is no DNA or RNA from other sources.
  • the other source DNA may, for example, comprise DNA from a yeast or bacterial genome, or a cloning vector such as pUC19. This term distinguishes from naturally occurring events, such as viral infection, or tumor type growths, in which the level of one mRNA may be naturally increased relative to other species of mRNA. That is, the term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid.
  • nucleotide sequence be in purified form.
  • purified in reference to nucleic acid molecule does not require absolute purity (such as a homogeneous preparation) ; instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e . g. , in terms of mg/ml) .
  • the nucleic acid molecule may be constructed from an existing VRP nucleotide sequence by modification using, for example, oligonucleotide site-directed mutagenesis, or by deleting sequences using restriction enzymes, or as described herein.
  • Standard recombinant techniques for mutagenesis such as in vitro site-directed mutagenesis (Hutchinson et al . , J ⁇ Biol. Chem. 253:6551, (1978), Sambrook et al . , Chapter 15, supra ) , use of TAB® linkers (Pharmacia) , and PCR-directed mutagenesis can be used to create such mutations.
  • the nucleic acid molecule may also be synthesized by the triester method or by using an automated DNA synthesizer.
  • the invention also features recombinant DNA vectors and recombinant DNA expression vectors preferably in a cell or an organism.
  • the recombinant DNA vectors may contain a sequence coding for a truncated VRP or a functional derivative thereof in a vector containing a promoter effective to initiate transcription in a host cell.
  • the recombinant DNA vector can contain a transcriptional initiation region functional in a cell and a transcriptional termination region functional in a cell.
  • the present invention also relates to a cell or organism that contains the above-described nucleic acid molecule or recombinant DNA vector and thereby is capable of expressing a truncated VRP peptide.
  • the polypeptide may be purified from cells which have been altered to express the polypeptide.
  • a cell is said to be "altered to express a desired polypeptide" when the cell, through genetic manipulation, is made to produce a protein which it normally does not produce or which the cell normally produces at lower levels.
  • One skilled in the art can readily adapt procedures for introducing and expressing either genomic, cDNA, or synthetic sequences into either eukaryotic or prokaryotic cells.
  • a nucleic acid molecule such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are “operably linked” to nucleotide sequences which encode the polypeptide.
  • the precise nature of the regulatory regions needed for gene sequence expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal synthesis initiation.
  • Such regions will normally include those 5 ' -non- coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • the entire coding sequence of a truncated VRP subunit or a fragment thereof may be combined with one or more of the following in an appropriate expression vector to allow for such expression: (1) an exogenous promoter sequence (2) a ribosome binding site (3) a polyadenylation signal (4) a secretion signal.
  • Modifications can be made in the 5'- untranslated and 3 ' -untranslated sequences to improve expression in a prokaryotic or eukaryotic cell; or codons may be modified such that while they encode an identical amino acid, that codon may be a preferred codon in the chosen expression system.
  • codons may be modified such that while they encode an identical amino acid, that codon may be a preferred codon in the chosen expression system.
  • the use of such preferred codons is described in, for example, Grantham et al., Nuc. Acids Res. , 9:43-74 (1981), and Lathe, J. Mol. Biol., 183:1-12 (1985) hereby
  • the non-coding region 3' to the genomic VRP sequence may be operably linked to the nucleic acid molecule encoding such VRP subunit.
  • This region may be used in the recombinant DNA vector for its transcriptional termination regulatory sequences, such as termination and polyadenylation.
  • the transcriptional termination signals may be provided.
  • a 3' region functional in the host cell may be substituted.
  • An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene sequence expression.
  • Two DNA sequences (such as a promoter region sequence and a truncated VRP sequence) are said to be operably linked if the nature of the linkage between the two DNA sequences does not
  • a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
  • Examples 2 and 3 describe the expression and purification of novel truncated VRP sequences of the present invention as expressed in baculovirus systems. Those skilled in the art will recognize that the truncated VRPs of the present invention may also be expressed in other cell systems, both prokaryotic and eukaryotic, all of which are within the scope of the present invention. Examples 4-6 provide examples of suitable assays for functional activity of the novel truncated VRPs.
  • the truncated VRPs of the present invention may be expressed in prokaryotic cells, which are generally very efficient and convenient for the production of recombinant proteins, the truncated VRPs produced by such cells will not be glycosylated and therefore may have a shorter half-life in vivo.
  • Prokaryotes most frequently are represented by various strains of E. coli. However, other microbial strains may also be used, including other bacterial strains. Recognized prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like. The prokaryotic host must be compatible with the replicon and control sequences in the expression plasmid.
  • plasmid vectors that contain replication sites and control sequences derived from a species compatible with the host may be used.
  • suitable plasmid vectors may include pBR322, pUCll ⁇ , pUC119 and the like; suitable phage or bacteriophage vectors may include ⁇ gtlO, ⁇ gtll and the like; and suitable virus vectors may include pMAM- neo, pKRC and the like.
  • the selected vector of the present invention has the capacity to replicate in the selected host cell.
  • truncated VRP polypeptides or subunits or a functional derivative thereof in a prokaryotic cell
  • a functional prokaryotic promoter may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible) .
  • constitutive promoters include the int promoter of bacteriophage ⁇ , the bla promoter of the ⁇ -lactamase gene sequence of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene sequence of pPR325, and the like.
  • inducible prokaryotic promoters examples include the major right and left promoters of bacteriophage ⁇ (P L and P R ) , the trp, recA, ⁇ acZ, ⁇ acl, and gal promoters of E. coli, the ⁇ -amylase (Ulmanen et al., J. Bacteriol. 162: 176- 182(1985)) and the ⁇ -28-specific promoters of B.
  • subtilis (Gilman et at., Gene sequence 32:11-20(1984)), the promoters of the bacteriophages of Bacillus (Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)), and Streptomyces promoters (Ward et at., Mol. Gen. Genet. 203:468- 478(1986)).
  • Prokaryotic promoters are reviewed by Glick ( J. Ind. Microbiot. 2:277-282(1987)); Cenatiempo (Biochimie 68:505- 516(1986)); and Gottesman (Ann. Rev. Genet. 18:415-442 (1984)).
  • ribosome binding site upstream of the gene sequence-encoding sequence.
  • ribosome binding sites are disclosed, for example, by Gold et at. (Ann. Rev. Microbiol. 35:365-404(1981)).
  • the ribosome binding site and other sequences required for translation initiation are operably linked to the nucleic acid molecule coding for the truncated VRP by, for example, in frame ligation of synthetic oligonucleotides that contain such control sequences.
  • no signal peptide sequence is required for expression in prokaryotic cells. The selection of control sequences, expression vectors, transformation methods, and the like, are dependent on the type of host cell used to express the gene.
  • Truncated VRP peptides expressed in prokaryotic cells are expected to comprise a mixture of properly truncated VRP peptides with the N-terminal sequence predicted from the sequence of the expression vector, and truncated VRP peptides which have an N-terminal methionine resulting from inefficient cleaving of the initiation methionine during bacterial expression.
  • Both types of truncated VRP peptides are considered to be within the scope of the present invention as the presence of an N-terminal methionine is not expected to affect biological activity. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. However, as defined, mutant progeny have the same functionality as that of the originally transformed cell.
  • Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coil (such as, for example, pBR322, ColEl, pSClOl, pACYC 184, ⁇ VX.
  • plasmids are, for example, disclosed by Sambrook (cf. "Molecular Cloning: A Laboratory Manual", second edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, (1989)).
  • Bacillus plasmids include pC194, pC221, pT127, and the like. Such plasmids are disclosed by Gryczan (In: The Molecular Biology of the Bacilli, Academic Press, NY (1982), pp. 307- 329) .
  • Suitable Streptomyces plasmids include plJlOl (Kendall et al., J. Bacteriol. 1 69 : 4171-4183 (1987)), and streptomyces bacteriophages such as ⁇ C31 (Chater et al., In: Sixth International Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54). Pseudomonas plasmids are reviewed by John et al. (Rev. Infect. Pis. 8:693- 704(1986)), and Izaki (Jpn. J. Bacteriol. 33:729-742(1978)).
  • Eukaryotic host cells which may be used in the expression systems of the present invention are not strictly limited, provided that they are suitable for use in the expression of the truncated VRP peptide.
  • Preferred eukaryotic hosts include, for example, yeast, fungi, insect cells, mammalian cells either in vivo, or in tissue culture.
  • Mammalian cells which may be useful as hosts include HeLa cells, cells of fibroblast origin such as VERO or CHO-Kl, or cells of lymphoid origin and their derivatives .
  • the truncated VRPs of the present invention may also be expressed in human cells such as human embryo kidney 293EBNA cells which express Epstein-Barr virus nuclear antigen 1, as described, for example, in Olofsson, B.
  • the cells are transfected with the expression vectors of Example 2 by using calcium phosphate precipitation, and the cells are then incubated for at least 48 hours.
  • the truncated VRP peptides may then be purified from the supernatant as described in Example 3.
  • plant cells are also available as hosts, and control sequences compatible with plant cells are available, such as the cauliflower mosaic virus 35S and 19S, and nopaline synthase promoter and polyadenylation signal sequences.
  • Another preferred host is an insect cell, for example the Drosophila larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase promoter can be used. Rubin, Science 240:1453-1459(1988) .
  • yeast gene sequence expression systems can be utilized which incorporate promoter and termination elements from the actively expressed gene sequences coding for glycolytic enzymes are produced in large quantities when yeast are grown in mediums rich in glucose.
  • Known glycolytic gene sequences can also provide very efficient transcriptional control signals.
  • Yeast provides substantial advantages in that it can also carry out post-translational peptide modifications.
  • Yeast recognizes leader sequences on cloned mammalian gene sequence products and secretes peptides bearing leader sequences (i.e., pre-peptides) .
  • leader sequences i.e., pre-peptides
  • transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host.
  • the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, cytomegalovirus, simian virus, or the like, where the regulatory signals are associated with a particular gene sequence which has a high level of expression.
  • promoters from mammalian expression products such as actin, collagen, myosin, and the like, may be employed.
  • Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the gene sequences can be modulated.
  • eukaryotic regulatory regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis.
  • Preferred eukaryotic promoters include, for example, the promoter of the mouse metallothionein I gene sequence (Hamer et al . , J. Mol. Appl . Gen.
  • eukaryotic mRNA Translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic promoter and a DNA sequence which encodes a truncated VRP (or a functional derivative thereof) does not contain any intervening codons which are capable of encoding a methionine (i.e., AUG). The presence of such codons results either in a formation of a fusion protein (if the AUG codon is in the same reading frame as the truncated VRP coding sequence) or a frame-shift mutation (if the AUG codon is not in the same reading frame as the truncated VRP coding sequence) .
  • a truncated VRP nucleic acid molecule and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic cell either as a nonreplicating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the gene may occur through the transient expression of the introduced sequence. Alternatively, permanent expression may occur through the integration of the introduced DNA sequence into the host chromosome.
  • a vector may be employed which is capable of integrating the desired gene sequences into the host cell chromosome.
  • Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector.
  • the marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like.
  • the selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of single chain binding protein mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.
  • cDNA expression vectors incorporating such elements include those described by Okayama, Molec. Cell. Biol. 3:280 (1983).
  • the introduced nucleic acid molecule can be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
  • Preferred eukaryotic plasmids include, for example, BPV, vaccinia, SV40, 2-micron circle, and the like, or their derivatives.
  • Such plasmids are well known in the art (Botstein et al., Miami Wntr. Symp. 19:265-274(1982); Broach, In: "The Molecular Biology of the Yeast Saccharomyces : Life Cycle and Inheritance", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470 (1981); Broach, Cell 28:203-204 (1982); Bollon et al., J. Clin. Hematol . Oncol. 10:39-48 (1980); Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression, Academic Press, NY, pp. 563- 608(1980) .
  • the DNA construct (s) may be introduced into an appropriate host cell by any of a variety of suitable means, i.e., transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, lipofection, calcium phosphate precipitation, direct microinjection, DEAE-dextran transfection, and the like.
  • suitable means i.e., transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, lipofection, calcium phosphate precipitation, direct microinjection, DEAE-dextran transfection, and the like.
  • the most effective method for transfection of eukaryotic cell lines with plasmid DNA varies with the given cell type.
  • recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells.
  • Expression of the cloned gene molecule (s) results in the production of truncated VRP or fragments thereof. This can take place in the transformed cells as such, or following the induction of these cells to differentiate (for example, by administration of bromodeoxyuracil to neuroblastoma cells or the like) .
  • a variety of incubation conditions can be used to form the peptide of the present invention. The most preferred conditions are those which mimic physiological conditions.
  • Production of the stable transfectants may be accomplished by, for example, transfection of an appropriate cell line with an eukaryotic expression vector, such as pCEP4, in which the coding sequence for the truncated VRP polypeptide or subunit has been cloned into the multiple cloning site.
  • pCEP4 eukaryotic expression vector
  • These expression vectors contain a promoter region, such as the human cytomegalovirus promoter (CMV) , that drive high-level transcription of desired DNA molecules in a variety of mammalian cells.
  • CMV human cytomegalovirus promoter
  • these vectors contain genes for the selection of cells that stably express the DNA molecule of interest.
  • the selectable marker in the pCEP4 vector encodes an enzyme that confers resistance to hygromycin, a metabolic inhibitor that is added to the culture to kill the nontransfected cells.
  • One object of this invention is to provide truncated VRP in a pharmaceutical composition suitable for therapeutic use.
  • the invention provides a method for stimulating angiogenesis in a patient by administering a therapeutically effective amount of pharmaceutical composition comprising a truncated VRP.
  • terapéuticaally effective amount is meant an amount of a compound which produces the desired therapeutic effect in a patient.
  • a disease or disorder it is the amount which reduces to some extent one or more symptoms of the disease or disorder, and returns to normal, either partially or completely, physiological or biochemical parameters associated or causative of the disease or disorder.
  • therapeutically treat a patient it is an amount expected to be between 0.1 mg/kg to 100 mg/kg, preferably less than 50 mg/kg, more preferably less than 10 mg/kg, more preferably less than 1 mg/kg.
  • the amount of compound depends on the age, size, and disease associated with the patient.
  • the optimal formulation and mode of administration of compounds of the present application to a patient depend on factors known in the art such as the particular disease or disorder, the desired effect, and the type of patient. While the compounds will typically be used to treat human patients, they may also be used to treat similar or identical diseases in other vertebrates such as other primates, farm animals such as swine, cattle and poultry, and sports animals and pets such as horses, dogs and cats.
  • the therapeutically effective amount is provided as a pharmaceutical composition.
  • a pharmacological agent or composition refers to an agent or composition in a form suitable for administration into a multicellular organism such as a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should allow the agent or composition to reach a target cell whether the target cell is present in a multicellular host or in culture. For example, pharmacological agents or compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms which prevent the agent or composition from exerting its effect.
  • compositions can also be formulated as pharmaceutically acceptable salts (e . g. , acid addition salts) and/or complexes thereof.
  • Pharmaceutically acceptable salts are non-toxic salts at the concentration at which they are administered. The preparation of such salts can facilitate the pharmacological use by altering the physical-chemical characteristics of the composition without preventing the composition from exerting its physiological effect. Examples of useful alterations in physical properties include lowering the melting point to facilitate transmucosal administration and increasing the solubility to facilitate the administration of higher concentrations of the drug.
  • Pharmaceutically acceptable salts include acid addition salts such as those containing sulfate, hydrochloride, phosphate, sulfonate, sulfamate, sulfate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclolexylsulfonate, cyclohexylsulfamate and quinate.
  • acid addition salts such as those containing sulfate, hydrochloride, phosphate, sulfonate, sulfamate, sulfate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclolexylsulfonate, cyclohe
  • Pharmaceutically acceptable salts can be obtained from acids such as hydrochloric acid, sulfuric acid, phosphoric acid, sulfonic acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclcohexylsulfonic acid, cyclohexylsulfamic acid, and quinic acid.
  • acids such as hydrochloric acid, sulfuric acid, phosphoric acid, sulfonic acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclcohexylsulfonic acid, cycl
  • Such salts may be prepared by, for example, reacting the free acid or base forms of the product with one or more equivalents of the appropriate base or acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water which is then removed in vacuo or by freeze-drying or by exchanging the ions of an existing salt for another ion on a suitable ion exchange resin.
  • Carriers or excipients can also be used to facilitate administration of the compound. Examples of carriers and excipients include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose, or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents.
  • compositions or pharmaceutical composition can be administered by different routes including intravenously, intraperitoneal, subcutaneous, and intramuscular, orally, topically, or transmucosally .
  • the desired isotonicity may be accomplished using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, polyols (such as mannitol and sorbitol) , or other inorganic or organic solutes.
  • sodium chloride is preferred particularly for buffers containing sodium ions.
  • the compounds of the invention can be formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remington ' s Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton, PA, 1990. See also Wang, Y.J. and Hanson, M.A. "Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers," Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42:2S (1988). A suitable administration format may best be determined by a medical practitioner for each patient individually.
  • injection is preferred, e . g. , intramuscular, intravenous, intraperitoneal, subcutaneous, intrathecal, or intracerebroventricular .
  • the compounds of the invention are formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution.
  • the compounds of the invention are formulated in one or more excipients (e.g., propylene glycol) that are generally accepted as safe as defined by USP standards. They can, for example, be suspended in an inert oil, suitably a vegetable oil such as sesame, peanut, olive oil, or other acceptable carrier.
  • compositions are suspended in an aqueous carrier, for example, in an isotonic buffer solution at a pH of about 5.6 to 7.4.
  • aqueous carrier for example, in an isotonic buffer solution at a pH of about 5.6 to 7.4.
  • the compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH buffering agents.
  • Useful buffers include for example, sodium acetate/acetic acid buffers.
  • a form of repository or "depot" slow release preparation may be used so that therapeutically effective amounts of the preparation are delivered into the bloodstream over many hours or days following transdermal injection or delivery.
  • the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
  • Systemic administration can also be by transmucosal or transdermal means, or the molecules can be administered orally.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, bile salts and fusidic acid derivatives.
  • detergents may be used to facilitate permeation.
  • Transmucosal administration may be, for example, through nasal sprays or using suppositories.
  • the molecules are formulated into conventional oral administration dosage forms such as capsules, tablets, and liquid preparations.
  • the compounds of the invention are formulated into ointments, salves, gels, or creams, as is generally known in the art.
  • solutions of the above compositions may be thickened with a thickening agent such as methyl cellulose.
  • a thickening agent such as methyl cellulose.
  • They may be prepared in emulsified form, either water in oil or oil in water.
  • emulsifying agents including, for example, acacia powder, a non-ionic surfactant (such as a Tween) , or an ionic surfactant (such as alkali polyether alcohol sulfates or sulfonates, e.g., a Triton) .
  • compositions useful in the invention are prepared by mixing the ingredients following generally accepted procedures.
  • the selected components may be simply mixed in a blender or other standard device to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tonicity.
  • the amounts of various compounds of this invention to be administered can be determined by standard procedures.
  • a therapeutically effective amount is between about 1 nmole and 3 ⁇ mole of the molecule, preferably between about 10 nmole and 1 ⁇ mole depending on the age and size of the patient, and the disease or disorder associated with the patient.
  • it is an amount between about 0.1 and 50 mg/kg, preferably 1 and 20 mg/kg of the animal to be treated.
  • compositions will be provided in dosage unit form containing an amount of a truncated VRP, VRP polypeptide, or VRP subunit.
  • VRP truncated VRP or its genetic sequences will also be useful in gene therapy (reviewed in Miller, Nature 357:455-460 (1992)). Miller states that advances have resulted in practical approaches to human gene therapy that have demonstrated positive initial results.
  • the basic science of gene therapy is described in Mulligan, Science 260:926-931 (1993) .
  • One example of gene therapy is presented in Example 7, which describes the use of adenovirus-mediated gene therapy.
  • an expression vector containing the truncated VRP coding sequence may be inserted into cells, the cells are grown in vi tro and then injected or infused in large numbers into patients.
  • a DNA segment containing a promoter of choice (for example a strong promoter) is transferred into cells containing an endogenous truncated VRP in such a manner that the promoter segment enhances expression of the endogenous truncated VRP gene (for example, the promoter segment is transferred to the cell such that it becomes directly linked to the endogenous truncated VRP gene) .
  • the gene therapy may involve the use of an adenovirus vector including a nucleotide sequence coding for a truncated VRP subunit, or a naked nucleic acid molecule coding for a truncated VRP subunit.
  • engineered cells containing a nucleic acid molecule coding for a truncated VRP subunit may be injected.
  • Example 7 illustrates a method of gene therapy using an adenovirus vector to provide angiogenesis therapy.
  • Expression vectors derived from viruses such as retroviruses, vaccinia virus, adenovirus, adeno-associated virus, herpes viruses, several RNA viruses, or bovine papilloma virus, may be used for delivery of nucleotide sequences (e.g. , cDNA) encoding recombinant truncated VRP subunit into the targeted cell population.
  • nucleotide sequences e.g. , cDNA
  • Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors containing coding sequences. See, for example, the techniques described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.
  • nucleic acid molecules encoding protein sequences can be used as naked DNA or in reconstituted system e.g., liposomes or other lipid systems for delivery to target cells (See e.g. , Feigner et al., Nature 337:387-8, 1989).
  • gene transfer can be performed by simply injecting minute amounts of DNA into the nucleus of a cell, through a process of microinjection.
  • Capecchi MR Cell 22:479-88 (1980).
  • genes Once recombinant genes are introduced into a cell, they can be recognized by the cells normal mechanisms for transcription and translation, and a gene product will be expressed.
  • Other methods have also been attempted for introducing DNA into larger numbers of cells. These methods include: transfection, wherein DNA is precipitated with calcium phosphate and taken into cells by pinocytosis (Chen C. and Okayama H, Mol. Cell Biol. 7:2745-52 (1987)); electroporation, wherein cells are exposed to large voltage pulses to introduce holes into the membrane (Chu G.
  • adenovirus proteins are capable of destabilizing endosomes and enhancing the uptake of DNA into cells.
  • the admixture of adenovirus to solutions containing DNA complexes, or the binding of DNA to polylysine covalently attached to adenovirus using protein crosslinking agents substantially improves the uptake and expression of the recombinant gene.
  • adeno-associated virus vectors may be used for gene delivery into vascular cells (Gnatenko, D., J. of Invest. Med. 45:87-97, (1997)).
  • gene transfer means the process of introducing a foreign nucleic acid molecule into a cell. Gene transfer is commonly performed to enable the expression of a particular product encoded by the gene.
  • the product may include a protein, polypeptide, anti-sense DNA or RNA, or enzymatically active RNA. Gene transfer can be performed in cultured cells or by direct administration into animals.
  • gene transfer involves the process of nucleic acid molecule contact with a target cell by non-specific or receptor mediated interactions, uptake of nucleic acid molecule into the cell through the membrane or by endocytosis, and release of nucleic acid molecule into the cytoplasm from the plasma membrane or endosome.
  • Expression may require, in addition, movement of the nucleic acid molecule into the nucleus of the cell and binding to appropriate nuclear factors for transcription.
  • gene therapy is a form of gene transfer and is included within the definition of gene transfer as used herein and specifically refers to gene transfer to express a therapeutic product from a cell in vivo or in vi tro .
  • Gene transfer can be performed ex vivo on cells which are then transplanted into a patient, or can be performed by direct administration of the nucleic acid molecule or nucleic acid- protein complex into the patient.
  • a vector having nucleic acid molecule sequences encoding a truncated VRP is provided in which the nucleic acid molecule sequence is expressed only in a specific tissue. Methods of achieving tissue-specific gene expression as set forth in International Publication No. WO 93/09236, filed November 3, 1992 and published May 13, 1993.
  • Gene replacement means supplying a nucleic acid molecule sequence which is capable of being expressed in vivo in an animal and thereby providing or augmenting the function of an endogenous gene which is missing or defective in the animal.
  • nucleic acid sequence contained in the vector may include additions, deletions or modifications to some or all of the sequence of the nucleic acid, as defined above.
  • Example 1 To assist in understanding the present invention, the following Examples are included which describes the results of a series of experiments. The experiments relating to this invention should not, of course, be construed as specifically limiting the invention and such variations of the invention, now known or later developed, which would be within the purview of one skilled in the art are considered to fall within the scope of the invention as described herein and hereinafter claimed.
  • Example 1 To assist in understanding the present invention, the following Examples are included which describes the results of a series of experiments. The experiments relating to this invention should not, of course, be construed as specifically limiting the invention and such variations of the invention, now known or later developed, which would be within the purview of one skilled in the art are considered to fall within the scope of the invention as described herein and hereinafter claimed.
  • Example 1 Example 1
  • a cDNA construct is created in the following manner:
  • a DNA encoding human VEGF-B is amplified from a human heart or skeletal muscle cDNA),or a human fetal brain cDNA library, or a cDNA preparation from another suitable human tissue source by PCR with oligonucleotides corresponding to the published sequence of human VEGF-B.
  • a DNA fragment then is generated that encodes at its 5' end the signal sequence of human VEGF-B, followed by a codon for proline, the first amino acid residue in mature VEGF-B, and then followed by codons corresponding to amino acids from residues 22 to the C-terminus of human VEGF-B, followed by a stop codon.
  • Appropriate additional non-coding nucleotide sequences are added to the 5' and 3' ends of this DNA construct so as to allow insertion of the DNA into an appropriate expression vector.
  • the cleavage site for the signal peptide is preserved in a manner identical to that found in native VEGF- B.
  • this strategy results in a change in the new N- terminal amino acid of the truncated VEGF-B.
  • the normal N-terminal amino acid residue in des (1-20) -VEGF-B is a tyrosine residue: mspllrrlllvallqlartqa[PVSQFDGPSHQKKVVPWIDV] TRAT
  • the new N-terminal amino acid is proline
  • the resulting truncated VEGF-B is equivalent to des (2-21) -VEGF-B) : mspllrrlllvallqlartqaPTRAT...
  • the change from the native amino acid of the truncated VEGF-B (tyrosine in the case of a a)20-residue truncation) is not expected to have any effect on the biological activity of the truncated VEGF-B.
  • the advantage of this strategy is that the signal peptide sequence is maintained thus ensuring efficient cleavage of the signal peptide from the precursor during protein processing/secretion.
  • truncated VEGF-B des (1-15) -VEGF-B, is constructed by deleting the first 15 amino acids.
  • the signal peptide cleavage site would be preserved in this case because residue#16 and residue#l (the new and old N-termini) are identical (proline) : mspllrrillvallqlartqa[PVSQFDGPSHQKKVV]PWIDVYTRAT...
  • signal peptides may be used in the present invention.
  • the signal peptide of VEGF-B or VEGF-C could be used which would require that the first amino acid of the truncated protein be an alanine or glycine, respectively, in order to preserve the respective signal peptide cleavage sites.
  • a further alternative would be to use signal peptide sequences from other known proteins; some of these may have cleavage sites compatible with the N-terminal tyrosine of the truncated des (1-20) -VEGF-B.
  • Another alternative would be to generate a construct that encodes a precursor protein with a cleavage site that incorporates two, rather than one, amino acids from the N- terminus of the original VEGF-B protein sequence.
  • the purpose of this strategy would be to ensure more fully that the cleavage site is compatible with signal peptidase function. This would introduce two new amino acids at the N-terminus of the truncated VEGF-B sequence but such a change would not be expected to alter biological function of the truncated peptide.
  • the strategy described to generate DNA for expression of des (1-20) -VEGF-B is useful for generation in an analogous manner of VEGF-B mutants with N-terminal truncations of other desired lengths. Further, the strategy is useful to generate N-terminal truncations of other desired lengths in other VEGF- related forms and their isoforms of other species.
  • Example 2 Expression Of N-Terminally Truncated VEGF-B Subunits
  • the DNA fragment encoding truncated VEGF-B from Example 1 may be cloned into a suitable plasmid vector.
  • Sf9 (Sporoptera frugiperda) cells are co-transfected with baculovirus transfer vector pAcUW51 containing cDNA encoding truncated VEGF-B and baculovirus (Baculogold, Pharmingen, San Diego, CA) .
  • Selection and plaque purification of recombinant virus are performed according to established protocols using Blue agar overlays (Gibco BRL) . High stock of recombinant virus is produced in exponentially growing Sf9 cells using a multiplicity of infection of 0.05.
  • VEGF-B For expression of truncated VEGF-B, Sf9 cells (1x106 cells/ml) growing in serum free medium are infected with recombinant virus at a multiplicity of 10. Supernatant is collected after 72 hours post infection.
  • VEGF expression in baculovirus-infected insect cells, which can be used to express the truncated VRPs of the present invention is also described in Fiebich et al., (Eur. J. Biochem. 211: 19-26, 1993) . In this system, VEGF has been shown to be produced in high yield, with efficient glycosylation similar to that seen in mammalian cells.
  • VEGF isoforms have been expressed in E. Coli (Wilting et al., Dev. Biol. 176, 76-85, 1996) from inclusion body by refolding according to the procedure described previously for homo- and heterodimers of PDGF (Schneppe et al., Gene 143, 201-09, 1994) and in yeast (Mohanraj et al., Biochem. Biophys. Res. Commun. 215:750-56, 1995) .
  • Example 3 Purification Of Recombinant Truncated VRPS
  • a number of standard techniques can be used. These techniques include, but are not limited to ammonium sulfate precipitation, acetone precipitation, ion-exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, reverse-phase HPLC, concanavalin A affinity chromatography, isoelectric focusing, and chromatofocusing.
  • Other standard protein purification techniques are readily obvious to one skilled in the art.
  • proteins with specific tags such as histidine tags, antigen tags, etc.
  • proteins with specific tags could be produced by engineering DNA encoding such tags into the VEGF-B DNA such that proteins containing said tags in a manner compatible with the protein's biological activity would be expressed and purified by affinity chromatography directed at the tag.
  • affinity chromatography directed at the tag.
  • a preferred purification method for truncated forms of VEGF-B is described in the following: Sf9 Cell supernatant is centrifuged at 10000 rpm for 30 minutes to remove cell debris and viral particles. Supernatant is then concentrated and dialyzed against 20 mM Tris (pH 8.3) for 24 hours. The dialyzed supernatant is centrifuged again to remove insoluble material and loaded onto a Sepharose Q anion exchange column. Protein is eluted from the column by gradient elution using a gradient of NaCl (0 - 1 M NaCl) . Chromatography fractions are analyzed by SDS polyacrylamide gel electrophoresis and by ELISA using an antibody that recognizes VEGF-B.
  • Fractions with VEGF- B immunoreactivity are pooled, concentrated, and dialyzed overnight against 0.1% trifluoroacetic acid. Material so prepared is further purified by reverse phase HPLC. Typically approximately 2-5 mg of protein is loaded on a semipreparative C4 column and eluted with a gradient of acetonitrile in 0.1% trifluoracetic acid as described in Esch et al., Meth. Enzymol. 103, 72-89, 1983. Fractions containing truncated VEGF-B are pooled and stored at -80 degrees Celsius until further use.
  • a preferred method of purification of the basic and heparin-binding N-terminally truncated forms of VEGF-related protein subunits and analogs thereof includes the combined use of heparin-Sepharose affinity chromatography and cation- exchange chromatography, optionally followed by reverse-phase HPLC, essentially as described in Connolly et al., J. Biol. Chem. 264:20017-24, 1989, Gospodarowicz et al., (Proc. Natl. Acad. Sci. USA, 86:7311-15, 1989), or Plouet et al . , (Embo J. 8:3801-06, 1989).
  • VRP- like material Purification is monitored by following the elution of VRP- like material using a number of techniques including radioreceptor assay using 125 I-labeled VRP and receptor preparations consisting of cells or cell membrane preparations in functional assays as described in Examples 4-6.
  • the truncated VRPs expressed in other eukaryotic cell systems such as yeast or mammalian cells, may be purified in the same manner.
  • Truncated VRPs expressed in prokaryotic cells will likely need to undergo a re-folding step for proper dimerization of subunits, as described in, for example, Schneppe et al., (Gene 143:201-09, 1994).
  • VRP binding can be assessed in various ways.
  • Useful methods include the determination of the ability of VRP analogs to bind to endothelial cells or to cells artificially transfected with KDR, or to soluble forms of the KDR receptor (for example, a KDR/alkaline phosphatase fusion protein (Gitay-Goren et al., J. Biol. Chem. 271:5519-23 (1996)).
  • KDR/alkaline phosphatase fusion protein for example, a KDR/alkaline phosphatase fusion protein (Gitay-Goren et al., J. Biol. Chem. 271:5519-23 (1996)).
  • KDR/alkaline phosphatase fusion protein for example, a KDR/alkaline phosphatase fusion protein (Gitay-Goren et al., J. Biol. Chem. 271:5519-23 (1996).
  • a preferred procedure has been described by Ter
  • KDR cDNA is transfected into CMT-3 monkey kidney cells by the DEAE-dextran method by incubating plated cells with DMEM containing 1 ⁇ g/ml DNA, 0.5 ⁇ g/ml DEAE dextran, and 100 ⁇ M chloroquine. Following incubation for 4 hours at 37 degrees Celsius, the medium is aspirated and cells are exposed to 10% DMSO in PBS for one minute. The cells are then washed once with DMEM containing 10% calf serum and then incubated for 40 hours at 37 degrees Celsius in DMEM/10% calf serum containing 100 ⁇ M ZnCl 2 and 1 ⁇ M CdCl 2 .
  • VEGF-B is radioiodinated using either the Iodogen method or the chloramine T method. Radiolabelled VEGF-B is separated from excess free iodine-125 using gel filtration on a Sephadez G25 column or a heparin-Sepharose column. Specific activity of radiolabelled 125 I-VEGF-B analog should typically be in the order of 10 5 cpm/ng.
  • CMT-3 (10 5 cells/well) are plated in 12-well plates. Twenty four hours later, cells are washed twice with PBS, and 0.5 ml of DMEM containing 0.15% gelatin and 25 mM HEPES, pH 7.4 is added.
  • 125 I-VEGF-B at concentrations ranging from 1-500 pM, is then added. Binding experiments are done in the presence or absence of 0.5 nM unlabeled VEGF-B for the determination of specific binding. After a 90-minute incubation at room temperature, a 50 ⁇ l sample of the media from each well is used to determine the concentration of free radioligand, and the wells are washed 3 times with ice cold PBS containing 0.1% BSA. Cells are extracted from the wells by incubation for 30 minutes with 1% Triton X100 in 100 mM sodium phosphate, pH 8.0, and the radioactivity of the extract is determined in a gamma counter.
  • the mitogenic activity of truncated VRPs on endothelial cells of human or mammalian origin can be determined by a number of different procedures, including assays where cell proliferation is measured by growth of cell numbers or by incorporation of radioactive DNA precursors (thymidine incorporation) or otherwise appropriately labeled DNA precursors (bromo-deoxyuridine incorporation) .
  • thymidine incorporation thymidine incorporation
  • bromo-deoxyuridine incorporation bromo-deoxyuridine incorporation
  • Bovine aortic arch endothelial cells maintained in stock cultures in the presence of Dulbecco' s modified Eagle's medium supplemented with 10% calf serum and antibiotics (gentamycin at 50 ⁇ g/ml and fungizine at 0.25 ⁇ g/ml) and basic fibroblast growth factor (1- 10 ng/ml, added every 48 h) are passaged weekly at a split ratio of 1:64.
  • Dulbecco' s modified Eagle's medium supplemented with 10% calf serum and antibiotics (gentamycin at 50 ⁇ g/ml and fungizine at 0.25 ⁇ g/ml) and basic fibroblast growth factor (1- 10 ng/ml, added every 48 h) are passaged weekly at a split ratio of 1:64.
  • cell monolayers from stock plates are dissociated using trypsin. Cells are then seeded at a density of approximately 8000 cells/well in 24-well plates in the presence of DMEM and antibiotics as described
  • Samples to be assayed (1-10 ⁇ l) , appropriately diluted in DMEM/0.1% bovine serum albumin), are added six hours after plating of cells and again after 48 hours. After 4 days of culture, endothelial cells are detached from plates with trypsin and counted using a Coulter particle counter.
  • Fresh cell culture conditioned medium containing [ 3 H] thymidine (Amersham; 10 ⁇ Ci/ml) is added to the cells and stimulation is continued for another 48 hours. Cells are washed with PBS and trypsinized and the incorporated radioactivity is determined by liquid scintillation counting. The activity of truncated VRP is compared to the activity of non-truncated VRP.
  • bovine capillary endothelial (BCE) cells are seeded into 24-well plates and grown until confluence in minimal essential medium (MEM) supplemented with 10% fetal calf serum. Cells are starved in MEM supplemented with 3% fetal calf serum for 72 hours, after which conditioned medium diluted into serum-free medium is added to the cells and the cells are stimulated for 24 hours.
  • MEM minimal essential medium
  • thymidine is included during the last 4 hours of the stimulation (1 ⁇ Ci/ml) .
  • Cells are washed with PBS and lysed with NaOH, and incorporated radioactivity is determined by liquid scintillation counting.
  • the activity of truncated VRP is compared to that of non-truncated VRP.
  • Bovine fibroblast growth factor (b-FGF) may be used as an additional control for mitogenic activity, and may also be used to measure its potentiating activity of truncated VRP activity.
  • the angiogenic activity of substances can be determined using a variety of in vivo methods. Commonly used methods include the chick chorioallantoic membrane assay, the corneal pouch assay in rabbits, rats, or mice, the matrigel implant assay in mice, the rabbit ear chamber angiogenesis assay, the hamster cheek pouch assay, the Hunt-Schilling chamber model and the rat sponge implant model. Other assay methods to assess the formation of new blood vessels have been described in the literature and are considered to be within the scope of this invention.
  • a preferred method for demonstrating the angiogenic activity of truncated VRPs is the rabbit corneal pouch assay.
  • Elvax (ethylene vinyl acetate) polymer pellets containing approximately 1-1000 ng of the growth factor and a constant amount of rabbit serum albumin as carrier is implanted into a surgical incision in the cornea as described in more detail in Phillips and Knighton, Wound Rep. Reg. 3, 533-539, 1995; Gimbrone et al., J. Natl. Cane. Inst. 52:413-27, 1974; Risau, Proc. Natl. Acad. Sci. USA 83:3855-59, 1986).
  • Growth factor-induced vascularization of the cornea is then observed over a period of 2 weeks.
  • Semiquantitative analysis is possible with morphometric and image analysis techniques using photographs of corneas.
  • Example 7 Gene-Transfer-Mediated Angiogenesis Therapy Using Truncated VRPS
  • Truncated VRPs are used for gene-transfer-mediated angiogenesis therapy as described, for example, in PCT/US96/02631, published September 6, 1996 as W096/26742, hereby incorporated by reference herein in its entirety.
  • a helper independent replication deficient human adenovirus 5 system may be used for gene-transfer.
  • a nucleic acid molecule coding for a truncated VRP subunit may be cloned into the polylinker of plasmid ACCMVPLPA which contains the CMV promoter and SV40 polyadenylation signal flanked by partial adenoviral sequences from which the E1A and E1B genes (essential for viral replication) have been deleted.
  • This plasmid is co-transferred (lipofection) into 293 cells with plasmid JM17 which contains the entire human adenoviral 5 genome with an additional 4.3 kb insert making pJM17 too large to be encapsidated.
  • Homologous rescue recombination results in adenoviral vectors containing the transgene in the absence of E1A/E1B sequences. Although these recombinants are nonreplicative in mammalian cells, they can propagate in 293 cells which have been transformed with E1A/E1B and provided these essential gene products in trans. Transfected cells are monitored for evidence of cytopathic effect which usually occurs 10-14 days after transfection.
  • cell supernatant from plates showing a cytopathic effect is treated with proteinase K (50 mg/ml with 0.5% sodium dodecyl sulfate and 20 mM EDTA) at 56°C for 60 minutes, phenol/ chloroform extracted and ethanol precipitated.
  • proteinase K 50 mg/ml with 0.5% sodium dodecyl sulfate and 20 mM EDTA
  • the truncated VRP genes, driven by the CMV promoter and with the SV40 polyadenylation sequences are well within the packaging constraints.
  • Recombinant vectors are plaque purified by standard procedures.
  • the resulting viral vectors are propagated on 293 cells to titers in the 10 10 -10 12 viral particles range. Cells are infected at 80% confluence and harvested at 36-48 hours.
  • the cellular debris is pelleted by standard centrifugation and the virus further purified by double CsCl gradient ultracentrifugation (discontinuous 1.33/1.45 CsCl gradient; cesium prepared in 5 mM Tris, 1 mM EDTA (pH 7.8); 90,000 x g (2 hr) , 105,000 x g (18 hr) ) .
  • the viral stocks Prior to in vivo injection, the viral stocks are desalted by gel filtration through Sepharose columns such as G25 Sephadex. The resulting viral stock has a final viral titer approximately in the 10 10 -10 12 viral particles range.
  • the adenoviral construct should thus be highly purified, with no wild-type (potentially replicative) virus.
  • a left thoracotomy is performed on domestic pigs (30-40 kg) under sterile conditions for instrumentation.
  • Catheters are placed in the left atrium and aorta, providing a means to measure regional blood flow, and to monitor pressures. Wires are sutured on the left atrium to permit ECG recording and atrial pacing.
  • an ameroid constrictor (ameroid) , a metal ring including an ameroid substance, is placed around the proximal left circumflex coronary artery (LCx) (Hammond et al. J.
  • the treatment group receives an adenoviral construct that includes a truncated VRP gene driven by a CMV promoter.
  • Control animals receive gene transfer with an adenoviral construct that includes a reporter gene, lacZ, driven by a CMV promoter.
  • Saline is delivered through the brachycephalic artery cannula (120 mmHg pressure), thereby perfusing the coronary arteries.
  • Glutaraldehyde solution (6.25%, 0.1 M cacodylate buffer) was perfused (120 mmH pressure) until the heart is well fixed (10- 15 min) .
  • the heart is then removed, the beds identified using color-coded dyes injected anterograde through the left anterior descending (LAD) , left circumflex (LCx) , and right coronary arteries.
  • LAD left anterior descending
  • LCx left circumflex
  • the ameroid is examined to confirm closure. Samples taken from the normally perfused and ischemic regions are divided into thirds and the endocardial and epicardial thirds are plastic-imbedded.
  • PCR and RT-PCR may be used to detect transgenic truncated VRP DNA and mRNA in myocardium from animals that have received truncated VRP gene transfer.
  • a sense primer to the CMV promoter may be used to detect transgenic truncated VRP DNA and mRNA in myocardium from animals that have received truncated VRP gene transfer.
  • PCR is used to amplify the expected 500 bp fragment.
  • RT-PCR is used to amplify the expected 400 bp fragment.
  • truncated VRP expression may be demonstrated 48 hours as well as 14 ⁇ 1 days after gene transfer in cells and myocardium from animals that have received gene transfer with a truncated VRP gene.
  • the helper independent replication deficient human adenovirus 5 system is used to prepare transgene containing vectors.
  • the material injected in vivo should be highly purified and contain no wild-type (replication competent) adenovirus. Thus adenoviral infection and inflammatory infiltration in the heart are minimized.
  • Injection of the construct (4.0 ml containing about 10 11 viral particles of adenovirus) is performed by injecting 2.0 ml into both the left and right coronary arteries (collateral flow to the LCx bed appeared to come from both vessels) .
  • Animals are anesthetized, and arterial access acquired via the right carotid by cut-down; a 5F Cordis sheath is then placed.
  • a 5F Multipurpose (A2) coronary catheter is used to engage the coronary arteries. Closure of the LCx ameroid is confirmed by contrast injection into the left main coronary artery.
  • the catheter tip is then placed 1 cm within the arterial lumen so that minimal material is lost to the proximal aorta during injection. This procedure is carried out for each of the pigs.
  • Some constructs may include a reporter gene (lacZ) ; (2) myocardium from the relevant beds is sampled, and immunoblotting is performed to quantitate the presence of truncated VRP and (3) PCR is used to detect truncated VRP mRNA and DNA.
  • lacZ reporter gene
  • the regional contractile function data obtained should show that control pigs show a similar degree of pacing-induced dysfunction in the ischemic region before and 14 + 1 days after gene transfer.
  • pigs receiving truncated gene transfer should show an increase in wall thickening in the ischemic region during pacing, demonstrating that truncated VRP subunit gene transfer in accordance with the invention is associated with improved contraction in the ischemic region during pacing.
  • Wall thickening in the normally perfused region should be normal during pacing and unaffected by gene transfer.
  • the percent decrease in function measured by transthoracic echocardiography should be very similar to the percentage decrease measured by sonomicrometry during atrial pacing in the same model (Hammond, et al. J. Clin. Invest. 92:2644, 1993), documenting the accuracy of echocardiography for the evaluation of ischemic dysfunction.
  • Lys Glu Ser Ala Val Lys Pro Asp Ser Pro Arg lie Leu Cys Pro Pro 130 135 140
  • MOLECULE TYPE Protein
  • Tyr Pro Asp Glu lie Glu Tyr lie Phe Lys Pro Ser Cys Val Pro Leu 65 70 75 80 Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro
  • Gin Ser Gin His lie Gly Glu Met Ser Phe Leu Gin His Ser Lys Cys 115 120 125
  • MOLECULE TYPE Protein
  • MOLECULE TYPE Protein

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EP98919794A 1997-04-25 1998-04-20 Truncated vegf-related proteins Withdrawn EP0977854A2 (en)

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Families Citing this family (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6040157A (en) 1994-03-08 2000-03-21 Human Genome Sciences, Inc. Vascular endothelial growth factor 2
US7153827B1 (en) 1994-03-08 2006-12-26 Human Genome Sciences, Inc. Vascular endothelial growth factor 2 and methods of use
US7109308B1 (en) 1994-03-08 2006-09-19 Human Genome Sciences, Inc. Antibodies to human vascular endothelial growth factor 2
ES2249762T3 (es) 1994-03-08 2006-04-01 Human Genome Sciences, Inc. Factor de crecimiento del endotelio vascular 2.
US7186688B1 (en) 1994-03-08 2007-03-06 Human Genome Sciences, Inc. Methods of stimulating angiogenesis in a patient by administering vascular endothelial growth factor 2
US6734285B2 (en) 1994-03-08 2004-05-11 Human Genome Sciences, Inc. Vascular endothelial growth factor 2 proteins and compositions
US6608182B1 (en) 1994-03-08 2003-08-19 Human Genome Sciences, Inc. Human vascular endothelial growth factor 2
US5932540A (en) 1994-03-08 1999-08-03 Human Genome Sciences, Inc. Vascular endothelial growth factor 2
US7727761B2 (en) 1995-08-01 2010-06-01 Vegenics Limited Vascular endothelial growth factor C (VEGF-C) protein and gene, mutants thereof, and uses thereof
PT848755E (pt) 1995-09-08 2003-12-31 Genentech Inc Proteina relacionada com o vegf
US7125714B2 (en) 1997-02-05 2006-10-24 Licentia Ltd. Progenitor cell materials and methods
CA2322748A1 (en) * 1998-03-13 1999-09-16 Human Genome Sciences, Inc. Vascular endothelial growth factor 2
WO1999049882A2 (en) * 1998-03-27 1999-10-07 Eicher Dorothea J Vegf and vegf-c as infant formula supplements
DE19813774A1 (de) * 1998-03-27 1999-09-30 Max Planck Gesellschaft Parapockenvirus-kodierter vaskulärer Endothelzell Wachstumsfaktor (PPV-VEGF)
AU760008B2 (en) 1998-09-08 2003-05-08 Agouron Pharmaceuticals, Inc. Modifications of the VEGF receptor-2 protein and methods of use
US6958147B1 (en) 1998-10-26 2005-10-25 Licentia Ltd Use of VEGF-C to prevent restenosis
NZ510121A (en) * 1998-10-26 2003-02-28 Licentia Ltd Use of VEGF-C or VEGF-D gene or protein to prevent restenosis of blood vessels
EP1568375A1 (en) * 1998-10-26 2005-08-31 Ludwig Institute For Cancer Research Use of VEGF-C or VEGF-D gene or protein to prevent restenosis
ES2316194T3 (es) 1998-10-28 2009-04-01 Cornell Research Foundation, Inc. Metodos para la regulacion de la angiogenesis y la integridad vascular utilizando los ligandos bdnf, nt-3 y nt-4.
US6783953B1 (en) 1998-12-22 2004-08-31 Janssen Pharmaceutica N.V. Vascular endothelial growth factor-X
US7223724B1 (en) 1999-02-08 2007-05-29 Human Genome Sciences, Inc. Use of vascular endothelial growth factor to treat photoreceptor cells
US6492169B1 (en) 1999-05-18 2002-12-10 Crucell Holland, B.V. Complementing cell lines
WO2001057181A2 (en) * 2000-02-04 2001-08-09 Carmeliet, Peter Use of vascular endothelial growth factor, placenta growth factor or both for preventing or treating ischemic diseases or stroke
US7727971B2 (en) 2000-02-04 2010-06-01 Life Sciences Research Partners Vzw Use of placental growth factor for treating ischemic muscle disease
CA2405291A1 (en) 2000-04-06 2001-10-18 Wayne P. Franco Methods of using growth factors for treating heart disease
EP1157999A1 (en) * 2000-05-24 2001-11-28 Introgene B.V. Methods and means for enhancing skin transplantation using gene delivery vehicles having tropism for primary fibroblasts, as well as other uses thereof
NZ518077A (en) 2000-08-04 2003-11-28 Human Genome Sciences Inc Biologically active fragments, analogues and derivatives of VEGF-2 for the treatment of peripheral artery diseases such as critical limb ischemia and coronary disease
WO2002014367A1 (fr) 2000-08-10 2002-02-21 Center For Advanced Science And Technology Incubation,Ltd. Facteur de croissance chimere de cellules endotheliales vasculaires de type humain
EP1191104A1 (en) * 2000-09-26 2002-03-27 Introgene B.V. Gene delivery vehicles and use thereof in the preparation of a medicament and/or vaccine
US7067317B2 (en) 2000-12-07 2006-06-27 Sangamo Biosciences, Inc. Regulation of angiogenesis with zinc finger proteins
CA2431308C (en) 2000-12-07 2010-04-13 Sangamo Biosciences, Inc. Regulation of angiogenesis with zinc finger proteins
US7402312B2 (en) 2001-04-13 2008-07-22 Human Genome Sciences, Inc. Antibodies to vascular endothelial growth factor 2 (VEGF-2)
EP2228389B1 (en) 2001-04-13 2015-07-08 Human Genome Sciences, Inc. Antibodies against vascular endothelial growth factor 2
US7709450B2 (en) 2001-06-20 2010-05-04 Ludwig Institute For Cancer Research Stimulation of vascularization with VEGF-B-186
ATE345799T1 (de) 2001-10-15 2006-12-15 Janssen Pharmaceutica Nv Substituierte 4-phenyl-4-(1h-imidazol-2-yl)- piperidinderivate zur verringerung von ischämischer schädigung
EP1827494A4 (en) * 2004-11-18 2010-02-17 Wayne Franco COMBINATION GROWTH THERAPY AND CELL THERAPY FOR THE TREATMENT OF ACUTE AND CHRONIC ORGANIC DISEASES
WO2010078624A1 (en) * 2009-01-07 2010-07-15 Vegenics Limited Materials and methods for the treatment of hypertension

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT1242149B (it) * 1990-09-27 1994-02-16 Consiglio Nazionale Ricerche Sequenza di nucleotidi codificante per una proteina umana con proprieta' regolative dell'angiogenesi
ATE212665T1 (de) * 1992-11-18 2002-02-15 Arch Dev Corp Adenovirus gelenkter gen-transfer zum herz- und glatten vaskularen muskel
US6645933B1 (en) * 1995-08-01 2003-11-11 Helsinki University Licensing Ltd. Oy Receptor ligand VEGF-C
CN1136920C (zh) * 1995-02-28 2004-02-04 加利福尼亚大学董事会 基因转移介导的血管形成疗法
US5928939A (en) * 1995-03-01 1999-07-27 Ludwig Institute For Cancer Research Vascular endothelial growth factor-b and dna coding therefor
HU223438B1 (hu) * 1995-03-02 2004-07-28 Amrad Operations Pty. Ltd. Új növekedési faktor és ezt kódoló génszekvencia
CA2224002A1 (en) * 1995-06-06 1996-12-12 Human Genome Sciences, Inc. Human vascular endothelial growth factor 3

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9849300A2 *

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CA2287538A1 (en) 1998-11-05
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EA199900861A1 (ru) 2000-06-26

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