MXPA01003065A - Use of specific hybrid promoters for controlling tissue expression - Google Patents

Abstract

The invention concerns a hybrid promoter comprising:all or part of the enhancer region of a strong and ubiquitous promoter/enhancer;a promoter region enabling specific expression in smooth muscle cells, and all vector or cell containing said promoter, and their uses.

Description

USE OF SPECIFIC HYBRID PROMOTERS TO CONTROL TISSUE EXPRESSION FIELD OF THE INVENTION The present invention relates to the domain of biology, in particular to the domain of the regulation of gene expression. It describes in particular the novel constructs and the novel vectors that allow a directed and strong expression of the genes. The present invention is useful in several domains, and in particular for the production of recombinant proteins, for the creation of transgenic animal models, for the creation of cell lines, for the implementation of screening tests, or even in genetic and cellular therapy . The possibility of controlling and directing the expression of genes is a very important position in the development of biotechnology. In vitro, it can allow to improve the production conditions of recombinant proteins, using particular populations of cells. Always in vitro, it can allow the detection or the demonstration of the presence of specific populations of cells in a sample, or equally to prove the properties of a product or the regulation of a gene in a specific population of cells. The control of gene expression is also very important for Ref: 127505 therapeutic approaches ex vivo or in vivo, in which the possibility of selectively controlling the production of a therapeutic molecule is essential. In fact, according to the applications, according to the gene to be transferred, it is important to be able to target certain tissues or only certain parts of an organism, in order to concentrate the therapeutic effect and limit the spread and effects secondary The direction of the expression of a given nucleic acid can be carried out according to different approaches or methods. A first method consists, for example, of using the vectors or transfer agents that have a given cellular specificity. However, the specificity conferred by this type of vector is generally quite coarse and does not allow targeting in precise populations of cells. Another method is to use the expression signals specific to certain cell types. In this regard, so-called "specific" promoters have been described in the literature, such as the promoter of the genes encoding pyruvate kinase, villin, GFAP, the intestinal protein promoter of fatty acid binding, the actin a promoter of smooth muscle cells, the SM22 promoter or even the promoter of the human albumin gene for example. However, if these promoters present a tissue specificity, they present likewise, in counterpart, a relatively weak power. Thus, the vast majority of these promoters have levels of activity without reaching those of the promoters called "strong", generally by a factor between 10 and 100 at least. On the other hand, it is generally considered that the specificity of a promoter is inversely proportional to its strength, and that, the more the force is increased, the more important is the level of non-specific activity. It will be, therefore, particularly advantageous to be able to have promoters that are at the same time specific to certain tissues and forces. The object of the present invention is precisely to provide new constructs that allow the strong and directed expression of the genes. The invention describes in particular novel chimeric promoters that allow expression of the strong and specific genes of smooth muscle cells. The invention also describes vectors containing such promoters and their use for gene transfer in cells in vitro, ex vivo and in vivo. The constructs of the invention make it possible for the first time to combine the opposite properties, namely high selectivity and high transcriptional activity. The present invention thus offers a means particularly suitable for the directing of the expression of genes in the cells of the smooth muscle, in vivo or in vitro, and for the regulation of this expression. The invention is based, more particularly, on the construction of chimeric (or hybrid) promoters, comprising regions of different origin and function. More particularly, a first object of the invention resides in a hybrid promoter comprising: - all or part of the promoter region of a strong and ubiquitous promoter / enhancer, and - a region of the promoter that allows specific expression in the smooth muscle cells. The association of the enhancer and promoter regions has already been described in the prior art. Thus, it is known, for example, how to couple the CMV enhancer region with non-specific promoters, such as the promoter of the chicken β actin gene (W096 / 13597) in order to increase its strength. However, such constructs have not been described or suggested in order to try to obtain a strong and specific expression of smooth muscle cells. The invention is based, in part, on the selection and combination of particular "enhancer" elements and particular "promoter" elements. The invention is also based on the demonstration that this combination of elements allows an expression to be obtained at high levels, without affecting the selectivity of the promoter for smooth muscle target cells. The invention therefore provides particularly advantageous constructs, because they allow directed production and with important levels of molecules in smooth muscle cells. On the other hand, the present application also shows that these constructs can also be used in vitro or in vivo. In the hybrid promoters according to the invention, the enhancer region and the promoter region are functionally associated, ie, such that the enhancer region exerts a stimulating activity on the activity of the promoter region. Generally, these two regions are, therefore, genetically linked and are sufficiently close to one another to allow the enhancer region to activate the promoter region. Preferably, the distance separating the promoter region and the promoter region is less than 1 kb, more preferably less than 500 bp. In particularly preferred constructs according to the invention, these two regions are separated by less than 400 bp, more preferably by less than 200 bp. On the other hand, as shown in the examples, the respective orientation of the two regions has no significant influence on the activity of the hybrid promoters of the invention. Because of this fact, the region of the enhancer can be positioned in the same orientation or in reverse orientation with respect to the sense of transcription of the promoter region. In a preferred mode of implementation, the enhancer region is chosen from the immediate precursor gene region of the cyto egalovirus (CMV-IE), the LTR enhancer region of Rous sarcoma virus (LTR-RSV), the SV40 virus enhancer region, and the EFIa gene enhancer region. More preferably, in the hybrid promoters of the invention, the enhancer region is the enhancer region of the immediate cytomegalovirus immediate gene (CMV-IE), preferably the human cytomegalovirus (hCMV-IE). A region of the particular enhancer is constituted, for example, of the fragment -522 to -63 of the hCMV-IE gene, or of any fragment comprising at least a part thereof and having an enhancing activity. If it is the promoter region, a region comprising all or part of the promoter of the gene encoding the smooth muscle cell actin-a (SMact) or the SM22 gene is advantageously used for the implementation of the invention. The promoter of these genes has been described for its specific character of smooth muscle cells (see particularly Ueyama H. et al., Mol. Cell. Biol., 4 (1984) 1073-1078; Solway J. et al., J. Biol. Chem. , 270 (1995) 13460-13469). A first particularly advantageous variant of the present invention is constituted by a hybrid promoter comprising: - all or part of the enhancer region of the immediate prior gene of the human cytomegalovirus (hCMV-IE), and - all or part of the promoter of the gene which encodes for smooth muscle cell actin-a (SMact), preferably the human Smact gene. A second particularly advantageous variant of the present invention is constituted by a hybrid promoter comprising: - all or part of the enhancer region of the immediate prior gene of the human cytomegalovirus (hCMV-IE), and all or part of the promoter of the SM22 gene, preferably of the mouse SM22 alpha gene. Furthermore, in a particular embodiment of the invention, the promoter region used is a chimeric region comprising a basal promoter and a sequence that confers tissue specificity, said sequence is derived from the SMact promoter or the SM22 promoter, or from a combination of both. In this embodiment, the basal promoter can be a "minimal" promoter, ie comprising only the sequences essential for the activity of the transcriptional promoter (for example, a TATA cassette). This basal promoter can be the basal promoter of the SMact gene or SM22, or of heterologous origin (ß-globin, HSV-TK, SV40 or EFI-a for example). The sequence conferring tissue specificity advantageously comprises a part of the sequence of the SMact promoter (RT Shimizu et al., J. Biol. Chem. 270 (1995) 7634-7643) and / or of the SM22 promoter (L.Li et al. J Cell Biol. 132 (1996) 849-859; S. Kim et al., J. Clin, Invest 100 (1997) 1006-1014; Kemp et al., Biochem. J. 310 (1995) 1037-1043). A particular type of the hybrid promoter according to the invention therefore comprises: all or part of the enhancer region of a strong and ubiquitous promoter / enhancer, a basal promoter and a sequence that confers the tissue specificity comprising all or part of the SMact promoter and / or the SM22 promoter. For the construction of the hybrid promoters of the invention, the molecular biology techniques known to the person skilled in the art can be applied. Thus, the enhancer region and the promoter region (comprising the basal promoter and the sequence that confers the specificity tissue) can be isolated by classical techniques from nucleic acid libraries or from total cellular DNA, for example, by amplification by means of specific probes. These fragments can also be artificially synthesized using information from the sequences available in the prior art. When these fragments are obtained, they can be easily combined among them by means of ligases and other restriction enzymes, for general hybrid promoters of the invention. On the other hand, these fragments can be modified by digestion, mutation, insertion or addition of base pairs, either in order to facilitate their cloning, either in order to modify their functional properties. Furthermore, as indicated hereinabove, the fragments can be directly associated with one another, or on the contrary, separated by base pairs that have no significant influence on the activity of the hybrid promoter. The hybrid promoters of the invention thus possess the ability to express a nucleic acid of interest, specifically in smooth muscle cells. The "specific" nature of the expression means that the activity of the promoter is significantly much higher in smooth muscle cells. Although a non-specific expression may exist in other cells, the level of corresponding activity usually remains very weak (negligible) with respect to that observed in smooth muscle cells, generally less than a factor of at least 10. The results presented in the examples show in this respect, an expression differential that can reach a factor of 140, which attests to the important selectivity of the promoters of the invention. In this regard, the results presented also show a strong specificity with respect to the smooth muscle cells since no expression has been detected in the endothelial cells that are in the vicinity, in the blood vessels, particularly the arteries. The results presented in the examples also show that the strength of the promoters of the invention is much higher than that of the specific non-hybrid promoters, the differential can exceed a factor of 100. These elements therefore illustrate the advantageous properties of the hybrid promoters of the invention, in terms of strength and specificity, for the expression of nucleic acids of interest in smooth muscle cells. In this regard, another object of the invention relates to an expression cassette comprising a nucleic acid encoding an RNA or a polypeptide of interest, placed under the control of a hybrid promoter, as defined above. Advantageously, the The cassette of the invention comprises, on the other hand, a transcription termination signal, placed in the 3 'position of the nucleic acid. Taking into account the white cell populations by the cassettes of the invention, the nucleic acid can encode, for example, for a protein chosen from: the proteins involved in the cell cycle, such as, for example, p21 or any other protein inhibiting the cyclin-dependent kinases, (cdk), the gene product of retinoblastoma (Rb), GAX, GAS-1, GAS-3, GAS-6, Gadd 45, Gadd 153, cyclins A, B and D. proteins that induce apoptosis, such as, for example, p53, the members of the family of apoptosis inducers, such as Bas, Bcl-Xs, Bad or any other antagonist of Bcl2 and Bcl-X ?. proteins capable of modifying the proliferation of smooth muscle cells, such as, for example, an intracellular antibody or a ScFv that inhibits the activity of proteins involved in cell proliferation, such as, for example, the Ras protein, the kinase, or the receptors of tyrosine kinase or growth factors. the labeling proteins (LacZ, GFP, Luc, secreted alkaline phosphatase (SeAP), hormone growth (GH) etc.), for the purpose of conducting proliferation studies or diagnostic studies, the proteins that induce angiogenesis, such as, for example, members of the VEGF family, members of the FGF family and more particularly FGFI, FGF2, FGF4, FGFS, angiogenin, EGF, TGFa, TGFβ, TNFα, Factor Scatter / HGF, members of the family of angiopoietins, cytokines and in particular interleukins IL-1, IL-2, IL-8, angiotensin-2, plasminogen activator (TPA) ), urokinase (uPA), the molecules involved in the synthesis of active lipids (prostaglandins, Cox-1). transcription factors, such as, for example, natural or chimeric nuclear receptors, comprising a DNA binding domain, a ligand-binding domain and an activating or transcriptional-inhibiting domain, such as, for example, proteins tetR-NLS-VP16 fusion proteins, fusion proteins derived from oestrogenic receptors, fusion proteins derived from steroid hormone receptors, fusion proteins derived from progesterone receptors, proteins from the CID system (Chemical Inducer of Dimerization) described by Rivera et al. (Rivera et al. (1996), A humanized system for pharmacologic control of gene expression, Na ture Medecine, 2: 1028-1032). It is understood that the present invention is not limited to particular examples of proteins or RNA, but that it can be used by the person skilled in the art for the expression of any nucleic acid in smooth muscle cells, by ordinary simple experimentation operations. Another object of the invention relates, on the other hand, to any vector comprising a hybrid promoter or a cassette as defined above. The vector of the invention can be, for example, a plasmid, a cosmid or any DNA not encapsulated by a virus, a phage, an artificial chromosome, a recombinant virus, etc. It is preferably a plasmid or a recombinant virus. Among the plasmid type vectors, mention may be made of all cloning and / or expression plasmids known to those skilled in the art and generally comprising a duplication origin. Mention may also be made of the new generation plasmids, which carry replication origins and / or improved markers, such as those described, for example, in applications W096 / 26270 and PCT / FR96 / 01414.

Among the vectors of the recombinant virus type, there can be mentioned, preferably, the adenovirus, retrovirus, herpes virus or recombinant adeno-associated virus. The construction of this type of defective recombinant viruses for duplication has been widely described in the literature, as well as the infection properties of these vectors (see in particular S. Baeck et K.L. March (1998), Circuit Research vol. 82, pp. 295-305), T. Shenk, B.N. Fields, D.M. Knipe, P.M. Howley et al (1996), Adenoviridae: the viruses and their replication (in virology). Pp 211-2148, EDS - Ravenspublishers / Philadelphia, P. Yeh and M. Perricaudet (1997), FASEB Vol. 11, pp 615-623. A particularly preferred recombinant virus for the application of the invention is a defective recombinant adenovirus. Adenoviruses are linear double-stranded DNA viruses, approximately 36 (kilobases) kb in size. There are different serotypes, whose structure and properties vary a little, but which have a comparable genetic organization. More particularly, the recombinant adenoviruses may be of human or animal origin. With respect to adenoviruses of human origin, those classified in group C, in particular adenoviruses of type 2 (Ad2), 5 (Ad5), 7 (Ad7) or 12 (Adl2), can be cited preferably. Among the different adenoviruses of origin animal, can be cited preferably, adenoviruses of canine origin, and particularly all strains of adenovirus CAV2 [strain manhattan or A26 / 61 (ATCC VR-800) for example]. Other adenoviruses of animal origin are cited in particular in the application W094 / 26914 incorporated herein by reference. The genome of the adenoviruses especially comprises a repeated reverse sequence (ITR) at each end, an encapsulation sequence (Psi), early genes and late genes. The main early genes are contained in the El, E2, E3 and E4 regions. Among them, the genes contained in the El region, in particular, are necessary for viral propagation. The main late genes are contained in regions Ll to L5. The Ad5 adenovirus genome has been completely sequenced and is accessible in a database (see especially Genebank M73260). Likewise, part, even the totality of other adenoviral genomes (Ad2, Ad7, Adl2, etc.), have also been sequenced. For their use as recombinant vectors, different constructs derived from adenoviruses have been prepared, which incorporate different therapeutic genes. In each of these constructs, the adenovirus has been modified so that it becomes incapable of duplication in the infected cell. Thus, the constructs described in Prior art are the adenoviruses with the deleted region, essential for viral duplication, at which level the heterologous DNA sequences are inserted (Levrero et al., Gene 101 (1991) 195; Gosh-Choudhury et al., Gene 50 (1986) 161). In addition, to improve the properties of the vector, it has been proposed to create other deletions or modifications in the adenovirus genome. Thus, a thermosensitive point mutation has been introduced into the tsl 25 mutant, which allows inactivating the 72 kDa DNA binding protein (DBP) (Van der Vliet et al., 1975). Other vectors comprise a deletion of another region essential for duplication and / or viral propagation, the E region. The E4 region is, in effect, involved in the regulation of the expression of late genes, in the stability of late nuclear RNAs, in the extinction of the expression of the host cell proteins and in the efficiency of the duplication of the Viral DNA The adenoviral vectors in which the regions El and E4 are deleted, therefore possess a background noise of transcription and an expression of very reduced viral genes. Such vectors have been described, for example, in applications W094 / 28152, WO95 / 02697, W096 / 22378). On the other hand, vectors carrying a modification at the level of the IVa2 gene have also been described (WO96 / 10088).

In a preferred embodiment of the invention, the recombinant adenovirus is a human adenovirus of group C. More preferably, it is an Ad2 or Ad5 adenovirus. Advantageously, the recombinant adenovirus used in the context of the invention comprises a deletion in the El region of its genome. Even more particularly, it comprises a deletion of the Ela and Elb regions. By way of a precise example, we can mention the deletions that affect nucleotides 454-3328; 382-3446 or 357-4020 (with reference to the Ad5 genome). According to a preferred variant, the recombinant adenovirus used in the context of the invention comprises, on the other hand, a deletion in the E4 region of its genome. More particularly, the deletion in the E4 region affects the set of the open phases. The deletions 33466-35535 or 33093-35535 can be cited as a precise example. Other types of deletions in the E4 region are described in the applications WO95 / 02697 and W096 / 22378, incorporated herein by reference. The expression cassette can be inserted in different sites of the recombinant genome. It can be inserted at the level of the region El, E3 or E4, replacing the suppressed or excess sequences. It can also be inserted in any other place, outside the sequences necessary in cis for the production of viruses (ITR sequences and encapsulation sequence). The recombinant adenoviruses are produced in an encapsulation line, ie a line of cells capable of trans-complementing one or more of the deficient functions in the recombinant adenoviral genome. Among the encapsulation lines known to the person skilled in the art, there may be mentioned, for example, line 293 in which a part of the adenovirus genome has been integrated. More precisely, line 293 is a line of human kidney embryonic cells, containing the left end (approximately 11-12%) of the adenovirus serotype 5 (Ad5) genome, comprising the left ITR, the encapsulation region , the El region, including Ela and Elb, the region encoding the pIX protein and a part of the region encoding the pIVa2 protein. This line is capable of trans-complementing the recombinant adenoviruses defective for the El region, ie, devoid of all or part of the El region, and of producing viral patterns having high titers. This line is also capable of producing, at permissive temperature (32 ° C), the virus patterns that comprise, on the other hand, the thermosensitive E2 mutation. Other cell lines capable of complementing the El region have been described, based particularly on human lung carcinoma cells A549 (W094 / 28152) or on human retinoblasts (Hum. Gen. Ther. (1996) 215). In addition, lines capable of trans-complementing various adenovirus functions have also been described. In particular, the lines complementing the regions El and E4 (Yeh et al., J. Virol. Vol. 70 (1996) pp 559-565; Cancer Gen. Ther.2 (1995) 322; Krougliak et al. ., Hum. Gen. Ther.6 (1995) 1575) and the lines that complement the El and E2 regions (W094 / 28152, WO95 / 02697, WO95 / 27071). Recombinant adenoviruses are usually produced by the introduction of viral DNA into the encapsulation line, followed by a lysate of the cells after approximately 2 or 3 days (the kinetics of the adenoviral cycle is from 24 to 36 hours). For the implementation of the process, the introduced viral DNA can be the complete recombinant viral genome, eventually constructed in a bacterium (WO96 / 25506) or in a yeast (WO95 / 03400), transfected in the cells. It can also be a recombinant virus used to infect the encapsulation line. The viral DNA can also be introduced in the form of fragments that each carry a part of the recombinant viral genome and a zone of homology that allows, after introduction into the encapsulation cell, to reconstitute the recombinant viral genome by homologous recombination. between the different fragments.

After lysate of the cells, the recombinant viral particles are isolated by centrifugation in a gradient of cesium chloride. An alternative method has been described in the application FR96: 08164 incorporated herein by reference. The invention also relates to a composition comprising a vector as defined above and a chemical or biochemical transfer agent. The term "chemical transfer or biochemical agent" is understood to be any compound (ie, different from a recombinant virus), which facilitates the penetration of a nucleic acid into a cell. It can be cationic non-viral agents, such as cationic lipids, peptides, polymers (Polyethylene Imine, Polylysine), nanoparticles; or non-cationic non-viral agents such as non-cationic liposomes, polymers or non-cationic nanoparticles. Such agents are well known to the person skilled in the art. The invention also relates to a composition comprising a recombinant virus as defined above and to a physiologically acceptable carrier. The invention also relates to a pharmaceutical composition comprising a vector such as that described above. The pharmaceutical compositions of invention can be formulated in view of topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. administration. Preferably, the pharmaceutical composition contains pharmaceutically acceptable carriers for an injectable formulation, in particular for an intravenous injection or smooth muscle tissues. In particular, it is possible to treat saline solutions (monosodium phosphate, disodium, sodium chloride, potassium, calcium or magnesium, etc., or mixtures of such salts), sterile, isotonic, or dry compositions, especially lyophilized, which the addition, according to the case, of sterilized water or physiological saline, allows the constitution of injectable solutions. Other excipients may be used such as, for example, a hydrogel. This hydrogel can be prepared from any polymer (homo or hetero) biocompatible and non-cytotoxic. Such polymers have been, for example, described in the application WO93 / 08845. Certain among them, as particularly those obtained from ethylene oxide and / or propylene are commercial. The use of a hydrogel is particularly advantageous for the transfer of nucleic acids in the vascular walls, and especially in the smooth muscle cells of the vascular walls. The doses used for the injection they can be adapted according to different parameters, and especially depending on the mode of administration used, the object to be followed (marking, pathology, diagnosis, etc.), the gene to be expressed, or even the duration of the expression sought. In a general manner, the recombinant viruses according to the invention are formulated and administered in the form of doses comprised between 104 and 1014 pfu, and preferably 106 to 1010 pfu. The term pfu ("plaque forming unit") corresponds to the infectious power of a viral solution, and is determined by the infection of an appropriate cell culture, and the measurement of the number of plaques of infected cells. The techniques for determining the pfu titer of a viral solution are well documented in the literature. For an in vitro or ex vivo use, the cassettes, vectors or compositions of the invention can be incubated at classical doses in the presence of selected cell populations. These incubations can be carried out in culture boxes, in flasks, in fermenters, or in any other chosen device. In addition, the invention also relates to any cell modified by a cassette or a vector (especially an adenovirus) such as those described above. "Modified" cell is understood to mean any cell that contains a construct according to the invention. These cells can be used for the production of recombinant proteins in vitro. They can also be used for an implantation in an organization, according to the methodology described in the application W095 / 14785. These cells are preferably human smooth muscle cells. The invention also relates to the use of a hybrid promoter such as defined above for the specific expression of a nucleic acid in smooth muscle cells, in vitro, ex vivo or in vivo. The invention also relates to the use of a hybrid promoter as defined above for the preparation of a composition intended for the expression of a nucleic acid in smooth muscle cells in vivo and not in the endothelial cells found in the neighborhood of the artery. Due to the specific character of the smooth muscle cells, the constructs according to the invention are equally usable for the creation of animated models of vascular pathologies or for carrying out labeling studies or in methods of detection or diagnosis of the presence of cells smooth muscles in the samples. The subject of the present invention is a process for the production of recombinant proteins that comprises the introduction into a cell population of a vector such as defined above, the culture of said recombinant cell population, and the recovery of said produced protein. Advantageously, for the implementation of the method of the invention, smooth muscle cells are used. It can be established lines or primary crops. The present application will be described in more detail with the help of the following examples, which should be considered as illustrative and not as limiting.

LEGENDS OF THE FIGURES Table I Relative activities of the hybrid promoters (hSMa-actin) evaluated in transient transfections in the smooth muscle cells of rabbit in primary culture (rabbit SMC), in ECV304 cells, in C2C12 myoblasts, in HeLa cells, in NIH 3T3 cells, in TU182 carcinoma cells, as well as in renal cells 293. The relative activity of each promoter is expressed as a percentage of the luciferase activity obtained with the plasmid pCMV-leaderTK. Enh-X: the enhancer sequence of hCMV-IE is cloned upstream of the X promoter according to its normal orientation. HnE-X: the The enhancer sequence of hCMV-IE is cloned upstream of the X promoter according to the opposite orientation. Table II: Relative activities of hybrid promoters (mSM22) evaluated in transient transfections in vi tro in rabbit smooth muscle cells in primary culture (rabbit SMC), in ECV304 cells, in C2C12 myoblasts, in HeLa cells, in NIH cells 3T3, in the TU182 carcinoma cells, as well as in the renal cells 293. The relative activity of each promoter is expressed as a percentage of the luciferase activity obtained with the pCMV-leadTK plasmid. Enh-X: the enhancer sequence of hCMV-IE is cloned upstream of the X promoter according to its normal orientation. HnE-X: the enhancer sequence of hCMV-IE is cloned upstream of the X promoter according to the opposite orientation. Figure 1: Schematic representations of the plasmids whose expression cassette contains the hSMa-actin hybrid promoter. Figure 2: Schematic representations of the plasmids whose expression cassette contains the mSM22a hybrid promoter. Figure 3: Activities of the hybrid promoters evaluated in transient transfections in vi tro in the smooth muscle cells of rabbit in primary culture (SMC of rabbit), in endothelial cells from a human umbilical cord carcinoma (ECV304), in mouse myoblasts (C2C12), as well as in epithelial cells from a carcinoma of the human cervix (HeLa). The relative activity of each promoter is expressed as a percentage of the activity of the luciferase obtained with the plasmid pCMV-leadTK. Enh-X: the enhancer sequence of hCMV-IE is cloned upstream of the X promoter according to its normal orientation. hnE-X: the enhancer sequence of hCMV-IE is cloned upstream of the X promoter according to the opposite orientation. Figure 4: Activities of the hybrid promoters evaluated in transient transfections in vi tro in mouse embryonic fibroblasts (NIH 3T3), in the cells derived from a human ORL carcinoma (TU182), as well as in the transformed human embryonic kidney cells (293) . The relative activity of each promoter is expressed as a percentage of the activity of the luciferase obtained with the plasmid pCMV-leadTK. Enh-X: the enhancer sequence of hCMV-IE is cloned upstream of the X promoter according to its normal orientation. hnE-X: the enhancer sequence of hCMV-IE is cloned upstream of the X promoter according to the opposite orientation. Figure 5: Activities of the hybrid promoters evaluated in the gene transfer in vivo in the muscle warm cranial mouse CS7BL6. The relative activity of each promoter is expressed as a percentage of the activity of the luciferase obtained with the plasmid pCMV-leadTK. Figure 6: Description of the construction of the AVI.OS A.Gax virus MATERIALS AND METHODS The methods used classically in molecular biology, such as the preparative extractions of plasmid DNA, the centrifugation of plasmid DNA in a gradient of cesium chloride, the electrophoresis on agarose gels, the purification of DNA fragments by electroelution, the precipitation of plasmid DNA in saline medium by ethanol or isopropanol, the transformation in Escherichia coli are well known to those skilled in the art and are abundantly described in the literature (Sambrook et al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). Plasmid pGL3-Basic, used for the cloning of different promoter regions, is of origin commercial (Promega Corporation). Plasmids pCMVß (Clontech Laboratories Inc.) and pUC18 (Boehringer Mannheim) are also of commercial origin. The enzymatic amplification of DNA fragments or the ACP technique (Amplification in Chain by the Polymerase) can be performed using a thermal DNA cycler (Perkin Elmer Cetus) MR according to the manufacturer's recommendations. The electroporation of the plasmid DNA in Escherichia coli cells can be performed with the help of an electroporator (Bio-Rad) according to the manufacturer's recommendations. The verification of the nucleotide sequences can be carried out by the method developed by Sanger et al. (Proc. Na ti. Acad. Sci. USA, 74 (1977) 5463-5467) using the equipment distributed by Applied Biosystems according to the manufacturer's recommendations.

EXAMPLES EXAMPLE 1: Construction of hybrid promoters and expression plasmids containing them. 1.1. HSMa-actin hybrid promoters. Plasmid phSMact. The high molecular weight genomic DNA has been prepared according to the method described by Sambrook et al. ("Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989) from a primary culture of human aortic smooth muscle cells (Clonetics).

This DNA has been used as a matrix for a first amplification by ACP using the following primers: - Primer 6417 (5 'GATGGTCCCTACTTATGCTGCTA 3') (SEQ ID 1) starting at position -1034 (promoter region) of the a-gene. human-specific smooth muscle actin (Ueyama H. et al., Mol Cell Biol., 4 (1984) 1073-1078, Genbank Access D00618). Primer 6418 (5 'CTTCCATCATACCAAACTACATA 3') (SEQ ID 2) at position 1974 of sequence D00618 is located inside the first intron of the hSMact gene. The reaction mixture comprises 1 mg of genomic DNA, 10 pmol of each of the two primers (6417 and 6418), 100 mM of each deoxyribonucleotide (dATP, dCTP, dGTP, dTTP), 2 mM MgCl2 and 5 units of Taq. DNA polymerase (PerkinElmer). The reaction volume is completed to 50 ml adjusted to the optimum concentration of the ACP buffer recommended by Perkin Elmer. The ACP amplification is performed in Micoamp ™ tubes (Perkin Elmer) with the help of a PTC-100MR thermocycler (MJ Research, Inc.). This amplification consists of a denaturation step at 95 ° C for 2 min followed by 30 cycles comprising a denaturation step of 15 sec at 95 ° C, a hybridisation step of 30 sec at 60 ° C and a step extension from 1 min to 72 ° C. These thirty cycles are followed by a supplementary extension of 5 min, then the ACP reactions are conserved at 10 ° C. One microliter of this reaction has been taken from this first reaction, then diluted in 10 ml of water. Next, 1 ml of this dilution is used to make a second ACP under the same conditions as the first (above), but with a different pair of primers: - Primer 6453 (5 'CTGCTAAATTGctcgagGACAAATTAGACAAA 3') (SEQ ID 3), this primer introduces an Xhol site (underlined lower case) upstream of the hSMact promoter (position -680). 6456 Primer (5 ' CCCTGACAaagcttGGCTGGGCTGCTCCACTGG 3 ') (SEQ ID 4), this primer introduces a HindIII site in the +30 position of hSMact. After analysis on an agarose gel after purification, the DNA fragment amplified by ACP is digested for 3 hours at 37 ° C by Xhol and HindIII, then cloned into the vector pGL3-Basic (Proméga) previously digested by these same restriction enzymes, to generate the plasmid phSMact (Figure 1).

Plasmids pXL3130 and pXL3131. A fragment of DNA that corresponds to the promoter region of the IE gene of human cytomegalovirus (hCMV-IE) between positions -522 and -63 with respect to the site of transcription initiation, has been amplified by ACP using plasmid PCMVß as matrix and oligonucleotides 8557 (5 'ATC GAC GCG TGC CCG TTA CAT AAC TTA CGG 3 ') (SEQ ID 5) and 8558 (5 'ATC GAC GCG TCC GCT CGA GCG TCA ATG GGG CGG AGT TG 3') (SEQ D 6) as primers. This fragment has been digested by Mlul, then it has been cloned in the plasmid phSMact previously digested by Mlul and treated by alkaline phosphatase. According to the direction of insertion of the fragment, two different plasmids are obtained: pXL3130 and pXL3131. The schematic representations of these plasmids are represented in the figure (figure 1). These plasmids comprise, in the form of a Mlul-Ncol fragment, a hybrid promoter constituted by the hCMV-IE gene promoter enhancer and the hSMa-actin gene promoter. 1. 2. mSM22 hybrid promoters. Plasmid pmSM22. The high molecular weight genomic DNA has been prepared according to the method described by Sambrook et al. ("Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989) from Balbc mouse liver.

This DNA has been used as a matrix for an ACP amplification using the following primers: - Primer 6517: (5 'CCAGGTGCActcgagACTAGTTCCCACCAACTCGA 3') (SEQ ID 7), this primer introduces an Xhol site (lower case underlined) at position -436 of the promoter of mouse SM22 alpha gene (Solway J. et al., J. Biol. Chem., 270 (1995) 13460-13469; Genbank access L41161). -Cobador 6518: (5 'TCGTTTGaagcttGGAAGGAGAGTAGCTTCGGTGTC 3') (SEQ ID 8), this primer introduces a HindIII site at position +43 of mSM22 alpha. A reaction mixture comprising 1 mg of mouse genomic DNA, and 10 pmol of each of these two primers (6517 and 6518) has been prepared with the same reagents as for hSMact and at the same concentrations, followed by an ACP amplification performed in the same conditions (see example 1.1.). After analysis on an agarose gel followed by purification, the DNA fragment amplified by ACP is digested for 3 hours at 37 ° C by Xhol and HindIII, then cloned in the vector pGL3-Basic (Proméga) previously digested by these same restriction enzymes. The resulting plasmid has been designated as pmSM22 (Figure 2).

Plasmid pXL3152 and pXL3153. A DNA fragment corresponding to the promoter region of the hCMV-IE gene promoter between positions -522 and -63 with respect to the transcription initiation site, has been amplified by ACP using the pCMVβ plasmid as the matrix and the oligonucleotides 8557 (5 'ATC GAC GCG TGC CCG TTA CAT AAC TTA CGG 3') (SEQ ID 5) and 8558 (5 * ATC GAC GCG TCC GCT CGA GCG TCA ATG GGG CGG AGT TG 3 ') (SEQ ID 6) as primers . This fragment has been digested by Mlul, then it is cloned into the plasmid pmSM22 previously digested by Mlul and treated by alkaline phosphatase. According to the insertion direction of the fragment, two different plasmids have been obtained: pXL3152 and pXL3153. The schematic representations of these plasmids are represented in figure 2. These plasmids comprise, in the form of a Mlul-NeoI fragment, a hybrid promoter constituted by the promoter of the hCMV-IE gene promoter and of the mSM22 gene promoter. 1. 3. Control plasmid pCMV-leaderTK. The pCGN expression vector described above by Tanaka et al. (Cell, 60 (1990) 375-386) contains the CMV promoter (-522 / + 72) fused to the "leader" of the HSV tk gene (+ 51 / + 101) upstream of a sequence coding for the epitope of hemagglutinin. Plasmid pCGN (10 ng) has been used as a matrix for an ACP amplification. The ACP reaction, as well as the amplification, were carried out under the same conditions as those used for hSMact and mSM22 (examples 1.1 and 1.2). The primers that have been used are the following: Primer 6718 (5 'CCCGTTACATAACTTACGGTAAATGGCCCG 3') (SEQ ID 9), this primer hybridized with the CMV promoter at position -522 (8 nucleotides downstream of the EcoRI site of pCGN) . - Primer 6719 (5 'gGGACGCGCTTCTACAAGGCGCTGGCCGAA 3 ') (SEQ ID 10), this primer was hybridized to position 101 of "leader" tk. The first G nucleotide in bold is intended to restore the Ncol site of pGL3-Basic as will be explained later. The ACP fragment thus obtained is purified, then phosphorylated with the help of the T4 phage polynucleotide kinase (New England Biolabs). In parallel, the pGL3-Basic vector (Proméga) has been linearized by Ncol, purified, then treated by Klenow DNA polymerase (Boehringer Manheim), in order to fill the Ncol site. This vector is then dephosphorylated with the aid of alkaline phosphatase (Boehringer Manheim), then used for the insertion of the phosphorylated ACP fragment. Thus, guanosine (G) from primer 6719 allows the Ncol site to be restored only when the CMV-leader tk fragment is oriented with the 5 'part (primer 6718, position -522 of the CMV) downstream of the HindIII site of pGL3-Basic and its 3' end (primer 6719, leader tk) is ligated to the Ncol site of pGL3-Basic (first ATG of luciferase). The plasmid thus obtained is designated pCMV-leaderTK.

EXAMPLE 2: Specificity of hybrid promoters In vi tro. This example illustrates the tissue specificity properties of the hybrid promoters of the invention in vitro. 2. 1. Cell cultures. The smooth muscle cells (SMC) of rabbit are cultured in DMEMMR medium (Life Technologies Inc.), supplemented with 20% fetal calf serum (SVF). ECV304 cells are grown in 199 ™ medium (Life Technologies Inc.), supplemented with 10% SVF. The C2C12 myoblasts, HeLa cells, NIH 3T3 cells, as well as TU182 cells, are cultured in DMEMMR medium supplemented with 10% SVF. 293 cells are cultured in MEMMR medium (Life Technologies Inc.), supplemented with pyruvate, non-essential amino acids and 10% SVF. All the cultures are carried out in an oven at 37 ° C, in humid atmosphere and under a partial pressure of C02 of 5%. 2. 2. Transfections in vi tro. Transfections are performed in 24-well plates and each transfection is performed three times. Twenty-four hours before transfection, the cells are 4 seeded: (i) 5x10 cells per well for the smooth muscle cells of rabbit, the ECV304, NIH 313 and HeLa cells, (ii) 105 cells per well for the TU182 cells, ( iii) Four. Five 3x10 cells per well for C2C12 cells, and (iv) 2x10 cells per well for 293 cells. For each well, 500 ng of plasmid DNA (250 ng of the plasmid of interest and 250 ng of pUC18) are mixed with cationic lipid RPR120535 B (WO 97/18185) at a rate of 6 nmoles of lipid per μg of DNA in DMEMMR medium (final 20 μl) comprising 150 mM NaCl and 50 mM bicarbonate. After 20 minutes at room temperature, the 20 μl DNA / lipid mixture is contacted with the cells, in the absence of SVF, for 2 hours. The culture medium is then supplemented in SVF so that the percentage of SVF required for the culture of each cell type is obtained. Forty-eight hours after transfection, the culture medium is removed and the cells are rinsed with PBS (Life Technologies Inc.). The activity of Luciferase is now determined with the help of the Luciferase Assay SystemTM (Promega Corporation) team, according to the supplier's recommendations. 2. 3. Specific activities of the hybrid promoters. The activities of the relative luciferases of the hybrid promoters (with respect to the promoter pCMV-leader TK) measured in vi tro in seven different cell types, are represented in the 3 and 4, as well as in Tables I and II. The results show that for the promoters hSMact (Table I) and mSM22 (Table II), the relative activity is a value that renders a good account of the specificity of these promoters for smooth muscle cells; specificity that has already been described in the literature (Skalli et al., J. Histochem, Cytochem., 37 (1989) 315-321).; Shimizu et al., J. Biol. Chem. , 270 (1995) 7631-7643; Li et al., J. Cell Biol. , 132 (1996) 849-859). Indeed, the relative activity in rabbit SMCs is at least 5 times higher than those observed in the other cell types: (i) 5 to 20 times higher for the hSMact promoter (Table I), and (ii) 5 25 fold higher for the mSM22 promoter (Table II). The results presented in Tables I and II clearly show that in smooth muscle cells, the activity of the four hybrid promoters according to the invention (Enh-hSMact, hnE-hSMact, Enh-mSM22, and hnE-mSM22) is comparable, in terms of strength, to that of the CMV promoter. On the other hand, the relative activity of each of these promoters, in another cell type, is at least 10 times lower for the hybrid promoters hSMact, and at least 4 times lower for the hybrid promoters mSM22, than that observed in the muscle cells smooth: (i) from 10 to 140 times for the hybrid promoters hSMact, and (ii) from 4 to 55 times for the hybrid promoters mSM22. These hybrid promoters therefore retain the same tissue specificity as that observed for the specific promoters hSMact and mSM22. These results show, on the other hand, that the orientation of the enhancer region in the hybrid promoters of the invention have no significant influence on its activity. The four hybrid promoters therefore possess, in vitro, an activity in smooth muscle cells as important as that of the CMV promoter (which has the reputation of being a strong promoter), retaining comparable, or even superior, tissue specificity to that promoter. of the hSMact and mSM22 promoters.

EXAMPLE 3: Specificity of the hybrid promoters in vivo. This example illustrates the tissue specificity properties of the hybrid promoters of the invention in vivo. 3. 1. Gene transfer in skeletal muscle. The different plasmids have been injected, intramuscularly, into the cranial tibial muscle of female C57BL6 mice, 5 weeks of age. Each plasmid, diluted in a final 150 mM NaCl solution, is injected at a rate of 10 μg per muscle. Three days after the injection, the muscles are placed in 2 ml of Cell Culture MR Lysis Reagent buffer (Promega Corporation), and crushed with the aid of a Diax homogenizer (Heidolph). The grinding is then centrifuged for 15 minutes at 4000 g, then the activity of the luciferase is evaluated with the help of a Luciferase Assay SystemTM (Promega Corporation) equipment, according to the supplier's recommendations. 3. 2. Activities of the hybrid promoters in skeletal muscle in vivo.

The relative activities of two specific promoters (hSMact and mSM22), such as those of two of the hybrid promoters of the invention (Enh-hSMact and Enh-mSM22) have also been evaluated in vivo after the transfer of DNA only in the warm muscle. cranial mouse. The results depicted in Figure 5 show that the activity of the Enh-hSMact promoter is 100 times lower than that of the CMV promoter. Similarly, the activity of the Enh-mSM22 promoter is 17 times lower than that of the CMV promoter. Thus, the tissue specificity observed in vi tro, and especially in the C2C12 cells that constitute the closest model thereof used in vivo, is, therefore, conserved in vivo.

EXAMPLE 4: Construction of recombinant adenoviruses expressing the GAX protein under the control of specific hybrid promoters. The purpose of this example is to describe an adenoviral vector carrying the gene coding for the GAX protein operationally linked to the hybrid promoter of the invention, composed of the CMV enhancer and the SMa-actin promoter (enh-hSMact). The human gax gene comprises 912 base pairs and codes for a transcription factor of 303 amino acids involved in the arrest of cell growth (growth-arrest-specific homeobox) and have a role on the proliferation of human smooth muscle cells. This gene with a homeodomain has been initially isolated from the aorta and is expressed, in particular, in adult cardiovascular tissues (Gorski et al., 1993). The human gax gene sequence has been cloned from a skeletal muscle cDNA library by PCR (Polymerase Chain Reaction), using as primer a sequence derived from the human gax gene and published by Walsh et al. (Genomics (1994), 24, p535). The sequence below is cloned into the expression vector pXL3297.

This plasmid is derived from the Bluescript plasmid (Stratagene) containing the human CMV IE promoter / enhancer (-522 / + 72) (Cell (1985), 41, p521) and SV poly A 40 (2538-2759) (locus GenBank SV4CG). The construct uses the plasmid pXL3130 described in Example 1 (Figure 1), in which the SMa promoter actin had previously been introduced. Plasmid pXL3297 is an expression vector that contains the human gax gene. It has been digested by the enzymes HindIII and Avrll in order to introduce the human gax gene into the preceding plasmid pXL3282 equally digested by the enzymes HindIII and Avrll to provide the plasmid pXL 3300. As indicated in figure 6, in which the different stages of the The construction of the plasmids described above are detailed, the final plasmid, pXL3310 comprises an expression cassette consisting of the CMV-IE enhancer, the SMa-actin promoter (pSMA), associated according to the invention and operationally linked to the gene coding for the human GAX protein, as well as the SV40 poly A termination signal. The expression cassette of the human gax gene is then introduced into a recombinant human adenovirus of serotype 5 (Ad5) with the El and E3 regions suppressed by cotransfection and homologous recombination between the plasmid carrying the gax gene expression cassette and the adenovirus, in encapsulation cells. These cells are preferably line 293. The production of an adenovirus standard containing the expression cassette of the human gax gene results from the lysing of the encapsulation cells 2 or 3 days after infection and the isolation of the particles. recombinant virals, by centrifugation in a cesium chloride gradient. The viral particles are then used to study the expression of the human gax gene under the control of the promoter of the invention in smooth muscle cells. The expression of the GAX protein is verified 24 hours after the infection of the smooth muscle primary cells by immunofluorescence or by spotting western, using polyclonal rabbit anti-gax antibodies. The expression of the messenger RNAs is analyzed 24 hours after the infection of the smooth muscle cells by spotting and northern spotting, using an oligonucleotide whose sequence is present in the gax gene. The analysis of the biological activity of the adenovirus coding for the gax gene is carried out in the following manner: Smooth muscle cells in exponential growth phase are infected with an adenovirus containing the gene coding for the GAX protein, under the control of the enh-hSMact promoter, in the absence and presence of 125 ng of lipofectin (48-well plates). Adenoviruses (in varying dilutions) and lipofectamine are incubated for 30 minutes at ambient temperatures in a serum-deprived medium. The mixture or virus is only contacted with the cells for one hour at 37 ° C. At the end of the infection period, the medium containing the virus is removed and the cells are incubated in DMEM medium containing 0.5% SVF. In the 24 hours following the infection, the culture medium is replaced by a growth medium for half of the cultures and the incubation is continued 48 hours to allow the cells to enter the S phase. For the other half of the crops, a means weakly mitogen is added to keep the cells at rest. Viable cells are counted 72 hours after infection using the Alamar protocol.

TABLE 1 TABLE II It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

LIST OF SEQUENCES < 110 > RHONE-POULENC RORER < 120 > Use of specific hybrid promoters to control tissue expression < 130 > Sequence list of the PCT application < 140 > < 141 > < 150 > FR9812000 < 151 > 1998-09-25 < 160 > 10 < 170 > Patentln Ver. 2.1 < 210 > 1 < 211 > '23 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: oligonucleotide < 220 > < 221 > misc_feature < 222 > (1) .. (23) < 400 > 1 gatggtccct acttatgctg cta 23 < 210 > 2 < 211 > 23 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: oligonucleotide < 220 > '< 221 > misc_feature < 222 > (1) •• (23) < 400 > cttccatcat accaaactac ata 23 < 210 > 3 < 211 > 32 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: oligonucleotide < 220 > < 221 > misc_feature < 222 > (1) .. (32) < 400 > 3 ctgctaaatt gctcgaggac aattagaca aa 32 < 210 > 4 < 211 > 33 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: oligonucleotide < 220 > < 221 > misc_feature < 222 > (1) .. (33) < 400 > 4 ccctgacaaa gcttggctgg gctgctccac tgg 33 < 210 > 5 < 211 > 30 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence oligonucleotide < 220 > < 221 > mise feature < 222 > (1) .. (30) < 400 > 5 atcgacgcgt gcccgttaca taacttacgg 30 < 210 > 6 < 211 > 38 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: oligonucleotide < 220 > < 221 > misc_feature < 222 > (1) .. (38) < 400 > 6 atcgacgcgt ccgctcgagc gtcaatgggg cggagttg 38 < 210 > 7 < 211 > 36 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: oligonucleotide < 220 > < 221 > mise feature < 222 > (1) • • (36) < 400 > 7 ccaggctgca ctcgagacta gttcccacca actcga 36 < 210 > < 211 > 36 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: oligonucleotide < 220 > < 221 > misc_feature < 222 > (1) •• (36) < 400 > 8 tcgtttgaag cttggaagga gagtagcttc ggtgtc 36 < 210 > 9 < 211 > 30 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: oligonucleotide < 220 > < 221 > mise feature < 222 > (1) .. (30) < 400 > 9 cccgttacat aacttacggt aaatggcccg 30 < 210 > 10 < 211 > 30 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: oligonucleotide < 220 > < 221 > misc_feature < 222 > (1) .. (23) < 400 > 10 gggacgcgct tctacaaggc gctggccgaa 30

Claims (17)

46 CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A hybrid promoter, characterized in that it comprises: all or part of the enhancer region of a strong and ubiquitous promoter / enhancer, and - a region of the promoter that allows specific expression in smooth muscle cells.

2. The hybrid promoter according to claim 1, characterized in that the enhancer region is chosen from the enhancer region of the cytomegalovirus immediate early gene (CMV-IE), the LTR enhancer region of Rous sarcoma virus ( LTR-RSV), the SV40 virus enhancer region, and the EFIa gene enhancer region.

3. The hybrid promoter according to claim 2, characterized in that the enhancer region is the enhancer region of the immediate early cytomegalovirus gene (CMV-IE), preferably the human cytomegalovirus (hCMV-IE). 47 .

The hybrid promoter according to claim 1, characterized in that the promoter region comprises all or part of the promoter of the gene encoding the smooth muscle cell actin-a (SMact) or the SM22 gene.

5. A hybrid promoter, characterized in that it comprises: - all or part of the enhancer region of the immediate early gene of the human cytomegalovirus (hCMV-IE), and - all or part of the promoter of the gene coding for the actin-a of the smooth muscle cells (SMact).

6. A hybrid promoter, characterized in that it comprises: - all or part of the enhancer region of the immediate early human cytomegalovirus gene (hCMV-IE), and all or part of the promoter of the SM22 gene.

7. The hybrid promoter according to claim 1, characterized in that the promoter region comprises a basal promoter and a sequence conferring tissue specificity, said sequence is derived from the SMact promoter and / or the SM22 promoter.

8. An expression cassette, characterized in that it comprises a nucleic acid encoding an RNA or a polypeptide of interest, placed under the control of an 48 hybrid promoter, according to one of claims 1 to 7.

9. A cassette according to claim 8, characterized in that it comprises, on the other hand, a transcription termination signal.

10. The cassette according to claim 8 or 9, characterized in that the nucleic acid encodes a protein chosen among the proteins involved in the cell cycle, the proteins that induce apoptosis, the proteins capable of modifying the proliferation of muscle cells smooth, the proteins that induce angiogenesis and transcription factors.

11. A vector, characterized in that it comprises a hybrid promoter according to claim 1 or a catheter according to claim 8.

12. The vector according to claim 11, characterized in that it is a plasmid, a cosmid or any other DNA not encapsulated by a virus.

13. The vector according to claim 11, characterized in that it is a recombinant virus, preferably derived from an adenovirus, a retrovirus, a herpes virus or an adeno-associated virus.

14. A composition, characterized in that it comprises a vector according to claim 12 and a chemical or biochemical transfer agent.

15. A composition, characterized in that it comprises a recombinant virus according to claim 13 and a physiologically acceptable carrier.

16. A cell modified by a cassette according to claim 8 or a vector according to claim 11.

17. The use of a hybrid promoter according to one of claims 1 to 7, for the preparation of a composition intended for for the selective expression of a nucleic acid in smooth muscle cells. The use of a hybrid promoter according to one of claims 1 to 7, for the preparation of a composition intended for the expression of a nucleic acid in smooth muscle cells and not in endothelial cells that are in the vicinity of the blood vessels.