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WO2002072814A2 - Baculovirus genetiquement modifies et utilisation - Google Patents

Baculovirus genetiquement modifies et utilisation Download PDF

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Publication number
WO2002072814A2
WO2002072814A2 PCT/GB2002/001115 GB0201115W WO02072814A2 WO 2002072814 A2 WO2002072814 A2 WO 2002072814A2 GB 0201115 W GB0201115 W GB 0201115W WO 02072814 A2 WO02072814 A2 WO 02072814A2
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Prior art keywords
baculovirus
cells
genes
vector
gene
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PCT/GB2002/001115
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WO2002072814A3 (fr
Inventor
Seppo Yla-Herttuala
Kari Juhani Airenne
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Ark Therapeutics Ltd
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Ark Therapeutics Ltd
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Priority to AU2002242820A priority Critical patent/AU2002242820A1/en
Publication of WO2002072814A2 publication Critical patent/WO2002072814A2/fr
Publication of WO2002072814A3 publication Critical patent/WO2002072814A3/fr
Priority to ZA200406921A priority patent/ZA200406921B/en
Priority to JP2003576633A priority patent/JP2005519620A/ja
Priority to CA002478692A priority patent/CA2478692A1/fr
Priority to AU2003212521A priority patent/AU2003212521B2/en
Priority to MXPA04008753A priority patent/MXPA04008753A/es
Priority to PCT/GB2003/001029 priority patent/WO2003078641A1/fr
Priority to KR10-2004-7014275A priority patent/KR20040095280A/ko
Priority to CNA038057697A priority patent/CN1643153A/zh
Priority to EP03708341A priority patent/EP1483391A1/fr
Priority to IL16380003A priority patent/IL163800A0/xx
Priority to PL03372972A priority patent/PL372972A1/xx
Priority to US10/507,268 priority patent/US20050201983A1/en
Anticipated expiration legal-status Critical
Priority to NO20044106A priority patent/NO20044106L/no
Priority to US12/327,423 priority patent/US20090176660A1/en
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • 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
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14122—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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14145—Special targeting system for 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
    • C12N2810/6009—Vectors comprising as targeting moiety peptide derived from defined protein from viruses dsDNA viruses

Definitions

  • This invention relates to engineered baculoviruses and their use, and especially to libraries and peptide display provided in baculovirus.
  • DNA microarrays allow high throughput analysis of transcriptome (the complement of mRNAs transcribed from a cell's genome at any one time), genes may be present, they may be mutated, but they are not necessarily transcribed. Some messengers are transcribed but not translated, and the number of mRNA copies does not necessarily reflect the number of functional protein molecules.
  • Proteomics (the complete set of proteins encoded by a cell at any one time) addresses problems that cannot be approached by DNA analysis, namely, relative abundance of the protein product, post-translational modification, subcellular localisation, turnover, interaction with other proteins as well as functional aspects.
  • Baculoviruses have long been used as biopesticides and as tools for efficient recombinant protein production in insect cells. They are generally regarded as safe, due to their naturally high species-specificity and because they are not known to propagate in any non-invertebrate host.
  • the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), containing an appropriate eukaryotic promoter, is able to efficiently transfer and express target genes in several mammalian cell types in vitro. Further, as reported in WO-A-01/90390, baculoviruses are able to mediate in vivo gene transfer comparable to adenoviruses (see also Airenne et al, Gene Ther. 7:1499-1504, 2000).
  • recombinant (re-)baculoviruses The ease of manipulation and rapid construction of recombinant (re-)baculoviruses, the lack of cytotoxicity in mammalian cells, even at a high multiplicity of infection, an inherent incapability to replicate in mammalian cells, and a large capacity (no known insert limit) for the insertion of foreign sequences, are features of baculovirus.
  • Vp39 is a major capsid protein of baculovirus.
  • Baculovirus enters the cells via receptor-mediated endocytosis.
  • the virus is efficiently internalised by many mammalian cell lines, but not able to enter nucleus in non-permissive cells. It has been previously suggested that the block of an efficient transduction of mammalian cells is not the lack of penetration of the baculovirus into the cells by receptor-mediated endocytosis, but the incapability of the virus to reach the nucleus (Boyce, PNAS USA 93:2348-2352, 1996; Barsoum, Hum. Gene Ther. 8:2011-2018, 1997). There is a general assumption that the block of transduction is in the virus escape from the endosomes.
  • a method for selecting a target gene comprises the steps of:
  • Baculoviral genomic or cDNA libraries offer a powerful tool for phenomics, by enabling the functional screening of the constructed libraries in eukaryotic cells both in vitro and in vivo. Addition of a bacterial promoter into a baculovirus donor vector will also allow expression screening of cDNA libraries in bacterial cells.
  • Baculovirus libraries may be constructed from suitable validated full-length clones and sequences from human and other vertebrate sources. This will allow integration of the efficient infection (insect cells) and transduction (vertebrate cells) of target cells by baculoviruses, and application to phenomics.
  • the baculovirus capsid is modified to display one or more heterologous proteins.
  • the major block in baculovirus transduction of mammalian cells is not in endosome escape, but in nuclear transport of the virus capsid.
  • baculovirus thus provides a versatile tool for real-time analysis of the transduction route of AcMNPV in mammalian cells and intact animals as well as infection mechanism in insect cells. Capsid-modified baculoviruses also hold a great promise for the nuclear and subcellular targeting of transgenes and as a new peptide display system for eukaryotic cells.
  • the capsid display system has many advantages compared to a gp64 envelope display system.
  • vp39 In vp39, no structural motifs have been recognised either for association with molecules within the stromal matter or for capsid assembly, nor is it responsible for infectivity of the virus. In addition, irnrnunoelectron microscopy shows that vp39 is randomly distributed on the surface of the capsid as opposed to gp64 on the virus envelope. Baculovirus envelope display system allows only fusions to N-terminal end of the gp64, whereas vp39 allows tagging to both terminus. Although it remains to be shown how large proteins can be, displayed on the baculovirus capsid, results suggest that at least 27 kDa protein can be efficiently expressed.
  • vp39 is also compatible with larger proteins, e.g. up to 100 kDa or higher. Random display of peptides or proteins on the capsid may allow the discovery of moieties capable of transporting the capsid into the nucleus or other intracellular organels.
  • modified baculovirus includes any form of "capsid therapy".
  • proteins can be used as a system for the transport of peptides or proteins directly into the nucleus. Description of the Invention
  • an expression cassette may be constructed, based on a hybrid or other suitable promoter which allows high level expression of target genes both in prokaryotic and eukaryotic cells.
  • Target site for cre-recombinase may be included into the expression cassette, to allow easy construction of baculovirus libraries using site-specific recombination in vitro (Sauer, Methods 14:381-392, 1998).
  • AttR and ccdB sites can be included into expression cassette.
  • This enables facile conversion of Life Technologies Gibco BRL ® GatewayTM Cloning Technology (Life Technologies) compatible libraries to the novel baculovirus library.
  • the expression cassette can allow traditional library construction by several unique restriction enzymes available in vector MCS after modifications such as those described above.
  • the constructed expression cassette may be cloned into any suitable baculovirus plasmid or baculovirus system which can act as a donor vector.
  • pFastBac-1 is a preferred backbone plasmid since it is compatible with Bac-To- BacTM baculovirus expression system (Gibco BRL) which allows rapid and easy preparation of re-baculoviruses by site-specific transposition in Escherichia coli.
  • the cassette can also be integrated to any desired plasmid/expression system, e.g. into a version of Bac-TO-BacTM baculovirus expression system that permits more efficient and direct construction of baculoviruses (Leusch et al, Gene 160:191-194, 1995).
  • the expression cassette can also be cloned as part of the baculovirus genome and library construction then performed directly to it by cre/lox, Gateway or direct cloning methods.
  • baculovirus libraries will be screened for expression/phenotype effect(s) in suitable E. coli strain(s) (library in donor plasmid format), insect cells and vertebrate cells. Selected viruses or whole libraries can also be used directly for in vivo studies. This alleviates the great and unique potential of the new baculovirus libraries; the same library can be used for prokaryotic and eukaryotic cells and in cell (in vitro) and animal (in vivo) studies.
  • baculovirus capsid display system By way of example, and in order to allow intracellular targeting of AcMNPV, a baculovirus capsid display system has been developed. The system is based on a versatile donor vector which allows efficient production of desired proteins as N- or C-terminal fusion to the baculovirus major capsid protein, vp39 (Thiem & Miller, J. Virol. 63:2008-2018, 1989). Alternative baculovirus capsid proteins which are potential targets for peptides or proteins include p24 and p80.
  • a construct of high titre re-AcMNPV can display a high concentration of a foreign protein in its capsid.
  • the tagged virus is a facile tool to study the route of baculovirus transduction in mammalian cells from the cell surface into the nucleus and transfection capacity of baculovirus in vivo.
  • the system provides at the same time a powerful tool to study the bottlenecks of AcMNPV transduction of non-permissible cell lines and a possibility to improve nuclear or subcellular targeting by incorporation of specific sequences in vp39 protein.
  • AcMNPV may also allow double-targeting at the cell surface level by insertion of specific ligands or antibodies to the envelope, followed by intracellular targeting by vp39 modification.
  • a transfer plasmid was constructed which enables fusion of desired entities either into N- or C-terminus of the vp39 (Fig. 1 ). Fusion protein production is driven by a strong polyhedrin promoter, e.g. as disclosed by O'Reilly et al, supra. Since computer prediction showed that vp39 had low complexity at C-terminus but was constrained at N-terminus, a linker sequence (e.g. GGGGS) may be added to the N-terminus, to give distance and flexibility for N-terminal fusion proteins to fold correctly. An option to tag the vp39 fusion proteins with a His-tag may also be preferred.
  • GGGGS linker sequence
  • the pBACcap-1 plasmid produces vp39 with His-tag at the N-terminus.
  • the same transfer plasmid can be used for N- or C-terminal fusions with or without His-tag.
  • the system is compatible with transporon-mediated virus preparation.
  • the expression cassette in the pBACcap-1 can be easily moved to any desired baculovirus vector.
  • the present invention includes the possibility of double-targeting, as an extension of the conventional targeting working primarily at tissue or cell surface level.
  • tissue targeting is to add a specific ligand on the surface of the gene transfer vector to achieve specific binding to desired cells or tissues. It is well known that a specific ligand-receptor interaction does not guarantee efficient transduction of the target cell. Internalisation, escape from endosomes and transport of the genetic material into nucleus are also required. Although the transduction can be improved by selection of cell membrane targeting moieties, the route from cytosol to nucleus remains difficult to achieve. Enveloped viruses hold a promise for an efficient double-targeting at the tissue and intracellular levels.
  • nt nucleotides 469-1506 of vp 39
  • PCR polymerase chain reaction
  • the forward primer was 5' - TT G AA AGA TCT GAA TTC A TG CAC CAC CATCAC CAT CAC GGATCC GGC GGC GGC GGC TCG GCG GOT AGT GCC CGT GGG T - 3' (specific sequence for nt 469-486 of vp39 gene in bold; Bgfll, EcoRI, BamHL, sites underlined; 6 X Histidine tag with start codon in italics); the reverse primer was 5' -TT CTG GGTACC GCt tta A TG GTG ATGATG GTG GTG TCT AGA GCt ta ACT AGT GAC GGC TAT TCC TCC ACC - 3' (specific sequence for nt 1489- 1506 of vp39 gene in bold; Kpril, Xbal and Spel sites underlined; 6 X Histidine tag in italics; stop codon in small caps).
  • the PCR was performed essentially as described by Airenne et al, Gene 144:75-80, 1994, except annealing was set to 58° C.
  • Amplified fragment was digested with Sg I and Kpnl enzymes and purified as described in Airenne et al, supra.
  • the purified PCR product was cloned into BamHI+Kpnl -digested pFastBACI vector (Invitrogen, Carlsbad, USA).
  • the resulted plasmid was named as pBACcap-1.
  • the nucleotide sequence was confirmed by sequencing (ALF; Amersham Pharmacia Biotech, Uppsala, Sweden).
  • EGFP Displaying Viruses cDNA encoding EGFP (enhanced green fluorescent protein) was amplified from the pEGFP-N1 plasmid (Genbank:U55762, Clontech, Palo Alto, USA) by PCR and cloned into the pBACcap-1. Two sets of primers were used to enable EGFP fusion both to N- and C-terminal ends of the vp39.
  • the forward primer was 5' - CGG GAT GAA TTC GTC GCC ACC ATG GTG AGC AAG GGC GAG GAG - 3' (specific sequence for nt 670- 699 of pEGFP-N1 in bold; EcoRI site in italics), and the reverse primer 5' - GCG GCC GGA TCC CTT GTA CAG CTC GTC CAT GCC - 3' (specific sequence for nt 1375-1395 of pEGFP-N1 in bold; BamHI site in italics).
  • the amplified fragment which corresponded to nt 670-1395 of pEGFP-N1 was cloned into EcoR-t/BamHI site ofthe Spel Xi al deleted pBACcap-1.
  • the resulting plasmid was named EGFPvp39.
  • the forward primer was 5' - GTC GCC ACT
  • Viruses were generated using Bac-To-Bac systemTM according to manufacturer's instructions (Invitrogen). Viruses were concentrated and gradient-purified, as described by Airenne et al, Gene Ther. 7:1499-1504, 2000. Virus titre was determined by end-point dilution assay on Sf9 cells. Sterility test was performed for virus preparations and they were analysed to be free of lipopolysaccha de and mycoplasma contamination. Immunoblotting
  • vp39EGFP baculovirus particles were bound to formwar-coated metal grids treated with 5% foetal calf serum in PBS. Grids were then treated with protein A gold for 25 min (5 nm in diameter, G. Posthuma and J. Slot, Utrecht, The Netherlands) and washed with PBS for 25 min. The grid was fixed with 2.5% glutaraldehyde and contrasted and embedded using 0.3% uranyl acetate in 1.5% methyl cellulose. The HepG2 and EAHY cells infected with the virus were fixed with 2.5% glutaraldehyde for 1 h at room temperature and then with 1% osmium tetroxide for 1 h at +4°C.
  • Subconfluent human hepatoma cell line HepG2 and human endothelial aortic hyb doma (EaHy926, Dr. Edgell, University of North Carolina, USA) cultures were infected by vp39EGFP baculovirus as follows: cells were first washed with PBS on ice, the virus was added on the cells in DMEM containing 1 % foetal calf serum using multiplicity of transduction of 80-100 and incubated for 1 h on ice. Cells were washed with PBS containing 0.5% BSA.
  • Live confocal microscopy on HepG2 and EAHY cells was performed as follows: cells were plated on chambered coverglasses (Nalge NUNC, Naperville, Illinois). After virus binding on ice, cells were transferred to confocal microscope with a heated working stage and objective controlled by Tempcontrol 37-2 (Carl Zeiss, Jena, Germany). Cells that were positive for EGFP were scanned with various time intervals using the programme in LSM 510 software (version 2.3; Carl Zeiss, Jena, Germany). In vivo Injection into Rat Brain
  • mice Male Wistar rats (320-350 g) were anaesthetised intraperitoneally with a solution (0.150 ml/100 g) containing fentanyl-fluanisone (Janssen-Cilag, Hypnorm®, Buckinghamshire, UK) and midazolame (Roche, Dormicum®, Espoo, Finland) and placed into stereotaxic apparatus (Kopf Instruments). A burr hole was done into the following stereotaxic coordinates: 1 mm to the midline and +1 mm to bregma.
  • Sf9 cells infected with EGFPvp39 or vp39EGFP encoding viruses produced expected 67 kDa bands in immunoblots.
  • the same results were obtained from purified virus preparations. The results suggested that both vp39 variants were efficiently produced in insect cells and incorporated into virus particles.
  • vp39EGFP virus was incubated with anti-EGFP followed by immunogold labelling and examined by immunoelectron microscopy. Viruses showed a typical rod-shaped morphology surrounded by a characteristically loose-fitting envelope and the surfaces of the unenveloped capsids were heavily gold- labelled.
  • the high incorporation rate was also supported by Coomassie-stained SDS-PAGE, according to which a high proportion of the capsid was made of the vp39EGFP and by easy visualisation of the virus preparations by confocal microscopy. Assembly of the viruses was not affected by the fusion protein, since the titres of the EGFPvp39 and vp39EGFP viruses were 9.5 x 10 9 and 8.8 x 10 9 pfu/ml, respectively.
  • vp39EGFP virus The intracellular route of vp39EGFP virus was followed by monitoring EGFP-tagged capsids and fluorescently labelled cellular compartments by confocal microscopy. Both EAHY and HepG2 cells were transduced for various time periods and the co-localisation of the virus with an early endosome antigen 1 (EEA1 ) was studied.
  • EA1 early endosome antigen 1
  • the virus particles were present in nuclei 4 h p.t, shpowing that intact capsids were translocated to the nucleus after the release from the early endosomes. Electron microscopy of EAHY cells confirmed that no virus particles were present in the nuclei 4 h p.t. Visualisation in Rat Brain
  • vp39EGFP baculovirus can be used for immediate analysis of virus localisation in vivo.

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Abstract

L'invention concerne une méthode de sélection d'un gène cible, consistant (i) à générer une bibliothèque de gènes ou de fragments génomiques clonés dans un baculovirus utilisé en tant que vecteur; (ii) à transformer une cellule hôte à l'aide du vecteur; et (iii) à détecter l'expression génique dans des conditions prédéterminées. L'invention concerne également des baculovirus dont la capside a été modifiée pour afficher un ou plusieurs peptides ou protéines hétérologues.
PCT/GB2002/001115 2001-03-12 2002-03-12 Baculovirus genetiquement modifies et utilisation Ceased WO2002072814A2 (fr)

Priority Applications (15)

Application Number Priority Date Filing Date Title
AU2002242820A AU2002242820A1 (en) 2001-03-12 2002-03-12 Engineered baculoviruses and their use
PL03372972A PL372972A1 (en) 2002-03-12 2003-03-12 Engineered baculoviruses and their use
US10/507,268 US20050201983A1 (en) 2002-03-12 2003-03-12 Engineered baculoviruses and their use
KR10-2004-7014275A KR20040095280A (ko) 2002-03-12 2003-03-12 조작된 바쿨로바이러스 및 그것의 용도
CNA038057697A CN1643153A (zh) 2002-03-12 2003-03-12 工程杆状病毒及其用途
CA002478692A CA2478692A1 (fr) 2002-03-12 2003-03-12 Baculovirus mis au point et utilisation associee
AU2003212521A AU2003212521B2 (en) 2002-03-12 2003-03-12 Engineered baculoviruses and their use
MXPA04008753A MXPA04008753A (es) 2002-03-12 2003-03-12 Baculovirus disenados y su uso.
PCT/GB2003/001029 WO2003078641A1 (fr) 2002-03-12 2003-03-12 Baculovirus mis au point et utilisation associee
ZA200406921A ZA200406921B (en) 2002-03-12 2003-03-12 Engineered baculoviruses and their use
JP2003576633A JP2005519620A (ja) 2002-03-12 2003-03-12 遺伝子操作したバキュロウィルスおよびその使用
EP03708341A EP1483391A1 (fr) 2002-03-12 2003-03-12 Baculovirus mis au point et utilisation associee
IL16380003A IL163800A0 (en) 2002-03-12 2003-03-12 Engineered baculoviruses and their use
NO20044106A NO20044106L (no) 2002-03-12 2004-09-27 Omkonstruerte baculoviruser og anvendelse derav
US12/327,423 US20090176660A1 (en) 2002-03-12 2008-12-03 Engineered Baculoviruses and Their Use

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GB0106063.1 2001-03-12
GB0106063A GB0106063D0 (en) 2001-03-12 2001-03-12 Libraries and their use

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