WO1997049824A1 - Systeme de production de vecteurs de virus aav - Google Patents

Systeme de production de vecteurs de virus aav 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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WO
WIPO (PCT)
Prior art keywords
aav
rep
sequences
vector
agent
Prior art date
Application number
PCT/DE1997/001333
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German (de)
English (en)
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WO1997049824B1 (fr
Inventor
Christoph Bogedain
Gerd Maass
Michael Hallek
Original Assignee
Medigene Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Medigene Ag filed Critical Medigene Ag
Priority to JP10502116A priority Critical patent/JP2000512501A/ja
Priority to US09/214,151 priority patent/US6294370B1/en
Priority to EP97930352A priority patent/EP0954592A1/fr
Publication of WO1997049824A1 publication Critical patent/WO1997049824A1/fr
Publication of WO1997049824B1 publication Critical patent/WO1997049824B1/fr

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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

Abstract

L'invention concerne un système comprenant un vecteur de virus AAV, qui comprend un ADN étranger et des séquences 68/78 répétitives de virus AAV, dont l'expression est retardée, ces séquences étant présentes (a) en cis ou (b) en trans. L'invention concerne également l'utilisation d'un tel système pour la production de vecteurs de virus AAV.
PCT/DE1997/001333 1996-06-24 1997-06-24 Systeme de production de vecteurs de virus aav WO1997049824A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP10502116A JP2000512501A (ja) 1996-06-24 1997-06-24 Ａａｖベクターの生産系
US09/214,151 US6294370B1 (en) 1997-06-24 1997-06-24 System for the production of AAV vectors
EP97930352A EP0954592A1 (fr) 1996-06-24 1997-06-24 Systeme de production de vecteurs de virus aav

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE1996125188 DE19625188A1 (de) 1996-06-24 1996-06-24 System zur Herstellung von AAV-Vektoren
DE19625188.5 1996-06-24

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US09/214,151 A-371-Of-International US6294370B1 (en) 1996-06-24 1997-06-24 System for the production of AAV vectors
US09/908,660 Division US6541012B2 (en) 1996-06-24 2001-07-18 System for the production of AAV vectors

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WO1997049824A1 true WO1997049824A1 (fr) 1997-12-31
WO1997049824B1 WO1997049824B1 (fr) 1998-02-19

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JP (1) JP2000512501A (fr)
DE (1) DE19625188A1 (fr)
WO (1) WO1997049824A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000047757A1 (fr) * 1999-02-10 2000-08-17 Medigene Ag Procede de fabrication d'un virus adeno-associe recombine, moyens adaptes a cette fabrication et utilisation dudit virus pour la fabrication d'un medicament
US7115391B1 (en) 1999-10-01 2006-10-03 Genovo, Inc. Production of recombinant AAV using adenovirus comprising AAV rep/cap genes

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6037177A (en) * 1997-08-08 2000-03-14 Cell Genesys, Inc. Method for increasing the efficiency of recombinant AAV production
CA3042853A1 (fr) 2016-11-07 2018-05-11 Crossbeta Biosciences B.V. Nouvelle molecule de liaison specifique d'oligomere beta-amyloide

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995013392A1 (fr) * 1993-11-09 1995-05-18 Medical College Of Ohio Lignees cellulaires stables aptes a exprimer le gene de replication du virus adeno-associe
WO1995034670A2 (fr) * 1994-06-06 1995-12-21 Children's Hospital, Inc. Genome du virus adeno-associe recombine codant une proteine du virus de l'immunodeficience
WO1996017947A1 (fr) * 1994-12-06 1996-06-13 Targeted Genetics Corporation Lignees cellulaires d'encapsidation utilisees pour la generation de titres hauts de vecteurs aav recombinants

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995013392A1 (fr) * 1993-11-09 1995-05-18 Medical College Of Ohio Lignees cellulaires stables aptes a exprimer le gene de replication du virus adeno-associe
WO1995034670A2 (fr) * 1994-06-06 1995-12-21 Children's Hospital, Inc. Genome du virus adeno-associe recombine codant une proteine du virus de l'immunodeficience
WO1996017947A1 (fr) * 1994-12-06 1996-06-13 Targeted Genetics Corporation Lignees cellulaires d'encapsidation utilisees pour la generation de titres hauts de vecteurs aav recombinants

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
FLOTTE, T.R. ET AL.: "An improved system for packaging recombinant adeno-associated virus vectors capable of in vivo transduction", GENE THERAPY, vol. 2, no. 1, January 1995 (1995-01-01), pages 29 - 37, XP002043509 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000047757A1 (fr) * 1999-02-10 2000-08-17 Medigene Ag Procede de fabrication d'un virus adeno-associe recombine, moyens adaptes a cette fabrication et utilisation dudit virus pour la fabrication d'un medicament
US6846665B1 (en) 1999-02-10 2005-01-25 Medigene Aktiengesellschaft Method of producing a recombinant adeno-associated virus, suitable means for producing the same and use thereof for producing a medicament
US7115391B1 (en) 1999-10-01 2006-10-03 Genovo, Inc. Production of recombinant AAV using adenovirus comprising AAV rep/cap genes

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JP2000512501A (ja) 2000-09-26
DE19625188A1 (de) 1998-01-08
EP0954592A1 (fr) 1999-11-10

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