JPH07505773A - Gene therapy using targeted viral vectors - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] TECHNICAL FIELD This invention relates to a gene therapy method.

Gene therapy can be used in a wide range of applications by directly inducing therapeutic genes into the human body. It is an approach to treating disease. could be used with gene therapy Diseases include (1) cancer, heart disease, Alzheimer's disease, hypertension, and atherosclerosis. (2) acquired immunodeficiency syndrome (AI); DS) and viral infections such as herpes; (3) diabetes, hemophilia, These include genetic diseases, such as capsular fibrosis, and muscular dystrophy.

A recent method for inducing new genetic information into cells using ispitrt is cell fusion method, chromosome-mediated insertion method (chromosome-sedlated 1n sertion), microcell-mediated gene transfer - (sicro cell-mediated gene transfer), liposome D NA carriers (llposome DNA carriers), Sufuji Blast fusion, DNA-mediated gene transfer, microinjection, recombination Infection caused by RNA virus and recombinant DNA virus injection (Mart [n, J, C,, l984.110/, (”e//　l /for/1w, 59:3-10). However, these technologies are It is not commonly used. It has little effect when used on animals or humans ( , the introduced genes are unstable and may transfer unnecessary or unwanted genetic information. This is because the target specificity may be lost.

In one example, the preferred approach for human gene therapy is to Transplanting cells that have been changed into a patient into a patient (Rosenberg, et al. l,,198g,,,ReilI/7/] Thin'rije 323: 570-57 8). This approach is used to isolate target cells from non-target cells. , cells must be surgically removed from each patient. genes are viruses This genetic vector is then introduced into the cell via a genetic vector or other means. The transformed cells are then reimplanted into the patient. This approach (e.g. For transplantation into patients of cells that secrete hormones that the cells can no longer secrete. Although it is good for purposes such as enzyme replacement therapy, it is useful for treating diseased cells such as cystic fibrosis and cancer. Transplant strategies are unsuitable for dealing with diseases that must themselves be corrected. Probably. Other problems commonly encountered with this approach are that transduction of stem cells is Select cells that are inefficient, have low expression of metastatic genes, and are inherently prone to becoming cancerous. It is a technical problem that requires growing cells in tissue culture. last In fact, inappropriate expression of the transplanted gene in non-target cells may actually be harmful to the patient. That would be harmful.

Another approach to gene therapy is to directly transfer the gene to the target tissue at 1lsjltt. This includes direct introduction. jty 5iru for gene introduction Two methods (biological transfer method (Blolistlc transfer) and dub The balloon catheterization method) has been developed. The biological transfer method of genes is DN It involves driving a small platinum or gold bullet coated with A directly into the target tissue. nothing. Biological transfer methods involve the use of ears, skin, and surgically exposed living mice. We have succeeded in transiently expressing genes in the liver (Johnsto n, S.A., 1990. Jk/yrg376:77B-777;Witfl aws, R, S, et al, +1991. +c Nil/It, tt f Sd 113181++2726-2730) A double Balloon catheterization allows genetic transduction in cells of well-circumscribed arterial wall sections. Introduce. In this approach, the distal end of the catheter reaches the target area. Insert a double balloon catheter into the artery until at the end of this catheter When the two existing balloons are inflated, a closed space is created, in which the retro virus is placed. Inject either DNA-loaded liposomes or DNA-loaded liposomes. This method is used in pig ileum. Transient expression of the β-galactosidase gene in well-defined sections of the femoral artery (Nabel E, G, et al, 1990.sc/ etity 249:12g5-1288), however, biological metastases Although both catheterization and double-balloon catheterization are location-specific, It must be non-specific in the individual cells transduced, and the transduced gene is the target. When expressed in cells that are not arise. Furthermore, both of these methods are effective in treating the lungs, muscles, tumors, or systemic circulatory system. It does not appear to be effective in processing tissues that occupy large volumes such as cells. child Is this not possible to subject the majority of cells to //75ittt gene transfer? It is et al.

The third approach to gene therapy is to induce genes into cells with //7 yjyo. It is to guide. This approach can be done by injection method, spray method or involves introducing the viral vector directly into the patient by other means. many kinds A virus is engineered to introduce genes into cells that the virus normally infects.

For example, adenoviruses (which normally infect lung cells) carry genes that target lung cells. It has been developed as a child vector (Rosenfleld, et al., 1 992. rg//88143-155). However, most virus vectors Since vectors can only induce genes into specific cells, vectors designed to achieve a single purpose are - becomes. The target cell specificity of a viral vector depends on the normal tropism of the virus. Viral vectors generally target a wide range of cell types because of their limited Either it infects widely, or it doesn't infect certain types of cells at all. Limited.

Liposomes deliver genes or drugs to specific target cells at //7y/π. Designed to guide. Chemically conjugate antibodies or ligands to liposomes By doing so, the liposomes are targeted to specific cells. This method smells By using anti-CD3-conjugated liposomes, antisense~e 17V-RNA is attracted to lymphocytes infected with human immunodeficiency virus (HIV). (Rennefsen, et al., 1990.) 17 /c/l’kz 265:18337-16342), chloramphenicol The transacetylase (CAT) gene is linked to anti-H2Kk-conjugated liposomes. H2Kk-positive H2-negative nude mice CWang, C. et al., 1987, which is induced by lymphomas. /)/-or #// lea/Scj 1ls184 Near 851-71155 ). And xanthine guanine phosphoribosyltransferase ( XGPRT) gene using Staphylococcal protein A-conjugated liposomes. and is induced in immunoglobulin-coated cells (Machy P , , et al, , 19118. Proc, hl/1est/S r/l/3185:8027-80R1), this The main drawback of the technology is the expense of producing large quantities of ligand-conjugated liposomes. It is.

Wu et al. report a method for targeting naked DNA to specific cells. Asia Loglycoprotein DNA1! Coalescence expresses asialoglycoprotein receptors (Wu G, Y., et al., 1991) i! t’of7er, gpy3:87-95).

Similar problems are encountered with immunotoxins, but in most cases this strategy is Therefore, a receptor that can internalize DNA DNA can only be induced into cells that express the body.

Antisense DNA technology suppresses the expression of specific genes using complementary DNA. CMOffaL, 1991. Scl'ence 253:51G- 511) 6 However, antisense DNA is specific for the cell type into which it is introduced, but non-specific for the type of cell into which it is introduced. Ru. This creates a problem in /77t410. Because in normal cells Endogenous genes can be shadowed by antisense DNA (e.g. proto-oncogenes). This is because it is desirable to be in a state where no noise is received. Furthermore, antisense DNA The manufacturing and administration costs would be high. It uses antiserum to pass through the plasma membrane. Half of the phosphate esters in the DNA have to be chemically modified, so This is because expensive organic chemistry cannot be avoided. to get the effect When antisense DNA is concentrated to millimolar order, /ly　r It may also suffer from toxicity problems at t″Vθ.

Human gene therapy is limited by the technology required to induce the gene. transplant The strategy (which requires surgery) is to provide expensive services for a small number of diseases. Gene therapy can only be carried out in the industry. //7 Perform positional transduction with s/lu Targeting genes by doing so is generally not precise enough. Wi The virus vector induces gene induction in lγP/yo, which the virus is normally sensitive to. Restrict staining to cells. Liposome technology is not viable due to high production costs. be. In a simple ligand-DNA complex, the receptor to which the ligand is directed is Genes cannot be introduced into cells unless they are internalized within cells. like this In addition, recent useful gene induction systems have expanded the range of diseases that can be treated with gene therapy. It is extremely narrowly limited.

Disclosure of invention The present invention is a method for expressing a nucleic acid to be expressed in a heterologous host cell. Features. This method requires 1) a nucleic acid to be expressed, and 2) a hybrid envelope. The present invention includes providing a virus having a rope gene in its chromosome. The hybrid The coded gene encodes an envelope fragment combined with a targeting ligand. do. This allows the envelope fragment to be recognized by its normal host cell. or enable effective incorporation of the virus into virus mature particles to facilitate binding. Make it g. This also allows the targeting ligand to target the foreign host. Facilitates targeting and binding of mature virus particles to the surface of host cells. how to do that involves administering the virus so that the virus can infect the cell.

Here, "effective incorporation" means that the hybrid envelope protein is The corresponding wild-type envelope protein is added to the mature virus particle. means that it is incorporated at least 25% of the time Ru.

In another aspect, the invention provides a virus, its hybrid envelope protein. It is characterized by a genome encoding. Here, the hybrid protein is Contains an envelope fragment bound to a targeting ligand in a molecule.

This ensures that this envelope fragment is also recognized by its normal host cell. The hybrid envelope protein does not facilitate binding or binding. Facilitates effective incorporation into mature virus particles. Also, this , non-viral proteins are normally sensitive to mature virus particles depending on the virus. The product targets and binds to the surface of unstained cells.

In a third aspect, the present invention provides methods for inducing a nucleic acid to be expressed into a heterologous host cell. It is characterized by being a method for This method consists of 1) the nucleic acid to be expressed and 2) Hybrid envelope gene (this hybrid gene is a targeting a virus containing an envelope fragment (encoding an envelope fragment bound to a ligand) including providing. This allows the envelope fragment to maintain its normal does not facilitate recognition or binding to host cells and does not allow virus development into mature virus particles. facilitate the effective incorporation of Additionally, this allows the target sotengrid to Gands allow targeting and binding of mature virus particles to the surface of the foreign host cell. make it easier This method involves administering a virus to infect cells. including

In various preferred embodiments, the virus is an enveloped virus. preferably Herpetoviridae, Baaramyxoviridae, or Retroviridae, most preferably Moloney murine leukemia virus; is Hepadnaviridae, Poxviridae, or Iridaviidae. Rusaceae may also be preferred as viruses. Similarly, as a virus, Togawi Rusufamily, Rhabdoviridae, Coronaviridae, Rhabdoviridae, Fil.

viridae, Orthomyxoviridae, Bunyaviridae, or other It may also be a virus with an envelope that has not yet been classified.

In various other preferred embodiments, the nucleic acid to be expressed may be limited to antisense oncogenes, tumor suppressor genes (e.g., the gene encoding p53) or the gene encoding the retinoblastoma protein Rb), toxin gene ( For example, diphtheria toxin gene) or a gene encoding a cytokine (e.g. tumor necrosis factor or contains interferon). The nucleic acid to be expressed may be DNA or RNA. . For example, antisense DNA or antisense RNA, or antisense It may also be a nucleic acid encoding RNA. The nucleic acid you want to express is one that confers intracellular immunity. genes or nucleic acids for the treatment of genetic diseases (e.g. insulin genes or cystic fibrosis transmembrane regulator gene (cystic fibrosis transmembrane regulator gene) BROSTS TRANSMEMBRANE REGULATOR GENE) ) is also fine. “Genes that confer intracellular immunity” are dominant negative resistance genes. A gene that confers a phenotype in a cell that causes the cell to be immersed in drugs. be protected from.

the xenogeneic host cell is a cell that has a pre-existing mutation that results in a disease state; Preferably, it may be a cancer cell (eg, a colon cancer cell). This heterologous host cell infected with a virus (e.g. human immunodeficiency virus (HIV)) Even if the infection is caused by a living organism, it may be infected with bacteria or parasites. It may also be a chromogen. This infectious agent may be unicellular or multicellular. heterologous host cells Also, cells affected by genetic diseases (e.g. pancreatic beta cells or lung cells) bacteria, preferably hormones or immunoglobulins, more preferably antibodies an anti-tumor immunoglobulin, most preferably an anti-carcinoembryonic antigen antibody specifically bound to Foreign immunoglobulin or anti-HIV gp120 antibody-specific immunoglobulin can be included. The targeting ligand may be a carbohydrate or a lipid. It may be. The hybrid envelope fragment has receptor binding. synthesis region, oligomerization region, transmembrane region, virus budding region, sorting signal, symbol It may be composed of a signal sequence and preferably a fusion region. in many cases , the fusion activity of the envelope fragment can also be enhanced by a second protein. stomach.

Therefore, the second protein would fuse the virus with the outer coat of the target cell. cormorant.

Methods of administration include 1) direct injection of purified virus, or 2) direct injection of virus-containing This may include, but is not limited to, implantation of a container into a patient.

When the virus is administered in a container, the virus is inside the packaging cell. It is preferable that the “Packaging cells” are cells that contain viral vectors. A cell that supplies virus proteins necessary for production. "Container" means virus-permeable virus inclusions or packages in which the virus is present; Virus-permeable inclusions that contain bacterial cells.

As used herein, "normal host cell" refers to a naturally occurring virus. Refers to the cell type that is more commonly infected. In contrast, “heterologous host cells” or "Targeted cells" as used herein refers to hybrid enveloped cells. Although recognized as a function of the targeting ligand moiety of proteins, Not recognized as a function of the envelope portion of the lid envelope protein Refers to cells. "Targeting ligand" refers to a desired cell that is targeted. A molecule that binds with affinity to molecules on the surface of "Hybrid envelope" In this specification, “protein” refers to a protein that is covalently bonded to a targeting ligand. a viral envelope protein (or biologically active analogue thereof) that A protein that contains a portion of For example, hybrid immunoglobulin-e r-protein is an immunoglobulin that covalently binds to parts of envelope proteins. means the part of “Hybrid envelope gene” is a hybrid envelope gene. A nucleic acid that provides genetic instructions to envelope proteins. “Hybrid anti-cancer embryo ``antigen antibody-specific immunoglobulin'' refers to hybrid immunoglobulin-e A V protein that specifically binds to carcinoembryonic antigen antibodies.

The word "fragment" is also used as envelope fragment. Sea urchins contain some, but not all, envelope proteins.

Fragments usually have at least about 20 amino acids, typically at least also about 30 amino acids, usually at least about 40 contiguous amino acids, preferably is at least about 50 amino acids, and most preferably at least about 60 to Consists of about 80 or more contiguous amino acids. envelope protein Fragments can be constructed using conventional methods (e.g., these are described herein) by those skilled in the art. It can be made according to

Biologically active viral envelope protein fragments are shown below. It has any one of the following activities.

a) Can bind to the cell envelope given the appropriate targeting ligand.

b) May allow fusion with the cell membrane. or C) proteins into mature virus particles. may enable the incorporation of quality.

These three biological activities are associated with several envelope protein fragments. Alternatively, this can be achieved by separating the envelope protein fragment into two parts. I can do it. As mentioned above, the envelope fragment according to the present invention does not facilitate recognition or binding of normal host cells by the virus. this mutations that abolish the activity of the normal Sebuter-binding region or This is achieved by physically removing the Newly recombined or setter A binding site is added at that location. Biology of viral envelope proteins The ability of candidate fragments to exhibit activity can be assessed by methods well known to those skilled in the art. can be done.

Envelope fragments are amino acid sequences of naturally occurring viral envelopes. sequences and biologically active analogs thereof.

The biological activity of envelope analogs is determined by the envelope analog as described herein. It is evaluated by a method that tests the activity of envelope fragments.

Applicant has identified specific animal, plant or human cell types or infectious agents. Effect of specifically inducing therapeutic genes or antisense nucleic acids to cells provides accurate and reliable means. These methods are useful for mutagenic and acquired The products must be handled with infectious or genetic diseases. For example, it is This is due to either. 1) Neutralize the effects present on cellular genes, 2) supplement existing defects in cellular genes, and 3) introduce new genetic material. or 4) introducing new genetic material. changes the phenotype of target cells. cause specific interactions with specific target cells hybrid envelope protein (e.g., envelope antibody or envelope protein) (velope-ligand hybrid) is used to guide specific target cells. It will be done. The virus itself, through its efficient internalization mechanism, Perform effective internalization of the product. Such viral vectors carry genes, RNA or adapted to deliver the drug to cell surface proteins that do not normally internalize it.

Another advantage of the present invention is that it overcomes gene regulation problems encountered with other gene therapies. That is. Genetically selected cells are placed in the right place, at the right time, and at the right time. Not only must the gene product be synthesized in the correct amount, but it must also be synthesized in the same amount as in the native tissue. must be adjusted according to the method. Therefore, transduced cells have extracellular must have all the inherent signal transduction machinery to respond to the signals of Must be. This would be a problem in gene therapy for diabetes. for example, Depending on where the fibroblasts containing the insulin gene are transplanted, there may or may not be any effect. or even potentially harmful. Fibroblasts live in the same lysate as beta cells in the pancreas. Since pigs do not have the signal transduction mechanism, the insulin gene is expressed. It would be difficult to do so. The target gene for the correct cell is ensure that it is adjusted accordingly.

In a further aspect of the invention, a virus comprising hybrid envelope protein A selection plan was drawn up to create Rus. This strategy is based on immunoglobulin young or any ligand and any protein that recognizes a specific receptor on the cell. It would be possible to run to fuse.

Other features and advantages of the invention will be apparent from the following preferred embodiments and from the claims. It will be.

detailed description First, a brief explanation of the drawing will be given.

drawing Figure 1 shows the construction plan for the retroviral vector pLNCX.

Figure 2 shows the construction plan for plasmid LNCenvpA.

Figure 3 shows the construction plan for plasmid LNCetiv.

Figure 4 shows the construction plan for plasmid pUCStar-3ig.

Figure 5 shows the construction plan for plasmid LNG-3ig.

FIG. 6 shows the construction plan of plasmid LNG-7/7/;CEA.

Figure 7 shows the plasmid used to construct the targeted virus. Ru.

Figure 8 shows hybrid immunoglobulin-1''/ A plan for producing the targeted retrovirus, including the construction of the 7V gene. It is.

Figure 9. Targeted Retroviruses Containing Generation of Pooled Virus Constructs This shows the generation strategy.

Figure 10 shows the targeted retroviral process, including selection and identification of targeted viruses. This shows the generation strategy of russ.

Figure 11 shows a plasmid containing a targeted retroviral vector. be.

Figure 12 shows the construction plan for plasmid pUC5tar-anticEA. be.

Figure 13 shows another construction plan for plasmid LNC-ant tcEA. be.

Below is a demonstration of the gene expression of targeted cells using targeted viral vectors. This is a method of guiding children. Viral envelope to create and target viruses The receptor recognition region of the protein binds to a ligand for a specific cell surface receptor. Replaced. The hybrid envelope protein protects the viral envelope during the germination process. integrated into the lobe and generates a hybrid virus in l’n VI’Vθ do. When infecting a host, hybrid viruses specifically recognize target cells.

The fusion with the cell then allows the internalization of the viral genes (into the target cell). For simplicity, this gene contains a therapeutic gene introduced into the viral genome. is included. Such internalization will be extremely important. For example, immunotoxins Despite being effective in directing toxin molecules to target cells, The surface molecules of the vesicles are not internalized and this internalization is necessary for the toxin molecules to enter the cell. Therefore, immunotoxins are often clinically ineffective (Waldsann, T. , 1991, 5science 252, 1657-1622). Targeted cormorant The virus itself has the necessary cell fusion machinery, so the virus can avoid the need for internalization (Gllbert, J. M., eLal, 199 0. J Vlrol 64:5106-5113; Rolzman B, et al., 19900. In BN Field Katana et al.

Generally, the target virus specifies the receptor recognition region of the viral envelope protein. composed by replacing the ligand directed to the cell surface receptor of . Ligands include, but are not limited to, immunoglobulins (e.g., FAb, dA bSFd, or FC), hormones, or target viruses on the cell surface. It can be any other synthetic or natural protein that can be directed to the molecule.

Ligands are involved in virus assembly and germination in the normal viral envelope Biologically integrated into the viral envelope by genetic fusion with a protein moiety It is rare. The envelope part of the hybrid protein is envelope 11 sufficient enzymes to guide efficient incorporation of the target protein into the viral envelope. Consists of a fragment of a envelope (or something similar). envelope hybrin It is desired that the protein portion does not lead to interaction between the virus and its normal host cell. Delicious.

As previously demonstrated, altering the receptor specificity of the viral envelope This changes the affinity of the virus. For example, if the virus envelope is retroviral, The vesicular stomatitis virus (VSV) busdotype, which has been replaced by that of the acquisition of the ability to infect infectious cells (Schnltzer ■ T, J, et al, 1977, J Gen l/Irol 23:4 49-454; Zavada, J., et at., 1972.　J　 Mr. Ge@Vlrol 15 :183-191), the species-specific relationship between the core protein and envelope protein of the virus. protein content interactions are important for viral fusion and entry, at least in these cases. This is shown to be inconclusive. Furthermore, previous experiments also showed that virus envelopes Ropes are lengths induced within envelope proteins by e.g. conjugated ligands. and conformational changes. For example, Glt■an et al, is a viral envelope glycoprotein chemically cross-linked to anti-erythrocyte antibodies. Reconstituted Sendai virus envelope strips away normal virus receptors It has been shown that the cells acquire the ability to bind to red blood cells that have been removed. Similarly, insulin Sendai virus envelope reconstituted with envelope protein chemically cross-linked with molecules. The rope expresses insulin receptors and can bind to receptor-removed red blood cells. did it. In both cases, envelope binding with receptor-depleted red blood cells occurs. However, no fusion occurred. However, between the bound envelope and the red blood cell The fusion occurred because the bound envelope was normal hemagglutinin/neurami This was when the core was formed by Nidase and the fusion protein (Gitsan, ^, G , et al., 1985. Biochem 24:2762-2788 ).

Preferably, the target virus comprises: 1) derived from the host cell membrane; Viral Envelope: 2) A mechanism that directs virus binding and entry to specific target cells. membrane hybrid envelope protein; 3) cell membrane of target cell for virus entry; transmembrane envelope proteins that lead to fusion of the target virus with (e.g., the target protein itself) or other envelope protein); 4) Virus core protein; 5) Target foreign gene - and 6) all necessary for the introduction and expression of the genes contained in the viral genome. Viral and genetic components. Transmembrane 11 hybrid envelope proteins are: 1) the hybrid protein is processed and imported into the viral envelope; 2) a determinant that enables the fusion of the viral envelope with the target cell membrane; enabling determinants, these are essential for target virus entry; 3) target virus Ligand determination that allows cells to recognize and bind to specific receptors on target cells. Teiko. The viral genome contains bacterial selection markers (e.g. ampicillin resistance) and and/or mammalian cell selection markers (e.g., nemomycin resistance).

The transmembrane hybrid protein connects the cell surface receptor binding region of the ligand gene to the virus. Genetically constructed by joining part of the envelope protein gene . Transmembrane hybrid proteins are capable of effective post-translational processing (post-1 translational processing). onal prOCeSSing), classification and protein viral envelope The part of the envelope protein that directs import into the cell must be retained.

The following areas must be taken into account in hybrid ligand envelope construction: Must be... 1. Receptor binding region The receptor binding region is an envelope that recognizes and binds to cell surface receptors. It is a type of protein. In the hybrid envelope protein, the envelope protein This protein in white is replaced by the Gantt sequence. retrovirus envelope The receptor-binding region of the protein was restricted to the SU subunit (Coin, J., M. ,, 1990, In BN Fields, at al,, eds, V [rology, Raven Press, Ltd, New xork ).

Since the retrovirus SU protein is coded 5' to the transmembrane protein, Replacement of the amino-terminal sequence of the envelope protein with the ligand sequence creates a functional hive. There are no problems in producing lidligand envelope proteins.

2. Proteolytic cleavage site Retroviral envelope proteins form SU and TM heterodimers Therefore, it is synthesized as a protein that is later proteolytically cleaved. l) Ibri Sodrigande Due to the construction of envelope proteins, protein cleavage sites must be removed. protein The cleavage site must be removed by deletion or site-directed mutagenesis. No. Perez and 1lunter are made by removing protein cleavage sites. demonstrated that it does not block the migration or surface expression of tumor virus envelope proteins. (Perez, L, G.

The envelope proteins of many animal viruses combine to form trimers (Fie fds, B, N, et at, 1991. Vlrology, 2 nd ed, , Raven Press, Ltd, , N ■ Bath@York) .

Trimerization of envelope proteins occurs when envelope proteins enter the viral envelope. considered essential for proper movement and immigration (Slngh, J. et al. 　1990. Ecbo J 2:631-639: Krejs T, E,... et al, 1986. Cel 46:929-937), therefore , it is important that this region is retained within the hybrid-ligand envelope protein. It is. The trimerization region appears to be within the transmembrane TM protein of retroviruses (Eln feld, D, etal, 1911g, Proc Natl Ac ad Scl USA 85:86gg-8692); Therefore, SU subunit Create a functional hybrid ligand-enveloped retroviral protein that lacks It is a powerful ability to put out. Some viral envelope proteins are made into low polymers and have 3 amounts. will form stoichiometric combinations other than bodies.

4. Fusion area The fusion region is a hydrophobic stretch of amino acids involved in the fusion of the viral envelope and cell membrane. Nagabe (Vlley, D, C, et at, 1990. In F ields, B, N, et al,, eds, V■ rology, 2nd ed, Raven Press, t, td,, NeV York) o Virus fusion is when the virus core protein and genome enter the cell. make it possible to In influenza virus, envelope HA2 protein The fusion region located at the amino terminus of The hemagglutinin is sequestered within the hemagglutinin trimer until it is capable of being presented to the cell membrane. envelope The trimerization of the peptide proteins constitutes fusion activity by sequestering them within an internal hydrophobic sac. It is possible to prevent the development of symptoms. The potential fusion region is gp3 of Rous' sarcoma virus. Attached within the extracellular portion of the 71M protein (Hunter E, et, at ,, 1983. J Virol 46:920-936), similar hydrophobic The sex fusion sequence is Mol.

Also reported in the p15E protein of Mr. Ney's mouse leukemia virus o-MuLV. (Chasbers, P., et al., 1990. J. Gen Vlrol 71:3075-30110).

When constructing a hybrid ligand envelope protein, the infection activity of the target virus is It may be necessary to remove constitutive fusion activity, a condition that may impair Not possible. Therefore, targeting two proteins within the viral envelope of the virus. It may be transferred to The first protein directs the targeting of the virus / 1 Evelynd Although it is a ligand envelope protein, it lacks fusion activity. The second protein is It is an envelope protein that has synthetic activity but lacks a receptor binding region. child A situation like this occurs when one envelope protein is dedicated to targeting while the other carries out the fusion. found in paramyxoviruses (Klngsbury, D. 1990. B, N, Piled When there is no need to block fusion activity, both Containing activity within one protein allows the transmembrane region to bind the envelope protein to the viral envelope. It is a stretch of approximately 20 or more amino acids that anchors the envelope. That's Mo Located within the p15E protein of Mr. Loney's murine leukemia virus (Chabe rs, et at, supra). Preservation of the transmembrane area is considered essential; Synthetic envelope proteins are secreted as a result of deletion of the transmembrane region. The amino acid sequence contains the exclusion of viral envelope proteins within the viral envelope. Infection and virus germination may be involved.

The virus budding region inserts the hybrid envelope protein into the virus envelope. turn towards These sequences are located in the inward-facing envelope protein of the virus. special protein content interactions between envelope proteins and virus core or substrate proteins. It may be accompanied by Perez et al, Rous sarcoma virus env. Deletion of the carboxy-terminal sequence of the protein results in normal germination of the mutant virus. (Perez, L.G., et al., 1987. J.V. lrol 61:29111-29118), other U.S. For viruses, the interaction between the envelope proteins and the virus core is May lead to assembly and envelopes (BN Fields, et al. al,, eds classification signals are subjected to post-translation processing (post-translation directing envelope proteins to fixed intercellular locations during It is a determinant that These sequences indicate that the envelope protein is located in the cytoplasmic network and Golgi. through the body and other organelles, ultimately reaching the viral envelope. guaranteed. Other information that should be retained in the hybrid-ligand envelope protein It is involved in the glycosylation sequence and efficient conformation of envelope proteins. There are arrays (e.g., binary combinations).

8. Signal array (signal array) The signal sequence is an amino-terminal chain of amino acids that directs the envelope protein into the cytoplasmic retinal structure. It is an aqueous extension. The signal sequence that is later cleaved by proteolysis is a hybrid Essential for locating the ligand envelope proteins within the membrane.

Due to the variety of signals and regions that must be considered in the construction of the target virus, the ligand It is necessary that precise and accurate joining of the envelope gene and the envelope gene occur. present invention A target virus is constructed in which the gant gene is spliced to an envelope gene fragment. describe a selection scheme for the hybrid gene to Efficiently import hybrid envelope ligand proteins into mature virus particles The code designates the envelope protein required to direct half of the protein. How to choose According to the formula, the cell surface receptor binding region of the ligand gene is located within the viral envelope. are randomly linked to stepwise deletions of multiple genes. Ligand and envelope sequence positive The exact combination is determined by the selection strategy for the generation of biologically active target viruses. determined. The selection method not only generates target viruses, but also generates future target viruses. Simplify the configuration.

Specific examples of targeted retroviruses Targeting specific host cells for the purpose of delivering the desired therapeutic gene to the cells Examples of recombinant retroviruses used for infection are listed below. This example illustrates the invention. These are listed for clarity and are not intended to be limiting.

Moloney murine leukemia virus (No-MuLV) is a mouse ecotype. It's a retrovirus. Recombinant No-MuLV system targeting colon cancer cells A torovirus vector is constructed. The target retroviral vector is neomycin The tumor resistance gene is provided to colon cancer cells. Targeting human colon cancer cells Hybrid immunoglobulins directed against carcinoembryonic antigens within the virus envelope This is achieved by populating the envl white. Carcinoembryonic antigen (CEA) is human associated with tumors, expressed on the surface of colon cancer cells, but not on the surface of normal adult cells. It is an antigen. CEA glycoprotein, which has numerous membrane-crosslinked alpha helices, is sensitive to ligands. (Benchlmol, S., et al. + 1989) ．． Ce1l 57:327-334), a protein homologous to carcinoembryonic antigen is It has recently been shown to be a receptor for hepatitis viruses (Dveksler, G. .

S., et al., 1991. J Vlrol 65:6881-68 91).

For the purpose of this illustration, the single variable region of the anti-CEA heavy chain is part of the env gene. fused into. A single variable heavy chain fragment (dAb) combines the heavy and light chains in antigen binding. Proven to be as effective as fragmented antibodies (FAbs) containing both fragments (Ward, E. S., et al., Nature 341 :544-546) o However, the function of immunoglobulin-env protein is Not limited to the use of Abs, but also FAbs.

Can be applied with Fv and mAb.

Modification of retroviral vector LNCX LNCX is contained within plasmid pLNCX. based on retroviral vectors (Moloney murine leukemia virus) (Miller, ^, D,, et at,, 19g9. Bolec hnlques 1, 980-990). pLNCX contains the inserted Unique ll[ndlll and Clal sites, giant cell Wis. rus (CMV) promoter, polyadenylation site (pA), retrovirus RN Retroviral long terminal repeat (LTR) for A transcription and reverse transcription, neomycin The bacterial neomycin resistance gene ( Neo), bacterial origin of replication (Or), bacterial ampicillin resistance gene (Amp) and retroviral RNA filling sequence (A+). LNCX is shown in Figure 1. It has been modified to include the unique 5all site as shown in Figure 1. pLCNX is Linearized with Xbal, Xb of phagemid Bluescrlptll SK+ subcloned within the a1 site (Stratagene, LaJoll a, CA), - terminal strand DNA is generated and the unique Bst11 site of LNCX is Ligonucleotide 5'-GCAGAAGGTCGACCCAACG-3° ( within the 5all site by site-directed mutagenesis with SEQ ID NO: 1). be converted. BstF, site 11 is an extended filling signal of No-MuLV RNA. Positioned within the number (W+) (Bender, M, ^,, et at,, 1987. J Virol 81:1639-+649; Adam, M , ^,, ■Soot@al,.

198g, J Vlrol B2:31102-3806: Ar1enta no, D, et at, 1987. J Vlrogeki@61:1647 − 1650). Conversion of the BsLEII site to 5all does not affect filling, but it since it was determined that this region is not important for effective filling (Schv artzberg, P., et al., but it is not the 5all site. , BsLEI 1 site frequently occurs within heavy chain genes (066-1070). plastic 5all containing sumid recirculates with Xbal and DNA ligase to make a positive Form pLNCX*.

Cloning of No-MuLV e nv protein in pLNCX* No-MuLV The env gene is cloned into pt, Ncxs as shown in Figure 2. o No-MuLV e nv gene is 1, 9 k b 5 cal-Nh It is excised from plasmid p8゜2 as an el fragment (Shoemaker, C. , et al., 1980.　Proc　Na1l^cad　Aci　US A 77:3932-3936), 1. 9kb 5eal-Nhel fragment is the complete coding region for the p15E transmembrane protein and the coding region for the gp70 SU protein. Includes most of the area. The 5′-protruding end was digested with Sl-nuclease. and Hlndllll linker (5-CCAAGCTTGG-3'; SEQ ID NO. number = 2) is added. env gene is pLNCX* as H1ndlll fragment was cloned into the HIndl11 site of do. The orientation of the Hlndlll env fragment is similar to that of the cytomegalovirus (CMV) protein. It is the direction as it is transferred and expressed from the motor.

Modification of LNCenvpA to LNCenv LNCenvpA has an additional site Phagemid pBIuescripi It for controlled mutagenesis It is cloned as an XbaI fragment into SK+ (Fig. 3). enV encoding H1 The ndlll fragment interacts with the polyadenylation signal provided by the viral vector. contains a polyadenylation signal that may interfere with AAUAAA polyadeni Therefore, the oligonucleotide 5° -GTTTTCTTTTTATC- AAGAAA by site-directed mutagenesis at 3' (SEQ ID NO: 3) can be changed to The Bindl11 site located at the 3° end of the env gene is Ligonucleotide 5-CAAGCATGGCTTGCC-3' (SEQ ID NO: 4 ) is eliminated by site-directed mutation. Retrovirus containing env The vector is recycled by Xbal restriction and ligation to create the plasmid LNCe n Form v.

Molecular cloning of anti-CEA immunoglobulin gene Maturation of anti-CEA H chain gene The cDNA encoding the variable region domain is produced using the polymerase chain reaction (PCR). and cloned as an Xhol-8pel fragment using an RNA template. RNA was obtained from the spleen of a mouse immunized against purified carcinoembryonic antigen. Instead In addition, the RNA was isolated from a hybridoma cell line secreting monoclonal antibodies against CEA. For example, 1.116NS-3d (＾merlcan Type Cu1tu re　C.

11ectlon crllll (119) or CEA 66-E 3 CWa Gener, C., et al., 1983. J1+nuno1130: 2308-2315).

The following PCR primers were used for the mature H chain gene (Stratacyte, Inc. ) is hybridized for cDNA coding. Degenerate plastic The timer introduces the underlined Xho1 site.

5' AGGTGCAACTG Roe Ei 1. QTCTGG]F (from SEQ No: 8) □5’ AGGTCCAGCTG Loe a ring TCTGG 3’ ( From SEQ No.: 9) The following PCR primers coded the J region. 5pel and BstE11 hybridize to immunoglobulin heavy chain mRNA within Introduce the parts.

5' CTAlrT Kinuta sλπGAC weak com x GTGGTCCCTrGGCCCC A 3' (No. 10 from SEQ) Spar BstE engineering The amplified anti-CEA gene DNA was transferred to the immune ZAP H vector (Stratacy te, Inc.) as an Xhol-3pel fragment (Mu lilnax, R,1, et al, 1990, Proc Natl^ cad Act USA It7:11095-1t099). Immune ZAP H is behind the pe IB signal sequence and E is within the eoll immunoglobulin variable heavy chain fragment. Fixed to appear.

The procedure is carried out by expressing immunoglobulin light chain fragments in packed cell cell lines. The same could be done.

Identification of high affinity anti-CEA clones Clones expressing high affinity anti-CEA antibodies were identified by filter binding analysis. Ru. The anti-CEA phage library was constructed using <25! conjugated to CEA as described above. ] Screened by nitrocellulose plaque pulling with bovine serum albumin () fuse, W, D,, et al, + 1989.5science 2 4B:1275-1281) High and intermediate affinity anti-CEA clones were selected. , further manipulated.

Plasmid LNG - Constituent of immunoglobulin Plasmid LNC - Immunoglobulin ( In this example, LNC-anti-CEA, which codes for the anti-CEA immunoglobulin gene. ) are provided. LNG-immunoglobulin vector codes for an immunoglobulin peptide fused to an amino-terminal signal sequence. mature Some amino acids at the amino-terminal end of immunoglobulin peptides are modified by the PCR used to generate the library. Furthermore, LNC- The design of the anti-CEA plasmid resulted in the insertion of extra amino acids at the amino-terminal end.

These amino acid changes do not affect antigen binding because 1) the amino acid changes are conservative; 2) the affected amino acids are usually variable at that site; and 3) The affected amino acids have been shown not to be involved in antigen binding or antibody conformation. occurs within the framework of immunoglobulins (Relchman et al. , 1988 Nature 332:323-327). child For the same reason, cleavage of the signal sequence from the mature peptide is unaffected. Dew.

The strategy for creating LNG-immunoglobulin, the structure of which is introduced below, is as follows: It relies on the use of plasmid pU CStar-31g.

The immune signal sequence was modified to create pUCStar-81g as follows: It is cloned into the Pucl19 vector (Fig. 4). pUC is a polylinker - A phagemid containing the cloning site. Multiple cloning of pUc119 Insert the following polylinker into the Hlndlll and Xba1 sites of pUc119. A new restriction site is substituted by entering the restriction site.

HlndHI　P+str　Xhol　BcL　Spa　Not　(La X Xba15'-AGCTTCTGCAGGCTCGAGTGATCAACT AGTGCGGCCGCATCGATT-3' (From SEQ No: 11) The modified pUc119 is called pUC5tar-1 (Figure 4). restriction part positions may be restricted by small linkers if adjacent restriction sites interfere with each other during digestion. It is also possible to separate them further.

The signal sequence from the anti-NP immunoglobulin H chain gene is a 330 bp Pstl fragment. as plasmid pc DF L, 1 (Ucker, D, S,, etc. al, +1985. J Iginol 135:l404-4314) isolated from 330bp Pstl fragment is plasmid pUC3tar-31g is subcloned into pUC5tar-1 to purify it (Fig. 4).

The Pstl fragment is oriented such that the signal sequence is expressed.

A, LNG immunoglobulin via immunoexpression vector, plasmid LNG-3rg Composition of phosphorus LNCX* is transformed into a eukaryotic immunoglobulin expression vector (Figures 4 and 5). immunity Globulin H product signal sequence and Xhol-8pel cloning site is plasmid L PCR amplified immunoglobulin gene inserted behind the CMV promoter of NCX* Allows the expression of children. The conversion of LNCX* is as follows.

Immunoglobulin H product signal sequence is pU CStar-8dg to Hindl 1lindllll-Cla of LNCX* was recovered as a l-C1al restriction fragment. cloned into the l site. The resulting plasmid LNC-8ig is a retrow with immunoglobulin heavy chain signals under the control of the CMV promoter. contains a virus vector (Figure 5).

The anti-CEA gene from the immune ZAP library was then subcloned into LNG-3ig. ligated to form the plasmid LNC anti-CEA. This includes the signal array. Generate an anti-CEA variable heavy chain gene (Figure 6). The anti-CEA gene first appeared in the immune Z Extracted from AP phage DNA as Bluescrlpt SK-phagemid (see IalbdaZAP protocols, Stratagen e, Inc., La Jolla, CA) 6 anti-CEA gene is Xhol- Purified as an 8pel fragment and ligated to Xhol-Sps+restricted LNG-5ig. It will be done.

LNG-3ig contains three 5pel sites. Therefore, plasmid L To generate NC anti-CEA, the ligation mixture is neomycin-sensitive, ampicillin-sensitive neomycin-resistant and ampicillin-resistant transformants. selected. Plasmid LNC anti-CEA was used as a probe in Bluescript Neo using the 5pel-Xhol anti-CEA restriction fragment from SK-anti-CEA. Selected from mycin-resistant and ampicillin-resistant transformants. Use 5pel site This is because it depends on the available sites within the immune ZAP expression vector.

To simplify LNC anti-CEA construction, we immunized ZAPH with a unique Not1 site. into an expression vector and use the Not1 site instead of the 5pel site. I can do it.

B, Construction of LNC-immunoglobulin with plasmid pU CStar-81g Immune ZAP library to form the construction plasmid pUcstar-anti-CEA The anti-CEA gene from be converted into This generates an anti-CEA variable H chain gene containing a signal sequence (Fig. 12). The anti-CEA gene was immunized as a Bluescrpt SK-phagemid. Asbda ZAP prot is isolated from the ZAP phage DNA (see ocols, Stratagene, Inc., La Jolla, CA ), B■ uescript SK-anti-CEA double-stranded DNA is prepared and combined with Xhol and 5pel limited by. Xhol-3pel fragment containing anti-CEA was purified by electroelution and Xhol-3pel to create plasmid pUcstar-anti-CEA. The antigene ligated to the restricted pU CStar-31g (Figure 12) is a plasmid. pUC5t as 1lindllll-C1al to generate LNC anti-CEA ar anti-CEA to LNCX* (Figure 13). 1 nd containing anti-CEA The lll-Clal fragment was purified from pUC3tar anti-CEA by electroelution. Ru. Phosphotase-treated Hindll-Clal restricted LNCX* is an anti-LNC Combined with purified 11indlll-C1al anti-CEA fragment to produce CEA. ligated (Figure 13).

Strategies for generating targeted viruses The starting material for generating the target virus is the LNCe nv shown in Figure 7. LNC immunoglobulin (in this example, LNC anti-CEA) plasmid. Figure 8 ~11 illustrates the general principles of primary generation of target viruses. anti-carcinoma virus /X hybrid immunoglobulin-env protein targets cells expressing the generated. For envelope protein classification (S), trimerization (T), fusion (F) Since the location of important determinants for immunoglobulin genes is known with certainty, immunoglobulin genes linked to stepwise deletion of the v gene, resulting in functional immunoglobulin end/'ib1 selected.

Useful envelope fragments or analogs The envelope portion of the fusion protein is derived from the envelope fusion protein (from the production cell line). (in the germination of recombinant viruses) directs efficient transfer into the viral envelope. can consist of any portion of an envelope protein (or analogue thereof) sufficient to Wear. Such fragments or analogs can be used to identify the cell surface receptor binding domain of the ligand gene. Stepwise fPROGEsslVEl deletion of the main viral envelope gene can be determined using the general selection scheme described below, which generally involves ligation of the ligation.

Precise recombination of ligand and envelope allows for the production of biologically active target viruses. determined by the selection method for production. The selection method produces the target virus. In addition to simplifying the configuration of future target viruses.

In VILrO hybrid immunoglobulin-env gene constituent plasmid LNCe nv is the cryptographic designated area for No-MuLV env polyprotein (Figure 8). LNCe n v was initially linearized. A series of extended deletions within the env gene develop over time as exonuclei. Recover the Rease 111-treated DNA (7) aliquot and 5° with Sl-nuclease. -PROCESS I is created by removing the VE end (Guo, 1.tL , et al., 1983. Methods Enzy++ol 100 :60; and 5asbrook, J., er al., 1989. Mo 1ecular Cloning, Co1d Spring 1 (arb shichichi @ Lab oratory Press, Cod Spring 1labor) o Notl linker (5' AGCGGCCGCT 3' SEQ ID NO: 13) is blunt The sensitive end is ligated and restricted with Notl. As a result of this, the Not I limit is The 5'-boundary of each deletion within the env gene is prominent. No. on the other end of the molecule The Ll overhang is removed by 5all restriction of the reaction mixture. The reaction mixture is then heated Treatment with sulfodase prevents circulation of reaction products.

The reaction mixture contains 5alt- from LNC-anti-CEA containing the anti-CEA variable heavy chain gene. ligated to the Not1 restriction fragment. This allows a series of anti-env deletions to be fused to Creation of a pool of functional retroviral vectors encoding the CEA peptide be done.

Generation of stored virus structures The total reaction mixture from above was converted into ampicillin sensitive E, Co11, Picillin resistance is selected (Figure 9). Recombinant containing functional retroviral vectors The prototypes are selected because they are the only ones that contain the ampicillin resistance gene. It is from. Plasmid DNA is prepared from transformants grown in liquid culture and A collection of retroviral vectors containing immunoglobulin env fusion genes. produce.

DNA was transfected into the Clip2 retrovirus-filled cell line. Then, the DNA was transfected into a packed cell line that did not code for the wild-type env protein. be affected. Transfected filling cell lines are different hybrids Immune env protein and wild type env protein (env gene contained within the cell line) (cipher specified by the gene). transfected The filled cell line contains a hybrid immunoglobulin-env gene encoding variant. secretes an enveloped reservoir of retroviruses containing retroviral genomes. Ru.

Hybrid immunoglobulin-env proteins efficiently attach to the viral envelope. The hybrid-env protein retains all the determinants necessary for transfer. White can be imported into the virus envelope. By filling cell line The coded wild-type env protein is also imported into the viral envelope. this contains both wild-type and hybrid env proteins within the viral envelope. A virus is created. Therefore, this system will upload its gene product. Immunoglobulin-env hybrids that can be transferred into the virus envelope Select the option.

Virus pools are harvested from 0.45μ filtered media and contaminated with G418. -Remove resistant filling cells.

Target virus selection and characterization G418-sensitive target cells are exposed to viral reservoirs using standard procedures and 0418-resistant Sex cells are selected. The target cells are non-mouse cell lines (wild) that express carcinoembryonic antigens. type Mo-MuLV). For example, ATCCC 0LO205, a human cell line isolated from the ascites of a patient with colon cancer (A, T .

C, C, #CCL 222); LR-73CEA, mouse carcinoembryonic antigen gene. Transfected Chinese hamster ovary cells (Benchl of, S, et al.

, supra); and HCT48, a human colon adenocarcinoma cell line (Shl, Z, R, . et al, 11183, Cancer Res 43:4045-40 49).

G418-resistant cells are generated from transduction of the neomycin resistance gene by target cells. I just lost it. Therefore, this system is essential for virus targeting and fusion. Contains a hybrid immunoglobulin-env protein that retains all essential determinants Select recombinant virus.

Rescue of integrated immunoglobulin-env genes after infection with target virus As a result, the hybrid envelope gene that created the targeted protein was It will be done. The integrated hybrid immunoglobulin-env gene is Rescued from host DNA by polymerase chain reaction: PCR5’ rescue primer: 5'-GGTTq; λ included green CTGCTGAGGGGC-3' (No from SEQ :15) PCR amplification using these primers Generate an immunoglobulin-env gene bounded by one site. amplified DN A is restricted by Hindll and Xbal to create sticky edges and DN A is ligated into the old ndlll-Xbal cut LNCX. Clip 2 filling thin When transfected intravecally, this induces cell surface cancer embryonic antigens (e.g., colon cancer Generate retroviral vectors targeted to cells expressing the virus.

Retroviral vectors generated by the above selection method are generated in Clip2 packed cells. Human cells expressing CEA at birth (controlled by hybrid envelope protein) and normal mouse cells. A virus that infects only target cells To create viruses, retroviruses first use a hybrid envelope protein. It will be tested whether the transfer alone is sufficient to direct virus induction. this is wild Transfecting DNA into a modified packed cell line that does not code env type This is achieved by

Once it is determined that the fusion function is provided by the wild-type envelope protein, it is possible to A virus can be created as follows. env gene within the receptor binding domain. Create a filled cell line that code-specifies the mutation containing the gene. target virus vector When transfected with vector DNA, the hybrid ligand-env protein A target virus that expresses both a white and an env protein with a mutated binding site is generated. The virus will exclusively infect target cells.

The target virus vector is a universal vector, which is constructed using the above procedure. vector is a universally targeted vector (Figure 11). Targeting to other cells is Xh Xh code designating ol-3pel anti-CEA fragment as in-frame immunoglobulin ol-8pel fragments or can be directed to specific cell surface proteins or substituted for Gant. This is realized by For example, Xhol-3peI immunization from the immunization ZAP library The globulin-containing fragment is fused into the frame behind the signal sequence, as outlined above. and (, subcloned into LNCXs via the pUCStar-3ig plasmid can do. 5all-N from another LNG-immunoglobulin plasmid By substituting otl into the universal vector, another target viral vector Generate any encirclement as a vector for targeted delivery of therapeutic genes virus can be used. Specific examples include herpes slyrib Both NA and RNA viruses, e.g. herpes simplex 1 or 2, Paramyxola Iribu, Retrorairib, Hebadnaviride, Boxwillide, Iridrairib , Togarai Ribs, Frawiwiride, Corona Rai Ribs, Rabo Drai Ribs, Filo Lai Rib, Orthomixo Lai Rib, Bunya Willide or Arena Willide, also Other, unclassified, besieged viruses include:

A comprehensive selection of these viruses is, for example, American Type Cu. It can be obtained from 1ture Col 1ectlon.

targeting ligand Molecules that can direct specific interactions with target host cells (e.g. host cell (by specific recognition and binding of vesicle surface proteins) allows targeting of envelope fusion proteins. Can be used as a gund part. Preferably, such protein is a ligand:receptor pair. It originates from one side. Targeting ligands are not limited to proteins. The carbohydrate and fat parts are attached to envelope proteins via protein fragments containing common sequences for lycolylation and lipidation. can be done.

As shown in the example above, immunoglobulin gene can be used as a glue gun. . Genes for high-affinity immunoglobulins were conjugated to antigens as described above [ 12511 Lambda or Ba by filter binding analysis with bovine serum albumin (t(use, et al,, supra)) .

Cell surface molecules such as integrins, adhesion molecules or regression receptors are cell-specific triggers. They can be used as intercellular receptors through receptors on other cells. This is because they are involved in interaction. The gene that specifies these molecules is 5e. It can be identified by the panning method of ed and Aruffo (Seed, B, et al, 1911? .

Proc NaLl^cad Scl LISA 84:3365-3369) .

Hormones that bind to specific receptors can be used as targeting ligands as well as Viral proteins such as envelope protein gp120, as a modification of naturally occurring ligands Can be used.

therapeutic gene Therapeutic genes useful in the present invention include the following: 1) Therapeutic effect on cancer cells Some genes include a) antisense oncogenes; b) tumor suppressor genes such as p53. gene suppression or disruption of the retinoblastoma gene product Rb5c) such as the diphtheria toxin gene. necrotoxin genes; d) cytokines such as tumor necrosis factor or interferon; or e) other-cut therapeutic genes. 2) Persons infected with HIV Therapeutic genes targeted to cells. As a specific example, HIV such as polymerase antisense DNA that complements the essential gene for, and destruction of diphtheria toxin Necrotoxin genes: and HIV enhancer sequences that titrate and remove HIV regulatory proteins Genes that utilize intercellular immunity, such as (Baltimore, D., 198 8. Nature 335:395-396). 3) Genetic deletion correction gene.

Illustrative examples include, but are not limited to: insulin gene, or delivered to appropriate lung cells in cystic fibrosis patients. Examples include the cystic fibrosis transmembrane regulator (CFTR) gene. Target gene expression is Furthermore, tissue-specific enhancers that regulate transgenes − is realized through the use of

For the gene therapy described in this document, suitable recombinant viruses, such as those described above, are The drug is administered to a patient in a physiologically acceptable buffer (eg, physiological saline). therapeutic preparations is administered depending on the treatment condition. For example, in treating individuals infected with HIV, The virus is stabilized at doses that provide adequate targeting and lysis of HIV-infected host cells. Administered by direct injection, such as intramuscular injection, intramuscular injection, or intracellular injection. As an alternative, target virus to the appropriate target tissue externally or via catheter; or It may also be necessary to administer via video. The therapeutic agent may be e.g. a liquid or It may be convenient to administer orally, nasally, or topically in the form of a spray. here However, the appropriate dose is the amount of therapeutic virus that results in reduced disease. be.

Therapeutic viruses can also be administered by implanting virus-filled cells into patients. . Cells are confined in a semi-permeable container that allows the passage of e.g. viruses but not the filled cells. You can The embedded container may also be removable. with an alternative into the patient's veins so that the virus enters the patient via the needle or catheter. You can also hook it inside. In this way, patients can benefit from viral gene therapy. Continuous doses can be received.

Other implementations Other embodiments are limited by the claims below.

For example, a virus capable of replication may be used in some cases. replication deletion In other cases where virus was required, modified packed cells were administered to the patient rather than the target virus. Sometimes it's more effective. According to this method, a non-proliferating dose of recombinant virus is Once delivered to a localized area, the virus then locates specifically labeled cells. example For example, tumor-infiltrating lymphocytes (TIL) that surround cancer cells have a locally high concentration of cancer cells. can be modified to secrete cell-targeted viruses. Repeat treatment as needed can do. The immune response to the target virus can be overcome by immunosuppressants. Wear.

In addition to colon cancer cells, the viruses of the invention can be used to target other cancer cells, such as ovarian, breast, or Affected by lung cancer cells or genetic diseases such as muscular dystrophy or Huntington's disease It can also be used to target cells or cells that are deficient in adenosine diaminase. I can do it. Herpes slime virus is herpes simplex type 1 or 2, Eps Also included is Teln-Barr virus or cytomegalovirus. Sen Dai viruses and vaccination viruses can also be adapted to this method.

array list (1) General information.

(1) Applicant: Alenxander T. Young (1. Title of invention: Mark Gene therapy using targeted viral vectors (i　i　i) Number of arrays, 15 (iv) Corresponding address: (A) Addressee 2 Fish & Richardson (B) 4 Frank 225 Lynn Street (C) City; Boston (D) State: Massachusetts (E) Country/USA (F) Postal code: 02110-2804 (V) Computer readout format (A) Media format: 3.5 disks, 1.44 Mb (B) Computer: IBM PS/2 Model 50Z or 55SX (C) Operating system: MS-DO3 (version 5.0) (D) Software : Word Perfect (version 5゜(vi) current application data: (A) Application number: (B) Filing date.

(C) Classification: (vii) Priority application data: (A) Application number: 07/862,795 (B) Application date: April 3, 1992 (vii) Agent information (A) Name - Ball Tee Clark (B) Registration number: 30.162 (C) Reference number: 051401002002 (ix) Telecommunications information.

(A) Phone number: (617) 542-5070 (B) Fax number: (6 17) 542-8906 (C) Telex number: 200154 (2) Sequence number 1 information/ (1) Array characteristics・ (A) Sequence length: 19 (B) Sequence type: Nucleic acid (C) Number of chains: - terminal chain (D) Topology/linear (x) Sequence description: Sequence number 1: GCAGAAGGTCGACCCAACG 19(2) Information of SEQ ID NO. 2・ (1) Characteristics of array.

(A) Array length: 10 (B) Sequence type: Nucleic acid (C) Number of strands: 2 Double strands (D) Topology linear (xi) Sequence description: Sequence number 2: CCAAGCTTGG 10 (2) Information on sequence number 3: (i) Characteristics of array: (A) Array length = 13 (B) Sequence type: Nucleic acid (C) Number of chains: - terminal chain (D) Topology: Linear (xi) Sequence description: Sequence number 3: GTTTTCTTTT ATC13 (2) Information on sequence number 4: (1) Characteristics of array = (A) Sequence length; 15 (B) Sequence type: Nucleic acid (C) Number of chains: - terminal chain (D) Topology: Linear (xi) Sequence description: Sequence number 4: CAAGCATGGCTTGCC15 (2) Information on sequence number 5: (1) Characteristics of array: (A) Array length = 22 (B) Sequence type: Nucleic acid (C) Number of chains, -terminal chain (D) Topology: Linear (x i) Sequence description: Sequence number 5: CT GCTCGAG to AGGTGCA TCG GG 22 (2) Information on sequence number 6: (i) Characteristics of array: (A) Array length: 22 (B) Sequence type: Nucleic acid (C) Number of chains: - terminal chain (D) Topology: Linear (x i) Sequence description Sequence number 6: AGGTGCAACT GCTCGAG TCT GG 22 (2) Information on sequence number 7 (i) Characteristics of array (A) Array length 22 (B) Sequence type, nucleic acid (C) Number of chains: - terminal chain (D) Topology = linear (x i) Sequence description Sequence number 7: AGGTGCAGCT GCTCGAG TCT GG 22(2) Information on sequence number 8: (i) Characteristics of array (A) Array length: 22 (B) Sequence type: Nucleic acid (C) Number of chains: - terminal chain (D) Topology/linear (x i) Sequence description: SEQ ID NO: 8・AGGTGCAACT GCTCGAG TCT GG 22(2) Information on sequence number 9: (1) Characteristics of array: (A) Array length: 22 (B) Sequence type = Nucleic acid (C) Number of chains: - terminal chain (D) Topology: Linear (xi) Sequence description: Sequence number 9: AGGTCCAGCT GCTCGAGTCT GG 22 (2) SEQ ID NO: 10 Information: (1) Characteristics of array.

(A) Array length = 39 (B) Sequence type: Nucleic acid (C) Number of chains: - terminal chain (D) Topology: Linear (x) Sequence description: Sequence number 10: CTATTAACTA GTGACGGTTA CCGTGGTCCCTTGG CCCCA 39 (2) Information on sequence number 11・ (1) Characteristics of array: (A) Array length: 45 (B) Sequence type; Nucleic acid (C) Number of strands: 2 Double strands (D) Topology/linear (x) Sequence description Sequence number 11: AGCTTCTGCA GGCTCGAGTG ATCAACTAGTGCGGG CCGCAT CGATT 45 (2) Information on sequence number 12: (i) Characteristics of array: (A) Array length: 45 (B) Sequence type: Nucleic acid (C) Number of strands; double strands (D) Topology: Linear (x) Sequence description; Sequence number 12. AGACGTCCGA GCTCACTAGT TGATCACGCCGGCG TAGCTA AGATC45(2) Information on sequence number 13: (i) Characteristics of array: (A) Array length: 10 (B) Sequence type: Nucleic acid (C) Number of strands: double strands (D) Topology: Linear (x) Sequence description: Sequence number 13: AGCGGCCGCT 10 (2) Information on sequence number 14: (1) Characteristics of array: (A) Array length = 28 (B) Sequence type: Nucleic acid (C) Number of chains 2 - terminal chain (D) Topology: Linear (x) Sequence description: Sequence number 14: CCAGCCTCCG　CGGCCCCAAG　CTTCTGCA　28(2) Information on sequence number 15: (i) Characteristics of array: (A) Array length: 24 (B) Sequence type: Nucleic acid (C) Number of chains: - terminal chain (D) Topology; linear (x) Sequence description: Sequence number 15: GGTTCTCTA GAAAACTGCTG AGGGC24HirwlIll l Special Table Sen7-505773 (13) H1kaku111 FIG. 9 Targeted Retrovirus Production Strategy 11, Production of Pooled Viral Constructs Transformation of adult ampicillin-sensitive E. coll with ampicillin-resistant cells in liquid medium. FIG. 11 pUc Star-Sig Bluescript 5K-antiCEAFI G, 12 Continuation of front page (51) Int, C1,6 identification symbol Internal office reference number C12N 7101 (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE) , 0A (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN.

TD, TG), AT, AU, BB, BG, BR, CA.

CH, CZ, DE, DK, ES, FI, GB, HU, JP, KP, KR, L K, LU, MG, MN, MW, NL, No, NZ, PL, PT, RO , RU, SD, SE.

SK, UA, VN I

Claims (39)

[Claims] 1. A method for expressing a nucleic acid to be expressed in a heterologous host cell, the method comprising: a) supplying a virus containing in its genome: (i) said expressed virus; a nucleic acid, and (ii) Hybrid envelope gene (this hybrid gene is a target encodes an envelope fragment bound to a tinging ligand (which includes Thus, the envelope fragment is susceptible to its normal host cell recognition or does not facilitate binding of the virus to the mature virion. and the targeting ligand is Facilitating the targeting and binding of the mature virus particles to the surface of host cells)) and, (b) administering said virus so as to be amenable to viral infection of said heterologous host cell; the process of giving method including. 2. The method according to claim 1, wherein the virus is an enveloped virus. This is an envelope. 3. The method according to claim 2, wherein the enveloped virus is a herpes virus. It is a member of the Viridae family. 4. The method according to claim 2, wherein the enveloped virus is a retrovirus. It is a member of the Viridae family. 5. 5. The method according to claim 4, wherein the Retroviridae is Murine leukemia virus (Moloney murine leukemia virus) irus). 6. In the method according to claim 1, the nucleic acid to be expressed is DNA. Ru. 7. In the method according to claim 1, the nucleic acid to be expressed is RNA. Ru. 8. The method of claim 1, wherein the heterologous host cell is an infectious one. It is. 9. The method of claim 1, wherein the hybrid envelope flap The receptor binding region, oligomerization region (oligomeriza tion domain), transmembrane region, virus budding region, sorting signal (s ortin signals), and signal sequence (signal sequence) nce). 10. The method of claim 9, wherein the envelope fragment is , further consists of a fusion area. 11. The method of claim 1, wherein the envelope fragment is Fusion activity is due to another protein. 12. The method of claim 1, wherein the administering comprises encapsulating the virus. The container is then transplanted into the patient. 13. 13. The method of claim 12, wherein the virus is packaged. It is inside the packing cell. 14. virus, the genome of this virus contains hybrid envelope proteins The hybrid protein encodes a targeting trigger in frame. containing an envelope fragment bound to the said envelope fragment. The rope fragment facilitates its normal host cell recognition or binding rather than the hybrid envelope protein into mature virus particles. the non-viral protein facilitates effective incorporation, and the non-viral protein Targeting the mature virus particles to the surface of cells that are not normally infected by facilitate integration and combination. 15. 15. The method of claim 14, wherein the virus is enveloped. It is an envelope virus. 16. The method according to claim 15, wherein the enveloped virus is It is a member of the Lupesviridae family. 17. The method according to claim 15, wherein the enveloped virus is It is a member of the Trosviridae family. 18. 18. The method according to claim 17, wherein the Retroviridae is Morroviridae. Moloney murine leukemia virus virus). 19. In the method according to claim 14, the nucleic acid to be expressed is DNA. It is. 20. In the method according to claim 14, the nucleic acid to be expressed is RNA. It is. 21. 15. The method of claim 14, wherein the heterologous host cell is an infectious cell. It is something. 22. 15. The method of claim 14, wherein the hybrid envelope The protein has a receptor binding domain, an oligomerization domain (oligomerizat ion domain), transmembrane region, virus budding region, separation type signal (s ortin signals), and signal sequence (signal sequence) nce). 23. 23. The method of claim 22, wherein the envelope fragment further comprises a fusion region. 24. 15. The method of claim 14, wherein the envelope fragment The fusion activity of is due to another protein. 25. 15. The method of claim 14, wherein the administering seals the virus. The container is then transplanted into the patient. 26. 26. The method of claim 25, wherein the virus is packaged. It is inside the packing cell. 27. A method for expressing a nucleic acid to be expressed in a heterologous host cell, the method comprising: (a) providing a virus containing in its genome: (i) expressing said virus; a nucleic acid, and (ii) Hybrid envelope gene (this hybrid gene is a target encodes an envelope fragment bound to a tinging ligand (which includes Thus, the envelope fragment is susceptible to its normal host cell recognition or does not facilitate binding of the virus to the mature virion. and the targeting ligand is Facilitating the targeting and binding of the mature virus particles to the surface of host cells)) and, (b) administering said virus such that said cell is amenable to viral infection; and, method including. 28. 28. The method of claim 27, wherein the virus is enveloped. It is an envelope virus. 29. 29. The method according to claim 28, wherein the enveloped virus is It is a member of the Lupesviridae family. 30. 29. The method of claim 28, wherein the enveloped virus is It is a member of the Trosviridae family. 31. 31. The method according to claim 30, wherein the Retroviridae is Morroviridae. Moloney murine leukemia virus virus). 32. In the method according to claim 27, the nucleic acid to be expressed is DNA. It is. 33. In the method according to claim 27, the nucleic acid to be expressed is RNA. It is. 34. 28. The method of claim 27, wherein the heterologous host cell is an infectious It is something. 35. 28. The method of claim 27, wherein the hybrid envelope Fragment sites include the receptor binding region, oligomerization region zationdomain), transmembrane region, virus budding region, sorting signal ( sorting signals), and signal sequences ence). 36. 36. The method of claim 35, wherein the envelope fragment further comprises a fusion region. 37. 28. The method of claim 27, wherein the envelope fragment The fusion activity of is due to another protein. 38. 28. The method of claim 27, wherein the administering seals the virus. The container is then transplanted into the patient. 39. 39. The method of claim 38, wherein the virus is packaged. It is inside the packing cell.