EP0716711A1 - Vector for gene therapy of the liver - Google Patents

Vector for gene therapy of the liver

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Publication number
EP0716711A1
EP0716711A1 EP94925337A EP94925337A EP0716711A1 EP 0716711 A1 EP0716711 A1 EP 0716711A1 EP 94925337 A EP94925337 A EP 94925337A EP 94925337 A EP94925337 A EP 94925337A EP 0716711 A1 EP0716711 A1 EP 0716711A1
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Prior art keywords
vector
hepatitis
liver
gene
vector according
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EP94925337A
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German (de)
French (fr)
Inventor
Michael Strauss
Volker Sandig
Christian Hofmann
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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Priority claimed from DE4339922A external-priority patent/DE4339922C1/en
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Publication of EP0716711A1 publication Critical patent/EP0716711A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention relates to a vector for liver-specific gene therapy; Areas of application are medicine and genetic engineering.
  • the aim of the invention is to construct a vector which finds liver cells highly specific in vivo, is effectively absorbed by the cells and can introduce introduced therapeutic genes into the cell nucleus.
  • the vector is said to be usable for gene therapy in humans.
  • a therapeutic gene which is coupled to a promoter is packaged in a polypeptide envelope and chemically, enzymatically or via antibodies coupled to protein domains of the HBV.
  • the therapeutic gene used is the cDNA of a gene which is defective in the disease to be treated, i.e. H. is missing or changed by mutation. Examples of such genes are the LDL receptor gene, the absence of which causes the most common metabolic disease of the liver, familial hypercholesterolemia, and the alpha-1 antitrypsin gene.
  • Liver-specific promoters preferably promoters / enhancers of the hepatitis B virus, such as e.g. the combinations core-promoter / enhancer II. In addition to their specificity, they are also small enough to be easily incorporated into an expression vector. Promoters of liver-specific genes such as albumin, PEPCK (phosphoenolpyruvate carboxykinase) or OTC (ornithine transcarbamylase) are also suitable for the construction of the vector according to the invention.
  • promoters / enhancers of the hepatitis B virus such as e.g. the combinations core-promoter / enhancer II.
  • Promoters of liver-specific genes such as albumin, PEPCK (phosphoenolpyruvate carboxykinase) or OTC (ornithine transcarbamylase) are also suitable for the construction of the vector according to the invention.
  • the polypeptide envelope used for packaging preferably consists of chromosomal protein such as. B. purified HMG1.
  • Other DNA-binding proteins such as e.g. B. Protamine or hepatitis core protein. This protein is particularly suitable because, in addition to its DNA binding and DNA condensation ability, it is a natural component of the hepatitis B virus and therefore favors incorporation into virus envelopes.
  • the polypeptide shell can also be produced from polyamino acids of a type of basic amino acids, poly-L-lysine and poly-L-ornithine being primarily suitable.
  • the particulate pre-SI / S protein of the hepatitis B virus used as a coupled component according to the invention can be isolated from virus-producing cells.
  • pre-Sl / S protein is advantageously produced by genetic engineering for safety reasons.
  • nucleic acid-free particles represent a complete virus envelope when viewed from the outer surface.
  • the resulting vector thus has a high degree of homology to the natural hepatitis B virus and can therefore understand the infection mechanism.
  • the invention can also be implemented with liposomes as transport vehicles.
  • the surface of the liposomes used is modified by pre-Sl / S protein so that uptake via hepatitis B-specific mechanisms is possible.
  • the vectors are produced in accordance with claim 10, subclaims 11 to 13 are preferred variants.
  • An advantageous method is e.g. in that the coupling of the gene packaged in HMG1 to pre-S / S1 proteins of HBV takes place covalently by means of a transglutaminase reaction.
  • the vector according to the invention enables a desired gene to be introduced into the tissue, in particular the liver of a patient, and to optimally design its path to the functional site. This is done, for example, by making up the vector and administering it to a patient via the bloodstream, preferably via the portal vein of the liver.
  • the invention creates an essential prerequisite for therapy of genetic diseases of the liver.
  • the envelope of the HBV virus consists of three proteins. They are translation products of an open reading frame in the HBV genome with different initiation sites. While the large coat protein (L: P39, GP42) contains the domains pre-S1, pre-S2 and S, the middle (M: P33, GP36) consists of pre-S2 and S and the small coat protein (S: P24 , GP27) only from the domain S.
  • the genes of the small (S) and large (L) HBV coat protein are obtained by amplification from the genome of the hepatitis B virus (subtype ayw). Various variants are created for the L gene in order to facilitate the secretion of the protein.
  • the recombinant plasmids and baculovirus DNA are cotransfected with lipofectin in Spodoptera frugiperda cells (Sf9).
  • Sf9 Spodoptera frugiperda cells
  • recombinant baculoviruses are generated, which express the HBV envelope proteins under control of the polyhedrin promoter in infected Sf9 cells. The synthesis of these proteins is demonstrated in the Western blot. Electron microscopy shows that the coat proteins associate to form particles.
  • spinner cultures (10 9 Sf9 cells) are infected. 72 hours after infection, the cells are obtained by sedimentation and disrupted using ultrasound. After separation of membranes by sedimentation, the shell particles are cleaned either by centrifugation in the CsCl density gradient or by affinity chrootography. S-specific monoclonal antibodies are used here.
  • the gene of the rat non-histone protein HMG1 is taken from the vector for bacterial expression pT7RNHMGl [Bianchi, E., Gene, 104 (1991) 271-275] and cloned into the baculovirus transfer vector PVL941. Recombinant baculoviruses are generated according to the method described for HBV genes. The expression detection is carried out by Coomassie staining of the proteins after SDS polyacrylamide gel electrophoresis.
  • a cell lysate is generated by disruption using ultrasound, membranes are separated by sedimentation and the lysate is precipitated with 2% TCA. The supernatant is then subjected to acetone precipitation in an acidic medium. The resulting precipitate is dissolved and fractionated by ion exchange chromatography on a Mono Q column in a saline gradient. The fraction eluted at 1.7M NaCl contains electrophoretically pure HMG1.
  • the DNA-protein complexes are formed by gradually adding a 20-fold excess of HMG1 to a DNA solution of 50 ⁇ g / ml in 150mM NaCl 10MM Tris / HCl at pH 8.0. DNA binding and condensation are detected by gel retardation and sedimentation in the sucrose gradient.
  • HBV coat proteins are covalently cross-linked with HMG1 using transglutaminase.
  • e-amino groups of the lysines in the HMG molecule act as acyl acceptors and 7-carboxamide groups of the glutamine residues in HBV-L and S protein as acyl donors.
  • a confluent monolayer culture of human hepatocytes is infected with the transfer vector 5 days after installation for 12 hours.
  • the Plas id pBAG [Proc. Natl. Acad. Be. 84 (1987) 156-160], which contains the gene of the ⁇ -galactosidase from E. coli under the control of a retroviral LTR, is packed into the vector. The gene transfer is detected 48 hours after infection by the in tu enzyme test for ⁇ -galactosidase.

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Abstract

A liver-specific, gene therapy vector finds applications in medicine and genetic engineering. The object of the invention is to develop a vector that finds liver cells with high specificity, is effectively absorbed by the cells and may inject the imported therapeutic genes into the cell nucleus. The vector should be suitable for the gene therapy of human beings. The vector according to the invention is characterised in that a therapeutic gene coupled to a promoter is packed in a polypeptide sheath and coupled to components of hepatitis B virus.

Description

Vektor für Leber-GentherapieVector for liver gene therapy
Beschreibungdescription
Die Erfindung betrifft einen Vektor für die leberspezifische Gentherapie; Anwendungsgebiete sind die Medizin und die Gentechnik.The invention relates to a vector for liver-specific gene therapy; Areas of application are medicine and genetic engineering.
In den vergangenen Jahren sind zahlreiche Methoden und Vektoren für die Gentherapie entwickelt worden (Übersicht in Mulligan /1993/ Science 260, 926). Dabei werden viele Vektoren, vor allem solche, die von Retroviren oder Adenoviren abgeleitet sind, favorisiert. Beide Virus-Vektortypen sind relativ breit anwendbar, wobei retrovirale Vektoren nur bei teilungsfähigen Zellen effektiv sind und Adenoviren auch bei ruhenden Zellen funktionieren. Beide Vektortypen sind zwar für die Genübertragung in Leberzellen (Hepatozyten) in vitro geeignet, können aber für eine in vivo Anwendung zur Gentherapie beim Menschen kaum in Betracht gezogen werden. Während für die Anwendung retroviraler Vektoren eine Leberteilresektion zur Stimulierung von Zellteilung (Regeneration) erforderlich wird, ist der adenovirale Gentransfer nicht sehr stabil (keine Integration in das Genom) .Numerous methods and vectors for gene therapy have been developed in recent years (overview in Mulligan / 1993 / Science 260, 926). Many vectors are favored, especially those derived from retroviruses or adenoviruses. Both types of virus vector can be used relatively broadly, retroviral vectors being effective only with dividable cells and adenoviruses also working with resting cells. Both types of vector are suitable for gene transmission in liver cells (hepatocytes) in vitro, but can hardly be considered for in vivo use in gene therapy in humans. While a partial liver resection to stimulate cell division (regeneration) is required for the use of retroviral vectors, adenoviral gene transfer is not very stable (no integration into the genome).
Alternative Vektoren mit potentieller Anwendbarkeit für den Lebergentransfer basieren auf Liposomen oder auch auf Multikomponenten-Partikeln mit Proteindomänen, die spezifisch an bestimmte Rezeptoren der Leber (z.B. Asialoglykoprotein- Rezeptor) binden und durch deren Internalisierung in die Zelle aufgenommen werden können (Übersicht in: Versland et al /1992/ Seminors in Liver Disease 12, 332). Ein wesentlicher Nachteil dieser Vektoren besteht in der Aufnahme über den endozytokischen Weg, der zu einer Degration eines großen Teils der Vektoren und ihrer DNA in den Endosomen führt, so daß nur wenig funktionsfähige DNA den Zellkern erreichen kann. Eine Lösung für dieses Problem wurde zwar für die in vitro Anwendung gefunden; diese ist aber nicht auf die Situation in vivo (am Patienten) anwendbar. Sie basiert auf der gleichzeitigen Infektion der Zielzellen mit Adenovirus, was zur Auflösung der Endosomen und Freisetzung von Vektor (DNA) führt. (Curiel, D.T. , Agrawal, S., Wagner, E. und Cotten,M. 1991, PNAS 88, 8850-8854). Ziel der Erfindung ist die Konstruktion eines Vektors, der Leberzellen in vivo hochspezifisch findet, von den Zellen effektiv aufgenommen wird und eingebrachte therapeutische Gene in den Zellkern einschleusen kann. Der Vektor soll für die Gentherapie beim Menschen einsetzbar sein.Alternative vectors with potential applicability for liver transfer are based on liposomes or also on multicomponent particles with protein domains that bind specifically to certain receptors of the liver (e.g. asialoglycoprotein receptor) and which can be absorbed into the cell by internalizing them (overview in: Versland et al / 1992 / Seminors in Liver Disease 12, 332). A major disadvantage of these vectors is that they are taken up by the endocytocial route, which leads to a degration of a large part of the vectors and their DNA in the endosomes, so that only little functional DNA can reach the cell nucleus. A solution to this problem has been found for in vitro use; however, this is not applicable to the situation in vivo (on the patient). It is based on the simultaneous infection of the target cells with adenovirus, which leads to the dissolution of the endosomes and release of vector (DNA). (Curiel, DT, Agrawal, S., Wagner, E. and Cotten, M. 1991, PNAS 88, 8850-8854). The aim of the invention is to construct a vector which finds liver cells highly specific in vivo, is effectively absorbed by the cells and can introduce introduced therapeutic genes into the cell nucleus. The vector is said to be usable for gene therapy in humans.
Die Erfindung wird gemäß Anspruch 1 realisiert, die Unteransprüche 2 - 9 sind Vorzugsvarianten.The invention is implemented according to claim 1, subclaims 2-9 are preferred variants.
Erfindungsgemäß wird ein therapeutisches Gen, das an einen Promoter gekoppelt ist, in eine Polypeptidhülle verpackt und mit Proteindomänen des HBV chemisch, enzy atisch oder über Antikörper gekoppelt. Als therapeutisches Gen wird die cDNA eines Gens verwendet, das bei der zu behandelten Krankheit defekt ist, d. h. fehlt oder durch Mutation verändert ist. Beispiele für solche Gene sind das LDL-Rezeptor-Gen, dessen Fehlen die häufigste StoffWechselerkrankung der Leber, die familiäre Hypercholesterinämie, verursacht, und das Alpha-1- Antitrypsin-Gen.According to the invention, a therapeutic gene which is coupled to a promoter is packaged in a polypeptide envelope and chemically, enzymatically or via antibodies coupled to protein domains of the HBV. The therapeutic gene used is the cDNA of a gene which is defective in the disease to be treated, i.e. H. is missing or changed by mutation. Examples of such genes are the LDL receptor gene, the absence of which causes the most common metabolic disease of the liver, familial hypercholesterolemia, and the alpha-1 antitrypsin gene.
Als Promotoren dienen leberspezifische Promotoren, bevorzugt Promoter/Enhancer des Hepatitis-B-Virus wie z.B. die Kombinationen core-Promoter/ enhancer II. Sie sind neben ihrer Spezifität auch klein genug, um leicht in einen Expressionsvektor eingebaut werden zu können. Auch in Betracht für die Konstruktion des erfindungsgemäßen Vektors kommen Promotoren leberspezifischer Gene wie Albumin, PEPCK (Phosphoenolpyruvat-carboxykinase) oder OTC (Ornithintrans- carbamylase) .Liver-specific promoters, preferably promoters / enhancers of the hepatitis B virus, such as e.g. the combinations core-promoter / enhancer II. In addition to their specificity, they are also small enough to be easily incorporated into an expression vector. Promoters of liver-specific genes such as albumin, PEPCK (phosphoenolpyruvate carboxykinase) or OTC (ornithine transcarbamylase) are also suitable for the construction of the vector according to the invention.
Die für die Verpackung verwendete Polypeptidhülle besteht vorzugsweise aus chromosomalem Protein wie z. B. gereinigtem HMG1. Gleichermaßen geeignet sind auch andere DNA-bindende Proteine wie z. B. Protamine oder Hepatitis-core-Protein. Dieses Protein ist deshalb besonders geeignet, weil es außer seiner DNA-Bindungs- und DNA-Kondensationsfähigkeit eine natürliche Komponente des Hepatitis-B-Virus ist und deshalb den Einbau in Virushüllen begünstigt.The polypeptide envelope used for packaging preferably consists of chromosomal protein such as. B. purified HMG1. Other DNA-binding proteins such as e.g. B. Protamine or hepatitis core protein. This protein is particularly suitable because, in addition to its DNA binding and DNA condensation ability, it is a natural component of the hepatitis B virus and therefore favors incorporation into virus envelopes.
Die Polypeptidhülle kann gemäß der Erfindung auch aus Polyaminosäuren eines Typs basischer Aminosäuren hergestellt werden, wobei sich in erster Linie Poly-L-Lysin und Poly-L- Ornithin eignen. Das erfindungsgemäß als gekoppelte Komponente eingesetzte partikuläre prä-Sl/S-Protein des Hepatitis-B-Virus kann aus Virus-produzierenden Zellen isoliert werden. Prä-Sl/S-Protein wird aber aus Sicherheitsgründen vorteilhafterweise gentechnisch hergestellt. Derartige Nukleinsäure-freie Partikel stellen von der äußeren Oberfläche gesehen eine vollständige Virushülle dar. Damit hat der resultierende Vektor eine hohe Homologie zum natürlichen Hepatitis-B-Virus und kann deshalb den Infektionsmechanismus nachvollziehen.According to the invention, the polypeptide shell can also be produced from polyamino acids of a type of basic amino acids, poly-L-lysine and poly-L-ornithine being primarily suitable. The particulate pre-SI / S protein of the hepatitis B virus used as a coupled component according to the invention can be isolated from virus-producing cells. However, pre-Sl / S protein is advantageously produced by genetic engineering for safety reasons. Such nucleic acid-free particles represent a complete virus envelope when viewed from the outer surface. The resulting vector thus has a high degree of homology to the natural hepatitis B virus and can therefore understand the infection mechanism.
Anstelle der Verwendung der kompletten Virushüllproteine läßt sich die Erfindung auch mit Liposomen als Transportvehikeln realisieren. Dabei wird die Oberfläche der verwendeten Liposomen durch prä-Sl/S-Protein so modifiziert, daß eine Aufnahme über Hepatitis-B-spezifischen Mechanismen möglich ist. Die Herstellung der Vektoren erfolgt gemäß Anspruch 10, die Unteransprüche 11 - 13 sind Vorzugsvarianten. Ein vorteilhaftes Verfahren besteht z.B. darin, daß die Kopplung des in HMG1 verpackten Gens an prä-S/Sl-Proteine des HBV kovalent mittels Transglutaminase-Reaktion erfolgt.Instead of using the complete virus envelope proteins, the invention can also be implemented with liposomes as transport vehicles. The surface of the liposomes used is modified by pre-Sl / S protein so that uptake via hepatitis B-specific mechanisms is possible. The vectors are produced in accordance with claim 10, subclaims 11 to 13 are preferred variants. An advantageous method is e.g. in that the coupling of the gene packaged in HMG1 to pre-S / S1 proteins of HBV takes place covalently by means of a transglutaminase reaction.
Der erfindungsgemäße Vektor ermöglicht, ein gewünschtes Gen in das Gewebe, insbesondere die Leber eines Patienten einzuführen und seinen Weg zum Funktionsort optimal zu gestalten. Das erfolgt beispielsweise dadurch, daß der Vektor konfektioniert und einem Patienten über die Blutbahn, vorzugsweise über die Portalvene der Leber, verabreicht wird. Mit der Erfindung wird eine wesentliche Voraussetzung für eine Therapie genetischer Erkrankungen der Leber geschaffen.The vector according to the invention enables a desired gene to be introduced into the tissue, in particular the liver of a patient, and to optimally design its path to the functional site. This is done, for example, by making up the vector and administering it to a patient via the bloodstream, preferably via the portal vein of the liver. The invention creates an essential prerequisite for therapy of genetic diseases of the liver.
Die Erfindung soll nachfolgend durch ein Ausführungsbeispiel näher erläutert werden. The invention will be explained in more detail below using an exemplary embodiment.
AnwendungsbeispielApplication example
1. Expression von HBV-Hüllproteinen in Insektenzellen1. Expression of HBV coat proteins in insect cells
Die Hülle des HBV-Virus besteht aus drei Proteinen. Sie sind Translationsprodukte eines offenen Leserahmens im HBV-Genom-mit unterschiedlichen Initiationsstellen. Während das große Hüll¬ protein (L: P39, GP42) die Domänen Prä-Sl, Prä-S2 und S enthält, besteht das mittlere (M: P33, GP36) aus Prä-S2 und S und das kleine Hüllprotein (S: P24, GP27) nur aus der Domäne S. Die Gene des kleinen (S) und großen (L) HBV-Hüllproteins werden durch Amplifikation aus dem Genom des Hepatitis B Virus (Subtyp ayw) gewonnen. Für das L-Gen werden verschiedene Varianten er¬ stellt, um die Sekretion des Proteins zu erleichtern. Sie kodie¬ ren für ein N-terminal verkürztes L-Protein (Deletion der Amino¬ säuren 1-6) , ein L-Protein, dessen Myristilierungsort mutiert wird (Aminosäure 2 Gly—>Ala) bzw. eine Fusion mit der Melli- tin-Signalsequenz.The envelope of the HBV virus consists of three proteins. They are translation products of an open reading frame in the HBV genome with different initiation sites. While the large coat protein (L: P39, GP42) contains the domains pre-S1, pre-S2 and S, the middle (M: P33, GP36) consists of pre-S2 and S and the small coat protein (S: P24 , GP27) only from the domain S. The genes of the small (S) and large (L) HBV coat protein are obtained by amplification from the genome of the hepatitis B virus (subtype ayw). Various variants are created for the L gene in order to facilitate the secretion of the protein. They code for an N-terminally truncated L protein (deletion of amino acids 1-6), an L protein whose myristilation site is mutated (amino acid 2 Gly-> Ala) or a fusion with the mellitin Signal sequence.
Alle Gene werden einzeln in den Baculovirus-Transfervektor PVL941 (Pharmingen) kloniert und stehen unter Kontrolle des Polyhedrin-Promoters. Nachträglich wird ein DNA-Fragment, beste¬ hend aus dem Polyhedrin-Promoter und dem S-Gen in alle Vektoren, die Varianten des L-Gens enthalten, derart eingefügt, daß beide Expressionseinheiten in gleicher Orientierung vorliegen und von Baculovirus-Sequenzen flankiert sind.All genes are cloned individually into the baculovirus transfer vector PVL941 (Pharmingen) and are under the control of the polyhedrin promoter. Subsequently, a DNA fragment consisting of the polyhedrin promoter and the S gene is inserted into all vectors which contain variants of the L gene in such a way that both expression units are in the same orientation and are flanked by baculovirus sequences.
Die rekombinanten Plasmide und Baculovirus-DNA (BaculoGold, Pharmingen) werden mit Lipofectin in Spodoptera frugiperda-Zel- len (Sf9) kotransfiziert. Durch homologe Rekombination zwischen Plasmid und Virus-DNA werden rekombinante Baculoviren erzeugt, welche die HBV-Hüllproteine unter Kontrolle des Polyhedrinpromo- ters in infizierten Sf9-Zellen expremieren. Die Synthese dieser Proteine wird im Westernblot nachgewiesen. Elektronenmikrosko¬ pisch wird gezeigt, daß die Hüllproteine zu Partikeln assoziie¬ ren.The recombinant plasmids and baculovirus DNA (BaculoGold, Pharmingen) are cotransfected with lipofectin in Spodoptera frugiperda cells (Sf9). By homologous recombination between plasmid and virus DNA, recombinant baculoviruses are generated, which express the HBV envelope proteins under control of the polyhedrin promoter in infected Sf9 cells. The synthesis of these proteins is demonstrated in the Western blot. Electron microscopy shows that the coat proteins associate to form particles.
Nach wiederholter Passagierung der Viren auf Sf9-Zellen werden Spinnerkulturen (109 Sf9-Zellen) infiziert. 72 Stunden nach Infektion werden die Zellen durch Sedimentation gewonnen und mittels Ultraschall aufgeschlossen. Nach Abtrennung von Membra¬ nen durch Sedimentation erfolgt die Reinigung der Hüllpartikel wahlweise durch Zentifugation im CsCl-Dichtegradienten oder durch Affinitätschro atografie. Hierbei finden S-spezifische monoklonale Antikörper Anwendung.After repeated passage of the viruses on Sf9 cells, spinner cultures (10 9 Sf9 cells) are infected. 72 hours after infection, the cells are obtained by sedimentation and disrupted using ultrasound. After separation of membranes by sedimentation, the shell particles are cleaned either by centrifugation in the CsCl density gradient or by affinity chrootography. S-specific monoclonal antibodies are used here.
2. Expression und Reinigung von HMG12. Expression and Purification of HMG1
Das Gen des Nichthistonproteins HMG1 der Ratte wird aus dem Vektor zur bakteriellen Expression pT7RNHMGl [Bianchi, E., Gene, 104 (1991) 271-275] entnommen und in den Baculovirus-Transfer¬ vektor PVL941 kloniert. Rekombinante Baculoviren werden nach dem für HBV-Gene beschriebenen Verfahren erzeugt. Der Expressions¬ nachweis erfolgt durch Coomassiefärbung der Proteine nach SDS- Polyacrylamidgelelektrophorese.The gene of the rat non-histone protein HMG1 is taken from the vector for bacterial expression pT7RNHMGl [Bianchi, E., Gene, 104 (1991) 271-275] and cloned into the baculovirus transfer vector PVL941. Recombinant baculoviruses are generated according to the method described for HBV genes. The expression detection is carried out by Coomassie staining of the proteins after SDS polyacrylamide gel electrophoresis.
Zur Reinigung von Ratten-HMGl aus infizierten Sf9-Zellen wird ein Zelllysat durch Aufschluß mit Ultraschall erzeugt, Membranen durch Sedimentation abgetrennt und das Lysat mit 2% TCA gefällt. Der Überstand wird anschließend einer Azetonfällung im sauren Milieu unterzogen. Das entstehende Prazipitat wird gelöst und durch Ionenaustauschchromatographie über eine Mono Q-Säule im Kochsalzgradient fraktioniert. Die bei 1,7M NaCl eluierte Frak¬ tion enthält elektrophoretisch reines HMG1.To purify rat HMGl from infected Sf9 cells, a cell lysate is generated by disruption using ultrasound, membranes are separated by sedimentation and the lysate is precipitated with 2% TCA. The supernatant is then subjected to acetone precipitation in an acidic medium. The resulting precipitate is dissolved and fractionated by ion exchange chromatography on a Mono Q column in a saline gradient. The fraction eluted at 1.7M NaCl contains electrophoretically pure HMG1.
3. Herstellung von DNA-HMG1-Komplexen3. Preparation of DNA-HMG1 complexes
Nach Böttger et al. [Biochim. Biophys. Acta 950 (1988) 221-228] wird Plas id-DNA der Form 3 sequenzunabhängig von HMG1 gebunden und kondensiert.According to Böttger et al. [Biochim. Biophys. Acta 950 (1988) 221-228], Plas id DNA of form 3 is bound and condensed independently of the sequence by HMG1.
Die Bildung der DNA-Protein-Komplexe erfolgt durch schrittweise Zugabe eines zwanzigfachen Masseüberschusses an HMG1 zu einer DNA-Lösung von 50μg/ml in 150mM NaCl lOmM Tris/HCl bei pH 8,0. DNA-Bindung und Kondensation werden durch Gelretardation und Sedimentation im Sacharosegradienten nachgewiesen.The DNA-protein complexes are formed by gradually adding a 20-fold excess of HMG1 to a DNA solution of 50μg / ml in 150mM NaCl 10MM Tris / HCl at pH 8.0. DNA binding and condensation are detected by gel retardation and sedimentation in the sucrose gradient.
4. Bindung von DNA-HMG1-Komplexen an HBV-Partikel4. Binding of DNA-HMG1 complexes to HBV particles
HBV-Hüllproteine werden mittels Transglutaminase kovalent mit HMG1 vernetzt. In dieser Reaktion treten e-Aminogruppen der Lysine im HMG-Molekül als Acyl-Akzeptor und 7-Karboxamidgruppen der Glutaminreste im HBV-L und S-Protein als Acyldonor auf. Die Reaktion wird lh bei 37°C in 150ιrιM NaCl, 10mM CaCl2 2θ'mM Tris/HCl pH 8,0 mit 0,5 Einheiten Meerschweinchen-Transglutami- nase bei einem Masseverhältnis von HMG1 : HBVL+S = 10:1 durch¬ geführt.HBV coat proteins are covalently cross-linked with HMG1 using transglutaminase. In this reaction, e-amino groups of the lysines in the HMG molecule act as acyl acceptors and 7-carboxamide groups of the glutamine residues in HBV-L and S protein as acyl donors. The reaction is carried out at 37 ° C. in 150 μm NaCl, 10 mM CaCl 2 2θ ′ mM Tris / HCl pH 8.0 with 0.5 units of guinea pig transglutaminase with a mass ratio of HMG1: HBVL + S = 10: 1 guided.
5.Infektion primärer humaner Hepatozyten mit dem Gentransfervek¬ tor5. Infection of primary human hepatocytes with the gene transfer vector
Eine konfluente Monolayerkultur humaner Hepatozyten wird 5 Tage nach Anlage für 12 Stunden mit dem Transfervektor infiziert. Das Plas id pBAG [Proc. Natl. Acad. Sei. 84 (1987) 156-160], das das Gen der ß-Galaktosidase aus E. coli unter Kontrolle eines retro- viralen LTR enthält, wird in den Vektor verpackt. Der Nachweis des Gentransfers erfolgt 48 Stunden nach Infektion durch den in s tu-Enzymtest für ß-Galaktosidase. A confluent monolayer culture of human hepatocytes is infected with the transfer vector 5 days after installation for 12 hours. The Plas id pBAG [Proc. Natl. Acad. Be. 84 (1987) 156-160], which contains the gene of the β-galactosidase from E. coli under the control of a retroviral LTR, is packed into the vector. The gene transfer is detected 48 hours after infection by the in tu enzyme test for β-galactosidase.

Claims

Patentansprüche claims
1. Vektor für die gewebespezifische Gentherapie, gekennzeichnet dadurch, daß ein therapeutisches Gen, das an einen Promoter gekoppelt ist, mit einer Polypeptidhülle verpackt und an Komponenten des Hepatitis B-Virus gekoppelt wird.1. Vector for tissue-specific gene therapy, characterized in that a therapeutic gene which is coupled to a promoter is packaged with a polypeptide envelope and coupled to components of the hepatitis B virus.
2. Vektor für die leberspezifische Gentherapie nach Anspruch 1, gekennzeichnet dadurch, daß ein therapeutisches Gen, das an einen Promoter gekoppelt ist, mit einer Polypeptidhülle verpackt und an Komponenten des Hepatitis B-Virus gekoppelt wird.2. Vector for liver-specific gene therapy according to claim 1, characterized in that a therapeutic gene which is coupled to a promoter is packaged with a polypeptide envelope and coupled to components of the hepatitis B virus.
3. Vektor nach Anspruch 1 und 2, gekennzeichnet dadurch, daß als therapeutisches Gen die cDNA eines Gens verwendet wird, das bei der zu behandelnden Krankheit defekt ist.3. Vector according to claim 1 and 2, characterized in that the cDNA of a gene is used as a therapeutic gene, which is defective in the disease to be treated.
4. Vektor nach Anspruch 1 bis 3, gekennzeichnet dadurch, daß als Promotor ein leberspezifischer Promoter, bevorzugt ein Promoter/Enhancer von Hepatitis B verwendet wird.4. Vector according to claim 1 to 3, characterized in that a liver-specific promoter, preferably a promoter / enhancer of hepatitis B is used as a promoter.
5. Vektor nach Anspruch 1 bis 4, gekennzeichnet dadurch, daß die Polypeptidhülle aus einem chromosomalen Protein, vorzugsweise aus gereinigtem HMG1, besteht.5. Vector according to claim 1 to 4, characterized in that the polypeptide shell consists of a chromosomal protein, preferably of purified HMG1.
6. Vektor nach Anspruch 1 bis 5, gekennzeichnet dadurch, daß die Polypeptidhülle aus core-Protein von von Hepatitis B besteht.6. Vector according to claim 1 to 5, characterized in that the polypeptide envelope consists of core protein of hepatitis B.
7. Vektor nach Anspruch 1 bis 5, gekennzeichnet dadurch, daß die Polypeptidhülle aus Polyaminosäuren eines Typs basischer Aminosäuren hergestellt wird.7. Vector according to claim 1 to 5, characterized in that the polypeptide shell is made from polyamino acids of a type of basic amino acids.
8. Vektor nach Anspruch 1 bis 7, gekennzeichnet dadurch, daß als gekoppelte Komponente partikuläres prä-Sl/S-Protein des Hepatitis B-Virus eingesetzt wird. 8. Vector according to claim 1 to 7, characterized in that particulate pre-Sl / S protein of the hepatitis B virus is used as the coupled component.
9. Vektor nach Anspruch 1 bis 8, gekennzeichnet dadurch, daß als gekoppelte Komponente Liposomen mit gebundenem prä-Sl-Peptid eingesetzt werden.9. Vector according to claim 1 to 8, characterized in that liposomes with bound pre-Sl peptide are used as the coupled component.
10. Verfahren zur Herstellung des Vektors nach Anspruch 1 bis 9, gekennzeichnet dadurch, daß die Kopplung des verpackten Gens an die Komponente(n) des Hepatitis B-Virus chemisch, enzymatisch oder über Antikörper durchgeführt wird.10. The method for producing the vector according to claim 1 to 9, characterized in that the coupling of the packaged gene to the component (s) of the hepatitis B virus is carried out chemically, enzymatically or via antibodies.
11. Verfahren zur Herstellung nach Anspruch 10, gekennzeichnet dadurch, daß die Kopplung mit einem bifunktionellen Linker, 3bevorzugt SPDP, durchgeführt wird.11. The method for producing according to claim 10, characterized in that the coupling is carried out with a bifunctional linker, preferably 3 SPDP.
12. Verfahren zur Herstellung nach Anspruch 10, gekennzeichnet dadurch, daß die Kopplung mit Transaminase durchgeführt wird.12. A method for producing according to claim 10, characterized in that the coupling is carried out with transaminase.
13. Verfahren zur Herstellung nach Anspruch 10, gekennzeichnet dadurch, daß die Kopplung mit bispezifischen Antikörpern durchgeführt wird.13. The method for producing according to claim 10, characterized in that the coupling is carried out with bispecific antibodies.
14. Verfahren zur Anwendung des Vektors nach Anspruch 1 bis 9, gekennzeichnet dadurch, daß der Vektor konfektioniert und einem Patienten über die Blutbahn, vorzugsweise über die Portalvene der Leber, verabreicht wird. 14. A method for using the vector according to claims 1 to 9, characterized in that the vector is made up and administered to a patient via the bloodstream, preferably via the portal vein of the liver.
EP94925337A 1993-09-03 1994-08-24 Vector for gene therapy of the liver Withdrawn EP0716711A1 (en)

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DE4329811 1993-09-03
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DE4339922A DE4339922C1 (en) 1993-09-03 1993-11-19 Vector for gene therapy of the liver
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DE4339922C1 (en) * 1993-09-03 1994-10-06 Max Planck Gesellschaft Vector for gene therapy of the liver
US5846782A (en) 1995-11-28 1998-12-08 Genvec, Inc. Targeting adenovirus with use of constrained peptide motifs
US6465253B1 (en) 1994-09-08 2002-10-15 Genvec, Inc. Vectors and methods for gene transfer to cells
US5770442A (en) * 1995-02-21 1998-06-23 Cornell Research Foundation, Inc. Chimeric adenoviral fiber protein and methods of using same
US6127525A (en) * 1995-02-21 2000-10-03 Cornell Research Foundation, Inc. Chimeric adenoviral coat protein and methods of using same
US20030017138A1 (en) 1998-07-08 2003-01-23 Menzo Havenga Chimeric adenoviruses
US6929946B1 (en) 1998-11-20 2005-08-16 Crucell Holland B.V. Gene delivery vectors provided with a tissue tropism for smooth muscle cells, and/or endothelial cells
US6913922B1 (en) 1999-05-18 2005-07-05 Crucell Holland B.V. Serotype of adenovirus and uses thereof
US6492169B1 (en) 1999-05-18 2002-12-10 Crucell Holland, B.V. Complementing cell lines
US7235233B2 (en) 2000-09-26 2007-06-26 Crucell Holland B.V. Serotype 5 adenoviral vectors with chimeric fibers for gene delivery in skeletal muscle cells or myoblasts
SI1497438T1 (en) 2002-04-25 2010-03-31 Crucell Holland Bv Means and methods for the production of adenovirus vectors

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