CA2214451A1 - Conditional expression system - Google Patents

Abstract

The present invention relates to a new conditional gene expression system. The system is based on the creation and expression 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 transrepressor or a transactivator or transrepressor complex.

Description

CA 022144 ~ 1 1997-09-22 W O96 / 30512 PCTA ~ R96 / 00477 CONDITIONAL EXPRESSION SYSTEM
The present invention relates to a new system for Conditional gene expression. It also concerns the use of this system in gene or cell therapy, to target in a very 5 selective expression of genes of interest.
Gene and cell therapy involves correcting a deficiency or abnormality (mutation, outlier, etc.) or ensure the expression of a protein of therapeutic interest by introduction genetic information in the affected cell or organ. This 10 genetic information can be introduced either ex vivo in a cell ~ extracted from the organ, the modified cell then being reintroduced into the organism (cell therapy), either directly in vivo in the tissue appropriate (gene therapy). Different techniques exist to perform gene transfer, including various techniques of transfection involving chemical or biochemical, natural or synthetics such as DNA and DEAE-dextran complexes (Pagano and 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), the use of 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 have been tested for their ability to infect certain cell populations. In in particular, retroviruses (RSV, HMS, MMS, etc.), HSV virus, viruses adeno-associated and adenoviruses. One of the major difficulties for the development of these gene and cell therapies however resides in treatment selectivity. Depending on the applications, depending on the gene to be transferred, it is important to be able to target certain tissues or certain parts only from the body in order to concentrate the therapeutic effect and limit dissemination and side effects. This targeting can be done by CA 022l44 ~ l l997-09-22 W O96 / 30512 PCT ~ R96 / 00477 using vectors having a given cell specificity. Another approach is to use specific expression signals of certain cell types. In this regard, so-called specific promoters have been described in the literature, such as the promoter of genes encoding pyruvate 5 kinase, villin, GFAP, the intestinal binding protein promoter fatty acids, the actin promoter has smooth muscle cells, or the promoter of genes for apo-AI, apo-AII, human albumin, etc.
However, these promoters have certain drawbacks and particular they present a certain transcriptional background noise which can be 10 genes 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 new system conditional gene expression, particularly selective and effective. A
advantageous characteristics of the system of the invention resides in its 15 ability 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 a situation particular physiological. This system uses molecules bispecific chimeras comprising a domain capable of binding selectively a defined DNA sequence and a detector domain capable 20 to specifically bind a transactivator or a transactivator complex.
A first aspect of the present invention lies more particularly in the creation and expression of chimeric molecules bispecific comprising a domain capable of selectively binding a defined sequence of DNA and a domain capable of specifically binding a 25 transactivator or transrepressor or a transactivator complex or transrepressor.
Another aspect of the present invention resides in a sequence of nucleic acid encoding a chimeric molecule as defined above CA 022144 ~ 1 1997-09-22 before, as well as a whole expression vector comprising said sequence nucleic acid.
Another aspect of the invention consists of a conditional system expression of genes comprising (i) a chimeric molecule such as 5 defined above and (ii) an expression cassette comprising a sequence of regulation, a minimal promoter (whose activity depends on the presence of a transactivator) and said gene.
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Another aspect of the invention also resides in a vector of expression including - a nucleic sequence coding for a chimeric molecule such as defined above and - said expression cassette.
The conditional expression system of the invention is particularly suitable for use in gene therapy or 15 cell, to very selectively target gene expression 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 20 domain capable of specifically binding a transactivator or a complex transactivator. The bi-specificity of the molecules of the invention resides on the one hand in their capacity to link a defined DNA sequence (generally designated as regulatory or operator sequence) and secondly in their ability to specifically recruit a transactivating protein domain or 25 transrepressor for inducing or repressing gene expression.
The invention relates particularly to the development of molecules bispecific chimeras allowing the recruitment of any factor transcriptional whose activation or inactivation leads to a situation CA 022144 ~ 1 1997-09-22 WO g6 / 30512 PCT / liR96 / 00477 pathophysiological. Bispecific chimeric molecules according to the invention thus allow the selective recruitment of transactivators specific transcription of a pathophysiological state, the fixation of these transcriptional factor to promoters via fixation 5 of these molecules to defined DNA sequences located near these promoters (regulatory sequences or operator), and thus the expression conditional of genes (Figure 1).
The invention also relates to the development of molecules bispecific chimeras allowing the recruitment not of a molecule 10 carrying a transactivating domain but of a transactivating complex transcriptional, i.e. a complex formed between a target molecule present in a cell and a molecule carrying a domain transactivator (Figure 2). In this case, the transactivating complex is preferably formed by means of a second chimeric molecule Bispecific comprising a transactivating domain and a binding domain selective for said cell molecule. The fixation of this second molecule allows the formation of a transcriptional transactivator binary complex, which complex then being recruited by the detector system of the invention.
The fixation of this ternary complex near promoters thus allows 20 regulated gene expression. This type of construction allows advantageously to broaden the conditions of use of the 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 allows, thanks to a system of very selective detection ("sensor") to activate the expression of genes of interest only in the presence of target proteins. These may be factors transcriptional appearing during physiological situations or pathophysiological, or any endogenous molecule or of infectious origin CA 022144 ~ 1 1997-09-22 WO 96/30512 PCTA ~ R96 / 00477 for example. The system of the invention indeed contains an element ~ very sensitive and very selective detector allowing to condition the expression of a gene to the presence, appearance or disappearance of any molecule in ~ a cell.
In the context of the present invention, the term transactivator denotes any transactivating factor of transcription or any protein having a transcriptional transactivating domain. The complex transactivator designates the complex formed between a molecule present in a cell and a bispecific molecule of the invention comprising a transactivating domain and a binding domain specific to said molecule. The expression system of the invention can be used for recruit any transaetivator or domain-carrying protein transactivator, and in particular any protein of viral, parasitic, mycobacterial or cellular origin having a transactivating activity transcriptional. Among the transcriptional factors of viral origin one can include the Tat protein of the HIV virus, the E6 / E7 proteins of the papilloma or 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, i.e. in specific pathophysiological conditions. The expression system conditional according to the invention advantageously allows detection of this physiological situation (the appearance of these specific transactivators viral infection) and induction of selective expression of gene (s) given. Among the cellular proteins, mention may preferably be made of 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 (about 1-100 residues). In some situations, wild p53 is CA 022144 ~ 1 1997-09-22 W O96 / 30512 PCTn ~ R96 / 00477 also capable of inducing apoptosis (Yonish-Rouach et al., Nature, 352, 345-347, 1991). These properties appear 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 highlights the probably crucial role that mutations in this gene play 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 this invention it is possible to selectively recruit the transactivator domain of the protein p53 and thus of inducing the controlled expression of gene (s) only in cells containing this protein. lt is particularly interesting according to the invention to specifically recruit mutated forms of the p53 protein which, as indicated above, appears in pathophysiological situations of cellular hyperproliferation (cancer type).
This targeting can preferably be carried out by means of a domain of specific binding to mutated forms of the p53 protein. However, there are 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 protein detected 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 including the HIV virus, hepatitis, herpes, etc. It can also be proteins specific to a state of cellular hyperproliferation such as including myc, fos, jun proteins, cyclins, etc.
One of the properties of the chimeric molecules of the invention resides therefore in their ability to bind to specific regions of DNA (regions CA 022l44 ~ l l997-09-22 regulators or operator). This link makes it possible to bring the domain - transactivator near the promoter and thereby activating the expression a gene placed under the control of said promoter.
The domain capable of selectively binding a defined DNA sequence 5 present in the molecules of the invention is essentially of origin protein. More preferably, this domain derives from a protein prokaryotic or eukaryotic capable of interacting with DNA sequences. Of numerous genetic and structural studies have now made it possible to define precisely, within proteins interacting with sequences 10 double stranded DNA, the domains responsible for these interactions.
Among the prokaryotic proteins interacting with sequences double stranded DNA, there may be mentioned in particular bacterial repressors and, preferentially, the tetracycline repressor of E. Coli and the repressor Cro of the bacteriophage Lambda.
E.coli's tetracycline repressor (tetR) is a 210 protein amino acids approx. 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 level of a specific sequence (designated operator sequence or Tetop) and suppresses transcription of the resistance gene. On the contrary, in the presence tetracycline, the tetR repressor no longer attaches 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 W094 / 04682). The specific double-strand DNA sequence of tetR to DNA (Tetop) is composed of the following motif:
TCTCTATCACTGATAGGGA (SEQ ID n ~ 1).
This pattern can be repeated several times to increase affinity and The efficiency of the system. So the regulatory sequence can include -CA 022144 ~ 1 1997-09-22 W O96 / 30512 PCTnFR96 / 00477 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). The cloning of the cro gene allowed the identification of a protein of 66 acids amines (SEQ ID No. 21; Roberts; T et al. (1977) Nature 270, pp274). Cro exercises its physiological role by fixing itself preferentially to the operator OR3 of Lambda.
The specific double strand DNA sequence of Cro binding to DNA (region designated OR3) is composed of the following bases:
TATCACCGCAAGGGATA (SEQ ID n ~ 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 sequences double stranded DNA, we prefer to use for the construction of molecules of the invention the proteins or domains derived from the STAT, p53 or NFkB (Inoue et al., PNAS 89 (1992) 4333). Regarding 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 sequence of DNA present in the molecules of the invention is preferentially derived from a prokaryotic or eukaryotic protein capable 25 to interact with a double-stranded DNA region. The domain used for the construction of the molecules of the invention may consist of the whole of the protein or a fragment thereof comprising the interaction region with DNA. This domain has been identified for different proteins and in particular TetR (see for example Berens et al., J. Biol. Chem. 267 (1992) 30 1945). It can also consist of a derivative of this protein or of the CA 022l44 ~ l l997-09-22 W O96 / 30512 PCTA ~ R96 / 00477 fragment having retained the properties of DNA linkage. Such derivatives are in particular proteins with modifications of one or more amino acids, for example to allow their fusion with others fields of the molecules of the invention, prepared according to the techniques 5 classics of molecular biology. TetR and Cro protein derivatives for example have been described in the literature, possessing mutations punctual eVou 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 ability to 10 binding of these derivatives to a defined DNA sequence can then be tested by incubation of the derivative prepared with the regulatory sequence and detection complexes formed. In addition, the derivatives can also be proteins with improved DNA binding properties (specificity, affinity, etc.).
According to a preferred mode of implementation, the domain capable of binding selectively a defined sequence of DNA 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, 20 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 binding selectively a defined sequence of DNA present in the molecules of The invention is derived from the tetR or Cro proteins. It is particularly advantageous to use the complete tetR or Cro proteins (SEQ ID n ~ 21).
The domain capable of specifically binding the transactivator transcriptional or the transcriptional transactivator complex present in the molecules of the invention can be of different types. It can be in 30 particular of an oligomerization domain in the case where the transactivator CA 022l44 ~ l l997-09-22 W O96 / 30512 PCT ~ R96 / 00477 or the targeted transactivator complex also has such a domain. he can also be any synthetic or natural field known for interact with said transactivator or transactivator complex. he can still be an antibody or a fragment or derivative of an antibody directed 5 against the transactivator or transactivator complex.
Among the areas of oligomerization that can be used in the context of the invention may more particularly be cited leucine-zippers, domains SH2 or domains SH3 for example. Leucine-zippers are propellers cc amphipatics which contain 4 or 5 leucines every 7 amino acids.
10 This periodicity allows the location of leucines at roughly the same position on the propeller c ~. Dimerization is underpinned by interactions hydrophobic between the side chains of leucines from two domains contiguous zipper (Vogt et al., Trends In Bioch. Science 14 (1989) 172). The SH2 domains are known to interact with peptide sequences 15 specific phosphorylated tyrosine. SH3 domains can be used to form an oligomer with any transactivator or complex transactivator containing the corresponding proline-rich peptide (Pawson et al., Current Biology 3 (1993) 434). You can also use protein regions known to induce oligomerization, such as In particular the C-terminal region of the p53 protein. The use of this region allows to selectively recruit the p53 proteins present in a cell. Preferably used in the context of the invention a region of p53 between amino acids 320-393 (SEQ ID n ~ 3), 302-360 or 302-390.
Among the synthetic or natural domains known to interact with the molecule comprising the targeted transactivating element, mention may be made of example the region of the MDM2 protein interacting with the p53 protein.
This type of construction thus makes it possible to recruit as a transactivator the wild-type or mutated p53 protein.

CA 022144 ~ 1 1997-09-22 WO 96/30512 PCTtFR96tOO477 A specific binding domain for the transcriptional transactivator - preferred of the invention consists of an antibody or a fragment or derivative of antibodies. 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 else 5 single chain antibodies (ScFv) comprising a VH region linked to a LV region by an arm. This type of domain is particularly advantageous since it can be directed against any molecule.
Antibodies, molecules of the immunoglobulin superfamily, consist of different chains (2 heavy (H) and 2 light (L)) they 10 memes made up of different domains (variable domain (V) domain junction (J), etc.). The domain of binding to the transactivator or complex transactivator present in the molecules of the invention is advantageously consisting of a fragment or derivative of antibodies comprising at least the antigen binding site. This fragment can be either the 15 variable domain of a light (VL) or heavy (VH) chain, possibly in the form of a Fab or F (ab ') 2 fragment or, preferably, in the form single chain antibody (ScFv). Single chain antibodies used for construction of the molecules of the invention consist of a peptide corresponding to the binding site of the light chain variable region of a 20 antibody linked by a peptide arm to a peptide corresponding to the site of binding of the variable region of the heavy chain of an antibody. The construction of nucleic acid sequences encoding such antibodies modified according to the invention has been described for example in the patent US4,946,778 or in applications WO94 / 02610, W094 / 29446. She is 25 illustrated in the examples.
A preferred construction according to the present invention comprises a binding domain to a p53 protein. It is more preferably a derived from an antibody to a p53 protein. An embodiment particular is constituted by a single chain antibody directed against p53. AT

CA 022144 ~ 1 1997-09-22 W O96 / 30512 PCT ~ R96 / 00477 As a particular example, we use the ScFv of sequence SEQ ID n ~ 4, the construction is described in the examples.
The DNA binding domain and the DNA binding domain transactivators are usually linked together through a arms. This arm generally consists of a peptide conferring a sufficient flexibility for the two domains of the molecules of the invention can be functional independently. This peptide is generally composed of uncharged amino acids, not interfering with the activity of the molecules of the invention, such as for example glycine, serine, tryptophan, Iysine or proline. The arm includes generally 5 to 30 amino acids and preferably 5 to 20 acids amines. Examples of peptide arms usable for construction molecules of the invention are for example:

- GGGGSGGGGSGGGGS (SEQ ID n ~ 5) - PKPSTPPGSS (SEQ ID n ~ 6) whose coding sequence is CCCAAGCCCAGTACCCCCCCAGGTTCTTCA (SEQ ID n ~ 6).
Preferred examples of a molecule according to the invention are in particular the following molecules:
a) ScFv-tag-Hinge-TET or Cro (Figure 5A) This type of molecule includes:
- a binding domain to a transactivator consisting of an antibody single chain, - 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 n ~ 7) whose coding sequence is ATGAACCGGCTGGGCAAG (SEQ ID n ~ 7), or The myc sequence EQKLISEEDLN (SEQ ID n ~ 8) whose sequence coding is GAACAAAAACTCATCTCAGAAGAGGATCTGAAT (SEQ ID n ~
8), recognized by the antibody 9E10.

CA 022144 ~ 1 1997-09-22 W O96 / 30512 PCTA ~ R96100477 - a peptide arm of sequence SEQ ID n ~ 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 SB) This type of molecule has the same elements as the molecule a) with the exception of the tag sequence which is absent.
c) ScFv-TET or Cro (Figure 5C) This type of molecule simply has a binding domain to a transactivator consisting of a single chain antibody and a domain of DNA binding consisting of the TET or Cro protein. It has neither arms nor tag sequence. In this construction, the binding domain to transactivator 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 areas: the area of binding 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 the molecule b) above. The difference essentially lies in the arrangement of the domains: the transactivator binding domain is now localized 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 domain oligomerization with the p53 protein of sequence SEQ ID n ~ 3. This molecule allows the recruitment of mutated p53 proteins appearing in cells tumor.

CA 022144 ~ 1 1997-09-22 W O96 / 30512 PCTn ~ R96 / 00477 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 domain oligomerization with the p53 protein of sequence SEQ ID n ~ 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 domain oligomerization with the p53 protein of sequence SEQ ID n ~ 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 domain oligomerization with the p53 protein of sequence SEQ ID n ~ 3.
j) TET or Cro-Hinge-Oligom (Figure 5E) This type of molecule is similar to type e), except for the 15 transactivator binding domain which is replaced by the domain oligomerization with the p53 protein of sequence SEQ ID n ~ 3.
Furthermore, in each of these molecules, the peptide arm can be easily replaced by the sequence (G4S) 3 (SEQ ID n ~ 5).
Another object of the present invention resides in a sequence 20 of nucleic acid encoding a chimeric molecule as defined above before. It is advantageously a DNA sequence, in particular cDNA.
It can also be an RNA. The sequences of the invention are generally constructed by assembly, within a cloning vector, sequences coding for the different fields according to the techniques 25 classics of molecular biology. The nucleic acid sequences of the invention may possibly be modified chemically, enzymatic or genetic, in order to generate stabilized domains, eVou multifunctional, small eVou, eVou in order to promote their localization in this or that intracellular compartment. So the sequences 30 of nucleic acids of the invention may include sequences CA 022l44 ~ l l997-09-22 W O96 / 30512 PCTn ~ R96 / 00477 encoding nuclear localization peptides (NLS). In particular, it is ~ possible to merge the sequences of the invention with the coding sequence for the SV40 virus NLS, 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 make part of an expression vector, which may be plasmid in nature or viral.
Another object of the present invention resides in a protein of fusion comprising a transcriptional transactivating domain and a domain specific binding to a given molecule, possibly linked by a peptide arm, as well as any nucleic acid sequence encoding a such merger. The transactivating domain can be derived from any protein transcriptional transactivator, such as p53, VP16, EBNA, E6 / E7, Tat, etc.
Another object of the invention consists of a conditional system expression of genes 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 complex transactivator recruited by the bispecific molecule. So the sequence regulator is the DNA binding sequence of the chimeric molecule expressed. When the DNA binding domain of the chimeric molecule - is represented by all or part of TetR, the regulatory sequence includes the sequence SEQ ID n ~ 1 or a derivative thereof, optionally repeated several times. It is preferably the op2 sequence (comprising 2 repeated Tetop patterns) or Op7 (comprising 7 repeated Tetop patterns such as CA 022144 ~ 1 1997-09-22 W O96 / 30512 PCT ~ R96 / 00477 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 includes the sequence SEQ ID n ~ 2 or a derivative thereof, possibly repeated 5 several times. It is preferably the OR3 sequence. Drifts sequences SEQ ID n ~ 1 and 2 can be any sequence obtained by modification of a genetic nature (mutation, deletion, addition, repetition, etc.) and retaining the ability to specifically bind a protein. Such derivatives have been described in the literature (Baumeister et al supra, Tovar et al., Mol.
Gen. Broom. 215 (1988) 76, W094 / 04672).
Regarding the minimum transcriptional promoter, this is a promoter whose activity depends on the presence of a transactivator. From this in fact, in the absence of the chimeric molecule, the promoter is inactive and the gene is little or not expressed. However, in the presence of the molecule 15 chimeric, the recruited transactivator or transactivator complex allows 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 box or INR. These elements are indeed the minimum elements necessary to expression of a gene in the presence of a transactivator. Promoter 20 minimum can be prepared from any promoter by modification genetic. As a preferred example of a candidate promoter, mention may be made of promoter of the thymidine kinase gene. Interesting results have specifically obtained with a minimal promoter derived from TK promoter composed of nucleotides -37 to +19. The minimum promoter 25 can also be derived from human CMV. In particular, it can be made up of the fragment between nucleotides -53 to +75 or -31 to +75 of CMV.
Any conventional promoter can however be used such as for example the promoter of the genes coding for chloramphenicol acetyl transferase, ~ -galactosidase or luciferase.

CA 022144 ~ 1 1997-09-22 W 096/30512 PCTtFR96tO0477 The expression cassette advantageously consists of following items:
- As a regulatory sequence, a sequence comprising the sequence SEQ ID n ~ 1 or 2 or a derivative thereof, optionally 5 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 minimum promoter consists of 10 -37 to +19 region of the thymidine kinase gene promoter.
Advantageously, the expression cassette is chosen from among the Tetop2.TK-Gene structure cassettes; Tetop7.TK-Gene and OR3.TK-Gene.
Another aspect of the invention resides in an expression vector comprising a nucleic acid sequence encoding a molecule 15 chimeric and an expression cassette as defined above. In the vectors of the invention, the nucleic acid sequence encoding the chimeric molecule and the expression cassette can be inserted into the same orientation or in opposite orientations. In addition, the vector can be plasmid or viral in nature.
Among the viral vectors, there may be mentioned more preferably the adenoviruses, retroviruses, herpes viruses or adeno viruses associated. The viruses according to the present invention are defective, that is to say unable to replicate autonomously in the target cell.
Generally, the genome of defective viruses used in the context of 25 present invention therefore lacks at least the necessary sequences 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 CA 022144 ~ 1 1997-09-22 W O96 / 30512 PCTA ~ R96 / 00477 sequences of its genome which are necessary for the packaging of viral particles.
As regards more particularly adenoviruses, different serotypes, whose structure and properties vary somewhat, have been 5 characterized. Among these serotypes, it is preferred to use in the context of present invention human adenoviruses type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin (see application WO94 / 26914). Among adenoviruses of animal origin usable in the context of this invention include 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) by example]. Preferably, in the context of the invention, 15 adenovirus of human or canine or mixed origin.
Preferably, the genome of the recombinant adenoviruses the invention comprises at least the ITRs and the packaging region of a adenovirus, and the nucleic acid sequence encoding a molecule chimeric and an expression cassette as defined above. More 20 preferably, in the genome of the adenoviruses of the invention, the region E1 at least is non-functional. The viral gene considered can be rendered non-functional by any technique known to those skilled in the art trade, and in particular by total deletion, substitution (for example by the sequences of the invention), partial deletion, or addition of one or 25 several bases in the genes considered. Such modifications can be obtained in vitro (from isolated DNA) or in situ, for example, using genetic engineering techniques, or by treatment by means of mutagens. Other regions may also be modified, and in particular the region E3 (WO 95/02697), E2 (WO 94/28938), CA 022144 ~ 1 1997-09-22 W O96 / 30512 PCT ~ FR96 / 00477 E4 (W0 94/28152, W0 94/12649, W0 95/02697) and L5 (W0 95/02697).
- According to a preferred embodiment, the adenovirus according to the invention includes a deletion in regions E1 and E4. According to another mode of ~ preferred embodiment, it includes a deletion in region E1 at the level of which are inserted the E4 region and the sequences of the invention (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, EMB0 J. 3 (1984) 2917). In particular, they can be prepared by recombination homologous between an adenovirus and a plasmid carrying inter alia the DNA sequences of the invention (sequence coding for the molecule chimeric + expression cassette). Homologous recombination occurs produced after co-transfection of said adenovirus and plasmid in a appropriate cell line. The cell line used should preferably (i) be transformable by said elements, and (ii), include the sequences capable of complementing the part of the genome of the defective adenovirus, preferably in integrated form to avoid the risk of recombination. As an example of a line, one can mention the line kidney, human vonnary 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains in particular, integrated into its genome, the left part the genome of an Ad5 adenovirus (12%) or lines capable of complement functions E1 and E4 as described in particular in applications no W0 94/26914 and W095 / 02697.
Then the multiplied adenoviruses are recovered and purified according to conventional molecular biology techniques, such as illustrated in the examples.

Concerning adeno-associated viruses (AAV), these are viruses with DNA of relatively small size, which integrate into the genome of W O96 / 30512 PCTA ~ R96 / 00477 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. They also do not seem to be involved in pathologies in humans. The 5 MV genome has been cloned, sequenced and characterized. He understands about 4700 bases, and contains at each end a repeat region inverted (ITR) of about 145 bases, serving as the 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 10 rep gene involved in viral replication and gene expression viral; the right part of the genome, which contains the cap gene coding for virus capsid proteins.
The use of vectors derived from MV for the transfer of genes in vitro and in vivo has been described in the literature (see in particular WO 91/18088; WO 93/09239; US 4,797,368, US 5,139,941, EP 488,528).
These requests describe various constructions derived from MV, in which rep eVou 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 in an organism) said gene of interest. AAVs 20 defective recombinants according to the invention can be prepared by co-transfection, in a cell line infected with a helper virus human (for example an adenovirus), a plasmid containing the nucleic acid sequences of the invention (sequence coding for the molecule chimeric + expression cassette) bordered by two repeated regions 25 inverted (ITR) of MV, and of a plasmid carrying the packaging genes (rep and cap genes) of MV. The recombinant AAVs produced are then purified by conventional techniques.
Regarding herpes viruses and retroviruses, the construction of recombinant vectors has been widely described in the literature: see especially Breakfield et al., New Biologist 3 (1991) 203; EP 453242, CA 022l44 ~ l l997-09-22 WO 96/30512 PCT ~ R96 / 00477 EP178220, Bernstein et al. Broom. Eng. 7 (1985) 235; McCormick, ~ BioTechnology 3 (1985) 689, etc.
In particular, retroviruses are integrative viruses, infecting selectively 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 an acid sequence heterologous nucleic acid of interest. These vectors can be made from different types of retroviruses such as in particular MoMuLV ("murine moloney leukemia virus "; also known as MoMLV), MSV (" murine moloney sarcoma virus "), HaSV (" harvey sarcoma virus "); SNV
("spleen necrosis virus"); RSV ("rous sarcoma virus") or the virus from Friend.
To construct recombinant retroviruses according to the invention, a plasmid comprising in particular the LTRs, the packaging sequence and the sequences of the invention (sequence coding for the molecule chimeric + expression cassette) is generally constructed, then used to transfect a cell line called packaging, capable to bring 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 PsiCRlP line (WO90 / 02806) and the GP + envAm-12 line (WO89 / 07150). In addition, recombinant retroviruses can - include modifications to the LTRs to remove the activity transcriptional, as well as extended packaging sequences, containing part of the gag gene (Bender et al., J. Virol. 61 (1987) CA 022144 ~ 1 1997-09-22 W O96 / 30512 PCTA ~ R96 / 00477 1639). The recombinant retroviruses produced are then purified by classic techniques.
An example of construction of a defective recombinant virus according to the invention (retrovirus) is described in Figure 8. This figure highlights a 5 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 complex transactivator recruited by the system of the invention, the promoter is inactive and the gene is not expressed in the production cell shown 10 8A). It is only when the virus has effectively infected a target cell, i.e. a cell in which the transactivator or transactivating complex recruited by the system of the invention, that the gene is effectively expressed (Figure 8B). This is particularly advantageous for the construction of viruses containing genes whose 15 the expression would be toxic to cells (genes Grb3-3, IL-2, Toxin diphtheria, etc.).
For the implementation of the present invention, it is all particularly advantageous to use an adenovirus or a retrovirus defective recombinant. These vectors indeed have properties 20 of particular interest for the transfer of genes into cells tumor.
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 indeed be incorporated into a non-viral agent 25 capable of promoting 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, security and also by the absence of theoretical limit as regards the size of the DNA to be transfected.

CA 022l44 ~ l l997-09-22 W O96 / 30512 PCT ~ R96 / 00477 These synthetic vectors have two main functions, - compact the nucleic acid to be transfected and promote its fixation cell as well as its passage through the plasma membrane and, the if necessary, the two nuclear membranes. To compensate for nature 5 polyanionic nucleic acids, non-viral vectors have all polycationic charges.
Among the synthetic vectors developed, polymers polylysine type cationics, (LKLK) n, (LKKL) n, immine polyethylene and DEAE dextran or the cationic lipids or lipofectants are the 10 more advantageous. They have the property of condensing DNA and promote its association with the cell membrane. Among these the latter include lipopolyamines (lipofectamine, transfectam, etc.) and different cationic or neutral lipids (DOTMA, DOGS, DOPE, etc.).
More recently, the concept of targeted transfection has been developed, 15 mediated by a receptor, which takes advantage of the principle of condensing DNA
thanks to the cationic polymer while directing the attachment 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 type cell that we want to transplant. Targeting the transferrin receptor, 20 insulin or hepatocyte asialoglycoprotein receptor a been described.
The present invention also relates to any composition pharmaceutical comprising a vector as defined above. These compositions can be formulated for administration by route Topical, oral, parenteral, intranasal, intravenous, intramuscular, sub-cutaneous, intraocular, etc. Preferably, the composition according to the invention contains pharmaceutically acceptable vehicles for a - injectable formulation. They may in particular be saline solutions (sodium phosphate, disodium, sodium chloride, potassium, calcium 30 or magnesium, etc., or mixtures of such salts), sterile, isotonic, or CA 022144 ~ 1 1997-09-22 W O96 / 30S12 PCTA ~ R96 / 00477 dry compositions, in particular lyophilized, which, by addition according to the sterilized water or physiological saline, allow the constitution of injectable solutions. Retroviruses can be beneficial to directly use the packaging cells or infected cells ex5 vivo for their reimplantation in vivo, possibly in the form of neo organs (WO 94/24298).
The vector doses used for injection can be adapted according to different parameters, and in particular according to the method of administration used, the pathology concerned or even 10 the duration of the treatment sought. Generally speaking, viruses recombinants according to the invention are formulated and administered in the form of doses between 104 and 1014 pfulml. For MV and adenovirus, doses of 106-101 ~ pfu / ml may also be used. The term pfu ("plaque forming unit") corresponds to the power 15 infectious with a suspension of virions, and is determined by infection of a appropriate cell culture, and measurement, usually after 48 hours, the number of ranges of infected cells. The techniques of determination of the pfu titer of a viral solution are well documented in the litterature.
The expression system according to the invention and the vectors correspondents are particularly useful for controlling the expression of genes of interest in cell or gene therapy. They can thus be used to control the expression of any sequence 25 coding unit of interest, and in particular of a sequence coding for a product therapeutic, whether it is a peptide, polypeptide, protein, acid ribonucleics, etc. More specifically, the gene is a sequence DNA (cDNA, gDNA, synthetic DNA, human, animal, plant, etc.) encoding a protein product such as enzymes, derivatives 30 blood, hormones, Iymphokines: interleukins, interferons, TNF, CA 022144 ~ 1 1997-09-22 W O96 / 30512 PCTA ~ R96 / 00477 etc (FR 9203120), growth factors, neurotransmitters or Their precursors or synthetic enzymes, trophic factors:
BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, etc; the apolipoproteins: ApoAI, ApoAlV, ApoE, etc. (FR 93 05125), the dystrophin or a minidystrophin (FR 9111947), the genes tumor suppressors: p53, Rb, RaplA, DCC, k-rev, etc. (FR 93 04745), genes coding for factors involved in coagulation : Factors Vll, Vlll, IX, etc., or all or part of an immunoglobulin natural or artificial (Fab, ScFv, etc.), a ligand RNA (W091 / 19813) etc.
The gene of interest can also be an antisense sequence, whose expression in the target cell makes it possible to control the expression of genes or transcription of cellular mRNAs. Such sequences can for example be transcribed, in the target cell, into RNA
complementary to cellular mRNAs and thus block their translation into protein, according to the technique described in patent EP 140,308.
The present invention is particularly suited to the expression of sequences encoding toxic factors. It can be in particular of poisons for cells (diphtheria toxin, toxin pseudomonas, ricin A, etc.) of a product inducing sensitivity to an agent external (suicide genes: Thymidine kinase, cytosine deaminase, etc.) or still killer genes capable of inducing cell death (Grb3-3 (PCT / FR94 / 00542), ScFv anti-ras (W094 / 29446), etc). The system of the invention indeed makes it possible to produce vectors, notably viral containing these sequences without toxicity for the production cells, then to induce the expression of these toxic molecules selectively in target cells with the transactivator or desired transactivating complex. This type of construction is therefore particularly suitable for anti-tumor therapy strategies by example, in which the objective is to selectively destroy the affected cells. This system is also particularly interesting -CA 022144 ~ 1 1997-09-22 for the expression of cytokines, interferons, TNF or TGF for example, of which uncontrolled production can have very side effects marked.
This application will be described in more detail using the 5 examples which follow, which should be considered as illustrative and not limiting.

Legend of Figures Fi ~ ure 1: Representation of the conditional expression system according to the invention allowing the selective recruitment of a transactivator by means 10 of an oigomerization domain (A) or of an ScFv (B).
Fi ~ ure 2: Representation of the conditional expression system according to the invention allowing the selective recruitment of a transactivating complex.
Fi ~ ure 3: Representation of an expression cassette according to the invention containing a regulatory sequence Tetop7, a minimal promoter (box 15 TATA) 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).
Fi ~ ure 5: Representation of bispecific chimeric molecules according to 20 the invention.
Fi ~ ure 6: Construction of DNA sequences encoding molecules bispecific chimeras according to the invention.
Figure 7: Representation of control chimeric constructions.
Figure 8: Structure and functioning of a viral vector (retrovirus) according to 25 the invention.
Fi ~ ure 9: Study of the interaction between the hybrid molecules of the invention and a regulatory sequence.
Fi ~ ure 10: Study of the interaction between the hybrid molecules of the invention anddifferent forms of the p53 protein.

CA 022l44 ~ l l997-09-22 W O96 / 30512 PCT ~ FR96 / 00477 Fi ~ ure 11: Highlighting the activation of the Tet-Luc cassette in the ~ SAOS-2 cells.
Fi ~ ure 12: Highlighting the activation of the Tet-Luc cassette in the H358 cells.
5 General Molecular Biology Techniques Classical molecular biology methods such as centrifugation of plasmid DNA in cesium chloride-bromide gradient ethidium, restriction enzyme digestion, electrophoresis on gel, transformation in E. coli, precipitation of nucleic acids etc, 10 are described in the literature (Maniatis et al., 1989).
The enzymes were provided by New-England Biolabs (Beverly, MA).
For ligations the DNA fragments are separated according to their size on 0.8 to 1.5% agarose gels, purified by GeneClean (Bl0101, LaJolla CA) and incubated overnight at 14 ~ C in 50 mM Tris-HCl pH 7.4 buffer, MgCl2 10 15 mM, 10 mM DTT, 2 mM ATP, in the presence of phage T4 DNA ligase.
Amplification by PCR, (Polymerase Chain Reaction), also was carried out according to Maniatis et al., 1989, with the following specifications:
- MgCI2 concentration increased to 8mM.
- Denaturation temperature 95 ~ C, hybridization temperature 55 ~ C, 20 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 B by a cyanoethyl group, (Sinha al., 1984, Giles 1985), with the Applied DNA automatic synthesizer 25 Biosystem model 394, (Applied Biosystem, Foster City CA), according to - manufacturer's recommendations.
The sequencing was carried out on double-stranded matrices by the chain termination method using fluorescent primers.
We used the Taq Dye Primer Kit from Applied CA 022144 ~ 1 1997-09-22 W O96 / 30512 PCT ~ R96100477 Biosystem (Applied Biosystem, Foster City CA) according to the specifications of the maker.

Examples Example 1: Constructing expression cassettes including a 5 re ~ ulation sequence. a minimal transceiver, otional and a ~ ene.
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 Tetop motifs (SEQ ID
10 n ~ 1) repeated;
- a minimal promoter derived from the promoter of the thymidine gene kinase (region -37 to + 19 carrying the TATA box);
- the sequence coding for chloramphenicol acetyl transferase (CAT) under the control of said minimum promoter.
This plasmid was constructed by cloning the Smal-Bglll fragment of plasmid pUHD10-7 (WO 94/29442) in plasmid pKK232-8 (Pharmacia) previously digested with Smal and BamHI.

1.2. Construction of the plasmid pOR3 / CAT
The pOR3 / CAT plasmid contains the following elements (Figure 4):
- A regulatory sequence consisting of an OR3 sequence interaction with the repressor Cro (SEQ ID n ~ 2);
- a minimal promoter derived from the promoter of the thymidine gene kinase (region -37 to + 19 carrying the TATA box);
- the sequence coding for chloramphenicol acetyl transferase (CAT) 25 under the control of said minimum promoter.
This plasmid was constructed as follows: The OR3 sequence interaction with the repressor Cro has been artificially synthesized. For that, the following two oligonucleotides were synthesized:

CA 022144 ~ 1 1997-09-22 W O96t30512 PCTA ~ R96 / 00477 Oligo 5533 (SEQ ID n ~ 22): 5'-GATCCTATCACCGCMGGGATAA-3 ' ~ Oligo 5534 (SEQ ID n ~ 23): 3'-GATAGTGGCGTTCCCTA III CGA-5 ' These two oligonucleotides were then hybridized to reconstitute the double stranded OR3 sequence bordered by sequences allowing its cloning 5 oriented as follows:
GATCCTATCACCGCAAGGGATAA

GATAGTGGCGTTCCCTATTTCGA

1.3. Construction of toxic gene expression cassettes The toxic gene expression cassettes are obtained from 10 of the plasmids described above (1.1. And 1.2.) By replacing the CAT sequence by 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 specific D53 specific chain anticorD
This single chain antibody was constructed according to the following protocol:
- The cDNAs coding for the VH and VL regions were obtained from of the pAb421 hybridoma producing an anti-p53 antibody. For this, the RNAs hybridoma totals were extracted and subjected to a reaction of reverse transcription using random hexamers as primers.
20 The use of this type of primer avoids the use of primers specific for immunoglobulins. The cDNA clones obtained have a sufficient length to clone V regions. However, insofar as they represent a small fraction of the total cDNA present, a reaction amplification must be carried out to produce sufficient 25 DNA for cloning. For this, the cDNAs coding for the VH and VL regions have been amplified separately. The primers used are oligonucleotides hybridizing at opposite ends of regions variables of each chain (H and L). The amplification product using the specific heavy chain primers is a fragment of 340 pairs of CA 022144 ~ 1 1997-09-22 W O96 / 30512 PCTA ~ R96100477 bases approx. The amplification product using the specific primers of the light chains is a fragment of approximately 325 base pairs.
- After purification, the cDNAs encoding the VH and VL regions of The antibodies were assembled in a single chain by means of an arm 5 nucleotide (L). The nucleotide arm was constructed so that one end binds to the 3 'end of the cDNA encoding the VH region and the other at the 5 'end of the cDNA encoding the VL region. The sequence of arm code for the peptide SEQ ID n ~ 5. The assembled sequence of 700 bp approximately contains, in the form of an Ncol-NotI fragment, the VH-L- chain VL whose sequence is represented SEQ ID n ~ 4 (amino acids 9 to 241).
This sequence also includes in C-terminal the tag sequence of myc (residues 256 to 266).
FxemDle 3: Construction of nucleic acid sequences coding for chimeric molecules ~ bispecified ~ ues containing a binding domain to a 15 transactivator consisting of a single chain anticorDs (ScFv).
3.1. Construction of a plasmid comprising a ScFv-myc- sequence Hinge-TetR or Cro (Figures 5A and 6) The Ncol-NotI fragment containing the cDNA coding for the anti-p53 ScFv was first cloned into a plasmid of the pUC19 type. The sequence 20 coding for the VSV epitope (SEQ ID n ~ 7) or myc (SEQ ID n ~ 8) is inserted downstream of the fragment (Figure 6).
The sequences encoding the TetR and Cro proteins were then obtained as follows:
- The sequence coding for TetR was obtained by amplification at 25 from a template plasmid carrying the sequence tetR by means of the following oligonucleotides:
Oligo 5474 (SEQ ID n ~ 10):
GGCTCTAGACCCAAGCCCAGTACCCCCCCAGGTTCTTCAACGCG
TGGATCCATGTCCAGATTAGATAAAAGTAAAG

CA 022144 ~ 1 1997-09-22 Oligo 5475 (SEQ ID n ~ 11):
CGTACGGAATTCGGGCCCTTACTCGAGGGACCCACI I ICACAI l I
AAGTTG
These oligonucleotides also provide the sequence coding for the 5 Hinge peptide arms connecting the two functional areas of the molecules.
The amplified fragment therefore contains the sequence coding for the arm peptide and for the tetR DNA binding domain. This fragment was cloned at the Xbal-EcoRI sites of the plasmid obtained above to generate a 10 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 template of the bacteriophage Lambda using oligonucleotides following:
Oligo 5531 (SEQ ID n ~ 12):
GGCTCTAGACCCAAGCCCAGTACCCCCCCAGGTTCTTCAACGCG
TGGATCCATGGAACAACGCATAACCCTGAAAG
Oligo 5532 (SEQ ID n ~ 13):
CGTACGGAATTCGG GCCCTTACTCGAGTGCTGTTG ~ GTT

These oligonucleotides also provide the sequence coding for the Hinge peptide arm connecting the two functional areas of the molecules.
The amplified fragment therefore contains the sequence coding for the arm 25 peptide and for the Cro 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-Cro (Figure 6).
3.2. Construction of a plasmid comprising a ScFv- sequence 30 Hinge-TetR or Cro (Figure 5B) CA 022144 ~ 1 1997-09-22 W O96 / 30512 PCT ~ FR96 / 00477 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 piasmids were obtained from the plasmids described in 3.1.
above by digestion with the enzymes Notl and Xbal. This digestion allows to excise the fragment carrying the region coding for the tag myc.
3.3. Construction of a plasmid comprising a ScFv-TetR sequence or Cro (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 tag sequence.
These plasmids were obtained from the plasmids described in 3.1.
above by digestion with the enzymes Notl and BamHI. This digestion allows to excise the fragment carrying the region coding for the tag myc and for the arm Hinge peptide.
Example 4 Construction of Nucleic Acid Sequences Coding for bispecific chimeric molecules containing a binding domain to a transactivator consisting of a domain of oli ~ omerization.
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 n ~ 3) was obtained by PCR amplification on a plasmid carrying the cDNA of human wild-type p53 using the oligonucleotides following:
Oligo 5535 (SEQ ID n ~ 14):
CAGGCCATGGCATGAAGAAACCACTGGATGGAGAA
(the underlined part represents an Ncol site) Oligo 5536 (SEQ ID n ~ 15):
CGTCGGATCCTCTAGATGCGGCCGCGTCTGAGTCAGGCCCTTC

CA 022144 ~ 1 1997-09-22 W O96 / 30512 PCT ~ FR96100477 (Underlined part: BamHI site; Double-underlined: Xbal site: Bold: site Notl).
4.2. Plasmids p53 320/393-myc-Hinge-TetR or Cro (Figure 5A) were obtained by cloning the amplified fragment above in the form of a Ncol-Notl 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) have were obtained by cloning the fragment amplified in 4.1. in the form of a Ncol-Xbal fragment in the corresponding sites of the plasmids described in Example 3.1., replacing the region coding for ScFv and tag.
4.4. Plasmids p53 320/393-TetR or Cro (Figure 5C) were obtained by cloning the fragment amplified in 4.1. as a fragment Ncol-BamHI 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 fragments amplified by PCR on a plasmid carrying the p53 cDNA
wild human using oligos 5537/5539 or 5538/5539 digested by Xhol / EcoRI in the plasmids described in 3.1., Previously digested with Xhol / EcoRI.
Oligo 5537 (SEQ ID n ~ 16):
CAGGCTCGAGAAGAAACCACTGGATGGAGAA
Oligo 5538 (SEQ ID n ~ 17):
- CAGGCTCGAGCCCAAGCCCAGTACCCCCCCAGGTTCTTCAAAGA
AACCACTGGATGGAGAA
Oligo 5539 (SEQ ID n ~ 18):
GGTCGAATTCGGGCCCTCAGTCTGAGTCAGGCCCTTC

CA 022144 ~ 1 1997-09-22 Example 5 Construction of a Control Plasmid Sleeping a Coding Sequence for a chimeric molecule having a DNA binding domain (TetR or Cro) and the transactivating domain of the protein D53 (re ~ ion 1-73).
Plasmids p53 1/73 - TetR or Cro with or without tag (myc or VSV) and 5 Hinge (Figures 7 A, B and C) were obtained by cloning fragments amplified by PCR from a plasmid carrying the cDNA of wild-type p53 human using oligos 5661/5662 then digested with Ncol / Notl, 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 n ~ 20):
CGTCGGATCCTCTAGATGCGGCCGCCACGGGGGGAGCAGCCTC
TGG
15 Example 6: Construction of expression plasmids of different hybrid molecules of the invention The plasmids used for the expression of hybrid molecules have been obtained by cloning of the fragments containing the cDNA coding for these molecules at Ncol / EcoRI sites of the eukaryotic expression vector pcDNA3 20 (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 Figure 6 according to Example 3.1 - TET02: hybrid protein containing the chain p53 (320/393) -VSV-25 Hinge-TetR described in FIG. 5A according to example 4.3 - TET03: hybrid protein containing the chain p53 (320/393) -Hinge-TetR described in Figure 5B according to Example 4.4 .

CA 022144 ~ 1 1997-09-22 W O96t30S12 PCTAFR96 / 00477 - TET04: hybrid protein containing the chain p53 (320/393) -TetR
described in Figure 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 Iysate of the molecules described in Example 6 using the TNT Coupled Reticulocyte Iysate Systems kit (Promega) following the experimental protocol described by the supplier for a total reaction volume of 50, ul.
7.2 Construction of the specific double-stranded DNA sequence The specific double stranded DNA sequence used in this experiment is consisting of two synthetic oligonucleotides whose sequence is next:
Oligo 5997 (SEQ ID n ~ 24):
GATCCGACTTTCAC ~ CTCTATCACTGATAGTGAGTGGTAAACTCA
Oligo 5998 (SEQ ID n ~ 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, ul of the following reaction medium:
Tris-HCI pH7.6 50mM
MgCI2 1 OmM
5mM dithiothreitol CA 022144 ~ 1 1997-09-22 W O96130512 PCT ~ R96 / 00477 Spermidine 1 00, uM
EDTA 1 00, uM
ATP ~ P (Amersham) 50, uCi (1000-3000 Ci / mmole) T4 kinase (Boehringer) 10U
Then the two oligonucleotides thus labeled were hybridized in the presence of 400mM NaCI to reconstitute the following double strand TetO sequence (SEQ ID n ~ 26):
GATCCGACTTTCACTTTTCTCTATCACTGATAGTGAGTGGTAAACTCA
CTAGGCTCAAAGTGAAAAGAGATAGTGACTATCACTCACCATTTGAGT
7.3 Recognition of the TetO double strand sequence by the different hybrid molecules of the invention The DNA binding reaction is carried out in 50 ~ I of reaction medium (Tris-HCI pH7.4 10mM, MgC12 10mM, KCI 10mM, ~ -mercaptoethanol 6mM, 0.1 mM EDTA, 0.5 mg / ml BSA) by addition of the TetO sequence (10- '~ M) prepared according to Example 7.2, 10 μl of the translation product prepared according to Example 7.1 and 1 0 ~ M of the cold competitor oligonucleotide AP2 (Promega) used to eliminate non-specific fixation. The specificity of the interaction is verified by shifting the equilibrium by adding tetracycline 10, uM (Sigma) in the reaction medium. Mixtures Reactions are incubated for 15 min at 20 ° C. and then added with 10 μl of glycerol at 50%, 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 hybrid molecules TET19, TET02 and TET07 is shown in Figure 9. Under these conditions, the binding of these three molecules to the specific double-stranded DNA sequence TetO is observed by a delay in migration thereof, and the specificity of CA 022144 ~ 1 1997-09-22 WO 96 / 30S12 PCTA ~ R96 / 00477 this interaction is demonstrated by the inhibition of this delay by addition tetracycline.
This result therefore confirms that the hybrid molecules of the invention are capable of specifically binding to the TetO nucleotide sequence 5 Example 8 ~ specific ison of the hybrid molecules of the invention to a molecule with a transcriptional transactivating domain.
8.1. Production of the hybrid molecules of the invention and of molecules whether or not having 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 into using the experimental protocol according to example 7.1 in the presence of 44, uCi 35S-methionine (Amersham) (1175 Ci / mmol) to generate these molecules radioactive labeled hybrids.
The cDNAs of molecules with or without a domain 15 transcriptional transactivators were cloned into the vector pBlueBaclll (Invitrogen) at the BamHI site. From these vectors, baculoviruses recombinants have been produced and purified according to the manufacturer's instructions. The molecules are produced by infection with the recombinant baculovirus sf9 insect cells following the manufacturer's experimental protocol. Of 20 protein extracts at the final protein concentration of 1 Omg / 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 25 to 320 and therefore devoid of its oligomerization domain and the domain recognized by the monoclonal antibody pAb421.

W O96 / 30S12 PCT ~ R96 / 00477 8.2. Binding of the hybrid molecules of the invention to the molecules presenting or not a transcriptional transactivating domain 5 11 of each of the in vitro translation products prepared according to the example 8.1 were incubated with 5111 of the baculovirus extract prepared according to the example 5 8.1 and 2119 of the monoclonal antibody DO1 (Oncogene Sciences) which recognizes the N-terminus of the p53 protein for 16 hours at 4 ~ C in 100111 of modified RIPA buffer ((K. Ory, EMBO J., 13, 3496-3504, 1994).
Immunoprecipitation is carried out as described by K. Ory et al. (K. Ory, EMBO J., 13, 3496-3504, 1994). The complexes retained by the antibody are 10 eluted by incubation for 10 min at 80 ~ C in the presence of 30, u1 of buffer migration (Laemmli UK, Nature, 227, 680-685, 1970) and subject to electrophoresis on 10% polyacrylamide gel in denaturing medium at 200V
following the previously described protocol (Laemmli UK, Nature, 227, 680-685, 1970). The gel is then dried and revealed using an instantimager 15 (Packard instruments) which quantifies the quantities of molecules hybrids linked to the molecule with or without a transactivating domain transcriptional. The results of this experiment are shown on the figure 10.
Under these conditions, it clearly appears that the hybrid molecule 20 presenting ScFv 421 (TET19) clearly recognizes the p53 molecule (1/393) of equivalent to ScFv 421 alone, and the hybrid molecule presenting the domain 320/393 (TET02) has the same properties but with a much greater p53 retention capacity (1/393). Furthermore, No signal observed during incubation with the p53 molecule (1/320) 25 shows that these interactions are very specific and mediated by the end C-terminal of the p53 protein (amino acids 320 to 393) as expected.
These results therefore confirm that the hybrid molecules of the invention are capable of recruiting a transcriptional transactivator domain by a molecule in which they are specific partners.

CA 022l44 ~ l l997-09-22 W O96 / 30512 PCT ~ FR96 / 00477 Example 9: Functional recruitment of a transactivating domain tr ~ nscriDtionnel Dar hybrid molecules of the invention Functional recruitment of a transcriptional transactivator domain by the hybrid molecules of the invention was evaluated in a system of 5 in vivo transactivation in SAOS-2 cells (human osteosarcoma) deficient for the two alleles of the p53 protein, in the tumor line H358 deficient for the two alleles of the p53 protein (Maxwell & Roth, Oncogene 8, 3421, 1993) and in the tumor line HT29 deficient for a of the two alleles of the p53 protein and having a mutated allele (mutation 10 H273). This system is based on the use of a dosable reporter gene enzymatically and placed under the dependence of a promoter containing the nucleotide motifs for 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 and their genotype bound to the protein p53 and the culture medium used for their growth are 20 reported in the Table below:
Table: cell lines P53 line Culture medium n ~ ATCC
DMEM medium (Gibco BRL) supplemented with SAOS-2 - / - 10% fetal calf serum (Gibco BRL) HTB 85 RPMI1640 medium (Gibco BRL) supplemented with H358 - / - 10% fetal calf serum (Gibco BRL) DMEM medium (Gibco BRL) supplemented with HT29 - / H27310% fetal calf serum (Gibco BRL) CA 022144 ~ 1 1997-09-22 W O96 / 30512 PCTA ~ R96100477 9.2. Plasmids for expression of molecules with a domain transcriptional transactivator Molecules with a transcriptional transactivating domain 5 used in this experiment are the wild-type p53 protein (wt) and the mutant G281 and H175 of this protein. The cDNA encoding these three proteins were inserted at the BamHI site of the pcDNA3 vector (Invitrogen).
9.3. Intracellular expression of the hybrid molecules of the invention The hybrid molecules of the invention are expressed in cells in 10 culture by transient transfection using the following protocol:
The cells (3.5 105) are seeded in 6-well plates containing

2 ml of culture medium, and grown overnight in a CO2 incubator (5%) at 37 ~ C. The different constructions are then transfected into using lipofectAMlNE (Gibco BRL) as a blood transfection agent as follows: 1.5 μg of total plasmid are incubated (of which 0.25 μg of plasmid reporter) with 5 μl of lipofectAMlNE 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 it is aspirated and replaced with 2 ml 20 of culture medium supplemented with 10% fetal calf serum inactivated with heat and the cells returned to grow for 48 h at 37 ~ C.
9.4. Transcription activation detection Transcription activation linked to functional recruitment of the transcriptional activator is detected and quantified by measuring 25 luciferase activity encoded by the LUC gene using the Luciferase Assay kit System (Promega) according to the manufacturer's experimental protocol.

CA 022l44 ~ l l997-09-22 WO 96 / 30S12 PCTn ~ R96 / 00477 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 TET07 molecule serves as positive control since it has its own transactivating domain transcriptional.
The results obtained in SAOS-2 cells and presented in the figure 11 show that the TET07 molecule alone is capable of activating the transcription of the LUC gene under the control of the operator Tet unlike the construction TET02. This is consistent with the fact that this cell line does not contain endogenous p53 protein which cannot therefore be recruited by the TET02 molecule. However, the introduction of the wild-type p53 protein or its mutant G281 which do not produce signal alone, is capable of generating transcriptional activity in the presence of the TET02 molecule. Such a result is not observable with the mutant H175 described in the literature as having a domain non-functional transcriptional transactivator.
This result obtained in the SAOS-2 cell line with the molecule TET02 could be reproduced in a tumor line also containing no endogenous p53 (H358 cells) and could be extended to TET03 molecules and TET04 ffigure 12).
In order to confirm these two results in a cellular context different, the TET02 molecule as well as the TET07 positive control were expressed in HT29 cells which have an endogenous p53 protein mutant (H273), the negative control of this experiment consisting of transfect the empty pcDNA3 vector. The result of this experiment, presented in the Table below, shows that the TET02 molecule is well capable CA 022144 ~ 1 1997-09-22 WO 96/30512 PCTA ~ R96 / 00477 to recruit the transcriptional transactivator domain of the p53 protein endogenous.
Table: Transcriptional activation of the hybrid molecules of the invention in HT29 cells pcDNA3 TET07 TET02 All of these experiments therefore show that the hybrid molecules of the invention are well able to link both double DNA sequences specific strand and proteins with a transactivating domain 10 transcriptional, and that these molecules are capable of inducing conditional expression of genes of interest.

CA 022144 ~ 1 1997-09-22 WO 96/30512 PCT ~ R96 / 00477 LIST OF SEQUENCES

(1) GENERAL INFORMATION:

(i) DEPOSITOR:
(A) NAME: RHONE POULENC RORER SA
(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:
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Claims (57)

1. Bispecific chimeric molecule comprising a domain capable of binding selectively a defined DNA sequence and a detector domain capable of to specifically bind a transactivator or transrepressor or a complex transactivator or transrepressor characteristic of a physiological state or physiopathological. 2. Molecule according to claim 1 characterized in that the domain capable of selectively binding a defined sequence of DNA 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 sequence of DNA derives from a eukaryotic protein. 4. Molecule according to claim 3 characterized in that the domain capable of selectively binding a defined sequence of DNA derives from p53, STAT or NFkB proteins. 5. Molecule according to claim 2 characterized in that the domain capable of selectively binding a defined sequence of DNA derives from a prokaryotic protein. 6. Molecule according to claim 5, characterized in that the protein prokaryote is a bacterial repressor. 7. Molecule according to claim 6 characterized in that the domain capable of selectively binding a defined sequence of DNA derives the protein tetR. 8. Molecule according to claim 6 characterized in that the domain capable of selectively binding a defined sequence of DNA 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 sequence of DNA
comprises the DNA-interacting domain of said protein. 10. Molecule according to one of claims 2 to 8 characterized in that the domain capable of selectively binding a defined sequence of DNA is consisting of the complete protein. 11. Molecule according to claim 10, characterized in that the domain capable of selectively binding a defined sequence of DNA consists of the tetR protein. 12. Molecule according to claim 10, characterized in that the domain capable of selectively binding a defined sequence of DNA 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 a domain of oligomerization. 14. Molecule according to claim 13, characterized in that the domain of oligomerization is a leucine-zipper, an SH3 or SH2 domain. 15. Molecule according to claim 13, characterized in that the domain of oligomerization capable of specifically binding the transactivator is consisting of the C-terminal part of the p53 protein. 16. Molecule according to claim 15, characterized in that the domain of oligomerization consists of the C-terminal part of the protein p53 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 domain or 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 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 complex transactivator consists of an antibody Fab or F(ab)'2 fragment 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 complex transactivator 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 domain of DNA binding and transactivator binding domain are linked between them through an arm. 22. Molecule according to claim 21 characterized in that the arm is consisting of a peptide comprising 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 the peptides of sequence SEQ ID No. 5 or SEQ ID No. 6. 24. Molecule according to one of the preceding claims, characterized in that that the DNA-binding domain is located in the N-terminal position and the transactivator binding domain 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 at the C-terminal position and the domain of binding to the transactivator is located in the N-terminal position. 26. Bispecific chimeric molecule of structure ScFv-VSV/myc-Hinge-TET
or Cro (Figure 5A). 27. Bispecific chimeric molecule of structure ScFv-Hinge-TET or Cro (Figure 5B). 28. Bispecific chimeric molecule of ScFv-TET or Cro structure (Figure 5C). 29. Bispecific chimeric molecule of TET or Cro-ScFv structure (Figure 5D). 30. Bispecific chimeric molecule of TET or Cro-Hinge-ScFv structure (Figure 5E). 31. Bispecific chimeric molecule of structure Oligom-VSV/myc-Hinge-TET or Cro (Figure 5A), Oligom-Hinge-TET or Cro (Figure 5B) or Oligom-TET or Cro (Figure 5C). 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 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 at 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 any or part of TetR and the regulatory sequence comprises the sequence SEQ ID
No. 1 or a derivative thereof, 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 any or part of Cro and the regulatory sequence comprises the sequence SEQ ID no.
2 or a derivative thereof, optionally repeated several times. 38. Conditional system according to one of claims 35 to 37 characterized in that the minimal promoter comprises a TATA or INR box. 39. Conditional system according to claim 38 characterized in that the minimal promoter is derived from the thymidine kinase gene promoter. 40. Conditional system according to claim 39 characterized in that the minimal promoter is composed of nucleotides -37 to +19 of the promoter of thymidine kinase gene. 41. Vector including:
- a nucleic acid sequence coding for a molecule chimeric 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 promoter minimal transcriptional is defined according to claims 38 to 40. 43. Vector according to claim 41, characterized in that the domain of DNA binding 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 derived from it, possibly repeated several times. 44. Vector according to claim 41, characterized in that the domain of DNA binding 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 derived from it, 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 product therapeutic. 46. Vector according to claim 45 characterized in that the product therapeutic is a toxic peptide or polypeptide. 47. Vector according to claim 46 characterized in that the product toxic therapeutic is selected from diphtheria toxin, toxin pseudomonas, ricin A, thymidine kinase, cytosine deaminase, Grb3-3 protein, or the ScFv Y28. 48. Vector according to one of claims 41 to 47 characterized in that it it is a plasmid vector. 49. Vector according to one of claims 41 to 47 characterized in that it 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 possessing 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 virus HIV, the E6/E7 proteins of the papilloma virus and the EBNA protein of the virus by Epstein Barr. 56. Molecule according to claim 54, characterized in that the transactivator is a cellular protein, preferably p53 protein mutated or wild. 57. Molecule according to claim 1, characterized in that the transactivator or transactivator complex characteristic of a state physiological or physiopathological is a protein appearing in a infected or hyperproliferative cell