JPH10511951A - Production and administration of high titer recombinant retrovirus - Google Patents

Abstract

Abstract: A method is provided for obtaining measurable levels of a protein, nucleic acid molecule, or enzymatic product in a human body fluid or cell. This method, a recombinant retrovirus preparations with 10 ⁵ cfu / ml greater than titers in HT1080 cells comprising administering to a human, wherein the recombinant virus preparation, human body fluids or cells Can be directed to the expression of an enzyme that produces a protein, nucleic acid molecule, or enzymatic product, such that measurable levels of the protein, nucleic acid molecule, or enzymatic product can be obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION                Production and administration of high titer recombinant retrovirusDescription   The present invention relates generally to recombinant retroviruses, and more particularly to various applications. High-titer recombinant retrovirus particle preparations suitable forBackground of the Invention   Diseases have been developed by recombinant DNA technology that has been in use since the discovery of DNA in the 1940s, Recognize that the course of life can be affected by the interaction of living organisms with nucleic acids Substantial investigations have been conducted in order to do so. Most recently, genes (eg, retro Virus, adenovirus, vaccinia virus, herpes virus, and Includes viral vectors derived from deno-associated virus (Jolly, Cancer Gene Ther apy 1 (1): 51-64, 1994))) And direct transfer techniques (eg, Lipofectin (Felgner et al., Proc. Natl. A cad. Sci. USA 84: 7413-7417, 1989), direct DNA injection (Acsadi et al., Nature 352: 815-). 818, 1991), microprojectile bombardment (Williams et al., PNAS 8 8: 2726-2730, 1991), several types of liposomes (eg, Wang et al., PNAS 84: 785). 1-7855, 1987) and administration of nucleic acid alone (WO 90/11092)) A wide variety of methods have been described.   Of these technologies, the recombinant retroviral gene delivery method is the most widely used. Have been. This includes, in part: (1) the efficiency of genetic material (vector genome) in cells (2) Active and efficient invasion process into target cell nucleus; (3) Relatively high level (4) Control of vector-target cell binding and tissue expression of gene expression Possibility to target specific cell subtype through differential control; (5) Pre-existing A general lack of host immunity; and (6) the substance obtained using such vectors. Due to technical knowledge and clinical experience.   Briefly, retroviruses replicate twice via an integrated DNA intermediate. It is a body positive strand RNA virus. More specifically, , The retroviral genome is a virus-encoded reverse transcriptase (which (Transported as a protein in each retrovirus) You. The viral DNA is then pseudo-randomly integrated into the host cell genome of the infected cell. And form a "provirus" that is inherited by daughter cells.   Wild-type retroviral genomes (and their proviral copies) (Gag, pol, and env genes). Packages preceding these Signal (Ψ) and two long terminal repeat (LTR) sequences flanking both ends There is. Briefly, the gag gene is an internal structure (nucleocapsid) protein Code. The pol gene is an RNA-dependent DNA polymerase that reverse transcribes the RNA genome. And the env gene encodes the retroviral envelope glycoprotein. To load. The 5 'and 3' LTRs regulate transcription and polyadenylation of retroviral RNA. Includes cis-acting elements required to promote.   Sequence required for reverse transcription of the genome adjacent to the 5 'LTR (tRNA primer binding site) And sequences required for efficient encapsidation of retroviral RNA into particles (Ψ sequence ). Removal of the packaging signal involves encapsidation of genomic RNA (infectious Packaging of retroviral RNA into virions), but also However, the resulting mutants still contain all the proteins encoded in the viral genome. Can be oriented.   Recombinant retroviruses and their various uses are described, for example, in Mann et al. 3: 153, 1983), Cane and Mulligan (Proc. Nat'l. Acad. Sci. USA 81: 6349, 198). 4), Miller et al., Human Gene Therapy 1: 5-14, 1990, U.S. Patent No. 4,405,712; No. 4,861,719; No. 4,980,289, and PCT applications WO 89 / 02,468, WO 89/05, 349, and WO 90 / 02,806. Briefly The foreign gene of interest is in the retrovirus instead of the normal retroviral RNA Can be taken into account. When a retrovirus injects its RNA into cells, The gene is also introduced into the cell, then it is as if it is the retrovirus itself Can be integrated into the host cell DNA. Expression of this foreign gene in the host Results in the expression of the foreign protein by the host cell.   However, one drawback of recombinant retroviruses is that they are primarily Infects only cells, making it difficult to perform efficient direct gene transfer It is. In fact, some scientists say that other, more efficient Transfer methods (eg, direct administration of pure plasmid DNA) have been suggested (Davis et al., Human Gene Therapy 4: 733-740, 1993).   To increase the efficacy of a recombinant retrovirus, Methods that have been suggested to increase force are primarily to replicate in desired target cells. Have been sought to induce and thereby infect cells with retroviruses. This For example, a chemical treatment using 10% carbon tetrachloride in mineral oil (Kaleko et al.) , Human Gene Therapy 2: 27-32, 1991). Alternatively, hepatocyte rush In order to stimulate rapid division and thereby increase the infection of such cells, the liver (Eg, 70% in Rettinger et al., PNAS 91: 1460-1464, 1994; Moscion i et al., suggest that 70% in Surgery 113: 304-311, 1993; Was.   Another further disadvantage of recombinant retroviruses is that primates (eg, humans) Derived serum is known to cause inactivation by the antibody-independent complement lysis method It is that you are. In particular, retroviruses of bird, mouse, cat and monkey origin Are all inactivated and lysed by normal human serum (Welsh et al., Nat. ure 257: 612-614, 1975; Welsh et al., Virology 74: 432-440, 1976; Banapour et al. Virology 152: 268-271, 1986; and Cooper et al.Immunology of the Comple ment System , American Press, Inc., pp. 139-162, 1986). By scientific literature , A replication-competent mouse amphotropic retrovirus injected intravenously into primates Luss will be wiped out within 15 minutes, and this disappearance is caused by primate complement. It has also been reported to be wholly or partially mediated (Cornetta et al., Huma n Gene Therapy 2: 5-14, 1991; Cornetta et al., Human Gene Therapy 1: 15-30, 1 990; Banapour et al., Virology 152: 268-271, 1986).   In order to increase the effects of recombinant retroviruses delivered in vivo, Ming provides recombinant retroviral compositions that can withstand inactivation in human serum I do. In addition, the present invention relates to treatment by routes not previously considered possible. Allow the delivery of substances or palliatives, and by chemical treatment or as in the liver High-titer recombinant retroviruses without the need to induce cell replication by excision of the target organ A virus composition is provided. The present invention provides these and other related advantages. .Summary of the Invention   Briefly, the present invention relates to a high titer composition comprising a recombinant retrovirus. , As well as methods for utilizing these compositions. One station of the present invention In terms of measurable levels of protein, nuclear Methods are provided for obtaining acid molecules, or enzymatic products. This method uses HT1080 10 in cells^Fivea recombinant retrovirus preparation with a titer greater than cfu / ml Administering to a human, wherein the recombinant retroviral preparation comprises Measurable levels of proteins, nucleic acid molecules, or Or a protein, nucleic acid molecule, or enzymatic product such that an enzymatic product can be obtained. Can be directed to the expression of an enzyme that produces In certain embodiments, the titer is 10⁶c fu / ml, 10⁷cfu / ml, 10⁸cfu / ml, 10⁹cfu / ml, 10^Tencfu / ml, or 10¹¹over cfu / ml I can get it.   In another aspect of the invention, a measurable level in a human body fluid or cell. Methods for obtaining a protein, nucleic acid molecule, or enzymatic product of the invention are provided. This Was performed in human serum and HT1080 cells using heat-inactivated serum and HT1080. A recombinant retrovirus preparation having a titer equivalent to that in cells Wherein the recombinant retroviral preparation comprises the human Measurable levels of proteins, nucleic acid molecules, or enzymes in body fluids or cells To produce a protein, nucleic acid molecule, or enzymatic product such that Expression of the enzyme.   As used in the context of the present invention, "measurable levels" of proteins, nucleic acids The molecule, or enzymatic product, can be obtained by any suitable technique (eg, antibody-mediated Sex detection, PCR analysis for the presence of nucleic acid molecules, or visualization of enzymatic products). As used herein, refers to the detection of a level that is statistically significant relative to the background. further, As used in the context of the present invention, an "equivalent" titer is substantially the same in a given assay. And are generally considered to be titers within about three times the range of each other.   In certain embodiments of the invention, the recombinant retrovirus is cerebrospinal fluid, bone marrow, , Joints, arterial endothelial cells, rectum, buccal / sublingual, vaginal, lymphatic system, lung, liver An organ selected from the group consisting of the spleen, spleen, skin, blood, and brain, or a tumor It is administered to a site selected from the group consisting of a tumor and a stromal lumen. In another embodiment Recombinant retrovirus is available intraocular, intranasal, sublingual, oral, topical, intravesical Can be administered intrathecally, topically, intravenously, intraperitoneally, intracranial, intramuscularly, and subcutaneously .   In yet other embodiments of the invention, the protein is a virus (eg, an influenza Enzavirus, RS virus, HPV, HBV, HCV, EBV, HIV, HSV, FeLV, FIV, C Virus, HTLVI, HTLV II, and CMV). other In some embodiments, the protein is a cytokine (eg, IL-1, IL-2, IL-3, IL-3). -4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL- 15, γ-IFN, G-CSF, and GM-CSF) or any of these cytokines Putter.   In another embodiment, the nucleic acid molecule is an antisense sequence, a non-coding non-heterologous sense sequence. Sequence, and ribozyme sequence. In yet another aspect, the protein is toxic It is.   These and other aspects of the invention are described in the following detailed description and accompanying drawings. It will be clear if you refer to Further, certain procedures or compositions (e.g., , Plasmids, etc.) are described below; The entirety of these references is incorporated by reference as each is individually incorporated by reference. Used.BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 is a schematic diagram of p31N2R5 (+).   FIG. 2 is a schematic diagram of pN2R3 (−).   FIG. 3 is a schematic diagram of p31N25Δ (+).   FIG. 4 is a schematic diagram of pN2R3 (+).   FIG. 5 is a schematic diagram of pN2R5 (−).   FIG. 6 is a schematic diagram of p31N25Δ (+).   FIG. 7 is a schematic diagram of pTKΔA.   FIG. 8 is a schematic diagram of pPrTKΔA.   FIG. 9 is a schematic diagram of pTK-1 and pTK-3.   FIG. 10 shows gansik against CT26 cells, CT26 bgal cells, and CT26TK Neo cells. 5 is a bar graph showing the effect of Robil.   FIG. 11 shows tumors in a ganciclovir dose study of mice injected with CT26TK Neo. 4 is a graph showing the effect of capacity over time.   FIG. 12 shows the effect of different dose regimens of ganciclovir on intraperitoneal tumor growth 4 is a series of four photographs of the mouse showing   FIG. 13 shows the effect of different dose regimens of ganciclovir on subcutaneous tumor growth. 4 is a series of four photographs of the mouse shown.   FIG. 14 shows the effect of ganciclovir in CT26 cells on CT26TK Neo cells. This is a graph.   FIG. 15 shows a lyophilized representative recombinant in a formulation buffer containing mannitol. 4 is a graph showing the retention of virus activity upon reconstitution of a retrovirus.   FIG. 16 shows a lyophilized representative recombinant in a formulation buffer containing lactose. Fig. 4 is a graph showing the retention of virus activity upon reconstitution of a retrovirus.   FIG. 17 shows a lyophilized representative recombinant in a formulation buffer containing trehalose. 4 is a graph showing the retention of virus activity upon reconstitution of a retrovirus.   FIGS.18A-18D show lyophilized formulation recombinants stored at −20 ° C. using various sugars. Liquid, non-lyophilized recombinant letto stored at -80 ° C against retrovirus 4 is a representative graph comparing the stability of rovirus.   FIG. 19 shows the results of the reverse transcriptase assay on samples cut from the gel. It is a bar graph shown. Slice 1 is from the bottom of the gel and 8 is from the top.   FIG. 20 shows an 8% to 25% gradient polyacrylamide gel. Lane 1 is S- Lane 2 is the crude supernatant of DA / βgal; Lane 3 is 500 purified DA / βgal; Is the crude supernatant of HIV-IT; lane 4 is the crude supernatant of DAC 6A3; lane 5 is , The crude supernatant of HBc / SA2; and lane 5 is the molecular weight marker.   FIG. 21 is a schematic diagram of pDHF811.   FIGS. 22A-22D show the total lactate (lower and higher seeding cultures). FIGS. 22A and 22B) and one day in low and high seeded cultures, respectively. FIG. 22 is a graph showing the level of lactate production per unit (FIGS. 22C and 22D, respectively) .   FIG. 23 is a bar graph showing the titer of cell line 2X-β-gal under different initial seeding conditions. is there.Detailed description of the invention   Before describing the present invention, the definitions of certain terms used in this specification must be stated. May be useful for understanding the present invention.   "Vector constructs", "retroviral vectors", "recombinant vectors", And "recombinant retroviral vectors" carry a nucleic acid molecule of interest and Refers to a nucleic acid construct capable of directing its expression. Retro virus The vector comprises at least one transcription promoter / enhancer or locus. A locus defining element or other means (another splice) Sing, nuclear RNA transport, messenger post-translational modification, or protein transcription Other elements that control gene expression by post-modification) must be included. Such vector constructs also include a packaging signal, a long terminal repeat (LTR). ), Or portions thereof, as well as the appropriate positives for the retrovirus used. And negative strand primer binding sites (already included in the retroviral vector). If not present). Optionally, recombinant retrovirus vector Also include signals that direct polyadenylation, selectable markers (eg, neo- Mycin resistance, TK, hygromycin resistance, phleomycin resistance, histidinow Resistance, or DHFR), and one or more restriction sites and translation termination sequences obtain. By way of example, such vectors typically include a 5 'LTR, a tRNA binding site. , Packaging signal, origin of second strand DNA synthesis, and 3 ′ LTR or their Including parts.   "Recombinant retrovirus", "retroviral gene delivery vehicle", and "Retroviral vector particles", as utilized in the present invention, Both are retroviruses having one gene of interest. Retroviruses also , A selection marker. Recombinant retroviruses carry their genetic Can reverse transcribe material into DNA and incorporate this genetic material into host cell DNA .   As noted above, the present invention provides a high titer recombinant retrovirus suitable for human administration. Provide a luz preparation. Such preparations have not previously been easy for gene therapy To provide effective gene therapy for various diseases (and by various routes) Provide unexpected results. In addition, the present invention provides Can withstand activation, thereby enabling more efficient gene transfer over long periods of time To provide a recombinant retrovirus. A.Recombinant retroviral vectors, packaging cells, producer cells And recombinant retrovirus preparation   As described above, the present invention has or expresses a selected nucleic acid molecule of interest. A recombinant retrovirus constructed as described above. Simply put, many retro An irs gene delivery vehicle may be utilized within the context of the present invention. These include examples For example, EP 0,415,731; WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Patent No. 5,219,740; WO 9311230; WO 9310218; Vile and Hart, Cancer Re. s. 53: 3860-3864, 1993; Vile and Hart, Cancer Res. 53: 962-967, 1993; Ra m et al., Cancer Res. 53: 83-88, 1993; Takamiya et al., J. Neurosci. Res. 33: 493-50 3, 1992; Baba et al., J. Neurosurg. 79: 729-735, 1993 (U.S. Pat.No. 4,777,127; GB 2,200,651, EP 0,345,242, and vehicles described in WO 91/02805). I will. Particularly preferred recombinant retroviruses are those described in WO 91/02805. Is included.   The retroviral gene delivery vehicles of the present invention are compatible with a wide variety of retroviruses. Viruses (eg, retroviruses B, C, and D, and spumaviruses and And RNA lentiviruses (including RNA Tumor Viruses, No. 2) Edition, Cold Spring Harbor Laboratory, 1985). Retrow of the present invention Preferred retroviruses for preparing or constructing an ils gene delivery vehicle include Chicken leukemia virus, bovine leukemia virus, mouse leukemia virus, mink cell Vesicle focus-forming virus, mouse sarcoma virus, reticuloendotheliosis virus, and A retrovirus selected from the group consisting of Rous sarcoma virus is included. Especially Preferred mouse leukemia viruses include 4070A and 1504A (Hartley and Rowe, J. .Virol. 19: 19-25, 1976), Abelson (ATCC No. VR-999), Friend (ATC) C-No.VR-245), GRAPHICS, Gloss (ATCC No.VR-590), Kirsten, Harvey Sarcoma virus, and Lausher (ATCC VR-998), and Moloney mouse white Hematologic virus (ATCC No. VR-190). Such retroviruses are Trustor or American Type Culture Collection ("ATCC"; Rockville, Marylan d) can be easily obtained from a collection such as or ordinary available It can be isolated from known sources using techniques.   Any of the retroviruses described above are compatible with the disclosure and standard provided herein. Recombinant techniques (eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989; Kunkle, PNAS 82: 488. , 1985) to assemble or construct a retroviral gene delivery vehicle It can be easily utilized to Further, in certain embodiments of the present invention, Some parts of the torovirus gene delivery vehicle have different retroviruses Can be derived. For example, in one embodiment of the present invention, the retrovector LTR is Packaging for mouse sarcoma virus and tRNA binding site for Rous sarcoma virus Signal is mouse leukemia virus, and origin of second strand synthesis is chicken leukemia It can be derived from a virus.   In a preferred aspect of the present invention, the recombinant retrovirus comprises the vector construct described above. The construct was required for production of infectious recombinant retrovirus and was missing in the vector construct. Transfection into cells that contain the elements (called "packaging cells") Can be produced. A wide variety of retroviral constructs are contemplated in the present invention. It can be used to prepare a recombinant retrovirus. For example, one station of the present invention In terms of 5 'LTR, tRNA binding site, packaging signal, one or more heterologous sequences , A starting point for second strand DNA synthesis, and a retrovector construct comprising a 3 ′ LTR are provided. . Here, the gag / pol or env coding sequence is missing in this vector construct I ing. Briefly, long terminal repeats ("LTRs") consist of three, termed U5, R, and U3 Is further divided into elements. These elements include the retroviral Various signals responsible for biological activity (for example, promoters located in U3 and And enhancer elements). LTR is the end of the genome Exact duplication at the ends can easily be identified in proviruses. This specification As used herein, the 5'LTR is a 5 'promoter element as well as reverse transcription and And contain sufficient LTR sequences to allow integration of the DNA form of the vector. It should be understood. The 3 'LTR is a polyadenylation signal and reverse transcription and Contain sufficient LTR sequences to allow integration of the DNA form of the vector Should be understood.   The tRNA binding site and the origin of second strand DNA synthesis are also Is important for being active in and can be easily identified by those skilled in the art. An example For example, retroviral tRNAs bind at the tRNA binding site by Watson-Crick base pairing. Position and is carried into the virion along with the retroviral genome. The tRNA is then used by reverse transcriptase as a primer for DNA synthesis. You. The tRNA binding site can be easily identified based on its position just downstream of the 5 'LTR. You. Similarly, the origin of second-strand DNA synthesis is, as the name implies, retrovirus Important for the synthesis of second strand DNA. This area also called polyprint lacto Is located just upstream of the 3 'LTR.   In addition to the 5 'and 3' LTRs, the tRNA binding site and the origin of second strand DNA synthesis, Certain preferred retrovector constructs provided in Signals, as well as one or more nucleic acid molecules (eg, heterologous sequences), which Each is discussed in more detail below.   In one aspect of the invention, both gag / pol and env coding sequences are lacking A retro vector construct is provided. As used herein, "gag / pol Or lacks the env coding sequence "indicates that the retrovector is gag / pol or en v found in the gene, especially for packaging At least in the gag / pol or env expression cassette used to construct the cell line Also 20, preferably at least 15, more preferably at least 10, and most preferably Also preferably does not contain at least 8 contiguous nucleotides Should be understood.   Packaging cells suitable for use with the above retrovector constructs Strains can be readily prepared (US patent application Ser. No. 08 / 240,030, filed May 9, 1994). See also WO 92/05266) and the production of recombinant vector particles. A producer cell line (also known as a vector cell line or "VCL") Can be used to In a particularly preferred embodiment of the present invention, the packaging The cell line may be made from human (eg, HT1080 cells) or a mink parental cell line. This allows for the production of recombinant retroviruses that can withstand inactivation in human serum. Make it work. Determine if recombinant retrovirus is inactivated in human serum One representative assay that can be utilized to determine the activity is described in more detail in Example 11 below. It is described in detail.   Utilizing the method of the invention as disclosed herein, (in the crude supernatant)⁶Ma Or 10⁷Produce more recombinant retroviral particles at titers greater than cfu / ml Packaging cell lines can be readily obtained. Moreover, such titers are In general, HT1080 produces a titer three times lower than that obtained in mouse 3T3 cells It should be noted that this results from a titer assay on cells. B.Recombinant retrovirus carrying and / or expressing a desired nucleic acid molecule   A wide variety of nucleic acid molecules are carried and carried by the recombinant retroviruses of the present invention. And / or can be expressed. Generally, the nucleic acid molecule described herein is a nucleic acid molecule Does not occur naturally in recombinant retroviruses carrying This means that some The desired benefit, typically the ability to fight the disease, or a pathogenic agent or condition I will provide a. As used herein, a "pathogenic substance" is a cell that is responsible for a disease state. Refers to a vesicle. Representative examples of pathogenic substances include tumor cells, autoreactive immune cells, Hormone secreting cells, cells deficient in normal functions, usually those cells Cells that additionally have inappropriate gene expression that does not occur in the mold; And cells infected with irs or other intracellular parasites. further As used herein, "pathogenic agent" also refers to a recombinant retrovirus ( An example Cells that over- or inappropriately express (eg, the wrong cell type) Refers to cells that have become tumorigenic due to improper insertion into the host cell genome .   Can be carried and / or expressed by a recombinant retrovirus of the invention Examples of nucleic acid molecules that can be obtained are those incorporated into substances, as well as certain intracellular nucleic acid molecules, and Biologically active nucleic acid molecules such as inactivating sequences that inactivate the molecule Encoding genes and other nucleic acid molecules are included. Nucleic acid molecules increase the expression of a substance Biologically active if the molecule itself provides the desired benefits without need it is conceivable that. For example, a biologically active nucleic acid molecule can be a specific intracellular nucleic acid molecule. May be an inactivating sequence that integrates and inactivates the molecule, or The molecule is a tRNA, rRNA, or mRNA with an arrangement that provides binding capacity obtain.   Substances that can be encoded by the nucleic acid molecules described herein include proteins (Eg, antibodies containing single-chain molecules), immunostimulatory molecules (eg, antigens), immunosuppressants Probes, blocking agents, emollients (eg, toxins, antisense ribonucleic acids, Zyms, enzymes and other substances that can interfere with the function of pathogenic substances), cytoca In, various polypeptide or peptide hormones, and agonists or Include antagonists. Here, these hormones are Pituitary, hypothalamus, kidney, endothelial cells, liver, pancreas, bone, hematopoietic medulla, and adrenal glands) Can be derived. Such polypeptides can be used to induce proliferation, tissue regression, Suppression, apoptosis, gene expression, blocking of receptor-ligand interaction, Can be used for immune response and specific anemia, diabetes, infection, hypertension, abnormalities Blood chemistry (eg, elevated blood cholesterol, lack of blood coagulation factors, LDL elevation with falling), Alzheimer-related amyloid protein levels, bone Erosion / calcification and various metabolites, such as steroid hormones (Lumon, purines and pyrimidines).   With respect to a moderating agent, “capable of inhibiting function” when used in the context of the present invention, For example, the presence of cells in a cell from substances that do not normally By mitigating the presence of a substance, the moderator can either directly or indirectly perform its function. It should be understood that the inhibition. Of this function against viral diseases Examples include adsorption, replication, gene expression, assembly, and virus from infected cells. And the emission of loose. Examples of such functions for cancerous diseases include: Cell replication, sensitivity to external signals (eg contact inhibition), and anti-oncogy Deficiency in protein production. Such features for cardiovascular disease Examples of inappropriate blood or substance accumulation in blood vessels, high blood pressure, unwanted blood Fluid level factors (cholesterol or low density lipotan Parkin), localized hypoxia, as well as inappropriately high and damage tissue Levels of free radicals. Of such functions for neurological conditions Examples include pain, lack of dopamine production, inability to replace damaged cells, physical Insufficient motor control of activity, incompatibility from neurological tissue such as the pituitary or hypothalamus Severely low levels of various peptide hormones, amyloid associated with Alzheimer's disease Plaque protein accumulation and the inability to regenerate damaged nerve connections It is. Examples of such functions for autoimmune or inflammatory diseases include: Inappropriate production of itokines and lymphokines, autoimmune antibodies and cellular immunity Inappropriate production and presence of a response, of tissue by proteases and collagenases Usually due to improper disruption, inhibition of the normal activity of proteases, destroyed cells Lack of production of the supplied factor and excessive regeneration of tissue under autoimmune attack or Is abnormal regeneration.   In one aspect of the invention, a recombinant retrovirus that directs expression of a palliative Are provided. In various embodiments, the moderator is DNA It can be a molecule, an RNA molecule, or some combination of the two.   Representative examples of palliatives that act directly on cell growth inhibition include: Toxins. For example, lysine (Lamb et al., Eur. J. Biochem. 148: 265). -270, 1985), Abrin (Wood et al., Eur. J. Biochem. 198: 723-732, 1991; Even). sen et al. of Biol. Chem. 266: 6848-6852, 1991; Collins et al. of Biol. Che m. 265: 8665-8669, 1990; Chen et al., Fed. of Eur. Biochem Soc. 309: 115-118, 1 992), diphtheria toxin (Tweten et al., J. Biol. Chem. 260: 10392-10394, 198). 5), cholera toxin (Mekalanos et al., Nature 306: 551-557, 1983; Sanchez & Ho) lmgren, PNAS 86: 481-485, 1989), gelonin (Stirpe et al., J. Biol. C). hem. 255: 6947-6953, 1980), and the American pokeweed (Irvin, Pharmac. Ther) 21: 371-387, 1983), antiviral proteins (Barbieri et al., Biochem. J. 203: 5). 5-59, 1982; Irvin et al., Arch. Biochem. & Biophys. 200: 418-425, 1980; Irvin,  Arch. Biochem. & Biophys. 169: 522-528, 1975), tritin, dysentery Xin (Calderwood et al., PNAS 84: 4364-4368, 1987; Jackson et al., Microb. Path. 2: 147-153, 1987), and Pseudomonas exotoxin A (Carroll and Collier, J. . Biol. Chem. 262: 8707-8711, 1987). Express Russel's Viper Venom A detailed description of recombinant retroviruses can be found in U.S. Patents filed December 30, 1994. Application number No. (Attorney No. 930049.440).   In another aspect of the invention, the recombinant retrovirus is not itself toxic But proteins such as proteases specific for viruses or other pathogens To identify products that are converted to toxic forms when processed or modified by Have a gene. For example, a recombinant retrovirus encodes a proprotein chain Gene. This proprotein is processed by HIV protease. Toxic if treated. More specifically, recognizing the appropriate "pro" element Arrange the protease encoded by the HIV virus to cleave it Toxic lysine in the form of an inactive synthetic inactive proprotein or The diphtheria A chain can be cleaved to the active form.   In yet another aspect of the invention, the recombinant retrovirus is an inactive precursor Which can be activated by an active inhibitor of a pathogenic substance or a conditional safener Directs the expression of quality. These are moderators that are toxic to cells expressing the pathogenic condition. It is a Japanese medicine. Broadly, as will be apparent from certain disclosures provided herein. A variety of inactive precursors can be converted into active inhibitors of pathogenic females. For example, To specifically inhibit Torovirus reverse transcriptase (and therefore virus replication) Antiviral nucleoside analogs (eg, AZT or ddI) may be activated by cellular machinery. Metabolized to the nucleotide triphosphate form (Furmam et al., Proc. Natl. Acad. Sci.  USA 83: 8333-8337, 1986). Their activity of antiviral nucleoside analogs Herpes simplex virus thymidine kinase (HSVTK), which assists metabolism into forms Or gene products such as varicella-zoster virus thymidine kinase (VZVTK) Recombinant retrovirus that directs the expression of Activating an osido analog precursor (eg, AZT or ddC) to its active form Useful for This makes AZT or ddI treatment more effective and Lower doses, lower systemic toxicity, and higher for virus-producing infections Enable the ability to work well. Nucleotide triphosphate form is retroviral reverse transcriptase Showing selectivity for nucleosides, but substrates for cellular nucleosides and nucleoside kinases As a result of the specificity, another non-phosphorylated nucleoside analog is more effective You.   In one embodiment of the present invention, the HSVTK gene comprises a constitutive macrophage. Alternatively, it can be expressed under the control of a T cell-specific promoter, and Di or T cells. By constitutive expression of HSVTK, AZT or ddI Such nucleotide analogs to biologically active nucleotide triphosphate forms Resulting in more effective metabolism of the drug, which results in better efficacy, lower dose delivery , Less systemic toxicity, and higher potency against virus-producing infections provide. Nucleotide triphosphate forms select for retroviral reverse transcriptase , But results in substrate specificity of cellular nucleosides and nucleotide kinases Other nucleoside analogs that are not phosphorylated as Can be used.   In a related aspect of the invention, the cytotoxicity in the presence of Or a set that directs the expression of a substance that activates another compound that has no at all to a toxic product A modified retrovirus is provided. Local treatment for pathogenic substances To achieve. In this case, the gene product from the recombinant retrovirus Expression is associated with pathogenic agents such as intercellular signals that identify the pathogenic state. Limited to situations where the body is present, thereby preventing the destruction of non-pathogenic cells. Pathogenic disease Label recombinant retroviruses against cells that have or are susceptible to By targeting, this cell type specificity can also be conferred at the infection level.   In a related aspect of the invention, the method has little or no cytotoxicity. Recombinant retrovirus directing expression of a gene product that activates the compound to a toxic product Is provided. Briefly, compounds with little or no cytotoxicity A wide variety of genes that activate toxic products, either directly or indirectly, Things may be utilized within the context of the present invention. Representative examples of such gene products Selectively purine arabinoside and substituted pyrimidine compounds Phosphorylates these compounds into cytotoxic or cytostatic metabolites HSVTK and VZVTK. More specifically, ganciclovir, Such as acyclovir, or any analog thereof (eg, FIAC.DHPG) Exposure of a drug to HSVTK causes the drug to be converted to the corresponding active nucleotide triphosphate form To phosphorylation.   For example, in one embodiment of the present invention, the recombinant retrovirus is Motor (which is not activated unless activated by the HIV tat protein). Herpes simplex under the transcriptional control of what is known to be Directs expression downstream of the illthymidine kinase ("HSVTK") gene. Simply saying For example, tat in human cells infected with HIV and carrying a recombinant retrovirus Expression of the gene product causes an increase in HSVTK production. Then (in vitro or Or in vivo) cells with ganciclovir, acyclovir, or Exposure to drugs such as analogs (FIAC, DHPG). As mentioned above, these drugs Is phosphorylated by HSVTK (but not by cellular thymidine kinase). (Not oxidized), is known to be the corresponding active nucleotide triphosphate form ing. Acyclovir triphosphate generally inhibits cellular polymerases and Leads to specific destruction of HSVTK-expressing cells in transgenic mice (Bor relli et al., Proc. Natl. Acad. Sci. USA 85: 7572, 1988). Recombinant Those cells containing retroviruses and expressing the HIV tat protein Vesicles are selectively killed by the presence of certain doses of these drugs.   In one embodiment of the invention, the cis-acting component is converted to a transcript ("CRS / CAR"). ), The expression of the conditional lethal gene of HSVTK can be made even more HIV-specific. It can be. This requires another HIV gene product, rev, for optimal activity. (Rosen et al., Proc. Natl. Acad. Sci. USA 85: 2071, 1988). More general Typically, cis elements present in mRNA modulate the stability or translatability of the mRNA. A clause is indicated in some cases. An array of this type That is, post-translational regulation of gene expression) is an event-specific regulation of vector gene expression or Can be used for tissue-specific regulation. In addition, these sequences (ie, Rev responsive “CRS / CAR” element or tat responsive “TAR” element) Quantization can be utilized to produce even better specificity.   In a manner similar to the embodiment described above, the purine or pyrimidine based drug phosphorus Preserve genes for oxidation, phosphoribosylation, ribosylation, or other metabolism A recombinant retrovirus can be produced. Such genes are Have no equivalent in the vesicles and are likely to be viruses, bacteria, fungi, or protozoa. Can be derived from such organisms. Typical examples are: thioxanthine to thioxanthine E. Convert to monophosphate coli guanine phosphoribosyltransferase ("gpt ") Gene products (see Besnard et al., Mol. Cell. Biol. 7: 4139-4141, 1987). And) such as mitomycin phosphate and doxorubicin phosphate. Alkaline phosphatars convert active phosphorylated compounds to toxic dephosphorylated compounds -A protein that converts 5-fluorocytosine to the toxic compound 5-fluorouracil Fungal (eg Fusarium oxysporum) or bacterial cytosine deaminase (Mullen, PNAS 89:33, 1992), para-N-bis (2-chloroethyl) aminobenzoylglutami Cleaves glutamic acid from acetic acid, thereby producing toxic mustard benzoate Carboxypeptidase G2; and doxorubicin and melphalan Penicili converts phenoxyacetabide derivatives into toxic compounds. V-amidase and the like. This type of conditional lethal gene product is Or applied to many currently known anti-cancer drugs based on pyrimidine. This includes intracellular ribosylation or phosphorylation to become an effective cytotoxic agent. Often needed. The conditional lethal gene product may also be purine or pyrimidine. Nontoxic analogs that are not analogs can be metabolized into cytotoxic forms (Searle et al., Brit. J. Cancer 53: 377-384, 1986).   Further, when the target pathogen is a mammalian virus, the mammalian virus In addition, it is necessary for the subsequent transcriptional activation of other viral promoter elements. Taking advantage of the fact that they tend to have "immediate" genes, recombinant retroviral vectors May be built. Gene products of this nature are used as intracellular signals of viral infection. (Or “identifying substance”). Therefore, such an "immediate Transcription from a transcriptional promoter element responsive to "time" viral gene products Conditional lethal gene specifically kills cells infected with any particular virus Can be destroyed. In addition, the human α and β interferon promoter elements Is transcriptionally activated in response to infection by a wide variety of unrelated viruses For example, from these viral responsive elements (VREs), conditional Infection with a variety of different viruses by introducing a vector expressing the lethal gene product Destruction of damaged cells can occur.   In another embodiment of the present invention, an inhibitor that inhibits viral assembly A recombinant retrovirus producing a substance such as a palliative is provided. Related to this As a result, recombinant retroviruses inhibit viral particle assembly in a preferential manner. Harm, incomplete gag, pol. env or other virion proteins or Encodes a peptide. Such inhibition is due to the normal subunit Interaction is disrupted by interaction with imperfect subunits Occurs.   One way to increase the efficiency of inhibitory mitigants is by the virus in question. Along with the expression of genes that increase the probability of infection of resistant cells To express an inhibitory gene such as a gene. The result is the viral productivity A non-productive "dead end" event that antagonizes the staining event. Specific cases of HIV In HIV replication (by expressing antisense tat or the like as described above) A recombinant retrovirus that inhibits production can be administered, and this can also Proteins required for such infections can also be overexpressed. Thus, comparison A very small number of vector-resistant HIV-resistant cells are free virus or virus-infected cells "Sink" or "magnet" for multiple non-productive fusion events with (magnet) ".   In another embodiment of the present invention, an inhibitory peptide specific for a viral protease is used. Recombinant retrovirus for expressing substances such as peptides or proteins Is provided. Viral proteases include viral gag and gag / pol proteins Is cleaved into a number of smaller peptides. In all cases, this disconnection failure Leads to complete inhibition of the production of infectious retroviral particles. HIV protease , Aspartyl protease. And these are proteins Or be inhibited by peptides made from amino acids derived from analogs It has been known. Recombinant retroviruses that inhibit HIV have such peptides Express one or more fusion copies of the inhibitor.   Administration of the recombinant retroviruses described above has been associated with many virus-related diseases, cancer, and Should be effective against other pathogenic substances.   In yet another embodiment of the present invention, a recombinant retrovirus expressing a moderating agent Is provided. Here, the palliative has a membrane anchor and acts as an antitumor agent. To use. Such palliatives can be used, for example, with antitumor agent-membrane anchor fusion proteins. And can be built. Briefly, the aspect of the fusion protein membrane anchor is, for example, For example, it can be selected from various sequences including the transmembrane domain of a known molecule. Generally, membrane An anchor sequence is a region of a protein that binds the protein to a membrane. As usual, There are two types of anchor sequences that attach proteins to the outer surface of the cell membrane: ( 1) Transmembrane spanning the lipid bilayer of the cell membrane and interacting with the hydrophobic central region Region (a protein containing such a region is called an integral membrane protein) (2) and (2) domain interaction with integral membrane proteins or polar surfaces of the membrane. In (such proteins are called peripheral proteins or exogenous proteins) Is).   The membrane anchor used in the present invention is a transmembrane domain that crosses the membrane one or more times. May be included. For example, in glycophorin and guanylyl cyclase, The binding region crosses the membrane once, whereas the transmembrane domain of rhodopsin crosses the membrane seven times. And the transmembrane domain of the photosynthetic reaction center of Rhodopseudomonas viridis Cross the membrane 11 times (Ross et al., J. Biol. Chem. 257: 4152, 1982; Garbers, Pharmac.  Ther. 50: 337-345, 1991; Engelman et al., Proc. Natl. Acad. Sci. USA 77: 2023,  1980; Heijne and Manoil, Prot. Eng. 4: 109-112, 1990). Book Membrane anchors for use in the invention may also include, for example, phosphoinositol anchors. It contains. Regardless of the number of times the protein passes through the membrane, the transmembrane region is representative And have a similar structure. More specifically, 20 to 20 domains located inside the membrane The 25 amino acid residue portion generally consists almost entirely of hydrophobic residues (Ei senberg et al., Ann. Rev. Biochem. 53: 595-623, 1984). For example, 28-34 residues in the transmembrane region of lycophorin are hydrophobic (Ross et al., Supra). See Tomita et al., Biochemistry 17: 4756-4770, 1978). Furthermore, β Structures such as sheet and beta barrel occur, but X-ray diffraction, crystal structure analysis, and The transmembrane region is typically an α-helical structure, as determined by (See Eisenberg et al., Supra; Heijne and Manoil, supra). Place The location of these transmembrane helices within a given sequence can be determined based on the hydrophobicity plot. Can often be predicted. Stryer et al., Biochemistry, 3rd edition 304, 1988. In the present invention Membrane anchors that are particularly preferred for use in Naturally occurring cellular proteins that have been shown to function as (Eg, glycophorin).   In a preferred embodiment of the present invention, the membrane anchor gamma interferon fusion tan A DNA sequence encoding a protein is provided. In one embodiment, the fusion The protein encodes a sequence encoding the membrane anchor of the Fc receptor -Feron can be constructed by gene fusion to a sequence encoding the same.   In yet another aspect, the RNA molecule corresponding to the pathogenic function is cleaved, and Activating one or more ribozymes (RNA enzymes) (Haseloff and Gerlach, Nature  334: 585, 1989). Lus provided. Ribozymes recognize specific sequences in target RNA. And since this sequence is usually 12-17 bp, Specific recognition of specific RNA sequences (eg, HIV tat) corresponding to pathogenic conditions And the toxicity is specific for such pathogenic conditions. Further specificity However, in some cases, ribozymes may be used as conditional safeners as described above. Can be achieved by   In yet another aspect, a biologically active nucleic acid molecule that is an antisense sequence (An antisense sequence may also comprise a nucleic acid sequence And can then be produced in a host cell by transcription). Preferred In another embodiment, the antisense sequence comprises an influenza virus, HIV, HSV , It is selected from the group consisting of sequences encoding HPV, CMV, and HBV. This anti A sense sequence is also an antisense RNA that is complementary to an RNA sequence required for virulence. possible. Alternatively, a biologically active nucleic acid molecule is converted to an RNA sequence required for virulence. It can be complementary sense RNA (or DNA).   More specifically, the biologically active nucleic acid molecule can be an antisense sequence. Simple Simply put, antisense sequences are designed to bind RNA transcripts, To prevent the cell synthesis of a particular protein or its RNA sequence by the cell Prevent the use of. Representative examples of such sequences include antisense thymidine Kinase, antisense dihydrofolate reductase (Maher and Dolnick, Arch Biochem. & Biophys. 253: 214-220, 1987; Bzik et al., PNAS 84: 8360-8364, 1987. ), Antisense HER2 (Coussens et al., Science 230: 1132-1139, 1985), antisense Sense ABL (Fainstein et al., Oncogene 4: 1477-1481, 1989), antisense Myc (S tanton et al., Nature 310: 423-425, 1984), and antisense ras; Antisense sequences blocking any enzyme in the nucleotide biosynthetic pathway I can do it.   Further, in a further embodiment of the invention, a strong class I restricted response is elicited. To do so, antisense RNA may be utilized as an antitumor agent. Simply put, R In addition to binding NA and thereby preventing translation of certain mRNAs, Bell's specific antisense sequence is interrupted by the formation of large amounts of double-stranded RNA. Is thought to induce increased expression of erythron (including γ-interferon) I have. Increased expression of gamma interferon in turn increases MHC class I antigen expression You. Preferred antisense sequences used in this regard include actin RNA , Myosin RNA, and histone RNA. Mismatch with actin RNA Are particularly preferred.   In another embodiment, the substance of the invention comprises a surface which is itself therapeutically beneficial. Contains protein. For example, especially in the case of HIV, especially in HIV infected cells Expression of the human CD4 protein can be beneficial in two ways.   1. Soluble CD4 inhibits free virion formation against free virus. Intracellular binding of CD4 to HIV env is a systemic clear run. It can inhibit this without the problem of potential and potential immunogenicity. Because this The protein remains bound to the membrane and is structurally endogenous CD4 (patients Should be immunologically tolerant to).   2. Since the CD4 / HIV env complex has been implicated as a cause of cell death, Further expression of CD4 (in the presence of excess HIV-env present in the cells) It leads to rapid cell death and thus inhibits virus seeding. This is induced by HIV Cytotoxicity (this cytotoxicity is then apparent in the relative loss of CD4 on the cell surface) Result of their relative refractility (relatively due) And macrophages act as carrier of pathogens for virus production It may be particularly applicable to devices.   Yet a further aspect of the invention relates to a recombinant retrovirus capable of effecting immunostimulation. I do. Simply put, recognize foreign pathogens and protect against them Ability is essential for the functioning of the immune system. In particular, the immune system has a host defense mechanism "Self" and "Self" to be oriented towards the invading entity rather than to the organization We must be able to distinguish between "non-self" (ie, extrinsic). Cytolytic T phosphorus Papillocytes (CTL) are typically associated with MHC class I or class II cell surface proteins. Bound, cell surface recognition structures (eg, processed pathogen-specific peptides) Is induced or stimulated by the presentation of   Diseases suitable for treatment include viral infections (eg, influenza virus , RS virus, HPV, HBV, HCV, EBV, HIV, HSV, FeLV, FIV, Hantavirus, H TLV I, HTLV II, and CMV), cancer (eg, melanoma, renal carcinoma, breast cancer, Ovarian cancer, and other cancers), and heart disease.   In one embodiment, the present invention relates to a method for treating an antigen or its antigen in a susceptible target cell. By using a recombinant retrovirus that directs the expression of a modified form, Methods are provided for stimulating an immune response and inhibiting viral spread. here The antigen may (1) mount an immune response against a viral antigen, or (2) virus occupation by occupying cell receptors required for virus interaction Can prevent diffusion. Protein expression can be transient or transgenic. Can be stable over time. Where the immune response should be stimulated against a pathogenic antigen If so, the recombinant retrovirus is preferably a modified form of the antigen (the antigen being Stimulating the response and reduced pathogenicity compared to the natural antigen) Designed to. This immune response indicates that cells are in the correct mode (ie, CD3, ICAM-1). , ICAM-2, LFA-1, or analogs thereof (eg, Altmann et al., Nature 338: 5). MHC class I and / or II molecules with accessory molecules such as This is achieved when the antigen is presented in the situation described above.   The immune response can also be determined by: (a) (if appropriate, in the context of appropriate MHC molecules) Specific T cell receptor gene that recognizes the target antigen, (b) recognizes the target antigen (C) CTL response in the absence of the MHC context The gene of the hybrid of the two genes provided is transferred to a suitable immune cell (eg, T (Lymphocytes). Therefore, recombinant retrowi Ruth can also be used as an immunostimulant, immunomodulator, or vaccine, etc. .   If immune stimulation is desired in certain disease cases caused by HIV infection, Antigens generated from recombinant retroviruses are HLA class I or class II restricted It may be in a form that elicits one or both of the epidemic responses. For example, HIV En In the case of the envelope antigen, the antigen is gp160, g, which has been modified to reduce its pathogenicity. It is preferably selected from p120 and gp41. In particular, the selected antigen Reduce the likelihood of titanium and enhance the expression of epitopes leading to diseases that enhance the immune response. Eliminate or eliminate immunodominant but lineage-specific epitopes Modified to present several strain-specific epitopes, and most HIV Or allow a response that can eliminate infected cells in all strains. This strain-specific Animals that further select for specific epitopes and cross-react with other strains of HIV Can promote the stimulation of the immune response. Other HIV genes or gene combinations Antigens of origin (eg, gag, pol, rev, vif, nef, prot, gag / pol, gag prot, etc.) ) May also provide protection in certain cases.   HIV is just one example. This approach uses a unique antigen (membrane protein Many virus-related diseases or cancers (eg, HP V and cervical vertebral carcinoma, HTLV-I induced leukemia, prostate specific antigen (PSA) and prostate cancer, Mutant p53 and colon carcinoma and melanoma, melanoma-specific antigen (MAGE) and melanoma Ma, mucin and breast cancer).   According to the immunostimulatory aspect of the present invention, it is protected by the recombinant retrovirus of the present invention. Substances possessed and / or expressed are also “immunomodulators” (often described above). Have been included). An immunomodulator is one or more cells involved in the immune response Or when exogenously added to cells. An immune response that occurs in the absence of offspring is an immune response that differs in quality or potency. Refers to factors that occur. This factor is also expressed from non-recombinant retrovirus-derived genes However, expression is driven or controlled by the recombinant retrovirus. response Quality or potency can be determined, for example, by in vitro assays that measure cell proliferation (eg, If^ThreeH-thymidine incorporation), and in vitro cytotoxicity assays (eg, ,⁵¹(Cr release measurement). (See Warner et al., AIDS Res. And Human Retroviruses 7: 645-655, 1991. ). Immunomodulators can be active both in vivo and ex vivo. this Representative examples of such factors include IL-1, IL-2 (Karupiah et al., J. Immunology 14). 4: 290-298, 1990; Weber et al. Exp. Med. 166: 1716-1733, 1987; Gansbacher et al. , J. et al. Exp. Med. 172: 1217-1224, 1990; U.S. Patent No. 4,738,927), IL-3, IL-4 (Tepper et al., Cell 57: 503-512, 1989; Golumbek et al., Science 254: 713-716, 1991. U.S. Patent No. 5,017,691), IL-5, IL-6 (Brakenhof et al., J. Immunol. 139: 4116). -4121, 1987; WO 90/06370), IL-7 (U.S. Pat. No. 4,965,195), IL-8, IL-9 , IL-10, IL-11, IL-12, IL-13 (Cytokine Bulletin, summer 1994), IL-14, and And IL-15, especially IL-2, IL-4, IL-6, IL-12, and IL-13, alpha interferon (F inter et al., Drugs 42 (5): 749-765, 1991; U.S. Pat. No. 4,892,743; U.S. Pat. No. 66,843; WO No. 85/02862; Nagata et al., Nature 284: 316-320, 1980; Familletti Et al., Methods in Enz. 78: 387-394, 1981; Twu et al., Proc. Natl. Acad. Sci. USA 86: 2046-2050, 1989; Faktor et al., Oncogene 5: 867-872, 1990), beta interface Ron (Seif et al., J. Virol. 65: 664-671, 1991), gamma interferon (Radford Et al., The American Society of Hepatology 2008 2015, 1991; Watanabe et al., PNAS 86: 9456-9460, 1989; Gansbacher et al., Cancer Research 50: 7820-7825, 1990; Ma. io et al., Can. Immunol. Immunother. 30: 34-42, 1989; U.S. Patent No. 4,762,791 No. 4,727,138), G-CSF (US Pat. Nos. 4,999,291 and 4,810) No. 643), GM-CSF (WO 85/04188), tumor necrosis factor (TNF) (Jayaraman et al., J. . Immunology 144: 942-951, 1990), CD3 (Krissanen et al., Immunogenetics 26:25). 8-266, 1987), ICAM-1 (Altman et al., Nature 338: 512-514, 1989; Simmons et al., Na 331: 624-627, 1988), ICAM-2, LFA-1, LFA-3 (Wallner et al., J. Exp. Med. 166 (4): 923-932, 1987), MHC class I molecule, MHC class II molecule, B7.1-.3, β_TwoMi Globulins (Parnes et al., PNAS 78: 2253-2257, 1981), chaperones (eg, , Calnexin), MHC binding transporter protein or its analog (P owis et al., Nature 354: 528-531, 1991). One In a preferred embodiment, the gene encodes gamma interferon. Immunomodulators can also be agonists, antagonists, or the ligands of these molecules. Can be For example, the soluble form of the receptor is itself a mutant form of the factor. As antagonists to these types of factors, Works often.   One example of an immunomodulator cited above is a B7 family molecule (eg, B7.1-.3 Co-stimulator). Simply put, the full functional activity of T cells Sexualization requires two signals. One signal is an antigen-specific T cell receptor. Interaction between the peptide and a peptide that binds to a major histocompatibility complex (MHC) molecule. A second signal, provided by the antigen presenting cell and called co-stimulation, To be delivered to T cells. The second signal is that interleukin-2 ( Required for the production of IL-2) and B7.1-.3 molecules on antigen presenting cells and on T lymphocytes Interaction with CD28 and CTLA-4 receptors (Linsley et al., J. Ex. p.Med., 173: 721-730,1991a and J.Exp.Med., 174: 561-570,1991). One of the present invention In an embodiment, B7.1-.3 comprises CD8⁺T cells expand and become fully activated CD8 to produce enough IL-2⁺Tumor to cause costimulation of T cells It can be introduced into a cell. Because co-stimulation is no longer needed for further CTL function , These CD8⁺T cells can kill tumor cells that do not express B7. Simultaneous stab Vectors that express both intense B7.1-.3 factors and, for example, immunogenic HBV core proteins. The reactor may be made utilizing the methods described herein. These vectors The cells transduced with the enzyme become more effective antigen-presenting cells. HBV core Specific CTL responses were fully activated by costimulatory ligand B7.1-.3 CD8⁺T thin Increased from vesicles.   The choice of which immunomodulator to include in the recombinant retrovirus depends on the factors It can be based on known therapeutic effects or can be determined experimentally. For example, chronic B A known therapeutic effector in hepatitis B infection is alpha interferon. You. This compensates for the patient's immunodeficiency, and thereby helps to recover from the disease. Has been found to be effective in removing Alternatively, implement appropriate immunomodulators Can be determined empirically. Briefly, a blood sample is first taken from a patient with liver disease. You. Recombinant retro-directed expression of the immunogenic portion of hepatitis antigens and immunomodulators Autologous cells transduced with the virus or HLA-compatible cells (eg, EBV transformed cells ) To restimulate peripheral blood lymphocytes (PBL) in vitro. Then, HLA-compatible transduction These stimulated cells are used as effectors in CTL assays that target cells. Use PBL. Formed with HLA-compatible stimulants and vectors encoding only the antigen The increase in CTL response seen in the same assay performed with transfected target cells Indicates a useful immunomodulator. In one embodiment of the invention, immunomodulation The factor gamma interferon is particularly preferred.   The present invention also provides a method for producing a desired antigen (eg, a viral, bacterial, or parasitic antigen). (Including recombinant origin). example Immunity when administered by one of the recombinant retroviruses described herein. The various immunogenic portions of the HBV S antigen can be combined to show a response. In addition, Region of the HBV S antigen open reading frame Due to the large immunological diversity, specific areas may require specific sets of antigens for administration. A combination may be preferred. Briefly, all human hepatitis B virus S samples The epitope found therein is defined as antigenic determinant "a". But each other Exclusive subtype antigenic determinants also provide two-dimensional double immunodiffusion (Ouchterlony, Prog r. Allergy 5: 1, 1958). These antigenic determinants are up to "d" Or "y", and "w" or "r" (LeBouvier, J. Infe ct. 123: 671, 1971; Bancroft et al. Immunol. 109: 842, 1972; Courouce et al. Bibl. Haematol. 42: 1-158, 1976). This immunological diversity is based on the hepatitis B virus S A single nucleotide substitution in two regions of the antigen open reading frame Causes the following amino acid changes: (1) hepatitis B virus S antigen open Conversion of lysine-122 to arginine in the reading frame is subtype Is shifted from d to y, and (2) the conversion of arginine-160 to lysine , Shift the subtype from r to w. Subtype ayw among African blacks Is dominant, while in the US and Northern Europe the subtype adw_TwoIs more abundant (Molecular Biology of the Hepatitis B Virus, edited by McLachlan, CRC Press, 1991). As will be apparent to those skilled in the art, certain B prevalent in the area of administration. It is generally preferable to construct vectors for administration appropriate for the hepatitis B virus subtype. Good. Can the specific region subtype be determined by two-dimensional double immunodiffusion? , Preferably the S antigen library of HBV virus isolated from individuals in that region. The reading frame can be determined by sequencing.   Those indicated by HBV also include pol ("HBV pol"), ORF5, and ORF6 antibodies. There is a field. Simply put, the polymerase open reading frame of HBV is Reverse transcriptase activity found in virions and core-like particles in infected liver tissue Code sex. Polymerase protein consists of at least two domains : Amino-terminal domain encoding a protein that initiates reverse transcription, and reversal Carboxyl-terminal domain encoding transcriptase and RNase H activity. HBV pol An immunogenic moiety is a group that expresses an antigen of interest to generate an immune response in an animal. By introducing the recombinant retrovirus into an animal, it can be administered to a warm-blooded animal. same And others such as ORF5 and ORF6 (Miller et al., Hepatology 9: 322-327, 1989). HBV antigens can be expressed using the recombinant retroviruses described herein.   As mentioned above, at least one immunogenic portion of the hepatitis B antigen is Virus. Immunogenicity incorporated into recombinant retrovirus The portion can be of various lengths, but generally this portion will be at least 9 amino acids long. It is preferably long and may contain the entire antigen. Immunogenicity of a specific sequence Is often difficult to predict, but T-cell epitopes are e 351: 290, 1991) using the HLA A2.1 motif. This From the analysis of the peptide can be synthesized and in an in vitro cytotoxicity assay Can be used as a target. However, other assays may also be utilized, and such As an assay, for example, to detect the presence of an antibody against a newly introduced vector ELISA and gamma interferon assay, IL-2 production assay, and amplification Assays that test T helper cells, such as the proliferation assay.   In one embodiment of the invention, at least one immunogenic part of a hepatitis C antigen Can be incorporated into a recombinant retrovirus. Preferred immunogenic portions of hepatitis C are C and can be found in the NS3-NS4 region. Because these areas are Because it is the most conserved among the various types of flame viruses (Houghton et al., Hepat ology 14.381-388, 1991). Particularly preferred immunogenic moieties can be determined in various ways. obtain. For example, as described above for hepatitis B virus, The identity of the original moiety can be predicted based on the amino acid sequence. Simply put, this business CTL epitopes are predicted using various computer programs known to obtain. For example, HLAA2. Described by Falk et al. (Nature 351: 290, 1991). 1 motif can be used to predict CTL epitopes for HLA A2.1 haplotype You. From this analysis, the peptides are synthesized and used in in vitro cytotoxicity assays. Use as a target in   In another aspect of the invention, a method is provided for destroying hepatitis B carcinoma cells. . This method directs the expression of an immunogenic portion of antigen X such that an immune response is generated. Administering the recombinant retrovirus to a warm-blooded animal. Provided herein According to the content of the disclosure provided, the sequence encoding HBxAg is readily accessible to those skilled in the art. Can be handled. Briefly, in one embodiment of the present invention, from ATCC 45020 to 64 2 bp of NcoI-TaqI was recovered and assembled as described above for the other hepatitis B antigens. Insert into the replacement retrovirus.   However, the X antigen is similar to other potential oncogenes (eg, EIA) A known transactivator that can function in the formula. Therefore, generally first After altering the X antigen so that the gene product is non-tumorigenic, it is recombined. Preferably, it is inserted into a torovirus. For example, truncation, point mutation, premature termination Various methods, including addition of codons or modification of the phosphorylation site, can be used to The progenitor can be non-tumorigenic. In one embodiment, the eye encoding the X antigen Cut the end of the target sequence or gene. Various fragments due to truncation Can be produced, but it is generally preferable to retain 50% or more of the coding gene sequence. Good. In addition, after any truncation, the complete immunogenic sequence of the gene product It is necessary to keep it as it is. Alternatively, in another embodiment of the present invention, A number of translation stop codons can be introduced into a gene. Insertion of a stop codon is Stops expression of the transformed part of the protein because expression is stopped before maturation .   The tumorigenicity of the X gene or a modified version thereof can be tested in various ways. Typical assays include tumor formation in nude mice and colony in soft agar. Transgenic animals such as Ronnie formation and transgenic mice Preparation of   In another aspect of the invention, a recombinant that directs the expression of an immunogenic portion of a hepatitis C antigen Destroying hepatitis C carcinoma cells, comprising administering a retrovirus to a warm-blooded animal A method is provided for: Preferred immunogenic portions of the hepatitis C antigen are the core antigen and And polypeptides containing the NS1-NS5 region (Choo et al., Proc. Natl. Acad. Sci. USA 88: 2451-2455, 1991). Particularly preferred immunogenic moieties are It can be determined by various methods. For example, as described above, Thus, a preferred immunogenic portion can be predicted. Briefly, as is known to those skilled in the art. Various computer programs can be used to predict CTL epitopes. For example (Nature 351: 290, 1991) described by Falk et al. This can be used to predict CTL epitopes for the HLA A2.1 haplotype. Immunogen Another method that can be used to predict sex parts is to use retroviruses Is to determine in the mouse which part has CTL inducing properties (W arner et al., AIDS Res. and Human Retroviruses 7: 645-655, 1991) . As described in Warner et al., CTL induction in mice Cell immunogenicity can be predicted. Preferred immunogenic moieties are also polypeptide anti- Which fragment of the original or peptide is present after vector transduction of the corresponding gene Self-patient lymphocytes (eg, autologous EBV- Transforming lymphocytes) to determine if obtain.   Preferred immunogenic moieties can also be selected as follows. Simply put HCV to determine which antigenic fragments are present in the patient's serum Blood samples from patients suffering from a target disease such as Antibodies against peptide regions (eg, HCV core, E1, E2 / SN1, and NS2-NS5 regions) Analyze by body. Patients treated with alpha interferon to obtain transient remission In some cases, some antigenic determinants are lost and endogenous antibodies to that antigen are replaced. Wrong. Such antigens are useful as immunogenic portions in the context of the present invention (Ha yata et al., Hepatology 13: 1022-1028, 1991; Davis et al. Eng. J. Med. 321: 1501 -1506, 1989).   By cutting off the ends of the coding sequence, Additional immunogenic portions of the selected antigen, such as antigens, can be obtained. For example, HBV To cut the ends of the following sites: Bst UI, SspI, Ppu M1 and MspI. (Valenzuela et al., Nature 280: 815-19, 1979; Valenzuela et al., Animal Virus Genetics: ICN / UCLA Symp. Mol. Cell Biol., 1980, B.N. Fields and R.S. Jaen isch (eds.), pp. 57-70, New York: Academic). Appropriate immunogenic moieties and methods Further methods for determining are also described below in the context of hepatitis C.   Particularly preferred for incorporation into recombinant retroviruses for treatment of HBV Immunogenic moieties include HBeAg, HBcAg, and HBsAgs. In addition, 2 The immunogenic portion (and immunomodulator, if desired) can be Ruth can be taken up. For example, in one embodiment, the immunogenicity of a hepatitis B antigen Set directed to co-expression of both a portion and an immunogenic portion of a hepatitis C polypeptide A recombinant retrovirus can be prepared. Such constructs are of type B or C May be administered to prevent or treat any of the acute or chronic hepatitis infections. You. Similarly, in other embodiments, the immunogenic portion of hepatitis B X antigen and hepatitis C Recombinant retrovirus directing co-expression of both immunogenic portions of the polypeptide Can be prepared. Similarly for such constructs, hepatitis B or C It can be administered to treat hepatocellular carcinoma associated with any. In addition, liver type B Individuals chronically infected with hepatitis C and hepatitis C are at increased risk of developing hepatocellular carcinoma. As such, such vectors are also useful as prophylactic treatments for this disease. Can be used.   Immunogenic moieties can also be selected by other methods. For example, HLA A2.1 / K^b Transgenic mice serve as a model for human T cell recognition of viral antigens. Is shown. Simply put, influenza and hepatitis B In the ills system, the mouse T cell receptor repertoire relies on human T cells. Recognize the same antigenic determinant as that recognized. HLA A2.1 in both systems / K^bCTL responses generated in transgenic mice were analyzed by HLA A2.1 haplota To an epitope substantially identical to the epitope recognized by the human CTL (Vitiello et al., J. Exp. Med. 173: 1007-1015, 1991; Vitiello et al., Abst ract of Molecular Biology of Hepatitis B Virus Symposia, 1992).   The immunogenic proteins of the invention also make them more immunogenic It can be operated by various methods known in the art. Such person As a typical example of the method, an amino acid sequence corresponding to the T helper epitope was added. Promoting cell uptake by adding hydrophobic residues; Promoting cellular uptake by forming child structures; or (In general, Hart, supra, Milich et al., Proc. Natl. A. cad. Sci. USA 85: 1610-1614, 1988; Willis, Nature 340: 323-324, 1989; Grif fiths et al. Virol. 65: 450-456, 1991).   The present invention also provides for the detection of various feline diseases (eg, feline leukemia virus (“FeLV”)). And the treatment of feline immunodeficiency virus ("FIV") infections and the like. And methods, as well as vaccines for the prevention of these diseases. These ui Ruth is discussed more fully in PCT Application No. US 93/09070.   Briefly, feline leukemia virus (FeLV) is an oncornavirus Millie's retrovirus. Currently, FeLV has its envelope antigens gp70 and And that there are three subgroups A, B or C, distinguished by Have been. FeLV also has many resources that are highly conserved in all FeLV subgroups. A antigen (including p15, p12, p27, and p10) (Geering et al., Vir. 36 : 678-680,1968; Hardy et al., JAVA 158: 1060-1069,1971; Hardy et al., Science 166: 1. 019-1021, 1969). In one embodiment of the present invention, the recombinant retro Viruses include p15gag, p12gag, p27gag, p10gag, p14pol, p80pol, p46pol, gp70 less feline leukemia virus antigens selected from the group consisting of env and p15env Both lead to partial expression. In a particularly preferred embodiment, the recombinant retrovirus Leads to the expression of gp85env. The sequences encoding these antigens are provided in the description. Provided disclosure (Donahue et al., J. Vir. 62 (3): 722-731, 1988; Stewart et al., J. Vir. 58 (3): 825-834,1986; Kumar et al., J. Vir. 63 (5): 2379-2384,1989; Elder et al., J. Vir. 46 (3): 87. 1-880, 1983; Berry et al., J. Vir. 62 (10): 3631-3641, 1988; Laprevotte et al., J. Vir. 50 (3 ): 884-894, 1984).   Feline immunodeficiency virus (FIV) has a requirement for magnesium in reverse transcriptase (RT) and Retroviruses of the lentivirus subfamily based on morphology of viral particles (Pederesen et al., Science 235: 790-793, 1987. ). Feline immunodeficiency virus is morphologically and antigenically similar to other feline retroviruses ( Feline leukemia virus, oncornavirus type C (RD-114), and cat (Including Yamamoto et al., “Efficacy of exper imental FIV vaccines, '' (abstract), First International Conference of Feline Immunodeficiency Virus Researchers, University of California, Davis, CA, Sep .4-7, 1991). In one embodiment of the present invention, Ils is p15gag, p24gag, p10gag, p13pol, p62pol, p15pol, and p36pol At least one immunogenicity of a feline immunodeficiency virus antigen selected from the group consisting of: Guides expression of the moiety. In a particularly preferred embodiment, the recombinant retrovirus is Directs the expression of gp68env, gp27env, and rev. In the context of the present invention, "rev" is Is understood to refer to the antigen corresponding to the rev open reading frame (Philli ps et al., First International Conference, supra). Arrangements encoding these antigens The columns correspond to the disclosure provided herein (Phillips et al., J. Vir. 64 (10): 4605-4613, 1990). ; Olmsted et al., PNAS 86: 2448-2452,1989; Talbott et al., PNAS 86: 5743-5747,1989. Can be readily obtained by one of ordinary skill in the art given.   Still other examples are non-tumorigenic modified genes (eg, ras (ras^*) Gene (WO Includes recombinant retroviruses that direct expression of You. Briefly, ras^*Genes are causally linked to neoplastic phenotypes It is an attractive target because it is certainly a widespread clear cancer (eg pancreatic carcinoma, colon carcinoma) , And pulmonary adenocarcinoma). Furthermore, ra s^*The gene is found in preneoplastic tumors, so that immune-mediated May be performed prior to the detection of   The normal ras gene is non-tumorigenic and is ubiquitous in all mammals. this is It is highly conserved during evolution and is important for maintaining the cell cycle and normal growth characteristics. Seems to play a key role. Normal ras protein binds to GTP and increases GTPase activity G-protein that transmits signals from the external environment to the inside of the cell To enable cells to respond to the environment. one Ras^*Genes help normal neoplastic cells by decoupling their behavior from the environment. Alters growth regulation, thereby leading to uncontrolled growth of neoplastic cells. ras remains Gene mutations are thought to be an early event in carcinoma formation (Kumar et al., Activation o f ras Oncogenes Preceding the Onset of Neoplasia "Science 248: 1101-1104 , 1990), which may prevent tumor formation if treated early.   ras^*Genes include a wide range of cancers, including pancreatic, colorectal, and adenocarcinoma of the lung. ). However, ras found in various cancers^*Range of mutations occurring in genes The enclosure is very limited. These mutations constitute a normal on / off switch GTPase activity of ras protein is changed by switching to the on position Let ras^*The oncogenic mutations in are mainly (in vivo) three codons: 12, 13, And only happens at 61. Codon 12 mutations in both human and animal tumors Is the most.   In another embodiment of the present invention, the modified p53 (p53^*) Recombination to direct gene expression Eh, retroviruses are provided. Briefly, p53 is used to extract transformed cells It is the first nuclear phosphoprotein found in an object, and therefore (Linzer and Levine, Cell 17: 43-52, 1979; Lane and Craw ford, Nature 278: 261-263, 1979). Later, the original p53 cDNA clone is It was found to be in body form (Hinds et al., J. Virol. 63: 739-746, 1989). now P53 is a tumor suppressor gene that negatively regulates the cell cycle, and It appears that mutations in offspring can lead to tumor formation. 75% to 80% of colorectal carcinomas studied % Indicate deletion of both p53 alleles (one due to deletion, the other to point mutation) According). Similar mutations are found in lung and brain and breast tumors.   Many p53 mutations (eg, p53^{* 1}, P53^{* 2}Etc.) between amino acid residues 130-290 (See Levine et al., Nature 351: 453-456, 1991; and See also the following references which describe specific mutations in more detail: Baker et al., Science.  244: 217-221, 1989; Nigro et al., Nature 342: 705-708, 1989 (p53 mutations Focus on four “hot spots” that fit highly conserved gene regions and These mutations have been observed in human brain, breast, lung, and colon tumors Vogelstein, Nature 348: 681-682, 1990; Takahashi et al., Science 246: 4. 91-494, 1989; Iggo et al., Lancet 335: 675-679, 1990; James et al., Proc. Natl. Ac ad. Sci. USA 86: 2858-2862, 1989; Mackay et al., Lancet 11: 1384-1385, 1988; Kelman et al., Blood 74: 2318-2324, 1989; Malkin et al., Science 250: 1233-1238, 1 990; Baker et al., Cancer Res. 50: 7717-7722, 1991; Chiba et al., Oncogene 5: 1603. -1610, 1990 (Early etiology of early non-small cell lung cancer was between codons 132 and 283 of the p53 gene. Prosser et al., Oncogene 5: 1573-1579, 1990 ( A mutation in the p53 gene encoding amino acids 126-224 was identified in early breast cancer); C heng and Hass, Mol. Cell. Biol. 10: 5502-5509, 1990; Bartek et al., Oncogene.  5: 893-899, 1990; Rodrigues et al., Proc. Natl. Acad. Sci. USA 87: 7555-7559 Menon et al., Proc. Natl. Acad. Sci. USA 87: 5435-5439, 1990; Mulliga n et al., Proc. Natl. Acad. Sci. USA 87: 5863-5867, 1990; and Romano et al., Onc. ogene 4: 1483-1488, 1990 (p53 modification of codon 156 of human osteosarcoma-derived cell line HOS-SL) Differentiation).   Certain modifications of the p53 gene may be due to certain specific toxins. For example Bresac et al. (Nature 350: 429-431, 1991) reported that patients with hepatocellular carcinoma had A specific G to T mutation at don 249 is described. One suggestion of this mutation The known causative agent is a liver carcinogen known as a food contaminant in Africa Aflatoxin B is quality₁It is.   The four regions of the gene that are particularly affected are residues 132-145, 171-179, 239-248 , And 272-286. Three particularly interesting “hot spots” are residues 17 5, 248, and 273 (Levine et al., Nature 351: 453-456, 1991). these Protein containing a novel coding sequence by modification of Is produced. The novel proteins encoded by these sequences are It can be used as a marker for tumorigenic cells and binds to these novel coding regions. The immune response directed by the modified sequence (p53^*Destroys tumorigenic cells containing Can be used to   In another embodiment of the present invention, the modified Rb (Rb^*) Recombination directing gene expression A retrovirus is provided. Briefly, retinoblastoma is a chromosome van Ocular cancer in children associated with deletion of a locus called Rb located at 13q14 It is. A gene from this region has been cloned, which is approximately 110 kd. Produces nuclear phosphoproteins (Friend et al., Nature 323: 643, 1986; Lee et al., Scien ce 235: 1394, 1987; and Fung et al., Science 236: 1657, 1987).   Rb is thought to be a negative regulator of cell growth, and regulates transcriptional regulation and It has a role in cell cycle regulation. Rb is at least found in the nuclear Binds to seven proteins, especially E2F (Bagchi et al., Cell 62: 659-669, 1990) and And DRTF (Shivji and La Thangue, Mol. Cell. Biol. 11: 1686-1695, 1991). It appears to be involved with a cellular transcription factor referred to as both. Rb is involved in cell proliferation To limit cell proliferation by isolating various nuclear proteins Have been.   A deletion in the Rb gene has been detected. This indicates that the Rb gene may be responsible for tumorigenicity. Demonstrate that. These deletions include, for example, prostate cancer and bladder cancer cells Deletion in exon 21 of the strain (Bookstein et al., Science 247: 712-715, 1990; Horo witz et al., Science 243: 937, 1989), exon 16 deletion in small cell carcinoma of the lung (Shew). Cell Growth and Diff. 1:17, 1990), and the deletion between exons 21 and 27 (Shew et al., Proc. Natl. Acad. Sci. USA 87: 6, 1990). These d Deleted exon contains new coding sequence at junction of deleted exon This results in the production of proteins that This novel protein coding sequence is Can be used as a marker for forming cells, and to this novel coding region A directed immune response may eliminate tumorigenic cells containing the Rb exon deletion.   In another embodiment of the invention, the expression of a modified gene responsible for Wilms tumor is described. A recombinant retrovirus directed to the present is provided. Briefly, Wilm Tumors are typically found in children younger than 16 years. This tumor affects one child in 10,000 Tumors develop, which make up about 5% of children's cancer. This tumor is usually Presents itself as a large abdominal mass surrounded by a fibrous pseudocapsule. About 7 of the tumor % Are multifocal in one kidney and 5.4% involve both kidneys You. The Wilms oncogene is located on chromosome 11p13 and is specific for tumor suppressor genes. A characteristic cDNA clone (wt1) has been isolated (Call et al., Cell 60: 509, 1990; Gess ler et al., Nature 343: 744, 1990; Rose et al., Cell 60: 495, 1990; and Haber et al. Cell 61: 1257, 1990). The wt1 gene has four zinc fingers, glutamine and And a protein having a proline-rich amino terminus. like this These structures are likely to be involved in transcription and regulatory functions.   Mutations in the Wilms oncogene include the third and fourth zinc fingers. Insertion of lysine, threonine, and serine between the Such an insertion The wt1 protein containing the input does not bind to the EGR-1 site. The second alternative When a difference occurs, about 17 amino acids are located just NH_TwoTerminal Inserted into the region (Madden et al., Science 253: 1550-1553, 1991; Call et al., Cell 60 : 509, 1990; Gessler et al., Nature 343: 744, 1990; Rose et al., Cell 60: 495, 1990; Haber et al., Cell 61: 1257, 1990; and Buckler et al., Mol. Cell. Biol. 11: 1707, 1 991).   In another embodiment of the present invention, a recombinant retrovirus that directs the expression of a modified mucin Irus is provided. Briefly, mucin contains about 50% carbohydrates High molecular weight glycoprotein. Epithelial mucin (PEM) is a serum from cancer patients Tumor-associated mucins found therein (Girling et al., Int. J. Cancer 43: 1072-1076, 1989). A full-length cDNA sequence has been identified (Gendler et al., J. Biol. Chem. 26 5 (25): 15286-15293, 1990; Lan et al. Biol. Chem. 265 (25): 15294-15299, 1990 And Ligtenberg et al. Biol. Chem. 265: 5573-5578, 1990). Breast tumors and All pancreatic tumors were tandem repeats of 20 amino acids (Jerome et al., Cancer Res. 51: 2 908-2916, 1991) with the same core sequence. Developed in breast tumor CTL lines that reach and cross-react with pancreatic tumor targets Appear to specifically recognize system repeats (Jerome et al., Supra). 20 amino acid tande A sequence encoding one or more of the Expressed by the recombinant retrovirus of the present invention to produce an immune response Can be   In another embodiment of the invention, a modified DCC (deleted in colorectal carcinoma) inheritance Recombinant retroviruses are provided that direct expression of the offspring. Briefly, the conclusion A very common region of allelic deficiency in colorectal tumors is chromosome 18q, It is missing in more than 70% of carcinomas and almost 50% of late stage adenomas. this A putative tumor suppressor gene (DCC) from the region has been identified (Fearon et al., 1990 ). This includes neuronal cell adhesion molecules (NCAM) and contactin (contactin). Encodes a protein with significant homology to cell surface adhesion molecules (Edelma n, Biochem 27: 3533-3543, 1988). This protein is Possibly in normal cell-cell and / or cell-extracellular matrix interactions May play a role in the development of colorectal tumors You.   The DCC gene is expressed in normal colon mucosa, but its expression is in colorectal carcinoma Is reduced or absent in most of the population (Solomon, Nature 343: 412-414,  1990). This loss of expression may be associated with somatic mutations in the DCC gene . A continuous stretch of DNA having 370 kb has been cloned. This is Encodes a 750 amino acid protein (Fearon et al., “Identification of a C hromosome 18q Gene That Is Altered in Colorectal Cancers, Science 247: 49 -56, 1990).   In another embodiment of the invention, the expression of MCC (mutated in colorectal cancer) or APC Are provided. MCC and APC are both tumor suppressors It has been identified as a regulatory gene (Kinzler et al., Science 251: 1366-1370, 1991). It is mutated in familial sarcoma polyposis (FAP). FAP Is considered to be the most common autosomal dominant disease causing At least one in 5,000 individuals is affected in the United States (Nishiho et al., Scienc e 253: 665-669, 1991). In affected individuals, usually several hundred to several in the colon and rectum Thousands of glandular polyps are formed, which can progress to carcinomas. Gardner Syndrome ("GS", a variant of FAP), along with multiple glandular species of the colon and rectum, Present tendonomas, osteomas, and other neoplasms. This growth occurs on chromosome 5q Deficiency or missing familial adenoma polyposis genes (especially MCC and APC) It is thought to be induced by inactivation.   For example, in Nishiho et al. (Supra), the following APC remains in FAP and GS patients: Germline mutations in the gene were found: (1) Codon 280, serine to termination mutation (Patient with mandibular osteoma), (2) codon 302, two separate patients (one is tendonoid Arginine to termination mutation in (3) codon 414, mandibular osteoma Arginine to cysteine mutation in one patient, (5) codon 713, lower jaw Serine to termination mutation in another patient with osteoma (Nishiho et al., Science 253 : 665-669, 1991). In addition, six point mutations have MCC codon numbers 12, 145, 267, 490, 506, and 698, and four more somatic mutations in APC (Codon numbers 289, 332, 438, and 1338).   In another embodiment of the present invention, the device is functionally locked in an "on" or "off" mode. Recombinant retrovirus directing expression of a stacked or stacked modified receptor Is provided. Briefly, many cell receptors monitor the external environment. And participate in cell growth by sending signals to respond appropriately to cells doing. Lack of either the monitor or the signal transduction mechanism Cells no longer respond to the external environment and may exhibit uncontrolled growth . Many different receptors or receptor-like structures function as altered cellular components obtain. This includes, for example, neu and mutant or modified thyroid hormone receptors. Puter, PDGF receptor, insulin receptor, interleukin receptor -(Eg, receptors for IL-1, -2, -3, etc.) or G-CSF, GM-CSF, or M-C CSF receptors such as SF receptors are included.   For example, neu (human epidermal growth factor receptor “HER” or epidermal growth factor “EGF "Also called the receptor) is found in at least 28% of women who have breast cancer. Modified receptor. A cDNA clone encoding this protein has been isolated (Slamon et al., Science 244: 707-712, 1989; Slamon et al., Cancer Cells 7: 371-380, 1989; Shih et al., Nature 290: 261, 1981). This clone is extracellular Encodes a protein with a domain, transmembrane domain, and intracellular domain (Schechter, Nature 312: 513, 1984; Coussens et al., Science 230: 1132, 198 5) Therefore, it is thought to encode the neu receptor.   Studies of the rat neu gene isolated from chemically induced glioblastoma cells have Gene contains a single mutation from valine to glutamic acid at position 664 (Bar gmann et al., EMBO J .; 7: 2043, 1988). In other studies, N-ethyl-N-nitrosoureas Baby rats developed malignant tumors in the nervous system. 47 tridents that occurred Schwannomas and 12 schwannomas are both T to A bases at position 664 of the neu gene. (Nikitin et al., Proc. Natl. Acad. Sci. USA 88: 9939-9943, 1991).   Other modified receptors are also engineered to destroy selected tumor cells. Can be expressed by retroviruses. For example, a deletion in chromosomes 3p21-p25 Loss is associated with small cell lung carcinoma (Leduc et al., Am. J. Hum. Genet. 44: 282-287). , 1989). Deletion must encode DNA-bound thyroid hormone receptor (THR) For example, it may occur in the ERBAb gene.   By modifying the receptor as described above, a protein containing a novel coding sequence ( Or receptor). Coded by these arrays Novel proteins can be used as markers for tumorigenic cells, and these An immune response directed against a novel coding region of It can be used to destroy tumorigenic cells that have.   If the altered cellular component is involved in making the cells tumorigenic, It is necessary that the resulting cellular components be non-tumorigenic. For example, one embodiment The sequence or gene of interest encoding the altered cellular component is a gene product The ends are cut off to make the object non-tumorigenic. Code for altered cellular components Genes can be truncated to various sizes, but preserve the altered cellular components as much as possible. It is preferable to carry it. Furthermore, even if the end is cut, the immunogen of altered cellular components At least some of the sexual sequences need to be intact. Or multiple The translation termination codon is located below the immunogenic region in the gene encoding the altered cellular component. Can be introduced into the stream. Insertion of a stop codon terminates protein expression early and therefore To prevent expression of the transformed portion of the protein.   In one embodiment, ras^*The gene is ras^*Make proteins non-tumorigenic The ends are cut off. Briefly, ras^*The carboxy terminal amino acid of Functionally binds the protein to the cell membrane. Truncations at the ends of these sequences can change The resulting cellular components are rendered non-tumorigenic. Preferably, ras^*Genes form purine rings Here, for example, the end is cut near the sequence encoding amino acid number 110. About 20 amino acids (including modified amino acids) are encoded by recombinant retrovirus Ras as^*The ends of the gene sequence may be truncated, but preferably Of amino acids should be expressed (while remaining non-tumorigenic).   In another embodiment, p53 is used to render the cellular component non-tumorigenic.^*protein Are modified by truncation of the ends. As mentioned earlier, not all p53 proteins Mutations are not tumorigenic, so not all mutations need to be truncated There is no. Nevertheless, in a preferred embodiment, p53^*Replaces amino acids 100-300 Code, thereby terminating an array containing all four major "hot spots" Is disconnected.   Other altered cellular components that are tumorigenic also make them non-tumorigenic To be cut off. For example, both neu and bcr / abl The ends can be cut off to make them ulcerative. Non-tumorigenic, truncated as described above It can be confirmed by assaying the altered cellular components.   However, the altered cellular components are generally only associated with non-tumorigenic cells, and If it is not necessary or not necessary to make the cells tumorigenic, It should be noted that the minutes need not be non-tumorigenic. Not tumorigenic A representative example of such altered cellular components is Rb^*, Ubiquitin^*, And mucin^*To Including.   As mentioned above, in order to generate an appropriate immune response, the altered cellular components also Must be immunogenic. T cell epitopes are often immunogenic and amphipathic However, in many cases the immunogenicity of a particular sequence is not predicted. And difficult. However, it is generally preferable to determine immunogenicity in the assay. No. Typical assays detect the presence of antibodies against newly introduced vectors ELISA, and gamma interferon assay, IL-2 production assay, and Assays that test T helper cells, such as cell proliferation and proliferation assays. Immunogen A particularly preferred method for determining gender is the CTL assay.   Once the sequence encoding at least one anti-tumor agent is obtained, this sequence Preferably encodes a non-tumorigenic protein. specific Various assays for assessing the tumorigenicity of cellular components are known and readily accessible. Can be achieved. Representative assays include tumorigenesis in nude mice or rats. , Colonization in soft agar, and transgenic animals (eg, transgenic Genetic mouse).   For this and many other aspects of the invention, a nude mouse or rat Tumorigenesis is particularly important and sensitive in determining the tumorigenicity of antitumor agents It is an easy method. Nude mice lack a functional cellular immune system (ie, Have no CTL), and thus are useful for testing the potential of cells for tumorigenicity. Provide an in vivo model. Normal non-tumorigenic cells are injected in nude mice When displayed, it does not exhibit uncontrolled growth properties. However, transformed cells are Proliferates rapidly in mice and gives rise to tumors. Briefly, one In embodiments, the recombinant retrovirus is delivered to cells of a syngeneic mouse, Administered to nude mice. 2-8 weeks after administration to determine tumor growth, The mice are observed visually. Mouse to determine if tumors are present (Giovanella et al., J. Natl. Cancer Inst. 48: 1531-1533, 1). 972; Furesz et al., “Tumorigenicity testing of cell lines considered for pro Abduction Cells, New Products and Risk, Hopp s and Petricciani eds., Tissue Culture Association, 1985; and Levenbook J. et al. Biol. Std. 13: 135-141, 1985). Tumorigenicity is also due to the It can also be assessed by visualizing knee formation (MacPherson and Montagnie r, Vir. 23: 291-294, 1964). Briefly, normal non-tumorigenic cells One property is “contact inhibition” (ie, cells proliferate upon contact with adjacent cells). Stop). When cells are plated in semi-solid agar support medium, normal cells Is rapidly inhibited and stops growing, but tumorigenic cells continue to grow and soft agar Form colonies in it.   Transgenic animals, such as transgenic mice, also have antitumor agents. (See, eg, Stewart et al., Cell 38: 627). -637, 1984; Quaife et al., Cell 48: 1023-1034, 1987; and Koike et al., Proc. Natl.  Acad. Sci. USA 86: 5615-5619, 1989). In transgenic animals, eyes The target gene can be expressed in all tissues of the animal. Such a transgene Unregulated expression provides a model for the tumorigenic potential of newly introduced genes. Can be helpful.   In addition to tumorigenicity studies, recombinant retroviral venom is generally It is preferred to determine the gender. Using various methods well known to those skilled in the art, Can be measured. As an example, for example, various proteins and Clinical chemistry assays to measure systemic levels of enzymes and blood cell volume and number Is mentioned.   The present invention also provides a recombinant retrovirus capable of immune downregulation. Offer. Briefly, specific downregulation of inappropriate or unwanted immune responses Is an autoimmune disease or a pseudo-autoimmune disease (eg, chronic hepatitis, sugar Urine, rheumatoid arthritis, graft-versus-host disease, and Alzheimer's disease), or Immunization that suppresses the surface expression of transplantation (MHC) antigens in transplantation of xenogeneic tissue Manipulations can be performed using the epidemic suppressor virus gene product, or an active portion thereof. Departure In fact, the “active part” of a gene product is retained for biological activity. A fragment of the gene product that must be Such fragments Or active domains systematically remove nucleotide sequences from their protein sequences Transform target cells with the resulting recombinant retrovirus, and perform FACS analysis or Using other immunological assays (eg, CTL assays), MHC It can be easily identified by measuring Las I presentation. These fragments Indicates that the size of the sequence encoding the entire protein is the capacity of the virus carrier. It is particularly useful when the distance exceeds the threshold. Alternatively, MHC antigen-presenting inhibitor protein The active domain of proteins can be digested enzymatically and the active moiety Thus, it can be purified. For example, a monoblock that blocks the active portion of this protein Lonal antibodies are used to isolate and purify the active portion of the cleaved protein. (Harlow et al., Antibodies: A Laboratory Manual, Cold Springs Ha rbor, 1988).   In one embodiment, the suppression is achieved with accessory molecules (eg, CD8, cells). Intracellular adhesion molecule-1 (ICAM-1), ICAM-2, ICAM-3, leukocyte functional antigen-1 (LFA-1) (Altmann Et al., Nature 338: 521, 1989), B7.1-.3 molecule (Freeman et al., J. Immunol. 143: 2714). , 1989), LFA-3 (Singer, Science 255: 1671, 1992; Rao, Crit. Rev. Immunol). 10: 495, 1991) or other cell adhesion molecules) as well as self-MHC molecules. By specifically inhibiting the activation of presentation of the modified peptide You. Antigen peptide presentation associated with MHC class I molecules leads to CTL activation. MHC antigen Transfer and stability of specific sequences that can express products that are expected to inhibit presentation Built-in, CD8⁺Block activation of T cells such as CTLs, and thereby transfer Suppresses rejection of plants. A standard CTL assay is used to detect this response Can be As components of the antigen presentation pathway, 45Kd MHC class I heavy chain, β_Two-Micro gro Processing enzymes such as Blin, Protease, Accessory molecules, Carnequin Chaperones such as Singh (Gaczynska et al., Nature, 365: 264-282, 1993), and Transporter proteins (eg, PSF1, TAP1, and TAP2 (Driscoll et al., Nature, 365: 262-263, 1993)).   In another example, β_Two-Gene products or genes capable of binding to microglobulin Recombinant retroviruses that direct the expression of the active portion of the product are provided. Easy theory To elucidate, the transfer of MHC class I molecules to the cell surface for antigen presentation requires β_Two-Miku Requires association with logoglobulin. Thus, β_Two-Binds with microglobulin And proteins that inhibit its association with MHC class I are indirectly Inhibits Las I antigen presentation. Suitable proteins include the H301 gene product Can be Briefly, H30 obtained from human cytomegalovirus (CMV) One gene is the β on the heavy chain of the MHC class I molecule._Two-Microglobulin binding site and sequence phase Encodes a homosexual glycoprotein (Browne et al., Nature 347: 770, 1990). H3 01 is β_Two-Binds microglobulin, thereby preventing MHC class I molecule maturation To render the transformed cells unrecognizable by cytotoxic T cells, Escape I-restricted immune surveillance.   In another embodiment, binds a newly synthesized MHC class I molecule intracellularly Recombinant retrovirus directing expression of a protein or active portion of a protein Is provided. This binding prevents the transfer of MHC class I molecules from the endoplasmic reticulum, Inhibition of the terminal glycosylation occurs. This is the movement of these molecules to the cell surface To prevent cell recognition and lysis by CTL. For example, the E3 gene One of the products is used to inhibit the transfer of MHC class I molecules to the surface of transformed cells. Can be used. More specifically, E3 is transcribed from the E3 region of the adenovirus 2 genome Encodes the 19kD transmembrane glycoprotein E3 / 19K. In the context of the present invention, Woven cells are recombinant retroviruses containing E3 / 19K sequences that upon expression produce E3 / 19K proteins. Transformed with virus. The E3 / 19K protein is expressed on the surface of MHC class I surface molecules. Inhibits expression and allows cells transformed by the recombinant retrovirus to escape the immune response You. As a result, donor cells can be transplanted with reduced risk of graft rejection, Even minimally immunosuppressive measures may be required in transplanted patients. this is, That an acceptable donor-recipient chimera condition exists with fewer complications And enable. Similar treatments include systemic lupus erythematosus, multiple sclerosis, chronic To treat so-called autoimmune diseases, including rheumatoid arthritis or chronic hepatitis B infection Can be used for   Alternative immunosuppressive methods include antisense messages, ribozymes, or existing Of other gene expression inhibitors specific for a self-reactive T cell clone Use. These are the T cells of specific unwanted clones responsible for the autoimmune response. Block vesicle receptor expression. Antisense, ribozyme, or other Can be introduced using a viral vector delivery system.   Inhibits or suppresses MHC class I antigen presentation with other proteins different from those described above Or proteins with down-regulating functions have also been identified. And may be utilized within the context of the present invention. Such proteins, especially Feeding Identify proteins from animal pathogens (and, alternatively, their active parts) Therefore, a protein or its activity that is considered to be able to inhibit MHC class I antigen presentation Recombinant retrovirus expressing the sex part transforms a tester cell line such as BC Is replaced. Tests with and without sequences encoding the candidate proteins The cell line to the stimulator and / or target in the CTL assay Are compared. A decrease in cell lysis corresponding to the transformed tester cells is Figure 4 shows that proteins can inhibit MHC presentation.   For many infectious diseases, cancer, autoimmune diseases, and other diseases, viral particles And cell, cell to cell, or cell to factor interactions. Virus infection In general, viruses enter cells through receptors on the surface of susceptible cells. In cancer, cells may inappropriately respond to signals from other cells or factors Or no response. Inappropriate "self" marker in autoimmune diseases There is a keen perception. In the present invention, such an interaction is referred to as an interaction. Recombinant in vivo producing analogs for any of its partners It can be blocked by using ills.   This blocking effect can occur intracellularly, on the cell membrane, or extracellularly. Bro Of viruses (especially recombinant retroviruses) that carry genes for Blocking action can be from inside susceptible cells or localize pathogenic interactions Mediated by either blocking or secreting a type of blocking protein Can be done.   For example, in the case of HIV, the two interacting substances are the gp120 / gp41 envelope proteins. Proteins and CD4 receptor molecules. Therefore, a suitable blocker may HIV env analog or CD4 receptor that blocks HIV entry without triggering A recombinant retrovirus that expresses any of the analogs. CD4 analog Gp120 / gp41 is secreted and functions to protect adjacent cells, while gp120 / gp41 Secreted or produced only intracellularly to protect You. Human immunoglobulin heavy chain added to CD4 for increased stability or complement solubility Or it may be advantageous to add other components. Such a hybrid possible Delivery of a recombinant retrovirus encoding soluble CD4 to the host is a stable hybrid. Results in a continuous supply of pad molecules.   Vector particles that direct the expression of HIV env can also be constructed. Which part is obvious disease It is obvious to a person skilled in the art whether virus adsorption can be prevented without the side effects of the origin (Wille y et al., J. Virol. 62: 139,1988; Fisher et al., Science 233: 655,1986).   Another aspect of the invention is when a cell type is deleted, mutated, or not expressed. And delivery of suppressor genes that lead to tumor formation in that cell type. Virus The reintroduction of the deleted gene by the vector will result in a tumor phenotype in these cells Leads to involution. Malignant tumors are thought to inhibit terminal differentiation of cells compared to cell proliferation As such, the delivery and expression of gene products that lead to tumor differentiation may also generally be It should lead to involution.   Sequences encoding the above nucleic acid molecules can be obtained from a variety of sources. For example, Plasmids containing sequences encoding modified cell products are, for example, American Type Cult ure Collection (ATCC, Rockville, Maryland) or Advan obtained from commercial sources such as ced Biotechnologies (Columbia, Maryland) obtain. Representative examples of plasmids containing some of the above sequences include ATCC No. 41 000 (including the G to T mutation at the 12th codon of ras), and ATCC No. 41 049 (including the G to A mutation at the twelfth codon).   Other nucleic acid molecules encoding the above substances, as well as for use in the present invention Other nucleic acid molecules that are advantageous for are available from various sources, such as the American Type Culture Collec. contractors (ATCC, Rockville, Maryland) or British Bio-Tec readily available from commercial sources such as hnology Limited (Cowley, Oxford, England) can get. A typical example is BBG12 (GM encoding a 127 amino acid mature protein). -Contains the CSF gene), BBG6 (containing the sequence encoding gamma interferon) ATCC No. 39656 (including the sequence encoding TNF), ATCC No. 20663 (α No. 31902 and No. 39517 (including β-interferon). ATCC No. 67024 (including interleukin-1b). No. 39405, No. 39452, No. 39516, No. 39626, and N o.39673 (including the sequence encoding interleukin-2), ATCC No.59399, No.59 398, and No. 67326 (including the sequence encoding interleukin-3), ATC C No. 57592 (including the sequence encoding interleukin-4), ATCC No. 59394 and And No.59395 (including the sequence encoding interleukin-5), and ATCC No.671 53 (including the sequence encoding interleukin-6).   The genome that encodes the molecularly cloned hepatitis B virus is Sources such as the American Type Culture Collection (ATCC, Rockville, Maryland ). For example, ATCC No. 45020 is based on pBR322 (Moriarty et al., Proc. Nat. l. Acad. Sci. USA 78: 2606-2610, 1981) Whole genomic DNA of hepatitis B at the BamHI site (Extracted from purified Dane particles) (Blum et al., TIG 5 (5): 154-158, 1989) 3). (Note that correctable mistakes occur in the ATCC No. 45020 sequence. Meaning).   Alternatively, a cDNA sequence for use in the present invention expresses or comprises that sequence It can be obtained from cells. Briefly, in one embodiment, the gene of interest is MRNA from cells expressing the offspring is reversed using oligo dT or random primers. Reverse transcribed by transcriptase. Then, the single-stranded cDNA is replaced with any of the desired sequences. PCR using oligonucleotide primers complementary to the sequence on the side of Nos., 683,202, 4,683,195 and 4,800,159. PCR Te chnology: Principles and Applications for DNA Amplification, Erlich (eds.) ), See also Stockton Press, 1989). In particular, double-stranded DNA Stable Taq polymerase, sequence-specific DNA primers, ATP, CTP, GTP and TTP Denatured by heating in the presence. When the synthesis is completed, double-stranded DNA is produced. This cycle can be repeated many times, and the desired DNA is amplified multiple times.   Retained and / or retained by a recombinant retrovirus described herein. The expressed nucleic acid molecule is, for example, a DNA synthesizer (for example, Applied Biosystems Inc.) For example, it can be synthesized using APB DNA synthesizer model 392 (Foster City, California). C.Pretreatment of target tissue:   Retroviral vectors are known to preferentially infect dividing cells (See Miller et al. (1990) Molec. Cell Biol. 10: 4239). In the target tissue Increase the number of dividing cells, thereby marking with the retroviral vector of the invention. There are various techniques that can be used to increase the efficiency of targeted cell infection. example For example, growth factors stimulate target cells to allow for retrovector integration. Can be used to penetrate part of the cell cycle. Such growth factors and their targets Objective organizations include, but are not limited to, the following:   Proteins S and Gas6 acting on nervous system cells and smooth muscle cells; smooth muscle cells , Gastrointestinal epithelium, liver obesity storage cells, dental pulp cells, fibroblasts, endothelial cells, mesangiu Thrombin acts on astrocytes and astrocytes; Xa clotting factor; nerve growth factor acting on nervous system cells; acting on placenta and endometrium CSF-1; kidney, bone, skin, adipose tissue, airway smooth muscle cells, gastrointestinal epithelium, nervous tissue, IGF-1 acts on muscle and vesicle cells; acts on gastrointestinal epithelium, skin, and adipose tissue Insulin for use; urothelium (urotherium), mammalian epithelium, skin, liver, and KGF acting on gastrointestinal epithelium; gastrointestinal epithelium, dental pulp, nerve tissue, fibroblasts, connective tissue, TGs acting on inner ear sensory epithelium, colon, bone, type II alveolar cells, cornea, and smooth muscle cells F; acts on kidneys, smooth muscle cells, melanocytes, myocardium, and astrocytes Endothelin; in kidney, airway smooth muscle cells, gastrointestinal epithelium, nervous tissue, and connective tissue Acting PDGF; kidney, skin, nerve tissue, inner ear sensory epithelium, connective tissue, fibroblasts, EGF acting on the endometrium, liver, and intestine; liver, kidney, mammalian epithelium, GI HGF acting on skin, alveolar epithelium, melanocytes, placenta, and type II alveolar cells; prostate PSA acting on glands, breast, lung, colon, ovary, liver, and kidney; liver, kidney, and And HGF activators acting on human and mammalian epithelium; Transcellular, kidney, endothelium, fibroblast, skin, skeletal muscle, connective tissue, melanocyte, horn Membrane, bone marrow, dental pulp cells, liver, melanocytes, smooth muscle cells, and thyroid follicle cells Acts on FGF; VEGF acts on endothelial cells; acts on liver, kidney, and fibroblasts Arg vasopressin used; thyroid hormone acting on bone; azo acting on colon Ximethane; Prostaglandin acting on liver and dental pulp; Acting on fibroblasts IL1 which acts on T cells; IL15 which acts on muscle; Triyo which acts on liver Dothyronine; LIF acting on muscle and bone; amphiregulin acting on skin (am phiregulin); soluble thrombomodulin acting on fibroblasts; erythroid precursor Cell factor acting on osteoblasts; chondrocytes and osteogenic protein 1 acting on bone; liver Bone morphogenetic protein acting on the kidney; MGF acting on melanocytes; Works on mammalian epithelium, keratinocytes, and Schwann cells Heregulin; and melanotropins acting on melanocytes. Growth factors It can also be used in combination, but in particular one or several EGF, IGF, PDGF , FGF, or KGF. In particular, the details of a particular target tissue Several growth factors known to stimulate mitosis are found in dividing cells in tissues Can be used in combination to increase the proportion of Above and other growth factors The action of dextran (dextan) sulfate, heparin, and other sulfated Cosaminoglycan, FBP, leukotriene, prostaglandin, oleic acid, H GF activator, androgen, estrogen, ethanol, PF4, and T Can be fortified with substances including but not limited to GFβ antagonists . Treatment with the growth factors or other substances described above also involves administering the substance in vivo. Administration, or can be used in other treatment preparations, such as ex vivo treatments. Can also be used.   The liver is an attractive organ for gene therapy. Because the circulation Various proteins involved in genetic abnormalities (including factor VIII) )). Liver using the retroviral vector of the present invention Targeted gene therapy involves pre-treatment to induce benign hyperplasia of the liver. Or without. Pretreatment for inducing benign hyperplasia of the liver is, for example, Transforms growth factor alpha by treatment with liver growth factor (hGF) and / or (See, for example, Liu et al., (1994) Hepatology 19: 1521. When). In addition to growth factor treatment, liver cell growth can also be stimulated by nutritional manipulation You. A variety of different nutritional regimens can be used. For example, protein for a certain period Consumption of high protein diets after deficiency may stimulate DNA synthesis and cell division (Mead et al., (1990) Cancer Res. 50: 7023-7030).   Another example of stimulating cell division in certain tissues is the use of cyclooxidase inhibitors. (Eg, indomethacin that induces proliferation in the gastric mucosa). In particular, indomethacin is involved in DNA synthesis and duodenal and jejunal mucus. It is known to increase cell clearance (Uribe et al., (1992) Dig.Dis.Sc i.37: 403-408). Therefore, indomethacin or nonsteroidal anti-inflammatory Pretreatment with symptomatic agents can be used to increase cell proliferation in the gastric mucosa You. In particular, in addition to prostaglandins, prostaglandin E2 is transduced. Can be used after introduction of a gene therapy vehicle to increase the retention of isolated enterocytes You. D.Cell culture   As mentioned above, the present invention provides a high titer recombinant retrovirus suitable for administration to humans. Provide an ils preparation. To produce such high titer preparations, Cell culture methods are provided to be able to produce high titers of recombinant retrovirus. Briefly, a wide variety of methods (eg, fermenters or bioreactors, Roller bottles, cell hotels or cell factories, and hollow fiber media (Including the use of nutrition).   Particularly for bioreactors or fermenters, the cells are preferably Carrier (ie, Cytodex 1 or Cytodex 2; Pharmacia, Piscataway, N.J. 3-15). g / L microcarriers). Appropriate medium and growth medium The case is described by a representative illustration of Example 17.   For roller bottles, the appropriate condition is not to use microcarriers With the exception of the conditions described above for the bioreactor. In general, cells 850 cm containing DMEM medium supplemented with 15% to 20% FBS^TwoRoller bottle ("FALCON" Corning , Corning New York). Preferably, bottles avoid contamination Bottles are also closed under appropriate conditions (eg, 5% CO2)._Two ). Generally, roller bottles are rotated at 0.5 rpm / min at 37 ° C. Incubate at temperature.   Cell factories also support large-scale cell culture and production of recombinant retroviruses. Can be used for raw. In short, Cell Factory ("Cell Hotel") ) Typically contain 2, 10, or 40 trays, and Molded from untreated polystyrene and treated to provide a Nuclon D surface And assembled by ultrasonic bonding together. Generally, these Some factories contact the chamber for reagent addition or media removal. It has two port tubes to allow access. 10-layer factory About 6000cm^TwoSurface area, which is approximately equal to a 27 T-225 flask . Cell factories are available from a variety of manufacturers, including, for example, Nunc. You.   Most cell types can produce high-titer vectors in media for 3-6 days, To harvest. Each cell type has an optimal harvest time after seeding the culture, and an optimal yield. Tested to determine harvest days. Cells are typically seeded into a cell factory. Until the required cell number for seeding is obtained, first 2-20 in roller bottles Grow in DMEM supplemented with% FBS. The cells are then seeded in the factory And culture supernatant containing 2 L of vector is harvested daily for 4 days. Fresh Replenish the culture with DMEM / FBS.   In another aspect of the present invention, the hollow fiber culture method comprises Provided for the production of Briefly, cells are denser in reduced media High-titer retrovirus production using hollow fiber cultures Is based on increasing virus concentration. Cells are nourished and waste is numerous Diluted with a large volume of fresh medium circulating through the capillary fiber lumen. Cells are promoted by the diffusion of cellular waste through 30,000 dalton holes in capillary fibers. The space outside the capillary fibers in the bioreactor chamber that is exchanged for nutrients Cultured in 30,000 retroviruses produced from cell lines are very large It cannot pass through the membrane of Dalton holes. Therefore, this retrovirus is hollow on the cell side. It is concentrated in the fiber bioreactor. The volume of medium cultured on the cell side is For equivalent cell density grown in tissue culture dishes or flasks About 10-100 times lower than the required capacity. Tissue capillary retrovirus titer Compared to titers in culture dishes or in flasks, The decrease is inversely correlated with the fold induction of the titer. Individual retroviral producer cells If the strain is suitable for capillary fiber growth conditions, a 10-100 fold induction in titer is shown . In order to achieve maximum cell density, individual cell lines should be very close and Must be able to proliferate at each other's tips. Many cell lines grow in this fashion. And retroviral packaging cells based on these types of cell lines The stock is You may not achieve a 10-fold increase in titer. Cells that grow very well The strain is a non-adherent cell line, and a retrovirus based on the non-adherent cell line Sproducer strains have a titer compared to tissue culture dishes or flasks. And achieve a 100-fold increase.   The harvesting procedure in the original design involves harvesting the produced vector, In this procedure, the culture supernatant is replaced with a new one. This syringe procedure There is an associated high risk potential contamination, which is associated with a significant number of manual connections in the media flow path. Requires binding and non-binding. However, it reduces the risk of contaminating the culture and reduces When needed to increase the daily capacity that can be harvested and to operate the culture system A convenient procedure to reduce the time has been devised. Currently, harvesting procedures are Using a batch peristaltic pump to deliver the same volume and exchange for the same volume of harvested material It is delivered through a thin wire tube to a collection bottle, which is then stored at 4 ° C. Po The batch array is activated by a timer that can be set at a specific time, and Pump to harvest a set volume of any harvested material 24 hours a day Can be adjusted. The recovered supernatant is then frozen and pooled with the previous crop. Or processed as described elsewhere. This recovery procedure is optional Empty fiber system (Cellco (Rockville including ceramic matrix high-density culture system) , Maryland), Unisyn (Tustin, CA), or Cellex (Coon Rapids, MN) Can be used for E. FIG.Enrichment and purification of recombinant retroviral particles   As noted above, the present invention is directed to increasing the purity of a therapeutic preparation, as well as administering To increase the titer of recombinant retroviruses that can be A method for the concentration and purification of a solution is provided. A wide variety of methods (eg, sulfuric acid Precipitation of recombinant retrovirus using ammonium, polyethylene glycol (" PEG ”) concentrated, in the presence of a gradient such as PERCOLL or a“ buffer ”such as sucrose. Or by centrifugation, either in the absence or presence, using a concentration filter (eg, , Amicon filtration), and two-phase separation) to increase virus concentration and purity Can be used for Each of these methods is described in further detail below.   Briefly, precipitation of recombinant retrovirus using ammonium sulfate Add ammonium sulfate slowly to the appropriate concentration to achieve Followed by centrifugation and separation on either a dialysis or hydrophobic column. Remove ammonium sulfate depending on However, the difficulty with this method is that In addition to retrovirus enrichment, other protein disruptions can also be enriched. And   Alternatively, the recombinant retrovirus can be concentrated from the culture medium using PEG ( Green et al., PNAS 67: 385-393, 1970; Syrewicz et al., Appl. Micro. 24: 488-494, 1972). Such a method is quick, simple, and inexpensive. However, sulfuric acid Like ammonium precipitation, the use of PEG also enriches other proteins from solution. You.   In another embodiment, the recombinant retrovirus is centrifuged, more particularly, It can be concentrated by low speed centrifugation. Briefly, low-speed centrifugation is Can concentrate Torovirus, while related to pelleting associated with high speed centrifugation Avoid difficulties (eg, virus destruction or inactivation). By low speed centrifugation A particularly preferred method of virus concentration is described in further detail below in Example 15. You.   In yet another aspect of the invention, the recombinant retrovirus is used in an aqueous two-phase separation method. It can be more concentrated. Briefly, an aqueous two-phase system of a polymer has two distinct Prepared by dissolving an incompatible polymer. Many water-soluble polymers Pairs are used to construct such two-phase systems (eg, polyethylene glycol (“PEG”)). Or methylcellulose and dextran or dextran sulfate) (Walter and Johansson, Anal.Biochem. 155: 215-242, 1986; Albertss on, “Partiton of Cell Particles and Macromolecules” Wiley, New York, 1960 See). As described in more detail in Example 13 below, PEG is 5 % To 8% (preferably 6.5%), and dextran sulfate at 0.4%. % To 1% (preferably, 0.4%). Aqueous two-phase systems suitable for the purification of can be established. Using such a procedure, about 1.4 One liter of crude research grade supernatant recovers about 50% of total starting retrovirus While it can be reduced to a 10 mL volume.   One exemplary enrichment combining several enrichment stages for illustrative purposes The process is shown below. Briefly, recombinant retroviruses must be , From a roller bottle, cell factory, or bioreactor Can be prepared. Preferably, each recombinant retrovirus from the producer cells is Day harvesting followed by addition of fresh medium is preferred. Contains recombinant retrovirus The removed medium can be frozen at -70 ° C or, more preferably, treated. Can be stored at 2 ° C. to 8 ° C. in a large pooled batch before   For materials obtained from bioreactors, the recombinant retrovirus pool Was first connected to a 0.8um filter (Sartorious) in series with a 0.8um filter (Sartorious). 1.2um glass fiber prefilter, 0.8um cellulose acetate) Clarified. This filter array provides about 2 square feet of filter, And about 15-20 liters of pooled material can be processed before clogging. roller -For substances obtained from bottles or cell factories, one 0.65um Cartridges (2 square feet) typically handle volumes up to 40 liters. 80 l A 5-square-foot filter is required for the Tuttle cell factory process. May be required.   Preferably, after clarification, filters are buffered (150 mM NaCl, 25 mM Tris (pH 7.2- Rinse with 7.5)). By this step, a range of 80% to 120% of the recombinant retrovirus Could be recovered.   After clarification, recombinant retrovirus is cut with Filtron unit and 300,000 mw Tangential flow ultrafiltration using off Sigma Screen cassette Concentrate. For bioreactor materials (including 12% -16% FBS) Five liters of material can be concentrated per cassette. Roller with 12-16% FBS For bottles or cell factories, 5-6 liters of material should be placed in the cassette. Can be concentrated. Finally, about a cell factory containing 10% FBS Can concentrate 8-9 liters of material per cassette. Filtrate (8psi) and residual Using a 2 psi pressure difference between the liquid (10 psi) and up to 80 liters of material for 2 hours Below it can be concentrated to a volume of less than 500 mL. This process also yields about 80% I will provide a.   Following the ultrafiltration step, DNAse is added to a concentration of 50 U / mL and closed for 30 minutes. Recirculate at a lower pump speed using a filtered filtration line. Retro virus This step can be performed if the gel is trapped in the gel layer formed during ultrafiltration. Destroy retroviruses capped and improve recovery.   Then discontinuous diafiltration adds 4 liters of additional buffer. Addition and achieved by utilizing the same cross-differential pressure as described above. In general, The recovery after this step is about 70%.   The concentrated material was then reduced to a concentration of minimum salt and ionic strength of 50 mM NaC Phamacia S-500 HG size exclusion gel utilizing l and 25 mM Tris (pH 7.2-7.5) For column chromatography. Generally, recombinant retroviruses are first Elute at the peak of   Tangential flow filtration once again reduces the volume to less than 200 ml. Can be used to reduce. Finally, the concentrated material is Sterilized by filtration through a filter (PVDF or Sterivex). F.Assay   In another aspect of the present invention, a method for evaluating or quantifying the obtained product ( For example, staining with Coomassie blue or silver stain, followed by densitometry Along with the non-denaturing gel (e.g., 4-15% gradient A method for quantifying retroviral particles using acrylamide gel) has been proposed. Provided. Such methods involve the interaction between viable and non-viable vector particles. Although indistinguishable, they are advantageous because they are relatively simple and fast. this One representative example of such a method is shown in more detail in Example 10 below.   In another aspect of the invention, the assay comprises detecting the recombinant retrovirus in the sample. Provided for measuring titer. Typically, such assays are Or the formation of blue clones. But certain implementations , A recombinant retrovirus that does not contain a gene encoding a selectable marker Is provided. Therefore, antibodies and PCR assays (the latter of which are described below) ) Can be used to determine retroviral titers. Using PCR Thus, to amplify sequences unique to the recombinant retroviruses described herein, Various primers are required. Such primers are readily available to those skilled in the art. Retroviral vector backbones that can be designed and used in And the particular region desired to be amplified. Specific primer pairs Typical examples are LTR sequences, packaging signal sequences or retroviruses. The nucleic acid molecule of interest (ie, non-heterologous sequence) contains sequences specific to other regions of the backbone. ) Is included.   Briefly, in one embodiment of the invention, the PCR titration assay is Using recombinant retroviruses in 6-well plates for at least 16 hours before harvest It is performed by growing a known number of transduced cells. One way per plate Is sacrificed for counting. Lyse cells from other wells, and Isolate the contents. Transfer the DNA to the QUIAmp DNA isolation kit (QUIAgen, Inc., Chatsworth, (CA). 5 x 10 DNA per sample⁶Resuspend in single cell equivalent / μL I do.   To calculate the titer, the standard curve was 5 × 10⁶Non-transduced HT 1080 cells Negative control) and a known vector. 5 × 10 with 1 copy of the vector per system⁶HT 1080 cells (positive (For example, neomycin) Transduced with a recombinant retrovirus encoding a selectable marker such as resistance Can be prepared from packaging cell lines). This standard curve represents different amounts of positive Combination of active and negative control DNA and recombinant retrovirus PCR using primers specific to specific regions of Created by amplifying a column. The representative group of the mixture for preparing the standard curve is as follows. Shown below:   5 μl from each tube is placed in one of the eight reaction tubes (duplicates are also preferred). And save the rest at -20 ° C. Duplicate 5 μl from each sample DNA preparation And put it in its own reaction tube. The PCR reaction (50 μL total volume) was then tested Add 45.0 μL of reaction mixture containing the following components per tube to be performed Started by: 24.5 μL water, 5 μL 10 × reaction PCR buffer, 4 μL 25 mM  MgCl_Two4 μL of dNTPs (including 2.5 mM of each dATP. DGTP, dCTP and dTTP), 5 μL of primer mix (100 ng or each primer), 0.25 μL of TaqStart Mono Clonal antibody (Clontech Laboratories, Inc., Palo Alto, CA), 1.00 μL TaqStar t buffer (Clontech Labs, Inc.) and 0.25 μL AmpliTaq DNA polymerase (Perk in-Elmer, Inc., Norwalk, CN). Immediately before aliquoting the reaction mixture into reaction tubes, μL α-³²P dCTP (250 μCi; 3000 C / mmol, 10 mCi / mL, Amersham Corp., Arlingto n Heights, IL) is added to the reaction mixture. Add 45.0 μL of the reaction mixture to each reaction tube. After aliquoting, cap tube and place in thermocycler. specific Denaturation, annealing, extension time and temperature, and the number of thermocycles It will vary depending on the size of the primer pair used and the nucleic acid composition. Then 20 Perform ~ 25 amplification thermocycles. Then, 5 μL of each reaction was added to DE81 ion exchange. Spotted on exchange chromatography paper (Whatman, Maidstone, England), Then air dry for 10 minutes. The filters were then washed with 100 mL 50 mM Na per wash._TwoPO_Four Washed 5 times with 200 mM NaCl, pH 7, and then air-dried. To switch. Quantification was performed using PhosphoImager SI (Molecular Dynamics, Sunnyvale, CA) Done in Typically, the filter is exposed to a phosphor screen. This screen Lean provides energy from ionizing radiation for an appropriate period of time, typically about 120 minutes. Store. After the exposure, the phosphor screen is scanned, which restores the light Emitted in proportion to the radioactivity on the filter. Then down the scan results Load and cpm (ordinate) vs.% of positive control DNA (abscissa) Plot on a log scale. The titer of each sample (infection unit / mL) is The recombinant retrovirus used to transduce the cells to the number of cells isolated Percentage determined from a standard curve based on the detected radioactivity divided by the volume of lus Calculated by multiplying (converted to decimal form). Approved by those skilled in the art As will be appreciated, other detection methods, such as colorimetry, also label the amplification product Can be used for G. FIG.Prescription   In another aspect of the invention, the recombinant retrovirus is a mammalian Methods are provided for storing infectious recombinant retroviruses so that they can infect cells. (See US patent application Ser. No. 08 / 153,342). Briefly, as above The purified or concentrated recombinant retrovirus forms an aqueous suspension First, a sufficient amount of formulation buffer is added to the medium containing the recombinant retrovirus. It can be preserved by adding it to the ground. The formulation buffer contains saccharides in water, high An aqueous solution containing a molecular weight structure additive and a buffer component. Range related to the present invention As utilized in, "buffer compounds" or "buffer components" are used in aqueous suspensions. It should be understood that this refers to a substance that functions to maintain a desired pH. The aqueous solution may also include one or more amino acids.   Recombinant retroviruses can also be stored in purified form. Further details Before the addition of the formulation buffer, the crude recombinant retrovirus was filtered The impurities can be removed by passing through It can be concentrated by a system (Filtron Technology Corp., Nortborough, MA). 1 In one embodiment, DNase is added to the concentrate to digest the exogenous DNA. This digest is then diafiltered to remove excess media components. And establish a recombinant retrovirus in a more desirable buffered solution. You. The diafiltrate is then passed through a Sephadex S-500 gel column, and The purified recombinant retrovirus is eluted. Make sure that a sufficient amount of formulation buffer The desired final concentrations of the components added to the eluate (see, eg, Example 9) ) And dilute the recombinant retrovirus to a minimum. Then the aqueous suspension Is stored (preferably at -70 ° C) or immediately dried. As described above, The formulation buffer contains saccharide, high molecular weight structural additives, and buffer components in water. It is an aqueous solution. The aqueous solution may also include one or more amino acids.   Crude recombinant retrovirus can also be purified by ion-exchange column chromatography. Can be purified. This method is described in further detail in U.S. Patent Application Serial No. 08 / 093,436. Will be posted. Generally, the crude recombinant retrovirus is passed through a filter. The impurities are removed by filtration and the filtrate is a highly sulfonated cellulose Loaded on a column containing the tricks. This recombinant retrovirus has a high salt buffer The solution is eluted from the column in a purified form. Next, high salt relaxation The buffer is further exchanged for the desired buffer by passing through a molecular exclusion column. . Then, as described above, a sufficient amount of the formulation buffer was And the aqueous suspension is immediately dried or stored (preferably Or -70 ° C).   Aqueous suspensions in crude or purified form may be lyophilized or evaporated at ambient temperature. Can be dried. In particular, lyophilization is carried out below the glass transition temperature or in aqueous suspensions. Cooling the aqueous suspension below the eutectic temperature of Removing water by sintering to form a lyophilized retrovirus. simply In particular, an aliquot of the formulated recombinant retrovirus was placed in a lyophilizer (Super Modulyo 12K) in Edwards cooling chamber (3 shelf RC3S unit) I will The multi-stage lyophilization procedure described in Phillips et al. (Cryobiology 18; 414, 1981) Using the formulated recombinant retrovirus, preferably at a temperature of -40 ° C to -45 ° C. Lyophilize. The resulting composition is less than 10% by weight of the lyophilized retrovirus. Contains no water. Once lyophilized, the recombinant retrovirus is stable, And stored at -20 ° C to 25 ° C as described in more detail below.   In the evaporation method, water is removed from the aqueous suspension at ambient temperature by evaporation . In one embodiment, the water is removed by spray drying (EP 520,748) . In the spray drying procedure, the aqueous suspension is preheated gas (usually air ) Where the water rapidly evaporates from the suspension droplets. Spray drying equipment The device is available from a number of manufacturers (eg, Drytec, Ltd., Tonbridge, Engl. and; Lab-Plant, Ltd., Huddersfield, England). Once dehydrated, recombinant retro The virus is stable and can be stored at -20C to 25C. Described in this specification The dry or freeze-dried retrovirus resulting in water content The amount is based on Karl-Fischer equipment (EM Science Aquastar^TMVIB volumetric titrator, C herry Hill, NJ) or by gravimetry.   Aqueous solutions used in formulations as described previously include saccharides, high molecular weight structures Consists of additives, buffer components, and water. The aqueous solution may also contain one or more amino acids May be included. Combinations of these compositions can be obtained by freezing and lyophilization, or evaporation. It acts to maintain the activity of the recombinant retrovirus when dried. Preferred suckers The ride is lactose, but other saccharides (eg, sucrose, man) Nitol, glucose, trehalose, inositol, fructose, maltou Or galactose) can also be used. Furthermore, the combination of saccharides For example, lactose and mannitol, or sucrose and mannitol Can be used. A particularly preferred lactose concentration is 3% to 4% by weight. You. Preferably, the concentration of the saccharide varies from 1% to 12% by weight.   High molecular weight structural additives help prevent virus aggregation during freezing, and Provides structural support in a dry or dry state. Within the scope related to the present invention, A build additive is considered to be "high molecular weight" if the molecular weight is greater than 5000. A preferred high molecular weight structural additive is human serum albumin. But other substances (For example, hydroxyethyl cellulose, hydroxymethyl cellulose, dex (Tolan, cellulose, gelatin, or povidone) may also be used. Especially good The preferred concentration of human serum albumin is 0.1% by weight. Preferably, high molecular weight The concentration of the structural additive varies from 0.1% to 10% by weight.   If amino acids are present, this is the virus in the cooling and thawing of the aqueous suspension. It functions to further maintain infectivity. In addition, amino acids can be To further maintain viral infectivity during suspension sublimation and during lyophilization Function. A preferred amino acid is arginine, but other amino acids (eg, Ricin, ornithine, serine, glycine, glutamine, asparagine, glutami Acid or aspartic acid) can also be used. Particularly preferred arginine concentration The degree is 0.1% by weight. Preferably, the amino acid concentration is between 0.1% and 10% by weight. Change.   The buffer component works to buffer the solution by maintaining a relatively constant pH. To use. Various buffers may be used depending on the desired pH range, preferably 7.0 And between 7.8. Suitable buffers include phosphate and citrate buffers. No. A particularly preferred pH of the recombinant retroviral formulation is 7.4, and A new buffer is tromethamine.   In addition, the aqueous solution can be used to convert the final formulated recombinant retrovirus into a suitable isotonic salt. It is preferable to include a neutral salt used to adjust the concentration. A suitable neutral salt is , Sodium chloride, potassium chloride, or magnesium chloride. Preferred salt Is sodium chloride.   An aqueous solution containing the desired concentrations of the components described above is prepared as a concentrated stock solution Can be done.   Particularly preferred of recombinant retroviruses in lyophilized state for later reconstitution The preservation method involves the following steps: (a) dissolving the infectious recombinant retrovirus in water; Mixing with the liquid to form an aqueous suspension. This aqueous suspension contains 4% by weight Glucose, 0.1% by weight human serum albumin, 0.03% by weight or less NaCl, 0.1% by weight Nin, and a tromethamine buffer effective to provide an aqueous suspension at a pH of about 7.4 Amount, thereby stabilizing the infectious recombinant retrovirus; (b) -40 ° C to -4 Cooling the suspension to a temperature of 5 ° C. to form a frozen suspension; (c) freezing by sublimation Lyophilization to remove water from the suspension and to have less than 2% water by weight of the lyophilized composition Forming a composition. This composition can infect mammalian cells in reconstitution . Recombinant retrovirus is replication-deficient and administered to humans upon reconstitution It is preferable to be suitable.   Lyophilized mannitol and lactose as illustrated in FIGS. Recombinant retroviral formulations can be used at different storage temperatures to allow the virus to function as a functional Assayed for conservation of activity. Similarly, FIG. 3 shows that at various storage temperatures, Trehalose recombinant retrovirus for conservation of viral activity as active period 5 illustrates the results of assaying a pharmaceutical formulation. Fig. 4 shows a frozen prescription recombinant as a liquid. Virus infectivity of torovirus (-80 ° C) and lyophilized recombinant virus stored at -20 ° C Figure 4 shows a comparison of torovirus with viral infectivity. Mannitol prescription freeze-dried Can lose significant activity (5-6 fold), but after a lyophilization event, Seems to keep. Although not preferred, such a loss is sufficient for retrovirus If any amount is present in the aqueous solution, it is acceptable.   If the lyophilized retrovirus is intended for storage at room temperature, It is provided herein that it is preferred to utilize a particular saccharide of The disclosure will be apparent to those skilled in the art. More specifically, disaccharides (eg, , Lactose or trehalose), especially for storage at room temperature. preferable.   The lyophilized or dehydrated retrovirus of the present invention can be reconstituted using various substances. However, it is preferable to reconstitute using water. In some cases, the final prescription Dilute salt solutions that render the substance isotonic can also be used. In addition, the reconstructed Use aqueous solutions containing components known to increase the activity of Torovirus It can be advantageous to do so. Such components include cytokines (eg, IL-2), Transduction of recations (eg, protamine sulfate) or reconstituted retroviruses Contains other components that increase entry efficiency. Lyophilized or dehydrated recombinant retrovirus Any suitable volume of water, or substantially and preferably a lyophilized or dehydrated sample Can be reconstituted using the reconstituting agents described above, which can totally solubilize H.Administration   As described above, the high titer recombinant retroviral particles of the present invention can Location (eg, cerebrospinal fluid, bone marrow, joints, arterial endothelial cells, rectum, buccal / sublingual, vaginal, Selected from the group consisting of lung, liver, spleen, skin, blood, and brain. Selected organ, or site selected from the group consisting of tumor and intestinal space) Can be administered. In another embodiment, the recombinant retrovirus is intraocular, intranasal , Sublingual, oral, topical, intravesical, subarachnoid, topical, intravenous, intraperitoneal, intracranial, muscle Of which Or it can be administered subcutaneously. Other typical routes of administration include complete surgical procedures and Gastroscopy, ECRP, and conclusions that do not require a hospital but may require the presence of medical personnel Including enteroscopy.   Considerations for administering the compositions of the present invention include:   Oral administration is easy, convenient, economical (no sterilization required) and cheap. All (overdose can be treated in most cases), and this set Enables controlled release of the active ingredient of the product (recombinant retrovirus). on the other hand, Local irritation (eg, nausea, vomiting or diarrhea, vagus for poorly soluble drugs) Recombinant retrovirus may be associated with liver metabolism and gastric acid and enzyme Suffers the "first pass effect" due to physical decomposition. In addition, there may be a slow onset of action, Effective plasma levels may not be achieved, require patient cooperation, and Things can affect absorption. A preferred embodiment of the present invention provides erythropoietin, Insulin, GM-CSF, cytokines, various polypeptides or peptide Mon, its agonists or antagonists (where these hormones are Tissue (eg, pituitary, hypothalamus, kidney, endothelial cells, liver, pancreas, bone, hematopoietic bone marrow, And a recombinant retrovirus expressing a gene encoding Including oral administration of Luth. Such polypeptides can induce growth, tissue regression, Suppression of immune response, apoptosis, gene expression, receptor-ligand interaction Blocks, can be used for immune response, and certain anemias, saccharideuria , Infection, high blood pressure, abnormal blood chemistry (eg, increased blood cholesterol, blood clotting Solid factor deficiency, increased LDL with decreased HDL), Alzheimer-related amyloid Protein levels, bone erosion / calcification, and various metabolites (eg, In treatment for controlled levels of steroid hormones, purines, and pyrimidines) possible. Preferably, the recombinant retrovirus is first lyophilized and then Filled and dosed into the capsule.   Buccal / sublingual administration is a convenient method of administration that provides rapid onset of the active ingredients of the composition And avoid first-pass metabolism. Therefore, there is no stomach acid or enzymatic degradation, Thus, recombinant retrovirus absorption is feasible. High bioavailability exists , And substantially immediate treatment cessation is possible. On the other hand, such administration Target Limited to low doses (typically about 10-15 mg), and at the same time food, drink Or cannot eat what to swallow. A preferred embodiment of the present invention is a Encoding self and / or exogenous MHC or immune modulators for placement Buccal / sublingual administration of recombinant retroviruses containing the following genes; Treatment of Syndrome syndrome involves the use of such recombinant genes containing IgA or IgE antisense genes. Via buccal / sublingual administration of torovirus; and treatment of gingivitis and periodontitis Or via buccal / sublingual administration of cytokine antisense genes.   Rectal administration can overcome first-pass metabolic effects (good vascular / lymphatic supply And the absorbed material drains directly into the inferior vena cava) and provides The method is suitable for children, vomiting patients and unconscious persons. This method uses stomach acid and yeast Avoids elemental degradation and ionization of the composition is due to the fact that rectal fluid has no buffer capacity No change (pH 6.8; charged composition absorbs best). On the other hand, slow or incomplete Vagal absorption, irritation, microbial flora degradation can be present, and small absorption surfaces Exists (about 0.05m^Two). In addition, lipophilic and water-soluble compounds are Is preferred for absorption and requires absorption enhancers (e.g., salts, EDTA, NSAIDs) obtain. Preferred embodiments of the present invention include colon cancer antigens, autologous and / or foreign MHC , Or a recombinant retrovirus containing a gene encoding an immune modulator Including rectal administration of luz.   Nasal administration avoids first-pass metabolism and gastric acid and enzymatic degradation and It is profitable. In a preferred embodiment, nasal administration is compatible with recombinant retroviral administration. Is useful, wherein the recombinant retrovirus is a polypeptide having the above-mentioned properties. It can express tide. On the other hand, such administration can cause local irritation, Thus, absorption can depend on the condition of the nasal mucosa.   Pulmonary administration also avoids first-pass metabolism and gastric acid and enzymatic degradation, and And convenient. In addition, pulmonary administration provides localized and minimal effects with minimal systemic side effects. And the dosage required for efficacy and rapid onset of action and Self-medication may be present. On the other hand, sometimes, a small portion of the administered composition is bronchiole / pulmonary Vesicles may be reached, local irritation may be present, and overdose may occur. In addition, The cooperation and understanding of the practitioner is preferred, and the propellant for administration may have toxic effects. You. A preferred embodiment of the present invention is asthma, hay fever, allergic alveolitis, or IgA or IgE for the treatment of conditions such as fibrosis alveolitis, cystic fibrosis CFTR gene for treatment, and protease and collagen for treatment of emphysema Gene encoding a genease inhibitor (eg, α-1-antitrypsin) Pulmonary administration of a recombinant retrovirus expressing Or about oral administration Many of the same types of polypeptides or peptides listed above can be used. You.   Ophthalmic administration provides a local effect and allows for a long-term effect. The administration here Through the insert. In addition, avoids first-pass metabolism and gastric acid and enzymatic degradation And allows self-administration by using eye drops or contact lens-like inserts I do. On the other hand, administration is not always effective. Because administration induces lacrimation Because you do. A preferred embodiment of the present invention is a hay fever or spring conjunctivitis. Encoding IgA or IgE for the treatment of and conjunctivitis atomic Ocular administration of offspring expressing recombinant retrovirus; and melanoma-specific antigens (eg, For example, high molecular weight melanoma-associated antigens), autologous and / or foreign MHC, or immune Including ocular administration of recombinant retrovirus containing the gene encoding the epidemic modulator No.   Transdermal administration allows for rapid treatment cessation and long-term effects and good compliance Lead the dance. In addition, local effects are possible, and first-pass metabolism, as well as Avoid gastric acid and enzymatic degradation. On the other hand, such administration may cause local irritation. Occur, especially susceptible to development of tolerance, and typically have a very strong composition Not good for things. A preferred embodiment of the present invention relates to atopic dermatitis and Encodes IgA or IgE for the treatment of conditions such as and other skin allergies Transdermal administration of recombinant retrovirus expressing the gene; and melanoma specific anti- Original (eg, high molecular weight melanoma associated antigen), autologous and / or foreign MHC, Or a recombinant retrovirus encoding a gene encoding an immune modulator Percutaneous administration.   Vaginal administration provides one preferred route for local treatment and hormonal administration I do. In addition, such administration may include first-pass metabolism and gastric acid and enzymatic Solution and avoiding the case where the recombinant retrovirus expresses the peptide Preferred for administration of the composition. A preferred embodiment of the present invention provides for self and / or exogenous M Recombinant retrovirus expressing the gene encoding HC or immunomodulator Including vaginal administration of ils. Another preferred embodiment promotes the production of sperm-specific antibodies Sperm components (eg histones, flagellin) Vaginal administration of the gene encoding the same. This effect can be reversed, and / Or the immunoglobulin antisense gene (the gene that is used to produce sperm-specific antibodies) By delivering a recombinant retroviral vector encoding Pregnancy in some women may be enhanced.   Intravesical administration allows local treatment for genitourinary problems and reduces systemic side effects Avoid and avoid first-pass metabolism, as well as gastric acid and enzymatic degradation. on the other hand This method requires urinary catheterization, and requires highly skilled staff I need. A preferred embodiment of the present invention relates to an antitumor gene (eg, thymidine) Prodrug activating genes such as Nase) or various immunomodulatory molecules (eg, , Cytokines).   Endoscopic retrograde gallbladder pancreatography (ERCP) (pass through mouth; does not require skin penetration) I) is advantageous for extensive gastroscopy and has selective access to the bile and pancreatic ducts. Allow access. On the other hand, this method requires highly skilled staff, Discomfort to the patient.   Many of the routes of administration described herein (eg, intra-CSF, intra-bone marrow, intra-articular, intravenous) Intra-arterial, intracranial, intramuscular, subcutaneous, various organs, tumor, intestinal space, intraperitoneal , Within the lymph glands (intraalymphatic) or within the capillary bed), needles, catheters, Or it can be easily achieved by direct administration using an associated device. In particular, the book In certain embodiments of the invention, one or more doses are administered directly in the indicated manner. Can be: 10 for cerebrospinal fluid^Five,Ten⁶,Ten⁷,Ten⁸,Ten⁹,Ten^TenOr 10¹¹more than cfu Or equivalent dose; 10 for bone marrow^Five,Ten⁶,Ten⁷,Ten⁸,Ten⁹,Ten^TenOr 10¹¹cfu Higher or equal dose; 10 for joints^Five,Ten⁶,Ten⁷,Ten⁸,Ten⁹,Ten^TenOr 1 0¹¹Dose greater than or equal to cfu; 10 intravenously⁸,Ten⁹,Ten^TenOr 10¹¹cf Dose greater than or equal to u; 10 into arteries^Five,Ten⁶,Ten⁷,Ten⁸,Ten⁹,Ten^TenMa Or 10¹¹cfu Higher or equal dose; 10 intracranial⁹,Ten^TenOr 10¹¹more than cfu Or equivalent dose; 10 intramuscularly^TenOr 10¹¹Throws greater than or equal to cfu Dosage: 10 into eyes^Five,Ten⁶,Ten⁷,Ten⁸,Ten⁹,Ten^TenOr 10¹¹more than cfu or Equal dose; 10 for lung^Five,Ten⁶,Ten⁷,Ten⁸,Ten⁹,Ten^TenOr 10¹¹more than cfu Or equivalent dose; 10 nose^Five,Ten⁶,Ten⁷,Ten⁸,Ten⁹,Ten^TenOr 10¹¹from cfu High or equal dose; 10 sublingually^Five,Ten⁶,Ten⁷,Ten⁸,Ten⁹,Ten^TenOr 10^{1 1} Dose greater than or equal to cfu; 10 for rectum^Five,Ten⁶,Ten⁷,Ten⁸,Ten⁹,Ten^Ten Or 10¹¹Dose greater than or equal to cfu; 10 by mouth^Five,Ten⁶,Ten⁷,Ten⁸,Ten⁹ ,Ten^TenOr 10¹¹Dose greater than or equal to cfu; 10 topical^Five,Ten⁶,Ten⁷, Ten⁸,Ten⁹,Ten^TenOr 10¹¹Dose greater or equal to cfu; 10 for vagina^Five,Ten⁶ ,Ten⁷,Ten⁸,Ten⁹,Ten^TenOr 10¹¹Dose greater or equal to cfu; subcutaneously Ten⁹,Ten^TenOr 10¹¹Dose greater than or equal to cfu; 10 into bladder^Five,Ten⁶, Ten⁷,Ten⁸,Ten⁹,Ten^TenOr 10¹¹Dose greater than or equal to cfu; organ (eg 10 for lung, liver, spleen, skin, blood, or brain)^Five,Ten⁶,Ten⁷,Ten⁸,Ten⁹, 1 0^TenOr 10¹¹Dose greater than or equal to cfu; 10 into tumor⁸,Ten⁹,Ten^TenMa Or 10¹¹Dose greater than or equal to cfu; 10 intraperitoneally⁸,Ten⁹,Ten^TenOr 1 0¹¹Dose greater than or equal to cfu; 10 to intestinal space^TenOr 10¹¹more than cfu Equal or equal dose; 10 into lymph glands^Five,Ten⁶,Ten⁷,Ten⁸,Ten⁹,Ten^TenOr 1 0¹¹Dose greater than or equal to cfu; 10 for capillary bed^Five,Ten⁶,Ten⁷,Ten⁸,Ten⁹ ,Ten^TenOr 10¹¹Dose greater than or equal to cfu; or intrathecally Ten^Five,Ten⁶,Ten⁷,Ten⁸,Ten⁹,Ten^TenOr 10¹¹Dose greater than or equal to cfu.   Recombinant retroviruses can be delivered from outside the body (as an outpatient procedure) or As a procedure (where the vector is administered as part of a procedure with other purposes) Or specifically delivered as a procedure designed to administer the vector. You. Other routes of administration and methods include non-pareneral routes and multisite Via administration.   The following examples are provided by way of illustration, not by way of limitation.                                  Example                                 Example 1                     Preparation of retroviral vector backbone A.Preparation of retrovirus backbone KT-1 and KT-3B   N2 vector (Armentano et al., J. Vir. 61: 1647-1650, 1987; Eglitas et al., Science). 230: 1395-1398,1985) 5 'long terminus of Moloney murine leukemia virus (MoMLV) Repeat (LTR) EcoRI-EcoRI fragment (containing gag sequence)⁺(Stra tagene, La Jolla, CA). The resulting construct is named N2R5. This N2 The R5 construct is transformed by site-directed in vitro mutagenesis to change the ATG start codon to ATT. To prevent gag expression. This mutated fragment is 200 base pairs (bp ) Long, flanked by PstI restriction sites. SK PstI-PstI mutant fragment⁺Step Purified from the plasmid and place it at the PstI site of the N2 MoMLV5 'LTR in plasmid pUC31. The insertion replaces the non-mutated 200 bp fragment. Plasmid pUC31 Is derived from pUC19 (Stratagene, La Jolla, CA) with additional restriction sites XhoI, BglII, BssHII and NcoI are inserted between the EcoRI and SacI sites of the polylinker. You. This construct is named pUC31 / N2R5gM.   Purify the 1.0 kilobase (Kb) MoMLV3 'LTR EcoRI-EcoRI fragment from N2 Sumid SK⁺Cloned into N2R3^-To obtain a construct called 1.0Kb ClaI-Hi The ndIII fragment is purified from this construct.   SV40 drives expression of neomycin (neo) phosphotransferase gene PAFVXM retroviral vector containing the early promoter (Kriegler et al., Cell 3 8: 483, 1984; St. Louis et al., PNAS 85: 3150-3154,1988) from ClaI-ClaI dominant selection. Selectable marker gene fragment, SK⁺Clone into plasmid. this SK construct⁺SV_TwoName it -neo. SK the 1.3 Kb ClaI-BstBI gene fragment⁺SV_Two Purify from the -neo plasmid.   XhoI-ClaI fragment containing target gene and 1.0 Kb MoMLV3 'LTR ClaI-HindI Part III which inserts the II fragment into the XhoI-HindIII site of the pUC31 / N2R5gM plasmid KT-3B or KT-1 vectors are constructed by split ligation. It has a KT-1 skeleton This gives the vector referred to as Next, the pAFVXM retrovirus vector-derived Insert the 1.3 Kb ClaI-BstBI neo gene fragment into the ClaI site of this plasmid. To obtain a vector referred to as having a KT-3B backbone.                                 Example 2              Oral administration of recombinant retrovirus-expressed factor VIII A.Construction of full length and B domain deleted factor VIII cDNA RETROVECTOR ^™   Below are several retroviral vectors encoding full length factor VIII cDNA The construction of the key is described. Further discussion is also made in the U.S. Patent Application filed December 30, 1994. License application number No. [Attorney Reference Number 930049.438]. Retro ui Due to the packaging constraints of the lus vector, and the choice of transduced cells is therapeutic A retroviral scaffold lacking a selectable marker gene because it is not required (eg, KT-1). 1.Construction of plasmid vector encoding full length factor VIII   The gene encoding full length factor VIII can be obtained from a variety of sources. One Such a source for the plasmid pCIS-F8 (see EP 0260148) ). This plasmid contains the full-length factor VIII cDNA and its expression is Under the control of the immediate (CMV MIE) promoter and enhancer. Factor VIII The child cDNA has an approximately 80 bp 5 ′ untranslated sequence from the factor VIII gene and an approximately 500 bp 3 ′ non-translated sequence. Includes translation region. In addition, a CMV promoter is inserted between the CMV promoter and the factor VIII sequence. There is an intron sequence, or "cis" element. This cis element , Spanning about 280 bp, about 1 of splice acceptors from immunoglobulin genes Includes a splice donor site from the CMV major immediate early promoter 40 bp upstream.   More specifically, retroviral vectors for expressing full-length factor VIII Construction of the encoding plasmid (designated pJW-2) using the KT-1 backbone derived from pKT-1 I do. Briefly, directional cloning of the factor VIII cDNA insert into pKT-1 is possible. Convert the unique XhoI site to a NotI site by site-directed mutagenesis for ease of use You. Next, the obtained plasmid vector is cleaved with NotI and ClaI. pCIS-F8 Was completely digested with ClaI and EagI (for these two sites are present). To release the fragment encoding full length factor VIII. Next, this flag The plasmid was ligated to a vector digested with NotI / ClaI and the plasmid designated pJW-2. Generate 2.Construction of truncated factor VII retrovector (ND-5)   Encodes a truncated form of about 80% (about 370 bp) of the 3 'untranslated region of Factor VIII cDNA A plasmid (designated pND-5) is constructed with the pKT-1 vector as follows. pJ The XhoI restriction site of the pKT-1 vector used was restricted to NotI as described for W-2. Replace with the site. Factor VIII insert digests pCIS-F8 with ClaI and XbaI It is produced by The latter enzyme cleaves 5 'of the factor VIII stop codon. Next, an approximately 7 kb fragment containing all but the 3 'coding region of the factor VIII gene. Purify the components. pCIS-F8 is also digested with XbaI and PstI to terminate the gene. Release the 121 bp fragment containing the connection codon. This fragment was also purified And, in turn, a larger fragment encoding the rest of the factor VIII gene. N PND-2) and a plasmid called pND-2. Make it.   Next, a unique SmaI site in pND-2 was added under reduced conditions to a ClaI linker (New Engla nd Biolabs, Beverly, MA) to blunt ends generated by SmaI digestion. Change to a ClaI site. After recircularization and ligation, a plasmid containing two ClaI sites The sumid was identified and designated as pND-3.   Factor VIII sequence within pND-3 (linked by a ClaI site and of the 3 'untranslated region Containing many truncated full-length genes) as follows: Plasmid backbone from the ClaI digest (cut at the XhoI site and blunted with Klenow) And pKT-1 induction by inserting a NotI linker (New England Biolabs) Body). This plasmid backbone is a 5.2 kb NotI / ClaI fragment Is generated. pCIS-F8 is cleaved with EagI and EcoRV and the resulting fragment of approximately 4.2 kb Isolate (encoding the 5 'portion of the full length factor VIII gene). Eco pND-3 Digest with RV and ClaI and isolate the 3.1 kb fragment. Next, the VIII These two fragments containing the part of the factor gene were ligated to NotI / ClaI digested vector bone. The plasmid named pND-5 is ligated. 3.Construction of B domain deletion vector   B-deleted FVIII precursor DNA is obtained from Miles Laboratory. This expression vector Is named p25D. It has exactly the same backbone as pCISF8 above. in p25D The HpaI site at 3 'of the FVI118 cDNA is modified to ClaI by an oligo linker. AccI ~ C The laI fragment is excised from the modified p25D plasmid. This fragment Extends to the B domain deletion and contains two thirds of the complete 3 'of the cDNA. AccI ~ Cl aI fragment from the Retrovector^TMRemoved from JW-2 and modified B above Replace with domain deleted fragment. This is named B-del-1. B.Assay for factor VIII expression 1.KT-ND5 vector generation by transient packaging and transduction of mouse cells Current assay   Cell line, L33 (Dennert, USC Comprehensive Cancer Center, Los Angeles, CA Patek et al., Int. J. of Cancer 24: 624-628, 1979), BCIOME (Patek et al., Cell Immunol 72: 113, 1982, ATCC number TIB85), L33env, and BCenv (L33env and And BCenv express HIV-1 IIIBenv, Warner et al., AIDS Res. and Human Retrovir us 7: 645, 1991), using amphotropic or VSVG envelope proteins And transduced with the KT-ND5 vector containing Intangible Transduced cells were also analyzed for Factor VIII expression and KT-ND5 transduced cells. To determine the effect of transduction on protein expression.   Mouse cell line, L33-KT-ND5, L33env-KT-ND5, L33env, L33, BC10ME, BC10ME-K T-ND5, BCenv, and BCenv-KT-ND5 are tested for expression of the KT-ND5 molecule. Fine The cells are grown to subconfluent density and the supernatant is centrifuged at 200 xg. (Rum Diagnostica, Molndal, Sweden).   The assay is performed as follows. 100 μl of culture medium sample is included in the kit. Mix with 200 μl of the working buffer to which it belongs. The mixture is incubated at 37 ° C for 4-5 minutes. And then 0.025M CaCl_TwoAdd 100 μL of stock solution. afterwards, Incubate for 5 minutes at 37 ° C. Next, 200 μL of the coloring reagent (20 mg S-2222, 335 μg of synthetic thrombin inhibitor (I-2581)). 5 minutes at 37 ° C After incubation, add 100 μL of 20% acetic acid or 2% citric acid and react. To stop. Next, absorbance was measured using 50 mM Tris (pH 7.3) and 0.2% bovine serum Measure against blank containing Bumin (BSA). Normal human plasma (1.0 IU A standard curve based on a dilution of factor VIII / mL) is used. And add an assay Should be done in a tick tube. Factor VIII serum levels in non-hemophilic patients , Within the range of 200 ng / mL.   If this assay is to be used on patient samples, 9 volumes of blood will be collected at neutral pH. Mix with 1 volume of 0.1 M sodium citrate at 20-25 ° C. for 5-20 minutes Centrifuge at 00xg to pellet cells. Factor VIII was thermally unstable Plasma samples should be tested within 30 minutes of isolation or immediately at -70 ° C. Should be saved. However, during freezing and thawing, as much as 20% of the activity of Factor VIII Can be lost. 2.KT-ND5 vector expression by transient packaging and transduction of human cells Assay   Cell lines transduced with KT-ND5 are examined for factor VIII expression. Non-transduction The incoming cells are analyzed and compared to KT-ND5 transduced cells, and transduction is Determine the effect you have.   Two human cell lines (JY and JY-KT-ND5) are tested for KT-ND5 expression. 1 0⁶The suspended cells grown to cells / ml are pipetted out of the culture flask, Then, pelletize by centrifugation at 200 × g. Remove and dilute the supernatant And Coatest as described in Example 2B1 above.^RFactor VIII assay Assay. C.Transient transfer of packaging cell lines HX and DA with the vector construct KT-ND5 Transfection and transduction 1.Plasmid DNA transfection   On day 1, Dulbecco's modified Eagle packaging cell line HX (WO92 / 05266) 10 cm tissue culture dish with medium (DMEM) and 10% fetal bovine serum (FBS) 5.0 × 10^FiveSeed with cells. On day two, four hours before transfection, Replace the ground with 5.0 ml of fresh medium. 40.0 μl of 2.5 M CaCl_Two, 10 μg of plasmid D NA and deionized H_TwoStandard mixing by mixing O to a total volume of 400 μl Perform calcium phosphate-DNA coprecipitation. 400 μl DNA-CaCl_TwoStir the solution constantly 400 μl of precipitation buffer (50 mM HEPES-NaOH, pH 7.1; 0.25 M NaCl, and 1.5 mM M Na_TwoHPO_Four-NaH_TwoPO_Four). Incubate this mixture for 10 minutes at room temperature You. The resulting fine precipitate is added to a cell culture dish. DNA precipitate cells And 37 ° C overnight. On the third day, aspirate the medium and Add fresh medium. Remove the supernatant on day 4 and pass through a 0.45 μl filter. And store at -80 ° C.   Alternatively, 29 23 cells (WO92 / 05266), which express gag and pol 293 Cells) are vector DNA and plasmid pMLP-VSVG (or other VSVG Transfected with a VSVG pseudotyped vector Obtain the tar particles, which are collected and stored as described above. 2.Packaging cell line transduction   On day 1, DA (D17 cell line ATCC deposit no. Cells, 10 ml DMEM and 10% FBS, 4 μg / ml polybrene (Sigma). , St. Louis × MO) 5.0 × 10^FiveSeed in cells / 10 cm tissue culture dish. Two days Eyes receive 3.0 ml, 1.0 ml and 0.2 ml of freshly harvested virus-containing HX medium Add. The cells are incubated with the virus overnight at 37 ° C. On the third day , Remove medium and add 1.0 ml DMEM, 10% FBS containing 800 μg / ml G418 to the plate. Les Add to the list. Vector transduced and contains neomycin selectable marker Only the cells that survive survive. G418 resistant pools are generated over a week. Cells Remove the cell suspension, count the cell suspension, and transfer the cell suspension to 10 cells / ml. Dilute and add 0.1 ml to each well of a 96-well plate (Corning, Corning, NY) (1 cells / well) to dilute and clone this pool of cells You. Cells at 37 ° C, 10% CO_TwoAnd incubate for 14 days. Select 24 clones, And expanded to 24 well plate, 6 well plate, then 10cm plate, At this point, the clones are assayed for expression, the supernatant is collected, and the virus Assay for titer.   The titer of individual clones was used to infect HT1080 cells (ATCC Deposit No. CCL 121). Therefore, it is determined. On the first day, 5.0 × 10^FiveHT1080 cells, 3.0 ml DMEM, 10% FBS, And 4 μg / ml polybrene in each well of a 6-well microtiter plate Plate. On day two, serially dilute the supernatant from each clone 10-fold and This is used to infect HT1080 cells in 1.0 ml aliquots. Culture medium with fresh DMEM, 1 Replace with 0% FBS medium and cells with the vector at 37 ° C., 10% CO 2_TwoIn overnight Cubate. On the third day, the medium was replaced with fresh DMEM containing 800 μg / ml G418, 10% FBS. Selection of transduced cells is performed by replacing the medium. Cells at 37 ° C, 10% CO_TwoAt which point G418-resistant colonies at each dilution were And determine the virus titer of each clone as colony forming units (cfu) / ml I do.   Using these procedures, 10⁶Induce cell lines that produce more than cfu / ml You.   In the same manner as described for transduction of DA cells with HX supernatant, DA vector Packaging cell line HX using vectors generated from cell lines that produce Introduce quality.   Transduction of DA or HX cells with a vector lacking the neo selectable marker (Example 1 ) Was performed as described above. However, instead of adding G418 to the cells on day 3, Cells are cloned by limiting dilution. The titer is analyzed as described above. 3.Generation of producer cell lines through one packaging cell line   In some situations, in the process of producing a producer strain, more than one cell line It may be desirable to avoid the use of In this case, on the first day, DA cells were 5.0 cm in a 10 cm tissue culture dish with 10% irradiated (minimum 2.5 Mrad) FBS × 10^FiveSeed with cells. On day 2, four hours before transfection, the medium was Replace with 0.0 ml of fresh medium. 60 μl 2.0 M CaCl_Two, 10 μg of MLP-G plasmid, 1 0 μg of KT-ND5 retroviral vector plasmid, and deionized H_TwoMix with O Perform standard calcium phosphate-DNA co-precipitation with a volume of 400 μl . 400 μl DNA-CaCl_TwoWhile stirring the solution constantly, 400 μl of 2 × precipitation buffer (50 mM HEPES-NaOH, pH 7.1, 0.25 M NaCl, and 1.5 mM Na_TwoHPO_Four-NaH_TwoPO_Four). This Incubate the mixture at room temperature for 10 minutes. The fine precipitate obtained is Add to plated DA cell culture dish. Cells at 37 ° C with DNA precipitate And culture overnight. On the third day, the medium is removed and fresh medium is added. G-fake type The supernatant containing immobilized virus was removed on day 4 and passed through a 0.45 μl filter. And used to infect DA packaging cells.   On day 1, DA cells were harvested with 10 ml of DMEM and 10% FBS, 4 mg / ml polybrene (Sigma, St. Louis × MO) 5.0 × 10^FiveSeed the cells with a 10 cm tissue culture dish. Two days In the eyes, 2.0 ml, 1.0 ml, or 0.5 ml of freshly collected and filtered G-pseudotyped mouse Add the ils containing supernatant to the cells. Incubate the cells with the virus overnight at 37 ° C. Lab. On day 3, the medium was removed and 800 μg / ml G41 was added to the plate. Add 10 ml of DMEM containing 10 and 10% irradiated FBS. Transduced by the vector Only cells containing the neo selectable marker survive. G418 resistant pool 1-2 Produce over a week. Test the pool for expression, then play cells Remove the cell suspension, count the cell suspension, and dilute the cell suspension to 10 cells / ml. And add 0.1 ml (1 cell / well) to each well of a 96-well plate. And clone by dilution. Cells at 37 ° C, 10% CO_TwoIncubate for 2 weeks To Select 24 clones, 24 well plate, then 6 well plate, And finally expanded to a 10 cm plate, at which point the clones were assayed for expression. And collect the supernatant and assay for virus titer as described above. D.Detection of replicable retrovirus (RCR) 1.Expanded S ⁺ L ^- assay   Expansion S⁺L^-Assays include replicable infectious virus present in the supernatant of the cell line of interest. Decide if you want to. The assay uses an infectious retrovirus to indicate the cell line MI Cl₁Empirical observation of generating focus on (ATCC Deposit No. CCL 64.1) Based on MICl₁Cell line for transduction using mouse sarcoma virus (MSV) Thus, it is induced in the MvlLu mink cell line (ATCC Deposit No. CCL 64). this is, Contains replication defective mouse sarcoma provirus (S⁺) Is a replicable mouse leukemia Contains no virus (L^-) Non-producer non-transformed revertant clone is there. MiCl by replicable retrovirus₁Cell infection "activates the MSV genome" To induce "transformation" resulting in foci formation.   Remove supernatant from cell line to be tested for the presence of replicable retrovirus And remove all cells by passing through a 0.45μ filter. On the first day, MvlLu Cells were plated at 1.0 × 10 5 in 2 ml DMEM, 10% FBS, and 8 μg / ml polybrene.^FiveCell / C Seed wells (1 well per sample to be tested) into 6-well plates You. For positive and negative controls, separate MvlLu cells in the same 6 wells Plate on plate. Cells at 37 ° C, 10% CO_TwoAnd incubate overnight. 2 On the day, add 1.0 ml of the test supernatant to the MvlLu cells. Negative control plate Incubate with 1.0 ml of medium. The positive control was the MA virus (M iller et al., Molec. and Cell Biol. 5: 431, called pAM in 1985) Different dilutions (200 foci, 1.0 ffu, 20 ffu, And 2ffu), which are added to the cells in the positive control wells. One cell Incubate overnight. On day 3, the medium is aspirated and 3.0 ml of fresh DMEM and And 10% FBS to the cells. Cells are grown to confluence and 6 days Amplify any replicable retroviruses by splitting 1:10 on eyes and day 10 I do. On day 13, aspirate media on MvlLu cells and add 2.0 ml DMEM and 10% FB Add S to the cells. In addition, MiCl₁Cells were grown in 2.0 ml DMEM, 10% FBS, and 8 μg / ml 1.0 × 10 in polybrene^FivePlate with cells / well. On day 14, from MvlLu cells Supernatant MiCl₁Transfer to corresponding wells of cells, and 37 ° C, 10% CO_TwoIncubate overnight To On day 15, the medium is aspirated and 3.0 ml of fresh DMEM and 10% FBS are Add to the vesicles. On day 21, focus cells on a monolayer of cells (cover the monolayer, (Appearing as confluent refractory cells that remain attached). Foca Is MiCl₁Once on the cell, the test article is contaminated with a replicable retrovirus. Is determined to be. Using these procedures, the HBV core producer cell line It may indicate that it is not contaminated with a replicable retrovirus. 2.Producer strain co-culture and MdH marker rescue assay   Another method to test for the presence of the RCR in cell lines producing the vector Producer cells with the same number of Mus dunni (NIH NIAID, Bethesda, MD) cells Both are co-cultured. On day 0, 5.0 × 10^Five5.0 x 10 Mus dunni cells^FiveProde And mix the mixture with a 10 cm plate (10 ml of standard culture medium). / Plate, 4 μg / ml polybrene) for small-scale co-culture. Nourish. Effectively dilute out the producer cell line, and Split the culture every 3-4 days in a 1:10 ratio to provide maximal amplification of the RCR, And 5.0 × 10^FiveOf Mus dunni cells is added to each culture plate. On day 14, culture Collect the clarification, pass through a 0.45μ cellulose-acetate filter, and Test with car rescue assay. 1.0 × 10⁸1.0 × 10 with Mus dunni cells⁸No Mix the mixture with the reducer cells in a total of 20 T-150 flasks (30 ml of standard culture medium / floor). Large scale co-culture by inoculating Rasco (4 μg / ml polybrene). Now. Cultures were grown at a 1:10 ratio on days 3, 6, and 13 and on day 9 Divide by a ratio of 1:20. On day 15, the final supernatant is collected, filtered, and each Some of them are tested in the MdH marker rescue assay.   MdH marker rescue cell line was transformed with LHL (Hygromycin B resistance gene Retrovirus vector) (Palmer et al., PNAS 84: 1055-1059, 1987). Clones from the pool of introduced Mus dunni cells. This retrovirus The vector may be rescued from MdH cells upon infection of the cells with the RCR. 4μg / ml Standard culture medium containing polybrene (10% FBS, 1% 200 mM L-glutamine, 1% DMEM containing 10 amino acids)^FiveWells of a 6-well plate containing MdH cells Add 1 ml of test sample to After 24 hours, replace the medium with a standard without polybrene. Replace with culture medium. Two days later, the entire volume of the MdH culture supernatant was added to 0.45 μl of cellulose-acetate. 5.0 x 10 in 2 ml of standard culture medium containing polybrene^Four Transfer to wells of a 6-well plate containing the target cells of Mus dunni. 24 hours later, supernatant Was replaced with a standard culture medium containing 250 μg / ml hygromycin B, and then On days 2 and 5, replace with medium containing 200 μg / ml hygromycin B . Colonies resistant to hygromycin B appeared and they were 9 days after selection , Visualized by staining with 0.2% Coomassie Blue. F.KT-ND5 Transduction of human cells with vector constructs   On the first day, HT1080 cells contain 2 ml of standard growth medium (DME + 10% FBS). 2x10 in a 6-well tissue culture plate^FourAdjust to cells / well. Two days The ND-5 FVIII retroviral vector from a confluent vector-producing cell line The collector particles are collected as HX-ND-5 clones. Place them in a 0.45 μm syringe Filter through a filter and then test the supernatant. (Or filtered media The supernatant may be frozen at -80 ° C in aliquots for subsequent use. ) Polybrene Is added to each well so that the final concentration is 8 μg / ml. After 30 minutes, Add any undiluted retroviral vector supernatant to duplicate wells . Typical volumes and dilutions are 0.5 ml / well and 4 or more steps in growth media 1: 3 serial dilution. As a control, replicate two wells in equal volume Transduce with medium alone. On the third day, the wells were re-fed with 2 ml of fresh medium and To reach confluency. This is typically around 4 It can be day 5 or day 5. On that day, cells were added to 1 ml of fresh growth medium per well. Resupply the ground. After 24 hours, the medium is collected and filtered as described above. G. FIG.FVIII Expression of transduction vector for   Expression of the vector transduced human cell line for FVIII is described in Example 2B1 above. Like, Coatest^RDetect by assay. Activity in two controls The cells per well of the well were counted and 1 × 10⁶Per cell Normalize FVIII expression data to compare with control well supernatant And assay. H.Administration of vector constructs 1.Animal dosing protocol   Intestinal epithelial cells form a rapidly growing tissue population and Lieberkuh (Lieberkuh). n) The known location of the stem cells in the crypts makes them attractive for gene delivery. Part. The deep role of stem cells in this crypt and the protective role of the mucus gel layer For this reason, retroviruses are relatively inaccessible to tissue cells. But retro ui The accessibility of the Lus vector to these stem cells has been demonstrated in animal models by Sanberg,  J. et al. (Human Gene Therapy 5: 3232-329, 1994). Can be improved.   Male Sp from Charles River Breeding Laboratories (Portage, MD) The rague-Dawley rats are anesthetized and the cecum is confirmed by opening the peritoneal cavity. 3cm times Intestinal segment from last Peyer's patch in terminal ileum Isolate and ligate at each end. Plastic connected to a syringe A catheter is inserted into this segment and a mucolytic, dithiothreito And 2 ml of N-acetylcysteine dropwise under gentle pressure for 2 minutes Note, then remove. Repeat this procedure again, then⁶~Ten^Tencfu / ml 0.2 ~ Fill the segment with 2.0 ml of retroviral vector particles. Ligation 1 to 4 o'clock Shortly afterwards, and the abdominal cavity is sutured. Inject control animals with prescription buffer only I do.   Blood was collected from the tail vein and (Zatloukal, K. et al., PNAS 91: 5148-5152, 199). According to a modified procedure of 4) Factor VIII by a sandwich ELISA specific for human Factor VIII Assay for factor production. This ELISA provides two assays for human factor VIII. Based on monoclonal antibodies (ESH4 and ESH8: American Diagonistica). ESH 4 (1.0M NaHCO_Three/ 25M NaCl (pH9.0) 25μg / ml) at 4 ℃ overnight ELISA Bind, wash with 0.1% Tween20 in PBS, and block with 1% BSA in PBS. Click. Samples were prepared in 0.05M Tris-HCl / 1M NaCl / 2% BSA (pH 7.5) Apply at room temperature for 4 hours. Wash plate and add N-hydroxys ESH8 (0.05M) biotinylated with succinimide biotin (Pierce, Rockford, IL)  2.5 μg / ml in Tris-HCl / 1M NaCl / 2% BSA, pH 7.5) for 2 hours at room temperature You. The color reaction is performed by using peroxidase-conjugated streptavidin (Boehringer Mannhei m, Indianapolis, IN) and o-phenylenediamine dihydrochloride as substrate Do it. Human factor VIII: c preparation (National Institute for Biological Standa rds and Control, from Hertfordshire, U.K.) and normal rat plasma as controls To use. 2.Human dosing protocol   Lyophilized recombinant retrovirus containing the gene for factor VIII expression, Formulation into enteric tablets or gel capsules according to methods known in the art. this Are described in the following patents: US 4,853,230, EP 225,189, AU 9,224,296. No. AU9,230,801 and WO 92144,52.   The capsule is administered orally and is targeted to the jejunum. Recombinant retrovirus One to four days after oral administration, the expression of factor VIII is determined by CoCo as described in Example 2B1. atest^RIt is measured in plasma and blood by the factor VIII assay.                                 Example 3                Intracapsular administration of recombinant TK expressing retrovirus A.TK Construction of vector constructs 1.Construction of plasmid containing vector LTR sequence   All of the following retroviral vectors are N2 vectors (Keller et al., Nature 3 18: 149-154, 1985). Briefly, 5 'and 3' EcoRI LTR fragments (2.8 Kb and 1.0 Kb, respectively) (Armentano, J. Vir. 61: 1647, 1987; Eglit is, Science 230: 1395, 1985), and⁺(Stratagene, San Di ego, CA) and pUC31 at the EcoRI site. pUC31 is polyline Additional restriction sites (XhoI, BglII, BssHII) between the EcoRI and SacI sites of Kerr And NcoI) with modification of pUC19 (Stratagene, San Diego, CA). It Allows the plasmid N2R3 +/-⁺Between the plasmid and the 1.0 Kb 3 'LTR fragment Made from the connection. Plasmids p31N2R5 +/- and p31N2R3 +/- 5 'LTR and packaging signal (Y) of b, or 1.0 Kb of 3' LTR fragment Constructed from the ligation of pUC31 with pUC31. In each case, N2 is a vector Where R indicates the fact that the fragment is an EcoRI fragment. And 3 indicate the 5 'LTR or 3' LTR, and + or-indicates the orientation of the insert ( See Figures 1-6 for examples of LTR subclones).   In one case, a 1.2 Kb ClaI / EcoRI 5'LTR from N2 and W fragment SK⁺Subclone at the same site in the vector. Call this vector pN2CR5 Name. In another case, the 5 'LTR containing the 6 bp deletion of the splice donor sequence (Y ee et al., Cold Spring Harbor, Quantitative Biology, 51: 1021, 1986) Subcloned into pUC31 as an EcoRI fragment. Transfer this vector to p31N25 D [+] (FIG. 6). 2.HSVTK Of a plasmid containing   HSV-1 thymidine kinase gene (HSVTK) coding region and transcription termination signal The plasmid 322TK (3.5 kb BamH of HSV-1 cloned into BamHI of pBR322) 1.8 Kb BglII / PvuII from I fragment (McKnight et al.) (ATCC No. 31344) Isolated as a fragment and cloned into BglII / SmaI digested pUC31 Become This construct is named pUCTK. Requires termination of terminator signal PUCTK was digested with Smal and BamHI, and (A)_nSigna Excluding the 0.3 Kb fragment containing Remaining coding sequence and cohesive end BamHI bump Was reconstructed using double-stranded oligonucleotides made from the following oligomers: Accomplish:   The resulting construct is named pTKDA (Figure 7).   For diagnostic purposes, retain the AvaI site without altering the translated protein While designing the oligonucleotide to destroy the SmaI site.   Plasmid pPrTKDA (Figure 8), which contains the HSVTK promoter and coding sequence ( (A)_nWhich lacks the signal) is constructed as follows.   1. pTKDA was linearized with BglII, treated with alkaline phosphatase, and Purify.   2. The 0.8 Kb fragment containing the HSVTK transcription promoter was converted from p322TK to BamHI / Bg Isolated as a II fragment.   3. The products from (1) and (2) are ligated, transformed into bacteria and positive Clones are screened for the proper orientation of the promoter region. Generated The resulting clone is named pPrTKDA (FIG. 8). 3.Construction of a retroviral provector expressing HSVTK from a constitutive promoter Construction   Retroviral provectors (pTK-1 and pTK-3) were constructed essentially as described below. Build on the street.   The 1.5 Kb XhoI / HindIII 5 ′ LTR and plasmid sequence were isolated from p31N2R5 (+) Release (Fig. 1).   2. The HSVTK coding sequence lacking the transcription termination sequence was replaced with a 1.2 kb XhoI / BamHI plasmid from pTKDA. Isolate as a fragment (FIG. 2).   3. The 3 ′ LTR sequence was simply ligated as a 1.0 Kb BamHI / HindIII fragment from pN2R3 (−). Release (Figure 2).   4. The fragments from steps 1 to 3 are mixed, ligated, transformed into bacteria, and And individual clones are identified by restriction enzyme analysis. Name this construct TK-1 (FIG. 9).   5. pTK-3, TK-1 was linearized with BamHI, 5 'overhangs were filled in, and pAFVXM Retroviral vectors (Krieger et al., Cell 39: 483, 1984; St. Louis et al., PNAS  85: 3150, 1988), the bacterial lac UV5 promoter, SV40 early promoter. Tar, + Tn5 neo^rBlunt 5 'fill-in ClaI / ClaI fragment containing gene Constructed by end ligation. Isolating kanamycin resistant clones, and Screen individual clones for appropriate orientation by restriction enzyme analysis ( (FIG. 9).   These constructs can be used to sensitize with packaging cell lines such as DA above. Dyeable recombinant vector particles were produced. B.TK-3 Ganshik against mouse colon cancer cells in the presence or absence of the vector Determining the effect of Robil   Ganciclovir was transformed with DA / TK-3 transduced CT26 cells (colon tumor 26, Brattai n, Baylor College of Medicine, Houston, TX) An experiment was performed to determine. Transduce CT26 cells with G pseudotyped TK-3 vector . 24 hours after addition of virus supernatant, place CT26 cells under G-418 selection (4501 g / ml) . After 10 days of incubation, a G-418 selection pool was obtained, which was transferred to CT26 TK Neo. Name. CT26TK Neo cells, 2.5 × 10⁶Six 10cm at a density of pieces / plate^Twoplay Sow the seeds. As controls, each of the other two cell types, CT26 and CT26β-gal (this cell line is a reporter gene β-galactosidase from E. coli) Transduced with a retroviral vector containing And six 10cm^TwoSeeded on plates. 5 plates for each cell type Gangsik at concentrations of μg / ml, 50 μg / ml, 25 μg / ml, 12.5 μg / ml, and 6.25 μg / ml Treatment was carried out twice daily for 4 consecutive days with media containing Rovir. One step for each cell type The rate was left untreated. The cells were then transferred to each cell using trypsin-EDTA. Removed from the dish, resuspended in DMEM with 10% FBS and counted. The data in FIG. 10 show that even the lowest dose of ganciclovir Have a dramatic cytotoxic effect. This dose of ganciclovir (6.25 μg / ml) or even the next higher dose (12.5 μg / ml) It had no effect on any of the β-gal cells. However, 25 μg / ml ganshi Starting with a dose of clovir, a dose-dependent decrease in cell proliferation could be seen, CT26 TK Neo cells were always more sensitive to the drug. C.CT26TKNeo Ganciclovir dosing for treatment of mice injected with cells   Testing whether disease can be treated using in vivo transduction of mouse tumors Transduced in vitro to ensure 100% transduction, and Experiments were performed to determine the optimal concentration of ganciclovir needed to remove selected tumors. went. 2 × 10 mice in 12 groups of 3 mice each^FiveOne CT26 TK Neo cell is injected. Six groups of mice were injected with these cells intraperitoneally (I.P.) and six groups of mice Inject these cells subcutaneously (S.C.). The other three mice, two groups each, 2 × 10^FiveUnmodified CT26 cells (as control), either I.P. or S.C. Inject with   Ten days after injection of these groups of mice with CT26 or CT26 TK Neo cells, Initiate several concentrations of ganciclovir treatment. Each dose regimen is given twice daily, Consisting of morning and afternoon I.P. injections of ganciclovir. The experiments are summarized in Table A below I do.   After 5 days, all mice in the 125 mg / Kg, 250 mg / Kg, and 500 mg / Kg treatment groups Died due to the toxic effects of nciclovir. 15.63mg / Kg, 31.25mg / Kg and 62.5 Mice in the mg / Kg treatment group were treated for an additional 7 days and these were resistant to treatment Obtained. Tumor measurements were taken for 23 days (FIG. 11). The growth of CT26 TK Neo is In the absence of the compound, it was only slightly slower than the unmodified CT26. Complete tumor regression , In a group of mice treated with the 62.5 mg / Kg regimen. Partial tumor Regression was noted in the 31.25 mg / Kg treatment group. In the 15.63 mg / Kg treatment group, two Little or no effect compared to untreated control group . This is life threatening although some toxicity was observed in the 62.5 mg / Kg group And was reversible upon discontinuation of the treatment. So this Concentrations were used in further studies (FIG. 11). After 24 days, the I.P.injected animal was sacrificed, and evaluated. As shown in FIGS. 12 and 13, the optimal concentration of the antitumor effect, Or when grown on S.C. D.CT26 Ratio of cytotoxicity to tumor growth of in vivo and CT26TK Neo in vivo Comparison   Ganciclovir has an effect on the growth of unmodified CT26 tumor cells in vivo To determine if the mice were 2 × 10 × 7 mice in two groups of 7^FiveUnmodified C Inject T26 cells. The mice in two groups of 7 were 2 × 10 in S.C.^FiveCT26TK  Inject Neo cells. Seven days after tumor implantation, one group of CT26 injected mice and CT26TK N One group of eo-injected mice received 62.5 mg / Kg twice daily (am and pm) Apply a Roville regimen. These mice were injected for 12 days or CT26TK Neo injected animals Are treated until there is no detectable tumor burden. 3 weeks of tumor growth Monitor over time. Mice injected with CT26 and treated with ganciclovir Had tumors somewhat smaller than untreated mice injected with CT26. This means It shows some HSVTK-dependent inhibition of tumor growth (FIG. 14). But CT26 TKneo If the containing mice were treated with ganciclovir, and only in those cases, A dramatic decrease in burden was observed. E. FIG.TK-3 AY-27 rat cancer cells with vector or A without TK-3 vector Determination of the effect of ganciclovir on Y-27 rat cancer cells   Does ganciclovir have an effect on DA / TK-3 transduced AY-27 cells? An experiment is performed to determine no. AY-27 cells contain N- [4- (5-nitro-2-furyl) ) -2-Thiazol] formamide induced rat cancer cells (Selman, S. et al., J Urol 136: 141, 1986). Transduce AY-27 cells with G pseudotyped TK-3 vector Enter. 24 hours after addition of virus supernatant, AY-27 cells were selected by G-418 (450 μg / ml) Put it down. After 10 days of incubation, a G-418 selected pool was obtained and This is named AY-27TKNeo. AY-27TKNeo cells, 6 10cm^Two2.5 on plate × 10⁶Seed at a density of pieces / plate. As control, the other two cell types , AY-27 and AY-27β-gal (this cell line is a reporter gene β- (Transduced with the virus carrying galactosidase) 10cm^TwoSeed on plate as control. 5 plates for each cell type , 100 μg / ml, 50 μg / ml, 25 μg / ml, 12.5 μg / ml, and 6.25 μg / ml Treat twice daily for 4 consecutive days with medium containing rovir concentration. Of each cell type One plate is left untreated. Thereafter, the cells were treated with trypsin-EDTA. Out of each dish, resuspend in DMEM containing 10% FBS, and count I do. Using the obtained data, AY-27, AY-27β-gal, or AY-27TKNeo cells The concentration with the highest cytotoxic effect can be determined. F.AY-27TKNeo Determination of ganciclovir dose for treatment of rats injected with cells   Testing whether disease can be treated using in vivo transduction of mouse tumors Transduced to ensure 100% transduction, and in vitro Experiments were performed to determine the optimal concentration of ganciclovir needed to remove selected tumors. went. 2 x 10 rats in 12 groups of 3 rats each^FiveAY-27TKNeo cells are injected. 6 Groups of rats are injected intrathecally with these cells. The other three groups of rats, each in six groups, 2 × 10^FiveTwo unmodified AY-27 cells (as control) are injected intracapsularly.   Ten days after injection of AY-27 cells or AY-27TKNeo cells into rats in these groups, Initiate several concentrations of ganciclovir treatment. Each dose regimen is given twice daily, Consisting of morning and afternoon I.P. injections of ganciclovir. The experiments are summarized in Table B below I do.   Rats are treated for 12 days and rats that die from higher concentrations of ganciclovir except. Tumor measurements were evaluated for tumor regression to determine optimal ganciclovir concentrations. Perform for 20 days while paying. G. FIG.Dosing protocol 1.Rat dosing protocol   AY-27 rat bladder cancer cells were cultured in 20 male Fischer 344 rats (Charles River B (Reeding Laboratories, Portage, MD). 1-14 days after transplantation Anesthetize rats weighing between 200 g and 300 g, with tumors, The bladder is surgically removed from the body and urine is excreted using a 27 gauge needle. Virus Particles in 200-2,000 μl of formulation buffer.⁶~Ten^Tencfu of 5 rats Inject into the bladder. Addition of 4-8 μg / ml polybrene increases transduction efficiency Let it. Repair the cystotomy with a 7-zero non-absorbable suture to prevent leakage To recover. The virus keeps the animal under anesthesia, thereby preventing excretion Incubate for 0.5-1.0 hours in the presence of tumor cells. 10 cons Troll rats receive only 500 μl of formulation buffer.   Alternatively, after urinary excretion and washing with saline, 200-2,000 μl of vector Can be instilled directly into the bladder by catheterization through the urethra.   At 24 to 72 hours after vector treatment, rats are twice daily for 4 to 12 days (morning and PM), ganciclovir I at the optimal dose determined previously (eg, 62.5 mg / Kg body weight) Perform an injection. Finally, rats are given a single daily dose until the end of the experiment (1-10 weeks). Receive an amount of ganciclovir. Remove whole bladder and measure tumor growth . 2.Human dosing protocol   A urine (Foley) catheter is inserted through the urethra into the bladder and fixed in place. Set. Drain urine from the bladder, and flush the bladder with 100-500 ml of sterile saline I do. A recombinant retrovirus containing the gene for thymidine kinase expression was ~ 500 ml of formulation buffer (preferably 4-8 μl / ml polybrene or other potentiator 10 in^Five~Ten¹¹With the cfu, instill the bladder through the catheter. Will The particles are allowed to incubate for 0.25-12 hours, after which the catheter is removed. 1-7 days Thereafter, ganciclovir was administered every 12 hours for 2-21 days (at a constant rate for 1 hour). ) Administer 1-5 mg / Kg I.V. The vector is re-administered multiple times (2-20). So After that, ganciclovir is administered. Frequency in patients receiving ganciclovir Due to prolonged granulocytopenia and thrombocytopenia, neutrophils and It is recommended to perform a platelet count. Tumor regression is assessed by x-ray and / or Monitor by iopsy and repeat treatment if necessary.                                 Example 4            Multiple administration of recombinant retrovirus expressing factor VIII A.Construction of Factor VIII cDNA retroviral vectors ^TM of full-length and B-domain deleted   Construction of a full length and B domain deleted Factor VIII retrovector is described in Example 2A. It is described in. B.Aerosolization of recombinant retrovirus expressing factor VIII   Formulation buffer with 4-8 μg / ml polybrene or other transduction enhancing excipient KT-ND5 virus supernatant in a De designed to produce 0.3-0.5 μm particles. Use Vilbiss # 15 Atomizer (DeVilbiss Health Care Division, Somerset, PA) And atomize (Rousculp, M., Human Gene Therapy 3: 471-477, 1992). This The aerosols produced by the nebulizers use compressed air in a laminar flow hood. The mist is guided into a polypropylene tube and the liquefied steam (and control Virus supernatant) is again sterilized by 0.22 μm filtration. Retroviral particles Pass through this filter without any significant loss of functional activity. C.Administration of vector constructs 1.Rat dosing protocol   Rats are anesthetized and the trachea is exposed by an anterior midline incision. Factor VIII remains Recombinant retroviral particles expressing the gene product are reconstituted with 4-8 μg / ml polybrene. Or in the presence or absence of other transduction enhancing excipients, 10^Five~Ten^Ten300 μl at cfu / ml Diluted in the formulation buffer and intratracheally instilled through a small gauge needle. Con Troll animals receive only 300 μl of formulation buffer. Suture the incision and To recover Blood is drawn from the tail vein 2-14 days after virus instillation and And test for Factor VIII production as described in Example 2Hi. 2.Human dosing protocol   Recombinant retrovirus was purified using DeVilbiss # 15 Atomizer (above) for 2-60 minutes. Administer in between. 2-14 days after administration of the recombinant retrovirus, expression of factor VIII , Coatest^RMeasured in blood and plasma by the factor VIII assay (Example 2B1).                                 Example 5                      Transdermal administration of recombinant retrovirus A.TK-3 Construction of vector constructs   Construction and confirmation of TK-3 vector constructs and recombinant retroviruses are described in Examples 3A to 3F. B.Administration of vector constructs 1.Animal dosing protocol   Cottontail rabbit papillomavirus (CRPV), a highly carcinogenic human papilloma virus Animal model for HPV (HPV). Papillomas can be induced in cottontail rabbits You. And viral infection leads to three different consequences in these rabbits (Lin, Y. et al., J Virol 67: 382, 1993). First, papillomas appear and It lasts for the life of the giant. Second, papillomas regress spontaneously 2-3 months after infection . And third, papillomas progress to cancer after 8 to 15 months.   In this experiment, 12 cottontail rabbits (E. Johnson, Rago, KA) were given Steven s, J. et al. (J Virol 30: 891, 1979) by intradermal injection of the B strain of CRPV. (Stevens, J. et al., J Virol 30: 891, 1979). 4-6 months after the injection ( At this point papillomas form), the animals are divided into two groups. In the first group, 4-8μ In the presence or absence of g / ml polybrene or other transduction enhancing excipients, 10^Five~ 1 0^Ten25-100 μl of formulation buffer is passed through a small gauge needle at cfu / ml into 6 animal milk Inject into the head tumor. In the second group, control animals received 25-100 μl of formulation buffer. Administer only Rabbits are given daily for 4 to 12 days 24 to 72 hours after vector treatment. Times (morning and afternoon) at the optimal dose determined previously (eg, 62.5 mg / Kg body weight). An i.p. injection of niciclovir is given. Finally, rabbits are allowed to reach the end of the experiment (1-10 weeks ), Receive a single dose of ganciclovir daily. Papilloma regression for 2-14 days Monitor visually. 2.Human dosing protocol   Clinical skin lesions caused by human papillomavirus (HPV) include vulgaris vulgaris, Includes filamentous warts, plantar warts, and genital warts (Cobb, M. et al., J. Am. Aca d. Derm. 22: 547, 1990). In this experiment, patients were divided into two groups You. In the first group, 100-500 μl of the recombinant retroviral particles were added to the prescribed ointment (Preferably containing 4-8 μg / ml polybrene or other transduction enhancing excipient) In 10⁹At a concentration of cfu / ml, it is applied to the wart using a transdermal delivery system (TDS).   Transdermal delivery systems (TDS) provide a systemic system in which a drug is delivered in sufficient quantities to be therapeutically beneficial. The drug may be delivered through intact skin to reach the annulus. TDS is extensive Provides benefits, including the elimination of gastrointestinal absorption problems and the first-pass effect of the liver, medication Includes reduced amounts and dosing intervals, and improved patient compliance. The main components of TDS consist of polymers, drugs to be administered, excipients, and enhancers Controlled release device, as well as a solid for securing the device to the skin. It is a fixed system. Numerous polymers have been described, including gelatin, Arabic Contains rubber, paraffin wax, and cellulose acetate phthalate (Sogibayashi, K. et al., J. Controlled Release, 29: 177-185, 1994).   The second group receives only 100-500 μl of vector particles in formulation buffer. Area Cover and allow the virus particles to incubate for 0.25-12 hours, after which the TDS is removed. Good. After 1 to 7 days, ganciclovir is administered (preferably at a constant rate for one hour). ) Administer 1 to 5 mg / Kg I.V. every 12 hours for 2 to 21 days. Vector multiple times ( 2-20) can be administered again. Thereafter, ganciclovir is administered. Ganciclovir Due to the frequency of granulocytopenia and thrombocytopenia in patients receiving It is recommended that neutrophil and platelet counts be performed every two days. Regression of warts Monitor visually for 2-14 days.                                 Example 6                Ocular administration of recombinant retrovirus for E3 / 19K A.E3 / 19K Cloning of gene into KT-3B 1.Adenovirus isolation and purification   Adenovirus isolation and purification is described in Green et al., Methods in Enzymology 58: 4. 25, 1979). Specifically, 5 liters of Hela cells (3-6.0 × 10^Five Cells / ml) were transformed with 100-500 plaque forming units (pfu) / ml of adenovirus type 2 (Ad 2) Infect with virions (ATCC number VR-846). Ink for 30-40 hours at 37 ° C After incubation, the cells were placed on ice and centrifuged at 230 xg for 20 minutes at 4 ° C. And resuspend in Tris-HCl buffer (pH 8.1). Ultrasonic pellet Crushed mechanically and homogenized in trichlorotrifluoroethane Then centrifuge at 1,000 xg for 10 minutes. Take out the upper water layer, and 10m l CsCl (1.43g / cm^Three) And centrifuged at 20,000 rpm for 1 hour in a SW27 rotor. Let go. Remove the milky virus band and add 1.34 g / cm with CsCl^ThreeAdjust to And further centrifuged at 30,000 rpm for 16-20 hours in a Ti50 rotor. Gradient Remove the central visible virus band and purify the adenovirus DNA Store at 4 ° C until 2.Isolation and purification of adenovirus DNA   Incubate adenovirus band with protease for 1 hour at 37 ° C To digest the protein. After centrifugation at 7,800 × g for 10 minutes at 4 ° C., the particles were Solubilize in 5% SDS for 30 minutes at room temperature, then extract with an equal volume of phenol. The upper aqueous layer is removed, re-extracted with phenol, extracted three times with ether, and Dialyze for 24 hours in is buffer. The virus Ad2 DNA is precipitated with ethanol and Wash with knol and resuspend in Tris-EDTA buffer (pH 8.1). About 0.5mg Viral Ad2 DNA is isolated from the virus obtained in 1.0 L of cells. 3.E3 / 19K Gene isolation   Virus Ad2 DNA was digested with EcoRI and electrophoresed on a 1% agarose gel. Separate more. Generation containing the E3 / 19K gene, located in the Ad2 coordinate region of 75.9 to 83.4 2.7 Kb Ad2 EcoRI D fragment (Herisse et al., Nucleic Acids Research 8: 2173, 1980; Cladaras et al., Virology 140: 28, 1985). Out, phenol extract, ethanol precipitate and in Tris-EDTA (pH 8.1) Dissolve. 4.E3 / 19K Cloning of gene into KT-3B   The E3 / 19K gene is cloned into the EcoRI site of PUC1813. PUC1813, essentially , Kay et al. (Nucleic Acids Research 15: 2778, 1987) and Gray et al. (PNAS 80: 5842). , 1983). E3 / 19K is recovered by EcoRI digestion and The released fragment was inserted into the EcoRI site of the phosphatase-treated pSP73 plasmid. Clone. This construct is named SP-E3 / 19K. SP-E3 / 19K cDNA direction Confirm using appropriate restriction enzyme digests and DNA sequencing. In sense direction, cDN The 5 'end of A is adjacent to the XhoI site of the pSP73 polylinker, and the 3' end is Adjacent. X containing E3 / 19K cDNA in either sense or antisense orientation The hoI to ClaI fragment was recovered from the SP-E3 / 19K construct and Clone into the XhoI-ClaI site of Lus. This construct is named KT-3B / E3 / 19K You. B.PCR Cloning of amplified E3 / 19K gene into KT-3B 1.E3 / 19K PCR amplification of gene   Ad2 DNA E3 / 19K gene (amino terminal signal sequence followed by E3 / 19K protein Intraluminal domains and caps that allow the matrix to be embedded in the endoplasmic reticulum (ER) Containing the ruboxyl-terminal cytoplasmic tail) are the viral nucleotides 28,812 and 29,2. Located between 88 and. Isolation of the Ad2 E3 / 19K gene from viral genomic DNA Achieved by PCR amplification using the primer pairs shown below: Forward primer for Ad2 nucleotide sequence 28,812-28,835 Reverse primer for Ad2 nucleotide sequence 29,241-29,213   In addition to the complementary sequence of Ad2, both primers are used for the efficiency of the PCR amplicon product. Contains a 5 nucleotide "buffer sequence" at its 5 'end for efficient enzymatic digestion You. This sequence in the forward primer is followed by an XhoI recognition site, and vice versa. direction The primer is followed by a ClaI recognition site. Therefore, in the direction from 5 'to 3', E3 / 19K The gene is flanked by XhoI and ClaI recognition sites. E3 / 19K gene from Ad2 DNA Is achieved using the following PCR cycling protocol: 2.PCR Ligation of amplified E3 / 19K gene to KT-3B   The E3 / 19K gene (about 780 bp in length) from the SP-E3 / 19K construct was removed and 1 Isolate by% agarose / TBE gel electrophoresis. Next, the XhoI-ClaI E3 / 19K The fragment is linked to the KT-3B retroviral backbone. KT-3B / E3 / 19K Called. The KT-3B / E3 / 19K construct was transformed with E. coli, DH5α bacterial strain (Bethesda Research La bs, Gaithersburg, Maryland). For details, The bacteria are transformed with 1-100 ng of the ligation mixture DNA. Transformed bacterial cells On an LB plate containing ampicillin. Plate at 37 ° C overnight Incubate, select bacterial colonies, and prepare DNA from them. This DNA Is digested with XhoI and ClaI. Expected end of plasmid containing E3 / 19K gene The sizes of the nuclease restriction cleavage fragments are 780 bp and 1,300 bp. C.Recombinant retrovirus vector KT-3B / E3 / 19K of packaging cell line DA     Transduction 1.Plasmid DNA transfection   293 2-3 cells (cell line derived from 293 cells, ATCC Deposit No. CRL 1573, (WO 92/0526 6)) 5.0 × 10^FiveIndividual cells at approximately 50% confluence on a 6 cm tissue culture dish Sowing The following day, 4 hours before transfection, the medium was reconstituted in 4 ml of fresh medium. Replace with the ground. 10.0 μg of KT-3B / E3 / 19K with standard calcium phosphate-DNA co-precipitation Plasmid and 10.0 μg MLPG plasmid were replaced with 2M CaCl_TwoBy mixing with the solution And add 1X HEPES buffered saline solution (pH 6.9) and incubate for 15 minutes at room temperature. Cubate. Transfer the calcium phosphate-DNA coprecipitate to 293 2-3 cells, then 7 ℃, 5% CO_TwoAnd incubate overnight. The next morning, cells are washed three times in 1 × PBS (pH 7.0) Wash. Add fresh medium to the cells, then 37 ° C, 10% CO_TwoIncubate overnight To The next day, harvest the cells from the medium and pass through a 0.45μ filter. This supernatant is used for transduction of packaging cells and tumor cell lines. Other bees Transient vector supernatants for the vector are prepared in a similar manner. 2.Transduction of packaging cell lines   Transduction of the packaging cell line is performed as described in Example 2C. 3.Detection of replicable retroviruses   Detection of the replicable retrovirus is performed as described in Example 2D. D.Transduction of cell line with recombinant retrovirus vector KT-3B / E3 / 19K   The following adherent human and mouse cell lines were^FiveIn a cell / 10cm dish Seed with 4 μg / ml polybrene: HT 1080, Hela, and BC10ME. Next day On the day, add 1.0 ml of the filtered supernatant from the DA E3 / 19K pool to each cell culture plate. The next day, 800 μg / ml G418 is added to all cell culture media. Select this culture Selections are completed and sufficient cell numbers are tested for gene expression. To be generated. The transduced cell lines were each designated HT 1080-E3 / 19K. Hela-E3 / Named 19K and BC10ME-E3 / 19K.   EBV transformed cell lines (BLCL) and other suspension cell lines were Transduction is performed by co-culture with the irradiated producer cell line. For details Irradiated (10,000 rad) producer cell line with 4 μg / ml polybrene 5.0 × 10 in culture medium^FivePlate on individual cells / 6 cm dish. Cells 2 to After attaching for 24 hours, 10⁶Add individual suspended cells. After 2-3 days, remove the suspended cells. And pelleted by centrifugation and resuspended in growth medium containing 1 mg / ml G418. Turbid and seed 10 wells of a round bottom 96 well plate. This culture is Plate and then into a T-25 flask. E. FIG.Expression of E3 / 19K in the recombinant retroviral vector construct KT3B-E3 / 19K 1.Western blot analysis   Radioimmunoprecipitation assay (RIPA) lysates were selected for analysis of E3 / 19K expression From the culture. Prepare RIPA lysate from confluent plate of cells To make. Specifically, the medium is first aspirated from the cells. Culture plate containing cells 100-500 ml of ice-cold RIPA lysis buffer (10 mM Tris, pH 7.4; 1% Noni det P40; 0.1% SDS; 150 mM NaCl) is added to the cells. Using a micropipette The cells are removed from the plate and the mixture is transferred to a microcentrifuge tube. This Centrifuge the tube for 5 minutes to pellet cell debris, and discard the supernatant in another tube. Transfer to The supernatant is electrophoresed on a 10% SDS polyacrylamide gel. And The protein band was treated with CAPS buffer (10 mM CAPS (Aldrich, Milwaukee, Wis.), PH 11.0; 1 (0% methanol) at 10-60 volts for 2-18 hours. You. This membrane was washed from CAPS buffer with 5% Blotto (5% skim milk; 50 mM Tris, p). H7.4; 150 mM NaCl; 0.02% sodium azide, and 0.05% Tween 20) and transfer to E3 / 19K (Severinsson et al., J. Cell. Bio l. 101: 540-547,1985). Binding of antibody to membrane¹²⁵Detected by I-Protein A I do. 2.To demonstrate reduced levels of class I expression compared to non-transduced cells                 KT3B-E3 / 19K FACS analysis of vector transduced cells   A cell line transduced with the KT3b-E3 / 19K vector was analyzed by FACS analysis for MHC class I Test for expression of offspring. Non-transduced cells also express MHC class I molecules To analyze and determine the transduction effect on the expression of MHC class I molecules And compared with E3 / 19K transduced cells.   Mouse cell lines, BC10ME, BC10ME-E3 / 19K, P815 (ATCC No.TIB64), and P815 -E3 / 19K, H-2D on cell surface^dTest for expression of the molecule. Sub confluence The cells that have grown to their densities are removed from the culture dish by treatment with Versene. And cold (4 ° C.) PBS containing 1% BSA and 0.02% sodium azide (wash (Buffer solution) and washing twice by centrifugation at 200 g. 2 million cells Microcentrifuge at 200 g before removing the supernatant. To let. Transfer cell pellet to H-2D^dSpecific Mab34-2-12s (50 ml 1: 100 dilution) (Purified antibody, ATCC No. HB87) and mix occasionally for 30 minutes at 4 ° C. Incubate. Wash the antibody-labeled cells with 1 ml of washing buffer before removing the supernatant. Wash twice with buffer (4 ° C.). Cells are harvested with biotinylated goat anti-mouse κ light chain Mab (50 ml (1: 100 dilution of purified antibody) (Amersham, Arlington Height, IL). And incubate at 4 ° C for 30 minutes. Wash cells and add 50 ml avidin-conjugated FITC (Pierce, Rockford, IL) and incubate at 4 ° C. for 30 minutes You. The cells are washed once more, resuspended in 1 ml of wash buffer, and They were kept on ice prior to analysis on an analyzer (Becton Dickinson, Los Angeles, CA). Trait The average fluorescence intensity of the transfected cells was determined by the E3 / 19K protein versus surface MHC class I molecule expression. In order to determine the effect, the average fluorescence intensity of non-transduced cells is compared. F.Administration of vector constructs   1.Rat dosing protocol   Rats are anesthetized and one eye is treated with 4-8 μg / ml polybrene or other In a formulation buffer in the presence or absence of^Five~Ten^Tencfu / m 5-100 μl of the recombinant retroviral particles at a concentration of 1 l are instilled. Of prescription buffer 5-100 μl of the solution containing only Add to eyes. This solution was allowed to incubate for 1 hour, after which each eye was Rinse three times with 100 μl saline. Two to seven days after treatment, the rats are sacrificed and the cornea is Taking Remove and homogenize in 2 ml of ice-cold RIPA lysis buffer. Expression of E3 / 19K Is detected by Western blot analysis as described in Example 6El. 2.Human dosing protocol   Although corneal transplant rejection rates are relatively low, current treatments for those with rejection Requires continuous treatment of steroid compounds. It eventually requires surgery Leads to the formation of cataracts. Therefore, introduction of recombinant retrovirus expressing E3 / 19K Incorporation hinders the need for such steroid regimens. 4 in formulation buffer In the presence or absence of ８8 μg / ml polybrene or other transduction enhancing excipients , 10 in prescription buffer^Five~Ten^Ten10-500 μl of recombinant retrovirus at a concentration of cfu / ml The particles are administered to the eyes of a prone patient. Incubate this solution for 15-30 minutes. Cuvate and then wash with saline.   Alternatively, the cornea can be treated with 4-8 μg / ml polybrene or just prior to surgical adhesion. 10 in formulation buffer with or without other transduction enhancing excipients^Five~Ten^Tencfu / m One hour can be incubated with 1 ml of retroviral vector particles at a concentration of l. In any of the above cases, the progress of the transplant is visually observed for tissue viability. Monitor by doing                                 Example 7           Intranasal administration of recombinant retrovirus expressing factor VIII A.Construction of full-length and B-domain deleted Factor VIII cDNA Retrovector ^™   Construction of a full length and B domain deleted Factor VIII retrovector is described in Example 2A. be written. B.Administration of vector constructs   1.Rat dosing protocol   The nasal passage is often due to the extensive network of capillaries located below the nasal mucosa. Have been shown to be effective in the administration of molecules. This is an effective systemic absorption And when the drug is administered with an absorption enhancer, absorption is highly biological. Occur rapidly with global availability (Gizurarson et al., Acta Pharm 2: 105, 1990). (Review)   One group of six Fischer-344 rats was treated with Factor VIII retroviral vector particles. Used for nasal administration of pups. 4-8 μg / ml polybrene or other transduction 10 in formulation buffer with or without enhancing excipients⁹1-50 μl at cfu / ml Using a pipette with retroviral vector particles inserted into each nostril about 3-5 mm Apply. Another group receives the vector-free formulation buffer in the same manner. blood Samples were collected from the jugular or tail vein 1-14 days later, and as described in Example 2Hi. Assay for Factor VIII production as described. 2.Human dosing protocol   There are several types of drug delivery devices for the nasal cavity (Chien, Y et al., Crit Rev  Therap Drug Carr Sys, 4:67, 1987). These systems use a nasal sp Including laye, nasal drip, wet cotton gauze, aerosol spray, and inhaler. Me The needle-dispensing nebulizer can deliver a predetermined volume of the formulation to the nasal cavity.   Two groups of patients are used in this study. One group of patients may receive a nasal spray or Or 4-8 μg / ml polybrene or other form applied to each nostril via nasal instillation 10 in formulation buffer in the presence or absence of⁹100-50 at cfu / ml Receive 0 μl of retroviral vector particles. The other group is applied in the same way Receive only the prescribed formulation buffer. Blood samples are collected after 1-14 days and performed Assay for Factor VIII as described in Example 2B1.                                 Example 8         Preparation of recombinant retrovirus for delivery of human growth hormone A.hGH Preparation of a vector containing   The vector pDHF828 containing the full-length human growth hormone gene is essentially Build as below. Briefly, plasmid pDHF811 was replaced with the KT-1 Remove the XhoI-ClaI fragment of the virus vector and connect the cohesive ends. It was constructed by inserting the following oligonucleotide linkers. Linker sequence: Specifically, the linker was added at 65 ° C for 20 minutes, 42 ° C for 20 minutes, 37 ° C for 20 minutes, and room temperature. For 2 hours. The concentration of both oligonucleotides was 18 mM and the salt concentration was 1 00 mM NaCl. After annealing, add 50 ml of 1.8 mM annealed linker. , Digested with ClaI overnight to generate ClaI ends. 3 nM KT-1 X for coupling The hoI-ClaI fragment was mixed with 90 nM linker and the resulting mixture was Incubated for 3 hours at ° C. Transfer the ligated DNA sample to DH-5α competent And transformed cells were screened.   Plasmid chGH800 containing the full-length cDNA of the hGH gene (Martial, R.A., et al., Science  205: 602,1979), digested with HindIII and blunt-ended with Klenow fragment enzyme And cloned into the SrfI site of pDHF811. The resulting plasmid was cloned into pDHF828 It was called. B.hGH Preparation of expressed recombinant retrovirus   The pDHF828 plasmid is then transferred to HX packaging cells using standard procedures. According to the preferred modification of the kit protocol. Assay (HGH I00T) (Nichols Institute, San Juan Capistrano, CA) did. On day 1, warm kit components to room temperature and gently invert by inversion And then open some vials. The test sample is Centrifuge in a volume centrifuge for 5 minutes, then remove fibrin and other debris Used them. All samples were measured in quadruplicates, including standards. Incubation was performed in 12 x 17 polypropylene tubes stored in the dark. U. Aliquot 150 μl of sample or standard into each tube and add Of antibody was added and the samples were mixed gently. One bead in the kit Were added to each well using the tweezers provided in. Cover the tube , Covered with foil and shaken on an orbital shaker at room temperature for 24 hours. Stan Dard contains 530 pg / ml (STD D) and serial dilutions are given at 250, 100, 50, 25, 1 0, 5, and 2.5 pg / ml were made in a zero standard of Std D.   After 24 hours, open the tubes and add 0.5 ml of wash buffer. Was. Combine these washing solutions with enough force so that the beads come off the bottom of the tube. added. The samples are washed three times with 2.0 ml of nanopure water and thoroughly each time Aspirated. Luminometer measurements were taken on 12 x 75 polycarbonate (transparent) stored in the dark. Light plastic) in a tube. Luminometer for performance control Preliminary tests were performed using a darn.   Using this assay, HX / HGH retrovector producing cell lines were transformed to 4.8 x 10⁶cfu / ml Produced at a titer of Introduction of plasmid into DX packaging cells is 1.6 × 10⁷cf This resulted in the generation of clonogenic cells with a titer of u / ml.                                 Example 9                        Preservation of recombinant retrovirus A.Lactose formulation of recombinant retrovirus   Crude recombinant retrovirus is bound to beads in a bioreactor matrix. Celligan cells containing the combined recombinant retrovirus-transformed DA cells Obtained from Io Reactor (New Brunswick, New Brunswick, NJ). Cells are continuous In a growth medium that traverses cells in a continuous flow process Next, the recombinant retrovirus is released. Collect the medium leaving the bioreactor and Through a 0.8 micron filter and then a 0.65 micron filter To clarify the crude recombinant retrovirus. Crossflow enrichment system (Filtron , Boston, MA). About 50 units of DNa per ml of concentrate The exogenous DNA is digested by the addition of se (Intergen, New York, NY). Digestion, Using the same cross-flow system, 150 mM NaCl, 25 mM tromethamine, pH 7.2 Diafiltrate against. In 50 mM NaCl, 25 mM tromethamine, pH 7.4 On a Sephadex S-500 gel column (Pharmacia, Piscataway, NJ) equilibrated with Load the diafiltrate into Purified recombinant retrovirus at 50 mM Elution from Sephadex S-500 gel column in NaCl, 25mM tromethamine, pH 7.4 I do.   A formulation buffer containing lactose was prepared as a 2x concentrated stock solution. This formulation buffer contains 25 mM tromethamine, 70 mM NaCl, 2 mg / ml arginine, 10 mg / ml HSA and 100 mg / ml lactose in a final volume of 100 ml to pH 7.4 Contained.   1 part of S-500 purified recombinant retrovirus plus 1 part of 2x lactose formulation buffer To form a purified recombinant retrovirus. Prescribed recombination Storing or drying the retrovirus at -70 ° C to -80 ° C Can be.   Supermodulyo 12K freeze dryer (Edwards High Vacuum, Tonawanda, NY) In the attached Edwards Refrigerated Chamber (3 Shelf RC3S unit), Lyophilized retrovirus. When the lyophilization cycle is complete, After the release of nitrogen gas, the vial is stoppered under vacuum. Remove vials when removing Crimp with a luminium seal.   In a given lactose study, the formulated liquid product was stored at -80 ° C and -20 ° C. Stored in both circulation freezer. In FIG. 15, the virus of these samples Infectivity was compared to the virus infectivity of the lyophilized sample. Lyophilized sample , -20 ° C, refrigerated, and room temperature. Sample activity during reconstitution Determined by a titration assay.   The lyophilized recombinant retrovirus is reconstituted with 1.0 ml of water. Reconstructed The infectivity of the recombinant retrovirus is determined by a titer activity assay. Assay , HT1080 fibroblast or 3T3 mouse fibroblast cell line (ATCC Deposit No. CCL 163) It went in. Specifically, 1 × 10^FivePlate cells on a 6cm plate And 37 ° C, 10% CO_TwoIncubate overnight at. Reconstituted recombination Les Ten microliters of serial dilutions of Torovirus were added to 4 mg / ml polybrene (Sigma, (St. Louis, MO) and added to the cells at 37 ° C., 10% CO 2_TwoIncubate overnight at Beate. Following incubation, cells are neomycined in medium containing G418. Select for thin tolerance, and 37 ° C, 10% CO_TwoIncubate for 5 days at . Following the initial selection, these cells are re-fed with fresh medium containing G418. And incubate for 5-6 days. After the last selection, for colony detection Next, the cells are stained with Coomassie blue. Determine the sample titer based on the number of colonies, Determine from the label and the volume used.   FIG. 15 shows that storage in a lyophilized form at −20 ° C. to refrigerated temperature was −80 ° C. to −80 ° C. Similar virus activity to recombinant retrovirus stored in liquid at 20 ℃ Retain and allow less stringent temperature control during storage. And B.Mannitol formulation of recombinant retrovirus   The recombinant retrovirus utilized in this example was purified as described in Example 9A. did.   Formulation buffer containing mannitol was prepared as a 2x concentrated stock solution . This formulation buffer contains 25 mM tromethamine, 35 mM NaCl, 2 mg / ml arginine , 10 mg / ml HSA, and 80 mg / ml mannitol in a final volume of 100 ml, pH 7.4.  Contained in   1 part S-500 purified recombinant retrovirus, 1 part mannitol formulation buffer To form a purified recombinant retrovirus. Prescribed recombination The retrovirus is stored at -70 ° C to -80 ° C at this stage, or Can be dried.   Edwards Refrigerated Chambe mounted on a Supermodulyo 12K freeze dryer Dry the formulated retrovirus in r (3 Shelf RC3S units). freeze drying When the cycle is complete, release the nitrogen gas to 700 mbar and place in a vial under vacuum. Stopper. Upon removal, the vial is crimped with an aluminum seal.   In a given mannitol study, the prescribed liquid product was stored at -80 ° C and -2 ° C. Stored in both 0 ° C. circulating freezer. FIG. The virus transmission power of the lyophilized sample was compared to the virus transmission power of the ills. Lyophilized sump Were stored at −20 ° C., refrigerated temperature, and room temperature. Sample activity during reconstitution Is determined using the titer assay described in Example 9A.   FIG. 16 shows that storage in lyophilized form at −20 ° C. to refrigerated temperatures is −80 ° C. Significant virus, comparable to recombinant retrovirus stored in liquid at -20 ° C Temperature control that retains virus activity and is less stringent during storage Is allowed. C.Trehalose formulation of recombinant retrovirus   The recombinant retrovirus utilized in this example was prepared as described in Example 9A. Was purified.   Formulation buffer containing trehalose was prepared as a 2x concentrated stock solution . This formulation buffer contains 25 mM tromethamine, 70 mM NaCl, 2.0 mg / ml arginine , 10.0 mg / ml HSA, and 100 mg / ml trehalose in a final volume of 100 ml, pH 7 Contained in .2.   Add 1 part of trehalose formulation buffer to 1 part of S-500 purified recombinant retrovirus By addition, a purified recombinant retrovirus is formulated. Prescribed recombination The retrovirus is now stored at -70 ° C to -80 ° C or dried. Can be dried.   Edwards Refrigerated Chambe mounted on a Supermodulyo 12K freeze dryer Dry the formulated retrovirus in r (3 Shelf RC3S units). freeze drying When the cycle is complete, release the nitrogen gas to 700 mbar and place in a vial under vacuum. Stopper. Upon removal, the vial is crimped with an aluminum seal.   In a given trehalose study, the formulated liquid product was stored at -80 ° C and -20 Stored in both circulating freezer at ° C. In FIG. 17, the windows of these samples are shown. Russ transmission was compared to the virus transmission of lyophilized samples. Lyophilized sump Were stored at −20 ° C., refrigerated temperature, and room temperature. Sample activity during reconstitution Determined using the titer assay described in Example 9A.   FIG. 17 shows that storage in a lyophilized form at −20 ° C. to refrigerated temperature was Similar virus compared to recombinant retrovirus stored in solution at 20 ° C Retention of activity and allow for less stringent temperature control during storage. It indicates that it is acceptable.   Virus in solution formulated recombinant retrovirus sample stored at -80 ° C Infectivity was determined by lyophilized recombinant retrovirus storage at -20 ° C. Compared to Lus infectivity. First, obtain a large amount of recombinant retrovirus, and then Formulated in four different ways as shown in Then, the prescribed recombinant retro Irus is frozen in bulk for 1.5 months, then quickly thawed and lyophilized . The lyophilized positive control was stored at -80 ° C and then lyophilized at -20 ° C. Compare with sample. The prescriptions are listed below:   In the graph of FIG. 18, the Y axis in each of the four graphs (A, B, C, D) is It represents the normalized titer. At the starting time point after freeze-drying (t = 0), Established for both 80 ° C liquid samples and -20 ° C lyophilized samples. At each time point in the stability study, the titer obtained was divided by the titer value at the zero time point. And the original percentage was plotted on the graph.   This data shows that the activity after lyophilization is comparable to that of liquid samples (stored at -80 ° C). To demonstrate that it is maintained in lyophilized samples (stored at -20 ° C). The formulated lyophilized recombinant retrovirus is stored in a -20 ° C freezer (frost-free). (Circulation freezer). Formulated liquid recombinant leto stored at -80 ° C Lyophilized forms, compared to virus, are less stringent of storage conditions. Tolerant controls.                                 Example 10                 Analysis of crude and purified recombinant retrovirus   Crude and purified solutions of recombinant retroviral particles can be used, for example, with PHASTGEL system. (Pharmacia Biotech) to separate on a gradient polyacrylamide gel . Briefly, samples were run with 4-15% untreated polyacrylamide. Run on gel and run at 250V for 35 minutes. The gel is then removed and Stained with Coomassie Blue to detect viruses and other protein components To color. This gel is then used to determine the contents of the virus and other components. Scan by laser densitometry.   Viral bands are identified by their relative molecular weight and reverse transcriptase activity (RT). Can be determined. The purpose of this assay is to reverse transcriptase (RT) (this enzyme (Associated exclusively with the virus). In the sample The relative amount of retrovirus can be determined by measuring the activity of this enzyme in a given preparation. And can be determined.   Briefly, Moloney murine leukemia virus reverse transcriptase (Pharmacla, Newar k, NJ) in 1 × Tris / EDTA buffer solution containing 10 mM Tris-HCl and 1 mM EDTA (pH 8.0) To a concentration of 1 μg / ml. Add 100 μl of this solution to 6. 84ml sterile dH_TwoO, 500 μl 1 M Tris HCl (pH 8.0), 10 μl 0.1 M MnCl_Two200 μl 1 M dithiothreitol, 50 μl of 10% Nonidet P40 (NP40), 2 μl of 100 μM dNTP ( Pharmacia, Newark, NJ, dNTP Ultrapure Kit^TM), And 300 μl of methyl-^ThreeH Chimiji To 5'-triphosphate (30-50 Ci / mmol). Place this mixture in a water bath Incubate at 37 ° C for 1 hour. After incubation, place samples on ice. Good. About 1.0 ml of 2N HCl is added to the cooled sample. Precipitated radiolabeled D The NA fragment was subjected to Millipore sampling manifold (Millipore, Philadelphia, PA) Filter with suction on a glass fiber filter using. Wash this filter Clean, dry, place in scintillation cocktail, and Beckman LS5000 Count on a TD scintillation counter (Beckman, Dallas, TX).                                 Example 11             Analysis of complement resistance in various packaging cell lines A.Preparation and transduction of packaging cell lines   Four different packaging cell lines were packaged with pCBβ-gal in infectious virions. Used for caging. Two cell lines are D17 dog cells ("D") (ATCC C CL 183) and two cell lines are derived from the human embryonic kidney cell line 293 ("2") (ATC C CRL 1573). For both canine and human packaging cells, One cell line expresses an amphotropic envelope derived from 4070A virus (Ie, DA and 2A, respectively), and the other is xenobiotic derived from the NZB9-1 virus. Nootropic envelopes (ie, DX and 2X, respectively) were expressed. all Of 10% heat-inactivated fetal bovine serum (Hyclone, Salt Lake City, (UT) supplemented in DMEM medium (Irvine Scientific, Santa Ana, CA).   Each of the four packaging cell lines was transduced with G pseudotyped pCBβ-gal (pseudotyped For procedures, see Burns et al., (1993) Proc. Nat'l. Acad. Sci. USA, Vol. 90, 8033-8037. See page.) After G418 selection, a pool of selected cells is diluted and cloned (Amplified vector maintained as a non-clonal pool) Producing human cell lines). Highest on HT1080 cells (ATCC deposit number CCL 121) Cell lines producing the vector producing high titers were selected. G418 roller Determined about 2 days after transduction by X-gal staining of knee forming units or monolayers ( For a more detailed titration assay, see Current Protocols in Molecular Bio logy, Ausubel et al.).   The retrovirus-containing supernatant was transferred to individual amphotropic envelopes (DA and And 2A) or xenotropic envelope (DX or 2X) vectors Collect from each confluent monolayer of cell lines and filter with a 0.45 μm After filtration over, aliquoted and stored at -70 ° C. B.Serum inactivation assay   The serum inactivation titer assay was performed as follows: at least two sera From different human volunteers, chimpanzees, baboons, and macaque monkeys . Approximately 20-70 mL of blood is separated into serum separation tube (Becton Dickinson, Los Angeles, CA) Within each donor. Blood is allowed to clot for 20-30 minutes at room temperature, The samples were then centrifuged at 2000 xg for 10 minutes at 4 ° C. Serum was added to about 1.1 ml And frozen at -80 ° C. All classic vials from each batch For complement activity (Quantiplate, Kallestad Labs, Inc., Chaska, MN) Only batches with "normal" levels of complement activity were used. 100% fresh A 1 ml aliquot of serum was used for each inactivation assay. Complement of each serum Was prepared by heat inactivation at 56 ° C for 30 minutes. Was. Undiluted and diluted preparations of supernatants containing recombinant retroviral particles (10^Three/ ml blood BCFU) or medium control, serum, or heat-inactive The mixture was mixed 1:10 with the immobilized serum. These mixtures are then incubated at 37 ° C for 30 minutes. I was vate. The treated vector particles were then transformed into standard blue colony forming cells. Titration by position assay (Current Protocols in Molecular Biology, supra) did.   The results of this experiment are shown below in Table 1 as BCFU / ml.   Briefly, these results were generated in a human packaging cell line. Recombinant retrovirus is a thermolabile product of human serum in an in vitro assay. Show no detectable sensitivity to inactivation by minutes (probably complement) Prove. In contrast, this result indicates that inactivation by chimpanzee serum Partially susceptible to This order shows that susceptibility to baboon serum and macaque serum increases. In addition, these in vitro results have been demonstrated in D17-derived packaging cell lines. The recombinant retrovirus produced contains heat-labile components (probably human, chimpanzee). , Baboon, and macaque serum). Demonstrate sensitivity.   Any of the above two cell lines (canine cell line DA and human cell line 2A), and Packaging in different human cell lines (HX, HT1080-derived packaging cell lines) Other experiments performed with another recombinant retroviral genome performed; Confirm that the conclusions obtained are not vector dependent. In addition, 29 3 and HT1080-derived human producer cell lines are also equally complement resistant vectors. Generated.   A package derived from human HT1080 expressing the envelope tropism of different mice, poly Further generated using recombinant retroviral particles generated in zing cells. Experimental data also indicate a lack of sensitivity for thermolabile human serum components, and The argument was further confirmed.                                 Example 12           Production of recombinant retrovirus from hollow fiber culture A.Start culture   To begin hollow fiber culture, first 48 hours before seeding, 100-200 ml Hollow fibers can be simulated at 37 ° C operating conditions in complete growth media. Bioreactor (HFB) conditions were set. The growth medium has been Should be adapted. Aspirate all liquid in HFB sent first And should be replaced with complete growth medium. Place to sow in bioreactor If this is the case, the cells should not be split faster than 48 hours and the cells Should be in exponential phase at the time of harvest for seeding. The cells are Collected by centrifugation and pelleted by centrifugation. Then this cell The pellet is resuspended in 4 mL of 25% unconditioned medium and found on HFB Extra-capillary space with a syringe using the syringe port on the side Deliver to After seeding the HFB, allow the cells to sit for 20-30 minutes before starting the circulation pump. To wear. During this time, the medium used to prepare the HFB must be 100-200 mL before the 25% preparation. Replace with medium. Open the circulation pump at the starting flow rate set at 25 ml / min. Start (set to 5 with 2 long pump needles). When the pump is turned on One hour after starting, a 1 mL sample of the medium was used to reduce the initial lactate and ammonia levels. Collect for recording. Take 1ml samples every 24 hours as daily plan And assay daily lactate and ammonia production. Lactate levels are 2. When starting to reach 0 g / L (or equivalent to 22 mM / L), add 100-200 mL of the first old medium Replace with fresh medium. The same until the culture reaches the daily level of 20 mmol / L Replace volume of medium. When the daily lactate level reaches 20 mmol / L, a storage bottle Increase size to 500 mL bottle containing 500 mL fresh medium. The culture is then: When the production of lactate at 2.2 mmol / day begins, the flow rate is increased to 50 mL / min. 500 If the volume of mL reaches 20 mmol / L lactate daily, the storage supplied to the original Cellco Replace the liquid cap with the male luer lock fitting. Larger storage caps (Unisy Replace with n-vender part # 240820). This storage cap is a 2 liter Cornin g Corresponds to the size of the bottle (to avoid changing the storage cap during the culture operation, Cultures can be transported with larger storage caps that can also support smaller bottle sizes. Start rolling). When assaying and recording daily lactate readings, When the culture reaches maximum cell density (lactate percentage is reduced and levels are The daily lactate production level of the culture can be calculated to determine . B.2X- Seeding density for β-gal   Establish two hollow fiber runs to establish specific sowing requirements . One run seeds a small number of cells and the other seeds a large number of cells You. The progress of the culture can be monitored for daily glucose consumption and lactate production levels. And track by. FIG. 22 shows the vector producer cell line 2X-β-GAL_17-142 5 is a representative graph of data generated over a period of weeks.   In this experiment, one HFB contained 1.3 × 10⁷Seed cells (low seeding culture 1.6) for the other HFB⁸Cells were seeded. In this experiment, the cell line (2x-β-GAL_17-14) Is a suitable hollow fiber under low and high seeding conditions -I could start driving. The ability to incubate HFB with fewer cells is To reduce the effort required to generate the number of cells needed to start feeding It is only convenient. However, a low seeding start is also usually optimal to produce the highest titers. of Extend the time required to reach cell density. In this experiment, the details used were The spore strain is a hollow fiber that ultimately requires a daily medium change of 500 mL per day. Very well adapted to culture. C.Cell culture health and maximum cell density:   In the initial Cellco design, the initial media storage cap was a standard 100 mL And not suitable for larger bottles other than 500 mL media bottles Was observed. This allows more than 500 mL of daily medium exchange for vigorous growth cultures. If replacement is necessary, it is a problem. A large number of medium changes daily Increasing the probability of culture contamination by increasing daily handling time . If you do not choose to perform multiple daily medium changes, the culture will run To the daily toxicity level of waste that can affect cell expansion in the system as well as how long it survives Exposed. Fig. 22-B shows that 500 mL of fresh medium must be replaced every day after culturing on day 7. The lactate concentration of the required culture is shown. Figure 22-D tracks the amount of lactate produced daily This is one indication of the health of the culture. The graph shows that the culture is No longer expanded based on reaching stable levels of lactate production Is shown. Another indicator of cell culture health is a decrease in peak titer production ( See FIG. 23). This also correlates with high levels of daily lactate exposure . These findings suggest that the optimal titer is determined by the maximum cell density and the relative health of the culture. It shows that it can be correlated with D.Optimal titer concentration, frequency of recovery, and total recovery   Β-gal titers for the above experiments were determined from frozen samples and Titration was performed in 293 cells assayed 48 or 72 hours after transfection. Transduction Entered cells are stained for βGAL activity and individual cells are counted with a hemocytometer. The titer was obtained based on the number of blue cells / mL (BCT / mL). As shown in FIG. Optimal titers were 72 h blue cell titers in 293 cells on day 7 of high seed culture. From 1.8 × 10⁸Obtained at BCT / mL. Duplicate cultures initially seeded at 10 times lower seeding density The product was 5.2 x 10 from the 48 hour blue cell titer.⁷Peak reached at BCT / ml. 2X b-GAL_{17- 14} Previous planar stock cultures of the cultures were blue cell titers for 48 hours on HT1080 cells. And the number is 5 × 10⁶Calculated as BCT / mL Have been. If using the value obtained from the 48 hour blue cell titer, use the hollow fiber The increase in titer due to the use of the system 10 times higher than the crude supernatant obtained from Ko. These maximum titers include Reaches lactate with a daily toxicity level of 20 mmol / L that appears to reduce potency Reached before. Crude supernatant was collected every 9 hours without any loss of titer (See FIG. 2). Three recoveries per day achieved with minimal loss of titer It is expected that it can be done. In addition, continuous hollow fiber cultures can be maintained for several weeks. obtain. When the titers were compared between low and high seeding cultures, two seeding cultures were used. There is almost no difference between the nutrients on day 11 and the average is 4 × 10⁷BCT / mL.                                 Example 13                      Two-phase purification of recombinant retrovirus A.DA / ND-7 Enrichment of recombinant particles   3.5 × 10⁸cfu / ml (total 1.75 × 10⁹DX / DN-7 recombination at a titer of cfu) Five milliliters of retroviral particles were added to 1400 ml of medium (5% fetal bovine serum Diluted DMEM). 300 ml of two-phase partitioning components (PEG-8000 (autoclaved) ), Dextran sulfate, and NaCl) to a final concentration of 6.5% PEG, 0.4% Add kisstran and 0.3 M NaCl. The resulting solution is Place in a separating funnel and leave in the cold room for 24 hours (approximately 6-16 (Including two mixing steps at time intervals).   After 24 hours, carefully withdraw the lower layer (about 20 ml) and remove the intermediate phase (about 1 ml). Collect in a 15 ml conical FALCON tube. Add mesophase containing vector with PBS Dilute to 10 ml by addition and incubate at 37 ° C to bring the solution to room temperature. Incubated and destabilized micelles.   To half of the diluted mesophase, add KCl to a final concentration of 0.4M and add And mix well. The tube is then placed on ice for 10 minutes and Spin at 2,000 rpm for 2 minutes with a heart separator. The supernatant is removed and 0.45 Filter with a μm syringe filter.   The other half of the mesophase containing the vector was purified by S-500 Sephadex chromatography in 1X PBS. Separate by chromatography.   The results of these enrichment processes, as determined in the BCFU assay, are described below. Shown in Table 1 of:   In summary, if KCl isolation is used as a final step, recombinant retrovirus Reduce 1.4 liters of crude research grade supernatant containing particles to a volume of 10 ml. About 50% (+/- 20%) is recovered. S-500 chromatography as the final step If used, only about 10% of the first recombinant retroviral particles are in 14 ml Will be collected.   MY membrane Amicon for complete retroviral vector particle enrichment Utilizing a filter, the vector-containing solution may be subjected to further concentration, resulting in a volume The volume 10-14 ml was reduced to less than 1 ml.                                 Example 14   Production of DX / ND7βGAL clone 87-derived vector using cell factory   DX / ND7βGAL clone 87 (expression vector) was propagated in a cell factory . Enough cells to seed 20 10-layer cell factories (NUNC) at 1: 3 dilution Cells were grown in DMEM supplemented with fetal calf serum in roller bottles until obtained . Each 10-layer cell factory is seeded with approximately 0.8 liter of cell culture medium.   Place the medium containing cells in the factory so that the suspension is evenly filled to 10 layers. Cells were seeded into the cell factory by injection. Then this file To prevent redistribution of the suspension into common tubes. Carefully tilt away from it. Finally, this cell factory is Circulate in the upright position. Attach hepavent filters to each doorway. Then , This factory CO_TwoPlaced in incubator.   On day 3 and each of the following 3 days, the supernatant containing the vector was collected. Cell The factory is placed in a tissue culture hood. Take out one filter, and Connect a sterile transport tube to the opening. To drain the supernatant into a tube Lift this factory. About 2 liters of supernatant from each factory to recover. Refill the lost media with fresh DMEM / FBS. Transport tube Remove and put the factory back in the incubator. 20 cells About 90 liters of the crude supernatant containing the vector was obtained from the factory.   Vector validation was performed by transduction of HT1080 cells. These cells Collected after days and stained for β-gal protein. The supernatant was titrated to 2 × 10⁷/ ml.                                 Example 15               Concentration of recombinant retrovirus by low-speed centrifugation A.Preparation of supernatant of retro vector   Producer cell lines DA / βgal and HX / DN-7 were combined with 10% fetal bovine serum and 1 mM L- Added glutamine, sodium pyruvate, non-essential amino acids and antibiotics Culture flask containing Dulbecco's modified Eagle's medium (DMEM), and roller bottle Respectively. Transfer virus supernatant from flask and roller bottle. Collected and filtered through a 0.45 μm syringe filter. This filtered supernatant Is stored at 4 ° C (HX / ND7) or frozen at -70 ° C (DAβ-gal) Or did. B.Virus concentration   Transfer the virus supernatant to a 50 ml sterile OAKRIDGE screw cap tube. And placed in an SS34 rotor using a Sorvall centrifuge. This tu The tube was spun at 16,000 rpm (25,000 g force) for 1 hour at 4 ° C. When the spin is completed Remove tube, decant supernatant, and place small opaque pellet on top The cells were resuspended in the DMEM medium described above. C.Virus titer determination   Concentrated virus was placed in a 6-well plate at 2 x 10^FiveCell density HT108 cells plated 24 h before with 4 μg / ml polybrene. Was. Briefly, the virus preparation was diluted 1/10 to 1 / 10,000 and 50 μl One of each dilution was used to infect one of the 6-well plates. Plate, 37 Incubated overnight at ° C. After 48 hours, cells were fixed and stained with X-gal Was. The results are shown in Table 1 below. As is evident from Table 1, virus recovery rates ranged from 30% to 99%. High recovery was obtained from human producer cells (HX / ND7; recovery was 91% ~ 99% range).                                 Example 16                 Enrichment of recombinant retrovirus by ultrafiltration   Purified on S-500 containing β-gal expressing recombinant retrovirus DX / CB-βgal The supernatant and partially concentrated supernatant containing the same virus were each added to a 0.45 μm filter. Filter through filter and CENTRIPREP-100 filter (Product # 4308, Amicon, MA) Loaded. The supernatant was kept at a temperature of 4 ° C. throughout the procedure, including during centrifugation. . CENTRIPREP filters were spun three times at 500 × G for 45-60 minutes each. The filtrate was decanted between each spin. The retentate is then continuously reduced As a result, the volume that was initially 15 ml (or 10 ml) is reduced to about 0.6 ml per unit. Diminished.   The resulting titer is transferred to one well 24 hours before transduction of the virus sample. 1 × 10 per^FiveBy assaying HT1080 target cells set at the cell concentration Decided. Cells were treated with 8 μg / ml polybrene and 2 ml growth medium per well Transduction was performed in the presence of (DMEM + 10% FBS). This procedure is shown in Table 1 below. Approximately 100% of the virus was recovered (note that the titer is BCFU / ml) That).                                 Example 17            Preparation of recombinant retrovirus in bioreactor A.Freezing protocol   1 × 10⁷At a concentration of cells / ml / vial, 10% to 20% of FBS, And frozen in DMEM medium containing 5% to 15% DMSO. Freezer for speed control of cells ー (Series PC, Controlled Rate Freezing System, Custom Biogenic Systems, Wa Freeze at 1 ° C to 10 ° C per minute with rren MI). Keep frozen cells in liquid nitrogen Exist. B.Bioreactor protocol   Thaw cells from frozen vials at 37 ° C and wash once with medium to remove DMSO And 850cm^TwoExpanded in FALCON roller bottles (Corning, Corning, N.Y.) I do. Using the expanded cell culture, a “CELLIGEN PLUS” bioreactor (5 The working volume of the turtle; New Brunswick, Edison, NJ) was inoculated. 3-15 g of these cells Microcarriers (ie, Cytodex 1 or Cyt odex 2; Pharmacia, Piscataway, NJ). The initial inoculation density is 2 4 to 9 cells / bead in half to full volume in 24 hours. For virus products The medium components of FBS (10% -20%), glutamine (8-15 mM), glucose (4.5- 6.5 g / l), non-essential amino acids (IX), RPMI 1640 amino acids (0.2-9.6X), 10 mM HEPES, R DMEM high glucose basal medium supplemented with PMI 1640 vitamins (0.2-5X) (Irvine Scie ntific, SantaAna, CA.).   During the cultivation, the pH (6.9-7.6) and dissolved oxygen ("DO" 5-90%) are removed by air, oxygen, nitrogen. It is controlled by the use of a four gas system containing sulfur and carbon dioxide. Then After several days of batch growth, the perfusion was increased stepwise at a rate of 0.5-2.5 volumes / day, The nutrients are collected and continuously perfused with fresh medium at the same time. Cell retention, decant FIG. 3 is the result of differential sedimentation of cells covering the beads in the column.   During the operation, the bioreactor is replaced with live cells, titers, glucose, lactate, Monitor for monia level and no contamination. Living cells and power Price is 1 × 10^FiveCells / ml-1 x 10⁷In the range of cells / ml. Glucose is 6-0.25 g / l, lactate ranges from 1 to 25 mM, and ammonia ranges from 0.5 to It is in the range of 30 mM. Incubate cells in bioreactor for 5-25 days You.   From the foregoing, certain embodiments of the present invention have been described herein for purposes of illustration. However, various modifications may be made without departing from the spirit and scope of the invention. It will be appreciated that it will gain. Accordingly, the invention is not limited except as by the appended claims. , But not limited to.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 39/00 ADY A61K 37/02 AED (C12N 15/09 ZNA C12R 1:92) (C12P 21/02 C12R 1:92) ( 81) Designated country EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), AM, AT, AU, BB, BG , BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LT, L U, LV, MD, MG, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TT, A, UG, UZ, VN (72) inventor Chan, Stephen M.. W. California 92064, Poway, Camino Del Valle 12912 (72) Inventor Respeth, James G. United States of America 92109, San Diego, Lamont Street 4966 (72) Inventor Allen, John Earl. United States of America 92083, Vista Ridge, Elm Ridge Drive 1907 (72) Inventor Bodner, Moldechai United States of America California 92104, San Diego, Juniper Street 3480 (72) Inventor Chong, Kimberly United States of America California 128128, San Diego, Avenue Venue Number 7 14955 (72) Inventor de la Vega, Dan Jr. United States of America California 92104, Grimm Avenue, San Diego, 3243 (72) Inventor Depolo, Nicholas Jay. California, USA 926 05, Solana Beach, Santa Estrela 904 (72) Inventor, David Chi-Tan United States California 92122, San Diego, Camino Tranquilo 8012 (72) Inventor Ivanes, Carlos E. United States California 92129, San Diego, Milpondway 13592 (72) Inventor Mittelstade, Dennis M. United States 92129, Quinton Road, San Diego, 13687 (72) Inventor Pulsack, Charles E. California 92124, San Diego, Camino Playa Malaya 5121

Claims (1)

[Claims] 1. Protein measurable levels in human body fluids or cells, a method for obtaining a nucleic acid molecule or enzymatic products, the recombinant retroviral preparations with 10 ⁵ cfu / ml greater than titers in HT1080 cells Administering to a human, wherein the recombinant retroviral preparation is prepared such that a measurable level of a protein, nucleic acid molecule, or enzymatic product is obtained in the human body fluid or cells. A method that can direct the expression of a protein, nucleic acid molecule, or enzyme that produces an enzymatic product. 2. The titer is greater than 10 ⁶ cfu / ml, The method of claim 1. 3. The titer is greater than 10 ⁷ cfu / ml, The method of claim 1. 4. The titer is greater than 10 ⁸ cfu / ml, The method of claim 1. 5. The titer is greater than 10 ⁹ cfu / ml, The method of claim 1. 6. 2. The method of claim 1, wherein said titer is greater than ¹⁰ < ¹⁰ > cfu / ml. 7. 2. The method of claim 1, wherein said titer is greater than 10 < ¹¹ > cfu / ml. 8. A method for obtaining measurable levels of proteins, nucleic acid molecules, or enzymatic products in human body fluids or cells, which is equivalent in human serum and HT1080 cells to a titer in heat-inactivated serum and HT1080 cells Administering to a human a recombinant retroviral preparation having a high titer, wherein the recombinant retroviral preparation comprises measurable levels of proteins, nucleic acid molecules, in the human body fluids or cells. Or a method that can direct the expression of the protein, nucleic acid molecule, or enzyme that produces the enzymatic product, such that an enzymatic product can be obtained. 9. 9. The method of claim 1 or 8, wherein the recombinant retrovirus is administered to a site selected from the group consisting of cerebrospinal fluid, bone marrow, joints, arterial endothelial cells, rectum, vagina, and lymphatic system. 10. The method of claim 1, wherein the recombinant retrovirus is administered in a manner selected from the group consisting of intraocular, intranasal, sublingual, oral, topical, intravesical, and intrathecal. 11. 9. The method according to claim 1 or 8, wherein the recombinant retrovirus is administered to an organ selected from the group consisting of lung, liver, spleen, skin, blood, and brain. 12. 9. The method according to claim 1 or 8, wherein the recombinant retrovirus is administered intravenously. 13. 9. The method of claim 1 or 8, wherein the recombinant retrovirus is administered in a manner selected from the group consisting of intracranial, intramuscular, and subcutaneous. 14． 9. The method of claim 1 or 8, wherein said recombinant retrovirus is administered to a site selected from the group consisting of a tumor and a stromal lumen. 15. A virus antigen obtained from a virus selected from the group consisting of influenza virus, RS virus, HPV, HBV, HCV, EBV, HIV, HSV, FeLV, FIV, hantavirus, HTLVI, HTLV II, and CMV; The method according to claim 1 or 8, wherein 16. The protein is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL- The method according to claim 1 or 8, which is a cytokine selected from the group consisting of 12, IL-13, IL-14, IL-15, γ-IFN, G-CSF, and GM-CSF. 17． 9. The method according to claim 1, wherein the nucleic acid molecule is selected from the group consisting of an antisense sequence, a non-coding non-heterologous sense sequence, and a ribozyme sequence. 18. 9. The method according to claim 1, wherein the protein is a toxin.