WO2003006657A2 - Elements de regulation du gene destines a la therapie genique pour la prevention et le diagnostic de metastases ainsi que le traitement des tumeurs par therapie genique - Google Patents

Elements de regulation du gene destines a la therapie genique pour la prevention et le diagnostic de metastases ainsi que le traitement des tumeurs par therapie genique Download PDF

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WO2003006657A2
WO2003006657A2 PCT/EP2002/007857 EP0207857W WO03006657A2 WO 2003006657 A2 WO2003006657 A2 WO 2003006657A2 EP 0207857 W EP0207857 W EP 0207857W WO 03006657 A2 WO03006657 A2 WO 03006657A2
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tissue
promoter
gene
tumor
enhancer
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PCT/EP2002/007857
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German (de)
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WO2003006657A3 (fr
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Karsten Brand
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Custos Biotechnologie Gmbh
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Priority claimed from DE10159128A external-priority patent/DE10159128A1/de
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Priority to AU2002328897A priority Critical patent/AU2002328897A1/en
Publication of WO2003006657A2 publication Critical patent/WO2003006657A2/fr
Publication of WO2003006657A3 publication Critical patent/WO2003006657A3/fr

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    • 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
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • 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
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • 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
    • 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

Definitions

  • Gene regulatory elements for gene therapy for the prevention and diagnosis of metastases and for gene therapy of tumors
  • the present invention relates to a method for producing promoters / enhancers which can be induced by tumor tissue and / or metastases. Furthermore, the present invention relates to promoters / enhancers, the activity of which is regulated by at least one factor from tumor tissue or metastases, and to the use of the promoters / enhancers in diagnostics and cancer therapy.
  • liver metastases of colorectal origin are the leading cause of death for patients with colorectal cancer. Since they are often the only manifestation of the disease over a long period of time after surgical removal of the primary tumor, they are a possible target for curative therapeutic approaches (Dreben, JA and Niederhuber, JE. (1993) Cancer of the lower gastrointestinal tract - colon cancer. In : Niederhuber, JE ed. Current Therapy in Oncology. St. Louis, MO: Decker, 426-431). The potentially curative surgical removal of liver metastases is only possible for a small percentage of the patients and temporary remissions are shown for chemotherapy but no life extension. There is therefore an urgent need for alternative forms of therapy.
  • the method is characterized in that the normal organ tissue is equipped with defense functions directly at the site of a potential or existing metastasis, which prevent the establishment or further growth of the metastases. The further spread of an inoperable primary tumor can also be prevented.
  • This strategy of impregnating healthy tissue differs fundamentally from all gene therapy and non-gene therapy approaches carried out to date.
  • liver metastasis shows that the cells of normal tissue react to the invasion in a variety of ways.
  • the hepatocytes react with vacuole formation, fat storage or massive deformation of the cell shape.
  • Such changes which are to be interpreted as defense reactions or at least reactions to the invasion process, depend on the transcription of the corresponding genes involved in these processes (invasion-induced genes).
  • genes in distant normal tissue also react to tumor activity (genes that can be induced to spread).
  • the invention was based on the problem of providing gene regulatory elements which can be activated by spread or invasion and which make gene therapy of metastases and primary tumors more effective and safer or enable early diagnosis of activated micrometastases. Furthermore, the invention was based on the problem of specifying a method for producing the elements mentioned.
  • enhancers / promoters are used to place antitumor genes such as tumor suppressor genes, suicide genes, angiostatic and antiproteolytic genes under their control. This then enables the activation of these genes in normal tissue at exactly the desired time of active tumor invasion.
  • the promoters / enhancers in question can also be used diagnostically.
  • reporter genes are used as transgenes, their gene products be secreted into the blood when e.g. B.
  • Micrometastases become active and activate the promoters / enhancers in the surrounding normal tissue. In this way, active micrometastases can be identified, which at least would not yet be recognizable with imaging methods, and which accordingly react therapeutically.
  • Viral, liposomal and other non-viral vectors are used as the gene transfer vehicle.
  • a promoter / enhancer which is more active in the invasion front by at least factor 2, preferably by at least factor 3, than in normal tissue further away from the tumor, and by a method for producing a promoter / enhancer which at least one factor is regulated from tumor tissue and / or metastases, and / or the influence of the tumor tissue or metastases on the surrounding normal tissue is regulated either directly or indirectly, comprising the following steps:
  • the tissue for identifying suitable promoters is selected from liver tissue, lung tissue, nerve tissue, bone tissue, lymph nodes and skin.
  • the tissue is a material that has been obtained by means of biopsy.
  • the desired material is obtained by means of microdissection techniques, with laser microdissection being particularly preferred.
  • This technology is described, for example, in M.R. Emmert-Buck et al., Laser capture microdissection. Science 274 (1996), 998-1001.
  • At least two cell layers are isolated for the tissue of type I, the material being removed from the contact area with the tumor in the direction of the normal tissue.
  • Preferably at least three cell layers of material are isolated.
  • at least three cell layers are removed for the type II tissue, but these are located further from the contact area with the tumor than the type I tissue, so that the type II tissue is removed from healthy tissue located further away. At least five cell layers of this type are preferably removed.
  • the removed tissue of type I or type II each comprises approximately 10,000 cells.
  • RNA preferably mRNA
  • RNA is isolated both from the type I tissue and from the type II tissue. This is then analyzed using microarrays. The respective RNA sample is placed on a microarray with known probes and after hybridization under suitable conditions it is determined whether and if so which species from the RNAs of the respective tissue types hybridize with probes of the microarray and thus give a signal.
  • gene expression analysis is, for example, described in detail in M. Noordewier et al., Gene expression microarrays and the Integration of biological knowledge, Trends in Biotechnology 19 (2001), 412-415.
  • the RNA is first amplified again after its isolation, before it is then fed to the analysis described above by means of microarrays.
  • amplification including in vitro transcription and the known PCR techniques. Care must be taken to ensure that the RNA of both tissue types is amplified to the exact same degree, in order to avoid artificial results in the gene expression analysis. A different degree of amplification of RNA of tissue type I compared to the RNA of tissue type II would lead to incorrect conclusions regarding a possible activation of the respective DNA by the tumor tissue or the metastases.
  • the isolated RNA is provided with a label which delivers the measurement signal in the later analysis by means of microarrays.
  • This marker can, for example, coincide with the amplification of the RNA done. Suitable markers for this purpose are well known to the person skilled in the art, as described, for example, in: http://www.affymetrix.com/products/reagents/specific/enzotranscript.affx.
  • the expression patterns of the genes in tissue type I and tissue type II are compared with one another, for which purpose in particular the comparison of the gene expression patterns obtained by means of microarrays is used. Since these gene expression patterns provide not only qualitative, but also at least semi-quantitative information about individual RNA species, a comparison of the patterns obtained with tissue type I and tissue type II enables conclusions to be drawn as to which RNA species is present in tissue of type I in a different concentration than in type II tissue.
  • the difference in the amount of RNA of a particular species should be at least a factor of 3 when comparing tissue type I and tissue type II.
  • Such a difference is highly significant and shows the influence of the tumor tissue, or one or more factors thereof, on the activity of the corresponding gene.
  • genes and their promoters of particular interest are those which are expressed more strongly by a factor of 3 in type I tissue than in type II tissue.
  • the promoter is then identified, characterized and possibly isolated, which promoter is responsible for the changed RNA expression of a particular RNA species.
  • the promoter can be isolated, for example, from genomic banks, the sequence serving as a probe from the microarray, for example, also being used to isolate the complete gene, including the regulatory sequences from a gene bank.
  • the corresponding promoter can also be identified using the bioinformatics in corresponding databases.
  • the present invention also relates to a promoter / enhancer which can be obtained by the process described above and which is regulated by at least one factor from tumor tissue and / or metastases.
  • "Regulated" in this context means that the promoter / enhancer activity changes under the influence of tumor tissue or metastases. The activity of a particular promoter / enhancer can be reduced under the influence of the factor mentioned, while in the case of another promoter / Enhancers the activity is increased.
  • promoters / enhancers in which the activity is increased by at least a factor of 2 when the promoters come to lie in the invasion front compared to their activity in normal tissue which is further away from the tumor tissue or the This activity preferably differs by at least a factor of 3, the activity preferably being increased in the invasion front compared to normal tissue located further away.
  • the promoter / enhancer controls a heat shock gene, an apoptosis inducer gene or a tissue transglutaminase gene, these genes representing the natural environment of the promoter / enhancer.
  • the promoter / enhancer according to the invention can be induced by metabolic stress and / or hypoxia.
  • Metabolic stress includes e.g. following metabolic states: lack of nutrients (especially glucose), lack of removal of toxic metabolites.
  • Hypoxia is characterized, for example, by the presence of reactive oxygen molecules, such as oxyl, peroxyl or hydroxyl radicals or non-radical oxygen molecules such as oxygen, ozone, peroxyl nitrite, by nitrogen oxide, lipid peroxides and others, as well as by the enzymes and other proteins which are responsible for their formation or their degradation and as a defense reaction, such as superoxide dismutase, catalase, NAD (P) H oxidase or glutathione.
  • reactive oxygen molecules such as oxyl, peroxyl or hydroxyl radicals or non-radical oxygen molecules such as oxygen, ozone, peroxyl nitrite, by nitrogen oxide, lipid peroxides and others, as well as by the enzymes and other proteins which are responsible
  • the promoter / enhancer is induced in hepatocytes or copper star cells or sinus endothelial cells when these come into contact with a tumor and / or metastases or when these cells are located in the invasion front.
  • the invasion front encompasses at least 2 Cell positions calculated from the point of contact with tumor tissue into healthy normal tissue.
  • the promoter / enhancer is induced in lung tissue or nerve tissue or bone tissue or lymph nodes or skin when these cell types come into contact with a tumor and / or metastases.
  • the present invention also relates to a vector containing the promoter / enhancer according to the invention.
  • a vector containing the promoter / enhancer according to the invention.
  • Common plasmids, viruses and non-viral vectors are suitable as vectors.
  • the person skilled in the art is familiar with inserting the promoter / enhancer into a vector, cf. e.g. Sambrook, J., Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989).
  • the vector is constructed in such a way that it is used in the context of gene therapy.
  • Suitable gene therapy vector systems are described, for example, in Brand, K., Strauss, M. (1998) Molecular basis of gene transfer and application for gene therapy, in: Handbook of Molecular Medicine, Vol. 2: Tumor Diseases (Ganten, D., Ruckpaul, K., ed.), Springer, Berlin, Heidelberg, New York, pp. 110-145.
  • the vector contains a gene which is under the control of the promoter / enhancer according to the invention. Suitable arrangements are familiar to the person skilled in the art.
  • the gene encodes a reporter molecule and / or an active pharmaceutical ingredient.
  • a reporter molecule is of particular interest in diagnostics. Switching on the promoter / enhancer under the influence of a tumor or metastases leads to the production of a reporter molecule, for example a protein, which either directly or indirectly generates a detectable signal.
  • a reporter molecule for example a protein
  • Such well-known reporter molecules include, for example, the well-known Lac-Z system. More suitable Reporter molecules / detection systems include, for example, the luciferase system, the green fluorescent protein system or the alpha 1 antitrypsin system.
  • the gene encodes a factor that can limit tumor growth, destroy the tumor or protect normal tissue from tumor invasions.
  • the gene codes for a metalloprotease inhibitor, TIMP-1, TIMP-2, TIMP-3, TIMP-4, PAI-1, PAI-2 or a C-terminally shortened TIMP-2, an extracellular matrix protein or a cell adhesion - molecule.
  • a metalloprotease inhibitor TIMP-1, TIMP-2, TIMP-3, TIMP-4, PAI-1, PAI-2 or a C-terminally shortened TIMP-2, an extracellular matrix protein or a cell adhesion - molecule.
  • the factor is a protein which is selected from the polypeptide chains, collagen, fibronectin and laminins, claudins, occludins, cadherins and an integrin.
  • the gene codes for a protein which comprises a membrane anchor sequence or a secretory leader sequence.
  • the gene is a suicide gene.
  • the protein is particularly preferably a cytosine deaminase or nitroreductase.
  • the gene product of the reporter gene is a product which is secreted into the blood or which can be detected in tissue sections.
  • the activation of the promoter, and thus the spread of the tumor or the metastases can be recognized by the fact that the reporter gene product increases in the blood.
  • the protein can be detected, for example, by means of antibodies in tissue sections.
  • the protein can also be an enzyme, the activity of which can then be detected in tissue sections.
  • the vector according to the invention encodes both a reporter molecule and an active pharmaceutical ingredient.
  • the vector is a vector suitable for gene therapy, which is selected in particular from an adenovirus, an adeno-associated virus, a minimal adenovirus, an HSV and lentivirus-based vector.
  • a vector suitable for gene therapy which is selected in particular from an adenovirus, an adeno-associated virus, a minimal adenovirus, an HSV and lentivirus-based vector.
  • Other possible vectors are described, for example, in Brand, K., Strauss, M. (1998) Molecular Foundations of Gene Transfer and Application for Gene Therapy, in: Handbook of Molecular Medicine, Vol. 2: Tumor Diseases (Ganten, D., Ruckpaul, K., ed.), Springer, Berlin, Heidelberg, New York, pp. 110-145.
  • the present invention also relates to a cell, preferably a mammalian cell, which contains a promoter / enhancer according to the invention and / or a vector according to the invention, the promoter or the vector being integrated into the cell's genome at a position which is not its own corresponds to natural position. This is usually the result of the transformation of the cell with a corresponding construct, the construct being randomly integrated into the genome.
  • the present invention also relates to a kit for diagnosing tumors and / or metastases, the kit containing the vector according to the invention together with further reagents, such as, for example, buffer solutions, reagents for the detection of a reporter molecule, and corresponding instructions for use.
  • further reagents such as, for example, buffer solutions, reagents for the detection of a reporter molecule, and corresponding instructions for use.
  • the present invention also relates to a pharmaceutical composition containing the promoter or vector according to the invention or the cell genetically modified with the products mentioned.
  • a pharmaceutical composition usually contains other auxiliaries which are however familiar to the person skilled in the field of galenics.
  • the promoter according to the invention is particularly suitable for producing a medicament for the treatment of cancer.
  • the promoter according to the invention is administered, for example, together with a gene which produces a tumor-toxic substance. After administration of the medicament to the patient, the spreading of a tumor or metastases leads to an activation of the promoter according to the invention, which in turn produces tumor-toxic product which is suitable for inhibiting tumor growth or eliminating the tumor.
  • the promoter according to the invention or the vector containing the promoter is used to diagnose cancer or to monitor the course of cancer therapy. With the means mentioned, it can be tracked, for example, whether tumor spread or metastatic spread can be reduced when cancer therapy is started.
  • an enhancer / promoter is selected which is activated in the normal tissue when the tumor spreads into the surrounding normal tissue.
  • the enhancer / promoter is the enhancer / promoter of the tissue transglutaminase. 5. Means in which the enhancer / promoter can be induced by metabolic stress.
  • Agent containing a modified transgene the antitumor effect of which was enhanced by this modification.
  • Agent containing a transgene of the extracellular matrix 15. Agent containing at least two polypeptide chains of collagen or fibronectin or laminin or genes whose products are responsible for the synthesis of non-protein components of the ECM.
  • Agent containing a modified transgene of the extracellular matrix that it is difficult or not degradable.
  • Agent containing a transgene coding for an adhesion molecule 18.
  • Means in which the adhesion molecule in question is claudin or occludin or a cadherin or an integrin or a gene from the immunoglobulin superfamily is a selectin or a mucin.
  • Agent for the prophylaxis and treatment of tumor diseases containing an antitumor transgene or sequences thereof, which is provided with a membrane anchor sequence.
  • a method for the prophylaxis and treatment of tumor diseases in which an antitumor transgene or sequences thereof, which is provided with a membrane anchor sequence or secretion sequence, is transferred.
  • Agent containing at least 1 transgene, the gene product of which is secreted into the blood such as alpha fetoprotein or alpha 1 antitrypsin.
  • the virus is a first generation adenovirus or an adeno-associated virus or a minimal adenovirus or an HSV or a lentivirus.
  • agent in which the virus is a lentivirus / minimal adenovirus hybrid.
  • agent in which the vector is a non-human mammalian adenovirus.
  • 35 Means in which the vector is not a virus.
  • 36 Agent in which the vector is a liposomal formulation or carrier proteins are used.
  • non-tumorous organ tissue which is adjacent to tumors, is isolated by laser microdissection (LCM), mRNA isolated, amplified by in vitro transcription or unamplified on genomic microarrays (Affimetrix, Santa Clara, California, US). Differential gene expression is determined in comparison to organ tissue not adjacent to the tumor. Promoters of genes that are differentially upregulated in the invasion front are candidate promoters for invasion-regulated gene expression (see example of use).
  • liver metastases are surgically removed
  • Regions containing the invasion front were cut into 1 cm pieces and frozen as described above. 2. Preparation of tissue sections
  • Cryopreserved tissue is placed in a cryotome at an object temperature of -18 ° C in a section thickness of 5 ⁇ m on a slide.
  • the sections are dried for 3-5 minutes at approx. 50 ° C on a hot plate or 1-2 minutes at 80 ° C in the hybridization oven.
  • liver part of the invasion front L1
  • the number of cut cells is approximately 10,000.
  • An equal number of cells which are at least 5 cell layers and further away from the invasion front are isolated as comparative tissue.
  • RNA of the microdissected tissue is isolated using the standard Chomczynski method (Chomczinski and Sacchi, 1987, AnalBiochem, 162, 156).
  • the synthesized cDNA is transcribed in vitro using T7 RNA polymerase, for which a commercially available kit is used (Ambion Megascript T7, Austin,
  • the biotinylation reaction corresponds to another round of amplification.
  • biotinylated UTP nucleotides are used for the labeling (Enzo-Kit, Farmingdale, New York, USA).
  • RNA antisense RNA
  • RNA columns RNeasy kit from Qiagen, Hilden, Germany
  • 35-200 bp fragments to avoid steric interactions due to their size
  • Affymetrix protocol http: //www.affvmetrix .com / products / reagents / specific / enzotranscript.affx .. Affimetrix, Santa Clara, CA, USA).
  • the biotinylated aRNA is applied to microarrays (MG U74Av2, Affimetrix) for hybridization. These are glass supports onto which 25 nucleotide long oligos have been synthesized, which represent a total of over 12,000 mouse genes and ESTs. The hybridization takes place at 45 ° C. in the buffer described by Affimetrix
  • non-amplified mRNA is isolated from the invasion front and the non-invasion front liver. This mRNA is first reverse transcribed and then subjected to a SYBRgreen-based quantitative PCR (Röche, Mannheim, Germany) according to the manufacturer's instructions. Genes whose differential regulation is confirmed in qPCR are candidate genes for invasive gene expression.
  • the promoters of those differentially regulated genes of the invasion front that are found in steps 1-10 are determined in the following manner. a) Promoters that have already been known and characterized are requested from the responsible laboratories and can be integrated into a suitable gene therapy vector without modification. b) Known promoters which are not yet sufficiently characterized for gene therapy use (size limitation, for example viral vectors) are initially characterized using promoter restriction digests and reporter gene assays. Then proceed as described under a).
  • the genomic fragment of promoters that have not yet been cloned is isolated from a genomic library using the corresponding cDNA probe, the transcription start is determined by a primary extension assay, and a sequence up to 8 kB in size, 5 'of the transcription start is analyzed as described under b).
  • Reporter gene assays are carried out for cases b) and c) under a kind of stimulation that simulates the in vivo situation.
  • Such stimuli can e.g. be pro-apoptotic (e.g. transfection of a caspase gene or p53), hypoxic (oxygen deprivation) or anti-metabolic (serum deprivation). If such a stimulus does not lead to promoter activation, a 4 kB fragment is cloned into an adenoviral vector without further in vitro testing and tested according to the application example in vivo.
  • Gene transfer vectors are constructed which contain the HSP 70 promoter or the TG promoter as a promoter / enhancer, the TIMP-2 gene (inhibits tumor spread) as a transgene or either carry the reporter gene ⁇ -galactosidase or ⁇ antitrypsin and a first-generation adenovirus as a vector component to have.
  • colon carcinoma cells eg LS174 T
  • LS174 T colon carcinoma cells
  • recombinant adenoviruses according to item 1 are administered via the tail vein of the animals.
  • a dose of 3x10 10 infectious units about 50% of the liver cells are infected.
  • the concentration of the protein in the serum of animals with tumor induction and transfer of the ⁇ rAntitrypsin adenovirus is several times higher than in the serum of animals which either had only tumor induction or only gene transfer. The concentration will increase with increasing size of the tumor invasion front, i.e. with increasing spread of the tumor.
  • ß-galactosidase adenovirus Similar results are obtained when ß-galactosidase adenovirus is used, except that it is not the serum concentration that is measured here, but the intracellular ß-galactosidase concentration, which is visualized by a histochemical reaction (blue color). Liver cells in the immediate vicinity of spreading tumors are stained more blue than more distant cells. No blue staining is seen in tumor animals without gene transfer. Little or no blue coloration can be seen in gene transfer without tumor induction.
  • TIMP-2 gene as a transgene.
  • Tumor-bearing animals that received a TIMP-2 adenovirus are killed at different times, the tumor volumes are determined and compared with those of animals that received reporter gene viruses.
  • the TIMP-2 animals have significantly smaller tumors.

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Abstract

La présente invention concerne un procédé de production de promoteurs/amplificateurs qui peuvent être induits par des tissus tumoraux et/ou des métastases. Les promoteurs/amplificateurs sont adaptés à une utilisation dans des systèmes vectorettes de thérapie génique, notamment aux fins de traitement de cancers et métastases.
PCT/EP2002/007857 2001-07-13 2002-07-15 Elements de regulation du gene destines a la therapie genique pour la prevention et le diagnostic de metastases ainsi que le traitement des tumeurs par therapie genique WO2003006657A2 (fr)

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DE10133560 2001-07-13
DE10133560.1 2001-07-13
DE10159128.4 2001-12-01
DE10159128A DE10159128A1 (de) 2001-07-13 2001-12-01 Genregulatorische Elemente zur Gentherapie, zur Prävention und Diagnose von Metastasen bzw. zur Gentherapie von Tumoren

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000028092A1 (fr) * 1998-11-05 2000-05-18 Ortho-Mcneil Pharmaceutical, Inc. Methode permettant de generer des profils d'expression genique
WO2000061806A2 (fr) * 1999-04-09 2000-10-19 Arcturus Engineering, Inc. Reseau generique d'adnc ou de proteines destine a des bioanalyses personnalisees
WO2000066176A2 (fr) * 1999-04-30 2000-11-09 Karsten Brand Agent pour la therapie genique et la prevention de metastases ainsi que pour la therapie genique de tumeurs

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000028092A1 (fr) * 1998-11-05 2000-05-18 Ortho-Mcneil Pharmaceutical, Inc. Methode permettant de generer des profils d'expression genique
WO2000061806A2 (fr) * 1999-04-09 2000-10-19 Arcturus Engineering, Inc. Reseau generique d'adnc ou de proteines destine a des bioanalyses personnalisees
WO2000066176A2 (fr) * 1999-04-30 2000-11-09 Karsten Brand Agent pour la therapie genique et la prevention de metastases ainsi que pour la therapie genique de tumeurs

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BRAND, K. ET AL.: "Treatment of Colorectal Liver Metastases by Adenoviral Transfer of Tissue Inhibitor of Metalloproteinases-2 into the Liver Tissue" CANCER RESEARCH, Bd. 60, Nr. 20, 15. Oktober 2000 (2000-10-15), Seiten 5723-5730, XP002235668 *
HAROON, Z.A. ET AL.: "Tissue Transglutaminase Is Expressed as a Host Response to Tumor Invasion and Inhibits Tumor Growth" LABORATORY INVESTIGATION, Bd. 79, Nr. 12, Dezember 1999 (1999-12), Seiten 1679-1686, XP008017379 *
HAYASHI, S. ET AL.: "Two promoters in expression of estrogen receptor messenger RNA in human breast cancer" CARCINOGENESIS, Bd. 18, Nr. 3, M{rz 1997 (1997-03), Seiten 459-464, XP002235667 *
WIDSCHWENDTER, M. ET AL.: "Loss of retinoic acid receptor beta expression in breast cancer and morphologically normal adjacent tissue but not in the normal breast tissue distant from the cancer" EUROPEAN JOURNAL OF CANCER, Bd. 34, Februar 1998 (1998-02), Seite S17 XP004284726 *

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