+

WO1993020195A1 - Procede et cellules recombinees conferant a des cellules souches hematopoietiques une resistance accrue a la toxicite d'agents chimiotherapeutiques - Google Patents

Procede et cellules recombinees conferant a des cellules souches hematopoietiques une resistance accrue a la toxicite d'agents chimiotherapeutiques Download PDF

Info

Publication number
WO1993020195A1
WO1993020195A1 PCT/US1993/002929 US9302929W WO9320195A1 WO 1993020195 A1 WO1993020195 A1 WO 1993020195A1 US 9302929 W US9302929 W US 9302929W WO 9320195 A1 WO9320195 A1 WO 9320195A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
hematopoietic progenitor
progenitor cells
glutathione
patient
Prior art date
Application number
PCT/US1993/002929
Other languages
English (en)
Inventor
Thomas C. Hamilton
Andrew K. Godwin
Alton Meister
Mary E. Anderson
Chin Shiou Huang
Original Assignee
Fox Chase Cancer Center
Cornell Research Foundation
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 Fox Chase Cancer Center, Cornell Research Foundation filed Critical Fox Chase Cancer Center
Publication of WO1993020195A1 publication Critical patent/WO1993020195A1/fr

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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/104—Aminoacyltransferases (2.3.2)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y108/00—Oxidoreductases acting on sulfur groups as donors (1.8)
    • C12Y108/01—Oxidoreductases acting on sulfur groups as donors (1.8) with NAD+ or NADP+ as acceptor (1.8.1)
    • C12Y108/01007—Glutathione-disulfide reductase (1.8.1.7), i.e. glutathione reductase (NADPH)
    • 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

Definitions

  • the present invention relates to cancer chemotherapy.
  • methods and recombinant cells are provided for increasing cellular resistance of hematopoietic progenitor cells to toxic effects of chemotherapeutic agents used in treating patients • having malignant tumors.
  • BACKGROUND Several forms of malignant tumors, including ovarian cancer, are effectively treated by anti cancer drugs, including alkylating agents and platinum- containing drugs. Unfortunately, the initial positive response to therapy is often limited by development of resistance to these chemotherapeutic agents.
  • Glutathione is synthesized intracellularly by the successive actions of ⁇ - glutamylcysteine synthetase and glutathione synthetase. ⁇ -glutamylcysteine synthetase catalyzes the rate-limiting step in glutathione synthesis.
  • ⁇ -glutamyltranspeptidase functions in cellular recovery of cysteine moieties, which are required for continued glutathione synthesis. Thus, these two enzymes are essential catalysts in glutathione metabolism. See generally, eister and Anderson, Ann. Rev. Biochem., 52.: 711-60 (1983). It has been shown that glutathione is associated with cellular resistance to radiation and DNA reactive drugs (platinum analogs and classical alkylating agents) . Treatment of resistant tumor cells with D,L-buthionine- (S,R) -sulfoximine (BSO) , a selective inhibitor of ⁇ -glutamylcysteine synthetase (Griffith and Meister, J. Biol.
  • Glutathione-mediated drug resistance is but one of several types of drug resistance arising from exposure of cells to various agents.
  • resistance to neomycin and analogs thereof is mediated in bacteria by neomycin phosphotransferase.
  • Methotrexate resistance appears to be associated with dihydrofolate reductase activity.
  • Classical multiple drug resistance in many cell types has been found to involve the m rl gene. These cancerous cells acquire resistance to drugs, such as colchicine, vinblastine and adriamycin, by repeated exposure, which results in enhanced expression of the multiple drug resistance gene.
  • the human MDR1 gene has been found to encode a 4.5 kb mRNA which is overexpressed in resistant cell lines. This mRNA encodes a drug efflux pump glycoprotein (P-glycoprotein) , which is believed to function in removal of the cytotoxic drugs from cells.
  • P-glycoprotein drug efflux pump glycoprotein
  • genes encoding drug resistance factors also have potential utility in cancer treatment.
  • a cloned mouse MDR gene when introduced via an expression vector into cultured tumor cells, is sufficient to confer multi-drug resistance to those cells. Gros et al., Nature, 323: 728-31 (1986).
  • a full length cDNA of the human MDR1 gene is capable of conferring multi-drug resistance when integrated into the genome of drug-susceptible recipient cultured tumor cells.
  • NTIS Publication No. PB87-218434 filed June 16, 1987.
  • a human MDR1 cDNA in the bone marrow of transgenic mice conferred resistance to daunomycin, a drug that induces bone marrow toxicity.
  • bone marrow toxicity is the main dose-limiting factor for many commonly used anti- cancer drugs.
  • the ability to impart drug resistance selectively to bone marrow stem cells would be particularly advantageous in cancer therapy because it would enable dosages of various chemotherapeutic agents to be increased, thereby enhancing the efficacy of chemotherapy without compromising viability of hematopoietic progenitor cells.
  • This may be accomplished by genetically altering the cells to produce more of the protein or other factors responsible for conferring drug resistance.
  • genes encoding factors involved in drug resistance must be efficiently transferred to isolated human hematopoietic progenitor cells, so that the resultant cells exhibit drug resistance upon infusion into a patient requiring therapy.
  • glutathione-mediated resistance to various chemotherapeutic agents may be imparted to drug- sensitive cells by genetically altering the cells to increase their intracellular production of reduced glutathione (GSH) .
  • GSH reduced glutathione
  • a method is provided for increasing the resistance of hematopoietic progenitor cells to chemotherapeutic agents used in treating a patient having a malignant tumor.
  • healthy hematopoietic progenitor cells are obtained from suitable donors. These cells are genetically altered by introducing into the cells, for stable incorporation in the genome, at least one segment of DNA that encodes at least one protein capable of increasing the intracellular production of glutathione in the cells.
  • the genetically altered cells are then infused into a recipient patient for whom chemotherapy has been prescribed.
  • These genetically altered hematopoietic cells and their progeny possess enhanced resistance to chemotherapeutic agents that would be toxic to hematopoietic progenitor cells not genetically altered in this manner. For this reason, a patient requiring anti-cancer chemotherapy may be treated with larger doses of chemotherapeutic agents than would normally be tolerable due to bone marrow toxicity.
  • the ' above-described method specifically comprises providing healthy hematopoietic progenitor cells, preferably from a cancer patient prescribed to receive chemotherapy with drugs whose dosage is limited by bone marrow toxocity.
  • the cells are then genetically altered by infection with a replication- incompetent retroviral shuttle vector which comprises at least one cDNA encoding a biologically active protein capable of increasing intracellular glutathione production.
  • the cDNA encodes an enzymatically active, preferably human, ⁇ -glutamylcysteine synthetase or a cDNA encoding an enzymatically active glutamyltranspeptidase.
  • the cDNA becomes stably incorporated into the genome of the hematopoietic progenitor cells, thereby imparting to the cells the capability of producing increased amounts of intracellular glutathione.
  • These cells are infused into a patient requiring chemotherapy, such that increased dosages of chemotherapeutic agents may be administered without the bone marrow toxicity that would result without genetically altering the hematopoietic progenitor cells.
  • the types of chemotherapeutic agents that may be administered in increased dosages in the practice of the present invention include platinum- containing agents such as cisplatin and carboplatin, classical bifunctional alkylating agents such as melphalan, subclasses of natural products such as anthracyclines and epipodophilotoxins and compounds such as vinblastine.
  • radiation therapy may be administered in increased dosages, either alone or in combination with the chemotherapeutic agents such as those mentioned above, in accordance with the practice of the invention.
  • recombinant hematopoietic progenitor cells are provided for use in conjunction with chemotherapeutic agents and/or radiation therapy in treatment of patients having a malignant tumor.
  • These cells comprise a genome having at least one introduced segment of DNA that encodes at least one protein capable of increasing intracellular production of glutathione.
  • these cells have enhanced resistance to concentrations of chemotherapeutic agents that would be toxic to non-recombinant hematopoietic progenitor cells.
  • recombinant hematopoietic progenitor cells which comprise a genome containing at least one introduced segment of DNA that encodes an expressible, enzymatically active form of ⁇ - glutamylcysteine synthetase capable of increasing intracellular production of glutathione.
  • These cells express 7-gluta ⁇ nylcysteine synthetase in an amount effective to substantially negate the inhibitory effect of a standard inhibitory dosage of BSO, which as noted above, is a specific inhibitor of 7- glutamylcysteine synthetase.
  • BSO which as noted above, is a specific inhibitor of 7- glutamylcysteine synthetase.
  • These cells have enhanced resistance to normally toxic concentrations of chemotherapeutic agents used for treatment of malignant tumors.
  • a method for treating a patient having a malignant tumor by administration of chemotherapeutic agents used in treatment of such tumors comprises infusing into the patient recombinant hematopoietic progenitor cells comprising a genome containing a DNA segment that encodes an expressible, enzymatically form of y- glutamylcysteine synthetase capable of increasing intracellular production of glutathione.
  • the 7- glutamylcysteine synthetase is expressed in an amount sufficient to substantially negate the inhibitory effect of a standard inhibitory dosage of BSO and the cells have enhanced resistance to normally toxic concentrations of the above-mentioned chemotherapeutic agents.
  • the method further comprises administering to the patient at least one of the above-mentioned chemotherapeutic agents at a dosage that would be toxic to the patient's non- recombinant hematopoietic progenitor cells.
  • Increased dosages which could provide effective irradication of these refractory solid tumors or the ability to treat initially with higher dosages, could not be administered heretofore because of the resultant toxicity to normal tissues, including hematopoietic stem cells present in bone marrow.
  • the present invention provides a way to increase the resistance of bone marrow stem cells to these chemotherapeutic agents, thereby permitting administration of increased dosages which could effectuate a cure. This strategy has particularly significant implications in the treatment of advanced solid tumor cancers, where high- dosage chemotherapy may be the only treatment available.
  • the methods set forth in the present invention provide a potentially life-saving therapeutic treatment for patients suffering from cancer.
  • ovarian tumor cell lines demonstrating high resistance to the platinum- containing chemotherapeutic agent, cisplatin, as well as cross resistance to carboplatin and melphalan possess markedly elevated levels of reduced glutathione (GSH) , and a close correlation exists between drug resistance and the level of GSH present in resistant cell lines.
  • GSH reduced glutathione
  • GSH elevation in the cell lines corresponds to a steady state increase in the amount of messenger RNA encoding two enzymes involved in the glutathione biosynthetic/metabolic pathway: y- glutamylcysteine synthetase (7-GCS) , which catalyzes the controlling (and feedback-inhibited) steps of GSH synthesis, and 7-glutamyltranspeptidase (7-GT) , which functions in cellular recovery of cysteine moieties (salvage pathways) . Resistance of the above-described ovarian cancer cell lines is reduced when the cells are treated with BSO, a specific inhibitor of 7-GCS. Griffin and Meister, J. Biol. Chem., 254: 7558-60 (1979) .
  • a hematopoietic progenitor cell i.e., stem cell
  • stem cell may be defined as a pleuripotent cell that gives rise to progeny in all defined hematolymphoid lineages.
  • Stem cells have the ability to restore, when transplanted, the production of hematopoietic and lymphoid cells to an individual who has lost such production due, e.g., to immunocompromising disease, radiation or chemotherapy.
  • a cDNA encoding human 7- GCS is isolated and cloned into a mammalian expression vector, pLTR-2.
  • transfection of drug-sensitive cell lines with the 7-GCS cDNA-containing vector may impart to these cell lines resistance to various alkylating chemotherapeutic agents and irradiation.
  • Resistance is correlated with expression of the recombinant 7-GCS protein and with intracellular production of GSH.
  • genetic alteration of cell lines with a key regulatory enzyme involved in glutathione biosynthesis can confer resistance to previously drug-sensitive cells.
  • other enzymes involved in the biosynthesis and metabolism of glutathione either alone or in combination are contemplated in conferring or enhancing bone marrow resistance to several classes of anti-cancer drugs.
  • Bone marrow comprising hematopoietic progenitor cells (sometimes referred to herein as stem cells)
  • stem cells may be obtained from autologous, allogeneic or syngeneic donors by methods well known to physicians and others skilled in the art. See, e.g., E. Beutler, Clinical Bone Marrow Transplantation, K. G. Blume and L. D. Petz, eds., Churchill Livingstone, pp. 1-13 (1983) .
  • bone marrow for transplantation is obtained by multiple aspirations from the iliac crest of a donor.
  • the aspirate is mixed with an anticoagulant (e.g., heparin) and a tissue culture medium.
  • an anticoagulant e.g., heparin
  • bone marrow cells are subjected to any desired manipulations, such as those described in further detail below.
  • Hematopoietic progenitor cells may be further purified from the bone marrow aspirate, if desired, as described herein.
  • the bone marrow cells are then infused intraveneously into the recipient (i.e., a patient requiring bone marrow transplantation) .
  • the cells migrate through the bloodstream to the recipient's bone marrow, where they establish colonies and continue to divide.
  • Glutathione-Affecting Enzymes Any DNA segment encoding a biologically active protein involved in the synthesis and/or metabolism of glutathione has potential utility in the practice of the present invention. Human cDNAs are preferred, though genes may also be useful in some cases. In a preferred embodiment, a cDNA encoding an enzymatically active 7-glutamylcysteine synthetase (7- GCS) is employed. In an alternative preferred embodiment, a cDNA encoding enzymatically active 7- glutamyltranspeptidase is utilized, either alone or in combination with 7-GCS. DNA segments encoding other glutathione-regulating enzymes may also be used, either alone or in combination.
  • cDNAs encoding the above-mentioned enzymes are obtainable by a variety of methods known in the art.
  • an appropriate cDNA library e.g., human, in a preferred embodiment
  • cDNA expression libraries may be screened with antibodies raised against the glutathione-affecting enzyme of interest. See, e.g.,.Huynh et al. , DNA Cloning: A Practical Approach, Vol. 1, D. M. Glover, ed. , pp. 49-78 (1985) for general methods of screening a lambda gt 11 expression library.
  • genomic libraries may be screened with DNA probes or antibodies; however, cDNAs, because of their size and lack of introne containing repetitive sequences, are more likely to be useful in the practice of the present invention.
  • Retrovirus-mediated DNA transfer is a preferred method in the practice of the present invention. Retrovirus-mediated DNA transfer is defined as a method by which replication- defective retroviruses and retroviral vectors are used to transfer segments of foreign DNA into host cells infectable by such viruses, and to effect stable integration of those DNA segments into the genomes of such host cells. The use of retroviral vectors is the method of choice because of the high efficiency of gene transfer that may achieved. Exogenous genes carried within the retrovirus genome are brought into target cells by the virus.
  • Retroviral vectors have been constructed so they do not contain any viral protein-encoding genes but only the desired exogenous genes.
  • the missing viral functions i.e., the synthesized gag, pol , and env proteins
  • packaging cell lines that act as helper viruses, but cannot themselves propagate.
  • retroviral mediated gene transfer has been applied successfully for the insertion and expression of several different genes in hematopoietic stem cells of several mammalian species; thus, methods for accomplishing such gene transfer are well known in the art.
  • retroviral shuttle vectors examples include the SVX-neo (Cepko et al., Cell, 3_7: 1053- 62 (1984)) and the N2 (Keller et al., Nature, 318: 275-77 (1985)) vectors and derivatives thereof.
  • SVX- neo is a highly transmissible replication-defective retroviral shuttle vector.
  • This vector consists of the Moloney murine leukemia virus (MoMuLV) from which the viral structural genes have been removed, leaving both long terminal repeat sequences (LTR) , the retroviral encapsidation signal sequence (Psi) , a portion of the gag coding sequence, including the splice donor and the splice acceptor sites, and the SV40 and pBR322 ori sequences.
  • LTR long terminal repeat sequence
  • Psi retroviral encapsidation signal sequence
  • gag coding sequence including the splice donor and the splice acceptor sites
  • SV40 and pBR322 ori sequences a portion of the gag coding sequence, including the splice donor and the splice acceptor sites
  • the bacterial gene Neo R (derived from the TN5 transposon) conferring resistance to the antibiotic neomycin (or its analogue G418 that is toxic to mammalian cells) has been inserted as a dominant selectable marker
  • this vector (referred to as SVX/SV40) has been modified so that it contains two promoters, the viral LTR and the SV40 early promoter, in opposing transcriptional orientations.
  • the Neo R gene is driven by the retroviral 5' LTR, and the cDNA encoding the enzymatically active subunit of 7-GCS is driven by the SV40 early promoter. Additional DNA segments described above may be substituted for 7-GCS into the SVX/SV40 vector for transfer into bone marrow.
  • Safe DNA transfer into mammalian cells using retroviral vectors requires the availability of safe amphotrophic retrovirus packaging cell lines, which are incapable of producing wild type virus.
  • appropriate producer cell lines capable of safely and efficiently mediating retroviral gene transfer are well known in the art.
  • an amphotropic packaging cell line, "GP+ envAm-12” in which the viral gag and pol functions are present in one plasmid, and the env function on another plasmid, is disclosed by Markowicz et al., Virology, 167: 400-06 (1988) .
  • a high-titer retrovirus producer cell line capable of mediating gene transfer into primate hematopoietic stem cells, has been disclosed, and may be used advantageously in the practice of the present invention. Bodine et al. , Proc. Natl. Acad. Sci.- (USA), 87: 3738-42 (1990).
  • This producer cell line which secretes gibbon IL-3 and human IL-6 and produces greater than 10 10 functional viral particles per ml of culture medium, has been used to reproducibly transfer genes into bone marrow stem cells of rhesus monkeys.
  • cDNAs or genes encoding the above-mentioned glutathione-affecting proteins may be cloned into mammalian expression vectors, then utilized to transfect cultured mammalian cells.
  • LTR long terminal repeat
  • This construct can be used with pKOneo, a plasmid encoding resistance to the neomycin analog, G418, to co-transfect cultured mammalian cells, e.g., ovarian tumor cells.
  • Transformed cells are selected by resistance to G418 and verified for genomic integration and expression through DNA, RNA and protein blotting.
  • G418 a plasmid encoding resistance to the neomycin analog
  • G418 a plasmid encoding resistance to the neomycin analog
  • G418 a plasmid encoding resistance to the neomycin analog
  • Transformed cells are selected by resistance to G418 and verified for genomic integration and expression through DNA, RNA and protein blotting.
  • genetic alteration of hematopoietic progenitor cells may be accomplished by means of transfection (via standard mammalian expression vectors) or high-efficiency infection (via replication-incompetent retroviral vectors) .
  • Promoters should be constructed so that DNA segments encoding the above-described glutathione-affecting proteins are positioned downstream from a viral or cellular promoter, thereby enabling high expression of the DNA segment.
  • Many constitutive and inducible promoters are known in the art, and may be suitable for expression of recombinant proteins in genetically altered hematopoietic stem cells.
  • the expression vector features a hormone inducible promoter, the mouse mammary tumor virus long terminal repeat, which controls the expression of a cDNA encoding 7-GCS.
  • This vector also features the neoR (neomycin phosphotransferase) gene under the control of the MoMuLV LTR.
  • Other promoters contemplated for use in the present invention include a human IL3 inducible promoter.
  • Constitutive promoters which may be used to advantage include a human 7-globin promoter, a ⁇ -actin promoter, as well as others.
  • High-efficiency retrovirus-mediated DNA transfer for the purpose of genetically altering hematopoietic stem cells, may be accomplished by infection protocols known in the art. See, e.g., Bodine et al. , Proc. Natl. Sci. (USA), 81_ 3638-42
  • a selected DNA segment can be used in clinical application, its effectiveness in increasing intracellular glutathione should be determined. This may be accomplished by introducing, e.g., by transfection, mammalian expression vectors containing selected DNA segments into cell lines with low levels of GSH. Genetic alteration may also be accomplished by other methods known in the art, such as calcium phosphate precipitation, liposome-mediated transfer or electroporation. Genetically altered cultured cell lines may then be tested for the presence of the selected DNA segment, as well as its expression, by standard methods including DNA blotting, RNA blotting and immunoblotting. Suitably altered cell lines should be analyzed for overproduction of intracellular GSH.
  • a cDNA encoding 7-GCS is used to genetically alter cisplatin-sensitive cell lines.
  • Overexpression of 7-GCS in these cell lines may be tested by treatment with the specific 7-GCS inhibitor, BSO. If overexpression of 7-GCS has been achieved in these cell lines, they should be able to produce elevated levels of GSH, even when treated with standard inhibitory doses of BSO (e.g., 25-100 ⁇ M) .
  • standard inhibitory doses of BSO e.g. 25-100 ⁇ M
  • This effect i.e., production of elevated levels of GSH in the presence of 25-100 ⁇ M BSO
  • substantial negation of the inhibitory effect of a standard inhibitory dosage of BSO is sometimes referred to herein as substantial negation of the inhibitory effect of a standard inhibitory dosage of BSO.
  • RNA and protein blotting After overexpression of the selected glutathione-affecting enzyme (e.g., 7-GCS) has been confirmed by RNA and protein blotting, cellular glutathione levels and resistance to cytotoxicity of selected alkylating chemotherapeutic agents is determined. Methods for making these determinations are described in the examples below.
  • the selected glutathione-affecting enzyme e.g., 7-GCS
  • Cell lines overexpressing 7-GCS are further tested with the 7-GCS inhibitor, BSO, according to the following general procedure.
  • Potentially cytotoxic agents such as melphalan, cisplatin or radiation, are administered to the transfected cell line in the presence or absence of 7-GCS-inhibitory concentrations of BSO (e.g., 25-100 ⁇ M) .
  • Procedures for testing transfected cell lines using BSO are more fully set forth in the examples below.
  • Cell lines that overexpress 7-GCS will show higher resistance to the cytotoxic agents than cell lines which were not genetically altered to overproduce 7-GCS.
  • Hematopoietic progenitor cells that have not been genetically altered in accordance with the present invention are sometimes referred to herein as non- recombinant hematopoietic progenitor cells.
  • a particular DNA segment encoding a glutathione- affecting protein is effective in increasing intracellular glutathione production in cultured cells, it may be developed for high-efficiency infection of hematopoietic progenitor cells.
  • the DNA segment of interest may be cloned into replication- defective retroviral shuttle vectors by the methods mentioned above. Healthy, hematopoietic progenitor cells are obtained from suitable donors, including autologous donation from a patient for whom chemotherapy has been prescribed, also as described above.
  • Producer cells as described above in Section II, are grown to confluence.
  • confluent plates of producer cells are split 1:10 in DMEM/10% fetal calf serum (FCS) containing 6 micrograms/ml Polybrene (Sigma Chemical Co.) .
  • FCS fetal calf serum
  • bone marrow is aspirated from donor individuals as described in the preceding section, into DMEM/2% FCS containing 10 units/ml heparin.
  • Mononuclear cells are isolated from the bone marrow by centrifugation through a lymphocyte separation medium, e.g., Organon Teknika-Cappel, according to the supplier's instructions. Approximately 1-2 x 10 7 mononuclear cells are added to each plate of producer cells, and co-cultivation is allowed to proceed for three days. During this time, the recombinant retrovirus vectors, containing the DNA segments of interest, are transferred from the producer cells to the mononuclear stem cells by infection. The replication incompetent vector then becomes stably integrated with the genome of the recipient cells. On Day 3, an additional quantity of confluent producer cell plates are split 1:10 in DMEM/10%- FCS.
  • a lymphocyte separation medium e.g., Organon Teknika-Cappel
  • the non-adherent cells are collected from the original plates and reseeded on the fresh plates for an additional three days.
  • the non-adherent co-cultivated mononuclear cells are collected, washed in Hanks' balanced salt solution and infused into the recipient in 50 ml of, e.g., phosphate buffered saline containing heparin at 10 units/ml.
  • Human IL-3 (approximately 200 units/ml) may be added to the cultures to facilitate colony development after reinfusion.
  • chemotherapeutic agents include, but are not limited to, platinum-containing agents such as cisplatin and carboplatin, classical bifunctional alkylating agents such as melphalan and cyclophosphamide, natural products subclasses including anthracyclines and epipodophilotoxins, as well as vinca alkaloids such as vinblastine.
  • Increased dosages of radiation therapy may also be administered to patients treated in accordance with the present invention.
  • the amount to which these dosages may be increased for an individual chemotherapeutic agent may be determined by in vi tro testing of cultured cells as described in Part III-A above. These elevated dosages may be tested in vivo in animal model systems, according to common methods known in the art. Such a method is described in detail in the examples below. Once it has been determined that a selected genetic alteration, such as insertion of a 7-GCS-encoding cDNA in the bone marrow cell genome, allows a particular chemotherapeutic agent to be administered at a known increased rate, the method may be applied clinically to human patients. Dosages may be adjusted to individual patients, using methods routine to pharmacologists and physicians. The following examples are provided to describe the invention in further detail. These examples are intended merely to illustrate and not to limit the invention.
  • A2780 and 1A9 Two ovarian cancer cell lines, A2780 and 1A9, have been determined suitable for transfection/ expression of 7-GCS, as it has been established, by methods described herein, and routine methods known in the art, that both of these cell lines possess very low levels of endogenous glutathione (relative to a number of cell lines examined) , express limited amounts of 7-GCS, GSH synthetase, and 7-GT, and are highly sensitive to cisplatin and its related analogs. It has also been determined that A2780 and 1A9 cells are readily "transfectable", which will allow a number of clonal cell lines carrying the desired constructs to be obtained.
  • a retroviral LTR is placed 5' to the 7-GCS cDNA.
  • This construct is used with pKOneo, a plasmid encoding resistance to the neomycin analogue, G418, in co- transfection with A2780 cells. Clonal populations are selected and verified for genomic integration and expression through combination of DNA (Southern) , RNA (Northern) and protein (Western) blotting. Each clone is expanded, the amount of 7-GCS quantitated and the cells examined for increased intracellular levels of GSH and for altered sensitivity to plantinum drugs, alkylating agents and irradiation. As a control, these cell lines are compared to the parental A2780 cell line and a vector (pLTR-2 and pKOneo)-only G418 resistance transfectant.
  • a full length cDNA clone for 7-GCS was obtained from a library prepared from highly cisplatin resistant human ovarian tumor cells.
  • a No I fragment of the 7-GCS cDNA, corresponding to the 5' untranslated region, the coding region, and 251 nucleotides of the 3' untranslated region, may be subcloned (by blunt-end ligation) into the Bam HI site of the expression vector pLTR-2.
  • This expression vector utilizes the long terminal repeat (LTR) of the Moloney murine leukemia virus to drive transcription and has successfully been used to express a number of other cDNAs in the ovarian tumor cell line, A2780.
  • LTR long terminal repeat
  • an expression vector which features an inducible promoter (the heavy metal- inducible mouse metallothionein I promoter) , the SV40 polyadenylation signal, and the bacterial neomycin phosphotransferase gene under the control of the SV40 late promoter.
  • Transfection Exponentially growing cells are trypsinized and seeded at a density of 5xl0 5 cells/lOmm dish and grown overnight at 37°C. The cells are washed twice with serum free RPMI medium and refed with 7 mis of RPMI medium prior to transfection.
  • plasmid DNA 10 ⁇ g of plasmid DNA (pLTR-2/7-GCS) and 1 ⁇ g of pKOneo (a plasmid containing the gene for geneticin (G418, Gibco BRL) resistance) in a total volume of 50 ⁇ l of TE (10 mM Tris-HCl, 0.1 mM EDTA) are mixed with 50 ⁇ l of Lipofectin Reagent (Gibco BRL) and incubated at room temperature for 15 minutes. The mixture is then added dropwise to the cells and after an overnight incubation at 37°C, the cells are fed with an equal volume of RPMI medium supplemented with 20% fetal bovine serum, and incubated for another 48 hours.
  • TE 10 mM Tris-HCl, 0.1 mM EDTA
  • Lipofectin Reagent Gibco BRL
  • the cells are refed with RPMI medium containing G418 at a concentration of 500 ⁇ g/ml.
  • Individual G418 resistant clones are picked 2 to 3 weeks later using sterile cotton tipped applicators and expanded.
  • Lipofectin Reagent mediated transfection of A2780 cells has provided more efficient transfection than the standard method of Graham and van der Eb employing calcium phosphate.
  • Transfected cell lines are checked for integration by standard Southern blotting techniques.
  • RNA is extracted by a modified one step guanidinium isothiocyanate-phenol-chloroform extraction procedure.
  • Total RNA (16 ⁇ g per lane) is denatured in 50% formamide containing 7.4% formaldehyde and separated by electrophoresis on an agarose (1% agarose/2.2M formaldehyde) gel.
  • RNA is blotted by capillary action on to Magna NT membrane filters (Micron Separations, Inc., Westboro, MA) in 10X SSC (1.5 M NaCl, 0.15 M sodium citrate, pH 7.0) and hybridized (>12h) with 32 P-labeled probes.
  • the 7-GCS cDNA (-3.7 kb) probe is 32 P labeled using the Prime-It random primer kit (Stratagene) to a specific activity of >5 x 10 8 dpm/ ⁇ g.
  • RNA gels are stained with ethidium bromide and examined to ascertain that equivalent amounts of RNA are analyzed. Filters are washed twice for 30 min.
  • the expression vector directs the transcription of a mRNA that is -1.8 kb longer than the cDNA and so the size difference may be used to distinguish it from endogenous mRNA.
  • the filters are incubated with a polyclonal antibody to y- glutamylcysteine synthetase (rabbit antirat) diluted 1:250 in TBS-A for at least 12 h.
  • the filters are then washed three times with TBS-T (10 min.) and treated with a secondard antibody ( 125 I-donkey anti- rabbit IgG; diluted 1:300 in TBS-A) for 1 h. After thorough washing with TBS-T, the filters are air dried. Autoradiography is performed at -70°C for 1 week.
  • Cytotoxicitv Assays Cisplatin, carboplatin, melphalan and BSO sensitivity is determined by the tetrazolium salt assay.
  • the cells are lysed with 100 ⁇ l per well of extraction buffer (20% (w/v) sodium dodecyl sulfate, 50% N,N-dimethyl formamide, pH 4.7). After incubation overnight, the absorbance at 570 nm is measured (Bio Rad Microplate multiscanner) employing the wells without cells as blanks.
  • extraction buffer 20% (w/v) sodium dodecyl sulfate, 50% N,N-dimethyl formamide, pH 4.7.
  • the absorbance at 570 nm is measured (Bio Rad Microplate multiscanner) employing the wells without cells as blanks.
  • the 7-GCS inhibitor is added as a concentrated solution directly to the culture medium, and the cells are treated 24h later by the addition of cisplatin, carboplatin, or melphalan.
  • Glutathione Determinations Cells (2 to 5 x 10 6 ; about 60% confluent) are lysed by sonication in 1 ml of phosphate buffered saline (PBS) (4°C) . The supernatant is obtained for assay after centrifugation (10,000xg, 10 min, 4°C) . The protein is precipitated by adding 12% 5-sulfosalicylic acid (SSA) (1 vol SSA to 3 vols of sample) . After standing on ice for 1 to 4h, the samples are centrifuged (10,000 g, 10 min) . The SSA extract is assayed as described by Griffith, Anal.
  • PBS phosphate buffered saline
  • 7-Glutamylcvsteine synthetase assay The spectrophotometric assay of Seelig et al., J. Biol. Che . , 259: 9345-47 (1984) is used. This method measures the rate of formation of ADP, which is derived from the decrease in absorbance of NADH (monitored at 340 nm) in a coupled enzymatic reaction.
  • Activity is determined at 37°C in reaction mixtures (final volume, 1.0 ml) containing 0.1 M Tris-HCl buffer (pH 8.0), 150 mM KC1, 5 mM Na 2 ATP, 2mM phosphoenolpyruvate, 10 mM L-glutamate, 10 mM L- ⁇ .- aminobutyrate, 20 mM MgCl 2 , 2 mM Na 2 EDTA, 0.2 mM NADH, 17 ⁇ g pyruvate kinase, and 17 ⁇ g lactate dehydrogenase.
  • the reaction is initiated by the addition of sample containing 7-glutamylcysteine synthetase.
  • One unit of enzyme activity is defined as the amount that results in the formation of 1 ⁇ ol of product per hour at 37°C, and expressed as nmol/min. Specific activity is expressed on the basis of protein determined by the Bradford assay (Bio-Rad) .
  • ⁇ -GCS Protein Cells are labeled in 60-mm Nunc tissue culture dishes by refeeding logarithmically growing cultures with 2 mis of methionine-free RPMI 1640 (Sigma) containing 10% dialyzed fetal calf serum (Gibco) and lOO ⁇ Ci/ml [ 35 S] -methionine (-lOOOCi/mmol, NEN, DuPont) . Labeling is carried out at 37°C for between 3 and 24 hours and the cell lysates are prepared as follows. The radioactive medium is removed, and the cell monolayer is washed twice in ice cold lxPBS.
  • the cells are scraped from the plate into 2 mis of PBSTDS buffer (10 mM dibasic sodium phosphate, 7.2, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 0.2% sodium azide, 0.004% sodium fluoride (pH 7.25)) at 4°C.
  • Tissue culture dishes are rinsed with an additional 1 ml of ice cold PBSTDS and combined with original extract.
  • the cells are incubated at 4°C for 10 minutes followed by vigorous vortexing to facilitate lysis, and the extracts are centrifuged for 1 hour at 35,000 rpm (l00,000xg) in Beckman 75Ti at 4°C.
  • the supernatant (S100) is recovered and either used immediately or stored at -70°C.
  • Each immunoprecipitation reaction contains approximately lxl0 7 trichloroacetic acid-precipitable cpm of [ 35 S] -labelled cell extract. Immuno ⁇ precipitation is carried out in PBSTDS. Each 300 ⁇ l reaction mixture contains labeled extract and 10 ⁇ l of polyclonal rabbit anti-rat 7-glutamylcysteine synthetase antibody. The reaction is incubated at 4°C for 4 hours, samples are constantly rotated during the incubation time. To render insoluble the antigen- antibody complexes, 100 ⁇ l of 10% (vol/vol) protein-A agarose is added, and the tube is rotated overnight at 4°C.
  • the immunoprecipitates are collected by centrifugation and washed extensively, 5 to 6 times in PBSTDS. Immunoprecipitates are dried, dissolved in IX SDS sample buffer (see Western Blot Analysis) and subjected to electrophoresis on a 10% SDS- polyacrylamide gel. Protein bands are identified by fluorography utilizing EN 3 HANCE (NEN, DuPont) .
  • mice may be used to determine in vivo dosages of chemotherapeutic agents, in the practice of the present invention. See, e.g., Dunbar et al., Oncogene Research, 6.: 39-51 (1991); Bodine et al., Proc. Nat'l. Acad. Sci. (USA), 86.: 8897-8901 (1989). Mice may be obtained from any commercial source. 6- to 10-week-old mice are suitable for in vivo testing.
  • Mice may be divided into four suitable test populations: (i) untreated (control); (ii) bone marrow removed and replaced without further manipulation; (iii) bone marrow removed and cells infected with retroviral vectors containing no DNA segments encoding glutathione-affecting proteins; and (iv) bone marrow removed and infected with retroviral vectors containing DNA segments encoding glutathione- affecting proteins.
  • Bone marrow cells may be harvested from the hind limbs of donor mice 48 hours after they have received 150 mg/kg 5-fluorouracil (5-FU) (Fluka) intraveneously. Preconditioning of the donor marrow with 5-FU, which increases both stem cell cycling and the relative number of stem cells, has been shown to facilitate retroviral gene transfer. Alternatively, hematopoietic cells from mouse fetal liver may be used in these and subsequent protocols.
  • 5-FU 5-fluorouracil
  • the isolated stem cells are washed and resuspended at a concentration of 5 x 10 5 cells/ml in Dulbecco's modified Eagle's medium (DMEM, Biofluids, Rockville, MD) containing 15% (vol/vol) fetal calf serum (FCS, HyClone) , 0.3 mg/ml L-glutamine and appropriate growth factors, such as
  • IL-3 200 units/ml
  • IL-6 20-200 units/ml
  • 10 ml of cell suspension may be plated on Sarstedt plates and incubated at 37°C (95% air/5% C0 2 ) for 2 days. Cells may be recovered by centrifugation, quantitated and resuspended in DMEM containing the same appropriate growth factors and 6 micrograms/ml of Polybrene (Sigma) . Approximately 1-5 x 10 6 cells may be added to plates containing appropriate producer cells split 1:5-1:10 24 hours previously. Marrow cells and producer cells are co-cultivated 48 hours.
  • the cells are recovered by aspirating the non-adherent cells from the plates, washing, and resuspending in phosphate-buffered saline (PBS) .
  • PBS phosphate-buffered saline
  • Approximately 1-3 x 10 6 cells are introduced to recipient mice (in this case, an autologous transplant wherein the donor mouse receives its own marrow cells) by injection through the tail vein.
  • recipient mice in this case, an autologous transplant wherein the donor mouse receives its own marrow cells
  • genetically anemic W/W v mice can be used for these procedures eliminating the need for lethal irradiation; transplantable bone marrow cells competitively repopulate W/W v mice due to their functional stem cell deficiency.
  • mice After successful recolonization of recipient bone marrow (as determined when acceptable neutrophil levels are achieved) with genetically manipulated and unmanipulated bone marrow cells, mice may be subjected to treatment with varying doses of selected chemotherapeutic agents.
  • sample groups of the four above-mentioned test populations may be treated with dosages of carboplatin ranging from 0 to 160 mg/kg body weight.
  • Animals in test populations 1- 3 should exhibit significantly greater bone marrow sensitivity to higher doses of carboplatin, while animals from test population 4 should exhibit higher bone marrow resistance.
  • caxboplatin 160 mg/kg
  • the maximum dose of caxboplatin is highly toxic to bone marrow of mice and humans resulting in leukopenia.
  • Recipient mice may be observed daily and samples of blood withdrawn for WBC counts, including platelets, and Wright's stained smears before and 1, 3, 8, and 14 days after injection of the various dosages of carboplatin.
  • a cDNA encoding 7-GCS is utilized in the above-described in vivo test. Elevated resistance of recipients of such genetically altered hematopoietic cells may be further examined through the use of BSO. Animals from test population 4, which will exhibit higher bone marrow resistance to carboplatin because of overexpression of 7-GCS, may be subjected to treatment with BSO (1500-4500 mg/m 2 /q 12h x 6) . This should result in transient inhibition in 7-GCS overexpression and a temporary concomitant return to sensitivity of bone marrow to carboplatin.
  • a suitable infection protocol is described supra in the Detailed Description of the Invention.
  • Animals may be divided to 4 test populations, as described for mice above, and subjected to similar manipulations of the bone marrow. After replacement of the appropriately- treated bone marrow cells into recipient monkeys, the animals may be subjected to treatment with varying concentrations of chemotherapeutic agents, e.g., 0-200 mgs/m 2 carboplatin, as described for mice above.
  • chemotherapeutic agents e.g., 0-200 mgs/m 2 carboplatin

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention se rapporte à des procédés et des cellules recombinées permettant d'accroître la résistance de cellules souches hématopoïétiques aux effets toxiques d'agents chimiothérapeutiques et autres agents thérapeutiques utilisés pour traiter des tumeurs solides et d'autres malignités. Des cellules souches hématopoïétiques sont obtenues de donneurs de moelle osseuse appropriés. Les cellules sont génétiquement modifiées par l'introduction et l'incorporation stable, dans le génome, d'au moins un segment d'ADN codant une protéine susceptible d'augmenter la production de glutathion intracellulaire, ce qui confère aux cellules la capacité de produire du glutathion intracellulaire en plus grandes quantités. Les cellules souches hématopoïétiques génétiquement modifiées sont introduites chez un patient auquel on a prescrit une chimiothérapie. Le patient peut être traité avec des agents chimiothérapeutiques à des doses qui seraient toxiques pour les cellules souches hématopoïétiques non recombinées du patient.
PCT/US1993/002929 1992-04-01 1993-03-30 Procede et cellules recombinees conferant a des cellules souches hematopoietiques une resistance accrue a la toxicite d'agents chimiotherapeutiques WO1993020195A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US86252592A 1992-04-01 1992-04-01
US07/862,525 1992-04-01

Publications (1)

Publication Number Publication Date
WO1993020195A1 true WO1993020195A1 (fr) 1993-10-14

Family

ID=25338689

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/002929 WO1993020195A1 (fr) 1992-04-01 1993-03-30 Procede et cellules recombinees conferant a des cellules souches hematopoietiques une resistance accrue a la toxicite d'agents chimiotherapeutiques

Country Status (2)

Country Link
AU (1) AU3969993A (fr)
WO (1) WO1993020195A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0648261A1 (fr) * 1993-04-01 1995-04-19 The Trustees Of Columbia University In The City Of New York Vecteur retroviral capable d'introduire par transduction le gene de l'aldehyde deshydrogenase-1 et utilisations dudit vecteur
EP0693122A1 (fr) * 1993-04-08 1996-01-24 Cornell Research Foundation, Inc. Sous-unite lumineuse de la synthetase glutamylcysteine
FR2723588A1 (fr) * 1994-08-12 1996-02-16 Rhone Poulenc Rorer Sa Adenovirus comprenant un gene codant pour la glutathion peroxydase
WO1997033975A1 (fr) 1996-03-12 1997-09-18 Rhone-Poulenc Rorer S.A. Milieu pour la conservation de materiel biologique
US5750360A (en) * 1995-06-07 1998-05-12 Lxr Biotechnology Inc. Method for quantitatively measuring apoptosis
WO2000060094A1 (fr) * 1999-04-07 2000-10-12 Institut National De La Recherche Agronomique (Inra) Lactocoques modifies exprimant une catalase et leurs utilisations
WO2002083168A1 (fr) * 2001-04-10 2002-10-24 Medestea Research & Production S.R.L. Combinaisons d'enzymes servants d'adjuvants dans le traitement par interferon
WO2001073056A3 (fr) * 2000-03-27 2002-11-07 Bayer Ag Regulation de l'enzyme humaine analogue a oxoprolinase
EP4096649A4 (fr) * 2020-01-31 2024-04-17 G Tech Bio Llc Composés et procédés pour fournir des effets chimoprotecteurs

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4497796A (en) * 1980-03-26 1985-02-05 The Regents Of The University Of California Gene transfer in intact mammals

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4497796A (en) * 1980-03-26 1985-02-05 The Regents Of The University Of California Gene transfer in intact mammals

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
BLOOD CELLS, Volume 13, issued 1987, KOHN et al., "Retroviral-Mediated Gene Transfer into Mammalian Cells", pages 285-298. *
EUR. J. BIOCHEM., Volume 208, issued 1992, ROEMER et al., "Concepts and Strategies for Human Gene Therapy", pages 211-255. *
NATURE, Volume 318, issued 14 November 1985, KELLER et al., "Expression of a Foreign Gene Myeloid and Lymphoid Cells Derived from Multipotent Haematopoietic Precursors", pages 149-154. *
NCI MONOGRAPHS, Number 6, issued 1988, OZOLS et al., "Keynote Address: Mechanisms of Cross-Resistance Between Radiation and Antineoplastic Drugs", pages 159-165. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES USA, Volume 87, issued May 1990, BODINE et al., "Development of a High-Titer Retrovirus Producer Cell Line Capable of Gene Transfer into Rhesus Monkey Hematopoietic Stem Cells", pages 3738-3742. *
SEMINARS IN ONCOLOGY, Volume 18, No. 3, issued June 1991, OZOLS et al., "Chemotherapy of Ovarian Cancer", pages 222-232. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 265, No. 3, issued 25 January 1990, YAN et al., "Amino Acid Sequence of Rat Kidney - Glutamylcysteine Synthetase", pages 1588-1593. *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0648261A4 (fr) * 1993-04-01 1997-10-22 Univ Columbia Vecteur retroviral capable d'introduire par transduction le gene de l'aldehyde deshydrogenase-1 et utilisations dudit vecteur.
EP0648261A1 (fr) * 1993-04-01 1995-04-19 The Trustees Of Columbia University In The City Of New York Vecteur retroviral capable d'introduire par transduction le gene de l'aldehyde deshydrogenase-1 et utilisations dudit vecteur
EP0693122A4 (fr) * 1993-04-08 1997-10-22 Cornell Res Foundation Inc Sous-unite lumineuse de la synthetase glutamylcysteine
EP0693122A1 (fr) * 1993-04-08 1996-01-24 Cornell Research Foundation, Inc. Sous-unite lumineuse de la synthetase glutamylcysteine
WO1996005320A1 (fr) * 1994-08-12 1996-02-22 Rhone-Poulenc Rorer S.A. Adenovirus comprenant un gene codant pour la glutathion peroxydase
FR2723588A1 (fr) * 1994-08-12 1996-02-16 Rhone Poulenc Rorer Sa Adenovirus comprenant un gene codant pour la glutathion peroxydase
US7241591B2 (en) 1994-08-12 2007-07-10 Aventis Pharma S.A. Adenovirus comprising a gene coding for glutathione peroxidase
US5750360A (en) * 1995-06-07 1998-05-12 Lxr Biotechnology Inc. Method for quantitatively measuring apoptosis
WO1997033975A1 (fr) 1996-03-12 1997-09-18 Rhone-Poulenc Rorer S.A. Milieu pour la conservation de materiel biologique
WO2000060094A1 (fr) * 1999-04-07 2000-10-12 Institut National De La Recherche Agronomique (Inra) Lactocoques modifies exprimant une catalase et leurs utilisations
FR2791998A1 (fr) * 1999-04-07 2000-10-13 Agronomique Inst Nat Rech Lactocoques modifies exprimant une catalase et leurs utilisations
WO2001073056A3 (fr) * 2000-03-27 2002-11-07 Bayer Ag Regulation de l'enzyme humaine analogue a oxoprolinase
WO2002083168A1 (fr) * 2001-04-10 2002-10-24 Medestea Research & Production S.R.L. Combinaisons d'enzymes servants d'adjuvants dans le traitement par interferon
EP4096649A4 (fr) * 2020-01-31 2024-04-17 G Tech Bio Llc Composés et procédés pour fournir des effets chimoprotecteurs

Also Published As

Publication number Publication date
AU3969993A (en) 1993-11-08

Similar Documents

Publication Publication Date Title
Spencer et al. A gene transfer strategy for making bone marrow cells resistant to trimetrexate
Bunnell et al. High-efficiency retroviral-mediated gene transfer into human and nonhuman primate peripheral blood lymphocytes.
Belmont et al. Expression of human adenosine deaminase in murine hematopoietic cells
RU2174846C2 (ru) Опосредованный вирусом усиленный перенос днк
US5219740A (en) Retroviral gene transfer into diploid fibroblasts for gene therapy
US5837510A (en) Methods and polynucleotide constructs for treating host cells for infection or hyperproliferative disorders
US11649455B2 (en) Micro RNA expression constructs and uses thereof
EP0832980A1 (fr) Thérapie recombinante des infections et désordres hyperprolifératifs
WO1992014829A1 (fr) Particules virales agissant sur une gamme d'hotes modifiee
Havenga et al. Retroviral stem cell gene therapy
US6472212B1 (en) Methods and compositions for genetically modifying primate bone marrow cells
JPH06335392A (ja) Ｈｉｖ阻害性アンチセンスおよび他のヌクレオチド配列を含有する発現構成体、レトロウイルスのベクターおよびそれを含有する組換えレトロウイルス
WO1993020195A1 (fr) Procede et cellules recombinees conferant a des cellules souches hematopoietiques une resistance accrue a la toxicite d'agents chimiotherapeutiques
US5837536A (en) Expression of human multidrug resistance genes and improved selection of cells transduced with such genes
JPH11514209A (ja) 組換えレトロウイルス調製物による造血幹細胞の高効率エクスビボ形質導入
US6743631B1 (en) Use of human serum resistant vector particles and cell lines for human gene therapy
KR20220063156A (ko) 암세포 요법을 위한 p21 발현 단핵구
Shaw et al. Retroviral expression of Escherichia coli thymidylate synthase cDNA confers high-level antifolate resistance to hematopoietic cells
JP2003525607A (ja) 変異サイクリンｇ１タンパク質
WO1997032025A1 (fr) Procede de greffage selectif de cellules souches hematopoietiques resistant aux medicaments
JPH11509735A (ja) 遺伝子導入、遺伝子治療及びタンパク質合成のための陽性選択マーカーとしてのシチジンデアミナーゼｃＤＮＡ
US6130089A (en) Materials and methods for gene transfer
AU687765B2 (en) Retroviral mediated transfer of the human multiple drug resistance gene
EP0859854B1 (fr) Vecteurs retroviraux porteurs d'inhibiteurs 1 derives de cellules senescentes (sdi-1) ou de sequences de nucleotides de sdi-1 antisens
AU663470C (en) Viral particles having altered host range

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

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

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载