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• • A—HUMAN NECESSITIES
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  • 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
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
  • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/60—Vector systems having a special element relevant for transcription from viruses

Definitions

• the present invention relates to recombinant vectors of viral origin and their use for the treatment of cancers. More particularly, it relates to recombinant adenoviruses comprising a heterologous DNA sequence under the control of expression signals active specifically in tumor cells. The invention also relates to the preparation of these vectors, the pharmaceutical compositions containing them and their use in gene therapy.
• Gene therapy consists of correcting a deficiency or an anomaly (mutation, aberrant expression, etc.) by introducing genetic information into the affected cell or organ.
• This genetic information can be introduced either in vitro into a cell extracted from the organ, the modified cell then being reintroduced into the organism, or directly in vivo into the appropriate tissue.
• Different techniques have been described for the introduction of this genetic information, among which various transfection techniques involving complexes of DNA and DEAE-dextran (Pagano et al., J. Virol.
• viruses as vectors for gene transfer has emerged as a promising alternative to these physical transfection techniques.
• retroviruses RSV, HMS, MMS, etc.
• the HSV virus adeno-associated viruses
• adenoviruses adenoviruses
• EP 259 212 describes the preparation of vaccines intended for the treatment of cancers, comprising a modified virus capable of expressing a tumor-specific antigen, making it possible to generate an immune response against these cells.
• application WO91 / 15580 describes the construction of retroviruses containing a gene coding for a ribozyme, the expression of which in cell culture can make it possible to destroy an mRNA of an oncogene. It is also known from the application WO 93/10814 to use vectors expressing immunogenic, non-tumorigenic forms of cellular oncogen involved in the development of cancers. Application WO 93/02556 finally describes the use of cells taken from the tumor, genetically modified ex vivo by the introduction of a toxic gene, then re-administered to the patient. However, this approach requires surgical steps, and moreover, the stability of the toxic gene in the transformed cell ex vivo has not been demonstrated.
• the present invention provides an advantageous solution to these problems. It indeed provides vectors capable of directing the expression of a given gene selectively in tumor cells.
• the present invention is based in particular on the demonstration that certain transcription control signals are active (or activated) specifically in tumor cells, and that they can be used for the selective expression of heterologous genes.
• adenoviruses constitute particularly effective vectors for the transfer and expression of therapeutic genes in tumor cells.
• adenoviruses have the advantage of not integrating into the genome of the cells they infect, of staying there in a very stable manner which makes it possible to obtain a lasting therapeutic effect, and of having a spectrum very broad host, which allows application to the treatment of cancers affecting all types of cells.
• adenoviruses can be obtained with high titer, which allows work at high multiplicities of infection, and introduce multiple copies of the heterologous gene per cell.
• the invention is also based on the demonstration that adenovirus type viruses are capable of incorporating heterologous sequences comprising such promoters, of transferring these sequences into tumor cells, and of expressing desired genes under the control of signals specific directly to the level of tumors.
• a first object of the invention therefore resides in a defective recombinant adenovirus comprising a heterologous DNA sequence under the control of expression signals active specifically in tumor cells.
• the invention also relates to the use of such a defective recombinant adenovirus for the preparation of a pharmaceutical composition intended for the treatment or prevention of cancers.
• the defective adenoviruses according to the invention are adenoviruses incapable of replicating autonomously in the target cell.
• the genome of the defective adenoviruses used in the context of the present invention is therefore devoid of at least the sequences necessary for the replication of said virus in the infected cell. These regions can be either eliminated (in whole or in part), or made non-functional, or substituted by other sequences and in particular by the inserted gene.
• the defective virus nevertheless retains the sequences of its genome which are necessary for the packaging of the viral particles.
• adenovirus There are different serotypes of adenovirus, the structure and properties of which vary somewhat. However, these viruses are not pathogenic for humans, especially non-immunocompromised people. Among these serotypes, it is preferred to use, within the framework of the present invention, human adenoviruses of type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin (see application FR 93 05954).
• the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus [Manhattan strain or A26 / 61 (ATCC VR-800) for example].
• adenoviruses of human or canine or mixed origin are used.
• the adenoviruses of the invention carry a heterologous DNA sequence.
• This heterologous DNA sequence allows the expression of a biological activity sought in tumor cells.
• the heterologous DNA sequence comprises at least one gene chosen from a gene toxic to the infected cell, a gene whose expression makes it possible to at least partially inhibit cell division, or a gene coding for a lymphokine.
• the adenoviruses of the invention can also comprise several of these sequences, in order to obtain in certain cases a synergistic anti-tumor effect.
• the genes toxic to the infected cell mention may preferably be made of genes whose expression product gives the cell sensitivity to a therapeutic agent. More preferably, the toxic gene is chosen from the thymidine kinase gene, the expression product of which gives mammalian cells sensitivity to certain therapeutic agents such as ganciclovir or acyclovir.
• the herpes simplex virus thymidine kinase is capable of phosphorylating nucleoside analogs such as acvclovir and ganciclovir. These modified molecules can be incorporated into a DNA chain in the process of elongation, which results in the cessation of DNA synthesis, leading to cell death (FL Moolten, Cancer Res. 46 (1986) 5276 ).
• hTK HSV-1 human herpes virus thymidine kinase gene
• cytosine deaminase gene the expression product of which confers on mammalian cells a sensitivity to 5-fluorocytosine (5-FC).
• 5-FC 5-fluorocytosine
• the genes whose expression makes it possible to at least partially inhibit cell division there may be mentioned more particularly the tumor suppressor (or anti-oncogene) genes or any active derivative thereof; the antisense sequences or the ribozymes, the expression of which in the target cell makes it possible to at least partially inhibit the expression of genes promoting cell division.
• tumor suppressor genes which can be used in the context of the present invention, there may be mentioned more particularly the p53 gene (Baker et al., Science 244 (1989) 217); the Rb gene (Friend et al., Nature 323 (1986) 643; Huang et al., Science 242 (1988) 1563), the rap 1A gene (Kitayama et al., Cell 56 (1989) 77); the DCC gene (Fearon et al., Science 247 (1990) 49), the k-rev2 and k-rev3 genes; or any other tumor suppressor gene described in the literature (see example WO 91/15580).
• the heterologous DNA sequence can also comprise an antisense sequence, the expression of which in the target cell makes it possible to control the expression of genes promoting cell proliferation. This control can intervene at the level of transcription, splicing of the premessager, degradation of the messenger, its translation into protein, or post-translational modifications.
• the heterologous DNA sequence comprises a gene coding for an antisense RNA capable of controlling the translation of a target mRNA (EP 140 308).
• antisense sequences which can be used in the context of the invention, there may be mentioned more particularly any antisense sequence making it possible to reduce the production levels of ras, myc, fos, c-erb B oncogenes, etc.
• the gene coding for lymphokine generally comprises, upstream of the coding sequence, a signal sequence directing the polypeptide synthesized in the secretory pathways of the target cell.
• This signal sequence may be the natural signal sequence of the lymphokine, but it may also be any other functional signal sequence, or an artificial signal sequence.
• the heterologous DNA sequence is placed under the control of expression signals active specifically in tumor cells.
• the gene used is only expressed and produces its effect when the virus has actually infected a tumor cell.
• these are expression signals induced by or active in the presence of viruses responsible for or associated with tumors. Even more preferably, it is used in the context of the present invention an expression signal inducible by the Epstein-Barr virus (EBV) or by the papilloma virus.
• EBV Epstein-Barr virus
• Epstein-Barr virus is associated with two types of human cancer: Burkitt's lymphoma and nasopharyngeal cancer.
• the use of a recombinant adenovirus comprising a toxic gene under the control of a promoter inducible by EBV advantageously makes it possible to specifically express this toxic gene in tumor cells of the nasopharynx.
• EBNA1 nuclear antigen is regularly present, which is involved in the maintenance of the viral genome in cells infected with EBV in latent phase, and which activates the viral promoter BCR2.
• a particular object of the invention therefore resides in the use, for the specific expression of a gene in nasopharyngeal cancer cells, of a sequence responding to EBNA1 (EBNA1-RE: EBNA1 "responsive element").
• the invention relates to an adenovirus comprising, as expression signal, a chimeric promoter comprising a sequence responding to EBNA1 fused upstream of another viral promoter, the promoter of the terminal protein 1 gene (TPI).
• TPI terminal protein 1 gene
• Papilloma viruses are responsible for 90% of cervical cancer in women and have been identified in pre-cancerous epithelial lesions (Riou et al., Lancet 335 (1990) 117).
• the E6 gene product leads to the formation of tumors by greatly reducing the amount of wild-type p53, an anti-oncogene, in HPV-positive cells (Wrede et al., Mol. Carcinog. 4 (1991) 171).
• the use of a recombinant adenovirus comprising a toxic gene under the control of a promoter inducible by HPV advantageously makes it possible to express this toxic gene specifically in the corresponding tumor cells.
• ⁇ -fetoprotein promoter Alpha-fetoprotein promoter
• P3 promoter e.g., IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, Lignin, Lignin, Lignin, Lignin, Lignin, ase, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12,
• the defective recombinant adenoviruses according to the invention can be prepared by any technique known to those skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3 (1984) 2917). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid carrying inter alia the heterologous DNA sequence. Homologous recombination occurs after co-transfection of said adenovirus and plasmid in an appropriate cell line.
• the cell line used must preferably (i) be transformable by said elements, and (ii), contain the sequences capable of complementing the part of the genome of the defective adenovirus, preferably in integrated form to avoid the risks of recombination.
• a line mention may be made of the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains in particular, integrated into its genome, the left part of the genome an Ad5 adenovirus (12%).
• Strategies for constructing vectors derived from adenoviruses have also been described in applications No. FR 93 05954 and
• the adenoviruses which have multiplied are recovered and purified according to conventional techniques of molecular biology, as illustrated in the examples.
• the present invention also relates to a pharmaceutical composition
• a pharmaceutical composition comprising one or more defective recombinant adenoviruses as described above.
• the pharmaceutical compositions of the invention contain a pharmaceutically acceptable vehicle for a formulation directly injectable into the tumors to be treated. They may in particular be sterile, isotonic solutions, or dry compositions, in particular lyophilized, which, by addition as appropriate of sterilized water or physiological saline, allow the constitution of injectable solutes. Direct injection into the tumor to be treated is advantageous because it allows the therapeutic effect to be concentrated in the affected tissues. However, it is also possible to use pharmaceutical compositions formulated for topical, oral, parenteral, intra-nasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. administration.
• adenoviruses recombinants according to the invention are formulated and administered in the form of doses of between 10 4 and 10 14 pfu / ml, and preferably 10 6 to 10 10 pfu / ml.
• pfu plaque forming unit
• plaque forming unit corresponds to the infectious power of a virus solution, and is determined by infection of an appropriate cell culture, and measures, generally after 48 hours, the number of plaques of infected cells. The techniques for determining the pfu titer of a viral solution are well documented in the literature.
• the present invention thus provides a very effective means for the treatment or prevention of cancers. It is particularly suitable for the treatment of cancers of the nasopharynx or hepatocarcinomas.
• this treatment can concern both humans and any animal such as sheep, cattle, domestic animals (dogs, cats, etc.), horses, fish, etc.
• Figure 1 Representation of the vector pONT-tk
• Figure 2 Representation of the vector pONT- ⁇ -gal
• the plasmids of the pBR322, pUC type and the phages of the Ml 3 series are of commercial origin (Bethesda Research Laboratories).
• the DNA fragments can be separated according to their size by electrophoresis in agarose or acrylamide gels, phenol extracts or by a phenol / chloroform mixture, precipitated with ethanol and then incubated in the presence of ADX ligase from phage T4 (Biolabs) according to the supplier's recommendations.
• the filling of the protruding 5 ′ ends can be carried out by the Klenow fragment of DNA Polymerase I of E. coli (Biolabs) according to the supplier's specifications.
• the destruction of the protruding 3 ′ ends is carried out in the presence of the DNA polymerase of phage T4 (Biolabs) used according to the manufacturer's recommendations.
• the destruction of the protruding 5 ′ ends is carried out by gentle treatment with nuclease SI.
• Mutagenesis directed in vitro by synthetic oligodeoxynucleotides can be carried out according to the method developed by Taylor et al. [Nucleic Acids Res. 13 (1985) 8749-8764] using the kit distributed by Amersham.
• Verification of the nucleotide sequences can be carried out by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467] using the kit distributed by Amersham.
• Example 1 Construction of the Ad-ONT-tk vector carrying the tk gene under the control of a chimeric promoter EBNA1-RE / TP1 (FIG. 1).
• This example describes the construction of a recombinant adenovirus comprising the thymidine kinase gene of the herpes simplex virus (tk) under the control of a promoter specifically active in cells infected with the EBV virus (chimeric promoter EBNA1-RE TP1).
• This example describes the construction of the plasmid p7tkl containing the open reading phase of the tk gene of 1131 base pairs (ATG 114-116 and stop codon TGA 1242-1244), inserted into a cloning multisite.
• the BglII-NcoI fragment containing the thymidine kinase (tk) gene of the herpes simplex virus type 1 was isolated from the plasmid pHSV-106 (marketed by Gibco BRL), repaired by the action of the klenow fragment and then inserted into the site. Smal of plasmid pGEM7zf (+) (marketed by Promega). The Smal and BglII sites are destroyed during this step, the Ncol site is preserved. The plasmid obtained was designated p7tkl.
• the EcoRI (7315) -SmaI (8191) fragment of the EBV virus was isolated from the B95-8 strain. The complete sequence of the EBV virus has been described by Baer et al. (Nature 310 (1984) 207). This fragment contains the sequences necessary for transactivation by nuclear antigen 1 (EBNA1) (D. Reisman & B. Sugden, 1986, Molecular and Cellular Biology, vol. 6 pp. 3838-3846). This fragment was then fused to the Nrul (166,241) -PstI (166,559) fragment of EBV B95-8 (the PstI site was digested with T4 polymerase), containing the TPI promoter. The chimeric promoter thus obtained was then inserted into the multisite for cloning the plasmid pBluescript II SK.
• EBNA1 nuclear antigen 1
• the plasmid obtained was designated pONTl.
• the plasmid pONTtk contains the thymidine kinase gene of the herpes simplex virus (tk) cloned in the plasmid p7tkl, under the control of the chimeric promoter EBNA1-RE / TP1 cloned in the plasmid pONTl.
• plasmid pAd.RSV ⁇ gal contains, in the orientation 5 '-> 3',
• the PvuII fragment corresponding to the left end of the Ad5 adenovirus comprising: the ITR sequence, the origin of replication, the packaging signals and the E1A amplifier; - the gene coding for ⁇ -galactosidase under the control of the RSV promoter
• the plasmid obtained was designated pONTtk ( Figure 1).
• the vector pONTtk was linearized and cotransfected with a deficient adenoviral vector, in helper cells (line 293) providing in trans the functions coded by the El regions (El A and E11B) of adenovirus.
• the adenovirus Ad-ONT-tk was obtained by homologous in vivo recombination between the mutant adenovirus Ad-dl324 (Thimmappaya et al., Cell 31 (1982) 543) and the vector pONTtk, according to the following protocol: the plasmid pONTtk, linearized by Xmnl, and the adenovirus dl324, linearized by the enzyme ClaI, were cotransfected in line 293 in the presence of calcium phosphate, to allow homologous recombination. The recombinant adenoviruses thus generated were selected by plaque purification.
• the DNA of the recombinant adenovirus was amplified in the cell line 293, which leads to a culture supernatant containing the unpurified recombinant defective adenovirus having a titre of approximately 10 10 pfu / ml.
• the viral particles are generally purified by centrifugation on a cesium chloride gradient according to known techniques (see in particular Graham et al., Virology 52 (1973) 456).
• the Ad-ONT-tk adenovirus can be stored at -80 ° C in 20% glycerol.
• This example describes the construction of a recombinant adenovirus comprising the E.coli beta-galactosidase gene ( ⁇ gal) under the control of a promoter specifically active in cells infected with the EBV virus (chimeric promoter EBNA1-RE / TP1).
• the Xba ⁇ - (Hind ⁇ II) fragment of the plasmid pONTl which contains the chimeric promoter transactivated by EBNA-1 and EBNA-2 and the (Stul) -Kpnl fragment of the plasmid pAd.RSV ⁇ gal which contains the ⁇ -galactosidase gene have been cloned to Xbal sites
• the vector pONT- ⁇ gal obtained in example 2.1 was used, by homologous recombination according to the protocol described in example 1.4., To prepare a recombinant adenovirus comprising the gene of oli beta-galactosidase ( ⁇ gal) under the control of the chimeric promoter EBNAl-RE / TPl.
• the Ad-ONTl- ⁇ gal adenovirus thus obtained can be stored at -80 ° C in 20% glycerol.
• This example describes the construction of a vector comprising the chloramphenicol acetyl transferase (CAT) gene under the control of a promoter specifically active in cells infected with the EBV virus (chimeric promoter EBNAl-RE / TP1).
• CAT chloramphenicol acetyl transferase
• NruI (166241) -PstI (1665S9) from EBV This fragment was then fused to the CAT gene, and inserted, in the form of a NruI-BamHI fragment, into the plasmid pGem7ZF (Promega).
• the resulting plasmid was designated pTP1-CAT.
• the NruI-BamHI fragment of the plasmid pTP1-CAT was then fused, downstream of the EcoRI (7315) - Smal (8191) fragment of the EBV strain B95-8, containing the sequences necessary for transactivation by EBNA1 (cf. example 1.2 .).
• the fragment obtained comprising the CAT gene under the control of the chimeric promoter EBNAl-RE / TP1, was inserted at the EcoRI and BamHI sites of the plasmid pBluescript SK to generate the plasmid pONT-CAT.
• a plasmid has also been constructed in which the response elements to the TNA promoter EBNA2 antigen have been deleted.
• the TPI promoter was isolated in the form of the Nrul (166375) -Pstl (1 66559) fragment of EBV. This plasmid was designated pOST-CAT.
• the pONT-CAT, pOST-CAT and pTP1-CAT vectors were transfected by electroporation into a line of EBV " B lymphocytes (DG75 cells), either alone, either in the presence of expression vectors of the viral antigens EBNAl, EBNA2 or EBNAl + EBNA2. 48 hours after transfection, the cells were lyzed by freezing / thawing, the cellular debris eliminated, then the extracts obtained were normalized according to the quantity of proteins. The CAT activity was then assayed in these extracts by enzymatic assay. The results obtained are as follows:

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