WO1997049824A1 - System for the production of aav vectors - Google Patents

System for the production of aav vectors Download PDF

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Publication number
WO1997049824A1
WO1997049824A1 PCT/DE1997/001333 DE9701333W WO9749824A1 WO 1997049824 A1 WO1997049824 A1 WO 1997049824A1 DE 9701333 W DE9701333 W DE 9701333W WO 9749824 A1 WO9749824 A1 WO 9749824A1
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Prior art keywords
aav
rep
sequences
vector
agent
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PCT/DE1997/001333
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German (de)
French (fr)
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WO1997049824B1 (en
Inventor
Christoph Bogedain
Gerd Maass
Michael Hallek
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Medigene Ag
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Priority to US09/214,151 priority Critical patent/US6294370B1/en
Priority to EP97930352A priority patent/EP0954592A1/en
Priority to JP10502116A priority patent/JP2000512501A/en
Publication of WO1997049824A1 publication Critical patent/WO1997049824A1/en
Publication of WO1997049824B1 publication Critical patent/WO1997049824B1/en

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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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N2750/14122—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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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
    • 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
    • C12N2750/14151—Methods of production or purification of viral material
    • C12N2750/14152—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles

Definitions

  • the present invention relates to a system suitable for the production of AAV vectors and its use.
  • AAVs 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
  • AAVs integrate into the host cell genome, particularly at a specific location on chromosome 19.
  • the genome of AAVs is linear and has a length of approximately 4680 nucleotides. This comprises two reading frames which code for a structural and a non-structural gene.
  • the structural gene is called the cap gene. This is under the control of the P40 promoter and codes for three capsid proteins.
  • the non-structural gene is referred to as the rep gene and codes for the Rep proteins, Rep 78, Rep 68, Rep 52 and Rep 40. The former two are expressed under the control of the P5 promoter, while the expression of Rep 52 and Rep 40 is under the control of the P19 promoter.
  • the functions of the Rep proteins include regulating the replication and transcription of the AAV genome. However, the production of AAVs presents itself as extremely problematic.
  • rAAVs recombined AAVs
  • adenoviruses as vectors for rAAVs does not lead to any convincing results.
  • the present invention is therefore based on the object of providing a means by which rAAVs can be provided in large quantities.
  • the present invention thus relates to a system comprising an AAV vector containing a foreign DNA, and rep 68/78 sequences of AAV, their expression with respect to the DNA replication of the AAV vector or one
  • the present invention is based on the knowledge of the applicant that the Rep proteins 68 and 78 of AAV impair the replication of AAV DNA, but this impairment can be prevented by delayed expression of the sequences encoding the Rep proteins 68 and 78 .
  • AAV vector relates to any AAV vector which does not contain any rep 68/78 sequences of AAV which are not delayed in terms of DNA replication of the AAV vector or a part thereof.
  • the term "part of the AAV vector” refers to any part of the AAV vector, in particular its cap sequences and the sequences coding for the Rep proteins 40 and 52.
  • rep 68/78 sequences from AAV indicates that the system according to the invention rep 68 and / or rep 78 sequences from AAV summarizes whose expression is delayed with respect to the DNA replication of the AAV vector or a part thereof.
  • the term "foreign DNA” refers to any foreign DNA that can be integrated in the above AAV vector.
  • the foreign DNA can be non-coding or coding.
  • the foreign DNA can be a regulatory element of DNA replication and / or transcription.
  • the foreign DNA can code for a diagnostic and / or therapeutic protein. Examples of a therapeutic protein are tumor necrosis factor, interferons, interleukins, lymphokines, growth factors, plasma proteins, such as coagulation factors and metabolic enzymes, and receptors.
  • the foreign DNA can code for proteins that can increase the immunogenicity of cells.
  • proteins that tumor cells lack e.g. Cytokines such as IL-2, interferons and GM-CSF, and costimulatory molecules such as B7-1, tumor associated antigens e.g. MAGE 1, tyrosinases, lymphon specific idiotypes and viral proteins e.g. Human papilloma virus E7 protein and Epstein-Barr virus EBNA 3 protein.
  • the foreign DNA can be integrated anywhere in the AAV vector. It can be advantageous if there are several foreign DNAs in one AAV vector.
  • a system comprises in (a) an AAV vector and rep 68/78 sequences of AAV present in eis, ie on the AAV vector, which ex. Ex - be primed.
  • Such an AAV vector can be obtained by conventional methods. It is favorable to start from an AAV vector which has a DNA coding for Rep proteins from AAV.
  • the endogenous P5 promoter of the rep 68 and rep 78 sequences of AAV can be replaced by one which is active after DNA replication of the AAV vector or a part thereof.
  • a promoter is, for example, the "major late Aden ⁇ virus promoter or a derivative thereof.
  • An inducible promoter for example the metailothionein promoter or a derivative thereof, can also be used instead of the endogenous P5 promoter of the rep 68 and rep 78 sequences of AAV.
  • the AAV vector is present in (a) in an agent (I).
  • agent (I) is a vector, e.g. a virus or a plasmid vector, or a cell
  • a system according to the invention comprises in (b) an AAV vector and in trans, i.e. Rep 68/78 sequences of AAV which are present separately from the AAV vector and which are delayed in terms of DNA replication of the AAV vector or a part thereof.
  • the rep 68/78 sequences of AAV are in an average ( II) before This can be any means which does not impair the functionality of the rep 68/78 sequences of AAV, in particular their delayed expression. The latter can be obtained as described in (a).
  • An agent (II) is advantageously a vector, e.g. a virus or a plasmid vector, or a cell.
  • agent (b) the AAV vector is present in an agent (I) and the rep 68/78 sequences of AAV are in an agent (II).
  • Agents (I) and (II) can have the meanings given above, but at the same time they cannot be cells. It is advantageous if the means (I) and (II) virus vectors, e.g. Adenovirus vectors or a combination of adenovirus and
  • Vaccinia virus vectors are. It is particularly favorable if the virus vectors can complement each other. Virus vectors as means (I) and (II) are described below. This can be seen as an example.
  • Agent (I) is an adenovirus vector which, instead of the E1 sequences from Adenovirus, contains an AAV vector with ITR sequences from AAV and a foreign DNA.
  • Agent (II) is an adenovirus vector which contains rep and cap sequences from AAV instead of the E3 sequences from Adenovirus, the rep 68/78 sequences from AAV being under the control of one after DNA replication of the AAV vector or a part thereof active promoter, for example the "major late promoter" of adenovirus, the rep 40 and rep 52 sequences of AAV under the control of the endogenous p1 9 promoter, and the cap sequences of AAV under the Control of a constitutive promoter, for example the CMV promoter; or
  • Agent (II) is a combination of two adenovirus vectors, one of which contains rep sequences from AAV instead of the E3 sequences from adenovirus, the rep 68/78 sequences from AAV being under the control of the "major late promoter" of adenovirus and the rep 40 and rep 52 sequences of AAV are under the control of the endogenous p 19 promoter, and which contains other portions of the E4 sequences of adenovirus cap sequences of AAV which are under the control of the CMV Promoters stand;
  • Agent (II) is a combination of an adenovirus vector and a vaccinia virus vector, the former of which instead of the E3 sequences of adenovirus rep 40 and rep 52 sequences of AAV under the control of the endogenous p 19 promoter and contains AAV cap sequences under the control of the CMV promoter, and the second rep contains AAV 68/78 sequences.
  • the vacciniavirus vector is only added after DNA replication of the adenovirus
  • a system according to the invention is suitable for producing recombined AAVs (rAAVs) in large quantities.
  • the system according to the invention is introduced into cells which allow the replication of rAAVs. These are e.g.
  • helper viruses such as adenoviruses.
  • helper viruses such as adenoviruses.
  • adenoviruses helper viruses
  • the system according to the invention all for the replica rAAVs necessary elements.
  • the system according to the invention is in alternative (b), in which the means (I) and (II) virus vectors, in particular adenovirus vectors or a combination of adenovirus and vaccinia virus vectors, are that complement each other.
  • the complementation can also be given if one of the agents is a virus vector and the other agent is a cell which provides the protein whose DNA has been deleted in the virus vector.
  • Such measures can be taken by selecting the foreign DNA.
  • the present invention is illustrated by the example.
  • Rep 68/78 sequences from AAV are placed under the control of the major late promoter in order to achieve delayed expression.
  • Rep 40/52 sequences from AAV remain under the control of the endogenous p1 9 promoter.
  • Cap sequences from AAV are under the control of the
  • cap sequences in the 5'-untranslated region of the mRNAs contain sequences from the 5'-untranslated region of the late adenoviral mRNAs.
  • a partial sequence of the rep gene (positions 1882 to 2280) is through PCR with the primers 5'-CGCCGGAAGCTTCGATCAACTACGCAGACAG-3 'and 5'-GCGGGCGTCGACTTTGAGCTTCCACCACTGTCTTAT-3' amplified from a template containing the AAV genome (eg pSVO ⁇ ' AAV), cut with Hindlll / Sall and cut the same with the same enzyme Plasmid pUC 19 inserted. PUC1 9Repdupl is obtained.
  • the cDNA sequence of the 5 'untranslated leaders of the late adenovirus mRNAs is PCR by using the primers 5'-CGGGGTACCCAG-CTGACTCTCTTCCGCATCGCTG-3' and 5 '-CGCGGATC CGAATT-CAAGCTTCTCGAGAGGTTMTPCVG2CidCan 3 and the 1 984) amplified, with suitable flanking restriction sites being formed for further ionizations.
  • the amplificate is cut with Kpnl / BamHI and inserted into the plasmid pCEP4 (Invitrogen) cut with the same enzymes, the vector pCEP4-CMVL being produced.
  • the CMV promoter is cut out together with the leader from pCEP4-CMV leader and inserted into pUC1 9Repdupl linearized with the same restriction enzymes; pRepdupl-CMVL is created.
  • the region with the rep partial sequence and the CMV promoter and the adeno leader is cut with Hindlll from the resulting pRepdupl-CMV leader and inserted in pSVOriAAV (Chiorini et al., 1 995) partially cut with Hindlll .
  • PRepCMVLCap is created. The insertion at the right place in the right orientation is checked with adequate restriction cleavages.
  • this procedure inserts the CMV promoter between the rep and the cap sequences, the interruption of the rep reading frame being prevented by duplicating a partial sequence and the CMV promoter in the HindIII interface at position 1 883 of the AAV sequence of the pSVOriAAV is inserted.
  • This HindIII interface is located between the transcription start point of the p40 promoter and the 5 'splicing signal of the cap sequences.
  • pRepCMVLCap is linearized with Spei and treated with T4 exonuclease delt.
  • a time series of the T4 exonuclease treatment enables different sizes of the deleted area to arise.
  • the work continues with a construct in which the 5 'region of the AAV genome has been deleted up to a position between positions 288 and 321, so that the remaining sequence between the start of transcription of the rep68 / 78 sequences, pos. 288, and the start codon of these sequences, item 321, begins.
  • a polylinker sequence with Xbal, Notl, Spei, and Bglll interfaces is built in with synthetic linkers, so that pRepCMVLCap ' is formed.
  • the major late promoter is by using the primer 5'-CGTCTA-
  • pRepCMVLCap * is cut with Bglll / Xbal and the major late promoter is inserted in the correct orientation so that pMLPRepCMVLCap is created.
  • the rep cassette has the endogenous p19 promoter, which is embedded in the rep sequence and continues to control the expression of the proteins rep40 and rep52, while only the sequences for rep68 / 78 under the
  • the rep / cap expression cassette is cut out of pMLPRep ⁇ CMVLCap with Xbal / Clal and inserted into the vector p ⁇ E1sp1A (Bett el al., 1995) linearized with the same enzymes.
  • This plasmid contains the sequences of type 5 adenovirus between card units 0 and 0.9 as well as 9.8 and 15.8, the E1A region being substituted by a polylinker sequence for inserting foreign genes.
  • Genomes of infectious recombinant adenoviruses can by in vivo recombination between overlapping areas of adenoviral sequences on pBHG IO, which the adenovirus sequences between the card units 0 and 0.5, from 3.7 to 78.3 and from 85.8 to 100, as well as the p ⁇ E 1 sp 1 A derivative, which contains rep / cap sequences.
  • the cotransfection of the two plasmids is carried out in 293 cells.
  • a cytopathic effect that manifests itself a few days after the transfection indicates the development of recombinant adenoviruses.
  • the 293 cells which show a cytopathic effect, are broken up by freezing and thawing and recombinant viruses (adrep / cap) are separated from the lysate by double plaque cleaning. Individual clones are amplified by successive passage over 293 cultures. The expression patterns of rep and cap are characterized by Western blotting.
  • a foreign DNA for example a luciferase reporter gene with flanking AAV-ITR sequences, is integrated into the E1 A region of an adenovirus.
  • the expression cassette with the ITR- Sequences cut out with Pvull. This fragment is integrated into EcoRV-cut p ⁇ E1 sp 1 A via the smooth ends. P ⁇ E1 sp1 A-Luc is formed.
  • the recombinant Ade ⁇ ovire ⁇ (AdAALuc) is produced by cotransfection of p ⁇ E1 sp 1 A-Luc and pBHG IO in 293 cells by in vivo recombination, as described under (a).
  • 4 x 10 a 293 cells are cultivated to 80% confluence and infected with a moi of 10 at a time with Adrep / cap and AdAAVLuc in a small volume of serum-free medium. Complete medium is added 2 hours after infection. 3 days after infection, the cells are removed from the surface of the culture vessels with a cell scratch; the suspension is centrifuged off at 200 g and room temperature for 5 min. The cell pellets are in 28 ml TD buffer (140 mM NaCl, 5 mM KCI, 0.7 mM

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Abstract

The invention concerns a system comprising an AAV vector, which contains a foreigh DNA, and rep 68/78 sequences of AAV with delayed expression, these sequences being present (a) in cis or (b) in trans. The invention also concerns the use of such a system for the production of AAV vectors.

Description

"System zur Herstellung von AAV-Vektoren""System for the Production of AAV Vectors"
Beschreibungdescription
Die vorliegende Erfindung betrifft ein System, das sich zur Herstellung von AAV- Vektoren eignet, und seine Verwendung.The present invention relates to a system suitable for the production of AAV vectors and its use.
Zur Durchführung gentherapeutischer Maßnahmen ist es wichtig, Vektoren zu haben, die Fremd-Gene in das Genom von Zellen einführen können und für diese nicht toxisch sind. Ein Beispiel für solche Vektoren sind Adeno-assoziierte Viren (AAVs) .To carry out gene therapy measures, it is important to have vectors that can introduce foreign genes into the genome of cells and are not toxic to them. An example of such vectors are adeno-associated viruses (AAVs).
AAVs sind einzelsträngige, zur Familie der Parvoviren gehörende DNA-Viren. Für ihre Replikation benötigen AAVs Helferviren, insbesondere Adenoviren oderAAVs are single-stranded DNA viruses belonging to the parvovirus family. For their replication, AAVs need helper viruses, in particular adenoviruses or
Herpesviren. In Abwesenheit von Helferviren integrieren AAVs in das Wirtszell¬ genom, insbesondere an einer spezifischen Stelle von Chromosom 19.Herpes viruses. In the absence of helper viruses, AAVs integrate into the host cell genome, particularly at a specific location on chromosome 19.
Das Genom von AAVs ist linear und weist eine Länge von ca. 4680 Nukleotiden auf. Dieses umfaßt zwei Leserahmen, die für ein strukturelles und ein nicht¬ strukturelles Gen kodieren. Das strukturelle Gen wird mit cap-Gen bezeichnet. Dieses steht unter der Kontrolle des P40-Promotors und kodiert für drei Capsid- Proteine. Das nicht-strukturelle Gen wird mit rep-Gen bezeichnet und kodiert für die Rep-Proteine, Rep 78, Rep 68, Rep 52 und Rep 40. Die beiden ersteren werden unter der Kontrolle des P5 Promotors exprimiert, während die Expres¬ sion von Rep 52 und Rep 40 unter der Kontrolle des P19 Promotors steht. Die Funktionen der Rep-Proteine liegen u.a. in der Regulation der Replikation und Transkription des AAV-Genoms. Die Herstellung von AAVs stellt sich allerdings als äußerst problematisch dar. Insbesondere ist es schwierig, rekombinaπte AAVs (rAAVs), d.h. AAVs, die eine Fremd-DNA enthalten, in großen Mengen herzustellen. Selbst der Versuch, Adenoviren als Vektoren für rAAVs zu verwenden, führt zu keinen befπedigen- den Ergebnissen.The genome of AAVs is linear and has a length of approximately 4680 nucleotides. This comprises two reading frames which code for a structural and a non-structural gene. The structural gene is called the cap gene. This is under the control of the P40 promoter and codes for three capsid proteins. The non-structural gene is referred to as the rep gene and codes for the Rep proteins, Rep 78, Rep 68, Rep 52 and Rep 40. The former two are expressed under the control of the P5 promoter, while the expression of Rep 52 and Rep 40 is under the control of the P19 promoter. The functions of the Rep proteins include regulating the replication and transcription of the AAV genome. However, the production of AAVs presents itself as extremely problematic. In particular, it is difficult to produce recombined AAVs (rAAVs), ie AAVs that contain foreign DNA, in large quantities. Even the attempt to use adenoviruses as vectors for rAAVs does not lead to any convincing results.
Der vorliegenden Erfindung liegt somit die Aufgabe zugrunde, ein Mittel bereitzu¬ stellen, mit dem rAAVs in großen Mengen bereitgestellt werden können.The present invention is therefore based on the object of providing a means by which rAAVs can be provided in large quantities.
Erfmdungsgemaß 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 vorliegenden Erfindung ist somit ein System, umfassend einen, eine Fremd-DNA enthaltenden AAV-Vektor, und rep 68/78-Sequenzen von AAV, deren Expression bezüglich der DNA-Replikation des AAV-Vektors oder einesThe present invention thus relates to a system comprising an AAV vector containing a foreign DNA, and rep 68/78 sequences of AAV, their expression with respect to the DNA replication of the AAV vector or one
Teils davon verzögert ist, wobei die rep 68/78 Sequenzen (a) in eis oder (b) in trans vorliegen.Part of it is delayed, with the rep 68/78 sequences (a) in ice or (b) in trans.
Die vorliegende Erfindung beruht auf der Erkenntnis des Anmelders, daß die Rep- Proteine 68 und 78 von AAV die Replikation von AAV-DNA beeinträchtigen, diese Beeinträchtigung aber durch eine verzögerte Expression der die Rep-Protei¬ ne 68 und 78 kodierenden Sequenzen verhindert werden kann.The present invention is based on the knowledge of the applicant that the Rep proteins 68 and 78 of AAV impair the replication of AAV DNA, but this impairment can be prevented by delayed expression of the sequences encoding the Rep proteins 68 and 78 .
Der Ausdruck "AAV-Vektor" betrifft jeglichen AAV-Vektor, der keine rep 68/78- Sequenzen von AAV enthält, die bezüglich der DNA-Replikation des AAV-Vek¬ tors oder eines Teils davon nicht verzögert expπmiert werden. Der Ausdruck "ein Teil des AAV-Vektors" betrifft jeglichen Teil des AAV-Vektors, insbesondere seine cap-Sequenzen und die für die Rep-Proteine 40 und 52 kodierenden Sequenzen.The expression “AAV vector” relates to any AAV vector which does not contain any rep 68/78 sequences of AAV which are not delayed in terms of DNA replication of the AAV vector or a part thereof. The term "part of the AAV vector" refers to any part of the AAV vector, in particular its cap sequences and the sequences coding for the Rep proteins 40 and 52.
Der Ausdruck "rep 68/78-Sequenzeπ von AAV" weist darauf hin, daß das erfindungsgemäße System rep 68- und/oder rep 78-Sequenzen von AAV um- faßt, deren Expression bezüglich der DNA-Replikation des AAV-Vektors oder eines Teils davon verzögert ist.The expression "rep 68/78 sequences from AAV" indicates that the system according to the invention rep 68 and / or rep 78 sequences from AAV summarizes whose expression is delayed with respect to the DNA replication of the AAV vector or a part thereof.
Der Ausdruck "Fremd-DNA" betrifft jegliche Fremd-DNA, die in einem vorstehen- den AAV-Vektor integriert sein kann. Die Fremd-DNA kann nicht-kodierend oder kodierend sein. In ersterem Fall kann die Fremd-DNA ein Regulator-Element der DNA-Replikation und/oder Transkription sein. In letzterem Fall ist es günstig, wenn die Fremd-DNA exprimierbar ist, wobei es besonders vorteilhaft ist, wenn die Expression unter der Kontrolle eines konstitutiven oder induzierbaren Promo- tors, wie eines Gewebe- oder Tumor-spezifischen Promotors, steht. Ferner kann die Fremd-DNA für ein diagnostisches und/oder therapeutisches Protein kodie¬ ren. Beispiele eines therapeutischen Proteins sind Tumornekrosefaktor, Inter- ferone, Interleukine, Lymphokine, Wachstumsfaktoren, Plasmaproteine, wie Gerinnungsfaktoren und Stoffwechselenzyme, und Rezeptoren. Insbesondere kann die Fremd-DNA für Proteine kodieren, welche die Immunogenität von Zellen steigern können. Dies können Proteine sein, die Tumorzellen fehlen, z.B. Zytoki- ne, wie IL-2, Interferoπe und GM-CSF, und kostimulierende Moieküle, wie B7-1 , tumorassoziierte Antigene, z.B. MAGE 1 , Tyrosinasen, Lymphon-spezifische Idiotypen und virale Proteine, z.B. E7-Protein von humanem Papillomvirus und EBNA 3-Protein von Epstein-Barr-Virus. Die Fremd-DNA kann an beliebiger Stelle des AAV-Vektors integriert sein. Günstig kann es sein, wenn mehrere Fremd- DNAs in einem AAV-Vektor vorliegen.The term "foreign DNA" refers to any foreign DNA that can be integrated in the above AAV vector. The foreign DNA can be non-coding or coding. In the first case, the foreign DNA can be a regulatory element of DNA replication and / or transcription. In the latter case, it is advantageous if the foreign DNA is expressible, it being particularly advantageous if the expression is under the control of a constitutive or inducible promoter, such as a tissue or tumor-specific promoter. Furthermore, the foreign DNA can code for a diagnostic and / or therapeutic protein. Examples of a therapeutic protein are tumor necrosis factor, interferons, interleukins, lymphokines, growth factors, plasma proteins, such as coagulation factors and metabolic enzymes, and receptors. In particular, the foreign DNA can code for proteins that can increase the immunogenicity of cells. These can be proteins that tumor cells lack, e.g. Cytokines such as IL-2, interferons and GM-CSF, and costimulatory molecules such as B7-1, tumor associated antigens e.g. MAGE 1, tyrosinases, lymphon specific idiotypes and viral proteins e.g. Human papilloma virus E7 protein and Epstein-Barr virus EBNA 3 protein. The foreign DNA can be integrated anywhere in the AAV vector. It can be advantageous if there are several foreign DNAs in one AAV vector.
Ein erfindungsgemäßes System umfaßt in (a) einen AAV-Vektor und in eis, d.h. auf dem AAV-Vektor, vorliegende rep 68/78-Sequenzen von AAV, die bezüglich der DNA-Replikation des AAV-Vektors oder eines Teils davon, verzögert ex- primiert werden. Ein solcher AAV-Vektor kann durch übliche Verfahren erhalten werden. Günstig ist es, von einem AAV-Vektor auszugehen, der eine für Rep- Proteine von AAV kodierende DNA aufweist. In einem solchen AAV-Vektor kann der endogene P5-Promotor der rep 68- und rep 78-Sequenzen von AAV durch einen solchen ersetzt werden, der nach der DNA-Replikation des AAV-Vektors oder eines Teils davon aktiv ist. Ein solcher Promotor ist z.B. der "major late Promoter" von Adenσvirus oder ein Derivat davon. Auch kann ein iπduzierbarer Promotor, z.B. der Metailothionein-Promotor oder ein Derivat davon, anstelle des endogenen P5-Promotors der rep 68- und rep 78-Sequenzen von AAV eingesetzt werden.A system according to the invention comprises in (a) an AAV vector and rep 68/78 sequences of AAV present in eis, ie on the AAV vector, which ex. Ex - be primed. Such an AAV vector can be obtained by conventional methods. It is favorable to start from an AAV vector which has a DNA coding for Rep proteins from AAV. In such an AAV vector, the endogenous P5 promoter of the rep 68 and rep 78 sequences of AAV can be replaced by one which is active after DNA replication of the AAV vector or a part thereof. Such a promoter is, for example, the "major late Adenσvirus promoter or a derivative thereof. An inducible promoter, for example the metailothionein promoter or a derivative thereof, can also be used instead of the endogenous P5 promoter of the rep 68 and rep 78 sequences of AAV.
In bevorzugter Ausführungsform liegt der AAV-Vektor in (a) in einem Mittel (I) vor. Dieses kann jegliches Mittel sein, das die Funktionalität des AAV-Vektors, insbesondere seine Replikation, nicht beeinträchtigt. Günstigerweise ist das Mittel (I) ein Vektor, z.B. ein Virus- oder ein Plasmid-Vektor, oder eine ZelleIn a preferred embodiment, the AAV vector is present in (a) in an agent (I). This can be any means that does not impair the functionality of the AAV vector, in particular its replication. Conveniently the agent (I) is a vector, e.g. a virus or a plasmid vector, or a cell
Ein erfindungsgemäßes System umfaßt in (b) einen AAV-Vektor und in trans, d.h. getrennt von dem AAV-Vektor, vorliegende rep 68/78-Sequenzen von AAV, die bezüglich der DNA-Replikation des AAV-Vektors oder eines Teils davon, verzögert expπmiert werden Vorzugsweise liegen die rep 68/78-Sequenzen von AAV in einem Mittel (II) vor Dieses kann jegliches Mittel sein, das die Funktiona¬ lität der rep 68/78-Sequenzen von AAV, insbesondere ihre verzögerte Expres¬ sion, nicht beeinträchtigt. Letztere kann, wie in (a) beschrieben, erhalten wer¬ den. Ein Mittel (II) ist vorteilhafterweise ein Vektor, z.B. ein Virus- oder ein Plasmid-Vektor, oder eine Zelle.A system according to the invention comprises in (b) an AAV vector and in trans, i.e. Rep 68/78 sequences of AAV which are present separately from the AAV vector and which are delayed in terms of DNA replication of the AAV vector or a part thereof. Preferably, the rep 68/78 sequences of AAV are in an average ( II) before This can be any means which does not impair the functionality of the rep 68/78 sequences of AAV, in particular their delayed expression. The latter can be obtained as described in (a). An agent (II) is advantageously a vector, e.g. a virus or a plasmid vector, or a cell.
Besonders günstig ist es, wenn in (b) der AAV-Vektor in einem Mittel (I) und die rep 68/78- Sequenzen von AAV in einem Mittel (II) vorliegen. Die Mittel (I) und (II) können die vorstehenden Bedeutungen haben, wobei sie gleichzeitig aber nicht Zellen sein können. Vorteilhaft ist es, wenn die Mittel (I) und (II) Virus- Vektoren, z.B. Adenovirus-Vektoren oder eine Kombination aus Adenovirus- undIt is particularly favorable if in (b) the AAV vector is present in an agent (I) and the rep 68/78 sequences of AAV are in an agent (II). Agents (I) and (II) can have the meanings given above, but at the same time they cannot be cells. It is advantageous if the means (I) and (II) virus vectors, e.g. Adenovirus vectors or a combination of adenovirus and
Vacciniavirus-Vektoren sind. Besonders günstig ist es, wenn sich die Virus- Vektoren komplementieren können. Nachstehend werden Virus-Vektoren als Mittel (I) und (II) beschrieben. Dies ist beispielhaft anzusehen.Vaccinia virus vectors are. It is particularly favorable if the virus vectors can complement each other. Virus vectors as means (I) and (II) are described below. This can be seen as an example.
Mittel (I) ist ein Adenovirus-Vektor, der anstelle der E1 -Sequenzen von Adenovi¬ rus einen AAV-Vektor mit ITR-Sequenzen von AAV und einer Fremd-DNA enthält. Mittel (II) ist ein Adenovirus-Vektor, der anstelle der E3-Sequenzen von Adenovi¬ rus rep- und cap-Sequenzen von AAV enthält, wobei die rep 68/78-Sequenzen von AAV unter der Kontrolle eines nach der DNA-Replikation des AAV-Vektors oder eines Teils davon aktiven Promotors, z.B. des "major late promoter" von Adenovirus, die rep 40- und rep 52-Sequenzen von AAV unter der Kontrolle des endogenen p1 9-Promotors, und die cap-Sequenzen von AAV unter der Kontrolle eines konstitutiven Promotors, z.B. des CMV-Promotors, stehen; oderAgent (I) is an adenovirus vector which, instead of the E1 sequences from Adenovirus, contains an AAV vector with ITR sequences from AAV and a foreign DNA. Agent (II) is an adenovirus vector which contains rep and cap sequences from AAV instead of the E3 sequences from Adenovirus, the rep 68/78 sequences from AAV being under the control of one after DNA replication of the AAV vector or a part thereof active promoter, for example the "major late promoter" of adenovirus, the rep 40 and rep 52 sequences of AAV under the control of the endogenous p1 9 promoter, and the cap sequences of AAV under the Control of a constitutive promoter, for example the CMV promoter; or
Mittel (II) ist eine Kombination aus zwei Adenovirus-Vektoren, von denen der eine anstelle der E3-Sequenzen von Adenovirus rep-Sequenzeπ von AAV enthält, wobei die rep 68/78-Sequenzen von AAV unter der Kontrolle des "major late promoter" von Adenovirus und die rep 40- und rep 52-Sequenzeπ von AAV unter der Kontrolle des endogenen p 1 9-Promotσrs stehen, und der andere ansteile der E4-Sequenzen von Adenovirus cap-Sequenzen von AAV enthält, die unter der Kontrolle des CMV-Promotors stehen;Agent (II) is a combination of two adenovirus vectors, one of which contains rep sequences from AAV instead of the E3 sequences from adenovirus, the rep 68/78 sequences from AAV being under the control of the "major late promoter" of adenovirus and the rep 40 and rep 52 sequences of AAV are under the control of the endogenous p 19 promoter, and which contains other portions of the E4 sequences of adenovirus cap sequences of AAV which are under the control of the CMV Promoters stand;
oderor
Mittel (II) ist eine Kombination aus einem Adenovirus-Vektor und einem Vacci- niavirus-Vektor, von denen ersterer anstelle der E3-Sequenzen von Adenovirus rep 40- und rep 52-Sequenzen von AAV unter der Kontrolle des endogenen p 1 9- Promotors und cap-Sequenzen von AAV unter der Kontrolle des CMV-Promotors enthält, und der zweite rep 68/78-Sequenzen von AAV enthält. Der Vacciniavi- rus-Vektor wird erst zugefügt, nachdem die DNA-Replikation der Adenovirus-Agent (II) is a combination of an adenovirus vector and a vaccinia virus vector, the former of which instead of the E3 sequences of adenovirus rep 40 and rep 52 sequences of AAV under the control of the endogenous p 19 promoter and contains AAV cap sequences under the control of the CMV promoter, and the second rep contains AAV 68/78 sequences. The vacciniavirus vector is only added after DNA replication of the adenovirus
Vektoren ganz oder teilweise abgelaufen ist.Vectors has expired in whole or in part.
Ein erfindungsgemäßes System eignet sich, rekombinaπte AAVs (rAAVs) in großen Mengen herzustellen. Hierzu wird das erfindungsgemäße System in Zellen eingebracht, welche die Replikation von rAAVs erlauben. Dies sind z.B.A system according to the invention is suitable for producing recombined AAVs (rAAVs) in large quantities. For this purpose, the system according to the invention is introduced into cells which allow the replication of rAAVs. These are e.g.
293-Zellen. Die Zellen werden dann mit Helferviren, wie Adenoviren, infiziert. Dies kann unterbleiben, wenn das erfindungsgemäße System alle für die Replika- tion von rAAVs notwendigen Elemente bereitstellt. Solches findet sich insbeson¬ dere dann, wenn das erfindungsgemäße System in der Alternative (b) vorliegt, in der die Mittel (I) und (II) Virus-Vektoren, insbesondere Adenovirus-Vektoren oder eine Kombination aus Adenovirus- und Vacciniavirus-Vektoren, sind, die sich komplementieren. Auch kann die Komplementation gegeben sein, wenn eines der Mittel ein Virus-Vektor und das andere Mittel eine Zelle ist, die das Protein bereitstellt, dessen DNA im Virus-Vektor deletiert ist.293 cells. The cells are then infected with helper viruses, such as adenoviruses. This can be omitted if the system according to the invention all for the replica rAAVs necessary elements. This is particularly the case if the system according to the invention is in alternative (b), in which the means (I) and (II) virus vectors, in particular adenovirus vectors or a combination of adenovirus and vaccinia virus vectors, are that complement each other. The complementation can also be given if one of the agents is a virus vector and the other agent is a cell which provides the protein whose DNA has been deleted in the virus vector.
Mit der vorliegenden Erfindung ist es möglich, rAAVs bereitzustellen, die für diagnostische und/oder therapeutische Maßnahmen verwendet werden können.With the present invention it is possible to provide rAAVs which can be used for diagnostic and / or therapeutic measures.
Durch die Auswahl der Fremd-DNA können gezielt solche Maßnahmen ergriffen werden.Such measures can be taken by selecting the foreign DNA.
Die vorliegende Erfindung wird durch das Beispiel erläutert.The present invention is illustrated by the example.
Beispiel: Hersteilung eines erfindungsgemäßen SystemsExample: Production of a system according to the invention
(a) Herstellung eines rekombinanten Adenovirus, in den rep/cap-Sequeπzen von AAV (rep/cap-Expressionskassette) in den E1 -Bereich des Adeno- viralen Genoms integriert sind. Es wird auf die Methode von Bett et ai.,(a) Production of a recombinant adenovirus in which rep / cap sequences from AAV (rep / cap expression cassette) are integrated into the E1 region of the adenoviral genome. It is based on the method of Bett et ai.,
Proc. Natl. Acad. Sei., USA 91 , 1 994, 8802-8806 verwiesen. Rep 68/78-Sequenzen von AAV werden unter die Kontrolle des "major late promoter" gestellt, um eine verzögerte Expression zu erreichen. Rep 40/52-Sequeπzen von AAV verbleiben unter der Kontrolle des endogenen p1 9-Promotors. Cap-Sequenzen von AAV werden unter die Kontrolle desProc. Natl. Acad. Sci., USA 91, 1 994, 8802-8806. Rep 68/78 sequences from AAV are placed under the control of the major late promoter in order to achieve delayed expression. Rep 40/52 sequences from AAV remain under the control of the endogenous p1 9 promoter. Cap sequences from AAV are under the control of the
CMV-Promotors gestellt. Ferner enthalten die cap-Sequenzen im 5'-un- translatierten Bereich der mRNAs Sequenzen aus dem 5'-untranslatierten Bereich der späten Adeno-viralen mRNAs.CMV promoter. Furthermore, the cap sequences in the 5'-untranslated region of the mRNAs contain sequences from the 5'-untranslated region of the late adenoviral mRNAs.
Herstellung einer rep/cap-ExpressionskassetteProduction of a rep / cap expression cassette
Eine Teilsequeπz des rep-Gens (Positionen 1882 bis 2280) wird durch PCR mit den Primern 5'-CGCCGGAAGCTTCGATCAACTACGCAGACAG- 3' und 5'-GCGGGCGTCGACTTTGAGCTTCCACCACTGTCTTAT-3' von einer das AAV-Genom enthaltenden Matritze (z.B. pSVOπ'AAV) amplifi- ziert, mit Hindlll/Sall geschnitten und in das mit den gleichen Enzymen geschnittene Plasmid pUC 1 9 inseriert. Es wird pUC1 9Repdupl erhalten.A partial sequence of the rep gene (positions 1882 to 2280) is through PCR with the primers 5'-CGCCGGAAGCTTCGATCAACTACGCAGACAG-3 'and 5'-GCGGGCGTCGACTTTGAGCTTCCACCACTGTCTTAT-3' amplified from a template containing the AAV genome (eg pSVOπ ' AAV), cut with Hindlll / Sall and cut the same with the same enzyme Plasmid pUC 19 inserted. PUC1 9Repdupl is obtained.
Die cDNA-Sequenz der 5' untranslatierten Leader der späten Adenovirus- mRNAs wird mit PCR unter Verwendung der Primer 5'-CGGGGTACCCAG- CTGACTCTCTTCCGCATCGCTG-3 ' und 5 '-CGCGGATC CGAATT- CAAGCTTCTCGAGAGGTTTTCCGATC-3' aus dem Plasmid pMPCV2 (Logan und Shenk, 1 984) amplifiziert, wobei für weitere Kionierungen passende flankierende Restriktionsschnittstellen entstehen. Das Amplifikat wird mit Kpnl/BamHI geschnitten und in das mit den gleichen Enzymen geschnittene Plasmid pCEP4 (Firma Invitrogen) inseriert, wobei der Vektor pCEP4-CMVL entsteht. Mit EcoRI/Sall wird der CMV-Promotor zusammen mit dem Leader aus pCEP4-CMV-Leader herausgeschnitten und in das mit den selben Restriktioπsenzymen linearisierte pUC1 9Repdupl inseriert; es entsteht pRepdupl-CMVL. Der Bereich mit der rep-Teilsequenz und dem CMV-Promotor und dem Adeno-Leader wird mit Hindlll aus dem resultie¬ renden pRepdupl-CMV-Leader geschnitten und in partiell mit Hindlll ge- schnittenen pSVOriAAV (Chiorini et al., 1 995) inseriert. Es entsteht pRepCMVLCap. Die Insertion an der richtigen Stelle in der richtigen Orien¬ tierung wird mit adäquaten Restriktionsspaltungen überprüft.The cDNA sequence of the 5 'untranslated leaders of the late adenovirus mRNAs is PCR by using the primers 5'-CGGGGTACCCAG-CTGACTCTCTTCCGCATCGCTG-3' and 5 '-CGCGGATC CGAATT-CAAGCTTCTCGAGAGGTTMTPCVG2CidCan 3 and the 1 984) amplified, with suitable flanking restriction sites being formed for further ionizations. The amplificate is cut with Kpnl / BamHI and inserted into the plasmid pCEP4 (Invitrogen) cut with the same enzymes, the vector pCEP4-CMVL being produced. With EcoRI / Sall, the CMV promoter is cut out together with the leader from pCEP4-CMV leader and inserted into pUC1 9Repdupl linearized with the same restriction enzymes; pRepdupl-CMVL is created. The region with the rep partial sequence and the CMV promoter and the adeno leader is cut with Hindlll from the resulting pRepdupl-CMV leader and inserted in pSVOriAAV (Chiorini et al., 1 995) partially cut with Hindlll . PRepCMVLCap is created. The insertion at the right place in the right orientation is checked with adequate restriction cleavages.
In pRepCMVLCap wird durch dieses Vorgehen der CMV-Promotor zwi- sehen die rep- und die cap-Sequenzen inseriert, wobei die Unterbrechung des rep-Leserasters durch Duplikation einer Teilsequenz verhindert wird und der CMV-Promotor in die Hindlll-Schnittstelle an Pos. 1 883 der AAV- Sequenz des pSVOriAAV inseriert wird. Diese Hindlll-Schnittstelle befin¬ det sich zwischen dem Transkriptionsstartpunkt des p40-Promotors und dem 5'-Splicing-Signal der cap-Sequenzen.In pRepCMVLCap, this procedure inserts the CMV promoter between the rep and the cap sequences, the interruption of the rep reading frame being prevented by duplicating a partial sequence and the CMV promoter in the HindIII interface at position 1 883 of the AAV sequence of the pSVOriAAV is inserted. This HindIII interface is located between the transcription start point of the p40 promoter and the 5 'splicing signal of the cap sequences.
pRepCMVLCap wird mit Spei linearisiert und mit T4-Exonuklease behan- delt. Eine Zeitreihe der T4-Exonuklease-Behandlung ermöglicht das Ent¬ stehen unterschiedlicher Größen des deletierten Bereichs. Es wird mit einem Konstrukt weitergearbeitet, bei dem der 5'-Bereich des AAV-Ge- noms bis zu einer Stelle zwischen den Position 288 und 321 deletiert ist, so daß die verbleibende Sequenz zwischen dem Transkriptionsstart der rep68/78-Sequenzen, Pos.288, und dem Startkodon dieser Sequenzen, Pos. 321, beginnt. In den deletierten Bereich wird mit synthetischen Linkern eine Polylinkersequenz mit Xbal, Notl, Spei, und Bglll-Schnitt- stellen eingebaut, so daß pRepCMVLCap' entsteht. Der major-late-Promo- tor wird durch Verwendung der Primer 5'-CGTCTA-pRepCMVLCap is linearized with Spei and treated with T4 exonuclease delt. A time series of the T4 exonuclease treatment enables different sizes of the deleted area to arise. The work continues with a construct in which the 5 'region of the AAV genome has been deleted up to a position between positions 288 and 321, so that the remaining sequence between the start of transcription of the rep68 / 78 sequences, pos. 288, and the start codon of these sequences, item 321, begins. In the deleted area, a polylinker sequence with Xbal, Notl, Spei, and Bglll interfaces is built in with synthetic linkers, so that pRepCMVLCap ' is formed. The major late promoter is by using the primer 5'-CGTCTA-
GAGCGGCCGCCCGCGGTCCTCCTCGTATAGAAACT-3' und 5'-GCA- GATCTACTAGTCTCGAGAGGTTTTCCGATCCGGTCG-3' aus pMPCV2 (Logan und Shenk, 1984) amplifiziert und mit Bglll/Xbal nachgeschnitten. pRepCMVLCap* wird mit Bglll/Xbal geschnitten und der major late-Promo- tor wird in der richtigen Orientierung inseriert, so daß pMLPRepCMVLCap entsteht.GAGCGGCCGCCCGCGGTCCTCCTCGTATAGAAACT-3 'and 5'-GCA-GATCTACTAGTCTCGAGAGGTTTTCCGATCCGGTCG-3' from pMPCV2 (Logan and Shenk, 1984) amplified and trimmed with Bglll / Xbal. pRepCMVLCap * is cut with Bglll / Xbal and the major late promoter is inserted in the correct orientation so that pMLPRepCMVLCap is created.
Die rep-Kassette weist den endogenen p19-Promotor auf, der in die rep- Sequenz eingebettet ist und weiterhin die Expression der Proteine rep40 und rep52 kontrolliert, während nur die Sequenzen für rep68/78 unter derThe rep cassette has the endogenous p19 promoter, which is embedded in the rep sequence and continues to control the expression of the proteins rep40 and rep52, while only the sequences for rep68 / 78 under the
Kontrolle des major late-Promotors stehen.Check the major late promoter.
Herstellung eines rekombinanten Adenovirus mit rep/cap-Expressions- kassetteProduction of a recombinant adenovirus with rep / cap expression cassette
Die rep/cap-Expressionskassette wird mit Xbal/Clal aus pMLPRep¬ CMVLCap ausgeschnitten und in den mit den gleichen Enzymen lineari- sierten Vektor pΔE1sp1A (Bett el al., 1995) inseriert. Dieses Plasmid enthält die Sequenzen des Adenovirus Typ 5 zwischen den Karteneinhei¬ ten 0 und 0,9 sowie 9,8 und 15,8 wobei der E1A-Bereich durch eine Polylinker-Sequenz zur Insertion von Fremdgenen substituiert wurde. Genome infektiöser rekombinanter Adenoviren können durch in vivo- Rekombination zwischen überlappenden Bereichen Adeno-viraler Sequen¬ zen auf pBHG I O, welches die Adenovirus-Sequenzen zwischen den Karteneinheiten 0 und 0,5, von 3,7 bis 78,3 und von 85,8 bis 100, sowie dem pΔE 1 sp 1 A-Derivat, welches rep/cap-Sequenzen enthält, entstehen.The rep / cap expression cassette is cut out of pMLPRep ¬ CMVLCap with Xbal / Clal and inserted into the vector pΔE1sp1A (Bett el al., 1995) linearized with the same enzymes. This plasmid contains the sequences of type 5 adenovirus between card units 0 and 0.9 as well as 9.8 and 15.8, the E1A region being substituted by a polylinker sequence for inserting foreign genes. Genomes of infectious recombinant adenoviruses can by in vivo recombination between overlapping areas of adenoviral sequences on pBHG IO, which the adenovirus sequences between the card units 0 and 0.5, from 3.7 to 78.3 and from 85.8 to 100, as well as the pΔE 1 sp 1 A derivative, which contains rep / cap sequences.
Zur Komplementation des E1 A-Gens wird die Kotransfektion der beiden Plasmide in 293-Zellen durchgeführt. Ein zytopathischer Effekt, der sich einige Tage nach der Transfektion manifestiert, deutet auf das Entstehen rekombinanter Adenoviren hin. Die 293-Zellen, die einen zytopathischen Effekt zeigen, werden durch Einfrieren und Auftauen aufgebrochen und aus dem Lysat werden durch zweifache Plaque-Reinigung rekombinante Viren (Adrep/cap) vereinzelt. Einzelklone werden durch sukzessive Passa¬ ge über 293-Kulturen amplifiziert. Die Expressionsmuster von rep und cap werden durch Western-Blotting charakterisiert.To complement the E1 A gene, the cotransfection of the two plasmids is carried out in 293 cells. A cytopathic effect that manifests itself a few days after the transfection indicates the development of recombinant adenoviruses. The 293 cells, which show a cytopathic effect, are broken up by freezing and thawing and recombinant viruses (adrep / cap) are separated from the lysate by double plaque cleaning. Individual clones are amplified by successive passage over 293 cultures. The expression patterns of rep and cap are characterized by Western blotting.
(b) Herstellung eines rekombinanten Adenovirus, in den ein rAAV-Genom integriert ist(b) Production of a recombinant adenovirus in which an rAAV genome is integrated
Es wird eine Fremd-DNA, z.B. ein Luciferase-Reportergen mit flankie- renden AAV-ITR-Sequenzen, in die E1 A-Region eines Adenovirus inte¬ griert. Aus dem Vektor pAAVCMVLuc (vgl. Maass et al., in Vorbereitung), der das Luciferase-Reportergen unter der Kontrolle des CMV-Promotors, flankiert von AAV-ITR-Sequenzen in einem pEMBL8-Vektor enthält, wird die Expressionskassette mit den ITR-Sequenzen mit Pvull herausgeschnit- ten. Dieses Fragment wird in EcoRV-geschnittenen pΔE1 sp 1 A über die glatten Enden integriert. Es entsteht pΔE1 sp1 A-Luc. Die Herstellung der rekombinanten Adeπovireπ (AdAALuc) erfolgt durch Kotransfektion von pΔE1 sp 1 A-Luc und pBHG I O in 293-Zellen durch in vivo-Rekombination, wie unter (a) beschrieben. (c) Hersteilung von rekombinanten AAV-Vektoren durch Infektion von 293- Zellen mit Adrep/cap und AdAAVLucA foreign DNA, for example a luciferase reporter gene with flanking AAV-ITR sequences, is integrated into the E1 A region of an adenovirus. The expression cassette with the ITR- Sequences cut out with Pvull. This fragment is integrated into EcoRV-cut pΔE1 sp 1 A via the smooth ends. PΔE1 sp1 A-Luc is formed. The recombinant Adeπovireπ (AdAALuc) is produced by cotransfection of pΔE1 sp 1 A-Luc and pBHG IO in 293 cells by in vivo recombination, as described under (a). (c) Production of recombinant AAV vectors by infection of 293 cells with Adrep / cap and AdAAVLuc
4 x 10a 293-Zellen werden zu 80% Koπfluenz kultiviert und mit einer m.o.i. von jeweils 10 gleichzeitig mit Adrep/cap und AdAAVLuc in einem geringen Volumen serumfreien Mediums infiziert. 2 Stunden nach Infek¬ tion wird Vollmedium zugegeben. 3 Tage nach Infektion werden die Zellen mit einem Zell-Kratzer von der Oberfläche der Kulturgefäße entfernt; die Suspension wird 5 min bei 200g und Raumtemperatur abzentrifugiert. Die Zellpellets werden in 28 ml TD-Puffer (140 mM NaCI, 5 mM KCI, 0,7 mM4 x 10 a 293 cells are cultivated to 80% confluence and infected with a moi of 10 at a time with Adrep / cap and AdAAVLuc in a small volume of serum-free medium. Complete medium is added 2 hours after infection. 3 days after infection, the cells are removed from the surface of the culture vessels with a cell scratch; the suspension is centrifuged off at 200 g and room temperature for 5 min. The cell pellets are in 28 ml TD buffer (140 mM NaCl, 5 mM KCI, 0.7 mM
K2HP04/ 25 mM Tris/Cl pH 7,4) suspendiert und es werden 2 ml 10% (w/v) Natriumdesoxycholat und 2 ml 0,25% Trypsinlösung zugegeben. Das Lysat wird zum Zeilaufschluß 30 min bei 37°C inkubiert und an¬ schließend mit einem Dounce-Homogenisatσr behandelt. Mit CsCI wird eine Dichte von 1 ,4 g/ml eingestellt und das Lysat wird 24 h einer iso- pyknischen Dichtegradientenzentrifugatioπ bei 1 30000 g und 20°C unterzogen. Fraktionen mit einem refraktorischen Index zwischen 1 ,3705 und 1 ,3750 werden mit einem Fraktionssammler gesammelt, vereinigt und einer weiteren Dichtegradientenzentrifugation unter den gleichen Bedingungen unterzogen. Fraktionen mit einem refraktorischen Index zwischen 1 ,3705 und 1 ,3750 werden erneut gesammelt und gegen 0,9%ige NaCI-Lösung dialysiert. Das resultierende AAV-Lysat kann bei biologischen Tests, z.B. einem Luciferase-Expressionstest, verwendet werden. K 2 HP0 4/25 suspended 7.4) mM Tris / Cl pH and 2 ml of 10% (w / v) sodium deoxycholate and 2 ml of 0.25% trypsin was added. The lysate is incubated for 30 min at 37 ° C. and then treated with a dounce homogenizer. A density of 1.4 g / ml is set with CsCI and the lysate is subjected to an isopycnic density gradient centrifugation at 130000 g and 20 ° C. for 24 h. Fractions with a refractive index between 1, 3705 and 1, 3750 are collected with a fraction collector, pooled and subjected to a further density gradient centrifugation under the same conditions. Fractions with a refractive index between 1, 3705 and 1, 3750 are collected again and dialyzed against 0.9% NaCl solution. The resulting AAV lysate can be used in biological tests, for example a luciferase expression test.

Claims

Ansprüche Expectations
1 . System, umfassend einen, eine Fremd-DNA enhaltenden AAV-Vektor, und rep 68/78-Sequenzen von AAV, deren Expression verzögert ist, wobei diese Sequenzen (a) in eis oder (b) in trans vorliegen.1 . A system comprising an AAV vector containing a foreign DNA, and rep 68/78 sequences of AAV, the expression of which is delayed, these sequences being present in (a) in eis or (b) in trans.
2. System nach Anspruch 1 , dadurch gekennzeichnet, daß die Fremd-DNA exprimierbar ist.2. System according to claim 1, characterized in that the foreign DNA is expressible.
3. System nach Anspruch 1 oder 2, dadurch gekennzeichnet, daß die Fremd- DNA unter der Kontrolle eines konstitutiveπ oder induzierbaren Promotors steht.3. System according to claim 1 or 2, characterized in that the foreign DNA is under the control of a constitutiveπ or inducible promoter.
4. System nach einem der Ansprüche 1 -3, dadurch gekennzeichnet, daß in4. System according to any one of claims 1 -3, characterized in that in
(a) der AAV-Vektor und die rep 68/78-Sequeπzen von AAV in einem Mittel (I) vorliegen.(a) the AAV vector and the rep 68/78 sequences of AAV are present in an agent (I).
5. System nach Anspruch 4, dadurch gekennzeichnet, daß das Mittel (I) eine Zelle oder ein Vektor ist.5. System according to claim 4, characterized in that the means (I) is a cell or a vector.
6. System nach Anspruch 5, dadurch gekennzeichnet, daß der Vektor ein Virus- oder ein Plasmid-Vektor ist.6. System according to claim 5, characterized in that the vector is a virus or a plasmid vector.
7. System nach einem der Ansprüche 1 -3, dadurch gekennzeichnet, daß in7. System according to any one of claims 1 -3, characterized in that in
(b) der AAV-Vektor in einem Mittel (I) und die rep 68/78-Sequenzen von AAV in einem Mittel (II) vorliegen.(b) the AAV vector is in an agent (I) and the rep 68/78 sequences of AAV are in an agent (II).
8. System nach Anspruch 7, dadurch gekennzeichnet, daß das Mittel (I) und das Mittel (II) eine Zelle und/oder ein Vektor sind, wobei das Mittel (!) und das Mittel (II) gleichzeitig keine Zelle sind.8. System according to claim 7, characterized in that the means (I) and the agent (II) is a cell and / or a vector, the agent (!) and the agent (II) at the same time not being a cell.
9. System nach Anspruch 8, dadurch gekennzeichnet, daß der Vektor ein Virus- und/oder ein Plasmid-Vektor ist.9. System according to claim 8, characterized in that the vector is a virus and / or a plasmid vector.
1 0. System nach Anspruch 9, dadurch gekennzeichnet, daß der Virus-Vektor ein Adenovirus-Vektor ist.1 0. System according to claim 9, characterized in that the virus vector is an adenovirus vector.
1 1 . System nach Anspruch 9, dadurch gekennzeichnet, daß der Virus-Vektor in Mittel (I) ein Adenovirus-Vektor und in Mittel (II) ein Vacciniavirus- Vektor ist.1 1. System according to claim 9, characterized in that the virus vector in agent (I) is an adenovirus vector and in agent (II) is a vaccinia virus vector.
1 2. Verwendung des Systems nach einem- der Ansprüche 1 -1 1 zur Herstel- lung von AAV-Vektoren. 1 2. Use of the system according to one of claims 1 -1 1 for the production of AAV vectors.
PCT/DE1997/001333 1996-06-24 1997-06-24 System for the production of aav vectors WO1997049824A1 (en)

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