Mixed-cell gene therapy

Abstract

To provide a mixed cell composition for generating hyaline cartilage at a target site for a method of treating osteoarthritis by injecting a cell mixture into mammalian connective tissue.SOLUTION: Provided is a mixed cell composition for generating a therapeutic protein at a target site by providing a first population of mammalian cells transfected or transduced with a gene that is sought to be expressed and a second population of mammalian cells that have not been transfected or transduced with the gene, wherein endogenously existing forms of the second population of mammalian cells are decreased at the target site, and wherein generation of the therapeutic protein by the first population of mammalian cells at the target site stimulates the second population cells to induce a therapeutic effect.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the use of a cell mixture for somatic gene therapy. The present invention also relates to a cell mixture comprising mammalian cells transformed or transduced with a gene encoding a member of the transforming growth factor β superfamily and connective tissue cells not transformed or transduced with a gene encoding a member of the transforming growth factor β superfamily. The present invention also relates to a method for regenerating cartilage by injecting the cell mixture into mammalian connective tissue. Furthermore, the present invention relates to a method for treating osteoarthritis by injecting the cell mixture into mammalian connective tissue. [Background technology]

[0002] In orthopedics, degenerative arthritis, or osteoarthritis, is the most common cartilage damage-related disease. It affects almost every joint in the body (knees, hips, shoulders, and even the wrist). The etiology of the disease is the degeneration of articular hyaline cartilage (Mankin et al., J Bone Joint Surg, 52A:460-466, 1982). The hyaline cartilage in the joints becomes distorted, fibrillates, and eventually collapses. If the degenerated cartilage could somehow be regenerated, most patients would be able to live lives free of debilitating pain.

[0003] Traditional drug delivery routes (e.g., oral, intravenous, or intramuscular administration) for delivering drugs to joints are inefficient. Drugs injected intra-articularly generally have a short half-life. Another disadvantage of intra-articular drug injections is the need for repeated, frequent injections to achieve acceptable drug concentrations in the joint cavity for the treatment of chronic conditions (e.g., arthritis). Because traditional therapeutic agents cannot selectively target joints, achieving a sustained intra-articular therapeutic dose requires systemic exposure of the mammalian host to high drug concentrations. This exposure to non-target organs exacerbates the tendency of anti-arthritic drugs to cause severe side effects in the mammalian host, including gastrointestinal disorders and alterations in the hematologic, cardiovascular, hepatic, and renal systems.

[0004] In the orthopedic field, several cytokines have been considered as potential candidates for the treatment of orthopedic diseases. Bone morphogenetic proteins are considered to be effective stimulators of bone formation (Ozkaynak et al., EMBO J, 9:2085-2093, 1990; Sampath and Rueger, Complications in Ortho, 101-107, 1994), and TGF-β has been reported to be a stimulator of bone and chondrogenesis (Joyce et al., J Cell Biology, 110:2195-2207, 1990).

[0005] Transforming growth factor beta (TGF-β) is considered to be a multifunctional cytokine (Sporn and Roberts, Nature (London), 332:217-219, 1988), and plays a regulatory role in cell proliferation, differentiation, and extracellular matrix protein synthesis (Madri et al., J Cell Biology, 106:1375-1384, 1988). TGF-β inhibits the proliferation of epithelial and osteoclast-like cells in vitro (Chenu et al., Proc Natl Acad Sci, 85:5683-5687, 1988), but stimulates endochondral bone formation and ultimately bone formation in vivo (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 is mediated by stimulation of subperiosteal pluripotent cells, which 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).

[0006] The biological effects of TGF-β in orthopedics have been reported (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, staining has revealed that TGF-β is closely associated with mesenchymal-derived tissues, such as connective tissue, cartilage, and bone. In addition to embryological findings, TGF-β is present at sites of bone formation and chondrogenesis. TGF-β promotes fracture healing in rabbit tibiae. 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), but its short-term efficacy and high cost limit its clinical application.

[0007] Intra-articular injection of TGF-β for the treatment of arthritis is undesirable because the action of injected TGF-β is short-lived due to in vivo degradation of TGF-β to an inactive form. Therefore, a novel method for long-term sustained release of TGF-β is needed for hyaline cartilage regeneration.

[0008] Although there have been reports of articular cartilage regeneration using autologous chondrocyte transplantation (Brittberg et al., New Engl J Med 331:889-895, 1994), this procedure requires two surgeries, including extensive resection of soft tissue. If treatment for degenerative arthritis could be achieved simply by intra-articular injection, this would be economically and physically advantageous for patients.

[0009] Gene therapy, which involves the delivery of specific proteins to specific sites, may be an answer to this problem (Wolff and Lederberg, Gene Therapeutics ed. Jon A. Wolff, 3-25, 1994; and Jenks, J Natl Cancer Inst, 89(16):1182-1184, 1997).

[0010] U.S. Patent Nos. 5,858,355 and 5,766,585 disclose the creation of viral or plasmid constructs of the IRAP (interleukin-1 receptor antagonist protein) gene; the transformation of synovial cells (5,858,355) and bone marrow cells (5,766,585) with the constructs; and the injection of the transduced cells into rabbit joints, but do not disclose the use of genes belonging to the TGF-β superfamily in connective tissue regeneration.

[0011] U.S. Patent Nos. 5,846,931 and 5,700,774 disclose the injection of a composition containing a bone morphogenetic protein (BMP) belonging to the TGFβ "superfamily" in combination with a truncated parathyroid hormone-related peptide to induce and maintain cartilage formation, but do not disclose gene therapy using BMP genes.

[0012] U.S. Patent No. 5,842,477 discloses the implantation of a combination of a scaffold, periosteal / perichondral tissue, and stromal cells (including chondrocytes) into a cartilage defect area. The patent disclosure requires the presence of all three elements in the implantation system, and therefore the reference does not disclose or suggest the simple gene therapy method of the present invention, which does not require the implantation of a scaffold or periosteal / perichondral tissue.

[0013] U.S. Patent No. 6,315,992 discloses the production of hyaline cartilage in defective knee joints of mammals by injecting fibroblasts transformed with TGF-β1 into the defective knee joints, but this patent does not disclose the advantages of using a mixed cell composition such as that of the present invention.

[0014] Lee et al. (Human Gene Therapy, 12:1085-1813, 2001) disclose that hyaline cartilage can be produced in defective knee joints of mammals by injecting fibroblasts transformed with TGF-β1 into the defective joint. However, Lee et al. do not disclose the use of a mixed cell composition such as that of the present invention.

[0015] Despite these prior art disclosures, there remains a very real and substantial need for more effective and potent therapeutic methods, not only for regenerating connective tissue in a mammalian host, but also for better and more effective somatic gene therapy methods. Summary of the Invention

[0016] The present invention fulfills the above needs.

[0017] The present invention provides a mixed cell composition for use in producing a therapeutic protein at a target site, the mixed cell composition comprising: (a) a first population of mammalian cells transformed or transduced with the gene to be expressed; (b) a second population of mammalian cells that have not been transformed or transduced with the gene; and (c) a pharmaceutically acceptable carrier thereof; Includes; wherein the endogenous form of the second mammalian cell population is reduced at the target site; wherein production of a therapeutic protein by a first mammalian cell population at a target site stimulates induction of a therapeutic effect by a second cell population; Mixed cell compositions are of interest.

[0018] In the present invention, the mixed cell composition may be an injectable composition.

[0019] The present invention further provides a hyaline cartilage-forming effective amount of: (a) a first mammalian cell population transformed or transduced with a gene encoding transforming growth factor β (TGF-β) or bone morphogenetic protein (BMP); (b) a second fibroblast or chondrocyte population that has not been transformed or transduced with a gene encoding TGF-β or a BMP; and (c) a pharmaceutically acceptable carrier thereof; The present invention relates to a mixed cell composition comprising:

[0020] In a more specific embodiment, the present invention provides a hyaline cartilage-forming effective amount of: (a) a first mammalian cell population transformed or transduced with a gene encoding TGF-β or a BMP; (b) a second chondrocyte population that has not been transformed or transduced with genes encoding TGF-β or BMP; and (c) a pharmaceutically acceptable carrier thereof; The present invention relates to a mixed cell composition comprising:

[0021] The composition comprises a hyaline cartilage-forming effective amount of: (a) a first mammalian cell population transformed or transduced with a gene encoding TGF-β or a BMP; (b) a second chondrocyte population that has not been transformed or transduced with genes encoding TGF-β or BMP; and (c) a pharmaceutically acceptable carrier thereof; may include:

[0022] In the above composition, the gene may be, but is not limited to, TGF-β1, TGF-β2, TGF-β3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, or BMP-9. In particular, the gene may be TGF-β1 or BMP-2.

[0023] In the above compositions, the first mammalian cell population to be transformed or transduced may comprise epithelial cells, preferably human epithelial cells or human 293 embryonic kidney cells (also known as HEK293, HEK-293, or 293 cells).

[0024] Furthermore, in the composition, the ratio of the second population of fibroblasts or chondrocytes not transformed or transduced with a gene encoding TGF-β or BMP to the first population of mammalian cells transformed or transduced with a gene encoding TGF-β or BMP is about 1 to 20 to 1. In particular, the ratio may be about 1 to 10 to 1, or even about 1 to 3 to 1.

[0025] In the above compositions, the first cell population transformed or transduced with the gene may be irradiated, and in particular, the first mammalian cell population transformed or transduced with the gene encoding TGF-β or BMP is irradiated.

[0026] The cells of the mixed cell population may be derived from different organisms. In particular, in certain embodiments, the first mammalian cell population transformed or transduced with a gene encoding TGF-β or BMP and the second fibroblast or chondrocyte population not transformed or transduced with a gene encoding TGF-β or BMP are derived from different organisms. The first cell population and the second cell population may be derived from different mammalian sources. In particular, the first mammalian cell population transformed or transduced with a gene encoding TGF-β or BMP and the second fibroblast or chondrocyte population not transformed or transduced with a gene encoding TGF-β or BMP are derived from different mammalian sources.

[0027] The present invention also provides a method for producing a therapeutic protein at a target site in a mammal, the method comprising: (a) constructing a recombinant vector comprising a DNA sequence operably linked to a promoter, the DNA sequence encoding a therapeutic protein; (b) in vitro transforming or transducing a population of cells with said recombinant vector; and (c) a protein-producing effective amount of: (i) a first population of cells transformed or transduced with the gene; (ii) a second population of cells that has not been transformed or transduced with the gene; and (iii) a pharmaceutically acceptable carrier thereof; injecting a mixed cell composition comprising: A method comprising: wherein the endogenous form of the second mammalian cell population is reduced at the target site; wherein production of a therapeutic protein by a first mammalian cell population at a target site stimulates induction of a therapeutic effect by a second cell population; Target method.

[0028] In particular, in the above methods, there is provided a method of producing hyaline cartilage in a mammal, the method comprising: (a) constructing a recombinant vector comprising a DNA sequence operably linked to a promoter, the DNA sequence encoding transforming growth factor beta (TGF-β) or bone morphogenetic protein (BMP); (b) in vitro transforming or transducing a population of mammalian cells with the recombinant vector; and (c) a hyaline cartilage-forming effective amount of: (i) a first mammalian cell population transformed or transduced with a gene encoding TGF-β or a BMP; (ii) a second fibroblast or chondrocyte population that has not been transformed or transduced with a gene encoding TGF-β or a BMP; and (iii) a pharmaceutically acceptable carrier thereof; an injectable mixed cell composition comprising injecting into a joint cavity of a mammal; A method comprising: The injection results in expression of a DNA sequence encoding TGF-β or BMP in the joint cavity, resulting in hyaline cartilage formation in the joint cavity. It is a method.

[0029] In the above method, the gene may be, but is not limited to, TGF-β1, TGF-β2, TGF-β3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, or BMP-7. In particular, the gene may be TGF-β1 or BMP-2.

[0030] In the above methods, the first mammalian cell population to be transformed or transduced may comprise epithelial cells, preferably human epithelial cells, or human 293 embryonic kidney cells (also known as HEK293, HEK-293, or 293 cells).

[0031] Additionally, the method may include mixing the cells in a ratio of about 3 to 20:1 of a second population of fibroblasts or chondrocytes that have not been transformed or transduced with a gene encoding TGF-β or BMP to a first population of mammalian cells that have been transformed or transduced with a gene encoding TGF-β or BMP. The ratio may be about 3 to 10:1. Further, the ratio may be about 10:1.

[0032] Also in the present invention, in the above method, the first population of mammalian cells transformed or transduced with a gene encoding TGF-β or BMP is irradiated.

[0033] With respect to the cell sources in the above methods, the first mammalian cell population transformed or transduced with a gene encoding TGF-β or a BMP and the second fibroblast or chondrocyte population not transformed or transduced with a gene encoding TGF-β or a BMP may be syngeneic, allogeneic, or xenogeneic to the recipient host.

[0034] The above methods may use a recombinant vector, such as a viral vector. The recombinant vector may be, but is not limited to, a plasmid vector. Furthermore, the transformation or transduction may be achieved by liposome encapsulation, calcium phosphate co-precipitation, electroporation, DEAE-dextran-mediated or viral-mediated.

[0035] In the practice of the present invention, cells may be stored prior to transplantation. Additionally, cells may be stored in a cryopreservative prior to transplantation.

[0036] In another embodiment, the present invention provides a method of treating osteoarthritis, the method comprising: (a) constructing a recombinant vector comprising a DNA sequence operably linked to a promoter, the DNA sequence encoding transforming growth factor beta (TGF-β) or bone morphogenetic protein (BMP); (b) in vitro transforming or transducing a population of mammalian cells with the recombinant vector; and (c) a hyaline cartilage-forming effective amount and an osteoarthritis-treating effective amount of: (i) a first mammalian cell population transformed or transduced with a gene encoding TGF-β or a BMP; (ii) a second fibroblast or chondrocyte population that has not been transformed or transduced with a gene encoding TGF-β or a BMP; and (iii) a pharmaceutically acceptable carrier thereof that is not a non-biological three-dimensional structure; injecting into a joint cavity of a mammal an injectable mixed cell composition comprising: thereby resulting in expression of a DNA sequence encoding TGF-β or BMP in the joint cavity, resulting in the formation of bone and cartilage tissue in the joint cavity. Target method.

[0037] In the above method, the gene may be, but is not limited to, TGF-β1, TGF-β2, TGF-β3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, or BMP-7. In particular, the gene may be TGF-β1 or BMP-2.

[0038] In the above methods, the first mammalian cell population to be transduced or transformed may comprise epithelial cells, preferably human epithelial cells, or human 293 embryonic kidney cells (also known as HEK293, HEK-293, or 293 cells).

[0039] The present invention further provides a hyaline chondrogenic effective amount and osteoarthritis treating amount of: (a) a first mammalian cell population transformed or transduced with a gene encoding transforming growth factor β (TGF-β) or bone morphogenetic protein (BMP); (b) a second fibroblast or chondrocyte population that has not been transformed or transduced with a gene encoding TGF-β or a BMP; and (c) a pharmaceutically acceptable carrier thereof; The present invention is directed to an injectable mixed cell composition comprising:

[0040] In the above method, the gene may be, but is not limited to, TGF-β1, TGF-β2, TGF-β3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, or BMP-7. In particular, the gene may be TGF-β1 or BMP-2.

[0041] In the above methods, the first mammalian cell population to be transduced or transformed may comprise epithelial cells, preferably human epithelial cells, or human 293 embryonic kidney cells (also known as HEK293, HEK-293, or 293 cells).

[0042] In another embodiment of the present invention, the present invention provides a storage container for storing cells at a temperature of about −70° C. to about −196° C., the storage container containing a mixed cell composition for producing a protein in a target site, the mixed cell composition comprising: (a) a first population of mammalian cells transformed or transduced with the gene to be expressed; (b) a second population of mammalian cells that have not been transformed or transduced with the gene; and (c) a pharmaceutically acceptable carrier thereof; Including, wherein the endogenous form of the second mammalian cell population is reduced at the target site; wherein production of a therapeutic protein by a first mammalian cell population at a target site stimulates induction of a therapeutic effect by a second cell population; mixed cell composition Storage containers, including to provide.

[0043] In particular, the present application provides a storage container for storing cells at a temperature of about −70° C. to about −196° C., the storage container containing a hyaline cartilage-forming effective amount of: (a) A mammalian cell population transformed or transduced with a gene encoding TGF-β or a BMP; (b) a fibroblast or chondrocyte population that has not been transformed or transduced with genes encoding TGF-β or BMP; and (c) a pharmaceutically acceptable carrier thereof; an injectable mixed cell composition comprising: Storage containers, including to provide.

[0044] These and other objects of the present invention will be more fully understood from the following description of the invention, the accompanying reference drawings and the claims.

[0045] The present invention will be more fully understood from the detailed description set forth herein below and the accompanying drawings, which are provided by way of illustration and therefore not by way of limitation. [Brief explanation of the drawings]

[0046] [Figure 1] Figure 1 shows mRNA expression of TGF-β1. Total RNA was isolated from NIH3T3 cells or NIH3T3 cells stably transformed with the TGF-β1 expression vector pmTβ1, grown in the presence or absence of zinc. Total RNA (15 mg) was probed using TGF-β1 cDNA or control β-actin cDNA. [Figure 2] Figures 2A and 2B show BMP2 expression in NIH3T3-BMP2 cells. Figures 2A and 2B show control NIH3T3-metallothionein (A) and NIH3T3-BMP2 cells (B). The blue color in panel (B) indicates BMP2 protein expression. [Figure 3] Figures 3A-3D show cartilage regeneration after injection of a cell mixture (human chondrocytes and NIH3T3-TGF-β1 cells) into rabbits with a partial defect. Figures 3A and 3C show photographs taken 6 weeks after injection of either a mixture of hChon (human chondrocytes) and NIH3T3-TGF-β1 cells (A) or hChon alone (C) into the femoral condyle. Figures 3B and 3D show Masson's trichrome stained sections of femoral condyles injected with either a mixture of hChon and NIH3T3-TGF-β1 cells (B) or hChon alone (D). Original magnification: (B and D) x12.5. [Figure 4]Figures 4A-4E show cartilage regeneration in rabbits with full-thickness defects after injection of a cell mixture (human chondrocytes and NIH3T3-TGF-β1 cells). Figures 4A and 4D show photographs taken 12 weeks after injection of either a mixture of hChon and NIH3T3-TGF-β1 cells (A) or hChon alone (D) into the femoral condyle. Figures 4B and 4E show Masson's trichrome stained sections of femoral condyles injected with either a mixture of hChon and NIH3T3-TGF-β1 cells (B and C) or hChon alone (E), while Figure 4C shows Safranin O staining. Original magnification: (B, C, and E) x12.5. [Figure 5] Figures 5A-5D show cartilage regeneration in rabbits with partial defects after injection of a cell mixture (human chondrocytes and NIH3T3-BMP2 cells). Figures 5A and 5C show photographs taken 6 weeks after injection of either a mixture of hChon and NIH3T3-BMP2 cells (A) or hChon alone (C) into the femoral condyle. Figures 5B and 5D show Masson's trichrome stained sections of femoral condyles injected with either a mixture of hChon and NIH3T3-BMP2 cells (B) or hChon alone (D). Original magnification: (B and D) x12.5. [Figure 6] Figures 6A-6E show cartilage regeneration in rabbits with full-thickness defects after injection of a cell mixture (human chondrocytes and NIH3T3-BMP2 cells). Figures 6A and 6D show photographs 12 weeks after injection of either a mixture of hChon and NIH3T3-BMP2 cells (A) or hChon alone (D) into the femoral condyle. Figures 6B and 6E show Masson's trichrome stained sections of femoral condyles injected with either a mixture of hChon and NIH3T3-BMP2 cells (B and C) or hChon alone (E), while Figure 6C shows Safranin O staining. Original magnification: (B, C, and E) x12.5. [Figure 7]Figures 7A-7D show cartilage regeneration in rabbits with full-thickness defects after injection of a cell mixture (human chondrocytes and human chondrocyte-TGF-β1 cells). Figures 7A and 7C show photographs taken 6 weeks after injection of either a mixture of hChon and 293-TGF-β1 cells (A) or hChon alone (C) into the femoral condyle. Figures 7B and 7D show Masson's trichrome stained sections of femoral condyles injected with either a mixture of hChon and 293-TGF-β1 cells (B) or hChon alone (D). [Original magnification: (B and D) x12.5] [Figure 8] Figures 8A-8D show cartilage regeneration in rabbits with partial defects after injection of a cell mixture (human chondrocytes and human 293-TGF-β1 cells). Figures 8A and 8C show photographs taken 6 weeks after injection of a mixture of hChon and 293-TGF-β1 cells (3:1 ratio) (A) or a mixture of hChon and 293-TGF-β1 cells (5:1 ratio) (C) into the femoral condyle. Figures 8B and 8D show Masson's trichrome stained sections of femoral condyles injected with a 3:1 mixture of hChon and 293-TGF-β1 cells (B) or a 5:1 mixture of hChon and 293-TGF-β1 cells (D). [Original magnification: (B and D) x12.5] DETAILED DESCRIPTION OF THE INVENTION

[0047] As used herein, the term "patient" includes members of the animal kingdom, including humans, but is not limited to humans.

[0048] The term "mammalian cell population" as used herein in reference to transformed or transduced cells includes all types of mammalian cells, particularly human cells, including but not limited to connective tissue cells such as fibroblasts or chondrocytes, or stem cells, and particularly human embryonic kidney cells, more particularly human 293 embryonic kidney cells, or epithelial cells.

[0049] As used herein, the term "mammalian host" includes members of the animal kingdom, including humans, but is not limited to humans.

[0050] As used herein, the term "connective tissue" means tissue that connects or supports other tissues or organs, including, but not limited to, ligaments, cartilage, tendons, bones, and synovial membranes of a mammalian host.

[0051] As used herein, the terms "connective tissue cells" and "cells of connective tissue" include cells present in connective tissue, such as fibroblasts, chondrocytes, and bone cells (osteoblasts / osteocytes), which secrete a gelatinous extracellular matrix, as well as adipocytes and smooth muscle cells. Preferably, the connective tissue cells are fibroblasts, chondrocytes, and bone cells. It will be understood that the present invention can be practiced using a single type of cell, or even a mixed culture of connective tissue cells. It will also be understood that the tissue cells may be pretreated with a compound or irradiation before injection into the joint cavity so that the tissue cells stably express a gene of interest in the host organism. Preferably, the connective tissue cells do not provoke an adverse immune response when injected into the host organism. In this regard, it will be understood that not only autologous cells but also allogeneic cells may be used for cell-mediated gene therapy or somatic cell therapy.

[0052] A "connective tissue cell line" as described herein comprises multiple connective tissue cells derived from a common parent cell.

[0053] "Depletion" of cells as used herein refers to a reduction in the cell population compared to the normal amount at the site, which may refer to a percentage reduction in the cell population, such as at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the normal cell population at the location, or may refer to damage or depletion of cells at the location.

[0054] As used herein, "helper cells" refers to helper cells mixed with cells that have been transformed or transduced with a gene of interest. The helper cells are not themselves transformed or transduced with the gene of interest. In particular, the cells transformed or transduced with the gene of interest produce a protein that activates the helper cells. Administration of this mixture to a subject site that endogenously produces helper cells but is depleted at the time of administration provides effective somatic gene therapy with benefits at the subject site.

[0055] In one embodiment, "helper cells" may refer to connective tissue cells for generating a cell mixture that have been transformed or transduced with a gene encoding a member of the transforming growth factor β superfamily. Such helper cells may include any connective tissue cell. Generally, these cells are not transformed or transduced with a gene encoding a member of the transforming growth factor β superfamily. In particular, these cells are not transformed or transduced with any gene, and these cells are usually located in cartilage regions. Typically, the cells are fibroblasts or chondrocytes.

[0056] As used herein, "histocompatibility" of donor cells and recipient host means that they share a sufficient variety of histocompatibility factors so that the transplant will be accepted and functionally maintained in the host mammal. In particular, the donor and recipient combination should be compatible with respect to human leukocyte antigens (HLA), such as HLA-A, B, and C (Class I) and HLA-DR (Class II).

[0057] As used herein, "hyaline cartilage" refers to connective tissue that covers joint surfaces, including, but not limited to, articular cartilage, costal cartilage, and nasal cartilage.

[0058] In particular, hyaline cartilage is known to self-renew and responds to deformation, allowing for more stable, frictionless movement. Although hyaline cartilage in different joints, and even within the same joint, differs in thickness, cell density, matrix composition, and mechanical properties, it is comparable in general structure and function. Some of the functions of hyaline cartilage include its surprising stiffness and resilience to compression, its excellent ability to distribute weight load, its ability to minimize peak stresses in the subchondral bone, and its high durability.

[0059] Macroscopically and histologically, hyaline cartilage is a smooth, rigid surface that resists deformation. The extracellular matrix of cartilage contains chondrocytes but does not contain blood vessels, lymphatic vessels, or nerves. The elaborate, highly ordered structure that maintains the interactions between chondrocytes and the matrix maintains the structure and function of hyaline cartilage while maintaining a low level of metabolic activity. Reference: O'Driscoll, J. Bone Joint Surg., 80A:1795-1812, 1998, provides a detailed description of the structure and function of hyaline cartilage, which is incorporated herein by reference in its entirety.

[0060] As used herein, "injectable" compositions do not include various three-dimensional scaffold, framework, mesh, or felt structures, which may be made of a material or form to which cells can adhere and grow in two or more layers, and which are generally implanted rather than injected. In one embodiment, the injection method of the present invention is typically performed using a syringe. However, any method for injecting the subject composition may be used. For example, a catheter, a sprayer, or a temperature-dependent polymer gel may also be used.

[0061] As used herein, "mixed cells" or "cell mixture" or "cell mixture" refers to a combination of multiple cells, wherein the combination includes a first cell population that has been transformed or transduced with a gene of interest, wherein expression of the gene of interest is beneficial to helper cells, and the helper cells are a second cell population.

[0062] In one embodiment of the present invention, the term "mixed cells" refers to a combination of multiple mammalian cells, including cells transformed or transduced with a gene or DNA encoding a member of the transforming growth factor β superfamily and helper cells that have not been transformed or transduced with a gene encoding a member of the transforming growth factor β superfamily. The ratio of cells that have not been transformed or transduced with a gene encoding a member of the transforming growth factor β superfamily to cells that have been transformed or transduced with a TGF superfamily gene may typically be in the range of about 3 to 20:1. The range may also be about 3 to 10:1. In particular, the range may be about 10:1 in terms of cell number. However, it is understood that the ratio of these cells does not need to be fixed within a specific range, as long as the combination of these cells is effective in producing hyaline cartilage in a partial or complete joint defect.

[0063] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier known in the art that promotes the delivery efficiency of the composition of the present invention and prolongs the effect of the composition.

[0064] As used herein, "somatic cell" or "cell" generally refers to a cell of the body other than an egg or sperm.

[0065] As used herein, "preserved" cells refer to a composition of mixed cells that have been stored individually or together prior to administration to the joint cavity. The cells may be stored in a freezer. Alternatively, the cells may be frozen in a liquid nitrogen tank or equivalent storage device at about -70°C to about -196°C, and the cells may be stored for later administration to the joint cavity. The cells may be thawed using known protocols. Cryopreservation and thawing can be performed in a variety of ways, so long as they maximize the viability and potency of the cells.

[0066] As used herein, the terms "transformation" and "transduction" refer to specific methods of introducing DNA into a host cell, resulting in subsequent integration into the chromosomal DNA of the recipient cell. In practicing the present invention, any method of introducing foreign DNA into a host cell, including non-viral or viral gene transfer methods, can be used, so long as the foreign gene is introduced into the host cell and stably expressed in the host cell. Therefore, the terms "transformed" and "transduced" as used herein include any method of introducing genes into cells, such as calcium phosphate precipitation, DEAE-dextran, electroporation, liposomes, or viral mediation.

[0067] The "transforming growth factor beta (TGF-β) superfamily," as used herein, includes a group of structurally related proteins that affect a wide range of embryonic differentiation processes. This family includes: Müllerian inhibitory substance (MIS), which is required for normal male sexual development (Behringer et al., Nature, 345:167, 1990); the Drosophila decapentaplegic (DPP) gene product, which is required for dorsoventral axis formation and imaginal disc morphogenesis (Padgett et al., Nature, 325:81-84, 1987); the Xenopus Vg-1 gene product, which specifies the position of the vegetal pole of the egg (Weeks et al., Cell, 51:861-867, 1987); Activins (Mason et al., Biochem. Biophys. Res. Commun., 135:957-964, 1986) can induce mesoderm formation and anterior structures in Xenopus embryos (Thomsen et al., Cell, 63:485, 1990); and bone morphogenetic proteins (BMPs, such as BMP-2, 3, 4, 5, 6, and 7, osteogenin, and OP-1) can induce de novo chondrogenesis and osteogenesis (Sampath et al., J. Biol. Chem., 265:13198, 1990). TGF-β gene products can affect a variety of differentiation processes, including adipogenesis, myogenesis, chondrogenesis, hematopoiesis, and epithelial cell differentiation (for a review, see Massague, Cell, 49:437, 1987; this reference is incorporated herein by reference in its entirety).

[0068] Proteins in the TGF-β family are initially synthesized as large precursor proteins, which subsequently undergo proteolytic cleavage at a cluster of basic residues approximately 110–140 amino acids from the C-terminus. The C-terminal regions of these proteins are structurally related, allowing family members to be divided into distinct subgroups based on the degree of homology. Within a particular subgroup, homology ranges from 70% to 90% amino acid sequence identity, but between subgroups, homology is much lower, typically only 20%–50%. In both cases, the active species appears to be a disulfide-bridged dimerization of the C-terminal fragment. For most of the family members studied to date, the homodimeric species have been found to be biologically active, but for other family members, such as the inhibins (Ung et al., Nature, 321:779, 1986) and TGF-βs (Cheifetz et al., Cell, 48:409, 1987), heterodimers have also been detected, which appear to have biological properties distinct from the individual homodimers.

[0069] Members of the TGF-β gene superfamily include TGF-β3, TGF-β2, TGF-β4 (chicken), TGF-β1, TGF-β5 (Xenopus), BMP-2, BMP-4, Drosophila DPP, BMP-5, BMP-6, Vgr1, OP-1 / BMP-7, Drosophila 60A, GDF-1, Xenopus Vgf, BMP-3, inhibin-βA, inhibin-βB, inhibin-α, and MIS. These genes are described in Massague, Ann. Rev. Biochem. 67:753-791, 1998, which is incorporated herein by reference in its entirety.

[0070] Preferably, the members of the TGF-β gene superfamily are TGF-β and BMP. More preferably, the members are TGF-β1, TGF-β2, TGF-β3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, or BMP-7. More preferably, the members are human or porcine TGF-β1 or BMP-2.

[0071] As used herein, a "selectable marker" includes a gene product expressed by cells that stably maintain introduced DNA and that causes the cell to undergo a phenotypic change, such as morphological transformation, or to express an enzymatic activity. Isolation of cells expressing the transformed or transduced gene can be achieved by optionally introducing into the same cells a second gene encoding a selectable marker, such as an enzymatic activity that confers resistance to antibiotics or other drugs. Examples of selectable markers include, but are not limited to, thymidine kinase, dihydrofolate reductase, aminoglycoside phosphotransferases that confer resistance to aminoglycoside antibiotics (e.g., kanamycin, neomycin, and geneticin), hygromycin B phosphotransferase, xanthine-guanine phosphoribosyltransferase, CAD (a single protein containing the first three enzymatic activities of de novo uridine biosynthesis (i.e., carbamyl phosphate synthetase, aspartate carbamyltransferase, and dihydroorotase)), adenosine deaminase, and asparagine synthetase (Sambrook et al., Molecular Cloning, Chapter 16, 1989); this reference is incorporated herein by reference in its entirety. It will be understood that the use of a selectable marker is not required to practice the present invention. Indeed, in one embodiment, the genetic constructs of the present invention do not incorporate a selectable marker.

[0072] As used herein, a "promoter" refers to any DNA sequence that is active and controls transcription in eukaryotic cells. The promoter may be active in either or both eukaryotic and prokaryotic cells. Preferably, the promoter is active in mammalian cells. The promoter may be constitutively expressed or inducible. Preferably, the promoter is inducible. Preferably, the promoter is inducible by an external stimulus. More preferably, the promoter is inducible by hormones or metals. Most preferably, the promoter is a metallothionein gene promoter or a promoter inducible by glucocorticoids. Similarly, "enhancer elements," which also control transcription, can be inserted into DNA vector constructs and used with the constructs of the present invention to enhance expression of a gene of interest.

[0073] The term "DC-chol" as used herein refers to a cationic liposome containing a cationic cholesterol derivative. The "DC-chol" molecule contains a tertiary 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).

[0074] "SF-chol" as described herein is defined as a type of cationic liposome.

[0075] As used herein, the term "biologically active" as used in reference to liposomes refers to the ability to deliver functional DNA and / or proteins to target cells.

[0076] The term "biologically active," as used herein in reference to a nucleic acid, protein, protein fragment, or derivative thereof, is defined as the ability of a nucleic acid sequence or amino acid sequence to mimic a known biological function performed by the wild-type nucleic acid or wild-type protein.

[0077] As used herein, the term "maintenance" in the context of liposome delivery refers to the ability of introduced DNA to remain present in a cell. In other contexts, "maintenance" refers to the ability of target DNA to remain present in a target cell or tissue so as to exert a therapeutic effect.

[0078] The present invention involves administering a mixture of cells to a mammal at a site in need thereof, wherein a first cell population is transformed or transduced with a gene of interest to be expressed at the mammalian target site. In performing somatic cell gene therapy, the present invention provides for the use of a second cell population that has not been transformed or transduced with the gene of interest, the second cell population being endogenously depleted at an injured, diseased, or weakened target site, and which requires the activation of expression of the gene of interest at the target site as well as the second cell population to activate and expand the endogenously produced or exogenously administered second cell population type.

[0079] In particular, the present invention discloses techniques for ex vivo and in vivo transfer of a DNA sequence of interest into mammalian cells in a mammalian host, including culturing the target mammalian cells, in vitro transformation or transduction with the DNA sequence, transfer of a DNA vector or other transfer vehicle of interest into mammalian cells, and subsequent implantation of the modified mammalian cells into a target joint in the mammalian host for in vivo expression of the gene product of interest.

[0080] It should be understood that in the gene therapy protocols of the present invention, various foreign tissues may be co-implanted with materials such as scaffolds or frameworks, but such scaffolds or tissues may not be included in the injection system of the present invention. In one preferred embodiment, the present invention is directed to a simple method for expressing exogenous TGF superfamily proteins within a joint cavity by injecting a transformed or transduced mammalian cell population into the joint cavity for cell-mediated gene therapy or somatic cell therapy.

[0081] One ex vivo method for treating connective tissue disorders disclosed herein involves first generating a recombinant viral or plasmid vector containing a DNA sequence encoding the protein or a biologically active fragment thereof. This recombinant vector is then used to infect or transform a population of mammalian cells cultured in vitro, resulting in a mammalian cell population containing the vector. The mammalian cells are then implanted into the target joint cavity of a mammalian host, either as a mixture or individually injected into the joint cavity to form a mixture within the joint, whereupon the protein or protein fragment is expressed within the joint cavity. Expression of the target DNA sequence is useful for substantially alleviating at least one deleterious joint pathology associated with connective tissue disorders.

[0082] The source of cells for treating a human patient can be the patient's own cells, such as autologous cells, although one skilled in the art will appreciate that allogeneic and xenogeneic cells can also be utilized regardless of the histocompatibility of the cells. Alternatively, in one embodiment of the present invention, allogeneic cells that are histocompatible with the mammalian host can be utilized. More specifically, the histocompatibility of the donor and patient is determined, and the histocompatible cells are administered to the mammalian host.

[0083] More preferably, the method comprises using as said gene a gene capable of encoding a member of the transforming growth factor β superfamily or a biologically active derivative or fragment thereof, and a selectable marker or a biologically active derivative or fragment thereof.

[0084] A further embodiment of the present invention includes using as the gene a gene capable of encoding at least one member of the transforming growth factor β superfamily or a biologically active derivative or fragment thereof, and using as the DNA plasmid vector any DNA plasmid vector known to those skilled in the art that can be stably maintained in target cells or tissues when introduced, regardless of the method of introduction used.

[0085] Another embodiment of the present invention provides a method for introducing at least one gene encoding a product into at least one cell of connective tissue, wherein the cell is intended for use in treating a mammalian host. The method comprises using a non-viral method to introduce the gene encoding the product into the connective tissue cell. More preferably, the method comprises liposome encapsulation, calcium phosphate co-precipitation, electroporation, or DEAE-dextran mediation, and comprises using a gene capable of encoding a member of the transforming growth factor superfamily, or a biologically active derivative or fragment thereof, and a selectable marker, or a biologically active derivative or fragment thereof.

[0086] In another embodiment of the present invention, there is provided a method for introducing at least one gene encoding a product into at least one cell of a mammalian tissue, wherein the cell is used to treat a mammalian host. This method involves using a virus as a biological tool to introduce a DNA vector molecule into a target cell or tissue. Preferably, the virus is a pseudovirus, whose genome has been modified so that it is only capable of pseudovirus introduction and stable maintenance within the target cell, but does not retain the ability to replicate within the target cell or tissue. The modified viral genome is further engineered by recombinant DNA techniques so that it acts as a DNA vector molecule containing a heterologous gene of interest that is expressed in the target cell or tissue.

[0087] A preferred embodiment of the present invention is a method for delivering TGF-β or BMP to a target joint cavity by introducing the TGF-β or BMP gene into the connective tissue of a mammalian host using the ex vivo techniques and retroviral vectors disclosed herein. In other words, a DNA sequence of interest encoding a functional TGF-β or BMP protein or protein fragment is incorporated into a selected transducing retroviral vector. The transduced mammalian cells, preferably autologous cells, are then implanted into the target joint by intra-articular injection along with an untransformed or untransduced sample of mammalian cells.

[0088] Another preferred method of the present invention involves direct in vivo delivery of a TGF-β superfamily gene to the connective tissue of a mammalian host using either a retroviral vector, an adenoviral vector, an adeno-associated virus (AAV) vector, or a herpes simplex virus (HSV) vector. In other words, a DNA sequence of interest encoding a functional TGF-β or BMP protein or protein fragment is incorporated into the respective viral vector. The recombinant virus containing the TGF-β or BMP is then propagated to an appropriate titer and introduced into the joint cavity, preferably by intra-articular injection.

[0089] Methods for introducing DNA molecules into target connective tissues in the joint include, but are not limited to, encapsulating the DNA molecules in cationic liposomes, incorporating the desired DNA sequence into retroviral or plasmid vectors, or directly injecting the DNA molecules themselves into the joint. Regardless of the mode of introduction into the knee joint, the DNA molecules are preferably introduced as DNA vector molecules, i.e., recombinant viral or recombinant DNA plasmid vector molecules. Expression of the desired heterologous gene is achieved by directly inserting a promoter fragment active in eukaryotic cells upstream of the coding region of the heterologous gene. Those skilled in the art can utilize known strategies and techniques for vector construction to achieve appropriate expression levels after introduction of the DNA molecules into connective tissues.

[0090] In a preferred embodiment, mammalian cells are cultured in vitro for subsequent use as a delivery system for gene therapy. It will be apparent that the applicants are not limited to the use of the specific tissues disclosed. Other tissue sources may be utilized for in vitro culture techniques. The gene-based methods of the present invention can be used both preventatively and therapeutically for osteoarthritis and wound healing. It will also be apparent that the present invention is not limited to preventative or therapeutic use in knee joint therapy alone. The present invention may be utilized for preventative or therapeutic treatment of damage in osteoarthritis of susceptible joints or injuries resulting from cartilage tearing or deterioration.

[0091] Another embodiment of the present invention provides a compound comprising a gene encoding a protein of the TGF-β superfamily and a suitable pharmaceutical carrier, for parenteral administration to a patient in a therapeutically effective amount.

[0092] Another embodiment of the present invention provides a compound comprising a gene encoding a protein of the TGF-β superfamily and a suitable pharmaceutical carrier, which is administered parenterally to a patient in a prophylactically effective amount.

[0093] In a further embodiment of the present invention, the cells are preserved prior to administration into the joint cavity. The transformed or transduced cells may be preserved alone, or the untransformed helper cells may be preserved alone, or they may be preserved as a mixture, but not necessarily simultaneously. Furthermore, the preservation period need not be the same. Thus, separately preserved cells may be mixed prior to injection. Alternatively, the cells may be preserved and then injected separately to form a cell mixture within the joint cavity. Those skilled in the art will appreciate that the cells may be cryopreserved in liquid nitrogen in a cryopreservative (such as, but not limited to, a composition of about 10% DMSO) or an equivalent preservation medium.

[0094] In another embodiment of the present invention, there is provided a method for introducing at least one gene encoding a product into at least one cell of a mammalian tissue, wherein the cell is intended for use in treating the mammalian host, the method comprising in vivo infection of the cell by directly introducing a viral vector containing the gene encoding the product into the mammalian host. Preferably, the method comprises direct introduction into the mammalian host by intra-articular injection. The method includes use in a method for substantially preventing the onset of arthritis in a mammalian host highly susceptible to the development of arthritis. The method also includes use in a method for therapeutic use in a mammalian host with arthritis. Furthermore, the method also includes use in a method for repairing and regenerating connective tissue as defined hereinabove.

[0095] Those skilled in the art will understand that viral vectors using liposomes do not have the cell division restriction required for infection and internalization into mammalian cells as is the case with retroviruses. This method using the above-mentioned non-viral means includes using a gene capable of encoding a member of the TGF-β superfamily, and optionally including a selectable marker gene (such as an antibiotic resistance gene). It will also be understood that a selectable marker gene is not a requirement for carrying out the present invention.

[0096] Another embodiment of the present invention is the introduction, by the methods disclosed herein, of a DNA sequence encoding a member of the TGF-β superfamily into mammalian host connective tissue, thereby resulting in the in vivo expression of collagen for regenerating connective tissue such as cartilage.

[0097] Connective tissues are difficult organs to target therapeutically. Drug delivery via intravenous or oral routes known in the art has the drawback of poor access to these connective tissues and exposing the entire mammalian host body to the therapeutic agent. More preferably, known intra-articular protein injection methods provide direct access to the joint. However, most injectable drugs in the form of encapsulated proteins have a short intra-articular half-life. The present invention solves these problems by introducing into the connective tissue of a mammalian host a gene encoding a protein that can be used to treat the mammalian host. More preferably, the present invention provides a method for introducing into the connective tissue of a mammalian host a gene encoding a protein with anti-arthritic properties.

[0098] In the examples described herein, collagen synthesis in joints was stimulated by NIH3T3-TGF-β1 and NIH3T3-BMP2 cells mixed with untransduced chondrocyte helper cells. In these examples, the ratio of transduced cells to helper cells was 1:10, and 2 x 10 293-TGF-β1, NIH3T3-TGF-β1, or NIH3T3-BMP2 cells and untransduced chondrocyte helper cells were mixed. 6 The cells were injected into the joint at a concentration of 1000 cells / ml. Samples were collected 6 to 12 weeks after injection. The cells moved freely within the joint and migrated to areas with specific affinity for these cells. Synovial membrane defects, meniscal defects, and cartilage defects were potential sites for cell adhesion. Six and 12 weeks after injection, regenerated tissue was observed in both partially and completely damaged cartilage defects. This specific affinity for the damaged area is another advantage of clinical application of the mixed cells. If degenerative arthritis could be cured simply by injecting cells into the joint without the need for various physical substrates such as scaffolds or other three-dimensional structures, patients could receive a convenient treatment without major surgery.

[0099] Regardless of the mechanism of action, and without being bound by any particular theory, the findings regarding hyaline cartilage formation using the mixed cell compositions of the present invention indicate that sustained, long-term high concentrations of TGF-β or BMP can stimulate hyaline cartilage regeneration. The characteristics of the newly formed tissue were determined by histological methods. Masson's trichrome staining and Safranin O staining demonstrated that the newly formed tissue was identical to the surrounding hyaline cartilage (Figures 3-7).

[0100] The following examples are presented for the purpose of illustrating the present invention, but are not intended to be limiting thereof.

[0101] Example Example 1: Materials and Methods Plasmid construction A 1.2-kb BglI fragment containing the TGF-β1 coding sequence, which contained a growth hormone poly(A) site at its 3' end, was subcloned into the BamHI site of pMTMLV to generate plasmid pMTMLVβ1. A 1.2-kb SalI-NotI fragment containing the BMP2 coding sequence was subcloned into the SalI-NotI site of pMTMLV to generate plasmid pMTBMP2. The pMTMLV vector was constructed by deleting the entire gag and env sequences and a portion of the Ψ packaging sequence from the retroviral vector MFG.

[0102] Cell culture and transduction TGF-β and BMP-2 cDNA cloned into retroviral vectors were individually transduced into fibroblasts (NIH3T3-TGF-β1 and NIH3T3-BMP-2) and mammalian cells (293-TGF-β1). Cells were cultured in Dulbecco's modified Eagle's medium (GIBCO-BRL, Rockville, MD) containing 10% fetal bovine serum.

[0103] To select for cells carrying the transgene sequence, neomycin (300 μg / ml) was added to the medium. Cells expressing TGF-β1 and BMP-2 were stored in liquid nitrogen and cultured immediately before injection.

[0104] Transformation with the TGF-β gene was performed using the calcium phosphate coprecipitation method (Figure 1). Approximately 80% of the surviving colonies expressed the transgene mRNA. These selected TGF-β1-producing cells were incubated in a zinc sulfate solution. When cultured in a 100 mM zinc sulfate solution, the cells produced mRNA. The TGF-β secretion rate was approximately 32 ng / 10 6 cells / 24 hours.

[0105] To test whether NIH3T3 fibroblasts infected with a retroviral vector containing BMP2 cDNA produced biologically active BMP2 protein, alkaline phosphatase (ALP) activity assays were performed using control NIH3T3-metallothionein cells (Figure 2A) and NIH3T3-BMP2 cells (Figure 2B). Blue in Figure 2B indicates BMP2 protein expression.

[0106] 1.5x10 6 NIH3T3 cells were grown overnight in 6-well tissue culture plates. 0.5x10 cells were added as indicated. 5Cells (MC3T3E1) were placed in tissue culture inserts and grown overnight. The medium was aspirated from the inserts, and the inserts were transferred to 6-well plates and incubated for 48-72 hours. The medium was aspirated from the inserts. 5 ml of 1X phosphate-buffered saline (PBS) was added to wash the cells. 4 ml of 3.7% formaldehyde / 1X PBS solution was added to each insert, and the cells were fixed at 4°C for 20 minutes. The cells were washed twice with 1X PBS. 3 ml of alkaline phosphatase staining solution was added to each insert, and the inserts were incubated at room temperature for approximately 20 minutes to 1 hour in the dark to allow the blue color to develop. The ALP staining solution was 0.1 M Tris-HCl (pH 8.5) containing 0.1 mg / ml naphthol AS-MX phosphate (Sigma N5000), 0.5% N-dimethylformamide (Sigma D8654), 2 mM MgCl2, and 0.3 mg / ml Fast Blue BB salt (Sigma F3378).

[0107] Example 2: Experimental Methods and Results Regeneration in rabbit articular cartilage defects: New Zealand white rabbits weighing 2.0–2.5 kg were selected for animal testing. These rabbits were mature and had tide marks. The knee joints were exposed, and partial cartilage defects (3 mm x 6 mm, 1–2 mm deep) or full-thickness defects (3 mm x 6 mm, 2–3 mm deep) were created in the hyaline cartilage layer of the femoral condyle with a scalpel. Either control human chondrocytes (hChon), a mixture of hChon and NIH3T3-TGF-β1 cells, or a mixture of hChon and NIH3T3-BMP2 cells were injected into the defective knee joints of the rabbits. These cells (2 x 10 6 A volume of 15–20 μl of cells / ml was injected into the upper and then the left side of the defect, allowing the cells to infiltrate the wound for 15–20 minutes before suturing. In experiments in which a mixture of hChon and NIH3T3-BMP2 cells was injected into rabbits with full-thickness defects, the mixed cell compositions were injected into the defect 3 weeks after defect creation. At 6 or 12 weeks after cell injection, femoral condyles were harvested and examined.

[0108] Cartilage regeneration by injection of a cell mixture (human chondrocytes and NIH3T3-TGF-β1 cells) into rabbits with partial defects Either control hChon or a composition containing a mixture of hChon and NIH3T3-TGF-β1 cells was injected into the knee joints of rabbits containing a partial cartilage defect (3 mm x 5 mm, 1-2 mm deep) in the femoral condyle. The cell mixture (2 x 10 6 15–20 μl of cells / ml (hChon:NIH3T3-TGF-β1 ratio 10:1) was injected into the upper and then the left side of the defect. The cells were allowed to infiltrate the wound for 15–20 minutes before suturing. Six weeks after injection, specimens were collected and examined microscopically. Figures 3A and 3C show photographs of femoral condyles 6 weeks after injection with either a mixture of hChon and NIH3T3-TGF-β1 cells (A) or hChon alone (C). Figures 3B and 3D show Masson's trichrome stained sections of femoral condyles injected with a mixture of hChon and NIH3T3-TGF-β1 cells (B) or hChon alone (D). [Original magnification: (B and D) ×12.5]

[0109] Cartilage regeneration by injection of a cell mixture (human chondrocytes and NIH3T3-TGF-β1 cells) in full-thickness defects in rabbits Either control hChon or a mixture of hChon and NIH3T3-TGF-β1 cells was injected into the knee joints of rabbits containing full-thickness cartilage defects (3 mm x 5 mm, 2-3 mm deep) in the femoral condyle. The cell mixture (2 x 10 6Twenty to 25 μl of cells / ml (Chon:NIH3T3-TGF-β1 ratio 10:1) was injected into the upper and then the left side of the defect. The cells were allowed to infiltrate the wound for 15 to 20 minutes before suturing. At 12 weeks after injection, specimens were collected and examined microscopically. Figures 4A and 4D show photographs of femoral condyles 12 weeks after injection with either a mixture of hChon and NIH3T3-TGF-β1 cells (A) or hChon alone (D). Figures 4B, 4C, and 4E show Masson's trichrome stained (B and E) and Safranin O stained (C) sections of femoral condyles injected with either a mixture of hChon and NIH3T3-TGF-β1 cells (B and C) or hChon alone (E). [Original magnification: (B, C, and E) x12.5]

[0110] Cartilage regeneration by injection of a cell mixture (human chondrocytes and NIH3T3-BMP-2 cells) into rabbits with partial defects Either control hChon or a mixture of hChon and NIH3T3-BMP2 cells was injected into the knee joints of rabbits containing a partial cartilage defect (3 mm x 5 mm, 1-2 mm deep) in the femoral condyle. The cell mixture (2 x 10 6 15–20 μl of cells / ml (hChon:NIH3T3-BMP-2 ratio 10:1) was injected into the upper and then the left side of the defect. The cells were allowed to infiltrate the wound for 15–20 minutes before suturing. Six weeks after injection, specimens were collected and examined microscopically. Figures 5A and 5C show photographs of femoral condyles 6 weeks after injection with either a mixture of hChon and NIH3T3-BMP2 cells (A) or hChon alone (C). Figures 5B and 5D show Masson's trichrome stained sections of femoral condyles injected with either a mixture of hChon and NIH3T3-BMP2 cells (B) or hChon alone (D). [Original magnification: (B and D) ×12.5]

[0111] Cartilage regeneration by injection of a cell mixture (human chondrocytes and NIH3T3-BMP-2 cells) into full-thickness defects in rabbits Either control hChon or a mixture of hChon and NIH3T3-BMP2 cells was injected into the knee joints of rabbits containing full-thickness cartilage defects (3 mm x 5 mm, 2-3 mm deep) in the femoral condyle. In this study, cell injection occurred 3 weeks after defect creation. The cell mixture (2 x 10 6 Twenty to 25 μl of cells / ml (hChon:NIH3T3-BMP-2 ratio 10:1) was injected into the upper and then the left side of the defect. The cells were allowed to infiltrate the wound for 15 to 20 minutes before suturing. Six weeks after injection, specimens were collected and examined microscopically. Figures 6A and 6D show photographs of femoral condyles 12 weeks after injection with either a mixture of hChon and NIH3T3-BMP2 cells (A) or hChon alone (D). Figures 6B and 6E show Masson's trichrome stained sections of femoral condyles injected with either a mixture of hChon and NIH3T3-BMP2 cells (B and C) or hChon alone (E), while Figure 6C shows Safranin O staining. [Original magnification: (B, C, and E) x12.5]

[0112] Cartilage regeneration by injection of a cell mixture (human chondrocytes and human 293-TGF-β1 cells) into full-thickness defects in rabbits Either control human chondrocytes (hChon) or a mixture of hChon and 293-TGF-β1 cells was injected into the knee joints of rabbits containing full-thickness cartilage defects (3 mm x 5 mm, 2-3 mm deep) in the femoral condyle. The cell mixture (2 x 10 6 Twenty to 25 μl of cells / ml (at a 1:1 ratio of hChon to 293-TGF-β1) were injected into the upper and then left sides of the defect. The cells were allowed to infiltrate the wound for 15 to 20 minutes before suturing. Six weeks after injection, specimens were collected and examined microscopically. Figures 7A and 7C show photographs of femoral condyles 6 weeks after injection with either a mixture of hChon and 293-TGF-β1 cells (A) or hChon alone (C). Figures 7B and 7D show Masson's trichrome stained sections of femoral condyles injected with either a mixture of hChon and 293-TGF-β1 cells (B) or hChon alone (D). [Original magnification: (B and D) ×12.5]

[0113] Cartilage regeneration by injection of a cell mixture (human chondrocytes and human 293-TGF-β1 cells) into rabbits with partial defects A mixture of hChon and 293-TGF-β1 cells was injected into the knee joints of rabbits containing a partial cartilage defect (3 mm x 5 mm, 1-2 mm deep) in the femoral condyle. The cell mixture (2 x 10 6 15–20 μl of cells / ml (hChon:293-TGF-β1 ratios of 3:1 or 5:1) were injected into the upper and then the left side of the defect. The cells were allowed to infiltrate the wound for 15–20 minutes before suturing. Six weeks after injection, specimens were collected and examined microscopically. Figures 8A and 8C show photographs of femoral condyles injected with a mixture of hChon and 293-TGF-β1 cells (3:1 ratio) (A) or a mixture of hChon and 293-TGF-β1 cells (5:1 ratio) (C) 6 weeks after injection. Figures 8B and 8D show Masson's trichrome stained sections of femoral condyles injected with a mixture of hChon and 293-TGF-β1 cells (3:1 ratio) (B) or a mixture of hChon and 293-TGF-β1 cells (5:1 ratio) (D). [Original magnification: (B and D) ×12.5]

[0114] All references cited herein are incorporated by reference in their entirety.

[0115] While particular embodiments of the present invention have been described above for purposes of illustration, it will be obvious to those skilled in the art that many changes can be made in the details of the invention without departing from the invention as defined by the appended claims.

Claims

1. A mixed cell composition for producing hyaline cartilage at a target site, comprising: (a) a first mammalian cell population transformed or transduced with a gene encoding TGF-β or a BMP; (b) a second mammalian cell population that has not been transformed or transduced with a gene encoding said TGF-β or BMP; and (c) a pharmaceutically acceptable carrier thereof; Including, the endogenous morphology of said second mammalian cell population is reduced at said target site; production of TGF-β or BMP by said first mammalian cell population at said target site stimulates said second mammalian cell population to induce a therapeutic effect; a ratio of the second mammalian cell population not transformed or transduced with a gene encoding a TGF-β or a BMP to the first mammalian cell population transformed or transduced with a gene encoding a TGF-β or a BMP of about 1 to 20 to 1; the first mammalian cell population is HEK293 cells and the second mammalian cell population is chondrocytes; The TGF-β is TGF-β1 and the BMP is BMP-2. Mixed cell composition.

2. The mixed cell composition of claim 1, which is an injectable composition.

3. The mixed cell composition of any one of claims 1 to 2, wherein the gene encodes TGF-β1.

4. The mixed cell composition of any one of claims 1 to 3, wherein the ratio is about 1 to 10 to 1.

5. 5. The mixed cell composition of claim 4, wherein said ratio is about 1-3 to 1.

6. 6. The mixed cell composition of any one of claims 1 to 5, wherein the first population of mammalian cells transformed or transduced with a gene encoding TGF-β or BMP is irradiated.

7. 7. The mixed cell composition of any one of claims 1 to 6, wherein the first mammalian cell population and the second mammalian cell population are derived from the same or different source organisms.

8. 8. The mixed cell composition of any one of claims 1 to 7, wherein the first mammalian cell population is a mammalian cell population that has been transformed or transduced in vitro with a recombinant vector comprising a DNA sequence encoding the gene operably linked to a promoter.

9. 9. The mixed cell composition of any one of claims 1 to 8, wherein the first mammalian cell population and the second mammalian cell population are syngeneic or xenogeneic with respect to the recipient host.

10. The mixed cell composition of claim 8, wherein the recombinant vector is a viral vector or a plasmid vector.

11. 9. The mixed cell composition of claim 8, wherein the transformation or transduction is achieved by liposome encapsulation, calcium phosphate co-precipitation, electroporation, DEAE-dextran mediation, or viral mediation.

12. 12. The mixed cell composition of any one of claims 1 to 11 for use in a method for treating osteoarthritis, said method comprising injecting said mixed cell composition into a joint cavity of a mammal.

13. A storage container for storing cells at temperatures between -70°C and -196°C, comprising the mixed cell composition of any one of claims 1 to 12.

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