EP1066221A2 - Vectors and viruses used in gene therapy - Google Patents
Vectors and viruses used in gene therapyInfo
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- EP1066221A2 EP1066221A2 EP99919057A EP99919057A EP1066221A2 EP 1066221 A2 EP1066221 A2 EP 1066221A2 EP 99919057 A EP99919057 A EP 99919057A EP 99919057 A EP99919057 A EP 99919057A EP 1066221 A2 EP1066221 A2 EP 1066221A2
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P35/00—Antineoplastic agents
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- 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
Definitions
- the present invention relates to vectors and viruses which are suitable for gene therapy, processes for their preparation and their use.
- a common tumor therapy includes the most complete surgical removal of the tumor and the treatment of the patient by means of radiation and / or systemic or regional application of cytostatics (chemotherapy). This radiation and / or chemotherapy can be carried out before or after surgical removal of the tumor and in some cases makes it more inoperable
- Tumors are often the only therapeutic option. Radiation and / or chemotherapy attempts to kill any tumor tissue that is still present or metastases that have formed. These therapy modalities induce DNA damage / inhibition of DNA synthesis in the tumor cells and are intended to kill the tumor cells. Because of strong side effects on healthy
- Tissue is given the total dose planned for the therapy, usually fractionally over weeks and months. In those tumor cells that survive such a cytotoxic therapy shock, however, this treatment can lead to genomic instability or exacerbate it. Genomic instability means that structural and numerical chromosome aberrations, gene rearrangements, complete or partial gene deletions, gene amplifications, point mutations etc. occur. Of course, these genetic changes can influence the gene expression of the cell to a considerable extent by overexpressing, underexpressing or deregulated expression of normal proteins or expressing abnormal proteins
- genomic instability also occurs in cells that are still considered healthy (“normal cells”) and may even be seen as the driving force behind tumor development. This is supported by the following facts: a) There are a number of different, monogenic hereditary disorders, the common feature of which is increased genomic instability in normal cells even in young subjects (e.g. Bloom's syndrome, Werner syndrome, etc.). These syndromes are all associated with an increased risk of cancer. b) Chemical and physical carcinogens can cause genomic instability, which can be seen as the use of "error-producing" DNA repair systems due to the overloading of initially activated "error-free” repair systems. Certain tumor virus gene products can also cause genomic instability in host cells.
- the present invention is therefore based on the object of providing a means with which conventional tumor therapies can be improved, prophylaxis carried out against tumor development and development and, in particular, the generation of genomic instability in tumor cells and normal cells can be avoided.
- a vector suitable for gene therapy which comprises an expressible insert DNA which codes for the essentially complete genetic information of the poly (ADP-ribose) polymerase (hereinafter referred to as PARP).
- PARP is a highly conserved nuclear enzyme in higher eukaryotes, which catalyzes the covalent modification of nuclear proteins with poly (ADP-ribose) in the presence of DNA strand breaks. This reaction contributes to efficient base excision repair and the recovery of proliferating cells from the cytotoxic effects of DNA damage from alkylating agents,
- Oxidants or ionizing radiation This finding has so far been used to prevent cellular poly (ADP-ribose) production in tumor cells by inhibiting PARP, as a result of which a sensitization of the cells to harmful agents has been found and the tumor cells should die.
- This therapeutic approach is for example in
- an expressible insert DNA which codes for the DNA binding domain of PARP or an at least partially catalytically inactive PARP is inserted into a vector suitable for gene therapy and introduced into tumor cells in order to inhibit the PARP present there in a trans-dominant manner , which drastically reduces the rate of repair of DNA damage and the tumor cell is very likely to die.
- the present invention is now based on the additional finding that to avoid genomic instability in tumor cells it is not PARP that is to be inhibited, but rather that PARP must be overexpressed in tumor cells. This finding is of course based on
- the foregoing "gene therapy vector” includes any vector that can be used alone or with other agents in gene therapy. These are, for example, plasmid vectors and virus vectors, which can be integrating or non-integrating vector systems.
- Virus vectors are in particular those of adenovirus, herpes simplex virus, adeno-associated virus (hereinafter referred to as AAV), "minute virus of mice” (hereinafter referred to as MVM) and retroviruses.
- Virus vectors from AAV for example AAV-sub201 (cf. Samulski, RJ et al., J. Virology 61, (1 987), 3096-3101), from MVM z. PSR2 (see Russell, SJ et al., J.
- Non-viral vector systems can also be used, such as, for example, complexing the foreign DNA with a GAL4 / invasin fusion protein (Paul et al., Gene Ther. 8, pp. 1 253-1 262 (1 997) ) or with peptides (Hart et al., Gene Ther. 2, pp. 552-554 (1 995); Gottschalk et al., Gene Ther. 3, pp. 48-57 (1 996)); improved lipid-based vectors (eg DNA association with cationic liposomes; DNA packaging in neutral or anionic liposomes; liposome-packed, polycation-condensed DNA;
- an insert DNA which codes for the essentially complete genetic information of PARP is inserted into a vector above.
- essentially complete genetic information means that insertions, deletions, base changes or modifications may have taken place, but which do not change the function of the PARP.
- Such a functional PARP must be catalytically active and must be able to be activated by DNA strand breaks.
- An insert DNA above can come from any organism, for example from humans or animals or plants.
- An insert DNA from humans, in particular human cDNA and particularly preferably that from FIG. 1 or a DNA different therefrom by one or more nucleotides is preferably used.
- DNA different from it by one or more nucleotides means that the function of the PARP has been retained despite base changes. This also includes allelic variants.
- the above insert DNA is inserted into the vector in such a way that the insert DNA can be expressed. This can be achieved by using the insert
- DNA is inserted into an expression unit in phase in the vector.
- elements of the existing expression unit such as enhancers, promoters or polyadenylation signals, with others.
- a promoter which is specific for a tissue type is preferably introduced into an expression unit, as a result of which the expression of the insert DNA which is under the control of the promoter becomes tissue-specific.
- a promoter that is active in tumor cells is particularly preferred.
- An example of such a promoter is the MVM P4 promoter (see Russell, S.J. et al., Supra).
- the expression of the above insert DNA can also be achieved in an expression unit which has to be introduced into the vector for this purpose.
- the above statements also apply to this expression unit.
- virus vectors In the case of virus vectors, it often proves advantageous to insert the insert DNA into an expression unit present in the vector.
- the associated removal or partial removal of virus DNA present in the expression unit then leads to a virus vector which has a defect in a virus function.
- This defect can be used as a selection marker.
- the defect can be compensated for by conventional methods, such as complementation in trans.
- virus vectors are preferred in which the insert DNA is inserted in such a way that the virus vectors alone are no longer able to form the viruses encoded by them.
- Vectors preferred according to the invention are shown in FIG. 3.
- viruses encoded by the virus vectors can also be formed by conventional complementation methods.
- an AAV rep " vector containing the PARP insert is transfected into cells which are co-transfected at the same time with a DNA construct expressing the rep gene. Viruses are obtained. These are well suited for gene therapy since they do not multiply in the patient can.
- the virus encoded by the virus vector is obtained. This is also very suitable for gene therapy.
- the present invention thus also relates to viruses which are encoded by the above virus vectors.
- Vectors and viruses according to the invention are distinguished by the fact that they are identical to each other
- tumor cells treated with the vectors and viruses according to the invention show an increased tendency to die. It is particularly important here that the viruses and vectors according to the invention can be tissue-specific (tumor) -active.
- the efficiency of radiation and chemotherapy can thus be increased in an excellent way.
- the tumor cells are systematically or intratumorally applied to the gene therapy before the planned radiation or chemotherapy. transduced tors.
- ADP ribosyDation as a cellular response to DNA damage caused by radiation or chemotherapy
- the phenomenon of "genomic instability" is specifically inhibited in those tumor cells that survive the therapy shock. As expected, there is no further Tumor cell
- 1 shows the nucleotide sequence of the cDNA of human poly (ADP-ribose) polymerase
- Example 1 Preparation of the vector AAV r ⁇ p / cap -PARP according to the invention
- the vector AAV-sub201 (Samulski, R.J. et al., Supra) is assumed. This vector is cleaved with the restriction enzyme Xbal and all AAV components except the inverted terminal repetitions are removed. The ends of the vector fragment are "smoothed".
- An insert, P4-PARP-poly A, is inserted into this, which comprises the following sequences from 5 'to 3', each with smoothed ends: (I) a 259 bp BamHI / Ncol- Fragment representing the
- P4 promoter This fragment originates, for example, from plasmid pEG61 8 (cf. Astell, C. et al., J. Virology 57, (1 986), 656-669); (II) a 3.6 kb Smal / Hindlll fragment from pPARP31 generated by partial digest (cf. van Gool et al., Eur. J. Biochem. 244 (1 997), 1 5-20), which both 3.0 kb open reader frame as well as the 630 bp HSV thymidine kinase poly A signal.
- the vector AAV ' ep - ⁇ P -PARP is obtained.
- the vector N2 (cf. Keller, G. et al., Above) is used as the starting point.
- This vector is opened with EcoRI partial digest 3 'of the neomycin gene.
- the following sequences are then inserted into the vector from 5 'to 3' at the 3 'end of the neomycin gene: (I) the 0.7 kb poly-A signal of the ⁇ -globin gene (for example the EcoRI / Sall- Fragment from pECV; see Berg, BGM et al., Gene 4 (1 989), 407-41 7); (II) a 259 bp BamHI / Ncol fragment which contains the P4 promoter (cf.
- Example 1 (I)); (III) a 3.6 kb Smal / Hindlll fragment from pPARP31 produced by partial digestion (cf. Example 1, (II)).
- the retroviral PARP vector is obtained.
- the transfectant HertTA was obtained by stable transfection of the human cervical carcinoma cell line HeLa with an expression plasmid for the tetracycline-sensitive transactivator rtTA (plasmid pUHD1 72-1 neo; see Gossen et al., Science 268, pp. 1 766-1 769, 1 995).
- the complete human cDNA for PARP was cloned into plasmid pUHD10-3 and is under the control of a tetracycline / rtTA-sensitive promoter
- HelNDc The doxycycline-inducible transfectant HelNDc was obtained by stable transfection of HertTa cells with this expression plasmid.
- This cell line additionally contains the hygromycin resistance plasmid pTKHygro (Küpper et al., Mol. Cell. Biol. 1 5, pp. 31 54-31 63,
- the control cell line HertTAKon was obtained by stable transfection of HertTA with pTKHygro (without PARP expression cassette).
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Abstract
The invention relates to a vector suitable for gene therapy, comprising an insert DNA that can be expressed coding substantially the entire genetic information on the poly(ADP ribose)-polymerase. The invention also relates to a method for the production of said vectors and viruses which are suitable for gene therapy.
Description
Vektoren und Viren zur Gentherapie Vectors and viruses for gene therapy
Die vorliegende Erfindung betrifft Vektoren und Viren, die sich zur Gentherapie eignen, Verfahren zu ihrer Herstellung und ihre Verwendung.The present invention relates to vectors and viruses which are suitable for gene therapy, processes for their preparation and their use.
Eine gängige Tumortherapie umfaßt die möglichst vollständige operative Entfernung des Tumors und die Behandlung des Patienten mittels Bestrahlung und/- oder systemischer oder regionaler Applikation von Zytostatika (Chemotherapie). Diese Strahlen- und/oder Chemotherapie kann dabei vor oder nach operativer Entfernung des Tumors durchgeführt werden und stellt in Fällen inoperablerA common tumor therapy includes the most complete surgical removal of the tumor and the treatment of the patient by means of radiation and / or systemic or regional application of cytostatics (chemotherapy). This radiation and / or chemotherapy can be carried out before or after surgical removal of the tumor and in some cases makes it more inoperable
Tumoren oft sogar die einzige Therapiemöglichkeit dar. Durch die Strahlen- und/oder Chemotherapie wird versucht, noch vorhandenes Tumorgewebe bzw. gebildete Metastasen abzutöten. Diese Therapiemodalitäten induzieren DNA- Schädigung/Hemmung der DNA-Synthese in den Tumorzellen und sollen die Tumorzellen dadurch abtöten. Wegen starker Nebenwirkungen auf gesundesTumors are often the only therapeutic option. Radiation and / or chemotherapy attempts to kill any tumor tissue that is still present or metastases that have formed. These therapy modalities induce DNA damage / inhibition of DNA synthesis in the tumor cells and are intended to kill the tumor cells. Because of strong side effects on healthy
Gewebe wird die für die Therapie geplante Gesamtdosis zumeist über Wochen und Monate hin fraktioniert gegeben. In denjenigen Tumorzellen, die einen solchen zytotoxischen Therapiestoß jedoch überleben, kann diese Behandlung zu genomischer Instabilität führen bzw. eine solche verstärken. Genomische In- Stabilität bedeutet, daß es zu strukturellen und numerischen Chromosomenaberrationen, Genumlagerungen, kompletten oder partiellen Gendeletionen, Gen- amplifikationen, Punktmutationen etc. kommt. Selbstverständlich können diese genetischen Veränderungen die Genexpression der Zelle in erheblichem Umfang beeinflussen, indem es zur Überexpression, Unterexpression oder deregulierten Expression von normalen Proteinen bzw. zur Expression abnormer ProteineTissue is given the total dose planned for the therapy, usually fractionally over weeks and months. In those tumor cells that survive such a cytotoxic therapy shock, however, this treatment can lead to genomic instability or exacerbate it. Genomic instability means that structural and numerical chromosome aberrations, gene rearrangements, complete or partial gene deletions, gene amplifications, point mutations etc. occur. Of course, these genetic changes can influence the gene expression of the cell to a considerable extent by overexpressing, underexpressing or deregulated expression of normal proteins or expressing abnormal proteins
(einzelne Aminosäurenaustausche, Trunkierung von Proteinen etc.) kommt. Wenn solche Proteine betroffen sind, die das Zellwachstum, den Differenzierungszustand, den programmierten Zelltod oder die Expression von Oberflächenproteinen steuern, kann dies direkt zum typischen "malignen Phänotyp" von Tumorzellen einschließlich gestörter Proliferationskontrolle, Immortalisierung,
Differenzierungsstörung, "Immun-Escape", Metastasierung und pathologischer Angiogenese führen. Ein Herbeiführen oder eine Verstärkung von genomischer Instabilität in Tumorzellen, die einen zytotoxischen Therapiestoß überleben, kann also dazu führen, daß der Prozeß der Malignisierung ungewollt beschleunigt wird, daß sich z.B. Therapieresistenz oder eine Metastasierungsneigung einstellen und dadurch die Heilung der Patienten letzlich unmöglich gemacht wird. Dies steht auch im Einklang mit der klinischen Erfahrung, daß bei sehr vielen Krebspatienten durch Strahlen- bzw. Chemotherapie initial zwar Remissionen induziert werden können, daß aber im weiteren Therapieverlauf die Tumoren therapieresistent werden und die Patienten schließlich daran versterben. Der(individual amino acid exchanges, truncation of proteins etc.). If such proteins are affected which control cell growth, the state of differentiation, programmed cell death or the expression of surface proteins, this can directly lead to the typical "malignant phenotype" of tumor cells including impaired proliferation control, immortalization, Differentiation disorder, "immune escape", metastasis and pathological angiogenesis lead. Causing or intensifying genomic instability in tumor cells that survive a cytotoxic therapy shock can therefore lead to the process of malignancy being accelerated unintentionally, that therapy resistance or a tendency to metastasis develop, for example, which ultimately makes it impossible for the patient to heal. This is also in line with the clinical experience that remissions can initially be induced in a large number of cancer patients by radiation or chemotherapy, but that in the further course of therapy the tumors become resistant to therapy and the patients eventually die. The
Erfolg dieser gängigen Tumortherapien ist deshalb gering.The success of these common tumor therapies is therefore low.
Aber auch bei noch als gesund anzusehenden Zellen ("Normalzellen") tritt genomische Instabilität auf und kann vielleicht sogar als treibende Kraft der Tumorentwicklung gesehen werden. Dies wird durch folgende Tatsachen unterstützt: a) Es gibt eine Anzahl verschiedener, monogener Erbleiden, deren gemeinsames Merkmal eine verstärkte genomische Instabilität in normalen Zellen bereits bei jungen Probanden ist (z.B. Bloom's Syndrom, Werner-Syndrom etc.). Diese Syn- drome sind alle mit einem erhöhten Krebsrisiko assoziiert. b) Chemische und physikalische Karzinogene können genomische Instabilität hervorrufen, was als Inanspruchnahme von "fehlerproduzierenden" DNA-Reparatursystemen infolge Überlastung von zunächst aktivierten "fehlerfreien" Reparatursystemen angesehen werden kann. Ebenso können bestimmte Genprodukte von Tumorviren genomische Instabilität in den Wirtszellen herbeiführen.But genomic instability also occurs in cells that are still considered healthy ("normal cells") and may even be seen as the driving force behind tumor development. This is supported by the following facts: a) There are a number of different, monogenic hereditary disorders, the common feature of which is increased genomic instability in normal cells even in young subjects (e.g. Bloom's syndrome, Werner syndrome, etc.). These syndromes are all associated with an increased risk of cancer. b) Chemical and physical carcinogens can cause genomic instability, which can be seen as the use of "error-producing" DNA repair systems due to the overloading of initially activated "error-free" repair systems. Certain tumor virus gene products can also cause genomic instability in host cells.
Der vorliegenden Erfindung liegt somit die Aufgabe zugrunde, ein Mittel bereitzustellen, mit dem gängige Tumortherapien verbessert, eine Prophylaxe gegen die Tumorentstehung und -entwicklung durchgeführt und insbesondere die Erzeu- gung von genomischer Instabilität in Tumorzellen und Normalzellen vermieden werden kann.
Erfindungsgemäß wird dies durch einen zur Gentherapie geeigneten Vektor erreicht, der eine exprimierbare Insert-DNA umfaßt, die für die im wesentlichen vollständige genetische Information der Poly(ADP-Ribose)-Polymerase (nachstehend mit PARP bezeichnet) codiert.The present invention is therefore based on the object of providing a means with which conventional tumor therapies can be improved, prophylaxis carried out against tumor development and development and, in particular, the generation of genomic instability in tumor cells and normal cells can be avoided. According to the invention, this is achieved by a vector suitable for gene therapy which comprises an expressible insert DNA which codes for the essentially complete genetic information of the poly (ADP-ribose) polymerase (hereinafter referred to as PARP).
Die PARP ist ein hochkonserviertes nukleares Enzym bei höheren Eukaryonten, welches in Anwesenheit von DNA-Strangbrüchen die kovalente Modifikation von Zellkernproteinen mit Poly(ADP-Ribose) katalysiert. Diese Reaktion trägt zur effizienten Basenexzisionsreparatur und zur Erholung proliferierender Zellen von den zytotoxischen Auswirkungen von DNA-Schädigungen durch Alkylantien,PARP is a highly conserved nuclear enzyme in higher eukaryotes, which catalyzes the covalent modification of nuclear proteins with poly (ADP-ribose) in the presence of DNA strand breaks. This reaction contributes to efficient base excision repair and the recovery of proliferating cells from the cytotoxic effects of DNA damage from alkylating agents,
Oxidantien bzw. ionisierender Strahlung bei. Diese Erkenntnis wurde bisher benutzt, um die zelluläre Poly(ADP-Ribose)-Produktion in Tumorzellen durch Hemmung der PARP zu unterbinden, wobei als Konsequenz eine Sensibilisierung der Zellen gegen schädigende Agentien festgestellt wurde und ein Absterben der Tumorzellen eintreten sollte. Dieser therapeutische Ansatz ist beispielsweise inOxidants or ionizing radiation. This finding has so far been used to prevent cellular poly (ADP-ribose) production in tumor cells by inhibiting PARP, as a result of which a sensitization of the cells to harmful agents has been found and the tumor cells should die. This therapeutic approach is for example in
DE-44 44 949 C1 beschrieben. Dort wird in einen zur Gentherapie geeigneten Vektor eine exprimierbare Insert-DNA, die für die DNA-Bindungsdomäne von PARP oder eine zumindest teilweise katalytisch nicht aktive PARP codiert, insertiert und in Tumorzellen eingebracht, um die dort vorhandene PARP in trans- dominanter Weise zu hemmen, wodurch die Reparatur von DNA-Schäden drastisch in ihrer Geschwindigkeit vermindert wird und die Tumorzelle mit großer Wahrscheinlichkeit abstirbt. Die vorliegende Erfindung beruht nun auf der darüber hinausgehenden Erkenntnis, daß zur Vermeidung von genomischer Instabilität in Tumorzellen nicht die PARP zu hemmen ist, sondern für eine Überexpres- sion von PARP in Tumorzellen zu sorgen ist. Diese Erkenntnis ist natürlich aufDE-44 44 949 C1 described. There, an expressible insert DNA which codes for the DNA binding domain of PARP or an at least partially catalytically inactive PARP is inserted into a vector suitable for gene therapy and introduced into tumor cells in order to inhibit the PARP present there in a trans-dominant manner , which drastically reduces the rate of repair of DNA damage and the tumor cell is very likely to die. The present invention is now based on the additional finding that to avoid genomic instability in tumor cells it is not PARP that is to be inhibited, but rather that PARP must be overexpressed in tumor cells. This finding is of course based on
Normalzellen übertragbar, wo dieser Ansatz im Sinne einer "Krebsprophylaxe" wirken soll.Normal cells can be transferred where this approach is supposed to work in the sense of "cancer prophylaxis".
Der vorstehende Ausdruck "zur Gentherapie geeigneter Vektor" umfaßt jegliche Vektoren, die alleine oder zusammen mit anderen Mitteln in der Gentherapie verwendet werden können. Dies sind z.B. Plasmid-Vektoren und Virus-Vektoren, die integrierende oder nicht-integrierende Vektorsysteme sein können. Von den
Virus-Vektoren sind insbesondere solche von Adenovirus, Herpes Simplex Virus, Adeno-assoziiertem Virus (nachstehend mit AAV bezeichnet), "Minute virus of mice" (nachstehend mit MVM bezeichnet) und Retroviren zu nennen. Ganz besonders bevorzugt werden Virus-Vektoren von AAV, z.B. AAV-sub201 (vgl. Samulski, R.J. et al., J. Virology 61 , ( 1 987), 3096 - 3101 ), von MVM z. B. pSR2 (vgl. Russell, S.J. et al., J. Virology 66, ( 1 992), 2821 - 2828) und von Retroviren, z. B. N2 (vgl. Keller, G. et al., Nature 31 8, ( 1 985), 149 - 1 54). Es können auch nicht-virale Vektorsysteme zum Einsatz kommen, wie z.B. die Komplexierung der Fremd-DNA mit einem GAL4/Invasin-Fu- sionsprotein (Paul et al., Gene Ther. 8, S. 1 253-1 262 ( 1 997)) oder mit Peptiden (Hart et al., Gene Ther. 2, S. 552-554 ( 1 995); Gottschalk et al., Gene Ther. 3, S. 48-57 ( 1 996)); verbesserte lipidbasierte Vektoren (z.B. DNA-Assoziation an kationische Liposomen; DNA-Verpackung in neutralen oder anionischen Liposomen; liposomen-verpackte, Polykationen-kondensierte DNA;The foregoing "gene therapy vector" includes any vector that can be used alone or with other agents in gene therapy. These are, for example, plasmid vectors and virus vectors, which can be integrating or non-integrating vector systems. Of the Virus vectors are in particular those of adenovirus, herpes simplex virus, adeno-associated virus (hereinafter referred to as AAV), "minute virus of mice" (hereinafter referred to as MVM) and retroviruses. Virus vectors from AAV, for example AAV-sub201 (cf. Samulski, RJ et al., J. Virology 61, (1 987), 3096-3101), from MVM z. PSR2 (see Russell, SJ et al., J. Virology 66, (1 992), 2821-2828) and of retroviruses, e.g. B. N2 (see Keller, G. et al., Nature 31 8, (1 985), 149 - 1 54). Non-viral vector systems can also be used, such as, for example, complexing the foreign DNA with a GAL4 / invasin fusion protein (Paul et al., Gene Ther. 8, pp. 1 253-1 262 (1 997) ) or with peptides (Hart et al., Gene Ther. 2, pp. 552-554 (1 995); Gottschalk et al., Gene Ther. 3, pp. 48-57 (1 996)); improved lipid-based vectors (eg DNA association with cationic liposomes; DNA packaging in neutral or anionic liposomes; liposome-packed, polycation-condensed DNA;
Lee et al., Crit. Rev. Ther. Drug Carrier Syst. 14, S. 1 73-206 ( 1 997))Lee et al., Crit. Rev. Ther. Drug carrier system. 14, p. 1 73-206 (1 997))
T7-RNA-Polymerase-basierte Vektoren (Chen et al., Cancer Gene Ther. 2, S. 281 -289 ( 1 995); - die "Transferrinfektion" (Zatloukal et al., Proc. Natl. Acad. Sei USAT7 RNA polymerase-based vectors (Chen et al., Cancer Gene Ther. 2, pp. 281-289 (1,995); the "transfer infection" (Zatloukal et al., Proc. Natl. Acad. Sei USA
91 , S. 5148-51 52 ( 1 994)); die direkte Injektion der gereinigten DNA von Expressionsplasmiden ohne weitere Zusätze (Wolff, Neuromuscul. Disord. 7, S. 314-31 8 ( 1 997)).91, pp. 5148-51 52 (1,994)); the direct injection of the purified DNA from expression plasmids without further additives (Wolff, Neuromuscul. Disord. 7, pp. 314-31 8 (1 997)).
Erfindungsgemäß wird in einen vorstehenden Vektor eine Insert-DNA eingefügt, die für die im wesentlichen vollständige genetische Information von PARP codiert. Der Begriff "im wesentlichen vollständige genetische Information" bedeutet, daß Insertionen, Deletionen, Basenaustausche oder Modifikationen stattgefunden haben können, die jedoch die Funktion der PARP nicht verändern.According to the invention, an insert DNA which codes for the essentially complete genetic information of PARP is inserted into a vector above. The term "essentially complete genetic information" means that insertions, deletions, base changes or modifications may have taken place, but which do not change the function of the PARP.
Eine solche funktionelle PARP muß katalytisch aktiv sein und muß durch DNA- Strangbrüche aktivierbar sein.
Eine vorstehende Insert-DNA kann aus einem beliebigen Organismus, z.B. aus Mensch oder Tier oder Pflanzen, stammen. Vorzugsweise wird eine Insert-DNA aus dem Menschen, insbesondere menschliche cDNA und besonders bevorzugt jene von Fig. 1 oder eine durch ein oder mehrere Nukleotide davon unterschiedli- ehe DNA verwendet. Eine davon "durch ein oder mehrere Nukleotide davon unterschiedliche DNA" bedeutet, daß die Funktion der PARP trotz Basenaustausche erhalten geblieben ist. Dies schließt auch allelische Varianten ein.Such a functional PARP must be catalytically active and must be able to be activated by DNA strand breaks. An insert DNA above can come from any organism, for example from humans or animals or plants. An insert DNA from humans, in particular human cDNA and particularly preferably that from FIG. 1 or a DNA different therefrom by one or more nucleotides is preferably used. One of them "DNA different from it by one or more nucleotides" means that the function of the PARP has been retained despite base changes. This also includes allelic variants.
Die Einfügung vorstehender Insert-DNA in den Vektor erfolgt derart, daß die Insert-DNA exprimiert werden kann. Dies kann erreicht werden, indem die Insert-The above insert DNA is inserted into the vector in such a way that the insert DNA can be expressed. This can be achieved by using the insert
DNA in eine im Vektor vorhandene Expressionseinheit in Phase inseriert wird. Dazu kann es notwendig sein, eine in der Expressionseinheit vorliegende DNA zumindest teilweise zu entfernen. Auch kann es vorteilhaft sein, Elemente der vorhandenen Expressionseinheit, wie Enhancer, Promotor oder Polyadenylie- rungssignale, zumindest teilweise durch andere zu ersetzen. Vorzugsweise wird in eine Expressionseinheit ein Promotor eingeführt, der spezifisch für eine Gewebe-Art ist, wodurch die Expression der unter der Kontrolle des Promotors stehenden Insert-DNA gewebespezifisch wird. Besonders bevorzugt ist ein Promotor, der in Tumorzellen aktiv ist. Ein Beispiel eines solchen Promotors ist der P4- Promotor von MVM (vgl. Russell, S.J. et al., vorstehend).DNA is inserted into an expression unit in phase in the vector. For this purpose, it may be necessary to at least partially remove a DNA present in the expression unit. It can also be advantageous to at least partially replace elements of the existing expression unit, such as enhancers, promoters or polyadenylation signals, with others. A promoter which is specific for a tissue type is preferably introduced into an expression unit, as a result of which the expression of the insert DNA which is under the control of the promoter becomes tissue-specific. A promoter that is active in tumor cells is particularly preferred. An example of such a promoter is the MVM P4 promoter (see Russell, S.J. et al., Supra).
Die Expression vorstehender Insert-DNA kann ferner in einer Expressionseinheit erreicht werden, die hierzu in den Vektor eingeführt werden muß. Für diese Expressionseinheit gelten auch die vorstehenden Ausführungen.The expression of the above insert DNA can also be achieved in an expression unit which has to be introduced into the vector for this purpose. The above statements also apply to this expression unit.
Im Falle von Virus-Vektoren erweist es sich oftmals als günstig, die Insert-DNA in eine im Vektor vorhandene Expressionseinheit einzufügen. Die hiermit u.U. verbundene Entfernung oder Teilentfernung von in der Expressionseinheit vorliegender Virus-DNA führt dann zu einem Virus-Vektor, der in einer Virus-Funk- tion einen Defekt aufweist. Dieser Defekt kann als Selektionsmarker genutzt werden. Andererseits kann der Defekt, wenn nötig, durch übliche Verfahren, wie Komplementation in trans, ausgeglichen werden.
Erfindungsgemäß werden Virus- Vektoren bevorzugt, in denen die Insert-DNA so eingefügt ist, daß die Virus-Vektoren alleine nicht mehr zur Bildung der durch sie codierten Viren in der Lage sind. Erfindungsgemäß bevorzugte Vektoren sind in Fig. 3 gezeigt.In the case of virus vectors, it often proves advantageous to insert the insert DNA into an expression unit present in the vector. The associated removal or partial removal of virus DNA present in the expression unit then leads to a virus vector which has a defect in a virus function. This defect can be used as a selection marker. On the other hand, if necessary, the defect can be compensated for by conventional methods, such as complementation in trans. According to the invention, virus vectors are preferred in which the insert DNA is inserted in such a way that the virus vectors alone are no longer able to form the viruses encoded by them. Vectors preferred according to the invention are shown in FIG. 3.
Durch übliche Komplementationsverfahren können allerdings auch die durch die Virus-Vektoren codierten Viren gebildet werden. Beispielsweise wird ein das PARP-Insert enthaltender AAV rep"Vektor in Zellen transfiziert, die gleichzeitig mit einem das rep-Gen exprimierenden DNA-Konstrukt cotransfiziert sind. Es werden Viren erhalten. Diese eignen sich gut zur Gentherapie, da sie sich im Patienten nicht vermehren können.However, the viruses encoded by the virus vectors can also be formed by conventional complementation methods. For example, an AAV rep " vector containing the PARP insert is transfected into cells which are co-transfected at the same time with a DNA construct expressing the rep gene. Viruses are obtained. These are well suited for gene therapy since they do not multiply in the patient can.
Durch Transfektion eines Retrovirus-Vektor, der das PARP-Insert enthält, in eine übliche Packaging-Zellinie wird das durch den Virus-Vektor codierte Virus erhal- ten. Dieses eignet sich ebenfalls gut zur Gentherapie.By transfecting a retrovirus vector containing the PARP insert into a conventional packaging cell line, the virus encoded by the virus vector is obtained. This is also very suitable for gene therapy.
Gegenstand der vorliegenden Erfindung sind somit auch Viren, die durch vorstehende Virus-Vektoren codiert werden.The present invention thus also relates to viruses which are encoded by the above virus vectors.
Erfindungsgemäße Vektoren und Viren zeichnen sich dadurch aus, daß sie dieVectors and viruses according to the invention are distinguished by the fact that they
Entstehung von genomischer Instabilität in Normal- und Tumorzelien hemmen können. Sie eignen sich daher bestens, in Therapien eingesetzt zu werden, wo einer genomischen Instabilität vorgebeugt werden soll. Ganz besonders ist die Eignung der erfindungsgemäßen Vektoren und Viren in der Behandlung von Tumoren, insbesondere zusammen mit konventionellen Bestrahlungs- und/oderCan inhibit genomic instability in normal and tumor cells. They are therefore ideally suited for use in therapies where genomic instability is to be prevented. The suitability of the vectors and viruses according to the invention is very particularly suitable for the treatment of tumors, in particular together with conventional radiation and / or
Zytostatika-Verfahren, zu sehen. Mit den erfindungsgemäßen Vektoren und Viren behandelte Tumorzellen zeigen erfreulicherweise eine vermehrte Tendenz zum Absterben. Hierbei schlägt besonders zu Buche, daß die erfindungsgemäßen Viren und Vektoren gewebe(tumor)-spezifisch aktiv sein können. Die Effizienz von Strahlen- und Chemotherapie kann damit in hervorragender Weise gesteigert werden. Dazu werden die Tumorzellen vor der geplanten Strahlen- bzw. Chemotherapie durch systemische bzw. intratumorale Applikation des Gentherapievek-
tors transduziert. Infolge der dann zu erwartenden übersteigerten Poly(ADP- RibosyDierung als zelluläre Antwort auf die DNA-Schädigung durch Strahlenbzw. Chemotherapie kommt es zur spezifischen Hemmung des Phänomens "genomischer Instabilität" in denjenigen Tumorzellen, die den Therapiestoß überleben. Erwartungsgemäß kommt es zum Ausbleiben von weiterer Tumorzell-Cytostatics procedure, see. Fortunately, tumor cells treated with the vectors and viruses according to the invention show an increased tendency to die. It is particularly important here that the viruses and vectors according to the invention can be tissue-specific (tumor) -active. The efficiency of radiation and chemotherapy can thus be increased in an excellent way. For this purpose, the tumor cells are systematically or intratumorally applied to the gene therapy before the planned radiation or chemotherapy. transduced tors. As a result of the exaggerated poly (ADP ribosyDation as a cellular response to DNA damage caused by radiation or chemotherapy), the phenomenon of "genomic instability" is specifically inhibited in those tumor cells that survive the therapy shock. As expected, there is no further Tumor cell
Malignisierung und der gefürchteten Therapie-Resistenz. Auch für gesundes Gewebe bei Krebspatienten bzw. für gesunde Personen empfiehlt sich, insbesondere wenn es bereits Hinweise auf ein erhöhtes Krebsrisiko gibt, eine wiederholte Transduktion der Zellen durch systemische Applikation des Gentherapie- vektors. Die vorliegende Erfindung ist richtungsweisend für die gentherapeutische "Krebsprophylaxe" und die Behandlung schwerster Erkrankungen.Malignation and the dreaded resistance to therapy. Repeated transduction of the cells by systemic application of the gene therapy vector is also recommended for healthy tissue in cancer patients or for healthy people, especially if there is already evidence of an increased risk of cancer. The present invention is trend-setting for gene therapy "cancer prophylaxis" and the treatment of the most serious diseases.
Kurze Beschreibung der Zeichnungen:Brief description of the drawings:
Fig. 1 zeigt die Nukleotidsequenz der cDNA der humanen Poly(ADP-Ribo- se)-Polymerase1 shows the nucleotide sequence of the cDNA of human poly (ADP-ribose) polymerase
Fig. 2 zeigt die Aminosäuresequenz der humanen Poly(ADP-Ribose)-Poly- merase2 shows the amino acid sequence of the human poly (ADP-ribose) polymerase
Fig. 3 erfindungsgemäßer AAV rep cap -PARP-Vektor und retroviraler PARP-3 AAV rep cap -PARP vector and retroviral PARP- according to the invention
Vektorvector
Fig. 4 a, b SCE-Frequenzen in COMF-ZellenFig. 4 a, b SCE frequencies in COMF cells
Fig. 4 c, d SCE-Frequenzen in COR4-ZellenFig. 4 c, d SCE frequencies in COR4 cells
Fig. 5 a, b SCE-Frequenzen in menschlichen HelNDc-ZellenFig. 5 a, b SCE frequencies in human HelNDc cells
Fig. 5 c, d SCE-Frequenzen in menschlichen HertTAKon-KontrollzellenFig. 5 c, d SCE frequencies in human HertTAKon control cells
Die folgenden Beispiele erläutern die Erfindung.
BeispieleThe following examples illustrate the invention. Examples
Beispiel 1 : Herstellung des erfindungsgemäßen Vektors AAVrβp /cap -PARPExample 1: Preparation of the vector AAV rβp / cap -PARP according to the invention
Es wird von dem Vektor AAV-sub201 (Samulski, R.J. et al., vorstehend) ausgegangen. Dieser Vektor wird mit dem Restriktionsenzym Xbal gespalten und sämtliche AAV-Anteile außer den invertierten terminalen Repetitionen werden entfernt. Die Enden des Vektor-Fragments werden "geglättet" In dieses wird ein Insert, P4-PARP-poly A, eingefügt, das von 5' nach 3' folgende Sequenzen mit jeweils geglätteten Enden umfaßt: (I) ein 259 bp BamHI/Ncol-Fragment, das denThe vector AAV-sub201 (Samulski, R.J. et al., Supra) is assumed. This vector is cleaved with the restriction enzyme Xbal and all AAV components except the inverted terminal repetitions are removed. The ends of the vector fragment are "smoothed". An insert, P4-PARP-poly A, is inserted into this, which comprises the following sequences from 5 'to 3', each with smoothed ends: (I) a 259 bp BamHI / Ncol- Fragment representing the
P4-Promotor enthält. Dieses Fragment stammt z.B. aus dem Plasmid pEG61 8 (vgl. Astell, C. et al., J. Virology 57, ( 1 986), 656-669); (II) ein durch Partialver- dau erzeugtes 3,6 kb Smal/Hindlll-Fragment aus pPARP31 (vgl. van Gool et al., Eur. J. Biochem. 244 (1 997), 1 5-20), das sowohl den 3,0 kb offenen Leser- ahmen als auch das 630 bp HSV-Thymidinkinase Poly-A-Signal aufweist. Es wird der Vektor AAV 'ep-^P -PARP erhalten.Contains P4 promoter. This fragment originates, for example, from plasmid pEG61 8 (cf. Astell, C. et al., J. Virology 57, (1 986), 656-669); (II) a 3.6 kb Smal / Hindlll fragment from pPARP31 generated by partial digest (cf. van Gool et al., Eur. J. Biochem. 244 (1 997), 1 5-20), which both 3.0 kb open reader frame as well as the 630 bp HSV thymidine kinase poly A signal. The vector AAV ' ep - ^ P -PARP is obtained.
Beispiel 2: Herstellung des erfindungsgemäßen retroviralen PARP-VektorsExample 2: Production of the retroviral PARP vector according to the invention
Es wird von dem Vektor N2 (vgl. Keller, G. et al., vorstehend) ausgegangen.The vector N2 (cf. Keller, G. et al., Above) is used as the starting point.
Dieser Vektor wird mit EcoRI-Partialverdau 3' des Neomycin-Gen geöffnet. In den Vektor werden dann von 5' nach 3' folgende Sequenzen am 3'-Ende des Neomycin-Gens inseriert: (I) das 0,7 kb poly-A-Signal des ß-Globin-Gens (z.B. das EcoRI/Sall-Fragment aus pECV; vgl. Berg, B.G.M. et al., Gene 4 ( 1 989), 407 - 41 7); (II) ein 259 bp BamHI/Ncol-Fragment, das den P4-Promotor enthält (vgl.This vector is opened with EcoRI partial digest 3 'of the neomycin gene. The following sequences are then inserted into the vector from 5 'to 3' at the 3 'end of the neomycin gene: (I) the 0.7 kb poly-A signal of the β-globin gene (for example the EcoRI / Sall- Fragment from pECV; see Berg, BGM et al., Gene 4 (1 989), 407-41 7); (II) a 259 bp BamHI / Ncol fragment which contains the P4 promoter (cf.
Beispiel 1 , (I)); (III) ein durch Partialverdau erzeugtes 3,6 kb Smal/Hindlll-Fragment aus pPARP31 (vgl. Beispiel 1 , (II)). Es wird der retrovirale PARP-Vektor erhalten.Example 1, (I)); (III) a 3.6 kb Smal / Hindlll fragment from pPARP31 produced by partial digestion (cf. Example 1, (II)). The retroviral PARP vector is obtained.
Beispiel 3: Hemmung der Entstehung von genomischer InstabilitätExample 3: Inhibition of Genomic Instability
Anhand von induzierbar PARP-überexprimierenden Transfektanten wird die Aus-
Wirkung der PARP-Überexpression auf die genomische Stabilität von Zellen unter Bedingungen von genotoxischem Streß untersucht. Als Meßparameter dient die Häufigkeit von Schwesterchromatid-Austausch (SCE)-Vorgängen, ein allgemein anerkannter Marker für genomische Instabilität und ein wichtiger Mechanismus sowohl für die Entstehung von Gendeletionen als auch Genamplifikationen ("un- equal sister-chromatid exchange") .Using inducible PARP-overexpressing transfectants, the Effect of PARP overexpression on the genomic stability of cells under conditions of genotoxic stress was examined. The frequency of sister chromatid exchange (SCE) processes, a generally recognized marker for genomic instability and an important mechanism for both the generation of gene deletions and gene amplifications ("unequal sister-chromatid exchange") serve as measurement parameters.
a) Durch stabile Transfektion der Hamsterzellinie CO60 mit einem Expres- sionsplasmid für den menschlichen Glucocorticoidrezeptor unter der Kontrolle des frühen SV40-Promotors (Kumar, V. et al., Cell 51 , S. 941 -a) By stable transfection of the hamster cell line CO60 with an expression plasmid for the human glucocorticoid receptor under the control of the early SV40 promoter (Kumar, V. et al., Cell 51, p. 941 -
951 , 1 987) wurde die Transfektante COR4 erhalten. Durch stabile Transfektion von COR4-Zellen mit einem auf pPARP31 basierenden Expres- sionsplasmid für die vollständige menschliche PARP unter der Kontrolle des "Mouse Mammary Tumor Virus Long Terminal Repeat (MMTV-LTR)- Promotors" ( = pPARP 93) wurde die Glucocorticoid-induzierbare Transfektante COMF erhalten.951, 1 987) the transfectant COR4 was obtained. Stable transfection of COR4 cells with an expression plasmid based on pPARP31 for complete human PARP under the control of the "Mouse Mammary Tumor Virus Long Terminal Repeat (MMTV-LTR) promoter" (= pPARP 93) made the glucocorticoid-inducible Obtained transfectant COMF.
Es wurden 3x105 Zellen COMF bzw. COR4 in Kulturflaschen (75 cm2) mit 20 ml D-MEM, 10% FKS mit 1 % Penicillin/Streptomycin ausgesät (t = -24h). Nach 24 Stunden Kultur (t = Oh) erfolgte die Zugabe von Dexame- thason (Endkonzentration 10"7 M) zur Induktion der Transgenexpression. Danach weitere 24h Kultur. Dann Zugabe von 460 μl Brom-Desoxyuridin (BrdU) Gebrauchslösung ( = 270 μg/ml BrdU in PBS) zur 20 ml Kultur (t = + 24h). Nachfolgend Zugabe von MNNG-(N-Methyl-N'-Nitro-N-Nitrosogua- nidin; Endkonzentration 2 / M) bei t = + 32h. Zum Zeitpunkt t = + 51 h erfolgte Zugabe von 20 μ\ Demecolcine-Stammlösung ( 1 Oμg/ml Demecol- cine; Fa. Sigma)/20 ml Kultur, dann Inkubation für weitere 4h im Brutschrank. Die Ernte der mitotischen Zellen sowie die weitere Behandlung zur Bestimmung der SCE pro Mitose erfolgte nach dem bekannten Stan- dardverfahren (Perry et al., Nature 258, S. 1 21 -1 25 ( 1 974)) .3x10 5 cells COMF or COR4 were sown in culture bottles (75 cm 2 ) with 20 ml D-MEM, 10% FCS with 1% penicillin / streptomycin (t = -24h). After 24 hours of culture (t = Oh), dexamethasone (final concentration 10 "7 M) was added to induce transgene expression. Then another 24 hours of culture. Then addition of 460 μl bromo-deoxyuridine (BrdU) working solution (= 270 μg / ml BrdU in PBS) for 20 ml culture (t = + 24h), followed by addition of MNNG- (N-methyl-N'-nitro-N-nitrosoguanidine; final concentration 2 / M) at t = + 32h t = + 51 h, 20 μ \ Demecolcine stock solution (1 μg / ml Demecolcine; Sigma) / 20 ml culture was added, then incubation in the incubator for a further 4 h, harvesting the mitotic cells and further treatment for The SCE per mitosis was determined using the known standard method (Perry et al., Nature 258, p. 1 21-1 25 (1 974)).
Zusammenfassend ist festzustellen, daß es nach Inkubation von COMF-
Zellen mit Dexamethason ( 100 nM) über 24 Std. zur starken Überexpression der menschlichen PARP in den Zellkernen, zu einer Steigerung des zellulären Gehalts an Poly(ADP-Ribose) nach Gamma-Bestrahlung der Zellen auf das 2- bis 5-fache (gemessen 10 Min. nach Bestrahlung) sowie zu einer signifikant verminderten Häufigkeit von MNNG-induzierten SCEsIn summary, it can be stated that after incubation of COMF- Cells with dexamethasone (100 nM) for 24 hours for the strong overexpression of the human PARP in the cell nuclei, for an increase in the cellular content of poly (ADP-ribose) after the gamma irradiation of the cells 2 to 5 times (measured 10 min after radiation) and to a significantly reduced frequency of MNNG-induced SCEs
(s. Fig. 4a und 4b) kommt. In den Ausgangszellen COR4, in denen es nicht zur PARP-Überexpression kommt, ist die Dexamethason-Behandlung jeweils folgenlos (Fig. 4c und 4d).(see Fig. 4a and 4b) comes. In the starting cells COR4, in which there is no PARP overexpression, the dexamethasone treatment has no consequences (FIGS. 4c and 4d).
Durch stabile Transfektion der menschlichen Zervixkarzinomzellinie HeLa mit einem Expressionsplasmid für den tetrazyklinsensitiven Transaktivator rtTA (Plasmid pUHD1 72-1 neo; vgl. Gossen et al., Science 268. S. 1 766- 1 769, 1 995) wurde die Transfektante HertTA erhalten. Die vollständige menschliche cDNA für PARP wurde in Plasmid pUHD10-3 kloniert und steht dort unter der Kontrolle eines Tetrazyklin/rtTA-sensitiven PromotorsThe transfectant HertTA was obtained by stable transfection of the human cervical carcinoma cell line HeLa with an expression plasmid for the tetracycline-sensitive transactivator rtTA (plasmid pUHD1 72-1 neo; see Gossen et al., Science 268, pp. 1 766-1 769, 1 995). The complete human cDNA for PARP was cloned into plasmid pUHD10-3 and is under the control of a tetracycline / rtTA-sensitive promoter
(pUHD10-3; vgl. Gossen et al., Science 268, S. 1 766-1 769, 1 995) . Durch stabile Transfektion von HertTa-Zellen mit diesem so erhaltenen Expressionsplasmid wurde die Doxycyclin-induzierbare Transfektante HelNDc erhalten. Diese Zellinie enthält zusätzlich das Hygromycinresi- stenzplasmid pTKHygro (Küpper et al., Mol. Cell. Biol. 1 5, S. 31 54-31 63,(pUHD10-3; see Gossen et al., Science 268, pp. 1 766-1 769, 1 995). The doxycycline-inducible transfectant HelNDc was obtained by stable transfection of HertTa cells with this expression plasmid. This cell line additionally contains the hygromycin resistance plasmid pTKHygro (Küpper et al., Mol. Cell. Biol. 1 5, pp. 31 54-31 63,
1 995). Die Kontrollzellinie HertTAKon wurde durch stabile Transfektion von HertTA mit pTKHygro (ohne PARP-Expressionskassette) erhalten.1 995). The control cell line HertTAKon was obtained by stable transfection of HertTA with pTKHygro (without PARP expression cassette).
Es wurden 3x1 06 Zellen HelNDc- bzw. HertTAKon in Kulturflaschen (75 cm2) mit 20 ml D-MEM, 10% FKS mit 1 % Penicillin/Streptomycin ausgesät (t = -24h). Nach 24 Stunden Kultur (t = Oh) erfolgte die Zugabe von Doxycyclin ( 1 μg/ml Endkonzentration) zur Induktion der Transgenexpression. Danach weitere 28h Kultur. Dann Zugabe von 460 μl Brom-De- soxyuridin (BrdU) Gebrauchslösung ( = 270 μg/ml BrdU in PBS) zur 20 ml Kultur (t = + 28h). Nachfolgend Zugabe von MNNG (N-Methyl-N'-Nitro-N-3x1 0 6 cells of HelNDc- or HertTAKon were sown in culture bottles (75 cm 2 ) with 20 ml D-MEM, 10% FCS with 1% penicillin / streptomycin (t = -24h). After 24 hours of culture (t = Oh), doxycycline (1 μg / ml final concentration) was added to induce transgene expression. Then another 28 hours of culture. Then add 460 μl bromo-de-soxyuridine (BrdU) working solution (= 270 μg / ml BrdU in PBS) to the 20 ml culture (t = + 28h). Then add MNNG (N-methyl-N'-nitro-N-
Nitrosoguanidin; 0, 1 μM Endkonzentration) bei t = + 48h. Zum Zeitpunkt t = + 72h erfolgte Zugabe von 20 μl Demecolcine-Stammlösung ( 10μg-
/ml Demecolcine; Fa. Sigma)/20 ml Kultur, dann Inkubation für weitere 4h im Brutschrank. Die Ernte der mitotischen Zellen sowie die weitere Behandlung zur Bestimmung der SCE pro Mitose erfolgte nach dem bekannten Standardverfahren (Perry et al. , Nature 258, S. 1 21 -1 25 ( 1 974)) .Nitrosoguanidine; 0.1 μM final concentration) at t = + 48h. At the time t = + 72h, 20 μl of Demecolcine stock solution (10μg- / ml demecolcine; Sigma) / 20 ml culture, then incubation for another 4 hours in the incubator. The mitotic cells were harvested and the further treatment to determine the SCE per mitosis was carried out using the known standard method (Perry et al., Nature 258, pp. 1 21-1 25 (1 974)).
Zusammenfassend ist festzustellen, daß es nach Inkubation von HelNDc- Zellen mit Doxycyclin über 48 Stunden zur Überexpression der menschlichen PARP in den Zellkernen, zu einer Steigerung des zellulären Gehalts an Poly(ADP-Ribose) nach Gamma-Bestrahlung der Zellen auf das 1 ,5- bis 1 ,8-fache (gemessen 10 Min. nach Bestrahlung) sowie zu einer signifikant verminderten Häufigkeit von MNNG-induzierten SCEs (Fig. 5a und 5b) kommt. In den Kontrollzellen HertTAkon, in denen es nicht zur PARP- Überexpression kommt, ist die Doxycyclin-Anwendung jeweils folgenlos (Fig. 5c und 5d).In summary, it can be stated that after incubation of HelNDc cells with doxycycline for 48 hours, overexpression of the human PARP in the cell nuclei, an increase in the cellular content of poly (ADP-ribose) after gamma irradiation of the cells on the 1st, 5th - up to 1.8 times (measured 10 minutes after irradiation) and to a significantly reduced frequency of MNNG-induced SCEs (FIGS. 5a and 5b). In the control cells HertTAkon, in which PARP overexpression does not occur, the use of doxycycline is without consequences (FIGS. 5c and 5d).
Als Ergebnis wird festgestellt, daß durch die Überexpression der PARP sowohl in den Hamsterzellen (s. Fig. 4 a, b) als auch in den humanen Zellen (s. Fig. 5 a, b) die durch das Alkylans N-Methyl-N'-Nitro-N-Nitrosoguanidin (MNNG) induzierte Häufung von SCEs pro Chromosom signifikant vermindert wird. In den ent- sprechenden Kontrollzellinien ohne PARP-Fremdgen (Fig. 4 c, d und Fig. 5 c, d) bewirkt die Einwirkung der Fremdgen-Indikatorsubstanzen Dexamethason bzw. Doxycyclin keinerlei Veränderung der SCE-Rate. Hierdurch wird die Spezifität des Effekts der SCE-Hemmung durch die PARP-Überexpression bewiesen. Die Zytotoxizität der MNNG-Behandlung wird weder in den COMF-Hamsterzellen noch in den menschlichen HelNDc durch die PARP-Überexpression vermindert.As a result, it is found that the overexpression of the PARP both in the hamster cells (see FIG. 4 a, b) and in the human cells (see FIG. 5 a, b) caused by the alkylane N-methyl-N '-Nitro-N-nitrosoguanidine (MNNG) induced clustering of SCEs per chromosome is significantly reduced. In the corresponding control cell lines without PARP foreign gene (FIGS. 4 c, d and 5 c, d), the action of the foreign gene indicator substances dexamethasone or doxycycline does not cause any change in the SCE rate. This demonstrates the specificity of the effect of SCE inhibition due to PARP overexpression. The cytotoxicity of the MNNG treatment is not reduced by the PARP overexpression neither in the COMF hamster cells nor in the human HelNDc.
Letzteres beweist, daß die verminderte SCE-Rate nicht durch eine verbesserte DNA-Reparatur bedingt ist.
The latter proves that the reduced SCE rate is not due to an improved DNA repair.
Claims
Vektor mit einer exprimierbaren Insert-DNA, die für die im wesentlichen vollständige genetische Information der Poly(ADP-Ribose)-Polymerase codiert, wobei der Vektor zur Gentherapie geeignet ist.Vector with an expressible insert DNA encoding the substantially complete genetic information of the poly (ADP-ribose) polymerase, the vector being suitable for gene therapy.
Vektor nach Anspruch 1 , dadurch gekennzeichnet, daß der Vektor auf einem Virus-Vektor beruht.Vector according to claim 1, characterized in that the vector is based on a virus vector.
Vektor nach Anspruch 2, dadurch gekennzeichnet, daß der Virus-Vektor ein Adeno-assoziierter Virus- oder ein Retrovirus-Vektor ist.Vector according to claim 2, characterized in that the virus vector is an adeno-associated virus or a retrovirus vector.
4. Vektor nach einem der Ansprüche 1 - 3, dadurch gekennzeichnet, daß die für die Poly(ADP-Ribose)-Polymerase codierende DNA die Sequenz von4. Vector according to one of claims 1-3, characterized in that the coding for the poly (ADP-ribose) polymerase DNA the sequence of
Fig. 1 oder eine durch ein oder mehrere Nukleotide davon unterschiedliche Sequenz aufweist.1 or a sequence that is different from one or more nucleotides thereof.
5. Vektor nach einem der Ansprüche 1 - 4, dadurch gekennzeichnet, daß die Insert-DNA in einem Tumor und/oder in Normalgewebe exprimierbar ist.5. Vector according to one of claims 1-4, characterized in that the insert DNA can be expressed in a tumor and / or in normal tissue.
6. Verfahren zur Herstellung des Vektors nach Anspruch 1 , umfassend die Einfügung der Insert-DNA nach Anspruch 1 in einen Vektor, der zur Gentherapie geeignet ist, derart, daß eine Expression der Insert-DNA durch eine bereits im Vektor vorhandene oder ebenfalls inserierte Expressionseinheit erfolgen kann.6. A method for producing the vector according to claim 1, comprising inserting the insert DNA according to claim 1 into a vector which is suitable for gene therapy such that expression of the insert DNA by an expression unit already present in the vector or also inserted can be done.
7. Virus, codiert durch den Vektor nach Anspruch 2 oder 3.7. Virus encoded by the vector of claim 2 or 3.
8. Verwendung des Vektors nach einem der Ansprüche 1 bis 5 oder des8. Use of the vector according to one of claims 1 to 5 or of
Virus nach Anspruch 7 zur Gentherapie.
Verwendung nach Anspruch 8 zur Unterstützung der Therapie und/oder Prophylaxe von Tumorerkrankungen.
Virus according to claim 7 for gene therapy. Use according to claim 8 to support the therapy and / or prophylaxis of tumor diseases.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19808889A DE19808889A1 (en) | 1998-03-03 | 1998-03-03 | Vectors and viruses for gene therapy |
DE19808889 | 1998-03-03 | ||
PCT/DE1999/000647 WO1999044943A2 (en) | 1998-03-03 | 1999-03-03 | Vectors and viruses used in gene therapy |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1066221A2 true EP1066221A2 (en) | 2001-01-10 |
Family
ID=7859479
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP99919057A Withdrawn EP1066221A2 (en) | 1998-03-03 | 1999-03-03 | Vectors and viruses used in gene therapy |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1066221A2 (en) |
JP (1) | JP2002505855A (en) |
AU (1) | AU3698699A (en) |
DE (1) | DE19808889A1 (en) |
WO (1) | WO1999044943A2 (en) |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU688749B2 (en) * | 1992-11-30 | 1998-03-19 | Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The | Mammalian muscle NAD:arginine ADP-ribosyltransferase |
DE4433130C2 (en) * | 1994-09-16 | 1997-02-06 | Deutsches Krebsforsch | Poly (ADP-ribose) polymerase overexpressing animal cell lines and methods for the identification of DNA damaging substances |
DE4444949C1 (en) * | 1994-12-16 | 1996-11-21 | Deutsches Krebsforsch | Vectors and viruses for gene therapy |
-
1998
- 1998-03-03 DE DE19808889A patent/DE19808889A1/en not_active Ceased
-
1999
- 1999-03-03 EP EP99919057A patent/EP1066221A2/en not_active Withdrawn
- 1999-03-03 JP JP2000534497A patent/JP2002505855A/en active Pending
- 1999-03-03 AU AU36986/99A patent/AU3698699A/en not_active Abandoned
- 1999-03-03 WO PCT/DE1999/000647 patent/WO1999044943A2/en not_active Application Discontinuation
Non-Patent Citations (1)
Title |
---|
See references of WO9944943A2 * |
Also Published As
Publication number | Publication date |
---|---|
DE19808889A1 (en) | 1999-09-09 |
JP2002505855A (en) | 2002-02-26 |
WO1999044943A2 (en) | 1999-09-10 |
AU3698699A (en) | 1999-09-20 |
WO1999044943A3 (en) | 1999-12-09 |
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