WO2004098534A2 - Techniques d'apport de modulateurs angiogeniques au moyen de poxvirus - Google Patents

Techniques d'apport de modulateurs angiogeniques au moyen de poxvirus Download PDF

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WO2004098534A2
WO2004098534A2 PCT/US2004/005376 US2004005376W WO2004098534A2 WO 2004098534 A2 WO2004098534 A2 WO 2004098534A2 US 2004005376 W US2004005376 W US 2004005376W WO 2004098534 A2 WO2004098534 A2 WO 2004098534A2
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vector
angiogenesis
virus
pox
gene
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WO2004098534A3 (fr
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Dennis L. Panicali
Gail P. Mazzara
Linda R. Gritz
Patricia Greenhalgh
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Therion Biologics Corporation
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    • 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
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to methods of delivery of angiogenic modulators to an individual, constructs comprising a recombinant pox virus comprising at least one nucleic acid sequence encoding an angiogenic modulating agent and methods of treating disorders associated with abnormal angiogenesis using recombinant poxviruses. More particularly, the invention relates to a custom- designed recombinant poxvirus vectors carrying nucleic acid sequences encoding one or more angiogenesis modulating agents, the agents being selected to address a particular angiogenesis abnormality according to the need of an individual.
  • Angiogenesis is the growth of new blood vessels from existing endothelium.
  • growth factors e.g. VEGF and Angiopoietins
  • ECM surface protein-extra cellular matrix
  • TIMP tissue inhibitor metalloproteinases
  • Mitogenic endothelial cells sprout into the matrix and migrate with controlled matrix degradation as the tip. Proliferation occurs proximal to migration with formation of a primitive tube. Extensive remodeling ensues until the new capillary matures and fuses or joins with other sprouts (Risau, Nature, 386:671-674, 1997; Risau and Flamme, Ann. Rev. Cell Dev. Biol. 11:73-91, 1995). [003] In mammals, normal angiogenesis is confined to the reproductive system, embryogenesis, development, and repair after injury.
  • Undesirable or pathological neovascularization has been associated with disease states including diabetic retinopathy, psoriasis, cancer, rheumatoid arthritis, atheroma, Kaposi's sarcoma and haemangioma (Fan et al, Trends Pharmacol. Sci. 16: 57-66 (1995); Folkman, Nature Medicine 1 : 27-31 (1995)).
  • Alteration of vascular permeability is thought to play a role in both normal and pathological physiological processes (Cullinan-Bove et al, Endocrinology 133: 829-837 (1993); Senger et al, Cancer and Metastasis Reviews. 12: 303-324 (1993)).
  • Intraocular neovascularization is usually associated with diabetic retinopathy and retinopathy of prematurity (King and Brownlee, 1996).
  • the new blood vessels are leaky and rupture easily, which may result in blindness.
  • chronic inflammatory diseases such as rheumatoid arthritis, new vessels invade the joint surfaces and degrade the cartilage by proteolysis (Battegay, 1995).
  • angiogenesis is essential to the progression of cancer because it is a prerequisite for tumor growth and metastasis (Folkman, 1992). Without vasculanzation, tumors may remain for years as small (less than a few millimeters) asymptomatic lesions. (Weidner et al. New England J. of Med. 324:1-8 (1991)). Tumors which become vascularized receive increased oxygen and nutrients through perfusion. Thus, tumors which are vascularized can grow and proliferate. A tumor must constantly stimulate the growth of new capillary blood vessels in order for it to continue to grow. Additionally, angiogenesis allows the tumor cells access to the host animal's circulatory system.
  • the new blood vessels provide a gateway for tumor cells to enter the circulation and metastasize to distant sites.
  • gene therapy has been used to describe a wide variety of methods using recombinant biotechnology techniques to deliver a variety of different materials to a cell. Such methods include, for example, the delivery of a gene, antisense RNA, a cytotoxic agent, etc., by a vector to a mammalian cell, preferably a human cell either in vivo or ex vivo. Most of the initial work in gene therapeutics has focused on the use of retroviral vectors to transform these cells.
  • the retroviral vector is typically a modified Moloney Murine Leukemia Virus (MMLV), which has had its packaging sequences deleted to prevent packaging of the entire retroviral genome.
  • MMLV Moloney Murine Leukemia Virus
  • AAV adenoassociated virus
  • Retroviruses typically enter cells through cell surface receptors and if such receptors are not present on the cell, or not present in sufficient numbers, then infection may not be possible or may be inefficient. These viruses are also relatively labile in comparison to other viruses. Outbreaks of wild-type virus from recombinant virus- producing cell lines have also been reported with the vector itself causing a disease. Moreover, these viruses only express in dividing cells.
  • viruses have been proposed as vectors such as herpes virus.
  • non-viral vectors such as ligand-DNA-conjugates have been proposed. Nevertheless, these vectors all pose certain problems. For example, the vector itself must not become a potential source for infection to the individual treated. However, as already mentioned, outbreaks of wild-type retroviruses have been reported in some cell lines. Similarly, the use of herpes virus as a vector has been found to result in persistence of the virus. Furthermore, many of these vectors can contain and express only a relatively small amount of genetic material. This is undesirable for numerous situations in which the ability to express multiple products is preferred.
  • Poxviruses have been used for many years as vectors, particularly with respect to providing a foreign antigen or self-antigen to generate an immune response in a host.
  • the advantages of the poxvirus vectors include: (i) ease of generation and production; (ii) the large size of the genome permitting insertion of multiple genes, (iii) efficient delivery of genes to multiple cell types, including antigen-presenting cells; (iv) high levels of protein expression; (v) optimal presentation of antigens to the immune system; and (vi) the ability to elicit cell- mediated immune responses as well as antibody responses; and (vii) the long-term experience gained with using this vector in humans as a small pox vaccine.
  • MVA was derived from the Ankara vaccinia strain CVA, which was used in the 1950s as a smallpox vaccine.
  • attenuation experiments were initiated in the laboratory of Dr. Anton Mayr (University of Kunststoff) comprising terminal dilution of CVA in chicken embryo fibroblast (CEF) cells that ultimately resulted in over 500 passages.
  • CEF chicken embryo fibroblast
  • pox viruses can be genetically engineered to contain and express foreign DNA with or without impairing the ability of the virus to replicate.
  • foreign DNA can encode protein antigens that induce protection against one or more infectious agents.
  • vaccinia viruses have been engineered to express immunizing antigens of herpesvirus, hepatitis B, rabies, influenza, human immunodeficiency virus (HIV), and other viruses
  • herpesvirus hepatitis B, rabies, influenza, human immunodeficiency virus (HIV)
  • HAV human immunodeficiency virus
  • other viruses Zagury et al, Nature 326:249-50 (1987); Cooney et al, Lancet 337:567-72 (1991); Graham et al., J Infect. Dis.
  • pox viruses are cytoplasmic viruses and thus DNA does not integrate into the cell's chromosomes, pox vectors are generally not the first choice for gene therapy where an agent is needed for an extended period of time.
  • Gene therapy can also be applied to delivery of angiogenesis modulating factors.
  • Gene therapy approaches for delivery of angiogenesis modulating factors have several advantages over conventional administration, including chronic production, lack of peak-and-trough pharmacokinetics, and potential economics of production of vectors versus protein.
  • the present invention is directed to a system for regulating angiogenesis.
  • the system of the present invention uses recombinant poxvirus vectors to deliver angiogenesis modulating agents to cells, such as angiogenesis inhibitors, e.g., endostatin and angiostatin, or angiogenesis inducers or enhancers, e.g., VEGF, to effectively and non-cytotoxically regulate angiogenesis.
  • angiogenesis inhibitors e.g., endostatin and angiostatin
  • angiogenesis inducers or enhancers e.g., VEGF
  • the poxviruses used are not capable of sustained replication in the target cell.
  • a preferred recombinant poxvirus useful according to the present invention for human cells is an attenuated orfhopox such as modified vaccinia Ankara (MVA), NYNAC, TROYNAC, an avipox, such as fowl pox or canary pox, or suipox.
  • one or more angiogenesis modulating agents are delivered using the same vector.
  • a system comprising two or more pox virus vectors can be used.
  • the angiogenesis modulating agents are selected to address the needs of the subject in need of treatment.
  • the angiogenesis modulating agent encoding nucleic acid to be delivered into the target cells are inserted into the vector at a non-essential site by standard means, such as homologous recombination or use of a unique insertion site.
  • the vector is then introduced into the target cells by any known method.
  • the vector system used does not integrate into the target's chromosomes. Therefore, the risks of long term exposure, either from the viral vector or the added gene, are substantially reduced.
  • the invention provides a method of treating disease associated angiogenesis by administering to a subject individual in need thereof a recombinant poxvirus vector comprising a custom-designed combination of nucleic acids encoding angiogenic modulators.
  • custom-designed as used herein is meant to describe a selection of agents that are known or suspected to be effective in improving or correcting a condition diagnosed in a subject in need of the therapeutic intervention.
  • the term encompasses phenotypically diagnosing the abnormal angiogenic problem or disease associated problem in angiogenesis in the subject individual and/or determining a gene and/or genome defect and/or defects in the subject and/or testing the defective cells/tissue taken from the subject individual for responses to a panel of angiogenesis modulators.
  • the angiogenic modulators are then selected based upon the need to correct and/or supplement the abnormal angiogenesis in the subject based upon the phenotypic and/or genotypic and/or angiogenic modulator response analysis.
  • the method of the present invention uses a recombinant poxvirus vector comprising nucleic acids encoding one or more anti- angiogenic agents.
  • anti-angiogenic agent and “angiogenesis inhibiting agent” are used interchangeably in the specification and are meant to include an agent which has the capacity to inhibit angiogenesis including anti-angiogenic protein or an angiogenesis inhibiting fragment thereof, an antisense nucleic acid, a ribozyme, an interference RNA (iRNA) or the like.
  • the method of the present invention uses a recombinant poxvirus vector comprising nucleic acids encoding one or more angiogenesis enhancing or "angiogenic" agents.
  • the angiogenic modulators are delivered in two or more pox virus vectors administered either simultaneously or consecutively.
  • the pox virus vectors administered consecutively are from different genus.
  • the vectors can be administered at intervals ranging from hours to days or even a few weeks.
  • the method of treatment is combined with treatment with an angiogenesis modulating drug or small inorganic molecule.
  • the system of the present invention is combined with therapeutic antibody production using immunization.
  • the method of treatment can be combined with traditional methods of treatment such as surgery, chemotherapy and/or radiation therapy in combination with cancer targeting antiangiogenic agents.
  • the present invention relates to methods of treating disorders associated with abnormal angiogenesis using pox virus vectors as a non-cytotoxic and preferably non-integrating vehicle to deliver preferably several angiogenesis modulating agents to cells.
  • the invention further relates to custom-designed pox virus vectors wherein the angiogenic modulators are selected according to the need of a subject affected with abnormal angiogenesis.
  • the viral vector used in the present invention is based upon using a pox virus that is non-lytic to the target cell in a 48 hour time span, more preferably a 96 hour time span, and still more preferably, a 120 hour or longer time span.
  • a non-lytic, virus is one that will not kill most target cells in the host animal or a tissue culture in a short period of time during which the viable infected cells will be expressing the gene product. For example, it preferably will not kill more than about 25% of the target cells and it is being used within 48 hours, more preferably 72 hours, still more preferably, 96 hours. More preferably, it will not kill more than about 10% of the target cells in the host animal or tissue culture and it is used in within 48 hours, more preferably 72 hours, and still more preferably 96 hours. Even niore preferably, such a transformed target cell population will be expressing the delivered gene product for a period of 1 to 2 weeks after initial infection. This can readily be determined by assaying samples of the target cell for viability, e.g., by staining with trypan blue, and gene expression, e.g., measuring protein production with ELISA.
  • the term "short-term" delivery system described herein is preferably directed to the use of vector systems that although capable of expressing the desired genetic material for at least about 1 week will result in the transient expression of the gene product. Preferably, the expression will be for less than about 2 months, more preferably, less than about 1 month. In addition, by preferably using an avirulent virus for the selected animal host the virus will not cause serious disease in the host. If any adverse effects are observed, such effects can be further curtailed as described below. Moreover, the delivery system described herein is capable of "controlled release" of a desired protein by continuously expressing specific amounts of the desired protein over a given period of time.
  • Preferred non-integrating viruses are poxviruses such as, avipox (e.g. fowl pox, canary pox), orthopox (e.g. vaccinia, ectomelia, rabbit pox), suipox (e.g. swine pox), capripox (e.g. sheppox), and leporipox.
  • avipox e.g. fowl pox, canary pox
  • orthopox e.g. vaccinia, ectomelia, rabbit pox
  • suipox e.g. swine pox
  • capripox e.g. sheppox
  • leporipox e.g. sheppox
  • avipox or avipoxvirus a genus of closely related pox viruses which infect fowl.
  • the genus avipox includes, for example, the species fowlpox, canary pox, junco pox, pigeon pox, quail pox, sparrow pox, starling pox, and turkey pox.
  • the genus avipox shares many characteristics with other pox viruses and is a member of the same subfamily, poxviruses of vertebrates, as vaccinia. These viruses are distinguished by their large size, complexity, and by the cytoplasmic site of replication. However, different genera of poxviruses, e.g.
  • orthopox and avipox are dissimilar in their respective molecular weights, their antigenic determinants, and their host species (Intervirology Vol. 17, pages 42-44, Fourth Report of the International Conmmittee on Taxonomy of Viruses (1982)).
  • Another preferred group is a highly attenuated vaccinia viruses such as modified vaccinia Ankara (MNA) (Sutter and Moss, Proc. ⁇ at'l Acad. Sci. U.S.A.. 89:10847-10851; Sutter et al Virology 1994) or ⁇ YNAC, replicating vaccinia virus (Perkus et al Science 229:981-984, 1985; Kaufman et al Int. J. Cancer 48:900- 907, 1991, Moss Science 252:1662, 1991), Wyeth.
  • MNA was derived from the Ankara vaccinia strain CNA.
  • CNA was used in the Federal Republic of Germany as a smallpox vaccine.
  • attenuation experiments were initiated in the laboratory of Dr. Anton Mayr (University of Kunststoff) comprising terminal dilution of CNA in chicken embryo fibroblast (CEF) cells. Over 500 passages were performed in CEF cells, resulting in an attenuated, replication-defective virus. The virus is restricted to replication primarily in avian cells.
  • MNA has been administered to numerous animal species, including monkeys, mice, swine, sheep, cattle, horses and elephants with no local or systemic adverse effects. Over 120,000 humans have been safely vaccinated with MNA by intradermal, subcutaneous or intramuscular injections. MNA has also been reported to be avirulent among normal and immunosuppressed animals (Mayr et al., Monb. Bakte ⁇ ol 167:375-90 (1978).
  • One preferred vaccinia virus is a Wyeth strain or derivative thereof.
  • a derivative of the Wyeth strain includes but is not limited to vTBC33 which lacks a functional K1L gene and the like.
  • the virus is Dry-Nax available as a smallpox vaccine from the Centers for Disease Control, Atlanta, GA.
  • the poxvirus is a strain of fowlpox, for example POXNAC-TC (Schering-Plough Corporation), and the like
  • Pox viruses are well known cytoplasmic viruses. Thus, genetic material expressed by such viral vectors typically remains in the cytoplasm and does not have the potential for inadvertent integration of the genetic material carried into host cell genes, unless specific steps are taken such as described above.
  • the vector system will not result in long term persistence in other cells.
  • the vector and transformed cells will not adversely affect cells in the host animal at locations distant from where the target cell is.
  • poxvirus vectors have a large genome, they can readily be used to deliver a wide range of genetic material including multiple genes (i.e., act as a multivalent vector).
  • the sizes of the poxvirus genomes range between about 130-300 kbp with up to about 300 genes, depending on the strain of the virus. Therefore, it is possible to insert large fragments of foreign D ⁇ A into these viruses and yet maintain stability of the viral genome.
  • At least one nucleic acid fragment encoding an angiogenesis modulating agent is inserted into a poxvirus vector. In another embodiment at least two and up to about 10 different nucleic acids encoding different angiogenesis modulating agents are inserted into the poxvirus vector.
  • poxvirus vectors encoding different angiogenesis modulating agents are administered sequentially.
  • the poxvirus vectors are selected from different poxvirus genera.
  • the homology between the nucleic acid sequences and the resulting protein homology is generally low between different genera of poxviruses. Therefore, to avoid host immune reaction during the subsequent administration encoding one or more different angiogenesis modulating agents it is preferred to use a pox of a different family for the second or third administration.
  • initial administrations by vaccinia or avipox would preferably be followed by an avipox or vaccinia, respectively, or by a suipox.
  • the angiogenesis modulating treatment using a poxvirus vector may also be combined with other treatment methods. For example, if antiangiogenic agent is delivered to an individual affected with cancer, traditional chemotherapeutic or radiotherapeutic interventions may be used before, simultaneously or after treatment with the poxvirus encoding a set of angiogenesis inhibiting molecules. One may also administer pox viruses encoding a tumor associated antigen to induce an immune reaction against the tumor in combination with administering the angiogenesis modulating agents.
  • Angiogenesis inducing agents useful according to the present invention include, but are not limited to a NEGF protein, and more preferably, the angiogenic peptide is NEGF 1 j, NEGF] 45 , NEGF 165 , NEGF] 89 , or a mammalian counterpart, which are variously described in U.S. Pat. No. 5,332,671 (Ferrara et al.), U.S. Pat. No. 5,240,848 (Keck et al.); and U.S. Pat. No. 5,219,739 (Tischer et al.).
  • the angiogenic peptide is VEGF] j or VEGF] 65 , particularly VEGFm.
  • VEGF] 2 ⁇ and VEGF ⁇ 65 A notable difference between VEGF] 2 ⁇ and VEGF ⁇ 65 is that VEGF 12 ⁇ does not bind to heparin with a high degree of affinity, as does VEGF 165 .
  • VEGF moieties are advantageous over other angiogenic peptides because VEGF proteins do not induce the growth of tissues not involved in the production of new vasculature.
  • angiogenic peptides include VEGF II, VEGF-C, fibroblast growth factors (FGFs) (e.g., aFGF, bFGF, and FGF-4), angiopoiteins, angiogenin, angiogenin-2, and PIGF, which are variously described in U.S. Pat. No. 5,194,596 (Tischer et al.), U.S. Pat. No. 5,219,739 (Tischer et al.), U.S. Pat. No. 5,338,840 (Bayne et al.), U.S. Pat. No. 5,532,343 (Bayne et al.), U.S. Pat. No.
  • FGFs fibroblast growth factors
  • Anti-angiogenic proteins are also referred to herein as angiogenesis inhibitors or anti- angiogenic agents.
  • Anti-angiogenic proteins for purposes of the present invention, also include anti-angiogenic fragments of those proteins.
  • the antiangiogenic, or angiogenesis inhibiting proteins useful in the present method include, but are not limited to, VEGF inhibitors such as antibodies against VEGF (e.g., anti-VEGF) or antigenic epitopes thereof, and soluble VEGF receptors such as Fit- 1 , Flk /KDR, Flt-4, neuropilin-1 and -2; VEGF receptor inhibitors or antibodies against such receptors such as DC101 [ImClone Systems, Inc., NY]; tyrosine kinase inhibitors; prolactin, angiostatin, endostatin, somatostatin; protamine; interleukin-12; troponin-1; platelet factor 4; thrombospondin-1; interferon alfa; basic fibroblast derived growth factor (bFGF) inhibitors such as a soluble bFGF receptor; transforming growth factor beta; epidermal-derived growth factor inhibitors; platelet derived growth factor inhibitors; an intergrin blocker; tissue fibroblast
  • angiogenesis modulating agents can be delivered using the system of the present invention.
  • One preferred group of nucleic acids encoding angiogenesis inhibitors encode antibodies.
  • Antibodies have long been used in biomedical science as in vitro tools for the identification, purification and functional manipulation of target antigens. Antibodies have been exploited in vivo for both diagnostic and therapeutic applications. Recent advances in antibody engineering have now allowed the gene encoding antibodies to be manipulated so that the antigen biding domain can also be expressed intracellularly.
  • intracellular antibodies are called "intrabodies” (Marasco et al. Gene Therapy, 4:11-15, 1997).
  • nucleic acids encoding angiogenesis modulating intrabodies encode a single chain, humanized antibody.
  • Diseases, disorders, or conditions, associated with abnormal angiogenesis that can be treated with the method of the present invention include, but are not limited to of solid tumors, tumor growth, retinal neovascularization, hemangioma, leukemia, metastasis, psoriasis, neovascular glaucoma, diabetic retinopathy, arthritis, endometriosis, and retinopathy of prematurity (ROP), vascular atherosclerotic disease, coronary artery disease, and myocardial ischemia.
  • any angiogenesis-dependent solid tumor will be a potential target for treatment using angiogenesis inhibitors encoded by a poxvirus vector.
  • solid tumors which will be particularly amenable to gene therapy applications include, but are not limited to, (a) neoplasms of the central nervous system such as, but again not necessarily limited to glioblastomas, astrocytomas, neuroblastomas, meningiomas, ependymomas; (b) cancers of hormone-dependent, tissues such as prostate, testicles, uterus, cervix, ovary, mammary carcinomas including but not limited to carcinoma in situ, medullary carcinoma, tubular carcinoma, invasive (infiltrating) carcinomas and mucinous carcinomas; (c) melanomas, including but not limited to cutaneous and ocular melanomas; (d) cancers of the lung which at least include squamous cell carcinoma, spindle carcinoma, small cell carcinoma, adenocarcinoma and
  • a preferred embodiment of the present invention relates to a method of inhibiting angiogenesis associated with solid tumors to inhibit or prevent further tumor growth and eventual metastasis and to reduce the size of a preexisting tumor.
  • preferably more than one anti-angiogenic agent may be combined either by inserting nucleic acids encoding the anti-angiogenic molecules into the same poxvirus vector or in different vectors.
  • the antiangiogenic treatment may also be combined with surgery, chemotherapeutic agents or radiation therapy and can be administered before, during or after surgical intervention, chemotherapy or radiotherapy treatment.
  • the treatment method in accordance with the present invention includes treating a subject affected with cancer, for example breast cancer, ovarian cancer or prostate cancer, with a first poxvirus vector encoding at least one antiangiogenic agent and a second vector designed to elicit a cytotoxic T- cell response to a tumor-associated antigen, such as PSA, CEA or MUC.
  • Cytotoxic T-cells specific for the desired cancer-associated antigen can be generated by administering between about 10 5 -10 9 pfu of a recombinant poxvirus carrying a sequence encoding a tumor-associated antigen to the individual affected with the tumor.
  • a tumor-associated antigen such as PSA, CEA or MUC.
  • cytokines e.g., IL-2
  • co-stimulatory molecules e.g., B7.1, B7.2, ICAM-1, LFA-3, CD72, OX40L (with or without OX40), CD40, CD40L, and the like may be used as biologic adjuvants and can be administered systemically to the host via inserting nucleic acids encoding such into same or different recombinant poxvirus vectors.
  • cytokines and growth factors encompassed by the present invention include but are not limited to: granulocyte macrophage-colony stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), macrophage-colony stimulating factor (M-CSF), tumor necrosis factors (TNF. alpha, and TNF.beta.), transforming growth factors (TGF.alpha.
  • GM-CSF granulocyte macrophage-colony stimulating factor
  • G-CSF granulocyte-colony stimulating factor
  • M-CSF macrophage-colony stimulating factor
  • TGF. alpha tumor necrosis factors
  • TNF.beta. transforming growth factors
  • EGF epidermal growth factors
  • SCF stem cell factor
  • PDGF platelet-derived growth factors
  • NGF nerve growth factor
  • FGF insulin-like growth factors
  • growth hormone interleukins 1 to 15 (IL-1 to IL-15), interferons .alpha., .beta, and gamma.
  • IFN-.alpha., IFN- .beta, and IFN-.gamma. brain-derived neurotrophic factor, neurotrophins 3 and 4, hepatocyte growth factor, erythropoictin, EGF-like mitogens, TGF-like growth factors, PDGF-like growth factors, melanocyte growth factor, mammary-derived growth factor 1, prostate growth factors, cartilage-derived growth factor, chondrocyte growth factor, bone-derived growth factor, osteosarcoma-derived growth factor, glial growth-promoting factor, colostrum basic growth factor, endothelial cell growth factor, tumor angiogenesis factor, hematopoietic stem cell growth factor, B-cell stimulating factor 2, B-cell differentiation factor, leukemia- derived growth factor, myelomonocytic growth factor, macrophage-derived growth factor, macrophage-activating factor, erythroid-potentiating activity, keratinocyte growth factor, ciliary neurotrophic growth factor, Schwann cell
  • inducing angiogenesis or “induction of angiogenesis” it is meant that angiogenesis is either initiated or enhanced. Therefore, for example, when a nonischemic skeletal muscle is not already undergoing angiogenesis, the present method provides for initiation of angiogenesis in the nonischemic skeletal muscle. However, when the nonischemic skeletal muscle is already undergoing angiogenesis, the present method provides a means by which the level of angiogenesis is enhanced or heightened. Preferably, the pox viral vector is injected into muscle tissue or the liver.
  • Revascularization with angiogenic inducing agents is useful, for example in diseases such as vascular atherosclerotic disease, also known as peripheral arterial occlusive disease, which is a major health problem, especially in the elderly. Its prevalence increases with age from 3% in individuals younger than 60 years old to over 20% in individuals 75 years or older. Treatment of patients suffering from peripheral arterial occlusive disease remains a considerable clinical issue despite advances in both surgical and percutaneous revascularization techniques. Many patients cannot benefit from these therapies because of the anatomic extent and distribution of arterial occlusion. In such patients, new therapeutic strategies have been sought to prevent the development of disabling symptoms related to ischemia such as claudication, resting pain and loss of tissue integrity in the distal limbs. The latter can ultimately lead to limb loss.
  • diseases such as vascular atherosclerotic disease, also known as peripheral arterial occlusive disease, which is a major health problem, especially in the elderly. Its prevalence increases with age from 3% in individuals younger than 60 years old to over 20% in individuals 75
  • the gene delivery system described herein can be used for any host or subject to either inhibit or induce angiogenesis.
  • the host will be a mammal.
  • Preferred mammals include primates such as humans and chimpanzees, domestic animals such as horses, cows, pigs, etc. and pets such as dogs and cats.
  • the host animal is a primate or domestic animal.
  • the host animal is a primate such as a human.
  • the pox virus vector used for a particular host animal is avirulent in that animal
  • one method is looking at a pox virus 'natural host range.
  • the virus vector can be selected from a virus whose primary range of infection is for a different host animal than the animal that the gene delivery system is to be used in.
  • swinepox can be used as a viral vector when the host is a primate such as a human.
  • the host is a pig it would not be preferable.
  • Another approach is to modify or select for a virus that is attenuated regardless of the host range.
  • certain highly attenuated or modified strains such as modified orthopoxvirus (e.g., the MNA or ⁇ YVAC strain of vaccinia) or strains genetically modified or selected to be non- virulent in their normal host range or in a desired host cell).
  • Tissue specificity also can be used to preliminarily screen for infectivity and replication efficiency.
  • preferred vectors include pox vectors, for example, suipox [Feller, et al., Virology 183:578-585 (1991)], such as swinepox, avipox such as fowlpox, canary pox, or pigeon pox, and capripoxvirus.
  • pox vectors for example, suipox [Feller, et al., Virology 183:578-585 (1991)]
  • swinepox such as fowlpox, canary pox, or pigeon pox
  • capripoxvirus iridoviruses
  • iridoviruses such as frog virus, and African swine fever virus are also preferred.
  • Preferred viral vectors for use with human cells are non-lytic, avirulent pox viruses such as avipox [Taylor, et al., Vaccine, 6:497-503 (1985) and Jenkins, et al., AIDS Research And Human Retroviruses 7:991-998 (1991)] and attenuated orthopox.
  • the basic techniques of inserting genes into viruses are known to the skilled artisan and involve, for example, recombination between the viral DNA sequences flanking a gene in a donor plasmid and homologous sequences present in the parental virus (Mackett, et al., Proc. Natl. Acad. Sci. USA 79:7415-7419 (1982)).
  • a recombinant virus such as a poxvirus for use in delivering the gene can be constructed in two steps known in the art and analogous to the methods for creating synthetic recombinants of the fowlpox virus described in U.S. Pat. No. 5,093,258, the disclosure of which is incorporated herein by reference.
  • Other techniques include using a unique restriction endonuclease site that is naturally present or artificially inserted in the parental viral vector.
  • the DNA gene sequence to be inserted into the virus can be placed into a plasmid, e.g., an E. coli plasmid construct, into which DNA homologous to a section of DNA such as that of the poxvirus has been inserted.
  • a plasmid e.g., an E. coli plasmid construct
  • the DNA gene sequence to be inserted is ligated to a promoter.
  • the promoter-gene linkage is positioned in the plasmid construct so that the promoter-gene linkage is flanked on both ends by DNA homologous to a DNA sequence flanking a region of pox DNA which is the desired insertion region.
  • the resulting plasmid construct is then amplified by growth within E. coli bacteria and isolated.
  • the plasmid also contains an origin of replication such as the E. coli origin of replication, and a marker such as an antibiotic resistance gene for selection and propagation in E. coli.
  • the isolated plasmid containing the DNA gene sequence to be inserted is transfected into a cell culture, e.g., chick embryo fibroblasts, along with the poxvirus. Recombination between homologous pox DNA in the plasmid and the viral genome respectively results in a poxvirus modified by the presence of the promoter-gene construct in its genome, at a site which does not affect virus viability.
  • the gene is inserted into a site or region (insertion region), in the virus which does not affect virus viability of the resultant recombinant virus.
  • Novel insertion sites can be identified by analyzing a poxvirus genome to identify sequences with the following characteristics. First, the insertion site should lie in an intergenic space, preferably between non-essential genes. Second, the insertion of foreign DNA at the insertion site should not disrupt.any cryptic ORFs in the region, or promoters of adjacent genes or other regulatory elements.
  • thymidine kinase gene One region that can readily be used and is present in many viruses is the thymidine kinase gene. For example, it has been found in all pox virus genomes examined [leporipoxvirus: Upton, et al., J. Virology, 60:920 (1986) (shope fibroma virus); capripoxvirus: Gershon, et al., J. Gen. Virol., 70:525 (1989) (Kenya sheep-1); orthopoxvirus: Weir, et al., J.
  • One preferred poxvirus useful according to the present invention is an avipox, including but not limited to fowlpox, and canary pox, including ALVAC.
  • a particularly preferred avipoxvirus is fowlpox.
  • insertion regions include, for example, BamHI J [Jenkins, et al., AIDS Research and Human Retroviruses 7:991-998 (1991)] the EcoRI-Hindlll fragment, BamHI fragment, EcoRV-Hindlll fragment, BamHI fragment and the Hindlll fragment set forth in EPO Application No. 0 308 220 Al. [Calvert, et al., J. of Nirol. 67:3069-3076 (1993); Taylor, et al., Vaccine 6:497-503 (1988); Spehner, et al., (1990) and Boursnell, et al., J. of Gen. Nirol. 71:621-628 (1990)].
  • fowlpox insertion sites useful according to the present invention are designated the LUS insertion site, the FP14 insertion site, and the 43K insertion site. These sites are also referred to sometimes as FPN006/FPN007 (LUS insertion site), FPN254/FPN255 (LUS insertion site), FPN060/FPN061 (FP14 insertion site), and FPN107/FPN108 (43K insertion site).
  • the insertion site in fowlpox is designated the LUS insertion site.
  • LUS long unique sequences
  • the LUS insertion site at the left end of the genome is between positions 7470 - 7475 in the fowlpox genomic sequence, and lies 3' of FPN006 and 5' of FPN007 125L.
  • the LUS insertion site at the right end of the genome is between positions 281065 and 281070 in the fowlpox genomic sequence, and lies 5' of FPN254 and 3' of FPN255.
  • an insert representing a sequence of interest can be inserted at any position within the specified insertion site.
  • the insertion site in fowlpox is designated the FP14 insertion site. This site is between positions 67080 - 67097 in the fowlpox genomic sequence, and lies 5' of FPV060 and 3' of FPV061.
  • the D ⁇ A sequence at the specified insertion site i.e. between the nucleotides, is deleted in the recombinant virus and replaced with defined inserts representing a sequence of interest.
  • the insertion site in fowlpox is designated the 43K insertion site. This site is at position 128178 of the fowlpox genomic sequence, and lies 5' of FP VI 07 and 5' of FPV108. These genes are divergently transcribed, and the insertion site lies between the two promoter elements for the two ORFs. In this embodiment, an insert representing a sequence of interest can be inserted at this position within the fowlpox genome.
  • the fowlpox is a vaccinia virus.
  • Particularly preferred vaccinia viruses include attenuated vaccinia viruses such as MVA, ⁇ YVAC (attenuated), and Wyeth strain, as well as non-attenuated strains such as TROY VAC.
  • MVA contains 6 natural deletion sites, which have been demonstrated to serve as insertion sites. See e.g. U.S. Patent No. 5,185,146, and U.S. Patent No. 6,440,422.
  • Insertion sites in vaccinia are described in U.S. serial number 60/448,591 , which is hereby incorporated by reference.
  • the insertion site in vaccinia is designated insertion site 44/45.
  • insertion site 44/45 lies between ORFs 044L and 045L, and the insertion site is between positions 37346-37357 in the MVA genomic sequence (Genbank Accession # U94848).
  • This region is 5 'of the translational start codon of MVA 044L and 3 'of the translational stop codon of MNA 045 L.
  • the corresponding ORFs are F14L (homologous to MNA 044L) and F15L (MNA 045L), and the insertion site is 5' of the translational start codon of vaccinia F14L and 3' of the translations stop codon of vaccinia F15L.
  • Vaccinia Copenhagen which contains this region and has its sequence available as Genbank Accession number M35027, is a prototypical vaccinia.
  • Insertion site analogous to sites such as 44/45 can also be used in other vaccinia strains including vaccinia Wyeth, ⁇ YNAC (where the insertion site is not known to be modified) and TROYNAC.
  • the D ⁇ A sequence at the specified insertion site i.e. between the nucleotides, contains defined inserts representing a sequence of interest; the flanking nucleotides on both sides remain unchanged.
  • the insertion site in vaccinia is designated insertion site 49/50.
  • insertion site 49/50 lies between ORFs 049L and 050L, and the insertion site is between positions 42687 - 42690 in the MVA genomic sequence (Genbank Accession # U94848). This region is 5' of the translational start codon of MVA 049L and 3' of the translational stop codon of MVA 050L.
  • insertion site 49/50 for insertion site 49/50 the corresponding ORFs are E2L (homologous to MVA 049L) and E3L (MVA 050L), and the insertion site is 5' of the translational start codon of vaccinia E2L and 3' of the translations stop codon of vaccinia E3L.
  • Vaccinia Copenhagen is a prototypical vaccinia.
  • insertion site 49/50 can also be used in other vaccinia strains including ⁇ YVAC (where the insertion site is not known to be modified) and TROYVAC.
  • the D ⁇ A sequence at the specified insertion site i.e.
  • the insertion site in vaccinia is designated insertion site 124/125.
  • insertion site 124/125 lies between ORFs 124L and 125L, and the insertion site is between positions 118481 - 118482 in the MVA genomic sequence (Genbank Accession # U94848). This region is 5' of the translational start codon of MVA 124L and 3' of the translational stop codon of MNA 125L.
  • insertion site 124/125 the corresponding ORFs are A13L (homologous to MNA 124L) and A14L (MNA 125L), and the insertion site is 5' of the translational start codon of vaccinia A13L and 3' of the translation stop codon of vaccinia A14L.
  • insertion site 124/125 can also be used in other vaccinia strains including ⁇ YNAC (where the insertion site is not known to be modified) and TROYNAC.
  • the D ⁇ A sequence at the specified insertion site i.e. between the nucleotides, is deleted in the recombinant virus and replaced with defined inserts representing a sequence of interest.
  • the vaccinia virus is a Modified vaccinia virus Ankara (MNA) or derivative thereof.
  • MNA has been generated by long-term serial passages of the Ankara strain of vaccinia virus (CNA) on chicken embryo fibroblasts (for review see Mayr, A., et al., Infection, 3:6-14 (1975).
  • the MNA virus itself may be obtained from a number of public repository sources.
  • MVA was deposited in compliance with the requirements of the Budapest Treaty at C ⁇ CM (Institut Pasteur, Collection ⁇ ationale de Cultures Microorganisms, 25, rue du Dondel Roux, 75724 Paris Cedex 15) on Dec. 15, 1987 under Depositary No.
  • Therion Biologies MVA products identified by the tradenames Therion-MVA(tm), Therion Prifree(tm) Vectors and Therion M-Series Vectors(tm), are products of Therion Biologies Corporation, Cambridge, Massachusetts, United States.
  • Another preferred poxvirus useful according to the treatment system of the present invention is an avipox, including but not limited to fowlpox, and canary pox, including ALNAC.
  • a particularly preferred avipoxvirus is fowlpox.
  • Particularly preferred fowlpox insertion sites of the present invention are designated the LUS insertion site, the FP14 insertion site, and the 43K insertion site. These sites are also referred to sometimes as FPV006/FPN007 (LUS insertion site), FPN254/FPN255 (LUS insertion site), FPN060/FPN061 (FP14 insertion site), and FPN107/FPN108 (43K insertion site), as described in U.S. serial number 60/448,591, which is hereby incorporated by reference.
  • the insertion site in fowlpox is designated the LUS insertion site.
  • fowlpox there are two long unique sequences (LUS) at each end of the viral genome (Genbank Accession # AF198100), and thus two LUS insertion sites in each genome.
  • the LUS insertion site at the left end of the genome is between positions 7470 - 7475 in the fowlpox genomic sequence, and lies 3' of FPN006 and 5' of FPN007 125L.
  • the LUS insertion site at the right end of the genome is between positions 281065 and 281070 in the fowlpox genomic sequence, and lies 5' of FPN254 and 3' of FPV255.
  • an insert representing a sequence of interest can be inserted at any position within the specified insertion site.
  • the insertion site in fowlpox is designated the FP14 insertion site. This site is between positions 67080 - 67097 in the fowlpox genomic sequence, and lies 5' of FPV060 and 3' of FPV061.
  • the D ⁇ A sequence at the specified insertion site i.e. between the nucleotides, is deleted in the recombinant virus and replaced with defined inserts representing a sequence of interest.
  • the novel insertion site in fowlpox is designated the 43K insertion site.
  • This site is at position 128178 of the fowlpox genomic sequence, and lies 5' of FPV107 and 5' of FPV108. These genes are divergently transcribed, and the insertion site lies between the two promoter elements for the two ORFs.
  • an insert representing a sequence of interest can be inserted at this position within the fowlpox genome.
  • swinepox preferred insertion sites include the thymidine kinase gene region.
  • a promoter operably linked to the desired gene i.e., in the proper relationship to the inserted gene.
  • the promoter must be placed so that it is located upstream from the gene to be expressed. Promoters are well known in the art and can readily be selected depending on the host and the cell type one wishes to target. For example in poxviruses, poxviral promoters should be used, such as the vaccinia 7.5K, 40K, fowlpox. Enhancer elements can also be used in combination to increase the level of expression. Furthermore, the use of inducible promoters, which are also well known in the art, in some embodiments are preferred.
  • Promoters useful according to the present invention include but are not limited to poxvirus promoters such as, e.g. an entomopox promoter, an avipox promoter, or a vaccinia promoter, e.g., HH, 1 IK or Pi.
  • poxvirus promoters such as, e.g. an entomopox promoter, an avipox promoter, or a vaccinia promoter, e.g., HH, 1 IK or Pi.
  • the Pi promoter from the Ava I H region of vaccinia, is described in Wachsman et al., J. of Inf. Dis. 155, 1188-1197 (1987). More particulary, this promoter is derived from the Ava I H(Xho I G) fragment of the L- variant WR vaccinia strain, in which the promoter directs transcription from right to left.
  • the map location of the promoter is approximately 1.3 Kbp (kilobase pair) from the 5' end of Ava IH, approximately 12.5 Kbp from the 5' end of the vaccinia genome, and about 8.5 Kbp 5' of the Hind III C N junction.
  • the Hind III H promoter (also "HH" and "H6" herein) sequence is an up-stream of open reading frame H6 by Rosel et al., J. Nirol. 60, 436-449 (1986).
  • the 1 IK promoter is as described by Wittek, J. Nirol. 49, 371-378 (1984) and Bertholet, C. et al., Proc. ⁇ atl. Acad. Sci. USA 82, 2096-2100 (1985).
  • promoter is modulated by an external factor or cue, and in turn to control the level of polypeptide being produced by the vectors by activating that external factor or cue.
  • heat shock proteins are proteins encoded by genes in which the promoter is regulated by temperature.
  • the promoter of the gene which encodes the metal-containing protein metallothionine is responsive to Cd + ions. Incorporation of this promoter or another promoter influenced by external cues also make it possible to regulate the production of the proteins.
  • the poxvirus genome is modified to carry a nucleic acid encoding at least one angiogenesis modulating agent which is operably linked to an "inducible” promoter.
  • inducible systems allow careful regulation of gene expression. See, Miller and Whelan, Human Gene Therapy, 8:803-815 (1997).
  • the phrase "inducible promoter” or “inducible system” as used herein includes systems wherein promoter activity can be regulated using an externally delivered agent.
  • Such systems include, for example, systems using the lac repressor from E. coli as a transcription modulator to regulate transcription from lac operator-bearing mammalian cell promoters (Brown et al.
  • ecdysone inducible system see, e.g. Karns et al, MBC Biotechnology 1:11, 2001. Inducible systems are available, e.g., from Invitrogen, Clontech, and Ariad. Systems using a repressor with the operon are preferred. One would adapt these promoters by substituting portions of pox promoters for the mammalian promoter.
  • a "transcriptional regulatory element”, or “TRE” is a polynucleotide sequence, preferably a DNA sequence, that regulates (i.e., controls) transcription of an operably-linked polynucleotide sequence by an RNA polymerase to form RNA.
  • a TRE increases transcription of an operably linked polynucleotide sequence in a host cell that allows the TRE to function.
  • the TRE comprises an enhancer element and/or pox promoter element, which may or may not be derived from the same gene.
  • the promoter and enhancer components of a TRE may be in any orientation and/or distance from the coding sequence of interest, and comprise multimers of the foregoing, as long as the desired transcriptional activity is obtained. As discussed herein, a TRE may or may not lack a silencer element.
  • An "enhancer” is a term well understood in the art and is a polynucleotide sequence derived from a gene which increases transcription of a gene which is operably-linked to a promoter to an extent which is greater than the transcription activation effected by the promoter itself when operably-linked to the gene, i.e. it increases transcription from the promoter.
  • enliancer activity is a term well understood in the art and means what has been stated, i.e., it increases transcription of a gene which is operably linked to a promoter to an extent which is greater than the increase in transcription effected by the promoter itself when operably linked to the gene, i.e., it increases transcription from the promoter.
  • a TRE derived from a specific gene is referred to by the gene from which it was derived and is a polynucleotide sequence which regulates transcription of an operably linked polynucleotide sequence in a host cell that expresses said gene.
  • a "pox glandular kallikrein transcriptional regulatory element”, or “hpKLK2-TRE” is a polynucleotide sequence, preferably a DNA sequence, which increases transcription of an operably linked polynucleotide sequence in a host cell that allows an hKLK2-TRE to function, such as a cell (preferably a mammalian cell, even more preferably a human cell) that expresses androgen receptor.
  • An hKLK2-TRE is thus responsive to the binding of androgen receptor and comprises at least a portion of a pox promoter, an hKLK2 promoter and/or an hKLK2 enhancer (i.e., the ARE or androgen receptor binding site).
  • PB-TRE is a polynucleotide sequence, preferably a DNA sequence, which selectively increases transcription of an operably-linked polynucleotide sequence in a host cell that allows a PB-TRE to function, such as a cell (preferably a mammalian cell, even more preferably a human cell) that expresses androgen receptor.
  • a PB-TRE is thus responsive to the binding of androgen receptor and comprises at least a PB enhancer (i.e., the ARE or androgen receptor binding site) as well as the pox promoter.
  • a "prostate-specific antigen (PSA) transcriptional regulatory element” is polynucleotide sequence, preferably a DNA sequence, which selectively increases transcription of an operably linked polynucleotide sequence in a host cell that allows a PSA-TRE to function, such as a cell (preferably a mammalian cell, even more preferably a human cell) that expresses androgen receptor.
  • a PSE-TRE is thus responsive to the binding of androgen receptor and comprises at least a portion of a PSA promoter and/or a PSA enhancer (i.e., the ARE or androgen receptor binding site).
  • the PSA promoter consists of the sequence from about nt -540 to nt +8 relative to the transcription start site. To work with pox vectors, one use a pox promoter in place of, or in addition to, the PSA promoter.
  • a TRE generally depends upon the presence of transcriptional regulatory factors and/or the absence of transcriptional regulatory inhibitors. Transcriptional activation can be measured in a number of ways known in the art (and described in more detail below), but is generally measured by detection and/or quantization of mRNA or the protein product of the coding sequence under control of (i.e., operatively linked to) the TRE. As discussed herein, a TRE can be of varying lengths, and of varying sequence composition. By transcriptional activation, it is intended that transcription will be increased above basal levels in the target cell by at least about 2-fold, preferably at least about 5 -fold, preferably at least about 10-fold, more preferably at least about 20-fold.
  • Basal levels are generally the level of activity, if any, in a non-target cells, or the level of activity (if any) of a reporter construct lacking the TRE of interest as tested in a target cell type.
  • a "functionally-preserved" variant of a TRE is a TRE which differs from another TRE, but still retains ability to increase transcription of an operably linked polynucleotide, especially cell-specific transcription activity.
  • the difference in a TRE can be due to differences in linear sequence, arising from, for example, single or multiple base mutation(s), addition(s), deletion(s), and/or modification(s) of the bases.
  • the difference can also arise from changes in the sugar(s), and/or linkage(s) between the bases of a TRE.
  • Certain point mutations within sequences of TREs have been shown to decrease transcription factor binding and gene activation.
  • One of skill in the art would recognize that some alterations of bases in and around known the transcription factor binding sites are more likely to negatively affect gene activation and cell-specificity, while alterations in bases which are not involved in transcription factor binding are not as likely to have such effects.
  • Certain mutations are also capable of increasing TRE activity. Testing of the effects of altering bases may be performed in vitro or in vivo by any method known in the art, such as mobility shift assays, or transfecting vectors containing these alterations in TRE functional and TRE non-functional cells. Additionally, one of skill in the art would recognize that point mutations and deletions can be made to a TRE sequence without altering the ability of the sequence to regulate transcription.
  • a cell which allows a TRE to function or a cell in which the function of a TRE is "sufficiently preserved" or "a cell in which a TRE is functiona ' is a cell in which the TRE, when operably linked to a promoter (if not included in the TRE) and a reporter gene, increases expression of the reporter gene at least about 2-fold, preferably at least about 5-fold, preferably at least about 10-fold, more preferably at least about 20-fold, more preferably at least about 50-fold, more preferably at least about 100-fold, more preferably at least about 200-fold, even more preferably at least about 400- to about 500-fold, even more preferably at least about 1000-fold, when compared to the expression of the same promoter and reporter gene when not operably linked to said TRE.
  • Methods for measuring levels (whether relative or absolute) of expression are known in the art and are described herein.
  • AR refers to a protein whose function is to specifically bind to androgen and, as a consequence of the specific binding, recognize and bin to an androgen response element (ARE), following which the AR is capable of regulating transcriptional activity.
  • the AR is a nuclear receptor that, when activated, binds to cellular androgen-responsive element(s). In normal cells the AR is activated by androgen, but in non-normal cells (including malignant cells) the AR may be activated by non-androgenic agents, including hormones other than androgens.
  • androgen receptor are mutant forms of an androgen receptor, as long as the function is sufficiently preserved.
  • Mutants include androgen receptors with amino acid additions, insertions, truncations and deletions, as long as the function is sufficiently preserved.
  • a functional androgen receptor is one that binds both androgen and, upon androgen binding, an ARE.
  • human glandular kallikrein encoding the hK2 protein
  • hKLK2 human glandular kallikrein
  • hK2 found in various tumors and in the serum of patients with prostate cancer differ substantially from those of PSA and indicate that hK2 antigen may be a significant marker for prostate cancer. Circulating hK2 in different relative proportions to PSA has been detected in the serum of patients with prostate cancer. Charlesworth et al. (1997) Urology 49:487-493. Expression of hK2 has been detected in each of 257 radical prostatectomy specimens analyzed. Darson et al. (1997) Urology 49:857-862.
  • hKLK2 5 ' promoter The activity of the hKLK2 5 ' promoter has been previously described and a region up to -2256 relative to the transcription start site was previously disclosed. Schedlich et al. (1987) DNA 6:429-437.
  • the hKLK2 promoter is androgen responsive and, in plasmid constructs wherein the promoter alone controls the expression of a reporter gene, expression of the reporter gene is increased approximately 10-fold in the presence of androgen.
  • hKLK2 enhancer activity is found within a polynucleotide sequence approximately nt -12,014 to nt -2257 relative to the start of transcription and, when this sequence is operably linked to an hKLK2 promoter and a reporter gene, transcription of operably-linked sequences in prostate cells increases in the presence of androgen at levels approximately 30- to approximately 100-fold over the level of transcription in the absence of androgen. This induction is generally orientation independent and position independent.
  • Enhancer activity has also been demonstrated in the following regions (all relative to the transcription start site): about nt -3993 to about nt -3643, about nt -4814 to about nt -3643, about nt -5155 to about nt -3387, about nt -6038 to about nt -2394.
  • ah hKLK2 enhancer can be operably linked to a pox promoter to form an hKLK2 transcriptional regulatory element (hKLK2-TRE).
  • An hKLK2-TRE can then be operably linked to a heterologous polynucleotide to confer hKLK2-TRE-specific transcriptional regulation on the linked angiogenesis modulating agent encoding gene, thus increasing its expression.
  • the poxvirus vectors directed at specific target cells may also be generated with the use of TREs that are preferentially functional in the target tumor cells.
  • tumor cell-specific heterologous TREs include TREs from the following genes: .alpha.-fetoprotein (AFP) (liver cancer), mucin-like glycoprotein DF3 (MUCl) (breast carcinoma), carcinoembryonic antigen (CEA) (colorectal, gastric, pancreatic, breast, and lung cancers), plasminogen activator urokinase (uPA) and its receptor gene (breast, colon, and liver cancers), E2F1 (cell cycle S-phase specific promoter) (tumors with disrupted retinoblastoma gene function), HER-2/neu (c-erbB2/neu) (breast, ovarian, stomach, and lung cancers).
  • AFP .alpha.-fetoprotein
  • MUCl mucin-like glycoprotein DF3
  • tumor-specific TREs may be used in conjunction with tissue-specific TREs from the following exemplary genes (tissue in which the TREs are specifically functional are in parentheses): hypoxia responsive element, vascular endothelial growth factor receptor (endothelium), albumin (liver), factor Nil (liver), fatty acid synthase (liver), Non Willebrand factor (brain endothelium), alpha-actin and myosin heavy chain (both in smooth muscle), synthetast I (small intestine), ⁇ a— K--C1 transporter (kidney). Additional tissue specific TREs are known in the art.
  • the cell specific, heterologous TRE is tumor cell specific.
  • both heterologous TREs are tumor cell specific and functional in the same cell.
  • one of the first heterologous TREs is tumor cell specific and the second heterologous TRE is tissue specific, whereby both TREs are function in the same cell.
  • Introduction of the viral vector carrying the nucleic acid encoding angiogenesis modulating agents to be delivered to the target host cell may be effected by any method known to those of skill in the art.
  • Administration of the recombinant poxvirus of the invention can be either “prophylactic” or “therapeutic” depending on the subject.
  • the recombinant pox virus of the present invention is provided in advance of any symptom, but when one believes the subject is at risk.
  • the prophylactic administration of the recombinant pox virus serves to prevent or ameliorate any subsequent angiogenesis related condition.
  • the recombinant poxvirus is provided at or after the onset of a symptom of the disease.
  • the present invention may be provided to either prior the anticipated disease state or after the initiation of a disease.
  • unit dose refers to physically discrete units suitable as unitary dosages for mammals, each unit containing a predetermined quantity of recombinant poxvirus calculated to produce the desired immunogenic effect in association with the required diluent.
  • the specifications for the novel unit dose of an inoculum of this invention are dictated by and are dependent upon the unique characteristics of the recombinant virus and the particular immunologic effect to be achieved.
  • the inoculum is typically prepared as a solution in tolerable (acceptable) diluent such as saline, phosphate-buffered saline or other physiologically tolerable diluent and the like to form an aqueous pharmaceutical composition.
  • the route of inoculation may be scarification, intravenous (I.N.), intramuscular (I.M.), subcutaneous (S.C.), intradermal (I.D.), intraperitoneal (I.P.), intratumor and the like, which results in eliciting a protective response against the disease causing agent.
  • the dose is administered at least once. Subsequent doses may be administered as indicated.
  • heterologous prime-boost regimens are employed.
  • the host is immunized at least once with a first vector such as a nucleic acid-based vector. Subsequent immunizations are performed with a poxvirus vector.
  • the host is first immunized with a first poxvirus vector and then with a second poxvirus vector of a different genus.
  • the dosage of administered recombinant pox virus will vary depending upon such factors as the mammal's age, weight, height, sex, general medical condition, previous medical history, disease progression, tumor burden and the like.
  • a dosage of recombinant virus in the range of about 10 5 to about 10 10 plaque forming units, although a lower or higher dose may be administered.
  • Examples of methods for administering the recombinant pox virus into mammals include, but are not limited to, exposure of tumor cells to the recombinant virus ex vivo, or injection of the recombinant pox virus into the affected host by intravenous, S.C., I.D. or I.M. administration of the virus.
  • the recombinant pox virusor combination of recombinant vectors may be administered locally by direct injection into the cancerous lesion or tumor or topical application in a pharmaceutically acceptable carrier.
  • the quantity of recombinant pox virus carrying the nucleic acid sequence encoding one or more angiogenesis modulating agents be administered is based on the titer of virus particles.
  • a preferred range of the virus particles to be administered is 10 5 to 10 10 virus particles per mammal, preferably a human.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a recombinant poxvirus comprising at least one, preferably more than one nucleic acid sequence encoding an angiogenesis modulating agent and a pharmaceutically acceptable carrier.
  • the effect of the genetic material delivered can be carefully monitored and regulated using this system.
  • Preferred vectors such as swinepox will only express the genetic material for about two weeks.
  • additional dosages will be needed, additional administration of the material can be accomplished by repeating the injection.
  • the addition of a second, third, etc. material can also be added with these vectors.
  • a number of pox viruses have been developed as live viral vectors for the expression of heterologous proteins.
  • Representative vaccinia virus strains such as Wyeth and MNA have been disclosed previously. (Cepko et al., Cell 37:1053-1062 (1984); Morin et al, Proc. ⁇ atl. Acad. Sci. USA 84:4626-4630 (1987); Lowe et al., Proc. ⁇ atl. Acad. Sci. USA, 84:3896-3900 (1987); Panicali & Paoletti, Proc. ⁇ atl. Acad. Sci. USA, 79:4927-4931(1982); Mackett et al., Proc. ⁇ atl. Acad.
  • D ⁇ A Vectors For In Vivo Recombination With A Parent Virus Genes that code for desired angiogenesis modulating proteins are inserted into the genome of a pox virus in such a manner as to allow them to be expressed by that virus along with the expression of the normal complement of parent virus proteins. This can be accomplished by first constructing a D ⁇ A donor vector for in vivo recombination with a pox virus.
  • the D ⁇ A donor vector contains the following elements:
  • all DNA fragments for construction of the donor vector can be obtained from genomic DNA or cloned DNA fragments.
  • the donor plasmids can be mono-, di-, or multivalent (i.e., can contain one or more inserted foreign gene sequences).
  • the donor vector preferably contains an additional gene which encodes a marker which will allow identification of recombinant viruses containing inserted foreign DNA. Several types of marker genes can be used to permit the identification and isolation of recombinant viruses.
  • genes that encode antibiotic or chemical resistance include genes that encode antibiotic or chemical resistance (e.g., see Spyropoulos et al., J. Virol., 62:1046 (1988); Falkner and Moss., J. Virol., 62:1849 (1988); Franke et al., Mol. Cell. Biol., 5:1918 (1985), as well as genes such as the E. coli lacZ gene, that permit identification of recombinant viral plaques by colorimetric assay (Panicali et al., Gene, 47:193-199 (1986)).
  • Homologous recombination between donor plasmid DNA and viral DNA in an infected cell results in the formation of recombinant viruses that incorporate the desired elements.
  • Appropriate host cells for in vivo recombination are generally eukaryotic cells that can be infected by the virus and transfected by the plasmid vector. Examples of such cells suitable for use with a pox virus are chick embryo dermal (CED) cells, HuTK143 (human) cells, and CV-1 and BSC-40 (both monkey kidney) cells. Infection of cells with pox virus and transfection of these cells with plasmid vectors is accomplished by techniques standard in the art (Panicali and Paoletti, U.S. Patent No.
  • the donor DNA can be directly ligated into the parental virus genome at a unique restriction site (Scheiflinger, et al. (1992) Proc. Natl. Acad. Sci. (USA) 89:9977-9981).
  • recombinant viral progeny can be identified by one of several techniques. For example, if the DNA donor vector is designed to insert foreign genes into the parent virus thymidine kinase (TK) gene, viruses containing integrated DNA will be TK " and can be selected on this basis (Mackett et al., Proc. Natl. Acad. Sci.
  • TK thymidine kinase
  • co- integration of a gene encoding a marker or indicator gene with the foreign gene(s) of interest, as described above, can be used to identify recombinant progeny.
  • One preferred indicator gene is the E. coli lacZ gene: recombinant viruses expressing ⁇ - galactosidase can be selected using a chromogenic substrate for the enzyme (Panicali et ah, Gene, 47:193 (1986)).

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Abstract

La présente invention concerne des techniques d'apport de modulateurs angiogéniques à un individu, des construits comprenant un poxvirus de recombinaison lequel comprenant au moins une séquence d'acide nucléique codante pour un agent modulateur angiogénique et des techniques de traitement de pathologies associées à une angiogenèse anormale au moyen de ces poxvirus de recombinaison.
PCT/US2004/005376 2003-02-24 2004-02-24 Techniques d'apport de modulateurs angiogeniques au moyen de poxvirus WO2004098534A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1601333A2 (fr) * 2003-02-20 2005-12-07 Therion Biologics Corporation Nouveaux sites d'insertion dans des vecteurs de variole
WO2008100292A3 (fr) * 2006-10-16 2009-05-14 Genelux Corp Souches du virus de la vaccine modifié pour une utilisation dans des procédés diagnostiques et thérapeutiques
EP2062023B2 (fr) † 2006-08-25 2016-11-09 The Government of the United States of America, as represented by the Secretary, Department of Health and Human Services Sites intergéniques entre des gènes conservés dans le génome du virus de la vaccine vaccinia ankara modifié (mva)
US10463730B2 (en) 2003-06-18 2019-11-05 Genelux Corporation Microorganisms for therapy

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Publication number Priority date Publication date Assignee Title
US6121246A (en) * 1995-10-20 2000-09-19 St. Elizabeth's Medical Center Of Boston, Inc. Method for treating ischemic tissue
US6258791B1 (en) * 1997-05-29 2001-07-10 Transgene S.A. Combination product for enhanced gene delivery comprising a hyaluronidase
US20020025941A1 (en) * 2000-06-14 2002-02-28 Olivier Meyer Combination product intended for carrying out a cytotoxic treatment, in particular an antitumour treatment, in a mammal

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6121246A (en) * 1995-10-20 2000-09-19 St. Elizabeth's Medical Center Of Boston, Inc. Method for treating ischemic tissue
US6258791B1 (en) * 1997-05-29 2001-07-10 Transgene S.A. Combination product for enhanced gene delivery comprising a hyaluronidase
US20020025941A1 (en) * 2000-06-14 2002-02-28 Olivier Meyer Combination product intended for carrying out a cytotoxic treatment, in particular an antitumour treatment, in a mammal

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1601333A2 (fr) * 2003-02-20 2005-12-07 Therion Biologics Corporation Nouveaux sites d'insertion dans des vecteurs de variole
EP1601333A4 (fr) * 2003-02-20 2008-08-13 Us Gov Health & Human Serv Nouveaux sites d'insertion dans des vecteurs de variole
US7638134B2 (en) 2003-02-20 2009-12-29 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Insertion sites in fowlpox vectors
EP2363493A1 (fr) * 2003-02-20 2011-09-07 The Government of the United States of America, as represented by the Secretary, Department of Health and Human Services Nouveaux sites d'insertion dans des vecteurs de variole
US10463730B2 (en) 2003-06-18 2019-11-05 Genelux Corporation Microorganisms for therapy
EP2062023B2 (fr) † 2006-08-25 2016-11-09 The Government of the United States of America, as represented by the Secretary, Department of Health and Human Services Sites intergéniques entre des gènes conservés dans le génome du virus de la vaccine vaccinia ankara modifié (mva)
WO2008100292A3 (fr) * 2006-10-16 2009-05-14 Genelux Corp Souches du virus de la vaccine modifié pour une utilisation dans des procédés diagnostiques et thérapeutiques
US9944903B2 (en) 2006-10-16 2018-04-17 Genelux Corporation Modified vaccinia virus strains for use in diagnostic and therapeutic methods
US10584317B2 (en) 2006-10-16 2020-03-10 Genelux Corporation Modified vaccinia virus strains for use in diagnostic and therapeutic methods

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