JPH11505128A - Chimeric integrase protein-mediated site-specific integration of vector constructs into eukaryotic genomes - Google Patents

Abstract

(57) SUMMARY Compositions and methods are provided that direct the site-specific integration of a vector construct encoding one or more desired genes into a particular region of a target eukaryotic genome. Such compositions and methods are useful for performing germ cells and germ cell gene therapy. The advantages provided by site-specific integration are a more constant level of expression of the desired gene and the substantial elimination of the possibility of insertional mutagenesis.

Description

DETAILED DESCRIPTION OF THE INVENTION                 Mediated by chimeric integrase protein           Site-specific integration of vector constructs into eukaryotic genomesTechnical fields   The present invention relates generally to the field of gene therapy, and more particularly to vectors Compositions and compositions useful for introducing the construct into specific regions of the target eukaryotic cell genome And methods.Background of the Invention   Since the discovery of DNA as genetic material in the 1940s, and Recent advances in recombinant DNA technology have enabled disease processes to interact with living organisms' nucleic acids. Substantial research has been undertaken to realize the possibility of being affected by effects. I have been. Most recently, very widespread approaches to altering or affecting genes An exemplary method has been described. These include, for example, retroviruses, adenowis From Rus, Vaccinia virus, Herpes virus, and Adeno-associated virus Viral vectors (see Jolly, Cancer Gene Therapy 1 (1): 51-64, 1994). And lipofection (Felgner et al., Proc Natl. Acad. Sci. USA 84:74). 13-7417, 1989), direct DNA injection (Acsadi et al., Nature 352: 815-818, 1991), microphone Microprojectile bombardment (Williams Et al., PNAS 88: 2726-2730, 1991), several types of liposomes (e.g., Wang et al., PN AS 84: 7851-7855, 1987), and administration of nucleic acids alone (WO 90/11092). Such physical methods of gene transfer are included.   Among the technologies studied to date, recombinant retroviral gene delivery is the most widely used method. It has been partially used for: (1) cells of genetic material (vector genome) Efficient and efficient process of entry into the target cell nucleus; (3) comparison High level of gene expression; (4) regulation of vector-target cell binding and gene expression Possibility of targeting specific cell subtypes through tissue-specific control; (5) pre-existing General deficiency in host immunity; and (6) the substantial deficiency obtained with such vectors. Due to extensive knowledge and clinical experience.   Briefly, retroviruses replicate via an integrated DNA intermediate. It is a diploid positive strand RNA virus. Typically, retroviruses are viruses Protein-containing lipids surrounding a core encapsidated with a genome-containing protein Including the envelope. Retroviral infection involves the transfer of viral particles to eukaryotic cells. It begins by binding to a specific receptor on the surface. Then, cells and c The irs membrane fuses and releases the genome-containing capsid into the cytoplasm of the cell. Then leto Rovirus genome contains the components packaged within each infectious retroviral particle. Into double-stranded linear DNA by the reverse transcriptase encoded by the virus Reverse transcribed. In order to remove two nucleotides from each 3 'end of the DNA, Teglase (IN) protein (also encoded by a retrovirus and Packaged in retroviral particles with reverse transcriptase during virion assembly After processing of the linear DNA, the nucleoprotein complex enters the nucleus. And then, here, by staggered cutting of the host cell genome and strand transfer. Ligation of viral DNA to host cell DNA, retroviral genome inherited by daughter cells Virus DNA is pseudo-randomly incorporated You. Processing and ligation are mediated by IN and are pseudo-random pairs. The location of the embedding is mainly determined by the host DNA accessibility, not by the nucleotide sequence. (Kulkosky et al., (1994), Pharmac. Ther., Vol. 61: 185-20). 3).   Wild-type retroviral genomes (and their proviral copies) Both contain three genes (gag, pol, and env genes), which are packaged Signal (y) and a long terminal repeat (LTR) sequence located adjacent to both ends. You. Briefly, the gag gene encodes an internal core structural protein. pol The gene encodes an RNA-dependent DNA polymerase that reverse transcribes the RNA genome, and IN. And the env gene encodes a retroviral envelope glycoprotein You. 5 'and 3' LTRs promote retroviral RNA transcription and polyadenylation Cis-acting elements required for   Adjacent to the 5 'LTR, reverse transcription of the genome (tRNA primer binding site) and retro There are sequences required for efficient encapsidation of irs RNA into particles (y-sequence). Package The removal of the aging signal allows encapsidation of genomic RNA (retroviral RNA Packaging into infectious virions), but the resulting mutants are still It can direct the synthesis of all encoded proteins in the viral genome.   Recombinant retroviruses and their various uses are described in numerous references. Have been. See, for example, Mann et al. (Cell 33: 153, 1983), Cane and Mulligan (Proc. N at'l. Acad. Sci. USA 81: 6349, 1984), Miller et al., Human Gene Therapy 1: 5-14, 1990, U.S. Patent Nos. 4,405,712; 4,861,719; 4,980,289, PCT Application WO 89 / 02,468; WO89 / 05,349, and WO90 / 02,806, and U.S. Patent Application 08 / 116,82. No. 7 (filed on September 3, 1993), No. 08 / 116,828 (filed on September 3, 1993), No. 08 / 116,98 No. 3 (filed on September 3, 1993), No. 08 / 366,851 (filed on December 30, 1994), No. 08 / 425,18 No. 0 (filed on April 20, 1995) and No. 08 / 425,762 (filed on April 20, 1995). Simple In other words, one or more foreign genes of interest contain most of the normal retroviral RNA. Alternatively, it can be incorporated into a retrovirus. The resulting recombinant retroviral vector Constructs the essential retroviral genes necessary to form infectious virions. Proteins (i.e., various proteins encoded by gag, pol, and env genes) Using one of several systems to provide infectious retroviral particles. Packaged. After infection of cells with such a recombinant retrovirus, A recombinant genome that encodes a foreign gene It can be incorporated into host cell DNA as if it were itself. In the host of this foreign gene Results in expression of the foreign protein by the host cell.   Despite the utility of recombinant retroviruses as gene delivery vehicles, Several disadvantages are known. These include those that infect only replicating cells. Their general capabilities, limitations on the size of their genomic packaging, and insertions Potential mutagenesis of the input (i.e., due to the random nature of retroviral integration) Required for activation of oncogenes or survival of tumor suppressor genes or cells Disruption of other essential genes), and various packaging cell lines or prod Between vectors used to produce recombinant virions in a tumor cell line Wild-type replicable retrovirus that can result from one or more recombination events of Contamination of the recombinant retrovirus preparation with the virus (RCR).   Due to the apparent ability of retroviruses to infect only replicating cells, Methods have been developed to increase the efficacy of torovirus. Such a method , Typically, induces cell replication, thereby causing the retrovirus to infect the cells The purpose is to be able to do. As such a method, for example, in mineral oil Treatment with 10% carbon tetrachloride (Kaleko et al., Human Gene Therapy 2: 27-32,1991 ) And remove certain tissues, thereby stimulating rapid cell division Surgery to Increase the Infectivity of the Mouse (Rettinger et al., PNAS 91: 1460-1464, 1994; Moscio ni et al., Surgery 113: 304-311, 1993;). In addition, standard transduction Cells that are resistant to the technology (e.g., stem and non-dividing cells) can be substantially replicated. Use a high-titer recombinant retroviral particle preparation that is free of torovirus contamination. To transduce (i.e., infect cells with the recombinant retrovirus) Other methods have recently been developed. See U.S. Patent Application No. 08 / 425,180, supra. That.   Concerns about contamination of recombinant retrovirus preparations with RCRs hinder RCR production Virtually eliminated by the development of various packaging cell lines designed to Have been or have been excluded. Such systems are described in more detail below. .   Despite these and other advances in retroviral gene delivery technology, Other potentially more efficient methods of transferring a child (e.g., direct injection of pure plasmid DNA) (Davis et al., Human Gene Therapy 4: 733-740, 1993), or other viral Gene delivery vehicles (e.g., based on DNA viruses or non-integrated RNA viruses) Some scientists suggested that viral gene delivery vehicles could be used . However, these alternative systems also have disadvantages (e.g., only providing transient expression). Therefore, the treatment of many diseases susceptible to gene therapy approaches Requires multiple doses). But another vial like this Gene delivery vehicle to the wild-type form of the strain, or in the case of naked nucleic acids. The immunogenicity that exists in many potential patients against its own immunogenicity Therapy may not be possible with repeat therapy using the same gene delivery vehicle .   With regard to gene delivery vehicles, existing survivors are considered in light of the current state of the art. Stable and long-term stability of one or more desired genes without many of the disadvantages of gene delivery technology There is a need to provide a gene delivery vehicle that can provide efficient expression. This eye For purpose, eukaryotic genes of vector constructs encoding one or more desired genes It is an object of the present invention to provide compositions and methods that allow for site-specific introduction into a nome. This is the purpose of Ming. Thus, the present invention is in the area of somatic and germ cell gene therapy. Related.Summary of the Invention   Briefly, the present invention provides a defined eukaryotic genome of a vector construct. Chimeric integrase proteins capable of directing integration into regions Provided are methods for making and utilizing a La Integrase protein. this In one embodiment of the aspect, the chimeric integrase protein is an RNA poly Vector construction into regions flanked by eukaryotic genes transcribed by Merase III Oriented for building incorporation. Preferably, such incorporation typically comprises Occurs within less than about 1,000 bp of the RNA pol III transcription start site and less than about 100 bp of the transcription start site. Fullness is more preferred and less than 10 bp of the transcription start site (ie, 9, 8, 7 , 6, 5, 4, 3, 2, and 1 bp).   In one embodiment of this aspect, the location of the chimeric integrase protein Specificity is mediated by the domain from Ty3 integrase. In another embodiment, And chimeric integrase is derived from retroviral integrase protein It is. In a preferred embodiment, the chimeric integrase protein is Moro It is derived from the knee mouse leukemia virus. Such a chimeric retrovirus inte Representative examples of glases include, from the amino terminus to the carboxy terminus, an A domain, A B domain, and a C domain, wherein at least the One is from Ty3 integrase. Such a chimeric integrase Specific examples of consist of AmBmCt, AmBtCm, AmBtCt, AtBtCm, and AtBmCm Integrase selected from the group, where "m" is MoMLV integrator And "t" indicates a derivative of Ty3 integrase. Other practices In embodiments, the chimeric integrase protein can be isolated or purified Can be done.   Yet another embodiment of this aspect is a marker incorporated in a gene delivery vehicle. Key to the integration of vector constructs into defined regions of the eukaryotic genome For the mela integrase protein, the gene delivery vehicle is Further comprising a vector construct encoding a seed gene product, wherein the heterologous gene product is , Polypeptides, antisense RNAs, sense RNAs, and ribozymes Selected.   In another aspect, a vector encoding the chimeric integrase protein of the present invention. A constructor construct is provided. Such constructs are also described as "Chimeric integrase An expression vector, a gene encoding a chimeric integrase protein Functional association (i.e., to effect regulation of gene expression) At least one element that regulates expression (eg, prokaryotic or eukaryotic) Transcription promoter, enhancer, or locus defining element, or other Means (eg, alternative splicing, nuclear RNA export, post-transcription of messenger RNA) Modification, or post-translational modification of the protein) Ment).   A related aspect of the invention is a vector construct encoding a chimeric integrase Can be transformed, transfected, transduced, or nucleated into a cell, for example. Host cells introduced by any other technique useful for introducing acids. This Such host cells include both prokaryotic and eukaryotic host cells. This Such cells can be used for various purposes (e.g., production of chimeric integrase proteins). Can be used for Chimeric integrase is a chimeric integrase protein In a manner that allows expression of the protein (It varies depending on the host cell, the expression system used, etc.) Can thus be generated. The resulting chimeric integrase is, for example, Affinity using antibodies reactive to melaintegrase epitopes Can be isolated or purified as required by chromatography. One In an embodiment, the chimeric integrase protein thus generated is Incorporated into a gene delivery vehicle assembled by the in vitro process You. In another embodiment, such a host cell is capable of producing recombinant viral particles. For packaging cells. Others required for assembly of infectious virus particles In addition to the components of the packaging cells, the packaging cells also Produces mela integrase protein.   In another aspect of the present invention, vectors into defined regions of the target eukaryotic genome. Chimeric integrase proteins that direct integration of A gene delivery vehicle is provided, comprising a protein construct. One embodiment is a target Of recombinant retroviral vector constructs into defined regions of the eukaryotic genome Chimeric retroviral integrase proteins directing integration and sets A recombinant retroviral particle, including a recombinant retroviral vector construct. . In a preferred embodiment, such recombinant retroviral particles are transduced. And, more preferably, substantially stained with a replicable retrovirus. There is no dye. In another preferred embodiment, in a defined region of the target eukaryotic genome The region is the region adjacent to the eukaryotic gene transcribed by RNA polymerase III is there. Eukaryotic genes transcribed by RNA polIII include tRNA gene and 5SRN Including the A gene. In another embodiment, a wild-type retroviral integrase Recombination mediated by transducible recombinant retroviral particles with proteins Recombination into eukaryotic genomes compared to integration of retroviral vector constructs Of the rate of insertional mutagenesis caused by integration of the retroviral vector construct Transducible recombinant retroviral particles that lead to a reduction are provided. Another embodiment Is a wild-type retroviral integrator in transfected eukaryotic cells. Transduced with a transducible recombinant retroviral particle with a ze protein Compared to the expression of the gene of interest in eukaryotic cells, Transduction, leading to reduced changes in expression of the gene of interest carried by the constructor Includes possible recombinant retroviral particles.   In another aspect of the present invention, a pharmaceutical set comprising the gene delivery vehicle of the present invention Compositions and pharmaceutically acceptable carriers are provided. Another embodiment is freezing. Dried or dehydrated gene delivery vehicles, such as recombinant viruses Particles (e.g., recombinant retroviral particles, recombinant alphavirus particles (e.g., , (Recombinant Symbidos virus particles), recombinant adenovirus particles, recombinant adenovirus Associated virus particles, recombinant herpesvirus particles, and recombinant poxvirus (Particles) are provided. In a preferred embodiment, the gene transfer The delivery vehicle is transducible. A particularly preferred embodiment is lyophilized Transducable recombinant retroviral particles.   In yet another aspect of the invention, a vector construct integrated within a defined region. The eukaryotic cell genome containing the building (and the corresponding transduced eukaryotic cell) Vesicle) is provided. In one embodiment of this aspect, the vector construct is The defined region in which eukaryotic organisms are transcribed by RNA polymerase III This is the area adjacent to the gene.   In another aspect of the invention, a vector construct is introduced into a eukaryotic cell genome. , Resulting in integration of vector constructs with wild-type integrase protein Vector into the eukaryotic cell genome compared to the rate of insertional mutagenesis caused by Methods for reducing the rate of insertional mutagenesis caused by integration of a construct Is provided. This method uses the chimeric integrase protein of the present invention. And introducing the vector construct into the genome of a eukaryotic cell.   In a related aspect, the vector construct is transformed into a defined region of a eukaryotic cell genome. Into the region, and as a result, compared to the expression of the gene of interest in eukaryotic cells , The purpose of the vector construct in the eukaryotic cell into which the vector construct is introduced Provided is a method for reducing changes in the expression of a gene of The construct is introduced using a wild-type integrase protein. This way Transforms a vector construct into a eukaryotic using the chimeric integrase protein of the present invention. And introduction into the genome of a biological cell.   Other aspects of the invention include genetic diseases, cancer, infectious diseases, degenerative diseases, inflammatory diseases. For treating a disease selected from the group consisting of a disease, a cardiovascular disease, and an autoimmune disease About. In one embodiment, the method comprises defining a target eukaryotic genome. Gene Delivery Vehicles Directing the Integration of Vector Constructs into the Restricted Region In vivo administration. In another embodiment, the method comprises the steps of: Gene delivery vias that direct integration of the vector construct into defined regions of the nom Administering to the patient the cells treated ex vivo with the vehicle. like this Ex vivo methods are preferably performed using transduced autologous cells. Is done.BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 illustrates the Ty-3 and MoMLV genomes.   FIG. 2 shows “A”, “B”, and Ty-3 and MoMLV integrase proteins. And the "C" domain. The number of amino acids indicates the boundary of each domain.   Figure 3 shows the primary sequence between Ty-3 integrase (top) and MoMLV integrase (bottom). Amino acid alignment of Boundary between "A", "B", and "C" domains The field is indicated by an arrow. Conserved H-H-C-C and D-D-E models for each protein Show the chief. Was used to make the chimeric integrase protein described in Example 1. Reference is generally made to expression vectors.   FIG. 5 illustrates each of the seven chimeric MoMLV / Ty3 integrase proteins. .   6 (a) and (b) show the chimeric integrase protein described in Example 1. The plasmid construct used for construction is illustrated.   FIG. 7 shows five of the seven chimeric integrase proteins described in Example 1. Provide the nucleotide sequence of the oligonucleotide used to make one . Each of the above oligonucleotide sequences is complementary to the MLV and Ty3 integrase genes. It is a strategic arrangement. “//” indicates the location of the loop-out that occurred in each mutagenesis reaction. Show.   FIG. 8 shows infectious reticulum containing chimeric integrase protein in NC10 cells. Figure 4 shows the pseudotyping procedure used to generate virus particles.   FIG. 9 shows how the integrated library described in Example 1 is created. IndicatesDetailed description of the invention   Before presenting the invention, the definitions of certain terms used hereinafter are given. Can be helpful in understanding that.   A "vector construct" is one or more genes of interest (sometimes referred to herein as "heterologous" (Referred to as a "sequence"). Baek The vector construct can direct the expression of the gene of interest, as well as the heterologous sequence (i.e., (`` Functionally related '') promoter, preferably Expression products have therapeutic or prophylactic value. The expression products of such genes are , Proteins, polypeptides, antisense RNA, sense RNA, and ribozymes including. Optionally, the vector construct may include a transcription termination site, a splice recognition site. One or more genes encoding a polyadenylation addition site, and a selectable marker May be included. Multivalent vector constructs (i.e., co- Promoter that directs the expression of each heterologous sequence. Can, but need not, be present. Multivalent vector constructs are used for more than one purpose In a preferred embodiment encoding the expression of the gene, the expression downstream of the first gene is It is mediated by an internal ribosome entry site ("IRES") sequence.   “Virus vector”, “Recombinant virus vector”, “Virus vector structure” "Construct" and "recombinant viral vector construct" are the vector constructs of the present invention. And, in certain embodiments, directs expression of the vector constructs of the invention. Refers to a nucleic acid construct that can be directed. The viral vector contains at least one transcription vector. Motor / enhancer, or locus defining element, or another sp Licensing, nuclear RNA export, messenger post-translational modification, or protein Including other elements that control gene expression by other means, such as post-transcriptional modification I have to wait. In addition, for inclusion in the intended specific type of virus particle Essential other optional nucleic acid components are included. If necessary, recombinant retrovirus The vector also contains a signal that directs polyadenylation, a selectable marker (e.g., Resistance to neomycin, hygromycin, phleomycin and histidinol DHFR (confers methotrexate resistance) and HSVTK (ganciclovir) And one or more restriction sites, and And translation termination sequences.   “Retroviral vector construct”, “Retroviral vector”, “Recombinant "Retroviral vectors" and "recombinant retroviral vector constructs" Having the vector construct of the present invention, and in certain embodiments, A nucleic acid construct capable of directing the expression of a vector construct. Retro virus vector -Means at least one transcription promoter / enhancer or gene locus Constant element or alternative splicing, nuclear RNA export, messenger translation Gene expression by post-modification or other means such as post-transcriptional modification of proteins Must include other elements that control Such a vector construct Can also be a sequence encoding a packaging signal, a long terminal repeat (LTR), or Some of them, and (if they are not already present in the retroviral vector A) Positive and negative strand primers appropriate for the retrovirus used -It must contain a binding site. If necessary, recombinant retrovirus vector A signal that directs polyadenylation, a selectable marker (e.g., (Resistance to mycin, hygromycin, phleomycin, histidinol) Or DHFR (to confer methotrexate resistance) and HSVTK (ganciclovir sensitivity) As well as one or more restriction sites, and translation It may include a translation termination sequence. By way of example, such vectors are typically 5 ′ LTR A sequence encoding a tRNA binding site, a sequence encoding a packaging signal, The origin of second strand DNA synthesis, and the 3 'LTR, or a portion thereof.   A “recombinant DNA molecule” is a “vector construct” or “retroviral vector Sometimes used instead of "construct."   A “gene delivery vehicle” can deliver a vector construct to a eukaryotic cell, To facilitate site-specific integration of vector constructs into the genome of biological cells , A composition further comprising the chimeric IN protein of the present invention. Gene delivery vehicle Representative examples of recombinant virus vectors (e.g., Favirus), physical systems (e.g. ELVS), other viral systems (e.g. Virus, adeno-associated virus, and poxvirus), nucleic acid vectors (e.g., (E.g., plasmids), naked nucleic acid molecules (e.g., genes), Complexes with polycationic molecules that can integrate and condense nucleic acid molecules into dense molecules body (See WO 93/03709), liposome-bound nucleic acid (Wa ng et al., PNAS 84: 7851, 1987), bacteria, and nucleic acid components having one or more desired properties. Certain eukaryotic organisms, such as producer cells, that can deliver offspring to host cells in an organism Cell. As discussed below, desired properties include proteins, The ability to express a desired substance, such as an enzyme, or an antibody, and / or Activity (this is because the nucleic acid molecule carried by GDV itself requires expression of the desired substance) Which is the active substance without the active substance). Such raw One example of a physical activity includes gene therapy, where the delivered nucleic acid The molecule is incorporated into the designated gene, thereby inactivating this gene, And the product made by this gene is "turned off". Another example is nucleic acid delivery. If the sequence is an antisense molecule that binds to mRNA and inhibits translation . If the nucleic acid sequence encodes a ribozyme, the ribozyme binds to the mRNA, and Cleave it, thereby inhibiting translation.   "Defined regions of the target eukaryotic genome" are the The region into which the vector construct of the present invention is integrated due to the positional specificity of gluse. You. A typical example is the adjacency of eukaryotic genes transcribed by RNA polymerase III. It is an area that touches.   As described above, the present invention provides for the use of chimeric integrase proteins Compositions directed to site-specific integration of vector constructs into the genome of biological cells And methods. Such compositions include various eukaryotes, particularly mammals, and And particularly suitable for administration to warm-blooded animals, including humans. Eukaryotic vector constructs Incorporation at specific locations within the nom, in particular, allows the expression level of the gene of interest Cell-to-cell variability and potential impairments such as insertional mutagenesis in It can be substantially avoided or eliminated. As a result, such a composition Is useful for effective gene therapy for various diseases by various routes. A.Preparation of chimeric integrase protein   The enzyme responsible for the catalysis of the integration phenomenon is retrovirus IN, which is a 3 ' Detachment of nucleotides from the end of extrachromosomal retroviral vector DNA, characteristic (4-6) Cleavage of target site producing 5 'overhang of 5' and retroviral vector Mediates ligation of the 3 'end of the DNA to the 5' end of the host chromosomal DNA (Fujiwara et al., (1988) , Cell, vol. 54: 497-504; Brown et al., (1989), Proc. Natl. Acad. Sci. USA, vol .86: 2525-2529). MoMLV intracellular viral core particles containing the replicated DNA are integrated (Brown et al., (1987), Cell, vol. 49: 347-356; Fuj). Iwara et al., (1989), Proc. Natl. Acad. Sci. USA, vol. 86: 3065-3069). More recently Thus, the purified recombinant IN is a linear molecule representing the replicated viral DNA or Oligos representing the ends of the virus replicated in the presence of buffer and divalent cation Enough to catalyze nicking and ligation, with nucleotide duplexes And mimicked the integration reaction (Katz et al., (1990), Cell, vol. 63: 87-95; C raigie et al., (1990), Cell, vol. 62: 829-837; Bushman et al., (1990), Science, vol. 249: 1555-1558). In addition, IN is used for the integration reaction on oligonucleotide substrate DNA. (Chow et al., (1992), Science, vol. 255: 723-726).   Ty3 is a yeast retrotransposon, except for the absence of the env gene. Are systematically and functionally similar to animal retroviruses (Hansen et al., (19 88), Mol. Cell. Biol., Vol. 8: 5245-5256; Hansen et al., (1990), J. Am. Virol., Vol .64: 2599-2607; Hansen et al., (1992), J. Am. Virol., Vol. 66: 1414-1424.). See FIG. Teru. Ty3 IN nicks the 3 'end of the replicated Ty3 DNA by 2 bp Required for Ty3 integration. Ty3 is a 4.7kbp internal domain Consists of a 340 bp LTR adjacent to Transcription of the 5.2 kbp genomic RNA is 5 'LTR and 3 'Start and end, respectively, in the LTR. Internal domain is a retrovirus Contains two ORFs (GAG3 and POL3) corresponding to the gag and pol genes . GAG3 gene is a precursor polyprotein Pr38<sup>GAG3</sup>And this is a 26kDa cap Processed to Sid (CA) species and 9 kDa nucleocapsid (NC) species. these Species have conserved motifs found in these retroviral counterparts And are functionally equivalent to these proteins. GAG3-POL3<sup>173</sup>Fusion polypep Pide is the GAG3 protein described above, 16 kDa aspartyl protease (PR), IN Three p115 POLs composed of reverse transcriptase (RT), including mains, 55 kDa RT, and 6 Processed into 1 and 58 kDa IN species. These proteins are Ty3 RNA With virus-like particles (VLPs) about 50 nm in diameter and 156S in size. RNA And in addition to having the Ty3 protein, the particle fraction exhibited RT activity and And contains full-length, replicated Ty3 DNA. The primer for Ty3 replication is the initiator tRNA<sup>Met</sup>This is a Ty3 primer that starts 2 bp downstream of the U5 internal domain junction. Complementary to the mer binding site. As with retroviruses, this DNA is short Conserved, ends with inverted repeats, and 2 bp compared to the integrated form Has terminal extensions. Therefore, it is the replicated extrachromosomal intermediate of the retrovirus It is structurally similar to the body.   In one embodiment of the invention, the regiospecificity of the yeast Ty3 element is Molon ey mouse leukemia virus-based retroviral vector integrase (I N). Integrase protein contains at least three domains Known to include: amino terminal domains, chain transfer and metal chelating activities Core domain having sex ("B" in FIG. 2). On the other hand, the carboxyl terminal domain ( “C” in FIG. 2 relates to DNA binding. Known to give site-specific integration One or more of these separate domains of an integrase (eg, Ty3 IN) Corresponding domain of integrase incorporated into a gene delivery vehicle according to the invention Can be replaced with Vector constructs of the invention encoding chimeric integrase Are the codes for the amino-terminal, core, and carboxyl-terminal regions of Ty3 IN Region to a similar coding region for MoMLV non-specific integrase activity. Have commutations.   The full length protein sequences of Ty3 IN and MoMLV IN are aligned (FIG. 3). Ty3 Mutations in the D-D-E region conserved in retrovirus IN and in region B Mutations affect 3 'nicking and strand transfer (Kirchner, J. and Sandmeyer , S. (1992). Proteolytic processing of Ty3 protein is necessary. J. Virol. 67: 1, 19-28. Kulkosky et al., (1992), Mol. Cell. Biol. , Vol. 12: 2331-2338; Engelman et al., (1992), J. Am. Virol, vol. 66: 6361-6369). IN The amino- and carboxyl-terminal regions of Although required for strand transfer activity, these domains also appear to be functionally distinct. (Bushman et al., (1993), Proc. Natl. Acad. Sci. USA, vol. 90: 3428-3432; Gei duschek et al., (1988), Annu. Rev. Biochem., Vol.57: 873-914). Recombinant protein Subdomains of this core region from quality expressed HIV (Kassavetis et al., (1992). 0), Cell, vol. 60: 235-245) is a reversal of incorporation (reversa l of integration) has been shown to be sufficient. Saved The acidic residues appear to chelate divalent cations, and this region Domain containing the active site.   Seven representative chimeric constructs are described in Example 1 below. Of the transcription start site The Ty3 insert and the RNA polIII gene sequence within some nucleotides are highly Preserved and not regulated in a tissue-specific manner, so An almost unchanged expression level can be achieved. Assess the level of expression of the gene of interest Techniques that can be used to do so include Northern analysis and PCR, among others.   Despite the similarities between Ty3 IN and its retroviral counterpart, Ty3 remains It is integrated with a position specificity not observed in such retroviruses. Ty3 is In particular, genes transcribed by RNA polymerase III, such as 5S, U6, and It is integrated into the transcription initiation region of the tRNA gene. Genetics transcribed by polymerase III Offspring tRNA classes are distinguished by the internal promoter elements of boxA and boxB. (Chalker et al., (1990), Genetics, vol. 126: 837-850). tRNA gene code arrangement These regions of the sequence direct the binding of the transcription factor TFIIIC. The transcription factor TFIIIC continues To bind TFIIIB to the 5 'flanking region of the tRNA gene. Binds to the DNA template TFIIIB is sufficient to cause RNA polIII to initiate multiple rounds of transcription (Chalker et al. , (1992), Genes Dev., Vol 6: 117-128). Consensus DNA binding to TFIIIB No sequence has been observed, and the region upstream of this gene contains a conserved protein. Does not include motor elements.   The in vivo specificity of Ty3 was tested directly using a plasmid targeting assay (Nats oulis et al. (1989), Genetics, vol. 123: 269-279), tRNA, 5S, and U6 genes Has been shown to be a target for Ty3 integration. When the embedding phenomenon occurs, the embedding At the transcription site-the twisted nick of the closest member is one of the transcription initiation sites. Or within 2 bp immediately downstream of the position of TFIIIB. Ty3 pair to target tRNA gene Integration requires a functional tRNA gene promoter element (Natsoulis et al., (1989), Genetics, vol. 123: 269-279), and transcription factors are inherited during integration. It appears to bind upstream of the offspring (Kinsey et al. (1991), Nucleic Acids Res., Vol. 9: 1317-1324).   Since the RNA polIII promoter element is internal, An upstream pair of the present invention by a chimeric integrase having Ty3 IN position specificity Integration of the recombinant DNA molecule does not disrupt tRNA gene expression. Ty3 element or One t in both directions under conditions where the expression level of the LTR sequence varies by more than 50-fold Analysis of RNA gene alone and transcription of tRNA gene with Ty3 showed that Ty3 Had only a slight positive effect on the expression level of ngelman et al., (1993), EMBO J., vol.12: 3269-3275). T of eukaryotic cells tested RNA genes are highly redundant, thereby minimizing insertion effects.   Recently, an in vitro assay has been developed that uses the components of the Ty3 integration reaction to Researched. This reaction can be used in in vitro retroviral core integration assays. (Brown et al., (1987), Cell, vol. 49: 347-356). Ty3 VLP is in addition to other ingredients Containing IN and full length Ty3 DNA, and can provide Ty3 DNA to a plasmid target. VLPs were isolated from cells overexpressing Ty3, and RNA polIII transcript extracts or Means that purified factors (IIIC and IIIB) and polymerase III are added Mixed with mid. The plasmid harbors the modified SUP2 tRNA gene into a wild-type or Contains with any of the mutant (G56) boxB promoter elements. Built-in anti On ice, 5-20 mM MgCl<sub>Two</sub>Mixed together in a buffer containing Incubate at 30 ° C for 30 minutes. The DNA is then extracted and the fluorometric assay Quantified by Assay. To a target plasmid containing the tRNA gene of Ty3 DNA Can be monitored by a PCR assay. Start of tRNA gene target Incorporation of Ty3 DNA into the region yields a product that can be amplified into a diagnostic fragment. It is.   Differences in codon usage between eukaryotic retroviruses and retrotransposons Therefore, those skilled in the art will be able to insert the site specificity domain into each vector construct. Modified using degenerate codons preferred by the particular integrase to be done I can do it. Host (s) susceptible to infection by the retrovirus (or Cell lines used for virion or integrase expression) Information on frequency of use can be obtained if abundantly expressed tanks are not available in the literature. Determined by examining codon usage in the gene encoding the protein Can be One or more preferred codons can then be obtained by standard techniques, e.g. Introduction into chimeric integrase gene by mutagenesis or solid-state nucleic acid synthesis Can be done. A.Recombinant retroviral vectors, packaging cells, producer cells And recombinant retrovirus preparation   As noted above, one embodiment of the present invention provides for one or more selected nucleic acids of interest. Deliver molecules, or "genes," to the genome of eukaryotic cells in a site-specific manner To provide a recombinant retrovirus constructed to Position-specific integration Mediated by chimeric IN protein incorporated into recombinant retroviral particles Is done. Briefly, a number of retroviral gene delivery vehicles have been identified in the context of the present invention. Can be used within. These include, for example, EP 0,415,731; WO 90/07936; WO 94/0 WO 93/25698; WO 93/25234; U.S. Patent No. 5,219,740; WO 9311230; WO 93 10218; Vile and Hart, Cancer Res. 53: 3860-3864, 1993; Vile and Hart, C ancer Res. 53: 962-967, 1993; Ram et al., Cancer Res. 53: 83-88, 1993; Takami ya et al., J. Neurosci. Res. 33: 493-503, 1992; Baba et al., J. Neurosurg. 79: 729-73 5,1993 (U.S. Patent No. 4,777,127, GB 2,200,651, EP 0,345,242, and WO 91 / 02805). Particularly preferred recombinant retrovirus Include those described in WO 91/02805.   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-inducing 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 like or normally available It can be isolated from known sources using techniques.   Any of the retroviruses described above may be any of the disclosures provided herein and Standard recombination techniques (eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989; Kunkle, PNAS 82: 488, 1985), whether to construct a retroviral gene delivery vehicle. , Or can be easily utilized to build. Further, certain embodiments of the present invention In some parts of the retroviral gene delivery vehicle, different Virus. For example, in one embodiment of the present invention, Ils vector LTR is mouse sarcoma virus, tRNA binding site is Rous sarcoma virus In addition, the packaging signal is to the murine leukemia virus and to second-strand synthesis. The origin may be from chicken leukemia virus.   In a preferred embodiment of the invention, a recombinant retrovirus useful in the practice of the invention is provided. The virus described above contains the elements necessary for the production of infectious recombinant retrovirus. By introducing the constructor into cells (referred to as "packaging cells"). Can be made. This element is a position-specific set of recombinant retroviral genomes. Vector construct that mediates integration, but transcribes the recombinant retroviral genome Is lacking. A wide variety of retroviral vector constructs are described in the present invention. And can be used to prepare recombinant retroviruses. For example, the present invention In one aspect, the 5 'LTR, a tRNA binding site, a packaging signal, one or more Retroviral vector constructs containing heterologous sequences, origin of second strand DNA synthesis, and 3 'LTR A structure is provided. Where the vector construct is the gag / pol or env code Missing sequence. Briefly, long terminal repeats ("LTRs") consist of U5, R, and U3 Are further divided into three elements called. These elements include Various signals responsible for the biological activity of rovirus (for example, pro- Motor and enhancer elements). LTR is Easily identified in proviruses by accurate replication at either end obtain. As used herein, a 5 ′ LTR is a 5 ′ promoter element. Sufficient LTR sequence to allow reverse transcription and integration of the DNA form of the vector. It should be understood that it includes. The 3 'LTR has a polyadenylation signal and Contains sufficient LTR sequences to allow reverse transcription and integration of the DNA form of the vector It should be understood that.   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.   Because site-specific integration is an essential aspect of the invention, retroviral vectors Construct is a reverse transcribed double-stranded form of the retroviral genome to be integrated When deployed, processed by intact chimeric IN protein It is important to include the sequence. After reverse transcription of the retroviral RNA genome, the LTR Present at each end of a linear double-stranded DNA molecule. Some nucleotides, typical Are removed by chimeric IN from the 3'-OH end of the retroviral DNA. In a particularly preferred embodiment, the substrate DNA is the 3 'end of each of two complementary DNA strands. It has two base pairs of cytosine and adenosine (5'-CA-3 ') dinucleotide derived from the end You. See Kulkosky et al., Supra. But either or both nucleos Contains substitutions of tides or dinucleotides from one or both ends of the DNA The resulting retroviral DNA recessed can also be used. But Leto Processing and binding of rovirus DNA (to a truncated eukaryotic genome) (jo ining) does not seem to be very effective.   In addition to the 5 'and 3' LTRs, the tRNA binding site and the origin of second strand DNA synthesis, Certain preferred retroviral vector constructs provided in Zing signals, as well as one or more nucleic acid molecules (eg, heterologous sequences). This Each of these is discussed in more detail below.   In a preferred embodiment of the invention, both the gag / pol and env coding sequences are A missing retroviral vector construct is provided. Used in this specification The term "lacking the gag / pol or env coding sequence" Vector is a gag / pol or env gene, especially retroviral vector construction Gag / pol or env used to construct a packaging cell line for the product At least 20, preferably at least 15, found in the expression cassette of More preferably, at least 10, and most preferably, at least 8 consecutive rows. Should be understood to mean not containing any nucleotides.   Packages suitable for use with the retroviral vector constructs described above Cell lines can be readily prepared (US Patent Application No. 08 / 240,030, May 1994). See application filed on Jan. 9, 2009; see also WO 92/05266), and site-specific integration Producer cell lines (vectors) for the production of recombinant vector particles Cell line or “VCL”). Features of the present invention In a particularly preferred embodiment, the packaging cell line is the resulting recombinant retrovirus. Derived from a cell line obtained from the same species as that treated with Rus particles. For example, (For example, HT1080 cells), a recombinant retroviral particle produced from It has been found that inactivated forms in human serum can survive. U.S. Patent Application No. See 08 / 367,071 (filed December 30, 1994).   In a preferred embodiment of the invention, in a crude supernatant<sup>6</sup>Or 10<sup>7</sup>from cfu / ml Packaging cell lines that produce recombinant retroviral particles at high titers It can be easily obtained. In addition, such titers are generally higher in mouse 3T3 cells. From a titer assay on HT1080 cells, yielding a titer three times lower than the titer obtained It should be noted that it can be obtained. 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 vector constructs 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 gene delivery vehicles that carry This is in several places The desired benefits, typically the ability to fight or stop the disease, or other Provide a pathogenic agent or condition. As used herein, "pathogenicity" "Substance" refers to a cell responsible for a disease state. Typical examples of pathogenic substances include tumors Cells, self-reactive immune cells, hormone-secreting cells, lack normal functions Cells, cells with inappropriate gene expression that would not normally occur in that cell type, And cells infected with bacteria, viruses, or other intracellular parasites Can be   Can be carried and / or expressed by a gene delivery vehicle of the invention Examples of nucleic acid molecules include, but are not limited to, substances such as polypeptides, antisense RNAs, sense RNAs. Or ribozymes, as well as being incorporated into certain intracellular nucleic acid molecules and Encodes a biologically active nucleic acid molecule, such as an inactivating sequence that inactivates other molecules Genes and other nucleic acid molecules. Nucleic acid molecules require expression of a substance Without being considered biologically active if the molecule itself provides the desired benefits. Can be For example, a biologically active nucleic acid molecule is incorporated into a specific intracellular nucleic acid molecule. And may be an inactivating sequence that inactivates the molecule, or The molecule can be a tRNA, rRNA, or mRNA having a form that provides binding ability.   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 (eg, steroid hormones) Mon, purine and pyrimidine).   With respect to a moderating agent, “capable of inhibiting function” when used in the context of the present invention, For example, a substance that does not normally inhibit the function of a pathogenic substance By converting existing substances, the moderator can either directly or indirectly function. It should be understood that it inhibits with. Such a function against viral diseases Examples include virus adsorption, replication, gene expression, and assembly of virus from infected cells. , And emissions. Examples of such functions for cancerous diseases include: Cell replication, sensitivity to external signals (eg, contact inhibition), and anti-oncogene Deficiency in the production of protein proteins. Such a machine for cardiovascular disease Examples of noses include inappropriate growth or accumulation of substances in blood vessels, high blood pressure, undesired Blood level factors (such as cholesterol or Degree lipoprotein), localized hypoxia, as well as inappropriately high and tissue Damage levels of free radicals. This for neurological conditions Examples of such functions include pain, loss of dopamine production, and inability to replace damaged cells. And lack of motor control of physical activity, neurological tissue such as the pituitary or hypothalamus Low levels of various peptide hormones of different origin, associated with Alzheimer's disease Amyloid plaque protein accumulation and inability to regenerate damaged nerve connections It is mentioned. Examples of such functions for autoimmune or inflammatory diseases These include inappropriate production of cytokines and lymphokines, autoimmune antibodies and Production and presence of immune and cellular immune responses, proteases and collagenases Inappropriate disruption of tissue due to, production of factors normally supplied by destroyed cells And excessive or abnormal regeneration of tissue under an autoimmune attack It is.   In one aspect of the present invention, a method for administering a gene delivery vehicle is provided. Provided. The nucleic acid encodes at least one moderating agent and modulates its expression. Be oriented. In various embodiments, the moderating agent is a DNA molecule, an RNA molecule, Or a specific combination of   Representative examples of palliatives that act directly on cell growth inhibition include: Toxins. For example, ricin (Lamb et al., Eur. J. Biochem. 148: 265 -270, 1985), Abrin (Wood et al., Eur. J. Biochem. 198: 723-732, 1991; Evens en et al. of Biol. Chem. 266: 6848-6852, 1991; Collins et al. of Biol. Chem . 265: 8665-8669, 1990; Chen et al., Fed. of Eur. Biochem Soc. 309: 115-118, 19 92), diphtheria toxin (Tweten et al., J. Mol. Biol. Chem. 260: 10392-10394,1985 ), Cholera toxin (Mekalanos et al., Nature 306: 551-557, 1983; Sanchez & Hol). mgren, PNAS 86: 481-485, 1989), gelonin (Stirpe et al., J. Biol. Biol. Che m. 255: 6947-6953, 1980), and the pokeweed burdock (Irvin, Pharmac. Ther. 21: 371-387, 1983), antiviral proteins (Barbieri et al., Biochem. J. 203: 55 -59, 1982; Irvin et al., Arch. Biochem. & Biophys. 200: 418-425, 1980; Irvin, Arch. Biochem. & Biophys. 169: 522-528, 1975), tritin, dysentery ibis Shin (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. Mol. Biol. Chem. 262: 8707-8711, 1987). Express Russel's Viper Venom A detailed description of the recombinant retrovirus was published in a U.S. patent filed December 30, 1994. Provided in application Ser. No. 08 / 368,574.   In another embodiment of the present invention, the gene delivery vehicle is not itself toxic. However, proteins such as proteases specific for viruses or other pathogens Products that, when processed or modified by quality, are converted to toxic forms Carry the specific gene. For example, a recombinant retrovirus contains a proprotein It may carry a gene encoding a chain. This proprotein is an HIV protease Toxic if processed by More specifically, the appropriate “professional” HIV-encoded proteases to recognize and cleave elements Use of lysine to synthesize toxic lysine or diphtheria A chains. The active proprotein form may be cleaved into the active form.   In yet another embodiment of the invention, a vehicle carried by a gene delivery vehicle. The construct protects the inactive precursor from the active inhibitor or condition of the pathogenic substance. And the expression of a substance that can be activated by an effective safener. These develop pathogenic conditions It is a moderating agent that is toxic to the appearing cells. Disclosure provided herein As is evident, a wide variety of inactive precursors can be used to Can be converted to For example, retroviral reverse transcriptase (and thus viral replication) To specifically inhibit, antiviral nucleoside analogs (eg, AZT or Is ddI) is metabolized to the nucleotide triphosphate form by cellular machinery (Furmam Proc. Natl. Acad. Sci. USA 83: 8333-8337, 1986). Antiviral nucleo Herpes simplex virus virus, which supports the metabolism of sid analogs into their active forms Midine kinase (HSVTK) or varicella-zoster virus thymidine kinase (VZVTK) Recombinant retrovirus that directs the expression of gene products (eg, proteins) such as Lus therefore removes the nucleoside analog precursor (eg, AZT or ddC). It is useful for activating to an active form. This allows AZT or ddI treatment Are more effective, lower doses, lower systemic toxicity, and Allows a higher potency for dyeing. Retronucleotide triphosphate form Shows selectivity for irs reverse transcriptase, but shows cellular nucleoside and nucleotidic Another nucleoside analog that is not phosphorylated as a result of the substrate specificity of Logs are more effective.   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 Biologically active nucleotide triphosphates of such nucleotide analogs Resulting in more effective metabolism of the Delivers delivery, less systemic toxicity, and greater potency against productive infections I do. Nucleotide triphosphate forms selectivity for retroviral reverse transcriptase Shown as a result of the substrate specificity of cellular nucleosides and nucleotide kinases. Additional nucleoside analogs that are not phosphorylated are also useful within the context of the present invention. Can be used.   In a related aspect of the invention, the cytotoxicity in the presence of Or a vector that directs the expression of a substance that activates another compound that has no A constructor construct is provided. Achieving local treatment for pathogenic substances I do. In this case, expression of the gene product from the recombinant retrovirus will There is an entity associated with the pathogenic substance, such as an intercellular signal to identify, Limited to situations that prevent destruction of non-pathogenic cells. This cell type specificity also The vector construct is maintained against cells that are in the original state or sensitive to it. Granted at the infection level by targeting a recombinant gene DNA vehicle Can be done.   In a related embodiment, the compound has little or no cytotoxicity Vector constructs that direct the expression of gene products that activate You. Briefly, compounds with little or no cytotoxicity are directly or A wide variety of gene products that activate either directly or indirectly into toxic products It can be used within the context of light. Representative examples of such gene products include: Selective phosphorylation of selected purine arabinoside and substituted pyrimidine compounds To convert these compounds into cytotoxic or cytostatic metabolites HSVTK and VZVTK. More specifically, ganciclovir, acyclovir HSVT for drugs such as le, or any of their analogs (eg, FIAC, DHPG) Exposure to K phosphorylates this drug to the corresponding active nucleotide triphosphate form I do. For example, a vector construct may contain a vector activated by the HIV tat protein. Transcriptional regulation of the HIV promoter, which is known to be transcriptionally silent except for Under control, it can direct the expression of the HSVTK gene. Simply put, HIV infected vector Expression of the tat gene product in human cells harboring the construct results in HSVTK production. cause. The cells are then transferred (either in vitro or in vivo) to the cancer For drugs like cyclovir, acyclovir, or their analogs (FIAC, DHPG) Expose. As mentioned above, these drugs are phosphorylated by HSVTK (but Is not phosphorylated by cellular thymidine kinase) and its corresponding active It is known to be in the form of nucleotide triphosphate. Acyclovir triphosphate is Generally, it inhibits cellular polymerases and causes HSVTK in transgenic mice. To Leads to specific destruction of expressing cells (Borrelli et al., Proc. Natl. Acad. Sci . USA 85: 7572, 1988). Containing the vector construct and HIV tat Those cells that express the protein are selected in the presence of an appropriate dose of these drugs. Alternatively killed.   In another embodiment of the present invention, the cis-acting element is transferred from the vector construct. Included in the mRNA to be transcribed ("CRS / CAR") Expression may be even more HIV-specific. This is due to the additional H Requires an IV gene product, such as rev (Rosen et al., Proc. Natl. Acad. Sci. USA  85: 2071, 1988). More generally, cis elements present in the mRNA It has been shown in some cases to regulate the stability or translatability of I have. Sequences of this type (ie, for post-translational regulation of gene expression) -Can be used for event-specific or tissue-specific regulation of gene expression. further, These sequences (ie, the rev responsive “CRS / CAR” element for HIV or t at responsive “TAR” element) can lead to even better specificity Can be used for   In a manner similar to the embodiments described above, purine or pyrimidine-based drug Genes for phosphorylation, phosphoribosylation, ribosylation, or other metabolism A retained gene delivery vehicle can be generated. 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 ("g pt ") gene product (Besnard et al., Mol. Cell. Biol. 7: 4139-4141, see 1987. Such as mitomycin phosphate and doxorubicin phosphate Alkaline phosphat converts inactive phosphorylated compounds into toxic dephosphorylated compounds Vatase converts 5-fluorocytosine to the toxic compound 5-fluorouracil Fungal (eg, Fusarium oxysporum) or bacterial cytosine deaminase (Mull en, PNAS 89:33, 1992), para-N-bis (2-chloroethyl) aminobenzoyl glu Cleaves glutamic acid from tamic acid, thereby toxic benzoic mustard Carboxypeptidase G2 that produces lipase; and doxorubicin and melphala To convert phenoxyacetabide derivatives of Nisillin V amidase and the like. This type of conditional lethal gene product is Applied to many currently known anticancer drugs based on phosphorus or pyrimidine These include intracellular ribosylation or ribosylation to become effective cytotoxic agents. Often requires phosphorylation. The conditional lethal gene product may also be purine or A nontoxic drug that is not an analog of pyrimidine can be metabolized into a cytotoxic form (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, vector constructs can be constructed. You. Gene products of this nature are used to signal intracellular signals of viral infection (or A good candidate for "quality"). Therefore, such virus "immediate type" Conditional transcription transcribed from a transcription promoter element responsive to the gene product Death genes can specifically kill cells infected with any particular virus. Further In addition, the human α and β interferon promoter elements are widely diverse. Because it is transcriptionally activated in response to infection by an unrelated virus, The production of conditional lethal genes such as HSVTK from their viral responsive elements (VRE) Disruption of cells infected with a variety of different viruses by introducing a vector that expresses Breakage can occur.   In another embodiment of the present invention, an inhibitor that inhibits viral assembly Vectors for incorporation into gene delivery vehicles producing substances such as palliatives -A construct is provided. In this context, the recombinant DNA molecule is the virus particle Incomplete gag, pol, env or other cue that inhibits assembly in a preferential manner Encodes an irs particle protein or peptide. Such an inhibition is Interaction of normal subunits of particles with interaction of incomplete subunits It is caused by being destroyed by.   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. This results in a productive infection event A non-productive "dead end" event that antagonizes In the specific case of HIV To replicate HIV (by expressing antisense tat or the like as described above). Inhibiting vector constructs can be administered and this can also lead to infections such as CD4 Can be overexpressed. Thus, a relatively small number of vectors Infected HIV-resistant cells may have multiple non-productive with free virus or virus-infected cells. Created as a 'sink' or 'magnet' for biotic fusion events To use.   In another embodiment of the present invention, an inhibitory peptide specific for a viral protease is used. Provides vector constructs for expressing substances such as peptides or proteins Is done. Viral proteases abundant in viral gag and gag / pol proteins To smaller peptides. In all cases, this failure to disconnect is This leads to a complete inhibition of the production of infectious retroviral particles. HIV protease Also known as party protease. And these are proteins or Known to be inhibited by peptides made from amino acids derived from analogs Have been. Vector constructs that inhibit HIV are such peptide inhibitors Express one or more fusion copies of   Administration of the above-described vector constructs in a suitable gene delivery vehicle can be accomplished by a number of Should be effective against lupus-related diseases, cancer, or other virulence factors You.   In yet another embodiment of the present invention, a vector construct encoding the expression of a moderator is provided. A structure is provided. Here, the palliative has a membrane anchor, and as an antitumor agent Works. Such moderating agents include, for example, antitumor agent-membrane anchor fusion proteins Can be constructed as Simply stated, the properties of the fusion protein membrane anchor are For example, it can be selected from a variety of sequences including the transmembrane domain of a known molecule. In general The membrane anchor sequence is the region of the protein that binds the protein to the membrane. Through For example, there are two types of anchor sequences that attach proteins to the outer surface of the cell membrane : (1) spans the lipid bilayer of the cell membrane and interacts with the hydrophobic central region Transmembrane domain (a protein containing such a domain is called a complete membrane protein) Interacts with integral membrane proteins or polar surfaces of the membrane Do Main (such proteins are called peripheral or exogenous proteins) ). However, proteins also directly covalently bind to lipid components of the cell membrane. Can be determined.   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. Mol. Biol. Chem. 257: 4152, 1982; Garbers, Pharmac . Ther. 50: 337-345, 1991; Engelman et al., Proc. Natl. Acad. Sci. USA 77: 2023 Heijne and Manoil, Prot. Eng. 4: 109-112, 1990). Ta Regardless of the number of times the protein passes through the membrane, the area across the membrane is typically of a type It has a similar structure. More specifically, 20-25 members of the domain located inside the membrane The amino acid residue portion generally consists almost entirely of hydrophobic residues (Eisenberg Ann. Rev. Biochem. 53: 595-623, 1984). For example, Glycoho Of the 34 residues in the region across the phosphorus membrane, 28 are hydrophobic (Ross et al. See Tomita et al., Biochemistry 17: 4756-4770, 1978). further , Β-sheet and β-barrel-like structures occur, but X-ray diffraction, crystal structure analysis, The region across the membrane, as determined by and cross-linking studies, is typically α Has a helical structure (see Eisenberg et al., Supra; Heijne and Manoil, supra). thing). The location of these transmembrane helices within a given sequence is indicated by a hydrophobic plot. Can often be predicted based on Stryer et al., Biochemistry, 3rd edition 304, 1988. . Particularly preferred membrane anchors for use in the present invention include membrane signal A naturally occurring cellular protein that has been shown to function as an anchor (this It is non-immunogenic) (eg, glycophorin).   In a preferred embodiment of the present invention, the membrane anchor-γ interferon fusion tag A vector construct encoding a protein is provided. In one embodiment, This fusion protein has a sequence encoding the membrane anchor of the γ chain of the Fc receptor, constructed by genetically fusing to a sequence encoding gamma interferon Can be   In yet another embodiment, the RNA molecule corresponding to the pathogenic function is cleaved and Therefore, one or more ribozymes (RNA enzymes) that inactivate (Haseloff and Gerlach, (Nature 334: 585, 1989) by construction of a therapeutically effective vector Things are provided. Ribozymes recognize specific sequences in target RNA And this sequence is usually 12-17 bp, Specific recognition of specific RNA sequences (eg, HIVtat) corresponding to the primordial state And the toxicity is specific for such pathogenic conditions. Further anomalies Sex, in some cases, makes ribozymes a conditional safener as described above. Can be achieved.   In yet another embodiment, a biologically active nucleic acid that is an antisense sequence A vector construct comprising the molecule is provided (the antisense sequence is also included in the nucleic acid sequence). Thus, it can be encoded and then produced in a host cell by transcription). preferable In embodiments, 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., Cncogene 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, Specific antisense sequences can interfere with the formation of large amounts of double-stranded RNA. Ron (including γ-interferon) is thought to induce increased expression . Increased expression of gamma interferon in turn increases expression of MHC class I antigen. This Preferred antisense sequences used in this regard include actin RNA, myo Syn RNA, and histone RNA. Form a mismatch with actin RNA Antisense RNA is 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 live virus particle formation against free virus It has been shown that intracellular binding of CD4 to HIVenv This can be inhibited without the problem of immunological and potential immunogenicity. Because this Of the protein remain bound to the membrane and are structurally endogenous CD4 (patients Should be immunologically tolerant).   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 cells) is more rapid Cell death, thus inhibiting virus seeding. This is a monocyte and macro It may be particularly applicable to phage, which are capable of HIV-induced cytotoxicity. Cytotoxicity is then apparently due to the relative loss of CD4 on the cell surface) Virus production as a result of their relative refractility to Acts as a holder of goods.   Yet a further embodiment of the present invention relates to a vector construct capable of effecting immunostimulation. You. In short, recognizes and protects against foreign pathogens Competence is essential for the functioning of the immune system. In particular, the immune system is responsible for host defense mechanisms. "Self" and "non- We must be able to distinguish from "self" (ie, extrinsic). Cytolytic T lymphocyte Spheres (CTLs) typically bind to MHC class I or class II cell surface proteins. Combined, cell surface recognition structures (eg, processed pathogen-specific peptides) Induced or stimulated by presentation.   Diseases suitable for treatment with immunostimulation include viral infections (eg, influenza Enzavirus, respiratory syncytial virus, HPV, HBV, HCV, EBV, HIV, HSV , FeLV, FIV, Hantavirus, HTLV I, HTLV II, and CMV), cancer (eg , Melanoma, renal carcinoma, breast, ovarian, and other cancers), and heart Disease.   In one example of this embodiment, the present invention provides that the antigen or antigen may be present in sensitive target cells. Or by using a vector construct that directs the expression of A method for stimulating the immune response and inhibiting the spread of the virus is provided. Here, the antigen can (1) initiate an immune response against the viral antigen, or Or (2) by occupying cell receptors required for virus interaction, Either can hinder the spread of ills. Protein expression can be transient or May be stable over time. The immune response should be stimulated against a pathogenic antigen If a vector construct is used, a modified form of the antigen, preferably an immune response Stimulates and reduces the pathogenicity compared to the natural antigen) Designed. This immune response indicates that the cells are in the correct mode (ie, CD3, ICAM-1, I CAM-2, LFA-1, or analogs thereof (eg, Altmann et al., Nature 338: 512, 1989) with information on MHC class I and / or II molecules with accessory molecules. This is achieved when presenting the antigen in the context.   The immune response can also be determined by: (a) (if appropriate, in the context of appropriate MHC molecules) A specific T cell receptor gene recognizing a target antigen, and (b) a target antigen CTL response in the absence of an immunoglobulin gene or (c) MHC context The genes of the two hybrids that provide for By embedding the spheres. Therefore, the vector construct is In addition, it can be used as an immunostimulant, immunomodulator, vaccine or the like.   If immunostimulation is desired in certain cases caused by HIV infection, vectors -Antigens generated from the constructs are responsible for HLA class I or class II restricted immune responses. It may be in a form that induces one or both. For example, HIV envelope anti In the case of the original, the antigen is preferably gp160 which has been modified to reduce its pathogenicity , Gp120, and gp41. In particular, the selected antigen was syncytium Avoiding the expression of epitopes that lead to disease by enhancing the immune response, Remove immunodominant but strain specific epitopes, or Modified to present a lineage-specific epitope, and most or All lines allow a response that can eliminate infected cells. This strain-specific epi Further selection of topes and immunity in animals that cross-react with other strains of HIV It may facilitate stimulation of a response. From other HIV genes or gene combinations Antigens (eg, gag, pol, rev, vif, nef, prot, gag / pol, gag prot, etc.) It 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 carcinoma, HTLV-I-induced leukemia, prostate specific antigen (PSA) and prostate cancer, Different p53 and colon carcinoma and melanoma, melanoma specific antigen (MAGE) and melanoma , Mucins and breast cancer).   According to the immunostimulation aspect of the invention, it is carried by the vector construct of the invention. And / or the expressed substance may also be referred to as an "immunomodulator" (often described above). Included). An immunomodulator is activated by one or more cells involved in the immune response. When manufactured or added exogenously to cells, Causes of an immune response that differs in quality or potency from the immune response that occurs in presence A child. This factor can also be expressed from a gene delivery vehicle-derived gene, Expression is driven or controlled by the recombinant retrovirus. Quality of response or crop The application intensity may be determined, for example, by an in vitro assay for measuring cell proliferation (eg,<sup>Three</sup>H Chimiji And in vitro cytotoxicity assays (eg,<sup>51</sup>Cr release (Warning), including a variety of assays known to those skilled in the art (Warn er et al., AIDS Res. and Human Retroviruses 7: 645-655, 1991). Exemption Epidemiological regulators can be active both in vivo and ex vivo.   Representative examples of such factors include IL-1, IL-2 (Karupiah et al., J. Immunol. ogy 144: 290-298, 1990; Weber et al. Exp. Med. 166: 1716-1733, 1987; Gansba cher 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. U.S. Patent No. 5,017,691), IL-5, IL-6 (Brakenhof et al., J. Immunol. 13). 9: 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 IL-15, especially IL-2, IL-4, IL-6, IL-12, and IL-13, alpha interface Ron (Finter et al., Drugs 42 (5): 749-765, 1991; U.S. Patent No. 4,892,743; No. 4,966,843; WO 85/02862; Nagata et al., Nature 284: 316-320, 1980; Fam. illetti et al., Methods in Enz. 78: 387-394, 1981; Twu et al., Proc. Natl. Acad. Sc i. USA 86: 2046-2050, 1989; Faktor et al., Oncogene 5: 867-872, 1990), β-in. Terferon (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, 1 990; Maio et al., Can. Immunol. Immunother. 30: 34-42, 1989; U.S. Patent No. 4,762,7 No. 91; U.S. Pat. No. 4,727,138); G-CSF (U.S. Pat. Nos. 4,999,291 and 4,8 10,643), 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, b<sub>Two</sub>Miku Logoglobulin (Parnes et al., PNAS 78: 2253-2257, 1981), chaperones (eg, Calnexin), MHC binding transporter protein or its analog (Pow is et al., Nature 354: 528-531, 1991). One In a preferred embodiment, the gene encodes gamma interferon. Exemption Epidemic regulators may also be agonists, antagonists, or ligands of these molecules. Can be For example, the soluble form of the receptor is Often act as antagonists to these types of factors so that they can act. Can be used.   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 of Ster with Peptide Bound to Major Histocompatibility Complex (MHC) Molecule A second signal, provided by and referred to as costimulation, Thus, it is delivered to T cells. The second signal is interleukin by T cells. B7.1-.3 molecule required for the production of IL-2 (IL-2) and on antigen presenting cells It is likely to involve interaction with CD28 and CTLA-4 receptors on papocytes (Linsley et al. J. Exp. Med., 173: 721-730, 1991a and J. Exp. Med., 174: 561-570, 1991). The present invention In one embodiment, B7.1-.3 comprises CD8<sup>+</sup>Cause co-stimulation of T cells, Result, CD8<sup>+</sup>T cells produce enough IL-2 to expand and be fully activated To be introduced into tumor cells. Co-stimulation is no longer necessary for further CTL function It's not necessary, so these CD8<sup>+</sup>T cells kill tumor cells that do not express B7 I can do it. Both costimulatory B7.1-.3 factor and, for example, immunogenic HBV core protein A vector expressing one can be made utilizing the methods described herein. This Cells transduced with these vectors become more effective antigen presenting cells. HB V core-specific CTL response was fully activated by costimulatory ligand B7.1-.3 CD8<sup>+</sup> Increased from T cells.   The choice of which immunomodulator to use can be based on the known therapeutic effects of the factor Or can be determined experimentally. For example, known cures in chronic hepatitis B infection The therapeutic effector is alpha interferon. This compensates for the patient's immunodeficiency , And thereby found to be effective in assisting recovery from the disease Have been. Alternatively, appropriate immunomodulators can be determined experimentally. Simply put First, a blood sample is collected from a patient with liver disease. Peripheral blood lymphocytes (PBLs) Recombinant retrovirus directing expression of the immunogenic portion of the inflammatory antigen and immunomodulators Autologous or HLA-compatible cells (eg, EBV-transformed cells) Restimulate in vitro. Next, CTL using HLA-compatible transduced cells as targets Use these stimulated PBLs as effectors in the assay. HLA compatible stab Using target cells transduced with excitatory substances and vectors encoding only antigens The increase in CTL response relative to the CTL response seen in the same assay performed Key Indicates a nodal factor. In one embodiment of the present invention, the immune modulator Mamma interferon is particularly preferred.   The present invention also provides a method for producing a desired antigen (eg, a viral antigen, a bacterial antigen, or a parasite). (Including antigens). For example, Immune response when administered by one of the recombinant retroviruses described herein The different immunogenic portions of the HBV S antigen can be combined to give an answer. further, HBV S antigen open reading frame found in different regions Due to the widespread immunological variability of certain antigens, Combinations may be suitable. Briefly, all human hepatitis B virus S sumps The epitope found in the antigen is defined as the antigenic determinant "a". But each other A subtype antigenic determinant that is exclusive to is also two-dimensional double immunodiffusion (Ouchterlony, Pr. ogr. Allergy 5: 1, 1958). These antigenic determinants are "d" Or "y" and "w" or "r" (LeBouvier, J. In. fect. 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 For single nucleotide substitutions in two regions of the S antigen open reading frame Causes the following amino acid changes: (1) Hepatitis B virus S antigen The conversion of lysine-122 to arginine in the pun reading frame is subtype Is shifted from d to y, and (2) the conversion of arginine-160 to lysine is Shift subtype from r to w. African black among subtype ayw Is dominant, while in the United States and Northern Europe the subtype adw<sub>Two</sub>Is 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 hepatitis B It is generally preferred to construct vectors for administration appropriate for the viral subtype . The subtype of a particular region can be determined by two-dimensional double immunodiffusion, or Is preferably the S antigen open library of the 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. The polymerase protein consists of at least two domains: reverse Amino-terminal domain encoding a protein that initiates transcription, and reverse transcriptase Carboxyl-terminal domain encoding elemental and RNase H activities. HBV pol immunogen The sexual moiety is a recombinant that expresses the antigen of interest to produce an immune response in the animal. The retrovirus can be administered to a warm-blooded animal by introducing it into the animal. Likewise HBV, such as, ORF5 and ORF6 (Miller et al., Hepatology 9: 322-327, 1989) Antigens can be expressed using the vector constructs described herein.   As described above, at least one immunogenic portion of the hepatitis B antigen comprises a vector construct. Can be incorporated into structures. The immunogenic moiety incorporated into the vector construct can vary It can be long, but in general this part should be at least 9 amino acids long. And may contain the entire antigen. Predict the immunogenicity of a particular sequence Although often difficult, epitopes on T cells have been described by Falk et al. (Nature 351: 290, 1991) using the HLA A2.1 motif. From this analysis Can be synthesized and targeted in in vitro cytotoxicity assays Can be used. However, other assays may also be utilized, and such assays For example, an ELISA that detects the presence of an antibody against a newly introduced vector, such as Laby and gamma interferon assay, IL-2 production assay, and proliferation assay Assays for testing helper T cells such as   In one embodiment of the invention, at least one immunogenic part of a hepatitis C antigen Can be incorporated into vector constructs destined for site-specific integration. Good The preferred immunogenic portion of hepatitis C can be found in the C and NS3-NS4 regions. Because these regions are the most conserved among the various types of hepatitis C virus. (Houghton et al., Hepatology 14: 381-388, 1991). Particularly preferred immunogen The sex part can be determined in various ways. For example, as described above for the hepatitis B virus As described above, the identification of the immunogenic portion of the polypeptide is based on the amino acid sequence. Can be measured. Briefly, various computer programs known to those skilled in the art are utilized. Can be used to predict CTL epitopes. For example, as described by Falk et al. (Nature 351: 290, 1991) Using HLA A2.1 motif, HLA A2.1 haplotype Can predict the CTL epitope for a group. From this analysis, the peptide was synthesized and As a target in an in vitro cytotoxicity assay.   In another aspect of the invention, a method is provided for destroying hepatitis B carcinoma cells. . This method comprises a vector construct that directs the expression of an immunogenic portion of antigen X. Administering the gene delivery vehicle to the warm-blooded animal, resulting in an immune response. Include. According to the disclosure provided herein, HBxAg is The coding sequence is readily available. Briefly, one embodiment of the present invention Now, 642 bp of NcoI-Taq I was recovered from ATCC 45020, and other hepatitis B antigens were recovered. Is inserted into the recombinant retrovirus as described above.   However, the X antigen does not resemble other potential oncogenes (eg, E1A) 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 The X antigen can be prepared using various methods, including addition of codons or modification of the phosphorylation site. It can be non-tumorigenic. In one embodiment, the X antigen encodes an antigen of interest. Trim the ends of the sequence or gene. Various fragments are produced by cutting off the ends. But it is generally preferable to retain at least 50% of the coding gene sequence. No. In addition, after any cleavage some of the immunogenic sequence of the gene product remains intact It is necessary to keep it. Alternatively, in another embodiment of the present invention, a plurality of A translation termination codon can be introduced into the gene. Insertion of a stop codon can result in protein expression Is stopped before maturation, which prevents expression of the transformed portion of the protein.   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 doing so. The preferred immunogenic portion of the hepatitis C antigen is And polypeptides containing the NS1-NS5 region (Choo et al., Proc. Natl. Acad. Sci. USA 88: 2451-2455, 1991). Particularly preferred immunogenic part The minutes can be determined by various methods. For example, as described above, the amino acid sequence The preferred immunogenic portion can be predicted based on the Briefly, known to those skilled in the art Various computer programs can be used to predict CTL epitopes. An example For example, HLA A2.1 Mochy described by Falk et al. (Nature 351: 290, 1991) Can be used to predict CTL epitopes for the HLA A2.1 haplotype. Exemption Another method that can be used to predict the epidemiological portion uses retroviruses To determine which parts have CTL-inducing properties in mice (See Warner et al., AIDS Res. And Human Retroviruses 7: 645-655, 1991. When). As described by Warner et al. Cell immunogenicity in a cell. Preferred immunogenic moieties are also polypep Which fragment of the peptide antigen or peptide is the vector trait of the corresponding gene Autologous patient lymphocytes (eg, autologous EBs) of target cells that express the fragment after transduction V-transformed lymphocytes) to determine if they can induce lysis. Can be   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 recovered. Instead of Such antigens are useful as immunogenic portions in the context of the present invention. (Hayata et al., Hepatology 13: 1022-1028, 1991; Davis et al., N. Eng. J. Med. 321: 1501-1506, 1989).   By truncating the coding sequence, the hepatitis B or C virus Additional immunogenic portions of the selected antigen, such as antigens, can be obtained. For example, HBV Can be used to cut the following sites: Bst UI, Ssp I, Ppu M1 and Msp I (Vale nzuela 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. Jaenisch (ed.), 57-70, New York: Academic). To determine appropriate immunogenic moieties and methods Further methods for this are also described below in connection with hepatitis C.   Particularly preferred immunity for uptake into recombinant retroviruses for treatment of HBV Genogenic moieties include HBeAg, HBcAg, and HBsAgs. More than 1 Many immunogenic moieties (and immunomodulators, if desired) are incorporated into the vector construct. It can be inserted. For example, in one embodiment, the immunogenic portion of the hepatitis B antigen and Vector vector that directs the simultaneous expression of both the immunogenic portion of hepatitis C polypeptide and hepatitis C polypeptide A structure can be prepared. Such constructs may be of type B or type C acute and And prevent or treat chronic hepatitis infection. Similarly, other implementations In an aspect, an immunogenic portion of a hepatitis B X antigen and an immunogen of a hepatitis C polypeptide Vector constructs can be prepared that direct co-expression of both sex moieties. Such a structure The structure also treats hepatocellular carcinoma associated with either hepatitis B or hepatitis C To be administered. In addition, chronic infection with hepatitis B and C Because some individuals are at higher risk of developing hepatocellular carcinoma, Can also be used as a prophylactic treatment for this disease.   Immunogenic moieties can also be selected by other methods. For example, HLA A2.1 / K<sup>b</sup> Transgenic mice serve as a model for human T cell recognition of viral antigens. Is shown. Briefly, influenza virus and B In the hepatitis B virus system, the mouse T cell receptor repertoire contains human T cells. Recognizes the same antigenic determinants as those recognized by the vesicle. In both systems, HLA A2.1 / K<sup>b</sup>The CTL response generated in transgenic mice was Epitopes substantially identical to those recognized by human CTLs (Vitiello et al., J. Exp. Med. 173: 1007-1015, 1991; Vitiello et al., Abstract 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 A typical example of the method is: Add amino acid sequence corresponding to helper T epitope 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 It is a family retrovirus. Currently, FeLV has its envelope antigen gp70 And that there are three subgroups A, B or C, distinguished by Has been obtained. FeLV is also highly conserved in all FeLV subgroups. Core antigens (including p15, p12, p27, and p10) (Geering et al., Vi r. 36: 678-680, 1968; Hardy et al., JAVA 158: 1060-1069, 1971; Hardy et al., Science 166. : 1019-1021, 1969). In one embodiment of the present invention, the vector construct The structures are p15gag, p12gag, p27gag, p10gag, p14pol, p80pol, p46pol, gp70env , And at least a feline leukemia virus antigen selected from the group consisting of p15env Directs partial expression. In a particularly preferred embodiment, the vector construct comprises Directs gp85env expression. Sequences encoding these antigens are provided herein. 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): 871-880, 1983; Berry et al., J. Vir. 62 (10): 3631-3641, 1988; Laprevotte et al., J. Vi. r.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, type C oncornavirus (RD-114), and cat (Including the FeSFV) (see Yamamoto et al., “Experimental FIV vaccines”). Efficacy of Chin '', (Summary), First International Conference of Feline Immunodef iciency Virus Researchers, University of California, Davis, CA, Sep.4-7,1991 checking). In one embodiment of the present invention, the vector construct comprises p15gag Selected from the group consisting of p24gag, p10gag, p13pol, p62pol, p15pol, and p36pol Directing the expression of at least one immunogenic portion of a subject feline immunodeficiency virus antigen I do. In a particularly preferred embodiment, the vector construct comprises gp68env, gp27env, And rev expression. In the context of the present invention, "rev" means rev open It is understood to refer to the antigen corresponding to the reading frame (Phillips et al., Fir st International Conference, supra). Encoding these antigens The sequence described in 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: 5743-574. 7,1989) can be readily obtained by those skilled in the art.   Still other examples are non-tumorigenic modified genes (eg, ras (ras<sup>*</sup>) Gene (WO93 / 10814; U.S. patent application Ser. No. 08 / 367,071, supra)), modified p53 (p53).<sup>*</sup>) Genes (Linzer and Levine, Cell 17: 43-52, 1979; Lane and Crawford, Nature  278: 261-263,1979; Hinds et al., J. Virol. 63: 739-746,1989; U.S. Patent Application No. 08 /. 367,071, supra), modified Rb (Rb<sup>*</sup>) Gene (Friend et al., Nature 323: 643,1986; Lee Et al., Science 235: 1394,1987; Fung et al., Science 236: 1657,1987; U.S. Patent Application No. 08 / 367,071, supra), a modified gene that causes Wilms tumor (Call et al., Cel l 60: 509,1990; Gessler et al., Nature 343: 744,1990; Rose et al., Cell 60: 495,1990; Ha ber et al., Cell 61: 1257,1990; U.S. Patent Application No. 08 / 367,071, supra), modified whip Gene (Girling et al., Int. J. Cancer 43: 1072-1076, 1989; Gendler et al., J. Biol. Ch. em. 265 (25): 15286-15293, 1990; Lan et al., J. Biol. Chem. 265 (25): 15294-15299,199 0; Ligtenberg et al., J. Biol. Chem. 265: 5573-5578, 1990; Jerome et al., Cancer Res. 51 : 2908-2916,1991; and U.S. Patent Application No. 08 / 367,071, supra), a modified DCC (conclusion). Gene (deleted in colorectal carcinoma) (Edelman, Biochem 27: 3533-3543,1988; Sol) omon, Nature 343: 412-414,1990; Fearon et al., Science 247: 49-56,1990; U.S. Patent Application No. 08 / 367,071, supra), MCC (mutated in colorectal cancer Or APC). MCC and APC remains Both genes (Kinzler et al., Science 251: 1366-1370, 1991; Nishiho et al., Sciencec e 253: 665-669, 1991), for example, thyroid hormone receptor, PDGF receptor , Insulin receptor, interleukin receptor (eg, IL-1, IL-2, A receptor such as IL-3), or a CSF receptor (eg, G-CSF, GM-CSF, or Are M-CSF receptors (U.S. patent application Ser. No. 08 / 367,071, supra, Slamon et al., Scienc e 244: 707-712,1989; Slamon et al., Cancer Cells 7: 371-380,1989; Shih et al., Nature. 290: 261,1981; Schechter, Nature 312: 513,1984; Coussens et al., Science 230: 1132. Leduc et al., Am. J. Hum. Genet. 44: 282-287, 1989)) Is a modified cell receptor containing a modified form. Modifications in such receptors Has led to the production of proteins (or receptors) containing novel coding sequences. Be sent. Novel proteins encoded by these sequences are It can be used as a marker for cells, and Utilizes the directed immune response to destroy tumorigenic cells containing altered sequences or genes Can break.   If the altered cellular component is involved in making the cells tumorigenic, It is necessary that the cellular components that have become non-tumorigenic. For example, in one embodiment The sequence or gene of interest encoding the altered cellular component is a gene product The ends are truncated to make them non-tumorigenic. Remains encoding changed cellular components Genes can be truncated to various sizes but retain as much of the altered cellular components as possible Is preferred. In addition, the immunogenicity of altered cellular components, even when the ends are truncated At least some of the sequences need to be intact. Or, multiple Number of translation termination codons to alter the immunogenic region in the gene encoding the altered cellular component Downstream. Insertion of a termination codon terminates protein expression early, Thus, expression of the transformed portion of the protein is prevented. U.S. Patent Application No. 08/367 No., 071, supra. However, altered cellular components are generally non-tumorigenic cells. Is only associated with the vesicle and is not necessary to make the cell tumorigenic, or Note that if not essential, the cellular components do not need to be non-tumorigenic It is. A representative example of such altered cellular components that are not oncogenic is Rb<sup>*</sup>, Ubi Kitchen<sup>*</sup>, And mucin<sup>*</sup>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, the immunogenicity of a particular sequence is often predictive. Is difficult to do. However, it is generally preferable to determine immunogenicity in the assay. Good. A typical assay tests for the presence of antibodies against a newly introduced vector. ELISA, gamma interferon assay, IL-2 production assay, And assays that test T helper cells, such as proliferation assays. Immunogenicity A particularly preferred method for determining is the CTL assay. U.S. Patent Application No. 0 See 8 / 367,071, supra.   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 tumor formation in nude mice or rats, Colonization in soft agar, and transgenic animals (eg, trans Transgenic mice).   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 into nude mice. Does not show uncontrolled growth properties. However, transformed cells are Proliferates rapidly in the mouse and gives rise to tumors. Briefly, one implementation In embodiments, the vector construct encoding the altered cellular component is derived from a syngeneic mouse. The cells are delivered and then administered to nude mice. To determine tumor growth The mice are observed visually for 2-8 weeks after administration. Also determine if a tumor is present Mice can be sacrificed and necropsied (Giovanella et al., J. Natl. Cancer Inst. . 48: 1531-1533, 1972; Furesz et al., Abnormal Cells, New Products and Risk, Hopps and Pe tricciani ed., Tissue Culture Association, 1985; and Levenbook et al. Bio l. Std. 13: 135-141, 1985). Tumorigenicity also depends on colony formation in soft agar. Can also be evaluated by visualizing the composition (MacPherson and Montagnier, Vir . 23: 291-294, 1964). Briefly, one property of normal non-tumorigenic cells Is a "contact inhibition" (ie, cells stop growing when they come into contact with adjacent cells) ). When cells are plated on a semi-solid agar support medium, normal cells are quickly contacted. The tumorigenic cells continue to proliferate and colony in soft agar To form   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, The gene of interest can be expressed in all tissues of the animal. This transgene Unregulated expression provides a model for the tumorigenic potential of newly introduced genes. Can be helpful.   In addition to studies on tumorigenicity, the toxicity of gene delivery vehicles is generally Is preferably determined. Using various methods well known to those skilled in the art, Such toxicity can be measured. This includes, for example, systemic levels of various tampering. Clinical chemistry assays that measure the volume and number of proteins and enzymes and blood cells No.   The present invention also provides one or more gene products capable of immune downregulation. An encoding vector construct is provided. Simply put, for example, autoimmune diseases Or pseudo-autoimmune diseases (eg, chronic hepatitis, diabetes, rheumatoid arthritis, transplantation) Transplantation of heterologous tissues such as uni-versus-host disease, and Alzheimer's), or bone marrow Specific down regulation of inappropriate or unwanted immune responses in transplantation (MHC) An immunosuppressive viral gene product that suppresses antigen surface expression, or its activity. It can be manipulated using the sex part. In the present invention, the "active portion" of the gene product is Fragmentation of gene products that must be retained for biological activity It is. Such a fragment or active domain may comprise a nucleotide sequence Systematic removal from the protein sequence and the resulting vector construct For FACS analysis or other immunological assays (eg, CTL assays) To Can be easily identified by measuring MHC class I presentation on the cell surface You. These fragments have the size of the sequence encoding the entire protein It is particularly useful when the capacity of the loose carrier is exceeded. Alternatively, MHC antigen presentation The active domain of the inhibitor protein can be digested enzymatically and the active moiety Can be purified by biochemical methods. For example, the active part of this protein Blocking monoclonal antibodies isolate and isolate the active portion of the cleaved protein. And used for purification (Harlow et al., Antibodies: A Laboratory Manua) 1, Cold Springs Harbor, 1988).   For example, inhibition can be achieved with accessory molecules (e.g., CD8, intracellular adhesion molecule-1 (ICAM-1) , ICAM-2, ICAM-3, leukocyte functional antigen-1 (LFA-1) (Altmann et al., Nature 338: 521, 198). 9), 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 Processed pepti associated with self MHC molecules in addition to other cell adhesion molecules) This is achieved by specifically inhibiting the activation of presentation of the drug. MHC class I molecule Presentation of antigenic peptides associated with CTLs leads to CTL activation. Inhibit MHC antigen presentation The transfer and stable integration of certain sequences that can express the expected product is CD8<sup>+</sup>Block activation of T cells, such as CTL, and thereby suppress graft rejection You. Standard CTL assays can be used to detect this response. Antigen Components of the indicated pathway, 45Kd MHC class I heavy chain, b<sub>Two</sub>-Microglobulin, protea Processing enzymes, accessory molecules, calnexin (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, b<sub>Two</sub>-Gene products or genes capable of binding to microglobulin Vector constructs are provided that direct the expression of the active portion of the product. Simply put For transport of MHC class I molecules to the cell surface for antigen presentation, b<sub>Two</sub>-Microglob Meeting with phosphorus is required. Thus, b<sub>Two</sub>-Binds with microglobulin and Proteins that inhibit its association with MHC class I indirectly provide MHC class I antigens. Inhibit the indication. Suitable proteins include the H301 gene product. Simple To In other words, the H301 gene obtained from human cytomegalovirus (CMV) B on the heavy chain of Las I molecule<sub>Two</sub>-A glycoprotein with sequence homology to the microglobulin binding site Encodes proteins (Browne et al., Nature 347: 770, 1990). H301 is b<sub>Two</sub>-Micro gro Binds to Brin, thereby preventing the maturation of MHC class I molecules and MHC class I-restricted immune surveillance that cannot be recognized by cytotoxic T cells Escape.   In another embodiment, binds a newly synthesized MHC class I molecule intracellularly Provides vector constructs that direct the expression of proteins or active portions of proteins Is done. Alternatively, antisense RNA or the like that inhibits the translation of MHC class I proteins Or the ribozyme is encoded by the vector construct. These activities are small Prevents migration of MHC class I molecules from the endoplasmic reticulum, resulting in inhibition of termination glycosylation Brought. This blocks the transport of these molecules to the cell surface, Cell recognition and lysis by For example, one of the products of the E3 gene It can be used to inhibit the transport of ras I molecules to the surface of transformed cells. More details Specifically, E3 is a 19 kD transmembrane glycoprotein transcribed from the E3 region of the adenovirus 2 genome. Encodes the protein E3 / 19K. In the context of the present invention, tissue cells are expressed as E3 Transformed with a vector construct containing the E3 / 19K sequence producing the / 19K protein. The E3 / 19K protein inhibits surface expression of MHC class I surface molecules and Cells transformed by Irus escape the immune response. As a result, the donor cells Transplantation with reduced risk of graft rejection and minimal immunity in transplant patients Epidemiological control measures may only be required. This is an acceptable donor recipe Ant chimerism is present with fewer complications. A similar treatment is used for systemic lupus erythema Including Todes, multiple sclerosis, rheumatoid arthritis or chronic hepatitis B infection It can be used to treat any autoimmune disease.   Other alternative immunosuppression methods include antisense messages, ribozymes, or Or other gene expression inhibitors specific for existing autoreactive T cell clones. Use of bitter. These are specific unwanted clones responsible for the autoimmune response. Block T cell receptor expression. Antisense, ribozyme, also Other genes can be introduced using a gene delivery vehicle.   MHC class I (MHC class II presentation) other proteins different from those described above A protein that functions to inhibit, suppress, or down regulate antigen presentation Proteins can also be identified and utilized within the context of the present invention. like this Proteins, especially those from mammalian pathogens (and, instead, May be able to inhibit MHC class I antigen presentation in order to identify Recombinant retrovirus expressing a protein or an active part thereof, such as BC Transformed into a tester cell line. Has a sequence encoding the candidate protein Tester cell line with and without stimulator in CTL assay And / or compared to the target. Cells corresponding to transformed tester cells Reduced lysis indicates that the candidate protein can inhibit MHC presentation.   Many infectious diseases, cancer, autoimmune diseases, and other diseases are associated with viral particles. Including the interaction of cells, cells and cells, or cells and factors. Virus infection smell Thus, the virus usually enters cells via receptors on the surface of susceptible cells. gun In some cases, cells may respond inappropriately to signals from other cells or factors. Or do not respond at all. Inappropriate "self" markers in autoimmune diseases Recognition exists. In the present invention, such interactions are referred to as Of vectors encoding in vivo expression of analogs for any of the toners It can be blocked by using things. Such analogs are blockin It is known as a bulking agent. This blocking effect can occur intracellularly, on cell membranes, or in cells. It can happen outside. The blocking action of this blocking agent is From or blocking proteins that block pathogenic interactions locally It can be mediated by either secretion.   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 have HIV env analog, or CD4 receptor that blocks HIV entry without causing It is a tar analog. CD4 analogs are secreted to protect adjacent cells And functions, while gp120 / gp41 protects only vector-containing cells, It is excreted or produced only intracellularly. Increases stability or complement solubility To add human immunoglobulin heavy chains or other components to CD4 so possible. Position of the vector construct encoding such hybrid soluble CD4 Delivery in a position-specific manner results in a continuous supply of stable hybrid molecules .   Recombinant DNA molecules that mediate the expression of HIV env can also be constructed. Which part is obvious It is clear to a person skilled in the art whether virus adsorption can be blocked without (Willey et al., J. Virol. 62: 139,1988; Fisher et al., Science 233: 655,1986).   Another embodiment of the present invention is directed to a method for removing, mutating, or not expressing in a cell type. And the delivery of suppressor genes that lead to tumor formation in that cell type. Deletion Reintroduction of the resulting gene leads to a regression of the tumor phenotype in these cells. Malignant Tumors can be considered to be an inhibition of terminal differentiation of cells as compared to cell growth Delivery and expression of gene products that lead to tumor differentiation also generally lead to regression Should be.   Sequences encoding the above nucleic acid molecules can be obtained from a variety of sources. For example, Plasmids containing sequences encoding altered cell products are, for example, American Typ Advan, such as e Culture Collection (ATCC, Rockville, Maryland) ced Biotechnologies (Columbia, Maryland) or British Bio-Technology It can be obtained from commercial sources such as Limited (Cowley, Oxford England). There Is the cDNA sequence for use in the present invention a cell that expresses or contains the sequence? Can be obtained. Briefly, in one embodiment, the gene of interest is expressed Cell-derived mRNA is reverse transcribed using oligo dT or random primers. For reverse transcription. The single-stranded cDNA is then sequenced on either side of the desired sequence. PCR using oligonucleotide primers complementary to the columns (US Pat. No. 4,683,202). Nos. 4,683,195 and 4,800,159. Also PCR Technology : Principles and Applications for DNA Amplification, Erlich (ed.), Stock ton 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. Upon completion of synthesis, double-stranded DNA is produced. This cycle can be repeated many times, and the desired DNA is amplified multiple times. further Genes of known nucleotide sequence useful in the practice of the invention are available cDNA or Clones the desired gene from an mRNA library using standard techniques This And can be obtained by Carried and / or by the recombinant DN molecule described herein. Alternatively, the expressed nucleic acid molecule is, for example, a DNA synthesizer (Applied Biosystems Inc.) For example, APB DNA synthesizer model 392 (Foster City, California)) In part, it can be synthesized. C.Cell culture   As mentioned above, certain embodiments of the present invention provide for the preparation of viral preparations suitable for human administration. Provide products.   In a particularly preferred embodiment, a high titer recombinant retrovirus preparation is used. It is. To produce such high titer preparations, U.S. patent application Ser. No. 7,071, supra, a cell culture method is used. In short, a wide variety Methods (eg, fermenters or bioreactors, roller bottles, cell hotels) Or use of cell factories and hollow fiber cultures) obtain. For a bioreactor or fermentor, cells are collected in 1 liter of appropriate medium. At a concentration in the range of 3 to 15 g microcarriers per microgram, preferably micro Carrier (ie, Cytodex1 or Cytodex2; Pharmacia, Piscataway, N.J.) Proliferate. For roller bottles, the appropriate conditions are microcarrier beads Includes the conditions used for the bioreactor, except that no is utilized. Fine Cell factories are also used for recombinant retroviruses from large cell cultures and adherent cells. It can be used for the production of ills. In short, the cell factory (" Typically referred to as “cell hotels”) is to ultrasonically couple one to the other Molded from more assembled uncontaminated polystyrene, then Nuclon  D includes a plurality of trays that have been treated to provide a surface, and For example, it is available from various manufacturers including Nunc. The hollow fiber culture method also It can be used for production of recombinant retrovirus. Simply put, hollow fan High titer retrovirus production using ivver cultures is based on reduced cell volumes of medium. Based on the increase in virus concentration as it is cultured to high density in. cell The volume of medium to be cultured on the side should be cultivated in a tissue culture dish or flask. Approximately 10-100 times lower than required for equivalent cell density. It Allows individual retroviral producer cell lines to propagate hollow fibers If the conditions are followed, a 10 to 100-fold increased titer results. Achieve maximum cell density Individual cell lines are very close together and grow so that they touch each other Must be able to do it. Many cell lines do not grow in this manner and these Retroviral packaging cell lines based on cell types You may not achieve a 10-fold increase. Very good growing cell lines are non-adherent Retroviral producer based on non-adherent and non-adherent cell lines -The strain has a 100-fold increase in titer compared to tissue culture dishes and flasks. It is believed that addition can be achieved. D.Enrichment and purification of recombinant retroviral particles   When a retroviral gene delivery vehicle is used in the practice of the present invention, Increasing the purity of a therapeutic preparation, as well as recombinant retroviruses that can be administered It is preferred to increase the titer of the loose. A wide variety of methods (eg, Precipitation of recombinant retrovirus using monium, polyethylene glycol (“PE G ") enrichment, in the presence of a gradient such as PERCOLL or a" buffer "such as sucrose or Is concentrated by centrifugation in the absence of any, using a concentration filter (e.g., A micon filtration), and two-phase separation) to increase virus concentration and purity. Can be used for U.S. Patent Application No. 08 / 367,071, supra and U.S. Patent Application See No. 08 / 153,342. E. FIG.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 Scanning) along with non-denaturing gels (eg, (4-15% gradient polyacrylamide gel for separation) A method is provided for quantifying a vehicle. Such methods are useful for surviving gene delivery Although it is not possible to distinguish between vehicles and non-viable gene delivery vehicles, they are relatively simple. This is advantageous because it is fast. One typical example of such a method is Further details are shown in Example 10 below.   In another aspect of the invention, the recombinant virus (eg, recombinant An assay is provided for measuring the titer of Torovirus). Typically, this Assays based on the presence of a selectable marker or the formation of blue colonies obtain. However, in certain embodiments, including a gene encoding a selectable marker. A gene delivery vehicle is provided. Therefore, antibodies and PCR assays (that The latter of which is described below) can be used to determine the titer. PC Use R to create unique sequences for the vector constructs carried in the gene delivery vehicle. To amplify, appropriate amplification primers are required. Such primers Vector constructs that can be readily designed and used by those skilled in the art, , The particular region desired to be amplified, and the like. Recombinant retro When irus is used for gene delivery, a typical PCR primer pair is an LTR Specific to the sequence, packaging signal sequence or other regions of the retroviral backbone And may include primers specific for the gene of interest.   Briefly, in one embodiment of the invention, the PCR titration assay is Used to determine the titer of the recombinant retroviral preparation. This asse B. Recombinant retrovirus in a 6-well plate for at least 16 hours before harvest This is accomplished by growing a known number of cells transduced with the cells. One per plate Wells are sacrificed for counting. Lyse cells from other wells, Then the contents are isolated. Use the QUIAmp DNA isolation kit (QUIAgen, Inc., Chats worth, CA). 5 x 10 DNA per sample<sup>6</sup>Individual cell equivalent / μL Resuspend.   To calculate the titer, the standard curve was 5 × 10<sup>6</sup>Non-transduced HT 1080 cells Negative control) and a known vector. 5x10 with one integrated copy of the vector per system<sup>6</sup>HT 1080 Produced using DNA isolated from cells (positive control) A recombinant retrovirus encoding a selectable marker such as neomycin resistance Can be prepared from a packaging cell line transduced with This standard curve is Combine and recombine different amounts of positive and negative control DNA PCR using primers specific to specific regions of the retrovirus Produced by amplifying a specific sequence from Mix for standard curve generation Representative groups of the compounds are 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 Into its own reaction tube. Next, the PCR reaction (50 μL total volume) was Add 45.0 μL reaction mixture containing the following components per tube to be tested: Start with: 24.5 μL water, 5 μL 10 × reaction PCR buffer, 4 μL 25 mM MgCl<sub>Two</sub>4 μL of dNTP (containing 2.5 mM of each dATP, dGTP, dCTP, and dTTP), 5 μL primer mix (100 ng each primer), 0.25 μL TaqStart Monochrome Antibody (Clontech Laboratories, Inc., Palo Alto, CA), 1.00 μL TaqStart buffer Buffer (Clontech Labs, Inc.) and 0.25 μL AmpliTaq DNA polymerase (Perkin- Elmer, Inc., Norwalk, CN). Immediately before aliquoting the reaction mixture into reaction tubes, add 1 μL Α-<sup>32</sup>P dCTP (250 μCi; 3000 C / mmol, 10 mCi / mL, Amersham Corp., Arlington He ights, IL) is added to the reaction mixture. 45.0 μL of reaction mixture to each reaction tube After aliquoting, cap tube and place in thermocycler. Specific degeneration , Annealing, extension time and temperature, and the number of thermocycles It will vary depending on the size and nucleotide composition of the primer pair used. Then Perform 20 to 25 amplification thermocycles. Then, 5 μL of each reaction was added to DE81 ion Spot on exchange chromatography paper (Whatman, Maidstone, England) And air dry for 10 minutes. The filter was then filtered with 50 mM Na<sub>Two</sub>PO<sub>Four</sub>, PH 7, 200 mM Wash 5 times with 100 mL per wash in NaCl, then air dry, then dry At Sandwich. Quantification was performed using PhosphoImager SI (Molecular Dynamics, Sunnyvale, CA). Typically, the filter is exposed to a phosphor screen. this The screen is exposed to energy from ionizing radiation for a suitable period of time, typically about 120 minutes. Store. After exposure, the phosphor screen is scanned, which allows the light to 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 Recombinant retrovirus used to transduce cells to the number of cells isolated Percentage determined from a standard curve based on the detected radioactivity divided by the volume of (Converted to base form). Recognized by those skilled in the art As described, other detection methods, such as colorimetry, can also be used to label amplification products. Can be used for F.Prescription   Another aspect of the present invention relates to the formulation of a gene delivery vehicle according to the present invention.   A preferred embodiment of this aspect of the invention is the preparation of an infectious recombinant retrovirus. Concerning the preservation of things. Recombinant retrovirus infects eukaryotic cells during reconstitution (See US patent application Ser. No. 08 / 153,342), and locate the vector construct. It can be delivered to the genome of such cells in an unusual manner. Conveniently purified and If necessary, first concentrate the recombinant retrovirus with sufficient formulation buffer. Solution to the medium containing the recombinant retrovirus to form an aqueous suspension. And can be held by Preferably, the formulation buffer is a saccharide, high molecular weight structural An aqueous solution containing additives and buffer components in water. Used within the context of the present invention The "buffer compound" or "buffer component" is used to maintain an aqueous suspension at the desired pH. It should be understood to refer to a substance that functions as follows. The aqueous solution may also contain one or more Contains amino acids.   Recombinant retroviruses can also be stored in purified form. More specifically Prior to the addition of the formulation buffer, the crude recombinant retrovirus described above is, for example, Cross flow enrichment system (Filtron Technology Corp., Nortborough, MA) Yo Clarify by passing the retrovirus through a filter, then concentrate Can be done. In one embodiment, DNase is added to the concentrate to eliminate exogenous DNA. Become The digest is then diafiltrated to remove excess media components. And establish the recombinant retrovirus in a more desirable buffered solution. Then The dialysis filtrate is passed through a Sephadex S-500 gel column, and the purified recombinant retrovirus is purified. Elute the virus. Add sufficient formulation buffer to this eluate to obtain the desired components (See, eg, Example 9) and recombinant retrovirus Dilute to a minimum. The aqueous suspension is then added, preferably at -70 ° C or immediately. Dry and store. As mentioned above, the formulation buffer contains saccharides, high molecular weight structural An aqueous solution containing additives and buffer components in water. The aqueous solution may also contain one or more It may contain amino acids.   Crude recombinant retrovirus can also be used for ion-exchange column chromatography. It can be more purified. This method is described in more detail in US patent application Ser. No. 08 / 093,436. It is described in. Generally, the crude recombinant retrovirus is recovered through a filter. Clarified by passage of torovirus and highly sulfonated Load the filtrate into the column containing the cellulose matrix. Recombinant retro The virus is eluted from the column in purified form by using a high salt buffer. You. The high salt buffer is then more desirably passed by passing the eluate through a molecular exclusion column. Replace with a new buffer. A sufficient amount of the formulation buffer is then purified as described above. Added to the recombinant retrovirus, and immediately dry the aqueous suspension. Or preferably stored at -70 ° C.   The aqueous suspension in crude or purified form can be lyophilized or evaporated at any temperature. It can be dried by poration. Specifically, freeze drying is below the glass transition temperature Or cooling the aqueous suspension below the eutectic temperature of the aqueous suspension, and sublimation Removing the water from the more cooled suspension to form a lyophilized retrovirus. Include. Conveniently, aliquots of the formulated recombinant retrovirus are lyophilized. Refrig chamber (Edwards Refrig) connected to a machine (Supermodulyo 12K) erated Chamber) (3 shelf RC33S unit). Phillips et al. (Cryobiolo gy 18: 414, 1981), using a multi-stage freeze-drying procedure. The isolated recombinant retrovirus is lyophilized, preferably from a temperature of -40 ° C to -45 ° C. You. The resulting composition contains less than 10% water by weight of lyophilized retrovirus. It contains. Once lyophilized, the recombinant retrovirus is stable and And may be stored at -20 ° C to 25 ° C, as discussed in more detail below.   In the evaporation method, water is removed from the aqueous suspension at any temperature by evaporation Is done. In one embodiment, the water is spray-dried (EP 520,748). No.). In the spray drying process, the aqueous suspension is preheated Delivered into a stream of gas (usually air). So the water is a droplet of the suspension Evaporates quickly from. Spray-drying equipment is available from many manufacturers (eg, Drytec , Ltd., Tonbridge, England; LabPlant, Ltd., Huddersfield, England) Available. Once dehydrated, the recombinant retrovirus is stable, -20 ° C to 2 ° C. Can be stored at 5 ° C. In the method described herein, the resulting dried or Indicates the water content of the lyophilized retrovirus using the Karl-Fischer instrument (EM Science  Aquastar “V1B volumetric titrator”, Cherry Hill, NJ) Can be determined gravimetrically.   As mentioned above, the aqueous solutions used for the formulation are sugars, high molecular weight structural Contains additives, buffer components and water. This solution also contains one or more amino acids . Combinations of these components can be frozen and lyophilized, or evaporated. When dried by, it acts to preserve the activity of the recombinant retrovirus. preferable The saccharide is lactose, but other saccharides such as sucrose, mannitol, Lucose, trehalose, inositol, fructose, maltose or gala Ktose may be used. In addition, combinations of sugars, such as lactose and man Nitol, or sucrose and mannitol may be used. Especially lactose The preferred concentration is 3% to 4% by weight. Preferably, the concentration of the saccharide is The ratio ranges from 1% to 12%.   High molecular weight structural additives help prevent virus aggregation during freezing, and Provides structural support in a lyophilized or dry state. In the context of the present invention Structural additives, if they have a molecular weight above 5000 Is considered to be "amount". A preferred high molecular weight structural additive is human serum alcohol. This is Bumin. However, other substances, such as hydroxyethyl cellulose, hydrid Roxymethylcellulose, dextran, cellulose, gelatin, or povid Can also be used. A particularly preferred concentration of human serum albumin is by weight 0.1%. Preferably, the concentration of high molecular weight structural additives is 0.1% by weight It is in the range of ~ 10%.   The amino acids (if present) are added to the aqueous suspension upon cooling and thawing. It functions to further maintain the infectivity of the virus. In addition, amino acids are chilled aqueous Keep the virus infectious during sublimation of the suspension and during lyophilization. Works like A preferred amino acid is arginine. But other amino acids For example, lysine, ornithine, serine, glycine, glutamine, asparagine , Glutamic acid, or aspartic acid may also be used. Particularly preferred al Guinine concentration is 0.1% by weight. Preferably, the amino acid concentration is 0% by weight. It is in the range of .1% to 10%.   The buffer component works to buffer the solution by maintaining a relatively constant pH. To use. The various buffers are adjusted to the desired pH range (preferably between 7.0 and 7.8). Can be used depending. Suitable buffers include phosphate and citrate buffers. Including. A particularly preferred pH of the recombinant retroviral formulation is 7.4 and is preferred. Another buffer is tromethamine.   In addition, the aqueous solution can be used to transfer the final formulated recombinant retrovirus to a suitable isotonic saline solution. It preferably contains a neutral salt used for adjusting the temperature. Suitable neutral salt Includes sodium chloride, potassium chloride, or magnesium chloride. Like A new salt is sodium chloride.   An aqueous solution containing the desired concentrations of the above components is prepared as a concentrated stock solution. Can be made.   Particularly preferred is the storage of the lyophilized recombinant retrovirus for subsequent reconstitution. A preferred method involves the following steps: (a) culturing the infectious recombinant retrovirus in water; Mixing with the solution to form an aqueous suspension, wherein the aqueous suspension is 4 wt. % Lactose, 0.1% human serum albumin by weight, N less than 0.03% by weight Provides aCl, 0.1% arginine by weight, and pH of aqueous suspension of about 7.4 An effective amount of a tromethamine buffer, thereby providing an infectious recombinant retrovirus. (B) cooling the suspension to a temperature of -40 ° C to -45 ° C and freezing; Forming a suspended suspension; and (c) removing water from the frozen suspension by sublimation. To form a lyophilized composition having less than 2% water by weight of the lyophilized composition. Wherein the composition is capable of infecting mammalian cells upon reconstitution. . Recombinant retroviruses are replication deficient and, upon reconstitution, are Preferably, it is suitable.   Lyophilized, as will be apparent to those skilled in the art from the disclosure provided herein. Retroviruses intended for storage at room temperature use specific saccharides in aqueous solution. May be preferred. More specifically, disaccharides (eg, lactose or Is trehalose), but it is particularly preferable to use it for storage at room temperature.   The lyophilized or dehydrated retrovirus of the present invention can be regenerated using various substances. It can be constituted, but is preferably reconstituted using water. In one example, the final A dilute salt solution that renders the formulation isotonic can also be used. Furthermore, it is restructured An aqueous solution containing a component known to enhance the activity of Use may be advantageous. Such components include cytokines (eg, IL -2), polycations (eg, protamine sulfate), or reconstituted retro Includes other components that increase the efficiency of transduction of irus. Lyophilized or dehydrated Recombinant retrovirus is used in any convenient volume of water or lyophilized or dehydrated. Reconstruction as described above to allow substantial, and preferably all, dissolution of the watered sample It can be reconstituted with an agent. G. FIG.Administration   In another aspect of the present invention, eukaryotes (especially warm-blooded animals, And especially humans). Diseases include genetic diseases, cancer, infectious diseases, Includes autoimmune and inflammatory diseases, cardiovascular diseases, and degenerative diseases. Remains Examples of transmission include, but are not limited to, thalassemia, phenylketonuria, Lesch-Nyan syndrome, SCID, hemophilia A and B, cystic line Fibrosis, Duchenn muscular dystrophy, hereditary emphysema, familial hypercholesterol Examples include rollemia, and Gaucher's disease. An example of a cancer is Examples include, but are not limited to, solid tumors, leukemias and lymphomas. Typical Examples are melanoma, colorectal carcinoma, lung carcinoma (large cell, small cell, squamous and adenocarcinoma) ), Renal cell carcinoma, cervical cancer, adult T-cell lymphocytic leukemia, and breast adenocarcinoma No. Infectious diseases include, but are not limited to, hepatitis, tuberculosis, malaria A, human immunodeficiency virus, herpes virus, tetanus, dysentery, Shigella (shigel la), FeLV, and FIV. Degenerative diseases are not limited to But Alzheimer's disease, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis No. Inflammatory diseases include rheumatoid arthritis, spinal meningitis, pancreatitis . Autoimmune diseases include diabetes, uveitis, HIV, and SCID. heart Vascular disease includes chronic rheumatic heart disease, atherosclerosis, stenosis of the mitral valve and aorta, myocardium Inflammation, pericarditis, Marfan syndrome, Ehlers-Danlos syndrome, Churg-Strauss syndrome, and scleroderma.   Each of these methods involves the administration of a gene delivery vehicle according to the present invention. You. As a result, the gene of interest transported by the gene delivery vehicle encodes A therapeutically effective amount of the gene product is produced. As used herein, according to the present invention A “therapeutically effective amount” of a gene product expressed from a vector construct is To a greater extent than observed when not treated with offspring products, The amount that achieves the desired therapeutic benefit. For example, if the gene product is factor VIII, Where "therapeutically effective amount" is the amount of factor VIII required to produce a therapeutically beneficial coagulation Say. Accordingly, a “therapeutically effective amount” is generally determined by the attending physician of each patient. As determined, serum levels of about 0.2 ng / mL (about 0.1% of "normal" levels) or More than that is typically therapeutically beneficial. Gene product is an antisense RNA Or if it is an RNA molecule with intrinsic biological activity such as a ribozyme, "Therapeutically effective amount" refers to reduced expression of a target gene product (often a protein) Is sufficient to achieve a clinically relevant change in the patient's condition. Like In a preferred embodiment, an RNA molecule having endogenous biological activity (ie, Antisense RNA or ribozyme) is the target RNA molecule in the transduced cells. And expressed from its position-specific position. Heterologous RNA and target R The expression level of NA can be determined by various assays, eg, PCR analysis.   Typical doses for ex vivo treatment of hematopoietic stem cells are generally about 10<sup>Five</sup>,Ten<sup>6</sup>, Ten<sup>7</sup>,Ten<sup>8</sup>,Ten<sup>9</sup>,Ten<sup>Ten</sup>, I0<sup>11</sup>~Ten<sup>12</sup>A range of infectious gene delivery vehicles; And ten<sup>7</sup>~Ten<sup>Ten</sup>Is preferred.   The gene delivery vehicles of the present invention can be administered to a wide range of sites. For example, cerebral spine Cerebrospinal fluid, bone marrow, joints, arterial endothelial cells, rectum, buccal / sublingual, vaginal, lymphatic system And organs selected from the group consisting of lung, liver, spleen, skin, blood and brain. Or a site selected from the group consisting of tumor and interstitial space. Other fruit In embodiments, the recombinant retrovirus is intraocular, intranasal, sublingual, oral, topical Intravesical, intravesical, topical, intravenous, intraperitoneal, intracranial, intramuscular, or transdermal Can be given. Other typical routes of administration include gastroscopy, ECRP, and rectal examination I do. They do not require complete operating procedures and hospitalization, but must be attended by a physician. May be required.   For administration of the compositions of the present invention, the following considerations are made:   Oral administration is easy and convenient, economical (does not require bactericidal), Is safe (often can be treated in overdose) and the active ingredient of the composition (Gene delivery vehicle). Conversely, local hypersensitivity, For example, there is nausea, vomiting or diarrhea, unstable absorption of poorly soluble drugs. And gene delivery vehicles rely on hepatic metabolism and gastric acid and enzymatic degradation to " It is susceptible to a "first pass effect". In addition, the action is slow Effective plasma levels may not be reached and require patient cooperation. In short, and food can affect absorption. A preferred embodiment of the present invention Lopoietin, insulin, GM-CSF cytokine, various polypeptides or peptides Genes encoding the hormones of pide, their agonists or antagonists Oral administration of a gene delivery vehicle that expresses In this case, these Mont is a pituitary, hypothalamus, kidney, endothelial cell, liver, pancreas, bone, hematopoietic bone marrow, And may be from tissues such as the adrenal gland. Such polypeptides can be used to induce proliferation, tissue Regression, suppression of immune response, apoptosis, gene expression, receptor-ligand phase It can be used for blocking interactions, for immune responses, and for certain anemias, diabetes, Staining, high blood pressure, abnormal blood chemistry (s) (eg, elevated blood choles) Terol, blood clotting factor deficiency, elevated LDL with low HDL), Alzheimer's Relevant amyloid protein levels, bone erosion / calcification, and various Levels of metabolites (eg, steroid hormones, purines, and pyrimidines) Can be a treatment for the control of Preferably, the gene delivery vehicle is first frozen Dried and then filled into capsules and administered.   Buccal / sublingual administration provides a rapid onset of action of the active ingredient (s) of the composition Convenient administration to provide and avoid first pass metabolism Is the law. Thus, there is no gastric acid degradation or enzymatic degradation and the gene delivery The absorption of the vehicle is easy. High bioavailability and virtually immediate treatment Suspension is possible. Conversely, such administration requires relatively low doses (typically, about 10-15 mg) and cannot have concurrent eating, drinking, or swallowing No. A preferred embodiment of the present invention involves buccal / sublingual administration of a gene delivery vehicle. The gene delivery vehicle may be self- and / or exogenous for the following treatments: Contains genes encoding MHC or immune modulators: treatment of oral cancer Such a gene delivery vehicle containing an IgA or IgE antisense gene For Sjogren's syndrome via buccal / sublingual administration of IgG; and IgG or Indicates gingivitis and periodontitis via buccal / sublingual administration of cytokine antisense genes Flame treatment.   Rectal administration provides negligible first-pass metabolic effects (good vascular / lymphatic vessels There is a supply and the absorbed material is excreted directly into the inferior vena cava). this The method is suitable for children, vomiting patients and unconscious patients. This method is useful for gastric acid degradation and And avoids enzymatic degradation and does not change the ionicity of the composition. Because Rectal fluid has no buffering capacity (pH 6.8; charged compositions are best absorbed). Collected). Conversely, slow, poor or unstable absorption, irritability, Degradation can be present and the absorbing surface is small (about 0.05 m<sup>Two</sup>). In addition, lipophilic and water Compounds that are soluble in water are preferred for absorption by the rectal mucosa and absorption enhancers (eg, , Salt, EDTA, NSAID). A preferred embodiment of the present invention is a colon cancer Encodes an antigen, self and / or foreign MHC, or immune modulator Rectal administration of a gene delivery vehicle containing the gene.   Nasal administration avoids first-pass metabolism and gastric acidolysis and enzymatic degradation, It is convenient. In a preferred embodiment, the nasal administration is performed using a gene delivery vehicle. Useful for administration. Here, the gene delivery vehicle is the sex delivery vehicle described herein. It has a nucleic acid capable of expressing a quality polypeptide. Conversely, such administration May cause local hypersensitivity. And absorption can depend on the condition of the nasal mucosa.   Pulmonary administration also avoids first-pass metabolism, and gastric acidolysis and enzymatic degradation, And it is convenient. In addition, pulmonary administration allows for a localized effect, Minimize doses required for effective side effects and efficacy. And action and self-medication Self-medication can be started quickly. Conversely, most of the administered composition When small amounts of cancer reach the bronchioles / alveoli, local hypersensitivity can occur and Excess dosing is possible. In addition, patient cooperation and understanding are preferred and Propulsion can have toxic effects. Preferred embodiments of the present invention include asthma, hay fever, IgA or IgE for the treatment of conditions such as allergic or fibrous alveolitis Gene, CFTR gene for treatment of cystic fibrosis, and treatment of emphysema Protease and collagen inhibitors (eg, 1-antitri Psin). Alternatively, for oral administration Many of the above polypeptides or peptides of the same type can be used.   Ocular administration provides a local effect and is prolonged when administration is through insertion Enable the action. In addition, first-pass metabolism, and gastric acidolysis and enzymatic Avoid solutions and self through the use of eye drops or contact lens-like inserts Allow dosing. Conversely, administration is not always efficient. Because the administration This is because it includes tearing. In a preferred embodiment of the present invention, IgA for the treatment of conjunctivitis or spring conjunctivitis and atopic conjunctivitis Ocular administration of a gene delivery vehicle that expresses a gene encoding IgE or Ranoma-specific antigens (eg, high molecular weight melanoma-associated antigens), autologous and / or Or a gene containing a foreign MHC, or a gene encoding an immune modulator Ocular administration of a child delivery vehicle.   Transdermal administration provides rapid treatment and prolonged action leading to good compliance Enable the suspension of In addition, local treatment is possible and first-pass metabolism, And avoid gastric acid degradation and enzymatic degradation. Conversely, such administration is topical And can be particularly susceptible to developmental tolerance, and typically It is not preferred for high potency compositions. A preferred embodiment of the present invention is a transdermal Including administration: symptoms (eg, atopic dermatitis and other skin allergies) Transdermal administration to express a gene encoding IgA or IgE for the treatment of And melanoma-specific antigens (eg, high molecular weight melanoma-associated antigens), autologous And / or a gene encoding a foreign MHC or an immune modulator. Transdermal administration of a gene delivery vehicle to be administered.   Vaginal administration provides a local treatment for hormone administration and one preferred route. Offer. In addition, such administration may involve first-pass metabolism and gastric acid degradation and enzymes Of a composition that avoids chemical degradation and wherein the gene delivery vehicle expresses the peptide Preferred. Preferred embodiments of the present invention include self and / or exogenous MHC, Or vaginal administration to express a gene encoding an immune modulator . Another preferred embodiment enhances the production of sperm-specific antibodies, thereby Vaginal administration of genes encoding sperm components such as histones and flagellin Include. This effect is due to the immune (where the gene interferes with the production of sperm-specific antibodies) Gene delivery vehicle having a vector encoding a globulin antisense gene By delivering a drug, the pregnancy of some women may be reversed and / or Can be increased.   Intravesical administration allows local treatment of urological problems and eliminates systemic side effects. Avoid and avoid first-pass metabolism and gastric acidolysis and enzymatic degradation. vice versa The method requires the insertion of a urinary catheter, and highly skilled staff Need. A preferred embodiment of the present invention relates to a prodrug such as thymidine kinase. Such as various anti-tumor genes or various cytokines such as lag-activating genes And intravesical administration to deliver various immunomodulatory molecules.   Endoscopic retrograde cystopancreatography (ERCP)) (through the mouth; does not require skin penetration) is an enlarged gastroscopy Utilization and allows selective access to the bile and pancreatic ducts. Conversely, the book The method requires highly skilled staff and is uncomfortable for the patient.   Many of the routes of administration described herein (eg, intra-CSF, intra-bone marrow, intra-articular, Intravenous, intraarterial, intracranial, intramuscular, subcutaneous, in various organs, in tumors, in interstitial space, Into the abdominal cavity, into the lymph, or into the capillary bed). It can be conveniently achieved by direct administration using a device. In particular, the features of the present invention In certain embodiments, one or more doses are administered in the indicated manner directly as follows: Can be administered: 10<sup>Five</sup>,Ten<sup>6</sup>,Ten<sup>7</sup>,Ten<sup>8</sup>,Ten<sup>9</sup>,Ten<sup>Ten</sup>Or 10<sup>11</sup>is equal to cfu Cerebrospinal fluid at or above dose; 10<sup>Five</sup>,Ten<sup>6</sup>,Ten<sup>7</sup>,Ten<sup>8</sup>,Ten<sup>9</sup>,Ten<sup>Ten</sup>,Also Is 10<sup>11</sup>into bone marrow at doses equal to or greater than cfu; 10<sup>Five</sup>,Ten<sup>6</sup>,Ten<sup>7</sup>,Ten<sup>8</sup>, 1 0<sup>9</sup>,Ten<sup>Ten</sup>Or 10<sup>11</sup>Joints (single or single) at doses equal to or greater than cfu Are plural); 10<sup>8</sup>,Ten<sup>9</sup>,Ten<sup>Ten</sup>Or 10<sup>11</sup>For use equal to or greater than cfu Intravenously in quantity; 10<sup>Five</sup>,Ten<sup>6</sup>,Ten<sup>7</sup>,Ten<sup>8</sup>,Ten<sup>9</sup>,Ten<sup>Ten</sup>Or 10<sup>11</sup>equal to or equal to cfu Or higher doses into arteries; 10<sup>9</sup>,Ten<sup>Ten</sup>Or 10<sup>11</sup>equal to cfu or Intracranial at higher doses; 10<sup>Ten</sup>Or 10<sup>11</sup>greater than or equal to cfu Intramuscularly at the dose; 10<sup>Five</sup>,Ten<sup>6</sup>,Ten<sup>7</sup>,Ten<sup>8</sup>,Ten<sup>9</sup>,Ten<sup>Ten</sup>Or 10<sup>11</sup>equal to cfu or Or higher dose into eyes; 10<sup>Five</sup>,Ten<sup>6</sup>,Ten<sup>7</sup>,Ten<sup>8</sup>,Ten<sup>9</sup>,Ten<sup>Ten</sup>Or 10<sup>11</sup>c into the lung at a dose equal to or greater than fu; 10<sup>Five</sup>,Ten<sup>6</sup>,Ten<sup>7</sup>,Ten<sup>8</sup>,Ten<sup>9</sup>,Ten<sup>Ten</sup> Or 10<sup>11</sup>intranasally at doses equal to or greater than cfu; 10<sup>Five</sup>,Ten<sup>6</sup>,Ten<sup>7</sup> ,Ten<sup>8</sup>,Ten<sup>9</sup>,Ten<sup>Ten</sup>Or 10<sup>11</sup>sublingually at doses equal to or greater than cfu ;Ten<sup>Five</sup>,Ten<sup>6</sup>,Ten<sup>7</sup>,Ten<sup>8</sup>,Ten<sup>9</sup>,Ten<sup>Ten</sup>Or 10<sup>11</sup>greater than or equal to cfu Rectal at a dose of 10;<sup>Five</sup>,Ten<sup>6</sup>,Ten<sup>7</sup>,Ten<sup>8</sup>,Ten<sup>9</sup>,Ten<sup>Ten</sup>Or 10<sup>11</sup>equal to cfu or Orally in higher doses; 10<sup>Five</sup>,Ten<sup>6</sup>,Ten<sup>7</sup>,Ten<sup>8</sup>,Ten<sup>9</sup>,Ten<sup>Ten</sup>Or 10<sup>11</sup>c topically at a dose equal to or greater than fu; 10<sup>Five</sup>,Ten<sup>6</sup>,Ten<sup>7</sup>,Ten<sup>8</sup>,Ten<sup>9</sup>, 1 0<sup>Ten</sup>Or 10<sup>11</sup>Intravaginally at doses equal to or greater than cfu; 10<sup>9</sup>,Ten<sup>Ten</sup>, Or 10<sup>11</sup>subcutaneously at a dose equal to or greater than cfu; 10<sup>Five</sup>,Ten<sup>6</sup>,Ten<sup>7</sup>,Ten<sup>8</sup> ,Ten<sup>9</sup>,Ten<sup>Ten</sup>Or 10<sup>11</sup>into the bladder at a dose equal to or greater than cfu; 1 0<sup>Five</sup>,Ten<sup>6</sup>,Ten<sup>7</sup>,Ten<sup>8</sup>,Ten<sup>9</sup>,Ten<sup>Ten</sup>Or 10<sup>11</sup>greater than or equal to cfu Dose to organs (eg, lung, liver, spleen, skin, blood, or brain); 10<sup>8</sup>,Ten<sup>9</sup>, Ten<sup>Ten</sup>Or 10<sup>11</sup>In a tumor at a dose equal to or greater than cfu; 10<sup>8</sup>,Ten<sup>9</sup> ,Ten<sup>Ten</sup>,Also Is 10<sup>11</sup>intraperitoneally at doses equal to or greater than cfu; 10<sup>Ten</sup>Or 10<sup>11</sup>cfu Into the interstitial space at equal or greater doses; 10<sup>Five</sup>,Ten<sup>6</sup>,Ten<sup>7</sup>,Ten<sup>8</sup>,Ten<sup>9</sup>,Ten<sup>1 0</sup> Or 10<sup>11</sup>Intralymph at doses equal to or greater than cfu; 10<sup>Five</sup>,Ten<sup>6</sup>, Ten<sup>7</sup>,Ten<sup>8</sup>,Ten<sup>9</sup>,Ten<sup>Ten</sup>Or 10<sup>11</sup>Capillary at doses equal to or greater than cfu On the vascular bed; or 10<sup>Five</sup>,Ten<sup>6</sup>,Ten<sup>7</sup>,Ten<sup>8</sup>,Ten<sup>9</sup>,Ten<sup>Ten</sup>Or 10<sup>11</sup>equal to cfu or Or to the dura at higher doses. "Cfu" means "colony forming unit" And therefore, any of a variety of gene delivery vehicles useful in the practice of the present invention. Can also be applied. Colony-forming units are used to identify specific modes of detection (eg, drug resistance, antibody Gene expression detected by the reaction with PCR, PCR of the transduced gene, etc.) Mean number of cells transduced in vitro.   Gene delivery vehicles may serve other purposes, e.g., during surgical procedures for other purposes. As part of a procedure that has or specifically to administer the gene delivery vehicle. As a metered procedure, it can be delivered from outside the organism to be treated. For administration Other routes and methods are described in US patent application Ser. No. 08 / 366,788, filed Dec. 30, 1994. Non-parenteral route disclosed in US Patent Application No. 08/3 , Issue (December 1994 (Filed on the 30th) through multiple sites as disclosed in [Attorney Docket No. 930049.427] Administration.   In addition to in vivo administration, the gene delivery vehicles of the present invention can also be used in an ex vivo format. Can be delivered.   The following examples are provided by way of illustration and not by way of limitation.                                  Example   The following examples are provided to more fully illustrate the invention. Furthermore, this These examples provide preferred embodiments of the present invention, and It is not intended to limit the scope. Many procedures described in the examples below or Standard methods for other suitable procedures are described, for example, in "Molecular Cloning" 2nd edition. (Sambrook et al., Cold Spring Harbor Laboratory Press, 1987) and "Current Protocols in Molecular Biology '' (edited by Ausubel et al., Greene Associates / Wiley In) terscience, NY, 1990). Provided to                                 Example 1                 Preparation of chimeric retrovirus integrase   This example demonstrates that the positional specificity of the yeast Ty3 element (US Pat. No. 5,292,662) How to add to the integrase (IN) protein from loney leukemia virus Describe the law. Then, under appropriate conditions, the resulting chimeric integrase is Once packaged into a gene delivery vehicle, the vector Genome of eukaryotic cells transduced or otherwise transformed thereby Site-specific incorporation may be provided.   As previously described, Ty3 IN is a retroviral IN containing MoMLV IN. It is a functional and structural analog of protein. UWGCG Bestfit algorithm (De Vereux et al., (1984), Nucl.Acids Res., Vol. 12, 387-395) using Ty3 and MoMLV. Amino acid sequence alignment (see FIG. 3) was performed using retrovirus IN protein. Show approximately 25% identity within the core region containing the D-D-E motif conserved between proteins did. MoMLV IN contains 408 amino acids. The core region corresponds to about 220 residues, The no-terminal domain contains the first about 40 amino acids, and the remaining carboxy terminus has 14 Contains about 0 amino acids. Ty3 IN consists of 536 amino acids. Amino-terminal domain Over the first approximately 60 residues, the core domain contains the next 240 amino acids, The ruboxyl-terminal domain contains about 230 amino acids. For each protein, Figure 2 The amino terminal domain is the "A" domain, and the carboxy terminal The ends are referred to as the "C" domain and the core domain as the "B" domain. Each domain To further identify the source of the in, a single capitalization about it is "M" of the domain derived from V IN or "t" of the domain derived from Ty3 Can follow. For example, "At" means A domain derived from Ty3 and AmBmCr. The chimeric IN protein or its gene (or For example, if the A domain and B domain derived from MoMLV IN And a chimera containing a C domain derived from Ty3. Each of the following chimeric IN proteins And the actual AB breakpoint (MoML) for the purpose of designing their corresponding genes. At residue 41 in V IN and residue 61 in Ty3), and the BC breakpoint (MoMLV IN and At amino acids 263 and 304 in Ty3), respectively, And in the area of the random coil to minimize tertiary structure destruction So selected. A.Construction of recombinant retroviral vector encoding chimeric IN protein   Each chimeric IN retrovirus vector is a retrovirus vector exemplified in FIG. Based on pRgpKan. pRgpKan is responsible for all cis Contains elements. In addition, pRgpKan provides functional gag and pol gene products. When expressed and introduced into a cell line that expresses the appropriate env gene product, pRgpKan This results in the production of recombinant retroviral particles containing genomic RNA. pRgpKan Contains the neomycin phosphotransferase gene from lanpozone Tn5 This gene is expressed as kanamycin resistance in bacteria and It is expressed as G418 resistant in cells (using the SV40 early promoter). Retro Viral vectors also allow the plasmid to grow in E. coli. ColE1 origin of replication. Properties of pRgpKan are important for selection of transduced cells, and Rescue vector DNA from these cells in bacteria after kanamycin selection to enable. Genes expressed from retroviral vectors based on pRgpKan , Splices found in the MoMLV IN gene because env is supplied in trans Does not require an acceptor sequence. The plasmid type of pRgpKan contains only one LTR contains. However, when expressed in cells, the RNA genome is transcribed in two LTRs. pRgpKan is derived from the BAG vector (Price et al., (1987), Proc. Natl. Acad.sci. US A, 84: 156-160).   The seven chimeric retroviral vectors shown in FIG. 5 were constructed using pRgpKa as follows. Constructed with n skeleton. First, a 2.8 kb sequence containing the IN coding region was PMLV-K encoding the LV genome (Miller et al., J. Mol. Cell Biol. 5: 431, 1985) or Excised from plasmids such as 2xMLV, flanked by LTRs at each end (See FIG. 6). Excision was performed using SalI and BamHI. occured The agarose gel purified fragment was digested with SalI-BamHI to generate pMLVIN. Cloned into pIBI-20 (FIG. 6). Next, the Ty3 IN coding region from pVB193 was The containing 4.5 kb SalI-ScaI fragment from Ty3 (3132 of the Ty3 element itself) (Hansen et al. (1990) J. Virol. 64: 2599-2607)). pMLVIN was cloned into a gel-purified 4.4 kb ScaI fragment and 8.8 kb The rasmid vector pMLV / Ty3IN was obtained. This plasmid vector contains the heterologous MLV and And the Ty3 integrase gene, arranged in tandem as follows: AmB mCm-AtBtCt. Subsequently, Kunkel mutagenesis (Kunkel, T.A. (1985), Proc. Natl. Acad. Sci. USA, 82: 488-492; oligonucleotides complementary to the desired junction region in MoMLV and Ty3 IN Loop-out of single-stranded phage DNA using nucleotide bridges) with pMLV / Ty3IN. Thus, the first three codons of MLV IN and the Ty3 integrase All but the 5 'immediately upstream were deleted to produce plasmid pfTY3IN (Figure 6). . An oligo that is the nucleic acid sequence of the oligonucleotide used to perform this mutagenesis 1 (SEQ ID NO: 5) is provided in FIG. MLV and Ty3 sequences complementary to oligo 1 sequence The columns are also shown.   Then, pfTY3 / MLVIN, the XmnI-EcoRI fragment from pfTY3IN, pMLVIN It was generated by ligating to the 3.8 Kb XmnI fragment from E. coli. pfTY3 / MLVIN Shows Ty3 and MLV integrase aligned with AtBtCt-AmBmCm as shown in FIG. It contains. As designed, the pfTY3 / MLVIN construct was designed to address the Ty3-IN coding region. Contains the proteolytic cleavage site of MLV at the mino terminus. Next, pMLV / Ty3IN and Kunkel mutagenesis of each of the two phagemid constructs, and pfTY3IN / MLVIN Four chimeric IN genes were generated using as substrates for development. In particular, oligo 4 and Using oligo 5 (FIG. 7; SEQ ID NO: 14 and SEQ ID NO: 17, respectively), pMLV / Ty3  PAmBtCt and pAmBmCt were prepared from IN, respectively. Oligo 2 (FIG. 7; Sequence No. 8) was used to generate pAmBmCm by mutagenizing pfTY3 / MLVIN while oligo 3 PyTy3 / MLVIN was mutagenized using (FIG. 7; SEQ ID NO: 11) to produce pAtBtCm.   B domain from one integrase between the other A and C domains The remaining two chimeras containing the chitin were made as follows. HindII to pAtBtCm And cut with MscI to release the BtCm-containing region. Cut this fragment as well. It was cloned into pAmBtCt which had been cut and the BtCt region was removed. The resulting construct (pAmBtCm Has an A region and a C region derived from MLV and a B domain derived from Ty3. Chimeric IN. Coding of A and C domains from Ty3 PAtBmCt containing the B domain coding region derived from the Was made by digesting with HindIII and gel purifying the BmCt-containing region, This was cloned into pAtBmCm from which the BmCm region had been removed by HindIII digestion.   Then, each of the seven plasmids encoding the chimeric IN protein, or `` Phagemid '' (pAmBmCt, pAtBmCm, pAtBmCt, pAmBtCm, pAtBtCm, pAmBtCt and And pfTy3IN) were digested with either Sal1-Xho1 or Sal1-BamHI The coding region encoding teglase was released. Then contains the coding region Approximately 2.7 kb of each fragment was combined with the 4.1 kb NheI to SalI plasmid from pRgpKan. The fragment and the ligation are combined in three separate ligations. See FIG. . The resulting construct is called pRgpAxByCz, where x, y, and z are either t or m. Depending on the source of the phagemid). It depends on the origin of the A, B and C domains in the lase gene. For each construct Two independent kanamycin resistant clones were analyzed by restriction digestion and And confirmed by sequencing analysis.   In addition to the seven chimeric retroviral vector constructs described above, a negative control Retroviral vector (designated pRgpAmBtCt (-)) in the antisense orientation with A Prepared by inserting the mBtCt DNA sequence. pRgpAmBtCt (-) is an appropriate package When introduced into a Zing cell line (eg, NC10 or 292 2-3), Virus particles are produced, but these particles are Contains no special IN. PRgpKan originally obtained from the MoMLV IN coding region Used as troll. B.A set using a recombinant retroviral vector encoding a chimeric IN protein Production of recombinant retroviral particles   For biological activity and site-specific integration in human and other mammalian cells Recombinant retrovirus carrying chimeric integrase to test for Cultures that produce particles can be produced as described herein below. At first, Produced from human adenovirus 5-transformed embryonic kidney cell line 293 (ATCC # CRL1573), 293-2 cells (AKA 293-GP, Burns et al.) Expressing MoMLV gag and pol genes Proc. Natl. Acad. Sci. 90: 8033 1993, WO 92/05266). Four NC10 cells expressing the 070A amphotropic envelope (see WO 92/05266) Is generated from the HT1080 human fibrosarcoma strain (ATCC # CCL121). HT1080 and 292 Both cells may lack DNA sequences that hybridize to the MoMLV genome. It is shown. The plasmid construct pMLP-G is vesicular stomatitis virus G protein. MoML expressing protein and complementary to pseudotyped retroviral vector particles V Used for gag-pol proteins. FIG. 8 produces a producer culture. The method is illustrated. In each case, the chimeric IN retroviral vector was The encoding plasmid was co-transfected into the 293 2-3 cell line with pMLP-G at a 1: 1 ratio. Transfect (Graham et al., (1973), Virology, 52: 456-467). 293 2-3 In cells, the retroviral RNA genome encoding chimeric IN is pol protein Encapsulates into particles formed by the gag gene product along with the quality. These grains Offspring also contain the VSV-G protein on its outer surface. 48 hours later, filtration (.45 μm The supernatant liquid (cellulose acetate) is placed on NC10 cells. After another 24 hours, transduction (Emi et al., (1991), J. Virol., 65: 1202-1207) NC10 cultures were cultured with G418 (600 μg / ml Move under the selection of). Select until no viable cells are present in the non-transduced NC10 control culture. Continue with. The resulting culture produces retroviral particles consisting of: PNA genome encoding chimeric IN protein; chimeric retroviral vector construct Gag and pol gene products expressed from the building; an amphotropic envelope; And chimeric IN protein.   Filter the functionality of these retroviral particles onto target cells (eg, HT1080) Layered supernatant and subsequently evaluated by selecting G418 resistant transductants I do. Using all the chimeric retroviral vector particles produced successfully, NC10 producer cells (ie, produce infectious recombinant retroviral particles Cells). By making decisions based on relative transient titers, The retroviral genome encoding the la IN protein was observed for RgpKan Packaging at the same rate as   Additional NC10 producer cells confirm biological activity of gag and pol components NC10 cell lysates and supernatants were analyzed by Northern analysis (Sambrook et al., Supra) for characterization. C) to generate a retroviral construct encoding chimeric integrase. The current level and packaging are tested against the control RgpKan construct. It is determined by comparison with the expression level obtained. Required for transfer, packaging, or duplication The missing cis-acting sequences have been modified so that Results are expected to be similar to those determined for controls .   In addition, NC10 cell lysates and supernatants are separately prepared according to standard techniques. MoML using anti-p30, anti-MOMLV IN, and anti-Ty3 IN rabbit polyclonal antibodies Tested by Western blot analysis for V gag and IN protein production I do. RagKan for control of gag protein level and construction of 7 chimeras These are internal controls for the level of pol-derived IN protein for comparison with Act as a roll. Mobility of chimeric integrase generated from each construct And the amount detected is the Ty3 sequence for those chimeras containing the At domain. MoMLV protein at the MoMLV IN processing site, three amino acids before the junction of The occurrence of ase indicates whether it is correctly processed. Les The torovirus processing site requires about seven residues (Pettit et al., ( 1991), J. Biol. Chem., 266: 14539-14547) have been clearly and locally determined, And because it is similar to the Ty3 protease processing site, the function of the MoMLV IN site A unique processing site should also occur for the AtBtCt construct.   Further, the NC10 cell supernatant was concentrated by centrifugation and reverse transcriptase activity (Goff (1981), J. Virol. 38: 329-248), and Determine whether incorporation into the torovirus particle disrupts reverse transcriptase function. The activity of the test construct was compared to the activity of the RgpKan supernatant, and both were Standardize the quality level.   Two clones of the seven chimeric IN constructs and two control constructs. For each of RgpKan and RgpAmBtCt (-), three G418 resistant cultures were derived. And collect the supernatant. C.Target cell transduction   As described above, recombinant retroviral vectors generated from the NC10 producer Eukaryotic cell lines (e.g., , HT1080) is used. HT1080 cells are unphotopiped Susceptible to infection by a retroviral vector (As determined by G418 resistance) showed integrase function in 292 2-3 cells. The retroviral vector introduced by the VSV-g pseudotyped virion produced Virion assembly in NC10 producer cells when encoded by NC cells Machiyo is mediated by chimeric IN incorporated into retroviral particles during yellowtail Depends on the integration.   The transduction assay is performed as follows; about 10 ml of filtration from the NC10 producer. Excess (0.45 μm) supernatant solution for about 10⁶Put in fresh HT1080 cells. 24 hours later, the medium is DM Replace with EM + 10% FBS. After another 24 hours, the medium was changed again and at this point To contain 600 μg / ml G418. Non-transduction control of HT1080 cells G418-resistant colonies were successfully harvested when the cultures could no longer survive under G418 selection. Recorded as a new transduction event. Then HT1080 G418 resistance from the chimeric construct Number of clones, as well as HT1080 G418 resistant clones from control constructs The number is determined by the amount of gag protein in the NC10 supernatant, as determined by Western blot. Based on the relative infectivity of control and chimeric constructs Standardize for production. Results of a transduction assay using the chimeric construct described above Is shown in Table 1 below and the Ty3 sequence for the corresponding domain of MoMLV IN Shows that the substitution of the integrase results in an integrase that can mediate integration.   Four hours after infection, to analyze reverse transcription and viral DNA processing, Cells are harvested and Hirt supernatant is obtained (Roth et al., (1989), Cell, 58: 47-54). each The amount of supernatant used in the sample is normalized based on gag protein levels. This These supernatants were produced by reverse transcription of the retroviral RNA genome following cell entry. Should contain extrachromosomal viral DNA. After RNase digestion, DNA levels determined by Southern blot analysis using MoMLV-specific probes I do. Viral DNA processing is assayed by examining the ends of extrachromosomal DNA . Both the retrovirus and the Ty3 IN protein were replicated before integration Remove 2 bp from the 3 'end of the retroviral DNA. Hi near the end of the virus Digest the DNA in the rt supernatant with restriction enzymes and dissolve the Fractionation on a gel suitable for fragmentation (this depends on the nucleic acid sequence and the selected restriction enzymes). Dependent), transfer to nitrocellulose, and end processing Probe with an end-strand specific probe to determine if it occurs. this The information is used as described below (Section D) to optimize another set of constructs. You. D.Analysis of site-specific integration   An integrated library to determine the location of the provirus in the eukaryotic genome Can be constructed from the DNA of the transduced cells. Below, we will discuss the provirus The analysis is described. The integration of the provirus into the genome of HT1080 target cells is described above. It is mediated by the chimeric IN protein. Chimeric integrase and pRgpKan co Each of the retroviral vectors encoding a control Point (ori), as well as bacterial control genes expressing the kanamycin resistance gene in E. coli. Contains Rement. As a result, integration with the host DNA adjacent to the integration site Incorporated proviruses can be cloned directly in bacteria (Cepko et al., (1984) Cell, Vol. 37, 1053-1062).   FIG. 9 shows the results described herein for isolating integrated proviral DNA. The technique is illustrated. HT1080 target cells selected for transduction by G418 treatment Later, genomic DNA from the pooled culture was isolated and incorporated into proviral DNA. Is not present (eg, present in chimeric vector constructs that are ScaI, Bst1107I). No restriction recognition sites) Digest completely with restriction enzymes. Next, this DNA is inserted into the integration section. Preferred cyclization conditions so that host DNA on either side of the position is included in the circular molecule. Connect with. Then, using the circular DNA, E. coli DH12s C strain (Gibco BRL), or Transform with another E. coli strain set up for cloning non-bacterial DNA . Kanamycin resistant colonies were then isolated and pooled, Remove the Smid DNA.   Ty3 integrase is a gene transcribed by RNA polymerase III (tRNA gene Mediates integration adjacent to the tRNA gene, so the approximate frequency of insertion next to the tRNA gene Animal cell tRNA was purified, labeled (Goddard et al., (1983), Nuc. Acids Res., Vol. 11: 2551-62), and pooled from kanamycin-resistant bacteria Used to probe Southern blots of plasmid DNA. LTR-specific professional Parallel blots probed with the probe show proviral DNA and adjacent tRN Determine the relative abundance of the A gene. Expected about RgpKan control As such, non-specific integration was the average of the 3 kb cloned integration fragment. 10 with size and about 1,300 tRNA genes⁶1,00 kb based on total genomic DNA This results in a frequency of binding of the tRNA gene with less than one provirus in 0 clones ( Hatlen et al. (1971), J. Mol. Biol., 56: 535-553). Pooled DNA and rollers Knee was also screened in a similar fashion using 5S and U6 sequence probes. Can be Because these genes are conserved in the sequence, (Sorensen et al., (1991), Nuc.Acids.Res., Vol. 19, 4147-51). For U6, 200 (Hayashi, K. (1981), Nuc. Acids Res., Vol. 9, 3379-88) times, This is because it is repeated in the genome.   Clones and pools from the integrated library were also radiolabeled Alu It can be probed with a specific probe. Alu elements generally lack transcriptional activity Contains the pol III promoter element.   Preferably, at least 10 plasmids for each construct are sequenced Analysis to identify potential target genes unrelated to probe reactivity (Pavesi et al., ( 1994), Nuc. Acids Res., Vol. 22: 1247-56). Sequencing from the LTR is transferred with pol III Allows determination of proviral positioning with respect to the element being transcribed. Insertion The 5 'end of the mature tRNA coding region (or the coding region of another RNA pol III gene) Within 500 bp, preferably within 100 bp, more preferably within 20 bp of Only is thought to be accompanied by position specificity. However, about 10-20 position-specific events were analyzed Only after that, the chimeric IN function is considered to be position specific.   Furthermore, the chimeric integrase of the present invention incorporates it in a position-specific manner. The level of expression of the gene of interest from the provirus may also be analyzed. Preferred Alternatively, expression levels are determined for a given gene of interest integrated in a site-specific manner. To the same gene randomly integrated into the comparable eukaryotic cell genome Less than 100 times, preferably less than 50 times, and most preferably less than 10 times. Become Typically, the target cells are transformed cells produced from the integrated provirus. The transcripts are tested for expression levels by quantitative Northern blot analysis. statistics At least 10 independent clones per original construct for biological analysis. test.                                 Example 2                     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) The repeat (LTR) EcoRI-EcoRI fragment (containing the gag sequence) was⁺(St ratagene, La Jolla, CA). The resulting construct is named N2R5. this The N2R5 construct was modified by site-directed in vitro mutagenesis to change the ATG start codon to ATT. To prevent gag expression. This mutagenized fragment contains 200 bases. Pair (bp) long and flanked by PstI restriction sites. PstI-PstI mutation flag SK⁺Purified from plasmid and N2 MoMLV5'L in plasmid pUC31 Place the unmutated 200 bp fragment by inserting it into the PstI site of TR. Replace. Plasmid pUC31 is derived from pUC19 (Stratagene, La Jolla, CA). In the additional restriction sites XhoI, BglII, BssHII and NcoI are the EcoRI part of the polylinker Inserted between the position and the SacI site. This construct is named pUC31 / N2R5gM.   A 1.0 kilobase (Kb) MoMLV3 'LTR EcoRI-EcoRI fragment from N2 was Rasmid SK⁺Cloned into N2R3^-To obtain a construct called 1.0Kb ClaI- The HindIII 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. Louls 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 Insert the II fragment into the XhoI-HindIII site of the pUC31 / N2R5gM plasmid 3 The KT-3B or KT-1 vector is constructed by partial ligation. This is the KT-1 skeleton A vector is provided that is referred to as having. Next, the pAFVXM retrovirus vector Of the 1.3 Kb ClaI-BstBI neo gene fragment into the ClaI site of this plasmid. Insertion in the sense direction yields a vector referred to as having a KT-3B backbone.                                 Example 3              Construction of recombinant retrovirus expressing factor VIII A.Construction of full length and B domain deleted factor VIII cDNA retroviral vectors   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. No. 08 / 366,851. Retrovirus vector package Due to signaling constraints and because the selection of transduced cells is not required for treatment, A retroviral backbone lacking a selectable marker gene (eg, KT-1) is used. 1.Construction of a plasmid vector encoding full-length factor VIII   The gene encoding full length factor VIII can be obtained from a variety of sources. One One such source for this is plasmid pCIS-F8 (see EP 0 260 148). And). This plasmid contains the full-length factor VIII cDNA and its expression is Under immediate control of the immediate early (CMV MIE) promoter and enhancer. No. V Factor III cDNA has approximately 80 bp of 5 'untranslated sequence from the factor VIII gene and approximately 500 bp. Includes 3 'untranslated region. In addition, between the CMV promoter and the factor VIII sequence, C There is an MV intron sequence, or "cis" element. This cis element The splice acceptor derived from the immunoglobulin gene spans approximately 280 bp. About 140 bp upstream of the splice donor site from the CMV major immediate early promoter Including.   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-F 8 is completely digested with ClaI and EagI (for these two sites are present ) To release the full length factor VIII-encoding fragment. Next, Fragment was ligated to a vector digested with NotI / ClaI and called pJW-2. Produces mid. 2.Construction of truncated factor VII retroviral vector (ND-5)   A coding sequence for a truncated form of about 80% (about 370 bp) of the 3 'untranslated region of Factor VIII cDNA. A rasmid vector (designated pND-5) is constructed with the pKT-1 vector as follows. You. As described for pJW-2, the XhoI restriction site of the pKT-1 vector used was NotI To the restriction site. Insert the Factor VIII insert with pCIS-F8 with ClaI and XbaI. Prepared by digestion. The latter enzyme cuts the factor VIII stop codon 5 '. I do. Next, an approximately 7 kb fragment containing all but the 3 'coding region of the factor VIII gene. Refine the fragment. pCIS-F8 was also digested with XbaI and PstI and The 121 bp fragment containing the gene termination codon is released. This fragment Again purified, and then the larger fragment encoding the rest of the factor VIII gene. ratagene, supra) and three-way ligation, termed pND-2 Make a plasmid.   The unique SmaI site in pND-2 was then replaced with a ClaI linker (New Engla nd Biolabs, Beverly, MA) to blunt ends generated by SmaI digestion. Change to ClaI site. After recircularization and ligation, a plasmid containing two ClaI sites A rasmid was identified and designated as pND-3.   Factor VIII sequence in pND-3 (linked by a ClaI site and of the 3 'untranslated region NotKT / ClaI digestion of pKT-1 as follows: Plasmid backbone (cut at the XhoI site, blunted with Klenow, and NotI linker (pKT-1 derivative by inserting a New England Biolabs) Make a loan. This plasmid backbone yields a 5.2 kb NotI / ClaI fragment. . pCIS-F8 is cut with EagI and EcoRV and the resulting approximately 4.2 kb fragment (Which encodes the 5 'portion of the full length factor VIII gene). pND-3 Is digested with EcoRV and ClaI, and the 3.1 kb fragment is isolated. Then These two fragments containing the part of the factor VIII gene were digested with NotI / ClaI. Ligation into the vector backbone produces a plasmid designated pND-5. 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 FVIII8 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 The aI fragment was removed from the retroviral vector JW-2 described above and modified as described above. Replace with the altered B domain deletion fragment. This is named B-del-1 .   Then, using the vectors described herein, the appropriate retrovirus Within a packaging cell line (preferably a human packaging cell line), Infectious, non-replicable recombinant retrovirus that incorporates integrase protein Produces irs particles.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 creating focus on (ATCC Accession No. CCL 64.1) Based on MiCl₁Cell line for transduction using mouse sarcoma virus (MSV) Thus, it is derived from the Mv1Lu mink cell line (ATCC Accession No. CCL 64). This is Containing the defective mouse sarcoma provirus (S⁺) But replicable mouse leukemia pro Virus free (L^-) Non-producer non-transformed revertant clone You. MiCl by replicable retrovirus₁Cell infection "activates" MSV genome To induce "transformation" that leads to focus formation.   Remove supernatant from cell line to be tested for the presence of replicable retrovirus And remove all cells by passing through a 0.45μ filter. On the first day, Mv1Lu Cells were plated at 1.0 × 10 5 in 2 ml DMEM, 10% FBS, and 8 μg / ml polybrene.^FiveCell / C Seed wells (1 well per sample to be tested) into 6-well plates You. For positive and negative controls, Mv1Lu cells were transferred to another 6 wells in the same manner. Lup Plate to rate. Cells at 37 ° C, 10% CO_TwoAnd incubate overnight. Two days In the eye, add 1.0 ml of the test supernatant to the Mv1Lu cells. Place the negative control plate in 1. Incubate with 0 ml medium. The positive control was MA virus (Mil ler et al., Molec. and Cell Biol. 5: 431, called pAM in 1985) Dilutions (200 focus forming units (ffu), 20 ffu, and 2ffu), which is added to the cells in the positive control wells. Cells overnight Incubate. 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 on day 6 And amplify any replicable retroviruses by splitting 1:10 on day 10 You. On day 13, the medium on the Mv1Lu cells is aspirated and 2.0 ml of DMEM and 10% FBS Is added to the cells. In addition, MiCl₁Cells were grown in 2.0 ml DMEM, 10% FBS, and 8 μg / ml 1.0 × 10 in polybrene^FiveSeed cells / well. On day 14, the Mv1Lu cells 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 added. Add to cells. On day 21, focus cells on a monolayer of cells (cover the monolayer , And appear as confluent refractory cells that remain attached). Pho Focus is MiCl₁If present on cells, the test article is contaminated with a replicable retrovirus Is determined to be. Using these procedures, HBV core producer cells It may indicate that the strain 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.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 example, retroviruses do not allow relatively close access to tissue cells. But retro ui The accessibility of the Lus vector to these stem cells has been demonstrated in animal models by Sandberg. , Revised by the in vivo mucus removal method of 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 container connected to syringe The catheter is inserted into this segment and 2 ml of the mucolytic, Thiothreitol and N-acetylcysteine are instilled under gentle pressure for 2 minutes And then remove. Repeat this procedure again, then⁶~Ten^Tencfu / ml 0.2-2. Fill the segment with 0 ml of retroviral vector particles. Ligation for 1-4 hours Remove later and suture the abdominal cavity. Inject control animals with prescription buffer only You.   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 is directed against human factor VIII. Based on two monoclonal antibodies (ESH4 and ESH8: American Diagonistica) Follow. ESH4 (1.0M NaHCO_Three/ 25Mg / ml in 0.5M NaCl (pH 9.0) at 4 ° C overnight Bind to ISA plate, wash with 0.1% Tween20 in PBS, and 1% BSA in PBS Block with. Samples were prepared using 0.05M Tris-HCl / 1M NaCl / 2% BSA (pH 7.5) For 4 hours at room temperature. Wash plate and add N-hydro ESH8 biotinylated with xysuccinimide biotin (Pierce, Rockford, IL) ( 2.5 μg / ml in 0.05 M Tris-HCl / 1 M NaCl / 2% BSA (pH 7.5) for 2 hours at room temperature During the addition. The color reaction is performed using peroxidase-conjugated streptavidin (Boehringer  Mannheim, Indianapolis, IN) and o-phenylenediamine disalt as substrate This is performed using an acid. Human factor VIII: c preparation (National Institute for Biologica l Standards and Control, from Hertfordshire, U.K.) and normal rat plasma Use as a control. 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. AU 9,230,801 and WO 92,14452.   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 4                Intracapsular administration of recombinant TK expressing retrovirusTK 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 flags (2.8 Kb and 1.0 Kb, respectively) (Armentano, J. Vir. 61: 1647, 1987) Eglitis, Science 230: 1395, 1985) and⁺(Stratagene , San Diego, CA) and pUC31 at the EcoRI site. pUC31 Additional restriction sites (XhoI, BglII, BsI) between the EcoRI and SacI sites of the relinker modification of pUC19 (Stratagene, San Diego, CA) harboring sHII and Ncol). You. Thereby, plasmid N2R3 +/-⁺Plasmid and 1.0 Kb 3 'LTR fragment It is made from the connection with the client. Plasmids p31N2R5 +/- and p31N2R3 +/- 2.8 Kb 5 'LTR and packaging signal (Y), or 1.0 Kb 3' LTR Constructed from the connection of fragment and pUC31. In each case, N2 is a vector Indicates the feed, R indicates the fact that the fragment is an EcoRI fragment , 5 and 3 indicate the 5 'LTR or 3' LTR, and + or-indicates the orientation of the insert (See FIGS. 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. Obtained The clone obtained 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. And Tn5 neo^r5 ′ fill-in ClaI / ClaI fragment containing the gene Constructed by blunt end ligation. Kanamycin resistant clones were isolated and 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.                                 Example 5         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 And this is introduced into the appropriate packaging cell line and Incorporates a chimeric integrase protein that confers site-specific integration into the system Recombinant retroviral particles can be produced.                                 Example 6                 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 prepared with 4-15% polyacrylic without pre-treatment. Load on amide gel and electrophorese at 250V for 35 minutes. The gel is then removed Coomassie to extract and detect viruses and other protein components Stain in blue. This gel is then used to determine the content of virus and other components Scanning 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 (Pharmacia, 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 LS5000TD Count with a scintillation counter (Beckman, Dallas, TX).   The invention has been described above, both generally and with respect to preferred embodiments. However, it is recognized that changes and modifications are possible to those skilled in the art in light of the above description. Is done. It is therefore intended that the appended claims be within the scope of the invention as claimed. It is intended to encompass all such changes.   Furthermore, to clarify the background of the invention, and in particular, the detailed description and To provide further details on its implementation, as described in the examples Publications and other materials cited in this specification are hereby incorporated by reference in their entirety. Be used as a helper.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // A61K 31/70 ABB A61K 39/21 ABN 38/43 ABE C12N 5/00 B 39/21 ABN A61K 37/48 ABE (72 Inventor Respeth, James G. United States of America 92109, San Diego, Lamont Street 4966 (72) Inventor Sandmeyer, Susan Be. United States of America 92625, Corona del Mar, Jasmine 226 (72) Inventor Jolly, Douglas J. United States of America 92024 , Encinitas, Hillcrest Drive 277 (72) Inventor Bilanchoon, Virginia W. USA 92714, Irvine, Sandown Scan 13 (72) inventor Dirudain, Sandra El. United States California 92677, Laguna Niguel, Toraura 26

Claims (1)

[Claims] 1. Directs integration of vector constructs into defined regions of the target eukaryotic genome A chimeric integrase protein. 2. To regions adjacent to eukaryotic genes transcribed by RNA polymerase III 2. The chimeric integrase of claim 1, wherein the chimeric integrase is directed to integration of a vector construct. protein. 3. The site specificity of integration is mediated by the domain from Ty3 integrase, The chimeric integrase protein according to claim 2. 4. A chimeric retrovirus integrase protein according to claim 3. 5. The chimeric retrovirus according to claim 4, which is derived from Moloney mouse leukemia virus. Ruth integrase protein. 6. The integrase has a domain A, a domain from the amino terminus to the carboxy terminus. Including main B and domain C, at least one domain has Ty3 integration The chimeric retroviral integrase tannin according to claim 5, which is derived from an enzyme. Park quality. 7. AmBmCt, AmBtCm, AmBtCt, AtBtCm, and selected from the group consisting of AtBmCm, Where "m" indicates the domain from MoMLV integrase and "t" is the Ty3 7. The chimeric retrovirus according to claim 6, which shows a domain derived from integrase. Integrase protein. 8. 2. The chimeric integrator of claim 1, wherein the chimera integrator is incorporated into a gene delivery vehicle. Wherein the gene delivery vehicle is a polypeptide, Heterologous inheritance selected from the group consisting of chisense RNA, sense RNA, and ribozyme A chimeric integrase protein further comprising a vector construct encoding a progeny product. Quality. 9. The chimeric integrase protein according to any one of claims 1 to 7 is encoded. At least one gene that is operatively linked to the gene to regulate expression of the gene. Vector constructs, including the elements. 10. A host cell into which the vector construct according to claim 9 has been introduced. 11. A method for producing a chimeric integrase protein, said method comprising: Under suitable nutrient conditions, transform the host cell of claim 10 into the chimeric integra Growing the protein in a manner that allows expression of the protease protein. 12. 2. The chimeric integrase protein of claim 1, which is isolated. 13. A packaging cell for producing a recombinant virus particle, comprising: Wherein the packaging cell comprises the chimeric integrase protein according to claim 1. Producing packaging cells. 14． (i) integration of the vector construct into a defined region of the target eukaryotic genome And (ii) the vector construct And a gene delivery vehicle. 15. (i) Regions adjacent to eukaryotic genes transcribed by RNA polymerase III Retro to direct integration of a recombinant retroviral vector construct into the region A viral integrase, and (ii) the recombinant retroviral vector construct. A transducible recombinant retroviral particle, comprising: 16. 16. The transducible recombinant retroviral particle of claim 15, wherein Transducible recombinant leto with live retroviral integrase protein B virus particle mediated integration of the recombinant retroviral vector construct Combining the recombinant retroviral vector construct into a eukaryotic genome as compared to Transducible recombination, leading to a reduced rate of insertional mutagenesis caused by Virus particles. 17． 16. The transducible recombinant retroviral particle of claim 15, wherein In transfected eukaryotic cells, wild-type retrovirus integrase tan Eukaryotes transduced with transducible recombinant retroviral particles with protein Comparing the expression of the gene of interest in the cells Transducable, leading to reduced changes in expression of the gene of interest carried by the construct Recombinant retroviral particles. 18. A gene delivery vehicle according to claim 14, and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising a rear. 19. 16. The transducible recombinant retroviral particle according to claim 15, and pharmaceutical. A pharmaceutical composition comprising a chemically acceptable carrier. 20. Pharmaceutical set comprising lyophilized transducible recombinant retroviral particles Adult. 21. A eukaryotic cell culture containing a vector construct integrated into a defined region. Nom. 22. A eukaryote wherein the defined region is transcribed by RNA polymerase III 22. The eukaryotic cell genome of claim 21, which is a region adjacent to the gene. 23. Pairs in the region adjacent to the gene transcribed by RNA polymerase III A transduced eukaryotic cell containing the recombinant retroviral vector construct. Here, the integration of the recombinant retroviral vector construct is performed by RNA polymerization. Said recombination into a region flanked by eukaryotic genes transcribed by Rase III Chimeric retroviral integrators for integration of roviral vector constructs Transduced eukaryotic cells mediated by the enzyme. 24. A method for introducing a vector construct into a eukaryotic cell genome, the method comprising: Integration of the vector construct by wild-type integrase protein Compared to the rate of insertional mutagenesis caused by the eukaryotic cell genome. The rate of insertional mutagenesis resulting from the integration of the vector construct is reduced, The vector construct uses the chimeric integrase protein of claim 1. And introducing into the genome of the eukaryotic cell. 25. Methods for introducing vector constructs into defined regions of the eukaryotic cell genome Wherein the vector construct comprises a wild-type integrase protein Expression of a gene of interest in eukaryotic cells introduced using Derived from the vector construct in a eukaryotic cell into which the vector construct is introduced. A change in the expression of the gene of interest is reduced, and the method comprises claiming the vector construct. Item 1. The eukaryotic cell genome using the chimeric integrase protein according to item 1. A method comprising the steps of: 26. Genetic disease, cancer, infectious disease, degenerative disease, inflammatory disease, cardiovascular disease, And a method for treating a disease selected from the group consisting of an autoimmune disease, The method comprises integrating the vector construct into a defined region of the target eukaryotic genome. Administering a directed gene delivery vehicle to a patient in vivo. . 27. Genetic disease, cancer, infectious disease, degenerative disease, inflammatory disease, cardiovascular disease, And a method for treating a disease selected from the group consisting of an autoimmune disease, The method comprises integrating the vector construct into a defined region of the target eukaryotic genome. Administering ex vivo treated cells to a patient with a directed gene delivery vehicle A method comprising: 28. 27. The method of claim 26, wherein the ex vivo treated cells are autologous cells. The described method.