WO2003082302A9 - Bioadhesive directed somatic cell therapy - Google Patents
Bioadhesive directed somatic cell therapyInfo
- Publication number
- WO2003082302A9 WO2003082302A9 PCT/US2003/009719 US0309719W WO03082302A9 WO 2003082302 A9 WO2003082302 A9 WO 2003082302A9 US 0309719 W US0309719 W US 0309719W WO 03082302 A9 WO03082302 A9 WO 03082302A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- tgf
- bmp
- cartilage
- gene
- Prior art date
Links
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Classifications
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
- A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1841—Transforming growth factor [TGF]
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- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
- A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
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- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
Definitions
- the present invention relates to a bioadhesive directed gene therapy composition for localized expression of a gene of interest, including various cytokines.
- the present invention also relates to a method of using the composition to regenerate cartilage.
- the present invention relates to a method of treating osteoarthritis by injecting the composition to a mammalian connective tissue.
- degenerative arthritis or osteoarthritis is the most frequently encountered disease associated with cartilage damage. Almost every joint in the body, such as the knee, the hip, the shoulder, and even the wrist, is affected. The pathogenesis of this disease is the degeneration of hyaline articular cartilage (Mankin et al., J Bone Joint Surg, 52A: 460-466, 1982). The hyaline cartilage of the joint becomes deformed, fibrillated, and eventually excavated. If the degenerated cartilage could somehow be regenerated, most patients would be able to enjoy their lives without debilitating pain.
- Bone mo ⁇ hogenetic protein has been considered to be an effective stimulator of bone formation (Ozkaynak et al, EMBO J, 9:2085-2093, 1990; Sampath and Rueger, Complications in Ortho, 101-107, 1994), and TGF- ⁇ has been reported as a stimulator of osteogenesis and chondrogenesis (Joyce et al., J Cell Biology, 1 10:2195- 2207, 1990).
- TGF- ⁇ Transforming growth factor- ⁇
- TGF- ⁇ Transforming growth factor- ⁇
- TGF- ⁇ inhibits the growth of epithelial cells and osteoclast-like cells in vitro (Chenu et al., Proc Natl Acad Sci, 85: 5683-5687, 1988), but it stimulates enchondral ossification and eventually 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- ⁇ -induced bone formation is mediated by its stimulation of the subperiosteal pluripotential cells, which eventually differentiate into cartilage-forming cells (Joyce et al., J Cell Biology, 110: 2195-2207, 1990; and Miettinen et al., J Cell Biology, 127-6: 2021-2036, 1994).
- TGF- ⁇ is present at the site of bone formation and cartilage formation. It can also enhance fracture healing in rabbit tibiae. Recently, the therapeutic value of TGF- ⁇ 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 effects and high cost have limited wide clinical application. [0009] Intraarticular injection of TGF- ⁇ for the treatment of arthritis is not desirable, because the injected TGF- ⁇ has a short duration of action, as TGF- ⁇ is degraded into inactive form in vivo. Therefore, a new method for long-term release of TGF- ⁇ is necessary for the regeneration of hyaline cartilage.
- United States Patents 5,858,355 and 5,766,585 disclose making a viral or plasmid construct of the IRAP (interleukin- 1 receptor antagonist protein) gene; transfecting synovial cells (5,858,355) and bone marrow cells (5,766,585) with the construct; and injecting the transfected cells into a rabbit joint, but there is no disclosure of using a gene belonging to the IRAP (interleukin- 1 receptor antagonist protein) gene; transfecting synovial cells (5,858,355) and bone marrow cells (5,766,585) with the construct; and injecting the transfected cells into a rabbit joint, but there is no disclosure of using a gene belonging to the IRAP (interleukin- 1 receptor antagonist protein) gene; transfecting synovial cells (5,858,355) and bone marrow cells (5,766,585) with the construct; and injecting the transfected cells into a rabbit joint, but there is no disclosure of using a gene belonging to the IRAP (interleukin- 1
- TGF- ⁇ superfamily to regenerate connective tissue.
- United States Patents 5,846,931 and 5,700,774 disclose injecting a composition that includes a bone mo ⁇ hogenesis protein (BMP), which belongs to the TGF ⁇
- BMP bone mo ⁇ hogenesis protein
- United States Patent 5,842,477 discloses implanting a combination of a scaffolding, periosteal/perichondrial tissue, and stromal cells, including chondrocytes, to a cartilage defected area. Since this patent disclosure requires that all three of these elements be present in the implanted system, the reference fails to disclose or suggest the simple gene therapy method of the invention which does not require the implantation of the scaffolding or the periosteal/perichondrial tissue.
- United States Patent 6,315,992 discloses that hyaline cartilage is generated in defected mammalian joint when fibroblast cells transfected with TGF- ⁇ 1 are injected into the defected knee joint.
- the patent does not disclose the advantages of using a bioadhesive composition as in the present invention.
- TGF- ⁇ 1 are injected into the defected knee joint.
- Lee et al. does not disclose using a bioadhesive composition as in the present invention.
- Mrowiec et al. Blood Cells, Molecules, and Diseases 21(3):25-33, 1995, shows a novel technique for preparing improved buffy coat platelet concentrates.
- Mrowiec et al. does not disclose mixing the purified buffy coat with cells for injection as a gene therapy composition.
- the present invention has met the hereinbefore described need.
- the present invention is directed to a composition comprising a treatment- effective amount of a population of mammalian somatic cells transfected or transduced with a therapeutic gene, and an adhesive-effective amount of bioadhesive material for administration into a mammalian site in need thereof.
- the bioadhesive material may be purified buffy coat.
- the gene may encode a cytokine. Further, the cytokine may belong to TGF- ⁇ superfamily.
- cytokine may be, without limitation, TGF- ⁇ l , TGF- ⁇ 2, TGF- ⁇ 3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, or BMP-9. Still further, the gene may encode TGF- ⁇ l or BMP-2.
- the cell may be a connective tissue cell.
- the connective tissue cell may be, without limitation, fibroblast or chondrocyte.
- the fibroblast or chondrocyte cell may be transfected or transduced with any therapeutic gene, including a cytokine, such as the genes encoding a member of the TGF- ⁇ superfamily.
- the ratio of the amount of the buffy coat to cells may be from about 1-5 to 1 in te ⁇ ns of volume. In addition, the ratio may be about 1-3 to 1. And still further, the ratio may be from about 1 to 1.
- the cells may be irradiated. And the cells may be mixed with cells that are not transfected or transduced with any DNA.
- the cells may be histocompatible with each other, in which case the cells may be derived from the same source organism.
- the cells may be also derived from different source organisms.
- the present invention is also directed to a storage container for storing the above- described cells at a temperature of about -70 ° C to about -196 ° C.
- the present invention is also directed to a method of localizing gene expression at a target site in a mammal, comprising mixing an adhesive-effective amount of a bioadhesive material with therapeutic somatic cells to form a composition, and administering the composition to the site in need thereof.
- the bioadhesive material may be purified buffy coat.
- the somatic cells may be transfected or transduced with a recombinant vector comprising a therapeutic gene.
- the therapeutic gene may be a cytokine, such as, but not limited to, the genes encoding a member of the TGF- ⁇ superfamily.
- the target site may be the joint space
- the gene may encode TGF- ⁇ l, TGF- ⁇ 2, TGF- ⁇ 3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, or BMP- 7.
- the gene may encode TGF- ⁇ l or BMP-2.
- the cells may be irradiated. Also, in the method described above, the cells may be syngeneic or allogeneic with respect to the host recipient.
- the present invention also encompasses the method above, and in particular, the somatic cells comprise a mixture of a first type of cells that are transfected or transduced with DNA encoding a therapeutic gene, and a second type of cells that are not transfected or transduced with DNA encoding a therapeutic gene.
- the cells may be stored prior to transplantation. And in particular, cells may be stored in a cryoperservative prior to transplantation.
- the transfection or transduction may be accomplished by liposome encapsulation, calcium phosphate coprecipitation, electroporation, DEAE-dextran mediation or viral mediation.
- the present invention is directed to a method of generating hyaline cartilage in a mammal comprising: a) generating a recombinant vector comprising a DNA sequence encoding transforming growth factor ⁇ (TGF- ⁇ ) or bone mo ⁇ hogenetic protein (BMP) operatively linked to a promoter; b) transfecting or transducing a population of fibroblast or chondrocyte cells in vitro with said recombinant vector; and c) injecting an injectable mixed cell composition comprising hyaline cartilage-generating effective amount of (i) a population of fibroblast or chondrocyte cells transfected or transduced with a gene encoding TGF- ⁇ or BMP; (ii) an adhesive effective amount of bioadhesive material; and (iii) a pharmaceutically acceptable carrier thereof, into a joint space of a mammal such that expression of the DNA sequence encoding TGF- ⁇ or BMP within the joint
- TGF- ⁇
- the invention is directed to a method of generating hyaline cartilage in a mammal comprising: a) generating a recombinant vector comprising a DNA sequence encoding transforming growth factor ⁇ (TGF- ⁇ ) or bone mo ⁇ hogenetic protein (BMP) operatively linked to a promoter; b) transfecting or transducing a population of fibroblast or chondrocyte cells in vitro with said recombinant vector; and c) injecting an injectable mixed cell composition comprising hyaline cartilage-generating effective amount of (i) a population of fibroblast or chondrocyte cells transfected or transduced with a gene encoding TGF- ⁇ or BMP; (ii) a population of fibroblast or chondrocyte cells that have not been transfected or transduced with a gene encoding TGF- ⁇ or BMP; (iii) an adhesive effective amount of bioadhes
- the invention is directed to a method of treating osteoarthritis comprising: a) generating a recombinant vector comprising a DNA sequence encoding transforming growth factor ⁇ (TGF- ⁇ ) or bone morphogenetic protein (BMP) operatively linked to a promoter; b) transfecting/transducing a population of fibroblast or chondrocyte cells in vitro with said recombinant vector; and c) injecting an injectable mixed cell composition comprising bone- and hyaline-generating effective amount of (i) a population of fibroblast or chondrocyte cells transfected or transduced with a gene encoding TGF- ⁇ or BMP; (ii) an adhesive effective amount of bioadhesive material; and (iii) a pha ⁇ naceutically acceptable earner thereof, into a joint space of a mammal such that expression of the DNA sequence encoding TGF- ⁇ or BMP within the joint space
- TGF- ⁇ transforming growth factor ⁇
- the invention is directed to a method of treating osteoarthritis comprising: a) generating a recombinant vector comprising a DNA sequence encoding transforming growth factor ⁇ (TGF- ⁇ ) or bone morphogenetic protein (BMP) operatively linked to a promoter; b) transfecting or transducing a population of fibroblast or chondrocyte cells in vitro with said recombinant vector; and c) injecting an injectable mixed cell composition comprising bone- and cartilage-generating effective amount of (i) a population of fibroblast or chondrocyte cells transfected or transduced with a gene encoding TGF- ⁇ or BMP; (ii) a population of fibroblast or chondrocyte cells that have not been transfected or transduced with a gene encoding TGF- ⁇ or BMP; (iii) an adhesive effective amount of bioadhesive material; and (iv) a pha ⁇ naceutically
- FIGS 1A-1C show expression of TGF- ⁇ l mRNA (A) and BMP2 (B and C).
- FIG 1A total RNA was isolated from NIH 3T3 cells or NIH 3T3 cells stably transfected with pmT ⁇ l , a TGF- ⁇ l expression vector, which was grown in the absence or presence of zinc. Total RNA (15 mg) was probed with either the TGF- ⁇ l cDNA or ⁇ actin cDNA as a control.
- Figures IB and IC show expression of BMP2 in NIH3T3-BMP2 cells.
- Figures IB and IC show control NIH3T3-methallothionein (B) and NIH3T3-BMP2 cells (C). Blue color in panel (C) shows expression of BMP2 protein.
- Figures 2A-2B show gross findings of regenerated cartilage.
- Figure 2A shows a rectangular partial cartilage defect was made on the femoral condyle and the knee joint was injected with NIH 3T3 cells without TGF- ⁇ l transfection. The defect was not covered.
- Figure 2B shows that at 6 weeks after injection of NIH 3T3-TGF- ⁇ l cells, the defect was covered by newly formed tissue. The color of the regenerated tissue was almost identical to that of the surrounding cartilage.
- Figures 3A-3D show microscopic findings of regenerated cartilage (X 200).
- Figure 3A and 3B show hematoxilin-eosine (H&E) analysis of defect area 4 and 6 weeks after injection with control cells. No tissue covered the initial defect area.
- Figures 3C and 3D show hematoxilin-eosine (H&E) analysis of defect area 4 and 6 weeks after injection of TGF- ⁇ l -transfected cells. At 4 weeks, partial defect area was covered by hyaline cartilage after injection of TGF- ⁇ 1 -transfected cells. At 4 weeks and 6 weeks after injection, the regenerated tissue became thicker and its height was almost, identical to normal cartilage at 6 weeks. Histologically, the regenerated cartilage (a ⁇ ow) was identical to the surrounding hyaline cartilage.
- Figures 4A-4B show immunohistochemical analysis for TGF- ⁇ l expression in rabbit joint x 200. Brown immunoperoxidase reaction product indicates high levels of recombinant TGF- ⁇ l expression in the NIH 3T3-TGF- ⁇ l cells ( Figure 4B).
- Figure 4A shows hyaline cartilage in a rabbit joint injected with control cells.
- Figures 5A-5B show microscopic findings (X 200) of regenerated tissues with H&E staining (A) and Safranin-0 staining (B).
- Figure 5A shows that in the partially damaged area, the regenerated hyaline cartilage is shown by H&E staining (black a ⁇ ow).
- Figure 5B shows that in the completely denuded cartilage area, the regenerated tissue (white arrow) was fibrous collagen.
- Figures 6A-6F and 6A'-6F' show regeneration of cartilage with irradiated
- NIH3T3- TGF- ⁇ l fibroblast cells NIH3T3- TGF- ⁇ l fibroblast cells.
- Figures 7A-7H show regeneration of cartilage with human foreskin fibroblast cells producing TGF- ⁇ 1.
- Figures 8A-8H show regeneration of cartilage with NIH3T3-TGF- ⁇ l cells in a dog model.
- Figures 9A-9C show immunohistochemical staining of regenerated cartilage with
- TGF- ⁇ l antibody at 3 weeks after injection of TGF- ⁇ l producing fibroblast cells.
- Figures 10A-10E show regeneration of cartilage with human chondrocyte cells producing TGF- ⁇ 1.
- Figure 11A-11D show regeneration of cartilage with mixed-cell (human chondrocytes and NIH3T3-TGF- ⁇ l cells) injection in rabbits with a partial defect.
- 1 1A and 1 IC show pictures of the femoral condyles 6 weeks post injection with either a mixture of hChon (human chondrocytes) and NIH3T3-TGF- ⁇ l cells (A) or hChon alone (C).
- Figures 1 IB and 1 ID show Mason's trichrome staining of sections from the femoral condyle injected with either a mixture of hChon and NIH3T3-TGF- ⁇ l cells (B) or hChon alone (D).
- Figures 12A-12E show regeneration of cartilage with mixed-cell (human chondrocytes and NIH3T3-TGF- ⁇ l cells) injection in rabbits with a full-thickness defect.
- Figures 12A and 12D show pictures of the femoral condyles 12 weeks post injection with either a mixture of hChon and NIH3T3-TGF- ⁇ l cells (A) or hChon alone (D).
- Figures 12B and 12E show Mason's trichrome staining
- Figure 12C shows Safranin-0 staining of sections from the femoral condyle injected with either a mixture of hChon and NIH3T3-TGF- ⁇ l cells (B & C) or hChon alone (E).
- Figures 13A-13D show regeneration of cartilage with mixed-cell (human chondrocytes and NIH3T3-BMP-2 cells) injection in rabbits with a partial defect.
- Figures 13A-13D show regeneration of cartilage with mixed-cell (human chondrocytes and NIH3T3-BMP-2 cells) injection in rabbits with a partial defect.
- FIGS. 13A and 13C show pictures of the femoral condyles 6 weeks post injection with either a mixture of hChon and NIH3T3-BMP-2 cells (A) or hChon alone (C).
- Figures 13B and 13D show Mason's trichrome staining of sections from the femoral condyle injected with either a mixture of hChon and NIH3T3-BMP-2 cells (B) or hChon alone (D).
- Figures 14A-14E show regeneration of cartilage with mixed-cell (human chondrocytes and NIH3T3-BMP-2 cells) injection in rabbits with a full-thickness defect.
- Figures 14A and 14D show pictures of the femoral condyles 12 weeks post injection with either a mixture of hChon and NIH3T3-BMP-2 cells (A) or hChon alone (D).
- Figures 14B and 14E show Mason's trichrome staining and Figure 14C shows Safranin-O staining of sections from the femoral condyle injected with either mixture of hChon and NIH3T3-BMP-2 cells (B & C) or hChon alone (E). Original magnification: (B, C & E) xl2.5.
- Figures 15A-15D show regeneration of cartilage with mixed-cell (human chondrocytes and human chondrocyte-TGF- ⁇ 1 cells) injection in rabbits with a full-thickness defect.
- Figures 15A and 15C show pictures of the femoral condyles 6 weeks post injection with either a mixture of hChon and hChon-TGF- ⁇ l cells (A) or hChon alone (C).
- Figures 15B and 15D show Mason's trichrome staining of sections from the femoral condyle injected with either mixture of hChon and hChon-TGF- ⁇ l cells (B) or hChon alone (D). [Original magnification: (B& D) xl2.5].
- Figures 16A-16F show regeneration of cartilage with injection of a mixture of human buffy coat and NIH3T3-TGF- ⁇ l cells in a rabbit knee joint with a partial defect.
- Figures 17A-17D show pictures of the femoral condyles 6 weeks post injection with a mixture of hChon and hChon-TGF- ⁇ 1 cells at either a partial (A) or full-thickness defect (C).
- Figures 17B and 17D show Mason's trichrome staining of sections from the femoral condyle injected with a mixture of hChon and hChon-TGF- ⁇ l cells at a partial (B) or full-thickness defect (D). [Original magnification: (B& D) xl2.5].
- bioadhesive or “bio-adhesive” composition, formulation, material and so on refer to a naturally occurring or synthetic compound that adheres, binds or interacts with a biological tissue, including, but not limited to, connective tissue, and in particular, bone and cartilage.
- the bioadhesive used with the invention results in prolonging the residence time of the therapeutic somatic cell at the site of contact.
- the bioadhesive material is mixed with the somatic cells to produce an adhesive effective mixture.
- biologically derived carrier molecule refers to molecules that are naturally found in a mammal, which is useful as a carrier. Examples of it include albumin, glycosaminoglycans, heparin, hyaluronic acid, collagens, buffy coat and the like. The somatic cells may be mixed with these carrier molecules.
- the term "buffy coat” or “buffy crust” refers to a thin yellowish layer that contains leukocytes and platelets overlying the packed red cells in centrifuged blood.
- connective tissue is any tissue that connects and supports other tissues or organs, and includes but is not limited to a ligament, a cartilage, a tendon, a bone, and a synovium of a mammalian host.
- connective tissue cell and “cell of a connective tissue” include cells that are found in the connective tissue, such as fibroblasts, cartilage cells
- the connective tissue cells are fibroblasts, cartilage cells, and bone cells.
- the invention can be practiced with a mixed culture of connective tissue cells, as well as cells of a single type.
- the tissue cells may be pretreated with chemical compounds or radiation before injecting them into the joint space so that the cells stably express the gene of interest within the host organism.
- the connective tissue cell does not cause a negative immune response when injected into the host organism. It is understood that allogeneic cells may be used in this regard, as well as autologous cells for cell-mediated gene therapy or somatic cell therapy.
- connective tissue cell line includes a plurality of connective tissue cells originating from a common parent cell.
- helper cells refer to somatic cells that are not transfected or transduced with the therapeutic gene. In one embodiment, these cells are not transfected or transduced with any therapeutic gene. Helper cells are mixed with therapeutic somatic cells that produce the therapeutic gene product. Such helper cells may include any cell at all, including connective tissue cells, such as fibroblasts and chondrocytes.
- hyaline cartilage refers to the connective tissue covering the joint surface.
- hyaline cartilage includes, but is not limited to, articular cartilage, costal cartilage, and nose cartilage.
- hyaline cartilage is known to be self-renewing, responds to alterations, and provides stable movement with less friction.
- Hyaline cartilage found even within the same joint or among joints varies in thickness, cell density, matrix composition and mechanical properties, yet retains the same general structure and function.
- Some of the functions of hyaline cartilage include suiprising stiffness to compression, resilience, and exceptional ability to distribute weight loads, ability to minimize peak stress on subchondral bone, and great durability.
- hyaline cartilage appears as a slick, fi ⁇ n surface that resists deformation.
- the extracellular matrix of the cartilage comprises chondrocytes, but lacks blood vessels, lymphatic vessels or nerves.
- An elaborate, highly ordered structure that maintains interaction between chondrocytes and the matrix serves to maintain the stmcture and function of the hyaline cartilage, while maintaining a low level of metabolic activity.
- O'Driscoll J. Bone Joint Surg., 80A: 1795-1812, 1998 describes the structure and function of hyaline cartilage in detail, which is incoiporated herein by reference in its entirety.
- injectable composition refers to a composition that excludes various three-dimensional scaffold, framework, mesh or felt stmcture may be made of any material or shape that allows cells to attach to it and allows cells to grow in more than one layer, and which stmcture is generally implanted, not injected.
- the injection is typically ca ⁇ ied out by a syringe.
- any mode of injecting the composition of interest may be used. For instance, catheters, sprayers, or temperature dependent polymer gels also may be used.
- mammalian host includes members of the animal kingdom including but not limited to human beings.
- the cells are used in cell- mediated gene therapy. More preferably, the cells are connective tissue cell that include cells that have been transfected or transduced with a gene or DNA encoding any therapeutic gene product. In particular, this gene product is a member of the transfo ⁇ ning growth factor ⁇ superfamily.
- the mixture includes helper cells that have not been transfected or transduced with the therapeutic gene.
- the ratio of cells that have not been transfected or transduced with a gene encoding a member of the transforming growth factor ⁇ superfamily to cells that have been transfected or transduced with a TGF superfamily gene may be in the range of about 3-20 to 1.
- the range may include about 3-10 to 1.
- the range may be about 10 to about 1 in terms of the number of cells.
- the ratio of these cells should not be necessarily fixed to a particular range so long as the combination of these cells is effective to produce hyaline cartilage in partially and fully defected joints.
- patient includes members of the animal kingdom including but not limited to human beings.
- pharmaceutically accepted carrier refers to any carrier that is known in the art to promote the efficiency of transport of the composition of the invention and prolong the effectiveness of the composition.
- “somatic cell” or “cell” in general refers to the cell of the body other than egg or spe ⁇ n.
- stored cells refer to a composition of mixed cells that have been either stored individually or together before they are administered to a mammalian host.
- the cells may be stored in a refrigeration unit. Or, the cells may be frozen at about -20° to -70°C so that the cells are preserved for later administration into the mammalian host.
- the cells may be thawed using known protocols. The duration of freezing and thawing may be carried out by any number of ways, so long as the viability and potency of the cells are optimized.
- TGF- ⁇ transforming growth factor- ⁇
- the "transforming growth factor- ⁇ (TGF- ⁇ ) superfamily” encompasses a group of stmcturally related proteins, which affect a wide range of differentiation processes during embryonic development.
- the family includes, M ⁇ llerian inhibiting substance (MIS), which is required for normal male sex development (Behringer, et al., Nature, 345:167, 1990), Drosophila decapentaplegic (DPP) gene product, which is required for dorsal-ventral axis formation and mo ⁇ hogenesis of the imaginal disks (Padgett, et al., Nature, 325:81-84, 1987), the Xenopus Ng-1 gene product, which localizes to the vegetal pole of eggs (Weeks, et al, Cell, 51:861-867, 1987), the activins (Mason, et al., Biochem, Biophys. Res.
- MIS M ⁇ llerian inhibiting substance
- DPP Drosophila decapentaplegic
- TGF- ⁇ gene products can influence a variety of differentiation processes, including adipogenesis, myogenesis, chondrogenesis, hematopoiesis, and epithelial cell differentiation.
- the proteins of the TGF- ⁇ family are initially synthesized as a large precursor protein, which subsequently undergoes proteolytic cleavage at a cluster of basic residues approximately 1 10-140 amino acids from the C-te ⁇ ninus.
- the C-terminal regions of the proteins are all stmcturally related and the different family members can be classified into distinct subgroups based on the extent of their homology. Although the homologies within particular subgroups range from 70% to 90% amino acid sequence identity, the homologies between subgroups are significantly lower, generally ranging from only 20% to 50%.
- the active species appears to be a disulfide-linked dimer of C-terminal fragments.
- the homodimeric species has been found to be biologically active, but for other family members, like the inhibins (Ung, et al., Nature, 321 :779, 1986) and the TGF- ⁇ 's (Cheifetz, et al., Cell, 48:409, 1987), heterodimers have also been detected, and these appear to have different biological properties than the respective homodimers.
- TGF- ⁇ genes include TGF- ⁇ 3, TGF- ⁇ 2, TGF- ⁇ 4 (chicken), TGF- ⁇ l, TGF- ⁇ 5 (Xenopus), BMP-2, BMP-4, Drosophila DPP, BMP-5, BMP-6, Vgrl, OP-l/BMP-7, Drosophila 60A, GDF-1, Xenopus Vgf, BMP-3, Inhibin- ⁇ A, Inhibin- ⁇ B, Inhibin- ⁇ , and MIS. These genes are discussed in Massague, Ann. Rev. Biochem. 67:753-791, 1998, which is inco ⁇ orated herein by reference in its entirety.
- the member of the superfamily of TGF- ⁇ genes is TGF- ⁇ and BMP. More preferably, the member is TGF- ⁇ l, TGF- ⁇ 2, TGF- ⁇ 3, BMP-2, BMP-3, BMP-4, BMP- 5, BMP-6, or BMP -7. Most preferably, the member is human or porcine TGF- ⁇ l or BMP-2.
- selectable marker includes a gene product that is expressed by a cell that stably maintains the introduced DNA, and causes the cell to express an altered phenotype such as mo ⁇ hological transformation, or an enzymatic activity.
- Isolation of cells that express a transfected gene is achieved by optional introduction into the same cells a second gene that encodes a selectable marker, such as one having an enzymatic activity that confers resistance to an antibiotic or other dmg.
- selectable markers include, but are not limited to, thymidine kinase, dihydrofolate reductase, aminoglycoside phosphotransferase, which confers resistance to aminoglycoside antibiotics such as kanamycin, neomycin and geneticin, hygromycin B phosphotransferase, xanthine-guanine phosphoribosyl transferase, CAD (a single protein that possesses the first three enzymatic activities of de novo uridine biosynthesis - carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase), adenosine deaminase, and asparagine synthetase
- a selectable marker is not a requirement to practice the claimed invention. In fact, in one embodiment, a selectable marker is not inco ⁇ orated into the genetic constmct of the claimed invention.
- a "promoter” can be any sequence of DNA that is active, and controls transcription in an eucaiyotic cell.
- the promoter may be active in either or both eucaiyotic and procaryotic cells.
- the promoter is active in mammalian cells.
- the promoter may be constitutively expressed or inducible.
- the promoter is inducible.
- the promoter is inducible by an external stimulus. More preferably, the promoter is inducible by ho ⁇ nones or metals. Still more preferably, the promoter is inducible by heavy metals. Most preferably, the promoter is a metallothionein gene promoter. Likewise,
- “enhancer elements”, which also control transcription, can be inserted into the DNA vector constmct, and used with the constmct of the present invention to enhance the expression of the gene of interest.
- DC-chol means a cationic liposome containing cationic cholesterol derivatives.
- the "DC-chol” molecule includes a tertiary amino group, a medium length spacer arai (two atoms) and a carbamoyl linker bond (Gao et al., Biochem. Biophys.
- SF-chol is defined as a type of cationic liposome.
- the te ⁇ n "biologically active" used in relation to liposomes denotes the ability to introduce functional DNA and/or proteins into the target cell.
- biologically active in reference to a nucleic acid, protein, protein fragment or derivative thereof is defined as an ability of the nucleic acid or amino acid sequence to mimic a known biological function elicited by the wild type fo ⁇ n of the nucleic acid or protein.
- the te ⁇ n “maintenance” when used in the context of liposome delivery, denotes the ability of the introduced DNA to remain present in the cell. When used in other contexts, it means the ability of targeted DNA to remain present in the targeted cell or tissue so as to impart a therapeutic effect.
- the therapeutic somatic cell may be fully or partially sunounded by bio-adhesive material.
- the bioadhesive material may affect the release profile of the therapeutic substance, and therefore the amount of the admixed bioadhesive material should not hinder or adversely alter the desired release profile of the therapeutic substance.
- suitable bio-adhesive materials should be bio-compatible, that is non-toxic, non-inflammatory, substantially non-immunogenic and hemo-compalible in the amounts employed.
- Bio-adhesive materials can either have non-specific or specific binding properties. Bio-adhesive materials with non-specific binding properties will adhere generally to the cells and the components of the extracellular matrix that fonu the tissue at the treatment site, through, for example, charge interactions. Examples of bio-adhesive material with nonspecific binding properties include, but are not limited to, CARBOPOL® (BF Goodrich Perfonnance Materials, Cleveland, OH) polymers, homopolymers and copolymers. The CARBOPOL® polymers include high molecular weight, crosslinked, acrylic acid-based polymers.
- CARBOPOL® BF Goodrich Perfonnance Materials, Cleveland, OH
- CARBOPOL® homopolymers include polymers of aciylic acid crosslinked with allyl sucrose or allylpentaerythritol.
- CARBOPOL® copolymers include polymers of acrylic acid, modified by long chain (C10-C30) alkyl acrylates, and crosslinked with allylpentaerythritol.
- Carbopol-poloxamer gels hydroxypropylmethylcellulose, sodium carboxymethyl cellulose, Guar gum, polyvinyl pyrrolidone, chitosan, polyacrylic acid, hydroxypropyl cellulose, polycarbophil, sodium starch glycolate, alginate, and their mixtures and/or copolymers, as well as mixtures, copolymers or graft constructs with polyethylene glycol.
- Bio-adhesive materials with specific binding properties adhere to tissue through specific intermolecular interactions with molecules exposed on the surface of the cells and matrix of the tissue at the treatment site.
- bio-adhesive materials with specific binding properties include, but are not limited to, lectins, ligands and antibodies to receptor proteins such as cell adhesion molecules and integrins, and albumin.
- bio-adhesive material will depend on the intended use and placement of the somatic cells, and in particular, the characteristics of the treatment site to which the cells need to adhere. For example, in cartilaginous or bony areas, buffy coat may be effectively used as an adhesive material, which may preferentially bind to such a solid or semi-solid surface. It is also contemplated that if the target tissue area has a positive charge, then a negatively charged polymeric molecule may be used to aid in the adhesiveness of the therapeutic somatic cells to the target tissue, and vice versa.
- a bio-adhesive material with specific binding properties can be chosen for use in the carrier composition.
- a bio-adhesive material that binds to molecules exposed only on tissues in specific regions of the body can be used, thus directing the somatic cells to bind to those tissues.
- the treatment site is a region of tissue that is diseased or injured
- the somatic cells can be targeted to the treatment site by using a bio-adhesive material that binds to molecules that are exposed on that region of tissue due to the disease or injury.
- Lectins are proteins or glyco-protein conjugates that bind to sugar moieties, and therefore, lectins will bind to glycosylated cell surface. If the target treatment site is the heavily glycosylated cells, lectins can be used as a bio-adhesive material that will adhere to the tissue. Lectins are also advantageous because they are generally non-immunogenic.
- lectins include, but are not limited to, Lycopersicon eculantum agglutinin, wheat germ agglutinin, Urtica dioica agglutinin, peanut agglutinin, tomato lectin, and Ulex europaeus isoagglutinin.
- Adhesion molecules mediate cell-cell binding by specifically recognizing and binding to molecules on the surface of other cells.
- ligands or antibodies that bind to adhesion molecules exposed on the surface of cells at a particular treatment site can be used as a specific bio-adhesive material to a treatment site.
- a bio-adhesive mixture may be made of those having nonspecific binding properties and those bio-adhesive material having specific binding properties. Such a mixture may improve adhesion of therapeutic somatic cell-bioadhesive mixture to a particular treatment site by using, for example, both non-specific charge interactions and specific intermolecular interactions between the bio-adhesive materials and the tissue at the treatment site.
- the above-described bioadhesive-somatic cell mixture may be delivered to a desired treatment site, which is typically an internal body tissue including, but not limited to, a joint space.
- the bioadhesive- somatic cell may be delivered using any apparatus or technique, such as by injection, so long as the contents are effectively delivered to or near the target tissue.
- the invention is directed to using a composition comprised of fibrinogen and thrombin, preferably from a human blood semm.
- the composition may act like a liquid-type glue when fibrinogen and thrombin solutions are mixed. Further, if the fibrinogen and thrombin are isolated from human se m, the composition should be non- toxic.
- a composition may be commercially available through for example GreenplastTM (Greencross, Korea). Without being limited to any specific process. Without being bound by any particular process of preparing the composition, an example may be as follows:
- Buffy coat may be mixed with therapeutic somatic cells before administering the mixture to a site in the body that would benefit from the expression of the therapeutic gene product made from the therapeutic somatic cells.
- Buffy coat possesses the physical property of being adhering to solid substrates, such as bone and cartilage, as well as semi-solid substrates such as muscle and other tissue, and thus may be used to provide a type of temporary "glue" to hold the therapeutic somatic cell in place at the site of administration so that localized delivery of the therapeutic gene product is achieved.
- buffy coat may be mixed with connective tissue cells that are then injected into the joint space so that a therapeutic gene, such as a cytokine is expressed resulting in a prolonged and effective delivery of the gene product to regenerate cartilage or bone.
- a therapeutic gene such as a cytokine is expressed resulting in a prolonged and effective delivery of the gene product to regenerate cartilage or bone.
- Buffy coat is the middle layer in the centrifuge tube when a sample of blood is centrifuged. Top layer is plasma, and the bottom layer contains erythrocytes. However, buffy coat contains leukocytes and platelets. There are various methods of purifying the contents of the buffy coat layer. According to the present invention, this layer may be extracted from a blood sample and mixed with cells that are used in somatic cell therapy protocols.
- buffy coat assists in binding, fixing or detaining the cells to physiological stmcture within the mammalian host such as a bone or cartilage so that these cells are expressed.
- the ratio of amount of buffy coat to cells may be in the range of about 1-5 to 1 by volume percent of the injectable composition. Typically, the range may include about 1-3 to 1. In particular, the range may be about 1 to 1 in terms of the volume of buffy coat and cells injected.
- buffy coat may be made by centrifuging the anti-coagulated blood in a narrow test tube, then carefully removing as much as possible of the plasma without disturbing the buffy coal. Buffered 2% Glutaraldehyde is then very gently layered on top and the tube left to stand in the fridge for about a couple of hours. This gives a buffy coat which is embedded in solid plasma and can be removed from the tube with the help of a thin wooden stick or similar object. The resultant disk can then be trimmed and the pieces processed to resin for use with normal tissue.
- the slender tube is cut with a razor blade above and below the buffy coat (on a piece of Paraf ⁇ lm), making a short log with open ends. Then, with a paper clip or applicator stick (depending on the diameter of the tube) the packed buffy coat is pushed out. 1% molten agar is sometimes used to keep it together. The pellet is then processed.
- the components of a buffy coat are not well characterized, but the buffy coat of the present invention encompasses the middle layer of a centrifuged blood sample.
- the bioadhesive material of the invention may be mixed with any cell that may be administered to a mammalian host for somatic cell gene therapy.
- the buffy coat may be used with connective tissue cells, and in particular cartilage generating cells, such as fibroblasts and chondrocytes.
- the bioadhesive material of the invention may be mixed with a further mixture of cells that have been transfected or transduced with a gene encoding a cytokine and cells that have not been so engineered.
- the combination is more adhesive to the defect than with cells alone.
- the buffy coat method provides greater independence between the location of administration of the cell-buffy coat combination and the location of the defect.
- the cell-buffy coat combination also provides a higher success rate for the generation of cartilage in animal tests.
- the average quality of newly generated cartilage is substantially better than from injecting cells alone. In other words, the percentage of normal-like cartilage generated was greater when the cell-buffy coat combination composition was used. Thus, the amount of fibrous cartilage observed was lower for the cell- buffy coat composition than using cells alone.
- the present invention discloses ex vivo and in vivo techniques for delivery of a DNA sequence of interest to the connective tissue cells of the mammalian host.
- the ex vivo technique involves culture of target connective tissue cells, in vitro transfection of the DNA sequence, DNA vector or other delivery vehicle of interest into the connective tissue cells, followed by transplantation of the modified connective tissue cells to the target joint of the mammalian host, so as to effect in vivo expression of the gene product of interest.
- the invention in a cell- mediated gene therapy or somatic cell therapy, is directed to a simple method of injecting a population of transfected or transduced connective tissue cells to the joint space so that the exogenous TGF superfamily protein is expressed in the joint space.
- One ex vivo method of treating a connective tissue disorder disclosed throughout this specification comprises initially generating a recombinant viral or plasmid vector which contains a DNA sequence encoding a protein or biologically active fragment thereof. This recombinant vector is then used to infect or transfect a population of in vitro cultured connective tissue cells, resulting in a population of connective cells containing the vector. These connective tissue cells together with buffy coat are then transplanted to a target joint space of a mammalian host either as a mixture of transfected and untransfected cells or separately into the joint space so as to cause a mixture of cell types inside the joint, thus effecting subsequent expression of the protein or protein fragment within the joint space.
- the source of cells for treating a human patient may be the patient's own connective tissue cells, such as autologous fibroblast or chondrocyte cells, but that allogeneic cells as well as xenogeneic cells may also be used without regard to the histocompatibility of the cells.
- this method includes employing as the gene 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.
- a further embodiment of the present invention includes employing as the gene a gene capable of encoding at least one member of the transforming growth factor ⁇ superfamily or a biologically active derivative or fragment thereof, and employing as the DNA plasmid vector any DNA plasmid vector known to one of ordinary skill in the art capable of stable maintenance within the targeted cell or tissue upon delivery, regardless of the method of delivery utilized.
- Another embodiment of this invention provides a method for introducing at least one gene encoding a product into at least one cell of a connective tissue for use in treating the mammalian host.
- This method includes employing non-viral means for introducing the gene coding for the product into the connective tissue cell. More specifically, this method includes a liposome encapsulation, calcium phosphate coprecipitation, electroporation, or DEAE- dextran mediation, and includes employing as the gene a gene capable of encoding a member of transfo ⁇ ning growth factor superfamily or biologically active derivative or fragment thereof, and a selectable marker, or biologically active derivative or fragment thereof.
- Another embodiment of this invention provides an additional method for introducing at least one gene encoding a product into at least one cell of a connective tissue for use in treating the mammalian host.
- This additional method includes employing the biologic means of utilizing a vims to deliver the DNA vector molecule to the target cell or tissue.
- the vims is a pseudo-virus, the genome having been altered such that the pseudovims is capable only of delivery and stable maintenance within the target cell, but not retaining an ability to replicate within the target cell or tissue.
- the altered viral genome is further manipulated by recombinant DNA techniques such that the viral genome acts as a DNA vector molecule which contains the heterologous gene of interest to be expressed within the target cell or tissue.
- a preferred embodiment of the invention is a method of delivering TGF- ⁇ or BMP to a target joint space by delivering the TGF- ⁇ or BMP gene to the connective tissue of a mammalian host through use of a retroviral vector with the ex vivo technique disclosed within this specification.
- a DNA sequence of interest encoding a functional TGF- ⁇ or BMP protein or protein fragment is subcloned into a retroviral vector of choice.
- the recombinant viral vector is then grown to adequate titer and used to infect in vitro cultured connective tissue cells, and the transduced connective tissue cells, preferably autografted cells, are mixed with buffy coat and are transplanted into the joint of interest with optionally an untransfected sample of connective tissue cell such as chondrocytes preferably by intra-articular injection.
- fibroblasts are cultured in vitro for subsequent utilization as a delivery system for gene therapy. It will be apparent that Applicants are not limited to the use of the specific connective tissue disclosed. It would be possible to utilize other tissue sources for in vitro culture techniques. The method of using the gene of this invention may be employed both prophylactically and in the therapeutic treatment of arthritis. It will also be apparent that Applicants are not limited to prophylactic or therapeutic applications in treating only the knee joint. It would be possible to utilize the present invention either prophylactically or therapeutically to treat arthritis in any susceptible joint.
- a compound for parenteral administration to a patient in a therapeutically effective amount contains a gene encoding a TGF- ⁇ superfamily protein and a suitable pha ⁇ naceutical carrier.
- Another embodiment of this invention provides for a compound for parenteral administration to a patient in a prophylactically effective amount that includes a gene encoding a TGF- ⁇ superfamily protein and a suitable pharmaceutical carrier.
- the cells are stored before administration to the joint space. The transfected or transduced cells alone may be stored, or optionally the untransfected helper cells alone may be stored, or the mixture may be stored, but not necessarily simultaneously.
- the duration of storage need not be for the same time period.
- the individually stored cells may be optionally mixed prior to injection.
- the cells may be stored and injected separately to form a mixture of cells within the joint space. It will be appreciated by those skilled in the art that these cells may be stored frozen in about 10 percent DMSO in liquid nitrogen.
- the buffy coat may be included in the storage composition.
- Another embodiment of this invention includes a method of introducing at least one gene encoding a product into at least one cell of a connective tissue of a mammalian host for use in treating the mammalian host as hereinbefore described including effecting in vivo the infection of the cell by introducing the viral vector containing the gene coding for the product directly into the mammalian host.
- this method includes effecting the direct introduction into the mammalian host by intra-articular injection.
- This method includes employing the method to substantially prevent the development of arthritis in a mammalian host having a high susceptibility of developing arthritis.
- This method also includes employing the method on an arthritic mammalian host for therapeutic use. Further, this method also includes employing the method to repair and regenerate the connective tissue as hereinbefore defined.
- the viral vectors employing a liposome are not limited by cell division as is required for the retrovimses to effect infection and integration of connective tissue cells.
- This method employing non-viral means as hereinbefore described includes employing as the gene a gene capable of encoding a member belonging to the TGF- ⁇ superfamily and optionally, a selectable marker gene, such as an antibiotic resistance gene. And it is also understood that using a selectable marker gene is not a requirement to practicing the claimed invention.
- Another embodiment of the present invention is delivery of a DNA sequence encoding a member of the TGF- ⁇ superfamily to the connective tissue of a mammalian host by any of the methods disclosed within this specification so as to effect in vivo expression of collagen to regenerate connective tissue, such as cartilage.
- Connective tissues are difficult organs to target therapeutically.
- Intravenous and oral routes of dmg delivery that are known in the art provide poor access to these connective tissues and have the disadvantage of exposing the mammalian host body systemically to the therapeutic agent.
- known intra-articular injection of proteins to joints provides direct access to a joint.
- most of the injected dmgs in the fo ⁇ n 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 genes coding for proteins that may be used to treat the mammalian host. More specifically, this invention provides a method for introducing into the connective tissue of a mammalian host genes coding for proteins with anti-arthritic properties.
- various cytokine producing cells such as fibroblasts and chondrocytes as well as mixtures of various cell types stimulated collagen synthesis in the joint. Compositions that included buffy coat also showed stimulation of collagen synthesis.
- the joint was generally injected with about 10 cells/ml concentration. The specimens were harvested from 2 weeks to 12 weeks after injection. The cells move freely within the joint, and move to the area with specific affinity for these cells. The synovium, meniscus and cartilage defect areas may be possible sites for cellular adhesion. At two to twelve weeks after injection, the regenerated tissues were observed in both the partially and completely damaged cartilage defect areas. This specific affinity for the damaged area is another advantage of using mixed cells for clinical application. If degenerative arthritis can be cured with just injection of cells into the joint without including various physical apparatuses such as scaffolding or any other three-dimensional stmcture, the patients can be treated conveniently without major surgery.
- the finding of hyaline cartilage synthesis by using the cytokine producing cells, mixed cell compositions and buffy coat containing compositions of the invention indicate that a long duration of high TGF- ⁇ or BMP concentration can stimulate hyaline cartilage regeneration.
- the properties of newly formed tissue were determined by histological methods. Through H & E staining, Mason's trichrome staining and Safranin-O, it was indicated that the newly formed tissue was identical to the su ⁇ ounding hyaline cartilage.
- 660/+63 was generated by polymerase chain amplification using genomic DNA using Xba I and Bam HI restriction sites built into the oligonucleotides used for amplification.
- the amplified fragment was subcloned into Xba I-Bam HI sites of pBluescript (Stratagene, La
- the plasmid pmT ⁇ l was generated by subcloning a 1.2-kb Bgl II fragment containing the TGF- ⁇ l coding sequence and a growth hormone poly A site at the 3' end into the Bam Hi-Sal I sites of pM.
- the plasmid pMTBMP2 was generated by subcloning a 1.2-kb
- pMTMLV vector was derived from the retroviral vector MFG by deleting entire gag and env sequences as well as some of ⁇ packaging sequence.
- TGF- ⁇ l cDNA was transfected into fibroblasts (NIH 3T3-TGF- ⁇ l), human foreskin fibroblasts (human foreskin-TGF- ⁇ l), and chondrocytes (hChon-TGF- ⁇ l). They were cultured in Dulbecco's Modified Eagle's Medium
- TGF- ⁇ l cDNA sequence was added into the pmT ⁇ l vector with a metallothionein gene promoter.
- a neomycin resistance gene sequence was also inserted into the vector.
- RNA was electrophoresed on a 1.0 % agarose gel containing 0.66M formaldehyde, transfe ⁇ ed to a DURALON-UN membrane, and cross linked with a UN STRATALI ⁇ KER (STRATAGE ⁇ E). Blots were prehybridized and hybridized in a solution of 1% bovine semm albumin, 7% (w/v) SDS, 0.5 M sodium phosphate, and 1 mM EDTA at 65°C. Hybridized blots were washed in 0J % SDS, 1 X SSC for 20 minute periods at 50°C before film exposure. R ⁇ A blots were hybridized with 32P- labelled cD ⁇ A probes for human TGF- ⁇ l . A probe for ⁇ -actin was used to control for sample loading.
- Stable cell line - Transfection was earned out by using the calcium phosphate coprecipitation method (Fig. 1A-1C). About 80% of the surviving colonies expressed the transgene mRNA. These selected TGF- ⁇ l -producing cells were incubated in a zinc sulfate solution. When the cells were cultured in 100 mM zinc sulfate solution, they produced mRNA. The TGF- ⁇ secretion rate was about 32 ng/10 6 cells/24 hr.
- NIH3T3 cells 1.5 x 10 NIH3T3 cells were grown overnight in a 6 well tissue culture plate. 0.5 x 10 indicating cells (MC3T3E1) were placed in tissue culture inserts and grown overnight. Culture medium was aspirated from the culture insert and the culture insert transferred into a 6 well plate and incubated for 48-72 hours. Culture medium was aspirated from the culture inserts. 5ml of IX phosphate buffered saline (PBS) was added to wash the cells. 4ml of 3.7% formaldehyde/ IX PBS solution was added to each insert, and the cells were fixed for 20min at 4 ° C. Cells were washed twice with IX PBS.
- PBS IX phosphate buffered saline
- ALP staining solution is 0J mg/ml naphthol AS-MX phosphate (Sigma N5000), 0.5% ,N-dimethylfo ⁇ namide (Sigma D8654), 2 mM MgCl 2 , 0.3 mg/ml Fast Blue BB salt (Sigma F3378) in 0.1 M Tris-HCl, pH 8.5.
- the contralateral side injected with normal fibroblasts without TGF- ⁇ l transfection was not covered by hyaline cartilage.
- the regenerated hyaline cartilage was colored red in Safranin-O staining (Fig. 4).
- the depth of newly formed cartilage was almost the same as that of the defect. This finding suggests that the injected cells activate the sui ⁇ ounding no ⁇ nal cartilage cells through a paracrine mode of action.
- Either control NIH3T3 or NIH3T3-TGF- ⁇ l cells (5-7 x 10 5 ) were irradiated with 6000 rad. and injected into rabbit knee joints. These i ⁇ adiated cells died completely in 3 weeks in a tissue culture dish. The injection procedure was the same as in the previous protocol with untreated cells. The knee joints were harvested at 3 or 6 weeks post injection. The specimens were fixed in formalin and decalcified with nitric acid. Sections of the specimens were made and embedded with paraffin and then cut into 0.5 mm thickness slices. In Fig.
- Either control human foreskin fibroblast (hFSF) or hFSF-TGF- ⁇ l cells were injected into the rabbit knee joint containing a partial cartilage defect (3mm x 5mm, 1.5mm deep) on the femoral condyle. These cells (0.5ml of 2 x 106 cells/ml) were injected as in the previous protocol, or 20-25 ml cells of the same concentration were loaded to the top of the defect. In the latter case, the cells were left in the defect for 15-20 min to let them settle down at the bottom of the defect before suturing. In both cases, a similar level of cartilage regeneration was obtained. The specimens were harvested at 6 weeks after injection and observed microscopically. Fig.
- FIG. 7A & B show pictures of the femoral condyles 6 weeks post injection with either hFSF (A) or hFSF-TGF- ⁇ l cells (B).
- C, E, & G show Safranin-O staining (C & E) and H&E staining (G) of sections from the femoral condyle injected with control hFSF cells.
- D, F, & H show Safranin-O staining (D & F) and H&E staining (H) of sections from the femoral condyle injected with hFSF-TGF- ⁇ l cells. (Original magnification: (C & D) ⁇ l2.5; (E-H) x400).
- Either control ⁇ IH3T3 or NIH3T3-TGF- ⁇ 1 cells was injected into the dog knee joint containing a partial cartilage defect (6mm x 6mm, 2mm deep) on the femoral condyle. These cells (4ml of 2 x 106 cells/ml) were injected as in the previous protocol, or 30-35 ml cells of the same concentration were loaded to the top of the defect. In the latter case, the cells were left in the defect for 15-20 min to let them settle down at the bottom of the defect before suturing. In both cases, a similar level of cartilage regeneration was obtained. The specimens were harvested at 6 weeks post injection and observed microscopically. Fig.
- a & B show pictures of the femoral condyles 6 weeks post injection with either NIH3T3 cells (A) or NIH3T3-TGF- ⁇ l cells (B).
- C, E, & G show Safranin-O staining (C & E) and H&E staining (G) of sections from the femoral condyle injected with control NIH3T3 cells.
- D, F, & H show Safranin-O staining (D & F) and H&E staining (H) of sections from the femoral condyle injected with NIH3T3-TGF- ⁇ l cells.
- the specimen was fixed in formalin and decalcified with nitric acid. Sections of the specimen were made and embedded with paraffin and then cut into 0.8 mm thickness slices. The sections were deparaffinized and hydrated by sequential incubations in xylene and ethanol. After washing in lx PBS for 2 min, the sections were blocked with 3% H 2 0 2 for 10 min. The primary antibody against TGF- ⁇ l protein was applied to the sections and incubated for 1 hour. The control sections were incubated in lx PBS without the primary antibody at this step. The sections were washed and blocked with 5% milk in lx PBS for 20 min before incubating with the HRP-conjugated secondary antibody.
- hChon-TGF- ⁇ l or control hChon cells were injected into the rabbit knee joint containing a partial cartilage defect (3mm x 5mm, 1.5mm deep) on the femoral condyle. These cells (15-20ul of 2 x 10° cells/ml) were loaded into the defect. Then the cells were left in the defect for 15-20 min to let them settle down in the defect before suturing. The specimens were harvested at 6 weeks after injection and observed microscopically (Figs. 10A-E). Figs. 10A & D show pictures of the femoral condyles 6 weeks after injection with either hChon-TGF- ⁇ l (A) or control hChon (D) cells. Figs.
- Fig. 1 1 A & C show pictures of the femoral condyles 6 weeks post injection with either mixture of hChon and NIH3T3-TGF- ⁇ l cells (A) or hChon alone (C). Figs.
- Figs. 12A & D show pictures of the femoral condyles 12 weeks post injection with either mixture of hChon and NIH3T3-TGF- ⁇ l cells (A) or hChon alone (D). Figs.
- Figs. 13A & C show pictures of the femoral condyles 6 weeks post injection with either mixture of hChon and NIH3T3-BMP-2 cells (A) or hChon alone (C).
- Figs. 13B & D show Mason's trichrome staining of sections from the femoral condyle injected with either mixture of hChon and NIH3T3-BMP-2 cells (B) or hChon alone (D). [Original magnification: (B & D) xl2.5]. [00161] EXAMPLE XII
- Figs. 14A & D show pictures of the femoral condyles 12 weeks post injection with either mixture of hChon and NIH3T3 -BMP-2 cells (A) or hChon alone (D). Figs.
- FIGS. 15A and 15C show pictures of the femoral condyles 6 weeks post injection with either a mixture of hChon and hChon-TGF- ⁇ l cells (A) or hChon alone (C).
- Figures 15B and 15D show Mason's trichrome staining of sections from the femoral condyle injected with either mixture of hChon and hChon-TGF- ⁇ l cells (B) or hChon alone (D). [Original magnification: (B& D) xl2.5]. [00165] EXAMPLE XIV [00166] Regeneration of Cartilage with Injection of Mixture of Rabbit Buffy Coat and NIH3T3-TGF- ⁇ l Cells in Rabbits with a Partial Cartilage Defect - New Zealand white rabbits weighing 2.0 - 2.5 kg were selected for the animal study. These rabbits were mature and had a tidemark.
- the knee joint was exposed and a partial cartilage defect (3mm x 6mm, l-2mm deep) was made on the hyaline cartilage layer of the femoral condyle with a surgical knife.
- the femoral condyles were harvested at 6 or 8 weeks post injection. [00167] The specimens were harvested at 6 or 8 weeks after loading and examined histologically.
- Figures 16A, C, and E show pictures of the femoral condyles 6 or 8 weeks post loading with either the mixture of rBC and NIH3T3-TGF- ⁇ l cells (A and C) or buffy coat alone (E).
- Figures 16 B, D, and F show Mason's trichrome staining of sections from the femoral condyle loaded with the mixture of rBC and NIH3T3-TGF- ⁇ l cells (B & D) or rBC alone (F). [Original magnification: (B, D, & F) xl2.5].
- FIGS. 17A and 17C show pictures of the femoral condyles 6 weeks post injection with a mixture of hChon and hChon-TGF- ⁇ 1 cells at either a partial (A) or full-thickness defect (C).
- Figures 17B and 17D show Mason's trichrome staining of sections from the femoral condyle injected with a mixture of hChon and hChon-TGF- ⁇ l cells at a partial (B) or full-thickness defect (D). [Original magnification: (B& D) xl2.5]. [00171] All of the references cited herein are incoiporated by reference in their entirety. ⁇ * ⁇ i ⁇ ⁇ ⁇ ⁇
Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
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EP03714466A EP1490075A4 (en) | 2002-03-29 | 2003-03-28 | Bioadhesive directed somatic cell therapy |
AU2003218464A AU2003218464B2 (en) | 2002-03-29 | 2003-03-28 | Bioadhesive directed somatic cell therapy |
CN038121778A CN1655802B (en) | 2002-03-29 | 2003-03-28 | Bioadhesive directed somatic cell therapy |
CA2480656A CA2480656C (en) | 2002-03-29 | 2003-03-28 | Bioadhesive directed somatic cell therapy |
JP2003579839A JP4451135B2 (en) | 2002-03-29 | 2003-03-28 | Bioadhesion-directed somatic cell therapy |
KR1020047015308A KR100572217B1 (en) | 2002-03-29 | 2003-03-28 | Bioadhesive directed somatic cell therapy |
Applications Claiming Priority (2)
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US36911102P | 2002-03-29 | 2002-03-29 | |
US60/369,111 | 2002-03-29 |
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WO2003082302A2 WO2003082302A2 (en) | 2003-10-09 |
WO2003082302A3 WO2003082302A3 (en) | 2003-12-18 |
WO2003082302A9 true WO2003082302A9 (en) | 2005-01-20 |
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PCT/US2003/009719 WO2003082302A2 (en) | 2002-03-29 | 2003-03-28 | Bioadhesive directed somatic cell therapy |
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US (1) | US7431922B2 (en) |
EP (1) | EP1490075A4 (en) |
JP (1) | JP4451135B2 (en) |
KR (1) | KR100572217B1 (en) |
CN (1) | CN1655802B (en) |
AU (1) | AU2003218464B2 (en) |
CA (1) | CA2480656C (en) |
WO (1) | WO2003082302A2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US9125888B2 (en) | 2003-09-08 | 2015-09-08 | Depuy Mitek, Llc | Chondrocyte therapeutic delivery system |
Families Citing this family (10)
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US7005127B2 (en) | 2002-03-29 | 2006-02-28 | Tissuegene, Inc. | Mixed-cell gene therapy |
EP1594973B1 (en) * | 2003-02-10 | 2011-12-07 | Max-Delbrück-Centrum für Molekulare Medizin (MDC) | Transposon-based targeting system |
BE1015610A3 (en) * | 2003-07-17 | 2005-06-07 | Corrutech Nv | Improved glue and method for manufacturing same. |
US7897384B2 (en) | 2003-09-08 | 2011-03-01 | Ethicon, Inc. | Chondrocyte therapeutic delivery system |
US8697139B2 (en) | 2004-09-21 | 2014-04-15 | Frank M. Phillips | Method of intervertebral disc treatment using articular chondrocyte cells |
WO2006086242A2 (en) * | 2005-02-07 | 2006-08-17 | Genenews, Inc. | Mild osteoarthritis biomarkers and uses thereof |
EP1904113B1 (en) * | 2005-06-23 | 2016-01-06 | TissueGene, Inc. | Neuroprotective effective compound |
WO2013063518A1 (en) * | 2011-10-28 | 2013-05-02 | Tufts University | A 3d culture system for culturing cells in synovial fluid, cells cultured in synovial fluid and their use |
KR20190003414A (en) | 2017-06-30 | 2019-01-09 | 코오롱생명과학 주식회사 | A Method For Evaluating Efficacy of Cell Therapeutic Agents |
WO2019004794A1 (en) * | 2017-06-30 | 2019-01-03 | 코오롱생명과학 주식회사 | Pharmaceutical composition for preventing or treating osteoarthritis |
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US4806523A (en) * | 1985-08-06 | 1989-02-21 | Collagen Corporation | Method of treating inflammation |
US5266480A (en) * | 1986-04-18 | 1993-11-30 | Advanced Tissue Sciences, Inc. | Three-dimensional skin culture system |
US6413511B1 (en) * | 1990-12-20 | 2002-07-02 | University Of Pittsburgh Of The Commonwealth System Of Higher Education | Cartilage alterations by administering to joints chondrocytes comprising a heterologous polynucleotide |
US5858355A (en) * | 1990-12-20 | 1999-01-12 | University Of Pittsburgh Of The Commonwealth System Of Higher Education | IRAP gene as treatment for arthritis |
CA2155929A1 (en) * | 1993-12-14 | 1995-06-22 | Christopher H. Evans | Systemic gene treatment of connective tissue diseases |
US5965125A (en) * | 1995-10-25 | 1999-10-12 | Transkaryotic Therapies, Inc. | Hybrid matrix implants and explants |
US5842477A (en) * | 1996-02-21 | 1998-12-01 | Advanced Tissue Sciences, Inc. | Method for repairing cartilage |
US5700774A (en) * | 1996-03-26 | 1997-12-23 | Genetics Institute, Inc. | Compositions comprising bone morphogenic proteins and truncated parathyroid hormone related peptide, and methods of inducing cartilage by administration of same |
US6151525A (en) * | 1997-11-07 | 2000-11-21 | Medtronic, Inc. | Method and system for myocardial identifier repair |
US6291240B1 (en) * | 1998-01-29 | 2001-09-18 | Advanced Tissue Sciences, Inc. | Cells or tissues with increased protein factors and methods of making and using same |
US6315992B1 (en) * | 1999-06-30 | 2001-11-13 | Tissuegene Co. | Generating cartilage in a mammal using fibroblasts transfected with a vector encoding TGF-β-1 |
AU2003217384A1 (en) * | 2002-02-13 | 2003-09-04 | Ludwig Institute For Cancer Research | Isolated peptides which bind to hla molecules and uses thereof |
US6811777B2 (en) * | 2002-04-13 | 2004-11-02 | Allan Mishra | Compositions and minimally invasive methods for treating incomplete connective tissue repair |
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2003
- 2003-03-05 US US10/382,190 patent/US7431922B2/en not_active Expired - Lifetime
- 2003-03-28 KR KR1020047015308A patent/KR100572217B1/en active IP Right Grant
- 2003-03-28 AU AU2003218464A patent/AU2003218464B2/en not_active Expired
- 2003-03-28 CA CA2480656A patent/CA2480656C/en not_active Expired - Lifetime
- 2003-03-28 WO PCT/US2003/009719 patent/WO2003082302A2/en active IP Right Grant
- 2003-03-28 EP EP03714466A patent/EP1490075A4/en not_active Withdrawn
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Cited By (1)
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US9125888B2 (en) | 2003-09-08 | 2015-09-08 | Depuy Mitek, Llc | Chondrocyte therapeutic delivery system |
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Publication number | Publication date |
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EP1490075A4 (en) | 2008-07-30 |
WO2003082302A2 (en) | 2003-10-09 |
CN1655802A (en) | 2005-08-17 |
JP2005537222A (en) | 2005-12-08 |
US7431922B2 (en) | 2008-10-07 |
KR20050002898A (en) | 2005-01-10 |
JP4451135B2 (en) | 2010-04-14 |
CA2480656C (en) | 2013-09-24 |
CA2480656A1 (en) | 2003-10-09 |
KR100572217B1 (en) | 2006-04-24 |
US20040018179A1 (en) | 2004-01-29 |
EP1490075A2 (en) | 2004-12-29 |
WO2003082302A3 (en) | 2003-12-18 |
AU2003218464A1 (en) | 2003-10-13 |
AU2003218464B2 (en) | 2006-09-21 |
CN1655802B (en) | 2011-02-23 |
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