MXPA97002132A - Use of a non-myamiferous dna virus to express an exogenous gene in a mamif cell - Google Patents

Abstract

The present invention relates to a method of expressing an exogenous gene in a mammalian cell, which involves infecting the cell with a non-mammalian virus, such as a baculovirus, whose genome carries an exogenous gene, and culturing the cell under such conditions that the gene is expressed. The exogenous genes are delivered to mammalian cells by the use of a transfer vector such as that described in the drawing. Also disclosed is a method of treating a gene deficiency disorder in a mammal by providing a cell with a therapeutically effective amount of a virus whose genome carries an exogenous gene and culturing the cell under conditions such that the exogenous gene is expressed in the mammal.

Description

USE OF A NON-MAMMABLE DNA VIRUS TO EXPRESS AN EXOGENOUS GENE IN A MAMMALIAN CELL BACKGROUND OF THE INVENTION This invention relates to the use of a non-mammalian DNA virus to express an exogenous gene in a mammalian cell. Current methods for expressing an exogenous gene in a mammalian cell include the use of viral mammalian vectors, such as those that are derived from retroviruses, adenoviruses, herpes viruses, vaccinia viruses, polioviruses, or associated viruses. with adeno. Other methods for expressing an exogenous gene in a mammalian cell include direct injection of DNA, the use of ligand-DNA conjugates, the use of adenovirus-ligand-DNA conjugates, calcium phosphate precipitation, and methods utilizing a liposome- or polycation-DNA complex. In some cases, the liposome- or polycation-DNA complex can direct the exogenous gene to a specific type of tissue, such as liver tissue. Some methods for targeting genes to liver cells use the asialoglycoprotein receptor (ASGP-R) that is present on the surface of hepatocytes (Spiess et al., 1990, Biochem 29: 10009-10018). ASGP-R is a lectin that has affinity for the terminal galactose residues of glycoproteins. In these cases, the DNA complexes are endocytosed by the cell after they bind to the ASGP-R on the cell surface. The construction of viruses that are commonly used in genetic expression methods (eg, gene therapy) is typically based on the principle of removing the unwanted functions of a virus known to infect, and replicate in, a mammalian cell. . For example, the genes involved in viral replication and packaging are often removed to create a defective virus, and then a therapeutic gene is added. This principle has been used to create gene therapy vectors from many types of animal viruses, such as retroviruses, adenoviruses and herpes viruses. This method has also been applied to the Sinbis virus, an RNA virus that normally infects mosquitoes, but which can replicate in humans, causing a rash and arthritis syndrome. Non-mammalian viruses have been used to express exogenous genes in non-mammalian cells. For example, viruses of the Baculoviri-dae family (commonly referred to as baculoviruses) have been used to express exogenous genes in insect cells. One of the most studied baculoviruses is Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). Although some species of baculoviruses infecting crustaceans have been described (Blissard et al., 1990, Ann. Rev. Entomology 35: 127), the normal host range of AcMNPV baculoviruses is limited to the order of Lepidoptera. It has been reported that baculoviruses enter mammalian cells, and baculoviral DNA has been detected in nuclear extracts from mammalian cells (Volkman and Goldsmith, 1983, Appl. And Environ.Microbial 45: 1085-1093, Carbonell and Miller, 1987, Appl. And Environ, Microbiol 53: 1412-1417, Brusca et al., 1986, Intervirology 26: 207-222, and Tjia et al., 1983, Virology 125: 107-117). Although a report of baculovirus-mediated gene expression in mammalian cells has appeared, the authors later attributed the apparent reporter gene activity to the reporter gene product that was carried into the cell after a prolonged incubation of the cell with the virus (Carbonell et al, 1985, J. Virol 56: 153-160, and Carbonell and Miller, 1987, Appl. And Environ.Microbiol 53: 1412-1417). These authors reported that, when the exogenous gene has access to the cell as part of the baculovirus genome, the exogenous de novo gene is not expressed. In addition to the Baculoviridae, other virus families multiply naturally only in invertebrates; some of these viruses are listed in Table 1. Currently, genetic therapy methods are being investigated for their usefulness in the treatment of a variety of disorders. Most genetic therapy methods involve supplying an exogenous gene to overcome a deficiency in the expression of a gene in the patient. Other methods of gene therapy are designed to counteract the effect of a disease. Still other methods of gene therapy involve supplying an anti-sense nucleic acid (e.g., RNA) to inhibit the expression of a host cell gene (e.g., an oncogene), or the expression of a gene from a pathogen (e.g. example, a virus). Certain methods of gene therapy are being examined for their ability to correct errors of birth of the urea cycle, for example, (see, for example, ilson et al., 1992, J. Biol. Chem. 267: 11483-11489). The urea cycle is the predominant metabolic pathway, by which nitrogen waste is removed from the body. The steps of the urea cycle are limited primarily to the liver, presenting the first two steps within the liver mitochondria. In the first step, carbamoyl phosphate is synthesized in a reaction that is catalyzed by carbamoyl phosphate synthetase I (CPS-I). In the second step, citrulline is formed in a reaction catalyzed by ornithine transcarbamylase (OTC). Then citrulline is transported to the cytoplasm, and condensed with aspartate to form arginosuccinate by the argininosuccinate synthetase (AS). In the next step, argininosuccinate lyase (ASL) dissociates arginosuccinate to produce arginine and fumarate. In the last step of the cycle, arginase converts arginine to ornithine and urea. A deficiency in any of the five enzymes involved in the urea cycle has significant pathological effects, such as lethargy, poor diet, mental retardation, coma, or death within the neonatal period (see, for example, Emery et al., 1990, in: Principies and Practice of Medical Genetics, Churchill Livingstone, New York). Deficiency of ornithine transcarbamylase usually manifests as a lethal hyperammonemic coma within the neonatal period. A deficiency in arginosuccinate synthetase results in citrullinemia which is characterized by high levels of citrulline in the blood. The absence of argininosuccinate lyase results in argininosuccinic aciduria (ASA), which results in a variety of conditions, including severe neonatal hyperammonemia and mild mental retardation. An absence of arginase results in hypertengymia, which can manifest as progressive spasticity and mental retardation during early childhood. Other therapies currently used for liver disorders include dietary restrictions; Liver transplant; and administration of free base of arginine, sodium benzoate, and / or sodium phenylacetate. SUMMARY OF THE INVENTION It has been discovered that the non-mammalian DNA virus, which carries an exogenous gene expression construct, can be used to express the exogenous gene in a mammalian cell. In accordance with the foregoing, in one aspect, the invention provides a method for expressing an exogenous gene in a mammalian cell, which involves introducing into the cell a non-mammalian DNA virus (also referred to herein as a "non-mammalian" DNA). "virion"), whose genome leads to the exogenous gene, and allow the cell to live under conditions such that the exogenous gene is expressed. In a second aspect, the invention provides a method for the treatment of a genetic deficiency disorder in a mammal (e.g., a human or a mouse), which involves introducing into a cell a therapeutically effective amount of a DNA virus. that is not mammalian, whose genome carries an exogenous gene, and allow the cell to live under conditions such that the exogenous gene is expressed in the mammal. The invention further provides a method for treating hepatocellular carcinoma in a mammal, which involves introducing into a cell (e.g., a hepatocyte), a non-mammalian DNA virus (e.g., a baculovirus), whose genome expresses a therapeutic gene for carcinoma (e.g., tumor necrosis factors, thymidine kinases, diphtheria toxin chimeras, and cytosine diamines). In general, in vivo or in vi tro methods can be used to introduce the virus into the cell. Preferably, the exogenous gene is operably linked to a promoter that is active in the carcinoma cells, but not in other cells of the mammal. For example, the α-fetoprotein promoter is active in hepatocellular carcinoma cells and in fetal tissue, but is otherwise not active in mature tissues. In accordance with the foregoing, the use of this promoter in this aspect of the invention is preferred. In each aspect of the invention, the non-mammalian DNA virus is preferably an invertebrate virus. For example, the DNA viruses mentioned in Table 1 in the invention can be used. Preferably, the invertebrate DNA virus is a baculovirus, for example, a nuclear polyhedrosis virus, such as a multiple nuclear polyhedrosis virus Autographa cali fornica. If desired, the nuclear polyhedrosis virus can be designed in such a way that it lacks a functional polyhedron gene. Either the occluded form or the yolk form of the virus (e.g., baculovirus) can be used.

TABLE 1. DNA VIRUSES NOT OF MAMMALS, WHICH CAN BE USED IN THE INVENTION.1 I. FAMILY: BACULOVIRUS BACULOVIRIDAE SUBFAMILIA: BACULOVIRUS VIRUS OCCUPIED EUBACULOVIRINAE Genus: Nuclear polyhedrosis virus (NPV) Subgenre: Multiple Nucleocapsid Virus (MNPV) Preferred species: Autographa californica nuclear polyhedrosis virus (AcMNPV). Other Members: Chroistoneura fumiferana (MNPV (Cf NPV) Mamestra brassicae MNPV (MbMNPV) Orgyia pseudotsugata MNPV (OpMNPV) and approximately 400 to 500 species isolated from seven orders of insects and crustaceans.

These viruses are mentioned in: "Fifth Report of the International Committee on Taxonotny of Viruses" ("Fifth Report of the International Committee on Virus Taxonomy") (ICTV) by Cornelia Buchen-Osmond, 1991, Research School of Biological Sciences, Canberra, Australia. Most of the viruses mentioned here are available in the American Type Culture Collection.

Subgenus: Simple Nucleocapsid Virus (SNPV) Preferred species: Bombyx mori S Nuclear Polyhedrosis Virus (BmSNPV) Other Members: Heliothis zea SNPV (HzSnpv) Trichoplusia or SNPV (TnSnpv) and similar viruses isolated from seven orders of insects and crustaceans. Genus: Granulosis virus (GV) Preferred species: Plodia interpunctella granulosis virus (PiGV) Other Members: Granulosis virus Trichoplusia (TnGV) Granulosis virus Pieris brassicae (PbGV) Granulosis virus Artogeia rapae (ArGV) Granulosa virus Cydia pomonella (CpGV) and similar viruses of approximately 50 species in the Lepidoptera.

SUBFAMILY: NON-OCCLIDED BACULOVIRUS VIRUSES NUDIBACULOVIRINAE Genus: Non-occluded Baculovirus (NOB) Preferred species: Heliothis zea NOB (HzNOB) Other Members: Oryctes rhinoceros Virus Additional viruses have been observed in a fungus. (Strongwellsea magna), a spider, the European crab. (Carcinus maenas), and the blue crab (Callinectes sapidus). II. FAMILY: ICOSAHÉDRICOS DESOXIRRIBOVIRUS ICOSAHÉDRICOS IRIDOVIRIDAE Genus: Group of insect virus Small Iridescent Iridovirus. Preferred species: Iridescent Chilo virus. Other Members: Iridescent insect virus 1 Iridescent insect virus 2 Iridescent insect virus 6 Iridescent insect virus 9 Iridescent insect virus 10 Iridescent insect virus 16 Iridescent insect virus 17 Iridescent insect virus 18 Iridescent insect virus 19 Iridescent virus insect 20 Iridescent insect virus 21 Iridescent insect virus 22 Iridescent insect virus 23 Iridescent insect virus 24 Iridescent insect virus 25 Iridescent insect virus 26 Iridescent insect virus 27 Iridescent insect virus 28 Iridescent insect virus 29 Iridescent virus of insect 30 Iridescent insect virus 31 Iridescent insect virus 32 Genus: Group of insect virus Large iridescent chloriridovirus. Preferred species: Iridescent mosquito virus (iridescent virus - type 3, regular strain). Other Members: Iridescent insect virus 3 Iridescent insect virus 4 Insect iridescent virus 5 Insect iridescent virus 7 Insect iridescent virus 8 Insect iridescent virus 11 Insect iridescent virus 12 Insect iridescent virus 13 Iridescent insect virus 14 Iridescent virus of insect 15 Putative member: Chironomus plumosus iridescent Genus: Frog virus group Ranavirus Preferred species: Frog virus 3 (FV3) Other Members: Frog virus 1 Frog virus 2 Frog virus 5 Frog virus 6 Frog virus 7 Frog virus 8 Frog virus 9 Frog virus 10 Frog virus 11 Frog virus 12 Frog virus 13 Frog virus 14 Frog virus 15 Frog virus 16 Frog virus 17 Frog virus frog 18 Frog virus 19 Frog virus 20 Frog virus 21 Frog virus 22 Frog virus 23 Frog virus 24 L2 L4 L5 LT 1 LT 2 LT 3 LT 4 T 21 T 6 T 7 T 8 T 9 T 10 T 11 T 12 T 13 T 14 T 15 T 16 T 17 T 18 T 19 T 20 Edema virus Tadpole of tritons Edema virus Tadpole of Rana catesbriana Tadpole edema virus of Xenopus Genus: Group of Lymphocystis disease virus. Lymphocystis virus Preferred Species: Flounder Isolate (LCDV-1) Other Members: Isolated dab of Linfocistis disease virus (LCDV-2). Putative member: Disease virus Octopus vulgaris Genus: Goldfish virus group Preferred species: Goldfish virus 1 (GFV-1) Other Member: Goldfish virus 2 (GF-2) III. FAMILY: PARVOVIRIDAE Genus: Densovirus from the insect parvovirus group Preferred species: Densovirus Galleria Other Members: Densovirus Junonia Densovirus Agraulis Densovirus Bombyx Densovirus Aedes Putative Members: Densovirus Acheta Densovirus Simulium Densovirus Diatraea Densovirus Euxoa Densovirus Leucorrhinia Densovirus Periplanata Densovirus Pieris Densovirus Sibine PC 84 (parvo virus of the crab Carcinus mediterraneus). Virus in the form of hepatopancreatic parvo of penaeid shrimp. IV. FAMILY: GROUP OF VIRUSES OF VARICELA POXVIRIDAE SUBFAMILIA: VIRUS OF VARICELA OF VERTEBRATES CHORODOPOXVIRINAE Genus: Subgroup Molluscum contagiosum Mollusc varicella virus Preferred species: Virus Molluscum contagiosum SUFAMILIA: VIRUS OF INSECT VARICELA ENTOMOPOXVIRINAE Genus Putative: Entomopoxvirus A Varicola virus of Colopterus Preferred species: Varicella virus of Melolontha melolontha Other Members: Coleoptera: Anomalous cuprea Aphodius tasmaniae Demodema boranensis Dermolepida albohirtum Figulus sublaevis Geotrupes sylvaticus Genus Putative: Entomopoxvirus B Varicella virus of Lepidoptera and Orthoptera. Preferred species: Varicella virus of Amsacta moorei (Lepidoptera) Other Members: Lepidoptera: Acrobasis zelleri Choristoneura biennis Choristoneura conflicta Choristoneura diversuma Chorizagrotis auxiliais Operophtera brumata Orthoptera: Arphia conspersa Locusta migratoria Melanoplus sanguinipes Oedaleus senuglaensis Schistocerca gregaria Genus Putative: Entomopoxvirus C Varicella virus Diptera Preferred species: Varicella virus Chironomus luridus (Diptera) Other Members: Diptera: Aedes aegypti Camptochironomus tentans Chironomus attenuatus Chironomus plumosus Goeldichironomus holoprasimus Other members of the Poxviridae family: Albatrosspox (Chickenpox virus) Cotia Embu Marmosetpox Marsupialpox ("quokkas" Australian) Varicella virus of hybrid deer (Odocoileus hemionus; Capripoxvirus). Volepox (Microtus oeconomus, Microtus pennsylvanicus). Skunk varicella virus (Mephitis mephitis; Orthopoxvirus). V. GROUP OF MOSAIC VIRUS CAULIMOVIRUS OF COLIFLOR Preferred Member: Cauliflower mosaic virus (CaMV) (col b, Davis isolate). Other Members: Berry red ring spot (327) Carnation branded ring (182) Dahlia mosaic (51) Fig wort mosaic Labano radish Mirabilis mosaic Peanut chlorotic streak (331) Chlorotic soybean speck Band strawberry vein (219) Thistle Speck Putative Members: Necrotic Mosaic Aquilegia Vein Mosaic Cassava Cestrum Virus Petunia Vein Release Plantago Virus 4 Mota Sonchus VI. GRUPO GEMINIVIRUS Subgroup I (ie, Gender) Maize striped virus Preferred Member: Maize striped virus (MSV) (133) Other Members: Chloris striated mosaic (221) Digrant Stripe Miscanthus Dwarf Wheat Putative Members: Bajra Stripe Striated Mosaic of Bromus Digitaria striated mosaic Oat chlorotic strip Paspalum striated mosaic Subgroup II (ie, Genus): Wavy upper beetle virus Preferred member: Wavy beet top virus (BCTV) (210) Other Members: Upper pseudo-curly virus Tomato Summer Death Virus Bean Dwarf Yellow Tobacco Virus Tomato Leaf Roll Virus Subgroup III (ie Gender): Bean Golden Mosaic Virus Preferred Member: Golden Bean Mosaic Virus (BGMV) (192 ) Other Members: Abutilon mosaic virus African cassava mosaic virus Cotton crumpled leaf virus Euphorbia mosaic virus Horsegram yellow mosaic virus Indian cassava mosaic virus Jatropha mosaic virus Lime seed golden mosaic virus Malvaceous chlorosis virus Melon curly hour virus Mung seed yellow mosaic virus Yellow potato mosaic virus Rhynochosia mosaic virus Chayote curly leaf virus Tiger disease virus Tobacco leaf curly virus Mosaic virus Tomato Grasp Tomato Curly Virus Tomato Yellow Dwarf Yellow Tomato Curly Leaf Virus Tomato Yellow Mosaic Virus Tomato Yellow Mosaic Wavy Watermelon Speck Virus Watermelon Chlorotic Stain Virus Yellow Honeysuckle Vein Mosaic Virus Putative Members: Curly cotton leaf virus Cowpea golden mosaic virus Aubergine yellow mosaic virus Eupatorium yellow vein virus Lupine curly leaf virus Crushed soybean leaf virus Solanum apical curly leaf virus Wissadula mosaic virus VII. FAMILY: dsDNA ALPS VIRUS PHYCODNAVIRIDAE Genus: Phycovirus dsDNA group Phycodnavirus Preferred species: Paramecium bursaria chlorella-1 (PBCV-1) virus Virus: Paramecium bursary Chlorella NC64A virus (NC64A virus). Paramecium bursaria Chlorella Pbi virus (Pbi virus) Hydra virdis Chlorella virus (HVCV) Other Members: Chlorella virus NC64A (thirty-seven NC64A viruses, including PBCV-1). Chlorella NE-8D virus (CV-NE8D, synonymous NE-8D) CV-NYbl CV-CA4B CV-AL1A CV-NY2C CV-NC1D CV-NC1C CV-CA1A CV-CA2A CV-IL2A CV-IL2B CV-IL3A CV- IL3D CV-SC1A CV-SC1B CV-NC1A CV-NE8A CV-AL2C CV-MA1E CV-NY2F CV-CA1D CV-NC1B CV-NYsl CV-IL5-2sl CV-AL2A CV-MA1D CV-NY2B CV-CA4A CV- NY2A CV-XZ3A CV-SH6A CV-BJ2C CV-XZ6E CV-XZ4C CV-XZ5C CV Chlorella Virus CVP-1 CVB-1 CVG-1 CVM-1 CVR-1 Virus Hydra viridis Chlorella HVCV-1 HVCV-2 HVCV-3 VIII. FAMILY: POLYDNAVIRUS GROUP POLYDNAVIRIDAE Genus: Ichnovirus Preferred species: Campoletis sonorensis virus (CsV) Other Member: Glypta sp. Gender : Bracovirus Preferred Species: Cotesia melanoscela Virus (CmV) The genome of the non-mammalian DNA virus can be designed to include one or more genetic elements, such as a promoter of a long terminal repeat of a transposable element, or a retrovirus (eg, Rous Sarcoma Virus). ); an integrative terminal repeat of a virus associated with adeno; and / or an immortalizing cell sequence, such as the EBNA-1 gene of the Epstein Barr Virus (EBV). If desired, the genome of the non-mammalian DNA virus may include a replication origin that functions in a mammalian cell (e.g., an EBV replication origin, or a mammalian replication origin). The non-mammalian DNA virus genome used in the invention can include a polyadenylation signal and an RNA splicing signal placed for proper processing of the exogenous gene product. In addition, the virus can be designed to encode a signal sequence for an appropriate address of the gene product. Where cell-type specific expression of the exogenous gene is desired, the genome of the virus can include a cell-type-specific promoter, such as a liver cell-specific promoter. For example, the liver cell-specific promoter may include a promoter from a gene encoding albumin, α-1-antitrypsin, pyruvate kinase, phospholene pyruvate carboxykinase, transferrin, transthyretin, α-fetoprotein, α-fibrinogen, or β-fibrinogen. Alternatively, a hepatitis B promoter may be used. If desired, a hepatitis B enhancer may be used in conjunction with a hepatitis B promoter. Preferably an albumin promoter is used. An α-fetoprotein promoter is particularly useful for driving the expression of an exogenous gene, when the invention is used to express a gene for the treatment of a hepatocellular carcinoma. Other promoters specific to the preferred liver include promoters of the genes encoding the low density lipoprotein receptor, a2-macroglobulin, atl-antichymotrypsin, a2-HS-glycoprotein, haptoglobin, ceruloplasmin, plasminogen, complement proteins (Clq, Clr, C2 , C3, C4, C5, C6, C8, C9, Complement factor I and Factor H), complement activator C3, β-lipoprotein, and α-acid glycoprotein. Essentially any mammalian cell can be used in the methods of the invention; preferably, the mammalian cell is a human cell. The cell can be a primary cell, or it can be a cell of an established cell line. If desired, the virus can be introduced into a primary cell approximately 24 hours after coating the primary cell to maximize the efficiency of the infection. Preferably, the mammalian cell is a hepatocyte, such as a HepG2 cell, or a primary hepatocyte; a cell of kidney 293 cell line; or a PC12 cell (for example, a differentiated PC12 cell induced by nerve growth factor). Other preferred mammalian cells are those that have an asialoglycoprotein receptor. Additional preferred mammalian cells include NIH3T3 cells, HeLa cells, Cos7 cells, and C2C12 cells. The virus can be introduced into the cell in vi tro 6 in vivo. Where the virus is introduced into an in vi tro cell, the cell can consequently be introduced into a mammal (e.g., portal vein or spleen), if desired. In accordance with the foregoing, the expression of the exogenous gene can be performed allowing the cell to live or cool in vi tro, in vivo or in vi tro and in vivo, in sequence. In a similar manner, where the invention is used to express an exogenous gene in more than one cell, a combination of in vi tro and in vivo methods can be employed to introduce the gene into more than one mammalian cell. If desired, the virus can be introduced into the cell by administering the virus to a mammal carrying the cell. For example, the virus can be administered intravenously or intraperitoneally to that mammal. If desired, a slow-release device, such as an implantable pump, can be used to facilitate delivery of the virus to a cell. Where the virus is administered to a mammal carrying the cell into which the virus is to be introduced, the cell can be engineered by modulating the amount of the virus administered to the mammal, and controlling the method of application. For example, intravascular administration of the virus to the portal phena or the hepatic artery may be employed to facilitate the targeting of the virus to a liver cell. In another method, the virus can be administered to a cell or organ of a donor individual, before transplantation of the cell or organ to a recipient. Where the cell is allowed to live under in vi tro conditions, conventional tissue culture conditions and methods can be employed. In a preferred method, the cell is allowed to live on a substrate containing collagen, such as Type I collagen, or rat tail collagen, or a matrix containing laminin. Implantable versions of these substrates are also suitable for use in the invention (see, for example, Hubbell et al., 1995, Bio / Technology 13: 565-576, and Langer and Vacanti, 1993, Science 260: 920-925). As an alternative to, or in addition to, allowing the cell to live under in vi tro conditions, the cell can be allowed to live under conditions in vivo (eg, in a human being). A variety of exogenous genes can be used to encode gene products, such as proteins, anti-sense nucleic acids (e.g., RNAs), or catalytic RNAs. If desired, the genetic product (e.g., protein or RNA) can be purified from the cell. Accordingly, the invention can be used in the manufacture of a wide variety of proteins that are useful in the fields of biology and medicine. The invention can also be used to treat a genetic deficiency disorder, and genes particularly suitable for expression include those genes that are expressed in normal cells of the cell type to be infected, but which are expressed at less than the level normal in the particular cell that is going to be infected. Particularly useful gene products include carbamoyl I synthetase, ornithine transcarbamylase; argininosuccinate synthetase, argininosuccinate lyase, and arginase. Other desirable genetic products include fumaryl acetoacetate hydrolase, phenylalanine hydroxylase, alpha-1-antitrypsin, glucose-6-phosphatase, low density lipoprotein receptor, and forfobilinogen-deaminase, factor VIII, factor IX, beta-synthase cystathion, branched-chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl carboxylase CoA, methylmalonyl-CoA mutase, glutaryl dehydrogenase CoA, insulin, β-glucosidase, pyruvate carboxylase, hepatic phosphorylase, phosphorylase kinase , glycine decarboxylase (also referred to as protein P), protein H, protein T, Menkes disease protein, and the product of the Wilson disease gene PWD. Other examples of genes desirable for expression include those encoding tumor suppressors (e.g., p53), insulin, or CFTR (e.g., for the treatment of cystic fibrosis). The therapeutic utility of the invention is not limited to correcting deficiencies in gene expression. Where the invention is used to express an anti-sense RNA, the preferred anti-sense RNA is complementary to a nucleic acid (e.g., mRNA) of a pathogen of the mammalian cell (e.g., a virus, a bacterium). or a mushroom). For example, the invention can be used in a method for the treatment of a hepatitis infection, by the expression of an anti-sense RNA that hybridizes to an mRNA of a genetic product of essential hepatitis virus (e.g., an mRNA) of polymerase). Other preferred anti-sense RNAs include those that are complementary to a gene that occurs naturally in the cell, but whose genes are expressed at an undesirably high level. For example, an anti-sense RNA can be designed to inhibit the expression of an oncogene in a mammalian cell. In a similar manner, the virus can be used to express a catalytic RNA (ie, a ribozyme) that inhibits the expression of an objective gene in the cell, by hydrolyzing an mRNA of the target gene product. Anti-sense RNAs and catalytic RNAs can be designed using conventional criteria. A "non-mammalian" DNA virus means a virus that has a DNA genome (instead of RNA), and that is naturally unable to replicate in an invertebrate, and specifically in a mammalian cell. Insect viruses are included (eg, baculovirus, bird virus, plant virus, and fungal viruses). Viruses that naturally replicate in prokaryotes are excluded. Examples of viruses that are useful in the practice of the invention are listed in Table 1. An "insect" DNA virus means a virus that has a DNA genome, and that is naturally capable of replicating in a cell. insect (for example, Baculoviridae, Iridoviri-dae, Poxviridae, Polydnaviridae, Densoviridae, Caulimoviridae and Phycodnaviridae). "Positioned for expression" means that the DNA molecule that includes the exogenous gene is placed adjacent to a DNA sequence that directs transcription and, if desired, the translation of DNA and RNA (ie, facilitates the production of the exogenous genetic product or an RNA molecule). "Promoter" means a minimum sequence sufficient to direct the transcription. Also useful in the invention are promoters that are sufficient to make it possible to control promoter-dependent gene expression for specificity of the cell type, specificity of the cell stage, or tissue specificity (eg, promoter-specific promoters). liver), and those promoters that are inducible by signals or external agents; these elements can be located in the 5 'or 3' regions of the native gene. "Operably linked" means that a gene and a regulatory sequence (eg, a promoter) are connected in such a way as to allow genetic expression when the appropriate molecules (eg, transcriptional activator proteins) are fixed to the regulatory sequences. "Exogenous" promoter or gene means any gene or promoter that is not normally part of the genome of the non-mammalian DNA virus (e.g., baculovirus). These genes include those genes that are normally present in the mammalian cell that is going to become infected; genes are also included that are not normally present in the mammalian cell to be infected (e.g., related and unrelated genes of other cells or species). "Immortalizing cell sequence" means a nucleic acid which, when present in a cell, is capable of transforming the cell for a prolonged inhibition of senescence. They include SV40 T antigen, c-myc, telomerase, and E1A. The invention is useful for expressing an exogenous gene in a mammalian cell (e.g., a HepG2 cell). This method can be used in the manufacture of proteins that are to be purified, such as proteins that are used pharmaceutically (eg, insulin). The invention can also be used therapeutically. For example, the invention can be used to express in a patient a gene encoding a protein that corrects a deficiency in gene expression. In alternative therapy methods, the invention can be used to express any protein, anti-sense RNA, or catalytic RNA in a cell. The non-mammalian viral expression system of the invention offers several advantages. The invention allows de novo expression of an exogenous gene; therefore, the detection of the exogenous protein (eg, β-galactosi-dasa) in an infected cell represents the protein that was actually synthesized in the infected cell, as opposed to a protein that is carried along with the virus in a manner aberrant. The non-mammalian viruses used in the invention are not normally pathogenic to humans; therefore, concerns regarding the safe handling of these viruses are minimized. In a similar manner, because the majority of naturally occurring viral promoters are normally not active in a mammalian cell, the production of undesired viral proteins is inhibited. For example, experiments based on polymerase chain reaction indicate that some viral late genes are not expressed. In accordance with the foregoing, in contrast to some gene therapy methods based on mammalian virus, the non-mammalian virus-based methods of the invention should not elicit a host immune response to the viral proteins. In addition, non-mammalian viruses can be propagated with cultured cells in a serum-free medium, eliminating the risk of adventitious infectious agents present in the serum contaminating the virus preparation. In addition, the use of a serum-free medium eliminates a significant expense faced by users of mammalian viruses. Certain non-mammalian viruses, such as baculoviruses, can be cultured to a high titration (i.e., 108 pfu / ml). In general, the virus genomes are large (for example, the baculovirus genome is 130 kbp); consequently, the viruses used in the invention can accept large exogenous DNA molecules. In certain embodiments, the invention employs a virus whose genome is designed to contain an exogenous replication origin (e.g., the EBV oriP). The presence of these sequences on the genome of the virus allows the episomal replication of the virus, increasing the persistence in the cell. Where the invention is used in the manufacture of proteins to be purified from the cell, the invention offers the advantage that it employs a mammalian expression system. In accordance with the foregoing, appropriate processing and modification after translation (eg, glycosylation) of the gene product can be expected. Other characteristics and advantages of the invention will become clearer from the following description of the preferred embodiments thereof, and from the claims. Detailed Description First the drawings will be described: Drawings Figure 1 is a schematic representation of the Z4 transfer plasmid of AcMNPV RSV-lacZ. Figure 2 is a schematic representation of the Z5 transfer plasmid of AcMNPV RSV-lacZ occluded. Figure 3 is a schematic representation of the episomal transfer plasmid Z-EBV # 1, a chimera of Baculovirus and Epstein Barr Virus sequences. A virus produced with this transfer plasmid is capable of replicating in a cell. Figure 4A is a schematic representation of a transfer plasmid allowing the separation of a genetic cassette. Figure 4B is a schematic representation of the genetic cassette separated by the transfer plasmid of Figure 4A. The separation of the genetic cassette is mediated by endogenous recombination. This strategy allows the persistence of an exogenous gene in the absence of viral sequences. Figure 5 is a schematic representation of the transfer plasmid, pBV-AVneo, a chimera of baculovirus sequences and adeno-associated virus. This plasmid is capable of integrating into the genome of the infected cell. Figures 6A-6D are photographs of cells that were stained with X-gal one day after infection, with an AcMNPV virus containing an RSV-lacZ cassette. Cells expressing the lacZ gene stain dark with X-gal. Figure 6A is a photograph of a typical field of HepG2 cells infected at a multiplicity of infections of 15. Figure 6B is a photograph of a typical field of infected HepG2 cells at a multiplicity of infections of 125; more than 25 percent of the cells were stained. Figure 6C is a typical field of Sk-Hep-1 cells infected at a multiplicity of infections of 125, which does not show positively stained cells. Figure 6D is a less typical field of Sk-Hep-1 cells infected at a multiplicity of infections of 125, which shows a positively stained cell. Bar = 55 microns. Figure 7 is a photograph of cells obtained following baculovirus-mediated gene transfer in primary cultures of rat hepatocytes. More than 70 percent of the cells were stained blue. Figure 8 is a graph showing the dose dependence of baculovirus-mediated gene transfer. Here, 106 HepG2 cells were seeded in 60 millimeter petri dishes, and one day after the cells were exposed to the indicated dose of an AcMNPV virus containing an RSV-lacZ cassette (viral titre = 1.4 x 109 pfu / ml). One day after infection, the cells were harvested, and extracts were prepared and assayed for the activity of the β-galactosidase enzyme. The activity of the extract is expressed in units of ß-galacto-sidase activity as defined above (Norton and Coffin, 1985, Mol Cell. Biol. 5: 281-290). The activity of the enzyme was normalized for the protein content of each extract. Each point is the average of three independent tests, with the error bars representing the standard deviation. Figure 9 is a graphic representation of the results obtained over time of baculovirus-mediated expression. HepG2 cells were infected with the AcMNPV virus containing an RSV-lacZ cassette (multiplicity of infection = 15) at time zero. After one hour, the medium containing the virus was removed and replaced with fresh medium. The infected cells were harvested at the indicated time points, and assayed for the β-galactosidase activity as described above. Each plotted point is expressed as the average of three independent tests, with the error bars representing the standard deviation. Virus expression peaked at 12 to 24 hours after infection, and then declined when normalized to total cellular protein. I. Genetic Manipulation of Viruses In contrast to conventional genetic expression methods, the invention relates to modification. of non-mammalian DNA viruses, which do not naturally infect or replicate in mammalian cells. Accordingly, the invention is based on the addition of new properties to a non-mammalian DNA virus, which allow it to deliver a gene to a mammalian cell, in contrast to conventional gene therapy vectors that are based on the principle of removing functions of the virus. In the present method, the viral particle serves as a 'shell' for delivery of the DNA to the mammalian cell. Viral DNA is designed to contain transcriptional control signals that are active in a mammalian cell, to allow expression of the gene of interest. Conventional recombinant DNA techniques can be used to insert these sequences. Because the non-mammalian DNA viruses used in the invention are not capable of replicating in mammalian cells, it is not necessary to suppress the essential viral functions to render them defective. Preferably, the genome of the virus used in the invention is normally transported to the nucleus in its natural host species, because the nuclear localization signals function in a similar manner in invertebrate cells and in mammalian cells. Preferably, the capsid or viral envelope contains a ligand that binds to the mammalian cells to facilitate entry. Viruses propagated in invertebrate species (eg, insects) typically do not end glycoproteins with sialic acid and therefore, the viral ligand is often an asialoglycoprotein that binds to mammalian lectins (eg, the hepatic asialoglucroprohein receptor), facilitating entry into mammalian cells. Alternatively, the viral particle can be modified by conventional methods (e.g., pseudotyping with VSV-G coat protein) to allow binding and entry into the mammalian cell. For example, the non-mammalian virus may contain a virion protein (e.g., the gp64 glycoprotein of AcMNPV), which facilitates fusion of the viral coat with the membrane of a mammalian cell, thereby allowing the entry of the viral particle in the cytosol. In addition, it is preferred that the virus replicate naturally in a eukaryotic species. Examples of viruses that can be designed to express the exogenous gene in the invention are listed in Table 1. Established methods for manipulating recombinant viruses can be incorporated into these new methods to express an exogenous gene in a cell. mammal. For example, the viral genes of the virus can be suppressed, and they can be delivered in trans lines by means of packaging. The suppression of these genes may be desired in order to: (1) suppress the expression of viral gene products that may elicit an immune response, (2) provide additional space for the viral vector, or (3) provide additional levels. of security in the maintenance of the virus in a cell. Because the majority of non-mammalian virus promoters are not active in mammalian cells, the exogenous gene must be operably linked to a promoter that is capable of directing gene expression in a mammalian cell. Examples of suitable promoters include the RSV LTR, the SV40 early promoter, the CMV IE promoter, the adenovirus major late promoter, and the Hepatitis B promoter. In addition, promoters that are cell type specific can be used, specific to the stage or specific to the tissue. For example, several liver-specific promoters, such as the albumin promoter / enhancer have been described (see, eg, Shen et al., 1989, DNA 8: 101-108.; Tan et al., 1991, Dev. Biol. 146: 24-37; McGrane et al., 1992, TIBS 17: 40-44; Jones et al., J. Biol. Chem. 265: 14684-14690; and Shimada et al., 1991, FEBS Letters 279: 198-200). Where the invention is used to treat a hepatocellular carcinoma, an α-fetoprotein promoter is particularly useful. This promoter is normally active only in the fetal tissue; however, it is also active in tumor cells of the liver (Huber et al., 1991, Proc. Nati, Acad. Sci. 88: 8039-8043). In accordance with the foregoing, an α-fetoprotein promoter can be used to direct the expression of a liver cancer therapet to liver tumor cells. If desired, the genome of the virus can be designed to carry a replication origin, in order to facilitate the persistence of the exogenous gene in the mammalian cell. The replication origins derived from mammalian cells have already been identified (Burhans et al., 1994, Science 263: 639-640). Other replication origins, such as the Epstein-Barr virus oriP, can also facilitate the maintenance of the expression of the presence of appropriate trans action factors, such as EBNA-1. If desired, the virus genome can be designed to express more than one exogenous gene (for example, the virus can be designed to express OTC and AS). Now follow the descriptions of several viruses that can be used in the invention. These examples are provided for illustrative purposes, and are not intended to limit the scope of the invention. Construction of Transfer Plasmid Z4: Genetic manipulation of a baculovirus can be performed for use in the invention, with commonly known recombination techniques originally developed to express proteins in baculovirus (see, for example, O'Reilly et al., 1992, in : Baculovirus expression vectors, WH Freeman, New York). In this example, an AcMNPV was constructed by interrupting the virus polyhedron gene with a cassette that directs the expression of a reporter gene. The reporter gene cassette included DNA sequences corresponding to the Rous Sarcoma Virus (RSV) promoter operably linked to the lacZ gene of E. coli (Figure 1). The reporter gene cassette also included sequences coding for RNA splicing of Simian Virus 40 (SV40), and polyadenylation signals. The AcMNPV transfer plasmid RSV-lacZ used in several examples stipulated below, is called Z4, and was constructed as follows. A 847 base pair fragment was separated from pRSVPL9, including the SV40 RNA splicing signal and the polyadenylation signal, using BglII and BamHI. The plasmid pRSVPL9 was derived from pRSVglobin (Gorman et al., Science 221: 551-553) by digestion of pRSVglobin with BglII, the addition of a HindIII linker, and then the dissociation of the DNA with HindIII. A double-stranded polylinker made by the hybridization of the 5 'oligonucleotides AGCTGTCGACTCGAGGTACCAGATCTCTAGA3' (SEQ ID NO: 1) and 5 'AGCTTCTAGAGATCTGGTACCTCGAGTCGAC3' (SEQ ID NO: 2) was ligated to the 4240 base pair fragment that had the RSV promoter and the splice and polyadenylation signals of SV40. The resulting plasmid has the polylinker in place of the globin sequences. The SV40 sequence of pRSVPL9 was cloned into the BamH1 site of pVL1392 (Invitrogen and Pharmingen) using standard techniques. The resulting intermediate plasmid was called pVL / SV40. An RSV-lacZ cassette was separated from pRSVlacZII (Lin et al., 1991, Biotechniques 11: 344-348, and 350-351) with BglII and Spel, and inserted into the BglII and Xbal sites of pVL / SV40. The RSV-lacZ AcMNPV virus, called Z4, was prepared by homologous recombination of the Z4 transfer plasmid with linearized AcMNPV DNA. The AcMNPV virus used to prepare this DNA was AcV-EPA (Hartig et al., 1992, J. Virol. Methods 38: 61-70). Construction of Transfer Plasmid Z5: Certain non-mammalian viruses (e.g., baculovirus) can be occluded in a protein inclusion body, or they can exist in a form with plasma membrane sprouts. Where an occluded virus is used in the invention, the virus can be first released from the protein inclusion body, if desired. Conventional methods employing alkali to release the virus can be employed (O'Reilly et al., 1992, in: Baculovirus expression vectors, W.H. Freeman, New York). An alkali-released baculovirus, occluded, can be recovered by a cell more easily than non-occluded virus with outbreaks (Volkman and Goldsmith, 1983, Appl. And Environ.Microbiol 45: 1085-1093). To construct the Z5 transfer plasmid (Figure 2), to use an occluded virus in the invention, the RSV-lacZ cassette was separated from the transfer plasmid Z4, using BglII and BamHI, and then inserted into the BglII site of pAcUWl ( Weyer et al., 1990, J. Gen. Virol. 71: 1525-1534). Construction of Transfer Plasmid Z-EBV # 1: The non-mammalian DNA viruses used in the invention can be designed to allow episomal replication of the virus in the mammalian cell. This virus would persist longer, thus optimizing the methods for the long-term expression of an exogenous gene in a cell. An example of this replication virus is Z-EBV # 1 (Figure 3), which was constructed as follows. EBV oriP and EBNA-1 region was separated from pREP9 (Invitrogen) using EcoRI and Xbal, and then inserted into the baculoviral transfer plasmid pBacPAK9 (Clontech) at its EcoRI and Xbal sites, yielding pEBVBP9. The RSV-lacZ cassette was then separated from the transfer plasmid Z4 with BglII and BamHI, and then inserted into the BamH1 site of pEBVBP9 to give the plasmid pZ-EBV # 1. Construction of Z41oxP: The Z4loxP viral genome is a substrate for recombination with the bacteriophage Pl recombinase. This virus can be used to insert genetic cassettes carrying a loxP site into the virus, or using conventional procedures (Patel et al., 1992, Nucí Acids Res. 20: 97-104). A variation of these insertion systems can be designed in such a way that the viral sequences are separated from the remaining gene expression sequences. For example, a self-separating transfer plasmid (Figure 4) can be constructed to express an exogenous gene in a mammalian cell. This plasmid contains loxP sequences that facilitate the separation of the baculoviral sequences. The Z4loxP transfer plasmid was constructed by inserting a synthetic loxP site into the Z4 transfer plasmid. Two loxP oligonucleotides were synthesized, and annealed with each other. The oligonucleotides were: 5 'GATCTGACCTAATAACTTCGTA TAGCATACAT-TATACGAAGTTATATTAAGG3' (SEQ ID NO: 3) and 5 'GATCCCTTAATATAACTTCGTA-TAATGTATGCTATACGAAGTTA TTAGGTCA3' (SEQ ID NO: 4). The oligonucleotides were annealed by heating at 80 ° C in the presence of 0.25 M NaCl, and then the mixture was allowed to cool slowly to room temperature, before being used in the ligation reactions. The tempered oligonucleotides were then ligated with the transfer plasmid Z4 which had been digested with BglII. The ligations and the analysis of the resulting clones were carried out with conventional cloning techniques. Then the recombinant Z4loxP baculovirus was generated with conventional methods for recombination in the linear baculoviral DNA. Construction of pBV-AVneo, an AAV Chimera Transfer Plasmid: A baculovirus genome capable of integrating into a chromosome of the host cell can also be used in the invention. This integrated virus can persist in the cell longer than a non-integrated virus. In accordance with the above, genetic expression methods involving these viruses can obviate the need for repeated administration of the virus to the cell, thus decreasing the possibility of provoking an immune response to the virus. The pBV-AVneo transfer plasmid (Figure 5) includes the integrative terminal repeats of a Adeno-associated virus (AAV). This transfer plasmid was constructed by separating the BglII-BamHI fragment from pFasV.neo, and inserting the fragment in the BamHl site of pAVgal instead of the lacZ gene. Plasmid pAVgal was constructed by replacing the rev and cap coding sequences of AAV with a CMV promoter and a lacZ gene. The resulting intermediate fragment, called pAV.neo, was digested with Pvul, and then the large Pvul fragment was inserted into the PacJ site of pBacPAK9. If desired, a suitable promoter can be inserted operably linked to a AAV rep gene in this construct (eg, between the AAV ITR and the polyhedron promoter) to facilitate separation and recombination in the genome. Examples of the rep genes that can be inserted in this construct include rep40, rep52, rep68 and rep78. Virus spreading: Conventional methods can be used to propagate the viruses used in the invention (see, for example, Burleson et al., 1992, Virology: A Laboratory Manual, Academic Press, Inc., San Diego, CA and Mahy, ed. , 1985, Virology: A Practical Aprroach, IRL Press, Oxford, UK). For example, for the baculoviruses used in the experiments described below, the virus was plaque purified, and amplified according to conventional procedures (see, for example, O'Reilly et al., Infra and Summers and Smith, 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experiment Station Bulletin No. 1555, College Station, Texas). AcMNPV and Sf21 cells were propagated by rotary culture in a Hinks TNM-FH medium (JRH Biosciences) containing 10 percent fetal bovine serum (FBS) and 0.1% PLURONIC F-dd1. The amplified virus can be concentrated by ultracentrifugation in a SW28 rotor (24,000 rpm, 75 minutes) with a 27 percent (w / v) sucrose cushion in 5 mM NaCl, and 10 mM Tris, pH 7.5, and EDTA 10 mM. The viral granule is then resuspended in phosphate-regulated serum (PBS), and sterilized by passing it through a 0.45 micron filter (Nalgene). If desired, the virus can be resuspended by sonification in a cup heater. The AcMNPV was titrated by plaque assay on Sf21 insect cells. II. Expression of an Ex Gene in a Mammalian Cell Almost all mammalian cells are potential targets of non-mammalian viruses, and any cultured cell can be tested quickly. Cell Culture: Conventional tissue culture methods can be used to culture mammalian cells that are to be infected (Freshney, 1987, Culture of Animal Cells; A Manual of Basic Techniques, 2nd edition, Alan R. Liss, Inc. New York, NY). In the following example, the ability of baculovirus Z4 to infect a variety of cells was tested. HepG2, Sk-Hep-1, NIH3T3, HeLa, CHO / dhfr ", 293, COS, Ramos, Jurkat, HL60, K-562, C2C12 myoblasts, C2C12 myotubes, and differentiated growth factor PC12 cells were included. The cells were cultured as follows: HepG2 and Sk-Hep-1 cells were cultured in minimal essential medium modified by Eagle (EMEM) containing 10 percent fetal bovine serum, NIH3T3, HeLa, 293, and COS cells were grown in the cells. DMEM containing 10 percent fetal bovine serum CHO / dhfr cells were cultured in MEM alpha containing 10 percent fetal bovine serum Ramos, Jurkat, HL60, and K-562 cells were cultured in RPMI 1640 medium containing fetal bovine serum. 10 percent HL60 cells were induced to differentiate by culture in the same medium containing 0.5 percent dimethyl sulfoxide and 1 μM retinoic acid (Sigma). C2C12 myoblasts were propagated in DMEM containing 20 percent fetal bovine serum, and they differed in myotubes durant e culture in DMEM containing 10 percent horse serum. PC12 was propagated in DMEM containing 5 percent fetal bovine serum and 10 percent horse serum, and induced to differentiate during culture in DMEM containing 10 percent fetal bovine serum, 5 percent horse serum, and 100 nanogra-mos / milliliter of nerve growth factor. The cells were plated one day before infection with AcMNPV, and the multiplicities of the infection were calculated assuming a duplication in the number of cells during this time. In Viral Cell Infection: Infection of mammalian cells can be performed with an in vi tro virus allowing the virus to adsorb in the cells for 0.1 to 6 hours; preferably, the adsorption proceeds for 1 to 2 hours. In general, a multiplicity and infection of 0.1 to 1,000 is adequate; preferably, the multiplicity of infection is 100 to 500. For relatively refractory cells, a multiplicity of infection of 100 to 1,000 is preferable. In general, a titration of 10 to 200 pfu / cell is desirable. For the viruses used in the invention, the titration can be determined by conventional methods employing non-mammalian cells to which the virus naturally infects. If desired, the mammalian cell to be infected can be maintained in a collagen-containing matrix (e.g., rat tail collagen Type I). Based on the cell count after culturing and infection of the cells on collagen-coated plates, and comparison with the cells grown in a conventional EHS matrix, I have found that a collagen matrix increases the susceptibility of the cells ( for example, liver cells) to infection by a non-mammalian virus 10 to 100 times, relative to a conventional EHS matrix. There are commercially available plates containing a collagen matrix (eg, BIO-COAT plates 11, Collaborative Research), and rat tail collagen (Sigma Chemical and Collaborative Research) is also commercially available. described below, standard conditions for infection used 2 x 10 6 cells and AcMNPV of RSV-lacZ at a multiplicity of infection of 15. Adherent cell lines were seeded one day before infection.The cells were exposed to the virus in 2 milliliters of medium for 90 minutes, and then the medium containing the virus was removed, and replaced with fresh medium.Fugidae cells were treated with 2 milliliters of medium lacking the viral inoculum.Infection Detection and Genetic Expression: application of a virus to a cell, and the expression of the exogenous gene, can be monitored using conventional techniques., the application of a virus (e.g., AcMNPV) to a cell can be measured by detection of the viral DNA or RNA (e.g., by Southern or Northern blot, slot or spot blot, or site hybridization, with or without amplification by polymerase chain reaction). One skilled in the art of molecular biology can conveniently prepare suitable probes that hybridize to virus nucleic acids, regulatory sequences (e.g., the promoter), or the exogenous gene. Where the invention is used to express an exogenous gene in a cell in vivo, the application of the virus to the cell can be detected by obtaining the cell in a biopsy. For example, where the infection is used to express a gene in a liver cell, a liver biopsy can be performed, and conventional methods can be used to detect the virus in a liver cell. Expression of an exogenous gene in a mammalian cell can also be followed by assay of a cell or fluid (eg, serum) obtained from the mammal for RNA or protein corresponding to the gene. The detection techniques commonly used by molecular biologists (eg, Northern or Western blot, site hybridization, slot or spot blot, polymerase chain reaction amplification, SDS-PAGE, immunoblotting, RIA and ELISA) are they can use to measure the expression of the gene. If desired, a reporter gene (eg, lacZ) can be used to mediate the ability of a particular baculovirus to direct gene expression to certain tissues or cells. Tissue examination may involve: (a) instant freezing of the tissue in ice-cold isopentane with liquid nitrogen; (b) assembly of the fabric on cork using O.C.T and freezing; (c) cutting the tissue on a cryostat in sections of 100 microns; (d) drying of the sections and their treatment with paraformaldehyde; (e) tissue staining with X-gal (0.5 milligrams / milliliter) / ferrocyanide (35 mM) / ferri-cyanide (35 mM) in phosphate-regulated serum; and (f) tissue analysis by the microscope. In the following example, I measured the ability of a baculovirus to infect fourteen different types of mammalian cells. In this example, the baculovirus was the Z4 virus, prepared by homologous recombination of the Z4 transfer plasmid with linearized AcMNPV DNA. The cells tested were HepG2, Sk-Hep-1, NIH3T3, HeLa, CH0 / dhfr ~, 293, COS, Ramos, Jurkat, HL60, K-562, C2C12 myoblasts, C2C12 myotubes and differentiated growth factor PC12 cells, and those cells were cultured and infected as described above. The C2C12 and PC12 cells may have increased their cell number during differentiation and, therefore, may reflect a slightly lower multiplicity of infection. To measure the expression of the reporter gene in the infected cells, colorimetric assays of ß-galactosidase enzyme activity were performed with conventional methods (Norton et al., 1985, Molecular &Cellular Biology 5: 281-290). Other conventional methods could be used to measure β-galactosidase activity instead of the methods employed in this example. Cell extracts were prepared on the first day after infection. The cell monolayers were rinsed three times with phosphate-buffered serum, scraped off the dish, and collected by low speed centrifugation. The cell granules were resuspended in 25 mM Tris, pH 7.4 / 0.1 mM EDTA, and then subjected to three cycles of freezing in liquid nitrogen and thawing in a 37 ° C water bath. The extracts were then rinsed by centrifugation at 14,000 x g for 5 minutes. Standard conditions for assaying ß-galactosidase activity used 0.1 milliliter of cell extract, 0.8 milliliter of regulator PM-2, and 0.1 milliliter of o-nitrophenyl-aD-galactopy-noside (4 milligrams / milliliter) in PM-2 regulator for 10 minutes at 37 ° C (Norton et al., 1985, Mol. &Cell. Biol. : 281-290). The reaction was stopped by the addition of 0.5 milliliters of 1 M sodium carbonate. The amount of hydrolyzed substrate was detected spectrophotometrically at 420 nanometers, and the enzymatic activity of β-galactosidase was calculated with conventional methods (Norton et al. 1985, Mol. &Cell. Biol. 5: 281-290). It was verified that the test was linear with respect to the concentration of the extract and the time. The protein concentrations of the extract were determined using the Coomassie Plus protein assay (Pierce) with bovine serum albumin as standard, and the level of β-galactosidase activity was expressed as units of β-galactosidase activity per milligram of protein. If desired, other conventional protein assays can be employed. For the istochemical staining of β-galactosidase activity, cells were fixed in formaldehyde at 2 percent (w / v) -for 0.2 percent formaldehyde (volume / volume) in serum regulated with phosphate, for 5 minutes. After several rinses with phosphate-buffered serum, the cells were stained by adding 0.5 milligrams / milliliter of X-gal (BRL) in phosphate-buffered serum for 2 to 4 hours at 37 ° C. Of the 19 mammalian cell lines examined, three of the cell lines (HepG2, 293 and PC12) showed a statistically significant higher β-galactosidase agility (P <0.05, Student's t-Test) in extracts after the exposure to the virus (Table 2). The human liver tumor line HepG2 exposed to RSV-lacZ baculovirus expressed more than 80-fold higher levels of β-galactosidase than fictitiously infected controls. The human embryonic kidney cell line transformed with adenovirus 293 expressed the lacZ reporter gene at a level approximately four times above the background. In addition, PC12 cells, which differentiated into a neuronal genotype with nerve growth factor, exhibited β-galactosidase levels approximately two times higher after infection with RSV-lacZ baculovirus. This difference was statistically significant (P = 0.019). By means of histochemical staining, a more sensitive assay, β-galactosidase activity was detected in 14 of the 19 cell lines exposed to the virus. Therefore, some of the cell lines that did not produce statistically significantly higher levels of β-galactosidase, measured in extracts, could in fact express β-galactosidase at low, but reproducible frequencies, as detected by the spotting procedure with X-gal more sensitive. This frequency could be increased by using higher multiplicities of infection, such that cells that at a low multiplicity of infection appear not to express the gene, stain blue at a higher multiplicity of infection. Examples of cell lines that could be transfected in this way include SK-Hep-1, NIH3T3, HeLa, CHO / dhfr ", 293, Cos and C2C12 cells In addition, the β-galactosidase activity was detected in myoblasts of Primary human muscle that was exposed to the virus This discovery indicates that the baculovirus could mediate the transfer of the gene to both the primary cells and the corresponding established cell line (C2C12), indicating that the expression of the exogenous gene in an established cell line has a predictive value for the results obtained with the primary cells, β-galactosidase activity was also detected in Hep3B cells treated with the virus, the level of expression in these cells was almost equivalent to the level detected with the HepG2 cells. , it was discovered that the activity of β-galactosidase in FT02B cells (rat hepatoma) and in Hepal-6 cells (human hepatoma) exposed to the virus. Β-galactosidase activity was also detected in NIH3T3 cells, which were designed to express the asialoglycoprotein receptor on the cell surface. These cells expressed approximately twice the level of β-galactosidase as normal NIH3T3 cells. This observation suggests that an asialoglycoprotein receptor can be used to increase the susceptibility to virus-mediated gene transfer. In the multiplicity of infection used, the Ramos, Jurkat, HL60 and K-562 cell lines did not express statistically significant levels of β-galactosidase, as revealed by β-galactosidase enzyme assays after infection. Based on the results with other mammalian cell lines, it is expected that β-galactosidase activity would be detected in these apparently refractory cell lines, when a higher dose (i.e., multiplicity of infection) of the virus is used, or a period of longer absorption time. Even when the exposure of the cells to the virus results in the expression of the exogenous gene in a relatively low percentage of cells (in vi tro or in vivo), the invention can be used to identify or confirm the specificity to the cell type or to the cell type. tissue type of the promoter that drives expression of the exogenous gene (eg, a reporter gene, such as a chloramphenicol acetyltransferase gene, an alkaline phosphatase gene, a luciferase gene or a green fluorescent protein gene). Once identified, this promoter can be used in any of the conventional methods of gene expression. In a similar manner, only relatively low expression levels are required to elicit an immune response (i.e., to produce antibodies) in a mammal against the heterologous gene product. Accordingly, the method of gene expression of the invention can be used in the preparation of antibodies against a preferred heterologous antigen, by expression of the antigen in a cell of a mammal. These antibodies can be used, inter alia, to purify the heterologous antigen. The method of gene expression can also be used to elicit an immunoprotective response in a mammal (i.e., it can be used as a vaccine) against a heterologous antigen. In addition, the invention can be used to make a permanent cell line from a cell where the virus mediates the expression of an immortalizing cell sequence (e.g., SV40 T antigen).

TABLE 2 - MEDIATED EXPRESSION BY BACULOVIRUS OF A REVERSE GENE RSV-lacZ IN MAMMALITY CELLULAR LINES. Histochemical staining using X-gal provided a highly sensitive method to detect β-galactosidase expression in cells exposed to modified AcMNPV. When HepG2 cells were exposed to the modified AcMNPV at a multiplicity of infection of 15, approximately 5 to 10 percent of the cells were stained with X-gal (Figure 6A). At a multiplicity of infection (moi) of 125, approximately 25 to 50 percent of the cells were stained (Figure 6B). No adverse effects of exposure to the virus were observed, such as nuclear swelling. These data demonstrate that the modified AcMNPV is highly effective in the genetic transfer to HpeG2 cells when a sufficient dose of the virus is used. When the Sk-Hep-1 line was exposed to the virus at a multiplicity of infection of 15, no stained cells were observed (data not shown). Although the majority of Sk-Hep-1 cells were exposed to the virus at a multiplicity of infection of 125, they were not stained blue (Figure 6C); it was discovered that a few cells were stained dark after treatment with these higher doses of the virus (Figure 6D). These data indicate that cells that appear to be refractory to the virus at a relatively low multiplicity of infection, in fact, can become infected, and can express the exogenous gene, at a higher multiplicity of infection. No stained cells were found in cultures with simulated infection (data not shown). The frequency of cells stained in the Sk-Hep-1 cell line was estimated to be 2,000 to 4,000 times lower than in HepG2 cells after exposure to equivalent doses of the modified virus, as determined by cell count. Therefore, the specificity to the cell type demonstrated by the modified AcMNPV is relative rather than absolute. These data also indicate that, when a mixture of cells is contacted with the virus. { in vi tro or in vivo), the dosage of the virus can be adjusted to target the virus to cells that are infected with a lower multiplicity of infection. Expression of an Exogenous Gene in Primary Cultures of Rat Hepatocytes: A non-mammalian DNA virus can also be used to express an exogenous gene at high levels in primary cultures of rat hepatocytes. In this experiment, freshly prepared rat hepatocytes were coated on dishes coated with rat tail collagen as described previously (Rana et al., 1994, Mol Cell. Biol. 14: 5858-5869). After 24 hours, the cells were fed fresh medium containing RSV-lacZ baculovirus at a multiplicity of infection of approximately 430. After an additional 24 hours, the cells were fixed and stained with X-gal. More than 70 percent of the cells were stained blue, indicating that they recovered and expressed the cassette of RSV-lacZ (Figure 7). The frequency of expression obtained in this example is higher than the frequency reported with conventional viral vectors used in gene therapy (eg, retroviral and Herpes Simplex Virus vectors). The cultures infected in a simulated manner did not contain positively stained cells (data not shown). Other preferred exogenous genes can be used in place of the lacZ gene. In addition, other primary cells can be easily coated and incubated with a non-mammalian cell in place of the primary rat hepatocytes. Response to the Dose of Genetic Transfer Baculovirus mediated: The histochemical data presented above indicate that increasing amounts of β-galactosidase are produced after exposure of mammalian cells to increasing amounts of the virus. To quantify the dose dependence of baculovirus-mediated gene expression, HepG2 cells were exposed to increasing doses of the virus, and tested for the activity of the enzyme β-galactosidase. The amount of enzyme produced was linearly related to the inoculum of the virus used over a wide range of doses (Figure 8). This suggests that the entry of each virus particle occurs independently of the entry of other virus particles. The maximum dose of virus used in this trial was limited by the titration and volume of the viral material, and a plain in the amount of expression was not observed using higher doses of the virus. In accordance with the foregoing, these data indicate that, in the practice of the invention, the level of expression (ie, the percentage of cells in which the endogenous gene is expressed) can be modulated by adjusting the dosage of the virus used. . Transition of the Time of the Genetic Transfer Baculovirus Mediated: HepG2 cells were exposed to the RSV-lacZ virus for 1 hour, after which the cells were harvested at different times, and quantitatively assayed for the β-galactosidase activity. As shown in Figure 9, β-galactosidase activity was detected as early as 6 hours after exposure to the virus, and expression reached its peak 12 to 24 hours after infection. As expected for an episomal DNA molecule, expression from the RSV-lacZ cassette was stopped gradually at a later time (Figure 9 and data not shown). LacZ expression remained detectable by staining with X-gal at 12 days after transfection, in less than 1 in 1,000 cells (data not shown). This expression of LacZ was not the result of viral extension, because culture supernatants taken from HepG2 cells 10 days after infection had titers of 10 pfu / milliliter, determined by plaque assay on Sf21 cells. These data suggest that, where the invention is used in the manufacture of proteins that are purified from HepG2 cells, it may be advisable to isolate the protein from the cell at the same time, not earlier than 6 hours after infection of the cell. . Depending on the half-life of the protein, it may be advisable to isolate the protein shortly after reaching the peak in protein expression (ie, after approximately 24 hours after infection for HepG2 cells). The optimal period of time to maximize the isolation of the manufactured protein can be easily determined for each protein, virus and cell. Expression Presented De novo in Mammalian Cells: To confirm that the reporter gene activity detected in mammalian cells was not simply the result of ß-galactosidase being physically associated with the AcMNPV virions as they entered the mammalian cell. In the mammalian cell, several experiments were carried out demonstrating that the observed expression of the lacZ reporter gene was the result of the de novo synthesis of β-galactosidase. First, the RSV-lacZ virus inoculum was assayed for β-galactosidase activity, and it was found that the level of β-galactosidase activity is less than 10 percent that that expressed after infection of the HepG2 cells. Second, HepG2 cells were infected with the RSV-lacZ virus, and then cultured in the presence of the cycloheximide protein synthesis inhibitor. The inclusion of cycloheximide after infection inhibited the accumulation of β-galactosidase enzyme activity by more than 90 percent (Table 3). Third, HepG2 cells were infected at a multiplicity of equivalent infection with BacPAK6 (Clontech), a baculovirus in which the lacZ gene was under the control of the viral polyhedron promoter rather than the RSV promoter (Table 3). This latter virus expresses extremely high levels of ß-galactosi-dasa activity in insect cells, where the promoter is active (data not shown). In mammalian cells, the viral polyhedron promoter is inactive, and the virus containing this promoter failed to provide enzymatic activity in the mammalian cells (Table 3). In contrast to previous studies of baculovirus interactions with mammalian cells, these data demonstrate that de novo synthesis of lacZ occurs after baculovirus-mediated gene transfer to a mammalian cell.

TABLE 3 - GENETIC TRANSFER MEASURED BY BACULOVIRUS IS PRESENT OF NOVO.

Baculovirus Mediated Gene Transfer is Inhibited by Lysomotropic Agents: To have a perspective on the mechanism by which baculoviruses express an exogenous gene in a mammalian cell, the susceptibility of gene expression to a lysomotropic agent was examined. Like other enveloped viruses, the yolk form of AcMNPV normally enters the cells via endocytosis, followed by fusion triggered by a low pH of the viral envelope with an endosomal membrane, thus allowing escape into the cytoplasm (Blissard and collaborators, 1993, J. Virol. 66: 6829-6835; Blissard et al., 1990, Ann. Rev. of Environment1. 35: 1227-155). To determine if acidification of the endosome was necessary for baculovirus-mediated gene transfer to mammalian cells, HepG2 cells were infected with RSV-lacZ AcMNPV in the presence of chloroquine, a lyomotropic agent. The HepG2 cells were exposed to the AcMNPV virus in a medium containing or lacking inhibitor for 90 minutes, and then the medium containing the virus was removed, and replaced with fresh medium containing or lacking the inhibitors mentioned. One day after infection, the cells were harvested, and the extracts were assayed for β-galactosidase activity and protein content. Each value in the table represents the average of three independent tests, assigning to the amount of β-galactosidase produced by the AcMNPV virus of RSV-lacZ in the absence of inhibitors, a value of 100 percent. The β-galactosidase activity was normalized for the protein content of each extract. When 25 μM chlorine was continuously present during and after exposure of the HepG2 cells to the virus, de novo expression of β-galactosidase was completely prevented (Table 3). This suggests that baculovirus-mediated gene transfer depends on endosomal acidification. When chloroquine was added to the cells 90 minutes after exposure to the virus, only partial inhibition of β-galactosidase expression was observed. Apparently a portion (approximately 22 percent) of the viral particles could proceed through the endosomal pathway during 90 minutes of exposure to the virus. The Baculovirus is Not Affected by the Complement of Guinea pig: The ability of certain retroviruses to infect mammalian cells is inhibited by the complement, which lyses the virion membrane (see, for example, Rother et al., 1995, Human Gene Therapy 6: 429-435). In accordance with the above, for intravascular administration of a virus to a mammal, it is preferred that the virus is not affected by the complement. To determine whether components of the guinea pig complement could inhibit the ability of the Z4 virus to infect cells, 24 microliters of the Z4 virus was diluted in 2 milliliters of restored guinea pig complement (BRL) or, as a control, 2 milliliters. of the restorative solution (6 percent sodium acetate, 2 percent boric acid, and 0.25 percent sodium azide; BRL) in the absence of complement. The mixture was then incubated at 37 ° C for 10 minutes, and then serially diluted (two 100-fold dilutions) in serum-free TNM-FH insect medium. Then a 100 microliter aliquot of each viral material was used to infect a monolayer of Sf9 insect cells, and the monolayer was overlaid with medium containing 1.5 percent low melting point agarose, according to the titration methods of conventional baculoviruses. After 7 days, the viral plaques were visualized by staining with MTT (see, for example, Shanafelt, 1991, Bio / Techniques 11: 330). It was estimated that the plate of cells containing the virus that was exposed to the complement contained 430 plates, representing a viral titre of 4.3xl09 pfu / milliliter. The control dish had approximately 260 plates, representing a viral titre of 2.6xl09 pfu / milliliter. These results indicate that no significant decrease in viral titre was found in the samples exposed to the complement, suggesting that the intravascular administration of the virus will be an effective means to apply the virus in vivo. Analysis of RNA Expression from Viral Promoters in HepG2 Cells: An advantage of using a non-mammalian virus to express an exogenous gene in a mammalian cell is that, due to the lack of appropriate host cell factors, the Non-mammalian viral promoters can not be active in the mammalian cell. To determine if the AcMNPV viral gene is expressed in HepG2 cells, the viral RNA was analyzed. In these experiments, HepG2 cells were infected with the Z4 virus at a multiplicity of infection of about 30. At 18 hours after infection, the cells were harvested, and the total cellular RNA was extracted from the cells. The total cellular RNA was analyzed by Northern blot for the expression of the viral genes. The probe included a Pacl-Sall fragment of 1.7 kbp from pAcUWl (Pharmingen), which contains the late viral gene p74, as well as the very late gene (overexpressed), plO. Total cellular RNA from Sf9 insect cells infected with Z4 was used as a positive control. Although extremely strong signals were detected for plO and p74, for the control insect cells no signal was observed for HepG2 cells infected with Z4 or uninfected control cells. Additional experiments that used reverse transcriptase polymerase chain reaction (RT-PCR), a highly sensitive method, provided further evidence that most viral genes are not transcribed in mammalian HepG2 cells. The analysis by RT-PCR was performed with RNA prepared from HepG2 cells infected with Z4, HepG2 uninfected, or Sf9 infected, at 6 or 24 hours after infection. HepG2 cells were infected at a multiplicity of infection of 10 or 100. At 6 hours after infection, no RT-PCR product was observed from the viral genes p39, ETL, LEF1, IE1 or IE- N, at any dose of virus in HepG2 cells infected with Z4. By contrast, RT-PCR products were rapidly detected in Sf9 cells infected with Z4. At 24 hours after infection, no expression of these genes was detected in infected HepG2 cells at a multiplicity of infection of 10. At 24 hours after infection, no expression of the p39 viral genes was observed, ETL or LEF1 in HepG2 cells infected at a multiplicity of infection of 100. However, in these high doses of virus, low levels of expression were observed from the viral genes IE1 and IE-N. However, the low level of expression detected at a multiplicity of infection of 100 was significantly lower than the level of expression in insect cells. Expression of these genes can result from recognition of the TATA viral picture by mammalian transcription factors (i.e., transcription of immediate early genes by RNA polymerase II (see, eg, Hoopes and Rorhamn, 1991, Proc. Nati, Acad. Sci. 88: 4513-4517.) In contrast to the immediate early genes, late viral genes are transcribed by a virally encoded RNA polymerase that, instead of requiring a TATA box, initiates transcription in a TAAG motif (O'Reilly et al., supra.) According to the above, the expression of viral late genes is naturally blocked in mammalian cells, if desired, the expression of immediate early genes can be blocked. By suppressing these genes, using conventional methods, the series of data stipulated above suggests that a receptor on the surface of the infected mammalian cell can mediate, although it may not necessarily be required for, mammalian cell infection. Candidate cell lines of particular interest include those that express a cell surface asialoglucoprotein receptor (ASGP-R). HepG2 cells differ from human hepatocytes Sk-Hep-1 and mouse fibroblast cells NIH3T3 by the presence of asialoglycoprotein receptor on the cell surface. In these studies, ß-galactosidase was expressed in fewer Sk-Hep-1 cells (Figure 6B) or NIH3T3 cells than in HepG2 cells. The lacZ gene was expressed in HepG2 cells at a frequency estimated to be more than 1,000 times higher than that of the Sk-Hep-1 cells, based on the quantitative counts of cells stained with X-gal. Normal hepatocytes have 100,000 to 500,000 asialoglycoprotein receptors, each receptor internalising up to 200 ligands per day. The asialoglycoprotein receptor can facilitate the entry of the virus into the cell by providing a cell surface receptor for the glycoproteins on the virion. The glycosylation patterns of insect and mammalian cells are different, the carbohydrate moieties lacking on the surface of the virion produced in terminal sialic acid insect cells. These carbohydrate moieties can mediate virion internalization and trafficking. In addition to the asialoglycoprotein receptor, there are other galactose-binding lectins in mammals (see, for example, Jung et al., 1994, J. Biochem. (Tokyo) 116: 547-553) that can mediate recovery of the virus. If desired, the asialoglycoprotein receptor can be expressed on the surface of a cell to be infected with the virus (eg, baculovirus). The genes encoding the asialoglycoprotein receptor have already been cloned (Spiess et al., 1985, J. Biol. Chem. 260: 1979 and Spiess et al., 1985, PNAS 82: 6465), and standard retroviral vectors, virus associated with adeno, or adenovirals, or chemical methods can be used, for the expression of the asialoglycoprotein receptor in the cell that is to be infected by a virus. Other receptors for ligands on the virion, such as receptors for insect carbohydrates, can also be expressed on the surface of the mammalian cell to be infected, to facilitate infection (see, for example, Monsigny et al., 1979, Biol. Cellulaire 33: 289-300). Alternatively, the virion can be modified through chemical elements (see, for example, Neda, et al., 1991, J. Biol. Chem. 266: 14143-14146) or other methods, such as pseudotyping (see, for example, Bruns et al., 1993, PNAS 90: 8033-8037), to make it possible for the virion to bind to other receptors on the mammalian cell. For example, viral coat proteins, such as vesicular stomatitis g virus glycoprotein (VSV-G), or influenza virus hemagglutinin protein, can be used for pseudotyping. Alternatively, fusions between viral coat proteins (e.g., gp64) and a targeting molecule, (e.g., VSV-G or VCAM) can be expressed on the virion. Overexpression of a membrane protein, such as a cell adhesion molecule (e.g., VCAM), in insect packaging cells, may also facilitate targeting the virus to a mammalian cell. In addition, events not mediated by receptor can mediate the recovery of the virus by the cell, leading to the expression of an exogenous gene in the mammalian cell. III. Therapeutic Use of a Non-Mammalian Virus Expressing an Exogenous Gene The discovery that a non-mammalian DNA virus efficiently expressed a lacZ reporter gene in several mammalian cells indicates that a DNA virus can be used that is not mammalian therapeutically to express an exogenous gene in a mammalian cell. For example, the method of the invention can facilitate the expression of an exogenous gene in a cell of a patient for the treatment of a disorder that is caused by a deficiency in gene expression. It is known that numerous disorders are caused by simple gene defects (see Table 4), and many of the genes involved in genetic deficiency disorders have been identified and cloned. Using conventional cloning techniques (see, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley &Sons (1989)), a non-mammalian virus can be designed to express a desired exogenous gene in a cell of a mammal (for example, a human cell).

TABLE 4 - EXAMPLES OF DISORDERS THAT CAN BE TREATED WITH THE INVENTION, AND GENETIC PRODUCTS THAT CAN BE MANUFACTURED WITH THE INVENTION.

The invention can also be used to facilitate the expression of a desired gene in a cell that does not have an obvious deficiency. For example, the invention can be used to express insulin in a hepatocyte of a patient, for the purpose of supplying the patient with insulin in the body. Other examples of proteins that can be expressed in a mammalian cell (e.g., a liver cell) to be applied to the circulation of the mammalian system, include hormones, growth factors, and interferons. The invention can also be used to express a regulatory gene or a gene encoding a transcription factor (eg, a VP16-tet gene repressor fusion) in a cell to control the expression of another gene (eg, genes that they are operably linked to a tet operator sequence, see for example, Gossen and colaboras, 1992, PNAS 89: 5547-5551). If desired, a tumor suppressor gene, such as the gene encoding p53, can be expressed in a cell, in a method for the treatment of cancer. In addition, the invention can be used to treat a hepatocellular carcinoma, by expressing in a cell a therapeutic gene of hepatocellular carcinoma. For example, genes, such as those encoding tumor necrosis factors, thymidine kinases, diphtheria toxin chimeras, and cytosine diamines, can be expressed in a method for the treatment of a hepatocellular carcinoma (see, for example, Vile. and Russell, 1994, Gene Therapy 1: 88-98). In the treatment of a hepatocellular carcinoma, it is particularly desirable to operably link the exogenous gene to an a-fetoprotein promoter, because this promoter is active in cells of hepatocellular carcinomas, but not in normal mature cells. Other useful genetic products include molecules of RNA for use in RNA decoy, anti-sense, or ribozyme-based methods to inhibit gene expression (see, for example, Yu et al., 1994, Gene Therapy 1: 13-26. invention can be used to express a gene, such as cytosine deaminase, which product will alter the activity of a drug or prodrug, such as 5-fluorocytosine, in a cell (see, for example, Harris et al., 1994, Gene Therapy 1 170-175) Methods such as the use of ribozymes, anti-sense RNAs, transdominant repressors, polymerase mutants, or surface or core antigen mutants can be used to suppress hepatitis viruses (e.g. , hepatitis A, B, C or D virus) in a cell Other disorders, such as hemagromatosis familiar with the invention, can also be treated by treatment with the normal version of the affected gene.Preferred genes for expression include those genes what they encode proteins that are expressed in normal mammalian cells (eg, hepatocytes or lung cells).

For example, genes encoding enzymes involved in the urea cycle, such as genes encoding carbamoyl phosphate synthetase (CPS-I), ornithine transcarbamylase (OTC), argininosuccinate synthetase (AS), argininosuccinate lyase ( ASL), and arginase are useful in this method. All these genes have already been cloned (for ornithine transcarbamylase, see Horwich et al., 1984, Science 224: 1068-1074, and Hata et al., 1988, J. Biochem (Tokyo) 103: 302-308; for arginosuccinate synthetase; , see Bock et al., 1983, Nucí Acids Res. 11: 6505, Surh et al., 1988, Nucí Acids Res. 16: 9252, and Dennis et al., 1989, PNAS 86: 7947; for arginosuccinate lyase, see O'Brien et al., 1986, PNAS 83: 7211; for carbamoyl phosphate synthetase, see Adcock et al., 1984 (Extract) Fed. Proc. 43: 1726; for arginase, see Haraguchi et al., PNAS 84: 412) . Subcloning of these genes in a baculovirus can be easily performed with common techniques. The therapeutic effectiveness of the expression of an exogenous gene in a cell can be evaluated by monitoring the patient for known signs or symptoms of a disorder. For example, the depletion of ornithine transcarbonylase deficiency, and carbamoyl phosphate synthetase deficiency, can be detected by monitoring plasma levels of ammonium or orotic acid. In a similar manner, plasma citrulline levels provide an indication of argininosuccinate synthetase deficiency, and argininosuccinate lyase deficiency can be followed by monitoring plasma levels of arginosuccinate. The parameters for evaluating treatment methods are known to those skilled in the art of medicine (see, for example, Maestri et al., 1991, J. Pediatrics, 119: 923-928). The non-mammalian virus (e.g., baculovirus) can be formulated into a pharmaceutical composition by mixing it with a non-toxic pharmaceutically acceptable excipient or carrier (e.g., serum) for administration to a mammal. In the practice of the invention, the virus can be prepared for parenteral administration (eg, for intravenous injection (eg, portal vein)), intra-arterial injection (eg, into the femoral artery or into the artery) hepatic), intraperitoneal injection, intrathecal injection or direct injection into an area (for example, intramuscular injection). In particular, the non-mammalian virus can be prepared in the form of liquid solutions or suspensions. The virus can also be prepared for intranasal or intrabronchial administration, particularly in the form of nasal drops or aerosols. In the practice of the invention, the virus can be used to infect a cell outside the mammal to be treated (e.g., a cell in a donor mammal, or an in vi tro cell), and then the infected cell is administered. to the mammal that is going to be treated. In this method, the cell can be autologous or heterologous to the mammal to be treated.

For example, an autologous hepatocyte obtained in a liver biopsy may be used (see, for example, Grossman et al., 1994, Nature Genetic 6: 335). The cell can then be administered to the patient by injection (for example, into the portal vein). In this method, a volume of hepatocytes tota from about 1 percent to 10 percent of the volume of the entire liver is preferred. Where the invention is used to express an exogenous gene in a liver cell, the liver cell can be applied to the spleen, and the cell subsequently can migrate to the liver in vivo (see, for example, Lu et al. 1995, Hepatology 21: 7752-759). If desired, the virus can be applied to a cell by using conventional techniques to perfuse fluids into organs, cells or tissues (including the use of infusion pumps and syringes). For perfusion, the virus is generally administered in a titre of lxlO6 at lxlO10 pfu / milliliter (preferably lxlO9 to lxlO10 pfu / milliliter) in a volume of 1 to 500 milliliters, for a period of time from 1 minute to 6 hours. The optimal amount of virus or the number of infected cells to be administered to a mammal, and the frequency of administration, depend on factors such as the sensitivity of the methods to detect the expression of the exogenous gene, the concentration of the promoter used, the safety of the disorder that is going to be treated, and the objective cells of the virus. In general, the virus is administered in a multiplicity of infection of about 0.1 to 1,000; preferably, the multiplicity of infection is from about 5 to 100; more preferably, the multiplicity of infection is from about 10 to 50. Use of a non-Mammalian Virus to Express a Exogenous Gene Live: In two experiments, I detected the expression of lacZ in vivo, followed by the administration of a non-mammalian virus to two different animal models. In the first experiment, 0.5 milliliters of Z4 virus (approximately 1.4 x 109 pfu / milliliter) (at a rate of about 1 milliliter per minute) was injected into the portal vein of a single rat. Approximately 72 hours after infection, lacZ expression could be detected in at least one liver cell from cryosections that were examined by conventional histochemical methods. The efficiency of expression can be increased by any, or a combination of, the following procedures: (1) pretreatment of the animal with growth factors; (2) partial hepatectomy, (3) administration of immunosuppressants to suppress any immune response to the virus; (4) use of a titration or higher dose of the virus; (5) Infusion of the virus by surgical perfusion to the liver (for example, in order to limit possible non-specific binding of the virus to red blood cells), - and / or (6) virus sonification to minimize virus accumulation .

In the second experiment, the Z4 virus was injected via the tail vein of 3 female 8-week-old BALB / c mice. In this case, the mice received either: (i) 6x107 pfu of virus in 0.15 milliliters of phosphate-buffered serum (PBS), or (ii) 6x108 pfu of virus in 0.15 milliliters of phosphate-buffered serum, or (iii) 2xl08 pfu of virus in 0.05 milliliters of serum regulated with phosphate. As a control, 150 microliters of phosphate-regulated serum without virus was injected into a mouse. At 24 hours after injection, the animals were sacrificed, and lacZ expression was detected by staining with X-gal tissue. A substantial number of blue cells were detected in the ten cryosections of mouse lung tissue that received 6xl08 pfu of Z4. No blue cells were detected in the lung tissue of the control mice or in the cryosections examined from mice that received lower amounts of the virus. As seen in vi tro, the expression of the exogenous gene depends on the dose; the lower doses of the virus did not produce a detectable expression. No blue cells were detected in sections of the liver tissue of the mice that received the virus. In summary, these data indicate that injection of a non-mammalian DNA virus, which expresses an exogenous gene in a mammal, can result in the production of the exogenous gene product in vivo. Other Modes Non-mammalian viruses other than the Autographa californica viruses described above may be used in the invention; these viruses are mentioned in Table 1. Nuclear polyhedrosis viruses are preferred, such as multiple nucleocapsid viruses (MNPV) or single nucleocapsid viruses (SNPV). In particular, MNPV from Choristoneura fumiferana, MNPV from Mamestra brassicae, nuclear polyhedrosis virus from Buzura suppressaria, MNPV from Orgyia pseudotsugata, SNPV from Bombyx mor i, SNPV from Helio tis zea, and SNPV from Trichoplusia ni. Also included are granulosis (GV) viruses, such as the following viruses, among those that can be used in the invention: GV of Cryptophlebia leucotreta, GV of Plodia interpunctella, GV of Trichoplusia ni, GV of Pieris brassicae, GV of Artogeia rapae, and Cydia pomonella granulosis virus (CpGV). Also, non-occluded baculoviruses (NOB), such as Heliothis zea NOB and Oryctes rhinoceros virus can be used. Other insect viruses (e.g., lepidoptera) and crustaceans can be used in the invention. Other examples of useful viruses include those that have infectious fungi (e.g., Strongwellsea magna) and spiders. Viruses that are similar to baculoviruses have been isolated from myths, Crustacea (eg, Carcinus maenas, Callinectes sapidus, the Yellow-headed Baculovirus of penaeid shrimp, and Baculovirus of the Penaeus monodon type), and Coleoptera. In the invention, the baculovirus Lymantria dispar is also useful. If desired, the virus can be designed to facilitate the targeting of the virus to certain cell types. For example, ligands that bind to cell surface receptors other than the asialoglucopro-tein receptor can be expressed on the surface of the virion. Alternatively, the virus can be chemically modified to target the virus to a particular receptor. If desired, the cell to be infected can be stimulated first to be mitotically active. In the culture, agents, such as chloroform can be used for this effect; in vivo, stimulation of liver cell division can be induced, for example, by partial hepatectomy (see, for example, Wilson et al., 1992, J. Biol. Chem. 267: 11283-11489). Optionally, the virus genome can be designed to carry a herpes simplex virus thymidine kinase gene; this would allow the cells that house the genome of the virus to be annihilated by ganciciclovir. If desired, the virus could be designed in such a way that it is defective in growth in its host cell other than a natural mammal (e.g., insect cell). These cell strains could provide additional security, and can be propagated on a complement packaging line. For example, a defective baculovirus could be made where an immediate early gene, such as IE1, has been deleted. This suppression can be done by directed recombination in yeast or E. coli, and the defective virus can be replicated in insect cells where the gene product IE1 is delivered in trans. If desired, the virus can be treated with neuraminidase to reveal additional terminal galactose residues before infection (see, eg, Morell et al., 1971, J. Biol. Chem. 246: 1461-1467).

LIST OF SEQUENCES (1) GENERAL INFORMATION: Title of the invention: USE OF A NON-MAMMALIAN DNA VIRUS TO EXPRESS AN EXOGENOUS GENE IN A MAMMALIAN CELL Number of sequences: 4 (2) INFORMATION FOR SEQ ID NO: 1 (i) ) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acids (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1 AGCTGTCGAC TCGAGGTACC AGATCTCTAG A 31 (2) INFORMATION FOR SEQ ID NO: 2 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acids (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2 AGCTTCTAGA GATCTGGTAC CTCGAGTCGA C 31 (2) INFORMATION FOR SEQ ID NO: 3 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 52 base pairs (B) TYPE: nucleic acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3 GATCTGACCT AATAACTTCG TATAGCATAC ATTATACGAA GTTATATTAA GG 52 (2) INFORMATION FOR SEQ ID NO: 4 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH : 52 base pairs (B) TYPE: nucleic acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 4 GATCCCTTAA TATAACTTCG TATAATGTAT GCTATACGAA GTTATTAGGT CA 52

Claims (103)

1. CLAIMS 1. A method of expressing an exogenous gene in a mammalian cell, said method comprising: a) introducing into a cell a non-mammalian DNA virus whose genome comprises said exogenous gene; b) allowing said cell to live under conditions such that said exogenous gene is expressed.
2. 2. The method of claim 1, wherein said virus is an invertebrate virus.
3. 3. The method of claim 2, wherein said invertebrate virus is an insect virus.
4. 4. The method of claim 3, wherein said insect virus is selected from the group consisting of Baculoviri-dae, Polydnaviridae, Iridoviridae, Poxiridae, Densoviridae, Caulimoviridae and Phycodnaviridae.
5. 5. The method of claim 4, wherein said virus is a baculovirus.
6. 6. The method of claim 5, wherein said baculovirus is a nuclear polyhedrosis virus. The method of claim 6, wherein said nuclear polyhedrosis virus is a multiple nuclear polyhedrosis virus of Autographa cali fornica. The method of claim 6, wherein said genome lacks a functional polyhedron gene. 9. The method of claim 5, wherein said baculovirus is in the occluded form. 10. The method of claim 5, wherein said baculovirus is in the bud form. The method of claim 1, wherein said genome further comprises a promoter of a long terminal repeat of a transposable element. The method of claim 1, wherein said genome further comprises a promoter of a long terminal repeat of a retrovirus. The method of claim 12, wherein said retrovirus is Rous sarcoma virus. The method of claim 1, wherein said genome further comprises an integrative terminal repeat of an adeno-associated virus. The method of claim 14, wherein said genome further comprises a rep gene of the adeno-associated virus. 16. The method of claim 1, wherein said genome further comprises a cell immortalization sequence. The method of claim 1, wherein said genome further comprises an origin of replication. 18. The method of claim 17, wherein said origin of replication comprises an origin of replication of the Epstein Barr virus. The method of claim 1, wherein said genome further comprises a polyadenylation signal and an RNA splicing signal. The method of claim 1, wherein said genome further comprises a promoter specific to the cell type. The method of claim 20, wherein said promoter specific to the cell type comprises a liver cell-specific promoter. 22. The method of claim 21, wherein said liver cell-specific promoter comprises a promoter selected from the group consisting of hepatitis B promoters, hepatitis A promoters, hepatitis C promoters, albumin promoters, alpha-1 promoters. -antitrypsin, pyruvate kinase promoters, fosfenol pyruvate carboxykinase promoters, transferrin promoters, transthyretin promoters, alpha-fetoprotein promoters, alpha-fibrinogen promoters, and beta-fibrinogen promoters. The method of claim 22, wherein said genome comprises a hepatitis B promoter and further comprises a hepatitis B enhancer. The method of claim 22, wherein said genome comprises an albumin promoter. The method of claim 21, wherein said liver cell specific promoter comprises a promoter of a gene selected from the group consisting of the low density lipoprotein receptor, alpha2-macroglobulin, alfal-antichymotrypsin, alpha2-HS glycoprotein, haptoglobulin, ceruloplasmin, plasminogen, complement proteins, complement activator C3, beta-lipoprotein, and alpha-acid glycoprotein. 26. The method of claim 1, wherein said cell is a hepatocyte. 27. The method of claim 26, where said hepatocyte is a primary hepatocyte. 28. The method of claim 26, wherein said hepatocyte is a HepG2 hepatocyte. 29. The method of claim 1, wherein said cell is a cell of the kidney cell line 293. 30. The method of claim 1, wherein said cell is a PC12 cell. The method of claim 1, wherein said cell is selected from the group consisting of Sk-Hep-1 cells, NIH3T3 cells, HeLa cells, CHO / dhfr "cells, 293 cells, COS cells and C2C12 cells. The method of claim 1, wherein said cell is a primary cell 33. The method of claim 32, wherein said virus is introduced into said primary cell approximately 24 hours after plating said primary cell. claim 32, further comprising introducing said primary cell into a mammal. 35. The method of claim 1, wherein said virus is introduced into said cell in vi tro. 36. The method of claim 1, further comprising allowing said cell to live in a collagen-containing substrate. 37. The method of claim 36, wherein said collagen is type I collagen from rat tail. 38. The method of claim 1, wherein said cell is allowed to live under in vi tro conditions. 39. The method of claim 1, wherein said cell is allowed to live under in vivo conditions. 40. The method of claim 39, wherein said cell is allowed to live in a human being. 41. The method of claim 1, wherein said mammalian cell is a human cell. 42. The method of claim 1, wherein said cell comprises an asialoglycoprotein receptor. 43. The method of claim 1, wherein said virus is introduced into said cell by administering said virus to a mammal comprising said cell. 44. The method of claim 43, wherein said virus is administered intravascularly. 45. The method of claim 43, further comprising targeting said cell by modulating the amount of said virus administered to said mammal. 46. The method of claim 1, wherein said gene encodes a protein. 47. The method of claim 46, further comprising purifying said protein from said cell. 48. The method of claim 46, wherein said protein comprises a protein selected from the group consisting of carbamoyl synthetase I; ornithine transcarbamylase; arginosuccinate synthetase; arginosuccinate lyase; and arginase. 49. The method of claim 46, wherein said protein comprises a protein selected from the group consisting of fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, low density lipoprotein receptor, and porphobilinogen deaminase. , factor VIII, factor IX, cystathion beta-synthase, branched-chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylase, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, Menkes disease protein, the pWD gene product of Wilson's disease, and CFTR. 50. The method of claim 46, wherein said protein comprises insulin. 51. The method of claim 1, wherein said protein comprises p53. 52. The method of claim 1, wherein said gene encodes an anti-sense RNA. 53. The method of claim 52, wherein said anti-sense RNA comprises a sequence that is complementary to a nucleic acid of a hepatitis virus. 54. The use of a non-mammalian DNA virus whose genome comprises an exogenous gene in the preparation of a medicament for treating a gene deficiency disorder in a mammal. 55. The use of claim 54, wherein said virus is an invertebrate virus. 56. The use of claim 55, wherein said invertebrate virus is an insect virus. 57. The use of claim 56, wherein said insect virus is a baculovirus. 58. The use of claim 54, wherein said gene encodes a gene product selected from the group consisting of fumarilacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, low density lipoprotein receptor, and porphobilinogen deaminase. , factor VIII, factor IX, cystathion beta-synthase, branched-chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylase, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, Menkes disease protein, the pWD gene product of Wilson's disease, and CFTR. 59. The use of a non-mammalian DNA virus whose genome comprises a therapeutic gene to carcinoma selected from the group consisting of a tumor necrosis factor, thymidine kinase, diphtheria toxin chimeras, and cytosine diaminase, in the preparation of a medicine to treat hepatocellular carcinoma in a mammal. 60. The use of claim 59, wherein said non-mammalian DNA virus is a baculovirus. 61. The use of claim 59, wherein said therapeutic gene to carcinoma is operably linked to an alpha-fetoprotein promoter. 62. The method of claim 1, wherein said exogenous gene is selected from the group consisting of lacZ genes, chloramphenicol acetyltransferase genes, alkaline phosphatase genes, luciferase genes, and green fluorescent protein genes. 63. A nucleic acid, comprising: a genome of a non-mammalian DNA virus; an exogenous mammalian gene; and an exogenous active promoter in a mammal, wherein said gene is operably linked to said promoter. 64. The nucleic acid of claim 63, wherein said genome is the genome of an insect virus. 65. The nucleic acid of claim 64, wherein said genome is the genome of a baculovirus. 66. The nucleic acid of claim 65, wherein said genome is the genome of a multiple nuclear polyhedrosis virus of Autographa cali fornica. 67. The nucleic acid of claim 63, wherein said mammalian active promoter is selected from the group consisting of mammalian promoters, retro-virus long-terminal repeat promoters, promoters of long-terminal repeats of transposable elements, the promoter early simian virus, the IE cytomegalovirus promoter, and the adenovirus major late promoter. 68. The nucleic acid of claim 67, wherein said promoter is a mammalian promoter. 69. The nucleic acid of claim 63, wherein said promoter is selected from the group consisting of cell-type specific promoters, step-specific promoters, inducible promoters, and tissue-specific promoters. 70. The nucleic acid of claim 69, wherein said promoter is a liver-specific promoter. 71. The nucleic acid of claim 70, wherein said liver-specific promoter is selected from the group consisting of hepatitis B promoters, hepatitis A promoters, hepatitis C promoters, albumin promoters, alpha-1-antitrypsin promoters, pyruvate kinase promoters, fosfenol pyruvate carboxykinase promoters, transferrin promoters, transthyretin promoters, alpha-fetoprotein promoters, alpha-fibrinogen promoters, and beta-fibrinogen promoters. 72. The nucleic acid of claim 70, wherein said liver-specific promoter is selected from the group consisting of low density lipoprotein receptor promoters, alpha2-macroglobulin promoters, alfal-anti-chymotrypsin promoters, alpha2-HS glycoprotein promoters. , haptoglobulin promoters, ceruloplasmin promoters, plasminogen promoters, complement protein promoters, C3 complement activator promoters, beta-lipoprotein promoters, and alpha-glycoprotein acid promoters. 73. The nucleic acid of claim 63, further comprising a mammalian origin of replication. 74. The nucleic acid of claim 63, further comprising an integrative terminal repeat. 75. The nucleic acid of claim 63, wherein said genome lacks a functional polyhedron gene. 76. The nucleic acid of claim 63, wherein said gene is a human gene. 77. The nucleic acid of claim 63, wherein said gene is a therapeutic gene. 78. The nucleic acid of claim 63, wherein said gene encodes a gene product selected from the group consisting of carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarilacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose -6-phosphatase, low density lipoprotein receptor, porphobilinogen deaminase, arginase, factor VIII, factor IX, cystathion beta synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase, and pyruvate carboxylase, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, Menkes disease protein, the pWD gene product of Wilson's disease, factors of growth, interferons, CFTR, tumor suppressors, thymidine kinase of the herpes simplex virus, and transcription factors. 79. A nucleic acid, comprising: a genome of a non-mammalian DNA virus; an exogenous anti-sense RNA gene, the RNA encoded by said gene being complementary to a nucleic acid of a gene that is expressed in a cell at an undesirably high level; and an exogenous active promoter in a mammal, wherein said gene is operably linked to said promoter. 80. The nucleic acid of claim 79, wherein said genome is the genome of an insect virus. 81. The nucleic acid of claim 79, wherein said genome is the genome of a baculovirus. 82. The nucleic acid of claim 79, wherein said promoter is selected from the group consisting of mammalian promoters, retro-virus long-terminal repeat promoters, and long-terminal repeat promoters of transposable elements, the early promoter of the promoter. simian virus 40, the IE cytomegalovirus promoter, and the adenovirus major late promoter. 83. A cell containing a nucleic acid, wherein said nucleic acid comprises: a genome of a non-mammalian DNA virus; an exogenous mammalian gene; and an exogenous active promoter in a mammal, wherein said gene is operably linked to said promoter. 84. The cell of claim 83, wherein said genome is the genome of an insect virus. 85. The cell of claim 85, wherein said genome is the genome of a baculovirus. 86. The cell of claim 83, wherein said promoter is selected from the group consisting of mammalian promoters, promoters of long-terminal retro-virus repeats, and long-terminal repeat promoters of transposable elements, the early promoter of the virus 40 simian, the IE cytomegalovirus promoter, and the adenovirus major late promoter. 87. The cell of claim 83, wherein said cell is a primary cell. 88. The cell of claim 83, wherein said cell is a human cell. 89. The cell of claim 83, wherein said cell is selected from the group consisting of hepatocytes, kidney cells, NIH3T3 cells, HeLa cells, Cos7 cells, C2C12 myotubes, C2C12 myoblasts, CHO / dhfr "cells, lung cells, and PC12 cells 90. The cell of claim 89, wherein said cell is a hepatocyte selected from the group consisting of HepG2 cells, Sk-Hep-1 cells, Hep3B cells, FT02B cells, and Hepa 1-6 cells. The cell of claim 83, wherein said cell is selected from the group consisting of Ramos cells, Jurkat cells, HL60 cells, and K-562 cells 92. The cell of claim 83, wherein said promoter is selected from the group consisting of promoters specific to cell type, tissue-specific promoters, stage-specific promoters, and inducible promoters 93. The cell of claim 83, wherein said promoter is a liver-specific promoter. 94. The cell of claim 83, wherein said gene is a human gene. 95. The cell of claim 83, wherein said gene encodes a gene product selected from the group consisting of carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetoacetate-hydroxylase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose- 6-phosphatase, low density lipoprotein receptor, porphobilinogen deaminase, arginase, factor VIII, factor IX, cystathion beta synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase, and pyruvate carboxylase, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, Menkes disease protein, the pWD gene product of Wilson's disease, growth, interferons, CFTR, tumor suppressors, herpes simplex virus thymidine kinase, and factors of transcription. 96. A nucleic acid, comprising: a genome of a non-mammalian DNA virus; an exogenous therapeutic gene for cancer selected from the group consisting of tumor necrosis factor genes, thymidine kinase genes, chimeric diphtheria toxin genes, and cytosine diaminase genes; and an exogenous active promoter in a mammal, wherein said gene is operably linked to said promoter. 97. The nucleic acid of claim 96, wherein said genome is the genome of an insect virus. 98. The nucleic acid of claim 97, wherein said genome is the genome of a baculovirus. 99. A nucleic acid, comprising: a genome of a non-mammalian DNA virus; an exogenous gene selected from the group consisting of RNA decoy genes and ribozyme genes; and an exogenous active promoter in a mammal. 100. The nucleic acid of claim 99, wherein said genome comprises the genome of a baculovirus. 101. A pharmaceutical composition, comprising: (A) a pharmaceutically acceptable excipient; and (B) a nucleic acid, said nucleic acid comprising: a genome of a non-mammalian DNA virus; an exogenous mammalian gene; and an exogenous active promoter in a mammal, wherein said gene is operably linked to said promoter. 102. The pharmaceutical composition of the claim 101, wherein said genome comprises the genome of an insect virus. 103. The pharmaceutical composition of the claim 102, wherein said genome comprises the genome of a baculovirus.