WO1996034955A1 - Procede de traitement de maladies du cartilage par chondrocytes modifies genetiquement - Google Patents

Procede de traitement de maladies du cartilage par chondrocytes modifies genetiquement Download PDF

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WO1996034955A1
WO1996034955A1 PCT/US1996/004902 US9604902W WO9634955A1 WO 1996034955 A1 WO1996034955 A1 WO 1996034955A1 US 9604902 W US9604902 W US 9604902W WO 9634955 A1 WO9634955 A1 WO 9634955A1
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chondrocytes
gene
cartilage
cytokine
cells
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PCT/US1996/004902
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Vijaykumar M. Baragi
Blake J. Roessler
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Warner-Lambert Company
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

  • the invention is concerned with the utilization of articular chondrocytes and the treatment of cartilaginous diseases.
  • the invention pertains to chondrocytes that have been subjected to recombinant engineering.
  • Chondrocyte-mediated cartilage degradation is a common end point in the pathogenesis of various human arthropathies including osteo- (0A) and rheumatoid arthritis (1-6) .
  • therapeutic strategies aimed at developing chondroprotective agents are faced with the challenge of targeting these agents to cartilage, an avascular tissue.
  • gene therapy offers a new approach for treating these diseases (7-11) .
  • Both in vitro and in vivo techniques using viral and non-viral vectors have been used to deliver lacZ, neo and Interleukin-1 Receptor Antagonist (IRAP) genes to synoviocytes/synovium (12) .
  • IRAP Interleukin-1 Receptor Antagonist
  • IL-1 a cytokine present in arthritic joint fluid (14, 15 ) and known to induce cartilage degradation (15,16), generate and maintain synovial inflammation (17) , up regulate matrix metalloproteinase (18) and prostaglandin production (19), is implicated in the pathophysiology of arthritic joints.
  • IRAP is considered to be a potentially therapeutic protein because of its ability to counteract many of the effects of IL-1.
  • There have been a number of in vitro and some in vivo studies demonstrating the beneficial effects of IRAP and an extensive review of this literature has been published (20) .
  • One of the issues with chondrocytes is that they are not readily accessible in the body in that they constitute a very small fraction of the cartilage matrix.
  • chondrocytes are cells that have no blood supply and rely upon materials to be diffused to them. This, therefore, makes them quite different from synovial cells which, in fact, do have a blood supply. Further, chondrocytes are embeded in the cartilage matrix whereas synovial cells are not. Thus drug delivery to chondrocytes is constrained by the matrix materials. Furthermore, the chondrocytes have a low replication rate.
  • U.S. Patent No. 5,310,759 teaches methods of protecting and preserving connective and support tissues.
  • the patent indicates that a pharmaceutically acceptable prostaglandin E 2 and cyclic adenosine monophosphate agonists and inducers can selectively inhibit collagenase gene expression in human and animal (mammal) cells.
  • the patent does not pertain to recombinantly modified chondrocytes.
  • PCT Publication 092/11359 published July 9, 1992 teaches truncated interleukin-l receptor gene for the treatment of arthritis.
  • the specification indicates that a major source of IL-1 in the joint is the synovium.
  • the specification does not teach the genetic modification of recombinant chondrocytes by gene therapy.
  • PCT Specification O94/01139 published January 20, 1994 pertains to targeting somatic gene therapy to joints.
  • the invention is particularly concerned with a transfected synovial gene.
  • the invention is not concerned with a recombinant or genetic modification of chondrocytes and the treatment of arthritic diseases with such chondrocytes.
  • German Application 4,219,626 published December 23, 1993 teaches incorporating a therapeutic gene via a vector into body cells in vivo and in vitro for subsequent expression and secretion of active protein, particularly for treating the diseases of spine and nerves.
  • the reference does not specifically teach recombinant articular chondrocytes for treatment of cartilaginous diseases.
  • the method involves genetic alteration of articular chondrocytes so as to contain at least one gene encoding for a protein with therapeutic or prophylactic activity for an arthritic condition.
  • transfected chondrocytes comprising articular chondrocytes genetically modified to contain at least one gene encoding for a protein with therapeutic or prophylactic activity for an arthritic condition.
  • Also disclosed is a method treating an arthritic condition comprising the steps of: subjecting a patient, in need thereof, to an effective amount of recombinant chondrocytes comprising mortal articular chondrocytes recombinantly modified with at least one gene which has therapeutic or prophylactic activity for an arthritic condition.
  • Also disclosed is a method of delivering a gene to cartilage comprising the steps of providing chondrocytes modified with at least one gene encoding for a protein which has therapeutic or prophylactic activity for an arthritic condition, and delivering the modified chondrocytes to a mammal having a diseased cartilaginous state.
  • FIGURE 1 is a photomicrograph reflecting the expression of transgenic LacZ by human chondrocytes.
  • Cells expressing the transgene have a blue staining nuclei (A) and nuclei of control cells are not stained
  • FIGURE 2 is a photomicrograph of expression of transgenic LacZ by human chondrocytes transplanted onto cartilage organ cultures.
  • Photograph 2A shows cells transduced with Ad.RSVntLacZ vector two days after transplantation onto cartilage organ cultures.
  • FIGURE 2B is the same as Figure 2A except after eight days after transplantation.
  • FIGURE 2C shows cartilage with cells subjected to mock transfection which did not stain.
  • FIGURE 2D shows a photomicrograph of chondrocytes transduced with Ad.CMVLacZ vector and transplanted onto the surface of intact cartilage. The cells exhibit cytoplasmic staining.
  • FIGURE 2E is a photomicrograph showing control cartilage with mock transfected cells which show no staining.
  • FIGURE 2F is a photomicrograph of scanning electron micrograph of cartilage showing transplanted chondrocytes embedded in territorial matrix.
  • FIGURE 2G is similar to Figure 2F but shows cells at a higher magnification.
  • FIGURE 3 is a graph demonstrating expression of human IL-lra by transduced chondrocytes transplanted onto cartilage organ cultures.
  • FIGURE 4 is a graph showing that transplantation of transduced chondrocytes overexpressing human IL-lra protects cartilage organ culture from IL-1 mediated proteoglycan degradation.
  • FIGURE 5 shows restriction maps for the generation of Ad.RSVntLacZ.
  • FIGURE 6 shows the restriction maps for the generation of Ad.CMVLacZ.
  • FIGURE 7 shows the restriction maps for the generation of Ad.RSVhIL-lra.
  • the present invention is concerned with the use of genetically modified chondrocytes.
  • the chondrocytes are generally obtained from a patient, subjected to genetic manipulation and then inserted back into the very same patient. This is called the "ex vivo" technique.
  • chondrocytes can be obtained from another source, subjected to genetic manipulation and then introduced into desired patients where there is not a significant immunological response by the host. This is also called the "ex vivo" technique.
  • autologous chondrocytes are used, whereas in the second example, allogenic chondrocytes are used.
  • mortal chondrocytes are used.
  • memory chondrocytes is meant chondrocytes that are not immortal but have senescence.
  • transduction which is the transfer of non-viral DNA by a virus to a cell.
  • transfection which is the transfer of DNA to a eukaryotic cell.
  • chondrocytes The technique for obtaining chondrocytes is well known in the art. It is likewise well known in the art as to how to subject some types of cells to genetic manipulation to insert appropriate genes which produce proteins and the like.
  • inserting DNA deoxyribonucleic acid
  • DNA deoxyribonucleic acid
  • the introduction of DNA can be done either transiently or permanently into cells.
  • the methods for introducing DNA into cells include calcium phosphate coprecipitation, cationic liposome or lipofection, electroporation, DEAE-Dextran, receptor mediated endocytosis, particle mediated gene transfer, or for some cell types, naked DNA can be utilized.
  • the appropriate genes can be introduced into any of the well known viral vectors preferably adenovirus, adeno- associated virus, retrovirus, alpha virus and herpes virus.
  • antisense or ribozyme expression vectors for controlling gene expression by cells. See Molecular Biotechnology, supra, and ribozyme patents listed, infra.
  • the therapies are to treat cartilaginous diseases, especially chondroprotection.
  • Applicant is interested in treating or controlling osteo arthritis, rheumatoid arthritis, metabolic bone disorders, infectious arthritis such as septic arthritic disease, inflammatory arthritic syndromes, e. g., ankylosing spondylitis and/or sports injuries which involve cartilage or arthritic symptoms.
  • Applicant is desirous of introducing one or more genes into a particular cell and to deliver multiple genes to the site of affliction. A first gene would be to cease or prevent the degradation of the ailment. The second gene would be used to treat or improve the ailment being treated.
  • the classes of potential anti arthritic proteins that may be introduced together or alone into cells are cited in the table below.
  • IRAP Antagonizes IL-1 A excluding IL-1 IL-4 Downregulates C,D,I IL-1, TNF- ⁇ induces IRAP
  • Ribozymes Reduce cytokine mRNA levels
  • Anticytokines Soluble Antagonize their G,H receptors cognate cytokine for IL-1, TNF- ⁇ etc.
  • Cartilage IGF-1 Promote M growth TGF- ⁇ Cartilage Repair factors
  • Matrix Meta- TIMP-1* Inhibit cartilage N lloproteinase TIMP-2 degradation mediated Inhibitors by Matrix Metallo- proteinase (MMP)
  • interleukin 4 suppreses interleukin 1 production but, upregulates gene expression and synthesis of interleukin 1 receptor antagonist. Proc. Natl. Acad. Sci. USA 89: 4076-4080, 1992.
  • ICAM-l-dependent pathway is critically involved in the pathogenesis of adjuvant arthritis in rats. J. Immunol. 147-4167-4171, 1991.
  • the most preferred is to insert the gene for IRAP into the cell. This gene would thereby bind IL-1 which would prevent further inflammation in an arthritic environment.
  • the delivery method for the recombinant chondrocyte can be either by ex vivo or in vivo delivery. See Molecular Biotechnology, supra, pp.402- 415.
  • a suitable viral or non- viral delivery system is used to administer the desirable gene to the patient.
  • This administration may be intravenous or intraarticular.
  • Another formulation could be, for example, using cationic liposomes (Philip R. et al. J. Biol.Chem., 268: 16087-16090 (1993)) where from 10 ⁇ g to 10 mg of a vector expressing the desired gene is delivered.
  • the articular cartilage used in this study was obtained from osteoarthritis patients undergoing total knee or hip replacement surgery. The ages of the patients ranged from 50 to 70 years.
  • uniform cartilage slices measuring approximately 11 mm by 11 mm were dissected from the underlying bone using a #20 scalpel blade and placed in isotonic saline. The cartilage slices were washed in Gey' s balanced salt solution and placed (one piece/well) in Falcon 24 well flat-bottom tissue culture plates (Becton Dickinson, Lincoln Park, NJ) .
  • the cartilage organ cultures were maintained in complete Ham's F-12 (supplemented with 10% fetal calf serum 6.5 mg /ml Hepes, 58.5 ⁇ g /ml glutamine, 200 ⁇ g/ml MgS0 4 , 100 units/ml Penicillin G sodium, 100 ⁇ g/ml streptomycin sulfate, and 0.25 ⁇ g/ml amphotericin B) at 37° c in a humidified atmosphere of 95% air and 5% C0 2 .
  • the chondrocytes needed for the study were isolated from the cartilage pieces separated from the underlying bone.
  • the cartilage slices were minced finely and then digested sequentially with 0.2 % testicular hyaluronidase (5 minutes at 37°C) , 0.2% trypsin (30 minutes at 37°C) , and 0.2 % collagenase (1-2 hours at 37°C) .
  • the resulting solutions were strained through sterile nylon mesh and centrifuged at 1,200 x g for 10 minutes.
  • the cell pellets were washed twice with Ham' s F-12 medium, resuspended, combined and centrifuged at 1,000 x g for 10 minutes. The resulting pellet was suspended in complete F-12 medium (described above) .
  • This cell suspension was used to establish cell cultures in 25-cm 2 falcon flasks at a density of 1-2 x 10 6 cells /flask.
  • the cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% C0 2#
  • the chondrocyte used in this study were maintained as monolayer cultures for no more than 2 passages, in order to maintain the differentiated chondrocyte phenotype.
  • adenoviral vectors used in this study are based on an adenoviral (serotype 5) genomic backbone deleted of sequences spanning E1A and E1B and a portion of E3 region (Restriction Map shown in Figures 5-7) . This impairs the ability of this virus to replicate or transform non-permissive cells (21) .
  • Gene transcription was driven by the early enhancer/promoter of the cyto arcadeovirus (CMV) in the Ad.CMVIacZ vector, and Rocus sarcoma virus (RSV) long terminal repeat (LTR) in Ad.RSVntlacZ and Ad.RSV lL-lra vectors.
  • Ad.RSVntlacZ contains a nuclear targeting (designated as nt) epitope linked to the lacZ gene, and as result, the cells transduced with this gene can be identified by the presence of blue nuclei after reaction with the chromogenic substrate X-gal (as described below) .
  • DMEM serum free Dulbec
  • Transduced allogeneic chondrocyte were treated with trypsin, suspended in 1-3 ml of complete Ham' s F-12 medium and added slowly onto the articular surface of cartilage organ cultures or intact cartilage (with the underlying subchondral bone) selected for transplantation experiments.
  • cartilage pieces were pretreated with 10-20% serum (Fetal calf, Life Technologies Inc., Grand Island, N.Y.), 0.5 % gelatin (Sigma, St Louis, Mo.) or 3.5 ⁇ g /cm 2 (surface area) Cell-Tak*(Collaborative Products, Bedford, Ma.).
  • Chondrocytes were washed in phosphate-buffered saline (PBS) and fixed in 2% paraformaldehyde in 100 mM PIPES pH 6.9 for 30 min. Cells were washed for 30 minutes in PBS/2 mM MgCl-. and reacted in 5 mM K 3 Fe(CN) 6 , 5 mM K Fe(CN) 6 , 2 mM MgCl 2 0.01% sodium deoxycholate, 0.02% NP-40, and 1 mg/ml 5-bromo-4-chloro-3-indolyl-b-D- galactoside (X-gal ) in PBS for 4 hours at 37°C (22) .
  • PBS phosphate-buffered saline
  • X-gal 5-bromo-4-chloro-3-indolyl-b-D- galactoside
  • Duplicate samples of transduced cells were analyzed for the co-expression of ntlacZ and type II or type I collagen using immunohistochemical staining.
  • Cells were fixed in cold methanol, then reacted with X- gal for 2 hours.
  • Cells were washed twice in PBS then incubated for 1 hour with PBS/10% FCS containing monoclonal murine anti-human type II or I collagen (1:100) at 37°C (Chemicon International, Temecula, Ca.) .
  • Cells were washed twice in PBS then incubated with goat anti-murine IgG conjugated to biotin (Histostain, Zymed Laboraties, Inc., San Francisco, Ca.).
  • Cells were then reacted with Streptavidin-horseradish peroxidase followed by aminoethylcarbazole and examined by light microscopy.
  • Cartilage slices used for transplantation experiments were examined by scanning electron microscopy (SEM) to identify integration of transduced cells into the articular surface.
  • Discs for SEM were trimmed to a width of 1 mm and fixed in 4% paraformaldehyde in PBS followed by 1% glutaraldehyde in 0.5 M cacodylate (Fluka Chemika, Swizerland) buffer.
  • Discs were dehydrated in graded alcohols and coated with gold, then imaged using a ISC DS-130 scanning electron microscope.
  • chondrocyte Twenty-four hours post-infection, the chondrocyte were treated with trypsin and plated onto human OA cartilage organ cultures and the cultures incubated in complete F-12 medium at 37°C Forty-eight hours post-transplantation of chondrocyte, cartilage organ cultures were rinsed with 1 mL F-12 Nutrient Mixture (without serum) in order to remove residual FCS. Each treatment group was done in triplicate. The controls received 1 ml F-12 medium + 10 ⁇ l PBS with 0.1% BSA (bovine serum albumin) and IL-1 treatment group received 1ml F-12 medium + 10 ⁇ L IL-l ⁇ (500 Units) . Cartilage organ cultures were incubated for 10 days.
  • BSA bovine serum albumin
  • cartilage pieces were subjected to papain digestion. Cartilage pieces were digested in 24 well plates by adding 50 ⁇ L papain in 1.0 mL cysteine buffer (0.1M phosphate buffer, pH 7.0 containing 0.01M L-cysteine HCI and 0.05M EDTA) and incubating at 56°C until the cartilage pieces were dissolved completely.
  • cysteine buffer 0.1M phosphate buffer, pH 7.0 containing 0.01M L-cysteine HCI and 0.05M EDTA
  • Effect of IL-1 on proteoglycan degradation was determined by analyzing the cartilage digests and media samples collected for chondroitin sulfate content, using the modified 1,9-Dimethylmethylene Blue (DMB) assay (23) .
  • the conditioned media were also analyzed by ELISA (R & D Systems, Minneapolis, MN) for the presence of endogenous and transgenic human IL-lra.
  • lacZ was used as a marker gene.
  • Two recombinant human serotype 5 (sub360 derivative) adenovirus vectors, Ad.RSVntlacZ and Ad.CMVIacZ were used to deliver the marker gene.
  • the vector Ad.RSVntlacZ contained a nuclear targeting epitope linked to the lacZ gene, thus transduced cells could be identified by the presence of blue nuclei after reaction with the chromogenic substrate X-gal. Greater than 90% of the infected cells exhibited blue nuclei, indicating expression of the E. coli lacZ gene mediated by adenoviral gene transfer (Fig 1 A ) .
  • chondrocytes transduced with the LacZ and IRAP genes were undertaken as a means to transfer genes to articular cartilage. Twenty-four hours post-infection, chondrocyte were transplanted onto the surface of cartilage organ cultures. Examination of cartilage cultures for the presence of lacZ transduced cells at various times (1-8 days) post-transplantation showed no evidence of a decline in number or intensity of blue staining cells on the surface of cartilage cultures, indicating that lacZ expression remained about the same during the 8-day period (Fig 2 A, B) .
  • Cartilage samples with mock-transfected cells showed no evidence of cells with blue staining nuclei (Fig 2C) , indicating that the presence of blue staining nuclei on the articular surface was specific for the Ad.RSVntlacZ transduced chondrocyte.
  • Examination of intact cartilage with the underlying bone also revealed the presence of blue staining cells on the articular surface (Fig 2 D) .
  • cartilage samples with mock-transfected cells were negative for blue staining cells (Fig 2E) .
  • transduced chondrocyte would adhere to the surface of human OA cartilage, remain viable, and continue to express transgenic protein.
  • Fig 2 F shows transplanted chondrocyte on two pieces of cartilage that are embedded within the territorial matrix.
  • Fig 2G is a higher magnification of cells adhered to cartilage. The cells display an irregular surface as well as interconnecting cytoplasmic processes, characteristics of viable, metabolically active cells. Bundles of collagen fibers are found surrounding each cell.
  • cartilage cultures were incubated in the presence or absence of exogenous IL-1 ( 500 units/ml) for 10 days and proteoglycan degradation monitored. On an average, 11.7 % of the total proteoglycan was degraded from IL-1 stimulated cultures transduced with the IRAP gene and 23.3 % from those transduced with the lacZ gene; whereas, proteoglycan degradation in control cultures(transduced with IRAP or LacZ gene, but not stimulated with IL-1) was 11.2 %.
  • exogenous IL-1 500 units/ml
  • the IL- lra is a soluble protein that can bind to type I and type II IL-1 receptors, but is unable to mediate signal transduction through these receptors.
  • transduced chondrocyte are able to adhere to the surface of articular OA cartilage. Further, the transduced cells are able to express, process and secrete a biologically active cytokine, and this cytokine has biological effects in the context of the microenvironment present across the surface of OA cartilage organ cultures.
  • chondrocyte transplantation has been successfully used to treat human patients with cartilage defects in their knees (13) .
  • this treatment was applied to more or less solitary lesions in an otherwise relatively normal joint.
  • Cartilage degradation in arthritic joints is an end result of catabolic processes outstripping anabolic processes (5-6) ; therefore, it is desirable for chondrocyte transplantation alone to be sufficient to heal arthritic cartilage.
  • a combination of chondrocyte transplantation and gene delivery techniques are used to deliver IRAP gene to OA articular cartilage.
  • IRAP is t reported to be a potentially therapeutic protein able to counteract the effects of IL-1, a cytokine recognized to function as a central mediator of inflammation and tissue destruction in arthritis.
  • the data presented here indicate that chondrocyte transduced with IRAP cDNA upon transplantation adhere to cartilage, and express quantities of biologically active IRAP that is sufficient to counteract IL-1 induced proteoglycan degradation from OA cartilage organ cultures over a period of 10 days. This is a noteworthy achievement in that it has not been possible to demonstrate chondroprotection by delivery of IRAP by traditional routes of delivery (27, 28) or by transfer of IRAP gene to synovium (10, 11) .
  • the present invention demonstrates that transplantation of transduced chondrocytes is a viable strategy that may lead to treatment of osteoarthritis and other disorder of joints.
  • FIG. 1 Expression of transgenic LacZ by human chondrocyte in culture.
  • chondrocytes infected with Ad.RSVntLacZ were fixed in 2% paraformaldehyde, reacted with 5-bromo-4-chloro-3- indolyl-b-D-galactoside (X-gal) , and counterstained with neutral red.
  • Cells expressing the transgene exhibited blue staining nuclei (A) and nuclei of control cells (mock-infected) did not stain (not shown) .
  • Immunohistochemical staining of transduced cells blue nuclei
  • B type II
  • C type I collagen
  • FIG. 1 Expression of transgenic LacZ by human chondrocyte transplanted onto cartilage organ cultures.
  • Chondrocytes transfected with Ad.CMVLa ⁇ Z vector and transplanted onto the surface of intact cartilage (with the underlying subchondral bone) exhibited blue cytoplasmic staining (D) ; whereas control cartilage (with mock-transfected cells) exhibited no blue staining cells (E) .
  • FIG. 3 Expression of IRAP by transduced chondrocytes transplanted on the cartilage surface.
  • Cartilage cultures containing chondrocytes transduced with LacZ or IRAP gene incubated in the presence or absence of 500 units of IL-l ⁇ for 10 days.
  • the conditioned medium collected from these cultures was analyzed for IRAP content by ELISA.
  • FIG. 4 Transplantation of chondrocytes transduced with IRAP gene protects cartilage from IL-1- induced proteoglycan degradation.
  • Cartilage cultures containing chondrocyte transduced with LacZ or IRAP gene incubated in the presence or absence of 500 units of IL- l ⁇ for 10 days.
  • the conditioned medium and cartilage from these cultures was analyzed for proteoglycan content using 1,9 dimethylmethylene blue dye-binding assay.
  • FIGURE 5 Generation of Ad.RSVntLacZ
  • the RSV promoter and a modified B-gal gene cloned into the proviral plasmid pADBglll to obtain pAdRSVntlacZ (nt sequences that direct transgenic protein to the nuclear membrane) .
  • Linearized pAdRSVntlacZ (Nhel digestion) and truncated Ad5 derivative sub360 (Clal digested to remove the E1A and E1B genes) DNA are co-transfected into 293 cells.
  • Linearized pAdCMVlacZ (Nhel digestion) and truncated Ad5 derivative sub360 (Clal digested to remove the E1A and E1B genes) DNA are co-transfected into 293 cells.
  • pEHX-L3 are plasmid backbone sequences.
  • X represents a partial E3 deletion present in the sub360 derivative.
  • FIGURE 7 Generation of Ad.RSVhlL-lra
  • the RSV promoter and the cDNA for human IL-lra are cloned into the proviral plasmid pADBglll to obtain pAdRSVhIL-lra.
  • Linearized pAdRSVhlL-lra (Nhel digestion) and truncated Ad5 derivative sub360 (Clal digested to remove the ElA and ElB genes) DNA are co-transfected into 293 cells.
  • a recombinant adenovirus containing the RSV promoter and the human IL-lra cDNA is produced.
  • pEHX-L3 are plasmid backbone sequences.
  • X represents a partial E3 deletion present in the sub360 derivative.
  • HAM F-12 A commercially available nutrient medium mixture of inorganic salts, amino acids, vitamins, D-glucose, hypoxanthine, linoleic acid, lipoic acid, phenol red, putrescine HCL, sodium pyruvate, and thymidine.
  • HEPES (N-[2-hydroxyethyl)piperazine-N•-[2- ethanesulfonic acid]); Buffer (Sigma Chemical, St. Louis, Mo.
  • TESTICULAR HYALURONIDASE enzyme from Worthington Biochemicals Corp., Freehold, N. J.
  • COLLAGENASE enzyme from Worthington Biochemicals Corp.
  • PIPES piperazine-N,N"-bis[2-ethanesulfonic acid]; buffer from Sigma Chemical
  • NP-40 nonyl phenoxy polyethoxy ethanol
  • EDTA Ethylenediaminetetraacetic acid.
  • DMEM Dulbecco's Modified Eagle Media, a commercially available medium containing inorganic salts, D-glucose, phenol red, sodium pyruvate, amino acids and vitamins.
  • Hanks Balanced Salt Solutions is a mixture of inorganic salts, D-glucose and phenol red (See Proc ⁇ Soc. Exp. Biol. Med. (1949) 71, 196, Modification - NIH) .

Abstract

L'invention décrit un procédé destiné à traiter un état arthritique, consistant à soumettre un patient nécessitant le traitement à une dose efficace de chondrocytes de recombinaison comprenant des chondrocytes articulaires modifiés par recombinaison avec un gène ayant une activité thérapeutique ou prophylactique en cas d'état arthritique.
PCT/US1996/004902 1995-05-03 1996-04-10 Procede de traitement de maladies du cartilage par chondrocytes modifies genetiquement WO1996034955A1 (fr)

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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
EP1512739A1 (fr) * 2003-09-08 2005-03-09 DePuy Mitek, Inc. Système therapeutique à base de chondrocytes
US7067144B2 (en) 1998-10-20 2006-06-27 Omeros Corporation Compositions and methods for systemic inhibition of cartilage degradation
US7091181B2 (en) 1994-12-12 2006-08-15 Omeros Corporation Method of inhibition of pain and inflammation during surgery comprising administration of soluble TNF receptors
USRE41286E1 (en) 1997-08-14 2010-04-27 Zimmer Orthobiologics, Inc. Compositions for regeneration and repair of cartilage lesions
US7927599B2 (en) 2003-09-08 2011-04-19 Ethicon, Inc. Chondrocyte therapeutic delivery system
USRE43714E1 (en) 1999-12-15 2012-10-02 Zimmer Orthobiologics, Inc. Preparation for repairing cartilage defects or cartilage/bone defects in human or animal joints
US8945535B2 (en) 2005-09-20 2015-02-03 Zimmer Orthobiologics, Inc. Implant for the repair of a cartilage defect and method for manufacturing the implant

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EP1932544A1 (fr) * 1995-06-06 2008-06-18 University Of Pittsburgh Of The Commonwealth System Of Higher Education Transfert de gène pour le traitement de tissus conjonctifs chez les mammifères
USRE41286E1 (en) 1997-08-14 2010-04-27 Zimmer Orthobiologics, Inc. Compositions for regeneration and repair of cartilage lesions
US7067144B2 (en) 1998-10-20 2006-06-27 Omeros Corporation Compositions and methods for systemic inhibition of cartilage degradation
WO2001034166A1 (fr) * 1999-11-11 2001-05-17 Sulzer Orthopedics Ltd Dispositif de greffe/implant et procede de production de celui-ci
JP2003513929A (ja) * 1999-11-11 2003-04-15 ズルツァー・オルトペディクス・リミテッド 移植／内植デバイスおよびその製造方法
EP1099443A1 (fr) * 1999-11-11 2001-05-16 Sulzer Orthopedics Ltd. Dispositif pour greffe/implant et sa méthode de production
USRE43714E1 (en) 1999-12-15 2012-10-02 Zimmer Orthobiologics, Inc. Preparation for repairing cartilage defects or cartilage/bone defects in human or animal joints
AU2004205266B2 (en) * 2003-09-08 2007-11-08 Depuy Mitek, Inc Chondrocyte therapeutic delivery system
JP2005082599A (ja) * 2003-09-08 2005-03-31 Depuy Mitek Inc 軟骨細胞治療薬配給システム
US7897384B2 (en) 2003-09-08 2011-03-01 Ethicon, Inc. Chondrocyte therapeutic delivery system
US7927599B2 (en) 2003-09-08 2011-04-19 Ethicon, Inc. Chondrocyte therapeutic delivery system
US8153117B2 (en) 2003-09-08 2012-04-10 Depuy Mitek, Inc. Chondrocyte therapeutic delivery system
JP2012077089A (ja) * 2003-09-08 2012-04-19 Depuy Mitek Inc 軟骨細胞治療薬配給システム
EP1512739A1 (fr) * 2003-09-08 2005-03-09 DePuy Mitek, Inc. Système therapeutique à base de chondrocytes
US9125888B2 (en) 2003-09-08 2015-09-08 Depuy Mitek, Llc Chondrocyte therapeutic delivery system
US8945535B2 (en) 2005-09-20 2015-02-03 Zimmer Orthobiologics, Inc. Implant for the repair of a cartilage defect and method for manufacturing the implant

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