WO1993001297A1 - Adenovirus recombinants servant a la detection d'une fonction genetique specifique de transactivation - Google Patents

Adenovirus recombinants servant a la detection d'une fonction genetique specifique de transactivation Download PDF

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Publication number
WO1993001297A1
WO1993001297A1 PCT/GB1992/001195 GB9201195W WO9301297A1 WO 1993001297 A1 WO1993001297 A1 WO 1993001297A1 GB 9201195 W GB9201195 W GB 9201195W WO 9301297 A1 WO9301297 A1 WO 9301297A1
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gene
recombinant adenovirus
trans
hiv
acting
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PCT/GB1992/001195
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English (en)
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Alan Akrigg
Gavin William Graham Wilkinson
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Public Health Laboratory Service Board
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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
    • C12N2710/10343—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
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16122—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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/38—Vector systems having a special element relevant for transcription being a stuffer

Definitions

  • This invention relates to recombinant adenoviruses and to the use thereof for detecting the presence in eukaryotic cells of specific gene expression products. Particularly, but not exclusively the invention relates to the use of recombinant adenoviruses for detecting metabolic states associated with infection of eukaryotic cells with viruses such as the HIV virus.
  • HIV infection generally involves assaying tissues, particularly body fluids such as blood, for antibodies to HIV proteins or for HIV nucleic acids using the polymerase chain reaction.
  • the present invention describes a novel and improved method for detecting viable HIV particles and actively infected cells.
  • the HIV tat gene product trans-activates the 5' long terminal repeat (LTR) promoter to stimulate expression of the viral gene products under its control.
  • the viral structural genes downstream of the LTR can be replaced by a reporter gene (e.g. the LacZ gene which encodes ⁇ -galactosidase) using recombinant DNA technology.
  • a reporter gene e.g. the LacZ gene which encodes ⁇ -galactosidase
  • infection with HIV induces expression from the LTR promoter driving the reporter gene.
  • the activated expression from the reporter gene can be used to monitor HIV infection.
  • Indicator cell lines have been constructed based on CD4+ Hela cell lines which carry copies of the LacZ gene under the control of the HIV 5' LTR promoter either on stably maintained episomal vectors or integrated into chromosomal DNA (Akrigg et al , 1990; Rocancourt et al , 1990; Akrigg et al , 1991).
  • Such HIV-infected indicator cells can be stained dark blue by the addition of a chromogenic substrate (e.g. X-gal) for ⁇ -galactosidase or a quantitative enzyme assay can be performed on extracts from the infected culture.
  • a chromogenic substrate e.g. X-gal
  • a quantitative enzyme assay can be performed on extracts from the infected culture.
  • a procedure is described for detecting infectious HIV particles which utilizes an indicator cell line based on CD4+ HeLa cells.
  • This indicator cell line is stably transfected with a recombinant plasmid containing the E. coli ⁇ -galactosidase gene ⁇ Lac-Z) under the control of the HIV-1 promoter from the long terminal repeat (LTR).
  • LTR long terminal repeat
  • indicator cell lines have serious practical limitations in that they are restricted to specific immortalised cell types which have to be specially constructed, selected and tested for each cell type. Additionally, indicator cell lines can only measure HIV infection in peripheral blood lymphocytes indirectly by co-cultivatio .
  • the present invention overcomes these disadvantages by providing a recombinant adenovirus useful for detecting the presence in eukaryotic cells of the expression products of specific gene products, particularly such products associated with HIV infection.
  • the adenovirus-based detection systems of the invention are immediately adaptable to a wide variety of cell types and have the potential to be used directly to detect HIV-infection in peripheral blood cells.
  • a recombinant adenovirus for use in the detection of a trans-acting gene function in a target eukaryotic cell
  • said adenovirus genome comprises nucleic acid capable of entering the target eukaryotic cell, said nucleic acid including a marker gene capable of being activated, directly or indirectly, in the presence of said trans-acting gene function, the activated marker gene mediating the formation of a detectable product whereby production of the detectable product is indicative of the presence of said trans-acting gene function in the target eukaryotic cell.
  • the present invention is applicable to the detection of a broad range of trans-acting gene functions.
  • trans-acting gene functions associated with virus infection of target eukaryotic cells include both trans-acting gene functions associated with virus infection of target eukaryotic cells and trans-acting gene functions occurring in non-infected cells.
  • the recombinant adenoviruses of the invention may thus be used to detect trans-acting gene functions associated with specific normal and abnormal metabolic states, including malignant and pre-malignant conditions as well as metabolic states associated with infection.
  • the invention is however particularly applicable to the detection of trans-acting gene functions which are associated with infection of the target eukaryotic cell by a virus, for example the HIV virus.
  • trans-acting gene function may thus refer to the formation of an expression product specifically associated with virus infection, for example, the formation of virus-associated nucleic acid or virus-associated protein.
  • the virus protein may comprise a structural protein of the virus or a protein associated with expression (including transcription and/or translation) of virus genes.
  • An example of such a protein is an activator protein which is capable of interacting with a promoter in order to effect expression of a gene product associated with a promoter.
  • an activator protein which is capable of interacting with a promoter in order to effect expression of a gene product associated with a promoter.
  • HIV tat gene product is the HIV tat gene product. During HIV replication in infected cells, the HIV tat gene product transactivates the 5'-long terminal repeat- (LTR) promoter to stimulate expression of the viral gene products under its control.
  • LTR 5'-long terminal repeat-
  • trans-acting gene functions envisaged herein include trans-acting gene functions which are non-chromosomal with respect to the genome of the target eukaryotic cell, e.g. trans-acting gene functions of genetic material introduced by tranfection or infection.
  • Another category includes trans-acting gene functions associated with chromosomal genes, particularly activated chromosomal genes.
  • the HIV genome may be integrated into the chromosome of the host cell after infection and the recombinant adenoviruses of the invention may be designed to detect gene products associated with this integrated genetic material.
  • Cellular genes encoding proteins involved in regulating transcription are further examples of transacting functions which could be detected using this invention.
  • Such cellular transacting functions include oncogenes and tumour suppressor genes specifically the retinoblastoma and p53 tumour suppressor genes.
  • the recombinant adenoviruses of the invention are particularly useful in detecting disease states including, but not limited to, virus infection and other abnormal cellular states including malignant and pre-malignant conditions in which, for example oncogenes and tumour suppressor genes exist in an abnormal, for example mutated states.
  • transactivation involves the formation of a so-called "transactivator".
  • a transactivator may be defined as a gene product which stimulates a second non-covalently associated molecule to perform a function.
  • Such a second non-covalently associated molecule may, for example, contain a promoter linked to a reporter gene, in which case the aforementioned function would comprise a factor that stimulates expression from the reporter gene by activating its associated promoter.
  • the marker gene component of the recombinant adenovirus according to the invention preferably comprises a gene which codes for an enzyme which catalyzes the conversion of a substrate to the aforementioned detectable product.
  • the marker gene may comprise the Lac-Z gene of E. coli ⁇ -galactosidase, in which case the detectable product may comprise a coloured product formed from a chromogenic substrate for ⁇ -galactosidase.
  • the recombinant adenovirus genome comprises a recombinant nucleic acid capable of entering the target eukaryotic cell, said nucleic acid including a marker gene capable of being transactivated by the product of the HIV tat gene, the activated marker gene mediating the formation of a detectable product whereby production of the detectable product is indicative of the presence of HIV infection.
  • the marker gene is associated with a promoter which is activated by the HIV tat gene product.
  • a promoter thus, for example, can comprise the HIV 5'-long terminal repeat (LTR) promoter.
  • the marker gene comprises the E. coli Lac-Z gene located downstream of the HIV long terminal repeat (LTR) promoter.
  • a further example of recombinant adenovirus according to the invention is adapted for detection of cytomegalovirus (CMV) infection.
  • CMV cytomegalovirus
  • Such adenoviruses may include a marker gene capable of being transactivated by a product of CMV infection.
  • the recombinant adenoviruses according to the invention are preferably defective in that they are incapable of replicating other than under specifically selected conditions.
  • the adenoviruses may be defective in the sense that they are incapable of replicating under conditions allowing repliction of non-defective (i.e. wild type) virus, but are capable of replicating under specifically selected conditions.
  • the defective virus may be derived from a wild type virus by mutation of one or more genes which are normally essential for virus replication.
  • the thus formed defective virus may then be capable of reproduction in specifically selected helper cells in which the effect of the aforementioned mutation or mutations is reversed.
  • Recombinant adenoviruses according to the invention may advantageously be used to detect HIV infection in a wide variety of different cell types, including primary lymphocytic cultures and non-immortalised cells.
  • Recombinant adenoviruses according to the invention may advantageously be used to detect HIV infection in a wide variety of different cell types, including primary lymphocytic cultures and non-immortalised cells.
  • the invention further provides a method for detecting a trans-acting gene function in a target eukaryotic cell, which comprises contacting the cell with a recombinant adenovirus as defined above and detecting the presence of said detectable substance. More specifically according to this aspect of the invention there is provided a method for detecting HIV infection in target eukaryotic cells, which comprises contacting the cell with a recombinant adenovirus, the genome of which comprises a recombinant nucleic acid capable of entering the target eukaryotic cell, said nucleic acid including a marker gene capable of being transactivated by the product of the HIV tat gene, the activated marker gene mediating the formation of a detectable product whereby production of the detectable product is indicative of the presence of HIV infection.
  • Figure la illustrates the production of a recombinant plasmid MV34
  • Figure lb illustrates the production of a recombinant adenovirus
  • Figures 2a and 2b illustrate the use of the recombinant adenovirus in the detection of HIV infection in lymphocytic cell culture.
  • Figure 3 illustrates the use of a further recombinant adenovirus in the detection of CMV infection
  • the HIV ' LTR promoter linked to the lacZ reporter gene was inserted into the genome of an adenovirus type 5 vector which carried deletions in the Ela and E3 gene so as to generate the recombinant virus RAd34 (Fig 1).
  • a short linker carrying a multiple cloning site was inserted into the unique Xbal site in the plasmid shuttle vector pXCX2 (McGrory et al, 1988) .
  • Numbers marked on the plasmid maps of pXCX2 and MV34 refer to adenovirus DNA sequences with respect to the complete adenovirus genomic sequence.
  • the HIV LTR promoter driving ⁇ -galactosidase expression was excised from the plasmid MTL23LTR lacterm (Akrigg et al, 1991) on a Xhol/Sall fragment and inserted into pXCX2 between the Xhol sites on the inserted linker, in the orientation shown, to produce the plasmid pMV34.
  • a polyadenylation signal (term) derived from the cytomegalovirus major immediate early gene.
  • Plasmid pMV34 was recombined with plasmid pJM17 «
  • the plasmid pJM17 (McGrory et al, 1988) contains a complete copy of the adenovirus type 5 Ela , E3 genome.
  • pJM17 When transfected into permissive 293 cells pJM17 seldom produces adenovirus plaques because a large 'stuffer' fragment of DNA has been inserted in place of the Ela gene which makes pJM17 too big to package into adenovirus nucleocapsids.
  • the adenovirus vector is defective so that it will not replicate when infected into target cells.
  • Replication of adenovirus Ela mutants require the provision of the Ela function in trans , this can be provided by a special helper cell line which contains an endogenous Ela gene (e.g. 293 cells). While adenovirus Ela mutants replicate in 293 cells, in cells lacking the Ela function early phase gene expression is not activated so there is no detectable adenovirus gene expression.
  • RAd34 can readily be grown on the helper line to an
  • Ad type 5 can infect an exceptionally wide range of human and animal cells. It is consequently relatively straightforward to efficiently infect a wide variety of cell types with RAd34 but because the virus is defective it will not replicate. Co-infection of the cell with HIV followed by tat expression from the integrated provirus will activate expression from the reporter gene in RAd34.
  • Figure 2 shows a CD4+ Hela cell onolayer infected with RAd34 (moi of approximately 10) and superinfected with HIV.
  • lacZ gene as a reporter gene has provided greater sensitivity in accordance with the invention compared to known immunological detection systems, for example those using immunofluorescence. Further, it is not necessary to fix cells before assaying for fS-galactosidase expression, it is possible to identify and select infected cells without killing the cell or destroying the virus. Thus using a defective adenovirus according to the invention it is possible to apply the detection system to a wide variety of cell types without the need to select any particular indicator cell lines.
  • a defective adenovirus is used in accordance with the invention, it can easily be propagated to high titres and has an extremely wide host range ensuring efficient infection of target cell populations. Further, because the adenovirus vector is defective it is not able to replicate in target cell populations.
  • the specificity of the detection system for HIV can be further enhanced according to the invention by incorporating a second transactivatable function.
  • the functional rev-responsive element RRE
  • the functional rev-responsive element can be incorporated into the construct and can be used as the target signal for a second HIV transactivator, rev.
  • Example 2 Production of an Indicator system for Cytomegalovirus - Construction of Recombinant Adenovirus RAd 8
  • Cytomegalovirus operates a cascade system of gene regulation conventionally divided into three phases: immediate early (IE), early and late.
  • the IE gene products are responsible for activating early and late phase gene expression.
  • CMV encodes a single abundant early RNA of approximately 2.7kb whose transcription is driven by an exceptionally strong early promoter. The DNA sequence of the promoter and its associated gene has been determined (Greenaway and Wilkinson, 1987) . Expression from the strong early promoter from CMV strain ADI69, and the analogous promoter from CMV strain Towne, can be stimulated by CMV infection but not by herpes simplex type 1 infection (Spaete and Mocarski, 198 ; Wilkinson, 1987). This CMV early promoter is therefore trans-activated specifically by CMV infection.
  • An adenovirus recombinant was constructed containing the CMV early promoter driving LacZ expression for use in anassay to detect CMV infection as follows. 2.1. Construction
  • the CMV early promoter was sub cloned upstream of the Esherichia colt LacZ gene and the polyadenylation signal from the CMV major IE promoter.
  • the CMV early promoter/LacZ expression cassette was inserted into the adenovirus shuttle vector pXCX2 (Spessot et al, 1989) to generate the plasmid pMV48.
  • pMV48 was co-transfeeted with pJM17 and following homologous recombination between the two plasmids the recombinant virus RAd48 was produced.
  • RAd48 is essentially identical to RAd34 except that the HIV LTR promoter is replaced by the CMV promoter for gene encoding the abundant 2.7kb early RNA. 2.2. CMV Detection
  • MRC5 cells Human fibroblasts (MRC5 cells) were co-infected with RAd48 (10 plaque forming units per cell) and ten-fold serial dilutions of a titrated CMV stock. 16 hours after infection cells were stained with a chromogenic substrate for ⁇ -galactosidase (X-Gal) . Cells infected with CMV stained dark blue. The number of blue cells was proportional to the size of the CMV inoculum. By counting the number of blue cells, it was possible to titrate the CMV stock.
  • Fig 3 shows a photograph of MRC5 cells co-infected with RAd48 and CMV for 16 hours and then stained with X-gal. CMV infected cells can be clearly be seen to be stained (originally a dark blue colour) , no such staining could be detected in cells infected by RAd48 alone. Deposits
  • Recombinant adenoviruses RAd48 and RAd34 have been deposited at the European Collection of Animal Cell Cultures under the following Accession Nos:
  • the invention has been specifically described with reference to the detection of HIV and CMV infections, it may also be used to detect other virus which respond to trans-acting signals, e.g. herpes simplex virus as well as other abnormal states including those associated with malignant and pre-malignant conditions.

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Abstract

L'invention décrit des adénovirus recombinants s'utilisant dans la détection d'une fonction génétique de transactivation dans une cellule eucaryote cible. Le génome des adénovirus recombinants comprend un acide nucléique capable de pénétrer dans la cellule eucaryote cible, ledit acide nucléique comportant un gène marqueur pouvant être activé, directement ou indirectement, en présence de ladite fonction génétique de transactivation, le gène marqueur activé provoquant la formation d'un produit détectable. De ce fait la production dudit produit détectable indique la présence de ladite fonction génétique de transactivation dans la cellule eucaryote cible. On peut utiliser les adénovirus dans la détection de différents états normaux et anormaux dans des cellules eucaryotes, y compris des infections par virus.
PCT/GB1992/001195 1991-07-03 1992-07-02 Adenovirus recombinants servant a la detection d'une fonction genetique specifique de transactivation WO1993001297A1 (fr)

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GB919114437A GB9114437D0 (en) 1991-07-03 1991-07-03 Recombinant adenoviruses and the use thereof for detecting the presence in eukaryotic cells of the expression products of specific genes
GB9114437.8 1991-07-03

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0586076A2 (fr) * 1992-08-07 1994-03-09 American Home Products Corporation Vaccins adénoviraux recombinant
FR2708283A1 (fr) * 1994-01-14 1995-02-03 Rhone Poulenc Rorer Sa Compositions pharmaceutiques et leur utilisation, notamment pour le traitement des maladies neurodégénératives.
WO1997038139A1 (fr) * 1996-04-10 1997-10-16 Signal Pharmaceuticals, Inc. Systeme de lignee cellulaire de marquage permettant de detecter la presence du cytomegalovirus et d'identifier des modulateurs de l'expression de genes viraux
WO1997045556A1 (fr) * 1996-05-31 1997-12-04 Onyx Pharmaceuticals, Inc. Procedes et compositions pour determiner l'etat des suppresseurs de tumeurs de cellules
EP0919626A2 (fr) * 1993-05-28 1999-06-02 Transgene S.A. Adénovirus défectifs et lignées de complémentation correpondantes
EP1454984A1 (fr) * 1993-06-30 2004-09-08 Aventis Pharma S.A. Compositions pharmaceutiques et leur utilisation, notamment pour le traitement des maladies neurodégénératives
KR100454265B1 (ko) * 2001-11-19 2004-10-26 엘지전선 주식회사 고내구성 고무조성물

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WO1991002797A1 (fr) * 1989-08-17 1991-03-07 Institut Pasteur Lignees de cellules infectables par un retrovirus et contenant un marqueur activable a l'occasion de l'infection
GB2244712A (en) * 1990-05-02 1991-12-11 Medical Res Council Plasmid for HIV indicator cell lines

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WO1989003878A1 (fr) * 1987-10-19 1989-05-05 Institut Pasteur Gene recombinant defectif et activable par un transactivateur
WO1991002797A1 (fr) * 1989-08-17 1991-03-07 Institut Pasteur Lignees de cellules infectables par un retrovirus et contenant un marqueur activable a l'occasion de l'infection
GB2244712A (en) * 1990-05-02 1991-12-11 Medical Res Council Plasmid for HIV indicator cell lines

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NUCLEIC ACIDS RESEARCH. vol. 20, no. 9, 11 May 1992, ARLINGTON, VIRGINIA US pages 2233 - 2239 WILKINSON, G.W.G. & AKRIGG, A. 'Constitutive and enhanced expression from the CMV major IE promoter in a defective adenovirus vector' *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA. vol. 87, no. 22, November 1990, WASHINGTON US pages 8746 - 8750 VENKATESH, L.K. ET AL. 'Selective induction of toxicity to human cells expressing human immunodeficiency virus type 1 tat by a conditionally cytotoxic adenovirus vector' *
SCIENCE vol. 239, 8 January 1988, LANCASTER, PA US pages 184 - 187 FELBER, B.K. & PAVLAKIS, G.N. 'A quantitative bioassay for HIV-1 based on trans-activation' *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0586076A2 (fr) * 1992-08-07 1994-03-09 American Home Products Corporation Vaccins adénoviraux recombinant
EP0586076A3 (fr) * 1992-08-07 1994-04-20 American Home Products Corporation Vaccins adénoviraux recombinant
EP0919626A3 (fr) * 1993-05-28 1999-08-18 Transgene S.A. Adénovirus défectifs et lignées de complémentation correpondantes
EP0919626A2 (fr) * 1993-05-28 1999-06-02 Transgene S.A. Adénovirus défectifs et lignées de complémentation correpondantes
EP0919625A2 (fr) * 1993-05-28 1999-06-02 Transgene S.A. Adénovirus défectifs et lignées de complémentation correspondantes
EP0919625A3 (fr) * 1993-05-28 1999-08-18 Transgene S.A. Adénovirus défectifs et lignées de complémentation correspondantes
EP1149916A3 (fr) * 1993-05-28 2007-08-29 Transgene S.A. Adénovirus défectifs et lignées de complémentation correspondantes
EP1454984A1 (fr) * 1993-06-30 2004-09-08 Aventis Pharma S.A. Compositions pharmaceutiques et leur utilisation, notamment pour le traitement des maladies neurodégénératives
FR2708283A1 (fr) * 1994-01-14 1995-02-03 Rhone Poulenc Rorer Sa Compositions pharmaceutiques et leur utilisation, notamment pour le traitement des maladies neurodégénératives.
WO1997038139A1 (fr) * 1996-04-10 1997-10-16 Signal Pharmaceuticals, Inc. Systeme de lignee cellulaire de marquage permettant de detecter la presence du cytomegalovirus et d'identifier des modulateurs de l'expression de genes viraux
US5945276A (en) * 1996-04-10 1999-08-31 Signal Pharmaceuticals, Inc. Reporter cell line system for detecting cytomegalovirus and identifying modulators of viral gene expression
WO1997045556A1 (fr) * 1996-05-31 1997-12-04 Onyx Pharmaceuticals, Inc. Procedes et compositions pour determiner l'etat des suppresseurs de tumeurs de cellules
US5876711A (en) * 1996-05-31 1999-03-02 Onyx Pharmaceuticals, Inc. Methods and compositions for determining the tumor suppressor status of cells
AU712816B2 (en) * 1996-05-31 1999-11-18 Onyx Pharmaceuticals Methods and compositions for determining the tumor suppressor status of cells
KR100454265B1 (ko) * 2001-11-19 2004-10-26 엘지전선 주식회사 고내구성 고무조성물

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