JP2001520503A - Methods and compositions for the treatment of solid tumors in vivo - Google Patents

Abstract

  (57) [Summary] The present invention provides compositions and methods for the treatment of solid tumors. This treatment employs gene therapy utilizing a recombinant viral vector expressing the polypeptide. This polypeptide selectively initiates the irreversible coagulation of blood in the tumor vasculature, inhibits tumor neovascularization, activates non-toxic drugs into toxic drugs within the tumor vessel wall, and increases the vascular bed. It can cause destruction and absorb or metabolize nutrients in the blood supplied to the tumor. Production of these polypeptides by transduced cells in or adjacent to the blood vessels of the tumor results in death of the tumor cells.

Description

DETAILED DESCRIPTION OF THE INVENTION          Methods and compositions for the treatment of solid tumors in vivoTechnical field   The present invention relates generally to the field of anti-cancer therapy, and more particularly, to recombinant cancer. And methods of inducing selected tumor cells using viral vectors.Background of the Invention   Many different therapies are available for the treatment of various types of human tumors. These include surgery, radiation therapy, chemotherapy, radioimmunotherapy, and neoadjuvant. Including neoadjuvant therapy. These treatments determine whether the tumor is metastatic or not. Or they can be used individually or in combination depending on whether they are non-metastatic.   Surgery is mainly used for non-metastatic tumors and, therefore, This is the treatment of choice for locally treatable cancers. Under certain circumstances, Surgery can be used to alleviate or reconstruct unrepairable cases and rehabilitation Can be used for In addition, surgery is performed locally prior to removal of the primary neoplasm. Diagnostic Role in Determining the Pathological Stage of Extent of Local and Local Invasion Play a part. However, for certain tumor types, surgery can be disadvantageous. Because surgery can lose shape, be disabled, or be ineffective It is. For inoperable tumors, primary local treatment with ionizing radiation , Is often the treatment of choice.   Two types of radiation therapy, brachytherapy and teleradiation, are It is used to treat. In brachytherapy, the radiation source is placed near the tumor. Is placed. This endoluminal approach is used for gynecological or oral neoplasms It is. In teleradiotherapy, ultra-high pressure radiation therapy usually uses a linear accelerator. Delivered, allowing more accurate beam localization, and concomitant skin radiation toxicity Work around. For tumor areas while minimizing toxicity to adjacent normal tissue Various approaches are used to increase the radiation dose. Radiation therapy , Usually delivered in a fractionated fashion, normal to sublethal damage during the treatment Radiation by allowing time for the recovery of healthy host tissues (not tumors) Has biological superiority. The fractionated radiation dose is usually Usually administered 5 days per week until delivered in a 4-6 week course You.   Unfortunately, radiation therapy is followed by acute and potential toxicity. Acute toxicity May cause generalized fatigue and malaise, anorexia, nausea and vomiting, local skin changes , Including diarrhea and mucosal ulceration of the irradiated area. Large area, especially pelvis and proximal length Irradiation of bone can cause significant myelosuppression. Long-term toxicity depends on skin color Causes elemental excess, reduced organ function, myelopathy, osteonecrosis and secondary malignancy Can wake up. These toxicities are dose related, but due to careful shielding and fractionation. Can be minimized or avoided.   Currently, more than 50% of all cancer patients receive radiation therapy during the disease. I Radiation therapy is often used with therapeutic purposes for certain types of tumors It is the only substance. For a wider range of cancers, irradiation is combined with surgery You. Irradiation is also useful for some patients with lymphoma or lung cancer and for some Used as an adjuvant to chemotherapy for pediatric cancer. Depending on the case In some cases, chemotherapy is used to make tumor cells sensitive to the toxic effects of irradiation. Can be used.   Localized (locoregional) hyperthermia, although its use is very limited, Can be used for some tumor sites. Hyperthermia is important as an aid to ionizing irradiation It is a form of non-ionizing irradiation between a certain 40-42 ° C. This treatment may also Also includes drugs that increase the response of the ulcer. The largest use of hyperthermia is in large vascular hyperplasia Is to control the tumor.   Chemotherapy makes surgery or irradiation impractical or only partially effective Used if Chemotherapy (cytokines, cytotoxic drugs, hormones, Including the use of antihormones and other biological agents) are increasingly It has become an effective means. Cytokines alone or with other cytokines or When used in combination with cytotoxic drugs, metastatic gas may be regulated by modulating the immune system. (Heaton, K. et al., Cancer Immunol.) Immunother 37: 213-219, 1993). Useful cytokines include interleukins (IL ) -2, IL-4, IL-6, tumor necrosis factor (TNF), α-interferon, and γ-I Interferon (α-IFN and γ-IFN). Intravenous IL-2 alone is metastatic It is effective in about 13-50% of patients with renal cell carcinoma or melanoma, and treats other tumors. It can be effective also in the position. In addition, α-IFN has been shown to be specific after resection of tumor tissue mass. It has been shown to be effective against solid tumors, which may be an adjuvant treatment Suggests a possible postoperative role (reviewed in Heaton, K et al., Supra).   Combining cytokines to enhance antitumor effects is a different mechanism Combinations of drugs that attack more neoplastic cells increase antitumor efficacy in vivo It is based on the assumption that it should be done. Therefore, drugs with different side effects In patients treated with the combination, toxicity should be reduced. Preclinical research laboratory Studies show that such a combination of cytokines may be effective in animal models (Reviewed in Heaton, K et al., Supra). Furthermore, cytokines are new forms Combined with other cytotoxic drugs to provide more effective treatment for adult diseases obtain. Such combinations may have different mechanisms of action and different toxicity profiles. To use. For example, α-IFN is a serum concentration of the active 5-fluorouracil (5-FU) metabolite. Increasing the degree appears to enhance the effect of 5-FU. In addition, IL-2 When α-IFN and cisplatin and dacarbazine are used in combination, Produced a much higher response rate than everything observed with the cytotoxic drug alone.   Most anticancer drugs are used systemically, but are not available for local or local administration. In some cases, there are indications to be selected. Topical administration may cause active chemotherapeutic tumors Injection directly into the site (eg, by embolizing the primary blood supply to the tumor (Intravesical treatment, intraperitoneal treatment, hepatic artery infusion). These treatments are remarkable Mitigation and improved survival.   However, many patients treated with chemotherapy have a limited response. That's it Such treatment may result in spontaneous genetic alterations in a subpopulation of cancer cells prior to chemotherapy exposure. It can be nullified due to drug resistance due to differences. Remove sensitive cells with chemotherapy After removal, the resistant subpopulation grows to a dominant cell type.   Radioimmunotherapy offers potential for anticancer activity for common tumors This is one form of treatment. This treatment is very experimental, but in particular hematopoietic malignancies In tumors, progress has been made in the development of useful clinical drugs. Radioimmunotherapy The success of multiple doses of selective tumors without damaging radiation-sensitive tissue Depending on the delivery. For solid tumors, multiple radiolabeled polyclonals And monoclonal antibodies in liver cancer, intrahepatic cholangiocarcinoma, melanoma, ovarian cancer, peritoneum It has been used in the treatment of patients with carcinomatosis and glioma (Larson, S. et al. Nucl Med Biol. 21: 785-792, 1994). In these cases,¹³¹ Iodine was the most commonly used label.   Administration of radiolabeled antibody into an enclosed space offers the greatest potential for successful treatment. It seems to offer. Intravenous administration of the compound is not effective. For hematopoietic progenitor cells Mouse monoclonal antibody M195 (anti-CD-33), colorectal, pancreatic, ovarian and (Anti-TAG-72) for primary and other human tumors and primary and metastatic in patients Numerous monoclonal antibodies, including 3F8 (anti-GDZ), are localized in It is used. But the real problem with this treatment is that The development of immunity to local antibodies and the associated hematopoietic toxicity.   Neoadjuvant therapy, a systemic procedure prior to local therapy, is a treatment for cancer. Are increasingly better in terms of location (Trimble, E. et al., Cancer Sup pl 72: 3515-3524, 1993). This treatment requires subsequent surgical intervention. Designed to reduce, increase local control, and improve long-term patient outcomes It is. This in part provides an effort to 1) be entirely successful with local control. 2) reduce the tumor burden at the primary site; Control the disease, 3) reduce the number of cancerous cells before surgery, and 4) surgery If administered earlier, less resection may be required, thereby reducing normal organ Achieved by potentially preserving function. Neoadjuvant therapy's latent Local disadvantages jeopardize the acute toxicity of treatment, local control or delay of final treatment Ineffectiveness to metastasize invasive local tumors, scarring and fibrosis, more difficult surgery Difficulty, delaying wound healing by reducing tissue vascularity, and Includes ineffectiveness leading to a higher percentage of quiescent tumor cells.   Despite all existing treatments for solid tumors, efficacy and current methods are not Substantially satisfied with a method that is more effective than treating the patient, both in terms of Unmet requirements still exist in this area. The present invention addresses this need. Satisfaction, and yet provide other related benefits.Summary of the Invention   It is an object of the present invention to provide a blood clot within a blood vessel that supplies blood to the tumor. Causing tumor death in vivo by inhibiting flow, inhibition in tumor Inhibiting angiogenesis in tumors by producing sex proteins Producing proteins that activate non-toxic drugs into toxic forms within the tumor or That compete for or metabolize nutrients in the blood supply of humans To produce.   In one aspect of the invention, clot formation within or adjacent to a tumor is inhibited. Recombinant vector comprising a nucleic acid molecule encoding a polypeptide capable of stimulating Transducing cells within the tumor or adjacent to the tumor. A method is provided for damaging tumor cells. In various embodiments, The nucleic acid molecule is a Russell's viper snake venom factor X activator, Snake venom factor V activator, thrombin and thrombin-like enzymes (eg, Crotal us adamanteus (Crotalus), Crotalus horridus horridus, Agkistrodon rhodo stoma (Ancrod), Agkistrondon contortrix contortrix, Agkisirodon acutus , Bothrops atrox (Batroxobin), Bothrops marajoensis, Bothrops moojeni, Trimeresurus gramineus, Trimeresurus okinavensis and Brirtis gabonica? Enzymes, tissue factors, truncated tissue factors, cancer procoagulant factors, and Riga that binds to endothelial cells with high affinity for partial or whole procoagulant protein Selected from the group consisting of fusion proteins comprising part or all of the protein. Departure In one embodiment, the nucleic acid molecules are von Willebrand's agent. Progeny antigen II, endothelial monocyte activating polypeptide I, endothelial activating polypeptide II, tumor Necrosis factor alpha, tumor necrosis factor beta, and tissue factor induction from Rickettsia rickettsii Encodes a protein selected from the group consisting of a lead factor. Yet another embodiment To In some cases, the nucleic acid molecule is a polypeptide capable of inhibiting fibrinolysis. Code. In one embodiment, the nucleic acid molecules are α2-antiplasmid Plasminogen activator inhibitor I, plasminogen activator Turnover inhibitor II, plasminogen activator inhibitor III and Ert hrina is selected from the group consisting of proteinase inhibitors.   In another aspect of the invention, a polypeptide having the ability to inhibit tumor angiogenesis A recombinant vector containing a peptide-encoding nucleic acid molecule within or next to a tumor Inhibits tumor angiogenesis in vivo, including transducing neighboring cells A method is provided for: In various embodiments, the nucleic acid molecule encodes Polypeptides include angiostatin, interferon alpha, interfero Β, platelet factor-4, metalloproteinase I tissue inhibitor, metalloproteinase Tissue inhibitor of inase II, tissue inhibitor of metalloproteinase III, Thrombosporin, angiogenic fragment of prolactin, heparinase, base Neutralizing antibody fragments against inflammatory fibroblast growth factor, vascular endothelial cell growth factor and And αVβ3 integrin.   In another aspect of the invention, a non-cytotoxic agent, which may be a cytotoxic agent, is activated. Tumor with a recombinant vector containing a nucleic acid molecule encoding a polypeptide having the ability to Transducing vascular cells within or adjacent to the arterial side of the tumor; and Administering to the animal a non-cytotoxic agent that is activated by the peptide into a cytotoxic agent. Provided are methods for damaging tumor cells in vivo, including. Various implementations In embodiments, the nucleic acid molecule is a herpes simplex virus thymidine kinase, varicella- Shingles virus thymidine kinase, cytosine deaminase, xanthine-gua Nin phosphoribosyltransferase, Escherichia coli purine nucleoside Phosphorylase, cytochrome p450 2B1 gene product, alkaline phosphatase, a polypeptide selected from the group consisting of β-glucosidase and nitroreductase Code the peptide.   In yet another aspect of the invention, nutrients are provided in the intervascular space around the tumor. A nucleus encoding a polypeptide capable of binding or metabolizing nutrients A recombinant vector containing an acid molecule that binds cells in blood vessels in or adjacent to a tumor. Method for depriving tumor cells of nutrients in vivo, comprising transducing Is provided. In various embodiments, a nucleic acid molecule encoding a polypeptide Are soluble folate receptor / folate binding protein, soluble transferrin receptor Fetal hemoglobin, fetal hemoglobin, oxygen binding of fetal hemoglobin Fragment or extracellular matrix-binding fragment derived from β3 integrin And folate receptor or transferrin receptor. Selected from the group consisting of fusion polypeptides with fragments of the scepter polypeptide Is done.   In various aspects, the recombinant vectors are recombinant viral vectors and non- A gene delivery vehicle selected from viral nucleic acid (DNA or RNA) vectors. You. In a preferred embodiment, the recombinant viral vector is an adenovirus , Poxvirus, poliovirus, rhinovirus, influenza virus Virus, parvovirus, adeno-associated virus, herpes virus, simian virus Virus 40, human immunodeficiency virus, measles virus, astrovirus or coronau Selected from the group consisting of Irus. Preferred viruses are retroviruses or Alpha virus. A particularly preferred retrovirus is avian leukemia virus , Bovine leukemia virus, mouse leukemia virus, mink cell focus formation window Ruth, mouse sarcoma virus, reticuloendotheliosis virus, gibbon ape leukemia virus, Mason-Petzer leukemia virus or Rous sarcoma virus. More Also preferred mouse retroviruses are Abelson, Friend, Graphy, Glo , Kirsten, Harvey sarcoma, Lausher, or Moloney leukemia It is. Particularly preferred alphaviruses are Sindbis virus, Semliki Forest Virus, Middelburg virus, Ross river virus, Aura virus, pho Tomorgan virus or Venezuelan equine encephalomyelitis virus. In addition, species In various aspects, the recombinant virus is replication defective.   Another aspect of the invention is a packaging cell, a plasmid containing a recombinant viral vector. Producer cells, virus particles produced by producer cells, and shapes Transfected Recombinant Virus Vector Target Cell, and Vector According to the Present Invention And a pharmaceutical composition comprising: Preferred compositions are lyophilized or dehydrated A composition comprising the recombinant virus.   These and other aspects are described with reference to the following detailed description and accompanying drawings. Will be clear.BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 is a schematic diagram of the coagulation path.   FIG. 2 is a schematic diagram of p31N2R5 (+).   FIG. 3 is a schematic diagram of pN2R3 (−).   FIG. 5 is a schematic diagram of pN2R3 (+).   FIG. 6 is a schematic diagram of pN2R5 (−).   FIG. 7 is a schematic diagram of p31N25V (+).   FIG. 8 is a schematic diagram of pTK_A.   FIG. 9 is a schematic diagram of pPrTK_A.   FIG. 10 is a schematic diagram of pTK-1 and pTK-3.   Figure II shows Sindbis basic vector and Sindbis luciferase vector FIG.   FIG. 12 is a schematic diagram of a representative embodiment of a eukaryotic cell overlay vector initiation system. is there.   Figure 13 shows the time course of luciferase expression from ELVIS-LUC and SINBV-LUC vectors. It is a graph which shows conversion.   Fig. 14 shows a schematic diagram of the mechanism for disabling the virus junction region using the "RNA loop-out". FIG.   FIG. 15 is a diagram of one method for modifying the Sindbis junction region.   FIG. 16 is a schematic diagram of a Sindbis packaging expression cassette.   FIG. 17 shows packaging of SIN-luc vector by a representative packaging cell line. FIG.   Figure 18 shows the packaging of SIN-luc vector with PCL clone # 18 over time. It is a bar graph.   FIG. 19 is a graph showing the expression and rescue of Sindbis-luciferase vector. It is rough.   FIG. 20 shows a representative recombinant retro lyophilized in mannitol-containing formulation buffer. 4 is a graph showing the retention of virus activity upon virus reconstitution.   FIG. 21 shows a representative recombinant retrovirus lyophilized in a lactose-containing formulation buffer. 4 is a graph showing the retention of virus activity upon reconstitution of virus.   FIG. 22 shows a representative recombinant retro lyophilized in trehalose-containing formulation buffer. 4 is a graph showing the retention of virus activity upon virus reconstitution.   Figure 23 shows a liquid non-lyophilized recombinant resin stored at -80 ° C using various saccharides. Stability of Torovirus vs. Lyophilized Recombinant Retrovirus Stored at -20 ° C It is a set of representative graphs to be compared. Standardize titers for easy comparison ing.   Figure 24 shows the effect of ganciclovir on CT26, CT26βgal and CT26TK Neo cells FIG.   FIG. 25 shows tumor volume in a ganciclovir dose study of mice injected with CT26TK Neo. 4 is a graph showing the effect of the product over time.   FIG. 26 is a series of four photographs of mice showing different doses for intraperitoneal tumor growth. 9 shows the effect of the regimen ganciclovir.   FIG. 27 is a series of four photographs of mice showing different dose levels for subcutaneous tumor growth. The effect of ganciclovir on dime is shown.   FIG. 28 is a schematic diagram of pHCMV-PA.Definition of terms   The following terms are used throughout the description and the claims. These terms are Unless otherwise indicated, they are defined as follows.   "High affinity binding pairIs 10^-yK less than M_DOf molecules that have the ability to bind to each other at A pair. Where y is selected from the group consisting of 8, 9, 10, 11, 12, 13, 14 and 15. Is done. As used herein, "K_D": Reaction, A + B = dissociation determination of AB A number. Here, A and B are members of a high affinity binding pair. (further, As the skilled artisan will appreciate, as the affinity of two molecules increases, K_DDecreases The dissociation constant is determined by, for example, Scatchard analysis (Scatchard, Ann. NY Acad). . Sci. 51: 660-672, 1949). Representative examples of suitable affinity binding pairs include biotin / avidin, cytostatin / papaya , Phosphonate / carboxypeptidase A, and 4CABP / RuBisCo.   "Targeting elementIs a molecule that can interact specifically with the selected cell type. Say. As used in connection with the present invention, the biological efficacy of the attached targeting element Results are found in the cell type, or when the targeting element is bound to the target cell. More than 10-fold difference between the binding of the If there is more than 25, 50 or 100 fold difference, the targeting element is selected Specific cell types. Generally, the targeting element is selected 10 for cell types^-FiveLess than M, preferably 10^-6M, more preferably 10^-7M, and Most preferably 10^-8K less than M_DIt is preferable to bind with (Scatchard Determined by analysis, Scatchard, Ann. N.Y. Acad. Sci. 51: 660-672, see 1949 thing). In addition, the cell type for which the targeting element has been selected is given the parent of the high affinity binding pair. Binding with an affinity at least 1 log (ie 10 times) lower than the sum constant Generally preferred (in other words, K_DValues are at least 1 log or 10 times greater). Suitable targeting elements are preferably non-immunogenic and may be proteolytically And is not captured by the immune system. Particularly preferred targeted elements (Bound to a member of a high affinity binding pair) for 10 minutes to 1 week Should have a half-life in the animal (in the absence of a clearing agent) It is. Representative examples of suitable targeting elements are described in more detail below.   "Clarified substance"Is covalently associated with a circulating linked targeting element or A molecule that can interact non-covalently. Preferably, the clarified substance is non-immune Primate, specific for the bound targeting element, and rapid kidney Large enough to avoid clearance in the offal. Further, clarifying substances are preferred Is not degraded by proteolysis and is not captured by the immune system. Book Particularly preferred clarifying substances for use within the scope of the invention are affinity binding members Other than those that bind to the bound targeting element, and Preferably, binds in a manner that blocks binding of the targeting element to its target Including things. With respect to the present invention, see, for example, Marshall et al., Brit. J. Cancer 69: 502 Many clarifying substances are used, including those described in -507, 1994. Can be   "Recombinant retrovirus vector"In a preferred embodiment of the present invention , Expression of the sequence (s) of interest or the gene (s) of interest Assembly. Preferably, the retroviral vector construct is 5 ' LTR, tRNA binding site, packaging signal, one or more heterologous sequences, second strand DNA The origin of the synthesis and the 3 'LTR should be included. A wide range of heterologous sequences can be used to construct vectors For example, proteins, such as cytotoxic proteins, A gene encoding a disease-associated antigen, an immune accessory molecule, or a replacement protein). Sequence, or a sequence that is naturally and per se useful (eg, a ribozyme or (Like antisense sequences). Alternatively, the heterologous sequence simply "Staff" that is large enough to allow the production of retroviral particles, including "Stuffer" or "filler" array. Preferably, different The seed sequence is at least 1, 2, 3, 4, 5, 6, 7 or 8 Kb in length.   Retroviral vector constructs may also include a transcription promoter / enhancer or Is the locus defining element (s) or the alternate splice Sing, nuclear RNA export, post-transcriptional modification of messengers or post-translational modification of proteins Other elements that control gene expression, such as by decoration, may be included. Optionally, retro Viral vector constructs can also be transduced or transformed with recombinant retroviral vectors. Gives transfected cells TK, hygromycin, phleomycin, A selectable marker that confers resistance to stidinol or DHFR, and one or more Specific restriction sites and translation termination sequences.   "Gene delivery vehicle'' Means a recombinant vehicle such as a recombinant virus vector, Naked nucleic acid molecules such as nucleic acid vectors (such as plasmids), genes, etc. Polycationic that can neutralize charge and condense nucleic acid molecules into compact molecules Nucleic acid molecules complexed to molecules, nucleic acids binding to liposomes (Wang et al., PNAS 84: 7851, 1987), bacteria, and certain eukaryotic cells, such as producer cells, Here it delivers a nucleic acid molecule having one or more desired properties to a host cell in an organism. Have the ability to As discussed further below, the desired property is the desired material ( (Eg, proteins, enzymes, or antibodies) and / or GDV An active substance whose nucleic acid molecule carried by itself does not require expression of the desired substance Where it includes the ability to provide a biological activity. Such biological activity One example is that the delivered nucleic acid molecule is integrated into a particular gene, and as a result Inactivates offspring and "turn off" the product produced by the gene "It's gene therapy.   "Zymogen" is an inactive enzyme that can be activated to a catalytic state by post-translational modification. It is a carnival. Briefly, some zymogens are enzymes that block enzyme availability. Includes a polypeptide chain. Enzymes are acid or enzymatic cleavages that remove inhibitory polypeptide chains Activated byDetailed description of the invention   The present invention relates to a recombinant vector based on the interruption of nutrient supply to tumor cells. Several methods are provided for gene therapy-mediated treatment of solid tumors. these Shows (i) some strong diffusion into intratumoral vascular endothelial cells or perivascular tumor cells By transferring the gene of any one of the sex procoagulant factor proteins, Selectively initiating irreversible coagulation of tumor vasculature; (ii) intratumoral vascular endothelial cells. Transfer genes encoding anti-angiogenic proteins to vesicles or perivascular tumor cells (Iii) transfer of the "suicide" gene Disruption of the tumor vascular bed by selective damage of endothelial cells in the tumor And (iv) perivascular regions that absorb or metabolize incoming nutrients required for life. By creating a microenvironment in the area, starving the tumor And soluble soluble extracellular matrix-bound nutrient receptors or specific metabolic enzymes. This is achieved by transferring the gene to be loaded into perivascular cells.   In one aspect of the invention, a procoagulant factor protein, at least a procoagulant With a high affinity for the procoagulant factor portion of the transcription factor protein and at least for endothelial cells. Fusion proteins containing a binding ligand moiety, Multiple genes for inducers or inhibitors of fibrinolysis Using the gene delivery vehicle of the present invention, the vascular endothelial cells or Selectively initiate coagulation of tumor vasculature by transferring to periductal tumor cells Or worsening methods are provided.   The establishment of an important hypercoagulable state in the tumor vascular bed is based on three broad classes of tampering. It can be achieved by inducing local secretion of proteins. The first group is physiological Procoagulant that directly activates one or more zymogens that make up the coagulation cascade Factor proteins (eg, Russell's viper snake venom factor X activator, Lasse Lux viper snake venom factor V activator, tissue factor, shortened tissue factor, cancer clotting Thrombin, thrombin-like enzymes, and endothelial cell membranes or associated with them The entire ligand or at least a portion thereof that binds with high affinity to the protein Whole or at least extracellular portion of operably fused procoagulant factor protein A fusion protein comprising The second group consists of host endothelial cells and monocytes / macros. Phage induces expression of key procoagulant factor proteins (such as tissue factor) Tumor cells, leukocytes and the like that lead to and indirectly cause local activation of the coagulation cascade And proteins produced by certain infectious organisms (eg, von Willebra) Factor antigen II, endothelial monocyte activating polypeptide I, endothelial monocyte activating polypeptide II, tumor necrosis factor α, tumor necrosis factor β, and a set from Rickettsia rickettsii Tissue factor inducer). The third group of proteins is the homeostatic response to coagulation ( Ie fibrin dissolution). For example, α2-antiplasmin, plasmin Nogen activator inhibitor I, plasminogen activator inhibitor II, plasminogen activator inhibitor III, and Erythrina pro Tainase inhibitor. Members of the third group of proteins are localized blood coagulation Act synergistically with procoagulant factor proteins in the induction of Is a procoagulant tumor by itself, between fibrin production and degradation In the absence of exogenous procoagulant activity in the tumor vascular bed Causes coagulation.   The central point in the coagulation cascade, where the intrinsic and extrinsic pathways are concentrated, Activation of factor X (FX) to FXa (see FIG. 1). In the extrinsic pathway And tissue factor (TF) activates FVII to FVIIa, which is then combined with FVIIa in endothelial cell membranes. Bind and activate FX to FXa. In the intrinsic pathway, to tissue wounds or air Exposure results in the activation of FXII to FXIIa, which in turn activates FXI to FXIa. Once this has been achieved, this in turn results in the surface of perturbed endothelial cells or platelets, FVIII and And FIX are converted to their active forms. FX is calcium ion and phosphorus fat Is activated to FXa by the FVIIIa / FIXa complex in the presence of the plasma membrane (Fig. 1 See). Following a common pathway, Ca in endothelial cells or platelet membranes²⁺I The complex of FXa with ON and FVa forms a prothrombinase complex. This It efficiently catalyzes the conversion of prothrombin to thrombin. Prothrombina The protease complex produces 1,500 moles (m) / min (m / min) of moles of thrombin / FXa. G Rombin breaks down plasma fibrinogen into fibrin and converts FXIII to FXIIIa Which in turn crosslinks fibrin molecules to form an insoluble matrix (See FIG. 1).   Procoagulant factors can act at several points in the coagulation cascade. Organization Factor (TF) and truncated TF bind to FVIIa and the phospholipid bilayer to activate FX. (Nemerson et al., Prog. Hemost. Thromb. 6: 237, 1982). Russell Kusa The snake venom FX activator (RVV-X) and cancer procoagulant (CP) are TF, FVII Activates FX directly, independently of FVIII or FIX (Gordon et al., Hemat. Onc. o. Clinics North America 6: 1359, 1992; Takeya et al. Biol. Chem. 267: 1410 9, 1992). Wide range of thrombin isolated from thrombin and many snake venoms (Doull et al., Toxicology. Ed. MacMillan Pub. Co., NY 1986) (for example, Cr otalus adamanteus (Crotalase), Crotalus horridus horridus, Agkistrodon Rhodostoma (Ancrod), Agkistrodon contortrix contortrix, Agkistrodon acu tus, Bothrops atrox (Batroxobin), Bothrops marajoensis, Bothrops moojen i, Trimeresurus gramineus, Trimeresurus okinavensis and Bitis gabonica ) All directly degrade fibrin and thus most coagulation cascades And independent. TF, RVV-X and CP are extremely powerful coagulation inducers It is. Because the effect is amplified by the next stage of the zymogen cascade This is because that. Another inducer of coagulation is Russell's viper snake venom factor X Includes activator and shortened tissue factor. Thrombin and thrombin-like enzymes , Requires fibrinogen and FXIII to produce solids. Many tumors are Induce local fibrin formation as part of the malignant process (Flier et al., New Eng. J. Med. 315: 1650, 1986) and immunohistochemically detected in the tumor space Coagulation factor (Zacharski et al., Cancer 60: 2675 1987) is a tumor-inducing local vascular Is thought to leak as a result of hyperpermeability of (Senger et al., Science 219: 993, 1993). TF targeting has been shown to be effective in one tumor model (Moloma et al., Posting). Thus, at present, TF, CP and RVV-X etc. The use of an early acting procoagulant is preferred.   In recent years, Coley has reported that several terminal cancer patients who have been injected with viable microbial preparations have become seriously ill. He developed sepsis but subsequently recovered and reported that the tumor was cured (Coley et al., Am. J. Med. Sci. 105: 487, 1983). This activity eventually leads to Me Bacterial endotoxin causing specific hemorrhagic necrosis in th-A fibrosarcoma In response, it is mediated by cytokines released by macrophages. And was therefore called tumor necrosis factor (TNF, Old et al., Scienc e 230: 630, 1986; Carswell et al., PNAS 72: 3666, 1975; Nawroth et al. Exp. Med . 168: 637, 1988). TNF injection may be similar in other animal models or clinical trials Unique when not effective (Kao et al., Behring Inst. Mitt. 92:92, 1993). Synergistic cofactor is produced by Meth-A cells, which sensitize tumors to TNF It was argued that they had sex. These two factors, endothelial monocyte-activating polypeptide Peptides I and II (EMAP-I, EMAP-II) have recently been isolated and cloned (Kao et al., Supra, Kao et al., J. Boil. Chem. 269: 9774, 1994). TNF and EMAP-I And II all induce weak tissue factor activity in endothelial cells and monocytes, Acts synergistically when used in combination (Kao et al., Supra, Clauss et al., J. Biol. Chem. 265: 70). 78, 1990). This synergistic effect is used for gene therapy of procoagulant factor in solid tumors Can be done. First, the EMAP-I or II gene (Kao et al., Behring Inst. Mitt. 92:92 1993; Kao et al. Boil. Chem. 269: 9774, 1994) in combination with the TNF gene. Generating a powerful bicistronic procoagulant factor gene therapy vector I can do it. Second, the increased specificity of the procoagulant effect is due to EMAP-I or II, TNF (TNF α or β) or the tissue factor inducer gene from Rickeitsia rickettsii It can be produced by introduction into separate vectors. This vector selectively Has little or no common cross-reactivity with tumor cells or tumor endothelial cells Targeting vehicles (eg, antibodies or other cell / tissue specific ligands, growth dependent Retroviral vectors). Third, select by vector Alternatively, EMAP-I or II produced at the tumor site can be recombinant TNF or flavone acetate (FAA) (Effective against Meth-A tumors, but less so in other systems (A low molecular weight antitumor agent) can be combined (Murray et al., In t. J. Cancer 49: 254, 1991). Another procoagulant factor that may be utilized in connection with the present invention The protein is von Willebrand factor antigen II.   Disruption of the balance between fibrin formation and fibrinolysis may further result in clot formation Can be promoted. In particular, many animal and human tumors have localized vascular destruction or Is the result of the production of TF, TF-like activity in tumor cells, or cancer procoagulant (Go rdon et al., supra), which is essentially prothrombotic (Flier et al., supra). This process may explain, at least in part, necrotic areas in solid tumors, Functional vascular integrity sufficient to support tumor growth in Is maintained in vivo by local fibrinolysis that keeps pace (Dvorak et al., Cancer Res. 44: 3348, 1984). Fibrin Degraded by the rotase, plasmin. This plasmin is plasmino Generated from zymogen plasminogen by the action of gen activator (Dane et al., Adv. Cancer Res. 44: 139, 1985). Tissue type plasminogen Activator (T-PA) and urokinase-type plasminogen activator (U- PA) are both secreted by endothelial cells and tumor cells (Dane et al., Supra). T- Genes encoding PA or U-PA antagonists can be transferred to endothelial cells or Transfer into peri-tumor cells results in reduced plasmin levels. Recombinant vector When delivered to tumors by some proteins, some candidate proteins also perform this function. Can demonstrate. Plasminogen activator inhibitors I, II and III (PAI -I, PAI-II and PAI-III) bind reversibly to T-PA and U-PA, A competitive inhibitor of plasminogen activator that blocks gen cleavage (Wun et al., Crit. Rev. Biotech. 8: 131, 1988). α2-antiplasmin is Major plasmin inhibitor, which is constitutively present at fairly low levels. If present, remove plasmin until plasmin is present in excess. It is reversibly inactivated (Aoki et al., Semin. Thromb. Hemostasis 10:24, 1984). Several specific protease inhibitors are found in the legume, Erythrina ca ffra and Erythrina latissima (Teixeira et al., Bioch em. Biophys. 1217: 23, 1994). These proteins are soybean trypsin inhibitors Have high homology with Bitter, and these are strong inhibitors of plasmin and T-PA. Inhibitors (Teixeira et al., Biochem. Biophys. 1217: 23, 1994). Tumor blood vessels In addition to promoting bed coagulation, plasmin antagonists such as α2 antiplasmin Has additional antitumor activity. Overexpression of U-PA by tumor cells leads to increased metastasis With potential and angiogenic activity (Meissauer et al., Exp. Cell Res. 192: 453, 199). 1). U-PA produces plasmin, which also contributes to fibrin degradation. To cleave procollagenase into collagenase, Release bioactive angiogenic growth factors from the tissue. Collagenase is interstitial It is thought to contribute to the breakdown of stromal and basement membranes, which (i) Invasion of surrounding tissues, and (ii) invasion of foreign substances into local blood vessels and primary tumors Escape from the site, (iii) essential for endothelial cell migration during angiogenesis (G oodson et al., PNAS 91: 7129, 1994). Therefore, inhibition of plasmin production or activity Shocks the malignancy pathology at several levels.   One embodiment of the present invention is directed to a capillary endothelial cell (EC) lining the blood vessels of a tumor vascular bed. ) Or in the subpopulation of tumor cells closest to these vessels Provides expression of a gene for a promoter or anti-fibrinolytic protein. these Procoagulant or antifibrinolytic tan from "perivascular" tumor cells (PTC) Expression of the gene (s) encoding the protein is important. Because , Such expression provides a concentration of plasma-derived clotting factor sufficient to cause clot formation And the coagulation process initiated at the tumor cell surface spreads close to the blood vessels Because it induces extensive ischemia of the tumor. In a preferred embodiment, As described below, perivascular tumor cells are treated with procoagulant or anti-fibrin. Target a gene delivery vehicle comprising a nucleic acid molecule encoding a lytic protein.   In another preferred embodiment of this approach, the direction of the gene delivery vehicle is Sex is combined with the specificity of the polypeptide it encodes and is operable Achieve tumor vascular specificity. For example, targeted or non-targeted gene delivery vehicles Is a fusion gene encoding a protein having procoagulant activity and tissue specificity Can be used to deliver a recombinant expression vector with perivascular tumor cells . In one example, the fusion gene comprises at least a procoagulant factor protein (eg, For example, tissue factor, truncated tissue factor, Russell's viper snake venom factor X activator And the domain responsible for the procoagulant activity of Ligand that binds with high affinity to a receptor present on at least the endothelial cell membrane Encodes a fusion protein comprising the receptor binding domain of the peptide. Perivascular tumor After expression in the cell, the fusion protein is secreted into the perivascular space. tumor After diffusion of blood vessels near endothelial cells, the receptor becomes the ligand part of the fusion protein. Membrane-bound form that can bind to and initiate thrombus formation on the luminal surface of endothelial cells in the tumor vascular bed Produces a multimolecular sequence.   In a particularly preferred embodiment, the fusion protein is mobile at its amino terminus. The oligopeptide spacer allows the receptor binding domain of VEGF [Peretz et al. , (1992), Biochem. Biophys. Res. Commun., Vol.182, no.3: 1340-1347] Truncated tissue factor (lacking transmembrane and intracellular domains, Ruf et al., ( 1994); Biol. Chem., Vol. 266: 2158). VEGF receptor is endothelial cell Is overexpressed in The gene encoding this fusion protein (tTF-VEGF) Assembled using standard recombinant techniques. Other preferred fusion proteins are , Instead of tTF, Russell's viper snake venom factor X activator or cancer clotting Many other proteins or portions thereof, including promoters, may also be used. Sa Furthermore, ligand-derived receptors that bind with high affinity to receptors on endothelial cells The binding domain can be replaced by the receptor binding domain of VEGF. Such a resource Gand is a TGF-β, FGF, PDGF, u-PA, u-PA receptor antagonist and Endoglin, endosialin, CD31, CD34, integrin αVβ3 ligand , As well as monochromes that are reactive to receptors found on endothelial cells Includes, but is not limited to, an antigen binding fragment of a null antibody.   The present invention further provides a vector mediated gene encoding an anti-angiogenic polypeptide. Inhibit tumor growth by inhibiting tumor angiogenesis via sexual transfer, Alternatively, a method is provided for causing regression of an established tumor.   The growth of solid tumors over 1-2 mm in diameter may cause new growth from surrounding host tissue into the tumor. (Folkman et al., J. Nat. Cancer Inst. 82: 4, 1990; Folkman et al. Engl. J. Med. 285: II82, 1971). Critical stages in malignant progression Is the acquisition of angiogenic activity by tumor cells (Folkman et al., Nature 339: 58,19). 89). Uncontrolled growth of tumor cells in confined spaces often results in tumors This causes physical breakdown of the inner capillaries. Blocking angiogenesis causes acute vascular depression And may lead to actual tumor regression rather than growth inhibition (Ingber et al., Nature 348: 5 55, 1990). Many proteins secreted by tumor cells are found in some in vivo Have been shown to exert angiogenic activity in assays (Bicknell et al., Eu r. J. Cancer 27: 785, 1991) has two cytokines (basic fibroblast growth) Factor (bFGF) and vascular endothelial cell growth factor (VEGF) It has been proven to function as a tube forming factor. Because bFGF or VEGF c Transduction with DNA confers tumorigenicity on non-tumor cell lines (Winer et al., Supra) and Blocking cytokine activity using monoclonal antibodies specific for It inhibits growth and angiogenesis (Kim et al., Supra).   Angiogenesis is a complex process containing at least four steps (Folkma n, J.B.Lippincott, Philadelphia, pp.42-62, 1985). Host endothelial cells (EC) Degrades (i) the underlying basement membrane and (ii) migrates toward angiogenic stimuli (Iii) proliferate and promote new capillary sprouts; and (iv) anastomize existing blood vessels. To create a new basement membrane and initiate blood flow through a new capillary loop ( Folkman, J.B. Lippincott, Philadelphia, pp. 42-62, 1985). Each stage is As essential for the process, inhibitors of angiogenesis can be effective in various ways. Can be demonstrated. Physiological angiogenesis, such as in wound healing, is an angiogenic factor Is regulated by the balance between offspring and anti-angiogenic factors (Fidler et al., Cell 79: 185, 1994). To date, at least 10 anti-angiogenic proteins have been described (Fidler et al., Supra, O'Reilly et al., Cell 79: 315, 1994, Ratinejad Cell 56: 345, 1989; Moses et al., J. Cell. Biochem. 47: 230, 1991, and Bic Knell et al., supra). These include angiostatin, α-IFN, β-IFN, platelet factor- 4. Tissue inhibitors of metalloproteinases (TIMP-I, TIMP-II, TIMP-III) and Glioma-derived angiogenesis inhibitor (Van Meir et al., Nature Genetics 8: 171,199 4), thrombospondin, anti-angiogenic fragment of prolactin, heparina Or directly or indirectly (for example, retinoic acid or its analogs) Cell migration, endothelial cell proliferation, basement membrane Any other suitable peptide protein or protein that inhibits the degradation or 3-D organization of new blood vessels Are polynucleotides (Aucrbach, W. and Aucrbach, R. Pharmac. Ther., 63:26). 5, 1994). bFGF, VEGE and αVβ_ThreeBlocking against integrins Monoclonal antibodies are also angiogenesis inhibitors. α-IFN is life threatening Complete regression of hemangiomas and highly vascular kaposi's sarcoma in young children (Ezekowitz et al., N. Engl. J. Med. 326: 1456, 1992). Real et al., J. Clin. Oncol. 4: 544, 1986).   The present invention relates to the use of a gene encoding an anti-angiogenic factor in tumor endothelial cells or vascular Antiangiogenic strategy delivered using recombinant vectors to any of the peritumoral cells Physiological polypeptide negative regulator of angiogenesis Provides one or more uses. For example, α-IFN down-converts bFGF transcription and translation. (Singh et al., Am. J. Pathol. 145: 365, 1994). It is most effective when produced in tumor cells. TIMP cells during EC migration Inhibits endothelial cell proteases that require extramatrix degradation (Moses et al., Supra), and is therefore active in solutions everywhere in the tumor stroma. Thrombospondin or blocking anti-FGF or anti-VEGF antibodies are used in endothelial cells Antagonize angiogenic cytokine activity at the surface (Kim et al., Supra; Fidler et al., Supra) ), And is most effective when expressed in the vasculature itself.   Anti-angiogenic polypeptide genes were obtained using second-generation antibody-targeted recombinant vectors. Can be selectively delivered to perivascular tumor cells. Appropriate targeted tumor Is c-erbB2 / p185^HER-2(Shepard et al., Supra), TAG-72 (Guadagni et al., Supra). ), Carcinoembryonic antigen (CEA, Guadagni et al., Supra), high-affinity folate receptor (B ottero et al., supra), polymorphic epithelial mucin (PEM, Rettig et al., supra), epidermal growth factor Receptors (Rettig et al., Supra), CD44 isoforms (Rettig et al., Supra) and prostate features And different antigens (Mulders et al., Supra).   Another approach is to deliver angiostatin ex vivo. A Ndiostatin is produced by cells in the primary tumor mass, but distant metastatic neoplasms Suppress angiogenesis. This is probably due to metastases affecting several patients after removal of the primary tumor. Explain the clinical observations that grew more rapidly in the elderly (O'Reilly et al., Supra). Obedience Therefore, angiotatin is powerful and safe enough in the circulation to exert its endocrine effects. Stable transduction with a recombinant vector containing the angiostatin gene By transplanting a population of normal, long-lived non-neoplastic cells It can mimic the presence of a tumor. In addition, the vector encoding angiostatin is Indirect administration is sufficient to prevent local or systemic growth or metastasis of the primary tumor To a different level.   Due to proven efficacy in clinical or animal models, α-IFN, TIMP-I Or angiostatin is a particularly preferred candidate for anti-angiogenic gene therapy However, recombinant single-chain Fv genes from anti-VEGE antibodies are also preferred.   The invention further provides for the selective killing of tumor endothelial cells using "suicide" gene therapy. Provide a way to harm. In general, the physiological activation of the coagulation cascade is A mechanism that stops blood flow and seals tissue damaged from exposure to the environment. You. As a result, damage to the vascular endothelial cells lining the blood vessels is caused by the intrinsic pathways of coagulation and And initiators of both the extrinsic pathway (Kao et al., Supra). Beneath Regression of damaged endothelial cells from subendothelial matrix exposes blood to procoagulant factors You. Tissue factor in the subendothelial space causes activation of FX by an extrinsic pathway (Dr. ake et al., Am J. Pathol. 134: 1087, 1989), and treated with matrix proteins Platelets that become attached and become activated by matrix proteins Start the road (Stern et al., Haemostasis 18: 202, 1988). Therefore, in the tumor vascular bed Therapeutic strategies that result in a large number of endothelial cell deaths in In addition, thrombosis and ischemic necrosis of the tumor parenchyma can occur. Recently, this has been Has been demonstrated in a mouse model of solid tumor therapy through bleb injury. Mouse tumor The strain was transduced with a retroviral vector containing the mouse γ-IFN gene. This When a number of transduced tumor cells are grown in nude mice, they Γ-IFN activates local endothelial cells (ECs) to express MHC class II antigens in a reactive manner (Burrows et al., Cancer Res. 52: 5954, 1992). Normal EC is Class II Being negative, the anti-clumps that specifically bind to tumor EC in this model Antibody was utilized (Burrows et al., Cancer Res. 52: 5954, 1992). Anti-class II An immunoassay constructed by chemically attaching a ricin A chain to a monoclonal antibody. Epitoxin caused a dramatic regression of large (1 cm diameter) solid tumors (Burrows et al., PNA S 90: 8996, 1993). A time-course study following treatment with an anti-tumor endothelial cell immunotoxin Necrosis of the tumor mass has been demonstrated to be secondary to classic intravascular thrombosis. Inside After exfoliation of dead endothelial cells from the subcutaneous matrix, activated blood cells Focal adhesion and clumping of the plate continues rapidly, forming immature platelet / polycytic clots, This was followed by fibrin bridging, which causes permanent fibrotic thrombosis (Burrows et al., J. Mol. Controlled Release 28: 195, 1994). All these events are due to the injection of immunotoxin. Completed within 4 hours later, but initial necrotic changes in tumor cells were not Not observed. This can occur after a cessation of blood flow, as a result of a lack of nutrients. Indicate that tumor cells were injured (Burrows et al., PNAS 90: 8996, 1993).   An effective way to damage tumor endothelial cells is to use a "suicide gene" To the tumor's vascular wall and then intravenously deliver the prodrug. Is to reach. This prodrug is trans-transduced in the cytoplasm of tumor endothelial cells. Activated by the corn products to produce cytotoxic substances (Moolten et al., Cancer Gene Therap. 1: 279, 1994). Many alternative "suicide genes" are the inheritance of cancer It may be useful for treating children (Moolten et al., Supra). Bacterial cytosine deamina The introduction of the gutase gene (Huber et al., Cancer Res. 53: 4619, 1993) into tumor cells It confers sensitivity to the fungal substance 5-fluorocytosine (5-FC). Sitoshi Deaminase converts 5-FC to 5-fluorouracil (5-FU, Nishiyama et al.) Cancer Res. 45: 1753, 1985). 5-FU is commonly used as a chemotherapy drug for breast cancer. Some groups use cytosine deaminase for this disease A "suicide gene" treatment model based on the Wikipedia has been developed. Tumor specificity, therapeutic trans C-erbB2 so that the gene is selectively transcribed in c-erbB2-overexpressing breast cancer tumor cells Promoter / enhancer element 5 'of cytosine deaminase gene Can be further increased by introduction (Harris et al., Gene Therap. 1: 170, 1994). ). Alkaline phosphatase is involved in antibody-directed enzyme prodrug therapy (ADEPT). In related fields, it has been widely explored as a prodrug activating enzyme. this Enzymes are anticancer substances that cannot cross cell membranes until the charged phosphate groups are cleaved (Eg mitomycin C, etoposide, etc.) And thus a single enzyme can be used to create a new mixture of chemotherapeutic agents in the tumor mass It has the advantage that it can be produced (Senter et al., Bioconjugate Chem. 4: 3, 1993). Other suicide genes include, for example, a polypeptide (s) such as Herpes simplex virus thymidine (with corresponding non-cytotoxic substances) Kinase (ganciclovir or acyclovir), varicella-herpes zoster virus Thymidine kinase (6 methoxypurine arabinonucleoside; Huber et al., PNAS 88: 8039, 1991); coli cytosine deaminase (fluorouracil; Mullen , C.A., et al., PNAS USA 89:33, 1992); coli xanthine-guanine phosphorylation Bosyltransferase (thioxanthine; Beshard, C. et al., Mol. Cell Biol . 7: 4139, 1987); coli or Leishmania pudding Reotide phosphorylase (various non-toxic purine deoxyadenosine, adenosine , Deoxyguanosine, or derivatives of guanosine) (Koszalka, G. and Kren itsky, T.A., J. Biol. Chem 254: 8185, 1979; Sorscher, E.J., et al., Gene Therapy. , 1: 233, 1994), cytochrome pla50 2B1 or cytochrome p450 reductase. (For example, 3 amino-1,2,4 benzotriazine 1,4-dioxide (Walton, M.I. et al. Biochem. Pharmacol., 44: 251, 1992), alkaline phosphatase on the cell surface (Eg, etoposide monophosphate; Senter, P.D., et al., PNAS USA, 85: 4842. , Nitro reductase (eg, metronidazole or nitrofurant) Hof, H et al., Immunat und Infektion, 16: 220, 1988), N-deoxyribo Syltransferase (1-deazapurine; Betbeder, D. et al., Nucleic Acid Res)  17: 4217, 1989), pyruvate ferrodoxin oxidoreductase (Metroni Dazol; Upcroft, J.A., et al., Int. J. Parasitology, 20: 489, 1990), Carboxypeptidase G2 (aminoacylate nitrogen mustard; Anto niw, P. et al., Brit J .; Cancer 62: 909, 1990), carboxypeptidase A (meth Trexate α-alanine; Haenseler, E. et al., Biochemistry 31: 891, 1992), β -Lactamases (cephalosporin derivatives; Mayer, D.L. et al., Cancer Res. 53: 395 6, 1993; and Vradhula, V.M. et al., Bioconjugate Chemistry 4: 334, 1993), Actinomycin D synthetase complex (synthetic pentapeptide lactone precursor; Katz, E. et al. Antibiotics 43: 231, 1990) and β-glucuronidase (eg Various glucuronide derivatives of toxic drugs such as doxorubicin; Bosslet, K Et al., Cancer Res. 54: 2151, 1994; Haeberlin, B. et al., Pharmaceutical Res. 10: 1 553, 1993).   The HSV-TK system has important advantages for anti-tumor endothelial cell therapy. In particular, HSV-TK Transduced tumor cells are most commonly located next to each other through appropriate intercellular gap junctions. Results of transfer to GCV triphosphate, a toxic ganciclovir metabolite in contact with cells (Bi et al., Human Gene Therap., 4: 725, 1993). Mediates the pronounced bystander damage effect of neighboring cells that are not transduced (Moolten et al., Supra; Freeman et al., Cancer Res. 53: 5274, 1993). Hair Endothelial cells in the vessel wall are connected by gap junctions and therefore Dramatic bystander effect, caused by transfer of GCV triphosphate between skin cells Strong amplification effect on clotting cascade and tumor to endothelial cell ratio (Burrows et al., Supra; Denekamp et al., Cancer Topics 6: 6, 1986; Denekamp et al., Prog. . Appl. Microcir. 4:28, 1984) can result. Recent evidence suggests that HSV- Incidental shape of tumor endothelial cells during intra-injury treatment with TK retroviral vector Suggest that transduction can account for a key component of the antitumor activity of the vector (Ra m et al. Neurosurg. 81: 256, 1994). In addition, suicide genes are And only conditionally (ie, only if GCV is provided). Thus, ex vivo administration methods may be utilized. Briefly, endothelial cells are used for tumor biopsy (Medzelewski et al., Cancer Res. 54: 336, 1994); Proliferation can be induced using a cleavage promoting factor (Ferrara et al., Supra) and T It can be transduced with K. Implanted ECs can be days to weeks after intratumoral injection Shortly after, it is incorporated into the neovasculature (Lal et al., Cancer Gene Therap. 1: 322, 1994). Thus, GCV treatment allows transduced ECs to be functionally integrated into tumor vasculature. After an appropriate "lag period" to be effective. This approach works after intravenous injection Avoids the need for vectors to target tumor endothelium. Finally, the loss to normal EC Pause of GCV infusion for (potentially very serious) complication event resulting from injury Blocks toxicity without the need to block transgene activity in situ As a result, a two-stage enzyme-prodrug system is more sensitive to subtle clinical operations. Provide flexibility.   The present invention also provides extremely active metabolic and uncontrolled metabolism that characterizes a malignant condition. To starve certain nutrients from tumor cells, which are necessary to maintain growth Provide a method for Briefly, recombinant vectors compete with nutrient receptors for Vascular perimeter containing the gene sequence of an inhibitor or an enzyme that degrades certain nutrients Introduced into surrounding tumor cells.   The growth and growth of tumor cells resulting from oxygen and catabolic metabolism in the intestine and liver And all other nutrients required for survival have settled through their blood supply. Enter the tumor mass. Binds or binds to nutrients entering the intervascular tissue space The selective expression of nutrient-degrading proteins is caused by the lack of nutrients Rapidly starve tumor cells.   Tumor growth is typical of tumor cells (and many normal cells) around central blood vessels The “cord” structure limits the radius according to the oxygen diffusion distance, thus increasing oxygen availability. Speed is more clearly limited (Denekamp et al., Cancer Topics 6: 6, 1986). acid Elemental diffusion is due to fetal hemoglobin or the oxygen-binding fragment of fetal hemoglobin. Therapeutic gene transfer and expression in tumor intervascular tissue spaces It can be disturbed.   Another way to reduce available oxygen is to use oxygen molecules in the oxygenated hemoglobin complex. Is to deliver a gene for an enzyme that produces a molecule that can bind and displace . Such enzymes are enzymes that synthesize cyanide (eg, Wissing F. et al., J. B. acteriol 121: 695, 1975; Cutler, A.J. et al., Arch. Biochem Biophys, 238: 272, 1 985), an enzyme that synthesizes carbon monoxide (heme oxygenase; Murphy, B.J. et al., Ca ncer Res. 53: 2700, 1993).   Reduced folic acid undergoes a one-carbon transfer reaction to synthesize nucleotide bases. Is an essential vitamin required by the cells in order to be compatible (Kamen et al., PNAS 83: 5983,198 6). Many tumor cells overexpress the high-affinity folate receptor (see below). ) And folate deficiency partially or completely inhibits growth (Matsue Et al., PNAS 89: 6006, 1992). Family of chemotherapeutic drugs including methotrexate Exert their antitumor activity by antagonizing the folate cycle (Koong-Nah et al., J. Cl. in. Invest. 91: 1289, 1993). Short and soluble form of folate binding protein / leaf The acid receptor has been cloned (Weltman et al., Cancer Res. 52: 3396, 1992). This is due to the perivascular tumor cells as a result of gene transfer using recombinant vectors. Competes with the cell's complete membrane receptor when overexpressed in Tumorous Additional polypeptides that can bind and use nutrients in the perivascular intercellular space Is a soluble transferrin receptor.   Expression of simple stoichiometric binding proteins between perivascular tumor tissues is It is possible that it can be overcome by putting nutrients into the tumor mass. This is for example , Folic acid (Huennekens, F. et al., NCI Monographs 5: 1, 1987; Huennekens, F. et al., Ad vances in Enzymol and Related Areas of Mol. Biol. 47: 313, 1978), gluco Source (hexokinase), NAD (NAD phosphorylase), base or nucleotide (Adenase, nucleotide hydrolase, or uricase; Wu, X. et al., PNAS  USA 91: 742, 1994; Leyoux, R. et al. Biol. Chem. 267: 8565, 1992; Ito, M. Decompose or remove important nutrients such as Genomics 11: 905, 1991). This could be improved by transferring a gene that confers resistance to cellular uptake.   Occurrence of soluble nutrient receptor binding proteins or degrading enzymes in surrounding tissues To prevent possible spread, these genes must be Can be engineered to contain a DNA sequence encoding the binding domain of like this The fusion or "chimeric" protein is secreted by perivascular tumor cells and Abundant extracellular matrix proteins that are components of the tumor stroma and subendothelial basement membrane Proteins (collagen, laminin, fibronectin, vitronectin, Binds rapidly (including immunoglobulin factor (vWF)). Integrins are all Superfamily of heterodimeric adhesion proteins expressed by different proteins Lee. In ectoderm-derived tissues (eg, epithelial and endothelial cells), Glin is used to anchor cells to each other and to tissue stromal elements. , Directing tissue organization and regulating cell growth and differentiation (Hyne s et al., Cell 69:11, 1992). Many integrins, especially the β1 and β3 families Integrins are often collagen, fibronectin, vitronectin , And through recognition of the common tripeptide Arg-Gly-Asp (RGD) present in vWF Specifically binds to extracellular matrix (ECM) proteins (Ruoslahti et al., Sc ience 238: 491, 1987). An RGD recognition sequence derived from one of these integrins Inclusion within a recombinant nutrient receptor or enzyme gene is essential for optimal activity. It must allow for the isolation of therapeutic proteins where needed. β3 in The RGD recognition site on tegulin was isolated and sequenced (Fitzgerald J. et al. Biol. Chem. 262: 3936, 1987). This domain is used by many different ECM Recognizes protein and is included in nutrient receptor antagonists that bind to ECM Can be Particularly preferred fusion proteins are extracellular proteins derived from β3 integrin. Trick binding fragment and folate or transferrin receptor Is a fusion protein containing a fragment of   One method of targeting vectors to tumor endothelial cells in animals is by avidin domain. (Which binds specifically to biotin), and then Some monochromes that are selective or specific for capillary endothelial cells in solid tumors Chimeric envelope that forms a complex with any biotinylated derivative of the internal antibody (MAb) This can be achieved using a rope protein. Some of these reagents are mentioned in the dissertation (Burrows et al., Pharmac. Ther. 64: 155, 1994). For example, like this The reagent was MAb FB5, which has low levels on tumor endothelial cells and reactive fibroblasts. Expressed at moderate to moderate levels, but absent from normal tissue vasculature Recognizes endosialin, a highly sialylated glycoprotein (Rettig et al., PNAS 89: 10832, 1989)); TEC-4 and TEC-11 (which Endoglin, a regulated type III TGFβ receptor (C And LM609, which is a human vitronectin receptor (Integrin αvβ_Three, CD51 / CD61) shaped by epitopes from the α and β chains Which are MAbs that recognize the structural determinants formed). Normal and malignant human skin In skin tissue samples, LM609 stained blood vessels in tumor samples (Bro oks et al., Science 264: 569, 1994). LM609 is angiogenic in some models Αvβ_ThreeIntegrins appear to be involved in angiogenesis (Br ooks et al., supra). A more specific and widely applicable tumor endothelial cell marker One is vascular endothelial cell growth factor (VEGF) bound to the receptor. VEGF is A potent EC mitogen involved in tumor angiogenesis in many models (Winer et al., J. Clin. Invest. 91: 160, 1993; Kim et al., Nature 362: 841, 1993). ). This cytokine not only regulates VEGF synthesis but also VEGF receptor expression. Also, if not tumor specific (Jakeman et al., J. Clin. Invest. 89: 244, 19 92; Ferrara et al. Cell Biochem. 47: 211, 1991), specifically in the tumor vasculature Accumulates (Dvorak et al., J. Exp. Med. 174: 1275, 1991). MAbs are human and molar Elicited for epitopes that do not bind to the Mott VEGF receptor (Th orpe et al., undisclosed). Nude mouse with human tumor xenograft or with tumor Each of these MAbs, when injected into guinea pigs, has almost no effect on the tumor vasculature. Exclusively localized (Thorpe et al., Undisclosed). Cells for transgene expression A retroviral vector that requires cleavage contains VEGF, a mitogenic factor. `` Piggy backing '' into the target cell and with high efficiency Integrated into the genome.   Suitable tumor markers for targeting include, but are not limited to: Not available: c-erbB2 / p185<sup>HER-2</sup>(Shepard et al., J. Clin. Immun. 11: 117, 1991), TAG- 72 (Guadagni et al., Int. J. Biol. Markers 9:53, 1994), carcinoembryonic antigen (CEA, Guad Agni et al., supra), high affinity folate receptor (Bottero et al., Cancer Res. 53: 5791, 19). 93), polymorphic epithelial mucin (PEM, Rettig et al., Current Opinion Immunology 4: 630, 1). 992), epidermal growth factor receptor (Rettig et al., Supra), CD44 isoform (Rettig et al., Supra) And prostate specific antigen (Mulders et al., Eur. J. Surg. Onco. 16: 371990). Mark Conjugate with a retroviral vector containing a chimeric env-avidin receptor This can be achieved using a suitable biotinylated MAb that has been embodied (US application Ser. No. 08 / 242,4 No. 07) or directly containing the env ligand chimera (eg, for c-erbB2) The ligand heregulin) recombinant vector may be used. Particularly preferred targets The ligating ligand is readily available and can be produced by relatively mild chemical procedures. Folic acid because it can be ligated into a recombinant vector (Lee et al., Cancer Res. 52: 5954 , 1992). Importantly, the serum half-life of the vector is limited without a lack of activity Can be done. Because the target subpopulation of tumor cells is the most easily accessible in the body This is one of the extravascular tissues. The site of action is "upstream" (ie, Procoagulant protein (in the lumen of adjacent capillaries) against perivascular tumor cells Targeting is achieved by coagulation factors through the hyperpermeable tumor vasculature (Senger et al., Supra) so that bispecific antimicrobial Shown using a tumor / anti-tissue factor antibody (Molema et al., Submitted for publication).   Alternatively, targeting may also involve heregulin (which binds to her-2-neu) or EPO (EP O-receptors) (WO93 / 25234) Can be achieved using a hybrid envelope with a ligand (WO92 / 14829) You. Such hybrid envelopes provide the required capabilities, It may be necessary to be combined with an unmodified envelope. Even if the receptor Ligand affinity may be preferred for monoclonal antibody targeting Many, if not all, ligand receptors interact with target cells. As they interact, they may be sufficient to target the vector. This "berg The "velcro" effect was 10%, as measured by Scatchard blot.<sup>-Ten</sup>From Ten<sup>-15</sup>(Yu, H. et al., P. et al., Supra). roceedings of 1st West Coast Retrovirus Meeting, H. Fan and M. Linial , 1994).   Several features of this strategy are optimized for recombinant vector gene therapy. Suitable. In particular, the proximity of the procoagulant protein to be synthesized to the vascular system Is important, and this means that the perivascular tumor cells Are the most physically accessible tumor cells, and grow rapidly, It occurs because it is particularly susceptible to recombinant virus infection.   Tumor cells are sensitive to integration due to their increased proliferation, and Gene therapy using recombinant retroviral vectors based on irs (eg MLV) Therapy can be used without targeting. This is because cells undergoing the cell cycle (c (ycling cell) only integrates the vector, and non-dividing normal cells are unaffected. Because it is. Other proliferating epithelial cells (eg, within the hair follicle and in the lumen of Inner) is isolated from vector particles that have extravasated by several layers of stromal tissue You. Thus, the vector is deep enough to be able to integrate into such cells. It is not desirable to penetrate tissues (Burrows et al., Cancer Res. 52: 5954, 1992). .   The present invention relates to recombinant vectors in various viral vector packaging systems. In this system, one or more essential functions of the parent virus Function (eg, genome replication) but lack packaging signals and And the ability to express heterologous inserted gene sequences ("genes of interest") So that it has been deleted. The missing essential function (s) are Can introduce a vector genome into a producer cell line Provided by the cell. The producer cell line then encapsulates the recombinant vector. It is sidified to produce replication defective virus particles. The recombinant vector is one of the above The above-described nucleic acid cassette may be further included. The vector genome is then transformed into the infectious event ( "Transduction") into the target cells but without further proliferation. Any In this situation, the diversity of the virus in the producer cell line Interfering with the provision of a replication-competent viral genome by partial recombination It is important to remove the cells in which this occurs. Such a vector, Many of the packaging cells and producer cells include, for example: Poliovirus (Evans et al., Nature 33). 9: 385, 1989, and Sabin et al. of Biol. Standardization 1: 115, 1973) (AT CC VR-58); rhinovirus (Arnold et al., J. Cell. Biochem. L401, 1990) (ATCC  VR-1110); Positive virus such as canarypox virus or vaccinia virus Virus (Fisher-Hoch et al., PNAS 86: 317, 1989; Flexner et al., Ann. NY Aca) d. Sci. 569: 86, 1989; Flexner et al., Vaccine 8:17, 1990; U.S. 4,603,112 and And U.S. 4,769,330; WO 89/01973) (ATCC VR-III; ATCC VR-2010); SV40 (Mulli gan et al., Nature 277: 108, 1979) (ATCC VR-305), (Madzak et al., J. Gen. Vir. 73: 1). 533, 1992); influenza virus (Luytjes et al., Cell 59: 1107, 1989; McM icheal et al., The New England Journal of Medicine 309: 13, 1983; and Yap et al. , Nature 273: 238, 1978) (ATCC VR-797); adenovirus (Berkner et al., Biotec). hniques 6: 616, 1988, and Rosenfeld et al., Science 252: 431, 1991) (ATCC VR- 1); parvoviruses such as adeno-associated virus (Samulski et al., J. Vir. 63: 382). 2, 1989, and Mendelson et al., Virology 166: 154, 1988) (ATCC VR-645); human Immunodeficiency virus; herpes simplex virus (Kit et al., Adv. Exp. Med. Biol. 215: 2 19, 1989) (ATCC VR-977; ATCC VR-260); Nature 277: 108, 1979); HIV (EPO 386, 882, Buchschacher et al. Vir. 66: 2731, 1992); Measles virus (EPO 440,219) ) (ATCC VR-24); Sindbis virus (ATCC VR-68; ATCC VR-1248; Xiong et al., S) cience 234: 1188, 1989), Semliki Forest virus (ATCC VR-67; ATCC VR-1247). ), Aura (ATCC VR-368), Beval virus (ATCC VR-600; ATCC VR-1240), Hippo Saw (Cabassou) (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCC VR- 1241), Fort Morgan (ATCC VR-924), Getah virus (ATCC VR-369; ATCC VR-1) 243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR-66), Muka Nombovirus (ATCC VR-580; ATCCVR-1244), Nusum (ATCC VR-371), Pixnawi Luz (ATCC VR-372; ATCCVR-1245), Tonate (ATCC VR-925), Trinite (ATCC VR-469), Yuna (ATCC VR-374), Watalloy (ATCC VR-926), Y-62-33 (ATCC  VR-375), Middelburg (ATCC VR-370), Ross River Virus (ATCC VR-373; ATCC) VR-1246), O'Nyog virus, Eastern equine encephalomyelitis virus (ATC C VR-65, ATCC VR-1242), Western equine encephalomyelitis virus (ATCC VR-70; ATCC VR-1251) ATCC VR-622; ATCC VR-1252) or Venezuelan equine encephalomyelitis virus (ATCC V R923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532), and coronavirus (Hamr e et al., Proc. Soc. Exp. Biol. Med. 121: 190, 1966) (ATCC VR-740). In particular, this Such vectors, packaging cells, and producer cells include the following: Can be constructed from a variety of retroviruses, including: Avian leukemia virus (ATCC No. . VR-535 and VR-247), BLV (VR-1315), bovine leukemia virus, MLV, Vesicle focus-forming virus (Koch et al., J. Vir. 49: 8281984; and Oliff et al. Vir. 48: 542, 1983), mouse sarcoma Irs (ATCC No. VR-844, 45010 and 45016), reticuloendotheliosis virus (ATCC No. V R-994, VR-770, and VR-45011), gibbon ape leukemia virus, Mason-pa Itzer leukemia virus and Rous sarcoma virus (ATCC Nos VR-772, VR-35 4, VR-270, VR-724, and VR-725). Particularly preferred retroviruses are: Such viruses are: murine leukemia virus 4070A and 1504A (Hartley, J.V. ir. 19:19, 1976), Abelson (ATCC No. VR-999), Friend (ATCC No. VR) -245), graphy (Ru et al., J. Vir. 67: 4722, 1993; and Yantchev, Neoplasma 2). 6: 397, 1979), Gross (ATCC No. VR-590), Kristen (Albino et al., J. Exp. Med. 164.1710, 1986), Harvey sarcoma virus (Manly et al., J. Vir. 62). : 3540, 1988; and Albino et al. Exp. Med. 164: 1710, 1986) (Raucher) (ATCC No. VR-998) and Moloney leukemia virus (ATCC No. . VR-190). Particularly preferred non-mouse retroviruses are the Rous sarcoma virus, Bratislava (Copeland et al., J. Neuropath. Exp. Neurology 34: N1. , P100, 1975), Brian high titers (eg, ATCC No. VR-334, VR-657, VR-726). , VR-659, and VR-728), Brian standard (ATCC No VR-140), Karzelber (Carr-Zilber) (Adgighitov et al., Neoplasma 27: 159, 1980), Engelbreth-Hol Engelbreth-Holm-Swarm (Laurent et al., Biochem Biophys Acta 908: 241, 1987), Harris, Plug (ATCC No. VR-772 and 45033). ), Schmidt-Ruppin (for example, ATCC No. VR-724, VR-725, And VR-354). Expression vectors are prepared using standard recombinant techniques (eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Labor atory Press, 1989), from any virus or retrovirus Can be assembled. Further description of retroviral vector construction can be found in US No. 07 / 586,603, which is incorporated herein by reference.   In a preferred embodiment of the invention, the recombinant vector is a recombinant retrovirus. It is a vector. These constructs contain the selected gene of interest (single or multiple). Number) or sequence (s) or is expressed. For example, A number of retroviral gene delivery vehicles are included in the context of the present invention, including: EP 415,731; WO 90/07936; WO 91/0285, WO 94/03622; WO 93/256 98; WO 93/25234; U.S. Patent No. 5,219,740; WO 93/11230; WO 9310218; Vile and Ha rt, Cancer Res. 53: 3860-3864, 1993; Vile and Hart, Cancer Res. 53: 962-96 7, 1993; Ram et al., Cancer Res. 53: 83-88, 1993; Takamiya et al. Neurosci. Res. 33: 493-503, 1992; Baba et al. Neurosurg. 79: 729-735, 1993 (U.S. Pat. 127, GB 2,200,651, EP 345,242, and WO 91/02805).   The retroviral gene delivery vehicles of the present invention include, for example, B, C, and D A wide range of toroviruses, including spumaviruses and lentiviruses (RNA oncovirus, 2nd edition, C old Spring Harbor Laboratory, 1985). Simply put, a virus Are often classified according to their morphology as seen under an electron microscope . “C” retroviruses have a central core, while “B” retroviruses are isolated. It seems to have a heart core. "D" type retroviruses are type B retroviruses And a form intermediate between the virus and the type C retrovirus. Representative of suitable retroviruses A good example is the retrovirus discussed in RNA Oncovirus, as well as various xeno Tropic retroviruses (eg, NZB-X1, NZB-X2, and NZB<sub>9-1</sub>(O'Neill et al. J. Vir. 53: 100-106, 1985)), and polytropic retro Ils (eg, MCF and MCF-MLV (see Kelly et al., J. Vir. 45 (1): 291-298, 1983). See))). Such retroviruses are available from American Type Culture C ollection (ATCC, Rockville, MD). But can be isolated from known sources using commonly available techniques. obtain.   Particularly preferred for the preparation or construction of the retroviral gene delivery vehicle of the present invention. Preferred retroviruses include retroviruses selected from the group consisting of: Do: chicken leukemia virus, bovine leukemia virus, mouse leukemia virus, Mink cell focus forming virus, mouse sarcoma virus, reticuloendotheliosis virus , And Rous sarcoma virus. Particularly preferred mouse leukemia viruses include the following: Contain: 4070A and 1504A (Hartley, and Rowa, J. Vir. 19: 19-25, 1976), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graphi Ross (ATCC No. VR-590), Kristen, Harvey's sarcoma virus, and Lausha -(ATCC No. VR-998) and Moloney leukemia virus (ATCC No. VR-190) . Particularly preferred Rous sarcoma viruses include: Bratislava, Bra Ian high titers (eg, ATCC Nos. VR-334, VR-657, VR-726, VR-659, and VR-72 8), Bryan Standard, Kerr-Zelber, Engel Breath-Holm-Swarm, Ha Squirrels, plugs (eg, ATCC Nos. VR-772 and 45033), and schmirets Lupine (eg, ATCC Nos. VR-724, VR-725, and VR-354).   Any of the above retroviruses may be obtained from the disclosure and standard set provided herein. Replacement techniques (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition). Edition, Cold Spring Harbor Laboratory Press, 1989; Kunkle, PNAS 82: 488, 1985. )) To assemble or construct a retroviral gene delivery vehicle. Can be easily utilized for Further, in certain embodiments of the present invention, Some parts of rovirus gene delivery vehicles are derived from different retroviruses I can do it. For example, in one embodiment of the present invention, a recombinant retroviral vector LTR is for mouse sarcoma virus, tRNA binding site is for Rous sarcoma virus, The caging signal is murine leukemia virus and the origin of second strand synthesis is chicken. It can be derived from avian leukemia virus.   In one aspect of the invention, a 5 ′ LTR, a tRNA binding site, a packaging signal Retro containing one or more heterologous sequences, origin of second strand DNA synthesis, and 3 'LTR A viral vector construct is provided. Here, the vector construct is gag / po Lack of l or env coding sequence. Briefly, the long terminal repeat ("LTR") It is further divided into three elements named 5, R, and U3. these Elements include promoter and enhancer elements located in U3 And various signals responsible for the biological activity of the retrovirus. LT R is a provirus (combination) with precise replication at either end of the genome. (In the form of an integrated DNA). I will use it here As such, the 5 'LTR is composed of the 5' promoter element, and the reverse transcription and D It is understood that it contains enough LTR sequence to allow for incorporation into the NA form. Should be. The 3 'LTR contains a polyadenylation signal, as well as reverse transcription and vector Understands that it contains enough LTR sequences to allow integration of the DNA form of the It should be.   The tRNA binding site and the origin of second-strand DNA synthesis can also Important for activity and can be easily identified by those skilled in the art. example For example, retroviral tRNA binds to the tRNA binding site by Watson-Crick base pairing. And are carried into the virus particle along with the retroviral genome. Next TRNA is used as a primer for DNA synthesis by reverse transcriptase. You. The tRNA binding site can be easily identified based on its position just downstream of the 5 'LTR. You. Similarly, the origin of second strand DNA synthesis, as its name implies, is retrovirus. Important for second-strand DNA synthesis. This area, also called polyprint lacto, , Located just upstream of the 3 'LTR.   In addition to the 5 'and 3' LTRs, the tRNA binding site and the origin of second strand synthesis, Certain preferred recombinant retroviral vector constructs provided in Caging signals, as well as one or more heterologous sequences, Discussed in more detail below.   In one aspect of the invention, lacking both gag / pol and env coding sequences Provided are recombinant retroviral vector constructs. As used herein As such, the term "lacking the gag / pol or env coding sequence" Virus vectors found in the gag / pol or env genes, And the recombinant retroviral vector described in more detail in Example 1. Gag / pol used to construct packaging cell lines for Or at least 20, preferably at least 15, and more in the env expression cassette. More preferably at least 10 and most preferably at least 8 contiguous It should be understood to mean not containing nucleotides.   As shown, in one embodiment of the invention, the gag / pol or env sequence is Construction of the missing recombinant retroviral vector construct will This can be achieved by preparing a vector construct that lacks the signaling. Honcho As used in the text, the term "spread packaging signal" A nucleotide sequence that extends beyond the minimum core sequence required for caging. And allows for increased viral titers due to enhanced packaging. As an example For mouse leukemia virus MoMLV, the smallest core packaging signal Starts at the end of the 5 'LTR (approximately nucleotide 144) and begins at the PstI site (nucleotide). 567). MoMLV's expanded packaging sig The null extends beyond nucleotide 567 to the beginning of the gag / pol gene (nucleotide 621). And contains more than 1560 nucleotides. Therefore, this implementation According to an embodiment, a recombinant retrovirus lacking an extended packaging signal Vector construct eliminates the packaging signal before nucleotide 567 Or can be constructed from MoMLV by trimming.   In another embodiment of the invention, the sequence extends to the gag / pol sequence of the retrovirus. Missing or truncated packaging signal Provided are recombinant retroviral vector constructs. For example, representative of MoMLV In a typical case, the packaging signal is the gag / pol gene (nucleotide 6 Deleted or truncated before 21) begins. In a preferred embodiment of the present invention, The packaging signal comprises nucleotides 570, 575, 580, 585, 590, 595, 600 , 610, 615, or 617.   In another aspect of the invention, the gag / pol gene extends beyond the start (eg, M oMLV contains a packaging signal (over nucleotide 621) A recombinant retroviral vector construct is provided. Such a recombinant retro If viral vector constructs are used, to produce recombinant viral particles The use of packaging cell lines is preferred, where the gag / pol expression cassette The 5'-terminus of the gag / pol gene to be deleted may contain a codon degenerate for gag. Has been modified.   In another aspect of the present invention, a 5 ′ LTR, a tRNA binding site, a packaging signal Retroviral Vector Containing the Origin of Second-Strand DNA Synthesis, and 3 'LTR A construct is provided wherein the recombinant retroviral vector construct comprises a 5'LT Does not contain retroviral nucleic acid sequences upstream of R. Used in the context of the present invention As used herein, the term "does not contain a retroviral nucleic acid sequence upstream of the 5 'LTR" e Retroviral vectors are found in retroviruses, and more particularly, At least one of the retroviruses that are homologous to the retroviral vector construct. 20, preferably at least 15, more preferably at least 10, and most Preferably means not containing at least 8 contiguous nucleotides Should be understood. In a preferred embodiment, the recombinant retroviral vector The vector construct does not contain the env coding sequence upstream of the 5 'LTR. Such recombination records A particularly preferred embodiment of the Torovirus vector construct is described below in Example 1. Will be described in more detail.   In a further aspect of the invention, a 5 'LTR, a tRNA binding site, a packaging sig Recombinant retroviral vector containing null, second strand DNA synthesis origin, and 3 'LTR A recombinant construct is provided wherein the recombinant retroviral vector construct comprises a 3 ' It does not contain the retroviral packaging signal sequence downstream of the LTR. This specification As used in, the term "packaging signal sequence" refers to the It should be understood to mean sufficient sequence to allow for packaging. You. Representative examples of such recombinant retroviral vector constructs are set forth below. This is described in more detail in Example 1.   Packages suitable for use with the above recombinant retroviral vector constructs Cell lines can be readily prepared (US application Ser. No. 08 / 240,030, May 9, 1994). See also US application Ser. No. 07 / 800,921, also filed Nov. 27, 1991. ) And producer cell lines (for producing recombinant vector particles) Vector cell line or “VCL”).   In another preferred aspect of the invention, the recombinant viral vector comprises Sindbis This is a viral vector of a recombinant alphavirus based on the virus. The ter may function as a gene delivery vehicle. Briefly, Sindbis virus , A prototype member of the alphavirus genus of the Togaviridae family. Sindbisui Genomic RNA (49S RNA), which has not been segmented into lus, has a length of approximately 11,703 nuclei. Leotide, containing a 5 'cap and a 3' polyadenylation tail, and Indicates the polarity of Sindbis virus, surrounded by an infectious envelope, Assembly of viral nucleocapsid proteins on viral genomic RNA in the You Through the cell membrane in which glycoproteins encoded by Produced by budding. Virus invasion into the cell is clathharin Through the coated hole, the viral membrane and the endosome Fusion, nucleocapsid release, and viral genome decoating You. During virus replication, genomic 49S RNA is used for the synthesis of the complementary negative strand Provided as a template. Second, this negative strand is used for genomic RNA and Provided as a template for the 26S subgenomic RNA starting from the inside and the inside. Shi Ndbis virus nonstructural proteins are translated from genomic RNA, The protein is translated from the subgenomic 26S RNA. All viral genes are Expressed as a polyprotein and by post-translational proteolytic cleavage Processed into individual proteins. The packaging sequence is a non-structural coding region And therefore only genomic 49S RNA is packaged in the virion. It is.   Several different alphavirus vector systems can be constructed in the present invention. , And can be utilized. A representative example of such a system is U.S. Pat. No. 5,091,309. , And 5,217,879.   A particularly preferred alphavirus vector for use in the present invention is WO 9 Alphavirus vectors described in 5/07994 and U.S. Application No. 08 / 405,627 including. Briefly, in one embodiment, the transcription of Sindbis virus is opened. 5 ′ sequence, nucleotide sequence encoding a Sindbis nonstructural protein, For example, inactivated so that viral transcription of subgenomic fragments is prevented Syndrome containing the viral junction region and the Sindbis RNA polymerase recognition sequence A Dobis vector construct is provided. In another embodiment, the virus junction region May reduce, increase, or maintain viral transcription of subgenomic fragments Has been modified. In another embodiment, the transcription of Sindbis virus is initiated. The resulting 5 'sequence, a nucleotide sequence encoding a Sindbis nonstructural protein, e.g., If inactivated such that viral transcription of the subgenomic fragment is One viral junction region reduces viral transcription of subgenomic fragments Modified second viral junction region and Sindbis RNA polymerase recognition Sindbis vector constructs containing sequences are provided. In yet another embodiment, 5 'promoter and transcription termination that can initiate viral RNA synthesis from cDNA Sindbis cDNA containing the above vector construct in addition to the 3 'sequence that controls A vector construct is provided.   In a still further embodiment, the vector construct is in a vector construct. Heterologous sequences that do not contain Sindbis structural proteins in the Region may be located downstream of the vector construct; The selected heterologous sequence can be located downstream of the second viral junction region; If the sequence is located downstream, the vector construct will have a viral junction with the heterologous sequence. And may contain a polylinker located between the rows, and preferably a polylinker Contains no wild-type Sindbis virus restriction endonuclease recognition sequence.   The Sindbis vector construct, like many similar vector constructs, No. 08 / 405,627, which is incorporated herein by reference in its entirety. As described qualitatively, it can be easily prepared.   In addition to the viral-based vectors described above, a number of non-viral gene delivery vehicles A circle is used within the context of the present invention as well. Such gene delivery Representative examples of vehicles include nucleic acid expression vectors, naked DNA alone (WO 90/11092), Cationic lipids and liposome-encapsulated nucleic acids (single- or double-stranded DNA or RNA ) Expression vector, dead adenovirus bound or unbound polycation condensed DNA (Cur iel et al., Hum. Gene Ther. 3: 147-154, 1992), with one of the above high affinity pairs Or DNA without ligand (Wu et al., J. of Biol. Chem 264: 16985- 16987, 1989) and certain eukaryotic cells (eg, producer cells, May 1994). U.S. patent application Ser. No. 08/240/030 and U.S. patent application Ser. 21)). All such DNA vectors are The first RNA pol II transcript is an RNA rep of a viral bacteriophage or other pictorial source. Encodes a lipase and encodes the desired gene sought for therapy The same or separate transcripts encoding the gene (in the cytoplasm, due to RNA replicase RNA message that can be amplified) as a gene delivery vehicle. (US Patent Application No. 08 / 404,796).   When the gene delivery vehicle of the present invention targets tumors, various strategies may be used. Available. According to one embodiment, targeting of a gene delivery vehicle to a tumor For the purpose of targeting, the targeting element may be a gene delivery vehicle for the selected cell type. Is used to specifically direct the file. Generally, the targeting element is a tan Proteins or peptides, but other non-protein molecules may also be targeted elements. Can function as A wide variety of targeting elements are known to those skilled in the art. For example According to one embodiment of the invention, an antibody for targeting a selected cell type (Generally, Wilchek and Bayer, Anal. Biochem 171: 1-32. , 1988). Representative examples include: anti-CD34 antigen on stem cells Anti-CD34 antibodies (eg, 12.8 (Andrews et al., Blood 67: 842, 1986), and My10 (Civin et al., J. Immunol. 133: 157, 1984; Bec called HPCA-2). ton Dickinson)), an anti-CD that can be used to target CD4 + T cells 4 antibodies, anti-CD8 antibody to target CD8 + cells, targeting ovarian and breast cells HER2 / neu monoclonal antibody 4D5 (Sarup et al., Growth Regul. 1:72, 19 91), c-erbB-2 monoclonal antibody GDF-OA-p185-1 (A lper et al., Cell Growth Differ. 1: 591, 1990), TAG72 monoclonal antibody: colon CC49 and B72.3 for targeting cells and milk cells (King et al., J. Biochem. 2 81: 317, 1992), and a cancer embryo antigen monoclonal for targeting colon carcinoma cells Antibody ZCE025 (Nap et al., Canc. Res. 52: 2329, 1992).   As an alternative to using targeting elements, high affinity binding pairs can be used. Extensive High-affinity binding pairs are known to those skilled in the art. Representative of appropriate affinity binding pairs Examples include: 10<sup>-15</sup>M affinity (K<sub>D</sub>) Biotin / avidin (Richar ds, Meth. Enz. 184: 3-5, 1990; Green, Adv. in Protein Chem. 29:85, 1985) ;Ten<sup>-14</sup>Cytostatin / papain (Bjork and Yli) with M affinity nenjarvi, Biochemistry 29: 1770-1776, 1990); 10<sup>-14</sup>Val-phospho with affinity for M Nate / carboxypeptidase A (Kaplan and Bartlett, Biochemistry 30: 8165-8170, 1991), 10<sup>-13</sup>4CABP-RuBisCo with M affinity (Schloss, J. Biol. Chem. . 263: 4145-4150, 1988); and 10<sup>-11</sup>M affinity tobacco caterpillar diuretic hor Mon / tobacco caterpillar diuretic hormone receptor (Reagan et al., Arch. Insect B) iochem. Physiol. 23: 135-145, 1993).   A wide range of other high affinity binding pairs are also available, e.g., to prepare antibodies that recognize the antigen of choice. By making and selecting, and to select those with high affinity Such antibodies can be developed by further screening (generally, U.S. Pat.Nos. RE 32,011, 4,902,614, 4,543,439, and 4,4 See No. 11,993; 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.), C See also old Spring Harbor Laboratory Press, 1988). Or anti Bodies or antigen fragments can also be produced and selected using recombinant technology. (See William D. Huse et al., Science 246: 1275-1281, December 1989; L . Sastry et al., Proc. Natl. Acad Sci. See also USA 86: 5728-5732, 1989. Michelle Alting-Mees et al., Strategies in Molecular Biology 3: 1-9, 1990 See also; these references enable the production of antibodies using recombinant technology. Describe commercial systems available from Stratacyte, La Jolla, California ).   As will be apparent to those skilled in the art from the disclosure provided herein, the affinity binding pair Either the gene delivery vehicle (or conversely, the targeted Element). Nevertheless, in a preferred embodiment of the invention, In addition, two broader affinity binding pairs (eg, avidin / biotin pair aviation) Gin) is coupled to the gene delivery vehicle.   As mentioned above, certain embodiments of the present invention provide for high affinity binding pairs ("bound genetic As well as high affinity binding pairs (also referred to as "binding Targeting element, also known as "targeted targeting element") And a gene delivery vehicle conjugated with the gene delivery vehicle. Use within the scope related to the present invention As such, the term “bound” may indicate non-covalent or covalent interactions, Generally, covalent bonds are preferred. One member of a high affinity binding pair and the gene To attach a delivery vehicle or targeting element, for example, A number of methods can be utilized including the use of various crosslinkers: N-succinimidyl-3- (2-Pyridyldithio) propionate (“SPDP”; Carlson et al., J. Biochem. 173: 7) 23, 1978); sulfosuccinimidyl 4-N-maleimidomethyl) cyclohexane-1- Carboxylate (“SulfoSMCC”); 1-ethyl-3 (3-dimethylaminopropyl) Carbodiimide ("EDC"); bis-diazobenzidine ("BDB"); Uric acid / Schiff base.   According to a preferred embodiment of the present invention, the members of the high affinity binding pair Expression outside the delivery vehicle (eg, the envelope) or gene delivery Whether it is included as a whole outside the vehicle (eg, the envelope) That's it. For example, according to one embodiment of the present invention, a member of an affinity binding pair -Envelope of viral gene delivery vehicle as hybrid protein Co-expressed with the protein.   As will be appreciated by those skilled in the art, other targeting technologies are also in the art. And, according to the invention, are inherited for a specific tumor or cell type. It can be used to target offspring delivery vehicles.   Some of the above gene delivery vehicles include one or more heterologous sequences , Contained (and / or expressed). A wide variety of heterologous sequences may be relevant to the present invention. Can be utilized in the enclosure, including, for example: poly that can cause clot formation Polynucleotide sequence encoding a peptide, a polynucleotide capable of inhibiting fibrinolysis Nucleotide sequence encoding peptide, polypeptide capable of inhibiting tumor angiogenesis Nucleotide sequence that encodes, has slight cytotoxicity or cytotoxicity Activates non-toxic compounds (ie, “prodrugs”) into toxic products Nucleotide sequence encoding the polypeptide to be obtained, and Encoding polypeptide capable of binding or metabolizing nutrients Array.   DNA molecules encoding such heterologous genes can, for example, recognize biological substances. To be cloned from plasmids stored in It can be obtained from various sources. Alternatively, a cytotoxic gene or other heterologous sequence Known cDNA sequences that encode can be obtained from cells that express or contain such sequences. Can be Briefly, according to one embodiment of the invention, a gene of interest is expressed. MRNA derived from the cells is treated with reverse transcriptase using oligo dT or random primers. For reverse transcription. The single-stranded cDNA is then sequenced upstream or downstream of the desired sequence. PCR using oligonucleotide primers complementary to the columns (U.S. 4,683,202, U.S. .S. 4,683,195, and U.S. See 4,800,159. PCR Technology: Princip les and Applications for DNA Amplification, Erlich, Stockton Press, 1989 See also, All of which are incorporated herein by reference. ) It can be amplified more. In particular, double-stranded DNA is thermostable Taq polymerase, sequence specific DNA primers and the presence of the nucleotide bases dATP, dCTP, dGTP, and dTTP Denatured by heating in the presence. After annealing and extension, synthesis is complete In this case, double-stranded DNA is produced. This cycle is repeated several times to obtain the desired DNA. It can be log-amplified.   The sequence encoding the gene of interest can also be used, for example, in Applied Biosystems I nc. Automatic DNA synthesizer (eg ABI, DNA synthesizer model 392 (FosteruCity, CA)) Can be partially or completely chemically synthesized. Such genes are naturally occurring Nucleotide sequence, "optimized" nucleotide sequence based on codon choice Or a combination (s) of the two.   According to another embodiment of the invention, the recombinant vector comprises two or more heterologous sequences. Can be oriented to the present. Such multiple sequences are combined into a single promoter (which is a vector Or in the genome into which the vector is integrated), or Or preferably, can be controlled by one or more additional promoters. Internal ribosomes, and if polycistronic messages are used A binding site ("IRBS") may also be included. Such sequences are short, typically about Multicistronies that are 300 bp and have a cistron starting at this sequence Easy integration into vectors to express multiple genes from a message (Jacejak and Sarnow, Nature 353: 90, 1991).   As mentioned above, some anti-tumor agents are selected according to the method of the invention. It can be administered either simultaneously or sequentially to inhibit tumor growth. An example For example, an anti-tumor agent (such as, for example, γ-IFN) can be, for example, an agent such as IL-2 or IL-12. Tumors in combination with the recombinant vectors described herein along with other anti-tumor agents The animals can be co-administered or sequentially administered to inhibit or destroy. like this Therapeutic compositions comprise a single vector that directs the expression of two or more anti-tumor agents Can be administered directly, i.e., the anti-tumor agent is expressed by an independent vector obtain. Alternatively, the antitumor agent of the present invention is obtained by a recombinant vector administered to an animal. While other tumor agents (eg, IL-2, γ-IFN) can be expressed in pharmaceutical compositions (Eg, intravenously).   According to another embodiment of the present invention, two genes carried by the gene delivery vehicle The recombinant vector expressing the anti-tumor agent can also be administered to a patient. This In such a vector, the first antitumor agent is the LTR present in the recombinant vector. The other agent may be an additional transcriptional promoter located between the LTRs. Can be used. Alternatively, the second antineoplastic agent may have one or more internal It can be expressed as a polycistronic mRNA that can incorporate a bososome binding site. Briefly, the untranslated region upstream of the immunoglobulin heavy chain binding protein with respect to IRBS Showed support for the interlocking of dual cistronic messages (Jace jak and Sarnow, Nature 353: 90, 1991). Such arrays are short (300 bp) and a multicistronic with a cistron starting at this sequence. Retroviral vectors to easily express multiple genes from Can be incorporated into A representative vector construct using IRBS is described in Example 5 below. (A) (i), 5 (B) (i), 5 (C) (i), and 5 (D) (i).   According to another embodiment of the present invention, a pharmaceutically acceptable carrier or diluent There is provided a pharmaceutical composition comprising the gene delivery vehicle in combination with This Pharmaceutical compositions such as are in liquid or solid form (eg, lyophilized) Can be prepared. The solid form is suspended in a solution before it is administered parenterally. Remains The therapeutically effective amount of the gene delivery vehicle is IV, intraarterial (I.A.), or intralymphatic Is administered through A therapeutically effective amount of gene delivery vehicle arrests tumor growth And is preferably sufficient to kill the tumor cells. Teens The representative dose depends on the vector with the selectable marker and the PCR plaque formation. Equivalent titers, or equivalent methods for vectors without a selectable marker. 10 for<sup>Five</sup>,Ten<sup>6</sup>,Ten<sup>7</sup>,Ten<sup>8</sup>,Ten<sup>9</sup>,Ten<sup>Ten</sup>Or 10<sup>11</sup>cfu. The nucleic acid vector is 0.1, It is added at a dose of 1.0, 10, 100, 1,000, or 10,000 μg.   The pharmaceutically acceptable carrier or diluent may be used at the dosages and concentrations Is non-toxic to recipients. Carrier for injectable solution Or representative examples of dilutions include: water, preferably buffered to physiological pH Saline solution (eg, phosphate buffered saline or Tris buffered saline) Saline), mannitol, lactose, dextrose, glycerol, and Ethanol, as well as polypeptides or proteins such as, for example, human serum albumin. Protein. A particularly preferred composition is 40 mg / ml mannitol or lactose, 5 m g / ml HSA, 25 mM Tris, pH 7.2, 1 mg / ml arginine, and 25-75 mM NaCl. Or recombinant viruses. The composition has at least 6 at -70 ° C. Stable for months.   A variety of tumors can be selected for treatment with the methods described herein. general In particular, solid tumors are preferred. However, leukemias and lymphomas also When it has progressed to a tumor mass or when the tumor cells are physically separated from normal non-pathogenic cells It can be treated if there is an appropriate tumor-associated marker that can be released. For example, acute Lymphocytic leukemia cells are differentiated from other lymphocytes using the leukemia-specific marker CALLA. Can be separated from cells. Representative examples of suitable tumors are melanoma, colon carcinoma, lung carcinoma (Including large cells, small cells, squamous cell and adenocarcinoma), renal cell carcinoma, And breast carcinoma.   The following examples are provided by way of illustration and provide preferred embodiments of the present invention. Provided, but is not meant to be limited to these ranges. In the following example Standard methods for many procedures mentioned or described, or appropriate alternative procedures See, for example, Sambrook et al., "Molecular Cloning" 2nd edition, Cold Spring Harbor Laboratory Press (1997) and Asubel et al. (Eds.), "Current Protocols in Molec ular Biology, "Greene Associates / Wiley Interscience, New York (1990) Provided in a widely recognized manual of various molecular biology.                                  Example                                 Example 1                           Construction of recombinant vector A.Preparation of retroviral backbone   i.Preparation of KT-1 and KT-3B retroviral scaffolds   N2 (Armentano et al., J. Vir. 61: 1647-1650, 1987; Eglitias et al., Science 230: 139. 5-1398,1985) 5 'long terminus of Moloney murine leukemia virus (MoMLV) derived from vector The repeat (LTR) EcoRI-EcoRI fragment (including the gag sequence) was inserted into plasmid SK.<sup>+</sup>( Stratagene, San Diego, CA). The resulting construct is named N2R5. This N2R5 construct is subjected to site-directed in vitro mutagenesis to change the ATG start codon to ATT To prevent gag expression. This suddenly induced fragment is 200bp Long, flanked by PstI restriction sites. SK PstI-PstI mutant fragment<sup>+</sup>Plastic Purified from the Smid, which was then cloned into the PstI portion of the N2 MoMLV5 'LTR in plasmid pUC31. The non-mutated 200 bp fragment is replaced by inserting at the position. Plasmid p UC31 is derived from pUC19 (Stratagene, San Diego, CA) with additional restriction sites XhoI, Bg lII, BssHII, and NcoI were inserted between the EcoRI and SacI sites of the polylinker. Have been. This construct is named pUC31 / N2R5gM.   The 1.0 Kb MoMLV3'LTR EcoRI-EcoRI fragment derived from N2 was transformed into plasmid SK.<sup>+</sup>inside Clone to N2R3<sup>-</sup>To obtain a construct called 1.0 kilobase (Kb) ClaI-HindI The II fragment is purified from this construct.   SV40 drives expression of neomycin (neo) phosphotransferase gene ClaI-ClaI dominance from pAFVXM retroviral vector, including early promoter Selectable marker gene fragment<sup>+</sup>Clone into plasmid. this SV construct<sup>+</sup>SV<sub>Two</sub>Name it -neo. SK the 1.3 Kb ClaI-BstBI gene fragment<sup>+</sup>SV<sub>Two</sub> Purify from the -neo plasmid.   XhoI-ClaI fragment containing the desired gene and 1.0 Kb of MoMLV3'LTR ClaI-HindI Part III which inserts the II fragment into the XhoI-HindIII site of the pUC31 / N2R5gM plasmid KT-3B or KT-1 vectors are constructed by split ligation. It has a KT-1 skeleton This gives the vector referred to as Next, the pAFVXM retrovirus vector-derived Insert the 1.3 Kb ClaI-BstBI neo gene fragment into the ClaI site of this plasmid. To obtain a vector referred to as having a KT-3B backbone.   ii.Preparation of retrovirus backbone KT-3BC   For use in transforming genes into cells that are already neomycin resistant, Another selectable marker, phleomycin resistance (Mulsant et al., Som. Cell and M A ruth skeleton KT-3BC can be made. Plasmid pUT507 (Mulsant et al., Som. Cell and M ol. Gen. 14: 243,1988), digested with NdeI, and digested with Klenow fragment. Smooth edges. Next, the sample was digested with HpaI and the ClaI linker was flagged. Ment mixture. This sample is then digested with ClaI and excess ClaI linker Remove And Rous sarcoma virus (RSV) LTR and phleomycin resistant Isolating the 1.2 Kb ClaI fragment with the gene by agarose gel electrophoresis, Thereafter, purification is performed using GeneCleanII (Bio101, San Diego, CA). This 1.2Kb Insert the fragment in place of the 1.3 Kb ClaI-BstBI neomycin resistant fragment To obtain skeleton KT-3BC. iii.Preparation of retroviral backbone containing polylinker   To facilitate insertion of heterologous sequences, each KT vector backbone (KT-1, KT-3B, KT-3BC ) Is inserted with a polylinker sequence. When hybridizing this polylinker First, two complementary oligonucleotides forming a compatible cohesive end duplex with XhoI and ClaI Constructed using nucleotides.   This oligonucleotide is phosphorylated with T4 polynucleotide kinase and Heat and cool slowly for hybridization to occur. Next The appropriate KT-1, obtained after digestion of this hybrid with Xho I and Cla I, Ligated to KT-3B or KT-3BC vector backbone fragment, then It is treated with a fatase and purified by agarose gel. The resulting constructs are Xho I, Eco47 III, Nsi I, Pme I, Mlu I, Not I, Pml I, and Cla I Contains a single sequence for insertion. These constructs were designated as pKT-1L, pKT-3B, respectively. L, and pKT-3BCL. B.HSVTK Of a retroviral vector containing   i.Construction of plasmid containing vector LTR sequence   The following retroviral vectors are based on the N2 vector. Briefly, 5 'and The 3 'EcoRI LTR fragment (2.8 Kb and 1.0 Kb, respectively) (Armentano, J.V. ir. 61: 1647, 1987; Eglitis, Science 230: 1395, 1985) Do SK<sup>+</sup>And subcloned into the EcoRI site of pUC31. pUC31 is a polylinker Additional restriction sites between the EcoRI and SacI sites (XhoI, BglII, BssHII, and NcoI) is a modification of pUC19. Thereby, plasmid N2R3 +/-<sup>+</sup>Step It is made from the ligation of a rasmid with a 1.0 Kb 3 'LTR fragment. Plasmid p31N2R 5 +/- and p31N2R3 +/- with a 2.8Kb 5 'LTR and packaging signal, respectively (Ψ), or the ligation of the 1.0 Kb 3 ′ LTR fragment with pUC31. each In each case, N2 represents the vector supply and R represents the fragment whose EcoRI 5 and 3 indicate 5 'LTR or 3' LTR, And + or-indicates the orientation of the insert (for the LTR subclone example, See Figures 2-7).   In one case, a 1.2 Kb ClaI / EcoRI 5'LTR from N2 and a Ψ fragment SK<sup>+</sup>Subclone at the same site in the vector. Call this vector pN2CR5 Name. In another case, a 5 splice donor sequence containing a 6 base (bp) deletion 'LTR (Yee et al., Cold Spring Harbor, Quantitative Biology, 51: 1021, 1986) To Subclone into pUC31 as a 1.8 Kb EcoRI fragment. P this vector It is named 31N25Δ [+] (FIG. 7).   ii.HSVTK Of a plasmid containing   HSV-1 thymidine kinase gene (HSVTK) coding region and transcription termination signal The plasmid 322TK (3.5 kb BamH of HSV-1 cloned into BamHI of pBR322) I fragment (McKnight et al. Nuc. Acids 8: 5949, 1980) (ATCC No. 31344)) And isolated as a 1.8 Kb BglII / PvuII fragment from BglII / SmaI Clone into digested pUC31. This construct is named pUCTK. terminator For constructs requiring signal loss, pUCTK was deleted with SmaI and BamHI. And (A)<sub>n</sub>The 0.3 Kb fragment containing the signal is removed. Remaining code Sequences and BamHI overhangs were converted to double-stranded oligonucleotides made from the following oligomers: Reconstruct using ochides:   The resulting construct is named pTKΔA (FIG. 8).   For diagnostic purposes, retain the AvaI site without altering the translated protein While designing the oligonucleotide to destroy the SmaI site.   Plasmid pPrTKΔA (FIG. 9), which contains the HSVTK promoter and coding sequence ((A)<sub>n</sub>Which lacks a signal) is constructed as follows.   1. pTKΔA is linearized with BglII, treated with alkaline phosphatase, and Purify.   2. The 0.8 Kb fragment containing the HSVTK transcription promoter was converted from p322TK to BamHI / Bg Isolated as a II fragment.   3. The products from (3) and (4) are ligated, transformed into bacteria, and positive Clones are screened for the proper orientation of the promoter region. Generated The clone obtained is named pPrTKΔA (FIG. 9).   iii.Retroviral provector expressing HSVTK from a constitutive promoter Construction   Retroviral provectors (pTK-1 and pTK-3) were constructed essentially as described below. Build on the street.   The 1.5 Kb XhoI / HindIII 5 ′ LTR and plasmid sequence were isolated from p31N2R5 (+) Release (Figure 2).   2. The HSVTK coding sequence lacking the transcription termination sequence was replaced with a 1.2 kb XhoI / BamHI plasmid from pTKΔA. Isolate as a fragment (FIG. 3).   3. The 3 ′ LTR sequence was simply ligated as a 1.0 Kb BamHI / HindIII fragment from pN2R3 (−). Release (Figure 3).   4. The fragments from steps 1-3 are mixed, ligated, transformed into bacteria, and And individual clones are identified by restriction enzyme analysis. Name this construct pTK-1 (Figure 10).   5. pTK-3, pTK-1 was linearized with BamHI, the 5 'overhang was filled in, and pAFVX M retroviral vectors (Krieger et al., Cell 39: 483, 1984; St. Louis et al., PNA S85: 3150, 1988), a bacterial lac UV5 promoter obtained from the SV40 early promoter. Data, + Tn5 neo<sup>r</sup>5 ′ fill-in ClaI / ClaI fragment containing the gene Constructed by ligation with blunt ends. Kanamycin resistant clones were isolated and Individual clones by restriction enzyme analysis for appropriate orientation. (Fig. 10).   These constructs can be used to sensitize with packaging cell lines such as DA below. Dyeable recombinant vector particles were produced. C.Generation of recombinant Sindbis vector   i.Cloning of Sindbis genome-length cDNA   The nature of the virus with the RNA genome having an anodism depends on the permissive host (permissive h When introduced into eukaryotic cells that act as ost), purified genomic nucleic acids It acts as a ssenger RNA (mRNA) molecule. Therefore, from virus This genomic RNA produced is characteristic for infection by wild-type virus from which RNA has been purified. The same infection cycle can be initiated.   For example, Sindbis virus strain AR-339 (mosquito, isolated from Culexus univittatus) ATCC @ VR-1248, Taylor et al., Am. J. Trop. Med. Hyg. 4: 844 1955) It can spread to bee hamster kidney-21 (BHK-21, ATCC ♯CCL10) cells and has low multiplicity (0.1 pfu / cell). Alternatively, Sindbis virus strain HR (Lee Biomole (cular, San Diego, CA) can also be used and propagated in the same manner. Rice As described in National Patent Application No. 08 / 198,450, 48% after infection, 10% (w / v ) Using polyethylene glycol-8000 (PEG) Precipitating the virions of Dobis. This Sindbis screw in the PEG pellet Are dissolved in 2% SDS and commercially available oligo dT columns (Invitrogen, Sa Polyadenylated mRNA is isolated by chromatography using I do.   Using an oligonucleotide primer containing the sequence shown below, Perform two rounds of first strand cDNA synthesis with the isolated mRNA:   Briefly, this primer has an efficient restriction endonuclease digest at the 5 'end. "Buffer sequence" for 5 nucleotides, followed by an XbaI recognition sequence, 25 contigs DT nucleotides and six nuclei exactly complementary to the 3'-most end of Sindbis Contains leotide. Therefore, the choice for the first round of cDNA synthesis is at two levels. Result: (1) a polyadenylation molecule essential for functional mRNA, and (2) multiple Selective priming from Sindbis mRNA molecules in a pool containing mRNA species. In addition, reverse transcription was performed in the presence of 10 mM McHgOH to determine the frequency of artificial termination during reverse transcription. To reduce   The first genome-length Sindbis cDNA was then used, using six pairs of overlapping primers. Amplify by PCR in six separate segments. In short, ui In addition to the complement of Rus, the Sindbis 5'-end forward primer was 19 nucleotide (nt) sequence corresponding to A polymerase promoter and 5 'end Constructed to contain the ApaI restriction endonuclease recognition sequence linked to Bacterial SP6 RNA polymerase has in vitro transcription corresponding to the authentic Sindbis 5 'end Contains only a single non-viral G ribonucleotide linked to A ribonucleotide It is kept so. The ApaI recognition sequence is contained in the plasmid vector pKSII.<sup>+</sup>(Strata gene, San Diego, CA) Facilitates insertion of PCR amplicons into polylinker sequences You. To enable efficient digestion, a "buffer sequence" of 5 nucleotides is also , Located before the ApaI recognition sequence. SP6-5 'Sindbis forward primer and complete The sequences of all primer pairs required to amplify the entire Sindbis genome are listed below. Show. The reference sequence (GenBank reference number SINCG) is described in Strauss et al., Virology 133: 92-110,19. According to 84.   PCR amplification of Sindbis cDNA using the above 6 primer sets Thermose thermostable DNA polymerase (Amresco Inc., Solon, OH) and 1.5mM MgCl provided by this supplier<sub>Two</sub>Separate reactions using a buffer containing Responsive. In addition, 5% DMSO and Hot Start Wax beads (Perkin-Elmer, Los A ngeles, CA) using the following PCR amplification protocol: After amplification, the six reaction products were first inserted into the pCRII vector (Invitrogen, San Diego, CA). Enter. This fragment is then stepped into pK using the appropriate enzyme described above. SII<sup>+</sup>Insert between the ApaI and XbaI sites of the vector. This clone is called pVGSP6 Called GEN.   The Sindbis genomic cDNA clone pVGSP6GEN was converted to a 25 nucleotide long poly dA : XbaI, which cuts pVGSP6GEN once at the downstream position immediately adjacent to the dT stretch It is linearized by digestion. GeneClean the linearized pVGSP6GEN clone<sup>TM</sup>Purification Adjust to a concentration of 0.5 mg / ml. The transcription of the linearized pVGSP6GEN clone was In vitro at 37 ° C. for 90 minutes: DNA 2 μl / H<sub>Two</sub>O 4.25 μl; 2.5 mM NTP (UTP, ATP, GTP, CTP) 10 μl; 20 mM Me<sup>7</sup>G (5 ') ppp (5') G cap analog 1.25μ l; 100 mM DTT 1.25 l; 5 x transfer buffer (Promega, Madison, WI) 5 l; RNasi n (Promega, Madison, WI) 0.5 μl; 10 mg / ml bovine serum albumin 0.25 μl; and And SP6 RNA polymerase (Promega, Madison, WI) 0.5 μl. In vitro transcription anti The reaction product is subsequently digested with DNaseI (Promega, Madison, WI) and a series of Knoll / CHCl<sub>Three</sub>And purified by ether extraction followed by ethanol precipitation Alternatively, use directly for transfection. In vitro transcription products Alternatively, the purified RNA can be prepared by using a commercially available cationic lipid compound (Lipofectin).<sup>TM</sup>, GIBCO-BRL, Ga ithersburg, MD) and 60 mM Petride at 75% confluence. Apply to BHK-21 cells retained in tissue. Transfected cells Incubate at 30 ° C. Extensive cellular disease 94 hours after transfection A physical effect (cpe) is observed. Receiving RNA transcribed from Sindbis cDNA clones No clear cpe is observed on the unloaded plate. In addition, transfer Harvested cells, added to a fresh monolayer of BHK-21 cells, and Alternatively, 1.0 ml of supernatant incubated at 37 ° C produces apparent CPE within 18 hours. Occurs. This confirms that the Sindbis cDNA clone pVGSP6GEN is truly infectious. Show.   The sequence analysis of pVGSP6GEN shown in Table 1 is based on the Sindbis described herein. Multiple clones between the genomic clone and the clone of the virus whose sequence is registered in Genbank The sequence differences are shown. Many sequence differences are due to non-conservation in Sindbis proteins This results in a substitution of an amino acid change. Which sequence changes correspond to the clones described herein. Issues unique to clones or introduced into cloning artifacts Virion RNA is amplified by RT-PCR as described above and Direct distribution of RT-PCR amplicon products using kits (Promega, Madison, WI) By sequencing, the sequence for the nucleotide of interest is determined and the corresponding pVGS Compare with P6GEN sequence. Table 2 shows the results of this study. To briefly explain, Three non-conservative amino acid changes, Gly to Glu, As, which are the result of artificial artifacts p to Gly and Tyr to Cys are the viral nucleotides 2,245, 6,1 Observed at 93, and 6,730. These nucleotides that cause non-conservative amino acid substitutions All of the nucleotide changes are related to the viral nonstructural protein (NSP) gene (nucleotide Nucleotide 2,245 maps to NSP2 and nucleotides 6,193 and 6,730 map to NSP4) Can be   Repair of the NSP2 and NSP4 genes was performed as described above for plaque purified Achieved by RT-PCR using virion RNA from cucumber. SP6-1A / 1B Limer pairs are used to repair nucleotide 2,245 changes. RT-PCR The amplicon product was digested with Eco47III and BglII and the 882 bp fragment was Purified by Galose / TBE gel electrophoresis and digested with Eco47III and BglII And the corresponding region of the pVGSP6GEN clone prepared by treatment with CIAP. Enter. The 3A / 7349R primer pair described above comprises nucleotide 6,193 and a nucleotide. 6,730 used to repair changes. Transfer the RT-PCR amplicon product to EcoRI Digested with HpaI, and the 1,050 bp fragment was subjected to 1% agarose / TBE gel electrophoresis. More purified and exchanged into the corresponding region of the pVGSP6GEN clone. This black The sequence is called pVGSP6GENrep. PVGSP6GENrep D linearized by digestion with XbaI Transfection of BHK-21 cells with RNA in vitro transcribed from NA 18 hours after transfection results in extensive cpe.   ii.Generation of a DNA vector that initiates alphavirus infection: eukaryotic cell overlay Initiation system   As described above, the present invention generally provides promoters capable of initiating 5 'synthesis of RNA from cDNA. Constructs that can replicate autonomously or autocatalytically in cells, as well as heterologous nucleic acids Constructs capable of expressing sequence (s), and 3 'sequences that control transcription termination The present invention provides a eukaryotic cell multilayer vector initiation system comprising: One embodiment According to such a construct may be constructed by the following sequence of elements: A 5 'eukaryotic cell vector that can initiate viral RNA synthesis at the authentic alphavirus 5' end Motor, a 5 'sequence capable of initiating alphavirus transcription, Nucleotide sequences encoding nonstructural proteins, viral junctions, heterologous Sequence, alphavirus RNA polymerase recognition sequence, and 3 'transcription termination / polyamide Nylation signal sequence. Such an alphavirus cDNA expression vector also contains Sequence (intron), which is pre-RN in the nucleus before being transported to the cytoplasm. For functional mRNA molecules spliced from A and transported to the cytoplasm The overall effect of the system can be improved. Intron splicing signal Is located, for example, between Sindbis and the heterologous gene region.   Sindbis clone pVGSP6GENrep and mammalian RNA polymerase II promoter The construction of eukaryotic cell multilayer vector initiation system using This can be achieved as follows. Briefly, plasmid pVGSP6GENrep was replaced with BglII and And XbaI, and the reaction products are electrophoresed on a 0.8% agarose / TBE gel. The resulting 9,438 bp fragment was excised, and GeneCleanII<sup>TM</sup>And pCDN A3 (Invitrogen, San Diego, CA) obtained from treatment with BglII, XbaI, and CIAP. To the resulting 4,475 bp vector fragment. This construct is called pCDNASINbgl Called / xba.   The U3 region of the LTRC from MoMLV, the first transcribed nucleotide is a single G residue, The 5 'viral end is capped in vivo and followed by the Sindbis 5' end. Position on the edge. The parallelization of MoMLV LTR and Sindbis 5 'end is described below As achieved by overlapping PCR. BAG vector (Price et al., PNAS 84: 156-160, 1987) ) And the following primer pairs in the first primary PCR reaction with MoMLV Achieve LTR amplification: Forward primer: BAGBg12F1 (buffer sequence / BglII recognition sequence / MoMLV LTR nt 1-22) : Reverse primer: BAGwt441R2 (SIN nt 5-1 / MoMLV LTR nt 441-406):   PCR amplification of MoMLV LTR with the above primer pair was performed using 1.5 mM MgCl provided by polymerase and supplier<sub>Two</sub>Use a buffer solution containing And use it. This PCR reaction containing 5% DMSO and Hot Start Wax beads is described below. Perform using the PCR amplification protocol:   Amplification of the 5 'end of Sindbis in the second primary PCR reaction was performed with pVGSP6GENrep clone. Achieved with a reaction containing the following primer pairs: Forward primer: (MoML VLT Rnt 421-441 / SINnt 1-16): Reverse primer: (SINnt 3,182-3,160):   The MoMLV LTR PCR amplification was performed using the primer pair and the amplification reaction conditions described below. Use a PCR amplification protocol:   The 457 bp and 3,202 bp products from the primary PCR reaction are<sup>TM</sup>Refined and Together in a PCR reaction using the following primer pairs: Forward primer: BAGBg12F1 (buffer sequence / BglII recognition sequence / MoMLV LTR nt 1-22) : Reverse primer: (SINnt 2,300-2,278):  PCR amplification of the primary PCR amplicon product is performed using this primer pair and the amplification reaction described above. In conditions, achieve using the following PCR amplification protocol:   The 25 primary 3 ′ terminal bases of the first primary PCR amplicon product Overlaps the 25 5 'terminal bases of the recon product; the resulting 2,752 bp overlapping secondary PCR primer The amplicon product is purified by 0.8% agarose / TBE electrophoresis and digested with BglII And the 2,734 bp product was converted to BglII and fetal calf intestinal alkaline phosphatase (CI Ligation into pcDNASINbgl / xba treated with AP). The resulting construct is 16,656 bp Yes, called pVGELVIS. Sindbis nucleotides are bases 1-11,7 of this sequence. Included in 00.   pVGELVIS plasmid DNA was transferred to Lipofectamine according to the conditions suggested by the supplier. (About 5 μg DNA / 8 μg lipid reagent) and at about 75% confluency Add to 35 mm wells containing BHK-21 cells. Wild-type Sindbis virus infection A characteristic cytopathological effect is observed 48 hours after infection. Fresh BHK-21 single layer Addition of 1 ml of transfected supernatant to CPA results in cpe within 16 hours. this Data is obtained from viral cDNA and RNA polymerase II expression cassette in pVGELVIS construct Signal was aligned correctly, indicating that the DNA expression Provides a new start for RNA viruses.   Wild-type syndrome after transfection of pHKELVIS plasmid DNA into BHK cells During infection, to test the relative efficiency of initiating Perform a heart assay. Briefly, 5 μg of pVGELVIS plasmid DNA was placed in a 35 mm well. Transfected into BHK-21 cells as described above, and transfected 1.5 hours after application, the cells are trypsinized and 5 x 10<sup>Five</sup>Unprocessed BHK-21 The cells are serially diluted 10,000-fold, 10,000-fold. Then this transfect BHK-21 cells and a mixture of untreated BHK-21 cells are added to 35 mm wells. The cells are allowed to attach to the plate and are subsequently superimposed on medium containing 1.0% Noble agar. Layer. 48 hours after transfection, cell lysis (Sindbis virus Plaques, which contain direct or neutral red staining Can be visualized by overlaying with layers. This experiment was performed with transfection into BHK-21 cells. PVGLEVIS Plasmid in Generation of Wild-type Sindbis Virus After Sfection Efficiency of about 1 × 10<sup>Three</sup>This indicates that the plasmid DNA is pfu / μg.   iii.Construction of Sindbis RNA and Plasmid DNA Basic Vectors   The first step in the construction of the Sindbis basic vector is the viral 5 'and 3' Production of two plasmid subclones containing individual elements from the ends You. These elements are then used to connect the basic gene transfer vector Can be used to assemble.   Briefly, the first plasmid subclone contains the 40 termini at the 3 'end of the virus. Contains a 25 bp stretch of nucleotides and contiguous dA: dT nucleotides Is built. In particular, the following oligonucleotide pairs are first synthesized. Forward primer: SIN11664F (buffer sequence / NotI site / SINnt 11,664 to 11,698): Reverse primer: SINSac11700R (buffer sequence / SacI site dT25 / SINnt 11,700 to 11,6 92):   Next, the oligonucleotide was added to 10 mM MgCl<sub>Two</sub>At equimolar concentration in the presence of And heat to 100 ° C. for 5 minutes and slowly cool to room temperature. Then this part Two double-stranded molecules using Klenow DNA polymerase and 50 μM dNTPs Make a chain. The resulting 89 bp molecule was then digested with NotI and SacI, and 2% NuSieve / 1% agarose gel and digested with NotI and SacI, 10: 1 Excess insert: pKSII + plasmid prepared by CIAP treatment at vector ratio Connect to This construct is called pKSII3'SIN.   The second plasmid subclone was cloned with the first 5 '7,643 nucleotides of Sindbis. Bacteriophage RNA polymerase promoter located at the 5 'end of DNA and virus And a single non-viral construct after in vitro transcription. Only sex nucleotides are added to the authentic viral 5 'end. Briefly, this black The 3 'end of the primer is standard with a reverse primer having the sequence shown below. Derived from 3-temperature PCR amplification. Reverse primer: SINXho7643R (buffer sequence / XhoI site / SINnt 7643-7621):   This reverse primer maps to viral nucleotides 7,643 to 7,621. And 41 bp downstream from the 3 'end of the mating core element. In addition, virus Nucleotide 7,643 is 4 nucleotides from the translation initiation codon of the structural protein gene Upstream. The first five 5 'nucleotides of this primer should be Contained to act as a "buffer sequence" for efficient digestion of Followed by six nucleotides containing the XhoI recognition sequence. The forward primer in this reaction was Primer 2A (see Example 1C (i)). And has the following sequence:   Using pVGSP6GENrep plasmid (see Example 1C (i)) as template The 4,510 bp amplicon product obtained from the above PCR amplification is combined with the enzymes SfiI and Xh Digest with oI. The resulting 2,526 bp fragment is gel purified. Sindbis cDN A clone pVGSP6GENrep was also digested with ApaI and SfiI and the resulting 5 ' Gel a 5,144 bp fragment containing the SP6 RNA polymerase promoter at the end Purify. This 5,144 bp fragment together with the above-mentioned 2,526 bp fragment was Ap pKSII digested with aI and XhoI and treated with CIAP<sup>+</sup>Connect to plasmid. pKSII<sup>+</sup>plus Syndrome containing an RNA polymerase promoter at the 5 'end contained in the mid vector A clone having Dovi nucleotides 1 to 7,643 is isolated. This construct is called pKSII Called 5'SIN.   Complete basic vector assembly, elimination of pKSII5'SIN with XhoI and SacI And treated with CIAP, and gel purification of the large 10,533 bp fragment. Achieve more. This 10,533 bp fragment was then ligated with the XhoI of pKSII3'SIN and Ligation with a small 168 bp fragment resulting from digestion with SacI. this The resulting construct is called pKSSINBV (shown in FIG. 11A).   iv.Construction of Sindbis luciferase vector   Transfected with RNA transcribed in vitro from a Sindbis vector clone To demonstrate heterologous gene expression in isolated cells and Firefly luciferase reporter gene Into the Sindbis Basic Vector.   Construction of the Sindbis luciferase vector was performed in three independent plasmids: pK Assemble the components of SII5'SIN, pKSII3'SIN, and pGL2-basic vector together. This is done by blowing. pGL-2 basic vector plasmid (Promega, Mad ison, WI) contains the entire firefly luciferase gene. Briefly, this The luciferase gene is first inserted into the pKSII3'SIN plasmid. This is the pG L2 is digested with BamHI and HindIII and the fragment containing 2,689 bp This is achieved by purification. This fragment was used for the BamHI and pKSII3'SIN And gel purified 3,008 bp obtained by digestion with HindIII and treatment with CIAP To the fragment. The resulting construct is called pKSII3'SIN-luc.   Final assembly of Sindbis luciferase butter, XKS pKSII5'SIN digested with oI and SacI, treated with CIAP, and digested a large 10,533 bp fragment. This is achieved by purification. This 10,533 bp fragment of pKS5'SIN was A small 2,854 bp fragment obtained by digestion of I3'SIN-luc with XhoI and SacI. Connect with the client. This construct contains the complete Sindbis nonstructural gene coding region and The 3 'viral elements required for genome replication, as well as the 5' Element and the firefly luciferase gene located between the 3 'elements. Have. This vector is called pKSSINBV-luc and is shown schematically in FIG. 11B.   v.Construction of plasmid DNA alphavirus expression vector   The SIN BV and SIN-BV-luciferase constructs described in section D (iii) above were After introducing the plasmid DNA directly into the cells, the Sindbis vector-derived The pVGELVIS vector described in section D (ii) is Insert into a column. As described in Section D (ii), the linearized template vector Cells directly to alpha without the first step consisting of in vitro transcription of Transfect the virus-based vector plasmid DNA and transfer the RNA Representative heterologous inheritance of transfection of alphavirus vector based The ability to produce offspring expression levels is based on an alphavirus-based expression vector system. Greatly enhances the utility and efficiency of certain embodiments of Figure 12 shows the plasmid DN One mechanism for the expression of heterologous genes from the A alphavirus expression (ELVIS) vector FIG. Primary transcription in the nucleus and transport of vector RNA to the cytoplasm Then acts as a template for the production of genomic and subgenomic mRNAs Alphavirus catalyzes the expansion of heterologous gene mRNA through antigenome intermediates Leads to the synthesis of nonstructural proteins. This ELVIS vector can be directly As a complex with various liposome preparations by physical means as a DNA molecule, Alternatively, alphavirus DNA vector molecules, polycations such as polylysine Compounds, receptor-specific ligands and, if necessary, Sendai virus Or DNA ligand conjugates containing psoralen-inactivated viruses such as adenovirus May be introduced as a body.   First step of constructing one representative plasmid DNA Sindbis expression vector Is a step of digesting pKSSINBV with SacI, a step of blunting with T4 polymerase, Digestion, isolating the 2,689 bp fragment, and digestion with XbaI. Digestion, blunting with T4 polymerase, digestion with SfiI, treatment with CIAP, and 1% agar 10,053 bp pVGELVIS vector fragment prepared by Loose / TBE gel electrophoresis Connecting to the This construct is called pVGELVIS-SINBV.   To insert the luciferase gene into the pVGELVIS-SINBV vector, The SV40 intron and transcription termination sequence at the 3 'end of the Pre-transferred RNA transcribed from plasmid DNA luciferase vector When processed, the 3 'end of the reporter gene is a Sindbis vector It must be removed so as not to separate from the 3 'end. pVGELVIS-SINBV vector The Sindbis 5 'and 3' ends contained within the Required by cis for sex. The 3 'end of the Sindbis vector is Or a template for the subgenomic RNA encoding the transporter protein, Necessary for the initiation of the synthesis of the thy genome strand.   The SV40 RNA processing signal located at the 3 'end of the luciferase gene It is removed from the SIN-BV-luc construct described in section D (iv) above. Then modified le The luciferase fragment is ligated with the pVGELVIS-SINBV Place in the construct. Modification of luciferase gene using the following primer pair Achieved. Forward primer 7328F (SINnt 7,328-7,349): Reverse primer LucStop (buffer sequence / NotI, XbaI recognition sequence / pGL-2nt 1,725 to 1,70 3):   The primer was used in a 3 temperature cycle program using an extension time of 3 minutes. Used for PCR reaction. GeneCleanII<sup>TM</sup>With XhoI and XbaI Digest and GeneCleanII<sup>TM</sup>Purify again. And the 2,037 bp fragment, PVGELVIS-SINBV obtained by digestion with XhoI and XbaI and treatment with CIAP Ligation to the 13,799 bp fragment. This construct was constructed using pVGELVIS-SINBV-luc (ELV IS-luc).   Lucifera in BHK-21 cells transfected with pVGELVIS-SINBV-luc DNA Expression of Sindbis showed that the Sindbis physical gene transfer vector was functional. To measure. Briefly, 5 μg of pVGELVIS-SINBV-luc DNA or the above section 5 μg of invite from the linearized SINBV-luc template described in section D (iv) B) Transcribe RNA with 10 μl of Lipofectamine<sup>TM</sup>Or Lipofectin<sup>TM</sup>And complex each 5 × 10 5 in 35 mM Petri dishes<sup>Five</sup>Tiger on BHK-21 cells Transfected. After transfection, the luciferase activity was measured as 2, Measure from three samples each at 4, 8, 16, 20, 28, 48, 72, 96, and 120 hours . The results of this study, shown in Figure 13, show the reporter genes from the pVGELVIS-SINBV-luc vector. The maximum level of gene expression is in vitro from the linearized SINBV-luc template. Similar to expression levels observed in cells transfected with transcribed RNA Is shown. However, lucifera expressed from the pVGELVIS-SINBV-luc vector The luciferase activity was similar to the luciferase activity observed using the SINBV-luc RNA vector. It is at the highest level at a later point in time, and its activity is derived from the RNA vector. Continue to high levels while begins to decrease.   The overall efficiency of the ELVIS vector, determined by the level of heterologous gene expression, is ( As detailed in US patent application Ser. No. 08 / 348,472), the pVGELVIS-SINBV-luc vector To some extent. These modifications are based on RNA polymerase II Modification of promoter and transcription termination signal, intron arrangement in vector construct Add row, insert ribozyme processing sequence adjacent to 3 'end of alphavirus And replacement with a smaller plasmid vector.   The linker sequence was changed to pKSSINBV and pVGELV to facilitate insertion of the heterologous sequence. Insert into IS-SINBV construct. When this linker was hybridized, Xh Two complementary oligonucleotides forming a duplex with oI and XbaI compatible cohesive ends Constructed using nucleotides. SINBVLinkF: SINBVLinkR:This oligonucleotide is phosphorylated with T4 polynucleotide kinase and heated to 90 ° C. Heat and cool slowly so that hybridization occurs. Next This hybrid was digested with XhoI and XbaI, followed by alkaline phosphatase. 10.6 kb of pKSSINBV-Luc obtained after treatment with agarose and purification on agarose gel To the fragment. The resulting construct has a Sindbis junction region and a Sindbis junction. XhoI, PmlI, PmeI, MluI, BclI, StuI, Xb Includes aI and NotI. This construct is called pKSSINBVLII.   This polylinker is also cloned into the pVGELVIS-SINBV construct. This resource The insert is inserted by digestion of pVGELVIS-SINBV-luc with SfiI and NotI. This 10 The .1 kb fragment was agarose gel purified, and the fragment was pKSSIN Ligation was performed on a gel-purified 2.6 kb fragment from SfiI / NotI digestion of BVLII. Profit The resulting construct has a unique site between the Sindbis junction region and the Sindbis 3 'end. And XhoI, PmlI, PmeI, MluI, and NotI. This construct is called pVGELVIS-S Called INBVLII.   Plasmid pKSSINBVLII was replaced with the 8 nucleotides flanking the SacI transcriptional run-off site It is further modified by the addition of the recognition sequence AscI. The relative rarity of this recognition sequence Allows linearization of a wide variety of heterologous gene-containing Sindbis vector constructs. Special Next, use self-annealing oligonucleotides with SacI compatible ends. AscI / SacI linker: This AscI / SacI linker is phosphorylated with T4 polynucleotide kinase and heated to 90 ° C. Heat and cool slowly so that self-annealing occurs. Then, PKSS digested with SacI and treated with calf intestinal alkaline phosphatase Ligation to INBVLII plasmid DNA. The resulting construct is called pKSSINBVLIIA.D. Generation of recombinant Sindbis CEA vector i.CEA Structure of recombinant Sindbis vector (SIN-CEA) depending on tumor marker expression Construction   The disabled joint loop-out model was selected, as described above and shown in FIG. Using the junction region of the vector flanked by inverted repeats homologous to the RNA Build. In this example, the CEA tumor antigen cDNA (Beauchemin et al., Molec. And Cel. l. Biol. 7: 3221, 1987) is used in the inverted repeat. CEARNA responsiveness To construct a Sindbis vector, a 6 bp hinge domain Thus, the two CEA antisense sequence domains (A<sup>1</sup>And B<sup>1</sup>) Precedes I do. A single 20 base pair CEA sense sequence (A2) that is complementary to A1 At the 3 'end. In selecting the correct A1 and B1 antisense sequences The only requirement is that they be specific for the target RNA sequence. RNA sequence domains separated by 3 nucleotides from each other and the antisense sequence Is to hybridize to This three nucleotide gap is The merase is hopped to switch the read strand and the non-structural protein Acts as a hinge domain for bridging the main to the junction region of the vector ( (Figure 15). Convenient restriction enzymes at the 5 'and 3' ends to construct such forms Two complementary oligonucleotides are used to generate a fragment insert containing the prime site. Synthesize lignonucleotides. This oligonucleotide fragment was then inserted. The input was made by combining the multiple cloning site of the Sindbis vector with the inactivating junction region. Ligated into a Sindbis vector. Is the sense oligonucleotide strand 5 ' 3 'to ApaI restriction site followed by A1 antisense domain, 6 bp hinged Main, B1 antisense domain, synthetic junction domain and A2 antisense It should contain a domain followed by a XhoI restriction site. CEA RNA-responsive syn The following oligonucleotide sequence is used to design a Dobis vector. The nucleotide number sequence is described in Beauchemin et al., Molec. and Cell Biol. 7: 3221,198 Obtained from 7.  The 5'-3 'CEA antisense strand is complementary to the above oligonucleotide. You. After synthesizing both oligonucleotides, the oligonucleotides were<sup>+2</sup> And heat to 100 ° C. for 5 minutes and slowly cool to room temperature. Next The oligonucleotide pair was digested with ApaI and XhoI restriction enzymes and pre- digested with the same enzymes. Insert into a digested plasmid (pCMV-SIN or pMET-SIN) at a molar ratio of 25: 1 Mix and connect. These constructs were designated pCMV / SIN-CEA and pMET / SIN-C, respectively. Called EA. ii.SIN-CEA vector expressing gamma interferon and producer cells ( (SIN-CEA / IFN) Building   Human gamma interferon (γ-IFN) gene is retroviral vector plus Xido and XhoI from mid pHu-IFN (Howard et al., Ann.N.Y.Acad. Subclone by digestion with ClaI. The coding sequence of the obtained γ-IFN Is isolated from a 1% agarose gel.   Alternatively, human γ-IFN cDNA can be obtained by guanidinium thiocyanate extraction followed by Isolated from PHA-stimulated Jurkat T cells by ultracentrifugation through a CsCl gradient. Derived from isolated RNA. Next, RNA (Sigma, St. Louis, MO) is reverse transcribed in vitro. And using Taq polymerase, using a pair of gene-specific oligonucleotides. Γ-IFN cDNA is amplified by the polymerase chain reaction. Transfer PCR DNA to T4 DNA PSK repaired with limerase and Klenow and treated with CIAP<sup>+</sup>HincII part of plasmid Cloned into place. In the sense direction, the 5 'end of the cDNA is pSK<sup>+</sup>Polylinker And the 3 'end is adjacent to the ClaI site. Human γ-IFN content The 512 bp fragment encoding the offspring was ligated into pCMV / SIN-CEA or pMET / SIN-CEA In any of the XhoI / ClaI sites of the vector. Add these new plasmids They are called pCMV / SIN-CEA / IFN or pMET / SIN-CEA / IFN, respectively.   iii.Construction of SIN-CEA vector expressing thymidine kinase (SIN-CEA / TK)   Contains HSVTK cDNA clones flanked by 5'XhoI and 3'ClaI restriction sites The PCR amplification product to be purified is pHS1TK3KB (McKnight et al., Nuc. Acids Res. 8: 5949, 198). 0) Obtain using clones as target DNA. Primer arrangement used for PCR amplification Columns are obtained from the published sequence (Wagner et al., PNAS 78: 1442, 1981). Then, 1, The 260 bp amplification product was digested with XhoI and ClaI, and pCMV / SIN-CEA or pMET / SI Ligation into each XhoI / ClaI site of N-CEA vector. These new plasmi Are called pCMV / SIN-CEA / HSVTK or pMET / SIN-CEA / HSVTK, respectively. E. FIG.Construction of recombinant vector containing human tissue factor gene   Specifically, a recombinant retroviral vector and a human tissue factor gene Cloning into the Nvidis vector uses two 38 base oligonucleotides. Achieved by PCR amplification. Oligonucleotides can be used as tissue factor 25 nucleotides corresponding to the gene template, plus a PmeI recognition site and It contains an additional 5 'flanking sequence containing an opposing sequence. HTFP-F: HTFP-R: PCR amplification uses these primers, tissue factor cDNA clone as template λHTF7 (Scarpati et al., Biochemistry 26: 5234, 1987), Thermalase thermostable DNA Polymerase, 1.5 mM MgCl provided by the supplier<sub>Two</sub>, 5% DMSO and Hot St It is performed using art Wax beads according to the following protocol: After amplification, an approximately 920 bp tissue factor gene amplicon is digested with PmeI and digested with PmeI. Sindbis vector digested and treated with CIAP (pVGELVIS-SINBVLII or pKS SINBVLIIA) or retroviral vector backbone (pKT-1L, pKT-3BL, or pKT- 3BCL). Proper orientation of the insert sequence will result in restriction endonuclease deletion. And the resulting constructs were pVGELVIS-HTF, pKSIN-HTF, pKT-1L- HTF, pKT-3BL-HTF, and pKT-3BCL-HTF. Packaged vector particles Is achieved using the method described in Example 2 below. In the practice of the present invention DNA that encodes other gene products that are useful in , Can be readily cloned into these or other vectors. F.Construction of recombinant vectors containing tissue inhibitors of metalloproteinases   Specifically, recombinant retroviral vectors and synthons of the human TIMP-1 gene Cloning into Dobis vector uses two 38-mer oligonucleotides Achieved by PCR amplification. Oligonucleotides are used for TIMP-1 Contains the 25 nt corresponding to the offspring template, and a PmeI recognition site and buffer sequence Contains additional 5 'flanking sequences: HTIMP-F: HTIMP-R: PCR amplification was performed using these primers, TIMP-1 cDNA clone as a template ( Docherty et al., Nature 318: 66, 1985), and Thermalase thermostable DNA polymerase 1.5 mM MgCl provided by the supplier<sub>Two</sub>, 5% DMSO, and Hot St Performed in a reaction containing art Wax beads according to the following protocol. RU: After amplification, the approximately 700 bp TIMP-I gene amplicon is digested with PmeI and Sindbis vector digested and treated with CIAP (pVGELVIS-SINBVLII or pKS SINBVLIIA) or retroviral vector backbone (pKT-1L, pKT-3BL, or pKT- 3BCL). Proper orientation of the insert sequence will result in restriction endonuclease deletion. And the resulting constructs were pVGELVIS-HTIMP, pKSIN-HTIM, respectively. P, pKT-1L-HTIMP, pKT-3BL-HTIMP, and pKT-3BCL-HTIMP. Package The production of the resulting vector particles is achieved using the method described in Example 2 below. . Other related gene products from this group are based on these approaches and Contain PmeI or other suitable restriction sites, and It is easily cloned using specific oligonucleotide primers. G. FIG.KT-3 Group containing extracellular matrix binding fragment derived from integrin β3 Construction of recombinant vector containing recombinant folate receptor   i.Folate receptor / integrin β3 with amino-terminal glycine binding tether Construction of fusion gene   Integrin β3 linked to its amino terminus by a glycine tether Having a ligand binding domain of the human folate receptor α-form Cloning is accomplished by overlapping PCR amplification. 2 pairs used for primary PCR reaction Oligonucleotides have 20 overlapping bases, and a PmeI recognition site and buffer sequence. Generate amplicons containing additional 5 'or 3' flanking sequences You. In addition, each oligonucleotide primer is added as follows (i nput) 25 corresponding to the sequence on the template DNA to enable priming Contains nucleotides.Folate receptor gene primer: GHFBP-F: HFBP-R: PCR amplification uses these primers, human folate receptor cDN as template A clone c32KB (Elwood et al., J. Biol. Chem. 264: 14893, 1989), and VENT<sup>TM</sup> Using DNA polymerase (New England Biolabs, Beverly, MA) Reactions containing supplied buffer, 5% DMSO, and Hot Start Wax beads In an object, according to the following protocol: Integrin β3 primer: Iβ3109F: GIβ3171R: PCR amplification uses these primers and human integrin β3 ( Glycoprotein IIIa) cDNA clone (Fitzgerald et al., J. Biol. Chem. 262: 3936, 1987), and VENT<sup>TM</sup>Performed as above using a polymerase.   The folate receptor and integrin β3 PCR reactions were GeneCle an II<sup>TM</sup>And Mermaid Kit<sup>TM</sup>(BIO 101, San Diego, CA). And combine about 5% of the product from each reaction and, as described above, Including immersion Iβ3109F and HFBP-R, and Thermalase DNA polymerase Used as a template in a second round of PCR using the following amplification protocol: Use:  After amplification, about 1000 bp integrin β3 / folate receptor gene fusion amplicon The gel was gel purified, digested with PmeI, and synthesized with PmeI and CIAP treated. Dobis vector (pVGELVIS-SINBVLII or pKSSINBVLIIA) or retrovirus Ligation to the vector backbone (pKT-1L, pKT-3BL, or pKT-3BCL). insert The proper orientation of the sequence was determined by restriction endonuclease digestion and the resulting construction The products were pVGELVIS-IF, pKSIN-IF, pKT-IL-IF, pKT-3BL-IF, and pKT-3BCL, respectively. -IF. ii.Arg <sub>227</sub> Ligand binding domain of integrin β3 between C and the C-terminal hydrophobic region Of folate receptor / integrin β3 fusion gene by insertion of in   Specifically, an integrin inserted between Arg residue 227 and the C-terminal hydrophobic region A sequence encoding a human folate receptor α-form having a ligand binding domain of β3 Row cloning is achieved by overlapping PCR amplification. Used for primary PCR reaction The two pairs of oligonucleotides have 21 bases overlapping each other, as well as a PmeI recognition site and Containing additional 5 'or 3' flanking sequences containing buffer and buffer sequences Generate a recon. In addition, each oligonucleotide primer is: Priming, corresponding to the sequence of the template DNA to be added Contains a minimum of 25 nucleotides for: Folate receptor gene primer: HFBP-F:HFBP-R / β3: PCR amplification uses these primers, human folate receptor cDN as template A clone c32KB (Elwood et al., J. Biol. Chem. 264: 14893, 1989), and VENT<sup>TM</sup> Using DNA polymerase, buffers provided by the supplier, 5% DMSO, and Perform the following protocol in a reaction containing Hot Start Wax beads. : Integrin β3 and β3 / C-terminal fusion primers: Iβ3LBD-F: Iβ3LBD-R / FC-TERM:PCR amplification uses these primers (Iβ3LBD-R / FC-TERM Human integrin β3 (sugar tan Protein IIIa) cDNA clone (Fitzgerald et al., J. Biol. Chem. 262: 3936, 1987) And VENT polymerase as described above.   The folate receptor and integrin β3 PCR reaction were performed using GENECLEAN. And combined about 5% of the product from each reaction and Primers HFBP-F and Iβ3 LBD-R / FC-TERM and Thermalase DNA A second round of PCR containing remerase and using the following amplification protocol Use as rate:   After amplification, about 1000 bp integrin β3 / folate receptor gene fusion amplicon The gel is gel purified, digested with PmeI, and digested with PmeI and calf intestinal alkaline Sindbis vector treated with phosphatase (pVGELVIS-SINBVLII or pKSSINB VLIIA) or retroviral vector backbone (pKT-1L, pKT-3BL, or pKT-3BCL) ). Proper orientation of the insert sequence is important for restriction endonuclease digestion. And determined the resulting constructs as pVGELVIS-FI, pKSIN-FI, pKT-1L-FI, pK T-3BL-FI and pKT-3BCL-FI. Production of packaged vector particles Performed using the method described in Example 2. H.TF-IRES-PAI Construction of retrovirus vector   i.Retroviral vector with internal ribosome entry site   Plasmid pBS-ECAT (Jang et al., J. Virol. 63: 1651, 1989) contains the viral genome. Contains the 5 'untranslated region of encephalomyocarditis virus (EMCV) derived from nt 260-848 . This 5 'untranslated region contains an internal ribosome entry site (IRES). EM CV nt 260-827 was amplified from pBS-ECAT by PCR using the following primer pairs: You. EMCV IRES forward primer (for insertion into the MluI site in pKT-3BCL): EMCV IRES reverse primer: The amplicon obtained by amplification using these primers has a 5 bp "buffer Flanking the MluI recognition site within the sequence. EMCV IRES sequence from pBS-ECAT plasmid Is achieved using the following PCR protocol. Digest the IRES amplicon with MluI for insertion into the pKT-3BCL vector backbone, Purified on a 1% agarose gel and ligated to a pre-MluI digested CIAP-treated vector I do. This construct is designated as pKT-3BCL (IR). The exact orientation of the insert, obtained Determined by plasmid sequencing. In addition, this amplicon can be Virus vector backbone, and Sindbis vector backbone pKSSINBVL II and It can be inserted into pVGELVIS-SINBVL II.   ii.pKT-3BCL (IR) Tissue factor and plasminogen activator-in in the skeleton Retroviral vector having inhibitor 1 gene   Human tissue factor protein cDNA for PCR amplification of cDNA from plasmid λHTF7 More synthetic (Scarpati et al., Supra). TF cDNA was prepared as described above in Example 1E. Amplification is performed using a pair of gene-specific primers (HTFP-F and HTFP-R). these The amplicon obtained by amplification using the primers Is adjacent to the PmeI recognition site.   In addition to the cDNA sequence of the TF protein, a primer pair was added to the effective sequence following the PmeI recognition sequence. Contains a 5 nucleotide "buffer sequence" for efficient enzymatic digestion. pKT-3BCL (I To insert into R), the amplicon is digested with PmeI, purified on 1% agarose, Then, it is ligated to a PAP-digested CIAP-treated vector. This construct is called pKT-3BCL (T F-IR). Alternatively, other restriction sites in the polylinker may be replaced with pKT containing IRES -3BCL, pKSSINBVL II and pVGELVIS-SINBVL II Can be incorporated into primers. Insert the correct orientation of the insert into the resulting plasmid. Determined by sequencing.   CDN of human plasminogen activator inhibitor 1 (PAI-1) protein A is synthesized by PCR amplification of cDNA derived from plasmid PAIB4 (Ginsburg et al., J. . Clin. Invest. 78: 1673, 1986). PAI-1 cDNA was ligated to the following gene-specific primers. Amplify using mer pairs. An amplifier obtained by amplification using these primers The precon is adjacent to the NotI recognition site within a 5 bp "buffer sequence". PAI-1 cDNA forward primer:PAI-1 cDNA reverse primer: Amplification of the TF and PAI-1 sequences is achieved using the following PCR protocol:   In addition to the cDNA sequence of the PAI-1 protein, a primer pair follows the NotI recognition site Contains a 5 nucleotide "buffer sequence" for efficient enzymatic digestion. pKT-3B The amplicon is cut with NotI and inserted with 1% agarose for insertion into CL (TF-IR). Purify and ligate to CIAP-treated vector previously digested with NotI. PK T-3BCL (TF-IR-PAI). Alternatively, other restriction sites in the polylinker are replaced with pKT-3 PCR primers for insertion into BCL, pKSSINBVL II and pVGELVIS-SINBVL II Can be incorporated. Correct orientation of insert for sequencing of resulting plasmid Yo To decide.                                 Example 2 Vector construction of packaging cell lines HX and DA Transient transfection and transduction A.Plasmid DNA transfection   The following procedure is suitable using a plasmid encoding a vector according to the invention. Can be used to transiently transfect various packaging cell lines. For example, on day one, packaging cell line HX (WO92 / 05266) was 5.0 × 10 in 10cm tissue culture dish with S<sup>Five</sup>Seed with cells. On the second day, the tiger Four hours before transfection, the medium is replaced with 5.0 ml of fresh medium. 40.0μl 2.5M CaCl<sub>Two</sub>, 10 μg of plasmid DNA (e.g., pkSIN-HTF, pkT-IL-HTIMP, pkT- 38L (TF-IR-PAI)) and deionized H<sub>Two</sub>O to make a total volume of 400 μl. And perform standard calcium phosphate-DNA co-precipitation. DNA-CaCl<sub>Two</sub>The solution is constantly stirred. 400 μl of precipitation buffer (50 mM HEPES-NaOH, pH 7.1; 0.25 M NaCl and 1.5 mM Na<sub>Two</sub>HPO<sub>Four</sub>-NaH<sub>Two</sub>PO<sub>Four</sub>). Incubate this mixture for 10 minutes at room temperature You. The fine precipitate obtained is added to the HX of the cells. Cells at 37 ° C with DNA precipitate And incubate overnight. On day 3, the medium is aspirated and fresh medium is added. You. The supernatant is removed on day 4, passed through a 0.45 μm filter, and kept at -80 ° C. Exist. B.Transduction of packaging cell lines and generation of producer strains   To increase the retroviral titer produced from the packaging cells, Another package using a retroviral vector transiently produced from another cell line It is desirable to transduce a soaking cell line. For example, on the first day, DA (D17 Cells from ATCC Deposit No. 183, WO92 / 05266) cells were combined with 10 ml DMEM and 1 ml 5.0 × 10 5 in 0% FBS, 4 μg / ml polybrene (Sigma, St. Louis, Mo.)<sup>Five</sup>Cell / 10cm Seed in tissue culture dish. On day 2, 3.0 ml, 1.0 ml and 0.2 ml of fresh Harvested virus-containing HX medium (produced as described in Example 2 (A) above) Is added to the cells. The cells are incubated with the virus overnight at 37 ° C. The retrovirus also encodes a selectable marker (eg, neomycin resistance). If necessary, on day 3, the medium is removed and 1.0 ml DMEM containing 800 μg / ml G418, Add 10% FBS to the plate. Transduced with vector and selectable marker Only cells that express the cell survive. G418 in the case of neomycin resistance A resistant pool can be generated over a week. Typically, the cells are then plated Out, count the cell suspension and dilute the cell suspension to 10 cells / ml And 0.1 ml (1) into each well of a 96-well plate (Corning, Corning, NY). Cells / well) to dilute and clone this pool of cells. You. Cells at 37 ° C, 10% CO<sub>Two</sub>And incubate for 14 days. Select about 24 clones And expanded to a 24-well plate, a 6-well plate, then a 10 cm plate, At this point, the clones are assayed for expression, the supernatant is collected, and the virus Assay for titer.   The titer of individual clones was determined by infection of HT1080 cells (ATCC Deposit No. CCL 121). To decide. On the first day, 5.0 × 10<sup>Five</sup>HT1080 cells, 3.0 ml DMEM, 10% FBS and Preplate each well of a 6-well microtiter plate in 4 μg / ml polybrene. To On day two, serially dilute the supernatant from each clone 10-fold and Infect HT1080 cells in 1.0 ml aliquots using. Culture medium with fresh DMEM, 10% F Replace with BS medium and replace cells with vector at 37 ° C, 10% CO<sub>Two</sub>Incubate overnight Beate. On the third day, the medium is recruited to the appropriate selection factor (eg, if neomycin resistance is present). By replacing with fresh DMEM containing 10% FBS medium containing 800 μg / ml G418). , Selection of transduced cells (assuming the selection marker is present in the recombinant vector) Make a selection. Cells at 37 ° C, 10% CO<sub>Two</sub>For 14 days, at which point each Evaluate G418-resistant colonies at dilutions and determine the virus titer of each clone by cfu / ml To be determined.   Using these procedures, 1.0 × 10<sup>6</sup>Induce cell lines producing cfu / ml or more Lead. In addition, as will be appreciated by those skilled in the art, selectable markers other than drug resistance Or the selectable marker is encoded by a recombinant vector. If not, other titer methods (eg, antibody-based assays, PCR assays) Say etc.) can be used.   In the same manner as described for transduction of DA cells with HX supernatant, DA vector Transduce packaging cell line HX with vector generated from producer cell line I do.   Transduction of DA or HX cells with a vector lacking the neo selectable marker as described above Was performed as follows. However, instead of adding G418 to the cells on day 3, the cells are Clone by digestion. The titer is analyzed as described above. C.Alternative methods for producing producer cell lines   In some situations, in the process of producing a producer strain, more than one cell line It may be desirable to avoid the use of In this case, on the first day, DA cells were 5.0% in a 10 cm tissue culture dish with 10% irradiated (minimum 2.5 Mrad) FBS. × 10<sup>Five</sup>Seed with cells. On day 2, four hours before transfection, the medium was Replace with 0.0 ml of fresh medium. 60 μl 2.0 M CaCl<sub>Two</sub>, 10 μg of pMLP-G (Hartman et al., Nuc. Acid Res. 16: 9345, 1988; Emi et al., J. Vir 65: 1202, 1991) Plasmid, 10 μg Of the recombinant viral vector plasmid and make up to 400 μl volume with deionized water. Perform a standard calcium phosphate-DNA co-precipitation. Then, DNA-Ca Cl<sub>Two</sub>The solution was mixed with 400 μl of 2 × precipitation buffer (50 mM HEPES-NaOH, pH 7) with constant stirring. .1,0.25M NaCl and 1.5mM Na<sub>Two</sub>HPO<sub>Four</sub>-NaH<sub>Two</sub>PO<sub>Four</sub>). At room temperature Incubate for 10 minutes. The resulting fine precipitate was collected on the DA Add to the vesicle culture dish. Incubate cells with DNA precipitate overnight at 37 ° C. To On day 3, the medium is removed and fresh medium is added. Including G-pseudovirus Remove the supernatant on day 4, pass through a 0.45 μm filter, and remove Used to infect soaking cells.   On day one, DA cells were placed in 10 ml of DMEM and 10% FBS, 4 mg / ml polybrene. 5.0 × 10<sup>Five</sup>Seed the cells with a 10 cm tissue culture dish. On the second day, 2.0 mL, 1.0 mL Alternatively, add 0.5 ml of freshly collected and filtered G-pseudovirus-containing supernatant to cells. Add. The cells are incubated with the virus overnight at 37 ° C. On the third day , Remove the medium and add 800 μg / ml G418 (neo<sup>R</sup>Have a gene set (In the case of a replacement vector), add 10 ml of DMEM, 10% irradiated FBS. By vector Only those cells that have been transduced and contain the neo selectable marker survive. G41 Eight resistant pools are generated over 1-2 weeks. The pool is tested for expression The cells are then removed from the plate, the cell suspension is counted, and the cell suspension The solution is diluted to 10 cells / ml, and 0.1 ml (1 cell) is added to each well of a 96-well plate. / Well) by dilution cloning. Cells at 37 ° C, 10% CO<sub>Two</sub> And incubate for 2 weeks. Then select 24 clones, 24 well plate, The plate is then expanded to a 6-well plate and finally to a 10 cm plate, at which point Assayed for expression, collected the supernatant, and Assay as              D. Construction of alphavirus packaging cell line 1.Selection of parental cell line for development of alphavirus packaging cell line   a.Cells that can be persistently or chronically infected   Of potential parental cell lines to create alphavirus packaging cell lines One important criterion in selection is the appropriateness of alphavirus vector particles. Selection of cell lines that show little or no cytopathological effects before production . This criterion indicates that long-term propagation and stable supply of vectors To develop alphavirus vector producer cell lines that can be used as a source Is indispensable. Alphavirus infection in most mammalian cells Has been found to result in cytopathology and cell lysis. But various This problem has been overcome by being derived from packaging cells derived from human insect cell lines. I can avoid it. For example, insect cell lines (eg, Aedes albopictus, Aedes aegypti, Sp odoptera frugiperda, and may be derived from Drosophila melanogaster cells) Can be utilized to construct alphavirus packaging cell lines. example For example, in one embodiment, the alphavirus packaging cell line comprises Under the control of an inducible or non-inducible promoter active in these cell types Stably transfects to allow expression of alphavirus structural proteins Aede containing a customized expression vector cassette and co-expressing a selectable marker s al It is provided using an insect parent cell line such as from bopictus cells.     b.Modification of cells to reduce susceptibility to alphavirus expression: Inhibition of apoptosis and cytopathology   Potential parental cell lines (eg, canine D-17 and CF2 cells; human HT1080 (ATCC Accession numbers CCL121) and 293 cells; quail QT-6 cells; BHK-21 cells; mouse neuroblastoma N18 Cells; and rat prostate adenocarcinoma AT-3 cells) overexpression of the bcl-2 gene product Thus, the packaging cell line can also be modified. To be able to sustain infection Transformation of these cells is also possible with recombinant retroviral vector producer strains. As well as alphavirus packaging cell lines and producer cells Enables their use as strains.   To construct such packaging cells, plasmid pB4 (Reed et al., Na 336: 259, 1988) from the constitutive promoter. Any commercially available expression vector containing a promoter and encoding a selectable marker (e.g., , PCDNA3 (Invitrogen, San Diego, CA)), using standard recombinant DNA techniques. Through the use of surgery, a bcl-2 expression vector is constructed. Alphavirus nucleic acid sequence To avoid any type of homology with other transduced vectors Must be carefully considered. Selectable markers in recombinant Sindbis particles Or recombination events that can lead to unwanted packaging of the bcl-2 oncogene This should be noted in order to prevent Alphau described herein This is important because the ills vector system is designed for use as a biological therapy. The point is important. Once the bcl-2 expression vector is constructed, the parent cell line (i.e., BH (K-21 cells) using any standard technique, and Select after 24 hours using a maker. Pool resistant colonies and then dilute And then clone individual clones and screen for bcl-2 expression. Training. Once expression is confirmed, persistent Sindbis infection is tested and Later, as a parent cell line for the development of an alphavirus packaging cell line It is used.   In addition to the bcl-2 oncogene, other gene products that suppress apoptosis are also In alphavirus packaging or producer cell lines same Can be expressed. Three particularly preferred viral genes are: Adenovirus E1B gene (Rao et al., PNAS 89: 7742-7746, 1992) Pesvirus type 1<sub>1</sub>34.5 genes (Chou and Roizman, PNAS 89: 3266-3270, 1992), And AcMNPV baculovirus p35 gene (Clem et al., Science 254: 1388-1390, 1991 )including. Control of appropriate constitutive eukaryotic transcription promoters using standard techniques Any commercially available plasmid expression vector under control and also containing a selectable marker These individual genes can be inserted into the cell. Subsequently, the expression vector construct is Transfect cell lines as described above and apply appropriate selections. this Selection for stable integration of these genes and constitutive expression of their products is Cells found to be susceptible to alphavirus-induced apoptotic events It must allow for a wider range of vector production in the cell line. In addition, each Gene products can inhibit apoptosis by their own unique mechanism It is. Therefore, the gene can also be used in various combinations to obtain stronger suppression effects. Can be introduced into a packaging cell line or a producer cell line. Finally Other gene products that have similar effects on apoptosis have also been identified It can be easily incorporated into such packaging cell lines.   Alphavirus vector packaging and producer cell lines Stable in both vector and vector packaging cassettes To be able to derive a genetically transformed producer cell line. Many approaches to control the expression of the lus gene can be used. these Approaches include inducible and / or cell differentiation sensitive promoters, anti- Mosquitoes or mosquitoes in which persistent viral infections can be established Includes the use of other cells. Alpha virus vector producer Regardless of the final structure of the cell line, persistent infection or Or at least the ability to establish a delay in cell death by inhibiting apoptosis Can be enhanced. For example, adenovirus, HPV, SV40, and mouse polio DNA tumor viruses, including the retinoblastoma (Py) virus, are the retinoblastoma (Rb) gene products p105 and And its closely related gene product p107, and cyclin A, p33<sup>cdk2</sup>And p 34<sup>cdc2</sup>And binds to other gene products involved in controlling the cell cycle, including The cells are then partially transformed by inactivating them. Excluding Py All these viruses bind to p53 and inactivate it Encode the product. Only Py is a membrane tyrosine kinase, src, and this Phosphatidylyl required for full transformation potential of virus An intermediate T antigen (mT) that also binds and activates nositol-3-kinase ) (Talmage et al., Cell 59: 55-65, 1989). Rb and p53 recessive oncology Binding to the inactivation product and its inactivation is formed by these DNA tumor viruses. Transformed cells are prevented from entering the apoptotic pathway. p53 is, in part, C-fos inhibiting the expression of proteins related to cell proliferation, including hsc-70 and bcl-2 Can stop cell division (Miyashita et al., Cancer R esearch 54: 3131-3135, 1994).   To prolong the duration of alphavirus vector production or Preferably, the packaging cells and producers are used to promote the staining state. -Transform cells with viral genomic DNA from Py or SV40. In particular , Establish cell lines transformed with SV40 and Py, and The kinetics and level of Dobis production and cytopathology are determined. Hamster fine If the apoptotic events characteristic of Sindbis growth in the vesicles are reduced, then Alphavirus packaging cell lines and producers for each prototype Sir cell lines increase the yield of packaged vectors from these cells To be transformed with Py or SV40.   c.Modification of cells to reduce susceptibility to alphavirus expression: Production of activation-dependent vector particles   Sindbis E2 glycoprotein is synthesized as precursor PE2. This PE2 precursor is Together with the two viral glycoproteins E1 in the endoplasmic reticulum and Up to the membrane of infected cells as a heterodimer for virion uptake Be transported. At some point during this processing, PE2 is a complex of E3 and the mature virion glycoprotein. It is cleaved into E2 protein. E3 corresponds to the 64 amino-terminal residues of PE2, and Lost during maturity. The larger cleavage product, E2, associates with E1 and Moored in what will be the envelope. Host cell protease is PE2 precursor A highly conserved, canonical four amino acid (aa) residue moieties responsible for body processing Cleaved at the site immediately following the basic-X-basic-basic amino acid. RPE.40 A mutant cell line derived from the CHO-K1 strain (Watson et al., J. Virol. 65: 2332-2339 1991) lacks the ability to process PE2 precursors into E3 and mature E2 forms. Are incomplete in the production of Sindbis virus strain AR339. Therefore, RPE.4 The Sindbis virion envelope produced in cell line 0 is PE2 / E1 Including rhodimer. RPE.40 cells are more susceptible to Sindbis virus infection than parental CHO-K1 It is at least 100 times more tolerant. Incompleteness produced by RPE.40 cell line Whole virions can be converted to a fully infectious form by treatment with trypsin. You.   Vectors and structures in packaging and producer cell lines Any wild-type alpha produced by recombination with the protein gene RNA The virus reinfects the cells and is rapidly amplified and packaged. Contamination of the reactor preparation and significantly reduces the titer. Opened from RPE.40 shares The generated packaging and producer cells are used for vector production and production. Inefficient amplification of any wild-type virus produced during packaging and packaging Thus, it is an alternative to other cell lines that are permissive for alphavirus infection. Obedience Thus, the vector preparation is not significantly contaminated by the wild-type virus. further, The benefit of this system is that "cells" within similar cellular proteases can be made using techniques known in the art. By developing "knockout" mutants, other packaging cell lines and Expanded to producer cell lines.   d. Hopping cell line development   Alphavirus hopping cell line is spoofed for different cell receptor tropism Used to transiently produce typed infectious RNA vector particles. Hoppin Once the cell line has produced the vector particles, it is no longer needed. Because it transduces the original alphavirus packaging cell line discussed above. This is because only the infectious culture supernatant is required for introduction. Therefore, For transient production of tar particles, hopping cell lines must be It is not necessary to show persistent infection due to this. In this case, the parent cell line is 24 hours to 72 hours after infection with the worm cell line or virus-producing alphavirus It can be any of the mammalian cell lines that appear to lyse into. The only criterion is The cell line will affect cell growth prior to transfection of the alphavirus RNA vector. With or without the appropriate alphavirus structural protein Just the VSV-G protein or the retroviral gag-pol and env proteins Can be expressed. Therefore, Alpha Will The hopping cell line was used for further discussions such as bcl-2 oncogene expression. Replication of either the alphavirus or the retrovirus It can be any of the parent cell lines described above that can support production.   Generation of VSV-G pseudotyped alphavirus vector particles requires at least three separate This can be achieved by an approach. Each of which is disclosed in U.S. Patent Application No. 08 / 348,472. Is described in detail.   Alphavirus vector fake in retrovirus packaging cell line For typing, the engineered engineered to include the retroviral packaging sequence. Retroviral gag-pol for packaging favirus vectors Any cell line that expresses the sequence and the env sequence can be used. In subgenomic RNA But only the genome-length vector is packaged by the viral envelope protein. The retroviral packaging sequence is inactivated, so that Between the joined junction region and the tandem repeat of the synthetic junction region. Alpha virus Virus-based particles containing vector RNA can be obtained in vitro using the procedures described above. By transfecting the transcribed alphavirus vector RNA Can be produced. Pseudotyped retroviral particles containing an alphavirus RNA vector Supernatants with are collected 24 hours after transfection, and then The clarified can be used to transduce the alphavirus packaging cell line.   e.Produces an alphavirus resistant to inactivation by human complement Of parent cell line   For successful intravenous administration of recombinant alphavirus particles, the vector is Must be resistant to inactivation. Sindbis grown on BHK-21 cells, Susceptibility to complement-mediated serum inactivation with respect to effective virus titer It is well known to those skilled in the art. Sindvi resistant to inactivation by human complement Sindvi grown on multiple cell types to identify parent cell lines that produce Test the level of serum inactivation of the virus. The cell type tested (eg, 293 Cells or HT1080 cells) are derived from many species, including humans.   As a source of human complement, a serum separation tube (Becton  Dickinson, Los Angeles, CA). Let the blood clot at room temperature for 30 minutes. Coagulation After solidification, the serum is centrifuged at 2000 xg for 10 minutes at 4 ° C. Collect serum and 15m Place in a conical tube (Corning, Corning, NY) and place on ice. About 1.1ml An aliquot of the serum in a 2 ml frozen vial and in a dry ice / ethanol bath And store at -70 ° C for subsequent serum inactivation assays. 56 Heat inactivation of control aliquots at 30 ° C for 30 min. Prepare rolls.   1.1 ml of 100% non-heat inactive to test Sindbis for serum inactivation Two vials containing humanized human serum are used for various virus preparations. You. Thaw the first serum vial rapidly at 37 ° C. Then the complement present in the serum Heat the serum at 56 ° C. for 30 minutes to heat inactivate. After inactivation, serum Put on ice. Thaw the second vial rapidly at 37 ° C. After thawing, place serum on ice Good.   About 1.0 ml of non-heat-inactivated serum, culture medium only, and heat-inactivated serum Into 1.5 ml tubes (Fisher Scientific, Pittsburgh, PA) and 1 x 10<sup>Five</sup>pfu And incubate at 37 ° C. for 1 hour. Inn After the cuvette, place the tube on ice.   Alphavirus parent cell line host resistant to human serum inactivation Non-heat-inactivated serum, medium, and heat-inactivated serum virus preparation to determine Is titrated by plaque assay on BHK-21 cells. Non-heat-inactivated serum, Medium or equivalent with or without incubation with heat-inactivated serum. Ruth titer produces Sindbis virus resistant to human complement inactivation The parent cell line host is shown. 2.Structural protein expression construct   a.Inducible and constitutive structural protein vector expression cassettes   Development of the alphavirus packaging cell line requires the necessary structural proteins That is, it synthesizes high intracellular levels of capsids, pE2 and / or E2 and E1) Depends on the ability to Unfortunately, these proteins, especially the envelope High level expression of glycoproteins E2 and E1 has associated cytopathology and consequences Can lead to cell death. Therefore, the structural protein expression cassette Regulates the level of gene expression in addition to others that maintain the target expression level It has been designed with inducible regulatory elements.   In the first structure, expression of the alphavirus structural protein is inducible Under the control of the RSV LTR in combination with a unique lac operon sequence. This is called pOP13 and p Viral structural proteins in OPRSV1 vector (Stratagene, San Diego, CA) This is achieved by insertion of the alphavirus cDNA corresponding to the gene. Used separately These vectors use the p3'SS vector (S tratagene, San Diego, CA). Inducer ( For example, in the absence of isopropyl-BD-thiogalactopyranoside (IPTG), The basic, that is, structural, of the luciferase reporter gene under the control of the lac operon. Adult expression levels have been reported to be 10-20 copies per cell. IPTG The addition results in a change in the conformation of the repressor protein, Reduces the affinity of the lac repressor protein for the lac-operator sequence And enables high-level expression of heterologous genes. 95x in the presence of IPTG Induction levels have been reported for heterologous genes contained in the pOP13 vector. You.   More specifically, the Sindbis structural protein gene (SP) cDNA was converted to pOP13 as follows. And inserted into the pOPRSV1 vector. SP code region is Sindbis nt 7,638 Surrounding nucleotides corresponding to Kozak consensus sequences for efficient translation initiation Map the authentic AUG translation start and UGA translation stop sites, including Amplified as a whole using a primer pair with a 5 'end to be pinged. square Directed primers are complementary to Sindbis nts 7,638-7,661 and reverse Directional primers are complementary to Sindbis nt 11,384-11,364. Structure PCR amplification of Sindbis cDNA corresponding to the protein gene was performed using the following oligonucleotides. Achieved by a standard three-temperature cycling protocol using a tide pair. Forward primer (7638F): Reverse primer (11384R):   In addition to its respective complementarity to the indicated Sindbis nucleotide sequence The 5 nucleotide "buffer sequence" followed by the NotI recognition sequence is the 5 'end of each primer. Included at the end. After PCR amplification, a 3,763 bp fragment in a 1% agarose gel Is purified and subsequently digested with NotI. Then, the obtained 3,749 bp flag Are ligated separately into the pOP13 and pOPRSV1 vectors. These vectors Has been digested with NotI and treated with calf intestinal alkaline phosphatase. These expression cassettes contain the entire coding capacity of Sindbis structural proteins. The vectors are called pOP13-SINSP and pOPRSV1-SINSP, respectively.   A variant form of the gene expression cassette for the lac operon-Sindbis structural protein Constructed using other viral, mammalian or insect based promoters Can be done. Using common molecular biology techniques known in the art, the lac Ron and RSV LTR promoter, or just RSV LTR promoter alone, Removed from tagene pOP13 and pOPRSV1 vectors, cytomegalovirus major Immediate promoter (pOPCMV-SINSP); adenovirus major late promoter (pOPAML P-SINSP); SV40 promoter (pOPSV-SINSP): or Drosophila metallothionein Inducible promoter (pMET-SINSP), Drosophila actin 5C distal promoter (pOP A5C-SINSP), insects containing the heat shock promoter HSP65 or HSP70 (pHSP-SINSP) Promoter sequence or baculovirus polyhedrin promoter It can be replaced with another promoter sequence such as (pPHED-SINSP).   b.Inducible expression of structural proteins via alphavirus vector   Due to potential cytotoxic effects from the expression of alphavirus structural proteins, Inducible packaging that expresses even the smallest basal levels of these proteins Establishment of a cell line may not always be preferred. Therefore, Alf Via a non-structural protein supplied in trans by a virus vector Contains regulatory elements for high-level induction of protein synthesis, but appropriate stimulation Packaging cell line expression without any basal level of synthesis until subjected to A cassette is built.   In this form, the structural A structural protein gene cassette has been constructed to allow transcription of the You. A key feature of such cassettes is the alphavirus nucleus whose transcription initiation is genuine. Close to nucleotide 1 of the alphavirus, starting with nucleotide 1 Adjacent RNA polymerase II promoter, transcriptase (tra nscriptase) 5 'terminal alphavirus sequence required for recognition, structural protein inheritance Alphavirus junction region sequence and alphavirus structure for mRNA expression in offspring Protein gene sequence, 3'-terminal alphavirus sequence required for replication, and transcription Transcription termination / polyadenylation sequence. Before the AUG start site of the structural protein gene Alphavirus structural protein for the upstream open reading frame that ends Quality expression depends on the synthesis of negative-strand RNA by nonstructural proteins supplied by the vector. After which mRNA transcription of the structural protein gene from the junction region occurs. Can come. Therefore, the inducibility of this system is dependent on the alphavirus vector itself. Supplied and transcribed in vitro RNA or appropriate promoter elements Complete with the presence of nonstructural proteins, introduced as any of the downstream cDNAs Depends on everything. In addition, the 5 'and 3' end alphavirus sequences have the structure Non-structural feed of this RNA transcript of the protein gene cassette into the same vector Allow the protein to amplify (see Figure 16).   Specifically, see the positive-sense vector-inducible Sindbis packaging cassette. The construction of the unit is accomplished as follows. Briefly, the pVGELVIS vector described above Is digested with the enzyme BspEI to obtain a nucleic acid containing most of the nonstructural gene coding sequence. Nucleotides 422 to 7054 were removed and the remaining 9925 bp fragment was replaced with 0.8% agar. Purified on Rose gel, then religated to itself and named pLTR / SindlBspE An object is generated (FIG. 16). This deletion is the genuine translation initiation codon at the 5 'end in nts 60-62. Leave the don as it is, and with nts 7,130-7,132 and 7,190-7,192, respectively Creates UAA and UGA stop codons downstream of the in-frame. Therefore, the downstream structure It prevents translation of open reading frames of protein genes. pLTR / S The lndlBspE packaging cassette construct is subsequently transferred into BHK-21 cells. And selecting transfectants using G418 at 400 μg / ml, Then, clone by limiting dilution. Of the transfected clone After expansion, screening for packaging activity was performed using the Sindbis It is performed by transfection of ferase (SIN-Luc) vector RNA. In FIG. According to the data shown, the recovered supernatant was purified from SIN-luc vector RNA from BHK-21 cells. Some of these cloned LTR / SindlB Transfection of SIN-luc vector RNA into spE packaging cells It is demonstrated to result in the production of infectious Sindbis particles containing IN-luc RNA.   A wide variety of variants of these packaging cassette constructs, for example, Substitution of other RNA polymerase promoters for MuLV LTR in a row, RNA polymerase One or more nucleosides between the co-promoter and the first Sindbis nucleotide Adding tides, replacing other ribozyme processing sequences, or transcriptors May or may not retain the 5 'terminal Sindbis sequence required for protease recognition. May not be present in Sindbis upstream of the structural protein gene sequence. Including open reading frame substitutions, which are provided herein. It can be made according to the disclosure. In addition, these constructs are compatible with other cell lines as described above. Can be transfected.   In another vector-inducible packaging form, the expression cassette has its native Alphavirus structural protein flanked by junctional and 3 'untranslated regions of It contains a cDNA copy of the gene sequence and primary transcription from the promoter is Inserted into the expression vector in an orientation that produces RNA molecules Has been entered. In addition, these constructs are located adjacent to the junction region, Alphavirus 5 'terminal sequence required for recognition by transcriptase, and Located immediately adjacent and adjacent to alphavirus nucleotide 1 of the 5 'end sequence Contains a catalytic ribozyme sequence. For this reason, this ribozyme is Cleavage the primary RNA transcript exactly after the viral nucleotides. This antisen In the orientation, the structural protein gene cannot be translated and precedes its expression For the transcription of the two positive strands into mRNA, the viral nonstructural It is completely dependent on the presence of protein. These nonstructural proteins are alpha Further provided by the viral vector itself. In addition, this form Since it contains the 5 'and 3' terminal sequences of the favirus genome, The transcript of the gene is derived from the same nonstructural protein provided by the alphavirus vector. It is amplified by utilizing the protein. Details of this construct can be found in U.S. Patent Application No. No. 08 / 348,472.   In addition, using the above approach, integration and expression of structural protein genes That separate the present and enable their transcription as independent, non-overlapping RNA molecules. A packaging cell line can also be generated. For example, with glycoproteins E2 and E1 Independent capsid protein expression, ie, each of the three proteins Independence indicates that recombination with vector RNA and subsequent contaminating wild-type Exclude the possibility of producing ills. Such a “split gene” expression cassette The construction of the protocol is described in detail in US patent application Ser. No. 08 / 348,472.   c.Assembling the components to create an alphavirus packaging cell line Re   For illustrative purposes, BHK-21 cell line and replicon-induced packaging expression The cassette is used to demonstrate assembly of the components. But other potential parent cells The strain can be used to make an alphavirus packaging cell line. About this Discussed earlier. Briefly, BHK-21 cells were incubated at 37 ° C with 5% CO<sub>Two</sub>And DMEM, Grow in 2 mM L-glutamine and 10% fetal bovine serum (optimal medium). Supply 35 mM Petri dishes in serum-free medium conditions, as suggested by About 5 × 10<sup>Five</sup>Of BHK-21 cells in 5 μl of Transfectam (Promega, Madison , WI) Transfect with 5 μg of pLTR / SindIBspE using cationic lipid reagent . However, any transfection method (ie, electroporation) By calcium phosphate precipitation or any readily known generally in the art. Quickly replaces the use of cationic liposome formulations and procedures available at Can be done. Twenty-four hours after transfection, cells are trypsinized, and As above, 100 mm dishes containing 10 ml of optimal medium supplemented with 400 μg / ml G418 The seeds are re-seeded and selected over a period of 5-7 days. Then G418 drug The colonies that are resistant to are pooled, the dilutions are cloned and expanded. Individual clones were cloned into the SacI linearized plasmid pKSSINBV-luc (see Example 1C (iv)). Sindbis luciferase vector RNA transcribed in vitro from -Level expression of Sindbis structural proteins after transfection using ATP , And for functional packaging. For details, see 60 Transfer with pLTR / SindlBspE from clones grown in mm Petri dishes 2 μg of BHK-21 cells (called LTR / SindI BspE or BK-Bsp cells) Transfected with Ndbis-luciferase vector RNA and 3 ml optimal Cover with medium (see above). Remove the supernatant 20 hours after transfection. Out, and centrifuged at 3,000 rpm for 30 minutes in a Sorvall RT6000B tabletop centrifuge. Make Qing transparent. Additionally, transfected as described by the manufacturer. Lysed cell monolayer in reporter lysis buffer (Promega, Madison, Wis.) And assay for luciferase expression as described above.   Transfer of luciferase activity (and functional packaging) to 60 mm dish Use 1.0 ml of the above supernatant to infect a fresh BHK-21 cell monolayer in the screen Test by: Twenty hours after infection, lyse the cell monolayer as described above, and Test for luciferase expression. As shown in FIG. 17, the three clones (# 13, 18 and 40) are packaged Sindbis luciferase vectors Produce. In addition, the transfected clone # 18 cells were Test for increased vector packaging over time post-fection I do. As described above, the supernatant from the transfected clone # 18 cells was Harvested at 20, 45 and 70 hours after transfection and fresh BHK-21 cells Used to infect monolayers. Figure 18 shows the system at 45 hours post-transfection. Shows significant increase in Ndbis-luciferase vector packaging . Expression was also determined by Western blot using a polyclonal rabbit anti-Sindbis antibody. Can be tested by a set analysis. 3.Inducible vectors and constructs for alphavirus producer cell lines Expression of protein   a.Use of viral promoters   Another approach to developing alphavirus vector producer cell lines If the persistence of the virus frequently occurs after infection, alpha Generating a viral vector producer strain. However, persistent The titer of infectious virus produced in mosquito cells infected with 0<sup>Four</sup>pfu / ml, which was seen after lytic infection of BHK-21 cells with Sindbis. At least 5 orders of magnitude lower than the given titer.   Vectors so that virus-producing cytolytic infections occur only after proper stimulation Inducible alphavirus vector containing both the viral and viral structural gene cassettes. Several strategies have been described for cell producer cell lines . Because these approaches operate at the “feed forward” level, systems "Leakage" in regulatory control of the virus initiates the alphavirus replication cycle And may possibly result in cell death. Therefore, a tightly regulated control mechanism Is necessary for such systems.   A salient feature of development is a differentiation state-dependent gene expression pattern. Briefly stated In general, the gene expression patterns differ greatly between the undifferentiated state and the terminally differentiated state. Has become. Therefore, cells whose differentiation state can be controlled are alphavirus vectors. It appears to be an ideal host to derive cell producer cell lines. like this In one form, the expression vector cassette, and in some cases, the structural component, According to the strategy described for ELVIS, the terminal differentiation-inducible promoter And is used to stably transform undifferentiated host cells. Terminal differentiation of host producer cells following induction with appropriate stimuli is simultaneously Induction of virus replication cycle and production of packaged vector Sprinkle. The present invention, including an antisense structural gene and a heterologous viral expression system The other strategies described in the section below are the cell differentiation status dependent promoters described below. Easily combined with tar.   This approach involves a virus pro- cess that is active only in the terminally differentiated cells. Four examples using either a motor or a cellular promoter are described.   Mouse py, SV40, and MoMLV all contribute to undifferentiated mouse embryo carcinoma (EC) cells They can infect and invade there, but their genes (and heterologous genes) Expression and establishment of virus-producing infections have been shown to be blocked. (Swartzendruber and Lehman, J. Cell.Physiol. 85: 179-188, 1975; Peries et al., J. .Natl. Cancer Inst, 59: 463-465, 1977). These viral growth characteristics also It has also been demonstrated in two cell lines derived from malignant teratocarcinoma stem cells, PCC4 and F9. Proven. Blocking viral growth occurs at the level of transcription and replication, And is mapped to an enhancer contained within the non-coding region of the virus (Linney et al., Nature 308: 470-472, 1984; Fujimura et al., Cell 23: 809-814, 1981; Kati nka and Yaniv, Cell 20: 393-399, 1980). Where MoMLV infects undifferentiated EC cells In that case, the viral DNA is integrated into the genome. However, as mentioned above, Expression of viral or heterologous genes is blocked. This of virus expression Blocks the final differentiation of EC cells by adding retinoic acid to the growth medium It will be released at the time.   To test the RNA expression properties of the pVGELVIS construct in EC cells, plasmid DNA was Lipofectamine<sup>TM</sup>And the conditions suggested by the supplier (about 5 g DNA / 8 g lipid reagent) Thus, undifferentiated PCC4 or F9 cells that are complexed and at about 75% confluency (Fujimur a, et al., 1981. Cell 23: 809-814). generation of cpe and Of Sindbis virus-producing infection determined by plaque assay of the culture supernatant Levels in undifferentiated and differentiated transfected PCC4 or F9 cells Measurements at regular intervals over 5 days. Differentiation of F9 and PCC4 cells is 1M At the final concentration of retinoic acid (Sigma Chemical Co., St. Louis, MO) Achieved.   Heterogeneous gene phase observed in undifferentiated EC cells infected with MoMLV vector It has been proposed that the hierarchy of opposing expression may be in part insertion dependent (Linney et al., 1987, J. Virol. 61; 3248-3253). Therefore, transfection with pVGELVIS Undifferentiated EC cells are transcribed from Sindbis genomic cDNA and In terms of the beginning of the life cycle, it seems likely to have different consequences. In this case, pV After G418 selection of undifferentiated EC cells transfected with GELVIS, the remaining cells Clone and expand. The cell clones were then transformed by the addition of retinoic acid. Test for Sindbis virus production after differentiation.   In the presence of Sindbis nonstructural proteins, the production of that structural protein To isolate vector packaging cell lines that are dependent on the state of differentiation, As described above, undifferentiated F9 or PCC4 cells were transfected using pLTR / SINdlBspE. , And make a G418 selection. Next, the packaged pSIN-BV-luc vector Due to the high multiplicity of infection used, clones sensitive to the state of differentiation are selected. Fine PSIN-BV-luc vector resistant to cell lysis or packaged Clones that do not produce particles are candidates for vector packaging clones . These candidate clones were isolated after terminal differentiation with retinoic acid as described. Test for SIN-luc vector particle production.   Mouse wild-type Py cannot replicate in teratocarcinoma cell lines PCC4 or F9. Not yet This replication block in transformed cells is responsible for the transcriptional level of the early region (ie, T antigen) gene. Occurs in the bell and is released by the induction of terminal differentiation with vitamin A. Undifferentiated Py mutants that can establish virus-producing infections in PCC4 and F9 cells of Maps to the enhancer region. Embryonic tissue-specific transcription enhancer element The production of these mutants has resulted in these variants. In undifferentiated teratocarcinoma cell lines To take advantage of this property of inhibiting Py replication, virus-regulated non- The coding region is ligated to the Sindbis virus genomic cDNA according to the ELVIS strategy. To join. The exact transcription start site of the Py early region has been determined (Tooze, DNA tumor Tumor Virus, 2nd Edition, Cold Spring Harbor, NY, 1981). PCC4 and And the F9 cell line are stably transformed with the Py-Sindbis vector. Culture After adding retinoic acid to the ground and inducing terminal differentiation, Sindbis virus Productive infection occurs. Construction of alphavirus vector with controlled differentiation state , It is described in detail in US patent application Ser. No. 08 / 348,472.   b.Use of cell promoter   A third example of this strategy uses the β-globin locus control region. β-globin multigene cluster contains five developmentally regulated genes I do. In the early stages of human development, the yolk sac of the embryo is a hematopoietic tissue and ε -Express the globin gene. After that, in the fetal liver, the γ-globin gene And in the adult bone marrow exchange for δ- and β-globin genes (Collins and And Weissman, 1984, Prog, Nucleic Acid Res. Mol. Biol. 31: 315).   At least two mouse erythroleukemia strains (MEL and Friend) Serves as a model for terminal differentiation-dependent expression. Β-globin in these strains Expression only occurs after induction of terminal differentiation by adding 2% DMSO to the growth medium. To be observed.   The entire β-globin locus is regulated by the locus control region (LCR). LCR Within the dominant control region (DCR) within the DNaseI hypersensitivity region, 5 'of the coding region Exists. DCR contains five DNaseI hypersensitive (HS1-HS5) sites. DCR Erythroleukemia (MEL) in sgenic and stably transfected mice ) Independent of integration for the human β-globin gene linked in the cell, Governs high level sites of copy number dependent expression (Grosveld et al., CSHSQB 58: 7-1 2, 1993). A recent study (Ellis et al., EMBO 12: 127-134 1993) found that the sequence in HS2 A matching synthetic core concatemer has been shown to function as a locus control region Was.   To achieve expression that depends on the differentiation state of the alphavirus vector, Genomic cDNA is converted to a promoter containing a tandem synthetic core corresponding to the LCR HS2 site. And juxtapose. Alternatively, the desired alphavirus vector construct is By replacement, it can be inserted downstream of the LCR in the endogenous β-globin gene. like this Can place the alphavirus vector exactly at the start site To this end, the β-globin transcription start site after terminal differentiation is first determined.   The initiation of the lytic virus life cycle is controlled by the differentiation state of the host cell. It has applications in other systems where control of virus-induced cytopathology is desired. Wear.   Regulation of alphavirus gene expression by differentiation-state-sensitive promoters Yet another approach involves retinoic acid receptor (RARA) and acute promyelocytic monocytes. The use of Aplastic Leukemia Cells (APL). APL cells are distinguished by high proliferation rates and differentiation arrest. It is a clonal myeloid precursor that has been characterized. Non-random chromosome translocation breakpoint, t (15; 17) (q22; 21) is present in almost all patients with APL. RARA gene Is located on chromosome 17q21. Most analysis of APL mRNA from patients APL breakpoint is in the second intron of the RARA gene and is aberrant fusion transcription To bring things. Simultaneous tracing using RARA and PML-RARA fused cDNA The transfection assay shows that the resulting fusion protein has the presence of retinoic acid. It has been demonstrated that it can antagonize wild-type RARA in the presence. These studies are based on the PML-RA RA fusion proteins are implicated in the molecular pathogenesis of APL. Importantly, so many Patients are completely sedated after all-trans retinoic acid treatment (ATRA). High concentration of AT RA can overcome RARA deficiencies leading to high levels of RA in the nucleus. Then, the APL cells Activation can be achieved by activation of a RARA-responsive gene. RA is compatible with many cell lines (human (Including the leukemia strain HL-60).   Retinoic acid receptors are thyroid hormone receptors and steroid hormones. It is a member of the nuclear receptor superfamily, including the mon receptor. Four Of different forms of human RAR have been identified, and their corresponding cDNAs have been cloned. And characterized. Depends on Sindbis vector differentiation status To achieve expression, the viral genomic cDNA was juxtaposed with a RARA DNA binding site and ELV Create IS-RARASIN. Proposal for ELVIS-PySIN expression in undifferentiated EC cells Like ELVIS-RARASIN expressing cells sensitive to differentiation .   c.Insertion of a vector construct into an inducible promoter controlled by the state of differentiation   Differential expression of heterologous genes from Sindbis vectors arranged in ELVIS form The generation of a state-dependent clone was performed on the pVGELVIS and pLTR / SindlBspE plasmids. As described above. The production of the vector particles is The generation of the clones described above was performed using the isolated By transfection of the Achieve. Desirable phenotype or vector after differentiation induced by retinoic acid Clones with ter production are isolated as described above.   E. FIG.CEA RNA Of cell line for Sindbis vector producer   Unlike the previous example for producing producer cell lines, packaging Only a single gene transfer into the cell line is possible by transfection of the vector. May be capable. These vectors are disabled and complete genomic vectors Reintegration of fresh layers of Sindbis packaging cell lines The infection results in infection failure. Because these vectors are now CE This is because they are activated depending on the presence of ARNA. Using RT-PCR technology, High titer by dilution and cloning of the infected producer cell line Can be achieved.                                 Example 3 Detection of replicable retroviruses A.Expanded S <sup>+</sup> L <sup>-</sup> assay   Expansion S<sup>+</sup>L<sup>-</sup>Assays are performed by replicating infectious virus (RCR) in the supernatant of the target cell line. Determine if it exists. This assay uses an infectious retrovirus Cater cell line MiCl<sub>1</sub>To generate focus on (ATCC deposit number CCL 64.1) Based on experimental observations. MiCl<sub>1</sub>Cell line in mouse sarcoma virus (MSV) form Derived from the Mv1Lu mink cell line (ATCC accession number CCL 64) by transduction. this is Contains replication defective mouse sarcoma provirus (S<sup>+</sup>) Is a replicable mouse leukemia Contains no virus (L<sup>-</sup>) Non-producer non-transformed revertant clone is there. MiCl in replicable retroviruses<sub>1</sub>Cell infection "activates" MSV genome To induce "transformation" that leads to focus formation.   Remove supernatant from cell line to be tested for the presence of replicable retrovirus And remove all cells by passing through a 0.45μ filter. On the first day, Mv1Lu Cells were plated at 1.0 × 10 5 in 2 ml DMEM, 10% FBS, and 8 μg / ml polybrene.<sup>Five</sup>Cell / W Plate (1 well per sample to be tested) in a 6-well plate . For positive and negative controls, Mv1Lu cells were separated into 6 separate wells in the same manner. Le Plate on plate. Cells at 37 ° C, 10% CO<sub>Two</sub>And incubate overnight. 2 On day, add 1.0 ml of test supernatant to Mv1Lu cells. Negative control plate Incubate with 1.0 ml of medium. The positive control was the MA virus (M iller et al., Molec. and Cell Biol. 5: 431, 1985, called pAM). Three types of dilutions (200 foci forming units (ffu), 20 ffu in 1.0 ml medium each) , And 2ffu), which are added to the cells in the positive control wells. Fine Incubate the cells overnight. On day 3, the medium is aspirated and 3.0 ml of fresh D Add MEM and 10% FBS to the cells. Grow cells to confluence, and A 1:10 split on days 6 and 10 to increase all replicable retroviruses Width. On day 13, aspirate the medium on Mv1Lu cells and add 2.0 ml DMEM and 10% Add FBS to the cells. In addition, MiCl<sub>1</sub>Cells were treated with 2.0 ml of DMEM, 10% FBS, and 8 μg / 1.0 × 10 in ml polybrene<sup>Five</sup>Seed in cells / well. On day 14, from Mv1Lu cells Of the supernatant<sub>1</sub>Transfer to corresponding wells of cells, and 37 ° C, 10% CO<sub>Two</sub>In overnight Cubate. On day 15, the medium was aspirated and 3.0 ml of fresh DMEM and 10 ml Add% FBS to the cells. On day 21, focus cells on a monolayer of cells (monolayer Overlying and appearing as confluent refractory cells that remain attached). Pho Focus is MiCl<sub>1</sub>If appearing on cells, the test article is contaminated with a replicable retrovirus. It is determined that it has been dyed. Using these procedures, recombinant virus vector May indicate that the reducer cell line is not contaminated with a replicable retrovirus You. B.Producer cell line co-culture and MdH marker rescue assay   Another method to test for the presence of RCR in vector producer cell lines Producer cells were used for the same number of Mus dunni (NIH NIAID, Bethesda, MD) cells. Co-culture with cells. On day 0, 5.0 × 10<sup>Five</sup>5.0 x 10 Mus dunni cells<sup>Five</sup>No Mix with the reducer cells and mix the mixture in a 10 cm plate (10 ml standard culture Medium / plate, 4 μg / ml polybrene) for small scale simultaneous Perform culture. Effectively dilute out the producer cell line and Splitting the culture at a 1:10 ratio every 3-4 days to provide maximum amplification of the RCR; And 5.0 × 10<sup>Five</sup>Add Mus dunni cells to each culture plate. On day 14, culture supernatant Collected, passed through a 0.45μ cellulose-acetate filter, and -Test with rescue assay. 1.0 × 10<sup>8</sup>1.0 × 10 with Mus dunni cells<sup>8</sup>Professional Mix the mixture with theducer cells in a total of 20 T-150 flasks (30 ml of standard culture medium / flask). Large scale co-culture by seeding on Sco, 4μg / ml polybrene) . Cultures were split 1:10 on days 3, 6, and 13 and 1:20 on day 9 Divide by the ratio of On day 15, the final supernatant is collected, filtered, and Portions are tested in the MdH marker rescue assay.   MdH marker rescue cell line was transformed with LHL (Hygromycin B resistance gene With a retroviral vector (Palmer et al., PNAS 84: 1055-1059, 1987) It is cloned from a pool of entered Mus dunni cells. This retrovirus vector May rescue from MdH cells upon infection of the cells by the RCR. 4 μg / ml Standard culture medium containing rivulene (10% FBS, 1% 200 mM L-glutamine, 1% non-essential 1 x 10 in 2 ml of DMEM containing amino acids)<sup>Five</sup>Of a 6-well plate containing MdH cells Add 1 ml of test sample to the sample. After 24 hours, the medium is Replace with sub-culture medium. Two days later, the entire volume of the MdH culture supernatant was 5.0x in 2 ml of standard culture medium containing polybrene Ten<sup>Four</sup>Transfer to wells of a 6-well plate containing the target cells of Mus dunni. 24 hours later, on The supernatant was replaced with a standard culture medium containing 250 μg / ml hygromycin B, and then 2 days On days 5 and 5, the medium is replaced with a medium containing 200 μg / ml hygromycin B. Selection Nine days after selection, colonies resistant to hygromycin B appeared and they were 0. Visualized by staining with 2% Coomassie Blue.                                 Example 4 Measuring protein expression A.Western blotting   i.RIPA Preparation of lysis solution   Cells transduced with the recombinant vector of the present invention are prepared using trypsin / EDTA. Collected and washed twice with cold PBS. The cells are mixed with 50-400 ml of RIPA buffer (10 m M 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. Lysate was centrifuged in an Eppendorf centrifuge for 5 minutes. Centrifuge at maximum speed for minutes. The supernatant was removed and stored at -20 ° C. Bradf An ord protein assay was performed to determine total protein concentration.   ii.SDS-PAGE   A sample of RIPA lysate containing a total protein concentration of 20 mg, and a commercially available molecular weight (M W) marker (Amersham, Chicago, IL) was added to 2 × 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% β-mercaptoethanol) at a ratio of 1: 1 and heated at 65 ° C for 10 minutes. Heated. Samples and MW markers were placed on ice after heating. Ready-made 7.5% Tri s HCl-based minigels (BioRad, Hercules, CA) with slots in running buffer (3 .0gm Tris-HCl pH 8.6, 1.0gm SDS and 14.4gm glycine to 1.0L) , And the sample and MW marker were loaded on the gel. Marker is the bottom of the gel Approximately 70-12 OV is applied to the gel until a.   iii.transfer   The protein band was reconstituted with CAPS buffer pH 11.0 containing 5% (v / v) methanol. Immobilon-P from gel by soaking for 5 minutes<sup>TM</sup>Transfer to membrane (Millipore, Bedford, MA) You. Hoefer HSI TTE transfer equipment (Hoefer Scientific Instruments, San Proteins were transferred from gels to membranes using Francisco, CA). About 70V for 1.5 hours Was applied to the gel.   iv.Immunodetection   Immobilon-P<sup>TM</sup>The membrane was treated with 0.5% BM blocking reagent containing 3% BSA (Boehringer Man (heim, Chicago, IL) for 30 minutes at room temperature and slowly add in the microwave. Heated. The membrane was then diluted 1: 2000 in BM blocking solution containing 3% BSA Probe with a primary mouse antibody that specifically reacts with the protein detected in Incubate for 1 hour at room temperature. The membrane was washed with 40 ml of PBST (0.2% Tween-2 in PBS). 0) And washed three times for 10 minutes each in a 3% BSA solution (Sigma, St. Louis, MO) at 1:20. Reaction with goat HRP-labeled secondary anti-mouse antibody (Jackson, Bar Harbor, MA) at 2,000 dilution Let it. The membrane is then washed three times for 10 minutes with 40 ml of PBST. After washing, remove the membrane Submerge in ECL color solution (Amersham, Chlcago, IL) for 1 minute and then plastic. Cover with claps and expose to Hyperfilm (Amersham, Chlcago, IL) for 5 seconds. Then The rum is developed and the protein bands are analyzed. B.Indirect immunofluorescence staining and FACS analysis   Indirect immunofluorescence staining, and Amlot et al. FACS analysis (Lymphocyte: A Practical App roach, GGB Klaus (eds.), IRL Press (1987), pp. 77-72). From the transduced cells, cell surface expression of the heterologous protein antigen is detected. 0.2%  BSA and 0.2% NaN<sub>Three</sub>Specific in FACS buffer PBSA containing 50 μl of the reactive primary antibody stock (62 μg / ml antibody) was added to a 96-well U-shaped Add to each well in the first column of the microtitre plate. Next, The antibody in the ram is serially diluted into the wells of the remaining column. About 50μ in FACS buffer l target cell suspension (2.0 × 10<sup>6</sup>~ 4.0 × 10<sup>6</sup>Cells / ml) in a microtiter plate Each well (final antibody concentration is 31, 10, 3.1, 0.31, and 0.1 μg / ml). And incubate at room temperature for 15 minutes. After incubation, 100 μl FACS buffer is added to each well and the microtiter plate is run at 2000 rpm. Centrifuge for 1 minute. Remove the supernatant and add 200 μl of fresh FACS buffer Add to wells. This process is repeated three more times. 85% after the last wash Secondary antibody containing FACS buffer, 10% normal mouse serum, and 5% FITC-labeled anti-mouse antibody Add body solution. Wash microtiter plate 4 times with fresh FACS buffer . After the last wash, add 150 μl from each well to a FACS tube (Falcon 2052 tube ) And add 100 μl of FACS buffer. FACScan counts fluorescent signal Detected by C.Simple indirect immunoperoxidase staining of frozen sections   Dry the frozen tissue section at room temperature for 30 minutes. Dry tissue sections are allowed to dry for 15 minutes at room temperature. Fix with seton and air dry. Rehydrate this section with PBS for a few minutes . The tissue sections were then detected at room temperature for 45-120 minutes in a humidified chamber with 50 μl detection. Primary antibodies that react specifically with the protein to be purified (Marek et al., Cancer 67: 1377, 19 Incubate with 91). The tissue sections are then washed with PBS and in PBS for several minutes. Soak in After soaking, the sections were removed for 50-120 minutes at room temperature for 50 μl of HRP-conjugated anti-mouse. Incubate antibody or HRP-conjugated streptavidin (DAKO, Carpenteria, CA) To The tissue sections are then washed with PBS and soaked in PBS for several minutes. Soaked Later, sections were taken from 60-100 μl of 0.01% H<sub>Two</sub>O<sub>Two</sub>And 3,3 diaminodendidine (DAB) 8 minutes or 100 μl of α-ethylcarbazole (60 mg in 25 ml DMF)  3-amino-9-ethylcarbazole) and 1 ml of acetate buffer (20 min) H<sub>Two</sub>0.68 g sodium acetate pH 5.2 in O to 250 ml volume) and 0.01% H<sub>Two</sub>O<sub>Two</sub>Inn in Cubate. The tissue sections are washed with water for 2-5 minutes and then with hematoxylin for 15 minutes. Stain for seconds. The tissue sections are then washed with water for 10 seconds and the manufacturer's instructions Follow Crystal Mount<sup>TM</sup>(Biomeda, Alameda, CA). D.Luciferase expression in transfected and infected BHK-21 cells   To test the functionality of the Sindbis basic vector, use the SacI linearized pKSSIN In cells transfected with RNA in vitro transcribed from BV-luc Test the expression of luciferase. In addition, most of the nonstructural gene regions were deleted Digestion of pVGSP6GENrep with BspEI And religation under dilution conditions. This construct called pVGSP6GENdlBsp Lacks the nonstructural gene sequence between bases 422-7,054. XbaI linearized pVGSP6GENd In vitro transcription of lBsp and SacI linearized pKSSINBV-luc was performed as described previously. Do. Transfection and co-transfection are performed in vitro. Lipofectin transfection product<sup>TM</sup>And applied to BHK-21 cells It is implemented by doing. Expression of luciferase in transfected cells Currently, it is tested 18 hours after transfection. In addition, 1.0 ml of transformer H Inject confluent monolayers of BHK-21 cells with the injection supernatant, and Test luciferase expression at 24 hours.   The results of this experiment are shown in FIG. 19, which shows that abundant reporter gene expression Transfection of BHK-21 cells with RNA in vitro transcribed from SSINBV-luc And RNs in vitro transcribed from pVGSP6GENdlBsp Transfer of expression activity (e.g., packaging) when co-transfected with A Show clearly.   To junction region promoters that increase, decrease, or inactivate activity Or insertion of a tandem copy of the junction region. , (US Patent Application Serial No. 08 / 348,472).   FACS analysis is performed to detect intracellular markers such as HSV TK. Simple In other words, HT1080 cells were transformed with a recombinant viral vector carrying the HSV TK gene. Transiently transduce with various dilutions. Centrifuge the sample and remove the cells, Ca<sup>+2</sup>Or Mg<sup>+2</sup>Resuspend in 2.0 ml of PBS without (CMF.PBS). About 0.2ml 37% The formaldehyde solution is added while gently shaking the sample. Then Incubate the sample for 20 minutes at room temperature with gentle agitation. Incube After the washing, the sample is washed three times with CMF.PBS. The cells are then transformed into CMF.PBS. 50mM NH<sub>Four</sub>Suspended in 2.0 ml of Cl and stirred for 20 minutes at room temperature with gentle stirring. Incubate. After incubation, wash the sample twice with CMF.PBS . The samples were then combined with 1% BVSA (Fraction V, Sigma, St. Louis, MO) and 1% Wash again with CMF.PBS containing% Saponin (CMF.PBS / BSA / Sap). About 100μl primary anti (Repligen, Cambridge, MA; 0.5 μg / Cl / sample) was added to this suspension and The mixture is then incubated for 1.0 hour at room temperature with gentle agitation. The mixture is then centrifuged and the pellet is washed three times with CMF.PBS / BSA / Sap I do. Approximately 100 μl of diluted secondary antibody (Cappel, Durh) resuspended in CMF.PBS / BSA / Sap am, NC; rabbit anti-mouse IgG-FITC 1: 1000) and the mixture while gently stirring. Incubate for 30 minutes at room temperature. After incubation, pellet the pellet with CMF.PBS / Wash 3 times with BSA / Sap. CMF.PB containing 25μg Propidium Iodide Resuspend in 0.5 ml of S / BSA / Sap and analyze by FACS.Example 5 Measuring the activity of expressed proteins A.Measurement of tissue factor activity   Cells are grown in 1 ml of 0.01 M sodium phosphate buffer, pH 7.0, containing 0.15 M NaCl. Remove from plate by resuspending. Adjust different cell densities on the plate To adjust, adjust the absorbance at 550 nm to 0.750. Sonicate the cells for 1 minute, Then, TritonX-100 is added to a final concentration of 0.1%. Sample at room temperature 90 Spin for minutes and remove cell debris by centrifugation at 10,000 xg . The detergent solubilized extract was mixed with 2 μl of the sample, and 0.1 M NaCl, 0.1% bovine Dilute in 0.8 ml of 0.05 M Tris-HCl, pH 7.5 (TBS buffer) containing serum albumin Thereby becoming relipidated. Phosphotide in 0.25% deoxycholic acid 50 μl of a 5 mg / ml solution of lecholine (lecithin) and CdCl<sub>Two</sub>Of 25 μl, and This solution is incubated at 37 ° C. for 30 minutes.   A chromogenic assay for tissue factor activity was performed using factor X in the presence of tissue factor and factor VII. Based on factor activation. Then, the formed Xth<sub>a</sub>The amount of the factor is No. X<sub>a</sub>Assessed by factor-catalyzed cleavage. Human factor X (Enzyme Research La 3 μl of a 0.4 mg / ml solution of boratories, South Bend, IN) and human factor VII (Sigm a 2 μl of a 200 U / ml solution of a Chemical Company, St. Louis, MO) with 0.025 M CaCl Add to 0 μl and 46 μl of TBS. Add the sample to be assayed, and The reaction mixture is incubated at 37 ° C. for 5 minutes. Chromogenic substrate S2222 (Helena Laborator ies, Beaumont, TX) (50 μl of a 2 mg / ml solution) and the reaction was allowed to continue for 10 minutes. You. The reaction is terminated by adding 100 μl of glacial acetic acid and absorbing at 405 nm. Measure the degree. Assay controls replace tissue factor containing samples with Consisted of the addition of the relipidation mixture to which TBS buffer was added.   For assay of COS-7 transfected cells, 2 μl of cell extract was added to the reaction mixture To be added. As a reference, rabbit brain reconstructed according to the manufacturer's instructions Construct standard curve using rhomboplastin (Sigma Chemical Co., St. Louis, MO) did. Dilute the thromboplastin solution 1:30 and add the indicated volume to the chromogenic assay. Lee (Fisher et al.,Thrombosis Research 48:89, 1987). B.Measurement of anti-angiogenic activity   Anti-angiogenic activity of various polypeptides useful according to certain aspects of the invention Is Takigawa et al. (Biochem.Int.14: 357,1987). Can be assayed on anoic membrane (CAM). Briefly, B16 melanoma cells were transformed into C Inject subcutaneously into the flank of 57BL / 6N mice. When the tumor reaches about 1.0 cm in diameter, it Were excised, cut into 2 mg sections, and sterilized Whatmann GF / B glass fiber Place on a filter disc (diameter 6 mm; Reeve-Angel, Clifton, NJ) and add 30 μl Of the transduced cells are added. They were made in eggshells on day 8 of inoculation. Upside down on the CAM of a 10-day-old chicken embryo through the open window. Embryos after 5 days Kill by injecting 10% formalin in PBS. Excision of CAM, 10% in PBS Fix oppositely in formalin and examine under a stereomicroscope. Angiogenesis is Assay by measuring the number and thickness of capillaries under the filter. Thick capillaries, medium-sized capillaries, small capillaries, and fine capillaries Gives 3, 2, 1 and 1 points, respectively, and the average of these points Is defined as pro-angiogenic activity. Measure the diameter of the tumor on the filter in three dimensions Tumor size and (π / 6) abc mm<sup>Three</sup>(a, b, and c: length, width, and And height). Average number of low points and reduced tumor size Shows anti-angiogenic activity. C.Measurement of tumor angiogenesis   For visualizing neovascularization of tumors treated according to the methods taught herein Four minutes before sacrifice, mice were injected with 300 μl of India ink and the skin was removed. And dry. Free blood flow through 10μm E-Z TRAC Ultrasphere (Interactive  Medical Technology, Los Angeles, CA). 10μm micro sphere 5 × 10<sup>Five</sup>Is injected into the left ventricle of the mouse and tumor tissue is harvested after 5 minutes You. After solubilizing the tumor tissue, the beads present in the tumor are counted and the tumor Expressed as beads per gram woven. D.Measurement of herpes simplex virus thymidine kinase activity   Disclosure using the sensitivity of HSVTK vector transduced cells to ganciclovir Of expressed HSVTK expressed in cells treated according to the method described . Briefly, cells transduced with pTK-3 were plated at 2.5 × 10<sup>6</sup>Dense Inoculate 6 plates at a time. In addition, non-transduced CT26 and CT26β-gal (this Is a strain containing a reporter gene β-galactosidase from E. coli. (Transduced with Rus) is also inoculated on 6 plates as a control . Five plates of each cell type were applied 100 μg / m twice a day for 4 consecutive days. l, 50 μg / ml, 25 μg / ml, 12.5 μg / ml, and 6.25 μg / ml concentrations of ganciclovir Treatment with a medium containing One plate of each cell type is left untreated. This After treatment, remove cells from each dish using trypsin / EDTA and add 10% FBS Resuspend in DMEM containing and measure. A similar protocol is used for other pro Can be used for egg activating enzymes. E. FIG.Measurement of folate integrin β3 activity   i.Cell surface folate binding assay   Seed MCF-7 stable transfectants and maintain in the same medium without G418 Carry. The seeding density should be such that the cells are 75% confluent for the folate binding assay. Adjust it. The form of radiolabeled folic acid used was obtained from New England Nuclear Is a histamine derivative, iodination of folic acid. Cells are washed with 3 ml ice cold pH 4.5 saline. Wash twice with water (10 mM Na-acetate, 150 mM NaCl) and remove from the cell surface folate receptor. Dissociates folic acid. The cell monolayer is then washed twice with 3 ml of ice-cold PBS, pH 7.4. Return the pH to neutral. For cell surface binding of radiolabeled folate, cells must be Histamine derivatives (total 20,000 cpm added to 50 nM non-radioactive folate) and In 2 ml of ice-cold DMEM (without FCS) containing 50 μg / ml BSA and ice-cold H<sub>Two</sub>15 minutes in in O bath Cubate. 1000x to determine specific cell surface binding (cpm / mg protein) A parallel experiment is performed in which excess non-radioactive folic acid is added. The monolayer was washed with ice cold PBS, pH 7.4. Washing Clean (3 ml each). Cells are solubilized and supernatant samples are gamma-treated with ~ 70% efficiency. Count using a counter (55B; Beckman Instruments, Inc., Dallas, T X). The protein concentration in each solubilized sample is determined as described above.   ii.[ <sup>3</sup> H] Folate binding assay   [<sup>Three</sup>[H] folate binding assay<sup>Three</sup>Direct binding of [H] folic acid to membranes and solution-phase assays Perform both. For direct binding assays, membrane samples (10-100 μg protein) With 3 pmol of [10 mM sodium phosphate buffer (pH 7.5) / 150 mM NaCl / 10 mM EDTA.<sup>Three</sup>H] Incubate with folic acid (40 Ci / mmol) at 37 ° C for 30 minutes with constant stirring. 12,000g of membrane For 15 minutes, wash once with the same buffer, and add 10 mM sodium phosphate buffer (pH 7). .5) / 150 mM NaCl / 1% Triton X-100 and dissolve in liquid scintillation cow To serve. [<sup>Three</sup>Nonspecific binding of [H] folate was determined for both assays in each case by [<sup>Three</sup> H] Incubate with 100 pmol of unlabeled folic acid for 5 minutes at room temperature prior to folic acid addition It is measured by performing the assay at the same time as the control.   iii.Methotrexate transport study   Stable transfectants are seeded, and the cell monolayer is washed with 3 ml of ice-cold pH 4. 5. Wash twice with saline and twice with 3 ml ice-cold PBS, pH 7.4. radiation Irradiated methotrexate (<sup>Three</sup>For internalization of (H-MTX), cells were harvested at 50 μg / ml BSA , And 2 μM [<sup>Three</sup>2 ml of pre-warmed (37 ° C.) DMEM containing [H] MTX (FCS or other additive None) at 37 ℃, 5% CO<sub>Two</sub>And incubate for 30 minutes. Specific MTX internalization (pmol / mg Parallel experiments in the presence of a 500-fold excess of non-radioactive MTX to determine Give. Reduced folate transport, mediated by human folate receptor (hFR) Molar excess (100-fold) of non-radioactive leaves inhibits transport through hFR to distinguish them from Perform parallel experiments in the presence of acid. The monolayer was washed once with 2 ml of ice-cold PBS (pH 7.5) for 2 ml (1) p Wash once with H4.5 saline, surface-bound<sup>Three</sup>Remove H-MTX and finally 2 ml ice Wash with cold PBS (pH 7.5). Solubilize the cells and dispense the sample (500 μl) with 10 ml Add to liquid scintillation cocktail, and liquid scintillation with ~ 50% efficiency (Tri-carb; Packard Instrument Co., Inc.D) o wners Grove, IL). Measure protein concentration in each solubilized sample as described above . F.Measurements of integrin B <sub>3</sub> activity   i.peptide   A fibrinogen γ chain peptide of 16 amino acids having the sequence KYGGHHLGGAKQAGDV (K16) on an Applied Biosystems model 430 peptide synthesizer (Foster City, CA) Phenylacetamide methyl resin and t-butane purchased from Applied Biosystems Prepared by solid-phase synthesis using xycarbonyl amino acids. Peptide homogeneity Using a C18μBondapak column, high performance liquid chromatography Analyze with a linear gradient of 0-60% acetonitrile in 1% trifluoroacetic acid,> Find 85% homogeneity. Peptide was added to PBS (0.15M NaCl, 0.01M sodium phosphate Buffer, pH 7.3) and modified lactoperoxidase-glucose Oxidase method (D'Souza et al., J. Biol. Chem. 263: 3943, 1988 and Lam et al., J.B. lol. Chem. 262: 947, 1987). Glucose (80 μl 0.2 M Sodium phosphate, pH 7.4, 40 μg), carrier-free Na<sup>125</sup>(15mCi), and Enzym Obead reagent (Bio-Rad, Hercules, CA) is added to 10-12 mg of peptide. Iodine Activated peptides were purified by gel filtration on a Bio-Gel P-2 column (Bio-Rad, Hercules, CA). , Free Na<sup>125</sup>Separate from Determine the concentration of the labeled peptide by its amino acid set. It is measured by the absorbance at 280 nm using the extinction coefficient derived from the formation.   ii.Platelet binding and cross-linking   Platelets were separated from fresh human blood by differential centrifugation, and 0.1% bovine serum albumin was added. For gel filtration on Sepharose 2B in Tyrode buffer, pH 7.3, containing no divalent ions More isolated. Briefly, platelets are 4 × 10<sup>8</sup>/ ml, not containing divalent ions In Tyrode's albumin buffer. Ca<sup>2+</sup>To a final concentration of 1 mM. for Platelet stimulants were 10 μM ADP, 0.5 units / ml α-thrombin, or 100 mM PM. A. Radiolabeled K16 peptide is added at a concentration of 30 μM and binding is Let go for 45 minutes. The main crosslinking reagent is bis (sulfosuccinimidyl) (BS<sup>Three</sup>) (Pierce Chemical Co., Rockford, IL). After 10 minutes at 22 ° C, crosslinking reaction Is terminated by the addition of 10 mM Tris, pH 7.0. 20% ligand bound to cells Harvest by centrifugation through sucrose and cells are harvested with 1% No. nidet P-40 and And 10 mM N-ethylmaleimide (Sigma, St. Louis, MO) in PBS. The extracted protein is precipitated with 10% trichloroacetic acid and obtained after centrifugation. Wash the pellet three times with cold 85% ethanol.   Polyacrylamide gel electrophoresis was performed using the Laemmli buffer system (Nature 227: 680, 19 Performed in the presence of sodium dodecyl sulfate in a vertical slab gel in 70). Change Use a percentage of the gel. For the first analysis of the crosslinked sample, the 7.5% gel was Run under reducing conditions. Gel used for amino acid sequencing has a 10-20% gradient It is a gel. The sample in Laemmli sample buffer is used for disulfide bond reduction. For treatment with 5% 2-mercaptoethanol. Dry the analytical gel, and Kod Develop autoradiograms using ak X-Omat AR film. The molecular weight is Di Based on electrophoretic mobility for prestained standards obtained from versified Biotech presume. Relative mobility (R<sub>F</sub>), Ovalbumin marker protein in the same gel Against moving<sup>125</sup>It is measured by measuring the migration of the I-K16-GPIIb band.                                 Example 6 Retroviral vector formulation A.Lactose formula   Crude recombinant retrovirus is bound to beads in a bioreactor matrix. Combined with a recombinant retrovirus (US patent application Ser. No. 07 / 395,932). Celligan bioreactor containing isolated DA cells (New Brunswick, New Brunswi ck, NJ). Cells are a continuous flow process Release the recombinant retrovirus into the growth medium that passes through the cells at Ba Collect the medium leaving the ioreactor, and first a 0.8 micron filter, then Clarify crude recombinant retrovirus by passing through a 0.65 micron filter with I do. Concentrate the filtrate using a cross-flow concentrator system (Filtron, Boston, MA) . Add about 50 units of DNase (Intergen, New York, NY) per ml of concentrate, Digest exogenous DNA. The digest was converted to 150 mM NaCl using the same cross-flow system. , Diafiltrate against 25 mM tromethamine, pH 7.2. 50 mM NaCl, 2 Sephadex S-500 gel column equilibrated in 5 mM tromethamine, pH 7.4 (Phar macia, Piscataway, NJ). Purified recombination The retrovirus was prepared using Sephadex S- in 50 mM NaCl, 25 mM tromethamine, pH 7.4. Elute from a 500 gel column.   A formulation buffer containing lactose was prepared in a 2x concentrated stock solution. This Formulation buffer was 25 mM tromethamine, 70 mM NaCl, 2 mg / ml arginine, 1 0 mg / ml human serum albumin (HSA) and 100 mg / ml lactose in 100 ml final Contains by volume at pH 7.4.   1 part S-500 purified recombinant retrovirus plus 1 part 2x lactose formulation buffer The purified recombinant retrovirus is formulated by adding the solution. Prescribed group The recombinant retrovirus can be stored at -70 ° C to -80 ° C or dried .   Supermodulyo 12K freeze dryer (Edwards High Vacuum, Tonawanda, NY) In the attached Edwards Refrigerated Chamber (3 Shelf RC3S unit), Lyophilized retrovirus. When the lyophilization cycle is complete, After the release of nitrogen gas, the vial is stoppered under vacuum. Remove vials when removing Crimp with a luminium seal.   In a given lactose study, the formulated liquid product was stored at -80 ° C and -20 ° C. Stored in both circulation freezer. In FIG. 20, the virus of these samples Infectivity was compared to the virus infectivity of the lyophilized sample. Lyophilized sample , -20 ° C, refrigerated, and room temperature. Sample activity during reconstitution Determined by a titration assay.   The lyophilized recombinant retrovirus is reconstituted with 1.0 ml of water. Reconstructed The infectivity of the recombinant retrovirus is determined by a titer activity assay. Assay Performed on HT1080 fibroblasts or 3T3 mouse fibroblast cell line (ATCC CCL 163) Was. In detail, 1.0 × 10<sup>Five</sup>Individual cells are plated on a 6 cm plate, and 37 ℃, 10% CO<sub>Two</sub>Incubate overnight at. Reconstituted recombinant retrovirus 10 microliters of a serial dilution of the solution in 4 μg / mL polybrene (Sigma, St. Louis , MO) and added to the cells at 37 ° C., 10% CO 2<sub>Two</sub>Incubate overnight at You. Following incubation, the recombinant vector encoding the neo resistance gene Transfected cells are selected for neomycin resistance in a medium containing G418 and 37 ° C, 10% CO<sub>Two</sub>And incubate for 5 days. Following the first choice, this The cells were refed with fresh medium containing G418 and incubated for 5-6 days. Beate. After the last selection, the cells are coomassie blue for colony detection. Stain with Determine the titer of the sample based on the number of colonies, dilution used, and volume. To decide.   FIG. 20 shows that storage in lyophilized form at −20 ° C. to refrigerated temperatures was Retains virus activity similar to recombinant retrovirus stored in solution at To allow less stringent temperature control during storage and storage Is shown. B.Mannitol prescription   The recombinant retrovirus used in this example was purified as described above.   A formulation buffer containing mannitol was prepared as a 2x concentrated stock solution. Was. This formulation buffer contains 25 mM tromethamine, 35 mM NaCl, 2 mg / ml Nin, 10 mg / ml HSA, and 80 mg / ml mannitol in a final volume of 100 ml Contains in 7.4.   1 part S-500 purified recombinant retrovirus plus 1 part mannitol formulation buffer The purified recombinant retrovirus is formulated by adding the solution. Prescribed group The recombinant retrovirus is stored at this stage at -70 ° C to -80 ° C, or Can be dried.   Edwards Refrigerated Chambe mounted on a Supermodulyo 12K freeze dryer Dry the formulated retrovirus in r (3 Shelf RC3S units). freeze drying When the sital is complete, release the nitrogen gas to 700 mbar and place in a vial under vacuum. Stopper. Upon removal, the vial is crimped with an aluminum seal.   In a given mannitol study, the prescribed liquid product was stored at -80 ° C and -20 ° C. Stored in both circulating freezer at ° C. In FIG. 21, the windows of these samples are shown. Russ infectivity was compared to the virus infectivity of the lyophilized sample. Lyophilized sump Were stored at −20 ° C., refrigerated temperature, and room temperature. Sample activity during reconstitution Is determined using the titer assay of A above.   FIG. 21 shows that storage in lyophilized form at −20 ° C. to refrigeration temperatures is −80 ° C. or Significant viruses compared to recombinant retrovirus stored in solution at -20 ° C Temperature control that retains virus activity and is less stringent during storage Is allowed. c.Trehalose prescription   The recombinant retrovirus used in this example was purified as in A above. Was.   A formulation buffer containing trehalose was prepared as a 2x concentrated stock solution. Was. This formulation buffer contains 25 mM tromethamine, 70 mM NaCl, 2.0 mg / ml Nin, 10.0 mg / ml HSA, and 100 mg / ml trehalose in a final volume of 100 ml , PH 7.2.   1 part S-500 purified recombinant retrovirus to 1 part trehalose formulation buffer To form a purified recombinant retrovirus. Prescribed recombination The retrovirus is stored at -70 ° C to -80 ° C at this stage, or Can be dried.   Edwards Refrigerated Chambe mounted on a Supermodulyo 12K freeze dryer Dry the formulated retrovirus in r (3 Shelf RC3S units). freeze drying When the cycle is complete, release the nitrogen gas to 700 mbar and place it in a vial under vacuum. Stopper. Upon removal, the vial is crimped with an aluminum seal.   In a given trehalose study, the formulated liquid product was stored at -80 ° C and -20 ° C. Stored in both circulating freezer at ° C. In FIG. 22, the windows of these samples Russ transmission was compared to the virus transmission of lyophilized samples. Lyophilized sump Were stored at −20 ° C., refrigerated temperature, and room temperature. Sample activity during reconstitution , Using the titer assay of A above.   FIG. 22 shows that storage in lyophilized form at −20 ° C. to refrigerated temperature was Similar virus compared to recombinant retrovirus stored in solution at 20 ° C Retention of activity and allow for less stringent temperature control during storage. It indicates that it is acceptable.   Virus in solution formulated recombinant retrovirus sample stored at -80 ° C Infectivity was determined by lyophilized recombinant retrovirus storage at -20 ° C. Compared to Lus infectivity. First, a large amount of recombinant retrovirus was obtained, shown below Formulated in four different ways. Then, the prescribed recombinant retrovirus Are frozen in bulk for 1.5 months, then rapidly thawed and lyophilized. Positive A lyophilized sample stored at −80 ° C. after control and lyophilized at −20 ° C. Compare with The prescriptions are listed below:  In the graph of FIG. 23, the Y-axis in each of the four graphs (A, B, C, D) is a mark. It represents the normalized titer. At the starting time point after lyophilization (t = 0), Established for both 80 ° C liquid samples and -20 ° C lyophilized samples. At each time point in the stability study, the titer obtained was divided by the titer value at the zero time point. And the original percentage was plotted on the graph.   This data shows that the activity after lyophilization is comparable to that of liquid samples (stored at -80 ° C). To demonstrate that it is maintained in lyophilized samples (stored at -20 ° C). The formulated lyophilized recombinant retrovirus is placed in a -20 ° C freezer (frost-free). (Circulation freezer). Formulated liquid recombinant retro stored at -80 ° C Lyophilized form is less stringent of storage conditions compared to virus Allow control. D.Sindbis prescription   Crude recombinant alphavirus vector, recombinant alphavirus vector Celligan containing transfected or transduced packaging cells Obtained from the ioreactor and bound to beads in the bioreactor matrix You. This cell is used to transform the recombinant alphavirus vector into cells during a continuous flow process. Release into growth medium passing over. Collect the medium exiting the bioreactor and First pass through a 0.8 micron filter, then a 0.65 micron filter Clarify the crude alphavirus vector. The filtrate is used for cross-flow concentration Concentrate using. Digest about 50 units of DNase per ml of concentrate to exogenous DNA To be added. This digest was converted to 150 mM NaCl, 25 using the same cross-flow system. Diafiltrate against mM tromethamine, pH 7.2. Diafil Sephad equilibrated in treated solution in 50 mM NaCl, 25 mM tromethamine, pH 7.4 Load on ex S-500 gel column. Purified recombinant alphavirus vector, Elute from a Sephadex S-500 gel column in 50 mM NaCl, 25 mM tromethamine, pH 7.4 You.   Formulation buffer containing lactose is prepared as a 2x concentrated stock solution. Prescription The buffer was 25 mM tromethamine, 70 mM NaCl, 2 mg / ml arginine, 10 mg / ml HSA, And 100 mg / ml lactose in a final volume of 100 ml at pH 7.4.   Purify the recombinant alphavirus vector with 1 part of S-500 purified recombinant Adding 1 part of 2x lactose formulation buffer to favirus vector Prescribed by The formulated recombinant alphavirus vector is -70 ° C to -80 ° C Can be stored or dried.   Take the prescribed alpha vector into a Supermodulyo 12K freeze dryer Lyophilize with attached Edwards Refrigerated Chamber (3 Shelf RC3S unit). At the end of the freeze-drying cycle, bypass under vacuum with a slight nitrogen flow. Plug Al. When removing, cover the vial with an aluminum seal . The lyophilized recombinant retrovirus may contain 1.0 ml of water or other physiologically Reconstitute with an acceptable diluent.Example 7 Dosing protocol A.mouse   i.Administration of cells transduced with a recombinant retroviral vector a.TK-3 Of the effect of ganshikuvir on CT26 in the presence or absence of ceramide   Colon tumor cells (CT26) (Brattain, Baylor College of Mediine, Houston, TX) Is transduced with DA / TK-3 (G-pseudotyped TK-3 vector). Will Twenty-four hours after addition of the supernatant, the CT26 cells are placed under G-418 selection (450 μg / ml). 10 days After incubation, a G-418 selection pool is obtained, which is named CT26 TKneo. (CT26 TKneo), CT26TKneo cells, 2.5 × 10<sup>6</sup>Six 10cm at a density of pieces / plate<sup>Two</sup> Seed on plate. As controls, each of the other two cell types, CT26 And CT26β-galactosidase (β-gal) (this cell line is a repo from E. coli) Transduced with a virus carrying the beta gene). As 6 10cm<sup>Two</sup>Seeded on plates. 5 plates for each cell type Ganci at concentrations of 00 μg / ml, 50 μg / ml, 25 μg / ml, 12.5 μg / ml, and 6.25 μg / ml The cells were treated twice a day with a medium containing Clovir for four consecutive days. One for each cell type Plates were left untreated. Thereafter, cells were harvested using trypsin-EDTA Removed from dish, resuspended in DMEM with 10% FBS and counted . The data in FIG. 24 shows that even the lowest dose of ganciclovir was effective against CT26 TK neo cells. Have a dramatic cytotoxic effect. This dose of ganciclovir (6. 25 μg / ml) or even the next higher dose (12.5 μg / ml) It had no effect on any of the 26β-gal cells. However, 25 μg / ml of cancer Starting with a dose of cyclovir, a dose-dependent decrease in cell proliferation could be seen. , CT26TK neo cells were always more sensitive to the drug. b.CT26TK neo Ganciclovir dosing for treatment of mice injected with cells   Whether in vivo transduction of mouse tumors can be used to treat disease To test for transduction in vitro to ensure 100% transduction To determine the optimal concentration of ganciclovir required to remove the selected tumor An experiment was performed for the purpose. Twelve groups of Balb / c mice (Harlan Spague Dawley, Ind. ianapolis, IN) 2 × 10<sup>Five</sup>Inject CT26 TK neo cells. Six groups of mice These cells were injected intraperitoneally (I.P.) and six groups of mice contained these cells. Is injected subcutaneously (S.C.). The other two groups of three mice were 2 × 10<sup>Five</sup>Unmodified Modified CT26 cells (as control) are injected with either I.P. or S.C.   Ten days after injection of these groups of mice with CT26 or CT26 TK neo cells, Initiate several concentrations of ganciclovir treatment. Each dose regimen should be taken twice a day at noon Consisting of an infusion of ganciclovir before and afternoon. The experiments are summarized in Table A below. You.  After 5 days, all mice in the 125 mg / Kg, 250 mg / Kg, and 500 mg / Kg treatment groups Died due to the toxic effects of nciclovir. 15.63mg / Kg, 31.25mg / Kg and 62.5 Mice in the mg / Kg treatment group were treated for an additional 7 days and these were resistant to treatment Obtained. Tumor measurements were taken for 23 days (FIG. 25). The growth of CT26 TK neo is higher than that of unmodified CT26. Rihon was slightly slower. Complete tumor regression was observed in mice treated with the 62.5 mg / Kg regimen. In the group of males. Partial tumor regression was seen in the 31.25 mg / Kg treatment group Admitted. In the 15.63 mg / Kg treatment group, compared to the two untreated control groups, Little or no effect was observed. 62.5mg / Kg Although toxicity was observed, this was not life threatening and Was reversible upon discontinuation. Therefore, this concentration was used for further research (see Figure twenty five). Twenty-four days later, the IP injected animals were sacrificed and evaluated. As shown in FIGS. 26 and 27 Thus, the optimal concentration of antitumor effect is similar whether the tumor is grown on I.P. or S.C. there were.ii. Administration of gene delivery vehicle   a.Transfer and expression of vector-encoded genes in tissue culture   Transmission and expression of genes encoded by specific vectors Tested by transduction followed by testing expression in these cells. Ten<sup>6</sup>HT1080 cells (human fibrosarcoma line) or other suitable cells (eg, from breast tumors) Other human tumor-derived strains such as cell line MCF-7; human primary endothelial cells HUVTEC; colon tumor Strain CT26, fibrosarcoma cell line L33, melanoma cell line B16, breast tumor cell line Tg-6-2, neuroblastoma Mouse tumor cell lines, such as cell line C1300; mouse endothelial cells, such as the SVEC cell line. Or a cell line) in a 10 cm dish with 1 to 10 cell transduction units (eg, , Colony forming units) with the gene delivery vector. After two to six days, Harvest cells and test for expression of transferred gene using appropriate assay You. Where appropriate antibodies are available, use the assays described in Examples 4A and 4B. obtain. Examples of specific activity assays include tissue factor (Example 5A), general anti-angiogenesis promotion Factor (Example 5B), herpes thymidine kinase (Example 5D), folate integrin B3 (Example 5E).   b.Administration to animals and detection of activity and biological effects   A major challenge in the use of the present invention is that other tumors or vasculature around Without any unacceptable toxic effects due to significant expression in place Novel like snake venom proteins, tissue factor, prodrug activating enzymes Effector proteins or metabolic modifying proteins such as uricase To achieve good expression.   This is assessed using the following mouse model: BalB / c mouse + L33 or C T26 cell line; C57B / 6 + B16 cell; FVB / N + TG-6-2 cell; C3H mouse + C1300 or SVE C cells; BalB / c nu / nu mice + any of these + human tumor cells (eg See, for example, the melanoma cell lines DM252, DM6, DM92 (see US patent application Ser. No. 08/032846). ) Or MCF-7).   The tumor cells are dosed at an appropriate dose (typically 2 x 10<sup>Five</sup>Cells), about B16 lung metastasis type Inject subcutaneously, I.V. or intrasplenic for CT26 liver metastasis. Injected Groups of mice (1-10 mice) were apparent for subcutaneous tumors at necropsy Until the appearance of a tumor (1-4 mm in diameter) or metastasis that is easily detectable Is maintained in the metastatic model over time. This is typically 7-14 Days. At this time, tumor angiogenesis was performed using the technique of Example 5C and tumor Various routes of administration tested to determine which route gives the best flow to the ulcer Can be visualized using.   10 vector adjustments<sup>Five</sup>,Ten<sup>6</sup>,Ten<sup>7</sup>,Ten<sup>8</sup>,Ten<sup>9</sup>,Ten<sup>Ten</sup>Or 10<sup>11</sup>Vector At the dose of the position (eg, colony forming unit, cfu), by the I.V. Alternatively, place polybrene (1-8 μg / ml) or DEAE dextran ( (2-30 μg / ml) with or without a transduction enhancer. note The shots are given daily for 1, 2, 3, 4, 5, 6, or 7 days, and 2 After ７7 days, tumors are excised and assayed for gain of activity of the delivered gene. (See, eg, Examples 4C, 4D, and Example 5). The controls are Irrelevant inactive vectors (eg, human gamma interfero inactive in mice) Mice that were injected with a Vector Encodes the type of gene described in the description and, in the case of a retroviral vector, It has a nonspecific envelope, such as a vegetative or xenotropic envelope. And small amounts of replicating cells in the general vascular endothelium, and new It relies on the over-representation of such replicating cells at the site of control angiogenesis. Ah Alternatively, the vector has a suitable targeting ligand to avoid non-specific adsorption and Allows binding and entry into target cell types. The specificity of this system is also It can be controlled by the inclusion of a control element.   Selected based on similar dosing protocol or the above experiments or other criteria Fewer dosing protocols measure antitumor effects in these mouse models. Used to Endpoints are tumor size versus time and growth / regression, death Extinction time, survival time, or other appropriate marker (for example, in these models) Number of metastases). c.Administration to humans   Keep patients on the same vector as the mouse model or on a vector resistant to human complement. Treatment with the same nucleic acid scaffold (eg, the comparion application “Produ ction and Administrasion of High Titer Recombinant Retroviruses Thing). Tumor masses that cannot be removed by surgery (eg, brain, prostate, cervix (cer vical), ovary, bladder) have a large amount of healthy tissue that cannot be replaced Need to be removed simultaneously (eg, amputation of limbs or removal of whole organs) Or metastases (eg, metastasis from colon to liver, black to brain, other subcutaneous sites and lungs) (Metastatic metastasis) may have been treated.   Patients are treated intravenously, intraarterially, in the local vasculature or around the tumor.<sup>6</sup>,Ten<sup>7</sup> ,Ten<sup>8</sup>,Ten<sup>9</sup>,Ten<sup>Ten</sup>Or 10<sup>11</sup>Vector unit (I.V.) dose, preferably 0.1-3 m Administered in a volume of l. This vector contains the clot enhancer encoding It has a gene or other gene selected from the types described above. This gene is non-toxic If the prodrug is to be converted to a toxic product, The final dose of the dose or vector specified in the Physicians Desk Reference The dose predicted from animal studies is administered in the time between 1 and 30 days after administration.   This vector is administered 1-20 times, at intervals of 1-15 days, and depends on the condition of the patient. Track routine clinical parameters and monitor tumor size by radiography, millis Monitor by monitoring by can, pet scan or other conventional methods Be monitored.Example 8 Targeting recombinant retroviral vectors to melanoma cells   The following examples illustrate the binding of retroviral vector particles to specific cell types. Note the use of the conjugated targeting element Ab-Biotin to target avidin. Put on. Generally, biotinylated melanoma cell stimulating hormone (MSH) is first injected into the patient I do. After a period of time (up to 3 days), after non-specific binding is reduced and A vector that expresses avidin on its surface when only the ligand complex remains Injection. The high affinity of avidin for biotin allows the vector to target tissues Focus.   Briefly, melanoma cell stimulating hormone (MSH) is a receptor on melanoma cells Is a 13 amino acid peptide specifically recognized by MSH is 10<sup>-8</sup>Range of M Receptor affinity (K<sub>D</sub>). A.Construction of expression cassette skeleton, pH CMV-PA   The vector first forms the backbone for both the gap / pol and env expression cassettes To make it. In short, pBluescript SK<sup>-</sup>Phagemid (Stratagene, San Diego, CA, GenBank accession number 52324, `` SK^-Is digested with SpeI, and Make blunt ends with Klenow. Then, the Dra I (bp 2,366) to Dra I (2,729) SV40 SK blunt-ended Dra I fragment (Fiers et al., Nature 273: 113-120, 1978)^-Insert into And constructs isolated from the SV40 late polyadenylation signal by SK^-LacZ remains Place it in the opposite direction to the gene. This construct is called SK-SV40A.   Human cytomegalovirus major immediate early promoter (HCMV-MIE; Boshart et al., Cell 41: 521-530, 1985) (Hinc II, bp 140 to Eag I, bp 814) was replaced with Hinc II and Eag I. After digestion with, and blunt the Eag I site. This 647 blunt-ended flag Ligation to SK-SV40A. The final construct, called pHCMV-PA, is then isolated (See FIG. 28). This construct is an HCMV that is placed in the opposite orientation of the LacZ gene. Contains the promoter, and the upstream from the SV40 late polyadenylation signal . B.pCMV-env ^eco Building   pCMV-env^ceoWith the Xba I-Nhe I fragment of MoMLV (bp 5,766 to bp 7,845 of MoMLV). Is inserted into the expression vector of pCMV-PA (Example 2A). Concisely For example, the Xba I-Nhe I envelope fragment was converted to pMLV-K (MiMi) on an agarose gel. ller et al. Vir. 49: 214, 1988). The fragment is then Was blunt-ended with T4 polymerase using a conventional method and ligated to pCMV-PA (Example 2). And digests at the Eco RV and Sma I sites. The product in the right direction is C MVMIE promoter followed by a complete ecotropic envelope coding sequence And the SV40 polyadenylation signal. C.Construction of avidin-envelope chimera   A part of the avidin DNA of bp 116 to 499 (SEQ ID NO: 27, GenBank number CHKAVIR) was MLV ecotropic envelope construct pCMV-env^ecoIncorporate in. In short, The following oligonucleotides are generated as follows: Using oligonucleotides, single-stranded pC by the method of Kunkle (PNAS 82: 488,1985). MV-env^ecoIs modified. This modification involves the variable A region of the envelope (Battini et al.). , J. Virol. 66: 1468-1475.1992). Change. The product is then digested with EcoRI and partially digested with FspI. Avi The Eco RI-Fsp I fragment of gin (bp 198-bp 485) is ligated into the vector. Final The products are CMV promoter, hybrid eco-avidin envelope and SV40 plasmid. Plasmid containing a rearenylation signal, pCMV-env^eco-avidinCall. D.Biotinylated MSH   MSH peptide S-Y-S-M-E-H-F-R-W-G-L-P-V-NH_Two(Chiron Corp., Emeryvi lle, CA), and NHS-biotin (Pierce, Rockford, IL) according to the manufacturer's instructions. To be biotinylated. E. FIG.Generation of a marker-recombinant retroviral vector displaying avidin   Β-galactosidase (C encoding a marker recombinant retroviral vector Bβ-gal) with pCMV-env^cco-avidinTogether with the 293 2-3 cell line (US Patent Application No. 07 / 800,921). Or luciferase, green Use equal amounts of vector encoding fluorescent protein (GFP) or other markers. obtain. Select clones (at G418) and screen for high RNA-containing products And then^ThreeScreening avidin surface expression using H-biotin linkage To Vector particles containing avidin¹⁴C-biotin (Amersham, Chlcago, IL) and And using the sucrose gradient method. F.In vitro targeting   Human melanoma cells (DM252, DM6, DM92) are grown in a suitable medium. Results in target cells The specificity of biotinylated MSH to be combined is determined by the addition of avidin-fluorescein and fluorescence. Test by microscope. Eco-avidin CBβ-gal transduction by staining or G4 Tested by 18 selections (see US Patent No. 08 / 032,846) and transduced Is compared to non-melanoma cells such as HT1080 human fibrosarcoma cells. G. FIG.In vivo targeting   Transplant one or more of the following human melanoma cell lines into the peritoneal cavity of nude mice: No .: DM252, DM6, DM92 (see US patent application Ser. No. 08 / 032,846). Targeting First, mice were injected with biotin-MSH, followed by 1 × 10^Five~ 1 × 10⁸ cfu eco-a Determined by injecting vidin CB β-gal recombinant retroviral vector You. Targeting is followed by dissecting melanoma tissue and staining for β-gal, Or assay for luciferase activity in melanoma and mouse tissues It is evaluated by doing. As a control, pCMV-env of 293-2-3 cells^ecoNo A similar vector encapsidated for transfection was used for the envelope plasmid. Without the addition of a drug, the mice are injected in parallel and the tissues of these mice are Say. H.In vivo targeting-human   Using the data obtained in the mouse system, a protocol for administering to patients To determine Patients can receive the vector encoding the desired gene in 3-20 doses. Administer I.P., I.V., intraarterial (I.A.), or intralymphatic. This dose depends on I.V., I.V. About 1. of gene delivery vehicles given by A., I.P., or intralymphatic administration. 0x10⁶~ 1.0 × 10^Tencfu range.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 35/00 C12N 15/00 ZNAA 43/00 111 5/00 B C12N 5/10 A61K 37/02 // A61K 35/76 37/52 (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), AM, AT, AU, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IS, JP, KE, KG, KP, KR, KZ , LK, LR, LT, LU, LV, MD, MG, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ , TM, TT, UA, UG, UZ, VN (72) Inventor Polo, John M. United States of America 92109, San Diego, Reed Avenue Number 4 1222 (72) Inventor Duvensky, Thomas W. Jr. United States of America 92014 , Del Mar, Via Felino 12729 (72) Inventor Jolly, Douglas Jay. United States California 92024, Luccadia, Hillcrest Drive 277

Claims (1)

[Claims] 1. A method of killing tumor cells in vivo, comprising: causing a nucleic acid molecule encoding a polypeptide capable of stimulating clot formation in a tumor or clot formation adjacent to a tumor. Transducing with an included gene delivery vehicle. 2. A method for inhibiting tumor angiogenesis in vivo, comprising transducing cells in or adjacent to a tumor with a gene delivery vehicle comprising a nucleic acid molecule encoding a polypeptide capable of inhibiting tumor angiogenesis. A method comprising: 3. A method for killing tumor cells in vivo, comprising the steps of: (a) removing vascular cells in a tumor or vascular cells adjacent to the arterial side of a tumor with a non-cytotoxic factor and a cytotoxic factor; Transducing with a gene delivery vehicle containing a nucleic acid molecule encoding a polypeptide that can be activated to the cell; and (b) administering to the animal a non-cytotoxic factor that can be activated by the polypeptide within the cytotoxic factor. . 4. A method of depriving a tumor cell of nutrients in vivo, wherein the nutrients can be bound by cells in blood vessels in the tumor or cells adjacent to blood vessels in the tumor in the intervascular space around the tumor. Transducing with a gene delivery vehicle comprising a nucleic acid molecule encoding a polypeptide that is capable of metabolizing the nutrient or conferring resistance to cellular uptake of the nutrient. 5. 5. The method of any of claims 1, 2, 3, or 4, wherein the gene delivery vehicle is a recombinant retroviral vector. 6. 2. The method of claim 1, wherein the gene delivery vehicle comprises a nucleic acid molecule encoding a polypeptide capable of stimulating the clot formation. 7. The polypeptide may be a Russell's viper snake venom factor X-activator, a Russell's viper snake venom factor X-activator, tissue factor, truncated tissue factor, truncated tissue factor vascular endothelial growth factor fusion protein, cancer coagulation promoting factor 7. The method of claim 6, wherein the method is selected from the group consisting of, thrombin, and a thrombin-like enzyme. 8. Wherein said polypeptide is von Willebrand factor antigen II, endothelial monocyte activation polypeptide I, endothelial monocyte activation polypeptide II, tumor necrosis factor α, tumor necrosis factor β, and tissue factor induction from Rickettsia rickettsil 7. The method of claim 6, wherein the method is selected from the group consisting of factors. 9. The thrombin-like enzymes are Crotalus adamanteus (Crotalase), Crotalus horridu s horridus, Agkistrodon Rhodostoma (Ancrod), Agkistrodon contortrix, Agki strodon acutus, Bothrops attox (Batroxobin), Bothrops marothenus, Tris, Bothrops marotheros 9. The method according to claim 8, wherein the method is selected from the group consisting of thrombin-like enzymes from s gabonica venom. 10. 2. The method of claim 1, wherein said gene delivery vehicle comprises a nucleic acid molecule encoding a polypeptide capable of inhibiting fibrinolysis. 11. The method according to claim 7, wherein the polypeptide is selected from the group consisting of α2-antiplasmin, plasminogen activator inhibitor I, plasminogen activation inhibitor II, plasminogen activation inhibitor III, and Erythrina proteinase inhibitor. 12. 3. The method of claim 2, wherein the recombinant retroviral vector comprises a nucleic acid molecule encoding a polypeptide capable of inhibiting tumor angiogenesis. 13. The polypeptide is angiostein, interferon α, interferon β, platelet factor 4, tissue inhibitor of metalloproteinase I, tissue inhibitor of metalloproteinase II, tissue inhibitor of metalloproteinase III, anti-angiogenic fragment of thrombospondin, prolactin, heparinase, neutralizing antibody fragments against the basic fibroblast growth factor, it is selected vascular endothelial cell growth factor, and from the group consisting of Arufabuibeta ₃ integrin, the method according to claim 12. 14． 4. The method of claim 3, wherein said gene delivery vehicle comprises a nucleic acid molecule encoding a polypeptide capable of activating a non-cytotoxic factor to a cytotoxic factor. 15. The polypeptide is herpes simplex virus thymidine kinase, varicella-zoster virus thymidine kinase, cytosine deaminase, Escherischia coli purine nucleoside phosphorylase, Leishmania purine nucleoside phosphorylase, Escherischia coli xanthine-guanine phosphoribosyltransferase, cytochrome P450 2B1 gene product. , Cytochrome P450 reductase, cell surface, alkaline phosphatase, β-glucosidase, N-deoxyribosyltransferase, ferredoxin oxidoreductase, carboxypeptidase G2, carboxypeptidase A, β-pactamase, actinomycin D synthetase complex, and nitrogen reductase 4. The method of claim 3, wherein the method is selected from: 16. A polypeptide wherein the gene delivery vehicle is capable of binding, metabolizing, or conferring resistance to cellular uptake of the nutrients in the intervascular space of the tumor 5. The method of claim 4, comprising a nucleic acid molecule encoding 17． A gene delivery vehicle comprising a nucleic acid molecule encoding a polypeptide capable of stimulating clot formation. 18. A gene delivery vehicle comprising a nucleic acid molecule encoding a polypeptide capable of inhibiting fibrinolysis. 19. A gene delivery vehicle comprising a nucleic acid molecule encoding a polypeptide capable of inhibiting tumor angiogenesis. 20. A gene delivery vehicle encodes a polypeptide that can bind, metabolize, or confer resistance to cellular uptake of the nutrient in the space of the perivascular space of the tumor A gene delivery vehicle comprising a nucleic acid molecule that comprises 21. A producer cell that produces the gene delivery vehicle of claim 17. 22. A producer cell that produces the gene delivery vehicle of claim 18. 23. A producer cell that produces the gene delivery vehicle of claim 19. 24. A producer cell that produces the gene delivery vehicle of claim 20. 25. A target cell transduced with the gene delivery vehicle of claim 17. 26. A target cell transduced with the gene delivery vehicle of claim 18. 27. 20. Target cells transduced with the gene delivery vehicle of claim 19. 28. 21. A target cell transduced with the gene delivery vehicle of claim 20. 29. The recombinant vector is adenovirus, retrovirus, poxvirus, alphavirus, poliovirus, rhinovirus, influenza virus, parvovirus, adeno-associated virus, herpes virus, SV40 virus, human immunodeficiency virus, measles virus, astrovirus, 21. A target cell transduced with a gene delivery vehicle according to any of claims 17, 18, 19, or 20, wherein the target cell is selected from the group consisting of: and a coronavirus. 30. The gene delivery vehicle according to claim 1, 2, 3, or 4, wherein the gene delivery vehicle is selected from the group consisting of a viral vector, a nucleic acid vector, a liposome, a polycation-enriched nucleic acid, or a recombinant vector. 31. A gene delivery vehicle produced by the producer cell of claim 21. 32. A gene delivery vehicle produced by the producer cell of claim 22. 33. A gene delivery vehicle produced by the producer cell of claim 23. 34. A gene delivery vehicle produced by the producer cell of claim 24. 35. The gene delivery vehicle is an adenovirus particle, a retrovirus particle, a poxvirus particle, an alphavirus particle, a poliovirus particle, a rhinovirus particle, an influenza virus particle, a parvovirus particle, an adeno-associated virus particle, a herpes virus particle, an SV40 virus particle. 35. The gene delivery vehicle of any one of claims 31, 32, 33, or 34, wherein the gene delivery vehicle is selected from the group consisting of: a human immunodeficiency virus particle, a measles virus particle, an astrovirus particle, and a coronavirus particle. 36. 36. The gene delivery vehicle of claim 35, wherein the alphavirus particles are selected from the group consisting of Sindbis virus, Semliki Forest virus, Middelburg virus, Ross River virus, and Venezuelan equine encephalomyelitis virus. 37. The gene delivery vehicle is derived from avian leukemia virus, bovine leukemia virus, mouse leukemia virus, mink cell focus-forming virus, mouse sarcoma virus, reticuloendotheliosis virus, gibbon leukemia virus, Mason-Petesar virus and Rous sarcoma virus. 36. The retroviral particle of claim 35, wherein the retroviral particle is selected from the group consisting of: 38. 38. The mouse of claim 37, wherein the retroviral particles are selected from the group consisting of Abelson virus, Friend virus, Graffy virus, Gross virus, Carsten virus, Harvey sarcoma virus, Lausher virus, and Moloney murine leukemia virus. Retroviral particles. 39. 35. The gene delivery vehicle according to any of claims 31, 32, 33, or 34, wherein the viral particle is replication defective. 40. A pharmaceutical composition comprising the gene delivery vehicle of any one of claims 31, 32, 33, or 34. 41. 35. The gene delivery vehicle according to any of claims 31, 32, 33, or 34, which is lyophilized. 42. 36. The gene delivery vehicle of claim 35, which is lyophilized. 43. 43. The lyophilized gene delivery vehicle of claim 42, wherein upon reconstitution, the gene delivery vehicle is suitable for administration to a human. 44. 35. The gene delivery vehicle according to any of claims 31, 32, 33, or 34, which is dehydrated. 45. 36. The gene delivery vehicle of claim 35, which is dehydrated.