KR19990067174A - Uses of G-EX Protein for Cancer Treatment - Google Patents

Abstract

A therapeutic use of nucleic acids encoding all or part of the protein GAX or variants thereof, especially in the treatment of cancer.

Description

Uses of G-EX Protein for Cancer Treatment

The present invention relates to a novel cancer treatment method. More specifically, the present invention provides for cancer prevention by blocking cell proliferation that may be uncontrolled in tumor cells expressing tumorigenic genes such as mutated ras genes or in cells lacking tumor suppressor genes such as p53. To a method of treatment. The present invention further relates to the use of the vector in gene therapy to enable the latter modulation, and to pharmaceutical compositions containing the vector.

The product of the Lars gene, commonly called the p21 protein, has shown an important role in controlling cell division in all eukaryotes studied. When these proteins are specifically modified, they lose their normal control and become tumorigenic. Thus, many of the human tumors are associated with the presence of modified Lars genes. Likewise, overexpression of this p21 protein results in deregulation of cell proliferation.

In a normal cellular environment, the proliferation of these tumorigenic genes will probably be checked, at least in part, by the generation of so-called tumor suppressor genes such as p53 and Rb. Thus, the p53 protein, at least the wild type of the protein, is a transcription factor that negatively regulates proliferation and cell division, and in some situations can cause death (Yonish-Rouach et al., Nature, 352, 345-347, 1991). . Assuming that this property is evident in stressful situations where the integrity of cellular DNA is threatened, p53 has been proposed as the "guardian of the genome." However, certain phenomena interfere with these cells' self-regulating mechanisms and promote the development of neoplastic states. One such case is a mutation of a tumor suppressor gene. Thus, the inactivated form of Rb gene has been associated with various tumors, especially mesenchymal cancers such as retinoblastoma, or osteosarcoma, and the presence of mutated p53 in about 40% of all types of human tumors is known. Montenarh, Oncogene, 7, 1673-1680, 1992; Oren, FASEB J., 6, 3169-3176, 1992; Zambetti and Levine, FASEB J., 7, 855-865, 1993.

In conclusion, the identification of biological compounds that can impede this type of deregulation of cell proliferation and / or can compensate for this type of deficiency of tumor suppressor genes is an important concern in carcinogenic therapy.

The present invention has been rigorously partially derived from evidence that GAX proteins constitute an effective inhibitor of cell proliferation induced by Ras proteins. In particular, the present invention has been made from evidence that uncontrolled cell proliferation in tumor cells can be restored by expressing GAX protein in the cells.

GAX protein is a protein consisting of 303 amino acids. Its sequence was characterized and its cDNA was cloned (Gorski et al., Mol, Cell. Biol. 1993, 6, 3722-3733). This gax (growth arrest specific homeobox) gene belongs to the homeotic gene family. Such genes encode transcriptional factors containing matched sequences (or homeodomains) that recognize specific regions of DNA (or homeoboxes). Gehring et al. Cell, 78; 211-223, 1994. The homeodomain of the rat GAX protein is located between the 185th and 245th amino acids. This gene appears to control G0 / G1 metastasis in the cell cycle and thus has similar characteristics to the gas and Gadd genes. Thus, gax mRNA levels in rat CMLV are lowered to factor 10 2 hours after PDGF exposure (Gorski et al., Mol. Cell. Biol. 1993, 6, 3722-3733). Thus, expression of the gax gene is inhibited during the cleavage-producing response of CMLV. This study concludes that endovascular hyperplasia caused by carotid artery inflammation in rats is associated with a high decrease in gax expression in vivo (Weir et al. J. Biol. Chem. 1995, 270, 5457). -5461).

The expression of the gax gene has been demonstrated in the cardiovascular system, especially the heart and the aorta, and so far the use of this gene in gene therapy has been limited to cells that naturally express it.

Unexpectedly, we have found it beneficial to promote the inhibitory activity of GAX proteins on cell proliferation in tumor cells, ie cells which do not endogenously express the gax gene.

The present invention thus provides a novel and very effective method of treating tumors associated with deregulation of cell proliferation, such as non-small cell lung tumors, pancreatic and colon carcinomas, osteosarcomas and the like.

The present invention also describes very efficacious systems that can deliver such compounds directly to tumors in vivo to inhibit cancer progression.

Accordingly, a first object of the present invention is the use of GAX proteins or variants thereof in the manufacture of pharmaceutical compositions for the treatment of cancer.

More particularly, the present invention relates to the use of one or more nucleic acid sequences encoding all or part of a GAX protein or variant thereof in the manufacture of a pharmaceutical composition for treating cancer.

As indicated above, the nucleic acid sequence will be a gene encoding all or part of a GAX protein or variant thereof. For the purposes of the present invention, the term variant refers to all variants, fragments or peptides having one or more biological properties of GAX, as well as all homologs of GAX obtained from other species. These fragments and variants are obtainable by any technique known to those of skill in the art and, in particular, by their genetic and / or chemical and / or enzymatic modifications, or by hybridization or expression cloning, to their biological activity. By selecting the variant according to the invention. Such genetic modifications include inhibition, deletion, mutation, and the like.

The nucleic acid sequence used may encode all or part of a rat GAX protein or variant thereof.

The gene used for the purpose of the present invention is preferably a gene encoding rat GAX protein or human homolog thereof. More preferably, it is cDNA or gDNA.

Nucleic acid sequences of the present invention may themselves be injected into the treatment site or directly incubated with the cells to be destroyed or treated. In fact, it has been described that the original nucleic acid can penetrate cells without a specific vector. However, in the present invention, it is preferable to use a dosage vector that can enhance (i) cell penetration efficiency, (ii) targeting, (iii) extracellular and intracellular stability.

Various types of vectors can be used. It can be a virus or a vector other than a virus.

The vector according to the invention may thus be a factor other than a virus which can facilitate the delivery and expression of the nucleic acid sequence of the invention in prokaryotic or eukaryotic cells. Chemical or biochemical vectors are particularly beneficial replacements for natural viruses because of the lack of theoretical limitations on convenience, certainty and also on the size of the DNA to be transfected.

Such synthetic vectors have two important functions: to compress the nucleic acid to be transfected and to facilitate the passage of the nucleic acid to both the plasma membrane and, optionally, the nuclear membranes, as well as the attachment of the nucleic acid to the cell. In order to offset the diionic nature of the nucleic acid, all non-viral vectors have a polyionic charge.

Among the synthetic vectors developed are cationic polymers or cationic lipids or lipofectants of the polylysine (LKLK) n, (LKKL) n, polyethyleneimide and DEAE-dextran types. These vectors have the property of concentrating DNA and facilitating association with cell membranes. Among these, lipopolyamine (lipopeptamine, transfectam, etc.) and various cationic or neutral lipids (DOTMA, DOGS, DOPE, etc.) or liposomes can be mentioned. More recently, the concept of receptor-mediated targeting transfection has been developed, which concentrates DNA by cationic polymers while catalyzing ligands and cationics of membrane receptors that are preferably grafted on the surface of the cell type. The principle of directing the attachment of the complex to the membrane by chemical coupling between the polymers is used. Targeting of receptors for transferrin or insulin, or receptors for neglected allo glycoproteins, has been described.

Nucleic acid sequences used in the present invention can be formulated for administration via the topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular or transdermal routes. Preferably in injectable form. It is therefore admixable with all pharmaceutically acceptable excipients for injectable formulations, in particular for formulations directly injectable to the site to be treated. It may specifically be an isotonic sterile liquid or a dry, especially lyophilized composition, which may optionally be made into an injectable solution by the addition of sterile water or physiological saline. Injecting nucleic acid sequences directly into a patient's tumor is advantageous because it can focus therapeutic effects on the infected tissue. The amount of the nucleic acid sequence used may be adjusted according to various parameters, and in particular, depending on the vector used, the mode of administration, the relevant etiology or the desired duration of treatment.

In order to carry out the invention, it is most particularly advantageous to use defective recombinant viruses.

Thus, it is possible to use adenoviruses, herpes viruses, retroviruses and more recently adeno-associated viruses. It has been found that these viruses are particularly efficient in terms of transfection.

A second object of the present invention is therefore the use of a defective recombinant virus containing at least one insert gene encoding all or part of a GAX protein or variant thereof. The present invention also encompasses the use of this virus in the treatment of cancer. In the vectors of the invention, the inserted gene and / or the existing gene consists of a complementary DNA (cDNA) or genomic DNA (gDNA) fragment, or a cDNA into which, for example, one or more introns can be inserted. Hybrid constructs. It may also be a synthetic or semisynthetic sequence.

In general, the insert gene also includes sequences that allow expression in infected cells. This sequence may be a sequence that is naturally responsible for the expression of a gene when the sequence can function in an infected cell. It may also be a sequence of different sources (of other proteins, or synthetic proteins, which may be responsible for expression). In particular, it may be a prokaryotic or viral gene sequence or an inducing sequence, which stimulates or inhibits the transcription of a gene in a specific manner or in another and inducible or otherwise. For example, it may be a promoter sequence obtained from the genome of the cell to be infected, or from the genome of the virus, in particular the adenovirus genes ElA and MLP, CMV and RSV-LTR promoters and the like. Among the prokaryotic promoters are also ubiquitous promoters (HPRT, Vinemtin, Actin, Tubulin, etc.), promoters for intermediate filaments (desmin, neurofilament, keratin, GFAP, etc.), promoters for therapeutic genes (MDR, CFTR and factor VIII types) Etc.), a tissue-specific promoter (a promoter for actin of smooth muscle cells), a promoter that is preferentially activated upon cell division, or a promoter (steroid hormone receptor, retinoic acid receptor, etc.) in response to stimulation. In addition, these expression sequences can be modified by adding activation or regulatory sequences and the like. However, if the insert gene does not contain an expression sequence, it can be inserted downstream of the expression sequence into the genome of the defective virus.

In addition, the insert gene may generally include a signal sequence that directs the polypeptide synthesized in the secretory pathway of the target cell upstream of the coding sequence. This signal sequence may be a native signal sequence of GAX, but may be any other functional signal sequence (eg, a sequence of thymidine kinase gene) or an artificial signal sequence.

Viruses according to the invention are defective, that is, they cannot spontaneously replicate in target cells. In general, the genome of a defective virus used in the present invention thus lacks at least the sequence necessary for replication of the virus in infected cells. This region can be removed (completely or partially), made nonfunctional, or replaced with other sequences, particularly with insertion genes. Nevertheless, it is desirable for the defective virus to possess the genomic sequence necessary for capsid formation of the viral particles.

There are various adenovirus serotypes that differ somewhat in structure and properties. Among these serotypes, it is preferred in the present invention to use type 2 or 5 human adenoviruses (Ad 2 or Ad 5) or human source adenoviruses (see WO 94/26914). Adenoviral from sources of murine (eg, MAV1, Beard et al., Virology 75 (1990) 81), sheep, swine, avian or apes (eg SAV). Preferably the adenovirus of animal origin is canine adenovirus, or more preferably CAV2 adenovirus (eg Manhattan strain or A26 / 61 (ATCC VR-800)). Preferably human or dog adenoviruses or mixed source adenoviruses are used in the present invention.

Preferably, the defective adenovirus of the present invention comprises an ITR and a corresponding nucleic acid, the sequence forming the capsid. More preferably, at least the El region in the genome of the adenovirus of the present invention is nonfunctional. The viral gene contemplated can be made nonfunctional by any technique known to those skilled in the art, in particular by total inhibition, substitution or partial deletion, or by adding one or more bases into the gene under consideration. Such modifications can be obtained in vitro or in situ (for isolated DNA), for example by genetic engineering techniques or by treatment with mutagenic agents. Other regions, in particular E3 (WO95 / 02697), E2 (WO95 / 28938), E4 (WO94 / 28152, WO94 / 12649, WO95 / 02697) and L5 (WO95 / 02697) can also be modified. According to a preferred embodiment, the adenovirus according to the invention has deletions in the El and E4 regions. According to another preferred embodiment, there is a deletion where the sequence encoding the E4 region and GAX is inserted in the El region. See FR94 / 13355. In the virus of the present invention, the deletion in the El region is preferably extended to the 455th to 3329th nucleotides of the sequence of adenovirus Ad5.

Defective recombinant adenoviruses according to the invention can be prepared by any technique known to those skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3 (1984) 2917). In particular, it can be produced by homologous recombination between the plasmid carrying the DNA sequence and the adenovirus, in particular. Homologous recombination occurs after simultaneous transfection of the adenovirus and plasmid into a suitable cell line. The cell line used should preferably contain (i) a sequence capable of transduction by said elements, and (ii) a sequence that can compensate for a portion of the defective adenovirus genome, preferably in one piece to avoid the risk of recombination. do. Examples of cell lines include the human fetal kidney system 293 (Graham et al., J. Gen. Virol, 36 (1977) 59), or internationally, which contains in particular the left hand portion of the genome of Ad5 adenovirus (12%) integrated within the genome. And cell systems capable of supplementing the E1 and E4 functions as described in detail in applications WO94 / 26914 and WO9502697.

The multilayered adenovirus is then recovered and purified according to conventional molecular biology techniques.

For adeno-associated virus (AAV), it is a small DNA virus that integrates in a stable, site-specific manner within the genome of the infecting cell. The virus can infect a wide range of cells without inducing action on cell proliferation, morphology or differentiation. In addition, the virus does not appear to be involved in human pathology. The genome of AAV has been cloned, sequenced and characterized. It consists of about 4700 bases, and at each end has an inverted repeat region (ITR) of about 145 bases, which serves as the starting point for the replication of the virus. The remaining regions of the genome are composed of two essential regions with capsid forming functions: the left hand of the genome containing the rep gene involved in viral replication and expression of viral genes; It is divided into the right hand portion of the genome containing the cap gene encoding the viral capsid protein.

The use of vectors derived from AAV for ex vivo and in vivo transfer of genes has been described in the literature (especially WO 91/18088, WO 93/09239; US 4,797,368, US 5,139,941, European Patents). No. 488528). The application discloses a variety of constructs derived from AAV, in which the rep and / or cap genes have been deleted and replaced with the gene of interest, and in vitro (cells in culture) or in vivo (directly in an organism) of the gene of interest. Its use is described for metastasis. Defective recombinant AAV according to the invention is a plasmid and AAV capsid forming gene containing the corresponding nucleic acid sequence bounded by two AAV inversion repeat regions (ITRs) into a cell line infected with a human helper virus (eg adenovirus). It can be prepared by cotransfecting a plasmid containing (rep and cap genes). The recombinant AAV produced is purified by conventional techniques. Thus, the present invention relates to a recombinant virus derived from AAV whose genome comprises a sequence encoding a GAX bounded by an AAV ITR. The invention also relates to a plasmid comprising a sequence encoding a GAX bordered by two ITRs from an AAV. Such plasmids can be used in themselves to transfer GAX sequences, optionally in liposome vectors (like viruses).

For retroviruses, the construction of the recombinant vector is widely described in the literature (especially in European Patent 453,242, European Patent 178,220, Bernstein et al. Genet. Eng. 7 (1985) 235; McCormick, BioTechnology 3 ( 1985) 689 et al. Specifically, retroviruses are monolithic viruses that infect dividing cells. The genome of retroviruses basically comprises two LTRs, capsid forming sequences and three coding regions (gag, pol and env). Recombinant vectors derived from retroviruses typically have the gag, pol, and env genes completely or partially deleted and replaced with the desired heterologous nucleic acid sequence. Such vectors include various types of retroviruses, in particular MoMuLV (mouse leukemia virus in mice, so-called MoMLV), MSV (mouse sarcoma virus in mice), HaSV (havey sarcoma virus), SNV (splenic necrosis virus), It can be prepared from RSV (Louse Sarcoma Virus) or Friends's Virus.

In order to prepare a recombinant retrovirus containing a sequence encoding the GAX according to the present invention, a plasmid containing the LTR, the capsid forming sequence and the coding sequence, in particular, is constructed, and then the plasmid is deleted using the plasmid. It is common to transfect so-called capsid forming cell lines that can automatically provide the function of a retrovirus. Generally, the capsid forming system can thus express gag, pol and env genes. Such capsid forming systems have been described in the prior art, in particular PA317 cell lines (US 4,861,719), PsiCRIP cell lines (WO 90/02806) and GP + envAm-12 cell lines (WO 89/07150). In addition, recombinant retroviruses may have modifications in the extended capsid forming sequences containing part of the gag gene as well as LTR to inhibit transcriptional activity (Bender et al., J. Viro. 61 (1987) 1639). Next, the produced recombinant retroviruses are purified by conventional techniques.

In the treatment of cancer, it is most advantageous to use defective recombinant adenoviruses. Relatively small amounts of active ingredient can be used (recombinant adenovirus), and can also be effective and very quickly acting on the site to be treated. The adenoviruses of the invention can also express high levels of the introduced gax gene, thereby providing a very effective therapeutic action. In addition, the adenoviruses of the present invention, due to their episomal properties, are maintained for a limited period of time in proliferative cells and thus have a short-term effect that exactly matches the desired therapeutic effect.

The injection amount of virus used is adapted depending on various parameters, in particular the mode of administration used and the duration of treatment desired. In general, the recombinant virus according to the invention is prepared and administered in a dosage form of 10 ⁴ to 10 ¹⁴ pfu per ml. In the case of AAV and adenovirus, dosages of 10 ⁶ to 10 ¹⁰ pfu per ml are also used. The pfu (“plaque forming unit”) corresponds to the infectivity of the virion suspension and is determined by infecting the appropriate cell culture and generally measuring the plaque number of infected cells after 48 hours. Methods of determining pfu titers of viral solutions are widely published in the literature.

The invention is advantageously used in vivo for the prevention of ongoing cell hyperproliferation (ie abnormal proliferation).

Thus, it can be used to destroy tumor cells. Activated tumorigenic genes are most appropriate for the treatment of cancers involved. For example, colon adenocarcinoma, thyroid cancer, lung carcinoma, myeloid leukemia, colorectal cancer, breast cancer, lung cancer, gastric cancer, esophageal cancer, B lymphoma, avian cancer, bladder cancer, glioblastoma, and the like.

In addition, this treatment is available to both humans and animals such as sheep, bovines, livestock (dogs, cats, etc.), horses, fish and the like.

The invention is described in more detail in the following examples, which are illustrative but not limiting.

1 shows the plasmid pCO1.

2 shows the plasmid pXL-CMV-GaxHA.

3 shows the effect of AdCMV gax expression on cell proliferation of human tumor cell H460.

4 shows tumor cells H460 treated with adenovirus AdCMV gax at MOI 1000 (FIG. 4A), MOI 100 (FIG. 4B) and MOI 1000 (FIG. 4C).

5 shows the effect of AdCMV gax on cell proliferation of human tumor cell Saos.

6 shows the effect of AdCMV gax on cell proliferation of human tumor cell H358.

General Molecular Biology Technology

Methods commonly used in molecular biology, such as preliminary extraction of plasmid DNA, centrifugation of plasmid DNA under a cesium chloride gradient, electrophoresis of agarose or acrylamide gels, purification of DNA fragments by electroelution, phenol or phenol of proteins Chloroform extraction, precipitation of ethanol or isopropanol of DNA in saline media, transformation in Escherichia coli and the like are well known to those skilled in the art and extensively described in the literature (Maniatis T. et al., "Molecular Cloning , a Laboratory Manual ", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982; Ausubel FM et al. (eds)," Current Protocols in Molecular Biology ", John Wiley & Sons, New York, 1987].

pBR332 and pUC plasmids and the M13 series phage are available from Bethesda Research Laboratories.

For ligation, DNA fragments are separated by size by agarose or acrylamide gel electrophoresis, extracted with phenol or with a phenol / chloroform mixture, precipitated with ethanol and phage according to the seller's recommendations. It can be cultured in the presence of T4 DNA ligase (Biolabs).

The protruding 5 'end is E. Klenow fragments of E. coli DNA polymerase I (Biorads) can be used and filled according to the vendor's instructions. The protruding 3 'end can be destroyed by the presence of phage T4 DNA polymerase (Biolabs) used according to the manufacturer's recommendations. Destruction of the protruding 5 'end is controllably performed with S1 nuclease treatment.

In vitro site-directed mutagenesis by synthetic oligodeoxynucleotides can be carried out using a kit sold by Amersham according to the method developed by Taylor (Taylor et al., Nucleic Acids Res. 13 ( 1985) 8939-8764).

So-called PCR techniques (Polymerase-catalysed Chain Reaction, Polymerase-catalysed Chain Reaction, Saiki RK et al., Science 239 (1985) 1350-1354; Mullis KB and Faloona FA, Meth. Enzym. 155 (1987) 335-350) Enzymatic amplification of the DNA fragments by means of DNA Perkin Elmer Cetus can be performed using the manufacturer's instructions.

Identification of the nucleotide sequence can be performed using a kit sold by Amersham, in a method developed by Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467).

Example 1: Construction of a vector pXL-CMV-GaxHA carrying a gene encoding a rat GAX protein under the control of a CMV promoter

This example describes the construction of a vector containing a cDNA encoding a GAX protein (species: rat) and an adenovirus sequence enabling recombination. An influenza virus hemagglutinin epitope (HA1 epitope) consisting of 18 amino acids is added to the N-terminus of the GAX protein (Field et al., Mol. Cell. Biol. 8: 2159-2165, 1988). This epitope addition process makes it possible to monitor the expression of gax with the help of antibodies directed against HA1 epitopes, in particular immunofluorescence techniques. In addition to this sensitivity, this method eliminates background noise corresponding to expression of endogenous GAX proteins in vitro and in vivo.

1.1. Construction of Plasmid pCO1 (FIG. 1)

Construction of A-plasmid pCE

The EcoRI-XbaI fragment corresponding to the left end of the adenovirus Ad5 genome was first cloned between the EcoRI and XbaI sites of vector pIC19H. This resulted in plasmid pCA. Plasmid pCA was cleaved with HinfI and its protruding 5 'terminus. It was filled with Klenow fragment of E. coli DNA polymerase and then digested with EcoRI. Thus, fragments produced from plasmid pCA containing the left end of the Ad5 adenovirus genome were cloned between the EcoRI and SamI sites of vector pIC20H (Marsh et al., Gene 32 (1984) 481). This gave plasmid pCB. The plasmid pCB was then cleaved with EcoRI and its protruding 5 'end. The clono fragment of E. coli's DNA polymerase I was filled and then digested with BamHI. Thus, fragments produced from plasmid pCB containing the left end of the Ad5 adenovirus genome were cloned between the NruI and BglII sites of vector pIC20H. This first produced a plasmid pCE with advantageous properties with 382 base pairs of Ad5 adenovirus followed by multiple cloning sites.

B. Construction of Plasmid pCD '

The plasmid pPY53 was prepared by first ligating the Sau3A (3346)-SstI (3645) fragment and the SstI (3645)-NarI (5519) fragment of the Ad5 adenovirus genome and cloning between the ClaI and BamHI sites of the vector pIC20H. SalI-TaqI fragment of plasmid pPY53 prepared from dam-environment containing part of Ad5 adenovirus genome between Sau3A (3346) and TaqI (5207) sites was cloned between SalI and ClaI sites of vector pIC20H pCA 'was prepared. TaqI (5207)-NarI (5519) fragments of Ad5 adenovirus prepared from the dam-environment and SalI-TaqI fragment of plasmid pCA 'were ligated and cloned between SalI and NarI sites of vector pIC20H. This gave plasmid pCC '. NarI (5519)-NruI (6316) fragments of the Ad5 adenovirus genome prepared from the dam-environment and SalI-NarI fragment of plasmid pCC 'were ligated and cloned between SalI and NruI sites of vector pIC20R. This produced plasmid pCD '.

C. Construction of Plasmid pC01

A restriction fragment containing an Ad5 adenovirus sequence from the Sau3A (3446) site to the NruI (6316) site was made by cleaving a portion with XhoI and completely cutting the plasmid pCD 'with SalI. This fragment was cloned into the SalI site of plasmid pCE. This produced plasmid pCO1 containing the left portion of Ad5 adenovirus, multiple cloning sites and Sau3A (3446) -NruI (6316) from Ad5 adenovirus up to the HinfI (382) site (FIG. 1).

1.2. Construction of the vector pXL-CMV-GaxHA (see Figure 2)

Gax cDNA was cloned into the XbaI-BamHI site of the vector pCGN (Tanaka and Herr, Cell 60; 375-386, 1990). The resulting vector pGCN-Gax encodes the early promoter and enhancer sequence of cytomegalovirus (CMV) (-522, +72; Boshart et al., Cell, 41: 521-530, 1985), HA1 epitope The leader sequence of herpes simplex virus thymidine kinase comprising the start codon AUG and the first three amino acids (+55, +104; Rusconi and Yamamoto, EMBO J., 6: 1309-1315, 1987), rat gax cDNA and finally the polyadenylation sequence of the rabbit β-globin gene (Paebo et al, cell, 35: 445-453).

The fragment containing the promoter, cDNA and polyadenylation sequence, which was then obtained by cleaving the vector pCGN-Gax with XmnI and SfiI and pretreatment with clenow, was then shuttle vector pCO1 containing the sequence of adenovirus essential for recombination. Was introduced at the EcoRV site. The resulting plasmid was named pXL-CMV-GaxHA (see FIG. 2).

Example 2: Construction of Recombinant Adenovirus Ad-CMVgax

Helper cells (cytosystem 293) that linearize the vector pXL-CMV-GaxHA prepared in Example 1 and automatically provide the function encoded by the adenovirus E1 regions (E1A and E1B), together with the missing adenovirus vector for recombination. At the same time.

Adenovirus Ad-CMVgax was obtained by homologous recombination in vivo between adenovirus Ad.RSVβgal (Startford-Perricaudet et al., J. Clin. Invest 90 (1992) 626) and vector pXL-CMV-GaxHA according to the following procedure . The vectors pXL-CMV-GaxHA linearized with the enzyme XmnI, and the adenovirus Ad.RESβgal linearized with ClaI were simultaneously transfected into 293 cell lines in the presence of calcium phosphate to allow homologous recombination. The recombinant adenovirus thus produced was selected by plaque purification. After isolation, the recombinant adenovirus was amplified in 293 cell lines to obtain a culture supernatant containing crude recombinant defective adenovirus with a titer of about 10 ¹⁰ pfu / ml.

Viral particles were purified by centrifugation under a cesium chloride gradient according to known techniques (see in particular Graham et al., Virology 52 (1973) 456). Adenovirus Ad-CMVgax was stored at -80 ° C in 10% glycerol.

Example 3: Control of Expression of Ad-CMVgax in Human Tumor Cells H460

Human tumor cell H460 derived from non-small cell lung cancer was infected by recombinant adenovirus Ad-CMV gax and by control virus expressing β-galactosidase (Ad-RSV-βgal) (MOI, multiplicity of infection) ) Transduced with increasing) (FIG. 3). After 1 hour 30 minutes of incubation in the presence of adenovirus solution, the culture medium containing 10% fetal serum was exchanged and the cells were incubated for 48 hours. The number of viable cells per culture well, which is the cell viability examined by trypan blue exclusion test, was determined. Two batches of adenoviruses AdCMVgax, Ad-gax and Ad-gax (2) were prepared, corresponding to two independent products of 293 cells. The activity of these two batches was comparable and found to be very different from the control adenovirus AdRSV βgal.

The use of Ad-RSV-βgal has previously demonstrated the activity of recombinant adenoviruses that efficiently transduce H460 cells. Thus, nuclear βgal activity was detected in at least 75% of cells treated with Ad-RSV-βgal (MOI 1000). Similarly, expression of GAX protein was also detected in cells transduced with Ad-CMV gax by immunofluorescence. Transformation efficiency of Ad-CMV gax and Ad-RSV-βgal virus was found to be comparable. Cell treatment with Ad-CMV gax is associated with a decrease in cell proliferation and also has large cytotoxicity detected 48 hours after transformation with adenovirus (see FIGS. 4A, 4B and 4C). This effect was not observed in the adenovirus of the control group. Thus, these data demonstrate that overexpression of GAX proteins involves disruption of proliferation of H460 cells.

It is beneficial for H460 cells to express mutated Ras protein (Ki-ras). The data of the present invention demonstrate that GAX protein can regain control of pre-deregulated cell proliferation by expression of modified Ras protein. This main property of the gax factor involved in oncogenic mutations of Lars is not limited to lung carcinoma. Similar results were obtained, ie, stopping the proliferation of AdCMVgax in HCT116 cells derived from colon carcinoma. Mutations in the Ras tumor gene (G13C) and in the DCC gene have been described in this cell line (Brattain et al., Cancer Research, 41: 1751-1856, 1981).

Example 5: Controlling Expression of Ad-CMV-gax in Human Tumor Cells with Null p53

In addition, H358 cells derived from non-small cell lung carcinoma constitute a defect model for the p53 protein. This cell system is virtually homologous to the p53 gene (Maxwell and Roth, Oncogene 8: 3421, 1993). Advantageously, Ad-gax adenovirus efficiently blocks proliferation of H358 cells, resulting in massive cytotoxicity 48 hours after transduction of adenoviruses (see FIG. 5). This cell death was not detected in the presence of the control adenovirus Ad-RSV βgal. This data demonstrates that overexpression of gax specifically and efficiently blocks the proliferation of p53-deficient tumor cells. Advantageously, this data is not limited to cells derived from lung carcinoma. In fact, proliferation of Saos human cells derived from osteosarcoma was also blocked after treatment with Ad-CMV gax. Saos cells constitute a cellular model with null p53 and pRb. Again, this proliferation arrest depends on virus use concentration and is not observed after transduction of cancer cells by control virus (FIG. 6). Thus, βgal activity is positive after at least 90% of Saos-2 cells are induced by AdRSV βgal at 250 infections. Under the same experimental conditions (number of infections 250), AdCMV gax results in a decrease in cell viability of at least 70%. Applicants have observed that Gax, initially described as a proliferation arrest gene, surprisingly leads to cell death similar to apoptosis after proliferation arrest.

In vivo compensation for cell death caused by overexpression of gax would advantageously be associated with therapeutic benefit in cancer patients.

Claims (13)

Use of a GAX protein or variant thereof in the manufacture of a pharmaceutical composition for treating cancer. Use of one or more nucleic acid sequences encoding all or part of a GAX protein or variant thereof in the manufacture of a pharmaceutical composition for treating cancer. 3. Use according to claim 2, characterized in that the nucleic acid gene is used in the form of liposomes in the form of complexes with DEAE-dextran, a receptor protein or a lipid or cationic polymer. 3. Use according to claim 2, wherein the nucleic acid sequence is part of a recombinant viral vector. Use according to claim 1, characterized in that the nucleic acid sequence is part of a viral vector selected from adenoviruses, retroviruses and adeno-associated viruses. Use according to claim 4 or 5, characterized in that adenoviruses, preferably adenoviruses of the type Ad5 or Ad2, are used. 7. Use according to any one of claims 4 to 6, characterized in that an animal, preferably a canine source adenovirus is used. 8. Use according to any one of claims 2 to 7, characterized in that the nucleic acid sequence used encodes all or part of the rat GAX protein or variant thereof. Use according to claim 8, characterized in that the nucleic acid sequence used encodes a rat GAX protein or human homologue thereof. The use according to any one of claims 2 to 9, characterized in that the nucleic acid sequence used is cDNA. Use according to any one of claims 2 to 9, characterized in that the nucleic acid sequence used is gDNA. Use according to any of claims 4 to 11, characterized in that the nucleic acid sequence used comprises a sequence which enables expression in infected cells. The use according to any one of claims 4 to 12, wherein the nucleic acid used comprises a signal sequence which directs the synthesized polypeptide into the secretory pathway of the target cell.