EP0954590A1 - Hex ii tumorspezifischer promotor und dessen anwendung für die krebstherapie - Google Patents

Hex ii tumorspezifischer promotor und dessen anwendung für die krebstherapie

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Publication number
EP0954590A1
EP0954590A1 EP97918865A EP97918865A EP0954590A1 EP 0954590 A1 EP0954590 A1 EP 0954590A1 EP 97918865 A EP97918865 A EP 97918865A EP 97918865 A EP97918865 A EP 97918865A EP 0954590 A1 EP0954590 A1 EP 0954590A1
Authority
EP
European Patent Office
Prior art keywords
promoter
gene
hex
tumor
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97918865A
Other languages
English (en)
French (fr)
Inventor
Gerald Batist
Maha Katabi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
McGill University
Original Assignee
McGill University
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 McGill University filed Critical McGill University
Publication of EP0954590A1 publication Critical patent/EP0954590A1/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the invention relates to a novel tumor-specific promoter for use in gene targeted therapy that is differentially regulated in cancer cells, such as to drive a suicide gene in cancer therapy.
  • a successful gene therapy approach is dependent upon two parameters: 1) efficiency of target cells transduction and 2) specificity of gene delivery.
  • Selective targeting is especially critical in the context of cancer therapy for gene directed enzyme prodrug therapy (GDEPT), where a suicide gene expressed in tumor cells encodes an enzyme that converts an otherwise non-toxic prodrug into its active form.
  • GDEPT gene directed enzyme prodrug therapy
  • Several methods have been explored to increase the specificity. They can be broadly divided into two categories: directed delivery of the gene of interest or its directed expression.
  • the ideal candidate for transcriptional targeting would be a tumor specific promoter and/or enhancer and its activation will be strong enough to achieve therapeutic levels of the desired transcript.
  • a wide range of promoters have been explored in this context. They were mostly characterized as tissue specific promoters as opposed to tumor selective.
  • Some examples are: surfactant protein SP-A promoter for non small cell lung carcinoma (NSCLC), immunoglobulin enhancer or 0 enhancer for B-cell lineage cancers, tyrosinase for melanomas, and MUC-l/Df3 for breast cancer.
  • NSCLC non small cell lung carcinoma
  • tyrosinase for melanomas
  • MUC-l/Df3 for breast cancer.
  • these promoters also direct gene expression in the normal tissue of origin of these neoplasms and other critical organs as well.
  • the erbB2 and a-fetoprotein promoters are activated to a greater extent in certain neoplasms. They have also been used in this strategy and have lead to promising results. Nonetheless, other promoters to further improve and optimize this strategy are needed.
  • Hexokinase ATP: D-hexose-6-phosphotransferase
  • HK Hexokinases
  • HK I to HK IV They exist in four molecular forms, HK I to HK IV, with distinct electrophoretic and kinetic properties (Wilson, J.E., (1985) In Regula tion of Carbohydra te Metabolism, Vol I, 45-85, CRC Press, Boca Raton).
  • the profile of these enzymes in tissues at different stages of malignancies shows an increase in HK II in tumor versus normal tis- sues.
  • rats the type I HK is expressed in brain, kidney and heart.
  • the type II HK was found in skeletal muscle and in AH130 hepatoma cells. In normal liver it is type IV HK that is most abundant (Mathupala, S.P., Rempel, A., and Pedersen, P.L. (1995) J. Biol . Chem. 270, 16918-16925).
  • One aim of the present invention is to provide a novel tumor-specific promoter for use in gene targeted therapy that is differentially regulated in cancer cells, such as to drive a suicide gene in cancer therapy.
  • a tumor-specific promoter for use in gene targeted therapy that is differentially regulated in cancer cells, which comprises Hex II reporter gene.
  • Hex II gene construct which comprises Hex II promoter in a vector selected from pCAT basic expression vector p ⁇ ElsplB and a shuttle plasmid.
  • the gene construct further comprises ⁇ -gal or HSV Tk.
  • the preferred gene construct based on pCAT vector is pHexIl4557-CAT.
  • the preferred gene constructs based on p ⁇ ElsplB are p ⁇ ElsplBHex-LacZ and p ⁇ ElsplBHex-TK.
  • Fig. 1 illustrates the Hex II reporter gene construct in pCAT basic expression vector in accordance with the present invention
  • Fig. 2 illustrates the Hex II promoter construct including ⁇ -galactosidase in the shuttle plasmid p ⁇ ElsplB in accordance with the present invention
  • Fig. 3 illustrates the Hex II promoter construct including HSV Tk in the shuttle plasmid p ⁇ ElsplB in accordance with the present invention
  • Fig. 4 illustrates a graph of the results of MUC-1 versus HexII promoters activation in normal bronchial and mammary epithelial cells
  • Fig. 5 illustrates a graph of the results of HexII promoter activation in normal bronchial epithelial cells versus non-small cell lung carcinomas.
  • the 3.74 kb lacZ gene (Hindlll-Sall ) from pSV2- ⁇ -galactosidase was cloned into the Hindlll and Sail polycloning sites of the shuttle vector p ⁇ ElsplB.
  • This shuttle plasmid contains Adenovirus 5 (Ad5) sequences from map unit 0 to 1, followed by the polycloning site, followed by Ad5 sequences from mu 9.8 to 15.8, and therefore allows recombination to take place with the adenoviral genome.
  • the Hex II promoter 4557 bp was released from the pHexII 4557/CAT with Xbal followed by an EcoRI digest and cloned into the Xbal site of the p ⁇ ElsplB.
  • Clone 10 p ⁇ ElsplBHexII
  • p ⁇ ElsplBLacZ was digested with Xhol followed with a partial digest with EcoRI.
  • p ⁇ ElsplBHexII was in turn digested with Xhol and EcoRI, and the purified 4.6 kb fragment was ligated into p ⁇ ElsplBLacZ.
  • tissue or tumor selective promoters in targeted gene therapy for cancer depends on strong promoters with specific activity.
  • the Muc-1/Df3 promoter has been used in the context of gene directed enzyme prodrug therapy (GDEPT) (Chen et al (1995) J. Clin . Invest . 96(6), 2775). However we have found that it has limited promoter activity and appears to be expressed in a wide range of normal cells (Fig. 4).
  • GDEPT gene directed enzyme prodrug therapy
  • Hexokinase type II catalyzes the first committed step of glycolysis and has been linked to this phenotype since it is overexpressed in tumors and is not responsive to the normal physiological inhibitors, e.g. glucagon (Mathupala, S.P., Rempel, A., and Pedersen, P.L. (1995) J. Biol . Chem . 270, 16918-16925).
  • the tumor HK II promoter was tested in variety of human tumor cell lines and in normal human cells. We studied the Hex II promoter by transfecting cells with the pHex II4557/CAT (Fig. 1) construct and performing a chloramphenicol acetyl transferase (CAT) reporter gene assay.
  • CAT chloramphenicol acetyl transferase
  • Transient transfections were performed using lipofectamine according to the manufacturer's recommendations (GIBCO-BRL). Cells were plated the day before transfection to give 60% confluency in 6-well plates. The pl583/+33MUCl.CAT or pHex4557.CAT vectors were transfected along with pSV2lacZ to determine promoter activity. 1 ug of each plasmid were used for each well. All conditions assayed were done in duplicate. The plasmids pRSV.CAT and promoterless pCAT were used as positive and negative controls, respectively. Cells extracts were prepared 48 hours after transfection and ⁇ -galactosidase activity was assayed to compensate for variations in transfection efficiency. CAT activity was determined from 75-100 ug of proteins. The reaction was carried out with 0.1 uCi of - ⁇ C-labeled chlo- ramphenicol in a 100 ul reaction at 37°C for 4 hrs .
  • the efficacy of this promoter in the context of GDEPT was tested by using the herpes thy id- ine kinase gene in combination with the prodrug gancyclovir .
  • the following suicide genes may be used in accordance with the Hex II promoter constructs of the present invention: Cytochrome P-450TM 2B1 with cyclo- phosphamide, penicillin, amidase and ⁇ -lactamase.
  • Cell survival was determined using a colorimet- ric assay which measures the ability of viable cells to reduce a soluble yellow tetrazolium salt (MTT) to an insoluble purple formazan precipitate.
  • MTT soluble yellow tetrazolium salt
  • Cells in the logarithmic phase of growth were resuspended at a concentration of 2x10 ⁇ cells/ml. 2ml/well were plated in 6-well plates. Plates were incubated for 24 h at 37 °C in 5% C02- Subsequently, cells were transfected with the p ⁇ ElsplB Hex TK plasmid as described above. 6 h after the transfection, cells were treated with the drug gancyclovir at concentrations of 10 or 25 ug/ml. Each condition was done in triplicate.
  • Controls are cells transfected with the plasmid alone or treated with the drug alone.
  • MTT assays was performed two days following treatment. The formazan crystals were dissolved in dimethyl sulfide (Fisher) and glycine buffer (0.1 M glycine- 0.1 M NaCl, pH 10.5). The formazan product formed by viable cells was quantitated by measuring the absorbance at a wavelength of 570 nm.
  • Hex II promoter with or without the metabolic manipulation of the normally express enzyme in muscle using glucagon will provide and important degree of selectivity to the anti-tumor effect. This represents a novel use of selective promoter, taking advantage of its abnormal regulation in tumor cells.
  • the p ⁇ ElspIBHex-LacZ may be used in tumor bearing rats for the in vivo localization of the suicide gene in pre-clinical testing of this novel targeting strategy.
  • the gene construct is going to be administered in adenovirus type 5 recombinant vector or in lipid-based delivery system.
  • Recombinant, replication deficient adenoviral vectors derived from type 5 adenovirus are constructed by the homologous recombination method in the human embryonic kidney cell line 293.
  • the recombinant shuttle plas ids and pBHGll, containing the adenoviral genome are co-transfected by calcium phosphate precipitation in 293 cells.
  • the viral DNA is isolated from a single plaque and analyzed by restriction enzyme digestion.
  • Recombinant adenovirus is expanded from a single plaque in 293 cells. Large scale production of the recombinant adenovirus is accomplished by growth in 293 spinner cells and purification by double cesium chloride gradient.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
EP97918865A 1996-09-25 1997-09-22 Hex ii tumorspezifischer promotor und dessen anwendung für die krebstherapie Withdrawn EP0954590A1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US2667896P 1996-09-25 1996-09-25
US26678P 1996-09-25
PCT/CA1997/000691 WO1998013507A1 (en) 1996-09-25 1997-09-22 Hex ii tumor-specific promoter and uses thereof in cancer therapy

Publications (1)

Publication Number Publication Date
EP0954590A1 true EP0954590A1 (de) 1999-11-10

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EP97918865A Withdrawn EP0954590A1 (de) 1996-09-25 1997-09-22 Hex ii tumorspezifischer promotor und dessen anwendung für die krebstherapie

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EP (1) EP0954590A1 (de)
AU (1) AU4292797A (de)
CA (1) CA2266846A1 (de)
WO (1) WO1998013507A1 (de)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1130106A1 (de) * 2000-03-01 2001-09-05 Rijksuniversiteit te Groningen Epithelium-spezifische Transkription in Nicht-Schuppenzellkarzinomen
JPWO2003102186A1 (ja) * 2002-05-31 2005-09-29 株式会社メディネット 癌細胞特異的発現誘導ｄｎａ、および癌細胞特異的発現ベクター

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Publication number Priority date Publication date Assignee Title
AU6676396A (en) * 1995-07-14 1997-02-18 Johns Hopkins University, The Tumor type ii hexokinase transcription regulatory regions

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9813507A1 *

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Publication number Publication date
CA2266846A1 (en) 1998-04-02
WO1998013507A1 (en) 1998-04-02
AU4292797A (en) 1998-04-17

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