EP0931158A1 - Procede inductible de production de virus adeno-associes recombines au moyen de la polymerase t7 - Google Patents

Procede inductible de production de virus adeno-associes recombines au moyen de la polymerase t7

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Publication number
EP0931158A1
EP0931158A1 EP97939829A EP97939829A EP0931158A1 EP 0931158 A1 EP0931158 A1 EP 0931158A1 EP 97939829 A EP97939829 A EP 97939829A EP 97939829 A EP97939829 A EP 97939829A EP 0931158 A1 EP0931158 A1 EP 0931158A1
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Prior art keywords
aav
host cell
vector
recombinant
rep
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EP97939829A
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German (de)
English (en)
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James M. Wilson
Nancie Chen
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University of Pennsylvania Penn
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University of Pennsylvania Penn
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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
    • 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

  • Adeno-associated virus is a replication- deficient parvovirus, the genome of which is about 4.6 kb in length, including 145 nucleotide inverted terminal repeats (ITRs) .
  • the single-stranded DNA genome of AAV contains genes responsible for replication (rep) and formation of virions (cap) .
  • rep nucleotide inverted terminal repeats
  • cap nucleotide inverted terminal repeats
  • AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells.
  • Various groups have studied the potential use of AAV in the treatment of disease states.
  • T7 RNA polymerase is the product of T7 gene 1, which can recognize its responsive promoter sequence specifically and exhibit a high transcriptase activity [M. Chamberlin et al, Nature - ££8_:227-231 (1970); J. Dunn and F. Studier, J. Mol.
  • the present invention provides an inducible method for efficient production of rAAV which makes use of T7 polymerase.
  • T7 Pol is derived from lambda phage and its promoter is not active in mammalian cells.
  • expression of rep/cap can be controlled by placing these genes under control of the T7 promoter and providing the T7 Pol in trans or under the control of an inducible promoter.
  • This method avoids the toxic effects of rep which rendered prior art methods of producing rAAV inefficient.
  • the method of the invention is particularly suitable for large scale production of rAAV, which is desired for rAAV vectors to be used in gene therapy.
  • the invention provides a method of producing rAAV which utilizes three vectors.
  • a first vector is capable of expressing T7 polymerase in the host cell following transfection or infection.
  • a second vector comprises the AAV rep and cap genes under the control of T7 promoter sequences (T7/rep/cap) .
  • the third vector comprises a cassette containing 5 • and 3 ' AAV inverted terminal repeats (ITRs) flanking a selected transgene.
  • ITRs inverted terminal repeats
  • the invention provides a method in which a host cell is stably transfected with one of the three components of the system used in the triple infection system. The remaining components are introduced into the host cell, as described above.
  • the invention provides method in which a vector containing T7 /rep /cap and a vector containing a cassette comprising a selected minigene flanked by 5 1 and 3' AAV ITRs are introduced into a host cell expressing T7 polymerase. The host cell is then cultured under conditions which permit production of rAAV.
  • this invention provides a method which utilizes a host cell stably transfected with a plasmid containing T7 /rep/cap.
  • a vector containing T7 pol and a vector containing a cassette comprising 5' AAV inverse terminal repeat (ITR), a selected minigene, and 3' AAV ITR are introduced into the host cell.
  • the host cell is cultured under conditions which permit production of rAAV.
  • the invention provides a method which utilizes a host cell stably transfected with a rescuable rAAV cassette.
  • a vector containing T7 pol and a vector containing T7 /rep/ cap are introduced into the host cell.
  • the host cell is cultured under conditions which permit production of rAAV.
  • the present invention provides a method which utilizes a host cell stably transfected with two of the three components of the system used in the triple infection system. The remaining component is then introduced into the host cell, as described above.
  • the present invention provides a method which utilizes a host cell stably transfected with the three components of the system used in the triple infection system.
  • the T7 Pol is controlled by an inducible promoter.
  • the invention provides a rAAV produced according to the method of the invention.
  • Fig. 1 provides a schematic illustration of the construction of a recombinant adenovirus containing the T7 polymerase gene.
  • Fig. 2 provides a schematic illustration of the construction of a recombinant plasmid containing the AAV rep/cap genes under control of a T7 promoter.
  • Fig. 3 provides a schematic illustration of the construction of a recombinant adenovirus containing the rep/cap genes under control of a T7 promoter.
  • Fig. 4 provides a schematic illustration of the construction of a recombinant hybrid Ad/AAV virus.
  • the invention provides an inducible method for efficient production of recombinant AAV vectors useful particularly for gene delivery and transfer. Specifically, the invention provides methods of AAV production in which expression of the toxic but necessary rep gene is controlled by the T7 promoter.
  • the method of the invention for production of rAAV involves introducing into a host cell the AAV rep and cap genes under control of a T7 promoter, and a recombinant adeno-associated virus (rAAV) cassette containing a selected minigene flanked by AAV ITRs.
  • rAAV adeno-associated virus
  • Upon introduction of a gene encoding T7 pol high level expression of rep protein from the T7/rep/cap construct is induced and cells may be grown on a large scale.
  • the cells are caused to express the T7 polymerase which acts on the T7 promoter. This facilitates the efficient replication and packaging of rAAV carrying a gene of interest.
  • a host cell may be triple transfected (or infected) with vectors containing the above elements.
  • a host cell which expresses one or more of the required elements and may be transfected/infected with the remaining elements is utilized.
  • a host cell which stably expresses all three elements of the system, and the T7 pol is placed under the control of an inducible promoter, which permits rep/cap expression to be controlled and the avoidance of toxic effects to the cell.
  • adenoviral constructs are currently preferred.
  • a different viral (adenoviral or non-adenoviral) or a plasmid vector which is capable of driving expression of the desired genes in the host cell.
  • vectors carrying the required elements of this system e.g., the T7 polymerase, may be readily constructed using retroviruses. Therefore, this invention is not limited by the virus or plasmid selected for purposes of introducing the T7 pol, T7/rep/cap, or AAV cassette into the host cell.
  • At least one of the vectors is a virus which provides the necessary helper functions to enable packaging.
  • the helper functions may be provided by a co-transfected adenovirus or herpesvirus. Suitable techniques for introducing these vectors into the host cell are discussed below and are known to those of skill in the art.
  • a "host cell” is any cell (cell line) , preferably mammalian, which permits expression of the T7 pol and T7/rep/cap and packaging of the rAAV containing the cassette, under the conditions described herein. Suitable packaging cells are known, and may be readily selected by the skilled artisan.
  • a host cell used for assembly and packaging of recombinant AAV may be transfected with plasmid vectors or infected with viral vectors containing the required components of the system.
  • a first vector contains the T7 Pol gene under the control of a suitable promoter.
  • the nuclear localized T7 Pol gene is obtained from a publicly available plasmid [M. Strauss, Nucleic Acid Res.. 12:8485-8493 (1989)]. However, the gene may alternatively be obtained from other commercial and academic sources, including the American Type Culture Collection (pTF7-3, Accession No. 484944). See, also GenBank accession number M30308. Desirably, the T7 pol gene is linked to a nuclear localization signal, such as that described in Dunn, Gene. 6JJ:259-266 (1988), using conventional techniques.
  • T7 Pol is under the control of a cytomegalovirus (CMV) immediate early promoter/enhancer [see, e.g., Boshart et al, Cell. 41:521-530 (1985)].
  • CMV cytomegalovirus
  • suitable promoters may be readily selected by one of skill in the art.
  • Useful promoters may be constitutive promoters or regulated (inducible) promoters, which will enable control of the amount of the transgene to be expressed.
  • another suitable promoter includes, without limitation, the Rous sarcoma virus LTR promoter/enhancer.
  • Still other promoter/enhancer sequences may be selected by one of skill in the art.
  • the vector also includes other conventional regulatory elements necessary to drive expression of T7 Pol in a cell transfected with the vector.
  • regulatory elements are known to those of skill in the art.
  • the second vector component of this system contains the rep and cap genes under control of a T7 promoter.
  • the rep and cap genes can be obtained from a variety of known sources. See, e.g., T. Shenk, J. Virol.. 61:3096-3101 (1987), which provides the AAV2 genome within the plasmid psub20l; E. W. Lusby et al, J. Virol. , 4_l: 518-526 (1982) and J. Smuda and B.J. Carter, Virology. 1 :310-318 (1991).
  • T7 promoter sequences can be obtained from a variety of known sources. See, e.g., T. Shenk, J. Virol.. 61:3096-3101 (1987), which provides the AAV2 genome within the plasmid psub20l; E. W. Lusby et al, J. Virol. , 4_l: 518-526 (1982) and J. Smuda and B.J. Carter,
  • the vector further contains the sequence of untranslated region (UTR) of encephalomyocarditis (EMCV) downstream of the T7 promoter.
  • UTR untranslated region
  • EMCV encephalomyocarditis
  • the vector also includes conventional regulatory elements necessary to drive expression of the rep/cap in a cell transfected with the vector.
  • regulatory elements are known to those of skill in the art.
  • the third vector component contains a rAAV cassette containing a minigene flanked by AAV ITRs.
  • a minigene contains a suitable transgene, a promoter, and other regulatory elements necessary for expression of the transgene.
  • the AAV sequences employed are preferably limited to the cis-acting 5 ' and 3 ' inverted terminal repeat (ITR) sequences [See, e.g., B. J. Carter, in "Handbook of Parvoviruses" , ed. , P. Tijsser, CRC Press, pp.155-168 (1990)]. Desirably, substantially the entire 143 bp sequences encoding the ITRs are used in the vectors.
  • AAV ITR sequences may be obtained from any known AAV, including presently identified human AAV types. Similarly, AAVs known to infect other animals may also be employed in the vector constructs of this invention. The selection of the AAV is not anticipated to limit the following invention.
  • a variety of AAV strains, types 1-4, are available from the American Type Culture Collection or available by request from a variety of commercial and institutional sources. In the following exemplary embodiment an AAV-2 is used for convenience.
  • the 5 • and 3 • AAV ITR sequences flank a minigene which is made up of a selected transgene sequence and associated regulatory elements.
  • the transgene sequence of the vector is a nucleic acid sequence heterologous to the AAV sequence, which encodes a polypeptide or protein of interest.
  • the transgene is operatively linked to regulatory components in a manner which permits transgene transcription.
  • transgene sequence will depend upon the use to which the resulting vector will be put.
  • one type of transgene sequence includes a reporter sequence, which upon expression produces a detectable signal.
  • reporter sequences include without limitation an E. coli beta- galactosidase (LacZ) cDNA, an alkaline phosphatase gene and a green fluorescent protein gene. These sequences, when associated with regulatory elements which drive their expression, provide signals detectable by conventional means, e.g., ultraviolet wavelength absorbance, visible color change, etc.
  • a more preferred transgene sequence includes a therapeutic gene which expresses a desired gene product in a host cell.
  • These therapeutic nucleic acid sequences typically encode products which may be administered to a patient in vivo or ex vivo to replace or correct an inherited or non- inherited genetic defect or treat an epigenetic disorder or disease.
  • the selection of the transgene sequence is not a limitation of this invention.
  • the minigene also includes conventional regulatory elements necessary to drive expression of the transgene in a cell transfected with the vector carrying the AAV cassette.
  • the minigene contains a selected promoter which is linked to the transgene and located within the minigene, between the AAV ITR sequences of the vector.
  • promoter which mediates expression of the transgene is a routine matter and is not a limitation of the vector.
  • Useful promoters include those which are discussed above in connection with the first vector component.
  • the minigene will also desirably contain heterologous nucleic acid sequences including sequences providing signals required for efficient polyadenylation of the transcript and introns with functional splice donor and acceptor sites.
  • a common poly-A sequence which is employed in the exemplary vectors of this invention is that derived from the papovavirus SV-40. The poly-A sequence generally is inserted following the transgene sequences and before the 3* AAV ITR sequence.
  • a common intron sequence is also derived from SV-40, and is referred to as the SV-40 T intron sequence.
  • a minigene of the present invention may also contain such an intron, desirably located between the promoter/enhancer sequence and the transgene. Selection of these and other common vector elements are conventional and many such sequences are available [see, e.g., Sambrook et al, and references cited therein].
  • the rAAV vector containing the AAV ITRs flanking the minigene may be carried on a plasmid backbone and used to transfect a selected host cell or may be flanked by viral sequences (e.g., adenoviral sequences) which permit it to infect the selected host cell.
  • viral sequences e.g., adenoviral sequences
  • Suitable Ad/AAV recombinant viruses may be produced in accordance with known techniques. See, e.g., WO 96/13598, WO 95/23867, and WO 95/06743, which are incorporated by reference herein.
  • a cell line stably transfected with T7 rep/cap may be double transfected (infected) with a vector carrying T7 pol and a vector carrying the rAAV cassette, as illustrated in the following table.
  • a cell line which contains a rescuable rAAV cassette may be double transfected (infected) with a vector containing T7 Pol and a vector containing T7/rep/cap, as illustrated in the following table.
  • a stable cell line of the invention can be produced by transfection of a desired cell, e.g., 293 cells or other packaging cell lines expressing required adenoviral genes, with a plasmid containing the desired gene, e.g., T7 Pol, using conventional techniques and selected via an accompanying resistant marker gene. Depending upon whether inducible or constitutive expression is desired, an appropriate promoter may be selected.
  • the cell may be transfected with a plasmid containing T7 Pol under control of a metallothionein promoter.
  • a host cell constitutively expressing T7 Pol it may be inserted under control of a RSV or CMV promoter. Similar techniques may be used for providing a host cell containing the T7/rep/cap and a host cell containing a rescuable rAAV. The examples below describe production of stable cell lines. However, one of skill in the art could readily produce such cell lines using other conventional techniques. See, generally, Ausubel et al, Current Protocols in Molecular Biology (Wiley Interscience 1987) .
  • a cell line which stably expresses two of the components of this system may be constructed, and then transfected (or infected) with a vector containing the remaining component of the system, as described above.
  • a cell line is utilized which is stably transfected with the T7/rep/cap and a rescuable rAAV.
  • the cell line is then transfected or infected with a vector containing the T7 pol.
  • the cell line is stably transfected with the T7 pol and a rescuable rAAV.
  • the cell line is then transfected or infected with a vector containing the T7 rep/cap.
  • a cell line which stably expresses all three of the components of this system may be constructed and utilized in the method of the invention.
  • a suitable packaging cell line is constructed which contains the rAAV, the T7/rep/cap and the T7 pol.
  • the T7 Pol is placed under the control of an inducible promoter. Suitable inducible promoters are known to those of skill in the art and are discussed herein.
  • T7 Pol may be placed under control of a metallothionein promoter. In this manner, expression of the T7 Pol, and thus the rep/cap, which are under control of the T7 promoter can be regulated and toxic effects to the cell avoided.
  • Assembly of the selected DNA sequences of the adenovirus, AAV and the reporter genes or therapeutic genes and other vector elements into the vectors described above utilize conventional techniques.
  • Such techniques include cDNA cloning such as those described in texts [Sambrook et al, cited above], use of overlapping oligonucleotide sequences of the adenovirus or AAV genome, polymerase chain reaction, and any suitable method which provides the desired nucleotide sequence.
  • HEK human embryonic kidney
  • Other conventional methods employed in this invention include homologous recombination of the viral genomes, plaquing of viruses in agar overlay, methods of measuring signal generation, and the like.
  • the host cell Following infection/transfection, the host cell is then cultured under standard conditions, to enable production of the rAAV. See, e.g., F. L. Graham and L. Prevec, Methods Mol. Biol.. 7:109-128 (1991). Desirably, once the rAAV is identified using conventional techniques, it may be isolated using standard techniques and purified.
  • Figure 1 provides a schematic of the construction of the recombinant adenovirus carrying the T7 polymerase.
  • pMTT7N The plasmid pMTT7N was obtained from Dr. Michael Strauss [A. Lieber et al, Nucl. Acids Res.. 12:8485-8493 (1989)].
  • pMTT7N contains a N-terminal nuclear location signal of SV40 large T antigen fused to the T7 Pol gene (T7N Pol) which is linked to the polyadenylation sequence of SV40. Expression is driven by the inducible mouse metallothionein promoter.
  • the pMTT7N plasmid DNA was digested with Bglll and PvuII restriction enzymes and the fragments separated on an agarose gel.
  • pAd.CMV.link.l is a plasmid containing the adenoviral sequences 0 to 16 map units deleted of Ela and Elb into which a CMV promoter-polylinker cassette was cloned. This is described in X. Ye et al, J. Biol. Chem.. 271:3639-3646 (1996).
  • the expression unit of T7 Pol is directed by the CMV promoter.
  • the promoter for the T7 Pol gene is linked to a PolyA tail as a cassette within the sequence of adenovirus 0-1 map unit (mu) and 9-16 mu.
  • the pAd.CMV.T7N is linearized by Nhe I digestion and co- transfected with Cla I linearized Addel327 backbone using Cellphate kit (Pharmacia) . Approximately 1 week post- transfection, the T7 Pol adenovirus can be isolated from the plaques for further purification.
  • a cell line stably expressing T7 Pol is established by co-transfection of plasmids pMTT7N and pMTCB6+ (which provides a selective marker) [K. H. Choo et al, SNA, 5:529-538; Eur. J. Biochem.. 124:417-424] into 293 cell at a ratio of 10:1 using calcium phosphate precipitation [F. Graham and A. van der Eb, Virol.. 5_2:456-467 (1973)].
  • Colony cloning is carried out by Geneticin selection at a concentration of 1 mg/ml. Each clone obtained is transfected with pT7 rep/cap plasmid [see. Example 3 below] and analyzed for its ability to induce the expression of Rep protein upon induction by supplementation with Zn ++ .
  • the T7N Pol obtained by Bglll/PvuII digestion of pMTT7N, as described above was subcloned downstream of RSV promoter at the cloning sites of BamHI and PvuII in the vector of pEBVhis [Invitrogen] .
  • the resulting plasmid, designated pEBVhisT7N was transfected into 293 cells and selected with Hygromycin at a concentration of 400 ⁇ g/ml. Each positive clone is analyzed for the presence of T7 Pol by its ability to produce expression of T7-LacZ or T7- rep/cap in cells transfected with these plasmids.
  • the plasmid pTMl [B. Moss et al, Nature. 348:91-92 (1990)], designed for expressing genes under control of the T7 promoter/EMCV UTR (untranslated region of encephalomyocarditis) , was used as the vector for expressing AAV rep/cap.
  • the entire coding sequence of rep/cap was separated into two portions by the unique Sad site and subcloned into the pTMl plasmid as described below.
  • N-rep the left end of the rep sequence (N-rep) was first amplified by PCR.
  • the sequence of the upper primer was SEQ ID NO: 2: TATTTAAGCCCGAGTGAGCT. (from position of 255 to 274) which introduced a nucleotide substitution A->T at position 274 (underlined) .
  • a Sad site was then generated to permit the cloning of N-rep into pTMl and in-frame expression of Rep protein from the EMCV UTR preferred initiation site (within the Ncol site) .
  • the PCR product (739 bp in length) was directly cloned into pCR2.1 vector (Invitrogen) and named pCR-N-rep.
  • the pTM-1 plasmid was digested with Sad and Stu I restriction enzymes and ligated with a 3.7 kb SacI/SnaBI fragment from psub201 [Samulski et al, J. Virol. ⁇ .51:3096-3101 (1987)] containing the right end of the AAV genome (without ITR sequence), i.e., the c- terminal portion of rep and full-length cap sequence.
  • This T7 promoter-driven rep/cap construct is named pT7-c- rep/cap.
  • the first 535 bp sequence of rep was removed from the pCR-N-Rep plasmid by Sad digestion and subcloned into pT7-C-rep/cap, which has similarly been digested with Sad and subjected to alkaline phosphatase treatment to prevent self-ligation of the vector.
  • the final construct was named pT7 rep/cap which contains the full length coding sequence of rep/cap downstream of T7 promoter/EMCV UTR, followed by the T7 terminating sequence.
  • Adenovirus pAd.link is a construct similar to pAd.CMV. link, a plasmid containing the adenoviral sequences 0 to 16 map units deleted of Ela and Elb as described in the other adenovirus vectors into which a CMV promoter-polylinker cassette was cloned and described in X. Ye et al, J. Biol. Chem.. 221:3639-3646 (1996). However, pAd.link contains no CMV promoter or polyA tail sequence.
  • the entire region including the T7 promoter, EMCV UTR, rep/cap and T7 terminating sequence was excised from pT7 rep/cap by digestion with Clal and EagI, and then subcloned into the adenoviral sequences of pAd.link, which had previously been subjected to Clal/Sail digestion, after filling in the sticky ends of EagI and Sail by Klenow polymerase.
  • the resulting plasmid is designated pAd.T7 rep/cap.
  • the pAd.T7 rep/cap is co-transfected with the Clal linearized Ad.del327 backbone DNA into 293 cell for the generation of T7 rep/cap adenovirus.
  • Example 4 - Cell Line Expressing rep/cap
  • a cell line stably transfected with pT7 rep/cap is established by transfection of pMTCB6+ into 293 cell at ratio of 10:1 and selected with Geneticin. Each clone is analyzed for the presence of rep protein by transfection with T7 Pol expressing plasmid.
  • Example 5 Production of Recombinant AAV Hybrid Vector Plasmid pAV.CMVLacZ serves as a template for rAAV to be replicated and packaged in the presence of AAV non-structural and capsid proteins.
  • Plasmid AV.CMVLacZ is a rAAV cassette in which rep and cap genes are replaced with a minigene expressing ⁇ -galactosidase from a CMV promoter.
  • the linear arrangement of AV.CMV acZ includes: (a) the 5 1 AAV ITR (bp 1-173) obtained by PCR using pAV2 [C. A. Laughlin et al, Gene. 23: 65-73 (1983)] as template [nucleotide numbers 365-538 of SEQ ID NO:l];
  • E. coli beta-galactosidase cDNA (nucleotide numbers 1356 - 4827 of SEQ ID N0:1), (e) an SV40 polyadenylation signal (a 237
  • Bglll fragment (nucleotide numbers 5053 - 5221 of SEQ ID N0:l) .
  • the LacZ gene can be replaced with a desired therapeutic or other transgene for the purpose of generating new rAAV. See, Fig. 4.
  • the sequence including CMV directed LacZ reporter cassette in between two AAV ITR sequences is excised from pAV.CMV.LacZ by PvuII digestion. This fragment is ligated with the EcoRV treated pAd.link to generate the plasmid pAd.AV.CMVLacZ.
  • This plasmid is co-transfected with Clal linearized Addel327 backbone DNA to generate an adeno-rAAV hybrid virus.
  • Example 6 Cell line containing rescuable. integrated rAAV template
  • 293 cells are transfected/infected with pAV.CMVLacZ/rAAV Ad hybrid virus to generate cell line that has incorporated rAAV, as determined by analysis of the geno ic DNA by Southern blot. The clone is examined for the rescue of rAAV template by transfection/infection with rep/cap expressing constructs.
  • CAAATCAAAG AACTGCTCCT CAGTGGATGT TGCCTTTACT TCTAGGCCTG TACGGAAGTG 1320
  • TCCAGTTCCG TTTATCCGGG CAAACCATCG AAGTGACCAG CGAATACCTG TTCCGTCATA 3420
  • AAACCTTATT TATCAGCCGG AAAACCTACC GGATTGATGG TAGTGGTCAA ATGGCGATTA 4080
  • CTTCTCGCTT CCGGCGGCAT CGGGATGCCC GCGTTGCAGG CCATGCTGTC CAGGCAGGTA 8100
  • AAACTCTCAA GGATCTTACC GCTGTTGAGA TCCAGTTCGA TGTAACCCAC TCGTGCACCC 10080
  • CAAAATGCCG CAAAAAAGGG AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC 10200
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:2: TATTTAAGCC CGAGTGAGCT 20

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Abstract

L'invention concerne des procédés de production efficace de Vaa recombinés. Dans un premier aspect de l'invention, trois vecteurs sont introduits dans une cellule hôte. Un premier vecteur dirige l'expression de la polymérase T7. Un deuxième vecteur porte des gènes rep et cap sous le contrôle du promoteur T7. Un troisième vecteur contient une cassette de Vaa recombinés qui comporte un minigène flanqué de RTi de Vaa adjacents. Dans un second aspect de l'invention, la cellule hôte est transfectée de façon stable de sorte qu'elle contient un plasmide renfermant un des constituants vectoriels appropriés et la cellule hôte est doublement transfectée/infectée.
EP97939829A 1996-09-06 1997-09-04 Procede inductible de production de virus adeno-associes recombines au moyen de la polymerase t7 Withdrawn EP0931158A1 (fr)

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US24699P 1996-09-06
PCT/US1997/015716 WO1998010088A1 (fr) 1996-09-06 1997-09-04 Procede inductible de production de virus adeno-associes recombines au moyen de la polymerase t7

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EP (1) EP0931158A1 (fr)
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AU4183397A (en) 1998-03-26
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WO1998010088A1 (fr) 1998-03-12
JP2001500015A (ja) 2001-01-09
AU722624B2 (en) 2000-08-10

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