WO1996030512A1 - Conditional expression system - Google Patents

Abstract

A novel conditional gene expression system particularly using the creation and expression of bispecific chimeric molecules including a domain capable of selectively binding a given DNA sequence and a sensing domain capable of specifically binding a transactivator or transrepressor or a transactivator or transrepressor complex.

Description

 CONDITIONAL EXPRESSION SYSTEM

The present invention relates to a new system for the conditional expression of genes. It also relates to the use of this system in gene or cell therapy, to very selectively target the expression of genes of interest.

Gene and cell therapies consist in correcting a deficiency or an anomaly (mutation, aberrant expression, etc.) or in ensuring the expression of a protein of therapeutic interest by introducing genetic information into the affected cell or organ. . This genetic information can be introduced either ex vivo in a cell extracted from the organ, the modified cell then being reintroduced into the body (cell therapy), or directly in vivo in the appropriate tissue (gene therapy). Different techniques exist for performing gene transfer, including various transfection techniques involving chemical or biochemical, natural or synthetic vectors such as DNA and DEAE-dextran complexes (Pagano et al., J. Virol. 1 (1967) 891), DNA and nuclear proteins (Kaneda et al., Science 243 (1989) 375), DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), employment liposomes (Fraley et al., J. Biol. Chem. 255 (1980) 10431), cationic lipids, etc. Another technique is based on the use of viruses as vectors for gene transfer. In this regard, different viruses have been tested for their ability to infect certain cell populations. In particular, retroviruses (RSV, HMS, MMS, etc.), the HSV virus, adeno-associated viruses and adenoviruses. One of the major difficulties for the development of these gene and cell therapies however lies in the selectivity of the treatment. Depending on the applications, depending on the gene to be transferred, it is important to be able to target certain tissues or only certain parts of the body in order to concentrate the therapeutic effect and limit dissemination and side effects. This targeting can be done by using vectors having a given cell specificity. Another approach is to use expression signals specific for certain cell types. In this regard, so-called specific promoters have been described in the literature, such as the promoter of genes coding for pyruvate kinase, villin, GFAP, the promoter of the intestinal fatty acid binding protein, the promoter of α actin of smooth muscle cells, or the promoter of the genes of apo-AI, apo-AII, human albumin, etc. However, these promoters have certain drawbacks and in particular they present a certain transcriptional background noise which can be troublesome for the expression of toxic genes and they are limited to certain cells and therefore cannot be used for any application.

The present invention now describes a novel conditional gene expression system, which is particularly selective and efficient. One of the advantageous characteristics of the system of the invention lies in its capacity to express a gene not as a function of a cell type, but as a function of the presence of a particular cellular element or of a particular physiological situation. This system makes use of bispecific chimeric molecules comprising a domain capable of selectively binding a defined DNA sequence and a detector domain capable of specifically binding a transactivator or a transactivator complex.

A first aspect of the present invention lies more particularly in the creation and expression of chimeric bispecific molecules comprising a domain capable of selectively binding a defined DNA sequence and a domain capable of specifically binding a transactivator or transrepressor or a transactivator complex or transrepressor.

Another aspect of the present invention resides in a nucleic acid sequence coding for a chimeric molecule as defined above. before, as well as a whole expression vector comprising said nucleic sequence.

Another aspect of the invention consists of a conditional gene expression system comprising (i) a chimeric molecule as defined above and (ii) an expression cassette comprising a regulatory sequence, a minimal promoter (including activity depends on the presence of a transactivator) and said gene.

Another aspect of the invention also resides in an expression vector comprising - a nucleic sequence coding for a chimeric molecule as defined above and

- said expression cassette.

The conditional expression system of the invention is particularly suitable for use in gene or cell therapy, for very selective targeting of the expression of genes of interest.

One of the components of the system of the invention therefore consists of particular bispecific chimeric molecules comprising a domain capable of selectively binding a defined DNA sequence and a domain capable of specifically binding a transactivator or a transactivator complex. The bispecificity of the molecules of the invention resides on the one hand in their capacity to link a defined DNA sequence (generally designated regulatory or operator sequence) and on the other hand in their capacity to specifically recruit a transactivating or transrepressant protein domain allowing induce or suppress gene expression.

The invention relates in particular to the development of bispecific chimeric molecules allowing the recruitment of any transcriptional factor whose activation or inactivation leads to a situation pathophysiological. The bispecific chimeric molecules according to the invention thus allow the selective recruitment of transcriptional transactivators specific for a pathophysiological state, the binding of these transcriptional factors to promoters by means of a binding of these molecules to defined DNA sequences. located near these promoters (regulatory or operator sequences), and thus the conditional expression of genes (Figure 1).

The invention also relates to the development of chimeric bispecific molecules allowing the recruitment not of a molecule carrying a transactivating domain but of a transcriptional transactivating complex, that is to say of a complex formed between a target molecule present in a cell and a molecule carrying a transactivating domain (Figure 2). In this case, the transactivator complex is preferably formed by means of a second bispecific chimeric molecule comprising a transactivator domain and a domain for selective binding to said cell molecule. The binding of this second molecule allows the formation of a transcriptional transactivating binary complex, which complex is then recruited by the detector system of the invention. The binding of this ternary complex near promoters thus allows the regulated expression of genes. This type of construction advantageously makes it possible to extend the conditions of use of the system of the invention to the detection of any intracellular molecule devoid of transactivating domain, whether it is an endogenous molecule or a molecule of infectious origin for example.

The system of the invention thus makes it possible, thanks to a very selective detection system ("sensor") to activate the expression of genes of interest only in the presence of target proteins. They can be transcriptional factors appearing during physiological or pathophysiological situations, or any endogenous molecule or of infectious origin for example. The system of the invention indeed contains a very sensitive and very selective detector element making it possible to condition the expression of a gene to the presence, the appearance or the disappearance of any molecule in a cell. In the context of the present invention, the term transactivator designates any transactivating factor for transcription or any protein comprising a transcriptional transactivating domain. The transactivating complex designates the complex formed between a molecule present in a cell and a bispecific molecule of the invention comprising a transactivating domain and a specific binding domain for said molecule. The expression system of the invention can be used to recruit any transactivating protein or carrying a transactivating domain, and in particular any protein of viral, parasitic, mycobacterial or cellular origin having a transcriptional transactivating activity. Among the transcriptional factors of viral origin, there may be mentioned in particular the Tat protein of the HIV virus, the E6 / E7 proteins of the papilloma virus or also the EBNA protein of the Epstein Barr virus. These proteins have a transcriptional transactivating domain and are present only in cells infected with these viruses, that is to say under special pathophysiological conditions. The conditional expression system according to the invention advantageously allows a detection of this physiological situation (the appearance of these specific transactivators of viral infection) and the induction of a selective expression of given gene (s) . Among the cellular proteins, mention may preferably be made of the mutated or wild-type p53 protein. The p53 protein consists of 393 amino acids. In its wild form, it is a tumor suppressor capable of negatively regulating growth and cell division. This activity is linked to the presence of a transcriptional transactivator domain in the structure of the p53 protein, located in the N-terminal region of the protein (residues 1-100 approximately). In some situations, wild p53 is also capable of inducing apoptosis (Yonish-Rouach et al., Nature, 352, 345-347, 1991). These properties appearing in stressful situations where the integrity of cellular DNA is threatened, p53 has been suggested to be a "guardian of the genome". The presence of mutated p53 in approximately 40% of human tumors, all types combined, reinforces this hypothesis and underlines the probably crucial role played by mutations of this gene in tumor development (for reviews, see Montenarh, Oncogene, 7, 1673- 1680, 1992; Oren, FASEB J., 6, 3169-3176, 1992; Zambetti and Levine, FASEB J., 7, 855-865, 1993). In the context of the present invention, it is possible to selectively recruit the transactivating domain of the p53 protein and thus to induce controlled expression of gene (s) only in cells containing this protein. It is particularly advantageous according to the invention to specifically recruit the mutated forms of the p53 protein which, as indicated above, appear in physiopathological situations of cellular hyperproliferation (cancer type). This targeting can preferably be carried out by means of a specific binding domain to the mutated forms of the p53 protein. There is, however, a de facto specificity linked to the accumulation of mutated forms which have a half-life much higher than the wild form. The system of the invention can also be used to induce selective expression of gene (s) by detection of any target molecule present in a cell. The detected protein is preferably a protein appearing in a cell in abnormal situations (infection, hyperproliferation, etc.). They may in particular be viral proteins such as proteins of structure or function of a virus, and in particular of the HIV virus, hepatitis, herpes, etc. They may also be proteins specific to a state of cellular hyperproliferation such as in particular the proteins myc, fos, jun, cyclins, etc.

One of the properties of the chimeric molecules of the invention therefore lies in their ability to bind to specific regions of DNA (regions regulators or operator). This bond makes it possible to bring the transactivating domain close to the promoter and therefore to activate the expression of a gene placed under the control of said promoter.

The domain capable of selectively binding a defined DNA sequence present in the molecules of the invention is essentially of protein origin. More preferably, this domain derives from a prokaryotic or eukaryotic protein capable of interacting with DNA sequences. Numerous genetic and structural studies have now made it possible to precisely define, within proteins interacting with double-stranded DNA sequences, the domains responsible for these interactions.

Among the prokaryotic proteins interacting with double-stranded DNA sequences, mention may be made in particular of bacterial repressors and, preferably, the tetracycline repressor of E. Coli and the repressor Cro of the bacteriophage Lambda. The tetracycline repressor (tetR) of E.coli is a protein of about 210 amino acids. In E.coli, tetR negatively controls the transcription of genes mediating resistance to this antibiotic within the tet operon. In the absence of tetracycline, the tetR repressor binds to DNA at the level of a specific sequence (designated operator sequence or Tetop) and represses the transcription of the resistance gene. On the contrary, in the presence of tetracycline, the tetR repressor no longer binds to the tetop operator allowing a constitutive transcription of the gene (Hillen.W and Wissmann.A. (1989) in Protein-Nucleic Acid Interaction. Topics in Molecular and Structural Biology.eds, Saenger.W. And Heinemann.U. (Macmillan, London), Vol. 10, pp. 143-162). The tetR sequence has been published (it is reproduced in particular in WO94 / 04682). The specific double-strand DNA sequence for binding of tetR to DNA (Tetop) is composed of the following motif: TCTCTATCACTGATAGGGA (SEQ ID No. 1). This pattern can be repeated several times to increase the affinity and efficiency of the system. So the regulatory sequence can include up to 10 patterns and preferably contains 2 patterns (Tetop2) or 7 patterns (Tetop7) (see Figure 3).

The Cro protein was originally defined as a regulator of the expression of the repressor Cl (Eisen.H. Et al. (1970) PNAS 66, pp855). Cloning of the cro gene made it possible to identify a protein of 66 amino acids (SEQ ID No. 21; Roberts; T et al. (1977) Nature 270, pp274). Cro exercises its physiological role by preferentially attaching itself to the operator OR3 of Lambda.

The specific double-strand DNA sequence for binding of Cro to DNA (region designated OR3) is composed of the following bases:

TATCACCGCAAGGGATA (SEQ ID No. 2) This region can also be repeated several times to increase the affinity and efficiency of the system (see Figure 4).

Among the eukaryotic proteins interacting with double-stranded DNA sequences, it is preferred to use for the construction of the molecules of the invention the proteins or domains derived from the STAT, p53 or NFkB proteins (Inoue et al., PNAS 89 (1992) 4333 ). Concerning the p53 protein, its DNA binding domain is located in the central region of the protein and, more precisely, in the region between amino acids 102 to 292 (Pavletich et al., Genes & Dev. 7 ( 1993) 2556).

As indicated above, the domain capable of selectively binding a defined DNA sequence present in the molecules of the invention is preferably derived from a prokaryotic or eukaryotic protein capable of interacting with a region of double-stranded DNA. The domain used for the construction of the molecules of the invention can consist of the whole of the protein or of a fragment thereof comprising the region of interaction with DNA. This domain has been identified for various proteins and in particular TetR (see for example Berens et al., J. Biol. Chem. 267 (1992) 1945). It can also consist of a derivative of this protein or of the fragment having retained the properties of DNA linkage. Such derivatives are in particular proteins having modifications of one or more amino acids, for example to allow their fusion with the other domains of the molecules of the invention, prepared according to conventional techniques of molecular biology. Derivatives of the TetR and Cro proteins for example have been described in the literature, having point mutations and / or deletions (Hecht et al., J. Bact. 175 (1993) p. 1206; Altschmied et al., EMBO J 7 (1988) 4011; Baumeister et al., Proteins 14 (1992) 168; Hansen et al., J. Biol. Chem. 262 (1987) 14030). The binding capacity of these derivatives to a defined DNA sequence can then be tested by incubating the derivative prepared with the regulatory sequence and detecting the complexes formed. In addition, the derivatives can also be proteins with improved DNA binding properties (specificity, affinity, etc.).

According to a preferred embodiment, the domain capable of selectively binding a defined DNA sequence present in the molecules of the invention is derived from a prokaryotic protein. This type of construction is particularly advantageous since these proteins, of non-human origin, recognize double stranded DNA sites of at least 14 nucleotides. The probability of finding the same sequence within the human genome is almost zero and therefore the expression system obtained is all the more selective.

In a preferred embodiment, the domain capable of selectively binding a defined DNA sequence present in the molecules of the invention is derived from the proteins tetR or Cro. It is particularly advantageous to use the complete tetR or Cro proteins (SEQ ID No. 21).

The domain capable of specifically binding the transcriptional transactivator or the transcriptional transactivator complex present in the molecules of the invention can be of different types. It may in particular be an oligomerization domain in the case where the transactivator or the targeted transactivator complex also has such a domain. It can also be any synthetic or natural domain known to interact with said transactivator or transactivator complex. It may also be an antibody or a fragment or derivative of an antibody directed against the transactivator or transactivator complex.

Among the oligomerization domains which can be used in the context of the invention, mention may be made more particularly of leucine-zippers, SH2 domains or SH3 domains for example. Leucine-zippers are amphipatic α-helices which contain 4 or 5 leucines every 7 amino acids. This periodicity allows the location of leucines at approximately the same position on the α helix. Dimerization is underpinned by hydrophobic interactions between the side chains of leucines from two contiguous zipper domains (Vogt et al., Trends In Bioch. Science 14 (1989) 172). The SH2 domains are known to interact with specific peptide sequences phosphorylated in tyrosine. The SH3 domains can be used to form an oligomer with any transactivator or transactivator complex comprising the corresponding proline-rich peptide (Pawson et al., Current Biology 3 (1993) 434). It is also possible to use regions of proteins known to induce oligomerization, such as in particular the C-terminal region of the p53 protein. The use of this region makes it possible to selectively recruit the p53 proteins present in a cell. Preferably, in the context of the invention, a region of p53 between amino acids 320-393 (SEQ ID No. 3), 302-360 or 302-390 is used.

Among the synthetic or natural domains known to interact with the molecule comprising the targeted transactivating element, mention may be made, for example, of the region of the MDM2 protein interacting with the p53 protein. This type of construction thus makes it possible to recruit, as transactivator, the wild-type or mutated p53 protein. A specific binding domain to the preferred transcriptional transactivator of the invention consists of an antibody or an antibody fragment or derivative. The fragments or derivatives of antibodies are for example the Fab or F (ab) ′ 2 fragments, the VH or VL regions of an antibody or also single chain antibodies (ScFv) comprising a VH region linked to a VL region by a arms. This type of domain is particularly advantageous since it can be directed against any molecule.

Antibodies, molecules of the immunoglobulin superfamily, are made up of different chains (2 heavy (H) and 2 light (L)) themselves made up of different domains (variable domain (V) junction domain (J), etc.). The transactivator binding domain or transactivating complex present in the molecules of the invention advantageously consists of a fragment or derivative of antibodies comprising at least the binding site of the antigen. This fragment can be either the variable domain of a light (V | _) or heavy (VH) chain, optionally in the form of a Fab or F (ab ') 2 fragment or, preferably, in the form of single chain antibodies (ScFv ). The single chain antibodies used for the construction of the molecules of the invention consist of a peptide corresponding to the binding site of the variable region of the light chain of an antibody linked by a peptide arm to a peptide corresponding to the binding site of the variable region of the heavy chain of an antibody. The construction of nucleic acid sequences encoding such modified antibodies according to the invention has been described for example in patent US Pat. No. 4,946,778 or in applications WO94 / 02610, WO94 / 29446. It is illustrated in the examples.

A preferred construct according to the present invention comprises a binding domain to a p53 protein. It is more preferably an antibody derivative directed against a p53 protein. A particular embodiment consists of a single chain antibody directed against p53. AT As a specific example, the ScFv of sequence SEQ ID No. 4 is used, the construction of which is described in the examples.

The DNA binding domain and the transactivator binding domain are generally linked together via an arm. This arm generally consists of a peptide conferring sufficient flexibility for the two domains of the molecules of the invention to be able to function independently. This peptide is generally composed of uncharged amino acids, which do not interfere with the activity of the molecules of the invention, such as for example glycine, serine, tryptophan, lysine or proline. The arm generally comprises from 5 to 30 amino acids and, preferably, from 5 to 20 amino acids. Examples of peptide arms which can be used for the construction of the molecules of the invention are for example:

- GGGGSGGGGSGGGGS (SEQ ID No. 5) - PKPSTPPGSS (SEQ ID No. 6) whose coding sequence is

CCCAAGCCCAGTACCCCCCCAGGTTCTTCA (SEQ ID No. 6).

Preferred examples of a molecule according to the invention are in particular the following molecules: a) ScFv-tag-Hinge-TET or Cro (FIG. 5A) This type of molecule comprises:

- a binding domain to a transactivator consisting of a single chain antibody,

- a tag peptide sequence recognized by a monoclonal antibody allowing the immunological detection of the molecule. This sequence can for example be the VSV epitope of sequence MNRLGK (SEQ ID No. 7) whose coding sequence is ATGAACCGGCTGGGCAAG (SEQ ID No. 7), or the myc epitope of sequence EQKLISEEDLN (SEQ ID No. 8) the coding sequence is GAACAAAAACTCATCTCAGAAGAGGATCTGAAT (SEQ ID No. 8), recognized by the antibody 9E10. - a peptide arm of sequence SEQ ID No. 6 (Hinge) and

- a DNA binding domain consisting of the TET or Cro protein. Preferably, the ScFv is directed against a p53 protein. b) ScFv-Hinge-TET or Cro (Figure 5B) This type of molecule has the same elements as molecule a) with the exception of the tag sequence which is absent. c) ScFv-TET or Cro (Figure 5C)

This type of molecule simply comprises a binding domain to a transactivator consisting of a single chain antibody and a DNA binding domain consisting of the TET or Cro protein. It has neither arms nor tag sequence. In this construction, the transactivator binding domain is located in the N-terminal part of the molecule and the DNA binding domain in the C-terminal part. d) TET or Cro-ScFv (Figure 5D) This type of molecule is similar to type c) above. The difference essentially lies in the arrangement of the domains: the binding domain to the transactivator is now located in the C-terminal part of the molecule and the DNA binding domain in the N-terminal part. e) TET or Cro-Hinge-ScFv (Figure 5E) This type of molecule has the same elements as molecule b) above. The difference essentially lies in the arrangement of the domains: the binding domain to the transactivator is now located in the C-terminal part of the molecule and the DNA binding domain in the N-terminal part. f) Oligom-tag-Hinge-TET or Cro (Figure 5A)

This type of molecule is similar to type a), except for the transactivator binding domain which is replaced by the oligomerization domain with the p53 protein of sequence SEQ ID No. 3. This molecule makes it possible to recruit the p53 proteins mutated cells appearing in tumor cells. g) Oligom-Hinge-TET or Cro (Figure 5B)

This type of molecule is similar to type b), except for the transactivator binding domain which is replaced by the oligomerization domain with the p53 protein of sequence SEQ ID No. 3. h) Oligom-TET or Cro ( Figure 5C)

This type of molecule is similar to type c), except for the transactivator binding domain which is replaced by the oligomerization domain with the p53 protein of sequence SEQ ID No. 3. i) TET or Cro-Oligom ( Figure 5D) This type of molecule is similar to type d), except for the transactivator binding domain which is replaced by the oligomerization domain with the p53 protein of sequence SEQ ID No. 3. j) TET or Cro -Hinge-Oligom (Figure 5E)

This type of molecule is similar to type e), except for the transactivator binding domain which is replaced by the oligomerization domain with the p53 protein of sequence SEQ ID No. 3.

Furthermore, in each of these molecules, the peptide arm can be easily replaced by the sequence (G4S) 3 (SEQ ID No. 5).

Another object of the present invention resides in a nucleic acid sequence coding for a chimeric molecule as defined above. It is advantageously a DNA sequence, in particular cDNA. It can also be an RNA. The sequences of the invention are generally constructed by assembling, within a cioning vector, sequences coding for the different fields according to the conventional techniques of molecular biology. The nucleic acid sequences of the invention can optionally be modified chemically, enzymatically or genetically, with a view to generating stabilized domains, and / or multifunctional domains, and / or of reduced size, and / or with the aim of promoting their location in this or that intracellular compartment. Thus, the nucleic acid sequences of the invention can include sequences encoding nuclear localization peptides (NLS). In particular, it is possible to merge the sequences of the invention with the sequence coding for the NLS of the SV40 virus, the peptide sequence of which is as follows: PKKKRKV (SEQ ID No. 9) (Kalderon et al, Cell 39 (1984 ) 499).

The nucleic acid sequences according to the invention advantageously form part of an expression vector, which can be of plasmid or viral nature.

Another object of the present invention resides in a fusion protein comprising a transcriptional transactivating domain and a binding domain specific to a given molecule, optionally linked by a peptide arm, as well as any nucleic acid sequence coding for such a fusion. The transactivating domain can come from any transcriptional transactivating protein, such as p53, VP16, EBNA, E6 / E7, Tat, etc.

Another subject of the invention consists of a conditional gene expression system comprising:

- a chimeric molecule as defined above and

- An expression cassette comprising a regulatory sequence, a minimal transcriptional promoter and said gene.

The expression cassette contains the elements necessary for activation of gene expression by the transactivator or transactivator complex recruited by the bispecific molecule. Thus, the regulatory sequence is the DNA binding sequence of the expressed chimeric molecule. When the DNA binding domain of the chimeric molecule is represented by all or part of TetR, the regulatory sequence comprises the sequence SEQ ID No. 1 or a derivative thereof, optionally repeated several times. It is preferably the sequence op2 (comprising 2 repeated Tetop motifs) or Op7 (comprising 7 repeated Tetop motifs such as described for example in Weinmann et al., The Plant Journal 5 (1994) 559). Likewise, when the DNA binding domain of the chimeric molecule is represented by all or part of Cro, the regulatory sequence comprises the sequence SEQ ID No. 2 or a derivative thereof, optionally repeated several times. It is preferably the OR3 sequence. The derivatives of sequences SEQ ID No. 1 and 2 can be any sequence obtained by modification of a genetic nature (mutation, deletion, addition, repetition, etc.) and retaining the capacity to specifically bind a protein. Such derivatives have been described in the literature (Baumeister et al cited above, Tovar et al., Mol. Gen. Genêt. 215 (1988) 76, WO94 / 04672).

Concerning the minimal transcriptional promoter, it is a promoter whose activity depends on the presence of a transactivator. Therefore, in the absence of the chimeric molecule, the promoter is inactive and the gene is little or not expressed. On the other hand, in the presence of the chimeric molecule, the recruited transactivator or transactivator complex makes it possible to induce the activity of the minimal promoter and thus the expression of the gene of interest. The minimum promoter generally consists of a TATA or INR box. These elements are in fact the minimum elements necessary for the expression of a gene in the presence of a transactivator. The minimal promoter can be prepared from any promoter by genetic modification. As a preferred example of a candidate promoter, mention may be made of the promoter of the thymidine kinase gene. Interesting results have more precisely been obtained with a minimal promoter derived from the TK promoter composed of nucleotides -37 to +19. The minimal promoter can also be derived from human CMV. In particular, it can consist of the fragment between the nucleotides -53 to +75 or -31 to +75 of the CMV. Any conventional promoter can however be used such as for example the promoter of the genes coding for chloramphenicol acetyl transferase, β-galactosidase or even luciferase. The expression cassette advantageously consists of the following elements:

As a regulatory sequence, a sequence comprising the sequence SEQ ID No. 1 or 2 or a derivative thereof, optionally repeated several times,

As a minimal promoter, a promoter derived from the promoter of the thymidine kinase (TK) gene,

- a coding sequence of interest.

Even more preferably, the minimal promoter consists of the region -37 to +19 of the promoter of the thymidine kinase gene.

Advantageously, the expression cassette is chosen from cassettes of Tetop2.TK-Gene structure; Tetop7.TK-Gene and OR3.TK-Gene.

Another aspect of the invention resides in an expression vector comprising a nucleic acid sequence coding for a chimeric molecule and an expression cassette as defined above. In the vectors of the invention, the nucleic acid sequence coding for the chimeric molecule and the expression cassette can be inserted in the same orientation or in the opposite orientations. Furthermore, the vector may be of plasmid or viral nature.

Among the viral vectors, there may be mentioned more preferably the adenoviruses, the retroviruses, the herpes viruses or even the associated adeno viruses. The viruses according to the present invention are defective, that is to say incapable of replicating autonomously in the target cell. Generally, the genome of the defective viruses used in the context of the present invention is therefore devoid of at least the sequences necessary for the replication of said virus in the infected cell. These regions can be either eliminated (in whole or in part), or made non-functional, or substituted by other sequences and in particular by the sequences of the invention. Preferably, the defective virus nevertheless retains the sequences of its genome which are necessary for the packaging of the viral particles.

With regard more particularly to adenoviruses, various serotypes, whose structure and properties vary somewhat, have been characterized. Among these serotypes, it is preferred to use, in the context of the present invention, human adenoviruses of type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin (see application WO94 / 26914). Among the adenoviruses of animal origin which can be used in the context of the present invention, mention may be made of adenoviruses of canine, bovine, murine origin (example: Mav1, Beard et al., Virology 75 (1990) 81), ovine, porcine , avian or even simian (example: after-sales service). 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, in the context of the invention, adenoviruses of human or canine or mixed origin are used.

Preferably, the genome of the recombinant adenoviruses of the invention comprises at least the ITRs and the packaging region of an adenovirus, and the nucleic acid sequence coding for a chimeric molecule and an expression cassette as defined above. before. More preferably, in the genome of the adenoviruses of the invention, the E1 region at least is non-functional. The viral gene considered can be made non-functional by any technique known to those skilled in the art, and in particular by total suppression, substitution (for example by the sequences of the invention), partial deletion, or addition of one or more bases in the gene (s) considered. Such modifications can be obtained in vitro (on isolated DNA) or in situ, for example, by means of genetic engineering techniques, or by treatment with mutagenic agents. Other regions can also be modified, and in particular the region E3 (WO 95/02697), E2 (WO 94/28938), E4 (WO 94/28152, WO 94/12649, WO 95/02697) and L5 (WO 95/02697). According to a preferred embodiment, the adenovirus according to the invention comprises a deletion in the E1 and E4 regions. According to another preferred embodiment, it comprises a deletion in region E1 at the level of which the region E4 and the sequences of the invention are inserted (Cf FR 94 13355).

The defective recombinant adenoviruses according to the invention can be prepared by any technique known to those skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3 (1984) 2917). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid carrying inter alia the DNA sequences of the invention (sequence coding for the chimeric molecule + expression cassette). Homologous recombination occurs after co-transfection of said adenovirus and plasmid in an appropriate cell line. The cell line used must preferably (i) be transformable by said elements, and (ii), contain the sequences capable of complementing the part of the genome of the defective adenovirus, preferably in integrated form to avoid the risks of recombination. As an example of a line, mention may be made of the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains in particular, integrated into its genome, the left part of the genome an Ad5 adenovirus (12%) or lines capable of complementing the E1 and E4 functions as described in particular in applications No. WO 94/26914 and WO95 / 02697. Then, the adenoviruses which have multiplied are recovered and purified according to conventional techniques of molecular biology, as illustrated in the examples.

Concerning adeno-associated viruses (AAV), these are DNA viruses of relatively small size, which integrate into the genome of cells they infect, in a stable and site-specific manner. They are capable of infecting a wide spectrum of cells, without inducing an effect on cell growth, morphology or differentiation. Furthermore, they do not seem to be involved in pathologies in humans. The AAV genome has been cloned, sequenced and characterized. It comprises approximately 4,700 bases, and contains at each end an inverted repeat region (ITR) of approximately 145 bases, serving as an origin of replication for the virus. The rest of the genome is divided into 2 essential regions carrying the packaging functions: the left part of the genome, which contains the rep gene involved in viral replication and the expression of viral genes; the right part of the genome, which contains the cap gene coding for the capsid proteins of the virus.

The use of vectors derived from AAVs for gene transfer 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 various constructs derived from AAVs, in which the rep and / or cap genes are deleted and replaced by a gene of interest, and their use for transferring in vitro (onto cells in culture) or in vivo (directly into an organism ) said gene of interest. The defective recombinant AAVs according to the invention can be prepared by cotransfection, in a cell line infected with a human helper virus (for example an adenovirus), of a plasmid containing the nucleic sequences of the invention (sequence coding for chimeric molecule + expression cassette) bordered by two inverted repeat regions (ITR) of AAV, and of a plasmid carrying the packaging genes (rep and cap genes) of AAV. The recombinant AAVs produced are then purified by conventional techniques.

Concerning 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; EP 453242, EP178220, Bemstein et al. Broom. Eng. 7 (1985) 235; McCormick, BioTechnology 3 (1985) 689, etc.

In particular, retroviruses are integrative viruses, selectively infecting dividing cells. They therefore constitute vectors of interest for cancer applications. The genome of retroviruses essentially comprises two LTRs, an encapsidation 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 produced from different types of retroviruses such as in particular MoMuLV ("murine moloney leukemia virus"; also designated MoMLV), MSV ("murine moloney sarcoma virus"), HaSV ("harvey sarcoma virus") ; SNV ("spleen necrosis virus"); RSV ("rous sarcoma virus") or the Friend virus.

To construct recombinant retroviruses according to the invention, a plasmid comprising in particular the LTRs, the encapsidation sequence and the sequences of the invention (sequence coding for the chimeric molecule + expression cassette) is generally constructed, then used to transfect a cell line known as packaging, capable of providing in trans retroviral functions deficient in the plasmid. Generally, the packaging lines are therefore capable of expressing the gag, pol and env genes. Such packaging lines have been described in the prior art, and in particular the line PA317 (US4,861,719); the PsiCRIP line (WO90 / 02806) and the GP + envAm-12 line (WO89 / 07150). Furthermore, the recombinant retroviruses may include modifications at the level of the LTRs to suppress transcriptional activity, as well as extended packaging sequences, comprising a part of the gag gene (Bender et al., J. Virol. 61 (1987) 1639). The recombinant retroviruses produced are then purified by conventional techniques.

An example of construction of a defective recombinant virus according to the invention (retrovirus) is described in FIG. 8. This figure highlights a second advantage of the constructions according to the invention which resides in the absence of expression of the gene of interest in the packaging lines. These lines being devoid of the transactivator or transactivator complex recruited by the system of the invention, the promoter is inactive and the gene is not expressed in the production cell (FIG. 8A). It is only when the virus has effectively infected a target cell, that is to say a cell in which the transactivator or transactivator complex recruited by the system of the invention is present, that the gene is effectively expressed (FIG. 8B). This is particularly advantageous for the construction of viruses containing genes whose expression would be toxic to cells (Grb3-3, IL-2 genes, diphtheria toxin, etc.).

For the implementation of the present invention, it is very particularly advantageous to use a defective recombinant adenovirus or retrovirus. These vectors indeed have properties which are particularly advantageous for the transfer of genes into tumor cells.

Different types of non-viral vectors can also be used in the context of the invention. The conditional expression system according to the invention can in fact be incorporated into a non-viral agent capable of promoting the transfer and expression of nucleic acids in eukaryotic cells. Chemical or biochemical vectors represent an interesting alternative to natural viruses, in particular for reasons of convenience, safety and also by the absence of theoretical limit as regards the size of the DNA to be transfected. 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, where appropriate, the two nuclear membranes. To compensate for the polyanionic nature of nucleic acids, the non-viral vectors all have polycationic charges.

Among the synthetic vectors developed, cationic polymers of polylysine type, (LKLK) n, (LKKL) n, polyethylene immine and DEAE dextran or else cationic lipids or lipofectants are the most advantageous. They have the property of condensing DNA and promoting its association with the cell membrane. Among the latter, mention may be made of lipopolyamines (lipofectamine, transfectam, etc.) and various cationic or neutral lipids (DOTMA, DOGS, DOPE, etc.). More recently, the concept of targeted transfection, mediated by a receptor, has been developed which takes advantage of the principle of condensing DNA thanks to the cationic polymer while directing the binding of the complex to the membrane thanks to a chemical coupling between the cationic polymer and the ligand of a membrane receptor, present on the surface of the cell type that we want to graft. The targeting of the transferrin, insulin receptor or the hepatocyte asialoglycoprotein receptor has thus been described.

The present invention also relates to any pharmaceutical composition comprising a vector as defined above. These compositions can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, etc. administration. Preferably, the composition according to the invention contains pharmaceutically acceptable vehicles for an injectable formulation. They may in particular be saline solutions (monosodium phosphate, disodium, sodium chloride, potassium, calcium or magnesium, etc., or mixtures of such salts), sterile, isotonic, or dry compositions, in particular lyophilized, which, by addition as appropriate of sterilized water or physiological saline, allow the constitution of injectable solutes. With regard to retroviruses, they may be advantageous to directly use packaging cells or cells infected ex vivo with a view to their reimplantation in vivo, possibly in the form of neo¬ organs (WO 94/24298).

The vector doses used for the injection can be adapted according to different parameters, and in particular according to the mode of administration used, the pathology concerned or the duration of the treatment sought. In general, the recombinant viruses according to the invention are formulated and administered in the form of doses of between 10 ⁴ and 10 ¹⁴ pfu / ml. For AAVs and adenoviruses, doses of 10 to 10 ¹⁰ pfu / ml can also be used. The term pfu ("plaque forming unit") corresponds to the infectious power of a suspension of virions, and is determined by infection of an appropriate cell culture, and measures, generally after 48 hours, the number of plaques of infected cells. The techniques for determining the pfu titer of a viral solution are well documented in the literature.

The expression system according to the invention and the corresponding vectors are particularly useful for controlling the expression of genes of interest in the context of cell or gene therapies. They can thus be used to control the expression of any coding sequence of interest, and in particular of a sequence coding for a therapeutic product, whether it is a peptide, polypeptide, protein, ribonucleic acid, etc. More particularly, the gene is a DNA sequence (cDNA, gDNA, synthetic, human, animal, plant DNA, etc.) coding for a protein product such as enzymes, blood derivatives, hormones, lymphokines: interleukins, interferons , TNF, etc (FR 9203120), growth factors, neurotransmitters or their precursors or synthetic enzymes, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, etc; apolipoproteins: ApoAl, ApoAIV, ApoE, etc (FR 93 05125), dystrophin or a minidystrophin (FR 9111947), tumor suppressor genes: p53, Rb, RaplA, DCC, k-rev, etc (FR 93 04745) , genes coding for factors involved in coagulation: Factors VII, VIII, IX, etc., or all or part of a natural or artificial immunoglobulin (Fab, ScFv, etc.), a ligand RNA (WO91 / 19813), etc. . The gene of interest can also be an antisense sequence, the expression of which in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNA. Such sequences can for example be transcribed, in the target cell, into RNAs complementary to cellular mRNAs and thus block their translation into protein, according to the technique described in patent EP 140308.

The present invention is particularly suitable for the expression of sequences coding for toxic factors. It can be in particular poisons for cells (diphtheria toxin, pseudomonas toxin, ricin A, etc.) of product inducing sensitivity to an external agent (suicide genes: Thymidine kinase, cytosine deaminase, etc.) or even killer genes capable of inducing cell death (Grb3-3 (PCT / FR94 / 00542), anti-ras ScFv (W094 / 29446), etc.). The system of the invention indeed makes it possible to produce vectors, in particular viral containing these sequences without toxicity for the production cells, then then to induce the expression of these toxic molecules selectively in target cells having the desired transactivator or transactivator complex . This type of construction is therefore particularly suitable for strategies of antitumor therapies for example, in which the objective is to selectively destroy the affected cells. This system is also particularly interesting for the expression of cytokines, interferons, TNF or TGF for example, whose uncontrolled production can have very marked side effects.

The present application will be described in more detail with the aid of the following examples, which should be considered as illustrative and not limiting.

Legend of Figures

Figure 1: Representation of the conditional expression system according to the invention allowing the selective recruitment of a transactivator by means of an oligomerization domain (A) or a ScFv (B).

Figure 2: Representation of the conditional expression system according to the invention allowing the selective recruitment of a transactivating complex.

Figure 3: Representation of an expression cassette according to the invention comprising a regulatory sequence Tetop7, a minimal promoter (TATA box) and a gene (CAT).

Figure 4: Representation of an expression cassette according to the invention comprising an OR3 regulatory sequence, a minimal promoter (box

TATA) and a gene (CAT).

Figure 5: Representation of bispecific chimeric molecules according to the invention.

Figure 6: Construction of DNA sequences coding for bispecific chimeric molecules according to the invention.

Figure 7: Representation of control chimeric constructions.

Figure 8: Structure and functioning of a viral vector (retrovirus) according to the invention.

Figure 9: Study of the interaction between the hybrid molecules of the invention and a regulatory sequence.

Figure 10: Study of the interaction between the hybrid molecules of the invention and different forms of the p53 protein. Figure 11: Demonstration of the activation of the Tet-Luc cassette in SAOS-2 cells.

Figure 12: Demonstration of the activation of the Tet-Luc cassette in H358 cells.

General Molecular Biology techniques

Classical molecular biology methods such as centrifugation of plasmid DNA in cesium chloride-ethidium bromide gradient, digestion with restriction enzymes, gel electrophoresis, transformation in E. coli. precipitation of nucleic acids, etc., is described in the literature (Maniatis EI al., 1989).

The enzymes were provided by New-England Biolabs (Beverly, MA). For the ligations, the DNA fragments are separated according to their size on 0.8 to 1.5% agarose gels, purified by GeneClean (BIO101, LaJolla CA) and incubated overnight at 14 ° C in Tris buffer. -HCI pH 7.4 50 mM, MgCl2 10 mM, DTT 10 mM, ATP 2 mM, in the presence of DNA ligase from phage T4.

Amplification by PCR, (Polymerase Chain Reaction), was also carried out according to Maniatis ei sJ., 1989, with the following specifications:

- MgCl2 concentration increased to 8mM.

- Denaturation temperature 95 ° C, hybridization temperature 55 ° C, elongation temperature 72 ° C. This cycle was repeated 25 times in one

PE9600 Thermalcycler (Perkin Elmer, Norwalk CO).

The oligonucleotides are synthesized using the chemistry of phosphoramidites protected in β by a cyanoethyl group, (Sinha __ al., 1984, Giles 1985), with the automatic DNA synthesizer Applied Biosystem model 394, (Applied Biosystem, Foster City CA) , according to the manufacturer's recommendations.

The sequencing was carried out on double-stranded templates by the chain termination method using fluorescent primers. We used the Taq Dye Primer Kit from Applied Biosystem (Applied Biosystem, Foster City CA) according to the manufacturer's specifications.

Examples

Example 1: Construction of expression cassettes comprising a regulatory sequence, a minimal transcriptional promoter and a gene.

1.1. Construction of the plasmid pTETop7 / CAT

The plasmid pTETop7 / CAT contains the following elements (Figure 3):

- A regulatory sequence consisting of an interaction sequence with the tetracycline tetR repressor composed of 7 repeated Tetop motifs (SEQ ID No. 1);

- a minimal promoter derived from the promoter of the thymidine kinase gene (region -37 to + 19 carrying the TATA box);

- The sequence coding for chloramphenicol acetyl transferase (CAT) under the control of said minimal promoter. This plasmid was constructed by cloning the Smal-Bglll fragment of the plasmid pUHD10-7 (WO 94/29442) into the plasmid pKK232-8 (Pharmacia) previously digested with Smal and BamHI.

1.2. Construction of the plasmid pOR3 / CAT

The plasmid pOR3 / CAT contains the following elements (Figure 4): - A regulatory sequence consisting of an OR3 sequence for interaction with the Cro repressor (SEQ ID No. 2);

- a minimal promoter derived from the promoter of the thymidine kinase gene (region -37 to + 19 carrying the TATA box);

- The sequence coding for chloramphenicol acetyl transferase (CAT) under the control of said minimal promoter.

This plasmid was constructed in the following manner: The OR3 sequence for interaction with the Cro repressor was synthesized artificially. For this, the following two oligonucleotides were synthesized: Oligo 5533 (SEQ ID n ° 22): 5'-GATCCTATCACCGCAAGGGATAA-3 'Oligo 5534 (SEQ ID n ° 23): 3'-GATAGTGGCGTTCCCTATTTCGA-5'

These two oligonucleotides were then hybridized to reconstitute the double-stranded sequence OR3 bordered with sequences allowing its cloning oriented as follows:

GATCCTATCACCGCAAGGGATAA

GATAGTGGCGTTCCCTATTTCGA

1.3. Construction of toxic gene expression cassettes

The toxic gene expression cassettes are obtained from the plasmids described above (1.1. And 1.2.) By replacing the CAT sequence with the sequence coding for the toxic product, preferably the Grb3-3 gene (PCT / FR94 / 00542), the thymidine kinase gene, the gene coding for diphtheria toxin or pseudomonas, etc.

Example 2: Construction of a single chain antibody specific for D53 This single chain antibody was constructed according to the following protocol:

- The cDNAs coding for the VH and VL regions were obtained from the pAb421 hybridoma producing an anti-p53 antibody. For this, the total RNAs of the hybridoma were extracted and subjected to a reverse transcription reaction using random hexamers as primers. The use of this type of primer makes it possible to avoid the use of specific primers of immunoglobulins. The cDNA clones obtained are of sufficient length to clone the V regions. However, since they represent a small fraction of the total cDNA present, a prior amplification reaction must be carried out to produce sufficient DNA for cloning. For this, the cDNAs encoding the VH and VL regions were amplified separately. The primers used are oligonucleotides hybridizing at the opposite ends of the variable regions of each chain (H and L). The amplification product using the primers specific for heavy chains is a fragment of 340 pairs of bases approx. The amplification product using the primers specific to light chains is a fragment of approximately 325 base pairs.

- After purification, the cDNAs encoding the VH and VL regions of the antibody were assembled into a single chain using a nucleotide arm (L). The nucleotide arm was constructed such that one end binds to the 3 'end of the cDNA encoding the VH region and the other to the 5' end of the cDNA encoding the region VL. The sequence of the arm code for the peptide SEQ ID No. 5. The assembled sequence of approximately 700 bp contains, in the form of an Ncol-NotI fragment, the VH-L-VL sequence whose sequence is represented SEQ ID No. 4 (amino acids 9 to 241). This sequence also includes in C-terminal the tag sequence of myc (residues 256 to 266).

Example 3: Construction of nucleic acid sequences encoding bispecific chimeric molecules containing a binding domain to a transactivator consisting of a single chain antibody (ScFv).

3.1. Construction of a plasmid comprising a ScFv-myc-Hinge-TetR or Cro sequence (Figures 5A and 6)

The Ncol-NotI fragment containing the cDNA coding for the anti-p53 ScFv was first of all cloned into a plasmid of the pUC19 type. The sequence coding for the VSV (SEQ ID No. 7) or myc (SEQ ID No. 8) epitope is inserted downstream of the fragment (FIG. 6).

The sequences coding for the proteins TetR and Cro were then obtained as follows:

- The sequence coding for TetR was obtained by amplification from a template plasmid carrying the tetR sequence using the following oligonucleotides:

Oligo 5474 (SEQ ID # 10):

GGCTCTAGACCCAAGCCCAGTACCCCCCCAGGTTCTTCAACGCG TGGATCCATGTCCAGATTAGATAAAAGTAAAG Oligo 5475 (SEQ ID # 11):

CGTACGGAATTCGGGCCCTTACTCGAGGGACCCACTTTCACATTT AAGTTG

These oligonucleotides also provide the sequence coding for the Hinge peptide arm connecting the two functional domains of the molecules.

The amplified fragment therefore contains the sequence coding for the peptide arm and for the tetR DNA binding domain. This fragment was cloned at the Xbal-EcoRI sites of the plasmid obtained above to generate a plasmid containing the sequence coding for the molecule ScFv-myc-Hinge-TetR

(Figure 6).

- The sequence coding for Cro was obtained by amplification on a DNA matrix of the bacteriophage Lambda using the following oligonucleotides: Oligo 5531 (SEQ ID No. 12):

GGCTCTAGACCCAAGCCCAGTACCCCCCCAGGTTCTTCAACGCG TGGATCCATGGAACAACGCATAACCCTGAAAG

Oligo 5532 (SEQ ID # 13):

CGTACGGAATTCGGGCCCTTACTCGAGTGCTGTTG I I I I I I I GTT ACTCGG

These oligonucleotides also provide the sequence coding for the Hinge peptide arm connecting the two functional domains of the molecules.

The amplified fragment therefore contains the sequence coding for the peptide arm and for the DNA binding domain Cro. This fragment was cloned at the Xbal-EcoRI sites of the plasmid obtained above to generate a plasmid containing the sequence coding for the molecule ScFv-myc-Hinge-Cro

(Figure 6).

3.2. Construction of a plasmid comprising a ScFv-Hinge-TetR or Cro sequence (Figure 5B) This example describes the construction of plasmids carrying a sequence coding for a bispecific chimeric molecule according to the invention devoid of tag sequence.

These plasmids were obtained from the plasmids described in 3.1. above by digestion with the enzymes Notl and Xbal. This digestion makes it possible to excise the fragment carrying the region coding for the tag myc.

3.3. Construction of a plasmid comprising a ScFv-TetR or Cro sequence (Figure 5C)

This example describes the construction of plasmids carrying a sequence coding for a bispecific chimeric molecule according to the invention devoid of arms and of tag sequence.

These plasmids were obtained from the plasmids described in 3.1. above by digestion with the enzymes Notl and BamHI. This digestion makes it possible to excise the fragment carrying the region coding for the myc tag and for the Hinge peptide arm.

Example 4: Construction of nucleic acid sequences encoding bispecific chimeric molecules containing a binding domain to a transactivator consisting of an oligomerization domain.

4.1. Cloning of the oligomerization region of the p53 protein (fragment 320-393).

The cDNA encoding the oligomerization region of the p53 protein (SEQ ID No. 3) was obtained by PCR amplification on a plasmid carrying the cDNA of human wild p53 using the following oligonucleotides: Oligo 5535 ( SEQ ID # 14):

CAGGCCATGGCATGAAGAAACCACTGGATGGAGAA (the underlined part represents an Ncol site) Oligo 5536 (SEQ ID n ° 15): CGTCGGATCCTCTAGATGCGGCCGCGTCTGAGTCAGGCCCTTC (Underlined part: BamHI site; Double-underlined: Xbal site: Bold: Notl site).

4.2. The plasmids p53 320/393-myc-Hinge-TetR or Cro (FIG. 5A) were obtained by cloning of the amplified fragment above in the form of an Ncol-NotI fragment in the corresponding sites of the plasmids described in Example 3.1 ., replacing the region coding for ScFv.

4.3. Plasmids p53 320/393-Hinge-TetR or Cro (Figure 5B) were obtained by cloning the amplified fragment in 4.1. in the form of an Ncol-Xbal fragment in the corresponding sites of the plasmids described in Example 3.1., in substitution for the region coding for ScFv and the tag.

4.4. Plasmids p53 320/393-TetR or Cro (Figure 5C) were obtained by cloning the amplified fragment in 4.1. in the form of an Ncol-BamHI fragment in the corresponding sites of the plasmids described in Example 3.1. , replacing the region coding for ScFv, the tag and the Hinge.

4.5. The plasmids tetR or Cro-p53 320/393 (Figure 5D) or tetR or Cro-Hinge-p53 320/393 (Figure 5E) were obtained by cloning of fragments amplified by PCR on a plasmid carrying the cDNA of wild human p53 using oligos 5537/5539 or 5538/5539 digested with Xhol / EcoRI in the plasmids described in 3.1. , previously digested with Xhol / EcoRI.

Oligo 5537 (SEQ ID # 16):

CAGGCTCGAGAAGAAACCACTGGATGGAGAA Oligo 5538 (SEQ ID n ° 17):

CAGGCTCGAGCCCAAGCCCAGTACCCCCCCAGGTTCTTCAAAGA AACCACTGGATGGAGAA

Oligo 5539 (SEQ ID # 18):

GGTCGAATTCGGGCCCTCAGTCTGAGTCAGGCCCTTC Example 5: Construction of a control plasmid carrying a sequence coding for a chimeric molecule comprising a DNA binding domain (TetR or Cro) and the transactivating domain of the P53 protein (region 1-73).

Plasmids p53 1/73 - TetR or Cro with or without tag (myc or VSV) and Hinge (Figures 7 A, B and C) were obtained by cloning of fragments amplified by PCR from a plasmid carrying the cDNA of human wild-type p53 using oligos 5661/5662 then digested with Ncol / NotI, Ncol / Xbal, Ncol / BamHI in the plasmids described in 3.1. , previously digested with Ncol / Notl, Ncol / Xbal or Ncol / BamHI. Oligo 5661 (SEQ ID n ° 19):

CAGGCCATGGAGGAGCCGCAGTCAGATCC

Oligo 5662 (SEQ ID # 20):

CGTCGGATCCTCTAGATGCGGCCGCCACGGGGGGAGCAGCCTC TGG

Example 6 Construction of Expression Plasmids of Different Hybrid Molecules of the Invention

The plasmids used for the expression of hybrid molecules were obtained by cloning of the fragments containing the cDNA coding for these molecules at the Ncol / EcoRI sites of the eukaryotic expression vector pcDNA3 (Invitrogen). The different constructions thus carried out are as follows:

- ScFv 421: SEQ IDn ° 4

- TET19: hybrid protein containing the chain ScFv421-VSV-Hinge- TetR described in FIG. 6 according to Example 3.1

- TET02: hybrid protein containing the chain p53 (320/393) - VSV- Hinge-TetR described in FIG. 5A according to example 4.3

- TET03: hybrid protein containing the chain p53 (320/393) -Hinge- TetR described in FIG. 5B according to example 4.4 - TET04: hybrid protein containing the chain p53 (320/393) -TetR described in FIG. 5C according to example 4.4

- TET07: hybrid protein containing the chain p53 (1/73) -VSV-Hinge- TetR described in FIG. 7A according to Example 5

Example 7 Recognition of Specific Double-Stranded DNA Sequences by the Hybrid Molecules of the Invention

7.1. Production of hybrid molecules

The different molecules used in this experiment were obtained by in vitro translation into reticulocyte lysate of the molecules described in Example 6 using the TNT Coupled Reticulocyte lysate Systems kit (Promega) according to the experimental protocol described by the supplier for a reaction volume. total of 50 μl.

7.2 Construction of the specific double-stranded DNA sequence

The specific double-stranded DNA sequence used in this experiment consists of two synthetic oligonucleotides, the sequence of which is as follows:

Oligo 5997 (SEQ ID # 24): GATCCGACTTTCACTTTTCTCTATCACTGATAGTGAGTGGTAAACTCA

Oligo 5998 (SEQ ID # 25): AGCTTGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCG

These two synthetic oligonucleotides were labeled with phosphorus 33 by incubation for 30 min at 37 ° C of 10 pmole of each oligonucleotide in 10 μl of the following reaction medium:

Tris-HCI pH7.6 50mM MgCI ₂ 10mM dithiothreitol 5mM Spermidine 100μM

EDTA 100μM

ATP-γ-∞P (Amersham) 50μCi (1000-3000 Ci / mmole)

T4 kinase (Boehringer) 10U

Then the two oligonucleotides thus labeled were hybridized in the presence of 400mM NaCl to reconstitute the following double strand TetO sequence (SEQ ID No. 26):

GATCCGACTTTCACTTTTCTCTATCACTGATAGTGAGTGGTAAACTCA CTAGGCTCAAAGTGAAAAGAGATAGTGACTATCACTCACCATTTGAGT

7.3 Recognition of the TetO double strand sequence by the various hybrid molecules of the invention

The DNA binding reaction is carried out in 50 μl of reaction medium (Tris-HCI pH 7.4 10 mM, MgCI _{2 10} mM, KCI 10 mM, β-mercaptoethanol 6 mM, EDTA 0.1 mM, BSA 0.5 mg / ml) by adding the TetO sequence (10 ¹⁰ M) prepared according to Example 7.2, 10 μl of the translation product prepared according to Example 7.1 and 10 ^ M of the cold competing oligonucleotide AP2 (Promega) used to remove the non-specific fixation. The specificity of the interaction is verified by shifting the equilibrium by adding 10 μM tetracycline (Sigma) to the reaction medium. The reaction mixtures are incubated for 15 min at 20 ° C. and then added with 10 μl of 50% glycerol, and the final mixtures are subjected to native electrophoresis on 5% polyacrylamide gel with migration at 200V and 16 ° C. The gel is then dried and autoradiographed. The result of this experiment carried out with the hybrid molecules TET19, TET02 and TET07 is presented in FIG. 9. Under these conditions. The binding of these three molecules to the specific double-stranded DNA sequence TetO is observed by a migration delay of this one, and the specificity of this interaction is demonstrated by the inhibition of this delay by the addition of tetracycline.

This result therefore confirms that the hybrid molecules of the invention are capable of binding specifically to the TetO nucleotide sequence

Example 8; Specific binding of the hybrid molecules of the invention to a molecule having a transcriptional transactivating domain.

8.1. Production of the hybrid molecules of the invention and of molecules with or without a transcriptional transactivating domain.

For this experiment, the hybrid molecules of the invention ScFv 421, TET19 and TET02 according to Example 6 were produced by in vitro translation using the experimental protocol according to Example 7.1 in the presence of 44 μCi of ^ S-methionine (Amersham ) (1175 Ci / mmol) to generate these radioactive labeled hybrid molecules.

The cDNAs of the molecules with or without a transcriptional transactivating domain were cloned into the vector pBlueBaclll (Invitrogen) at the BamHI site. From these vectors, recombinant baculoviruses were produced and purified according to the manufacturer's instructions. The molecules are produced by infection with the recombinant baculovirus of insect cells sf9 according to the manufacturer's experimental protocol. Protein extracts at the final protein concentration of 10 mg / ml are prepared according to the protocol described by K. Ory et al. (K. Ory, EMBO J., 13, 3496-3504, 1994). These molecules are as follows:

- p53 (1/393): wild p53 protein

- p53 (1/320): wild p53 protein limited to its amino acid sequence 1 to 320 and therefore devoid of its oligomerization domain and of the domain recognized by the monoclonal antibody pAb421. 8.2. Binding of the hybrid molecules of the invention to molecules with or without a transcriptional transactivating domain

5 μl of each of the in vitro translation products prepared according to example 8.1 were incubated with 5 μl of the baculovirus extract prepared according to example 8.1 and 2 μg of the monoclonal antibody D01 (Oncogene Sciences) which recognizes the N end -terminal of the p53 protein for 16 hours at 4 ° C. in 100 μl of modified RIPA buffer ((K. Ory, EMBO J., 13, 3496-3504, 1994). The immunoprecipitation is carried out as described by K. Ory and al. (K. Ory, EMBO J., 13, 3496-3504, 1994) The complexes retained by the antibody are eluted by incubation for 10 min at 80 ° C. in the presence of 30 μl of migration buffer (Laemmli UK, Nature , 227, 680-685, 1970) and subjected to electrophoresis on 10% polyacrylamide gel in denaturing medium at 200V according to the protocol described above (Laemmli UK, Nature, 227, 680- 685, 1970). The gel is then dried and revealed using an instantimager (Packard instruments) which quantifies the quantities of hybrid molecules ideas linked to the molecule with or without a transcriptional transactivating domain. The results of this experiment are shown in Figure 10.

Under these conditions, it clearly appears that the hybrid molecule presenting the ScFv 421 (TET19) recognizes well the molecule p53 (1/393) in an equivalent manner to the ScFv 421 alone, and the hybrid molecule presenting the domain 320/393 (TET02) the same properties but with a much greater p53 retention capacity (1/393). In addition, the absence of signal observed during incubation with the p53 molecule (1/320) shows that these interactions are very specific and mediated by the C-terminal end of the p53 protein (amino acids 320 to 393) as expected.

These results therefore confirm that the hybrid molecules of the invention are well capable of recruiting a transcriptional transactivating domain carried by a molecule of which they are specific partners. EXAMPLE 9 Functional Recruitment of a Transcriptional Transactivator Domain by the Hvhririps and the Invention

The functional recruitment of a transcriptional transactivating domain by the hybrid molecules of the invention was evaluated in an in vivo transactivation system in SAOS-2 cells (human osteosarcoma) deficient for the two alleles of the p53 protein, in the line. H358 tumor deficiency for the two alleles of the p53 protein (Maxwell & Roth, Oncogene 8, 3421, 1993) and in the HT29 tumor line deficient for one of the two alleles of the p53 protein and having a mutated allele (mutation H273). This system is based on the use of a reporter gene which can be assayed enzymatically and placed under the dependence of a promoter containing the nucleotide patterns of specific recognition by the Tet repressor. (Operator Tet).

In this test, the reporter gene LUC (luciferase) placed under the control of the operator Tet is contained in the plasmid pUHC13-3 (Gossen M. & Bujard H., Proc. Natl. Acad. Sci. USA, 89, 5547- 5551, 1992).

9.1 Cell lines used and culture conditions

The cell lines used in these experiments as well as their genotype linked to the p53 protein and the culture medium used for their growth are given in the Table below:

Table: cell lines

Line p53 Culture medium ATCC No. DMEM medium (Gibco BRL) supplemented with

SAOS-2 - / - HTB 85 10% fetal calf serum (Gibco BRL) RPMI1640 medium (Gibco BRL) supplemented with

H358 - / - 10% fetal calf serum (Gibco BRL) DMEM medium (Gibco BRL) supplemented with

HT29 - / H273 10% fetal calf serum (Gibco BRL) 9.2. Expression plasmids of molecules with a transcriptional transactivator domain

The molecules having a transcriptional transactivating domain used in this experiment are the wild-type p53 protein (wt) and the mutants G281 and H 175 of this protein. The cDNA coding for these three proteins were inserted at the BamHI site of the vector pcDNA3 (Invitrogen).

9.3. Intracellular expression of the hybrid molecules of the invention

The hybrid molecules of the invention are expressed in cells in culture by transient transfection using the following protocol:

The cells (3.5 × 10 ⁵ ) are seeded in 6-well plates containing 2 ml of culture medium, and cultured overnight in a CO ₂ (5%) incubator at 37 ° C. The different constructions are then transfected using lipofectAMINE (Gibco BRL) as transfection agent in the following manner: 1.5 μg of total plasmid are incubated (including 0.25 μg of the reporter plasmid) with 5 μl of lipofectAMINE for 30 min with 2 ml of culture medium without serum (transfection mixture). Meanwhile, the cells are rinsed twice with PBS and then incubated for 4 h at 37 ° C. with the transfection mixture, after which the latter is aspirated and replaced with 2 ml of culture medium added with 10% fetal calf serum. heat inactivated and cells returned to grow for 48 h at 37 ° C.

9.4. Transcription activation detection

The activation of the transcription linked to the functional recruitment of the transcriptional transactivator is detected and quantified by measuring the luciferase activity encoded by the LUC gene using the Luciferase Assay System kit (Promega) according to the manufacturer's experimental protocol. 9.5. Functional recruitment of a transcriptional transactivator domain by the molecules of the invention

This experiment was carried out using the molecules TET02, TET03 and TET07 according to example 6. In this experiment the molecule TET07 serves as positive control since it has its own transcriptional transactivator domain.

The results obtained in SAOS-2 cells and presented in FIG. 11 show that the TET07 molecule alone is well capable of activating the transcription of the LUC gene placed under the control of the operator Tet unlike the construction TET02. This is in agreement with the fact that this cell line does not contain an endogenous p53 protein which therefore cannot be recruited by the TET02 molecule. On the other hand, the introduction of the wild-type p53 protein or of its mutant G281 which do not produce a signal on their own, is capable of generating transcriptional activity in the presence of the molecule TET02. Such a result cannot be observed with the H 175 mutant described in the literature as having a non-functional transcriptional transactivating domain.

This result obtained in the SAOS-2 cell line with the TET02 molecule could be reproduced in a tumor line also containing no endogenous p53 (H358 cells) and could be extended to the TET03 and TET04 molecules (FIG. 12).

In order to confirm these two results in a different cellular context, the TET02 molecule as well as the TET07 positive control were expressed in HT29 cells which have an endogenous mutant p53 protein (H273), the negative control of this experiment consisting in transfecting the empty pcDNA3 vector. The result of this experiment, presented in the Table below, shows that the TET02 molecule is well capable to recruit the transcriptional transactivating domain of the endogenous p53 protein.

Table: Transcriptional activation of hybrid molecules of the invention in HT29 cells

pcDNA3 TET07 TET02

1 59 10

All of these experiments therefore show that the hybrid molecules of the invention are well capable of binding both specific double-stranded DNA sequences and proteins having a transcriptional transactivating domain, and that these molecules are capable of inducing conditionally the expression of genes of interest.

LIST OF SEQUENCES

(1) GENERAL INFORMATION:

(i) DEPOSITOR:

(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: (1) 40.91.70.36 (H) FAX: (1) 40.91.72.91 (ii) TITLE OF THE INVENTION: CONDITIONAL EXPRESSION SYSTEM

(iii) NUMBER OF SEQUENCES: 26

(iv) COMPUTER-DETACHABLE FORM: (A) TYPE OF SUPPORT: Tape

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: PatentIn Release # 1.0, Version # 1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1

(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 base pairs (B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:

TCTCTATCAC TGATAGGGA 19

(2) INFORMATION FOR SEQ ID NO: 2:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: TATCACCGCA AGGGATA 17

(2) INFORMATION FOR SEQ ID NO: 3:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 74 amino acids, s

(B) TYPE: amino acid,

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linen, area

(ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:

Lys Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gin Ile Arg Gly Arg 1 5 10 15

Glu Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys 20 25 30

Asp Ala Gin Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser 35 40 45

His Leu Lys Ser Lys Lys Gly Gin Ser Thr Ser Arg His Lys Lys Leu 50 55 60 Met Phe Lys Thr Glu Gly Pro Asp Ser Asp 65 70

(2) INFORMATION FOR SEQ ID NO: 4:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 768 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: TTACTCGCGG CCCAGCCGGC CATGGCCCAG GTGCAGCTGC AGCAGTCTGG GGCAGAGCTT 60 GTAAGGTCAG GGGCCTCAGT CAAGTTGTCC TGCACAGCTT CTGGCTTCAA CATTAAAGAC 120

TACTATATGC ACTGGGTGAA GCAGAGGCCT GAACAGGGCC TGGAGTGGAT TGGATGGATT 180

GATCCTAAGA ATGGTGATAC TGAATATGCC CCGAAGTTCC AGGGCAAGGC CACTATGACT 240

GCAGACACAT CCTCCAATAC AGCCTACCTG CAGCTCAGCA GCCTGGCATC TGAGGACACT 300 GCCGTGTATT ATTGTAATTT TTACGGGGAT GCTTTGGACT ATTGGGGCCA AGGGACCACG 360

GTCACCGTCT CCTCAGGTGG AGGCGGTTCA GGCGGAGGTG GCTCTGGCGG TGGCGGATCG 420

GATGTTTTGA TGACCCAAAC TCCACTCACT TTGTCGGTTA CCATTGGACA ACCAGCCTCC 480

ATCTCTTGCA AGTCAAGTCA GAGCCTCTTG GATAGTGATG GAAAAACATA TTTGAATTGG 540

TTGTTACAGA GGCCAGGCCA GTCTCCAAAG CGCCTAATCT ATCTGGTGTC TAAACTGGAC 600 TCTGGAGTCC CTGACAGGTT CACTGGCAGT GGATCAGGGA CAGATTTCAC ACTTAAAATC 660

AACAGAGTGG AGGCTGAGGA TTTGGGAGTT TATTATTGCT GGCAAGGTAC ACATTCTCCG 720

CTTACGTTCG GTGCTGGCAC CAAGCTGGAA ATTAAACGGG CGGCCGCA 768

(2) INFORMATION FOR SEQ ID NO: 5:

(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 15 amino acids, s

(B) TYPE: amino acid

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: peptide

(iii) HYPOTHETIC: NO

(iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:

Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 6:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: double (D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA

(iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO

(ix) CHARACTERISTIC:

(A) NAME / KEY: CDS

(B) LOCATION: 1..30

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:

CCC AAG CCC AGT ACC CCC CCA GGT TCT TCA 30

Pro Lys Pro Ser Thr Pro Pro Gly Ser Ser 1 5 10

(2) INFORMATION FOR SEQ ID NO: 7:

(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO

(ix) CHARACTERISTIC:

(A) NAME / KEY: CDS

(B) LOCATION: 1..18

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7:

ATG AAC CGG CTG GGC AAG 18 Met Asn Arg Leu Gly Lys

1 5

(2) INFORMATION FOR SEQ ID NO: 8

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: double (D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA

(iii) HYPOTHETIC: NO

(iv) ANTI-SENSE: NO

(ix) CHARACTERISTIC: (A) NAME / KEY: CDS

(B) LOCATION: 1..33

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8:

GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG AAT 33

Glu Gin Lys Leu Ile Ser Glu Glu Asp Leu Asn 1 5 10

(2) INFORMATION FOR SEQ ID NO: 9:

(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9:

Pro Lys Lys Lys Arg Lys Val 1 5

(2) INFORMATION FOR SEQ ID NO: 10:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 66 base pairs

(B) TYPE: nucleotide (C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO

(iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10: GGCTCTAGAC CCAAGCCCAG TACCCCCCCA GGTTCTTCAA CGCGTGGATC CATGTCCAGA 60 TTAGATAAAA GTAAAG 66

(2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 51 base pairs

(B) TYPE: nucleotide (C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO

(iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11: CGTACGGAAT TCGGGCCCTT ACTCGAGGGA CCCACTTTCA CATTTAAGTT G 51

(2) INFORMATION FOR SEQ ID NO: 12:

(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 66 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12: GGCTCTAGAC CCAAGCCCAG TACCCCCCCA GGTTCTTCAA CGCGTGGATC CATGGAACAA 60 CGCATAACCC TGAAAG 66

(2) INFORMATION FOR SEQ ID NO: 13:

(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 51 base pairs (B) TYPE: nucleotide

(C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13: CGTACGGAAT TCGGGCCCTT ACTCGAGTGC TGTTGTTTTT TTGTTACTCG G

(2) INFORMATION FOR SEQ ID NO: 14:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: double (D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14: CAGGCCATGG CATGAAGAAA CCACTGGATG GAGAA 35

(2) INFORMATION FOR SEQ ID NO: 15:

(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 43 base pairs (B) TYPE: nucleotide

(C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15: CGTCGGATCC TCTAGATGCG GCCGCGTCTG AGTCAGGCCC TTC 43

(2) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA

(iii) HYPOTHETIC: NO

(iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16: CAGGCTCGAG AAGAAACCAC TGGATGGAGA A 31

(2) INFORMATION FOR SEQ ID NO: 17:

(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 61 base pairs (B) TYPE: nucleotide

(C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17: CAGGCTCGAG CCCAAGCCCA GTACCCCCCC AGGTTCTTCA AAGAAACCAC TGGATGGAGA 60 A 61

(2) INFORMATION FOR SEQ ID NO: 18:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 37 base pairs

(B) TYPE: nucleotide (C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO

(iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18: GGTCGAATTC GGGCCCTCAG TCTGAGTCAG GCCCTTC 37 (2) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 19:

CAGGCCATGG AGGAGCCGCA GTCAGATCC 29

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18:

GGTCGAATTC GGGCCCTCAG TCTGAGTCAG GCCCTTC 37

(2) INFORMATION FOR SEQ ID NO: 20:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 46 base pairs

(B) TYPE: nucleotide (C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO

(iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 20: CGTCGGATCC TCTAGATGCG GCCGCCACGG GGGGAGCAGC CTCTGG 46

(2) INFORMATION FOR SEQ ID NO: 21:

(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 66 amino acids

(B) TYPE: amino acid

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 21:

Met Glu Gin Arg Ile Thr Leu Lys Asp Tyr Ala Met Arg Phe Gly Gin 1 5 10 15 Thr Lys Thr Ala Lys Asp Leu Gly Val Tyr Gin Ser Ala Ile Asn Lys

20 25 30

Ala Ile His Ala Gly Arg Lys Ile Phe Leu Thr Ile Asn Ala Asp Gly 35 40 45

Ser Val Tyr Ala Glu Glu Val Lys Pro Phe Pro Ser Asn Lys Lys Thr 50 55 60

Thr Ala 65

(2) INFORMATION FOR SEQ ID NO: 22: (i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 22:

GATCCTATCA CCGCAAGGGA TAA 23

(2) INFORMATION FOR SEQ ID NO: 23:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleotide (C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 23: GATAGTGGCG TTCCCTATTT CGA 23

(2) INFORMATION FOR SEQ ID NO: 24: (i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 48 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 24:

GATCCGACTT TCACTTTTCT CTATCACTGA TAGTGAGTGG TAAACTCA 48

(2) INFORMATION FOR SEQ ID NO: 25:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 48 base pairs

(B) TYPE: nucleotide (C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO

(iv) ANTI-SENSE: NO

(i) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 25: AGCTTGAGTT TACCACTCCC TATCAGTGAT AGAGAAAAGT GAAAGTCG 48

(2) INFORMATION FOR SEQ ID NO: 26:

(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 96 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: double

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA

(iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 26:

GATCCGACTT TCACTTTTCT CTATCACTGA TAGTGAGTGG TAAACTCACT AGGCTCAAAG 60 TGAAAAGAGA TAGTGACTAT CACTCACCAT TTGAGT 96

Claims

1. A bispecific chimeric molecule comprising a domain capable of selectively binding a defined DNA sequence and a detector domain capable of specifically binding a transactivator or transrepressor or a transactivator or transrepressor complex characteristic of a physiological or pathophysiological state.

2. Molecule according to claim 1 characterized in that the domain capable of selectively binding a defined DNA sequence derives from a protein capable of interacting with DNA.

3. Molecule according to claim 2 characterized in that the domain capable of selectively binding a defined DNA sequence derives from a eukaryotic protein.

4. Molecule according to claim 3 characterized in that the domain capable of selectively binding a defined DNA sequence derives from the proteins p53, STAT or NFkB.

5. Molecule according to claim 2 characterized in that the domain capable of selectively binding a defined DNA sequence derives from a prokaryotic protein.

6. Molecule according to claim 5 characterized in that the prokaryotic protein is a bacterial repressor.

7. Molecule according to claim 6 characterized in that the domain capable of selectively binding a defined DNA sequence derives from the tetR protein.

8. Molecule according to claim 6 characterized in that the domain capable of selectively binding a defined DNA sequence derives the protein

Cro.

9. Molecule according to one of claims 2 to 8 characterized in that the domain capable of selectively binding a defined DNA sequence comprises the domain of interaction with the DNA of said protein.

10. Molecule according to one of claims 2 to 8 characterized in that the domain capable of selectively binding a defined DNA sequence consists of the complete protein.

11. Molecule according to claim 10 characterized in that the domain capable of selectively binding a defined DNA sequence consists of the protein tetR.

12. Molecule according to claim 10 characterized in that the domain capable of selectively binding a defined DNA sequence consists of the Cro protein.

13. Molecule according to claim 1 characterized in that the domain capable of specifically binding the transactivator or transrepressor or the transactivator or transrepressor complex is an oligomerization domain.

14. Molecule according to claim 13 characterized in that the oligomerization domain is a leucine-zipper, an SH3 or SH2 domain.

15. Molecule according to claim 13 characterized in that the oligomerization domain capable of specifically binding the transactivator consists of the C-terminal part of the p53 protein.

16. Molecule according to claim 15 characterized in that the oligomerization domain consists of the C-terminal part of the p53 protein ranging from residue 320 to 393 (SEQ ID No. 3), 302-360 or 302-390.

17. Molecule according to claim 1 characterized in that the domain capable of specifically binding the transactivator or transrepressor or the transactivator or transrepressor complex is a synthetic or natural domain known to interact with said transactivator or transrepressor or transactivator or transrepressor complex.

18. Molecule according to claim 1 characterized in that the domain capable of specifically binding the transactivator or transrepressor or the transactivator or transrepressor complex is an antibody or a fragment or derivative of antibody directed against the transactivator or transrepressor or transactivator or transrepressor complex.

19. Molecule according to claim 18 characterized in that the domain capable of specifically binding the transactivator or the transactivator complex consists of a Fab or F (ab) '2 fragment of antibody or a VH or VL region of an antibody.

20. Molecule according to claim 18 characterized in that the domain capable of specifically binding the transactivator or the transactivator complex consists of a single chain antibody (ScFv) comprising a VH region linked to a VL region by an arm.

21. Molecule according to claim 1 characterized in that the DNA binding domain and the transactivator binding domain are linked together by means of an arm.

22. Molecule according to claim 21 characterized in that the arm consists of a peptide comprising from 5 to 30 amino acids and, preferably, from 5 to 20 amino acids.

23. Molecule according to claim 22 characterized in that the arm is chosen from peptides of sequence SEQ ID No. 5 or SEQ ID No. 6.

24. Molecule according to one of the preceding claims, characterized in that the DNA binding domain is located in the N-terminal position and the binding domain to the transactivator is located in the C-terminal position.

25. Molecule according to one of claims 1 to 23 characterized in that the DNA binding domain is located in the C-terminal position and the binding domain to the transactivator is located in the N-terminal position.

26. Bispecific chimeric molecule with ScFv-VSV / myc-Hinge-TET or Cro structure (Figure 5A).

27. Bispecific chimeric molecule with ScFv-Hinge-TET or Cro structure (Figure 5B).

28. Bispecific chimeric molecule with ScFv-TET or Cro structure (Figure 5C).

29. Bispecific chimeric molecule with TET or Cro-ScFv structure (Figure 5D).

30. Bispecific chimeric molecule with TET or Cro-Hinge-ScFv structure (Figure 5E).

31. Bispecific chimeric molecule with an Oligom-VSV / myc-Hinge-TET or Cro (Figure 5A), Oligom-Hinge-TET or Cro (Figure 5B) or Oligom- TET or Cro (Figure 5C) structure.

32. Nucleic acid sequence coding for a chimeric molecule according to one of claims 1 to 31.

33. Nucleic acid sequence according to claim 32 characterized in that it is a DNA sequence.

34. Nucleic acid sequence according to claim 32 or 33 characterized in that it is part of a vector.

35. Conditional gene expression system comprising:

- a chimeric molecule as defined in claims 1 to 31, and,

- An expression cassette comprising a regulatory sequence, a minimal transcriptional promoter and said gene.

36. Conditional system according to claim 35 characterized in that the DNA binding domain of the chimeric molecule is represented by all or part of TetR and the regulatory sequence comprises the sequence SEQ ID No. 1 or a derivative thereof. ci, possibly repeated several times.

37. Conditional system according to claim 35 characterized in that the DNA binding domain of the chimeric molecule is represented by all or part of Cro and the regulatory sequence comprises the sequence SEQ ID No. 2 or a derivative thereof. ci, possibly repeated several times.

38. Conditional system according to one of claims 35 to 37 characterized in that the minimum promoter comprises a TATA or INR box.

39. Conditional system according to claim 38 characterized in that the minimal promoter derives from the promoter of the thymidine kinase gene.

40. Conditional system according to claim 39 characterized in that the minimal promoter is composed of nucleotides -37 to +19 of the promoter of the thymidine kinase gene.

41. Vector comprising:

a nucleic acid sequence coding for a chimeric molecule according to one of claims 1 to 31, and

- an expression cassette comprising a regulatory sequence, a minimal transcriptional promoter and a coding sequence of interest.

42. Vector according to claim 41 characterized in that the minimal transcriptional promoter is defined according to claims 38 to 40.

43. Vector according to claim 41 characterized in that the DNA binding domain of the chimeric molecule is represented by all or part of TetR and the regulatory sequence comprises the sequence SEQ ID No. 1 or a derivative thereof , possibly repeated several times.

44. Vector according to claim 41 characterized in that the DNA binding domain of the chimeric molecule is represented by all or part of Cro and the regulatory sequence comprises the sequence SEQ ID No. 2 or a derivative thereof , possibly repeated several times.

45. Vector according to one of claims 41 to 44 characterized in that the coding sequence of interest is a DNA sequence coding for a therapeutic product.

46. Vector according to claim 45, characterized in that the therapeutic product is a toxic peptide or polypeptide.

47. A vector according ^'to claim 46 characterized in that the toxic therapeutic product is selected from diphtheria toxin, pseudomonas toxin, ricin A, thymidine kinase, cytosine deaminase, the Grb3-3 protein or ScFv Y28.

48. Vector according to one of claims 41 to 47 characterized in that it is a plasmid vector.

49. Vector according to one of claims 41 to 47 characterized in that it is a viral vector.

50. Vector according to claim 49, characterized in that it is a defective recombinant adenovirus.

51. Vector according to claim 49, characterized in that it is a defective recombinant retrovirus.

52. Pharmaceutical composition comprising at least one vector according to one of claims 41 to 51.

53. Nucleic acid comprising the sequence SEQ ID No. 4.

54. Molecule according to claim 1 characterized in that the transactivator characteristic of a physiological or physiopathological state is a protein of viral, parasitic, mycobacterial or cellular origin having a transcriptional transactivator activity.

55. Molecule according to claim 54 characterized in that the transactivator is a viral protein chosen from the Tat protein of the HIV virus, the E6 / E7 proteins of the papilloma virus and the EBNA protein of the Epstein Barr virus.

56. Molecule according to claim 54 characterized in that the transactivator is a cellular protein, preferably the mutated or wild-type p53 protein.

57. Molecule according to claim 1 characterized in that the transactivator or transactivating complex characteristic of a physiological or physiopathological state is a protein appearing in an infected or hyperproliferative cell