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WO1997024144A1 - Procedes de preparation de cellules de stroma de moelle osseuse utilisees en therapie genique - Google Patents

Procedes de preparation de cellules de stroma de moelle osseuse utilisees en therapie genique Download PDF

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Publication number
WO1997024144A1
WO1997024144A1 PCT/US1995/016991 US9516991W WO9724144A1 WO 1997024144 A1 WO1997024144 A1 WO 1997024144A1 US 9516991 W US9516991 W US 9516991W WO 9724144 A1 WO9724144 A1 WO 9724144A1
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cells
bone marrow
stromal cells
hgh
marrow stromal
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PCT/US1995/016991
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English (en)
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Joel S. Greenberger
David R. Hurwitz
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Alg Company
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Priority to PCT/US1995/016991 priority Critical patent/WO1997024144A1/fr
Priority to AU47435/96A priority patent/AU4743596A/en
Publication of WO1997024144A1 publication Critical patent/WO1997024144A1/fr

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    • 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
    • C12N5/0669—Bone marrow stromal cells; Whole bone marrow
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/113—Acidic fibroblast growth factor (aFGF, FGF-1)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/90—Polysaccharides
    • C12N2501/91—Heparin

Definitions

  • This invention relates to methods of preparing bone marrow stromal cells for use in ex vivo gene therapy.
  • Bone marrow is a complex and dynamic organ system comprised of hematopoietic cells, bone marrow stromal cells, and extracellular matrix. Pluripotent stem cells within the bone marrow proliferate and differentiate into numerous cell types including erythrocytes and leukocytes. It has been known for some time that association between stem cells and stromal cells is critical for this process. Studies in cell culture have shown that a layer of adherent stromal cells must be established before hematopoietic stem cells can grow and differentiate.
  • Bone marrow stromal cells are a heterogeneous population of cells that are defined by their morphology and function. In cell culture, they have a characteristic, spindle-shaped morphology and secrete growth factors and components that form an extracellular matrix. Stromal cells have been shown to divide in culture in response to epidermal growth factor (EGF;
  • Bone marrow and other biological substances can be frozen according to precalculated time and temperature curves (see, e.g., U.S. Pat. Nos. 4,107,937 and 4,117,881). Cryopreserved materials can be stored for extended periods of time with little degradation (Motta, M.R., (1993), Bone Marrow Trans. 12(2) :177).
  • U.S. Pat. No. 4,963,489 indicates that when a suspension -2- of fresh bone marrow and blood is mixed with cryoprotectants and frozen using computerized cryotechnological equipment, 90% of the cells remain viable when thawed. See also, Boswell et al., 1983, Exp. Hematol. 11:315-323; Gilabert et al., 1994, Eur. J. Haematol. 53:93-99; and Rowley, 1992, J. Hematother. Fall:l(3) :233-250.
  • Bone marrow transplantation is a promising therapy for a number of diseases that involve hematopoietic cells. Transplantation can serve to replace cells that have been damaged by an intrinsic disease, such as an anemia, or in instances where hematopoietic cells have been destroyed by chemotherapy or radiation therapy. Transplantation can be autologous, i.e., the patient can serve as his or her own donor. Alternatively, a patient could receive bone marrow from a histocompatible donor. To date, however, conditions for preserving bone marrow, particularly bone marrow stromal cells, which could be used in numerous gene therapies, have not been optimized.
  • a major obstacle to gene therapies based on the modification of stromal cells is the procurement and sustained availability of therapeutically useful numbers of stromal cells. Consequently, despite the success of bone marrow transplantation, gene therapies that require successful transplantation of bone marrow stromal cells have not yet been realized.
  • This invention relates to sequential methods of expanding and cryopreserving bone marrow stromal cells that are transfected and used for gene therapy.
  • the invention is based on the discovery that bone marrow stromal cells can be cryopreserved and then transfected, or transfected and then cryopreserved, and yet still maintain the ability to effectively secrete a desired polypeptide.
  • the cells are plated and expanded according to a particular regimen in complete bone marrow stromal cell medium. With these methods, populations of bone marrow stromal cells can be acquired that are large enough to be useful in a number of therapies. Further, these large populations can be stored for extended periods of time for immediate use when needed.
  • the invention features a method of preparing bone marrow stromal cells for implantation for gene therapy.
  • the method includes the steps of: (a) obtaining bone marrow stromal cells; (b) culturing the stromal cells to obtain an expanded number of cultured stromal cells; (c) transfecting cultured stromal cells with an exogenous gene to obtain transfected stromal cells; and (d) cryopreserving the transfected stromal cells until implantation.
  • the method includes the steps of: (a) obtaining bone marrow stromal cells; (b) culturing the stromal cells to obtain an expanded number of cultured stromal cells; (c) cryopreserving the cultured stromal cells; (d) thawing the cryopreserved stromal cells; and (e) transfecting the thawed stromal cells with an exogenous gene prior to implantation.
  • the method includes the steps of: (a) obtaining bone marrow cells, e.g., from a primary aspirate of bone marrow; (b) cryopreserving the bone marrow cells; (c) thawing the cryopreserved bone marrow cells; (d) culturing the thawed bone marrow cells to obtain an expanded number of cultured stromal cells; and (e) transfecting the cultured stromal cells with an exogenous gene prior to implantation.
  • the bone marrow stromal cells can be obtained from bone marrow, e.g., by a primary bone marrow aspirate, a core sample, or by scrapings from one or more bones, from a vertebrate, living or not, e.g., a primate such as a baboon or a human, or mammals in general including dogs, pigs, and cows, or other animals, or can be obtained from bones removed from a vertebrate.
  • the exogenous gene preferably encodes a secreted peptide such as a serum protein, a blood clotting factor, e.g., factor VIII or factor IX, a cytokine, a lymphokine, a growth factor, e.g., human growth hormone, a peptide hormone, a lipid binding protein, a metabolic enzyme, an antibacterial peptide, an antimicrobial peptide, an antifungal peptide, or a neurotransmitter.
  • the exogneous gene can also encode a cell surface molecule, e.g., V-CAM-1, I-CAM-1, N-CAM, or V-LAM.
  • the culturing or expanding steps can include the steps of: introducing the stromal cells into a vessel pre-coa ed on an inner surface with a gelatin, e.g., 1.0 percent gelatin in water, and containing a culture medium including an acidic fibroblast growth factor ("aFGF") polypeptide; and expanding the stromal cells in the culture medium under conditions and for a time sufficient to obtain an increased number of bone marrow stromal cells.
  • the culture medium further preferably includes at least 0.05 units/ml of a heparin polypeptide.
  • the inner surface of the vessel additionally can be precoated with fetal bovine serum prior to introducing the bone marrow stromal cells.
  • the culture medium used in these methods can include 1.0 to 50.0 percent by volume fetal bovine serum, 0.01 to 100.0 ng/ml aFGF polypeptide, and 0.05 to 100 units/ml heparin polypeptide.
  • the culture medium includes 16.0 percent by volume fetal bovine serum, 1.0 ng/ml aFGF polypeptide, and 5.0 units/ml heparin polypeptide.
  • the expansion step of the culturing method preferably includes the steps of: (i) removing culture medium and non-adherent cells from the vessel; (ii) adding an amount of fresh culture medium to the vessel; (iii) removing culture medium and non-adherent cells from the vessel and centrifuging the medium and non-adherent cells to form a pellet of non-adherent cells; (iv) resuspending the pellet of non-adherent cells in an amount of culture medium taken from the vessel to form a non-adherent cell mixture; and (v) returning the non- adherent cell mixture to the vessel.
  • an "aFGF polypeptide” is any polypeptide that has an amino acid sequence that is the same as, or substantially identical to, all or a portion of the naturally occurring aFGF protein and which has substantially the same function as the natural or full- length recombinant aFGF as described herein with respect to bone marrow stromal cells.
  • the term includes recombinant aFGF (e.g., as manufactured by Life Technologies, Inc., Grand Island, N.Y.; #13241-013),
  • aFGF analogs i.e., mutant forms of aFGF, and natural or synthetic polypeptide fragments of the full-length aFGF protein and analogs, as long as these analogs and fragments have substantially the same function as natural or full-length recombinant aFGF with respect to bone marrow stromal cells as described herein. These analogs and fragments can easily be tested for their function by using the culture methods described below. Acidic FGF analogs and fragments that do not provide at least 10 7 cells with these methods are not within the present invention.
  • heparin polypeptide is any polypeptide that has an amino acid sequence that is the same as, or substantially identical to, all or a portion of the naturally occurring heparin protein and which has substantially the same function as the natural heparin as described herein with respect to bone marrow stromal cells.
  • the term includes natural heparin or chemically modified natural heparin, e.g., sodium heparin (ElkinsSinn, Inc., Cherry Hill, NJ) , recombinant heparin, "heparin analogs," i.e., mutant forms of heparin, and natural or synthetic polypeptide fragments of the full- length heparin protein and analogs, as long as these analogs and fragments have substantially the same function as natural heparin with respect to bone marrow stromal cells as described herein.
  • the heparin function can be assayed using the culture methods described below.
  • polypeptide any chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation, phosphorylation, or chemical modification, and thus includes natural and synthetic peptides and proteins.
  • mutant form of aFGF or heparin is meant a polypeptide that includes any change in the amino acid sequence compared to the naturally occurring protein, as long as the mutant form has substantially the same function as the natural or full-length recombinant protein as described herein with respect to bone marrow stromal cells.
  • changes can arise, e.g., spontaneously by chemical energy, e.g.. X-ray, or by other forms of mutagenesis, by genetic engineering, or as a result of mating or other forms of exchange of genetic information encoding the aFGF polypeptide.
  • Mutations can include, for example, substitutions, deletions, insertions, inversions, translocations, or duplications.
  • the mutations are preferably conservative substitutions, e.g. , substitutions within the following groups: glycine alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, giutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • identity refers to the amino acid sequence similarity between two polypeptides. When an amino acid position in both of the polypeptides is occupied by identical amino acids, then they are identical at that position.
  • substantially identical is meant an amino acid sequence that is at least 80%, preferably 85%, more preferably 90%, and most preferably 95% identical to a reference amino acid sequence, and which retains the same functional activity as the reference sequence.
  • sequence analysis software e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705.
  • expansion or “expanding” of cells means culturing cells for a time and under conditions that allow the cells not only to grow and thrive, but to multiply to obtain a greater number of cells at the end of the expansion than at the beginning of the expansion.
  • a "passage" is the process whereby cells that have reached a given number, or a given density, up to and including or beyond confluence, are detached from the tissue culture vessel, collected in an aggregate, such as a pellet formed by centrifugation, and resuspended in tissue culture medium.
  • the suspension is then distributed to tissue culture vessels, such as plates or flasks, in such a way as to provide the cells with a greater total surface area on which to grow and divide than they had access to previously. This may be done by increasing the number of vessels. For example, the cells growing in one vessel may be detached, collected, resuspended, and distributed to two or more vessels.
  • This process also includes providing the cells with a volume of tissue culture medium that is able to support cellular growth and division.
  • Fig. 1 is a graph depicting the normalized plasma levels in dogs of human growth hormone (hGH) expressed by genetically modified canine bone marrow stromal cells.
  • hGH human growth hormone
  • the cells transplanted into dogs ALG-3, -9, -10, -11, and -15 were not cryopreserved at any stage of preparation.
  • the cells transplanted into dogs ALG-2 and ALG-4 were cryopreserved before they were transfected. Plasma levels of hGH for each data point were determined in quadruplicate.
  • Fig. 2 is a bar graph depicting the expression of hGH in vitro from transfected canine bone marrow stromal cells. These cultures were established from cells from a sample that was expanded, transfected, cryopreserved, and then thawed. The majority of the cells in the sample were autologously transplanted.
  • Fig. 3 is a graph that shows the presence of hGH inhibitors in dogs. The inhibitors are shown by comparing the level of hGH assayed by RIA with the amount of hGH "spiked" into the plasma of a dog (ALG-2) , over time, following intravenous administration of transfected hGH-expressing stromal cells. Plasma samples were obtained at the times indicated, beginning at day 0. This time point reflects hGH levels in dog plasma
  • Fig. 4 is a graph showing the normalized plasma level of hGH over time in dogs, following autologous transplantation of bone marrow stromal cells.
  • the cells infused into dogs ALG-3, -9, -10, -11, and -15 were not frozen at any stage of preparation.
  • the cells infused into dogs ALG-2 and ALG-4 were expanded, transfected, and then cryopreserved before they were thawed and transplanted.
  • the cells infused into dog ALG-8 were expanded, cryopreserved, thawed, recultured in vitro, and then transfected before implantation. Plasma levels of hGH for each data point were determined in quadruplicate. Fig.
  • FIG. 5 is a bar graph that shows the level of hGH in samples of stromal cell-conditioned medium in vitro . These cultures were established from cells from a sample that were expanded, cryopreserved, thawed, recultured in vitro, and then transfected. The majority of the cells in the sample were autologously transplanted.
  • Mongrel dogs were fully anesthetized and whole bone marrow was aspirated aseptically from the iliac crest.
  • the aspirate syringes contained heparin to prevent clotting.
  • the bone marrow was transferred from the syringe to a 50 ml conical tube containing 15 mis of a chilled tissue culture medium, such as RPMI or DMEM, and anti-fungal and antibiotic agents (50 ⁇ g/ml fungizone; 50 ⁇ g/ml gentamicin; 100 units/ml penicillin; 100 ⁇ g/ml steptomycin sulfate) .
  • a chilled tissue culture medium such as RPMI or DMEM
  • anti-fungal and antibiotic agents 50 ⁇ g/ml fungizone; 50 ⁇ g/ml gentamicin; 100 units/ml penicillin; 100 ⁇ g/ml steptomycin sulfate
  • Approximately 10-15 mis of bone marrow aspirate was added to
  • Nucleated cells were prepared from the bone marrow samples by a standard Ficoll cushion technique. Briefly, 15 ml of FICOLL-PAQUE ⁇ (Pharmacia Biotech) was placed in a 50 ml conical tube and one half of each of the marrow-medium samples was carefully layered on top of the Ficoll. The samples were centrifuged at 400 x g for 30 minutes at 18°C with the brake off so that the centrifuge head decelerated slowly after the elapsed time. The top layer of the resultant preparation, which contained cell-free medium, was removed and discarded. The middle layer, which contained nucleated cells, was carefully collected and placed into a fresh 50 ml tube containing 20 ml of tissue culture medium, as described above. Additional medium was added to bring the final volume to 50 ml.
  • the nucleated cells include the bone marrow stromal cells.
  • the stromal cells represent only a small fraction, i.e., one in a thousand, of the total number of nucleated bone marrow cells obtained in a bone marrow aspirate. Expansion
  • the nucleated cells were collected in a pellet by centrifugation at 100 x g for 10 minutes.
  • the cell pellet was washed with tissue culture medium (RPMI or DMEM with fungizone (25 ⁇ g/ml) , gentamicin (25 ⁇ g/ml) , penicillin (100 units/ml) and streptomycin sulfate (100 ⁇ g/ml) ) and resuspended in 5-10 ml of "complete bone marrow stromal cell medium" ("complete medium”) . After resuspension the cells were counted.
  • the complete bone marrow stromal medium contains the following ingredients in the following ranges of amounts or concentrations.
  • DMEM with 1 to 50% fetal bovine serum (FBS) (preferably greater than 12.5%) by volume; 0.01 to 100 ng/ml of an aFGF polypeptide, e.g., a recombinant aFGF; 0.05 to 100 units/ml of a heparin polypeptide, e.g., sodium heparin; 0.25 to 250 ⁇ g/ml of fungizone; 0.25 to 250 ⁇ g/ml of gentamicin; 1 to 1000 units/ml penicillin; and 1 to 1000 ⁇ g/ml of streptomycin sulfate.
  • FBS fetal bovine serum
  • the complete medium contained DMEM with 16 percent by volume heat-inactivated FBS, recombinant aFGF (1 ng/ml) by volume, heparin (5 units/ml) , fungizone (25 ⁇ g/ml) , gentamicin (25 ⁇ g/ml) , penicillin (100 units/ml) , and streptomycin sulfate (100 ⁇ g/ml) .
  • Tissue culture flasks (T150 cm 2 ) are preferably coated initially with gelatin and FBS. Specifically, a solution of gelatin (Sigma; 1% in water) was added to each flask until the bottom of the flask was just covered.
  • the excess was removed and the flasks were left undisturbed, bottom side down, at room temperature, for at least 30 minutes.
  • the flasks can be refrigerated at this point for later use.
  • Heat-inactivated FBS was then added to the gelatinized flasks. As before, the excess solution was removed and the flasks were left, bottom side down, at room temperature for at least 30 minutes. The flasks were either used at this point or refrigerated.
  • the cells were incubated in 15 mis of complete bone marrow medium, at 33°C, in the presence of 5% C0 2 .
  • 15 ml of complete medium was added to the cultures, dropwise, so the cells were not disturbed.
  • the so-called "conditioned medium,” i.e., the medium in the flask which contains non-adherent cells was removed, and 15 ml of fresh complete medium was added to the flask.
  • the non- adherent cells were pelleted by centrifugation at 500 x g for 5 minutes, resuspended in 15 ml of conditioned medium and returned to the original flask. Thus, the non- adherent cells were returned to the flask and the medium was changed in such a way that it contained one part fresh medium and one part conditioned medium.
  • the key to this regimen of cell culture is to: (1) coat the inner surface of the tissue culture vessel with a solution of gelatin, (2) keep returning the non-adherent cells to the culture when exchanging the medium, (3) add medium that contains sufficient nutrients to sustain growth without removing all of the substances secreted by the bone marrow cells, which enhance their growth, (4) supplement the tissue culture medium with aFGF, and (5) supplement the tissue culture medium with heparin.
  • This process where the non-adherent cells are removed, pelleted, and returned to the culture with equal parts of fresh and conditioned medium, is repeated once a week, for 2-3 weeks or until a monolayer of adherent cells has formed.
  • the cells were passaged by splitting them 1:2 or 1:3 into fresh flasks. At this point, and from this point on, the flasks were coated with gelatin but not with FBS. It is also no longer necessary to feed the established stromal cells with conditioned medium or to return non-adherent cells to the culture.
  • the cells may be passaged in this manner at least 8 times or more.
  • This method can be used to select and expand canine or human (or other vertebrate) bone marrow stromal cells, to develop a total cell number of more than IO 8 , and even more than 3 x IO 9 in vitro, from bone aspirates of individual subjects.
  • Other techniques of obtaining bone marrow can be used.
  • the bone marrow stromal cells obtained from dogs by this method exhibited the characteristic appearance of fibroblast-like bone marrow stromal cells. Given that the success of gene therapy depends on the cellular production of adequate levels of the transgene product, which can be quite low, the ability to expand cells in culture to IO 8 to l ⁇ 9 or more represents a substantial improvement.
  • the plasmid expression vector pETKhGH was prepared and transfected into canine stromal cells using standard techniques.
  • the dog model is an accepted animal model of the human bone marrow system, and results in dog studies are reasonably predictive of efficacy in human patients.
  • the vector was prepared from pTKGH (Selden et al., 1986, Mol. Cell. Biol., 6:3173-3179), which is comprised of the human growth hormone (hGH) gene, including introns, under the transcriptional regulation of HSV thymidine kinase (TK) promoter sequences (Nichols Institute Diagnostics, San Juan Capistrano, CA) .
  • TK thymidine kinase
  • TK thymidine kinase
  • vectors can be made that would express secreted proteins such as serum proteins, clotting factors, e.g., factor VIII and factor IX, cytokines, lymphokines, growth factors, e.g., human growth hormone, peptide hormones, lipid binding proteins, metabolic enzymes, antibacterial agents, antimicrobial agents, antifungal agents or neurotransmitters.
  • the vector could express cell surface components, e.g. receptors or cell surface adhesion molecules such as vascular cell adhesion molecule-1 (V-CAMl) , intercellular adhesion molecule-1 (I-CAM-1) , or V-LAM, or secreted gene products, such as collagen.
  • V-CAMl vascular cell adhesion molecule-1
  • I-CAM-1 intercellular adhesion molecule-1
  • V-LAM V-LAM
  • plasmid expression vectors Methods of engineering plasmid expression vectors are well known to persons of ordinary skill in the art, and many expression vectors that contain genes encoding proteins within the families listed above have been constructed and can be used to transfect bone marrow stromal cells. These include viral vectors that encode cytokines, such as IL-6 (Whartenby, et al., 1995, Pharmacology and Therapeutics, 66:175-190), and IL-7 (Kim et al., 1994, Human Gene Ther., 5: 1457-1466); clotting factors such as factor VIII (Dwarki et al., 1995, Proc. Natl. Acad.
  • metabolic enzymes such as aspartyl-glucosaminidase (Enomaa et al., 1995, Human Gene Ther. , 6:723-731), purine nucieoside phosphorylase (Jorisson et al., 1995, Human Gene Ther., 6:611-623), and uroporphyrinogen III synthase (Moreau- Gaudry et al., 1995, Human Gene Ther., 6:13-20); as well as cell surface adhesion molecules such as I-CAM-1 (Pilewski et al. , 1995, AM. J.
  • V-CAM-1 V-CAM-1
  • N-CAM neural cell adhesion molecule
  • canine bone marrow stromal cells were transfected with pETKhGH by either the CaP0 4 - DNA coprecipitation method, using the MBS Mammalian Transfection Kit (Stratagene Cloning Systems, LaJolla, CA) , or the cationic lipid-DNA complex method using LIPOFECTAMINE® reagent and OPTI-MEM® I reduced-serum medium (Life Technologies) according to the manufacturer's instruction.
  • the hGH expression is based on in vitro expression during the 24 hour period prior to cryopreservation. All cells derived from dog ALG-2 (data in rows 1-5 of Table 1) were transfected with the MBS Mammalian Transfection Kit from Stratagene or ProFection Mammalian Transfection Systems from Promega. The data in the last row, from dog ALG-4-derived cells were transfected with the LIPOFECTAMINE reagent from Life Technologies, Inc.
  • the expanded stromal cells were frozen. In preparation for cryopreservation, the cells were rinsed once with Dulbecco's Phosphate Buffered Saline (Gibco #14190-144) and detached with Trypsin-EDTA (0.05% Trypsin, 0.53 mM tetra-sodium-EDTA; Gibco #25300-062). The trypsinization was stopped by adding an equal volume of media (DMEM with antibacterial and anti-fungal agents at the concentrations given above) . The cells were pelleted by centrifugation at 500 x g for 5 minutes, resuspended in 3 ml of media, and counted. Cell density was adjusted to 1 x IO 6 cells/ml with media containing 10% Dimethyl Sulfoxide (DMSO; Sigma D-8779) , and 1 ml aliquots were added to sterile 2 ml cryogenic vials
  • the cells are pelleted by centrifugation at 500 x g in larger volumes of medium, e.g., 200 ml or more.
  • the cell pellet is suspended in 10 to 20 ml of medium, and cells are counted.
  • the suspension is then brought to a larger volume, e.g., 45 ml, with medium, and added to a transfer pack container (Baxter Fenwal, 4R2001) with a sterile syringe fitted with an 18 gauge sterile needle. Five ml of DMSO is then added, and the pack is stored at -80°C overnight.
  • the pack is transferred to liquid nitrogen tanks or to a -150°C freezer for long-term storage. Both of these cryopreservation methods can be used for stromal cells from humans as well as from dogs, primates, cows, pigs, and other animals.
  • the data shown in Table 2 were derived from cells that were reintroduced intravenously into a foreleg vein.
  • the hGH expression capacities, of 1,400 and 575 ⁇ g/24 hours, shown in the last column were based on in vitro expression during the 24 hour period prior to cryopreservation.
  • the value of 445 ⁇ g/24 hours, again in the last column, was also based on in vitro expression. In this instance, an aliquot of cells was taken from the pool of cells that were reinfused and returned to tissue culture. hGH was measured during the 24 hour period immediately after the remainder of the cells were reinfused.
  • Some cells derived from dog ALG-4 were returned to tissue culture, and hGH was measured in vitro for 24 hours after the sibling cells were reinfused. During this time, the cells in culture expressed and secreted 2.24 ⁇ g of hGH/1 x IO 6 cells/24 hours, which predicts an in vivo expression capacity of 445 ⁇ g of hGH/24 hr period (Table 2) . The same measurements were taken before cryopreservation. At that time, the cells in culture expressed and secreted 2.89 ⁇ g of hGH/l x 106 cells/24 hours, which predicts an expression capacity in vivo of 575 ⁇ g/24 hours. Note that the measurements taken before cryopreservation are very similar to those obtained after cryopreservation.
  • Table 3 the lowest hGH standards, shown in column 2, are statistically higher (t-test) than the expression of hGH in pre-infusion plasma at a confidence level of greater than 95%.
  • the data above the thick line in Table 3 pertains to ALG-2, and data below the thick line pertains to ALG-4.
  • Table 2 Reintroduction into Dogs of Thawed Autologous Bone Ma ⁇ ow Stromal Cells
  • hGH levels of hGH from transplanted stromal cells that were genetically modified as described here, but that were not cryopreserved at any stage were determined.
  • the normalized in vivo levels of hGH expression were comparable, whether or not the genetically modified stromal cells were cryopreserved before reinfusion.
  • Levels of hGH in the plasma of each dog, at each time point were normalized by dividing absolute hGH plasma levels (ng/ml) by the total hGH expression capacity ( ⁇ g/24 hr) of the cells infused into each dog, multiplied by the weight (kg) of the individual dog, and then by 100%.
  • Fig. 2 shows that the level of hGH peaked at 4 or 3 (flasks 1 and 2, respectively) ⁇ g/24 hr/10 6 cells, was still over 1 ⁇ g/24 hr/10 6 cells, and then dropped gradually until week 15.
  • Circulating stromal cells bearing the hGH vector were detected by a sensitive nested PCR assay.
  • This assay can detect 5-10 copies of the pETKhGH plasmid expression vector in a background of 1 x 10 5 control stromal cells and is effective in 90% of the reactions performed (9 positives/10 reactions) .
  • the first step 25 ⁇ l of cell lysate (2.5 X IO 5 cell equivalents) was mixed with 25 ⁇ l of 2X PCR reaction mix and subjected to an initial incubation at 94°C for 5 minutes and then 35 cycles of 94°C (30 seconds), 58°C (50 seconds), and 72°C (1.5 minutes) in the presence of a 5'-oligonucleotide primer that is homologous to exon 2 sequences in the hGH gene, and a 3'- primer that is homologous to exon 5 sequences in the hGH gene. Primers were obtained from Oligos, etc. Inc.
  • the final concentration of the PCR reaction mix was 50 mM Tris-HCl (pH 9.0), 50 mM NaCl, 2 mM MgCl 2 , 0.2 mM each of NTPs (dATP, dGTP, dCTP, and dTTP) , 0.5 ⁇ M each of 5'- and 3'-primers, and 0.5 units Taq DNA polymerase (Promega Corp., Madison, WI) .
  • this second set of oligos is homologous to gene sequences internal to the first set of oligos and did not overlap their sequences. All primers were chosen from regions of the template gene that did not contain internally repetitive sequences and that had a G-C content of less than 67%. Numerous other primer combinations can be developed by standard techniques for use in this nested PCR assay.
  • Thermocycling for the nested reaction was identical to the first reaction except that the annealing temperature was 56°C.
  • a 10 ⁇ l aliquot of each of the first and nested PCR reaction products was analyzed by electrophoresis on a 1% agarose gel and the amplified cDNA was visualized by ethidium bromide staining.
  • DNA molecular weight markers were comprised of phiX174 DNA digested with Haelll (Boehringer Mannheim) .
  • the expected size of the correct nested PCR product is 1,119 base pairs. Control analyses on canine cells demonstrated that the nested PCR reaction protocol did not detect canine growth hormone gene sequences.
  • hGH inhibitors developed in vivo (Fig. 3; dog ALG-2) . These inhibitors were detected by experiments in which plasma samples were spiked with 0.5 ng/ml of hGH at various times. As shown in Fig. 3, the first hGH measurement (indicated on Fig. 3 as day 0) was obtained prior to infusion of transduced cells. These measurements continued for 20 days following infusion of hGH-expressing stromal cells. Inhibitor activity was determined by comparing the level of hGH in each sample, which was empirically measured by RIA, with the amount of hGH (0.5 ng/ml) purposefully added to each sample.
  • bone marrow stromal cells in culture and cryopreserve them prior to transduction.
  • bone marrow stromal cells were aspirated from the primary iliac crest of dog ALG-8, and expanded in vitro by culturing methods described in detail above.
  • 2.12 x IO 8 cells were cryopreserved in media containing DMSO by the procedure described in detail above. From this frozen stock, 1 x 10 8 cells were subsequently thawed and placed in tissue culture as passage 3 cells. After one additional passage, 1.35 x IO 8 cells were transduced by transfection with the pETKhGH plasmid expression vector.
  • the level of in vitro expression and secretion of hGH into the tissue culture media was 1.22 ⁇ g/1 x IO 6 cells/24 hr period, as determined by radioimmunoassay.
  • the levels after infusion e.g. 0.189 ng/ml at 24 hours; Table 5
  • the hGH present in the plasma 3 days after modified stromal cell infusion must be due to de novo expression of hGH by these cells.
  • the hGH expression capacity shown in Table 4 was based on in vitro expression of hGH during the 24 hour period preceeding cryopreservation.
  • the lowest hGH standards, shown in column 2 are statistically higher (t-test) than the expression of hGH in pre-infusion plasma at a confidence level of greater than 95%.
  • the average plasma levels of hGH, shown in column 4 represent values that are statistically higher (t-test) than those seen in pre-infusion plasma at a confidence level of greater than 99%, except where indicated.
  • hGH normalized plasma levels of hGH were measured from bone marrow stromal cells that had never been cryopreserved (Fig. 4, samples from dogs ALG-3, -9, -10, -11, and 15). These levels were compared with those from stromal cells that were expanded in vitro, cryopreserved, thawed, and transduced with the hGH expression vector prior to reintroduction into animals (dog ALG-8) . The normalized in vivo expression levels were comparable, regardless of whether or not the cells had been cryopreserved.
  • hGH expression was comparable regardless of whether the cells were cryopreserved before (dog ALG-8) or after (dogs ALG-2 and ALG-4) they were genetically modified.
  • Plasma levels of hGH were detected in dog ALG-8 as soon as 15 minutes following the infusion of cells.
  • a 1 ml aliquot of the modified cells that were intended for reinfusion to ALG-8 was kept on ice while the rest of the cells were infused.
  • Complete medivim (5 ml) was added to the aliquot and the cells were kept at 37°C for 20 minutes. Subsequently the cells were washed, resuspended in tissue culture medium, and the amount of hGH expressed and secreted into the medium was determined at a time that reflected the 15 minute period post-infusion measurement from plasma.
  • the in vitro expression capacity was
  • FBS FBS at a density of 2-5 x IO 7 cells/ml.
  • 900 ⁇ l of this suspension was aliquoted into 2 ml sterile cryogenic vials (Corning #25704) with 100 ⁇ l of DMSO.
  • the vials were stored at -80°C for 24 hours and then transferred to a -150°C freezer or to liquid nitrogen tanks for long- term storage. Human primary bone marrow aspirates can be cryopreserved in the same manner.
  • Cryopreserved cells were subsequently thawed and bone marrow stromal cells established and expanded in culture as described above. After the fourth passage, two T25 flasks containing 4 x 10 5 stromal cells each were transfected with the pETKhGH expression vector using
  • LipofectAMINE® Transduced stromal cells expressed and secreted hGH into the media. Two weeks following transfection, hGH was expressed at 143 ng and 155 ng per 1 x IO 6 cells per 24 hours in two flasks and expression of hGH continued for 9 weeks.
  • bone marrow stromal cells can be expanded from primary bone marrow aspirates that have been cryopreserved and that these cells can be transduced at a later time and express transgene product.

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Abstract

Cette invention concerne des procédé séquentiels de préservation cryogénique de cellules de stroma de moelle osseuse, lesquelles sont transfectées et utilisées en thérapie génique lors de transplantations. Ces procédés comprennent les étapes suivantes dans des ordres divers: obtenir lesdites cellules; effectuer l'expansion des cellules dans un milieu de culture; transfecter les cellules; et enfin, préserver ces cellules par cryogénie. Ces procédés permettent d'obtenir des populations de cellules de stroma de moelle osseuse suffisamment importantes pour qu'elles puissent être utilisées dans un grand nombre de thérapies. Ces populations importantes peuvent en outre être stockées pour de longues durées afin d'être utilisées immédiatement en cas de besoin.
PCT/US1995/016991 1995-12-29 1995-12-29 Procedes de preparation de cellules de stroma de moelle osseuse utilisees en therapie genique WO1997024144A1 (fr)

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Cited By (11)

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WO1999038952A2 (fr) * 1998-01-29 1999-08-05 Advanced Tissue Sciences, Inc. Cellules ou tissus avec facteurs proteiques en quantite accrue et leurs methodes d'obtention et d'utilisation
US6241701B1 (en) 1997-08-01 2001-06-05 Genetronics, Inc. Apparatus for electroporation mediated delivery of drugs and genes
US6991787B1 (en) 1995-12-29 2006-01-31 Alg Company Methods of preparing bone marrow stromal cells for use in gene therapy
WO2019155031A3 (fr) * 2018-02-09 2019-09-26 General Electric Company Procédés de biotraitement pour thérapie cellulaire
US10889792B2 (en) 2018-02-09 2021-01-12 Global Life Sciences Solutions Usa Llc Cell expansion vessel systems and methods
US11332732B2 (en) 2017-12-01 2022-05-17 Global Life Sciences Solutions Usa Llc Magnetic cell isolation techniques
US11371007B2 (en) 2018-02-09 2022-06-28 Global Life Sciences Solutions Usa Llc System and method for fluid flow management in a bioprocessing system
US11414639B2 (en) 2018-02-09 2022-08-16 Global Life Sciences Solutions Usa Llc Bioprocessing vessel
US11920119B2 (en) 2018-02-09 2024-03-05 Global Life Sciences Solutions Usa Llc Systems and methods for bioprocessing
US11932842B2 (en) 2018-02-09 2024-03-19 Global Life Sciences Solutions Usa Llc Bioprocessing apparatus
US12077743B2 (en) 2018-02-09 2024-09-03 Global Life Sciences Solutions Usa Llc Apparatus for fluid line management in a bioprocessing system

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EP0381490A2 (fr) * 1989-02-02 1990-08-08 Joel S. Greenberger Thérapie génique utilisant des cellules du stroma

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6991787B1 (en) 1995-12-29 2006-01-31 Alg Company Methods of preparing bone marrow stromal cells for use in gene therapy
US6241701B1 (en) 1997-08-01 2001-06-05 Genetronics, Inc. Apparatus for electroporation mediated delivery of drugs and genes
WO1999038952A2 (fr) * 1998-01-29 1999-08-05 Advanced Tissue Sciences, Inc. Cellules ou tissus avec facteurs proteiques en quantite accrue et leurs methodes d'obtention et d'utilisation
WO1999038952A3 (fr) * 1998-01-29 1999-10-14 Advanced Tissue Sciences Inc Cellules ou tissus avec facteurs proteiques en quantite accrue et leurs methodes d'obtention et d'utilisation
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
US11332732B2 (en) 2017-12-01 2022-05-17 Global Life Sciences Solutions Usa Llc Magnetic cell isolation techniques
US10889792B2 (en) 2018-02-09 2021-01-12 Global Life Sciences Solutions Usa Llc Cell expansion vessel systems and methods
WO2019155031A3 (fr) * 2018-02-09 2019-09-26 General Electric Company Procédés de biotraitement pour thérapie cellulaire
US11371007B2 (en) 2018-02-09 2022-06-28 Global Life Sciences Solutions Usa Llc System and method for fluid flow management in a bioprocessing system
US11414639B2 (en) 2018-02-09 2022-08-16 Global Life Sciences Solutions Usa Llc Bioprocessing vessel
US11920119B2 (en) 2018-02-09 2024-03-05 Global Life Sciences Solutions Usa Llc Systems and methods for bioprocessing
US11932842B2 (en) 2018-02-09 2024-03-19 Global Life Sciences Solutions Usa Llc Bioprocessing apparatus
US12077743B2 (en) 2018-02-09 2024-09-03 Global Life Sciences Solutions Usa Llc Apparatus for fluid line management in a bioprocessing system

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