WO1995022612A2 - Genes et elements genetiques associes a la sensibilite aux medicaments a base de platine - Google Patents

Genes et elements genetiques associes a la sensibilite aux medicaments a base de platine Download PDF

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WO1995022612A2
WO1995022612A2 PCT/US1995/002303 US9502303W WO9522612A2 WO 1995022612 A2 WO1995022612 A2 WO 1995022612A2 US 9502303 W US9502303 W US 9502303W WO 9522612 A2 WO9522612 A2 WO 9522612A2
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gene
gse
nucleotide sequence
cells
cisplatin
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WO1995022612A3 (fr
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Deborah J. Kirschling
Andrei Gudkov
Igor B. Roninson
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Board Of Trustees Of The University Of Illinois
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1072—Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1079—Screening libraries by altering the phenotype or phenotypic trait of the host
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1082—Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to genetic factors associated with sensitivity to chemotherapeutic drugs. More particularly, the invention relates to methods for identifying such factors as well as to uses for such factors.
  • the invention specifically provides genetic suppressor elements derived from genes associated with sensitivity of mammalian cells to platinum-based chemotherapeutic drugs, such as cisplatin, and therapeutic and diagnostic uses related thereto.
  • Lippincott Company, Philadelphia, PA (1985) discloses as major antineoplastic agents the plant alkaloids vincristine, vinblastine, and vindesine; the antibiotics actinomycin-D, doxorubicin, daunorubicin, mithramycin, mitomycin C and bleomycin; the antimetabolites methotrexate, 5- fluorouracil, 5-fluorodeoxyuridine, 6-mercaptopurine, 6-thioguanine, cytosine arabinoside, 5-aza-cytidine and hydroxyurea; the alkylating agents cyclophosphamide, melphalan, busulfan, CCNU, MeCCNU, BCNU, streptozotocin, chlorambucil, bis- diaminedichloro-platinum, azetidinylbenzoquinone; and the miscellaneous agents dacarbazine, mAMSA and mitoxantrone.
  • the antibiotics actinomycin-D the
  • Chemotherapeutic agents have proven to be very useful in the treatment of cancer. Unfortunately, some tumor cells become resistant to specific chemotherapeutic agents, in some instances even to multiple chemotherapeutic agents. Such drug resistance or multiple drug resistance can theoretically arise from either the presence of genetic factors that confer resistance to the drags, or from the absence of genetic factors that confer sensitivity to the drugs. The former type of factors have been identified, and include the multiple drug resistance gene mdr-1 (see Chen et al., 1986, Cell 47: 381-389). However, the latter type of factor remains largely unknown, perhaps in part because the absence of such factors would tend to be a recessive trait.
  • platinum coordination complexes typified by cisplatin (ris-diamminedichloroplatinum (II)) (Reed, 1993, in Cancer. Principles and Practice of Oncology, ibid. , pp. 390-4001), have been described as "the most important group of agents now in use for cancer treatment". These agents, used as a part of combination chemotherapy regimens, have been shown to be curative for testicular and ovarian cancers and beneficial for the treatment of lung, bladder and head and neck cancers. DNA damage is believed to be the major determinant of cisplatin cytotoxicity, though this drug also induces other types of cellular damage.
  • this group of drugs includes carboplatin, which like cisplatin is used clinically, and other platinum-containing drugs that are under development. These compounds are believed to act by the same or very similar mechanisms, so that conclusions drawn from the study of the bases of cisplatin sensitivity and resistance are expected to be valid for other platinum-containing drugs.
  • Cisplatin is known to form adducts with DNA and to induce interstrand crosslinks. Adduct formation, through an as yet unknown signalling mechanism, is believed to activate some presently unknown cellular enzymes involved in programmed cell death (apoptosis), the process which is believed to be ultimately responsible for cisplatin cytotoxicity (see Eastman, 1990, Cancer Cells 2: 275-2802).
  • the first such mechanism involves decreased cellular accumulation of the drug. Observed changes in cisplatin transport in different cell lines may be related to the altered expression of specific membrane proteins.
  • a protein termed SQML that is decreased in some cisplatin-resistant cell lines (Bernal et al., 1990, Mol. Cell. Biochem. 95: 61-703).
  • Another such protein is a 200 kilodalton (kDa) glycoprotein which expression has been found to be increased in another such cell line having diminished intracellular accumulation of cisplatin (Kawai et al, 1990, J. Biol. Chem. 265: 13137-13142).
  • cytosolic inactivation (termed “quenching") of the drug by sulfhydryl-containing proteins or glutathione.
  • quenching cytosolic inactivation
  • some cisplatin-resistant cell lines have been found to exhibit increased expression of the sulfhydryl-containing proteins, the metallothioneins (Reed, ibid.).
  • the invention provides genetic suppressor elements (GSEs) that are random fragments derived from genes associated with sensitivity to platinum-based drugs, particularly cisplatin, and that confer resistance to platinum-based drugs upon cells expressing such GSEs.
  • GSEs genetic suppressor elements
  • the invention is based in part on the discoveries disclosed in co-pending U.S. Patent Applications, Serial No. 08/033,086, filed March 3, 1993 and Serial No. 008/177,157, filed January 5, 1994, incorporated by reference, providing a method for identifying and isolating GSEs that confer resistance to any chemotherapeutic drug for which resistance is possible.
  • the invention provides a method for identifying GSEs that confer resistance to platinum-based drugs, including cisplatin. This method utilizes cisplatin selection of cells that harbor clones from a random fragment expression library derived from total cDNA derived from cisplatin-sensitive cells, and subsequent rescue of library inserts from drug-resistant cells.
  • the invention provides a method for identifying and cloning genes that are associated with sensitivity to cisplatin, and also provides the genes themselves.
  • This method comprises the steps of screening a full length cDNA library with a GSE that confers upon cells resistance to cisplatin (or, alternatively, with an oligonucleotide or polynucleotide constituting a portion of such a GSE) and determining the nucleotide sequence of the cDNA insert of any positive clones obtained.
  • the technique of "anchored PCR" can be used to isolate cDNAs corresponding to cisplatin resistance-conferring GSEs.
  • isolation of genomic DNA encoding genes associated with sensitivity to platinum-based drugs for example from genomic libraries.
  • the invention provides a method for obtaining GSEs having optimized suppressor activity for a gene associated with sensitivity to platinum-based drugs. This method utilizes drug selection of cells that harbor clones from a random fragment expression library derived from DNA of a gene associated with sensitivity to a platinum-based drug such as cisplatin, and subsequent rescue of the library inserts from drag resistant cells.
  • the invention provides synthetic peptides and oligonucleotides that confer upon cells resistance to, for example, cisplatin. These synthetic peptides and oligonucleotides are designed based upon the sequence of drug-resistance conferring GSEs according to the invention.
  • the invention provides a diagnostic assay for tumor cells that are resistant to platinum-based drugs due to the absence of expression or underexpression of a particular gene.
  • This diagnostic assay comprises quantitating the level of expression of the particular gene product by a particular tumor cell sample to be tested.
  • One feature of this aspect of the invention is the development of antibodies specific for proteins whose underexpression or absence of expression is associated with such resistance to platinum-based drugs. Such antibodies have utility as diagnostic agents for detecting cisplatin resistant tumor cells in tumor samples.
  • the invention provides dominant selectable markers that are useful in gene co-transfer studies. These dominant selectable markers are drag resistance-conferring GSEs according to the invention operably linked to appropriate transcriptional control elements.
  • the invention provides in vivo selectable markers that are useful both for gene therapy and for enhanced chemotherapy for cancer. Such in vivo selectable markers are transferred, for example, into blood progenitor cells, which are then used to repopulate the patient's blood exclusively with cells that contain a co-transferred therapeutic gene, or, for chemotherapeutic purposes, just the resistance-conferring GSE.
  • the invention provides a starting point for in vitro drag screening and rational design of pharmaceutical products that are useful against tumor cells that are resistant to cisplatin and other platinum-based drags. By examining the structure, function, localization and pattern of expression of genes associated with sensitivity to cisplatin, strategies can be developed for creating pharmaceutical products that will overcome drag resistance in tumor cells in which such genes are either not expressed or underexpressed.
  • Also provided by the invention are cultures of mammalian cells which express the cisplatin resistance-conferring GSEs of the invention and are cisplatin resistant thereby. Such cells are useful for determining the physiological and biochemical basis for cisplatin toxicity and resistance in tumor cells. Such cells also have utility in the development of pharmaceutical and chemotherapeutic agents for overcoming such resistance, and thus are ultimately useful in establishing improved chemotherapeutic protocols to more effectively treat neoplastic disease.
  • Figure 1 shows the structure of the adaptor used in cDNA cloning.
  • the nucleotide sequences are shown for the ATG-sense (SEQ.ID.No.:l) and ATG- antisense (SEQ. ID. No. :2) strands of the adaptor.
  • Figure 2 shows the structure of the pLNCX vector used in cDNA cloning.
  • Figure 3 shows a scheme for selection of cisplatin resistance-conferring GSEs from a random fragment expression library (RFEL) from HeLa cell cDNA.
  • RFEL random fragment expression library
  • Figure 4 shows polyacrylamide gel electrophoretic analysis of PCR fragments enriched by selection for cisplatin resistance-conferring GSEs.
  • Figures 5 A through 5G shows resistance to various concentrations of cisplatin, conferred upon cells by the GSEs HL7.1, HL7.2, HL7.4 (Figure 5A), HL7.10, HL7.11, and HL7.12 ( Figure 5B), HL6.1, HL6.10 ( Figure 5C), H63.C8, HL4.1 ( Figure 5D), H91.E2, H91.E2, H93.G6 ( Figure 5E), H62.B2, H62.B3, H62.B5 ( Figure 5F). Clones that scored as inactive in this assay are exemplified by the elements H62.B3 ( Figure 5F) and H63.C7, HL4.4 and HL6.4 ( Figure 5G).
  • Figure 6 shows the nucleotide sequence of the GSE H62. B5 (SEQ ID No . : 3) .
  • Figure 7 shows the nucleotide sequence of the GSE H63.C8 (SEQ ID No.:4).
  • Figure 8 shows the nucleotide sequence of the GSE H91.E2 (SEQ ID No.: 5).
  • Figure 9 shows the nucleotide sequence of the GSE H93.G6 (SEQ ID No.: 6).
  • Figure 10 shows the nucleotide sequence of the GSE HL4.1 (SEQ ID No.: 7).
  • Figure 11 shows the nucleotide sequence of the GSE HL6.1 (SEQ ID No . : 8) .
  • Figure 12 shows the nucleotide sequence of the GSE HL7.2 (SEQ ID No.:9).
  • Figure 13 shows the nucleotide sequence of the GSE HL7.4 (SEQ ID No.: 10).
  • Figure 14 shows the nucleotide sequence of the GSE HL7.12 (SEQ ID No.:ll).
  • Figure 15 shows the nucleotide sequence of the GSE H62.B2 (SEQ ID No. : 12).
  • Figure 16 shows the nucleotide sequence of the GSE HL6.10 (SEQ ID No.: 13).
  • Figure 17 shows the nucleotide sequence of the GSE HL7.1 (SEQ ID NO: 13).
  • Figure 18 shows the nucleotide sequence of the GSE HL7.10 (SEQ ID No. : 15).
  • Figure 19 shows the nucleotide sequence of the GSE HL7.11 (SEQ ID No.: 16).
  • Figure 20 shows a comparison between the nucleotide sequence of GSE HL6.10 (SEQ ID No. : 13) and the XRCC1 gene sequence (SEQ ID No.: 17).
  • Figure 21 shows a comparison between the nucleotide sequence of GSE HL7.1 (SEQ ID No.: 14) and the TRPM2 gene sequence (SEQ ID No.: 18).
  • Figure 22 shows a comparison between the nucleotide sequence of GSE
  • Figure 23 shows a comparison between the nucleotide sequence complementary to that of GSE HL7.11 (SEQ ID No.: 15) and the DHOD gene sequence (SEQ ID No.: 20).
  • Figure 24 shows a comparison between the nucleotide sequence complementary to that of GSE H91.E2 (SEQ ID No.: 17) and the CaMKGB gene sequence (SEQ ID No. :21).
  • Figure 25 shows a comparison between the nucleotide sequence complementary to that of GSE H62.B2 (SEQ ID No.: 12) and the decorin gene sequence (SEQ ID No.:22).
  • Figure 26 shows the structure of the adaptors used for random fragment cDNA cloning described in Example 7 (SEQ ID Nos. 23 and 24).
  • the invention relates to means for suppressing specific gene functions that are associated with sensitivity to platinum-based drags, including cisplatin.
  • the invention provides genetic suppressor elements (GSEs) that have such suppressive effect and thus confer resistance to cisplatin on cells expressing such GSEs.
  • GSEs genetic suppressor elements
  • the invention further provides methods for identifying such GSEs, as well as methods for their use.
  • platinum-based drags is intended to encompass the clinically-approved platinum-based drags, such as cisplatin and carboplatin, as well as any platinum-based drags currently in development or to be developed that act with the same or similar mechanism as cisplatin.
  • the invention provides a method for identifying GSEs that confer resistance to cisplatin.
  • the GSEs identified by this method will be homologous to a gene that is associated with sensitivity to cisplatin.
  • the term "homologous to a gene" has two different meanings, depending on whether the GSE acts through an antisense or antigene mechanism, or through a mechanism of interference at the protein level.
  • a GSE that is an antisense or antigene oligonucleotide or polynucleotide is homologous to a gene if it has a nucleotide sequence that hybridizes under physiological conditions to the gene or its mRNA transcript by Hoogsteen or Watson-Crick base-pairing.
  • a GSE that interferes with a protein molecule is homologous to the gene encoding that protein molecule if it has an amino acid sequence that is the same as that encoded by a portion of the gene encoding the protein, or that would be the same, but for conservative amino acid substitutions.
  • whether the GSE is homologous to a gene is determined by assessing whether the GSE is capable of inhibiting or reducing the function of the gene.
  • the method according to this aspect of the invention comprises the step of screening a total cDNA or genomic DNA random fragment expression library phenotypically to identify clones that confer resistance to cisplatin.
  • the library of random fragments of total cDNA or genomic DNA is cloned into a retroviral expression vector.
  • retrovirus particles containing the library are used to infect cells and the infected cells are tested for their ability to show increased survival in the presence of cisplatin relative to uninfected cells.
  • the inserts in the library will range from about 100 bp to about 700 bp and more preferably, from about 200 bp to about 500 bp.
  • the random fragment library will be a normalized library containing roughly equal numbers of clones corresponding to each gene expressed in the cell type from which it was made, without regard for the level of expression of any gene.
  • normalization of the library is unnecessary for the isolation of GSEs that are homologous to abundantly or moderately expressed genes.
  • the library clone encoding the GSE is rescued from the cells.
  • the insert of the expression library may be tested for its nucleotide sequence.
  • the rescued library clone may be further tested for its ability to confer resistance to cisplatin in additional transfection or infection and selection assays, prior to nucleotide sequence determination. Determination of the nucleotide sequence, of course, results in the identification of the GSE. This method is further illustrated in Examples 1-4.
  • the invention provides a method for identifying and cloning genes that are associated with sensitivity to cisplatin, as well as the genes derived by this method. This is because GSEs, or portions thereof, can be used as probes to screen full length cDNA or genomic libraries to identify their gene of origin. In some cases, genes that are associated with sensitivity to chemotherapeutic drugs such as cisplatin will turn out to be quite surprising.
  • GSEs that abrogate cisplatin include GSEs derived from the following human genes: XRCC1 (X-ray repair cross-complementing- 1); TRPM-2 (testosterone-repressed prostatic message-2); phosphoglycerate mutase, isozyme B, non-muscle isoform; dihydroorotate dehydrogenase; calcium/calmodulin-dependent protein kinase, isoform 7B; and chondroitin/dermatan sulfate proteoglycan core protein (decorin), as well as nine GSEs from previously unidentified human genes.
  • GSEs conferring resistance to cisplatin may be derived from genes involved in programmed cell death.
  • the method according to this aspect of the invention therefore also provides valuable information about the genetic basis for senescence and programmed cell death.
  • GSEs isolated as disclosed herein for the human TRPM-2 gene which is known to be a common marker for programmed cell death (apoptosis) in different tissues (see Example 5 below).
  • the method according to this aspect of the invention and its use for studying genes identified thereby and their cellular effects are further illustrated in Example 6.
  • the invention provides a method for obtaining GSEs having optimized suppressor activity for a gene associated with sensitivity to cisplatin.
  • an initial GSE is obtained by the method according to the first aspect of the invention.
  • the gene from which the GSE is derived is identified and cloned by the method according to the second aspect of the invention.
  • This gene is then randomly fragmented and cloned into an expression vector, preferably a retroviral vector, to obtain a random fragment expression library derived exclusively from the gene of interest.
  • This library is then transferred to and expressed in mammalian cells, which are selected in the presence of cisplatin.
  • the invention provides synthetic peptides and oligonucleotides that are capable of inhibiting the function of genes associated with sensitivity to cisplatin. Synthetic peptides according to the invention have amino acid sequences that correspond to amino acid sequences encoded by GSEs according to the invention.
  • Synthetic oligonucleotides according to the invention have nucleotide sequences corresponding to the nucleotide sequences of GSEs according to the invention. Once a GSE is discovered and sequenced, and its orientation is determined, it is straightforward to prepare an oligonucleotide corresponding to the nucleotide sequence of the GSE (for antisense-oriented GSEs) or amino acid sequence encoded by the GSE (for sense-oriented GSEs). In certain embodiments, such synthetic peptides or oligonucleotides may have the complete sequence encoded by the GSE or may have only part of the sequence present in the GSE, respectively.
  • the peptide or oligonucleotide may have only a portion of the GSE-encoded or GSE sequence.
  • undue experimentation is avoided by the observation that many independent GSE clones corresponding to a particular gene will have the same 5' or 3' terminus, but generally not both. This suggests that many GSE's have one critical endpoint, from which a simple walking experiment will determine the minimum size of peptide or oligonucleotide necessary to inhibit gene function.
  • functional domains as small as 6-8 amino acids have been identified for immunoglobulin binding regions.
  • peptides or peptide mimetics having these or larger dimensions can be prepared as GSEs.
  • oligonucleotides having sufficient length to hybridize to their corresponding mRNA under physiological conditions oligonucleotides having about 12 or more bases will fit this description.
  • oligonucleotides will have from about 12 to about 100 nucleotides.
  • oligonucleotide includes modified oligonucleotides having nuclease-resistant internucleotide linkages, such as phosphorothioate, methylphosphonate, phosphorodithioate, phosphoramidate, phosphotriester, sulfone, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate and bridged phosphorothioate internucleotide linkages.
  • nuclease-resistant internucleotide linkages such as phosphorothioate, methylphosphonate, phosphorodithioate, phosphoramidate, phosphotriester, sulfone, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate and bridged phosphorothio
  • oligonucleotides also includes oligonucleotides having modified bases or modified ribose or deoxyribose sugars.
  • the invention provides a diagnostic assay for tumor cells that are resistant to cisplatin due to absence of expression or underexpression of a particular gene. By using the methods according to the first and second aspects of the invention such a gene is identified and cloned.
  • a first embodiment of a diagnostic assay according to this aspect of the invention utilizes an oligonucleotide or oligonucleotides that is/are homologous to the sequence of the gene for which expression is to be measured.
  • RNA is extracted from a tumor sample, and RNA specific for the gene of interest is quantitated by standard filter hybridization procedures, RNase protection assay, or by quantitative cDNA-PCR (see Noonan et al. , 1990, Proc. Natl Acad. Sci. USA 87: 7160-7164).
  • a diagnostic assay according to this aspect of the invention, antibodies are raised against a synthetic peptide having an amino acid sequence that is identical to a portion of the protein that is encoded by the gene of interest. These antibodies are then used in a conventional quantitative immunoassay (e.g.
  • the invention provides dominant selectable markers that are useful in gene co-transfer studies. Since GSEs according to the invention confer resistance to cisplatin, the presence of a vector that expresses the GSE can readily be selected by growth of a vector-transfected cell in a concentration of cisplatin that would be cytotoxic in the absence of the GSE. GSEs according to the invention are particularly well suited as dominant selectable markers because their small size allows them to be easily incorporated along with a gene to be co-transferred even into viral vectors having limited packaging capacity.
  • the invention provides in vt ' vo-selectable markers that are useful both in gene therapy and in enhancing the effectiveness of chemotherapy.
  • GSEs according to the invention can be co-transferred on a vector into human blood progenitor cells from a patient along with a therapeutic gene that, when expressed, will alleviate a genetic disorder.
  • the cells can be selected in vitro for resistance to cisplatin, thereby assuring successful transfer of the GSE, and by implication, of the therapeutic gene as well.
  • the progenitor cells containing the GSE and therapeutic gene can then be returned to the patient's circulation.
  • the cells containing the GSE and therapeutic gene can also be selected in vivo by administration of cisplatin (to which the GSE confers resistance) in a concentration that is cytotoxic to normal blood cells. In this way, those cells having the GSE and therapeutic gene will repopulate the patient's blood.
  • a GSE according to the invention can be transferred alone or with another gene on an expression vector into blood progenitor cells taken from a cancer patient. The cells are then returned to the patient's circulation and allowed time to begin repopulating the blood.
  • in vitro selection of the progenitor cells harboring the GSE is carried out before re- introduction of the cells into the patient.
  • aggressive chemotherapy with cisplatin or combination chemotherapy using cisplatin and other chemotherapeutic drags can be carried out, using higher than ordinary concentrations of cisplatin (to which the GSE confers resistance), since toxic side effects to the immune system, for example, will be avoided due to GSE expression in those cells.
  • the GSE may be desirable to have the GSE expressed in the progenitor cells (and subsequently in the blood cells), only when its expression is beneficial, i.e. , during in vivo selection or chemotherapy.
  • an inducible promoter can be used to express the GSE.
  • the appropriate inducing agent is added to the cells prior to and during in vitro selection or prior to and during in vivo selection or chemotherapy.
  • the GSE will not be expressed at any other time.
  • the invention provides a starting point for in vitro drag screening and rational design of pharmaceutical products that can counteract resistance by tumor cells to cisplatin.
  • the invention provides cultures of mammalian cells which express the cisplatin resistance-conferring GSEs of the invention and are cisplatin resistant thereby. Included within this aspect of the invention are cell cultures that are representative of almost any tissue or cell type for which cisplatin is normally toxic. Such cells are useful for determining the physiological and biochemical basis for cisplatin toxicity and resistance in tumor cells, as well as for screening pharmaceutical and chemotherapeutic agents for overcoming cisplatin resistance. Identification of such agents would lead to the development of improved chemotherapeutic protocols to more effectively treat neoplastic disease.
  • the protein sequence encoded by genes from which the GSEs were derived can be deduced from the cDNA sequence, and the function of the corresponding proteins may be determined by searching for homology with known genes or by searching for known functional motifs in the protein sequence. If these assays do not indicate the protein function, it can be deduced through the phenotypic effects of the GSEs suppressing the gene. Such effects can be investigated at the cellular level, by analyzing various growth-related, morphological, biochemical or antigenic changes associated with GSE expression. The GSE effects at the organism level can also be studied by introducing the corresponding GSEs as transgenes in transgenic animals
  • the gene function can also be studied by expressing the full-length cDNA of the corresponding gene, rather than a GSE, from a strong promoter in cells or transgenic animals, and studying the changes associated with overexpression of the gene.
  • Full-length or partial cDNA sequences can also be used to direct protein synthesis in a convenient prokaryotic or eukaryotic expression system, and the produced proteins can be used as immunogens to obtain polyclonal or monoclonal antibodies. These antibodies can be used to investigate protein localization and as specific inhibitors of the protein function, as well as for diagnostic purposes.
  • antibodies raised against a synthetic peptide encoded by part of the complement of the sequence of the GSEs HL7.11, H91.E2 or H62.B2, or the corresponding region of the human dihydroorotate dehydrogenase gene, the calcium calmodulin-dependent protein kinase gene or the decorin gene, respectively should be particularly useful, as should antibodies raised against an amino acid sequence encoded by part of the GSEs HL6.10, HL7.1 or HL7.10, or proteins encoded by the X-ray repair cross-complementing- 1 gene, the testosterone-repressed prostatic message-2 gene, or the phosphogly cerate mutase, isozyme B, non-muscle isoform gene, respectively (see Examples 3 and 4 and Figures 15-20).
  • a drag sensitivity gene identified through the GSE approach, would be to insert a full-length cDNA for such a gene into a gene therapy expression vector, for example, a retroviral vector.
  • a gene therapy expression vector for example, a retroviral vector.
  • Such a vector in the form of a recombinant retrovirus, will be delivered to tumor cells in vivo, and, upon integration, would sensitize such cells to the effects of the corresponding chemotherapeutic drag.
  • the selective delivery to tumor cells can be accomplished on the basis of the selectivity of retrovirus-mediated transduction for dividing cells.
  • the selectivity can be achieved by driving the expression of the drag sensitivity gene from a tissue-or tumor-specific promoter, such as, for example, the promoter of the carcinoembryonic antigen gene.
  • the protein structure deduced from the cDNA sequence can also be used for computer-assisted drag design, to develop new drugs that affect this protein in the same manner as the known anticancer drags.
  • the purified protein, produced in a convenient expression system can also be used as the critical component of in vitro biochemical screen systems for new compounds with anticancer activity.
  • mammalian cells that express cisplatin resistance-conferring GSEs according to the invention are useful for screening compounds for the ability to overcome drug resistance associated with down-regulation of the corresponding gene.
  • a normalized cDNA population was prepared using a modification of the protocol described in co-pending U.S. Patent Application Serial No. 08/033,086, filed March 3, 1993, which is incorporated by reference, and as described as follows.
  • Poly(A) + RNA was purified from total RNA extracted from exponentially- growing cultures of human HeLa adenocarcinoma cells (subline S3, obtained under
  • the ligated mixture was then size-fractionated by electrophoresis in a 6% polyacrylamide gel, and fragments ranging in size from approximately 200-600 basepairs (bps) were amplified by PCR, using the "sense" strand of the adaptor as a PCR primer. These PCRs were carried out in 23 separate reactions that were subsequently combined, to minimize random over-or under-amplification of specific sequences and to increase the yield of the product.
  • the cDNA preparation was denatured and reannealed, using the following time-points for reannealing: 0, 24, 48, 72, 96 and 120 hours.
  • the single-stranded and double-stranded DNAs from each reannealed mixture were then separated by hydroxyapatite chromatography.
  • DNA fractions from each time point of reannealing were PCR-amplified using adaptor-derived primers and analyzed by slot blot hybridization with probes corresponding to genes expressed at different levels in human cells, ⁇ -tubulin and c-myc probes were used to represent highly-expressed genes, adenosine deaminase and topoisomerase-II (using separate probes for the 5' and 3' ends of the latter cDNA) probes were used to represent intermediately-expressed genes, and a c-fos probe was used to represent low-level expressed genes. The greatest relative enrichment for low-level expressed genes was found in the single-stranded DNA fraction obtained after 120 hours of reannealing.
  • This fraction was operationally defined as normalized and was re-amplified by PCR.
  • the re-amplified DNA was then purified by gel electrophoresis, digested with Clal, size-fractionated by gel electrophoresis, and 200-600 bp fragments were used for constructing a random-fragment normalized cDNA library.
  • the normalized cDNA preparation was cloned into a Clal site of the MoMLV-based retroviral vector pLNCX, which carries the neo (G418 resistance) gene, expressed under the transcriptional control of the promoter contained in the retroviral long terminal repeat (LTR), and which expresses the cDNA insert sequences from a cytomegalovirus (CMV)-derived promoter (see Figure 2 and Miller and Rosman, 1989, Biotechniques 7: 980-986).
  • pLNCX contains translation termination codons in all three reading frames within 20 bp downstream of the cloning site.
  • this ligation mixture was used to transform a recombination-deficient strain of E. coli (strain inv ⁇ F', available under Accession No. from the ATCC) in 14 separate electroporation experiments, using conventional techniques and standard conditions for electroporation (see Sambrook et al. , ibid.). The results of these experiments are shown in Table I; a total of 2.3 x 10 7 independent clones were obtained. PCR analysis of 269 randomly-picked clones revealed that 70% contained cDNA inserts, thus providing a total library size of 1.6 x 10 7 recombinant clones. Plasmid DNA was isolated from colony-amplified libraries prepared from each of the independent electroporation experiments; the yield of plasmid DNA from each fraction is also shown in Table I.
  • the plasmid library prepared according to Example 1 was used to isolate GSEs inducing cisplatin resistance in human cells in three separate experiments. In each of the experiments, the LNCX vector itself was used in place of the library as a negative control. In the first two of these experiments, DNA from libraries prepared from individual electroporation experiments (Experiments 6 and 9 in Table
  • HeLa cells were plated in six PI 50 culture plates at a density of 1.25 x 10° cells per plate and infected (after overnight growth) by incubation in the presence of 4 ⁇ g/mL polybrene and 25 mL per plate of the filtered virus- containing supernatant from the transfected amphotrophic cells described above. Infection of these HeLa cell cultures was then repeated twice more at 24 hour intervals. Three days after the last infection, the infection efficiency was estimated by plating 10 4 cells in each of three PI 00 culture plates, growing the cells in media containing 600 ⁇ g/mL G418 and determining the percentage of G418-resistant cells that arose from these cultures. Using this assay, 1-2% of the HeLa cells were found to have been infected in the above-described experiments.
  • a 1: 1 mixture of ecotropic and amphotropic packaging cell lines (Markowitz et al, 1988, Virology 167: 400-406), each plated at 1.25 x 10 6 cells per PI 50 plate in a total of 16 culture plates, was transfected with a pooled fraction of the combined plasmid library.
  • Such a pooled population of library DNA was prepared by mixing individual DNA preparations from each of the 14 independent electroporation experiments described in Example 1 at a ratio corresponding to the number of clones in each fraction.
  • the retroviral population was collected as described above and used to infect cells of the cell line HT1080/pJET-2fTGH (clone 2a) (provided by G.
  • HT1080 human fibrosarcoma cells available under Accession No. from ATCC
  • a plasmid capable of expressing the murine ecotropic receptor Albritton et al. , 1989, Cell 57: 659-666
  • HT1080/ER cells were plated at a density of 1.25 x 10 6 cells per plate in sixteen P150 culture plates and infected as described above. Infection efficiency analysis (carried out at a concentration of 400 ⁇ g/mL G418) indicated that 60% of the HT1080/ER cells were infected in this experiment.
  • Proviruses integrated into the genomes of library-transduced cells that survived selection were rescued by fusion of populations of such cells with amphotropic (for the first two experiments described above and in Figure 3) or ecotropic (for the third experiment described above and in Figure 3) virus packaging cells.
  • amphotropic for the first two experiments described above and in Figure 3
  • ecotropic for the third experiment described above and in Figure 3
  • HL1 through HL8 three independent fusions were performed using cells from the second experiment (designated H91, H92 and H93), and eight independent fusions were performed using cells from the third experiment (designated HL1 through HL8).
  • the selected cells were mixed at ratios of 1: 1 or 1:2 with packaging cells.
  • PBS phosphate-buffered saline
  • DMEM serum-free Dulbecco 's Modified Eagle's Medium
  • Retroviral particles in the supernatant of the fiised cell cultures were collected at 24 and 48 hours after fusion and used to infect fresh HeLa cell cultures (for the first two experiments) or HT1080/ER cells (for the third experiment). These infected cells were then selected with G418 and subsequently with cisplatin, using the protocol illustrated in Figure 3. Most of the fusion-derived retroviral populations produced a clear increase in survival in the presence of cisplatin, relative to control cells transduced with insert-free virus.
  • DNA was extracted using conventional techniques from each of the cell populations that were infected with fusion cell-derived retrovirus showing increased cisplatin resistance, as described in Examples 2 and 3.
  • the proviral inserts present in each of the corresponding populations of cell-derived DNAs were recovered by
  • the cloned proviral inserts were first compared to each other on the basis of their electrophoretic mobility in a 6% polyacrylamide gel. Of these, six inserts having different electrophoretic mobilities, and isolated from the first two selection experiments described in Example 2, were chosen for biological activity tests. Each plasmid was transfected into a mixed cell population containing equal numbers of ecotropic and amphotropic packaging cells, and the resulting virus-containing supernatant used to infect HT1080/ER cells. After G418 selection of the transfected cells, each of the transduced cell populations was plated in PI 00 culture plates at a density of 2 x 10 5 cells per plate and grown in the presence of 500-700 ng/mL cisplatin, in triplicate cultures.
  • the remaining 48 inserts were subjected to partial DNA sequence analysis to determine how many different sequences they contained. DNA sequencing reactions were performed on each insert specific for G and T nucleotides, and the pattern of the sequence ladders for each insert compared. As a result of these analyses, 30 of the 48 clones were found to be non-identical.
  • Each of the clones was transfected into a ecotropic virus packaging cell line PE501 (see Miller and Rosman, ibid.), and virus-containing supernatants were collected 24 and 48 hours post-transfection, and were used to infect HT1080/ER cells. Infected cell populations were selected with G418 as described above to obtain populations in which all or almost all of the cells were infected with the retrovirus.
  • PE501 ecotropic virus packaging cell line
  • HL6.10, HL7.1, HL7.2, HL7.4, HL7.10, HL7.11 and HL7.12) produced elevated levels of cisplatin resistance, confirming their identity as cisplatin resistance- conferring GSEs.
  • GSE HL6.10 (SEQ ID No. 13) is a sense-oriented fragment (see Figure 20) of a previously-isolated cDNA encoding the XRCCl gene (SEQ ID No.: 17; X-ray repair cross-complementing- 1; Thompson et al, 1990, Mol. Cell. Biol. 10: 6160-6171). This gene is known to correct defective DNA strand break repair and sister chromatid exchange.
  • DNA damage is known to be a mechanism of cisplatin-mediated cytotoxicity (see Background of the Invention), so that DNA damage repair would be clearly important in cisplatin resistance, the types of DNA damage repaired by XRCCl have not been previously associated with exposure to cisplatin.
  • GSE HL7.1 (SEQ ID No . : 14) is a sense-oriented fragment (see Figure 21) of a previously-isolated cDNA encoding the TRPM-2 gene (SEQ ID No.: 18; testosterone-repressed prostatic message-2, also termed
  • FIG. 26 of a previously-isolated cDNA encoding human calcium/calmodulin (CaM) dependent protein kinase, isoform ⁇ B (CaMKGB, SEQ ID No.:21; Nghiem et al, 1993, J. Biol Chem. 268: 5471-5479).
  • CaM kinase is known to mediate various effects of signal compounds that utilize Ca + + as an intracellular second messenger.
  • CaM kinase antagonists have been shown to potentiate the cytotoxicity of cisplatin, this effect being related to increased intracellular cisplatin accumulation in the presence of the antagonists (Kikuchi et ⁇ /. , 1992, Gynecol. Oncol.
  • FIG 25 of a previously-isolated cDNA encoding human chondroitin/dermatan sulfate proteoglycan core protein PG40 (SEQ ID No.:22; Krasius & Ruoslahti, 1986, Proc. Natl. Acad. Sci. USA 83: 7683-7687), also known as decorin.
  • Decorin is a ubiquitous small proteoglycan and a component of the extracellular matrix, which acts as a specific biological ligand for a number of proteins (see Iozzo & Cohen, 1993, Experientia 49: 447-455).
  • Decorin is known to be capable of high affinity interactions with collagen Type I, Type II and Type VI, fibronectin, and transforming growth factor ⁇ (TGF-jS).
  • Transfection of decorin cDNA into Chinese hamster ovary (CHO) cells has been reported to result in decreased cell density and increased growth inhibition (Yamaguchi & Ruoslahti, 1988, Nature 336: 244-246). These effects are believed to result from the ability of decorin to bind to and neutralize TGF- ⁇ , which is an autocrine growth factor for CHO cells (Yamaguchi et al, 1990, Nature 346:
  • any additional nucleotides encoding amino acids from the amino terminus are then determined from 5 '-specific cDN isolated using the "anchored PCR” technique, as described by Ohara et al. (1989 Proc. Natl. Acad. Sci. USA 86: 5763-5677.) Additional missing 3' termina sequences are also isolated using this technique.
  • the "anchored PCR” technique ca also be used to isolate full-length cDNA starting directly from the GSE sequenc without library screening.
  • cDNAs corresponding to novel and heretofore unidentifie genes associated with cisplatin resistance as described in Example 5 above, as wel as cDNAs corresponding to the six GSEs derived from known genes (CaMKGB decorin, XRCCl, TRPM-2, PGAM-B, and DHOD) are used to isolate and identif additional GSEs, including GSEs having an optimal ability to provide cisplati resistance to human cells.
  • a library of random DNAasel-generated fragments of any particular full length human cDNA is generated essentially as described for topoisomerase II cDN (see Example 1 in co-pending U.S. Patent Application Serial No. 08/033,086 incorporated by reference) and the human oKHCS kinesin gene (in co-pending U.S
  • cDN fragments are amplified by PCR using sense and antisense strands of the first an second adaptor, respectively. PCR products are then digested with Clal and H dI and cloned into the corresponding sites of the pLNCX plasmid.
  • a plasmid library is obtained from this ligation mixture and is transfecte into ecotropic packaging cells using the calcium phosphate precipitation techniqu
  • a plasmid library is obtained from this ligation mixture and is transfected into ecotropic packaging cells using the calcium phosphate precipitation technique.
  • Virus released by transiently transfected cells is used to infect HT1080/ER. After infection and G418 selection, these cells are plated at 10 5 cells per 100 mm plate and cultivated for 3 days in different concentrations of cisplatin (500-700 ng/mL). After removal of the drug, cells are allowed to grow in media without drag for 9-10 more days. At this point, some of the plates are fixed and stained with crystal violet, to determine the number of surviving colonies.
  • DNA is prepared from populations of infected cells, and cisplatin resistance-associated inserts identified by PCR amplification as described above in Example 3. Individual amplified fragments are tested for biological activity after being recloned into the pLNCX vector, and those fragments capable of providing cisplatin resistance to infected human tumor cells are isolated and their nucleotide sequence and expression orientation determined. Relative levels of cisplatin resistance capacity are determined by plating experiments in increasing concentrations of cisplatin, as described above in Example 3 and more generally in co-pending U.S. Patent Application Serial No. 08/033,086, incorporated by reference.
  • GGTGGCATTA CAGGGATGAG GGAGTCACTG TGGGTGGCCA CAGGGCTATG GCCAAGCGTT 120

Abstract

L'invention concerne des éléments génétiques répresseurs qui confèrent à une cellule une résistance aux médicaments à base de platine, y compris le cisplatine, des procédés permettant d'identifier et d'obtenir ces éléments et des procédés permettant de les utiliser. L'invention concerne aussi des gènes clonés associés à la sensibilité au cisplatine.
PCT/US1995/002303 1994-02-22 1995-02-22 Genes et elements genetiques associes a la sensibilite aux medicaments a base de platine WO1995022612A2 (fr)

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US9687641B2 (en) 2010-05-04 2017-06-27 Corium International, Inc. Method and device for transdermal delivery of parathyroid hormone using a microprojection array
US9962534B2 (en) 2013-03-15 2018-05-08 Corium International, Inc. Microarray for delivery of therapeutic agent, methods of use, and methods of making
US10195409B2 (en) 2013-03-15 2019-02-05 Corium International, Inc. Multiple impact microprojection applicators and methods of use
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EP1925677A3 (fr) * 2002-07-04 2008-07-02 Novartis AG Marqueur de gènes
EP1925677A2 (fr) * 2002-07-04 2008-05-28 Novartis AG Marqueur de gènes pour déterminer la toxicité rénale
US10238848B2 (en) 2007-04-16 2019-03-26 Corium International, Inc. Solvent-cast microprotrusion arrays containing active ingredient
US9687641B2 (en) 2010-05-04 2017-06-27 Corium International, Inc. Method and device for transdermal delivery of parathyroid hormone using a microprojection array
US11419816B2 (en) 2010-05-04 2022-08-23 Corium, Inc. Method and device for transdermal delivery of parathyroid hormone using a microprojection array
US11052231B2 (en) 2012-12-21 2021-07-06 Corium, Inc. Microarray for delivery of therapeutic agent and methods of use
US10245422B2 (en) 2013-03-12 2019-04-02 Corium International, Inc. Microprojection applicators and methods of use
US11110259B2 (en) 2013-03-12 2021-09-07 Corium, Inc. Microprojection applicators and methods of use
US9962534B2 (en) 2013-03-15 2018-05-08 Corium International, Inc. Microarray for delivery of therapeutic agent, methods of use, and methods of making
US10384046B2 (en) 2013-03-15 2019-08-20 Corium, Inc. Microarray for delivery of therapeutic agent and methods of use
US10384045B2 (en) 2013-03-15 2019-08-20 Corium, Inc. Microarray with polymer-free microstructures, methods of making, and methods of use
US10195409B2 (en) 2013-03-15 2019-02-05 Corium International, Inc. Multiple impact microprojection applicators and methods of use
US11565097B2 (en) 2013-03-15 2023-01-31 Corium Pharma Solutions, Inc. Microarray for delivery of therapeutic agent and methods of use
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