[go: up one dir, main page]

EP1141362A1 - Gene transfer vector for the diagnosis and therapy of malign tumors - Google Patents

Gene transfer vector for the diagnosis and therapy of malign tumors

Info

Publication number
EP1141362A1
EP1141362A1 EP99968319A EP99968319A EP1141362A1 EP 1141362 A1 EP1141362 A1 EP 1141362A1 EP 99968319 A EP99968319 A EP 99968319A EP 99968319 A EP99968319 A EP 99968319A EP 1141362 A1 EP1141362 A1 EP 1141362A1
Authority
EP
European Patent Office
Prior art keywords
vector according
gene transfer
gene
transfer vector
promoter
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Withdrawn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
EP99968319A
Other languages
German (de)
French (fr)
Inventor
Bernd DÖRKEN
Gerhard Wolff
Hans-Dieter Royer
Christiane Woischwill
Martin Janz
Axel Schumacher
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.)
Dorken Bernd
Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Original Assignee
Dorken Bernd
Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
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 Dorken Bernd, Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft filed Critical Dorken Bernd
Publication of EP1141362A1 publication Critical patent/EP1141362A1/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/86—Viral vectors
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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 new gene transfer vector and its use, in particular for the treatment of chemoresistant tumor cells.
  • Such a resistant phenotype can result from overexpression of a transporter protein.
  • a transporter protein As a transmembrane protein, this so-called P-glycoprotein forms a kind of pump for, among other things. Chemotherapy drugs, which transport them back into the extracellular space.
  • the P-glycoprotein is encoded by the MDR-1 ("multi-drug resistance") gene, which is regulated at the transcriptional level by the YB-1 binding protein by this on the "Y-box" within the DNA sequence of the MDR -1 gene binds (Van Veen and Konings et al, 1997, Sem Cancer Biol, 8, 183-191).
  • the YB-1 promoter controls the expression of the YB-1 protein, which belongs to the family of "Y-box" binding proteins. These Y-box factors belong to a highly conserved class of proteins that play a role in the regulation of transcription and translation. The proteins bind to a sequence within the DNA of a target gene (the so-called Y-box sequence), which leads to the expression of this gene (Bargou et al, 1997, Nat Med, 3, 447-450).
  • YB-1 is increasingly expressed in the context of cell proliferation and can be affected by genotoxic substances, e.g. Chemotherapeutic agents, UV light and ionizing radiation can be induced (Koike et al, 1997, Febs Lett, 417, 390-394). It was also found that the expression of YB-1 is significantly increased in proliferating cells such as embryonic and regenerating tissues, while this state is reversed with tissue differentiation (Grant and Deeley, 1993, Mol Cell Biol, 12, 4186-4196, Spitkovsky et al, 1992, Nucleic Acido Res, 20, 797-803).
  • genotoxic substances e.g. Chemotherapeutic agents, UV light and ionizing radiation can be induced
  • adenoviruses can infect many types of tissue is, on the one hand, an advantage of this gene transfer system. Ordinary adenoviral strategies, on the other hand, are not sufficient for certain diseases / therapies. It is therefore necessary to develop methods in which gene expression is restricted to certain cells only. This can be achieved through tissue-specific promoters. By using e.g. tumor-specific promoters are given the opportunity to express the therapeutic gene only in the tumor tissue and not in adjacent, also infected, normal tissue (Robbins et al, 1998, Trends Biotechnol, 16, 35-40). This increases the accuracy of gene transfer, which makes it possible to use therapeutic genes that are harmful to normal cells.
  • the invention was therefore based on the object of developing a vector with an expression cassette which contains a tumor-specific promoter which expresses a relevant gene in a targeted manner only in chemoresistant tumor cells.
  • This gene therapy vector should therefore be used in tumor cells that are already chemoresistant and therefore no longer respond to conventional chemotherapy.
  • the cassette should be constructed in such a way that it can be inserted into different gene transfer vectors, e.g. can be cloned into adenoviral vectors.
  • it should at the same time be possible to clone the most diverse therapeutic genes "downstream" from the promoter into this cassette without great technical effort.
  • the object is achieved by a vector which has the following constituents: the YB-1 promoter, a transgene and two "multiple cloning sites" (MCS).
  • the tumor-specific YB-1 promoter is said to be incorporated into chemoresistive tumor cells by adenoviral gene transfer Bring transgene to expression.
  • This transgene can be a therapeutically relevant gene, such as an apoptosis-inducing gene, which initiates the death of the tumor cells. It can also be a "prodrug converting enzyme" which converts a certain externally added molecule (“prodrug”) into a pharmacologically active substance which then has its therapeutic effect on the tumor cells.
  • the YB-1 promoter is cloned into a correspondingly adapted MCS of a vector.
  • This is characterized in that it contains a number of selected restriction enzyme interfaces (RES) that allow a new therapeutic gene "downstream" of the promoter to be cloned into the expression cassette without the rest of the vector or the one already in the vector YB-1 promoter changes need to be made.
  • RES restriction enzyme interfaces
  • the new gene transfer vector can be used to treat tumors. It is preferably suitable for the treatment of chemoresistant tumors. Another application is in diagnostics (microlocalization of tumors).
  • the corresponding therapeutic gene is cloned behind the YB-1 promoter (nucleotide 259-294 of the MCS of the pCR2.1 vector from Invitrogen and nucleotide 453-2150 of the YB-1 promoter sequence, Genbank Acc. # X96666).
  • YB-1 promoter is cloned into an MCS of a vector specially adapted for this purpose in such a way that various therapeutic genes can be placed under the control of the promoter without the MCS having to be readjusted.
  • the TP is then transformed together with the "helper plasmid" (HP) into bacteria (BJ cells).
  • the helper plasmid has the entire adenoviral genome except for the El and E3 regions, the El deletion making the virus replication-deficient. Since the adenoviral genome sections that surround the EK in the TP have homology with certain sections of the genome of the HP, a recombinant adenoviral plasmid that contains the expression cassette is generated by homologous recombination in the BJ cells (see Fig. 3).
  • This recombinant adenoviral plasmid is then introduced into a production cell line (293; human, embryonic kidney cell line) by gene transfer techniques, such as calcium phosphate precipitation or liposomes, in order to lead to the production of replication-deficient viruses.
  • a production cell line (293; human, embryonic kidney cell line)
  • gene transfer techniques such as calcium phosphate precipitation or liposomes
  • These contain the therapeutic gene under the control of the tumor-specific YB-1 promoter.
  • tumors of chemoresistant cell lines eg epithelial origin
  • SCID and nude mice mouse strains
  • the measurement of transgene expression by, for example, ELISA and immunohistochemical techniques and its effect on the tumor are then analyzed with regard to a possible therapeutic approach.
  • the invention was checked in an animal model for the proliferation-specific activity of the YB-1 promoter in vivo. It is known that adenoviral vectors drive hepatocytes into proliferation on the third day after vector application. Therefore, two adenoviral vectors with human alpha 1-antitrypsin (hAAT), which differed only in the promoter (AdYB-l.hAAT and AdRSV.hAAT) were compared in a liver transfer model in SCID mice.
  • hAAT human alpha 1-antitrypsin
  • the constitutive promoter led to a continuous increase in the serum content of hAAT (Fig. 4A).
  • the ad vector with the YB-1 promoter led to a temporarily very strong expression with a maximum serum content of hAAT on the third day (FIG. 4B).
  • AdRSV.hAAT AdRSV.hAAT
  • AdYB-l.hAAT AdYB-l.hAAT
  • the serum content of human alpha 1-antitrypsin (hAAT) was determined by ELISA.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Biophysics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Molecular Biology (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Die Erfindung betrifft einen Gentransfervektor für die Diagnostik und die Therapie von malignen Tumoren, der für ein beliebiges Transgen unter Kontrolle des tumorspezifischen YB-1 Promotors kodiert. Er besteht aus dem YB-1 Promotor, einem Transgen und zwei zum Herausschneiden des Transgens geeigneten "Multiple cloning sites" für Restriktionsenzyme, die das Transgen umgeben. In das Konstrukt kann ein beliebiges Gen hineinkloniert werden, um gewebsspezifisch in chemoresistenten Tumorzellen exprimiert zu werden.

Description

Gentransfervektor für die Diagnostik und die Therapie von malignen TumorenGene transfer vector for the diagnosis and therapy of malignant tumors

Die Erfindung betrifft einen neuen Gentransfervektor und seine Verwendung, insbesondere zur Behandlung von chemoresistenten Tumorzellen.The invention relates to a new gene transfer vector and its use, in particular for the treatment of chemoresistant tumor cells.

Bekanntermaßen sind etwa 50% aller Tumoren nicht behandelbar, da diese "multi-drug resistant" sind. So können z.B. Brustkrebszellen entweder primär resistent gegen Chemotherapie sein oder können diese Resistenz nach anfangs erfolgreicher Behandlung in einer späteren Phase entwickeln (sekundäre Therapieresistenz).It is known that around 50% of all tumors cannot be treated because they are "multi-drug resistant". For example, Breast cancer cells are either primarily resistant to chemotherapy or can develop this resistance after successful treatment in the later phase (secondary therapy resistance).

Ein solcher resistenter Phänotyp kann durch die Überexpression eines Transporterproteins zustande kommen. Dieses sogenannte P-Glycoprotein bildet als Transmembranprotein eine Art Pumpe für u.a. Chemotherapeutika, wodurch diese zurück in den extrazellulären Raum transportiert werden. Das P- Glycoprotein wird durch das MDR-1 ("Multi-drug-resistance") Gen kodiert, das auf transkriptioneller Ebene durch das YB-1 Bindungsprotein reguliert wird, indem dieses an der "Y-box" innerhalb der DNA Sequenz des MDR-1 Gens bindet (Van Veen and Konings et al, 1997, Sem Cancer Biol, 8, 183-191).Such a resistant phenotype can result from overexpression of a transporter protein. As a transmembrane protein, this so-called P-glycoprotein forms a kind of pump for, among other things. Chemotherapy drugs, which transport them back into the extracellular space. The P-glycoprotein is encoded by the MDR-1 ("multi-drug resistance") gene, which is regulated at the transcriptional level by the YB-1 binding protein by this on the "Y-box" within the DNA sequence of the MDR -1 gene binds (Van Veen and Konings et al, 1997, Sem Cancer Biol, 8, 183-191).

Der YB-1 Promotor kontrolliert die Expression des YB-1 Proteins, welches zur Familie der " Y-box" Bindungsproteine gehört. Diese Y-box Faktoren gehören einer hoch konservierten Klasse von Proteinen an, die in der Regulation von Transkription und Translation eine Rolle spielen. Die Proteine binden an eine Sequenz innerhalb der DNA eines Zielgens (die sogenannte Y-box Sequenz) wodurch es zur Expression dieses Gens kommt (Bargou et al, 1997, Nat Med, 3, 447-450).The YB-1 promoter controls the expression of the YB-1 protein, which belongs to the family of "Y-box" binding proteins. These Y-box factors belong to a highly conserved class of proteins that play a role in the regulation of transcription and translation. The proteins bind to a sequence within the DNA of a target gene (the so-called Y-box sequence), which leads to the expression of this gene (Bargou et al, 1997, Nat Med, 3, 447-450).

YB-1 ist im Rahmen der Zeilproliferation verstärkt exprimiert und kann durch genotoxische Substanzen, z.B. Chemotherapeutika, UV Licht und ionisierende Strahlen induziert werden (Koike et al, 1997, Febs Lett, 417, 390-394). Außerdem konnte festgestellt werden, daß die Expression von YB-1 in proliferierenden Zellen wie embryonalen und regenerierenden Geweben wesentlich erhöht ist, während dieser Zustand sich bei Gewebedifferenzierung umkehrt (Grant and Deeley, 1993, Mol Cell Biol, 12, 4186-4196, Spitkovsky et al, 1992, Nucleic Acido Res, 20, 797-803).YB-1 is increasingly expressed in the context of cell proliferation and can be affected by genotoxic substances, e.g. Chemotherapeutic agents, UV light and ionizing radiation can be induced (Koike et al, 1997, Febs Lett, 417, 390-394). It was also found that the expression of YB-1 is significantly increased in proliferating cells such as embryonic and regenerating tissues, while this state is reversed with tissue differentiation (Grant and Deeley, 1993, Mol Cell Biol, 12, 4186-4196, Spitkovsky et al, 1992, Nucleic Acido Res, 20, 797-803).

Eigene Studien konnten zeigen, daß die Überexpression des MDR-1 Gens in Brustkrebszellen und die damit zusammenhängende intrinsische Multidrug Resistenz mit der Aktivität und Lokalisation des YB-1 Proteins zusammenhängen (Bargou et al, 1997, Nat Med, 3, 447-450). Bei vorhandener Chemoresistenz ist es also nötig, eine Alternative zum Gebrauch von Chemotherapeutika zu finden. Bekanntermaßen wird Gentherapie für die Behandlung von erworbenen und vererbten Krankheiten eingesetzt, wobei es um den Transfer eines therapeutischen Gens,wie zB. eines Tumorsuppressorgens, geht. Verschiedene Vektorsysteme stehen dabei zur Verfügung, um beim Gentransfer einen möglichst hohen Anteil der Zellen des Zielgewebes zu erreichen. Hierbei sind virale Vektoren bisher am geeignetsten. Adenovirale Vektoren werden zunehmend häufiger eingesetzt, da sie mit einer großen Effektivität eine Anzahl von Tumorgeweben infizieren können. Diese Vektoren enthalten eine jeweils spezifische Expressionskassette (EK), die durch die adenovirale Infektion in die Zielzelle gelangt. Diese Expressionskassette besteht aus einem Promotor und einem therapeutischem Gen, wobei der Promotor für die Expression des Gens in den Zielzellen sorgt. Häufig zum Einsatz kommende Promotoren sind z.B. SV40, RSV und CMV (Sandig et al, 1997, Nat med, 3, 313-319).Our own studies have shown that the overexpression of the MDR-1 gene in breast cancer cells and the associated intrinsic multidrug resistance are related to the activity and location of the YB-1 protein (Bargou et al, 1997, Nat Med, 3, 447-450). If chemoresistance is present, it is therefore necessary to find an alternative to using chemotherapeutic agents. As is known, gene therapy is used for the treatment of acquired and inherited diseases, with the transfer of a therapeutic gene such as, for example. a tumor suppressor morning. Various vector systems are available to achieve the highest possible proportion of cells in the target tissue during gene transfer. So far, viral vectors are the most suitable. Adenoviral vectors are increasingly used because they can infect a number of tumor tissues with great effectiveness. These vectors each contain a specific expression cassette (EK) which reaches the target cell as a result of the adenoviral infection. This expression cassette consists of a promoter and a therapeutic gene, the promoter ensuring the expression of the gene in the target cells. Promoters that are frequently used are, for example, SV40, RSV and CMV (Sandig et al, 1997, Nat med, 3, 313-319).

Die Tatsache, daß Adenoviren viele Gewebstypen infizieren können, ist einerseits ein Vorteil dieses Gentransfersystems. Gewöhnliche adenovirale Strategien sind andererseits bei gewissen Krankheiten/Therapien nicht ausreichend. Es ist also nötig, Verfahren zu entwickeln, bei denen die Genexpression nur auf bestimmte Zellen beschränkt wird. Dies kann durch gewebsspezifische Promotoren erreicht werden. Durch den Einsatz von z.B. tumorspezifischen Promotoren wird die Möglichkeit geschaffen, das therapeutische Gen nur im Tumorgewebe zu exprimieren und nicht in angrenzendem auch infizierten normalen Gewebe (Robbins et al, 1998, Trends Biotechnol, 16, 35-40). Hiermit erhöht sich die Zielgenauigkeit des Gentransfers, wodurch es möglich wird, auch solche therapeutischen Gene einzusetzen, die für die normalen Zellen schädlich sind.The fact that adenoviruses can infect many types of tissue is, on the one hand, an advantage of this gene transfer system. Ordinary adenoviral strategies, on the other hand, are not sufficient for certain diseases / therapies. It is therefore necessary to develop methods in which gene expression is restricted to certain cells only. This can be achieved through tissue-specific promoters. By using e.g. tumor-specific promoters are given the opportunity to express the therapeutic gene only in the tumor tissue and not in adjacent, also infected, normal tissue (Robbins et al, 1998, Trends Biotechnol, 16, 35-40). This increases the accuracy of gene transfer, which makes it possible to use therapeutic genes that are harmful to normal cells.

Der Erfindung lag deshalb die Aufgabe zugrunde, einen Vektor mit einer Expressionskassette zu entwickeln, die einen tumorspezifischen Promotor enthält, der zielgerichtet nur in chemoresistenten Tumorzellen ein relevantes Gen exprimiert. Dieser Gentherapievektor soll also in Tumorzellen zum Einsatz kommen, die schon chemoresistent und damit auf eine konventionelle Chemotherapie nicht mehr ansprechen.The invention was therefore based on the object of developing a vector with an expression cassette which contains a tumor-specific promoter which expresses a relevant gene in a targeted manner only in chemoresistant tumor cells. This gene therapy vector should therefore be used in tumor cells that are already chemoresistant and therefore no longer respond to conventional chemotherapy.

Die Kassette soll zum einen so konstruiert sein, daß sie in verschiedene Gentransfervektoren, wie z.B. in adenovirale Vektoren, hineinkloniert werden kann. Zum anderen soll es gleichzeitig möglich sein, die verschiedensten therapeutischen Gene ohne großen technischen Aufwand "downstream" vom Promotor in diese Kassette hineinzuklonieren.On the one hand, the cassette should be constructed in such a way that it can be inserted into different gene transfer vectors, e.g. can be cloned into adenoviral vectors. On the other hand, it should at the same time be possible to clone the most diverse therapeutic genes "downstream" from the promoter into this cassette without great technical effort.

Die Aufgabe wird gemäß den Patentansprüchen durch einen Vektor gelöst, der die folgenden Bestandteile hat: den YB-1 Promotor, ein Transgen und zwei "Multiple cloning sites" (MCS). Hierbei soll der tumorspezifische YB-1 Promotor in chemoresistenten Tumorzellen durch adenoviralen Gentransfer ein Transgen zur Expression bringen. Dieses Transgen kann ein therapeutisch relevantes, wie z.B. ein Apoptose-induzierendes Gen sein, wodurch ein Absterben der Tumorzellen eingeleitet wird. Es kann auch ein "Prodrug converting enzyme" sein, das ein bestimmtes von außen zugefügtes Molekül ("prodrug") in einen pharmakologisch aktiven Stoff umwandelt, der dann seine therapeutische Wirkung auf die Tumorzellen auswirkt. Weiterhin können auch zwei verschiedene therapeutisch relevante Gene in einem sogenanntem Doppelgentransfer unter die Kontrolle des YB-1 Promotors gestellt werden. Der YB-1 Promotor ist dabei in eine dementsprechend angepasste MCS eines Vektors kloniert. Diese ist dadurch gekennzeichnet, daß sie eine Anzahl ausgewählter Restriktionsenzym-Schnittstellen (RES) enthält, die es zulassen, ein neues therapeutisches Gen "downstream" des Promotors in die Expressionskassette zu klonieren, ohne daß am restlichen Vektor bzw. am sich schon im Vektor befindlichen YB-1 Promotor Veränderungen vorgenommen werden müssen.According to the patent claims, the object is achieved by a vector which has the following constituents: the YB-1 promoter, a transgene and two "multiple cloning sites" (MCS). The tumor-specific YB-1 promoter is said to be incorporated into chemoresistive tumor cells by adenoviral gene transfer Bring transgene to expression. This transgene can be a therapeutically relevant gene, such as an apoptosis-inducing gene, which initiates the death of the tumor cells. It can also be a "prodrug converting enzyme" which converts a certain externally added molecule ("prodrug") into a pharmacologically active substance which then has its therapeutic effect on the tumor cells. Furthermore, two different therapeutically relevant genes can be placed under the control of the YB-1 promoter in a so-called double gene transfer. The YB-1 promoter is cloned into a correspondingly adapted MCS of a vector. This is characterized in that it contains a number of selected restriction enzyme interfaces (RES) that allow a new therapeutic gene "downstream" of the promoter to be cloned into the expression cassette without the rest of the vector or the one already in the vector YB-1 promoter changes need to be made.

Der neue Gentransfervektor ist zur Behandlung von Tumoren einsetzbar. Bevorzugt eignet er sich zur Behandlung von chemoresistenten Tumoren. Eine weitere Anwendungsmöglichkeit besteht in der Diagnostik (Mikrolokalisation von Tumoren).The new gene transfer vector can be used to treat tumors. It is preferably suitable for the treatment of chemoresistant tumors. Another application is in diagnostics (microlocalization of tumors).

AUSFÜHRUNGSBEISPIEL 1EMBODIMENT 1

Zunächst wird das entsprechende therapeutische Gen hinter den YB- 1 Promotor (Nukleotid 259-294 der MCS des pCR2.1 Vektors von Invitrogen und Nukleotid 453-2150 der YB-1 Promotor-Sequenz, Genbank Acc.# X96666) kloniert. Diese beiden Elemente stellen die Expressionskassette dar (siehe Abb. 1). Hierbei wird der YB-1 Promotor so in eine speziell für diese Zwecke angepasste MCS eines Vektors kloniert, daß verschiedene therapeutische Gene unter die Kontrolle des Promotors gesetzt werden können, ohne daß die MCS erneut angepasst werden muß. Dabei handelt es sich um eine MCS, die eine Gruppe speziell ausgewählter RES enthält. Diese Schnittstellen sollen einen schnellen und unkomplizierten Austausch der therapeutischen Gene "downstream" vom Promotor in die Expressionskassette zulassen. Zusätzliche Veränderungen am restlichen Vektor bzw. am schon vorhandenen YB-1 Promotor werden hierdurch vermieden. Es entsteht somit eine Art "Baukastensystem", in dem die therapeutischen Gene unter geringem Aufwand auswechselbar sind (siehe Abb.2). Die den YB-1 Promotor und das therapeutische Gen enthaltende Expressionskassette wird dann in ein sogenanntes Transferplasmid (TP) hineinkloniert. Dieses Plasmid enthält einen Teil des adenoviralen Genoms. Für diesen Schritt werden die RES der MCS genutzt, die die EK umgeben und die auch im Transferplasmid vorhanden sind. Das TP wird dann zusammen mit dem "Helferplasmid" (HP) in Bakterien (BJ Zellen) transformiert. Das Helferplasmid besitzt bis auf die El- und E3-Region das gesamte adenovirale Genom, wobei die El Deletion den Virus replikationsdefizient macht. Da die adenoviralen Genomabschnitte, die die EK im TP umgeben, eine Homologie mit bestimmten Abschnitten des Genoms des HP aufweisen, wird durch homologe Rekombination in den BJ Zellen ein rekombinantes adenovirales Plasmid generiert, das die Expressionskassette enthält (siehe Abb.3). Durch Gentransfertechniken, wie z.B. Calciumphosphat-Präzipitation oder Liposomen, wird dieses rekombinante adenovirale Plasmid dann in eine Produktionszellinie (293; humane, embryonale Nierenzellinie) eingebracht, um zur Produktion von replikationsdefizienten Viren zu fuhren. Diese enthalten das therapeutische Gen unter der Kontrolle des tumorspezifischen YB-1 Promotors. Danach werden in unterschiedlichen Mausstämmen (SCID und nude Mäuse) Tumore chemoresistenter Zellinien (z.B. epithelialen Ursprungs) etabliert, die dann mit dem rekombinanten Virus infiziert werden. Die Messung der Transgenexpression durch z.B. ELISA- und immunhistochemische Techniken und seine Wirkung auf den Tumor werden dann in Hinsicht auf einen möglichen therapeutischen Ansatz analysiert.First, the corresponding therapeutic gene is cloned behind the YB-1 promoter (nucleotide 259-294 of the MCS of the pCR2.1 vector from Invitrogen and nucleotide 453-2150 of the YB-1 promoter sequence, Genbank Acc. # X96666). These two elements represent the expression cassette (see Fig. 1). Here, the YB-1 promoter is cloned into an MCS of a vector specially adapted for this purpose in such a way that various therapeutic genes can be placed under the control of the promoter without the MCS having to be readjusted. This is an MCS that contains a group of specially selected RES. These interfaces are intended to allow a rapid and uncomplicated exchange of the therapeutic genes "downstream" from the promoter into the expression cassette. This avoids additional changes to the remaining vector or to the YB-1 promoter already present. This creates a kind of "modular system" in which the therapeutic genes can be replaced with little effort (see Fig. 2). The expression cassette containing the YB-1 promoter and the therapeutic gene is then cloned into a so-called transfer plasmid (TP). This plasmid contains part of the adenoviral genome. The RES of the MCS that surround the EK and that are also present in the transfer plasmid are used for this step. The TP is then transformed together with the "helper plasmid" (HP) into bacteria (BJ cells). The helper plasmid has the entire adenoviral genome except for the El and E3 regions, the El deletion making the virus replication-deficient. Since the adenoviral genome sections that surround the EK in the TP have homology with certain sections of the genome of the HP, a recombinant adenoviral plasmid that contains the expression cassette is generated by homologous recombination in the BJ cells (see Fig. 3). This recombinant adenoviral plasmid is then introduced into a production cell line (293; human, embryonic kidney cell line) by gene transfer techniques, such as calcium phosphate precipitation or liposomes, in order to lead to the production of replication-deficient viruses. These contain the therapeutic gene under the control of the tumor-specific YB-1 promoter. Thereafter, tumors of chemoresistant cell lines (eg epithelial origin) are established in different mouse strains (SCID and nude mice), which are then infected with the recombinant virus. The measurement of transgene expression by, for example, ELISA and immunohistochemical techniques and its effect on the tumor are then analyzed with regard to a possible therapeutic approach.

AUSFÜHRUNGSBEISPIEL 2EMBODIMENT 2

Die Erfindung wurde in einem Tiermodell auf die proliferationsspezifische Aktivität des YB-1 Promotors in vivo überprüft. Es ist bekannt, daß adenovirale Vektoren Hepatozyten am dritten Tag nach Vektorapplikation in die Proliferation treiben. Daher wurden zwei adenovirale Vektoren mit humanem alpha 1-Antitrypsin (hAAT), die sich nur durch den Promotor unterschieden (AdYB-l.hAAT bzw. AdRSV.hAAT) in einem Lebergentransfermodell in SCID Mäuesen verglichen.The invention was checked in an animal model for the proliferation-specific activity of the YB-1 promoter in vivo. It is known that adenoviral vectors drive hepatocytes into proliferation on the third day after vector application. Therefore, two adenoviral vectors with human alpha 1-antitrypsin (hAAT), which differed only in the promoter (AdYB-l.hAAT and AdRSV.hAAT) were compared in a liver transfer model in SCID mice.

Der konstitutive Promotor führte zu einem kontinuierlichen Anstieg des Serumgehalts von hAAT (Abb. 4A). Im Gegensatz dazu führte der Ad- Vektor mit dem YB-1 Promotor zu einer temporär sehr starken Expression mit einem maximalen Serumgehalt von hAAT am dritten Tag (Abb. 4B).The constitutive promoter led to a continuous increase in the serum content of hAAT (Fig. 4A). In contrast, the ad vector with the YB-1 promoter led to a temporarily very strong expression with a maximum serum content of hAAT on the third day (FIG. 4B).

I,0xl09 pfu AdRSV.hAAT (A) bzw. AdYB-l.hAAT (B) wurde intravenös in SCID Mäuse injiziert (n=3 für A und B). Der Serumgehalt von humanem alpha 1-Antitrypsin (hAAT) wurde durch ELISA ermittelt.I, 0xl0 9 pfu AdRSV.hAAT (A) or AdYB-l.hAAT (B) was injected intravenously into SCID mice injected (n = 3 for A and B). The serum content of human alpha 1-antitrypsin (hAAT) was determined by ELISA.

Hiermit konnte die proliferationsspezifische Aktivität des YB-1 Promotors nachgewiesen werden. This enabled the proliferation-specific activity of the YB-1 promoter to be demonstrated.

Claims

PATENTANSPRÜCHE PATENT CLAIMS 1. Gentransfervektor, bestehend aus1. Gene transfer vector consisting of - dem YB-1 -Promoter, seinen Mutanten oder Deletionsvarianten,- the YB-1 promoter, its mutants or deletion variants, - einem Transgen oder der cDNA eines Transgens- a transgene or the cDNA of a transgene - zwei zum Herausschneiden des Transgens geeigneten Multicloningsites(MCS) für Restriktionsenzyme, die das Transgen umgeben.- Two multicloning sites (MCS) suitable for cutting out the transgene for restriction enzymes which surround the transgene. 2. Gentransfervektor nach Anspruch 1, dadurch gekennzeichnet, daß das Transgen ein therapeutisches Gen ist.2. Gene transfer vector according to claim 1, characterized in that the transgene is a therapeutic gene. 3. Gentransfervektor nach Anspruch 1, dadurch gekennzeichnet, daß das Transgen ein Reportergen ist.3. Gene transfer vector according to claim 1, characterized in that the transgene is a reporter gene. 4. Gentransfervektor nach Anspruch 1 und 2, dadurch gekennzeichnet, daß das therapeutische Gen ein zellzyklusregulierendes oder ein proapoptotisches Gen ist.4. gene transfer vector according to claim 1 and 2, characterized in that the therapeutic gene is a cell cycle regulating or a pro-apoptotic gene. 5. Gentransfervektor nach Anspruch 1, 2 und 4, dadurch gekennzeichnet, daß als therapeutisches Gen p 16, p21, p53 oder Bax eingesetzt wird.5. gene transfer vector according to claim 1, 2 and 4, characterized in that p 16, p21, p53 or Bax is used as therapeutic gene. 6. Gentransfervektor nach Anspruch 1-5, dadurch gekennzeichnet, daß zusätzlich ein regulierendes Element in den Vektor eingebracht wird.6. gene transfer vector according to claim 1-5, characterized in that in addition a regulating element is introduced into the vector. 7. Gentransfervektor nach Anspruch 1-6, dadurch gekennzeichnet, daß die Multicloningsites (MCS) für Restriktionsenzyme mindestens 3 Restriktionsenzymschnittstellen (RES) enthalten.7. Gene transfer vector according to claims 1-6, characterized in that the multicloning sites (MCS) for restriction enzymes contain at least 3 restriction enzyme interfaces (RES). 8. Gentransfervektor nach Anspruch 1-7, dadurch gekennzeichnet, daß die Multicloningsites (MCS) für Restriktionsenzyme 5-10 Restriktionsenzymschnittstellen (RES) enthalten.8. Gene transfer vector according to claims 1-7, characterized in that the multicloning sites (MCS) for restriction enzymes contain 5-10 restriction enzyme interfaces (RES). 9. Gentransfervektor nach Anspruch 1-8, dadurch gekennzeichnet, daß die Multicloningsites (MCS) für Restriktionsenzyme keine Restriktionsenzymschnittstellen (RES) enthalten, die innerhalb der Sequenzen des YB-1 -Promoters vorkommen.9. gene transfer vector according to claim 1-8, characterized in that the multicloning sites (MCS) for restriction enzymes contain no restriction enzyme interfaces (RES) that occur within the sequences of the YB-1 promoter. lO.Gentransfervektor nach Anspruch 1-9, dadurch gekennzeichnet, daß die Multicloningsites (MCS) für Restriktionsenzyme sticky-RES und blunt-RES enthalten.10th gene transfer vector according to claims 1-9, characterized in that the multicloning sites (MCS) for restriction enzymes contain sticky-RES and blunt-RES. 11. Verwendung des Vektors nach Anspruch 1-10 zur Behandlung von Tumoren. 11. Use of the vector according to claims 1-10 for the treatment of tumors. 12. Verwendung des Vektors nach Anspruch 1-10 zur Behandlung von chemoresistenten Tumoren.12. Use of the vector according to claims 1-10 for the treatment of chemoresistant tumors. 13. Verwendung des Vektors nach Anspruch 1-10 zur Behandlung von chemosensitiven Tumoren.13. Use of the vector according to claims 1-10 for the treatment of chemosensitive tumors. 14. Verwendung des Vektors nach Anspruch 1-10 zur Behandlung von Brustkrebs.14. Use of the vector according to claims 1-10 for the treatment of breast cancer. 15. Verwendung des Vektors nach Anspruch 1-10 zur Mikrolokalisation von Tumoren. 15. Use of the vector according to claims 1-10 for the microlocalization of tumors.
EP99968319A 1998-12-29 1999-12-27 Gene transfer vector for the diagnosis and therapy of malign tumors Withdrawn EP1141362A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19860602A DE19860602A1 (en) 1998-12-29 1998-12-29 Gene transfer vector for the diagnosis and therapy of malignant tumors
DE19860602 1998-12-29
PCT/DE1999/004099 WO2000039317A1 (en) 1998-12-29 1999-12-27 Gene transfer vector for the diagnosis and therapy of malign tumors

Publications (1)

Publication Number Publication Date
EP1141362A1 true EP1141362A1 (en) 2001-10-10

Family

ID=7893041

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99968319A Withdrawn EP1141362A1 (en) 1998-12-29 1999-12-27 Gene transfer vector for the diagnosis and therapy of malign tumors

Country Status (3)

Country Link
EP (1) EP1141362A1 (en)
DE (1) DE19860602A1 (en)
WO (1) WO2000039317A1 (en)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2002216500A1 (en) * 2000-11-28 2002-06-11 Genesis Research And Development Corporation Limited Methods for modulating apoptotic cell death
AU2002229679B2 (en) * 2000-12-28 2007-12-20 Per Sonne Holm Use of transcription factor YB-1 in adenoviral systems
CN1655827B (en) 2002-05-27 2010-06-23 佩尔·松内·霍尔姆 Novel use of adenoviruses and nucleic acids coding therefor
WO2005051430A1 (en) 2003-11-14 2005-06-09 Per Sonne Holm Novel use of adenoviruses and nucleic acids that code for said viruses
US20090081639A1 (en) * 2007-05-31 2009-03-26 Phil Hill Assay for sensitivity to chemotherapeutic agents

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4238778C2 (en) * 1992-11-12 1995-11-02 Max Delbrueck Centrum Vector for the expression of therapeutically relevant genes
AU5297496A (en) * 1995-02-17 1996-09-04 Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The Methods of preparation and use of recombinant adenoviral vectors
AU5253998A (en) * 1996-11-13 1998-06-03 Board Of Regents, The University Of Texas System Diminishing viral gene expression by promoter replacement

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0039317A1 *

Also Published As

Publication number Publication date
WO2000039317A1 (en) 2000-07-06
DE19860602A1 (en) 2000-07-06

Similar Documents

Publication Publication Date Title
DE69518910T3 (en) METHOD OF GENERATING VIRAL VECTORS AT LEAST 20KB BY INTERMOLECULAR HOMOLOGOUS RECOMBINATION IN A PROKARYOTIC CELL
DE69434781T2 (en) DEFECTIVE RECOMBINANT ADENOVIRES FOR TUMOR GENE THERAPY
DE69330774T2 (en) CYTOKIN EXPECTING DEFECTIVE RECOMBINANT ADENOVIRUS IN USE IN ANTITUMORAL TREATMENT
DE69834936T2 (en) VECTOR FOR TISSUE-SPECIFIC REPLICATION AND EXPRESSION
DE69230993T3 (en) RECOMBINANT VIRUS VECTORS FOR EXPRESSION IN MUSCLE CELLS
DE69616559T2 (en) AUXILIARY VIRUS FOR THE PRODUCTION OF RECOMBINANT VIRUS VECTORS
DE69636863T2 (en) USE OF IMMUNOMODULATORS FOR THE MANUFACTURE OF MEDICAMENTS FOR INHIBITING AN IMMUNE RESPONSE AGAINST SIMULTANEOUSLY RECOMBINANT VIRUSES
DE69733035T2 (en) CHIMERIC RETROVIRUS / ADENOVIRUS SYSTEM
DE60037189T2 (en) RECOMBINANT, SODIUM JODID SYMPORTER-CODING ADENOVIRES
DE69725882T2 (en) ADENOVIRUS E4 PROTEINS FOR INDUCTION OF CELL DEATH
EP1185279B1 (en) Agents for treating malignant diseases using e1a-deficient adenoviruses with yb-1 protein-dependent replication
EP1141362A1 (en) Gene transfer vector for the diagnosis and therapy of malign tumors
WO1996033272A1 (en) Vectors for the transfection of eucaryotic cells, their use and target cells transfected thereby
EP1436403B1 (en) Use of the adenoviral e2 late promoter
EP1346058B1 (en) Adenoviral systems and the uses thereof
EP1097234B1 (en) Method for improving the gene transfer of genetic material in mammalian cells through the use of p21 (waf-1)
DE19807265C2 (en) Adenoviral transfer vector for the gene transport of a DNA sequence
DE10039844A1 (en) New vector useful in the treatment of malignant disease and other hyperplasia
WO2002096468A2 (en) Method for the diagnosis and therapy of renal cell carcinoma
DE10109780A1 (en) Episomal vectors containing matrix attachment regions for cell-specific gene expression
DE10150945A1 (en) Adenoviral nucleic acid useful for preventing or treating malignant or benign tumors, by inducing cell lysis, comprises a sequence for the YB-1 protein
DE10055383A1 (en) Use of the human LRP / MVP promoter for a therapy-inducible vector
DE4436665C2 (en) Provision of rep-negative AAV mutants and cells that can be used for this
DE10048950A1 (en) Activating p53-responsive promoter, useful for treating tumors that contain mutant forms of p53, using an adaptor protein that links the promoter to mutated p53
DE10065504A1 (en) Adenoviral nucleic acid useful for preventing or treating malignant or benign tumors, by inducing cell lysis, comprises a sequence for the YB-1 protein

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20010718

AK Designated contracting states

Kind code of ref document: A1

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

17Q First examination report despatched

Effective date: 20040802

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20050804