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WO1996012010A1 - Preparation of rep-negative aav mutants and cells which can be used therefor - Google Patents

Preparation of rep-negative aav mutants and cells which can be used therefor Download PDF

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Publication number
WO1996012010A1
WO1996012010A1 PCT/DE1995/001429 DE9501429W WO9612010A1 WO 1996012010 A1 WO1996012010 A1 WO 1996012010A1 DE 9501429 W DE9501429 W DE 9501429W WO 9612010 A1 WO9612010 A1 WO 9612010A1
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Prior art keywords
rep
aav
negative
cells
dna
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Application number
PCT/DE1995/001429
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German (de)
French (fr)
Inventor
Christina HÖLSCHER
Alexander BÜRKLE
Jürgen KLEINSCHMIDT
Markus HÖRER
Harald Zur Hausen
Pierre Chambon
Regine Heilbronn
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Deutsches Krebsforschungszentrum DKFZ
Max Planck Gesellschaft zur Foerderung der Wissenschaften
Original Assignee
Deutsches Krebsforschungszentrum DKFZ
Max Planck Gesellschaft zur Foerderung der Wissenschaften
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Priority claimed from DE19944436664 external-priority patent/DE4436664A1/en
Priority claimed from DE19944436665 external-priority patent/DE4436665C2/en
Application filed by Deutsches Krebsforschungszentrum DKFZ, Max Planck Gesellschaft zur Foerderung der Wissenschaften filed Critical Deutsches Krebsforschungszentrum DKFZ
Priority to EP95934604A priority Critical patent/EP0785991A1/en
Priority to JP8512845A priority patent/JPH10507352A/en
Publication of WO1996012010A1 publication Critical patent/WO1996012010A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
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    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention relates to the provision of rep-negative AAV mutants and cells which can be used therefor.
  • the invention further relates to an expression plasmid which can be used to produce the cells.
  • Adeno-associated viruses are single-stranded DNA viruses belonging to the parvovirus family. For their replication, AAVs need helper viruses, in particular adenoviruses or herpes viruses. In the absence of helper viruses, AAVs integrate into the host cell genome, particularly at a specific location on chromosome 19 or 17.
  • the 145 bp "inverted repeats" serve as the origin of replication and as ice signals for integration and packaging.
  • the cap gene codes for three structural proteins and the rep gene for a family of multifunctional regulator proteins.
  • the mRNAs for Rep 78 and its C-terminal spliced version Rep 68 start at the p5 promoter.
  • Two N-terminally shortened versions of Rep 78 and Rep 68, namely Rep 52 and Rep 40, are expressed under the control of the p19 promoter.
  • Rep proteins are necessary for AAV DNA replication. They are also required for AAV gene regulation.
  • AAVs suppress tumor development in animals. Furthermore, they suppress the cell transformation caused by oncogenes as well as the induced DNA amplification. Furthermore, AAVs have an antiproliferative activity. AAV Rep proteins are held responsible for the above activities. However, these activities are not assigned to the individual Rep proteins or domains thereof. This would be necessary in order to use AAVs therapeutically. One way to achieve this assignment is to examine AAVs that have mutations in the Rep proteins. Many attempts have been made in this regard. So far, however, it has not been possible to provide rep-negative AAV mutants which are free from wild-type AAV. However, such are indispensable for the above investigations.
  • the present invention is therefore based on the object of providing a means by which rep-negative AAV mutants can be obtained without the above disadvantages.
  • the invention thus relates to cells which stably express the AAV Rep proteins 78 and 52 and 40 and / or 68. Cells which express Rep proteins 78, 52 and 40 are preferred.
  • Cells according to the invention can be produced in the usual way.
  • Cells of the known HeM1 line are advantageously used as starting material (cf. "5th Parvovirus Workshop", Chrystal River, Florida, USA, Nov. 10-14, 1993). These cells express the AAV Rep proteins 78 and 52, the Rep 78 expression being under the control of dexamethasone-inducible MMTV-LTR.
  • HeM1 cells are transfected with an expression plasmid coding for Rep 40 and / or an expression plasmid coding for Rep 68 or an expression plasmid which codes for both Rep proteins.
  • An expression plasmid for Rep 40 is preferably used, the expression plasmid.
  • pCMRep 40 is very particularly preferred.
  • the cells obtained by transfection of pCMRep 40 stably express the AAV-Rep proteins Rep 78, Rep 52 and Rep 40. These cells were identified as cell line He 10-1, He 22-2 and He 5-5 in the DSM under DSM ACC2193 , DSM ACC2192 and DSM ACC2191 on September 28, 1994. Furthermore, the cells were deposited as cell line HeCMI g at DSM under DSM ACC2185 on August 30, 1994. The above cells are also an object of the invention.
  • Another object of the invention is a method for providing rep-negative AAV mutants.
  • Such a process comprises the following process steps:
  • process step (a) involves cotransfection with an expression plasmid coding for a glucocorticoid receptor.
  • an expression plasmid coding for a glucocorticoid receptor Such is known to the person skilled in the art. For example, he knows the expression plasmid HGO (cf. Kumar, V., et al., Cell 51 (1987), 941-951).
  • method step (a) implies the infection of cells according to the invention with a rep-negative AAV mutant.
  • the expression "DNA of a rep-negative AAV mutant” encompasses an AAV genome, optionally present in a vector, which has mutations in the rep gene. Such mutations can in particular be deletions, insertions and / or substitutions of one or more nucleotides.
  • the AAV DNA can also have a deletion of the entire region coding for Rep.
  • the DNA encoding Rep can be partially or completely replaced by a DNA encoding a foreign protein (peptide) or by a DNA encoding an "antisense” RNA.
  • the foreign protein (peptide) or the “antisense” RNA is preferably suitable for gene therapy measures.
  • the expression "rep-negative AAV mutant” therefore also implies the term "rep-negative AAV vector”.
  • DNA of a rep-negative AAV mutant also includes a DNA which, in addition to the mutations indicated above, has further mutations in other areas of AAV DNA.
  • This can e.g. Mutations in the cap gene.
  • an expressible AAV-cap gene is present in the cells according to the invention. This can be done by the means enabling AAV replication, e.g. the AAV helper virus. Methods are known to those skilled in the art, an AAV-cap gene, e.g. to insert into an AAV helper virus.
  • AAV helper virus includes viruses that allow AAVs to replicate. These are in particular adenoviruses, such as adenovirus-2, and herpes viruses.
  • Rep-negative AAV mutants are free from wild-type AAV, since recombination events, such as those that occur in cells with transient expression of Rep proteins, are avoided.
  • the present invention thus represents the basis for restricting the activities attributed to the Rep proteins to individual Rep proteins or domains thereof. This enables the mechanism of action of AAV as a tumor suppressive principle to be investigated in detail, which is essential for the use of AAV in tumor therapy.
  • the Framework of gene therapy can be used as viral vectors.
  • Rep-negative AAV mutants according to the invention can carry genes or gene segments that can be used for this purpose. These can lie in particular in the rep gene and / or cap gene. The present invention thus represents a breakthrough in the field of the production of vectors which can be used for gene therapies.
  • FIG. 1 shows a schematic representation of the Rep-coding DNA in the expression plasmid pCMRep40.
  • the start ATG triplet and the termination codon TGA are given, they correspond to those of the wild-type AAV genome.
  • the intron (position: 1907-2227) is removed by directed mutagenesis, so that Rep 40 can be expressed without splicing.
  • He 10-1 cells are transfected with the DNA of the known AAV Rep mutant pTAV 2-3.
  • pTAV2-3 has a "frame shift" mutation at position 1045, whereby all four Rep proteins are inactivated (cf. Heilbronn, R., et al., J. Virol. 64 (1990), 3012-3018).
  • the cells are harvested and the total cell DNA is isolated. This is cleaved with the restriction enzymes Xbal or Dpnl and analyzed in a Southern blot.
  • 32P P-labeled AAV-DNA is used as a hybridization sample.
  • the restriction enzyme Xbal does not cut AAV DNA, nor does the restriction enzyme Dpnl cleave any DNA replicated in eukaryotes. Replicated pTAV2-3 DNA is obtained.
  • the supernatant of the cells not harvested is alternately frozen and thawed and subjected to an ultrasound treatment. The supernatant is titrated on He10-1 cells.
  • the detection of infectious AAVs is followed by hybridization with a 3 P-labeled probe which is specific for rep-negative AAVs. Infectious rep-negative AAV particles are detected.
  • HeCMI g cells are co-transfected with the above AAV-Rep mutant pTAV 2-3 and the expression plasmid HGO.
  • the cells are incubated until the adenovirus-induced cytopathic effect (approx. 48 h) and then harvested by freeze-thaw lysis and ultrasound treatment in a hypotonic buffer. The cell fragments are centrifuged off, the AAV particles are in the supernatant. These also show up as infectious.

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Abstract

The invention concerns the preparation of rep-negative AAV mutants and cells which can be used therefor. The invention also concerns an expression plasmid used to prepare the cells.

Description

Bereitstellung von rep-negativen AAV-Mutanten und hierfür verwendbare Zellen Provision of rep-negative AAV mutants and cells that can be used for this

Die Erfindung betrifft die Bereitstellung von rep-negativen AAV-Mutanten und hierfür verwendbare Zellen. Ferner betrifft die Erfindung ein zur Herstellung der Zellen verwendbares Expressionsplasmid.The invention relates to the provision of rep-negative AAV mutants and cells which can be used therefor. The invention further relates to an expression plasmid which can be used to produce the cells.

Adeno-assoziierte Viren (AAVs) sind einzelsträngige, zur Familie der Parvoviren gehörende DNA-Viren. Zu ihrer Replikation benötigen AAVs Helferviren, insbeson¬ dere Adenoviren oder Herpesviren. In Abwesenheit von Helferviren integrieren AAVs in das Wirtszell-Genom, insbesondere an einer spezifischen Stelle von Chromosom 19 bzw. 17.Adeno-associated viruses (AAVs) are single-stranded DNA viruses belonging to the parvovirus family. For their replication, AAVs need helper viruses, in particular adenoviruses or herpes viruses. In the absence of helper viruses, AAVs integrate into the host cell genome, particularly at a specific location on chromosome 19 or 17.

Auf dem 4,65-kb großen, linearen Genom von humanem AAV-Typ 2 wurden drei virale Funktionen lokalisiert. Die 145 bp langen "inverted repeats" dienen als Replikationsursprung und als eis Signale für Integration und Verpackung. Das cap Gen codiert für drei Strukturproteine und das rep Gen für eine Familie multifunktio¬ naler Regulatorproteine. Die mRNAs für Rep 78 und seine C-terminal gespleißte Version Rep 68 starten am p5 Promotor. Zwei N-terminal verkürzte Versionen von Rep 78 und Rep 68, nämlich Rep 52 bzw. Rep 40, werden unter der Kontrolle des p19 Promotors exprimiert. Rep Proteine sind für die DNA-Replikation von AAV notwendig. Ferner werden sie für die Genregulation von AAV benötigt.Three viral functions were localized on the 4.65 kb linear genome of human AAV type 2. The 145 bp "inverted repeats" serve as the origin of replication and as ice signals for integration and packaging. The cap gene codes for three structural proteins and the rep gene for a family of multifunctional regulator proteins. The mRNAs for Rep 78 and its C-terminal spliced version Rep 68 start at the p5 promoter. Two N-terminally shortened versions of Rep 78 and Rep 68, namely Rep 52 and Rep 40, are expressed under the control of the p19 promoter. Rep proteins are necessary for AAV DNA replication. They are also required for AAV gene regulation.

AAVs unterdrücken die Tumorentwicklung in Tieren. Ferner unterdrücken sie die durch Onkogene bedingte Zelltransformation wie auch die induzierte DNA-Am- plifikation. Desweiteren haben AAVs eine antiproliferative Wirksamkeit. Rep-Proteine von AAV werden für vorstehende Aktivitäten verantwortlich ge¬ macht. Eine Zuordnung dieser Aktivitäten zu den einzelnen Rep-Proteinen bzw. Domänen davon existiert jedoch nicht. Eine solche wäre aber notwendig, um AAVs therapeutisch einsetzen zu können. Eine Möglichkeit, diese Zuordnung zu errei¬ chen, liegt in der Untersuchung von AAVs, die Mutationen in den Rep-Proteinen aufweisen. Viele Versuche wurden diesbezüglich durchgeführt. Bisher ist es allerdings nicht gelungen, rep-negative AAV-Mutanten bereitzustellen, die frei von Wildtyp-AAV sind. Solche sind aber für vorstehende Untersuchungen unerläßlich.AAVs suppress tumor development in animals. Furthermore, they suppress the cell transformation caused by oncogenes as well as the induced DNA amplification. Furthermore, AAVs have an antiproliferative activity. AAV Rep proteins are held responsible for the above activities. However, these activities are not assigned to the individual Rep proteins or domains thereof. This would be necessary in order to use AAVs therapeutically. One way to achieve this assignment is to examine AAVs that have mutations in the Rep proteins. Many attempts have been made in this regard. So far, however, it has not been possible to provide rep-negative AAV mutants which are free from wild-type AAV. However, such are indispensable for the above investigations.

Der vorliegenden Erfindung liegt somit die Aufgabe zugrunde, ein Mittel bereitzu¬ stellen, mit dem rep-negative AAV-Mutanten ohne vorstehende Nachteile erhalten werden können.The present invention is therefore based on the object of providing a means by which rep-negative AAV mutants can be obtained without the above disadvantages.

Erfindungsgemäß wird dies durch die Gegenstände in den Patentansprüchen erreicht.According to the invention, this is achieved by the subject matter in the claims.

Gegenstand der Erfindung sind somit Zellen, welche die AAV-Rep-Proteine 78 und 52 sowie 40 und/oder 68 stabil exprimieren. Bevorzugt werden Zellen, welche die Rep-Proteine 78, 52 und 40 exprimieren.The invention thus relates to cells which stably express the AAV Rep proteins 78 and 52 and 40 and / or 68. Cells which express Rep proteins 78, 52 and 40 are preferred.

Erfindungsgemäße Zellen können in üblicher weise hergestellt werden. Günstiger¬ weise werden Zellen der bekannten Linie HeM1 als Ausgangsmaterial verwendet (vgl. "5th Parvovirus Workshop", Chrystal River, Florida, USA, Nov. 10-14, 1993). Diese Zellen exprimieren die AAV-Rep-Proteine 78 und 52, wobei die Rep 78- Expression unter der Kontrolle von Dexamethason-induzierbarem MMTV-LTR steht. Zellen von HeM1 werden mit einem für Rep 40 codierenden Expressionsplasmid und/oder einem für Rep 68 codierenden bzw. einem Expressionsplasmid trans- fiziert, das für beide Rep-Proteine codiert. Vorzugsweise wird ein Expressions¬ plasmid für Rep 40 verwendet, wobei das Expressionsplasmid. pCMRep 40 ganz besonders bevorzugt ist. In diesem liegt die für Rep 40 codierende DNA zwischen den Schnittstellen Notl und Xbal des bekannten Vektors pKEX-2-XL vor (vgl. Rittner, K.H. et al., Methods Mol. Cell. Biol. 2 (1991 ), 176-181 ). pCMRep 40 wurde bei der DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) unter DSM 9491 am 7. Okt. 1994 und DSM 9488 am 10. Okt. 1994 hinterlegt. Es stellt auch einen Gegenstand der Erfindung dar.Cells according to the invention can be produced in the usual way. Cells of the known HeM1 line are advantageously used as starting material (cf. "5th Parvovirus Workshop", Chrystal River, Florida, USA, Nov. 10-14, 1993). These cells express the AAV Rep proteins 78 and 52, the Rep 78 expression being under the control of dexamethasone-inducible MMTV-LTR. HeM1 cells are transfected with an expression plasmid coding for Rep 40 and / or an expression plasmid coding for Rep 68 or an expression plasmid which codes for both Rep proteins. An expression plasmid for Rep 40 is preferably used, the expression plasmid. pCMRep 40 is very particularly preferred. This contains the DNA coding for Rep 40 between the interfaces Notl and Xbal of the known vector pKEX-2-XL (cf. Rittner, KH et al., Methods Mol. Cell. Biol. 2 (1991), 176-181) . pCMRep 40 was deposited with the DSM (German Collection of Microorganisms and Cell Cultures GmbH) under DSM 9491 on October 7, 1994 and DSM 9488 on October 10, 1994. It is also an object of the invention.

Die durch Transfektion von pCMRep 40 erhaltenen Zellen exprimieren stabil die AAV-Rep-Proteine Rep 78, Rep 52 und Rep 40. Diese Zellen wurden als Zellinie He 10-1 , He 22-2 und He 5-5 bei der DSM unter DSM ACC2193, DSM ACC2192 bzw. DSM ACC2191 am 28. Sept. 1994 hinterlegt. Ferner wurden die Zellen als Zellinie HeCMI g bei der DSM unter DSM ACC2185 am 30. August 1994 hinter¬ legt. Vorstehende Zellen stellen ebenso einen Gegenstand der Erfindung dar.The cells obtained by transfection of pCMRep 40 stably express the AAV-Rep proteins Rep 78, Rep 52 and Rep 40. These cells were identified as cell line He 10-1, He 22-2 and He 5-5 in the DSM under DSM ACC2193 , DSM ACC2192 and DSM ACC2191 on September 28, 1994. Furthermore, the cells were deposited as cell line HeCMI g at DSM under DSM ACC2185 on August 30, 1994. The above cells are also an object of the invention.

Ein weiterer Gegenstand der Erfindung ist ein Verfahren zur Bereitstellung von rep- negativen AAV-Mutanten. Ein solches Verfahren umfaßt die folgenden Verfahrens¬ schritte:Another object of the invention is a method for providing rep-negative AAV mutants. Such a process comprises the following process steps:

(a) Transfektion erfindungsgemäßer Zellen mit der DNA einer rep-negativen AAV-Mutante,(a) transfection of cells according to the invention with the DNA of a rep-negative AAV mutant,

(b) Behandlung der transfizierten Zellen von (a) mit einem eine AAV-Replikation ermöglichenden Mittel, insbesondere einem AAV-Helfervirus, und(b) treating the transfected cells of (a) with an agent which enables AAV replication, in particular an AAV helper virus, and

(c) Isolierung der in (b) erhaltenen rep-negativen AAV-Mutanten.(c) Isolation of the rep-negative AAV mutants obtained in (b).

In bevorzugter Ausführungsform erfolgt in Verfahrensschritt (a) eine Cotrans- fektion mit einem für einen Glukokortikoid-Rezeptor codierenden Expressions¬ plasmid. Ein solches ist dem Fachmann bekannt. Beispielsweise kennt er das Expressionsplasmid HGO (vgl. Kumar, V., et al., Cell 51 (1987), 941 -951 ).In a preferred embodiment, process step (a) involves cotransfection with an expression plasmid coding for a glucocorticoid receptor. Such is known to the person skilled in the art. For example, he knows the expression plasmid HGO (cf. Kumar, V., et al., Cell 51 (1987), 941-951).

Ferner impliziert der Verfahrensschritt (a) die Infektion erfindungsgemäßer Zellen mit einer rep-negativen AAV-Mutante. Desweiteren kennt der Fachmann sämtliche zur Durchführung vorstehender Verfahrensschritte notwendigen. Techniken. Ergän¬ zend wird auf Maniatis et al., Molecular Cloning: A laboratory mannual (1982), Cold Spring Harbor, New York, verwiesen. Der Ausdruck "DNA einer rep-negativen AAV-Mutante" umfaßt ein, gegebenenfalls in einem Vektor vorliegendes, AAV-Genom, das Mutationen im rep-Gen hat. Solche Mutationen können insbesondere Deletionen, Insertionen und/oder Sub¬ stitutionen von ein oder mehreren Nukleotiden sein. Auch kann die AAV-DNA eine Deletion des gesamten für Rep codierenden Bereichs aufweisen. Desweiteren kann die Rep codierende DNA teilweise oder ganz durch eine für ein Fremdprotein (-peptid) codierende DNA bzw. durch eine für eine "antisense"-RNA kodierende DNA ersetzt sein. Vorzugsweise eignet sich das Fremdprotein(-peptid) bzw. die "antisense"-RNA für gentherapeutische Maßnahmen. Der Ausdruck "rep-negative AAV-Mutante" impliziert somit auch den Begriff "rep-negativer AAV- Vektor".Furthermore, method step (a) implies the infection of cells according to the invention with a rep-negative AAV mutant. Furthermore, the person skilled in the art knows all of the necessary steps for carrying out the above process steps. Techniques. In addition, reference is made to Maniatis et al., Molecular Cloning: A laboratory mannual (1982), Cold Spring Harbor, New York. The expression "DNA of a rep-negative AAV mutant" encompasses an AAV genome, optionally present in a vector, which has mutations in the rep gene. Such mutations can in particular be deletions, insertions and / or substitutions of one or more nucleotides. The AAV DNA can also have a deletion of the entire region coding for Rep. Furthermore, the DNA encoding Rep can be partially or completely replaced by a DNA encoding a foreign protein (peptide) or by a DNA encoding an "antisense" RNA. The foreign protein (peptide) or the “antisense” RNA is preferably suitable for gene therapy measures. The expression "rep-negative AAV mutant" therefore also implies the term "rep-negative AAV vector".

Desweiteren umfaßt der Ausdruck "DNA einer rep-negativen AAV-Mutante" auch eine DNA, die neben den vorstehend angegebenen Mutationen weitere Mutationen in anderen Bereichen der AAV-DNA aufweist. Dies können z.B. Mutationen im cap- Gen sein. Für einen solchen Fall ist es gefordert, daß ein exprimierbares AAV-cap- Gen in den erfindungsgemäßen Zellen vorliegt. Dies kann durch das die AAV- Replikation ermöglichende Mittel, z.B. dem AAV-Helfervirus, eingebracht sein. Dem Fachmann sind Verfahren bekannt, ein AAV-cap Gen, z.B. in ein AAV-Helfervirus zu inserieren.Furthermore, the expression "DNA of a rep-negative AAV mutant" also includes a DNA which, in addition to the mutations indicated above, has further mutations in other areas of AAV DNA. This can e.g. Mutations in the cap gene. In such a case, it is required that an expressible AAV-cap gene is present in the cells according to the invention. This can be done by the means enabling AAV replication, e.g. the AAV helper virus. Methods are known to those skilled in the art, an AAV-cap gene, e.g. to insert into an AAV helper virus.

Der Ausdruck "AAV-Helfervirus" umfaßt Viren, die eine Replikation von AAVs ermöglichen. Dies sind insbesondere Adenoviren, wie Adenovirus-2, und Herpes¬ viren.The term "AAV helper virus" includes viruses that allow AAVs to replicate. These are in particular adenoviruses, such as adenovirus-2, and herpes viruses.

Mit der vorliegenden Erfindung ist es möglich, rep-negative AAV-Mutanten bereit¬ zustellen. Diese sind frei von Wildtyp-AAV, da Rekombinationsereignisse, wie sie bei transienter Expression von Rep-Proteinen in Zellen eintreten, vermieden wer¬ den. Die vorliegende Erfindung stellt somit die Basis dar, die den Rep-Proteinen zugeschriebenen Aktivitäten auf einzelne Rep-Proteine bzw. Domänen davon zu beschränken. Damit ist die Möglichkeit gegeben, den Wirkungsmechanismus von AAV als tumorsuppressives Prinzip eingehend zu untersuchen, was für den Einsatz von AAV in der Tumortherapie unabdingbar ist. Desweiteren eröffnet die vor- Rahmen der Gentherapie als virale Vektoren eingesetzt werden können. Erfin¬ dungsgemäße rep-negative AAVMutanten können hierfür verwendbare Gene bzw. Genabschnitte tragen. Diese können insbesondere in dem rep-Gen und/oder cap- Gen liegen. Die vorliegende Erfindung stellt somit einen Durchbruch auf dem Gebiet der Herstellung von für Gentherapien verwendbaren Vektoren dar.With the present invention it is possible to provide rep-negative AAV mutants. These are free from wild-type AAV, since recombination events, such as those that occur in cells with transient expression of Rep proteins, are avoided. The present invention thus represents the basis for restricting the activities attributed to the Rep proteins to individual Rep proteins or domains thereof. This enables the mechanism of action of AAV as a tumor suppressive principle to be investigated in detail, which is essential for the use of AAV in tumor therapy. Furthermore, the Framework of gene therapy can be used as viral vectors. Rep-negative AAV mutants according to the invention can carry genes or gene segments that can be used for this purpose. These can lie in particular in the rep gene and / or cap gene. The present invention thus represents a breakthrough in the field of the production of vectors which can be used for gene therapies.

Kurze Beschreibung der Zeichnung:Brief description of the drawing:

Fig. 1 zeigt eine schematische Darstellung der Rep-codierenden DNA im Expres¬ sionsplasmid pCMRep40. Das Start-ATG-Triplett und das Terminationscodon TGA sind angegeben, sie entsprechen denen des Wildtyp-AAV-Genoms. Durch gerichte¬ te Mutagenese ist das Intron (Position: 1907-2227) entfernt, wodurch Rep 40 ohne Spleißen exprimiert werden kann.1 shows a schematic representation of the Rep-coding DNA in the expression plasmid pCMRep40. The start ATG triplet and the termination codon TGA are given, they correspond to those of the wild-type AAV genome. The intron (position: 1907-2227) is removed by directed mutagenesis, so that Rep 40 can be expressed without splicing.

Die Erfindung wird durch die Beispiele erläutert.The invention is illustrated by the examples.

Beispiel 1 : Bereitstellung einer AAV-Rep-Mutante unter Verwendung der Zellinie He10-1Example 1: Provision of an AAV Rep mutant using the He10-1 cell line

He 10-1 -Zellen werden mit der DNA der bekannten AAV-Rep-Mutante pTAV 2-3 transfiziert. pTAV2-3 weist eine "frameshift"-Mutation an der Position 1045 auf, wodurch alle vier Rep-Proteine inaktiviert sind (vgl. Heilbronn, R., et al., J.Virol. 64 (1990), 3012-3018). Die Zellen werden mit Adenovirus-2 (MOI = 10-20) infiziert. Danach werden sie mit 10 β M Dexamethason induziert.He 10-1 cells are transfected with the DNA of the known AAV Rep mutant pTAV 2-3. pTAV2-3 has a "frame shift" mutation at position 1045, whereby all four Rep proteins are inactivated (cf. Heilbronn, R., et al., J. Virol. 64 (1990), 3012-3018). The cells are infected with adenovirus-2 (MOI = 10-20). Then they are induced with 10 β M dexamethasone.

Nach ca. 30 h werden ein Teil der Zellen geerntet und die Gesamtzell-DNA isoliert. Diese wird mit den Restriktionsenzymen Xbal bzw. Dpnl gespalten und in einem Southern-Blot analysiert. Hierzu wird 32PP-markierte AAV-DNA als Hybridisierungs- probe verwendet. Das Restriktionsenzym Xbal schneidet AAV-DNA nicht, ebenso spaltet das Restriktionsenzym Dpnl keine in Eukaryoten replizierte DNA. Es wird replizierte pTAV2-3-DNA erhalten. Desweiteren wird der Überstand der nicht geernteten Zellen abwechselnd eingefro¬ ren und aufgetaut sowie einer Ultraschallbehandlung unterzogen. Der Überstand wird auf He10-1 -Zellen titriert. Der Nachweis infektiöser AAVs wird durch Hybri¬ disierung mit einer 3 P-markierten Sonde verfolgt, die für rep-negative AAVs spezifisch ist. Es werden infektiöse rep-negative AAV-Partikel nachgewiesen.After about 30 hours, part of the cells are harvested and the total cell DNA is isolated. This is cleaved with the restriction enzymes Xbal or Dpnl and analyzed in a Southern blot. For this purpose, 32P P-labeled AAV-DNA is used as a hybridization sample. The restriction enzyme Xbal does not cut AAV DNA, nor does the restriction enzyme Dpnl cleave any DNA replicated in eukaryotes. Replicated pTAV2-3 DNA is obtained. Furthermore, the supernatant of the cells not harvested is alternately frozen and thawed and subjected to an ultrasound treatment. The supernatant is titrated on He10-1 cells. The detection of infectious AAVs is followed by hybridization with a 3 P-labeled probe which is specific for rep-negative AAVs. Infectious rep-negative AAV particles are detected.

Vorstehendes Beispiel zeigt, daß mit erfindungsgemäßen Zellen rep-negative AAV- Mutanten bereitgestellt werden können.The above example shows that rep-negative AAV mutants can be provided with cells according to the invention.

Beispiel 2: Bereitstellung einer rep-negativen AAV-Mutante unter Verwendung der Zellinie HeCMIgExample 2: Provision of a rep-negative AAV mutant using the HeCMIg cell line

HeCMI g-Zellen werden mit der vorstehenden AAV-Rep-Mutante pTAV 2-3 und dem Expressionsplasmid HGO kotransfiziert. Die Zellen werden mit Adenovirus-2 (MOI = 10-20) infiziert. Danach werden sie mit 10 β (107) M Dexamethason indu¬ ziert.HeCMI g cells are co-transfected with the above AAV-Rep mutant pTAV 2-3 and the expression plasmid HGO. The cells are infected with adenovirus-2 (MOI = 10-20). Then they are induced with 10 β (10 7 ) M dexamethasone.

Die Zellen werden bis zum vollständigen, durch Adenovirus ausgelösten zytopathi- schen Effekt (ca. 48 h) inkubiert und anschließend durch Frier-Tau-Lyse und Ultraschallbehandlung in einem hypotonen Puffer geerntet. Die Zellfragmente werden abzentrifugiert, die AAV-Partikel befinden sich im Überstand. Diese zeigen sich auch als infektiös.The cells are incubated until the adenovirus-induced cytopathic effect (approx. 48 h) and then harvested by freeze-thaw lysis and ultrasound treatment in a hypotonic buffer. The cell fragments are centrifuged off, the AAV particles are in the supernatant. These also show up as infectious.

Vorstehendes Beispiel unterstreicht, daß mit erfindungsgemäßen Zellen rep-hegati- ve AAV-Mutanten bereitgestellt werden können. The above example underlines that rep-hegative AAV mutants can be provided with cells according to the invention.

Claims

Patentansprüche claims 1 . Zellen, stabil exprimierend die AAV-Rep-Proteine 78 und 52 sowie 40 und/oder 68.1 . Cells stably expressing the AAV Rep proteins 78 and 52 and 40 and / or 68. 2. Zellen nach Anspruch 1 , dadurch gekennzeichnet, daß sie die AAV-Rep- Proteine Rep 78,52 und 40 stabil exprimieren.2. Cells according to claim 1, characterized in that they stably express the AAV Rep proteins Rep 78, 52 and 40. 3. Zellen nach Anspruch 2, nämlich die Zellinien He 10-1 (DSM ACC2193), He 22-2 (DSM ACC2192), He 5-5 (DSM ACC2191 ) und HeCMI g (DSM ACC2185).3. Cells according to claim 2, namely the cell lines He 10-1 (DSM ACC2193), He 22-2 (DSM ACC2192), He 5-5 (DSM ACC2191) and HeCMI g (DSM ACC2185). 4. Expressionsplasmid, nämlich pCMRep 40 (DSM 9491 ; DSM 9488).4. Expression plasmid, namely pCMRep 40 (DSM 9491; DSM 9488). 5. Verfahren zur Bereitstellung von rep-negativen AAV-Mutanten, umfassend die folgenden Verfahrensschritte:5. A method for providing rep-negative AAV mutants, comprising the following process steps: (a) Transfektion der Zellen nach einem der Ansprüche 1 -3 mit der DNA einer rep-negativen AAV-Mutante,(a) transfection of the cells according to one of claims 1 -3 with the DNA of a rep-negative AAV mutant, (b) Behandlung der transfizierten Zellen von (a) mit einem eine AAV- Replikation ermöglichenden Mittel, insbesondere einem AAV-Helfervi¬ rus, und(b) treatment of the transfected cells of (a) with an agent which enables AAV replication, in particular an AAV helper virus, and (c) Isolierung der in (b) erhaltenen rep-negativen AAV-Mutanten.(c) Isolation of the rep-negative AAV mutants obtained in (b). 6. Verfahren nach Anspruch 5, dadurch gekennzeichnet, daß im Verfahrens¬ schritt (a) eine Kotransfektion mit einem für einen Glukokortikoidrezeptor codierenden Expressionsplasmid erfolgt.6. The method according to claim 5, characterized in that in the procedural step (a) co-transfection with an expression plasmid coding for a glucocorticoid receptor takes place. 7. Verfahren nach Anspruch 5 oder 6, dadurch gekennzeichnet, daß die DNA der rep-negativen AAV-Mutante von Verfahrensschritt (a) ein oder mehrere Deletionen, Insertionen und/oder Substitutionen im rep Gen aufweist. 7. The method according to claim 5 or 6, characterized in that the DNA of the rep-negative AAV mutant of step (a) has one or more deletions, insertions and / or substitutions in the rep gene. 8. Verfahren nach Anspruch 5 oder 6, dadurch gekennzeichnet, daß in der DNA der rep-negativen AAV-Mutante von Verfahrensschritt (a) das rep Gen deletiert ist.8. The method according to claim 5 or 6, characterized in that in the DNA of the rep-negative AAV mutant of step (a), the rep gene is deleted. 9. Verfahren nach Anspruch 5 oder 6, dadurch gekennzeichnet, daß in der DNA der rep-negativen AAV-Mutante von Verfahrensschritt (a) das rep Gen zumindest teilweise durch ein Fremd-Gen ersetzt ist.9. The method according to claim 5 or 6, characterized in that in the DNA of the rep-negative AAV mutant of process step (a), the rep gene is at least partially replaced by a foreign gene. 10. Verfahren nach einem der Ansprüche 5-9, dadurch gekennzeichnet, daß das AAV-Helfervirus ein Adenovirus oder Herpesvirus ist.10. The method according to any one of claims 5-9, characterized in that the AAV helper virus is an adenovirus or herpes virus. 1 1. Verfahren nach einem der Ansprüche 5-10, dadurch gekennzeichnet, daß die DNA der rep-negativen AAV-Mutante von Verfahrensschritt (a) eine weitere Mutation im cap-Gen aufweist, mit der Maßgabe, daß das die AAV- Replikation ermöglichende Mittel, insbesondere der AAV-Helfervirus, ein ex- primierbares cap-Gen enthält.1 1. The method according to any one of claims 5-10, characterized in that the DNA of the rep-negative AAV mutant of step (a) has a further mutation in the cap gene, with the proviso that the AAV replication enables Agent, in particular the AAV helper virus, contains an expressible cap gene. 12. rep-negative AAV-Mutante, erhalten durch das Verfahren nach einem der Ansprüche 5-1 1. 12. rep-negative AAV mutant obtained by the method according to any one of claims 5-1 1.
PCT/DE1995/001429 1994-10-13 1995-10-12 Preparation of rep-negative aav mutants and cells which can be used therefor Ceased WO1996012010A1 (en)

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