MXPA98007181A - Combinations of enzymes for the destruction of proliferati cells - Google Patents

Abstract

The present invention relates to combinations of enzymes, useful for the destruction of cells, in particular of proliferating cells. It also refers to the vectors that allow the transfer and intracellular expression of these combinations of enzymes, as well as their therapeutic use, in particular in anti-cancer gene therapy.

Description

Enzyme combinations for the destruction of proliferating cells The present invention relates to the domain of gene and cell therapy. It refers in particular to combinations of enzymes, useful for the destruction of cells, in particular proliferating cells. It also refers to the vectors that allow the transfer and intracellular expression of these combinations of enzymes, as well as their therapeutic use, in particular in anti-cancer gene therapy.

Gene therapy, which consists in introducing genetic information into an organism or a cell, has undergone extraordinary development in recent years. The identification of genes involved in pathologies, the preparation of gene delivery vectors, the development of control or tissue-specific expression systems in particular have contributed to the development of these new therapeutic approaches. Thus, in the course of the last 5 years, numerous clinical trials of gene or cell therapy have been undertaken in Europe as in the United States, in domains such as monogenic diseases (hemophilia, mucoviscidosis), cancer, cardiovascular diseases or even problems of the nervous system.

REF .: 27989 In the domain of pathologies linked to a cellular hyperproliferation (cancer, restenosis, etc.), different approaches have been developed. Certain based on the use of tumor suppressor genes (p53, Rb), others in the use of antisense directed against oncogenes (myc, Ras), others even in immunotherapy (administration of tumor antigens or specific immune cells, etc). Another approach consists in introducing into the referred cells a toxic or suicidal gene capable of inducing the destruction of said cells. Such genes are for example genes capable of sensitizing cells having a pharmaceutical agent. These are generally genes encoding non-mammalian and non-toxic enzymes which, when expressed in mammalian cells, transform a prodrug, initially little or non-toxic, into a highly toxic agent. Such mechanism of prodrug activation is advantageous in several examples: it allows to optimize the therapeutic index by adjusting the concentration of the prodrug or the expression of the enzyme, interrupts the toxicity by not administering more prodrug and evaluates the mortality rate. In addition, the use of these suicide genes offers the advantage of not being specific for a particular type of tumor, but of general application. Thus, strategies based on the use of tumor suppressor genes or antioncogene antisense apply only to tumors that have a deficiency in said suppressor gene or an overexpression of said oncogene. Also, approaches based on immunotherapy should be developed patient by patient, to take into account restrictions and immune competencies. On the contrary, a strategy based on the use of a suicide gene is applicable to all types of tumors, and, more generally, in practically every type of cell.

Numerous suicide genes are described in the literature, such as the genes that code for cytosine deaminase, purine nucleoside phosphorylase or thymidine kinase, such as the thymidine kinases of the varicella virus or the herpes simplex virus type 1.

The cytosine deaminase of Escherichia coli is able to catalyze the deamination of cytosine in uracil. Cells expressing the E. coli gene are consequently capable of converting 5-fluorocytosine to 5-fluorouracil, which is a toxic metabolite (Mullen et al., 1992 Proc. Nati. Acad. USA ü = p33).

The purine nucleoside phosphorylase of Escherichia coli allows the conversion of non-toxic analogs of deoxyadenosine into highly toxic adenine analogues. Since the eukaryotic enzyme does not exhibit this activity, if the mammalian cells express the bacterial gene, the deoxyadenine analogs such as 6-methyl-purine-2 * -deoxyrribo-nucleoside is transformed into a toxic product for these cells (Sorscher et al., 1994). Gene Therapy 1 p233).

Varicella virus thymidine kinase allows the monophosphorylation of 6-methoxypurine arabinoside. If the branched cells express this viral gene, this monophosphate is produced and then metabolized by the cellular enzymes into a toxic compound (Huber et al., 1991 Proc.Nat.Accid USA ££ p8039).

Among these genes, the gene coding for thymidine kinase (TK) is particularly interesting in the therapeutic plan because, unlike other suicide genes, it generates an enzyme capable of specifically eliminating cells in the course of division, since the Prodrug is transformed into a non-diffusible product that inhibits DNA synthesis. The viral thymidine kinase, and in particular the thymidine kinases of the varicella virus or the herpes simplex virus type 1 have a substrate specificity different from the cellular enzyme, and have been shown to be the target of guanosine analogues such as acyclovir or ganciclovir (Moolten 1986 Cancer Res. 4_ £ p5276). Thus, ganciclovir is phosphorylated in ganciclovir monophosphate only when mammalian cells produce the HSV1-TK enzyme, and then cell kinases allow the metabolism of ganciclovir monophosphate into diphosphate and then triphosphate which causes the stoppage of DNA synthesis and leads to cell death (Moolten 1986 Cancer Res. A £ p5276; Mullen 1994 Pharmac. Ther. £ pl99). The same mechanism occurs with other thymidine kinases and other guanosine analogues.

On the other hand, an effect of propagated toxicity (effect "by-stander") has been observed when the TK is used. This effect is manifested by the destruction not only of the cells that have been incorporated into the TK gene, but also of the neighboring cells. The mechanism of this process can be explained in three ways: i) the formation of apoptotic vesicles containing phosphorylated ganciclovir or thymidine kinase, which comes from dead cells, and then phagocytosis of these vesicles by neighboring cells; ii) the pae of the prodrug metabolized by thymidine kinase, by a process of metabolic cooperation of cells containing the suicide gene against cells that do not contain it and / or iii) an immune response linked to tumor regression (Marini et al. coll 1995 Gene Therapy 2 p655).

For the person skilled in the art, the use of the suicide gene coding for thymidine kinase of the herpes virus is widely documented. In particular, the first in vivo studies in rats having a glioma show tumor regressions when the HSV1-TK gene is expressed and that doses of 150 mg / kg of ganciclovir are injected [K. Culver et al. 1992 Science 256 pl550]. However, these doses are highly toxic in mice [T.

Osaki et al. 1994 Cancer Research 5_1 p5258] and therefore totally proscribed in gene therapy in the Man.

A certain number of therapeutic tests are also common in man, in which the TK gene is released to the cells by means of different vectors such as in particular the retroviral or adenoviral vectors. In clinical trials of gene therapy in men, these are much weaker doses that should be administered, in the order of 5 mg / kg and for a short treatment duration (14 days) (E. Oldfield et al., 1995 Human Gene Therapy £ p55). For higher doses or longer treatments over time, the undesirable side effects of toxicity are indeed observed.

To remedy these drawbacks, it was proposed to synthesize more specific or more active thymidine kinase derivatives to phosphorylate the guanosine analogs. They have described the derivatives obtained by directed mutagenesis. However, no precise biochemical characterization has been performed on the pure enzymes, no cell test has been published using these mutants and no functional improvement has been reported (O95 / 30007, Black et al., 1993 Biochemistry 32 pll618). In addition, the inducible expression of a HSV1-TK gene, which eliminated the first 45 codons, was carried out in eukaryotic cells, but the doses of prodrug used decreased in comparison with those described in all the tests in the literature (B. Salomon et al., 1995 Mol Cell Cell Biol. 15. p5322). As a consequence, none of the variants described thus far have an improved activity with respect to thymidine or ganciclovir.

The present invention proposes an improved method of gene therapy by suicide gene. The present invention describes in particular a method that improves the efficiency of phosphorylation of guanosine analogs by thymidine kinase and thus improve the therapeutic potential of this treatment. The present invention proposes in particular a method for triphosphorylating nucleoside analogs such as ganciclovir or acyclovir so that the triphosphorylation of these analogs is significantly increased at the doses of ganciclovir (resp. Acyclovir) i) significantly weaker; ii) or likely to cause a more pronounced "bystander" effect; iii) or else it does not lead to cellular toxicity that could occur when the wild type thymidine kinase is overexpressed.

This method can be applied to cancer, to cardiovascular diseases, or to any application that requires the death of certain cells such as cells infected by a virus; this virus can be a virus of type HIV (human immunodeficiency virus), CMV (cytomegalovirus) VCR (respiratory syncytial virus).

The present invention is based in particular on the use of a combination of enzymes, which make it possible to improve the phosphorylation reaction of the nucleoside analogues in vivo.

A first objective of the invention therefore resides in a composition comprising: an enzyme capable of phosphorylating a nucleoside analog, to generate a monophosphate analog, an enzyme capable of phosphorylating said monophosphate analog, to generate a diphosphate analog, and, an enzyme capable of phosphorylating said diphosphate analog, to generate a toxic triphosphate analog.

More particularly, the enzyme capable of phosphorylating a nucleoside analog, to generate a monophosphate analog is a thymidine kinase, the enzyme capable of phosphorylating said monophosphate analog, to generate a diphosphate analogue is a guanylate kinase and the enzyme capable of phosphorylating said diphosphate analog to generate an analog triphosphate is a nucleoside diphosphate kinase. On the other hand, the compositions according to the invention may comprise, not directly the enzyme, but a nucleic acid encoding the enzyme. In this regard, the invention also aims at a composition useful for the release and in vivo production of a combination of enzymes, comprising: - a first nucleic acid encoding an enzyme capable of phosphorylating a nucleoside analog, to generate a monophosphate analog, a second nucleic acid encoding an enzyme capable of phosphorylating said monophosphate analog, to generate a diphosphate analog, and, - a third nucleic acid encoding an enzyme capable of phosphorylating said diphosphate analog, to generate a toxic triphosphate analogue.

Advantageously, the first nucleic acid codes for a thymidine kinase, the second nucleic acid codes for a guanylate kinase and the third nucleic acid codes for a nucleoside diphosphate kinase.

The nucleoside analog is generally a guanosine analog, such as for example ganciclovir, acyclovir or penciclovir. Other nucleoside analogs are, for example, the compounds of the trifluorothymidine, l- [2-deoxy, 2-fluoro, beta-D-arabino furanosyl] -5-iodouracil, ara-A, 1-beta-D-arabinofuranosyl thymidine type (araT). ), 5-ethyl-2 'deoxyuridine, iodouridine, AZT, AIU, dideoxycytidine, AraC and bromovinyldeoxyuridine (BVDU). Preferred analogues are ganciclovir, acyclovir, penciclovir and BVDU, preferably ganciclovir and acyclovir. The triphosphate form is toxic in the sense that it causes, directly or indirectly, cell death.

When mammalian cells, modified to express thymidine kinase (HSV1-TK for example), are placed in the presence of a nucleoside analogue (ganciclovir for example), they become capable of effecting the phosphorylation of ganciclovir to carry ganciclovir monophosphate. Later, cell kinases allow the metabolization of this ganciclovir monophosphate successively in diphosphate and then triphosphate. The ganciclovir triphosphate thus generated, then produces toxic effects that are incorporated into the DNA and partially inhibits cellular alpha DNA polymerase that causes the stoppage of DNA synthesis and consequently leads to cell death. In this mechanism, the monophosphorylation step of the nucleoside analogue is considered as the limiting step. It is for this reason that different approaches have been described in the prior art to try to improve the intrinsic properties of thymidine kinase (creation of TK mutants, search for expression systems and administration of more functions, etc.).

The present invention now shows that it is possible to improve the efficiency of the treatment by administering, in combination with a thymidine kinase, other enzymes involved in the phosphorylation of nucleoside analogues. The present invention thus aims to different combinations of enzymes that optimize the intracellular reaction of triphosphorylation of nucleoside analogs. Another aspect of the present invention relates to vectors that allow the introduction and intracellular expression of these combinations of enzymes. It can be in particular of several vectors that each allows the production of an enzyme, or of one or several vectors that each allows the production of several enzymes or of all the enzymes. The present invention also relates to a process of triphosphorylation of nucleoside analogues in the presence of a combination of enzymes, optionally produced in situ by expression of the corresponding genes, as well as a method for the destruction of proliferating cells.

Phosphorylation of nucleoside monophosphates in nucleoside diphosphates and then triphosphates has been documented in vitro. These phosphorylations are carried out in the presence i) of lysate of human erythrocytes in the case of ganciclovir (Cheng et al., 1983 J. Biol. Chem. 258 pl2460) or ii) of enzymatic preparations of guanylate kinase and nucleoside diphosphate kinase of human erythrocytes , in the case of ganciclovir and acyclovir (Miller et al., 1980 J. Biol. Chem. 255 p720; Smée et al., 1985 Biochem. Pharmac. 34 pl049). Although phosphorylation of nucleoside monophosphates in diphosphates and then triphosphates has been demonstrated in mammalian cells, these conversions do not appear to be complete with ganciclovir monophosphate or acyclovir monophosphate (Agbaria et al., 1994 Mol.Pharmacol., 45 p777; Caruso et al. 1995 Virology 206 p495; Solomon et al., 1995 Mol Cell Cell Biol. 15 p5322).

The present application now describes the compositions that allow to improve the therapeutic efficiency of a thymidine kinase in vivo.

The first enzyme used in the compositions and methods according to the invention, capable of phosphorylating a nucleoside analog to generate a monophosphate analog, is advantageously a non-mammalian thymidine kinase. It is preferably a thymidine kinase of viral origin, and in particular herpetic. Among the herpetic thymidine kinases on can mention in particular the thymidine kinase of the herpes simplex virus type 1 (HSV1-TK), the thymidine kinase of the herpes simplex virus type 2 (HSV2-TK), the thymidine kinase of the virus of chicken pox (VZV-TK), thymidine kinase of Eppstein Barr virus (EBV-TK), or even thymidine kinase of herpetic viruses of bovine origin (Mittal et al., J. Virol 70 (1989) 2901), equine Robertson et al., NAR 16 (1988) 11303), feline (Nunberg et al., J. Virol., 63 (1989) 3240) or simian (Otsuka et al., Virology 135 (1984) 316).

The sequence of the gene encoding the thymidine kinase enzyme of herpes simplex virus type 1 has been described in the literature (see in particular McKnight 1980 Nucí Acids Res. £ p5931). There are natural variants that lead to proteins that have an enzymatic activity comparable to thymidine, or ganciclovir (M. Michael et al., 1995 Biochem, Biophys, Res. Commun 209, p966). The sequence of the gene encoding the herpes simplex virus type 2 thymidine kinase enzyme (Swain et al., J. Virol 46 (1983) 1045) has also been described.

The thymidine kinase used in the present invention can be a native thymidine kinase (the natural form of the enzyme or one of its natural variants), or a derivative form, that is, it results from the structural modification (s) ) of the native form. As indicated below, different mutants or derivatives have been described in the literature. Although their intrinsic properties do not seem significantly modified, these molecules can be used within the framework of the present invention. These are, for example, mutants which have a modification close to the DRH region of the nucleoside interaction site (WO95 / 30007). The DRH region corresponds to aspartic waste, arginine and histidine at positions 163, 164 and 165 of the TK. These three positions are three conserved among the herpetic TK. Different mutants have been described at positions 160-162 and 168-170 (WO95 / 30007). Other artificial variants possess a modification at the level of the binding site of ATP (FR96 01603). On the other hand, other TK derivatives can be prepared according to the classical techniques of molecular biology, and useful in the combinations of the invention. These mutants can be prepared, for example, by mutagenesis of a nucleic acid encoding a native thymidine kinase, preferably herpetic, or one of its variants. Numerous methods that allow to perform the directed or random mutagenesis are known by the person skilled in the art and can be mentioned the mutagenesis directed by PCR or by oligonucleotide, the mutagenesis at random in vitro by chemical agents such as for example hydroylamine or in vivo in strains of E. coli mutants (Miller "A short course in bacterial genetics", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1992). The sequences thus mutated are then expressed in a cellular or acellular system and the expression product is tested by the presence of a thymidine kinase-like activity, under the conditions described in particular in the examples. Any enzyme resulting from this process, which has the ability to phosphorylate a nucleoside analog, to generate a monophosphate analog, can be used in the present invention.

Preferably, a TK output of thymidine kinase from herpes simplex virus type 1 (HSV1-TK) or a corresponding encoding nucleic acid is used within the scope of the present invention. It is more particularly the HSVI-TK or one of its variants, such as natural variants or artificial variants. Among the artificial variants, the variants P15SA / F161V and F1611 (Biochemistry 32 (1993) p.11618), the variant A168S (Prot.Engin. 7 (1994) p.83) or the variants possessing one can be mentioned more particularly. modification at the level of the ATP binding site such as the M601 variant. Even more preferably, a nucleic acid encoding the herpes simplex virus type thymidine kinase is used 1 (HSV1-TK).

The second enzyme used in the compositions and methods according to the invention, capable of phosphorylating a nucleoside monophosphate analog to generate a diphosphate analog, is advantageously a guanylate kinase. The guanylate kinase (GMPK) was purified from different organisms (man, rat, ox, yeast). The gene coding for GMPK was also cloned into different cell types, and in particular in the yeast Saccharomvces cerevisae. GUK1 gene (M. Konrad 1992 J. Biol. Chem. 267 p25652). From this gene, the 20 kDa GMPK enzyme was also purified. The gene of GMPK, called amk. it was also isolated and overexpressed in Escherichia coli (D. Gentry et al., 1993 J. Biol. Chem. 6JJL pl4316). This enzyme is different from the S. cerevisae enzyme in terms of cooperativity and oligomerization, while the sequences have strong 46.2% identity regions in 182 residues. The sequence A11042, identified as coding for a factor having a hematopoietic cell growth potential activity (EP0274560), presents 51.9% identity in 180 residues with the GUK1 gene of S. cerevisae. and it seems to be the human homologue of the GMPK, although no biochemical data has been published.

The third enzyme used in the compositions and methods according to the invention, capable of phosphorylating a nucleoside diphosphate analog to generate a triphosphate analog, is advantageously a nucleoside diphosphate kinase. The nucleoside diphosphate kinase (NDPK) is an enzyme of high substrate specificity and was purified from very varied sources (M. Inouye et al., 1991 Gene 105 p31). For various organisms (Myxococcus xanthus, Drosophila melanogaster, Dictyostelium discoideum, rat, ox, man, E. coli and. Cerevisae) the gene coding for NDPK was cloned and the corresponding enzymes are very homologous (K.

Watanabe et al. 1993 Gene 29 pl41). However, only enzymes from higher eukaryotes possess a "leucine zyper" sequence. The described human genes encoding NDPK activity are in particular nm23-Hl and nm23-H2. It is suggested that the nm23-H2 gene codes for a bifunctional protein with two independent functions that are NDPK activity and transcription factor (E. Postel et al., 1994 J. Biol. Chem. 269 p8627). The YN & amp; de = ___ cerevisae is not an essential gene for yeast and it codes for NDPK which is probably a tetrameric protein in which the molecular weight of the monomers is 19 kDa (A. Jong et al 1991 Arch. Biochem. Biophys. 291 p241).

The nucleic sequences coding for GMPK or NDPK used in the framework of the invention can be of human, animal, viral, semi-synthetic synthetic origin.

In a general manner, the nucleic sequences of the invention can be prepared according to any technique known to the person skilled in the art. Illustrative of these techniques can be mentioned in particular: - the chemical synthesis, using the sequences described in the literature and for example a nucleic acid synthesizer, - the servant of banks by means of specific probes, in particular such as those described in the literature, or even - mixed techniques that include chemical modification (elongation, elimination, substitution, etc.) of sequences screened from banks.

Advantageously, the nucleic sequences used within the framework of the invention are cDNA or gDNA sequences. The cDNA sequences are sequences devoid of introns, obtained from RNA. The gDNA sequences are chromosome regions. In eukaryotes, they comprise one or more introns. The gDNA sequences used in the framework of the invention may comprise all or part of the introns present in the natural gene, or one or more introns artificially introduced into a cDNA to increase eg the efficiency of expression in mammalian cells. Nucleic acids can code for native enzymes or for variants or derivatives that have an activity of the same type. These analog nucleic acids can be obtained by the classical techniques of molecular biology, well known to the person skilled in the art. It can be mutagenesis, directed or not, hybridization from banks, elimination or insertion, the construction of hybrid molecules, etc. In general, the modifications carry at least 20% of the nucleic acid bases. The functionality of the analog nucleic acids was determined as described in the examples for the dosage of the enzymatic activity of the expression product.

A particular composition in the sense of the invention comprises a first nucleic acid encoding a thymidine kinase and a second nucleic acid encoding a nucleoside diphosphate kinase. In this embodiment, the nucleoside diphosphate kinase is preferably of non-human eukaryote origin. By non-human enzyme is meant an enzyme not naturally present in human cells. It can be a viral, animal, or exit from a lower eukaryote organism (such as a yeast). It can also be a non-natural derivative of a human enzyme, which has one or several structural modifications. More preferably, the NDPK used in the present invention is chosen from the NDPK of yeast or beef. These compositions may further comprise a nucleic acid encoding a guanylate kinase, such as a yeast guanylate kinase.

Another particular composition in the sense of the invention comprises a first nucleic acid encoding a thymidine kinase and a second nucleic acid encoding a non-human guanylate kinase. The non-human GMPK can be chosen from the rat, ox, yeast, bacterium GMPK or derivatives thereof. Preferably, the GMPK leaves a lower eukaryote, in particular yeast.

According to a particularly advantageous embodiment, the nucleoside diphosphate kinase used in the context of the present invention is of eukaryotic or animal origin. Even more preferably, it is a yeast or beef nucleoside diphosphate kinase. The applicant has indeed shown that, surprisingly, yeast nucleoside diphosphate kinase, and in particular Saccharomvces cerevisae or ox, allow to phosphorylate the nucleoside diphosphate analogs, such as ganciclovir diphosphate or acyclovir diphosphate, in nucleosides triphosphates. Furthermore, the results present in the examples clearly show that these enzymes have an activity far superior to the human enzyme on these substrates. Thus, in the presence of 0.675 μg of human enzyme, the percentage of GCV triphosphate obtained is 1.5%, however in the presence of 0.75 μg of yeast enzyme, it is 82.9%. Also, in the presence of 6.75 μg of human enzyme, the percentage of GCV 2 triphosphate obtained is 24%, however in the presence of 1.5 μg of yeast enzyme, it is 91.1% and in the presence of 5 μg of ox enzyme, is 92%. The same results are obtained with another nucleoside analogue, acyclovir. A) Yes, in the presence of 6.75 μg of human enzyme, the percentage of ACV triphosphate obtained is lower than 0.4%, however in the presence of 1.5 μg of yeast enzyme, it is 8%, in the presence of 15 μg of enzyme of yeast, is 81% and in the presence of 5 μg of ox enzyme, is 1.3%. These results clearly show the advantage of using, in the combinations according to the invention, a yeast or beef nucleoside diphosphate kinase. These results also show that the first stage of phosphorylation of the analogue in monophosphate is not necessarily the limiting step of the process and that the use of a combination of enzymes according to the invention allows to increase the therapeutic potential of the treatment.

On the other hand, the applicant also shows that yeast guanylate kinase, and in particular Saccharomvces cerevisae. it would also make it possible to phosphorylate nucleoside monophosphate analogs, such as ganciclovir monophosphate or acyclovir monophosphate, in nucleoside diphosphates with good activity. Thus, in the presence of 2.5 μg of yeast enzyme, the percentage of CCV diphosphate obtained can exceed 92% and in the presence of 74 μg of yeast enzyme, the percentage of ACV diphosphate obtained is 54%. In addition, the present results show that the yeast guanylate kinase has a phosphorylation rate of GCVMP 2 times higher than the human enzyme. Also, its affinity for GCVMP is higher by a factor at least equal to 2 in the affinity that the human enzyme presents for this substrate. In total, the Vmax / Km value of the yeast enzyme for GCVMP is 4.4 times higher than the value of the human enzyme. For the ACVMP, the Vmax / Km value of the yeast enzyme is 7 to 9 times higher than the value presented by the human enzyme by this substrate.

The Applicant has also demonstrated that a coupling of these two non-human enzymes with a thymidine kinase, for example the thymidine kinase of herpes virus type 1 would allow to phosphorylate nucleoside analogues such as ganciclovir or acyclovir in triphosphate derivatives, with a very important efficiency.

According to a preferred embodiment, the compositions of the invention comprise sequences coding for the guanylate kinase (EC-2.7.4.8) and / or the nucleoside diphosphate kinase (EC-2.7.4.6) of yeast. More preferably, these are enzymes of the yeast = __. cerevisae. These sequences are used simultaneously with a sequence (HSV1-TK) coding for thymidine kinase of herpes simplex virus type 1 (EC-2.7.1.21) to allow triphosphorylation of nucleoside analogues such as ganciclovir or acyclovir.

As indicated below, the compositions according to the invention may comprise a combination of enzymes or nucleic acids that allow the in vivo production of the enzymes. It is advantageously nucleic acids. This mode of use is preferred since it allows an in vivo production of higher levels of enzymes and thus a more important therapeutic effect.

According to a first embodiment, in the compositions of the invention, the nucleic acids are carried by the same expression vector. This embodiment is particularly advantageous because it is sufficient to introduce a single vector into a mammalian cell in order to obtain the desired therapeutic effect. In this embodiment, the different nucleic acids can constitute three different expression cassettes within the same expression vector. Thus, the different nucleic acids can each be placed under the control of a transcriptional promoter, a transcriptional terminator and different translation signals. It is also possible to insert several nucleic acids in the form of a policistron in which the expression is directed by a single promoter and a single transcriptional terminator. This can be done in particular by the use of IRES ("Internal Ribosome Entry Site") sequences positioned between the nucleic sequences. In this regard, the expression vectors of the invention may comprise a bicistronic unit that directs the expression of two nucleic acids, and optionally a separate nucleic acid encoding the third enzyme. The vectors of the invention may also comprise a tricistronic unit that directs the expression of three nucleic acids. These different embodiments are illustrated in the examples.

Of the preferred expression vectors in the sense of the invention are in particular: - A vector comprising: a first nucleic acid that codes for a thymidine kinase, and, a second nucleic acid encoding a non-human guanylate kinase. It is preferably a yeast guanylate kinase.

- A vector comprising: a first nucleic acid that codes for a thymidine kinase, and, a second nucleic acid encoding a nucleoside diphosphate kinase. It is preferably a non-human eukaryotic nucleoside diphosphate kinase. More preferably, it is an NDPK of bovine or yeast origin.

Advantageously, this vector further comprises a nucleic acid encoding a guanylate kinase.

The thymidine kinase used in the vectors of the invention is advantageously a thymidine kinase of viral origin, in particular herpetic. It is preferably a thymidine kinase exit from the TK of HSV1 or HSV-2 virus.

As indicated below, in the vectors according to the invention, the different nucleic acids can be placed under the control of different promoters, or constitute a polycistronic unit under the control of a single promoter.

In this regard, as indicated below, enzymes can also be produced in a coupled form, the different nucleic acids that are coupled to produce a protein that carries the different enzymatic activities. In particular, a particular embodiment of the vectors according to the invention is characterized in that the nucleic acid encoding the thymidine kinase of viral origin and the nucleic acid encoding the non-human guanylate kinase are coupled and encoded for a protein that carries both the TK and CUK activities. According to another variant, in the vectors of the invention, the nucleic acid coding for the thymidine kinase of viral origin and the nucleic acid coding for the nucleoside diphosphate kinase are coupled and encoded for a protein that carries both the activities TK and NDPK. By way of illustration, the coupling between the enzymes is carried out by means of a peptide linker, for example of structure (G4S) n.

According to another embodiment, in the compositions of the invention, the nucleic acids are carried by several expression vectors.

As indicated below, the expression vectors may be of plasmid or viral origin. These are vectors of viral origin, advantageously they are retroviruses or adenoviruses.

Different promoters can be used within the framework of the invention. These are sequences that allow the expression of a nucleic acid in a mammalian cell. The promoter is advantageously chosen from functional promoters in human cells. More preferably, it is a promoter that allows the expression of a nucleic acid sequence in a hyperproliferative cell (cancerous, restenosis, etc.). In this regard, different promoters can be used. It can be, for example, the promoter of the gene considered (TK, GMPK, NDPK). They can also be regions of different origin (responsible for the expression of other proteins, or the same synthetics). This can be any promoter or derived sequence that stimulates or represses the transcription of a gene in a specific way or not, inducible or not, strong or weak. Mention may be made in particular of the promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences exiting the genome of the target cell. Among eukaryotic promoters, ubiquitous promoters (promoter of the HPRT, PCK, a-actin, tubulin, DHFR etc), promoters of intermediate filaments (CFAP gene promoter, desmin, vimentin, neurofilaments, etc.) can be used in particular. keratin, etc.), promoters of therapeutic genes (for example the promoter of the MDR, CFTR, Factor VIII, ApoAI, etc. genes), of tissue-specific promoters (promoter of the pyruvate kinase gene, biline, intestinal protein binding of the fatty acids, alpha-smooth muscle actin, etc.), cell-specific promoters of dividing cells such as cancer cells or even promoters that respond to a stimulus (steroid hormone receptor, retinic acid receptor, glucocorticoid receptor, etc.) or inducible ones. Also, they may be promoter sequences exiting the genome of a virus, such as for example the promoters of the adenovirus ElA and MLP genes, the early CMV promoter, or even the RSV LTR promoter, etc. In addition, these promoter regions can be modified by the addition of sequences of activation, regulation, or allowing tissue-specific or majority expression.

Another object of the invention relates to a product comprising a combination of enzymes capable of triphosphorylating a guanosine analog, and said guanosine analog, for the purpose of simultaneous, separate, or extended administration in time.

The invention also relates to a composition for the in vivo production of a toxic nucleoside triphosphate analogue comprising separate conditions or together of: - a nucleoside analog - a nucleic acid that codes for a thymidine kinase - a nucleic acid encoding a guanylate kinase, and, - a nucleic acid encoding a nucleoside diphosphate kinase.

The invention also aims at a composition comprising a combination of enzymes involved in the phosphorylation of nucleosides, optionally generated in situ by expression of nucleic sequences, at least one of these enzymes is of non-human eukaryotic origin. The enzyme combination comprises in particular a TK and an NDPK; a TK and a GMPK or a TK, a GMPK and an NDPK.

The present invention still relates to a method for the destruction of proliferating cells comprising the administration in said cells of a combination of enzymes comprising a TK and an NDPK. Preferably, the combination further comprises a guanylate kinase. The invention also relates to a method for the destruction of proliferating cells comprising the administration in said cells of a combination of enzymes comprising a TK and a GMPK.

According to this method, the cells are contacted with a nucleoside analog, preferably a guanosine analog, which is converted into cells expressing the enzyme combination in a toxic compound.

According to the invention, the enzymes can be administered to the cells by administration of nucleic acids encoding said enzymes.

The invention also resides in the use of the nucleoside diphosphate kinase or of a nucleic acid encoding the same, in combination with a thymidine kinase or a nucleic acid encoding a thymidine kinase, for the preparation of a pharmaceutical composition intended for destruction of proliferating cells.

The invention still relates to a triphosphorylation process of a nucleoside analog comprising comprising said analog with a combination of enzymes, at least one of those of non-human eukaryotic origin.

The present invention now provides new therapeutic agents that allow interfering with numerous cellular dysfunctions. For this purpose, the nucleic acids or cassettes according to the invention can be injected as such at the site-to-treat level, or directly incubated with the cells to be destroyed or treated. Indeed, it has been described that naked nucleic acids could penetrate cells without a particular vector. However, it is preferred within the framework of the present invention to use an administration vector, which allows to improve (i) the efficiency of cell penetration, (ii) target (iii) extra- and intracellular stability. In a particularly preferred embodiment of the present invention, the nucleic sequences are incorporated into a transfer vector. The vector used can be of chemical, plasmid or viral origin.

By chemical vector, it is understood to cover, in the sense of the invention, any non-viral agent capable of promoting the transfer and expression of nucleic sequences in eukaryotic cells. These chemical or biochemical vectors, synthetic or natural, represent an interesting alternative to natural viruses in particular for reasons of convenience, safety and also due to the absence of a theoretical limit to which the size of the DNA to be transfected refers. These synthetic vectors have two main functions, to compact the nucleic acid to be transfected and to promote its cellular fixation as well as its passage through the plasma membrane and, if applicable, the two nuclear membranes. To alleviate the polyanionic nature of nucleic acids, non-viral vectors possess all polycationic charges. As representative of this type of non-viral transfection techniques, currently developed for the introduction of a genetic information, we can mention those involving the DNA and DEAE-dextran complexes (Pagano et al., J.Virol. (1967) 891), DNA and nuclear proteins (Kaneda et al., Science 243 (1989) 375), DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), the use of liposomes ( Fraley et al., J. Biol. Chem. 255 (1980) 10431), etc.

The use of viruses as vectors for gene transfer appeared as a promising alternative to 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.

The nucleic acid or the vector used in the present invention can be formulated for the purpose of topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. administration. Preferably, the nucleic sequence or the vector is used in an injectable form. Consequently, it can be mixed with any pharmaceutically acceptable carrier for an injectable formulation, in particular for a direct injection at the level of the site to be treated. It can be, in particular, sterile, isotonic solutions, or dry compositions, in particular lyophilised, which, by addition according to the case of sterilized water or physiological saline, allow the constitution of injectable solutes. A direct injection of the nucleic acid sequence in the tumor of the patient is interesting because it allows to concentrate the therapeutic effect at the level of the affected tissues. The doses of nucleic sequences used can be adapted according to different parameters, and in particular depending on the vector, the mode of administration used, the aforementioned pathology or even the duration of the investigated treatment.

The invention also relates to any pharmaceutical composition comprising a combination of enzymes as defined below.

It also refers to any pharmaceutical composition comprising at least one vector as defined below.

It also relates to the use of a NDPK of yeast origin or of a GMPK of yeast origin for the in vivo phosphorylation of nucleoside analogues.

Due to their antiproliferative properties, the pharmaceutical compositions according to the invention are all particularly suitable for the treatment of hyperproliferative disorders, such as in particular cancers and restinosis. The present invention thus provides a particularly effective method for the destruction of cells, in particular hyperproliferative cells. This is applicable to the destruction of tumor cells or smooth muscle cells of the vascular wall (restenosis). It is particularly appropriate in the treatment of cancers. By way of example, mention may be made of adenocarcinomas of the colon, cancers of the thyroid, lung carcinomas, myeloid leukemias, colorectal cancers, breast cancers, lung cancers, gastric cancers, esophageal cancers , B lymphomas, ovarian cancers, bladder cancers, glioblastomas, hepatocarcinomas, cancers of the bones, skin, pancreas or even cancers of the kidney and prostate, cancers of the esophagus, cancers of the larynx, cancers of the head and neck, HPV positive ano-genital cancers, EBV-positive nasopharyngeal cancers, etc.

It can be used in vitro or ex vivo. Ex vivo, consists essentially of incubating the cells in the presence of a nucleic sequence (or of a vector, or cassete or directly of the derivative). In vivo, it consists of administering to the organism an active amount of a vector (or of a cassette) according to the invention, preferably directly at the level of the site to be treated (tumor in particular), previously, simultaneously and / or after the injection of the prodrug considered ie ganciclovir or a nucleoside analog. In this regard, the invention also aims at a method of destroying hyperproliferative cells comprising contacting said cells or a part thereof with a combination of enzymes or nucleic sequences as defined below, in the presence of a nucleoside analogue. .

The present invention will be described more fully with the help of the examples and figures that follow, which should be considered as illustrative and not limiting.

DESCRIPTION OF THE FIGURES: Figure 1: Schematic representation of the pcDNA3-TK vector Figure 2: Schematic representation of the vector pXL2854 Figure 3: Schematic representation of the vector pXL2967 Figure 4: Schematic representation of the vector pXL3081 Figure 5: Demonstration of the expression of GMPK and NDPK proteins Figure 6: Schematic representation of the vector pXL3098 Table 1: Kinetic constants of yeast GMPK and human erythrocytes in the GCVMP and the ACVMP. Published values: [D.F. Smée et al. (1985) Bi ochem. Pharmacol. 34: 1049-1056] a; [R. E. Boehme (1984) J. Bi ol. Chem. 259: 12346-12349] b; [W. H. Miller and R. L. Miller (1980) J. Biol. Chem. 255: 7204-7207] = Table 2: TK-GMPK-NDPK coupling:% of products formed MATERIALS AND METHODS Aftryr- c4.?nmetneM ACV: acyclovir GCV: ganciclovir GMPK: guanylate kinase HSV1-TK: thymidine kinase of the herpes simplex virus type 1.

NDPK: nucleoside diphosphate kinase Molecular biology techniques aantralas The methods used classically in molecular biology such as preparative extractions of plasmid DNA, centrifugation of plasmid DNA in cesium chloride gradient, agarose or acrylamide gel electrophoresis, purification of DNA fragments by electroelution, protein extraction with phenol-chloroform, the precipitation of DNA in saline medium by ethanol or isopropanol, the transformation in Escherichia coli are well known to those skilled in the art and are abundantly described in the literature (Sambrook et al. "Molecular Cloning, a Laboratory Manual ", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989, Ausubel et al.," Current Protocols in Molecular Biology, "John Wiley &Sons, New York, 1987).

The pUC type plasmids and the phages of the M13 series are of commercial origin (Bethesda Research Laboratories), the pBSK or pBKS plasmids are from Stratagen.

Enzymatic amplification of DNA fragments by the so-called PCR technique [P_olymerase-catalyzed £ hain ßeaction] can be performed using a "DNA thermal cycler" (Perkin Elmer Cetus) according to the manufacturer's recommendations.

The electroporation of plasmid DNA in E. cpli cells can be performed with the help of an electroporator (Bio-Rad) according to the manufacturer's recommendations.

The verification of nucleotide sequences can be carried out by the method developed by Sanger et al.

[Proc. Nati Acad. Sci USA l (1977) 5463-5467] using the 4 case distributed by Amersham or the one distributed by Applied Biosystems. "ilr ^ TT? 1 ~ Construction of expression vectors of enzyme combinations This example describes different methods for the construction of expression and transfer vectors of nucleic sequences of the invention in vitro or in vivo. 1. 1 - Construction of plasmid vectors For the construction of plasmid vectors, different types of expression vectors can be used. 2 types of vectors are more particularly preferred: - The vector pSV2, described in DNA Cloning, A practical approach Vol.2; D.M. Glover (Ed) IRL Press, Oxford, Washington DC, 1985. This vector is a vector of eukaryotic expression. The nucleic acids encoding the enzyme combinations of the invention can be inserted into this vector at the Hpal EcoRV sites. They are thus placed under the control of the promoter of the SV40 virus enhancer.

- The vector pCDNA3 (Invitrogen). It is also a vector of eukaryotic expression. The nucleic sequences encoding the enzymes or combinations of enzymes of the invention were placed, in this vector, under the control of the early CMV promoter. 1. 2 - Construction of viral vectors According to a particular embodiment, the invention resides in the construction and use of viral vectors that allow the transfer and in vivo expression of nucleic acids as defined below.

It is more particularly adenovirus, different serotypes have been characterized, in which the structure and properties vary a little. Among these serotypes, it is preferred to use human adenovirus type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin within the framework of the present invention (see application W094 / 26914). Among the adenoviruses of animal origin useful in the context of the present invention can be mentioned adenoviruses of canine, bovine, murine origin (example: Mavl, Beard et al., Virology 75 (1990) 81), sheep, swine, aviary or even simian (example: SAV). Preferably, the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus [manhattan strain or A26 / 61 (ATCC VR-800) for example]. Preferably, adenoviruses of human or canine or mixed origin are used within the framework of the invention. Preferably, the defective adenoviruses of the invention comprise the ITRs, a sequence allowing encapsidation and a nucleic acid according to the invention. Even more preferably, in the genome of the adenoviruses of the invention, the E region is at least non-functional. The considered viral gene can be made non-functional by any technique known to the person skilled in the art, and in particular without total suppression, substitution, partial elimination, or addition of one or more bases in the gene (s) considered. Such modifications can be obtained in vitro (in the isolated DNA) or in situ, for example, by means of genetic engineering techniques, or even by treatment by means of mutagenesis agents. Other regions can also be modified, and in particular the region E3 (WO95 / 02697), E2 (W094 / 28938), E4 (W094 / 28152, W094 / 12649, WO95 / 02697) and L5 (WO95 / 02697). According to a preferred embodiment employed, the adenovirus according to the invention comprises a deletion in the El and E4 regions. According to another preferred embodiment, it comprises a deletion in the El region at which the E4 region and the nucleic sequence of the invention are inserted (Cf FR94 13355). In the viruses of the invention, deletion in the El region preferably extends nucleotides 455 to 3329 in the Ad5 adenovirus sequence.

The defective recombinant adenoviruses according to the invention can be prepared by any technique known to the person skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573, Graham, EMBO J.3 (1984) 2917). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid carrying among others a nucleic sequence or a combination of nucleic sequences of the invention. Homologous recombination occurs after co-transfection of said adenovirus and plasmid into an appropriate cell line. The cell line used should preferably (i) be transformable by said elements, and (ii), contain the sequences capable of complementing the defective adenovirus genome part, preferably integrally to avoid the risks of recombination. By way of a line example, mention may be made of the human embryo kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains, in particular, the left part of the genome integrated in its genome. genome of an Ad5 adenovirus (12%) or of the lines capable of complementing the El and E4 functions as described in particular in applications No. WO 94/26914 and WO95 / 02697 or in Yeh et al., J. Virol . 70 (1996) 559.

Next, the multiplying adenoviruses are recovered and purified according to the classical techniques of molecular biology, as illustrated in the examples.

With respect to adeno-associated viruses (AAV), it is DNA virus of relatively small size, which are integrated into the genome cells that are infected, stably and site-specific. They are capable of infecting a broad spectrum of cells, without inducing an effect on cell growth, morphology or differentiation. On the other hand, do not seem to be involved in pathologies in man. The AAV genome has been given, sequenced and characterized. They comprise approximately 4700 bases, and contain in each extremity an inverse repeat region (ITR) of approximately 145 bases, which serve as the origin of replication for the virus. The rest of the genome is divided into 2 essential regions that have the functions of encapsidation: the left part of the genome, which contains the rep gene involved in viral replication and expression of viral genes; the right part of the genome, which contains the cap gene that codes for the capsid proteins of the virus.

The use of AAV derived vectors for the transfer of genes in vitro and in vivo has been described in the literature (see in particular WO 91/18088, WO 93/09239, US 4,797,368, US 5,139,941, EP 488 528). These applications describe different constructions derived from AAV, in which the rep and / or cap genes are deleted and replaced by a gene of interest, and their use to transfer in vitro (in cells in culture) or in vivo (directly in an organism). ) said gene of interest. Defective recombinant AAVs according to the invention can be prepared by co-transfection, in a cell line infected by a human helper virus (for example an adenovirus), a plasmid containing a nucleic sequence or a combination of nucleic sequences of the invention bordered by two reverse repeating regions (ITR) of AAV, and of a plasmid carrying the encapsidation genes (rep and cap genes) of AAV. A usable cell line is for example line 293. Other production systems are described, for example, in applications W095 / 14771; W095 / 13365; W095 / 13392 or WO95 / 06743. The produced recombinant AAVs are then purified by the classical techniques.

With respect to herpes viruses and retroviruses, the construction of recombinant vectors has been widely described in the literature: see in particular Breakfield et al., New Biologist 3 (1991) 203; EF 453242, EP178220, Bernstein et al. Genet Eng. (1985) 235; MeCormick, BioTechnology 3 (1985) 689, etc. In particular, retroviruses are integrating viruses, which selectively infect dividing cells. They therefore constitute vectors of interest for cancer applications. The genome of retroviruses essentially comprises two LTRs, one packaging sequence and three coding regions (gag, pol and env). In recombinant vectors derived from retroviruses, the gag, pol and env genes are generally deleted, in whole or in part, and replaced by a heterologous nucleic acid sequence of interest. These vectors can be made from different types of retroviruses such as in particular the MoMuLV ("murine Moloney leukemia virus", still called MoMLV), the MSV ("murine Moloney sarcoma virus"), the HaSV ("Harvey sarcoma virus"); the SNV ("splice necrosis virus"); the RSV ("Rous sarcoma virus") or even the Friend virus.

To construct the recombinant retroviruses according to the invention containing a nucleic acid sequence or a combination of nucleic sequences according to the invention, a plasmid containing in particular the LTRs, the encapsidation sequence and said nucleic sequence is constructed, and then it is used to transfect a so-called encapsidation cell line, capable of trans-delivering retroviral functions deficient in the plasmid. In general, the encapsidation lines are consequently capable of expressing the gag, pol and env genes. Such encapsidation lines have been described in the prior art, and in particular the line PA317 (US 4,861,719); the PsiCRIP line (WO90 / 02806) and the GP + envAm-12 line (W089 / 07150). On the other hand, recombinant retroviruses may contain modifications at the LTR level to suppress transcriptional activity, as well as extended encapsidation sequences, which contain a part of the gag gene (Bender et al., J. Virol. 61 (1987) 1639 ). The recombinant retroviruses produced are then purified by classical techniques.

For use of the present invention, it is particularly advantageous to use an adenovirus or a defective recombinant retrovirus. These vectors in fact possess particularly interesting properties for the transfer of suicide genes in tumor cells. 1. 3 - Chemical vectors The nucleic acids or plasmid expression vectors described in this example (1.1) and in example 2 can be administered as they are in vivo or ex vivo. It has indeed been shown that naked nucleic acids could transfect the cells. Meanwhile, to improve the transfer efficiency, it is preferred to use a transfer vector in the framework of the invention. It can be a viral vector (example 1.2.) Or a synthetic transfection agent.

Among the developed synthetic vectors, it is preferred to use in the framework of the invention the cationic polymers of the polylysine type, (LKLK) n, (LKKL) n, (PCT / FR / 00098) polyethylene imine (WO96 / 02655) and DEAE dextran or even the cationic or lipofectant lipids. They have the property of condensing DNA and promoting its association with the cell membrane. Among the latter, lipopolyamines (lipofectamine, transfectam, etc.) different cationic or neutral lipids (DOTMA, DOGS, DOPE, etc.) as well as peptides of nuclear origin can be cited. In addition, the concept of directed transfection, mediated by a receptor, has been developed, which takes advantage of the principle of condensing DNA thanks to the cationic polymer that directs the fixation of the complex in the membrane thanks to a chemical coupling between the cationic polymer and the ligand. of a membranal receptor, present on the surface of the cell type that is to be inserted. Thus the target of the transferrin receptor, insulin or the receptor of the asialoglycoproteins of hepatocytes has been described. The preparation of a composition according to the invention using such a chemical vector is carried out according to any technique known to the person skilled in the art, generally only by contacting the different components.

EXAMPLE 2 - Cloning of HSV1-TK v / o of the cinnamate auanilate? / Or of the nucleoside diphosphocinase into an expression vector eμcarj.ote This example describes a particular embodiment of the invention, which uses a plasmid expression vector system to produce in situ the combinations of enzymes of the invention.

The expression of prokaryotic or eukaryotic genes in mammalian cells is known to the person skilled in the art. To optimize this expression, the vectors of the invention describe below the behavior of the following signals: i) a promoter / enhancer such as the CMV promoter that is well expressed in human cells; ii) a Kozak sequence, in which the consensus is (G / A) NNAUG (G / A); iii) the gene to be expressed; followed iv) of a polyadenylation sequence (V. Chisholm 1995 DNA cloning Vol.4, ed D. Clover et B. Hames pl). Such constructions are possible with the help of commercial vectors such as the vectors pZeoSV, pcDNA3 ... and have been carried out with the genes coding for HSV1-TK, GMPK and NDK of Sj_ cerevisae. 2. 1 - HSV1-TK expression vector The HSV1-TK gene encoding the herpes simplex virus type 1 thymidine kinase, derived from the plasmid pHSV-106 (Gibco-BRL) was cloned into the eukaryote expression vector pcDNA3 (Invitrogen). This PcDNA3-TK plasmid of 6936 bp was constructed by introducing the 1.5 kb EcoRI-Notl insert from pBTK1 between the EcoRI and Notl sites of pcDNA3, see figure 1. Plasmid pBTK1 was obtained as follows: having made the frank limbs, the BglII-NcoI insertion of 1.5 kb that comes from pHSV * -106 and that contains the HSV1-TK gene, of which the sequence was published by McKnight 1980 Nucí. Acids Res. 8 p5931, was cloned into the Smal site of pBSK.

The insertion of plasmid pcDNA3-TK contains i) 60 bp upstream of the HSV1-TK gene comprising the Kozak sequence (CCTATGC), ii) the gene sequence (1.13 kb) which is identical to that published by McKnight 1980 Nuci. Acids Res. £ p5931, iii) the 3 'sequence in the gene (0.3 kb) that is also described by McKnight. 2. 2 - Vector of guanylate kinase expression The 561 bp GUK1 gene that codes for the guanylate kinase of. cerevisae exited from pCUK-1 (see example 3), was cloned into the eukaryote expression vector pcDNA3 (Invitrogen) after having introduced a Kozak consensus. This plasmid pXL2854 was obtained in the following manner. The Xbal-BamHl insert of pGUK-1 containing the GUKl gene was cloned into the plasmid pSL301 (Invitrogen) between the Xbal-BamHl sites in such a way that the GUK1 gene can then be cut by the enzymes HindIII and BamHl and cloned between the HindIII and BamH1 sites of pcDNA3 to generate a PcDNA3-GUKl plasmid. Between the HindIII and P £ IMI sites of this plasmid, a 150 bp HindIII-PfIMI fragment containing a Kozak consensus and the 5 'region of GUK1 was cloned to form the 6001 bp pXL2854 plasmid see figure 2. The HindIII-PfIMI fragment 150 bp was isolated from a 280 bp fragment amplified by PCR with the help of plasmid pGUK-1 and sense oligonucleotides 6915 51 (GAG AAG CTT GCC ATG G.CC CGT CCT ATC GTA A) 3 ' (SEQ ID No. 1) and antisense 6916 5 '(GAC GAT CCG TTT GAC GGA AGC GAC AGT A) 3 * (SEQ ID NO: 2), the hybridization taking place at 45 ° C (the Kozak consensus is underlined in oligonucleotide 6915). The nucleic sequence amplified by PCR was sequenced and presented two differences with respect to the published sequence (M. Konrad 1992 J. Biol. Chem. 267 p25652) corresponding to the changes S2A (Serine in position 2 replaced by an alanine) and V34A (Valine in position 34 replaced by an alanine). 2. 3 - Vector of nucleoside diphosphokinase expression The YNK gene that codes for the nucleoside diphosphokinase of JL. cerevisae. from the pADl-YNK plasmid (K. Watanabe et al., 1993 Gene 29 pl41), was cloned into the eukaryote expression vector pcDNA3 (Invitrogen) after having introduced a Kozak consensus. This plasmid pXL2967 was obtained in the following manner. A PCR amplification was carried out with the plasmid pADI-YNK per matrix and the oligonucleotides sense 7017 5 '(AAG GAT CCA CGA TGG CTA GTC AAA CAG AAA) 3' (SEQ ID No. 3.) and antisense 7038 5 '(AAG AAT TCA GAT CTT CAT TCA TAA ATC CA) 3 '(SEQ ID No. 4) at the hybridization temperature of 40 ° C (the Kozak consensus is underlined in oligonucleotide 7017). The 477 bp amplified fragment was digested with BamHl and EcoRI and then cloned between the BamH1 and EcoRI sites of pCDNA3 to generate plasmid pXL2 67 of 5861 bp, see figure 3. The fragment sequence amplified by PCR is the same as the published for the YNK gene except in position 4 which corresponds to a change S2A S2A (Serine in position 2 replaced by an alanine) for the NDPK protein (K. Watanabe et al., 1993 Gene 29 pl41). 2. 4 - Vector for the co-expression of 2 genes The coexpression of genes can be performed in various ways known to the person skilled in the art. A preferred embodiment is to introduce, between the sequences to be expressed, the internal sites of entry of the ribosomes, IRES sequences (Mountford et al., TIG 11 (1995) 179).

An IRES sequence and the YNK gene are introduced in 3 'of the GUKl gene was cloned in pcDNA3 to generate a vector that allows the co-expression of GUK1 and YNK, in the form of a bicistronic unit (vector pGUK1-YNK). More precisely, the sequence of IRES (internal ribosome entry site) of EMCV (encephalomyocartis virus) that comes from the pCITE plasmid (Novagen) was recloned by PCR between the EcoRI and Ncol sites and introduced into the EcoRI and EcoRV sites of the pBluescript plasmid (Stratagene ) to generate the plasmid pXL3065. The 477 bp NcoI-EcoRV fragment containing the YNK gene from plasmid pXL2967 was cloned between the Ncol and EcoRV sites of pXL3065 to form the plasmid pXL3079. The 1 kb BamHI-EcoRV fragment containing the IRES and the YNK gene of the plasmid pXL3079 was then cloned between the BamH1 and EcoRV sites of the plasmid pXL2854 to generate the plasmid pXL3081. This plasmid contains the CMV and T7 promoters upstream of the GUK1 gene coding for the guanylate kinase of S. cerevisae followed by the IRES and the YNK gene coding for the nucleoside diphosphokinase of = _t- cerevisae see figure 4. The expression of the GMPK and NDPK proteins was tested with the help of the plasmids pXL2854, pXL2967 and pXL3081 in a reticulocyte transcription / translation system from Promega, see figure 5. The results obtained show that the GMPK and NDPK proteins are coexpressed with the plasmid? XL3081.

The same approach is used to generate a vector that coexpresses the TK and the YNK (vector pTK-YNK). or the TK and the GUKl (vector pTK-GUKl). 2. 5 - Vector for the co-expression of 3 genes The sequence coding for TK is inserted in the pGUKl-YNK vector of Example 2.4 above to generate a vector capable of expressing the 3 enzymatic activities (vector pTK-GUK1-YNK). 2. 6 - Vector for the expression of a BSVI-TK / S fusion. cerevisiae GMPK The construction of fusion protein is well known to the person skilled in the art and is carried out for the creation of a peptide linker between the C-terminal part of a protein and the N-terminal part of another protein (1989 Nature 339 p394). Such a protein allows cellular co-localization of the enzymes and can also favor substrate "tunelage" (Ljungcrantz et al 1989, Biochemistry 28 p8786).

A fusion protein was made with the help of the linker - (Gly) 4-Ser- (Gly) 4-Ser- (Gly) 4 (SEQ ID No. 9) the C-terminal sequence of the HSV1-TK (Asn376) and the N-terminal sequence of the GMPK of £ _s. cerevisiae (Ser2). The plasmid "XL3098" allowing the production of this fusion protein was constructed in the following manner. The 3 'sequence of the HSV1-TK gene (positions 1108 to 1128) was cloned by hybridization of the 5' sense oligonucleotides (CCG GGA GAT GGG GGC TAA CGG AGG TGG CGG TTC TGG TGG CGG AGG CTC CC) 3 '(SEQ. n ° 5) and 5 'antisense (GAT CCG GAG CCT CCG CCA CCA GAA CCG CCA CCT CCG TTA GCC TCC CCC ATC TC) 3 * (SEQ ID No. 6), such that the Asn codon of the HSV1-TK gene (position 1128 bp) is followed by codons coding for amino acids ((Gly) 4Ser) 2Gly. The 58 bp fragment was thus cloned between the Xmal and BamHl sites of pNEB193 (Biolabs) to generate the pTKL + plasmid. The 5 * sequence of the GUK1 gene of = ___ cerevisiae was amplified by PCR with the help of the pXL2854 matrix and the 5 'sense oligonucleotides (GAG AAT TCC GGA GGC GGT GGC TCC CGT CCT ATC GTA) 3' (SEQ ID No. 7) ) and 5 'antisense (GAG GAT CCG TTT GAC GGA AGC GAC AGT A) 3' (SEQ ID No. 8), such that the codon Ser in position 2 of the GMPK is precedent of the codons (Gly) 3Ser . This fragment of 0.27 kb was cloned between the BamHl and EcoRI sites of pUC19 (Biolabs) to form a plasmid which was then cut by PflMI and BamHl in order to introduce the 0.44 kb PflMI-BamHl fragment of pXL2854 containing the sequence 31 of GUKl, and generates the plasmid pGUKL-. The 0.58 kb BspEI-Xbal fragment from pGUKL- was inserted between the BspEI and Xbal sites of pTKL + to create pTKLGUK. The 0.63 kb XmaI-Ba Hl insert of pTKLGUK was then cloned between the Xmal and BamHl sites of the pETlla expression vector, to form the plasmid pXL3098, see figure 6. This plasmid was then introduced into the BL2IDE3met-strain and led to the production of the TK protein - ((Gly) 4Ser) 3-GMPK.

The fusion protein was then purified to homogeneity and the kinetic parameters of GCV and ACV phosphorylation of this enzyme were compared with the kinetic parameters obtained with the HSV1-TK and GMPK proteins of S. cerevisiae. 2. 7 - Transfer and expression in vivo The vectors described in Examples 2.1 to 2.6 were used for the transfer and in vivo expression of combinations of enzymes according to the invention. To this end, different compositions containing said vectors were prepared: - a composition containing the vector pcDNA3-TK, the vector pXL2854 and Lipofectamine, - a composition containing the vector pcDNA3-TK, the vector pXL2967 and Lipofectamine, - a composition containing the vector pcDNA3-TK, the vector pXL2854, the vector pXL2967 and Lipofectamine - a composition containing the vector pTK-GUK1 and Lipofectamine, optionally in combination with the vector pXL2967, - a composition containing the vector pTK-YNK and Lipofectamine, optionally in combination with the vector PXL2854, and, - a composition containing the vector pTK-GUK1-YNK and Lipofectamine Lipofectamine can be replaced by another chemical vector as described in example 1.3.

These different compositions are used in vivo or ex vivo for the intracellular transfer and expression of combinations of enzymes according to the invention. They can also be used in cell cultures, and for example in cultures of fibroblast cells NIH3T3 or of human colon carcinoma cells, HCT116. After the transfer of the vectors, the nucleoside analog is administered and cell destruction is demonstrated.

EXAMPLE 3 - Purification of the cinaaa suanilate The acellular extracts of the ís_coli strains that overexpress the GMPK of S. cerevisiae can be prepared in various ways, among which can be mentioned, lysis with lysozyme in the presence of EDTA, the use of grinding apparatuses of the Menton-Golin type , French Press, X-Press, or ultrasound action. More particularly, the acellular extracts of the E. coli strains overexpressing the GMPK of £ _. cerevisiae were prepared as follows: The E ^ coli strain BL21 (DE3) pGUK-1 was cultured as described by M. Konrad in J. Biol. Chem. 231 p25652 in 1992.

After centrifugation (5000 xg, 20 min), the cells obtained from 1 1 of culture were resuspended in 20 ml of 20 mM Tris / HCl buffer pH 7.5, containing 1 mM EDTA and sonicated for 4 min. 4 ° C. After centrifugation (50000 x g; lh) the supernatant was injected onto a MONO Q HR 10/10 column (Pharmacia) equilibrated with the 20 mM Tris / HCl buffer pH 7.5. Proteins were eluted with a linear gradient of 0 to 500 mM NaCl in the 20 mM Tris / HCl buffer pH 7.5. The fractions containing the GMPK activity were pooled and concentrated, and then chromatographed on a Superdex 75 HR16 / 10 column (Pharmacia) eluting with a 50 mM Tris / HCl buffer pH 7.5, 150 M NaCl. Fractions containing the activity GuK regrouped. After this step the preparation presents a single band visible on SDS-PAGE, and after the revelation with Cromassie Blue, and this band migrates with an apparent molecular weight of approximately 21,000.

The activity of GMPK is classically dosed using a dosing protocol described in the literature, Agarwal et al. 1978 Meth. Enzymol. vol.LI p 483.

EXAMPLE 4 - Determination of the kinetic constants of the qu * nj-ltt9 ginaaa of g, cerevwe.

The kinetic constants of purified yeast GMP kinase as described in example 3 were determined under the following enzymatic dosing conditions: The yeast GMP kinase is incubated for 10 min at 30 ° C in 100 μl of 50 mM Tris / HCl buffer pH 7.8 containing 4 mM ATP, 10 mM Mg C12, 100 mM KCl, 1 mg / ml BSA (albumin of bovine serum) and 5-100 μM [8-3H] GCVMP (40 nCi / nmol) or 200-3200 μM [8-3H] ACVMP (40 nCi / nmol). The reaction was stopped by heating the reaction mixture for 3 min at 80 ° C, 50 μl of 10 mM potassium phosphate buffer pH 3.5 was added, and after centrifugation for 2 min at 10,000 xg, 100 μl of Supernatant were analyzed by high pressure liquid chromatography (CLHP) in the following system: Phase ffftacjo yift Partisfera SAX (WHATMAN) - particle diameter: 5 μm - Dimensions: 4.6 x 125 mm.

F * ye p6vi »l: Shock absorber A: KH2P040.01 M pH 3.5 (adjusted with the help of concentrated H3PO4) Shock absorber B: KH2P040.75 M pH 3.5 (adjusted with the help of concentrated H3P04) Fluio: 1 ml / min Gradient: Detggcj: UV: 265 nm Radiochemistry: detection of tritium Scintillation flow (Optisafe 1 of Berthold) 1 ml / min or the calculation of the kinetic constants, the amount of yeast GMP kinase introduced in the enzymatic reaction was adjusted in the form to transform at the most 10% of substrate introduced at the start. The Michaelis curves were adjusted to the experimental points with the help of logiciel Grafit (Sigma). The results are presented in Table 1. It is shown that the yeast GMP kinase is capable of phosphorylating the GCVMP and the ACVMP. In addition, it presents a phosphorylation rate of GCVMP 2 times higher than the human enzyme. Also, its affinity for GCVMP is higher by a factor at least equal to 2 of the affinity that the human enzyme presents for this substrate. In total, the Vmax / Km value of the yeast enzyme for GCVMP is 4.4 times higher than the value of the human enzyme. For substrates that enter competition, the constant Vmax / Km determines the specificity of the enzyme versus the substrates. It is known as the "constant of specificity" [A. Fersht, Enzyme Structure and Mechanism, 1985, W. H. Fréeman and Co., London].

In the same way, the yeast GMP kinase has a capacity to phosphorylate the ACVMP much higher than the human enzyme. The Vmax / Km value of the yeast enzyme for the ACVMP is 7 to 9 times higher than the value presented by the human enzyme for this substrate.

GMPK and t K ' In a preferred embodiment of the coupling, the incubation was carried out in 100 μl of 50 mM Tris / HCl buffer pH 7.8, containing 1 mg / ml of BSA (bovine serum albumin), 5 mM ATP, 4 mM MgC12, 12 KM mM, 2 mM DTT, 600 μM EDTA, 100 μM [8-3H] -GCV (40 nCi / nmol) or 100 μM [2-3H] -ACV 40 nCi / nmol and different amounts of TK, GuK and NDPK (Cf) Table 2). The NDPK of 3 organisms (enzymes marketed by SIGMA) were used, as indicated in table 2.

The coupling of the enzymes HSV1-TK and GMPK of S. cerevisae allows a 90% phosphorylation of ganciclovir in ganciclovir diphosphate. And the nucleoside diphosphokinase of baker's yeast, ie £ _ ,. cerevisae - coupled to enzymes HSVI-TK and GMPK, allows the phosphorylation of ganciclovir triphosphate with a better activity that does not allow the human erythrocyte diphosphokinase nucleotide.

The comparable results were obtained with acyclovir. These results clearly demonstrate (a) that the combination of enzymes according to the invention provides a significant improvement in phosphorylation, indicating that the single modification of the TK could not suffice to improve the properties of the system, (b) that the system of the invention makes it possible to increase the efficiency of treatment by the suicide gene TK (c) that certain non-human enzymes possess a better activity for the nucleoside analogues, which make their use particularly advantageous.

EXAMPLE 6 - Purification of the fusion protein HSV1-TK / S. cerevisiae GMPK The acellular extracts of the strains of £ __. Coli overexpressing the fusion protein can be prepared in various ways, among which can be cited, lysis with lysozyme in the presence of EDTA, the use of crushing apparatuses of the Menton Golin type, French Press, X-Press, or stock of ultrasound. More particularly, the acellular extracts of the _____coli strains overexpressing the fusion protein have been prepared in the following manner: The E. coli strain BL21 DE3 pXL3098 was cultured in LB medium. After centrifugation (5000 xg, 20 min), the cells obtained from 1 1 were resuspended in 20 ml of buffer A: 50 mM Tris / HCl pH 7.8, containing 5 mM DTT, 24 mM MgCl, 10% Glycerol (v. / v), 2 mM Benzamidine, E64 50 μl / 1 (solution in 100 μg / 1 N- [N- (L-3-trans-carboxyoxyran-2-carbonyl) -L-leucyl] -4-aminobutylguanidine, Péfabloc 0.2 mM, STI (Soybeam trypsin inhibitor), Leupeptin 2 mg / ml, and were sonicated for 4 min at 4 ° C. After centrifugation (50000 xg, 1 h) the supernatant was injected into a column MONO Q HR 10/10 (Pharmacia) equilibrated with buffer A. Proteins were eluted with a linear gradient of 0 to 400 mM NaCl in the 20 mM Tris / HCl buffer pH 7.5 Fractions containing the TK and GMPK activity were regrouped and concentrated and then they were chromatographed on a Superdex 200 HILoad 26/60 column (Pharmacia) eluting with buffer A containing 150 mM NaCl Fractions containing the TK activity and GMPK regrouped. In this stage the preparation presents a single band visible on SDS-PAGE, after the revelation with Coomassie Blue, and this band migrated with an apparent molecular weight of about 61000.

EXAMPLE 7- Phosphorylation study for the HSV1-TK / GMPK fusion This example describes a study of the kinetic parameters of GCV phosphorylation and ACV of the fusion, compared with the kinetic parameters obtained with the proteins HSV1-TK and GMPK of _L__ cerevisiae. 7. 1. Dosage of the TK activity The activity of the TK was dosed as follows: an enzyme extract containing approximately 0.1 units of TK was incubated for 15 min at 37 ° C in 100 μl of buffer Tris / HCl 50 mm pH 7.8 containing 1 mg / ml of BSA (albumin of bovine serum), 5 mM ATP, 4 mM MgCl 2, 12 mM Kcl, 2 mM DTT, 600 μM EDTA and 100 μM GCV + [8-3H] -GCV 40 nCi / nmol. The reaction was stopped by the addition of 10 μl of 50 mM Tris / HCl buffer pH 7.8 containing 1 mM of non-radioactive thymidine. The phosphorylated species were fixed in a DEAE sephadex column (400μl gel) and then, the column was washed, these species were eluted with 2ml of IM HCl. The radioactivity in the sample was counted immediately by liquid scintillation. 7. 2. Dosage of GMPK activity The activity of GMPK was classically dosed using a dosing protocol described in the literature, K.C. Agarwal et al. (Methods In Enzymology (1978) Vol. Ll 483-490). 7. 3. Dosage of the activity of the fusion protein and co-incubation of the enzymes TK and GMPK The ability of the TK-GMPK fusion protein to transform the GCV or the ACV into GCVDP or ACVDP with respect to a synthetic mixture of TK and GMPK was determined as follows: Incubation took place in 200 μl of 50 mM Tris / HCl buffer pH 7.8 containing 1 mg / ml of BSA (bovine albumin serum), 5 mM ATP, 4 mM MgC12, 12 mM KCl, 2 mM DTT, EDTA 600 μM, 1 to 100 μM of GCV + [8-3H] GCV (40 nCi / nmol) wave 100 μM of ACV + [2-3H] ACV 40 nCi / nmol and of different amounts of TK, GMPK and the equivalent amounts of the fusion protein. The reaction was stopped by heating 3 min at 80 ° C, after centrifugation 100 μl of incubation was analyzed in the following system: Stationary phase: SAX Partisphere (WHATMAN) - particle diameter: 5 μm - Dimensions: 4.6 x 125 mm.

Mobile phase: Shock absorber A: KH2P040.01 M pH 3.5 (adjusted with the help of concentrated H3PO4) Shock absorber B: KH2P040.75 M pH 3.5 (adjusted with the help of H3P04 concentrate) Fluio: 1 ml / min SüdÁßnift: Detection: UV: 265 nm Radiochemistry: detection of tritium Scintillation flow (Optisafe 1 of Berthold) 1 ml / min The coupling and combination of the enzymes HSV1-TK and GMPK of S. cerevisiae allow the phosphorylation of ganciclovir in ganciclovir diphosphate (see table 3). The comparable results were obtained with acyclovir (see table 3).

These results clearly show that the TK-GMPK fusion protein retains the properties of two original enzymes. Furthermore, following the operating conditions, the TK-GMPK fusion protein provides a significant improvement of the phosphorylation a) that goes up to a factor of 1.8, for the GCV b) that goes up to a factor of 1.2 for ACV with respect to the coincubation of wild TK and GMPK enzymes.

The fusion protein allows in vivo a colocalization of the enzymatic activities in the cell, the separated enzymes are distributed, in the nucleus by the HSV1-TK and in the cytosol by the GMPK. This construction makes it possible to increase the efficiency of treatments due to the suicide gene.

The set of these results clearly demonstrates the therapeutic interest of the present invention, while allowing to decrease the dose of nucleoside and enzymes and obtain an important pharmacological benefit.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: RHONE POULENC RORER S.A. (B) STREET: 20, Avenue Raymond Aron (C) CITY: ANTONY (E) COUNTRY: FRANCE (F) POSTAL CODE: 92165 (G) TELEPHONE: 01. 55. 71. 70. 36 (H) TELEFAX: (01) 55. 71. 72. 91 +++ (ii) TITLE OF THE INVENTION: COMBINATION OF ENZYMES FOR THE DESTRUCTION OF PROLIFERATIVE CELLS (iii) NUMBER OF SEQUENCES: 9 (iv) COMPUTER READING FORM: (A) TYPE OF MEDIA: Tape (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PACKAGE: Patentln Relay # 1.0, Version # 1.30 (OEB) (2) INFORMATION FOR SEC ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 31 base pairs (B) TYPE: nucleotide (C) NUMBER OF HEBRAS: simple (D) CONFIGURATION: Linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: GAGAAGCTTG CCATGGCCCG TCCTATCGTA A 31 (2) INFORMATION FOR SEC ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 28 base pairs (B) TYPE: nucleotide (C) NUMBER OF HEBRAS: Simple (D) CONFIGURATION: Linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: GAGGATCCGT TTGACGGAAG CGACAGTA 28 12) INFORMATION FOR SEC ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleotide (C) NUMBER OF HEBRAS: simple (D) CONFIGURATION: Linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: AAGGATCCAC CATGGCTAGT CAAACAGAAA 30 (2) INFORMATION FOR SEC ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 29 base pairs (B) TYPE: nucleotide (C) NUMBER OF HEBRAS: simple (D) CONFIGURATION: Linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: AAGAATTCAG ATCTTCATTC ATAAATCCA 29 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 53pairs of bases (B) TYPE: nucleotide (C) NUMBER OF HEBRAS: simple (D) CONFIGURATION: Linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: CCGGGAGATG GGGGAGGCTA ACGGAGGTGG CGGTTCTGGT GGCGGAGGCT CCG 53 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 53 base pairs (B) TYPE: nucleotide (C) NUMBER OF HEBRAS: simple (D) CONFIGURATION: Linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: GATCCGGAGC CTCCGCCACC AGAACCGCCA CCTCCGTTAG CCTCCCCCAT CTC 53 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleotide (C) NUMBER OF HEBRAS: simple (D) CONFIGURATION: Linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: GAGAATTCCG GAGGCGGTGG CTCCCGTCCT ATCGTA 36 (2) INFORMATION FOR SEC ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 28 base pairs (B) TYPE: nucleotide (C) NUMBER OF HEBRAS: simple (D) CONFIGURATION: Linear (ü) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: GAGGATCCGT TTGACGGAAG CGACAGTA 28 (2) INFORMATION FOR SEC ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 14 amino acid (B) TYPE: amino acid (D) CONFIGURATION: Linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 9 : Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 Table 1. CONSTANT KINETICS OF GUANILATO KINASES OF YEAST AND OF HUMAN ERYTHROCYTES FOR GCVMP and ACVMP Table 2 - COUPLING TK-GMPK-NDPK:% FOR NONE OF THE PRODUCTS FORMED i-O Table 3: GCV and ACV phosphorylation by the coupling or combination of the TK and GMPK enzymes pmmol of GCVDP formed It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates.

Having described the invention as above, the content of the following is claimed as property.

Claims (44)

1. Useful composition for the release and in vivo production of a combination of enzymes, characterized in that it comprises: - a first nucleic acid encoding an enzyme capable of phosphorylating a nucleoside analog, to generate a monophosphate analog, a second nucleic acid encoding an enzyme capable of phosphorylating said monophosphate analog, to generate a diphosphate analog, and, - a third nucleic acid encoding an enzyme capable of phosphorylating said diphosphate analog, to generate a toxic triphosphate analogue.

2. Composition according to claim 1, characterized in that the first nucleic acid codes for a thymidine kinase.

3. Composition according to claim 1, characterized in that the second nucleic acid codes for a guanylate kinase.

4. Composition according to claim 1, characterized in that the third nucleic acid encodes a nucleoside diphosphate kinase.

5. Composition according to claim 1, characterized in that the nucleic acids are contained in the same vector.

6. Composition according to claim 1, characterized in that the nucleic acids are contained in several vectors.

7. Composition according to claim 5 or 6, characterized in that the vectors are plasmid or viral vectors.

8. Composition containing a nucleic acid encoding a thymidine kinase and a second nucleic acid encoding a nucleoside diphosphate kinase.

9. Composition according to claim 8, characterized in that the nucleoside diphosphate kinase is of non-human eukaryotic origin.

10. Composition according to claim 9, characterized in that the nucleoside diphosphate kinase is chosen from the NDPK of yeast or beef.

11. Composition according to claim 10, characterized in that the nucleic acid encoding the thymidine kinase and the nucleic acid encoding the nucleoside diphosphate kinase are coupled and encoded for a protein having both the TK and NDPK activities.

12. Composition according to claim 8, characterized in that it also comprises a nucleic acid encoding a guanylate kinase.

13. Composition according to claim 12, characterized in that the nucleic acid codes for a yeast guanylate kinase.

14. Composition containing a nucleic acid encoding a thymidine kinase and a second nucleic acid encoding a non-human guanylate kinase.

15. Composition according to claim 14, characterized in that the guanylate kinase is of yeast origin.

16. Composition according to claim 15, characterized in that the nucleic acid encoding the thymidine kinase and the nucleic acid encoding the guanylate kinase are coupled and encoded for a protein having both the TK and GUK activities.

17. Composition according to one of claims 1 to 16, characterized in that the thymidine kinase is of viral origin.

18. Vector comprising: - a first nucleic acid encoding a thymidine kinase, and, - a second nucleic acid encoding a non-human guanylate kinase.

.19. Vector comprising: - a first nucleic acid encoding a thymidine kinase, and, - a second nucleic acid encoding a nucleoside diphosphate kinase.

20. Vector according to claim 19, characterized in that the nucleoside diphosphate kinase is of non-human eukaryotic origin, preferably bovine or yeast.

21. Vector according to claim 19 or 20, characterized in that it also comprises a nucleic acid encoding a guanylate kinase.

22. Vector according to one of claims 18 to 21, characterized in that the thymidine kinase is of viral origin.

23. Vector according to claims 18 to 22, characterized in that the different nucleic acids are placed under the control of different promoters.

24. Vector according to claims 18 to 22, characterized in that the different nucleic acids form a polycistronic unit under the control of a single promoter.

25. Vector according to claims 18, 22 and 24, characterized in that it comprises a first nucleic acid encoding a thymidine kinase of viral origin and a second nucleic acid encoding a non-human guanylate kinase, said nucleic acids are coupled and encoded for a protein that has both TK and GUK activity.

26. Vector according to claims 18 to 25, characterized in that it is a plasmid or viral vector.

27. Composition comprising: - a combination of enzymes capable of triphosphorylating a nucleoside analog, and - said nucleoside analog, with the purpose of a simultaneous administration, separated, or extended in time.

28. Composition for the in vivo production of a nucleoside triphosphate analog comprising, under separate conditions or together of: - a nucleoside analog - a nucleic acid encoding a thymidine kinase - a nucleic acid encoding a guanylate kinase, Y, - a nucleic acid encoding a nucleoside diphosphate kinase.

29. Composition for the in vivo production of a nucleoside triphosphate analog comprising, under separate conditions or together of: - a nucleoside analog - a nucleic acid encoding a thymidine kinase, and - a nucleic acid encoding a nucleoside diphosphate cina3a.

30. Composition according to claims 27 to 29, characterized in that the nucleoside analog is a guanosine analogue.

31. Composition according to claim 30, characterized in that the guanosine analogue is chosen from ganciclovir, acyclovir and penciclovir.

32. Composition comprising a combination of enzymes involved in the phosphorylation of nucleosides, at least one of these enzymes is of non-human eukaryotic origin.

33. Method for the destruction of proliferating cells comprising the administration in said cells of a combination of enzymes comprising a TK and an NDPK and a nucleoside analogue.

34. Method according to claim 33, characterized in that the combination further comprises a guanylate kinase.

35. Method for the destruction of proliferating cells comprising the administration in said cells of a combination of enzymes comprising a TK and a non-human NDPK and a nucleoside analogue.

36. Method according to claims 33 to 35, characterized in that the nucleoside analogue is a guanosine analogue.

37. Method according to claim 36, characterized in that the guanosine analogue is chosen from ganciclovir, acyclovir and penciclovir.

38. Method according to claims 33 to 37, characterized in that the enzymes are administered in the cells by administration of nucleic acids encoding said enzymes.

39. A triphosphorylation process of a nucleoside analog comprising comprising said analogue with a combination of enzymes, at least one of which is of viral origin.

40. Use of the nucleoside diphosphate kinase or of a nucleic acid encoding the same, in combination with a thymidine kinase or a nucleic acid encoding a thymidine kinase and a nucleoside analog, for the preparation of a pharmaceutical composition intended for destruction of proliferating cells.

41. Nucleic acid that codes for a coupling protein between a thymidine kinase of viral origin and a guanylate kinase.

42. Nucleic acid according to claim 41, characterized in that the guanylate kinase is of yeast origin.

43. Nucleic acid that codes for a coupling protein between a thymidine kinase of viral origin and a nucleoside diphosphate kinase.

44. Protein encoded by a nucleic acid according to claims 41 to 43.