WO2004046364A1 - Adenoviral vectors for the transfer of specific genes into somatic cells - Google Patents

Adenoviral vectors for the transfer of specific genes into somatic cells Download PDF

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WO2004046364A1
WO2004046364A1 PCT/DE2003/003036 DE0303036W WO2004046364A1 WO 2004046364 A1 WO2004046364 A1 WO 2004046364A1 DE 0303036 W DE0303036 W DE 0303036W WO 2004046364 A1 WO2004046364 A1 WO 2004046364A1
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adenoviral vectors
adenovirus
vectors according
luciferase
coexpression
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PCT/DE2003/003036
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German (de)
French (fr)
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Andreas Block
Peter Schäfer
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Universitätsklinikum Hamburg-Eppendorf Körperschaft des Öffentlichen Rechts
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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
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid

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  • the invention relates to adenoviral vectors for the transfer of specific genes into body cells.
  • adenoviral vectors can attack a broad spectrum of target cells in a highly efficient manner both in humans and in experimental animals, usually the mouse. In contrast to the retroviruses, they also transfect cells that are not dividing because they do not integrate into the genome.
  • adenovirus vectors can be easily purified and produced in high titers, which are easily sufficient for in vivo applications.
  • a significant limitation when using adenovirus vectors is, however, that the transgene expression in most tissues drops significantly after 4 to 6 weeks at the latest and that a second application has little effect.
  • Such effects are described in Davidson BL, Allen ED, Kozarsky KF, Wilson JM, Roessler BJ (1993), A model syste for in vivo gene transfer into the central nervous system using an adenoviral vector, Nat Genet 3 219 to 223, and in Yang Y, Nunes FA, Berencsi K, Furth EE, Gonczol E, Wilson JM (1994), Cellular immunity to viral antigens limits El-deleted adenoviruses for gene the- rapy, Proc Natl Acad Sei USA 91: 4407-4411. The main reason for this is an immune response against the adenoviral proteins that are also expressed. However, this mechanism prevents prolonged transgene expression in the gene therapy of metabolic diseases in experimental therapy of cancer without significant relevance.
  • Another strategy leaves the vector structures for their advantage. Instead, the immune response of the host organism is suppressed. It is feasible in animal experiments to suppress the immune response systemically, but this is very problematic for clinical studies, since susceptibility to infections and the development of tumors can occur. In the long term, this strategy can only be successful if ways are found to prevent immunostimulation locally, ideally directly on the antigen-carrying, adenovirally transfected cell.
  • the object of the present invention is to generate adenoviral vectors of the type mentioned in the introduction in such a way that the change in a local immune stimulation is supported.
  • This object is achieved in that the specific gene is coexpressed with at least one immunomodulatory viral gene in the same adenoviral vector.
  • viruses which traditionally lead to persistent infections, specialize in breaking through the local network between immunological defense cells, especially natural killer [NK] cells and T-Ly phocytes, and their messenger substances, for example chemokines and cytokines.
  • chemokines and cytokines are also interested here.
  • herpes viruses for example the cytomegaloviruses (CMV), in which different gene products show immunomodulatory properties, even when they are expressed in isolation.
  • CMV cytomegaloviruses
  • the approach according to the invention is to provide adenoviral vectors with one or more immunomodulatory viral genes in addition to the specific gene to be expressed, for example a therapeutic transgene or a reporter gene, in order to avoid their elimination.
  • to co-express therapeutic genes or reporter genes with immunomodulatory genes of the herpes and POX virus family in the same adenoviral vector With such vectors it is possible to achieve a significantly prolonged expression of the therapeutic genes or the reporter genes as well as to enable renewed virus administration.
  • new approaches to support longer-term therapeutic gene expression in the treatment of metabolic or autoimmune diseases in humans can arise.
  • Ad5 serogroup 5
  • Ad5 recombinant serogroup 5
  • the necessary techniques for producing recombinant serogroup 5 (Ad5) adenovirus vectors have been established in the laboratory. Because of a deleted El region of the virus genome, these vectors are not capable of replication (only in 293 cells). Another deletion is in the E3 region.
  • the virus is constructed according to one of Graham et al. developed protocol.
  • the transgenes to be expressed are cloned into a pAd shuttle plasmid, the expression cassette being flanked by homologous sequences of the Ad-5.
  • This plasmid is co-transfected with pBHG 10, which contains essential parts of the Ad5 genome, according to the calcium chloride precipitation method in 293 cells, a human embryonic kidney cell line that is stably transfected with approximately 11% of the Ad5 genome. Homologous recombination integrates the expression cassette into the deleted El region of the virus genome, and recombinant infectious virus particles are created. The viruses from the culture supernatant are purified by cesium chloride density gradient centrifugation. The virus titer is determined by means of a plaque assay in 293 cells.
  • Fig. 1 is a schematic representation of recombinant adenoviruses.
  • the reporter gene luciferase was chosen as the transgene, which was cloned into the deleted El region of the adenovirus serotype 5 and was expressed there under the control of the CMV promoter.
  • the ml44 gene of murine CMV codes for a membrane protein which has homology to the MHC class I molecules of the host cells and in this way impairs NK ell function in vitro and in vivo.
  • the human CMV also has a similar gene (UL18), to which the corresponding properties are attributed.
  • An adenovirus vector was constructed that expresses the luc marker gene together with the mouse CMV ml44 protein under the control of a CMV promoter.
  • An internal ribosome binding site (IRES) is connected between the two transgenes. efficient translation of two genes. This is shown in Fig. 1.
  • a fragment was isolated from the plasmid pcDNA3ml44 (from Dr. Helen Farrell, Ani al Health Trust, Newmarket, Suffolk, UK) which contains the cDNA of the ml44 gene of the mouse CMV. This was cloned in front of the 5 'end of the coding IRES sequence in the plasmid pIRES (from Clontech). 5 'of the IRES, the luciferase marker gene isolated from the vector pluc (from Promega) was then inserted into pIRES-ml44.
  • the lucIRES-ml44 expression cassette was then cloned into the adenoviral expression plasmid under the control of the constitutive CMV promoter to construct the adenoviral expression vector. This is also illustrated in Fig. 1. This is how pAd.luc-IRES-ml44, which can be used for coprecipitation, was created.
  • the recombinant adenovirus Ad.luc-IRES-ml44 was generated.
  • the correctness of the individual cloning steps was confirmed by sequence analysis of the virus DNA extracted from the supernatant.
  • the expression of the luc reporter gene was demonstrated in a standard assay.
  • Second embodiment adenovirus for the coexpression of luciferase and the CC chemokine-homologous protein ml31 / 129 of the mouse CMV
  • An adenovirus vector was constructed which expresses the luciferase together with the ml31 / 129 protein of the mouse CMV under the control of a CMV promoter.
  • the ml3l / 129 belongs to the homologues of the CC chemokines and contributes significantly to the establishment of a persistent infection of mouse CMV in the liver.
  • Chemokine homologous genes are also known in human CMV.
  • the cDNA of the ml31 / 129 gene from the plasmid pcDNA3ml31 / 129 was isolated by suitable restriction enzymes and first cloned into plRES. The luc marker gene was then inserted into the resulting pIRESml31 / l29. The complete expression cassette was isolated from the resulting plasmid pluc-IRES-ral3l / l29 and cloned into the adenoviral expression plasmid under the control of the CMV promoter, so that the vector pAd.luc-IRES-ml3l / 129 which could be used for coprecipitation was formed.
  • the expression is bicistronic, which means that the luciferase and the immunomodulatory gene are transcribed on the same messenger RNA.
  • An internal ribosome binding site IVS enables the efficient translation of both proteins.
  • MCMV ml31 / 129 (CC homolog)
  • MCMV Murine cytomegalovirus
  • HCMV Human cytomegalovirus
  • HHV-6 Human herpes virus 6
  • HHV-7 Human herpes virus 7
  • HHV-8 Human herpes virus 8
  • MCV Molluscum contagiosum-irus

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Abstract

Adenoviral vectors are used in gene therapy for transferring specific genes into somatic cells, especially for transferring therapeutic genes or reporter genes. The specific gene is co-expressed with at least one immunomodulatory viral gene in the same adenoviral vector. The invention especially relates to a use of immunomodulatory viral genes from the herpes virus family or the poxvirus family.

Description

Adenovirale Vektoren zum Transfer von spezifischen Genen in Körpe zellen Adenoviral vectors for the transfer of specific genes into body cells
Die Erfindung betrifft adenovirale Vektoren zum Transfer von spezifischen Genen in Körperzellen hinein.The invention relates to adenoviral vectors for the transfer of specific genes into body cells.
Gemäß der Erläuterungen in Mountain A (2000) , Gene thera- py: the first decade, Trends Biotechnol .18: 119-128, sind in den letzten Jahren große Erwartungen in die Gentherapie als neuartigem Ansatz zur Behandlung von Krebs- und StoffWechselerkrankungen gesetzt worden. Eine zentrale Rolle als Vehikel für den Transfer der therapeutischen Gene in den bisher verfolgten Strategien spielen Viren, bei denen mit Hilfe gentechnologischer Verfahren die Fähigkeit zur Vermehrung in Körperzellen ausgeschaltet wurde, wie dies beispielsweise in Walther W, Stein u (2000) , Viral vectors for gene transfer: a review of their use in the treatment of human disease, Drugs 60 : 249-271, be- schrieben ist . Hierbei bietet die Gruppe der in Kozarsky KF, Wilson JM (1993) , Gene therapy: adenovirus vectors, Curr Opin Genet Dev 3_ 499-503, erläuterten Adenoviren mehrere große Vorteile gegenüber anderen häufig verwendeten viralen Systemen, beispielsweise Retroviren oder Ade- noassoziierten Viren.According to the explanations in Mountain A (2000), Gene therapy: the first decade, Trends Biotechnol .18: 119-128, great expectations have been placed in gene therapy in recent years as a novel approach to the treatment of cancer and metabolic diseases , Viruses play a central role as vehicles for the transfer of therapeutic genes in the strategies pursued so far, in which the ability to multiply in body cells was switched off with the help of genetic engineering methods, as described, for example, in Walther W, Stein u (2000), Viral vectors for gene transfer: a review of their use in the treatment of human disease, Drugs 60: 249-271, be is written. The group of adenoviruses explained in Kozarsky KF, Wilson JM (1993), Gene therapy: adenovirus vectors, Curr Opin Genet Dev 3_ 499-503, offers several major advantages over other frequently used viral systems, for example retroviruses or adeno-associated viruses.
Adenovirale Vektoren können in vivo sowohl beim Menschen als auch in Versuchstieren, meist der Maus, ein breites Spektrum von Zielzellen hocheffizient befallen. Im Gegensatz zu den Retroviren transfizieren sie auch Zellen, die sich nicht in Teilung befinden, da sie nicht ins Genom integrieren.In vivo, adenoviral vectors can attack a broad spectrum of target cells in a highly efficient manner both in humans and in experimental animals, usually the mouse. In contrast to the retroviruses, they also transfect cells that are not dividing because they do not integrate into the genome.
Adenovirusvektoren können im Vergleich zu den Adeno- assoziierten Viren leicht aufgereinigt und in hohen Titern hergestellt werden, die für in vivo-Applikationen problemlos ausreichen.Compared to the adeno-associated viruses, adenovirus vectors can be easily purified and produced in high titers, which are easily sufficient for in vivo applications.
Eine wesentliche Einschränkung bei der Verwendung von Adenovirusvektoren ist jedoch, daß die Transgenexpression in den meisten Geweben nach spätestens 4 bis 6 Wochen signifikant abfällt und auch eine zweite Applikation wenig Wirkung zeigt. Derartige Effekte sind in Davidson BL, Allen ED, Kozarsky KF, Wilson JM, Roessler BJ (1993) , A mo- del syste for in vivo gene transfer into the central nervous system using an adenoviral vector, Nat Genet 3 219 bis 223, und in Yang Y, Nunes FA, Berencsi K, Furth EE, Gonczol E, Wilson JM (1994) , Cellular immunity to viral antigens limits El-deleted adenoviruses for gene the- rapy, Proc Natl Acad Sei U S A 91: 4407-4411 beschrieben. Die Hauptursache hierfür liegt in einer Immunantwort gegen die ebenfalls exprimierten adenoviralen Proteine. Bei der experimentellen Therapie von Krebserkrankungen ohne wesentliche Relevanz, verhindert dieser Mechanismus jedoch eine längerdauernde Transgenexpression bei der Gentherapie von Stoffwechselkrankheiten.A significant limitation when using adenovirus vectors is, however, that the transgene expression in most tissues drops significantly after 4 to 6 weeks at the latest and that a second application has little effect. Such effects are described in Davidson BL, Allen ED, Kozarsky KF, Wilson JM, Roessler BJ (1993), A model syste for in vivo gene transfer into the central nervous system using an adenoviral vector, Nat Genet 3 219 to 223, and in Yang Y, Nunes FA, Berencsi K, Furth EE, Gonczol E, Wilson JM (1994), Cellular immunity to viral antigens limits El-deleted adenoviruses for gene the- rapy, Proc Natl Acad Sei USA 91: 4407-4411. The main reason for this is an immune response against the adenoviral proteins that are also expressed. However, this mechanism prevents prolonged transgene expression in the gene therapy of metabolic diseases in experimental therapy of cancer without significant relevance.
Um dieses Problem zu beherrschen, sind zwei grundsätzlich verschiedene Wege bereits beschritten worden. Zum einen wurden Vektoren entwickelt, bei denen das adenovirale Gerüst auf das zur Expression der Transgene absolut Notwendige reduziert ist ("gutless vectors"). Die Herstellung dieser Konstrukte ist jedoch technisch äußerst anspruchsvoll, und die Vermehrung ist aufwendig. Darüber hinaus sind die Effizienz der Transfektion und die Genexpression zum gegenwärtigen Zeitpunkt für therapeutische Applikationen unbefriedigend.In order to master this problem, two fundamentally different approaches have already been taken. On the one hand, vectors were developed in which the adenoviral scaffold is reduced to what is absolutely necessary for the expression of the transgenes ("gutless vectors"). However, the production of these constructs is extremely demanding technically and the multiplication is complex. In addition, the efficiency of transfection and gene expression are currently unsatisfactory for therapeutic applications.
Bei einer anderen Strategie werden die Vektorstrukturen ihrer Vorteile wegen belassen. Statt dessen wird die Immunantwort des Wirtsorganismus unterdrückt. Es ist zwar tierexperimentell machbar, die Immunantwort systemisch zu supprimieren, dies ist aber für klinische Studien sehr problematisch, da Infektanfälligkeiten sowie ein Hervorrufen von Tumoren auftreten können. Langfristig kann diese Strategie also nur dann Erfolg haben, wenn Wege gefunden werden, die Immunstimulation lokal zu verhindern, am besten direkt an der Antigen tragenden, adenoviral trans- fizierten Zelle. Aufgabe der vorliegenden Erfindung ist es, adenovirale Vektoren der einleitend genannten Art derart zu erzeugen, daß die Veränderung einer lokalen Immunstimulation unterstützt wird.Another strategy leaves the vector structures for their advantage. Instead, the immune response of the host organism is suppressed. It is feasible in animal experiments to suppress the immune response systemically, but this is very problematic for clinical studies, since susceptibility to infections and the development of tumors can occur. In the long term, this strategy can only be successful if ways are found to prevent immunostimulation locally, ideally directly on the antigen-carrying, adenovirally transfected cell. The object of the present invention is to generate adenoviral vectors of the type mentioned in the introduction in such a way that the change in a local immune stimulation is supported.
Diese Aufgabe wird erfindungsgemäß dadurch gelöst, daß das spezifische Gen mit mindestens einem immunmodulatorischen viralen Gen im gleichen adenoviralen Vektor koex- primiert wird.This object is achieved in that the specific gene is coexpressed with at least one immunomodulatory viral gene in the same adenoviral vector.
Viele Viren, die klassischerweise zu persistierenden Infektionen führen, sind geradezu darauf spezialisiert, das lokale Netzwerk zwischen immunologischen Abwehrzellen, insbesondere Natürlichen Killer [NK] -Zellen und T- Ly phozyten, und ihren Botenstoffen, beispielsweise Che- mokinen und Cytokinen, zu durchbrechen. Besonders interessant sind hierbei verschiedene Vertreter der Herpes- viren, beispielsweise die Cytomegaloviren (CMV) , bei denen verschiedene Genprodukte immunmodulatorische Eigenschaften zeigen, sogar dann, wenn sie isoliert exprimiert werden.Many viruses, which traditionally lead to persistent infections, specialize in breaking through the local network between immunological defense cells, especially natural killer [NK] cells and T-Ly phocytes, and their messenger substances, for example chemokines and cytokines. Of particular interest here are various representatives of the herpes viruses, for example the cytomegaloviruses (CMV), in which different gene products show immunomodulatory properties, even when they are expressed in isolation.
Der erfindungsgemäße Lösungsansatz besteht darin, adenovirale Vektoren zusätzlich zu dem zu exprimierenden spezifischen Gen, beispielsweise einem therapeutischen Transgen oder einem Reportergen, mit einem einzelnen oder mehreren immunmodulatorischen viralen Genen auszustatten, um ihre Elimination zu vermeiden.The approach according to the invention is to provide adenoviral vectors with one or more immunomodulatory viral genes in addition to the specific gene to be expressed, for example a therapeutic transgene or a reporter gene, in order to avoid their elimination.
Gemäß einer bevorzugten Ausführungsform ist vorgesehen, therapeutische Gene oder Reportergene mit immunmodulatorischen Genen der Familie der Herpes- und POX- Viren im gleichen adenoviralen Vektor zu koexprimieren . Mit derartigen Vektoren ist es möglich, eine signifikant verlängerte Expression der therapeutischen Gene oder der Reportergene zu erzielen sowie erneute Virusgaben zu ermöglichen. Im Hinblick auf die großen Ähnlichkeiten etwa zwischen dem Maus-CMV und dem humanen CMV in der Funktion einiger homologer Gene können sich daraus grundsätzlich neue Ansätze zur Unterstützung einer längerfristigen therapeutischen Genexpression bei der Therapie von Stoffwechsel- oder Autoimmunerkrankungen des Menschen ergeben.According to a preferred embodiment, to co-express therapeutic genes or reporter genes with immunomodulatory genes of the herpes and POX virus family in the same adenoviral vector. With such vectors it is possible to achieve a significantly prolonged expression of the therapeutic genes or the reporter genes as well as to enable renewed virus administration. In view of the great similarities between, for example, the mouse CMV and the human CMV in the function of some homologous genes, new approaches to support longer-term therapeutic gene expression in the treatment of metabolic or autoimmune diseases in humans can arise.
Die nötigen Techniken zur Herstellung rekombinanter Adenovirusvektoren der Serogruppe 5 (Ad5) sind im Labor etabliert. Wegen einer deletierten El-Region des Virusgenoms sind diese Vektoren nicht replikationsfähig (nur in 293- Zellen) . Eine weitere Deletion liegt in der E3-Region. Die Viruskonstruktion erfolgt gemäß einem von Graham et al. entwickelten Protokoll. Die zu exprimierenden Trans- gene werden in ein pAd-Shuttleplasmid kloniert, wobei die Expressionskassette von homologen Sequenzen des Ad-5 flankiert wird. Dieses Plasmid wird mit pBHG 10, welches wesentliche Anteile des Ad5-Genoms enthält, nach der Cal- ciumchloridPräzipitationsmethode in 293-Zellen kotransfi- ziert, eine humane embryonale Nieren-Zelllinie, die stabil mit ca. 11% des Ad5-Genoms transfiziert ist. Durch homologe Rekombination wird die Expressionskassette in die deletierte El-Region des Virusgenoms integriert, und es entstehen rekombinante infektiöse Viruspartikel . Die Aufreinigung der Viren aus dem Kulturüberstand erfolgt durch Cäsiumchlorid-Dichtegradientenzentri- fugation. Der Virus-titer wird mittels Plaque-Assay in 293-Zellen ermittelt.The necessary techniques for producing recombinant serogroup 5 (Ad5) adenovirus vectors have been established in the laboratory. Because of a deleted El region of the virus genome, these vectors are not capable of replication (only in 293 cells). Another deletion is in the E3 region. The virus is constructed according to one of Graham et al. developed protocol. The transgenes to be expressed are cloned into a pAd shuttle plasmid, the expression cassette being flanked by homologous sequences of the Ad-5. This plasmid is co-transfected with pBHG 10, which contains essential parts of the Ad5 genome, according to the calcium chloride precipitation method in 293 cells, a human embryonic kidney cell line that is stably transfected with approximately 11% of the Ad5 genome. Homologous recombination integrates the expression cassette into the deleted El region of the virus genome, and recombinant infectious virus particles are created. The viruses from the culture supernatant are purified by cesium chloride density gradient centrifugation. The virus titer is determined by means of a plaque assay in 293 cells.
In den Zeichnungen sind Ausführungsbeispiele der Erfindung schematisch dargestellt. Es zeigt:Exemplary embodiments of the invention are shown schematically in the drawings. It shows:
Fig. 1 Eine schematische Darstellung rekombinanter Adenoviren. Als Transgen ist das Reportergen Luciferase gewählt, das in die deletierte El-Region des Adenovirus Serotyp 5 kloniert und dort unter der Kontrolle des CMV-Promotors exprimiert wurde.Fig. 1 is a schematic representation of recombinant adenoviruses. The reporter gene luciferase was chosen as the transgene, which was cloned into the deleted El region of the adenovirus serotype 5 and was expressed there under the control of the CMV promoter.
Erstes Ausfuhrungsbeispiel: Adenovirus zur Koexpression von Luciferase und dem MHC I-homologen Protein ml44 des Maus-CMVFirst exemplary embodiment: adenovirus for the coexpression of luciferase and the MHC I-homologous protein ml44 of the mouse CMV
Das ml44-Gen des murinen CMV (MCMV) kodiert für ein Membranprotein, welches Homologie zu den MHC Klasse I- Molekülen der Wirtszellen aufweist und auf diese Weise die NK- ellfunktion in vitro und in vivo beeinträchtigt. Auch das humane CMV besitzt ein ähnliches Gen (UL18) , dem entsprechende Eigenschaften zugeschrieben werden.The ml44 gene of murine CMV (MCMV) codes for a membrane protein which has homology to the MHC class I molecules of the host cells and in this way impairs NK ell function in vitro and in vivo. The human CMV also has a similar gene (UL18), to which the corresponding properties are attributed.
Es wurde ein Adenovirusvektor konstruiert, welcher das luc-Markergen zusammen mit dem ml44-Protein des Maus-CMV unter der Kontrolle eines CMV-Promotors exprimiert. Zwischen die beiden Transgene ist eine interne Ribosomenbin- dungsstelle (IRES) geschaltet, die auf RNA-Ebene eine ef- fiziente Translation zweier Gene ermöglicht. Dies ist in Fig. 1 dargestellt.An adenovirus vector was constructed that expresses the luc marker gene together with the mouse CMV ml44 protein under the control of a CMV promoter. An internal ribosome binding site (IRES) is connected between the two transgenes. efficient translation of two genes. This is shown in Fig. 1.
Aus dem Plasmid pcDNA3ml44 (von Dr. Helen Farrell, Ani al Health Trust, Newmarket, Suffolk, UK) wurde ein Fragment isoliert, welches die cDNA des ml44-Gens des Maus-CMV enthält. Dieses wurde vor das 5' -Ende der kodierenden IRES-Sequenz im Plasmid pIRES (Fa. Clontech) kloniert. 5' der IRES wurde dann das aus dem Vektor pluc (Fa. Promega) isolierte Luciferase-Markergen in pIRES-ml44 eingefügt.A fragment was isolated from the plasmid pcDNA3ml44 (from Dr. Helen Farrell, Ani al Health Trust, Newmarket, Suffolk, UK) which contains the cDNA of the ml44 gene of the mouse CMV. This was cloned in front of the 5 'end of the coding IRES sequence in the plasmid pIRES (from Clontech). 5 'of the IRES, the luciferase marker gene isolated from the vector pluc (from Promega) was then inserted into pIRES-ml44.
Zur Konstruktion des adenoviralen Expressionsvektors wurde dann die Expressionskassette lucIRES-ml44 unter der Kontrolle des konstitutiven CMV-Promotors in das adenovirale Expressionsplasmid kloniert . Dies ist ebenfalls in Fig. 1 veranschaulicht. So entstand das für die Kopräzi- pitation verwendbare pAd.luc-IRES-ml44.The lucIRES-ml44 expression cassette was then cloned into the adenoviral expression plasmid under the control of the constitutive CMV promoter to construct the adenoviral expression vector. This is also illustrated in Fig. 1. This is how pAd.luc-IRES-ml44, which can be used for coprecipitation, was created.
Durch Kotransfektion von pAd. luc-IRES-ml44 und pBHGIO in 293-Zellen wurde das rekombinante Adenovirus Ad.luc-IRES- ml44 generiert . Die Richtigkeit der einzelnen Klonie- rungsschritte wurde durch Sequenzanalyse der aus dem Überstand extrahierten Virus-DNA bestätigt. Die Expression des luc-Reportergens wurde in einem Standardas- say nachgewiesen.By co-transfecting pAd. luc-IRES-ml44 and pBHGIO in 293 cells, the recombinant adenovirus Ad.luc-IRES-ml44 was generated. The correctness of the individual cloning steps was confirmed by sequence analysis of the virus DNA extracted from the supernatant. The expression of the luc reporter gene was demonstrated in a standard assay.
Zweites Ausführungsbeispiel: Adenovirus zur Koexpression von Luciferase und dem CC-Chemokin-homologen Protein ml31/129 des Maus-CMV Es wurde ein Adenovirusvektor konstruiert, welcher die Luciferase zusammen mit dem ml31/129-Protein des Maus-CMV unter der Kontrolle eines CMV-Promotors exprimiert. Das ml3l/129 gehört zu den Homologen der CC-Chemokine und trägt signifikant zur Etablierung einer persistenten Infektion von Maus-CMV in der Leber bei. Auch beim humanen CMV sind Chemokin-homologe Gene bekannt.Second embodiment: adenovirus for the coexpression of luciferase and the CC chemokine-homologous protein ml31 / 129 of the mouse CMV An adenovirus vector was constructed which expresses the luciferase together with the ml31 / 129 protein of the mouse CMV under the control of a CMV promoter. The ml3l / 129 belongs to the homologues of the CC chemokines and contributes significantly to the establishment of a persistent infection of mouse CMV in the liver. Chemokine homologous genes are also known in human CMV.
Die cDNA des ml31/129-Gens aus dem Plasmid pcDNA3ml31/129 (von Dr. Helen Farrell, Animal Health Trust, Newmarket, Suffolk, UK) wurde durch geeignete Restriktionsenzyme isoliert und zunächst in plRES einkloniert. In das resultierende pIRESml31/l29 wurde anschließend das luc- Markergen eingefügt. Aus dem entstandenen Plasmid pluc- IRES-ral3l/l29 wurde die vollständige Expressionskassette isoliert und unter der Kontrolle des CMV -Promotors in das adenovirale Expressionsplasmid kloniert, so daß der für die Kopräzipitation verwendbare Vektor pAd.luc-IRES- ml3l/129 entstand.The cDNA of the ml31 / 129 gene from the plasmid pcDNA3ml31 / 129 (from Dr. Helen Farrell, Animal Health Trust, Newmarket, Suffolk, UK) was isolated by suitable restriction enzymes and first cloned into plRES. The luc marker gene was then inserted into the resulting pIRESml31 / l29. The complete expression cassette was isolated from the resulting plasmid pluc-IRES-ral3l / l29 and cloned into the adenoviral expression plasmid under the control of the CMV promoter, so that the vector pAd.luc-IRES-ml3l / 129 which could be used for coprecipitation was formed.
In den obigen Ausfuhrungsbeispielen erfolgt die Expression bicistronisch, dies bedeutet, daß die Luciferase und das immunmodulatorische Gen auf der gleichen messenger- RNA transkribiert werden. Eine interne Ribosomenbindungs- stelle (IRES) erlaubt die effiziente Translation beider Proteine .In the above exemplary embodiments, the expression is bicistronic, which means that the luciferase and the immunomodulatory gene are transcribed on the same messenger RNA. An internal ribosome binding site (IRES) enables the efficient translation of both proteins.
Mögliche Realisierungsmöglichkeiten sind in der nachfolgenden Aufstellung zusammengefaßt . A) NK-Zell-InhibitionPossible implementation options are summarized in the following list. A) NK cell inhibition
MCMV: ml44 (MHCI-Homolog)MCMV: ml44 (MHCI homolog)
(Quelle: Farrell (1997: Nature 386:510)(Source: Farrell (1997: Nature 386: 510)
HCMV:HCMV:
1. ÜL18 (MHC-Homolog)1. ÜL18 (MHC homolog)
(Quelle: Reyburn (1997): Nature 386:514)(Source: Reyburn (1997): Nature 386: 514)
2. U 40 (Induktion von HLA-E)2. U 40 (induction of HLA-E)
(Quelle: Tomasec (2000): Science 287:1031)(Source: Tomasec (2000): Science 287: 1031)
HHV-8:HHV-8:
K5 (Downregulation von ICAM-1/B7-2)K5 (down regulation of ICAM-1 / B7-2)
(Quelle: Ishido (2000): Immunity 13:365)(Source: Ishido (2000): Immunity 13: 365)
B) Chemokin-homologe Agonisten / AntagonistenB) Chemokine homologous agonists / antagonists
MCMV: ml31/129 (CC-homolog)MCMV: ml31 / 129 (CC homolog)
(Quelle: Fleming (1999): J Virol 73:6800)(Source: Fleming (1999): J Virol 73: 6800)
HCMV:HCMV:
UL146 (vCXCL-1)UL146 (vCXCL-1)
(Quelle: Penfold (1999) : Proc Natl Acad Sei USA 96:9839)(Source: Penfold (1999): Proc Natl Acad Sei USA 96: 9839)
HHV-6: U83HHV-6: U83
(Quelle: Zou (1999): J Virol 73:5926) HHV- 8 :(Source: Zou (1999): J Virol 73: 5926) HHV- 8:
VMIP - IIVMIP - II
(Quelle: Kledal (1997): Science 227:1656(Source: Kledal (1997): Science 227: 1656
MCV:MCV:
MC1 8MC1 8
(Quelle: Lüttichau (2000) : J Exp Med 191:171)(Source: Liège (2000): J Exp Med 191: 171)
C) Chemokinrezeptor-homologe ProteineC) Chemokine receptor homologous proteins
MCMV:MCMV:
M33M33
(Quelle: Davis-Poynter (1997): j Virol 71:1521)(Source: Davis-Poynter (1997): j Virol 71: 1521)
HCMV:HCMV:
US28US28
(Quelle: Neote (1993): Cell 72:415)(Source: Neote (1993): Cell 72: 415)
HHV-6 :HHV-6:
1. U121. U12
(Quelle: Iseqawa (1998): J Virol 72:6104)(Source: Iseqawa (1998): J Virol 72: 6104)
2. U512. U51
(Quelle: Go pels (1996) : Virology 209:29)(Source: Go pels (1996): Virology 209: 29)
HHV-7:HHV-7:
1. U121. U12
(Quelle: Nicolas (1996): J. Virol 70:5975)(Source: Nicolas (1996): J. Virol 70: 5975)
2. U512. U51
(Quelle: Nicolas (1996): J. Virol 70:5975)(Source: Nicolas (1996): J. Virol 70: 5975)
HH -8: vGPCRHH -8: vGPCR
(Quelle: Cesarman (1996): J Virol 70:8218) In der obigen tabellarischen Zusammenstellung bedeuten:(Source: Cesarman (1996): J Virol 70: 8218) In the above table, they mean:
MCMV: Murines Cytomegalovirus HCMV: Humanes Cytomegalovirus HHV-6: Humanes Herpesvirus 6 HHV-7: Humanes Herpesvirus 7 HHV-8: Humanes Herpesvirus 8 MCV: Molluscum contagiosum- irusMCMV: Murine cytomegalovirus HCMV: Human cytomegalovirus HHV-6: Human herpes virus 6 HHV-7: Human herpes virus 7 HHV-8: Human herpes virus 8 MCV: Molluscum contagiosum-irus
Die obige tabellarische Zusammenfassung stellt keine abschließende Auflistung dar, sondern es werden lediglich beispielhaft Realisierungsmöglichkeiten zusammengefaßt. The above tabular summary does not represent a conclusive list, but rather only exemplary implementation options are summarized.

Claims

P a t e n t a n s p r ü c h e Patent claims
1. Adenovirale Vektoren zum Transfer von spezifischen Genen in Körperzellen hinein, dadurch gekennzeichnet, daß das spezifische Gen mit mindestens einem immunmodulatorische viralen Gen im gleichen adenoviralen Vektor koexprimiert wird.1. Adenoviral vectors for the transfer of specific genes into body cells, characterized in that the specific gene is co-expressed with at least one immunomodulatory viral gene in the same adenoviral vector.
2. Adenovirale Vektoren nach Anspruch 1, dadurch gekennzeichnet, daß das immunmodulatorische virale Gen zur Familie der Herpesviren gehört .2. Adenoviral vectors according to claim 1, characterized in that the immunomodulatory viral gene belongs to the family of herpes viruses.
3. Adenovirale Vektoren nach Anspruch 1, dadurch gekennzeichnet, daß das immunmodulatorische virale Gen zur Familie der POX-Viren gehört .3. Adenoviral vectors according to claim 1, characterized in that the immunomodulatory viral gene belongs to the family of POX viruses.
4. Adenovirale Vektoren nach einem der Ansprüche 1 bis 3, dadurch gekennzeichnet, daß die adenoviralen Vektoren zur Serogruppe 5 gehören. 4. Adenoviral vectors according to one of claims 1 to 3, characterized in that the adenoviral vectors belong to the serogroup 5.
5. Adenovirale Vektoren nach einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, daß das spezifische Gen als ein therapeutisches Gen ausgebildet ist.5. Adenoviral vectors according to one of claims 1 to 4, characterized in that the specific gene is designed as a therapeutic gene.
6. Adenovirale Vektoren nach einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, daß das spezifische Gen als ein Reportergen ausgebildet ist .6. Adenoviral vectors according to one of claims 1 to 4, characterized in that the specific gene is designed as a reporter gene.
7. Adenovirale Vektoren nach einem der Ansprüche 1 bis7. Adenoviral vectors according to one of claims 1 to
6, dadurch gekennzeichnet, daß das Adenovirus zur Realisierung einer NK-Zell-Inhibition vorgesehen ist.6, characterized in that the adenovirus is provided for the realization of an NK cell inhibition.
8. Adenovirale Vektoren nach einem der Ansprüche 1 bis8. Adenoviral vectors according to one of claims 1 to
7, dadurch gekennzeichnet, daß das Adenovirus zur Koexpression von Luciferase und dem MHC I-homologen Protein ml44 des Maus-CMV vorgesehen ist.7, characterized in that the adenovirus is provided for the coexpression of luciferase and the MHC I homologous protein ml44 of the mouse CMV.
9. Adenovirale Vektoren nach einem der Ansprüche l bis 7 dadurch gekennzeichnet, daß das Adenovirus zur Koexpression von Luciferase und dem MHC I-homologen Protein UL18 des Humanen CMV vorgesehen ist.9. Adenoviral vectors according to one of claims 1 to 7, characterized in that the adenovirus is provided for the coexpression of luciferase and the MHC I homologous protein UL18 of human CMV.
10. Adenovirale Vektoren nach einem der Ansprüche 1 bis 7 dadurch gekennzeichnet, daß das Adenovirus zur Koexpression von Luciferase und dem HLA-E induzierten Protein UL40 des Humanen CMV vorgesehen ist .10. Adenoviral vectors according to one of claims 1 to 7, characterized in that the adenovirus is provided for the coexpression of luciferase and the HLA-E-induced protein UL40 of human CMV.
11. Adenovirale Vektoren nach einem der Ansprüche 1 bis 7 dadurch gekennzeichnet, daß das Adenovirus zur Koexpression von Luciferase und dem ICAM-1/B7-2 downregulierenden Protein K5 des HHV-8 vorgesehen ist. 11. Adenoviral vectors according to one of claims 1 to 7, characterized in that the adenovirus is provided for the coexpression of luciferase and the ICAM-1 / B7-2 downregulating protein K5 of the HHV-8.
12. Adenovirale Vektoren nach einem der Ansprüche 1 bis 6, dadurch gekennzeichnet, daß das Adenovirus zur Expression eines chemokin- omologen Agonisten verwendet wird.12. Adenoviral vectors according to one of claims 1 to 6, characterized in that the adenovirus is used for the expression of a chemokinomologic agonist.
13. Adenovirale Vektoren nach einem der Ansprüche 1 bis 6, dadurch gekennzeichnet, daß das Adenovirus zur Expression eines chemoki -homologen Antagonisten verwendet wird.13. Adenoviral vectors according to one of claims 1 to 6, characterized in that the adenovirus is used to express a chemoki-homologous antagonist.
14. Adenovirale Vektoren nach einem der Ansprüche 1 bis 13, dadurch gekennzeichnet, daß das Adenovirus zur Koexpression von Luciferase und dem CC- Chemoki -homologen Protein ml31/l29 des Maus-CMV vorgesehen ist.14. Adenoviral vectors according to one of claims 1 to 13, characterized in that the adenovirus is provided for the coexpression of luciferase and the CC chemokine homologous protein ml31 / l29 of the mouse CMV.
15. Adenovirale Vektoren nach einem der Ansprüche 1 bis 13, dadurch gekennzeichnet, daß das Adenovirus zur Koexpression von Luciferase und dem vCXCL-1- homologen Protein UL146 des Humanen-CMV vorgesehen ist.15. Adenoviral vectors according to one of claims 1 to 13, characterized in that the adenovirus is provided for the coexpression of luciferase and the vCXCL-1 homologous protein UL146 of human CMV.
16. Adenovirale Vektoren nach einem der Ansprüche 1 bis 13, dadurch gekennzeichnet, daß das Adenovirus zur Koexpression von Luciferase und dem Protein vMlP-ll des HHV-8 vorgesehen ist.16. Adenoviral vectors according to one of claims 1 to 13, characterized in that the adenovirus is provided for the coexpression of luciferase and the protein vMlP-II of HHV-8.
17. Adenovirale Vektoren nach einem der Ansprüche 1 bis 13, dadurch gekennzeichnet, daß das Adenovirus zur Koexpression von Luciferase und dem Protein MC148 des Molluscum contagiosum-Virus vorgesehen ist.17. Adenoviral vectors according to one of claims 1 to 13, characterized in that the adenovirus is provided for the coexpression of luciferase and the protein MC148 of the Molluscum contagiosum virus.
18. Adenovirale Vektoren nach einem der Ansprüche 1 bis 6, dadurch gekennzeichnet, daß das Adenovirus zur Koexpression von Luciferase und einem chemokin- rezeptor-homologen Protein vorgesehen ist.18. Adenoviral vectors according to one of claims 1 to 6, characterized in that the adenovirus for coexpression of luciferase and a chemokine-receptor-homologous protein.
19. Adenovirale Vektoren nach einem der Ansprüche 1 bis 18, dadurch gekennzeichnet, daß das Adenovirus zur Koexpression von Luciferase und dem Protein M33 des Maus-CMV vorgesehen ist.19. Adenoviral vectors according to one of claims 1 to 18, characterized in that the adenovirus is provided for the coexpression of luciferase and the protein M33 of the mouse CMV.
20. Adenovirale Vektoren nach einem der Ansprüche 1 bis 18, dadurch gekennzeichnet, daß das Adenovirus zur Koexpression von Luciferase und dem Protein US28 des Humanen CMV vorgesehen ist.20. Adenoviral vectors according to one of claims 1 to 18, characterized in that the adenovirus is provided for the coexpression of luciferase and the protein US28 of human CMV.
21. Adenovirale Vektoren nach einem der Ansprüche 1 bis 18, dadurch gekennzeichnet, daß das Adenovirus zur Koexpression von Luciferase und dem Protein vGPCR des HHV-8 vorgesehen ist.21. Adenoviral vectors according to one of claims 1 to 18, characterized in that the adenovirus is provided for the coexpression of luciferase and the protein vGPCR of HHV-8.
22. Adenovirale Vektoren nach einem der Ansprüche 1 bis 18, dadurch gekennzeichnet, daß das Adenovirus zur Koexpression von Luciferase und dem Protein U12 des HHV-6 vorgesehen ist.22. Adenoviral vectors according to one of claims 1 to 18, characterized in that the adenovirus is provided for the coexpression of luciferase and the protein U12 of HHV-6.
23. Adenovirale Vektoren nach einem der Ansprüche 1 bis 18, dadurch gekennzeichnet, daß das Adenovirus zur Koexpression von Luciferase und dem Protein U51 des HHV-6 vorgesehen ist.23. Adenoviral vectors according to one of claims 1 to 18, characterized in that the adenovirus is provided for the coexpression of luciferase and the protein U51 of HHV-6.
24. Adenovirale Vektoren nach einem der Ansprüche 1 bis 18, dadurch gekennzeichnet, daß das Adenovirus zur Koexpression von Luciferase und dem Protein U12 des HHV-7 vorgesehen ist.24. Adenoviral vectors according to one of claims 1 to 18, characterized in that the adenovirus is provided for the coexpression of luciferase and the protein U12 of HHV-7.
25. Adenovirale Vektoren nach einem der Ansprüche 1 bis 18, dadurch gekennzeichnet, daß das Adenovirus zur Koexpression von Luciferase und dem Protein U51 des HHV-7 vorgesehen ist.25. Adenoviral vectors according to one of claims 1 to 18, characterized in that the adenovirus for coexpression of luciferase and the protein U51 of HHV-7 is provided.
26. Adenovirale Vektoren nach einem der Ansprüche 1 bis 25, dadurch gekennzeichnet, daß das Adenovirus statt des Luciferase-Genes mindestens ein anderes Reportergen oder mindestens ein therapeutisches Gen beinhaltet . 26. Adenoviral vectors according to one of claims 1 to 25, characterized in that the adenovirus contains at least one other reporter gene or at least one therapeutic gene instead of the luciferase gene.
PCT/DE2003/003036 2002-11-21 2003-09-12 Adenoviral vectors for the transfer of specific genes into somatic cells WO2004046364A1 (en)

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