EP0748374A1 - Mode de preparation de fibroplasts clonogenes, methode de transfection genique de fibroplasts et les fibroplasts genotransfectes ainsi obtenus - Google Patents

Mode de preparation de fibroplasts clonogenes, methode de transfection genique de fibroplasts et les fibroplasts genotransfectes ainsi obtenus

Info

Publication number
EP0748374A1
EP0748374A1 EP95909773A EP95909773A EP0748374A1 EP 0748374 A1 EP0748374 A1 EP 0748374A1 EP 95909773 A EP95909773 A EP 95909773A EP 95909773 A EP95909773 A EP 95909773A EP 0748374 A1 EP0748374 A1 EP 0748374A1
Authority
EP
European Patent Office
Prior art keywords
fibroblasts
cells
gene
transfected
csf
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95909773A
Other languages
German (de)
English (en)
Inventor
Felicia M. Rosenthal
Albrecht Lindemann
Thomas Boehm
Roland Mertelsmann
Hendrik Veelken
Peter Kulmburg
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universitaetsklinikum Freiburg
Albert Ludwigs Universitaet Freiburg
Original Assignee
Universitaetsklinikum Freiburg
Albert Ludwigs Universitaet Freiburg
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universitaetsklinikum Freiburg, Albert Ludwigs Universitaet Freiburg filed Critical Universitaetsklinikum Freiburg
Publication of EP0748374A1 publication Critical patent/EP0748374A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • 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
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the present invention relates to a process for the production of clonogenic fibroblasts, a process for gene transfecting fibroblasts and the gene-transfected fibroblasts obtained in this way.
  • SU 13 17 021 AI relates to diploid stem cells of human skin and embryonic muscle cells which have been isolated for the cultivation of viruses.
  • the SU 15 18 370 AI concerns embryonic stem cell cultures of human skin and muscles for the production of diagnostic preparations.
  • a method for the production of human, clonogenic fibroblasts is disclosed, as well as a method for gene transfection of fibroblasts.
  • tissue samples are first taken from the serosa and, in a preferred form, from the skin of the donor, wherein these samples can have a size of 0.5 to about 2 cm *.
  • Tissue samples of this type can be obtained from the biopsy using known routine surgical methods.
  • the samples obtained as part of the biopsy are then cut up into parts with a diameter of less than 2 mm using a scalpel or a comparable device. These parts can then be laid out on cell culture plates with the epidermis layer upwards.
  • Dulbecco's modified Eagles Medium (DMEM) with 101 fetal calf serum and the usual additives can preferably be used as the culture medium.
  • DMEM Dulbecco's modified Eagles Medium
  • Tissue samples are first crushed only to a size of about 0.5 cm and these parts are then incubated in culture medium (DMEM), the medium containing enzymes which promote cell separation.
  • DMEM culture medium
  • Collagenase, dispase (a neutral protease) or hyaluronidase are preferably used in the context of the present invention as such enzymes which promote cell separation, the enzymes being able to be used either individually or in various combinations.
  • the cells released by the enzymatic treatment are then washed with phosphate-buffered saline and sown at a density of about 2 ⁇ 10 ⁇ / crn- ⁇ in cell culture bottles.
  • the fibroblast cultures are usually provided with fresh growth medium twice a week. When the cultures have reached confluence, the cells can be harvested by treatment with trypsin / EDTA. After washing, counting, and again
  • Sowing at 1 x 10 cells / cm starts a new growth cycle.
  • feeder cells added to about 10 2 clonogenic fibroblast cells in dishes with a diameter of 10 cm.
  • the feeder cells are irradiated with an intensity of about 50 Gy.
  • Human embryonic WS-1 fibroblasts which are available from the American Type Culture Collection or mice NIH3T3 fibroblasts are preferably used for this purpose.
  • the tissue samples obtained by biopsy are first crushed into pieces that have no more than 0.5 cm ⁇ . These pieces of tissue are then digested with the enzyme dispase at a concentration of 2.5 units / ml for 16 hours at 4 ° C. After removing the epidermis, the skin cells are shredded into pieces that are only a few millimeters in size. The material thus obtained is further digested with a mixture of collagenase (200 units / ml) and hyaluronidase (300 units / ml) for 3 hours at 37 ° C. in a water shaking bath. Finally, the cells are sieved through a sieve with a mesh size of 70 ⁇ m, washed and transferred to the cell culture.
  • Figure 1 shows that the mean doubling time is about 4.3 ( ⁇ 0.6) days. The further growth was significantly slower in some cases and in some cases a plateau was reached, no increase in the number of cells was allowed. In some cultures, however, a cell count of more than 10 cells could be reached without a flattening of the curve being observed. It turned out that the von cultures from older donors have slower growth rates and tend to stagnate.
  • FIG. 2 shows a comparison of fibroblasts depending on the type of cell production.
  • the enzymatically produced fibroblasts clearly showed a better ability to proliferate than those obtained only by mechanical production from skin biopsies.
  • the cell cultures from different donors always showed better propagation behavior when the cells were produced after enzymatic treatment compared to those cells that only grew out of the biopsy samples.
  • FIG. 3 shows that autologous fibroblasts could also be obtained effectively from peritoneal cells using the enzymatic production method. Serosa and skin cultures initially grew at approximately the same rates, but the diploid fibroblasts from Serosa reached the plateau sooner than cultures of the same age.
  • GM-CSF colony stimulating factor
  • TNF ⁇ tumor necrosis factor
  • the diploid fibroblasts that can be produced by the method according to the invention can easily be obtained, for example, from cancer patients. These clonogenic fibroblasts serve as a suitable starting material for gene transfection. Such transfected fibroblasts can then be returned to the donors as autologous cells.
  • the fibroblasts obtainable by the method described in more detail above can be genetically manipulated by transfection.
  • Gene transfection involves introducing one or more genes into the clonogenic fibroblasts that code for a gene product that is therapeutically valuable.
  • gene products are growth factors, in particular hematopoietic growth factors, but also hormones, such as insulin, or coagulation factors, such as factor VIII, coagulation inhibitors, enzymes, such as lysosomal enzymes or adenosine deaminase.
  • hematopoietic growth factors such as G-CSF or erythropoietin are introduced into the fibroblasts by the transfection.
  • Suitable vectors are required for transfection.
  • a retroviral vector is used.
  • this vector can also contain a so-called suicide gene, such as the thymidine kinase gene derived from herpes simplex virus. Thereby selective cell killing is allowed in the presence of gancyclovir.
  • the vectors can contain inducible promoters which enable regulation of gene expression.
  • One vector that can preferably be used is the retroviral vector N2, which is derived from the genome of the Moloney Murine Leukemia Virus (MLV) and contains a bacterial neomycin resistance gene as a selective marker.
  • MMV Moloney Murine Leukemia Virus
  • the origin of the fragments and the restriction enzymes used to obtain DNA fragments encoding human interleukin-2, mouse interferon- ⁇ and herpes simplex virus thymidine kinase promoter and other genes have already been described [Gansbacher et al., J. Exp. Med., 172 (1990) pp. 1217-1224; Gansbacher et al., Cancer Res., 50 (1990) pp. 7820-7825 and Gansbacher et al., Blood, 80 (1992) pp. 2817-2825].
  • Another vector which can preferably be used in the present method has been described in Science, Vol. 256, April 1992, p. 445.
  • This is the outer shell of an adenovirus in which the adenoviral genes have either been deleted or otherwise inactivated.
  • An antibody with a lysine tail is bound to the viral envelope, the lysine tail binding to the DNA to be transfected.
  • the DNA is not packaged inside the viral envelope, but is appended to the outside of the viral envelope. This vector enables the transfection of larger DNA fragments.
  • the human clonogenic fibroblasts obtained by the method according to the invention can be transfected by the methods described above. Since only the clonogenic fibroblasts as target cells for stable gene transfection in Question, are the fibroblasts obtained in this way very suitable for the expression of foreign genes.
  • fibroblasts that have been transfected with cytokine genes can be used as so-called "bystander" cells in the context of vaccinations against tumors or for the prophylaxis of infections.
  • Fibroblasts transfected with other genes can be returned to the donor and the long-term care of the organism serve those molecules that are missing from the corresponding disease, for example, blood coagulation factor VIII, protein S, protein C, insulin, erythropoietin or other hematopoietic growth factors, or the provision of enzymes such as glucocerebrosidase or adenosine desaminase possible.
  • the fibroblasts according to the invention can either be used in the autologous system or also in the allologic system if this is possible from an immunological point of view.
  • the gene transfected fibroblasts are obtained by means of a physical gene transfer process.
  • Typical examples of such physical gene transfer methods are electroporation, microinjection, particle bombardment and anionic or cationic lipofection.
  • Electroporation is carried out as follows, for example: A quantity of 4 ⁇ 10 cells is washed twice with phosphate-buffered saline, in 0.5 ml of an electroporation buffer (20 mmol HEPES, 137 mmol sodium chloride, 5 mmol potassium chloride, 0.7 mmol Na 2 HP0 4, 6 mmol sucrose, 1 mg / ml bovine serum albumin, pH 7.0; (Goldstein et al, Nucleic. Acid Research, Volume 17 (1989), pages 3959 to 3971) and incubated for 10 minutes on ice with 20 ⁇ g of DNA in a 0.4 cm electroporation cuvette from Bio-Rad, Kunststoff, Germany.
  • an electroporation buffer (20 mmol HEPES, 137 mmol sodium chloride, 5 mmol potassium chloride, 0.7 mmol Na 2 HP0 4, 6 mmol sucrose, 1 mg / ml bovine serum albumin, pH 7.0; (Goldstein et al, Nu
  • the cells are then electroporated with a capacitance of 960 ⁇ F in an electroporation device that is charged with 250 V. After a second incubation on ice for 10 minutes, the cells are applied in a 50 ml cell culture vessel with a regular nutrient solution.
  • a typical cationic lipofection is as follows:
  • the cells are applied at a density of 10 per wall to a 35 ml petri dish, for example from Falcon.
  • 2 ⁇ g of the DNA with various proportions of lipofection agents (DOSPA / DOSPE 3: 1), available from Gibco in Germany, are added to 200 ⁇ l DMEM and incubated for 30 minutes at room temperature.
  • the cells are then washed twice with DMEM and the transfection complexes are added to the cells after dilution to 1 mm using DMEM. After 1 hour, 1 ml of a conventional culture medium is added, and 24 hours later the medium has changed completely.
  • the gene-transfected fibroblasts obtained according to the invention can be used as medicaments, in particular for in vivo treatment.
  • the gene-transfected fibroblasts serve, for example, to mobilize hematopoietic stem cells, provided there is a growth factor gene.
  • Another object of the present invention is a carrier made of biocompatible, preferably usable in endoprostheses Material containing the gene transfected fibroblasts discussed above.
  • the gene-transfected fibroblasts are first cocultivated with the biocompatible material in vitro, and the material covered in this way is then implanted.
  • the endoprosthesis is a vascular prosthesis made of a biocompatible, preferably human-compatible, synthetic material, for example fluoropolymers or a polyester, and the gene-transfected fibroblasts are implanted in vivo with this prosthesis.
  • the endoprosthesis in the form of a vascular prosthesis can consist of a biocompatible, preferably human-compatible, material derived from natural sources, for example a collagen fleece or a material obtained from bovine pericard, the gene-transfected fibroblasts being implanted with this prosthesis in vivo.
  • fibroblasts with collagen-coated polyvinylpyrrolidone matrices cocultured and applied as organoid intraperitoneally (ip) or subcutaneously (sc) in this form are known to be neovascularized in vivo.
  • the amount of gene product required in vivo is controlled by the amount of transfected cells and, if necessary, additionally by the inducible promoter.
  • suitable carriers such as fluoropolymer fibers, in particular polytetrafluoroethylene fibers, can also be used, which are then coated with a suitable coating agent such as collagen. These fibers can then be embedded in an extracellular gel matrix and applied intraperitoneally, for example by a small surgical procedure.
  • fluoropolymer fibers in particular polytetrafluoroethylene fibers
  • Skin and serosa samples of approximately 0.5-2 cm2 were obtained from 50 donors who had cancer. The biopsy samples were stored in Dulbecco's modified Eagles Medium (DMEM) and processed on the day of collection.
  • DMEM Dulbecco's modified Eagles Medium
  • DMEM Gibco BRL
  • fetal calf serum Boehringer Mannheim
  • the fibroblast cultures were grown at 37 C in a humid atmosphere containing 5% CO2, twice per
  • NIH3T3 added per culture plate.
  • the two cell lines available from ATCC, Rockville, MD served as so-called feeder cells after irradiation with 100 Gy. After 4
  • Example 1c The test conditions given in Example 1 were carried out using various enzymes (Example 1c) in order to determine the effectiveness for the creation of long-term cultures of diploid fibroblasts.
  • the best results were obtained when the cut biopsy samples were first digested with Dispase at a concentration of approximately 2.5 U / ml for 16 hours at 4 ° C. After this treatment, the epidermis could be easily removed from the cell fragments.
  • the skin cells were then further crushed into pieces of a few mm. This cell material was subjected to a second enzymatic treatment with a mixture of collagenase (200 U / ml) and hyaluronidase (300 U / ml) for 3 hours at 37 ° C. in a water shaking bath. Finally, the cells were separated by passage through a 70 ⁇ m sieve, washed and transferred to cell culture.
  • Figure 1 shows a plot of the donor cells (pieces) against the number of days of cultivation, the cells behind / after the curve indicate the age of the human donor.
  • Figure 2 shows a plot of donor cells (pieces) against the number of days of cultivation for human Donors of different ages.
  • ü denotes production of the fibroblasts by enzymatic treatment and ⁇ denotes only mechanical cell separation. It follows from this that the fibroblast cultures produced with the aid of the enzymatic method clearly had a better propagation capacity than those fibroblast cultures which were obtained only by mechanical treatment of the skin biopsy samples.
  • diploid fibroblast cultures can be obtained not only from skin cells but also from peritoneal cells. It was found that the cell cultures derived from the serosa initially grew similarly to the cell cultures derived from the skin, but the diploid fibroblasts derived from the serosa reached the plateau with a significantly lower cell number than the diploid fibroblast cultures of skin comparable in old age.
  • FIG. 3 shows a comparison of the cell numbers plotted against the number of days of cultivation of fibroblast cultures which originate either from skin (left diagram) or from Serosa (right diagram) from donors with an age of 45 to 59 years.
  • Figure 4 shows the influence of so-called feeder cells on the plating efficiency of the diploid fibroblast cultures.
  • Diploid fibroblast cultures of skin cells were prepared which were treated enzymatically. Their plating efficiency was determined by sowing the cells in a very low density on the cell culture bottles. While the diploid fibroblasts without feeder cells showed hardly any colony formation under these conditions, the plating efficiency could be increased to 9-24% by the addition of irradiated human fibroblasts from the WS-1 cell line.
  • Example 3 Example 3:
  • culture supernatants were taken from non-irradiated diploid fibroblasts and also from diploid fibroblasts that were irradiated with 20 or 100 Gy. The culture supernatants of the irradiated cells were removed on the 3rd and 8th day after irradiation.
  • the retroviral base vector N2 is derived from the genome of the Moloney Murine Leukemia Virus (MLV) and contains the bacterial neomycin resistance gene as a selection marker. DNA fragments coding for the Major Immediate Early Human CMV- Promoter, the promoter of adenosine deaminase (ADA promoter), the poly-A signal and the mouse GM-CSF hub cloned different sites of the N2 vector. The construct N2 vector / CMV promoter / mouse GM-CSF cloned into different sites of the N2 vector.
  • MMV Moloney Murine Leukemia Virus
  • N2 vector / CMV promoter / mouse GM-CSF was made by cloning the CMV-GM-CSF fusion product into an Xho I restriction site of the neomycin resistance gene in the N2 vector (Fig. 6, top).
  • the mouse GM-CSF cDNA was also cloned in reverse orientation into an interface generated by the restriction enzyme SnaB 1 and the poly-A signal into a Klenow-filled Apa I site in the 3'LTR polylinker.
  • the retroviral vectors were converted into the corresponding viruses by electroporation of the vector DNA into a helper-free echotropic packaging cell line (GP + E-86). After G418 selection (0.7 mg / ml genticin, Gibco Laboratory, Grand Island, NY), colonies were isolated and expanded into producer cell lines. Cell-free supernatant was tested for NIH 3T3 cells to determine the titer of the virus.
  • Figure 6 shows the structure of the retroviral vectors used.
  • CMS5 is a methylcholantrene-induced, non-metastatic fibrosarcoma from a BALB / c genetic background.
  • NIH 3T3 is a contact-inhibited fibroblast cell line established by NIH Swiss mouse embryos (CRL 1658).
  • BALB 3T3 clone A31 are contact-inhibited, non-tumorogenic fibroblasts established by BALB / c mouse embryos (CCL 163). Both fibroblast cell lines were purchased from ATCC.
  • the cells were cultured in Dulbecco's modified Eagles medium, supplemented with 10% fetal calf serum, 100 U / ml penicillin, 100 n3g / ml streptomycin and 2 mmol / 1 L-glutamine.
  • Virus-producing cell lines that have secreted high titers of the virus were used to infect the CMS5 cells and the fibroblast cells. Clones or bulk-infected cells were expanded into lines after G418 selection and further analyzed.
  • the secretion of GM-CSF in the supernatant of retrovirally infected CMS5 cells and fibroblasts and the GM-CSF serum concentration of treated mice were determined by means of a bioassay and confirmed by means of an ELISA test.
  • the supernatant from icon-confluent parenteral or GM-CSF-secreting cells was collected after 24 hours, the cell number was determined and the supernatant was examined for the production of mouse GM-CSF.
  • CMS5 cells irradiated with 50 Gy or cells transfected with the vector N2 / CMV / GM-CSF / CMS5 were examined.
  • the cells were plated into small tissue culture flasks on the day of irradiation and the amount of GM-CSF in the 24 hour culture supernatant was determined on the 3rd, 6th, 9th and 12th day after the irradiation.
  • the biological activity of the GM-CSF to be investigated was determined by determining the ability of the GM-CSF containing
  • GM-CSF activity was expressed as counts per minute (cpm) and production in ng per 24 hours of 10 cells was calculated by comparison with a standard curve of recombinant mouse GM-CSF. In addition, GM-CSF production was confirmed using an ELISA test.
  • GM-CSF-secreting cells were therefore first irradiated with 50 Gy and the GM-CSF secretion was determined up to 12 days after the irradiation.
  • Figure 7 it can be seen that the GM-CSF production after radiation was initially increased.
  • the values on the 3rd and 6th day after irradiation are about 130 ng / 10 6 cells / 24 hours.
  • mice Female BALB / c mice that were 7-10 weeks old were used for the experiments. On day 0 and day 2, all mice received 150 mg / kg cyclophosphamide intraperitoneally. A portion of the mice received no further injections, a second portion was subcutaneously injected on day 3 twice daily with 100 ng recombinantly produced mouse GM-CSF.
  • the last group received a total of 10 N2 / CMV / GM-CSF-CMS5 cells irradiated with 50 Gy subcutaneously.
  • blood from the tail veins of the mice was transferred daily to heparinized Eppendorf vessels.
  • the number of leukocytes was determined after lysing the erythrocytes in a Neubauer cell chamber. Differential blood count was taken every two days and the differential blood count was determined after staining with May-Grünwald-Gie sa.
  • Serum values of mouse GM-CSF were determined on day 1, 4 and 10 after injection of the irradiated GM-CSF-secreting fibrosarcoma cells.
  • mice Female BALB / c mice were treated as described in Example 4d. The peripheral leukocyte counts were determined daily from the 3rd day and a differential blood count was made every 2 days. The base leukocyte numbers in these mouse strains are around 9,000 to 10,000 leukocytes / ml. Through the application of cyclophosphamide, the leukocytes on the 3rd day are around 1,000 to 1,500
  • FIG. 8 shows the differential blood count of the various treatment groups as absolute leukocyte subpopulations (monocytes, granulocytes and lymphocytes). In untreated BALB / c mice, there are about 1% monocytes, 15-30% granulocytes and 70-85% lymphocytes in the differential blood count. On the 5th day after the start of cyclophosphamide treatment, i.e.
  • lymphocytes are almost exclusively found in all treatment groups in the differential blood count.
  • On day 7 when the absolute white blood cell count of 'treated with recombinant GM-CSF and GM-CSF-secreting cells mice is about twice as high as in the treated with cyclophosphamide animals, a clear difference is noted in the differential blood count.
  • the animals not treated with growth factor had only about 15% of the monocytes and 25% of the granulocytes of the mice treated subcutaneously with GM-CSF or with GM-CSF-secreting fibroblasts, which had already reached normal absolute granulocytes. There are no differences in the absolute lymphocyte values between the different treatment groups.
  • FIG. 8 therefore shows that a single injection of irradiated gene-transfected autologous cells already has biological effects on the blood count of mice treated with chemotherapy.
  • FIG. 9 shows the number of leukocytes that changes over time.
  • the mice were each treated with cyclophosphamide.
  • a control group received no GM-CSF.
  • recombinantly produced GM-CSF was injected subcutaneously.
  • gene transfected cells that produced GM-CSF were applied subcutaneously.
  • pCMV.GCSF.iresNEO contains the human G-CSF gene under control of the CMV promoter and the neomycin resistance gene via an IRES (internal ribosomal entry site). So G-CSF and neomycin phosphotransferase are to be expressed here.
  • pCMV.GCSF.iresTK / NEO contains the G-CSF gene under control of the CMV promoter and a herpes simplex virus thymidine kinase-neomycin resistance fusion gene via an IRES.
  • the TK are also to be expressed and thus, after administration of gancyclovir, this prodrug is to be phosphorylated and activated.
  • FIG. 10 shows on the left a plot of the number of leukocytes as pieces per nl (n nl) against the time (in days) after subcutaneous tumor inoculation of 2.5 ⁇ 10 unirradiated CMS-5 cells of the aforementioned construct 1 (far left) and the Constructs 2 (middle) in 3 BALB-c mice.
  • the diagrams at the bottom left and left in the middle show the time course of leukocyte development after tumor ⁇ oculation, where the prodrug Gancyclovir was also given ip twice daily.
  • the time development (in days) of the tumor size (mm) for three BALB-c test mice is again shown on the right-hand side of FIG. 10 for the same constructs.
  • mice The two groups of BALB-c mice were injected subcutaneously with 2.5 x 10 unirradiated CMS-5 cells each, which had been transfected with the above-mentioned vectors.
  • Each group of mice received gancyclovir (GCV) i.p. twice daily from the third day (D4). (see Figure 10, each of the four lower diagrams).
  • GCV gancyclovir
  • the experimental group which was treated with GCV and received CMS-5 cells transfected with p.CMV.GCSF.iresTK / NEO, shows a drop in the leukocytes and a regression of the tumor (see Figure 10, bottom center, and bottom right).
  • FIG. 11 shows a plot of the number of leukocytes (WBC / ⁇ l) against the time after the injection (in days) after the Chemotherapy and cytokine therapy in the presence / absence of transfected fibroblasts.
  • WBC / ⁇ l the number of leukocytes
  • rhG-CSF twice daily subcutaneous administration of the recombinant human G-CF
  • the diagram in the middle right shows the time course of the development of leukocytes after the addition of cyclophosphamide and the single injection of 5 x 10 G-CSF-transfected BALB-3T3 fibroblasts, and on the right side the time course of the development of leukocytes after the addition of Cyclophosphamide and a single injection of 5 x 10 irradiated G-CSF gene transfected B.ALB-3T3 fibroblasts.
  • the curves marked with a square, a diamond and a dot represent the development of the leukocyte contents of the three test mice used.
  • the three graphics show that the regeneration of the leukocytes in the groups with a single injection of the transfected fibroblasts (middle right as well as on the right) takes place at least as quickly as when the recombinant protein is injected twice a day subcutaneously (center left).
  • the following table shows the effect of GCSF application on the mobilization of hematopoietic stem cells in the peripheral blood.
  • the number of colony forming cells, ie the cells that provide information about the number of hematopoietic stem cells, is also called CFU in the following, and the value of CFU to leukocytes in the four different treatment groups.
  • Leukocytes ⁇ SE CFU / ul ⁇ SE CFU / leukocytes ⁇ SE leukocytes ⁇ SE CFU / ul ⁇ SE CFU / leukocytes ⁇ SE
  • Cyclophosphamide dO + d2 (i.p. 150 mg / kg)
  • G-CSF cyclophosphamide + rhG-CSF d3 - d7 (s.c, 125 ug / kg 2 x daily)
  • G-CSF / B ⁇ LB cyclophosphamide + hG-CSF gene lipofected BALB 3T3, d3 (sc, 5 x 10 6 )
  • G-CSF / B ⁇ LB Cyclophosphamide + irradiated (50 Gy) hG-CSF gene lipofected BALB 3T3, d3 (s.c, 5 x 10 ")
  • CD ro day x Day on which all mice in the group (3 mice / group each) had at least 3500 / ul leukocytes.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Rheumatology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Dans la méthode proposée pour préparer des fibroplasts clonogènes, on prélève un tissu sur un donneur et l'on en isole des cellules; la suspension cellulaire ainsi obtenue est passée au tamis; les cellules contenues dans la suspension sont lavées et transférée en culture tissulaire; une possibilité consiste à isoler les cellules par fragmentation mécanique et à les soumettre ensuite à un traitement enzymatique avec de la collagénase uniquement. La transfection dans les fibroblasts entraîne l'injection d'au moins un gène codant pour une protéine biologiquement active, de préférence une protéine active du point de vue thérapeutique, par exemple un facteur de croissance, une hormone, une enzyme, un facteur ou un inhibiteur de coagulation.
EP95909773A 1994-02-24 1995-02-23 Mode de preparation de fibroplasts clonogenes, methode de transfection genique de fibroplasts et les fibroplasts genotransfectes ainsi obtenus Withdrawn EP0748374A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE4406073A DE4406073A1 (de) 1994-02-24 1994-02-24 Verfahren zur Herstellung von humanen, klonogenen Fibroblasten, Verfahren zur Gentransfizierung von Fibroblasten und so erhaltene Fibroblasten
DE4406073 1994-02-24
PCT/EP1995/000660 WO1995023216A1 (fr) 1994-02-24 1995-02-23 Mode de preparation de fibroplasts clonogenes, methode de transfection genique de fibroplasts et les fibroplasts genotransfectes ainsi obtenus

Publications (1)

Publication Number Publication Date
EP0748374A1 true EP0748374A1 (fr) 1996-12-18

Family

ID=6511152

Family Applications (1)

Application Number Title Priority Date Filing Date
EP95909773A Withdrawn EP0748374A1 (fr) 1994-02-24 1995-02-23 Mode de preparation de fibroplasts clonogenes, methode de transfection genique de fibroplasts et les fibroplasts genotransfectes ainsi obtenus

Country Status (7)

Country Link
US (1) US6093393A (fr)
EP (1) EP0748374A1 (fr)
JP (1) JPH09509571A (fr)
AU (1) AU1811795A (fr)
CA (1) CA2183438A1 (fr)
DE (1) DE4406073A1 (fr)
WO (1) WO1995023216A1 (fr)

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5585115A (en) 1995-01-09 1996-12-17 Edward H. Mendell Co., Inc. Pharmaceutical excipient having improved compressability
US6277368B1 (en) 1996-07-25 2001-08-21 The Regents Of The University Of California Cancer immunotherapy using autologous tumor cells combined with cells expressing a membrane cytokine
DE19716098A1 (de) * 1997-04-17 1998-10-22 Univ Ludwigs Albert Fibroblasten mit einem Fremdgen enthaltende Zusammensetzung zur Behandlung von Wunden
US6328762B1 (en) * 1999-04-27 2001-12-11 Sulzer Biologics, Inc. Prosthetic grafts
ATE414141T1 (de) * 2000-08-09 2008-11-15 Hiroko Yanaga Verfahren zur kultivierung humaner chondrozyten
WO2006071794A2 (fr) * 2004-12-23 2006-07-06 Ethicon Incorporated Cellules postnatales derivees de tissus du cordon ombilical, et procedes de production et d'utilisation de celles-ci
US9592258B2 (en) 2003-06-27 2017-03-14 DePuy Synthes Products, Inc. Treatment of neurological injury by administration of human umbilical cord tissue-derived cells
US8790637B2 (en) 2003-06-27 2014-07-29 DePuy Synthes Products, LLC Repair and regeneration of ocular tissue using postpartum-derived cells
JP5148873B2 (ja) * 2003-06-27 2013-02-20 エチコン、インコーポレイテッド 臍帯組織由来の分娩後細胞、及びその作成及び使用方法
US9572840B2 (en) 2003-06-27 2017-02-21 DePuy Synthes Products, Inc. Regeneration and repair of neural tissue using postpartum-derived cells
ES2642844T3 (es) 2005-12-16 2017-11-20 DePuy Synthes Products, Inc. Composiciones y métodos para inhibir una respuesta inmune adversa en el trasplante de histocompatibilidad que no coinciden
US9125906B2 (en) 2005-12-28 2015-09-08 DePuy Synthes Products, Inc. Treatment of peripheral vascular disease using umbilical cord tissue-derived cells
US9102915B2 (en) 2006-11-13 2015-08-11 DePuy Synthes Products, Inc. In vitro expansion of postpartum-derived cells using microcarriers
US10179900B2 (en) 2008-12-19 2019-01-15 DePuy Synthes Products, Inc. Conditioned media and methods of making a conditioned media
CA2747794C (fr) 2008-12-19 2018-10-30 Advanced Technologies And Regenerative Medicine, Llc Traitement des poumons et des maladies et troubles pulmonaires
AU2010229651B2 (en) 2009-03-26 2014-05-08 Advanced Technologies And Regenerative Medicine, Llc Human umbilical cord tissue cells as therapy for Alzheimer' s disease
CN104837987B (zh) 2011-12-23 2018-10-02 德普伊新特斯产品公司 人脐带组织来源的细胞的检测
US20220401541A1 (en) * 2019-11-02 2022-12-22 Figene, Llc Intratumoral administration of immune cellular therapeutics

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4423145A (en) * 1981-05-07 1983-12-27 Stampfer Martha R Enhanced growth medium and method for culturing human mammary epithelial cells
US4804627A (en) * 1985-05-09 1989-02-14 Sloan-Kettering Institute For Cancer Research Method for cloning lymphoblastoid cells
ATE188125T1 (de) * 1991-10-25 2000-01-15 Sidney Kimmel Cancer Ct Lymphokine gentherapie bei krebs in kombination mit tumorantigenen

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9523216A1 *

Also Published As

Publication number Publication date
JPH09509571A (ja) 1997-09-30
CA2183438A1 (fr) 1995-08-31
AU1811795A (en) 1995-09-11
WO1995023216A1 (fr) 1995-08-31
DE4406073A1 (de) 1995-08-31
US6093393A (en) 2000-07-25

Similar Documents

Publication Publication Date Title
EP0748374A1 (fr) Mode de preparation de fibroplasts clonogenes, methode de transfection genique de fibroplasts et les fibroplasts genotransfectes ainsi obtenus
DE69636122T2 (de) Verfahren zum gentransfer in zielzellen mit einem retrovirus
EP1100878B1 (fr) Procede pour la fabrication des cellules souches hematopoietiques genetiquement modifiees cd34-negatives, croissantes par adhesion
DE69834257T2 (de) Natürliche killerzelllinien und verfahren zu ihrer verwendung
DE69832402T2 (de) Erzeugung von hämatopoietischen zellen aus multipotenten neuronalen stammzellen
DE69433168T2 (de) In vitro vermehrung von stammzellen
DE69837491T2 (de) Menschliche mesenchymale stammzellen aus peripherem blut
DE3851153T2 (de) Genetische modifizierung von endothelialen zellen.
DE69434708T2 (de) Antitumor-Gentherapie bei Immuno-und/oder Entzündungsmodulation
DE69012601T2 (de) Gentherapie durch stützgewebebezügliche Zellen.
DE69027123T2 (de) Proliferative wirkung des leukämie-hemmungsfaktors auf satellitzellen
DE69001484T2 (de) Verwendung von menschlichen BCDF für die Herstellung einer Zusammensetzung für die Behandlung von Thrombocytopenia.
DE10234192A1 (de) Verwendung von Erythropoetin
DE3855681T2 (de) Transkaryotische Einpflanzung
DE69531712T2 (de) Hybridgel, das eine biologisch aktive Substanz sekretiert
DE69637185T2 (de) Isolierte Stromazellen und Methoden zu deren Verwendung
DE69725317T2 (de) Implantierbare agarose-kollagenkügelchen enthaltende zellen, die ein diffusionsfähiges biologisches produkt bilden und ihre verwendung
DE10248141B4 (de) Nukleinsäuren und deren Verwendung für die Gentherapie
JPH03503167A (ja) 部位指定血管形成素子とその使用法
DE69432856T2 (de) Zufuhr von genprodukten mittels mesangium-zellen
EP1171587A1 (fr) Peptide derive de l'antigene muc-1 destine a declencher une reaction immunitaire contre des cellules tumorales
EP0607593B1 (fr) Procédé d'ontention de cultures cellulaires avec un taux amélioré de cytokines autogènes
DE69836315T2 (de) Verwendung von TCF-II zur Behandlung von durch Krebs verursachtem Gewichtsverlust, Anaemie und TNF-Erhöhung
DE69836853T2 (de) Etablierte Mikrogliazellen
DE60114193T2 (de) Transfektionssystem

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19960719

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL PT SE

17Q First examination report despatched

Effective date: 20020514

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20020925