JP4188986B2 - Gene therapy using TGF-β - Google Patents

Description

The present invention provides at least one gene encoding a factor of the transforming growth factor β superfamily for use in regenerating mammalian host connective tissue into at least one mammalian connective tissue. It relates to the method of introduction. The present invention also relates to a connective tissue cell line containing a DNA vector molecule comprising a gene encoding one of the transforming growth factor β superfamily.

  In the orthopedic field, degenerative arthritis or osteoarthritis is associated with cartilage defects and is the most frequent disease. It occurs in almost all joints of the body such as the knees, hips, shoulders and wrists. The cause of such diseases is regression of glass articular cartilage (Mankin et al., J Bone Joint Surg, 52A: 460-466, 1982). The glass cartilage of the joint is deformed, fibrillates and ultimately collapses. If regenerated cartilage can be regenerated no matter what, many patients will be able to live without pain that debilitates them. At present, no method for regenerating deficient glass cartilage has been reported.

  Traditional routes of drug delivery, such as oral, intravenous, and intramuscular administration, are ineffective at delivering drugs to the joints. In general, drugs injected into joints have a short half-life. Another disadvantage of injecting drugs into joints is that they do not frequently and repeatedly inject to obtain sufficient drug concentrations in the joint space to treat chronic conditions such as arthritis That means you can't. Traditional therapeutic agents have not been able to selectively reach the joints, so in order to obtain a sustained intra-therapeutic therapeutic capacity, it was necessary to expose the whole body of the mammalian host to a high concentration of drug. is there. In such methods, the exposure of non-target organs can cause anti-arthritic drugs to have more serious side effects such as gastrointestinal hypersensitivity, mammalian host blood, cardiovascular, liver, and renal system changes. There was a tendency to get worse.

  In the orthopedic field, several cytokines have been considered as candidates for therapeutic agents for orthopedic diseases. Bone morphogenic protein has been recognized as an effective promoter of bone formation (Ozkaynak et al., EMBO J, 9: 2085-2093, 1990; Sampath and Rueger, Complications in ortho, 101 -107, 1994), TGF-β has been reported as a promoter of bone formation and cartilage formation (Joyce et al., J Cell Biology, 110: 2195-2207, 1990).

  Transforming growth factor-β (TGF-β) is recognized by multifunctional cytokines (Sporn and Roberts, Nature (London), 332: 217-219, 1988), cell growth, differentiation, and extracellular matrix protein (Madri et al., J Cell Biology, 106: 1375-1384, 1988). TGF-β inhibits the growth of epithelial cells and osteoclast-like cells in vitro (Chenu et al., Proc Natl Acad Sci, 85: 5683-5687, 1988). Stimulates enchodral ossification and ultimately promotes bone formation (Critchlow et al., Bone, 521-527, 1995; Lind et al., A Orthop Scand, 64 (5): 553-556 , 1993; and Matsumoto et al., In vivo, 8: 215-220, 1994). TGF-β-induced bone formation stimulates subperiosteal multifunctional cells to ultimately differentiate into chondrogenic cells (Joyce et al., J Cell Biology, 110: 2195-2207 , 1990; and Miettinen et al., J Cell Biology, 127-6: 2021-2036, 1994.).

  Biological effects of TGF-β have been reported in orthopedic surgery (Andrew et al., Calcif Tissue In. 52: 74-78, 1993; Borque et al., Int J Dev Biol., 37 : 573-579, 1993; Carrington et al., J Cell Biology, 107: 1969-1975, 1988; Lind et al., A Orthop Scand. 64 (5): 553-556, 1993; Matsumoto et al., In vivo, 8: 215-220, 1994). In mouse embryos, the results of staining show that TGF-β is closely associated with tissue derived from liver lobes such as connective tissue, cartilage, and bone. In addition to developmental observations, TGF-β is present at sites of bone formation and cartilage formation. It can also improve the treatment of heel fractures of the heel. Recently, the therapeutic value of TGF-β has been reported (Critchlow et al., Bone, 521-527, 1995; and Lind et al., A Orthop Scand, 64 (5): 553-556, 1993), the short duration and the high cost of its effects have prevented its clinical application.

  Injecting TGF-β into the joint for the treatment of arthritis is not possible because the duration of action of the injected TGF-β is short due to the degradation of TGF-β into an inactive state in vivo. Not desirable. Therefore, a new method of releasing TGF-β for a long period is necessary for the regeneration of glass cartilage.

  Although it has been reported that cartilage cells are autografted to regenerate the cartilage of the joint (Brittberg et al., New Engl J Med 331: 889-895, 1994), such a method is used to regenerate soft tissue twice. It is accompanied by surgery to remove it widely. If intraarticular injection is sufficient to treat degenerative arthritis, it would be a great economic and physical benefit for the patient.

  Gene therapy, a method of transferring specific proteins to specific sites, can be a way to solve these problems (Wolff and Lederberg, Gene Therapeutics ed. Jon A. wolff, 3-25, 1994; and Jenks , J Natl Cancer Inst, 89 (16): 1182-1184, 1997).

  US Pat. Nos. 5,858,355 and 5,766,585 describe the production of viral or plasmid constructs of the IRAP (interleukin-1 receptor antagonist protein) gene; synovial cells ) (5,858,355), and bone marrow cells (5,766, 585) are transfected with the construct; and the transformed cells are injected into the heel joint. However, it has never been disclosed that a gene belonging to the TGF-β superfamily has been used to regenerate a binding organization.

  US Pat. Nos. 5,846,931 and 5,700,774 describe bones belonging to the TGF-β “superfamily” to maintain the formation of cartilage and to induce cartilage. It is disclosed to inject a composition comprising a forming protein (BMP) together with a truncated parathyroid hormone-related peptide. However, there is no disclosure of gene therapy using the BMP gene.

  Despite such prior art disclosure, in order to treat a mammalian host, at least one gene encoding an expression product in at least one cell of the mammalian host binding organization is obtained in an in vitro manner. Alternatively, there remains a real and substantial need for a method for in vivo introduction. There is also a need for a method that uses a gene encoding one of the transforming growth factor β superfamily to regenerate connective tissue in a host cell. More specifically, there is a need for a method for in vivo expression of a gene encoding a TGF-β superfamily protein in a host connective tissue cell.

  The present invention meets the above-mentioned requirements. The present invention provides a method for treating a mammalian host, wherein at least one gene encoding an expression product is introduced into at least one cell of mammalian connective tissue. The method comprises the steps of using recombinant technology to produce a DNA vector molecule comprising a gene encoding the expression product and introducing the DNA vector molecule comprising the gene encoding the expression product into connective tissue cells. Including. A DNA vector molecule is any DNA molecule that can be transmitted to and maintained in a target cell or tissue, and ultimately a gene encoding a target expression product can be stably expressed. In the present invention, a desirable DNA vector molecule to be used is a virus series or plasmid series DNA vector molecule. The method desirably includes introducing a gene encoding an expression product into a mammalian connective tissue cell for therapeutic use.

The present invention comprises: a) producing a recombinant virus or plasmid vector comprising a DNA sequence encoding one of the transforming growth factor protein superfamily and operably linked to a promoter;
b) producing a transformed connective tissue cell population by in vitro transfection of the cultured connective tissue cell population with the recombinant vector; and c) feeding the transformed connective tissue cell to a mammal. Regenerating connective tissue by expressing the DNA sequence in the joint space by implantation into an arthritic joint space of an animal host by intra-articular injection;
The present invention relates to a method for treating arthritis.

  The recombinant vector can be a retroviral vector, but is not limited thereto, and is preferably a retroviral vector. The vector may be a plasmid vector.

  The methods of the present invention include a step in which the transformed connective tissue cell population is stored prior to being transplanted. The cells can be stored in 10% DMSO under liquid nitrogen before being transplanted.

  Connective tissue cells include, but are not limited to, fibroblasts, mesenchymal cells, osteoblasts, or chondrocytes. The fibroblasts can be NIH 3T3 cells or human foreskin fibroblasts.

  Connective tissue includes but is not limited to cartilage, ligaments, or tendons. The cartilage may be glass cartilage.

  The method of the present invention uses one member of the transforming growth factor superfamily, including transforming growth factor β (TGF-β). One member of the transforming growth factor superfamily includes TGF-β1, TGF-β2, TGF-β3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, or BMP. It can be -7. Desirably, the TGF-β is human or porcine TGF-β1, TGF-β2, or TGF-β3.

The present invention also includes a) producing a recombinant virus or plasmid vector comprising a DNA sequence encoding a member of the transforming growth factor protein superfamily and operably linked to a promoter;
b) producing a transformed connective tissue cell population by in vitro transfection of the cultured connective tissue cell population with the recombinant vector; and c) feeding the transformed connective tissue cell to a mammal. Regenerating glass cartilage by expressing the DNA sequence in the joint space by intra-articular injection and implantation into an arthritic joint space of an animal host;
The present invention relates to a method for regenerating glass cartilage containing.

  The transfection method can be achieved by liposome capsule method, calcium phosphate coprecipitation method, electroporation method, DEAE-dextran mediated method.

  Preferably, the method of the invention comprises utilizing a pmTβ1 plasmid.

The present invention also relates to a connective tissue cell line containing a recombinant virus or plasmid vector comprising a DNA sequence encoding a member of the transforming growth factor superfamily. The connective tissue cell line can be, but is not limited to, a fibroblast cell line , a mesenchymal cell line , a chondrocyte cell line , an osteoblast cell line , or a bone cell line . The fibroblast cell line can be a human foreskin fibroblast cell line or an NIH 3T3 cell line .

Connective tissue cell line according to the invention comprises one of the transforming growth factor superfamily. Preferably, one factor of the transforming growth factor superfamily is TGF-β1, TGF-β2, TGF-β3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, or BMP. -7. More preferably, the member is human or porcine TGF-β1, TGF-β2, or TGF-β3.

Furthermore, the connective tissue cell line of the present invention can comprise cells containing the recombinant vector pmTβ1.

  The above object of the present invention and further objects different from the above object of the present invention are embodied by the following detailed description of the invention, the reference drawings attached thereto, and the claims.

  As used herein, “patient” includes not only humans but also members belonging to animal systems.

  As used herein, “mammalian host” includes not only humans but also members belonging to animal systems.

  As used herein, “connective tissue” is any organization that connects and supports other organizations and institutions, including mammalian ligaments, cartilage, tendons, bones, and synovium. It is not limited to this.

  As used herein, “connective tissue cells” or “cells of connective tissue” refers to fibroblasts and cartilage that secrete collagenous extracellular matrix as well as adipocytes and smooth muscle cells. Cells and cells present in connective tissue such as osteoblasts / osteocytes. Desirably, connective tissue cells are fibroblasts, chondrocytes, and bone cells. More desirably, the connective tissue cell is a fibroblast. Connective tissue cells also include hepatic lobe cells known as immature fibroblasts. It can be appreciated that the present invention can be used not only for a single type of cell, but also for a mixed culture of bound tissue cells.

As used herein, “connective tissue cell line ” includes a number of connective tissue cells derived from a common mother cell.

  As used herein, “glass cartilage” refers to connective tissue covering the joint surface. For example, hyaline cartilage includes, but is not limited to, articular cartilage, costal cartilage, and nasal cartilage.

  Specifically, glass cartilage is known to self-restore, react to changes, and provide a stable movement without friction. Glass cartilage of a joint has the same universal structure and function, even though it is glass cartilage in the same joint, although its thickness, cell density, matrix composition, and mechanical properties are various. Some functions of glass cartilage include sudden stiffness against compression, resilience, exceptional ability to distribute weight loads, ability to minimize maximum stress on subchondral bone, and excellent durability Including.

  Visually and histologically, the glass cartilage appears to be a smooth and hard surface that resists deformation. The extracellular matrix of cartilage includes chondrocytes, but there are no blood vessels, lymph vessels, or nerves. An elaborate and well-organized structure that maintains the interaction between chondrocytes and the matrix will maintain the structure and function of the glass cartilage while reducing metabolic activity. The document of Doriscoll (O'Driscoll, J. Bone Joint Surg., 80A: 1795-1812, 1988) discloses in detail the structure and function of glass cartilage, which is incorporated herein by reference in its entirety. Included as

  As used herein, the “transforming growth factor-β (TGF-β) superfamily” includes a group of structurally linked proteins that affect the differentiation process over a wide range during embryonic development. . The family includes Mullerian inhibitors (MIS) required for normal male development (Behringer, et al., Nature, 345: 167, 1990), dorsal-ventral axis formation and imaginal disk ) Drosophila decapentaplegic (DPP) gene product (Padgett, et al., Nature, 325: 81-84, 1987), which is required for morphogenesis in the egg vegetal pole Xenopus Vg-1 gene product (Weeks, et al., Cell, 51: 861-867, 1987), which can induce mesoderm and posterior germ layer structure formation in sea gall embryos (Thomsen, et al. , Cell, 63: 485, 1990) activines (Mason, et al., Biochem, Biophys. Res. Commun., 135: 957-964, 1986), and can induce de novo cartilage and bone formation Capable (Sampath, et al., J. Biol.) Bone morphogenetic proteins (BMPs such as BMP-2, 3, 4, 5, 6 and 7; osteogenin, OP-1). The TGF-β gene product can affect various differentiation processes including adipogenesis, myogenesis, chondrogenesis, hematopoiesis, and epithelial cell differentiation. (Massague, Cell 49: 437, 1987), which is incorporated herein by reference in its entirety.

  TGF-β family proteins are first synthesized as giant precursor proteins and then cleaved by proteolysis at a cluster of basic residues about 110-140 amino acids from the C-terminus. Protein C-terminal sites are all structurally related, and family members can be classified into different serve groups depending on their degree of similarity. While similarities within a particular serve group show 70% to 90% amino acid sequence homology, the homology between the serve groups is certainly low, typically 20% to 50% at best. In each case, the active species is seen as a disulfide bond dimer in the C-terminal section. In the majority of the family members studied, homomorphic dimeric species were found to be biologically active, but also inhibins (Ung, et al., Nature, 321: 779, 1986) and other family members such as TGF-β's (Cheifetz, et al., Cell, 48: 409, 1987) detected atypical dimers, which differ for each homodimer It appears to have biological properties.

  TGF-β gene superfamily members include TGF-β2, TGF-β3, TGF-β4 (chicken), TGF-β1, TGF-β5 (sea gal), BMP-2, BMP-4, Drosophila DPP, BMP- 5, BMP-6, Vgr1, OP-1 / BMP-7, Drosophila 60A, GDF-1, sea gall Vgf, BMP-3, inhibin-βA, inhibin-βB, inhibin-α, and MIS. Such genes are disclosed in Massague's literature (Massague, Ann. Rev. Biochem. 67: 735-791, 1988), which is incorporated herein by reference in its entirety.

  Desirably, the superfamily member of the TGF-β gene is TGF-β. More desirable are TGF-β1, TGF-β2, TGF-β3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, or BMP-7. More desirably, it is human or porcine TGF-β. More desirably, human or porcine TGF-β1, TGF-β2 or TGF-β3. Most preferred is human or porcine TGF-β1.

  As used herein, a “selectable marker” is expressed by a cell that stably maintains the introduced DNA, such that the cell expresses a modified phenotype such as morphogenic transformation or enzymatic activity. Including the gene product. Cells expressing the transfected gene introduce a second gene that encodes a selectable marker into the same cell that has enzymatic activity to confer resistance to antibiotics or other drugs on the cell. Can be separated. Examples of selectable markers include thymidine kinase, dihydrofolate reductase, aminoglycoside phosphotransferase (kanamycin, neomycin, and geneticin) The first three enzymes: hygromycin B phosphotransferase, xanthine-guanine phosphoribosyltransferase, CAD (de novo uridine biosynthesis-carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase) Active protein), adenosine diaminase, and asparagine synthetase, but are not limited to this (Sambrook et al. Molecular Cloning, Chapter 16. 1989). The above references are incorporated herein by reference in their entirety.

  As used herein, a “promoter” can be any DNA sequence that is active and regulates transcription in eukaryotic cells. Promoters are active in eukaryotic cells and / or prokaryotic cells. Desirably, the promoter is active in mammalian cells. A promoter can be constitutively expressed or induced. Desirably, the promoter is inducible. Desirably, the promoter can be induced by an external stimulus. More desirably, the promoter is induced by a hormone or metal. More preferably, the promoter is induced by heavy metal. Most desirably, the promoter is a metallothionein gene promoter. In addition, “enhancer elements” that regulate transcription can be inserted into DNA vector constructs and can be used with the constructs of the present invention to increase expression of the gene of interest.

  As used herein, “DC-col” refers to a cationic liposome containing a cationic cholesterol derivative. The “DC-chol” molecule includes a trivalent amino group, a medium length spacer arm (2 atoms), and a carbamoyl linker bond (Gao et al., Biochem. Biophys. Res, Commun. , 179: 280-285, 1991).

  “SF-chol” as used herein is defined as a form of cationic liposome.

  “Biologically active” as used herein in connection with liposomes refers to the ability to introduce functional DNA and / or proteins into a target cell.

  “Biologically active” as used herein in connection with nucleic acids, proteins, protein segments, or derivatives thereof is a biological function known by wild-type nucleic acids and proteins. Defined as the ability of a nucleic acid or amino acid sequence to mimic.

  “Maintenance” as used herein in the context of liposome delivery refers to the ability of the introduced DNA to remain in the cell. When used in other contexts, it means the ability to have target DNA present in the target cell or organization in order to have a therapeutic effect.

  The present invention discloses a technique for transferring a target DNA sequence in vivo or ex vivo to a binding tissue cell of a mammalian host. Ex vivo technology involves culturing target binding tissue cells, in vitro transfection of DNA sequences, DNA vectors or other intended transmission vehicles with binding tissue cells, and allowing the desired gene product to be expressed in vivo. Transplanting the modified tissue cells into a target joint of a mammalian host.

  As an alternative to in vitro growth of fibroblasts, a gene encoding the desired expression product is introduced into the liposome and injected directly into the joint site, where the liposome fuses with the connective tissue cells. As a result, gene products belonging to the TGF-β superfamily are expressed in vivo.

  As an alternative to in vitro growth of connective tissue cells, a gene encoding the desired expression product is introduced into the joint site in the naked DNA state. Naked DNA enters connective tissue cells and ultimately allows gene products belonging to the TGF-β superfamily to be expressed in vivo.

  One of the ex vivo methods of treating connective tissue abnormalities disclosed throughout this specification first produces a recombinant virus or plasmid vector containing a DNA sequence encoding a protein or biologically active section thereof. Including that. This recombinant vector is then used to infect or transfect a population of joined tissue cells cultured in vitro, eventually creating a population of joined cells containing the vector. Next, such binding tissue cells are transplanted into the target joint space of a mammalian host to cause expression of a protein or protein section in the joint space. Expression of the target DNA sequence is useful for substantially reducing harmful joint diseases associated with abnormal binding tissue.

  Those skilled in the art will know that the preferred source of cells for treating human patients is the patient's own connective tissue cells such as autologous fibroblasts.

  More specifically, the present method comprises a gene encoding a factor of the transforming growth factor β superfamily or a biologically active derivative or slice thereof, a selectable marker or a biological thereof. Use of active derivatives and slices.

  In another embodiment of the present invention, a gene encoding at least one member of the transforming growth factor β superfamily or a biologically active derivative or slice thereof is used as a gene. This includes using any DNA plasmid vector known to those skilled in the art as being able to be stably maintained in the target cell or tissue at the time of transmission, regardless of the transmission method utilized.

  One method is to directly transfer the DNA vector molecule to the target cell or organization, whether it is a viral DNA vector molecule or a plasmid DNA vector molecule. The method also includes utilizing a gene capable of encoding one of the transforming growth factor β superfamily or a biologically active derivative or slice thereof as a gene.

  Another embodiment of the invention provides a method for introducing at least one gene encoding an expression product into at least one cell of connective tissue for use in treating a mammalian host. The method includes utilizing a non-viral means to introduce a gene encoding an expression product into a connective tissue cell. More specifically, the method includes a liposome capsule method, a calcium-phosphate coprecipitation method, an electroporation method, or a DEAE-dextran mediated method, and the gene is one of the transforming growth factor β superfamily. Including the use of a gene capable of encoding a factor or biologically active derivative or slice thereof and a selectable marker or biologically active derivative or slice thereof.

  Another embodiment of the invention provides an additional method for introducing at least one gene encoding an expression product into at least one cell of connective tissue for use in treating a mammalian host. The additional method involves utilizing a biological means that utilizes a virus to transmit the DNA vector molecule to the target cell or organization. Desirably, the virus is a pseudovirus whose genome has been modified so that the pseudovirus is carried and stably maintained in the target cell, but does not have the ability to replicate within the target cell or organization. . If the modified viral genome is further amplified using recombinant DNA technology, the resulting viral genome is the DNA containing the desired heterologous gene that is expressed in the target cell or organization. Functions as a vector molecule.

  A preferred embodiment of the present invention is directed to the target joint space by transferring the TGF-β gene to the connective tissue of a mammalian host through the use of a retroviral vector utilizing the ex vivo technology described herein. This is a method for transmitting TGF-β. In other words, the DNA sequence of interest encoding a functional TGF-β protein or protein segment is subcloned with the selected retroviral vector, then the recombinant viral vector is cultured in the appropriate titer and the cultured binding set The craft cells are used to transfect in vitro and the transformed binding tissue cells, preferably autografted cells, are transplanted into the target joint, preferably by intra-articular injection.

  Another desirable method of the present invention is to use an adenovirus vector, an adeno-associated virus vector (AAV), or a herpes simplex virus vector (HSV), and a TGF-β superfamily gene in the connective tissue of a mammalian host. Direct transmission in vivo. In other words, a DNA sequence of interest encoding a functional TGF-β protein or protein segment is subcloned into the respective viral vector. Next, the TGF-β-containing virus vector is cultured in an appropriate titer and then injected into the joint cavity, preferably by intra-articular injection.

  Intra-articular injection of a DNA molecule containing the gene of interest directly into the joint causes transfection of receptor-bound tissue cells and thus contains a DNA vector to enhance stable expression of the heterologous target gene -The process of removal, in vitro culture, transfection, selection as well as fibroblast transplantation can be eliminated.

  DNA molecules can be introduced into the target connective tissue of the joint by encapsulating the DNA molecules in cationic liposomes, subcloning the desired DNA sequence into a retrovirus or plasmid vector, or the DNA molecule itself. Including, but not limited to, direct injection into the joint. Regardless of the form introduced into the knee joint, the DNA molecule is preferably introduced as a DNA vector molecule, ie, a recombinant viral DNA vector molecule or a recombinant DNA plasmid vector molecule. The expression of the target gene of the heterologous origin is ensured by inserting a promoter segment active in eukaryotic cells upstream of the coding site of the heterologous gene. One common technique in the art is to use existing strategies and techniques to construct vectors that ensure that DNA molecules enter connective tissue and are expressed at the proper level.

  In a preferred embodiment, fibroblasts removed from the knee joint are cultured in vitro for subsequent use as a transmission system for gene therapy. It is clear that culturable cells are not limited to the use of the specific connective tissue disclosed. Other organizational sources can be used for in vitro culture techniques. In the present invention, the method using a gene can be used for all methods for preventing or treating arthritis. It is clear that the field of application is not just limited to prophylactic or therapeutic application in the treatment of knee joints. The present invention can be utilized prophylactically or therapeutically to treat arthritis in any joint susceptible to disease.

  In another embodiment of the present invention, a composition comprising a gene encoding a TGF-β superfamily protein and a pharmaceutically acceptable carrier for parenteral administration of a therapeutically effective amount to a patient. provide.

  Another embodiment of the present invention provides a composition comprising a gene encoding a TGF-β superfamily protein and a pharmaceutically acceptable carrier for parenteral administration of a prophylactically effective amount to a patient. To do.

  In another embodiment of the present invention, there is provided a method as described above comprising introducing a gene into a cell in vitro. The method also includes the step of transplanting the transfected cells into a mammalian host. The method includes storing the transformed binding tissue cells after transfection of the binding tissue cells prior to transplanting the transfected cells into the mammalian host. It would be easy for one skilled in the art that the transfected binding tissue cells can be stored frozen in liquid nitrogen in 10% DMSO. This method comprises using the above method to substantially prevent the onset of arthritis in a mammalian host likely to develop arthritis.

  In another embodiment of the invention, as described above, by introducing a viral vector containing a gene encoding an expression product directly into a mammalian host for use in treating a mammalian host. A method of introducing at least one gene encoding an expression product into at least one cell of a connective tissue of a mammalian host, comprising causing in vivo transfection of the cell. Desirably, the method includes introducing directly into the mammalian host by intra-articular injection. This method comprises using the method described above to substantially inhibit arthritis pathogenesis in a mammalian host likely to develop arthritis. The method also includes using the method described above for an arthritic mammalian host for therapeutic use. In addition, the method includes using the method described above to repair and regenerate connective tissue as defined above.

  It is easy for those skilled in the art to understand that viral vectors utilizing liposomes are not limited by cell division as required for transfection and insertion of tissue cells that bind to retroviruses. I will. As described above, this method utilizing non-viral means utilizes a gene capable of encoding one member belonging to the TGF-β superfamily and a selectable marker gene such as an antibiotic resistance gene as genes. Including that.

  In another embodiment of the present invention, the TGF-β superfamily can be achieved via any of the methods disclosed herein to effect in vivo expression of collagen to regenerate connective tissue such as cartilage. The DNA sequence encoding one member of is transmitted to the connective tissue of the mammalian host.

  In a specific method disclosed by way of example only and not as a limitation of the present invention, a DNA plasmid vector containing a TGF-β coding sequence was ligated downstream of a metallothionein promoter. .

  Connective tissue is an institution that is difficult to treat. The venous and oral routes of drug delivery known in the art are inaccessible to such a binding organization and have the disadvantage that the entire body of the mammalian host must be exposed to the therapeutic agent. is there. More specifically, injecting protein into a joint with existing intra-articular injection provides a direct access to the joint. However, the majority of drugs injected in encapsulated protein form have a short half-life within the joint. The present invention solves such problems by introducing genes encoding proteins that can be used to treat mammalian hosts into the connective tissue of mammalian hosts. More specifically, the present invention provides a method for introducing a gene encoding a protein having anti-arthritic properties into the connective tissue of a mammalian host.

  In the present invention, gene therapy has been applied to solve the short duration of action and high costs associated with the administration of TGF-β. The transformed cells were able to survive over 6 weeks in tissue culture without morphological changes. Cells were injected into the Achilles tendon of the heel to confirm viability and duration of action. If the in vivo nutrient supply was sufficient for the cells, the cells could survive long enough to stimulate surrounding cells and could produce TGF-β. . The cells were functional for all intratendon and intraarticular environments.

The concentration of transfected cells is an important factor in local action. Previous experiments (Joyce et al., Supra, 1990) determined the type of organization in which the dose of TGF-β was formed. Specifically, the ratio of cartilage formation to intermembrane bone formation decreased with decreasing dose. TGF-β is also biphasic in stimulating early osteoblasts and MC3T3 cells (Centrella et al., Endocrinology, 119: 2306-2312, 1986). That is, it can be stimulated or inhibited by concentration (Chenu et al., Proc Natl Acad Sci, 85: 5683-5687, 1988). Thus, in the provided examples, NIH 3T3-TGF-β1 cells promoted collagen synthesis at concentrations of 10 ⁴ , 10 ⁵ , and 10 ⁶ cells / ml. The tendon was enlarged most at a concentration of 10 ⁶ cells / ml.

In the examples, 0.3 ml of cells with a concentration of 10 ⁶ cells / ml was injected into the joint. Samples were collected from 2 to 6 weeks after injection. The joint environment is different from the tendon environment. Cells can move more freely within the joint. They should move to sites that have selective affinity with cells. The synovium, meniscus and cartilage defect sites are sites where cells can attach. Six weeks after injection, the regenerated tissue was observed in partially and completely damaged cartilage defects, but not in the synovium or meniscus. Such selective affinity for damaged sites is a further advantage for clinical applications. If degenerative arthritis can be treated by injecting cells directly into the joint, the patient can be conveniently treated without major surgery.

  TGF-β secreted from injected cells can stimulate the regeneration of hyaline cartilage by two possible methods. One is that chondrocytes remaining in the damaged site produce TGF-β receptors on the surface of those cells (Brand et al., J Biol Chem, 270: 8274-8284, 1995). ; Cheifetz et al., Cell, 48: 409-415, 1987; Dumont et al., M Cell Endo, 111: 57-66, 1995; Lopez-Casillas et al., Cell, 67: 785-795, 1991; Miettinen et al., J Cell Biology, 127: 6, 2021-2036, 1994; and Wrana et al., Nature, 370: 341-347, 1994). Such receptors can be stimulated by TGF-β secreted from injected cells attached to the damaged site. Because TGF-β is secreted in an in vivo latent form (Wakefield et al., J Biol Chem, 263, 7646-7654, 1988), latent TGF-β requires an activation process. The other is that latent TGF-β or TGF-β secreted from transformed cells binds to TGF-β binding protein (LTBT) in the extracellular matrix of the partially defective cartilage layer. (Dallas et al., J Cell Biol, 131: 539-549, 1995).

  Regardless of the mechanism of action, observations on the synthesis of glass cartilage suggest that glass cartilage regeneration can be stimulated if TGF-β is sustained at high concentrations for a long time. The vehicle to achieve high local concentrations is not a decisive factor for local stimulation, but theoretically, chondrocytes can transmit TGF-β to the damaged site of the cartilage. It can be the most optimal vehicle for hesitation (Brittberg et al., New Engl J Med 331: 889-895, 1994). Collagen bilayer matrix is another possible vehicle for the local distribution of transformed cells (Frenkel et al., J Bone J Surg (Br) 79-B: 831-836, 1997) .

  The nature of the newly formed organization was investigated by an organizational method. In H & E staining, the newly formed organization was the same as the surrounding glass cartilage (FIG. 4). To assess the nature of the newly formed organization, the organization was safranin-O stained (Rosenburg, J Bone Joint Surg, 53A: 69-82, 1971). While the fibrous collagen is white, the newly formed tissue is stained red, indicating that it is glass cartilage (FIG. 5).

  The cells at the completely deficient site produced fibrillar collagen. Since the bone matrix for TGF-β stimulation acts as a barrier, surrounding osteoblasts may not be stimulated. Instead of stimulating surrounding cells, NIH 3T3-TGF-β1 cells produced fibrillar collagen by promoting autogeny. The fact that the cells were stimulated by autocrine and paracrine activation increases the probability that degenerative arthritis can be treated with stably transformed chondrocytes with a TGF-β1 expression construct.

Cell lines stably transformed with the TGF-β expression construct can survive tendons and knee joints. Cell lines produce fibrillar collagen at cartilage sites that are completely deficient with tendons. However, the cell line produces hyaline cartilage in partially defective articular cartilage. The stimulation mechanism by the action of autocrine and paracrine modes indicates that gene therapy using one member of the TGF-β superfamily gene is a new therapy for glass cartilage damage.

The present inventors made stable fibroblast (NIH 3T3-TGF-β1, and human foreskin fibroblast TGF-β1) cell lines by transfecting TGF-β1 expression constructs. Such TGF-β-producing cells maintained active TGF-β at high concentrations in vivo for a long time.

  With respect to gene therapy and, in particular, the potential of cell-mediated gene therapy, the first problem to be solved is the in vivo viability of the cells. Even though TGF-β can suppress disease cells in vitro, the cells may not be able to survive in other types of organizations with a highly efficient disease monitoring system. Second, the optimal concentration for in vivo gene expression should be determined. In order to solve this problem, the inventors injected cells into the Achilles tendon of the heel at three different concentrations. The concentration of intra-articular injection used was determined from the optimal concentration for intra-tendon injection. The third relates to a method in which cells stimulate cartilage regeneration within a joint.

  There are two modes of action for injected cells. One is activation of surrounding cells by secreted TGF-β (paracrine activation) (Snyder, Sci Am, 253 (4): 132-140, 1985), and the other is self -Activation (autocline activation). The concentration of cells can affect the pathway, but the surrounding environment can be the most important factor in determining the mode of action. The joint fluid between the joints and the inside of the ligament are two different environments in terms of blood supply, nutrient supply, and surrounding cells. To confirm the mode of action of the cells, the transfected cells were injected into two different environments. The overall objective of this study is to evaluate TGF-β-mediated gene therapy for orthopedic diseases and to confirm the mode of action in vivo.

  The following examples are intended to illustrate the present invention and are not intended to limit the present invention.

Example 1-Materials and Methods
(Plasmid structure)
In order to produce a metallothionein expression construct (pM), the Xba I and Bam HI restriction enzyme sites contained in the oligonucleotide for amplification were used to perform a metallothionein I promoter (− 660 / + 63). Amplified sections were subcloned at the Xba I-Bam HI site of pBluescript (Stratagene, La Jolla, Calif.). The plasmid pmTβ1 was prepared by subcloning the 1.2-kb BglII segment containing the TGF-β1 coding sequence and the 3 ′ end growth hormone poly A site into the Bam HI-SalI site of pM.

(Cell culture and transfection)
TGF-β cDNA was transfected into fibroblasts (NIH 3T3-TGF-β1) or human foreskin fibroblasts / TGF-β1. These were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco-BRL, Rockville, MD) containing 10% fetal calf serum. The TGF-β1 cDNA sequence was added to a pmTβ1 vector with a metallothionein gene promoter. A neomycin resistance gene sequence was also inserted into the vector.

  The calcium phosphate method was used to introduce this vector into the cells. Neomycin (300 μg / ml) was added to the medium to select for cells with the transfected gene sequence. Next, surviving colonies were selected, and TGF-β1 mRNA expression was confirmed by Northern analysis and TGF-β1 ELISA analysis method (R & D System). Cells expressing TGF-β1 were cultured in liquid nitrogen until just before injection.

(Northern Blot analysis)
Total RNA was isolated from cells using guanidinium isothiocyanate / phenol / chloroform. Total RNA was electrophoresed on 1.0% agarose-gel containing 0.66 M formaldehyde, transferred to a DURALON-UV membrane, and cross-linked with UV STRATALINKER (STRATAGENE). Blots were prehybridized and hybridized at 65 ° C. with a solution containing 1% calf serum albumin, 7% (w / v) SDS, 0.5 M sodium phosphate and 1 mM EDTA. Hybridized blots were washed with 0.1% SDS, 1 × SSC for 20 minutes at 50 ° C. before film exposure. RNA blots were hybridized with a ³² P-labeled cDNA probe of human TGF-β1. The β-actin probe was used as a control for sample loading.

(Cell injection into sputum)
New Zealand white cocoons weighing 2.0-2.5 kg were selected as the animal model. After intoxicating with Ketamine and roumpun, each moth was disinfected. The Achilles tendon was exposed, and 0.2 ml-0.3 ml of cells having a concentration of 10 ⁴ , 10 ^5, and 10 ⁶ cells / ml were injected into the middle part of the tendon. Zinc nitrate was added to the koji drink for the expression of the transfected DNA. In the Achilles tendon experiment, the optimal concentration was determined and then intra-articular injection was performed. The knee joint was exposed and a partial and complete cartilage defect was made with a knife. A partial defect was made in the glass cartilage layer, taking care not to expose the subchondral bone. For complete defects, the subchondral bone was exposed after removal of all the glass cartilage. After suturing the surgical wound, cells at a concentration of 10 ⁶ cells / ml were injected into the joint and zinc nitrate was added to the drink.

(Structural examination)
After collecting the tendon and knee joint, the specimen was fixed with formalin and decalcified with nitric acid. They were embedded in paraffin blocks and cut into 0.8 μm thick slices. Hematoxylin-eosin and safranin-O staining were used to observe the regenerated tissue under a microscope.

Example 2 Results
(Stable cell line )
Transfection was performed using the calcium-phosphate coprecipitation method (FIG. 1). Approximately 80% of the surviving colonies expressed transgene mRNA. Such sorted TGF-β1-producing cells were cultured in a zinc nitrate solution. Incubation of cells with 100 μM zinc nitrate solution produces mRNA. The TGF-β1 secretion rate was about 32 ng / 10 ⁶ cells / 24 hr.

(Regeneration of Achilles cartilage defect in acupuncture)
In order to confirm the survival of NIH 3T3-TGF-β1 cells, the Achilles tendon of the heel was observed. When observed with the naked eye, the 10 ⁶ cells / ml concentration had thicker tendons than the 10 ⁴ and 10 ⁵ concentrations. After partial and complete cartilage defect, 0.3 ml of NIH 3T3-TGF-β1 cells at a concentration of 10 ⁶ cells / ml was injected into the knee joint. Joints were examined from 2 to 6 weeks after injection. In partially defective cartilage, newly formed glass joints were observed; after 2 weeks, the glass joints appeared, and after 6 weeks, the cartilage defects were covered with glass cartilage (FIG. 2). The thickness of the regenerated cartilage increased with time (Figure 3). Injected cells secreted TGF-β1, which was confirmed by palliative tissue chemistry with TGF-β1 antigen (FIG. 3). The opposite side injected with normal fibroblasts without TGF-β1 transfection was not covered with glass cartilage. In the partially missing site, the regenerated glass cartilage was stained red by safranin-O staining (FIG. 4). (The newly formed cartilage depth is almost equal to the lost depth.) Such a result indicates that the injected cells activate surrounding normal chondrocytes via the paracrine mode of action. Show that.

  The organization that was regenerated with completely deficient cartilage was fibrous collagen, not glass cartilage. In safranin-O staining it stained white rather than the red color that appeared in the glass cartilage. (FIG. 5). The cartilage is covered with a fibrous tissue, which means that these cells are simply activated by autocrine mode. It appears that surrounding osteoblasts that can be stimulated by TGF-β are blocked from being stimulated by TGF-β by the presence of a thick demineralized bone matrix. The injected cells may not be able to stimulate osteoblasts due to this barrier.

  TGF-β1 transfected cells were injected into the Achilles tendon of the heel. The tendon was greatly enlarged and confirmed to be thicker than the control tendon (FIG. 7). Microscopic observation confirmed that the injected NIH 3T3-TGF-β1 cells survived in the Achilles tendon of the heel and produced fibrous collagen by H & E staining (FIG. 8). As a result of chemically staining the regenerated tendon tissue using TGF-β1 antibody, the expression of TGF-β1 was confirmed in the tendon (FIG. 9).

  While specific embodiments have been described above to illustrate the invention, it will be apparent to those skilled in the art that many variations of the invention can be made without departing from the scope of the invention as defined in the claims below. It is a true fact.

  All references mentioned herein are incorporated by reference in their entirety.

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Expression of TGF-β1 mRNA. Total RNA was isolated from NIH 3T3 cells or NIH 3T3 cells stably transformed into pmTβ1, which is a TGF-β1 expression vector that can grow regardless of the presence or absence of zinc. Total RNA (15 mg) was probed with TGF-β1 cDNA and β-actin cDNA used for control. Visual observation of regenerated cartilage. 2A. A quadrangular partial cartilage defect was created in the femoral protuberance (femoral condyle), and NIH 3T3 cells not transfected with TGF-β1 were injected into the knee joint. The deficiency was not recovered. 2B. Six weeks after NIH 3T3-TGF-β1 cell injection, the defect was recovered by the newly formed tissue. The color of the reconstructed organization was almost the same as the color of the surrounding cartilage. Microscopic observation of regenerated cartilage (X200). 3A and 3B. Hematoxylin-eosin (H & E) analysis of defect sites at 4 and 6 weeks after injection of control cells. There is no organization covering the initial defect. 3C and 3D. Hematoxylin-eosin (H & E) analysis of the defect site 4 and 6 weeks after injection of TGF-β1 transfected cells. Four weeks after the injection of TGF-β1 transfected cells, the defect site was partially covered with glass cartilage. At 4 weeks and 6 weeks after injection, the regenerated tissue became thicker, and after 6 weeks, the height was almost the same as normal cartilage. From the histological viewpoint, the regenerated cartilage (arrow) was almost identical to the surrounding glass cartilage. Criminal organization chemical analysis (X200) for TGF-β1 expression in the heel joint. The brown immuno-peroxidase reaction product indicates that recombinant TGF-β1 is expressed at high levels in NIH 3T3-TGF-β1 cells (4B). 4A shows the glass cartilage in the ankle joint injected with control cells. Microscopic observation (X200) of the regenerated tissue using H & E staining (A) and safranin-O staining (B) 5A. Regenerated glass cartilage is seen by H & E staining (black arrows) at the partially missing site. 5B. In the cartilage site completely peeled off, the regenerated tissue (white arrow) was fibrous collagen. Plasmid map of pmTβ1. Gross morphology of the Achilles tendon of the heel injected with cells transfected with TGF-β1. 7A. Tendon injected with control cells. 7B. Tendon injected with cells transformed into TGF-β1 6 weeks after injection. 7C. Sectional drawing of the tendon illustrated in 7A. 7D. Sectional drawing of the tendon illustrated in 7B. Microscopic observation of the organization regenerated on the Achilles tendon of the heels stained with H & E. 8A, 8B and 8C show tendons with control cells injected 6 weeks after injection. 8A X50 enlargement. 8B X200 enlargement. 8C X600 expansion. 8D, 8E, and 8F show tendons injected with TGF-β1 transfected cells 6 weeks after injection. 8D X50 enlargement. 8E X200 enlargement. 8F X600 enlargement. Cells transfected with TGF-β1 injected into the tendon appear slightly rounder than the endogenous tendon. Fibrous collagen was generated in autocrine and paracrine modes of action, and tendons became hypertrophied. The tendon became enlarged after the cells transfected with TGF-β1 were injected. A and B are microscopic observations of the tissue regenerated on the Achilles tendon of the heel using H & E staining (A) and the mortality tissue chemical staining (B) with TGF-β1 antibody. The brown immuno-peroxidase reaction product indicates that recombinant TGF-β1 is expressed at high levels in NIH 3T3-TGF-β1 cells.

Claims (10)

Encoding transforming growth factor (transforming growth factor) -β (TGF- β), comprising the operably linked DNA sequence probe motor, fibroblasts transfected with the recombinant viral or plasmid vector containing An injection for ex vivo treatment of arthritis characterized by being transplanted directly into the joint cavity .   The injection according to claim 1, wherein the recombinant viral vector is a retroviral vector.   The injection according to claim 1, wherein the recombinant vector is a plasmid vector.   The injection according to claim 1, wherein the fibroblasts are stored before transplantation.   The injection according to claim 4, wherein the fibroblast is stored in 10% DMSO under liquid nitrogen before being transplanted.   2. The injection according to claim 1, wherein the fibroblasts are NIH 3T3 cells or human foreskin fibroblasts.   The injection according to claim 1, wherein the TGF-β is human or porcine TGF-β1, TGF-β2, or TGF-β3. Encoding transforming growth factor-beta (TGF-beta), including operatively linked DNA sequence probe motor, it viewed including the transfected fibroblasts with recombinant viral or plasmid vector as an active ingredient, A cell composition for glass cartilage regeneration, which is directly administered to a deficient joint cavity .   The injection according to claim 1, wherein the transfection comprises a liposome capsule method, a calcium phosphate coprecipitation method, an electroporation method, and a DEAE-dextran mediated method.   The injection according to claim 3, wherein the plasmid is pmTβ1.