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  • C07K14/52—Cytokines; Lymphokines; Interferons
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  • C07K14/52—Cytokines; Lymphokines; Interferons
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  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • C12N15/86—Viral vectors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • 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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  • C12N2710/16011—Herpesviridae
  • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
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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
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  • C12N2830/60—Vector systems having a special element relevant for transcription from viruses

Definitions

• Adenoviruses comprising two therapeutic genes: suicide and immunostimulant
• the present invention relates to new viral vectors, their preparation and their use in gene therapy. It also relates to pharmaceutical compositions containing said viral vectors. More particularly, the present invention relates to recombinant adenoviruses as vectors for 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 exist, among which various transfection techniques involving complexes of DNA and DEAE-dextran (Pagano et al., J. Virol.
• adenoviruses have certain properties of interest for use in gene therapy. In particular, they have a fairly broad host spectrum, are capable of infecting quiescent cells, do not integrate into the genome of the infected cell, and have not been associated to date with significant pathologies in man.
• Adenoviruses are linear double-stranded DNA viruses approximately 36 kb in size. Their genome includes in particular a repeated inverted sequence (ITR) at their end, an encapsidation sequence, early genes and late genes (see FIG. 1).
• the main early genes are the El (Ela and Elb), E2, E3 and E4 genes.
• the main late genes are the L1 to L5 genes.
• adenoviruses Given the properties of the adenoviruses mentioned above, these have already been used for gene transfer in vivo. To this end, different vectors derived from adenoviruses have been prepared, incorporating different genes ( ⁇ -gal, OTC, ⁇ - 1 AT, cytokines, etc.). In each of these constructions, the adenovirus was modified so as to render it incapable of replication in the infected cell.
• the constructions described in the prior art are adenoviruses deleted from the El regions (El a and / or Elb) and optionally E3 into which a heterologous DNA sequence is inserted (Levrero et al., Gene 101 (1991) 195; Gosh-Choudhury et al., Gene 50 (1986) 161).
• the present invention relates to new vectors derived from adenoviruses which are particularly effective for gene therapy applications. More particularly, the present invention derives in part from the demonstration that it is possible to incorporate several genes of interest into adenoviruses, and to obtain a significant expression of these different genes in the infected cells
• the present invention follows also from the construction of adenoviral vectors capable of incorporating several therapeutic genes under conditions allowing their optimal expression. It also results from the demonstration of a synergistic effect of the vectors of the invention, linked to co-expression, in the same target cell, of complementary therapeutic genes
• the present invention thus provides viral vectors exhibiting therapeutic properties which are entirely advantageous with a view to their use in gene or cell therapy.
• the vectors of the invention have properties which are entirely advantageous for use in the treatment of pathologies presenting episodes of cellular hyperproliferation (cancers, restenosis, etc.)
• a first object of the present invention relates to a defective recombinant adenovirus comprising two therapeutic genes, in which one of the therapeutic genes is a suicide gene and the other is an immunostimulatory or tumor suppressor gene.
• the Applicant has indeed shown that the simultaneous co-expression of such genes in the same target cell produces a particularly advantageous anti-tumor therapeutic effect, much greater than the effect obtained by means of these genes alone or introduced separately.
• the therapeutic genes used in the context of the present invention may be a cDNA, a genomic DNA (gDNA), or a hybrid construct consisting, for example, of a cDNA in which one or more introns would be inserted. They can also be synthetic or semi-synthetic sequences. Particularly advantageously, a cDNA or a gDNA is used.
• the two therapeutic genes incorporated into the adenoviral vectors according to the present invention can be arranged in different ways.
• the two genes can first of all constitute a single transcriptional entity.
• the two genes are contiguous, arranged under the control of a single promoter, and give Heu to a single premessenger RNA.
• This arrangement is advantageous since it allows the use of a single transcriptional promoter.
• the two therapeutic genes can also be placed under the control of separate transcriptional promoters. This configuration makes it possible to obtain higher levels of expression, and offers better control of gene expression.
• the two therapeutic genes can be inserted in the same orientation or in the opposite orientations, in the same site of the genome of the denovirus or in different sites
• suicide gene use is preferably made of genes whose expression product confers on the cell a sensitivity to a therapeutic agent. More preferably, the suicide gene is 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 acyclovir 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 ). This strategy thus makes it possible to specifically eliminate the cells expressing the suicide gene.
• DNA synthesis is the target of toxicity, only the cells being divided are affected.
• the human herpes virus thymidine kinase gene (hTK HSV-1) is used.
• the sequence of this gene has been described in the literature (see in particular McKnight et al., Nucleic Acid. Res. 8 (1980) 5931). It is also possible to use derivatives of this sequence exhibiting greater substrate specificity or better kinase activity. Such derivatives can in particular be obtained by mutagenesis at the level of the binding site as described previously (Balasubramaniam et al., J. Gen. Virol. 71 (1990) 2979; Munir et al., JBC 267 (1992) 6584).
• cytosine deaminase gene the expression product of which gives mammalian cells sensitivity to 5-fluoro- cytosine (5-FC) or nitroreductases which give mammalian cells sensitivity to nitroaromatic products (J. Biol. Chem. 266 (1991) 4126).
• the immunostimulatory gene can be any gene whose expression product is capable of stimulating the body's defenses.
• it is a gene coding for a cytokine, such as in particular a lymphokine (IL-1 to IL-12), an interferon (alpha, beta, etc.), a tumor necrosis factor, a factor of colony stimulation (G-CSF, GM-CSF, M-CSF, SCF, etc.), etc.
• Interleukin 2 is synthesized essentially by lymphocytes, in response to the presence of antigens, in particular tumor antigens. It then acts on the development of the immune response against these antigens, in particular by local activation of cytotoxic and killer T cells (NK).
• NK cytotoxic and killer T cells
• GM-CSF is a factor for stimulating colonies of granocytes and macrophages. It therefore stimulates the proliferation of these cells of immunity and therefore makes it possible to strengthen the immune defenses.
• the GM-CSF gene and cDNA have been described in the literature. Its co-expression in a vector of the invention with a suicide gene produces a high synergistic anti-tumor effect
• tumor suppressor genes which can be used in the context of the present invention, there may be mentioned more particularly the p53, Rb, raplA, DDC, WAF and MTS genes. More particularly, the p53 gene or the Rb gene is used.
• the p53 gene codes for a 53 kDa nuclear protein.
• the mutated deletion and / or mutation form of this gene is involved in the development of most human cancers (Baker et al., Science 244 (1989) 217). Its mutated forms are also capable of cooperating with ras oncogenes to transform murine fibroblasts.
• the wild-type gene encoding native p53 inhibits the formation of foci of transformation in fibroblasts of rodents transfected with various combinations of oncogenes. Recent data highlights that the protein p53 could itself be a transcription factor and stimulate the expression of other tumor suppressor genes.
• the Rb gene determines the synthesis of a nuclear phosphoprotein of approximately 927 amino acids (Friend et al., Nature 323 (1986) 643) whose function is to suppress the division of cells by making them enter the quiescence phase.
• Inactivated forms of the Rb gene have been implicated in various tumors, and in particular in retinoblastomas or in mesenchymal cancers such as osteosarcomas. Reintroduction of this gene into tumor cells where it was inactivated produces a return to normal state and a loss of tumorigenicity (Huang et al., Science 242 (1988) 1563).
• the invention relates to a defective recombinant adenovirus comprising a gene coding for thymidine kinase and a tumor suppressor gene. More preferably, it relates to an adenovirus comprising a gene coding for the thymidine kinase of the herpes virus and the wild-type p53 gene (Ad-TK-p53).
• Ad-TK-p53 wild-type p53 gene
• the two genes are placed under the control of separate promoters, preferably the promoter of the LTR of the HSV virus. Even more preferably, the two genes are inserted at the level of the E1 region of the adenovirus genome.
• the invention relates to a defective recombinant adenovirus comprising a gene coding for thymidine kinase and a gene coding for a lymphokine. More preferably, it relates to an adenovirus comprising a gene coding for the thymidine kinase of the herpes virus and a gene coding for interleukin-2 (Ad-TK-EL2) or for GM-CSF (Ad-TK-GM -CSF).
• Ad-TK-EL2 interleukin-2
• Ad-TK-GM -CSF GM-CSF
• the two genes are placed under the control of separate promoters, preferably the LTR promoter of the HSV virus. Even more preferably, the two genes are inserted at the level of the E1 region of the adenovirus genome.
• transcriptional promoters used in the context of the present invention, they may be promoters which are naturally responsible for the expression of the therapeutic gene considered when these are capable of functioning in the infected cell. They may also be sequences of different origin (responsible for the expression of other proteins, or even synthetic). In particular, they may be promoter sequences of mammalian, eukaryotic or viral genes. For example, they may be promoter sequences originating from the genome of the cell which it is desired to infect. Likewise, they may be promoter sequences originating from the genome of a virus, including the adenovirus used. In this regard, mention may be made, for example, of the promoters of the El A, MLP, CMV, RSV, etc. genes.
• these expression sequences can be modified by adding activation, regulation or tissue-specific expression sequences.
• the inserted gene when it does not contain expression sequences, it can be inserted into the genome of the defective virus downstream of such a sequence
• a preferred promoter for producing the vectors of the invention consists of the LTR of the sorcoma virus (LTR-RSV).
• Other particularly preferred promoters are the specific promoters of proliferative or cancerous cells. These promoters indeed make it possible to target the therapeutic effect on a defined cell population.
• these are expression signals induced by or active in the presence of viruses responsible for or associated with tumors.
• an expression signal inducible by the Epstein-Barr virus (EBV) or by the papilloma virus is used.
• Epstein-Barr virus is associated with two types of human cancers, Burkitt 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.
• a single nuclear antigen is regularly present, EBNA1, which is involved in the maintenance of the viral genome in cells infected with EBV in the latent phase, and which activates the viral promoter BCR2.
• a particular object of the invention therefore lies in the use, for the specific expression of a gene in cancer cells of the nasopharynx, 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) 1 17) .
• 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 specifically express this toxic gene in the corresponding tumor cells.
• ⁇ -fetoprotein promoter Alpha-fetoprotein promoter
• P3 promoter e.g., 'IGF-II (Sussenbach et al, Growth Regulation 2 (1992) 1), which are active in adults, only in hepatocarcinomas. It is also possible to use promoters induced by hormones in the case of hormone-dependent or associated hormonal tumors (breast or prostate tumor for example).
• the vectors of the invention can first of all contain the two genes in the form of a single transcriptional entity.
• the two genes are contiguous, arranged under the control of a single promoter, and give rise to a single premessenger RNA.
• This configuration is advantageous since it makes it possible to use a single transcriptional promoter to regulate the expression of the 2 genes.
• this unique transcriptional entity can be incorporated into the adenoviral vector in the two possible orientations.
• the two genes can also be placed under the control of separate transcriptional promoters. This configuration provides levels expression, and provides better control of gene expression.
• the two therapeutic genes can be inserted in the same orientation or in the opposite orientations, in the same site of the genome of the denovirus or in different sites.
• the genes are inserted, at least in part, at the level of the El, E3 or E4 regions of the adenovirus genome.
• the regions E1 and E3 or E1 and E4 are preferred in the context of the invention to use the regions E1 and E3 or E1 and E4.
• a particularly advantageous embodiment is that in which two therapeutic genes are inserted at the level of the El region.
• the examples indeed show that this organization allows a high expression of the two genes, without interference between the two.
• the invention therefore also relates to any recombinant adenovirus comprising two genes of therapeutic interest inserted at the level of the E1 region of the genome
• the immunostimulant gene may also contain a signal sequence directing the product synthesized in the secretory pathways of the target cell.
• This signal sequence may be the natural signal sequence of the immunostimulant product, but it may also be any other signal sequence. functional, or an artificial signal sequence.
• the adenoviruses of the present invention are defective, that is to say that they are unable to replicate autonomously in the target cell.
• the genome of the defective adenoviruses according to 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 therapeutic genes.
• the defective nature of the adenoviruses of the invention is an important element, since it ensures the non-dissemination of the vectors of the invention after administration.
• the adenoviruses of the invention comprise the ITR sequences and a sequence allowing the encapsidation, and have a deletion of all or part of the E 1 gene.
• the inverted repeat sequences (ITR) constitute the origin of replication of adenoviruses. They are located at the 3 ′ and 5 ′ ends of the viral genome (cf. FIG. 1), from which they can be easily isolated according to the conventional techniques of molecular biology known to those skilled in the art.
• the nucleotide sequence of the ITR sequences of human adenoviruses is described in the literature, as well as of canine adenoviruses (in particular CAV1 and CAV2).
• the left ITR sequence corresponds to the region comprising nucleotides 1 to 103 of the genome
• the packaging sequence (also called Psi sequence) is necessary for the packaging of viral DNA. This region must therefore be present to allow the preparation of defective recombinant adenoviruses according to the invention.
• the packaging sequence is located in the adenovirus genome, between the left ITR (5 ') and the E1 gene (Cf figure 1) It can be isolated or artificially synthesized by standard molecular biology techniques
• the nucleotide sequence of the packaging sequence of human adenoviruses (in particular of serotypes Ad2 and Ad5) is described in the literature, as well as canine adenoviruses (in particular CAV1 and CAV2).
• Adenovirus Ad5 for example, the encapsidation sequence corresponds to the region comprising nucleotides 194 to 358 of the genome
• the adenoviruses of the invention comprise the ITR sequences and a sequence allowing the packaging, and have a deletion of all or part of the El and E4 genes
• the genome of the adenoviruses according to the invention is deleted from all or part of the El, E3 and E4 genes, and, even more preferably, from all or part of the El, E3, L5 and E4 genes
• adenoviruses of the invention can be prepared from adenoviruses of various origins. There are in fact different serotypes of adenovirus, the structure and properties of which vary somewhat, but which have a comparable genetic organization. Thus, the teachings described in the present application can be easily reproduced by a person skilled in the art for any type of adenovirus.
• the adenoviruses of the invention can be of human, animal, or mixed origin (human and animal) As regards adenoviruses of human origin, it is preferred to use those classified in group C. More preferably, among the various serotypes of human adenovirus, it is preferred to use, within the framework of the present invention, adenoviruses of type 2 or 5 (Ad 2 or Ad 5).
• the adenoviruses of the invention can also be of animal origin, or contain sequences derived from adenoviruses of animal origin.
• the Applicant has indeed shown that adenoviruses of animal origin are capable of infecting human cells with great efficiency, and that they are unable to propagate in the human cells in which they have been tested (see request FR 93 05954).
• the Applicant has also shown that adenoviruses of animal origin are in no way trans-complemented by adenoviruses of human origin, which eliminates any risk of recombination and of propagation in vivo, in the presence of a human adenovirus, which can lead to the formation of an infectious particle.
• the use of adenoviruses or adenovirus regions of animal origin is therefore particularly advantageous since the risks inherent in the use of viruses as vectors in gene therapy are even lower.
• the adenoviruses of animal origin which can be used in the context of the present invention can be of canine, bovine, murine origin (example: Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian or else simienne (example: after-sales service).
• serotypes 1 to 10 accessible to ATCC such as for example the strains Phelps (ATCC VR-432), Fontes (ATCC VR-280), P7-A (ATCC VR- 827), IBH-2A (ATCC VR-828), J2-A (ATCC VR-829), T8-A (ATCC VR-830), Kl l (ATCC VR-921) or the strains referenced ATCC VR-831 to 835.
• bovine adenoviruses it is possible to use the various known serotypes, and in particular those available at ATCC (types 1 to 8) under the references ATCC VR-313, 314, 639-642, 768 and 769. It is also possible to use cite the murine adenoviruses FL (ATCC VR-550) and E20308 (ATCC VR-528), the sheep adenovirus type 5 (ATCC VR-1343), or type 6 (ATCC VR-1340); porcine adenovirus 5359), or simian adenoviruses such as in particular adenoviruses referenced in the ATCC under the numbers VR-591-594, 941-943, 195-203, etc.
• adenoviruses or regions of adenoviruses of canine origin are used in the context of the invention, and in particular all the strains of adenoviruses CAV2 [Manhattan strain or A26 / 61 ( ATCC VR-800) for example].
• Canine adenoviruses have been the subject of numerous structural studies. . Thus, complete restriction maps of the CAV1 and CAV2 adenoviruses have been described in the prior art (Spibey et al., J. Gen. Virol.
• the defective recombinant adenoviruses according to the invention can be prepared in different ways.
• a first method consists in transfecting the DNA of the defective recombinant virus prepared in vitro (either by ligation or in the form of a plasmid) in a competent cell line, that is to say carrying in trans all the functions necessary for complementation of the defective virus. These functions are preferably integrated into the genome of the cell, which makes it possible to avoid the risks of recombination, and confers increased stability on the cell line.
• a second approach consists in co-transfecting into a suitable cell line the DNA of the defective recombinant virus prepared in vitro (either by ligation or in the form of a plasmid) and the DNA of a helper virus.
• a competent cell line capable of complementing all the defective functions of the recombinant adenovirus. Part of these functions is indeed complemented by the helper virus.
• This helper virus must itself be defective and the cell line carries in trans the functions necessary for its complementation.
• the human embryonic kidney line 293 there may be mentioned in particular the human embryonic kidney line 293, the cells KB, Hela cells, MDCK, GHK, etc. (see examples).
• the vectors which have multiplied are recovered, purified and amplified according to conventional techniques of molecular biology.
• the vectors of the invention advantageously have a deletion of all or part of certain viral genes, in particular the El, E3, E4 and / or L5 genes.
• This deletion can correspond to any type of deletion affecting the gene considered. It may especially be the suppression of all or part of the coding region of said gene, and / or of all or part of the promoter region of the transcription of said gene.
• Deletion is generally carried out on the DNA of the defective recombinant virus, for example by digestion using appropriate restriction enzymes, then ligation, according to molecular biology techniques, as illustrated in the examples.
• Therapeutic genes can then be inserted into this DNA by enzymatic cleavage then ligation, at the level of the selected regions and in the chosen orientation.
• the DNA thus obtained which therefore carries the appropriate deletions and the two therapeutic genes, makes it possible to directly generate the defective recombinant adenovirus carrying said deletions and therapeutic genes
• This first variant is particularly suitable for the production of recombinant adenoviruses in which the genes therapeutics are arranged in the form of a single transcriptional unit or, under the control of separate promoters but inserted at the same site of the genome
• the DNA of a first recombinant virus carrying the appropriate deletions (or part of said deletions) and one of the therapeutic genes is constructed, by ligation or in the form of a plasmid
• This DNA is then used to generate a first recombinant virus carrying said deletions and a therapeutic gene
• the DNA of this first virus is then isolated and co-transfected with a second plasmid or d DNA a second defective recombinant virus carrying the second therapeutic gene, the appropriate deletions (part not present on the first virus), and a region allowing homologous recombination
• This second step thus generates the defective recombinant virus carrying the two therapeutic genes
• This variant of preparation is particularly suitable for the preparation of recombinant virus nts carrying two therapeutic genes inserted in two different regions of the adenovirus genome.
• the present invention also relates to any pharmaceutical composition comprising one or more defective recombinant adenoviruses as described above.
• the pharmaceutical compositions of the invention can be formulated for administration by topical, oral, parenteral, intranasal, intravenous, intramuscular, -cutaneous, intraocular, transdermal, etc.
• the pharmaceutical composition contains pharmaceutically acceptable vehicles for an injectable formulation.
• pharmaceutically acceptable vehicles for an injectable formulation can be in particular saline solutions (monosodium phosphate, disodium, sodium chloride, potassium, calcium or magnesium, etc., or mixtures of such salts), sterile, isotonic, or dry compositions, in particular lyophilized, which, by addition depending on the case of sterilized water or physiological saline, allow the constitution of injectable solutions.
• the doses of virus used for the injection can be adapted according to various parameters, and in particular according to the mode of administration used, the pathology concerned, the gene to be expressed, or even the duration of the treatment sought.
• the recombinant adenoviruses 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
• the term pfu (“plaque forming unit ”) corresponds to the infectivity of a virus solution, and is determined by infection of an appropriate cell culture, and measures, generally after 5 days, 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 adenoviruses of the invention can be used for the treatment or prevention of many pathologies. They are particularly advantageous for the treatment of hyperproliferative pathologies (cancers, restenosis, etc.), by direct injection at the site concerned.
• the present invention also relates to a method for the destruction of proliferative cells comprising the infection of said cells or of a part of them with an adenoviral vector as defined above.
• the suicide gene is a gene which confers sensitivity to a therapeutic agent
• the method of destruction according to the invention then comprises the treatment of the cells with said therapeutic agent.
• the invention also relates to products comprising a recombinant adenovirus as defined above in which the suicide gene is a gene conferring sensitivity to a therapeutic agent; and said therapeutic agent, as a combination product for simultaneous, separate or spread over time use for the treatment of hyperproliferative pathologies.
• the suicide gene is a thymidine kinase gene and the therapeutic agent is gancyclovir or acyclovir or an analog.
• the plasmids of type pBR322, pUC 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, extracted with phenol or with a phenol / chloroform mixture, precipitated with ethanol and then incubated in the presence of the DNA ligase from phage T4 (Biolabs) according to the supplier's recommendations
• the filling of the prominent 5 'ends can be carried out by the Klenow fragment of DNA Polymerase I of E.
• Mutagenesis directed in vitro by synthetic oligodeoxynucleotides can be carried out according to the method developed by Taylor et al [Nucleic Acids Res H (1985) 8749-8764] using the kit distributed by Amersham Enzymatic amplification of DNA fragments by the so-called PCR technique
• 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.
• - Human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) This line contains in particular, integrated into its genome, the left part of the genome of the human adenovirus Ad5 (12%) .
• - Human cell line KB From a human epidermal carcinoma, this line is accessible to the ATCC (ref. CCL17) as well as the conditions allowing its culture.
• Hela Human cell line Hela: From a carcinoma of the human epithelium, this line is accessible to the ATCC (ref. CCL2) as well as the conditions allowing its culture.
• MDCK canine cell line The culture conditions for MDCK cells have been described in particular by Macatney et al. Science 44 (1988) 9.
• DBP6 cell line (Brough et al., Virology 190 (1992) 624). This line consists of Hela cells carrying the E2 gene of adenovirus under the control of the LTR of MMTV.
• Example 1 Construction of defective recombinant adenoviruses comprising the TK gene under the control of a cancer-specific promoter and the p53 gene under the control of the CMV promoter.
• adenoviruses were constructed by homologous recombination between a plasmid carrying the left part of the adenovirus Ad5, the two therapeutic genes and a region of the Ad5 adenovirus (corresponding to protein IX) and the DNA of a defective adenovirus carrying different deletions.
• 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 the plasmid pGEM7zf (+) (sold by Promega) The Smal and BglII sites are destroyed during this step, the Ncol site is preserved
• the plasmid obtained was designated p7tkl
• This example describes the construction of a plasmid containing a chimeric promoter consisting of a sequence necessary for transactivation by the EBNA1 antigen and of the TPI promoter of the EBV virus.
• the EcoRI (7315) -SmaI (8191) fragment of the EBV virus was isolated from the B95-8 strain.
• the complete sequence of the EBV virus was 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) 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.
• the plasmid obtained was designated pONT 1
• the plasmid pONTtk contains the gene for the thymidine kinase 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 To construct this plasmid, the BamHI-XhoI fragment from pONTl which contains the chimeric promoter transactivated by EBNA-1 and EBNA-2, and the fragment
• 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 El A amplifier;
• This example describes the construction of a vector carrying the left part of the adenovirus Ad5 (comprising the left ITR, the encapsidation region and the start of the E1 region), the tk gene under the control of the ONT promoter, the gene p53 under the control of the CMV promoter and a second fragment of the Ad5 genome (pIX protein) allowing homologous recombination with a view to the generation of the recombinant adenovirus (cf. example 1.3).
• This plasmid was constructed from the plasmid pONTtk, by insertion, downstream of the tk gene and in the same orientation, of a fragment carrying the p53 gene under the control of the CMV promoter and followed by the polyadenylation site of the SV40 virus. More specifically, the inserted fragment comprises:
• CMV cytomegalovirus
• the suppressor gene is in the form of c DNA, that is to say devoid of introns. This makes it possible in particular to reduce the size of the vector. Furthermore, it has been verified that the expression levels obtained are comparable in the presence or absence of introns - the polyadenylation signal from the late genes of the SV40 virus, which corresponds to a very effective polyadenylation signal.
• Two unique restriction sites SalI and HindIII are located downstream of the polyadenylation signal
• the vector obtained was designated pONTtkCMVp53
• the vector pONTtkCMVp53 was linearized and cotransfected with an adenoviral vector deficient in the El gene, in helper cells (line 293) providing in trans the functions coded by the El regions (El A and El B) of adenovirus.
• the adenovirus Ad-ONTtkCMVp53, ⁇ El is obtained by homologous recombination in vivo between the adenovirus Ad-RSV ⁇ gal (Cf Stratford-Perricaudet et al cited above) and the vector pONTtkCMVp53, according to the following protocol: the plasmid pONTtkCMVp53, linearized by Xmnl, and the adenovirus Ad-RSV ⁇ gal, linearized by the enzyme Clal, are co-transfected in line 293 in the presence of calcium phosphate, to allow homologous recombination. The recombinant adenoviruses thus generated are then selected by plaque purification.
• the DNA of the recombinant adenovirus is amplified in the line cell 293, which leads to a culture supernatant containing the unpurified recombinant defective adenovirus having a titer 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).
• Ad-ONT-tkCMVp53, ⁇ El adenovirus can be stored at -80 ° C in 20% glycerol
• the vector pONTtkCMVp53 was linearized and cotransfected with an adenoviral vector deficient in the El and E3 genes, in the helper cells (line 293) bringing in irons the functions coded by the El regions (El A and El B) of adenovirus
• the adenovirus Ad-ONTtkCMVp53, ⁇ El, ⁇ E3 is obtained by homologous recombination in vivo between the mutant adenovirus Ad-dll324 (Thimmappaya et al, Cell 3 1 (1982) 543) and the vector pONTtkCMVp53, according to the following protocol the plasmid pONTtkCMVp53, linearized by Xmnl, and the adenovirus Ad-dll324, linearized by the enzyme ClaI, are co-transfected in line 293 in the presence of calcium phosphate, to allow homologous recombination.
• the recombinant adenoviruses thus generated are then selected by plaque purification. After isolation, the DNA of the recombinant adenovirus is amplified in the cell line 293, which leads to a culture supernatant containing the non-purified recombinant defective adenovirus having a titer of approximately 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 adenovirus Ad-ONT-tkCMVp53, ⁇ El, ⁇ E3 can be stored at -80 ° C in 20% glycerol.
• adenoviruses in which the tk and p53 genes are positioned in the opposite orientations can be constructed starting from the plasmid pONTtkCMVp53inv.
• Example 2 Construction of defective recombinant adenoviruses comprising the TK gene under the control of the RSV virus LTR promoter and the p53 gene under the control of the CMV promoter.
• adenoviruses were constructed by homologous recombination between a plasmid carrying the left part of the adenovirus Ad5, the two therapeutic genes and a region of the adenovirus Ad5 (corresponding to protein IX) and the DNA of a defective adenovirus carrying different deletions.
• This plasmid was constructed from the plasmid pONTtk (Example 11), by substitution of the promoter transactivable by EBNA1 by the promoter of the LTR of RSV.
• the RSV promoter was isolated in the form of a BamHI-SalI fragment from the plasmid pAd.RSV. ⁇ gal (Stratford-Perricaudet et al., J. Clin. Invest. 90 (1992) 626), then cloned at the BamHI (478) and Sall (1700) sites of the plasmid pONTtk.
• the resulting plasmid was designated pRSVtk ( Figure 4).
• This example describes the construction of a vector carrying the left part of the adenovirus Ad5 (comprising the left ITR, the encapsidation region and the start of the E1 region), the tk gene under the control of the RSV promoter, the gene p53 under the control of the CMV promoter and a second fragment of the Ad5 genome (protein pIX) allowing homologous recombination with a view to the generation of the recombinant adenovirus (cf. example 2.3).
• This plasmid was constructed from the plasmid pRSVtk, by insertion, downstream of the tk gene, of a fragment carrying the p53 gene under the control of the CMV promoter and followed by the polyadenylation site of the SV40 virus. More specifically, the inserted fragment comprises:
• CMV cytomegalovirus
• the suppressor gene is in the form of cDNA, that is to say devoid of introns. This makes it possible in particular to reduce the size of the vector. Furthermore, it has been verified that the expression levels obtained are comparable in the presence or absence of introns.
• the polyadenylation signal of the late genes of the SV40 virus which corresponds to a very effective polyadenylation signal
• Two unique restriction sites Sali and HindIII are located downstream of the polyadenylation signal
• the vector obtained was designated pRSVtkCMVp53
• the adenovirus Ad-RSV-tkCMVp53, ⁇ El thus obtained can be stored at -80 ° C in 20% glycerol.
• This adenovirus is constructed according to the protocol described in Example 1 (3.2.).
• the adenovirus Ad-RSV-tkCMVp53, ⁇ El, ⁇ E3 thus obtained can be stored at -80 ° C in 20% glycerol.
• Example 3 Construction of defective recombinant adenoviruses comprising the TK gene under the control of the LTR promoter of the RSV virus and the interleukin-2 gene under the control of the same promoter. These adenoviruses were constructed by homologous recombination between a plasmid carrying the left part of the adenovirus Ad5, the two therapeutic genes and a region of the adenovirus Ad5 (corresponding to protein IX) and the DNA of a defective adenovirus carrying different deletions.
• This example describes the construction of a vector carrying the left part of the adenovirus Ad5 (comprising the left ITR, the encapsidation region and the start of the E1 region), the tk gene under the control of the RSV promoter, the gene interleukin-2 under the control of the RSV promoter and a second fragment of the Ad5 genome (pIX protein) allowing homologous recombination with a view to the generation of the recombinant adenovirus (cf. example 3.2).
• This plasmid was constructed from the plasmid pRSVtk (see example 2), by insertion, downstream of the tk gene, of a fragment carrying the interleukin-2 gene under the control of the RSV promoter and monitoring of the polyadenylation site of the virus. SV40. More specifically, the inserted fragment comprises:
• RSV virus LTR promoter isolated in the form of a BamHI-SalI fragment from the plasmid pAd.RSV. ⁇ gal (Stratford-Perricaudet et al., J. Clin. Invest. 90 (1992) 626),
• the therapeutic gene is in the form of cDNA.
• polyadenylation signal from late genes of the SV40 virus, which corresponds to a very effective polyadenylation signal.
• Two unique restriction sites SalI and HindIII are located downstream of the polyadenylation signal.
• the vector obtained was designated pRSVtkRSVIL-2.
• adenoviruses Two types of recombinant adenoviruses are constructed according to the protocol described in Example 1 or 2. These adenoviruses carry the two therapeutic genes inserted in the same orientation, at the level of the E1 region and have a deletion in the E1 region or in the El and E3 regions.
• the adenoviruses Ad-RSV-tkRSVIL-2, ⁇ El and Ad-RSV-tkRSVTL-2, ⁇ El, ⁇ E3 thus obtained can be stored at -80 ° C in 20% glycerol.
• Example 4 Construction of defective recombinant adenoviruses comprising the TK gene and the gene coding for the stimulation factor of the colonies of granocytes and macrophages (GM-CSF).
• defective adenoviruses carrying the tk gene (under the control of the ONT or RSV promoter for example) and the GM-CSF gene under the control of its own promoter or of the RSV promoter in particular can be constructed
• an intermediate vector carrying the two genes can be constructed from the pONTtk or pRSVtk vector by insertion, in the same orientation or in the reverse orientation, of a fragment carrying the GM-CSF gene under the control of the promoter.
• the gene coding for GM-CSF and constructs containing it have been described in particular in application WO86 / 03225.
• Example 5 Construction of defective recombinant adenoviruses comprising two genes of interest, one inserted at the level of the E1 region and the other inserted at the level of the E3 region.
• adenoviruses are constructed by homologous recombination between a DNA from a defective first virus carrying the first gene inserted at the E 1 region and DNA from a second defective adenovirus carrying the second gene inserted at the region E3.
• This virus is constructed from the Addl324 adenovirus (Thimmappaya et al.,
• This virus carries a deletion at the level of the E1 and E3 region (deleted Xbal-EcoRI fragment).
• the DNA of the Add1324 virus was isolated and purified. This DNA is then cut by the enzymes Xbal and EcoRI. A Xbal-EcoRI fragment is then derived from the plasmid pRSVtk carrying the sequence coding for the thymidine kinase under the control of the RSV promoter, then inserted at said sites in the DNA of Add1324 opened as above.
• the DNA thus obtained therefore comprises a deletion at the level of the E1 region and the TK gene inserted at the level of the E3 region.
• adenoviruses carrying the two genes The DNA of the recombinant virus prepared above and the DNA of a recombinant adenovirus carrying an immunostimulating or tumor suppressor gene inserted at the level of the E1 region, linearized with BamHI, are co-transfected into line 293 in the presence of calcium phosphate, to allow homologous recombination. The recombinant adenoviruses thus generated are then selected by plaque purification.
• the DNA of the recombinant adenovirus is amplified in the cell line 293, which leads to a culture supernatant containing the non-purified recombinant defective adenovirus having a titer of approximately 10 1 () 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)

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