WO2001005405A1 - Transfert du gene de folyl-polyglutamyl synthetase pour ameliorer la sensibilite au medicaments antifoliques - Google Patents

Transfert du gene de folyl-polyglutamyl synthetase pour ameliorer la sensibilite au medicaments antifoliques Download PDF

Info

Publication number
WO2001005405A1
WO2001005405A1 PCT/US2000/019162 US0019162W WO0105405A1 WO 2001005405 A1 WO2001005405 A1 WO 2001005405A1 US 0019162 W US0019162 W US 0019162W WO 0105405 A1 WO0105405 A1 WO 0105405A1
Authority
WO
WIPO (PCT)
Prior art keywords
fpgs
cells
gene
mtx
cancer
Prior art date
Application number
PCT/US2000/019162
Other languages
English (en)
Inventor
Manish Aghi
Christof M. Kramm
Xandra O. Breakefield
Original Assignee
The General Hospital Corporation
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 The General Hospital Corporation filed Critical The General Hospital Corporation
Publication of WO2001005405A1 publication Critical patent/WO2001005405A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/53—Ligases (6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2121/00—Preparations for use in therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/025—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a parvovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/028—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a herpesvirus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention is directed to the killing of neoplastic cells. More specifically, the present invention relates to the use of folylpolyglutamyl synthetase (FPGS) gene transfer to enhance antifolate drug sensitivity.
  • FPGS folylpolyglutamyl synthetase
  • folate antagonists have found widespread use as chemotherapeutic agents.
  • Methotrexate (MTX) a 4-aminofolate analogue, has been in clinical use for the treatment of various human malignancies, especially leukemias and breast cancer, for about 40 years.
  • MTX is a potent inhibitor of dihydro folate reductase (DHFR). Inhibition of this enzyme prevents the reduction of dihydrofolate that accumulates in cells actively synthesizing thymidylate via the thymidylate synthetase reaction. The cells subsequently become depleted of reduced folate cofactors. needed for synthesis of thymidylate and for de novo purine synthesis. The ensuing disruption of DNA replication leads to cell death in actively replicating cells found in tumors and some normal tissues.
  • DHFR dihydro folate reductase
  • Naturally occurring folates and some antifolates possess a single terminal benzoylglutamate residue and are converted intracellularly from monoglutamates into polyglutamates through the action of an enzyme, folylpolyglutamyl synthetase (FPGS), that attaches up to six glutamyl groups in ⁇ -peptide linkage to the terminal benzoylgutamate.
  • FPGS folylpolyglutamyl synthetase
  • Polyglutamylation of folates and antifolates causes two effects. First, polyglutamylation causes intracellular accumulation of folates and antifolates because the highly ionized polyglutamylated forms are not readily transported across cell membranes. For example, MTX polyglutamates efflux out of cells 70 times slower than the monoglutamylated drug (Balinska, M., et al , Cancer Research 41 :2151-2156
  • polyglutamylation enhances the affinity of folates for the enzymes that utilize them as cofactors. and increases the affinity (and inhibitory effect) of antifolates for their target enzymes, as well as expanding the range of enzymes which antifolates inhibit.
  • MTX is polyglutamylated much more inefficiently than naturally occurring folates, reductions in FPGS activity that have little effect on folate polyglutamate pools can have marked effects on the level of MTX polyglutamates and thus, on the cytotoxicity of MTX.
  • the ability to generate MTX polyglutamates correlates directly with sensitivity to MTX for both human and murine tumor cells (Samuels, L.L., etal., Cancer Research 45: 1488 (1985)).
  • antifolates exhibiting the most therapeutic selectivity in murine tumor models consistently display a greater differential in accumulation of the polyglutamylated drug in tumor compared to normal proliferative tissues (Rumberger, S. et al, Cancer Research 50:4639-4643 (1990)).
  • the phenotype of the target tumor cells is genetically altered to increase the tumor's drug sensitivity and responsiveness.
  • One strategy being actively investigated involves directly transferring a "chemosensitization" or "suicide” gene encoding a prodrug activation enzyme to malignant cells, in order to confer sensitivity to otherwise innocuous agents (Moolten, F.L., Cancer Gene Therapy 7:279-287 (1994); Freeman, S.M.. et al, Semin. Oncol. 25:31-45 (1996); Deonarain. M. P.. et al, Gene Therapy 2: 235-244 (1995)).
  • prodrug activation genes have been studied for application in cancer gene therapy.
  • the two most extensively investigated prodrug-activating enzymes are herpes simplex virus thymidine kinase (HSV-TK), which activates the prodrug ganciclovir. and E. coli cytosine deaminase (CD), which activates the prodrug 5-fluorocytosine (Roth, J.A., Cristiano, R.J., Journal of the National Cancer Institute 59:21-39 (1997); Aghi, M., et al, Journal of the National Cancer Institute 90:310-380 (1998)).
  • HSV-TK herpes simplex virus thymidine kinase
  • CD E. coli cytosine deaminase
  • HSV-TK phosphorylates the prodrug ganciclovir and generates nucleoside analogs that induce DNA chain termination and cell death in actively dividing cells.
  • Tumor cells transduced with HSV-TK acquire sensitivity to ganciclovir, a clinically proven agent originally designed for treatment of viral infections. Moolten, F.L. and Wells, J.M., J. Natl. Cancer Inst. 82:291-300 (1990);
  • the bacterial gene cytosine deaminase is a prodrug/enzyme activation system that has been shown to sensitize tumor cells to the antifungal agent 5-fluorocytosine as a result of its transformation to 5-flurouracil, a known cancer chemotherapeutic agent (Mullen, C.A., et al, Proc. Natl. Acad. Sci. USA
  • prodrug-activating enzyme systems have also been investigated (T. A. Connors, Gene Ther. 2:702-709 (1995)). These include the bacterial enzyme carboxypeptidase G2, which does not have a mammalian homolog, and can be used to activate certain synthetic mustard prodrugs by cleavage of a glutamic acid moiety to release an active, cytotoxic mustard metabolite (Marais, R., et al, Cancer Res. 56: 4735-4742 (1996)), and E.coli nitro reductase, which activates the prodrug CB 1954 and related mustard prodrug analogs (Drabek, D., et al , Gene Ther. -4:93-100 (1997); Green.
  • carboxypeptidase G2 which does not have a mammalian homolog
  • E.coli nitro reductase which activates the prodrug CB 1954 and related mustard prodrug analogs
  • HSV/TK non-mammalian nature of the HSV/TK and CD genes, whose gene products may elicit immune responses that interfere with prodrug activation; (b) their reliance on drugs which were initially developed as antiviral drugs (ganciclovir) or antifungal drugs (5-fluorocytosine) and whose cancer chemotherapeutic activity is uncertain; (c) the dependence of these gene therapy strategies on ongoing tumor cell DNA replication; and (d) the requirement, in the case of HSV-TK, for direct cell-cell contact to elicit an effective bystander cytotoxic response (Mesnil, M., et al, Proc. Natl. Acad.
  • the P450-based drug activation strategy utilizes a mammalian drug activation gene (rather than a bacterially or virally derived gene), and also utilizes established chemotherapeutic drugs ⁇ i.e., cyclophosphamide) widely used in cancer therapy. While MTX's well-established chemotherapeutic activity also distinguishes it from prodrugs such as ganciclovir and 5-fluorocytosine, MTX possesses troublesome toxicity to normal tissues and its effectiveness could be improved by a gene transfer strategy that enhances the drug's selective toxicity.
  • the inventors have discovered that by introducing a FPGS gene (and thus an FPGS gene product) into neoplastic cells, the enzymatic conversion of an antifolate drug to its therapeutically active metabolites is greatly enhanced within the cellular and anatomic locale of the tumor, thereby increasing both the selectivity and efficiency with which neoplastic cells are killed. At the same time, undesirable side-effects to normal host cells are minimized.
  • the inventors first determined if transfection of an experimental brain tumor cell line with an expression cassette bearing the FPGS cDNA would increase the cells' sensitivity to brief MTX pulses in culture. The ability of MTX to cause bystander killing of nontransfected tumor cells in cocultures of nontransfected and transfected cells was then ascertained. The next step involved determining if tumors formed by the transfected cells were more sensitive to reduced frequency of treatment in vivo than tumors formed by the nontransfected tumor cells. Finally, antifolates other than MTX were evaluated by treating the two cell lines and cocultures with brief pulses and by treating homogeneous and mixed tumors in vivo in order to determine what properties are desirable in drugs used in conjunction with FPGS gene delivery.
  • the present invention overcomes the disadvantages of the prior art by providing a method for killing neoplastic cells, the method comprising: (a) infecting the neoplastic cells with a vector for gene delivery, the vector comprising a folylpolyglutamyl synthetase (FPGS) gene; (b) treating the neoplastic cells with a chemotherapeutic agent that is activated by the product of the FPGS gene; and (c) killing the neoplastic cells.
  • FPGS folylpolyglutamyl synthetase
  • the invention also provides a preferred embodiment of the foregoing methods wherein the FPGS gene is a mammalian gene, although the FPGS gene from any species could be used .
  • the human FPGS gene is particularly preferred.
  • the FPGS- activated chemotherapeutic agent is a polyglutamylatable antifolate drug.
  • antifolate drugs include methotrexate (MTX), edatrexate (EDX), aminopterin, as well as antifolates which inhibit thymidylate synthetase.
  • MTX and EDX are particularly preferred.
  • the neoplastic cells are malignant cells that are sensitive to antifolate chemotherapy, such as breast cancer and colon cancer.
  • any neoplastic cell can be targeted since FPGS gene delivery will enhance the drug's anticancer effect.
  • neoplastic cells such as. e.g., central nervous system tumors (gliomas, astocytomas), lymphomas, lung cancer, melanoma, pancreatic cancer, ovarian cancer, prostate cancer, liver cancer, which are not typically treated with antifolate drugs, will be able to be used in the method of the invention.
  • the invention also provides a very particularly preferred embodiment of the foregoing methods, wherein the FPGS gene is the human FPGS gene and the chemotherapeutic agent is MTX.
  • the invention also provides a preferred embodiment of the foregoing methods, wherein the FPGS gene is delivered using a viral vector, preferably viral vectors whose use for gene therapy is well- established for those skilled in the art.
  • viral vectors include retrovirus (including lentivirus), adenovirus. adeno-associated virus, herpes virus (including herpes simplex virus I and II and Epstein Barr virus), poliovirus, papillomavirus, or hybrid vectors having attributes of two or more viruses.
  • retroviruses, adenoviruses. and herpes viruses are particularly preferred viral vectors.
  • the FPGS gene is delivered using any non-viral vector, preferably one whose use for gene therapy is well-established for those skilled in the art.
  • non-viral vectors for gene delivery include prokaryotic vectors (including tumor targeted bacterial vectors), cationic liposomes, DNA-protein complexes, non-viral T7 autogene vectors, fusogenic liposomes, direct injection of nucleic acid ("naked DNA"), particle or receptor-mediated gene transfer, hybrid vectors such as DNA-adenovirus conjugates or other molecular conjugates involving a non-viral and viral component, starburst polyamidoamine dendrimers, cationic peptides, and mammalian artificial chromosomes.
  • the present invention provides an embodiment of the foregoing methods wherein the FPGS gene is delivered using any cellular vector, preferably one whose use for gene therapy is well-established for those skilled in the art.
  • cellular vectors for gene therapy include endothelial cells and macrophages including tumor-infiltrating macrophages, each of which may be modified using viral or non-viral vectors to carry the FPGS gene, and thus express the FPGS gene product.
  • the present invention also provides an embodiment, whereby the FPGS drug activation system is combined with another established gene/prodrug activation system, such as ganciclovir/HSV-TKand 5-fluorocytosine/CD.
  • FPGS gene therapy may also be combined with other established cancer therapeutic genes, including tumor suppressor genes, such as p53; apoptotic factors, such as bax, tumor necrosis factor alpha, and caspases; and cytokines, such as interleukin 2, interleukin 4, and interleukin 12.
  • tumor suppressor genes such as p53
  • apoptotic factors such as bax, tumor necrosis factor alpha, and caspases
  • cytokines such as interleukin 2, interleukin 4, and interleukin 12.
  • the targetting specificity for FPGS gene delivery is facilitated by "transcriptional targeting," including the use of tumor-specific or tumor-selective DNA enhancer sequences.
  • sequences include those described for genes that encode tyrosinase (melanoma), ERBB2 (pancreatic cancer), carcinoembryonic antigen (lung and gastrointestinal cancer), DF3/MUC1 (breast cancer), alpha- fetoprotein (hepatoma), as well as synthetic gene regulation systems which allow for transcriptional control and other forms of regulated expression of the FPGS gene.
  • Targeting also includes sequences that control expression of genes induced by hypoxia (hypoxia response elements), or other tumor-specific conditions and factors.
  • FIG. 1 MTX dose-response curves for 9L and 9L/FPGS cells in culture. Percent cell survival was determined for 9L (open circles) and 9L/FPGS (closed circles) cells treated with varying doses of MTX for 4 hours, followed by 3 days of growth in drug-free medium. The x-axis represents the log of the molar concentration of MTX. Raw data points represent the averages of triplicate platings and were fit to the sigmoidal dose-response curves shown using SigmaPlot 3.0. Standard errors were ⁇ 6.3% cell survival and are shown.
  • FIG. 2 Growth of subcutaneous 9L and 9L FPGS tumors in response to different MTX regimens. Fold growth in volume from time treatment began was calculated for subcutaneous tumors formed by injecting 9L (solid lines) or 9L/FPGS (dashed lines) cells into the flanks of nude mice. Mice were treated with intraperitoneal injections of saline every day (closed circles), 9 mg MTX/kg body weight every day (open circles), or 9 mg MTX/kg body weight every third day (closed triangles). Standard errors were ⁇ 5.5-fold growth and are shown. Saline treated 9L and 9L/FPGS tumors achieved comparable fold growth (p >
  • FIGS.3A and 3B EDX dose-response curves for 9L and 9L/FPGS cells in culture. Percent cell survival was determined for 9L (open circles) and
  • 9L/FPGS (closed circles) cells plated in triplicate and treated with varying doses of EDX for 4 hours, followed by 3 days of growth in drug-free medium.
  • FIG.3A over a wide range of concentrations — raw data points were fit to the sigmoidal dose-response curves shown using SigmaPlot 3.0.
  • X-axis represents the log of the molar concentration of EDX. Standard errors were ⁇ 8 percent survival and are shown.
  • FIGS. 4A and 4B The bystander effect of FPGS gene transfer.
  • Data points for 9L (closed triangles), 9L/FPGS (closed circles), and a coculture containing 20% 9L/FPGS + 80% 9L cells (open triangles) were fit to the sigmoidal dose-response curves (solid) shown using SigmaPlot 3.0.
  • X-axis represents the log of the molar concentration of antifolate.
  • Triplicate plates were treated with 4 hour pulses of MTX (FIG. 4A) or EDX (FIG. 4B), followed by 3 days of growth in drug-free medium.
  • EDX pulses were also used to treat cocultures containing 10% 9L/FPGS cells (open squares) and 1 % 9L/FPGS cells (open diamonds) -the sigmoidal curves (dashed) that fit these data points are also shown. Standard errors were ⁇ 7 percent cell survival and are shown.
  • FIG. 5 Bystander effect is transmitted through the medium.
  • X-gal positive colonies formed by triplicate plating of 1000 9L/BAG cells in conditioned medium (CM) were counted.
  • a plus sign indicates that cells from which CM was taken were pulsed with 300 nM EDX during the first 4 hours of the 72 hours that the medium was conditioned.
  • Number of colonies formed was significantly less when CM was from EDX-treated 9L/FPGS cells than it was when CM was from EDX-treated 9L cells (p ⁇ 0.0005). Standard errors were ⁇ 10 colonies and are shown.
  • FIG. 6 Effect of EDX treatment on growth of subcutaneous 9L, 9L/FPGS, and mixed tumors.
  • Subcutaneous tumors were formed by injecting 9L cells (solid line), 9L/FPGS cells (long dash), and a mixture of 20%> 9L/FPGS cells + 80%) 9L cells (short dash) into the flanks of nude mice.
  • Mice were treated daily with intraperitoneal injections of saline (solid circles) or 3 mg EDX/kg body weight (open circles). Standard errors were ⁇ 5.5-fold growth and are shown.
  • Saline-treated tumors achieved similar fold growth except after 3 weeks of treatment, when 20%> 9L/FPGS tumors were slightly larger (p ⁇ 0.05) than 9L tumors.
  • the present invention is directed to the killing of neoplastic cells using FPGS-based gene therapy in combination with an antifolate drug.
  • neoplastic cells are intended cells whose normal growth control mechanisms are disrupted (typically by accumulated genetic mutations), thereby providing the potential for uncontrolled proliferation.
  • the term is intended to include both benign and malignant neoplastic cells in both the central nervous system and the periphery.
  • peripheral is intended to mean all other parts of the body outside of the brain or spinal cord.
  • neoplastic cells include cells of tumors, neoplasms, carcinomas, sarcomas, papillomas, leukemias, lymphomas, and the like.
  • the neoplastic cells can be malignant cells that are sensitive to antifolate chemotherapy, such as breast cancer and colon cancer. However, any neoplastic cell can be targeted since FPGS gene delivery will enhance the drug's anticancer effect.
  • neoplastic cells such as. e.g., central nervous system tumors
  • gliomas. astocytomas lymphomas, lung cancer, melanoma, pancreatic cancer, ovarian cancer, prostate cancer, liver cancer, which are not conventionally treated with antifolate drugs, will be able to be targeted in the method of the invention.
  • FPGS gene is intended a gene encoding the enzyme folylpolyglutamyl synthetase.
  • activating an antifolate drug is intended any metabolic reaction that increases the cytotoxic or cytostatic activity or otherwise increases the therapeutic efficacy of the antifolate drug; or that confers on the drug an additional mechanism of action beyond that which the drug exhibits in the absence of the metabolic reaction.
  • the FPGS gene may be from any species. FPGS cDNA from many species are known to those skilled in the art. and their nucleotide sequences can be obtained from the GenBank Sequence Database. The mammalian FPGS gene is preferred. The human FPGS gene is particularly preferred (see, GenBank accession number NM_004957; Garrow, T.A., et al, Proc. Natl. Acad. Sci. USA 59:9151-9155 (1992)).
  • gene product or “product of a particular gene” broadly refers to polypeptides or proteins encoded by a particular gene, but may also include transcription products of the particular gene.
  • chemotherapeutic agent that is activated by the product of said FPGS gene is meant a pharmaceutical agent that can be used in the treatment of neoplasms, and that is capable of being activated by FPGS.
  • activating or “bioactivating” an antifolate drug is intended any metabolic reaction that increases the cytotoxic or cytostatic activity or otherwise increases the therapeutic efficacy of the drug; or that confers on the drug an additional mechanism of action beyond that which the agent exhibits in the absence of the metabolic reaction.
  • cytotoxic or cytostatic is intended causing or leading to cell death or slower tumor cell growth.
  • antifolate drugs are MTX, EDX, aminopterin, and thymidylate synthetase inhibitors. Other antifolate drugs known to those skilled in the art can also be used in the present invention.
  • treating said neoplastic cells with a chemotherapeutic agent is intended to include both the local delivery of the prodrug into or near the site of the tumor by, e.g. , slow-release pellets, as well as the systemic administration of the chemotherapeutic agent, i. e. , through intraperitoneal, intravenous, parenteral, or intramuscular routes. Localized delivery of the drug is expected to increase the fraction of the drug activated within the tumor, and thus increase drug efficacy.
  • Dosages of a particular chemotherapeutic agent may be administered according to current standard clinical practice. See, e.g., Hubbard, S.M. and
  • Standard clinical practice may involve body surface area (BSA)-based dose calculations, as well as individualization of dosages based on pharmacokinetic optimization using plasma drug and metabolite concentrations ("therapeutic drug monitoring” or TDM).
  • concentrations may be obtained using limited sampling or other pharmacokinetic sampling and modeling techniques (van Warmerdam, L. J., et al. , Neth J. Med. 51:30-35 (1997); Desoize, B. and Robert, J., Eur. J. Cancer
  • the invention also provides preferred embodiments of the foregoing methods wherein the FPGS gene is the human FPGS gene and the chemotherapeutic agent is MTX.
  • the FPGS gene may be delivered to neoplastic cells using a viral vector, preferably one whose use for gene therapy is well known in the art. Techniques for the formation of vectors or virions are generally described in "Working Toward Human Gene Therapy," Chapter 28 in Recombinant DNA, 2nd Ed.,
  • Such vectors may be derived from viruses that contain RNA (Vile, R.G., et al, Br. Med Bull. 57: 12-30 (1995)) or DNA (Ali M., et al, Gene Ther. 7 :367- 384 (1994)).
  • RNA RNA
  • DNA DNA
  • Examples of viral vector systems utilized in the gene therapy art include the following: retroviruses (Vile, R.G., supra; U.S. Patent Nos. 5,741 ,486 and 5,763,242): adenoviruses (Brody, S.L., et al, Ann. N. Y.
  • Retroviruses, adenoviruses, and herpes viruses are the preferred viral vectors for gene delivery.
  • Other suitable viral vectors will be readily apparent to the skilled artisan.
  • the vector will include one or more promoters or enhancers, the selection of which will be known to those skilled in the art.
  • Suitable promoters include, but are not limited to. the retroviral long terminal repeat (LTR), the SV40 promoter, the human cytomegalovirus (CMV) promoter, and other viral and eukaryotic cellular promoters known to the skilled artisan.
  • enhancers include the tumor tissue-specific enhancers, described below.
  • the virus can be injected into a patient bearing a neoplasm, either at, into, or near the site of neoplastic growth.
  • the treatment will be by direct intraneoplastic inoculation.
  • MRI magnetic resonance imaging
  • CT computerized tomography
  • the tumor may also be resected prior to treatment with the vectors of the invention.
  • the pharmaceutical compositions of the present invention would be advantageously administered in the form of injectable compositions.
  • a typical composition for such purpose would comprise a pharmaceutically acceptable vehicle.
  • Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like. See, Remington 's Pharmaceutical Sciences (18th ed.), Mack Publishing Co. (1990).
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol. vegetable oil and injectable organic esters such as ethyloleate.
  • Aqueous carriers include water, aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.
  • Intravenous vehicles include fluid and nutrient replenishers. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine skills in the art (Goodman and Gilman, The Pharmacological Basis for Therapeutics (8th ed.) Pergamon Press (1990)).
  • the vector would be prepared as an injectable, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the preparation also may be emulsified.
  • the active immunogenic ingredient is often mixed with an excipient which is pharmaceutically-acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the preparation may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH-buffering agents, adjuvants or immunopotentiators.
  • the virus is provided in a therapeutically effective amount to infect and kill target cells.
  • the quantity of the vector to be administered will also depend on factors such as the clinical status, age, and weight of the subject to be treated, the capacity of the subject's immune system to synthesize antibodies, and available volume. Precise amounts of active ingredient required to be administered depend on the judgment of the gene therapist and will be particular to each individual patient.
  • the viral vector is administered in titers ranging from about l lO 5 to about lxl 0 9 colony forming units (cfu) per ml, although ranges may vary. Preferred titers will range from about lxlO 6 to about lxl0 8 cfu/ml.
  • a packaging cell line is transduced with a retroviral vector carrying the FPGS gene to form a producer cell line.
  • the packaging cells may be transduced by any means known in the art, including, e.g., electroporation, CaP0 4 precipitation, or the use of liposomes.
  • Examples of packaging cells that may be transfected include, but are not limited to, BOSC23, Bing, PE501, PA317, ⁇ -2, ⁇ -AM, PA12, T19-14X, VT-19-17-H2, ⁇ -CRE, ⁇ - CRIP, GP+E86, GP+envAml2, and DAN cell lines.
  • Guidance on retroviral producing packaging cells and how to construct them can be found in Short et al.
  • Retroviral vectors have also been successfully packaged with a vesicular stomatitis virus (VSV) envelope glycoprotein G ("pseudotyping"). These vectors are more stable and can be concentrated to 10 9 cfu/ml, allowing them to be injected directly (Burns, J.C., et al. Proc. Natl. Acad. Sci. USA 90:8033-8037 (1993)).
  • VSV vesicular stomatitis virus
  • the producer cells can then be grafted near or into the tumor in an amount effective to inhibit or kill the neoplastic cells.
  • Direct injection of high titer retroviral producer cells (Murdoch, B., et al, Gene Ther. 4:144-149 (1997); Onodera, M.. et al, Hum Gene Ther. 5: 1 189-1 194 (1997)) should allow for efficient in situ infection with the retroviral sequences (Rainov, N.G., et al , Cancer Gene Ther. 5:99-106 (1996); Ram. Z., et al, Cancer Res. 55:83-88 ( 1993)).
  • Producer cells injected intratumorally do not generally migrate from the site of injection.
  • vector producer cell (VPC) dosages range from about 2.5 x 10 8 VPCs to about lxl 0 9 VPCs. The exact amount of producer cells will ultimately be determined by the skilled artisan based on numerous factors, including, but not limited to, the available injectable volume, clinical status of the patient, and tumor type and size.
  • the viral genomes of the viral vectors used in the invention should be modified to remove or limit their ability to replicate, however, replication conditional viruses will also be useful in the present invention, as will replicating vectors that are capable of targeting certain cells. See, e.g. , Zhang, J., et al, Cancer Metastasis Rev. 75:385-401 (1996). Chase, M., et al (Nature Biotechnol.
  • the FPGS gene can also be delivered using non-viral methods for gene transfer, preferably those whose use in gene therapy is known in the art (Nakanishi, M., Crit. Rev. Therapeu. Drug Carrier Systems 72:263-310 (1995);
  • non-viral vectors for gene delivery include prokaryotic vectors, such as tumor targeted bacterial vectors (Pawelek, J.M.. e /., Cancer Res.
  • the present invention provides an embodiment of the foregoing methods wherein the FPGS gene is delivered using any cellular vector, preferably one whose use for gene therapy is well-established for those skilled in the art.
  • cellular vectors for gene therapy include endothelial cells (Rancourt, C., e/fl/., Clin. Cancer Res. 4:265-210 (1998); Ojeifo, J.O.. e/ ⁇ /., Cytokines Mol Ther. 2:89-101 (1996)) and macrophages including tumor-infiltrating macrophages (Zufferey, R., et al , Nat.
  • Biotechnol 75:871 -875 (1997): Naldini, L., et al, Science 272:263-267 (1996)), each of which may be modified using viral or non-viral vectors to carry the FPGS gene, and thus express the FPGS gene products.
  • viral or non-viral vectors to carry the FPGS gene, and thus express the FPGS gene products.
  • suitable non-viral vectors will be readily apparent to the skilled artisan.
  • the FPGS-based drug activation system is combined with established gene/prodrug activation systems, including ganciclovir/HSV-TK and 5-fluorocytosine/CD (Moolten, F.L., Cancer Gene
  • FPGS gene therapy may also be combined with other established cancer therapeutic genes, including tumor suppressor genes, such as p53 (Roth, J.A., et al, Nature Med. 2:985-991 (1996); Harris, M.P., et al, Cancer Gene Therap.
  • apoptotic factors such as bax (Bargou, R.C.. et al, J. Clin. Invest. 97:2651-2659 (1996)), tumor necrosis factor alpha (Gillio, T.A., et al, Blood 57:2486-2495 (1996)), and caspases (Kondo, S., etal, Cancer Research 58:962-961 (1998); Yu, J.S., e/ ⁇ /., Cancer Research 5(5:5423-5427 (1996)); and cytokines, such as interleukin 2 (Clary, B.M.. et al, Cancer Gene Ther.
  • the targetting specificity for FPGS gene delivery may be facilitated by targeted delivery or targeted expression (“transcriptional targeting"), including the use of tumor-specific or tumor-selective DNA enhancer sequences to selectively activate expression of the transduced gene in the tumor cell at either the primary tumor site or its metastases (Miller, N. and Whelan. J., Hum. Gene Ther. 5:803-815 (1997); Walther, W. and Stein, U., J. Mol Med 74:319-392 (1996); Schnierle, B.S. and Groner, B., Gene Therapy 5: 1069-1073 (1996); Lan, K-H., et al, Cancer Res. 57:4219-4284
  • DF3/MUC1 targeting to breast cancer
  • alpha- fetoprotein targeting to hepatoma
  • the use of synthetic gene regulation systems, which allow for transcriptional control and other forms of regulated expression of the FPGS gene, may also be used (Miller, N. and Whelan, J., Hum. Gene Ther. 5:803-815 (1997); Vile. R.G., Semin. Cancer Biol. 5:429-436 (1994); Hwang, J.J., et al, J. Virol.
  • DNA regulatory elements that are controlled by tumor-specific conditions and factors.
  • one unique aspect of solid tumors is their localized hypoxic environment (Brown, J.M. and
  • HRE hyperoxia response elements
  • HRE sequences can be used for the transcriptional targeting of FPGS genes to hypoxic neoplastic cells.
  • Exemplary candidates for treatment according to the present invention include, but are not limited to humans, other mammals, or non-mammal animals suffering from neoplasms, and in particular, malignant tumors of the central nervous system.
  • the method of the invention allows for greater localized tumor toxicity at a given drug concentration, leading to an enhanced chemotherapeutic response. It may also allow for lower doses of the drug to be given, thereby reducing toxic effects to the patient by decreasing exposure of normal cells and tissues to cytotoxic metabolites.
  • Amethopterin metalhotrexate or MTX
  • aminopterin were obtained from Sigma Biochemical (St. Louis, MO).
  • Edatrexate was provided by Dr. F. M. Sirotnak (Memorial Sloan-Kettering Cancer Center. New
  • the human FPGS cDNA was provided by Dr. B. Shane (University of California. Berkeley) (Garrow, T.A., etal, Proc.Natl.Acad. Sci. USA 59:9151-9155 (1992)). This cDNA was cloned into the plasmid pCDNA3.1 (Invitrogen Co.. Carlsbad, CA), where it could be expressed under control of the CMV promoter, while the gene for neomycin resistance would be expressed under control of the SV40 promoter.
  • Glioblastoma and astrocytoma tissue were obtained from Dr. D. Louis (Massachusetts General Hospital; Boston, MA); normal liver and liver tissue containing colon carcinoma metastases were obtained from Dr. K. Tanabe (Massachusetts General Hospital; Boston, MA). All cell lines were grown at 37 °C in a 5% C0 2 -95%> air atmosphere in Dulbecco's modified Eagle medium (Sigma) containing 10%> fetal bovine serum (Sigma), 100 U/mL. penicillin, and 100 ⁇ g/mL streptomycin (Sigma). The rat 9L gliosarcoma cell line has been described previously (Weizsaecker.
  • the transfectant 9L/FPGS was generated by lipofectamine-mediated (Life Technologies Inc., GIBCO BRL; Gaithersburg, MD) transfection of 9L cells with the plasmid pCDNA3.1 containing the human FPGS cDNA and was cloned under selection in medium containing 1 mg/mL G418 (neomycin analogue; Life Technologies, Inc.).
  • the cell line 9L/BAG expresses the histochemically detectable marker ⁇ -galactosidase (LacZ) due to infection with the BAG retrovirus (Scharf, J.M., et al, Transgenics 7:219-224 (1994)).
  • the human glioma cell line U87MG was purchased from American Tissue Culture Collection (Ponten, J., Macintyre, E.H., Acta Pathol Microbiol Scand 74:465-486 (1968)).
  • Enzymatic Assay FPGS activity was measured using a modification of a previously described protocol (Egan, M.G., et al, Journal of Biological
  • Frozen tissue was ground into small pieces using a mortar and pestle, suspended in lysis buffer (50 mM Tris pH 7.4, 250 mM NaCl, 0.1 % NP40, 5 mM EDTA, 2 ⁇ g/mL aprotinin, 1 ⁇ g/mL leupeptin), homogenized (Brinkmann Instruments Co.; Westbury, NY), and kept on ice for one hour to allow for completion of lysis.
  • lysis buffer 50 mM Tris pH 7.4, 250 mM NaCl, 0.1 % NP40, 5 mM EDTA, 2 ⁇ g/mL aprotinin, 1 ⁇ g/mL leupeptin
  • homogenized Brinkmann Instruments Co.; Westbury, NY
  • the samples were concentrated using an Integrated Speed Vac system (Savant Instruments Inc.; Farmingdale. NY), after which protein concentration was determined using the Bradford assay.
  • the FPGS enzymatic assay reaction mixture consisted of 1 M
  • [ 3 H] glutamate (1 ⁇ Ci/ ⁇ L; 50 Ci/mmol; DuPont New England Nuclear; Boston, MA), 5 mM ATP, 10 mM MgCl 2 , 20 mM KC1, 100 mM 2-mercaptoethanol, 0.1 ⁇ g/ ⁇ l BSA, and 0.4 to 0.8 mg cellular protein. Reaction was carried out in 500 ⁇ L at 37 °C for 2 hours, and was terminated by adding 1.5 mL ice-cold 30 mM 2-mercaptoethanol, 10 mM glutamate.
  • Free glutamate was separated from glutamate bound to aminopterin by chromatography on a 1-1.5 mL bed volume minicolumn (Poly-Prep Chromatography Column, Bio-Rad) containing DE52 Diethylaminoethyl (DEAE) cellulose (Whatman). The column was equilibrated with 5 mL 10 mM Tris (pH 7.5), 80 mM NaCl. The terminated reaction was then applied to the column. The column was washed with 5 mL equilibration buffer to remove unincorporated glutamate.
  • the glutamylated aminopterin was then eluted with 3 mL 0.1 N HC1 with the eluant collected in a scintillation vial containing 10 mL scintillation fluid (ScintiVerse II, Fisher), and subsequently counted on a scintillation counter.
  • Precipitated proteins were removed by centrifugation at 20,000 g for 30 min, and the supernatant was then concentrated to 30 ⁇ L under a stream of N 2 at 37°C on a Meyer N-Evap Analytical Evaporator (Organomation Associates Inc., Northborough, MA). A 1 ⁇ L aliquot was scintillation counted — cpm were converted into moles of drug taken up by determining the cpm of a known quantity of [ 3 H] MTX. MTX polyglutamates were separated by ascending thin layer chromatography (TLC) of samples on Baker Si250PA silica gel plates
  • MTX 0.66
  • MTX-G1 0.59
  • MTX-G2 0.44
  • MTX-G3 0.33
  • MTX-G4 0.27
  • MTX-G5 0.25.
  • Regions corresponding to where standards migrated were marked in the sample lane, scraped individually into scintillation vials, and counted.
  • the amount of each radioactive metabolite in a sample lane was determined as a percentage of the total radioactivity recovered from that sample lane, after subtraction of the background counts obtained from lanes lacking radioactive sample.
  • 10 5 9L or 9L/FPGS cells or a mixture of 8 X 10 5 9L + 2 X 10 5 9L/FPGS cells in 200 ⁇ L DMEM were injected subcutaneously into the flanks of six week old female nude mice (NCr/Sed, nu/nu, 20 gm; Massachusetts General Hospital breeding colony). After 14 days, when the tumors had reached an average volume of 60 mm 3 , the mice were randomly divided into experimental groups with five mice per group. Intraperitoneal injections of varying MTX or EDX doses dissolved in 200 ⁇ L 0.9% NaCl were administered either daily or every third day. Tumor size was measured weekly using calipers. Tumor volume was calculated as length X width X height, as described previously (Takamiya, Y., et al, Journal of Neuroscience Research
  • Gliomas have been used in numerous cancer gene therapy studies, but before using gliomas in this study of FPGS gene delivery, it was important to determine if their FPGS activities were low enough that they could potentially experience enhanced antifolate sensitivity upon transfection (Roth. J.A.. Cristiano, R.J., Journal of the National Cancer Institute 59:21-39 (1997); Aghi, M., et al, Journal of the National Cancer Institute 90:310-380 (1998)).
  • Cell lines derived from tumor tissue frequently have a higher replication fraction and rate than the primary tumor since rapidly dividing cells are selected for over time.
  • 9L cells were transfected with a plasmid containing the human FPGS cDNA under the control of the cytomegalovirus (CMV) immediate early gene promoter, as well as a gene conferring neomycin resistance.
  • CMV cytomegalovirus
  • the parental cell line and 16 G418-resistant clones selected after transfection were treated with 0.3 ⁇ M MTX for 4 hours, followed by 3 days of growth in drug-free medium. Susceptibility to 4 hour MTX pulses correlates somewhat with FPGS activity (Kim, J.S., et al, Journal of Biological Chemistry 2(55:21680-21685 (1993)).
  • Fourteen clones displayed approximately the 90%> survival seen with 9L cells, but two clones each displayed approximately 40% survival in response to the pulse.
  • One of these clones was selected for further study and designated 9L/FPGS.
  • the FPGS activity of 9L/FPGS cells was 1630 pmol aminopterin polyglutamylated/mg protein/hour (Table 1 ). a more than 4-fold increase over the FPGS activity of the parental 9L cell line.
  • the G418-resistant clone whose MTX pulse survival was comparable to 9L/FPGS possessed FPGS activity comparable to 9L/FPGS; while a G418-resistant clone whose MTX pulse survival was comparable to 9L possessed FPGS activity comparable to 9L (data not shown). Further evidence that the enhanced MTX pulse susceptibility of 9L/FPGS resulted from increased FPGS enzyme activity was offered by the fact that 9L and
  • 9L/FPGS cells displayed similar sensitivity to 4 hour pulses of the non- polyglutamylatable antifolate PT523 (Rosowsky, A., et al. , Journal of Medicinal Chemistry 31 : 1332-1337 (1988)).
  • 9L and 9L/FPGS cells proliferated at comparable rates in the absence of MTX.
  • the enhanced FPGS activity of 9L/FPGS cells did not affect their growth rate, nor did 9L/FPGS cells possess an altered replication rate arising through clonal variation.
  • the FPGS enzyme assay measures the amount of drug converted into any of the five polyglutamates. But, the attachment of each glutamate to the drug by FPGS has different enzyme kinetics. And, it is only after attachment of the last three glutamate residues that the drug's retention time and inhibitory properties improve substantially (Chu, E. and Allegra, C, Antifolates," in Cancer Chemotherapy and Biotherapy: Principles and Practice, Chabner et al. eds., Lippincott-Raven, Philadelphia (1996), pp. 109-148). Thus, it is important to determine a cell line's capacity to form each of the MTX polyglutamates.
  • TLC thin layer chromatography
  • 9L/FPGS extracts (Table 2). These results indicate that the higher FPGS enzyme activity of 9L/FPGS cells is indeed associated with greater formation of the higher order MTX polyglutamates. This greater formation of higher order polyglutamates in 9L/FPGS cells leads to greater cellular uptake of MTX. The greater capacity of 9L/FPGS cells to convert MTX into the highly retained polyglutamates lowers the intracellular concentration of monoglutamylated MTX, making MTX influx more favorable than efflux.
  • b Lysates were run on TLC plates in duplicate, along with MTX polyglutamate standards. Following elution, polyglutamate standards were visualized under ultraviolet light. In the sample lanes, the positions corresponding to the polyglutamate standards were scraped into scintillation vials and counted to determine radioactivity, Percentages shown represent percentages of total sample lane radioactivity found in each polyglutamate's position.
  • the ED 50 for cells grown in the presence of MTX for 72 hours was 40 nM for both 9L and 9L/FPGS cells (dose-response curves not shown).
  • 9L/FPGS cells display enhanced MTX sensitivity when exposed to the drug for brief periods.
  • a ED, 0 drug dose which caused 50% cell death, calculated by using Sigma Plot 3 0, which employs curve-fitting parameters based on the Marquardt-Levenberg method Marquardt, D W , Siam, J . J Soc Indust Appl Math 7/ 431-441 (1963)
  • the ED, 0 values for 4 hour MTX pulse given in the table are derived from the dose-response curve in Fig 1
  • This dose-response curve was generated twice, with identical results
  • the ED, 0 values for 4 hour EDX pulse given in the table are derived from the dose-response curve in Fig 3a This dose-response curve was generated one other time and produced ED 50 values of 1 2 ⁇ M (9L) and 26 nM (9L/FPGS), for a 46-fold shift d In cocultures.
  • the ED 50 values for MX treatment of 9L, 9L/FPGS, and a coculture are derived from the dose-response curves in Fig 14a Differences between the 9L and 9L/FPGS ED 50 values between Figs 1 and 4a can be attributed to interexperimental variation and the fact that the curve in Fig 1 covers a wider range of concentrations, which can slightly alter curve-fitting f
  • the ED, 0 values for EDX treatment of 9L, 9L/FPGS, and various cocultures are derived from the dose-response curves in Fig 4b Differences between these ED 50 values and those derived from Fig 3a can be attributed to interexperimental variation and the fact that the curve in Fig 3a covers a wide range of concentrations, which can slightly alter curve-fitting In Vivo Treatment of9L and 9L/FPGS Tumors with MTX
  • MTX is lethal to nude mice at lower doses than those used to treat humans
  • tumors implanted in nude mice are usually treated with the maximum tolerated (nonlethal) dose (MTD) of MTX.
  • MTD maximum tolerated dose
  • Nude mice bearing 9L or 9L/FPGS tumors were therefore treated with 9 mg MTX/kg body weight daily or every third day for 21 days. Tumor volume was measured once a week, and the fold growth of each tumor relative to its volume at the time treatment commenced was measured.
  • 9L/FPGS tumors responded slightly better to MTX treatment every third day than 9L tumors did to daily MTX treatment, meaning that a reduced frequency of treatment could be achieved by tumoral expression of the FPGS cDNA, which should translate into reduced toxicity to normal cells.
  • Antifolate drugs whose affinities for FPGS differ from MTX were tested in 4 hour pulses on 9L and 9L/FPGS cells in culture to determine if any drugs produced greater separation than MTX produced between the 9L and 9L/FPGS dose-response curves.
  • MTX has a relatively low affinity for FPGS.
  • EDX edatrexate
  • aminopterin two drugs with slightly higher affinity for FPGS, were investigated (Chu, E. and Allegra, C,
  • Pulsing with EDX concentrations between 1 nM and 10 ⁇ M generated statistically significant (p ⁇ 0.025) differences in the percent survival of the two cell lines. This represents a 10,000-fold range of concentrations for which the cell lines responded differently, a much wider range than the 100-fold range of concentrations seen with MTX (Fig. 1).
  • Fig. 3A In order to demonstrate more precisely the ability of EDX to induce cytotoxicity in 9L/FPGS cells while preserving 9L cells, the data in the middle of Fig. 3A was expanded by evaluating a larger number of concentrations between 100 and 900 nM using the same 4 hour pulse protocol that had been used to generate Fig. 3A. The resulting dose-response curves (Fig. 3B) were consistently separated by differences of 60 to 80 percent survival.
  • the cancer gene therapy strategies that have been the most successful to date have bystander effects.
  • the bystander effect refers to the ability of transduced tumor cells to cause cytotoxicity in neighboring nontransduced tumor cells.
  • drug-activating gene therapies the bystander effect results in part from transfer of the active metabolite to nontransduced cells via facilitated diffusion, gap junctions, or apoptotic vesicles (Huber, B.E., et al, Proceedings of the National Academy of Sciences 97:8302-8306 (1994); Freeman, S.M., et al, Cancer Research 55:5274-5283 (1993)).
  • the simplest way to investigate the bystander effect in culture is to coculture transfected and nontransfected cells in varying ratios, treat with a drug dose that kills only transfected cells, and determine if the resulting cytotoxicity exceeds that which would be expected if only the enzyme-expressing cells had died.
  • a more quantitative approach is to generate a dose-response curve for nontransfected cells, transfected cells, and a coculture and to calculate the transmission efficiency (TE) for the coculture (Friedlos. F., et al, Journal of
  • the superscripts 0, N, and 100 designate the ED 50 values for cultures containing 0, N, and 100% transfected cells.
  • the transmission efficiency indicates how well the drug's effect is transmitted from transfected to nontransfected cells, i.e. a TE of 50%) means the nontransfected cells in the coculture experience half the concentration of the active drug that the transfected cells experience.
  • the TE value obtained with EDX was high enough to merit treating cocultures containing 10 and 1%> 9L/FPGS cells with 4 hour pulses of the same
  • the next step was to determine whether the EDX bystander effect resulted from transfer of a metabolite through the medium.
  • Conditioned medium was harvested from 9L or 9L/FPGS cells that had been exposed to 0.3 ⁇ M EDX for 4 hours, washed extensively, and incubated for 3 days in drug-free medium. This conditioned medium was then added to 9L/BAG cells. A week later, the cells were stained for LacZ expression, which allowed colony formation to be assayed. Because multiple washes occur after EDX treatment, any EDX in the conditioned medium from treated cells at 3 days must derive from EDX that had been retained intracellularly and released after the 4 hour drug exposure. That is, even though the conditioned medium could be transferring EDX. it would have to be transferring EDX that had been released by the 9L/FPGS cells, not EDX that remained in the medium throughout the experiment.
  • 9L/BAG cells grown with conditioned medium from 9L cells that had been pulsed with EDX formed 83% as many colonies as those grown in conditioned medium taken from non-EDX treated 9L cells.
  • 9L/BAG cells grown with conditioned medium from 9L/FPGS cells that had been pulsed with EDX formed 47% as many colonies as 9L/BAG cells grown in conditioned medium taken from non-EDX treated 9L/FPGS cells. This suggests that part or all of the bystander effect observed during EDX treatment of cocultures results from release of a toxic metabolite from transfected cells into the medium.
  • 9L tumors After three weeks of daily EDX treatment, 9L tumors had grown 10-fold; 9L/FPGS tumors experienced some growth inhibition during the third week of treatment and had only doubled in volume; and treated mixed tumors had grown 5-fold. The disparity in growth between mixed and 9L/FPGS tumors that arose between the second and third weeks of treatment was not statistically significant
  • transfection of a gliosarcoma cell line with a plasmid bearing the FPGS cDNA was shown to enhance the sensitivity of these cells to antifolate pulses in culture and to antifolate treatment in vivo.
  • Slight bystander killing of nontransfected cells upon pulse treatment of a coculture containing transfected and nontransfected cells was also demonstrated.
  • Transfected cells displayed enhanced sensitivity in culture to 4 hour antifolate exposures, but not to 72 hour exposures.
  • a drug-enhancing gene therapy can be evaluated is the enhancement of drug sensitivity seen in clones resulting from transfection of a tumor cell line with the cDNA for the drug-activating enzyme.
  • the magnitude of this enhancement can be quantified by the shift in ED 50 observed between nontransfected and transected tumor cell lines.
  • HSV-TK herpes simplex virus thymidine kinase
  • CD coli cytosine deaminase
  • FPGS is a mammalian gene expressed to a certain extent by mammalian tumors, making relative increases in expression harder to achieve.
  • two advantages of the slight FPGS expression and slight antifolate sensitivity found in tumors that would be candidates for FPGS gene transfer are: (1) tumor cells expressing foreign enzymes like HSV-TK and CD can be killed by an immune response before generating enough active drug to mediate a bystander effect, while tumor cells expressing FPGS encounter no such response; and (2) transgene expression often shuts down over time during gene therapy — loss of HSV-TK or CD expression would render ganciclovir or 5-fluorocytosine completely ineffective, while loss of expression of delivered FPGS would leave behind native FPGS activity, allowing antifolates to retain a slight anticancer effect (Roth, J.A., Cristiano, R.J.. Journal of the National Cancer Institute 89:21-39 (1997)).
  • Enhanced chemosensitivity can also be appreciated by identifying drug doses which are almost completely cytotoxic to transfected cells but relatively nontoxic to nontransfected cells. In this study, such doses were not found with MTX treatment of 9L and 9L/FPGS, but 4 hours of treatment with 300 nM EDX was almost completely cytotoxic (below ⁇ 0% survival) to 9L/FPGS, while preserving (above 90%> survival) most 9L cells. Because antifolates can be toxic to normal tissues, having widely separated dose-response curves is important for the FPGS gene transfer approach (Chu, E. and Allegra, C.
  • FPGS gene transfer may be advantageous if it can render transduced tumor cells susceptible to antifolate doses that induce minimal damage to nontransduced tumor cells ⁇ such doses that are nontoxic to tumor cells prior to gene delivery should display minimal toxicity against normal tissues. Two factors should keep MTX toxicity directed towards tumor cells rather than normal tissues, making MTX a somewhat effective anticancer agent.
  • the greater replication rate of tumor cells compared to normal cells renders them more susceptible to S-phase specific antifolates.
  • the somewhat greater FPGS activities of tumor cells compared to normal cells confers tumor cells with a greater capacity for drug retention and possible toxicity after drug clearance.
  • normal bone marrow and gastrointestinal epithelium replicate rapidly enough that they are somewhat MTX sensitive, and normal liver tissue possesses enough FPGS activity that it is also susceptible (Chu, E. and Allegra, C, Antifolates," in Cancer Chemotherapy and Biotherapy: Principles and
  • Intratumoral FPGS gene transfer would enhance the disparity between tumor cells and normal cells in one of these two criteria for antifolate susceptibility and could therefore reduce the toxicity to normal tissue associated with antifolate therapy.
  • FPGS gene delivery could be improved by increasing the separation between the dose-response curves of transfected and nontransfected cells.
  • One way of increasing this separation would be to generate a mutant FPGS which preferentially polyglutamylates antifolates instead of natural folates.
  • MTX-treated tumor-bearing mice have higher levels of MTX polyglutamates in tumors than in many normal tissues and because FPGS purified from some leukemia and sarcoma cell lines has lower K m and higher V ma ⁇ values for antifolate drugs than FPGS purified from intestinal epithelium, it has been suggested that some tumors may express a tumor-specific
  • FPGS with a unique nucleotide sequence that confers enhanced antifolate affinity (Rumberger, S. et al, Cancer Research 50:4639-4643 (1990); Kim, J.S., et al, Journal of Biological Chemistry 2(55:21680-21685 (1993); Whitehead, V.M., et al, Cancer Research 55:2985-2990 (1975)). If such an altered form of FPGS were cloned, it could either be used directly in FPGS gene delivery or regions where it differs from wild type FPGS could be identified and subjected to site- specific mutagenesis to further enhance its affinity for antifolates.
  • Another way of increasing the separation between the dose-response curves of 9L and 9L/FPGS cells would be to find an antifolate that produces more widely separated dose-response curves than the drugs studied in this report.
  • the ideal antifolate for use with FPGS gene delivery would have its inhibitory activity enhanced tremendously after polyglutamylation, and would possess intermediate affinity for FPGS, causing the drug to experience a transition from minimal to extensive polyglutamylation as FPGS activity increased by the maximum amount attainable through gene transfer.
  • thymidylate synthetase instead of dihydrofolate reductase, which methotrexate inhibits
  • FPGS gene transfer through the following four step process: (1) the drug is retained intracellularly through polyglutamylation by transduced cells which survive the initial drug exposure; (2) after extracellular drug concentration drops following treatment, polyglutamates inside viable transduced cells are cleaved into the monoglutamylated form by ⁇ -glutamyl hydrolase; (3) monoglutamylated drug effluxes down its concentration gradient out of the transduced cells; and (4) extracellular drug released from transduced cells is taken up by nontransduced cells and given a second opportunity (although at a lower concentration) to kill the nontransduced tumor cells which survived the initial drug exposure.
  • TEs Transmission efficiencies
  • the prodrug CB 1954 displayed TE values between 90 and 99% for 20% cocultures (compared to 87% for EDX), above 70% for 10%> cocultures (compared to 64% for EDX), and above 50% for 1% cocultures (compared to 33% for EDX). The greatest bystander effect is believed to occur with the cytosine deaminase prodrug-activating system.
  • nude mice bearing subcutaneous tumors with the maximum tolerable antifolate dose did not eliminate tumors, but antifolate treatment in the context of FPGS gene transfer may prove more effective in humans because humans can tolerate larger antifolate doses than nude mice.
  • 9 mg/kg was the maximum nonlethal dose of MTX for nude mice, humans do not experience toxic side effects until 50 mg/kg — these doses correspond to peak plasma levels of 0.03 mM in a nude mouse and 0.1 mM in a human (Stoller, R.G., et al, New England Journal of Medicine 297:630-634 (1977)).
  • mice have shown that when tumors derived from cell lines with FPGS activity comparable to the 9L/FPGS cell line are treated with a single MTX injection, the tumors retain enough polyglutamates to occupy all tumor dihydrofolate reductase (DHFR) binding sites for up to 48 hours after the MTX injection (Fry, D.W., et al, Cancer
  • DHFR tumor dihydrofolate reductase
  • leucovorin 5-formyltetrahydrofolate
  • Leucovorin competitively displaces MTX from DHFR. This displacement reactivates DHFR and rescues cells from MTX- induced cytotoxicity. But leucovorin rescue is less likely to occur in tumor cells because, as mentioned earlier, tumor cells have slightly greater FPGS activity than normal cells, causing them to polyglutamylate MTX to a larger extent than most normal cells.
  • polyglutamates have a greater affinity for DHFR than the monoglutamylated drug, and are thus harder for leucovorin to displace from the enzyme (Matherly, L.H., et al, Cancer Research 46:588 (1986)).
  • Gene transfer of FPGS into tumor cells would enhance leucovorin's differential rescue by increasing the level of antifolate polyglutamates bound to DHFR in tumor cells.
  • MTX has been unsuccessful in glioma treatment — proposed explanations include: a low replicating fraction making gliomas less responsive to S-phase specific drugs like MTX; the inability of MTX to penetrate a spheroid mass of tumor cells (the form gliomas typically assume); and altered expression of FPGS, DHFR, or other enzymes important in MTX responsiveness.
  • MTX's hydrophilicity does not prevent it from penetrating the blood-brain barrier, as evidenced by the fact that high dose intravenous MTX therapy usually allows for sufficient drug penetration into the CNS.
  • CNS lymphoma is highly responsive to high dose MTX therapy (Chu, E. and Allegra, C, " Antifolates.” in Cancer Chemotherapy and Biotherapy: Principles and Practice, Chabner et al, eds., Lippincott-Raven, Philadelphia (1996), pp. 109-148).
  • CNS penetration of MTX during glioma treatment should be facilitated because the blood-brain barrier near gliomas is usually disrupted.
  • Enhanced penetration of MTX into the CNS for glioma treatment has been achieved by combining internal carotid artery drug infusions with agents, such as mannitol, that disrupt the blood-brain barrier.
  • agents such as mannitol
  • human glioma cells are not intrinsically resistant to antifolates, as they are sensitive to prolonged drug exposure in culture (Terzis, A.J.A., et al, International Journal of Cancer 54: 1 12-1 18 (1993)).
  • EDX the drug used in this study that produced slightly better results than MTX, is being investigated in phase II clinical trials in patients with malignant gliomas (Drinkard, L.. et al, Proceedings of the American Society of Clinical Oncology 75: 182 (1994)).
  • the approach described in this report could be applied to tumors other than gliomas.
  • cancer gene therapy using the prodrug-activating strategy has been studied in colon carcinoma, hepatocellular carcinoma, prostate cancer, ovarian cancer, melanoma, breast cancer, lung cancer, and pancreatic cancer (Roth, J.A., Cristiano, R.J., Journal of the National Cancer Institute 59:21-39 (1997)).
  • tumors such as breast and colon cancers
  • MTX melatonin-semiconductor
  • FPGS gene delivery could be evaluated as a means of enhancing the drug's efficacy; the other tumors are like gliomas in that MTX is not the drug of choice but could become a viable therapeutic if FPGS gene delivery substantially enhanced the drug's anticancer effect.
  • FPGS activity is just one variable contributing to antifolate susceptibility. Even when comparing different brain tumor cell lines, the correlation between FPGS activity and MTX susceptibility did not always hold.
  • the ED 50 for U87 cells treated with a 4 hour MTX pulse was 30 ⁇ M (Table 3), over 10-fold higher than that of 9L.
  • the greater sensitivity of 9L to MTX pulses was inconsistent with U87's slightly greater FPGS activity (Table 1), reflecting the fact that FPGS activity is probably not the only parameter affecting MTX sensitivity that may differ between these two cell lines - other differences could be levels of reduced folate carrier, replication rates, DHFR expression, or ⁇ -glutamyl hydrolase levels.
  • FPGS activity only correlates definitively with MTX pulse sensitivity in cell lines which express varying levels of FPGS activity but derive from the same parental cell line, such as 9L and 9L/FPGS (Rumberger, S. et al, Cancer Research 50:4639-4643 (1990)).
  • a prodrug is a chemical that is inert at all doses but can be converted into a highly toxic chemical by a specific prodrug-activating enzyme (Connors, T.A., Gene Therapy 2:702-709 (1995)).
  • prodrug-activating enzyme Connors, T.A., Gene Therapy 2:702-709 (1995)
  • all prodrugs become toxic at high enough doses, even in the absence of prodrug-activating enzyme, because cells have alternative enzymes which activate the prodrug to some extent at high enough doses. Therefore, all prodrug-activating gene therapies are in reality drug- enhancement systems.
  • MTX's chemotherapeutic activity makes it distinct from chemicals such as 5-fluorocytosine and ganciclovir which can only affect nontransduced tumors at doses that substantially exceed their maximum tolerable doses.
  • these prodrugs require gene delivery in order to become active at reasonable doses, gene delivery could enhance a tumor's existing antifolate sensitivity. This enhancement would increase the likelihood of a successful outcome with high dose antifolate therapy or, in patients experiencing antifolate toxicity, would permit reductions in dose or dose frequency without compromising therapeutic efficacy (Chu, E. and Allegra, C, Antifolates," in Cancer Chemotherapy and Biotherapy: Principles and Practice, Chabner et al, eds..

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne des procédés permettant de tuer des cellules néoplastiques, et traite de l'utilisation du transfert du gène de folyl-polyglutamyl synthétase (FPGS) destiné à améliorer la sensibilité de plusieurs types de cellules tumorales à des médicaments antifoliques polyglutamylables, tels que méthotrexate (MTX) et édatrexate (EDX).
PCT/US2000/019162 1999-07-16 2000-07-14 Transfert du gene de folyl-polyglutamyl synthetase pour ameliorer la sensibilite au medicaments antifoliques WO2001005405A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US14407499P 1999-07-16 1999-07-16
US60/144,074 1999-07-16

Publications (1)

Publication Number Publication Date
WO2001005405A1 true WO2001005405A1 (fr) 2001-01-25

Family

ID=22506940

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/019162 WO2001005405A1 (fr) 1999-07-16 2000-07-14 Transfert du gene de folyl-polyglutamyl synthetase pour ameliorer la sensibilite au medicaments antifoliques

Country Status (1)

Country Link
WO (1) WO2001005405A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102337297A (zh) * 2011-10-21 2012-02-01 南京医科大学 一种mbr-FPGS高效表达载体及其构建方法和应用
CN109689104A (zh) * 2016-08-12 2019-04-26 L.E.A.F.控股集团公司 α和γ-D聚谷氨酸化抗叶酸剂及其用途
US11534498B2 (en) 2016-08-12 2022-12-27 L.E.A.F. Holdings Group Llc Polyglutamated antifolates and uses thereof
US11730738B2 (en) 2018-02-07 2023-08-22 L.E.A.F. Holdings Group Llc Alpha polyglutamated pralatrexate and uses thereof
US11771700B2 (en) 2018-02-14 2023-10-03 L.E.A.F. Holdings Group Llc Gamma polyglutamated lometrexol and uses thereof
US11779584B2 (en) 2018-02-07 2023-10-10 L.E.A.F. Holdings Group Llc Alpha polyglutamated pemetrexed and uses thereof
US12048767B2 (en) 2018-02-14 2024-07-30 L.E.A.F. Holdings Group Llc Gamma polyglutamated pralatrexate and uses thereof
US12048766B2 (en) 2018-02-14 2024-07-30 L.E.A.F. Holdings Group Llc Gamma polyglutamated tetrahydrofolates and uses thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AGHI M. ET AL.: "Folypolyglutamyl synthetase gene transfer and glioma antifolate sensitivity in culture and in vivo", J. NATL. CANCER INST., vol. 91, no. 14, 21 July 1999 (1999-07-21), pages 1233 - 1241, XP002931163 *
FREEMANTLE S.J. ET AL.: "Transcription of the human folypoly-gamma-glutamate synthetase gene", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 272, no. 40, 3 October 1997 (1997-10-03), pages 25373 - 25379, XP002931165 *
MCGUIRE J.J. ET AL.: "Folypolyglutamate synthetase expression in antifolate-sensitive and resistant human cell lines", ONCOLOGY RESEARCH, vol. 10, no. 4, 1998, pages 193 - 200, XP002931164 *
TAYLOR S.M. ET AL.: "Structural organization of the human folypoly-gamma-glutamate synthetase geen: Evidence for a singlegenomic locus", CANCER RESEARCH, vol. 55, no. 24, 1995, pages 6030 - 6034, XP002931166 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102337297A (zh) * 2011-10-21 2012-02-01 南京医科大学 一种mbr-FPGS高效表达载体及其构建方法和应用
CN109689104A (zh) * 2016-08-12 2019-04-26 L.E.A.F.控股集团公司 α和γ-D聚谷氨酸化抗叶酸剂及其用途
EP3496757A4 (fr) * 2016-08-12 2020-04-15 L.E.A.F Holdings Group LLC Antifolates de polyglutamates et leurs utilisations.
US11344628B2 (en) 2016-08-12 2022-05-31 L.E.A.F. Holdings Group Llc Alpha polyglutamated antifolates and uses thereof
US11534498B2 (en) 2016-08-12 2022-12-27 L.E.A.F. Holdings Group Llc Polyglutamated antifolates and uses thereof
US11701432B2 (en) 2016-08-12 2023-07-18 Le.A.F. Holdings Group Llc Polyglutamated antifolates and uses thereof
US11730738B2 (en) 2018-02-07 2023-08-22 L.E.A.F. Holdings Group Llc Alpha polyglutamated pralatrexate and uses thereof
US11779584B2 (en) 2018-02-07 2023-10-10 L.E.A.F. Holdings Group Llc Alpha polyglutamated pemetrexed and uses thereof
US11771700B2 (en) 2018-02-14 2023-10-03 L.E.A.F. Holdings Group Llc Gamma polyglutamated lometrexol and uses thereof
US12048767B2 (en) 2018-02-14 2024-07-30 L.E.A.F. Holdings Group Llc Gamma polyglutamated pralatrexate and uses thereof
US12048766B2 (en) 2018-02-14 2024-07-30 L.E.A.F. Holdings Group Llc Gamma polyglutamated tetrahydrofolates and uses thereof

Similar Documents

Publication Publication Date Title
Aghi et al. Prodrug activation enzymes in cancer gene therapy
EP0776161B1 (fr) Utilisation de vecteur viral codant pour un cytochrome p450 en combinaison avec un agent chimiotherapique pour la destruction selective de cellules neoplastiques
JP4387059B2 (ja) Ｐ４５０に基づく抗癌遺伝子治療の増強のためのシトクロムｐ４５０レダクターゼを用いる方法
Spencer et al. A gene transfer strategy for making bone marrow cells resistant to trimetrexate
Flasshove et al. Ex vivo expansion and selection of human CD34+ peripheral blood progenitor cells after introduction of a mutated dihydrofolate reductase cDNA via retroviral gene transfer
Tiberghien Use of suicide genes in gene therapy
US6642043B1 (en) Double mutants of dihydrofolate reductase and methods of using same
WO2000002581A1 (fr) Peptides antigenes derivees de la telomerase
US6770632B1 (en) Folypolyglutamyl synthetase gene transfer to enhance antifolate
Li et al. Development of a retroviral construct containing a human mutated dihydrofolate reductase cDNA for hematopoietic stem cell transduction
KR100361254B1 (ko) 발현표적화를위한태아성암항원의전사조절서열
WO2001005405A1 (fr) Transfert du gene de folyl-polyglutamyl synthetase pour ameliorer la sensibilite au medicaments antifoliques
WO1997019183A2 (fr) Transcription specifique pour le tissu d'une sequence d'adn codant une enzyme heterologue connue pour etre utilisee dans une therapie a base d'un promedicament enzymatique pour traiter le cancer du poumon
WO1996026743A1 (fr) Therapie genique associee a une radiotherapie pour le traitement des tumeurs
JP2000507814A (ja) 増殖細胞破壊用酵素コンビネーション
Cao et al. In vivo gene transfer of a suicide gene under the transcriptional control of the carcinoembryonic antigen promoter results in bone marrow transduction but can avoid bone marrow suppression.
Maza et al. Establishing chemoresistance in hematopoietic progenitor cells
Hayashi et al. An experimental application of gene therapy for human retinoblastoma.
WO2003083052A2 (fr) Procede d'utilisation de facteurs anti-apoptotiques dans l'expression genetique
JP2000507554A (ja) 治療用アルキル化剤の毒性から保護する遺伝子治療のための変異型アルキルトランスフェラーゼの使用
Vinh et al. Selective expression of methotrexate-resistant dihydrofolate reductase (DHFR) activity in mice transduced with DHFR retrovirus and administered methotrexate.
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
Haas et al. Regulated gene expression after retroviral vector-mediated delivery of cancer-relevant therapeutic genes
JP2003530855A (ja) 遺伝子治療のためのベクター
Aghi Improving the efficacy of chemosensitizing brain tumor gene therapies

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

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

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

Ref country code: JP