EP0802801A2 - Verfahren und zusammensetzungen zur in vivo behandlung der festen tumoren - Google Patents

Verfahren und zusammensetzungen zur in vivo behandlung der festen tumoren

Info

Publication number
EP0802801A2
EP0802801A2 EP95944229A EP95944229A EP0802801A2 EP 0802801 A2 EP0802801 A2 EP 0802801A2 EP 95944229 A EP95944229 A EP 95944229A EP 95944229 A EP95944229 A EP 95944229A EP 0802801 A2 EP0802801 A2 EP 0802801A2
Authority
EP
European Patent Office
Prior art keywords
tumor
cells
vims
gene
delivery vehicle
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95944229A
Other languages
English (en)
French (fr)
Inventor
Francis J. Burrows
Timothy C Fong
John M. Polo
Thomas W. Dubensky, Jr.
Douglas J. Jolly
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novartis Vaccines and Diagnostics Inc
Original Assignee
Chiron Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chiron Corp filed Critical Chiron Corp
Publication of EP0802801A2 publication Critical patent/EP0802801A2/de
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • 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
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C07K14/555—Interferons [IFN]
    • C07K14/57—IFN-gamma
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8132—Plasminogen activator inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/8146—Metalloprotease (E.C. 3.4.24) inhibitors, e.g. tissue inhibitor of metallo proteinase, TIMP
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C12N9/1211—Thymidine kinase (2.7.1.21)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • C12N9/6491—Matrix metalloproteases [MMP's], e.g. interstitial collagenase (3.4.24.7); Stromelysins (3.4.24.17; 3.2.1.22); Matrilysin (3.4.24.23)
    • 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
    • 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
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus

Definitions

  • the present application relates generally the field of anti-cancer therapy, and more specifically, to methods of killing selected tumor cells using recombinant viral vectors.
  • brachytherapy Two types have been used to treat tumors: brachytherapy and teletherapy.
  • brachytherapy the radiation source is placed close to the tumor. This intracavitary approach is used for many gynecologic or oral neoplasms.
  • supervoltage radiotherapy is usually delivered with a linear accelerator, allowing for more precise beam localization and avoiding the complication of skin radiation toxicity.
  • Various approaches are used to increase the radiation dosage to the tumor area while minimizing toxicity to adjacent normal tissues.
  • Radiation therapy is normally delivered in a fractionated fashion, having radiobiologic superiority by permitting time for recovery of normal host tissues (but not the tumor) from sublethal damage during the period of treatment. Fractionated radiation doses are usually administered for 5 days per week until the desired total dose has been delivered, usually over the course of 4-6 weeks.
  • Acute toxicity may include generalized fatigue and malaise, anorexia, nausea and vomiting, local skin changes, diarrhea, and mucosal ulceration of the irradiated area. Radiation of large areas, especially the pelvis and proximal long bones, may result in significant bone marrow suppression. Long-term toxicity may result in hyperpigmentation of the involv skin, decreased function of the irradiated organ, myelopathy, bone necrosis, and seconda malignancies. These toxicities, which are dose-related, and may be minimized or avoid by careful shielding and fractionation. Currently, more than 50% of all patients with cancer receive radiation thera during the course of their illness.
  • Radiation therapy is frequently the sole agent used wi curative intent for certain tumors. For more extensive cancers, radiation is combined wi surgery. Radiation is also used as an adjuvant to chemotherapy for some patients wi lymphoma or lung cancer and for several cancers in children. Occasionally, chemothera may be used to sensitize tumor cells to the toxic effects of radiation.
  • Hyperthermia is a form of nonionizing irradiation between 40_ to 42 C that of value as an adjunct to ionizing irradiation.
  • the treatment may also involve drugs th increase a tumor's response to heat. Hyperthermias greatest use is controlling bul hypervascular tumors.
  • Chemotherapy is used when surgery or radiation is not practical or only partial effective. Chemotherapy, involving the use of cytokines, cytotoxic drugs, hormone antihormones, and other biological agents, has become an increasingly effective means treating cancer.
  • the use of a cytokine alone or in combination with other cytokines cytotoxic drugs has been effective in inducing the regression of metastatic cancers b modulating the immune system (Heaton, K., etal., Cancer Immunol Immother 37:213-21 1993).
  • Useful cytokines include interleukin (IL)-2, IL-4. IL-6, tumor necrosis fact (TNF), ⁇ -interferon, and ⁇ -interferon ( ⁇ -IFN and ⁇ -IFN).
  • Intravenous administration IL-2 alone has been effective in approximately 15-30% of patients with metastatic renal-ce carcinoma or melanoma and may be efficacious in the treatment of other tumor Additionally, ⁇ -IFN has been shown to be effective against certain solid tumors followin resection of the bulk of the tumor tissue, suggesting a possible post-operative role as a adjuvant therapy (reviewed in Heaton, K., et al. Supra).
  • cytokines to increase antitumor efficacy is based on th premise that combining agents that attack neoplastic cells by different mechanisms shoul increase antitumor effects in vivo. Therefore, toxicity should be lower in patients treate with combinations of agents that have different side-effects. Pre-clinical studies hav demonstrated that such cytokine combinations can be effective in animal models (reviewe in Heaton, K., et al., Supra). Additionally, cytokines can be combined with other cytotoxi agents in order to offer a more effective therapy for neoplastic diseases. Such combination take advantage of different mechanisms of action and different toxicity profiles.
  • ⁇ -IFN appears to potentiate the effects of 5-fluorouracil (5-FU) by increasin serum levels of active 5-FU metabolites.
  • EL-2 and ⁇ -IFN in combination with cisplatin and dacarbazine have produced response rates much higher than any observed with either cytokines or cytotoxic drugs alone.
  • Regional administration involves direct infusion of active chemotherapeutic agents into the tumor site (e.g., intravesical therapy, intraperitoneal therapy, hepatic artery infusion with or without embolization of the main blood supply of the tumor). These treatments can result in significant palliation and improved survival.
  • active chemotherapeutic agents e.g., intravesical therapy, intraperitoneal therapy, hepatic artery infusion with or without embolization of the main blood supply of the tumor.
  • Radioimmunotherapy is a form of treatment that offers the potential for anti-cancer activity with common tumors. Although this therapy is largely experimental, progress is being made in developing useful clinical agents, especially in the hematopoietic malignancies. The success of radioimmunotherapy depends on delivering a large dose of radiation to a selective tumor without injury to radiation sensitive tissue.
  • the administration of the radiolabeled antibody into a closed space seems to offer the greatest potential for successful treatment. Intravenous administration of the compound is not as effective.
  • a number of monoclonal antibodies have been used, including: Ml 95 (anti-CD-33), a murine monoclonal antibody against hematopoietic progenitors; CC49 (anti-TAG-72), against colorectal, pancreas, ovarian, and other human tumors; and 3F8 (anti-GDZ), localized to primary and metastatic neuroblastomas in patients.
  • Ml 95 anti-CD-33
  • CC49 anti-TAG-72
  • 3F8 anti-GDZ
  • Neoadjuvant therapy systemic treatment given before local therapy, has become increasingly more prominent in the treatment of cancer (reviewed in Trimble, £., et al., Cancer Suppl 72:3515-3524, 1993).
  • the therapy is designed to ease subsequent surgical intervention, increase local control, and improve long-term outcomes of patients. This is partially accomplished by 1) reducing the tumor burden at the primary site, rendering efforts at local control more likely to be completely successful , 2) controlling disease that is not initially amenable to local treatment, 3) reducing the number of cancerous cells befo surgery, and 4) when administered before surgery, less resection may be necessary, there potentially sparing normal organ function.
  • neoadjuva therapy includes: acute toxicity of treatment; ineffectiveness which may compromise loc control or delay of definitive therapy allowing for aggressive local tumors to metastasiz scarring and fibrosis; making surgery more difficult; retardation of wound healing decreasing tissue vascularity; and ineffectiveness leading to a higher proportion of quiesce tumor cells.
  • a method for killing tum cells in vivo comprising transducing cells in or adjacent to a tumor with a recombina vector comprising a nucleic acid molecule encoding a polypeptide capable of stimulatin blood clot formation in or adjacent to the tumor.
  • the nucleic aci molecules may be selected from the group consisting of Russell's viper venom factor X activating factor, Russell's viper venom factor V-activating factor, thrombin and thrombin like enzymes such as those extracted from Crotalus adamanteus (Crotalus), Crotalu ho ⁇ idus horridus, Agkistrodon rhodostoma (Ancrod), Agistrondon contortrix contortri Agk ⁇ strodon acutus, Bothrops atrox (Batroxobin), Bothrops marajoensis, Bothrop moojeni, Trimerersurus gramineus, Trimeresurus okinavensis and Brirtis gabonica, tissu factor, truncated tissue factor, cancer procoagulant, and a fusion protein comprising part o all of a procoagulant protein and part or all of a ligand that binds to endothelial cells wit high affinity.
  • these nucleic acid molecules encod a protein selected from the group consisting of von Willebrand factor antigen ⁇ endothelial-monocyte-activating polypeptide I, endothelial-monocyte-activatin polypeptide ⁇ , tumor necrosis factor ⁇ , tumor necrosis factor ⁇ , and the tissue factor- inducing factor from Rickettsia rickettsii.
  • the nucleic acid molecules encode a polypeptide capable of inhibiting fibrinolysis.
  • these nucleic acid molecules are selected from the group consisting of ⁇ 2-antiplasmin, plasminogen activator inhibitor I, plasminogen activator inhibitor ⁇ , plasminogen activator inhibitor m, and Erthrina proteinase inhibitors.
  • a method for inhibiting tumor angiogenesis in vivo comprising transducing cells in or adjacent to the tumor with a recombinant vector comprising a nucleic acid molecule encoding a polypeptide capable of inhibiting vascularization of the tumor.
  • the polypeptides encoded by the nucleic acid molecule are selected from the group consisting of angiostatin, interferon ⁇ , interferon ⁇ , platelet factor-4, tissue inhibitors of metalloproteinases I, tissue inhibitors of metalloproteinases ⁇ , tissue inhibitors of metalloproteinases IE, thrombospondin, the angiogenic fragment of prolactin, heparinase, neutralizing antibody fragments against basic fibroblast growth factor, vascular endothelial cell growth factor and ⁇ V ⁇ 3 integrin.
  • a method for killing tumor cells in vivo comprising transducing cells of a blood vessel in or adjacent to an arterial side of a tumor with a recombinant vector comprising a nucleic acid molecule encoding a polypeptide capable of activating a non-cytotoxic agent capable of being a cytotoxic agent and administering to the animal a non-cytotoxic agent activated by the polypeptide into a cytotoxic agent.
  • nucleic acid molecules encode polypeptides selected from the group consisting of Herpes simplex virus thymidine kinase, varicella-zoster virus thymidine kinase, cytosine deaminase, xanthine-guanine phosphoribosyl transferase, Escherischia coli purine nucleoside phophorelase, the cytochrome p450 2B1 gene product, alkaline phosphatase, ⁇ -glucosidase and nitroreductase.
  • polypeptides selected from the group consisting of Herpes simplex virus thymidine kinase, varicella-zoster virus thymidine kinase, cytosine deaminase, xanthine-guanine phosphoribosyl transferase, Escherischia coli purine nucleoside phophorelase, the cytochrome p450 2
  • a method for depriving tumor cells in vivo of nutrients comprising transducing cells in or adjacent to a blood vessel in a tumor with a recombinant vector comprising a nucleic acid molecule encoding a polypeptide capable of binding or metabolizing nutrients in the perivascular interstitial space of the tumor.
  • the nucleic acid molecule encoding the polypeptide is selected from the group consisting of soluble folate receptor/folate binding protein, soluble tranferrin receptor, fetal hemaglobin, oxygen-binding fragments of fetal hemoglobin, or a fusion polypeptide consisting of the extracellular matrix binding fragment derived from the ⁇ 3 integrin and a fragment of a receptor polypeptide selected from the folate receptor or the tranferrin receptor.
  • the recombinant vectors is a gene delivery vehicle is select from a recombinant viral vector and a non-viral nucleic acid (DNA or RNA) vector.
  • the recombinant viral vector is selected from the group consisting adenovirus, pox virus, poliovirus, rhinovirus, influenza vims, parvovi us, adeno-associat vims, Herpes vims, simian vims 40, human immunodeficiency vims, measles vim astroviros or corona vims.
  • Preferred viruses are retrovuuses and alphaviruses.
  • retroviruses are avian leukosis vims, bovine leukemia vims, murine leukem vims, mink-cell focus-inducing vims, murine sarcoma vims, reticuloendotheliosis vim gibbon ape leukemia vims, Mason-Pfizer leukemia vims or rous sarcoma vims.
  • the mo preferred murine retroviruses are Abelson, Friend, GrafB, Gross, Kristen, Harvey sarcom raucher, or Moloney leukemia.
  • alphaviruses are Sindbis vim Semliki Forest vims, Middleberg vims, Ross River vims Aura vims, Fort Morgan vims Venezuelan equine encephalitis vims.
  • the recombinant vi is replication defective.
  • Other aspects of the invention relate to packaging cells, producer cells containi the recombinant viral vectors, viral particles produced from the producer cells, a transduced recombinant viral vector target cells, as well as pharmaceutical compositio comprising vectors according to the invention.
  • Preferred compositions include tho comprising lyophilized or dehydrated recombinant vims.
  • Figure 1 is a diagramatic representation of the coagulation pathway.
  • Figure 2 is a schematic illustration of p31N2R5(+).
  • Figure 3 is a schematic illustration of pN2R3(-).
  • Figure 5 is a schematic illustration of pN2R3(+).
  • Figure 6 is a schematic illustration of pN2R5(-).
  • Figure 7 is a schematic illustration of p31N25V(+).
  • Figure 8 is a schematic illustration of pTK_A.
  • Figure 9 is a schematic illustration of pPrTK_A.
  • Figure 10 is a schematic illustration of pTK-1 and pTK-3.
  • Figure 11 is a schematic illustration of a SINDBIS Basic Vector and a SINDBI
  • FIG 12 is a schematic illustration of a representative embodiment of a Eukaryotic Layered Vector Initiation System.
  • Figure 13 is a graph which shows a time course for luciferase expression from ELVIS-LUC and SINBV-LUC vectors.
  • Figure 14 is a schematic illustration of the mechanism for disabling a viral junction region by "RNA loop-out.”
  • Figure 15 is an illustration of one method for modifying a Sindbis junction region.
  • Figure 16 is a schematic illustration of Sindbis Packaging Expression Cassettes.
  • Figure 17 is a bar graph which shows SIN-luc vector packaging by representative packaging cell lines.
  • Figure 18 is a bar graph which shows SIN-luc vector packaging by PCL clone #18 over time.
  • Figure 19 is a graph which illustrates expression and fescue of a Sindbis-luciferase vector.
  • Figure 20 is a graph demonstrating retention of viral activity upon reconstitution of a representative recombinant retrovims lyophilized in a formulation buffer containing mannitol.
  • Figure 21 is a graph demonstrating retention of viral activity upon reconstitution of a representative recombinant retrovims lyophilized in a formulation buffer containing lactose.
  • Figure 22 is a graph demonstrating retention of viral activity upon reconstitution of a representative recombinant retrovims lyophilized in a formulation buffer containing trehalose.
  • Figure 23 is a set of representative graphs comparing stability of liquid non- lyophilized recombinant retrovims stored at -80 C versus lyophilized formulated recombinant retrovims stored at -20 C, using various saccharides. For ease of comparison, the titers have been normalized.
  • Figure 24 is a bar graph which illustrates the effect of ganciclovir on CT26, CT26 ⁇ gal and CT26TK Neo cells.
  • Figure 25 is a graph which illustrates the effect of tumor volume over time in a ganciclovir dose study of mice injected with CT26TK Neo.
  • Figure 26 is a series of four photographs of mice, illustrating the effect of different dose regimens of ganciclovir on intraperitoneal tumor growth.
  • Figure 27 is a series of four photographs of mice, illustrating the effect of different dose regimens of ganciclovir on subcutaneous tumor growth.
  • Figure 28 is a schematic illustration of pHCMV-PA. Pefinjtion of Terms
  • High Affinity Binding Pair refers to a set a molecules which is capable of bindi one another with a KD of less than lO ⁇ M, wherein y is selected from the group consisti of 8, 9, 10, 11, 12, 13, 14 and 15.
  • the * K D " refers to the disassociatio constant of the reaction A + B - AB, wherein A and B are members of the high affini binding pair.
  • Disassociation constants may readily determined by a variety of techniques, including for example by a Scatchard analy (see Scatchard, Ann. N.Y. Acad. Sci. 57:660-672, 1949).
  • Representative examples suitable affinity binding pairs include biotin/avidin, cytostatin/papai phosphonate carboxypeptidase A, and 4CABP RuBisCo.
  • Targeting element refers to a molecule which is capable of specifically interacti with a selected cell type. As utilized within the context of the present invention, targeti elements are considered to specifically bind a selected cell type when a biological effect the coupled targeting element may be seen in that cell type, or, when there is greater than 10 fold difference, and preferably greater than a 25, 50 or 100 fold difference between t binding of the coupled targeting element to target cells and non-target cells. Generally, it preferable that the targeting element bind to the selected cell type with a K D of less th 10- 5 M, preferably less than lO ⁇ M, more preferably less than 10' 7 M, and most preferab less than 10 ⁇ °*M (as determined by a Scatchard analysis, see Scatchard, Ann.
  • the targeting element bi to the selected cell type with an affinity of at least 1 log (i.e., 10 times) less than the affini constant of the high affinity binding pair.
  • Suitable targeting elements are preferably non-immunogenic, n degraded by proteolysis, and not scavenged by the immune system.
  • Particularly preferr targeting elements (which are conjugated to a member of the high affinity binding pai should have a half-life (in the absence of a clearing agent) within an animal of between 1 minutes and 1 week. Representative examples of suitable targeting elements are set fort below in more detail.
  • “Clearing agent” refers to molecules which can covalently or non-covalently intera with circulating coupled targeting elements.
  • the clearing agent is no immunogenic, specific to the coupled targeting element, and large enough to avoid rapi renal clearance.
  • the clearing agent is preferably not degraded by proteolysi and not scavenged by the immune system.
  • Particularly preferred clearing agents for use within the present invention include those which bind to the coupled targeting element at a site other than the affinity binding member, and most preferably, which bind in a manner that blocks the binding of the targeting element to its target. Numerous cleaving agents may be utilized within the context of the present invention, including for example those described by Marshall et al. in Brit. J. Cancer 69:502-507, 1994.
  • Retroviral vector refers to an assembly which is, within preferred embodiments of the invention, capable of directing the expression of a sequences) or gene(s) of interest.
  • the retroviral vector construct should include a 5' LTR, a tRNA binding site, a packaging signal, one or more heterologous sequences, an origin of second strand DNA synthesis and a 3' LTR.
  • heterologous sequences may be included within the vector construct, including for example, sequences which encode a protein (e.g., cytotoxic protein, disease-associated antigen, immune accessory molecule, or replacement protein), or which are useful in and of themselves (e.g., as ribozymes or antisense sequences).
  • the heterologous sequence may merely be a "stuflfer” or “filler” sequence of a size sufficient to allow production of retroviral particles containing the RNA genome.
  • the heterologous sequence is at least 1, 2, 3, 4, 5, 6, 7 or 8 Kb in length.
  • the retroviral vector construct may also include transcriptional promoter/enhancer or locus defining element(s), or other elements which control gene expression by means such as alternate splicing, nuclear RNA export, post-translational modification of messenger, or post -transcriptional modification of protein.
  • the retroviral vector construct may also include selectable markers that confer resistance of recombinant retroviral vector, transduced or transfected, cells to TK, hygromycin, phleomycin, histidinol, or DHFR, as well as one or more specific restriction sites and a translation termination sequence.
  • a “gene delivery vehicle” is a recombinant vehicle, such as a recombinant viral vector, a nucleic acid vector (such as plasmid), a naked nucleic acid molecule such as genes, a nucleic acid molecule complexed to a polycationic molecule capable of neutralizing the negative charge on the nucleic acid molecule and condensing the nucleic acid molecule into a compact molecule, a nucleic acid associated with a liposome (Wang, et al, PNAS 5*/: 7851, 1987), a bacterium, and certain eukaryotic cells such as a producer cell, that are capable of delivering a nucleic acid molecule having one or more desirable properties to host cells in an organism.
  • a recombinant viral vector such as plasmid
  • a naked nucleic acid molecule such as genes
  • a nucleic acid molecule complexed to a polycationic molecule capable of neutralizing the negative charge on the nu
  • the desirable properties include the ability to express a desired substance, such as a protein, enzyme, or antibody, and/or the ability to provide a biological activity, which is where the nucleic acid molecule carried by the GDV is itself the active agent without requiring the expression of a desired substance.
  • nucleic ac molecule incorporates into a specified gene so as to inactivate the gene and "turn off* t product the gene was making.
  • Zvmogen is an inactive enzyme precursor capable of being activated to a catalyt state by post-translational modification. Briefly, some zymogens contain a polypepti chain which blocks enzyme utility. The enzyme is activated by acid or enzymatic cleava that removes the inhibitory polypeptide chain.
  • This invention provides several methods for gene therapy-mediated treatment solid tumors with recombinant vectors based on interference with nutrient supply to th tumor cells caused by (i) selectively initiating irreversible coagulation of the tum vasculature by transferring a gene for any one of several potent diffusible procoagula proteins to intratumoral vascular endothelial cells or perivascular tumor cells , (ii) inhibitio of tumor neovascularization by transfer of a gene encoding an anti-angiogenic protein t intratumoral vascular endothelial cells or perivascular tumor cells, (iii) destruction of th tumor vascular bed by selective killing of intratumoral vascular endothelial cells by transf of a 'suicide' gene and (iv) starving the tumor by creating a microenvironment i perivascular areas which absorbs or metabolizes vital incoming nutrients, accomplished b transfer of genes encoding soluble extracellular matrix-binding nutrient receptors or specifi
  • a method for selectively initiating o exacerbating coagulation of the tumor vasculature by transferring any of a number of gene for procoagulant proteins, fusion proteins comprising at least a procoagulant portion of procoagulant protein and at least that portion of a ligand which binds with high affinity t endothelial cells, inducers of procoagulant proteins or inhibitors of fibrinolysis t intratumoral vascular endothelial cells or perivascular tumor cells using a gene deliver vehicle according to the invention.
  • the establishment of a critical hypercoagulative state in the tumor vascular bed ca be achieved by inducing local secretion of three broad classes of proteins.
  • the first grou comprises procoagulant proteins (e.g., Russell's viper venom factor X-activating facto Russell's viper venom factor V-activating factor, tissue factor, truncated tissue facto cancer procoagulant, thrombin, thrombin-like enzymes, and fusion proteins comprising a or at least the extracellular portion of a procoagulant protein operatively fused to all or least that portion of a ligand which binds with high affinity to an endothelial cell membran or protein associated therewith) that directly activate one or more of the zymogens that make up the physiologic clotting cascade.
  • procoagulant proteins e.g., Russell's viper venom factor X-activating facto Russell's viper venom factor V-activating factor, tissue factor, truncated tissue
  • the second group comprises proteins produced by tumor cells, leukocytes and certain infectious organisms (e.g., von Willebrand factor santigen ⁇ , endothelial-monocyte-activating polypeptide I, endothelial-monocyte-activating polypeptide ⁇ , tumor necrosis factor ⁇ , tumor necrosis factor ⁇ , and the tissue factor- inducing factor from Rickettsia rickettsii) that induce the expression of a key procoagulant protein, such as tissue factor, by host endothelial cells and monocytes macrophages, indirectly resulting in local activation of the clotting cascade.
  • proteins produced by tumor cells, leukocytes and certain infectious organisms e.g., von Willebrand factor santigen ⁇ , endothelial-monocyte-activating polypeptide I, endothelial-monocyte-activating polypeptide ⁇ , tumor necrosis factor ⁇ , tumor necrosis factor ⁇ , and the tissue factor- inducing factor from
  • the third group of proteins inhibits the homeostatic response to coagulation (i.e., fibrinolysis), e.g., ⁇ 2-antiplasmin, plasminogen activator inhibitor I, plasminogen activator inhibitor ⁇ , plasminogen activator inhibitor IH, and Erythrina proteinase inhibitors.
  • coagulation i.e., fibrinolysis
  • procoagulant proteins e.g., ⁇ 2-antiplasmin, plasminogen activator inhibitor I, plasminogen activator inhibitor ⁇ , plasminogen activator inhibitor IH, and Erythrina proteinase inhibitors.
  • procoagulant proteins e.g., ⁇ 2-antiplasmin, plasminogen activator inhibitor I, plasminogen activator inhibitor ⁇ , plasminogen activator inhibitor IH, and Erythrina proteinase inhibitors.
  • FX Factor X
  • FXa Factor X
  • tissue factor TF
  • FVII tissue factor
  • FVTIa tissue factor
  • FVTla FVTla in the endothelial cell membrane
  • FXII is activated to FXIIa as a result of tissue injury or exposure to air and FXIIa in turn activates FXI to FXIa which then converts FVm and FIX on the surface of perturbed endothelial cells or platelets to their respective active forms.
  • FX is activated to FXa by FVI ⁇ a FTXa complexes in the presence of calcium ions and a phospholipid membrane (see Figure 1).
  • a complex of FXa with FVa in the endothelial or platelet membrane with Ca 2+ ions forms the prothrombinase complex, which efficiently catalyses the conversion of prothrombin to thrombin.
  • the prothrombinase complex will generate 1,500 moles(m)/minute(m/min) of thrombin/mole of FXa.
  • Thrombin degrades plasma fibrinogen to fibrin and activates FXm to FXT-Qa which, in turn, cross-links the fibrin molecules to form an insoluble matrix (see Figure 1).
  • Procoagulant factors can act at several points in the clotting cascade. Tissue factor
  • TF truncated TF combine with FVTIa and a phospholipid bilayer to activate FX
  • Russell's Viper venom FX- activating factor (RW-X) and cancer procoagulant (CP) activate FX directly, independently of TF, FVII, FVm, or FIX (Gordon, et al., Hemat. Onco. Clinics North America 6:1359, 1992; Takeya, et al., J. Biol. Chem. 267:14109, 1992).
  • thrombin-like enzymes that have been isolated from the venoms of numerous snakes (Doull, et al, Toxicology, eds. MacMillan Pub. Co., NY 1986), for example, Crotalus adamanteus (Crotalase), Crotalus horridus horridus, Agkistrodon Rhodostoma (Ancrod), Agkistrodon contortrix contortrix, Agkistrodon acutus, Bothrops atrox (Batroxobin), Bothrops marajoensis, Bothrops moojeni, Trimeresurus gramineus, Trimeresurus okinavensis and Bitis gabonica, all of which degrade fibrinogen directly and thus are independent of most of the clotting cascade.
  • TF, RW-X, and CP are extremely potent inducers of coagulation because their effects are amplified by subsequent stages o the zymogen cascade.
  • Other inducers of coagulation include Russell's viper venom factor V-activating factor and truncated tissue factor.
  • Thrombin and thrombin-like enzymes require fibrinogen and FXm to cause clotting.
  • Many tumors induce local fibrin formation as a part of the malignant process (Flier, et al., New Eng. J.
  • TF targeting has been shown to be effective in one tumor model (Moloma, et al. submitted for publication). As such, at present the use of early-acting procoagulants, such as TF, CP and RW-X, is preferred.
  • TNF and EMAP-I and II all induce weak tissue factor activity in endothelial cells and monocytes but act synergistically when combined (Kao, et al, supra, Clauss, et al., J. Biol Chem. 265:7078, 1990). This syngergistic effect may be utilized for procoagulant gene therapy of solid tumors.
  • the EMAP-I or - ⁇ genes Kao, et al, Behring Inst. Mitt. 92:92, 1993; Kao, et al., J. Biol. Chem. 269:9114, 1994
  • TNF potent bicistronic procoagulant gene therapy ve ⁇ ors.
  • EMAP-I or -H TNF TNF ⁇ or ⁇
  • tissue factor-inducing factor from Rickettsia ricketts ⁇ genes into separate vectors which are selectively targeted to tumor cells or tumor endothelial cells by targeting vehicles (e.g. antibodies or other cell tissue specific ligands, proliferation-dependent retroviral vectors) that have few or no common cross- reactivities.
  • targeting vehicles e.g. antibodies or other cell tissue specific ligands, proliferation-dependent retroviral vectors
  • EMAPs I or ⁇ selectively generated at the tumor site by vectors may be combined with systemic therapy with recombinant TNF or flavone acetic acid (FAA), a low molecular weight antitumor agent that is effective against Meth-A tumors but less so in other systems (Murray, et al., Int. J. Cancer 49:254, 1991).
  • FAA flavone acetic acid
  • Another procoagulant protein that may be utilized in the context of this invention is von Willebrand factor antigen II.
  • Dismption of the balance between fibrin formation and fibrinolysis may further promote clot formation.
  • many animal and human tumors are intrinsically prothrombotic (Flier, et al. supra) as a result of local vessel dismption or tumor cell production of TF, TF-like activity, or cancer procoagulant (Gordon, et al, supra.).
  • This process may account, at least in part, for the areas of necrosis in solid tumors and it is thought that functional vascular integrity sufficient to support tumor growth is maintained in vivo by local fibrinolysis that largely keeps pace with fibrin formation (Dvorak, et al, Cancer Res. 4 ⁇ /:3348, 1984).
  • Fibrin is degraded by the protease plasmin, which is generated from the zymogen plasminogen by the action of plasminogen activators (Dane, et al.. Adv. Cancer Res. 44:139, 1985). Both tissue-type (T-PA) and urokinase-type (U-PA) plasminogen activators are secreted by endothelial cells and tumor cells (Dane, et al, supra.). Transfer of genes encoding T-PA or U-PA antagonists to intratumoral endothelial cells or perivascular tumor cells would result in decreased plasmin levels. Several candidate proteins could also perform this function if delivered to tumors by recombinant vectors.
  • Plasminogen activator inhibitors I, ⁇ , and III are competitive inhibitors of plasminogen activators that bind to T-PA and U-PA reversibly, blocking plasminogen cleavage (Wun, et al, Crit. Rev. Biotech. 5:131, 1988).
  • ⁇ 2- antiplasmin is the major plasmin inhibitor in the blood, where it is constitutively present at fairly low levels, irreversibly inactivating plasmin until plasmin becomes present in excess (Aoki, et al, Semin. Thromb. Hemostasis 10:24, 1984).
  • U-PA Overexpression of U-PA by tumor cells is associated with increased metastatic potential and angiogenic activity (Meissauer, et al., Exp. Cell Res. 192:453, 1991).
  • U-PA generates plasmin which, in addition to degrading fibrin, also cleaves procollagenase to collagenase and releases bioactive angiogenic growth factor from the tumor's extracellular matrix.
  • Collagenase is thought to contribute to the degradation of interstitial stroma and basement membranes that is essential to (i) invasion of tumor cell into surrounding tissues (ii) intravasation into local blood vessels and escape from th primary tumor site and (iii) migration of endothelial cells during neovascularizatio (Goodson, et al, PNAS 91:7129, 1994).
  • inhibition of plasmin production or activit impacts upon the pathogenesis of malignancy at several levels.
  • One embodiment of the present invention provides for the expression of genes fo procoagulant or antifibrinolytic protons in capillary endothelial cells (EC) lining the vessel of the tumor vascular bed, or in the subpopulation of tumor cells closest to these vessels.
  • Expression of the gene(s) coding for procoagulant or antifibrinolytic proteins from thes 'perivascular 1 tumor cells (PTC) is important because such expression will provid concentrations of plasma-derived clotting factors sufficient to cause clot formation, and because the coagulation process initiated at the tumor cell surface will spread "back" t nearby vessels to induce widespread ischemia of the tumor.
  • a gene delivery vehicle containing a nucleic acid molecule encoding a procoagulant o antifibrinolytic protein is targeted to perivascular tumor cells, as described below.
  • the tropism of a gene delivery vehicle is combined with the specificity of a polypeptide encoded thereby to achieve operative tumor vascular specificity.
  • a targeted or untargeted gene delivery vehicle can be used to deliver to perivascular tumor cells a recombinant expression vector carrying a fusion gene which encodes a protein having procoagulant activity and tissue specificity.
  • the fusion gene encodes a fusion protein comprising at least the domain(s) responsible for procoagulant activity of a procoagulant protein (e.g., tissue factor, truncated tissue factor Russell's Viper Venom Factor X-activating factor, and cancer procoagulant) fused to at least the receptor binding domain(s) of a ligand which binds with high affinity to a receptor present on an endothelial cell membrane.
  • a procoagulant protein e.g., tissue factor, truncated tissue factor Russell's Viper Venom Factor X-activating factor, and cancer procoagulant
  • receptors After diffusion to nearby endothelial cells of the tumor vascular, receptors bind to the ligand portion of the fusion protein to produce a membrane-bound multimolecular array capable of initiating thrombus formation on the lumenal surface of endothelial cells of the tumor vascular bed.
  • the fusion protein comprises truncated tissue factor (lacking the transmembrane and intracellular domains; Ruf, et al. (1994), J. Biol. Chem., vol. 266:2158) at its amino terminus fused by way of a flexible oligopeptide spacer to the receptor binding domains of VEGF [Peretz, et al. (1992), Biochem. Biophys Res. Commun., vol. 182, no. 3:1340-1347], the receptor for which is overexpressed in endothelial cells.
  • tTF-VEGF truncated tissue factor
  • fusion proteins comprise Russell's Viper Venom Factor X-activating factor or cancer procoagulant in place of tTF, although many other proteins or portions thereof can also be used.
  • receptor binding domains from ligands which bind with high affinity to receptors on endothelial cells may be substituted for the receptor binding domains of VEGF.
  • ligands include, but are not limited to, TGF- ⁇ , FGF, PDGF, u-PA, u-PA receptor antagonist, and ligands for endoglin, endosialin, CD31, CD34, integrin ⁇ V ⁇ 3, and antigen binding fragments of monoclonal antibodies reactive against receptors found on endothelial cells.
  • the invention further provides methods for inhibiting tumor growth or causing regression of established tumors by inhibiting tumor angiogenesis via vector-mediated transfer of genes encoding anti-angiogenic polypeptides.
  • Blocking of angiogenesis may result in an acute hypovascular state and lead to actual tumor regression, rather than just inhibition of growth (Ingber, et al., Nature 348:555, 1990).
  • Numerous proteins secreted by tumor cells have been shown to exert angiogenic activity in several in vivo assays (Bicknell, et al., Eur. J.
  • cytokines basic fibroblast growth factor (bFGF) and vascular endothelial cell growth factor (VEGF) have been proven to function as tumor angiogenesis factors in situ, because transduction with bFGF or VEGF cDNA confers tumorigeneity on non-tumorgenic cell lines (Winer, et al, supra) and blocking of cytokine activity with specific monoclonal antibodies inhibits tumor growth and vascularization (Kim, et al, supra).
  • bFGF basic fibroblast growth factor
  • VEGF vascular endothelial cell growth factor
  • Angiogenesis is a complex process involving at least four steps (Folkman, eds. J.B. Lippincott, Philadelphia, pp. 42-62, 1985).
  • Host endothelial cells (EC) (i) degrade their underlying basement membrane (ii) migrate towards the angiogenic stimulus, (iii) proliferate to seed new capillary buds and (iv) anastamatose with an existing vessel, lay down a new basement membrane, and initiate blood flow through the new capillary loop (Folkman, eds. J.B. Lippincott, Philadelphia, pp. 42-62, 1985). Since each step is essential to the process, inhibitors of angiogenesis may exert their effects in a variety of ways.
  • Physiological angiogenesis is regulated by a balance between angiogenic and anti-angiogenic factors (Fidler, et al. Cell 79:185, 1994).
  • anti-angiogenic proteins include angiostatin, ⁇ -IFN, ⁇ -IFN, platelet factor-4, - 16 -
  • tissue inhibitor of metalloproteinases TIMP-III
  • glioma-deriv angiogenesis inhibiting factor Van Meir, et al, Nature Genetics 8:111, 1994 thrombospondin, the anti-angiogenic fragment of prolactin, heparinase or other appropria peptide proteins or poly nucleotides that directly or indirectly (e.g., by catalysis production of inhibitors such as retinoic acid or its analogs) inhibit cell migratio endothelial cell proliferation, basement membrane degradation or 3-D organization of ne blood vessels (Auerbach, W. and Auerbach, R. Pharmac. Ther,. 63:265, 1994).
  • Blocki monoclonal antibodies against bFGF, VEGF, and the ⁇ Vfl3 integrin are also angiogenes inhibitors.
  • ⁇ -IFN has been shown to induce complete regression of life-threateni hemangiomas in infants and highly vascular Kaposis sarcoma (Ezekowitz, et al, N. Engl. Med 326:1456, 1992; Real, et al, J. Clin. Oncol. 4:544, 1986).
  • the present invention provides for the use of any one of the physiologic polypeptide negative regulators of angiogenesis in anti-angiogenic strategies where t gene encoding the anti-angiogenic factor is delivered to either tumor endothelial cells perivascular tumor cells using a recombinant vector.
  • ⁇ -IFN downregulat bFGF transcription and translation (Singh, et al, Am. J. Pathol 145:365, 1994 consequently, it is most effective if produced within tumor cells.
  • TIMPs inhibit endotheli cell proteases required for extracellular matrix degradation during EC migration (Moses, al, supra) and therefore would be active in solution anywhere in the tumor interstitiu Thrombospondin or blocking anti-FGF or anti- VEGF antibodies antagonize angiogeni cytokine activity at the endothelial cell surface (Kim, et al, supra; Fidler, et al, supra) an are most effective if expressed in the vasculature itself.
  • Anti-angiogenic polypeptide genes may be delivered selectively to perivascul tumor cells with a second generation antibody-targeted recombinant vector.
  • Suitable tum markers for targeting include c-erbB ⁇ /pl ⁇ S 1 * 131 - 2 (Shepard, et al, supra), TAG-7 (Guadagni, et al, supra), carcinoembryonic antigen (CEA, Guadagni, et al, supra), th high affinity folate receptor (Bottero, et al, supra), polymorphic epithelial ucin (PE Rettig, et al, supra), epidermal growth factor receptor (Rettig, et al, supra), a CD4 isoform (Rettig, et al, supra) and prostate-specific antigen (Mulders, et al, supra).
  • angiostatin is produced by cell in primary tumor masses but suppresses neovascularization of distant metastases, perhap explaining the clinical observation that metastases grow more quickly in some patients afte the primary tumor is resected (O'Reilly, et al, supra).
  • angiostatin is sufficientl potent and stable in the circulation to exert endocrine effects, so it may be possible t mimic the presence of the primary tumor by transplantation of a population of normal long lived non-tumorigenic cells that have been stably transduced with recombinant vecto containing an angiostatin gene.
  • direct administration of a vector encodin angiostatin may lead to sufficient local or systemic levels to inhibit growth of primary tumors or metastases.
  • the present invention further provides a method for selectively killing tumor endothelial cells with 'suicide' gene therapy.
  • the physiologic activation of the clotting cascade is a mechanism to stop blood flow and seal the damaged tissue from exposure to the environment. Consequently, damage to vascular endothelial cells that line blood vessels is an initiator of both the intrinsic and extrinsic pathways of coagulation (Kao, et al, supra).
  • Tissue factor in the subendothelial space causes activation of FX by the extrinsic pathway (Drake, et al.. Am J. Pathol 134:10%!, 1989) and platelets that adhere to and become activated by matrix proteins intitiate the intrinsic pathway (Stem, et al., Haemostasis 18:202, 1988).
  • therapeutic strategies that result in the death of significant numbers of endothelial cells in the tumor vascular bed may produce a similar result, particularly, thrombosis and ischemic necrosis of the tumor parenchyma.
  • a mouse tumor line was transduced with a retroviral vector containing a murine ⁇ -IFN gene.
  • ⁇ -IFN which activated local endothelial cells
  • EC local endothelial cells
  • anti-Class II antibodies were utilized that bound specifically to tumor EC in this model (Burrows, et al, Cancer Res. 52:5954, 1992).
  • An effective method of killing tumor endothelial cells is to transfer 'suicide gene into the tumor vascular wall using gene delivery vehicles followed by intravenous delive of a prodrug that is activated by the transgene product withm the cytoplasm of the tum endothelial cell to produce a cytotoxic agent (Moolten, et al, Cancer Gene Therap. 7:27 1994).
  • a number of alternative 'suicide genes' may be useful for cancer gene therap (Moolten, et al, supra).
  • Introduction of the bacterial cytosine deaminase gene (Huber, al, Cancer Res. 53:4619, 1993) into tumor cells confers sensitivity to the antifungal age 5-fluorocytosine (5-FC).
  • Cytosine deaminase converts 5-FC to 5-fluorouracil (5-F Nishiyama, et al. Cancer Res. 45:1753, 1985). Since 5-FU is commonly use chemotherapeutic dmg for breast cancer, several groups have developed cytosin deaminase-based 'suicide gene' therapy models for this disease.
  • Tumor specificity may b further increased by introducing the c-erbB2 promoter/enhancer elements 5' to the cytosin deaminase gene, so that the therapeutic transgene is preferentially transcribed in c-erbB overexpressing breast tumor cells (Harris, et al, Gene Therap. 7:170, 1994).
  • Alkalin phosphatase has been widely explored as prodmg-activating enzyme in the related field antibody directed enzymne-prodmg therapy (ADEPT). This enzyme has the advantage th it can activate a wide range of phosphorylated derivatives of anticancer agents (e.
  • suicide genes may encode a polypeptide or polypeptides (with corresponding non-cytotoxic agent) such as Herpes Simpex vims thymidine kinas (gancyclovir or acyclovir), Varicella Zoster vims thymidine kinase (6 methoxypurin arabino nucleoside; Huber, et al, PNAS 55:8039, 1991), E.
  • E. coli cytosine deaminas fluorouracil; Mullen, C.A., et al, PNAS USA 89:33, 1992
  • E. coli xanthine-guanin phophoribosyl transferase thioxanthine; Beshard, C, etal. Mol. Cell Biol 7:4139, 1987
  • E. coli or Leishmania purine nucleotide phosphorylase variantous nontoxic purin deoxyadenosine, adenosine, deoxyguanosine, or guanosine derivaties (Koszalka, G. an Krenitsky, T.A., J.
  • cytochrome pla50 2B1 or cytochrome p450 reductase e.g., 3amino-l,2, benzotriazine 1,4-dioxide (Walton, M.I., et al, Biochem.
  • cel surface alkaline phosphatase e.g., etoposide monophosphate; Senter, P.D., et al, PNA USA, 55:4842, 1988
  • nhroreductase e.g., metronidazole or nitrofurantoin; Hof, H, et al Immunitat und Infetation, 16:220, 1988
  • N-deoxyribosy transferase (1-deazapurine Betbeder, D., et al, Nucleic Acids Res 77:4217, 1989
  • pymvate ferrodoxi oxidoreductase metalronidazol
  • carboxypepidase G2 aminoacylate nitrogen mustards; Antoniw, P., et al, Brit J. Cance 62:909, 1990
  • carboxypeptidase A metalhotrexate alpha alanine; Haenseler, E., et al, Biochemistry 31:891, 1992
  • ⁇ lactamase cephalosporin derivatives; Meyer, D.L., et al, Cancer Res 53:3956, 1993; and Vradhula, V.M., et al, Bioconj ⁇ gate Chemistry 4:334, 1993
  • Actinomycin D synthetase complex synthetic pentapeptide lactone precursors; Katz, E., et al., J.
  • ⁇ -glucuronidase variant glucuronide derivatives of toxic dmgs such as doxorubicin; Bosslet, K., et al, Cancer Res. 54:2151, 1994; Haeberlin, B., etal., Pharmaceutical Res. 70:1553, 1993.
  • HSV-TK transduced tumor cells can mediate a significant bystander killing effect on untransduced neighboring cells in vitro and in vivo (Moolten, et al, supra; Freeman, et al, Cancer Res. 53:5274, 1993), most commonly as a result of transfer to the toxic ganciclovir metabolite, GCV triphosphate, between adjacent cells through intercellular gap junctions (Bi, et al.. Human Gene Therap., 4:725, 1993).
  • HSV- TK retroviral vectors may account for a significant component of the antitumor activity of the vectors (Ram, et al, J. Neurosurg. 81:256, 1994).
  • the suicide gene is only conditionally cytotoxic to the target cells (i.e. only when GCV is given). Consequently, an ex vivo administration method may be utilized. Briefly, endothelial cells may be isolated from tumor biopsies (Medzelewski, et al, Cancer Res. 54:336, 1994), induced to proliferate with appropriate mitogens (Ferrara, et al, supra) and transduced with TK in vitro.
  • Transplanted EC become inco ⁇ orated into the neovasculature in days to weeks after intratumoral injection (Lai, et al.. Cancer Gene Therap. 7:322, 1994), so GCV treatment would follow a suitable 'lag phase' to allow the transduced EC to integrate functionally in to the tumor vasculature.
  • This approach circumvents the requirement for vectors to target the tumor endothelium after intravenous injection.
  • the two-step enzyme-prodrug system offers greater flexibility of delicate clinical management, because cessation of GCV infusion in the event of (potentially very serious) complications arising from damage to normal EC, would block toxicity without the need to block transgene activity in situ.
  • This invention also provides methods for starving tumor cells of specific nutrients that are required to sustain the hyperactive metabolism and uncontrolled proliferation that characterizes the malignant state. Briefly, recombinant vectors are introduced into perivascular tumor cells which contain gene sequences of competitive inhibitors of nutrie receptors or specific nutrient-degrading enzymes.
  • Tumor growth is clearly rate-limited by oxygen availability because the classic 'cor structure of tumor cells (and many normal cells) around a central vessel is defined in radiu by the diffusion distance of oxygen (Denekamp, et al. Cancer Topics 6:6, 1986) Diffusion of oxygen can be retarded in the perivascular interstitial space of the tumor b therapeutic gene transfer and expression of fetal hemoglobin, or oxygen-binding fragment of fetal hemoglobin.
  • Other methods of decreasing the available oxygen are to deliver the genes fo enzymes that produce molecules capable of binding and displacing the oxygen molecules i the oxygenated hemoglobin complex.
  • enzymes include those that synthesize cyanid (e.g., Wissing F., et al, J. Bacteriol 727:695, 1975; Cutler, A.J., et al., Arch. Bioche Biophys, 238:272, 1985) a carbon monoxide (heme oxygenases; Murphy, B.J., et al Cancer Res.
  • a truncated soluble form of the folate binding protein folate receptor ha was cloned (Weltman, et al, Cancer Res. 52:3396, 1992), which competes with cellula integral membrane receptors if overexpressed in perivascular tumor cells as a result o transfer of the gene with a recombinant vector.
  • Another polypeptide capable of bindin nutrients in the perivascular interstitial space of the tumor that may be used is solubl transferrin receptors.
  • these genes may be engineered to contain DNA sequences encoding binding domains for extracellular matrix proteins.
  • fusion, or "chimeric,” proteins would be secreted by perivascular tumor cells and then would be rapidly bound to the plentiful extracellular matrix proteins, including collagens, laminin, fibronectin, vitronectin and von Willebrands factor (vWF), that are part of the tumor stroma and subendothelial basement membrane.
  • the integrins are a superfamily of heterodimeric adhesion proteins expressed by all cells. In ectoderm-derived tissues (such as epithelial and endothelial cells) integrin function to anchor cells to each other and to tissue stromal elements, directing tissue organization and regulating cell growth and differentiation (Hynes, et al.. Cell 69:11, 1992).
  • integrins particularly those of the 61 and 63 families, bind specifically to extracellular matrix (ECM) proteins, often through recognition of a common tripeptide, Arg-CHy-Asp (RGD), present in collagen, fibronectin, vitronectin and vWF (Ruoslahti, et al., Science 238:491, 1987).
  • RGD extracellular matrix
  • Inclusion of an RGD recognition sequence from one of these integrins in a recombinant nutrient receptor or enzyme gene should permit sequestration of the therapeutic protein in the locale required for optimal activity.
  • the RGD recognition site in the ⁇ 3 integrin has been isolated and sequenced (Fitzgerald, et al, J. Biol Chem. 262:3936, 1987).
  • This domain may be included in ECM-binding nutrient receptor antagonists since it recognizes a number of different ECM proteins.
  • Particularly preferred fusion proteins are those comprising an extracellular matrix binding fragment derived from ⁇ 3 integrin and a fragment of the folate or transferrin receptors.
  • One way of targeting vectors to tumor endothelial cells in animals may be achieved using a chimeric envelope protein that has been engineered to contain an avidin domain (that binds specifically to biotin) and then complexed with biotinyl derivatives of any of several monoclonal antibodies (MAbs) that are selective or specific for capillary endothelial cells in solid tumors.
  • MAbs monoclonal antibodies
  • Such reagents include MAb FB5, recognizes a heavily sialylated glycoprotein, endosialin, that is expressed at low to moderate levels by tumor endothelial cells and reactive fibroblasts but is absent from the vasculature of normal tissues (Rettig, et al., PNAS 59:10832, 1989); TEC-4 and TEC-11, which react with endoglin (CD 105), a Type UI TGFfl receptor that is upregulated; and LM609, a MAb that recognizes a conformational determinant formed by epitopes from the ⁇ and ⁇ chains of the human vitronectin receptor (integrin ⁇ v ⁇ 3 , CD51/CD61).
  • MAb FB5 recognizes a heavily sialylated glycoprotein, endosialin, that is expressed at low to moderate levels by tumor endothelial cells and reactive fibroblasts but is absent from the vasculature of normal tissues (Rettig, et al.
  • LM609 stained vessels only in the tumor sample (Brooks, et al., Science 264:569, 1994).
  • the ⁇ v ⁇ 3 integrin appears to be involve in neovascularization since LM609 inhibited angiogenesis in several models (Brooks, et a supra).
  • VEGF receptor-bound vascular endothelial cell growth factor
  • VEGF is a potent E mitogen that is implicated in tumor angiogenesis in many models (Winer, et al, J. Cli Invest. 97:160, 1993; Kim, et al., Nature 362:841, 1993).
  • Retrovir vectors requiring cell division for transgene expression enter target cells 'piggybacking' on mitogenic factor, VEGF, and are integrated into the genome of target cells at hig efficiency.
  • Suitable tumor markers to target include, but are not limited to, c-erbB2/pl85 ER " (Shepard, et al., J. Clin. Immun. 77:117, 1991), TAG-72 (Guadagni, et al, Int. J. Bio Markers 9:53, 1994), carcinoembryonic antigen (CEA, Guadagni, et al, supra), the hig affinity folate receptor (Bottero, et al, Cancer Res. 53:5791, 1993), polymo ⁇ hic epitheli mucin (PEM, Rettig, et al.
  • Targeting may b achieved with the appropriate biotinylated MAbs complexed to retroviral vectors wit chimeric e ⁇ v-avidin receptors (U.S.S.N. 08/242,407), or a direct e ⁇ v-ligand Chimera containing (e.g., heregulin, the ligand for c-erbB2) recombinant vector could be used.
  • particularly preferred targeting ligand is folk acid because it is readily available and can b attached to recombinant vectors by a relatively mild chemical procedure (Lee, et al Cancer Res. 52:5954, 1992). Importantly, the semm half-life of the vector may be limited without loss of activity since the target subpopulation of tumor cells is one of the mos readily accessible extravascular tissues in the body.
  • Targeting to perivascular tumor cells o a procoagulant protein whose site of action is "upstream" (i.e., in the lumen of the adjacen capillary) has been shown to be effective in vivo using a bispecific anti-tumor/anti-tissu factor antibody (Molema, et al, submitted for publication) because clotting factors lea into the perivascular tumor interstium through the hype ⁇ ermeavle tumor vascularit (Senger, et al, supra).
  • targeting may also be achieved using hybrid envelopes (WO 92/14829) carrying other ligands for target receptors such as heregulin (bind her-2-neu) or EPO (binds the EPO receptor) (WO 93/25234).
  • Such hybrid envelopes may need to be combined with an unmodified envelope to give the required performance.
  • the receptor ligand affinity may not be of the order that is deemed desirable for monoclonal antibody targeting, it may be sufficient to target the vector because of the multiple ligand receptor interactions with the target cells.
  • This 'Velcro!' effect is known to give vector-cell affinities in 10 ⁇ 10 to 10 *15 range as measured by Scatchard blots (Yu, H., et al., Proceedings of 1st West Coast Retrovims Meeting, Eds. H. Fan and M. Linial, 1994)
  • the procoagulant protein be synthesized close to the vasculature and this will occur because the perivascular tumor cells are the most physically accessible tumor cells to circulating vector particles and are rapidly proliferating, thus being particularly susceptible to recombinant viral infection. Since tumor cells are suseptable to integration because of their increased proliferation, gene therapy using recombinant retroviral vectors based on the C-type viruses (e.g., MLV) may be used without targeting. Because only cycling cells will integrate the vector, non-dividing normal cells will be unaffected. Other proliferating epithelial cells (e.g., in hair follicles and the lumen of the gut) are separated from extravasating vector particles by several layers of stromal tissue. Consequently, it is unlikely that the vector would penetrate deeply enough into the tissue to be able to integrate in such cells (Burrows, et al, Cancer Res. 52:5954, 1992).
  • C-type viruses e.g., MLV
  • the present invention further provides recombinant vectors in a variety of viral vector packaging systems in which one or more essential functions of the parent vims has been deleted so that it is deficient in some function (e.g., genome replication), but retains a packaging signal and the ability to express a heterologous inserted gene sequence (the "gene of interest").
  • the deleted essential function or functions are provided by packaging cells into which the vector genome can be introduced to yield producer cell lines that then make replication defective viral particles encapsidating the recombinant vector.
  • the recombinant vector may further contain one or more nucleic acid cassettes described above. The vector genome is then introduced into target cells by an infection event ("transduction") but is incapable of further propagation.
  • influenza vim (Luytjes et al, Cell 59:1107, 1989; McM heal et al, The New England Journal o Medicine 309:13, 1983; and Yap et al, Nature 273:238, 1978) (ATCC VR-797) adenovirus (Berkner et al, Biotechniques 6:616, 1988, and Rosenfeld et al, Scienc 252:431, 1991) (ATCC VR-1); parvovims such as adeno-associated vims (Samulski et al, J. Vir.
  • such vectors, packaging cells and producer cells may be constmcted from a variety of retroviruses, including avian leukosis vims (ATCC Nos. VR-535 and VR-247), BLV (VR-1315), bovine leukemia vims, MLV, mink-cell focus-inducing vims (Koch et al, J. Vir. 49:828, 1984; and Oliff et al, J. Vir. 45:542, 1983), murine sarcoma vims (ATCC Nos.
  • VR-844, 45010 and 45016 reticuloendotheliosis vims (ATCC Nos VR-994, VR-770 and 45011), gibbon ape leukemia vims, Mason-Pfizer leukemia vims, and rous sarcoma vims (ATCC Nos VR-772, VR-354, VR-270, VR-724 and VR-725).
  • retroviruses are murine leukemia viruses 4070 A and 1504 A (Hartley, J. Vir. 79:19, 1976), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi (Ru et al., J. Vir.
  • the expression vector may be readily assembled from any vims or retrovims utilizing standard recombinant techniques (e.g., Sambrook et. al. Molecular Cloning: A Laboratory Manual, 2d ed. Cold Spring Harbor Laboratory Press, 1989). Further description of the construction of retroviral vectors is described in U.S.S.N. 07/586,603, herein inco ⁇ orated by reference.
  • the recombinant vector is a recombinant retroviral vector.
  • These constmcts carry or express a selected gene(s) or sequences) of interest.
  • Numerous retroviral gene delivery vehicles may be utilized within the context of the present invention, including for example EP 415,731; WO 90/07936; WO 91/0285, WO 94/03622; WO 93/25698; WO 93/25234; U.S. Patent No. 5,219,740; WO 93/11230; WO 9310218; Vile and Hart, Cancer Res. 53:3860-3864, 1993; Vile and Hart, Cancer Res.
  • Retroviral gene delivery vehicles of the present invention may be readily constmcted from a wide variety of retroviruses, including for example, B, C, and D type retroviruses as well as spumavimses and lentivimses (see RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985). Briefly, viruses are often classified according to their mo ⁇ hology as seen under electron microscopy. Type "B” retroviruses appear to have an eccentric core, while type “C” retroviruses have a central core. Type “D” retroviruses have a mo ⁇ hology intermediate between type B and type C retroviruses.
  • retroviruses include those discussed in RNA Tumor Vimses, supra, as well as a variety of xenotropic retrovinises (e.g., NZB-X1, NZB-X2 and NZBQ_I (see O'Neill et al., J. Vir. 53:100-106, 1985)) and polytropic retroviruses (e.g., MCF and MCF-MLV (see Kelly et al, J. Vir. 45(l):291-298, 1983)).
  • xenotropic retrovinises e.g., NZB-X1, NZB-X2 and NZBQ_I
  • polytropic retroviruses e.g., MCF and MCF-MLV (see Kelly et al, J. Vir. 45(l):291-298, 1983)
  • retroviruses may be readily obtained from depositories or collections such as the American Type Culture Collection (ATCC, Rockville, MD), or isolated from known sources using commonly available techniques.
  • retroviruses for the preparation or construction of retrovir gene delivery vehicles of the present invention include retroviruses selected from the gro consisting of Avian Leukosis Vms, Bovine Leukemia Virus, Murine Leukemia Vim Mink-Cell Focus-Inducing Vims, Murine Sarcoma Vims, Reticuloendotheliosis vims a Rous Sarcoma Vims.
  • retroviruses selected from the gro consisting of Avian Leukosis Vms, Bovine Leukemia Virus, Murine Leukemia Vim Mink-Cell Focus-Inducing Vims, Murine Sarcoma Vims, Reticuloendotheliosis vims a Rous Sarcoma Vims.
  • Particularly preferred Murine Leukemia Viruses include 4070A an 1504A (Hartley and Rowe, J. Virol 79:19-25, 1976), Abelson (ATCC No. VR-999 Friend (ATCC No. VR-24
  • Rous Sarcoma Viruses include Bratislav Bryan high titer (e.g., ATCC Nos. VR-334, VR-657, VR-726, VR-659, and VR-728 Bryan standard, Carr-Zilber, Engelbreth-Holm, Harris, Prague (e.g., ATCC Nos. VR-77 and 45033), and Schmidt-Ruppin (e.g. ATCC Nos. VR-724, VR-725, VR-354).
  • retroviral gene delivery vehicles Any of the above retroviruses may be readily utilized in order to assemble construct retroviral gene delivery vehicles given the disclosure provided herein, an standard recombinant techniques (e.g., Sambrook et al. Molecular Cloning: A Laborato Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Kunkle, PNAS 52:48 1985).
  • portions of the retrovir gene delivery vehicles may be derived from different retroviruses.
  • recombinant retroviral vector LTRs may be derived from Murine Sarcoma Vims, a tRNA binding site from a Rous Sarcoma Vims, a packagin signal from a Murine Leukemia Vims, and an origin of second strand synthesis from a Avian Leukosis Vims.
  • retroviral vector constmcts comprising a 5' LTR, a tRNA binding site, a packaging signal, one or more heterologou sequences, an origin of second strand DNA synthesis and a 3' LTR, wherein the vecto construct lacks gag/pol or env coding sequences.
  • LTRs Long Terminal Repeat
  • U5 Long Terminal Repeat
  • U3 LTRs Three elements, designated U5, R and U3.
  • These element contain a variety of signals which are responsible for the biological activity of a retrovim including for example, promoter and enhancer elements which are located within U3 LTRs may be readily identified in the provims (integrated DNA form) due to their precis duplication at either end of the genome.
  • a 5' LTR should be understoo to include a 5' promoter element and sufficient LTR sequence to allow revers transcription and integration of the DNA form of the vector.
  • the 3' LTR should b understood to include a polyadenylation signal, and sufficient LTR sequence to allo reverse transcription and integration of the DNA form of the vector.
  • the tRNA binding site and origin of second strand DNA synthesis are als important for a retrovims to be biologically active, and may be readily identified by one o skill in the art.
  • retroviral tRNA binds to a tRNA binding site by Watson- Crick base pairing, and is carried with the retrovims genome into a viral particle.
  • the tRNA is then utilized as a primer for DNA synthesis by reverse transcriptase.
  • the tRNA binding site may be readily identified based upon its location just downstream from the 5' LTR.
  • the origin of second strand DNA synthesis is, as its name implies, important for the second strand DNA synthesis of a retrovims. This region, which is also referred to as the poly-purine tract, is located just upstream of the 3' LTR.
  • certain preferred recombinant retroviral vector constructs which are provided herein also comprise a packaging signal, as well as one or more heterologous sequences, each of which is discussed in more detail below.
  • recombinant retroviral vector constmcts which lack both gag/pol and env coding sequences.
  • the phrase "lacks gag/pol or env coding sequences" should be understood to mean that the recombinant retroviral vector does not contain at least 20, preferably at least 15, more preferably at least 10, and most preferably at least 8 consecutive nucleotides which are found in gag/pol or env genes, and in particular, within gag/pol or env expression cassettes that are used to construct packaging cell lines for the recombinant retroviral vector constmcts are set forth in more detail below and in Example 1.
  • the phrase "extended packaging signal” refers to a sequence of nucleotides beyond the minimum core sequence which is required for packaging, that allows increased viral titer due to enhanced packaging.
  • the minimum core packaging signal is encoded by the sequence beginning from the end of the 5' LTR (approximately nucleotide 144), up through the Pst I site (nucleotide 567).
  • the extended packaging signal of MoMLV includes the sequence beyond nucleotide 567 up through the start of the gag/pol gene (nucleotide 621), and beyond nucleotide 1560.
  • recombinant retroviral vector constmcts which lack extended packaging signal may be constmcted from the MoMLV by deleting or truncating the packaging signal prior to nucleotide 567.
  • recombinant retroviral vector constmcts are provided wherein the packaging signal that extends into, or overlaps with, retroviral gag/pol sequence is deleted or tmncated.
  • the packaging signal is deleted or tmncated prior to the start of the gag/pol gene (nucleotide 621).
  • the packagin signal is terminated at nucleotide 570, 575, 580, 585, 590, 595, 600, 610, 615 or 617.
  • recombinant retroviral vector constmcts ar provided which include a packaging signal that extends beyond the start of the gag/po gene (e.g., for MoMLV, beyond nucleotide 621).
  • a packaging signal that extends beyond the start of the gag/po gene (e.g., for MoMLV, beyond nucleotide 621).
  • packaging cell lines for the production o recombinant viral particles wherein the 5' terminal end of the gag/pol gene in a gag/po expression cassette has been modified to contain codons which are degenerate for gag.
  • recombinant retroviral vecto constmcts comprising a 5' LTR, a tRNA binding site, a packaging signal, a origin of second strand DNA synthesis and a 3' LTR, wherein the recombinant retrovira vector constmct does not contain a retroviral nucleic acid sequence upstream of the 5 LTR.
  • the phrase "does not contai a retroviral nucleic acid sequence upstream of the 5' LTR" should be understood to mea that the recombinant retroviral vector does not contain at least 20, preferably at least 15 more preferably at least 10, and most preferably at least 8 consecutive nucleotides whic are found in a retrovims, and more specifically, in a retrovims which is homologous to th recombinant retroviral vector constmct.
  • the recombinan retroviral vector constmcts do not contain a env coding sequence upstream of the 5' LTR.
  • a particularly preferred embodiment of such recombinant retroviral vector constmcts is se forth in more detail below in Example 1.
  • recombinant retroviral vecto constmcts comprising a 5' LTR, a tRNA binding site, a packaging signal, a origin of second strand DNA synthesis and a 3' LTR, wherein the recombinant retroviral vector constmct does not contain a retroviral packaging signal sequence downstream of the 3' LTR.
  • packaging signal sequence should be understood to mean a sequence sufficient to allow packaging of the RNA genome.
  • packaging cell lines suitable for use with the above described recombinant retroviral vector constmcts may be readily prepared (see U.S. Serial No. 08/240,030, filed May 9, 1994; see also U.S. Serial No. 07/800,921, filed November 27, 1991), and utilized to create producer cell lines (also termed vector cell lines or "VCLs") for the production o recombinant vector particles.
  • the recombinant viral vector is a recombinant alphavims viral vector based on Sindbis vims, which may function as gene delivery vehicles.
  • Sindbis vims is the prototype member of the alphavims genus of the togavims family.
  • the unsegmented genomic RNA (49S RNA) of Sindbis virus is approximately 11,703 nucleotides in length, contains a 5' cap and a 3' poly- adenylated tail, and displays positive polarity.
  • Infectious enveloped Sindbis vims is produced by assembly of the viral nucleocapsid proteins onto the viral genomic RNA in the cytoplasm and budding through the cell membrane embedded with viral encoded glycoproteins. Entry of vims into cells is by endocytosis through clatharin coated pits, fusion of the viral membrane with the endosome, release of the nucleocapsid, and uncoating of the viral genome.
  • the genomic 49S RNA serves as template for synthesis of the complementary negative strand.
  • This negative strand in turn serves as template for genomic RNA and an internally initiated 26S subgenomic RNA.
  • the Sindbis viral nonstmctural proteins are translated from the genomic RNA while structural proteins are translated from the subgenomic 26S RNA.
  • AU viral genes are expressed as a polyprotein and processed into individual proteins by post translational proteolytic cleavage.
  • the packaging sequence resides within the nonstmctural coding region, therefore only the genomic 49S RNA is packaged into virions.
  • alphavims vector systems may be constmcted and utilized within the present invention. Representative examples of such systems include those described within U.S. Patent Nos. 5,091,309 and 5,217,879.
  • Sindbis vector constmcts comprising a 5' sequence which is capable of initiating transcription of a Sindbis vims, a nucleotide sequence encoding Sindbis non-structural proteins, a viral junction region which has been inactivated such that viral transcription of the subgenomic fragment is prevented, and a Sindbis RNA polymerase recognition sequence.
  • the viral junction region has been modified such that viral transcription of the subgenomic fragment is reduced, increased, or maintained.
  • Sindbis vector constmcts comprising a 5' sequence which is capable of initiating transcription of a Sindbis vims, a nucleotide sequence encoding Sindbis non-structural proteins, a first viral junction region which has been inactivated such that viral transcription of the subgenomic fragment is prevented, a second viral junction region which has been modified such that viral transcription of the subgenomic fragment is reduced, and a Sindbis RNA polymerase recognition sequence.
  • Sindbis cDNA vector constmcts are provided comprising the above-described vector constmcts, in addition to a 5' promoter which is capable of initiating the synthesis of viral RNA from cDNA, and a 3 * sequence which controls transcription termination.
  • the vector constmcts described above contain n Sindbis structural proteins in the vector constmcts the selected heterologous sequence ma be located downstream from the viral junction region; in the vector constmcts describe above having a second viral junction, the selected heterologous sequence may be locate downstream from the second viral junction region, where the heterologous sequence i located downstream, the vector constmct may comprise a polylinker located between th viral junction region and said heterologous sequence, and preferably the polylinker does no contain a wild-type Sindbis vims restriction endonuclease recognition sequence.
  • Sindbis vector constmcts as well as numerous similar vecto constructs, may be readily prepared essentially as described in U.S. Serial No. 08/405,627 which is incorporated herein by reference in its entirety.
  • gene delivery vehicles include direct delivery of nuclei acid expression vectors, naked DNA alone (WO 90/11092), polycationic lipids an liposomes encapsulating nucleic add (single or double stranded DNA or RNA) expressio vectors, polycation condensed DNA linked or unlinked to killed adenovirus (Curiel et al, Hum. Gene Ther. 3:147-154, 1992), DNA linked to a ligand with or without one of th high affinity pairs described above (Wu et al, J. of Biol.
  • RNA pol II transcription encodes a vira bacteriophage or other source RNA replicase and in the same or a separate transcript, a RNA message that can be amplified in the cytoplasm by the RNA replicase, encoding th desired gene to be explored for therapy (U.S.S.N. 08/404,796).
  • a targeting element is utilized to specifically direct a gene delivery vehicle to selected cell type.
  • targeting elements are proteins or peptides, although other non-proteinaceous molecules may also function as targeting elements.
  • a wide variety o targeting elements are known in the art.
  • antibodies may be utilized in order to target a selected cell type (see generally, Wilchek and Bayer, Anal Biochem 777:1-32, 1988).
  • anti-CD34 antibodies e.g., 12.8 (Andrews et al, Blood 67:842, 1986), and MylO (Civin et al, J. Immunol 733:157, 1984; commercially available from Becton Dickinson under the designation HPCA-2)
  • anti-CD34 antibodies e.g., 12.8 (Andrews et al, Blood 67:842, 1986)
  • MylO Cross-CD34 antigen on stem cells
  • anti-CD4 antibody which may be utilized to target CD4+ T-cells
  • the HER2/neu monoclonal antibody 4D5 (Samp, et al, Growth Regul 7:72, 1991) to target ovarian and breast cells
  • the c-erbB-2 monoclonal antibody GDF-OA-pl85-l (Alper, et al, Cell Growth Differ. 7:591, 1990) to target breast cells
  • the TAG72 monoclonal Ab: CC49 and B72.3 King, et al, J. Biochem. 257:317, 1992
  • the carcinoembryonic antigen monoclonal antibody ZCE025 (Nap, et al, Cane. Res. 52:2329, 1992) to target colon carcinoma cells.
  • a high affinity binding pair can be employed.
  • a wide variety of high affinity binding pairs are known in the art.
  • suitable affinity binding pairs include biotin/avidin with an affinity (K D ) of 10" 15 M (Richards, Meth. Era. 754:3-5, 1990; Green, Adv. in Protein Chem.
  • a wide variety of other high affinity binding pairs may also be developed, for example, by preparing and selecting antibodies which recognize a selected antigen, and by further screening of such antibodies in order to select those with a high affinity (see generally, U.S. Patent Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993; see also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988).
  • antibodies or antibody fragments may also be produced and selected utilizing recombinant techniques (see William D.
  • either member (or molecule) of the affinity binding pair may be coupled to the gene delivery vehicle (or conversely, the targeting element).
  • the larger of the two affinity binding pairs (e.g., avidin of the avidin/biotin pair) is coupled to gene delivery vehicle.
  • certain embodiments of the present invention provide gene delivery vehicles which have been coupled to a member of a high affinity binding pair (also referred to as the "coupled gene delivery vehicle"), as well as targeting elements which have coupled to a member of a high affinity binding pair (also referred to as the "coupled targetin element").
  • the term “coupled” ma refer to either noncovalent or covalent interactions, although generally covalent bonds ar preferred.
  • Numerous methods may be utilized in order to couple one member of a hig affinity binding pair to either a gene delivery vehicle or a targeting element, including fo example use of crosslinking agents such as N-succinimidyl-3-(2 -pyridyl dithio) propionat (“SPDP”; Carlson et al., J. Biochem. 173:723, 1978); Sulfosuccinimidyl 4-N maleimidomethyl) cyclohexane- 1 -carboxylate (“SulfoSMCC”); l-ethyl-3 (3 dimethylaminopropyl) carbodiimide (“EDC”); Bis-diazobenzidine (“BDB”); and Periodi acid / Schiffs base.
  • SPDP N-succinimidyl-3-(2 -pyridyl dithio) propionat
  • a member of the hig affinity binding pair is either expressed on, or included as an integral part of, the exterio (e.g., envelope) of the gene delivery vehicle.
  • the exterio e.g., envelope
  • a member of the affinity binding pair is co-expressed along with the envelop protein of a viral gene delivery vehicle, as a hybrid protein.
  • targeting technologies are also known i the art and may be employed to target a gene delivery vehicle according to the invention t a particular tumor or cell type.
  • any of the gene delivery vehicles described above may include, contain (and/o express) one or more heterologous sequences.
  • a wide variety of heterologous sequence may be utilized within the context of the present invention, including for example nucleotide sequences which encode polypeptides capable of causing blood clot formation inhibiting fibrinolysis, inhibiting tumor vascularization, activating a compound with little o no cytotoxicity (i.e., a "prodmg") into a toxic product, and binding or metabolizin nutrients in the perivascular interstitium.
  • DNA molecules encoding such heterologous genes may be obtained from a variet of sources, such as cloning from plasmids deposited at recognized depositories of biological materials.
  • known cDNA sequences encoding cytotoxic genes or othe heterologous sequences may be obtained from cells which express or contain such sequences.
  • mRNA from a cell expressing the gene of interest is reverse transcribed with reverse transcriptase using oligo dT or random primers.
  • the single-stranded cDNA may then be amplified by PCR (see U.S. 4,683,202, U.S. 4,683,195 and U.S. 4,800,159.
  • oligonucleotide primers complementary to sequences upstream or downstream of desired sequences are denatured by heating in the presence of heat stable Taq polymerase, sequence specific DNA primers, and nucleotide bases dATP, dCTP, dGTP and dTTP. After annealing and elongation, double-stranded DNA is produced when synthesis is complete. This cycle may be repeated many times, resulting in an exponential amplification of the desired DNA.
  • Sequences which encode the above-described genes of interest may also be partially or completely chemically synthesized, for example, on an Applied Biosystems Inc. automated DNA synthesizer (e.g., ABI, DNA synthesizer model 392 (Foster City, CA)). Such genes may comprise a naturally occurring nucleotide sequence, an "optimized" nucleotide sequence based on codon preference, or combination(s) of the two. In another embodiment of the present invention, recombinant vectors may direct the expression of more than one heterologous sequence.
  • Such multiple sequences may be controlled either by a single promoter (whether it is present in the vector or in the genome into which the vector is integrated), or preferably, by one or more additional promoters and may also include internal ribosome binding sites ("IRBS") in the event polycistronic messages are employed.
  • IRBS internal ribosome binding sites
  • Such sequence are small, typically about 300 bp, and may readily be inco ⁇ orated into a vector in order to express multiple genes from a multicistronic message whose cistrons begin with this sequence (Jacejak and Sarnow, Nature 353:90, 1991).
  • anti-tumor agents may be administered either concurrently or sequentially in order to inhibit the growth of a selected tumor in accordance with the methods of the present invention.
  • an anti-tumor agent such as ⁇ -IFN may be co-administered or sequentially administered to an animal along with other anti-tumor agents such as IL-2, or IL-12, in order to inhibit or destroy the tumor in conjunction with recombinant vectors described herein.
  • Such therapeutic compositions may be administered directly using a single vector which directs the expression of two or more anti-tumor agents or the anti-tumor agents may be expressed by independent vectors.
  • an anti ⁇ tumor agent according to the invention is expressed by a recombinant vector administered to the animal, while other tumor agents (e.g., TL-2, ⁇ -IFN) are administered (e.g., intravenously) as a pharmaceutical composition.
  • a recombinant vector carried by the gene delivery vehicle may also be administered to a patient that expresses two anti-tumor agents.
  • the first anti-tumor agent may be expressed from an LTR present in the recombinant vector and the other agent may utilize an additional transcriptional promoter located between the LTRs.
  • the second anti-tumor agent may be expressed as a polycistronic mRNA, which may inco ⁇ orate one or more internal ribosome binding sites.
  • a polycistronic mRNA which may inco ⁇ orate one or more internal ribosome binding sites.
  • IRBS the upstream untranslated region of the immunoglobulin heavy chain binding protein has been shown to support the internal engagement of a bicistronic message (see Jacejak and Sarnow, Nature 353:90, 1991). This sequence is small (300 bp), and may readily be incorporated into a retroviral vector in order to express multiple genes from a multi-cistronk message whose cistrons begin with this sequence.
  • a representative vector constmct utilizing IRBS is set forth in more detail below in Examples 5(A)(i), 5(BXi), 5(C)(i), and 5(D ⁇ i).
  • compositions comprising a gene delivery vehicle in combination with a pharmaceutically acceptable carrier or diluent.
  • Such pharmaceutical compositions may be prepared in liquid or solid form (e.g., lyophilized). The solid form is suspended in a solution prior to parenteral administration.
  • a the ⁇ eutically effective amount the gene delivery vehicle will be administered via I.V., intra-arterially (I. A ), or intralymphatically (I.L.).
  • a therapeutically effective amount of the gene delivery vehicle is an amount sufficient to halt tumor grwoth and preferably kill the tumor cells.
  • Typical dosages are 10 5 , lO 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 cfu for vectors that carry a selectable marker and equivalent titer by PCR plaque formation or equivalent methods for those that do not.
  • Nucleic acid vectors are delivered at doses of 0.1, 1.0, 10, 100, 1,000, or 10,000 ⁇ g.
  • Pharmaceutically acceptable carriers or diluents are non-toxic to recipients at the dosages and concentrations employed.
  • Representative examples of carriers or diluents for injectable solutions include water, isotonic saline solutions which are preferably buffered at a physiological pH (such as phosphate-buffered saline or Tris-buffered saline), mannitol, lactose, dextrose, glycerol, and ethanol, as well as polypeptides or proteins such as human serum albumin.
  • a particularly preferred composition comprises a vector or recombinant vims in 40 mg/ml mannitol or lactose, 5 mg/ml HSA, 25 mM Tris, pH 7.2, 1 mg ml arginine and 25 to 75 mM NaCl. This composition is stable at -70°C for at least six months.
  • tumors may be selected for treatment in accordance with the methods described herein.
  • solid tumors are preferred, although leukemias and lymphomas may also be treated if they have developed a solid mass, or if suitable tumor associated markers exist such that the tumor cells can be physically separated from nonpathogenic normal cells.
  • acute lymphocytic leukemia cells may be sorted from other lymphocytes with the leukemia specific marker 'CALLA".
  • suitable tumors include melanomas, colorectal carcinomas, lung carcinomas (including large cell, small cell, squamous and adeno-carcinomas), renal cell carcinomas and breast adeno-carcinomas.
  • the N2R5 constmct is mutated by site-directed in vitro mutagenesis to change the ATG start codon to ATT, preventing gag expression.
  • This mutagenized fragment is 200 bp in length and flanked by Pst I restriction sites.
  • the Pst I-Pst I mutated fragment is purified from the SK + plasmid and inserted into the Pst I site of N2 MoMLV 5' LTR in plasmid pUC31 to replace the non-mutated 200 bp fragment.
  • the plasmid pUC31 is derived from pUC19 (Stratagene, San Diego, CA) in which additional restriction sites Xho I, Bgl II, BssH II and Neo I are inserted between the EcoR I and Sac I sites of the polylinker. This construct is designated pUC3 l/N2R5gM.
  • a 1.0 Kb MoMLV 3' LTR EcoR I-EcoR I fragment from N2 is cloned into plasmid SK + resulting in a constmct designated N2R3 * .
  • a 1.0 kilobase (Kb) Cla I-Hind III fragment is purified from this constmct.
  • the Cla I-Cla I dominant selectable marker gene fragment from pAFVXM retroviral vector comprising a SV40 early promoter driving expression of the neomycin
  • (neo) phosphotransferase gene is cloned into the SK + plasmid. This constmct is designated SV+ SV2-W0 .
  • a 1.3 Kb Cla I-BstB I gene fragment is purified from the SK + SV2-neo plasmid.
  • KT-3B or KT-1 vectors are constmcted by a three part ligation in which the Xho I- Cla I fragment containing the gene of interest and the 1.0 Kb MoMLV 3' LTR Cla I-Hind m fragment are inserted into the Xho I-Hind UI site of pUC31/N2R5gM plasmid. Th gives a vector designated as having the KT-1 backbone.
  • the 1.3 Kb Cla I-BstB I neo ge fragment from the pAFVXM retroviral vector is then inserted into the Cla I site of th plasmid in the sense orientation to yield a vector designated as having the KT-3 backbone.
  • phleomycin resistance (Mulsant etal., Som. Ce and Mol Gen. 74:243, 1988, available from Cayla, Cedex, FR) may be used to make th retroviral backbone KT-3BC, for use in transforming genes to cells that are alread neomycin resistant.
  • the plasmid pUT507 (Mulsant et al., Som. Cell and Mol Ge 14:243, 1988) is digested with Nde I and the ends blunted with Klenow fragment. Th sample is then digested with Hpa I and Cla I linkers are ligated to the mix of fragment. Th sample is then digested with Cla I to remove excess Cla I linkers.
  • the 1.2 Kb Cla fragment carrying the rous sarcoma vims (RSV) LTR and the phleomycin resistance gen are isolated by agarose gel electrophoresis followed by purification using GeneClean ⁇ (Biol 01, San Diego, CA). This 1.2 Kb fragment is inserted into KT-1 instead of the 1. Kb Cla I-BstB I neomycin resistance fragment to give the backbone KT-3BC.
  • a polylinker sequence is inserted into each of the KT vector backbones (KT-1, KT 3B, KT-3BC) to facilitate the insertion of heterologous sequences.
  • the polylinker i constmcted using two complementary oligonucleotides that form a duplex with Xho I an Cla I compatible sticky ends when hybridized.
  • oligonucleotides are phosphorylated with T4 polynuclotide kinase, heated to 90_C, and slow cooled to allow hybridization to occur.
  • the hybrid is then ligated to the appropriate KT-1, KT-3B, or KT-3BC vector backbone fragment obtained after digestion with Xho I and Cla I, followed by treatment with alkaline phosphatase and agarose gel purification.
  • the resulting constmcts contain Xho I, Eco47 III, Nsi I, Pme I, Mlu I, Not I, Pml I, and Cla I, as unique sites for the insertion of heterologous sequences. These constmcts are designated pKT-lL, pKT-3BL, and pKT-3BCL, respectively.
  • the following retroviral vector is based on the N2 vector. Briefly, 5' and 3' Eco RI LTR fragment (2.8 and 1.0 Kb, respectively) (Armentano, J. Vir. 67:1647, 1987; Eglitis, Science 230:1395, 1985) are initially subcloned into the Eco RI site of plasmids SK+ and pUC31.
  • pUC31 is a modification of pUC19 carrying additional restriction sites (Xho I, Bgl ⁇ , BssH ⁇ , and Neo I) between the Eco RI and Sac I sites of the polylinker.
  • Plasmid N2R3+/- is thereby created from ligation of the SK + plasmid with the 1.0 Kb 3' LTR fragment.
  • the plasmids p31N2R5+/- and p31N2R3+/- are constmcted from the ligation of pUC31 with the 2.8 Kb 5' LTR and packaging signal ( ⁇ ) or the 1.0 Kb 3' LTR fragment, respectively.
  • N2 refers to the vector source
  • R refers to the fact that the fragment is an Eco RI fragment
  • 5 and 3 refer to 5' or 3' LTRs
  • + or - refers to the orientation of the insert (see Figures 2-7 for examples of LTR subclones).
  • a 1.2 Kb Cla I/Eco RI 5' LTR and ⁇ fragment from N2 is subcloned into the same sites of an SK vector.
  • This vector is designated pN2CR5.
  • the 5' LTR containing a 6 base pair (bp) deletion of the splice donor sequence (Yee et al, Cold Spring Harbor, Quantitative Biology, 51:1021, 1986) is subcloned as a 1.8 Kb Eco RI fragment into pUC31.
  • This vector is designated p31N25 ⁇ [+] ( Figure 7).
  • HSV-1 thymidine kinase gene The coding region and transcriptional termination signals of HSV-1 thymidine kinase gene (HSVTK) are isolated as a 1.8 Kb Bgl ⁇ /Pvu II fragment from plasmid 322TK (3.5 Kb Bam HI fragment of HSV-1 (McKnight et al, Nuc. Acids 5:5949, 1980) cloned into Bam HI of ⁇ BR322 (ATCC No. 31344)) and cloned into Bgl ⁇ /Sma I-digested pUC31. This constmct is designated pUCTK.
  • pUCTK is digested with Sma I and Bam HI and the 0.3 Kb fragment containing the (A) n signal is r ⁇ noved.
  • the remaining coding sequences and Bam H overiiang are reconstituted with a double-stranded oligonucleotide made from the followin oligomers:
  • the oligonucleotides are designed to destroy the Sma I sit while maintaining the Ava I she without changing the translated protein.
  • the plasmid pPrTK ⁇ A ( Figure 9), which contains the HSVTK promoter an coding sequence (lacking an (A) n signal), is constmcted as follows.
  • pTK ⁇ A is linearized with Bgl ⁇ , treated with alkaline phosphatase, and ge purified.
  • the retroviral provectors pTK-1 and pTK-3 are constmcted essentially as described below.
  • HSVTK coding sequences lacking transcriptional termination sequences are isolated as a 1.2 Kb Xho I Bam HI fragment from pTK ⁇ A ( Figure 3).
  • 3' LTR sequences are isolated as a 1.0 Kb Bam Hi/Hind IH fragment from pN2R3(-) ( Figure 3).
  • 4. The fragments from steps 1-3 are mixed, ligated, transformed into bacteria, and individual clones identified by restriction enzyme analysis. The constmct is designated pTK-1 ( Figure 10).
  • pTK-3 is constmcted by linearizing pTK-1 with Bam HI, filling in the 5' overhang and blunt-end ligating a 5'-filled Cla I/Cla I fragment containing the bacterial lac UV5 promoter, SV40 early promoter, plus Tn5 neo r gene obtained from pAFVXM retroviral vector (Krieger et al, Cell 39:483, 1984; St. Louis et al, PNAS 85:3150, 1988). Kanamycin-resistant clones are isolated and individual clones are screened for the proper orientation by restriction enzyme analysis (see Figure 10).
  • constmcts are used to generate infectious recombinant vector particles in conjunction with a packaging cell line such as DA described below.
  • viruses having an RNA genome with positive polarity is such that when introduced into a eukaryotic cell which serves as a permissive host, the purified genomic nucleic acid serves as a functional messenger RNA (mRNA) molecule.
  • mRNA messenger RNA
  • Sindbis vims strain AR-339 (ATCC #VR-1248, Taylor et al, Am. J. Trop. Med Hyg. 4:844 1955; isolated from the mosquito Culexus univittatus) may be propagated in baby hamster kidney-21 (BHK-21, ATCC # CCL10) cells, infected at low multiplicity (0.1 pfu/cell).
  • Sindbis vims strain HR (Lee Biomolecular, San Diego, CA) may also be used and propagated by the same methods. Sindbis virions are precipitated from a clarified lysate at 48 hours post-infection with 10% (w/v) of polyethylene glycol-8000 (PEG) at 0_C, as described in U.S.S.N.
  • Sindbis virions contained in the PEG pellet may be lysed with 2% SDS, and the poly-adenylated mRNA isolated by chromatography utilizing commercially available oligo dT columns (Invitrogen, San Diego, CA).
  • this primer contains at its 5' end a five nucleotide "buffer sequence' for efficien restriction endonuclease digestion, followed by the Xba I recognition sequence, 2 consecutive dT nucleotides and six nucleotides which are precisely complementary to th extreme Sindbis 3' end.
  • selection for first round cDNA synthesis occurs at tw levels: (1) polyadenylated molecules, a prerequisite for functional mRNA, and (2) selectiv priming from Sindbis mRNA molecules, in a pool containing multiple mRNA species Further, the reverse transcription is performed in the presence of 10 mM MeHgOH t mitigate the frequency of artificial stops during reverse transcription.
  • Sindbis cDNA Primary genomic length Sindbis cDNA is then amplified by PCR in six distinc segments using six pairs of overlapping primers.
  • the Sindbis 5' end forward primer is constructed to contain a 19 nucleotide (nt sequence corresponding to the bacterial SP6 RNA polymerase promoter and the Apa restriction endonuclease recognition sequence linked to its 5' end.
  • the bacterial SP6 RNA polymerase is poised such that transcription in vitro results in the inclusion of only a single non-viral G ribonucleotide linked to the A ribonucleotide, which corresponds to the authentic Sindbis 5' end.
  • Apa I recognition sequence facilitates insertion o the PCR amplicon into the plasmid vector pKS ⁇ + (Stratagene, San Diego, CA) polylinker sequence.
  • a five nt 'buffer sequence" is also inserted prior to the Apa I recognition sequence in order to permit efficient digestion.
  • the sequence of the SP6-5' Sindbis forward primer and all of the primer pairs necessary to amplify the entire Sindbis genome are shown below.
  • the reference sequence (GenBank accession no. SINCG) is from Strauss et al, Virology 133:92-110, 1984.
  • PCR amplification of Sindbis cDNA with the six primer sets shown above is performed in separate reactions, using the Thermalase thermostable DNA polymerase (Amresco Inc., Solon, OH) and the buffer containing 1.5 mM MgCt ⁇ , provided by the supplier. Additionally, the reactions, containing 5% DMSO and Hot Start Wax beads (Perkin-Elmer, Los Angeles, CA), are performed using the following PCR amplification protocol shown below:
  • the six reaction products are inserted first into the pCR II vector (Invitrogen, San Diego, CA). Then using the appropriate enzymes shown above, the fragments are inserted stepwise into the pKS IT 1" vector between the Apa I and Xba I sites. This clone is designated as pVGSP6GEN.
  • Sindbis genomic cDNA clone pVGSP6GEN is linearized by digestion with Xba I, which cuts pVGSP6GEN once, immediately adjacent and downstream of the 25 - 42 -
  • the linearized pVGSP ⁇ GEN clone is purified with GeneClean ⁇ TM and adjusted to a concentration of 0.5 mg ml. Transcription of the linearized pVG5P6GEN done it pqfiniued in vttro at 37_C for 90 minutes according to the following reaction conditions: 2 ⁇ l DNA/4.25 ⁇ l H 2 0; 10 ⁇ l 2.5 mM NTPs (UTP, ATP, GTP, CTP); 1.25 ⁇ l 20 mM Me 7 G(5')ppp(5')G cap analogue; 1.25 ⁇ l 100 mM DTT, 5 ⁇ l 5X transcription buffer (Promega, Madison, Wl); 0.5 ⁇ l RNasin (Promega, Madison, Wl); 0.25 ⁇ l 10 mg/ml bovine serum albumin; and 0.5 ⁇ l SP6 RNA polymerase (Promega, Madison, Wl).
  • the in vitro transcription reaction products are subsequently digested with DNase I (Promega, Madison, Wl) and purified by sequential phenol/CHC and ether extraction, followed by ethanol precipitation, or ahemativdy, are used directly for transfection.
  • the in vitro transcription reaction products or purified RNA are complexed with a commercial cationic lipid compound (LipofectinTM, GIBCO-BRL, Gaithersburg, MD), and applied to BHK-21 cells maintained in a 60 mM petri dish at 75% confluency. The transfected cells are incubated at 30_C. After 94 hours post-transfection, extensive cytopathic effeft (cpe) are observed. No obvious cpe is observed in plates not receiving DNase I (Promega, Madison, Wl) and purified by sequential phenol/CHC and ether extraction, followed by ethanol precipitation, or ahemativdy, are used directly for transfection.
  • RNA transcribed from the Sindbis cDNA done was 1.0 ml of supernatant taken from transfected cells, added to fresh monolayers of BHK-21 cells, and incubated at 30_C or 37_C results in obvious cpe within 18 hours.
  • Sequence analysis of pVGSP6GEN shown in Table 1, reveals multiple sequence differences between the Sindbis genomic done described herein and the viral done whose sequence is contained in Genbank. Many sequence differences result in non-conservative amino adds changes in the Sindbis proteins.
  • virion RNA is amplified by RT-PCR as described above, and sequence relating to the nucleotides in question is determined by direct sequencing of the RT-PCR amplicon product using a commercially available kit (Promega, Madison, Wl) and compared to the corresponding pVGSP6GEN sequence.
  • Table 2 The results of this study are given in Table 2. Briefly, three non- conservative amino add changes, Gly to Glu, Asp to Gly, and Tyr to Cys, which are a result of doning artifacts, are observed at viral nudeotides 2,245, 6,193, and 6,730, respectively. These nt changes result in non-conservative amino add substitutions which all map to the viral non-stmctural protein (NSP) genes, nt 2,245 to NSP 2, and nt 6,193 and 6,730 to NSP4.
  • NSP non-stmctural protein
  • NSP 2 and NSP 4 genes Repair of the NSP 2 and NSP 4 genes is accomplished by RT-PCR, as described above, using virion RNA from a 5 times plaque purified stock.
  • the SP6-1 A/IB primer pair described above is used to repair the nt 2,245 change.
  • the RT-PCR amplicon product is digested with Eco 47m and Bgl EL, and the 882 bp fragment is purified by 1% agarose/TBE - 43 -
  • Noncoding Region
  • the present invention provides eukaryotic layered vector initiation systems which generally comprise a promoter which is capable of initiating the 5' synthesis of RNA from cDNA, a constmct which is capable of autonomous or autocatalytic replication in a cell, the constmct also being capable of expressing a heterologous nucleic acid sequence(s), and a 3' sequence which controls transcription termination.
  • constmcts may be constmcted of the following ordered elements: a 5' eukaryotic promoter capable of initiating the synthesis of viral RNA at the authentic alphavims 5' end, a 5' sequence which is capable of initiating transcription of an alphavims, a nucleotide sequence encoding alphavims nonstmctural proteins, a viral junction region, a heterologous sequence, an alphavims RNA polymerase recognition sequence, and a 3' transcription termination/polyadenylation signal sequence.
  • a 5' eukaryotic promoter capable of initiating the synthesis of viral RNA at the authentic alphavims 5' end
  • a 5' sequence which is capable of initiating transcription of an alphavims a nucleotide sequence encoding alphavims nonstmctural proteins
  • a viral junction region a heterologous sequence
  • an alphavims RNA polymerase recognition sequence an alphavi
  • Such alphavims cDNA expression vectors also may include intervening sequences (introns), which are spliced from the pre-RNA in the nucleus prior to transport to the cytoplasm, and which may improve the overall efficiency of the system, in terms of molecules of functional mRNA transported to the cytoplasm.
  • the intron splicing signals are located, for example, between Sindbis and heterologous gene regions.
  • Plasmid pVGSP6GENrep is digested with Bgl II and Xba I, and the reaction products are electrophoresed in a 0.8% agarose/TBE gel.
  • the resulting 9,438 bp fragment is excised, purified with GeneClean ⁇ TM and ligated into the 4,475 bp vector fragment resulting from treatment of pCDNA3 (Invitrogen, San Diego,
  • the U3 region of the LTR from MoMLV is positioned at the 5' viral end such that the first transcribed nucleotide is a single G residue, which is capped in vivo, followed by the Sindbis 5' end. Juxtaposition of the MoMLV LTR and the Sindbis 5' end is accomplished by overlapping PCR as described below. Amplification of the MoMLV LTR in the first primary PCR reaction is accomplished in a reaction containing the BAG vector (Price et al , PNAS 84: 156- 160, 1987) and the following primer pair:
  • BAGBgl2Fl buffer sequence/Bgl II recoginition sequence/MoMLV LTR nt 1 to 22
  • BAGwt441R2 (SIN nt 5 to 1/MoMLV LTR nt 441 to 406):
  • PCR amplification of the MoMLV LTR with the primer pair shown above is performed using the Thermalase thermostable DNA polymerase and the buffer containing 1.5 mM MgCl 2 , provided by the supplier.
  • the PCR reaction, containing 5% DMSO and Hot Start Wax beads, is conducted using the following PCR amplification protocol:
  • Amplification of the Sindbis 5' end in the second primary PCR reaction is accomplished in a reaction containing the pVGSP6GENrep clone and the following primer pair:
  • Reverse primer (SIN nt 3,182 to 3,160):
  • PCR amplification of the MoMLV LTR is with the primer pair and amplification reaction conditions described above, utilizing the PCR amplification protocol shown below: Temperature (°C) Time (Min.) No. Cycles
  • the 457 bp and 3,202 bp products from the primary PCR reactions are purified with GeneClean IITM, and used together in a PCR reaction with the following primer pair:
  • BAGBgl2Fl buffer sequence/Bgl II recoginition sequence/MoMLV LTR nt 1 to 22
  • Reverse primer (SIN nt 2,300 to 2,278):
  • PCR amplification of the primer PCR amplicon products is with the primer pair and amplification reaction conditions described above, utilizing the following PCR amplification protocol shown below:
  • the 25 3' terminal bases of the first primary PCR amplicon product overlap with the 25 5' terminal bases of the second primary PCR amplicon product; the resultant 2,752 bp - 49 -
  • overlapping secondary PCR amplicon product is purified by 0.8% agarose TBE electrophoresis, digested with Bgl II, and the 2,734 bp product is ligated into pcDNASINbgl/xba treated with Bgl II and calf intestine alkaline phosphatase (CIAP).
  • the resulting construction is 16,656 bp and is designated pVGELVIS. Sindbis nucleotides are contained within bases 1 to 11,700 of the sequence.
  • pVGELVIS plasmid DNA is complexed with Lipofectamine according to the conditions suggested by the supplier (ca.
  • an infectious centers assay is performed. Briefly, 5 ⁇ g of pVGELVIS plasmid DNA is transfected onto BHK-21 cells in 35 mm wells as described above, and at 1.5 hours post- transfection the cells are trypsinized and serially diluted 10,000-fold, over 10-fold increments, into 5 x 10 5 untreated BHK-21 cells. This transfected and untreated BHK-21 cell mixture is then added to 35 mm wells. The cells are allowed to attach to the plate, and subsequently overlayed with media containing 1.0% Noble agar.
  • plaques due to cell lysis may be visualized either directly or after overlaying with a second layer containing neutral red stain.
  • This experiment reveals that the efficiency of the pVGELVIS plasmid in generating wild type Sindbis vims after transfection onto BHK-21 cells is approximately 1.0 x 10 3 pfu/ ⁇ g of plasmid DNA.
  • a first step in the construction of the Sindbis Basic Vector is the generation of two plasmid subclones containing separate elements from the viral 5' and 3' ends. These elements may then be utilized in order to subsequently assemble a basic gene transfer vector.
  • the first plasmid subclone is constmcted to contain the 40 terminal nucleotides of the viral 3' end and a 25 bp stretch of consecutive dA:dT nucleotides.
  • the following oligonucleotide pairs are first synthesized: Forward Primer: SIN11664F: (buffer sequence Not I site/ SIN nt 11,664 to 11,698):
  • oligonucleotides are then mixed together at equal molar concentrations in the presence of 10 mM MgCl2, heated to 100 C for 5 minutes and cooled slowly to room temperature.
  • the partially double-stranded molecule is then filled in using Klenow DNA polymerase and 50 ⁇ M dNTPs.
  • the resultant 89 bp molecule is then digested with Not I and Sac I, purified on a 2% NuSieve 1% agarose gd, and ligated into pKS 11+ plasmid, prepared by digestion with Not I and Sac I and treatment with CIAP, at a 10:1 molar excess of insert to vector. This construction is designated pKSID'SIN.
  • the second plasmid subclone is constmcted to contain the first 5' 7,643 nucleotides of Sindbis and a bacteriophage RNA polymerase promoter positioned at the viral 5' end such that only a single non-viral nucleotide is added to the authentic viral 5' end after in vitro transcription.
  • the 3' end of this clone is derived by a standard three temperature PCR amplification with a reverse primer having the sequence shown below.
  • the reverse primer maps to viral nucleotides 7,643 to 7,621 and is 41 bp downstream from the junction core element 3' end. Additionally, viral nucleotide 7,643 is 4 nucleotides upstream from the structural protein gene translation initiation codon. The first five 5' nucleotides in this primer are included to serve as a "buffer sequence' for the efficient digestion of the PCR amplicon products, and are followed by 6 nucleotides comprising the Xho I recognition sequence.
  • the forward primer in this reaction is primer 2 A (see Example lC(i)), having the following sequence:
  • the 4,510 bp amplicon product resulting from the PCR amplification shown above with pVGSP6GENrep plasmid (see Example lC(i)) as template, is digested with the enzymes Sfi I and Xho I. The resultant 2,526 bp fragment is gel purified. Sindbis cDNA clone pVGSP6GENrep is also digested with Apa I and Sfi I, and the resultant 5,144 bp fragment which includes the SP6 RNA polymerase promoter at its 5' end is gel purified.
  • the 5,144 bp fragment is ligated together with the 2,526 bp fragment from above, along with the Apa I and Xho I digested, CIAP treated pKS + plasmid.
  • a clone is isolated having the Sindbis nucleotides 1 to 7,643 and including an RNA polymerase promoter at its 5' end contained in the pKS ⁇ + plasmid vector. This construction is designated pKS ⁇ 5'SIN.
  • Assembly of the complete basic vector is accomplished by digesting pKSII5'SIN with Xho I and Sac I, treating with CLAP, and gel purifying a large 10,533 bp fragment.
  • pKSSINBV The 10,533 bp fragment is then ligated together with a 168 bp small fragment resulting from digestion of pKSII3'SIN with Xho I and Sac I. This resultant construction is designated pKSSINBV, and is shown in Figure 11 A.
  • the firefly luciferase reporter gene is inserted into the Sindbis Basic Vector in order to demonstrate the expression of a heterologous gene in cells transfected with RNA that is transcribed in vitro from the Sindbis vector clone, and to demonstrate the overall functionality of the Sindbis basic vector.
  • the Sindbis luciferase vector is performed by assembling together components of 3 independent plasmids: pKSII5'SIN, pKSH3'SIN, and pGL2-basic vector.
  • the pGL2-basic vector plasmid contains the entire firefly luciferase gene. Briefly, the luciferase gene is first inserted into the pKSID'SIN plasmid. This is accomplished by digesting pGL2 with Bam HI and Hind III and gel purifying a 2,689 bp containing fragment. This fragment is ligated with a gel purified 3,008 bp fragment resulting from digestion of pKS ⁇ 3'SIN with BamHI and Hind III and treatment with CIAP. The resultant construction is designated pKSID'SIN-luc.
  • a Sindbis luciferase vector is accomplished by digesting pKS ⁇ 5'SIN with Xho I and Sac I, treating with CIAP, and gel purifying the large 10,533 bp fragment.
  • the pKS 5'SIN 10,533 bp fragment is ligated together with the 2,854 bp small fragment resulting from digestion of pKS ⁇ 3 'SIN-luc with Xho I and Sac I.
  • This construction contains the entire Sindbis nonstmctural gene coding region and 3' viral elements necessary for genome replication, as well as the firefly luciferase gene positioned between these two viral 5' and 3' elements.
  • This vector is designated pKSSINBV-luc, and is shown schematically in Figure 1 IB.
  • the SIN BV and SIN-BV-luciferase constmcts described in Section D(iii), above, are inserted into the pVGELVIS vector configurations described in Se ⁇ ion D(ii), such that expression of the heterologous gene from Sindbis vectors occurs after direct introduction of the plasmid DNA into cells.
  • the ability to transfect alphavims-based vector plasmid DNA directly into cells and produce expression levels of heterologous genes typical of transfection of RNA-based alphavims vectors without a primary step consisting of in vitro transcription of linearized template vector DNA enhances greatly the utility and efficiency of certain embodiments of the alphavims-based expression vector system.
  • Figure 12 is a schematic representation of one mechanism of expression of heterologous genes from a plasmid DNA alphavims expression (ELVIS) vectors.
  • ELVIS plasmid DNA alphavims expression
  • the ELVIS vectors may be introduced into the target cells directly by physical means as a DNA molecule, as a complex with various liposome formulations, or as a DNA-ligand complex including the alphavims DNA vector molecule, a polycation compound such as polylysine, a receptor specific ligand, and, optionally, a psoralen inactivated vims such as Sendai or Adenovirus.
  • the first step of constructing a representative plasmid DNA Sindbis expression vector consists of digesting pKSSINBV with Sac I, blunting with T4 polymerase, digesting with Sfi I, isolating the 2,689 bp fragment, and ligating into the pVGELVIS 10,053 bp vector fragment prepared by digestion with XbaL blunting with T4 polymerase, digesting with Sfi I, treatment with CLAP, and 1% agarose/TBE gel electrophoresis. This constmction is designated pVGELVIS-SINBV.
  • the S V40 intron and transcription termination sequences at the 3' end of luciferase must be removed so that when the pre-RNA, transcribed from the plasmid DNA luciferase vector after transfection into cells, is processed the 3' end of the reporter gene is not separated from the Sindbis vector 3' end, as the Sindbis 5' and 3' ends contained within the pVGELVIS- SINBV vector are required in cis for the autocatalytic replication activity of the vector.
  • the Sindbis vector 3' end is required for initiation of synthesis of the antigenomic strand, which is the template for the subgenomic RNA ⁇ icoding the heterologous or reporter protein.
  • the SV40 RNA processing signals positioned at the 3' end of the luciferase gene are removed from the SIN-BV-luc construction described in Section D(iv) above.
  • the modified luciferase fragment is then placed in the pVGELVIS-SINBV constmction described above via unique restriction sites.
  • the alteration of the luciferase gene is accomplished with the primer pair shown bdow:
  • Reverse primer LucStop (buffer sequence/Not I, Xba I recognition sequences/pGL-2 nt 1,725 to 1,703):
  • the primers shown above are used in a PCR reaction with a three temperature cycling program using a 3 minute extension period.
  • the amplification products are purified with GeneClean ⁇ TM, digested with Xho I and Xba I, purified again with GeneClean HTM, and the 2,037 bp fragment is ligated into the 13,799 bp fragment of pVGELVIS-SINBV resulting from digestion with Xho I and Xba I, and treatment with CLAP.
  • This constmction is designated pVGELVIS-SINB V-luc (abbreviated as ELVIS-luc).
  • luciferase in BHK-21 cells transfected with pVGELVIS-SINBV- luc DNA is measured in order to demonstrate that the Sindbis physical gene transfer vector is functional. Briefly, 5 ⁇ g of pVGELVIS-SINBV-luc DNA or 5 ⁇ g of in vitro transcribed RNA from linearized SINBV-luc template as described in Section D(iv), above, are complexed with 10 ⁇ l of LipofectamineTM or LipofectinTM, respe ⁇ ively, and transferted into 5 x 10 5 BHK-21 cells contained in 35 mM petri plates. The luciferase activity is determined from each of three samples at 2, 4, 8, 16, 20, 28, 48, 72, 96, and 120 hours post transfection.
  • the overall efficiency of the ELVIS ve ⁇ or is enhanced by several modifications to the pVGELVIS-SINBV-luc ve ⁇ or, as described in detail in (U.S.S.N. 08/348,472). These modifications include alternate RNA polymerase II promoters and transcription termination signals, additions of intron sequences in the ve ⁇ or construrt, insertion of ribozyme processing sequences adjacent to the alphavims 3 '-end, and substitution with a smaller plasmid ve ⁇ or.
  • a linker sequence also is inserted into the pKSSINBV and pVGELVIS-SINBV constructs to facilitate the insertion of heterologous sequences.
  • the linker is constmcted using two complementary oligonucleotides that form a duplex with Xho I and Xba I compatible sticky ends when hybridized.
  • oligonucleotides are phosphorylated with T4 polynucleotide kinase, heated to 90_C, and slow-cooled to allow hybridization to occur.
  • the hybrid is then ligated to the 10.6 kb fragment of pKSSINBV-Luc obtained after digestion with Xho I and Xba I, followed by treatment with alkaline phosphatase and agarose gel purification.
  • constmct contains Xho I, Pml I, Pme I, Mlu I, B I, Stu I, Xba I, and Not I as unique sites between the Sindbis jun ⁇ ion region and the Sindbis 3' end. This constm ⁇ is designated
  • the polylinker also is cloned into the pVGELVIS-SINBV constm ⁇ s.
  • the linker is inserted by digestion of pVGELVIS-SINBV-luc with Sfi I and Not I.
  • the 10. lkb fragment is agarose gel purified, and this fragment was ligated to the gel purified 2.6kb fragment from a Sfi I/Not I digest of pKSSINBVLII.
  • the resulting constmct contains Xho I, Pml I, Pme I, Mlu I, and Not I as unique sites between the Sindbis jun ⁇ ion region and the Sindbis 3' end. This constm ⁇ is designated pVGELVIS-SINBVLu.
  • Plasmid pKSSINBVLII is modified further by the addition of an Asc I eight nucleotide recognition sequence adjacent to the Sac I transcription mn-off site.
  • the relative infrequency of this recognition sequence allows for linearization of a wide range of heterologous gene-containing Sindbis vector constm ⁇ s.
  • an oligonucleotide which has Sac I compatible termini when self-annealed, is used.
  • the AscI/SacI linker is phosphorylated with T4 polynucleotide kinase, heated to 90 C, and slow-cooled to allow self-annealing to occur.
  • the duplex is then ligated into pKSSINBVLII plasmid DNA which has been digested with Sac I and treated with calf intestinal alkaline phosphatase.
  • the resulting constm ⁇ is designated pKSSINBVLIIA.
  • the disabled jun ⁇ ion loop out model is constru ⁇ ed with the junction region of the ve ⁇ or flanked by inverted repeat sequences which are homologous to the RNA of choice.
  • sequences from the CEA tumor antigen cDNA (Beauchemin et al, Molec. and Cell. Biol. 7:3221, 1987) are used in the inverted repeats.
  • the jun ⁇ ion region is preceded by two CEA anti-sense sequence domains (A 1 and B 1 ) separated by a six bp hinge domain.
  • A2 which is complementary to Al
  • the only two requirements are that they be specific for the targ ⁇ ed RNA sequence and that the anti-sense sequences hybridize to two RNA sequence domains separated by three nucleotides. This three nucleotide gap will serve as a hinge domain for the polymerase to hop and switch reading strands bridging the non-structural protein domain of the ve ⁇ or to the jun ⁇ ion region of the ve ⁇ or ( Figure 15).
  • two oligonucleotides are synthesized complementing each other to create a fragment insert containing convenient restriction enzyme sites at the extreme 5' and 3' ends.
  • the oligonucleotide fragment insert is then ligated into the Sindbis ve ⁇ or between the disabled jun ⁇ ion region and the multiple cloning sites of the Sindbis ve ⁇ or.
  • the sense oligonucleotide strand from 5' to 3', should contain an Apa I restri ⁇ ion site, followed by the Al anti-sense domain, a six bp hinge domain, a Bl anti-sense domain, a synthetic junction region domain, and the A2 sense domain, followed by a Xho I restriction enzyme she.
  • the following oligonucleotide sequence is used to design a CEA RNA responsive Sindbis ve ⁇ or.
  • the nucleotide number sequence is obtained from Beauchemin et al, Molec. and Cell Biol. 7:3221, 1987.
  • CEA sense strand (Sequence ID No.: 30) CEA 618 CEA 589
  • the 5'-3' CEA anti-sense strand is complementary to the above oligonucleotide.
  • the oligonucleotides are mixed together in the presence of 10 mM Mg +2 , heated to 100_C for 5 minutes and cooled slowly to room temperature.
  • the oligonucleotide pair is then digested with the Apa I and Xho I restriction enzymes, mixed and ligated at a 25:1 molar ratio of insert to plasmid (pCMV-SIN or pMET-SIN) predigested with the same enzymes.
  • pCMV-SIN or pMET-SIN pMET-SIN
  • the human gamma interferon ( ⁇ -IFN) gene is subcloned from the retroviral ve ⁇ or plasmid pHu-IFN (Howard et al, Ann N.Y. Acad Sci. 716:167-187, 1994) by digesting with Xho I and Cla I. The resulting 500 bp fragment containing the coding sequences of ⁇ - IFN is isolated from a 1% agarose gel.
  • the human ⁇ -IFN cDNA is derived from RNA isolated from PHA- stimulated Jurkat T cells by guanidinium thiocyanate extraction followed by ultracentrifugation through a CsCl gradient.
  • the RNA (Sigma, St. Louis, MO) is then reverse-transcribed in vitro and a gene-specific oligonucleotide pair is used to amplify ⁇ - IFN cDNA by polymerase chain rea ⁇ ion using Taq polymerase.
  • the PCR DNA was repaired with T4 DNA polymerase and Klenow and cloned into the Hinc II site of pSK + plasmid treated with CIAP.
  • the 5' end of the cDNA is adjacent to the Xho I site of the pSK + polylinker and the 3' end adjacent to the Cla I site.
  • the 512 base pair fragment encoding the human ⁇ -IFN molecule is placed into the Xho I/Cla I site of either the pCMV/SIN-CEA or pMET/SIN-CEA ve ⁇ ors.
  • These new plasmids are designated pCMV/SIN-CEA/TFN or pMET/SIN-CEA/TFN, respe ⁇ ively.
  • a PCR amplified produ ⁇ containing the cDNA clone of the HSVTK, flanked with 5' Xho I and 3' Cla I restriction enzyme sites is obtained using the pHSlTK3KB (McKnight et al, Nuc. Acids Res. 5:5949, 1980) clone as target DNA.
  • the sequences for the primers used for the PCR amplification are obtained from published sequences (Wagner et al, PNAS 78:1442, 1981).
  • the 1,260 bp amplified produ ⁇ is then digested with Xho I and Cla I ligated into the Xho I / Cla I site of either the pCMV/SIN-CEA or pMET/SIN-CEA ve ⁇ ors.
  • These new plasmids are designated pCMV/SIN-CEA/HSVTK or pMET/SIN- CEA/HSVTK, respectively.
  • oligonucleotides contain 25 nucleotides corresponding to the tissue fa ⁇ or gene template, plus additional 5'-flanking sequences comprising a Pme I recognition site and buffer sequence, as follows.
  • the approximately 920 bp tissue fa ⁇ or gene amplicon is digested with Pme I and ligated into Sindbis ve ⁇ or (pVGELVIS-SEMBVLII or pKSSINBVLIIA), or r ⁇ roviral ve ⁇ or backbones (pKT-lL, pKT-3BL, or ⁇ KT-3BCL) that have been digested with Pme I and treated with CLAP.
  • Sindbis ve ⁇ or pVGELVIS-SEMBVLII or pKSSINBVLIIA
  • r ⁇ roviral ve ⁇ or backbones pKT-lL, pKT-3BL, or ⁇ KT-3BCL
  • Proper orientation of the insert sequence is determined by restriction endonuclease digests and the resulting constm ⁇ s are designated pVGELVIS- HTF, pKSIN-HTF, pKT-lL-HTF, pKT-3BL-HTF, and pKT-3BCL-HTF, respe ⁇ ively.
  • Produ ⁇ ion of packaged ve ⁇ or particles is accomplished using the methods described in Example 2 below. DNA encoding other gene produ ⁇ s useful in the practice of this invention may be readily cloned into these or other ve ⁇ ors using these approaches.
  • oligonucleotides contain 25 nt corresponding to the TIMP-1 gene template, plus additional 5'-flanking sequences comprising a Pme I recognition site and buffer sequence, as follows:
  • TIMP-1 cDNA clone Docherty et al, Nature 318:66, 1985
  • Thermalase thermostable DNA polymerase is performed in a rea ⁇ ion containing 1.5 mM MgCl2 provided by the supplier, 5% DMSO, and Hot Start Wax beads according to the following protocol:
  • the approximately 700 bp TIMP-I gene amplicon is digested with Pme I and ligated into Sindbis ve ⁇ or (pVGELVIS-SINBVLII or pKSSINBVLIIA), or retroviral vector backbones (pKT-lL, pKT-3BL, or pKT-3BCL) that have been digested with Pme I and treated with CLAP.
  • Sindbis ve ⁇ or pVGELVIS-SINBVLII or pKSSINBVLIIA
  • retroviral vector backbones pKT-lL, pKT-3BL, or pKT-3BCL
  • each oligonucleotide primer contains 25 nucleotides that correspond to sequences on the input template DNA to allow priming, as follows:
  • GHFBP-F (Sequence ID No.: 37)
  • the approximately 1000 bp integrin ⁇ 3/folate receptor gene fusion amplicon is gel purified, digested with Pme I and ligated into Sindbis ve ⁇ or (pVGELVIS-SINBVL ⁇ or pKSSINBVLIIA), or r ⁇ roviral ve ⁇ or backbones (pKT-lL, pKT-3BL, or pKT-3BCL) that have been digested with Pme I and treated with CIAP.
  • Sindbis ve ⁇ or pVGELVIS-SINBVL ⁇ or pKSSINBVLIIA
  • r ⁇ roviral ve ⁇ or backbones pKT-lL, pKT-3BL, or pKT-3BCL
  • Proper orientation of the insert sequence is determined by restri ⁇ ion endonuclease digests and the resulting constm ⁇ s are designated pVGELVIS-IF, pKSIN-EF, pKT-lL-IF, pKT- 3BL-IF, and pKT-3BCL-IF, respe ⁇ ively.
  • each oligonucleotide primer contains a minimum of 25 nucleotides that correspond to sequences on the input template DNA to allow priming, as follows:
  • HFBP-F (Sequence ID No. : 41 )
  • I ⁇ 3LBD-R/FC-TERM (Sequence ID No. 44)
  • the folate receptor and integrin ⁇ 3 PCR reactions from above are purified using GENECLEAN and approximately 5% of the produ ⁇ from each reaction is combined and used as template in a second round of PCR, containing primers HFBP-F and I ⁇ 3LBD- R/FC-TERM and Thermalase DNA polymerase, as described previously and using the following amplification protocol:
  • the approximately 1000 bp integrin ⁇ 3/folate receptor gene fusion amplicon is gel purified, digested with Pme I and ligated into Sindbis vector (pVGELVIS-SINBVL ⁇ or pKSSINBVLBA), or retroviral ve ⁇ or backbones (pKT-lL, pKT-3BL, or pKT-3BCL) that have been digested with Pme I and treated with calf intestinal alkaline phosphatase.
  • Sindbis vector pVGELVIS-SINBVL ⁇ or pKSSINBVLBA
  • retroviral ve ⁇ or backbones pKT-lL, pKT-3BL, or pKT-3BCL
  • Proper orientation of the insert sequence is determined by restriction endonuclease digests and the resulting constm ⁇ s are designated pVGELVIS-FI, pKSIN-FI, pKT-lL-FI, pKT-3BL-FI, and pKT-3BCL-FI, respe ⁇ ively.
  • Produ ⁇ ion of packaged vector particles is accomplished using the methods described in Example 2.
  • the plasmid pBS-ECAT (Jang, et al, J. Virol 63:1651, 1989) includes the 5' nontranslated region of encephalomyocarditis vims (EMCV) from nt 260-848 of the viral genome, which contains the internal ribosome entry site (IRES).
  • EMCV nt 260-827 are amplified from pBS-ECAT by PCR using the following primer pair: EMCV IRES forward primer (for insertion into the Mlul site in pKT-3BCL): (Sequence ID No.: 45)
  • the amplicon resulting from amplification with these primers is flanked by Mlul recognition sites, inside a 5 bp "buffer sequence'.
  • Amplification of the EMCV IRES sequence from the pBS-ECAT plasmid is accomplished with the following PCR protocol:
  • the IRES amplicon is digested with Mlul, purified on a 1% agarose gel, and ligated into a C AP treated ve ⁇ or previously digested with Mlul. This constm ⁇ is designated pKT-3BCL(IR). Correct orientation of the insert is determined by sequendng the resulting plasmid.
  • this amplicon may be inserted into other retroviral ve ⁇ or backbones and the Sindbis vector backbones pKSSINBVL II and pVGELVIS-SINBVL II.
  • the cDNA for the human Tissue Fa ⁇ or protein is synthesized by PCR amplification of the cDNA from plasmid ⁇ HTF7 (Scarpati, et al., supra).
  • the TF cDNA is amplified using the gene sperific primer pair as previously described in Example IE (HTFP-F and HTFP-R).
  • the amplicon resulting from amplification with these primers is flanked by Pme I recognition sites, inside a 5 bp "buffer sequence'.
  • the primer pair contains 5 nucleotide "buffer sequences" for effi ⁇ ent enzyme digestion followed by the Pmel recognition sequence.
  • the amplicon is digested with Pmel, purified on a 1% agarose gel, and ligated into a CLAP treated ve ⁇ or previously digested with Pmel. This constm ⁇ is designated pKT-3BCL(TF-IR).
  • other restriction sites within the polylinker may be inco ⁇ orated into the PCR primers for insertion into pKT-3BCL, pKSSINBVL ⁇ and pVGELVIS-SINBVL ⁇ , ve ⁇ ors containing IRES. Corre ⁇ orientation of the insert is determined by sequencing the resulting plasmid.
  • the cDNA for the human plasminogen a ⁇ ivator inhibitor -1 (PAI-1) protein is synthesized by PCR amplification of the cDNA from plasmid PALB4 (Ginsburg, et al, J.
  • the PAI-1 cDNA is amplified using the following gene specific primer pair.
  • the amplicon resulting from amplification with these primers is flanked by NotI recognition sites, inside a 5 bp 'buffer sequence'.
  • PAI-1 cDNA forward primer (Sequence ID No.: 47)
  • PAI-1 cDNA reverse primer (Sequence ID No.: 48)
  • the primer pair contains 5 nucleotide "buffer sequences' for efficient enzyme digestion followed by the NotI recognition sequence.
  • the amplicon is digested with NotI, purified on a 1% agarose gel, and ligated into a CIAP treated vector previously digested with NotI. This constm ⁇ is designated pKT-3BCL(TF-IR-PAI).
  • pKT-3BCL(TF-IR-PAI) Restri ⁇ ion sites within the polylinker may be inco ⁇ orated into the PCR primers for insertion into pKT-3BCL, pKSSINBVL II and pVGELVIS-SINBVL II. Correct orientation of the insert is determined by sequencing the resulting plasmid(s).
  • the packaging cell line HX (WO 92/05266) is seeded at 5.0 x 10 5 cells on a 10 cm tissue culture dish on day 1 with DMEM and 10% FBS. On day 2, the media is replaced with 5.0 ml fresh media 4 hours prior to transfe ⁇ ion.
  • a standard calcium phosphate-DNA co- precipitation is performed by mixing 40.0 ⁇ l 2.5 M CaCt ⁇ , 10 ⁇ g plasmid DNA, (e.g., pkSIN-HTF, pkT-IL-HTIMP, pkT-38L (TF-IR-PAI)) and deionized H2O in a total volume of 400 ⁇ l.
  • the DNA-CaCt ⁇ solution is added dropwise with constant agitation to 400 ⁇ l precipitation buffer (50 mM HEPES-NaOH, pH 7.1; 0.25 M NaCl and 1.5 mM Na2HPO 4 - NaH2PO4). This mixture is incubated at room temperature for 10 minutes.
  • the resultant fine predpitate is added to the HX of cells.
  • the cells are incubated with the DNA precipitate overnight at 37_C.
  • the media is aspirated and fresh media is added.
  • the supernatant is removed on day 4, passed through a 0.45 ⁇ l filter, and stored at -80_C.
  • DA an amphotropic cell line derived from D 17 cells ATCC No. 183, WO 92/05266
  • cells are seeded at 5.0 x 10 5 cells/10 cm tissue culture dish in 10 ml DMEM and 10% FBS, 4 ⁇ g/ml polybrene (Sigma, St. Louis, MO) on day 1.
  • FBS 4 ⁇ g/ml polybrene
  • 3.0 ml, 1.0 ml and 0.2 ml of the freshly collected vims-containing HX media (produced as described in Example 2(A) above) is added to the cells.
  • the cells are incubated with the vims overnight at 37_C.
  • the retroviral also encodes a sele ⁇ able marker, e.g., neomycin resistance
  • the media is removed and 1.0 ml DMEM, 10% FBS with 800 ⁇ g ml G418 is added to the plate. Only cells that have been transduced with the ve ⁇ or and expressing the sele ⁇ able marker will survive. In the case of neomycin resistance, G418 resistant pools can be generated over a period of a week.
  • a pool of cells is then dilution cloned by removing the cells from the plate and counting the cell suspension, diluting the cells suspension down to 10 cells/ml and adding 0.1 ml to each well (1 cell/well) of a 96 well plate (Corning, Corning, NY). Cells are incubated for 14 days at 37_C, 10% CO2. As many as twenty-four clones are selected and expanded up to 24 well plates, 6 well plates then 10 cm plates at which time the clones are assayed for expression and the supernatant are collected and assayed for viral titer.
  • the titer of the individual clones is determined by infection of HT1080 cells, (ATCC No. CCL 121). On day 1, 5.0 x 10 5 HT1080 cells are plated on each well of a 6 well microtiter plate in 3.0 ml DMEM, 10% FBS and 4 ⁇ g/ml polybrene. On day 2, the supernatant from each clone is serially diluted 10 fold and used to infect the HT1080 cells in 1.0 ml aliquots. The media is replaced with fresh DMEM, 10% FBS media, and the cells incubated with the vector overnight at 37_C, 10% CO2.
  • transduced cells On day 3, selection of transduced cells is performed (assuming the presence of a sele ⁇ able marker in the recombinant vector) by replacing the media with fresh DMEM, 10% FBS media containing the appropriate sele ⁇ ion agent, for instance, 800 ⁇ g/ml G418 in the case of neomycin resistance. Cells are incubated at 37_C, 10% CO2 for 14 days at which time G418 resistant colonies are scored at each dilution to determine the viral titer of each clone as cfu/ml. Using these procedures, cell lines are derived that produce greater than or equal to
  • the packaging cell line HX is transduced whh ve ⁇ or generated from the DA ve ⁇ or producing cell line in the same manner as described for transdu ⁇ ion of the DA cells from HX supernatant.
  • Transduction of the DA or HX cells with ve ⁇ ors lacking a neo sele ⁇ able marker was performed as described above. However, instead of adding G418 to the cells on day 3, the cells are cloned by limiting dilution. Titer is analyzed as described above.
  • DA cells are seeded at 5.0 x 10 s cells on a 10 cm tissue culture dish on day 1 with DMEM and 10% irradiated (2.5 megarads minimum) FBS. On day 2, the media is replaced with 5.0 ml fresh media 4 hours prior to transfe ⁇ ion.
  • a standard calcium phosphate-DNA copre pitation is performed by mixing 60 ⁇ l 2.0 M CaCb, 10 ⁇ g pMLP-G (Hartinan, et al., Nuc. Acid Res. 16:9345, 1988; Emi, et al, J.
  • plasmid 10 ⁇ g recombinant viral ve ⁇ or plasmid, and deionized water to a volume of 400 ⁇ l.
  • the DNA-CaCt ⁇ solution is then added dropwise with constant agitation to 400 ⁇ l 2x precipitation buffer (50 mM HEPES-NaOH, pH 7.1, 0.25 M NaCl and 1.5 mM Na2HPO4-NaH2PO4). This mixture is incubated at room temperature for 10 minutes. The resultant fine precipitate is added to a culture dish of DA cells plated the previous day. The cells are incubated with the DNA precipitate overnight at 37_C. On day 3, the medium is removed and fresh medium is added.
  • 2x precipitation buffer 50 mM HEPES-NaOH, pH 7.1, 0.25 M NaCl and 1.5 mM Na2HPO4-NaH2PO4
  • DA cells are seeded at 5.0 x 10 s cells on a 10 cm tissue culture dish in 10 ml
  • DMEM and 10% FBS 4 mg/ml polybrene on day 1.
  • 2.0 ml, 1.0 ml or 0.5 ml of the freshly colie ⁇ ed and filtered G-pseudotyped vims containing supernatant is added to the cells.
  • the cells are incubated with the vims overnight at 37_C.
  • the medium is removed and 10 ml DMEM, 10% irradiated FBS with 800 ⁇ g/ml G418 (in the case of recombinant ve ⁇ ors carrying the ne R gene) is added to the plate. Only cells that have been transduced with the ve ⁇ or and contain the neo selectable marker will survive.
  • a G418 resistant pool is generated over the period of 1-2 weeks.
  • the pool is tested for expression and then dilution cloned by removing the cells from the plate, counting the cell suspension, diluting the cell suspension down to 10 cells ml and adding 0.1 ml to each well (1 cell/well) of a 96-well plate.
  • Cells are incubated for 2 weeks at 37_C, 10% CO2 Twenty-four clones are sele ⁇ ed and expanded up to 24-well plates, then 6-well plates, and finally 10 cm plates, at which time the clones are assayed for expression and the supernatant are colle ⁇ ed and assayed for viral titer as described above.
  • alphavims packaging cell lines An important criteria in sele ⁇ ing potential parent cell lines for the creation of alphavims packaging cell lines is the choice of cell lines that exhibit little or no cytopathological effects prior to the appropriate production of alphavims ve ⁇ or particles. This criteria is essential for the development of an alphavims ve ⁇ or producer cell line which can be propagated for long periods of time and used as a stable source of vector. It is known that alphavims infection of most mammalian cells results in cytopathology and lysis of the cell. However, the derivation of packaging cells from various insect cell lines may circumvent this problem.
  • inse ⁇ cell lines may be derived from Aedes albopictus, Aedes aegypti, Spodoptera frugiperda, and Drosophila melanogaster cells, may be utilized to constmct alphavims packaging cell lines.
  • alphavims packaging cell lines are provided using an insect parent cell line, such as from Aedes albopictus cell, containing a stably transfected expression vector cassette which allows for expression of alphavims structural proteins under the control of inducible or non-inducible promoters a ⁇ ive in these cell types, and co-expressing a sele ⁇ able marker.
  • Packaging cell lines may also be modified by overexpressing the bcl-2 gene product in potential parent cell lines, such as canine D- 17 and CF2 cells; human HT1080 (ATCC
  • a bcl-2 expression ve ⁇ or is constmcted by using standard recombinant DNA techniques in order to insert the 910 bp Eco RI cDNA fragment derived from the plasmid pB4 (Reed, et. al., Nature 336:259, 1988) into any commercially available expression ve ⁇ or containing a constitutive promoter and encoding a sele ⁇ able marker, for example, pCDNA3 (Invhrogen, San Diego, CA). Careful consideration must be taken to avoid any type of homology between alphavims nucleic acid sequences and other transduced ve ⁇ ors.
  • the alphavims ve ⁇ or system described herein is designed for use as a biological therapeutic.
  • the parent cell line i.e., BHK-21 cells
  • Resistant colonies are pooled, followed by dilution cloning, and then individual clones are propagated and screened for bcl-2 expression. Once expression is verified, persistent Sindbis infection is tested, followed by its use as a parent cell line for alphavims packaging cell line development.
  • genes in addition to the bcl-2 oncogene which suppress apoptosis may likewise be expressed in an alphavims packaging or producer cell line.
  • Three viral genes which are particularly preferred include: the adenovirus E1B gene encoding the 19- kD protein (Rao et al, PNAS -59:7742-7746, 1992), the he ⁇ es simplex vims type 1 - . 34.5 gene (Chou and Roizman, PNAS 59:3266-3270, 1992), and the AcMNPV baculovirus p35 gene (Clem et al, Science 25* :1388-1390, 1991).
  • genes may be inserted into any commercially available plasmid expression ve ⁇ ors under the control of appropriate constitutive eukaryotic transcriptional promoters, and also containing a selectable marker using standard techniques.
  • the expression ve ⁇ or constmcts are subsequently transfected into cell lines as described above, and the appropriate selection is applied. Selection for stable integration of these genes and constitutive expression their produ ⁇ s should allow for more extended ve ⁇ or production in cell lines found to be susceptible to alphavirus-induced apoptotic events.
  • each gene product inhibits apoptosis by its own unique mechanism. Therefore, the genes may also be introduced into packaging or producer cell lines in various combinations in order to obtain a stronger suppressive effect.
  • other gene produ ⁇ s having similar effe ⁇ s on apoptosis can also be readily inco ⁇ orated into packaging cell lines as they are identified.
  • ve ⁇ or lines stably transformed with both ve ⁇ or and ve ⁇ or packaging cassettes can be derived. These approaches include indu ⁇ ble and/or cellular differentiation sensitive promoters, antisense structural genes, h ⁇ erologous control systems, and use of mosquito or other cells in which viral persistent infections can be established. Regardless of the final configuration for the alphavims ve ⁇ or producer cell line, the ability to establish persistent infection, or at least delay cell death as a result of viral gene expression, may be enhanced by inhibiting apoptosis.
  • the DNA tumor viruses including adenovirus, HPV, SV40, and mouse polyomavims (Py), transform cells in part by binding to and ina ⁇ ivating the retinoblastoma (Rb) gene produ ⁇ pi 05 and its closely related gene produ ⁇ , pi 07, and other gene produ ⁇ s involved in the control of the cell cycle including cyclin A, p33 cdk2 and p34cdc2 All of these viruses, except for Py, encode gene produ ⁇ s which bind to and ina ⁇ ivate p53.
  • adenovirus including adenovirus, HPV, SV40, and mouse polyomavims (Py)
  • Rb retinoblastoma
  • mT middle T antigen
  • src membrane tyrosine kinase
  • phosphatidylinositol-3-kinase which is required for the full transformation potential of this vims (Talmage etal, Cell 59:55-65, 1989).
  • the binding to and inactivation of the Rb and p53 recessive oncogene products prevents cells transformed by these DNA tumor viruses from entering the apoptotic pathway.
  • packaging and producer cells are preferably transformed with viral genomic DNA from Py or SV40.
  • SV40 and Py transformed cell lines are established and the kinetics and level of Sindbis production and cytopathology after viral infection determined. If apoptic events chara ⁇ eristic of Sindbis proliferation in hamster cells are diminished, each prototype alphavims packaging and producer cell line subsequently is transformed with Py or SV40 in order to increase the yield of packaged vector from these cells.
  • the Sindbis E2 glycoprotein is synthesized as a precursor, PE2.
  • This PE2 precursor along with the second viral glycoprotein, El, associate in the endoplasmic reticulum and are processed and transported to the infe ⁇ ed cell membrane as a heterodimer for virion inco ⁇ oration.
  • PE2 is cleaved into E3 and the mature virion glycoprotein E2.
  • E3 corresponds to the 64 amino-terminal residues of PE2 and is lost during maturation.
  • the larger cleavage produ ⁇ , E2 is associated with El and anchored in what becomes the viral envelope.
  • Host cell protease(s) is responsible for processing of the PE2 precursor, cleaving at a site that immediately follows a highly conserved canonical four amino add (aa) residue moti ⁇ basic-X-basic-basic aa.
  • a mutant cell line derived from the CHO-K1 strain designated RPE.40 (Watson et al, J. Virol 65:2332-2339, 1991), is defective in the production of Sindbis vims strain AR339 through its inability to process the PE2 precursor into the E3 and mature E2 forms.
  • the envelopes of Sindbis virions produced in the RPE.40 cell line therefore contain a PE2/E1 heterodimer.
  • RPE.40 cells are at least 100-fold more resistant to Sindbis vims infection than the parental CHO-K1.
  • the defective virions produced by the RPE.40 cell line can be converted into a fully infe ⁇ ious form by treatment with trypsin.
  • any wild-type alphavims that is produced by recombination between ve ⁇ or and structural protein gene RNAs will re-infect cells and be rapidly amplified, significantly contaminating and decreasing the titer of packaged vector preparations.
  • Packaging and producer cells developed from the RPE.40 line are an alternative to other cell lines permissive for alphavims infe ⁇ ion due to the inefficient amplification of any wild-type vims generated during ve ⁇ or produ ⁇ ion and packaging.
  • ve ⁇ or preparations are not significantly contaminated with wild-type vims.
  • the benefits of this system are extended to other packaging and producer cell lines by developing "knock-out" mutants in their analogous cellular protease(s), using techniques known in the art.
  • Alphavims hopping cell lines are used transiently to produce infectious RNA vector particles which have been pseudotyped for a different cellular receptor tropism. Once the hopping cell line produces vector particles, it is no longer required because only the infe ⁇ ious culture supernatants are needed to transduce the original alphavims packaging cell lines discussed above. Therefore, the hopping cell line need not exhibit persistent infe ⁇ ion by alphavims in order to transiently produce ve ⁇ or particles. In this instance, the parent cell line can be dther an inse ⁇ cell line that exhibits persistent infection, or a mammalian cell line which is likely to lyse within 24-72 hours after a productive alphavims infe ⁇ ion.
  • the alphavims hopping cell line can be any of the aforementioned parent cell lines able to support either alphavims or retroviral replication, without the additional cell modifications discussed previously, such as bcl-2 oncogene expression.
  • the generation of VSV-G pseudotyped alphavims ve ⁇ or particles can be accomplished by at least three alternative approaches, each of which is described in detail in U.S.S.N. 08/348,472.
  • any cell line which expresses retroviral gag-pol and env sequences may be used to package alphaviral ve ⁇ ors that have been engineered to contain a retroviral packaging sequence.
  • the r ⁇ rovirus ⁇ packaging sequence is inserted between the ina ⁇ ivated jun ⁇ ion region and a synthetic jun ⁇ ion region tandem repeat, such that only genomic-length vector, and not subgenomic RNA, is packaged by the viral envelope proteins.
  • Viral-based particles containing alphavims ve ⁇ or RNA may be produced by transfe ⁇ ing in vitro transcribed alphavims ve ⁇ or RNA using procedures described previously.
  • Supematants with pseudotyped retroviral particles containing alphavims RNA ve ⁇ ors are harvested 24 hours post-transfe ⁇ ion, and these supematants can then be used to transduce an alphavims packaging cell line.
  • the blood is allowed to clot for one half hour at room temperature. After clotting the semm is centrifuged at 2000 xg for 10 minutes at 4_C. The semm is collected and placed into a 15 ml conical tube (Corning, Corning, NY) and placed on ice. Approximately, 1.1 ml aliquots of the semm are placed in 2 ml cryovials, frozen in a dry ice/ethanol bath and stored at - 70_C for subsequent semm ina ⁇ ivation assays. Complement ina ⁇ ivated controls are prepared by heat inactivation of control aliquots for 30 minutes at 56_C.
  • non-heat inactivated semm, culture medium only, and heat-ina ⁇ ivated semm are placed in separate 1.5 ml tubes (Fisher Scientific, Pittsburgh, PA) and mixed with 1x10 s pfu of Sindbis vims and incubated at 37_C for 1 hour. After incubation the tubes are placed on ice.
  • the non-heat inactivated semm, medium, and heat-ina ⁇ ivated semm vims preparations are titered by plaque assay on BHK-21 cells. Equivalent vims titers regardless of incubation with non-heat ina ⁇ ivated semm, medium, or heat-inactivated semm are indicative of parent cell line hosts which produce Sindbis vims is resistant to human complement ina ⁇ ivation.
  • alphavims packaging cell lines is dependent on the ability to synthesize high intracellular levels of the necessary stmctural proteins: capsid, pE2 and/or E2, and El.
  • capsid a necessary stmctural proteins
  • pE2 and/or E2 a necessary stmctural proteins
  • El a necessary stmctural proteins
  • high level expression of these proteins, in particular, the envdope glycoproteins E2 and El may lead to concomitant cytopathology and eventual cell death. Therefore, stmctural protein expression cassettes have been designed with inducible regulatory elements which control the levels of gene expression, in addition to others which maintain constitutive levels of expression.
  • alphavims stm ⁇ ural proteins is under control of the RSV LTR, in conjun ⁇ ion with the inducible lac operon sequences. This is achieved by insertion of alphavims cDNA corresponding to the viral stm ⁇ ural protein genes into the pOP13 and pOPRSVl ve ⁇ ors (Stratagene, San Diego, CA). These vectors, used separately, are co-transfe ⁇ ed with the p3'SS ve ⁇ or (Stratagene, San Diego, CA), which expresses the lac repressor protein.
  • IPTG Isopropyl-B-D-thiogala ⁇ opyranoside
  • Sindbis stmctural protein gene (SP) cDNA is inserted into the pOP13 and pOPRSVl ve ⁇ ors as follows.
  • the SP coding region is amplified in toto with a primer pair whose 5' ends map, respectively, to the authentic AUG translational start and UGA translational stop sites, including the surrounding nucleotides corresponding to the Kozak consensus sequence for efficient translational initiation at Sindbis nt 7,638.
  • the forward primer is complementary to Sindbis nts 7,638 to 7,661
  • the reverse primer is complementary to Sindbis nt 11,384 to 11,364.
  • PCR amplification of Sindbis cDNA corresponding to the stmctural protein genes is accomplished by a standard three- temperature cycling protocol using the following oligonucleotide pair:
  • Reverse primer (11384R): (Sequence ID No.: 50)
  • a 5 nucleotide "buffer sequence” followed by the Not I recognition sequence is included in the 5* end of each primer.
  • the 3,763 bp fragment is purified in a 1% agarose gel, then subsequently digested with Not I.
  • the resulting 3,749 bp fragment is then ligated separately into the pOP13 and pOPRSVl vectors which have been digested with Not I and treated with calf intestine alkaline phosphatase.
  • These expression ve ⁇ or cassettes which contain the entire coding capacity of the Sindbis stmctural proteins, are designated pOP13-SINSP and pOPRSVl -SINSP, respectively.
  • Variations of the lac operon-Sindbis stmctural protein gene expression cassettes also can be constm ⁇ ed using other viral, mammalian, or insect-based promoters.
  • the lac operon and the RSV LTR promoter, or just the RSV LTR promoter can be switched out of the Stratagene pOP13 and pOPRSVl ve ⁇ ors and replaced by other promoter sequences, such as the cytomegalovirus major immediate promoter (pOPCMV-SINSP); the adenovirus major late promoter (pOPAMLP-SINSP); the SV40 promoter (pOPSV-SINSP): or insect promoter sequences, which include the Drosophila metallothionein inducible promoter (pMET- SINSP), Drosophila a ⁇ in 5C distal promoter (pOPA5C-SINSP), heat shock promoters HSP65 or HSP70 (pHSP-S
  • packaging cell line expression cassettes are constm ⁇ ed which contain regulatory elements for the high level induction of stmctural protein synthesis via nonstmctural proteins supplied in trans by the alphavims ve ⁇ or, but with no basal level of synthesis until appropriately stimulated.
  • a stmctural protein gene cassette is constmcted whereby transcription of the stmctural protein genes occurs from an adjacent alphavims jun ⁇ ion region sequence.
  • the primary features of such a cassette are: an RNA polymerase II promoter positioned immediately adjacent to alphavims nucleotide 1 such that transcription initiation begins with authentic alphavims nucleotide 1; the 5'-end alphavims sequences required for transcriptase recognition; the alphavims junction region sequence for expression of the stmctural protein gene mRNA; the alphavims stmctural protein gene sequences; the 3 '-end alphavims sequences required for replication; and a transcription termination/polyadenylation sequence.
  • RNA transcript of the stmctural protein gene cassette is amplified by the same ve ⁇ or-supplied nonstmctural proteins (see Figure 16).
  • constmction of a positive-sense, vector-inducible Sindbis packaging cassette is accomplished as follows.
  • the pVGELVIS ve ⁇ or described previously is digested with the enzyme Bsp El to remove nucleotides 422 to 7054, including most of the nonstmctural gene coding sequences, and the remaining 9925 bp fragment is purified in a 0.8% agarose gel, and subsequently re-ligated to itself to generate the constmct designated pLTR/SindlBspE ( Figure 16).
  • This deletion leaves the 5'-end authentic translation start codon at nts 60-62 inta ⁇ and creates in-frame downstream UAA and UGA stop codons at nts 7,130 to 7,132 and 7,190 to 7,192, respectively, thus preventing translation of the downstream stmctural protein gene open-reading frame.
  • the pLTR/SindlBspE packaging cassette constm ⁇ is subsequently transfe ⁇ ed into BHK-21 cells and transfe ⁇ ants are sde ⁇ ed using G418 at 400 ⁇ g/ml and cloned by limiting dilution.
  • SIN-luc vector RNA Sindbis-luciferase vector RNA as described previously.
  • the data shown in Figure 17 demonstrate that transfection of SIN-luc vector RNA into several of these clonal LTR/SindlBspE packaging cells results in the production of infectious Sindbis particles containing the SIN-luc RNA, as the recovered supematants are shown to transfer SIN-luc ve ⁇ or RNA to fresh monolayers of BHK-21 cells.
  • these packaging cassette constructions can be made given the disclosure provided herein, including, for example, the substitution of other RNA polymerase promoters for the current MuLV LTR, the addition of one or more nucleotides between the RNA polymerase promoter and the first Sindbis nucleotide, the substitution of other ribozyme processing sequences, or the substitution of a non-Sindbis-encoded open reading frame upstream of the stmctural protein gene sequences, which may or may not retain the 5'-end Sindbis sequences required for transcriptase recognition.
  • these constm ⁇ s can be transfe ⁇ ed into other cell lines, as discussed previously.
  • expression cassettes contain a cDNA copy of the alphavims stmctural protein gene sequences flanked by their natural jun ⁇ ion and 3 '-untranslated regions, and are inserted into an expression ve ⁇ or in an orientation such that primary transcription from the promoter produces antisense stmctural protein gene RNA molecules. Additionally, these constm ⁇ s contain adjacent to the jun ⁇ ion region alphavims 5'-end sequences necessary for recognition by the viral transcriptase and a catalytic ribozyme sequence positioned immediately adjacent to alphavims nucleotide 1 of the 5'-end sequence.
  • this ribozyme cleaves the primary RNA transcript precisely after the first alphavims nucleotide.
  • the stmctural protein genes cannot be translated, and are dependent entirely on the presence of alphavims vims nonstmctural proteins for transcription into positive-strand mRNA prior to their expression. These nonstmctural proteins again are provided by the alphavims ve ⁇ or itself.
  • the stmctural protdn gene transcripts undergo amplification by utilizing the same nonstmctural proteins provided by the alphavims vector. Details of this constm ⁇ ion are described in (U.S.S.N. 08/348,472).
  • packaging cell lines also are generated which segregate tht integration and expression of the stmctural protein genes, allowing for thdr transcription as non- overlapping, independent RNA molecules, using the approaches described above.
  • the expression of capsid protein independently of glycoproteins E2 and El, or each of the three proteins independent of each other eliminates the possibility of recombination with ve ⁇ or RNA and subsequent generation of contaminating wild-type vims.
  • the constmction of such "split gene" expression cassettes is detailed in (U.S.S.N. 08/348,472).
  • BHK-21 cell line and replicon-inducible packaging expression cassette are used to demonstrate assembly of the components.
  • other possible parent cell lines can be used to create alphavims packaging cell lines and have been discussed previously.
  • BHK-21 cells are grown at 37_C in 5% CO2 in DMEM, 2 mM L-glutamine, and 10% fetal bovine semm (optimal media).
  • transfe ⁇ ed Approximately 5 x 10 5 BHK-21 cells, grown in a 35 mM petri dish, are transfe ⁇ ed with 5 ⁇ g pLTR/SindlBspE using 5 ⁇ l of the Transfectam (Promega, Madison, Wl) cationic lipid reagent in semm-free media conditions, as suggested by the supplier.
  • Transfectam Promega, Madison, Wl
  • any method of transfe ⁇ ion can be rapidly substituted, i.e., electroporation, caldum phosphate precipitation, or by using any of the readily available cationic liposome formulations and procedures commonly known in the art.
  • the cells are trypsinized and reseeded in 100 mm dishes in 10 ml of optimal media, as described above, supplemented with 400 ⁇ g/ml of G418 and selected over a period of 5 to 7 days. Colonies displaying resistance to the G418 dmg are then pooled, dilution cloned, and propagated. Individual clones are screened for high levels of Sindbis stmctural protein expression and functional packaging after transfection with Sindbis-luciferase ve ⁇ or RNA transcribed in vitro from Sad linearized plasmid pKSSINBV-luc (see Example lC(iv)).
  • LTR SindlBspE or BK-Bsp cells clonally-derived pLTR/SindlBspE transfe ⁇ ed BHK-21 cells grown in 60 mm petri dishes are transfe ⁇ ed with 2 ⁇ g of Sindbis-luciferase vector RNA and overlayed with 3 ml of optimal media (see above).
  • the supematants are removed and clarified by centrifugation for 30 minutes at 3,000 ⁇ m in a Sorvall RT6000B tabletop centrifuge.
  • the transfe ⁇ ed cell monolayer is lysed in reporter lysis buffer (Promega, Madison, Wl) as described by the manufacturer and assayed for luciferase expression as described previously.
  • luciferase a ⁇ ivity (and thus fun ⁇ ional packaging) is tested by using 1.0 ml of the above supe atants to infert fresh monolayers of BHK-21 cells in 60 mm dishes. At 20 hours post-infe ⁇ ion, the cell monolayers are lysed as described above and tested for luciferase expression. As shown in Figure 17, three clones (#13, 18, and 40) produce packaged Sindbis-lu ⁇ ferase ve ⁇ or. In addition, transfe ⁇ ed clone #18 cells are tested for increased ve ⁇ or packaging over a timecourse following transfection.
  • Supematants from transfe ⁇ ed clone #18 cells are harvested at 20, 45, and 70 hours post- transfection, as described above, and used to infe ⁇ fresh monolayers of BHK-21 cells.
  • Figure 18 shows that Sindbis-luciferase vector packaging increases significantly at 45 hours post-transfection. Expression also can be tested by Western blot analysis using polyclonal rabbit anti-Sindbis antibodies.
  • Another approach to developing an alphavims ve ⁇ or producer cell line is to generate alphavims ve ⁇ or producer lines from mosquito cells, where viral persistence often results after infe ⁇ ion.
  • the titer of infectious vims produced in persistently infe ⁇ ed mosquito cells is only about 1.0 x 10 pfu/ml, at least five orders of magnitude less than that observed after lytic infection of BHK-21 cells by Sindbis.
  • a cell whose differentiation state can be controlled is likely an ideal host in which to derive an alphavims ve ⁇ or producer cell line.
  • the expression ve ⁇ or cassette and, in some instances, stmctural components are coupled to terminal differentiation state-inducible promoters, according to the strategy described for ELVIS, and used to transform stably an undifferentiated host cell. Terminal differentiation of the host producer cell after induction with the appropriate stimuli coincident-ally results in induction of the alphavims replication cycle and production of packaged ve ⁇ or.
  • plasmid DNA is complexed with LipofectamineTM according to the conditions suggested by the supplier (ca., 5 g DNA/8 g lipid reagent) and added to 35 mm wells containing undifferentiated PCC4 or F9 cells (Fujimura et al., 1981, Cell 23:809-814) at approximately 75% confluency.
  • cpe and the level of Sindbis produ ⁇ ive infe ⁇ ion, quantitated by plaque assay of media supernatant is determined at regular intervals over 5 days in undifferentiated and differentiated transfe ⁇ ed PCC4 or F9 cells. Differentiation of F9 and PCC4 cells is accomplished by addition of retinoic acid (Sigma Chemical Co., St. Louis, MO), to a final concentration of 1 M.
  • retinoic acid Sigma Chemical Co., St. Louis, MO
  • undifferentiated F9 or PCC4 cells are transfe ⁇ ed with pLTR SINdlBspE and G 18 sele ⁇ ed, as described above.
  • Differentiation state-sensitive clones are then sele ⁇ ed by infe ⁇ ion at high multiplidty with packaged pSIN-BV-luc ve ⁇ or.
  • Clones which are resistant to cell lysis or do not produce packaged pSIN-BV-luc ve ⁇ or particles are candidate ve ⁇ or packaging clones. These candidate clones are tested for SIN-luc vector particle produ ⁇ ion following terminal differentiation with retinoic acid, as described.
  • the murine wild type Py is unable to replicate in the teratocarcinoma cell lines PCC4 or F9.
  • This block of replication in undifferentiated cells occurs at the level of transcription of early region (i.e., T antigen) genes, and is released by indu ⁇ ion of terminal differentiation with vitamin A.
  • Py mutants which are able to establish produ ⁇ ive infe ⁇ ion in undifferentiated PCC4 and F9 cells map to the viral enhancer region. The genesis of an embryonic tissue specific transcriptional enhancer dement has resulted in these mutants.
  • the viral regulatory non-coding region is coupled to the genomic cDNA of Sindbis vims according to the ELVIS strategy.
  • the precise transcriptional start site of the Py early region has been determined (see Tooze, DNA Tumor Vimses, 2nd Ed. Cold Spring Harbor, NY, 1981).
  • the PCC4 and F9 cell lines are stably transformed with the Py-Sindbis ve ⁇ ors. Sindbis produ ⁇ ive infection occurs after addition of retinoic add to the culture medium and indu ⁇ ion of terminal differentiation.
  • the constmction of differentiation state-controlled alphavims vectors is detailed in (U.S.S.N. 08/348,472).
  • the third example of this strategy uses the ⁇ -globin locus control region.
  • the ⁇ - globin multigene cluster contains five developmentally regulated genes. In the early stages of human development, the embryonic yolk sac is the hematopoietic tissue and expresses the ⁇ -globin gene. This is followed by a switch to the ⁇ -globin gene in the fetal liver and the ⁇ - and ⁇ -globin genes in adult bone marrow (Collins and Weissman, 1984, Prog. Nucleic Acid Res. Mol. Biol. 31:315).
  • At least two mouse erythroleukemia lines serve as models for terminal differentiation dependent expression of ⁇ -globin. Expression of ⁇ -globin is observed in these lines only after indu ⁇ ion of terminal differentiation by addition of 2% DMSO to the growth medium.
  • the entire ⁇ -globin locus is regulated by the locus control region (LCR).
  • LCR locus control region
  • DCR dominant control region
  • the DCR contains five DNase I hypersensitive (HS1- HS5) sites.
  • the DCR dire ⁇ s high level site of integration independent, copy number dependent expression on a linked human ⁇ -globin gene in transgenic mice and stably transfe ⁇ ed mouse erythroleukemia (MEL) cells (Grosveld etal, CSHSQB 58:1-12, 1993).
  • MEL mouse erythroleukemia
  • the viral genomic cDNA is juxtaposed with a promoter containing a tandem synthetic core corresponding to the LCR HS2 site.
  • the desired alphavims ve ⁇ or constm ⁇ can be inserted downstream of the LCR in the endogenous ⁇ -globin gene by homologous recombination.
  • the ⁇ -globin transcription initiation site after terminal differentiation would be first determined, in order that the alphavims vector could be placed precisely at the start site. Initiation of a lytic viral life cycle is controlled by the differentiation state of the host cell is applicable to other systems, where the control of viral induced cytopathology is desired.
  • Y ⁇ another approach to regulating alphavims gene expression through a differentiation state sensitive promoter is the use of the r ⁇ inoic acid receptor a (RARA) and acute promyelomonocytic leukemia cdls (APL).
  • RARA r ⁇ inoic acid receptor a
  • APL acute promyelomonocytic leukemia cdls
  • APL cells are clonal myeloid precursors chara ⁇ erized by high growth rate and differentiation arrest.
  • a non-random chromosomal translocation breakpoint, t(15;17Xq22;21) occurs in almost all patients with APL.
  • the RARA gene has been localized to chromosome 17q21. Analysis of APL mRNA from patients has shown that most APL breakpoints occur within the second intron of the RARA gene and result in abnormal fusion transcripts.
  • the r ⁇ inoic add receptor is a member of a nuclear receptor superfamily that includes the thyroid and steroid hormone receptors.
  • Four different forms of the human RAR have been identified, and the corresponding cDNAs cloned and chara ⁇ erized.
  • viral genomic cDNA is juxtaposed with the RARA DNA binding site, creating ELVIS- RARASIN.
  • ELVIS-PySIN As with the strategy proposed for ELVIS-PySIN expression in undifferentiated EC cells, differentiation sensitive ELVIS-RARASIN expressing cells are isolated.
  • ve ⁇ or transfe ⁇ ion Since these ve ⁇ ors will be disabled and prevented in the synthesis of full genomic vectors, re-infe ⁇ ion of a fresh layer of Sindbis packaging cell lines will end in an aborted infection since these vectors are now dependent on the presence of the CEA RNA to become active. Higher titers may be achieved by dilution cloning transfe ⁇ ed producer cell lines using the RT-PCR technique.
  • the extended S + L assay determines whether replication competent, infectious vims (RCR) is present in the supernatant of the cell line of interest.
  • the assay is based on the empirical observation that infectious retroviruses generate foci on the indicator cell line MiCli (ATCC No. CCL 64.1).
  • the MiCli cell line is derived from the MvlLu mink cell line (ATCC No. CCL 64) by transduction with murine sarcoma vims (MSV). It is a non- producer, non-transformed, revertant clone containing a replication defective murine sarcoma provims, S + , but not a replication competent murine leukemia provirus, L".
  • MvlLu cells are seeded at 1.0 x 10 5 cells per well (one well per sample to be tested) of a 6 well plate in 2 ml DMEM, 10% FBS and 8 ⁇ g ml polybrene. MvlLu cells are plated in the same manner for positive and negative controls on separate 6 well plates. The cells are incubated overnight at 37_C, 10% CO2.
  • 1.0 ml of test supernatant is added to the MvlLu cells.
  • the negative control plates are incubated with 1.0 ml of media.
  • the positive control consists of three dilutions (200 focus forming units (ff ⁇ ), 20 fiu and 2 ffu each in 1.0 ml media) of MA vims (refered to as pAM in Miller et al, Molec. and Cell Biol. 5:431, 1985) which is added to the cells in the positive control wells.
  • the cells are incubated overnight.
  • the media is aspirated and 3.0 ml of fresh DMEM and 10% FBS is added to the cells.
  • the cells are allowed to grow to confluency and are split 1 : 10 on day 6 and day 10, amplifying any replication competent retrovims.
  • the media on the MvlLu cells is aspirated and 2.0 ml DMEM and 10% FBS is added to the cells.
  • the MiCli cells are seeded at 1.0 x 10 s cells per well in 2.0 ml DMEM, 10% FBS and 8 ⁇ g/ml polybrene.
  • the supernatant from the MvlLu cells is transferred to the corresponding well of the MiCli cells and incubated overnight at 37 C, 10% CO2.
  • the media is aspirated and 3.0 ml of fresh DMEM and 10% FBS is added to the cells.
  • the cells are examined for focus formation (appearing as clustered, refra ⁇ ile cells that overgrow the monolayer and remain attached) on the monolayer of cells.
  • the test article is determined to be contaminated with replication competent retrovims if foci appear on the MiCli cells. Using these procedures, it can be shown that the recombinant viral ve ⁇ or producer cell lines are not contaminated with replication competent retroviruses.
  • muscle dunni As an alternate method to test for the presence of RCR in a vector-producing cell line, producer cells are cocultivated with an equivalent number of Mus dunni (NTH NIAID Bethesda, MD) cells. Small scale cocultivations are performed by mixing of 5.0 x 10 5 Mus dunni cells with 5.0 x 10 5 producer cells and seeding the mixture into 10 cm plates (10 ml standard culture media/plate, 4 ⁇ g/ml polybrene) at day 0. Every 3 to 4 days the cultures are split at a 1 :10 ratio and 5.0 x 10 s Mus dunni cells are added to each culture plate to eflfe ⁇ ively dilute out the producer cell line and provide maximum amplifcation of RCR.
  • culture supernatant is harvested, passed through a 0.45 ⁇ cellulose-acetate filter, and tested in the MdH marker rescue assay.
  • Large scale co-cultivations are performed by seeding a mixture of 1.0 x 10 8 Mus dunni cells and 1.0 x 10 s producer cells into a total of twenty T-150 flasks (30 ml standard culture media/flask, 4 ⁇ g/ml polybrene). Cultures are split at a ratio of 1 : 10 on days 3, 6, and 13 and at a ratio of 1 :20 on day 9.
  • the final supernatant is harvested, filtered and a portion of each is tested in the MdH marker rescue assay.
  • the MdH marker rescue cell line is cloned from a pool of Mus dunni cells transduced with LHL, a retroviral ve ⁇ or encoding the hygromycin B resistance gene (Palmer et al, PNAS 84: 1055-1059, 1987).
  • the retroviral ve ⁇ or can be rescued from MdH cells upon infe ⁇ ion of the cells with RCR.
  • One ml of test sample is added to a well of a 6-well plate containing 1.0 x 10 s MdH cells in 2 ml standard culture medium (DMEM with 10% FBS, 1% 200 mM L-glutamine, 1% non-essential amino acids) containing 4 ⁇ g/ml polybrene.
  • DMEM standard culture medium
  • Cells transduced with the recombinant ve ⁇ ors of this invention are harvested with trypsin/EDTA and washed twice with cold PBS.
  • the cells are lysed with 50 to 400 ml R PA buffer (10 mM Tris-HCl pH 7.0; 1% (v/v) NP40; 0.1% (w/v) SDS; and 150 mM NaCl) and incubated for 15 minutes at room temperature.
  • the lysate is centrifuged at full speed in an Eppendorf centrifuge for 5 minutes. The supernatant is removed and stored at -20_C.
  • a Bradford protein assay is performed to ddermine the total protein concentration.
  • a sample of RIPA lysate containing a total protein concentration of 20 mg and a commercial molecular weight (MW) marker (Amersham, Chicago, IL) are mixed 1 : 1 with 2x sample buffer (4% (w v) SDS, 50 mM Tris-HCl pH 7.0, 24% (v/v) glycerol, 0.1% (w/v bromophenol blue and 0.05% ⁇ -mercaptoethanol) and heated to 65 C for 10 minutes. After heating the sample and MW marker are placed on ice.
  • the slots of a precast 7.5 Tris HCl based minigel (BioRad, Hercules, CA) are rinsed with running buffer (3.0 g Tris-HCl pH 8.6, 1.0 gm SDS and 14.4 gm glycine to 1.0 L) and the sample and M marker are loaded onto the gel. Approximately 70 to 120V is applied to the gel until the marker reaches the bottom of the gd.
  • the protein bands are transferred from the gel to an Immobilon-PTM membrane
  • the Immobilon-PTM membrane is blocked for 30 minutes at room temperature with 0.5% BM blocking reagent (Boehringer Mannheim, Chicago, IL) containing 3% BSA, heated slowly in a microwave.
  • the membrane is then probed with a primary mouse antibody that reacts specifically with the protein being dete ⁇ ed at a 1 :2000 dilution in BM blocking solution containing a 3% BSA and incubated for 1 hour at room temperature.
  • the membrane is washed three times with 40 ml of PBST (0.2% Tween-20 in PBS) for 10 min and a secondary goat anti-mouse, HRP-labeled antibody (Jackson, Bar Harbor, MA) at a 1:20,000 dilution in 3% BSA solution (Sigma, St Louis, MO).
  • the membrane is then washed three times with 40 ml of PBST for 10 minutes.
  • the membrane is submersed in ECL devdoping solution (Amersham, Chicago, IL) for 1 minute and then wrapped in plastic wrap and exposed to Hyperfilm (Amersham, Chicago, IL) for 5 seconds. The film is then developed and the protein bands are analyzed.
  • the secondary antibody solution containing 85% FACS buffer, 10% normal mouse semm, and 5% FTTC labeled anti-mouse antibody is added.
  • the microtiter plate is washed four times with fresh FACS buffer.
  • 150 ⁇ l from each well is transferred to a FACS tube (Falcon 2052 tubes) and 100 ⁇ l of FACS buffer is added.
  • the fluorescent signal is drte ⁇ ed by a FACScan counter.
  • Frozen tissue se ⁇ ions are dried at room temperature for 30 minutes.
  • the dried tissue se ⁇ ions are fixed with anhydrous acetone for 15 minutes at room temperature and allowed to air dry.
  • the sections are rehydrated with PBS for several minutes.
  • Tissue se ⁇ ions are then incubated in a humid chamber for 45 to 120 minutes at room temperature with 50 ⁇ l of primary antibody that specifically reads with the protein to be detected (Marek, et al, Cancer 67:1377, 1991).
  • the tissue se ⁇ ions are then washed with PBS and allowed to soak in PBS for several minutes.
  • the sections are incubated with 50 ⁇ l of HRP conjugated anti-mouse antibody or HRP-conjugated strept-avidin (DAKO, Ca ⁇ enteria, CA) for 45 to 120 minutes at room temperature.
  • the tissue sections are then washed with PBS and allowed to soak in PBS for several minutes.
  • the sections are incubated with 60 to 100 ⁇ l substrate solution containing 0.01% H2O2 and 3,3 diaminodenzidine (DAB) for 8 minutes or for 20 minutes with substrate solution containing 100 ⁇ l of ⁇ -ethyl carbazole (60 mg of 3-amino-9-ethylcarbazole in 25 ml DMF) in 1 ml of acetate buffer (0.68 g sodium acetate in H2O pH 5.2 adjusted to a volume of 250 mis) and 0.01% H2O2.
  • the tissue se ⁇ ions are washed with water for 2-5 minutes and then stained with haemotoxylin for 15 seconds.
  • pVGSP6GENdlBsp lacks nonstmctural gene sequences between bases 422-7,054.
  • Transfe ⁇ ions and co-transfections are performed by complexing in vitro transcription products with Lipofe ⁇ inTM and applying to BHK-21 cells.
  • the expression of luciferase in transfe ⁇ ed cells is tested 18 hours after transfection. Additionally, 1.0 ml of the transfection supernatant is used to infe ⁇ a confluent monolayer of BHK-21 cells and the expression of luciferase is tested at 24 hours post-infe ⁇ ion.
  • HT1080 cells are transiently transduced with varying dilutions of the recombinant viral ve ⁇ or carrying the HSV TK gene.
  • the samples are centrifuges and the cells are resuspended in 2.0 ml of PBS that does not contain Ca +2 or Mg +2 (CMF.PBS).
  • CMF.PBS Ca +2 or Mg +2
  • Approximately 0.2 ml of 37% formaldehyde solution is added while the sample is gently shaken.
  • the sample is then incubated for 20 minutes at room temperature with gentle agitation. Following incubation, the sample is washed three times with CMF.PBS.
  • the cells are then suspended in 2.0 ml of 50 mM NH4CI in CMF.PBS and incubated for 20 minutes at RT with gentle agitation. Following incubation, the sample is washed twice with CMF.PBS. The sample is then washed again with CMF.PBS containing 1% BVSA (Fra ⁇ ion V, Sigma, St. Louis, MO) and 1% saponin (CMF.PBS BSA Sap). Approximately 100 ⁇ l of primary antibody (Repligen, Cambridge, MA; 1C1 0.5 ⁇ g/sample) is added to this suspension and the mixture is incubated for 1.0 hour at RT with gentle agitation.
  • primary antibody Repligen, Cambridge, MA; 1C1 0.5 ⁇ g/sample
  • the mixture is then centrifuged and the pellet is washed three times with CMF.PBS/BSA/Sap.
  • Approximately 100 ⁇ l of dilute secondary antibody (Cappel, Durham, NC; rabbit anti-mouse IgG-FTTC 1:1000) resuspended in CMF.PBS/BSA/Sap and the mixture is incubated for 30 minutes at RT with gentle agitation. Following incubation, the pellet is washed three times with CMF.PBS/BSA Sap.
  • the sample is resuspended in 0.5 ml of CMF.PBS BSA Sap containing 25 ⁇ g of Propidium Iodide and analyzed by FACS.
  • Cells are removed from plates by suspension in 1 ml of 0.01 M sodium phosphate buffer, pH 7.0, containing 0.15 M NaCl. The absorbance at 550 nM is adjusted to 0.750 in order to adjust for differential cell density on the plates. The cells are sonicated for 1 minute and Triton X- 100 is added to a final concentration of 0.1 percent. The samples are rotated at room temperature for 90 minutes and cellular debris removed by centrifugation at 10,000 x g. D ⁇ ergent solubilized extra ⁇ s are relipidated by diluting 2 ⁇ l of the sample into 0.8 ml 0.05 M Tris-HCl, pH 7.5, containing 0.1 M NaCl, 0.1 percent bovine semm albumin (TBS buffer).
  • TBS buffer bovine semm albumin
  • chromogenic assay for tissue fa ⁇ or a ⁇ ivity is based upon the activation of fa ⁇ or X in the presence of tissue fa ⁇ or and fa ⁇ or VH.
  • the amount of fa ⁇ or X a formed is then assessed by the fa ⁇ or X a catalyzed cleavage of the chromogenic substrate S2222.
  • the rea ⁇ ion is terminated by the addition of 100 ⁇ l of gladal acetic add and the absorbance at 405 nm is measured.
  • An assay control consisted of the addition of a relipidation mixture to which TBS buffer is added instread of a tissue fa ⁇ or containing sample.
  • TBS buffer is added instread of a tissue fa ⁇ or containing sample.
  • 2 ⁇ l of cell extra ⁇ is added to the reaction mixture.
  • a standard curve was constm ⁇ ed using rabbit brain thromboplastin
  • thromboplastin solution is diluted 1:30 and the indicated volumes are added to the chromogenic assay (Fisher, et al, Thrombosis Research 48 ⁇ 9, 1987).
  • Anti-angiogenic a ⁇ ivity of various polypeptides useful in accordance with certain aspe ⁇ s of this invention may be assayed on the chorioallanoic membrane (CAM) as described by Takigawa, et al., (Biochem. Int. 14:357, 1987). Briefly, B16 melanoma cells are inoculated subcutaneously into the loins of C57BIJ6N mice. When the tumors reach approximately 1 cm in diameter, they are exdsed, cut into pieces of 2 mg and placed on sterile Whatman GF/B glass fiber filter disks (6 mm in diam ⁇ er; Reeve-Angel, Clifton, NJ) to which 30 ⁇ l of transduced cells have been added.
  • CAM chorioallanoic membrane
  • CAM 10-day-old chicken embryos through windows made in the egg shells on day 8 of inoculation.
  • the embryos are killed 5 days later by injection of 10% fomalin in PBS.
  • the CAM is exdsed, fixed in 10% formalin in PBS inverted, and examined under a stereo- microscope.
  • Angiogenesis is assayed by measuring the number and thickness of capillaries beneath the filter. A thick capillary, a middle sized capillary, a small capillary, and 5 minute capillaries are given 3, 2, 1 and 1 points, repe ⁇ ivdy, and the average number of points is defined as the angiogenic a ⁇ ivity.
  • the diameters of tumors on the filters are measured in three dimensions and the tumor size is calculated as ( ⁇ /6) abc mm 3 (a, b, and c: length, width, and height, respe ⁇ ively). Low average number of points and decreased tumor size indicates anti-angiogenic a ⁇ ivity.
  • mice are inje ⁇ ed with 300 ⁇ l of India ink at 4 minutes before sacrifice and the skin is taken and dried.
  • the free blood flow is estimated using 10 ⁇ m E-Z TRAC ultraspheres (Interactive Medical Technology, Los Angeles, CA). 5 x 10 5 of 10 ⁇ m microspheres are inje ⁇ ed into the left ventricle of a mouse and the tumor tissue harvested after 5 minutes. After dissolving the tumor tissue, the beads present in the tumor are counted and expressed as beads per gram tumor tissue.
  • D Determination of the Herpes Simplex Vims Thvmidine Kinase A ⁇ ivitv
  • the sensitivity of HSVTK ve ⁇ or transduced cells to gancyclovir may be used to determine the a ⁇ ivity of expressed HSVTK expressed in cells treated according to the disclosed methods. Briefly, cells that are tranduced with pTK-3 are seeded into six plates at a density of 2.5 x 10 6 per plate. In addition, untransduced CT26 and CT26 ⁇ -gal (this cell line was transduced with a vims carrying the reporter gene ⁇ -galactosidase from E. coli.), are also seeded into six plates as controls.
  • MCF-7 stable transfe ⁇ ants are plated and maintained in the same media without G418. Plating densities are adjusted so that cells are 75% confluent for the folic acid binding assays.
  • the form of radiolabeled folic add used is an iodinated, histamine derivative of folic acid obtained from New England Nuclear. Cells are washed twice with 3 ml ice-cold pH 4.5 saline (10 mM Na-acetate, 150 mM NaCl) to dissociate surface-bound folates from cell surface folate receptor. Cell monolayers are subsequently washed twice with 3 ml ice cold PBS, pH 7.4, to return the pH to neutral.
  • the [ 3 H] folic add binding assay is performed both by dire ⁇ binding of [ 3 H] folic acid to membranes and by a solution-phase assay.
  • membrane samples (10-100 ⁇ g of protdn) are incubated with 3 pmol of [ 3 H] folic acid (40 Ci/mmol) in 10 mM sodium phosphate buffer (pH 7.5)/ 150 mM NaCl/ 10 mM EDTA for 30 minutes at 37 C with constant agitation.
  • the membranes are sedimented at 12,000 g for 15 minutes, washed once with the same buffer, dissolved in 10 mM sodium phosphate buffer (pH 7.5)/150 mM NaCl 1% Triton X-100, and subjeded liquid scintillation counting.
  • Nonspecific binding of [ 3 H] folic add is determined for both the assays by performing the assay in each case simultaneously with controls in which the addition of [ 3 H] folic acid is preceded by incubation for 5 minutes at room temperature with 100 pmol of unlabeled folic add.
  • Stable transfectants are plated and cell monolayers are washed twice with 3 ml ice- cold pH 4.5 saline and twice with 3 ml ice-cold PBS, pH 7.4.
  • 3 H-MTX irradiated m ⁇ hotrexate
  • cells are incubated in 2 ml of prewarmed (37_C) DMEM (without FCS or other additives), containing 50 ⁇ g/ml BSA, and 2 ⁇ M [ 3 H] MTX, for 30 minutes at 37_C, 5% CO2.
  • 37_C prewarmed
  • DMEM without FCS or other additives
  • a 16-amino acid fibrinogen ⁇ chain peptide (K16), having the sequence KYGGHHLGGAKQAGDV, is prepared by solid phase synthesis on an Applied Biosystems model 430 peptide synthesizer (Foster City, CA) using phenylacetamidomethyl resins and r-butoxycarbonyl amino adds purchased from Applied Biosystems.
  • the peptide is analyzed for homogendty by high performance liquid chromatography using a C18 ⁇ Bondapak column with a linear gradient of 0-60% acetonitrile in 0.1% trifluoroacetic acid and is found to be >85% homogeneous.
  • the peptide is dissolved in PBS (0.15 M NaCl, 0.01 M sodium phosphate buffer, pH 7.3) and radioiodinated by a modified la ⁇ operoxidase-glucose oxidase method (D'Souza, et al, J. Biol. Chem. 263:3943, 1988 and Lam, et al, J. Biol Chem. 262:947, 1987).
  • Glucose 40 ⁇ g in 80 ⁇ l of 0.2 M sodium phosphate, pH 7.4
  • carrier-free Na 125 I 15 m Ci
  • Enzymobead reagent Bio-Rad, Hercules, CA
  • the iodinated peptide is separated from free Na 125 I by gel filtration on a Bio-Gel P-2 column (Bio-Rad, Hercules, CA).
  • the concentration of the labeled peptide is d ⁇ ermined by absorbance at 280 nm using in extin ⁇ ion coefficient derived from its amino acid composition.
  • Platel ⁇ s are isolated from fresh human blood by differential centrifugation followed by gel filtration on Sepharose 2B in divalent ion-free Tyrode's buffer, pH 7.3, containing 0.1% bovine semm albumin. Briefly, platel ⁇ s are suspended at 4 x 10 8 /ml in divalent ion- gree Tyrode's-albumin buffer. Ca 2+ is added to a final concentration of 1 mM.
  • the platelet stimuli used are 10 ⁇ M ADP, 0.5 unit/ml ⁇ -thrombin, or 100 mM PMA.
  • the radiolabeled K16 peptide is added at a concentration of 30 ⁇ M, and binding proceeded for 45 minutes at 22 C.
  • the primary cross-linking agent is bis(sulfosuccinimidyl)suberate (BS 3 ) (Pierce Chemical Co., Rockford, IL).
  • the cross-linking rea ⁇ ions are terminated after 10 minutes at 22_C by addition of 10 mM Tris, pH 7.0.
  • the cell-bound ligand is recovered by centrifugation through 20% su ⁇ ose, and the cells are extra ⁇ ed in PBS containing 1% Nonidet P-40 and 10 mM N-ethylmaleimide (Sigma, St. Louis, MO). Extracted proteins are precipitated with 10% trichloroacetic acid, and the pellet obtained after centrifugation is washed three times with cold 85% ethanol.
  • Polyacrylamide gel electrophoresis is performed in the presence of sodium dodecyl sulfate in vertical slab gels in the buffer system of Laemmli (Nature 227:680, 1970) Gels of varying percentages are used. For initial analysis of cross-linked samples 7.5% gels are mn under nonreducing conditions. Gels utilized for amino acid sequencing are 10-20% gradient gels. Samples in Laemmli sample buffer are treated with 5% 2-mercaptoethanol for disulfide bond redu ⁇ ion. Analytical gels are dried, and autoradiograms are developed with Kodak X-Omat AR films. Molecular weight is estimated on the basis of electrophoretic mobility relative to prestained standards obtained from Diversified Biotech. - 96 -
  • the relative mobility (RF) is d ⁇ ermined by measuring the migration of the 125 I-K16-GPII band relative to the migration of the ovalbumin marker protein in the same gel.
  • Cmde recombinant retrovirus is obtained from a Celligan biorea ⁇ or (Ne Brunswick, New Brunswick, NJ) containing DA cells transformed with the recombinan retrovims (U.S.S.N. 07/395,932) bound to the beads of the biorea ⁇ or matrix.
  • the cell release the recombinant retrovims into the growth media that is passed over the cells in continuous flow process.
  • the media exiting the biorea ⁇ or is colle ⁇ ed and passed initiall through a 0.8 micron filter then through a 0.65 micron filter to clarify the cmd recombinant retrovims.
  • the filtrate is concentrated utilizing a cross flow concentratin system (Filtron, Boston, MA).
  • DNase Intergen, New York, NY
  • the digest is diafiltrate using the same cross flow system to 150 mM NaCl, 25 mM tromethamine, pH 7.2.
  • Th diafiltrate is loaded onto a Sephadex S-500 gel column (Pharmacia, Piscataway, NJ), equilibrated in 50 mM NaCl, 25 mM tromethamine, pH 7.4.
  • the purified recombinan retrovims is eluted from the Sephadex S-500 gel column in 50 mM NaCl, 25 m tromethamine, pH 7.4.
  • the formulation buffer containing la ⁇ ose was prepared at a 2x concentrated stock solution.
  • the formulation buffer contains 25 mM tromethamine, 70 mM NaCl, 2 mg ml arginine, 10 mg ml human semm albumin (HSA), and 100 mg/ml la ⁇ ose in a final volume of 100 mis at a pH 7.4.
  • the purified recombinant retrovirus is formulated by adding one part 2x lactose formulation buffer to one part S-500 purified recombinant retrovims.
  • the formulated recombinant retrovims can be stored at -70°C to -80°C or dried.
  • the formulated retrovirus is lyophilized in an Edwards Refrigerated Chamber (3 Shelf RC3S unit) attached to a Supermodulyo 12K freeze dryer (Edwards High Vacuum, Tonawanda, NY).
  • a Supermodulyo 12K freeze dryer Edwards High Vacuum, Tonawanda, NY.
  • the vials are stoppered under a vacuum following a slight nitrogen gas bleeding.
  • vials are crimped with aluminum seals.
  • formulated liquid produ ⁇ was stored at both -80°C and at -20°C cycling freezer.
  • viral infectivity of these samples were compared to the viral infe ⁇ ivity of lyophilized samples.
  • the lyophilized samples were stored at -20°C, refrigerator temperature and room temperature. A ⁇ ivity of the samples upon reconstitution are d ⁇ ermined by titer assay.
  • the lyophilized recombinant retrovims is reconstituted with 1.0 ml water.
  • the infe ⁇ ivity of the reconstituted recombinant r ⁇ rovims is d ⁇ ermined by a titer activity assay.
  • the assay is conducted on HT 1080 fibroblasts or 3T3 mouse fibroblast cell line (ATCC CCL 163). Specifically, 1.0 x 10 5 cells are plated onto 6 cm plates and incubated overnight at 37°C, 10% CO2. Ten microliters of a dilution series of reconstituted recombinant retroviruses are added to the cells in the presence of 4 ⁇ g/mL polybrene (Sigma, St.
  • cells that have been transduced with a recombinant ve ⁇ or which encodes the neo resistance gene are sele ⁇ ed for neomycin resistance in G418 containing media and incubated for 5 days at 37°C, 10% CO2.
  • the cells are re-fed with fresh media containing G418 and incubated for 5 to 6 days.
  • the cells are stained with Commassie blue for colony dete ⁇ ion. The titer of the sample is determined from the number of colonies, the dilution and the volume used.
  • Figure 20 demonstrates that storage in lyophilized form at -20°C to refrigerator temperatures r ⁇ ains similar viral a ⁇ ivity as a recombinant r ⁇ rovims stored in liquid at -80° to -20°C permitting less stringent temperature control during storage.
  • the formulation buffer containing mannitol was prepared as a 2x concentrated stock solution.
  • the formulation buffer contains 25 mM tromethamine, 35 mM NaCl, 2 mg/ml arginine, 10 mg/ml HSA and 80 mg ml mannitol at a final volume of 100 mis at a pH 7.4.
  • the purified recombinant retrovims is formulated by adding one part mannitol formulation buffer to one part S-500 purified recombinant r ⁇ rovims.
  • the formulated recombinant r ⁇ rovims can be stored at this stage at -70°C to -80°C or dried.
  • the formulated r ⁇ rovims is dried in an Edwards Refrigerated Chamber (3 Shelf RC3S unit) attached to a Supermodulyo 12K freeze dryer.
  • the vials are stoppered under a vacuum following nitrogen gas bleeding to 700 mbar.
  • vials are crimped with aluminum seals.
  • formulated liquid produ ⁇ was stored at both -80°C and at -20°C in cycling freezers. The viral infectivity of these samples were compared to the viral infectivity of lyophilized samples, Figure 21. The lyophilized samples were stored at -
  • Figure 21 demonstrates that storage in lyophilized form at -20°C to refrigerator temperature retains significant viral activity as compared to recombinant retrovims stored in liquid at -80°C or -20°C, permitting less stringent temperature control during storage.
  • the formulation buffer containing trehalose was prepared as a 2x concentrated stock solution.
  • the formulation buffer contains 25 mM tromethamine, 70 mM NaCl, 2.0 mg/ml arginine, 10.0 mg/ml HSA and 100 mg/ml trehalose at a final volume of 100 mis at a pH 7.2.
  • the purified recombinant retrovims is formulated by adding one part trehalose formulation buffer to one part S-500 purified recombinant retrovims.
  • the formulated recombinant r ⁇ rovims can be stored at this stage at -70°C to -80°C or dried.
  • the formulated r ⁇ rovims is dried in an Edwards Refrigerated Chamber (3 Shelf RC3S unit) attached to a Supermodulyo 12K freeze dryer. When the freeze drying cycle is completed, the vials are stoppered under a vacuum following nitrogen gas bleeding to 700 mbar. Upon removal, vials are crimped with aluminum seals.
  • formulated liquid produ ⁇ was stored at both -80°C and at -20°C in cycling freezers. The viral infe ⁇ ivity of these samples was compared to the viral infe ⁇ ivity of lyophilized samples, Figure 22. The lyophilized samples were stored at - 20°C, refrigerator temperature and room temperature.
  • a ⁇ ivity of the samples upon reconstitution are determined using the titer assay as described in A above.
  • Figure 22 demonstrates that storage in lyophilized form at -20°C to refrigerator temperature retains similar viral a ⁇ ivity as compared to recombinant retrovims stored in liquid at -80°C to -20°C permitting less stringent temperature control during storage.
  • Viral infe ⁇ ivity of liquid formulated recombinant r ⁇ rovims samples stored at -80°C was compared to viral infe ⁇ ivity of lyophilized formulated recombinant r ⁇ rovims stored at -20°C.
  • a bulk of recombinant r ⁇ rovims was received and formulated in four different ways as shown below.
  • the formulated recombinant retrovims was then frozen in bulk for 1.5 months subsequent to being quick thawed and freeze dried. Positive controls were stored at -80°C for comparison with lyophilized samples which were stored at -20°C after freeze-drying.
  • the formulations are listed bdow:
  • Trehalose 50 25 60 . 1 5
  • the y-axis on each of the 4 graphs (A, B, C, D) represent the normalized titer.
  • t 0, a titer value was established for both the -80°C hquid sample and the -20°C lyophilized sample.
  • the titer obtained was divided by the zero time point titer value and the percentage of original entered onto the graph.
  • the data demonstrates that post-lyphilization activity is maintained in the lyophilized sample (stored at -20°C) relative to the liquid sample (stored at -80°C).
  • the formulated lyophilized recombinant retrovims was stored in a -20°C freezer (a frost-free cycling freezer). Comparison to the formulated liquid recombinant retrovims stored at -
  • Cmde recombinant alphavims ve ⁇ or is obtained from a Celligan bioreactor containing packaging cells transfe ⁇ ed or transduced with the recombinant alphavims ve ⁇ or, and bound to the beads of the biorea ⁇ or matrix.
  • the cells release the recombinant alphavims ve ⁇ or into the growth media that is passed over the cells in a continuous flow process.
  • the media exiting the biorea ⁇ or is colle ⁇ ed and passed initially through a 0.8 mi ⁇ on filter then through a 0.65 mi ⁇ on filter to clarify the cmde recombinant alphavims ve ⁇ or.
  • the filtrate is concentrated utilizing a cross flow concentrating system.
  • Approximately 50 units of DNase per ml of concentrate is added to digest exogenous DNA.
  • the digest is diafiltrated using the same CTOSS flow system to 150 mM NaCl, 25 mM trom ⁇ hamine, pH 7.2.
  • the diafiltrate is loaded onto a Sephadex S-500 gel column, equilibrated in 50 mM NaCl, 25 mM tromethamine, pH 7.4.
  • the purified recombinant alphavims ve ⁇ or is eluted from the Sephadex S-500 gel column in 50 mM NaCl, 25 mM tromethamine, pH 7.4.
  • the formulation buffer containing la ⁇ ose is prepared as a 2X concentrated stock solution.
  • the formulation buffer contains 25 mM tromethamine, 70 mM NaCl, 2 mg/ml arginine, 10 mg/ml HSA, and 100 mg/ml la ⁇ ose in a final volume of 100 mis at a pH 7.4.
  • the purified recombinant alphavims ve ⁇ or is formulated by adding one part 2X lactose formulation buffer to one part S-500 purified recombinant alphavims ve ⁇ or.
  • the formulated recombinant alphavims ve ⁇ or can be stored at -70°C to -80°C or dried.
  • the formulated alphavims ve ⁇ or is lyophilized in an Edwards Refrigerated
  • Chamber (3 Shelf RC3S unit) attached to a Supermodulyo 12K freeze dryer.
  • the vials are stoppered under a, vacuum following a slight nitrogen gas bleeding.
  • vials are crimped with aluminum seals.
  • the lyophilized recombinant r ⁇ rovims is reconstituted with 1.0 ml water or other physiologically acceptable diluent.
  • Colon tumor cells (CT 26) (Brattain, Baylor College of Mediine, Houston, TX) are transduced with DA/TK-3, a G-pseudotyped TK-3 ve ⁇ or. Twenty-four hours after adding the viral supernatant, the CT26 cells are placed under G-418 sele ⁇ ion (450 ⁇ g/ml). After 10 days incubation, a G-418 sele ⁇ ed pool is obtained and designated CT26 TKweo. (CT26 TKneo), CT26 TKweo cells were seeded into six 10 cm 2 plates at a density of 2.5 X 10 6 per plate.
  • each of two other cell types CT26 and CT26 ⁇ -gala ⁇ osidase, ( ⁇ -gal ), (this cell line was transduced with a vims carrying the reporter gene ⁇ -gal from E. coli.), were also seeded into six 10 cm 2 plates as controls. Five plates of each cell type were treated twice per day for four consecutive days with medium containing ganciclovir - 101 -
  • mice In order to test whether in vivo transdu ⁇ ion of a murine tumor could be used to treat the disease, an experiment was performed to determine the optimal concentration of ganciclovir necessary to eliminate a tumor that was transduced and sele ⁇ ed in vitro to assure 100% transdu ⁇ ion. Twelve groups of 3 Balb/c (Harlan Spague Dawley, Indianapolis, IN) mice each are inje ⁇ ed with 2.0 X 10 5 CT26 TKneo cells. Six groups of mice are injected with these cells intraperitoneally (I.P.) and six groups of mice are injected subcutaneously (S.C.). Two other groups of 3 mice each are inje ⁇ ed with 2.0 X 10 5 unmodified CT26 cells (as a control) either I.P. or S.C..
  • mice in the 125 mg/Kg, 250 mg/Kg and 500 mg Kg treated groups were dead due to the toxic effects of gandclovir.
  • Mice in the 15.63 mg/Kg, 31.25 mg/Kg and 62.5 mg/Kg treated groups were treated for an additional 7 days and were able to tolerate the treatment. Tumor measurements were made for 23 days ( Figure 25).
  • CT26TK neo grew only slightly slower than unmodified CT26. Complete tumor regression was seen in the groups of mice treated with the 62.5 mg/Kg regimen. Partial tumor regression was seen in the 31.25 mg/Kg treated groups. Little or no effect was seen in the 15.63 mg/Kg treated groups as compared to the 2 untreated control groups.
  • HT1080 cells a human fibrosarcoma line
  • other appropriate cells e.g. other human tumor derived lines such as MCF-7, a breast tumor derived cell line; primary human endothelial cells, HTJVEC; mouse tumor cell lines such as the colon tumor line CT26, the fibrosarcoma line L33, the melanoma cell line B 16, the breast tumor cell line Tg-6-2, the neuroblastoma cell line C1300; mouse endothelial cells or cell lines such as the SVEC cell line in a 10 cm dish are treated with the gene delivery ve ⁇ or at a multiplicity of cell transdu ⁇ ion units (e.g.
  • Example 5A Tissue Fa ⁇ or
  • Example 5B general antiangiogenic fa ⁇ ors
  • Example 5D He ⁇ es thymidine kinase
  • Example 5E folate- integrin B3
  • the major issues in the use of the invention is to achieve sufficient expression of the novel effe ⁇ or protein such as the Russel viper venom protein, tissue factor, prodmg activating enzyme, or metabolism altering protein such as uricase, in the tumor or the peritumoral vascular system, without having unacceptable toxic effe ⁇ s due to significant expression elsewhere.
  • novel effe ⁇ or protein such as the Russel viper venom protein, tissue factor, prodmg activating enzyme, or metabolism altering protein such as uricase
  • mice plus the L33 or CT26 cell lines; C57B/6 plus B 16 cells;FVB/N plus TG-6-2 cells; C3H mice plus C 1300 or SVEC cells; BalB/c nu/nu mice plus any of these, or plus human tumors such as the melanoma lines DM252, DM 6, DM92 (see U.S.S.N. 08/032846) or MCF-7 cells.
  • Tumor cells are inje ⁇ ed at an appropriate dose (typically 2 x 10 5 cells) either subcutaneously, I.V. for the B16 lung metastases model, or intra-splenically for the CT26 liver metastases model.
  • Groups (1-10 mice) of the inje ⁇ ed mice are maintained until palpable tumors (1-4 mm in diameter) are apparent for subcutaneous tumors, or for the length of time that leads to easily dete ⁇ able metastases, upon necropsy, in the metastases models. This is typically 7 to 14 days.
  • the tumor vascularization can be visualized using the techniques in Example 5C and various routes of administration examined to determine the one that gives best flow into the tumors.
  • the ve ⁇ or preparations are inje ⁇ ed at the previously determined I.V. or peritumoral site at doses of 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 ve ⁇ or units (e.g. colony forming units, cfu) with or without transduction enhancers such as polybrene (1-8 ⁇ g/ml) or DEAE dexran (2 - 30 ⁇ g/ml).
  • Inje ⁇ ions are given daily for 1, 2, 3, 4, 5, 6, or 7 days, and 2 to 7 days after the last injection, the tumors are exdsed and assayed for acquisition of the a ⁇ ivity of the delivered gene (see, for example, Example 4C, 4D, and Example 5) .
  • Controls are mice inje ⁇ ed with an irrelevant, inert ve ⁇ or (e.g. encoding human gamma interferon which is ina ⁇ ive in mice).
  • the ve ⁇ or encodes genes of the types described in the detailed description and in the case of a retroviral ve ⁇ or carries a non-specific envelope such as the amphotropic or xenotropic envelopes, and relies on the paucity of replicating cells in the general vascular endothelium, and the over-representation of such replicating cells in sites of neovascularization such as tumors.
  • the ve ⁇ or carries an appropriate targeting ligand that avoids non-specific adsorption, and allows binding and entry into the target cell type.
  • the specificity of the system may also be controlled by the inclusion of appropriate expression control elements
  • the same administration protocols or a smaller number chosen on the bases of the above experiments or other criteria, are used to measure the antitumor effect in these mouse models.
  • the endpoints are tumor size and growth/regression over time, time to death, survival, or other appropriate marker (e.g. number of metastases in those models).
  • Patients are treated with the same ve ⁇ ors as the mouse models or the same nucleic acid backbone carried in a ve ⁇ or that is resistant to human complement (see, for example, the companion application "Produ ⁇ ion and Administration of High Titer Recombinant Retroviruses")
  • Patients with tumor masses that are not removable surgically e.g. brain, prostate, cervical, ovarian, bladder), for which removal requires simultaneous removal of large amounts of irreplaceable healthy tissue (e.g. amputation of limbs or removal of whole organs), or where metastasis may have taken place (e.g. colon metastasis to the liver, melanoma metastasis to the brain, other subcutaneous sites and the lung), can be treated.
  • ve ⁇ or units I.V. intra- arterially, in the local vasculature or peritumorally in a volume of 0.1 to 3 ml preferably.
  • the ve ⁇ or carries a gene that encodes a clotting enhancer or other gene chosen from the types described above. If the gene is one that converts a non-toxic precursor (prodmg) into a toxic product , the prodmg is administered at doses defined in the Physicians Desk Reference or those predi ⁇ ed from animal experiments at times of between 1 to 30 days after the last administration of ve ⁇ or.
  • the ve ⁇ ors are administered from 1 to 20 times at intervals of 1 to 15 days and the patient status is monitored by following normal clinical parameters and monitoring tumor sizes by radiography, mri scans, pet scans or other conventional means
  • the following example describes the use of the coupled targeting element Ab-biotin to targ ⁇ the coupled retroviral ve ⁇ or particle-avidin to a specific cell type.
  • biotinylated melanocyte stimulating hormone (MSH) is first inje ⁇ ed into the patient. After a period of time (up to 3 days) after which non-specific binding has decayed and only specific ligand complexes remain, a ve ⁇ or expressing avidin on its surface is injected. The high affinity of avidin for biotin focuses the ve ⁇ or to the target tissue.
  • MSH melanocyte-stimulating hormone
  • MSH has a receptor affinity (Kp) in the range of lO ⁇ M.
  • a vector is first created in order to form the backbone for both the gag/pol and env expression cassettes. Briefly, pBluescript SK- phagemid (Stratagene, San Diego, CA, GenBank accession number 52324, referred to as "SK"”) is digested with Spe I and blunt ended with Klenow. A blunt end Dra I fragment of SV40 (Fiers, et al, Nature 273: 1 13- 120, 1978) from Dra I (bp 2,366) to Dra I (bp 2,729) is then inserted into SK", and a constmct isolated in which the SV40 late polyadenylation signal is oriented opposite to the LacZ gene of SK'. This constmct is designated SK-SV40A.
  • HCMV-MIE human cytomegalovirus major immediate early promoter
  • Boshart, et al, Cell ⁇ 7:521-530, 1985 (Hinc ⁇ , bp 140 to EagI, bp 814) is isolated after digestion with Hinc II and Eag I, and the Eag I site blunt ended.
  • the 674 blunt ended fragment is ligated into SK-SV40A.
  • the final constm ⁇ , designated pHCMV-PA is then isolated (see Figure 28). This constm ⁇ contains the HCMV promoter oriented in opposite orientation to the LacZ gene, and upstream from the late polyadenylation signal of SV40.
  • pCMV-env* 500 is created by inserting the Xba I-Nhe I fragment of MoMLV (bp 5,766 through bp 7,845 of MoMLV) into pCMV-PA (Example 2A) expression ve ⁇ or. Briefly the Xba I-Nhe I envelope fragment is isolated from pMLV-K (Miller et al, J. Vir. 49:214, 1988) on an agarose gel. The fragment is then blunt-ended with T4 polymerase using standard methods, Ugated into pCMV-PA (Example 2), and digested at the Eco RV and Sma I sites. The produ ⁇ in the corre ⁇ orientation has a CMV MIE promoter followed by the complete ecotropic envelope coding sequence and an SV40 polyadenylation signal.
  • the oligonucleotide is used to modify single stranded pCMV-e *600 by the method of Kunkle (PNAS 52:488, 1985). This modification replaces a portion of the variable A region of envelope (Battini, et al, J. Virol. 66:1468-1475, 1992) with the sequence of the oligonucleotide.
  • the produ ⁇ is then digested with EcoRl and partially digested with Fsp I.
  • the Eco RI-Fsp I fragment of avidin (bp 198 through bp 485) is ligated into the vector
  • the final produ ⁇ is a plasmid containing CMV promoter, hybrid eco-avidin envelope and SV40 polyadenylation signal, called pCMV-env ec ⁇ * v d n .
  • the MSH peptide S-Y-S-M-E-H-F-R-W-G-L-P-V-NH 2 is synthesized (Chiron Co ⁇ ., Emeryville, CA), and biotinylated with NHS-Biotin (Pierce, Rockford, IL) according to the manufacturer's instm ⁇ ions.
  • CB ⁇ -gal The ⁇ -gala ⁇ osidase encoding marker recombinant retroviral ve ⁇ or, CB ⁇ -gal is cotransfe ⁇ ed into cell Une 293 2-3 (see U.S.S.N. 07/800,921) along with pCMV-env 1500 - av idi n
  • equivalent ve ⁇ ors encoding luciferase, green fluorescent protein (GFP) or other markers can be used.
  • Clones are sele ⁇ ed (with G418) and screened for high produ ⁇ ion of RNA containing particles and screened for surface expression of avidin using 3 H-biotin binding. Vector particles containing avidin are tested utilizing 14 C-biotin (Amersham, Chicago, IL) and a sucrose gradient.
  • Human melanoma cells DM252, DM6, DM92 are grown in appropriate medium.
  • the specificity of biotinylated MSH binding to targ ⁇ cells is tested by addition of avidin- fluorescein and fluorescence microscopy.
  • Transduction of eco-avidin CB ⁇ -gal is tested either by staining or by G418 sele ⁇ ion (see U.S.S.N. 08/032,846), and the efficiency of tranduction compared to non-melanoma cells such as HT1080 human fibrosarcoma cells
  • Nude mice are implanted with one or more of the following human melanoma cell lines: DM252, DM6, DM92 (see U.S.S.N. 08/032,846) in the peritoneal cavity.
  • Targeting is determined by first injecting biotin-MSH into the mouse, followed by injection of 1 x 10 5 to 1 x 10 8 cfu eco-avidin CB ⁇ -gal recombinant retroviral vectors. Targeting is assessed by subsequently disse ⁇ ing the melanoma tissue, and staining for ⁇ -gal, or assaying for luciferase a ⁇ ivity in the melanoma and mouse tissue.
  • the data generated in the mouse system is used to d ⁇ ermine the protocol for administration in patients.
  • the patients receive three to twenty doses of ve ⁇ or encoding the desired gene I.P., I.V., inter-arterial (I.A.), or into the lymphatic system.
  • the dosage will range from about 1.0 x 10 6 to about 1.0 x 10 10 cfu of the gene delivery vehicle given
  • MOLECULE TYPE cDNA (2) INFORMA ⁇ ON FOR SEQ ID NO: 11: CCTTTTCAGG GGATCCGCCA C 21 (2) INFORMATION FOR SEQ ID NO: 12:
  • MOLECULE TYPE cDNA
  • SEQUENCE DESCRIP ⁇ ON SEQ ID NO: 12: GTGGCGGATC CCCTGAAAAG G 21

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Toxicology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Cell Biology (AREA)
  • Hematology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
EP95944229A 1994-12-30 1995-12-22 Verfahren und zusammensetzungen zur in vivo behandlung der festen tumoren Withdrawn EP0802801A2 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US368574 1989-06-20
US36857494A 1994-12-30 1994-12-30
PCT/US1995/016855 WO1996021416A2 (en) 1994-12-30 1995-12-22 Methods and compositions for treatment of solid tumors in vivo

Publications (1)

Publication Number Publication Date
EP0802801A2 true EP0802801A2 (de) 1997-10-29

Family

ID=23451821

Family Applications (1)

Application Number Title Priority Date Filing Date
EP95944229A Withdrawn EP0802801A2 (de) 1994-12-30 1995-12-22 Verfahren und zusammensetzungen zur in vivo behandlung der festen tumoren

Country Status (4)

Country Link
EP (1) EP0802801A2 (de)
JP (1) JP2001520503A (de)
AU (1) AU4608296A (de)
WO (1) WO1996021416A2 (de)

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0659209A1 (de) * 1991-07-26 1995-06-28 The University Of Rochester Krebstherapie unter verwendung maligner zellen
GB9601640D0 (en) * 1996-01-26 1996-03-27 Cancer Res Campaign Tech Ligand directed enzyme prodrug therapy
ATE340259T1 (de) * 1996-07-16 2006-10-15 Archibald James Mixson Kationisches vehikel: dns komplexe und ihre verwendung in gentherapie
US6080728A (en) * 1996-07-16 2000-06-27 Mixson; A. James Carrier: DNA complexes containing DNA encoding anti-angiogenic peptides and their use in gene therapy
US20020052309A1 (en) * 1996-09-11 2002-05-02 Athanasius A. Anagnostou Method of treating endothelial injury
WO1998012332A1 (en) * 1996-09-17 1998-03-26 Chiron Corporation Compositions and methods for treating intracellular diseases
FR2757178B1 (fr) * 1996-12-13 1999-03-05 Centre Nat Rech Scient Utilisation d'un vecteur exprimant l'adn polymerase beta comme medicament
DE19701141C1 (de) * 1997-01-16 1998-04-09 Hoechst Ag Genkonstrukte für durch Proteasen aktivierbare Wirksubstanzen
US6844188B1 (en) 1998-04-08 2005-01-18 University Of North Carolina At Chapel Hill Methods and modified cells for the treatment of cancer
US7862829B2 (en) 2004-07-09 2011-01-04 University Of North Carolina At Chapel Hill Viral adjuvants
EP1856250B1 (de) 2005-02-15 2013-07-24 The University of North Carolina At Chapel Hill Neue lebende virus-impfstoffe
ES2620702B2 (es) * 2017-03-29 2017-12-18 Universidad De Cantabria Construcción génica y su empleo en el tratamiento de la fibrosis cardíaca

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5091309A (en) * 1986-01-16 1992-02-25 Washington University Sindbis virus vectors
DE3716722A1 (de) * 1987-05-19 1988-12-01 Hoechst Ag Gentechnologisches verfahren zur herstellung von angiogeninen
US5217879A (en) * 1989-01-12 1993-06-08 Washington University Infectious Sindbis virus vectors
FR2683226B1 (fr) * 1991-10-30 1995-06-09 Univ Paris Curie Cellules piegees a l'egard d'antigenes provoquant le dysfonctionnement du systeme immunitaire, medicaments pour la prevention ou le traitement de maladies induites par ces dysfonctionnements.
WO1993013807A1 (en) * 1992-01-10 1993-07-22 Georgetown University A method of administering genetically engineered endothelial cells to sites of angiogenesis for effecting genetic therapy
JPH07508400A (ja) * 1992-03-03 1995-09-21 ザ　ソーク　インスティチュート　フォア　バイオロジカル　スタディーズ 受容体のインターナライゼーション・シグナル
WO1993018794A1 (en) * 1992-03-26 1993-09-30 Gensia, Inc. In vivo peptide therapy
BE1006437A3 (fr) * 1992-12-10 1994-08-30 Company S A Z Sequence nucleotidique destinee au traitement du cancer et des infections.
WO1994021118A1 (en) * 1993-03-24 1994-09-29 The Uab Research Foundation Gene-therapy composition and method for treating carcinomas

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9621416A2 *

Also Published As

Publication number Publication date
AU4608296A (en) 1996-07-31
WO1996021416A2 (en) 1996-07-18
JP2001520503A (ja) 2001-10-30
WO1996021416A3 (en) 1996-10-10

Similar Documents

Publication Publication Date Title
EP0694070B1 (de) Rekombinanter alphavirus vektor
JP3941963B2 (ja) 全長第ｖｉｉｉ因子のレトロウイルス送達
EP1997900A2 (de) Rekombinante Vektoren auf Alphavirus-Basis mit reduzierter Hemmung der zellulären Makromolekularsynthese
WO1997024447A1 (en) Immunostimulation mediated by gene-modified dendritic cells
EP0802801A2 (de) Verfahren und zusammensetzungen zur in vivo behandlung der festen tumoren
WO1999018226A2 (en) Recombinant alphavirus-based vectors with reduced inhibition of cellular macromolecular synthesis
US5643770A (en) Retroviral vector particles expressing complement inhibitor activity
JPH06335392A (ja) Ｈｉｖ阻害性アンチセンスおよび他のヌクレオチド配列を含有する発現構成体、レトロウイルスのベクターおよびそれを含有する組換えレトロウイルス
EP0800403A2 (de) Nichttraumatische erteilung von genbeförderungsmitteln
JP2002538182A (ja) 新脈管形成を阻害するための方法および試薬
US6960469B2 (en) Adenoviral vectors encoding an antibody fused to a CD4 extracellular domain
WO1997023608A1 (en) Compositions and methods for targeting gene delivery vehicles using covalently bound targeting elements
US20020123479A1 (en) Immunostimulation mediated by gene-modified dendritic cells
JP2002523053A (ja) 標的化されたアルファウイルスおよびアルファウイルスベクター
JPH11504802A (ja) 組換えアルファウイルスベクター
Gordon et al. Genetic engineering of targeted retroviral vectors
AU780164B2 (en) Novel implant and novel vector for the treatment of acquired diseases
AU738995B2 (en) Novel implant and novel vector for the treatment of acquired diseases
Perri Exploiting the use of plasminogen kringle domains for cancer gene therapy
WO1998039449A9 (en) COMPOSITIONS AND USE OF M-CSF-alpha
EP0973904A1 (de) Zusammensetzungen und Verwendungen von M-CSF-alpha
JP2000503532A (ja) 遺伝子送達ビヒクル標的化リガンド
HK1026920A (en) Recombinant alphavirus vectors
JPH10511951A (ja) 高力価組換えレトロウイルスの産生および投与

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19970709

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL PT SE

17Q First examination report despatched

Effective date: 20000825

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20030917