SK131197A3 - 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

Technical field

The invention relates to a novel system for the conditional expression of genes. The invention also relates to the use of this system in gene or cell therapy to achieve very selective expression of the gene of interest.

BACKGROUND OF THE INVENTION

Gene and cell therapy consist of correcting a deficiency or anomaly (mutation, aberrant expression, and the like) or ensuring expression of a therapeutic protein of interest by introducing genetic information into the organ or cell to be treated. This genetic information can be introduced either ex vivo into a cell isolated from an organ, the cell thus modified being then reintroduced into the body (cell therapy) or directly in vivo into the appropriate tissue (gene therapy).

There are various techniques for carrying out gene transfer, including, but not limited to, various transfection techniques in which natural or synthetic, chemical or biochemical vectors, such as DNA and DEAE-dextran complexes are used (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), use of liposomes (Fraley et al., J Biol. Chem. 255 (1980) 10431) as well as cationic lipids and the like. Another transfection technique involves the use of viruses as well as gene transfer vectors. In this regard, various viruses have been tested to determine their ability to infect certain cell populations. In particular, retroviruses (RSV, HMS, MMS and the like), HSV, adeno-associated viruses and adenoviruses.

However, one of the major difficulties encountered in the development of gene and cell therapy is the selectivity of treatment. Depending on the application and the gene to be transmitted, it is very important to be able to target certain tissues or only certain parts of the organism in order to direct the therapeutic effect only to these tissues or parts of the organism and to reduce the spread of this effect and secondary side effects.

Such targeting of a therapeutic effect can be accomplished using vectors that exhibit the desired cell specificity, i. j. vectors that are selectively specific only to the cells of interest.

Another approach to solving this problem is to use specific expression signals of some cell types. In this regard, specific promoters such as those encoding pyruvate kinase have been described in the literature for example by the vi11in, GFAP, intestinal fatty acid binding promoter, smooth muscle actin alpha promoter or the apo-AI, apo-AII, human albumin promoter. and so on. However, these promoters have some drawbacks and, in particular, exhibit some transcriptional noise, which may be unpleasant in the expression of toxic genes. Thus, the use of these promoters is limited to some cells and cannot be used for any application.

SUMMARY OF THE INVENTION

The invention now describes a novel conditional gene expression system that is particularly selective and effective. A preferred feature of this system of the invention is its ability to express the gene not depending on the cell type but on the presence of a specific cell element or specific physiological situation. In fact, this system relates to chimeric bispecific molecules comprising a region that is capable of selectively binding a defined DNA sequence and a detection region that is capable of specifically binding a transactivator or transactivation complex.

A first aspect of the invention is to provide and express chimeric bispecific molecules comprising a region that is capable of selectively binding a defined DNA sequence and a region that is capable of specifically binding a transactivator or transrepressor or a transactivating or transrepressor complex.

Another feature of the invention resides in a nucleic acid sequence encoding a chimeric molecule as defined above, as in any expression vector comprising said nucleic sequence.

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

Another feature of the invention also resides in an expression vector comprising

a nucleic acid sequence encoding 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 to achieve very selective expression of the genes of interest.

Thus, one component of the system of the invention is composed of chimeric bispecific molecules comprising a region which is capable of selectively binding a defined DNA sequence and a region that is capable of selectively binding a transactivator or transactivation complex. The bispecificity of the molecules of the invention lies in both their ability to bind a defined DNA sequence (commonly referred to as a regulatory or operative sequence) and their ability to specifically form a transactivating or transrepressive protein region that allows to induce or suppress gene expression.

The invention resides in the generation of chimeric bispecific molecules enabling one to obtain any transcription factor whose activation or inactivation leads to a physiopathological situation. Thus, the chimeric bispecific molecules of the invention allow selectively obtaining specific transcriptional transactivators of the physiopathological state, fixing these transcription factors to promoters by binding these molecules to defined DNA sequences located near these promoters (regulatory or operative sequence) and thereby achieving conditional gene expression (Fig. 1).

The invention also relates to chimeric bispecific molecules enabling the generation not only of a molecule carrying a transcriptional region but also of a transcriptional transactivation complex, i. j. a complex formed between the target molecule present in the cell and the molecule carrying the transactivation region (Fig. 2). In this case, the transactivation complex is preferably formed using a second chimeric bispecific molecule comprising a transactivation region and a selective binding region to said cell molecule. The fixation of the second molecule allows the formation of a transcriptional transactivation binary complex, which complex is thus formed by the detection system of the invention. The fixation of this ternary complex in the vicinity of the promoters thus permits the regulated expression of the genes. This type of construction advantageously extends the field of application of the system according to the invention to detect any intracellular molecule deprived of the transactivation region, whether it be an endogenous molecule or, for example, a molecule of infectious origin.

Thus, the system according to the invention makes it possible to activate exclusively the expression of the genes of interest in the presence of the target proteins due to the highly selective detection system (sensor). These may be transcription factors occurring in physiological or physiopathological situations, or any endogenous molecule or, for example, a molecule of infectious origin.

Within the scope of the invention, the term transactivator refers to any transactivation factor of transcription or any protein comprising a transcriptional transactivation region. The term transactivation complex refers to a complex formed between a molecule present in a cell and a bispecific molecule of the invention comprising a transactivation region and a specific binding region to said molecule.

The expression system of the invention may be used to obtain any transactivating protein or protein carrying a transactivating region, and in particular any protein of viral, parasitic or mycobacterial origin, as well as of cellular origin, having transcriptional transactivating activity.

Transcription factors of viral origin include, but are not limited to, the HIV Tat protein, the E6 / E7 papilloma virus proteins, or the Epstein-Barr virus EBNA protein. These proteins have a transcriptional transactivation region and are present exclusively in cells infected with these viruses, i. j. under specific physiopathological conditions. The conditional expression system of the invention preferably allows for the detection of such a physiological situation (occurrence of said specific transactivators of viral infection) and induction of selective expression of the gene or genes.

Of the cellular proteins, mutated or wild-type p53 protein may be mentioned. This p53 protein consists of 393 amino acids. In its wild form, this protein is a tumor suppressor capable of negatively regulating cell growth and division. This activity is associated with the presence of a transcriptional transactivation region in the structure of the p53 protein, located in the N-terminal region of the protein (about residues 1-100). In some situations, the wild-type p53 protein is also capable of inducing apoptosis (Yonish-Rouach et al., Nature, 352, 345-347, 1991). These properties manifest themselves in stressful situations where the integrity of cellular DNA is compromised and the p53 protein is thus considered to be a genome guardian. The presence of mutated p53 proteins in about 40% of human tumors of all types reinforces this hypothesis and underlines probably the key role that mutation of this gene plays in tumor development (see: Montenarh, Oncogene, 7, 1673-1680, 1992, Oren, FASEB J 6, 3169-3176, 1992, Zambetti and Levine, FASEB J., 7, 855-865, 1993). Within the scope of the invention, it is possible to selectively recruit the transactivating region of the p53 protein and thereby induce regulated expression of the gene or genes exclusively in cells containing the protein. According to the invention, it is of particular interest to specifically recruit mutated forms of the p53 protein which, as mentioned above, occur in physiopathological situations of cell proliferation (cancer type). Such targeting can advantageously be accomplished using a specific binding region to mutated forms of the p53 protein. However, there is a specificity associated with the accumulation of mutated forms having a half-life significantly longer than that of the wild-type.

The system of the invention can also be used to induce selective expression of a gene or genes by detecting each target molecule present in the cell. Preferably, the protein detected is a protein occurring in the cell in abnormal situations (infection, hyperproliferation, and the like). In particular, they may be viral proteins, such as structural or functional proteins of the virus, in particular HIV, hepatitis, herpes and the like. They may also be specific proteins of the cell hyperproliferative state, such as, in particular, myc, fos, jun, cyclins and the like.

Thus, one of the properties of the chimeric molecules of the invention lies in their ability to bind to specific DNA regions (regulatory or operational regions). This binding makes it possible to bring the transactivation region close to the promoter and in this way activate the expression of the gene inserted under the control of said promoter.

A region capable of selectively binding a defined DNA sequence present in the molecules of the invention is essentially of protein origin. More preferably, this region is derived from a prokaryotic or eukaryotic protein capable of interacting with DNA sequences. Numerous genetic and structural studies now make it possible to precisely define in the proteins interacting with the double-stranded DNA sequences that are responsible for these interactions.

Among the prokaryotic proteins interacting with the double-stranded DNA sequences, mention may be made in particular of bacterial repressors and more preferably the tetracycline repressor derived from E. coli and the repressor Cro derived from bacteriophage Lambda.

Said E.coli tetracycline repressor (tetR) is a protein of about 210 amino acids. In E. coli, this tetR tetracycline suppressor negatively regulates the transcription of genes mediating resistance to this antibiotic in the tet operon. In the absence of tetracycline, the tetR repressor is fixed to DNA at the level of a specific sequence (referred to as an operative sequence or Tetop) and suppresses the transcription of a resistant gene. Conversely, in the presence of tetracycline, the tetR repressor is no longer fixed to the tetop operator, thus allowing the constitutive transcription of the gene (Hillen. W and Wissman A. (1989) in Protein-Nucleic Acid Interaction. Topics in Molecular and Structural Biology, ed., Saenger W. and Heinemann U. (Macmillan, London), vol.10, pp 143-162). The sequence of the tetR tetracycline suppressor has already been published (particularly disclosed in WO94 / 04682). A double stranded DNA sequence that is specific for the binding of the tetR tetracycline suppressor to DNA (Tetop) is formed by the following motif:

TCTCTATCACTGATAGGGA (SEQ ID NO: 1).

This motif can be repeated several times to increase the affinity and efficiency of the system. The regulatory sequence may thus comprise 10 of said motifs, preferably comprising two motifs (Tetop2) or seven motifs (Tetop7) (see Fig. 3).

The Cro protein was originally defined as a regulator of CI repressor expression (Eisen H. et al. (1970) PNAS 66, page 855). Cloning of the cro gene allowed the identification of a protein of 66 amino acids (SEQ ID NO: 21, Roberts T. et al. (1977), Nature 270, p. 274). Cro performs its physiological role that is preferably fixed on the operator 0r ₃ derived from bacteriophage lambda.

The double stranded DNA sequence specific for Cro binding to DNA (the region designated OR ₃ ) is made up of the following bases:

TATCACCGCAAGGGATA (SEQ ID NO: 2).

This region can be repeated several times to achieve increased affinity and efficiency of the system (see Fig. 4).

Of the eukaryotic proteins interacting with the double-stranded DNA sequences, proteins or regions derived from STAT, p53 or NFκB proteins are preferably used to construct the molecules of the invention (Inoue et al., PNAS 89 (1992) 4333). With respect to the p53 protein, its DNA binding region is located in the central region of the protein, more precisely in the region between amino acids 102-292 (Pavletich et al., Genes and Dev. 7 (1993) 2556).

As mentioned above, the region is capable of selectively binding a defined DNA sequence present in the molecules of the invention preferably derived from a prokaryotic or eukaryotic protein capable of interacting with the double-stranded DNA region. The region used to construct the molecules of the invention may consist of all or part of a protein comprising a DNA interaction region. This region has been identified for various proteins, and in particular for TetR (see, for example, Berens et al., J.Biol.Chem267 (1992) 1945). This region may also consist of a derivative of the protein or a fragment thereof that retains the ability to bind to DNA. Such derivatives are, in particular, proteins with a modification of one or more amino acids, for example in order to allow their fusion with other regions of the molecules of the invention which can be prepared by conventional molecular biology techniques. For example, such TetR and Cro protein derivatives have already been described in the literature and have precise local mutations and / or deletions (Hecht et al., J. Bact.175 (1993), p. 1206, Atlschmied et al., EMBO J 7 (1988) 4011, Baumister et al., Proteins 14 (1992) 168, Hansen et al., J.Biol.Chem. 26 (1987) 14030). The ability of these derivatives to bind to a defined DNA sequence can then be tested by incubating the prepared derivative with a regulatory sequence and detecting the formed complexes. In addition, said derivatives may also be proteins having improved DNA binding ability (specificity, affinity, and the like).

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

In another preferred embodiment, the region is capable of selectively binding a defined DNA sequence present in the molecules of the invention derived from the TetR or Cro proteins. It is particularly preferred to use the TetR or Cro proteins (SEQ ID NO: 21) in the complete state.

A region capable of specifically binding a transcriptional transactivator or a transcriptional transactivating complex present in the molecules of the invention may be of various types. In particular, it may be an oligomerization region where the transactivator or transactivation complex to be targeted also contains such an region. It may also be any synthetic or natural region known to interact with said transactivator or transactivation complex. It may also be an antibody or a fragment or derivative of an antibody directed against said transactivator or transactivating complex.

Among the oligomerization regions useful in the present invention, mention may be made in particular of leucine zippers, for example SH2 or SH3 regions. Leucine zippers are alpha-amphipathic helices that contain 4 or 5 leucines every 7 amino acids. This periodicity allows the localization of leucines at almost the same position on the alpha helix. The dimerization is due to hydrophobic interactions between the leucine side chains of two adjacent zipper regions (Vogt et al., Trend In Bioch.Science 14 (1989). SH2 regions are known to interact with phosphorylated specific peptide regions in tyrosine. be used to form an oligomer with each transactivator or transactivation complex containing the corresponding proline-rich peptide (Pawson et al., Current Biology 3) (1993) 434). Protein regions known to induce oligomerization, such as, in particular, the C-terminal region of the p53 protein, may also be used. The use of this region makes it possible to selectively recruit p53 proteins present in the cell. Preferably, the p53 region comprised between amino acids 320-393 (SEQ ID NO: 3) 302-360 or 302-390 is used in the present invention.

Synthetic or natural regions known to interact with a molecule containing a target transactivating element include, for example, a region of the MDM2 protein interacting with the p53 protein. This type of construction thus makes it possible to recruit wild-type or mutated p53 protein as a transactivator.

A preferred region of specific binding to a transcriptional transactivator of the invention is an antibody or antibody fragment or derivative. For example, antibody fragments or derivatives are Fab or F (ab) '2 fragments, a VH or VL region of a single chain antibody (ScFv) comprising a VH region linked to a VL arm region. This type of region is particularly advantageous in that it can be directed against the entire molecule.

Antibodies formed by molecules of the immunoglobulin family are made up of different chains (2 heavy (H) and 2 light (L)), these chains being made up of different regions (variable region (V), linker region (J) and the like). The region of binding to a transactivator or transactivating complex present in the molecules of the invention is preferably comprised of an antibody fragment or derivative comprising at least the binding site responsible for antigen binding. This fragment may be either a light chain (V) or heavy chain (V) variable region, optionally in the form of a fab or F (ab ') 2 fragment, or preferably in the form of a single chain antibody (single chain antibody) (ScFv). The single chain antibodies used to construct the molecules of the invention are composed of a peptide corresponding to the antibody light chain variable region binding site and an attached peptide arm to the peptide corresponding to the antibody heavy chain variable region binding site. The construction of nucleic acid sequences encoding such modified antibodies of the invention has been described, for example, in US Patent 4,946,778 or in WO94 / 02610 and WO94 / 29446. This construction is illustrated in the examples below.

A preferred construct of the invention comprises a p53 binding region. Preferably, it is an antibody derivative directed against the p53 protein. A preferred form of this region is a single chain antibody directed against p53. By way of illustration, the use of a ScFv having the sequence of SEQ ID NO: 4, the construction of which is described in the Examples.

The DNA binding region and the transactivator binding region are connected to each other via an arm. This arm is generally made up of a peptide providing the entire complex with the flexibility necessary for both regions of the molecules of the invention to be functional in an autonomous manner. The peptide is generally composed of unloaded amino acids that do not interfere with the activity of the molecules of the invention and are, for example, glycine, serine, tryptophan, lysine or proline. This arm generally comprises 5 to 30 amino acids, preferably 5 to 20 amino acids. Examples of peptide arms useful for the construction of the molecules of the invention are, for example:

-GGGGSGGGGSGGGGS (SEQ ID NO: 5),

-PKPSTPPGSS (SEQ ID n ° 6), whose coding sequence is CCCAAGCCCAGTACCCCCCCAGGTTCTTCA (SEQ ID n ° 6),

In particular, the following molecules are preferred examples of the molecules of the invention:

a) ScFv-tag-Hinge-TET or Cro (Fig. 5A).

This type of molecule includes:

- a single chain antibody binding region of the transactivator,

- a peptide tag sequence recognized by a monoclonal antibody allowing immunological detection of the molecule. This sequence may be, for example, an epitope of the VSV sequence of MNRLGK (SEQ ID n °

7), whose coding sequence is ATGAACCGGCTGGGCAAG (SEQ ID n ° 7), or myc epitope with sequence EQKLISEEDLN (SEQ ID n °

8), the coding sequence of which is

GAACAAAAACTCATCTCAGAAGAGGATCTGAAT (SEQ ID NO: 8) and which is recognizable by 9E10.

a peptide arm having the sequence of SEQ ID No. 6 (Hinge), and

a DNA binding region formed by the TET or Cro protein.

The ScFv is preferably directed against the p53 protein.

b) ŠcFv-Hinge-TET ¹ or Cro (Fig. 5B)

This type of molecule contains the same elements as molecule a) except that the tag sequence is absent.

c) ScFv-TET or Cro (Fig. 5V).

This simplified type of molecule contains only a single chain antibody transactivator binding region and a DNA binding region consisting of a TET or Cro protein. This molecule contains neither arm nor tag sequence. In this construction, the transactivator-binding region is located in the N-terminal part of the molecule and the DNA-binding region is located in the C-terminal part of the molecule.

d) TET or Cro-ScFv (Fig. 5D)

This type of molecule is similar to the above type c). The only difference lies essentially in the arrangement of the regions: the transactivator-binding region is now located in the C-terminal part of the molecule and the DNA-binding region is located in the N-terminal region of the molecule.

e) TET or Cro-Hinge-ScFv (Fig. 5E)

This type of molecule contains the same elements as the above type b) molecule. The difference lies essentially in the arrangement of the regions: the transactivator-binding region is now located in the C-terminal part of the molecule, while the DNA-binding region in this case is located in the N-terminal part of the molecule.

f) 01igom-tag-Hinge-TET or Cro (Figure 5A)

This type of molecule is similar to the above type a) except that the transactivator-binding region is replaced by the p53 oligomerization region of SEQ ID NO: 3. This molecule makes it possible to recruit mutated p53 proteins occurring in tumor cells.

g) Oligo-Hinge-TET or Cro (Fig. 5B)

This type of molecule is similar to the aforementioned type b) except that the transactivator-binding region in this case is replaced by an oligomerization region with the p53 protein of SEQ ID n ° 3.

h) 01igom-TET or Cro (Figure 5C)

This type of molecule is similar to the above type c) except that the transactivator-binding region in this case is replaced by an oligomerization region with the p53 protein of SEQ ID NO: 3.

i) TET or Cro-Olig (Fig. 5D)

This type of molecule is similar to the aforementioned type d) except that the transactivator-binding region is replaced here by the oligomerization region of the p53 protein of SEQ ID NO: 3.

j) TET or Cro-Hinge-Oligo (Fig. 5E)

This type of molecule is similar to the aforementioned type e) except that the transactivator-binding region is replaced here by oligomerization with the p53 protein of SEQ ID NO: 3.

Alternatively, in each of these molecules, the peptide arm can be readily replaced by (G4S) 3 (SEQ ID NO 5).

Another object of the invention is a nucleic acid sequence encoding a chimeric molecule as defined above. It is preferably a DNA sequence, in particular a cDNA. It may also be RNA. These sequences of the invention are generally constructed by assembling sequences in a cloning vector encoding different regions by molecular biology techniques. Said nucleic acid sequences according to the invention may optionally be modified by chemical, enzymatic or genetic means to generate stabilized regions and / or multifunctional regions and / or regions of reduced length and / or in order to favorably influence their localization in one or another intracellular region. The nucleic acid sequences of the invention may thus comprise sequences encoding nuclear localization peptides (NLS). In particular, it is possible to fuse the sequences of the invention with the sequence encoding the NLS of SV40 virus having the following peptide sequence:

PKKKRKV (SEQ ID NO: 9) (Kalderon et al., Cell 39 (1984) 499).

The nucleic sequences of the invention preferably form part of an expression vector, which may be plasmid or viral in nature.

A further object of the invention is a fusion protein comprising a transcriptional transactivator region and a specific binding region to a given molecule, the regions of which are optionally linked to a peptide arm, as well as any nucleic acid sequence encoding such a fusion. The transactivating region may be derived from any transcriptional transactivating protein, such as p53, VP16, EBNA, E6 / E7, Tat and the like.

Another object of the invention is a conditional gene expression system comprising:

a chimeric molecule as defined above, and

an expression cassette comprising a regulatory sequence, a minimum transcriptional promoter, and said gene.

Said expression cassette contains elements necessary for activating gene expression by a transactivator or transactivation complex recruited by a bispecific molecule. The regulatory sequence is thus the binding sequence responsible for binding to the DNA of the expressed chimeric molecule. Where the DNA binding region of the chimeric molecule is represented in whole or in part by the tetetrotycline suppressor TetR, then the regulatory sequence comprises the sequence of SEQ ID NO: 1 or a derivative thereof, optionally repeated several times. Preferably, it is an op2 sequence (comprising 7 Tetop motifs that are repeated as described, for example, by Weinmann et al., In The Plant Journal 5 (1994) 559). Likewise, when the DNA binding region of the chimeric molecule is represented in whole or in part by Cro, the regulatory sequence comprises the sequence of SEQ ID NO: 2 or a derivative thereof, which is optionally repeated several times. Preferably it is an OR3 sequence. Derivatives of SEQ ID N ° 1 and N ° 2 may be made up of any sequence obtained by modification of a genetic nature (mutation, deletion, addition, repeat, and the like) that retains the ability of a derivative to specifically bind a protein. Such derivatives have been described in the literature (Baumeister et al. (Cited above), Tovar et al., Mol.Gen.Genet.215 (1988) 76, WO94 / 04672).

As regards the minimal transcriptional promoter, it is a promoter whose activity depends on the presence of the transactivator. Accordingly, the promoter is inactive in the absence of the chimeric molecule and there is no or only very limited expression of the gene. Conversely, in the presence of a chimeric molecule, a recombinant transactivator or transactivation complex allows inducing minimal promoter activity as well as expression of the gene of interest.

Said minimum promoter is generally comprised of a TATA or INR box. These elements are, in fact, the minimum necessary elements for gene expression in the presence of a transactivator. This minimal promoter can be prepared from each promoter by genetic modification. An example of a preferred promoter candidate is the thymidine kinase gene promoter. Interesting results were obtained with a minimal promoter derived from the TK promoter consisting of nucleotides -37 to +19. Said minimal promoter may also be derived from human CMV. In particular, it may be comprised of fragmentary between nucleotides -53 to +75 or -31 to +75 of CMV. However, any conventional promoter, such as the promoter of genes encoding chloramphenicol acetyl transferase, betagalactosidase, or even luciferase, can be used.

The expression cassette is preferably comprised of the following elements:

- regulatory sequence: a sequence comprising the sequence of SEQ ID n ° 1 or n ° 2 or a derivative thereof, optionally repeated several times,

- minimal promoter: a promoter derived from the thymidine kinase (TK) gene promoter,

- the coding sequence of interest.

Even more preferably, the minimal promoter is comprised of the -37 to +19 regions of the thymidine kinase gene promoter.

Preferably, the expression cassette is selected from the group consisting of the structural cassettes Tetop2.TK-Gen, Tetop7.TK-Gen and OR3.TK-Gen.

Another feature of the invention is an expression vector comprising a nucleic acid sequence encoding a chimeric molecule, and the above-defined invention may be a chimeric molecule expression cassette. In vectors according to the nucleic acid sequence encoding and expression cassette inserted in the same orientation or in opposite orientations. Alternatively, said vector may be plasmid or viral in nature.

Among the viral vectors, adenoviruses, retroviruses, herpesviruses or adeno-associated viruses may be mentioned. The viruses according to the invention are detective viruses, i. no. viruses that are unable to replicate autonomously in the target cell. In general, therefore, the genome of the defective viruses used in the invention is devoid of at least the sequences necessary for the replication of said virus in the infected cell. These regions may either be eliminated (in whole or in part) or rendered inoperative or replaced by other sequences, in particular those of the invention. Preferably, however, the defective virus retains those genome sequences that are necessary for encapsulating the viral particles.

With regard to adenoviruses, different serotypes have been characterized, whose structure and properties vary somewhat. Of these serotypes, it is preferred to use human adenoviruses of type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin (see WO94 / 26914). Adenoviruses of animal origin useful in the present invention include canine, bovine, murine adenoviruses (example: Mavl, Beard et al., Virology 75 (1990) 81), sheep, porcine, avian or also monkey (example: SAV) origin. Preferably, the animal-derived adenovirus is a canine adenovirus, in particular a CAV2 adenovirus (Manhattan strain or A26 / 61 (ATCC VR-800), for example). Preferably, adenoviruses of human or canine or mixed origin are used in the present invention.

Preferably, the genome of the recombinant adenoviruses of the invention comprises at least the ITR sequences and the adenovirus encapsidation region and the nucleic acid sequence encoding the chimeric molecule as well as the expression cassette as defined above. Preferably, at least the E1 region is non-functional in the genome of adenoviruses according to the invention. The viral gene contemplated may be rendered inoperative by any technique known per se, in particular by total suppression, substitution (e.g., sequences of the invention), partial deletion or addition of one or more bases in the gene or genes under consideration. Such modifications may be achieved in vitro (on isolated DNA) or in situ, for example using genetic engineering techniques or also by treatment with mutagenic agents. Likewise, other regions may be modified, in particular Area E3 (WO95 / 02697), Area E2 (WO94 / 28938), Area E4 (WO94 / 28152, WO94 / 12649, WO95 / 02697), and Area L5 (WO95 / 02697).

In a preferred embodiment, the adenovirus according to the invention comprises a deletion in the E1 and E4 regions. In another preferred embodiment, the adenovirus comprises a deletion in the E1 region at which the E4 region and the sequences of the invention are inserted (see FR 9413355).

The defective recombinant adenoviruses of the invention may be prepared by any technique known in the art (Lavrero et al., Gene 101 (1991) 195, EP185573, Graham, EMBO J.

3 (1984) 2917th In particular, these adenoviruses can be prepared by homologous recombination between an adenovirus and a plasmid carrying, inter alia, the DNA sequences of the invention (sequences encoding the chimeric molecule + expression cassette). Said homologous recombination is performed after co-transfection of said adenoviruses and plasmid into the respective cell strain. The usable cell strain must (preferably 1) be transformable with said elements and 2) it must contain sequences capable of complementing a portion of the genome of the defective adenovirus, preferably in integrated form, in order to avoid recombinant risks. An example of such a cell strain is the human embryonic renal strain 293 (Graham et al., J. Gen. Virol. 36 (1977) 59), which contains, in its integrated form, the left-hand part of the genome of the adenovirus 5d5 (12%). or strains capable of complementing E1 and E4 functions, which have been described in particular in WO94 / 26914 and WO95 / 02697.

Subsequently, the multiplied and isolated adenoviruses are purified by conventional molecular biology techniques as illustrated in the Examples below.

As regards adeno-associated viruses (AkV), they are viruses with DNA having a relatively reduced size and integrating into the genome of cells that infect in a stable and site-specific manner. These viruses are capable of infecting a broad spectrum of cells without inducing any effect on cell growth, morphology or division. Otherwise, these viruses do not appear to be implicated in human pathologies. The genome of adeno-associated viruses has already been cloned, sequenced and characterized. It comprises about 4700 bases and contains, at each end, an inverted repeat region (ITR) of about 145 bases, serving as the origin of virus replication. The remainder of the genome is divided into two major regions carrying the encapsidation functions: the left part of the genome that contains the rep gene implicated in viral replication and viral gene expression, and the right part of the genome that contains the cap gene encoding the capsid proteins of the virus.

The use of adeno-associated virus (AAV) vectors for gene transfer in vitro and in vivo has already been described in the literature (see especially WO91 / 18088, WO93 / 09239, US 4,797,368, US

5,139,941, EP 488,528). These applications describe various constructs derived from adeno-associated AAVs in which the rep and / or cap genes are eliminated by deletions and replaced with the gene of interest, as well as their use for transfer in vitro (on cells in a cell strain) or in vivo ( directly into the organism) of the gene of interest. Defective recombinant adeno-associated adenoviruses by co-transfection into a cellular virus of human origin (e.g., adenovirus) with a plasmid containing the nucleic sequences of the invention (sequences

AAVs can be prepared strains infected with the helper encoding chimeric molecule + expression cassette) flanked by two inverted repeat regions (ITRs) derived from adeno-associated viruses and a plasmid carrying the encapsidation genes (rep and cap genes) derived from adeno-associated AAV viruses. The recombinant AAVs produced are then purified by conventional techniques.

With regard to herpesviruses and retroviruses, the construction of recombinant vectors has been extensively described in the literature. In particular, see: Breakfield et al., New Biologist 3 (1991) 203, EP 453242, EP 178220, Bernstein et al. Genet.Eng.7 (1985) 235, McCormick, BioTechnology 3 (1985) 689 and the like.

Retroviruses are integrative viruses that selectively infect cells at the stage of their division. These viruses thus form the desired vectors in cancer applications. The retrovirus genome essentially comprises two LTR sequences, 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 eliminated by deletion, in whole or in part, and replaced by the heterologous nucleic acid sequence of interest. These vectors can be obtained from various types of retroviruses, such as in particular MoMuLV (Murine Maloney Leukemia Virus, also referred to as MoMLV), MSV (Murine Maloney Sarcoma Virus), HaSV (Harvey Sarcoma Virus), SNV (Spleen Necrosis Virus), RSV. Rous sarcoma virus) or Friend virus.

In order to construct the recombinant retroviruses according to the invention, a plasmid containing, in particular, an LTR sequence, an encapsidation sequence and a sequence according to the invention (a sequence encoding a chimeric molecule + expression cassette) is generally constructed, which is then used to transfect a so-called encapsidating cell strain capable of delivering trans retroviral functions that are plasmid deficient. In general, the encapsidation strains are therefore capable of expressing the gag, pol and env genes. Such encapsidation strains have been described in the prior art and are in particular strains PA317 (US 4,861, -719), PsiCRIP (WO90 / 02806) and GP + envAm-12 (WO89 / 07150). Alternatively, recombinant retroviruses may include modifications of the suppressions made to target as well as the digested portions of the gag gene (Bender at the LTR transcriptional activity sequence level, including encapsidation sequences including et al., J. Virol. 61 (1987) 1639). The recombinant retroviruses produced are then purified by conventional techniques.

An example of the construction of a defective recombinant virus of the invention (retrovirus) is described in FIG. From this figure, a second advantage of the constructs of the invention is that the absence of expression of the gene of interest in the encapsidation strains is apparent. Since there is no transactivator or transactivation complex recruited by the system of the invention in these strains, the promoter is inactive and thus does not express the gene in the production cell (Fig. 8A). This occurs only when the virus has effectively infected the target cell, i. j. a cell in which a transactivator or transactivation complex is present recruited by the system of the invention, and where the gene is efficiently expressed (FIG. 8B). This is particularly advantageous for the construction of a virus containing genes whose expression would be toxic to cells (Grb3-3, IL-2, diphtheria toxin and the like).

In the practice of the invention, it is particularly advantageous to use a defective recombinant adenovirus or retrovirus. In fact, these vectors have properties that are of particular interest for gene transfer into tumor cells.

Various types of non-viral vectors can also be used in the invention. The conditional expression system of the invention may in fact be incorporated into a non-viral agent capable of promoting the transfer and expression of nucleic acids into eukaryotic cells. Chemical or biochemical vectors are an interesting alternative to natural viruses, especially in view of ease of handling, increased safety and the lack of a theoretical limit on the size of the DNA to be transfected.

These synthetic vectors have two basic functions, namely to compact the transfected nucleic acid and promote its cellular fixation, as well as its passage through the plasma membrane and possibly through both nuclear membranes. In order to avoid the polyanionic nature of the nucleic acids, non-viral vectors in all cases have polycationic charges.

Among the synthetic vectors developed, the most preferred are cationic polymers of the polylysine type, (LKLK) n, (LKKL) n, polyethyleneimine and DEAE-dextran, or else cationic and lipofectant lipids. These vectors have the ability to compact DNA and promote its association with the cell membrane. Among the latter, lipopolyamines (lipofectamine, transfectam and the like) and various cationic or neutral lipids (DOTMA, DOGS, DOPE and the like) can be mentioned. Recently, a receptor-mediated targeted transfection system has been developed that utilizes the principle of DNA condensation due to a cationic polymer controlling the fixation of the complex to the membrane due to chemical coupling between the cationic polymer and the membrane receptor ligand present on the cell type surface to be transformed. Alignment of the transferrin receptor, insulin or hepatocyte azialoglycoprotein receptor has already been described.

The present invention also provides any pharmaceutical composition comprising the above vector. These compositions may be formulated with respect to topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular and the like administration. Preferably, the composition of the invention comprises pharmaceutically acceptable carriers for injectable formulations. These may be, in particular, solutions of salts (sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride and the like, or mixtures of such salts) in a sterile and isotonic state or in a dry composition, in particular lyophilized compositions which water or saline allows for reconstitution of injectable fluids. For retroviruses, it may be advantageous to directly use the encapsidation cells or cells infected ex vivo and reimplanted in vivo, optionally in the form of a neo-organ.

The dosages of the vector used for injection may be adjusted depending on various parameters, in particular depending on the route of administration used, the pathology to be treated or also the desired duration of treatment. In general, the recombinant viruses of the invention are formulated and administered in the form of doses ranging between 10 ⁴ and 10 ¹⁴ pfu / ml. For adeno-associated viruses (AAV) and adenoviruses, doses ranging from 10 ⁶ to 10 ¹⁰ pfu / ml may also be used. The term pfu (plaque forming unit) corresponds to the infectious potency of the virion suspension and is determined by infection of the respective cell strain and evaluation (usually after 48 hours) of the number of infected cell plaques. Techniques for determining the pfu titre of a viral solution are extensively documented in the literature.

The expression system of the invention and the corresponding vectors are particularly suitable for regulating expression of the genes of interest in cellular and gene therapies. These vectors may also be used to regulate the expression of any of the coding sequences of interest, particularly the sequence encoding the therapeutic product, which may be a peptide, a polypeptide, a ribonucleic acid protein, and the like. The gene is a DNA sequence (cDNA, gDNA, synthetic, human, animal, plant and the like DNA) encoding protein products such as enzymes, blood derivatives, hormones, lymphokines: interleukins, interferons, TNF and the like (FR 9203120), growth factors , neurotransmitters synthetic enzymes, trophic GMF, aFGF, bFGF, NT3, NT5 and ApoAIV, ApoE and the like or precursors or factors thereof: BDNF, CNTF, NGF, IGF, like, apolipoproteins: ApoAI, (FR 93 05125), dystrophin or dystrophin (FR 9111947), tumor suppressor genes: p53, Rb, RaplA, DCC, k-rev and the like (FR 93 04745), genes encoding factors implicated in coagulation: factors VII, VIII, IX and the like, or all or part of natural or artificial immunoglobulin (Fab, ScFv and the like), RNA-ligand (WO91 / 19813) and the like.

The gene of interest may also be an antisense sequence whose expression in a target cell allows the expression of genes or the transcription of cellular mRNAs to be regulated. Such sequences may, for example, be transcribed into the target cell as complementary RNA of cellular mRNAs and may thus block their translation into a protein by the technique described in EP 140 308.

The invention is particularly suitable for the expression of sequences encoding toxic factors. In particular, they may be cellular poisons (diphtheria toxin, pseudomonas toxin, ricin A and the like) and products inducing sensitivity to the intrinsic agent (suicide genes: thymidine kinase, cytosine deaminase and the like) or murine genes capable of inducing cell death (Grb3). 3 (PCT / FR94 / 00542), ScFv anti-ras (WO94 / 29446) and the like). In fact, the system according to the invention makes it possible, in particular, to produce viral vectors containing these sequences without the risk of production cell poisoning and then to induce the expression of these toxic molecules selectively in target cells having the desired transactivator or transactivation complex. Thus, this type of construction is particularly suited to anti-tumor therapeutic strategies, for example, to selectively destroy invaded cells. This system is also of particular interest for the expression of cytokines, interferons, TNF, TGF, whose unregulated production could have significant side effects.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following parts of the description, the invention will be described in more detail by way of examples of a specific embodiment thereof, these examples being illustrative only and not in any way limiting the scope of the invention, which is clearly defined by the definition of the claims.

In the attached drawings:

Fig. 1 depicts an illustration of a conditional expression system according to the invention allowing selectively recruiting a transactivator using the oligomerization region (A) or

ScFv (B), FIG. 2 shows an illustration of a conditional expression system according to the invention allowing selective recruiting of the transactivation complex; FIG. Figure 3 shows an expression cassette according to the invention comprising a Tetop7 regulatory sequence, a minimal promoter (TATA box) and a gene (CAT); Fig. 4 shows an illustration of an expression cassette according to the invention comprising the OR3 regulatory sequence, the minimal promoter (TATA box) and the gene (CAT); Figure 5 shows the bispecific chimeric molecules of the invention; Figure 6 shows DNA sequences encoding the bispecific chimeric molecules of the invention; 7 shows chimeric control structures; FIG. Figure 8 shows the structure and function of a viral vector (retrovirus) according to the invention; Fig. 9 shows an interaction study between the hybrid molecules of the invention and the regulatory sequence; Fig. 10 shows an interaction study between hybrid molecules of the invention and different forms of the p53 protein; 11 shows demonstration of Tet-Luc cassette activation in SAOS-2 cells; FIG. 12 shows demonstration of Tet-Luc cassette activation in H358 cells.

General techniques of molecular biology

Classical molecular biology methods, including, in particular, centrifugation of plasmid DNA in cesium chloride-ethidium bromide, restriction enzyme digestion, gel electrophoresis, transformation into E. coli, nucleic acid precipitation and the like, are described in the literature (Maniatis et al., 1989) ).

Enzymes were provided by New-England Biolabs (Beverly, MA). For ligation, DNA fragments are separated according to their size on a 0.8-1.5% agarose gel, purified by GeneClean (BI0101, LaJolla CA) and incubated overnight at 14 ° C in Tris-HCl buffer, pH 7.4 , 50 mM, MgCl 4 10 mM, DTT 10 mM, ATP 2 mM, in the presence of phage T4 DNA ligase.

Polymerase Chain Reaction (PCR) amplification was also performed according to Maniatis et al. (1989) under the following conditions:

- MgCl2 concentration adjusted to 8mM,

- denaturation temperature 95 ° C, hybridization temperature 55 ° C, elongation temperature 72 ° C. This cycle was repeated 25 times in a PE9600 Thermalcycler (Perkin Elmer, Norwalk CO).

Oligonucleotides are synthesized using the cyanoethyl-protected phosphoramidite chemistry (Sinha et al., 1984, Giles 1985) and an Applied Biosystem model 394 automatic DNA synthesizer (Applied Biosystem, Foster City CA) following the manufacturer's recommendations.

Sequencing was performed on double-stranded matrices by the chain termination method using fluorescent labeling reagents. The Taq Sye Primer Kit available from Applied Biosystem (Applied Biosystem, Foster City CA) was used herein following the manufacturer's instructions.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Construction of an expression cassette comprising a regulatory sequence, a minimum transcriptional promoter, and a gene

1.1. Construction of plasmid pTETop7 / CAT

Plasmid pTETop7 / CAT contains the following elements (Fig. 3):

- a regulatory sequence consisting of an interaction sequence with the tetR tetracycline suppressor consisting of seven repeated Tetop motifs (SEQ ID n ° 1),

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

a sequence encoding chloramphenicol acetyl transferase (CAT) under the control of said minimal promoter.

This plasmid was constructed by cloning the SmaI-BglII fragment of plasmid pUHD10-7 (WO94 / 29442) into plasmid pKK232-8 (Pharmacia), which had been pre-digested with the restriction enzymes SmaI and BamHI.

1.2. Construction of plasmid pOR ₃ / CAT

Plasmid pOR ^ / CAT contains the following elements (Fig. 4):

- a regulatory sequence formed by the sequence of the OR ^ interaction with the Cro repressor (SEQ ID n ° 2),

- minimal promoter derived from the thymidine kinase gene promoter (region -37 to 19 carrying the TATA box),

a sequence encoding chloramphenicol acetyl transferase (CAT) under the control of said minimal promoter.

This plasmid was constructed as follows: the sequence 0R ₃ interacting with the repressor Cro was synthesized artificially. To this end, the following two oligonucleotides were synthesized:

Oligo 5533 (SEQ ID NO: 22): 5'-GATCCTATCACCGCAAGGGATAA-3 '

Oligo 5534 (SEQ ID NO: 23): 3'-GATAGTGGCGTTCCCTATTTCGA-5 '.

Both of these oligonucleotides were then hybridized in order to reconstitute the double stranded OR ₃ sequence flanked by sequences allowing its cloning, oriented as follows:

CATCCTATCACCGCAAGGGATAA

GATAGTGGCGTTCCCTATTTCGA.

1.3

Construction of the toxic cassette expression cassette

The expression cassettes of the described plasmids with the sequence encoding the toxic genes are obtained from above (1.1 and 1.2) by replacing the CAT sequence with a toxic product, preferably the Grb3-3 gene (PCT / FR94 / 00542), the thymidine kinase gene, the diphtheria toxin coding gene or pseudomonas.

Example 2

Construction of p53-specific single-stranded antibodies

This single-stranded antibody is constructed according to the following protocol:

The cDNAs encoding the VH and VL regions were obtained from the anti-p53 antibody producing hybridoma pAb421. To this end, they were extracted and subjected to random subjects. Use this

However, in the case of total cDNAs, the total RNA of said hybridoma inverse transcription reaction of hexamers in the function of the primary type of the primer subject makes it possible to avoid the use of specific primer subjects of immunoglobulins. The cDNA clones obtained were of sufficient length to clone the V regions.

If they represent a small fraction of those present then a preliminary amplification reaction must be performed to produce an amount of DNA sufficient for cloning. To this end, cDNAs encoding the VH and VL regions are amplified separately. The primary subjects used are oligonucleotides hybridizing at the opposite ends of the variable regions of each chain (H and L). An amplification product utilizing specific primer entities of the heavy chains is a fragment containing about 340 base pairs. An amplification product using specific primer light chain entities consists of a fragment containing about 325 base pairs.

After purification, the cDNA coding region of the VH and VL antibodies is assembled into a single strand using a nucleotide arm (L). This nucleotide arm is constructed in such a way that one end binds to the end of the 3 'cDNA encoding the VH region and the other end binds to the end of the 5' cDNA encoding the VL region. The arm sequence encodes the peptide of SEQ ID NO: 5. The assembled sequence of about 700 base pairs contains, in the form of an NcoI-NotI fragment, the VH-L-VL chain, the sequence of which is represented by SEQ ID NO: 4 (amino acids 9 to 241). This sequence also includes a myc-derived tag at the end of the C sequence (residues 256-266).

Example 3

Construction of Nucleic Acid Sequences Encoding Bispecific Chimeric Molecules Containing a Single-Fiber Antibody (ScFv) Transactivator Binding Region

3.1. Construction of a plasmid containing the ScFv-myc sequence

Hinge-TetR or Cro (Fig. 5A and Fig. 6)

First, the NcoI-NotI fragment containing the cDNA encoding the ScFv anti-p53 was cloned into a pUC19 type plasmid. Then, a sequence encoding a VSV epitope (SEQ ID No. 7) or myc (SEQ ID No. 8) is inserted after this fragment.

The TetR and Cro coding sequences are then obtained as follows:

-TetR coding sequence was obtained by amplification from a parent plasmid carrying the TetR sequence using the following oligonucleotides:

Oligo 5474 (SEQ ID NO. 10):

GGCTCTAGACCCAAGCCCAGTACCCCCCCAGGTTCTTCAACGCG

TGGATCCATGTCCAGATTAGATAAAAGTAAAG

Oligo 5475 (SEQ ID NO. 11):

CGTACGGAATTCGGGCCCTTACTCGAGGGACCCACTTTCACATTT

AAGTTG

These oligomers also provide a sequence encoding the Hinge peptide arm linking the two functional regions of the molecules.

Thus, the amplified fragment contains the sequence encoding the peptide arm and the tetT DNA binding region. This fragment was cloned into the XbaI-EcoRI sites of the obtained plasmid to generate a plasmid containing the sequence encoding the ScFv-myc-Hinge-TetR molecule (Fig. 6).

- the Cro coding sequence was obtained by amplification on the DNA matrix of the bacteriophage Lambda using the following oligonucleotides:

Oligo 5531 (SEQ ID NO. 12):

GGCTCTAGACCCAAGCCCAGTACCCCCCCAGGTTCTTCAACGCG

TGGATCCATGGAACAACGCATAACCCTGAAAG

Oligo 5532 (SEQ ID NO. 13):

CGTACGGAATTCGGGCCCTTACTCGAGTGCTGTTGTTTTTTTGTT

ACTCGG

These oligonucleotides also provide a sequence encoding the Hinge peptide arm linking both functional regions of the molecule.

Thus, the amplified fragment comprises a sequence encoding a peptide arm and a Cro DNA binding region. This fragment was cloned into the XbaI-EcoRI sites of the above-obtained plasmid to generate a plasmid containing a sequence encoding a ScFv-myc-Hinge-Cro molecule (Fig. 6).

2.3 Construction of a plasmid containing the sequence ScFvHinge-TetR or Cro (Fig. 5B)

This example describes the construction of plasmids bearing a sequence encoding a bispecific chimeric molecule of the invention devoid of tag sequence.

These plasmids were obtained from the plasmids described in 3.1. digestion of NotI and XbaI. This cleavage makes it possible to cleave the fragment carrying the myc tag coding region.

3.3 Construction of a plasmid comprising the ScFv sequence

TetR or Cro (Fig. 5C)

This example describes the construction of plasmids carrying a sequence encoding a bispecific chimeric molecule of the invention lacking an arm and a tag sequence.

These plasmids were obtained from the plasmids described in 3.1. by digestion with the enzymes Nôti and BamH1. This cleavage makes it possible to cleave the fragment carrying the region encoding the myc tag and the Hinge peptide arm.

Example 4

Construction of Nucleic Acid Sequences Coding for Bispecific Chimeric Molecules Containing a Transactivator Binding Region Comprised by an Oligomeric Region

4.1. Cloning of the p53 oligomerization region (fragment 320-393) The cDNA encoding the p53 oligomerization region (SEQ ID NO: 3) was obtained by amplification by PCR reaction on a plasmid carrying wild-type human p53 cDNA using the following oligonucleotides:

Oligo 5535 (SEQ ID NO 14):

CAGGCCATGGCATGAAGAAACCACTGGATGGAGAA (underlined is Ncol)

Oligo 5536 (SEQ ID NO 15):

CGTCGGATCCTCTAGATGCGGCCGCGTCTGAGTCAGGCCCTTC (underlined part: BamH1 site, twice underlined: Xbal site, bold part: Nots).

2.4 Plasmids p53 320/393-myc-Hinge-TetR or Cro (Fig. 5A) were obtained by cloning the above amplified fragment as a fragment

Ncol-NotI to the sites corresponding to the plasmids described in Example 3.1. replacing it with the ScFv coding region.

3.4 Plasmids p53 320/393-Hinge-TetR or Cro (Fig. 5B) were obtained by cloning the amplified fragment of 4.1. in the form of the NcoI-XbaI fragment to the sites corresponding to the plasmids described in 3.1. replacing it with the ScFv coding region and tag.

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

4.5 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 wild-type human p53 cDNA using oligo 5537/5539 or 5538/5539 digested with XhoI / EcoRI into the plasmids described in 3.1, pre-digested with XhoI / EcoRI.

Oligo 5537 (SEQ ID NO 16):

CAGGCTCGAGAAGAAACCACTGGATGGAGAA

Oligo 5538 (SEQ ID NO 17):

CAGGCTCGAGCCCAAGCCCAGTACCCCCCCAGGTTCTTCAAAGA

AACCACTGGATGGAGAA

Oligo 5539 (SEQ ID NO 18):

GGTCGAATTCGGGCCCTCAGTCTGAGTCAGGCCCTTC

Example 5

Construction of a control plasmid carrying a sequence encoding a chimeric molecule comprising a DNA binding region (TetR or Cro) and a p53 transactivator region (1-73 region)

Plasmids p53 1/73-TetR or Cro with or without tag (myc or VSV) and Hinge (Figs. 7A, 7B and 7C) were obtained by cloning fragments amplified by PCR from a plasmid carrying wild-type human p53 cDNA using oligo 3661/5662 and then digested with NcoI / NotI, NcoI / XbaI, NcoI / BamHI into the plasmids described in 3.1., pre-digested with NcoI / NotI, NcoI / XbaI or NcoI / BamHI.

Oligo 5661 (SEQ ID NO 19):

CAGGCCATGGAGGAGCCGCAGTCAGATCC

Oligo 5662 (SEQ ID NO. 20):

CGTCGGATCCTCTAGAŤGCGGCCGCCACGGGGGGAGCAGCCTC

TGG

Example 6

Construction of Expression Plasmids of Various Hybrid Molecules of the Invention

The plasmids used to express the hybrid molecules were obtained by cloning fragments containing the cDNA encoding these molecules into the NcoI / EcoRI sites of the eukaryotic expression vector pcDNA3 (invitrogen). In this way, the following constructions were made:

-ScFv 421: SEQ ID NO: 4,

-TET19: a hybrid protein comprising the ScFv421-VSV-HingeTetR chain described in FIG. 6 according to example 3.1.,

-ΤΕΤ02: hybrid protein containing the p53 (320/393) -VSVHinge-TetR chain described in FIG. 5A according to Example 4.3.,

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

-TET04: a hybrid protein comprising the p53 (320/393) -TetR chain described in FIG. 5C according to Example 4.4.,

-TET07: a hybrid protein comprising the p.53 (1/73) -VSVHinge-TetR chain described in FIG. 7A according to Example 5.

Example 7

Recognition of double stranded DNA sequences by hybrid molecules of the invention

7.1. Production of hybrid molecules

The various molecules used in this experiment were obtained by in vitro translation in a reticulocyte lysate of the molecules described in Example 6 using the TNT Coupled Reticulocyte Lysate Systems (Promega) kit and following the experimental protocol described by the supplier for a total reaction volume of 50 microliters.

2.7 Construction of a specific double stranded DNA sequence

The specific double-stranded DNA sequence used in this experiment consists of two synthetic oligonucleotides having the following sequences:

Oligo 5997 (SEQ ID NO 24):

GATCCGACTTTCACTTTTCTCTATCACTGATAGTGAGTGGTAAACTCA

Oligo 5998 (SEQ ID NO 25):

AGCTTGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCG

Both of these synthesis oligonucleotides were labeled with phosphorus 33 by incubating the following mixture for 30 minutes at 37 ° C and using 10 pmoles of each of the oligonucleotides:

Tris-HCl pH 7.6 MgCl _from dithiothreitol Spermidine EDTA ATP-gamma- ³³ P (Amersham) T4-kinase (Boehringer) 50 10 5 100 100 50 10 mM mM mM μΜ Ci / mmol) μΜ μθί (1000-3000 U. Then they both follow marked oligonucleotides hybridize in the presence of 400 mM NaCl to reconstitute the double-stranded TetO sequence (SEQ ID n ° 26): following

GATCCGACTTTCACTTTTCTCTATCACTGATAGTGAGTGGTAAACTCA

CTAGGCTCAAAGTGAAAAGAGATAGTGACTATCACTCACCATTTGAGT

7.3.

Recognition of the TetO double stranded sequence by various hybrid molecules of the invention

The DNA binding reaction is performed in a 50 μΐ reaction mixture (Tris-HCl pH 7.4 10 mM, MgCl 4 10 mM, KCL 10 mM, betamercaptoethanol 6 mM, EDTA 0.1 mM, BSA 0.5 mg / ml) by adding a TetO sequence (10 ^-1θ Μ) prepared in Example 7.2, 10 μΐ of the translation product prepared according to Example 7.1. and 10 ^"to a competition-cold M AP2 oligonucleotide (Promega) used to eliminate nonspecific binding. The specificity of the interaction is verified by shifting the equilibrium by adding 10 μΜ (Sigma) of tetracycline to the reaction mixture. The reaction mixtures are then incubated for 15 minutes while 10 μΐ 50% glycerol of native electrophoresis is added to 5%, migration is performed at 200 V and 16 and subjected to autoradiography.

at 20 ° C, whereupon it and the final mixtures are subjected to a polyacrylamide gel, wherein

C. The gel is then dried

The result of this experiment carried out with hybrid molecules TET19, TET02 and TET07 is shown in FIG. 9. Under these conditions, the binding of the three molecules to the specific double-stranded DNA sequence of TetO was observed due to the migration delay of the molecules thus bound, and the specificity of this interaction was demonstrated by the inhibition of said migration delay by the addition of tetracycline.

This result thus confirms that the hybrid molecules of the invention are capable of binding in a specific manner to the nucleotide sequence of TetO.

Example 8

Specific Binding of Hybrid Molecules of the Invention to a Molecule Having a Transcriptional Transactivation Region

8.1. Production of Hybrid Molecules of the Invention and Molecules Having or Not having a Transcriptional Transactivation Region

For the purpose of this experiment, the hybrid molecules of the invention ScFv 421, TET19 and TET02 of Example 6 were produced by in vitro translation using the experimental protocol of Example 7.1. in the presence of 44 pci of ^3S S-methionine (Amersham) (1175 Ci / mmol) to give radiolabeled hybrid molecules.

The cDNAs of the molecules having or lacking the transcriptional transactivation region were cloned into the pBlueBacIII vector (Invitrogen) at the BamHI site. Recombinant baculoviruses were produced and purified from these vectors according to the manufacturer's instructions. Said molecules are produced by infection with sf9 insect cell recombinant baculovirus according to the manufacturer's experimental protocol. Protein extracts were prepared with a final concentration of the protocol described by K-Ory et al.

3496-3504, 1994). These molecules are:

protein of 10 mg / ml according to (K. Ory, EMBO J., 13,

- ρ53 (1/393): wild-type p53 protein,

- p53 (1/320): wild-type p53 protein restricted to amino acid sequence 1 to 320, and thus deprived of the oligomerization region and the region recognizable by the monoclonal antibody pAb421.

2.8 Binding of the hybrid molecules of the invention to molecules having or not having a transcriptional transactivation region μΐ of each of the translation products prepared according to Example 8.1. Incubate with 5 μΐ of baculovirus extract prepared according to Example 8.1. and 2 μΐ of monoclonal antibody D01 (Oncogene Sciences), which recognizes the N-terminal portion of p53, for 16 hours at 4 ° C in 100 μΐ of modified RIPA buffer (K. Ory, EMBO J., 13, 3496-3504, 1994). Immunoprecipitation is performed as described by K. Ory et al. (K. Ory, EMBO J., 1994, 13, 346-3504). The antibody-retained complexes are eluted by incubating at 80 ° C for 10 minutes in the presence of 30 μΐ migration buffer (Laemmli UK, Nature, 227, 680-685, 1970) and subjected to 10% polyacrylamide gel electrophoresis in a denaturing medium at 200 V above. described protocol (Laemmli UK, Nature, 227, 680-685, 1970). The gel is then dried and developed using an instant imager (Packard Instruments), which makes it possible to quantitatively determine the amount of hybrid molecules having or lacking the transcriptional transactivation region. The results of this experiment are shown in FIG. 10th

Under these conditions, it is clear that a hybrid molecule having a ScFv 421 (TET19) recognizes the p53 (1/393) molecule as well as the ScFv 421 itself and that the hybrid molecule having a 320/393 (TET02) region exhibits the same properties but has a more potent ability retention of the p53 protein (1/393). In addition, the absence of the signal observed upon incubation with the p53 molecule (1/320) shows that these interactions are truly specific and are mediated by the C-terminal portion of the p53 protein (amino acids 320-393) as expected.

Thus, these results confirm that the hybrid molecules of the invention are indeed capable of recruiting the transcriptional transactivation region carried by the molecule of which they are specific partners.

Example 9

Functional recombination of the transcriptional transactivation region by the hybrid molecules of the invention

Functional recruitment of the region by hybrid molecules in the transactivation system of the transcriptional transactivation of the invention was evaluated in vivo in SAOS-2 (human osteosarcoma) cells deficient in both p53 alleles in a tumor cell line deficient in both p53 alleles (Maxwell Roth, Oncogene 8, 3421, 1993) and in the HT29 tumor strain deficient in one of the two alleles of the p53 protein and with one mutated allele (H273 mutation). This system is based on the use of a reporter gene that is detectable enzymatically and placed under the control of a promoter containing nucleotide motifs specifically recognizable by the Tet repressor (Tet operator).

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

9.1. Cell strains used and culture conditions

The cell strains used in these experiments as well as their genotype attached to the p53 protein and the culture conditions used for their growth are shown in the following table:

table

Cell strains tribe p53 Culture environment no. ATCC SAOS DMEM (Gibco BRL) HTB 85 environment enriched with 10% fetal calf serum (Gibco BRL) H358 Environment RPMI1640 (Gibco BRL) enriched with 10% fetal calf serum (Gibco BRL) HT29 - / H237 DMEM (Gibco BRL) enriched with 10% fetal calf serum (Gibco BRL) 2.9 Express plasmids of molecules having transcriptional

transactivation region

Molecules with transcriptional region used in this experiment are wild-type p53 protein (Wt) and mutants G281 and H175 of this protein. The cDNA encoding these three proteins was inserted into the BamHI sites of the pcDNA3 vector (introgen).

9.3. Intracellular Expression of Hybrid Molecules of the Invention

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

Cells (3.5. 10 ^sec. ) Are seeded into six-well titration plates containing 2 ml of culture medium per well and cultured overnight in an incubator at 37 ° C with a 40% atmosphere of 5% carbon dioxide. The individual constructs are then transfected using lipofectAMIN (Gibco BRL) as transfection agent as follows: 1.5 µg of total plasmid is incubated (of which 0.25 µg reporter plasmid) with 5 µΐ lipofectAMIN for 30 minutes with 2 ml culture medium (transfection mixture). During this time, the cells are washed twice with PBS and then incubated for 4 hours at 37 ° C with the transfection mixture, after which the transfection mixture is aspirated and replaced with 2 ml of culture medium enriched with 10% heat-inactivated fetal calf serum. 48 hours at 37 ° C.

9.4. Detection of transcriptional activation

Transcriptional activation associated with functional recombinant transactivator recruitment is detected and quantitatively evaluated by measuring luciferase activity encoded by the LUC gene using the Luciferase Assay System (Promega) according to the protocol supplied by the manufacturer.

9.5. Functional recruitment of the transcriptional transactivation region by the molecules of the invention

This experiment was performed using the TETO 2, TETO 3, and TET07 molecules of Example 6. In this experiment, the TET07 molecule serves as a positive control subject due to its own transcriptional transactivator region.

The results obtained in SAOS-2 cells shown in FIG. 11, show that the TET07 molecule is indeed self-capable of activating the transcription of the LUC gene placed under the control of the Tet operator, unlike the TET02 construct. This is consistent with the fact that this cell strain does not contain the endogenous p53 protein, which thus cannot be recruited by the TET02 molecule. Conversely, the introduction of the wild-type p53 protein or mutant thereof, which do not themselves produce a signal, is capable of generating transcriptional activity in the presence of TET02 molecules. Such a result cannot be observed with the mutant H175, which is described in the literature as a mutant having a non-functional transcriptional transactivation region.

This result obtained in the cell line SAOS-2 with the TET02 molecule could be reproduced in a tumor cell line also free of endogenous p53 (H358 cells) and could be extended to the TET03 and TET04 molecules (Fig. 12).

To confirm these results in the context of different cell strains, the TET02 molecule as well as the positive control subject TET07 were expressed in HT29 cells having mutant endogenous p53 protein (H273), while the negative control subject in this experiment consisted in transfection of the empty pcDNA3 vector. The result of this experiment, presented in the table below, shows that the TET02 molecule is indeed capable of recruiting the transcriptional transactivation region of the endogenous p53 protein.

table

Transcriptional Activation of Hybrid Molecules of the Invention in HT29 Cells

pcDNA3 TET07 TET02 1 59 10 The file of these experiments shows that hybrid

indeed, the molecules of the invention are capable of binding both double stranded DNA sequences of a specific nature as well as proteins having a transcriptional transactivation region and that these molecules are capable of conditionally inducing expression of the genes of interest.

Sequence list

Sequence Information SEQ ID NO: 1:

Sequence characteristics:

length: 19 base pairs type: nucleotide number of strands: 1 configuration: linear

Molecule type: cDNA

Sequence description: SEQ ID NO: 1:

TCTCTATCAC TGATAGGGA

Sequence Information SEQ ID NO: 2:

Sequence characteristics:

length: 17 base pairs type: nucleotide number of strands: 1 configuration: linear

Molecule type: cDNA

Sequence description: SEQ ID NO: 2:

TATCACCGCA AGGGATA 17

Sequence Information SEQ ID NO: 3:

Sequence characteristics:

length: 74 amino acids type: amino acid number of fibers: 1 configuration: linear

Molecule type: peptide

Hypothetical: no

Antisense: no

Sequence description: SEQ ID NO: 3:

Lys 1 Lys Pro Leu Asp Gly Glu Tyr r Π S Thr io Leu Glrt 11 e Arg Gly 1 Ó ’ Arg - u Aro Phe Glu *, Λ 4th Xe: Phe Aru Glu L. c U 2 5 same time Glu Ala L · - ’* Glu 39 l. e u Lys r.SP A1ä Gin But Gly Lys for Gly Gly Ser Arg But 4 5 his Ser Ser His Leu Lys Ser Gly gin Ser Thr Ser A r g 60 His d. y s Lys u e u

Ys— 7 Ρr.e Lys Thr Glu Glv For A.so Ser A.so ó 5 70

Sequence Information SEQ ID n: 4: Sequence Characteristics: Length: 768 base pairs Type: Nucleotide Number of Fibers: 1 Configuration: Linear

Molecule type: cDNA

Hypothetical: no

Antisense: no

Sequence description: SEQ ID NO: 4:

TTACTCGCGG CCCAGCCC-C-C CATC-CCCCAG GTC-CAGCTGC AGCAGTCTGG GGCAGAGCTT 60 GTAAGGTCAG GGGCCTCAGT CAAGTTGTCC TGCACAGGTT CTGGCTTCAA CATTAAAGAC 1 20 TACTATATGC ACTGGGTGA-A GCAGAGGGCT GAACAGGGCC TGGAGTGGAT TGGATGGATT 1 SO C-ATCCTAAGA ATGGTGATAC TGAATATGCC CCGAAGTTCC AGGGCAAGGC CACTATGACT 240 GCAGACACAT CCTGCA-ATAC AGCCTACCTG CAGCTCAGCA GCCTGGCATC TGAGGACACT 300 GCCGTGTATT TA i ^r T ^, 'T' ^rn TTACGGGGAT GCTTTGGACT ATTGGGGCCA AGGGACCACG 360 GTCACCGTCT CCTCAGGTGG AGGCGGTTCA GGCGGAGGTG GCTCTGGCGG TGGCGGATCG 420 GATGTTTTGA TGACCCAAAC TCCACTCACT TTGTCGGTTA CCATTGGACA ACCAGCCTCC 460 ATCTCTTGCA AGTCAAGTCA GA GCC ^m C ^ G GATAGTGATG GA-AAAACATA TTTGAATTGG 540 TTGTTACAGA GGCCAG 'C \ N GTCTCCAAAG CGCCTAATCT ATCTGGTGTC TÄÄÄCTGGAC 600 TCTGGAGTGC CTGACAGGTT CACTGGGAGT GGATCAGGGA CAGATTTCAC ACTTAAAATC 660 Acac-AGTGG AGGCTGAGGA TTTGGGAGTT TATTATTGCT GGCAAGGTAC ACATTCTCCG 720 CTTACGTTCG GTGCTGGCAC CAAGCTGGAA ÄTTAAACGGG CGGCCGCA 7 6S

Sequence Information SEQ ID NO: 5:

Sequence characteristics:

length: 15 amino acids type: amino acid number of fibers: 1 configuration: linear

Molecule type: peptide

Hypothetical: no

Antisense: no

Sequence description: SEQ ID NO: 5:

Gly Gly Gly Ser Gly Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 15

Sequence Information SEQ ID NO: 6:

Sequence characteristics:

length: 30 base pairs type: nucleotide number of strands: 2 configuration: linear

Molecule type: cDNA

Hypothetical: no

Antisense: no

Sequence description SEQ : id N °: 6: CCC AGAG ACC CCC CCA GGT TCT TCA 30 Pro Lys Pro Ser Thr for for Gly Ser Ser 1 5 10

Sequence Information SEQ ID NO: 7: Sequence Characteristics: Length: 18 base pairs Type: Nucleotide Number of Fibers: 2 Configuration: Linear

Molecule type: cDNA

Hypothetical: no

Antisense: no

Sequence description: SEQ ID NO: 7:

ATG AAC CGG

Met Asn Arg Leu Gly Lys

SEQ ID NO: 8: Sequence characteristics: length: 33 base pairs type: nucleotide number of strands: 2 configuration: linear

Molecule type: cDNA

Hypothetical: no

Antisense: no

Sequence description: SEQ ID NO: 8:

GAA CTIA AAA CTC ATC TCA GAA GAG GAT GAT Glu Gin Lys Leu White Ser Glu Glu Asp Leu Asn

10

SEQ ID NO: 9: Sequence characteristics: length: 7 amino acids type: amino acid number of threads: 1 configuration: linear

Molecule type: peptide

Hypothetical: no

Antisense: no

Sequence description: SEQ ID NO: 9:

For Lys Lys Lys Arg Lys Val

5

Sequence Information SEQ ID NO: 10: Sequence Characterization: Length: 66 Base Pairs Type: Nucleotide Number of Fibers: 2 Configuration: Linear

Molecule type: cDNA

Hypothetical: no

Antisense: no

Sequence description: SEQ ID NO: 10:

GGCTCTAGAC CCAJAGCCCAG TACCCCCCCA GGTTCTTCAA CGCG7GGATC CATGTCCAGA \ ag;

G7AAAC

Sequence Information SEQ ID NO: 11:

Sequence characteristics:

length: 51 base pairs type: nucleotide number of strands: 2 configuration: linear

Molecule type: cDNA

Hypothetical: no

Antisense: no

Sequence description: SEQ ID NO: 11:

CGTACGGAAT TCGGGCCCTT ACTCGAGGGA CCCACTTTCA CAT 51

Sequence Information SEQ ID NO: 12:

Sequence characteristics:

length: 66 base pairs type: nucleotide number of strands: 2 configuration: linear

Molecule type: cDNA

Hypothetical: no

Antisense: no

Sequence description: SEQ ID NO: 12:

GGCTCTAGAC CCAAGCCCAG TACCCCCCCA GGTTCTTCAA CGCGTGGATC CATGGAACAA

CGCATAACCC TGAAAG

Sequence Information SEQ ID NO: 13:

Sequence characteristics:

length: 51 base pairs

4Ί type: nucleotide number of fibers: 2 configuration: linear

Molecule type: cDNA

Hypothetical: no

Antisense: no

Sequence description: SEQ ID NO: 13:

CGTACGGAAT TCGGGCCCTT ACTCGAGTGC TGTTGTTTTT TTGTTACTCG G 51

Sequence Information SEQ ID NO: 14:

Sequence characteristics:

length: 35 base pairs type: nucleotide number of strands: 2 configuration: linear

Molecule type: cDNA

Hypothetical: no

Antisense: no

Sequence description: SEQ ID NO: 14:

CAGGCCATGG CATGAAGAAA CCACTGGATG GAGAA 35

Sequence Information SEQ ID NO: 15:

Sequence characteristics:

length: 43 base pairs type: nucleotide number of strands: 2 configuration: linear

Molecule type: cDNA

Hypothetical: no

Antisense: no

Sequence description: SEQ ID NO: 15:

CGTCGGATCC TCTAGATGCG GCCGCGTCTG AGTCAGGCCC TTC 43

Sequence Information SEQ ID NO: 16:

Sequence characteristics:

length: 31 base pairs type: nucleotide number of strands: 2 configuration: linear

Molecule type: cDNA

Hypothetical: no

Antisense: no

Sequence description: SEQ ID NO: 16:

CAGGCTCGAG AAGAAACCAC TGGATGGAGA

Sequence Information SEQ ID NO: 17:

Sequence characteristics:

length: 61 base pairs type: nucleotide number of strands: 2 configuration: linear

Molecule type: cDNA

Hypothetical: no

Antisense: no

Sequence description: SEQ ID NO: 17:

CAC-GCTCGAC - CCCAAGCCCA GTACCCCCCC AGGTTCTTCA AAGAAACCAC TGGATGGAGA

A

Sequence Information SEQ ID NO: 18:

Sequence characteristics:

length: 37 base pairs type: nucleotide number of strands: 2 configuration: linear

Molecule type: cDNA

Hypothetical: no

Antisense: no

Sequence description: SEQ ID NO: 18:

GGTCGAATTC GGGCCCTCAG TCTGAGTCAG GCCCTTC

Sequence Information SEQ ID NO: 19:

Sequence characteristics:

length: 29 base pairs type: nucleotide number of strands: 2 configuration: linear

Molecule type: cDNA

Hypothetical: no

Antisense: no

Sequence description: SEQ ID NO: 19:

CAGGCCATGG AGGAGCCGCA GTCAGATCC 29

Sequence Information SEQ ID NO: 20:

Sequence characteristics:

length: 46 base pairs type: nucleotide number of strands: 2 configuration: linear

Molecule type: cDNA

Hypothetical: no

Antisense: no

Sequence description: SEQ ID NO: 20:

CGTCGGATCC TCTAGATGCG GCCGCCACGG GGGGAGCAGC CTCTGG 46

Sequence Information SEQ ID NO: 21:

Sequence characteristics:

length: 66 amino acids type: amino acid number of fibers: 1 configuration: linear

Molecule type: peptide

Hypothetical: no

Antisense: no

Pop: Ls SEQ enc. ie: SEQ ID N °: : 21 Met Glu gin Arg Ile Thr Leu Lys Asp TVR Ala Met Arg FHE G1 v gin 1 5 1o 1 5 * Thr Lys Thr Ala Lys Asp Leu Gly wall TVR gin Ser Ala ill same time Lys 20 25 30 Ala Ile His Ala Gly Arg Lys T T_Q Phe Leu Thr ill same time Ala Asp Gly 35 40 45

Ser Val Ty

About Ser Asr. Lys Lys Thr

Sequence Information SEQ ID NO: 22:

Sequence characteristics:

length: 23 base pairs type: nucleotide number of strands: 2 configuration: linear

Molecule type: cDNA

Hypothetical: no

Antisense: no

Sequence description: SEQ ID NO: 22:

GATCCTATCA CCGCAAGGGA TAA 23

Sequence Information SEQ ID NO: 23:

Sequence characteristics:

length: 23 base pairs type: nucleotide number of strands: 2 configuration: linear

Molecule type: cDNA

Hypothetical: no

Antisense: no

Sequence description: SEQ ID NO: 23:

GATAGTGGCG TTCCCTATTT CGA

Sequence Information SEQ ID NO: 24:

Sequence characteristics:

length: 48 base pairs type: nucleotide number of strands: 2 configuration: linear

Molecule type: cDNA

Hypothetical: no

Antisense: no

Sequence description: SEQ ID NO: 24:

GATCCGACTT TCACTTTTCT CTATCACTGA TAGTGAGTGG TAAACTCA 48

Sequence Information SEQ ID NO: 25:

Sequence characteristics:

length: 48 base pairs type: nucleotide number of strands: 2 configuration: linear

Molecule type: cDNA

Hypothetical: no

Antisense: no

Sequence description: SEQ ID NO: 25:

AGCTTGAGTT TACCACTCCC TATCAGTGAT AGAGAAAAGT GAAAGTCG 48

Sequence Information SEQ ID NO: 26:

Sequence characteristics:

length: 96 base pairs type: nucleotide number of strands: 2 configuration: linear

Molecule type: cDNA

Hypothetical: no

Antisense: no

Sequence description: SEQ ID NO: 26:

GATCCGACTT TCACTTTTCT CTATCACTGA TAGTGAGTGG TAAACTCACT AGGCTCAAAG

TGAAAAGAGA TAGTGACTAT CACTCACCAT TTGAGT

Claims (57)

PATENT CLAIMS A bispecific chimeric molecule comprising a region capable of selectively binding a defined DNA sequence and a detection region capable of specifically binding a transactivator or transrepressor or a transactivator or transrepressor complex that is characteristic of a physiological or physiopathological state. 2. A molecule according to claim 1, wherein the region capable of selectively binding a defined DNA sequence is derived from a protein capable of interacting with said DNA. 3. The molecule of claim 2, wherein the region capable of selectively binding a defined DNA sequence is derived from a eukaryotic protein. 4. The molecule of claim 3, wherein the region capable of selectively binding a defined DNA sequence is derived from the p53, STAT, and NFκB proteins. 5. The molecule of claim 2, wherein the region capable of selectively binding a defined DNA sequence is derived from a prokaryotic protein. 6. The molecule of claim 5, wherein the prokrayotic protein is a bacterial repressor. 7. The molecule of claim 6, wherein the region capable of selectively binding a defined DNA sequence is derived from the tetR protein. 8. The molecule of claim 6, wherein the region capable of selectively binding a defined DNA sequence is derived from the Cro protein. A molecule according to any one of claims 2 to 8, wherein the region capable of selectively binding a defined DNA sequence comprises a region of interaction with the DNA of said protein. A molecule according to any one of claims 2 to 8, characterized in that the region capable of selectively binding a defined DNA sequence is made up of a complete protein. 11. The molecule of claim 10, wherein the region capable of selectively binding a defined DNA sequence is the tetR protein. 12. The molecule of claim 10, wherein the region capable of selectively binding a defined DNA sequence is the Cro protein. 13. The molecule of claim 1 wherein the region capable of specifically binding a transactivator or transrepressor or a transactivator or transrepressor complex is an oligomerization region. 14. The molecule of claim 13, wherein the oligomerization region is a leucine zipper, an SH3 region, or an SH2 region. 15. The molecule of claim 13, wherein the oligomeric region capable of specifically binding transactivator is a C-terminal portion of the p53 protein. 16. The molecule of claim 15, wherein the oligomerization region is a C-terminal portion of the p53 protein beginning at residue 320 and ending at residue 393 (SEQ ID n ° 3) or beginning at residue 302 and ending at residue 360 or starting at residue 302 and ending in remainder 390. 17. The molecule of claim 1 wherein the region capable of specifically binding a transactivator or transrepressor or transactivator or transrepressor complex is a synthetic or natural region known to interact with said transactivator or transrepressor or transactivator or transrepressor complex. 18. The molecule of claim 1, wherein the region capable of specifically binding a transactivator or transrepressor or transactivator or transrepressor complex is an antibody or antibody fragment or derivative that is directed against said transactivator or transrepressor or transactivator or transrepressor complex. 19. The molecule of claim 18, wherein the region capable of specifically binding a transactivator or transactivator complex is comprised of a Fab or F (ab) '2 antibody fragment or a VH or VL antibody region. 20. The molecule of claim 18, wherein the region capable of specifically binding a transactivator or transactivator complex is a single-stranded antibody (ScFv) comprising a VH region linked to a VL arm region. 21. The molecule of claim 1, wherein the DNA binding region and transactivator binding region are connected to each other via an arm. A molecule according to claim 21, characterized in that the arm is composed of a peptide of 5 to 30 amino acids, preferably 5 to 20 amino acids. 23. The molecule of claim 22, wherein the arm is selected from the group consisting of peptides of SEQ ID NO: 5 and SEQ ID NO: 6. 24. The molecule of any one of the preceding claims, wherein the DNA binding region is situated at the N-terminal position while the transactivator binding region is situated at the C-terminal position. 25. A molecule according to any one of claims 1 to 23, wherein the DNA binding region is situated at the C-terminal position while the transactivator binding region is situated at the N-terminal position. 26. A bispecific chimeric molecule having the structure ScFv-VSV / myc-Hinge-TET or Cro (Fig. 5A). A bispecific chimeric molecule having the structure ScFv-Hinge-TET or Cro (Fig. 5B). A bispecific chimeric molecule having the structure ScFv-TET or Cro (Fig. 5C). A bispecific chimeric molecule having the structure of TET or Cro-ScFv (Fig. 5D). A bispecific chimeric molecule having the structure of TET or Cro-Hinge-ScFv (Fig. 5E). A bispecific chimeric molecule having the structure Oligom-VSV / myc-Hinge-TET or Cro (Figure 5A), Oligom-Hinge-TET or Cro (Figure 5B) or Oligom-TET or Cro (Figure 5C). A nucleic acid sequence encoding a chimeric molecule according to any one of claims 1 to 31. 33. The nucleic acid sequence of claim 32, wherein said nucleic acid sequence is a DNA sequence. 34. The nucleic acid sequence of claim 32 or 33, which is part of a vector. 35. A 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. The conditional expression according to claim 35, characterized in that the binding region of DNA chimeric molecule is made up of all or part of a Tet regulatory sequence comprises the sequence of SEQ ID No. 1 or a derivative of the ^P of the sequence to be optionally repeated several times. 37. The conditional expression system of claim 35, wherein the DNA binding region of the chimeric molecule is formed in whole or in part by Cro and the regulatory sequence comprises the sequence of SEQ ID NO: 2 or a derivative thereof, optionally repeated several times. The conditional expression system of any one of claims 35 to 37, wherein the minimal promoter comprises a TATA or INR box. 39. The conditional expression system of claim 38, wherein the minimal promoter is derived from a thymidine kinase gene promoter. 40. The conditional expression system of claim 39, wherein the minimal promoter consists of nucleotides -37 to +19 of the thymidine kinase gene promoter. 41. A vector containing: a nucleic acid sequence encoding a chimeric molecule according to any one of claims 1 to 31, and an expression cassette comprising a regulatory sequence, a minimum transcriptional promoter, and a coding sequence of interest. 42. The vector of claim 41, wherein the minimum transcriptional promoter is as defined in claims 38-40. 43. The vector of claim 41, wherein the DNA binding region of the chimeric molecule is formed in whole or in part by TetR and the regulatory sequence comprises the sequence of SEQ ID NO: 1 or a derivative thereof, optionally repeated several times. 44. The vector of claim 41, wherein the DNA binding region of the chimeric molecule comprises all or part of a Cro and the regulatory sequence comprises the sequence of SEQ ID NO: 2 or a derivative thereof, optionally repeated several times. 45. The vector of any one of claims 41 to 44, wherein the coding sequence of interest is a DNA sequence encoding a therapeutic product. 46. The vector of claim 45, wherein the therapeutic product is a toxic peptide or polypeptide. 47. The vector of claim 46, wherein the toxic therapeutic product is selected from the group consisting of diphtheria toxin, pseudomonas toxin, ricin A, thymidine kinase, cytosine deaminase, Grb3 protein, and ScFv Y28. 48. The vector of claim 41, wherein said vector is a plasmid vector. 49. The vector of any one of claims 41 to 47, wherein said vector is a viral vector. 50. The vector of claim 49, wherein said vector is a defective recombinant adenovirus. 51. The vector of claim 49, wherein said vector is a recombinant defective retrovirus. A pharmaceutical composition comprising at least one vector according to any one of claims 41 to 51. 53. A nucleic acid comprising the sequence of SEQ ID NO: 4. 54. The molecule of claim 1, wherein the transactivator characteristic of the physiological or physiopathological state is a protein of viral, parasitic, mycobacterial or cellular origin having transcriptional transactivating activity. 55. The molecule of claim 54 wherein the transactivator is a viral protein selected from the group consisting of HIV Tat protein, Papilloma virus E6 / EZ proteins, and Epstein-Barr virus EBNA protein. 56. The molecule of claim 54, wherein the transactivator is a cellular protein, preferably a mutated or wild-type p53 protein. 57. The molecule of claim 1, wherein the transactivator or transactivator complex characteristic of a physiological or physiopathological state is a protein occurring in an infected or hyperproliferative cell.