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WO1999053084A1 - Preparation d'adenovirus recombines portant un gene rep de virus adeno-associe - Google Patents

Preparation d'adenovirus recombines portant un gene rep de virus adeno-associe Download PDF

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Publication number
WO1999053084A1
WO1999053084A1 PCT/EP1999/002384 EP9902384W WO9953084A1 WO 1999053084 A1 WO1999053084 A1 WO 1999053084A1 EP 9902384 W EP9902384 W EP 9902384W WO 9953084 A1 WO9953084 A1 WO 9953084A1
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Prior art keywords
helper
cells
aav
rep
adenoviral
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PCT/EP1999/002384
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English (en)
Inventor
Gennaro Ciliberto
Stefano Colloca
Nicola La Monica
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Istituto di Ricerche di Biologia Molecolare P Angeletti SpA
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Istituto di Ricerche di Biologia Molecolare P Angeletti SpA
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Priority claimed from PCT/IT1998/000082 external-priority patent/WO1998045462A1/fr
Application filed by Istituto di Ricerche di Biologia Molecolare P Angeletti SpA filed Critical Istituto di Ricerche di Biologia Molecolare P Angeletti SpA
Priority to CA002326847A priority Critical patent/CA2326847A1/fr
Priority to US09/647,795 priority patent/US6521426B1/en
Priority to EP99922090A priority patent/EP1068343A1/fr
Priority to AU39265/99A priority patent/AU752811B2/en
Publication of WO1999053084A1 publication Critical patent/WO1999053084A1/fr
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
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10344—Chimeric viral vector comprising heterologous viral elements for production of another viral 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
    • C12N2750/14141—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 present invention relates to viral vectors which may be used in gene delivery, such as in gene therapy, processes for viral replication, and viral vectors, cells and cell lines useful in preparation of viral vectors which may be used in gene delivery.
  • the invention is not concerned with gene therapy itself, rather with the provision and production of vectors which may be used in gene delivery, such as in gene therapy.
  • Adeno-associated virus has the unique capacity of preferentially integrating its viral DNA within a defined region of the cellular genome, thus reducing the risks of insertional mutagenesis associated with other viruses such as retroviruses that integrate at random positions.
  • AAV is a non-pathogenic human parvovirus which usually requires Adenovirus (Ad) or Herpes virus as a helper to replicate efficiently.
  • Ad Adenovirus
  • Herpes virus Herpes virus as a helper to replicate efficiently.
  • the AAV genome integrates into host-cell genomic DNA at high frequency.
  • Analysis of flanking sequences from latently infected cells of human origin have revealed integration of 2 the AAV genome into a specific locus in 60-70% of cases.
  • the integration locus (aavsl) has been sequenced and localised to human chromosome 19ql3.3-qter (Kotin, RM, et al , 1990; Samulski. RJ et al , 1991).
  • Ad HDV Ad helper dependent vector
  • recombinant AAV vectors in which rep and cap genes have been deleted do not integrate into the AAVS1 locus but they do so in a random fashion.
  • Targeting of integration involves the AAV rep gene products.
  • the larger polypeptides Rep 78 and Rep 68 have been shown to bind in vi tro the AAV ITRs and the aavsl, and possess helicase and site-specific endonuclease activities 5 which may be required for AAV replication as well as AAV integration. See for example Shelling, A. N.et al (1994); Balague, C, M. et al . (1997); Surosky, R. T. et al . (1997).
  • the present invention is founded on the surprising realisation that a much simpler approach can be taken to produce the desired vectors in large amounts, as successfully demonstrated experimentally .
  • the present invention provides in various aspects new procedures for the efficient preparation of recombinant helper dependent adenoviral vectors comprising AAV rep genes, based on the finding that it is possible to avoid rep inhibition of replication of viral vectors by keeping on separate replicating units the viral replication functions required for vector amplification and the AAV rep genes.
  • AAV rep 78 and/or rep 68 may be included in the helper- dependent adenoviral vector (Surosky, R. T. et al. 1997) .
  • rep ⁇ O and/or rep42 may additionaly be included.
  • Modified forms of a rep gene which may encode a modified protein may be used, provided the ability of the protein to promote nucleic acid integration into the chromosome is retained.
  • a modified form which is inactive or substantially inactive unless activated by means of an appropriate stimulus or signal may be employed.
  • Adenoviral particles that are produced may be harvested, may be isolated and/or purified, and may be used as desired, e.g. in the formulation of a composition which may include one or more additional components, such as a composition (pharmaceutical) which includes one or more pharmaceutically acceptable excipients, vehicles or carriers (e.g. see below) .
  • a composition pharmaceutically acceptable excipients, vehicles or carriers
  • a substantially improved and preferred method further includes an additional step of pre-incubating the helper virus vector containing cells, for a period of time sufficient to allow 8 expression of viral proteins required for Ad genome replication, before introducing the helper-dependent adenovirus vectors including an AAV rep gene.
  • Methods in accordance with the present invention are generally applicable to the preparation of vectors that by carrying one or more AAV rep genes have the property to inhibit adenovirus replication.
  • helper is a helper adenovirus, and may be AdLC ⁇ cluc (Parks et al . , 1996) .
  • the optimal concentration of helper to be used in the method may vary, and if the helper virus genome is introduced into the host cell by DNA transfer methods, for each method a range of concentration may be used. If the helper genome is delivered by the use of infective viral particles, generally the range of concentration may be between 1 and 100 m.o.i. (multiplicity of infection) , the preferred concentration being between 1 and 5 m.o.i..
  • the rep gene is or rep genes are generally under the control of a promoter, and most preferably under the control of a regulatable promoter. Suitable promoters include the ⁇ l-anti trypsin promoter, T7 promoter, and tissue-specific promoters. T7 promoter has a very low activity in the absence of the phage T7 polymerase and is an appropriate promoter to rescue adenovectors encoding toxic genes. 9
  • the promoter is a tissue specific promoter that is known to be active in the cells of the target tissue (e.g. liver) but less active in the cell chosen as packaging cells for the production of the viral adenovectors .
  • the cell line that may be used as host is any cell line capable to sustain the replication of the helper and of the vector to be amplified.
  • a preferred cell line is 293 for its ability to sustain defective adenovirus replication, in particular the 293 derived line 293cre (Chen L., et al . , 1996) .
  • Use of this cell line selectively favors the packaging and amplification of helper dependent vectors which do not contain loxP sites at either end of the Adenovirus packaging signal.
  • helper adenovirus such as AdLc ⁇ cluc
  • helper 10 vectors may be employed based on the use of different recombinases and their specific substrate, and of suitable host cell lines.
  • yeast FLP recombinase and its recombinase sites may be used (0' Gorman et al . 1991) .
  • Standard conditions may be used, such as wherein infected 293cre cells are maintained in minimal essential medium (MEM) supplemented with 5% Horse serum (HS) , 2mM glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin. Cells are grown in tissue culture dishes (Falcon) at 37°C in 5% C0 2 .
  • MEM minimal essential medium
  • HS Horse serum
  • 2mM glutamine 100 units/ml penicillin
  • streptomycin 100 mg/ml
  • Cells may be cultured for approximately between 0.5 and 12 hours, more preferably at least about 2 hours, maybe about 2-6 hours, or about 3-7 hours, and more preferably about 4 hours.
  • Optimal timing may be established by monitoring the final titre in defective vector obtained, for example by setting up a series of growth experiment in parallel.
  • the viral vector genome is usually delivered to the helper infected cells by transfection in a first cycle, and by infection with infective viral particles containing the defective virus genome in successive cycles of amplification of the viral vector.
  • Other DNA transfer method may be used, such as electroporation, DEAE-dextran, calcium phosphate, DNA gun, liposomes, etc.
  • the viral vector is a recombinant Ad/AAV vector including one or more of the AAV rep genes, alone or in combination, under the control of a single or multiple regulatory elements.
  • a preferred vector may be one selected from the group consisting of pRS1032; pRS1033; pRA1034 and pSTK (Schiedner et al . , 1998) (see experimental section below) .
  • the present invention also provides a system for replication and packaging in cultured cells of recombinant DNA into mature virions.
  • Viral stocks containing recombinant DNA encapsidated into mature virions obtained by the methods here described represent further aspects of the present invention and can be used to transfer genetic information into any cell or tissue 12 of choice .
  • Nucleic acid sequences can be readily prepared and manipulated by the skilled person using the information and references contained herein and techniques known in the art .
  • the sequences can be incorporated in a vector in accordance with the present invention having control sequences operably linked to the nucleic acid to control its expression.
  • the vectors may include sequences such as promoters or enhancers to drive the expression of the inserted nucleic acid, and may include one or more further sequences so that the polypeptide is produced as a fusion and/or nucleic acid encoding secretion signals so that the polypeptide produced in the host cell is secreted from the cell.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.
  • a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.
  • Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the particles may be administered to an individual, particularly human or other primate.
  • This may be for a therapeutic purpose, e.g. in delivery of a functional gene encoding an authentic biologically active product in a method of gene therapy, to treat a patient who is unable to synthesize that product or unable to synthesize it at the normal level or in normal form, thereby providing the effect provided by the wild-type and ameliorating one or more symptoms of the relevant disease.
  • a therapeutic purpose e.g. in delivery of a functional gene encoding an authentic biologically active product in a method of gene therapy, to treat a patient who is unable to synthesize that product or unable to synthesize it at the normal level or in normal form, thereby providing the effect provided by the wild-type and ameliorating one or more symptoms of the relevant disease.
  • Examples include provision of Factor VIII to a haemophiliac, or erythropoietin to an individual with chronic anaemia, chronic renal failure or sickle cell anaemia.
  • Other therapeutically useful genes include those encoded Factor IX coagulation factor, L
  • Administration is preferably in a "prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions 15 on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners.
  • a composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • Figure IB shows the results of serial passage of Ad Rep vectors.
  • pRP1030 was amplified as a positive control.
  • 16 In the construction of viral vector useful for gene delivery such as in gene therapy, i.e. the transfer of genetic information into cells or tissues, one of the advantages offered by the AAV derived vectors is that the genetic material to be transfered can be directed to preferentially integrate in specific sites on the host chromosome. For this to occur the presence of at least one of the rep peptides is required, as discussed.
  • helper dependent Ad vectors it has been possible to separate the viral replication functions 17 required for vector amplification from the vector itself.
  • the present invention is based on the discovery that if a rep gene from AAV is carried by a helper dependent Ad vector, its expression can be suppressed or delayed during the helper replication stage thus bypassing the inhibitory effect due to the rep gene expression.
  • helper dependent Ad vectors carrying a rep gene here described for particular embodiments of the present invention is based on the exploitation of the helper dependent Ad system described by F. Graham and coworkers (Parks et al . 1996) .
  • the helper dependent Ad system is based on the development of recombinant helper adenovirus that, when they are replicating in suitable cells, are able to produce all the proteins and RNA necessary to perform helper functions, but whose genome cannot be packaged in infective viral particles. This can be achieved by inserting artificial sites for specific recombinases (e.g. loxP sites target of Cre recombinase) on both sides of the helper packaging signals sequences.
  • the AAV2-ITRGFP-HygroR cassette was constructed by inserting the humanized version of green fluorescent protein (GFP) gene driven by HCMV promoter from the pGreen Lantern plasmid (GIBCO BRL) and the Hygromicin B resistance gene fused to the Tk promoter between AAV2 ITRs in the context of plasmid pLITMUS28 (New England Biolabs) generating pITRGFP Hygro.
  • GFP green fluorescent protein
  • Plaques were visible after 10 days of incubation at 37°C. Picking, screening and amplification of the isolated plaques were performed as described in Hitt, M. et al . 1995.
  • the Rep gene of AAV (Srivastava et al . , 1983) was modified by point mutations to encode only the large polypeptide rep78 under the transcriptional control of the ⁇ l-anti trypsin or T7 20 promoter (Horer, M., et al . , 1995; De Simone, V., et al . , 1987) .
  • a hd Ad/Rep virus was constructed by insertion of the appropriately modified Rep gene into a hd Ad genome by restriction digestion.
  • Plasmids were constructed using standard protocols (J. Sambrook, et al . 1989).
  • Plasmid pRP1030 was used as vector for the insertion of Rep expression cassette. It was derived from pRPlOOl (Parks, et al . 1996) by deleting all Ad5 coding sequence and substituting it with a lambda phage DNA stuffer.
  • the helper dependent plasmid pRS1032 was derived from pRP1030. It contains the insertion of a cassette constituted by Rep 78 ATG deletion mutant fused to T7 promoter. Rep 78 gene was obtained as described in Horer et al . (1995) by mutating the ATG start codon for Rep 52/40 to GGA (methionine>glycine, amino acid 225) and the G of the splice donor site for expression of spliced versions Rep 68/40 (nucleotide 1907) to A.
  • T7Rep cassette was constructed following the strategy reproted for T7Rep78 ⁇ ATG and inserted into the unique BamHI cloning site of pABS .4 , generating pABT7Rep .
  • pABT7Rep was converted in a Rep 78 expression vector by substituting the Sfil-Xbal DNA fragment with the Sfil-Xbal restriction fragment obtained from pCIIIRep78 plasmid.
  • This fragment contains a mutation of the first ATG of Rep 52/40 (ATG-GGA) and a G>A mutation in the splice donor sequence present in the Rep ORF.
  • T7Rep78 cassette was than excised from pABT7Rep78 by PvuII digestion and inserted into the StuI site of pRP1030 generating pRS1033.
  • the helper dependent plasmid pRA1034 was derived from pRP1030 pRA1034 contains rep 78 gene fused to a ⁇ 137 ⁇ l-antitrypsin promoter.
  • ⁇ l-antitrypsin ( ⁇ l-at) -Rep78 cassette was constructed by fusing rep78 gene to the ⁇ 137 ⁇ l-at minimal promoter in the context of pABS-4 shuttle plasmid generating pRA1034. 22 (b) The ⁇ lat-Rep78 cassette was then excised by PvuII digestion from pAB ⁇ lat-Rep78 and cloned into pRP1030 generating pRA1034.
  • Amplification of a hd Ad/Rep virus was obtained with a modification of the established protocol for the amplification of hd Ad viruses (Parks, R., et al . , 1996) .
  • Semiconfluent monolayers of 293cre cells (Chen L, et al 1996) in 60 mm dishes were infected with AdLC ⁇ cLuc at a multiplicity of infection (m.o.i.) of 5 plaque-forming units (pfu)/cell.
  • the cells were scraped into the medium and the virus released by freezing and thawing. 23
  • the resulting crude lysate was serially passaged on 60 -mm dishes of 293cre cells as follows. During each round of amplification of the three different helper-dependent vectors, the 293cre monolayers were infected with AdLC ⁇ cLuc at an m.o.i. of 1 pfu/cell and incubated at 37°C for 4 hr before the medium was removed and cells were incubated at 30°C with an aliquot (500 ⁇ l) of the crude lysate obtained from previous passage .
  • Amplification was monitored at each passage by infecting 293 cells and counting lac-Z positive cells (blue forming unit (bfu) , as described in Parks et al . (1996) ( supra) .
  • Figure IB shows the amplification results of the three Rep containing Helper-Dependent vectors in comparison with the non-recombinant vector RP1030.
  • Rep gene did not affect the amplification of the AdHD vector up to 1.8xl0 7 b.f.u./ml (corresponding to 60- 100 Rep78 expressing viral particles per cells) .
  • Time course of CPE was not affected by Rep virus infection and full CPE was usually achieved after 48-72 hours post-infection even during late passages in the presence of an increasing titre of Rep expressing viruses.
  • the inhibition of Ad replication is mediated by Rep expression and dependent on the ratio of the multiplicity of infection of the two viruses and the temporal order of addition (Berns,K.I, 1996).
  • the Rep gene was fused to ⁇ -137 DNA fragment of ⁇ l -antitrypsin promoter or to the T7 promoter. Although a residual Rep enzymatic activity was detected with both constructs by transfecting 293 cells, the protein was not detectable using a standard Western blot. Furthermore, to rescue the Ad viruses 293cre cells were first infected with the helper virus and than transfected with Rep plasmids 3-5 hr post-infection when the adenovirus early gene expression has already reached the peak (Sharp P. , 1984) . The same protocol was followed during the serial passages of the HD viruses.
  • the episomal DNA extracted from cell lysate at different passages was digested with Dral and analyzed by Southern blot using a Rep DNA probe.
  • the detection of a single band of the expected size (6.3 Kb for AdHDRA1034, about 2.1 Kb for AdHDRS1032 and AdHDRS1033) demonstrated the integrity of the Rep cassette. No minor bands were detected after 48 hr exposure indicating that vector rearrangement did not occur during the serial passages .
  • Cells were harvested 36 hours post- infection and a Western blot analysis was performed on proteins extracted using a rabbit antiserum.
  • pCIII-Rep78 containing the same gene fused HCMV promoter was transfected as positive control.
  • Rep 78 expression was detected in Hep3B but not in HeLa cells infected with HDRA1034 as expected. This result confirms the tissue specificity of the Rep78 gene fused to the liver specific ⁇ lat promoter (De Simone et al . , 1987) .
  • the Rep78 protein is able to support in vi tro the replication of AAV DNA in presence of an adenovirus-infected cell extract (Ni TH, et al . 1994) .
  • An assay of Rep 78 expression based on the rescue and replication of an AAV- ITRs flanked DNA in cells infected by adenovirus was used to demonstrate the functionality of HD Rep vectors amplified by serial passages in 293cre cells.
  • 293 and 293cre monolayer were infected with about 0.5xl0 6 b.f.u. using a crude lysate obtained from 293cre cells after 8 serial passages of vectors HDRP1030, HDRS1032, HDRS1033, HDRA1034. 4 hr later the cells were infected with the first generation vector AdLBGITR-GFP/Hy carrying the AAV ITR flanked transgene.
  • AdLBGITR-GFP/Hy carrying the AAV ITR flanked transgene.
  • pCIII-Rep 78 a plasmid containing an Human Cytomegalovirus (HCMV) promoter driven Rep 78 gene expression.
  • HCMV Human Cytomegalovirus
  • HepG2 or Huh7 cells were infected first at an moi of 1 bfu/cell with HdRS1032 or HdRA1034 and 3-4 hours later with moi 1 pfu/cell of AdLB-ITRGFP/Hy .
  • Cells were harvested by scraping 48 hours after infection and total DNA extracted by standard techniques (Sambrook J. et al . T. 1989) Nested PCR was performed on 1 ⁇ g of cellular DNA in a reaction mixture containing 1.25 U of AmpliTaq Gold (Perkin Helmer) , lx ampliTaq Gold buffer, 2.5 M MgCl 2 , 200 mM of deoxynucleoside triphosphates and 50 pmol of each primer.
  • AAVSl primers were:
  • AAVSl DNA probe was labelled by random priming with 32 P (Multiprime, Amersham) .
  • AAVITR probe was obtained with two primers derived from pSUB201 (nts 1-119) labelled with 32 P by fill-in reaction in presence of 5 U of Klenow fragment of DNA polymerase and 50 ⁇ Ci of 32PdATP and 32PdCTP (NEN) .
  • HepG2 and HuH7 cells were coinfected with HdRA1034 and Ad LBITR-GFP/Hy. 48 hours post-infection cells were harvested and genomic DNA extracted. A nested PCR-based assay on genomic DNA was set up to detect junctions between AAVSl and viral ITRs. Two pairs of primers specific for AAVSl and AAVITRs were used. AAVSl primer sequences was chosen in the DNA 2 9 sequence located downstream the 100-bp AAVSl region identified as "hot spot" for site specific integration (Samulski R.J. et al . 1991) . Amplified DNA was loaded on agarose gel in duplicate and analyzed by Southern blot using either AAVSl or AAVITR specific probes.
  • Amplified DNA was eluted from the gel, cloned and sequenced.
  • the sequence data confirm that the amplified DNA contain the junction between the AAVSl genomic region and the AAV ITR.
  • HepG2 cells were prepared by standard cytogenetic techniques (Lawrence et al . , 1988) .
  • Cytogenetic preparations were pre-treated with RNase A solution (10 mg/ml in 2x SSC), with Pepsin solution (0.005 % in 10 mM HC1) and dehydrated through 70, 90 and 100% ethanol. The preparations were then denatured using a 70% deionized formamide solution and dehydrated again through cold ethanol at increasing concentration.
  • HepG2 cells were co-infected with 5 transducing unit/cell of HDRA1034 or HDRS1032, the lowest moi compatible with 80/90% cell infection, in combination with the same moi of HdFBl. 32
  • FISH analysis was performed after several passaging of cells in presence of hygromicin B.
  • AAVITR transgene was detected in 6 out of 16 different metaphase spreads on one allele of chromosome 19.
  • the present inventors have succeeded in providing an approach for generation of novel hybrid Ad/AAV vectors encoding the AAV Rep78 gene, which are able to mediate site specific integration of a ITR-flanked DNA.

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Abstract

L'invention concerne la production de particules adénovirales par des cellules d'incubation contenant un vecteur d'adénovirus auxiliaire et un vecteur adénoviral dépendant du virus auxiliaire incluant un gène rep de virus adéno-associé (AAV), tel que rep78. L'invention concerne des cellules contenant un vecteur d'adénovirus auxiliaire. Un vecteur adénoviral dépendant du virus auxiliaire qui inclut un gène rep d'AAV est introduit dans les cellules, par exemple par infection à l'aide de particules virales infectieuses. Les cellules sont incubées de manière à produire des particules adénovirales contenant un acide nucléique qui inclut le gène rep d'AAV. Il est avantageux de préincuber les cellules contenant le vecteur d'adénovirus auxiliaire pendant 0,5-12 heures, de préférence environ 4 heures, pour permettre l'expression de protéines virales nécessaires à la réplication du génome adénoviral avant d'introduire le vecteur d'adénovirus dépendant du virus auxiliaire qui inclut un gène rep d'AAV.
PCT/EP1999/002384 1998-04-08 1999-04-08 Preparation d'adenovirus recombines portant un gene rep de virus adeno-associe Ceased WO1999053084A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CA002326847A CA2326847A1 (fr) 1998-04-08 1999-04-08 Preparation d'adenovirus recombines portant un gene rep de virus adeno-associe
US09/647,795 US6521426B1 (en) 1998-04-08 1999-04-08 Preparation of recombinant adenovirus carrying a rep gene of adeno-associated virus
EP99922090A EP1068343A1 (fr) 1998-04-08 1999-04-08 Preparation d'adenovirus recombines portant un gene rep de virus adeno-associe
AU39265/99A AU752811B2 (en) 1998-04-08 1999-04-08 Preparation of recombinant adenovirus carrying a rep gene of Adeno-Associated Virus

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
ITPCT/IT98/00082 1998-04-08
PCT/IT1998/000082 WO1998045462A1 (fr) 1997-04-08 1998-04-08 Vecteurs de recombinaison derives d'un virus associe a un adenovirus adaptes a la therapie genique
GBGB9813670.8A GB9813670D0 (en) 1998-04-08 1998-06-24 Preparation of recombinant adenovirus carrying a rep gene of adeno-associated virus
GB9813670.8 1998-06-24

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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US6627617B1 (en) 1999-10-01 2003-09-30 University Of North Carolina At Chapel Hill Temperature-sensitive regulation of viral vector production

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1151123A1 (fr) * 1999-02-10 2001-11-07 MediGene Aktiengesellschaft Procede de fabrication d'un virus adeno-associe recombine, moyens adaptes a cette fabrication et utilisation dudit virus pour la fabrication d'un medicament
US6627617B1 (en) 1999-10-01 2003-09-30 University Of North Carolina At Chapel Hill Temperature-sensitive regulation of viral vector production

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CA2326847A1 (fr) 1999-10-21
GB9813670D0 (en) 1998-08-26
AU3926599A (en) 1999-11-01
AU752811B2 (en) 2002-10-03

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