MXPA97002078A - Adenovirus that comprise two therapeutic genes: suicide and immunoestimula - Google Patents

Abstract

The present invention relates to the new viral vectors derived from adenovirus, to their preparation and to their use in gene therapy. This relates more particularly to defective recombinant adenoviruses comprising two therapeutic genes, the former being a suicide gene and the latter an immunostimulatory gene or a tumor suppressor gene.

Description

ADENOVIRUS THAT COMPRISE TWO THERAPEUTIC GENES: SUICIDAL AND IMMUNOSTIMULANT The present invention relates to the new viral vectors, their preparation and their use in gene therapy. This also refers 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 a genetic information into the cell or into the affected organ. This genetic information can be introduced either in vi tro into a cell extracted from the organ, the modified cell being then reintroduced into the organism, or directly into the appropriate tissue. In this second case, there are different techniques, among which the various transfection techniques involve the DNA and DEAE-dextran complexes (Pagano et al., J. Virol. 1 (1967) 891), DNA and nuclear proteins ( Kaneda et al., Science 243 (1989) 375), DNA and lipids (Felgner et al., PNAS 84 (1987) REF: 24194 7413), the use of liposomes (Fraley et al., J. Biol. Chem. .. 55 (1980) 10431), etc. More recently, the use of viruses as vectors for gene transfer has appeared as a promising alternative for these physical transfection techniques. In this regard, different viruses have been tested for their ability to infect certain cell populations. In particular, retroviruses (RSV, HMS, MMS, etc.), HSV virus, adeno-associated viruses, and adenoviruses. Among these viruses, adenoviruses have certain interesting properties for use in gene therapy. Primarily, they have a very large host spectrum, are capable of infecting cells at rest, do not integrate into the genome of the infected cell, and have not been associated to date with the important pathologies in man. Adenoviruses are linear double-stranded DNA viruses, approximately 36 kb in size. Its genome comprises mainly an inverted, repeated sequence (ITR) at its end, an encapsidation sequence, early genes and late genes (see Figure 1). The main early genes are the El genes (Ela and Elb), E2, E3 and E4. The major late genes are the Ll to L5 genes.

Taking into account the properties of the adenoviruses mentioned above, these have already been used for the transfer of genes in vi ve. For this purpose, different vectors derived from adenoviruses have been prepared, which incorporate different genes (β-gal, OTC, α-IAT, cytokines, etc.). In each of these constructions, the adenovirus has been modified in a manner to render it incapable of replication in the infected cell. Thus, the constructions described in the prior art are the deleted adenoviruses of the El (Ela and / or Elb) regions and eventually E3 at 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, particularly effective for gene therapy applications. More particularly, the present invention stems in part from the disclosure, that it is possible to incorporate several genes of interest in the adenoviruses, and obtain an important expression of these different genes in the infected cells. The present invention also derives from the construction of adenoviral vectors capable of incorporating several therapeutic genes, under the conditions that allow their optimal expression. This is derived even from the demonstration of a synergistic effect of the vectors of the invention, linked to the coexpression, in the same target cell, of complementary therapeutic genes. The present invention thus provides viral vectors which have too advantageous therapeutic properties with a view to their use in gene or cell therapy. In particular, the vectors of the invention have very advantageous properties for use in the treatment of pathologies that present episodes of cellular hyperproliferation (cancers, restenosis, etc.). A first objective 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 coexpression of such genes in the same target cell will produce a particularly advantageous antitumor therapeutic effect, far superior to the effect obtained by means of these genes alone or separately introduced.

The therapeutic genes used in the context of the present invention can be a cDNA, a genomic DNA (gDNA), or a hybrid construct consisting, for example, of a cDNA in which one or more introns will be inserted. It can also be synthetic or semi-synthetic sequences. In a particularly advantageous manner, a cDNA or a gDNA is used. The two therapeutic genes incorporated in the adenoviral vectors according to the present invention can be arranged in different ways. These can first of all constitute a unique transcriptional entity. In this configuration, the two genes are contiguous, placed under the control of a single promoter, and give rise to a single pre-messenger RNA. This arrangement is advantageous, since it permits the use of a single transcriptional promoter. The two therapeutic genes can also be placed under the control of separate transcriptional promoters. This configuration allows to obtain higher expression levels, and to offer a better control of the expression of the genes. In this case, the two therapeutic genes can be inserted in the same orientation or in opposite orientations, in the same site of the adenovirus genome or in different sites. As a suicide gene, the genes whose expression product gives the cell a sensitivity for a therapeutic agent are preferably used. More preferably, the suicide gene is the thymidine kinase gene, whose expression product confers to mammalian cells a sensitivity to certain therapeutic agents such as ganciclovir or acyclovir. The herpes simplex virus thymidine kinase is able to phosphorylate nucleoside analogs, such as acyclovir and ganciclovir. These modified molecules can be incorporated into a strand of DNA in the course of lengthening, which results in the arrest of DNA synthesis, involving the death of the cell (F.L. Moolten, Cancer Res. 46 (1986) 5276). This strategy thus allows to specifically eliminate the cells that express the suicide gene. Furthermore, in the synthesis of DNA being the target of toxicity, only cells in the process of division are affected. More preferably, the thymidine kinase gene of human herpes virus (hTK HSV-1) is used in the context of the present invention. The sequence of this gene has been described in the literature (see mainly McKnight et al., Nucleic Acid, Res. 8 (1980) 5931). It is also possible to use derivatives of this sequence, which have a very high substrate specificity or a better kinase activity. Such derivatives can in particular be obtained by mutagenesis at the binding site level as described above (Balasubramaniam et al., J. Gen. Virol. 71 (1990) 2979; Muñir et al., JBC 267 (1992) 6584). It is also possible to use the cytokine deaminase gene, whose expression product confers on the mammalian cells a sensitivity to 5-fluoro-cytosine (5-FC) or to the nitroreductases that give mammalian cells sensitivity to nitroaromatic products (J. Biol. Chem. 266 (1991) 4126). As indicated above, it is more particularly advantageous to associate the suicide gene with an immunostimulatory or tumor suppressor gene. In this regard, the immunostimulatory gene can be any gene whose expression product is capable of stimulating the body's defenses.

Preferably, it is a gene coding for a cytokine, mainly such as a lymphokine (IL-1 to IL-12), an interferon (alpha, beta, etc.), a tumor necrosis factor, a stimulation factor of colonies (G-CSF, GM-CSF, M-CSF, SCF, etc.), etc. Still more preferably, it is the gene encoding interleukin 2 or G-CSF. Interleukin 2 is essentially synthesized by lymphocytes, in response to the presence of antigens, mainly of tumor antigens. This acts immediately on the development of the immune response to the encounter of these antigens, in particular by local activation of killer and cytotoxic T cells (NK). This lymphokine thus plays an important role in antitumor immunity. Thanks to the vectors of the present invention, it is now possible to obtain a synergistic antitumor effect resulting from a simultaneous expression, in the same tumor cell, of interleukin 2 and of a suicide gene, such as the thymidine kinase gene. The GM-CSF gene is a stimulation factor of granulocyte and macrophage colonies. This then stimulates the proliferation of these cells of immunity, and then allows to strengthen the immune defenses. The gene and cDNA of the GM-CSF gene have been described in the literature. Its coexpression in a vector of the invention with a suicide gene produces a high synergistic antitumor effect. Among the tumor suppressor genes that can be used in the context of the present invention, p53, Rb, raplA, DDC, AF and MTS genes can be more particularly mentioned. More particularly, the p53 gene or the Rb gene is used. The p53 gene codes for a nuclear protein of 53 kDa. The mutated form by deletion and / or mutation of this gene is involved in the development of most human cancers (Baker et al., Science 244 (1989) 217). Their mutated forms are equally capable of cooperating with ras oncogenes to transform murine fibroblasts. The wild-type gene coding for native p53, on the contrary, inhibits the formation of transforming foci in rodent fibroblasts transfected with various combinations of oncogenes. Recent data underscore that the p53 protein may itself be a transcription factor and stimulate the expression of other tumor suppressor genes. On the other hand, a p53 effect on the proliferation of vascular smooth muscle cells has been recently evidenced (Epstein et al., Science 151 (1994)). 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 repress the division of the cells, making them enter the resting phase. The inactive forms of the Rb gene have been set in motion in different tumors, and mainly in retinoblastomas or in mesenchymal cancers such as osteosarcomas. The reintroduction of this gene into tumor cells where it will be inactive, produces a return to the normal state and a loss of tumorigenicity (Huang et al., Science 242 (1988) 1563). Recently, it has been shown that the Rb protein, but not its mutated forms, represses the expression of the proto-oncogene c-fos, an essential gene in cell proliferation. The WAF and MTS genes and their antitumor properties have been described in the literature (Cell 75 (1993) 817; Science 264 (1994) 436). In a particularly preferred mode of operation, the invention relates to a defective recombinant adenovirus comprising a gene encoding thymidine kinase, and a tumor suppressor gene. More preferably, it relates to an adenovirus comprising a gene encoding the thymidine kinase of the herpes virus and the wild type p53 gene (Ad-TK-p53). In a particularly advantageous manner, the two genes are placed under the control of separate promoters, preferably the LTR promoter of the HSV virus. Still more preferably, the two genes are inserted at the level of the El region of the adenovirus genome. In another particularly preferred embodiment, the invention relates to a defective recombinant adenovirus comprising a gene encoding thymidine kinase and a gene encoding a lymphokine. More preferably, it relates to an adenovirus comprising a gene coding for herpes virus thymidine kinase, and a gene coding for interleukin 2 (Ad-TK-IL2) or for GM-CSF (Ad-TK-GM-CSF). In a particularly advantageous manner, the two genes are placed under the control of separate promoters, preferably the LTR promoter of the HSV virus. Still more preferably, the two genes are inserted at the level of the El region of the adenovirus genome. Regarding the transcriptional promoters used in the framework of the present invention, it can be promoters that are naturally responsible for the expression of the considered therapeutic gene, when they are capable of functioning in the infected cell. It can also be sequences of different origin (responsible for the expression of other proteins, or even synthetic). Primarily, it can be promoter sequences of mammalian, eukaryotic or viral genes. For example, it can be promoter sequences from the genome of the cell to be infected. Likewise, it can be promoter sequences from the genome of a virus, including the adenovirus used. In this regard, mention may be made, for example, of the promoters of the genes E1A, MLP, CMV, RSV, etc. In addition, these expression sequences can be modified by the addition of activation, regulatory, or tissue-specific expression sequences. On the other hand, when the inserted gene does not include expression sequences, it can be inserted into the genome of the defective virus in the downward direction of such a sequence. A preferred promoter for carrying out the vectors of the invention consists of the LTR of rous sarcoma virus (LTR-RSV). Other particularly preferred promoters are the cell-proliferating or cancerous cell-specific promoters. These promoters indeed allow to direct the therapeutic effect on a defined cell population. In a preferred embodiment of the invention, these are expression signals induced by or activated in the presence of viruses responsible or associated with the tumors. Still more preferably, an expression signal that is inducible by the Epstein-Barr virus (EBV) or the papilloma virus is used within the framework of the present invention. Epstein-Barr virus (EBV) is associated with two types of human cancers: Burkitt's lymphoma and nasopharyngeal cancer. The use of a recombinant adenovirus comprising a toxic gene under the control of an EBV-inducible promoter makes it possible to express advantageously and specifically this toxic gene in the tumor cells of the nosopharynx. In biopsies of nasopharyngeal cancers, only one nuclear antigen, EBNA1, is regularly present, which is involved in the maintenance of the viral genome in the cells infected by EBV in the latent phase, and which transactivates the BCR2 viral promoter. A particular object of the invention lies therefore in the use, for the specific expression of a gene in the cells of cancers of the nasopharynx, of a sequence that responds to EBNA1 (EBNA1-RE: EBNA1"responder element"). In particular, the invention relates to an adenovirus comprising as an expression signal a chimeric promoter comprising an EBNA1-responsive sequence fused upstream of another viral promoter, the promoter of the 1-terminal protein gene (TP1). The examples described in the present application show very well that this chimeric promoter is inducible by EBNA1. Papilloma viruses (mainly HPV 16 and 18 viruses) are responsible for 90% of cervical cancers in women, and have been identified in precancerous epithelial lesions (Riou et al., Lancet 335 (1990) 117). The product of the E6 gene leads to the formation of tumors that strongly decrease the amount of wild-type p53, an antioncogene, 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 an HPV inducible promoter (e.g. E6 protein), advantageously allows to specifically express this toxic gene in the corresponding tumor cells. It can even be inactive expression signals in normal cells, and active in tumor cells. In particular, within the framework of the present invention, the α-fetoprotein promoter (Alpert E., in Hepatocellular carcinoma, Okuda &; Peters (eds), New York, 1976, 353) or the P3 promoter of IGF-II (Sussenbach et al., Growth Regulation 2 (1992) 1), which are activated in adults, only in hepatocarcinomas. It is also possible to use promoters induced by hormones, in the case of hormone-dependent or hormone-associated tumors (breast or prostate tumor, for example). As indicated above, different configurations for the realization of the vectors of the invention can be considered. The vectors of the invention can first of all contain the genes in the form of a single transcriptional entity. In this configuration, the two genes are contiguous, placed under the control of a single promoter, and give rise to a single pre-messenger RNA. This configuration is advantageous, since it allows the use of a single transcriptional promoter to regulate the expression of 2 genes. On the other hand, 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 allows to obtain higher expression levels, and to offer a better control of the expression of the genes. In this case, the two therapeutic genes can be inserted in the same orientation or in opposite orientations, in the same site of the adenovirus genome or in different sites. Preferably, the genes are inserted, at least in part, at the level of the El, E3 or E4 regions of the adenovirus genome. When these are inserted in two different sites, it is preferred, within the framework of the invention, to use the regions El and E3 or El and E4. A particularly advantageous embodiment is that in which two therapeutic genes are inserted at the level of the El region. The examples show that this organization allows a high expression of two genes, without interference between the two. The invention thus also relates to any recombinant adenovirus comprising two genes of therapeutic interest, inserted at the level of the El region of the genome. On the other hand, the immunostimulatory gene can also have a signal sequence that directs the synthesized product in the secretion pathways of the target cell. This signal sequence may be the natural signal sequence of the immunostimulatory product, but may also be any other signal sequence, functional, or an artificial signal sequence. As indicated above, the adenoviruses of the present invention are defective, ie they are unable to replicate autonomously in the target cell. In general, the genome of the defective adenoviruses according to the present invention is devoid of at least the sequences necessary for the replication of said virus in the infected cell. These regions can be either deleted (totally or in part), either made non-functional, or substituted by other sequences, and mainly by therapeutic genes. The defective character of the adenoviruses of the invention is an important element, since it ensures the non-dissemination of the vectors of the invention after administration. In a preferred embodiment, the adenoviruses of the invention comprise the ITR sequences and a sequence that permits encapsidation, and possesses a total or partial deletion of the El gene. The inverted, repeated sequences (ITR) constitute the origin of adenovirus replication. These are located at the 3 'and 5 ends? of the viral genome (see Figure 1), from where these can be easily isolated according to the classical techniques of molecular biology known to the person skilled in the art. The nucleotide sequence of the ITR sequences of human adenoviruses (in particular Ad2 and Ad5 serotypes) is described in the literature, as well as canine adenoviruses (mainly CAVÍ and CAV2). With respect to Ad5 adenovirus for example, the left ITR sequence corresponds to the region comprising nucleotides 1 to 103 of the genome. The encapsidation sequence (also referred to as the Psi sequence) is necessary for the encapsidation of the viral DNA. This region must then be present to allow the preparation of defective recombinant adenoviruses, according to the invention. The encapsidation sequence is located in the adenovirus genome, between the left ITR (5f) and the El gene (see Figure 1). This can be isolated or artificially synthesized by the classical techniques of molecular biology. The nucleotide sequence of the encapsidation sequence of human adenoviruses (in particular Ad2 and Ad5 serotypes) is described in the literature, as well as canine adenoviruses (mainly CAVÍ and CAV2). With respect to Ad5 adenovirus, for example, the encapsidation sequence corresponds to the region comprising nucleotides 194 to 358 of the genome. More preferably, the adenoviruses of the invention comprise ITR sequences and a sequence that permits encapsidation, and possess a total or partial suppression of the El and E4 genes. In a particularly preferred embodiment, the genome of the adenoviruses according to the invention is completely or partially deleted from the El, E3 and E4 genes, and, even more preferably, from all or part of the El, E3, L5 genes. and E4. The adenoviruses of the invention can be prepared from the adenovirus of various origins. There are in fact different serotypes of adenoviruses, whose structure and properties vary a little, but which have a comparable genetic organization. In this way, the teachings described in the present application can be easily reproduced by the expert in the field for any type of adenovirus. More particularly, the adenoviruses of the invention can be of human, animal, or mixed (human and animal) origin. With respect to human rrigen adenoviruses, it is preferred to use those classes in group C. More preferably, among the different serotypes of human adenovirus, it is preferred to use adenovirus type 2 or 5 (Ad2 or T2) in the context of the present invention. Ad5). As indicated above, the adenoviruses of the invention can also be of animal origin, or possess adenovirus-derived sequences 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 French application FR 93 05954) . The Applicant has also shown that adenoviruses of animal origin are not nullly trans-complemented by adenoviruses of human origin, which eliminates any risk of recombination and of in vivo propagation, in the presence of a human adenovirus, which can lead to the formation of an infectious particle. The use of adenoviruses or regions of adenoviruses of animal origin is therefore particularly advantageous, since the risks inherent in the use of viruses as vectors in gene therapy are even more scarce. Adenoviruses of animal origin, usable within the framework of the present invention, can be of canine, bovine, murine origin (examples: Mavl, Beard et al., Virology 75 (1990) 81), sheep, swine, avian or even of ape (example: SAV). More particularly, among avian adenoviruses, serotypes 1 to 10 accessible in the ATCC may be mentioned, such as, for example, the Phelps strains (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), K-ll (ATCC VR-921) or even the strains with the reference ATCC VR-831 to 835. Among the bovine adenoviruses, the different known serotypes can be used, and mainly those available in the ATCC (types 1 to 8) under the ATCC references VR-313, 314, 639-642, 768 and 769 Mention may also be made of murine adenoviruses FL (ATCC VR-550) and E20308 (ATCC VR-528), ovine adenovirus type 5 (ATCC VR-1343), or type 6 (ATCC VR-1340); porcine adenovirus 5359), or simian adenoviruses mainly such as adenoviruses under the reference of the ATCC under the numbers VR-591-594, 941-943, 195-203, etc. Preferably, among the various adenoviruses of animal origin, adenoviruses or adenoviral regions of canine origin are used within the framework of the present invention, and especially all CAV2 adenovirus strains [Manhattan strain or A26 / 61 (ATCC VR- 800) for example]. Canine adenoviruses have been the subject of numerous structural studies. Thus, the complete restriction cards of the CAVÍ and CAV2 adenoviruses have been described in the prior art (Spibey et al., J. Gen. Virol. 70 (1989) 165), and the Ela, E3 genes as well as the sequences ITRs have been cloned and sequenced (see mainly Spibey et al., Virus Res. 14 (1989) 241; Linné, Virus Res. 23 (1992) 119, W091 / 11525). The defective recombinant adenoviruses according to the invention can be prepared in different ways. A first method consists of transfecting the DNA of the defective recombinant virus prepared in vitro (either by ligation or the plasmid form) in a competent cell line, that is to say that it possesses in the trans position all the functions necessary for the complementation of the defective virus. These functions are preferably integrated into the genome of the cell, which allows to avoid the risks of recombination, and confers increased stability for the cell line. A second procedure consists of co-transfecting the defective recombinant virus DNA prepared in vi tro into an appropriate cell line. (either by ligation, or in the form of a plasmid), and the DNA of an auxiliary virus. According to this method, it is not necessary to have a competent cell line capable of complementing all the defective functions of the recombinant adenovirus. A part of these functions is in fact complemented by the auxiliary virus. This auxiliary virus must by itself be defective, and the cell line possess in the trans position the functions necessary for its complementation. Among the cell lines which can be used mainly in the context of this second procedure, mention may be made in particular of the human embryonic kidney line 293, the KB cells, the Hela cells, MDCK, GHK, etc. (see examples). Then, the vectors that have been multiplied are recovered, purified and amplified according to the classical techniques of molecular biology. According to a variant embodiment, it is possible to prepare in vi tro, either by ligation, or in the form of a plasmid, the DNA of the defective recombinant virus that includes the appropriate deletions and the two therapeutic genes. As indicated above, the vectors of the invention advantageously possess a suppression of all or part of certain viral genes, mainly the genes El, E3, E4 and / or L5. This suppression can correspond to any type of suppression that affects the considered gene. It can be mainly the suppression of all or part of the region encoding 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 by means of appropriate restriction enzymes, then ligation, according to molecular biology techniques, as illustrated in the examples. The therapeutic genes can then be inserted into this DNA by enzymatic cleavage and then ligation, at the level of the selected regions and in the chosen orientation. The DNA obtained in this way, which then possesses the appropriate deletions and the two therapeutic genes, makes it possible to directly generate the defective recombinant adenovirus possessing said deletions and the therapeutic genes. This first variant is particularly adapted for carrying out recombinant adenoviruses, in which the therapeutic genes are placed in the form of a single transcriptional unit or, under the control of separate promoters but inserted in the same site of the genome. It is also possible to prepare the recombinant viruses in two stages, which allow the successive introduction of two therapeutic genes. In this way, the DNA of a first recombinant virus possesses the appropriate deletions (or a part of said deletions) and one of the therapeutic genes is constructed, by ligation or in the form of a plasmid. This DNA is used immediately to generate a first recombinant virus that includes such deletions and a therapeutic gene. The DNA of this first virus is then isolated and cotransfected with a second plasmid, or the DNA of a second defective recombinant virus that includes the second therapeutic gene, the appropriate deletions (portion not present on the first virus), and a region that allows the homologous recombination. This second stage thus generates the defective recombinant virus that possesses the two therapeutic genes. This preparation variant is particularly suitable for the preparation of recombinant viruses that include 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, such as those described above. The pharmaceutical compositions of the invention can be formulated with a view to administration by topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, infraocular, transdermal, etc. Preferably, the pharmaceutical composition contains pharmaceutically acceptable carriers for an injectable formulation. In particular, it may be 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, they allow the constitution of injectable solutes. The doses of the viruses used for the injection can be adapted according to different parameters, and mainly depending on the mode of administration used, the pathology in question, the gene to be expressed, or even the duration of the treatment sought. In a general manner, the recombinant adenoviruses according to the invention are formulated and administered in the form of doses between 10 and 1014 pfu / ml, and preferably 10"to 1010 pfu / ml. plaque ") corresponds to the infectious power of a virus solution, and is determined by infection of an appropriate cell culture, and is measured, generally after 5 days, by the number of plaques of infected cells. PFU titre of a viral solution are well documented in the literature The adenoviruses of the invention can be used for the treatment or prevention of numerous pathologies.These are particularly advantageous for the treatment of hyperproliferative pathologies (cancers, restenosis, etc.). ), by direct injection at the level of the site in question In this regard, the present invention also relates to a method for the destruction of pro cells. liferatives, which comprises the infection of said cells or a part of them with an adenoviral vector as defined above. In the case where the suicide gene is a gene that confers a sensitivity to a therapeutic agent, the method of destruction according to the invention immediately comprises the treatment of the cells by said therapeutic agent. For putting this method into operation, the invention also aims at products comprising a recombinant adenovirus as defined above, in which the suicide gene is a gene that confers a sensitivity to a therapeutic agent; and said therapeutic agent, as a combination product for simultaneous, separate or stepwise use, for the treatment of hyperproliferative pathologies. More particularly, the suicide gene is a thymidine kinase gene and the therapeutic agent is ganciclovir or acyclovir or an analogue. The present invention will be more fully described with the help of the following examples, which should be considered as illustrative and not as limiting.

Description of the Figures Figure 1: Genetic organization of Ad5 adenovirus. The complete sequence of Ad5 is available based on the data and allows the expert in the art to select or create any restriction site, and thus isolate any region of the genome.

Figure 2: Restriction diagram of adenovirus CAV2 Manhattan strain (from Spibey et al mentioned above).

Figure 3: Representation of the pONTtk vector Figure 4: Representation of the vector pRSVtk General Molecular Biology Techniques The methods classically used in molecular biology, such as the preparative extractions of plasmid DNA, the centrifugation of plasmid DNA in cesium chloride gradient, the electrophoresis in agarose or acrylamide gels, the purification of DNA fragments by electroelution, extractions of proteins with phenol or with phenol-chloroform, the precipitation of DNA in saline medium with ethanol or with isopropanol, the transformation in Escheri chi to col i, etc ... are well known to those skilled in the art and are abundantly described in Literature [Maniatis T. et al., "Molecular Cloning, a Laboratory Manual ", Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., 1982; Ausubel F. M. et al. (eds), "Current Protocols in Molecular Biology", John Wiley & Sons, New York, 1987], Plasmids of the type pBR322, pUC and phages of the M13 series are of commercial origin (Bethesda Research Laboratories). For ligatures, the DNA fragments can be separated according to their size by electrophoresis in agarose or acrylamide gels, extracted with phenol or with a mixture of phenol / chloroform, precipitated with ethanol and then incubated in the presence of DNA- T4 phage ligase (Biolabs) according to the supplier's recommendations.

The filling of the prominent 5 'ends can be effected by the Klenow fragment of E DNA polymerase I. col i (Biolabs) according to the supplier's specifications. The destruction of the 3 'protruding ends is carried out in the presence of the phage T4 DNA polymerase (Biolabs) used according to the manufacturer's recommendations. The destruction of the 5 'protruding ends is effected by a treatment provided by the nuclease SI. The directed mutagenesis in vi tro by the synthetic oligodeoxynuclees, can be carried out according to the method developed by Taylor et al. [Nucleic Acids Res. 1_3 (1985) 8749-8764] using the equipment distributed by Amersham. Enzymatic amplification of DNA fragments by the technique called PCR [Polymerase-catalyzed Chain Reaction, Saiki R.K. et al., Science 230 (1985) 1350-1354; Mullís K.B. and Faloona F.A., Meth. Enzym. 155 (1987) 335-350] can be performed using a "DNA thermal cycler" (Perkin Elmer Cetus) according to the manufacturer's specifications. The verification of the nuclee sequences can be carried out by the method developed by Sanger et al. [Proc. Nati Acad. Sci. USA, 74 ^ (1977) 5463-5467] using the equipment distributed by Amersham.

Cell Lines Used In the following examples, the following cell lines have been or may be used: - Human embryonic kidney line 293 (Graha et al., J. Gen. Virol. 36 (1977) 59).

This line contains mainly, integrated in its genome, the left part of the genome of human adenovirus Ad5 (12%). - Line of human cells KB: From a human epidermal carcinoma, this line is accessible in the ATCC (ref CCL17) as well as the conditions that allow its cultivation. Line of human cells Hela: From a human epithelium carcinoma, this line is accessible in the ATCC (ref CCL2) as well as the conditions that allow its cultivation. Canine MDCK cell line: The culture conditions of MDCK cells have been described mainly by Macatney et al., Science 44 (1988) 9. DBP6 gm cell line (Brough et al., Virology 190 (1992) 624). This line is composed of Hela cells that include the E2 gene of adenovirus under the control of MMTV LTR.

EXAMPLES 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.

These adenoviruses have been constructed by homologous recombination between a plasmid that includes the left part of Ad5 adenovirus, the two therapeutic genes and a region of Ad5 adenovirus (corresponding to protein IX) and the DNA of a defective adenovirus that includes different deletions. 1. Construction of the pONT-tk vector 1. 1. Construction of plasmid p7tkl This example describes the construction of plasmid p7tkl containing the open reading phase of the t31 gene of 1131 base pairs (ATG 114-116 and the stop codon TGA 1242-1244), inserted in a multiple site of cloning. The BglII-NcoI fragment containing the thymidine kinase (tk) gene of the herpes simplex virus type 1, has been isolated from the plasmid pHSV-106 (marketed by Gibco BRL), repaired by the action of the klenow fragment and then inserted in the Smal site of the plasmid pGEM7zf (+) (marketed by Promega). The Smal and BglII sites are destroyed after this stage, the Ncol site is conserved. The obtained plasmid has been designated p7tkl 1. 2. Construction of plasmid pONTl This example describes the construction of a plasmid containing a chimeric promoter consisting of a sequence necessary for transactivation by the EBNA1 antigen and the TP1 promoter of the EBV virus. The EcoRI (7315) -Smal fragment (8191) of the EBV virus has been isolated from strain B95-8.

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 has been fused immediately to the fragment Nrul (166 2 1) -PstI (166 559) of EBV B95-8 (the site PstI has been digested by polymerase T4), which contains the TP1 promoter. The chimeric promoter obtained in this way was then inserted into the multiple cloning site of plasmid pBluescript II SK. The obtained plasmid has been designated pONTl. 1. 3. Construction of plasmid pONTtk Plasmid pONTtk includes the thymidine kinase gene of herpes simplex virus (tk) cloned in the plasmid p7tkl, under the control of the chimeric promoter EBNAl-RE / TPl cloned in the pONTl plasmid. To construct this plasmid, the BamHI-XhoI fragment of pONTl, which contains the chimeric promoter transactivated by EBNA-1 and EBNA-2, and the Xhol-Clal fragment of p7tkl, which contains the open reading phase of tk, have been cloned in the BamHl (478) and Clal (4550) sites of the pAd.RSVßgal plasmid. Plasmid pAd.RSVßGal contains, in the orientation 5'- > 3 ', the PvuII fragment corresponding to the left end of Ad5 adenovirus comprising: the ITR sequence, the origin of replication, the encapsidation signals and the E1A amplifier; - the gene coding for ß-galactosidase under the control of the RSV promoter (from the Rous sarcoma virus), a second fragment of the Ad5 adenovirus genome, which allows homologous recombination between the plasmid pAd.RSVßGal and the adenovirus dl324. Plasmid pAd.RSVßGal has been described by Stratford-Perricaudet et al. (J. Clin.Invest.90 (1992) 626). All cloning sites are conserved. The obtained plasmid has been designated pONTtk (figure 3). 2. Construction of the plasmid pQNTtkCMVp53 This example describes the construction of a vector that includes the left part of Ad5 adenovirus (comprising the left ITR, the encapsidation region and the beginning of the El region), the tk gene under the control of the ONT promoter, the low p53 gene the control of the CMV promoter and a second fragment of the Ad5 genome (pIX protein) that allows homologous recombination with a view to the generation of the recombinant adenovirus (see example 1.3). This plasmid has been constructed from the pONTtk plasmid, by insertion, downstream of the tk gene and in the same orientation, of a fragment that includes the p53 gene under the control of the CMV promoter and followed by the polyadenylation site of the SV40 virus. More precisely, the inserted fragment comprises: a promoter region of viral origin corresponding to the early cytomegalovirus promoter (CMV). This region is surrounded in the vector of unique restriction sites EcoRI-Sphl for binding CMV / pONTtk and BamHl for CMV / p53 binding. The presence of unique sites flanking the promoter region allows the CMV region to be replaced by any other promoter. A second series of vectors is obtained in this way, in which the p53 gene is placed under the control of an inducible promoter: the promoter of the metallothionine, inducible by heavy metals (cadmium and zinc). a sequence of 1173 base pairs corresponding to the cDNA encoding the mouse p53 protein, in its wild form (Zakut-Houri et al., Natura 36 (1983) 594). In this construction, the suppressor gene is in the form of cDNA, ie devoid of introns. This allows mainly to reduce the size of the vector. On the other hand, it has been verified that the levels of expression obtained are comparable in the presence or absence of introns. - the polyadenylation signal of the SV40 virus late genes, which correspond to a very efficient polyadenylation signal. Two unique SalI and HindIII restriction sites are located downstream of the polyadenylation signal. The obtained vector has been designated pONTtkCMVp53. It is understood that the insertion of said fragment can be effected in a similar manner in the reverse orientation, leading to a plasmid in which the tk gene and the p53 gene are in opposite orientations (pONTtkCMVp53inv). 3. Construction of recombinant adenoviruses 3. 1. Construction of a deleted recombinant adenovirus in the El region, which includes the two therapeutic genes inserted in the same orientation, at the level of the El region. The vector pONTtkCMVp53 has been linearized and cotransfected with an adenoviral vector deficient in the El gene, in the helper cells (line 293) that include in the trans position the functions encoded by the El regions (E1A and E1B) of the adenovirus. More precisely, the adenovirus AdONTtkCMVp53,? El is obtained by homologous recombination in vi vo between the adenovirus Ad.RSVßgal (see Stratford-Perricaudet et al. Cited above) and the vector pONTtkCMVp53, according to the following protocol: the plasmid pONTtkCMVp53, linearized with Xmnl, and Ad-RSVßgal adenovirus, lined with Clal enzyme, are cotransfected in line 293 in the presence of calcium phosphate, to allow homologous recombination. The recombinant adenoviruses generated in this way are then selected by plaque purification. After isolation, the DNA of the recombinant adenovirus is amplified in the 293 cell line, which leads to a culture supernatant containing the recombinant, non-purified defective adenovirus, which contains a titer of about -pfu / ml. The viral particles are generally purified by centrifugation on cesium chloride gradient according to known techniques (see mainly Graham et al., Virology 52 (1973) 456). The Ad-ONT-tkCMVp53 adenovirus can be preserved at -80 ° C in 20% glycerol. 3. 2. Construction of a deleted recombinant adenovirus in the El and E3 region, which includes the two therapeutic genes inserted in the same orientation, at the level of the El region. The vector pONTtkCMVp53 has been linearized and cotransfected with an adenoviral vector deficient in the genes El and E3, in l.ss helper cells (line 293) including in trans position the functions encoded by the El regions (E1A and E1B) of the adenovirus. More precisely, the adenovirus Ad-ONTtkCMVp53,? El,? E3 is obtained by homologous recombination in vi vo between the mutant adenovirus Ad-dll324 (Thimmappaya et al., Cell 31 (1982) 543) and the vector pONTtkCMVp53, in accordance to the following protocol: the plasmid pONTtkCMVp53, linearized with Xmnl, and the adenovirus Ad-dll324, linearized by the Clal enzyme, are cotransfected in line 293 in the presence of calcium phosphate, to allow homologous recombination. The recombinant adenoviruses generated in this way are then selected by plaque purification. After isolation, the DNA of the recombinant adenovirus is amplified in the 293 cell line, which leads to a culture supernatant containing the unpurified recombinant defective adenovirus, which has a titer of approximately 10i0 pfu / ml. The viral particles are generally purified by centrifugation on cesium chloride gradient according to known techniquesEAR (see mainly Graham et al., Virology 52 (1973) 456). The adenovirus Ad-ONT-tkCMVp53,? El,? E3 can be conserved at -80 ° C in 20% glycerol. 3. 3. Construction of adenoviruses in which the tk and p53 genes are placed in opposite orientations. According to the protocols described in the examples 3.1. and 3.2. above, adenoviruses in which the tk and p53 genes are placed 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 LTR promoter of the RSV virus, and the p53 gene under the control of the CMV promoter.

These adenoviruses have been constructed by homologous recombination between a plasmid that includes the left part of Ad5 adenovirus, the two therapeutic genes and a region of Ad5 adenovirus. (corresponding to protein IX) and the DNA of a defective adenovirus that contains different suppressions. 1. Construction of the vector pRSVtk This plasmid has been constructed from the plasmid pONTtk (example 1.1.), By substitution of the transactivatable promoter by EBNA1 by means of the RSV LTR promoter. For this, the RSV promoter has been isolated in the form of a BamHI-SalI fragment from the plasmid pAd.RSV.ßgal (Stratford-Perricaudet et al J. Clin.Resh 90 (1992) 626), and then cloned into the sites BamHl (478) and Salí (1700) of the plasmid pONTtk. The resulting plasmid has been designated pRSVtk (Figure 4). 2. Construction of plasmid pRSVtkCMVp53 This example describes the construction of a vector that includes the left part of Ad5 adenovirus (comprising the left ITR, the encapsidation region and the beginning of the El region), the tk gene under the control of the RSV promoter, the p53 gene under the control of the CMV promoter, and a second fragment of the Ad5 genome (pIX protein) that allows homologous recombination with a view to the generation of the recombinant adenovirus (see example 2.3.). This plasmid has been constructed from the plasmid pRSVtk, by insertion, downstream of the tk gene, of a fragment including the p53 gene under the control of the CMV promoter, and followed by the polyadenylation site of the SV40 virus. More precisely, the inserted fragment comprises: a promoter region of viral origin corresponding to the y cytomegalovirus (CMV) promoter. This region is surrounded in the vector of unique restriction sites EcoRI-Sphl for CMV / pONTtk binding and BamHl for CMV / p53 binding. The presence of unique sites flanking the promoter region allows the CMV region to be replaced by any other promoter. A second series of vectors obtained in this way, in which the p53 gene is placed under the control of an inducible promoter: the promoter of the etalothionin, inducible by heavy metals (cadmium and zinc). a sequence of 1173 base pairs corresponding to the cDNA encoding the mouse p53 protein, in its wild form (Zakut-Houri et al., Nature 36 (1983) 594). In this construction, the suppressor gene is in the form of cDNA, ie devoid of introns. This allows mainly to reduce the size of the vector. On the other hand, it has been verified that the levels of expression 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 efficient polyadenylation signal. Two unique SalI and HindIII restriction sites are located downstream of the polyadenylation signal.

The obtained vector has been designated pRSVtkCMVp53. 3. Construction of recombinant adenoviruses 3. 1. Construction of a deleted recombinant adenovirus in the El region, which includes the two therapeutic genes inserted in the same orientation, at the level of the El region. This adenovirus is constructed according to the protocol described in Example 1 (3.1.). The adenovirus Ad-RSV-tkCMVp53,? The one obtained in this way can be stored at -80 ° C in 20% glycerol. 3. 2. Construction of a deleted recombinant adenovirus in the El and E3 regions, which includes the two therapeutic genes inserted in the same orientation, at the El region level. This adenovirus is constructed according to the protocol described in Example 1 (3.2. ). The Ad-RSV-tkCMVp53 adenovirus, E1, E3 obtained in this way, 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 have been constructed by homologous recombination between a plasmid that includes the left part of Ad5 adenovirus, the two therapeutic genes and a region of adenovirus Ad5 (corresponding to protein IX) and the DNA of a defective adenovirus that includes different suppressions. 1. Construction of plasmid pRSVtkRSVIL-2 This example describes the construction of a vector that includes the left part of Ad5 adenovirus (comprising the left ITR, the encapsidation region and the beginning of the El region), the tk gene under the control of the RSV promoter, the gene of the interleukin-2 under the control of the RSV promoter and a second fragment of the Ad5 genome (pIX protein) that allows homologous recombination with a view to the generation of the recombinant adenovirus (see example 3.2.).

This plasmid has been constructed from the pRSVtk plasmid (see example 2), by insertion, downstream of the tk gene, of a fragment possessing the interleukin-2 gene under the control of the RSV promoter, and followed by the site Polyadenylation of the SV40 virus. More precisely, the inserted fragment comprises: - the LTR promoter of the RSV virus, isolated in the form of a BamHI-SalI fragment from the plasmid pAd.RSV.ßgal (Stratford-Perricaudet et al., J. Clin. 90 (1992) 626); - a sequence corresponding to the cDNA encoding human interleukin-2 (EP91 539). In this construction, the therapeutic gene is in the form of cDNA. - the polyadenylation signal of the late genes of the SV40 virus, which corresponds to a very efficient polyadenylation signal. Two unique SalI and HindIII restriction sites are located downstream of the polyadenylation signal. The obtained vector has been designated pRSVtkRSVIL-2 2. Construction of recombinant adenoviruses Two types of recombinant adenoviruses are constructed according to the protocol described in example 1 or 2. These adenoviruses include the two therapeutic genes inserted in the same orientation, at the level of the El region and present a deletion in the El region or regions. The and E3. Ad-RSV-tkRSVIL-2,? El and Ad-RSV-tkRSVIL-2,? E1,? E3 adenoviruses obtained in this way can be stored at -80 ° C in 20% glycerol.

Example 4. Construction of defective recombinant adenoviruses including the TK gene and the gene encoding the granulocyte and macrophage colony stimulation factor (GM-CSF).

Following the protocols described in the preceding examples, the defective adenoviruses including the tk gene (under the control of the ONT or RSV promoter for example) and the GM-CSF gene under the control of their own promoter or the RSV promoter can be constructed. mainly. For this, an intermediate vector that has the two genes can be constructed from the vector pONTtk or pRSVtk by inserting, in the same orientation or in the reverse orientation, a fragment that possesses the gene of GM-CSF under the control of the promoter. The gene coding for GM-CSF and the constructions containing it, has been described mainly in the application WO86 / 03225.

Example 5. Construction of defective recombinant adenoviruses comprising two genes of interest, one inserted at the level of the El region and the other inserted at the level of the E3 region.

These adenoviruses are constructed by homologous recombination between a DNA of a defective first virus that includes the first gene inserted at the level of the El region, and the DNA of a second defective adenovirus that includes the second gene inserted at the level of the E3 region. 1. Construction of a defective virus that includes the gene inserted at the level of the E3 region.

This virus is constructed from adenovirus Addl324 (Thimmappaya et al., Cell 31 (1982) 543). This virus has a deletion at the level of the El region and E3 (deleted Xbal-EcoRI fragment). The DNA of the Addl324 virus has been isolated and purified. This DNA is then cut with the enzymes Xbal and EcoRI. An Xbal-EcoRI p -induced fragment extracted from the plasmid pRSVtk which includes the sequence encoding the thymidine kinase, under the control of the RSV promoter, and then inserted at the level of the sites in the Addl324 DNA, open, as described previously. The DNA obtained in this way thus includes a deletion at the level of the El region and the TK gene inserted at the level of the E3 region. 2. Construction of adenoviruses that include both genes.

The DNA of the recombinant virus prepared above and the DNA of a recombinant adenovirus that includes an immunostimulatory or tumor suppressor gene, inserted at the level of the El region, and linearized with BamHI, are cotransfected in line 293 in the presence of calcium phosphate. , to allow homologous recombination. The recombinant adenoviruses generated in this way are then selected by plaque purification.

After isolation, the DNA of the recombinant adenovirus is amplified in the 293 cell line, which leads to a culture supernatant containing the recombinant, non-purified defective adenovirus, which has a titer of approximately 101 'pfu / ml. 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).

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (28)

1. A defective recombinant adenovirus, characterized in that it comprises two therapeutic genes, the first being a suicide gene and the second an immunostimulatory gene or a tumor suppressor gene.

2. The adenovirus according to claim 1, characterized in that the two genes constitute a unique transcriptional entity, under the control of a single promoter.

3. The adenovirus according to claim 1, characterized in that the two genes are under the control of separate transcriptional promoters.

4. The adenovirus according to claim 3, characterized in that the two genes are inserted in the same orientation.

5. The adenovirus according to claim 3, characterized in that the two genes are inserted in opposite orientations.

6. The adenovirus according to any of claims 1 to 5, characterized in that the two therapeutic genes are inserted in the same site of the genome, preferably at the level of the El, E3 or E4 regions.

7. The adenovirus according to claim 6, characterized in that the two genes are inserted at the level of the El region.

8. The adenovirus according to any of claims 1, 3, 4 and 5, characterized in that the two therapeutic genes are inserted at different sites of the genome.

9. The adenovirus according to claim 8, characterized in that one of the genes is inserted at the level of the El region and the other at the level of the E3 or E4 region.

10. The defective recombinant adenovirus according to any one of claims 1 to 9, characterized in that it comprises the ITR sequences, a sequence that allows encapsidation, and because it includes a deletion of all or part of the El and E4 genes.

11. The adenovirus according to claim 10, characterized in that it comprises the ITR sequences, a sequence that allows encapsidation, and because it includes a deletion of all or part of the El, E3 and E4 genes.

12. The adenovirus according to any of claims 1 to 11, characterized in that its genome is totally or partially deleted from the El, E3, L5 and E4 genes.

13. The adenovirus according to claim 1, characterized in that it is of human, animal or mixed origin.

14. The adenovirus according to claim 13, characterized in that the adenoviruses of human origin are chosen from those classes in group C, preferably between adenoviruses of type 2 or 5 (Ad2 or Ad5).

15. The adenovirus according to claim 13, characterized in that the adenoviruses of animal origin are chosen from adenoviruses of canine, bovine, murine, ovine, porcine, avian and simian origin.

16. The adenovirus according to claim 1, characterized in that the suicide gene is a thymidine kinase gene, preferably the thymidine kinase gene of the herpes virus HSV-I.

17. The adenovirus according to claim 1, characterized in that it includes a gene coding for thymidine kinase and a tumor suppressor gene.

18. The adenovirus according to claim 1, characterized in that it includes a gene coding for thymidine kinase and a gene coding for a lymphokine.

19. The defective recombinant adenovirus, characterized in that it comprises a gene coding for the thymidine kinase of the herpes virus, and the wild type p53 gene (Ad-TK-p53).

20. The defective recombinant adenovirus, characterized in that it comprises a gene coding for the thymidine kinase of the herpes virus, and a gene coding for interleukin-2 (Ad-TK-IL2).

21. The defective recombinant adenovirus, characterized in that it comprises a gene coding for thymidine kinase of the herpes virus, and a gene coding for GM-CSF (Ad-TK-GM-CSF).

22. The adenovirus according to any of claims 2 or 3, characterized in that the transcriptional promoter (s) are chosen from mammalian, eukaryotic or viral promoters.

23. The adenovirus according to any of the preceding claims, characterized in that the immunostimulatory gene comprises a signal sequence that directs the synthesized therapeutic product, in the secretion pathways of the target cell.

24. The pharmaceutical composition, characterized in that it comprises at least one defective recombinant adenovirus according to any of claims 1 to 23.

25. The pharmaceutical composition according to claim 24, characterized in that it comprises a pharmaceutically acceptable carrier for an injectable formulation.

26. The products, characterized in that they comprise: - one or more recombinant adenoviruses as defined according to any one of claims 1 to 23, wherein the suicide gene is a gene that confers a sensitivity to a therapeutic agent, and, - said therapeutic gene as a combination product for simultaneous, separate or alternating use over time, for the treatment of hyperproliferative pathologies.

27. The product according to claim 26, characterized in that the suicide gene is a thymidine kinase gene, and the therapeutic agent is ganciclovir or acyclovir or an analogue.

28. The defective recombinant adenovirus, characterized in that it comprises two genes of therapeutic interest, inserted at the level of the El region of the genome.