KR19980703439A - Condition expression system - Google Patents

Abstract

Novel, using the generation and expression of bispecific chimeric molecules comprising a region capable of selectively binding to a corresponding DNA sequence and a sensing region capable of specifically binding to a transactivator or trans-inhibitor or a trans- activator or trans-inhibitor complex One genetic condition expression system.

Description

Condition expression system

Gene and cell therapy consists of correcting deletions or abnormalities (mutations, aberrant expression, etc.) or ensuring the expression of the therapeutic protein of interest by introducing genetic information into the cell or defective organ. The genetic information can be introduced ex vivo into the cells extracted from the organ and then by reintroducing the modified cells into the body (cell therapy) or directly in vivo into the appropriate tissue (gene therapy). Various techniques exist for introducing genes, among which are complexes of natural or synthetic chemical or biochemical vectors, such as DNA and DEAE-dextran [Ref. Pagano et al., J. Virol. 1 (1967) 891], complexes of DNA and nuclear proteins [References; Kaneda et al., Science 243 (1989) 375], complexes of DNA and lipids [Ref. Felgner et al., PNAS 84 (1987) 7413], the use of liposomes [Ref. Fraley et al., J. Biol. Chem. 255 (1980) 10431, various transfection techniques involving cationic lipids and the like. Another technique is based on the use of viruses as vectors for introducing genes. In this regard, various viruses have been tested for their ability to infect certain cell populations. In particular, retroviruses (RSV, HMS, MMS, etc.), HSV viruses, adeno-associated viruses and adenoviruses were tested. However, one of the major problems for the development of such gene and cell therapies is the selectivity of treatment. Depending on the application, it is important to be able to target only certain tissues or portions of the body in order to concentrate the therapeutic effect and limit metastasis and side effects, depending on the gene introduced. Such targeting can be accomplished by using a vector having specificity for a given cell. Another approach consists in using expression signals specific to certain cell types. In this respect, so-called specific promoters have been described in the literature, which include pyruvate kinases, bilins, promoters of genes encoding GFAP, promoters for fatty acid-binding intestinal proteins, promoters for α-actin of smooth muscle cells or promoters of apo-AI, apo-AII and human albumin genes. However, these promoters have certain disadvantages, in particular they have peripheral transcriptional effects that can be disrupted on the expression of virulence genes and are limited to some cells and therefore cannot be used in any application.

The present invention describes a novel condition system for particularly selective and effective gene expression. Advantageous features in the system of the present invention reside in the ability to express genes, not by cell type, but by the presence of specific intracellular elements or specific physiological situations. This system consists of a bispecific chimeric molecule consisting of a domain that can selectively bind to a defined DNA sequence and a detection region that can specifically bind to a transactivator and a transactivating complex. bispecific chimeric molecules).

More specifically, a first feature of the invention is a bispecific consisting of a region capable of selectively binding to a defined DNA sequence and a typical region capable of specifically binding to a transactivator or trans-inhibitor or trans-activating or trans-inhibiting complex. The creation and expression of enemy chimeric molecules.

Another feature of the invention consists of a nucleic acid sequence encoding a chimeric molecule as defined above and any expression of a vector comprising such nucleic acid sequence.

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

A feature of the present invention also consists of an expression vector consisting of the nucleic acid sequence encoding the chimeric molecule as defined above and the expression cassette.

Conditional expression systems of the present invention are particularly suitable for use in gene or cell therapy in order to highly selectively target expression of the genes of interest.

Thus, one component of the system of the present invention is a bispecific chimeric molecule consisting of a region capable of binding to a defined DNA sequence and a region capable of specifically binding to a transactivator or transactive complex. The bispecific of the present invention is, on the one hand, their ability to bind a defined DNA sequence (generally designated operator sequences or regulatory sequences) and, on the other hand, a trans that can induce or inhibit the expression of a gene. It lies in their ability to specifically recycle active or trans-suppressive proteins.

More specifically, the present invention relates to the development of bispecific chimeric molecules that allow the recovery of any transcription factor that induces a physiological pathology by activity or inactivation. Thus, the bispecific chimeric molecules of the invention allow selective recovery of trans transcriptional activators specific for physiological pathologies and attach these transcription factors to the promoter through attachment to a defined DNA sequence located near the promoter. And thus permit expression of the condition of the gene.

In addition, the present invention is not a molecule carrying a transactive region, but rather a molecule carrying a transactive transcription complex, ie, a complex formed between a target molecule present in a cell and a molecule carrying a transactive region (FIG. 2). It also relates to the development of specific chimeric molecules. In this case, the transactive complex is preferably formed by means of secondary bispecific chimeric molecules consisting of a transactive region and an optional region that binds to the cell molecule. The attachment of this secondary molecule allows the formation of a transactive transcriptional binary complex, which is then restored by the detection system of the present invention. In this way, the attachment of these ternary complexes near the promoter allows for regulated expression of the gene. Constructs of this type can advantageously extend the conditions using the system of the invention to detect any intracellular molecule (whether endogenous or infectious), which lacks a transactive region.

Thus, the system of the present invention can activate the expression of the gene only by the presence of the target protein by a highly selective detection system (sensor). These may be transcription factors that appear during physiological or physiological pathologies, or may be for example any endogenous molecule or any infectious molecule. Moreover, the system of the present invention includes very sensitive and highly selective detection components that allow expression of genes to be based on the presence, appearance or disappearance of any molecule in the cell.

Within the scope of the present invention, the term transactivator means any protein comprising any transactive transcription factor or transactive transcriptional region. A transactive complex refers to a complex formed between a molecule present in a cell and a bispecific molecule including a transactive region and a region that specifically binds to the molecule. The expression system of the present invention can be used to recover any transactive protein or any protein carrying a transactive region, and in particular, any protein of viral, parasitic, mycobacteria or cellular origin with transactive transcriptional activity Can be used to recover. Among the transcription factors of viral origin, mention may be made in particular of the Tat protein of HIV virus, the E6 / E7 protein of papilloma virus or the EBNA protein of Epstein Barr virus. These proteins have a transactive transcriptional region and are present only in cells infected with these viruses, ie only under special physiological pathologies. Conditional expression systems according to the present invention conveniently allow detection of physiological situations (appearance of these transfectants specific for viral infection) and allow for selective expression of a given gene (s). Among the cellular proteins, variant or wild type p53 proteins may be mentioned preferably. p53 protein consists of 393 amino acids. Its wild type is a tumor suppressor that can reversely regulate (ie, inhibit) cell division and growth. Its activity is linked to the presence of a transactive transcriptional region located in the N-terminal region (approximately 1-100 residues) in the structure of the p53 protein. In certain circumstances, wild type p53 may also be described by apoptosis [Ref. Yonish-Rouach et al., Nature, 352, 345-347, 1991. These properties are given to be seen in stressful situations where the state of cellular DNA is threatened, and p53 is proposed as a protector of the genome. The presence of mutated p53 in about 40% of human tumors covering all types reinforces and emphasizes the hypothesis that mutations in these genes probably played an important role in tumor development [Ref. Montenarh, Oncogene, 7, 1673-1680, 1992; Oren, FASEB J., 6, 3169-3175, 1992; Zambetti and Levine, FASEB J., 7, 855-865, 1993]. Within the framework of the present invention, it is possible to selectively restore the transactive region of the p53 protein and thus induce regulated expression of the gene (s) only in cells containing such protein. It is particularly advantageous of the present invention to specifically recover the modified p53 protein type as indicated above, which exists in the physiological pathology of hyperproliferation of the cell (cancer type). Such targeting may preferably be accomplished by binding regions specific for the mutated form of the p53 protein. However, there is a practical particularity in the accumulation of variants that have a longer lifespan than the wild type.

In addition, the system of the present invention may be used to induce selective expression of gene (s) by detecting any target molecule present in a cell. The detected protein is preferably a protein that appears in cells in abnormal situations (infection, hyperproliferation, etc.). They may be in particular viral proteins such as structural or functional proteins such as viruses, in particular HIV, hepatitis or Huff's virus. They may also be special proteins, especially for hyperproliferative states of cells, such as myc, fos and jun proteins, cyclins and the like.

Thus, one of the properties of the chimeric molecules of the present invention lies in their ability to bind to particular DNA regions (operating or regulatory regions). The binding allows the transactive region to be in close proximity to the promoter, thereby activating the expression of the gene located under the control of the promoter.

The regions capable of selectively binding to the defined DNA sequences present in the molecules of the invention have essentially protein origin. More preferably, the region is derived from prokaryotic or eukaryotic cell proteins that can interact with DNA sequences. Numerous genetic and structural studies have allowed us to elaborate on the regions responsible for these interactions in proteins that interact with double-stranded DNA sequences.

Among prokaryotic proteins that interact with double stranded DNA sequences, mention may in particular be made of bacterial inhibitors, preferably tetracycline inhibitors of E. coli and Cro inhibitors of bacteriophage lambda.

Tetracycline inhibitors (tetR) of E. coli are proteins consisting of about 210 amino acids. In E. coli, tetR reversely regulates the transcription of genes that mediate resistance to these antibiotics in tet operon. In the absence of tetracycline, tetR inhibitors attach to DNA at specific sequence levels and inhibit the transcription of resistance genes. In contrast, when tetracycline is present, the tetR inhibitor is no longer attached to the tetop working region, allowing for continuous transcription of the genes [Ref. Hillen, W. and Wissman, 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. tetR sequences have been published [Reference; WO 94/04682]. The specific double-stranded DNA sequence for binding tetR to DNA (Tetop) consists of the following units:

TCTCTATCACTGATAGGGA (SEQ ID NO. 1)

The unit may be repeated several times to increase the affinity and efficiency of the present system. Thus, the regulatory sequence may comprise up to 10 units, preferably 2 units (Tetop2) or 7 units (Tetop7) (FIG. 3).

Cro protein was initially defined as a regulator of expression of CI inhibitors [Ref. Eisen, H. et al (1970) PNAS 66, pp. 855]. Cloning of the cro gene allows for the identification of a 66 amino acid protein (SEQ ID NO. 21). Roberts, T. et al (1977) Nature 270, pp. 274]. Cro plays a physiological role by preferably attaching to a lambda OR ₃ operator.

The specific double stranded DNA sequence for Cro to bind to DNA (region named OR ₃ ) consists of the following bases:

TATCACCGCAAGGGATA (SEQ ID NO. 2)

The region can also be repeated several times to increase the affinity and efficiency of the present system (FIG. 4).

Of eukaryotic cell proteins that interact with double stranded DNA sequences, proteins or regions derived from STAT, p53 or NFkB proteins are preferably used to construct the molecules of the present invention [Reference; Inoue et al., PNAS 89 (1992) 4333. With respect to the p53 protein, the DNA-binding region is located in the center of the protein, more specifically the site between amino acids 102 and 292 [reference; Pavletich et al., Genes Dev. 7 (1993) 2556.

As noted above, the regions capable of specifically binding to the defined DNA sequences present in the molecules of the present invention are preferably derived from prokaryotic or eukaryotic cell proteins capable of interacting with double stranded DNA regions. The region used to construct the molecules of the present invention may consist of fragments thereof, including regions capable of interacting with the entire protein or DNA. This region is identified by various proteins and in particular TetR [Ref. Berens et al., J. Biol. Chem. 267 (1992) 1945]. It may also consist of derivatives of this protein or fragments with conserved DNA-binding properties. Such derivatives are in particular proteins which exhibit modification of one or more amino acids and are prepared according to conventional molecular biological techniques, for example, to allow fusion with other regions of the molecules of the invention. Derivatives of the TetR and Cro proteins are described in the literature as having, for example, point mutations and / or deletions [Reference; 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 of these derivatives to bind defined DNA sequences can be tested by preparing derivatives with regulatory sequences and incubating them to detect whether complexes are formed. In addition, the derivative may also be a protein with increased DNA-binding properties (especially affinity, etc.).

According to a preferred embodiment, the region capable of selectively binding to the defined DNA sequence present in the molecule of the invention is derived from prokaryotic protein. The type of construct is particularly advantageous because it is a protein of non-human origin and recognizes a double stranded DNA site of at least 14 nucleic acids. The likelihood of finding the same sequence in the human genome is indeed zero, thus the expression system obtained is more selective.

In a preferred embodiment, the region capable of selectively binding to a defined DNA sequence present in the molecule of the invention is derived from a tetR or Cro protein. It is most advantageous to use the complete tetR or Cro protein (SEQ ID NO. 21).

The region capable of specifically binding to the trans-transcriptional activator or trans-transcriptional active complex present in the molecules of the invention can be of various types. It may in particular be an oligomeric region, in which case the targeted transactivator or transactive complex also comprises said region. It may also be any synthetic or natural region known to interact with the transactivator or transactive complex. Alternatively, it may be an antibody or antibody fragment or derivative directed to a transactivator or transactive complex.

Among the oligomer regions which can be used within the scope of the invention, mention may be made, for example, of leucine zippers, SH2 regions and SH3 regions. Leucine zippers are bidirectional α helix structures containing 4 or 5 leucine every 7 amino acids. This periodicity allows for the placement of leucine at approximately the same location on the α helix. Dimerization is maintained by hydrophobic interactions between bilateral leucine chains of two adjacent zipper regions [Ref. Vogt et al., Trends in Bioch. Science 14 (1989) 172. The SH2 region is known to interact with the phosphorylated specific peptide sequence of tyrosine. The SH3 region can be used to form oligomers with any transactivator or transactive complex comprising a corresponding proline-rich peptide [Reference; Pawson et al., Current Biology 3 (1993) 434]. It is also possible to use protein sites known to induce oligomerization, such as in particular the C-terminal site of the p53 protein. By using this site, it is possible to selectively recover p53 protein present in the cell. P53 sites between 320 and 393 (SEQ ID NO. 3), 302 and 360 or 302 and 290 are preferably used within the scope of the present invention.

Among the synthetic or natural regions known to interact with molecules containing the target transactive component, mention may be made of examples of sites of MDM2 protein that interact with p53 protein. This type of construct allows for the recovery of wild type or variant of the p53 protein as a transactivator.

Preferred regions that specifically bind to the trans transcriptional activator of the invention consist of an antibody or antibody fragment or derivative. An antibody fragment or derivative is, for example, a single chain antibody (ScFv) comprising a Fab or F (ab) '2 fragment, a VH or VL site of the antibody or alternatively a VH site linked by an arm to the VL site. . This type of region is particularly advantageous because it can be directed to any molecule.

The molecules of the antibody, immunoglobulin upstream are composed of different chains (two heavy chains (H) and two light chains (L)) consisting of different regions (variable regions (V), connecting regions (J), etc.). The region that binds to a transactivator or transactive complex present in a molecule of the invention consists of an antibody fragment or derivative comprising at least an antigen binding locus, the fragment comprising a variable region of the light chain (V _L ) or a variable region of the heavy chain. (V _H ) and may optionally be a type of Fab or F (ab) ′ 2 fragment, or preferably a type of single-chain antibody (ScFv) Single-chain antibody used to construct the molecules of the invention Is composed of a peptide corresponding to the binding site of the variable region of the light chain of the antibody, linked by a peptide arm to the peptide corresponding to the variable binding site of the heavy chain of the antibody. Depending construct of the nucleic acid sequence encoding the modified antibodies, for example, are described in U.S. Patent 4,946,778 or application WO94 / 02610, WO94 / 29446 call. This is illustrated as an example.

Preferred constructs according to the invention comprise a region capable of binding to the p53 protein. It is more preferably an antibody derivative directed to the p53 protein. Specific embodiments consist of a single-chain antibody directed against p53. According to a specific example, SEQ ID NO. ScFv of 4 is used in the constructs described in the examples.

DNA binding regions and transactivator-binding regions are generally linked to one another via arms. This arm generally consists of a peptide that provides sufficient flexibility for the two regions of the molecule of the invention to function automatically. Such peptides generally consist of uncharged amino acids that do not interfere with the activity of the molecules of the invention, for example glycine, serine, tryptophan, lysine or proline. The arm generally consists of 5 to 30 amino acids, preferably 5 to 20 amino acids. Examples of peptide arms that can be used in the construction of the molecules of the invention include

-GGGGSGGGGSGGGGS (SEQ ID NO. 5)

-PKPSTPPGSS (SEQ ID NO. 6), the sequence encoding the latter

CCCAAGCCCAGTACCCCCCCAGGTTCTTCA (SEQ ID NO. 6).

Preferred examples of molecules according to the invention are in particular the following molecules:

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

This type of molecule,

A region that binds to a transactivator that constitutes a single-chain antibody,

Tag peptide sequences recognized by monoclonal antibodies that allow immunological detection of the molecule. This sequence is, for example, the VSV epitope or EQKLISEEDLN sequence (SEQ ID NO. 8) of the MNRLGK sequence (SEQ ID NO. 7) (the sequence encoding it is ATGAACCGGCTGGGCAAG SEQ ID NO. (moic) epitope, which is recognized by 9E10 antibodies.

-Sequence number. Peptide arm of 6 (Hinge) sequence and

DNA-binding region consisting of TET or Cro protein. Preferably, the ScFv is directed to the p53 protein.

b) ScFv-Hinge-TET or Cro (FIG. 5B)

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

c) ScFv-TET or Cro (FIG. 5C)

This type of molecule simply comprises a transactivator consisting of a single-chain antibody and a region for binding to a DNA-binding region consisting of a TET or Cro protein. This molecule does not contain both arm and tag sequences. In such constructs, the transactivator-binding region is located in the N-terminus of the molecule and the DNA-binding region is located in the C-terminus.

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

This type of molecule is similar to type c) above. If the difference is essentially in the arrangement of the regions, the transactivator-binding region is located in the C-terminus of the molecule and the DNA-binding region is located in the N-terminus.

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

Molecules of this type comprise the same elements as molecules b) above. If the difference is essentially in the arrangement of the regions, the transactivator-binding region is located in the C-terminus of the molecule and the DNA-binding region is located in the N-terminus.

f) Oligom-tag-Hinge-TET or Cro (FIG. 5A)

A molecule of this type is SEQ ID NO. It is similar to type a) except that the transactivator-binding region is substituted by a region for oligomerization of p53 protein of 3. This molecule can repair mutated p53 proteins that appear in tumor cells.

g) Oligom-Hinge-TET or Cro (FIG. 5B)

A molecule of this type is SEQ ID NO. It is similar to type b) except for the transactivator-binding region substituted by the region for oligomerization of p53 protein of 3.

h) Oligom-TET or Cro (FIG. 5C)

A molecule of this type is SEQ ID NO. It is similar to c) type except for the transactivator-binding region substituted by the region for oligomerization of p53 protein of 3.

i) TET or Cro-Oligom (FIG. 5D)

A molecule of this type is SEQ ID NO. Similar to type d) except for the transactivator-binding region substituted by the region for oligomerization of p53 protein of 3.

j) TET or Cro-Hinge-Oligom (FIG. 5E)

A molecule of this type is SEQ ID NO. Similar to e) type except for the transactivator-binding region substituted by the region for oligomerization of p53 protein of 3.

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

Another subject of the invention resides in a nucleic acid sequence encoding a chimeric molecule as mentioned above. DNA, especially cDNA sequences, is advantageous. It may also be RNA. Sequences of the invention can generally be constructed by assembling sequences encoding various regions into cloning vectors according to conventional molecular biology techniques. Nucleic acid sequences of the invention can be optionally modified chemically, enzymatically or genetically to create regions that are stable, multifunctional and / or reduced in size, and / or have the purpose of promoting their position in intracellular fractions. have. Thus, the nucleic acid sequences of the present invention may comprise sequences encoding nuclear position peptides (NLS). In particular, sequences encoding the SV40 virus NLS, whose peptide sequence is PKKKRKV (SEQ ID NO. 9), can be fused into the sequences of the present invention [Reference; Kalderon et al., Cell 39 (1984) 499].

The nucleic acid sequence according to the invention is advantageously part of an expression vector, which may be plasmid or viral characteristic.

Another subject of the invention is a fusion protein comprising a trans transcriptional activator region optionally linked by a peptide arm and a region that specifically binds to the molecule, as well as any nucleic acid sequence encoding said fusion. The transactivator region can be derived from any trans transcriptional activator such as p53, VP16, EBNA, Et / e7, Tat and the like.

Another subject of the invention consists of a conditional expression system of a gene comprising a chimeric molecule as defined above and a regulatory sequence, a minimal transcriptional promoter and an expression vector comprising said gene.

Expression cassettes contain components necessary for the activity of expression of a gene by a transactivator or transactive complex restored by a bispecific molecule. Thus, the regulatory sequence is the DNA binding sequence of the chimeric molecule to be expressed. When the DNA-binding region of a chimeric molecule is represented by all or part of TetR, the regulatory sequence is optionally repeated several times. 1 or derivatives thereof. It is preferably op2 (including two repeated Tetop units) or Op7 sequences (including seven repeated Tetop units) [Reference; Weinmann et al., The Plant Journal 5 (1994) 559]. Similarly, when the DNA-binding region of a chimeric molecule is expressed in whole or in part of Cro, the regulatory sequence is optionally repeated several times. 2 or derivatives thereof. It is preferably an OR3 sequence. SEQ ID NO. Derivatives of the sequences of 1 and 2 may be any sequence obtained by modification of genetic characteristics (mutations, deletions, additions, repetitions, etc.) and having the ability to specifically bind a protein. Such derivatives are described in the literature; Baumeister et al., Cited above, Tovar et al., Mol. Gen. Genet. 215 (1988) 76, WO 94/04672.

As regards the minimal transcriptional promoter, its activity is a promoter which is governed by the presence of a transactivator. As such, under the bonsai of the chimeric molecule, the promoter is inactive, and therefore the gene is not expressed or is very minimally expressed. On the other hand, in the presence of a chimeric molecule, a restored transactivator or transactive complex can induce minimal promoter activity and thus allow expression of the gene of interest. Minimal promoters typically consist of an INR or TATA box. These components are also the minimum components necessary for the expression of the gene in the presence of a transactivator. Minimal promoters can be made from any promoter by genetic modification. According to a preferred example of the candidate promoter, mention may be made of the promoter of the thymidine kinase gene. More particularly, advantageous results are obtained from minimal promoters derived from the TK promoter consisting of -37 to +19 nucleotides. Minimal promoters can also be derived from human CMV. In particular, it may consist of -53 to +75 or -31 to +75 nucleotide fragments of CMV. However, any conventional promoter may be used, for example, chloramphenicol acetyltransferase, β-galactosidase or alternatively a promoter of a gene encoding luciferase.

The expression cassette advantageously consists of the following elements:

A regulatory sequence, optionally repeated several times. A sequence consisting of one or two or derivative sequences thereof,

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

The sequence encoding the corresponding entity.

More preferably, the minimal promoter consists of a region from -37 to +19 of the promoter of the thymidine kinase gene.

Advantageously, the expression cassette is Tetop2. TK-gene; Tetop7. TK-gene or OR3.TK-gene structure cassette.

Another feature of the invention lies in an expression vector consisting of a nucleic acid sequence encoding a chimeric molecule and an expression vector as defined above. In the vectors of the invention, nucleic acid sequences encoding chimeric molecules and expression cassettes can be inserted in the forward or reverse direction. Furthermore, the vector may be plasmid or viral characteristic.

Among the viral vectors, more particularly adenoviruses, retroviruses, Huffs viruses or alternatively adeno-bound viruses can be mentioned. The virus according to the invention is defective, i.e. cannot automatically propagate in the target cell. Thus, in general, the genome of a defective virus used within the scope of the present invention does not have at least the necessary sequence for replication of the virus in infected cells. These sites can be removed (completely or partially) or nonfunctionalized or substituted, in particular by the sequences of the invention and other sequences. Nevertheless, defective viruses preferably preserve the genomic sequence necessary for encapsulation of viral particles.

More particularly with respect to adenoviruses, various serotypes have been identified which differ somewhat in structure and properties. Among these serotypes, the use of human adenovirus 2 or 5 type (Ad 2 or Ad 5) or adenovirus of animal origin (cf. WO94 / 26914) is preferred within the scope of the present invention. Among adenoviruses of animal origin that can be used within the scope of the present invention are canine, bovine, and murine [reference; MAV1, Beard et al., Virology 75 (1990) 81], ovine, forsine, avian or alternatively adenoviruses of simian (eg SAV) origin may be mentioned. Adenoviruses of animal origin are preferably canine adenoviruses, more preferably CAV2 adenoviruses such as manhattan strain or A26 / 61 (ATCC VR-800). Preferably, adenoviruses of human or canine or mixed origin are used within the scope of the present invention.

Preferably, the genome of the recombinant adenovirus of the invention comprises at least the ITRs and encapsulation sites of the adenovirus, and nucleic acid sequences and expression cassettes encoding the chimeric molecules as defined above. More preferably, in the adenovirus genome of the invention, at least the El region is nonfunctional. The viral gene under consideration will be rendered nonfunctional by any technique known in the art, in particular by total repression, substitution (eg, by the sequences of the invention), partial deletion, addition of one or two bases into the gene under consideration. Can be. Such modifications can be obtained in vitro (on isolated DNA) or in situ, for example by genetic engineering or alternatively by treatment with a mutagenic agent. Other sites, in particular the E3 (WO95 / 02697), E2 (WO94 / 28938), E4 (WO94 / 28152, WO94 / 12649, WO95 / 02697) and L5 (WO95 / 02697) sites can also be modified. According to a preferred embodiment, the adenovirus according to the invention comprises a deletion in the E1 or E4 site. According to another embodiment, a deletion is in E1 at the level inserted into the E4 site and the sequences of the invention (see FR 94 13355).

Defective recombinant adenoviruses according to the present invention can be prepared by any technique known in the art. Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO. J. 3 (1984) 2917]. In particular, they can be produced by homologous recombination via an alia between an adenovirus and a plasmid carrying the DNA sequence (sequence encoding an chimeric molecule + expression cassette) of the invention. Homologous recombination occurs after co-transfection of the adenovirus and plasmid into a suitable cell line. The cell line used should preferably be (i) transformable by said element, and (ii) comprise a sequence that can compensate for the defective adenovirus moiety, preferably in an integrated form to avoid the risk of recombination. shall. Examples of such cell lines include, in particular, the human embryonic kidney line 293 comprising the left part (12%) of the Ad5 adenovirus in the genome in an integrated form. Graham et al., J. Gen. Virol. 36 (1977) 59 or in particular Application No. Mention may be made of cell lines which can complement the functions of El and E4 as described in WO 94/26914 or WO 95/02697.

The multiplied adenovirus is then recovered and purified according to conventional molecular biology techniques as exemplified in the Examples.

Regarding adeno-bound viruses (AAV), they are relatively small DNA viruses that integrate in a stable and left-specific manner into the genome of the cells they infect. They can infect a wide range of cells without inducing a random effect on cell growth, morphology or differentiation. Furthermore, they do not appear to be associated with pathogenicity in humans. The genome of AAV has been cloned, sequenced and characterized. It contains approximately 4700 bases at both ends containing an inverted repeat sequence (ITR) of 145 bases used as a replicator of the virus. The residues in the genome are divided into two essential regions that perform encapsulation functions: the left side of the genome contains the rep gene involved in the replication of the virus and the expression of the viral gene, and the right side of the genome contains the gene encoding the viral capsid protein. Include.

The use of AAV-derived vectors for introduction of genes in vitro and in vivo is described in reference; WO 91/18088, WO 93/09329, US 4,797,368, US 5,139,941, EP 488 528. These applications provide for the introduction of various AAV-derived constructs and the in vitro (on cells in culture) or in vivo (directly in vitro) of a variety of AAV-derived constructs in which the rep and / or cap genes have been deleted and replaced by that gene. Describe their use for this purpose. The defective recombinant AAV according to the invention is a nucleic acid sequence of the invention (sequence encoding a chimeric molecule + surrounded by two AAV inversion repeat sequences (ITRs), into a cell line infected by a human helper virus (eg, adenovirus). Plasmids containing the cassette) and plasmids carrying the AAV encapsulation genes (rep and cap genes). The recombinant AAV thus produced is then purified by conventional techniques.

Regarding Huffs and retroviruses, the construction of recombinant vectors is widely described in the literature: Breakfield et al., Bernstein et al., Genet. Eng. 7 (1985) 235; McCormick, Bil Technology 3, (1985) 689 et al.

Retroviruses in particular are integrated viruses that selectively infect dividing cells. Thus, they construct the corresponding vector for application to cancer. The genome of a retrovirus consists essentially of two LTRs, encapsulation sequences, 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 corresponding heterologous nucleic acid sequences. These vectors are especially MoMuLV (murine molony leukemia virus, also called MoMLV), MSV (murine molony sarcoma virus), HaSV (harvey sarcoma virus); Spleen necrosis virus (SNV); It can be produced from various types of retroviruses such as RSV (Rous sarcoma virus) or alternatively Friend's virus.

In order to construct recombinant retroviruses according to the invention, in particular plasmids containing LTRs, encapsulation sequences and sequences of the invention (sequences encoding sequences of chimeric molecules + expression cassettes) are generally constructed and then retroviruses defective in plasmids. It is used to transfect with so-called encapsulated cell lines that can provide the function of trans. Thus, in general, encapsulated cell lines can express gag, pol and env genes. Such encapsulated cell lines described in the prior art, in particular, PA317 cell line (US 4,861,719); PsiCRIP cell line (WO90 / 02806) and GP + envAm-12 cell line (WO89 / 07150). Furthermore, recombinant adenoviruses may include modifications in the LTR to inhibit transcriptional activity and prolonged encapsulation sequences that include a portion of the gag gene. Bender et al., J. Virol. 61 (1987) 1639. The resulting recombinant retroviruses are then purified by conventional techniques.

One example of a construct of a defective recombinant virus according to the invention is shown in FIG. 8. This figure highlights the second advantage of the constructs according to the present invention constructed in the absence of expression of the genes of interest in encapsulated cell lines. In these cell lines that lack a activator or transactive complex recovered by the system of the present invention, the promoter is inactive and the gene is not expressed in the producing cell (FIG. 8A). Only when the virus is effectively infected with the target cell, ie, when there is a transactivator or transactive complex recovered by the system of the present invention in the cell, the gene is effectively expressed (FIG. 8B). Particularly advantageous are constructs of viruses containing genes whose expression is toxic in cells (such as the Grb3-3, IL-2 and diphtheria toxin genes).

For the implantation of the invention, it is most particularly advantageous to use defective retroviruses or adenoviruses. These vectors also have particularly advantageous properties for introducing genes into tumor cells.

In addition, various types of non-viral vectors can be used within the scope of the present invention. Conditional expression systems according to the present invention may also be incorporated into non-viral agents that can facilitate the introduction and expression of nucleic acids in eukaryotic cells. Chemical or biochemical vectors are a convenient alternative to native viruses due to their ease of use, safety and lack of theoretical limitations on the size of the DNA to be introduced.

These synthetic vectors have two main functions: condensing the nucleic acid to be introduced and attaching it to the cell and passing it through the cell membrane and the two nuclear membranes at appropriate locations. In order to overcome the ionicity of nucleic acids, all non-viral vectors have polycationic charges.

Of the synthetic vectors developed, the polymers of polylysine, (LKLK) n, (LKKL) n, poly (ethyleneimine) and DEAD dextran types or alternatively cationic lipids or lipofectants are most advantageous. They have the property of condensing ADN and promoting binding to the cell membrane. Among the latter, mention may be made of lipopolyamines (lipofectamine, transfectam, etc.) and various cationic or neutral lipids (DOTMA, DOGS, DOPE, etc.). More recently, the principle of attaching complexes to the membrane by chemical linkage between the cationic polymer and the ligands of the membrane receptor present on the cell surface of the type desired to be grafted, while condensing DNA by the nature of the cationic polymer The concept of target transfection mediated by receptors has been developed with the advantage of: Thus, a target for a transferrin or insulin receptor or a receptor for asialoglycoprotein of hepatosite has been described.

Also subject of the invention are any pharmaceutical composition comprising a vector as defined above. These compositions can be formulated for topical, oral, parenteral, endocrine, intravenous, intramuscular, subcutaneous or intraocular administration and the like. Preferably, the composition according to the invention contains a pharmaceutically acceptable vehicle in the infusion composition. These are possible in sterile saline of isotonic solution (sodium phosphate, disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc. and mixtures of these salts), or solutions injectable by the addition of sterile water or physiological saline in each case. Dry, in particular cold-dried composition. With respect to retroviruses, this may be advantageous to be used to directly encapsulate a cell or an ex vivo infected cell and to be transplanted in vivo, in particular in the form of a renal organ (WO 94/24298).

The dosage of the vector used for infusion can be tailored to various variables, in particular the mode of administration utilized, relative pathogenicity or alternatively the desired duration of treatment. In general, the recombinant virus according to the invention is formulated and administered in the form of a dose of 10 ⁴ to 10 ¹⁴ pfu / ml. For AAV and adenovirus, dosages of 10 ⁶ to 10 ¹⁰ pfu / ml may be used. pfu (plaque forming units) corresponds to the infectivity of suspensions of virions and is determined by measuring the number of plaques of infected cells, usually 48 hours after infection in an appropriate cell culture. Techniques for investigating pfu titration of viral solutions are described in detail in the literature.

The expression system and the corresponding vector according to the invention are particularly useful for regulating the expression of the gene of interest in cell or gene therapy. Thus, they can be used to modulate the expression of any coding sequence of interest, especially a sequence encoding a therapeutic product (peptide, polypeptide, protein, RNA, etc.). In more detail, genes may include enzymes, blood derivatives, hormones, lymphokines: protein products such as interleukin, interferon, TNF, etc. (FR 9,203,120), growth factors, neurotransmitters or precursors or synthetic enzymes thereof, nutritional factors: BDNF, CNTF , NF, IGF, aFGF, bFGF, NT3, NT5 and the like; Alloproteins: ApoAI, ApoAIV, ApoE, etc. (FR 93 05125), dystrophin or minidstropin (FR 9,111,947), tumor suppressor genes: p53, Rb, RaplA, DCC, k-rev, etc. (FR 93 04745), involved in aggregation Genes that encode factors such as: VII, VIII, IX, etc., or alternatively DNA sequences (human, animal, plant origin) encoding natural or artificial immunoglobulins (Fab, ScFv, etc.), RNA ligands (WO91 / 19813) CDNA, gDNA, synthetic DNA, etc.).

The gene of interest may also be an antisense sequence in which expression in the target cell can regulate expression of the gene or transcription of cellular mRNA. The sequence can be transcribed into RNA complementary to the cell mRNA in the target cell, for example, according to the technique described in EP 140 308 to block the translation of these cell mRNA into proteins.

The present invention is particularly adapted to the expression of sequences encoding virulence factors. They may be particularly toxic to cells (diphtheria toxin, Pseudomonas toxin, Lycine A, etc.) and the product may be exogenous factors (suicide genes: thymidine kinase, cytosine deaminase, etc.) or alternatively cause cell death. Induce sensitivity to killer genes (Grb3-3 (PCT / FR94 / 00542), ScFv anti-ras (WO04 / 29446), etc.). In addition, the system of the present invention can produce vectors comprising these sequences, in particular viral vectors, without toxicity to the producing cells, and then express the expression of these toxic molecules in the target cell with the desired transactivator or transactive complex. May be selectively induced. Thus, this type of construct is particularly suitable for anticancer therapies, for example, to selectively destroy only damaged cells. This system is also particularly advantageous for the expression of cytokines, interferons, TNF or TGF, for example, where unregulated production causes many side effects.

The invention will be described in more detail with the aid of the following examples, which are illustrative only and should be regarded as non-limiting.

The present invention relates to a novel system for conditional expression of genes. It also relates to the use of such systems in gene or cell therapy to highly selectively target the expression of the gene of interest of interest.

1 is a representative view of a conditional expression system according to the present invention, which allows selective recovery of a transactivator by oligomerization region (A) or ScFv (B).

2 is a representative view of a conditional expression system according to the present invention, which allows for selective recovery of trans active complexes.

3 is a representative representation of an expression cassette according to the present invention comprising the regulatory sequence Tetop7, minimal promoter (TATA box) and gene (CAT).

4 is a representation of an expression cassette according to the invention, comprising regulatory sequence OR3, minimal promoter (TATA box) and gene (CAT).

5 is a representation of a bispecific chimeric molecule in accordance with the present invention.

6 shows a construct of a DNA sequence encoding a bispecific chimeric molecule according to the present invention.

7 is a representative view of a control chimeric construct.

8 shows the structure and function of a viral vector (retrovirus) according to the present invention.

Figure 9 shows the results of the study of the interaction between the hybrid molecule and the regulatory sequence of the present invention.

Figure 10 shows the results of the study of the interaction between the hybrid molecule of the present invention and various forms of p53 protein.

11 shows activation of Tet-Luc cassettes in SAOS-2 cells.

12 shows activation of the Tet-Luc cassette in H358 cells.

Common molecular biology techniques

General molecular biology methods such as centrifugation of plasmid DNA in cesium chloride ethidium bromide gradient, digestion with restriction enzymes, gel electrophoresis, transformation into E. Coli , nucleic acid precipitation, and the like are described in the literature [Reference; Maniatis et al ., 1989].

The enzyme is provided by New-England Biolabs (Beverly, MA). For ligation, DNA fragments were isolated according to size in 0.8-1.5% agarose gel, purified by GeneClean (BIO101, LaJolla CA), at pH 14 in pH 7.4 with T4 phage DNA ligase at 14 ° C. Incubate overnight in mM tris HCL buffer.

PCR (polymerase chain reaction) amplification is also performed under the following conditions according to Maniatis et al., 1989:

MgCl ₂ , adjusted to -8 mM concentration,

Denaturation temperature of -95 ℃,

Hybridization temperature of -55 ° C,

Synthetic temperature of -72 ℃,

25 cycles in PE9600 Thermal cycler (Perkin Elmer, Norwalk CO).

Oligonucleotides are made using chemicals of phosphoramidites that are protected at the β position by cyanoethyl groups [Ref. Sinha et al., 1984, Giles 1985], were synthesized with a biosystem model 394 automated DNA synthesizer (Applied Biosystem, Foster City CA) applied according to the manufacturer's recommendations.

Sequencing is performed on double stranded models by chain termination methods using fluorescent primers. We use the sequence Taq Dye Primer Kit from Applied Biosystem according to the manufacturer's specifications.

Example 1 Construction of Expression Cassettes Containing Regulatory Sequences, Minimal Transcription Promoters and Genes

1.1. Construction of plasmid pTETop7 / CAT

The plasmid pTETop7 / CAT contains the following components (FIG. 3):

Regulatory sequences consisting of a sequence for interacting with a tetracycline inhibitor TetR consisting of a -7 repeated Tetop motif (SEQ ID NO: 1);

Minimal promoter derived from the thymidine kinase gene (-37 to +19 site carrying the TATA box);

A sequence encoding chloramphenicol acetyltransferase (CAT), which is placed under said minimal promoter control.

The plasmid is constructed by cloning the SmaI-BglII fragment of plasmid pUHD10-7 (WO 94/29442) into plasmid pKK232-8 (Pharmacia), already cut with SmaI and BamHI.

1.2. Construction of plasmid pOR ₃ / CAT

The plasmid pOR3 / CAT contains the following components (FIG. 4):

Regulatory sequences consisting of the sequence OR3 for interacting with a -Cro inhibitor (SEQ ID NO: 2);

Minimal promoter derived from the thymidine kinase gene (-37 to +19 site carrying the TATA box);

A sequence encoding chloramphenicol acetyltransferase (CAT), which is placed under said minimal promoter control.

The plasmid is constructed in the following way: The sequence OR3 for interacting with the Cro inhibitor is artificially synthesized. For example, two oligonucleotides are synthesized:

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

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

These two oligonucleotides are then hybridized to reconstruct double stranded sequence OR3 rimmed by some sequence, resulting in biased cloning as follows:

GATCCTATCACCGCAAGGGATAA

GATAGTGGCGTTCCCTATTTCCA

1.3. Construction of Cassettes for the Expression of Virulence Genes

Cassettes of expression of virulence genes are characterized by the sequence of the CAT sequence of plasmids (1.1 and 1.2) described above encoding the virulence product, preferably the Grb3-3 gene (PCT / FR94 / 00542), thymidine kinase gene, diphtheria or pseudomonas. Obtained by replacing the toxin with a gene encoding the toxin.

Example 2: Construction of Single-Chain Antibodies Specific to p53

Single-chain antibodies are constructed according to the following procedure:

CDNA encoding the VH and VL sites is obtained from hybridoma pAb421 producing anti-p53 antibodies. For example, after extracting the entire RNA from the hybridoma, reverse transcription reaction using a random hexamer as a primer. The use of primers of this type avoids the use of primers specific for immunoglobulins. The cDNA clone obtained has a sufficient length to clone the V site. However, since they correspond to a small amount of total cDNA, a primary amplification reaction must be performed to produce enough DNA for cloning. For example, cDNAs encoding VH and VL sites are amplified separately. Primers used are oligonucleotides that hybridize at the level of the opposite end of the variable region of each chain (H and L). Amplification products using primers specific for the heavy chain are fragments of about 340 base pairs. Amplification products using primers specific for the light chain are fragments of about 325 base pairs.

After purification of the cDNA encoding the VH and VL sites of the antibody, it is assembled into a single chain by the nucleotide arm (L). The nucleotide arm is constructed such that one end binds to the 3 'end of the cDNA encoding the VH site and the other end binds to the 5' end of the cDNA encoding the VL site. The sequence of the arm is SEQ ID NO. Encode the peptide of 5. The assembled sequence of about 700 bp is in the form of an NcoI-NotI fragment, SEQ ID NO. VH-L-VL chain having a sequence represented by 4 (amino acids 9 to 241). This sequence also contains the tag sequence of myc (residues of 256 to 266) at the C-terminus.

Example 3: Construction of Nucleic Acid Sequences Encoding Bispecific Chimeric Molecules Containing a Region for Binding to a Transient Comprised of Single-Chain Antibodies (ScFv)

3.1. Construction of Plasmids Containing ScFv-myc-Hinge-TetR or Cro Sequences (FIGS. 5A and 6)

NcoI-NotI fragments containing cDNA encoding anti-p53 ScFv are first cloned into plasmids of pUC19 type. The sequence encoding the VSV epitope (SEQ ID NO. 7) or the sequence encoding the myc epitope (SEQ ID NO. 8) is inserted at the bottom of the fragment (FIG. 6).

The sequence encoding TetR and Cro proteins is then prepared as follows:

From the model plasmid carrying the -TetR sequence, the sequence encoding TetR is obtained by amplification using the following oligonucleotides:

Oligo 5474 (SEQ ID NO. 10):

GGCTCTAGACCCAAGCCCAGTACCCCCCCAGGTTCTTCAACG

CGTGGATCCATGTCCAGATTAGATAAAAGTAAAG

Oligo 5475 (SEQ ID NO. 11):

CGTACGGAATTCGGGCCCTTACTCGAGGGACCCACTTTCACATT

TAAGTTG

In addition, these oligonucleotides provide a sequence that encodes a hinge peptide arm that connects the dual functional regions of the molecule.

Thus, the amplified fragment comprises a sequence encoding a peptide arm and a region for binding to tetR DNA. This fragment is cloned into the XbaI-EcoRI locus of the plasmid obtained above to generate a plasmid containing the sequence encoding the ScFv-myc-Hinge-TetR molecule (FIG. 6).

The sequence encoding Cro is obtained by amplifying the DNA model of lambda bacteriophage with the following oligonucleotides:

Oligo 5531 (SEQ ID NO. 12):

GGCTCTAGACCCAAGCCCAGTACCCCCCCAGGTTCTTCAACGCG

TGGATCCATGGAACAACGCATAACCCTGAAAG

Oligo 5532 (SEQ ID NO. 13):

CGTACGGAATTCGGGCCCTTACTCGAGTGCTGTTGTTTTTTTGTT

ACTCGG

In addition, these oligonucleotides provide a sequence encoding a hinge peptide arm that connects two functional regions of the molecule.

Thus, the amplified fragment comprises a sequence encoding a region for binding to the peptide arm and Cro DNA. This fragment is cloned into the XbaI-EcoRI locus of the plasmid obtained above to generate a plasmid containing the sequence encoding the ScFv-myc-Hinge-TetR molecule.

3.2. Construction of plasmids comprising ScFv-Hinge-TetR or Cro sequences (FIG. 5B).

This example relates to the construction of a plasmid carrying a sequence encoding a bispecific chimeric molecule according to the invention, lacking a tag sequence.

These plasmids are derived by digesting the plasmids described in 3.1. Above with NotI and XbaI enzymes. This cleavage allows the incision of fragments carrying the site encoding the myc tag.

3.3. Construction of plasmids comprising ScFv-TetR or Cro sequences (FIG. 5C).

This example relates to the construction of plasmids carrying sequences encoding bispecific chimeric molecules according to the invention, lacking arm and tag sequences.

These plasmids are derived by digesting the plasmids described in 3.1. Above with NotI and BamHI enzymes. This digestion allows for the cleavage of fragments carrying sites encoding the myc tag and Hinge peptide arms.

Example 4 Construction of Nucleic Acid Sequences Encoding Bispecific Chimeric Molecules Containing a Region for Binding to a Transient Comprised of Oligomeric Region

4.1. Cloning of the oligomerization site of p53 protein (fragments 320 to 393).

PCR amplification with the help of the following oligonucleotides on plasmids carrying the cDNA of human wild type p53 yields a cDNA encoding the oligomerization region of the p53 protein (SEQ ID NO: 3):

Oligo 5535 (SEQ ID NO. 14):

CAGG CCATGG CATGAAGAAACCACTGGATGGAGAA

(Underlined parts represent NcoI loci)

Oligo 5536 (SEQ ID NO. 15):

CGTC GGATCC TCTAGA T GCGGCCGC GTCTGAGTCAGGCCCTTC

(Underlined portion: BamHI left; dark portion: XbaI left; dark and underlined portion: NotI left).

4.2. Plasmid p53 320 / 393-myc-Hinge-TetR or by cloning the fragment amplified above in the form of an NcoI-NotI fragment into the corresponding locus by substituting the site encoding the ScFv of the plasmid described in Example 3.1. Cro (FIG. 5a) is obtained.

4.3. The plasmid p53 320 / 393-Hinge-TetR was cloned into the corresponding locus by substituting the fragment amplified in 4.1 in the form of an NcoI-XbaI fragment in place of the region encoding the ScFv and tag of the plasmid described in Example 3.1. Or Cro (FIG. 5B) is obtained.

4.4. The fragment amplified in 4.1 in the form of a NcoI-BamHI fragment was cloned into the corresponding locus by replacing it with a site encoding the ScFv, tag and Hinge of the plasmids described in Example 3.1, to plasmid p53 320 / 393-TetR. Or Cro (FIG. 5C) is obtained.

4.5. Fragments amplified in PCR with the help of oligo 5537/5539 or 5538/5539 digested with XhoI / EcoRI on plasmids carrying cDNA for wild-type human p53 were previously cloned into plasmids described in 3.1. Previously digested with XhoI / EcoRI. Obtain plasmid tetR or Cro-p53 320/393 (FIG. 5D) or tetR or Cro-Hinge-p53 320/393 (FIG. 5E).

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 regulatory plasmids carrying sequences encoding chimeric molecules comprising DNA binding regions (TetR or Cro) and transactivator regions of p53 protein (regions 1-73)

Fragments amplified using plasmids carrying cDNA against human wild type p53 were digested with NcoI / NotI, NcoI / XbaI, NcoI / BamHI to be previously digested with NcoI / NotI, NcoI / XbaI or NcoI / BamHI as described in Cloning into the plasmid yields plasmid p53 1 / 73-TetR or Cro (FIGS. 7A, b and c) with or without tag (myc or VSV) and Hinge.

Oligo 5661 (SEQ ID NO. 19):

CAGGCCATGGAGGAGCCGCAGTCAGATCC

Oligo 5662 (SEQ ID NO. 20):

CGTCGGATCCTCTAGATGCGGCCGCCACGGGGGGAGCAGCCTC

TGG

Example 6 Construction of Plasmids to Express Various Hybrid Molecules of the Invention

The fragment containing the cDNA encoding the hybrid molecule is cloned into the NcoI / EcoRI locus of the eukaryotic expression vector pcDNA3 (Invitrogen) to obtain the plasmid used for expression of the hybrid molecule. Thus, various constructs are produced as follows:

ScFv 421: SEQ ID NO. 4

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

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

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

-TET04: hybrid protein containing p53 (320/393) -TetR chain shown in FIG. 5C according to Example 4.4

-TET02: hybrid protein containing p53 (1/73) -VSV-Hinge-TetR chain shown in FIG. 7A according to Example 5

Example 7: Recognition of specific double-stranded DNA sequences by hybrid molecules of the present invention

7.1. Generation of hybrid molecules

In accordance with the experimental procedure described by the provider using a TNT Coupled Reticulocyte Lysate System kit (Promega) with 50 μl total reaction volume, the molecule described in Example 6 was detoxified in erythrocyte lysate in vitro. Obtain various molecules used.

7.2. Construction of Specific Double-Stranded DNA Sequences

The specific double stranded DNA sequence used in this experiment consists of two synthetic oligonucleotides:

Oligo 5997 (SEQ ID NO. 24):

GATCCGACTTTCACTTTTCTCTATCACTGATAGTGAGTGGTAAACTCA

Oligo 5998 (SEQ ID NO. 25):

AGCTTGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCG

These two synthetic oligonucleotides are labeled with phosphorus-33 by incubating 10 pmol of oligonucleotides each at 10O < 0 > C for 10 min in 10 [mu] l of the following reaction medium:

Tris-HCL pH 7.6 50 mM

MgCl ₂ 10 mM

Dithiothreitol 5 mM

Spermidine 100 mM

EDTA 100 mM

[γ- ³³ P] ATP (Amersham) 50 μCi (1000-3000 Ci / mmol)

T4 Kinase (Boehringer) 10 U

Thus, the two labeled oligonucleotides hybridize in the presence of 400 mM NaCl to reconstruct the following double-stranded sequence TetO (SEQ ID NO: 26):

GATCCGACTTTCACTTTTCTCTATCACTGATAGTGAGTGGTAAACTCA

CTAGGCTCAAAGTGAAAAGAGATAGTGACTATCACTCACCATTTGAGT

7.3. Construction of the double-stranded sequence TetO by various hybrid molecules of the present invention.

TetO prepared according to Example 7.2 in 50 μl of reaction medium (10 mM Tris-HCL pH 7.4, 10 mM MgCl ₂ , 10 mM KCL, 6 mM β-mercaptoethanol, 0.1 mM EDTA, 0.5 mg / ml BSA) performs (10 ^-10 M) sequence of example 7.1 cold competitor oligonucleotide used to remove the manufactured product 10 to decrypt ㎕ and non-specific binding in accordance with the added nucleotides AP2 (Promega) 10 ^-8 M DNA- binding reaction. The specificity of the interaction is checked by shifting the equilibrium by adding 10 μM tetracycline (Sigma) to the reaction medium. The reaction mixture is incubated at 20 ° C. for 15 minutes, 10 μl of 50% glycol is added and the final mixture is transferred by 200V pure 5% polyacrylamide gel electrophoresis at 16 ° C.

The results of this experiment performed with the TET19, TET02 and TET07 hybrid molecules are shown in FIG. Under these conditions, the delayed late migration indicates that these three molecules bound to specific double-stranded DNA sequences, and the specificity of this interaction was expressed by the interruption of these delays by the addition of tetracycline. do.

Thus, these results assure that the hybrid molecules of the present invention can specifically bind to TetO nucleotide sequences.

Example 8 Specific Binding of Hybrid Molecules of the Invention with Molecules Having Trans Transcription Activator Regions

For this experiment, the hybrid molecules of ScFv 421, TET19 and TET02 of the present invention according to Example 6 were subjected to in vitro detoxification procedure using the experimental procedure according to Example 7.1 in the presence of 44 μCi with 35S-methionine (Amersham). By means of production of these radially labeled hybrid molecules.

The cDNA of the molecule with or without the trans transcriptional activator region is cloned into the BamHl locus of the pBlueBacIII (Invitrogen) vector. Using these vectors, recombinant baculoviruses are produced and purified according to the manufacturer's instructions. These molecules are produced by infecting recombinant baculovirus with sf9 insect cells according to the manufacturer's experimental procedures. Protein extracts at 10 mg / ml dml final protein concentration are prepared according to the procedure described by K. Ory et al. K. Ory, EMBO J., 13, 3496-3504, 1994]. These molecules are as follows:

-p53 (1/393): wild type p53 protein

-p53 (1/320): wild type p53 protein, lacking an oligomerization region and a region recognized by monoclonal antibody pAb421.

8.2. Bonding between a molecule with or without a transtranscriptional activator and a hybrid molecule of the present invention

5 μl of each in vitro detoxification product prepared according to Example 8.1 was combined with 5 μl of the baculovirus extract according to Example 8.1 and 2 μg of the monoclonal antibody DO1 (Oncogene Science) recognizing the N-terminus of the p53 protein. Incubated in 100 μl of modified RIPA buffer for 16 h at 4 ° C. [Ref. K. Ory, EMBO J., 13, 3496-3504, 1994]. The complex retained by the antibody was eluted by incubating at 80 ° C. for 10 minutes in the presence of 30 μl of transfer buffer [Reference; Laemmli U.K., Nature, 227, 680-685, 1970] are electrophoresed on 10% polyacrylamide gel in denaturing medium at 200V [Ref. Laemmli U.K., Nature, 227, 680-685, 1970]. The gel is then dried and visualized by Instantimager (Packard Instruments), which allows to determine the amount of hybrid molecules bound with or without a trans transcriptional activator. The experimental results are shown in FIG.

Under these conditions, hybrid molecules representing ScFv 421 (TET19) recognize p53 molecules (1/393) in the same manner as ScFv 421 alone, while hybrid molecules representing 320/393 region (TET02) are more p53 (1). / 393) Except for holding power, the same property is clearly shown. Moreover, the absence of signals observed during incubation with p53 (1/320) molecules is very specific as their interactions were expected, mediated by the C-terminus of the p53 protein (amino acids 320 to 393). It is displayed.

Thus, these results assure that the hybrid molecules of the present invention can significantly recover trans transcriptases carried by molecules that are specific partners.

Example 9 Functional Recovery of Trans Transcription Activator Regions by Hybrid Molecules of the Invention

Functional recovery of the trans transcriptional activator region by the hybrid molecule of the present invention is defective for the SAOS-2 cell line (osteosarcoma), which is defective for the two alleles of the p53 protein, and for the two alleles of the p53 protein. One of the two alleles of the tumor lines H358 and p53 protein, which is defective, is measured in an in vivo transactivation system in tumor line HT29 with one mutated allele (H273 mutation). This system is based on the use of reporter genes that can be enzymatically analyzed and placed under the control of a promoter containing nucleotide units for specific recognition by a Tet inhibitor.

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

9.1. Cell Lines Used and Culture Conditions

The cell lines used in these experiments, as well as the culture medium used for genotyping and culture linked to the p53 protein, are shown in Table 1 below.

Cell line Cell line p53 Culture medium ATCC No. SAOS-2 -/- DMEM medium (Gibco BRL) supplemented with 10% bovine embryo serum (Gibco BRL) HTB 85 H358 -/- RPMI 1640 medium (Gibco BRL) supplemented with 10% bovine embryo serum (Gibco BRL) HT29 -/ H273 DMEM medium (Gibco BRL) supplemented with 10% bovine embryo serum (Giboc BRL)

9.2. Plasmids Expressing Molecules Having Trans Transcription Activator Regions

Molecules with trans-transcriptional activators used in this experiment are the wild type (wt) p53 protein and its variants G281 and H175. CDNA encoding these three proteins is inserted into the BamHI locus of the pcDNA3 (Invitrogen) vector.

9.3. Intracellular Expression of the Hybrid Molecules of the Invention

Using the following procedure, the hybrid molecules of the present invention are expressed in cells in culture by transient transfection:

Cells (3.5 × 10 ⁵ ) are seeded into 6-well plates containing 2 ml of culture medium and incubated overnight in a CO ₂ (5%) incubator at 37 ° C. The various constructs are then transfected using lipofectant AMINE (Gibco BRL) as a transfection agent in the following manner: 1.5 μg total plasmid with 2 ml of non-serum culture medium (transfection mixture) for 30 minutes. Incubate with 5 μl of lipofectant AMINE (containing 0.25 μg of reporter plasmid). During this time, cells were washed twice with PBS, incubated with the transfection mixture for 4 hours at 37 ° C. and subsequently replaced with 2 ml of culture medium supplemented with 10% bovine embryo serum neutralized by heat. The cells are allowed to grow again at 37 ° C. for 48 hours.

9.4. Detection of transcriptional activity

Activation of transcription associated with functional recovery of the trans transcriptional activator is detected and quantified by measuring luciferase activity encoded by the LUC gene using the Luciferase Assay System Kit (Promega) according to the manufacturer's experimental procedures.

9.5. Functional Recovery of Trans Transcription Activators by Molecules of the Invention

This experiment is performed using the TET02, TET03 and TET07 molecules of Example 6. In this experiment, the TET07 molecule plays a positive regulatory role because it has its own trans transcriptional active region.

The results obtained in SAOS-2 cells and shown in FIG. 11 show that, unlike the TET02 construct, the TET07 molecule can activate itself the transcription of the LUC gene under the control of the Tet operator. This is consistent with the fact that these cell lines do not contain endogenous p53 proteins that cannot be recovered by the TET02 molecule. In contrast, the introduction of the G281 variant, which does not produce any signal in the wild type p53 protein or itself, can generate transcriptional activity in the presence of the TET02 molecule. This result has a nonfunctional trans transcriptional active region and cannot be observed in the H175 variant described in the literature.

These results observed in SAOS-2 cell lines with TET02 molecules cannot be reproduced into tumor cell lines containing no endogenous p53 (H358 cells) or extended to TET03 and TET04 molecules (FIG. 12).

With the aim of assuring these results within different cell categories, the positive regulatory TET07 as well as the TET02 molecule were directed to the mutant endogenous p53 protein (H273), a negative regulator for this experiment consisting of transfecting an empty pcDNA3 vector. It is expressed in expressing HT29 cells. The experimental results shown in the table below show that the TET02 molecule can significantly restore the trans transcriptional active region of the endogenous p53 protein.

Transcriptional Activity of Hybrid Molecules of HT29 Cells of the Present Invention pcDNA3 TET07 TET02 One 59 10

Thus, these experiments as a whole indicate that the hybrid molecules of the present invention are able to bind fairly well both to double-stranded DNA sequences and to proteins that represent trans transcriptional active regions, and that these molecules may conditionally induce expression of the genes of interest. Shows that it can.

Sequence list

(1) General Information:

(i) Applicant:

(A) Name: Long Franc Laura Societe Annoim

(B) Street: Avenue Lamont Along 20

(C) City: Anthony

(E) Country: France

(F) Zip code: 92165

(G) Phone: (1) 40.91.70.36

(H) Telefax: 40.91.72.91

(ii) Name of the Invention: Conditional Expression System

(iii) SEQ ID NO: 26

(iv) computer readable form:

(A) Medium type: tape

(B) Computer: IBM PC compatible model

(C) operating system: PC-DOS / MS-DOS

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

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 19 base pairs

(B) Type: Nucleotide

(C) strand form: single

(D) Phase: Linear

(ii) Molecular Type: cDNA

(xi) sequence description: SEQ ID NO: 1:

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 17 base pairs

(B) Type: Nucleotide

(C) strand form: single

(D) Phase: Linear

(ii) Molecular Type: cDNA

(xi) sequence description: SEQ ID NO: 2:

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 74 amino acids

(B) Type: Amino Acid

(C) strand form: single

(D) Phase: Linear

(ii) Molecular Type: Peptide

(iii) Hypothesis: None

(iv) anti-sense: none

(xi) sequence description: SEQ ID NO: 3:

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 768 base pairs

(B) Type: Nucleotide

(C) strand form: single

(D) Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(iv) anti-sense: none

(xi) sequence description: SEQ ID NO: 4:

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 15 amino acids

(B) Type: Amino Acid

(C) strand form: single

(D) Phase: Linear

(ii) Molecular Type: Peptide

(iii) Hypothesis: None

(iv) anti-sense: none

(xi) sequence description: SEQ ID NO: 5:

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 30 base pairs

(B) Type: Nucleotide

(C) strand form: double

(D) Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(iv) anti-sense: none

(ix) Features:

(A) Name / key: CDS

(B) Location: 1..30

(xi) sequence description: SEQ ID NO: 6:

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 18 base pairs

(B) Type: Nucleotide

(C) strand form: double

(D) Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(iv) anti-sense: none

(ix) Features:

(A) Name / key: CDS

(B) Location: 1..18

(xi) sequence description: SEQ ID NO: 7:

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 33 base pairs

(B) Type: Nucleotide

(C) strand form: double

(D) Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(iv) anti-sense: none

(ix) Features:

(A) Name / key: CDS

(B) Location: 1..33

(xi) sequence description: SEQ ID NO: 8:

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 7 amino acids

(B) Type: Amino Acid

(C) strand form: single

(D) Phase: Linear

(ii) Molecular Type: Peptide

(iii) Hypothesis: None

(iv) anti-sense: none

(xi) sequence description: SEQ ID NO: 9:

(2) Information about SEQ ID NO: 10:

(i) Sequence Features:

(A) Length: 66 base pairs

(B) Type: Nucleotide

(C) strand form: double

(D) Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(iv) anti-sense: none

(xi) sequence description: SEQ ID NO: 10:

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 51 base pairs

(B) Type: Nucleotide

(C) strand form: double

(D) Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(iv) anti-sense: none

(xi) sequence description: SEQ ID NO: 11:

(2) Information about SEQ ID NO: 12:

(i) Sequence Features:

(A) Length: 66 base pairs

(B) Type: Nucleotide

(C) strand form: double

(D) Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(iv) anti-sense: none

(xi) sequence description: SEQ ID NO: 12:

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 51 base pairs

(B) Type: Nucleotide

(C) strand form: double

(D) Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(iv) anti-sense: none

(xi) sequence description: SEQ ID NO: 13:

(2) Information about SEQ ID NO: 14:

(i) Sequence Features:

(A) Length: 35 base pairs

(B) Type: Nucleotide

(C) strand form: double

(D) Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(iv) anti-sense: none

(xi) sequence description: SEQ ID NO: 14

(2) Information about SEQ ID NO: 15:

(i) Sequence Features:

(A) Length: 43 base pairs

(B) Type: Nucleotide

(C) strand form: double

(D) Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(iv) anti-sense: none

(xi) Sequence description: SEQ ID NO: 15

(2) Information about SEQ ID NO:

(i) Sequence Features:

(A) Length: 31 base pairs

(B) Type: Nucleotide

(C) strand form: double

(D) Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(iv) anti-sense: none

(xi) sequence description: SEQ ID NO: 16:

(2) Information about SEQ ID NO: 17:

(i) Sequence Features:

(A) Length: 61 base pairs

(B) Type: Nucleotide

(C) strand form: double

(D) Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(iv) anti-sense: none

(xi) sequence description: SEQ ID NO: 7:

(2) Information about SEQ ID NO: 18:

(i) Sequence Features:

(A) Length: 37 base pairs

(B) Type: Nucleotide

(C) strand form: double

(D) Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(iv) anti-sense: none

(xi) sequence description: SEQ ID NO: 18:

(2) Information about SEQ ID NO: 19:

(i) Sequence Features:

(A) Length: 29 base pairs

(B) Type: Nucleotide

(C) strand form: double

(D) Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(iv) anti-sense: none

(xi) sequence description: SEQ ID NO: 19:

(xi) sequence description: SEQ ID NO: 18:

SEQ ID NO: 18

(2) Information about SEQ ID NO: 20:

(i) Sequence Features:

(A) Length: 46 base pairs

(B) Type: Nucleotide

(C) strand form: double

(D) Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(iv) anti-sense: none

(xi) sequence description: SEQ ID NO: 20:

(2) Information about SEQ ID NO: 21:

(i) Sequence Features:

(A) Length: 66 amino acids

(B) Type: Amino Acid

(C) strand form: single

(D) Phase: Linear

(ii) Molecular Type: Peptide

(iii) Hypothesis: None

(iv) anti-sense: none

(xi) sequence description: SEQ ID NO: 21:

(2) Information about SEQ ID NO: 22:

(i) Sequence Features:

(A) Length: 23 base pairs

(B) Type: Nucleotide

(C) strand form: double

(D) Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(iv) anti-sense: none

(xi) sequence description: SEQ ID NO: 22:

(2) Information about SEQ ID NO: 23:

(i) Sequence Features:

(A) Length: 23 base pairs

(B) Type: Nucleotide

(C) strand form: double

(D) Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(iv) anti-sense: none

(xi) sequence description: SEQ ID NO: 23:

(2) Information about SEQ ID NO: 24:

(i) Sequence Features:

(A) Length: 48 base pairs

(B) Type: Nucleotide

(C) strand form: double

(D) Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(iv) anti-sense: none

(xi) sequence description: SEQ ID NO: 24:

(2) Information about SEQ ID NO: 25:

(i) Sequence Features:

(A) Length: 48 base pairs

(B) Type: Nucleotide

(C) strand form: double

(D) Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(iv) anti-sense: none

(xi) sequence description: SEQ ID NO: 25:

(2) Information about SEQ ID NO: 26:

(i) Sequence Features:

(A) Length: 96 base pairs

(B) Type: Nucleotide

(C) strand form: double

(D) Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(iv) anti-sense: none

(xi) sequence description: SEQ ID NO: 26:

Claims (57)

Bispecific chimeric molecules comprising a region capable of selectively binding a defined DNA sequence and a detection region capable of specifically binding to a transactivator or transinhibitor or a transactive or trans-inhibitory complex in a physiological or physiological pathology state. . The molecule of claim 1, wherein the region capable of selectively binding a defined DNA sequence is derived from a protein capable of interacting with DNA. The molecule of claim 2, wherein the region capable of selectively binding a defined DNA sequence is derived from a eukaryotic cell protein. 4. The molecule of claim 3 wherein the region capable of selectively binding a defined DNA sequence is derived from a p53, STAT or NFkB protein. 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 prokaryotic protein is a bacterial inhibitor. 7. The molecule of claim 6 wherein the region capable of selectively binding a defined DNA sequence is derived from a tetR protein. 7. The molecule of claim 6 wherein the region capable of selectively binding a defined DNA sequence is derived from a Cro protein. The molecule of claim 2, wherein the region capable of selectively binding a defined DNA sequence comprises a region that interacts with the DNA of the protein. 9. A molecule according to any one of claims 2 to 8, wherein the region capable of selectively binding a defined DNA sequence consists of an intact protein. The molecule of claim 10, wherein the region capable of selectively binding a defined DNA sequence consists of a tetR protein. The molecule of claim 10, wherein the region capable of selectively binding a defined DNA sequence consists of Cro proteins. 2. A molecule according to claim 1 wherein the region capable of specifically binding to the transactivator or trans-inhibitor or trans-active or trans-inhibitory complex is an oligomerization region. The molecule of claim 13, wherein the oligomerization region is a leucine zipper, SH3 or SH2 region. 14. The molecule of claim 13, wherein the oligomerization region capable of specifically binding to the transactivator consists of the C-terminus of the p53 protein. 16. The molecule of claim 15, wherein the oligomerization region consists of the C-terminus of p53 protein spanning 320 to 393 (SEQ ID NO. 3), 302 to 360 or 302 to 390 residues. The synthetic or natural composition of claim 1, wherein a region capable of specifically binding to a transactivator or trans-inhibitor or trans-active or trans-inhibitory complex is known to interact with the transactivator or trans-inhibitor or trans-active or trans-inhibitory complex. Molecules characterized in that the area. The method of claim 1, wherein the region capable of specifically binding to the transactivator or trans-inhibitor or trans-active or trans-inhibitory complex is an antibody or antibody fragment or derivative directed to the trans- activator or trans-inhibitor or trans-active or trans-inhibitory complex. Characterized by the molecule. 19. The antibody of claim 18, wherein the region capable of specifically binding to a transactivator or trans-inhibitor or trans-active or trans-inhibitory complex consists of a Fab or F (ab) '2 fragment of the antibody or a VH or VL region of the antibody. Molecule. 19. The antibody according to claim 18, wherein the region capable of specifically binding to a transactivator or trans-inhibitor or trans-active or trans-inhibitory complex consists of a single-chain antibody (ScFv) comprising a VH moiety that is linked to the VL moiety by the arm. Molecules characterized by. The molecule of claim 1, wherein the DNA-binding region and the transactivator-binding region are linked to each other via an arm. The molecule according to claim 21, wherein the arm consists of a peptide consisting of 5 to 30 amino acids, preferably 5 to 20 amino acids. The method of claim 22, wherein the arm is SEQ ID NO. 5 or SEQ ID NO. Molecule selected from the peptide sequences of 6. 24. The molecule of claim 1, wherein the DNA-binding region is in the N-terminal position and the transactivator-binding region is in the C-terminal position. 24. The molecule of claim 1, wherein the DNA-binding region is at the C-terminal position and the transactivator-binding region is at the N-terminal position. Bispecific chimeric molecules of ScFv-VSV / myc-Hinge-TET or Cro (FIG. 5A) structure. Bispecific chimeric molecules of ScFv-Hinge-TET or Cro (FIG. 5B) structure. Bispecific chimeric molecules of ScFv-TET or Cro (FIG. 5C) structure. Bispecific chimeric molecules of TET or Cro-ScFv (FIG. 5D) structure. Bispecific chimeric molecules of TET or Cro-Hinge-ScFv (FIG. 5E) structure. Bispecific chimeric molecules of the structure Oligom-VSV / myc-Hinge-TET or Cro (FIG. 5A), Oligom-Hinge-TET or Cro (FIG. 5B) or Oligom-TET or Cro (FIG. 5C). 32. 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 which is a DNA sequence. 34. A nucleic acid sequence according to claim 32 or 33 which is part of a vector. A chimeric molecule according to claim 1, and A genetic condition expression system comprising an expression cassette comprising regulatory sequences, minimal transcriptional promoters and genes. The method of claim 35, wherein the DNA-binding region of the chimeric molecule is represented by all or part of TetR and the regulatory sequence is SEQ ID NO. A condition system, comprising optionally repeating the sequence of 1 or derivatives thereof several times. 36. The chimeric molecule of claim 35, wherein the DNA-binding region of the chimeric molecule is represented by all or a portion of Cro and the regulatory sequence is SEQ ID NO. A condition system, comprising optionally repeating the sequence of 2 or a derivative thereof several times. 38. The condition system of any of claims 35 to 37, wherein the minimum promoter comprises an INR or TATA box. The condition system of claim 38, wherein the minimal promoter is derived from a promoter of the thymidine kinase gene. 40. The condition system of claim 39, wherein the minimal promoter consists of -37 to +19 nucleotides of thymidine kinase. A nucleic acid sequence encoding a chimeric molecule according to any one of claims 1 to 31, and A vector comprising an expression cassette comprising a regulatory sequence, minimal transcriptional promoter and corresponding coding sequence. 42. The vector of claim 41 wherein the minimum transcriptional promoter is defined according to claims 38-40. The method of claim 41, wherein the DNA-binding region of the chimeric molecule is represented by all or part of TetR and the regulatory sequence is SEQ ID NO. A vector comprising the sequence of 1 or a derivative thereof optionally optionally repeated several times. The method of claim 41, wherein the DNA-binding region of the chimeric molecule is represented by all or a portion of Cro and the regulatory sequence is SEQ ID NO. A vector comprising the sequence of 2 or a derivative thereof optionally optionally repeated many times. 45. The vector of any one of claims 41 to 44 wherein the coding sequence is a DNA sequence encoding a therapeutic product. 46. The vector of claim 45, wherein the therapeutic product is a toxic polypeptide or peptide. 49. The vector of claim 46, wherein the toxic therapeutic product is diphtheria toxin, pseudomonas toxin, lysine A, thymidine kinase, cytosine deaminase, Grb3-3 or ScFv Y28 protein. 48. The vector of any one of claims 41 to 47, which is a plasmid vector. 48. The vector of any one of claims 41 to 47, which is a viral vector. 50. The vector of claim 49 which is a defective recombinant adenovirus. The vector of claim 49, wherein the vector is a defective recombinant retrovirus. A pharmaceutical composition comprising at least one vector according to any one of claims 41 to 51. SEQ ID NO. Nucleic acid comprising the sequence of 4. The molecule of claim 1 wherein the physiological or physiological pathological state of the transactivator is a protein of virus, parasite, mycobacteria or cellular origin with transcriptional transactivation activity. 55. The molecule of claim 54 wherein the activator is a viral protein selected from HIV viral Tat protein, papilloma virus E6 / E7 protein and Epstein-Barr virus EBNA protein. 55. The molecule of claim 54 wherein the transactivator is a variant or wild type of cellular protein, preferably p-53 protein. The molecule of claim 1, wherein the activator or transactive complex in a physiological or physiological pathology is a protein that appears in an infected or hyperproliferated cell.